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Sample records for amazonensis recombinant antigen

  1. Immune responses induced by a Leishmania (Leishmania amazonensis recombinant antigen in mice and lymphocytes from vaccinated subjects

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    Ana Paula FERNANDES

    1997-03-01

    Full Text Available In the search for Leishmania recombinant antigens that can be used as a vaccine against American Cutaneous Leishmaniasis, we identified a Leishmania (Leishmania amazonensis recombinant protein of 33 kD (Larp33 which is recognized by antibodies and peripheral blood leukocytes (PBL from subjects vaccinated with Leishvacin ®, Larp33 was expressed in Escherichia coli after cloning of a 2,2 kb Sau3A digested genomic fragment of L. (L. amazonensis into the pDS56-6 His vector. Immunoblotting analysis indicated that Larp33 corresponds to an approximately 40-kD native protein expressed in promastigotes of L.(L. amazonensis and L. (Viannia braziliensis. Northern blots of total RNA also demonstrated that the gene coding for this protein is expressed in promastigotes of the major lineages of Leishmania causing American Cutaneous Leishmaniasis. Larp33 induced partial protection in susceptible mouse strains (BALB/c and C57BL/10 against L. (L. amazonensis after vaccination using Bacille Calmette-Guerin (BCG as adjuvant. In vitro stimulation of splenocytes from BALB/c protected mice with Larp33 elicited the secretion of IL-2 and IFN-g, suggesting that a Th1 cell-mediated protective response is associated with the resistance observed in these mice. As revealed by its immunogenic and antigenic properties, this novel recombinant antigen is a suitable candidate to compose a vaccine against cutaneous leishmaniasisA resposta imune induzida por uma proteína recombinante de Leishmania (Leishmania amazonensis de 33 kD (Larp33 foi avaliada em linfócitos de indivíduos vacinados com a Leishvacin® e em camundongos através de vacinação. Larp33 foi expressa em Escherichia coli após clonagem de um fragmento genômico de L. (L. amazonensis de 2,2 kb no vetor pDS56-6His. Larp33 foi reconhecida por anticorpos IgG presentes no soro de indivíduos vacinados com Leishvacin® e induziu proliferação em linfócitos desses indivíduos em níveis comparáveis ao ant

  2. Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA Induce Protective Immune Responses in Dogs.

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    Elodie Petitdidier

    2016-05-01

    Full Text Available Preventive vaccination is a highly promising strategy for interrupting leishmaniasis transmission that can, additionally, contribute to elimination. A vaccine formulation based on naturally excreted secreted (ES antigens was prepared from L. infantum promastigote culture supernatant. This vaccine achieved successful results in Phase III trials and was licensed and marketed as CaniLeish. We recently showed that newly identified ES promastigote surface antigen (PSA, from both viable promastigotes and axenically-grown amastigotes, represented the major constituent and the highly immunogenic antigen of L. infantum and L. amazonensis ES products. We report here that three immunizations with either the recombinant ES LaPSA-38S (rPSA or its carboxy terminal part LaPSA-12S (Cter-rPSA, combined with QA-21 as adjuvant, confer high levels of protection in naive L. infantum-infected Beagle dogs, as checked by bone marrow parasite absence in respectively 78.8% and 80% of vaccinated dogs at 6 months post-challenge. The parasite burden in infected vaccinated dogs was significantly reduced compared to placebo group, as measured by q-PCR. Moreover, our results reveal humoral and cellular immune response clear-cut differences between vaccinated and control dogs. An early increase in specific IgG2 antibodies was observed in rPSA/QA-21- and Cter-rPSA/QA-21-immunized dogs only. They were found functionally active in vitro and were highly correlated with vaccine protection. In vaccinated protected dogs, IFN-γ and NO productions, as well as anti-leishmanial macrophage activity, were increased. These data strongly suggest that ES PSA or its carboxy-terminal part, in recombinant forms, induce protection in a canine model of zoonotic visceral leishmaniasis by inducing a Th1-dominant immune response and an appropriate specific antibody response. These data suggest that they could be considered as important active components in vaccine candidates.

  3. Activity of recombinant and natural defensins from Vigna unguiculata seeds against Leishmania amazonensis.

    Science.gov (United States)

    Souza, Géssika Silva; do Nascimento, Viviane Veiga; de Carvalho, Laís Pessanha; de Melo, Edésio José Tenório; Fernandes, Keysson Vieira; Machado, Olga Lima Tavares; Retamal, Claudio Andres; Gomes, Valdirene Moreira; Carvalho, André de Oliveira

    2013-09-01

    Antimicrobial peptides (AMPs), which are differentiated from other antibiotic peptides, such as gramicidins and polymyxins, because they are synthesized by large enzymatic complex and bear modified amino acids including d-amino acids, are short polymers of l-amino acids synthesized by ribosomes upon which all living organisms rely to defend themselves from invaders or competitor microorganisms. AMPs have received a great deal of attention from the scientific community as potential new drugs for neglected diseases such as Leishmaniasis. In plants, they include several families of compounds, including the plant defensins. The aim of the present study was to improve the expression of recombinant defensin from Vigna unguiculata seeds (Vu-Defr) and to test its activity against Leishmania amazonensis promatigotes. Recombinant expression was performed in LB and TB media and under different conditions. The purification of Vu-Defr was achieved by immobilized metal ion affinity and reversed-phase chromatography. The purified Vu-Defr was analyzed by circular dichroism (CD), and its biological activity was tested against L. amazonenis promastigotes. To demonstrate that the recombinant production of Vu-Defr did not interfere with its fold and biological activity, the results of all experiments were compared with the results from the natural defensin (Vu-Def). The CD spectra of both peptides presented good superimposition indicating that both peptides present very similar secondary structure and that the Vu-Defr was correctly folded. L. amazonensis treated with Vu-Defr led to the elimination of 54.3% and 46.9% of the parasites at 24 and 48h of incubation time, respectively. Vu-Def eliminated 50% and 54.8% of the parasites at 24 and 48 h, respectively. Both were used at a concentration of 100 μg/mL. These results suggested the potential for plant defensins to be used as new antiparasitic substances.

  4. The T-cell anergy induced by Leishmania amazonensis antigens is related with defective antigen presentation and apoptosis

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    Roberta O. Pinheiro

    2004-09-01

    Full Text Available Leishmania amazonensis is the main agent of diffuse cutaneous leishmaniasis, a disease associated with anergic immune responses. In this study we show that the crude antigen of Leishmania amazonensis (LaAg but not L. braziliensis promastigotes (LbAg contains substances that suppress mitogenic and spontaneous proliferative responses of T cells. The suppressive substances in LaAg are thermoresistant (100ºC/1h and partially dependent on protease activity. T cell anergy was not due to a decreased production of growth factors as it was not reverted by addition of exogenous IL-2, IL-4, IFN-gamma or IL-12. LaAg did not inhibit anti-CD3-induced T cell activation, suggesting that anergy was due to a defect in antigen presentation. It was also not due to cell necrosis, but was accompanied by expressive DNA fragmentation in lymph node cells, indicative of apoptosis. Although pre-incubation of macrophages with LaAg prevented their capacity to present antigens, this effect was not due to apoptosis of the former. These results suggest that the T cell anergy found in diffuse leishmaniasis may be the result of parasite antigen-driven apoptosis of those cells following defective antigen presentation.A Leishmania amazonensis é o principal agente etiológico da leishmaniose cutânea difusa, uma doença associada a respostas imunes anérgicas. Neste estudo nós mostramos que o extrato bruto de promastigotas de Leishmania amazonensis (LaAg, mas não de L. braziliensis (LbAg, contém substâncias que suprimem respostas proliferativas, espontâneas e mitogênicas, de células T. As substâncias supressoras no LaAg são termo-resistentes (100°C/1h e parcialmente dependentes da atividade de proteases. A anergia de células T não foi devida à diminuição na produção de fatores de crescimento, uma vez que não foi revertida pela adição de: IL-2, IL-4, IFN-gama ou IL-12. O LaAg não inibiu a ativação de células T induzida por anti-CD3, sugerindo que a anergia

  5. Glycosphingolipid antigens from Leishmania (L. amazonensis amastigotes: Binding of anti-glycosphingolipid monoclonal antibodies in vitro and in vivo

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    A.H. Straus

    1997-03-01

    Full Text Available Specific glycosphingolipid antigens of Leishmania (L. amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC. Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L. amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues

  6. DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis.

    Science.gov (United States)

    Lira, C B B; Gui, K E; Perez, A M; da Silveira, R C V; Gava, L M; Ramos, C H I; Cano, M I N

    2009-02-01

    Replication protein A (RPA) is a single-stranded DNA-binding protein that has been implicated in DNA metabolism and telomere maintenance. Subunit 1 of RPA from Leishmania amazonensis (LaRPA-1) has previously been affinity-purified on a column containing a G-rich telomeric DNA. LaRPA-1 binds and co-localizes with parasite telomeres in vivo. Here we describe the purification and characterization of native recombinant LaRPA-1 (rLaRPA-1). The protein was initially re-solubilized from inclusion bodies by using urea. After dialysis, rLaRPA-1 was soluble but contaminated with DNA, which was removed by an anion-exchange chromatography of the protein solubilized in urea. However, rLaRPA-1 precipitated after dialysis to remove urea. To investigate whether the contaminating DNA was involved in chaperoning the refolding of rLaRPA-1, salmon sperm DNA or heparin was added to the solution before dialysis. The addition of either of these substances prevented the precipitation of rLaRPA-1. The resulting rLaRPA-1 was soluble, correctly folded, and able to bind telomeric DNA. This is the first report showing the characterization of rLaRPA1 and of the importance of additives in chaperoning the refolding of this protein. The availability of rLaRPA-1 should be helpful in assessing the importance of this protein as a potential drug target.

  7. Enhancement of Leishmania amazonensis infection in BCG non-responder mice by BCG-antigen specific vaccine

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    Kátia da Silva Calabrese

    1992-01-01

    Full Text Available Different patterns of cutaneous leishmaniasis can be induced when a challenge of alike dose of Leishmania amazonensis amastigotes in various inbred strains was applied. Two strains of mice, the Balb/c and C57 BL/10J, showed exceptional suscepbility, and 10(elevado a sexta potência amastigotes infective dose lead, to ulcerative progressive lesions with cutaneous metastasis and loss by necrosis of leg on wich the footpad primary lesion occured. Lesions were also progressive but in a lower degree when C3H/HeN and C57BL/6 were infected. Lesions progress slowly in DBA/2 mice presenting lesions wich reach a discreet peack after 12 weeks, do not heal but do not uncerate. DBA/2 mice is, therefore, a good model for immunomodualtion. In attempt to determine the influence of BCG in vaccination schedule using microsomal fraction, DBA/2 became an excellent model, since it is also a non-responder to BCG. Vaccination of DBA/2 mice, receiving the same 10(elevado a sexta potência BCG viable dose and 10 *g or 50 *g of protein content of microsomal fraction, lead to a progressive disease with time course similar to those observed in susceptible non-vaccinated C57BL/10J mice after 6 months of observation. An enhancement of infection in BCG non-responder mice suggests that use of BCG as immunostimulant in humans could be critical for both vaccination and immunoprophylactic strategies.

  8. Use of Recombinant Antigens for the Diagnosis of Invasive Candidiasis

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    Ana Laín

    2008-01-01

    Full Text Available Invasive candidiasis is a frequent and often fatal complication in immunocompromised and critically ill patients. Unfortunately, the diagnosis of invasive candidiasis remains difficult due to the lack of specific clinical symptoms and a definitive diagnostic method. The detection of antibodies against different Candida antigens may help in the diagnosis. However, the methods traditionally used for the detection of antibodies have been based on crude antigenic fungal extracts, which usually show low-reproducibility and cross-reactivity problems. The development of molecular biology techniques has allowed the production of recombinant antigens which may help to solve these problems. In this review we will discuss the usefulness of recombinant antigens in the diagnosis of invasive candidiasis.

  9. Serological diagnosis with recombinant N antigen for hantavirus infection.

    Science.gov (United States)

    Yoshimatsu, Kumiko; Arikawa, Jiro

    2014-07-17

    Hantaviruses are causative agents of two rodent-borne zoonoses, hemorrhagic fever with renal syndrome (HFRS) and nephropathia epidemica (NE) in the Old World and hantavirus pulmonary syndrome (HPS) in the New World. Serological examinations to detect hantavirus antibodies have been most widely used for surveillance among humans and rodent reservoirs. Here, we will review antigenic structure of nucleocapsid (N) protein of hantaviruses and application of recombinant N protein as diagnostic antigen for screening and serotyping.

  10. Recombinant antigens for immunodiagnosis of cystic echinococcosis

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    Li Jun

    2004-01-01

    Full Text Available Three cDNAs, termed EpC1, TPxEg and EgG5, were isolated by immunoscreening from an Echinococcus granulosus cDNA library. The recombinant phages exhibited strong reactivity with sera from humans with confirmed cystic echinococcosis (CE and with sera from mice infected with E. granulosus oncospheres. The cDNAs were subcloned into a pET vector, expressed as fusion proteins tagged with GST and affinity purified against the GST tag. Of the three recombinant proteins, EpC1 achieved the highest performance for serodiagnosis of CE in Western blot analysis using a panel of clinically defined human sera to initially address the sensitivity and specificity of the molecules. The protein yielded an overall sensitivity of 92.2% and specificity of 95.6%, levels unprecedented taking into account the large panel of 896 human sera that were tested. The strategy used may also prove suitable for improved immunodiagnosis of other parasitic infections.

  11. Antigenic structures stably expressed by recombinant TGEV-derived vectors.

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    Becares, Martina; Sanchez, Carlos M; Sola, Isabel; Enjuanes, Luis; Zuñiga, Sonia

    2014-09-01

    Coronaviruses (CoVs) are positive-stranded RNA viruses with potential as immunization vectors, expressing high levels of heterologous genes and eliciting both secretory and systemic immune responses. Nevertheless, its high recombination rate may result in the loss of the full-length foreign gene, limiting their use as vectors. Transmissible gastroenteritis virus (TGEV) was engineered to express porcine reproductive and respiratory syndrome virus (PRRSV) small protein domains, as a strategy to improve heterologous gene stability. After serial passage in tissue cultures, stable expression of small PRRSV protein antigenic domains was achieved. Therefore, size reduction of the heterologous genes inserted in CoV-derived vectors led to the stable expression of antigenic domains. Immunization of piglets with these TGEV vectors led to partial protection against a challenge with a virulent PRRSV strain, as immunized animals showed reduced clinical signs and lung damage. Further improvement of TGEV-derived vectors will require the engineering of vectors with decreased recombination rate.

  12. Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis

    Science.gov (United States)

    2011-09-01

    Army Research Laboratory: 2010; p 19. 18. Sambrook, J.; Fritsch, E. F.; Maniatis , T. Molecular Cloning : A Laboratory Manual. 2nd ed.; Cold Spring...on a rotating platform until not viscous (adapted from Molecular Cloning : A Laboratory Manual) (18). Each solution was centrifuged at 45,000 x g for... Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis by Deborah A. Sarkes, Joshua M. Kogot, Irene Val-Addo

  13. Enhancing the Immune Response to Recombinant Plague Antigens

    Science.gov (United States)

    2007-05-01

    this is an effective means of inducing serum antibody against the antigen (i.e., tetanus and diphtheria toxoid). Recently, a great deal of attention...and bronchioalveolar lavage (BAL) fluid were examined for the presence of anti-F1–V, anti-F1 or anti- V antibodies by ELISA . Briefly, ELISA plates were...anti-V antibodies by en- zyme-linked immunosorbent assay ( ELISA ) on plates that were coated with 0.1 g per well of recombinant F1-V, F1, or V in 100

  14. A recombinant vaccinia virus expressing human carcinoembryonic antigen (CEA).

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    Kaufman, H; Schlom, J; Kantor, J

    1991-07-30

    Carcinoembryonic antigen (CEA) is a 180-kDa glycoprotein expressed on most gastrointestinal carcinomas. A 2.4-kb cDNA clone, containing the complete coding sequence, was isolated from a human colon tumor cell library and inserted into a vaccinia virus genome. This newly developed construct was characterized by Southern blotting, DNA hybridization studies, and polymerase chain reaction analysis. The CEA gene was stably integrated into the vaccinia virus thymidine kinase gene. The recombinant was efficiently replicated upon serial passages in cell cultures and in animals. The recombinant virus expresses on the surface of infected cells a protein product recognized by a monoclonal antibody (COL-I) directed against CEA. Immunization of mice with the vaccinia construct elicited a humoral immune response against CEA. Pilot studies also showed that administration of the recombinant CEA vaccinia construct was able to greatly reduce the growth in mice of a syngeneic murine colon adenocarcinoma which had been transduced with the human CEA gene. The use of this new recombinant CEA vaccinia construct may thus provide an approach in the specific active immunotherapy of human GI cancer and other CEA expressing carcinoma types.

  15. Exploring the Association of Surface Plasmon Resonance with Recombinant MHC:Ig Hybrid Protein as a Tool for Detecting T Lymphocytes in Mice Infected with Leishmania (Leishmania amazonensis

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    Lenilton Silva da Silveira-Júnior

    2017-01-01

    Full Text Available A surface plasmon resonance- (SPR- based recognition method applying H-2 Ld:Ig/peptides complexes for ex vivo monitoring cellular immune responses during murine infection with Leishmania (Leishmania amazonensis is described. Lymphocytes from lesion-draining popliteal lymph nodes were captured on a carboxylated sensor chip surface previously functionalized with H-2 Ld:Ig (DimerX protein bound to synthetic peptides derived from the COOH-terminal region of cysteine proteinase B of L. (L. amazonensis. In computational analysis, these peptides presented values of kinetic constants favorable to form complexes with H-2 Ld at neutral pH, with a Gibbs free energy ΔG°<0. The assayed DimerX:peptide complexes presented the property of attaching to distinct T lymphocytes subsets, obtained from experimentally infected BALB/c mice, in each week of infection, thus indicating a temporal variation in specific T lymphocytes populations, each directed to a different COOH-terminal region-derived peptide. The experimental design proposed herein is an innovative approach for cellular immunology studies of a neglected disease, providing a useful tool for the analysis of specific T lymphocytes subsets.

  16. Maturation of recombinant hepatitis B virus surface antigen particles.

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    Zhao, Qinjian; Wang, Yang; Freed, Daniel; Fu, Tong-Ming; Gimenez, Juan A; Sitrin, Robert D; Washabaugh, Michael W

    2006-01-01

    The major surface antigen of Hepatitis B virus (HBsAg) is a cysteine-rich, lipid-bound protein with 226 amino acids. Recombinant HBsAg (rHBsAg) with associated lipids can self-assemble into 22-nm immunogenic spherical particles, which are used in licensed Hepatitis B vaccines. Little is known about the structural evolvement or maturation upon assembly beyond an elevated level of disulfide formation. In this paper, we further characterized the maturation of HBsAg particles with respect to their degree of cross-linking, morphological changes, and changes in conformational flexibility. The lipid-containing rHBsAg particles undergo KSCN- and heat-induced maturation by formation of additional intra- and inter-molecular disulfide bonds. Direct measurements with atomic force microscopy (AFM) revealed morphological changes upon maturation through KSCN-induced and heat-/storage-incurred oxidative refolding. Particle uniformity and regularity was greatly improved, and protrusions formed by the protein subunits were more prominent on the surface of the mature particles. Decreased conformational flexibility in the mature rHBsAg particles was demonstrated by millisecond-scale unfolding kinetics in the presence of an environment-sensitive conformation probe. Both the accessible hydrophobic cavities under native conditions and the changeable hydrophobic cavities upon denaturant-induced unfolding showed substantial decrease upon maturation of the rHBsAg particles. These changes in the structural properties may be critical for the antigenicity and immuno-genicity of this widely-used vaccine component.

  17. Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein.

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    Deirdre R Ducken

    Full Text Available Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to

  18. Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein.

    Science.gov (United States)

    Ducken, Deirdre R; Brown, Wendy C; Alperin, Debra C; Brayton, Kelly A; Reif, Kathryn E; Turse, Joshua E; Palmer, Guy H; Noh, Susan M

    2015-01-01

    Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to recombinant vaccines

  19. Protection of pigs against Taenia solium cysticercosis by immunization with novel recombinant antigens.

    Science.gov (United States)

    Gauci, Charles G; Jayashi, César M; Gonzalez, Armando E; Lackenby, Julia; Lightowlers, Marshall W

    2012-06-06

    Recombinant antigens from the oncosphere stage of the parasite Taenia solium were expressed in Escherichia coli. The TSOL16, TSOL45-1A and TSOL45-1B recombinant antigens, each consisting of fibronectin type III (FnIII) domain S, were produced as fusion proteins with glutathione S-transferase (GST) and maltose binding protein (MBP). Groups of pigs were immunized twice with the GST fusions of the antigens and boosted a third time with the MBP fusions prior to receiving a challenge infection with T. solium eggs. The TSOL16 antigen was found to be capable of inducing high levels of immunity in pigs against a challenge infection with T. solium. Immunological investigations identified differences in immune responses in the pigs vaccinated with the various antigens. The results demonstrate that the TSOL16 antigen could be a valuable adjunct to current porcine vaccination approaches and may allow the further development of new vaccination strategies against T. solium cysticercosis.

  20. [Detection and antigenic characteristics of the recombinant nucleocapsid proteins of Lassa and Marburg viruses].

    Science.gov (United States)

    Vladyko, A S; Scheslenok, E P; Fomina, E G; Semizhon, P A; Ignat'ev, G M; Shkolina, T V; Kras'ko, A G; Semenov, S F; Vinokurov, N V

    2012-01-01

    Two plasmid vectors, which allow the recombinant polypeptides of Lassa and Marburg viruses to be expressed in prokaryotic cells E. coli strain BL21 (DE3), were produced. The two recombinant polypeptides are able to bind specific antibodies. This provides an opportunity to use them as antigenic components of immunoassay diagnostic test kits.

  1. The physical stability of the recombinant tuberculosis fusion antigens h1 and h56

    DEFF Research Database (Denmark)

    Hamborg, Mette; Kramer, Ryan; Schanté, Carole E;

    2013-01-01

    The recombinant fusion proteins hybrid 1 [H1 (Ag85B-ESAT-6)] and hybrid 56 [H56 (Ag85B-ESAT-6-Rv2660c)] derived from Mycobacterium tuberculosis are promising antigens for subunit vaccines against tuberculosis. Both antigens are early batches of antigens to be enrolled in human clinical trials...... increased; however, the physical stabilities of the bound and the unbound antigens were comparable. This study provides important information about the biophysical properties of H1 and H56 and highlights the analytical challenges of characterizing complex vaccine formulations....

  2. Microbial antigenic variation mediated by homologous DNA recombination

    OpenAIRE

    2012-01-01

    Pathogenic microorganisms employ numerous molecular strategies in order to delay or circumvent recognition by the immune system of their host. One of the most widely used strategies of immune evasion is antigenic variation, in which immunogenic molecules expressed on the surface of a microorganism are continuously modified. As a consequence, the host is forced to constantly adapt its humoral immune response against this pathogen. An antigenic change thus provides the microorganism with an opp...

  3. Intragastric immunization with recombinant Lactobacillus casei expressing flagellar antigen confers antibody-independent protective immunity against Salmonella enterica serovar Enteritidis

    NARCIS (Netherlands)

    Kajikawa, A.; Satoh, E.; Leer, R.J.; Yamamoto, S.; Igimi, S.

    2007-01-01

    A recombinant Lactobacillus casei expressing a flagellar antigen from Salmonella enterica serovar Enteritidis was constructed and evaluated as a mucosal vaccine. Intragastric immunization of the recombinant strain conferred protective immunity against Salmonella infection in mice. This immunization

  4. A study of recombinant protective H. Pylori antigens

    Institute of Scientific and Technical Information of China (English)

    Zheng Jiang; Xiao-Hong Tao; Ai-Long Huang; Pi-Long Wang

    2002-01-01

    AIM: To construct a recombinant vector which can express Mr 26 000 outer membrane protein (OMP) from Helicobacter pylori (Hp), and to obtain the vaccine protecting against Hp infection and a diagnostic reagent kit quickly detecting Hp infection.METHODS: The gene encoding the structural Mr 26000 outermembrane protein of Hp was amplified from Hpchromosomal DNA by PCR, and inserted in the prokaryoticexpression vector pET32a ( + ), which was transformed intothe Topl0 E. coli strain. Recombinant vector was selected,identified and transformed into BL-21(DE3) E. coli strain.The recombinant fusion proteins were expressed. Theantigenicity of recombinant protein was studied by ELISA orimmunoblotting and immunized Balb/c mice.RESULTS: The gene of Mr 26 000 OMP was amplified to be594 base pairs, 1.1% of the cloned genes was mutated and1.51% of amino acid residues was changed, but there washomogeneity between them. The recombinant fusion proteinencoded objective polypeptides of 198 amino acid residues,corresponding to calculated molecular masses of Mr 26000.The level of soluble expression products was about 38.96 %of the total cell protein. After purification by Ni-NTA agaroseresin columniation, the purity of objective protein becameabout 90 %. The EESA results showed that recombinantfusion protein could be recognized by patient serum infectedwith Hp and rabbit serum immunized with the recombinantprotein. Furthermore, Balb/ c mice immunized with therecombinant proteln were protected against H. pyloriinfection.CONCLUSION: Mr 26 000 OMP may be a candidate vaccinepreventing Hp infection.

  5. Development of recombinant antigen array for simultaneous detection of viral antibodies.

    Directory of Open Access Journals (Sweden)

    Yi Liu

    Full Text Available Protein microarrays have been developed to study antibody reactivity against a large number of antigens, demonstrating extensive perspective for clinical application. We developed a viral antigen array by spotting four recombinant antigens and synthetic peptide, including glycoprotein G of herpes simplex virus (HSV type 1 and 2, phosphoprotein 150 of cytomegalovirus (CMV, Rubella virus (RV core plus glycoprotein E1 and E2 as well as a E1 peptide with the optimal concentrations on activated glass slides to simultaneously detect IgG and IgM against HSV1, HSV2, CMV and RV in clinical specimens of sera and cerebrospinal fluids (CSFs. The positive reference sera were initially used to measure the sensitivity and specificity of the array with the optimal conditions. Then clinical specimens of 144 sera and 93 CSFs were tested for IgG and IgM antibodies directed against HSV1, HSV2, CMV and RV by the antigen array. Specificity of the antigen array for viral antibodies detection was satisfying compared to commercial ELISA kits but sensitivity of the array varied relying on quality and antigenic epitopes of the spotting antigens. In short, the recombinant antigen array has potential to simultaneous detect multiple viral antibodies using minute amount (3 µl of samples, which holds the particularly advantage to detect viral antibodies in clinical CSFs being suspicious of neonatal meningitis and encephalitis.

  6. Microbial antigenic variation mediated by homologous DNA recombination

    NARCIS (Netherlands)

    C. Vink (Cornelis); L. Rudenko (Larisa); H.S. Seifert (H. Steven)

    2012-01-01

    textabstractPathogenic microorganisms employ numerous molecular strategies in order to delay or circumvent recognition by the immune system of their host. One of the most widely used strategies of immune evasion is antigenic variation, in which immunogenic molecules expressed on the surface of a mic

  7. Recombinant measles AIK-C vaccine strain expressing heterologous virus antigens.

    Science.gov (United States)

    Nakayama, Tetsuo; Sawada, Akihito; Yamaji, Yoshiaki; Ito, Takashi

    2016-01-04

    Further attenuated measles vaccines were developed more than 50 years ago and have been used throughout the world. Recombinant measles vaccine candidates have been developed and express several heterologous virus protective antigens. Immunogenicity and protective actions were confirmed using experimental animals: transgenic mice, cotton rats, and primates. The recent development of measles vaccine-based vectored vaccine candidates has been reviewed and some information on recombinant measles vaccines expressing respiratory syncytial virus proteins has been shown and discussed.

  8. Trypanosome Surface Antigen Genes: Analysis Using Recombinant DNA.

    Science.gov (United States)

    1984-06-15

    different VATs. These cDNAs were used to screen the genomic libraries . The cloned probes were examined by nucleotide sequence analysis and used to...examination of the consequence of - antigenic variation on the structure of the 1.1, 1.D and 1.11 VSG -, gene sequences. In addition, all genomic libraries have...immunoprecipitated by heterologous VAt specific antisera (preprint 2, figure 3; preprint 3, figure 1). These data presented in preprints 2 and Genomic

  9. Construction of Recombinant Modified Vaccinia Ankara (MVA) Expressing Hepatitis B Virus Surface Antigen

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The T lymphocyte response has been shown to be the determinant in the clearance of many viral infections.Hence, therapeutic vaccine candidates against HBV are designed to enhance this response of the immune system.Vaccinia virus vector-based vaccines have been proposed as excellent candidates to elicit long-term and strong T lymphocyte mediated immune responses. In this study, the recombinant MVA expressing HBV surface antigen has been constructed, which can elicit a potent T cell mediated response. The ELISA results for the surface protein in the medium of the recombinant MVA, strongly indicate that the recombinant virus has been successfully obtained.

  10. Use of Recombinant Antigens for Sensitive Serodiagnosis of American Tegumentary Leishmaniasis Caused by Different Leishmania Species

    Science.gov (United States)

    Sato, Camila Massae; Sanchez, Maria Carmen Arroyo; Celeste, Beatriz Julieta; Duthie, Malcolm S.; Guderian, Jeffrey; Reed, Steven G.; de Brito, Maria Edileuza Felinto; Campos, Marliane Batista; de Souza Encarnação, Helia Valeria; Guerra, Jorge; de Mesquita, Tirza Gabrielle Ramos; Pinheiro, Suzana Kanawati; Ramasawmy, Rajendranath; Silveira, Fernando Tobias; de Assis Souza, Marina

    2016-01-01

    ABSTRACT American tegumentary leishmaniasis (ATL) (also known as cutaneous leishmaniasis [CL]) is caused by various species of protozoa of the genus Leishmania. The diagnosis is achieved on a clinical, epidemiological, and pathological basis, supported by positive parasitological exams and demonstration of leishmanin delayed-type hypersensitivity. Serological assays are not routinely used in the diagnosis because many are considered to have low sensitivity and the particular Leishmania species causing the disease can lead to variable performance. In the present study, we generated recombinant versions of two highly conserved Leishmania proteins, Leishmania (Viannia) braziliensis-derived Lb8E and Lb6H, and evaluated both in enzyme-linked immunosorbent assays (ELISA). Recombinant Lb6H (rLb6H) had better performance and reacted with 100.0% of the ATL and 89.4% of the VL samples. These reactions with rLb6H were highly specific (98.5%) when compared against those for samples from healthy control individuals. We then assessed rLb6H against sera from ATL patients infected with different species of Leishmania prevalent in Brazil [Leishmania (Leishmania) amazonensis, L. (Viannia) braziliensis, and L. (V.) guyanensis] and samples from patients with other infectious diseases. In analyses of 500 sera, ELISA using rLb6H detected all 219 ATL samples (sensitivity of 100.0%) with an overall specificity of 93.9% (considering healthy individuals and other infectious diseases patients). Only a minority of samples from Chagas disease patients possessed antibodies against rLb6H, and all of these responses were low (with a highest reactivity index of 2.2). Taken together, our data support further evaluation of rLb6H and the potential for its routine use in the serological diagnosis of ATL. PMID:27927927

  11. DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

    DEFF Research Database (Denmark)

    Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas Salhøj

    2014-01-01

    -wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum......-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers...... of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens....

  12. Application of recombinant antigen 5 allergens from seven allergy-relevant Hymenoptera species in diagnostics

    DEFF Research Database (Denmark)

    Schiener, Maximilian; Eberlein, Bernadette; Moreno Aguilar, Carmen;

    2017-01-01

    , and BAT. This cross-reactivity was more pronounced in ImmunoCAP measurements with venom extracts than in sIgE analyses with recombinant antigens 5. Dose-response curves with the allergens in BAT allowed a differentiated individual dissection of relevant sensitization. CONCLUSIONS: Due to extensive cross...

  13. Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen

    Science.gov (United States)

    Smith, Geoffrey L.; Mackett, Michael; Moss, Bernard

    1983-04-01

    Potential live vaccines against hepatitis B virus have been produced. The coding sequence for hepatitis B virus surface antigen (HBsAg) has been inserted into the vaccinia virus genome under control of vaccinia virus early promoters. Cells infected with these vaccinia virus recombinants synthesize and excrete HBsAg and vaccinated rabbits rapidly produce antibodies to HBsAg.

  14. Several recombinant capsid proteins of equine rhinitis a virus show potential as diagnostic antigens.

    Science.gov (United States)

    Li, Fan; Stevenson, Rachel A; Crabb, Brendan S; Studdert, Michael J; Hartley, Carol A

    2005-06-01

    Equine rhinitis A virus (ERAV) is a significant pathogen of horses and is also closely related to Foot-and-mouth disease virus (FMDV). Despite these facts, knowledge of the prevalence and importance of ERAV infections remains limited, largely due to the absence of a simple, robust diagnostic assay. In this study, we compared the antigenicities of recombinant full-length and fragmented ERAV capsid proteins expressed in Escherichia coli by using sera from experimentally infected and naturally exposed horses. We found that, from the range of antigens tested, recombinant proteins encompassing the C-terminal region of VP1, full-length VP2, and the N-terminal region of VP2 reacted specifically with antibodies present in sera from each of the five experimentally infected horses examined. Antibodies to epitopes on VP2 (both native and recombinant forms) persisted longer postinfection (>105 days) than antibodies specific for epitopes on other fragments. Our data also suggest that B-cell epitopes within the C terminus of VP1 and N terminus of VP2 contribute to a large proportion of the total reactivity of recombinant VP1 and VP2, respectively. Importantly, the reactivity of these VP1 and VP2 recombinant proteins in enzyme-linked immunosorbent assays (ELISAs) correlated well with the results from a range of native antigen-based serological assays using sera from 12 field horses. This study provides promising candidates for development of a diagnostic ERAV ELISA.

  15. Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli.

    Science.gov (United States)

    Yee, Siew Fung; Chu, Chia Huay; Poili, Evenni; Sum, Magdline Sia Henry

    2017-02-01

    Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes.

  16. Evaluating Recombinant Antigen ROP1 Efficacy in Diagnosis of Toxoplasma Gondii Infection

    Directory of Open Access Journals (Sweden)

    F Keshavarzi

    2015-07-01

    Full Text Available Introduction:Toxoplasma gondii is a ubiquitous obligate intracellular parasite with a relatively broad host range infecting both mammals and birds. Toxoplasma proteins are strong antigens that can begin strong immune reactions, among which Rhoptry protein 1 (ROP1 can be named discharging from rhoptry cell-organ. ROP1 is regarded as a competitor for recombinant vaccines against toxoplasmosis. Therefore, the main objective of the current study was to evaluate the cloning and expression of ROP1 Toxoplasma gondii in a cloning vector as well as to create this recombinant antigen in order to be applied for later uses. Methods:Genomic DNA of Toxoplasma gondii was removed and reproduced by PCR, then the PCR product was cloned into the EcoR1 and BamH1 sites of cloning vector, pUET1, and transformed into Escherichia coli BL21 plysS strain. Moreover, pcROP1 was sub-cloned into the HindIII and EcoRI sites of the pcDNA3 in order to produce recombining eukaryotic declaration vector. The cloned ROP1 was verified by PCR, limitation enzymes (HindIII and BglΙ digestion and nucleotide sequencing. Then, this recombinant antigen was covered applying IgM and ELISAIgG. Results:The study results demonstrated that a fragment of 757 bp was separated. In addition, nucleotide sequence analysis of the ROP1 cloned in pUET1vector revealed high homology (96% with RH strain Gene Bank Accession (No. M71274. Conclusion:The recombinant ROP1 antigen in an IgM Rec-ELISA test can be replaced with the tachyzoite antigen in IgG and IgM serologic tests.

  17. Microplate Agglutination Test for Canine Brucellosis Using Recombinant Antigen-Coated Beads.

    Science.gov (United States)

    Castillo, Yussaira; Tachibana, Masato; Kimura, Yui; Kim, Suk; Ichikawa, Yasuaki; Endo, Yasuyuki; Watanabe, Kenta; Shimizu, Takashi; Watarai, Masahisa

    2014-01-01

    Brucella canis, a facultative intracellular pathogen, is the causative agent of canine brucellosis. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods, including agglutination and gel diffusion tests. In this study, four recombinant antigens, heat shock protein 60, rhizopine-binding protein, Cu-Zn superoxide dismutase, and hypothetical protein (Ag 4), were constructed. These antigens were coated on latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. All recombinant antigens showed specific reaction with sera from B. canis-infected dogs in Western blotting. In a microplate agglutination test, mixing sera from B. canis-infected dogs, but not sera from B. canis-free dogs, with single or multiple antigens-coated latex beads produced clear agglutination. Moreover, the antigen-coated latex beads did not show nonspecific agglutination in hemolyzed serum samples. A survey of canine serum samples conducted by the microplate agglutination test using single antigen-coated latex beads showed that this method would be useful in the serological diagnosis of canine brucellosis. Further investigations using more serum samples are required to confirm the usefulness of our method.

  18. Use of MAG1 recombinant antigen for diagnosis of Toxoplasma gondii infection in humans.

    Science.gov (United States)

    Holec, Lucyna; Hiszczyńska-Sawicka, Elzbieta; Gasior, Artur; Brillowska-Dabrowska, Anna; Kur, Józef

    2007-03-01

    This paper describes the cloning, purification, and serological applications of matrix antigen MAG1 of Toxoplasma gondii. The expression system used allows the production of a large amount of T. gondii recombinant protein, which was assessed for its potential use in an enzyme-linked immunosorbent assay (ELISA) for detection of T. gondii infection in humans. Serum samples from 117 patients with different stages of infection, along with 10 serum samples from seronegative patients obtained for routine diagnostic tests, were used. The results were compared with those of an ELISA that uses a native T. gondii antigen extract. The MAG1 antigen detected antibodies more frequently from the acute stage (97.3%) than from the chronic stage (7.5%) of toxoplasmosis. Hence, this antigen may be used as a tool for detection of T. gondii immunoglobulin G antibodies in persons with acute toxoplasmosis.

  19. A role for RAD51 and homologous recombination in Trypanosoma brucei antigenic variation.

    Science.gov (United States)

    McCulloch, R; Barry, J D

    1999-11-01

    Antigenic variation is an immune evasion strategy used by African trypanosomes, in which the parasites periodically switch the expression of VSG genes that encode their protective variant surface glycoprotein coat. Two main routes exist for VSG switching: changing the transcriptional status between an active and an inactive copy of the site of VSG expression, called the bloodstream VSG expression site, or recombination reactions that move silent VSGs or VSG copies into the actively transcribed expression site. Nothing is known about the proteins that control and catalyze these switching reactions. This study describes the cloning of a trypanosome gene encoding RAD51, an enzyme involved in DNA break repair and genetic exchange, and analysis of the role of the enzyme in antigenic variation. Trypanosomes genetically inactivated in the RAD51 gene were shown to be viable, and had phenotypes consistent with lacking functional expression of an enzyme of homologous recombination. The mutants had an impaired ability to undergo VSG switching, and it appeared that both recombinational and transcriptional switching reactions were down-regulated, indicating that RAD51 either catalyzes or regulates antigenic variation. Switching events were still detectable, however, so it appears that trypanosome factors other than RAD51 can also provide for antigenic variation.

  20. Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

    Directory of Open Access Journals (Sweden)

    N.S. Sato

    2004-07-01

    Full Text Available Three recombinant antigens of Treponema pallidum Nichols strain were fused with GST, cloned and expressed in Escherichia coli, resulting in high levels of GST-rTp47 and GST-rTp17 expression, and supplementation with arginine tRNA for the AGR codon was needed to obtain GST-rTp15 overexpression. Purified fusion protein yields were 1.9, 1.7 and 5.3 mg/l of cell culture for GST-rTp47, GST-rTp17 and GST-rTp15, respectively. The identities of the antigens obtained were confirmed by automated DNA sequencing using ABI Prism 310 and peptide mapping by Finningan LC/MS. These recombinant antigens were evaluated by immuno-slot blot techniques applied to 137 serum samples from patients with a clinical and laboratory diagnosis of syphilis (61 samples, from healthy blood donors (50 samples, individuals with sexually transmitted disease other than syphilis (3 samples, and from individuals with other spirochetal diseases such as Lyme disease (20 samples and leptospirosis (3 samples. The assay had sensitivity of 95.1% (95% CI, 86.1 to 98.7% and a specificity of 94.7% (95% CI, 87.0 to 98.7%; a stronger reactivity was observed with fraction rTp17. The immunoreactivity results showed that fusion recombinant antigens based-immuno-slot blot techniques are suitable for use in diagnostic assays for syphilis.

  1. A new MIC1-MAG1 recombinant chimeric antigen can be used instead of the Toxoplasma gondii lysate antigen in serodiagnosis of human toxoplasmosis.

    Science.gov (United States)

    Holec-Gąsior, Lucyna; Ferra, Bartłomiej; Drapała, Dorota; Lautenbach, Dariusz; Kur, Józef

    2012-01-01

    This study presents an evaluation of the MIC1 (microneme protein 1)-MAG1 (matrix antigen 1) Toxoplasma gondii recombinant chimeric antigen for the serodiagnosis of human toxoplasmosis for the first time. The recombinant MIC1-MAG1 antigen was obtained as a fusion protein containing His tags at the N- and C-terminal ends using an Escherichia coli expression system. After purification by metal affinity chromatography, the chimeric protein was tested for usefulness in an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-T. gondii immunoglobulin G (IgG). One hundred ten sera from patients at different stages of infection and 40 sera from seronegative patients were examined. The results obtained for the MIC1-MAG1 chimeric antigen were compared with those of IgG ELISAs using a Toxoplasma lysate antigen (TLA), a combination of recombinant antigens (rMIC1ex2-rMAG1) and single recombinant proteins (rMIC1ex2 and rMAG1). The sensitivity of the IgG ELISA calculated from all of the positive serum samples was similar for the MIC1-MAG1 chimeric antigen (90.8%) and the TLA (91.8%), whereas the sensitivities of the other antigenic samples used were definitely lower, at 69.1% for the mixture of antigens, 75.5% for the rMIC1ex2, and 60% for rMAG1. This study demonstrates that the MIC1-MAG1 recombinant chimeric antigen can be used instead of the TLA in the serodiagnosis of human toxoplasmosis.

  2. A role for RAD51 and homologous recombination in Trypanosoma brucei antigenic variation

    OpenAIRE

    1999-01-01

    Antigenic variation is an immune evasion strategy used by African trypanosomes, in which the parasites periodically switch the expression of VSG genes that encode their protective variant surface glycoprotein coat. Two main routes exist for VSG switching: changing the transcriptional status between an active and an inactive copy of the site of VSG expression, called the bloodstream VSG expression site, or recombination reactions that move silent VSGs or VSG copies into the actively transcribe...

  3. Isolation, Cloning, Expression and Purification of Recombinant RhD Antigen from Cord Blood

    Directory of Open Access Journals (Sweden)

    M Habibi Roudkenar

    2008-09-01

    Full Text Available "nBackground: Rh (Rhesus is a highly complex blood group system in man deeply rooted in transfusion medicine. Isolation of RhD from cord blod, cloning and expression of recombinant RhD antigen in bacterial expression system was the aim of this study."nMethods: Total RNAs were extracted from cord blood (O+.  The quality of RNA was determined by electrophoresis. In or­der to obtain coding sequence of RhD antigen cDNA was synthesized and Rh D gene was amplified by RT-PCR. The iso­lated RhD gene was   cloned to pUC18 vector and transformed to DH5α. The confirmed construct was sub cloned into expres­sion vector, pBADgIII/A, and expressed in Top10 E.coli. The expressed protein was characterized by SDS-PAGE and western blot analysis. Antigenicity of the expressed protein was assessed by ELISA using commercially available hu­man anti-RhD polyclonal   antibody with   peroxidase conjugated goat anti-human IgG, IgM, IgA as secondary antibody. "nRe­sults: RhD gene was successfully cloned and expressed. The expected size of recombinant RhD protein was detected in SDS-PAGE, and confirmed by dot and western blot analysis. RhD antibody reacted with recombinant RhD antigen as well as with RhD polypeptide extracted from RBCs membrane."nConclusion: The recombinant RhD may be helpful to further investigate the molecular basis of RhD protein and could be applica­ble for production anti- D antibody in an animal model.

  4. MHC-restricted antigen presentation and recognition: constraints on gene, recombinant and peptide vaccines in humans

    Directory of Open Access Journals (Sweden)

    Cunha-Neto E.

    1999-01-01

    Full Text Available The target of any immunization is to activate and expand lymphocyte clones with the desired recognition specificity and the necessary effector functions. In gene, recombinant and peptide vaccines, the immunogen is a single protein or a small assembly of epitopes from antigenic proteins. Since most immune responses against protein and peptide antigens are T-cell dependent, the molecular target of such vaccines is to generate at least 50-100 complexes between MHC molecule and the antigenic peptide per antigen-presenting cell, sensitizing a T cell population of appropriate clonal size and effector characteristics. Thus, the immunobiology of antigen recognition by T cells must be taken into account when designing new generation peptide- or gene-based vaccines. Since T cell recognition is MHC-restricted, and given the wide polymorphism of the different MHC molecules, distinct epitopes may be recognized by different individuals in the population. Therefore, the issue of whether immunization will be effective in inducing a protective immune response, covering the entire target population, becomes an important question. Many pathogens have evolved molecular mechanisms to escape recognition by the immune system by variation of antigenic protein sequences. In this short review, we will discuss the several concepts related to selection of amino acid sequences to be included in DNA and peptide vaccines.

  5. Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors.

    Science.gov (United States)

    Hart, Bryan E; Asrican, Rose; Lim, So-Yon; Sixsmith, Jaimie D; Lukose, Regy; Souther, Sommer J R; Rayasam, Swati D G; Saelens, Joseph W; Chen, Ching-Ju; Seay, Sarah A; Berney-Meyer, Linda; Magtanong, Leslie; Vermeul, Kim; Pajanirassa, Priyadharshini; Jimenez, Amanda E; Ng, Tony W; Tobin, David M; Porcelli, Steven A; Larsen, Michelle H; Schmitz, Joern E; Haynes, Barton F; Jacobs, William R; Lee, Sunhee; Frothingham, Richard

    2015-07-01

    The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10(24)-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10(68)-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches.

  6. Mismatch repair regulates homologous recombination, but has little influence on antigenic variation, in Trypanosoma brucei.

    Science.gov (United States)

    Bell, Joanna S; McCulloch, Richard

    2003-11-14

    Antigenic variation is critical in the life of the African trypanosome, as it allows the parasite to survive in the face of host immunity and enhance its transmission to other hosts. Much of trypanosome antigenic variation uses homologous recombination of variant surface glycoprotein (VSG)-encoding genes into specialized transcription sites, but little is known about the processes that regulate it. Here we describe the effects on VSG switching when two central mismatch repair genes, MSH2 and MLH1, are mutated. We show that disruption of the parasite mismatch repair system causes an increased frequency of homologous recombination, both between perfectly matched DNA molecules and between DNA molecules with divergent sequences. Mismatch repair therefore provides an important regulatory role in homologous recombination in this ancient eukaryote. Despite this, the mismatch repair system has no detectable role in regulating antigenic variation, meaning that VSG switching is either immune to mismatch selection or that mismatch repair acts in a subtle manner, undetectable by current assays.

  7. Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs.

    Directory of Open Access Journals (Sweden)

    Ryuichi Miura

    Full Text Available Canine distemper virus (CDV vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV-LACK, rCDV-TSA, and rCDV-LmSTI1, respectively. Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV-LACK showed markedly smaller nodules without ulceration. Although the rCDV-TSA- and rCDV-LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV-LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs.

  8. Production of Recombinant Echinococcus granulosus Antigen B Subunits, In Order to Using Of Them in Serodiagnostic Tests of Hydatidosis

    Directory of Open Access Journals (Sweden)

    R Pazoki

    2007-06-01

    Full Text Available Background: Hydatidosis is one of the most important helminthiasis, and is a public health problem in many regions of the world. Methods: With the aim of production of recombinant subunits of antigen B, two different sequences of Echinococcus granulosus Antigen B, acquired from Gene Bank and amplified with specific primers via RT-PCR reaction. The amplified fragments (HI, HII cloned into pTZ57R T.vector, and then subcloned into pGEMEX-1 expression vector. Resaults: The SDS-PAGE performed after induction of cloned genes, and production of about 35 K.Da recombinant fusion proteins were confirmed for either two cloned genes. The immunogenicity of the recombinant fusion proteins were tested using double diffusion and immunoblotting. Both recombinant fusion proteins derived from lysate of transformed bacteria, were reactive for antibodies in serum of cystic hydatid patient. Conclusion: The produced recombinant antigen B subunits can be use in seroldiagnostic tests of hydatidosis, after purification.

  9. Protection of aouts monkeys after immunization with recombinant antigens of Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Burhard Enders

    1992-01-01

    Full Text Available The genus Aotus spp. (owl monkey is one of the WHO recommended experimental models for Plasmodium falciparum blood stage infection, especially relevant for vaccination studies with asexual blood stage antigens of this parasite. For several immunization trials with purified recombinant merozoite/schizont antigens, the susceptible Aouts kenotypes II, III, IV and VI were immunized with Escherichia coli derived fusion proteins containg partial sequences of the proteins MSAI (merozoite surface antigen I, SERP (serine-strech protein and HRPII (histidine alanine rich protein II as well as with a group of recombinant antigens obtained by an antiserum raised against a protective 41 kD protein band. The subcutaneous application (3x of the antigen preparations was carried out in intact animals followed by splenectomy prior to challange, in order to increase the susceptibility of the experimental hosts to the parasite. A partial sequence of HRPII, the combination of three different fusion proteins of the 41 kD group and mixture of two sequences of SERP in the presence of the modified Al(OH3 adjuvant conferred significant protection against a challange infection with P. falciparum blood stages (2-5 x 10 (elevado a sexta potência i. RBC. Monkey immunized with the MS2-fusion protein carrying the N-terminal part of the 195 kD precursor of the major merozoite surface antigens induced only marginal protection showing some correlation between antibody titer and degree of parasitaemia. Based on the protective capacity of these recombinant antigens we have expressed two hybrid proteins (MS2/SERP/HRPII and SERP/MSAI/HRPII in E. coli containing selected partial sequences of SERP, HRPII and MSAI. Antibodies raised against both hybrid proteins in rabbits and Aotus monkeys recognize the corresponding schizont polypeptides. In two independent immunization trials using 13 animals (age 7 months to 3 years we could show that immunization of Aotus monkeys with either of the

  10. Molecular players of homologous recombination in protozoan parasites: implications for generating antigenic variation.

    Science.gov (United States)

    Bhattacharyya, Mrinal Kanti; Norris, Douglas E; Kumar, Nirbhay

    2004-06-01

    A major impediment to vaccine development against infections caused by protozoan parasites such as Plasmodium falciparum and Trypanosoma is the extraordinary ability of these parasites to rapidly change their surface molecules, a phenomenon known as antigenic variation. A prominent determinant of antigenic variation in these organisms is associated with rearrangements of genes, especially those known as var in P. falciparum and vsg in Trypanosoma. However, mechanisms underlying generation of anitgenic diversities among these protozoan parasites are poorly understood. The hypothesis that links all the different sections in this review is that antigenic variations in the protozoan parasites is coupled with genetic rearrangements, which occur during the course of DNA break repair. Here, we provide comprehensive and up-to-date information on Rad51 in these organisms, an eukaryotic homologue of bacterial RecA, and homologous recombination mechanisms. In trypanosomes both Rad51-dependent and -independent mechanisms have been suggested to play roles in antigenic variation. Finally, we speculate on how might similar DNA repair mechanisms contribute to genetic rearrangement associated with antigenic variation in the apicomplexan Plasmodium parasites, an immune evasion strategy.

  11. Antigenic variation by Borrelia hermsii occurs through recombination between extragenic repetitive elements on linear plasmids.

    Science.gov (United States)

    Dai, Qiyuan; Restrepo, Blanca I; Porcella, Stephen F; Raffel, Sandra J; Schwan, Tom G; Barbour, Alan G

    2006-06-01

    The relapsing fever agent Borrelia hermsii undergoes multiphasic antigenic variation through gene conversion of a unique expression site on a linear plasmid by an archived variable antigen gene. To further characterize this mechanism we assessed the repertoire and organization of archived variable antigen genes by sequencing approximately 85% of plasmids bearing these genes. Most archived genes shared with the expressed gene a UHS), that surrounded the start codon. The 59 archived variable antigen genes were arrayed in clusters with 13 repetitive, 214 nt long downstream homology sequence (DHS) elements distributed among them. A fourteenth DHS element was downstream of the expression locus. Informative nucleotide polymorphisms in UHS regions and DHS elements were applied to the analysis of the expression site of relapse serotypes from 60 infected mice in a prospective study. For most recombinations, the upstream crossover occurred in the UHS's second half, and the downstream crossover was in the DHS's second half. Usually the closest archival DHS element was used, but occasionally a more distant DHS was employed. The downstream extragenic crossover site in B. hermsii contrasts with the upstream [corrected] extragenic crossover site for antigenic variation in African trypanosomes.

  12. Genes encoding homologous antigens in taeniid cestode parasites: Implications for development of recombinant vaccines produced in Escherichia coli.

    Science.gov (United States)

    Gauci, Charles; Lightowlers, Marshall W

    2013-01-01

    Recombinant vaccine antigens are being evaluated for their ability to protect livestock animals against cysticercosis and related parasitic infections. Practical use of some of these vaccines is expected to reduce parasite transmission, leading to a reduction in the incidence of neurocysticercosis and hydatid disease in humans. We recently showed that an antigen (TSOL16), expressed in Escherichia coli, confers high levels of protection against Taenia solium cysticercosis in pigs, which provides a strategy for control of T. solium parasite transmission. Here, we discuss the characteristics of this antigen that may affect the utility of TSOL16 and related antigens for development as recombinant vaccines. We also report that genes encoding antigens closely related to TSOL16 from T. solium also occur in other related species of parasites. These highly homologous antigens have the potential to be used as vaccines and may provide protection against related species of Taenia that cause infection in other hosts.

  13. The expression and antigenicity identification of recombinant rat TGF-β1 n bacteria

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In order to study structure-function details of TGF-β1,the recombinant mature form of rat TGF-β1 was expressed in bacteria.Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-β1(amino acid 279390)was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase.This system allowed an active and selective synthesis of recombinant TGF-β1.The molecular weight of expressed TGF-β1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD.Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity.Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data.In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-β1 antibody.The mature recombinant rat TGF-β1 expressed in this study provides a useful tool for future detailed structural and functional studies.

  14. Antigen Gene Transfer to Human Plasmacytoid Dendritic Cells Using Recombinant Adenovirus and Vaccinia Virus Vectors

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    Hetty J. Bontkes

    2005-01-01

    Full Text Available Recombinant adenoviruses (RAd and recombinant vaccinia viruses (RVV expressing tumour-associated antigens (TAA are used as anti-tumour vaccines. It is important that these vaccines deliver the TAA to dendritic cells (DC for the induction of a strong immune response. Infection of myeloid DC (MDC with RAd alone is relatively inefficient but CD40 retargeting significantly increases transduction efficiency and DC maturation. Infection with RVV is efficient without DC maturation. Plasmacytoid dendritic cells (PDC play a role in the innate immune response to viral infections through the secretion of IFNα but may also play a role in specific T-cell induction. The aim of our study was to investigate whether PDC are better targets for RAd and RVV based vaccines. RAd alone hardly infected PDC (2% while CD40 retargeting did not improve transduction efficiency, but it did increase PDC maturation (25% CD83 positive cells. Accordingly, specific CTL activation by RAd infected PDC was limited (the number of IFNγ producing CTL was reduced by 75% compared to stimulation with peptide loaded PDC. RVV infected PDC specifically stimulated CTL but PDC were not activated. These Results indicate that PDC are not ideal targets for RAd and RVV based vaccines. However, PDC induced specific CTL activation after pulsing with recombinant protein, indicating that PDC can also cross-present antigens released from surrounding infected cells.

  15. Characterisation of antibody responses in pigs induced by recombinant oncosphere antigens from Taenia solium.

    Science.gov (United States)

    Jayashi, César M; Gonzalez, Armando E; Castillo Neyra, Ricardo; Kyngdon, Craig T; Gauci, Charles G; Lightowlers, Marshall W

    2012-12-14

    Recombinant antigens cloned from the oncosphere life cycle stage of the cestode parasite Taenia solium (T. solium) have been proven to be effective as vaccines for protecting pigs against infections with T. solium. Previous studies have defined three different host protective oncosphere antigens, TSOL18, TSOL16 and TSOL45. In this study, we evaluated the potential for combining the antigens TSOL16 and TSOL18 as a practical vaccine. Firstly, in a laboratory trial, we compared the immunogenicity of the combined antigens (TSOL16/18) versus the immunogenicity of the antigens separately. Secondly, in a field trial, we tested the ability of the TSOL16/18 vaccine to induce detectable antibody responses in animals living under environmental stress and traditionally reared in areas where T. solium cysticercosis is endemic; and finally, we characterised the immune response of the study population. Pigs of 8-16 weeks of age were vaccinated with 200 μg each of TSOL16 and TSOL18, plus 5mg of Quil-A. Specific total IgG, IgG(1) and IgG(2) antibody responses induced by TSOL16 and TSOL18 were determined with ELISA. The immunogenicity of both antigens was retained in the combined TSOL16/18 vaccine. The combined vaccine TSOL16/18 induced detectable specific anti-TSOL18 antibody responses in 100% (113/113) and specific anti-TSOL16 in 99% (112/113) of the vaccinated animals measured at 2 weeks following the booster vaccination. From the two IgG antibody subtypes analysed we found there was stronger response to IgG(2).

  16. Evaluation of a recombinant parasite antigen for the diagnosis of lymphatic filariasis.

    Science.gov (United States)

    Dissanayake, S; Zheng, H; Dreyer, G; Xu, M; Watawana, L; Cheng, G; Wang, S; Morin, P; Deng, B; Kurniawan, L

    1994-06-01

    We evaluated the usefulness of a recombinant parasite antigen (recSXP1) for the serologic diagnosis of lymphatic filariasis. A large proportion of sera from microfilaremic donors living in five different endemic countries (356 of 446 [80%]) contained IgG antibodies to recSXP1, as do sera from approximately 33% of amicrofilaremic patients with acute filarial disease and/or indirect evidence of active filarial infection. Exposure to filarial worms per se does not appear sufficient to elicit an anti-SXP1 antibody response. Thus, this serologic test identifies a large proportion of persons with active lymphatic filariasis among residents of endemic areas.

  17. Delivery of Echinococcus granulosus antigen EG95 to mice and sheep using recombinant vaccinia virus.

    Science.gov (United States)

    Dutton, S; Fleming, S B; Ueda, N; Heath, D D; Hibma, M H; Mercer, A A

    2012-06-01

    The tapeworm Echinococcus granulosus is the causative agent of hydatid disease and affects sheep, cattle, dogs and humans worldwide. It has a two-stage life cycle existing as worms in the gut of infected dogs (definitive host) and as cysts in herbivores and humans (intermediate host). The disease is debilitating and can be life threatening where the cysts interfere with organ function. Interruption of the hydatid life cycle in the intermediate host by vaccination may be a way to control the disease, and a protective oncosphere antigen EG95 has been shown to protect animals against challenge with E. granulosus eggs. We explored the use of recombinant vaccinia virus as a delivery vehicle for EG95. Mice and sheep were immunized with the recombinant vector, and the result monitored at the circulating antibody level. In addition, sera from immunized mice were assayed for the ability to kill E. granulosus oncospheres in vitro. Mice immunized once intranasally developed effective oncosphere-killing antibody by day 42 post-infection. Antibody responses and oncosphere killing were correlated and were significantly enhanced by boosting mice with either EG95 protein or recombinant vector. Sheep antibody responses to the recombinant vector or to EG95 protein mirrored those in mice.

  18. Detection of Q Fever Specific Antibodies Using Recombinant Antigen in ELISA with Peroxidase Based Signal Amplification

    Directory of Open Access Journals (Sweden)

    Hua-Wei Chen

    2014-01-01

    Full Text Available Currently, the accepted method for Q fever serodiagnosis is indirect immunofluorescent antibody assay (IFA using the whole cell antigen. In this study, we prepared the recombinant antigen of the 27-kDa outer membrane protein (Com1 which has been shown to be recognized by Q fever patient sera. The performance of recombinant Com1 was evaluated in ELISA by IFA confirmed serum samples. Due to the low titers of IgG and IgM in Q fever patients, the standard ELISA signals were further amplified by using biotinylated anti-human IgG or IgM plus streptavidin-HRP polymer. The modified ELISA can detect 88% (29 out of 33 of Q fever patient sera collected from Marines deployed to Iraq. Less than 5% (5 out of 156 of the sera from patients with other febrile diseases reacted with the Com1. These results suggest that the modified ELISA using Com1 may have the potential to improve the detection of Q fever specific antibodies.

  19. Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae.

    Science.gov (United States)

    Khulape, S A; Maity, H K; Pathak, D C; Mohan, C Madhan; Dey, S

    2015-09-01

    The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding  sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine.

  20. Further development of a recombinant feline herpesvirus type 1 vector expressing feline calicivirus immunogenic antigen.

    Science.gov (United States)

    Yokoyama, N; Fujita, K; Damiani, A; Sato, E; Kurosawa, K; Miyazawa, T; Ishiguro, S; Mochizuki, M; Maeda, K; Mikami, T

    1998-06-01

    We previously reported the attenuation of thymidine kinase (TK) deficient mutant (C7301dlTK) of feline herpesvirus type 1 (FHV-1) in cats and the construction of a recombinant FHV-1 (C7301dlTK-Cap) inserted a precursor capsid gene of feline calcivirus (FCV) into the TK deletion locus of the C7301dlTK. In this study, we constructed a further improved recombinant FHV-1 (dlTK(gCp)-Cap) carrying a putative FHV-1 gC promoter sequence upstream of the FCV precursor capsid gene of the C7301dlTK-Cap. Growth kinetics of the dlTK(gCp)-Cap in cell cultures was similar to those of C7301dlTK and C7301dlTK-Cap. A strong expression of FCV immunogenic antigen by dlTK(gCp)-Cap was confirmed by indirect immunofluorescence and enzyme-linked immunosorbent assays. In addition, one vaccination with dlTK(gCp)-Cap protected cats more effective against subsequent virulent FCV challenge than that with C7301dlTK-Cap.

  1. Protection against Taenia pisiformis larval infection induced by a recombinant oncosphere antigen vaccine.

    Science.gov (United States)

    Chen, L; Yang, D Y; Xie, Y; Nong, X; Huang, X; Fu, Y; Gu, X B; Wang, S X; Peng, X R; Yang, G Y

    2014-02-13

    Taenia pisiformis larvae cause significant health problems to rabbits. At present, it is not known whether the recombinant antigen from the T. pisiformis oncosphere is able to confer protective immunity against T. pisiformis larval infection. The full-length cDNA was cloned into a pET32a (+) vector, and the recombinant protein was then expressed in BL21 (DE3) cells. Vaccination with the purified rTpUbc2 coupled with QuilA was carried out in New Zealand rabbits to evaluate the immunoprotective effect against T. pisiformis infection. The full-length open reading frame of the TpUbc2 gene was 444 bp, and encoded a 16.63-kDa protein. Finally, rTpUbc2 was used to evaluate the ability to induce immunoprotective responses in rabbits. A 79.3-90.8% reduction (P 0.05). Our data support the use of rTpUbc2 as a potential candidate to develop a vaccine against T. pisiformis larvae.

  2. ION-EXCHANGE IMMUNOAFFINITY PURIFICATION OF A RECOMBINANT BACULOVIRUS PLASMODIUM-FALCIPARUM APICAL MEMBRANE ANTIGEN, PF83/AMA-1

    NARCIS (Netherlands)

    NARUM, DL; WELLING, GW; THOMAS, AW

    1993-01-01

    A two-step purification regime has been developed for a quantitatively minor, putatively transmembrane, M(r) 83 000, apical membrane blood stage vaccine candidate antigen of Plasmodium falciparum (PF83/AMA-1), that has been expressed as a full-length baculovirus recombinant protein, PF83-FG8-1. The

  3. A systematic approach for the identification of novel, serologically reactive recombinant Varicella-Zoster Virus (VZV antigens

    Directory of Open Access Journals (Sweden)

    Lueking Angelika

    2010-07-01

    Full Text Available Abstract Background Varicella-Zoster virus causes chickenpox upon primary infection and shingles after reactivation. Currently available serological tests to detect VZV-specific antibodies are exclusively based on antigens derived from VZV-infected cells. Results We present a systematic approach for the identification of novel, serologically reactive VZV antigens. Therefore, all VZV open reading frames were cloned into a bacterial expression vector and checked for small scale recombinant protein expression. Serum profiling experiments using purified VZV proteins and clinically defined sera in a microarray revealed 5 putative antigens (ORFs 1, 4, 14, 49, and 68. These were rearranged in line format and validated with pre-characterized sera. Conclusions The line assay confirmed the seroreactivity of the identified antigens and revealed its suitability for VZV serodiagnostics comparable to commercially available VZV-ELISA. Recombinant ORF68 (gE proved to be an antigen for high-confidence determination of VZV serostatus. Furthermore, our data suggest that a serological differentiation between chickenpox and herpes zoster may be possible by analysis of the IgM-portfolio against individual viral antigens.

  4. Assessment of the adjuvant activity of mesoporous silica nanoparticles in recombinant Mycoplasma hyopneumoniae antigen vaccines

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    Veridiana Gomes Virginio

    2017-01-01

    Full Text Available The adjuvant potential of two mesoporous silica nanoparticles (MSNs, SBa-15 and SBa-16, was assessed in combination with a recombinant HSP70 surface polypeptide domain from Mycoplasma hyopneumoniae, the etiological agent of porcine enzootic pneumonia (PEP. The recombinant antigen (HSP70212-600, previously shown as immunogenic in formulation with classic adjuvants, was used to immunize BALB/c mice in combination with SBa-15 or SBa-16 MSNs, and the effects obtained with these formulations were compared to those obtained with alum, the adjuvant traditionally used in anti-PEP bacterins. The HSP70212-600 + SBa-15 vaccine elicited a strong humoral immune response, with high serum total IgG levels, comparable to those obtained using HSP70212-600 + alum. The HSP70212-600 + SBa-16 vaccine elicited a moderate humoral immune response, with lower levels of total IgG. The cellular immune response was assessed by the detection of IFN-γ, IL-4 and IL-10 in splenocyte culture supernatants. The HSP70212-600 + SBa-15 vaccine increased IFN-γ, IL-4 and IL-10 levels, while no stimulation was detected with the HSP70212-600 + SBa-16 vaccine. The HSP70212-600 + SBa-15 vaccine induced a mixed Th1/Th2-type response, with an additional IL-10 mediated anti-inflammatory effect, both of relevance for an anti-PEP vaccine. Alum adjuvant controls stimulated an unspecific cellular immune response, with similar levels of cytokines detected in mice immunized either with HSP70212-600 + alum or with the adjuvant alone. The better humoral and cellular immune responses elicited in mice indicated that SBa-15 has adjuvant potential, and can be considered as an alternative to the use of alum in veterinary vaccines. The use of SBa-15 with HSP70212-600 is also promising as a potential anti-PEP subunit vaccine formulation.

  5. Development of Ss-NIE-1 recombinant antigen based assays for immunodiagnosis of strongyloidiasis.

    Science.gov (United States)

    Rascoe, Lisa N; Price, Courtney; Shin, Sun Hee; McAuliffe, Isabel; Priest, Jeffrey W; Handali, Sukwan

    2015-04-01

    Strongyloides stercoralis is a widely distributed parasite that infects 30 to 100 million people worldwide. In the United States strongyloidiasis is recognized as an important infection in immigrants and refugees. Public health and commercial reference laboratories need a simple and reliable method for diagnosis of strongyloidiasis to identify and treat cases and to prevent transmission. The recognized laboratory test of choice for diagnosis of strongyloidiasis is detection of disease specific antibodies, most commonly using a crude parasite extract for detection of IgG antibodies. Recently, a luciferase tagged recombinant protein of S. stercoralis, Ss-NIE-1, has been used in a luciferase immunoprecipitation system (LIPS) to detect IgG and IgG4 specific antibodies. To promote wider adoption of immunoassays for strongyloidiasis, we used the Ss-NIE-1 recombinant antigen without the luciferase tag and developed ELISA and fluorescent bead (Luminex) assays to detect S. stercoralis specific IgG4. We evaluated the assays using well-characterized sera from persons with or without presumed strongyloidiasis. The sensitivity and specificity of Ss-NIE-1 IgG4 ELISA were 95% and 93%, respectively. For the IgG4 Luminex assay, the sensitivity and specificity were 93% and 95%, respectively. Specific IgG4 antibody decreased after treatment in a manner that was similar to the decrease of specific IgG measured in the crude IgG ELISA. The sensitivities of the Ss-NIE-1 IgG4 ELISA and Luminex assays were comparable to the crude IgG ELISA but with improved specificities. However, the Ss-NIE-1 based assays are not dependent on native parasite materials and can be performed using widely available laboratory equipment. In conclusion, these newly developed Ss-NIE-1 based immunoassays can be readily adopted by public health and commercial reference laboratories for routine screening and clinical diagnosis of S. stercoralis infection in refugees and immigrants in the United States.

  6. Toxoplasma gondii: Recombinant GRA5 antigen for detection of immunoglobulin G antibodies using enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Holec-Gasior, Lucyna; Kur, Józef

    2010-03-01

    In this study, for the first time, the evaluation of Toxoplasma gondii full-length recombinant GRA5 antigen for the serodiagnosis of human toxoplasmosis is shown. The recombinant GRA5 antigen as a fusion protein containing His-tag at both terminals was obtained using an Escherichia coli expression system. The usefulness of rGRA5 for the diagnosis of toxoplasmosis in an ELISA was tested on a total of 189 sera from patients with different stages of the infection and 31 sera from sero-negative individuals, obtained during routine diagnostic tests. Anti-GRA5 IgG antibodies were detected in 70.9% of all seropositive serum samples. This result was comparable to ELISA using a Toxoplasma lysate antigen (TLA) and six combinations of recombinant antigens. The sensitivity of IgG ELISA calculated from all positive serum samples was similar for TLA (94.2%), rMAG1+rSAG1+rGRA5 (92.6%), rGRA2+rSAG1+rGRA5 (93.1%) and rROP1+rSAG1+rGRA5 (94.2%) cocktails, whereas the sensitivity of cocktails without rGRA5 antigens was lower giving 82.0%, 86.2% and 87.8%, respectively. Thus, the present study showed that the full-length rGRA5 is suitable for use as a component of an antigen cocktail for the detection of anti-T. gondii IgG antibodies.

  7. Small-angle neutron scattering study of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particle

    Science.gov (United States)

    Sato, M.; Ito, Y.; Kameyama, K.; Imai, M.; Ishikawa, N.; Takagi, T.

    1995-02-01

    The overall and internal structure of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particles was investigated by small-angle neutron scattering using the contrast variation method. The vaccine is a nearly spherical particle, and its contrast-matching point was determined to be at about 24% D 2O content, indicating that a large part of the vaccine particle is occupied by lipids and carbohydrates from the yeast. The Stuhrmann plot suggests that the surface antigens exist predominantly in the peripheral region of the particle, which is favorable to the induction of anti-virus antibodies.

  8. Evaluation of three recombinant multi-antigenic vaccines composed of surface and secretory antigens of Toxoplasma gondii in murine models of experimental toxoplasmosis.

    Science.gov (United States)

    Dziadek, Bozena; Gatkowska, Justyna; Brzostek, Anna; Dziadek, Jaroslaw; Dzitko, Katarzyna; Grzybowski, Marcin; Dlugonska, Henryka

    2011-01-17

    The great clinical and economical impact of Toxoplasma gondii infections makes the development of an effective vaccine for controlling toxoplasmosis an extremely important aim. In the presented study, we evaluate the protective and immunogenic properties of three recombinant subunit vaccines composed of rROP2+rGRA4+rSAG1, rROP2+rROP4+rGRA4 and rROP2+rROP4+rSAG1 proteins of T. gondii in an experimental toxoplasmosis model in the C3H/HeJ and C57BL/6 mouse strains. All three recombinant vaccines induced partial protection as measured by the reduction of brain cyst burden following challenge with five tissue cysts of the low virulence DX T. gondii strain. The level of protection was dependent on the antigen composition of the vaccine and the genetic background of the laboratory animals. The strongest protection against chronic toxoplasmosis was induced in both C3H/HeJ and C57BL/6 mice by the mixture of rhoptry proteins rROP2 and rROP4 combined with tachyzoite major protein rSAG1. The average parasite burden in these groups of mice was reduced by 71% and 90%, respectively, compared to non-vaccinated mice. The observed protective effect was related to the vaccine-induced cellular and humoral immune responses, as measured by the antigen-induced release of the Th1 cytokines IFN-γ and IL-2, the antigen-stimulated proliferation of spleen cells of vaccinated animals in comparison to control animals and the development of systemic antigen-specific IgG1 and IgG2a (C3H/HeJ) or IgG2c (C57BL/6) antibodies. Our studies show that recombinant rROP2, rROP4, rGRA4 and rSAG1 antigens may be promising candidates for a subunit vaccine against toxoplasmosis. Additionally, we demonstrate that the ideal composition of vaccine antigens can be equally effective in mice with different genetic backgrounds and variable levels of innate resistance to toxoplasmosis, resulting in strong protection against T. gondii invasion.

  9. Effects of Brazilian propolis on Leishmania amazonensis

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    Diana Copi Ayres

    2007-03-01

    Full Text Available Leishmaniasis, an endemic parasitosis that leads to chronic cutaneous, mucocutaneous or visceral lesions, is part of those diseases, which still requires improved control tools. Propolis has shown activities against different bacteria, fungi, and parasites. In this study we investigated the effect of four ethanolic extracts of typified propolis collected in different Brazilian states, on Leishmania amazonensis performing assays with promastigote forms, extracellular amastigotes, and on infected peritoneal macrophages. Ethanolic extracts of all propolis samples (BRG, BRPG, BRP-1, and BRV were capable to reduce parasite load as monitored by the percentage of infected macrophages and the number of intracellular parasites. BRV sample called red propolis, collected in the state of Alagoas, and containing high concentration of prenylated and benzophenones compounds, was the most active extract against L. amazonensis. The anti-Leishmania effect of BRV sample was increased in a concentration and time dependent manner. BRV treatment proved to be non-toxic to macrophage cultures. Since BRV extract at the concentration of 25 µg/ml reduced the parasite load of macrophages while presented no direct toxic to promastigotes and extracellular amastigotes, it was suggested that constituents of propolis intensify the mechanism of macrophage activation leading to killing of L. amazonensis. Our results demonstrate, for the first time, that ethanolic extracts of Brazilian propolis reduce L. amazonensis infection in macrophages, and encourage further studies of this natural compound in animal models of leishmaniasis.

  10. Construction of Recombinant Baculoviruses Expressing Infectious Bursal Disease Virus Main Protective Antigen and Their Immune Effects on Chickens

    OpenAIRE

    Jingping Ge; Qi An; Shanshan Song; Dongni Gao; Wenxiang Ping

    2015-01-01

    In order to overcome the limitations of conventional vaccines for infectious bursal disease virus (IBDV), we constructed recombinant dual expression system baculoviruses with VP2 and VP2/4/3, the main protective antigens of IBDV. We compared the immune effects of the baculoviruses in avian cells and detected their control effects on chickens with infectious bursal disease. We used Western blot analysis to measure VP2 protein and VP2/4/3 polyprotein expression in avian cells infected using the...

  11. A recombinant raccoon poxvirus vaccine expressing both Yersinia pestis F1 and truncated V antigens protects animals against lethal plague.

    Science.gov (United States)

    Rocke, Tonie E.; Kingstad-Bakke, B; Berlier, W; Osorio, J.E.

    2014-01-01

    In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307-a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis.. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas.

  12. Recombinant gp19 as a potential antigen for detecting anti-Ehrlichia canis antibodies in dog sera

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    Rômulo Silva de Oliveira

    Full Text Available The canine monocytic ehrlichiosis, caused by Ehrlichia canis, is endemic in several regions of Brazil. Some serological diagnostic techniques using immunodominant proteins of E. canis as antigens are available, but their specificities and sensitivities are questionable. Based on this, the objective of this study was to test the antigenic potential of the recombinant gp19 protein (rGP19 for subsequent use in diagnostic tests. The rGP19 expressed in the Escherichia coli strain BL21 (DE3 C41 was recognized in the sera from experimentally infected dogs using ELISA and Western blotting. Thus, it was possible to obtain a promising antigen with the ability to differentiate between E. canis-positive and -negative animals, even 1 week after infection.

  13. Cryogenic transmission electron microscopy of recombinant tuberculosis vaccine antigen with anionic liposomes reveals formation of flattened liposomes

    Directory of Open Access Journals (Sweden)

    Fox CB

    2014-03-01

    Full Text Available Christopher B Fox,1 Sean K Mulligan,2 Joyce Sung,2 Quinton M Dowling,1 H W Millie Fung,1 Thomas S Vedvick,1 Rhea N Coler1 1Infectious Disease Research Institute, Seattle, WA, USA; 2NanoImaging Services, La Jolla, CA, USA Abstract: Development of lipid-based adjuvant formulations to enhance the immunogenicity of recombinant vaccine antigens is a focus of modern vaccine research. Characterizing interactions between vaccine antigens and formulation excipients is important for establishing compatibility between the different components and optimizing vaccine stability and potency. Cryogenic transmission electron microscopy (TEM is a highly informative analytical technique that may elucidate various aspects of protein- and lipid-based structures, including morphology, size, shape, and phase structure, while avoiding artifacts associated with staining-based TEM. In this work, cryogenic TEM is employed to characterize a recombinant tuberculosis vaccine antigen, an anionic liposome formulation, and antigen–liposome interactions. By performing three-dimensional tomographic reconstruction analysis, the formation of a population of protein-containing flattened liposomes, not present in the control samples, was detected. It is shown that cryogenic TEM provides unique information regarding antigen–liposome interactions not detectable by light-scattering-based methods. Employing a suite of complementary analytical techniques is important to fully characterize interactions between vaccine components. Keywords: vaccine adjuvant; cryo-TEM; antigen-adjuvant interactions; vaccine physical characterization; vaccine formulation morphology; 3D tomographic reconstruction

  14. Adjuvant activity of chicken interleukin-12 co-administered with infectious bursal disease virus recombinant VP2 antigen in chickens.

    Science.gov (United States)

    Su, Bor Sheu; Chiu, Hua Hsien; Lin, Cheng Chung; Shien, Jui Hung; Yin, Hsien Sheng; Lee, Long Huw

    2011-02-15

    A recombinant fowlpox virus (rFPV/VP2) expressing infectious bursal diseases virus (IBDV) VP2 gene has been constructed. After purification and identification of rFPV/VP2, the adjuvant activity of the recombinant chicken IL-12 (rchIL-12), synthesized by our previous construct of rFPV/chIL-12, in rFPV/VP2-expressed rVP2 antigen was assessed in one-week-old specific-pathogen free chickens. The results indicated that rchIL-12 alone or rchIL-12 plus mineral oil (MO) co-administered with rVP2 antigen significantly enhanced the production of serum neutralization (SN) antibody against IBDV, compared to those with MO alone. The SN titers in groups receiving rVP2 antigen with MO alone were more inconsistent after vaccination. On the other hand, rchIL-12 significantly stimulated IFN-γ production in serum and in splenocyte cultured supernatant, suggesting that rchIL-12 alone or plus MO significantly induced a cell-mediated immune response. Finally, bursal lesion protection from very virulent IBDV (vvIBDV) challenge in chickens receiving rVP2 antigen with rchIL-12 alone or plus MO was much more effective than that with MO alone at two weeks after boosting. Taken together, rchIL-12 alone augmented in vivo the induction of a primary and also a secondary SN antibody production and a cell-mediated immunity against IBDV rVP2 antigen, which conferred the enhancement of bursal lesion protective efficacy from vvIBDV challenge. These data indicated that a potential for chIL-12 as immunoadjuvant for chicken vaccine development such as IBDV rVP2 antigen.

  15. Differential reactivity of mouse monoclonal anti-HBs antibodies with recombinant mutant HBs antigens

    Institute of Scientific and Technical Information of China (English)

    Azam Roohi; Yaghoub Yazdani; Jalal Khoshnoodi; Seyed Mohammad Jazayeri; William F Carman; Mahmood Chamankhah; Manley Rashedan; Fazel Shokri

    2006-01-01

    AIM: To investigate the reactivity of a panel of 8 mouse anti-hepatitis B surface antigen (HBsAg) monoclonal antibodies (mAbs) using a collection of 9 recombinant HBsAg mutants with a variety of amino acid substitutions mostly located within the "a" region.METHODS: The entire HBs genes previously cloned into a mammalian expression vector were transiently transfected into COS7 cells. Two standard unmutated sequences of the ayw and adw subtypes served as controls. Secreted mutant proteins were collected and measured by three commercial diagnostic immunoassays to assess transfection efficiency. Reactivity of anti-HBs mAbs with mutated HBsAgs was determined by sandwich enzyme-linked immunosorbent assay (ELISA).RESULTS: Reactivity of anti-HBs mAbs with mutated HBsAgs revealed different patterns. While three mutants reacted strongly with all mAbs, two mutants reacted weakly with only two mAbs and the remaining proteins displayed variable degrees of reactivity towards different mAbs. Accordingly, four groups of mAbs with different but overlapping reactivity patterns could be envisaged. One group consisting of two mAbs (37C5-S7 and 35C6-S11) was found to recognize stable linear epitopes conserved in all mutants. Mutations outside the "a"determinant at positions 120 (P→S), 123(T→N) and 161(M→T) were found to affect reactivity of these mAbs.CONCLUSION: Our findings could have important implications for biophysical studies, vaccination strategies and immunotherapy of hepatitis B virus (HBV) mutants.

  16. Immunogenicity and protective efficacy of recombinant Modified Vaccinia virus Ankara candidate vaccines delivering West Nile virus envelope antigens.

    Science.gov (United States)

    Volz, Asisa; Lim, Stephanie; Kaserer, Martina; Lülf, Anna; Marr, Lisa; Jany, Sylvia; Deeg, Cornelia A; Pijlman, Gorben P; Koraka, Penelope; Osterhaus, Albert D M E; Martina, Byron E; Sutter, Gerd

    2016-04-07

    West Nile virus (WNV) cycles between insects and wild birds, and is transmitted via mosquito vectors to horses and humans, potentially causing severe neuroinvasive disease. Modified Vaccinia virus Ankara (MVA) is an advanced viral vector for developing new recombinant vaccines against infectious diseases and cancer. Here, we generated and evaluated recombinant MVA candidate vaccines that deliver WNV envelope (E) antigens and fulfil all the requirements to proceed to clinical testing in humans. Infections of human and equine cell cultures with recombinant MVA demonstrated efficient synthesis and secretion of WNV envelope proteins in mammalian cells non-permissive for MVA replication. Prime-boost immunizations in BALB/c mice readily induced circulating serum antibodies binding to recombinant WNV E protein and neutralizing WNV in tissue culture infections. Vaccinations in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice elicited WNV E-specific CD8+ T cell responses. Moreover, the MVA-WNV candidate vaccines protected C57BL/6 mice against lineage 1 and lineage 2 WNV infection and induced heterologous neutralizing antibodies. Thus, further studies are warranted to evaluate these recombinant MVA-WNV vaccines in other preclinical models and use them as candidate vaccine in humans.

  17. Pro-recombination role of Srs2 protein requires SUMO (small ubiquitin-like modifier) but is independent of PCNA (proliferating cell nuclear antigen) interaction

    DEFF Research Database (Denmark)

    Kolesar, Peter; Altmannova, Veronika; Pinela da Silva, Sonia Cristina;

    2016-01-01

    -interacting motif (SIM) of Srs2 is important for the interaction with several recombination factors. Lack of SIM, but not proliferating cell nuclear antigen (PCNA)-interacting motif (PIM), leads to increased cell death under circumstances requiring homologous recombination for DNA repair. Simultaneous mutation...

  18. Screening of human tumor antigens for CD4 T cell epitopes by combination of HLA-transgenic mice, recombinant adenovirus and antigen peptide libraries.

    Directory of Open Access Journals (Sweden)

    Wolfram Osen

    Full Text Available BACKGROUND: As tumor antigen-specific CD4+ T cells can mediate strong therapeutic anti-tumor responses in melanoma patients we set out to establish a comprehensive screening strategy for the identification of tumor-specific CD4+ T cell epitopes suitable for detection, isolation and expansion of tumor-reactive T cells from patients. METHODS AND FINDINGS: To scan the human melanoma differentiation antigens TRP-1 and TRP-2 for HLA-DRB1*0301-restricted CD4+ T cell epitopes we applied the following methodology: Splenocytes of HLA-DRB1*0301-transgenic mice immunized with recombinant adenovirus encoding TRP-1 (Ad5.TRP-1 or TRP-2 (Ad5.TRP-2 were tested for their T cell reactivity against combinatorial TRP-1- and TRP-2-specific peptide libraries. CD4+ T cell epitopes thus identified were validated in the human system by stimulation of peripheral blood mononuclear cells (PBMC from healthy donors and melanoma patients. Using this strategy we observed that recombinant Ad5 induced strong CD4+ T cell responses against the heterologous tumor antigens. In Ad5.TRP-2-immunized mice CD4+ T cell reactivity was detected against the known HLA-DRB1*0301-restricted TRP-2(60-74 epitope and against the new epitope TRP-2(149-163. Importantly, human T cells specifically recognizing target cells loaded with the TRP-2(149-163-containing library peptide or infected with Ad5.TRP-2 were obtained from healthy individuals, and short term in vitro stimulation of PBMC revealed the presence of epitope-reactive CD4+ T cells in melanoma patients. Similarly, immunization of mice with Ad5.TRP-1 induced CD4+ T cell responses against TRP-1-derived peptides that turned out to be recognized also by human T cells, resulting in the identification of TRP-1(284-298 as a new HLA-DRB1*0301-restricted CD4+ T cell epitope. CONCLUSIONS: Our screening approach identified new HLA-DRB1*0301-restricted CD4+ T cell epitopes derived from melanoma antigens. This strategy is generally applicable to target

  19. Expression of recombinant Araraquara Hantavirus nucleoprotein in insect cells and its use as an antigen for immunodetection compared to the same antigen expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Wolff Jose LC

    2011-05-01

    Full Text Available Abstract Background Antigens for Hantavirus serological tests have been produced using DNA recombinant technology for more than twenty years. Several different strategies have been used for that purpose. All of them avoid the risks and difficulties involved in multiplying Hantavirus in the laboratory. In Brazil, the Araraquara virus is one of the main causes of Hantavirus Cardio-Pulmonary Syndrome (HCPS. Methods In this investigation, we report the expression of the N protein of the Araraquara Hantavirus in a Baculovirus Expression System, the use of this protein in IgM and IgG ELISA and comparison with the same antigen generated in E. coli. Results The protein obtained, and purified in a nickel column, was effectively recognized by antibodies from confirmed HCPS patients. Comparison of the baculovirus generated antigen with the N protein produced in E. coli showed that both were equally effective in terms of sensitivity and specificity. Conclusions Our results therefore indicate that either of these proteins can be used in serological tests in Brazil.

  20. Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding.

    Science.gov (United States)

    Pavan, María Elisa; Pavan, Esteban Enrique; Cairó, Fabián Martín; Pettinari, María Julia

    2016-01-01

    Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenic protein that is the basis for both anthrax vaccines and diagnostic methods. Properly folded antigenic PA is necessary for these applications. In this study a high level of PA was obtained in recombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, which facilitated its efficient purification by simple washing steps; however, it could not be recognized by specific antibodies. Refolding conditions were subsequently analyzed in a high-throughput manner that enabled nearly a hundred different conditions to be tested simultaneously. The recovery of the ability of PA to be recognized by antibodies was screened by dot blot using a coefficient that provided a measure of properly refolded protein levels with a high degree of discrimination. The best refolding conditions resulted in a tenfold increase in the intensity of the dot blot compared to the control. The only refolding additive that consistently yielded good results was L-arginine. The statistical analysis identified both cooperative and negative interactions between the different refolding additives. The high-throughput approach described in this study that enabled overproduction, purification and refolding of PA in a simple and straightforward manner, can be potentially useful for the rapid screening of adequate refolding conditions for other overexpressed antigenic proteins.

  1. Recombinant Varicella-Zoster Virus Vaccines as Platforms for Expression of Foreign Antigens

    Directory of Open Access Journals (Sweden)

    Wayne L. Gray

    2013-01-01

    Full Text Available Varicella-zoster virus (VZV vaccines induce immunity against childhood chickenpox and against shingles in older adults. The safety, efficacy, and widespread use of VZV vaccines suggest that they may also be effective as recombinant vaccines against other infectious diseases that affect the young and the elderly. The generation of recombinant VZV vaccines and their evaluation in animal models are reviewed. The potential advantages and limitations of recombinant VZV vaccines are addressed.

  2. Stable antigen is most effective for eliciting CD8+ T-cell responses after DNA vaccination and infection with recombinant vaccinia virus in vivo.

    Science.gov (United States)

    Schliehe, Christopher; Bitzer, Annegret; van den Broek, Maries; Groettrup, Marcus

    2012-09-01

    The induction of strong CD8(+) T-cell responses against infectious diseases and cancer has remained a major challenge. Depending on the source of antigen and the infectious agent, priming of CD8(+) T cells requires direct and/or cross-presentation of antigenic peptides on major histocompatibility complex (MHC) class I molecules by professional antigen-presenting cells (APCs). However, both pathways show distinct preferences concerning antigen stability. Whereas direct presentation was shown to efficiently present peptides derived from rapidly degraded proteins, cross-presentation is dependent on long-lived antigen species. In this report, we analyzed the role of antigen stability on DNA vaccination and recombinant vaccinia virus (VV) infection using altered versions of the same antigen. The long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) can be targeted for degradation by N-terminal fusion to ubiquitin or, as we show here, to the ubiquitin-like modifier FAT10. Direct presentation by cells either transfected with NP-encoding plasmids or infected with recombinant VV in vitro was enhanced in the presence of short-lived antigens. In vivo, however, the highest induction of NP-specific CD8(+) T-cell responses was achieved in the presence of long-lived NP. Our experiments provide evidence that targeting antigens for proteasomal degradation does not improve the immunogenicity of DNA vaccines and recombinant VVs. Rather, it is the long-lived antigen that is superior for the efficient activation of MHC class I-restricted immune responses in vivo. Hence, our results suggest a dominant role for antigen cross-priming in DNA vaccination and recombinant VV infection.

  3. Evaluation of rabbit antibody response against 8 and 16 kDa recombinant subunits of antigen B fromEchinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    Jahangir Abdi; Bahram Kazemi; Mohammad Hasan Karimfar; Mohammad Bagher Rokni

    2012-01-01

    ABSTRACT Objective:To immunize rabbits with12 and16 kDa recombinant subunits of antigenB from Echinococcus granulosus (E. granulosus) and measuring polyclonal antibody and humoral immune response usingELISA and gel diffusion.Methods:Two mentioned antigens were cloned and expressed in expression vector and purified by affinity chromatography.Four young rabbits were selected and challenged intradermally with yielded recombinant antigens.Rabbits’ sera were collected post infection and were tested usingELISA and gel diffusion for polyclonal antibody detection10 days after last injection.Results:The specific antibody against the recombinant peptides was efficiently produced within4 weeks post infection.Conclusions:Produced recombinants proteins could induce the immune response of the rabbits successfully. This process might improve the clarification of diagnosis and vaccination as regards hydatidosis.

  4. Enhanced protective efficacy of nonpathogenic recombinant leishmania tarentolae expressing cysteine proteinases combined with a sand fly salivary antigen.

    Directory of Open Access Journals (Sweden)

    Farnaz Zahedifard

    2014-03-01

    Full Text Available BACKGROUND: Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. CONCLUSION/SIGNIFICANCE: The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15 and represents a novel promising vaccination approach against leishmaniasis.

  5. Enhanced Protective Efficacy of Nonpathogenic Recombinant Leishmania tarentolae Expressing Cysteine Proteinases Combined with a Sand Fly Salivary Antigen

    Science.gov (United States)

    Taheri, Tahereh; Taslimi, Yasaman; Doustdari, Fatemeh; Seyed, Negar; Torkashvand, Fatemeh; Meneses, Claudio; Papadopoulou, Barbara; Kamhawi, Shaden; Valenzuela, Jesus G.; Rafati, Sima

    2014-01-01

    Background Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. Methodology/Principal Findings Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. Conclusion/Significance The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis. PMID:24675711

  6. Heat-shock protein 70 from plant biofactories of recombinant antigens activate multiepitope-targeted immune responses.

    Science.gov (United States)

    Buriani, Giampaolo; Mancini, Camillo; Benvenuto, Eugenio; Baschieri, Selene

    2012-04-01

    Although a physiological role of heat-shock proteins (HSP) in antigen presentation and immune response activation has not been directly demonstrated, their use as vaccine components is under clinical trial. We have previously demonstrated that the structure of plant-derived HSP70 (pHSP70) can be superimposed to the mammalian homologue and similarly to the mammalian counterpart, pHSP70-polypeptide complexes can activate the immune system. It is here shown that pHSP70 purified from plant tissues transiently expressing the influenza virus nucleoprotein are able to induce both the activation of major histocompatibility complex class I-restricted polyclonal T-cell responses and antibody production in mice of different haplotypes without the need of adjuvant co-delivery. These results indicate that pHSP70 derived from plants producing recombinant antigens may be used to formulate multiepitope vaccines.

  7. Recombinant Salmonella Expressing Burkholderia mallei LPS O Antigen Provides Protection in a Murine Model of Melioidosis and Glanders.

    Science.gov (United States)

    Moustafa, Dina A; Scarff, Jennifer M; Garcia, Preston P; Cassidy, Sara K B; DiGiandomenico, Antonio; Waag, David M; Inzana, Thomas J; Goldberg, Joanna B

    2015-01-01

    Burkholderia pseudomallei and Burkholderia mallei are the etiologic agents of melioidosis and glanders, respectively. These bacteria are highly infectious via the respiratory route and can cause severe and often fatal diseases in humans and animals. Both species are considered potential agents of biological warfare; they are classified as category B priority pathogens. Currently there are no human or veterinary vaccines available against these pathogens. Consequently efforts are directed towards the development of an efficacious and safe vaccine. Lipopolysaccharide (LPS) is an immunodominant antigen and potent stimulator of host immune responses. B. mallei express LPS that is structurally similar to that expressed by B. pseudomallei, suggesting the possibility of constructing a single protective vaccine against melioidosis and glanders. Previous studies of others have shown that antibodies against B. mallei or B. pseudomallei LPS partially protect mice against subsequent lethal virulent Burkholderia challenge. In this study, we evaluated the protective efficacy of recombinant Salmonella enterica serovar Typhimurium SL3261 expressing B. mallei O antigen against lethal intranasal infection with Burkholderia thailandensis, a surrogate for biothreat Burkholderia spp. in a murine model that mimics melioidosis and glanders. All vaccine-immunized mice developed a specific antibody response to B. mallei and B. pseudomallei O antigen and to B. thailandensis and were significantly protected against challenge with a lethal dose of B. thailandensis. These results suggest that live-attenuated SL3261 expressing B. mallei O antigen is a promising platform for developing a safe and effective vaccine.

  8. Evaluation on a Streptococcus suis vaccine using recombinant sao-l protein manufactured by bioreactors as the antigen in pigs.

    Science.gov (United States)

    Hsueh, K-J; Lee, J-W; Hou, S-M; Chen, H-S; Chang, T-C; Chu, C-Y

    2014-12-01

    Streptococcus suis (S. suis) can be classified into 33 serotypes based on the structure of capsular polysaccharides. Recent research indicated that a new surface protein designated as Sao (surface antigen one) reacts with 30 serotypes of convalescent-phase sera during S. suis infections, which makes Sao a good potential antigen for developing S. suis vaccines. The objectives of this study were to produce recombinant Sao-L protein (rSao-L) from a strain of S. suis serotype 2 by a prokaryotic expression system in bioreactors and to use rSao-L as the antigen for a S. suis vaccine in mouse and swine models. The antibody titres in mice and pigs immunized with rSao-L were significantly (P bacteria, the anatomical lesions in pigs immunized with rSao-L were reduced by 60%. These data indicated that immunization with rSao-L can confer cross-serotype protection against S. suis. Moreover, percentages of CD8(+) and CD4(+) /CD8(+) double-positive T cells in immunized pigs were significantly higher than those of the control group (P < 0.01). Using bioreactors to produce rSao-L as the antigen for S. suis vaccines may broaden protective efficacy and reduce production costs.

  9. Recombinant Salmonella Expressing Burkholderia mallei LPS O Antigen Provides Protection in a Murine Model of Melioidosis and Glanders.

    Directory of Open Access Journals (Sweden)

    Dina A Moustafa

    Full Text Available Burkholderia pseudomallei and Burkholderia mallei are the etiologic agents of melioidosis and glanders, respectively. These bacteria are highly infectious via the respiratory route and can cause severe and often fatal diseases in humans and animals. Both species are considered potential agents of biological warfare; they are classified as category B priority pathogens. Currently there are no human or veterinary vaccines available against these pathogens. Consequently efforts are directed towards the development of an efficacious and safe vaccine. Lipopolysaccharide (LPS is an immunodominant antigen and potent stimulator of host immune responses. B. mallei express LPS that is structurally similar to that expressed by B. pseudomallei, suggesting the possibility of constructing a single protective vaccine against melioidosis and glanders. Previous studies of others have shown that antibodies against B. mallei or B. pseudomallei LPS partially protect mice against subsequent lethal virulent Burkholderia challenge. In this study, we evaluated the protective efficacy of recombinant Salmonella enterica serovar Typhimurium SL3261 expressing B. mallei O antigen against lethal intranasal infection with Burkholderia thailandensis, a surrogate for biothreat Burkholderia spp. in a murine model that mimics melioidosis and glanders. All vaccine-immunized mice developed a specific antibody response to B. mallei and B. pseudomallei O antigen and to B. thailandensis and were significantly protected against challenge with a lethal dose of B. thailandensis. These results suggest that live-attenuated SL3261 expressing B. mallei O antigen is a promising platform for developing a safe and effective vaccine.

  10. Use of Strep-tag II for rapid detection and purification of Mycobacterium tuberculosis recombinant antigens secreted by Streptomyces lividans.

    Science.gov (United States)

    Ayala, Julio C; Pimienta, Elsa; Rodríguez, Caridad; Anné, Jozef; Vallín, Carlos; Milanés, María T; King-Batsios, Emmanuel; Huygen, Kris; Van Mellaert, Lieve

    2013-09-01

    Recent results with respect to the secretory production of bio-active Mycobacterium tuberculosis proteins in Streptomyces have stimulated the further exploitation of this host as a bacterial cell factory. However, the rapid isolation of a recombinant protein by conventional procedures can be a restrictive step. A previous attempt to isolate recombinant antigens fused to the widely used 6His-tag was found to be relatively incompatible with secretory production in the Streptomyces host. As an alternative, the eight-residue Strep-tag® II (WSHPQFEK), displaying intrinsic binding affinity towards streptavidin, was evaluated for the secretory production of two M. tuberculosis immunodominant antigens in Streptomyces lividans and their subsequent downstream processing. Therefore, the genes ag85A (Rv3804c, encoding the mycolyl-transferase Ag85A) and Rv2626c (encoding hypoxic response protein 1), were equipped with a 3'-Strep-tag® II-encoding sequence and placed under control of the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) transcriptional, translational and signal sequences. Strep-tagged Ag85A and Rv2626c proteins were detected in the spent medium of recombinant S. lividans cultures at 48h of growth, and purified using a Strep-Tactin Superflow® matrix. Recombinant Ag85A appeared as a 30-kDa protein of which the N-terminal amino acid sequence was identical to the expected one. Rv2626c was produced in two forms of 17 and 37kDa respectively, both with the same predicted N-terminal sequence, suggesting that the 37-kDa product is an Rv2626c dimer. The obtained results indicate that the Strep-tagII is proteolytically stable in Streptomyces and does not interfere with the membrane translocation of Ag85A and Rv2626c. A comparison of reactivity of serum from tuberculosis patients versus healthy persons by ELISA showed that both S. lividans-derived antigens were recognized by sera of individuals infected with M. tuberculosis, indicating that they remained

  11. Identification and Molecular Characterization of a Gene Encoding a Protective Leishmania amazonensis Trp-Asp (WD) Protein

    OpenAIRE

    2004-01-01

    Several Leishmania proteins have been identified and characterized in pursuit of understanding pathogenesis and protection in cutaneous leishmaniasis. In the present study, we utilized sera from infected BALB/c mice to screen a Leishmania amazonensis amastigote cDNA expression library and obtained the full-length gene that encodes a novel Trp-Asp (WD) protein designated LAWD (for Leishmania antigenic WD protein). The WD family of proteins mediates protein-protein interactions and coordinates ...

  12. Development of a sandwich Dot-ELISA for detecting bovine viral diarrhea virus antigen with E2 recombinant protein

    Institute of Scientific and Technical Information of China (English)

    Yuelan ZHAO; Yuzhu ZUO; Lei ZHANG; Jinghui FAN; Hanchun YANG; Jianhua QIN

    2009-01-01

    The IgG antibodies of rabbit anti-E2 protein of the bovine viral diarrhea virus were prepared by a general method from high efficiency serum immunized by E2 recombinant protein antigen expressed in E. coli prokaryotic expression system and were labeled to make enzymelabeled antibody with the method of NaIO4. A sandwich Dot enzyme-linked immunosorbent assay (Dot-ELISA) for the detection of BVDV was developed. The optimal reaction conditions of Dot-ELISAwere determined. The results show that optimal coating antibody was 300 μg·mL-1, the working concentration of HRP-labeled antibody was 1:50. The optimal blocking reagent and time were 5% bovine serum and 45 rain. The minimum detection of the content of antigen reached 1.35μg·mL-1. Compared with the routine IDEXX ELISA test kit with the whole virus, its specificity, sensitivity and coincidence rate were 90.48%, 96.55% and 95.24%, respectively. Compared with the sandwich Dot-ELISA with the negative staining electron microscope and RT-PCR, the coincidence rates were 90.9% and 93.1%, respectively. In addition, Bovine viral diarrhea virus (BVDV) antigen of 178 samples collected from cow farms in the Hebei Province, China, were detected by the developed Dot-ELISA and the IDEXX BVDV antigen Test Kit simultaneously, BVDV antigen positive rate was 39.89%-41.01%. The result of detecting clinical samples demonstrated that the established method showed its specificity, sensitivity and repeatability, whereas the results were easily interpreted without an ELISA reader.

  13. Development and comparative evaluation of a plate enzyme-linked immunosorbent assay based on recombinant outer membrane antigens Omp28 and Omp31 for diagnosis of human brucellosis.

    Science.gov (United States)

    Tiwari, Sapana; Kumar, Ashu; Thavaselvam, Duraipandian; Mangalgi, Smita; Rathod, Vedika; Prakash, Archana; Barua, Anita; Arora, Sonia; Sathyaseelan, Kannusamy

    2013-08-01

    Brucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genus Brucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling of Brucella species for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) of Brucella for diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and

  14. Immune responses to a recombinant attenuated Salmonella typhimurium strain expressing a Taenia solium oncosphere antigen TSOL18.

    Science.gov (United States)

    Ding, Juntao; Zheng, Yadong; Wang, Ying; Dou, Yongxi; Chen, Xiaoyu; Zhu, Xueliang; Wang, Shuai; Zhang, Shaohua; Liu, Zhenyong; Hou, Junling; Zhai, Junjun; Yan, Hongbin; Luo, Xuenong; Cai, Xuepeng

    2013-01-01

    A tapeworm, Taenia solium, remains a great threat to human health, particularly in developing countries. The life cycle of T. solium is thought to be terminated via vaccination of intermediate hosts. In this study, we constructed a recombinant attenuated Salmonella typhimurium live vaccine strain χ4558 expressing a TSOL18 antigen. SDS-PAGE and Western blot confirmed the expression of the interest protein and its antigenic property. The recombinant strain stably propagated in vitro, of which the growth was not reversely influenced by TSOL18 protein expressed. It was also shown that mice survived 10(12) CFU of S. typhimurium χ4558, while all mice infected with 10(7) CFU of the wild-type died within five days. The mouse experiment indicated that vaccine strain χ4558 induced a high titer of specific antibody for a long time. In contrast to the controls, the vaccinated mice had an obvious augment of CD4(+) and CD8(+) T lymphocytes and the percentage of helper CD4(+)/CD8(+) T lymphocytes was significantly increased (psolium.

  15. Recombinant Pvs48/45 antigen expressed in E. coli generates antibodies that block malaria transmission in Anopheles albimanus mosquitoes.

    Directory of Open Access Journals (Sweden)

    Myriam Arévalo-Herrera

    Full Text Available Transmission of malaria parasites from humans to Anopheles mosquitoes can be inhibited by specific antibodies elicited during malaria infection, which target surface Plasmodium gametocyte/gamete proteins. Some of these proteins may have potential for vaccine development. Pvs48/45 is a P. vivax gametocyte surface antigen orthologous to Pfs48/45, which may play a role during parasite fertilization and thus has potential for transmission blocking (TB activity. Here we describe the expression of a recombinant Pvs48/45 protein expressed in Escherichia coli as a ∼60kDa construct which we tested for antigenicity using human sera and for its immunogenicity and transmission blocking activity of specific anti-mouse and anti-monkey Pvs48/45 antibodies. The protein reacted with sera of individuals from malaria-endemic areas and in addition induced specific IgG antibody responses in BALB/c mice and Aotus l. griseimembra monkeys. Sera from both immunized animal species recognized native P. vivax protein in Western blot (WB and immunofluorescence assays. Moreover, sera from immunized mice and monkeys produced significant inhibition of parasite transmission to An. Albimanus mosquitoes as shown by membrane feeding assays. Results indicate the presence of reactive epitopes in the Pvs48/45 recombinant product that induce antibodies with TB activity. Further testing of this protein is ongoing to determine its vaccine potential.

  16. Dot-ELISA Rapid Test Using Recombinant 56-kDa Protein Antigens for Serodiagnosis of Scrub Typhus.

    Science.gov (United States)

    Rodkvamtook, Wuttikon; Zhang, Zhiwen; Chao, Chien-Chung; Huber, Erin; Bodhidatta, Dharadhida; Gaywee, Jariyanart; Grieco, John; Sirisopana, Narongrid; Kityapan, Manerat; Lewis, Michael; Ching, Wei-Mei

    2015-05-01

    We developed a rapid dot-enzyme-linked immunosorbent assay (dot-ELISA) using the combination of recombinant 56-kDa protein antigens that exhibited broad reactivity with serum antibodies against the four most prevalent strains (Karp, Kato, Gilliam, and TA763) of Orientia tsutsugamushi. The assay is rapid (30 minutes), and can be done at room temperature, and results can be read by the naked eye. Only a simple shaker is required to wash the membrane. Sera from 338 patients suspected of being ill with scrub typhus from rural hospitals around Thailand were tested using this dot-ELISA. Seventy-five (22.2%) patients were found to be positive. The sensitivity and specificity of dot-ELISA were determined using the indirect immunofluorescent assay (IFA) test as the gold standard, with the cutoff titer of immunoglobulin peroxidase conjugate M (IgM)/G (IgG) greater than 1:400/1:400. The dot-ELISA had a sensitivity of 98.5%, a specificity of 96.3%, a positive predictive value of 86.7%, and a negative predictive value of 99.6% for the acute-phase specimens. The results indicate that dot-ELISA rapid test using recombinant 56-kDa protein antigen was comparable with the IFA test and may be very useful for the diagnosis of scrub typhus in rural hospitals, where IFA is not available.

  17. Isolation of a novel gene from Photobacterium damselae subsp. piscicida and analysis of the recombinant antigen as promising vaccine candidate.

    Science.gov (United States)

    Andreoni, Francesca; Boiani, Romina; Serafini, Giordano; Amagliani, Giulia; Dominici, Sabrina; Riccioni, Giulia; Zaccone, Renata; Mancuso, Monique; Scapigliati, Giuseppe; Magnani, Mauro

    2013-01-21

    Photobacterium damselae subsp. piscicida (PDP) is the causative agent of fish pasteurellosis, a bacterial disease causing important losses in marine aquaculture. Vaccines against the pathogen can be a way to control the infection and avoid antibiotic treatments. However, a satisfactory protective vaccine against fish pasteurellosis is not commercially available. In this study, a biotechnogical approach based on reverse vaccinology has been used to identify potential vaccine candidates for the development of a recombinant subunit vaccine. Genome sequencing of clones from a genomic cosmid library of PDP and in silico selection of the surface exposed proteins were the initial steps in vaccine candidate identification. From 370 open reading frames (ORF) eight potential antigens were selected, expressed as recombinant proteins and purified. These vaccine candidates were used to generate specific polyclonal antibodies in mice. Each antibody was then screened in vitro by inhibition adherence assay of live PDP on chinook salmon embryo cells (CHSE-214). A lipoprotein, found to be involved in the adherence of the bacterium to epithelial cells and annotated as PDP_0080, was then selected. The recombinant protein was further investigated in fish vaccination and challenge experiments to assess its ability to protect sea bass, Dicentrarchus labrax, against PDP infection. Immunisation with PDP_0080 recombinant protein elicited high specific antibody titres. Furthermore, the survival rate of fish immunized with the 25 μg dose of protein was significantly higher compared to the control group. The results of the study suggest that the PDP_0080 protein could be a promising candidate for the design of a recombinant vaccine against pasteurellosis.

  18. Production of Toxocara cati TES-120 Recombinant Antigen and Comparison with its T. canis Homolog for Serodiagnosis of Toxocariasis.

    Science.gov (United States)

    Zahabiun, Farzaneh; Sadjjadi, Seyed Mahmoud; Yunus, Muhammad Hafiznur; Rahumatullah, Anizah; Moghaddam, Mohammad Hosein Falaki; Saidin, Syazwan; Noordin, Rahmah

    2015-08-01

    Toxocariasis is a cosmopolitan zoonotic disease caused by the infective larvae of Toxocara canis and T. cati. Diagnosis in humans is usually based on clinical symptoms and serology. Immunoglobulin G (IgG)-enzyme-linked immunosorbent assay kits using T. canis excretory-secretory (TES) larval antigens are commonly used for serodiagnosis. Differences in the antigens of the two Toxocara species may influence the diagnostic sensitivity of the test. In this study, T. cati recombinant TES-120 (rTES-120) was cloned, expressed, and compared with its T. canis homolog in an IgG4-western blot. The diagnostic sensitivity and specificity of T. cati rTES-120 were 70% (33/47) and 100% (39/39), respectively. T. canis rTES-120 showed 57.4% sensitivity and 94.4% specificity. When the results of assays using rTES-120 of both species were considered, the diagnostic sensitivity was 76%. This study shows that using antigens from both Toxocara species may improve the serodiagnosis of toxocariasis.

  19. Effect of context and adjuvant on the immunogenicity of recombinant proteins and peptide conjugates derived from the polymorphic malarial surface antigen MSA2.

    Science.gov (United States)

    Jones, G L; Spencer, L; Lord, R; Saul, A J

    1996-01-01

    We have identified a 51 kDa glycosylated myristylated merozoite surface antigen (MSA2) as the target of a number of monoclonal antibodies which inhibit in vitro invasion of the human malarial parasite Plasmodium falciparum. This antigen has been shown to exist in a limited number of strain specific forms but despite wide variation in the sequences of the internal repeat regions both N and C terminal elements of the protein are almost totally conserved. Accordingly, we prepared a large number of overlapping peptide constructs and demonstrated that one peptide SNTFINNA (E71) from the N terminus and two peptides, QHGHMHGS (G5) and NTSDSQKE (G12) from the C terminus could, when suitably conjoined to the carrier protein diphtheria toxoid (DT), elicit antibodies reactive with MSA2 from diverse strains of P. falciparum. Here we compare the immunogenicity of these peptide constructs with two recombinant proteins containing the entire amino acid sequence of MSA2 from the FCQ-27/PNG strain (1609) and the 3D7 strain (1623). We have formulated these recombinant and peptide antigens with Freund's adjuvant, Alum and Algammulin. Both recombinant and peptide antigens elicit high titre antibodies when tested by ELISA against the immunogens themselves. Although both recombinant proteins include the constant region peptide sequences E71, G5 and G12, the extent of ELISA cross reaction between antibody raised against recombinant and peptide antigen or antibody raised against peptide and recombinant antigen is small and sporadic, and depends to an extent on the adjuvant employed. Antisera against both recombinant proteins 1609 and 1623 detected either recombinant on Western blots, as well as detecting native MSA2 in whole protein extracts from both FCQ-27/PNG and 3D7 strains. Antisera against peptide construct E71 recognized recombinant 1609 but not 1623 but recognized the native MSA2 in both strains studied. Antisera against peptide construct G5 showed a similar pattern of recognition

  20. In vivo near-infrared fluorescence imaging of Leishmania amazonensis expressing infrared fluorescence protein (iRFP) for real-time monitoring of cutaneous leishmaniasis in mice.

    Science.gov (United States)

    Oliveira, Janaina Correia; da Silva, Aline Caroline; Oliveira, Renato Antonio Dos Santos; Pereira, Valéria Rêgo Alves; Gil, Laura Helena Vega Gonzales

    2016-11-01

    The use of Leishmania amazonensis-infected BALB/c mice is an important model for the study of experimental cutaneous leishmaniasis. Here we report the development of a non-invasive method to directly evaluate and measure parasite burden during the course of the infection, based on the near-infrared fluorescence detection of a recombinant L. amazonensis strain. So, we generated a L. amazonensis strain that stably expresses the near-infrared protein (iRFP) gene and compared the maintenance of its vitro and in vivo characteristics, such as fitness, pathogenicity and fluorescence emission. After that, we followed the disease development, as well as the parasite burden in BALB/c mice footpads infected with L. amazonensis-iRFP, by using an in vivo near-infrared fluorescence scanner. In vitro results showed a linear correlation between the fluorescence emission and the number of parasites. The in vivo study showed that the use of iRFP-transfected L. amazonensis enables the monitoring of parasite burden by measuring fluorescence signals. Therefore, this technique can be confidently used to directly monitor parasitic load and infection overtime and could be an excellent tool for in vitro and in vivo screening of anti-leishmanial drugs and vaccine efficiency. This is the first report of the use of the near-infrared fluorescence imaging technique for monitoring in vivo cutaneous leishmaniasis.

  1. Immunogenicity of recombinant attenuated Salmonella enterica serovar Typhimurium vaccine strains carrying a gene that encodes Eimeria tenella antigen SO7.

    Science.gov (United States)

    Konjufca, Vjollca; Jenkins, Mark; Wang, Shifeng; Juarez-Rodriguez, Maria Dolores; Curtiss, Roy

    2008-12-01

    Recombinant attenuated Salmonella vaccines against avian coccidiosis were developed to deliver Eimeria species antigens to the lymphoid tissues of chickens via the type 3 secretion system (T3SS) and the type 2 secretion system (T2SS) of Salmonella. For antigen delivery via the T3SS, the Eimeria tenella gene encoding sporozoite antigen SO7 was cloned downstream of the translocation domain of the Salmonella enterica serovar Typhimurium sopE gene in the parental pYA3868 and pYA3870 vectors to generate pYA4156 and pYA4157. Newly constructed T3SS vectors were introduced into host strain chi8879 (Delta phoP233 Delta sptP1033::xylE Delta asdA16), an attenuated derivative of the highly virulent UK-1 strain. The SopE-SO7 fusion protein was secreted by the T3SS of Salmonella. The vector pYA4184 was constructed for delivery of the SO7 antigen via the T2SS. The SO7 protein was toxic to Salmonella when larger amounts were synthesized; thus, the synthesis of this protein was placed under the control of the lacI repressor gene, whose expression in turn was dependent on the amount of available arabinose in the medium. The pYA4184 vector was introduced into host strain chi9242 (Delta phoP233 Delta asdA16 Delta araBAD23 Delta relA198::araC P(BAD) lacI TT [TT is the T4ipIII transcription terminator]). In addition to SO7, for immunization and challenge studies we used the EAMZ250 antigen of Eimeria acervulina, which was previously shown to confer partial protection against E. acervulina challenge when it was delivered via the T3SS. Immunization of chickens with a combination of the SO7 and EAMZ250 antigens delivered via the T3SS induced superior protection against challenge by E. acervulina. In contrast, chickens immunized with SO7 that was delivered via the T2SS of Salmonella were better protected from challenge by E. tenella.

  2. Antigenic Profiles of Recombinant Proteins from Mycobacterium avium subsp paratuberculosis in Sheep with Johne's Disease

    Science.gov (United States)

    Methods to improve the ELISA test to detect Mycobacterium avium subsp paratuberculosis have been explored over several years. Previously, selected recombinant proteins of M. avium subspecies paratuberculosis were found to be immunogenic in cattle with Johne’s disease. In the present study, antibo...

  3. Evaluation of recombinant Neospora caninum antigens purified from silkworm larvae for the protection of N. caninum infection in mice.

    Science.gov (United States)

    Yoshimoto, Mai; Otsuki, Takahiro; Itagaki, Kohei; Kato, Tatsuya; Kohsaka, Tetsuya; Matsumoto, Yumino; Ike, Kazunori; Park, Enoch Y

    2015-12-01

    Three antigens (NcSAG1, NcSRS2 and NcMIC3) from Neospora caninum were expressed using the BmNPV bacmid system in silkworm larvae and purified from the hemolymph. From 20 silkworm larvae, 1.5, 1.2 and 1.4 mg of purified recombinant NcSAG1, NcSRS2 and NcMIC3 were obtained, respectively. When each purified recombinant antigen was immunized with Freund's incomplete adjuvant (FIA) to mice, recombinant NcSAG1 induced a Th2 immune response in immunized mice and produced a SAG1-specific antibody. In the experiment where NcSAG1-immunized mice were challenged with N. caninum, the cerebral N. caninum burden was significantly reduced compared with that of either the FIA- or PBS-immunized mice. Recombinant NcSRS2 or NcMIC3 induced both Th1 and Th2 immune responses, but NcMIC3-immunization did not induce significant production of NcMIC3-specific antibodies. These results suggest that the silkworm can produce recombinant antigens of N. caninum, which can be used as a recombinant vaccine against N. caninum.

  4. Immune responses and protective efficacy induced by 85B antigen and early secreted antigenic target-6 kDa antigen fusion protein secreted by recombinant bacille Calmette-Guérin.

    Science.gov (United States)

    Shi, Changhong; Wang, Xiaowu; Zhang, Hai; Xu, Zhikai; Li, Yuan; Yuan, Lintian

    2007-04-01

    In an attempt to improve immune responses and protective efficacy, we constructed two recombinant bacille Calmette-Guérin (rBCG) strains expressing an 85B antigen (Ag85B) and early secreted antigenic target-6 kDa antigen (ESAT6) of Mycobacterium tuberculosis (MTB) fusion protein. Both rBCG strains have the same protein insertion but in a different order (Ag85B-ESAT6 and ESAT6-Ag85B). The cultured supernatant of rBCG strains and the sera from the mice immunized with the fusion protein Ag85B-ESAT6 or ESAT6-Ag85B formed a band with a fraction size of 37 kDa, equalivalent to the sum of Ag85B and ESAT6. Six weeks after BALB/c mice were immunized with BCG or rBCG, spleen lymphocytes showed significant proliferation in response to culture filtrate protein of MTB. Compared with the BCG group, mice vaccinated with rBCG elicited a high level increase of immunoglobulin G antibodies to culture filtrate protein in the serum. The gamma-interferon levels in the lymphocyte culture medium supernatants increased remarkably in the rBCG1 group, significantly higher than that of the BCG immunized group (p0.05).

  5. Antigenicity and Hemaglutination Activity of a Recombinant Hemagglutinin-Neuraminidase of Paramyxovirus Tianjin Strain

    Institute of Scientific and Technical Information of China (English)

    Mei LI; Li-jun YUAN; Li-ying SHI; Xiao-mian LI; Qing WANG; Wen-xiu WANG

    2008-01-01

    Paramyxovirus Tianjin strain, a new genotype of Sendal virus, was isolated from the lungs of common cotton-eared marmoset that died of severe respiratory infection in the marmoset colonies. The 19.28% IgM positive rate in the young children with acute respiratory tract infection suggested a close relationship between Tianjin strain and humans. Hemagglutinin-neuraminidase (HN) is its major transmembrane glycoprotein responsible for viral attachment, penetration and release. To clear the relationship between HN structure and function of paramyxovirus Tianjin strain, rHN1, rHN2 and rHN3 overlapping the ectodomain of HN protein were expressed. Their antigenicity and hemaglutination activity, as well as cross reactivity to standard antisera against influenza virus type A, type B were analyzed. The results indicated expressed rHNs have the natural antigenicity.The segment rHN2 possesses more linear epitopes exposed on the surface of the native I-IN protein than found in segments rHN3 and rHN1. The hemagglutination activity of segment rHN3 is higher than that of segments rHN2 and rHN1, and partially dependent on the three-dimensional conformation of HN3 protein. Cross-reactivity between rHNs and standard antisera against influenza virus type A, type B suggested that rHNs might not be the best alternative as specific antigens to detect virus in clinicalserum specimens.

  6. New skin test for detection of bovine tuberculosis on the basis of antigen-displaying polyester inclusions produced by recombinant Escherichia coli.

    Science.gov (United States)

    Chen, Shuxiong; Parlane, Natalie A; Lee, Jason; Wedlock, D Neil; Buddle, Bryce M; Rehm, Bernd H A

    2014-04-01

    The tuberculin skin test for diagnosing tuberculosis (TB) in cattle lacks specificity if animals are sensitized to environmental mycobacteria, as some antigens in purified protein derivative (PPD) prepared from Mycobacterium bovis are present in nonpathogenic mycobacteria. Three immunodominant TB antigens, ESAT6, CFP10, and Rv3615c, are present in members of the pathogenic Mycobacterium tuberculosis complex but absent from the majority of environmental mycobacteria. These TB antigens have the potential to enhance skin test specificity. To increase their immunogenicity, these antigens were displayed on polyester beads by translationally fusing them to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen-displaying inclusions in recombinant Escherichia coli. The most common form of these inclusions is poly(3-hydroxybutyric acid) (PHB). The respective fusion proteins displayed on these PHB inclusions (beads) were identified using tryptic peptide fingerprinting analysis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The surface exposure and accessibility of antigens were assessed by enzyme-linked immunosorbent assay (ELISA). Polyester beads displaying all three TB antigens showed greater reactivity with TB antigen-specific antibody than did beads displaying only one TB antigen. This was neither due to cross-reactivity of antibodies with the other two antigens nor due to differences in protein expression levels between beads displaying single or three TB antigens. The triple-antigen-displaying polyester beads were used for skin testing of cattle and detected all cattle experimentally infected with M. bovis with no false-positive reactions observed in those sensitized to environmental mycobacteria. The results suggested applicability of TB antigen-displaying polyester inclusions as diagnostic reagents for distinguishing TB-infected from noninfected animals.

  7. Neutralizing antibodies respond to a bivalent dengue DNA vaccine or/and a recombinant bivalent antigen.

    Science.gov (United States)

    Zhang, Zhi-Shan; Weng, Yu-Wei; Huang, Hai-Long; Zhang, Jian-Ming; Yan, Yan-Sheng

    2015-02-01

    There is currently no effective vaccine to prevent dengue infection, despite the existence of multiple studies on potential methods of immunization. The aim of the present study was to explore the effect of DNA and/or recombinant protein on levels of neutralizing antibodies. For this purpose, envelope domain IIIs of dengue serotypes 1 and 2 (DEN-1/2)were spliced by a linker (Gly‑Gly‑Ser‑Gly‑Ser)3 and cloned into the prokaryotic expression plasmid pET30a (+) and eukaryotic vector pcDNA3.1 (+). The chimeric bivalent protein was expressed in Escherichia coli, and one‑step purification by high‑performance liquid chromatography was conducted. Protein expression levels of the DNA plasmid were tested in BHK‑21 cells by indirect immunofluorescent assay. In order to explore a more effective immunization strategy and to develop neutralizing antibodies against the two serotypes, mice were inoculated with recombinant bivalent protein, the DNA vaccine, or the two given simultaneously. Presence of the specific antibodies was tested by ELISA and the presence of the neutralizing antibodies was determined by plaque reduction neutralization test. Results of the analysis indicated that the use of a combination of DNA and protein induced significantly higher titers of neutralizing antibodies against either DEN‑1 or DEN‑2 (1:64.0 and 1:76.1, respectively) compared with the DNA (1:24.7 and 1:26.9, DEN‑1 and DEN‑2, respectively) or the recombinant protein (1:34.9 and 1:45.3 in DEN‑1 and DEN‑2, respectively). The present study demonstrated that the combination of recombinant protein and DNA as an immunization strategy may be an effective method for the development of a vaccine to prevent dengue virus infection.

  8. Epitope-based recombinant diagnostic antigen to distinguish natural infection from vaccination with hepatitis A virus vaccines.

    Science.gov (United States)

    Su, Qiudong; Guo, Minzhuo; Jia, Zhiyuan; Qiu, Feng; Lu, Xuexin; Gao, Yan; Meng, Qingling; Tian, Ruiguang; Bi, Shengli; Yi, Yao

    2016-07-01

    Hepatitis A virus (HAV) infection can stimulate the production of antibodies to structural and non-structural proteins of the virus. However, vaccination with an inactivated or attenuated HAV vaccine produces antibodies mainly against structural proteins, whereas no or very limited antibodies are produced against the non-structural proteins. Current diagnostic assays to determine exposure to HAV, such as the Abbott HAV AB test, detect antibodies only to the structural proteins and so are not able to distinguish a natural infection from vaccination with an inactivated or attenuated virus. Here, we constructed a recombinant tandem multi-epitope diagnostic antigen (designated 'H1') based on the immune-dominant epitopes of the non-structural proteins of HAV to distinguish the two situations. H1 protein expressed in Escherichia coli and purified by affinity and anion exchange chromatography was applied in a double-antigen sandwich ELISA for the detection of anti-non-structural HAV proteins, which was confirmed to distinguish a natural infection from vaccination with an inactivated or attenuated HAV vaccine.

  9. Latex-protein complexes from an acute phase recombinant antigen of Toxoplasma gondii for the diagnosis of recently acquired toxoplasmosis.

    Science.gov (United States)

    Peretti, Leandro E; Gonzalez, Verónica D G; Marcipar, Iván S; Gugliotta, Luis M

    2014-08-01

    The synthesis and characterization of latex-protein complexes (LPC), from the acute phase recombinant antigen P35 (P35Ag) of Toxoplasma gondii and "core-shell" carboxylated or polystyrene (PS) latexes (of different sizes and charge densities) are considered, with the aim of producing immunoagglutination reagents able to detect recently acquired toxoplasmosis. Physical adsorption (PA) and chemical coupling (CC) of P35Ag onto latex particles at different pH were investigated. Greater amounts of adsorbed protein were obtained on PS latexes than on carboxylated latexes, indicating that hydrophobic forces govern the interactions between the protein and the particle surface. In the CC experiments, the highest amount of bound protein was obtained at pH 6, near the isoelectric point of the protein (IP=6.27). At this pH, it decreased both the repulsion between particle surface and protein, and the repulsion between neighboring molecules. The LPC were characterized and the antigenicity of the P35Ag protein coupled on the particles surface was evaluated by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results from ELISA showed that the P35Ag coupled to the latex particles surface was not affected during the particles sensitization by PA and CC and the produced LPC were able to recognize specific anti-P35Ag antibodies present in the acute phase of the disease.

  10. CLINICAL EVALUATION OF FOUR RECOMBINANT TREPONEMA PALLIDUM ANTIGEN-BASED RAPID TESTS IN THE DIAGNOSIS OF SYPHILIS

    Institute of Scientific and Technical Information of China (English)

    Lin-na Wang; Lei Yang; He-yi Zheng

    2007-01-01

    To assess the sensitivity, specificity, and feasibility of 4 recombinant Treponema pallidum antigenbased rapid tests in the diagnosis of syphilis.Methods A total of 970 outpatients were selected from the Sexually Transmitted Diseases Centre of Peking Union Medical College Hospital. Venous blood was collected and serum was extracted. T. pallidum antibodies in whole blood, anticoagulant whole blood, and serum were detected using 4 recombinant T. pallidum antigen-based rapid tests.T. pallidum haemagglutination test (TPHA) was considered as the gold standard for the detection of T. pallidum specific antibodies in serum. The sensitivities and specificities of four methods were analyzed.Results The sensitivities and specificities of Abbott Determine Syphilis TP test, SD-BIOLINE Syphilis 3.0 test,VISITECT-SYPHILIS test, and Syphicheck-WB test for serum specimens were 100% and 98.9%, 95.7% and 98.0%, 94.6% and 98.2%, 68.1% and 98.9%; for whole blood were 74.1% and 99.5%, 87.9% and 99.4%,73.2% and 99.7%, 64.7% and 99.7%. The observed sensitivities of the 4 rapid diagnosis tests were not significantly different with TPHA ( P>0.05 ).Conclusions The 4 rapid tests show good performance and characteristics in the diagnosis of syphilis. Furthermore, they are more sensitive for serum specimens than whole blood.

  11. Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

    Directory of Open Access Journals (Sweden)

    Tam Yew

    2012-10-01

    Full Text Available Abstract Background Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg from Pichia pastoris expression cells were optimized using response surface methodology (RSM based on the central composite design (CCD. The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells. Results The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR and HBsAg concentration of 29.20 mg/L respectively. Conclusions The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing.

  12. Construction of Recombinant Baculoviruses Expressing Infectious Bursal Disease Virus Main Protective Antigen and Their Immune Effects on Chickens.

    Directory of Open Access Journals (Sweden)

    Jingping Ge

    Full Text Available In order to overcome the limitations of conventional vaccines for infectious bursal disease virus (IBDV, we constructed recombinant dual expression system baculoviruses with VP2 and VP2/4/3, the main protective antigens of IBDV. We compared the immune effects of the baculoviruses in avian cells and detected their control effects on chickens with infectious bursal disease. We used Western blot analysis to measure VP2 protein and VP2/4/3 polyprotein expression in avian cells infected using the Bac-to-Bac baculovirus expression system. The recombinant baculoviruses were used to vaccinate specific pathogen-free chickens, which produced specific protective antibodies and strong cellular immune responses. The results of the virus challenge experiment revealed that the protective efficiency of VP2 and VP2/4/3 virus vaccines were 95.8% and 100%, respectively, both of which were higher than the vaccine group (87.5%, and significantly higher than the control group (50%. The results demonstrated that the immune effect of BV-S-ITRs-VP2/4/3 was superior to that of BV-S-ITRs-VP2. Compared with traditional attenuated vaccine and genetically engineered live vector vaccine, the dual expression viral vector vaccine has good bio-safety. The results of this study provide a foundation for the further development of poultry vaccines, in addition to providing a useful reference for developing non-replicating live vaccines against other viral diseases.

  13. Virus-Like Particles of Chimeric Recombinant Porcine Circovirus Type 2 as Antigen Vehicle Carrying Foreign Epitopes

    Directory of Open Access Journals (Sweden)

    Huawei Zhang

    2014-12-01

    Full Text Available Virus-like particles (VLPs of chimeric porcine circovirus type 2 (PCV2 were generated by replacing the nuclear localization signal (NLS; at 1–39 aa of PCV2 capsid protein (Cap with classical swine fever virus (CSFV T-cell epitope (1446–1460 aa, CSFV B-cell epitope (693–716 aa and CSFV T-cell epitope conjugated with B-cell epitope. The recombinant proteins were expressed using the baculovirus expression system and detected by immunoblotting and indirect immunofluorescence assay. The abilities to form PCV2 VLPs were confirmed by transmission electron microscopy. Immunogenicities of the three recombinant proteins were evaluated in mice. Our Results indicated that Cap protein NLS deletion or substitution with CSFV epitopes did not affect the VLPs assembly. Three chimeric Cap proteins could form VLPs and induce efficient humoral and cellular immunity against PCV2 and CSFV in mice. Results show that PCV2 VLPs can be used as an efficient antigen carrier for delivery of foreign epitopes, and a potential novel vaccine.

  14. Usefulness of Toxoplasma gondii recombinant antigens (GRA1, GRA7 and SAG1) in an immunoglobulin G avidity test for the serodiagnosis of toxoplasmosis

    DEFF Research Database (Denmark)

    Pietkiewicz, H; Hiszczyńska-Sawicka, E; Kur, J;

    2007-01-01

    The precise diagnosis of an acute and recent Toxoplasma infection in pregnant women and the newborn child is important before treatment. This study describes a new Toxoplasma gondii IgG avidity test based on a combination of recombinant GRA1, GRA7 and SAG1 antigens and shows that this test is use...

  15. Protective antibodies against Taenia taeniaeformis in rats infected with eggs or injected with non-viable oncospheres or recombinant antigens of oncospheres.

    Science.gov (United States)

    Ito, A; Asano, K; Okamoto, K

    1994-09-01

    Antibody responses against Taenia taeniaeformis in rats infected with eggs or injected with non-viable oncospheres or recombinant antigens of oncospheres were analysed by passive transfer of serum and Western blotting. When recipient rats were injected with 1 ml serum from donors infected with eggs (infected serum), they all showed complete resistance to oral egg challenge, whereas those injected with 1 ml serum from donors injected with either oncospheres or recombinant antigens (vaccinated serum) showed no resistance. IgG and IgG subclass responses detected by Western blotting revealed that antibody responses to oncosphere antigens in infected serum thoroughly differed from those in vaccinated serum. It is suggested that IgG2 alpha responses in infected serum should be used for screening of epitopes for candidate vaccine.

  16. Multi-isotype antibody responses against the multimeric Salmonella Typhi recombinant hemolysin E antigen.

    Science.gov (United States)

    Ong, Eugene Boon Beng; Ignatius, Joshua; Anthony, Amy Amilda; Aziah, Ismail; Ismail, Asma; Lim, Theam Soon

    2015-01-01

    The detection and measurement of different antibody isotypes in the serum provide valuable indicators of the different stages of typhoid infection. Here, the ability of S. Typhi recombinant hemolysin E (HlyE) to detect multi-isotype antibody responses in sera of patients with typhoid and paratyphoid A was investigated using an indirect antibody immunoassay. Nanogram amounts of HlyE were found to be sufficient for detection of IgG and IgA isotypes and, in a study of individuals' sera (n = 100), the immunoassay was able to distinguish between typhoid and non-typhoid sera. The overall sensitivity, specificity and efficiency of the ELISA were 70% (39/56), 100% (44/44) and 83% respectively.

  17. Immune Responses and Protective Efficacy Induced by 85B Antigen and Early Secreted Antigenic Target-6 kDa Antigen Fusion Protein Secreted by Recombinant Bacille Calmette-Guérin

    Institute of Scientific and Technical Information of China (English)

    Changhong SHI; Xiaowu WANG; Hai ZHANG; Zhikai XU; Yuan LI; Lintian YUAN

    2007-01-01

    In an attempt to improve immune responses and protective efficacy, we constructed two recombinant bacille Calmette-Guérin (rBCG) strains expressing an 85B antigen (Ag85B) and early secreted antigenic target-6 kDa antigen (ESAT6) of Mycobacterium tuberculosis (MTB) fusion protein. Both rBCG strains have the same protein insertion but in a different order (Ag85B-ESAT6 and ESAT6-Ag85B). The cultured supernatant of rBCG strains and the sera from the mice immunized with the fusion protein Ag85B-ESAT6 or ESAT6-Ag85B formed a band with a fraction size of 37 kDa, equalivalent to the sum of Ag85B and ESAT6. Six weeks after BALB/c mice were immunized with BCG or rBCG, spleen lymphocytes showed significant proliferation in response to culture filtrate protein of MTB. Compared with the BCG group, mice vaccinated with rBCG elicited a high level increase of immunoglobulin G antibodies to culture filtrate protein in the serum. The γ-interferon levels in the lymphocyte culture medium supernatants increased remarkably in the rBCG1 group, significantly higher than that of the BCG immunized group (P<0.05). Four weeks after vaccination, mice were infected with M. tuberculosis H37Rv and a dramatic reduction in the numbers of MTB colony forming units in the spleens and lungs was observed in the two rBCG immunization groups.Although these rBCG strains were more immunogenic, their protective effect was comparable to the classical BCG strain, and there were no significant differences between two rBCG groups (P>0.05).

  18. An oral recombinant Salmonella enterica serovar Typhimurium mutant elicits systemic antigen-specific CD8+ T cell cytokine responses in mice

    Directory of Open Access Journals (Sweden)

    Chin'ombe Nyasha

    2009-04-01

    Full Text Available Abstract Background The induction of antigen-specific CD8+ T cell cytokine responses against an attenuated, oral recombinant Salmonella enterica serovar Typhimurium vaccine expressing a green fluorescent protein (GFP model antigen was investigated. A GFP expression plasmid was constructed in which the gfp gene was fused in-frame with the 5' domain of the Escherichia coli β-galactosidase α-gene fragment with expression under the lac promoter. Groups of mice were orally immunized three times with the bacteria and systemic CD8+ T cell cytokine responses were evaluated. Results High level of the GFP model antigen was expressed by the recombinant Salmonella vaccine vector. Systemic GFP-specific CD8+ T cell cytokine (IFN-γ and IL-4 immune responses were detected after mice were orally vaccinated with the bacteria. It was shown that 226 net IFN-γ and 132 net IL-4 GFP-specific SFUs/10e6 splenocytes were formed in an ELISPOT assay. The level of IFN-γ produced by GFP peptide-stimulated cells was 65.2-fold above background (p Conclusion These results suggested that a high expressing recombinant Salmonella vaccine given orally to mice would elicit antigen-specific CD8+ T cell responses in the spleen. Salmonella bacteria may, therefore, be used as potential mucosal vaccine vectors.

  19. Use of dried blood spots to define antibody response to the Strongyloides stercoralis recombinant antigen NIE.

    Science.gov (United States)

    Mounsey, Kate; Kearns, Therese; Rampton, Melanie; Llewellyn, Stacey; King, Mallory; Holt, Deborah; Currie, Bart J; Andrews, Ross; Nutman, Thomas; McCarthy, James

    2014-10-01

    An approach to improve the diagnosis of Strongyloides stercoralis infection is the use of serologic assays utilising the NIE antigen from S. stercoralis, with good diagnostic sensitivity and excellent specificity reported. Detection of antibody eluted from dried blood spots (DBS) has shown utility in large-scale seroepidemiological studies for a range of conditions and is appealing for use with children where sample collection is difficult. We adapted an existing NIE-enzyme linked immunosorbent assay (ELISA) for the testing of strongyloides antibody response on DBS, and evaluated it in a population screening and mass drug administration programme (MDA) for strongyloidiasis conducted in an Australian indigenous community. Study participants were treated with 200 μg/kg ivermectin (>15 kg) or 3× 400 mg albendazole (stercoralis NIE-DBS prior to MDA treatment, and 6 of 50 participants (12.0%) tested positive after treatment. These prevalence values are similar to those documented by standard serology in the same community. For 30 participants where a DBS was collected at both MDA 1 and 2, a significant decline in ELISA values was evident post treatment (0.12-0.02, p=0.0012). These results are in agreement with previous studies documenting the high seroprevalence of S. stercoralis in remote Australian Indigenous communities, and suggest that collection of dried blood spots may be a useful approach for field diagnosis of S. stercoralis seroprevalence.

  20. Comparative evaluation of recombinant LigB protein and heat-killed antigen-based latex agglutination test with microscopic agglutination test for diagnosis of bovine leptospirosis.

    Science.gov (United States)

    Nagalingam, Mohandoss; Thirumalesh, Sushma Rahim Assadi; Kalleshamurthy, Triveni; Niharika, Nakkala; Balamurugan, Vinayagamurthy; Shome, Rajeswari; Sengupta, Pinaki Prasad; Shome, Bibek Ranjan; Prabhudas, Krishnamsetty; Rahman, Habibur

    2015-10-01

    This study aimed to develop latex agglutination test (LAT) using recombinant leptospiral immunoglobulin-like protein (LigB) (rLigB) antigen and compare its diagnostic efficacy with LAT using conventional heat-killed leptospiral antigen and microscopic agglutination test (MAT) in diagnosing bovine leptospirosis. The PCR-amplified 1053-bp ligB gene sequences from Leptospira borgpetersenii Hardjo serovar were cloned in pET 32 (a) vector at EcoRI and NotI sites and expressed in BL21 E. coli cells as fusion protein with thioredoxin (-57 kDa) and characterized by SDS-PAGE and immunoblot. Out of 390 serum samples [cattle (n = 214), buffaloes (n = 176)] subjected to MAT, 115 samples showed reciprocal titre≥100 up to 1600 against one or more serovars. For recombinant LigB protein/antigen-based LAT, agglutination was observed in the positive sample, while no agglutination was observed in the negative sample. Similarly, heat-killed leptospiral antigen was prepared from and used in LAT for comparison with MAT. A two-sided contingency table was used for analysis of LAT using both the antigens separately against MAT for 390 serum samples. The sensitivity, specificity and positive and negative predictive values of recombinant LigB LAT were found to be 75.65, 91.27, 78.38 and 89.96 %, respectively, and that of heat-killed antigen-based LAT were 72.17, 89.82, 74.77 and 88.53 %, respectively, in comparison with MAT. This developed test will be an alternative/complementary to the existing battery of diagnostic assays/tests for specific detection of pathogenic Leptospira infection in bovine population.

  1. Comparison of purified 12 kDa and recombinant 15 kDa Fasciola hepatica antigens related to a Schistosoma mansoni fatty acid binding protein

    Directory of Open Access Journals (Sweden)

    George V. Hillyer

    1995-04-01

    Full Text Available Vaccines in schistosomiasis using homologous antigens have been studied extensively in experimentally infected mammalian hosts. Vaccines using heterologous antigens have received comparatively less attention. This review summarizes recent work on a heterologous 12 kDa Fasciola hepatica antigenic polypeptide which cross reacts with Schistosoma mansoni. A cDNA has been cloned and sequenced, and the predicted amino acid sequence of the recombinant protein has been shown to have significant (44 identity with a 14 kDa S. mansoni fatty acid binding protein. Thus in the parasitic trematodes fatty acid binding proteins may be potential vaccine candidates. The F. hepatica recombinant protein has been overexpressed and purified and denoted rFh15. Preliminary rFh15 migrates more slowly (i.e. may be slightly larger than nFh12 on SDS-PAGE and has a predicted pI of 6.01 vs. observed pI of 5.45. Mice infected with F. hepatica develop antibodies to nFh12 by 2 weeks of infection vs. 6 weeks of infection to rFh15; on the other hand, mice with schistosomiasis mansoni develop antibodies to both nFh12 and rFh15 by 6 weeks of infection. Both the F. hepatica and S. mansoni cross-reactive antigens may be cross-protective antigens with the protection inducing capability against both species.

  2. Study on the Preparation of Recombinant Human HSP70 and Its Presenting-Antigen Function

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Guimei(

    2001-01-01

    [1]Yasuaki T Norioki T Noriyuki S et al.70 kDa heat shock cognate protein is a transformation-associated antigen and a possible target for the host's anti-tumor immunity.J Immunol 1993 151:5516[2]Yasuaki T Ping P Kang L et al.Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations.Science 1997 278:117[3]Heiichiro V Pramod K S.Heat shock protein 70-associated peptides elicit specific cancer immunity.J Exp Med 1998 178:1391[4]Sambrook J Fritch E F Maniatis T.Molecular Cloning a laboratory manu 2nd ed.New York:Cold Spring Harbor Laboratory Press 1989.253[5]Satish J Peter M Stanley R et al.Human stress protein hsp70:overexpression in E.coli purification and characterization.Bio/Technology 1995 113:1105[6]哈密斯B.D. 利克伍德D.著.刘毓秀 程桂芳译.蛋白质的凝胶电泳实践方法.北京:科学出版社 1994.151[7]Anne-marie T C Max P Carey L O et al.Immunization with a lymphocytic choriomeningitis virus peptide mixed with heat shock protein 70 results in protective antiviral immunity and specific cytotoxic T lymphocytes.J Exp Med 1998 187:685[8]Nandan D Daubenberger C Mpimbaza G.A rapid single-step purification method for immunogenic members of the Hsp70 family:validation and application.J Immunol Methods 1994 176(2) :255

  3. Immunization of Bos taurus steers with Babesia bovis recombinant antigens MSA-1, MSA-2c and 12D3.

    Science.gov (United States)

    Antonio Alvarez, J; Lopez, U; Rojas, C; Borgonio, V M; Sanchez, V; Castañeda, R; Vargas, P; Figueroa, J V

    2010-04-01

    The purpose of this research was to evaluate the recombinant proteins MSA-1, MSA-2c and 12D3 as a combined immunogen for cattle. Fifteen steers were randomly assigned into three groups of five animals each (I, II and III). On day 0, cattle in group I were injected with 50 microg each of rMSA-1, rMSA-2c and r12D3 with the adjuvant Montanide 75; cattle in Group II received adjuvant-PBS, and Group III were untreated controls. On day 14, cattle in Group I received a second injection of the three recombinant proteins in adjuvant and cattle in Group II again received adjuvant alone. On day 28, all groups of cattle were challenged with a field strain of Babesia bovis. After challenge, the experimental cattle were clinically and serologically monitored. Three of the five steers immunized with the combined recombinant B. bovis proteins seroconverted on day 14 post-immunization (P.I.) and the maximum titre was 1 : 1600. All five immunized steers presented strong seropositivity to B. bovis antigens at day 21 P.I. The prepatent periods of vaccinated cattle were delayed until day 10 post-challenge exposure versus 8 and 7 days in Groups II and III, respectively. Cattle in all groups had fever above 41 degrees C; the reduction in packed cell volume was not significantly different (P > 0.05) in vaccinated group I compared with Groups II and III (29% versus 26% and 31%, respectively). Treatment was required for one steer in the control group. During the period of the study, the weight of cattle in Groups I and II increased an average of 9 and 7 kg, whereas the weight of the control cattle was reduced on average 4 kg. Immunization with rMSA-1-rMSA-2c-r12D3 proteins was not sufficient to prevent clinical symptoms against challenge, but the immunologic response was sufficient to protect steers against a mild virulent strain of B. bovis.

  4. [Development of a new hydrophobic interaction chromatography absorbent and its application to the purification of recombinant hepatitis B surface antigen].

    Science.gov (United States)

    Wang, Yang-Mu; Bi, Jing-Xiu; Zhao, Lan; Zhou, Wei-Bin; Li, Yan; Huang, Yong-Dong; Zhang, Yan; Lin, Hai; Su, Zhi-Guo

    2006-03-01

    A new hydrophobic absorbent based on homemade highly cross-linked agarose beads was synthesized by immobilizing butyl derivative onto the matrix linkage. The density of ligand was controlled by adjusting the concentration of butanethiol and the synthesis route was optimized by evaluating the purification efficiency of recombinant Hepatitis B surface antigen (HBsAg) expressed by Chinese hamster ovary (CHO) cell line. A high performance absorbent was finally screened out with up to 80% of HBsAg recovery and purification-fold (PF) about 20. Furthermore, the column pressure was about 0.06 MPa under the flow rate of 500cm/h, and no leaked butyl were detected after exposing the gel in common buffers, chaotropic agents, high concentrations of denaturing agents such as guanidine hydrochloride, urea and polar organic solvents. These results demonstrated that the absorbent have high physico-chemical stability, so it was available for the downstream process. Finally, after scaled up to 2L wet gel/batch, the absorbent was applied to the integration of three-step chromatography and obtained the purified CHO-HBsAg with 95% purity by SDS-PAGE and HPLC, which meet the requirements of SFDA. The purification efficiency and the reproducible ability of the absorbents were also evaluated from batch-to-batch. The results demonstrated that the absorbent met the requirement of scalable, reproducible, economic effect as well. This absorbent is a promising alternative exported HIC gel for wildly being used in Chinese pharmaceutical industries.

  5. Fasciola hepatica - the pilot study of in vitro assessing immune response against native and recombinant antigens of the fluke.

    Science.gov (United States)

    Bąska, Piotr; Zawistowska-Deniziak, Anna; Zdziarska, Anna M; Wasyl, Katarzyna; Wiśniewski, Marcin; Cywińska, Anna; Klockiewicz, Maciej; Januszkiewicz, Kamil; Wędrychowicz, Halina

    2013-12-01

    Fasciola hepatica is a liver fluke that infects 2.4 million of people and causes great economical loss in animal production. To date a 100% effective vaccine has not been developed and the disease is controlled by drug therapy. Great efforts are put into development of effective vaccine against parasite what is difficult since Fasciola spp. (like other helmints) during evolutionary process has developed sophisticated and efficient methods to evade immune response. During preliminary experiments it is convenient to use cell lines which are relatively cheap and allow for reproducible comparison of results between laboratories. We stimulated BOMA (bovine monocyte/macrophage cell line) and BOMAC (bovine macrophage cell line) with native or recombinant antigens of Fasciola hepatica and assessed IFN-γ, IL-4 and TNF-α level upon stimulation. We observed diminished secretion of proinflammatory TNF-α in LPS activated BOMA cells stimulated with Excretory/Secretory products of adult fluke (Fh-ES). We also observed greater changes in gene expression in LPS activated BOMA cells than in non activated BOMA cells upon stimulation using Fh-ES. The results show possibility of using cell lines for in vitro research of bovine immune response against liver fluke, although this model still requires validation and further characterization.

  6. Recombinant 35-kDa inclusion membrane protein IncA as a candidate antigen for serodiagnosis of Chlamydophila pecorum.

    Science.gov (United States)

    Mohamad, Khalil Yousef; Rekiki, Abdessalem; Berri, Mustapha; Rodolakis, Annie

    2010-07-14

    Chlamydophila pecorum strains are commonly found in the intestine and vaginal mucus of asymptomatic ruminants and may therefore induce a positive serological response when the animals are tested for C. abortus. They have also been associated with different pathological diseases in ruminants, swine and koala. The aim of this study was to identify specific C. pecorum immunodominant antigens which could be used in ELISA tests allowing to distinguish between animals infected with C. pecorum and those infected with other chlamydial species. A gene encoding 35-kDa inclusion membrane protein incA of C. pecorum was isolated by immunoscreening of the C. pecorum DNA library using ovine anti-C. pecorum antibodies. The recombinant IncA protein did not react with a murine serum directed against C. abortus but did react with a specific monoclonal antibody of C. pecorum and toward several ovine serum samples obtained after experimental infection with different C. pecorum strains. This protein could be a good candidate for specific diagnosis of C. pecorum infection.

  7. Advax-adjuvanted recombinant protective antigen provides protection against inhalational anthrax that is further enhanced by addition of murabutide adjuvant.

    Science.gov (United States)

    Feinen, Brandon; Petrovsky, Nikolai; Verma, Anita; Merkel, Tod J

    2014-04-01

    Subunit vaccines against anthrax based on recombinant protective antigen (PA) potentially offer more consistent and less reactogenic anthrax vaccines but require adjuvants to achieve optimal immunogenicity. This study sought to determine in a murine model of pulmonary anthrax infection whether the polysaccharide adjuvant Advax or the innate immune adjuvant murabutide alone or together could enhance PA immunogenicity by comparison to an alum adjuvant. A single immunization with PA plus Advax adjuvant afforded significantly greater protection against aerosolized Bacillus anthracis Sterne strain 7702 than three immunizations with PA alone. Murabutide had a weaker adjuvant effect than Advax when used alone, but when murabutide was formulated together with Advax, an additive effect on immunogenicity and protection was observed, with complete protection after just two doses. The combined adjuvant formulation stimulated a robust, long-lasting B-cell memory response that protected mice against an aerosol challenge 18 months postimmunization with acceleration of the kinetics of the anamnestic IgG response to B. anthracis as reflected by ∼4-fold-higher anti-PA IgG titers by day 2 postchallenge versus mice that received PA with Alhydrogel. In addition, the combination of Advax plus murabutide induced approximately 3-fold-less inflammation than Alhydrogel as measured by in vivo imaging of cathepsin cleavage resulting from injection of ProSense 750. Thus, the combination of Advax and murabutide provided enhanced protection against inhalational anthrax with reduced localized inflammation, making this a promising next-generation anthrax vaccine adjuvanting strategy.

  8. Comparison of a recombinant-antigen enzyme immunoassay with Treponema pallidum hemagglutination test for serological confirmation of syphilis.

    Science.gov (United States)

    Rodríguez, Islay; Alvarez, Elvio L; Fernández, Carmen; Miranda, Alina

    2002-04-01

    A recombinant-antigen enzyme immunoassay (EIA), BioSCREEN anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38), secondary (n = 10), early latent (n = 28) and congenital syphilis (n = 2), patients with leptospirosis ( n= 8), infectious mononucleosis (n = 7), hepatitis (n = 9), diabetes mellitus (n = 11), rheumatoid arthritis (n = 13), leprosy (n = 11), tuberculosis (n = 9), HIV/Aids ( n= 12), systemic lupus erythematosus (n = 4), rheumatic fever (n = 3), old-persons (n = 9), pregnant women (n = 29) and blood donors (n = 164). The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.

  9. Comparison of a Recombinant-antigen Enzyme Immunoassay with Treponema pallidum Hemagglutination Test for Serological Confirmation of Syphilis

    Directory of Open Access Journals (Sweden)

    Islay Rodríguez

    2002-04-01

    Full Text Available A recombinant-antigen enzyme immunoassay (EIA, BioSCREEN TM anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38, secondary (n = 10, early latent (n = 28 and congenital syphilis (n = 2, patients with leptospirosis ( n= 8, infectious mononucleosis (n = 7, hepatitis (n = 9, diabetes mellitus (n = 11, rheumatoid arthritis (n = 13, leprosy (n = 11, tuberculosis (n = 9, HIV/Aids ( n= 12, systemic lupus erythematosus (n = 4, rheumatic fever (n = 3, old-persons (n = 9, pregnant women (n = 29 and blood donors (n = 164. The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.

  10. Establishment of an in vivo potency assay for the recombinant hepatit is B surface antigen in monovalent and combined vaccines

    Directory of Open Access Journals (Sweden)

    Mabel Izquierdo-López

    2014-12-01

    Full Text Available In this paper the development of potency assay in animals (mice was made, with the objective of demonstrating the immunogenic power of the recombinant Hepatitis B surface antigen in monovalent and combined vaccines, produced at the Center of Genetic Engineering and Biotechnology. The potency test is a parameter in quality control and it is also a tool to demonstrate the consistency of the production process. Parameters such as duration of the test, number of animals in the test, as well as different areas for the maintenance of the animals were evaluated. The results on the applicability of the potency test, to two presentations of the vaccines; monovalent Heberbiovac HB and pentavalent liquid in one vial Heberpenta-L are shown, for which specificity studies, evaluating different vaccine lots, the behavior of linearity, and parallelism, as well as establishing quality specification of the test were performed. This assay led to the obtainment of reliable results for the vaccines evaluated, the consistent evaluation of the immunogenic power and the monitoring of different production processes.

  11. Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells.

    Directory of Open Access Journals (Sweden)

    Dale O Starkie

    Full Text Available Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive. These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking

  12. Biophysical and formulation studies of the Schistosoma mansoni TSP-2 extracellular domain recombinant protein, a lead vaccine candidate antigen for intestinal schistosomiasis.

    Science.gov (United States)

    Cheng, Weiqiang; Curti, Elena; Rezende, Wanderson C; Kwityn, Clifford; Zhan, Bin; Gillespie, Portia; Plieskatt, Jordan; Joshi, Sangeeta B; Volkin, David B; Hotez, Peter J; Middaugh, C Russell; Bottazzi, Maria Elena

    2013-11-01

    A candidate vaccine to prevent human schistosomiasis is under development. The vaccine is comprised of a recombinant 9 kDa antigen protein corresponding to the large extracellular domain of a tetraspanin surface antigen protein of Schistosoma mansoni, Sm-TSP-2. Here, we describe the biophysical profile of the purified, recombinant Sm-TSP-2 produced in the yeast PichiaPink, which in preclinical studies in mice was shown to be an effective vaccine against intestinal schistosomiasis. Biophysical techniques including circular dichroism, intrinsic and extrinsic fluorescence and light scattering were employed to generate an empirical phase diagram, a color based map of the physical stability of the vaccine antigen over a wide range of temperatures and pH. From these studies a pH range of 6.0-8.0 was determined to be optimal for maintaining the stability and conformation of the protein at temperatures up to 25 °C. Sorbitol, sucrose and trehalose were selected as excipients that prevented physical degradation during storage. The studies described here provide guidance for maximizing the stability of soluble recombinant Sm-TSP-2 in preparation of its further development as a vaccine.

  13. Immune responses elicited by Mycoplasma hyopneumoniae recombinant antigens and DNA constructs with potential for use in vaccination against porcine enzootic pneumonia.

    Science.gov (United States)

    Virginio, Veridiana Gomes; Gonchoroski, Taylor; Paes, Jéssica Andrade; Schuck, Desirée Cigaran; Zaha, Arnaldo; Ferreira, Henrique Bunselmeyer

    2014-10-07

    Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (PEP) and causes major economic losses to the pig industry worldwide. Commercially available vaccines provide only partial protection and are relatively expensive. In this study, we assessed the humoral and cellular immune responses to three recombinant antigens of M. hyopneumoniae. Immune responses to selected domains of the P46, HSP70 and MnuA antigens (P46102-253, HSP70212-601 and MnuA182-378), delivered as recombinant subunit or DNA vaccines, were evaluated in BALB/c mice. All purified recombinant antigens and two DNA vaccines, pcDNA3.1(+)/HSP70212-601 and pcDNA3.1(+)/MnuA182-378, elicited a strong humoral immune response, indicated by high IgG levels in the serum. The cellular immune response was assessed by detection of IFN-γ, IL-10 and IL-4 in splenocyte culture supernatants. The recombinant subunit and DNA vaccines induced Th1-polarized immune responses, as evidenced by increased levels of IFN-γ. All recombinant subunit vaccines and the pcDNA3.1(+)/MnuA182-378 vaccine also induced the secretion of IL-10, a Th2-type cytokine, in large quantities. The mixed Th1/Th2-type response may elicit an effective immune response against M. hyopneumoniae, suggesting that P46102-253, HSP70212-601 and MnuA182-378 are potential novel and promising targets for the development of vaccines against PEP.

  14. The ue of polysiloxane/polyvinyl alcohol beads as solid phase in IgG anti-Toxocara canis detection using a recombinant antigen

    Directory of Open Access Journals (Sweden)

    Raquel de Andrade Lima Coêlho

    2003-04-01

    Full Text Available Immunodetection of human IgG anti-Toxocara canis was developed based on ELISA and on the use of polysiloxane/polyvinyl alcohol (POS/PVA beads. A recombinant antigen was covalently immobilized, via glutaraldehyde, onto this hybrid inorganic-organic composite, which was prepared by the sol-gel technique. Using only 31.2 ng antigen per bead, a peroxidase conjugate dilution of 1:10,000 and a serum dilution of 1:200 were adequate for the establishment of the procedure. This procedure is comparable to that which utilizes the adsorption of the antigen to conventional PVC plates. However, the difference between positive and negative sera mean absorbances was larger for this new glass based assay. In addition to the performance of the POS/PVA bead as a matrix for immunodetection, its easy synthesis and low cost are additional advantages for commercial application.

  15. Expression of recombinant West Nile virus prM protein fused to an affinity tag for use as a diagnostic antigen.

    Science.gov (United States)

    Setoh, Y X; Hobson-Peters, J; Prow, N A; Young, P R; Hall, R A

    2011-07-01

    Previous studies have concluded that the Flavivirus prM protein is a suitable viral antigen to distinguish serologically between infections with closely related Flaviviruses (Cardosa et al., 2002). To express the recombinant West Nile virus (WNV) prM antigen fused to a suitable affinity tag for purification, a series of prM-His-tag and prM-V5-tag fusion proteins were generated. Analysis of the prM-His-tag fusion proteins revealed that either prM epitopes were disrupted or the His-tag was not presented properly depending on the location of the His tag and the presence of the prM transmembrane domains in these constructs. This identified domains critical for proper folding of prM, and arrangements that allowed the correct presentation of the His-tag. However, the inclusion of the V5 epitope tag fused to the C terminus of prM allowed formation of the authentic antigenic structure of prM and the proper presentation of the V5 epitope. Capture of tagged recombinant WNV(NY99) prM antigen to the solid phase with anti-V5 antibody in ELISA enabled the detection of prM-specific antibodies in WNV(NY99)-immune horse serum, confirming its potential as a useful diagnostic reagent.

  16. 重组抗原在弓形虫病诊断中的应用%Use of recombinant antigens to diagnose toxoplasmosis

    Institute of Scientific and Technical Information of China (English)

    黄泽智; 赵晋英; 李艳伟; 侯玉英

    2011-01-01

    Toxoplasma gondii infection can cause severe harm. Thus, highly sensitive and highly specific detection of T. Gondii infection is crucial to the treatment of toxoplasmosis. Toxoplasmosis is generally diagnosed by demonstrating specific immunoglobulin M (IgM) and IgG antibodies to Toxoplasma antigens in a patient's serum sample. Most of the commercially available tests use T. Gondii native antigens and vary widely in accuracy. Recombinant antigens have been developed as diagnostic reagents for assays to detect toxoplasmosis. Thus, this review discusses recent use of recombinant antigens to diagnose toxoplasmosis.%刚地弓形虫感染危害严重,研究敏感性高和特异性好的弓形虫感染检测方法对于弓形虫病的诊断至关重要.弓形虫病的诊断一般依赖于检测患者血清中弓形虫特异性IgM和IgG.目前,商业试剂盒大多采用天然抗原,其检测精确性较差.重组抗原已经被发展用来诊断弓形虫病,且具有潜在的应用价值.本综述主要介绍重组抗原在弓形虫病诊断中的应用.

  17. Development of a single-antigen magnetic bead assay (SAMBA) for the sensitive detection of HPA-1a alloantibodies using tag-engineered recombinant soluble β3 integrin.

    Science.gov (United States)

    Skaik, Younis; Battermann, Anja; Hiller, Oliver; Meyer, Oliver; Figueiredo, Constanca; Salama, Abdulgabar; Blasczyk, Rainer

    2013-05-31

    Timely and accurate testing for human platelet antigen 1a (HPA-1a) alloantibodies is vital for clinical diagnosis of neonatal alloimmune thrombocytopenia (NAIT). Current antigen-specific assays used for the detection of HPA-1 alloantibodies are technically very complex and cumbersome for most diagnostic laboratories. Hence, we designed and applied recombinant soluble (rs) β3 integrins displaying HPA-1a or HPA-1b epitopes for the development of a single-antigen magnetic bead assay (SAMBA). Soluble HPA-1a and HPA-1b were produced recombinantly in human embryonic kidney 293 (HEK293) cells and differentially tagged. The recombinant soluble proteins were then immobilized onto paramagnetic beads and used for analysis of HPA-1 alloantibodies by enzyme-linked immunosorbent assay (ELISA). HPA-1a serum samples (n=7) from NAIT patients, inert sera and sera containing non-HPA-1a antibodies were used to evaluate the sensitivity and specificity of the SAMBA. Fusion of V5-His or GS-SBP-His tags to the rsβ3 integrins resulted in high-yield expression. SAMBA was able to detect all HPA-1a and -1b alloantibodies recognized by monoclonal antibody-specific immobilization of platelet antigens assay (MAIPA). No cross-reactions between the sera were observed. Two out of seven of the HPA-1a alloantibody-containing sera demonstrated weak to moderate reactivity in MAIPA but strong signals in SAMBA. SAMBA provides a very reliable method for the detection of HPA-1 antibodies with high specificity and sensitivity. This simple and rapid assay can be adapted for use in any routine laboratory and can be potentially adapted for use on automated systems.

  18. Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

    OpenAIRE

    Anbouhi, Mahdi Habibi; Baraz, Aida Feiz; Bouzari, Saeid; Abolhassani,Mohsen; Khanahmad, Hossein; Golkar, Majid; Aghasadeghi, Mohammad Reza; Behdani, Mahdi; Najafabadi, Ali Jahanian; Shokrgozar, Mohammad Ali

    2012-01-01

    Background: Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length ...

  19. Enhanced and durable protective immune responses induced by a cocktail of recombinant BCG strains expressing antigens of multistage of Mycobacterium tuberculosis.

    Science.gov (United States)

    Liang, Jinping; Teng, Xindong; Yuan, Xuefeng; Zhang, Ying; Shi, Chunwei; Yue, Tingting; Zhou, Lei; Li, Jianrong; Fan, Xionglin

    2015-08-01

    Although Bacillus Calmette-Guérin (BCG) vaccine confers protection from Mycobacterium tuberculosis infection in children, its immune protection gradually wanes over time, and consequently leads to an inability to prevent the reactivation of latent infection of M. tuberculosis. Therefore, improving BCG for better control of tuberculosis (TB) is urgently needed. We thus hypothesized that recombinant BCG overexpressing immunodominant antigens expressed at different growth stages of M. tuberculosis could provide a more comprehensive protection against primary and latent M. tuberculosis infection. Here, a novel cocktail of recombinant BCG (rBCG) strains, namely ABX, was produced by combining rBCG::85A, rBCG::85B, and rBCG::X, which overexpressed respective multistage antigens Ag85A, Ag85B, and HspX of M. tuberculosis. Our results showed that ABX was able to induce a stronger immune protection than individual rBCGs or BCG against primary TB infection in C57BL/6 mice. Mechanistically, the immune protection was attributed to stronger antigen-specific CD4(+) Th1 responses, higher numbers of IFN-γ(+) CD4(+) TEM and IL-2(+) CD8(+) TCM cells elicited by ABX. These findings thus provide a novel strategy for the improvement of BCG efficacy and potentially a promising prophylactic TB vaccine candidate, warranting further investigation.

  20. Evaluation of recombinant LigB antigen-based indirect ELISA and latex agglutination test for the serodiagnosis of bovine leptospirosis in India.

    Science.gov (United States)

    Deneke, Yosef; Sabarinath, T; Gogia, Neha; Lalsiamthara, Jonathan; Viswas, K N; Chaudhuri, Pallab

    2014-08-01

    Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of the genus Leptospira, causing febrile infection characterized by multi-organ failure in humans and animals. Leptospiral Ig-like protein B (LigB) is a surface-expressed antigen that mediates host cell invasion or attachment. In this study, N-terminal conserved region of LigB protein (46 kDa) was evaluated for its diagnostic potential to detect anti-leptospiral antibodies in the sera of various animal species. Dot blot analysis revealed immunoreactivity of Leptospira-positive sera of cattle, buffalo, dog, sheep and goat to purified LigB protein. We have analyzed 1126 bovine serum samples, collected from Northern and Eastern part of India, by microscopic agglutination test (MAT) and recombinant LigB (rLigB) based ELISA and latex agglutination test (LAT). The sensitivity of rLigB based ELISA for 554 MAT positive sera was 96.9% and the specificity with 572 MAT negative sera was 91.08% whereas LAT showed sensitivity and specificity of 93.68% and 92.31%, respectively. Kappa values of 0.879 and 0.860 for recombinant antigen based ELISA and LAT indicate excellent agreement with the gold standard serological test, MAT, for the detection of anti-leptospiral antibodies in sera. Further, LAT based on rLigB antigen is a simple and rapid test, suitable for serodiagnosis of leptospirosis under field conditions, owing to its portability and longer shelf life.

  1. Expression, purification, immunogenicity, and protective efficacy of a recombinant Tc24 antigen as a vaccine against Trypanosoma cruzi infection in mice.

    Science.gov (United States)

    Martinez-Campos, Viridiana; Martinez-Vega, Pedro; Ramirez-Sierra, Maria Jesus; Rosado-Vallado, Miguel; Seid, Christopher A; Hudspeth, Elissa M; Wei, Junfei; Liu, Zhuyun; Kwityn, Cliff; Hammond, Molly; Ortega-López, Jaime; Zhan, Bin; Hotez, Peter J; Bottazzi, Maria Elena; Dumonteil, Eric

    2015-08-26

    The Tc24 calcium binding protein from the flagellar pocket of Trypanosoma cruzi is under evaluation as a candidate vaccine antigen against Chagas disease. Previously, a DNA vaccine encoding Tc24 was shown to be an effective vaccine (both as a preventive and therapeutic intervention) in mice and dogs, as evidenced by reductions in T. cruzi parasitemia and cardiac amastigotes, as well as reduced cardiac inflammation and increased host survival. Here we developed a suitable platform for the large scale production of recombinant Tc24 (rTc24) and show that when rTc24 is combined with a monophosphoryl-lipid A (MPLA) adjuvant, the formulated vaccine induces a Th1-biased immune response in mice, comprised of elevated IgG2a antibody levels and interferon-gamma levels from splenocytes, compared to controls. These immune responses also resulted in statistically significant decreased T. cruzi parasitemia and cardiac amastigotes, as well as increased survival following T. cruzi challenge infections, compared to controls. Partial protective efficacy was shown regardless of whether the antigen was expressed in Escherichia coli or in yeast (Pichia pastoris). While mouse vaccinations will require further modifications in order to optimize protective efficacy, such studies provide a basis for further evaluations of vaccines comprised of rTc24, together with alternative adjuvants and additional recombinant antigens.

  2. Development and evaluation of two truncated recombinant NP antigen-based indirect ELISAs for detection of bovine parainfluenza virus type 3 antibodies in cattle.

    Science.gov (United States)

    Yang, Yong; Wang, Feng-Xue; Sun, Na; Cao, Li; Zhang, Shu-Qin; Zhu, Hong-Wei; Guo, Li; Cheng, Shi-Peng; Wen, Yong-Jun

    2015-09-15

    Bovine parainfluenza virus type 3 (BPIV3) is one of the most important viral respiratory pathogens in both young and adult cattle. Nucleocapsid protein (NP) is the most abundant viral protein and the main regulator of virus replication and transcription. In this study, amino acid sequence data of BPIV3 NP was used to identify potential linear epitopic regions, which were subsequently used to design truncated recombinant NP antigens. The amino-terminal region (aa 9-157, NP-N) and the carboxy-terminal region (aa 391-500, NP-C) were selected, and these two truncated recombinant BPIV3 NP proteins were expressed in Escherichia coli based on the results of prediction studies. Furthermore, Enzyme-Linked Immunosorbent Assays (ELISAs) were established using the truncated recombinant BPIV3-N proteins as antigens, and 154 clinical samples were used to evaluate the newly established ELISA systems in comparison with a virus neutralisation test (VNT) as a reference. The results showed that a high coincidence rate was observed for the data that were obtained by the two methods. The sensitivity of NP-N ELISA and NP-C ELISA were 98.4% and 94.6%, respectively, and the specificity of both ELISAs was 100% with reference to the VNTs. Our data indicated that both ends of NP have high immunogenicity during BPIV3 infection and that they were good targets for serodiagnosis. The ELISAs based on the two truncated proteins were especially suitable for use in large-scale epidemiological investigations.

  3. The promastigote surface antigen gene family of the Leishmania parasite: differential evolution by positive selection and recombination

    Directory of Open Access Journals (Sweden)

    Bañuls Anne-Laure

    2008-10-01

    Full Text Available Abstract Background PSA (promastigote surface antigen is one of the major classes of membrane proteins present at the surface of the parasitic protozoan Leishmania. While it harbours leucine rich repeats, which are suggestive of its involvement in parasite-to-host physical interactions, its exact role is largely unknown. Furthermore, the extent of diversity of this gene family, both in copy number and sequence has not been established. Results From the newly available complete genome sequences of L. major, L. infantum and L. braziliensis, we have established the complete list of PSA genes, based on the conservation of specific domain architecture. The latter includes an array of leucine rich repeats of unique signature flanked by conserved cysteine-rich domains. All PSA genes code either for secreted or membrane-anchored surface proteins. Besides the few previously identified PSA genes, which are shown here to be part of a relatively large subclass of PSA genes located on chromosome 12, this study identifies seven other PSA subtypes. The latter, whose genes lie on chromosomes 5, 9, 21 and 31 in all three species, form single gene (two genes in one instance subfamilies, which phylogenetically cluster as highly related orthologs. On the other hand, genes found on chromosome 12 generally show high diversification, as reflected in greater sequence divergence between species, and in an extended set of divergent paralogs. Moreover, we show that the latter genes are submitted to strong positive selection. We also provide evidence that evolution of these genes is driven by intra- and intergenic recombination, thereby modulating the number of LRRs in protein and generating chimeric genes. Conclusion PSA is a Leishmania family of membrane-bound or secreted proteins, whose main signature consists in a specific LRR sequence. All PSA genes found in the genomes of three sequenced Leishmania species unambiguously distribute into eight subfamilies of orthologs

  4. Low frequency of anti-SLA/LP autoantibody in Japanese adult patients with autoimmune liver diseases: analysis with recombinant antigen assay.

    Science.gov (United States)

    Miyakawa, Hiroshi; Kawashima, Yumi; Kitazawa, Eriko; Kawaguchi, Naomi; Kato, Takashi; Kikuchi, Kentaro; Imai, Erika; Fujikawa, Hirotoshi; Hashimoto, Etsuko; Schlumberger, Wolfgang

    2003-08-01

    Anti-soluble liver antigen/liver pancreas (SLA/LP) autoantibody has been proposed to be one of the autoantibodies characterizing autoimmune hepatitis (AIH). Recently, one of the autoantigens to anti-SLA/LP was identified as a UGA suppressor tRNA-associated protein. Although the function of this protein remains unknown, the recombinant protein has been prokaryotically expressed. Using this protein as an antigen, a recombinant immunoassay for anti-SLA/LP autoantibody has been established and the frequency and significance of this autoantibody have been discussed in European countries. So, in the present study, we investigated anti-SLA/LP autoantibodies in Japanese patients with autoimmune liver diseases using the recombinant antigen ELISA and Western blot assay. Seventy-five patients with AIH type 1, 5 with AIH type 2, 46 with primary biliary cirrhosis, 10 with primary sclerosing cholangitis, 47 with chronic hepatitis C, 48 with systemic lupus erythematosus, 3 with cryptogenic hepatitis, and 40 normal controls were the subjects of the present study. Anti-SLA/LP autoantibodies were detected in only 5 of 75 (6.7%) patients with AIH type 1, but in none of the other 159 patients or 40 normal controls. The clinicopathologic features of anti-SLA/LP-positive AIH type 1, including carriers of HLA DR locus variations, were not significantly different from anti-SLA/LP-negative patients except for the mortality rate. Anti-SLA/LP autoantibody was detected at a low frequency in Japanese patients with AIH type 1 and did not significantly influence clinical features. However, since it has high disease-specificity to AIH type 1, further analysis of SLA/LP may contribute to help clarify the pathogenesis of AIH type 1.

  5. Expression, purification, and improved antigenic specificity of a truncated recombinant bp26 protein of Brucella melitensis M5-90: a potential antigen for differential serodiagnosis of brucellosis in sheep and goats.

    Science.gov (United States)

    Liu, Wen-xing; Hu, Sen; Qiao, Zu-jian; Chen, Wei-ye; Liu, Lin-tao; Wang, Fang-kun; Hua, Rong-hong; Bu, Zhi-gao; Li, Xiang-rui

    2011-01-01

    Antibodies produced in animals vaccinated using live attenuated vaccines against Brucella spp. are indistinguishable using current conventional serological tests from those produced in infected animals. One potential approach is to develop marker vaccines in which specific genes have been deleted from parental vaccine strains that show good immunogenicity and vaccine efficacy. Corresponding methods of detection for antibodies raised by the marker vaccine should also be developed. A specific fragment of the bp26 gene of Brucella melitensis M5-90 was cloned into vector pQE32 to construct the recombinant plasmid (pQE32-rΔbp26). It was used to transform Escherichia coli M15 (pREP4) host cells, which expressed the rΔbp26 protein. Subsequently, the recombinant protein was purified by immobilized metal affinity chromatography and size-exclusion chromatography. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified rΔbp26 protein was represented by only one band, with a molecular weight of 14 kDa, and it showed good antigenic specificity on western blot and enzyme-linked immunosorbent assay (ELISA). The purified rΔbp26 protein was intended to be used as an antigen to develop a novel ELISA to differentiate animals vaccinated with bp26 mutants of Brucella spp. from those infected naturally and those vaccinated with the parental vaccine strains.

  6. APPLICATION OF COMBINATIED RECOMBINANT ANTIGENS IN TOXOPLASMOSIS%联合重组抗原在弓形虫病中的应用

    Institute of Scientific and Technical Information of China (English)

    赵前峰; 国天义; 傅克勤

    2013-01-01

    Toxoplasma gondii,an important opportunistic intracellular parasitic protozoa,is world-widely spread and getting damage to pregnant women or immunodeficient patient. With the continuous development of molecular biology techniques, obtaining high purity and specificity of genetic engineering recombinant antigens is the new direction for treating the T. gondii infection,toxoplasmosis diagnosis and vaccine-preventable. To improve diagnostic accuracy and effectiveness of prevention,a"cocktail" joint combined by Toxoplasma gondii recombinant antigens has a broad prospect.%刚地弓形虫( Toxoplasma gondii)是一种重要的机会致病性专性有核细胞内寄生原虫,在世界范围内广泛分布,对孕妇或免疫缺陷病人损伤很大。随着分子生物学技术的不断发展,获得纯度高、特异性强的基因工程重组抗原是用于弓形虫感染、弓形虫病诊断和疫苗防治的新方向。而由多个弓形虫重组抗原组合成“鸡尾酒冶式的联合重组抗原来提高诊断的准确性以及防治的有效性更有着广阔的前景。

  7. Recombinant Measles AIK-C Vaccine Strain Expressing the prM-E Antigen of Japanese Encephalitis Virus.

    Science.gov (United States)

    Higuchi, Akira; Toriniwa, Hiroko; Komiya, Tomoyoshi; Nakayama, Tetsuo

    2016-01-01

    An inactivated Japanese encephalitis virus (JEV) vaccine, which induces neutralizing antibodies, has been used for many years in Japan. In the present study, the JEV prM-E protein gene was cloned, inserted at the P/M junction of measles AIK-C cDNA, and an infectious virus was recovered. The JEV E protein was expressed in B95a cells infected with the recombinant virus. Cotton rats were inoculated with recombinant virus. Measles PA antibodies were detected three weeks after immunization. Neutralizing antibodies against JEV developed one week after inoculation, and EIA antibodies were detected three weeks after immunization. The measles AIK-C-based recombinant virus simultaneously induced measles and JEV immune responses, and may be a candidate for infant vaccines. Therefore, the present strategy of recombinant viruses based on a measles vaccine vector would be applicable to the platform for vaccine development.

  8. Serological Evaluation of EgAgB16 kDa, a Recombinant Antigen from Echinococcus Granulosus for Diagnosis of Human Hydatidosis

    Directory of Open Access Journals (Sweden)

    L Maghen

    2010-09-01

    Full Text Available Background: Regarding that accurate diagnosis of human hydatidosis still needs more investigations, the present study was conducted to clone, express, and evaluate the gene encoding AgB subunits (EgAgB16 kDa from Echinococcus granulosus (Iranian G1 strain and its evaluation by ELISA test.Methods: DNA was extracted from protoscoleces and was utilized by PCR for strain identification. Total RNA was prepared with RNeasy protect mini kit from E. granulosus (Iranian G1 strain protoscoleces collected from naturally infected sheep with hydatid cyst. Recombinant AgB16 kDa was produced using pETDuet as vector and evaluated by ELISA method. A panel of sera including hydatid cyst-infected individu­als (n=72, healthy individual (n=48, toxoplasmosis (n=4, strongyloidosis (n=4, kala-azar (n=5 and tuberculosis (n=5 were examined using this recombinant antigen.Results: Recombinant protein was purified by affinity chromatography using His-Tag column. After purifica­tion, recombinant protein was confirmed by western blot analysis using His Tag monoclonal anti­body or hydatid positive human serum. The sensitivity, specificity; positive and negative predictive values were calculated as 93.5%, 95.6%, 96% and 92.9%, in that order. The cut-off point was detected 0.3 for rAgB16. Conclusion: While the produced recombinant AgB16 kDa showed promising results in diagnosing human hy­datidosis, but more investigations should be implemented to reach an accurate gold standard.

  9. Bacterial fermentation of recombinant major wasp allergen Antigen 5 using oxygen limiting growth conditions improves yield and quality of inclusion bodies.

    Science.gov (United States)

    Kischnick, Stefanie; Weber, Bernhard; Verdino, Petra; Keller, Walter; Sanders, Ernst A; Anspach, F Birger; Fiebig, Helmut; Cromwell, Oliver; Suck, Roland

    2006-06-01

    A process for bacterial expression and purification of the recombinant major wasp allergen Antigen 5 (Ves v 5) was developed to produce protein for diagnostic and therapeutic applications for type 1 allergic diseases. Special attention was focused on medium selection, fermentation conditions, and efficient refolding procedures. A soy based medium was used for fermentation to avoid peptone from animal origin. Animal-derived peptone required the use of isopropyl-beta-D-thiogalactopyranoside (IPTG) for the induction of expression. In the case of soy peptone, a constitutive expression was observed, suggesting the presence of a component that mimics IPTG. Batch cultivation at reduced stirrer speed caused a reduced biomass due to oxygen limitation. However, subsequent purification and processing of inclusion bodies yielded significantly higher amount of product. Furthermore, the protein composition of the inclusion bodies differed. Inclusion bodies were denatured and subjected to diafiltration. Detailed monitoring of diafiltration enabled the determination of the transition point. Final purification was conducted using cation-exchange and size-exclusion chromatography. Purified recombinant Ves v 5 was analyzed by RP-HPLC, CD-spectroscopy, SDS-PAGE, and quantification ELISA. Up to 15 mg highly purified Ves v 5 per litre bioreactor volume were obtained, with endotoxin concentrations less than 20 EU mg(-1) protein and high comparability to the natural counterpart. Analytical results confirm the suitability of the recombinant protein for diagnostic and clinical applications. The results clearly demonstrate that not only biomass, but especially growth conditions play a key role in the production of recombinant Ves v 5. This has an influence on inclusion body formation, which in turn influences the renaturation rate and absolute product yield. This might also be true for other recombinant proteins that accumulate as inclusion bodies in Escherichia coli.

  10. LDL uptake by Leishmania amazonensis: involvement of membrane lipid microdomains.

    Science.gov (United States)

    De Cicco, Nuccia N T; Pereira, Miria G; Corrêa, José R; Andrade-Neto, Valter V; Saraiva, Felipe B; Chagas-Lima, Alessandra C; Gondim, Katia C; Torres-Santos, Eduardo C; Folly, Evelize; Saraiva, Elvira M; Cunha-E-Silva, Narcisa L; Soares, Maurilio J; Atella, Georgia C

    2012-04-01

    Leishmania amazonensis lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this lipid from the host environment. In this study we show that the L. amazonensis takes up and metabolizes human LDL(1) particles in both a time and dose-dependent manner. This mechanism implies the presence of a true LDL receptor because the uptake is blocked by both low temperature and by the excess of non-labelled LDL. This receptor is probably associated with specific microdomains in the membrane of the parasite, such as rafts, because this process is blocked by methyl-β-cyclodextrin (MCBD). Cholesteryl ester fluorescently-labeled LDL (BODIPY-cholesteryl-LDL) was used to follow the intracellular distribution of this lipid. After uptake it was localized in large compartments along the parasite body. The accumulation of LDL was analyzed by flow cytometry using FITC-labeled LDL particles. Together these data show for the first time that L. amazonensis is able to compensate for its lack of lipid synthesis through the use of a lipid importing machinery largely based on the uptake of LDL particles from the host. Understanding the details of the molecular events involved in this mechanism may lead to the identification of novel targets to block Leishmania infection in human hosts.

  11. Freeze-thaw stress of Alhydrogel ® alone is sufficient to reduce the immunogenicity of a recombinant hepatitis B vaccine containing native antigen.

    Science.gov (United States)

    Clapp, Tanya; Munks, Michael W; Trivedi, Ruchit; Kompella, Uday B; Braun, LaToya Jones

    2014-06-24

    Preventing losses in vaccine potency due to accidental freezing has recently become a topic of interest for improving vaccines. All vaccines with aluminum-containing adjuvants are susceptible to such potency losses. Recent studies have described excipients that protect the antigen from freeze-induced inactivation, prevent adjuvant agglomeration and retain potency. Although these strategies have demonstrated success, they do not provide a mechanistic understanding of freeze-thaw (FT) induced potency losses. In the current study, we investigated how adjuvant frozen in the absence of antigen affects vaccine immunogenicity and whether preventing damage to the freeze-sensitive recombinant hepatitis B surface antigen (rHBsAg) was sufficient for maintaining vaccine potency. The final vaccine formulation or Alhydrogel(®) alone was subjected to three FT-cycles. The vaccines were characterized for antigen adsorption, rHBsAg tertiary structure, particle size and charge, adjuvant elemental content and in-vivo potency. Particle agglomeration of either vaccine particles or adjuvant was observed following FT-stress. In vivo studies demonstrated no statistical differences in IgG responses between vaccines with FT-stressed adjuvant and no adjuvant. Adsorption of rHBsAg was achieved; regardless of adjuvant treatment, suggesting that the similar responses were not due to soluble antigen in the frozen adjuvant-containing formulations. All vaccines with adjuvant, including the non-frozen controls, yielded similar, blue-shifted fluorescence emission spectra. Immune response differences could not be traced to differences in the tertiary structure of the antigen in the formulations. Zeta potential measurements and elemental content analyses suggest that FT-stress resulted in a significant chemical alteration of the adjuvant surface. This data provides evidence that protecting a freeze-labile antigen from subzero exposure is insufficient to maintain vaccine potency. Future studies should

  12. Alphavirus replicon DNA expressing HIV antigens is an excellent prime for boosting with recombinant modified vaccinia Ankara (MVA or with HIV gp140 protein antigen.

    Directory of Open Access Journals (Sweden)

    Maria L Knudsen

    Full Text Available Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 μg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 μg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 μg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose.

  13. Alphavirus replicon DNA expressing HIV antigens is an excellent prime for boosting with recombinant modified vaccinia Ankara (MVA) or with HIV gp140 protein antigen.

    Science.gov (United States)

    Knudsen, Maria L; Ljungberg, Karl; Tatoud, Roger; Weber, Jonathan; Esteban, Mariano; Liljeström, Peter

    2015-01-01

    Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP) vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA) and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF) adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 μg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 μg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 μg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose.

  14. A recombinant adenovirus expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis elicits strong antigen-specific immune responses in mice.

    Science.gov (United States)

    Li, Wu; Deng, Guangcun; Li, Min; Zeng, Jin; Zhao, Liping; Liu, Xiaoming; Wang, Yujiong

    2014-11-01

    Tuberculosis (TB) is caused by an infection of Mycobacterium tuberculosis (Mtb) and remains an enormous and increasing health burden worldwide. To date, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only licensed anti-TB vaccine worldwide, which provides an important but limited protection from the Mtb infection. The development of alternative anti-TB vaccines is therefore urgently needed. Here we report, the generation of Ad5-CEAB, a recombinant adenovirus expressing Mtb antigens of CFP10, ESAT6, Ag85A and Ag85B proteins in a form of mixture. In order to evaluate the immunogenicity of Ad5-CEAB, mice were immunized with Ad5-CEAB by intranasal instillation three times with 2-week intervals. The results demonstrated that Ad5-CEAB elicited a strong antigen-specific immune response, particularly of the Th1 immune responses that were characterized by an increased ratio of IgG2a/IgG1 and secretions of Th1 type cytokines, IFN-γ, TNF-α, IL-2 and IL-12. In addition, the Ad5-CEAB also showed an ability to enhance humoral responses with a dramatically augmented antigen-specific serum IgG. Furthermore, an elevated sIgA were also found in the bronchoalveolar lavage fluid of the immunized mice, suggesting the elicitation of mucosal immune responses. These data indicate that Ad5-CEAB can induce a broad range of antigen-specific immune responses in vivo, which provides a promising and novel route for developing anti-TB vaccines and warrants further investigation.

  15. The Immunomodulatory Role of Adjuvants in Vaccines Formulated with the Recombinant Antigens Ov-103 and Ov-RAL-2 against Onchocerca volvulus in Mice.

    Directory of Open Access Journals (Sweden)

    Jessica A Hess

    2016-07-01

    Full Text Available In some regions in Africa, elimination of onchocerciasis may be possible with mass drug administration, although there is concern based on several factors that onchocerciasis cannot be eliminated solely through this approach. A vaccine against Onchocerca volvulus would provide a critical tool for the ultimate elimination of this infection. Previous studies have demonstrated that immunization of mice with Ov-103 and Ov-RAL-2, when formulated with alum, induced protective immunity. It was hypothesized that the levels of protective immunity induced with the two recombinant antigens formulated with alum would be improved by formulation with other adjuvants known to enhance different types of antigen-specific immune responses.Immunizing mice with Ov-103 and Ov-RAL-2 in conjunction with alum, Advax 2 and MF59 induced significant levels of larval killing and host protection. The immune response was biased towards Th2 with all three of the adjuvants, with IgG1 the dominant antibody. Improved larval killing and host protection was observed in mice immunized with co-administered Ov-103 and Ov-RAL-2 in conjunction with each of the three adjuvants as compared to single immunizations. Antigen-specific antibody titers were significantly increased in mice immunized concurrently with the two antigens. Based on chemokine levels, it appears that neutrophils and eosinophils participate in the protective immune response induced by Ov-103, and macrophages and neutrophils participate in immunity induced by Ov-RAL-2.The mechanism of protective immunity induced by Ov-103 and Ov-RAL-2, with the adjuvants alum, Advax 2 and MF59, appears to be multifactorial with roles for cytokines, chemokines, antibody and specific effector cells. The vaccines developed in this study have the potential of reducing the morbidity associated with onchocerciasis in humans.

  16. Immunization with recombinant DNA and modified vaccinia virus Ankara (MVA) vectors delivering PSCA and STEAP1 antigens inhibits prostate cancer progression.

    Science.gov (United States)

    Krupa, Magdalena; Canamero, Marta; Gomez, Carmen E; Najera, Jose L; Gil, Jesus; Esteban, Mariano

    2011-02-04

    Despite recent advances in early detection and improvement of conventional therapies, there is an urgent need for development of additional approaches for prevention and/or treatment of prostate cancer, and the use of immunotherapeutic modalities, such as cancer vaccines, is one of the most promising strategies. In this study, we evaluated the prophylactic efficacy of an active immunization protocol against prostate cancer associated antigens mPSCA and mSTEAP1 in experimental prostate cancer. Two antigen delivery platforms, recombinant DNA and MVA vectors, both encoding either mPSCA or mSTEAP1 were used in diversified DNA prime/MVA boost vaccination protocol. Antitumour activity was evaluated in TRAMP-C1 subcutaneous syngeneic tumour model and TRAMP mice. DNA prime/MVA boost immunization against either mPSCA or mSTEAP1, delayed tumour growth in TRAMP-C1 cells-challenged mice. Furthermore, simultaneous vaccination with both antigens produced a stronger anti-tumour effect against TRAMP-C1 tumours than vaccination with either mPSCA or mSTEAP1 alone. Most importantly, concurrent DNA prime/MVA boost vaccination regimen with those antigens significantly decreased primary tumour burden in TRAMP mice without producing any apparent adverse effects. Histopathological analysis of prostate tumours from vaccinated and control TRAMP mice revealed also that mPSCA/mSTEAP1 based-vaccination was effective at reducing the severity of prostatic lesions and incidence of high-grade poorly differentiated prostate cancer. Suppression of the disease progression in TRAMP mice was correlated with decreased proliferation index and increased infiltration of T-cells in prostate tissue. Active immunization against PSCA and STEAP1 using DNA prime/MVA boost strategy is a promising approach for prevention and/or treatment of prostate cancer.

  17. Use of Novel Recombinant Antigens in the Interferon Gamma Assay for Detection of Mycobacterium Avium Subsp. Paratuberculosis Infection in Cattle

    DEFF Research Database (Denmark)

    Mikkelsen, Heidi; Aagaard, Claus; Nielsen, Søren Saxmose;

    Early stage Mycobacterium avium subsp. paratuberculosis (MAP) infection can be detected by measuring antigen specific cell mediated immune responses by the interferon gamma (IFN-γ) assay. Available IFN-γ assay use purified protein derivate of Johnin (PPDj) leading to low specificity. The objectives...

  18. Use of novel recombinant antigens in the interferon gamma assay for detection of Mycobacterium avium subsp. paratuberculosis infection in cattle

    DEFF Research Database (Denmark)

    Mikkelsen, Heidi; Aagaard, C.; Nielsen, Søren Saxmose;

    2012-01-01

    Early stage Mycobacterium avium subsp. paratuberculosis (MAP) infection may be detected by measuring antigen specific cell-mediated immune responses by the interferon-gamma (IFN-¿) assay. Available IFN-¿ assay use purified protein derivate of Johnin (PPDj) leading to low specificity. The objectives...

  19. The Expression of Sperm Membrane Peptide-Hepatitis B Surface Antigen Fusion Protein with Recombinant Vaccinia Virus

    Institute of Scientific and Technical Information of China (English)

    杨晓鸣; 赵峰; 严缘昌; 李光地; 汪垣

    1998-01-01

    A synthetic oligonucleotide, HSD-2a, encoding a peptide segment of the extracellular domain of a human sperm membrane protein, YWK-Ⅱ, was fused with hepatitis B surface antigen gene (HBs gene). The fused gene was then cloned to pUC18 plasmid.

  20. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S;

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...

  1. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were us...

  2. Induction of partial protection against infection with Toxoplasma gondii genotype II by DNA vaccination with recombinant chimeric tachyzoite antigens

    DEFF Research Database (Denmark)

    Rosenberg, Carina Agerbo; De Craeye, S.; Jongert, E.

    2009-01-01

    complications. Although several strategies have been suggested for making a vaccine, none is currently available. Here, we investigate the protection conferred by DNA vaccination with two constructs, pcEC2 (MIC2-MIC3-SAG1) and pcEC3 (GRA3-GRA7-M2AP), encoding chimeric proteins containing multiple antigenic...

  3. Efficacy of vaccination with recombinant vaccinia and fowlpox vectors expressing NY-ESO-1 antigen in ovarian cancer and melanoma patients.

    Science.gov (United States)

    Odunsi, Kunle; Matsuzaki, Junko; Karbach, Julia; Neumann, Antje; Mhawech-Fauceglia, Paulette; Miller, Austin; Beck, Amy; Morrison, Carl D; Ritter, Gerd; Godoy, Heidi; Lele, Shashikant; duPont, Nefertiti; Edwards, Robert; Shrikant, Protul; Old, Lloyd J; Gnjatic, Sacha; Jäger, Elke

    2012-04-10

    Recombinant poxviruses (vaccinia and fowlpox) expressing tumor-associated antigens are currently being evaluated in clinical trials as cancer vaccines to induce tumor-specific immune responses that will improve clinical outcome. To test whether a diversified prime and boost regimen targeting NY-ESO-1 will result in clinical benefit, we conducted two parallel phase II clinical trials of recombinant vaccinia-NY-ESO-1 (rV-NY-ESO-1), followed by booster vaccinations with recombinant fowlpox-NY-ESO-1 (rF-NY-ESO-1) in 25 melanoma and 22 epithelial ovarian cancer (EOC) patients with advanced disease who were at high risk for recurrence/progression. Integrated NY-ESO-1-specific antibody and CD4(+) and CD8(+) T cells were induced in a high proportion of melanoma and EOC patients. In melanoma patients, objective response rate [complete and partial response (CR+PR)] was 14%, mixed response was 5%, and disease stabilization was 52%, amounting to a clinical benefit rate (CBR) of 72% in melanoma patients. The median PFS in the melanoma patients was 9 mo (range, 0-84 mo) and the median OS was 48 mo (range, 3-106 mo). In EOC patients, the median PFS was 21 mo (95% CI, 16-29 mo), and median OS was 48 mo (CI, not estimable). CD8(+) T cells derived from vaccinated patients were shown to lyse NY-ESO-1-expressing tumor targets. These data provide preliminary evidence of clinically meaningful benefit for diversified prime and boost recombinant pox-viral-based vaccines in melanoma and ovarian cancer and support further evaluation of this approach in these patient populations.

  4. Immunogenicity and protective efficacy of recombinant Modified Vaccinia virus Ankara candidate vaccines delivering West Nile virus envelope antigens

    NARCIS (Netherlands)

    Volz, Asisa; Lim, Stephanie; Kaserer, Martina; Pijlman, Gorben P.

    2016-01-01

    West Nile virus (WNV) cycles between insects and wild birds, and is transmitted via mosquito vectors to horses and humans, potentially causing severe neuroinvasive disease. Modified Vaccinia virus Ankara (MVA) is an advanced viral vector for developing new recombinant vaccines against infectious

  5. Brucella abortus Omp19 recombinant protein subcutaneously co-delivered with an antigen enhances antigen-specific T helper 1 memory responses and induces protection against parasite challenge.

    Science.gov (United States)

    Coria, Lorena M; Ibañez, Andrés E; Pasquevich, Karina A; Cobiello, Paula L González; Frank, Fernanda M; Giambartolomei, Guillermo H; Cassataro, Juliana

    2016-01-20

    The discovery of effective adjuvants for many vaccines especially those with limited commercial appeal, such as vaccines to poverty-related diseases, is required. In this work, we demonstrated that subcutaneous co-administration of mice with the outer membrane protein U-Omp19 from Brucella spp. plus OVA as antigen (Ag) increases Ag-specific T cell proliferation and T helper (Th) 1 immune responses in vitro and in vivo. U-Omp19 treated dendritic cells promote IFN-γ production by specific CD4(+) T cells and increases T cell proliferation. U-Omp19 co-administration induces the production of Ag specific effector memory T cell populations (CD4(+) CD44(high) CD62L(low) T cells). Finally, subcutaneous co-administration of U-Omp19 with Trypanosoma cruzi Ags confers protection against virulent parasite challenge, reducing parasitemia and weight loss while increasing mice survival. These results indicate that the bacterial protein U-Omp19 when delivered subcutaneously could be a suitable component of vaccine formulations against infectious diseases requiring Th1 immune responses.

  6. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...... for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific...... Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively....

  7. Protection Conferred by recombinant Yersinia pestis Antigens Produced by a Rapid and Highly Scalable Plant Expression System

    Science.gov (United States)

    2006-01-24

    signal; CT, synthetic chloroplast targeting sequence; PhiC31, integrase; 6% HIS, histidine tag; MP, movement protein. Fig. 2. Coomassie-stained gels...The level of F1 targeted to chloroplasts was lower, but still detectable on a Coomassie-stained gel (Fig. 2A). We con- Fig. 1. Constructs used in the...apoplastic targeting (lanes 5), chloroplast -targeted antigens (lanes 6), cytosolic-targeted translational fusions with GFP (lanes 7), cytosolic- targeted

  8. Evaluation of CD4+CD25+ T lymphocyte response time kinetics in patients with chronic Chagas disease after in vitro stimulation with recombinant Trypanosoma cruzi antigens

    Directory of Open Access Journals (Sweden)

    Suellen Carvalho de Moura Braz

    2013-05-01

    Full Text Available Introduction CD4+CD25+ T lymphocytes have been implicated in the regulation of host inflammatory response against Trypanosoma cruzi, and may be involved in the clinical course of the disease. Methods Peripheral blood mononuclear cells from patients with chronic Chagas disease were cultured in the presence of T. cruzi recombinant antigens and assayed for lymphocytes at distinct time points. Results It was possible to differentiate clinical forms of chronic Chagas disease at days 3 and 5 according to presence of CD4+CD25+ T cells in cell cultures. Conclusions Longer periods of cell culture proved to be potentially valuable for prospective evaluations of CD4+CD25+ T lymphocytes in patients with chronic Chagas disease.

  9. Expression and immunogenicity of recombinant Mycobacterium bovis Bacillus Calmette-Guérin strains secreting the antigen ESAT-6 from Mycobacterium tuberculosis in mice

    Institute of Scientific and Technical Information of China (English)

    WANG Li-mei; SHI Chang-hong; FAN Xiong-lin; XUE Ying; BAI Yin-lai; XU Zhi-kai

    2007-01-01

    Background Tuberculosis remains the leading cause of human death. Currently, Bacillus Calmette-Guérin (BCG) is the only available vaccine against tuberculosis but its efficacy is highly variable. Thus, developing new tuberculosis vaccines becomes an urgent task. In this study, we evaluated in BALB/c mice the humoral and cellular immune responses of recombinant BCG expressing the antigen ESAT-6 from Mycobacterium tuberculosis.Methods Escherichia coli-BCG shuttle plasmid named pDE22-esat-6 was constructed by inserting the BamHI/EcoRI digested esat-6 gene PCR product into the similarly digested parental plasmid pDE22. BCG cells were transformed with pDE22-esat-6, which was named recombinant BCG (rBCG). BALB/c mice were immunized subcutaneously on the back with 100 μl normal saline containing 106 CFU of BCG or rBCG. They were sacrificed after 4 weeks to detect their humoral and cellular responses.Results There was no any significant differences in the growth characteristics between the conventional BCG and rBCG. In immunized mice, the IgG antibody titres of rBCG group were as high as 1:8000, which was significantly higher than that in BCG group (1:1400, P<0.05). The elicited IFN-γ level of rBCG group was (1993±106) pg/ml, which was also significantly higher than that in BCG group ((1463±105) pg/ml, P<0.05). The splenocyte proliferation index of rBCG group reached 4.34±0.31, which was higher than that of BCG group (3.79±0.24, P<0.05).Conclusion rBCG secreted expressing antigen ESAT-6 stimulated stronger humoral and cellular immune responses than BCG did, and, therefore may be the better vaccine against mycobacterium tuberculosis.

  10. Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26.

    Science.gov (United States)

    Anderson, John P; Rascoe, Lisa N; Levert, Keith; Chastain, Holly M; Reed, Matthew S; Rivera, Hilda N; McAuliffe, Isabel; Zhan, Bin; Wiegand, Ryan E; Hotez, Peter J; Wilkins, Patricia P; Pohl, Jan; Handali, Sukwan

    2015-01-01

    The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag.

  11. Immunization against Lamb Haemonchosis with a Recombinant Somatic Antigen of Haemonchus contortus (rHcp26/23)

    Science.gov (United States)

    García-Coiradas, Leticia; Angulo-Cubillán, Francisco; Valladares, Basilio; Martínez, Enrique; de la Fuente, Concepción; Alunda, José María; Cuquerella, Montserrat

    2010-01-01

    Haemonchosis, caused by the abomasal nematode Haemonchus contortus, is a common parasitic disease of sheep. Our previous results showed that a soluble fraction from adult stages of the nematode (p26/23) induced partial protection against challenge. Recombinant DNA technology was applied to obtain a synthetic protein (rHcp26/23). Immunological assays (ELISA, Western blotting, and immunolocalization), using sera from lambs immunized with p26/23, confirmed the identity of the recombinant protein and demonstrated that the synthetic protein is equivalent to the purified protein employed in the previous immunoprophylaxis studies. Vaccination of lambs with 300 μg of rHcp26/23 and Freund's adjuvant elicited a notable specific antibody response. Immunization did not induce any significant protection after challenge with 16000 infective larvae of H. contortus, and comparable values for parasite faecal egg output, packed cell volume, and abomasal parasite burdens were found in vaccinated and control animals. PMID:20631899

  12. Immunization against Lamb Haemonchosis with a Recombinant Somatic Antigen of Haemonchus contortus (rHcp26/23

    Directory of Open Access Journals (Sweden)

    Leticia García-Coiradas

    2010-01-01

    Full Text Available Haemonchosis, caused by the abomasal nematode Haemonchus contortus, is a common parasitic disease of sheep. Our previous results showed that a soluble fraction from adult stages of the nematode (p26/23 induced partial protection against challenge. Recombinant DNA technology was applied to obtain a synthetic protein (rHcp26/23. Immunological assays (ELISA, Western blotting, and immunolocalization, using sera from lambs immunized with p26/23, confirmed the identity of the recombinant protein and demonstrated that the synthetic protein is equivalent to the purified protein employed in the previous immunoprophylaxis studies. Vaccination of lambs with 300 μg of rHcp26/23 and Freund's adjuvant elicited a notable specific antibody response. Immunization did not induce any significant protection after challenge with 16000 infective larvae of H. contortus, and comparable values for parasite faecal egg output, packed cell volume, and abomasal parasite burdens were found in vaccinated and control animals.

  13. Immuno-epidemiology of human Schistosoma haematobium infection: preferential IgG3 antibody responsiveness to a recombinant antigen dependent on age and parasite burden

    Directory of Open Access Journals (Sweden)

    Fernandez Cecilia

    2006-06-01

    Full Text Available Abstract Background Schistosomiasis is a major parasitic disease affecting over 200 million people in the developing world with a further 400 million people at risk of infection. The aim of this study was to identify a single antigen from adult Schistosoma haematobium worms and subsequently use this antigen to study the development of schistosome-acquired immunity in a human population. Methods The full-length cDNA sequence of a S. haematobium protein, a putative orthologue of the S. mansoni tegumental antigen Sm13, was obtained from a cDNA library of adult S. haematobium worms and named Sh13 following a small-scale expressed sequence tags (EST project. The recombinant Sh13 protein expressed in E. coli, was used to investigate immuno-epidemiological patterns in 147 Zimbabweans (7–18 years old exposed to S. haematobium. Results Sequence analysis of the full-length cDNA sequence of the S. haematobium protein Sh13, indicated that the protein has an N-terminal signal peptide and encodes an 85-amino acid mature protein with a highly conserved predicted transmembrane domain (86 % identity with the S. mansoni tegumental antigen Sm13. The recombinant Sh13 protein was used in ELISA assays to determine the reactivity of sera from the study participants. Antibody responses against Sh13 were predominantly IgG3 isotype compared to responses against crude worm antigens which were predominantly IgG1 and IgG4. The relationship between anti-Sh13 IgG3 levels and infection intensity varied significantly with host age. The youngest children (7–10 years old had relatively low levels of both infection and anti-Sh13 IgG3. In older children (11–12 years old rising infection levels were accompanied by a significant increase in anti-Sh13 IgG3 levels. Subsequently, infection intensity declined significantly in 13–18 year olds but levels of the antibody continued to rise. The changing relationship between infection intensity and anti-Sh13 IgG3 levels with host age

  14. Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection.

    Directory of Open Access Journals (Sweden)

    Matthew D Reed

    Full Text Available Protective antigen (PA, one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax. Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel, elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg.

  15. Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection.

    Science.gov (United States)

    Reed, Matthew D; Wilder, Julie A; Mega, William M; Hutt, Julie A; Kuehl, Philip J; Valderas, Michelle W; Chew, Lawrence L; Liang, Bertrand C; Squires, Charles H

    2015-01-01

    Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg.

  16. Macrophage Immune Response Suppression by Recombinant Mycobacterium tuberculosis Antigens, the ESAT-6, CFP-10, and ESAT-6/CFP-10 Fusion Proteins

    Science.gov (United States)

    Seghatoleslam, Atefeh; Hemmati, Mina; Ebadat, Saeedeh; Movahedi, Bahram; Mostafavi-Pour, Zohreh

    2016-01-01

    Background: Macrophage immune responses are affected by the secretory proteins of Mycobacterium tuberculosis (Mtb). This study aimed to examine the immune responses of macrophages to Mtb secretory antigens, namely ESAT-6, CFP-10, and ESAT-6/CFP-10. Methods: THP-1 cells (a human monocytic cell line) were cultured and differentiated to macrophages by phorbol 12-myristate 13-acetate. The cytotoxicity of the recombinant Mtb proteins was assessed using the MTT assay. Two important immune responses of macrophages, namely NO and ROS production, were measured in response to the ESAT-6, CFP-10, and ESAT-6/CFP-10 antigens. The data were analyzed using one-way ANOVA with SPSS, version 16, and considered significant at P<0.05. Results: The results showed that the ESAT-6, CFP-10, and ESAT-6/CFP-10 proteins markedly reduced macrophage immune response. The treatment of the THP-1-differentiated cells with ESAT-6, CFP-10, and ESAT-6/CFP-10 reduced NO and ROS production. The treated THP-1-differentiated cells exhibited less inducible NO synthase activity than did the untreated cells. No toxic effect on macrophage viability was observed for the applied proteins at the different concentrations. Conclusion: It seems that the decline in macrophage immune response is due to the suppression of NO and ROS production pathways without any effect on cell viability. PMID:27365551

  17. Characterization of Leishmania (Leishmania) amazonensis promastigotes resistant to pentamidine.

    Science.gov (United States)

    Coelho, Adriano C; Gentil, Luciana G; da Silveira, José Franco; Cotrim, Paulo C

    2008-09-01

    Pentamidine is a second-line agent used in the treatment of leishmaniasis and its mode of action and mechanism of resistance is not well understood. It was previously demonstrated that transfection of promastigotes and amastigotes with the ABC transporter PRP1 gene confers resistance to pentamidine. To further clarify this point, we generated Leishmania amazonensis mutants resistant to pentamidine. Our results indicated that this ABC transporter is not associated with pentamidine resistance in lines generated by drug pressure through amplification or overexpression mechanisms of PRP1 gene.

  18. Nanobiotechnologic approach to a promising vaccine prototype for immunisation against leishmaniasis: a fast and effective method to incorporate GPI-anchored proteins of Leishmania amazonensis into liposomes.

    Science.gov (United States)

    Colhone, Marcelle Carolina; Silva-Jardim, Izaltina; Stabeli, Rodrigo Guerino; Ciancaglini, Pietro

    2015-01-01

    Liposomes are known to be a potent adjuvant for a wide range of antigens, as well as appropriate antigen carriers for antibody generation response in vivo. In addition, liposomes are effective vehicles for peptides and proteins, thus enhancing their immunogenicity. Considering these properties of liposomes and the antigenicity of the Leishmania membrane proteins, we evaluated if liposomes carrying glycosylphosphatidylinositol (GPI)-anchored proteins of Leishmania amazonensis promastigotes could induce protective immunity in BALB/c mice. To assay protective immunity, BALB/c mice were intraperitoneally injected with liposomes, GPI-protein extract (EPSGPI) as well as with the proteoliposomes carrying GPI-proteins. Mice inoculated with EPSGPI and total protein present in constitutive proteoliposomes displayed a post-infection protection of about 70% and 90%, respectively. The liposomes are able to work as adjuvant in the EPSGPI protection. These systems seem to be a promising vaccine prototype for immunisation against leishmaniasis.

  19. Evaluation of immune effect of recombinant fusion protein targeting the prostate stem cell antigen based on PSCA and HSP70

    Directory of Open Access Journals (Sweden)

    Lei DONG

    2014-10-01

    Full Text Available Objective To explore the immune effect and antitumor activity of recombinant prostate stem cell protein (PSCA and heat shock protein 70 (HSP70 in a murine model of prostate cancer. Methods Twenty-five healthy male C57BL/6 mice were randomly divided into 5 groups (5 each: PSCA, HSP, PSCA+HSP, PSCA-HSP and control group. Mice in the first 4 groups were vaccinated with the corresponding proteins, and those in control group were faked with injection of phosphate buffer saline (PBS. After immunization with recombinant proteins, the PSCA-specific cellular immune responses were monitored with ELISPOT, intracellular cytokine staining assay, and flow cytometry, and ELISA assay was used to detect humoral immune responses. The tumor growth and survival of vaccined mice were observed. Results ELISPOT revealed that the mice in PSCA-HSP group generated much more IFN-γ spot-forming cells than those in other groups (P<0.05, and they could generate strong anti-PSCA antibody response. Results of flow cytometry showed that the number of CD8+/IFN-γ+ T cells was significantly higher in PSCAHSP group than that in other groups (P<0.05. ELISA results revealed that all the mice in PSCA, PSCA+HSP and PSCA-HSP group were induced to generate the PSCA-specific humoral immune response, and no statistical difference was found on the antibody levels among the three groups. Animal experiment showed that PSCA-HSP could inhibit the growth of PSCA-expressing tumors and prolong the survival time of vaccinated mice. Conclusion HSP70 is a chaperone with significant effect for protein vaccines, and the recombinant fusion protein PSCA-HSP70 could be of potential value for prostate cancer treatment. DOI: 10.11855/j.issn.0577-7402.2014.09.08

  20. Recombinant DNA technology for melanoma immunotherapy: anti-Id DNA vaccines targeting high molecular weight melanoma-associated antigen.

    Science.gov (United States)

    Barucca, A; Capitani, M; Cesca, M; Tomassoni, D; Kazmi, U; Concetti, F; Vincenzetti, L; Concetti, A; Venanzi, F M

    2014-11-01

    Anti-idiotypic MK2-23 monoclonal antibody (anti-Id MK2-23 mAb), which mimics the high molecular weight melanoma-associated antigen (HMW-MAA), has been used to implement active immunotherapy against melanoma. However, due to safety and standardization issues, this approach never entered extensive clinical trials. In the present study, we investigated the usage of DNA vaccines as an alternative to MK2-23 mAb immunization. MK2-23 DNA plasmids coding for single chain (scFv) MK2-23 antibody were constructed via the insertion of variable heavy (V H) and light (V L) chains of MK2-23 into the pVAC-1mcs plasmids. Two alternative MK2-23 plasmids format V H/V L, and V L/V H were assembled. We demonstrate that both polypeptides expressed by scFv plasmids in vitro retained the ability to mimic HMW-MAA antigen, and to elicit specific anti-HMW-MAA humoral and cellular immunoresponses in immunized mice. Notably, MK2-23 scFv DNA vaccines impaired the onset and growth of transplantable B16 melanoma cells not engineered to express HMW-MAA. This pilot study suggests that optimized MK2-23 scFv DNA vaccines could potentially provide a safer and cost-effective alternative to anti-Id antibody immunization, for melanoma immunotherapy.

  1. Recombinant Lactobacillus plantarum induces immune responses to cancer testis antigen NY-ESO-1 and maturation of dendritic cells.

    Science.gov (United States)

    Mobergslien, Anne; Vasovic, Vlada; Mathiesen, Geir; Fredriksen, Lasse; Westby, Phuong; Eijsink, Vincent G H; Peng, Qian; Sioud, Mouldy

    2015-01-01

    Given their safe use in humans and inherent adjuvanticity, Lactic Acid Bacteria may offer several advantages over other mucosal delivery strategies for cancer vaccines. The objective of this study is to evaluate the immune responses in mice after oral immunization with Lactobacillus (L) plantarum WCFS1 expressing a cell-wall anchored tumor antigen NY-ESO-1. And to investigate the immunostimulatory potency of this new candidate vaccine on human dendritic cells (DCs). L. plantarum displaying NY-ESO-1 induced NY-ESO-1 specific antibodies and T-cell responses in mice. By contrast, L. plantarum displaying conserved proteins such as heat shock protein-27 and galectin-1, did not induce immunity, suggesting that immune tolerance to self-proteins cannot be broken by oral administration of L. plantarum. With respect to immunomodulation, immature DCs incubated with wild type or L. plantarum-NY-ESO-1 upregulated the expression of co-stimulatory molecules and secreted a large amount of interleukin (IL)-12, TNF-α, but not IL-4. Moreover, they upregulated the expression of immunosuppressive factors such as IL-10 and indoleamine 2,3-dioxygenase. Although L. plantarum-matured DCs expressed inhibitory molecules, they stimulated allogeneic T cells in-vitro. Collectively, the data indicate that L. plantarum-NY-ESO-1 can evoke antigen-specific immunity upon oral administration and induce DC maturation, raising the potential of its use in cancer immunotherapies.

  2. Photodynamic vaccination of hamsters with inducible suicidal mutants of Leishmania amazonensis elicits immunity against visceral leishmaniasis

    Science.gov (United States)

    Kumari, Shraddha; Samant, Mukesh; Khare, Prashant; Misra, Pragya; Dutta, Sujoy; Kolli, Bala Krishna; Sharma, Sharad; Chang, Kwang Poo; Dube, Anuradha

    2016-01-01

    Leishmania, naturally residing in the phagolysosomes of macrophages, is a suitable carrier for vaccine delivery. Genetic complementation of these trypanosomatid protozoa to partially rectify their defective heme-biosynthesis renders them inducible with δ-aminolevulinate to develop porphyria for selective photolysis, leaving infected host-cells unscathed. Delivery of released “vaccines” to antigen-presenting cells is thus expected to enhance immune response, while their self-destruction presents added advantages of safety. Such suicidal-L. amazonensis was found to confer immunoprophylaxis and immunotherapy on hamsters against L. donovani. Neither heat-killed nor live parasites without suicidal induction were effective. Photodynamic vaccination of hamsters with the suicidal-mutants reduced the parasite loads by 99% and suppressed the development of disease. These suppressions were accompanied by an increase in Leishmania-specific delayed-type hypersensitivity and lymphoproliferation as well as in the levels of splenic iNOS, IFN-γ and IL-12 expressions and of Leishmania-specific IgG2 in the serum. Moreover, a single intravenous administration of T-cells from vaccinated hamsters was shown to confer on naïve animals an effective cellular immunity against L. donovani challenges. The absence of lesion development at vaccination sites and parasites in the draining lymphnodes, spleen and liver further indicates that the suicidal mutants provide a safe platform for vaccine delivery against experimental visceral leishmaniasis. PMID:19053149

  3. Metacyclic promastigotes of Leishmania amazonensis selection using gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Bonetti, Franco C.; Nascimento, Nanci do [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil). Centro de Biologia Molecular]. E-mail: fbonetti@usp.br; Andrade Junior, Heitor Franco de [Instituto de Medicina Tropical de Sao Paulo, SP (Brazil). Lab. de Protozoologia

    2005-07-01

    Leishmania spp. causes a spectrum of human diseases, ranging from self-healing skin lesions to severe and lethal visceral disease. In previous work we demonstrated that the protein and nucleic acid metabolism and oxidative respiration were severely affected by irradiation, in a dose response way, but a small but representative fractions are relatively radio resistant, surviving after 800 Gy of {sup 60}Co irradiation. The best explanation could be a selection of metacyclic promastigotes. In these forms, the G0 state allows the adequate correction of DNA repair after the irradiation insult. In this work, we are looking for the ideal radiation dose to select the higher proportion of metacyclic forms of L.. (L.) amazonensis in culture. Parasites were grown in RPMI 1640 medium, plus 20% fetal calf serum, than they were irradiated with different doses ranging between 25 and 400 Gy. Parasites irradiated at 400 Gy infected, proportionally, more cells than parasites irradiated at other doses. To confirm this metacyclogenesis, a complement lysis assay was performed with 5, 10 and 20% of male guinea pig blood serum at 20 deg C for 3 hours, and parasites counted. Guinea pig serum a 10% promotes more lysis, with 200 Gy irradiated parasites being less affected, probably due to metacyclic selection. These preliminary results suggests that the ionizing radiation, specially between 200 and 400 Gy, could be a alternative tool for the selection of metacyclic forms of Leishmania amazonensis in culture. (a0011uth.

  4. The yeast Scheffersomyces amazonensis is an efficient xylitol producer.

    Science.gov (United States)

    Cadete, Raquel M; Melo-Cheab, Monaliza A; Viana, Adriana L; Oliveira, Evelyn S; Fonseca, César; Rosa, Carlos A

    2016-12-01

    This study assessed the efficiency of Scheffersomyces amazonensis UFMG-CM-Y493(T), cultured in xylose-supplemented medium (YPX) and rice hull hydrolysate (RHH), to convert xylose to xylitol under moderate and severe oxygen limitation. The highest xylitol yields of 0.75 and 1.04 g g(-1) in YPX and RHH, respectively, were obtained under severe oxygen limitation. However, volumetric productivity in RHH was ninefold decrease than that in YPX medium. The xylose reductase (XR) and xylitol dehydrogenase (XDH) activities in the YPX cultures were strictly dependent on NADPH and NAD(+) respectively, and were approximately 10% higher under severe oxygen limitation than under moderate oxygen limitation. This higher xylitol production observed under severe oxygen limitation can be attributed to the higher XR activity and shortage of the NAD(+) needed by XDH. These results suggest that Sc. amazonensis UFMG-CM-Y493(T) is one of the greatest xylitol producers described to date and reveal its potential use in the biotechnological production of xylitol.

  5. Leishmania amazonensis Engages CD36 to Drive Parasitophorous Vacuole Maturation.

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    Kendi Okuda

    2016-06-01

    Full Text Available Leishmania amastigotes manipulate the activity of macrophages to favor their own success. However, very little is known about the role of innate recognition and signaling triggered by amastigotes in this host-parasite interaction. In this work we developed a new infection model in adult Drosophila to take advantage of its superior genetic resources to identify novel host factors limiting Leishmania amazonensis infection. The model is based on the capacity of macrophage-like cells, plasmatocytes, to phagocytose and control the proliferation of parasites injected into adult flies. Using this model, we screened a collection of RNAi-expressing flies for anti-Leishmania defense factors. Notably, we found three CD36-like scavenger receptors that were important for defending against Leishmania infection. Mechanistic studies in mouse macrophages showed that CD36 accumulates specifically at sites where the parasite contacts the parasitophorous vacuole membrane. Furthermore, CD36-deficient macrophages were defective in the formation of the large parasitophorous vacuole typical of L. amazonensis infection, a phenotype caused by inefficient fusion with late endosomes and/or lysosomes. These data identify an unprecedented role for CD36 in the biogenesis of the parasitophorous vacuole and further highlight the utility of Drosophila as a model system for dissecting innate immune responses to infection.

  6. Efficacy of a diarylheptanoid derivative against Leishmania amazonensis

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    Alves Luciana Vignólio

    2003-01-01

    Full Text Available The activity of several diarylheptanoid derivatives (curcuminoids was previously evaluated against Leishmania amazonensis promastigotes and among them the most active compound was the [1-(4-methoxy-phenyl-7-(3,4-methoxy-4-hydroxy-phenyl-1,6-heptadien-3, 5-dione]. This derivative was chosen to be assayed in vivo in a treatment trial. For these experiments, the curcuminoid compound was used in a concentration equivalent to the IC50/24 h, obtained from the previous study. Balb/c mice were inoculated subcutaneously in the footpad with L. amazonensis infective promastigotes and 4 weeks after the inoculation, the animals were treated with different schemes, varying from 1 to 3 doses. In all the experiments, Pentamidine Isethionate was used as reference drug under the same experimental conditions. The results showed that one dose was not enough to heal the lesion, however, with 2 and 3 doses the efficiency of the assayed compound was clear. On the other hand, treatment with Pentamidine Isethionate using the three different schemes was not satisfactory when compared to the curcuminoid derivative.

  7. Prime-boost vaccination with toxoplasma lysate antigen, but not with a mixture of recombinant protein antigens, leads to reduction of brain cyst formation in BALB/c mice.

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    Angelika Wagner

    Full Text Available Infection with the ubiquitous parasite Toxoplasma gondii is a threat for immunocompromised patients and pregnant women and effective immune-prophylaxis is still lacking.Here we tested a mixture of recombinant T. gondii antigens expressed in different developmental stages, i.e., SAG1, MAG1 and GRA7 (SMG, and a lysate derived from T. gondii tachyzoites (TLA for prophylactic vaccination against cyst formation. Both vaccine formulations were applied systemically followed by an oral TLA-booster in BALB/c mice.Systemic priming with SMG and oral TLA-booster did not show significant induction of protective immune responses. In contrast, systemic priming and oral booster with TLA induced higher levels of Toxoplasma-specific IgG, IgG1 and IgG2a in sera as well as high levels of Toxoplasma-specific IgG1 in small intestines. Furthermore, high levels of Toxoplasma-specific Th1-, Th17- and Th2-associated cytokines were only detected in restimulated splenocytes of TLA-vaccinated mice. Importantly, in mice orally infected with T. gondii oocysts, only TLA-vaccination and booster reduced brain cysts. Furthermore, sera from these mice reduced tachyzoites invasion of Vero cells in vitro, indicating that antibodies may play a critical role for protection against Toxoplasma infection. Additionally, supernatants from splenocyte cultures of TLA-vaccinated mice containing high levels of IFN-γ lead to substantial production of nitric oxide (NO after incubation with macrophages in vitro. Since NO is involved in the control of parasite growth, the high levels of IFN-γ induced by vaccination with TLA may contribute to the protection against T. gondii.In conclusion, our data indicate that prime-boost approach with TLA, but not with the mixture of recombinant antigens SMG, induces effective humoral and cellular Toxoplasma-specific responses and leads to significant reduction of cerebral cysts, thereby presenting a viable strategy for further vaccine development against T

  8. Induction of antigen-presenting capacity in tumor cells upon infection with non-replicating recombinant vaccinia virus encoding murine MHC class II and costimulatory molecules.

    Science.gov (United States)

    Marti, W R; Oertli, D; Meko, J B; Norton, J A; Tsung, K

    1997-01-15

    The possibility of inducing antigen-presenting capacity in cells normally lacking such capacity, currently represents a major goal in vaccine research. To address this issue we attempted to generate 'artificial' APC able to stimulate CD4+ T cell responses when tumor cells were infected with a single, recombinant, vaccinia virus (rVV) containing the two genes encoding murine MHC class II I-Ak and a third gene encoding the murine B7-1 (mB7-1) costimulatory molecule. To minimize the cytopathic effect and to improve safety, in view of possible in vivo applications, we made this rVV replication incompetent by Psoralen and long wave UV treatment. Tumor cells infected with rVV encoding I-Ak alone, pulsed with hen egg white lysozyme peptide (HEL46-61), induced IL-2 secretion by an antigen-specific T hybridoma. Tumor cells infected with the rVV encoding mB7-1 provided costimulation for activating resting CD4+ T cells in the presence of ConA. Tumor cells infected with the rVV encoding I-Ak and mB7-1, and pulsed with chicken ovotransferrin peptide (conalbumin133-145), induced a significantly higher response in a specific Th2 cell clone (D10.G4.1) as compared to cells infected with rVV encoding I-Ak molecules only. Thus, this replication incompetent rVV represents a safe, multiple gene, vector system able to confer in one single infection step effective APC capacity to non-professional APCs.

  9. Immune protection conferred by recombinant MRLC (myosin regulatory light chain) antigen in TiterMax Gold® adjuvant against experimental fasciolosis in rats.

    Science.gov (United States)

    Henker, Luan C; Schwertz, Claiton I; Lucca, Neuber J; Piva, Manoela M; Prior, Keila C; Baska, Piotr; Norbury, Luke; Januszkiewicz, Kamil; Dezen, Diogenes; Duarte, Marta M M F; Moresco, Rafael N; Bertagnolli da Rosa, Liana; Mendes, Ricardo E

    2017-01-23

    Protection against experimental fasciolosis in rats immunized with recombinant myosin regulatory light chain (MRLC) in TiterMax Gold® adjuvant was assessed. The experimental trial consisted of four groups of 15 animals; group 1 was unimmunized and infected, group 2 was immunized with MRLC in adjuvant and infected, group 3 was infected and immunized with adjuvant only and group 4 was unimmunized and uninfected. Immunization with MRLC in TiterMax Gold® adjuvant (group 2) induced a reduction in fluke burdens of 51.0% (p<0.001) when compared with the adjuvant control group, and 61.5% (p<0.001) when compared with the unimmunized infected controls. There was a reduction in fecal egg output in group 2 of 44.8% and 37.3% compared with group 1 and group 3, respectively; although this difference was not statistically significant. Measurement of cytokine levels revealed higher levels of TNF-alpha and IL-2 as well as lower levels of IL-4 in group 2 during the chronic stage of infection (p<0.05), along with higher levels of IFN-gamma during early stages of infection (p<0.05). These results suggest a mixed Th1/Th2 phenotype immune response; however predominance of Th1 cytokines was observed. Levels of anti-MRLC serum IgG in group 2 were significantly higher than controls at the time of euthanasia (p<0.05). This is the first report of immunization with recombinant MRLC in rats, demonstrating that this antigen significantly reduces fluke burdens, increases the Th1 immune response and encourages further studies to improve the vaccine's efficacy.

  10. In Vivo Synthesis of Cyclic-di-GMP Using a Recombinant Adenovirus Preferentially Improves Adaptive Immune Responses against Extracellular Antigens.

    Science.gov (United States)

    Alyaqoub, Fadel S; Aldhamen, Yasser A; Koestler, Benjamin J; Bruger, Eric L; Seregin, Sergey S; Pereira-Hicks, Cristiane; Godbehere, Sarah; Waters, Christopher M; Amalfitano, Andrea

    2016-02-15

    There is a compelling need for more effective vaccine adjuvants to augment induction of Ag-specific adaptive immune responses. Recent reports suggested the bacterial second messenger bis-(3'-5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) acts as an innate immune system modulator. We recently incorporated a Vibrio cholerae diguanylate cyclase into an adenovirus vaccine, fostering production of c-di-GMP as well as proinflammatory responses in mice. In this study, we recombined a more potent diguanylate cyclase gene, VCA0848, into a nonreplicating adenovirus serotype 5 (AdVCA0848) that produces elevated amounts of c-di-GMP when expressed in mammalian cells in vivo. This novel platform further improved induction of type I IFN-β and activation of innate and adaptive immune cells early after administration into mice as compared with control vectors. Coadministration of the extracellular protein OVA and the AdVCA0848 adjuvant significantly improved OVA-specific T cell responses as detected by IFN-γ and IL-2 ELISPOT, while also improving OVA-specific humoral B cell adaptive responses. In addition, we found that coadministration of AdVCA0848 with another adenovirus serotype 5 vector expressing the HIV-1-derived Gag Ag or the Clostridium difficile-derived toxin B resulted in significant inhibitory effects on the induction of Gag and toxin B-specific adaptive immune responses. As a proof of principle, these data confirm that in vivo synthesis of c-di-GMP stimulates strong innate immune responses that correlate with enhanced adaptive immune responses to concomitantly administered extracellular Ag, which can be used as an adjuvant to heighten effective immune responses for protein-based vaccine platforms against microbial infections and cancers.

  11. Intrarectal vaccination with recombinant vaccinia virus expressing carcinoembronic antigen induces mucosal and systemic immunity and prevents progression of colorectal cancer.

    Science.gov (United States)

    Kim-Schulze, Seunghee; Kim, Hong Sung; Wainstein, Alberto; Kim, Dae Won; Yang, Wein Cui; Moroziewicz, Dorota; Mong, Phyllus Y; Bereta, Michal; Taback, Bret; Wang, Qin; Kaufman, Howard L

    2008-12-01

    The gastrointestinal mucosa contains an intact immune system that protects the host from pathogens and communicates with the systemic immune system. Absorptive epithelial cells in the mucosa give rise to malignant tumors although the interaction between tumor cells and the mucosal immune system is not well defined. The pathophysiology of colorectal cancer has been elucidated through studies of hereditary syndromes, such as familial adenomatous polyposis, a cancer predisposition syndrome caused by germline mutations in the adenomatous polyposis coli tumor suppressor gene. Patients with FAP develop adenomas and inevitably progress to invasive carcinomas by the age of 40. To better delineate the role of mucosal immunity in colorectal cancer, we evaluated the efficacy of intrarectal recombinant vaccinia virus expressing the human carcinoembryonic Ag (CEA) in a murine FAP model in which mice are predisposed to colorectal cancer and also express human CEA in the gut. Mucosal vaccination reduced the incidence of spontaneous adenomas and completely prevented progression to invasive carcinoma. The therapeutic effects were associated with induction of mucosal CEA-specific IgA Ab titers and CD8(+) CTLs. Mucosal vaccination was also associated with an increase in systemic CEA-specific IgG Ab titers, CD4(+) and CD8(+) T cell responses and resulted in growth inhibition of s.c. implanted CEA-expressing tumors suggesting communication between mucosal and systemic immune compartments. Thus, intrarectal vaccination induces mucosal and systemic antitumor immunity and prevents progression of spontaneous colorectal cancer. These results have implications for the prevention of colorectal cancer in high-risk individuals.

  12. Trypanosoma cruzi Differentiates and Multiplies within Chimeric Parasitophorous Vacuoles in Macrophages Coinfected with Leishmania amazonensis.

    Science.gov (United States)

    Pessoa, Carina Carraro; Ferreira, Éden Ramalho; Bayer-Santos, Ethel; Rabinovitch, Michel; Mortara, Renato Arruda; Real, Fernando

    2016-05-01

    The trypanosomatids Leishmania amazonensis and Trypanosoma cruzi are excellent models for the study of the cell biology of intracellular protozoan infections. After their uptake by mammalian cells, the parasitic protozoan flagellates L. amazonensis and T. cruzi lodge within acidified parasitophorous vacuoles (PVs). However, whereas L. amazonensis develops in spacious, phagolysosome-like PVs that may enclose numerous parasites, T. cruzi is transiently hosted within smaller vacuoles from which it soon escapes to the host cell cytosol. To investigate if parasite-specific vacuoles are required for the survival and differentiation of T. cruzi, we constructed chimeric vacuoles by infection of L. amazonensis amastigote-infected macrophages with T. cruzi epimastigotes (EPIs) or metacyclic trypomastigotes (MTs). These chimeric vacuoles, easily observed by microscopy, allowed the entry and fate of T. cruzi in L. amazonensis PVs to be dynamically recorded by multidimensional imaging of coinfected cells. We found that although T. cruzi EPIs remained motile and conserved their morphology in chimeric vacuoles, T. cruzi MTs differentiated into amastigote-like forms capable of multiplying. These results demonstrate that the large adaptive vacuoles of L. amazonensis are permissive to T. cruzi survival and differentiation and that noninfective EPIs are spared from destruction within the chimeric PVs. We conclude that T. cruzi differentiation can take place in Leishmania-containing vacuoles, suggesting this occurs prior to their escape into the host cell cytosol.

  13. Oral Delivery of a Novel Recombinant Streptococcus mitis Vector Elicits Robust Vaccine Antigen-Specific Oral Mucosal and Systemic Antibody Responses and T Cell Tolerance.

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    Emily Xie

    Full Text Available The pioneer human oral commensal bacterium Streptococcus mitis has unique biologic features that make it an attractive mucosal vaccine or therapeutic delivery vector. S. mitis is safe as a natural persistent colonizer of the mouth, throat and nasopharynx and the oral commensal bacterium is capable of inducing mucosal antibody responses. A recombinant S. mitis (rS. mitis that stably expresses HIV envelope protein was generated and tested in the germ-free mouse model to evaluate the potential usefulness of this vector as a mucosal vaccine against HIV. Oral vaccination led to the efficient and persistent bacterial colonization of the mouth and the induction of both salivary and systemic antibody responses. Interestingly, persistently colonized animals developed antigen-specific systemic T cell tolerance. Based on these findings we propose the use of rS. mitis vaccine vector for the induction of mucosal antibodies that will prevent the penetration of the mucosa by pathogens such as HIV. Moreover, the first demonstration of rS. mitis having the ability to elicit T cell tolerance suggest the potential use of rS. mitis as an immunotherapeutic vector to treat inflammatory, allergic and autoimmune diseases.

  14. Development of EMA-2 recombinant antigen based enzyme-linked immunosorbent assay for seroprevalence studies of Theileria equi infection in Indian equine population.

    Science.gov (United States)

    Kumar, Sanjay; Kumar, Rajender; Gupta, Ashok K; Yadav, Suresh C; Goyal, Sachin K; Khurana, Sandip K; Singh, Raj K

    2013-11-15

    Equine piroplasmosis is a tick-transmitted protozoan disease caused by Theileria equi and/or Babesia caballi. In the present study, we expressed a 53kDa protein from the truncated EMA-2 gene of T. equi (Indian strain) and developed EMA-2ELISA using this expressed protein. This ELISA is able to detect T. equi-specific antibodies in experimentally infected animals as early as 9 days post-infection. The assay developed was validated with the OIE recommended competitive ELISA (cELISA) on 120 serum samples and significant agreement (kappa=0.93) was observed between results of both the ELISAs which indicates suitability of EMA-2ELISA for use in sero-diagnosis. Diagnostic sensitivity and specificity of EMA-2ELISA - as compared with cELISA - were 0.97 and 0.96, respectively. Analysis of 5651 equine serum samples - collected during 2007-2012 from 12 states of India representing eight agro-climatic zones - by EMA-2ELISA revealed 32.65% seroprevalence of T. equi in India. In conclusion, the EMA-2ELISA developed using the T. equi EMA-2 recombinant protein as antigen for detecting T. equi-specific antibodies has good diagnostic potential for sero-epidemiological surveys.

  15. Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

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    Virgínia MG Silva

    2006-08-01

    Full Text Available Indirect enzyme-linked immunosorbent assays (ELISAs based on recombinant major surface protein 5 (rMSP5 and initial body (IB antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2% and specificities (100% for rMSP5 and 93.8% for IB ELISA which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA to 15% (IB ELISA of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

  16. Transcriptional upregulation of fortilin in shrimp, Penaeus (Metapenaeus japonicus fed diets containing recombinant VP28, an antigenic protein of white spot syndrome virus (WSSV

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    Christopher Marlowe A. Caipang

    2013-07-01

    Full Text Available White spot syndrome virus (WSSV is considered as one of the serious viral pathogens ofshrimp. There are several preventive measures that have been developed to curb the devastating effectsof this virus in shrimp aquaculture. Juvenile shrimps, Penaeus (Metapenaeus japonicus were fed withcommercial feeds that were mixed with recombinant VP28, a structural protein antigen of WSSV for aperiod of 14 days. The immune response of the shrimp during oral administration of the medicated feedwas determined by expression analysis of fortilin, a gene that is involved in the antiviral response. Therewas a significant increase in the level of expression of fortilin both in the gut and the gills in the fedgroup during the duration of feeding. The level of expression gradually decreased in the fed group,whereby at the 7th and 14th day after the last day of feeding with medicated feeds, no significantdifferences were observed between the fed and control groups in the expression at the gills and gut,respectively.

  17. Development of an enzyme-linked immunoelectrotransfer blot (EITB) assay using two baculovirus expressed recombinant antigens for diagnosis of Taenia solium taeniasis.

    Science.gov (United States)

    Levine, Min Z; Lewis, Melissa M; Rodriquez, Silvia; Jimenez, Juan A; Khan, Azra; Lin, Sehching; Garcia, Hector H; Gonzales, Armando E; Gilman, Robert H; Tsang, Victor C W

    2007-04-01

    Taeniasis diagnosis is an important step in the control and elimination of both cysticercosis and taeniasis. We report the development of 2 serological taeniasis diagnostic tests using recombinant antigens rES33 and rES38 expressed by baculovirus in insect cells in an EITB format. In laboratory testing with defined sera from nonendemic areas, rES33 has a sensitivity of 98% (n = 167) and a specificity of 99% (n = 310) (J index: 0.97); rES38 has a sensitivity of 99% (n = 146) and a specificity of 97% (n = 275) (J index: 0.96). Independent field testing in Peru showed 97% (n = 203) of the taeniasis sera were positive with rES33, and 100% of the nontaeniasis sera (n = 272) were negative with rES33; 98% (n = 198) of taeniasis sera were positive with rES38, and 91% (n = 274) of the nontaeniasis sera were negative with rES38. Among the Peruvian sera tested, 17 of 26 Peruvian Taenia saginata sera were false positive with rES38 test. Both tests were also examined with cysticercosis sera, with a positive rate ranging from 21% to 46%. rES33 and rES38 tests offer sensitive and specific diagnosis of taeniasis and easy sample collection through finger sticks that can be used in large-scale studies. They are currently being used in cysticercosis elimination programs in Peru.

  18. 量子点荧光探针用于汉坦病毒重组抗原的检测%Detection of recombinant Hantavirus antigens by using water-solubility quantum dots fluorescent probe

    Institute of Scientific and Technical Information of China (English)

    陈琼; 张云; 郑锦峰; 孙军红; 姚苹苹; 王洁; 李晶; 邓小昭; 王长军; 朱函坪

    2012-01-01

    Objective To detect recombinant Hantavirus antigens by using a quantum dots ( QDs) fluorescent probe. Methods The probe of water-solubility QDs fluorescence nanoparticle was synthesized and modified with protein G and anti-HV Antibodies. We detected recombinant Hantavirus antigens by probe combining immune method,and optimized the detecting conditions. Results The optimum reaction time,pH and goat antibodies concentration for conjugating the QDs were 2 h ,6.0, and 20 μg/ml, respectively. The limit of detection of recombinant antigens was 5 ng/ml. Conclusion The fluorescent probe could effectively recognize HV antigens. The method was specific, sensitive and convenient. We developed a new method for HV antigens detection.%目的 应用量子点荧光探针对汉坦病毒(Hantavirus,HV)重组抗原进行检测.方法 合成水溶性量子点荧光纳米颗粒,并在其表面修饰G蛋白和anti-HV抗体作为量子点荧光探针,对HV重组抗原进行检测并优化检测条件.结果 量子点与抗体的最佳偶联条件:pH 6.0、反应时间2h、抗体浓度为20 μg/ml.用本方法检测HV重组抗原的最低检测值为5 ng/ml.结论 该探针能有效的识别HV抗原,且操作简便快速,为HV重组抗原的检测和肾出血热综合征的诊断提供了新方法.

  19. A new human IgG avidity test, using mixtures of recombinant antigens (rROP1, rSAG2, rGRA6), for the diagnosis of difficult-to-identify phases of toxoplasmosis.

    Science.gov (United States)

    Drapała, Dorota; Holec-Gąsior, Lucyna; Kur, Józef; Ferra, Bartłomiej; Hiszczyńska-Sawicka, Elżbieta; Lautenbach, Dariusz

    2014-07-01

    The preliminary diagnostic utility of two mixtures of Toxoplasma gondii recombinant antigens (rROP1+rSAG2 and rROP1+rGRA6) in IgG ELISA and IgG avidity test has been evaluated. A total of 173 serum samples from patients with toxoplasmosis and seronegative people were examined. The sensitivity of IgG ELISA for rROP1+rSAG2 and rROP1+rGRA6 was 91.1% and 76.7%, respectively, while the reactivity for sera from patients where acute toxoplasmosis was suspected was higher, at 100% and 95.4%, respectively, than for people with chronic infection, at 88.2% and 70.6%. In this study a different trend in avidity maturation of IgG antibodies for two mixtures of proteins in comparison with native antigen was observed. The results suggest that a new IgG avidity test using the mixtures of recombinant antigens may be useful for the diagnosis of difficult-to-identify phases of toxoplasmosis. For this reason, selected mixtures after the additional tests on groups of sera with well-defined dates of infection could be used as a better alternative to the native antigens of the parasite in the serodiagnosis of human T. gondii infection.

  20. Live recombinant Salmonella Typhi vaccines constructed to investigate the role of rpoS in eliciting immunity to a heterologous antigen.

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    Huoying Shi

    Full Text Available We hypothesized that the immunogenicity of live Salmonella enterica serovar Typhi vaccines expressing heterologous antigens depends, at least in part, on its rpoS status. As part of our project to develop a recombinant attenuated S. Typhi vaccine (RASTyV to prevent pneumococcal diseases in infants and children, we constructed three RASTyV strains synthesizing the Streptococcus pneumoniae surface protein PspA to test this hypothesis. Each vector strain carried ten engineered mutations designed to optimize safety and immunogenicity. Two S. Typhi vector strains (chi9639 and chi9640 were derived from the rpoS mutant strain Ty2 and one (chi9633 from the RpoS(+ strain ISP1820. In chi9640, the nonfunctional rpoS gene was replaced with the functional rpoS gene from ISP1820. Plasmid pYA4088, encoding a secreted form of PspA, was moved into the three vector strains. The resulting RASTyV strains were evaluated for safety in vitro and for immunogenicity in mice. All three RASTyV strains were similar to the live attenuated typhoid vaccine Ty21a in their ability to survive in human blood and human monocytes. They were more sensitive to complement and were less able to survive and persist in sewage and surface water than their wild-type counterparts. Adult mice intranasally immunized with any of the RASTyV strains developed immune responses against PspA and Salmonella antigens. The RpoS(+ vaccines induced a balanced Th1/Th2 immune response while the RpoS(- strain chi9639(pYA4088 induced a strong Th2 immune response. Immunization with any RASTyV provided protection against S. pneumoniae challenge; the RpoS(+ strain chi9640(pYA4088 provided significantly greater protection than the ISP1820 derivative, chi9633(pYA4088. In the pre-clinical setting, these strains exhibited a desirable balance between safety and immunogenicity and are currently being evaluated in a Phase 1 clinical trial to determine which of the three RASTyVs has the optimal safety and

  1. Immunogenic salivary proteins of Triatoma infestans: development of a recombinant antigen for the detection of low-level infestation of triatomines.

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    Alexandra Schwarz

    Full Text Available BACKGROUND: Triatomines are vectors of Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. The most effective vector, Triatoma infestans, has been controlled successfully in much of Latin America using insecticide spraying. Though rarely undertaken, surveillance programs are necessary in order to identify new infestations and estimate the intensity of triatomine bug infestations in domestic and peridomestic habitats. Since hosts exposed to triatomines develop immune responses to salivary antigens, these responses can be evaluated for their usefulness as epidemiological markers to detect infestations of T. infestans. METHODOLOGY/PRINCIPAL FINDINGS: T. infestans salivary proteins were separated by 2D-gel electrophoresis and tested for their immunogenicity by Western blotting using sera from chickens and guinea pigs experimentally exposed to T. infestans. From five highly immunogenic protein spots, eight salivary proteins were identified by nano liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS/MS and comparison to the protein sequences of the National Center for Biotechnology Information (NCBI database and expressed sequence tags of a unidirectionally cloned salivary gland cDNA library from T. infestans combined with the NCBI yeast protein sub-database. The 14.6 kDa salivary protein [gi|149689094] was produced as recombinant protein (rTiSP14.6 in a mammalian cell expression system and recognized by all animal sera. The specificity of rTiSP14.6 was confirmed by the lack of reactivity to anti-mosquito and anti-sand fly saliva antibodies. However, rTiSP14.6 was recognized by sera from chickens exposed to four other triatomine species, Triatoma brasiliensis, T. sordida, Rhodnius prolixus, and Panstrongylus megistus and by sera of chickens from an endemic area of T. infestans and Chagas disease in Bolivia. CONCLUSIONS/SIGNIFICANCE: The recombinant rTiSP14.6 is a suitable and promising

  2. Development of an Indirect Enzyme-Linked Immunosorbent Assay for Seromonitoring Contagious Bovine Pleuropneumonia Using Recombinant Lipoprotein LppQ of Mycoplasma mycoides subsp mycoides SC as Antigen

    Institute of Scientific and Technical Information of China (English)

    XIN Jiu-qing; GAO Yun-long; LI Yuan; WANG Yan-fan; QIAN Ai-dong

    2007-01-01

    Mycoplasma mycoides subsp mycoides SC (MmmSC) is the etiological agent of contagious bovine pleuropneumonia (CBPP). The lipoprotein LppQ encoded by lppQ gene is specific to MmmSC and is found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. No serological cross-reactions were observed with the related mycoplasmas of the Mycoplasma mycoides cluster. The N-terminal domain of the mature lipoprotein LppQ is hydrophilic, and it induces a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. Mycoplasma-specific TGA (Trp) codons are utilized as stop codons in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI Ⅹ strain, the Chinese strain for CF antigen production, was mutated with one-step overlapping extension PCR. Sequence analysis confirmed the successful mutation from A to G in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expression vector. The recombinant protein with molecular weight of 42 kDa was purified using the Ni-NTA His.Bind purification kit, with a purity of up to 95%. Western blot indicated that the standard positive serum of CBPP could react with the recombinant protein. The purified protein was diluted to 0.35 μg mL-1, and coated to microtiter enzyme-linked immunosorbent assay (ELISA) plates. Indirect ELISA reaction conditions were optimized. The value of P/N was determined to be 4.8 (0.934/0.193), the sensitivity to be 95.8% (46/48), and the specificity to be 98.9% (161/163). 3 817 cattle serum samples from three different provinces were detected by the indirect ELISA and CFT. The Kappa value is 0.63, which is middle or high agreement between the two methods.

  3. A new ELISA kit which uses a combination of Plasmodium falciparum extract and recombinant Plasmodium vivax antigens as an alternative to IFAT for detection of malaria antibodies

    Directory of Open Access Journals (Sweden)

    Doderer Cecile

    2007-02-01

    Full Text Available Abstract Background The methods most commonly used to measure malarial antibody titres are the Indirect Fluorescence Antibody Test (IFAT, regarded as the gold standard, and the Enzyme-Linked ImmunoSorbent Assay (ELISA. The objective here was to assess the diagnostic performance, i.e. the sensitivity and specificity, of a new malaria antibody ELISA kit in comparison to IFAT. This new ELISA kit, the ELISA malaria antibody test (DiaMed, uses a combination of crude soluble Plasmodium falciparum extract and recombinant Plasmodium vivax antigens. Methods Two groups were used: 95 samples from malaria patients to assess the clinical sensitivity and 2,152 samples from blood donors, who had not been exposed to malaria, to assess the clinical specificity. Results The DiaMed ELISA test kit had a clinical sensitivity of 84.2% and a clinical specificity of 99.6% as compared with 70.5% and 99.6% respectively, using the IFAT method. The ELISA method was more sensitive than the IFAT method for P. vivax infections (75% vs. 25%. However, in 923 malaria risk donors the analytical sensitivity of the ELISA test was 40% and its specificity 98.3%, performances impaired by large numbers of equivocal results non-concordant between ELISA and IFAT. When the overall analytical performances of ELISA was compared to IFAT, the ELISA efficiency J index was 0.84 versus 0.71 for IFAT. Overall analytical sensitivity was 93.1% and the analytical specificity 96.7%. Overall agreement between the two methods reached 0.97 with a reliability k index of 0.64. Conclusion The DiaMed ELISA test kit shows a good correlation with IFAT for analytical and clinical parameters. It may be an interesting method to replace the IFAT especially in blood banks, but further extensive investigations are needed to examine the analytical performance of the assay, especially in a blood bank setting.

  4. Phase I study of safety and immunogenicity of an Escherichia coli-derived recombinant protective antigen (rPA vaccine to prevent anthrax in adults.

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    Bruce K Brown

    Full Text Available BACKGROUND: The fatal disease caused by Bacillus anthracis is preventable with a prophylactic vaccine. The currently available anthrax vaccine requires a lengthy immunization schedule, and simpler and more immunogenic options for protection against anthrax are a priority for development. In this report we describe a phase I clinical trial testing the safety and immunogenicity of an anthrax vaccine using recombinant Escherichia coli-derived, B. anthracis protective antigen (rPA. METHODOLOGY/PRINCIPAL FINDINGS: A total of 73 healthy adults ages 18-40 were enrolled and 67 received 2 injections separated by 4 weeks of either buffered saline placebo, or rPA formulated with or without 704 µg/ml Alhydrogel® adjuvant in increasing doses (5, 25, 50, 100 µg of rPA. Participants were followed for one year and safety and immunologic data were assessed. Tenderness and warmth were the most common post-injection site reactions. No serious adverse events related to the vaccine were observed. The most robust humoral immune responses were observed in subjects receiving 50 µg of rPA formulated with Alhydrogel® with a geometric mean concentration of anti-rPA IgG antibodies of 283 µg/ml and a toxin neutralizing geometric 50% reciprocal geometric mean titer of 1061. The highest lymphoproliferative peak cellular response (median Lymphocyte Stimulation Index of 29 was observed in the group receiving 25 µg Alhydrogel®-formulated rPA. CONCLUSIONS/SIGNIFICANCE: The vaccine was safe, well tolerated and stimulated a robust humoral and cellular response after two doses. TRIAL REGISTRATION: ClinicalTrials.gov NCT00057525.

  5. Protection of C57BL/10 mice by vaccination with association of purified proteins from Leishmania (Leishmania amazonensis

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    MORA Ana Mariela

    1999-01-01

    Full Text Available In the past few years, induction of protective immunity to cutaneous leishmaniasis has been attempted by many researchers using a variety of antigenic preparations, such as living promastigotes or promastigote extracts, partially purified, or defined proteins. In this study, eleven proteins from Leishmania (Leishmania amazonensis (LLa with estimated molecular mass ranging from 97 to 13.5kDa were isolated by polyacrylamide gel electrophoresis and electro-elution. The proteins were associated as vaccine in different preparations with gp63 and BCG (Bacilli Calmette-Guérin. The antigenicity of these vaccines was measured by their ability to induce the production of IFN-g by lymphocyte from subjects vaccinated with Leishvacinâ . The immunogenicity was evaluated in vaccinated mice. C57BL/10 mice were vaccinated with three doses of each vaccine consisting of 30 mg of each protein at 15 days interval. One hundred mg of live BCG was only used in the first dose. Seven days after the last dose, they received a first challenge infection with 105 infective promastigotes and four months later, a second challenge was done. Two months after the second challenge, 42.86% of protection was obtained in the group of mice vaccinated with association of proteins of gp63+46+22kDa, gp63+13.5+25+42kDa, gp63+46+42kDa, gp63+66kDa, and gp63+97kDa; 57.14% of protection was demonstrated with gp63+46+97+13.5kDa, gp63+46+97kDa, gp63+46+33kDa, and 71.43% protection for gp63 plus all proteins. The vaccine of gp63+46+40kDa that did not protect the mice, despite the good specific stimulation of lymphocytes (LSI = 7.60 and 10.77UI/ml of IFN-g production. When crude extract of L. (L. amazonensis was used with BCG a 57.14% of protection was found after the first challenge and 28.57% after the second, the same result was observed for gp63. The data obtained with the vaccines can suggest that the future vaccine probably have to contain, except the 40kDa, a cocktail of proteins that

  6. Immunogenicity of three recombinant hepatitis B vaccines administered to students in three doses containing half the antigen amount routinely used for adult vaccination

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    Baldy José Luís da Silveira

    2004-01-01

    Full Text Available We evaluated the immunogenicity of three recombinant hepatitis B vaccines, one Brazilian (Butang, Instituto Butantan and two Korean vaccines (Euvax-B, LG Chemical Ltd. and Hepavax-Gene, Greencross Vaccine Corp., administered intramuscularly to students aged 17 to 19 years in three 10-µg doses (corresponding to half the amount of antigen routinely used for adult vaccination at intervals of one month between the first and second dose, and of four months between the second and third dose. A total of 316 students non-reactive for any serological marker of hepatitis B virus infection were vaccinated: 77 (24.4% with the Butang vaccine, 71 (22.5% with Euvax-B, 85 (26.9% with Hepavax-Gene and, for comparison, 83 (26.2% with Engerix-B (GlaxoSmithKline, whose efficacy in young adults at the dose used here has been confirmed in previous studies. Similar seroconversion rates (anti-HBs > 10 mIU/mL about one month after application of the third dose were obtained for the Butang, Euvax-B, Hepavax-Gene and Engerix-B vaccines (96.2%, 98.6%, 96.5% and 97.6%, respectively. The frequency of good responders (anti-HBs > 100 mIU/mL was also similar among students receiving the four vaccines (85.8%, 91.6%, 89.4% and 89.2%, respectively. The geometric mean titers (GMT of anti-HBs about one month after the third dose obtained with these vaccines were 727.78 ± 6.46 mIU/mL, 2009.09 ± 7.16 mIU/mL, 1729.82 ± 8.85 mIU/mL and 2070.14 ± 11.69 mIU/mL, respectively. The GMT of anti-HBs induced by the Euvax-B and Engerix-B vaccines were higher than those obtained with the Butang vaccine (p < 0.05; this difference was not significant when comparing the other vaccines two-by-two. No spontaneous adverse effects attributable to the application of any dose of the four vaccines were reported.

  7. Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG.

    Science.gov (United States)

    Logan, Michael; Law, John; Wong, Jason Alexander Ji-Xhin; Hockman, Darren; Landi, Amir; Chen, Chao; Crawford, Kevin; Kundu, Juthika; Baldwin, Lesley; Johnson, Janelle; Dahiya, Anita; LaChance, Gerald; Marcotrigiano, Joseph; Law, Mansun; Foung, Steven; Tyrrell, Lorne; Houghton, Michael

    2017-01-01

    A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography.

  8. Prime-boost vaccination with Bacillus Calmette Guerin and a recombinant adenovirus co-expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis induces robust antigen-specific immune responses in mice.

    Science.gov (United States)

    Li, Wu; Li, Min; Deng, Guangcun; Zhao, Liping; Liu, Xiaoming; Wang, Yujiong

    2015-08-01

    Tuberculosis (TB) remains to be a prevalent health issue worldwide. At present, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the singular anti-TB vaccine available for the prevention of disease in humans; however, this vaccine only provides limited protection against Mycobacterium tuberculosis (Mtb) infection. Therefore, the development of alternative vaccines and strategies for increasing the efficacy of vaccination against TB are urgently required. The present study aimed to evaluate the ability of a recombinant adenoviral vector (Ad5-CEAB) co-expressing 10-kDa culture filtrate protein, 6-kDa early-secreted antigenic target, antigen 85 (Ag85)A and Ag85B of Mtb to boost immune responses following primary vaccination with BCG in mice. The mice were first subcutaneously primed with BCG and boosted with two doses of Ad5-CEAB via an intranasal route. The immunological effects of Ad5-CEAB boosted mice primed with BCG were then evaluated using a series of immunological indexes. The results demonstrated that the prime-boost strategy induced a potent antigen-specific immune response, which was primarily characterized by an enhanced T cell response and increased production of cytokines, including interferon-γ, tumor necrosis factor-α and interleukin-2, in mice. In addition, this vaccination strategy was demonstrated to have an elevated humoral response with increased concentrations of antigen-specific bronchoalveolar lavage secretory immunoglobulin (Ig)A and serum IgG in mice compared with those primed with BCG alone. These data suggested that the regimen of subcutaneous BCG prime and mucosal Ad5-CEAB boost was a novel strategy for inducing a broad range of antigen-specific immune responses to Mtb antigens in vivo, which may provide a promising strategy for further development of adenoviral-based vaccine against Mtb infection.

  9. 对嗜T淋巴细胞病毒Ⅰ型重组env的研究%Study on the expression of recombinant antigen env in human T-cell lymphotropic virus type into prokaryotic vector I

    Institute of Scientific and Technical Information of China (English)

    朱庆华; 王梅芬

    2012-01-01

    Objective To investigate the expression of recombinant antigen env in human T-cell lymphotropic virus type I. Methods The target gene of HTLV-I env were analyzed and selected, cloned into prokaryotic vector pQE80L and identified it through PCR and restriction methods. Then the expressed recombinant env protein antigen was induced and purified by affinity chromatography. Western—blot method was adapted to test the activity of the recombinant env antigen expressed by prokaryotic, and ELISA method was adapted to test its specificity. Results The positive recombinants pQE80L-env was selected through PCR amplification and restriction. SDS-PAGE suggested the relative molecular mass of the recombinant protein was approximately 25KDa,which was in line with the expected molecular weight,and Western-blot showed an obvious specific band at 25KDa. The transfected cells were cultured for 48h,then the corresponding SDS-PAGE indicated the relative molecular mass of the recombinant protein was approximately 27KDa,which coincided with the expected molecular weight,and Western-blot revealed an obvious specific band at 27KDa. The reference sera of normal, HIV-positive and HTLV- II -positive people were all detected negative, while HTLV- I -positive people was strongly positive. Conclusion 5915-6545nt area of HTLV-I env gene can be cloned into prokaryotic and eukaryotic vector to express the specific recombinant antigen HTLV-I. There are no significant differences between the two antigens,and it holds the potential to be used as test kits.%目的 探讨人类嗜T淋巴细胞病毒( Human T-cell Lymphotropic Virus,HTLV)I型重组env抗原的表达.方法 分析和选择HTLV-Ⅰ env目的基因,将目的基因片段分别克隆人原核表达载体pQE80L,PCR和酶切鉴定重组子,诱导并亲和层析纯化表达重组env蛋白抗原,Western-blot检测重组env蛋白抗原活性,ELISA方法测试重组env蛋白抗原的特异性.结果 PCR扩增和酶切筛

  10. Interleukin- 2 production during murine infection by Leishmania mexicana amazonensis

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    Manoel Barral-Netto

    1986-03-01

    Full Text Available Highly susceptible BALB/c mice, resistant C57B1/6 and their F1 progeny (BDF1 were infected subcutaneously in the foot pad with Leishmania mexicana amazonenesis. At various times after infection, spleen or draining popliteal lymph node cells were assayed for their capacity to generate Interleukin-2 (I1-2 by Concanavalin A (ConA stimulation. In both BALB/c and C57B1/6 strains there was a transient increase in their capacity to produce I1-2, from the 3rd to the 10th week post-infection. Return to pre-infection levels ocurred between 13th to 16th week post-infection in all three strains. BALB/c mice always produced higher titers of 11-2 than C57B1/6, but such differences were statistically significant only at 3 and 10 weeks post-infection. BDF1 mice had titers similar to those observed in BALB/c mice. I1-2 production by ConA-stimulated lymph node cells was lower as compared to the spleen, but with a similar pattern among the three mice strains. Our data show that susceptibility to infection by l. mexicana amazonenesis is not associated with deficient ConA-stimulated I1-2 production.Camundongos BALB/c (susceptíveis, C57B1/6 (resistentes ou sua geração F1 (BDF foram infectados subcutaneamente na pata traseira com Leishmania mexicana amazonensis. Avaliamos, em diferentes períodos de infecção, a capacidade de células do baço ou de linfonodo poplíteo, de produzir Interleucina-2 (I1-2 em resposta à estimulação por Conconavalina A (ConA. Nos camundongos BALB/c e C57B1/6 observamos, da 3ª à 10ª semana pós-infecção transitória da capacidade de produzir I1-2. Da 13ª à 16ª semana pós-infecção houve um retorno dos níveis de produção pré-infecção. Camundongos BALB/c produziram títulos mais elevados de I2- que os C57B1/6, mas tais diferenças só foram estatisticamente significantes na 3ª e 10ª semanas pós-infecção. Camundongos BDF1 apresentaram títulos semelhantes aos dos BALB/c. Os níveis de I1-2 (estimulada por Con

  11. 泰勒焦虫重组蛋白 TaSP-Tams1-SPAG1间接ELISA 检测方法的建立%Development of ELISA with Recombinant Protein Antigens for Theileria annulata

    Institute of Scientific and Technical Information of China (English)

    魏玉荣; 吴国梁; 祁巧芬; 埃尼瓦尔·太外库力; 黄炯; 易忠; 马文戈; 郭会玲; 向银珍; 米晓云; 陈智; 魏婕; 苗书魁

    2013-01-01

    以真核表达的焦虫表面抗原重组蛋白 TaSP-Tams1-SPAG1作为 ELISA 板包被抗原,通过方阵实验确定抗原包被浓度,探索抗原包被时间、温度、一抗及二抗血清稀释度及作用时间,建立牛环形泰勒焦虫表面抗原重组蛋白 ELISA 方法.结果得出 TaSP-Tams1-SPAG1的最佳包被浓度为10μg/mL,血清最佳稀释度为1∶100, ELISA 阳性反应的临界值为 OD 492nm ≥0.348,重复试验变异系数小于12%,所建立的 ELISA 方法不与牛巴贝斯虫及牛瑟氏泰勒虫血清发生反应.TaSP-Tams1-SPAG1为抗原建立的 ELISA 方法特异性强,敏感度高,重复性好,为牛环形泰勒焦虫病的准确检测及防治提供技术支持.%The objective of this paper was to discuss the antigen coating time,temperature,serum dilution of first antibody and second antibody and action time,and develop antigen recombinant protein ELISA method against Theileria annulata in cattle,to determine antigen coating concentration on the square ex-periment by using antigen recombinant protein TaSP-Tams1-SPAG1 against Theileria annulata expressed by eukarya as ELISA coating antigen.The results indicated that the optimal concentration of coating anti-gen was 10 μg/mL and the optimal blocking dilution of serum sample was 1 ∶100 in the cross assay.The critical value reacted by ELISA positive was an OD 492nm ≥ 0.348.The variation coefficient of intra-batch and the inter-batch in the repeating tests was less than 12%.No cross reactions were found among the piro-plasms.The improved indirect TaSP-Tams1-SPAG1 ELISA Was highly sensitive specific and reproducible, which provided a technical basis for accurate detection and control of Theileria annulata diseases in cattle.

  12. Propolis reduces Leishmania amazonensis-induced inflammation in the liver of BALB/c mice.

    Science.gov (United States)

    da Silva, Suelen S; Mizokami, Sandra S; Fanti, Jacqueline R; Miranda, Milena M; Kawakami, Natalia Y; Teixeira, Fernanda Humel; Araújo, Eduardo J A; Panis, Carolina; Watanabe, Maria A E; Sforcin, José M; Pavanelli, Wander R; Verri, Waldiceu A; Felipe, Ionice; Conchon-Costa, Ivete

    2016-04-01

    Experimental models of mouse paw infection with L. amazonensis show an induction of a strong inflammatory response in the skin, and parasitic migration may occur to secondary organs with consequent tissue injury. There are few studies focusing on the resolution of damage in secondary organs caused by Leishmania species-related cutaneous leishmaniasis. We investigated the propolis treatment effect on liver inflammation induced by Leishmania amazonensis infection in the mouse paw. BALB/c mice were infected in the hind paw with L. amazonensis (10(7)) promastigote forms. After 15 days, animals were treated daily with propolis (5 mg/kg), Glucantime (10 mg/kg), or with propolis plus Glucantime combined. After 60 days, mice were euthanized and livers were collected for inflammatory process analysis. Liver microscopic analysis showed that propolis reduced the inflammatory process compared to untreated infected control. There was a decrease of liver myeloperoxidase and N-acetyl-β-glucosaminidase activity levels, collagen fiber deposition, pro-inflammatory cytokine production, and plasma aspartate transaminase and alanine transaminase levels. Furthermore, propolis treatment enhanced anti-inflammatory cytokine levels and reversed hepatosplenomegaly. Our data demonstrated that daily low doses of Brazilian propolis reduced the secondary chronic inflammatory process in the liver caused by L. amazonensis subcutaneous infection in a susceptible mice strain.

  13. Heme uptake by Leishmania amazonensis is mediated by the transmembrane protein LHR1.

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    Chau Huynh

    Full Text Available Trypanosomatid protozoan parasites lack a functional heme biosynthetic pathway, so must acquire heme from the environment to survive. However, the molecular pathway responsible for heme acquisition by these organisms is unknown. Here we show that L. amazonensis LHR1, a homolog of the C. elegans plasma membrane heme transporter HRG-4, functions in heme transport. Tagged LHR1 localized to the plasma membrane and to endocytic compartments, in both L. amazonensis and mammalian cells. Heme deprivation in L. amazonensis increased LHR1 transcript levels, promoted uptake of the fluorescent heme analog ZnMP, and increased the total intracellular heme content of promastigotes. Conversely, deletion of one LHR1 allele reduced ZnMP uptake and the intracellular heme pool by approximately 50%, indicating that LHR1 is a major heme importer in L. amazonensis. Viable parasites with correct replacement of both LHR1 alleles could not be obtained despite extensive attempts, suggesting that this gene is essential for the survival of promastigotes. Notably, LHR1 expression allowed Saccharomyces cerevisiae to import heme from the environment, and rescued growth of a strain deficient in heme biosynthesis. Syntenic genes with high sequence identity to LHR1 are present in the genomes of several species of Leishmania and also Trypanosoma cruzi and Trypanosoma brucei, indicating that therapeutic agents targeting this transporter could be effective against a broad group of trypanosomatid parasites that cause serious human disease.

  14. Reprogramming neutral lipid metabolism in mouse dendritic leucocytes hosting live Leishmania amazonensis amastigotes.

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    Hervé Lecoeur

    Full Text Available BACKGROUND: After loading with live Leishmania (L amazonensis amastigotes, mouse myeloid dendritic leucocytes/DLs are known to undergo reprogramming of their immune functions. In the study reported here, we investigated whether the presence of live L. amazonensis amastigotes in mouse bone marrow-derived DLs is able to trigger re-programming of DL lipid, and particularly neutral lipid metabolism. METHODOLOGY/PRINCIPAL FINDINGS: Affymetrix-based transcriptional profiles were determined in C57BL/6 and DBA/2 mouse bone marrow-derived DLs that had been sorted from cultures exposed or not to live L. amazonensis amastigotes. This showed that live amastigote-hosting DLs exhibited a coordinated increase in: (i long-chain fatty acids (LCFA and cholesterol uptake/transport, (ii LCFA and cholesterol (re-esterification to triacyl-sn-glycerol (TAG and cholesteryl esters (CE, respectively. As these neutral lipids are known to make up the lipid body (LB core, oleic acid was added to DL cultures and LB accumulation was compared in live amastigote-hosting versus amastigote-free DLs by epi-fluorescence and transmission electron microscopy. This showed that LBs were both significantly larger and more numerous in live amastigote-hosting mouse dendritic leucocytes. Moreover, many of the larger LB showed intimate contact with the membrane of the parasitophorous vacuoles hosting the live L. amazonensis amastigotes. CONCLUSIONS/SIGNIFICANCE: As leucocyte LBs are known to be more than simple neutral lipid repositories, we set about addressing two related questions. Could LBs provide lipids to live amastigotes hosted within the DL parasitophorous vacuole and also deliver? Could LBs impact either directly or indirectly on the persistence of L. amazonensis amastigotes in rodent skin?

  15. Leishmania donovani Nucleoside Hydrolase Terminal Domains in Cross-Protective Immunotherapy Against Leishmania amazonensis Murine Infection

    Science.gov (United States)

    Nico, Dirlei; Gomes, Daniele Crespo; Palatnik-de-Sousa, Iam; Morrot, Alexandre; Palatnik, Marcos; Palatnik-de-Sousa, Clarisa Beatriz

    2014-01-01

    Nucleoside hydrolases of the Leishmania genus are vital enzymes for the replication of the DNA and conserved phylogenetic markers of the parasites. Leishmania donovani nucleoside hydrolase (NH36) induced a main CD4+ T cell driven protective response against L. chagasi infection in mice which is directed against its C-terminal domain. In this study, we used the three recombinant domains of NH36: N-terminal domain (F1, amino acids 1–103), central domain (F2 aminoacids 104–198), and C-terminal domain (F3 amino acids 199–314) in combination with saponin and assayed their immunotherapeutic effect on Balb/c mice previously infected with L. amazonensis. We identified that the F1 and F3 peptides determined strong cross-immunotherapeutic effects, reducing the size of footpad lesions to 48 and 64%, and the parasite load in footpads to 82.6 and 81%, respectively. The F3 peptide induced the strongest anti-NH36 antibody response and intradermal response (IDR) against L. amazonenis and a high secretion of IFN-γ and TNF-α with reduced levels of IL-10. The F1 vaccine, induced similar increases of IgG2b antibodies and IFN-γ and TNF-α levels, but no IDR and no reduction of IL-10. The multiparameter flow cytometry analysis was used to assess the immune response after immunotherapy and disclosed that the degree of the immunotherapeutic effect is predicted by the frequencies of the CD4+ and CD8+ T cells producing IL-2 or TNF-α or both. Total frequencies and frequencies of double-cytokine CD4 T cell producers were enhanced by F1 and F3 vaccines. Collectively, our multifunctional analysis disclosed that immunotherapeutic protection improved as the CD4 responses progressed from 1+ to 2+, in the case of the F1 and F3 vaccines, and as the CD8 responses changed qualitatively from 1+ to 3+, mainly in the case of the F1 vaccine, providing new correlates of immunotherapeutic protection against cutaneous leishmaniasis in mice based on T-helper TH1 and CD8+ mediated immune responses

  16. HIV-1 Gp41 recombinant antigen: expression and immunoreactivity analysis%HIV-1gp41重组抗原的表达及其免疫反应性检测

    Institute of Scientific and Technical Information of China (English)

    张昕; 梁权; 王缦

    2012-01-01

    为了在大肠杆菌中表达具有良好免疫反应性的HIV-1 gp41重组抗原,本实验运用基因工程技术,经PCR扩增gp41的主要抗原表位序列,BamH I、XhoI双酶切后与E3质粒连接,转化克隆宿主菌DH5α,再提取重组质粒进一步转化表达宿主菌BL21(DE3),经IPTG诱导表达重组蛋白,纯化后标记HRP,通过双抗原夹心酶联免疫方法检测其免疫反应性和特异性.结果表明,获得的HIV-1 gp41重组抗原能够与相应抗体特异性结合,与多种无关抗体间无交叉反应,对825份HIV阴性标本检测无错检.检测结果说明该重组抗原具有良好的免疫反应性,在HIV-1抗体诊断试剂中具有潜在的应用价值,为进一步研究gp41抗原奠定了基础.%To express HIV-1 transmembrane glycoprotein gp41 that has fine immunoreactivity in prokaryotic system of E. coli, the target DNA sequence encoding for major epitopes of HIV-1 gp41 was amplified through PCR. The product was digested by BamH I and Xho I , and inserted into E3 vector. Then the joint product was transported into cloning host DH5a from which we extracted recombinant plasmid that was further transported into BL21(DE3) host. The recombinant protein was expressed when induced with IPTG. And after being purified and labelled with HRP, the immunoreactivity and specificity of the protein was evaluated through double-antigen sandwich ELISA. Results proved that, the HIV-1 gp41 recombinant antigen bound corresponding antibody specifically, and had no cross reactivity with various irrelevant antibodies and showed no false positive results when testing with 825 HIV negative specimens. So, this recombinant antigen has fine immunoreactivity and potentcial application value in HIV antibody detection kit.

  17. Immunogenicity and antigenicity of the recombinant EMA-1 protein of Theileria equi expressed in the yeast Pichia pastoris Imunogenicidade e antigenicidade da proteína recombinante EMA-1 de Theileria equi expressa em Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Leandro Q. Nizoli

    2009-06-01

    Full Text Available The equine piroplasmosis caused by Theileria equi is one of the most important parasitic diseases of the equine, causing damage to animal health and economic losses. In T. equi, 2 merozoite surface proteins, equi merozoite antigen EMA-1 and EMA-2, have been identified as the most immunodominant antigens. This suggests that these antigens might be used as immunobiological tools. The EMA-1 of Theileria equi was cloned and expressed in the yeast Pichia pastoris. The transformed yeast was grown at high cell density, expressing up to 389 mg.L-1 of recombinant protein. The protein was concentrated and detected in Dot blot. The recombinant product was antigenically similar to the native protein as determined using monoclonal antibodies, and polyclonal antibodies obtained from equines naturally infected with T. equi. The immunogenicity of rEMA-1 protein was demonstrated by IFAT using sera from recombinant-protein-immunized mice using aluminum hydroxide as adjuvant. All animals vaccinated with rEMA-1 developed a high specific antibody response. This results suggest that rEMA-1expressed in P. pastoris might be a strong candidate to be used as an antigen for immune diagnostics as well as a vaccine antigen.A piroplasmose equina causada por Theileria equi é uma das mais importantes doenças parasitárias de equídeos, causando danos a saúde animal e perdas econômicas. Em T. equi, 2 proteínas de superfície de merozoítos, equi merozoite antigen EMA-1 e EMA-2, têm sido identificadas como antígenos imunodominantes. Sugerindo que estes antígenos possam ser usados como produtos imunobiológicos. O gene EMA-1 de T. equi foi clonado e expressado na levedura Pichia pastoris. As leveduras transformadas foram cultivadas a altas densidades celulares expressando 389 mg.L-1 de proteína recombinante. A proteína foi concentrada e detectada em Dot blot. O produto recombinante foi antigenicamente similar à proteína nativa quando determinado usando anticorpo

  18. Recombinant Adenovirus Delivery of Calreticulin-ESAT-6 Produces an Antigen-Specific Immune Response but no Protection Against a Mycobacterium Tuberculosis Challenge

    NARCIS (Netherlands)

    Esparza-Gonzalez, S. C.; Troy, A.; Troudt, J.; Loera-Arias, M. J.; Villatoro Hernandez, Julio; Torres-Lopez, E.; Ancer-Rodriguez, J.; Gutierrez-Puente, Y.; Munoz-Maldonado, G.; Saucedo-Cardenas, O.; Montes-de-Oca-Luna, R.; Izzo, A.

    2012-01-01

    Bacillus CalmetteGuerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuber

  19. Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.

    Science.gov (United States)

    Crestani, Sandra; Leitolis, Amanda; Lima, Lucianna Freitas Oliveira; Krieger, Marco A; Foti, Leonardo

    2016-08-01

    Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI.

  20. Cloning and expression of merozoite surface antigen 2 locus(MSA-2c) of Babesia bovis isolated in Xinjiang and antigenicity of its recombinant protein%牛巴贝斯虫新疆株MSA-2c基因的克隆表达及其重组蛋白免疫原性

    Institute of Scientific and Technical Information of China (English)

    简子健; 袁江玲; 马素贞; 黄家雨; 沈炯玉

    2009-01-01

    本试验将新疆株牛巴贝斯虫的MSA-2c基因克隆,并构建重组质粒pGEX-4T-2/MSA-2c.利用大肠杆菌原核表达系统进行外源蛋白的表达,并成功诱导出GST-MSA-2c融合蛋白.通过优化诱导条件,得到了较高的可溶性表达;利用亲和层析技术纯化后的重组蛋白皮下免疫试验小鼠.间接ELISA检测发现,免疫接种56 d后,抗重组蛋白的抗体效价达到1∶220 000以上.用获得的抗血清进行Western-blot试验,可获得清晰的免疫反应条带.结果表明,本试验所表达的MSA-2c蛋白与文献报道的目的蛋白相符,并具有免疫原性.同时本试验也为建立以MSA-2c重组蛋白作为抗原的血清学诊断体系奠定了基础.%The merozoite surface antigen 2 locus gene(MSA-2c) of Babesia bovis found in Xinjiang was cloned and ligated into pGEX-4T-2 to construct a recombinant plasmid,pGEX-4T-2/MSA-2c.A fusion protein,GST-MSA-2c,was successfully expressed through prokaryotic expression system in E.coli.The high quantity of soluble expression was obtained by improving the induction conditions.The recombinant protein purified by affinity chromatography was inoculated into mice subcutaneously.The antibody titers of the recombinant protein in murine sera were above 1∶220 000 at 56 days postinoculation by indirect ELISA.The antisera were then examined by Western-blot and a clear reaction strip was found.The results showed that of the expressed MSA-2c protein was identified with interest protein reported in records,and was highly immunogenic(antigenic).It laysa foundation of construction serologically diagnostic system using MSA-2c recombinant protein as a diagnostic antigen.

  1. INTRACELLULAR Leishmania amazonensis KILLING INDUCED BY THE GUANINE NUCLEOSIDE 8-BROMOGUANOSINE

    Directory of Open Access Journals (Sweden)

    GIORGIO Selma

    1998-01-01

    Full Text Available In this study we investigated the effect of 8-Bromoguanosine, an immunostimulatory compound, on the cytotoxicity of macrophages against Leishmania amazonensis in an in vitro system. The results showed that macrophages treated with 8-Bromoguanosine before or after infection are capable to reduce parasite load, as monitored by the number of amastigotes per macrophage and the percentage of infected cells (i.e. phagocytic index. Since 8-Bromoguanosine was not directly toxic to the promastigotes, it was concluded that the ribonucleoside induced macrophage activation. Presumably, 8-Bromoguanosine primed macrophages by inducing interferon alpha and beta which ultimately led to L. amazonensis amastigote killing. The results suggest that guanine ribonucleosides may be useful to treat infections with intracellular pathogens.

  2. Eupomatenoid-5 Isolated from Leaves of Piper regnellii Induces Apoptosis in Leishmania amazonensis

    Directory of Open Access Journals (Sweden)

    Francielle Pelegrin Garcia

    2013-01-01

    Full Text Available Leishmania spp. are protozoa responsible for leishmaniasis, a neglected disease that kills up to 50,000 people every year. Current therapies mainly rely on antimonial drugs that are inadequate because of their poor efficacy and safety and increased drug resistance. An urgent need exists to find new and more affordable drugs. Our previous study demonstrated the antileishmanial activity of eupomatenoid-5, a neolignan obtained from leaves of Piper regnellii var. pallescens. The aim of the present study was to clarify the mode of action of eupomatenoid-5 against L. amazonensis. We used biochemical and morphological techniques and demonstrated that eupomatenoid-5 induced cell death in L. amazonensis promastigotes, sharing some phenotypic features observed in metazoan apoptosis, including increased reactive oxygen species production, hypopolarization of mitochondrial potential, phosphatidylserine exposure, decreased cell volume, and G0/G1 phase cell cycle arrest.

  3. Leishmania amazonensis Promastigotes Present Two Distinct Modes of Nucleus and Kinetoplast Segregation during Cell Cycle

    Science.gov (United States)

    da Silva, Marcelo Santos; Monteiro, Jomar Patrício; Nunes, Vinícius Santana; Vasconcelos, Elton José; Perez, Arina Marina; Freitas-Júnior, Lúcio de Holanda; Elias, Maria Carolina; Cano, Maria Isabel Nogueira

    2013-01-01

    Here, we show the morphological events associated with organelle segregation and their timing in the cell cycle of a reference strain of Leishmania (L.) amazonensis promastigotes, the main causative agent of Tegumentary leishmaniasis in the Americas. We show evidences that during the cell cycle, L. amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation, which occur in different temporal order in different proportions of cells. We used DAPI-staining and EdU-labeling to monitor the segregation of DNA-containing organelles and DNA replication in wild-type parasites. The emergence of a new flagellum was observed using a specific monoclonal antibody. The results show that L. amazonensis cell cycle division is peculiar, with 65% of the dividing cells duplicating the kinetoplast before the nucleus, and the remaining 35% doing the opposite or duplicating both organelles concomitantly. In both cases, the new flagellum appeared during S to G2 phase in 1N1K cells and thus before the segregation of both DNA-containing organelles; however, we could not determine the exact timing of flagellar synthesis. Most of these results were confirmed by the synchronization of parasites using hydroxyurea. Altogether, our data show that during the cell cycle of L. amazonensis promastigotes, similarly to L. donovani, the segregation of nucleus and kinetoplast do not follow a specific order, especially when compared to other trypanosomatids, reinforcing the idea that this characteristic seems to be species-specific and may represent differences in cellular biology among members of the Leishmania genus. PMID:24278433

  4. Bioactivity of flavonoids isolated from Lychnophora markgravii against Leishmania amazonensis amastigotes.

    Science.gov (United States)

    Salvador, Marcos José; Sartori, Fabiana Terezinha; Sacilotto, Ana Claudia B C; Pral, Elizabeth M F; Alfieri, Silvia Celina; Vichnewski, Walter

    2009-01-01

    The bioactivity of the flavonoids pinostrobin (1), pinocembrin (2), tectochrysin (3), galangin 3-methyl ether (4), and tiliroside (5) isolated from Lychnophora markgravii aerial parts was investigated in vitro against amastigote stages of Leishmania amazonensis. The compounds were isolated by several chromatographic techniques and their chemical structures were established by ESI-MS and NMR spectroscopic data. The flavonoids 1 and 3 were the most active compounds; they markedly reduced the viability of Leishmania amastigotes.

  5. Leishmania in synanthropic rodents (Rattus rattus): new evidence for the urbanization of Leishmania (Leishmania) amazonensis.

    Science.gov (United States)

    Caldart, Eloiza Teles; Freire, Roberta Lemos; Ferreira, Fernanda Pinto; Ruffolo, Bruno Bergamo; Sbeghen, Mônica Raquel; Mareze, Marcelle; Garcia, João Luis; Mitsuka-Breganó, Regina; Navarro, Italmar Teodorico

    2017-02-06

    This study aimed to detect parasites from Leishmania genus, to determine the prevalence of anti-Leishmania spp. antibodies, to identify circulating species of the parasite, and to determine epidemiological variables associated with infection in rats caught in urban area of Londrina, Paraná, Brazil. Animal capture was carried out from May to December 2006, serological and molecular methods were performed. DNA was extracted from total blood, and nested-PCR, targeting SSu rRNA from Leishmania genus, was performed in triplicate. The positive samples were sequenced twice by Sanger method to species determination. In total, 181 rodents were captured, all were identified as Rattus rattus and none showed clinical alterations. Forty-one of the 176 (23.3%) animals were positive for Leishmania by ELISA and 6/181 (3.3%) were positive by IFAT. Nine of 127 tested animals (7.1%) were positive by PCR; seven were identified as L. (L.) amazonensis, one as L. (L.) infantum. Four rats were positive using more than one test. This was the first description of synanthropic rodents naturally infected by L. (L.) amazonensis (in the world) and by L. (L.) infantum (in South Brazil). Regarding L. (L.) amazonensis, this finding provides new evidence of the urbanization of this etiological agent.

  6. Activity of the Lupane isolated from Combretum leprosum against Leishmania amazonensis promastigotes

    Energy Technology Data Exchange (ETDEWEB)

    Teles, Carolina B.G.; Silva-Jardim, Izaltina; Silva, Alexandre de A.E.; Zuliani, Juliana P.; Stabeli, Rodrigo G., E-mail: izaltina.jardim@pq.cnpq.b [Instituto de Pesquisas em Patologias Tropicais de Rondonia (IPEPATRO), Porto Velho, RO (Brazil); Moreira, Leandro S.; Facundo, Valdir A. [Universidade Federal de Rondonia, Porto Velho, RO (Brazil)

    2011-07-01

    This paper describes the activity of the ethanolic extract (EE), obtained from the fruits of Combretum leprosum, the triterpene 3{beta}, 6{beta}, 16{beta}-trihydroxylup-20(29)-ene (1) and its synthetic derivatives 1a-1d on Leishmania Amazonensis promastigotes. The EE displayed leishmanicidal activity and the IC{sub 50} was 24.8 {mu}g mL{sup -1}. However, the triterpene 3{beta}, 6{beta}, 16{beta}-trihydroxylup-20(29)-ene (1), at a concentration of 5.0 {mu}g mL{sup -1}, showed a potent inhibitory activity on promastigotes proliferation (IC{sub 50} = 3.3 {mu}g mL{sup -1}). Among the synthetic derivatives, only (1b) and (1d) were active against promastigotes (IC{sub 50} = 3.48 {mu}g mL{sup -1} and 5.8 {mu}g mL{sup -1}, respectively). Moreover, the synthetic derivative 1a showed no activity on promastigotes of L. Amazonensis. EE, (1) and the synthetic derivatives 1a-1d showed no cytotoxic effect on mice peritoneal macrophages. These results provide evidence that the ethanolic extract and the lupane isolated from C. leprosum was active against promastigotes of L. amazonensis, and may be used as a tool in the studies of new antileishmanial drugs. (author)

  7. 猪囊尾蚴重组抗原和基因工程疫苗的研究进展%Research Progress for Recombinant Antigen and Genetic Engineering Vaccine of Cysticercosis

    Institute of Scientific and Technical Information of China (English)

    冯金瑞; 刘立军

    2012-01-01

    猪囊尾蚴病(Cysticercosis)是由猪带绦虫的幼虫囊尾蚴寄生于人或猪等而引起的人畜共患寄生虫病,是公认的世界经济病之一。严重威胁着人体健康,并给畜牧业造成重大经济损失,猪囊尾蚴病的免疫防治势在必行。然而在猪囊尾蚴病疫苗研究中,疫苗抗原的选择和来源一直困扰着兽医工作者。该文就近年来猪囊尾蚴病诊断重组抗原和基因工程疫苗的分子生物学研究进展进行了综述。%Cysticercosis,which is known as a social-economic disease in the world,is an important zoonosis infecting human and pig caused by Taenia solium larval Cysticercus cellulosae.This disease must be prevented for its influence on international competition of the meat product and the great economic loss.However,the choice and source of vaccinal antigen always puzzles veterinarians.This paper reviewed recent research progress in molecular biology of vaccine for Recombinant antigen and genetic engineering vaccine of cysticercosis.

  8. A novel recombinant Mycobacterium bovis bacillus Calmette-Guerin strain expressing human granulocyte macrophage colony-stimulating factor and Mycobacterium tuberculosis early secretory antigenic target 6 complex augments Th1 immunity

    Institute of Scientific and Technical Information of China (English)

    Xiaoling Yang; Lang Bao; Yihao Deng

    2011-01-01

    Since Mycobacterium bovis bacillus Calmette-Guerin strain (BCG) fails to protect adults from pulmonary tuberculosis (TB), there is an urgent need for developing a new vaccine. In this study, we constructed a novel recombinant BCG strain (rBCG) expressing human granulocyte macrophage colony-stimulating factor (GM-CSF) and the 6 kDa early secretory antigenic target (ESAT6) of Mycobacteriutn tuberculosis, named rBCG:GE (expressing GMCSFESAT6 complex), and evaluated the immunogenicity of the construct in BALB/c mice. Our results indicated that the rBCG:GE was able to induce higher titer of antibody than the conventional BCG, the rBCG:G (expressing GM-CSF)and the rBCG:E (expressing ESAT6). Moreover, the rBCG:GE also elicited a longer-lasting and stronger Thl cellular immune responses than the other groups, which was confirmed by the incremental proliferation of splenocytes, the increased percentages of CD4+ and CD8+ T cells of spleen, the elevated level of interferon-γ in splenocyte culture after tuberculin-purified protein derivative stimulation, and the increased concentration of GM-CSF in serum. The data presented here suggested the possibility that the recombinant BCG:GE might be a good vaccine candidate to TB.

  9. Advances in the study of multivalent recombinant DNA vaccines utilizing the hepatitis B virus surface antigen gene%乙肝表面抗原载体多价重组核酸疫苗研究进展

    Institute of Scientific and Technical Information of China (English)

    肖婷; 郭根灵; 辛宪云; 魏庆宽

    2012-01-01

    研究表明,乙肝病毒的包膜蛋白HBsAg不仅可以作为疫苗的理想候选分子,还可作为基因工程疫苗的理想载体,用来成功构建多种重组核酸疫苗.本文概述了以乙肝表面抗原为载体,重组或联合其他病毒、寄生虫、细胞因子等其他基因制作多价核酸疫苗的研究进展.%Studies have shown that hepatitis B virus envelope protein HBsAg can be used as an ideal candidate molecule for vaccines and also as an ideal vehicle for genetically engineered vaccines to successfully build a variety of recombinant DNA vaccines. This article provides an overview of advances in recombinant DNA vaccines prepared by using hepatitis B virus surface antigen as a carrier to restructure or join it to other viruses, parasites, cell factors, or other genes.

  10. Recombinant Rhipicephalus appendiculatus gut (Ra86 and salivary gland cement (Trp64 proteins as candidate antigens for inclusion in tick vaccines: protective effects of Ra86 on infestation with adult R. appendiculatus

    Directory of Open Access Journals (Sweden)

    Saimo M

    2011-11-01

    Full Text Available Margaret Saimo1,2,*, David O Odongo3,4,*, Stephen Mwaura3, Just M Vlak1, Anthony J Musoke5, George W Lubega2, Richard P Bishop3, Monique M van Oers11Laboratory of Virology, Wageningen University, Wageningen, The Netherlands; 2School of Veterinary Medicine, Makerere University, Kampala, Uganda; 3International Livestock Research Institute, Nairobi, Kenya; 4School of Biological Sciences, University of Nairobi, Nairobi, Kenya; 5Onderstepoort Veterinary Institute, Onderstepoort, Pretoria, South Africa *These two authors made an equal contribution to this workAbstract: Rhipicephalus appendiculatus gut protein Ra86 (variants Ra85A and Ra92A and the salivary gland cement protein (Trp64 were expressed in the baculovirus-insect cell system. The recombinant gut proteins expressed as soluble proteins and the recombinant cement protein, as insoluble inclusion bodies, were used to immunize rabbits, which were then challenged with larval, nymphal, and adult stages of R. appendiculatus ticks. High tick mortality (23.3% occurred on adult ticks that fed on rabbits vaccinated with the gut proteins, compared with 1.9% mortality in ticks that fed on unvaccinated naïve control rabbits. The mean weight of engorged female ticks was significantly reduced by 31.5% in rabbits vaccinated with the Ra86 recombinant protein compared with controls, as was egg production. Marked effects on these parameters were also observed in adult ticks as a result from vaccination using Trp64, but these were not statistically significant. For both antigens, there was no demonstrable effect on larval or nymphal ticks. This study demonstrates for the first time the protective efficacy of a homolog of Boophilus microplus Bm86 in reducing tick infestation by the adult stage of the three-host tick R. appendiculatus. The results demonstrate the potential of Ra86 for vaccine development against this tick and for the control of East Coast fever.Keywords: baculovirus, Ra85A, Ra92A, Boophilus

  11. Vaccination against schistosomiasis and fascioliasis with the new recombinant antigen Sm14: potential basis of a multi-valent anti-helminth vaccine?

    Directory of Open Access Journals (Sweden)

    Miriam Tendler

    1995-04-01

    Full Text Available Molecular cloning of components of protective antigenic preparations have suggested that related parasite fatty acid binding proteins could form the basis of the well documented protective, immune cross reactivity between the parasitic trematode worms Fasciola hepatica and Schistosoma mansoni. We have now confirmed the cross protective potential of parasite fatty acid binding proteins and suggest that it may be possible to produce a single vaccine that would be effective against at least two parasites, F. hepatica and S. mansoni of veterinary and human importance respectively.

  12. Human T Cell and Antibody-Mediated Responses to the Mycobacterium tuberculosis Recombinant 85A, 85B, and ESAT-6 Antigens

    Directory of Open Access Journals (Sweden)

    Gilson C. Macedo

    2011-01-01

    Full Text Available Tuberculosis remains a major health problem throughout the world causing large number of deaths. Effective disease control and eradication programs require the identification of major antigens recognized by the protective responses against M. tuberculosis. In this study, we have investigated humoral and cellular immune responses to M. tuberculosis-specific Ag85A, Ag85B, and ESAT-6 antigens in Brazilian patients with pulmonary (P, n=13 or extrapulmonary (EP, n=12 tuberculosis, patients undergoing chemotherapy (PT, n=23, and noninfected healthy individuals (NI, n=7. Compared to NI, we observed increased levels of IgG1 responses to Ag85B and ESAT-6 in P and PT groups. Regarding cellular immunity, Ag85A and ESAT-6 were able to discriminate P, PT, and EP patients from healthy individuals by IFN-γ production and P and PT groups from EP individuals by production of TNF-α. In summary, these findings demonstrate the ability of Ag85A, Ag85B, and ESAT-6 to differentiate TB patients from controls by IgG1, IFN-γ and TNF-α production.

  13. Recombinant ROP2, ROP4, GRA4 and SAG1 antigen-cocktails as possible tools for immunoprophylaxis of toxoplasmosis: what's next?

    Science.gov (United States)

    Dziadek, Bozena; Brzostek, Anna

    2012-01-01

    Toxoplasmosis is a globally distributed foodborne zoonosis caused by a protozoan parasite Toxoplasma gondii. Usually asymptomatic in immunocompetent humans, toxoplasmosis is a serious clinical and veterinary problem often leading to lethal damage in an infected host. In order to overcome the exceptionally strong clinical and socio-economic impact of Toxoplasma infection, the construction of an effective vaccine inducing full immunoprotection against the parasite is an urgent issue. In the last two decades many live attenuated, subunit and DNA-based vaccines against toxoplasmosis have been studied, however only partial protection conferred by vaccination against chronic as well as acute infection has been achieved. Among various immunization strategies, no viable subunit vaccines based on recombinant secretory (ROP2, ROP4 and GRA4) and surface (SAG1) T. gondii proteins have been found as attractive tools for further studies. This is due to their high, but still partial, protective efficacy correlated with the induction of cellular and humoral immune responses.

  14. Recombinant viral-vectored vaccines expressing Plasmodium chabaudi AS apical membrane antigen 1: mechanisms of vaccine-induced blood-stage protection.

    Science.gov (United States)

    Biswas, Sumi; Spencer, Alexandra J; Forbes, Emily K; Gilbert, Sarah C; Holder, Anthony A; Hill, Adrian V S; Draper, Simon J

    2012-05-15

    Apical membrane Ag 1 (AMA1) is one of the leading candidate Ags for inclusion in a subunit vaccine against blood-stage malaria. However, the efficacy of Ab-inducing recombinant AMA1 protein vaccines in phase IIa/b clinical trials remains disappointing. In this article, we describe the development of recombinant human adenovirus serotype 5 and modified vaccinia virus Ankara vectors encoding AMA1 from the Plasmodium chabaudi chabaudi strain AS. These vectors, when used in a heterologous prime-boost regimen in BALB/c mice, are capable of inducing strong transgene-specific humoral and cellular immune responses. We show that this vaccination regimen is protective against a nonlethal P. chabaudi chabaudi strain AS blood-stage challenge, resulting in reduced peak parasitemias. The role of vaccine-induced, AMA1-specific Abs and T cells in mediating the antiparasite effect was investigated by in vivo depletion of CD4(+) T cells and adoptive-transfer studies into naive and immunodeficient mice. Depletion of CD4(+) T cells led to a loss of vaccine-induced protection. Adoptive-transfer studies confirmed that efficacy is mediated by both CD4(+) T cells and Abs functioning in the context of an intact immune system. Unlike previous studies, these results confirm that Ag-specific CD4(+) T cells, induced by a clinically relevant vaccine-delivery platform, can make a significant contribution to vaccine blood-stage efficacy in the P. chabaudi model. Given that cell-mediated immunity may also contribute to parasite control in human malaria, these data support the clinical development of viral-vectored vaccines that induce both T cell and Abs against Plasmodium falciparum blood-stage malaria Ags like AMA1.

  15. Clonging and Expression of Leptospiral Outer Membrane Protein LipL32 Gene and Application of Recombinant Antigen in Enzyme-linked Immunosorbent Assays%钩端螺旋体外膜脂蛋白LipL32基因的克隆和表达及其在ELISA检测中的应用

    Institute of Scientific and Technical Information of China (English)

    范薇; 于长明; 杨敬; 隋丽华; 战大伟; 贺争鸣; 孙岩松

    2003-01-01

    Objective To construct L32-pQE32 recombinant expression vectors, and to induce the expression of recombinant Leptospiral outer membrane protein LipL32. Establish method of recombinant Leptospiral outer membrane proteinbased ELISA. Method Gene coding of Leptospiral LipL32 protein was amplified by PCR, then recombinant cloning vectors pGEM-T/L32 and expression vectors L32-pQE32 were constructed. Recombinant expression vector was transformed into the competent host E. coli. DH-5α and E. coli. M15. Recombinant Leptospiral LipL32 protein was expressed by IPTG induced method. Immulon microtiter plates were coated at 37℃ overnight with 100 ng of purified recombinant protein per well, 3 positive and 4 negative sera were used in indirect ELISA. Results Mature Leptospiral LipL32 gene fragment about 750 bp was amplified by PCR. LipL32 gene was inserted into expression vectors pQE32, the molecular weight of fusion protein was corresponding to the estimated molecular size of mature Leptospiral LipL32 protein. Results of Western-blot and ELISA demonstrated intense LipL32 reactivity with anti-Leptospira sera. Conclusion findings indicate that recombinant Leptospiral LipL32 may be an important, useful antigen for the serodiagnosis of Leptospira.

  16. Two amino acid substitutions in apolipoprotein B are in complete allelic association with the antigen group (x/y) polymorphism: Evidence for little recombination in the 3 prime end of the human gene

    Energy Technology Data Exchange (ETDEWEB)

    Dunning, A.M.; Renges, H.H.; Xu, Chunfang; Peacock, R.; Humphries, S.E.; Talmud, P.; Laxer, G. (Bickbeck Coll., London (England)); Brasseur, R. (Free Univ. of Brussels (Belgium)); Tikkanen, M.J. (Univ. of Helsinki (Switzerland)); Buetler, R. (Swiss Red Cross, Berne (Switzerland)); Saha, N. (National Univ. of Singapore (Singapore)); Hamsten, A. (Karolinska Hospital, Stockholm (Sweden)); Rosseneu, M. (A.Z. St-Jan, Brugge (Belgium))

    1992-01-01

    The authors report the identification of an A-to-G base change, in exon 29 of the apolipoprotein B (apo B) gene, that results in the substitution of serine for asparagine at residue 4,311 of mature apo B100. In a recent publication, Huang et al. have reported a C-to-T base change in exon 26 that causes the substitution of leucine for proline at residue 2712 of apo B. The authors have found complete linkage disequilibrium between the alleles at both these sites and an immunochemical polymorphism of LDL designated antigen group (x/y) (Ag(x/y)) in a sample of 118 Finnish individuals. This implies that either one of these substitutions - or both of them combined - could be the molecular basis of the Ag(x/y) antigenic determinants, with the allele encoding serine{sub 4311} plus leucine{sub 2,712} representing the Ag(x) epitope, and that encoding asparagine{sub 4,311} plus proline{sub 2,712} the Ag(y) epitope. In a sample of 90 healthy Swedish individuals the Leu{sub 2,712}/Ser{sub 4,311} allele is associated both with reduced serum levels of LDL cholesterol and apo B and with raised levels of HDL. They have also genotyped 523 individuals from European, Asian, Chinese, and Afro-Caribbean populations and have found complete association between the sites encoding residues 2,712 and 4,311 in all of these samples, although there are large allele frequency differences between these populations. Taken together, these data suggest that, since the divergence of the major ethnic groups, there has been little or no recombination in the 3' end of the human apo B gene.

  17. New recombinant chimeric antigens, P35-MAG1, MIC1-ROP1, and MAG1-ROP1, for the serodiagnosis of human toxoplasmosis.

    Science.gov (United States)

    Drapała, Dorota; Holec-Gąsior, Lucyna; Kur, Józef

    2015-05-01

    The aim of the study was to evaluate the usefulness of 3 chimeric Toxoplasma gondii antigens, P35-MAG1, MIC1-ROP1 and MAG1-ROP1, in the serodiagnosis of an acute toxoplasmosis in humans. Proteins were produced as fusion proteins containing His tags ends and then further purified by metal affinity chromatography. Their application for the diagnosis of recently acquired T. gondii infection was tested in IgG and IgM enzyme-linked immunosorbent assays (ELISAs). At 100%, 77.3%, and 86.4%, respectively, the reactivity of the IgG ELISA using P35-MAG1, MIC1-ROP1, and MAG1-ROP1 for sera from patients where acute toxoplasmosis was suspected was significantly higher than for the samples from people with a chronic infection, at 26.2%, 36.1%, and 32.8%, respectively. Moreover, P35-MAG1, MIC1-ROP1, and MAG1-ROP1 detected IgM antibodies with a reactivity at 81.8%, 72.7%, and 59.1%, respectively. The results presented in the article show that, particularly, P35-MAG1 may be useful in the preliminary detection of recent T. gondii infection.

  18. Oral effectiveness of PMIC4, a novel hydroxyethylpiperazine analogue, in Leishmania amazonensis

    Directory of Open Access Journals (Sweden)

    Mariela Ferreira de Vasconcelos

    2014-12-01

    Full Text Available Pentavalent antimonials have saved the lives of thousands of Leishmania-infected patients more than seventy years but, unfortunately, they are highly toxic and require parenteral delivery. Therefore, the search for safer and orally delivered alternative is a need. This paper describes the antileishmanial properties of PMIC4, a novel hydroxyethylpiperazine analogue. PMIC4 showed potent activity against intracellular amastigotes of Leishmania amazonensis, with IC50 of 1.8 μM and selectivity index higher than 100-fold, calculated in relation to the toxicity on the host cell. Following laboratory animal welfare policies, we analyzed the absorption, distribution, metabolism, excretion and toxicity (ADMET properties and calculated the Lipinski’s rule of five of PMIC4 before proceeding to in vivo tests. PMIC4 satisfied Lipinski’s rule of five and presented high probability of human intestinal absorption, suggesting a good chance of druglikeness and oral bioavailability. For in vivo studies, PMIC4 was administered via intralesional injection (3.4 mg/kg/day, three times a week or orally (34.0 mg/kg/day, five times a week to L. amazonensis-infected BALB/c mice throughout the 98 day experiments. At the end of the treatment period, serum markers of toxicity were measured. When administered orally, PMIC4 controlled the lesions in L. amazonensis-infected BALB/c mice without altering serological markers of toxicity. These results demonstrate that PMIC4 is a promising molecular scaffold, orally effective against experimental leishmaniasis.

  19. Evaluation of Macroalgae Sulfated Polysaccharides on the Leishmania (L. amazonensis Promastigote

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    Marcos Hikari Toyama

    2013-03-01

    Full Text Available The sulfated polysaccharides from Solieria filiformis (Sf, Botryocladia occidentalis (Bo, Caulerpa racemosa (Cr and Gracilaria caudata (Gc were extracted and extensively purified. These compounds were then subjected to in vitro assays to evaluate the inhibition of these polysaccharides on the growth of Leishmania (L. amazonensis promastigotes. Under the same assay conditions, only three of the four sulfated polysaccharides were active against L. amazonensis, and the polysaccharide purified from Cr was the most potent (EC50 value: 34.5 μg/mL. The polysaccharides derived from Bo and Sf demonstrated moderate anti-leishmanial activity (EC50 values of 63.7 μg/mL and 137.4 μg/mL. In addition, we also performed in vitro cytotoxic assays toward peritoneal macrophages and J774 macrophages. For the in vitro cytotoxicity assay employing J774 cells, all of the sulfated polysaccharides decreased cell survival, with CC50 values of 27.3 μg/mL, 49.3 μg/mL, 73.2 μg/mL, and 99.8 μg/mL for Bo, Cr, Gc, and Sf, respectively. However, none of the sulfated polysaccharides reduced the cell growth rate of the peritoneal macrophages. These results suggest that macroalgae contain compounds with various chemical properties that can control specific pathogens. According to our results, the assayed sulfated polysaccharides were able to modulate the growth rate and cell survival of Leishmania (L. amazonensis promastigotes in in vitro assays, and these effects involved the interaction of the sulfated polysaccharides on the cell membrane of the parasites.

  20. A Trypanosomatid Iron Transporter that Regulates Mitochondrial Function Is Required for Leishmania amazonensis Virulence.

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    Bidyottam Mittra

    2016-01-01

    Full Text Available Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast and mitoferrin-1 (human that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1 showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes.

  1. IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu

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    Flábio R Araújo

    2008-03-01

    Full Text Available Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5. Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu. VirB9, VirB10 are considered part of the type IV secretion system (TFSS, which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.

  2. Synthesis and biological evaluation against Mycobacterium tuberculosis and Leishmania amazonensis of a series of diaminated terpenoids.

    Science.gov (United States)

    Dos Reis, Dijovani Batista; Souza, Thalita Cristina Alves; Lourenço, Maria Cristina S; de Almeida, Mauro Vieira; Barbosa, Adriano; Eger, Iriane; Saraiva, Mauricio Frota

    2016-12-01

    We report the synthesis of a series of diaminated terpenoids containing, as side-chain of the diamine core, the "head-to-tail" prenyl derivatives, with amino amino spacers of variable length. In vitro biological activity of these compounds was evaluated against Mycobacterium tuberculosis and Leishmania amazonensis, and the structure-activity relationships are discussed. Different biological results were observed depending on the terpenic side-chain length. The best results were obtained for trans,trans-farnesol derivatives. Moreover, these results demonstrated that the stereochemistry of the double bond could play an important role in determining antitubercular and antileishmanial activities of these compounds.

  3. Tamoxifen is effective in the treatment of Leishmania amazonensis infections in mice.

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    Danilo C Miguel

    Full Text Available BACKGROUND: Chemotherapy is still a critical issue in the management of leishmaniasis. Until recently, pentavalent antimonials, amphotericin B or pentamidine compounded the classical arsenal of treatment. All these drugs are toxic and have to be administered by the parenteral route. Tamoxifen has been used as an antiestrogen in the treatment and prevention of breast cancer for many years. Its safety and pharmacological profiles are well established in humans. We have shown that tamoxifen is active as an antileishmanial compound in vitro, and in this paper we analyzed the efficacy of tamoxifen for the treatment of mice infected with Leishmania amazonensis, an etiological agent of localized cutaneous leishmaniasis and the main cause of diffuse cutaneous leishmaniasis in South America. METHODOLOGY/PRINCIPAL FINDINGS: BALB/c mice were infected with L. amazonensis promastigotes. Five weeks post-infection, treatment with 15 daily intraperitoneal injections of 20 mg/kg tamoxifen was administered. Lesion and ulcer sizes were recorded and parasite burden quantified by limiting dilution. A significant decrease in lesion size and ulcer development was noted in mice treated with tamoxifen as compared to control untreated animals. Parasite burden in the inoculation site at the end of treatment was reduced from 10(8.5+/-0.7 in control untreated animals to 10(5.0+/-0.0 in tamoxifen-treated mice. Parasite load was also reduced in the draining lymph nodes. The reduction in parasite number was sustained: 6 weeks after the end of treatment, 10(15.5+/-0.5 parasites were quantified from untreated animals, as opposed to 10(5.1+/-0.1 parasites detected in treated mice. CONCLUSIONS/SIGNIFICANCE: Treatment of BALB/c mice infected with L. amazonensis for 15 days with tamoxifen resulted in significant decrease in lesion size and parasite burden. BALB/c mice infected with L. amazonensis represents a model of extreme susceptibility, and the striking and sustained reduction

  4. Subversion of Immunity by Leishmania amazonensis Parasites: Possible Role of Phosphatidylserine as a Main Regulator

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    Joao Luiz Mendes Wanderley

    2012-01-01

    Full Text Available Leishmania amazonensis parasites cause progressive disease in most inbred mouse strains and are associated with the development of diffuse cutaneous leishmaniasis in humans. The poor activation of an effective cellular response is correlated with the ability of these parasites to infect mononuclear phagocytic cells without triggering their activation or actively suppressing innate responses of these cells. Here we discuss the possible role of phosphatidylserine exposure by these parasites as a main regulator of the mechanism underlying subversion of the immune system at different steps during the infection.

  5. Serodiagnosis of Lyme disease by kinetic enzyme-linked immunosorbent assay using recombinant VlsE1 or peptide antigens of Borrelia burgdorferi compared with 2-tiered testing using whole-cell lysates.

    Science.gov (United States)

    Bacon, Rendi Murphree; Biggerstaff, Brad J; Schriefer, Martin E; Gilmore, Robert D; Philipp, Mario T; Steere, Allen C; Wormser, Gary P; Marques, Adriana R; Johnson, Barbara J B

    2003-04-15

    In a study of US patients with Lyme disease, immunoglobulin (Ig) G and IgM antibody responses to recombinant Borrelia burgdorferi antigen VlsE1 (rVlsE1), IgG responses to a synthetic peptide homologous to a conserved internal sequence of VlsE (C6), and IgM responses to a synthetic peptide comprising the C-terminal 10 amino acid residues of a B. burgdorferi outer-surface protein C (pepC10) were evaluated by kinetic enzyme-linked immunoassay. At 99% specificity, the overall sensitivities for detecting IgG antibody to rVlsE1 or C6 in samples from patients with diverse manifestations of Lyme disease were equivalent to that of 2-tiered testing. When data were considered in parallel, 2 combinations (IgG responses to either rVlsE1 or C6 in parallel with IgM responses to pepC10) maintained high specificity (98%) and were significantly more sensitive than 2-tiered analysis in detecting antibodies to B. burgdorferi in patients with acute erythema migrans. In later stages of Lyme disease, the sensitivities of the in parallel tests and 2-tiered testing were high and statistically equivalent.

  6. 弓形虫病免疫诊断用重组抗原及检测方法研究进展%Research progress on recombinant antigen and diagnostic technique for immunological diagnosis of toxoplasmosis

    Institute of Scientific and Technical Information of China (English)

    杨永刚; 曹利民; 朱荫昌

    2010-01-01

    Toxoplasma is a kind of parasitic protozoa that can infect all warm blooded animals including humans and cause toxoplasmosis manifesting various symptoms. Diagnosis for toxoplasmosis include etiology,immunology, and molecular biology methods. Immunological diagnostic methods are common for Toxoplasma infection. This article reviewed research progress on recombinant antigen and diagnostic technique development for immunological diagnosis of toxoplasmosis.%弓形虫是一种寄生原虫,可感染人在内的所有温血动物,并会引发表现为多种症状的弓形虫病.弓形虫病的诊断方法有病原学、免疫学、分子生物学等方法.免疫学诊断是进行弓形虫感染检测的常用方法.该文就弓形虫感染的免疫诊断用重组抗原及检测方法的应用和研究进展作一综述.

  7. Comparing Enterovirus 71 with Coxsackievirus A16 by analyzing nucleotide sequences and antigenicity of recombinant proteins of VP1s and VP4s

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    Sun Yu

    2011-11-01

    Full Text Available Abstract Background Enterovirus 71 (EV71 and Coxsackievirus A16 (CA16 are two major etiological agents of Hand, Foot and Mouth Disease (HFMD. EV71 is associated with severe cases but not CA16. The mechanisms contributed to the different pathogenesis of these two viruses are unknown. VP1 and VP4 are two major structural proteins of these viruses, and should be paid close attention to. Results The sequences of vp1s from 14 EV71 and 14 CA16, and vp4s from 10 EV71 and 1 CA16 isolated in this study during 2007 to 2009 HFMD seasons were analyzed together with the corresponding sequences available in GenBank using DNAStar and MEGA 4.0. Phylogenetic analysis of complete vp1s or vp4s showed that EV71 isolated in Beijing belonged to C4 and CA16 belonged to lineage B2 (lineage C. VP1s and VP4s from 4 strains of viruses expressed in E. coli BL21 cells were used to detect IgM and IgG in human sera by Western Blot. The detection of IgM against VP1s of EV71 and CA16 showed consistent results with current infection, while none of the sera were positive against VP4s of EV71 and CA16. There was significant difference in the positive rates between EV71 VP1 and CA16 VP1 (χ2 = 5.02, P 2 = 15.30, P 2 = 26.47, P 2 = 16.78, P Conclusions EV71 and CA16 were highly diverse in the nucleotide sequences of vp1s and vp4s. The sera positive rates of VP1 and VP4 of EV71 were lower than those of CA16 respectively, which suggested a less exposure rate to EV71 than CA16 in Beijing population. Human serum antibodies detected by Western blot using VP1s and VP4s as antigen indicated that the immunological reaction to VP1 and VP4 of both EV71 and CA16 was different.

  8. Antiproliferative, Ultrastructural, and Physiological Effects of Amiodarone on Promastigote and Amastigote Forms of Leishmania amazonensis

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    Sara Teixeira de Macedo-Silva

    2011-01-01

    Full Text Available Amiodarone (AMIO, the most frequently antiarrhythmic drug used for the symptomatic treatment of chronic Chagas' disease patients with cardiac compromise, has recently been shown to have also specific activity against fungi, Trypanosoma cruzi and Leishmania. In this work, we characterized the effects of AMIO on proliferation, mitochondrial physiology, and ultrastructure of Leishmania amazonensis promastigotes and intracellular amastigotes. The IC50 values were 4.21 and 0.46 μM against promastigotes and intracellular amastigotes, respectively, indicating high selectivity for the clinically relevant stage. We also found that treatment with AMIO leads to a collapse of the mitochondrial membrane potential (ΔΨm and to an increase in the production of reactive oxygen species, in a dose-dependent manner. Fluorescence microscopy of cells labeled with JC-1, a marker for mitochondrial energization, and transmission electron microscopy confirmed severe alterations of the mitochondrion, including intense swelling and modification of its membranes. Other ultrastructural alterations included (1 presence of numerous lipid-storage bodies, (2 presence of large autophagosomes containing part of the cytoplasm and membrane profiles, sometimes in close association with the mitochondrion and endoplasmic reticulum, and (3 alterations in the chromatin condensation and plasma membrane integrity. Taken together, our results indicate that AMIO is a potent inhibitor of L. amazonensis growth, acting through irreversible alterations in the mitochondrial structure and function, which lead to cell death by necrosis, apoptosis and/or autophagy.

  9. Isolation of an enriched plasma membrame subpellicular microtubule fraction of Leishmania mexicana amazonensis

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    Solange L. Timm

    1980-01-01

    Full Text Available A cell fractionation procedure previously developed for Trypanosoma cruzi was applied to isolated the plasma membrane of promastigotes of Leishania mexicana amazonensis. The cell, swollen in an hypotonic mediun, were disrupted in the presence of a nonionic detergent and the membrane fraction isolated by differencial centrifugation. Electron microscopy showed that the fraction consisted of pieces of the plasma membrane associated with subpellicular microtubules. It was also shown that this fraction is able to induce cell-mediated immune response in mice.Um método de fracionamento subcelular, previamente desenvolvido para Trypanosoma cruzi, foi aplicado para isolar a membrana plasmática de promastigotas de Leishmania mexicana amazonensis. As células, após turgimento em meio hipotônico, foram rompidas na presença de um detergente não iônico e a fração de membrana isolada por centrifugação diferencial. A microscopia eletrônica mostrou consistir a fração de fragmentos de membrana plasmática associados com microtúbulos subpeliculares. Foi também mostrado que esta fração era capaz de induzir resposta celular em camundongos.

  10. Natural canine infection by Leishmania infantum and Leishmania amazonensis and their implications for disease control

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    Letícia da Cruz Sanches

    Full Text Available Abstract Leishmaniasis is a major public health problem worldwide. Because Leishmania can adapt to new hosts or vectors, knowledge concerning the current etiological agent in dogs is important in endemic areas. This study aimed to identify the Leishmania species detected in 103 samples of peripheral blood from dogs that were naturally infected with these protozoa. The diagnosis of leishmaniasis was determined through parasitological examination, the indirect enzyme-linked immunosorbent assay (ELISA and the polymerase chain reaction (PCR. The Leishmania species were identified by means of PCR-restriction fragment length polymorphism (PCR-RFLP. The samples were subjected to PCR using oligonucleotide primers that amplify the intergenic region ITS1 of the rRNA gene in order to identify the species. The amplified DNA was digested using the restriction enzyme HaeIII. A restriction profile identical to L. amazonensis was shown in 77/103 samples and the profile was similar to L. infantum in 17/103. However, a mixed profile was shown in 9/103 samples, which impeded species identification. In conclusion, the infection in these dogs was predominantly due to L. amazonensis, thus indicating that diagnosing of cases of canine leishmaniasis needs to be reexamined, since the causative agent identified is not restricted to L. infantum.

  11. Benzaldehyde thiosemicarbazone derived from limonene complexed with copper induced mitochondrial dysfunction in Leishmania amazonensis.

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    Elizandra Aparecida Britta

    Full Text Available BACKGROUND: Leishmaniasis is a major health problem that affects more than 12 million people. Treatment presents several problems, including high toxicity and many adverse effects, leading to the discontinuation of treatment and emergence of resistant strains. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the in vitro antileishmanial activity of benzaldehyde thiosemicarbazone derived from limonene complexed with copper, termed BenzCo, against Leishmania amazonensis. BenzCo inhibited the growth of the promastigote and axenic amastigote forms, with IC(50 concentrations of 3.8 and 9.5 µM, respectively, with 72 h of incubation. Intracellular amastigotes were inhibited by the compound, with an IC(50 of 10.7 µM. BenzCo altered the shape, size, and ultrastructure of the parasites. Mitochondrial membrane depolarization was observed in protozoa treated with BenzCo but caused no alterations in the plasma membrane. Additionally, BenzCo induced lipoperoxidation and the production of mitochondrial superoxide anion radicals in promastigotes and axenic amastigotes of Leishmania amazonensis. CONCLUSION/SIGNIFICANCE: Our studies indicated that the antileishmania activity of BenzCo might be associated with mitochondrial dysfunction and oxidative damage, leading to parasite death.

  12. Transcriptional signatures of BALB/c mouse macrophages housing multiplying Leishmania amazonensis amastigotes

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    Lang Thierry

    2009-03-01

    Full Text Available Abstract Background Mammal macrophages (MΦ display a wide range of functions which contribute to surveying and maintaining tissue integrity. One such function is phagocytosis, a process known to be subverted by parasites like Leishmania (L. Indeed, the intracellular development of L. amazonensis amastigote relies on the biogenesis and dynamic remodelling of a phagolysosome, termed the parasitophorous vacuole, primarily within dermal MΦ. Results Using BALB/c mouse bone marrow-derived MΦ loaded or not with amastigotes, we analyzed the transcriptional signatures of MΦ 24 h later, when the amastigote population was growing. Total RNA from MΦ cultures were processed and hybridized onto Affymetrix Mouse430_2 GeneChips®, and some transcripts were also analyzed by Real-Time quantitative PCR (RTQPCR. A total of 1,248 probe-sets showed significant differential expression. Comparable fold-change values were obtained between the Affymetrix technology and the RTQPCR method. Ingenuity Pathway Analysis software® pinpointed the up-regulation of the sterol biosynthesis pathway (p-value = 1.31e-02 involving several genes (1.95 to 4.30 fold change values, and the modulation of various genes involved in polyamine synthesis and in pro/counter-inflammatory signalling. Conclusion Our findings suggest that the amastigote growth relies on early coordinated gene expression of the MΦ lipid and polyamine pathways. Moreover, these MΦ hosting multiplying L. amazonensis amastigotes display a transcriptional profile biased towards parasite-and host tissue-protective processes.

  13. First Report of Heterorhabditis amazonensis from Venezuela and Characterization of Three Populations.

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    Morales, Naiyulin; Morales-Montero, Patricia; Puza, Vladimir; San-Blas, Ernesto

    2016-09-01

    During a survey in western Venezuela in 2011, three new populations of Heterorhabditis amazonensis (LPV081, LPV156, and LPV498) were isolated. Some differences were found in terms of morphometry compared with the original description; however, the distance from the anterior end to the excretory pore is the most variable character; significantly shorter in all infective juveniles and in other developmental stages depending on the population. According to a Principal Component Analysis, LPV498 possesses more differences in morphometric characteristics and can be separated from the other two. Those intraspecific differences could be attributed to the geographic origin of the nematode. Molecular studies of ITS regions demonstrated that the sequences of the Venezuelan strains were identical to those of the type species originally isolated in the Brazilian Amazonian forest. This is an interesting fact because in several studies on heterorhabditids, intraspecific variability has been recorded. Herein, we present the first report of H. amazonensis in Venezuela and the characterization of three populations of this species.

  14. Development of a Procedure for Preparation of Recombinant Yersinia pestis LcrV Antigen%重组鼠疫耶尔森菌LcrV抗原制备工艺的建立

    Institute of Scientific and Technical Information of China (English)

    胡丽娜; 王秉翔; 常娅莉; 魏东; 万艳; 马维民; 傅林锋; 王革; 吴智远; 韩少波

    2011-01-01

    目的 建立稳定、高效的重组鼠疫耶尔森菌LcrV抗原工程菌的发酵与纯化工艺.方法 研究LcrV工程菌pET-V/BL21 (DE3)在试管和三角摇瓶中的生长和表达规律,对种子培养基、IPTG诱导时机、诱导时间及诱导浓度进行优化,并放大至30 L发酵罐培养,建立稳定的发酵工艺.收获菌体,经冻融、离心收集蛋白溶液,高压匀浆处理后,分别采用Q.Sepharose HP阴离子交换层析、Phenyl Sepharose 6 FF(hs)疏水层析及Superdex 75 pg凝胶过滤层析三步柱层析纯化,建立稳定的纯化工艺,连续纯化3批,并按《中国药典》三部(2010版)的相关要求进行全面检定.结果 经优化的条件培养及诱导表达,工程菌的收菌密度(A600值)可达34,LcrV抗原的表达量达36%,含量为1.6 g/L.发酵产物经柱层析纯化,LcrV产量高于140 mg,纯度大于95%,蛋白总回收率达8.5%以上.各项检定指标均符合《中国药典》三部(2010版)的相关要求.结论 已建立了稳定的、适合规模化生产的LcrV制备工艺,为新型鼠疫组分疫苗的研制奠定了基础.%Objective To develop a stable and effective procedure for fermentation of recombinant Yersinia pestis and purification of LcrV antigen. Methods The regularity of growth of recombinant E. Coli strain pET-V/BL21 (DE3) in tube and in conical flask as well as expression of LcrV antigen were investigated, based on which the medium, time point and duration for induction with IPTG and IPTG concentration were optimized and scaled up to a 30 L fermenter to develop a stable fermentation procedure. The harvested bacteria were subjected to freeze-thawing, from which protein solution was collected by centrifugation and treated by high pressure homogenization, the purified by Q. Sepharose HP anion exchange chromatography, Phenyl Sepharose 6 FF (hs) hydrophobic chromatography and Superdex 75 pg gel filtration chromatography, based on which a stable purification procedure was developed

  15. Inhibition of Leishmania (Leishmania amazonensis and rat arginases by green tea EGCG, (+-catechin and (--epicatechin: a comparative structural analysis of enzyme-inhibitor interactions.

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    Matheus Balduíno Goncalves dos Reis

    Full Text Available Epigallocatechin-3-gallate (EGCG, a dietary polyphenol (flavanol from green tea, possesses leishmanicidal and antitrypanosomal activity. Mitochondrial damage was observed in Leishmania treated with EGCG, and it contributed to the lethal effect. However, the molecular target has not been defined. In this study, EGCG, (+-catechin and (--epicatechin were tested against recombinant arginase from Leishmania amazonensis (ARG-L and rat liver arginase (ARG-1. The compounds inhibit ARG-L and ARG-1 but are more active against the parasite enzyme. Enzyme kinetics reveal that EGCG is a mixed inhibitor of the ARG-L while (+-catechin and (--epicatechin are competitive inhibitors. The most potent arginase inhibitor is (+-catechin (IC50 = 0.8 µM followed by (--epicatechin (IC50 = 1.8 µM, gallic acid (IC50 = 2.2 µM and EGCG (IC50 = 3.8 µM. Docking analyses showed different modes of interaction of the compounds with the active sites of ARG-L and ARG-1. Due to the low IC50 values obtained for ARG-L, flavanols can be used as a supplement for leishmaniasis treatment.

  16. Bovine immunoprotection against Rhipicephalus (Boophilus microplus with recombinant Bm86-Campo Grande antigen Imunoproteção de bovinos contra Rhipicephalus (Boophilus microplus com antígeno recombinante Bm86-Campo Grande

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    Rodrigo Casquero Cunha

    2012-09-01

    Full Text Available The southern cattle fever tick, Rhipicephalus (Boophilus microplus, is no doubt the most economically important ectoparasite of cattle globally. The inappropriate use of chemical acaricides has driven the evolution of resistance in populations of R. (B. microplus. Anti-tick vaccines represent a technology that can be combined with acaricides in integrated control programs to mitigate the impact of R. (B. microplus. The recombinant form of Bm86 antigen from the Campo Grande (rBm86-CG strain of R. (B. microplus was produced using the Pichiapastoris expression system to test its ability to immunoprotect cattle against tick infestation. Secretion of rBm86-CG by P. pastoris through the bioprocess reported here simplified purification of the antigen. A specific humoral immune response was detected by ELISA in vaccinated cattle. Immunoblot results revealed that polyclonal antibodies from vaccinated cattle recognized a protein in larval extracts with a molecular weight corresponding to Bm86. The rBm86-CG antigen showed 31% efficacy against the Campo Grande strain of R. (B. microplus infesting vaccinated cattle. The rBm86-CG is an antigen that could be used in a polyvalent vaccine as part of an integrated program for the control of R. (B. microplus in the region that includes Mato Grosso do Sul.O carrapato Rhipicephalus (Boophilus microplus é, sem dúvidas, o ectoparasito economicamente mais importante para o gado a nível mundial. A utilização inadequada de acaricidas tem impulsionado a evolução da resistência em populações de R. (B. microplus. Vacinas contra o carrapato representam uma tecnologia que pode ser combinada com acaricidas em programas de controle integrado para diminuir o impacto de R. (B. microplus. A forma recombinante da Bm86 da cepa Campo Grande (rBm86-CG de R. (B. microplus foi produzido utilizando o sistema de expressão em Pichia pastoris para testar sua capacidade de imunoproteção ao gado contra a infestação de

  17. Increasing the ex vivo antigen-specific IFN-γ production in subpopulations of T cells and NKp46+ cells by anti-CD28, anti-CD49d and recombinant IL-12 costimulation in cattle vaccinated with recombinant proteins from Mycobacterium avium subspecies paratuberculosis.

    Science.gov (United States)

    Thakur, Aneesh; Riber, Ulla; Davis, William C; Jungersen, Gregers

    2013-10-01

    T cells, which encounter specific antigen (Ag), require additional signals to mount a functional immune response. Here, we demonstrate activation of signal 2, by anti-CD28 mAb (aCD28) and other costimulatory molecules (aCD49d, aCD5), and signal 3, by recombinant IL-12, enhance Ag-specific IFN-γ secretion by CD4, CD8, γδ T cells and NK cells. Age matched male jersey calves, experimentally infected with Mycobacterium avium subsp. paratuberculosis (MAP), were vaccinated with a cocktail of recombinant MAP proteins or left unvaccinated. Vaccine induced ex vivo recall responses were measured through Ag-specific IFN-γ production by ELISA and flow cytometry. There was a significant increase in production of IFN-γ by T cell subsets or NKp46+ cells cultured in the presence of Ag and aCD28/aCD49d. The increase was accompanied by an increase in the integrated median fluorescence intensity (iMFI) of activated T cells. Addition of rIL-12 induced a significant additive effect leading to a maximum increase in responder frequency of Ag-specific T cell subsets or NKp46+ cells with a heavy bias toward IFN-γ production by CD4 T cells. We provide the first description of using aCD28/aCD49d costimulation to potentiate an Ag-specific increase in the production of IFN-γ in bovine immunology. The study also shows the degree of signaling in T cells is regulated by the costimulatory environment.

  18. Modulation of Na+/K+ ATPase Activity by Hydrogen Peroxide Generated through Heme in L. amazonensis.

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    Nathália Rocco-Machado

    Full Text Available Leishmania amazonensis is a protozoan parasite that occurs in many areas of Brazil and causes skin lesions. Using this parasite, our group showed the activation of Na+/K+ ATPase through a signaling cascade that involves the presence of heme and protein kinase C (PKC activity. Heme is an important biomolecule that has pro-oxidant activity and signaling capacity. Reactive oxygen species (ROS can act as second messengers, which are required in various signaling cascades. Our goal in this work is to investigate the role of hydrogen peroxide (H2O2 generated in the presence of heme in the Na+/K+ ATPase activity of L. amazonensis. Our results show that increasing concentrations of heme stimulates the production of H2O2 in a dose-dependent manner until a concentration of 2.5 μM heme. To confirm that the effect of heme on the Na+/K+ ATPase is through the generation of H2O2, we measured enzyme activity using increasing concentrations of H2O2 and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 μM H2O2. To investigate the role of PKC in this signaling pathway, we observed the production of H2O2 in the presence of its activator phorbol 12-myristate 13-acetate (PMA and its inhibitor calphostin C. Both showed no effect on the generation of H2O2. Furthermore, we found that PKC activity is increased in the presence of H2O2, and that in the presence of calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. 100 μM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na+/K+ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme. The modulation of L. amazonensis Na+/K+ ATPase by H2O2 opens new possibilities for understanding the signaling pathways of this parasite.

  19. The activity of azithromycin against Leishmania (Viannia braziliensis and Leishmania (Leishmania amazonensis in the golden hamster model A atividade da azitromicina contra a Leishmania (Viannia braziliensis e a Leishmania (Leishmania amazonensis no modelo golden hamster

    Directory of Open Access Journals (Sweden)

    Ángel Sinagra

    2007-12-01

    Full Text Available New therapeutic alternatives against leishmaniasis remain a priority. The activity of azithromycin against Leishmania (Leishmania major has been previously demonstrated. Different responses among species of Leishmania make species-specific drug screening necessary. The activity of azithromycin against Leishmania (Viannia braziliensis and Leishmania (Leishmania amazonensis was evaluated in golden hamsters infected through footpad injections of metacyclic promastigotes, and compared with untreated controls and animals treated with meglumine antimoniate. Footpad thickness, lesion cultures and dissemination sites were analyzed. Treatment of golden hamsters with oral azithromycin at 450mg/kg had no activity against infections with Leishmania (Leishmania amazonensis. For infections due to Leishmania (Viannia braziliensis, azithromycin demonstrated significant activity relative to untreated controls, but inferior to meglumine antimoniate, for controlling lesion size. Neither drug was able to totally eliminate parasites from the lesions. It was concluded that azithromycin has activity against Leishmania (Viannia braziliensis but not against Leishmania (Leishmania amazonensis in this model.Novas alternativas terapêuticas contra a leishmaniose são ainda uma prioridade. A atividade da azitromicina contra a Leishmania (Leishmania major foi anteriormente demonstrada. Diferentes respostas entre as espécies de Leishmania fazem com que um screening de drogas específicas para espécies seja necessário. A atividade da azitromicina contra a Leishmania (Viannia braziliensis e a Leishmania (Leishmania amazonensis foi avaliada em Golden hamsters infectados a través de injeções de promastigotas metacíclicas e comparando com controles sem tratamento e animais tratados com antimoniato de N-metil-glucamina. Foram analisadas a espessura da pata, a cultura das lesões e disseminação para órgãos internos. A azitromicina oral em dose de 450mg/kg não teve

  20. An alternative in vitro drug screening test using Leishmania amazonensis transfected with red fluorescent protein✩

    Science.gov (United States)

    Rocha, Marcele N.; Corrêa, Célia M.; Melo, Maria N.; Beverley, Stephen M.; Martins-Filho, Olindo Assis; Madureira, Ana Paula; Soares, Rodrigo P.

    2013-01-01

    Fluorescent and colorimetric reporter genes are valuable tools for drug screening models, since microscopy is labor intensive and subject to observer variation. In this work, we propose a fluorimetric method for drug screening using red fluorescent parasites. Fluorescent Leishmania amazonensis were developed after transfection with integration plasmids containing either red (RFP) or green fluorescent protein (GFP) genes. After transfection, wild-type (LaWT) and transfected (LaGFP and LaRFP) parasites were subjected to flow cytometry, macrophage infection, and tests of susceptibility to current antileishmanial agents and propranolol derivatives previously shown to be active against Trypanosoma cruzi. Flow cytometry analysis discriminated LaWT from LaRFP and LaGFP parasites, without affecting cell size or granulosity. With microscopy, transfection with antibiotic resistant genes was not shown to affect macrophage infectivity and susceptibility to amphotericin B and propranolol derivatives. Retention of fluorescence remained in the intracellular amastigotes in both LaGFP and LaRFP transfectants. However, detection of intracellular RFP parasites was only achieved in the fluorimeter. Murine BALB/c macrophages were infected with LaRFP parasites, exposed to standard (meglumine antimoniate, amphotericin B, Miltefosine, and allopurinol) and tested molecules. Although it was possible to determine IC50 values for 4 propranolol derivatives (1, 2b, 3, and 4b), all compounds were considered inactive. This study is the first to develop a fluorimetric drug screening test for L. amazonensis RFP. The fluorimetric test was comparable to microscopy with the advantage of being faster and not requiring manual counting. PMID:23312610

  1. In vitro activity of the antifungal azoles itraconazole and posaconazole against Leishmania amazonensis.

    Science.gov (United States)

    de Macedo-Silva, Sara Teixeira; Urbina, Julio A; de Souza, Wanderley; Rodrigues, Juliany Cola Fernandes

    2013-01-01

    Leishmaniasis, caused by protozoan parasites of the Leishmania genus, is one of the most prevalent neglected tropical diseases. It is endemic in 98 countries, causing considerable morbidity and mortality. Pentavalent antimonials are the first line of treatment for leishmaniasis except in India. In resistant cases, miltefosine, amphotericin B and pentamidine are used. These treatments are unsatisfactory due to toxicity, limited efficacy, high cost and difficult administration. Thus, there is an urgent need to develop drugs that are efficacious, safe, and more accessible to patients. Trypanosomatids, including Leishmania spp. and Trypanosoma cruzi, have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. Inhibition of ergosterol biosynthesis is increasingly recognized as a promising target for the development of new chemotherapeutic agents. The aim of this work was to investigate the antiproliferative, physiological and ultrastructural effects against Leishmania amazonensis of itraconazole (ITZ) and posaconazole (POSA), two azole antifungal agents that inhibit sterol C14α-demethylase (CYP51). Antiproliferative studies demonstrated potent activity of POSA and ITZ: for promastigotes, the IC50 values were 2.74 µM and 0.44 µM for POSA and ITZ, respectively, and for intracellular amastigotes, the corresponding values were 1.63 µM and 0.08 µM, for both stages after 72 h of treatment. Physiological studies revealed that both inhibitors induced a collapse of the mitochondrial membrane potential (ΔΨm), which was consistent with ultrastructural alterations in the mitochondrion. Intense mitochondrial swelling, disorganization and rupture of mitochondrial membranes were observed by transmission electron microscopy. In addition, accumulation of lipid bodies, appearance of autophagosome-like structures and alterations in the kinetoplast were also observed. In conclusion, our results indicate that ITZ and POSA are potent

  2. In vitro activity of the antifungal azoles itraconazole and posaconazole against Leishmania amazonensis.

    Directory of Open Access Journals (Sweden)

    Sara Teixeira de Macedo-Silva

    Full Text Available Leishmaniasis, caused by protozoan parasites of the Leishmania genus, is one of the most prevalent neglected tropical diseases. It is endemic in 98 countries, causing considerable morbidity and mortality. Pentavalent antimonials are the first line of treatment for leishmaniasis except in India. In resistant cases, miltefosine, amphotericin B and pentamidine are used. These treatments are unsatisfactory due to toxicity, limited efficacy, high cost and difficult administration. Thus, there is an urgent need to develop drugs that are efficacious, safe, and more accessible to patients. Trypanosomatids, including Leishmania spp. and Trypanosoma cruzi, have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. Inhibition of ergosterol biosynthesis is increasingly recognized as a promising target for the development of new chemotherapeutic agents. The aim of this work was to investigate the antiproliferative, physiological and ultrastructural effects against Leishmania amazonensis of itraconazole (ITZ and posaconazole (POSA, two azole antifungal agents that inhibit sterol C14α-demethylase (CYP51. Antiproliferative studies demonstrated potent activity of POSA and ITZ: for promastigotes, the IC50 values were 2.74 µM and 0.44 µM for POSA and ITZ, respectively, and for intracellular amastigotes, the corresponding values were 1.63 µM and 0.08 µM, for both stages after 72 h of treatment. Physiological studies revealed that both inhibitors induced a collapse of the mitochondrial membrane potential (ΔΨm, which was consistent with ultrastructural alterations in the mitochondrion. Intense mitochondrial swelling, disorganization and rupture of mitochondrial membranes were observed by transmission electron microscopy. In addition, accumulation of lipid bodies, appearance of autophagosome-like structures and alterations in the kinetoplast were also observed. In conclusion, our results indicate that ITZ and

  3. In Vitro Activity of the Antifungal Azoles Itraconazole and Posaconazole against Leishmania amazonensis

    Science.gov (United States)

    de Macedo-Silva, Sara Teixeira; Urbina, Julio A.; de Souza, Wanderley; Rodrigues, Juliany Cola Fernandes

    2013-01-01

    Leishmaniasis, caused by protozoan parasites of the Leishmania genus, is one of the most prevalent neglected tropical diseases. It is endemic in 98 countries, causing considerable morbidity and mortality. Pentavalent antimonials are the first line of treatment for leishmaniasis except in India. In resistant cases, miltefosine, amphotericin B and pentamidine are used. These treatments are unsatisfactory due to toxicity, limited efficacy, high cost and difficult administration. Thus, there is an urgent need to develop drugs that are efficacious, safe, and more accessible to patients. Trypanosomatids, including Leishmania spp. and Trypanosoma cruzi, have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. Inhibition of ergosterol biosynthesis is increasingly recognized as a promising target for the development of new chemotherapeutic agents. The aim of this work was to investigate the antiproliferative, physiological and ultrastructural effects against Leishmania amazonensis of itraconazole (ITZ) and posaconazole (POSA), two azole antifungal agents that inhibit sterol C14α-demethylase (CYP51). Antiproliferative studies demonstrated potent activity of POSA and ITZ: for promastigotes, the IC50 values were 2.74 µM and 0.44 µM for POSA and ITZ, respectively, and for intracellular amastigotes, the corresponding values were 1.63 µM and 0.08 µM, for both stages after 72 h of treatment. Physiological studies revealed that both inhibitors induced a collapse of the mitochondrial membrane potential (ΔΨm), which was consistent with ultrastructural alterations in the mitochondrion. Intense mitochondrial swelling, disorganization and rupture of mitochondrial membranes were observed by transmission electron microscopy. In addition, accumulation of lipid bodies, appearance of autophagosome-like structures and alterations in the kinetoplast were also observed. In conclusion, our results indicate that ITZ and POSA are potent

  4. Effects of medicinal plant extracts on growth of Leishmania (L. amazonensis and Trypanosoma cruzi Efeito de extratos de plantas medicinais no crescimento de Leishmania (L. amazonensis e Trypanosoma cruzi

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    Patrícia Shima Luize

    2005-03-01

    Full Text Available This study describes the screening of extracts obtained from 19 species of plants used in Brazilian traditional medicine for treatment of a variety of diseases. The extracts were tested against axenic amastigote and promastigote forms of Leishmania (L. amazonensis, and epimastigote forms of Trypanosoma cruzi in vitro at a concentration of 100 mg/ml. Baccharis trimera, Cymbopogon citratus, Matricaria chamomilla, Mikania glomerata, Ocimum gratissimum, Piper regnellii, Prunus domestica, Psidium guajava, Sambucus canadensis, Stryphnodendron adstringens, Tanacetum parthenium, and Tanacetum vulgare showed significant effects against one or both parasites, with a percentage of growth inhibition between 49.5 and 99%. The extracts showed no cytotoxic effect on sheep erythrocytes. These medicinal plants may be sources of new compounds that are clinically active against L. amazonensis and T. cruzi.Este estudo descreve a triagem de extratos obtidos de 19 espécies de plantas usadas na medicina tradicional brasileira para o tratamento de várias doenças. Os extratos foram testados contra formas amastigota axênica e promastigota de Leishmania (L. amazonensis, e formas epimastigota de Trypanosoma cruzi in vitro na concentração de 100 mg/ml. Baccharis trimera, Cymbopogon citratus, Matricaria chamomilla, Mikania glomerata, Ocimum gratissimum, Piper regnellii, Prunus domestica, Psidium guajava, Sambucus canadensis, Stryphnodendron adstringens, Tanacetum parthenium, e Tanacetum vulgare apresentaram efeito significante contra um ou ambos parasitas, com a porcentagem de inibição de crescimento entre 49,5 e 99%. Os extratos não mostraram efeito citotóxico em hemácias de carneiro. Essas plantas medicinais podem ser fontes alternativas de novos compostos clinicamente ativos contra L. amazonensis e T. cruzi.

  5. Genotyping and preparation of the recombinant nucleocapsid protein antigen of hantavirus%汉坦病毒核壳蛋白重组抗原的制备和基因分型研究

    Institute of Scientific and Technical Information of China (English)

    唐家琪; 操敏; 唐堂; 王长军; 魏春宝; 雷万里

    2001-01-01

    目的研制新的汉坦病毒核衣壳工程抗原,建立肾综合征出血热病毒检测和基因分型方法。 方法以一组引物克隆全长S基因片段和N端的部分S基因片段,并使它们在T7系统进行融合表达和非 融合表达。用另组引物建立了逆转录-聚合酶链式反应(RT-PCR),检测我国不同地区由8种主要宿主分 离的37个汉坦毒株,2个阳性标准对照毒株和5个阴性对照标本。对其中20个毒株的PCR扩增产物先 后用Rsa Ⅰ和HindⅢ作二级酶切,建立了逆转录-聚合酶链式反应-限制性片段长度多态性分析(RT-PCR- RFLP)分型法。 结果非融合表达产量虽不及融合表达高,但以非融合表达的两个S基因片段产物作间接ELISA的包被 抗原,其工作浓度均达1:10000,显示出良好的生物活性。RT-PCR检测结果表明,所有的毒株扩增出汉坦 特异性核酸组份(299 bp或577 bp)。用RT-PCR-RFLP法分型,上述毒株被定为汉滩型的9株,汉城型的 8株,余3株未能定型。 结论非融合表达的小分子抗原生物活性较高,有望替代天然抗原用于HV抗原抗体检测。RT-PCR法与 cELISA法比较,二者阳性检出率分别为100%和84.6%,符合率为84.6%,但前者比后者敏感性高15.4%。 RT-PCR-RFLP分型法与血清学分型法所得结果具有很高的符合率,但RFLP法的分型率为85%(17/20), 血清法的分型率为55%(11/20),前者比后者高30%。%Objective To identify new recombinant antigens with potential for diagnosis of hemorrhagic fever with renal syndrom (HFRS) and establish reverse transcription-polymerase chain reaction-restriction fragments length polymorphism (RT-PCR-RFLP) for genotyping of hantavirus. Methods One group of primers was used to clone the full-length S genome segment and the partial S genorme segment of the N-terminal. The two cloned genes were both fusionally expressed and non fusionally expressed in the T7 system

  6. Inhibitory effects of Zanthoxylum rhoifolium Lam. (Rutaceae) against the infection and infectivity of macrophages by Leishmania amazonensis

    OpenAIRE

    BERNARDO MELO NETO; JOSEANA M.S.R. LEITÃO; OLIVEIRA,LUCIANO G.C.; SANTOS,SÉRGIO E.M.; SABRINA M.P. CARNEIRO; Rodrigues, Klinger A. F.; Chaves, Mariana H.; DANIEL D.R. ARCANJO; CARVALHO,FERNANDO A.A.

    2016-01-01

    ABSTRACT Zanthoxylum rhoifolium Lam. (Rutaceae) has been traditionally used in the treatment of microbial infections and parasitic diseases. In the present study, the antileishmanial effect induced by the ethanol extract of stem barks from Z. rhoifolium (ZR-EEtOH) and its n-hexane fraction (ZR-FHEX) on infection and infectivity of murine macrophages by promastigote forms of Leishmania amazonensis were investigated. In different set of experiments, macrophages or promastigotes were pretreated ...

  7. N-acetyl-cysteine inhibits liver oxidative stress markers in BALB/c mice infected with Leishmania amazonensis

    Science.gov (United States)

    Gasparotto, Juciano; Kunzler, Alice; Senger, Mario Roberto; de Souza, Celeste da Silva Freitas; de Simone, Salvatore Giovanni; Bortolin, Rafael Calixto; Somensi, Nauana; Dal-Pizzol, Felipe; Moreira, José Claudio Fonseca; Abreu-Silva, Ana Lúcia; Calabrese, Kátia da Silva; Silva, Floriano Paes; Gelain, Daniel Pens

    2017-01-01

    BACKGROUND Leishmaniasis is a parasitosis caused by several species of the genus Leishmania. These parasites present high resistance against oxidative stress generated by inflammatory cells. OBJECTIVES To investigate oxidative stress and molecular inflammatory markers in BALB/c mice infected with L. amazonensis and the effect of antioxidant treatment on these parameters. METHODS Four months after infection, oxidative and inflammatory parameters of liver, kidneys, spleen, heart and lungs from BALB/c mice were assessed. FINDINGS In liver, L. amazonensis caused thiol oxidation and nitrotyrosine formation; SOD activity and SOD2 protein content were increased while SOD1 protein content decreased. The content of the cytokines IL-1β, IL-6, TNF-α, and the receptor of advanced glycation endproducts (RAGE) increased in liver. Treatment with the antioxidant N-acetyl-cysteine (20 mg/kg b.w) for five days inhibited oxidative stress parameters. MAIN CONCLUSIONS L. amazonensis induces significant alterations in the redox status of liver but not in other organs. Acute antioxidant treatment alleviates oxidative stress in liver, but it had no effect on pro-inflammatory markers. These results indicate that the pathobiology of leishmaniasis is not restricted to the cutaneous manifestations and open perspectives for the development of new therapeutic approaches to the disease, especially for liver function. PMID:28177049

  8. Leishmania (L). amazonensis induces hyperalgesia in balb/c mice: Contribution of endogenous spinal cord TNFα and NFκB activation.

    Science.gov (United States)

    Borghi, Sergio M; Fattori, Victor; Ruiz-Miyazawa, Kenji W; Miranda-Sapla, Milena M; Casagrande, Rúbia; Pinge-Filho, Phileno; Pavanelli, Wander R; Verri, Waldiceu A

    2017-02-17

    Cutaneous leishmaniasis (CL) is the most common form of the leishmaniasis in humans. Ulcerative painless skin lesions are predominant clinical features of CL. Wider data indicate pain accompanies human leishmaniasis, out with areas of painless ulcerative lesions per se. In rodents, Leishmania (L.) major infection induces nociceptive behaviors that correlate with peripheral cytokine levels. However, the role of the spinal cord in pain processing after Leishmania infection has not been investigated. Balb/c mice received intraplantar (i.pl.) injection of Leishmania (L). amazonensis and hyperalgesia, edema, parasitism, and spinal cord TNFα, TNFR1 and TNFR2 mRNA expression, and NFκB activation were evaluated. The effects of intrathecal (i.t.) injection of morphine, TNFα, TNFα inhibitors (etanercept and adalimumab) and NFκB inhibitor (PDTC) were investigated. The present study demonstrates that Leishmania (L.) amazonensis infection in balb/c mice induces chronic mechanical and thermal hyperalgesia in an opioid-sensitive manner. Spinal cord TNFα mRNA expression increased in a time-dependent manner, peaking between 30 and 40 days after infection. At the peak of TNFα mRNA expression (day 30), there was a concomitant increase in TNFR1 and TNFR2 mRNA expression. TNFα i.t. injection enhanced L. (L.) amazonensis-induced hyperalgesia. Corroborating a role for TNFα in L. (L.) amazonensis-induced hyperalgesia, i.t. treatment with the TNFα inhibitors, etanercept and adalimumab inhibited the hyperalgesia. L. (L.) amazonensis also induced spinal cord activation of NFκB, and PDTC (given i.t.), also inhibited L. (L.) amazonensis-induced hyperalgesia, and spinal cord TNFα, TNFR1 and TNFR2 mRNA expression. Moreover, L. (L.) amazonensis-induced spinal cord activation of NFκB was also inhibited by etanercept and adalimumab as well as PDTC i.t.

  9. HIV aspartyl peptidase inhibitors interfere with cellular proliferation, ultrastructure and macrophage infection of Leishmania amazonensis.

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    Lívia O Santos

    Full Text Available BACKGROUND: Leishmania is the etiologic agent of leishmanisais, a protozoan disease whose pathogenic events are not well understood. Current therapy is suboptimal due to toxicity of the available therapeutic agents and the emergence of drug resistance. Compounding these problems is the increase in the number of cases of Leishmania-HIV coinfection, due to the overlap between the AIDS epidemic and leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: In the present report, we have investigated the effect of HIV aspartyl peptidase inhibitors (PIs on the Leishmania amazonensis proliferation, ultrastructure, interaction with macrophage cells and expression of classical peptidases which are directly involved in the Leishmania pathogenesis. All the HIV PIs impaired parasite growth in a dose-dependent fashion, especially nelfinavir and lopinavir. HIV PIs treatment caused profound changes in the leishmania ultrastructure as shown by transmission electron microscopy, including cytoplasm shrinking, increase in the number of lipid inclusions and some cells presenting the nucleus closely wrapped by endoplasmic reticulum resembling an autophagic process, as well as chromatin condensation which is suggestive of apoptotic death. The hydrolysis of HIV peptidase substrate by L. amazonensis extract was inhibited by pepstatin and HIV PIs, suggesting that an aspartyl peptidase may be the intracellular target of the inhibitors. The treatment with HIV PIs of either the promastigote forms preceding the interaction with macrophage cells or the amastigote forms inside macrophages drastically reduced the association indexes. Despite all these beneficial effects, the HIV PIs induced an increase in the expression of cysteine peptidase b (cpb and the metallopeptidase gp63, two well-known virulence factors expressed by Leishmania spp. CONCLUSIONS/SIGNIFICANCE: In the face of leishmaniasis/HIV overlap, it is critical to further comprehend the sophisticated interplays among Leishmania

  10. Diagnostic Antigens of Leishmania.

    Science.gov (United States)

    1994-01-31

    L. major (LTM p-2), L. major (Friedlander), and Trypanosoma cruzi (MHOM/CH/00/Tulahuen C2) were used. Leishmania promastigotes and T. cruzi ...some weak hybridization was observed with L. amazonensis, but none was seen with L. braziliensis, L. guyanensis, or T cruzi . A similar, overlapping... cruzi (8) have been previously isolated by us. To address this possibility in rLt-1, a portion of the repeat was expressed separately as rLt-lr. The

  11. Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori

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    Leila Hasanzadeh

    2013-07-01

    Full Text Available Objective(s: Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity.   Materials and Methods: The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify a highly antigenic region of VacA gene from chromosomal DNA of H. pylori. The eluted product was cloned into the prokaryotic expression vector pET32a. The target protein was expressed in the Escherichia coli BL21 (DE3 pLysS. The bacteria including pET32a-VacA plasmids were induced by IPTG. The antigenicity was finally studied by western blotting using sera of 15 H. pylori infected patients after purification. Results: Enzyme digestion analysis, PCR and DNA sequencing results showed that the target gene was inserted correctly into the recombinant vector. The expressed protein was purified successfully via affinity chromatography. Data indicated that antigenic region of VacA protein from Helicobacter pylori was recognized by all 15 patient’s sera. Conclusion : Our data showed that antigenic region of VacA protein can be expressed by in E. co.li. This protein was recognized by sera patients suffering from H. pylori infection. the recombinant protein has similar epitopes and close antigenic properties to the natural form of this antigen. Recombinant antigenic region of VacA protein also seems to be a promising antigen for protective and serologic diagnosis .

  12. In Vitro Assessment of Plants Growing in Cuba Belonging to Solanaceae Family Against Leishmania amazonensis.

    Science.gov (United States)

    Monzote, Lianet; Jiménez, Jenny; Cuesta-Rubio, Osmany; Márquez, Ingrid; Gutiérrez, Yamile; da Rocha, Cláudia Quintino; Marchi, Mary; Setzer, William N; Vilegas, Wagner

    2016-11-01

    In this study, an in vitro antileishmanial assessment of plant extracts from 12 genera and 46 species growing in Cuba belonging to Solanaceae family was performed. A total of 226 extracts were screened against promastigotes of Leishmania amazonensis, and cytotoxicity of active extracts [median inhibitory concentration (IC50 ) promastigotes 5 were then assayed against intracellular amastigote. Metabolomics analysis of promissory extracts was performed using chemical profile obtained by ultra performance liquid chromatography. Only 11 extracts (4.9%) from nine plants were selected as potentially actives: Brunfelsia cestroides A. Rich, Capsicum annuum L., Capsicum chinense Jacq., Cestrum nocturnum L., Nicotiana plumbaginifolia Viv., Solanum havanense Jacq., Solanum myriacanthum Dunal, Solanum nudum Dunal and Solanum seaforthianum And., with IC50  5. Metabolomics analysis demonstrated significant differences in the chemical profiles with an average of 42.8 (range 31-88) compounds from m/z 104 to 1477, which demonstrated the complex mixture of compounds. In addition, no common markers among active extracts were identified. The results demonstrate the importance of the Solanaceae family to search new antileishmanial agents, particularly in unexplored species of this family. Copyright © 2016 John Wiley & Sons, Ltd.

  13. Degranulating Neutrophils Promote Leukotriene B4 Production by Infected Macrophages To Kill Leishmania amazonensis Parasites.

    Science.gov (United States)

    Tavares, Natália; Afonso, Lilian; Suarez, Martha; Ampuero, Mariana; Prates, Deboraci Brito; Araújo-Santos, Théo; Barral-Netto, Manoel; DosReis, George A; Borges, Valéria Matos; Brodskyn, Cláudia

    2016-02-15

    Neutrophils mediate early responses against pathogens, and they become activated during endothelial transmigration toward the inflammatory site. In the current study, human neutrophils were activated in vitro with immobilized extracellular matrix proteins, such as fibronectin (FN), collagen, and laminin. Neutrophil activation by FN, but not other extracellular matrix proteins, induces the release of the granules' contents, measured as matrix metalloproteinase 9 and neutrophil elastase activity in culture supernatant, as well as reactive oxygen species production. Upon contact with Leishmania amazonensis-infected macrophages, these FN-activated neutrophils reduce the parasite burden through a mechanism independent of cell contact. The release of granule proteases, such as myeloperoxidase, neutrophil elastase, and matrix metalloproteinase 9, activates macrophages through TLRs, leading to the production of inflammatory mediators, TNF-α and leukotriene B4 (LTB4), which are involved in parasite killing by infected macrophages. The pharmacological inhibition of degranulation reverted this effect, abolishing LTB4 and TNF production. Together, these results suggest that FN-driven degranulation of neutrophils induces the production of LTB4 and TNF by infected macrophages, leading to the control of Leishmania infection.

  14. Determination of femto Newton forces and fluid viscosity using optical tweezers: application to Leishmania amazonensis

    Science.gov (United States)

    Fontes, Adriana; Giorgio, Selma; de Castro, Archimedes B., Jr.; Neto, Vivaldo M.; Pozzo, Liliana d. Y.; Marques, Gustavo P.; Barbosa, Luiz C.; Cesar, Carlos L.

    2005-03-01

    The objective of this research is to use the displacements of a polystyrene microsphere trapped by an optical tweezers (OT) as a force transducer in mechanical measurements in life sciences. To do this we compared the theoretical optical and hydrodynamic models with experimental data under a broad variation of parameters such as fluid viscosity, refractive index, drag velocity and wall proximities. The laser power was measured after the objective with an integration sphere because normal power meters do not provide an accurate measurement for beam with high numerical apertures. With this careful laser power determination the plot of the optical force (calculated by the particle displacement) versus hydrodynamic force (calculated by the drag velocity) under very different conditions shows an almost 45 degrees straight line. This means that hydrodynamic models can be used to calibrate optical forces and vice-versa. With this calibration we observed the forces of polystyrene bead attached to the protozoa Leishmania amazonensis, responsible for a serious tropical disease. The force range is from 200 femto Newtons to 4 pico Newtons and these experiments shows that OT can be used for infection mechanism and chemotaxis studies in parasites. The other application was to use the optical force to measure viscosities of few microliters sample. Our result shows 5% accuracy measurements.

  15. The in Vitro Biological Activity of the Brazilian Brown Seaweed Dictyota mertensii against Leishmania amazonensis

    Directory of Open Access Journals (Sweden)

    Amanda Silva dos Santos Aliança

    2014-09-01

    Full Text Available Seaweeds present a wide variety of interesting bioactive molecules. In the present work we evaluated the biological activity of the dichloromethane/methanol (2:1 extract (DME from the brown seaweed Dictyota mertensii against Leishmania amazonensis and its cytotoxic potential on mammalian cells. The extract showed significant inhibitory effect on the growth of promastigote forms (IC50 = 71.60 μg/mL and low toxicity against mammalian cells (CC50 = 233.10 μg/mL. The DME was also efficient in inhibiting the infection in macrophages, with CC50 of 81.4 μg/mL and significantly decreased the survival of amastigote forms within these cells. The selectivity index showed that DME was more toxic to both promastigote (SI = 3.25 and amastigote (SI = 2.86 forms than to macrophages. Increased NO production was observed in treated macrophages suggesting that besides acting directly on the parasites, the DME also shows an immunomodulatory effect on macrophages. Drastic ultrastructural alterations consistent with loss of viability and cell death were observed in treated parasites. Confocal microscopy and cytometry analyzes showed no significant impairment of plasma membrane integrity, whereas an intense depolarization of mitochondrial membrane could be observed by using propidium iodide and rhodamine 123 staining, respectively. The low toxicity to mammalian cells and the effective activity against promastigotes and amastigotes, point to the use of DME as a promising agent for the treatment of cutaneous leishmaniasis.

  16. The in vitro biological activity of the Brazilian brown seaweed Dictyota mertensii against Leishmania amazonensis.

    Science.gov (United States)

    dos Santos Aliança, Amanda Silva; dos Anjos, Keicyanne Fernanda Lessa; de Vasconcelos Reis, Thiago Nogueira; Higino, Taciana Mirely Maciel; Brelaz-de-Castro, Maria Carolina Accioly; Bianco, Éverson Miguel; de Figueiredo, Regina Celia Bressan Queiroz

    2014-09-09

    Seaweeds present a wide variety of interesting bioactive molecules. In the present work we evaluated the biological activity of the dichloromethane/methanol (2:1) extract (DME) from the brown seaweed Dictyota mertensii against Leishmania amazonensis and its cytotoxic potential on mammalian cells. The extract showed significant inhibitory effect on the growth of promastigote forms (IC50=71.60 μg/mL) and low toxicity against mammalian cells (CC50=233.10 μg/mL). The DME was also efficient in inhibiting the infection in macrophages, with CC50 of 81.4 μg/mL and significantly decreased the survival of amastigote forms within these cells. The selectivity index showed that DME was more toxic to both promastigote (SI=3.25) and amastigote (SI=2.86) forms than to macrophages. Increased NO production was observed in treated macrophages suggesting that besides acting directly on the parasites, the DME also shows an immunomodulatory effect on macrophages. Drastic ultrastructural alterations consistent with loss of viability and cell death were observed in treated parasites. Confocal microscopy and cytometry analyzes showed no significant impairment of plasma membrane integrity, whereas an intense depolarization of mitochondrial membrane could be observed by using propidium iodide and rhodamine 123 staining, respectively. The low toxicity to mammalian cells and the effective activity against promastigotes and amastigotes, point to the use of DME as a promising agent for the treatment of cutaneous leishmaniasis.

  17. Opsonization modulates Rac-1 activation during cell entry by Leishmania amazonensis.

    Science.gov (United States)

    Morehead, J; Coppens, I; Andrews, N W

    2002-08-01

    Lesions caused by Leishmania amazonensis normally heal, but relapses occur due to parasite persistence in host tissues. It has been proposed that infection of fibroblasts plays an important role in this process by providing the parasites with a safe haven in which to replicate. However, most previous studies have focused on the entry of Leishmania into macrophages, a process mediated by serum opsonins. To gain insight into a possible role of nonopsonic entry in the intracellular persistence of amastigotes, we examined the invasion of Chinese hamster ovary (CHO) cells. Amastigotes entered CHO cells by a cytochalasin D, genistein, wortmannin, and 2,3-butanedione monoxime-sensitive pathway and replicated within phagolysosomes. However, unlike most phagocytic processes described to date, amastigote internalization in CHO cells involved activation of the GTPases Rho and Cdc42 but not Rac-1. When uptake was mediated by fibronectin or when amastigotes were opsonized with immunoglobulin G and internalized by Fc receptor-expressing CHO cells, Rac-1 activation was restored and found to be required for parasite internalization. Given the essential role of Rac in assembly of the respiratory burst oxidase, invasion through this nonopsonic, Rac-1-independent pathway may play a central role in the intracellular survival of Leishmania in immune hosts.

  18. Impact of tumor necrosis factor receptor p55 deficiency in susceptibility of C57BL/6 mice to infection with Leishmania (Leishmania) amazonensis.

    Science.gov (United States)

    Cargnelutti, Diego Esteban; Salomón, María Cristina; Celedon, Verónica; Cuello-Carrión, Fernando Darío; Gea, Susana; Di Genaro, María Silvia; Scodeller, Eduardo Alberto

    2016-04-01

    Tumor necrosis factor (TNF) is involved in host resistance to several intracellular pathogens. Although the critical role of TNF receptor (TNFR)p55 in Leishmania (Leishmania) major infection has been demonstrated, the impact of TNFRp55 deficiency on L. (L.) amazonensis infection has not been explored. L. (L.) amazonensis-infected TNFRp55(-/-) mice failed to resolve lesions, whereas C57BL/6 wild-type mice completely healed. The susceptibility of the TNFRp55(-/-) mice was characterized by higher lesion size and histopathological damage in comparison with the wild-type mice. A marked increased of the splenic index was observed in the TNFRp55(-/-) mice after 15 weeks infection. These results show that in the absence of TNFRp55, L. (L.) amazonensis-infected knockout mice fail to resolve lesions, whereas wild-type mice completely heal.

  19. Recombinant vaccines: experimental and applied aspects

    DEFF Research Database (Denmark)

    Lorenzen, Niels

    1999-01-01

    Development of vaccines for aquaculture fish represent an important applied functional aspect of fish immunology research. Particularly in the case of recombinant vaccines, where a single antigen is usually expected to induce immunity to a specific pathogen, knowledge of mechanisms involved in in......, these fields will open up a number of interesting research objectives of mutual benefit. Recent aspects of recombinant protein vaccines, live recombinant vaccines and DNA vaccines are discussed.......Development of vaccines for aquaculture fish represent an important applied functional aspect of fish immunology research. Particularly in the case of recombinant vaccines, where a single antigen is usually expected to induce immunity to a specific pathogen, knowledge of mechanisms involved...... in induction of a protective immune response may become vital. The few recombinant vaccines licensd so far, despite much research during the last decade, illustrate that this is not a straightforward matter. However, as vaccine technology as well as our knowledge of the fish immune system is steadily improved...

  20. Distinct Macrophage Fates after in vitro Infection with Different Species of Leishmania: Induction of Apoptosis by Leishmania (Leishmania amazonensis, but Not by Leishmania (Viannia guyanensis.

    Directory of Open Access Journals (Sweden)

    Jarina Pena DaMata

    Full Text Available Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6, whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection.

  1. Leishmania amazonensis promastigotes in 3D Collagen I culture: an in vitro physiological environment for the study of extracellular matrix and host cell interactions

    Directory of Open Access Journals (Sweden)

    Debora B. Petropolis

    2014-04-01

    Full Text Available Leishmania amazonensis is the causative agent of American cutaneous leishmaniasis, an important neglected tropical disease. Once Leishmania amazonensis is inoculated into the human host, promastigotes are exposed to the extracellular matrix (ECM of the dermis. However, little is known about the interaction between the ECM and Leishmania promastigotes. In this study we established L. amazonensis promastigote culture in a three-dimensional (3D environment mainly composed of Collagen I (COL I. This 3D culture recreates in vitro some aspects of the human host infection site, enabling the study of the interaction mechanisms of L. amazonensis with the host ECM. Promastigotes exhibited “freeze and run” migration in the 3D COL I matrix, which is completely different from the conventional in vitro swimming mode of migration. Moreover, L. amazonensis promastigotes were able to invade, migrate inside, and remodel the 3D COL I matrix. Promastigote trans-matrix invasion and the freeze and run migration mode were also observed when macrophages were present in the matrix. At least two classes of proteases, metallo- and cysteine proteases, are involved in the 3D COL I matrix degradation caused by Leishmania. Treatment with a mixture of protease inhibitors significantly reduced promastigote invasion and migration through this matrix. Together our results demonstrate that L. amazonensis promastigotes release proteases and actively remodel their 3D environment, facilitating their migration. This raises the possibility that promastigotes actively interact with their 3D environment during the search for their cellular “home”—macrophages. Supporting this hypothesis, promastigotes migrated faster than macrophages in a novel 3D co-culture model.

  2. Recombination instability

    DEFF Research Database (Denmark)

    D'Angelo, N.

    1967-01-01

    A recombination instability is considered which may arise in a plasma if the temperature dependence of the volume recombination coefficient, alpha, is sufficiently strong. Two cases are analyzed: (a) a steady-state plasma produced in a neutral gas by X-rays or high energy electrons; and (b) an af...

  3. Leishmania (Viannia braziliensis growth in vitro culture relies more on folic acid availability than Leihsmania (Leishmania amazonensis

    Directory of Open Access Journals (Sweden)

    Andrea Niño

    2005-05-01

    Full Text Available We compared the in vitro growth of promastigotes from two Leishmania species in TC-100 and Schneider media. Leishmania (Leishmania amazonensis replication rates were similar in both tissue culture media and reached maximum rates by 48 h. In contrast Leishmania (Viannia braziliensis growth was significantly greater in TC-100 but maximum rates were achieved by 96 h. Folic acid appears to be the limiting factor and supplementation of Schneider media with this nutrient improved L. (V. braziliensis replication rates and decreased the time of maximum replication to 48 h.

  4. Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents.

    Science.gov (United States)

    Verissimo-Villela, Erika; Kitahara-Oliveira, Milene Yoko; Reis, Ana Beatriz de Bragança Dos; Albano, Rodolpho Mattos; Da-Cruz, Alda Maria; Bello, Alexandre Ribeiro

    2016-05-01

    During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

  5. Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

    Science.gov (United States)

    Verissimo-Villela, Erika; Kitahara-Oliveira, Milene Yoko; dos Reis, Ana Beatriz de Bragança; Albano, Rodolpho Mattos; Da-Cruz, Alda Maria; Bello, Alexandre Ribeiro

    2016-01-01

    During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins. PMID:27223868

  6. Identification of specific antigenic epitope at N-terminal segment of enterovirus 71 (EV-71) VP1 protein and characterization of its use in recombinant form for early diagnosis of EV-71 infection

    OpenAIRE

    Zhang, Jianhua; Jiang, Bingfu; Xu, Mingjie; Dai, Xing; Purdy, Michael A.; Meng, Jihong

    2014-01-01

    Human enterovirus 71 (EV-71) is the main etiologic agent of hand, foot and mouth disease (HFMD). We sought to identify EV-71 specific antigens and develop serologic assays for acute-phase EV-71 infection. A series of truncated proteins within the N-terminal 100 amino acids (aa) of EV-71 VP1 was expressed in Escherichia coli. Western blot (WB) analysis showed that positions around 11–21 aa contain EV-71-specific antigenic sites, whereas positions 1–5 and 51–100 contain epitopes shared with hum...

  7. 17-AAG Kills Intracellular Leishmania amazonensis while Reducing Inflammatory Responses in Infected Macrophages

    Science.gov (United States)

    Petersen, Antonio Luis de Oliveira Almeida; Guedes, Carlos Eduardo Sampaio; Versoza, Carolina Leite; Lima, José Geraldo Bomfim; de Freitas, Luiz Antônio Rodrigues; Borges, Valéria Matos; Veras, Patrícia Sampaio Tavares

    2012-01-01

    Background Leishmaniasis is a neglected endemic disease with a broad spectrum of clinical manifestations. Pentavalent antimonials have been the treatment of choice for the past 70 years and, due to the emergence of resistant cases, the efficacy of these drugs has come under scrutiny. Second-line drugs are less efficacious, cause a range of side effects and can be costly. The formulation of new generations of drugs, especially in developing countries, has become mandatory. Methodology/Principal Findings We investigated the anti-leishmanial effect of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), an HSP90 inhibitor, in vitro. This inhibitor is currently in clinical trials for cancer treatment; however, its effects against intracellular Leishmania remain untested. Macrophages infected with L. amazonensis were treated with 17-AAG (25–500 nM) and parasite load was quantified using optical microscopy. Parasite load declined in 17-AAG-treated macrophages in a dose- and time-dependent manner. Intracellular parasite death became irreversible after 4 h of treatment with 17-AAG, and occurred independent of nitric oxide (NO) and superoxide (O2−) production. Additionally, intracellular parasite viability was severely reduced after 48 h of treatment. Interestingly, treatment with 17-AAG reduced pro-inflammatory mediator production, including TNF-α, IL-6 and MCP-1, yet IL-12 remained unaffected. Electron microscopy revealed morphological alterations, such as double-membrane vacuoles and myelin figures at 24 and 48 h after 17-AAG treatment. Conclusions/Significance The HSP90 inhibitor, 17-AAG, possesses high potency under low dosage and reduces both pro-inflammatory and oxidative molecule production. Therefore, further studies are warranted to investigate this inhibitor’s potential in the development of new generations of anti-leishmanials. PMID:23152914

  8. Determination of fluid viscosity and femto Newton forces of Leishmania amazonensis using optical tweezers

    Science.gov (United States)

    Fontes, Adriana; Giorgio, Selma; de Castro, Archimedes, Jr.; Neto, Vivaldo M.; de Y. Pozzo, Liliana; de Thomaz, Andre A.; Barbosa, Luiz C.; Cesar, Carlos L.

    2005-08-01

    The displacements of a polystyrene microsphere trapped by an optical tweezers (OT) can be used as a force transducer for mechanical measurements in life sciences such as the measurement of forces of living microorganisms or the viscosity of local fluids. The technique we used allowed us to measure forces on the 200 femto Newtons to 4 pico Newtons range of the protozoa Leishmania amazonensis, responsible for a serious tropical disease. These observations can be used to understand the infection mechanism and chemotaxis of these parasites. The same technique was used to measure viscosities of few microliters sample with agreement with known samples better than 5%. To calibrate the force as a function of the microsphere displacement we first dragged the microsphere in a fluid at known velocity for a broad range of different optical and hydrodynamical parameters. The hydrodynamical model took into account the presence of two walls and the force depends on drag velocity, fluid viscosity and walls proximities, while the optical model in the geometric optics regime depends on the particle and fluid refractive indexes and laser power. To measure the high numerical (NA) aperture laser beam power after the objective we used an integration sphere to avoid the systematic errors of usual power meters for high NA beams. After this careful laser power measurement we obtained an almost 45 degrees straight line for the plot of the optical force (calculated by the particle horizontal displacement) versus hydrodynamic force (calculated by the drag velocity) under variation of all the parameters described below. This means that hydrodynamic models can be used to calibrate optical forces, as we have done for the parasite force measurement, or vice-versa, as we did for the viscosity measurements.

  9. In Vitro and In Vivo Activities of 2,3-Diarylsubstituted Quinoxaline Derivatives against Leishmania amazonensis.

    Science.gov (United States)

    Kaplum, Vanessa; Cogo, Juliana; Sangi, Diego Pereira; Ueda-Nakamura, Tânia; Corrêa, Arlene Gonçalves; Nakamura, Celso Vataru

    2016-06-01

    Leishmaniasis is endemic in 98 countries and territories worldwide. The therapies available for leishmaniasis have serious side effects, thus prompting the search for new therapies. The present study investigated the antileishmanial activities of 2,3-diarylsubstituted quinoxaline derivatives against Leishmania amazonensis The antiproliferative activities of 6,7-dichloro-2,3-diphenylquinoxaline (LSPN329) and 2,3-di-(4-methoxyphenyl)-quinoxaline (LSPN331) against promastigotes and intracellular amastigotes were assessed, and the cytotoxicities of LSPN329 and LSPN331 were determined. Morphological and ultrastructural alterations were examined by electron microscopy, and biochemical alterations, reflected by the mitochondrial membrane potential (ΔΨm), mitochondrial superoxide anion (O2·(-)) concentration, the intracellular ATP concentration, cell volume, the level of phosphatidylserine exposure on the cell membrane, cell membrane integrity, and lipid inclusions, were evaluated. In vivo antileishmanial activity was evaluated in a murine cutaneous leishmaniasis model. Compounds LSPN329 and LSPN331 showed significant selectivity for promastigotes and intracellular amastigotes and low cytotoxicity. In promastigotes, ultrastructural alterations were observed, including an increase in lipid inclusions, concentric membranes, and intense mitochondrial swelling, which were associated with hyperpolarization of ΔΨm, an increase in the O2·(-) concentration, decreased intracellular ATP levels, and a decrease in cell volume. Phosphatidylserine exposure and DNA fragmentation were not observed. The cellular membrane remained intact after treatment. Thus, the multifactorial response that was responsible for the cellular collapse of promastigotes was based on intense mitochondrial alterations. BALB/c mice treated with LSPN329 or LSPN331 showed a significant decrease in lesion thickness in the infected footpad. Therefore, the antileishmanial activity and mitochondrial mechanism of

  10. Combined effect of the essential oil from Chenopodium ambrosioides and antileishmanial drugs on promastigotes of Leishmania amazonensis Efeito combinado do óleo de essência de Chenopodium ambrosioides e drogas anti-leishmaniose nos promastigotas de Leishmania amazonensis

    Directory of Open Access Journals (Sweden)

    Lianet Monzote

    2007-08-01

    Full Text Available To date, there are no vaccines against Leishmania, and chemotherapy remains the mainstay for the control of leishmaniasis. The drugs of choice used for leishmaniasis therapy are significantly toxic, expensive and with a growing frequency of refractory infections. Because of these limitations, a combination therapy is the better hope. This work demonstrates that the essential oil from Chenopodium ambrosioides shows a synergic activity after incubation in conjunction with pentamidine against promastigotes of Leishmania amazonensis. However, an indifferent effect has been found for combinations of meglumine antimoniate or amphotericin B and the essential oil.Até hoje não temos vacina contra a Leishmania e a quimioterapia é a indicação para o controle desta doença. Os remédios que hoje utilizamos são tóxicos e muito caros e além disso o resultado não é sempre o desejado. Por isso, uma terapia de combinação é a melhor opção. Este trabalho mostra que o óleo de essência de C. ambrosioides tem atividade sinérgica junto com a pentamidina sobre os promastigotas de L. amazonensis, diferente do resultado da combinação de antimônio de meglumine e anfotericina B e o óleo de essência.

  11. Regulation of Leishmania (L.) amazonensis protein expression by host T cell dependent responses: differential expression of oligopeptidase B, tryparedoxin peroxidase and HSP70 isoforms in amastigotes isolated from BALB/c and BALB/c nude mice.

    Science.gov (United States)

    Teixeira, Priscila Camillo; Velasquez, Leonardo Garcia; Lepique, Ana Paula; de Rezende, Eloiza; Bonatto, José Matheus Camargo; Barcinski, Marcello Andre; Cunha-Neto, Edecio; Stolf, Beatriz Simonsen

    2015-02-01

    Leishmaniasis is an important disease that affects 12 million people in 88 countries, with 2 million new cases every year. Leishmania amazonensis is an important agent in Brazil, leading to clinical forms varying from localized (LCL) to diffuse cutaneous leishmaniasis (DCL). One interesting issue rarely analyzed is how host immune response affects Leishmania phenotype and virulence. Aiming to study the effect of host immune system on Leishmania proteins we compared proteomes of amastigotes isolated from BALB/c and BALB/c nude mice. The athymic nude mice may resemble patients with diffuse cutaneous leishmaniasis, considered T-cell hyposensitive or anergic to Leishmania's antigens. This work is the first to compare modifications in amastigotes' proteomes driven by host immune response. Among the 44 differentially expressed spots, there were proteins related to oxidative/nitrosative stress and proteases. Some correspond to known Leishmania virulence factors such as OPB and tryparedoxin peroxidase. Specific isoforms of these two proteins were increased in parasites from nude mice, suggesting that T cells probably restrain their posttranslational modifications in BALB/c mice. On the other hand, an isoform of HSP70 was increased in amastigotes from BALB/c mice. We believe our study may allow identification of potential virulence factors and ways of regulating their expression.

  12. Multiplex assay (Mikrogen recomBead) for detection of serum IgG and IgM antibodies to 13 recombinant antigens of Borrelia burgdorferi sensu lato in patients with neuroborreliosis

    DEFF Research Database (Denmark)

    Dessau, Ram Benny; Møller, Jens K.; Kolmos, Birte

    2015-01-01

    A multiplex-bead-based assay for the detection of serum antibodies to Borrelia burgdorferi sensu lato was evaluated. The assay contained 13 different antigens in both the IgG and the IgM assay; thus, a total of 26 measurement results were available from each sample. A total of 49 Danish patients...

  13. Recombination monitor

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, S. Y. [Brookhaven National Lab. (BNL), Upton, NY (United States); Blaskiewicz, M. [Brookhaven National Lab. (BNL), Upton, NY (United States)

    2017-02-03

    This is a brief report on LEReC recombination monitor design considerations. The recombination produced Au78+ ion rate is reviewed. Based on this two designs are discussed. One is to use the large dispersion lattice. It is shown that even with the large separation of the Au78+ beam from the Au79+ beam, the continued monitoring of the recombination is not possible. Accumulation of Au78+ ions is needed, plus collimation of the Au79+ beam. In another design, it is shown that the recombination monitor can be built based on the proposed scheme with the nominal lattice. From machine operation point of view, this design is preferable. Finally, possible studies and the alternative strategies with the basic goal of the monitor are discussed.

  14. Physalis angulata induces death of promastigotes and amastigotes of Leishmania (Leishmania) amazonensis via the generation of reactive oxygen species.

    Science.gov (United States)

    Da Silva, B J M; Da Silva, R R P; Rodrigues, A P D; Farias, L H S; Do Nascimento, J L M; Silva, E O

    2016-03-01

    Leishmaniasis are a neglected group of emerging diseases that have been found in 98 countries and are caused by protozoa of the genus Leishmania. The therapy for leishmaniasis causes several side effects and leads to drug-resistant strains. Natural products from plants have exhibited activities against Leishmania in various experimental models. Physalis angulata is a widely used plant in popular medicine, and in the literature it has well-documented leishmanicidal activity. However, its mechanism of action is still unknown. Thus, this study aims to evaluate the mechanism driving the leishmanicidal activity of an aqueous extract of P. angulata root (AEPa). AEPa was effective against both promastigotes and intracellular amastigote forms of Leishmania amazonensis. This effect was mediated by an increase of reactive oxygen species (ROS), but not of nitric oxide (NO). The increased production of ROS induces cell death by phenotypes seems by apoptosis cell death in Leishmania, but not autophagy or necrosis. In addition, morphological analysis of macrophages showed that AEPa induced a high number of cytoplasmic projections, increased the volume of cytoplasm and number of vacuoles, caused cytoskeleton alterations and resulted in high spreading ability. AEPa also promoted superoxide anion (O2(-)) production in both uninfected macrophages and those infected with Leishmania. Therefore, these results revealed that AEPa causes cell death by phenotypes seems by apoptosis cell death in L. amazonensis and modulates macrophage activation through morphofunctional alterations and O2(-) generation to induce Leishmania death.

  15. Nematodes of Amazonian birds, with a description of Hoazinstrongylus amazonensis n.gen.n.sp. (Trichostrongylidae, Libyostrongylinae

    Directory of Open Access Journals (Sweden)

    R. Magalhães Pinto

    1985-06-01

    Full Text Available During recent studies of the parasites of birds from the Amazonian Regio, the following nematodes were recovered: Hoazinstrongylus amazonensis n.gen.n.sp. from Opisthocomus hoazin (Muller, 1776; Ascaridia columbae (Gmel., 1790 Travassos, 1913, from Leptotila r. rufaxilla (Richard & Bernard, 1712 representing a new host record; Inglisakis ibanezi Freitas, Vicente & Santos, 1969, Cyrnea (C. semilunaris (Molin, 1860 Seurat, 1914 and Thelazia digitata Travassos, 1918. A compelte description is restrained to the new genus and new species here proposed. The other known and well described species are listed and accounted.Durante recentes estudos dos parasitas de aves capturadas na Região Amazônica, foram identificadas as seguintes espécies de nematóides: Hoazinstrongylus amazonensis n.gen.n.sp, de Opisthocomus hoazin (Muller, 1776, Ascaridia columbae (Gmel., 1790 Travassos, 1912, de Leptotila r. rufaxilla (Richard & Bernard, 1712 que se constitui em um novo hospedeiro para a espécie, Inglisakis ibanezi Freitas, Vicente & Santos, 1969, Cyrnea (C. semilunaris (Molin, 1860 Seurat, 1914 e Thelazia digitata Travassos, 1918.

  16. Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania amazonensis promastigotes

    Directory of Open Access Journals (Sweden)

    José Andrés Morgado-Díaz

    2005-07-01

    Full Text Available Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME as substrate, phenylmethylsulphone fluoride (PMSF and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate, but also in a crude plasma membrane fraction (2.0-fold. Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP, with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

  17. Western blot diagnosis of vivax malaria with multiple stage-specific antigens of the parasite

    OpenAIRE

    Son, Eui-Sun; Kim, Tong Soo; Nam, Ho-Woo

    2001-01-01

    Western blot analysis was performed to diagnose vivax malaria using stage-specific recombinant antigens. Genomic DNA from the whole blood of a malaria patient was used as templates to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), merozoite surface protein (MSP-1), apical merozoite antigen (AMA-1), serine repeat antigen (SERA), and exported antigen (EXP-1) of Plasmodium vivax. Each amplified DNA fragment was inserted into a pGEX-4T plasmid to induce ...

  18. Expression.purification and antigenicity of a recombinant respiratory syncyttal virus fusion gene encoded protein(amino acids 168-289)in the insect cells%呼吸道合胞病毒F蛋白第168~289氨基酸片段的昆虫细胞表达及其抗原性分析

    Institute of Scientific and Technical Information of China (English)

    单志娟; 项金忠

    2009-01-01

    目的 研究人呼吸道合胞病毒(RSV)F基因第546~881碱基编码的融合蛋白(即F蛋白)主要抗原性区域第168~289氨基酸片段的抗原性初步探讨其作为诊断抗原的应用价值.方法 用逆转录(RT)-PCR的方法扩增出RSV Long株F基因546~881 bp片段(F'),将其插入到转移载体质粒pBacPAK9中,获得相应的重组质粒pBacRSV F',与线性杆状病毒BacPAK6 DNA(Bsu36 Ⅰ酶切)共转染Sf9昆虫细胞获得重组病毒BacPAK F',并在昆虫细胞中表达.重组蛋白用Ni2+螫合柱亲和层析纯化,用免疫印迹(Western blot,WB)法检测重组蛋白的抗原活性.并用该方法检测33例急性下呼吸道感染患儿血清特异性抗体,同时用间接免疫荧光法检测患儿的鼻咽分泌物中RSV抗原.对两种方法检测标本的阴性率进行比较.结果 重组病毒BacPAK F'在昆虫细胞中表达相对分子质量约13 000的重组蛋白.纯化后的重组蛋白能与特异性抗RSV抗体结合.WB检测33例急性下呼吸道患儿血清特异性抗体,结果显示阳性11例,阳性率为33.3%,免疫荧光法检测结果阳性9例,阳性率为27.3%,2种检测方法阳性率差异无统计学意义(x2=0.29,P>0.05).结论 纯化后的在昆虫细胞中表达的RSV F基因546~881 bp片段编码的融合蛋白片段具有较强的抗原活性,对RSV感染的快速诊断具有一定的临床应用价值.%Objective To explore the antigenicity of the recombinant respiration syncytial virus (RSV)fusion protein (amino acids 168-289) encoded by 546-881 bases of the fusion gene expressed in insect baculoviruses expression system.Methods The fragment F' of fusion gene 546-881 bases was amplified from viral RNA( Long strain ) by the reverse transcription-polymerase chain reaction(RT-PCR).F' was inserted into transfer vector pBacPAK9 and the recombinant plasmid pBacRSV F' was constructed.Sf9 insect cells were then co-transfeeted with a mixture of recombinant plasmids pBacRSV F' and linearized BacPAK6 viral

  19. Recombinant Toxins for Cancer Treatment

    Science.gov (United States)

    Pastan, Ira; Fitzgerald, David

    1991-11-01

    Recombinant toxins target cell surface receptors and antigens on tumor cells. They kill by mechanisms different from conventional chemotherapy, so that cross resistance to conventional chemotherapeutic agents should not be a problem. Furthermore, they are not mutagens and should not induce secondary malignancies or accelerate progression of benign malignancies. They can be mass-produced cheaply in bacteria as homogeneous proteins. Either growth factor-toxin fusions or antibody-toxin fusions can be chosen, depending on the cellular target.

  20. The Effect of Ursolic Acid on Leishmania (Leishmania) amazonensis Is Related to Programed Cell Death and Presents Therapeutic Potential in Experimental Cutaneous Leishmaniasis

    Science.gov (United States)

    Yamamoto, Eduardo S.; Campos, Bruno L. S.; Jesus, Jéssica A.; Laurenti, Márcia D.; Ribeiro, Susan P.; Kallás, Esper G.; Rafael-Fernandes, Mariana; Santos-Gomes, Gabriela; Silva, Marcelo S.; Sessa, Deborah P.; Lago, João H. G.; Levy, Débora; Passero, Luiz F. D.

    2015-01-01

    Among neglected tropical diseases, leishmaniasis is one of the most important ones, affecting more than 12 million people worldwide. The available treatments are not well tolerated, and present diverse side effects, justifying the search for new therapeutic compounds. In the present study, the activity of ursolic acid (UA) and oleanolic acid (OA) were assayed in experimental cutaneous leishmaniasis (in vitro and in vivo). Promastigote forms of L. amazonensis were incubated with OA and UA for 24h, and effective concentration 50% (EC50) was estimated. Ultraestructural alterations in Leishmania amazonensis promastigotes after UA treatment were evaluated by transmission electron microscopy, and the possible mode of action was assayed through Annexin V and propidium iodide staining, caspase 3/7 activity, DNA fragmentation and transmembrane mitochondrial potential. The UA potential was evaluated in intracellular amastigotes, and its therapeutic potential was evaluated in L. amazonensis infected BALB/c mice. UA eliminated L. amazonensis promastigotes with an EC50 of 6.4 μg/mL, comparable with miltefosine, while OA presented only a marginal effect on promastigote forms at 100 μg/mL. The possible mechanism by which promastigotes were eliminated by UA was programmed cell death, independent of caspase 3/7, but it was highly dependent on mitochondria activity. UA was not toxic for peritoneal macrophages from BALB/c mice, and it was able to eliminate intracellular amastigotes, associated with nitric oxide (NO) production. OA did not eliminate amastigotes nor trigger NO. L. amazonensis infected BALB/c mice submitted to UA treatment presented lesser lesion size and parasitism compared to control. This study showed, for the first time, that UA eliminate promastigote forms through a mechanism associated with programed cell death, and importantly, was effective in vivo. Therefore, UA can be considered an interesting candidate for future tests as a prototype drug for the treatment

  1. Evaluating the Use of Commercial West Nile Virus Antigens as Positive Controls in the Rapid Analyte Measurement Platform West Nile Virus Assay.

    Science.gov (United States)

    Burkhalter, Kristen L; Savage, Harry M

    2015-12-01

    We evaluated the utility of 2 types of commercially available antigens as positive controls in the Rapid Analyte Measurement Platform (RAMP®) West Nile virus (WNV) assay. Purified recombinant WNV envelope antigens and whole killed virus antigens produced positive RAMP results and either type would be useful as a positive control. Killed virus antigens provide operational and economic advantages and we recommend their use over purified recombinant antigens. We also offer practical applications for RAMP positive controls and recommendations for preparing them.

  2. Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

    Science.gov (United States)

    Hattori, Takamitsu; Lai, Darson; Dementieva, Irina S.; Montaño, Sherwin P.; Kurosawa, Kohei; Zheng, Yupeng; Akin, Louesa R.; Świst-Rosowska, Kalina M.; Grzybowski, Adrian T.; Koide, Akiko; Krajewski, Krzysztof; Strahl, Brian D.; Kelleher, Neil L.; Ruthenburg, Alexander J.; Koide, Shohei

    2016-01-01

    Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. This “antigen clasping” produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody–antigen recognition and suggests a strategy for developing extremely specific antibodies. PMID:26862167

  3. Intranasal vaccination with leishmanial antigens protects golden hamsters (Mesocricetus auratus against Leishmania (Viannia Braziliensis infection.

    Directory of Open Access Journals (Sweden)

    Luzinei da Silva-Couto

    2015-01-01

    Full Text Available Previous results have shown that oral and intranasal administration of particulate Leishmania (Leishmania amazonensis antigens (LaAg partially protects mice against L. amazonensis infection. However, vaccination studies on species of the subgenus Viannia, the main causative agent of cutaneous and mucosal leishmaniasis in the Americas, have been hampered by the lack of easy-to-handle bio-models that accurately mimic the human disease. Recently, we demonstrated that the golden hamster is an appropriate model for studying the immunopathogenesis of cutaneous leishmaniasis caused by L. (Viannia braziliensis. Using the golden hamster model, our current study investigated whether the protective effect of intranasal immunisation with LaAg can be extended to L. braziliensis infection.Golden hamsters vaccinated with either two intranasal (IN doses of LaAg (10 µg or two intramuscular doses of LaAg (20 µg were challenged 2 weeks post-vaccination with L. braziliensis. The results showed that IN immunisation with LaAg significantly reduced lesion growth and parasitic load as well as serum IgG and IgG2 levels. At the experimental endpoint on day 114 post-infection, IN-immunised hamsters that were considered protected expressed IFN-γ and IL10 mRNA levels that returned to uninfected skin levels. In contrast to the nasal route, intramuscular (IM immunisation failed to provide protection.These results demonstrate for the first time that the nasal route of immunisation can induce cross protection against L. braziliensis infection.

  4. Identification of Antigenic Proteins from Lichtheimia corymbifera for Farmer’s Lung Disease Diagnosis

    Science.gov (United States)

    Rognon, Bénédicte; Barrera, Coralie; Monod, Michel; Valot, Benoit; Roussel, Sandrine; Quadroni, Manfredo; Jouneau, Stephane; Court-Fortune, Isabelle; Caillaud, Denis; Fellrath, Jean-Marc; Dalphin, Jean-Charles; Reboux, Gabriel; Millon, Laurence

    2016-01-01

    The use of recombinant antigens has been shown to improve both the sensitivity and the standardization of the serological diagnosis of Farmer’s lung disease (FLD). The aim of this study was to complete the panel of recombinant antigens available for FLD serodiagnosis with antigens of Lichtheimia corymbifera, known to be involved in FLD. L. corymbifera proteins were thus separated by 2D electrophoresis and subjected to western blotting with sera from 7 patients with FLD and 9 healthy exposed controls (HEC). FLD-associated immunoreactive proteins were identified by mass spectrometry based on a protein database specifically created for this study and subsequently produced as recombinant antigens. The ability of recombinant antigens to discriminate patients with FLD from controls was assessed by ELISA performed with sera from FLD patients (n = 41) and controls (n = 43) recruited from five university hospital pneumology departments of France and Switzerland. Forty-one FLD-associated immunoreactive proteins from L. corymbifera were identified. Six of them were produced as recombinant antigens. With a sensitivity and specificity of 81.4 and 77.3% respectively, dihydrolipoyl dehydrogenase was the most effective antigen for discriminating FLD patients from HEC. ELISA performed with the putative proteasome subunit alpha type as an antigen was especially specific (88.6%) and could thus be used for FLD confirmation. The production of recombinant antigens from L. corymbifera represents an additional step towards the development of a standardized ELISA kit for FLD diagnosis. PMID:27490813

  5. 刚地弓形虫P30(SAG1)重组抗原的高效表达和体外纯化%Over-expression and purification of the recombinant p30 antigen of Toxoplasma gondii

    Institute of Scientific and Technical Information of China (English)

    张佃波; 宰德富; 公茂庆; 李瑾; 魏庆宽; 崔勇; 黄炳成; 刘克义

    2005-01-01

    目的表达和纯化弓形虫P30(SAG1)蛋白,为弓形虫病快速诊断试剂盒及蛋白质疫苗的研制奠定基础.方法 PCR法从弓形虫基因组DNA 中扩增 P30基因片段,P30产物克隆到表达质粒pET-30a(+) 构建重组载体,将其转化到DH5α中.经PCR扩增和质粒酶切及基因测序鉴定后,阳性重组质粒转化到大肠埃氏菌BL21(DE3)中,经IPTG诱导,表达产物用SDS-PAGE和Western blot进行鉴定.大量的诱导表达产物用SNBC 3S NTA Resin方法纯化并进行复性.结果扩增的P30基因片段为750bp, 重组表达融合蛋白量单位为30ku,与理论值相符.结论成功构建重组体,获得纯化和复性的弓形虫主要表面抗原P30的高效表达产物,为弓形虫病的诊断和疫苗研究奠定了基础.%To provide the basis for preparation of diagnostic kits and vaccines in Toxoplasma gondii infection, the gene coding for the qualified recombinant p30 protein (SAG1) of this parasite was amplified by PCR, and the amplified gene was cloned into prokaryotic expression vector pET-30a(+) to construct the recombinant plasmid, and then transformed to E.coli DH5α. The positive recombinant plasmid was screened by PCR and double enzymes digestion, and the nucleotide sequence of p30 gene was determined by automated DNA sequencing. Meanwhile, the identified recombinant plasmid was transformed to E.coli BL21(DE3) with the expression of p30 on bacteria induced by IPTG and the expressed protein was identified by SDS-PAGE. The protein obtained was then further purified and refolded, and its biological activity was checked by Western blotting. It was shown that the size of the amplified gene was 750 bp with molecular weight of 30 ku, and this protein could specifically react with monoclonal antibody against p30 protein.

  6. Evaluation of the immunogenicity of recombinant replicative DNA vaccines expressing multiple anti-gens of hepatitis C virus in a mice model%表达丙型肝炎病毒多个融合抗原的复制型DNA 疫苗在小鼠体内免疫原性的研究

    Institute of Scientific and Technical Information of China (English)

    邓瑶; 管洁; 殷霄; 文波; 陈红; 王文; 谭文杰

    2015-01-01

    目的:探索新型的基于丙型肝炎病毒( HCV)多个靶抗原的复制型DNA疫苗的免疫效果。方法本研究构建了两种表达基于HCV中国1b代表株(河北株)多个靶抗原的复制型DNA疫苗:pSCK-CE1E2Y(核心蛋白Core、包膜蛋白E1、E2抗原)和pSCK-H155(Core、E1、E2、非结构蛋白NS3融合抗原),蛋白印迹试验分析靶抗原表达,皮内电转法免疫BALB/c小鼠后评价其免疫应答,采用表达异型( JFH1,2 a型) HCV多聚蛋白的重组痘苗病毒接种的小鼠攻击替代模型进行保护效果分析。结果蛋白印迹试验表明DNA疫苗中靶抗原体外可有效表达;两种DNA疫苗均可诱导多抗原特异的抗体反应及T细胞免疫应答;未检测到明显的交叉免疫保护。结论两种复制型DNA疫苗均可诱导针对多抗原的体液及细胞免疫应答,但不能诱导有效的交叉保护。本研究为HCV新型疫苗研制提供了一定参考。%Objective To investigate the immunogenicity and cross protective effects of two novel HCV DNA vaccines in a mice model.Methods Two self-replicating alphavirus vector-based HCV DNA vaccines, pSCK CE1E2Y and pSCK H155, were constructed based on the genes encoding the structural pro-teins (Core, E1 and E2) and structural and NS3 fusion proteins (Core, E1 , E2 and NS3) of a HCV strain isolated from a Chinese patient (genotype 1b, Hebei strain), respectively.Western blot analysis was per-formed to detect the expression of fusion antigens.The BALB/c mice were intradermally immunized with the recombinant DNA vaccines by using electroporation.The immune responses induced in mice and the cross protective effects of the recombinant DNA vaccines were evaluated.Results The DNA vaccines effectively expressed the target antigens in vitro.The antigen-specific antibody responses and specific T cell immune re-sponses were induced in mice by the immunization of replicative DNA vaccines.However, no effective cross

  7. Recombinant horseradish peroxidase: production and analytical applications.

    Science.gov (United States)

    Grigorenko, V G; Andreeva, I P; Rubtsova, M Yu; Egorov, A M

    2015-04-01

    Horseradish peroxidase is a key enzyme in bio- and immunochemical analysis. New approaches in functional expression of the peroxidase gene in E. coli cells and the subsequent refolding of the resulting protein yield a recombinant enzyme that is comparable in its spectral and catalytic characteristics to the native plant peroxidase. Genetic engineering approaches allow production of recombinant peroxidase conjugates with both protein antigens and Fab antibody fragments. The present article reviews the use of recombinant horseradish peroxidase as the marker enzyme in ELISA procedures as well as in amperometric sensors based on direct electron transfer.

  8. In vitro activity of the hydroethanolic extract and biflavonoids isolated from Selaginella sellowii on Leishmania (Leishmania) amazonensis

    Science.gov (United States)

    Rizk, Yasmin Silva; Fischer, Alice; Cunha, Marillin de Castro; Rodrigues, Patrik Oening; Marques, Maria Carolina Silva; Matos, Maria de Fátima Cepa; Kadri, Mônica Cristina Toffoli; Carollo, Carlos Alexandre; de Arruda, Carla Cardozo Pinto

    2014-01-01

    This study is the first phytochemical investigation of Selaginella sellowii and demonstrates the antileishmanial activity of the hydroethanolic extract from this plant (SSHE), as well as of the biflavonoids amentoflavone and robustaflavone, isolated from this species. The effects of these substances were evaluated on intracellular amastigotes of Leishmania (Leishmania) amazonensis, an aetiological agent of American cutaneous leishmaniasis. SSHE was highly active against intracellular amastigotes [the half maximum inhibitory concentration (IC50) = 20.2 µg/mL]. Fractionation of the extract led to the isolation of the two bioflavonoids with the highest activity: amentoflavone, which was about 200 times more active (IC50 = 0.1 μg/mL) and less cytotoxic than SSHE (IC50 = 2.2 and 3 μg/mL, respectively on NIH/3T3 and J774.A1 cells), with a high selectivity index (SI) (22 and 30), robustaflavone, which was also active against L. amazonensis (IC50 = 2.8 µg/mL), but more cytotoxic, with IC50 = 25.5 µg/mL (SI = 9.1) on NIH/3T3 cells and IC50 = 3.1 µg/mL (SI = 1.1) on J774.A1 cells. The production of nitric oxide (NO) was lower in cells treated with amentoflavone (suggesting that NO does not contribute to the leishmanicidal mechanism in this case), while NO release was higher after treatment with robustaflavone. S. sellowii may be a potential source of biflavonoids that could provide promising compounds for the treatment of cutaneous leishmaniasis. PMID:25591109

  9. 重组多价人精子表位肽的原核表达及其条件优化%Prokaryotic expression and expression condition optimizing on recombinant multi-epitopes peptide of human sperm antigens

    Institute of Scientific and Technical Information of China (English)

    谢琦; 林军; 白玲; 石青峰; 叶元; 杨冰; 刘永明

    2012-01-01

    目的 构建重组多价人精子表位肽原核表达质粒,优化其在大肠杆菌中的表达条件.方法 用化学合成法合成多价人精子表位肽基因.该基因经PCR扩增,BamHI和XhoI双酶切后,克隆至GST融合表达载体PGEX-4T-1获得重组质粒.将该重组质粒转化E.coli BL21 (DE3),经IPTG诱导表达.利用SDS-PAGE电泳和AlphaEase凝胶电泳图像分析系统,观察在摇瓶发酵条件下改变培养基组成、诱导温度、诱导时机、诱导剂浓度和诱导时间等条件对GST-多价人精子表位肽融合蛋白表达量的影响.结果 构建了重组多价人精子表位肽原核表达载体.在摇瓶实验中,优化的表达条件为:TB培养基,诱导温度37℃,在菌体对数生长的中后期进行诱导,IPTG诱导终浓度0.05 mmol/L,诱导时间5h.优化后GST-多价人精子表位肽融合蛋白的表达量占菌体总蛋白的30.2%,且主要以可溶形式表达.结论 成功构建重组多价人精子表位肽原核表达质粒,重组表达载体在E.coli BL21 (DE3)内主要表达可溶形式的GST-多价人精子表位肽融合蛋白,在优化条件下可获得较高的表达量.%OBJECTIVE To construct the prokaryotic expression recombinant plasmid PGEX-4T-l/AG10-IR9-YLP12 and optimize the expression conditions of GST- AG10-IR9-YLP12 fusion protein in E.coli. METHODS The AG10-IR9-YLP12 gene was synthesized chemically. The target gene was amplified by polymerase chain reaction ( PCR). After being digested with BamHI and Xhol, the target gene was cloned into GST fusion expression vector PGEX-4T-1. E. coli BL21 (DE3) was trans-fected with the recombinant plasmid and GST- AG10-1R9-YLP12 fusion protein was induced by IPTG. Under the shake flask fermentation conditions, the factors that may be involved in the recombinant gene expression such as culture medium, induction temperature, induction period, inducer concentration and induction time on the expression level of target protein were investigated with SDS

  10. Dengue Type-2 Virus Envelope Protein Made Using Recombinant Baculovirus Protects Mice Against Virus Challenge

    Science.gov (United States)

    1994-01-01

    Spodoptera frugiperda (Sf9) cells, approximately I mg of recombinant E antigen was made per 10’ cells. This antigen reacted with polyclonal, anti...entry by fusion at acidic pH with host cell mem- in Spodoptera frugiperda (Sf9) cells brane.Ř The E antigen contains both T and B cell epitopes that

  11. Treatment of Leishmania (Leishmania) Amazonensis-Infected Mice with a Combination of a Palladacycle Complex and Heat-Killed Propionibacterium acnes Triggers Protective Cellular Immune Responses

    Science.gov (United States)

    Paladi, Carolina S.; da Silva, Danielle A. M.; Motta, Priscila D.; Garcia, Daniel M.; Teixeira, Daniela; Longo-Maugéri, Ieda M.; Katz, Simone; Barbiéri, Clara L.

    2017-01-01

    Palladacycle complex DPPE 1.2 was previously reported to inhibit the in vitro and in vivo infection by Leishmania (Leishmania) amazonensis. The aim of the present study was to compare the effect of DPPE 1.2, in association with heat-killed Propionibacterium acnes, on L. (L.) amazonensis infection in two mouse strains, BALB/c and C57BL/6, and to evaluate the immune responses of the treated animals. Foot lesions of L. (L.) amazonensis-infected mice were injected with DPPE 1.2 alone, or associated with P. acnes as an adjuvant. Analysis of T-cell populations in the treated mice and in untreated controls was performed by FACS. Detection of IFN-γ-secreting lymphocytes was carried out by an ELISPOT assay and active TGF-β was measured by means of a double-sandwich ELISA test. The treatment with DPPE 1.2 resulted in a significant reduction of foot lesion sizes and parasite burdens in both mouse strains, and the lowest parasite burden was found in mice treated with DPPE 1.2 plus P. acnes. Mice treated with DPPE 1.2 alone displayed a significant increase of TCD4+ and TCD8+ lymphocytes and IFN-γ secretion which were significantly higher in animals treated with DPPE 1.2 plus P. acnes. A significant reduction of active TGF-β was observed in mice treated with DPPE 1.2 alone or associated with P. acnes. Moreover, DPPE 1.2 associated to P. acnes was non-toxic to treated animals. The destruction of L. (L.) amazonensis by DPPE 1.2 was followed by host inflammatory responses which were exacerbated when the palladacycle complex was associated with P. acnes. PMID:28321209

  12. 基因重组HCV抗原斑点酶免疫法检测血清中抗-HCV%Immunoblotting Assay Using Recombinant Antigens for Detecting Antibodies against HCV

    Institute of Scientific and Technical Information of China (English)

    王百锁; 胡兴菊; 向前; 邢爱华; 王敬军

    2001-01-01

    目的 建立检测抗-HCV的斑点酶免疫法。方法 将基因重组HCV核心、NS3/NS4、NS5抗原和马抗人IgG、人IgG以最佳浓度固定在硝酸纤维膜条带上,用辣根过氧化酶和改进的底物进行斑点酶免疫反应。结果 检测了14份经ORTHO第三代抗-HCVELISA检测的血清,符合率为100%;72份经UBI第三代抗-HCVELISA监测的血清,符合率95.83%;103份经国产厦门新创抗-HCVELISA监测的血清,符合率95.14%。54份自然人群血清,阳性率3.47%。89份阳性标本中,三种抗体同时阳性的占30.34%,两种抗体阳性占69.66%,没有检出单一抗体阳性标本。核心抗体检出率最高87.64%,NS3/NS4抗体次之76.40%,NS5抗体最低61.80%。结论 用基因重组抗原建立的斑点酶免疫法与国内外广泛应用的ELISA试剂有较高的符合率,对进一步研究HCV感染诊断的确认试剂有意义。%Objective To set up a configuration of immunoblo tting assay forcletecting serum antibodies against HCV.Methods The proteins of the said virus that Core and NS3/NS4 and NS5 expres sed in E.coli cells were immobilized separately on nitrocellulose filter as antigens.HRP and a modified preparation of chromogenic substrate were used in the following procedure.Results Among 189 samples of sera tested by this Blotting Assay,14 were tested also by using ORTHO 3rd Generation ELISA regent and the coincident rate was 100% between the two assays,72 were tested by UBI 3rd Generation ELISA and their coincident rate was 95.83%,103 were tested by Xia Meng Xin Chuang ELISA and the coincident rate was 95.14%,Among 89 posi tive sera determined by Blotting Assay,those showed positive for all three antig ens accounted for 30.34%,and those positive for two of three antigens accounted for 69.66%,positive for only one antigen was not found.Among the 3 antibodies of Core was most efficient that 78 out of 89 positive sera reacted to it,the next was NS3/NS4with that number of 68

  13. Insulin-Like Growth Factor-I Induces Arginase Activity in Leishmania amazonensis Amastigote-Infected Macrophages through a Cytokine-Independent Mechanism

    Directory of Open Access Journals (Sweden)

    Celia Maria Vieira Vendrame

    2014-01-01

    Full Text Available Leishmania (Leishmania amazonensis exhibits peculiarities in its interactions with hosts. Because amastigotes are the primary form associated with the progression of infection, we studied the effect of insulin-like growth factor (IGF-I on interactions between L. (L. amazonensis amastigotes and macrophages. Upon stimulation of infected macrophages with IGF-I, we observed decreased nitric oxide production but increased arginase expression and activity, which lead to increased parasitism. However, stimulation of amastigote-infected macrophages with IGF-I did not result in altered cytokine levels compared to unstimulated controls. Because IGF-I is present in tissue fluids and also within macrophages, we examined the possible effect of this factor on phosphatidylserine (PS exposure on amastigotes, seen previously in tissue-derived amastigotes leading to increased parasitism. Stimulation with IGF-I induced PS exposure on amastigotes but not on promastigotes. Using a PS-liposome instead of amastigotes, we observed that the PS-liposome but not the control phosphatidylcholine-liposome led to increased arginase activity in macrophages, and this process was not blocked by anti-TGF-β antibodies. Our results suggest that in L. (L. amazonensis amastigote-infected macrophages, IGF-I induces arginase activity directly in amastigotes and in macrophages through the induction of PS exposure on amastigotes in the latter, which could lead to the alternative activation of macrophages through cytokine-independent mechanisms.

  14. Preliminary X-ray diffraction analysis of crystals from the recombinantly expressed human major histocompatibility antigen HLA-B*2704 in complex with a viral peptide and with a self-peptide

    Energy Technology Data Exchange (ETDEWEB)

    Loll, Bernhard [Institut für Chemie/Kristallographie, Freie Universität Berlin, Takustrasse 6, 14195 Berlin (Germany); Zawacka, Anna [Institut für Immungenetik, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Humboldt-Universität zu Berlin, Spandauer Damm 130, 14050 Berlin (Germany); Biesiadka, Jacek [Institut für Chemie/Kristallographie, Freie Universität Berlin, Takustrasse 6, 14195 Berlin (Germany); Petter, Cordula; Rückert, Christine [Institut für Immungenetik, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Humboldt-Universität zu Berlin, Spandauer Damm 130, 14050 Berlin (Germany); Saenger, Wolfram [Institut für Chemie/Kristallographie, Freie Universität Berlin, Takustrasse 6, 14195 Berlin (Germany); Uchanska-Ziegler, Barbara; Ziegler, Andreas, E-mail: andreas.ziegler@charite.de [Institut für Immungenetik, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Humboldt-Universität zu Berlin, Spandauer Damm 130, 14050 Berlin (Germany); Institut für Chemie/Kristallographie, Freie Universität Berlin, Takustrasse 6, 14195 Berlin (Germany)

    2005-10-01

    Crystallization of HLA-B*2704 in complex with two peptides. The product of the human leukocyte antigen (HLA) gene HLA-B*2704 differs from that of the prototypical subtype HLA-B*2705 by three amino acids at heavy-chain residues 77 (Ser instead of Asp), 152 (Glu instead of Val) and 211 (Gly instead of Ala). In contrast to the ubiquitous HLA-B*2705 subtype, HLA-B*2704 occurs only in orientals. Both subtypes are strongly associated with spondyloarthropathies and the peptides presented by these subtypes are suspected to play a role in disease pathogenesis. HLA-B*2704 was crystallized in complex with a viral peptide and with a self-peptide using the hanging-drop vapour-diffusion method with PEG as a precipitant. Both crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}. Data sets were collected to 1.60 Å (complex with the self-peptide pVIPR) or to 1.90 Å (complex with the viral peptide pLMP2) resolution using synchrotron radiation. With HLA-B*2705 complexed with pVIPR as a search model, unambiguous molecular-replacement solutions were found for the complexes of HLA-B*2704 with both peptides.

  15. Immunological response to parenteral vaccination with recombinant hepatitis B virus surface antigen virus-like particles expressing Helicobacter pylori KatA epitopes in a murine H. pylori challenge model.

    Science.gov (United States)

    Kotiw, Michael; Johnson, Megan; Pandey, Manisha; Fry, Scott; Hazell, Stuart L; Netter, Hans J; Good, Michael F; Olive, Colleen

    2012-02-01

    Virus-like particles (VLPs) based on the small envelope protein of hepatitis B virus (HBsAg-S) are immunogenic at the B- and T-cell level. In this study, we inserted overlapping sequences encoding the carboxy terminus of the Helicobacter pylori katA gene product into HBsAg-S. The HBsAg-S-KatA fusion proteins were able to assemble into secretion-competent VLPs (VLP-KatA). The VLP-KatA proteins were able to induce KatA-specific antibodies in immunized mice. The mean total IgG antibody titers 41 days post-primary immunization with VLP-KatA (2.3 × 10(3)) were significantly greater (P < 0.05) than those observed for vaccination with VLP alone (5.2 × 10(2)). Measurement of IgG isotypes revealed responses to both IgG1 and IgG2a (mean titers, 9.0 × 10(4) and 2.6 × 10(4), respectively), with the IgG2a response to vaccination with VLP-KatA being significantly higher than that for mice immunized with KatA alone (P < 0.05). Following challenge of mice with H. pylori, a significantly reduced bacterial load in the gastric mucosa was observed (P < 0.05). This is the first report describing the use of VLPs as a delivery vehicle for H. pylori antigens.

  16. Cloning and expression of recombinant human testis specific antigen - 1 related to infertility in E. coli%重组人睾丸特异性抗原-1(TSA-1)的基因克隆及原核表达

    Institute of Scientific and Technical Information of China (English)

    周晓宁; 武婕; 张宏斌; 王捷

    2008-01-01

    目的 为了阐明血清中存在的抗精子抗体引起不孕不育的原因,构建重组人睾丸特异性抗原-1(recombinanthuman testis specie antigen-1,rhTSA-1)的原核表达质粒并在大肠杆菌中诱导表达.方法 采用RT-PCR法,从人睾丸组织总RNA中扩增获得人TSA-1的cDNA,将其克隆入表达载体pGEX-4T-2,构建TSA-1的重组原核表达质粒pGEX/TSA-1.重组质粒经酶切和测序鉴定后,转化大肠杆菌JM109并在大肠杆菌中诱导表达目的 蛋白.结果 测序表明,重组基因序列与人TSA-1基因完全一致.SDS-PAGE电泳显示,表达产物的相对分子量与预期结果相符.结论 获得了人TSA-1的编码基因,并在大肠杆菌中可表达人TSA-1.

  17. Vaccination with a recombinant protein encoding the tumor-specific antigen NY-ESO-1 elicits an A2/157-165-specific CTL repertoire structurally distinct and of reduced tumor reactivity than that elicited by spontaneous immune responses to NY-ESO-1-expressing Tumors.

    Science.gov (United States)

    Bioley, Gilles; Guillaume, Philippe; Luescher, Immanuel; Bhardwaj, Nina; Mears, Gregory; Old, Lloyd; Valmori, Danila; Ayyoub, Maha

    2009-01-01

    In a recent vaccination trial assessing the immunogenicity of an NY-ESO-1 (ESO) recombinant protein administered with Montanide and CpG, we have obtained evidence that this vaccine induces specific cytolytic T lymphocytes (CTL) in half of the patients. Most vaccine-induced CTLs were directed against epitopes located in the central part of the protein, between amino acids 81 and 110. This immunodominant region, however, is distinct from another ESO CTL region, 157-165, that is a frequent target of spontaneous CTL responses in A2+ patients bearing ESO tumors. In this study, we have investigated the CTL responses to ESO 157-165 in A2+ patients vaccinated with the recombinant protein. Our data indicate that after vaccination with the protein, CTL responses to ESO 157-165 are induced in some, but not all, A2+ patients. ESO 157-165-specific CTLs induced by vaccination with the ESO protein were functionally heterogeneous in terms of tumor recognition and often displayed decreased tumor reactivity as compared with ESO 157-165-specific CTLs isolated from patients with spontaneous immune responses to ESO. Remarkably, protein-induced CTLs used T-cell receptors similar to those previously isolated from patients vaccinated with synthetic ESO peptides (Vbeta4.1) and distinct from those used by highly tumor-reactive CTLs isolated from patients with spontaneous immune responses (Vbeta1.1, Vbeta8.1, and Vbeta13.1). Together, these results demonstrate that vaccination with the ESO protein elicits a repertoire of ESO 157-165-specific CTLs bearing T-cell receptors that are structurally distinct from those of CTLs found in spontaneous immune responses to the antigen and that are heterogeneous in terms of tumor reactivity, being often poorly tumor reactive.

  18. Evaluation of the immune response to CRA and FRA recombinant antigens of Trypanosoma cruzi in C57BL/6 mice Avaliação da resposta imune em camundongos C57BL/6 imunizados com os antígenos recombinantes CRA e FRA de Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Valéria Rêgo Alves Pereira

    2003-07-01

    Full Text Available Humoral and cellular immune responses were evaluated in 44 C57BL/6 mice immunized with the Trypanosoma cruzi recombinant antigens CRA and FRA. Both antigens induced cutaneous immediate-type hypersensitivity response. The levels of IgG1, IgG2a, IgG2b and IgG3 were high in CRA immunized mice. IgG3 was the predominant isotype. Although no difference in antibody levels was observed in FRA-immunized mice when compared to control mice, both antigens were able to induce lymphoproliferation in immunized mice. Significant differences were observed between incorporation of [³H]- thymidine by spleen cell stimulated in vitro with CRA or FRA and the control group. These results suggest that CRA and FRA could be involved in mechanisms of resistance to Trypanosoma cruzi infection.As respostas imune humoral e celular foram avaliadas em 44 camundongos C57Bl/6 imunizados com os antígenos recombinantes CRA e FRA de Trypanosoma cruzi. Ambos antígenos induziram reação de hipersensibilidade do tipo imediato. Os níveis de IgG1, IgG2a, IgG2b e IgG3 foram elevados nos camundongos imunizados com CRA. IgG3 foi o isotipo predominante. Nenhuma diferença nos níveis de anticorpos foi observada em camundongos imunizados com FRA em relação aos animais controle. No entanto, ambos antígenos foram capazes de induzir proliferação de linfócitos em camundongos imunizados. Diferenças significativas foram observadas entre a incorporação da timidina - [³H] pelas células esplênicas estimuladas com CRA ou FRA e o grupo controle. Esses resultados sugerem que CRA e FRA poderão estar envolvidos nos mecanismos de resistência à infecção pelo Trypanosoma cruzi.

  19. Gorse GJ, et al. "Immunogenicity and tolerance of ascending doses of a recombinant protective antigen (rPA102) anthrax vaccine: a randomized, double-blinded, controlled, multicenter trial" [Vaccine 24 (2006) 5950-5959].

    Science.gov (United States)

    Zink, Thomas K

    2007-04-12

    In the study reported by Gorse et al. a unique, educational opportunity was lost. The vaccine and biodefense communities almost experienced the rare chance in a Phase I study to scientifically compare head-to-head an early-stage, investigational recombinant anthrax vaccine (rPA102) with the safe, effective and already FDA-licensed anthrax vaccine, AVA (BioThrax). The authors take a stab at making safety and immunogenicity comparisons between the candidate vaccine and AVA (BioThrax) but the study design and analytical approach makes this inappropriate. Inaccurate and poorly substantiated editorial comments in the paper's introduction compound these methodological problems. The reader is presented with a series of false and misleading statements about AVA (BioThrax). Out-of-date sources are relied upon and these references are offered to the reader as the best evidence available when more current papers with up-to-date information and data exist. Additionally, the conclusions in several original contributions are misrepresented in this paper by Gorse et al. Issues with protocol and bias notwithstanding, the single most compelling observation from this trial could be that the response of those subjects in this study population (n=19) who received AVA on the altered schedule and route of two doses of AVA (BioThrax) delivered intramuscularly (IM) in just 4 weeks mounted a robust immune response. Given the more than 30 year history of the safe and effective use of AVA (BioThrax) as well as the more current data on AVA (BioThrax) a strong case can be made for continued funding to investigate the feasibility of adding another route of delivery (IM) and optimizing the schedule for this already FDA-licensed vaccine.

  20. Effects of priming with recombinant human granulocyte colony-stimulating factor on conditioning regimen for high-risk acute myeloid leukemia patients undergoing human leukocyte antigen-haploidentical hematopoietic stem cell transplantation: a multicenter randomized controlled study in southwest China.

    Science.gov (United States)

    Gao, Lei; Wen, Qin; Chen, Xinghua; Liu, Yao; Zhang, Cheng; Gao, Li; Kong, Peiyan; Zhang, Yanqi; Li, Yunlong; Liu, Jia; Wang, Qingyu; Su, Yi; Wang, Chunsen; Wang, Sanbin; Zeng, Yun; Sun, Aihua; Du, Xin; Zeng, Dongfeng; Liu, Hong; Peng, Xiangui; Zhang, Xi

    2014-12-01

    HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is an effective and immediate treatment for high-risk acute myeloid leukemia (HR-AML) patients lacking matched donors. Relapse remains the leading cause of death for HR-AML patients after haplo-HSCT. Accordingly, the prevention of relapse remains a challenge in the treatment of HR-AML. In a multicenter randomized controlled trial in southwestern China, 178 HR-AML patients received haplo-HSCT with conditioning regimens involving recombinant human granulocyte colony-stimulating factor (rhG-CSF) or non-rhG-CSF. The cumulative incidences of relapse and graft-versus-host disease (GVHD), 2-year leukemia-free survival (LFS), and overall survival (OS) were evaluated. HR-AML patients who underwent the priming conditioning regimen with rhG-CSF had a lower relapse rate than those who were treated with non-rhG-CSF (38.2%; 95% confidence interval [CI], 28.1% to 48.3% versus 60.7%, 95% CI, 50.5% to 70.8%; P priming group and 31 patients in the non-rhG-CSF-priming group were still alive at the median follow-up time of 42 months (range, 24 to 80 months). The 2-year probabilities of LFS and OS in the rhG-CSF-priming and non-rhG-CSF-priming groups were 55.1% (95% CI, 44.7% to 65.4%) versus 32.6% (95% CI, 22.8% to 42.3%) (P priming group (67.4%; 95% CI, 53.8% to 80.9% versus 41.9%; 95% CI, 27.1% to 56.6%; P priming conditioning regimen is an acceptable choice for HR-AML patients, especially for the patients with no M4/M5/M6 subtype who achieved CR before transplantation.

  1. Streptomyces as host for recombinant production of Mycobacterium tuberculosis proteins.

    Science.gov (United States)

    Vallin, Carlos; Ramos, Astrid; Pimienta, Elsa; Rodríguez, Caridad; Hernández, Tairí; Hernández, Ivones; Del Sol, Ricardo; Rosabal, Grisel; Van Mellaert, Lieve; Anné, Jozef

    2006-01-01

    The 45/47 kDa APA protein (Rv1860) of Mycobacterium tuberculosis was produced by Streptomyces lividans. The recombinant protein could be recovered from the culture medium of an S. lividans clone containing the apa gene under control of the promoter and signal sequence of the Streptomyces coelicolor agarase gene. The recombinant protein production was further scaled-up using fermentation conditions. The APA protein was subsequently purified from the culture supernatant by means of immunochromatography. About 80 mg of recombinant protein were obtained per liter of culture media. In vivo tests with the APA protein purified from S. lividans TK24/pRGAPA1 revealed that the recombinant protein was antigenic and could induce high titers of specific antibodies in the mouse biological model. Results obtained concerning heterologous production of APA, its immunogenic and antigenic capacity, demonstrated the potential of S. lividans as a valuable host for the production of recombinant proteins from M. tuberculosis.

  2. Sequence analysis of secretory antigen P53 and immunological identification of the recombinant product of Trichinella spiralis%旋毛形线虫分泌抗原P53的序列分析及重组表达产物的免疫学鉴定

    Institute of Scientific and Technical Information of China (English)

    徐鸿绪; 武伟华; 毛玉玲; 梁健; 谌嘉嘉; 胡旭初

    2010-01-01

    Objective To analyze the immunological characteristics of Trichinella spiralis secretory antigen P53 and to evaluate its value in diagnosis of trichinellosis. Methods An open read frame of secretory antigen P53 was cloned from Trichinella spiralis by reverse transcriptasepolymerase chain reaction (RT-PCR) and then sequenced. Bioinformatics analysis was performed to search for its homologues in other helminths and predict its potential linear B cell epitopes and T cell epitopes. The sequence coding mature peptide was inserted into prokaryotic expression vector pET28a(+) and the purified recombinant product was identified by Western blot using serum samples of patients infected with Trichinella spiralis or other helminth. Results Bioinformaties analysis results showed that there was no P53 homologue in other helminths, which indicated that there were many linear B cell epitopes and T cell epitopes in TsP53. The recombinant P53 antigen only reacted with the serum samples of patients infected with Trichinella spiralis without any cross-reaction with the serum of patients infected with other helminths. Conclusion P53 has strong immunogenicity and immunoreactivity, which may be a promising candidate for developing Trichinella spiralis specific diagnostic method.%目的 分析旋毛形线虫分泌抗原P53的免疫学特性,评价其免疫诊断价值.方法 克隆并测序分析旋毛形线虫P53的编码区序列,应用生物信息学分析其与其他蠕虫同源蛋白的相似性以及潜在的线性B细胞和T细胞表位;并将成熟肽编码区克隆至pET28a(+)原核表达质粒中,纯化的重组蛋白用旋毛形线虫感染血清和其他寄生蠕虫感染血清经Western印迹法鉴定其免疫反应性.结果 生物信息学分析未发现其他寄生蠕虫的P53同源基因,表明TsP53具有多个T细胞和B细胞识别表位,Western印迹法显示TsP53抗原只与旋毛形线虫感染血清反应,与其他几种蠕虫感染血清无反应.结论 P53具有

  3. Lipophosphoglycans from Leishmania amazonensis Strains Display Immunomodulatory Properties via TLR4 and Do Not Affect Sand Fly Infection

    Science.gov (United States)

    Nogueira, Paula M.; Assis, Rafael R.; Torrecilhas, Ana C.; Saraiva, Elvira M.; Pessoa, Natália L.; Campos, Marco A.; Marialva, Eric F.; Ríos-Velasquez, Cláudia M.; Pessoa, Felipe A.; Secundino, Nágila F.; Rugani, Jerônimo N.; Nieves, Elsa; Turco, Salvatore J.; Melo, Maria N.

    2016-01-01

    The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly. PMID:27508930

  4. Exploring the unbinding of Leishmania (L.) amazonensis CPB derived-epitopes from H2 MHC class I proteins.

    Science.gov (United States)

    Brandt, Artur M L; Batista, Paulo Ricardo; Souza-Silva, Franklin; Alves, Carlos Roberto; Caffarena, Ernesto Raul

    2016-04-01

    New strategies to control Leishmania disease demand an extensive knowledge about several aspects of infection including the understanding of its molecular events. In murine models, cysteine proteinase B from Leishmania amazonensis promotes regulation of immune response, and fragments from its C-terminus extension (cyspep) can play a decisive role in the host-parasite interaction. The interaction between cyspep-derived peptides and major histocompatibility complex (MHC) proteins is a crucial factor in Leishmania infections. Seven cyspep-derived peptides, previously identified as capable of interacting with H-2 (murine) MHC class I proteins, were studied in this work. We established a protocol to simulate the unbinding of these peptides from the cleft of H-2 receptors. From the simulations, we estimated the corresponding free energy of dissociation (ΔGd ) and described the molecular events that occur during the exit of peptides from the cleft. To test the reliability of this method, we first applied it to a calibration set of four crystallographic MHC/peptide complexes. Next, we explored the unbinding of the seven complexes mentioned above. Results were consistent with ΔGd values obtained from surface plasmon resonance (SPR) experiments. We also identified some of the primary interactions between peptides and H-2 receptors, and we detected three regions of influence for the interaction. This pattern was systematically observed for the peptides and helped determine a minimum distance for the real interaction between peptides and H-2 proteins occurring at ∼ 25 Å.

  5. Inhibitory effects of Zanthoxylum rhoifolium Lam. (Rutaceae) against the infection and infectivity of macrophages by Leishmania amazonensis.

    Science.gov (United States)

    Melo, Bernardo; Leitão, Joseana M S R; Oliveira, Luciano G C; Santos, Sérgio E M; Carneiro, Sabrina M P; Rodrigues, Klinger A F; Chaves, Mariana H; Arcanjo, Daniel D R; Carvalho, Fernando A A

    2016-01-01

    Zanthoxylum rhoifolium Lam. (Rutaceae) has been traditionally used in the treatment of microbial infections and parasitic diseases. In the present study, the antileishmanial effect induced by the ethanol extract of stem barks from Z. rhoifolium (ZR-EEtOH) and its n-hexane fraction (ZR-FHEX) on infection and infectivity of murine macrophages by promastigote forms of Leishmania amazonensis were investigated. In different set of experiments, macrophages or promastigotes were pretreated with ZR-EEtOH or ZR-FHEX at non-lethal concentrations for 24 hours, and then macrophages were submitted to infection by promastigotes. Moreover, their effects on activation of macrophages, as well as on the DNA content, size and number of promastigotes by flow cytometry were also evaluated. The infection rate and the number of internalized amastigote forms were markedly decreased after pretreatment of macrophages or promastigotes when compared with non-treated cells. The increase in phagocytic capability and nitrite content was also observed. Furthermore, the decrease of DNA content, size and number of promastigotes was also observed. In conclusion, ZR-EEtOH and ZR-FHEX promoted a markedly significant antileishmanial effect and reduction of infection of macrophages, probably underlying defense mechanisms activation in macrophages. These findings reinforce the potential application of Z. rhoifolium in the treatment of leishmaniasis.

  6. Inhibitory effects of Zanthoxylum rhoifolium Lam. (Rutaceae against the infection and infectivity of macrophages by Leishmania amazonensis

    Directory of Open Access Journals (Sweden)

    BERNARDO MELO NETO

    2016-01-01

    Full Text Available ABSTRACT Zanthoxylum rhoifolium Lam. (Rutaceae has been traditionally used in the treatment of microbial infections and parasitic diseases. In the present study, the antileishmanial effect induced by the ethanol extract of stem barks from Z. rhoifolium (ZR-EEtOH and its n-hexane fraction (ZR-FHEX on infection and infectivity of murine macrophages by promastigote forms of Leishmania amazonensis were investigated. In different set of experiments, macrophages or promastigotes were pretreated with ZR-EEtOH or ZR-FHEX at non-lethal concentrations for 24 hours, and then macrophages were submitted to infection by promastigotes. Moreover, their effects on activation of macrophages, as well as on the DNA content, size and number of promastigotes by flow cytometry were also evaluated. The infection rate and the number of internalized amastigote forms were markedly decreased after pretreatment of macrophages or promastigotes when compared with non-treated cells. The increase in phagocytic capability and nitrite content was also observed. Furthermore, the decrease of DNA content, size and number of promastigotes was also observed. In conclusion, ZR-EEtOH and ZR-FHEX promoted a markedly significant antileishmanial effect and reduction of infection of macrophages, probably underlying defense mechanisms activation in macrophages. These findings reinforce the potential application of Z. rhoifolium in the treatment of leishmaniasis.

  7. Time-course analysis of the Shewanella amazonensis SB2B proteome in response to sodium chloride shock

    Energy Technology Data Exchange (ETDEWEB)

    Parnell, John J.; Callister, Stephen J.; Rompato, Giovanni; Nicora, Carrie D.; Pasa-Tolic, Ljiljana; Williamson, Ashley; Pfrender, Michael E.

    2011-06-29

    Organisms in the genus Shewanella have become models for response to environmental stress. One of the most important environmental stresses is change in osmolarity. In this study, we experimentally determine the response mechanisms of Shewanella amazonensis SB2B during osmotic stress. Osmotic stress in SB2B was induced through exposure to NaCl, and the time-course proteomics response was measured using liquid chromatography mass spectrometry. Protein trends were qualitatively compared to gene expression trends and to phenotypic characterization. Osmotic stress affects motility, and has also been associated with a change in the membrane fatty acid composition (due to induction of branched chain amino acid degradation pathways); however, we show this is not the case for SB2B. Although proteins and genes involved with branched chain amino acid degradation are induced, fatty acid degradation pathways are not induced and no change in the fatty acid profile occurs in SB2B as a result of osmotic shock. The most extensive response of SB2B over the time course of acclimation to high salt involves an orchestrated sequence of events comprising increased expression of signal transduction associated with motility and restricted cell division and DNA replication. After SB2B has switched to increased branched chain amino acid degradation, motility, and cellular replication proteins return to pre-perturbed levels.

  8. Extended recombinant bacterial ghost system.

    Science.gov (United States)

    Lubitz, W; Witte, A; Eko, F O; Kamal, M; Jechlinger, W; Brand, E; Marchart, J; Haidinger, W; Huter, V; Felnerova, D; Stralis-Alves, N; Lechleitner, S; Melzer, H; Szostak, M P; Resch, S; Mader, H; Kuen, B; Mayr, B; Mayrhofer, P; Geretschläger, R; Haslberger, A; Hensel, A

    1999-08-20

    Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri

  9. Protamine-based nanoparticles as new antigen delivery systems.

    Science.gov (United States)

    González-Aramundiz, José Vicente; Peleteiro Olmedo, Mercedes; González-Fernández, África; Alonso Fernández, María José; Csaba, Noemi Stefánia

    2015-11-01

    The use of biodegradable nanoparticles as antigen delivery vehicles is an attractive approach to overcome the problems associated with the use of Alum-based classical adjuvants. Herein we report, the design and development of protamine-based nanoparticles as novel antigen delivery systems, using recombinant hepatitis B surface antigen as a model viral antigen. The nanoparticles, composed of protamine and a polysaccharide (hyaluronic acid or alginate), were obtained using a mild ionic cross-linking technique. The size and surface charge of the nanoparticles could be modulated by adjusting the ratio of the components. Prototypes with optimal physicochemical characteristics and satisfactory colloidal stability were selected for the assessment of their antigen loading capacity, antigen stability during storage and in vitro and in vivo proof-of-concept studies. In vitro studies showed that antigen-loaded nanoparticles induced the secretion of cytokines by macrophages more efficiently than the antigen in solution, thus indicating a potential adjuvant effect of the nanoparticles. Finally, in vivo studies showed the capacity of these systems to trigger efficient immune responses against the hepatitis B antigen following intramuscular administration, suggesting the potential interest of protamine-polysaccharide nanoparticles as antigen delivery systems.

  10. Conformational dynamics and antigenicity in the disordered malaria antigen merozoite surface protein 2.

    Directory of Open Access Journals (Sweden)

    Christopher A MacRaild

    Full Text Available Merozoite surface protein 2 (MSP2 of Plasmodium falciparum is an abundant, intrinsically disordered protein that is GPI-anchored to the surface of the invasive blood stage of the malaria parasite. Recombinant MSP2 has been trialled as a component of a malaria vaccine, and is one of several disordered proteins that are candidates for inclusion in vaccines for malaria and other diseases. Nonetheless, little is known about the implications of protein disorder for the development of an effective antibody response. We have therefore undertaken a detailed analysis of the conformational dynamics of the two allelic forms of MSP2 (3D7 and FC27 using NMR spectroscopy. Chemical shifts and NMR relaxation data indicate that conformational and dynamic properties of the N- and C-terminal conserved regions in the two forms of MSP2 are essentially identical, but significant variation exists between and within the central variable regions. We observe a strong relationship between the conformational dynamics and the antigenicity of MSP2, as assessed with antisera to recombinant MSP2. Regions of increased conformational order in MSP2, including those in the conserved regions, are more strongly antigenic, while the most flexible regions are minimally antigenic. This suggests that modifications that increase conformational order may offer a means to tune the antigenicity of MSP2 and other disordered antigens, with implications for vaccine design.

  11. Conformational Dynamics and Antigenicity in the Disordered Malaria Antigen Merozoite Surface Protein 2

    Science.gov (United States)

    Andrew, Dean; Krishnarjuna, Bankala; Nováček, Jiří; Žídek, Lukáš; Sklenář, Vladimír; Richards, Jack S.; Beeson, James G.; Anders, Robin F.; Norton, Raymond S.

    2015-01-01

    Merozoite surface protein 2 (MSP2) of Plasmodium falciparum is an abundant, intrinsically disordered protein that is GPI-anchored to the surface of the invasive blood stage of the malaria parasite. Recombinant MSP2 has been trialled as a component of a malaria vaccine, and is one of several disordered proteins that are candidates for inclusion in vaccines for malaria and other diseases. Nonetheless, little is known about the implications of protein disorder for the development of an effective antibody response. We have therefore undertaken a detailed analysis of the conformational dynamics of the two allelic forms of MSP2 (3D7 and FC27) using NMR spectroscopy. Chemical shifts and NMR relaxation data indicate that conformational and dynamic properties of the N- and C-terminal conserved regions in the two forms of MSP2 are essentially identical, but significant variation exists between and within the central variable regions. We observe a strong relationship between the conformational dynamics and the antigenicity of MSP2, as assessed with antisera to recombinant MSP2. Regions of increased conformational order in MSP2, including those in the conserved regions, are more strongly antigenic, while the most flexible regions are minimally antigenic. This suggests that modifications that increase conformational order may offer a means to tune the antigenicity of MSP2 and other disordered antigens, with implications for vaccine design. PMID:25742002

  12. SEREX技术筛选及鉴定食管癌肿瘤抗原%Human Esophageal Carcinoma Antigens Screened by Serologic Analysis of Recombinant cDNA Expression Libraries (SEREX)

    Institute of Scientific and Technical Information of China (English)

    遇珑; 胡海; 冉宇靓; 彭良平; 李江伟; 杨治华

    2007-01-01

    背景与目的:正常细胞向癌细胞转化过程中,突变的基因或各种异常表达的蛋白可以成为肿瘤抗原诱导机体的免疫反应,因此肿瘤患者的血清中存在着与肿瘤相关的自身抗体.重组cDNA表达文库血清学分析法(serological analysis of recombinant cDNA expression libraries,SEREX)是利用肿瘤患者血清中的自身抗体筛选、鉴定肿瘤抗原的技术.本研究拟采用SEREX的方法寻找食管癌自身抗体的相关肿瘤抗原,鉴定与食管癌发生、发展相关的基因和免疫治疗分子靶点,并为食管癌的诊断提供候选血清标志物.方法:用食管癌组织建立库容量达1.6×106 pfu的cDNA表达文库,SEREX筛选获得21个不同cDNA序列的阳性克隆,进一步使用SADA法分析其中4个抗原在10例食管癌及10例正常人血清中的反应.结果:在Homosapiens desmin(DES)等21个阳性克隆中,4个克隆与已知EST序列明显无同源性,另外17个克隆与已知基因高度同源.Ribosomal protein S4等4个抗原与食管癌患者和正常人血清反应阳性率分别为40%和0%、60%和10%、70%和20%、30%和20%.结论:Ribosomal protein S4等4个抗原普遍参与了食管癌患者的体液免疫反应,与食管癌患者血清的反应阳性率明显高于正常人的血清.本研究发现的21个食管癌抗原可作为食管癌治疗的潜在分子靶点和食管癌诊断新的候选血清学标志物.

  13. Construction and identification of recombinant Mycobacterium smegmatis vaccine expressing Cysticercus cellulosae cC1 antigen%猪囊尾蚴特异性抗原cC1重组耻垢分枝杆菌疫苗的构建与鉴定

    Institute of Scientific and Technical Information of China (English)

    王雪梅; 骆江坤; 李倩; 李江艳; 陈勇; 陶志勇; 夏惠; 方强

    2014-01-01

    Objective To construct recombinant Mycobacterium smegmatis vaccine expressing Cysticercus cellulosae cC1 anti-gen. Methods The recombinant pET28a-cC1 plasmid was extracted and double digested by Xho I and BamH I restriction en-zymes,and shuttle plasmid pMV261 was extracted and double digested by Hind III and BamH I restriction enzymes. Both frag-ments were modified by Klenow fragment to form blunt end,then the large fragments of cC1 and pMV261 plasmid were purified and ligated by T4 ligase enzyme. The recombinant pMV261-cC1 plasmid was constructed and sequenced. Then the pMV261-cC1 plasmid was transformed into Mycobacterium smegmatis by the electrotransformation method. The recombinant cC1-Mycobacterium smegmatis was induced by heat and identified by the Western blotting method with the sera of cysticercosis patients. In addition, the growth states of the Mycobacterium smegmatis and the recombinant cC1-Mycobacterium smegmatis were compared and the growth curves were drawn. Results The restriction enzyme and sequencing results showed that the recombinant pMV261-cC1 plasmid was successfully constructed. After heat induction,a 40 kD band was showed by PAGE analysis of cC1-Mycobacterium smegmatis. The Western blotting results showed that the sera of cysticercosis patients could recognize the 40 kDa band,which sug-gested that cC1 protein was expressed in Mycobacterium smegmatis. Compared with the Mycobacterium smegmatis,the recombi-nant cC1-Mycobacterium smegmatis showed no significant difference in proliferation characteristics. Conclusion The recombi-nant cC1-Mycobacterium smegmatis vaccine has been successfully constructed.%目的:构建表达猪囊尾蚴cC1抗原的重组耻垢分枝杆菌疫苗。方法提取pET28a-cC1质粒DNA,用xho I和BamH I双酶切,用Klenow酶补平xho I酶切末端,同时提取pMV261穿梭质粒载体,用Hind III和BamH I双酶切,用Klenow酶补平Hind III酶切末端,纯化后的酶切产物通过T4

  14. Two pathways of homologous recombination in Trypanosoma brucei.

    Science.gov (United States)

    Conway, Colin; Proudfoot, Chris; Burton, Peter; Barry, J David; McCulloch, Richard

    2002-09-01

    African trypanosomes are unicellular parasites that use DNA recombination to evade the mammalian immune response. They do this in a process called antigenic variation, in which the parasites periodically switch the expression of VSG genes that encode distinct Variant Surface Glycoprotein coats. Recombination is used to move new VSG genes into specialised bloodstream VSG transcription sites. Genetic and molecular evidence has suggested that antigenic variation uses homologous recombination, but the detailed reaction pathways are not understood. In this study, we examine the recombination pathways used by trypanosomes to integrate transformed DNA into their genome, and show that they possess at least two pathways of homologous recombination. The primary mechanism is dependent upon RAD51, but a subsidiary pathway exists that is RAD51-independent. Both pathways contribute to antigenic variation. We show that the RAD51-independent pathway is capable of recombining DNA substrates with very short lengths of sequence homology and in some cases aberrant recombination reactions can be detected using such microhomologies.

  15. Sand fly captures with Disney traps in area of occurrence of Leishmania (Leishmania) amazonensis in the state of Mato Grosso do Sul, mid-western Brazil Capturas de flebotomíneos com armadilhas de Disney em área de ocorrência de Leishmania (Leishmania) amazonensis no estado de Mato Grosso do Sul, região Centro-Oeste do Brasil

    OpenAIRE

    Maria Elizabeth Cavalheiros Dorval; Tulia Peixoto Alves; Geucira Cristaldo; Hilda Carlos da Rocha; Murilo Andrade Alves; Elisa Teruya Oshiro; Alessandra Gutierrez de Oliveira; Reginaldo Peçanha Brazil; Eunice Aparecida Bianchi Galati; Rivaldo Venancio da Cunha

    2010-01-01

    INTRODUCTION: The work was conducted to study phlebotomine fauna (Diptera: Psychodidae) and aspects of American cutaneous leishmaniasis transmission in a forested area where Leishmania (Leishmania) amazonensis occurs, situated in the municipality of Bela Vista, State of Mato Grosso do Sul, Brazil. METHODS: The captures were conducted with modified Disney traps, using hamster (Mesocricetus auratus) as bait, from May 2004 to January 2006. RESULTS: Ten species of phlebotomine sandflies were capt...

  16. Therapeutic trial in experimental tegumentary leishmaniasis caused by Leishmania (Leishmania amazonensis. A comparative study between mefloquine and aminosidine Ensaio terapêutico na leishmaniose tegumentar experimental causada por Leishmania (Leishmania amazonensis. Um estudo comparativo entre mefloquina e aminosidine

    Directory of Open Access Journals (Sweden)

    Letícia Oba Galvão

    2000-08-01

    Full Text Available One hundred and eighty-two male inbred C57/BL/6 mice were infected with 3 x 106 Leishmania (Leishmania amazonensis promastigotes of the MHOM/BR/PH8 strain by means of a subcutaneous injection in the right ear. The animals were separated in three groups: 1 oral mefloquine hydrochloride treatment (16mg/kg/day/10 days, 2 intramuscular aminosidine (Paromomycin® treatment (20mg/kg/20 days and 3 control. Twenty six mice of each treated group were sacrificed, one at the end of treatment (nine weeks after inoculation, and one six weeks later (fifteen weeks after inoculation. Control Group animals were sacrificed at weeks six, nine and fifteen after inoculation. There was no significant difference between Group 1 (mefloquine and Group 3 (control subjects. Group 2 animals (aminosidine presented the smallest differences of all, both at the end of the treatment and six weeks later. The histopato-logical parameters have shown the following findings: a there was no significant difference between the mefloquine treated group and the control group; the group treated with aminosidine showed fewer of vacuolated macrophages than the control group, at week 9 (end of treatment. b both at the end of treatment and six weeks later, evaluation of tissue necrosis and tissue fibrosis revealed no differences between the treated groups. It was found that six weeks after the end of treatment, mice in the control group presented significantly more severe degrees of fibrosis than mice in the other groups. It can be concluded that mefloquine showed limited therapeutic effect in this experimental model, whereas aminosidine had a significant effect. Nevertheless, neither of them resulted in cure of the lesions.Foram utilizados 182 camundongos machos, isogênicos, da linhagem C57BL/6 inoculados na orelha direita com 3,0 x 10(6 formas promastigotas da cepa MHOM/BR/PH8 de Leishmania (Leishmania amazonensis. Os animais foram separados em três grupos: 1 52 animais tratados com

  17. Recent advances in the production of recombinant subunit vaccines in Pichia pastoris.

    Science.gov (United States)

    Wang, Man; Jiang, Shuai; Wang, Yefu

    2016-04-01

    Recombinant protein subunit vaccines are formulated using defined protein antigens that can be produced in heterologous expression systems. The methylotrophic yeast Pichia pastoris has become an important host system for the production of recombinant subunit vaccines. Although many basic elements of P. pastoris expression system are now well developed, there is still room for further optimization of protein production. Codon bias, gene dosage, endoplasmic reticulum protein folding and culture condition are important considerations for improved production of recombinant vaccine antigens. Here we comment on current advances in the application of P. pastoris for the synthesis of recombinant subunit vaccines.

  18. Morinda citrifolia Linn. Reduces Parasite Load and Modulates Cytokines and Extracellular Matrix Proteins in C57BL/6 Mice Infected with Leishmania (Leishmania) amazonensis.

    Science.gov (United States)

    Almeida-Souza, Fernando; Cardoso, Flávia de Oliveira; Souza, Bruno Vinicius da Conceição; do Valle, Tânia Zaverucha; de Sá, Joicy Cortez; Oliveira, Iara Dos Santos da Silva; de Souza, Celeste da Silva Freitas; Moragas Tellis, Carla Junqueira; Chagas, Maria do Socorro Dos Santos; Behrens, Maria Dutra; Abreu-Silva, Ana Lúcia; Calabrese, Kátia da Silva

    2016-08-01

    The absence of an effective vaccine and the debilitating chemotherapy for Leishmaniasis demonstrate the need for developing alternative treatments. Several studies conducted with Morinda citrifolia have shown various biological activities, including antileishmanial activity, however its mechanisms of action are unknown. This study aimed to analyze the in vivo activity of M. citrifolia fruit juice (Noni) against Leishmania (Leishmania) amazonensis in C57BL/6 mice. M. citrifolia fruit juice from the Brazilian Amazon has shown the same constitution of other juices produced around the world and liquid chromatography-mass spectrometry analysis identified five compounds: deacetylasperulosidic acid, asperulosidic acid, rutin, nonioside B and nonioside C. Daily intragastric treatment with Noni was carried out after 55 days of L. (L.) amazonensis infection in C57BL/6 mice. Parasitic loads, cytokine and extracellular protein matrix expressions of the lesion site were analyzed by qPCR. Histopathology of the lesion site, lymph nodes and liver were performed to evaluate the inflammatory processes. Cytokines and biochemical parameters of toxicity from sera were also evaluated. The Noni treatment at 500 mg.kg-1.day-1 for 60 days decreased the lesion size and parasitic load in the footpad infected with L. (L.) amazonensis. The site of infection also showed decreased inflammatory infiltrates and decreased cytokine expressions for IL-12, TNF-α, TGF-β and IL-10. On the other hand, Noni treatment enhanced the extracellular matrix protein expressions of collagen IV, fibronectin and laminin in the infected footpad as well collagen I and II, fibronectin and laminin in the mock-infected footpads. No toxicity was observed at the end of treatment. These data show the efficacy of Noni treatment.

  19. Morinda citrifolia Linn. fruit (Noni) juice induces an increase in NO production and death of Leishmania amazonensis amastigotes in peritoneal macrophages from BALB/c.

    Science.gov (United States)

    Almeida-Souza, Fernando; de Souza, Celeste da Silva Freitas; Taniwaki, Noemi Nosomi; Silva, João José Mendes; de Oliveira, Renata Mondêgo; Abreu-Silva, Ana Lúcia; Calabrese, Kátia da Silva

    2016-08-31

    Leishmaniasis is a complex disease that is considered a serious public health problem. Due to the absence of an effective vaccine and debilitating chemotherapy better therapies are urgently needed. This situation has stimulated the search for alternative treatments such as the use of herbal medicines. Several studies conducted with Morinda citrifolia Linn. have shown various biological activities such as antitumor, immunomodulation and antileishmanial activity, however its mechanisms of action are still unknown. This study aimed to analyze the activity of M. citrifolia fruit juice against Leishmania amazonensis and its action on peritoneal macrophages from BALB/c infected with L. amazonensis. Activity against the promastigote forms showed IC50 at 275.3 μg/mL. Transmission electron microscopy was used to evaluate the ultrastructural alterations in the promastigotes treated with the juice and the results showed cytoplasmic vacuolization, lipid inclusion and increased activity of exocytosis. The juice treatment presented an IC50 at 208.4 μg/mL against intracellular amastigotes and led to an increased nitrite production in infected and non-infected macrophages. When macrophages were pre-treated with iNOS inhibitors, aminoguanidine or 1400W, the intracellular amastigotes increased, demonstrating the important role of NO production in M. citrifolia fruit activity. In conclusion, our results reveal that treatment with M. citrifolia fruit juice can increase NO production in peritoneal macrophages and this ability has an important role in the killing of L. amazonensis intracellular amastigotes.

  20. In Vitro Activities of ER-119884 and E5700, Two Potent Squalene Synthase Inhibitors, against Leishmania amazonensis: Antiproliferative, Biochemical, and Ultrastructural Effects▿

    Science.gov (United States)

    Fernandes Rodrigues, Juliany Cola; Concepcion, Juan Luis; Rodrigues, Carlos; Caldera, Aura; Urbina, Julio A.; de Souza, Wanderley

    2008-01-01

    ER-119884 and E5700, novel arylquinuclidine derivatives developed as cholesterol-lowering agents, were potent in vitro growth inhibitors of both proliferative stages of Leishmania amazonensis, the main causative agent of cutaneous leishmaniasis in South America, with the 50% inhibitory concentrations (IC50s) being in the low-nanomolar to subnanomolar range. The compounds were very potent noncompetitive inhibitors of native L. amazonensis squalene synthase (SQS), with inhibition constants also being in the nanomolar to subnanomolar range. Growth inhibition was strictly associated with the depletion of the parasite's main endogenous sterols and the concomitant accumulation of exogenous cholesterol. Using electron microscopy, we identified the intracellular structures affected by the compounds. A large number of lipid inclusions displaying different shapes and electron densities were observed after treatment with both SQS inhibitors, and these inclusions were associated with an intense disorganization of the membrane that surrounds the cell body and flagellum, as well as the endoplasmic reticulum and the Golgi complex. Cells treated with ER-119884 but not those treated with E5700 had an altered cytoskeleton organization due to an abnormal distribution of tubulin, and many were arrested at cytokinesis. A prominent contractile vacuole and a phenotype typical of programmed cell death were frequently found in drug-treated cells. The selectivity of the drugs was demonstrated with the JC-1 mitochondrial fluorescent label and by trypan blue exclusion tests with macrophages, which showed that the IC50s against the host cells were 4 to 5 orders of magnitude greater that those against the intracellular parasites. Taken together, our results show that ER-119884 and E5700 are unusually potent and selective inhibitors of the growth of Leishmania amazonensis, probably because of their inhibitory effects on de novo sterol biosynthesis at the level of SQS, but some of our

  1. Morinda citrifolia Linn. Reduces Parasite Load and Modulates Cytokines and Extracellular Matrix Proteins in C57BL/6 Mice Infected with Leishmania (Leishmania) amazonensis

    Science.gov (United States)

    Almeida-Souza, Fernando; Cardoso, Flávia de Oliveira; Souza, Bruno Vinicius da Conceição; do Valle, Tânia Zaverucha; de Sá, Joicy Cortez; Oliveira, Iara dos Santos da Silva; de Souza, Celeste da Silva Freitas; Moragas Tellis, Carla Junqueira; Chagas, Maria do Socorro dos Santos; Behrens, Maria Dutra

    2016-01-01

    The absence of an effective vaccine and the debilitating chemotherapy for Leishmaniasis demonstrate the need for developing alternative treatments. Several studies conducted with Morinda citrifolia have shown various biological activities, including antileishmanial activity, however its mechanisms of action are unknown. This study aimed to analyze the in vivo activity of M. citrifolia fruit juice (Noni) against Leishmania (Leishmania) amazonensis in C57BL/6 mice. M. citrifolia fruit juice from the Brazilian Amazon has shown the same constitution of other juices produced around the world and liquid chromatography–mass spectrometry analysis identified five compounds: deacetylasperulosidic acid, asperulosidic acid, rutin, nonioside B and nonioside C. Daily intragastric treatment with Noni was carried out after 55 days of L. (L.) amazonensis infection in C57BL/6 mice. Parasitic loads, cytokine and extracellular protein matrix expressions of the lesion site were analyzed by qPCR. Histopathology of the lesion site, lymph nodes and liver were performed to evaluate the inflammatory processes. Cytokines and biochemical parameters of toxicity from sera were also evaluated. The Noni treatment at 500 mg.kg-1.day-1 for 60 days decreased the lesion size and parasitic load in the footpad infected with L. (L.) amazonensis. The site of infection also showed decreased inflammatory infiltrates and decreased cytokine expressions for IL-12, TNF-α, TGF-β and IL-10. On the other hand, Noni treatment enhanced the extracellular matrix protein expressions of collagen IV, fibronectin and laminin in the infected footpad as well collagen I and II, fibronectin and laminin in the mock-infected footpads. No toxicity was observed at the end of treatment. These data show the efficacy of Noni treatment. PMID:27579922

  2. Differential modulation of ATP-induced P2X7-associated permeabilities to cations and anions of macrophages by infection with Leishmania amazonensis.

    Directory of Open Access Journals (Sweden)

    Camila Marques-da-Silva

    Full Text Available Leishmania and other parasites display several mechanisms to subvert host immune cell function in order to achieve successful infection. The ATP receptor P2X7, an agonist-gated cation channel widely expressed in macrophages and other cells of the immune system, is also coupled to inflammasome activation, IL-1 beta secretion, production of reactive oxygen species, cell death and the induction of the permeabilization of the plasma membrane to molecules of up to 900 Da. P2X7 receptors can function as an effective microbicidal triggering receptor in macrophages infected with several microorganisms including Mycobacteria tuberculosis, Chlamydia and Leishmania. We have previously shown that its expression is up-regulated in macrophages infected with L. amazonensis and that infected cells also display an increase in P2X7-induced apoptosis and membrane permeabilization to some anionic fluorescent dyes. In an independent study we recently showed that the phenomenon of macrophage membrane permeabilization can involve at least two distinct pathways for cations and anions respectively. Here, we re-addressed the effects of ATP-induced P2X7-associated phenomena in macrophages infected with L. amazonensis and demonstrated that the P2X7-associated dye uptake mechanisms are differentially modulated. While the membrane permeabilization for anionic dyes is up-modulated, as previously described, the uptake of cationic dyes is strongly down-modulated. These results unveil new characteristics of two distinct permeabilization mechanisms associated with P2X7 receptors in macrophages and provide the first evidence indicating that these pathways can be differentially modulated in an immunologically relevant situation. The possible importance of these results to the L. amazonensis escape mechanism is discussed.

  3. Differential modulation of ATP-induced P2X7-associated permeabilities to cations and anions of macrophages by infection with Leishmania amazonensis.

    Science.gov (United States)

    Marques-da-Silva, Camila; Chaves, Mariana Martins; Rodrigues, Juliany Cola; Corte-Real, Suzana; Coutinho-Silva, Robson; Persechini, Pedro Muanis

    2011-01-01

    Leishmania and other parasites display several mechanisms to subvert host immune cell function in order to achieve successful infection. The ATP receptor P2X7, an agonist-gated cation channel widely expressed in macrophages and other cells of the immune system, is also coupled to inflammasome activation, IL-1 beta secretion, production of reactive oxygen species, cell death and the induction of the permeabilization of the plasma membrane to molecules of up to 900 Da. P2X7 receptors can function as an effective microbicidal triggering receptor in macrophages infected with several microorganisms including Mycobacteria tuberculosis, Chlamydia and Leishmania. We have previously shown that its expression is up-regulated in macrophages infected with L. amazonensis and that infected cells also display an increase in P2X7-induced apoptosis and membrane permeabilization to some anionic fluorescent dyes. In an independent study we recently showed that the phenomenon of macrophage membrane permeabilization can involve at least two distinct pathways for cations and anions respectively. Here, we re-addressed the effects of ATP-induced P2X7-associated phenomena in macrophages infected with L. amazonensis and demonstrated that the P2X7-associated dye uptake mechanisms are differentially modulated. While the membrane permeabilization for anionic dyes is up-modulated, as previously described, the uptake of cationic dyes is strongly down-modulated. These results unveil new characteristics of two distinct permeabilization mechanisms associated with P2X7 receptors in macrophages and provide the first evidence indicating that these pathways can be differentially modulated in an immunologically relevant situation. The possible importance of these results to the L. amazonensis escape mechanism is discussed.

  4. Antigenic variation with a twist--the Borrelia story.

    Science.gov (United States)

    Norris, Steven J

    2006-06-01

    A common mechanism of immune evasion in pathogenic bacteria and protozoa is antigenic variation, in which genetic or epigenetic changes result in rapid, sequential shifts in a surface-exposed antigen. In this issue of Molecular Microbiology, Dai et al. provide the most complete description to date of the vlp/vsp antigenic variation system of the relapsing fever spirochaete, Borrelia hermsii. This elaborate, plasmid-encoded system involves an expression site that can acquire either variable large protein (vlp) or variable small protein (vsp) surface lipoprotein genes from 59 different archival copies. The archival vlp and vsp genes are arranged in clusters on at least five different plasmids. Gene conversion occurs through recombination events at upstream homology sequences (UHS) found in each gene copy, and at downstream homology sequences (DHS) found periodically among the vlp/vsp archival genes. Previous studies have shown that antigenic variation in relapsing fever Borrelia not only permits the evasion of host antibody responses, but can also result in changes in neurotropism and other pathogenic properties. The vlsE antigenic variation locus of Lyme disease spirochaetes, although similar in sequence to the relapsing fever vlp genes, has evolved a completely different antigenic variation mechanism involving segmental recombination from a contiguous array of vls silent cassettes. These two systems thus appear to represent divergence from a common precursor followed by functional convergence to create two distinct antigenic variation processes.

  5. Prokaryotic expression of surface membrane antigen SAG1 gene from Toxoplasma Gondii and the diagnostic value of the recombinant protein%弓形虫膜表面抗原SAG1基因的原核表达及重组蛋白的免疫诊断价值

    Institute of Scientific and Technical Information of China (English)

    王朝兰; 汤冬生; 姚湧; 汪学龙; 王业梅

    2011-01-01

    目的 探讨重组弓形虫膜表面抗原SAG1基因的表达产物-原核表达蛋白(rSAG1)用于弓形虫病的免疫诊断价值.方法 用异丙基-B-D-硫代吡喃半乳糖苷(IPTG)诱导大肠埃希菌重组质粒pET28a-SAG1(pET28a-SAG1/BL21)表达,纯化重组pET28a-SAG1/BL21弓形虫膜表面抗原SAG1基因表达产物;用弓形虫缓殖子感染的鼠血清、正常鼠血清和10例弓形虫患者血清为一抗,基因表达产物rSAG1用免疫印迹法进行鉴定,比较rSAG1在弓形虫病免疫诊断中的价值.结果 纯化重组弓形虫膜表面抗原SAG1基因后获得了相对分子质量约38.5×103的表达产物rSAG1;表面抗原SAG1可被弓形虫缓殖子感染的鼠血清所识别;10例弓形虫患者血清在免疫印迹诊断中,有4例出现了弓形虫膜表面抗原SAG1基因表达产物rSAG1.结论 rSAG1具备一定的弓形虫病的免疫诊断价值.%Objective To investigate the diagnostic value of the recombinant surface antigen 1 (rSAG1) in immunodiagnosis of toxoplasmosis. Methods Isopropyl β-D- 1 -thio-galaetopyranoside (IPTG) was used to induce the expression of recombinant plasmid pET28a-SAG1 of Escherich coli(pET28a-SAG1/BL21 ). The expression products (rSAG1) of pET28a-SAG1/BL21 were identified by Western blotting. The serum of mice infected with Toxoplasma gondii tachyzoites, normal mouse serum and the serum from 10 toxoplasma gondii patients were used as primary anti-Toxoplasma gondii antibodies, and the rSAG1 gene products were identified by Western blotting, by which the diagnostic value of rSAG1 in Toxoplasmosis was compared. Results After induction and purification, rSAG1 protein was obtained and its relative molecular mass was 38.5 × 103. The fusion protein could be recognized by the serum of mouse infected with Toxoplasma gondii tachyzoites, rSAG1 of expression products of surface membrane antigen SAG1 gene from Toxoplasma Gondii could be detected in 4 cases from 10 patients by Westem blotting.Conclusion The r

  6. The experimental chemotherapy of leishmaniasis, VI. The development of rodent models for cutaneous infection with L. major and L. mexicana amazonensis.

    Science.gov (United States)

    Trotter, E R; Peters, W; Robinson, B L

    1980-06-01

    Two models have been established of leishmanial infections in random-bred TFW albino mice, one with Leishmania major LV39 and the other with L. mexicana amazonensis LV38. These parasites produce consistent lesions when injected subcutaneously a short distance anterior to the base of the tail. Consistent infections could not be produced in the mice with other lines of the L. mexicana group of skin-dwelling protozoa, nor with a line of L. braziliensis guyanensis (LV475). The development of procedures for the maintenance of these parasites, the routine infection of mice and drug testing are described. L. major inocula consist of 1--5 X 10(6) promastigotes derived from NNN culture. L. m. amazonensis inocula contain approximately 10(7) amastigotes obtained from lesions in hamsters. The effects of drugs on the rodent lesions are assessed by two methods. Method A provides a simple lesion score from 0 to 3 made when lesions in control animals begin to reach maximum development (about six weeks after infection). Method B summates earlier 'mean lesion scores' to provide a graphic analysis of doses giving 50 and 90% lesion suppression at the end of seven weeks after the start of treatment which is for five consecutive days (SD 50 and SD90). The activities of several compounds used for the treatment of human leishmanial infections (organic antimonials, diamidines, amphotericin B and primaquine) are compared in mice infected with L. major LV39, L. m. amazonensis LV78 and 'L. infantum LV9' (the latter as reported earlier). It is concluded that the rodent models provide a good parallel with the response to these compounds in man. The visceral organism is significantly more responsive to Pentostam and Glucantime than are L. major or L. m. amazonensis. Primaquine is active only against 'L. infantum LV9'. All three parasites in mice respond to Berenil, but not to pentamidine which is used in the treatment of kala-azar. Amphotericin B is effective against cutaneous and visceral

  7. Impacto de Leishmania amazonensis y la Sangre de Ave en el Potencial Biológico y Fecundidad de Lutzomyia migonei y Lutzomyia ovallesi (Diptera: Psychodidae)

    OpenAIRE

    2011-01-01

    En los flebótomos (Diptera: Psychodidae), la ingesta sanguínea es responsable de la inducción de varios procesos fisiológicos y es determinante en la transmisión de Leishmania Ross. El presente trabajo estudia la sangre de ave, de mamífero y mezclada con Leishmania amazonensis Lainson & Shaw sobre el potencial biológico de Lutzomyia migonei França y Lutzomyia ovallesi Ortiz. Se utilizaron hembras de ambas especies alimentadas arti...

  8. Recombinant Technology and Probiotics

    Directory of Open Access Journals (Sweden)

    Icy D’Silva

    2011-09-01

    Full Text Available Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecules offer the opportunity to further investigate their effects for food, nutrition, environment andhealth. This review highlights advances in native probiotics and recombinant probiotics expressing native and recombinant molecules for food, nutrition, environment and health.

  9. Analysis of the antibody repertoire of patients with mantle cell lymphoma directed against mantle cell lymphoma-associated antigens

    OpenAIRE

    Zwick, Carsten; Preuss, Klaus-Dieter; Kubuschok, Boris; Held, Gerhard; Ahlgrimm, Manfred; Bittenbring, Joerg; Schubert, Joerg; Neumann, Frank; Pfreundschuh, Michael

    2009-01-01

    Abstract Treatment results of mantle cell lymphomas (MCL) are not satisfactory and novel therapeutic approaches are warranted. Because ?shared? tumor antigens like the group of cancer testis antigens are only rarely expressed in MCL, we applied serological analysis of antigens using recombinant expression cloning (SEREX) to a complementary DNA library derived from five cases of MCL using the sera of eight patients with MCL in order to define MCL-associated antigens that are immunog...

  10. Recombinant Technology and Probiotics

    OpenAIRE

    Icy D’Silva

    2011-01-01

    Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecule...

  11. Mapping antigenic motifs in the trypomastigote small surface antigen from Trypanosoma cruzi.

    Science.gov (United States)

    Balouz, Virginia; Cámara, María de Los Milagros; Cánepa, Gaspar E; Carmona, Santiago J; Volcovich, Romina; Gonzalez, Nicolás; Altcheh, Jaime; Agüero, Fernán; Buscaglia, Carlos A

    2015-03-01

    The trypomastigote small surface antigen (TSSA) is a mucin-like molecule from Trypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathione S-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease.

  12. “Nothing is permanent but change”* -- Antigenic variation in persistent bacterial pathogens

    OpenAIRE

    Palmer, Guy H.; Bankhead, Troy; Lukehart, Sheila A.

    2009-01-01

    Pathogens persist in immunocompetent mammalian hosts using various strategies, including evasion of immune effectors by antigenic variation. Among highly antigenically variant bacteria, gene conversion is used to generate novel expressed variants from otherwise silent donor sequences. Recombination using oligonucleotide segments from multiple donors is a combinatorial mechanism that tremendously expands the variant repertoire, allowing thousands of variants to be generated from a relatively s...

  13. 细粒棘球绦虫Eg18基因的克隆表达和重组抗原免疫检测的初步评价%Cloning and expression of Echinococcus granulosus Eg18 and preliminary evaluation of immunological assay with recombinant antigen

    Institute of Scientific and Technical Information of China (English)

    王永顺; 韩秀敏; 王虎

    2011-01-01

    Objective To clone and express Echinococcus granulosus Egl8 gene and evaluate the immunoreactivity of the re-combinant protein. Methods Egl8 coding sequence was amplified by RT-PCR using primers designed according to the sequence of Egl8 in GenBank from total RNA extracted from Echinococcus granulosus protoscoleces isolated from infected sheep in Qinhai Province and cloned into the prokaryotic expression vector pET-28a( + ). The recombinant expression vector was transformed to Escherichia coli BL21 (DE) and induced to express by IPTG. The expressed products were analyzed by SDS-PAGE and purified with Ni-IDA agarose affinity chromatography. rEgl8 was evaluated for its reactivity with the sera from the patients infected with hy-datid and other helminthes by Western blotting and ELISA. Results The sequence of cloned Egl8 was completely identical with the original sequences of Egl8 and Em 18 deposited in GenBank. The recombinant protein strongly reacted to the sera from the patients with alveolar echinococcosis, cystic echinococcosis, and cysticercosis, and weakly reacted to the sera from the patients with nematodiasis, schistosomiasis and clonorchiasis. The detection of specific IgG4 was much more specific than that of IgG. Conclusion Egl8/Eml8 is the common antigen of cestode and its specific IgG4 is a serum marker of alveolar echinococcosis.%目的 克隆、表达细粒棘球绦虫Eg18基因,评价重组蛋白的免疫反应性.方法 根据已知Eg18基因序列,利用RT-PCR方法从青海绵羊肝棘球蚴原头节提取的总RNA中扩增目的基因,克隆到原核表达质粒pET-28a(+)中,转化大肠埃希菌BI21 (DE),IPTG诱导表达.表达产物经亲和层析纯化,用免疫印迹试验(Western blot)和酶联免疫吸附试验(ELISA)初步评价其与棘球绦虫及其他蠕虫感染血清的反应性.结果 RT-PCR扩增产物编码的蛋白质序列与GenBank中的Eg18和Em18完全相同.重组蛋白与多种蠕虫病人血清中的IgG和IgG4均

  14. Recombinant shark natural antibodies to thyroglobulin.

    Science.gov (United States)

    Schluter, Samuel F; Jensen, Ingvill; Ramsland, Paul A; Marchalonis, John J

    2005-01-01

    As cartilaginous fish are the vertebrates most distal from man to produce antibodies, fundamental information regarding conservation and variation of the antigen binding site should be gained by comparing the properties of antibodies directed against the same antigen from the two species. Since monoclonal cell lines cannot be generated using shark B cells, we isolated antigen binding recombinant single chain Fv antibodies (scFv) comprising of the complete variable regions from shark light and heavy chains. Thyroglobulin was used as the selecting antigen as both sharks and humans express natural antibodies to mammalian thyroglobulin in the absence of purposeful immunization. We report that recombinant sandbar shark (Carcharhinus plumbeus) scFvs that bind bovine thyroglobulin consist of heavy chain variable regions (VH) homologous to those of the human VHIII subset and light chain variable regions (VL) homologous to those of the human Vlambda6 subgroup. The homology within the frameworks is sufficient to enable the building of three-dimensional models of the shark VH/VL structure using established human structures as templates. In natural antibodies of both species, the major variability lies in the third complementarity determining region (CDR3) of both VH and VL.

  15. Antigenic modulation of metastatic breast and ovary carcinoma cells by intracavitary injection of IFN-alpha.

    Science.gov (United States)

    Giacomini, P.; Mottolese, M.; Fraioli, R.; Benevolo, M.; Venturo, I.; Natali, P. G.

    1992-01-01

    Antigenic modulation of major histocompatibility and tumour associated antigens was observed in neoplastic cells obtained from patients with pleural and abdominal effusions of breast and ovary carcinomas following a single intracavitary dose of 18 x 10(6) U recombinant IFN-alpha. This regimen resulted in antigenic modulation in seven out of 11 tested cases, suggesting a potential, although limited, responsiveness of at least a fraction of breast and ovary carcinoma cells to in situ biomodification with IFN-alpha. PMID:1503908

  16. Algae-based oral recombinant vaccines.

    Science.gov (United States)

    Specht, Elizabeth A; Mayfield, Stephen P

    2014-01-01

    Recombinant subunit vaccines are some of the safest and most effective vaccines available, but their high cost and the requirement of advanced medical infrastructure for administration make them impractical for many developing world diseases. Plant-based vaccines have shifted that paradigm by paving the way for recombinant vaccine production at agricultural scale using an edible host. However, enthusiasm for "molecular pharming" in food crops has waned in the last decade due to difficulty in developing transgenic crop plants and concerns of contaminating the food supply. Microalgae could be poised to become the next candidate in recombinant subunit vaccine production, as they present several advantages over terrestrial crop plant-based platforms including scalable and contained growth, rapid transformation, easily obtained stable cell lines, and consistent transgene expression levels. Algae have been shown to accumulate and properly fold several vaccine antigens, and efforts are underway to create recombinant algal fusion proteins that can enhance antigenicity for effective orally delivered vaccines. These approaches have the potential to revolutionize the way subunit vaccines are made and delivered - from costly parenteral administration of purified protein, to an inexpensive oral algae tablet with effective mucosal and systemic immune reactivity.

  17. Recombinant MVA vaccines: dispelling the myths.

    Science.gov (United States)

    Cottingham, Matthew G; Carroll, Miles W

    2013-09-06

    Diseases such as HIV/AIDS, tuberculosis, malaria and cancer are prime targets for prophylactic or therapeutic vaccination, but have proven partially or wholly resistant to traditional approaches to vaccine design. New vaccines based on recombinant viral vectors expressing a foreign antigen are under intense development for these and other indications. One of the most advanced and most promising vectors is the attenuated, non-replicating poxvirus MVA (modified vaccinia virus Ankara), a safer derivative of the uniquely successful smallpox vaccine. Despite the ability of recombinant MVA to induce potent humoral and cellular immune responses against transgenic antigen in humans, especially when used as the latter element of a heterologous prime-boost regimen, doubts are occasionally expressed about the ultimate feasibility of this approach. In this review, five common misconceptions over recombinant MVA are discussed, and evidence is cited to show that recombinant MVA is at least sufficiently genetically stable, manufacturable, safe, and immunogenic (even in the face of prior anti-vector immunity) to warrant reasonable hope over the feasibility of large-scale deployment, should useful levels of protection against target pathogens, or therapeutic benefit for cancer, be demonstrated in efficacy trials.

  18. Algae-based oral recombinant vaccines

    Directory of Open Access Journals (Sweden)

    Elizabeth A Specht

    2014-02-01

    Full Text Available Recombinant subunit vaccines are some of the safest and most effective vaccines available, but their high cost and the requirement of advanced medical infrastructure for administration make them impractical for many developing world diseases. Plant-based vaccines have shifted that paradigm by paving the way for recombinant vaccine production at agricultural scale using an edible host. However, enthusiasm for molecular pharming in food crops has waned in the last decade due to difficulty in developing transgenic crop plants and concerns of contaminating the food supply. Microalgae are poised to become the next candidate in recombinant subunit vaccine production, and they present several advantages over terrestrial crop plant-based platforms including scalable and contained growth, rapid transformation, easily obtained stable cell lines, and consistent transgene expression levels. Algae have been shown to accumulate and properly fold several vaccine antigens, and efforts are underway to create recombinant algal fusion proteins that can enhance antigenicity for effective orally-delivered vaccines. These approaches have the potential to revolutionize the way subunit vaccines are made and delivered – from costly parenteral administration of purified protein, to an inexpensive oral algae tablet with effective mucosal and system immune reactivity.

  19. Morphology and small subunit rDNA-based phylogeny of Ceratomyxa amazonensis n. sp. parasite of Symphysodon discus, an ornamental freshwater fish from Amazon.

    Science.gov (United States)

    Mathews, Patrick D; Naldoni, Juliana; Maia, Antonio A; Adriano, Edson A

    2016-10-01

    The specious genus Ceratomyxa Thélodan, 1892, infect mainly gallbladder of marine fishes, with only five species reported infecting species from freshwater environment. This study performed morphological and phylogenetic analyses involving a new Ceratomyxa species (Ceratomyxa amazonensis n. sp.) found in gallbladder of Symphysodon discus Heckel, 1840 (Perciformes: Cichlidae), an important ornamental fish endemic to Amazon basin. Mature spores were strongly arcuate shaped and measured 7.0 ± 0.3 (6.2-7.6) μm in length, 15.8 ± 0.4 (15.0-16.7) μm in thickness, and polar capsules 3.22 ± 0.34 (2.4-3.6) μm in length and 2.63 ± 0.17 (2.4-2.9) μm in width. This was the first small subunit ribosomal DNA (SS rDNA) sequencing performed to Ceratomyxa species parasite of freshwater fish, and the phylogenetic analysis showed C. amazonensis n. sp. clustering in the early diverging subclade of the ceratomyxids, together with species of parasites of amphidromous/estuaries fishes, suggesting some role of the transition of the fishes between marine/freshwater environments in the evolutionary history of these parasites.

  20. Eosinofil Sel Penyaji Antigen

    Directory of Open Access Journals (Sweden)

    Safari Wahyu Jatmiko

    2015-04-01

    Full Text Available Sel eosinofil merupakan jenis sel lekosit yang terlibat dalam berbagai patogenesis penyakit. Sel eosinofil pada awalnya dikenal sebagai sel efektor  dari sistem imunitas alamiah. Akan tetapi, kemampuan sel eosinofil dalam memfagositosis patogen menimbulkan dugaan bahwa sel eosinofil ikut berperan sebagai sel penyaji antigen. Hal ini dianalogikan dengan sel makrofag dan sel dendritik yang bisa memfagositosis dan menyajikan antigen sebagai hasil dari degradasi patogen yang difagositosis. Untuk menjawab permasalahan ini, penulis melakukan penelusuran artikel tentang eosinofil sebagai sel penyaji antigen melalui US National Library of Medicine National Institute of Healthdengan kata kunci eoshinophil dan antigen presenting cell. Hasil penelusuran adalah ditemukannya 10 artikel yang relevan dengan topik. Hasil dari sintesis kesepuluh jurnal tersebut adalah sel eosinofil mampu berperan sebagai sel penyaji antigen yang profesional (professionalantigenpresentng cell

  1. Utility of recombinant Fragment C for assessment of anti-tetanus antibodies in plasma

    Science.gov (United States)

    Ramakrishnan, Girija; Pedersen, Karl; Guenette, Denis; Sink, Joyce; Haque, Rashidul; Petri, William A.; Herbein, Joel; Gilchrist, Carol A.

    2016-01-01

    Anti-tetanus antibodies in biological samples are typically detected using an ELISA based on toxoided tetanus neurotoxin as antigen. We demonstrate that recombinantly produced Fragment C of the toxin heavy chain (rFragC) is an effective alternative antigen for assessment of tetanus- immune status in plasma samples. PMID:25749462

  2. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  3. Limited antigenic variation in the Trypanosoma cruzi candidate vaccine antigen TSA-1.

    Science.gov (United States)

    Knight, J M; Zingales, B; Bottazzi, M E; Hotez, P; Zhan, B

    2014-12-01

    Chagas disease (American trypanosomiasis caused by Trypanosoma cruzi) is one of the most important neglected tropical diseases in the Western Hemisphere. The toxicities and limited efficacies of current antitrypanosomal drugs have prompted a search for alternative technologies such as a therapeutic vaccine comprised of T. cruzi antigens, including a recombinant antigen encoding the N-terminal 65 kDa portion of Trypomastigote surface antigen-1 (TSA-1). With at least six known genetically distinct T. cruzi lineages, variability between the different lineages poses a unique challenge for the development of broadly effective therapeutic vaccine. The variability across the major lineages in the current vaccine candidate antigen TSA-1 has not previously been addressed. To assess the variation in TSA-1, we cloned and sequenced TSA-1 from several different T. cruzi strains representing three of the most clinically relevant lineages. Analysis of the different alleles showed limited variation in TSA-1 across the different strains and fit with the current theory for the evolution of the different lineages. Additionally, minimal variation in known antigenic epitopes for the HLA-A 02 allele suggests that interlineage variation in TSA-1 would not impair the range and efficacy of a vaccine containing TSA-1.

  4. Expression, purification and antigenicity of Neospora caninum-antigens using silkworm larvae targeting for subunit vaccines.

    Science.gov (United States)

    Otsuki, Takahiro; Dong, Jinhua; Kato, Tatsuya; Park, Enoch Y

    2013-02-18

    Infection of Neospora caninum causes abortion in cattle, which has a serious worldwide impact on the economic performance of the dairy and beef industries. Now, inexpensive and efficacious vaccines are required to protect cattle from neosporosis in livestock industry. In this study, N. caninum surface antigen 1 (SAG1) and SAG1-related sequence 2 (SRS2) were expressed in hemolymph of silkworm larvae as a soluble form. Expressed SAG1 and SRS2 clearly showed antigenicity against N. caninum-positive sera of cow. SAG1 and SRS2 were purified to near homogeneity from hemolymph of silkworm larvae using anti-FLAG M2 antibody agarose: approximately 1.7 mg of SAG1 from 10 silkworm larvae and 370 μg of SRS2 from 17 silkworm larvae. Mice that were injected by antigens induced antibodies against SAG1 and SRS2. This study indicates that it is possible that this silkworm expression system leads to a large-scale production of N. caninum-antigens with biological function and low production cost. Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expression system paves the way to produce largely and rapidly these recombinant antigens for its application to subunit vaccines against neosporosis in cattle.

  5. Pesquisa de IgA contra o antígeno recombinante HspX de Mycobacterium tuberculosis no diagnóstico de tuberculose pleural Determination of levels of specific IgA to the HspX recombinant antigen of Mycobacterium tuberculosis for the diagnosis of pleural tuberculosis

    Directory of Open Access Journals (Sweden)

    Loanda Carvalho Sant' Ana Limongi

    2011-06-01

    Full Text Available OBJETIVO: Avaliar a acurácia da dosagem de IgA contra o antígeno recombinante HspX no líquido pleural e no soro de pacientes com derrame pleural para o diagnóstico de tuberculose pleural. MÉTODOS: Estudo transversal de teste diagnóstico. Amostras de líquido pleural e de soro de pacientes com derrame pleural e suspeita de tuberculose pleural foram avaliadas para a determinação da densidade óptica de IgA contra HspX utilizando ELISA indireto. RESULTADOS: Foram avaliadas amostras de líquido pleural e de soro de 132 pacientes: 97 com tuberculose pleural (grupo de estudo e 35 com derrame pleural por outras causas (grupo controle. A dosagem de IgA em líquido pleural foi capaz de discriminar os pacientes com tuberculose pleural dos controles. A sensibilidade do teste em líquido pleural e em soro foi, respectivamente, de 69% e 30%, enquanto a especificidade foi de 83% e 84%, respectivamente. CONCLUSÕES: Os dados sugerem o potencial da utilização deste teste no diagnóstico de tuberculose pleural. Estudos com amostras maiores e em diferentes cenários epidemiológicos são necessáriosOBJECTIVE: To evaluate the accuracy of determining specific IgA to HspX recombinant antigen in pleural fluid and serum samples for the diagnosis of pleural tuberculosis in patients with pleural effusion. METHODS: This was a cross-sectional study. Serum and pleural fluid samples of patients with pleural effusion and suspected of having pleural tuberculosis were tested with indirect ELISA in order to determine the optical density of specific IgA to HspX. RESULTS: We evaluated serum and pleural fluid samples from 132 patients: 97 diagnosed with pleural tuberculosis (study group and 35 diagnosed with pleural effusion due to other causes (control group. The determination of IgA in pleural fluid satisfactorily discriminated between pleural tuberculosis patients and control patients. The sensitivity of the test in pleural fluid and in serum was 69% and 30

  6. Glycan-dependent immunogenicity of recombinant soluble trimeric hemagglutinin

    NARCIS (Netherlands)

    Vries, de R.P.; Smit, C.H.; Bruin, de E.; Rigter, A.; Vries, de E.; Cornelissen, A.H.M.; Eggink, D.; Chung, N.P.Y.; Moore, J.P.; Sanders, R.W.; Hokke, C.H.; Koopmans, M.P.G.; Rottier, P.J.M.; Haan, de C.A.M.

    2012-01-01

    Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA3) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA3 glycoprotein affects its immunogenicity. Different glycosylation forms of subtype H5 trimeric HA pro

  7. Detection of peste des petits ruminants virus antigen using immunofiltration and antigen-competition ELISA methods.

    Science.gov (United States)

    Raj, G Dhinakar; Rajanathan, T M C; Kumar, C Senthil; Ramathilagam, G; Hiremath, Geetha; Shaila, M S

    2008-06-22

    Peste des petits ruminants (PPR) is one of the most economically important diseases affecting sheep and goats in India. An immunofiltration-based test has been developed using either mono-specific serum/monoclonal antibodies (mAb) prepared against a recombinant truncated nucleocapsid protein of rinderpest virus (RPV) cross-reactive with PPR virus. This method consists of coating ocular swab eluate from suspected animals onto a nitrocellulose membrane housed in a plastic module, which is allowed to react with suitable dilutions of a mAb or a mono-specific polyclonal antibody. The antigen-antibody complex formed on the membrane is then detected by protein A-colloidal gold conjugate, which forms a pink colour. In the immunofiltration test, concordant results were obtained using either PPRV mAb or mono-specific serum. Another test, an antigen-competition ELISA which relies on the competition between plate-coated recombinant truncated 'N' protein of RPV and the PPRV 'N' protein present in ocular swab eluates (sample) for binding to the mono-specific antibody against N protein of RPV (in liquid phase) was developed. The cut-off value for this test was established using reverse transcription polymerase chain reaction (RT-PCR) positive and negative oculo-nasal swab samples. Linear correlation between percent inhibition (PI) values in antigen-competition ELISA and virus infectivity titres was 0.992. Comparison of the immunofiltration test with the antigen-competition ELISA yielded a sensitivity of 80% and specificity of 100%. These two tests can serve as a screening (immunofiltration) and confirmatory (antigen-competition ELISA) test, respectively, in the diagnosis of PPR in sheep or goats.

  8. A trans-Complementing Recombination Trap Demonstrates a Low Propensity of Flaviviruses for Intermolecular Recombination▿

    OpenAIRE

    Taucher, Christian; Berger, Angelika; Mandl, Christian W.

    2009-01-01

    Intermolecular recombination between the genomes of closely related RNA viruses can result in the emergence of novel strains with altered pathogenic potential and antigenicity. Although recombination between flavivirus genomes has never been demonstrated experimentally, the potential risk of generating undesirable recombinants has nevertheless been a matter of concern and controversy with respect to the development of live flavivirus vaccines. As an experimental system for investigating the a...

  9. Impacto de Leishmania amazonensis y la Sangre de Ave en el Potencial Biológico y Fecundidad de Lutzomyia migonei y Lutzomyia ovallesi (Diptera: Psychodidae

    Directory of Open Access Journals (Sweden)

    Elsa Nieves

    2011-03-01

    Resumo. Nos flebotomíneos (Diptera: Psychodidae o hábito pela hematofagia é responsável pela indução de vários processos fisiológicos também na transmissão de Leishmania Ross. O presente estudo compara o sangue de ave, de mamífero e com infecção por Leishmania amazonensis Lainson & Shaw sobre o potencial biológico de Lutzomyia migonei (França e de Lutzomyia ovallesi Ortiz. Foram utilizadas fêmeas das duas espécies alimentadas artificialmente com sangue de hamster (Mesocricetus auratus Waterhouse e frango (Gallus gallus Linnaeus, infectados com L. amazonensis. Os grupos controle foram alimentados somente com sangue, sem parasitas. Foram determinados o grau de repasto sanguíneo, o tempo de digestão, o padrão de diurese, o tempo de oviposição, a sobrevivencia a oviposição e a fecundidade. A espécie L. migonei quando alimentada com sangue de hamster e frango apresentaram maior fecundidade do que as fêmeas de L. ovallesi, a maior fecundidade foi com sangue de frango. A presença de Leishmania no sangue de frango ou sangue de hamster diminuiu significativamente o seu consumo, o que resultou na diminuição da sobrevida das fêmeas após a oviposição em L. migonei alimentados com sangue de frango e não com sangue de hamsters. Entretanto, não afetar a quantidade de sangue e a sobrevivência de oviposição de L. ovallesi. A infecção com L. amazonensis causo um aumento no número de ovos retidos e diminuiu o número de ovos postos por L. migonei e L. ovallesi, especialmente com sangue de frango e também reduz o tempo de digestão do sangue em ambas as espécies com sangue de frango, mas não com sangue de hamster. Embora o sangue de frango foi menos eficaz do que o sangue de hamster sobre o potencial biológico de L. migonei e L. ovallesi, não exclui o sangue de frango como uma fonte de sangue para a manutenção das populações de ambas as espécies nas casas.

  10. Duality of β-glucan microparticles: antigen carrier and immunostimulants

    Directory of Open Access Journals (Sweden)

    Baert K

    2016-05-01

    Full Text Available Kim Baert,1 Bruno G De Geest,2 Henri De Greve,3,4 Eric Cox,1,* Bert Devriendt1,* 1Department of Virology, Parasitology and Immunology, 2Department of Pharmaceutics, Ghent University, Merelbeke, Ghent, Belgium; 3Structural Biology Research Centre, VIB, Brussels, Belgium; 4Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium *These authors contributed equally to this work Abstract: Designing efficient recombinant mucosal vaccines against enteric diseases is still a major challenge. Mucosal delivery of recombinant vaccines requires encapsulation in potent immunostimulatory particles to induce an efficient immune response. This paper evaluates the capacity of β-glucan microparticles (GPs as antigen vehicles and characterizes their immune-stimulatory effects. The relevant infectious antigen FedF was chosen to be loaded inside the microparticles. The incorporation of FedF inside the particles was highly efficient (roughly 85% and occurred without antigen degradation. In addition, these GPs have immunostimulatory effects as well, demonstrated by the strong reactive oxygen species (ROS production by porcine neutrophils upon their recognition. Although antigen-loaded GPs still induce ROS production, antigen loading decreases this production by neutrophils for reasons yet unknown. However, these antigen-loaded GPs are still able to bind their specific β-glucan receptor, demonstrated by blocking complement receptor 3, which is the major β-glucan receptor on porcine neutrophils. The dual character of these particles is confirmed by a T-cell proliferation assay. FedF-loaded particles induce a significantly higher FedF-specific T-cell proliferation than soluble FedF. Taken together, these results show that GPs are efficient antigen carriers with immune-stimulatory properties. Keywords: β-glucan microparticles, FedF, antigen delivery vehicle, immunostimulants

  11. Sand fly captures with Disney traps in area of occurrence of Leishmania (Leishmania amazonensis in the state of Mato Grosso do Sul, mid-western Brazil Capturas de flebotomíneos com armadilhas de Disney em área de ocorrência de Leishmania (Leishmania amazonensis no estado de Mato Grosso do Sul, região Centro-Oeste do Brasil

    Directory of Open Access Journals (Sweden)

    Maria Elizabeth Cavalheiros Dorval

    2010-10-01

    Full Text Available INTRODUCTION: The work was conducted to study phlebotomine fauna (Diptera: Psychodidae and aspects of American cutaneous leishmaniasis transmission in a forested area where Leishmania (Leishmania amazonensis occurs, situated in the municipality of Bela Vista, State of Mato Grosso do Sul, Brazil. METHODS: The captures were conducted with modified Disney traps, using hamster (Mesocricetus auratus as bait, from May 2004 to January 2006. RESULTS: Ten species of phlebotomine sandflies were captured: Brumptomyia avellari, Brumptomyia brumpti, Bichromomyia flaviscutellata, Evandromyia bourrouli, Evandromyia lenti, Lutzomyia longipalpis, Psathyromyia campograndensis, Psathyromyia punctigeniculata, Psathyromyia shannoni and Sciopemyia sordellii. The two predominant species were Ev bourrouli (57.3% and Bi flaviscutellata (41.4%, present at all sampling sites. Two of the 36 hamsters used as bait presented natural infection with Leishmania. The parasite was identified as Leishmania (Leishmania amazonensis. CONCLUSIONS: Analysis of the results revealed the efficiency of Disney traps for capturing Bichromomyia flaviscutellata and the simultaneous presence of both vector and the Leishmania species transmitted by the same can be considered a predictive factor of the occurrence of leishmaniasis outbreaks for the human population that occupies the location.INTRODUÇÃO: O estudo foi realizado com o objetivo de estudar a fauna de flebotomíneos (Diptera: Psychodidae e aspectos ligados à transmissão da leishmaniose tegumentar americana em uma área florestal com ocorrência de Leishmania (Leishmania amazonensis, situada no município de Bela Vista, Estado do Mato Grosso do Sul, Brasil. MÉTODOS: As capturas de flebotomíneos foram realizadas utilizando-se armadilhas tipo Disney modificadas, com isca roedor, Mesocricetus auratus, no período de maio de 2004 a janeiro de 2006. RESULTADOS: As coletas resultaram na identificação de 10 espécies de Phlebotominae

  12. Dengue-1 Virus Envelope Glycoprotein Gene Expressed in Recombinant Baculovirus Elicits Virus-Neutralizing Antibody in Mice and Protects them from Virus Challenge

    Science.gov (United States)

    1991-01-01

    8217 terminus of E. When the recombinant virus was grown in Spodoptera frugiperda cells. about I mg of E antigen was made per 10’ cells. Recombinant E antigen...assay with DEN-I virus coprotein gene and its expression in Spodoptera hyperimmune mouse ascitic fluid. This heat-in- frugiperda cells activated...immunization, S. frugiperda cells infected with tion with BstNI (cuts at nucleotides 801 and recombinant baculovirus were pelleted. lysed by 2150). The

  13. Vaxvec: The first web-based recombinant vaccine vector database and its data analysis.

    Science.gov (United States)

    Deng, Shunzhou; Martin, Carly; Patil, Rasika; Zhu, Felix; Zhao, Bin; Xiang, Zuoshuang; He, Yongqun

    2015-11-27

    A recombinant vector vaccine uses an attenuated virus, bacterium, or parasite as the carrier to express a heterologous antigen(s). Many recombinant vaccine vectors and related vaccines have been developed and extensively investigated. To compare and better understand recombinant vectors and vaccines, we have generated Vaxvec (http://www.violinet.org/vaxvec), the first web-based database that stores various recombinant vaccine vectors and those experimentally verified vaccines that use these vectors. Vaxvec has now included 59 vaccine vectors that have been used in 196 recombinant vector vaccines against 66 pathogens and cancers. These vectors are classified to 41 viral vectors, 15 bacterial vectors, 1 parasitic vector, and 1 fungal vector. The most commonly used viral vaccine vectors are double-stranded DNA viruses, including herpesviruses, adenoviruses, and poxviruses. For example, Vaxvec includes 63 poxvirus-based recombinant vaccines for over 20 pathogens and cancers. Vaxvec collects 30 recombinant vector influenza vaccines that use 17 recombinant vectors and were experimentally tested in 7 animal models. In addition, over 60 protective antigens used in recombinant vector vaccines are annotated and analyzed. User-friendly web-interfaces are available for querying various data in Vaxvec. To support data exchange, the information of vaccine vectors, vaccines, and related information is stored in the Vaccine Ontology (VO). Vaxvec is a timely and vital source of vaccine vector database and facilitates efficient vaccine vector research and development.

  14. Strategies for designing and monitoring malaria vaccines targeting diverse antigens

    Directory of Open Access Journals (Sweden)

    Alyssa E Barry

    2014-07-01

    Full Text Available After more than 50 years of intensive research and development, only one malaria vaccine candidate, RTS,S, has progressed to Phase 3 clinical trials. Despite only partial efficacy, this candidate is now forecast to become the first licensed malaria vaccine. Hence, more efficacious second-generation malaria vaccines that can significantly reduce transmission are urgently needed. This review will focus on a major obstacle hindering development of effective malaria vaccines: parasite antigenic diversity. Despite extensive genetic diversity in leading candidate antigens, vaccines have been and continue to be formulated using recombinant antigens representing only one or two strains. These vaccine strains represent only a small fraction of the diversity circulating in natural parasite populations, leading to escape of non-vaccine strains and challenging investigators’ abilities to measure strain-specific efficacy in vaccine trials. Novel strategies are needed to overcome antigenic diversity in order for vaccine development to succeed. Many studies have now catalogued the global diversity of leading Plasmodium falciparum and Plasmodium vivax vaccine antigens. In this review, we describe how population genetic approaches can be applied to this rich data source to predict the alleles that best represent antigenic diversity, polymorphisms that contribute to it, and to identify key polymorphisms associated with antigenic escape. We also suggest an approach to summarise the known global diversity of a given antigen to predict antigenic diversity, how to select variants that best represent the strains circulating in natural parasite populations and how to investigate the strain-specific efficacy of vaccine trials. Use of these strategies in the design and monitoring of vaccine trials will not only shed light on the contribution of genetic diversity to the antigenic diversity of malaria, but will also maximise the potential of future malaria vaccine

  15. An efficient fusion protein system for expression ofBacillus anthracis protective antigen as immunogenic and diagnostic antigen

    Institute of Scientific and Technical Information of China (English)

    Vahid Bagheri; Hossein Motamedi; Masoud Reza Seifiabad Shapouri

    2010-01-01

    Objective:To produce high quantities of recombinant protective antigen (rPA) for human vaccine and diagnosis.Methods: ThePAgene was amplified byPCR with pXO1 plasmid as template. ThePCR product was cloned into pMAL-c2X vector using theBamHI andSalI restriction enzymes. The recombinant plasmid was transformed intoEscherichia coliDH5α strain and then screened for transformation. The expression of protective antigen was analyzed bySDS-PAGE and Western blotting after isopropyl β-D-thiogalactopyranoside(IPTG) induction.Results:The full-length PA gene (2.2kb) was cloned into pMAL vector system. The recombinant vector was confirmed by restriction enzyme andPCRanalysis. The expression of cytoplasmic maltose-binding protein-protective (MBP-P) antigen fusion protein was detected bySDS-PAGE and Western blotting, and obtained a125 kDa protein band, which was similar to expected size of fusion protein.Conclusions: This expression system can be used in the high production of rPA. After purification and immunization studies, the purified rPA may be used in the development of the human recombinant anthrax vaccine and also in diagnosis of anthrax disease.

  16. Ecological Niche Modelling Predicts Southward Expansion of Lutzomyia (Nyssomyia) flaviscutellata (Diptera: Psychodidae: Phlebotominae), Vector of Leishmania (Leishmania) amazonensis in South America, under Climate Change

    Science.gov (United States)

    Carvalho, Bruno M.; Ready, Paul D.

    2015-01-01

    Vector borne diseases are susceptible to climate change because distributions and densities of many vectors are climate driven. The Amazon region is endemic for cutaneous leishmaniasis and is predicted to be severely impacted by climate change. Recent records suggest that the distributions of Lutzomyia (Nyssomyia) flaviscutellata and the parasite it transmits, Leishmania (Leishmania) amazonensis, are expanding southward, possibly due to climate change, and sometimes associated with new human infection cases. We define the vector’s climatic niche and explore future projections under climate change scenarios. Vector occurrence records were compiled from the literature, museum collections and Brazilian Health Departments. Six bioclimatic variables were used as predictors in six ecological niche model algorithms (BIOCLIM, DOMAIN, MaxEnt, GARP, logistic regression and Random Forest). Projections for 2050 used 17 general circulation models in two greenhouse gas representative concentration pathways: “stabilization” and “high increase”. Ensemble models and consensus maps were produced by overlapping binary predictions. Final model outputs showed good performance and significance. The use of species absence data substantially improved model performance. Currently, L. flaviscutellata is widely distributed in the Amazon region, with records in the Atlantic Forest and savannah regions of Central Brazil. Future projections indicate expansion of the climatically suitable area for the vector in both scenarios, towards higher latitudes and elevations. L. flaviscutellata is likely to find increasingly suitable conditions for its expansion into areas where human population size and density are much larger than they are in its current locations. If environmental conditions change as predicted, the range of the vector is likely to expand to southeastern and central-southern Brazil, eastern Paraguay and further into the Amazonian areas of Bolivia, Peru, Ecuador, Colombia and

  17. Ecological Niche Modelling Predicts Southward Expansion of Lutzomyia (Nyssomyia flaviscutellata (Diptera: Psychodidae: Phlebotominae, Vector of Leishmania (Leishmania amazonensis in South America, under Climate Change.

    Directory of Open Access Journals (Sweden)

    Bruno M Carvalho

    Full Text Available Vector borne diseases are susceptible to climate change because distributions and densities of many vectors are climate driven. The Amazon region is endemic for cutaneous leishmaniasis and is predicted to be severely impacted by climate change. Recent records suggest that the distributions of Lutzomyia (Nyssomyia flaviscutellata and the parasite it transmits, Leishmania (Leishmania amazonensis, are expanding southward, possibly due to climate change, and sometimes associated with new human infection cases. We define the vector's climatic niche and explore future projections under climate change scenarios. Vector occurrence records were compiled from the literature, museum collections and Brazilian Health Departments. Six bioclimatic variables were used as predictors in six ecological niche model algorithms (BIOCLIM, DOMAIN, MaxEnt, GARP, logistic regression and Random Forest. Projections for 2050 used 17 general circulation models in two greenhouse gas representative concentration pathways: "stabilization" and "high increase". Ensemble models and consensus maps were produced by overlapping binary predictions. Final model outputs showed good performance and significance. The use of species absence data substantially improved model performance. Currently, L. flaviscutellata is widely distributed in the Amazon region, with records in the Atlantic Forest and savannah regions of Central Brazil. Future projections indicate expansion of the climatically suitable area for the vector in both scenarios, towards higher latitudes and elevations. L. flaviscutellata is likely to find increasingly suitable conditions for its expansion into areas where human population size and density are much larger than they are in its current locations. If environmental conditions change as predicted, the range of the vector is likely to expand to southeastern and central-southern Brazil, eastern Paraguay and further into the Amazonian areas of Bolivia, Peru, Ecuador

  18. Ecological Niche Modelling Predicts Southward Expansion of Lutzomyia (Nyssomyia) flaviscutellata (Diptera: Psychodidae: Phlebotominae), Vector of Leishmania (Leishmania) amazonensis in South America, under Climate Change.

    Science.gov (United States)

    Carvalho, Bruno M; Rangel, Elizabeth F; Ready, Paul D; Vale, Mariana M

    2015-01-01

    Vector borne diseases are susceptible to climate change because distributions and densities of many vectors are climate driven. The Amazon region is endemic for cutaneous leishmaniasis and is predicted to be severely impacted by climate change. Recent records suggest that the distributions of Lutzomyia (Nyssomyia) flaviscutellata and the parasite it transmits, Leishmania (Leishmania) amazonensis, are expanding southward, possibly due to climate change, and sometimes associated with new human infection cases. We define the vector's climatic niche and explore future projections under climate change scenarios. Vector occurrence records were compiled from the literature, museum collections and Brazilian Health Departments. Six bioclimatic variables were used as predictors in six ecological niche model algorithms (BIOCLIM, DOMAIN, MaxEnt, GARP, logistic regression and Random Forest). Projections for 2050 used 17 general circulation models in two greenhouse gas representative concentration pathways: "stabilization" and "high increase". Ensemble models and consensus maps were produced by overlapping binary predictions. Final model outputs showed good performance and significance. The use of species absence data substantially improved model performance. Currently, L. flaviscutellata is widely distributed in the Amazon region, with records in the Atlantic Forest and savannah regions of Central Brazil. Future projections indicate expansion of the climatically suitable area for the vector in both scenarios, towards higher latitudes and elevations. L. flaviscutellata is likely to find increasingly suitable conditions for its expansion into areas where human population size and density are much larger than they are in its current locations. If environmental conditions change as predicted, the range of the vector is likely to expand to southeastern and central-southern Brazil, eastern Paraguay and further into the Amazonian areas of Bolivia, Peru, Ecuador, Colombia and Venezuela

  19. Antigen, allele, and haplotype frequencies report of the ASHI minority antigens workshops: part 1, African-Americans.

    Science.gov (United States)

    Zachary, A A; Bias, W B; Johnson, A; Rose, S M; Leffell, M S

    2001-10-01

    HLA typing was performed on 977 African Americans residing throughout most of the United States. Class I and class II antigens and class II alleles were defined for all individuals and class I alleles were determined for a subset of individuals. The occurrence of 854 of the individuals in family groups permitted direct counting of allele and haplotype frequencies. The data were analyzed for antigen, allele, and haplotype frequencies; recombination frequencies; segregation distortion; distribution of haplotype frequencies; linkage disequilibria; and geographic distribution of DR antigens. Tables of the antigen, allele, the most common two and three point haplotypes, and 88 extended haplotypes that include class I and class II alleles are presented. Notable findings include a lower than expected frequency of recombination between the B and DR loci (theta= 0.0013), lower than expected frequency of inheritance (44.5% vs 54.5%) of the DRB1*1503; DQB1*0602 haplotype, lower than anticipated linkage disequilibrium values for DR; DQ haplotypes, and a skewed geographic distribution of DR antigens.

  20. Leishmaniose cutânea na Amazônia: registro do primeiro caso humano de infecção mista, determinado por duas espécies distintas de Leishmnias: Leishmania brasiliensis e Leishmania mexicana amazonensis Cutaneous leishmaniasis in Amazonia: the first record of a case of cutaneous leishmaniasis caused by two different parasites: Leishmania braziliensis braziliensis and Leishmania mexicana amazonensis

    Directory of Open Access Journals (Sweden)

    F. T. Silveira

    1984-10-01

    Full Text Available Fez-se o registro, na Amazônia, do primeiro caso humano de infecção cutânea mista determinada por duas espécies distintas de Leishmania: a Leishmania braziliensis braziliensis e a Leishmania mexicana amazonensis. As duas amostras, em questão, foram isoladas de lesões distintas de um mesmo paciente, e a caracterização das espécies foi feita com base em observações de infecção experimental em hamsters, comportamento em meios artificiais de cultura, desenvolvimento de infecção experimental em Lutzomyia longipalpis, e eletroforese de isoenzimas em gel de amido. Conclui-se ser de interesse o achado que, combinado com o fato já conhecido de ausência de imunidade cruzada entre a maioria das leishmânias, sugere a necessidade do emprego de uma vacina polivalente para a região.For the first time, in the Amazon region, a mixed infection of two Leishmania was found in a patient suffering from dermal leishmaniasis. L. mexicana amazonensis was isolated from one lesion and L. braziliensis braziliensis from another. Both parasites were characterized in sandflies, hamsters, in vitro cultures, by their morphology and by isoenzyme studies in starch gel. The Authors conclude that the occurrence of this case combined with the known lack of cross immunity between most leishmanial parasites means that a vaccine for this region must be polyvalent.

  1. Genetic diversity and antigenicity variation of Babesia bovis merozoite surface antigen-1 (MSA-1) in Thailand.

    Science.gov (United States)

    Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Takemae, Hitoshi; Simking, Pacharathon; Jittapalapong, Sathaporn; Igarashi, Ikuo; Yokoyama, Naoaki

    2016-07-01

    Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5-100%, and the similarity values among the translated amino acid sequences were 42.8-100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9-68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0-39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand.

  2. Recombinant viruses as vaccines against viral diseases

    Directory of Open Access Journals (Sweden)

    A.P.D. Souza

    2005-04-01

    Full Text Available Vaccine approaches to infectious diseases are widely applied and appreciated. Amongst them, vectors based on recombinant viruses have shown great promise and play an important role in the development of new vaccines. Many viruses have been investigated for their ability to express proteins from foreign pathogens and induce specific immunological responses against these antigens in vivo. Generally, gene-based vaccines can stimulate potent humoral and cellular immune responses and viral vectors might be an effective strategy for both the delivery of antigen-encoding genes and the facilitation and enhancement of antigen presentation. In order to be utilized as a vaccine carrier, the ideal viral vector should be safe and enable efficient presentation of required pathogen-specific antigens to the immune system. It should also exhibit low intrinsic immunogenicity to allow for its re-administration in order to boost relevant specific immune responses. Furthermore, the vector system must meet criteria that enable its production on a large-scale basis. Several viral vaccine vectors have thus emerged to date, all of them having relative advantages and limits depending on the proposed application, and thus far none of them have proven to be ideal vaccine carriers. In this review we describe the potential, as well as some of the foreseeable obstacles associated with viral vaccine vectors and their use in preventive medicine.

  3. Combination of two candidate subunit vaccine antigens elicits protective immunity to ricin and anthrax toxin in mice.

    Science.gov (United States)

    Vance, David J; Rong, Yinghui; Brey, Robert N; Mantis, Nicholas J

    2015-01-09

    In an effort to develop combination vaccines for biodefense, we evaluated a ricin subunit antigen, RiVax, given in conjunction with an anthrax protective antigen, DNI. The combination led to high endpoint titer antibody response, neutralizing antibodies, and protective immunity against ricin and anthrax lethal toxin. This is a natural combination vaccine, since both antigens are recombinant subunit proteins that would be given to the same target population.

  4. Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1

    OpenAIRE

    Yusibov, Vidadi; Modelska, Anna; Steplewski, Klaudia; Agadjanyan, Michail; Weiner, David; Hooper, D. Craig; Koprowski, Hilary

    1997-01-01

    The coat protein (CP) of alfalfa mosaic virus was used as a carrier molecule to express antigenic peptides from rabies virus and HIV. The antigens were separately cloned into the reading frame of alfalfa mosaic virus CP and placed under the control of the subgenomic promoter of tobacco mosaic virus CP in the 30BRz vector. The in vitro transcripts of recombinant virus with sequences encoding the antigenic peptides were synthesized from DNA constructs and used to inoculate tobacco plants. The p...

  5. Expression, purification and serological analysis of hepatocellular carcinoma associated antigen HCA587 in insect cells

    Institute of Scientific and Technical Information of China (English)

    Bing Li; Hong-Yan Wu; Xiao-Ping Qian; Yan Li; Wei-Feng Chen

    2003-01-01

    AIM: In order to assess hepatocellular carcinoma associated antigen HCA587 as a potential target for immunotherapy,the Bac-to-Bac expression system was used to express recombinant protein HCA587 in insect cells.METHODS: The cDNA encoding HCA587 gene was cloned into donor vector pFasBacHtb and recombinant pFasBac Htb587 was transformed into competent cells DH10Bac.Recombinant Bacmid-587 was transfected into Sf9 insect cells using CELLFECTIN, Recombinant HCA587 protein was produced in Sf9 insect cells after infection with recombinant baculovirus, and was purified using Ni-NTA resin. Sera from HCC patients were also screened using recombinant protein HCA587.RESULTS: The molecular weight of the recombinant protein HCA587 expressed in insect cells was approximately 43kd.Western blot results proved the recombinant protein HCA587had the similar antigenicity with its native counterpart.Serological analysis told that the rate of seroreactivity to HCA587 was not high in HCC patients.CONCLUSION: The recombinant protein HCA587 was successfully expressed and purified using Bac-to-Bac expression system. It paved the way for generation of specific antibody and investigation of immunohistochemical analysis and immune responses of HCC in the future.

  6. Antigen-expressed Recombinant Salmonella typhimurium Driven by an in Vivo-activated Promoter is Capable of Inducing Cellular Immune Response in Transgenic Mice%含体内激活启动子的重组减毒鼠伤寒沙门菌诱导转基因鼠细胞免疫应答

    Institute of Scientific and Technical Information of China (English)

    王宏卫; 张珉; 栾洁; 胡卫江; 赵平; 高军; 戚中田

    2003-01-01

    To explore the approaches and mechanisms for reversing the immune tolerance in transgenic mouse, and the pathogenicity of hepatitis G virus (HGV), the promoter of phoP-activated gene (PpagC) of Salmonella typhimurium was used as a transcriptionally regulating element to construct an attenuated S. typhimurium expressing HGV NS3. The recombinant S. typhimurium was orally administered to HGV transgenic mice. As the results, HGV antigen in serum and liver as well as HGV mRNA in liver were decreased significantly, although the serum anti-HGV NS3 remained undetectable as the control transgenic mice. The spleen cell proliferation, in vitro HGV NS3 specific CTL, and IFN-γ assays with the primed cultured splenocytes indicated the induction of Th1 immune responses in those administered transgenic mice. Adoptive transfer of fractionated primed spleen cells to the transgenic mice showed that T lymphocytes were responsible for, 05-be through IFN-γ, the down-regulation of HGV mRNA transcription. Histological examination found no significant inflammatory changes in liver of the transgenic mice. These findings suggested that the oral inoculation of the HGV NS3-expressed attenuated S. typhimurium driven by an in vivo-activated promoter should be a simple and effective approach for potential treatment of chronic viral infection.%以庚型肝炎病毒 (hepatitis G virus, HGV) 转基因小鼠为模型, 探讨逆转免疫耐受的方法及与HGV致病性的关系. 首先采用鼠伤寒沙门菌pagC基因的启动子(PpagC)为转录调控元件, 构建宿主体内表达HGV NS3抗原的重组减毒鼠伤寒沙门菌, 并口服接种免疫HGV转基因小鼠. 结果证明对诱导转基因小鼠血清HGV NS3抗体无明显影响, 但对小鼠血清HGV抗原含量、肝组织HGV抗原及HGV mRNA表达量有明显抑制作用. 体外培养脾细胞表现出针对HGV NS3抗原的细胞免疫反应, 并检测到Th1型细胞因子IFN-γ. 过继转移实验证明T细胞可能是通过IFN-γ

  7. Standardization of an ELISA test using a recombinant nucleoprotein from the Junin virus as the antigen and serological screening for arenavirus among the population of Nova Xavantina, State of Mato Grosso Padronização de um teste de ELISA utilizando a nucleoproteína recombinante de vírus Junin como antígeno e triagem sorológica para Arenavírus na população de Nova Xavantina, Estado do Mato Grosso

    Directory of Open Access Journals (Sweden)

    Alex Martins Machado

    2010-06-01

    Full Text Available INTRODUCTION: Arenavirus hemorrhagic fever is a severe emerging disease. METHODS: Considering that the levels of antibodies against arenavirus in the Brazilian population are completely unknown, we have standardized an ELISA test for detecting IgG antibodies using a recombinant nucleoprotein from the Junin virus as the antigen. This protein was obtained by inserting the gene of the Junin virus nucleoprotein into the genome of Autographa californica nucleopolyhedrovirus, using the Bac-to-Bac baculovirus expression system. This recombinant baculovirus was used to infect S. frugiperda cells (SF9. RESULTS: The infection resulted in synthesis of high concentrations of recombinant protein. This protein was detected on 12.5% polyacrylamide gel and by means of Western blot. Using the standardized ELISA test, 343 samples from the population of Nova Xavantina were analyzed. We observed that 1.4% of the serum samples (five samples presented antibody titers against arenavirus. CONCLUSIONS: These results show the population studied may present exposure to arenavirus infection.INTRODUÇÃO: A febre hemorrágica por Arenavirus é uma severa doença emergente. MÉTODOS: Considerando que os níveis de anticorpos contra Arenavirus na população brasileira é totalmente desconhecido, nos padronizamos um teste de ELISA para detecção de anticorpos IgG usando uma nucleoproteína recombinante do vírus Junin como antígeno. Esta proteína foi obtida pela inserção do gene da nucleoproteína do vírus Junin no genoma do vírus Autographa californica nucleopolyhedrovirus, utilizando o sistema de expressão em Baculovírus, Bac-To-Bac. Este baculovirus recombinante foi utilizado para infecção de células de S. frugiperda (Sf9. RESULTADOS: A infecção resultou na produção de altas concentrações de proteína recombinante. Esta proteína foi detectada em gel de poliacrilamida 12,5%, e em Western blot. Utilizando o teste de ELISA padronizado, foram analizadas 343

  8. Study on Sero-Diagnosis of Nasopharyngeal Carcinoma Using a Dual Antibody Test against Recombinant Epstein-Barr Virus Antigens%抗重组EB病毒抗原双重抗体检测血清学诊断鼻咽癌的研究

    Institute of Scientific and Technical Information of China (English)

    顾耀亮; 张昌卿; 吴子柏; 宗永生; 梁英杰; 陈跃龙

    2003-01-01

    BACKGROUND & OBJECTIVE: The aim of this study was tooptimize a dual-antibody assay for sero-diagnosis of nasopharyngeal carcinoma(NPC) by evaluating 4 Epstein-Barr virus(EBV) antigen-based enzyme-linkedimmunosorbent assays(ELISAs). METHODS: The serum samples of 57pretreated NPC patients and 58 apparently healthy adults in Guangzhou werecollected. The levels of anti-EBV antibody in the sera were tested by 4 ELISAs,which were developed using fusion proteins of glutathione transferase and EBVspecific recombinant antigens, namely, EBNA1-IgA, EBNA1-IgG, Zta-IgA, andZta-IgG. RESULTS: When evaluated individually, the sensitivity(0.9123) andnegative predictive value(0.9074) of EBNA1-IgA were the highest among the 4ELISAs tested. Zta-IgA test had the highest individual accuracy rate(n, 0.8870)and Youden index(J, 0. 7738) . The dual positives for EBNA1-IgA and Zta-IgAwere the highest among the 4 dual positives when paired ELISAs were evaluated.Five NPC patients with negative reaction to EBNA1-IgA showed positive reactionof Zta-IgA, and 7 NPC patients with negative reaction to Zta-IgA showed positivereaction of EBNA1-IgA. CONCLUSION: The EBNA1-IgA assay is more suitablethan the other 3 ELISAs(EBNA1-IgG, Zta-lgA, and Zta-IgG) when usedindividually for serological diagnosis of NPC. When two assays are combined,EBNA1-IgA and Zta-IgA have complementary effect on serological diagnosis forNPC and is thus an optimal combination of serum antibody assays.%背景与目的:在评估4种EB病毒抗原酶联免疫吸附法的基础上,探讨优化抗重组EB病毒抗原双重抗体检测应用于血清学诊断鼻咽癌.方法:收集广州地区57例治疗前鼻咽癌患者和58例健康成人的血清.应用EB病毒特异抗原(谷胱甘肽转移酶重组融合蛋白)为基础的4种免疫酶联吸附法,即EBNA1-IgA,EBNA1-IgG,Zta-IgA和Zta-IgG检测血清中抗EB病毒的抗体水平.结果:EBNA1-IgA的灵敏度(0.9123)和阴性预测值(0.9074)是单独使用4种ELISA实验中最高

  9. Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation.

    Science.gov (United States)

    Devlin, Rebecca; Marques, Catarina A; Paape, Daniel; Prorocic, Marko; Zurita-Leal, Andrea C; Campbell, Samantha J; Lapsley, Craig; Dickens, Nicholas; McCulloch, Richard

    2016-05-26

    Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating - a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility.

  10. The human parasite Leishmania amazonensis downregulates iNOS expression via NF-κB p50/p50 homodimer: role of the PI3K/Akt pathway

    Science.gov (United States)

    Calegari-Silva, Teresa C.; Vivarini, Áislan C.; Miqueline, Marina; Dos Santos, Guilherme R. R. M.; Teixeira, Karina Luiza; Saliba, Alessandra Mattos; Nunes de Carvalho, Simone; de Carvalho, Laís; Lopes, Ulisses G.

    2015-01-01

    Leishmania amazonensis activates the NF-κB transcriptional repressor homodimer (p50/p50) and promotes nitric oxide synthase (iNOS) downregulation. We investigated the role of PI3K/Akt in p50/p50 NF-κB activation and the effect on iNOS expression in L. amazonensis infection. The increased occupancy of p50/p50 on the iNOS promoter of infected macrophages was observed and we demonstrated that both p50/p50 NF-κB induction and iNOS downregulation in infected macrophages depended on PI3K/Akt activation. Importantly, the intracellular growth of the parasite was also impaired during PI3K/Akt signalling inhibition and in macrophages knocked-down for Akt 1 expression. It was also observed that the increased nuclear levels of p50/p50 in L. amazonensis-infected macrophages were associated with reduced phosphorylation of 907 Ser p105, the precursor of p50. Corroborating these data, we demonstrated the increased levels of phospho-9 Ser GSK3β in infected macrophages, which is associated with GSK3β inhibition and, consequently, its inability to phosphorylate p105. Remarkably, we found that the levels of pPTEN 370 Ser, a negative regulator of PI3K, increased due to L. amazonensis infection. Our data support the notion that PI3K/Akt activity is sustained during the parasite infection, leading to NF-κB 105 phosphorylation and further processing to originate p50/p50 homodimers and the consequent downregulation of iNOS expression. PMID:26400473

  11. 细粒棘球蚴重组抗原B诱导小鼠骨髓源树突状细胞表达吲哚胺2,3-双加氧酶的研究%Echinococcus granulosus Recombinant Antigen B Induced IDO Expression in Mouse Bone Marrow-derived Dendritic Cells in Vitro

    Institute of Scientific and Technical Information of China (English)

    单骄宇; 纪卫政; 吐尔洪江·吐逊; 李亮; 张传山; 林仁勇; 温浩

    2011-01-01

    目的 观察细粒棘球蚴重组抗原B (rAgB)体外诱导小鼠骨髓源树突状细胞表达吲哚胺2,3-双加氧酶(IDO)的情况.方法 从小鼠股骨中分离出骨髓细胞,进行小鼠重组巨噬细胞集落刺激因子(rmGM-CSF)的诱导,培养并获得CD11c+ 树突状细胞.应用倒置显微镜和扫描电镜观察树突状细胞形态,采用流式细胞术检测其表面标志物;用混合淋巴细胞反应(MLR)观察树突状细胞对T淋巴细胞的增殖能力.培养至第6天,收集未成熟树突状细胞进行流式细胞术检测;另向部分未成熟树突状细胞中加脂多糖(LPS)刺激24 h后,收集成熟树突状细胞,进行流式细胞术检测.在获得的未成熟树突状细胞中分别加入RPMI 1640完全培养液(为阴性对照组)、重组小鼠γ干扰素(rmIFN-γ,1 000 U/ml,为IFN-γ组)和rAgB(终浓度15 *9滋g/ml,为rAgB组),培养24 h后,通过细胞免疫组织化学和蛋白质印迹(Western blotting)检测各组树突状细胞IDO的表达情况.结果 获得纯度为80%的CD11c+ 树突状细胞,在倒置显微镜和扫描电镜下均观察到典型树突状细胞.经LPS刺激的成熟树突状细胞的CD40、CD80和主要组织相容性复合体(MHC)Ⅱ类分子I-A/I-E的阳性表达率与未成熟树突状细胞的相比,差异均有统计学意义(均P0.05).结论 细粒棘球蚴重组抗原B在体外具有诱导树突状细胞表达吲哚胺2,3-双加氧酶的功能.%Objective To observe the expression of indoleamine 2,3-dioxygenase (IDO) in mouse bone marrowderived dendritic cells (DCs) after adding Echinococcus granulosus recombinant antigen B (rAgB) in vitro. Methods CD11c+ DCs generated from bone marrow precursor cells of C57BL/6 mice and cultured in the presence of recombinant mouse GM-CSF (rmGM-CSF). The morphology of DCs was observed by inverted microscope and scanning electronic microscope. The level of I-A/I-E, CD40, CD80, and CD86 on DCs were determined by flow cytometry. T cell proliferation induced by

  12. A novel adjuvant-free H fusion system for the production of recombinant immunogens in Escherichia coli

    Science.gov (United States)

    Costa, Sofia J; Silva, Pedro; Almeida, André; Conceição, Antónia; Domingues, Lucília; Castro, António

    2013-01-01

    The production of recombinant antigens in Escherichia coli and specific polyclonal antibodies for diagnosis and therapy is still a challenge for world-wide researchers. Several different strategies have been explored to improve both antigen and antibody production, all of them depending on a successful expression and immunogenicity of the antigen. Gene fusion technology attempted to address these challenges: fusion partners have been applied to optimize recombinant antigen production in E. coli, and to increase protein immunogenicity. Taking a 12-kDa surface adhesion antigen from Cryptosporidium parvum (CP12) by example, the novel H fusion partner was presented in this work as an attractive option for the development of recombinant immunogens and its adjuvant-free immunization. The H tag (of only 1 kDa) efficiently triggered a CP12-specific immune response, and it also improved the immunization procedure without requiring co-administration of adjuvants. Moreover, polyclonal antibodies raised against the HCP12 fusion antigen detected native antigen structures displayed on the surface of C. parvum oocysts. The H tag proved to be an advanced strategy and promising technology for the diagnosis and therapy of C. parvum infections in animals and humans, allowing a rapid and simple recombinant production of the CP12 antigen. PMID:23941978

  13. THE SEARCH OF OPTIMAL COMBINATION OF ANTIGENS FOR SEROLOGICAL DIAGNOSTICS OF TUBERCULOSIS

    Directory of Open Access Journals (Sweden)

    E. V. Vasilyeva

    2013-01-01

    Full Text Available Abstract. The four chimeric recombinant antigens CBD-CFP10, CBD-ESAT6, ESAT6-CFP10 and CBD-P38 contained aminoacid sequences of full-size proteins ESAT6, CFP10 and matured protein P38 of M. tuberculosis, joined with aminoacid sequences of cellulose bind domain of endogluconase A (CBD from Cellumonas fimi have been obtained by gene engineering methods. Recombinant proteins were purified by affine chromatography in column with Ni-NTA-sepharose 6В-CL and as PPDN-3 were used for detection of their antigenic activity in indirect ELISA for TB serological diagnostics. The sera from patients with lung tuberculosis (n = 321, from persons who had professional contacts with TB patients (n = 42, from healthy blood donors (n = 366 and from patients with lung diseases of non-TB etiology were tested. It was detected that there was positive correlation between antibodies level for all studied antigens compared by pair. It has been demonstrated that although antigens were different by antigenic and immunobiological characteristics they add each other in the content of antigenic diagnostics compositions. Thus, all these antigens can be used in the test kits for serological diagnostics of TB. Using of these antigens will allow to detect persons infected by TB and patients with active tuberculosis. 

  14. Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Mustafa, A S; Amoudy, H A; Wiker, H G;

    1998-01-01

    GroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M. tuberculosis, M. tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well...... as the vaccine strain, Mycobacterium bovis bacillus Calmette-Guerin (BCG). In addition, M. tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T-cell lines tested for antigen-induced proliferation and IFN......We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon-gamma (IFN-gamma) secretion against a panel of purified recombinant (r) and natural (n) culture filtrate (rESAT-6, nMPT59, nMPT64 and nMPB70) and somatic-derived (r...

  15. Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Mustafa, A S; Amoudy, H A; Wiker, H G

    1998-01-01

    We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon-gamma (IFN-gamma) secretion against a panel of purified recombinant (r) and natural (n) culture filtrate (rESAT-6, nMPT59, nMPT64 and nMPB70) and somatic-derived (r......GroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M. tuberculosis, M. tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well...... as the vaccine strain, Mycobacterium bovis bacillus Calmette-Guerin (BCG). In addition, M. tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T-cell lines tested for antigen-induced proliferation and IFN...

  16. Optimization of the expression of surface antigen SAG1/2 of Toxoplasma gondii in the yeast Pichia pastoris.

    Science.gov (United States)

    Thiruvengadam, G; Init, I; Fong, M Y; Lau, Y L

    2011-12-01

    Surface antigens are the most abundant proteins found on the surface of the parasite Toxoplasma gondii. Surface antigen 1 (SAG1) and Surface antigen 2 (SAG2) remain the most important and extensively studied surface proteins. These antigens have been identified to play a role in host cell invasion, immune modulation, virulence attenuation. Recombinant SAG1/2 was cloned and expressed in yeast Pichia pastoris. We describe here optimization of critical parameters involved in high yield expression of the recombinant SAG1/2. Our results suggest that recombinant SAG1/2 were best expressed at 30ºC, pH 6 and 1% methanol as the carbon source by X33 Pichia cells. Additional optimizations included the downstream process such as ammonium sulphate precipitation and dialysis. The fusion protein was purified using Ni-NTA purification system with 80% recovery. The purified protein was 100% specific and sensitive in detection of toxoplasmosis.

  17. Antibodies to a recombinant glutamate-rich Plasmodium falciparum protein

    DEFF Research Database (Denmark)

    Hogh, B; Petersen, E; Dziegiel, Morten Hanefeld;

    1992-01-01

    A Plasmodium falciparum antigen gene coding for a 220-kD glutamate-rich protein (GLURP) has been cloned, and the 783 C-terminal amino acids of this protein (GLURP489-1271) have been expressed as a beta-galactosidase fusion protein in Escherichia coli. The encoded 783 amino acid residues contain two...... areas of repeated amino acid sequences. Antibodies against recombinant GLURP489-1271, as well as against a synthetic peptide corresponding to GLURP899-916, and against a synthetic peptide representing the major glutamate rich repeat sequence from the P. falciparum ring erythrocyte surface antigen (Pf155...

  18. Recombinant methods and materials

    Energy Technology Data Exchange (ETDEWEB)

    Roizman, B.; Post, L.E.

    1988-09-06

    This patent describes a method for stably effecting the insertion or deletion of a selected DNA sequence at a specific site in a viral genome. The method consists of: (1) isolating from the genome a linear DNA fragment comprising both (a) the specific site determined for insertion or deletion of selected DNA sequence and (b) flanking DNA sequences normally preceding and following the site; (2) preparing first and second altered genome fragments from the fragment isolated in step (1). (a) the first altered fragment comprising the fragment comprising a thymidine kinase gene in a position intermediate the ends of the fragment, and (b) the second altered fragment comprising the fragment having the selected DNA sequence inserted therein or deleted therefrom; (3) contacting the genome with the first altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome comprising the thymidine kinase gene, and isolating the recombinant genome; and (4) contacting the recombinant genome isolated in step (3) with the second altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome lacking the thymidine kinase gene, and isolating the recombinant genome product.

  19. Recombinant protein-based viral disease diagnostics in veterinary medicine.

    Science.gov (United States)

    Balamurugan, Vinayagamurthy; Venkatesan, Gnanavel; Sen, Arnab; Annamalai, Lakshmanan; Bhanuprakash, Veerakyathappa; Singh, Raj Kumar

    2010-09-01

    Identification of pathogens or antibody response to pathogens in human and animals modulates the treatment strategies for naive population and subsequent infections. Diseases can be controlled and even eradicated based on the epidemiology and effective prophylaxis, which often depends on development of efficient diagnostics. In addition, combating newly emerging diseases in human as well as animal healthcare is challenging and is dependent on developing safe and efficient diagnostics. Detection of antibodies directed against specific antigens has been the method of choice for documenting prior infection. Other than zoonosis, development of inexpensive vaccines and diagnostics is a unique problem in animal healthcare. The advent of recombinant DNA technology and its application in the biotechnology industry has revolutionized animal healthcare. The use of recombinant DNA technology in animal disease diagnosis has improved the rapidity, specificity and sensitivity of various diagnostic assays. This is because of the absence of host cellular proteins in the recombinant derived antigen preparations that dramatically decrease the rate of false-positive reactions. Various recombinant products are used for disease diagnosis in veterinary medicine and this article discusses recombinant-based viral disease diagnostics currently used for detection of pathogens in livestock and poultry.

  20. Dissociative recombination in aeronomy

    Science.gov (United States)

    Fox, J. L.

    1989-01-01

    The importance of dissociative recombination in planetary aeronomy is summarized, and two examples are discussed. The first is the role of dissociative recombination of N2(+) in the escape of nitrogen from Mars. A previous model is updated to reflect new experimental data on the electronic states of N produced in this process. Second, the intensity of the atomic oxygen green line on the nightside of Venus is modeled. Use is made of theoretical rate coefficients for production of O (1S) in dissociative recombination from different vibrational levels of O2(+).

  1. Trypanosoma cruzi as an effective cancer antigen delivery vector.

    Science.gov (United States)

    Junqueira, Caroline; Santos, Luara I; Galvão-Filho, Bruno; Teixeira, Santuza M; Rodrigues, Flávia G; DaRocha, Wanderson D; Chiari, Egler; Jungbluth, Achim A; Ritter, Gerd; Gnjatic, Sacha; Old, Lloyd J; Gazzinelli, Ricardo T

    2011-12-06

    One of the main challenges in cancer research is the development of vaccines that induce effective and long-lived protective immunity against tumors. Significant progress has been made in identifying members of the cancer testis antigen family as potential vaccine candidates. However, an ideal form for antigen delivery that induces robust and sustainable antigen-specific T-cell responses, and in particular of CD8(+) T lymphocytes, remains to be developed. Here we report the use of a recombinant nonpathogenic clone of Trypanosoma cruzi as a vaccine vector to induce vigorous and long-term T cell-mediated immunity. The rationale for using the highly attenuated T. cruzi clone was (i) the ability of the parasite to persist in host tissues and therefore to induce a long-term antigen-specific immune response; (ii) the existence of intrinsic parasite agonists for Toll-like receptors and consequent induction of highly polarized T helper cell type 1 responses; and (iii) the parasite replication in the host cell cytoplasm, leading to direct antigen presentation through the endogenous pathway and consequent induction of antigen-specific CD8(+) T cells. Importantly, we found that parasites expressing a cancer testis antigen (NY-ESO-1) were able to elicit human antigen-specific T-cell responses in vitro and solid protection against melanoma in a mouse model. Furthermore, in a therapeutic protocol, the parasites expressing NY-ESO-1 delayed the rate of tumor development in mice. We conclude that the T. cruzi vector is highly efficient in inducing T cell-mediated immunity and protection against cancer cells. More broadly, this strategy could be used to elicit a long-term T cell-mediated immunity and used for prophylaxis or therapy of chronic infectious diseases.

  2. Anti-HCV immunoassays based on a multiepitope antigen and fluorescent lanthanide chelate reporters.

    Science.gov (United States)

    Salminen, Teppo; Juntunen, Etvi; Khanna, Navin; Pettersson, Kim; Talha, Sheikh M

    2016-02-01

    There is a need for simple to produce immunoassays for hepatitis C virus (HCV) antibody capable of detecting all genotypes worldwide. Current commonly used third generation immunoassays use three to six separate recombinant proteins or synthetic peptides. We have developed and expressed in Escherichia coli a single recombinant antigen incorporating epitopes from different HCV proteins. This multiepitope protein (MEP) was used to develop two types of HCV antibody immunoassays: a traditional antibody immunoassay using a labeled secondary antibody (indirect assay) and a double-antigen assay with the same MEP used as capture binder and labeled binder. The secondary antibody assay was evaluated with 171 serum/plasma samples and double-antigen assay with 148 samples. These samples included an in-house patient sample panel, two panels of samples with different HCV genotypes and a seroconversion panel. The secondary antibody immunoassay showed 95.6% sensitivity and 100% specificity while the double-antigen assay showed 91.4% sensitivity and 100% specificity. Both assays detected samples from all six HCV genotypes. The results showed that combining a low-cost recombinant MEP binder antigen with a high sensitivity fluorescent lanthanide reporter can provide a sensitive and specific immunoassay for HCV serology. The results also showed that the sensitivity of HCV double-antigen assays may suffer from the low avidity immune response of acute infections.

  3. Indirect ELISA with Recombinant GP5 for Detecting Antibodies to Porcine Reproductive and Respiratory Syndrome Virus

    Institute of Scientific and Technical Information of China (English)

    Yan Chen; Hong Tian; Jian-Hui He; Jin-Yin Wu; You-jun Shang; Xiang-tao Liu

    2011-01-01

    Porcine reproductive and respiratory syndrome is caused by the PRRS virus(PRRSV), which has six structural proteins(GP2, GP3, GP4, GP5, M and N). GP5 and N protein are important targets for serological detection by enzyme-linked immunosorbent assay(ELISA)and other methods. Toward this goal, we developed an indirect ELISA with recombinant GP5 antigens and this method was validated by comparison to the LSI PRRSV-Ab ELISA kit. The results indicated that the optimal concentration of coated recombinant antigen was 0.2 μg/well for a serum dilution of 1:40. The rate of agreement with the LSI PRRSV-Ab kit was 88.7%(266/300). These results support the potential use of recombinant GP5 as an antigen for indirect ELISA to detect PRRSV antibodies in pigs.

  4. Construction and expression of recombined human AFP eukaryotic expression vector

    Institute of Scientific and Technical Information of China (English)

    Li-Wang Zhang; Yang-Lin Pan; Stephen M Festein; Jun Ren; Liang Zhang; Hong-Mei Zhang; Bin Jin; Bo-Rong Pan; Xiao-Ming Si; Yan-Jun Zhang; Zhong-Hua Wang

    2003-01-01

    AIM: To construct a recombined human AFP eukaryotic expression vector for the purpose of gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: The full length AFP-cDNA of prokaryotic vector was digested, and subcloned to the multi-clony sites of the eukaryotic vector. The constructed vector was confirmed by enzymes digestion and electrophoresis, and the product expressed was detected by electrochemiluminescence and immunofluorescence methods.RESULTS: The full length AFP-cDNA successfully cloned to the eukaryotic vector through electrophoresis, 0.9723 IU/ml AFP antigen was detected in the supernatant of AFPCHO by electrochemiluminescence method. Compared with the control groups, the differences were significant (P<0.05).AFP antigen molecule was observed in the plasma of AFPCHO by immunofluorescence staining.CONCLUSION: The recombined human AFP eukaryotic expression vector can express in CHO cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma.

  5. Cloning, Prokaryotic Expression, and Antigenicity Analysis of NS1 Gene of H9N2 Swine Influenza Virus

    Institute of Scientific and Technical Information of China (English)

    WANG Fang-kun; YUAN Xiu-fang; WANG Yi-cheng; ZHANG Cun; XU Li-huan; LIU Si-dang

    2008-01-01

    To obtain the NS1 gene of swine influenza virus H9N2 subtype expressed efficiently in E. coli, to develope the effective diagnostic methods for swine influenza virus H9N2 subtype, the NS1 gene of swine influenza virus H9N2 subtype was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and cloned into a prokaryotic expression vector, pET-28a(+), and overexpressed in E. coli BL21-DE3 after induction with 5 mmol L-1 lactose. The recombinant protein was purified by Ni-NTA and identified by western-blotting. An indirect enzyme-linked immunosorbent assay (ELISA) was used to analyze the antigenicity of the recombinant protein. The recombinant protein of NS1 was about 26 kD. The Western-blotting test showed that the recombinant protein reacted specifically with positive sera. The results of the ELISA test showed that the recombinant protein had good antigenicity.

  6. Unconventional T lymphocytes - recombinant MHC molecules pave the way

    OpenAIRE

    Walter, Steffen

    2005-01-01

    T cells are central orchestrators and effectors of the adaptive immune system. CD8+ T cells that recognize peptide antigens presented on MHC class I molecules are believed to play a central role in fighting viral infections, intracellular pathogens and cancer. The use of recombinant peptide-HLA class I complexes that mimic the natural ligands of human CD8+ T cells should greatly facilitate the manipulation and analysis of such cells, allowing further insight in their biology and opening thera...

  7. Surface expression of Helicobacter pylori HpaA adhesion antigen on Vibrio cholerae, enhanced by co-expressed enterotoxigenic Escherichia coli fimbrial antigens.

    Science.gov (United States)

    Tobias, Joshua; Lebens, Michael; Wai, Sun Nyunt; Holmgren, Jan; Svennerholm, Ann-Mari

    2017-02-17

    Helicobacter pylori infection can cause peptic ulceration and is associated with gastric adenocarcinoma. This study aimed to construct and characterize a non-virulent Vibrio cholerae O1 strain, which grows more rapidly than H. pylori, as vector for H. pylori antigens for possible use as a vaccine strain against H. pylori. This was done by recombinant expression of the H. pylori adhesion antigen HpaA alone or, as a proof of principle, together with different colonization factor (CF) antigens of enterotoxigenic Escherichia coli (ETEC) which may enhance immune responses against HpaA. A recombinant V. cholerae strain co-expressing HpaA and a fimbrial CF antigens CFA/I or CS5, but not the non-fimbrial CF protein CS6, was shown to express larger amounts of HpaA on the surface when compared with the same V. cholerae strain expressing HpaA alone. Mutations in the CFA/I operon showed that the chaperon, possibly together with the usher, was involved in enhancing the surface expression of HpaA. Oral immunization of mice with formaldehyde-inactivated recombinant V. cholerae expressing HpaA alone or together with CFA/I induced significantly higher serum antibody responses against HpaA than mice similarly immunized with inactivated HpaA-expressing H. pylori bacteria. Our results demonstrate that a non-virulent V. cholerae strain can be engineered to allow strong surface expression of HpaA, and that the expression can be further increased by co-expressing it with ETEC fimbrial antigens. Such recombinant V. cholerae strains expressing HpaA, and possibly also other H. pylori antigens, may have the potential as oral inactivated vaccine candidates against H. pylori.

  8. Generation of monoclonal antibodies against highly conserved antigens.

    Directory of Open Access Journals (Sweden)

    Hongzhe Zhou

    Full Text Available BACKGROUND: Therapeutic antibody development is one of the fastest growing areas of the pharmaceutical industry. Generating high-quality monoclonal antibodies against a given therapeutic target is very crucial for the success of the drug development. However, due to immune tolerance, some proteins that are highly conserved between mice and humans are not very immunogenic in mice, making it difficult to generate antibodies using a conventional approach. METHODOLOGY/PRINCIPAL FINDINGS: In this report, the impaired immune tolerance of NZB/W mice was exploited to generate monoclonal antibodies against highly conserved or self-antigens. Using two highly conserved human antigens (MIF and HMGB1 and one mouse self-antigen (TNF-alpha as examples, we demonstrate here that multiple clones of high affinity, highly specific antibodies with desired biological activities can be generated, using the NZB/W mouse as the immunization host and a T cell-specific tag fused to a recombinant antigen to stimulate the immune system. CONCLUSIONS/SIGNIFICANCE: We developed an efficient and universal method for generating surrogate or therapeutic antibodies against "difficult antigens" to facilitate the development of therapeutic antibodies.

  9. Molecular Modeling Approaches for Determining Gene Function: application to a Putative Poly-A Binding Protein from Leishmania amazonensis (LaPABP

    Directory of Open Access Journals (Sweden)

    Silva-Jr FP

    2002-01-01

    Full Text Available The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 aminoacids polypeptide (estimated 18 kDa and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy.

  10. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein

    Science.gov (United States)

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F.; Nam, Ho-Woo

    2016-01-01

    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection. PMID:27180586

  11. An Overview of Live Attenuated Recombinant Pseudorabies Viruses for Use as Novel Vaccines

    Directory of Open Access Journals (Sweden)

    Bo Dong

    2014-01-01

    Full Text Available Pseudorabies virus (PRV is a double-stranded, DNA-based swine virus with a genome approximating 150 kb in size. PRV has many nonessential genes which can be replaced with genes encoding heterologous antigens but without deleterious effects on virus propagation. Recombinant PRVs expressing both native and foreign antigens are able to stimulate immune responses. In this paper, we review the current status of live attenuated recombinant PRVs and live PRV-based vector vaccines with potential for controlling viral infections in animals.

  12. Regulation of Meiotic Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  13. Cancer testis antigen and immunotherapy

    Directory of Open Access Journals (Sweden)

    Krishnadas DK

    2013-04-01

    Full Text Available Deepa Kolaseri Krishnadas, Fanqi Bai, Kenneth G Lucas Department of Pediatrics, Division of Hematology/Oncology, University of Louisville, KY, USA Abstract: The identification of cancer testis (CT antigens has been an important advance in determining potential targets for cancer immunotherapy. Multiple previous studies have shown that CT antigen vaccines, using both peptides and dendritic cell vaccines, can elicit clinical and immunologic responses in several different tumors. This review details the expression of melanoma antigen family A, 1 (MAGE-A1, melanoma antigen family A, 3 (MAGE-A3, and New York esophageal squamous cell carcinoma-1 (NY-ESO-1 in various malignancies, and presents our current understanding of CT antigen based immunotherapy. Keywords: cancer testis antigens, immunotherapy, vaccine

  14. New approaches with different types of circulating cathodic antigen for the diagnosis of patients with low Schistosoma mansoni load.

    Directory of Open Access Journals (Sweden)

    Rafaella Grenfell

    Full Text Available BACKGROUND: Schistosomiasis mansoni is a debilitating and sometimes fatal disease. Accurate diagnosis plays a key role in patient management and infection control. However, currently available parasitological methods are laborious and lack sensitivity. The selection of target antigen candidates has turned out to be a promising tool for the development of more sensitive diagnostic methods. In our previous investigations, the use of crude antigens led to false-positive results. Recently, focus has been given to highly purified Schistosoma mansoni antigens, especially to circulating antigens. METHOD: Thus, our main goal was to test different types of circulating cathodic antigen glycoprotein (CCA, as "crude antigen," the protein chain of recombinant CCA and two individual peptides. These schistosome proteins/peptides were tested in a new diagnostic method employing immunomagnetic separation based on the improvement of antigen-antibody binding. PRINCIPAL FINDINGS: Use of recombinant CCA as a diagnostic antigen allowed us to develop a diagnostic assay with high sensitivity and specificity with no false-negative results. Interestingly, the "crude antigen" worked as a good marker for control of cure after praziquantel treatment. CONCLUSIONS/SIGNIFICANCE: Our new diagnostic method was superior to enzyme-linked immunosorbent assay in diagnosing low endemicity patients.

  15. Recombinant feline leukemia virus genes detected in naturally occurring feline lymphosarcomas.

    OpenAIRE

    Sheets, R L; Pandey, R.; Jen, W C; Roy-Burman, P

    1993-01-01

    Using a polymerase chain reaction strategy aimed at detecting recombinant feline leukemia virus (FeLV) genomes with 5' env sequences originating from an endogenous source and 3' env sequences resulting from FeLV subgroup A (FeLV-A), we detected recombinant proviruses in approximately three-fourths of naturally occurring thymic and alimentary feline lymphosarcomas (LSAs) and one-third of the multicentric LSAs from cats determined to be FeLV capsid antigen positive by immunofluorescence assay. ...

  16. High yield expression and single-step purification of Toxoplasma gondii SAG1, GRA1, and GRA7 antigens in Escherichia coli

    DEFF Research Database (Denmark)

    Hiszczynska-Sawicka, E.; Brillowska-Dabrowska, A.; Dabrowski, Slawomir;

    2003-01-01

    This report describes a simple, highly efficient and reproducible method for obtaining large quantities of highly pure recombinant Toxoplasma gondii antigens, which can be used for diagnostic application. The obtained T gondii SAG1, GRA1, and GRA7 antigens (as fusion proteins), expressed...... in the serodiagnosis of T gondii infection. (C) 2002 Elsevier Science (USA). All rights reserved....

  17. Assembly and expression of a synthetic gene encoding the antigen Pfs48/45 of the human malaria parasite Plasmodium falciparum in yeast.

    NARCIS (Netherlands)

    Milek, R.L.B.; Stunnenberg, H.G.; Konings, R.N.H.

    2000-01-01

    Pfs48/45 is an important transmission-blocking vaccine candidate antigen of the human malaria parasite Plasmodium falciparum. This study was aimed at synthesis of recombinant Pfs48/45 containing conformation-constrained epitopes of the native antigen in yeast. Since in the yeast Saccharomyces cerevi

  18. Comparative testing of six antigen-based malaria vaccine candidates directed toward merozoite-stage Plasmodium falciparum

    DEFF Research Database (Denmark)

    Arnot, David E; Cavanagh, David R; Remarque, Edmond J;

    2008-01-01

    Immunogenicity testing of Plasmodium falciparum antigens being considered as malaria vaccine candidates was undertaken in rabbits. The antigens compared were recombinant baculovirus MSP-1(19) and five Pichia pastoris candidates, including two versions of MSP-1(19), AMA-1 (domains I and II), AMA-1...

  19. Recombination experiments at CRYRING

    Energy Technology Data Exchange (ETDEWEB)

    Spies, W.; Glans, P.; Zong, W.; Gao, H.; Andler, G.; Justiniano, E.; Saito, M.; Schuch, R

    1998-11-15

    Recent advances in studies of electron-ion recombination processes at low relative energies with the electron cooler of the heavy-ion storage ring CRYRING are shown. Through the use of an adiabatically expanded electron beam, collisions down to 10{sup -4}eV relative energies were measured with highly charged ions stored in the ring at around 15 MeV/amu energies. Examples of recombination measurements for bare ions of D{sup +}, He{sup 2+}, N{sup 7+}, Ne{sup 10+} and Si{sup 14+} are presented. Further on, results of an experiment measuring laser-induced recombination (LIR) into n=3 states of deuterium with polarized laser light are shown.

  20. Recombinant Helicobacter pylori catalase

    Institute of Scientific and Technical Information of China (English)

    Yang Bai; Ya-Li Zhang; Jian-Feng Jin; Ji-De Wang; Zhao-Shan Zhang

    2003-01-01

    AIM: To construct a recombinant strain which highly expresses catalase of Helicobacter pylori(H.pylori) and assay the activity of H. pylori catalase.METHODS: The catalase DNA was amplified from H. pylori chromosomal DNA with PCR techniques and inserted into the prokaryotie expression vector pET-22b (+), and then was transformed into the BL21 (DE3) E. coli strain which expressed catalase recombinant protein. The activity of H.pylori catalase was assayed by the Beers & Sizers.RESULTS: DNA sequence analysis showed that the sequence of catalase DNA was the same as GenBank's research. The catalase recombinant protein amounted to 24.4 % of the total bacterial protein after induced with IPTG for 3 hours at 37 ℃ and the activity of H. pylori catalase was high in the BL21 (DE3) E. coli strain.CONCLUSION: A clone expressing high activity H. pylori catalase is obtained, laying a good foundation for further studies.

  1. Application of preparative disk gel electrophoresis for antigen purification from inclusion bodies.

    Science.gov (United States)

    Okegawa, Yuki; Koshino, Masanori; Okushima, Teruya; Motohashi, Ken

    2016-02-01

    Specific antibodies are a reliable tool to examine protein expression patterns and to determine the protein localizations within cells. Generally, recombinant proteins are used as antigens for specific antibody production. However, recombinant proteins from mammals and plants are often overexpressed as insoluble inclusion bodies in Escherichia coli. Solubilization of these inclusion bodies is desirable because soluble antigens are more suitable for injection into animals to be immunized. Furthermore, highly purified proteins are also required for specific antibody production. Plastidic acetyl-CoA carboxylase (ACCase: EC 6.4.1.2) from Arabidopsis thaliana, which catalyzes the formation of malonyl-CoA from acetyl-CoA in chloroplasts, formed inclusion bodies when the recombinant protein was overexpressed in E. coli. To obtain the purified protein to use as an antigen, we applied preparative disk gel electrophoresis for protein purification from inclusion bodies. This method is suitable for antigen preparation from inclusion bodies because the purified protein is recovered as a soluble fraction in electrode running buffer containing 0.1% sodium dodecyl sulfate that can be directly injected into immune animals, and it can be used for large-scale antigen preparation (several tens of milligrams).

  2. Identification, cloning, and purification of protein antigens of Treponema pallidum.

    Science.gov (United States)

    Stamm, L V; Dallas, W S; Ray, P H; Bassford, P J

    1988-01-01

    Difficulties in culturing the bacterium Treponema pallidum have greatly hindered syphilis research. In recent years, several laboratories have begun applying recombinant DNA technology to the study of this organism. Recent work is summarized concerning the expression of T. pallidum DNA in Escherichia coli. A number of E. coli clones expressing treponemal protein antigens have been identified. In one instance, a recombinant protein was purified to homogeneity and shown to be identical to a highly immunogenic, native T. pallidum membrane protein of molecular weight 39,000, which was designated the basic membrane protein (BMP) of this organism. In addition, recent experiments are described that were designed to identify cell-surface proteins that would serve as the primary focus of our cloning efforts. Results obtained with use of several different approaches strongly suggest that the outer membrane of T. pallidum is an antigenically inert structure largely devoid of protein. However, a class of low-molecular-weight protein antigens have been identified that are actively secreted into the extracellular medium. Attempts currently are being made to clone these secreted proteins and investigate their roles in the pathogenesis and immunobiology of syphilis.

  3. Antigen antibody interactions

    CERN Document Server

    DeLisi, Charles

    1976-01-01

    1. 1 Organization of the Immune System One of the most important survival mechanisms of vertebrates is their ability to recognize and respond to the onslaught of pathogenic microbes to which they are conti- ously exposed. The collection of host cells and molecules involved in this recognition­ 12 response function constitutes its immune system. In man, it comprises about 10 cells 20 (lymphocytes) and 10 molecules (immunoglobulins). Its ontogenic development is c- strained by the requirement that it be capable of responding to an almost limitless variety of molecular configurations on foreign substances, while simultaneously remaining inert to those on self components. It has thus evolved to discriminate, with exquisite precision, between molecular patterns. The foreign substances which induce a response, called antigens, are typically large molecules such as proteins and polysaccharides. The portions of these with which immunoglobulins interact are called epitopes or determinants. A typical protein epitope m...

  4. Recombineering linear BACs.

    Science.gov (United States)

    Chen, Qingwen; Narayanan, Kumaran

    2015-01-01

    Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.

  5. Recombinant renewable polyclonal antibodies.

    Science.gov (United States)

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  6. Recombinant DNA for Teachers.

    Science.gov (United States)

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  7. Antigenic Variation in Bacterial Pathogens.

    Science.gov (United States)

    Palmer, Guy H; Bankhead, Troy; Seifert, H Steven

    2016-02-01

    Antigenic variation is a strategy used by a broad diversity of microbial pathogens to persist within the mammalian host. Whereas viruses make use of a minimal proofreading capacity combined with large amounts of progeny to use random mutation for variant generation, antigenically variant bacteria have evolved mechanisms which use a stable genome, which aids in protecting the fitness of the progeny. Here, three well-characterized and highly antigenically variant bacterial pathogens are discussed: Anaplasma, Borrelia, and Neisseria. These three pathogens display a variety of mechanisms used to create the structural and antigenic variation needed for immune escape and long-term persistence. Intrahost antigenic variation is the focus; however, the role of these immune escape mechanisms at the population level is also presented.

  8. Radioimmunoassays of hidden viral antigens

    Energy Technology Data Exchange (ETDEWEB)

    Neurath, A.R. (Lindsley F. Kimbell Research Inst., New York, NY); Strick, N.; Baker, L.; Krugman, S.

    1982-07-01

    Antigens corresponding to infectious agents may be present in biological specimens only in a cryptic form bound to antibodies and, thus, may elude detection. We describe a solid-phase technique for separation of antigens from antibodies. Immune complexes are precipitated from serum by polyethylene glycol, dissociated with NaSCN, and adsorbed onto nitrocellulose or polystyrene supports. Antigens remain topographically separated from antibodies after removal of NaSCN and can be detected with radiolabeled antibodies. Genomes from viruses immobilized on nitrocellulose can be identified by nucleic acid hybridization. Nanogram quantities of sequestered hepatitis B surface and core antigens and picogram amounts of hepatitis B virus DNA were detected. Antibody-bound adenovirus, herpesvirus, and measles virus antigens were discerned by the procedure.

  9. Radioimmunoassays of hidden viral antigens.

    Science.gov (United States)

    Neurath, A R; Strick, N; Baker, L; Krugman, S

    1982-01-01

    Antigens corresponding to infectious agents may be present in biological specimens only in a cryptic form bound to antibodies and, thus, may elude detection. We describe a solid phase technique for separation of antigens from antibodies. Immune complexes are precipitated from serum by polyethylene glycol, dissociated with NaSCN, and adsorbed onto nitrocellulose or polystyrene supports. Antigens remain topographically separated from antibodies after removal of NaSCN and can be detected with radiolabeled antibodies. Genomes from viruses immobilized on nitrocellulose can be identified by nucleic acid hybridization. Nanogram quantities of sequestered hepatitis B surface and core antigens and picogram amounts of hepatitis B virus DNA were detected. Antibody-bond adenovirus, herpesvirus, and measles virus antigens were discerned by the procedure. Images PMID:6956871

  10. Recombinational joints in a simian virus 40 variant generated in a persistent infection.

    Science.gov (United States)

    Norkin, L C; Piatak, M

    1982-12-01

    SP1, a viable simian virus 40 (SV40) variant isolated from a persistent infection of rhesus monkey kidney cells, contains sequence rearrangements in the untranslated region of the SV40 genome which are transcribed into late mRNA leader sequences and in the region which encodes the large T antigen. Nucleotide sequences about the recombinational junctions in SP1 were determined. The sequence data show that in most instances there was not extensive homology between recombining sequences. The recombinant sequences are discussed with respect to the mechanisms by which they might have been generated.

  11. HA03 as an Iranian Candidate Concealed Antigen for Vaccination against Hyalomma anatolicum anatolicum: Comparative Structural and In silico Studies

    Directory of Open Access Journals (Sweden)

    Mohammadi, A.

    2013-12-01

    Full Text Available In the last decades researchers had focused on developing a vaccine against tick based on protective antigen. Recombinant vaccines based on concealed antigen from Boophilus microplus have been developed in Australia and Cuba by the name of TICKGARD and GAVAC (De La Fuente and Kocan, 2006. Further studies on this antigen have shown some extent of protection against other species (De Vos et al., 2001. In Iran most important species is Hyalomma anatolicum and limited information about its control are available. This paper reports structural and polymorphic analysis of HA03 as an Iranian candidate concealed antigen of H. a. anatolicum deposited in Gen-Bank .(Aghaeipour et al. GQ228820. The comparison between this antigen and other mid gut concealed antigen that their characteristics are available in GenBank showed there are high rate of similarity between them. The HA03 amino acid sequence had a homology of around 89%, 64%, 56% with HA98, BM86, BM95 respectively. Potential of MHC class I and II binding region indicated a considerable variation between BM86 antigen and its efficiency against Iranian H. a. anatolicum. In addition, predicted major of hydrophobisity and similarity in N-glycosylation besides large amount of cystein and seven EGF like regions presented in protein structure revealed that value of HA03 as a new protective antigen and the necessity of the development, BM86 homolog of H. a. anatolicum HA03 based recombinant vaccine.

  12. Herpesvirus glycoproteins undergo multiple antigenic changes before membrane fusion.

    Directory of Open Access Journals (Sweden)

    Daniel L Glauser

    Full Text Available Herpesvirus entry is a complicated process involving multiple virion glycoproteins and culminating in membrane fusion. Glycoprotein conformation changes are likely to play key roles. Studies of recombinant glycoproteins have revealed some structural features of the virion fusion machinery. However, how the virion glycoproteins change during infection remains unclear. Here using conformation-specific monoclonal antibodies we show in situ that each component of the Murid Herpesvirus-4 (MuHV-4 entry machinery--gB, gH/gL and gp150--changes in antigenicity before tegument protein release begins. Further changes then occurred upon actual membrane fusion. Thus virions revealed their final fusogenic form only in late endosomes. The substantial antigenic differences between this form and that of extracellular virions suggested that antibodies have only a limited opportunity to block virion membrane fusion.

  13. Proteomic selection of immunodiagnostic antigens for Trypanosoma congolense.

    Directory of Open Access Journals (Sweden)

    Jennifer R Fleming

    2014-06-01

    Full Text Available Animal African Trypanosomosis (AAT presents a severe problem for agricultural development in sub-Saharan Africa. It is caused by several trypanosome species and current means of diagnosis are expensive and impractical for field use. Our aim was to discover antigens for the detection of antibodies to Trypanosoma congolense, one of the main causative agents of AAT. We took a proteomic approach to identify potential immunodiagnostic parasite protein antigens. One hundred and thirteen proteins were identified which were selectively recognized by infected cattle sera. These were assessed for likelihood of recombinant protein expression in E. coli and fifteen were successfully expressed and assessed for their immunodiagnostic potential by ELISA using pooled pre- and post-infection cattle sera. Three proteins, members of the invariant surface glycoprotein (ISG family, performed favorably and were then assessed using individual cattle sera. One antigen, Tc38630, evaluated blind with 77 randomized cattle sera in an ELISA assay gave sensitivity and specificity performances of 87.2% and 97.4%, respectively. Cattle immunoreactivity to this antigen diminished significantly following drug-cure, a feature helpful for monitoring the efficacy of drug treatment.

  14. Expression and Purification of the Bacillus anthracis Protective Antigen Receptor-binding Domain

    Institute of Scientific and Technical Information of China (English)

    葛猛; 徐俊杰; 李冰; 董大勇; 宋小红; 郭强; 赵剑; 陈薇

    2004-01-01

    The aim of this study is to express the receptor-binding domain of Bacillus anthracis protective antigen in E. coli. Signal sequence of the outer membrane protein A (OmpA) of E. coli was attached to the 5' end of the gene encoding protective antigen receptor-binding domain (the 4th domain of PA, PALM). The plasmid carrying the fusion gene was then transformed into E. coli and induced to express recombinant PAlM by IFFG. The recombinant protein was purified by chromatography and then identified by N-terrainal sequencing and Western blot. The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ionexchange chromatography and gel filtration, about 10 mg of homogenous recombinant PAD4 was obtained from 1 L culture. Data from N-terminal sequencing suggested that the amino acid sequence of recombinant PAD4 was identical with its natural counterpart. And the result of Western blot showed the recombinant protein could bind with anti-PA serum from rabbit. High level secreted expression of PAD4 was obtained in E. coli. The results reported here are parts of a continuing research to evaluate PAD4 as a potential drug for anthrax therapy or a candidate of new vaccine.

  15. Selection of Ceratitis capitata (Diptera: Tephritidae) specific recombinant monoclonal phage display antibodies for prey detection analysis.

    Science.gov (United States)

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro

    2012-01-01

    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators.

  16. Recombinant baculovirus displayed vaccine

    Science.gov (United States)

    Prabakaran, Mookkan; Kwang, Jimmy

    2014-01-01

    The rapid evolution of new sublineages of H5N1 influenza in Asia poses the greatest challenge in vaccine development for pre-pandemic preparedness. To overcome the antigenic diversity of H5N1 strains, multiple vaccine strains can be designed based on the distribution of neutralizing epitopes in the globular head of H5 hemagglutinin (HA). Recently, we selected two different HAs of H5N1 strains based on the neutralizing epitopes and reactivity with different neutralizing antibodies. The HAs of selected vaccine strains were individually expressed on the baculovirus envelope (bivalent-BacHA) with its native antigenic configuration. Further, oral delivery of live bivalent-BacHA elicited broadly reactive humoral, mucosal and cell-mediated immune responses and showed complete protection against antigenically distinct H5N1 strains in mice. The strategy for the vaccine strain selection, vaccine design and route of administration will provide an idea for development of a widely protective vaccine against highly pathogenic H5N1 for pre-pandemic preparedness. PMID:23941989

  17. Phosphorylation of the Epstein-Barr virus nuclear antigen 2

    DEFF Research Database (Denmark)

    Grässer, F A; Göttel, S; Haiss, P

    1992-01-01

    A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK......-1. The CK-2 specific phosphorylation site was localized in the 140 C-terminal amino acids using a recombinant trpE-C-terminal fusion protein. In a similar experiment, the 58 N-terminal amino acids expressed as a recombinant trpE-fusion protein were not phosphorylated. Phosphorylation of a synthetic...

  18. COLONOSCOPY AND CARCINOEMBRYONIC ANTIGEN VARIATIONS

    Directory of Open Access Journals (Sweden)

    Rita G SOUSA

    2014-03-01

    Full Text Available Context Colonoscopy is essential for synchronous and metachronous cancer detection. Carcinoembryonic antigen is a colorectal cancer tumor marker, important as a follow-up tool in patients with previous colorectal cancer. False-positive carcinoembryonic antigen elevation results in multiples exams and in patient anxiety. In literature, there is reference to transient carcinoembryonic antigen increase with colonoscopy. Objective To evaluate the influence of bowel preparation and colonoscopy in carcinoembryonic antigen blood levels. Methods We prospectively studied subjects that underwent routine colonoscopy in our institution. Blood samples were collected (1 before bowel cleaning, (2 before colonoscopy and (3 immediately after colonoscopy. Blood carcinoembryonic antigen levels were determined by “Sandwich” immunoassay. The statistical methods used were the paired t-test and ANOVA. Results Thirty-seven patients (22M/15F were included; age range 28-84 (mean 56 years. Mean carcinoembryonic antigen values were 1.9, 2 and 1.8 for (1, (2 and (3, respectively. An increase in value (2 compared with (1 was observed in 20/37 patients (P = 0.018, mainly in younger patients and in patients requiring more endoluminal interventions. In 29/37 patients, the CEA value decreased from (2 to (3 (P = 1.3x10-7. Conclusions A trend for carcinoembryonic antigen increase after bowel cleaning was observed, especially in younger patients and in patients with more endoluminal interventions, but without clinical meaning.

  19. Recombinant T-cell receptors : An immunologic link to cancer therapy

    NARCIS (Netherlands)

    Calogero, A; de Leij, YFMH; Mulder, NH; Hospers, GAP

    2000-01-01

    Cytotoxic T cells can specifically kill target cells that express antigens recognized by the T-cell receptor. These are membrane-bound proteins that are not ubiquitous and thus are difficult to purify and study at the protein level. The advent of recombinant DNA technology has facilitated these obje

  20. Evaluation of Six Recombinant Proteins for Serological Diagnosis of Lyme Borreliosis in China

    Institute of Scientific and Technical Information of China (English)

    LIU Wei; LIU Hui Xin; ZHANG Lin; HOU Xue Xia; WAN Kang Lin; HAO Qin

    2016-01-01

    ObjectiveIn this study, we evaluated the diagnostic efficiency of six recombinant proteins for the serodiagnosis of Lyme borreliosis (LB) and screened out the appropriate antigens to support the production of a Chinese clinical ELISA (enzyme-linked immunosorbent assay) kit for LB. MethodsSix recombinant antigens, Fla B.g, OspC B.a, OspC B.g, P39 B.g, P83 B.g, and VlsE B.a, were used for ELISA to detect serum antibodies in LB, syphilis, and healthy controls. The ELISA results were used to generate receiver operating characteristic (ROC) curves, and the sensitivity and specificity of each protein was evaluated. All recombinant proteins were evaluated and screened by using logistic regression models. ResultsTwo IgG (VlsE and OspC B.g) and two IgM (OspC B.g and OspC B.a) antigens were left by the logistic regression model screened. VlsE had the highest specificity for syphilis samples in the IgG test (87.7%,P ConclusionThree recombinant antigens, OspC B.g, OspC B.a, and VlsE B.a, were useful for ELISAs of LB. Additionally, the interaction between OspC B.a and Fla B.g should be examined in future research.

  1. Cloning, expression and immunoreactivity of recombinant Toxoplasma gondii GRA5 protein

    Science.gov (United States)

    Arab-Mazar, Zahra; Fallahi, Shirzad; Koochaki, Ameneh; Mirahmadi, Hadi; Tabaei, Seyyed Javad Seyyed

    2016-01-01

    Background and Objectives: Toxoplasma gondii is an obligatory intracellular parasite which causes severe diseases in the fetus of pregnant women and immunocopmromised patients. Serological tests based on recombinant protein are one of the main diagnosis methods for the detection of specific antibodies in serum samples. Dense granule antigenic proteins derived from T. gondii (TgGRAs) are potential antigens for the development of diagnostic tools. Materials and Methods: DNA was extracted from T. gondii (RH-strain) tachyzoites and PCR reaction was done using corresponding primers for GRA5 antigen. The PCR product was purified and ligated into pTG19-t vector and then subcloned into XhoI and BamHI digested pGEX6p-1 expression vector. Recombinant plasmid was transformed into E. coli (BL21 DE3) and induced by 1mM IPTG and analyzed by 15% SDS-PAGE. Expressed protein was confirmed by western blot analysis. Results: There was no difference among the sequences of T. gondii GRA5 gene from different isolates. The recombinant plasmid pGEX-6p-1/GRA5 induced by IPTG was expressed in E. coli. It was a GST fusion protein and could react with human positive sera analyzed by western blot. Conclusion: The GRA5 gene of T. gondii isolates is highly conservative. This antigen as a recombinant protein was successfully expressed in E. coli, which showed high immunoreactivity. PMID:28149494

  2. SUMO Wrestles with Recombination

    Directory of Open Access Journals (Sweden)

    Lumír Krejčí

    2012-07-01

    Full Text Available DNA double-strand breaks (DSBs comprise one of the most toxic DNA lesions, as the failure to repair a single DSB has detrimental consequences on the cell. Homologous recombination (HR constitutes an error-free repair pathway for the repair of DSBs. On the other hand, when uncontrolled, HR can lead to genome rearrangements and needs to be tightly regulated. In recent years, several proteins involved in different steps of HR have been shown to undergo modification by small ubiquitin-like modifier (SUMO peptide and it has been suggested that deficient sumoylation impairs the progression of HR. This review addresses specific effects of sumoylation on the properties of various HR proteins and describes its importance for the homeostasis of DNA repetitive sequences. The article further illustrates the role of sumoylation in meiotic recombination and the interplay between SUMO and other post-translational modifications.

  3. Recombinant Human Enterovirus 71

    OpenAIRE

    2004-01-01

    Two human enterovirus 71 (HEV71) isolates were identified from hand, foot and mouth disease patients with genome sequences that had high similarity to HEV71 (>93%) at 5´ UTR, P1, and P2 and coxsackievirus A16 (CV-A16, >85%) at P3 and 3´UTR. Intertypic recombination is likely to have occurred between HEV71 and CV-A16 or an as-yet to be described CV-A16-like virus.

  4. Recombinant anti-human melanoma antibodies are versatile molecules.

    Science.gov (United States)

    Neri, D; Natali, P G; Petrul, H; Soldani, P; Nicotra, M R; Vola, R; Rivella, A; Creighton, A M; Neri, P; Mariani, M

    1996-08-01

    The low cost, high versatility, and reliable production of bacterially produced recombinant antibody fragments speeds up the development of tumor-targeting agents. High-quality recombinant anti-melanoma antibodies are much sought after in the scientific community. We cloned the murine antibody 225.28S, currently used in radioimmunoimaging of human melanoma lesions, in single-chain Fv configuration (scFv) for soluble expression in bacteria. The recombinant antibody fragment conserved the binding specificity of the parental antibody. In order to arm the scFv(225.28S) with biologically useful effector functions, we developed vectors for soluble expression of scFv(225.28S) in bacteria that allow both covalent and noncovalent chemical antibody modification at positions that do not interfere with antigen binding. An expression vector was developed that appends a cysteine residue at the C-terminal extremity of the recombinant antibody, thus allowing reaction with thiol-specific reagents, including 99mTc labeling, at a position that does not interfere with antigen binding. The scFv(225.28S) was also successfully expressed with a casein kinase II substrate tag that enables efficient and stable 32P labeling. For noncovalent antibody modification, we developed an expression vector that appends the human calmodulin gene at the C-terminal extremity of scFv(225.28S). The calmodulin domain is poorly immunogenic and can be targeted with chemically modified high-affinity calmodulin ligands. The recombinant anti-human melanoma antibodies described in this article should prove useful "building blocks" for the development of anti-melanoma diagnostic and therapeutic strategies.

  5. Immunoreactivity evaluation of a new recombinant chimeric protein against Brucella in the murine model

    Directory of Open Access Journals (Sweden)

    Abbas Abdollahi

    2016-10-01

    Full Text Available Background and Objectives: Brucellosis is an important health problem in developing countries and no vaccine is available for the prevention of infection in humans. Because of clinically infectious diseases and their economic consequences in human and animals, designing a proper vaccine against Brucella is desirable. In this study, we evaluated the immune responses induced by a designed recombinant chimera protein in murine model.Materials and Methods: Three immunodominant antigens of Brucella have been characterized as potential immunogenic and protective antigens including: trigger factor (TF, Omp31 and Bp26 were fused together by EAAAK linkers to produce a chimera (structure were designed in silico, which was synthesized, cloned, and expressed in E. coli BL21 (DE3. The purification of recombinant protein was performed using Ni-NTA agarose. SDS-PAGE and anti-His antibody was used for confirmation purified protein (Western blot. BALB/c immunization was performed by purified protein and adjuvant, and sera antibody levels were measured by ELISA. otted.Results: SDS-PAGE and Western blotting results indicated the similarity of in silico designing and in vitro experiments. ELISA result proved that the immunized sera of mice contain high levels of antibodies (IgG against recombinant chimeric protein.Conclusion: The recombinant chimeric protein could be a potential antigen candidate for the development of a subunit vaccine against Brucella. Keywords: Brucella, Vaccine, Immunity, Recombinant

  6. Recombinant protein- and synthetic peptide-based immunoblot test for diagnosis of neurocysticercosis.

    Science.gov (United States)

    Noh, John; Rodriguez, Silvia; Lee, Yeuk-Mui; Handali, Sukwan; Gonzalez, Armando E; Gilman, Robert H; Tsang, Victor C W; Garcia, Hector H; Wilkins, Patricia P

    2014-05-01

    One of the most well-characterized tests for diagnosing neurocysticercosis (NCC) is the enzyme-linked immunoelectrotransfer blot (EITB) assay developed at the CDC, which uses lentil lectin-bound glycoproteins (LLGP) extracted from Taenia solium cysticerci. Although the test is very reliable, the purification process for the LLGP antigens has been difficult to transfer to other laboratories because of the need for expensive equipment and technical expertise. To develop a simpler assay, we previously purified and cloned the diagnostic glycoproteins in the LLGP fraction. In this study, we evaluated three representative recombinant or synthetic antigens from the LLGP fraction, individually and in different combinations, using an immunoblot assay (recombinant EITB). Using a panel of 249 confirmed NCC-positive and 401 negative blood serum samples, the sensitivity of the recombinant EITB assay was determined to be 99% and the specificity was 99% for diagnosing NCC. We also tested a panel of 239 confirmed NCC-positive serum samples in Lima, Peru, and found similar results. Overall, our data show that the performance characteristics of the recombinant EITB assay are comparable to those of the LLGP-EITB assay. This new recombinant- and synthetic antigen-based assay is sustainable and can be easily transferred to other laboratories in the United States and throughout the world.

  7. Cloning and expression of the Legionella micdadei "common antigen" in Escherichia coli

    DEFF Research Database (Denmark)

    Bangsborg, Jette Marie; Collins, M T; Høiby, N;

    1989-01-01

    To study individual Legionella antigens, a Legionella micdadei genomic library in Escherichia coli SC181 was established. Partially Sau3A digested L. micdadei DNA fragments (15-25 kilobase pairs (kb] were cloned into the tetracycline resistance gene of the cosmid vector pHC79. Four thousand...... ampicillin resistant recombinants were obtained; seven hundred were screened for expression of Legionella antigens in Western blot analysis with a polyspecific E. coli-absorbed anti-L. micdadei rabbit antibody. One of the positive clones expressed a 60 kilodalton (K) antigen, which reacted strongly...... will provide important information with respect to genetic vs. antigenic relatedness among Legionellae and other Gram-negative species, as well as to CA structure and possible function....

  8. Two Mutually Exclusive Local Chromatin States Drive Efficient V(DJ Recombination

    Directory of Open Access Journals (Sweden)

    Daniel J. Bolland

    2016-06-01

    Full Text Available Variable (V, diversity (D, and joining (J (V(DJ recombination is the first determinant of antigen receptor diversity. Understanding how recombination is regulated requires a comprehensive, unbiased readout of V gene usage. We have developed VDJ sequencing (VDJ-seq, a DNA-based next-generation-sequencing technique that quantitatively profiles recombination products. We reveal a 200-fold range of recombination efficiency among recombining V genes in the primary mouse Igh repertoire. We used machine learning to integrate these data with local chromatin profiles to identify combinatorial patterns of epigenetic features that associate with active VH gene recombination. These features localize downstream of VH genes and are excised by recombination, revealing a class of cis-regulatory element that governs recombination, distinct from expression. We detect two mutually exclusive chromatin signatures at these elements, characterized by CTCF/RAD21 and PAX5/IRF4, which segregate with the evolutionary history of associated VH genes. Thus, local chromatin signatures downstream of VH genes provide an essential layer of regulation that determines recombination efficiency.

  9. Successive site translocating inoculation potentiates DNA/recombinant vaccinia vaccination.

    Science.gov (United States)

    Ren, Yanqin; Wang, Na; Hu, Weiguo; Zhang, Xiaoyan; Xu, Jianqing; Wan, Yanmin

    2015-12-15

    DNA vaccines have advantages over traditional vaccine modalities; however the relatively low immunogenicity restrains its translation into clinical use. Further optimizations are needed to get the immunogenicity of DNA vaccine closer to the level required for human use. Here we show that intramuscularly inoculating into a different limb each time significantly improves the immunogenicities of both DNA and recombinant vaccinia vaccines during multiple vaccinations, compared to repeated vaccination on the same limb. We term this strategy successive site translocating inoculation (SSTI). SSTI could work in synergy with genetic adjuvant and DNA prime-recombinant vaccinia boost regimen. By comparing in vivo antigen expression, we found that SSTI avoided the specific inhibition of in vivo antigen expression, which was observed in the limbs being repeatedly inoculated. Employing in vivo T cell depletion and passive IgG transfer, we delineated that the inhibition was not mediated by CD8(+) T cells but by specific antibodies. Finally, by using C3(-/-) mouse model and in vivo NK cells depletion, we identified that specific antibodies negatively regulated the in vivo antigen expression primarily in a complement depended way.

  10. Activity evaluation from different native or irradiated with {sup 60} Co gamma rays snake venoms and their inhibitory effect on Leishmania (Leishmania) amazonensis; Avaliacao da atividade de diferentes venenos de serpentes, nativos ou irradiados, com radiacao gama de {sup 60} Co, quanto ao poder inibitorio do crescimento de Leishmania (Leishmania) amazonensis

    Energy Technology Data Exchange (ETDEWEB)

    Lourenco, Cecilia de Oliveira

    2000-07-01

    Cutaneous leishmaniasis is a disease, caused by Leishmania parasites, that occurs frequently in tropical and sub-tropical regions of the world. Skin lesions that could results in disfiguring aspect characterize it. The treatment is based on few drugs as antimony salts or pentamidine that are toxic with increasing resistance by the parasite. Alternative forms of disease treatment are in constant search, including natural components as snake venoms. Previous studies demonstrate that some components of snake venoms have an inhibitory effect against those parasites, including Leishmania species. Although snake venoms presented high toxicity, several methods have been described to detoxify most or some of their toxic components, with favorable results by the use of gamma irradiation. In this report we tested several native and irradiated snake venoms for inhibitory effect against Leishmania (Leishmania) amazonensis parasite and LLCMK{sub 2} mammalian cells, with enzymatic tests and electrophoresis. There are significant activity in Acanthophis antarcticus, Agkistrodon bilineatus, Bothrops moojeni, Bothrops jararaca, Hoplocephalus stephensi, Naja melanoleuca, Naja mossambica, Pseudechis australis, Pseudechis colletti, Pseudechis guttatus and Pseudechis porphyriacus, venom being inactive Pseudonaja textilis, Notechis ater niger, Notechis scutatus. Oxyuranus microlepidotus and Oxyuranus scutellatus venoms. After 2 KGy of {sup 60}Co irradiation most venom loses significantly their activity. Venoms with antileishmanial activity presented L-amino acid oxidase (L-AO) activity and showed common protein with a molecular weight about 60kDa in SDS-PAGE. These results indicate that L-AO activity in those venoms are probably related with antileishmanial effect. (author)

  11. Study of ionizing radiation as a tool for select promastigotes forms of Leishmania Amazonensis, and the megalomaniac response in experimental models; Estudo do uso da radiacao ionizante como ferramenta de selecao de formas promastigotas metaciclicas de Leishmania amazonensis, e a inducao de resposta imunologica em modelos experimentais

    Energy Technology Data Exchange (ETDEWEB)

    Bonetti, Franco Claudio

    2006-07-01

    Actually, millions of people around the globe are under the risk of infection by a protozoan transmitted by a bit of a sand fly. This parasite is a Leishmania spp. This causes a wide spectrum disease, since a cutaneous disease to a visceral one. The cutaneous form is the major clinical manifestation (above 90%). The ionizing radiation, produced in a {sup 60}Co font, had being successes used to promote physical-chemical transformations on different protozoan, including Leishmania spp. In previous work was determined that promastigotes forms of Leishmania amazonensis, irradiated with different doses of radiation, lost their viability maintaining, however, their immunogenicity. In this work, was studied the use of ionizing radiation as a tool for selection of meta cyclic forms of the parasite in axenic culture, for a possible efficient irradiated immuno gene production. Our results shown that cultures irradiated with 400 Gy of gamma irradiation, has 75% of metacyclic form, which are capable to produce, in vitro, an infection that is similar the natural occurrence. These irradiated parasites have their internal cellular structure modified, maintaining their external structure intact. Susceptible strain of mice immunized with leishmania irradiated with different doses had high immunoglobulin production, and maintained this production after the challenge with naive parasites. In other strains this default was similar, however in lower titles. Immunodeficient mice didn't produce immunoglobulin nor on the immunization or on the challenge. (author)

  12. Oncogenic cancer/testis antigens

    DEFF Research Database (Denmark)

    Gjerstorff, Morten F; Andersen, Mads H; Ditzel, Henrik J

    2015-01-01

    Recent developments have set the stage for immunotherapy as a supplement to conventional cancer treatment. Consequently, a significant effort is required to further improve efficacy and specificity, particularly the identification of optimal therapeutic targets for clinical testing. Cancer....../testis antigens are immunogenic, highly cancer-specific, and frequently expressed in various types of cancer, which make them promising candidate targets for cancer immunotherapy, including cancer vaccination and adoptive T-cell transfer with chimeric T-cell receptors. Our current understanding of tumor...... immunology and immune escape suggests that targeting oncogenic antigens may be beneficial, meaning that identification of cancer/testis antigens with oncogenic properties is of high priority. Recent work from our lab and others provide evidence that many cancer/testis antigens, in fact, have oncogenic...

  13. Recombinant vaccines and the development of new vaccine strategies

    Directory of Open Access Journals (Sweden)

    I.P. Nascimento

    2012-12-01

    Full Text Available Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens. Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against few protective antigens. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. DNA vaccines, which consist of non-replicating plasmids, can induce strong long-term cellular immune responses. Prime-boost strategies combine different antigen delivery systems to broaden the immune response. In general, all of these strategies have shown advantages and disadvantages, and their use will depend on the knowledge of the mechanisms of infection of the target pathogen and of the immune response required for protection. In this review, we discuss some of the major breakthroughs that have been achieved using recombinant vaccine technologies, as well as new approaches and strategies for vaccine development, including potential shortcomings and risks.

  14. Recombinant vaccines and the development of new vaccine strategies.

    Science.gov (United States)

    Nascimento, I P; Leite, L C C

    2012-12-01

    Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens. Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against few protective antigens. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. DNA vaccines, which consist of non-replicating plasmids, can induce strong long-term cellular immune responses. Prime-boost strategies combine different antigen delivery systems to broaden the immune response. In general, all of these strategies have shown advantages and disadvantages, and their use will depend on the knowledge of the mechanisms of infection of the target pathogen and of the immune response required for protection. In this review, we discuss some of the major breakthroughs that have been achieved using recombinant vaccine technologies, as well as new approaches and strategies for vaccine development, including potential shortcomings and risks.

  15. An efficient depyrogenation method for recombinant bacterial outer membrane lipoproteins.

    Science.gov (United States)

    Basto, Afonso P; Morais, Joana; Marcelino, Eduardo; Leitão, Alexandre; Santos, Dulce M

    2014-06-01

    Bacterial outer membrane lipoproteins are anchored in the outer membrane lipid layer in close association with lipopolysaccharides (LPS) and with other hydrophobic membrane proteins, making their purification technically challenging. We have previously shown that a thorough delipidation of outer membrane preparations from the Escherichia coli expression host is an important step to eliminate contaminant proteins when purifying recombinant antigens expressed in fusion with the Pseudomonas aeruginosa OprI lipoprotein. Here we report the cloning and expression of three antigens in fusion with OprI (ovalbumin, eGFP and BbPDI) and our efforts to deal with the variable LPS contamination levels observed in different batches of purified lipoproteins. The use of polymyxin B columns or endotoxin removal polycationic magnetic beads for depyrogenation of purified lipoproteins resulted in high protein losses and the use of Triton X-114 or sodium deoxycholate during the course of affinity chromatography showed to be ineffective to reduce LPS contamination. Instead, performing a hot phenol/water LPS extraction from outer membrane preparations prior to metal affinity chromatography allowed the purification of the recombinant fusion lipoproteins with LPS contents below 0.02EU/μg of protein. The purified recombinant lipoproteins retain their capacity to stimulate bone marrow-derived dendritic cells allowing for the study of their immunomodulatory properties through TLR2/1. This is a simple and easy to scale up method that can also be considered for the purification of other outer membrane lipoproteins.

  16. Recombinant vaccines and the development of new vaccine strategies

    Energy Technology Data Exchange (ETDEWEB)

    Nascimento, I.P.; Leite, L.C.C. [Centro de Biotecnologia, Instituto Butantan, São Paulo, SP (Brazil)

    2012-09-07

    Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens. Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against few protective antigens. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. DNA vaccines, which consist of non-replicating plasmids, can induce strong long-term cellular immune responses. Prime-boost strategies combine different antigen delivery systems to broaden the immune response. In general, all of these strategies have shown advantages and disadvantages, and their use will depend on the knowledge of the mechanisms of infection of the target pathogen and of the immune response required for protection. In this review, we discuss some of the major breakthroughs that have been achieved using recombinant vaccine technologies, as well as new approaches and strategies for vaccine development, including potential shortcomings and risks.

  17. Immunodominant antigens of Leishmania chagasi associated with protection against human visceral leishmaniasis.

    Directory of Open Access Journals (Sweden)

    Daniel R Abánades

    Full Text Available BACKGROUND: Protection and recovery from visceral leishmaniasis (VL have been associated with cell-mediated immune (CMI responses, whereas no protective role has been attributed to humoral responses against specific parasitic antigens. In this report, we compared carefully selected groups of individuals with distinct responses to Leishmania chagasi to explore antigen-recognizing IgG present in resistant individuals. METHODOLOGY AND PRINCIPAL FINDINGS: VL patients with negative delayed-type hypersensitivity (DTH were classified into the susceptible group. Individuals who had recovered from VL and converted to a DTH+ response, as well as asymptomatic infected individuals (DTH+, were categorized into the resistant group. Sera from these groups were used to detect antigens from L. chagasi by conventional and 2D Western blot assays. Despite an overall reduction in the reactivity of several proteins after DTH conversion, a specific group of proteins (approximately 110-130 kDa consistently reacted with sera from DTH converters. Other antigens that specifically reacted with sera from DTH+ individuals were isolated and tandem mass spectrometry followed by database query with the protein search engine MASCO were used to identify antigens. The serological properties of recombinant version of the selected antigens were tested by ELISA. Sera from asymptomatic infected people (DTH+ reacted more strongly with a mixture of selected recombinant antigens than with total soluble Leishmania antigen (SLA, with less cross-reactivity against Chagas disease patients' sera. SIGNIFICANCE: Our results are the first evidence of leishmania proteins that are specifically recognized by sera from individuals who are putatively resistant to VL. In addition, these data highlight the possibility of using specific proteins in serological tests for the identification of asymptomatic infected individuals.

  18. Production of Brugia malayi BmSXP Recombinant Protein Expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Khoo, T. K.

    2010-01-01

    Full Text Available A rapid antibody detection test is very useful for detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. One such kit, panLF RapidTM (commercialized by Malaysian BioDiagnostic Research Sdn. Bhd. had been developed in our laboratory for the detection of all species of filarial infections. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant Brugia malayi antigens, BmR1 and BmSXP. In this study, the growth of recombinant bacteria that produce BmSXP was optimized under shake flask fermentation for high yield of the recombinant antigen. The optimizations involved selection of suitable growth medium, IPTG concentration and induction time. The medium that yielded the highest biomass as well as total protein was Terrific Broth (TB medium, which is an undefined medium. Initiation of induction of protein expression was found to be best at mid-log phase (OD600 = 1.5, with IPTG concentration of 1.0 mM, and harvest time at 9 h post-induction. This study showed that under the optimized conditions, the shake flask culture produced 4 g/L biomass (dry cell weight of recombinant Escherichia coli BmSXP/pPROEXHTa/TOP10F’, which yielded 2.42 mg/L of purified BmSXP recombinant antigen. The purified antigen was analyzed by SDS-PAGE and the antigenicity of protein was confirmed by Western blot.

  19. Presence of Human T-Cell Responses to the Mycobacterium leprae 45-Kilodalton Antigen Reflects Infection with or Exposure to M. leprae

    Science.gov (United States)

    Macfarlane, Anne; Mondragon-Gonzalez, Rafael; Vega-Lopez, Francisco; Wieles, Brigitte; de Pena, Josefina; Rodriguez, Obdulia; Suarez y de la Torre, Raul; de Vries, Rene R. P.; Ottenhoff, Tom H. M.; Dockrell, Hazel M.

    2001-01-01

    The ability of the 45-kDa serine-rich Mycobacterium leprae antigen to stimulate peripheral blood mononuclear cell (PBMC) proliferation and gamma interferon (IFN-γ) production was measured in leprosy patients, household contacts, and healthy controls from areas of endemicity in Mexico. Almost all the tuberculoid leprosy patients gave strong PBMC proliferation responses to the M. leprae 45-kDa antigen (92.8%; n = 14). Responses were lower in lepromatous leprosy patients (60.6%; n = 34), but some responses to the 45-kDa antigen were detected in patients unresponsive to M. leprae sonicate. The proportion of positive responses to the M. leprae 45-kDa antigen was much higher in leprosy contacts (88%; n = 17) than in controls from areas of endemicity (10%; n = 20). None of 15 patients with pulmonary tuberculosis gave a positive proliferation response to the 45-kDa antigen. The 45-kDa antigen induced IFN-γ secretion similar to that induced by the native Mycobacterium tuberculosis 30/31-kDa antigen in tuberculoid leprosy patients and higher responses than those induced by the other recombinant antigens (M. leprae 10- and 65-kDa antigens, thioredoxin, and thioredoxin reductase); in patients with pulmonary tuberculosis it induced lower IFN-γ secretion than the other recombinant antigens. These results suggest that the M. leprae 45-kDa antigen is a potent T-cell antigen which is M. leprae specific in these Mexican donors. This antigen may therefore have diagnostic potential as a new skin test reagent or as an antigen in a simple whole-blood cytokine test. PMID:11329466

  20. Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination.

    Science.gov (United States)

    Pines, Gur; Freed, Emily F; Winkler, James D; Gill, Ryan T

    2015-11-20

    The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing.

  1. Recombinant Collagenlike Proteins

    Science.gov (United States)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.