WorldWideScience

Sample records for capsid epitope immunoassays

  1. Epitope-distal effects accompany the binding of two distinct antibodies to hepatitis B virus capsids

    NARCIS (Netherlands)

    Bereszczak, J.Z.; Rose, R.J.; Duijn, E. van; Watts, N.R.; Wingfield, P.T.; Steven, A.C.; Heck, A.J.R.

    2013-01-01

    Infection of humans by hepatitis B virus (HBV) induces the copious production of antibodies directed against the capsid protein (Cp). A large variety of anticapsid antibodies have been identified that differ in their epitopes. These data, and the status of the capsid as a major clinical antigen, mot

  2. Analysis of epitopes in the capsid protein of avian hepatitis E virus by using monoclonal antibodies.

    Science.gov (United States)

    Dong, Shiwei; Zhao, Qin; Lu, Mingzhe; Sun, Peiming; Qiu, Hongkai; Zhang, Lu; Lv, Junhua; Zhou, En-Min

    2011-02-01

    Avian hepatitis E virus (HEV) is related genetically and antigenically to human and swine HEVs and capsid protein of avian HEV shares approximately 48-49% amino acid sequence identities with those of human and swine HEVs. Six monoclonal antibodies (MAbs) were produced and used to locate different epitopes in the ORF2 region of aa 339-570 of avian HEV Chinese isolate. The results showed that five epitopes were located in the aa 339-414 region and one in the aa 510-515 region. Two epitopes located in aa 339-355 and aa 384-414 regions are the immunodominant epitopes on the surface of the avian HEV particles as demonstrated by immune capture of viral particles and immunohistochemical detection of the ORF2 antigens with two MAbs.

  3. Human serum antibodies to a major defined epitope of human herpesvirus 8 small viral capsid antigen.

    Science.gov (United States)

    Tedeschi, R; De Paoli, P; Schulz, T F; Dillner, J

    1999-04-01

    The major antibody-reactive epitope of the small viral capsid antigen (sVCA) of human herpesvirus 8 (HHV-8) was defined by use of overlapping peptides. Strong IgG reactivity was found among approximately 50% of 44 human immunodeficiency virus-positive or -negative patients with Kaposi's sarcoma and 13 subjects who were seropositive by immunofluorescence assay (IFA) for the latent HHV-8 nuclear antigen. Only 1 of 106 subjects seronegative for both lytic and latent HHV-8 antigens and 10 of 81 subjects IFA-seropositive only for the lytic HHV-8 antigen had strong IgG reactivity to this epitope. Among 534 healthy Swedish women, only 1.3% were strongly seropositive. Comparison of the peptide-based and purified sVCA protein-based ELISAs found 55% sensitivity and 98% specificity. However, only 1 of 452 serum samples from healthy women was positive in both tests. In conclusion, the defined sVCA epitope was a specific, but not very sensitive, serologic marker of active HHV-8 infection. Such infection appears to be rare among Swedish women, even with sexual risk-taking behavior.

  4. Identification of two neutralization epitopes on the capsid protein of avian hepatitis E virus.

    Science.gov (United States)

    Zhou, E-M; Guo, H; Huang, F F; Sun, Z F; Meng, X J

    2008-02-01

    Avian hepatitis E virus (avian HEV) is genetically and antigenically related to human HEV, the causative agent of hepatitis E. To identify the neutralizing epitopes on the capsid (ORF2) protein of avian HEV, four mAbs (7B2, 1E11, 10A2 and 5G10) against recombinant avian HEV ORF2 protein were generated. mAbs 7B2, 1E11 and 10A2 blocked each other for binding to avian HEV ORF2 protein in a competitive ELISA, whereas 5G10 did not block the other mAbs, suggesting that 7B2, 1E11 and 10A2 recognize the same or overlapping epitopes and 5G10 recognizes a different one. The epitopes recognized by 7B2, 1E11 and 10A2, and by 5G10 were mapped by Western blotting between aa 513 and 570, and between aa 476 and 513, respectively. mAbs 1E11, 10A2 and 5G10 were shown to bind to avian HEV particles in vitro, although only 5G10 reacted to viral antigens in transfected LMH cells. To assess the neutralizing activities of the mAbs, avian HEV was incubated in vitro with each mAb before inoculation into specific-pathogen-free chickens. Both viraemia and faecal virus shedding were delayed in chickens inoculated with the mixtures of avian HEV and 1E11, 10A2 or 5G10, suggesting that these three mAbs partially neutralize avian HEV.

  5. Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection

    DEFF Research Database (Denmark)

    Brock, I; Weldingh, K; Leyten, EM;

    2004-01-01

    Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection.Brock I, Weldingh K, Leyten EM, Arend SM, Ravn P, Andersen P. Department of Infectious Disease Immunology, Statens Serum Institute, Artillerivej 5, DK-2300 Copenhagen S, Denmark. The currently used...... method for immunological detection of tuberculosis infection, the tuberculin skin test, has low specificity. Antigens specific for Mycobacterium tuberculosis to replace purified protein derivative are therefore urgently needed. We have performed a rigorous assessment of the diagnostic potential of four...... recently identified antigens (Rv2653, Rv2654, Rv3873, and Rv3878) from genomic regions that are lacking from the Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine strains as well as from the most common nontuberculous mycobacteria. The fine specificity of potential epitopes in these molecules...

  6. Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection

    DEFF Research Database (Denmark)

    Brock, I; Weldingh, K; Leyten, EM

    2004-01-01

    Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection.Brock I, Weldingh K, Leyten EM, Arend SM, Ravn P, Andersen P. Department of Infectious Disease Immunology, Statens Serum Institute, Artillerivej 5, DK-2300 Copenhagen S, Denmark. The currently used...... method for immunological detection of tuberculosis infection, the tuberculin skin test, has low specificity. Antigens specific for Mycobacterium tuberculosis to replace purified protein derivative are therefore urgently needed. We have performed a rigorous assessment of the diagnostic potential of four...... selected and combined the specific peptide stretches from the four proteins not recognized by M. bovis BCG-vaccinated individuals. These peptide stretches were tested with peripheral blood mononuclear cells obtained from patients with microscopy- or culture-confirmed tuberculosis and from healthy M. bovis...

  7. IgE and IgG4 Epitope Mapping of Food Allergens with a Peptide Microarray Immunoassay.

    Science.gov (United States)

    Martínez-Botas, Javier; de la Hoz, Belén

    2016-01-01

    Peptide microarrays are a powerful tool to identify linear epitopes of food allergens in a high-throughput manner. The main advantages of the microarray-based immunoassay are the possibility to assay thousands of targets simultaneously, the requirement of a low volume of serum, the more robust statistical analysis, and the possibility to test simultaneously several immunoglobulin subclasses. Among them, the last one has a special interest in the field of food allergy, because the development of tolerance to food allergens has been associated with a decrease in IgE and an increase in IgG4 levels against linear epitopes. However, the main limitation to the clinical use of microarray is the automated analysis of the data. Recent studies mapping the linear epitopes of food allergens with peptide microarray immunoassays have identified peptide biomarkers that can be used for early diagnosis of food allergies and to predict their severity or the self-development of tolerance. Using this approach, we have worked on epitope mapping of the two most important food allergens in the Spanish population, cow's milk and chicken eggs. The final aim of these studies is to define subsets of peptides that could be used as biomarkers to improve the diagnosis and prognosis of food allergies. This chapter describes the protocol to produce microarrays using a library of overlapping peptides corresponding to the primary sequences of food allergens and data acquisition and analysis of IgE- and IgG4-binding epitopes.

  8. Protection against P. aeruginosa with an adenovirus vector containing an OprF epitope in the capsid.

    Science.gov (United States)

    Worgall, Stefan; Krause, Anja; Rivara, Michael; Hee, Kyung-Kim; Vintayen, Enrico V; Hackett, Neil R; Roelvink, Peter W; Bruder, Joseph T; Wickham, Thomas J; Kovesdi, Imre; Crystal, Ronald G

    2005-05-01

    Pseudomonas aeruginosa is an important opportunistic pathogen that can cause chronic and often life-threatening infections of the respiratory tract, particularly in individuals with cystic fibrosis (CF). Because infections with P. aeruginosa remain the major cause of the high morbidity and mortality of CF, a vaccine against P. aeruginosa would be very useful for preventing this disorder. The outer membrane protein F (OprF) of P. aeruginosa is a promising vaccine candidate and various B cell epitopes within OprF have been identified. Given that adenovirus (Ad) vectors have strong immunogenic potential and can function as adjuvants for genetic vaccines, the present study evaluates the immunogenic and protective properties of a novel replication-deficient Ad vector in which the Ad hexon protein was modified to include a 14-amino acid epitope of P. aeruginosa OprF (Epi8) in loop 1 of the hypervariable region 5 of the hexon (AdZ.Epi8). Immunization of C57BL/6 mice with AdZ.Epi8 resulted in detectable serum anti-P. aeruginosa and anti-OprF humoral responses. These responses were haplotype dependent, with higher serum anti-OprF titers in CBA mice than in BALB/c or C57BL/6 mice. AdZ.Epi8 induced Epi8-specific IFN-gamma-positive CD4 and CD8 T cell responses and resulted in protection against a lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeated administration of AdZ.Epi8 resulted in boosting of the anti-OprF humoral and anti-Epi8 cellular response, whereas no boosting effect was present in the response against the transgene beta-galactosidase. These observations suggest that Ad vectors expressing pathogen epitopes in their capsid will protect against an extracellular pathogen and will allow boosting of the epitope-specific humoral response with repeated administration, a strategy that should prove useful in developing Ad vectors as vaccines where humoral immunity will be protective.

  9. Immunoassays

    Science.gov (United States)

    Hsieh, Y.-H. Peggy

    Immunochemistry is a relatively new science that has developed rapidly in the last few decades. One of the most useful analytical developments associated with this new science is immunoassay. Originally immunoassays were developed in medical settings to facilitate the study of immunology, particularly the antibody-antigen interaction. Immunoassays now are finding widespread applications outside the clinical field because they are appropriate for a wide range of analytes ranging from proteins to small organic molecules. In the food analysis area, immunoassays are widely used for chemical residue analysis, identification of bacteria and viruses, and detection of proteins in food and agricultural products. Protein detection is important for determination of allergens and meat species content, seafood species identification, and detection of genetically modified plant tissues. While immunoassays of all formats are too numerous to cover completely in this chapter, there are several procedures that have become standard for food analysis because of their specificity, sensitivity, and simplicity.

  10. Induction of mucosal immunity by intranasal immunization with recombinant adenovirus expressing major epitopes of Porcine circovirus-2 capsid protein.

    Science.gov (United States)

    Liu, Yu-feng; Guo, Quan-hai; Chen, Lu; Zhao, Jun; Chang, Hong-tao; Wang, Xin-wei; Yang, Xia; Wang, Chuan-qing

    2013-07-15

    Porcine circovirus-2 (PCV-2) is primarily transmitted through mucosa, thus the mucosal immunity may constitute an essential feature of vaccination strategies against PCV-2 infection. Mucosal immunity elicited by recombinant replication-deficient adenovirus expressing the major epitopes of PCV-2 capsid protein (rAd/Cap/518) via intranasal (i.n.), intramuscular (i.m.) or oral routes in mice were evaluated. Immunization with rAd/Cap/518 via i.n. route induced higher titers of IgA in saliva, bronchoalveolar and intestinal lavage fluid compared with those immunized via i.m. route. The proportions of CD3+, CD3+CD4+ and CD3+CD8+ T cells were significantly increased in mice immunized with rAd/Cap/518 via i.n. route compared with the control group. Higher levels of IFN-γ were detected in the spleen and mesenteric lymph nodes of mice immunized with rAd/Cap/518 via i.n. route compared with other groups, yet IL-4 was not detected in any group. Real-time PCR analysis confirmed viral DNA loads in the i.m. or i.n. immunization group was lower than that seen in the rAd immunization. These results indicate that i.n. administration of rAd/Cap/518 can elicit humoral and Th1-type cellular protective immunity in both systemic and mucosal immune compartments in mice, representing a promising mucosal vaccine candidate against PCV-2.

  11. Immunoassays

    NARCIS (Netherlands)

    Pas, Hendrikus; Jonkman, Marcel

    2016-01-01

    Immunoassays are helpful serological techniques for the laboratory diagnosis of autoimmune blistering diseases (AIBD) and for monitoring disease activity of individual patients. The three main immunoassaysare immunoblotting, immunoprecipitation, and ELISA. All three make use of the ability of the au

  12. An anti-phospholipase A2 receptor quantitative immunoassay and epitope analysis in membranous nephropathy reveals different antigenic domains of the receptor.

    Science.gov (United States)

    Behnert, Astrid; Fritzler, Marvin J; Teng, Beina; Zhang, Meifeng; Bollig, Frank; Haller, Hermann; Skoberne, Andrej; Mahler, Michael; Schiffer, Mario

    2013-01-01

    The phospholipase A2 receptor (PLA2R) was recently discovered as a target autoantigen in patients with idiopathic membranous nephropathy (IMN). Published evidence suggests that the autoantibodies directed towards a conformation dependent epitope are currently effectively detected by a cell based assay (CBA) utilizing indirect immunofluorescence (IIF) on tissue culture cells transfected with the PLA2R cDNA. Limitations of such IIF-CBA assays include observer dependent subjective evaluation of semi-quantitative test results and the protocols are not amenable to high throughput diagnostic testing. We developed a quantitative, observer independent, high throughput capture immunoassay for detecting PLA2R autoantibodies on an addressable laser bead immunoassay (ALBIA) platform. Since reactive domains of PLA2R (i.e. epitopes) could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA2R epitopes that bound autoantibodies of IMN patients. These studies confirmed that inter-molecular epitope spreading occurs in IMN but use of the cognate synthetic peptides in immunoassays was unable to conclusively distinguish between IMN patients and normal controls. However, combinations of these peptides were able to effectively absorb anti-PLA2R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA2R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA2R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA2R.

  13. An anti-phospholipase A2 receptor quantitative immunoassay and epitope analysis in membranous nephropathy reveals different antigenic domains of the receptor.

    Directory of Open Access Journals (Sweden)

    Astrid Behnert

    Full Text Available The phospholipase A2 receptor (PLA2R was recently discovered as a target autoantigen in patients with idiopathic membranous nephropathy (IMN. Published evidence suggests that the autoantibodies directed towards a conformation dependent epitope are currently effectively detected by a cell based assay (CBA utilizing indirect immunofluorescence (IIF on tissue culture cells transfected with the PLA2R cDNA. Limitations of such IIF-CBA assays include observer dependent subjective evaluation of semi-quantitative test results and the protocols are not amenable to high throughput diagnostic testing. We developed a quantitative, observer independent, high throughput capture immunoassay for detecting PLA2R autoantibodies on an addressable laser bead immunoassay (ALBIA platform. Since reactive domains of PLA2R (i.e. epitopes could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA2R epitopes that bound autoantibodies of IMN patients. These studies confirmed that inter-molecular epitope spreading occurs in IMN but use of the cognate synthetic peptides in immunoassays was unable to conclusively distinguish between IMN patients and normal controls. However, combinations of these peptides were able to effectively absorb anti-PLA2R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA2R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA2R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA2R.

  14. Characterization of the banana streak virus capsid protein and mapping of the immunodominant continuous B-cell epitopes to the surface-exposed N terminus.

    Science.gov (United States)

    Vo, Jenny N; Campbell, Paul R; Mahfuz, Nur N; Ramli, Ras; Pagendam, Daniel; Barnard, Ross; Geering, Andrew D W

    2016-12-01

    This study identified the structural proteins of two badnavirus species, Banana streak MY virus (BSMYV) and Banana streak OL virus (BSOLV), and mapped the distribution of continuous B-cell epitopes. Two different capsid protein (CP) isoforms of about 44 and 40 kDa (CP1 and CP2) and the virion-associated protein (VAP) were consistently associated with purified virions. For both viral species, the N terminus of CP2 was successfully sequenced by Edman degradation but that of CP1 was chemically blocked. De novo peptide sequencing of tryptic digests suggested that CP1 and CP2 derive from the same region of the P3 polyprotein but differ in the length of either the N or the C terminus. A three-dimensional model of the BSMYV-CP was constructed, which showed that the CP is a multi-domain structure, containing homologues of the retroviral capsid and nucleocapsid proteins, as well as a third, intrinsically disordered protein region at the N terminus, henceforth called the NID domain. Using the Pepscan approach, the immunodominant continuous epitopes were mapped to the NID domain for five different species of banana streak virus. Anti-peptide antibodies raised against these epitopes in BSMYV were successfully used for detection of native virions and denatured CPs in serological assays. Immunoelectron microscopy analysis of the virion surface using the anti-peptide antibodies confirmed that the NID domain is exposed on the surface of virions, and that the difference in mass of the two CP isoforms is due to variation in length of the NID domain.

  15. Sequence conservation, HLA-E-Restricted peptide, and best-defined CTL/CD8+ epitopes in gag P24 (capsid) of HIV-1 subtype B

    Science.gov (United States)

    Prasetyo, Afiono Agung; Dharmawan, Ruben; Sari, Yulia; Sariyatun, Ratna

    2017-02-01

    Human immunodeficiency virus type 1 (HIV-1) remains a cause of global health problem. Continuous studies of HIV-1 genetic and immunological profiles are important to find strategies against the virus. This study aimed to conduct analysis of sequence conservation, HLA-E-restricted peptide, and best-defined CTL/CD8+ epitopes in p24 (capsid) of HIV-1 subtype B worldwide. The p24-coding sequences from 3,557 HIV subtype B isolates were aligned using MUSCLE and analysed. Some highly conserved regions (sequence conservation ≥95%) were observed. Two considerably long series of sequences with conservation of 100% was observed at base 349-356 and 550-557 of p24 (HXB2 numbering). The consensus from all aligned isolates was precisely the same as consensus B in the Los Alamos HIV Database. The HLA-E-restricted peptide in amino acid (aa) 14-22 of HIV-1 p24 (AISPRTLNA) was found in 55.9% (1,987/3,557) of HIV-1 subtype B worldwide. Forty-four best-defined CTL/CD8+ epitopes were observed, in which VKNWMTETL epitope (aa 181-189 of p24) restricted by B*4801 was the most frequent, as found in 94.9% of isolates. The results of this study would contribute information about HIV-1 subtype B and benefits for further works willing to develop diagnostic and therapeutic strategies against the virus.

  16. Development of an in process control filtration-assisted chemiluminometric immunoassay to quantify foot and mouth disease virus (FMDV) non-capsid proteins in vaccine-antigen batches.

    Science.gov (United States)

    Capozzo, Alejandra Victoria; Martínez, Manuel Rosendo; Schielen, Wilhelmus Joseph Gerardus

    2010-09-14

    In many countries, foot and mouth disease (FMD) is controlled by vaccination and surveillance against non-capsid proteins (NCP); therefore vaccines are required not to induce antibodies against NCP. Vaccine purity is evaluated by repeated inoculation of naïve cattle, an expensive and time consuming protocol that raises several animal welfare concerns. We have developed an in process control filtration-assisted chemiluminometric immunoassay (FAL-ELISA), to detect and quantify NCP in vaccine-antigen batches regardless of its volume and composition. Samples are filtered through PVDF-filter microplates pre-coated with a monoclonal antibody against NCP. Filtration removes all unbound components in the sample and captured NCP are detected by anti-NCP conjugate followed by incubation with the substrate, luminol/peroxide. Analytical detection limit was 2 ng for purified NCP and 4 ng for vaccine-antigen batches spiked with NCP, which makes this assay sensitive enough to be applied to purity control of FMD vaccines. Vaccine components did not interfere with the antibody and substrate reactions in the assay. FAL-ELISA is an alternative for the in vivo tests, observing the objective to Replace, Reduce and Refine the use of animals for quality control of immunobiologicals.

  17. Immunodominant epitopes mapped by synthetic peptides on the capsid protein of avian hepatitis E virus are non-protective.

    Science.gov (United States)

    Guo, Hailong; Zhou, E M; Sun, Z F; Meng, X J

    2008-03-01

    Avian hepatitis E virus (avian HEV) was recently discovered in chickens with hepatitis-splenomegaly syndrome in the United States. The open reading frame 2 (ORF2) protein of avian HEV has been shown to cross-react with human and swine HEV ORF2 proteins, and immunodominant antigenic epitopes on avian HEV ORF2 protein were identified in the predicted antigenic domains by synthetic peptides. However, whether these epitopes are protective against avian HEV infection has not been investigated. In this study, groups of chickens were immunized with keyhole limpet hemocyanin (KLH)-conjugated peptides and recombinant avian HEV ORF2 antigen followed by challenge with avian HEV virus to assess the protective capacity of these peptides containing the epitopes. While avian HEV ORF2 protein showed complete protection against infection, viremia and fecal virus shedding were found in all peptide-immunized chickens. Using purified IgY from normal, anti-peptide, and anti-avian HEV ORF2 chicken sera, an in-vitro neutralization and in-vivo monitoring assay was performed to further evaluate the neutralizing ability of anti-peptide IgY. Results showed that none of the anti-peptide IgY can neutralize avian HEV in vitro, as viremia, fecal virus shedding, and seroconversion appeared similarly in chickens inoculated with avian HEV mixed with anti-peptide IgY and chickens inoculated with avian HEV mixed with normal IgY. As expected, chickens inoculated with the avian HEV and anti-avian HEV ORF2 IgY mixture did not show detectable avian HEV infection. Taken together, the results of this study demonstrated that immunodominant epitopes on avian HEV ORF2 protein identified by synthetic peptides are non-protective, suggesting protective neutralizing epitope on avian HEV ORF2 may not be linear as is human HEV.

  18. A pan-HPV vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing epitopes from the HPV minor capsid protein, L2.

    Directory of Open Access Journals (Sweden)

    Ebenezer Tumban

    Full Text Available BACKGROUND: Current human papillomavirus (HPV vaccines that are based on virus-like particles (VLPs of the major capsid protein L1 largely elicit HPV type-specific antibody responses. In contrast, immunization with the HPV minor capsid protein L2 elicits antibodies that are broadly cross-neutralizing, suggesting that a vaccine targeting L2 could provide more comprehensive protection against infection by diverse HPV types. However, L2-based immunogens typically elicit much lower neutralizing antibody titers than L1 VLPs. We previously showed that a conserved broadly neutralizing epitope near the N-terminus of L2 is highly immunogenic when displayed on the surface of VLPs derived from the bacteriophage PP7. Here, we report the development of a panel of PP7 VLP-based vaccines targeting L2 that protect mice from infection with carcinogenic and non-carcinogenic HPV types that infect the genital tract and skin. METHODOLOGY/PRINCIPAL FINDINGS: L2 peptides from eight different HPV types were displayed on the surface of PP7 bacteriophage VLPs. These recombinant L2 VLPs, both individually and in combination, elicited high-titer anti-L2 IgG serum antibodies. Immunized mice were protected from high dose infection with HPV pseudovirus (PsV encapsidating a luciferase reporter. Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs were nearly completely protected from both PsV16 and PsV18 challenge. Mice immunized with the mixture of eight L2 VLPs were strongly protected from genital challenge with PsVs representing eight diverse HPV types and cutaneous challenge with HPV5 PsV. CONCLUSION/SIGNIFICANCE: VLP-display of a cross-neutralizing HPV L2 epitope is an effective approach for inducing high-titer protective neutralizing antibodies and is capable of offering protection from a spectrum of HPVs associated with cervical cancer as well as genital and cutaneous warts.

  19. Enhancing mucosal immunity in mice by recombinant adenovirus expressing major epitopes of porcine circovirus-2 capsid protein delivered with cytosine-phosphate-guanosine oligodeoxynucleotides.

    Science.gov (United States)

    Chang, Hong-Tao; He, Xiu-Yuan; Liu, Yu-Feng; Chen, Lu; Guo, Quan-Hai; Yu, Qiu-Ying; Zhao, Jun; Wang, Xin-Wei; Yang, Xia; Wang, Chuan-Qing

    2014-01-01

    A recombinant replication-defective adenovirus expressing the major epitopes of porcine circovirus-2 (PCV-2) capsid protein (rAd/Cap/518) was previously constructed and shown to induce mucosal immunity in mice following intranasal delivery. In the present study, immune responses induced by intranasal immunization with a combination of rAd/Cap/518 and cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) were evaluated in mice. The levels of PCV-2-specific IgG in serum and IgA in saliva, lung, and intestinal fluids were significantly higher in the group immunized with rAd/Cap/518 and CpG ODN than animals immunized with rAd/Cap/518 alone. The frequencies of IL-2-secreting CD4⁺ T cells and IFN-γ-producing CD8⁺ T cells were significantly higher in the combined immunization group than mice immunized with rAd/Cap/518 alone. The frequencies of CD3⁺, CD3⁺CD4⁺CD8⁻, and CD3⁺CD4⁻CD8⁺ T cells in the combined immunization group were similar to that treated with CpG ODN alone, but significantly higher than mice that did not receive CpG ODN. PCV-2 load after challenge in the combined immunization group was significantly lower than that in the phosphate-buffered saline placebo group and approximately 7-fold lower in the group treated with CpG ODN alone. These results indicate that rAd/Cap/518 combined with CpG ODN can enhance systemic and local mucosal immunity in mice, and represent a promising synergetic mucosal vaccine against PCV-2.

  20. Identification of B-cell epitopes in the capsid protein of avian hepatitis E virus (avian HEV) that are common to human and swine HEVs or unique to avian HEV.

    Science.gov (United States)

    Guo, H; Zhou, E-M; Sun, Z F; Meng, X-J; Halbur, P G

    2006-01-01

    Avian hepatitis E virus (avian HEV) was recently discovered in chickens from the USA that had hepatitis-splenomegaly (HS) syndrome. The complete genomic sequence of avian HEV shares about 50 % nucleotide sequence identity with those of human and swine HEVs. The open reading frame 2 (ORF2) protein of avian HEV has been shown to cross-react with human and swine HEV ORF2 proteins, but the B-cell epitopes in the avian HEV ORF2 protein have not been identified. Nine synthetic peptides from the predicted four antigenic domains of the avian HEV ORF2 protein were synthesized and corresponding rabbit anti-peptide antisera were generated. Using recombinant ORF2 proteins, convalescent pig and chicken antisera, peptides and anti-peptide rabbit sera, at least one epitope at the C terminus of domain II (possibly between aa 477-492) that is unique to avian HEV, one epitope in domain I (aa 389-410) that is common to avian, human and swine HEVs, and one or more epitopes in domain IV (aa 583-600) that are shared between avian and human HEVs were identified. Despite the sequence difference in ORF2 proteins between avian and mammalian HEVs and similar ORF2 sequence between human and swine HEV ORF2 proteins, rabbit antiserum against peptide 6 (aa 389-399) recognized only human HEV ORF2 protein, suggesting complexity of the ORF2 antigenicity. The identification of these B-cell epitopes in avian HEV ORF2 protein may be useful for vaccine design and may lead to future development of immunoassays for differential diagnosis of avian, swine and human HEV infections.

  1. Vaccination of mice with plasmids expressing processed capsid protein of foot-and-mouth disease virus - Importance of dominant and subdominant epitopes for antigenicity and protection

    DEFF Research Database (Denmark)

    Frimann, Tine; Barfoed, Annette Malene; Aasted, Bent

    2007-01-01

    example of a dominant and variable site. This variability is a problem when designing vaccines against this disease, because it necessitates a close match between vaccine strain and virus in an outbreak. We have introduced a series of mutations into viral capsid proteins with the aim of selectively...

  2. Production of monoclonal antibodies specific to Macrobrachium rosenbergii nodavirus using recombinant capsid protein.

    Science.gov (United States)

    Wangman, Pradit; Senapin, Saengchan; Chaivisuthangkura, Parin; Longyant, Siwaporn; Rukpratanporn, Sombat; Sithigorngul, Paisarn

    2012-03-20

    The gene encoding the capsid protein of Macrobrachium rosenbergii nodavirus (MrNV) was cloned into pGEX-6P-1 expression vector and then transformed into the Escherichia coli strain BL21. After induction, capsid protein-glutathione-S-transferase (GST-MrNV; 64 kDa) was produced. The recombinant protein was separated using SDS-PAGE, excised from the gel, electro-eluted and then used for immunization for monoclonal antibody (MAb) production. Four MAbs specific to the capsid protein were selected and could be used to detect natural MrNV infections in M. rosenbergii by dot blotting, Western blotting and immunohistochemistry without cross-reaction with uninfected shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was 10 fmol µl-1 of the GST-MrNV, as determined using dot blotting. However, the sensitivity of the MAb on dot blotting with homogenate from naturally infected M. rosenbergii was approximately 200-fold lower than that of 1-step RT-PCR. Immunohistochemical analysis using these MAbs with infected shrimp tissues demonstrated staining in the muscles, nerve cord, gill, heart, loose connective tissue and inter-tubular tissue of the hepatopancreas. Although the positive reactions occurred in small focal areas, the immunoreactivity was clearly demonstrated. The MAbs targeted different epitopes of the capsid protein and will be used to develop a simple immunoassay strip test for rapid detection of MrNV.

  3. The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition.

    Science.gov (United States)

    Goh, Lucas Y H; Hobson-Peters, Jody; Prow, Natalie A; Baker, Kelly; Piyasena, Thisun B H; Taylor, Carmel T; Rana, Ashok; Hastie, Marcus L; Gorman, Jeff J; Hall, Roy A

    2015-06-08

    Chikungunya virus (CHIKV) is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs) previously generated towards the capsid protein (CP) of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1-35 and 140-210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

  4. The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition

    Directory of Open Access Journals (Sweden)

    Lucas Y. H. Goh

    2015-06-01

    Full Text Available Chikungunya virus (CHIKV is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs previously generated towards the capsid protein (CP of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1–35 and 140–210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

  5. Evaluation of the Architect Epstein-Barr Virus (EBV) viral capsid antigen (VCA) IgG, VCA IgM, and EBV nuclear antigen 1 IgG chemiluminescent immunoassays for detection of EBV antibodies and categorization of EBV infection status using immunofluorescence assays as the reference method.

    Science.gov (United States)

    Corrales, Isabel; Giménez, Estela; Navarro, David

    2014-05-01

    Commercial immunoassays for detecting IgG and IgM antibodies against Epstein-Barr virus (EBV), viral capsid antigens (VCA), and IgGs toward EBV nuclear antigen-1 (EBNA-1) are routinely used in combination to categorize EBV infection status. In this study, we evaluated the performances of the Architect EBV VCA IgG, VCA IgM, and EBNA-1 IgG chemiluminescent microparticle assays (CMIAs) in EBV serological analyses using indirect immunofluorescence assays and anticomplement immunofluorescence assays as the reference methods for VCA IgG, VCA IgM, and EBNA-1 IgG antibody detection, respectively. A total of 365 serum samples representing different EBV serological profiles were included in this study. The κ values (concordances between the results) obtained in the Architect CMIA and those in the reference assays were 0.905 (P EBV infection, and 92.42% and 97.82% for diagnosing the absence of an EBV infection. In summary, we demonstrated that the Architect EBV antibody panel performs very well for EBV antibody detection and correctly categorizes clinically relevant EBV infection states.

  6. Antigenic properties of avian hepatitis E virus capsid protein.

    Science.gov (United States)

    Zhao, Qin; Syed, Shahid Faraz; Zhou, En-Min

    2015-10-22

    Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease and hepatitis-splenomegaly syndrome in chickens, and is genetically and antigenically related to mammalian HEVs. HEV capsid protein contains immunodominant epitopes and induces a protective humoral immune response. A better understanding of the antigenic composition of this protein is critically important for the development of effective vaccine and sensitive and specific serological assays. To date, six linear antigenic domains (I-VI) have been characterized in avian HEV capsid protein and analyzed for their applications in the serological diagnosis and vaccine design. Domains I and V induce strong immune response in chickens and are common to avian, human, and swine HEVs, indicating that the shared epitopes hampering differential diagnosis of avian HEV infection. Domains III and IV are not immunodominant and elicit a weak immune response. Domain VI, located in the N-terminal region of the capsid protein, can also trigger an intense immune response, but the anti-domain VI antibodies are transient. The protection analysis showed that the truncated capsid protein containing the C-terminal 268 amino acid residues expressed by the bacterial system can provide protective immunity against avian HEV infection in chickens. However, the synthetic peptides incorporating the different linear antigenic domains (I-VI) and epitopes are non-protective. The antigenic composition of avian HEV capsid protein is altogether complex. To develop an effective vaccine and accurate serological diagnostic methods, more conformational antigenic domains or epitopes are to be characterized in detail.

  7. Identification of class I HLA T cell control epitopes for West Nile virus.

    Directory of Open Access Journals (Sweden)

    Saghar Kaabinejadian

    Full Text Available The recent West Nile virus (WNV outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1 the number of viral ligands presented by the HLA of infected cells, and 2 the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity.

  8. Expression and immunoreactivity of HCV/HBV epitopes

    Institute of Scientific and Technical Information of China (English)

    Xin-Yu Xiong; Xiao Liu; Yuan-Ding Chen

    2005-01-01

    AIM: To develop the epitope-based vaccines to prevent Hepatitis C virus (HCV)/Hepatitis B virus (HBV) infections.METHODS: The HCV core epitopes C1 STNPKPQRKTKRNTNRRPQD (residuals aa2-21) and C2 VKFPGGGQIVGGVYLLPRR (residuals aa22-40), envelope epitope E GHRMAWDMMMNWSP (residuals aa315-328) and HBsAg epitope S CTTPAQGNSMFPSCCCTKPTDGNC (residuals aa124-147) were displayed in five different sites of the flock house virus capsid protein as a vector, and expressed in E. coli cells (pET-3 system).Immunoreactivity of the epitopes with anti-HCV and anti-HBV antibodies in the serum from hepatitis C and hepatitis B patients were determined.RESULTS: The expressed chimeric protein carrying the HCV epitopes C1, C2, E (two times), L3C1-I2E-L1C2-L2E could react with anti-HCV antibodies. The expressed chimeric protein carrying the HBV epitopes S, I3S could react with anti-HBs antibodies. The expressed chimeric proteins carrying the HCV epitopes C1, C2, E plus HBV epitope S, L3C1-I2E-L1C2-L2E-I3S could react with antiHCV and anti-HBs antibodies.CONCLUSION: These epitopes have highly specific and sensitive immunoreaction and are useful in the development of epitope-based vaccines.

  9. The Astrovirus Capsid: A Review

    Science.gov (United States)

    Arias, Carlos F.; DuBois, Rebecca M.

    2017-01-01

    Astroviruses are enterically transmitted viruses that cause infections in mammalian and avian species. Astroviruses are nonenveloped, icosahedral viruses comprised of a capsid protein shell and a positive-sense, single-stranded RNA genome. The capsid protein undergoes dramatic proteolytic processing both inside and outside of the host cell, resulting in a coordinated maturation process that affects cellular localization, virus structure, and infectivity. After maturation, the capsid protein controls the initial phases of virus infection, including virus attachment, endocytosis, and genome release into the host cell. The astrovirus capsid is the target of host antibodies including virus-neutralizing antibodies. The capsid protein also mediates the binding of host complement proteins and inhibits complement activation. Here, we will review our knowledge on the astrovirus capsid protein (CP), with particular attention to the recent structural, biochemical, and virological studies that have advanced our understanding of the astrovirus life cycle. PMID:28106836

  10. A drug discovery platform: a simplified immunoassay for analyzing HIV protease activity.

    Science.gov (United States)

    Kitidee, Kuntida; Nangola, Sawitree; Hadpech, Sudarat; Laopajon, Witida; Kasinrerk, Watchara; Tayapiwatana, Chatchai

    2012-12-01

    Although numerous methods for the determination of HIV protease (HIV-PR) activity have been described, new high-throughput assays are required for clinical and pharmaceutical applications due to the occurrence of resistant strains. In this study, a simple enzymatic immunoassay to identify HIV-PR activity was developed based on a Ni(2+)-immobilized His(6)-Matrix-Capsid substrate (H(6)MA-CA) is cleaved by HIV protease-His(6) (HIV-PRH(6)) which removes the CA domain and exposes the free C terminus of MA. Following this cleavage, two monoclonal antibodies specific for either the free C-terminal MA or CA epitope are used to quantify the proteolytic activity using a standard ELISA-based system. Specificity for detection of the HIV-PRH(6) activity was confirmed with addition of protease inhibitor (PI), lopinavir. In addition, the assay was able to detect an HIV-PR variant activity indicating that this assay is capable of assessing viral mutation affect HIV-PR activity. The efficacy of commercially available PIs and their 50% inhibitory concentration (IC(50)) were determined. This assay provides a high-throughput method for both validating the efficiency of new drugs in vitro and facilitating the discovery of new PIs. In addition, it could serve as a method for examining the influence of various mutations in HIV-PRs isolated from drug-resistant strains.

  11. Identification of an antigenic domain in the N-terminal region of avian hepatitis E virus (HEV) capsid protein that is not common to swine and human HEVs.

    Science.gov (United States)

    Wang, Lizhen; Sun, Yani; Du, Taofeng; Wang, Chengbao; Xiao, Shuqi; Mu, Yang; Zhang, Gaiping; Liu, Lihong; Widén, Frederik; Hsu, Walter H; Zhao, Qin; Zhou, En-Min

    2014-12-01

    The antigenic domains located in the C-terminal 268 amino acid residues of avian hepatitis E virus (HEV) capsid protein have been characterized. This region shares common epitopes with swine and human HEVs. However, epitopes in the N-terminal 338 amino acid residues have never been reported. In this study, an antigenic domain located between amino acids 23 and 85 was identified by indirect ELISA using the truncated recombinant capsid proteins as coating antigens and anti-avian HEV chicken sera as primary antibodies. In addition, this domain did not react with anti-swine and human HEV sera. These results indicated that the N-terminal 338 amino acid residues of avian HEV capsid protein do not share common epitopes with swine and human HEVs. This finding is important for our understanding of the antigenicity of the avian HEV capsid protein. Furthermore, it has important implications in the selection of viral antigens for serological diagnosis.

  12. Structure of a Human Astrovirus Capsid-Antibody Complex and Mechanistic Insights into Virus Neutralization

    Energy Technology Data Exchange (ETDEWEB)

    Bogdanoff, Walter A.; Campos, Jocelyn; Perez, Edmundo I.; Yin, Lu; Alexander, David L.; DuBois, Rebecca M. (UCSC)

    2016-11-02

    ABSTRACT

    Human astroviruses (HAstVs) are a leading cause of viral diarrhea in young children, the immunocompromised, and the elderly. There are no vaccines or antiviral therapies against HAstV disease. Several lines of evidence point to the presence of protective antibodies in healthy adults as a mechanism governing protection against reinfection by HAstV. However, development of anti-HAstV therapies is hampered by the gap in knowledge of protective antibody epitopes on the HAstV capsid surface. Here, we report the structure of the HAstV capsid spike domain bound to the neutralizing monoclonal antibody PL-2. The antibody uses all six complementarity-determining regions to bind to a quaternary epitope on each side of the dimeric capsid spike. We provide evidence that the HAstV capsid spike is a receptor-binding domain and that the antibody neutralizes HAstV by blocking virus attachment to cells. We identify patches of conserved amino acids that overlap the antibody epitope and may comprise a receptor-binding site. Our studies provide a foundation for the development of therapies to prevent and treat HAstV diarrheal disease.

    IMPORTANCEHuman astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies in healthy adults protect them against reinfection. Here, we determined the crystal structure of a complex of the HAstV capsid protein and a virus-neutralizing antibody. We show that the antibody binds to the outermost spike domain of the capsid, and we provide evidence that the antibody blocks virus attachment to human cells. Importantly, our findings suggest that a subunit-based vaccine focusing the immune system on the HAstV capsid spike domain could be effective in protecting children against HAstV disease.

  13. CapsidMaps: protein-protein interaction pattern discovery platform for the structural analysis of virus capsids using Google Maps.

    Science.gov (United States)

    Carrillo-Tripp, Mauricio; Montiel-García, Daniel Jorge; Brooks, Charles L; Reddy, Vijay S

    2015-04-01

    Structural analysis and visualization of protein-protein interactions is a challenging task since it is difficult to appreciate easily the extent of all contacts made by the residues forming the interfaces. In the case of viruses, structural analysis becomes even more demanding because several interfaces coexist and, in most cases, these are formed by hundreds of contacting residues that belong to multiple interacting coat proteins. CapsidMaps is an interactive analysis and visualization tool that is designed to benefit the structural virology community. Developed as an improved extension of the φ-ψ Explorer, here we describe the details of its design and implementation. We present results of analysis of a spherical virus to showcase the features and utility of the new tool. CapsidMaps also facilitates the comparison of quaternary interactions between two spherical virus particles by computing a similarity (S)-score. The tool can also be used to identify residues that are solvent exposed and in the process of locating antigenic epitope regions as well as residues forming the inside surface of the capsid that interact with the nucleic acid genome. CapsidMaps is part of the VIPERdb Science Gateway, and is freely available as a web-based and cross-browser compliant application at http://viperdb.scripps.edu.

  14. Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

    Science.gov (United States)

    Tarasiuk, K; Woźniakowski, G; Holec-Gąsior, L

    2015-01-01

    The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.

  15. Multifunctional nanoparticles as simulants for a gravimetric immunoassay.

    Science.gov (United States)

    Miller, Scott A; Hiatt, Leslie A; Keil, Robert G; Wright, David W; Cliffel, David E

    2011-01-01

    Immunoassays are important tools for the rapid detection and identification of pathogens, both clinically and in the research laboratory. An immunoassay with the potential for the detection of influenza was developed and tested using hemagglutinin (HA), a commonly studied glycoprotein found on the surface of influenza virions. Gold nanoparticles were synthesized, which present multiple peptide epitopes, including the HA epitope, in order to increase the gravimetric response achieved with the use of a QCM immunosensor for influenza. Specifically, epitopes associated with HA and FLAG peptides were affixed to gold nanoparticles by a six-mer PEG spacer between the epitope and the terminal cysteine. The PEG spacer was shown to enhance the probability for interaction with antibodies by increasing the distance the epitope extends from the gold surface. These nanoparticles were characterized using thermogravimetric analysis, transmission electron microscopy, matrix-assisted laser desorption/ionization-time of flight, and (1)H nuclear magnetic resonance analysis. Anti-FLAG and anti-HA antibodies were adhered to the surface of a QCM, and the response of each antibody upon exposure to HA, FLAG, and dual functionalized nanoparticles was compared with binding of Au-tiopronin nanoparticles and H5 HA proteins from influenza virus (H5N1). Results demonstrate that the immunoassay was capable of differentiating between nanoparticles presenting orthogonal epitopes in real-time with minimal nonspecific binding. The detection of H5 HA protein demonstrates the logical extension of using these nanoparticle mimics as a safe positive control in the detection of influenza, making this a vital step in improving influenza detection methodology.

  16. Immunoassays in Biotechnology

    Science.gov (United States)

    Immunoassays have broad applications for a wide variety of important biological compounds and environmental contaminants. Immunoassays can detect the presence of an antigen in the human body, a pollutant in the environment, or a critical antibody in a patient’s serum to develop a...

  17. Topography of the Human Papillomavirus Minor Capsid Protein L2 during Vesicular Trafficking of Infectious Entry

    Science.gov (United States)

    DiGiuseppe, Stephen; Keiffer, Timothy R.; Bienkowska-Haba, Malgorzata; Luszczek, Wioleta; Guion, Lucile G. M.; Müller, Martin

    2015-01-01

    ABSTRACT The human papillomavirus (HPV) capsid is composed of the major capsid protein L1 and the minor capsid protein L2. During entry, the HPV capsid undergoes numerous conformational changes that result in endosomal uptake and subsequent trafficking of the L2 protein in complex with the viral DNA to the trans-Golgi network. To facilitate this transport, the L2 protein harbors a number of putative motifs that, if capable of direct interaction, would interact with cytosolic host cell factors. These data imply that a portion of L2 becomes cytosolic during infection. Using a low concentration of digitonin to selectively permeabilize the plasma membrane of infected cells, we mapped the topography of the L2 protein during infection. We observed that epitopes within amino acid residues 64 to 81 and 163 to 170 and a C-terminal tag of HPV16 L2 are exposed on the cytosolic side of intracellular membranes, whereas an epitope within residues 20 to 38, which are upstream of a putative transmembrane region, is luminal. Corroborating these findings, we also found that L2 protein is sensitive to trypsin digestion during infection. These data demonstrate that the majority of the L2 protein becomes accessible on the cytosolic side of intracellular membranes in order to interact with cytosolic factors to facilitate vesicular trafficking. IMPORTANCE In order to complete infectious entry, nonenveloped viruses have to pass cellular membranes. This is often achieved through the viral capsid protein associating with or integrating into intracellular membrane. Here, we determine the topography of HPV L2 protein in the endocytic vesicular compartment, suggesting that L2 becomes a transmembrane protein with a short luminal portion and with the majority facing the cytosolic side for interaction with host cell transport factors. PMID:26246568

  18. Updates in immunoassays: parasitology.

    Science.gov (United States)

    Josko, Deborah

    2012-01-01

    Although most clinical laboratories use microscopy and routine O&P procedures when identifying parasitic infections, there are several parasites that are better detected through serological means. Toxoplasma, Giardia, and Cryptosporidium were discussed along with immunoassays used for their detection. Immunoassays provide quick results and are less labor intensive than specimen concentration and slide preparation for microscopic examination. These assays are easy to use and provide sensitive and specific results. Some clinical laboratories no longer perform O&Ps in house and refer specimens to reference laboratories for evaluation. By using immunoassays, some of the more common parasites can be identified in a timely manner reducing turn-around times. Some controversy exists over the use of IIF and EIA tests used for ANA testing along with measuring CRPs and PCT as predictors of bacterial sepsis and septic shock. Regardless of the methodology discussed in this series of articles, there are pros and cons to the various immunoassays available. Determining the most appropriate assay based on patient population and volume is governed by the institution and its patients' needs. In conclusion, immunoassays, whether manual or automated, are easy to use, cost effective and allow the medical laboratory professional to provide quick and accurate results to the clinician so the most appropriate treatment can be administered to the patient. The ultimate goal of healthcare professionals is to provide the highest quality of medical care in a timely manner. The use of immunoassays in the clinical laboratory allows the healthcare team to successfully achieve this goal.

  19. Hydrogel nanoparticle based immunoassay

    Science.gov (United States)

    Liotta, Lance A; Luchini, Alessandra; Petricoin, Emanuel F; Espina, Virginia

    2015-04-21

    An immunoassay device incorporating porous polymeric capture nanoparticles within either the sample collection vessel or pre-impregnated into a porous substratum within fluid flow path of the analytical device is presented. This incorporation of capture particles within the immunoassay device improves sensitivity while removing the requirement for pre-processing of samples prior to loading the immunoassay device. A preferred embodiment is coreshell bait containing capture nanoparticles which perform three functions in one step, in solution: a) molecular size sieving, b) target analyte sequestration and concentration, and c) protection from degradation. The polymeric matrix of the capture particles may be made of co-polymeric materials having a structural monomer and an affinity monomer, the affinity monomer having properties that attract the analyte to the capture particle. This device is useful for point of care diagnostic assays for biomedical applications and as field deployable assays for environmental, pathogen and chemical or biological threat identification.

  20. Specificity of an anti-capsid antibody associated with Hepatitis B Virus-related acute liver failure.

    Science.gov (United States)

    Wu, Weimin; Chen, Zhaochun; Cheng, Naiqian; Watts, Norman R; Stahl, Stephen J; Farci, Patrizia; Purcell, Robert H; Wingfield, Paul T; Steven, Alasdair C

    2013-01-01

    Previously, the livers of patients suffering from acute liver failure (ALF), a potentially fatal syndrome arising from infection by Hepatitis B Virus (HBV), were found to contain massive amounts of an antibody specific for the core antigen (HBcAg) capsid. We have used cryo-electron microscopy and molecular modeling to define its epitope. HBV capsids are icosahedral shells with 25Å-long dimeric spikes, each a 4-helix bundle, protruding from the contiguous "floor". Of the anti-HBcAg antibodies previously characterized, most bind around the spike tip while one binds to the floor. The ALF-associated antibody binds tangentially to a novel site on the side of the spike. This epitope is conformational. The Fab binds with high affinity to its principal determinants but has lower affinities for quasi-equivalent variants. The highest occupancy site is on one side of a spike, with no detectable binding to the corresponding site on the other side. Binding of one Fab per dimer was also observed by analytical ultracentrifugation. The Fab did not bind to the e-antigen dimer, a non-assembling variant of capsid protein. These findings support the propositions that antibodies with particular specificities may correlate with different clinical expressions of HBV infection and that antibodies directed to particular HBcAg epitopes may be involved in ALF pathogenesis.

  1. Mass spectrometric immunoassay

    Science.gov (United States)

    Nelson, Randall W; Williams, Peter; Krone, Jennifer Reeve

    2007-12-04

    Rapid mass spectrometric immunoassay methods for detecting and/or quantifying antibody and antigen analytes utilizing affinity capture to isolate the analytes and internal reference species (for quantification) followed by mass spectrometric analysis of the isolated analyte/internal reference species. Quantification is obtained by normalizing and calibrating obtained mass spectrum against the mass spectrum obtained for an antibody/antigen of known concentration.

  2. Tetrodoxtoxin Immunoassays. Phase 2

    Science.gov (United States)

    1991-01-14

    TTX CIEIA warrants comment. Development of such a system was discussed with Dr. John Hewetson during a visit by James Raybould to Fort Detrick in...T.J.G. Raybould , Ph.D. - directed immunoassay development using T20G10 MAb and T20G10-AP conjugates. Gary S. Bignami, M.S. - responsible for hybridoma

  3. Repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response

    Institute of Scientific and Technical Information of China (English)

    LIU Zuqiang; WANG Zuguang; CHEN Yinghua

    2005-01-01

    Based on the hypothesis suggested by us that epitope-vaccine may be a new strategy against HIV mutation, we have studied several neutralizing epitopes on HIV envelope proteins. However we do not know whether a repeated epitope in a recombinant epitope-peptide can enhance epitope-specific antibody response or not. ELDKWA-epitope (aa669-674) on the C-domain of HIV-1 gp41 is a neutralizing epitope defined by the monoclonal antibody (mAb) 2F5 with broad neutralizing activity. In this study, we designed and prepared a series of the recombinant epitope-peptides bearing 1, 4 and 8 copies of ELDKWA-epitope respectively. In the comparison of the antisera induced by the three recombinant antigens, an obviously increased titre of ELDKWA-epitope-specific antibody was observed in the case of four and eight repeated epitopes. In flow cytometry analysis, the epitope-specific antibodies in both antisera showed stronger activity to bind the transfected CHO-WT cells that stably express HIV-1 envelope glycoprotein on the cell surfaces. These experimental results indicated that repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response, which could contribute to designing an effective recombinant epitope-vaccine.

  4. Updates in immunoassays: virology.

    Science.gov (United States)

    Josko, Deborah

    2012-01-01

    Virus identification is a challenge to the clinical microbiologist since growing viruses in traditional cell culture is labor intensive, time consuming, and subject to contamination. The advent of rapid and automated immunoassays has eliminated this problem by generating positive results in minutes to hours. For example, testing for infectious mononucleosis can yield a positive result in 3-8 minutes as seen with the Beckman Coulter, Inc. ICON Mono test or in 5-15 minutes with the MONO Mononucleosis Rapid Test Device marketed by ACON Laboratories, Inc. Fully automated immunoassay analyzers provide fast, accurate, sensitive results that aid in a prompt and accurate diagnosis for the patient. Turnaround times are shortened, allowing for timely medical intervention and treatment. The priority in any hospital or medical facility is to treat the patient as quickly and appropriately as possible. By using immunoassays, clinical laboratory professionals are able to report out correct results in a timely manner, ensuring overall positive patient outcomes and improved quality of healthcare.

  5. Dynamic pathways for viral capsid assembly

    OpenAIRE

    Hagan, Michael F.; Chandler, David

    2006-01-01

    We develop a class of models with which we simulate the assembly of particles into T1 capsid-like objects using Newtonian dynamics. By simulating assembly for many different values of system parameters, we vary the forces that drive assembly. For some ranges of parameters, assembly is facile, while for others, assembly is dynamically frustrated by kinetic traps corresponding to malformed or incompletely formed capsids. Our simulations sample many independent trajectories at various capsomer c...

  6. Critical Role of Autophagy in the Processing of Adenovirus Capsid-Incorporated Cancer-Specific Antigens.

    Directory of Open Access Journals (Sweden)

    Sarah R Klein

    Full Text Available Adenoviruses are highly immunogenic and are being examined as potential vectors for immunotherapy. Infection by oncolytic adenovirus is followed by massive autophagy in cancer cells. Here, we hypothesize that autophagy regulates the processing of adenoviral proteins for antigen presentation. To test this hypothesis, we first examined the presentation of viral antigens by infected cells using an antibody cocktail of viral capsid proteins. We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation. Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner. We then hypothesize that this mechanism can be utilized to generate an efficient cancer vaccine. To this end, we constructed an oncolytic virus encompassing an EGFRvIII cancer-specific epitope in the adenoviral fiber. Infection of cancer cells with this fiber-modified adenovirus resulted in recognition of infected cancer cells by a specific anti-EGFRvIII antibody. However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells. Our data suggest that combination of adenoviruses with autophagy inducers may enhance the processing and presentation of cancer-specific antigens incorporated into capsid proteins.

  7. 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.

  8. Protection of chickens against avian hepatitis E virus (avian HEV) infection by immunization with recombinant avian HEV capsid protein.

    Science.gov (United States)

    Guo, H; Zhou, E M; Sun, Z F; Meng, X J

    2007-04-12

    Avian hepatitis E virus (avian HEV) is an emerging virus associated with hepatitis-splenomegaly syndrome in chickens in North America. Avian HEV is genetically and antigenically related to human HEV, the causative agent of hepatitis E in humans. In the lack of a practical animal model, avian HEV infection in chickens has been used as a model to study human HEV replication and pathogenesis. A 32 kDa recombinant ORF2 capsid protein of avian HEV expressed in Escherichia coli was found having similar antigenic structure as that of human HEV containing major neutralizing epitopes. To determine if the capsid protein of avian HEV can be used as a vaccine, 20 chickens were immunized with purified avian HEV recombinant protein with aluminum as adjuvant and another 20 chickens were mock immunized with KLH precipitated in aluminum as controls. Both groups of chickens were subsequently challenged with avian HEV. All the tested mock-immunized control chickens developed typical avian HEV infection characterized by viremia, fecal virus shedding and seroconversion to avian HEV antibodies. Gross hepatic lesions were also found in portion of these chickens. In contrast, none of the tested chickens immunized with avian HEV capsid protein had detectable viremia, fecal virus shedding or observable gross hepatitis lesions. The results from this study suggested that immunization of chickens with avian HEV recombinant ORF2 capsid protein with aluminum as adjuvant can induce protective immunity against avian HEV infection. Chickens are a useful small animal model to study anti-HEV immunity and pathogenesis.

  9. Characterization of the antibody response against EV71 capsid proteins in Chinese individuals by NEIBM-ELISA.

    Science.gov (United States)

    Ding, Yingying; Chen, Xuguang; Qian, Baohua; Wu, Guorong; He, Ting; Feng, Jiaojiao; Gao, Caixia; Wang, Lili; Wang, Jinhong; Li, Xiangyu; Cao, Mingmei; Peng, Heng; Zhao, Chunyan; Pan, Wei

    2015-01-01

    Human enterovirus 71 (EV71) has become the major pathogen of hand, foot, and mouth disease (HFMD) worldwide, while the anti-EV71 antibody responses other than neutralizing epitopes have not been characterized. In this study, EV71 capsid proteins VP1, VP3, VP0 and various VP1 antigens were constructed to analyze anti-EV71 response in severe HFMD cases, non-HFMD outpatient children and normal adults using a novel evolved immunoglobulin-binding molecule (NEIBM)-based ELISA. The high prevalence of antibody responses against all three capsid proteins was demonstrated, and anti-EV71 VP1 showed the main antibody response. Anti-EV71 VP1 antibody response was found to predominantly target to epitopes based on the common enterovirus cross-reactive sequence. Moreover, inhibition pattern against anti-EV71 VP1 reactions in three groups was obviously different. Taken together, these results firstly characterized the anti-EV71 antibody responses which are predominantly against VP1 epitopes based on common enterovirus cross-reactive sequence. This finding could be helpful for the better understanding of anti-EV71 humoral immunity and useful for seroepidemiological surveillance.

  10. Identification of a linear B-cell epitope on the avian leukosis virus P27 protein using monoclonal antibodies.

    Science.gov (United States)

    Li, Xiaofei; Qin, Liting; Zhu, Haibo; Sun, Yingjun; Cui, Xuezhi; Gao, Yadong; Qi, Xiaole; Wang, Yongqiang; Gao, Honglei; Gao, Yulong; Wang, Xiaomei

    2016-10-01

    Avian leukosis virus (ALV) is an avian oncogenic retrovirus that can induce various clinical tumors. The capsid protein P27 is the group-specific antigen of ALV and has many viral antigen sites that are easy to detect. In this study, we produced a monoclonal antibody (mAb), 3A9, that is specific for the P27 protein. A series of partially overlapping peptides were screened to define (181)PPSAR(185) as the minimal linear epitope recognized by mAb 3A9. The identified epitope could be recognized by chicken anti-ALV and mouse anti-ALV P27 sera. The epitope was highly conserved among a number of ALV-A, ALV-B and ALV-J strains. MAb 3A9 might be a valuable tool for the development of new immunodiagnostic approaches for ALV, and the defined linear epitope might help further our understanding of the antigenic structure of the P27 protein.

  11. Dynamic pathways for viral capsid assembly

    Energy Technology Data Exchange (ETDEWEB)

    Hagan, Michael F.; Chandler, David

    2006-02-09

    We develop a class of models with which we simulate the assembly of particles into T1 capsid-like objects using Newtonian dynamics. By simulating assembly for many different values of system parameters, we vary the forces that drive assembly. For some ranges of parameters, assembly is facile, while for others, assembly is dynamically frustrated by kinetic traps corresponding to malformed or incompletely formed capsids. Our simulations sample many independent trajectories at various capsomer concentrations, allowing for statistically meaningful conclusions. Depending on subunit (i.e., capsomer) geometries, successful assembly proceeds by several mechanisms involving binding of intermediates of various sizes. We discuss the relationship between these mechanisms and experimental evaluations of capsid assembly processes.

  12. Crosslinking in viral capsids via tiling theory.

    Science.gov (United States)

    Twarock, R; Hendrix, R W

    2006-06-07

    A vital part of a virus is its protein shell, called the viral capsid, that encapsulates and hence protects the viral genome. It has been shown in Twarock [2004. A tiling approach to vius capsids assembly explaining a structural puzzle in virology. J. Theor. Biol. 226, 477-482] that the surface structures of viruses with icosahedrally symmetric capsids can be modelled in terms of tilings that encode the locations of the protein subunits. This theory is extended here to multi-level tilings in order to model crosslinking structures. The new framework is demonstrated for the case of bacteriophage HK97, and it is shown, how the theory can be used in general to decide if crosslinking, and what type of crosslinking, is compatible from a mathematical point of view with the geometrical surface structure of a virus.

  13. Viral capsids: Mechanical characteristics, genome packaging and delivery mechanisms

    NARCIS (Netherlands)

    Roos, W.H.; Ivanovska, I.L.; Evilevitch, A.; Wuite, G.J.L.

    2007-01-01

    The main functions of viral capsids are to protect, transport and deliver their genome. The mechanical properties of capsids are supposed to be adapted to these tasks. Bacteriophage capsids also need to withstand the high pressures the DNA is exerting onto it as a result of the DNA packaging and its

  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. Epitope prediction methods

    DEFF Research Database (Denmark)

    Karosiene, Edita

    Major histocompatibility complex (MHC) molecules play a crucial role in adaptive immunity by sampling peptides from self and non-self proteins to be recognised by the immune system. MHC molecules present peptides on cell surfaces for recognition by CD8+ and CD4+ T lymphocytes that can initiate...... immune responses. Therefore, it is of great importance to be able to identify peptides that bind to MHC molecules, in order to understand the nature of immune responses and discover T cell epitopes useful for designing new vaccines and immunotherapies. MHC molecules in humans, referred to as human...... on machine learning techniques. Several MHC class I binding prediction algorithms have been developed and due to their high accuracy they are used by many immunologists to facilitate the conventional experimental process of epitope discovery. However, the accuracy of these methods depends on data defining...

  16. Use of aptamers in immunoassays.

    Science.gov (United States)

    Nezlin, Roald

    2016-02-01

    Aptamers, short single-chain DNA or RNA oligonucleotides, react specifically with small molecules, as well as with proteins. Unlike antibodies, they may be obtained relatively easily. Aptamers are now widely employed in immunological studies and could replace antibodies in immunoassays. In this short review, methods for immobilizing aptamers on various insoluble materials (so-called apta-sorbents) are described. Recent findings on their use in the detection and isolation of immunoglobulins and their application in various immunoassays are also discussed.

  17. Characterization of the antibody response against EV71 capsid proteins in Chinese individuals by NEIBM-ELISA

    OpenAIRE

    Ding, Yingying; Chen, Xuguang; Qian, Baohua; Wu, Guorong; He, Ting; Feng, Jiaojiao; Gao, Caixia; Wang, Lili; Wang, Jinhong; Li, Xiangyu; Cao, Mingmei; Peng, Heng; Zhao, Chunyan; Pan, Wei

    2015-01-01

    Human enterovirus 71 (EV71) has become the major pathogen of hand, foot, and mouth disease (HFMD) worldwide, while the anti-EV71 antibody responses other than neutralizing epitopes have not been characterized. In this study, EV71 capsid proteins VP1, VP3, VP0 and various VP1 antigens were constructed to analyze anti-EV71 response in severe HFMD cases, non-HFMD outpatient children and normal adults using a novel evolved immunoglobulin-binding molecule (NEIBM)-based ELISA. The high prevalence o...

  18. Virus capsid dissolution studied by microsecond molecular dynamics simulations.

    Science.gov (United States)

    Larsson, Daniel S D; Liljas, Lars; van der Spoel, David

    2012-01-01

    Dissolution of many plant viruses is thought to start with swelling of the capsid caused by calcium removal following infection, but no high-resolution structures of swollen capsids exist. Here we have used microsecond all-atom molecular simulations to describe the dynamics of the capsid of satellite tobacco necrosis virus with and without the 92 structural calcium ions. The capsid expanded 2.5% upon removal of the calcium, in good agreement with experimental estimates. The water permeability of the native capsid was similar to that of a phospholipid membrane, but the permeability increased 10-fold after removing the calcium, predominantly between the 2-fold and 3-fold related subunits. The two calcium binding sites close to the icosahedral 3-fold symmetry axis were pivotal in the expansion and capsid-opening process, while the binding site on the 5-fold axis changed little structurally. These findings suggest that the dissociation of the capsid is initiated at the 3-fold axis.

  19. Impact of capsid conformation and Rep-capsid interactions on adeno-associated virus type 2 genome packaging.

    Science.gov (United States)

    Bleker, Svenja; Pawlita, Michael; Kleinschmidt, Jürgen A

    2006-01-01

    Single-stranded genomes of adeno-associated virus (AAV) are packaged into preformed capsids. It has been proposed that packaging is initiated by interaction of genome-bound Rep proteins to the capsid, thereby targeting the genome to the portal of encapsidation. Here we describe a panel of mutants with amino acid exchanges in the pores at the fivefold axes of symmetry on AAV2 capsids with reduced packaging and reduced Rep-capsid interaction. Mutation of two threonines at the rim of the fivefold pore nearly completely abolished Rep-capsid interaction and packaging. This suggests a Rep-binding site at the highly conserved amino acids at or close to the pores formed by the capsid protein pentamers. A different mutant (P. Wu, W. Xiao, T. Conlon, J. Hughes, M. Agbandje-McKenna, T. Ferkol, T. Flotte, and N. Muzyczka, J. Virol. 74:8635-8647, 2000) with an amino acid exchange at the interface of capsid protein pentamers led to a complete block of DNA encapsidation. Analysis of the capsid conformation of this mutant revealed that the pores at the fivefold axes were occupied by VP1/VP2 N termini, thereby preventing DNA introduction into the capsid. Nevertheless, the corresponding capsids had more Rep proteins bound than wild-type AAV, showing that correct Rep interaction with the capsid depends on a defined capsid conformation. Both mutant types together support the conclusion that the pores at the fivefold symmetry axes are involved in genome packaging and that capsid conformation-dependent Rep-capsid interactions play an essential role in the packaging process.

  20. Characterization of Immunodominant BK Polyomavirus 9mer Epitope T Cell Responses

    Science.gov (United States)

    Cioni, M.; Leboeuf, C.; Comoli, P.; Ginevri, F.

    2016-01-01

    Uncontrolled BK polyomavirus (BKPyV) replication in kidney transplant recipients (KTRs) causes polyomavirus‐associated nephropathy and allograft loss. Reducing immunosuppression is associated with clearing viremia and nephropathy and increasing BKPyV‐specific T cell responses in most patients; however, current immunoassays have limited sensitivity, target mostly CD4+ T cells, and largely fail to predict onset and clearance of BKPyV replication. To characterize BKPyV‐specific CD8+ T cells, bioinformatics were used to predict 9mer epitopes in the early viral gene region (EVGR) presented by 14 common HLAs in Europe and North America. Thirty‐nine EVGR epitopes were experimentally confirmed by interferon‐γ enzyme‐linked immunospot assays in at least 30% of BKPyV IgG–seropositive healthy participants. Most 9mers clustered in domains, and some were presented by more than one HLA class I, as typically seen for immunodominant epitopes. Specific T cell binding using MHC class I streptamers was demonstrated for 21 of 39 (54%) epitopes. In a prospective cohort of 118 pediatric KTRs, 19 patients protected or recovering from BKPyV viremia were experimentally tested, and 13 epitopes were validated. Single HLA mismatches were not associated with viremia, suggesting that failing immune control likely involves multiple factors including maintenance immunosuppression. Combining BKPyV load and T cell assays using immunodominant epitopes may help in evaluating risk and reducing immunosuppression and may lead to safe adoptive T cell transfer. PMID:26663765

  1. Identification of B cell epitopes of alcohol dehydrogenase allergen of Curvularia lunata.

    Directory of Open Access Journals (Sweden)

    Smitha Nair

    Full Text Available BACKGROUND/OBJECTIVE: Epitope identification assists in developing molecules for clinical applications and is useful in defining molecular features of allergens for understanding structure/function relationship. The present study was aimed to identify the B cell epitopes of alcohol dehydrogenase (ADH allergen from Curvularia lunata using in-silico methods and immunoassay. METHOD: B cell epitopes of ADH were predicted by sequence and structure based methods and protein-protein interaction tools while T cell epitopes by inhibitory concentration and binding score methods. The epitopes were superimposed on a three dimensional model of ADH generated by homology modeling and analyzed for antigenic characteristics. Peptides corresponding to predicted epitopes were synthesized and immunoreactivity assessed by ELISA using individual and pooled patients' sera. RESULT: The homology model showed GroES like catalytic domain joined to Rossmann superfamily domain by an alpha helix. Stereochemical quality was confirmed by Procheck which showed 90% residues in most favorable region of Ramachandran plot while Errat gave a quality score of 92.733%. Six B cell (P1-P6 and four T cell (P7-P10 epitopes were predicted by a combination of methods. Peptide P2 (epitope P2 showed E(X(2GGP(X(3KKI conserved pattern among allergens of pathogenesis related family. It was predicted as high affinity binder based on electronegativity and low hydrophobicity. The computational methods employed were validated using Bet v 1 and Der p 2 allergens where 67% and 60% of the epitope residues were predicted correctly. Among B cell epitopes, Peptide P2 showed maximum IgE binding with individual and pooled patients' sera (mean OD 0.604±0.059 and 0.506±0.0035, respectively followed by P1, P4 and P3 epitopes. All T cell epitopes showed lower IgE binding. CONCLUSION: Four B cell epitopes of C. lunata ADH were identified. Peptide P2 can serve as a potential candidate for diagnosis of allergic

  2. Microwave-based treatments of wheat kernels do not abolish gluten epitopes implicated in celiac disease.

    Science.gov (United States)

    Gianfrani, Carmen; Mamone, Gianfranco; la Gatta, Barbara; Camarca, Alessandra; Di Stasio, Luigia; Maurano, Francesco; Picascia, Stefania; Capozzi, Vito; Perna, Giuseppe; Picariello, Gianluca; Di Luccia, Aldo

    2017-03-01

    Microwave based treatment (MWT) of wet wheat kernels induced a striking reduction of gluten, up to gluten-free. In contrast, analysis of gluten peptides by G12 antibody-based ELISA, mass spectrometry-based proteomics and in vitro assay with T cells of celiac subjects, indicated no difference of antigenicity before and after MWT. SDS-PAGE analysis and Raman spectroscopy demonstrated that MWT simply induced conformational modifications, reducing alcohol solubility of gliadins and altering the access of R5-antibody to the gluten epitopes. Thus, MWT neither destroys gluten nor modifies chemically the toxic epitopes, contradicting the preliminary claims that MWT of wheat kernels detoxifies gluten. This study provides evidence that R5-antibody ELISA alone is not effective to determine gluten in thermally treated wheat products. Gluten epitopes in processed wheat should be monitored using strategies based on combined immunoassays with T cells from celiacs, G12-antibody ELISA after proteolysis and proper molecular characterization.

  3. Mutational Analysis of the Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Construction of AAV2 Vectors with Altered Tropism

    Science.gov (United States)

    Wu, Pei; Xiao, Wu; Conlon, Thomas; Hughes, Jeffrey; Agbandje-McKenna, Mavis; Ferkol, Thomas; Flotte, Terence; Muzyczka, Nicholas

    2000-01-01

    Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be β-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag

  4. Immune epitope database analysis resource

    DEFF Research Database (Denmark)

    Kim, Yohan; Ponomarenko, Julia; Zhu, Zhanyang

    2012-01-01

    The immune epitope database analysis resource (IEDB-AR: http://tools.iedb.org) is a collection of tools for prediction and analysis of molecular targets of T- and B-cell immune responses (i.e. epitopes). Since its last publication in the NAR webserver issue in 2008, a new generation of peptide...... and can also be downloaded as software packages....

  5. Automated Detection of Conformational Epitopes Using Phage Display Peptide Sequences

    Directory of Open Access Journals (Sweden)

    Surendra S Negi

    2009-01-01

    Full Text Available Background: Precise determination of conformational epitopes of neutralizing antibodies represents a key step in the rational design of novel vaccines. A powerful experimental method to gain insights on the physical chemical nature of conformational epitopes is the selection of linear peptides that bind with high affinities to a monoclonal antibody of interest by phage display technology. However, the structural characterization of conformational epitopes from these mimotopes is not straightforward, and in the past the interpretation of peptide sequences from phage display experiments focused on linear sequence analysis to find a consensus sequence or common sequence motifs.Results: We present a fully automated search method, EpiSearch that predicts the possible location of conformational epitopes on the surface of an antigen. The algorithm uses peptide sequences from phage display experiments as input, and ranks all surface exposed patches according to the frequency distribution of similar residues in the peptides and in the patch. We have tested the performance of the EpiSearch algorithm for six experimental data sets of phage display experiments, the human epidermal growth factor receptor-2 (HER-2/neu, the antibody mAb Bo2C11 targeting the C2 domain of FVIII, antibodies mAb 17b and mAb b12 of the HIV envelope protein gp120, mAb 13b5 targeting HIV-1 capsid protein and 80R of the SARS coronavirus spike protein. In all these examples the conformational epitopes as determined by the X-ray crystal structures of the antibody-antigen complexes, were found within the highest scoring patches of EpiSearch, covering in most cases more than 50% residues of experimental observed conformational epitopes. Input options of the program include mapping of a single peptide or a set of peptides on the antigen structure, and the results of the calculation can be visualized on our interactive web server.Availability: Users can access the EpiSearch from our web

  6. Kinetics versus Thermodynamics in Virus Capsid Polymorphism.

    Science.gov (United States)

    Moerman, Pepijn; van der Schoot, Paul; Kegel, Willem

    2016-07-07

    Virus coat proteins spontaneously self-assemble into empty shells in aqueous solution under the appropriate physicochemical conditions, driven by an interaction free energy per bond on the order of 2-5 times the thermal energy kBT. For this seemingly modest interaction strength, each protein building block nonetheless gains a very large binding free energy, between 10 and 20 kBT. Because of this, there is debate about whether the assembly process is reversible or irreversible. Here we discuss capsid polymorphism observed in in vitro experiments from the perspective of nucleation theory and of the thermodynamics of mass action. We specifically consider the potential contribution of a curvature free energy term to the effective interaction potential between the proteins. From these models, we propose experiments that may conclusively reveal whether virus capsid assembly into a mixture of polymorphs is a reversible or an irreversible process.

  7. Parvovirus capsid disorders cholesterol-rich membranes.

    Science.gov (United States)

    Pakkanen, Kirsi; Kirjavainen, Sanna; Mäkelä, Anna R; Rintanen, Nina; Oker-Blom, Christian; Jalonen, Tuula O; Vuento, Matti

    2009-02-06

    In this study canine parvovirus, CPV, was found to induce disorder in DPPC:cholesterol membranes in acidic conditions. This acidicity-induced fluidizing effect is suggested to originate from the N-terminus of the viral capsid protein VP1. In accordance with the model membrane studies, a fluidizing effect was seen also in the endosomal membranes during CPV infection implying an important functional role of the fluidization in the endocytic entry of the virus.

  8. Mechanostability of Proteins and Virus Capsids

    Science.gov (United States)

    Cieplak, Marek

    2013-03-01

    Molecular dynamics of proteins within coarse grained models have become a useful tool in studies of large scale systems. The talk will discuss two applications of such modeling. The first is a theoretical survey of proteins' resistance to constant speed stretching as performed for a set of 17134 simple and 318 multidomain proteins. The survey has uncovered new potent force clamps. They involve formation of cysteine slipknots or dragging of a cystine plug through the cystine ring and lead to characteristic forces that are significantly larger than the common shear-based clamp such as observed in titin. The second application involves studies of nanoindentation processes in virus capsids and elucidates their molecular aspects by showing deviations in behavior compared to the continuum shell model. Across the 35 capsids studied, both the collapse force and the elastic stiffness are observed to vary by a factor of 20. The changes in mechanical properties do not correlate simply with virus size or symmetry. There is a strong connection to the mean coordination number , defined as the mean number of interactions to neighboring amino acids. The Young's modulus for thin shell capsids rises roughly quadratically with - 6, where 6 is the minimum coordination for elastic stability in three dimensions. Supported by European Regional Development Fund, through Innovative Economy grant Nanobiom (POIG.01.01.02-00-008/08)

  9. Development of a sensitive and specific epitope-blocking ELISA for universal detection of antibodies to human enterovirus 71 strains.

    Directory of Open Access Journals (Sweden)

    Fang He

    Full Text Available BACKGROUND: Human Enterovirus 71 (EV71 is a common cause of hand, foot and mouth disease (HFMD in young children. It is often associated with severe neurological diseases and mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no efficient universal antibody test available to detect EV71 infections. METHODOLOGY/PRINCIPAL FINDING: In the present study, an epitope-blocking ELISA was developed to detect specific antibodies to human EV71 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (Mab 1C6 that specifically binds to capsid proteins in whole EV71 viruses without any cross reaction to any EV71 capsid protein expressed alone. The sensitivity and specificity of the epitope-blocking ELISA for EV71 was evaluated and compared to microneutralization using immunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses. Further, 200 serum sample from human individuals who were potentially infected with EV71 viruses were tested in both the blocking ELISA and microneutralization. Results indicated that antibodies to EV71 were readily detected in immunized animals or human sera by the epitope blocking ELISA whereas specimens with antibodies to other enteroviruses yielded negative results. This assay is not only simpler to perform but also shows higher sensitivity and specificity as compared to microneutralization. CONCLUSION: The epitope-blocking ELISA based on a unique Mab 1C6 provided highly sensitive and 100% specific detection of antibodies to human EV71 viruses in human sera.

  10. Classification and Evolutionary Trends of Icosahedral Viral Capsids

    Directory of Open Access Journals (Sweden)

    Richard Kerner

    2008-01-01

    Full Text Available A classification of icosahedral viral capsids is proposed. We show how the self-organization of capsids during their formation implies a definite composition of their elementary building blocks. The exact number of hexamers with three different admissible symmetries is related to capsids' sizes, labelled by their T-numbers. Simple rules determining these numbers for each value of T are deduced and certain consequences concerning the probabilities of mutations and evolution of viruses are discussed.

  11. A new EV71 VP3 epitope in norovirus P particle vector displays neutralizing activity and protection in vivo in mice.

    Science.gov (United States)

    Jiang, Liping; Fan, Rongjun; Sun, Shiyang; Fan, Peihu; Su, Weiheng; Zhou, Yan; Gao, Feng; Xu, Fei; Kong, Wei; Jiang, Chunlai

    2015-11-27

    Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16), as the main agents causing hand, foot and mouth disease (HFMD), have become a serious public health concern in the Asia-Pacific region. Recently, various neutralizing B cell epitopes of EV71 were identified as targets for promising vaccine candidates. Structural studies of Picornaviridae indicated that potent immunodominant epitopes typically lie in the hypervariable loop of capsid surfaces. However, cross-neutralizing antibodies and cross-protection between EV71 and CVA16 have not been observed. Therefore, we speculated that divergent sequences of the two viruses are key epitopes for inducing protective neutralizing responses. In this study, we selected 10 divergent epitope candidates based on alignment of the EV71 and CVA16 P1 amino acid sequences using the Multalin interface page, and these epitopes are conserved among all subgenotypes of EV71. Simultaneously, by utilizing the norovirus P particle as a novel vaccine delivery carrier, we identified the 71-6 epitope (amino acid 176-190 of VP3) as a conformational neutralizing epitope against EV71 in an in vitro micro-neutralization assay as well as an in vivo protection assay in mice. Altogether, these results indicated that the incorporation of the 71-6 epitope into the norovirus P domain can provide a promising candidate for an effective synthetic peptide-based vaccine against EV71.

  12. Large-scale functional purification of recombinant HIV-1 capsid.

    Directory of Open Access Journals (Sweden)

    Magdeleine Hung

    Full Text Available During human immunodeficiency virus type-1 (HIV-1 virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents.

  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. Impact of Capsid Conformation and Rep-Capsid Interactions on Adeno-Associated Virus Type 2 Genome Packaging

    OpenAIRE

    Bleker, Svenja; Pawlita, Michael; Kleinschmidt, Jürgen A.

    2006-01-01

    Single-stranded genomes of adeno-associated virus (AAV) are packaged into preformed capsids. It has been proposed that packaging is initiated by interaction of genome-bound Rep proteins to the capsid, thereby targeting the genome to the portal of encapsidation. Here we describe a panel of mutants with amino acid exchanges in the pores at the fivefold axes of symmetry on AAV2 capsids with reduced packaging and reduced Rep-capsid interaction. Mutation of two threonines at the rim of the fivefol...

  15. Structure of the small outer capsid protein, Soc: a clamp for stabilizing capsids of T4-like phages.

    Science.gov (United States)

    Qin, Li; Fokine, Andrei; O'Donnell, Erin; Rao, Venigalla B; Rossmann, Michael G

    2010-01-29

    Many viruses need to stabilize their capsid structure against DNA pressure and for survival in hostile environments. The 9-kDa outer capsid protein (Soc) of bacteriophage T4, which stabilizes the virus, attaches to the capsid during the final stage of maturation. There are 870 Soc molecules that act as a "glue" between neighboring hexameric capsomers, forming a "cage" that stabilizes the T4 capsid against extremes of pH and temperature. Here we report a 1.9 A resolution crystal structure of Soc from the bacteriophage RB69, a close relative of T4. The RB69 crystal structure and a homology model of T4 Soc were fitted into the cryoelectron microscopy reconstruction of the T4 capsid. This established the region of Soc that interacts with the major capsid protein and suggested a mechanism, verified by extensive mutational and biochemical studies, for stabilization of the capsid in which the Soc trimers act as clamps between neighboring capsomers. The results demonstrate the factors involved in stabilizing not only the capsids of T4-like bacteriophages but also many other virus capsids.

  16. Imaging of the alphavirus capsid protein during virus replication.

    Science.gov (United States)

    Zheng, Yan; Kielian, Margaret

    2013-09-01

    Alphaviruses are enveloped viruses with highly organized structures. The nucleocapsid (NC) core contains a capsid protein lattice enclosing the plus-sense RNA genome, and it is surrounded by a lipid bilayer containing a lattice of the E1 and E2 envelope glycoproteins. Capsid protein is synthesized in the cytoplasm and particle budding occurs at the plasma membrane (PM), but the traffic and assembly of viral components and the exit of virions from host cells are not well understood. To visualize the dynamics of capsid protein during infection, we developed a Sindbis virus infectious clone tagged with a tetracysteine motif. Tagged capsid protein could be fluorescently labeled with biarsenical dyes in living cells without effects on virus growth, morphology, or protein distribution. Live cell imaging and colocalization experiments defined distinct groups of capsid foci in infected cells. We observed highly motile internal puncta that colocalized with E2 protein, which may represent the transport machinery that capsid protein uses to reach the PM. Capsid was also found in larger nonmotile internal structures that colocalized with cellular G3BP and viral nsP3. Thus, capsid may play an unforeseen role in these previously observed G3BP-positive foci, such as regulation of cellular stress granules. Capsid puncta were also observed at the PM. These puncta colocalized with E2 and recruited newly synthesized capsid protein; thus, they may be sites of virus assembly and egress. Together, our studies provide the first dynamic views of the alphavirus capsid protein in living cells and a system to define detailed mechanisms during alphavirus infection.

  17. Molecular interactions of Epstein-Barr virus capsid proteins.

    Science.gov (United States)

    Wang, Wen-Hung; Chang, Li-Kwan; Liu, Shih-Tung

    2011-02-01

    The capsids of herpesviruses, which comprise major and minor capsid proteins, have a common icosahedral structure with 162 capsomers. An electron microscopic study shows that Epstein-Barr virus (EBV) capsids in the nucleus are immunolabeled by anti-BDLF1 and anti-BORF1 antibodies, indicating that BDLF1 and BORF1 are the minor capsid proteins of EBV. Cross-linking and electrophoresis studies of purified BDLF1 and BORF1 revealed that these two proteins form a triplex that is similar to that formed by the minor capsid proteins, VP19C and VP23, of herpes simplex virus type 1 (HSV-1). Although the interaction between VP23, a homolog of BDLF1, and the major capsid protein VP5 could not be verified biochemically in earlier studies, the interaction between BDLF1 and the EBV major capsid protein, viral capsid antigen (VCA), can be confirmed by glutathione S-transferase (GST) pulldown assay and coimmunoprecipitation. Additionally, in HSV-1, VP5 interacts with only the middle region of VP19C; in EBV, VCA interacts with both the N-terminal and middle regions of BORF1, a homolog of VP19C, revealing that the proteins in the EBV triplex interact with the major capsid protein differently from those in HSV-1. A GST pulldown study also identifies the oligomerization domains in VCA and the dimerization domain in BDLF1. The results presented herein reveal how the EBV capsid proteins interact and thereby improve our understanding of the capsid structure of the virus.

  18. Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus

    DEFF Research Database (Denmark)

    Martinez-Torrecuadrada, J.L.; Langeveld, J.P.M.; Venteo, A.;

    1999-01-01

    African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological...... immunodominant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151-200) and II (residues 83-120). The epitopes were further defined by PEPSCAN analysis with 12mer peptides, which determined eight antigenic sites in the N-terminal half of the molecule...

  19. Multivalent viral capsids with internal cargo for fibrin imaging.

    Directory of Open Access Journals (Sweden)

    Allie C Obermeyer

    Full Text Available Thrombosis is the cause of many cardiovascular syndromes and is a significant contributor to life-threatening diseases, such as myocardial infarction and stroke. Thrombus targeted imaging agents have the capability to provide molecular information about pathological clots, potentially improving detection, risk stratification, and therapy of thrombosis-related diseases. Nanocarriers are a promising platform for the development of molecular imaging agents as they can be modified to have external targeting ligands and internal functional cargo. In this work, we report the synthesis and use of chemically functionalized bacteriophage MS2 capsids as biomolecule-based nanoparticles for fibrin imaging. The capsids were modified using an oxidative coupling reaction, conjugating ∼90 copies of a fibrin targeting peptide to the exterior of each protein shell. The ability of the multivalent, targeted capsids to bind fibrin was first demonstrated by determining the impact on thrombin-mediated clot formation. The modified capsids out-performed the free peptides and were shown to inhibit clot formation at effective concentrations over ten-fold lower than the monomeric peptide alone. The installation of near-infrared fluorophores on the interior surface of the capsids enabled optical detection of binding to fibrin clots. The targeted capsids bound to fibrin, exhibiting higher signal-to-background than control, non-targeted MS2-based nanoagents. The in vitro assessment of the capsids suggests that fibrin-targeted MS2 capsids could be used as delivery agents to thrombi for diagnostic or therapeutic applications.

  20. Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation.

    Science.gov (United States)

    Wittkop, Linda; Schwarz, Alexandra; Cassany, Aurelia; Grün-Bernhard, Stefanie; Delaleau, Mildred; Rabe, Birgit; Cazenave, Christian; Gerlich, Wolfram; Glebe, Dieter; Kann, Michael

    2010-07-01

    Capsids of hepatitis B virus and other hepadnaviruses contain a cellular protein kinase, which phosphorylates the capsid protein. Some phosphorylation sites are shown to be essential for distinct steps of viral replication as pregenome packaging or plus strand DNA synthesis. Although different protein kinases have been reported to phosphorylate the capsid protein, varying experimental approaches do not allow direct comparison. Furthermore, the activity of a specific protein kinase has not yet been correlated to steps in the hepadnaviral life cycle. In this study we show that capsids from various sources encapsidate active protein kinase Calpha, irrespective of hepatitis B virus genotype and host cell. Treatment of a virion expressing cell line with a pseudosubstrate inhibitor showed that inhibition of protein kinase C phosphorylation did not affect genome maturation but resulted in capsid accumulation and inhibited virion release to the medium. Our results imply that different protein kinases have distinct functions within the hepadnaviral life cycle.

  1. Identification of linear B-cell epitopes on myotoxin II, a Lys49 phospholipase A₂ homologue from Bothrops asper snake venom.

    Science.gov (United States)

    Lomonte, Bruno

    2012-10-01

    Knowledge on toxin immunogenicity at the molecular level can provide valuable information for the improvement of antivenoms, as well as for understanding toxin structure-function relationships. The aims of this study are two-fold: first, to identify the linear B-cell epitopes of myotoxin II from Bothrops asper snake venom, a Lys49 phospholipase A₂ homologue; and second, to use antibodies specifically directed against an epitope having functional relevance in its toxicity, to probe the dimeric assembly mode of this protein in solution. Linear B-cell epitopes were identified using a library of overlapping synthetic peptides spanning its complete sequence. Epitopes recognized by a rabbit antiserum to purified myotoxin II, and by three batches of a polyvalent (Crotalidae) therapeutic antivenom (prepared in horses immunized with a mixture of B. asper, Crotalus simus, and Lachesis stenophrys venoms) were mapped using an enzyme-immunoassay based on the capture of biotinylated peptides by immobilized streptavidin. Some of the epitopes identified were shared between the two species, whereas others were unique. Differences in epitope recognition were observed not only between the two species, but also within the three batches of equine antivenom. Epitope V, located at the C-terminal region of this protein, is known to be relevant for toxicity and neutralization. Affinity-purified rabbit antibodies specific for this site were able to immunoprecipitate myotoxin II, suggesting that the two copies of epitope V are simultaneously available to antibody binding, which would be compatible with the mode of dimerization known as "conventional" dimer.

  2. Rapid homogenous time-resolved fluorescence (HTRF) immunoassay for anthrax detection.

    Science.gov (United States)

    Cohen, Noam; Mechaly, Adva; Mazor, Ohad; Fisher, Morly; Zahavy, Eran

    2014-05-01

    Infection with Bacillus anthracsis spores induces an acute anthrax disease that can cause casualties and death in untreated cases. Thus rapid diagnosis of anthrax at early stage of the disease is essential to allow an effective treatment. Here we present the development of rapid and sensitive homogenous time-resolved fluorescence (HTRF) immunoassays based on the energy transfer process of europium cryptate (EuK) donor to AlexaFluor647 acceptor. The energy transfer process is limited to d bacteremia in infected hosts, using two monoclonal anti-PA antibodies that specifically recognize two different epitopes on the PA molecule. The assay was sensitive enabling detection of 2 ng/ml PA in the serum of B. anthracsis-infected rabbits in only 15 min assay. Additionally, HTRF assay was developed for the detection of bacterial spores using polyclonal anti-spore antibodies that recognize many epitopes on the bacterial surface. The assay enabled the detection of 2 × 10(6) spores/ml in 30 min assay and was specific, showing no cross reactivity with closely related non-virulent bacillus cereus strain. This study describes the use of the HTRF assay for the detection of both singled-epitope (proteins) and multi-epitope (particles) as rapid, simple and sensitive method that can be used at the time that fast results are needed to allow an effective medical care.

  3. Recognition of Porphyromonas gingivalis gingipain epitopes by natural IgM binding to malondialdehyde modified low-density lipoprotein.

    Directory of Open Access Journals (Sweden)

    S Pauliina Turunen

    Full Text Available OBJECTIVE: Increased risk for atherosclerosis is associated with infectious diseases including periodontitis. Natural IgM antibodies recognize pathogen-associated molecular patterns on bacteria, and oxidized lipid and protein epitopes on low-density lipoprotein (LDL and apoptotic cells. We aimed to identify epitopes on periodontal pathogen Porphyromonas gingivalis recognized by natural IgM binding to malondialdehyde (MDA modified LDL. METHODS AND RESULTS: Mouse monoclonal IgM (MDmAb specific for MDA-LDL recognized epitopes on P. gingivalis on flow cytometry and chemiluminescence immunoassays. Immunization of C57BL/6 mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and apoptotic cells. Immunization of LDLR(-/- mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and diminished aortic lipid deposition. On Western blot MDmAb bound to P. gingivalis fragments identified as arginine-specific gingipain (Rgp by mass spectrometry. Recombinant domains of Rgp produced in E. coli were devoid of phosphocholine epitopes but contained epitopes recognized by MDmAb and human serum IgM. Serum IgM levels to P. gingivalis were associated with anti-MDA-LDL levels in humans. CONCLUSION: Gingipain of P. gingivalis is recognized by natural IgM and shares molecular identity with epitopes on MDA-LDL. These findings suggest a role for natural antibodies in the pathogenesis of two related inflammatory diseases, atherosclerosis and periodontitis.

  4. Virus capsid dissolution studied by microsecond molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Daniel S D Larsson

    Full Text Available Dissolution of many plant viruses is thought to start with swelling of the capsid caused by calcium removal following infection, but no high-resolution structures of swollen capsids exist. Here we have used microsecond all-atom molecular simulations to describe the dynamics of the capsid of satellite tobacco necrosis virus with and without the 92 structural calcium ions. The capsid expanded 2.5% upon removal of the calcium, in good agreement with experimental estimates. The water permeability of the native capsid was similar to that of a phospholipid membrane, but the permeability increased 10-fold after removing the calcium, predominantly between the 2-fold and 3-fold related subunits. The two calcium binding sites close to the icosahedral 3-fold symmetry axis were pivotal in the expansion and capsid-opening process, while the binding site on the 5-fold axis changed little structurally. These findings suggest that the dissociation of the capsid is initiated at the 3-fold axis.

  5. DNA Labeling Generates a Unique Amplification Probe for Sensitive Photoelectrochemical Immunoassay of HIV-1 p24 Antigen.

    Science.gov (United States)

    Zhao, Wei-Wei; Han, Ying-Mei; Zhu, Yuan-Cheng; Zhang, Nan; Xu, Jing-Juan; Chen, Hong-Yuan

    2015-06-01

    Photoelectrochemical (PEC) immunoassay is an attractive methodology as it allows for an elegant and sensitive protein assay. However, advanced PEC immunoassay remains challenging and the established amplifications rely almost exclusively on the labeling of various enzymes, which usually suffer the inferior stabilities. Here we report the development and validation of the DNA labeling that leads to a unique amplification probe for the sensitive PEC immunoassay of HIV-1 capsid protein, p24 antigen, an important biomarker of human immune deficiency virus (HIV). Following the sandwich immunobinding, the DNA tags could be released and the subsequent dipurinization of the oligonucleotide strands enables the easy oxidation of free nucleobases at a CdTe quantum dots (QDs) modified ITO transducer. Such DNA tags induced PEC amplification and readout permits the exquisite assay of HIV-1 p24 antigen with high sensitivity. As compared to the existing method of enzymatic labeling, the easy preparation and stability of these labels make them very suitable for PEC amplification. Another merit of this method is that it separates the immunobinding from the PEC transducer, which eliminates the commonly existing affection during the biorecognition processes. This work paves a new route for the PEC immunoassay of HIV-1 p24 antigen and provides a general format for the PEC biomolecular detection by means of the DNA labeling.

  6. Immune Epitope Database and Analysis Resource (IEDB)

    Data.gov (United States)

    U.S. Department of Health & Human Services — This repository contains antibody/B cell and T cell epitope information and epitope prediction and analysis tools for use by the research community worldwide. Immune...

  7. Structure of the Triatoma virus capsid

    Energy Technology Data Exchange (ETDEWEB)

    Squires, Gaëlle; Pous, Joan [Laboratoire de Virologie Moléculaire et Structurale, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette CEDEX (France); Agirre, Jon [Fundación Biofísica Bizkaia, Barrio Sarriena S/N, 48940 Leioa, Bizkaia (FBB) (Spain); Unidad de Biofísica (UBF, CSIC, UPV/EHU), PO Box 644, 48080 Bilbao (Spain); Rozas-Dennis, Gabriela S. [U.N.S., San Juan 670 (8000) Bahía Blanca (Argentina); U.N.S., Avenida Alem 1253 (8000) Bahía Blanca (Argentina); Costabel, Marcelo D. [U.N.S., Avenida Alem 1253 (8000) Bahía Blanca (Argentina); Marti, Gerardo A. [Centro de Estudios Parasitológicos y de Vectores (CEPAVE-CCT, La Plata, CONICET-UNLP), Calle 2 No. 584 (1900) La Plata (Argentina); Navaza, Jorge; Bressanelli, Stéphane [Laboratoire de Virologie Moléculaire et Structurale, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette CEDEX (France); Guérin, Diego M. A., E-mail: diego.guerin@ehu.es [Fundación Biofísica Bizkaia, Barrio Sarriena S/N, 48940 Leioa, Bizkaia (FBB) (Spain); Unidad de Biofísica (UBF, CSIC, UPV/EHU), PO Box 644, 48080 Bilbao (Spain); Rey, Felix A., E-mail: diego.guerin@ehu.es [Laboratoire de Virologie Moléculaire et Structurale, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette CEDEX (France)

    2013-06-01

    The crystallographic structure of TrV shows specific morphological and functional features that clearly distinguish it from the type species of the Cripavirus genus, CrPV. The members of the Dicistroviridae family are non-enveloped positive-sense single-stranded RNA (+ssRNA) viruses pathogenic to beneficial arthropods as well as insect pests of medical importance. Triatoma virus (TrV), a member of this family, infects several species of triatomine insects (popularly named kissing bugs), which are vectors for human trypanosomiasis, more commonly known as Chagas disease. The potential use of dicistroviruses as biological control agents has drawn considerable attention in the past decade, and several viruses of this family have been identified, with their targets covering honey bees, aphids and field crickets, among others. Here, the crystal structure of the TrV capsid at 2.5 Å resolution is reported, showing that as expected it is very similar to that of Cricket paralysis virus (CrPV). Nevertheless, a number of distinguishing structural features support the introduction of a new genus (Triatovirus; type species TrV) under the Dicistroviridae family. The most striking differences are the absence of icosahedrally ordered VP4 within the infectious particle and the presence of prominent projections that surround the fivefold axis. Furthermore, the structure identifies a second putative autoproteolytic DDF motif in protein VP3, in addition to the conserved one in VP1 which is believed to be responsible for VP0 cleavage during capsid maturation. The potential meaning of these new findings is discussed.

  8. A New Hapten for Immunoassay of Aldicarb

    Institute of Scientific and Technical Information of China (English)

    Yan Feng ZHANG; Zhi Xian GAO; Qing Min ZHANG; Shu Gui DAI

    2006-01-01

    A new hapten, aldicarb oxime succinic ester (AOSE), was synthesized for immunoassay of aldicarb. It was conjugated to proteins by active ester method. Polyclonal antibody was raised against AOSE-BSA (bovine serum albumin) conjugate. Enzyme-linked immunosorbent assays (ELISAs) showed that this antiserum had high affinity to aldicarb and can be used for sensitive and selective immunoassay of aldicarb.

  9. Nonlinear Finite Element Analysis of Nanoindentation of Viral Capsids

    CERN Document Server

    Gibbons, M M; Gibbons, Melissa M.; Klug, William S.

    2006-01-01

    Recent Atomic Force Microscope (AFM) nanoindentation experiments measuring mechanical response of the protein shells of viruses have provided a quantitative description of their strength and elasticity. To better understand and interpret these measurements, and to elucidate the underlying mechanisms, this paper adopts a course-grained modeling approach within the framework of three-dimensional nonlinear continuum elasticity. Homogeneous, isotropic, elastic, thick shell models are proposed for two capsids: the spherical Cowpea Chlorotic Mottle Virus (CCMV), and the ellipsocylindrical bacteriophage $\\phi 29$. As analyzed by the finite element method, these models enable parametric characterization of the effects of AFM tip geometry, capsid dimensions, and capsid constitutive descriptions. The generally nonlinear force response of capsids to indentation is shown to be insensitive to constitutive details, and greatly influenced by geometry. Nonlinear stiffening and softening of the force response is dependent on ...

  10. Prevalence and Genetic Variability in Capsid L1 Gene of Rare Human Papillomaviruses (HPV Found in Cervical Lesions of Women from North-East Brazil

    Directory of Open Access Journals (Sweden)

    Ana Pavla Almeida Diniz Gurgel

    2013-01-01

    Full Text Available The aim of this study was to examine the prevalence and genetic variability of the capsid L1 gene of rare HPV genotypes that were found in the cervical lesions of women from North-East Brazil. A total number of 263 patients were included in this study. HPV detection was performed using PCR followed by direct sequencing of MY09/11, as well as type-specific PCR to detect the Alpha-9 species. Epitope prediction was performed to determine whether or not the genetic variants are inserted in B-cell and T-cell epitopes. The prevalence of rare HPV types in cervical lesions was found to be 9.47%. The rare HPV genotypes that were detected were HPV-53, 54, 56, 61, 62, 66, 70, and 81. The genetic variability in the L1 gene of rare HPV types involved thirty nucleotide changes, eight of which were detected for the first time in this study. Moreover, some of these variants are embedded in B-cell or T-cell epitope regions. The results of this research suggest that rare HPV types might be involved in cervical lesions and some of these variants can be found in B-cell and T-cell epitopes. Data on the prevalence and variability of rare HPV types will assist in clarifying the role of these viruses in carcinogenesis.

  11. New Viral Vector for Superproduction of Epitopes of Vaccine Proteins in Plants

    Science.gov (United States)

    Tyulkina, L.G.; Skurat, E.V.; Frolova, O.Yu.; Komarova, T.V.; Karger, E.M.; Atabekov, I.G.

    2011-01-01

    The novel viral vectors PVX-CP AltMV and PVXdt-CP AltMV are superexpressors of the capsid protein (CP). These viral vectors were constructed on the basis of the potato virus X (PVX) genome andAlternantheramosaic virus (AltMV) CP gene. The expression, based on the hybrid viral vectors, is genetically safe, since the systemic transport and formation of infective viral particles are blocked. CP AltMV can self-assemble into virus-like particles (VLPs) in the absence of genomic RNA. The vectors can be used for the presentation of foreign peptides (including epitopes of human pathogens) on the surface of the VLP. The N-terminal extracellular domain (M2e) of the influenza virus A M2 protein and its truncated variant (ΔM2e) were used as model heterologous peptides for the construction of the chimeric CP AltMV. Chimeric CP AltMV retains its ability to self-assemble into VLP. The epitopes of the M2 influenza virus protein were not eliminated during the process of accumulation, polymerization and purification of chimeric VLP AltMV, providing evidence of the stability of chimeric VLP with C-terminal heterologous epitopes. It appears that VLP produced by the vectors PVX-CP AltMV and PVXdt-CP AltMV can be used in the field of biotechnology for the presentation of the epitopes of vaccine proteins on their surfaces. The chimeric VLP AltMV with the presented foreign epitopes can be used as candidate vaccines. PMID:22649706

  12. Polymorphism of DNA conformation inside the bacteriophage capsid

    OpenAIRE

    Leforestier, Amélie

    2013-01-01

    Double-stranded DNA bacteriophage genomes are packaged into their icosahedral capsids at the highest densities known so far (about 50 % w:v). How the molecule is folded at such density and how its conformation changes upon ejection or packaging are fascinating questions still largely open. We review cryo-TEM analyses of DNA conformation inside partially filled capsids as a function of the physico-chemical environment (ions, osmotic pressure, temperature). We show that there exists a wide vari...

  13. Photonic crystal enhanced cytokine immunoassay.

    Science.gov (United States)

    Mathias, Patrick C; Ganesh, Nikhil; Cunningham, Brian T

    2009-01-01

    Photonic crystal surfaces are demonstrated as a means for enhancing the detection sensitivity and resolution for assays that use a fluorescent tag to quantify the concentration of an analyte protein molecule in a liquid test sample. Computer modeling of the spatial distribution of resonantly coupled electromagnetic fields on the photonic crystal surface are used to estimate the magnitude of enhancement factor compared to performing the same fluorescent assay on a plain glass surface, and the photonic crystal structure is fabricated and tested to experimentally verify the performance using a sandwich immunoassay for the protein Tumor Necrosis Factor-alpha (TNF-alpha). The demonstrated photonic crystal fabrication method utilizes a nanoreplica molding technique that allows for large-area inexpensive fabrication of the structure in a format that is compatible with confocal microarray laser scanners. The signal-to-noise ratio for fluorescent spots on the photonic crystal is increased by at least five-fold relative to the glass slide, allowing a TNF-alpha concentration of 1.6 pg/ml to be distinguished from noise on a photonic crystal surface. In addition, the minimum quantitative limit of detection on the photonic crystal surface is one-third the limit on the glass slide - a decrease from 18 pg/ml to 6 pg/ml. The increased performance of the immunoassay allows for more accurate quantitation of physiologically relevant concentrations of TNF-alpha in a protein microarray format that can be expanded to multiple cytokines.

  14. Quantum dot-induced viral capsid assembling in dissociation buffer.

    Science.gov (United States)

    Gao, Ding; Zhang, Zhi-Ping; Li, Feng; Men, Dong; Deng, Jiao-Yu; Wei, Hong-Ping; Zhang, Xian-En; Cui, Zong-Qiang

    2013-01-01

    Viruses encapsulating inorganic nanoparticles are a novel type of nanostructure with applications in biomedicine and biosensors. However, the encapsulation and assembly mechanisms of these hybridized virus-based nanoparticles (VNPs) are still unknown. In this article, it was found that quantum dots (QDs) can induce simian virus 40 (SV40) capsid assembly in dissociation buffer, where viral capsids should be disassembled. The analysis of the transmission electron microscope, dynamic light scattering, sucrose density gradient centrifugation, and cryo-electron microscopy single particle reconstruction experimental results showed that the SV40 major capsid protein 1 (VP1) can be assembled into ≈25 nm capsids in the dissociation buffer when QDs are present and that the QDs are encapsulated in the SV40 capsids. Moreover, it was determined that there is a strong affinity between QDs and the SV40 VP1 proteins (KD=2.19E-10 M), which should play an important role in QD encapsulation in the SV40 viral capsids. This study provides a new understanding of the assembly mechanism of SV40 virus-based nanoparticles with QDs, which may help in the design and construction of other similar virus-based nanoparticles.

  15. Novel immunoassay formats for integrated microfluidic circuits: diffusion immunoassays (DIA)

    Science.gov (United States)

    Weigl, Bernhard H.; Hatch, Anson; Kamholz, Andrew E.; Yager, Paul

    2000-03-01

    Novel designs of integrated fluidic microchips allow separations, chemical reactions, and calibration-free analytical measurements to be performed directly in very small quantities of complex samples such as whole blood and contaminated environmental samples. This technology lends itself to applications such as clinical diagnostics, including tumor marker screening, and environmental sensing in remote locations. Lab-on-a-Chip based systems offer many *advantages over traditional analytical devices: They consume extremely low volumes of both samples and reagents. Each chip is inexpensive and small. The sampling-to-result time is extremely short. They perform all analytical functions, including sampling, sample pretreatment, separation, dilution, and mixing steps, chemical reactions, and detection in an integrated microfluidic circuit. Lab-on-a-Chip systems enable the design of small, portable, rugged, low-cost, easy to use, yet extremely versatile and capable diagnostic instruments. In addition, fluids flowing in microchannels exhibit unique characteristics ('microfluidics'), which allow the design of analytical devices and assay formats that would not function on a macroscale. Existing Lab-on-a-chip technologies work very well for highly predictable and homogeneous samples common in genetic testing and drug discovery processes. One of the biggest challenges for current Labs-on-a-chip, however, is to perform analysis in the presence of the complexity and heterogeneity of actual samples such as whole blood or contaminated environmental samples. Micronics has developed a variety of Lab-on-a-Chip assays that can overcome those shortcomings. We will now present various types of novel Lab- on-a-Chip-based immunoassays, including the so-called Diffusion Immunoassays (DIA) that are based on the competitive laminar diffusion of analyte molecules and tracer molecules into a region of the chip containing antibodies that target the analyte molecules. Advantages of this

  16. The Suramin Derivative NF449 Interacts with the 5-fold Vertex of the Enterovirus A71 Capsid to Prevent Virus Attachment to PSGL-1 and Heparan Sulfate.

    Directory of Open Access Journals (Sweden)

    Yorihiro Nishimura

    2015-10-01

    Full Text Available NF449, a sulfated compound derived from the antiparasitic drug suramin, was previously reported to inhibit infection by enterovirus A71 (EV-A71. In the current work, we found that NF449 inhibits virus attachment to target cells, and specifically blocks virus interaction with two identified receptors--the P-selectin ligand, PSGL-1, and heparan sulfate glycosaminoglycan--with no effect on virus binding to a third receptor, the scavenger receptor SCARB2. We also examined a number of commercially available suramin analogues, and newly synthesized derivatives of NF449; among these, NF110 and NM16, like NF449, inhibited virus attachment at submicromolar concentrations. PSGL-1 and heparan sulfate, but not SCARB2, are both sulfated molecules, and their interaction with EV-A71 is thought to involve positively charged capsid residues, including a conserved lysine at VP1-244, near the icosahedral 5-fold vertex. We found that mutation of VP1-244 resulted in resistance to NF449, suggesting that this residue is involved in NF449 interaction with the virus capsid. Consistent with this idea, NF449 and NF110 prevented virus interaction with monoclonal antibody MA28-7, which specifically recognizes an epitope overlapping VP1-244 at the 5-fold vertex. Based on these observations we propose that NF449 and related compounds compete with sulfated receptor molecules for a binding site at the 5-fold vertex of the EV-A71 capsid.

  17. A neutralizing epitope of human papillomavirus type 11 is principally described by a continuous set of residues which overlap a distinct linear, surface-exposed epitope.

    Science.gov (United States)

    Ludmerer, S W; Benincasa, D; Mark, G E; Christensen, N D

    1997-01-01

    A panel of monoclonal antibodies (MAbs) which neutralize human papillomavirus type 11 (HPV11) in the athymic mouse xenograph neutralization assay and bind HPV11 virus-like particles (VLPs) has been described. We recently presented evidence that the Gly131-Tyr132 residues of the major capsid protein L1 confer type 11-specific binding. However, residues distally located on the primary L1 sequence also were shown to affect binding. This poses the question whether the epitope is principally centered in the region of Gly131-Tyr132 or, alternatively, is comprised of diversely located residues which come into proximity only upon proper assembly. We analyzed the result of numerous substitutions located between Tyr123 and Val142 of the HPV11 L1 sequence. We show that substitutions at five positions result in loss of binding for one or more of these MAbs by an enzyme-linked immunosorbent assay which measures antibody binding to VLPs. We demonstrate that binding of these MAbs is redirected to HPV16 VLPs which harbor eight type 11-like substitutions within the homologous region. Three of these substitutions did not affect binding when individually substituted in HPV11 but yet were still required to transfer binding to substituted HPV16 VLPs. The results demonstrate that the epitope for this class of neutralizing MAbs, although conformational and requiring VLP assembly for presentation, principally lies along a 20-residue stretch of the L1 major capsid protein. This targets the region for evaluation of the possibility of receptor binding and suggests possibilities for the design of peptide inhibitors of virus infectivity. PMID:9094659

  18. Characterization of antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian hepatitis E virus.

    Science.gov (United States)

    Zhao, Qin; Sun, Ya-ni; Hu, Shou-bin; Wang, Xin-jie; Xiao, Yi-hong; Hsu, Walter H; Xiao, Shu-qi; Wang, Cheng-bao; Mu, Yang; Hiscox, Julian A; Zhou, En-Min

    2013-12-27

    Avian hepatitis E virus (HEV) is an emerging virus associated with the big liver and spleen disease or hepatitis-splenomegaly syndrome in chickens and subclinical infections by the virus are also common. The complete genome of avian HEV contains three open-reading frames (ORFs) in which ORF2 protein is part of virus particles and thus contains primary epitopes. Antigenic epitopes of avian HEV ORF2 protein have been described but those associated with the ORF3 have not. To analyze the antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian HEV (CaHEV), we generated a series of antigens comprised of the complete ORF3 and also five truncated overlapping ORF3 peptides. The antibodies used in this study were mouse antisera and monoclonal antibodies against ORF3, positive chicken sera from Specific Pathogen Free chickens experimentally infected with CaHEV and clinical chicken sera. Using these antigens and antibodies, we identified three antigenic domains at amino acids (aa) 1-28, 55-74 and 75-88 in which aa 75-88 was a dominant domain. The dominant domain contained at least two major epitopes since field chickens infected with avian HEV produced antibodies against the domain and epitopes. These results provide useful information for future development of immunoassays for the diagnosis of avian HEV infection.

  19. Production and characterization of monoclonal antibody specific to recombinant dengue multi-epitope protein.

    Science.gov (United States)

    Abhyankar, Ajay Vinayak; Bhargava, Rakesh; Jana, Asha Mukul; Sahni, Ajay Kumar; Rao, P V Lakshmana

    2008-06-01

    Monoclonal antibodies against novel dengue recombinant protein were produced following immunization of Balb/c mice with recombinant dengue multi-epitope protein (r-DMEP) expressed in Escherichia coli vector and purified in a single-step chromatography system. Antigenicity of r-DMEP was evaluated by dot enzyme immunoassay. Mice were immunized intraperitoneally with five doses each of 100 microg of this novel antigen at 1-week intervals and a final intravenous booster dose prior to the fusion. Hybridomas resulted from fusion of myeloma cells and splenocytes using PEG-1500 as an additive. Selection of the hybrids was done using HAT medium, and the hybrids thus selected were finally screened qualitatively and quantitatively by dot and plate immunoassays, respectively. Five antibody secretory hybrid clones exhibited specific reactivity against r-DMEP by dot-ELISA, whereas a lone clone was found to be cross-reactive with Japanese encephalitis virus (JEV). Monoclonal antibodies (MAbs) specific to r-DME protein recognized the envelope and non-structural epitopes by Western blot analysis. These MAbs were further checked for their diagnostic efficacy using dengue suspected clinical samples and found overall sensitivity and specificity for DRDE dipstick ELISA. MAb-based dipstick ELISA results were 85%, 75% and 85%, 90%, respectively.

  20. Diminished reovirus capsid stability alters disease pathogenesis and littermate transmission.

    Directory of Open Access Journals (Sweden)

    Joshua D Doyle

    2015-03-01

    Full Text Available Reovirus is a nonenveloped mammalian virus that provides a useful model system for studies of viral infections in the young. Following internalization into host cells, the outermost capsid of reovirus virions is removed by endosomal cathepsin proteases. Determinants of capsid disassembly kinetics reside in the viral σ3 protein. However, the contribution of capsid stability to reovirus-induced disease is unknown. In this study, we found that mice inoculated intramuscularly with a serotype 3 reovirus containing σ3-Y354H, a mutation that reduces viral capsid stability, succumbed at a higher rate than those infected with wild-type virus. At early times after inoculation, σ3-Y354H virus reached higher titers than wild-type virus at several sites within the host. Animals inoculated perorally with a serotype 1 reassortant reovirus containing σ3-Y354H developed exaggerated myocarditis accompanied by elaboration of pro-inflammatory cytokines. Surprisingly, unchallenged littermates of mice infected with σ3-Y354H virus displayed higher titers in the intestine, heart, and brain than littermates of mice inoculated with wild-type virus. Together, these findings suggest that diminished capsid stability enhances reovirus replication, dissemination, lethality, and host-to-host spread, establishing a new virulence determinant for nonenveloped viruses.

  1. Genetic mapping of a highly variable norovirus GII.4 blockade epitope: potential role in escape from human herd immunity.

    Science.gov (United States)

    Debbink, Kari; Donaldson, Eric F; Lindesmith, Lisa C; Baric, Ralph S

    2012-01-01

    Noroviruses account for 96% of viral gastroenteritis cases worldwide, with GII.4 strains responsible >80% of norovirus outbreaks. Histo-blood group antigens (HBGAs) are norovirus binding ligands, and antigenic and preferential HBGA binding profiles vary over time as new GII.4 strains emerge. The capsid P2 subdomain facilitates HBGA binding, contains neutralizing antibody epitopes, and likely evolves in response to herd immunity. To identify amino acids regulating HBGA binding and antigenic differences over time, we created chimeric virus-like particles (VLPs) between the GII.4-1987 and GII.4-2006 strains by exchanging amino acids in putative epitopes and characterized their antigenic and HBGA binding profiles using anti-GII.4-1987 and -2006 mouse monoclonal antibodies (MAbs) and polyclonal sera, 1988 outbreak human sera, and synthetic HBGAs. The exchange of amino acids 393 to 395 between GII.4-1987 and GII.4-2006 resulted in altered synthetic HBGA binding compared to parental strains. Introduction of GII.4-1987 residues 294, 297 to 298, 368, and 372 (epitope A) into GII.4-2006 resulted in reactivity with three anti-GII.4-1987 MAbs and reduced reactivity with four anti-GII.4-2006 MAbs. The three anti-GII.4-1987 MAbs also blocked chimeric VLP-HBGA interaction, while an anti-GII.4-2006 blocking antibody did not, indicating that epitope A amino acids comprise a potential neutralizing epitope for GII.4-1987 and GII.4-2006. We also tested GII.4-1987-immunized mouse polyclonal sera and 1988 outbreak human sera for the ability to block chimeric VLP-HBGA interaction and found that epitope A amino acids contribute significantly to the GII.4-1987 blockade response. Our data provide insights that help explain the emergence of new GII.4 epidemic strains over time, may aid development of norovirus therapeutics, and may help predict the emergence of future epidemic strains.

  2. The Immune Epitope Database 2.0

    DEFF Research Database (Denmark)

    Hoof, Ilka; Vita, R; Zarebski, L;

    2010-01-01

    The Immune Epitope Database (IEDB, www.iedb.org) provides a catalog of experimentally characterized B and T cell epitopes, as well as data on Major Histocompatibility Complex (MHC) binding and MHC ligand elution experiments. The database represents the molecular structures recognized by adaptive...... immune receptors and the experimental contexts in which these molecules were determined to be immune epitopes. Epitopes recognized in humans, nonhuman primates, rodents, pigs, cats and all other tested species are included. Both positive and negative experimental results are captured. Over the course...

  3. Coarse-grained simulation reveals key features of HIV-1 capsid self-assembly

    Science.gov (United States)

    Grime, John M. A.; Dama, James F.; Ganser-Pornillos, Barbie K.; Woodward, Cora L.; Jensen, Grant J.; Yeager, Mark; Voth, Gregory A.

    2016-05-01

    The maturation of HIV-1 viral particles is essential for viral infectivity. During maturation, many copies of the capsid protein (CA) self-assemble into a capsid shell to enclose the viral RNA. The mechanistic details of the initiation and early stages of capsid assembly remain to be delineated. We present coarse-grained simulations of capsid assembly under various conditions, considering not only capsid lattice self-assembly but also the potential disassembly of capsid upon delivery to the cytoplasm of a target cell. The effects of CA concentration, molecular crowding, and the conformational variability of CA are described, with results indicating that capsid nucleation and growth is a multi-stage process requiring well-defined metastable intermediates. Generation of the mature capsid lattice is sensitive to local conditions, with relatively subtle changes in CA concentration and molecular crowding influencing self-assembly and the ensemble of structural morphologies.

  4. Polymorphism of DNA conformation inside the bacteriophage capsid.

    Science.gov (United States)

    Leforestier, Amélie

    2013-03-01

    Double-stranded DNA bacteriophage genomes are packaged into their icosahedral capsids at the highest densities known so far (about 50 % w:v). How the molecule is folded at such density and how its conformation changes upon ejection or packaging are fascinating questions still largely open. We review cryo-TEM analyses of DNA conformation inside partially filled capsids as a function of the physico-chemical environment (ions, osmotic pressure, temperature). We show that there exists a wide variety of DNA conformations. Strikingly, the different observed structures can be described by some of the different models proposed over the years for DNA organisation inside bacteriophage capsids: either spool-like structures with axial or concentric symmetries, or liquid crystalline structures characterised by a DNA homogeneous density. The relevance of these conformations for the understanding of DNA folding and unfolding upon ejection and packaging in vivo is discussed.

  5. RNA-binding region of Macrobrachium rosenbergii nodavirus capsid protein.

    Science.gov (United States)

    Goh, Zee Hong; Mohd, Nur Azmina Syakirin; Tan, Soon Guan; Bhassu, Subha; Tan, Wen Siang

    2014-09-01

    White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20-29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein.

  6. Quantum dot-induced viral capsid assembling in dissociation buffer

    Directory of Open Access Journals (Sweden)

    Gao D

    2013-06-01

    Full Text Available Ding Gao,1,2 Zhi-Ping Zhang,1 Feng Li,3 Dong Men,1 Jiao-Yu Deng,1 Hong-Ping Wei,1 Xian-En Zhang,1 Zong-Qiang Cui1 1State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 2Graduate University of Chinese Academy of Sciences, Beijing, 3Division of Nanobiomedicine and i-Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, People's Republic of China Abstract: Viruses encapsulating inorganic nanoparticles are a novel type of nanostructure with applications in biomedicine and biosensors. However, the encapsulation and assembly mechanisms of these hybridized virus-based nanoparticles (VNPs are still unknown. In this article, it was found that quantum dots (QDs can induce simian virus 40 (SV40 capsid assembly in dissociation buffer, where viral capsids should be disassembled. The analysis of the transmission electron microscope, dynamic light scattering, sucrose density gradient centrifugation, and cryo-electron microscopy single particle reconstruction experimental results showed that the SV40 major capsid protein 1 (VP1 can be assembled into ≈25 nm capsids in the dissociation buffer when QDs are present and that the QDs are encapsulated in the SV40 capsids. Moreover, it was determined that there is a strong affinity between QDs and the SV40 VP1 proteins (KD = 2.19E-10 M, which should play an important role in QD encapsulation in the SV40 viral capsids. This study provides a new understanding of the assembly mechanism of SV40 virus-based nanoparticles with QDs, which may help in the design and construction of other similar virus-based nanoparticles. Keywords: quantum dots, simian virus 40, self-assembly, encapsulation, virus-based nanoparticles

  7. Sphingomyelin induces structural alteration in canine parvovirus capsid

    OpenAIRE

    Pakkanen, Kirsi; Karttunen, Jenni; Virtanen, Salla; Vuento, Matti

    2008-01-01

    One of the essential steps in canine parvovirus (CPV) infection, the release from endosomal vesicles, is dominated by interactions between the virus capsid and the endosomal membranes. In this study, the effect of sphingomyelin and phosphatidyl serine on canine parvovirus capsid and on the phospholipase A2 (PLA2) activity of CPV VP1 unique N-terminus was analyzed. Accordingly, a significant (P ≤ 0.05) shift of tryptophan fluorescence emission peak was detected at pH 5.5 in the presen...

  8. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    Science.gov (United States)

    Andoh, Y.; Yoshii, N.; Yamada, A.; Fujimoto, K.; Kojima, H.; Mizutani, K.; Nakagawa, A.; Nomoto, A.; Okazaki, S.

    2014-10-01

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 106 all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

  9. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    Energy Technology Data Exchange (ETDEWEB)

    Andoh, Y.; Yoshii, N.; Yamada, A.; Kojima, H.; Mizutani, K.; Okazaki, S., E-mail: okazaki@apchem.nagoya-u.ac.jp [Department of Applied Chemistry, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603 (Japan); Fujimoto, K. [Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Nakagawa, A. [Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565-0871 (Japan); Nomoto, A. [Institute of Microbial Chemistry, Kamiosaki, Shinagawa-ku, Tokyo 141-0021 (Japan)

    2014-10-28

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 10{sup 6} all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

  10. Crystal Structure of the Human Astrovirus Capsid Protein

    Science.gov (United States)

    Toh, Yukimatsu; Harper, Justin; Dryden, Kelly A.; Yeager, Mark; Méndez, Ernesto

    2016-01-01

    ABSTRACT Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. HAstV is a nonenveloped virus with a T=3 capsid and a positive-sense RNA genome. The capsid protein (CP) of HAstV is synthesized as a 90-kDa precursor (VP90) that can be divided into three linear domains: a conserved N-terminal domain, a hypervariable domain, and an acidic C-terminal domain. Maturation of HAstV requires proteolytic processing of the astrovirus CP both inside and outside the host cell, resulting in the removal of the C-terminal domain and the breakdown of the rest of the CP into three predominant protein species with molecular masses of ∼34, 27/29, and 25/26 kDa, respectively. We have now solved the crystal structure of VP9071–415 (amino acids [aa] 71 to 415 of VP90) of human astrovirus serotype 8 at a 2.15-Å resolution. VP9071–415 encompasses the conserved N-terminal domain of VP90 but lacks the hypervariable domain, which forms the capsid surface spikes. The structure of VP9071–415 is comprised of two domains: an S domain, which adopts the typical jelly-roll β-barrel fold, and a P1 domain, which forms a squashed β-barrel consisting of six antiparallel β-strands similar to what was observed in the hepatitis E virus (HEV) capsid structure. Fitting of the VP9071–415 structure into the cryo-electron microscopy (EM) maps of HAstV produced an atomic model for a continuous, T=3 icosahedral capsid shell. Our pseudoatomic model of the human HAstV capsid shell provides valuable insights into intermolecular interactions required for capsid assembly and trypsin-mediated proteolytic maturation needed for virus infectivity. Such information has potential applications in the development of a virus-like particle (VLP) vaccine as well as small-molecule drugs targeting astrovirus assembly/maturation. IMPORTANCE Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. As a nonenveloped virus

  11. Induction of multi-epitope specific antibodies against HIV-1 by multi-epitope vaccines

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Some neutralizing antibodies against HIV-1 envelope proteins were highly effective to inhibit the infection of different strains in vitro, and existed in the infected individuals with very low levels. We suggested multi-epitope-vaccine as a new strategy to increase levels of neutralizing antibodies and the abilities against HIV mutation in vivo. Two candidate multi-epitope-vaccines induced antibodies with predefined multi-epitope-specificity in rhesus macaque. These antibodies recognized corresponding neutralizing epitopes on epitope-peptides, gp41 peptides, V3 loop peptide, rsgp41 and rgp120. Besides, three candidate epitope-vaccines in combination (another kind of multi-epitopevaccines) showed similar potency to induce predefined multiple immune responses in rabbits. These results suggest that multi-epitope-vaccines may be a new strategy to induce multi-antiviral activities against HIV-1 infection and mutafions.

  12. Sphingomyelin induces structural alteration in canine parvovirus capsid.

    Science.gov (United States)

    Pakkanen, Kirsi; Karttunen, Jenni; Virtanen, Salla; Vuento, Matti

    2008-03-01

    One of the essential steps in canine parvovirus (CPV) infection, the release from endosomal vesicles, is dominated by interactions between the virus capsid and the endosomal membranes. In this study, the effect of sphingomyelin and phosphatidyl serine on canine parvovirus capsid and on the phospholipase A(2) (PLA(2)) activity of CPV VP1 unique N-terminus was analyzed. Accordingly, a significant (P< or =0.05) shift of tryptophan fluorescence emission peak was detected at pH 5.5 in the presence of sphingomyelin, whereas at pH 7.4 a similar but minor shift was observed. This effect may relate to the exposure of VP1 N-terminus in acidic pH as well as to interactions between sphingomyelin and CPV. When the phenomenon was further characterized using circular dichroism spectroscopy, differences in CPV capsid CD spectra with and without sphingomyelin and phosphatidyl serine were detected, corresponding to data obtained with tryptophan fluorescence. However, when the enzymatic activity of CPV PLA(2) was tested in the presence of sphingomyelin, no significant effect in the function of the enzyme was detected. Thus, the structural changes observed with spectroscopic techniques appear not to manipulate the activity of CPV PLA(2), and may therefore implicate alternative interactions between CPV capsid and sphingomyelin.

  13. L2, the minor capsid protein of papillomavirus

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Joshua W. [Department of Pathology, The Johns Hopkins University, Baltimore, MD 21287 (United States); Roden, Richard B.S., E-mail: roden@jhmi.edu [Department of Pathology, The Johns Hopkins University, Baltimore, MD 21287 (United States); Department of Oncology, The Johns Hopkins University, Baltimore, MD 21287 (United States); Department of Gynecology and Obstetrics, The Johns Hopkins University, Baltimore, MD 21287 (United States)

    2013-10-15

    The capsid protein L2 plays major roles in both papillomavirus assembly and the infectious process. While L1 forms the majority of the capsid and can self-assemble into empty virus-like particles (VLPs), L2 is a minor capsid component and lacks the capacity to form VLPs. However, L2 co-assembles with L1 into VLPs, enhancing their assembly. L2 also facilitates encapsidation of the ∼8 kbp circular and nucleosome-bound viral genome during assembly of the non-enveloped T=7d virions in the nucleus of terminally differentiated epithelial cells, although, like L1, L2 is not detectably expressed in infected basal cells. With respect to infection, L2 is not required for particles to bind to and enter cells. However L2 must be cleaved by furin for endosome escape. L2 then travels with the viral genome to the nucleus, wherein it accumulates at ND-10 domains. Here, we provide an overview of the biology of L2. - Highlights: • L2 is the minor antigen of the non-enveloped T=7d icosahedral Papillomavirus capsid. • L2 is a nuclear protein that can traffic to ND-10 and facilitate genome encapsidation. • L2 is critical for infection and must be cleaved by furin. • L2 is a broadly protective vaccine antigen recognized by neutralizing antibodies.

  14. Role of electrostatic interactions in the assembly of empty spherical viral capsids

    CERN Document Server

    Siber, Antonio

    2007-01-01

    We examine the role of electrostatic interactions in the assembly of empty spherical viral capsids. The charges on the protein subunits that make the viral capsid mutually interact and are expected to yield electrostatic repulsion acting against the assembly of capsids. Thus, attractive protein-protein interactions of non-electrostatic origin must act to enable the capsid formation. We investigate whether the interplay of repulsive electrostatic and attractive interactions between the protein subunits can result in the formation of spherical viral capsids of a preferred radius. For this to be the case, we find that the attractive interactions must depend on the angle between the neighboring protein subunits (i.e. on the mean curvature of the viral capsid) so that a particular angle(s) is (are) preferred energywise. Our results for the electrostatic contributions to energetics of viral capsids nicely correlate with recent experimental determinations of the energetics of protein-protein contacts in Hepatitis B ...

  15. Ca2+-Regulated Photoproteins: Effective Immunoassay Reporters

    Directory of Open Access Journals (Sweden)

    Ludmila A. Frank

    2010-12-01

    Full Text Available Ca2+-regulated photoproteins of luminous marine coelenterates are of interest and a challenge for researchers as a unique bioluminescent system and as a promising analytical instrument for both in vivo and in vitro applications. The proteins are comprehensively studied as to biochemical properties, tertiary structures, bioluminescence mechanism, etc. This knowledge, along with available recombinant proteins serves the basis for development of unique bioluminescent detection systems that are “self-contained”, triggerable, fast, highly sensitive, and non-hazardous. In the paper, we focus on the use of photoproteins as reporters in binding assays based on immunological recognition element—bioluminescent immunoassay and hybridization immunoassay, their advantages and prospects.

  16. Epitope discovery with phylogenetic hidden Markov models.

    LENUS (Irish Health Repository)

    Lacerda, Miguel

    2010-05-01

    Existing methods for the prediction of immunologically active T-cell epitopes are based on the amino acid sequence or structure of pathogen proteins. Additional information regarding the locations of epitopes may be acquired by considering the evolution of viruses in hosts with different immune backgrounds. In particular, immune-dependent evolutionary patterns at sites within or near T-cell epitopes can be used to enhance epitope identification. We have developed a mutation-selection model of T-cell epitope evolution that allows the human leukocyte antigen (HLA) genotype of the host to influence the evolutionary process. This is one of the first examples of the incorporation of environmental parameters into a phylogenetic model and has many other potential applications where the selection pressures exerted on an organism can be related directly to environmental factors. We combine this novel evolutionary model with a hidden Markov model to identify contiguous amino acid positions that appear to evolve under immune pressure in the presence of specific host immune alleles and that therefore represent potential epitopes. This phylogenetic hidden Markov model provides a rigorous probabilistic framework that can be combined with sequence or structural information to improve epitope prediction. As a demonstration, we apply the model to a data set of HIV-1 protein-coding sequences and host HLA genotypes.

  17. Increasing Type 1 Poliovirus Capsid Stability by Thermal Selection

    Science.gov (United States)

    Adeyemi, Oluwapelumi O.; Nicol, Clare

    2016-01-01

    ABSTRACT Poliomyelitis is a highly infectious disease caused by poliovirus (PV). It can result in paralysis and may be fatal. Integrated global immunization programs using live-attenuated oral (OPV) and/or inactivated (IPV) PV vaccines have systematically reduced its spread and paved the way for eradication. Immunization will continue posteradication to ensure against reintroduction of the disease, but there are biosafety concerns for both OPV and IPV. They could be addressed by the production and use of virus-free virus-like particle (VLP) vaccines that mimic the “empty” capsids (ECs) normally produced in viral infection. Although ECs are antigenically indistinguishable from mature virus particles, they are less stable and readily convert into an alternative conformation unsuitable for vaccine purposes. Stabilized ECs, expressed recombinantly as VLPs, could be ideal candidate vaccines for a polio-free world. However, although genome-free PV ECs have been expressed as VLPs in a variety of systems, their inherent antigenic instability has proved a barrier to further development. In this study, we selected thermally stable ECs of type 1 PV (PV-1). The ECs are antigenically stable at temperatures above the conversion temperature of wild-type (wt) virions. We have identified mutations on the capsid surface and in internal networks that are responsible for EC stability. With reference to the capsid structure, we speculate on the roles of these residues in capsid stability and postulate that such stabilized VLPs could be used as novel vaccines. IMPORTANCE Poliomyelitis is a highly infectious disease caused by PV and is on the verge of eradication. There are biosafety concerns about reintroduction of the disease from current vaccines that require live virus for production. Recombinantly expressed virus-like particles (VLPs) could address these inherent problems. However, the genome-free capsids (ECs) of wt PV are unstable and readily change antigenicity to a form not

  18. High capsid-genome correlation facilitates creation of AAV libraries for directed evolution.

    Science.gov (United States)

    Nonnenmacher, Mathieu; van Bakel, Harm; Hajjar, Roger J; Weber, Thomas

    2015-04-01

    Directed evolution of adeno-associated virus (AAV) through successive rounds of phenotypic selection is a powerful method to isolate variants with improved properties from large libraries of capsid mutants. Importantly, AAV libraries used for directed evolution are based on the "natural" AAV genome organization where the capsid proteins are encoded in cis from replicating genomes. This is necessary to allow the recovery of the capsid DNA after each step of phenotypic selection. For directed evolution to be used successfully, it is essential to minimize the random mixing of capsomers and the encapsidation of nonmatching viral genomes during the production of the viral libraries. Here, we demonstrate that multiple AAV capsid variants expressed from Rep/Cap containing viral genomes result in near-homogeneous capsids that display an unexpectedly high capsid-DNA correlation. Next-generation sequencing of AAV progeny generated by bulk transfection of a semi-random peptide library showed a strong counter-selection of capsid variants encoding premature stop codons, which further supports a strong capsid-genome identity correlation. Overall, our observations demonstrate that production of "natural" AAVs results in low capsid mosaicism and high capsid-genome correlation. These unique properties allow the production of highly diverse AAV libraries in a one-step procedure with a minimal loss in phenotype-genotype correlation.

  19. Characterization and specificity of the linear epitope of the enterovirus 71 VP2 protein

    Directory of Open Access Journals (Sweden)

    Kiener Tanja K

    2012-02-01

    Full Text Available Abstract Background Enterovirus 71 (EV71 has emerged as a major causative agent of hand, foot and mouth disease in the Asia-Pacific region over the last decade. Hand, foot and mouth disease can be caused by different etiological agents from the enterovirus family, mainly EV71 and coxsackieviruses, which are genetically closely related. Nevertheless, infection with EV71 may occasionally lead to high fever, neurologic complications and the emergence of a rapidly fatal syndrome of pulmonary edema associated with brainstem encephalitis. The rapid progression and high mortality of severe EV71 infection has highlighted the need for EV71-specific diagnostic and therapeutic tools. Monoclonal antibodies are urgently needed to specifically detect EV71 antigens from patient specimens early in the infection process. Furthermore, the elucidation of viral epitopes will contribute to the development of targeted therapeutics and vaccines. Results We have identified the monoclonal antibody 7C7 from a screen of hybridoma cells derived from mice immunized with the EV71-B5 strain. The linear epitope of 7C7 was mapped to amino acids 142-146 (EDSHP of the VP2 capsid protein and was characterized in detail. Mutational analysis of the epitope showed that the aspartic acid to asparagine mutation of the EV71 subgenogroup A (BrCr strain did not interfere with antibody recognition. In contrast, the serine to threonine mutation at position 144 of VP2, present in recently emerged EV71-C4 China strains, abolished antigenicity. Mice injected with this virus strain did not produce any antibodies against the VP2 protein. Immunofluorescence and Western blotting confirmed that 7C7 specifically recognized EV71 subgenogroups and did not cross-react to Coxsackieviruses 4, 6, 10, and 16. 7C7 was successfully used as a detection antibody in an antigen-capture ELISA assay. Conclusions Detailed mapping showed that the VP2 protein of Enterovirus 71 contains a single, linear, non

  20. Useful scars: Physics of the capsids of archaeal viruses

    Science.gov (United States)

    Perotti, L. E.; Dharmavaram, S.; Klug, W. S.; Marian, J.; Rudnick, J.; Bruinsma, R. F.

    2016-07-01

    We propose a physical model for the capsids of tailed archaeal viruses as viscoelastic membranes under tension. The fluidity is generated by thermal motion of scarlike structures that are an intrinsic feature of the ground state of large particle arrays covering surfaces with nonzero Gauss curvature. The tension is generated by a combination of the osmotic pressure of the enclosed genome and an extension force generated by filamentous structure formation that drives the formation of the tails. In continuum theory, the capsid has the shape of a surface of constant mean curvature: an unduloid. Particle arrays covering unduloids are shown to exhibit pronounced subdiffusive and diffusive single-particle transport at temperatures that are well below the melting temperature of defect-free particle arrays on a surface with zero Gauss curvature.

  1. Refinement of herpesvirus B-capsid structure on parallel supercomputers.

    Science.gov (United States)

    Zhou, Z H; Chiu, W; Haskell, K; Spears, H; Jakana, J; Rixon, F J; Scott, L R

    1998-01-01

    Electron cryomicroscopy and icosahedral reconstruction are used to obtain the three-dimensional structure of the 1250-A-diameter herpesvirus B-capsid. The centers and orientations of particles in focal pairs of 400-kV, spot-scan micrographs are determined and iteratively refined by common-lines-based local and global refinement procedures. We describe the rationale behind choosing shared-memory multiprocessor computers for executing the global refinement, which is the most computationally intensive step in the reconstruction procedure. This refinement has been implemented on three different shared-memory supercomputers. The speedup and efficiency are evaluated by using test data sets with different numbers of particles and processors. Using this parallel refinement program, we refine the herpesvirus B-capsid from 355-particle images to 13-A resolution. The map shows new structural features and interactions of the protein subunits in the three distinct morphological units: penton, hexon, and triplex of this T = 16 icosahedral particle.

  2. Automatic Generation of Validated Specific Epitope Sets

    Directory of Open Access Journals (Sweden)

    Sebastian Carrasco Pro

    2015-01-01

    Full Text Available Accurate measurement of B and T cell responses is a valuable tool to study autoimmunity, allergies, immunity to pathogens, and host-pathogen interactions and assist in the design and evaluation of T cell vaccines and immunotherapies. In this context, it is desirable to elucidate a method to select validated reference sets of epitopes to allow detection of T and B cells. However, the ever-growing information contained in the Immune Epitope Database (IEDB and the differences in quality and subjects studied between epitope assays make this task complicated. In this study, we develop a novel method to automatically select reference epitope sets according to a categorization system employed by the IEDB. From the sets generated, three epitope sets (EBV, mycobacteria and dengue were experimentally validated by detection of T cell reactivity ex vivo from human donors. Furthermore, a web application that will potentially be implemented in the IEDB was created to allow users the capacity to generate customized epitope sets.

  3. Antigen epitope of Helicobacter pylorivacuolating cytotoxin A

    Institute of Scientific and Technical Information of China (English)

    Xiu-Li Liu; Shu-Qin Li; Chun-Jie Liu; Hao-Xia Tao; Zhao-Shan Zhang

    2004-01-01

    AIM: To construct and select antigen epitopes of vacuolating cytotoxin A (VacA) for nontoxic VacA vaccine against Helicobacter pylori (H pylori) infection.METHODS: Eleven VacA epitopes were predicted according to VacA antigenic bioinformatics. Three candidates of VacA epitope were constructed through different combined epitopes. The candidate was linked with E. coli heat-labile enterotoxin B (LTB) by a linker of 7 amino acids, and cloned into plasmid pQE-60 in which fusion LTB-VacA epitope was efficiently expressed. To test the antigencity of the candidate, 6 BALB/c mice were treated with the fusion LTB-VacA epitope through intraperitoneal injection. To explore the ability of inhibiting the toxicity of VacA, cantiserum against the candidate was used to counteract VacA that induced HeLa cells to produce cell vacuoles in vitro.RESULTS: Serum IgG against the candidate was induced in the BALB/c mice. In vitro, the three antisera against the candidate efficiently counteracted the toxicity of VacA, and decreased the number of cell vacuoles by 14.17%, 20.20%and 30.41% respectively.CONCLUSION: Two of the three candidates, LZ-VacA1and LZ-VacA2, can be used to further study the mechanism of vacuolating toxicity of VacA, and to construct nontoxic VacA vaccine against H pylori infection.

  4. Immunization with Hexon modified adenoviral vectors integrated with gp83 epitope provides protection against Trypanosoma cruzi infection.

    Directory of Open Access Journals (Sweden)

    Anitra L Farrow

    2014-08-01

    Full Text Available Trypanosoma cruzi is the causative agent of Chagas disease. Chagas disease is an endemic infection that affects over 8 million people throughout Latin America and now has become a global challenge. The current pharmacological treatment of patients is unsuccessful in most cases, highly toxic, and no vaccines are available. The results of inadequate treatment could lead to heart failure resulting in death. Therefore, a vaccine that elicits neutralizing antibodies mediated by cell-mediated immune responses and protection against Chagas disease is necessary.The "antigen capsid-incorporation" strategy is based upon the display of the T. cruzi epitope as an integral component of the adenovirus' capsid rather than an encoded transgene. This strategy is predicted to induce a robust humoral immune response to the presented antigen, similar to the response provoked by native Ad capsid proteins. The antigen chosen was T. cruzi gp83, a ligand that is used by T. cruzi to attach to host cells to initiate infection. The gp83 epitope, recognized by the neutralizing MAb 4A4, along with His6 were incorporated into the Ad serotype 5 (Ad5 vector to generate the vector Ad5-HVR1-gp83-18 (Ad5-gp83. This vector was evaluated by molecular and immunological analyses. Vectors were injected to elicit immune responses against gp83 in mouse models. Our findings indicate that mice immunized with the vector Ad5-gp83 and challenged with a lethal dose of T. cruzi trypomastigotes confer strong immunoprotection with significant reduction in parasitemia levels, increased survival rate and induction of neutralizing antibodies.This data demonstrates that immunization with adenovirus containing capsid-incorporated T. cruzi antigen elicits a significant anti-gp83-specific response in two different mouse models, and protection against T. cruzi infection by eliciting neutralizing antibodies mediated by cell-mediated immune responses, as evidenced by the production of several Ig isotypes

  5. Assembly of recombinant Israeli Acute Paralysis Virus capsids.

    Directory of Open Access Journals (Sweden)

    Junyuan Ren

    Full Text Available The dicistrovirus Israeli Acute Paralysis Virus (IAPV has been implicated in the worldwide decline of honey bees. Studies of IAPV and many other bee viruses in pure culture are restricted by available isolates and permissive cell culture. Here we show that coupling the IAPV major structural precursor protein ORF2 to its cognate 3C-like processing enzyme results in processing of the precursor to the individual structural proteins in a number of insect cell lines following expression by a recombinant baculovirus. The efficiency of expression is influenced by the level of IAPV 3C protein and moderation of its activity is required for optimal expression. The mature IAPV structural proteins assembled into empty capsids that migrated as particles on sucrose velocity gradients and showed typical dicistrovirus like morphology when examined by electron microscopy. Monoclonal antibodies raised to recombinant capsids were configured into a diagnostic test specific for the presence of IAPV. Recombinant capsids for each of the many bee viruses within the picornavirus family may provide virus specific reagents for the on-going investigation of the causes of honeybee loss.

  6. Type II collagen C2C epitope in human synovial fluid and serum after knee injury

    DEFF Research Database (Denmark)

    Kumahashi, N; Swärd, P; Larsson, S;

    2015-01-01

    PURPOSE: Investigate in a cross-sectional study time-dependent changes of synovial fluid type II collagen epitope C2C concentrations after knee injury and correlate to other joint injury biomarkers. METHODS: Synovial fluid samples were aspirated between 0 days and 7 years after injury (n = 235......). Serum was collected from 71 of the knee injured patients. Synovial fluid from 8 knee-healthy subjects was used as reference. C2C was quantified by immunoassay and structural injury was determined from magnetic resonance images (MRI) of the injured knee acquired 1-38 days after injury (n = 98...... = 0.403, P type II collagen (r = 0.444, P = 0.003), ARGS-aggrecan (r = 0.337, P

  7. Intein-mediated peptide arrays for epitope mapping and kinase/phosphatase assays.

    Science.gov (United States)

    Xu, Ming-Qun; Ghosh, Inca; Kochinyan, Samvel; Sun, Luo

    2007-01-01

    Synthetic peptides are widely used for production and analysis of antibodies as well as in the study of protein modification enzymes. To circumvent the technical challenges of the existing techniques regarding peptide quantization and normalization, a new method of producing peptide arrays has been developed. This approach utilizes intein-mediated protein ligation that involves linkage of a carrier protein possessing a reactive carboxyl-terminal thioester to a peptide with an amino-terminal cysteine through a native peptide bond. Ligated protein substrates or enzyme-treated samples are arrayed on nitrocellulose membranes with a standard dot-blot apparatus and analyzed by immunoassay. This technique has improved sensitivity and reproducibility, and is suitable for various peptide-based applications. In this report, several experimental procedures including epitope mapping and the study of protein modifications were described.

  8. Crystallization and X-ray analysis of the T = 4 particle of hepatitis B capsid protein with an N-terminal extension

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Wen Siang [Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); McNae, Iain W.; Ho, Kok Lian; Walkinshaw, Malcolm D., E-mail: m.walkinshaw@ed.ac.uk [Institute of Structural and Molecular Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, King’s Buildings, Mayfield Road, Edinburgh EH9 3JR,Scotland (United Kingdom); Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia)

    2007-08-01

    Hepatitis B virus capsids have significant potential as carriers for immunogenic peptides. The crystal structure of the T = 4 particle of hepatitis B core protein containing an N-terminal extension reveals that the fusion peptide is exposed on the exterior of the particle. Hepatitis B core (HBc) particles have been extensively exploited as carriers for foreign immunological epitopes in the development of multicomponent vaccines and diagnostic reagents. Crystals of the T = 4 HBc particle were grown in PEG 20 000, ammonium sulfate and various types of alcohols. A temperature jump from 277 or 283 to 290 K was found to enhance crystal growth. A crystal grown using MPD as a cryoprotectant diffracted X-rays to 7.7 Å resolution and data were collected to 99.6% completeness at 8.9 Å. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 352.3, b = 465.5, c = 645.0 Å. The electron-density map reveals a protrusion that is consistent with the N-terminus extending out from the surface of the capsid. The structure presented here supports the idea that N-terminal insertions can be exploited in the development of diagnostic reagents, multicomponent vaccines and delivery vehicles into mammalian cells.

  9. Exploiting the yeast L-A viral capsid for the in vivo assembly of chimeric VLPs as platform in vaccine development and foreign protein expression.

    Directory of Open Access Journals (Sweden)

    Frank Powilleit

    Full Text Available A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8(+ memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i a heterologous model protein (GFP, (ii a per se toxic protein (K28 alpha-subunit, and (iii a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A. Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.

  10. Label free checkerboard assay to determine overlapping epitopes of Ebola virus VP-40 antibodies using surface plasmon resonance.

    Science.gov (United States)

    Anderson, George P; Liu, Jinny L; Zabetakis, Dan; Legler, Patricia M; Goldman, Ellen R

    2017-03-01

    Immunoassay formats, in which antibodies provide sensitivity and specificity, are often utilized to provide rapid and simple diagnostic tests. Surface plasmon resonance is frequently used to evaluate the suitability of antibodies by determining binding kinetics to agents or surrogate antigens. We used SPR to evaluate a number of commercial monoclonal antibodies as well as single domain antibodies produced in-house. All the antibodies targeted the Ebola virus viral protein 40 (VP40). We determined the ability of each antibody to bind to immobilized VP40, and ensured they did not bind Ebola glycoprotein or the nucleoprotein. A subset of the monoclonal antibodies was immobilized to characterize antigen capture in solution. It can be advantageous to utilize antibodies that recognize distinct epitopes when choosing reagents for detection and diagnostic assays. We determined the uniqueness of the epitope recognized by the anti-VP40 antibodies using a checkerboard format that exploits the 6×6 array of interactions monitored by the Bio-Rad ProteOn XPR36 SPR instrument. The results demonstrate the utility of surface plasmon resonance to characterize monoclonal and recombinant antibodies. Additionally, the analysis presented here enabled the identification of pairs of anti-VP40 antibodies which could potentially be utilized in sandwich type immunoassays for the detection of Ebola virus.

  11. Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly

    Science.gov (United States)

    Rodgers, R. E.; Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents.

  12. The Pseudorabies Virus VP1/2 Tegument Protein Is Required for Intracellular Capsid Transport†

    OpenAIRE

    Luxton, G.W. Gant; Lee, Joy I-Hsuan; Haverlock-Moyns, Sarah; Schober, Joseph Martin; Smith, Gregory Allan

    2006-01-01

    Transport of capsids in cells is critical to alphaherpesvirus infection and pathogenesis; however, viral factors required for transport have yet to be identified. Here we provide a detailed examination of capsid dynamics during the egress phase of infection in Vero cells infected with pseudorabies virus. We demonstrate that the VP1/2 tegument protein is required for processive microtubule-based transport of capsids in the cytoplasm. A second tegument protein that binds to VP1/2, UL37, was nec...

  13. Three-dimensional structure determination of capsid of Aedes albopicus C6/36 cell densovirus

    Institute of Scientific and Technical Information of China (English)

    CHENG Lingpeng; CHEN Senxiong; Jenifer M.Brannan; Joanita Jakana; ZHANG Qinfen; Z.H.Zhou; ZHANG Jingqiang

    2004-01-01

    The three-dimensional structure of capsid of Aedes albopictus C6/36 densovirus was determined to 14-(A) resolution by electron cryomicroscopy and computer reconstruction. The triangulation number of the capsid is 1. There are 12 holes in each triangular face and a spike on each 5-fold vertex. The validity of the capsid and nucleic acid densities in the reconstructions was discussed.

  14. A minimal representation of the self-assembly of virus capsids

    CERN Document Server

    Llorente, J M Gomez; Breton, J

    2013-01-01

    Viruses are biological nanosystems with a capsid of protein-made capsomer units that encloses and protects the genetic material responsible for their replication. Here we show how the geometrical constraints of the capsomer-capsomer interaction in icosahedral capsids fix the form of the shortest and universal truncated multipolar expansion of the two-body interaction between capsomers. The structures of many of the icosahedral and related virus capsids are located as single lowest energy states of this potential energy surface. Our approach unveils relevant features of the natural design of the capsids and can be of interest in fields of nanoscience and nanotechnology where similar hollow convex structures are relevant.

  15. Primate TRIM5 proteins form hexagonal nets on HIV-1 capsids

    Science.gov (United States)

    Li, Yen-Li; Chandrasekaran, Viswanathan; Carter, Stephen D; Woodward, Cora L; Christensen, Devin E; Dryden, Kelly A; Pornillos, Owen; Yeager, Mark; Ganser-Pornillos, Barbie K; Jensen, Grant J; Sundquist, Wesley I

    2016-01-01

    TRIM5 proteins are restriction factors that block retroviral infections by binding viral capsids and preventing reverse transcription. Capsid recognition is mediated by C-terminal domains on TRIM5α (SPRY) or TRIMCyp (cyclophilin A), which interact weakly with capsids. Efficient capsid recognition also requires the conserved N-terminal tripartite motifs (TRIM), which mediate oligomerization and create avidity effects. To characterize how TRIM5 proteins recognize viral capsids, we developed methods for isolating native recombinant TRIM5 proteins and purifying stable HIV-1 capsids. Biochemical and EM analyses revealed that TRIM5 proteins assembled into hexagonal nets, both alone and on capsid surfaces. These nets comprised open hexameric rings, with the SPRY domains centered on the edges and the B-box and RING domains at the vertices. Thus, the principles of hexagonal TRIM5 assembly and capsid pattern recognition are conserved across primates, allowing TRIM5 assemblies to maintain the conformational plasticity necessary to recognize divergent and pleomorphic retroviral capsids. DOI: http://dx.doi.org/10.7554/eLife.16269.001 PMID:27253068

  16. Comparison of a new serum topiramate immunoassay to fluorescence polarization immunoassay.

    Science.gov (United States)

    Snozek, Christine L H; Rollins, Lisa A; Peterson, Paul W; Langman, Loralie J

    2010-02-01

    Topiramate is a newer anticonvulsant used to treat epilepsy, migraines, bipolar disorder, posttraumatic stress, and other conditions. Serum topiramate concentrations are measured to determine optimal levels, address therapeutic failure or drug-drug interactions, and assess compliance. Two high-throughput assays for serum topiramate measurement were compared: the Seradyn fluorescence polarization immunoassay (FPIA) on an Abbott TDx/FLx instrument and a new immunoassay from ARK Diagnostics performed on an Olympus AU680 automated analyzer. Precision, linearity, limit of quantitation, carryover, spike recovery, and endogenous interferences were found to be acceptable for the ARK assay. These studies were complemented by comparison of 120 patient samples analyzed using both methods. The ARK immunoassay performed comparably to FPIA with minimal difference in serum topiramate concentrations within the therapeutic range (2.0-20 microg/mL). A slight systematic discordance was observed at higher concentrations (greater than 30 microg/mL) with ARK immunoassay results being on average 6% higher than FPIA. Thus, the ARK immunoassay appears to provide acceptable analytical performance and comparability to FPIA; furthermore, the assay is compatible with high-throughput autoanalyzers.

  17. Application of a Newly Developed High-Sensitivity HBsAg Chemiluminescent Enzyme Immunoassay for Hepatitis B Patients with HBsAg Seroclearance

    OpenAIRE

    2013-01-01

    We modified and automated a highly sensitive chemiluminescent enzyme immunoassay (CLEIA) for surface antigen (HBsAg) detection using a combination of monoclonal antibodies, each for a specific epitope of HBsAg, and by improving an earlier conjugation technique. Of 471 hepatitis B virus (HBV) carriers seen in our hospital between 2009 and 2012, 26 were HBsAg seronegative as determined by the Abbott Architect assay. The Lumipulse HBsAg-HQ assay was used to recheck those 26 patients who demonstr...

  18. Adeno-associated virus type 2 (AAV2) capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 capsid in vivo.

    Science.gov (United States)

    Li, Chengwen; Hirsch, Matthew; Asokan, Aravind; Zeithaml, Brian; Ma, Hong; Kafri, Tal; Samulski, R Jude

    2007-07-01

    A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kill a cell line stable for capsid expression in vitro and also in a mouse tumor xenograft model in vivo. Parent colon carcinoma (CT26) cells transduced with a large amount of AAV2 vectors in vitro were also destroyed by these CTLs. To determine the effect of CTLs on the elimination of target cells transduced by AAV2 vectors in vivo, we carried out adoptive transfer experiments. CTLs eliminated liver cells with endogenous AAV2 capsid expression but not liver cells transduced by AAV2 vectors, regardless of the reporter genes. Similar results were obtained for rAAV2 transduction in muscle. Our data strongly suggest that AAV vector-transduced cells are rarely eliminated by AAV2 capsid-specific CTLs in vivo, even though the AAV capsid can induce a CTL response. In conclusion, AAV capsid-specific CTLs do not appear to play a role in elimination of rAAV-transduced cells in a mouse model. In addition, our data suggest that the mouse model may not mimic the immune response noted in humans and additional modification to AAV vectors may be required for further study in order to elicit a similar cellular immune response.

  19. Adeno-Associated Virus Type 2 (AAV2) Capsid-Specific Cytotoxic T Lymphocytes Eliminate Only Vector-Transduced Cells Coexpressing the AAV2 Capsid In Vivo▿

    Science.gov (United States)

    Li, Chengwen; Hirsch, Matthew; Asokan, Aravind; Zeithaml, Brian; Ma, Hong; Kafri, Tal; Samulski, R. Jude

    2007-01-01

    A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kill a cell line stable for capsid expression in vitro and also in a mouse tumor xenograft model in vivo. Parent colon carcinoma (CT26) cells transduced with a large amount of AAV2 vectors in vitro were also destroyed by these CTLs. To determine the effect of CTLs on the elimination of target cells transduced by AAV2 vectors in vivo, we carried out adoptive transfer experiments. CTLs eliminated liver cells with endogenous AAV2 capsid expression but not liver cells transduced by AAV2 vectors, regardless of the reporter genes. Similar results were obtained for rAAV2 transduction in muscle. Our data strongly suggest that AAV vector-transduced cells are rarely eliminated by AAV2 capsid-specific CTLs in vivo, even though the AAV capsid can induce a CTL response. In conclusion, AAV capsid-specific CTLs do not appear to play a role in elimination of rAAV-transduced cells in a mouse model. In addition, our data suggest that the mouse model may not mimic the immune response noted in humans and additional modification to AAV vectors may be required for further study in order to elicit a similar cellular immune response. PMID:17475652

  20. Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes

    Directory of Open Access Journals (Sweden)

    Lin Na-Sheng

    2007-09-01

    Full Text Available Abstract Background Plant viruses can be employed as versatile vectors for the production of vaccines by expressing immunogenic epitopes on the surface of chimeric viral particles. Although several viruses, including tobacco mosaic virus, potato virus X and cowpea mosaic virus, have been developed as vectors, we aimed to develop a new viral vaccine delivery system, a bamboo mosaic virus (BaMV, that would carry larger transgene loads, and generate better immunity in the target animals with fewer adverse environmental effects. Methods We engineered the BaMV as a vaccine vector expressing the antigenic epitope(s of the capsid protein VP1 of foot-and-mouth disease virus (FMDV. The recombinant BaMV plasmid (pBVP1 was constructed by replacing DNA encoding the 35 N-terminal amino acid residues of the BaMV coat protein with that encoding 37 amino acid residues (T128-N164 of FMDV VP1. Results The pBVP1 was able to infect host plants and to generate a chimeric virion BVP1 expressing VP1 epitopes in its coat protein. Inoculation of swine with BVP1 virions resulted in the production of anti-FMDV neutralizing antibodies. Real-time PCR analysis of peripheral blood mononuclear cells from the BVP1-immunized swine revealed that they produced VP1-specific IFN-γ. Furthermore, all BVP1-immunized swine were protected against FMDV challenge. Conclusion Chimeric BaMV virions that express partial sequence of FMDV VP1 can effectively induce not only humoral and cell-mediated immune responses but also full protection against FMDV in target animals. This BaMV-based vector technology may be applied to other vaccines that require correct expression of antigens on chimeric viral particles.

  1. Intramolecular epitope spreading in Heymann nephritis.

    Science.gov (United States)

    Shah, Pallavi; Tramontano, Alfonso; Makker, Sudesh P

    2007-12-01

    Immunization with megalin induces active Heymann nephritis, which reproduces features of human idiopathic membranous glomerulonephritis. Megalin is a complex immunological target with four discrete ligand-binding domains (LBDs) that may contain epitopes to which pathogenic autoantibodies are directed. Recently, a 236-residue N-terminal fragment, termed "L6," that spans the first LBD was shown to induce autoantibodies and severe disease. We used this model to examine epitope-specific contributions to pathogenesis. Sera obtained from rats 4 weeks after immunization with L6 demonstrated reactivity only with the L6 fragment on Western blot, whereas sera obtained after 8 weeks demonstrated reactivity with all four recombinant fragments of interest (L6 and LBDs II, III, and IV). We demonstrated that the L6 immunogen does not contain the epitopes responsible for the reactivity to the LBD fragments. Therefore, the appearance of antibodies directed at LBD fragments several weeks after the primary immune response suggests intramolecular epitope spreading. In vivo, we observed a temporal association between increased proteinuria and the appearance of antibodies to LBD fragments. These data implicate B cell epitope spreading in antibody-mediated pathogenesis of active Heymann nephritis, a model that should prove valuable for further study of autoimmune dysregulation.

  2. Equivalent T cell epitope promiscuity in ecologically diverse human pathogens.

    Directory of Open Access Journals (Sweden)

    Kirsten E Wiens

    Full Text Available BACKGROUND: The HLA (human leukocyte antigen molecules that present pathogen-derived epitopes to T cells are highly diverse. Correspondingly, many pathogens such as HIV evolve epitope variants in order to evade immune recognition. In contrast, another persistent human pathogen, Mycobacterium tuberculosis, has highly conserved epitope sequences. This raises the question whether there is also a difference in the ability of these pathogens' epitopes to bind diverse HLA alleles, referred to as an epitope's binding promiscuity. To address this question, we compared the in silico HLA binding promiscuity of T cell epitopes from pathogens with distinct infection strategies and outcomes of human exposure. METHODS: We used computer algorithms to predict the binding affinity of experimentally-verified microbial epitope peptides to diverse HLA-DR, HLA-A and HLA-B alleles. We then analyzed binding promiscuity of epitopes derived from HIV and M. tuberculosis. We also analyzed promiscuity of epitopes from Streptococcus pyogenes, which is known to exhibit epitope diversity, and epitopes of Bacillus anthracis and Clostridium tetani toxins, as these bacteria do not depend on human hosts for their survival or replication, and their toxin antigens are highly immunogenic human vaccines. RESULTS: We found that B. anthracis and C. tetani epitopes were the most promiscuous of the group that we analyzed. However, there was no consistent difference or trend in promiscuity in epitopes contained in HIV, M. tuberculosis, and S. pyogenes. CONCLUSIONS: Our results show that human pathogens with distinct immune evasion strategies and epitope diversities exhibit equivalent levels of T cell epitope promiscuity. These results indicate that differences in epitope promiscuity do not account for the observed differences in epitope variation and conservation.

  3. Antigenic heterogeneity of capsid protein VP1 in foot-and-mouth disease virus (FMDV serotype Asia1

    Directory of Open Access Journals (Sweden)

    Alam SM

    2013-08-01

    Full Text Available SM Sabbir Alam,1 Ruhul Amin,1 Mohammed Ziaur Rahman,2 M Anwar Hossain,1 Munawar Sultana11Department of Microbiology, University of Dhaka, Dhaka, Bangladesh; 2International Centre for Diarrhoeal Disease Research, Dhaka, BangladeshAbstract: Foot and mouth disease virus (FMDV, with its seven serotypes, is a highly contagious virus infecting mainly cloven-hoofed animals. The serotype Asia1 occurs mainly in Asian regions. An in-silico approach was taken to reveal the antigenic heterogeneities within the capsid protein VP1 of Asia1. A total of 47 VP1 sequences of Asia1 isolates from different countries of South Asian regions were selected, retrieved from database, and were aligned. The structure of VP1 protein was modeled using a homology modeling approach. Several antigenic sites were identified and mapped onto the three-dimensional protein structure. Variations at these antigenic sites were analyzed by calculating the protein variability index and finding mutation combinations. The data suggested that vaccine escape mutants have derived from only few mutations at several antigenic sites. Five antigenic peptides have been identified as the least variable epitopes, with just fewer amino acid substitutions. Only a limited number of serotype Asia1 antigenic variants were found to be circulated within the South Asian region. This emphasizes a possibility of formulating synthetic vaccines for controlling foot-and-mouth disease by Asia1 serotypes.Keywords: protein modeling, antigenic sites, sequence variation

  4. Development of a Nanobody-Based Lateral Flow Immunoassay for Detection of Human Norovirus

    Science.gov (United States)

    Doerflinger, Sylvie Y.; Tabatabai, Julia; Schnitzler, Paul; Farah, Carlo; Rameil, Steffen; Sander, Peter; Koromyslova, Anna

    2016-01-01

    ABSTRACT Human noroviruses are the dominant cause of outbreaks of acute gastroenteritis. These viruses are usually detected by molecular methods, including reverse transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Human noroviruses are genetically and antigenically diverse, with two main genogroups that are further subdivided into over 40 different genotypes. During the past decade, genogroup 2 genotype 4 (GII.4) has dominated in most countries, but recently, viruses belonging to GII.17 have increased in prevalence in a number of countries. A number of commercially available ELISAs and lateral flow immunoassays were found to have lower sensitivities to the GII.17 viruses, indicating that the antibodies used in these methods may not have a high level of cross-reactivity. In this study, we developed a rapid Nanobody-based lateral flow immunoassay (Nano-immunochromatography [Nano-IC]) for the detection of human norovirus in clinical specimens. The Nano-IC assay detected virions from two GII.4 norovirus clusters, which included the current dominant strain and a novel variant strain. The Nano-IC method had a sensitivity of 80% and specificity of 86% for outbreak specimens. Norovirus virus-like particles (VLPs) representing four genotypes (GII.4, GII.10, GII.12, and GII.17) could be detected by this method, demonstrating the potential in clinical screening. However, further modifications to the Nano-IC method are needed in order to improve this sensitivity, which may be achieved by the addition of other broadly reactive Nanobodies to the system. IMPORTANCE We previously identified a Nanobody (termed Nano-85) that bound to a highly conserved region on the norovirus capsid. In this study, the Nanobody was biotinylated and gold conjugated for a lateral flow immunoassay (termed Nano-IC). We showed that the Nano-IC assay was capable of detecting at least four antigenically distinct GII genotypes, including the newly emerging GII.17. In the clinical

  5. Fast Diagnosis of Gonorrhea Witth Enhanced Luminescence Enzyme Immunoassay

    Institute of Scientific and Technical Information of China (English)

    ZHENG Heyi(郑和义); CAO Jingjiang(曹经江); SHAO Yanglin(邵燕玲)

    2002-01-01

    Objective:To evaluate the value of enhanced luminescence enzyme immunoassay in the diagnosis of Neisseria gonorrhea(NG) infection.Methods: Anti-catalase antibody for Neisseria gonorrheae combined with enhanced luminescence enzyme immunoassay were used to test for N. Gonorrhea.Results: A minimum of 1x104/CFU of GC in genital tract secretions or urine could be detected with the technique of luminescence enzyme immunoassay.Conclusion : The enhanced luninescence enzyme immunoassay has the advantage of high sensitivity and specificity for diagnosing NG from genitourinary tract secretion and urine.

  6. Production, purification, and capsid stability of rhinovirus C types.

    Science.gov (United States)

    Griggs, Theodor F; Bochkov, Yury A; Nakagome, Kazuyuki; Palmenberg, Ann C; Gern, James E

    2015-06-01

    The rhinovirus C (RV-C) were discovered in 2006 and these agents are an important cause of respiratory morbidity. Little is known about their biology. RV-C15 (C15) can be produced by transfection of recombinant viral RNA into cells and subsequent purification over a 30% sucrose cushion, even though yields and infectivity of other RV-C genotypes with this protocol are low. The goal of this study was to determine whether poor RV-C yields were due to capsid instability, and moreover, to develop a robust protocol suitable for the purification of many RV-C types. Capsid stability assays indicated that virions of RV-C41 (refractory to purification) have similar tolerance for osmotic and temperature stress as RV-A16 (purified readily), although C41 is more sensitive to low pH. Modification to the purification protocol by removing detergent increased the yield of RV-C. Addition of nonfat dry milk to the sucrose cushion increased the virus yield but sacrificed purity of the viral suspension. Analysis of virus distribution following centrifugation indicated that the majority of detectable viral RNA (vRNA) was found in pellets refractory to resuspension. Reduction of the centrifugal force with commiserate increase in spin-time improved the recovery of RV-C for both C41 and C2. Transfection of primary lung fibroblasts (WisL cells) followed by the modified purification protocol further improved yields of infectious C41 and C2. Described herein is a higher yield purification protocol suitable for RV-C types refractory to the standard purification procedure. The findings suggest that aggregation-adhesion problems rather than capsid instability influence RV-C yield during purification.

  7. Producing peptide arrays for epitope mapping by intein-mediated protein ligation.

    Science.gov (United States)

    Sun, Luo; Rush, John; Ghosh, Inca; Maunus, Jeremy R; Xu, Ming-Qun

    2004-09-01

    Peptide arrays are increasingly used to define antibody epitopes and substrate specificities of protein kinases. Their use is hampered, however, by ineffective and variable binding efficiency of peptides, which often results in low sensitivity and inconsistent results. To overcome these limitations, we have developed a novel method for making arrays of synthetic peptides on various membranes after ligating the peptide substrates to an intein-generated carrier protein. We have conducted screening for optimal carrier proteins by immunoreactivity and direct assessment of binding using a peptide derivatized at a lysine sidechain with fluorescein, CDPEK(fluorescein)DS. Ligation of a synthetic peptide antigen to a carrier protein, HhaI methylase, resulted in an improved retention of peptides and an increased sensitivity of up to 10(4)-fold in immunoassay- and epitope-scanning experiments. Denaturing the ligation products with 2% sodium dodecyl sulfate (SDS) or an organic solvent (20% methanol) prior to arraying did not significantly affect the immunoreactivity of the HhaI methylase-peptide product. Because the carrier protein dominates the binding of ligation products and contains one peptide reactive site, the amount of peptide arrayed onto the membranes can be effectively normalized. This technique was utilized in the alanine scanning of hemagglutinin (HA) antigen using two monoclonal antibodies, resulting in distinguishing the different antigen epitope profiles. Furthermore, we show that this method can be used to characterize the antibodies that recognize phosphorylated peptides. This novel approach allows for synthetic peptides to be uniformly arrayed onto membranes, compatible with a variety of applications.

  8. Epitope Mapping of Rhi o 1 and Generation of a Hypoallergenic Variant: A CANDIDATE MOLECULE FOR FUNGAL ALLERGY VACCINES.

    Science.gov (United States)

    Sircar, Gaurab; Jana, Kuladip; Dasgupta, Angira; Saha, Sudipto; Gupta Bhattacharya, Swati

    2016-08-19

    Efficacy of allergen-specific immunotherapy is often severely impaired by detrimental IgE-mediated side effects of native allergen during vaccination. Here, we present the molecular determinants for IgE recognition of Rhi o 1 and eventually converting the allergen into a hypoallergenic immunogen to restrain health hazards during desensitization. Rhi o 1 is a respiratory fungal allergen. Despite having cross-reactivity with cockroach allergen, we observed that non-cross-reactive epitope predominantly determined IgE binding to Rhi o 1. Denaturation and refolding behavior of the allergen confirmed that its IgE reactivity was not essentially conformation-dependent. A combinatorial approach consisting of computational prediction and a peptide-based immunoassay identified two peptides ((44)TGEYLTQKYFNSQRNN and (311)GAEKNWAGQYVVDCNK) of Rhi o 1 that frequently reacted with IgE antibodies of sensitized patients. Interestingly, these peptides did not represent purely linear IgE epitopes but were presented in a conformational manner by forming a spatially clustered surface-exposed epitope conferring optimal IgE-binding capacity to the folded allergen. Site-directed alanine substitution identified four residues of the IgE epitope that were crucial for antibody binding. A multiple mutant (T49A/Y52A/K314A/W316A) showing 100-fold lower IgE binding and reduced allergenic activity was generated. The TYKW mutant retained T-cell epitopes, as evident from its lymphoproliferative capacity but down-regulated pro-allergic IL-5 secretion. The TYKW mutant induced enhanced focusing of blocking IgG antibodies specifically toward the IgE epitope of the allergen. Anti-TYKW mutant polyclonal IgG antibodies competitively inhibited binding of IgE antibodies to Rhi o 1 up to 70% and suppressed allergen-mediated histamine release by 10-fold. In conclusion, this is a simple yet rational strategy based on epitope mapping data to develop a genetically modified hypoallergenic variant showing

  9. Immune epitope database analysis resource (IEDB-AR)

    DEFF Research Database (Denmark)

    Zhang, Qing; Wang, Peng; Kim, Yohan;

    2008-01-01

    We present a new release of the immune epitope database analysis resource (IEDB-AR, http://tools.immuneepitope.org), a repository of web-based tools for the prediction and analysis of immune epitopes. New functionalities have been added to most of the previously implemented tools, and a total...... of eight new tools were added, including two B-cell epitope prediction tools, four T-cell epitope prediction tools and two analysis tools....

  10. Adenovirus-based vaccine with epitopes incorporated in novel fiber sites to induce protective immunity against Pseudomonas aeruginosa.

    Science.gov (United States)

    Sharma, Anurag; Krause, Anja; Xu, Yaqin; Sung, Biin; Wu, Wendy; Worgall, Stefan

    2013-01-01

    Adenovirus (Ad) vector-based vaccines displaying pathogen-derived epitopes on Ad capsid proteins can elicit anti-pathogen immunity. This approach seems to be particularly efficient with epitopes incorporated into the Ad fiber protein. Here, we explore epitope insertion into various sites of the Ad fiber to elicit epitope-specific immunity. Ad vectors expressing the 14-mer Pseudomonas aeruginosa immune-dominant outer membrane protein F (OprF) epitope 8 (Epi8) in five distinct sites of the Ad5 fiber, loops CD (AdZ.F(CD)Epi8), DE (AdZ.F(DE)Epi8), FG (AdZ.F(FG)Epi8), HI (AdZ.F(HI)Epi8) and C terminus (AdZ.F(CT)Epi8), or the hexon HVR5 loop (AdZ.HxEpi8) were compared in their capacity to elicit anti-P. aeruginosa immunity to AdOprF, an Ad expressing the entire OprF protein. Intramuscular immunization of BALB/c mice with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 elicited higher anti-OprF humoral and cellular CD4 and CD8 responses as well as enhanced protection against respiratory infection with P. aeruginosa compared to immunization with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(CT)Epi8 or AdZ.HxEpi8. Importantly, repeat administration of the fiber- and hexon-modified Ad vectors boosted the OprF-specific humoral immune response in contrast to immunization with AdOprF. Strikingly, following three doses of AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 anti-OprF immunity surpassed that induced by AdOprF. Furthermore, in the presence of anti-Ad5 immunity, immunization with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8, but not with AdOprF, induced protective immunity against P. aeruginosa. This suggests that incorporation of epitopes into distinct sites of the Ad fiber is a promising vaccine strategy.

  11. The hepatitis B virus core protein intradimer interface modulates capsid assembly and stability.

    Science.gov (United States)

    Selzer, Lisa; Katen, Sarah P; Zlotnick, Adam

    2014-09-02

    During the hepatitis B virus (HBV) life cycle, capsid assembly and disassembly must ensure correct packaging and release of the viral genome. Here we show that changes in the dynamics of the core protein play an important role in regulating these processes. The HBV capsid assembles from 120 copies of the core protein homodimer. Each monomer contains a conserved cysteine at position 61 that can form an intradimer disulfide that we use as a marker for dimer conformational states. We show that dimers in the context of capsids form intradimer disulfides relatively rapidly. Surprisingly, compared to reduced dimers, fully oxidized dimers assembled slower and into capsids that were morphologically similar but less stable. We hypothesize that oxidized protein adopts a geometry (or constellation of geometries) that is unfavorable for capsid assembly, resulting in weaker dimer-dimer interactions as well as slower assembly kinetics. Our results suggest that structural flexibility at the core protein intradimer interface is essential for regulating capsid assembly and stability. We further suggest that capsid destabilization by the C61-C61 disulfide has a regulatory function to support capsid disassembly and release of the viral genome.

  12. Antigenic structure of the capsid protein of rabbit haemorrhagic disease virus

    DEFF Research Database (Denmark)

    Martinez-Torrecuadrada, Jorge L.; Cortes, Elena; Vela, Carmen;

    1998-01-01

    Rabbit haemorrhagic disease virus (RHDV) causes an important disease in rabbits. The virus capsid is composed of a single 60 kDa protein. The capsid protein gene was cloned in Escherichia coli using the pET3 system, and the antigenic structure of RHDV VP60 was dissected using 11 monoclonal...

  13. Varicella-zoster virus induces the formation of dynamic nuclear capsid aggregates

    Energy Technology Data Exchange (ETDEWEB)

    Lebrun, Marielle [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Thelen, Nicolas; Thiry, Marc [University of Liege (ULg), GIGA-Neurosciences, Laboratory of Cellular and Tissular Biology, Liege (Belgium); Riva, Laura; Ote, Isabelle; Condé, Claude; Vandevenne, Patricia [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Di Valentin, Emmanuel [University of Liege (ULg), GIGA-Viral Vectors Platform, Liege (Belgium); Bontems, Sébastien [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Sadzot-Delvaux, Catherine, E-mail: csadzot@ulg.ac.be [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium)

    2014-04-15

    The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains controversial. We created a recombinant VZV expressing ORF23 (homologous to HSV-1 VP26) fused to the eGFP and dually fluorescent viruses with a tegument protein additionally fused to a red tag (ORF9, ORF21 and ORF22 corresponding to HSV-1 UL49, UL37 and UL36). We identified nuclear dense structures containing the major capsid protein, the scaffold protein and maturing protease, as well as ORF21 and ORF22. Correlative microscopy demonstrated that the structures correspond to capsid aggregates and time-lapse video imaging showed that they appear prior to the accumulation of cytoplasmic capsids, presumably undergoing the secondary egress, and are highly dynamic. Our observations suggest that these structures might represent a nuclear area important for capsid assembly and/or maturation before the budding at the inner nuclear membrane. - Highlights: • We created a recombinant VZV expressing the small capsid protein fused to the eGFP. • We identified nuclear dense structures containing capsid and procapsid proteins. • Correlative microscopy showed that the structures correspond to capsid aggregates. • Procapsids and partial capsids are found within the aggregates of WT and eGFP-23 VZV. • FRAP and FLIP experiments demonstrated that they are dynamic structures.

  14. Modules for C-terminal epitope tagging of Tetrahymena genes

    Science.gov (United States)

    Kataoka, Kensuke; Schoeberl, Ursula E.; Mochizuki, Kazufumi

    2010-01-01

    Although epitope tagging has been widely used for analyzing protein function in many organisms, there are few genetic tools for epitope tagging in Tetrahymena. In this study, we describe several C-terminal epitope tagging modules that can be used to express tagged proteins in Tetrahymena cells by both plasmid- and PCR-based strategies. PMID:20624430

  15. Characterization of T cell epitopes in bovine α-lactalbumin

    NARCIS (Netherlands)

    Meulenbroek, Laura A P M; den Hartog Jager, Constance F; Lebens, Ans F M; Knulst, André C; Bruijnzeel-Koomen, Carla A F M; Garssen, Johan; Knippels, Léon M J; van Hoffen, Els

    2014-01-01

    BACKGROUND: Recent studies have indicated that peptides containing T cell epitopes may be used for immunotherapy. While for several cow's milk allergens the T cell epitopes have been described, the T cell epitopes in the major allergen α-lactalbumin (α-LAC) are unknown. Therefore, the aim of this st

  16. Competitive Enzyme Immunoassay for Diagnosis of Human Brucellosis

    Science.gov (United States)

    Lucero, Nidia E.; Foglia, Luis; Ayala, Sandra M.; Gall, David; Nielsen, Klaus

    1999-01-01

    The methods commonly used for human brucellosis serological testing are agglutination tests and the complement fixation test (CFT). Among the newer serological tests, primary binding assays were developed to improve sensitivity and specificity. The competitive enzyme immunoassay (CELISA) for the detection of serum antibody to Brucella is a multispecies assay which appears to be capable of differentiating vaccinal and cross-reacting antibodies from antibodies elicited by field infection in cattle. The competing monoclonal antibody used in this assay is specific for a common epitope of smooth lipopolysaccharide (S-LPS). In this study, we compared the CELISA to the classical tests for the diagnosis of human brucellosis. The CELISA cutoff value was determined to calculate its diagnostic specificity and sensitivity. A survey was performed with 911 sera. Of the sera, 341 were from an asymptomatic population that tested negative with conventional serological tests (screening and confirmatory). Based on these samples, the CELISA specificities were determined to be 99.7 and 100% with cutoff values of 28 and 30% inhibition (%I), respectively. In a further study with 393 additional sera from an asymptomatic population found negative by the conventional screening tests, the CELISA specificities were calculated to be 96.5 and 98.8% with cutoff values of 28 and 30%I. The CELISA sensitivities were determined to be 98.3 and 94.8% with cutoff values of 28 and 30%I, respectively, for sera from 116 individuals found positive by the classical tests. For the 51 culture-positive patients, CELISA was positive for 100%, the CFT was positive for 92%, and the standard tube agglutination test (TAT) was positive for 100%. The CELISA specificity was 100% for 31 sera from patients found negative by conventional serological tests but with brucellosis-like symptoms. The CELISA is fairly rapid to perform, somewhat faster than TAT, and cross-reacts less with other antigens (or antibodies) than the

  17. Single hepatitis-B virus core capsid binding to individual nuclear pore complexes in Hela cells.

    Science.gov (United States)

    Lill, Yoriko; Lill, Markus A; Fahrenkrog, Birthe; Schwarz-Herion, Kyrill; Paulillo, Sara; Aebi, Ueli; Hecht, Bert

    2006-10-15

    We investigate the interaction of hepatitis B virus capsids lacking a nuclear localization signal with nuclear pore complexes (NPCs) in permeabilized HeLa cells. Confocal and wide-field optical images of the nuclear envelope show well-spaced individual NPCs. Specific interactions of capsids with single NPCs are characterized by extended residence times of capsids in the focal volume which are characterized by fluorescence correlation spectroscopy. In addition, single-capsid-tracking experiments using fast wide-field fluorescence microscopy at 50 frames/s allow us to directly observe specific binding via a dual-color colocalization of capsids and NPCs. We find that binding occurs with high probability on the nuclear-pore ring moiety, at 44 +/- 9 nm radial distance from the central axis.

  18. Capsid modification of adeno-associated virus and tumor targeting gene therapy

    Institute of Scientific and Technical Information of China (English)

    XU ZengHu; ZHOU XiuMei; SHI WenFang; QIAN QiJun

    2008-01-01

    Targeting is critical for successful tumor gene therapy. The adeno-associated virus (AAV) has aroused wide concern due to its excellent advantages over other viral vectors in gene therapy. AAV has a broad infection spectrum, which also results in poor specificity towards tissues or cells and low transduction efficiency. Therefore, it is imperative to improve target and transduction efficiency in AAV-mediated gene therapy. Up to now, researchers have developed many strategies to modify AAV capsids for improving targeting or retargeting only desired cells. These strategies include not only traditional chemical modification, phage display technology, modification of AAV capsid genome, chimeric vectors and so on, but also many novel strategies involved in marker rescue strategy, direct evolution of capsid proteins, direct display random peptides on AAV capsid, AAVP (AAV-Phage), and etc. This review will summarize the advances of researches on the capsid modification of AAV to target malignant cells.

  19. Modeling of the rotavirus group C capsid predicts a surface topology distinct from other rotavirus species.

    Science.gov (United States)

    Eren, Elif; Zamuda, Kimberly; Patton, John T

    2016-01-01

    Rotavirus C (RVC) causes sporadic gastroenteritis in adults and is an established enteric pathogen of swine. Because RVC strains grow poorly in cell culture, which hinders generation of virion-derived RVC triple-layered-particle (TLP) structures, we used the known Rotavirus A (RVA) capsid structure to model the human RVC (Bristol) capsid. Comparative analysis of RVA and RVC capsid proteins showed major differences at the VP7 layer, an important target region for vaccine development due to its antigenic properties. Our model predicted the presence of a surface extended loop in RVC, which could form a major antigenic site on the capsid. We analyzed variations in the glycosylation patterns among RV capsids and identified group specific conserved sites. In addition, our results showed a smaller RVC VP4 foot, which protrudes toward the intermediate VP6 layer, in comparison to that of RVA. Finally, our results showed major structural differences at the VP8* glycan recognition sites.

  20. Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yoon Sik, E-mail: yumshak@naver.com; Seo, Hyun Wook, E-mail: suruk@naver.com; Jung, Guhung, E-mail: drjung@snu.ac.kr

    2015-02-13

    Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study that Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly. - Highlights: • We examined H{sub 2}O{sub 2} and GSH modulate HBV capsid assembly. • H{sub 2}O{sub 2} facilitates HBV capsid assembly in the presence of Hsp90. • GSH inhibits function of Hsp90 in facilitating HBV capsid assembly. • H{sub 2}O{sub 2} and GSH induce conformation change of Hsp90.

  1. Revised Mimivirus major capsid protein sequence reveals intron-containing gene structure and extra domain

    Directory of Open Access Journals (Sweden)

    Suzan-Monti Marie

    2009-05-01

    Full Text Available Abstract Background Acanthamoebae polyphaga Mimivirus (APM is the largest known dsDNA virus. The viral particle has a nearly icosahedral structure with an internal capsid shell surrounded with a dense layer of fibrils. A Capsid protein sequence, D13L, was deduced from the APM L425 coding gene and was shown to be the most abundant protein found within the viral particle. However this protein remained poorly characterised until now. A revised protein sequence deposited in a database suggested an additional N-terminal stretch of 142 amino acids missing from the original deduced sequence. This result led us to investigate the L425 gene structure and the biochemical properties of the complete APM major Capsid protein. Results This study describes the full length 3430 bp Capsid coding gene and characterises the 593 amino acids long corresponding Capsid protein 1. The recombinant full length protein allowed the production of a specific monoclonal antibody able to detect the Capsid protein 1 within the viral particle. This protein appeared to be post-translationnally modified by glycosylation and phosphorylation. We proposed a secondary structure prediction of APM Capsid protein 1 compared to the Capsid protein structure of Paramecium Bursaria Chlorella Virus 1, another member of the Nucleo-Cytoplasmic Large DNA virus family. Conclusion The characterisation of the full length L425 Capsid coding gene of Acanthamoebae polyphaga Mimivirus provides new insights into the structure of the main Capsid protein. The production of a full length recombinant protein will be useful for further structural studies.

  2. Development of an epitope conservancy analysis tool to facilitate the design of epitope-based diagnostics and vaccines

    Directory of Open Access Journals (Sweden)

    Fusseder Nicolas

    2007-09-01

    Full Text Available Abstract Background In an epitope-based vaccine setting, the use of conserved epitopes would be expected to provide broader protection across multiple strains, or even species, than epitopes derived from highly variable genome regions. Conversely, in a diagnostic and disease monitoring setting, epitopes that are specific to a given pathogen strain, for example, can be used to monitor responses to that particular infectious strain. In both cases, concrete information pertaining to the degree of conservancy of the epitope(s considered is crucial. Results To assist in the selection of epitopes with the desired degree of conservation, we have developed a new tool to determine the variability of epitopes within a given set of protein sequences. The tool was implemented as a component of the Immune Epitope Database and Analysis Resources (IEDB, and is directly accessible at http://tools.immuneepitope.org/tools/conservancy. Conclusion An epitope conservancy analysis tool was developed to analyze the variability or conservation of epitopes. The tool is user friendly, and is expected to aid in the design of epitope-based vaccines and diagnostics.

  3. Structure-based design and experimental engineering of a plant virus nanoparticle for the presentation of immunogenic epitopes and as a drug carrier.

    Science.gov (United States)

    Arcangeli, Caterina; Circelli, Patrizia; Donini, Marcello; Aljabali, Alaa A A; Benvenuto, Eugenio; Lomonossoff, George P; Marusic, Carla

    2014-04-01

    Biomaterials research for the discovery of new generation nanoparticles is one of the most active areas of nanotechnology. In the search of nature-made nanometer-sized objects, plant virus particles appear as symmetrically defined entities that can be formed by protein self-assembly. In particular, in the field of plant virology, there is plenty of literature available describing the exploitation of plant viral cages to produce safe vaccine vehicles and nanoparticles for drug delivery. In this context, we have investigated on the use of the artichoke mottled crinkle virus (AMCV) capsid both as a carrier of immunogenic epitopes and for the delivery of anticancer molecules. A dual approach that combines both in silico tools and experimental virology was applied for the rational design of immunologically active chimeric virus-like particles (VLPs) carrying immunogenic peptides. The atomic structures of wild type (wt) and chimeric VLPs were obtained by homology modeling. The effects of insertion of the HIV-1 2F5 neutralizing epitope on the structural stability of chimeric VLPs were predicted and assessed by detailed inspection of the nanoparticle intersubunit interactions at atomic level. Wt and chimeric VLPs, exposing on their surface the 2F5 epitope, were successfully produced in plants. In addition, we demonstrated that AMCV capsids could also function as drug delivery vehicles able to load the chemotherapeutic drug doxorubicin. To our knowledge, this is the first systematic predictive and empirical research addressing the question of how this icosahedral virus can be used for the production of both VLPs and viral nanoparticles for biomedical applications.

  4. Gliadin Detection in Food by Immunoassay

    Science.gov (United States)

    Grant, Gordon; Sporns, Peter; Hsieh, Y.-H. Peggy

    Immunoassays are very sensitive and efficient tests that are commonly used to identify a specific protein. Examples of applications in the food industry include identification of proteins expressed in genetically modified foods, allergens, or proteins associated with a disease, including celiac disease. This genetic disease is associated with Europeans and affects about one in every 200 people in North America. These individuals react immunologically to wheat proteins, and consequently their own immune systems attack and damage their intestines. This disease can be managed if wheat proteins, specifically "gliadins," are avoided in foods.

  5. [Enzyme immunoassay of usnic acid in lichens].

    Science.gov (United States)

    Burkin, A A; Kononenko, G P; Tolpysheva, T Iu

    2013-01-01

    An enzyme immunoassay for usnic acid in lichens was developed, the sensitivity of which was 0.1 microg/g of air-dried material (0.00001%). Polyclonal rabbit antibodies against bovine serum albumin conjugated to (+)-usnic acid under the conditions of formaldehyde condensation made it possible to determine the analyzed substance in solutions at concentrations from 1 ng/mL when it interacts with an immobilized gelatin conjugate homologous in the binding mode. Usnic acid in 2-26600 microg/g (0.0002-2.6%) amounts was found in all 236 studied samples of lichens belonging to 53 species and 8 families.

  6. Neutralization epitopes on HIV pseudotyped with HTLV-I: Conservation of carbohydrate Epitopes

    DEFF Research Database (Denmark)

    Sørensen, A M; Nielsen, C; Arendrup, M

    1994-01-01

    for pseudotypes to escape neutralization by the immune system in vivo. Previous reports have suggested that carbohydrate structures may be conserved neutralization epitopes on retroviruses. In this study, the neutralizing capacity of lectins and anti-carbohydrate monoclonal antibodies was found to block infection...... by cell-free pseudotypes in CD4-negative cells. We suggest that although viral cofactors might expand the tropism of HIV in vivo, HIV and HTLV-I seem to induce common carbohydrate neutralization epitopes....

  7. IgE-binding epitopes: a reappraisal

    NARCIS (Netherlands)

    R.C. Aalberse; R. Crameri

    2011-01-01

    Here, we discuss various questions related to IgE epitopes: What are the technical possibilities and pitfalls, what is currently known, how can we put this information into hypothetical frameworks and the unavoidable question: how useful is this information for patient care or allergenicity predicti

  8. Viral O-GalNAc peptide epitopes

    DEFF Research Database (Denmark)

    Olofsson, Sigvard; Blixt, Klas Ola; Bergström, Tomas

    2016-01-01

    on a novel three-step procedure that identifies any reactive viral O-glycosyl peptide epitope with respect to (i) relevant peptide sequence, (ii) the reactive glycoform out of several possible glycopeptide isomers of that peptide sequence, and (iii) possibly tolerated carbohydrate or peptide structural...

  9. B cell epitope spreading: mechanisms and contribution to autoimmune diseases.

    Science.gov (United States)

    Cornaby, Caleb; Gibbons, Lauren; Mayhew, Vera; Sloan, Chad S; Welling, Andrew; Poole, Brian D

    2015-01-01

    While a variety of factors act to trigger or initiate autoimmune diseases, the process of epitope spreading is an important contributor in their development. Epitope spreading is a diversification of the epitopes recognized by the immune system. This process happens to both T and B cells, with this review focusing on B cells. Such spreading can progress among multiple epitopes on a single antigen, or from one antigenic molecule to another. Systemic lupus erythematosus, multiple sclerosis, pemphigus, bullous pemphigoid and other autoimmune diseases, are all influenced by intermolecular and intramolecular B cell epitope spreading. Endocytic processing, antigen presentation, and somatic hypermutation act as molecular mechanisms that assist in driving epitope spreading and broadening the immune response in autoimmune diseases. The purpose of this review is to summarize our current understanding of B cell epitope spreading with regard to autoimmunity, how it contributes during the progression of various autoimmune diseases, and treatment options available.

  10. VLPs displaying a single L2 epitope induce broadly cross-neutralizing antibodies against human papillomavirus.

    Directory of Open Access Journals (Sweden)

    Ebenezer Tumban

    Full Text Available BACKGROUND: Virus-like Particles (VLPs display can be used to increase the immunogenicity of heterologous antigens. Here, we report the use of a bacteriophage MS2-based VLP display platform to develop a monovalent vaccine targeting a broadly neutralizing epitope in the minor capsid protein human papillomavirus (HPV that provides broad protection from diverse HPV types in a mouse pseudovirus infection model. METHODOLOGY/PRINCIPAL FINDINGS: Peptides spanning a previously described cross-neutralizing epitope from HPV type 16 were genetically inserted at the N-terminus of MS2 bacteriophage coat protein. Three of the four recombinant L2-coat proteins assembled into VLPs. L2-VLPs elicited high-titer anti-L2 antibodies in mice, similar to recombinant VLPs that we had previously made in which the L2 peptide was displayed on a surface-exposed loop on VLPs of a related bacteriophage, PP7. Somewhat surprisingly, L2-MS2 VLPs elicited antibodies that were much more broadly cross-reactive with L2 peptides from diverse HPV isolates than L2-PP7 VLPs. Similarly, mice immunized with L2-MS2 VLPs were protected from genital and cutaneous infection by highly diverse HPV pseudovirus types. CONCLUSION/SIGNIFICANCE: We show that peptides can be displayed in a highly immunogenic fashion at the N-terminus of MS2 coat protein VLPs. A VLP-based vaccine targeting HPV L2 elicits broadly cross-reactive and cross-protective antibodies to heterologous HPV types. L2-VLPs could serve as the basis of a broadly protective second generation HPV vaccine.

  11. Residues of the UL25 Protein of Herpes Simplex Virus That Are Required for Its Stable Interaction with Capsids

    OpenAIRE

    Cockrell, Shelley K.; Huffman, Jamie B.; Toropova, Katerina; James F Conway; Homa, Fred L.

    2011-01-01

    The herpes simplex virus 1 (HSV-1) UL25 gene product is a minor capsid component that is required for encapsidation, but not cleavage, of replicated viral DNA. UL25 is located on the capsid surface in a proposed heterodimer with UL17, where five copies of the heterodimer are found at each of the capsid vertices. Previously, we demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids. To further define the UL25 capsid binding domain, we generated reco...

  12. Structure of the capsid of Kilham rat virus from small-angle neutron scattering

    Energy Technology Data Exchange (ETDEWEB)

    Wobbe, C.R.; Mitra, S.; Ramakrishnan, V.

    1984-12-18

    The structure of empty capsids of Kilham rat virus, an autonomous parvovirus with icosahedral symmetry, was investigated by small-angle neutron scattering. From the forward scatter, the molecular weight was determined to be 4.0 x 10(6), and from the Guinier region, the radius of gyration was found to be 105 A in D2O and 104 A in H/sub 2/O. On the basis of the capsid molecular weight and the molecular weights and relative abundances of the capsid proteins, the authors propose that the capsid has a triangulation number of 1. Extended scattering curves and mathematical modeling revealed that the capsid consists of two shells of protein, the inner shell extending from 58 to 91 A in D2O and from 50 to 91 A in H/sub 2/O and containing 11% of the capsid scattering mass, and the outer shell extending to 121 A in H/sub 2/O and D2O. The inner shell appears to have a higher content of basic amino acids than the outer shell, based on its lower scattering density in D2O than in H/sub 2/O. The authors propose that all three capsid proteins contribute to the inner shell and that this basic region serves DNA binding and partial charge neutralization functions.

  13. Immobilization and One-Dimensional Arrangement of Virus Capsids with Nanoscale Precision Using DNA Origami

    Energy Technology Data Exchange (ETDEWEB)

    Stephanopoulos, Nicholas [Univ. of California, Berkeley, CA (United States); Liu, Minghui [Arizona State Univ., Tempe, AZ (United States); Tong, Gary J [Univ. of California, Berkeley, CA (United States); Li, Zhe [Arizona State Univ., Tempe, AZ (United States); Liu, Yan [Arizona State Univ., Tempe, AZ (United States); Yan, Hao [Arizona State Univ., Tempe, AZ (United States); Francis, Matthew B [Univ. of California, Berkeley, CA (United States)

    2010-06-24

    DNA origami was used as a scaffold to arrange spherical virus capsids into one-dimensional arrays with precise nanoscale positioning. To do this, we first modified the interior surface of bacteriophage MS2 capsids with fluorescent dyes as a model cargo. An unnatural amino acid on the external surface was then coupled to DNA strands that were complementary to those extending from origami tiles. Two different geometries of DNA tiles (rectangular and triangular) were used. The capsids associated with tiles of both geometries with virtually 100% efficiency under mild annealing conditions, and the location of capsid immobilization on the tile could be controlled by the position of the probe strands. The rectangular tiles and capsids could then be arranged into one-dimensional arrays by adding DNA strands linking the corners of the tiles. The resulting structures consisted of multiple capsids with even spacing (~100 nm). We also used a second set of tiles that had probe strands at both ends, resulting in a one-dimensional array of alternating capsids and tiles. This hierarchical self-assembly allows us to position the virus particles with unprecedented control and allows the future construction of integrated multicomponent systems from biological scaffolds using the power of rationally engineered DNA nanostructures.

  14. Controlling AAV Tropism in the Nervous System with Natural and Engineered Capsids

    Science.gov (United States)

    Castle, Michael J.; Turunen, Heikki T.; Vandenberghe, Luk H.; Wolfe, John H.

    2016-01-01

    More than one hundred naturally occurring variants of adeno-associated virus (AAV) have been identified, and this library has been further expanded by an array of techniques for modification of the viral capsid. AAV capsid variants possess unique antigenic profiles and demonstrate distinct cellular tropisms driven by differences in receptor binding. AAV capsids can be chemically modified to alter tropism, can be produced as hybrid vectors that combine the properties of multiple serotypes, and can carry peptide insertions that introduce novel receptor-binding activity. Furthermore, directed evolution of shuffled genome libraries can identify engineered variants with unique properties, and rational modification of the viral capsid can alter tropism, reduce blockage by neutralizing antibodies, or enhance transduction efficiency. This large number of AAV variants and engineered capsids provides a varied toolkit for gene delivery to the CNS and retina, with specialized vectors available for many applications, but selecting a capsid variant from the array of available vectors can be difficult. This chapter describes the unique properties of a range of AAV variants and engineered capsids, and provides a guide for selecting the appropriate vector for specific applications in the CNS and retina. PMID:26611584

  15. Structural rigidity in the capsid assembly of cowpea chlorotic mottle virus

    Science.gov (United States)

    Hespenheide, B. M.; Jacobs, D. J.; Thorpe, M. F.

    2004-11-01

    The cowpea chlorotic mottle virus (CCMV) has a protein cage, or capsid, which encloses its genetic material. The structure of the capsid consists of 180 copies of a single protein that self-assemble inside a cell to form a complete capsid with icosahedral symmetry. The icosahedral surface can be naturally divided into pentagonal and hexagonal faces, and the formation of either of these faces has been proposed to be the first step in the capsid assembly process. We have used the software FIRST to analyse the rigidity of pentameric and hexameric substructures of the complete capsid to explore the viability of certain capsid assembly pathways. FIRST uses the 3D pebble game to determine structural rigidity, and a brief description of this algorithm, as applied to body-bar networks, is given here. We find that the pentameric substructure, which corresponds to a pentagonal face on the icosahedral surface, provides the best structural properties for nucleating the capsid assembly process, consistent with experimental observations.

  16. Structural rigidity in the capsid assembly of cowpea chlorotic mottle virus

    Energy Technology Data Exchange (ETDEWEB)

    Hespenheide, B M [Department of Physics and Astronomy, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504 (United States); Jacobs, D J [Department of Physics and Astronomy, California State University, 18111 Nordhoff Street, Northridge, CA 91330-8268 (United States); Thorpe, M F [Department of Physics and Astronomy, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504 (United States)

    2004-11-10

    The cowpea chlorotic mottle virus (CCMV) has a protein cage, or capsid, which encloses its genetic material. The structure of the capsid consists of 180 copies of a single protein that self-assemble inside a cell to form a complete capsid with icosahedral symmetry. The icosahedral surface can be naturally divided into pentagonal and hexagonal faces, and the formation of either of these faces has been proposed to be the first step in the capsid assembly process. We have used the software FIRST to analyse the rigidity of pentameric and hexameric substructures of the complete capsid to explore the viability of certain capsid assembly pathways. FIRST uses the 3D pebble game to determine structural rigidity, and a brief description of this algorithm, as applied to body-bar networks, is given here. We find that the pentameric substructure, which corresponds to a pentagonal face on the icosahedral surface, provides the best structural properties for nucleating the capsid assembly process, consistent with experimental observations.

  17. Assembly of the small outer capsid protein, Soc, on bacteriophage T4: a novel system for high density display of multiple large anthrax toxins and foreign proteins on phage capsid.

    Science.gov (United States)

    Li, Qin; Shivachandra, Sathish B; Zhang, Zhihong; Rao, Venigalla B

    2007-07-27

    Bacteriophage T4 capsid is a prolate icosahedron composed of the major capsid protein gp23*, the vertex protein gp24*, and the portal protein gp20. Assembled on its surface are 810 molecules of the non-essential small outer capsid protein, Soc (10 kDa), and 155 molecules of the highly antigenic outer capsid protein, Hoc (39 kDa). In this study Soc, a "triplex" protein that stabilizes T4 capsid, is targeted for molecular engineering of T4 particle surface. Using a defined in vitro assembly system, anthrax toxins, protective antigen, lethal factor and their domains, fused to Soc were efficiently displayed on the capsid. Both the N and C termini of the 80 amino acid Soc polypeptide can be simultaneously used to display antigens. Proteins as large as 93 kDa can be stably anchored on the capsid through Soc-capsid interactions. Using both Soc and Hoc, up to 1662 anthrax toxin molecules are assembled on the phage T4 capsid under controlled conditions. We infer from the binding data that a relatively high affinity capsid binding site is located in the middle of the rod-shaped Soc, with the N and C termini facing the 2- and 3-fold symmetry axes of the capsid, respectively. Soc subunits interact at these interfaces, gluing the adjacent capsid protein hexamers and generating a cage-like outer scaffold. Antigen fusion does interfere with the inter-subunit interactions, but these interactions are not essential for capsid binding and antigen display. These features make the T4-Soc platform the most robust phage display system reported to date. The study offers insights into the architectural design of bacteriophage T4 virion, one of the most stable viruses known, and how its capsid surface can be engineered for novel applications in basic molecular biology and biotechnology.

  18. Non-enzymatic glucosylation induced neo-epitopes on human serum albumin: A concentration based study

    Science.gov (United States)

    Neelofar, Km; Arif, Zarina; Ahmad, Jamal; Alam, Khursheed

    2017-01-01

    Hyperglycaemia induced non enzymatic glycation is accelerated in diabetic patients and aggressively involved in diabetes progression. Human serum albumin (HSA) is the most abundant protein in blood circulation. In hyperglycaemia, it undergoes fast glycation and results in the impairment of structure. Our previous study has demonstrated structural alterations in Amadori-albumin modified with different glucose concentrations from physiological to pathophysiological range. Here, we focused on immunological characterization of Amadori-albumin. Immunogenicity of Amadori-albumin was analysed by direct binding and competitive ELISA. Amadori-albumin was found to be highly immunogenic (expect albumin modified with 5mM) and induced high titre antibodies depending upon the extent of modification. Very high titre antibodies were obtained with albumin modified with 75mM glucose as compared to native albumin. Anti-Amadori-albumin-IgG from rabbit sera exhibited increased recognition of Amadori-albumin than native albumin in competitive immunoassay. Alteration induced in albumin after glucosylation has made it highly immunogenic. Induced antibodies were quite specific for respective immunogens but showed cross-reaction with other Amadori/native proteins. It suggests that glucosylation has generated highly immunogenic epitopes on albumin. Formation of high molecular weight immune complex with retarded mobility further supports specificity of anti-Amadori-albumin-IgG towards Amadori-albumin. It may be concluded that due to early glycation, an array of modification occurred in HSA structure. Such gross structural changes might favour polymerization of most of the native epitopes into potent immunogenic neo-epitopes, but some original epitopes were still active and has contributed in the immunogenicity. It could be concluded that induction of anti-Amadori-albumin antibodies may be due to protection of glucose modified albumin from protiolytic breakdown. We assumed that this type of

  19. On the geometry of regular icosahedral capsids containing disymmetrons

    CERN Document Server

    Ang, Kai-Siang

    2016-01-01

    Icosahedral virus capsids are composed of symmetrons, organized arrangements of capsomers. There are three types of symmetrons: disymmetrons, trisymmetrons, and pentasymmetrons, which have different shapes and are centered on the icosahedral 2-fold, 3-fold and 5-fold axes of symmetry, respectively. In 2010 [Sinkovits & Baker] gave a classification of all possible ways of building an icosahedral structure solely from trisymmetrons and pentasymmetrons, which requires the triangulation number T to be odd. In the present paper we incorporate disymmetrons to obtain a geometric classification of icosahedral viruses formed by regular penta-, tri-, and disymmetrons. For every class of solutions, we further provide formulas for symmetron sizes and parity restrictions on h, k, and T numbers. We also present several methods in which invariants may be used to classify a given configuration.

  20. Assembly of bacteriophage T7. Dimensions of the bacteriophage and its capsids

    Energy Technology Data Exchange (ETDEWEB)

    Stroud, R.M.; Serwer, P.; Ross, M.J.

    1981-12-01

    The dimensions of bacteriophage T7 and T7 capsids have been investigated by small-angle x-ray scattering. Phage T7 behaves like a sphere of uniform density with an outer radius of 301 +/- 2 A (excluding the phage tail) and a calculated volume for protein plus nucleic acid of 1.14 +/- 0.05 x 10/sup -16/ ml. The outer radius determined of T7 phage in solution is approx.30% greater than the radius measured from electron micrographs, which indicates that considerable shrinkage occurs during preparation for electron microscopy. Capsids that have a phagelike envelope and do not contain DNA were obtained from lysates of T7-infected Escherichia coli (capsid II) and by separating the capsid component of T7 phage from the phage DNA by means of temperature shock (capsid IV). In both cases the peak protein density is at a radius of 275 A; the outer radius is 286 +/- 4 A, approx.5% smaller than the envelope of T7 phage. The thickness of the envelope of capsid II is 22 +/- 4 A, consistent with the thickness of protein estimated to be 23 +/- 5 A in whole T7 phage, as seen on electron micrographs in which the internal DNA is positively stained. The volume in T7 phage available to package DNA is estimated to be 9.2 +/- 0.4 x 10/sup -17/ ml. The packaged DNA adopts a regular packing with 23.6 A interplanar spacing between DNA strands. The angular width of the 23.6 A reflection shows that the mean DNA-DNA spacing throughout the phage head is 27.5 +/- <2.2 A. A T7 precursor capsid (capsid I) expands when pelleted for x-ray scattering in the ultracentrifuge to essentially the same outer dimensions as for capsids II and IV. This expansion of capsid I can be prevented by fixing with glutaraldehyde; fixed capsid I has peak density at a radius of 247 A, 10% less than capsid II or IV.

  1. Characterization of an isotype-dependent monoclonal antibody against linear neutralizing epitope effective for prophylaxis of enterovirus 71 infection.

    Directory of Open Access Journals (Sweden)

    Xiao Fang Lim

    Full Text Available BACKGROUND: Enterovirus 71 (EV71 is the main causative agent of Hand, Foot and Mouth disease (HFMD and is associated with severe neurologic complications and mortalities. At present, there is no vaccine or therapeutic available for treatment. METHODOLOGY/PRINCIPAL FINDING: In this study, we generated two mAbs, denoted as mAb 51 and 53, both targeting the same linear epitope on VP1 capsid protein, spanning amino acids 215-219. In comparison, mAb 51 belonging to isotype IgM possesses neutralizing activity in vitro, whereas, mAb 53 belonging to isotype IgG1 does not have any neutralizing ability, even towards its homologous strain. When mAb 51 at 10 µg/g of body weight was administered to the 2-week-old AG129 mice one day prior to lethal challenge, 100% in vivo passive protection was observed. In contrast, the isotype control group mice, injected with an irrelevant IgM antibody before the challenge, developed limb paralysis as early as day 6 post-infection. Histological examination demonstrated that mAb 51 was able to protect against pathologic changes such as neuropil vacuolation and neuronal loss in the spinal cord, which were typical in unprotected EV-71 infected mice. BLAST analyses of that epitope revealed that it was highly conserved among all EV71 strains, but not coxsachievirus 16 (CA16. CONCLUSION: We have defined a linear epitope within the VP1 protein and demonstrated its neutralizing ability to be isotype dependent. The neutralizing property and highly conserved sequence potentiated the application of mAb 51 and 53 for protection against EV71 infection and diagnosis respectively.

  2. AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating

    Energy Technology Data Exchange (ETDEWEB)

    Tenney, Rebeca M.; Bell, Christie L.; Wilson, James M., E-mail: wilsonjm@mail.med.upenn.edu

    2014-04-15

    Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8's robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII and IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype. - Highlights: • We construct chimeric vectors to identify determinants of AAV8 liver transduction. • An AAV2-based vector with 17 AAV8 residues exhibited high liver transduction in mice. • This vector also surpassed AAV2 in cell entry, nuclear entry and onset of expression. • Most chimeric vector particles were uncoated at 6 weeks, like AAV8 and unlike AAV2. • Chimera retained heparin binding and was antigenically distinct from AAV2 and AAV8.

  3. Competitive enzyme immunoassay for urinary vanillylmandelic acid.

    Science.gov (United States)

    Taran, F; Bernard, H; Valleix, A; Créminon, C; Grassi, J; Olichon, D; Deverre, J R; Pradelles, P

    1997-08-29

    An enzyme immunoassay for urinary vanillylmandelic acid (VMA) using polyclonal antiserum and VMA-acetylcholinesterase conjugate as enzymatic tracer is described. Two different strategies for immunogen preparation were developed and enantioselectivity was demonstrated. Selected EIA allowed direct measurement of urinary VMA using D(-)-VMA as standard with good sensitivity (MDC = 0.1 mumol/l) and precision (CV less than 7% in 0.2-2.25 mumol/l range). Cross-reactivity with homovanillic acid (HVA) was 0.8% and less than 0.4% with other structurally related catecholamine metabolites. Intra- and inter-assay repeatability were less than 10% and recovery was 97.3% +/- 3%. Good correlation was obtained for EIA and HPLC analysis with normal and pathologic human urine samples (EIA = 0.895 HPLC-7.085, r2 = 0.98, n = 47).

  4. Immunoassay of chemical contaminants in milk:A review

    Institute of Scientific and Technical Information of China (English)

    XU Fei; REN Kang; YANG Yu-ze; GUO Jiang-peng; MA Guang-peng; LIU Yi-ming; LU Yong-qiang; LI Xiu-bo

    2015-01-01

    The detection of chemical contaminants is critical to ensure dairy safety. These contaminants include veterinary medicines, antibiotics, pesticides, heavy metals, mycotoxins, and persistent organic polutants (POPs). Immunoassays have recently been used to detect contaminants in milk because of their simple operation, high speed, and low cost. This article describes the latest developments in the most important component of immunoassays—antibodies, and then reviews the four major substrates used for immunoassays (i.e., microplates, membranes, gels, and chips) as wel as their use in the detection of milk contaminants. The paper concludes with prospects for further applications of these immunoassays.

  5. Magic-angle spinning NMR of intact bacteriophages: Insights into the capsid, DNA and their interface

    Science.gov (United States)

    Abramov, Gili; Morag, Omry; Goldbourt, Amir

    2015-04-01

    Bacteriophages are viruses that infect bacteria. They are complex macromolecular assemblies, which are composed of multiple protein subunits that protect genomic material and deliver it to specific hosts. Various biophysical techniques have been used to characterize their structure in order to unravel phage morphogenesis. Yet, most bacteriophages are non-crystalline and have very high molecular weights, in the order of tens of MegaDaltons. Therefore, complete atomic-resolution characterization on such systems that encompass both capsid and DNA is scarce. In this perspective article we demonstrate how magic-angle spinning solid-state NMR has and is used to characterize in detail bacteriophage viruses, including filamentous and icosahedral phage. We discuss the process of sample preparation, spectral assignment of both capsid and DNA and the use of chemical shifts and dipolar couplings to probe the capsid-DNA interface, describe capsid structure and dynamics and extract structural differences between viruses.

  6. Remodeling nuclear architecture allows efficient transport of herpesvirus capsids by diffusion.

    Science.gov (United States)

    Bosse, Jens B; Hogue, Ian B; Feric, Marina; Thiberge, Stephan Y; Sodeik, Beate; Brangwynne, Clifford P; Enquist, Lynn W

    2015-10-20

    The nuclear chromatin structure confines the movement of large macromolecular complexes to interchromatin corrals. Herpesvirus capsids of approximately 125 nm assemble in the nucleoplasm and must reach the nuclear membranes for egress. Previous studies concluded that nuclear herpesvirus capsid motility is active, directed, and based on nuclear filamentous actin, suggesting that large nuclear complexes need metabolic energy to escape nuclear entrapment. However, this hypothesis has recently been challenged. Commonly used microscopy techniques do not allow the imaging of rapid nuclear particle motility with sufficient spatiotemporal resolution. Here, we use a rotating, oblique light sheet, which we dubbed a ring-sheet, to image and track viral capsids with high temporal and spatial resolution. We do not find any evidence for directed transport. Instead, infection with different herpesviruses induced an enlargement of interchromatin domains and allowed particles to diffuse unrestricted over longer distances, thereby facilitating nuclear egress for a larger fraction of capsids.

  7. Proof of principle for epitope-focused vaccine design

    Science.gov (United States)

    Correia, Bruno E.; Bates, John T.; Loomis, Rebecca J.; Baneyx, Gretchen; Carrico, Chris; Jardine, Joseph G.; Rupert, Peter; Correnti, Colin; Kalyuzhniy, Oleksandr; Vittal, Vinayak; Connell, Mary J.; Stevens, Eric; Schroeter, Alexandria; Chen, Man; MacPherson, Skye; Serra, Andreia M.; Adachi, Yumiko; Holmes, Margaret A.; Li, Yuxing; Klevit, Rachel E.; Graham, Barney S.; Wyatt, Richard T.; Baker, David; Strong, Roland K.; Crowe, James E.; Johnson, Philip R.; Schief, William R.

    2014-03-01

    Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza.

  8. Residues of the UL25 protein of herpes simplex virus that are required for its stable interaction with capsids.

    Science.gov (United States)

    Cockrell, Shelley K; Huffman, Jamie B; Toropova, Katerina; Conway, James F; Homa, Fred L

    2011-05-01

    The herpes simplex virus 1 (HSV-1) UL25 gene product is a minor capsid component that is required for encapsidation, but not cleavage, of replicated viral DNA. UL25 is located on the capsid surface in a proposed heterodimer with UL17, where five copies of the heterodimer are found at each of the capsid vertices. Previously, we demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids. To further define the UL25 capsid binding domain, we generated recombinant viruses with either small truncations or amino acid substitutions in the UL25 N terminus. Studies of these mutants demonstrated that there are two important regions within the capsid binding domain. The first 27 amino acids are essential for capsid binding of UL25, while residues 26 to 39, which are highly conserved in the UL25 homologues of other alphaherpesviruses, were found to be critical for stable capsid binding. Cryo-electron microscopy reconstructions of capsids containing either a small tag on the N terminus of UL25 or the green fluorescent protein (GFP) fused between amino acids 50 and 51 of UL25 demonstrate that residues 1 to 27 of UL25 contact the hexon adjacent to the penton. A second region, most likely centered on amino acids 26 to 39, contacts the triplex that is one removed from the penton. Importantly, both of these UL25 capsid binding regions are essential for the stable packaging of full-length viral genomes.

  9. SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption.

    Science.gov (United States)

    Ho, Michelle L; Adler, Benjamin A; Torre, Michael L; Silberg, Jonathan J; Suh, Junghae

    2013-12-20

    Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions.

  10. Identification of immunodominant epitopes in Trypanosoma cruzi trypomastigote surface antigen-1 protein that mask protective epitopes.

    Science.gov (United States)

    Wrightsman, R A; Dawson, B D; Fouts, D L; Manning, J E

    1994-10-01

    The gene that encodes trypomastigote surface Ag-1 (TSA-1), a major surface Ag of the bloodstream trypomastigote stage of Trypanosoma cruzi, was expressed in a baculovirus expression system. To determine the epitope(s) in TSA-1 that was recognized during T. cruzi infection and after immunization with TSA-1, subregions of the TSA-1 gene were expressed in a bacterial expression system. As seen by Western blotting, both mice and rabbits immunized with recombinant TSA-1 protein, as well as T. cruzi-infected mice, developed strong immune responses to the carboxyl-proximal region of TSA-1, but show no reaction to the amino-proximal portion of TSA-1. When mice were immunized with either recombinant TSA-1 protein or the carboxyl-proximal region of TSA-1, they did not survive challenge with 10(3) bloodstream trypomastigotes. However, 70% of the mice immunized with the amino-proximal portion of TSA-1 survived challenge with 10(3) bloodstream trypomastigotes. Thus, the immune responses elicited by recombinant TSA-1 or the carboxyl-proximal portion of TSA-1 are nonprotective during T. cruzi infection. In contrast, vaccination with the amino proximal region of TSA-1 elicits a protective immune response. These results suggest that responses to immunodominant epitope(s) within the carboxyl-proximal portion of TSA-1 mask epitopes within the amino-proximal portion that are capable of stimulating host-protective immune responses. It is suggested that immunodominant regions in surface molecules such as TSA-1 may provide a mechanism for the parasite to evade the host immune response by directing the response away from epitopes that have the potential to elicit a reaction that is damaging to the parasite.

  11. Packaging Double-Helical DNA into Viral Capsids: Structures, Forces, and Energetics

    OpenAIRE

    Petrov, Anton S.; Harvey, Stephen C.

    2008-01-01

    Small, icosahedral double-stranded DNA bacteriophage pack their genomes tightly into preformed protein capsids using an ATP-driven motor. Coarse-grain molecular-mechanics models provide a detailed picture of DNA packaging in bacteriophage, revealing how conformation depends on capsid size and shape, and the presence or absence of a protein core. The forces that oppose packaging have large contributions from both electrostatic repulsions and the entropic penalty of confining the DNA into the c...

  12. Conformational Changes in the Capsid of a Calicivirus upon Interaction with Its Functional Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Ossiboff, Robert J.; Zhou, Yi; Lightfoot, Patrick J.; Prasad, B.V. Venkataram; Parker, John S.L. (Baylor); (Cornell)

    2010-07-19

    Nonenveloped viral capsids are metastable structures that undergo conformational changes during virus entry that lead to interactions of the capsid or capsid fragments with the cell membrane. For members of the Caliciviridae, neither the nature of these structural changes in the capsid nor the factor(s) responsible for inducing these changes is known. Feline functional adhesion molecule A (fJAM-A) mediates the attachment and infectious viral entry of feline calicivirus (FCV). Here, we show that the infectivity of some FCV isolates is neutralized following incubation with the soluble receptor at 37 C. We used this property to select mutants resistant to preincubation with the soluble receptor. We isolated and sequenced 24 soluble receptor-resistant (srr) mutants and characterized the growth properties and receptor-binding activities of eight mutants. The location of the mutations within the capsid structure of FCV was mapped using a new 3.6-{angstrom} structure of native FCV. The srr mutations mapped to the surface of the P2 domain were buried at the protruding domain dimer interface or were present in inaccessible regions of the capsid protein. Coupled with data showing that both the parental FCV and the srr mutants underwent increases in hydrophobicity upon incubation with the soluble receptor at 37 C, these findings indicate that FCV likely undergoes conformational change upon interaction with its receptor. Changes in FCV capsid conformation following its interaction with fJAM-A may be important for subsequent interactions of the capsid with cellular membranes, membrane penetration, and genome delivery.

  13. Advances of Bioinformatics Tools Applied in Virus Epitopes Prediction

    Institute of Scientific and Technical Information of China (English)

    Ping Chen; Simon Rayner; Kang-hong Hu

    2011-01-01

    In recent years, the in silico epitopes prediction tools have facilitated the progress of vaccines development significantly and many have been applied to predict epitopes in viruses successfully. Herein, a general overview of different tools currently available, including T cell and B cell epitopes prediction tools, is presented. And the principles of different prediction algorithms are reviewed briefly. Finally, several examples are present to illustrate the application of the prediction tools.

  14. Atomic-level mapping of antibody epitopes on a GPCR.

    Science.gov (United States)

    Paes, Cheryl; Ingalls, Jada; Kampani, Karan; Sulli, Chidananda; Kakkar, Esha; Murray, Meredith; Kotelnikov, Valery; Greene, Tiffani A; Rucker, Joseph B; Doranz, Benjamin J

    2009-05-27

    Epitopes that define the immunodominant regions of conformationally complex integral membrane proteins have been difficult to reliably delineate. Here, a high-throughput approach termed shotgun mutagenesis was used to map the binding epitopes of five different monoclonal antibodies targeting the GPCR CCR5. The amino acids, and in some cases the atoms, that comprise the critical contact points of each epitope were identified, defining the immunodominant structures of this GPCR and their physicochemistry.

  15. Mapping of immunodominant B-cell epitopes and the human serum albumin-binding site in natural hepatitis B virus surface antigen of defined genosubtype.

    Science.gov (United States)

    Sobotta, D; Sominskaya, I; Jansons, J; Meisel, H; Schmitt, S; Heermann, K H; Kaluza, G; Pumpens, P; Gerlich, W H

    2000-02-01

    Twelve MAbs were generated by immunization of BALB/c mice with plasma-derived hepatitis B virus surface spherical antigen particles subtype ayw2 (HBsAg/ayw2 genotype D). Their epitopes were mapped by analysis of reactivity with plasma-derived HBsAg/ayw2 and HBsAg/adw2 (genotype A) in enzyme immunoassays and blots. Mapping was supported by nested sets of truncated preS2 proteins and preS2 peptides. Five antibodies were S domain-specific, seven were preS2-specific and 11 had a preference for genotype D. According to our data, group I of the three known epitope groups of preS2 has to be divided into IA and IB. Three preS2-specific MAbs forming the new group IA reacted with genotype D residues 3-15 which have not yet been described as an epitope region. IA antibodies strongly inhibited the binding of polymerized human serum albumin. Two antibodies (group II) reacted with the glycosylated N-terminal region of preS2 in plasma-derived HBsAg, but not with a preparation from transfected murine cells. One group III antibody was subtype-specific and reacted with the highly variable preS2 sequence 38-48. Only one antibody (group IB) mapped to the region (old group I) which was believed to be immunodominant and genotype-independent. Geno(sub)type-specific epitopes of preS2 are obviously the immunodominant components of natural HBsAg in BALB/c mice, but these epitopes may be masked by serum albumins in humans. The data may explain why it is difficult to detect anti-preS2 antibodies in human recipients of preS2-containing vaccines, in spite of the preS2 immunodominance in mice.

  16. Antibodies recognizing Mycobacterium avium paratuberculosis epitopes cross-react with the beta-cell antigen ZnT8 in Sardinian type 1 diabetic patients.

    Directory of Open Access Journals (Sweden)

    Speranza Masala

    Full Text Available The environmental factors at play in the pathogenesis of type 1 diabetes (T1D remain enigmatic. Mycobacterium avium subspecies paratuberculosis (MAP is transmitted from dairy herds to humans through food contamination. MAP causes an asymptomatic infection that is highly prevalent in Sardinian T1D patients compared with type 2 diabetes (T2D and healthy controls. Moreover, MAP elicits humoral responses against several mycobacterial proteins. We asked whether antibodies (Abs against one of these proteins, namely MAP3865c, which displays a sequence homology with the β-cell protein zinc transporter 8 (ZnT8 could be cross-reactive with ZnT8 epitopes. To this end, Ab responses against MAP3865c were analyzed in Sardinian T1D, T2D and healthy subjects using an enzymatic immunoassay. Abs against MAP3865c recognized two immunodominant transmembrane epitopes in 52-65% of T1D patients, but only in 5-7% of T2D and 3-5% of healthy controls. There was a linear correlation between titers of anti-MAP3865c and anti-ZnT8 Abs targeting these two homologous epitopes, and pre-incubation of sera with ZnT8 epitope peptides blocked binding to the corresponding MAP3865c peptides. These results demonstrate that Abs recognizing MAP3865c epitopes cross-react with ZnT8, possibly underlying a molecular mimicry mechanism, which may precipitate T1D in MAP-infected individuals.

  17. An alphavirus temperature-sensitive capsid mutant reveals stages of nucleocapsid assembly

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Yan, E-mail: yzheng15@students.kgi.edu; Kielian, Margaret, E-mail: margaret.kielian@einstein.yu.edu

    2015-10-15

    Alphaviruses have a nucleocapsid core composed of the RNA genome surrounded by an icosahedral lattice of capsid protein. An insertion after position 186 in the capsid protein produced a strongly temperature-sensitive growth phenotype. Even when the structural proteins were synthesized at the permissive temperature (28 °C), subsequent incubation of the cells at the non-permissive temperature (37 °C) dramatically decreased mutant capsid protein stability and particle assembly. Electron microscopy confirmed the presence of cytoplasmic nucleocapsids in mutant-infected cells cultured at the permissive temperature, but these nucleocapsids were not stable to sucrose gradient separation. In contrast, nucleocapsids isolated from mutant virus particles had similar stability to that of wildtype virus. Our data support a model in which cytoplasmic nucleocapsids go through a maturation step during packaging into virus particles. The insertion site lies in the interface between capsid proteins in the assembled nucleocapsid, suggesting the region where such a stabilizing transition occurs. - Highlights: • We characterize an alphavirus capsid insertion mutation. • These capsid mutants are highly temperature sensitive for growth. • The insertion affects nucleocapsid stability. • Results suggest that the nucleocapsid is stabilized during virus budding.

  18. Oral Administration of Astrovirus Capsid Protein Is Sufficient To Induce Acute Diarrhea In Vivo

    Directory of Open Access Journals (Sweden)

    Victoria A. Meliopoulos

    2016-11-01

    Full Text Available The disease mechanisms associated with the onset of astrovirus diarrhea are unknown. Unlike other enteric virus infections, astrovirus infection is not associated with an inflammatory response or cellular damage. In vitro studies in differentiated Caco-2 cells demonstrated that human astrovirus serotype 1 (HAstV-1 capsid protein alone disrupts the actin cytoskeleton and tight junction complex, leading to increased epithelial barrier permeability. In this study, we show that oral administration of purified recombinant turkey astrovirus 2 (TAstV-2 capsid protein results in acute diarrhea in a dose- and time-dependent manner in turkey poults. Similarly to that induced by infectious virus, TAstV-2 capsid-induced diarrhea was independent of inflammation or histological changes but was associated with increased intestinal barrier permeability, as well as redistribution of sodium hydrogen exchanger 3 (NHE3 from the membrane to the cytoplasm of the intestinal epithelium. Unlike other viral enterotoxins that have been identified, astrovirus capsid induces diarrhea after oral administration, reproducing the natural route of infection and demonstrating that ingestion of intact noninfectious capsid protein may be sufficient to provoke acute diarrhea. Based on these data, we hypothesize that the astrovirus capsid acts like an enterotoxin and induces intestinal epithelial barrier dysfunction.

  19. High Relaxivity Gadolinium Hydroxypyridonate-Viral Capsid Conjugates: Nano-sized MRI Contrast Agents

    Energy Technology Data Exchange (ETDEWEB)

    Meux, Susan C.; Datta, Ankona; Hooker, Jacob M.; Botta, Mauro; Francis, Matthew B.; Aime, Silvio; Raymond, Kenneth N.

    2007-08-29

    High relaxivity macromolecular contrast agents based on the conjugation of gadolinium chelates to the interior and exterior surfaces of MS2 viral capsids are assessed. The proton nuclear magnetic relaxation dispersion (NMRD) profiles of the conjugates show up to a five-fold increase in relaxivity, leading to a peak relaxivity (per Gd{sup 3+} ion) of 41.6 mM{sup -1}s{sup -1} at 30 MHz for the internally modified capsids. Modification of the exterior was achieved through conjugation to flexible lysines, while internal modification was accomplished by conjugation to relatively rigid tyrosines. Higher relaxivities were obtained for the internally modified capsids, showing that (1) there is facile diffusion of water to the interior of capsids and (2) the rigidity of the linker attaching the complex to the macromolecule is important for obtaining high relaxivity enhancements. The viral capsid conjugated gadolinium hydroxypyridonate complexes appear to possess two inner-sphere water molecules (q = 2) and the NMRD fittings highlight the differences in the local motion for the internal ({tau}{sub RI} = 440 ps) and external ({tau}{sub RI} = 310 ps) conjugates. These results indicate that there are significant advantages of using the internal surface of the capsids for contrast agent attachment, leaving the exterior surface available for the installation of tissue targeting groups.

  20. Simple immunoassay for detection of PCBs in transformer oil.

    Science.gov (United States)

    Glass, Thomas R; Ohmura, Naoya; Taemi, Yukihiro; Joh, Takashi

    2005-07-01

    A rapid and inexpensive procedure to detect polychlorinated biphenyls (PCBs) in transformer oil is needed to facilitate identification and removal of PCB contaminated transformers. Here we describe a simple two-step liquid-liquid extraction using acidic dimethyl sulfoxide in conjunction with an immunoassay for detecting PCBs in transformer oil. The process described is faster and simpler than any previous immunoassay while maintaining comparable detection limit and false negative rate. Cross reactivity data, characterizing the immunoassay response to the four Kanechlor technical mixtures of PCBs in oil, are presented. Forty-five used transformer oil samples were analyzed by gas chromatography-high-resolution mass spectrometry and were also evaluated using the immunoassay protocol developed. Results presented show zero false negatives at a 1.4 ppm nominal cutoff for the transformer oils analyzed.

  1. A Compact Immunoassay Platform Based on a Multicapillary Glass Plate

    OpenAIRE

    Shuhua Xue; Hulie Zeng; Jianmin Yang; Hizuru Nakajima; Katsumi Uchiyama

    2014-01-01

    A highly sensitive, rapid immunoassay performed in the multi-channels of a micro-well array consisting of a multicapillary glass plate (MCP) and a polydimethylsiloxane (PDMS) slide is described. The micro-dimensions and large surface area of the MCP permitted the diffusion distance to be decreased and the reaction efficiency to be increased. To confirm the concept of the method, human immunoglobulin A (h-IgA) was measured using both the proposed immunoassay system and the traditional 96-well ...

  2. Multiplex biosensor immunoassays for antibiotics in the food chain

    OpenAIRE

    2009-01-01

    The use of antibiotics in food-producing animals may result in unwanted residues in food products. The main objective of the present research was to study the development and application of fast and automated multiplex surface plasmon resonance (SPR)-based biosensor immunoassays (BIAs), based on multi-component antibodies and/or combined immunoassays in serially connected flow channels, for the detection of selected antibiotics in the food chain. The scientific challenges to deal with were: t...

  3. Polystyrene Based SPR Biosensor Chip for Use in Immunoassay

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    Biosensors are widely used in immunoassay.The biosensor chip carries a receptor which is used in immunoassay and the chip properties have an important influence on the detecting sensitivity of the biosensor.This paper describes a polystyrene-based biosensor chip developed and used as part of a surface plasmon resonance (SPR) biosensor.The SPR biosensor has a much higher detecting sensitivity than enzyme-linked immunoserbent assay (ELISA).

  4. A Sensitive Amphotericin B Immunoassay for Pharmacokinetic and Distribution Studies

    OpenAIRE

    Machard, Sophie; Theodoro, Frederic; Benech, Henri; Grognet, Jean-Marc; Ezan, Eric

    2000-01-01

    Since currently used assays of amphotericin B (AMB) lack sensitivity or are not easily adaptable in all laboratories, we have developed an enzyme immunoassay for AMB in biological fluids and tissues. Antibodies to AMB were raised in rabbits after administration of an AMB-bovine serum albumin conjugate. The enzymatic tracer was obtained by coupling AMB via its amino group to acetylcholinesterase (EC 3.1.1.7). These reagents were used for the development of a competitive immunoassay performed o...

  5. Epitope DNA vaccines against tuberculosis: spacers and ubiquitin modulates cellular immune responses elicited by epitope DNA vaccine

    Institute of Scientific and Technical Information of China (English)

    Wang QM; Sun SH; Hu ZL; Zhou FJ; Yin M; Xiao CJ; Zhang JC

    2005-01-01

    Cell-mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed epitope DNA vaccines (p3-M-38) encoding cytotoxic T lymphocyte (CTL) epitopes of MPT64 and 38 kDa proteins of Mycobacterium tuberculosis. In order to observe the influence of spacer sequence (Ala-Ala-Tyr) or ubiquitin (UbGR) on the efficacy of the two CTL epitopes, we also constructed DNA vaccines, p3-M-S(spacer)-38, p3-Ub (UbGR)-M-S-38 and p3-Ub-M-38. The immune responses elicited by the four DNA vaccines were tested in C57BL/6 (H-2b) mice. The cytotoxicity of T cells was detected by LDH-release method and by enzyme-linked immunospot assay for epitope-specific cells secreting interferon-gamma. The results showed that DNA immunization with p3-M-38 vaccine could induce epitope-specific CD8+ CTL response and that the spacer sequence (AAY) only enhanced M epitope presentation. The protein-targeting sequence (UbGR) enhanced the immunogenicity of the two epitopes. The finding that defined spacer sequences at C-terminus and protein-targeting degradation modulated the immune response of epitope string DNA vaccines will be of importance for the further development of multi-epitope DNA vaccines against tuberculosis.

  6. Structure of a novel shoulder-to-shoulder p24 dimer in complex with the broad-spectrum antibody A10F9 and its implication in capsid assembly.

    Directory of Open Access Journals (Sweden)

    Ying Gu

    Full Text Available Mature HIV-1 viral particles assemble as a fullerene configuration comprising p24 capsid hexamers, pentamers and dimers. In this paper, we report the X-ray crystal structures of the p24 protein from natural HIV-1 strain (BMJ4 in complex with Fab A10F9, which recognizes a conserved epitope in the C-terminal domain of the BMJ4 p24 protein. Our structures reveal a novel shoulder-to-shoulder p24 dimerization mode that is mediated by an S-S bridge at C177. Consistent with these structures, the shoulder-to-shoulder dimer that was obtained from the BMJ4 strain was also observed in p24 proteins from other strains by the introduction of a cysteine residue at position 177. The potential biological significance was further validated by the introduction of a C177A mutation in the BMJ4 strain, which then displays a low infectivity. Our data suggest that this novel shoulder-to-shoulder dimer interface trapped by this unique S-S bridge could represent a physiologically relevant mode of HIV-1 capsid assembly during virus maturation, although Cys residue itself may not be critical for HIV-I replication.

  7. Critical appraisal of four IL-6 immunoassays.

    Directory of Open Access Journals (Sweden)

    Dana K Thompson

    Full Text Available BACKGROUND: Interleukin-6 (IL-6 contributes to numerous inflammatory, metabolic, and physiologic pathways of disease. We evaluated four IL-6 immunoassays in order to identify a reliable assay for studies of metabolic and physical function. Serial plasma samples from intravenous glucose tolerance tests (IVGTTs, with expected rises in IL-6 concentrations, were used to test the face validity of the various assays. METHODS AND FINDINGS: IVGTTs, administered to 14 subjects, were performed with a single infusion of glucose (0.3 g/kg body mass at time zero, a single infusion of insulin (0.025 U/kg body mass at 20 minutes, and frequent blood collection from time zero to 180 minutes for subsequent Il-6 measurement. The performance metrics of four IL-6 detection methods were compared: Meso Scale Discovery immunoassay (MSD, an Invitrogen Luminex bead-based multiplex panel (LX, an Invitrogen Ultrasensitive Luminex bead-based singleplex assay (ULX, and R&D High Sensitivity ELISA (R&D. IL-6 concentrations measured with MSD, R&D and ULX correlated with each other (Pearson Correlation Coefficients r = 0.47-0.94, p<0.0001 but only ULX correlated (r = 0.31, p = 0.0027 with Invitrogen Luminex. MSD, R&D, and ULX, but not LX, detected increases in IL-6 in response to glucose. All plasma samples were measurable by MSD, while 35%, 1%, and 4.3% of samples were out of range when measured by LX, ULX, and R&D, respectively. Based on representative data from the MSD assay, baseline plasma IL-6 (0.90 ± 0.48 pg/mL increased significantly as expected by 90 minutes (1.29 ± 0.59 pg/mL, p = 0.049, and continued rising through 3 hours (4.25 ± 3.67 pg/mL, p = 0.0048. CONCLUSION: This study established the face validity of IL-6 measurement by MSD, R&D, and ULX but not LX, and the superiority of MSD with respect to dynamic range. Plasma IL-6 concentrations increase in response to glucose and insulin, consistent with both an early glucose-dependent response (detectable at 1

  8. Limited cross-reactivity of mouse monoclonal antibodies against Dengue virus capsid protein among four serotypes

    Directory of Open Access Journals (Sweden)

    Noda M

    2012-11-01

    Full Text Available Megumi Noda,1 Promsin Masrinoul,1 Chaweewan Punkum,1 Chonlatip Pipattanaboon,2,3 Pongrama Ramasoota,2,4 Chayanee Setthapramote,2,3 Tadahiro Sasaki,6 Mikiko Sasayama,1 Akifumi Yamashita,1,5 Takeshi Kurosu,6 Kazuyoshi Ikuta,6 Tamaki Okabayashi11Mahidol-Osaka Center for Infectious Diseases, 2Center of Excellence for Antibody Research, 3Department of Microbiology and Immunology, 4Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, Thailand; 5Graduate School of Life Science, Tohoku University, Sendai, Miyagi, 6Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, JapanBackground: Dengue illness is one of the important mosquito-borne viral diseases in tropical and subtropical regions. Four serotypes of dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4 are classified in the Flavivirus genus of the family Flaviviridae. We prepared monoclonal antibodies against DENV capsid protein from mice immunized with DENV-2 and determined the cross-reactivity with each serotype of DENV and Japanese encephalitis virus.Methods and results: To clarify the relationship between the cross-reactivity of monoclonal antibodies and the diversity of these viruses, we examined the situations of flaviviruses by analyses of phylogenetic trees. Among a total of 60 prepared monoclonal antibodies specific for DENV, five monoclonal antibodies stained the nuclei of infected cells and were found to be specific to the capsid protein. Three were specific to DENV-2, while the other two were cross-reactive with DENV-2 and DENV-4. No monoclonal antibodies were cross-reactive with all four serotypes. Phylogenetic analysis of DENV amino acid sequences of the capsid protein revealed that DENV-2 and DENV-4 were clustered in the same branch, while DENV-1 and DENV-3 were clustered in the other branch. However, these classifications of the capsid protein were different from those of the

  9. Transgenic tomatoes express an antigenic polypeptide containing epitopes of the diphtheria, pertussis and tetanus exotoxins, encoded by a synthetic gene.

    Science.gov (United States)

    Soria-Guerra, Ruth Elena; Rosales-Mendoza, Sergio; Márquez-Mercado, Crisóforo; López-Revilla, Rubén; Castillo-Collazo, Rosalba; Alpuche-Solís, Angel Gabriel

    2007-07-01

    A current priority of vaccinology is the development of multicomponent vaccines that protect against several pathogens. The diphtheria-pertussis-tetanus (DPT) vaccine prevents the symptoms of three serious and often fatal diseases due to the exotoxins produced by Corynebacterium diphteriae, Bordetella pertussis and Clostridium tetani. We are attempting to develop an edible DPT multicomponent vaccine in plants, based on the fusion of protective exotoxin epitopes encoded by synthetic genes. By means of Agrobacterium mediated transformation we generated transgenic tomatoes with a plant-optimised synthetic gene encoding a novel polypeptide containing two adjuvant and six DPT immunoprotective exotoxin epitopes joined by peptide linkers. In transformed tomato plants, integration of the synthetic DPT (sDPT) gene detected by PCR was confirmed by Southern blot, and specific transcripts of the expected molecular size were detected by RT-PCR. Expression of the putative polypeptide encoded by the sDPT gene was detected by immunoassay with specific antibodies to the diphtheria, pertussis and tetanus exotoxins. The sDPT gene is therefore integrated, transcribed and translated as the expected recombinant sDPT multiepitope polypeptide in transgenic tomatoes that constitute a potential edible vaccine.

  10. Characterization of the DNA binding properties of polyomavirus capsid protein

    Science.gov (United States)

    Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

  11. A new modular chemiluminescence immunoassay analyser evaluated.

    Science.gov (United States)

    Ognibene, A; Drake, C J; Jeng, K Y; Pascucci, T E; Hsu, S; Luceri, F; Messeri, G

    2000-03-01

    Thyrotropin (TSH), free thyroxine (fT4) and testosterone assays have been used as a probe to evaluate the performances of a new modular chemiluminescence (CL) immunoassay analyser, the Abbott Architect 2000. The evaluation was run in parallel on other systems that use CL as the detection reaction: DPC Immulite, Chiron Diagnostics ACS-180 and ACS Centaur (TSH functional sensitivity only). TSH functional sensitivity was 0.0012, 0.009, 0.033 and 0.039 mU/I for the Architect, Immulite, ACS Centaur and ACS-180, respectively. Testosterone functional sensitivity was 0.38, 3.7 and 2.0 nmol/l for Architect, Immulite and ACS-180, respectively. Good correlation was obtained between the ACS-180 and Architect for all assays. The Immulite correlation did not agree well with the Architect or ACS-180 for fT4 and testosterone but was in good agreement for TSH. Regarding fT4 and testosterone, equilibrium dialysis and isotopic dilution gas-chromatography mass-spectrometry (GC-MS) respectively were used as reference methods. For both within- and between-run precision, the Architect showed the best reproducibility for all three analytes (CV < 6%).

  12. Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis (Florida)

    2012-09-17

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  13. Structural studies of adeno-associated virus serotype 8 capsid transitions associated with endosomal trafficking.

    Science.gov (United States)

    Nam, Hyun-Joo; Gurda, Brittney L; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis

    2011-11-01

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  14. A xylogalacturonan epitope is specifically associated with plant cell detachment

    DEFF Research Database (Denmark)

    Willats, William George Tycho; McCartney, L.; Steele-King, C.G.;

    2004-01-01

    A monoclonal antibody (LM8) was generated with specificity for xyloglacturonan (XGA) isolated from pea (Pisum sativum L.) testae. Characterization of the LM8 epitope indicates that it is a region of XGA that is highly substituted with xylose. Immunocytochemical analysis indicates that this epitop...... that is specifically associated with a plant cell separation process that results in complete cell detachment....

  15. Analysis of cytotoxic T cell epitopes in relation to cancer

    DEFF Research Database (Denmark)

    Stranzl, Thomas

    kill the infected cells. The focus of my PhD project has been on improving a method for CTL epitope pathway prediction, on analyzing the epitope density in the alternative cancer exome, and on a study investigating minor histocompatibility antigens (mHags) associated with leukemia. Part I......CTL methods, the experimental effort to identify 90% of new epitopes can be reduced by 15% and 40%, respectively. Part III reports the results of an analysis investigating how the alternatively spliced cancer exome differs from the exome of normal tissue in terms of containing predicted MHC class I binding...... epitopes. We show that peptides unique to cancer splice variants comprise significantly fewer predicted HLA class I epitopes than peptides unique to spliced transcripts in normal tissue. We furthermore find that hydrophilic amino acids are significantly enriched in the unique carcinoma sequences, which...

  16. Autoantibody recognition mechanisms of p53 epitopes

    Science.gov (United States)

    Phillips, J. C.

    2016-06-01

    There is an urgent need for economical blood based, noninvasive molecular biomarkers to assist in the detection and diagnosis of cancers in a cost-effective manner at an early stage, when curative interventions are still possible. Serum autoantibodies are attractive biomarkers for early cancer detection, but their development has been hindered by the punctuated genetic nature of the ten million known cancer mutations. A landmark study of 50,000 patients (Pedersen et al., 2013) showed that a few p53 15-mer epitopes are much more sensitive colon cancer biomarkers than p53, which in turn is a more sensitive cancer biomarker than any other protein. The function of p53 as a nearly universal "tumor suppressor" is well established, because of its strong immunogenicity in terms of not only antibody recruitment, but also stimulation of autoantibodies. Here we examine dimensionally compressed bioinformatic fractal scaling analysis for identifying the few sensitive epitopes from the p53 amino acid sequence, and show how it could be used for early cancer detection (ECD). We trim 15-mers to 7-mers, and identify specific 7-mers from other species that could be more sensitive to aggressive human cancers, such as liver cancer. Our results could provide a roadmap for ECD.

  17. Broad epitope coverage of a human in vitro antibody library

    Science.gov (United States)

    Sivasubramanian, Arvind; Lynaugh, Heather; Yu, Yao; Miles, Adam; Eckman, Josh; Schutz, Kevin; Piffath, Crystal; Boland, Nadthakarn; Durand, Stéphanie; Boland, Todd; Vásquez, Maximiliano; Xu, Yingda; Abdiche, Yasmina

    2017-01-01

    ABSTRACT Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad “epitope coverage” increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or “bins” and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes. PMID:27748644

  18. B Epitope Multiplicity and B/T Epitope Orientation Influence Immunogenicity of Foot-and-Mouth Disease Peptide Vaccines

    Directory of Open Access Journals (Sweden)

    Esther Blanco

    2013-01-01

    Full Text Available Synthetic peptides incorporating protective B- and T-cell epitopes are candidates for new safer foot-and-mouth disease (FMD vaccines. We have reported that dendrimeric peptides including four copies of a B-cell epitope (VP1 136 to 154 linked to a T-cell epitope (3A 21 to 35 of FMD virus (FMDV elicit potent B- and T-cell specific responses and confer protection to viral challenge, while juxtaposition of these epitopes in a linear peptide induces less efficient responses. To assess the relevance of B-cell epitope multivalency, dendrimers bearing two (B2T or four (B4T copies of the B-cell epitope from type O FMDV (a widespread circulating serotype were tested in CD1 mice and showed that multivalency is advantageous over simple B-T-epitope juxtaposition, resulting in efficient induction of neutralizing antibodies and optimal release of IFNγ. Interestingly, the bivalent B2T construction elicited similar or even better B- and T-cell specific responses than tetravalent B4T. In addition, the presence of the T-cell epitope and its orientation were shown to be critical for the immunogenicity of the linear juxtaposed monovalent peptides analyzed in parallel. Taken together, our results provide useful insights for a more accurate design of FMD subunit vaccines.

  19. EPITOPE MAPPING OF SCLC-CLUSTER-2 MABS AND GENERATION OF ANTIBODIES DIRECTED AGAINST NEW EGP-2 EPITOPES

    NARCIS (Netherlands)

    HELFRICH, W; KONING, PW; THE, TH; DELEIJ, L

    1994-01-01

    Western blot analysis proved that all cluster-2 MAbs recognize identical or overlapping disulfide-bond-dependent epitopes, indicating the presence of a disulfide-bond-stabilized EGP-2 domain carrying highly immunodominant non-linear epitopes. The apparent immunodominance of this domain makes it diff

  20. Self-assembly of virus-like particles of canine parvovirus capsid protein expressed from Escherichia coli and application as virus-like particle vaccine.

    Science.gov (United States)

    Xu, Jin; Guo, Hui-Chen; Wei, Yan-Quan; Dong, Hu; Han, Shi-Chong; Ao, Da; Sun, De-Hui; Wang, Hai-Ming; Cao, Sui-Zhong; Sun, Shi-Qi

    2014-04-01

    Canine parvovirus disease is an acute infectious disease caused by canine parvovirus (CPV). Current commercial vaccines are mainly attenuated and inactivated; as such, problems concerning safety may occur. To resolve this problem, researchers developed virus-like particles (VLPs) as biological nanoparticles resembling natural virions and showing high bio-safety. This property allows the use of VLPs for vaccine development and mechanism studies of viral infections. Tissue-specific drug delivery also employs VLPs as biological nanomaterials. Therefore, VLPs derived from CPV have a great potential in medicine and diagnostics. In this study, small ubiquitin-like modifier (SUMO) fusion motif was utilized to express a whole, naturalVP2 protein of CPV in Escherichia coli. After the cleavage of the fusion motif, the CPV VP2 protein has self-assembled into VLPs. The VLPs had a size and shape that resembled the authentic virus capsid. However, the self-assembly efficiency of VLPs can be affected by different pH levels and ionic strengths. The mice vaccinated subcutaneously with CPV VLPs and CPV-specific immune responses were compared with those immunized with the natural virus. This result showed that VLPs can effectively induce anti-CPV specific antibody and lymphocyte proliferation as a whole virus. This result further suggested that the antigen epitope of CPV was correctly present on VLPs, thereby showing the potential application of a VLP-based CPV vaccine.

  1. A molecular thermodynamic model for the stability of hepatitis B capsids

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jehoon; Wu, Jianzhong, E-mail: jwu@engr.ucr.edu [Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521 (United States)

    2014-06-21

    Self-assembly of capsid proteins and genome encapsidation are two critical steps in the life cycle of most plant and animal viruses. A theoretical description of such processes from a physiochemical perspective may help better understand viral replication and morphogenesis thus provide fresh insights into the experimental studies of antiviral strategies. In this work, we propose a molecular thermodynamic model for predicting the stability of Hepatitis B virus (HBV) capsids either with or without loading nucleic materials. With the key components represented by coarse-grained thermodynamic models, the theoretical predictions are in excellent agreement with experimental data for the formation free energies of empty T4 capsids over a broad range of temperature and ion concentrations. The theoretical model predicts T3/T4 dimorphism also in good agreement with the capsid formation at in vivo and in vitro conditions. In addition, we have studied the stability of the viral particles in response to physiological cellular conditions with the explicit consideration of the hydrophobic association of capsid subunits, electrostatic interactions, molecular excluded volume effects, entropy of mixing, and conformational changes of the biomolecular species. The course-grained model captures the essential features of the HBV nucleocapsid stability revealed by recent experiments.

  2. CaPSID: A bioinformatics platform for computational pathogen sequence identification in human genomes and transcriptomes

    Directory of Open Access Journals (Sweden)

    Borozan Ivan

    2012-08-01

    Full Text Available Abstract Background It is now well established that nearly 20% of human cancers are caused by infectious agents, and the list of human oncogenic pathogens will grow in the future for a variety of cancer types. Whole tumor transcriptome and genome sequencing by next-generation sequencing technologies presents an unparalleled opportunity for pathogen detection and discovery in human tissues but requires development of new genome-wide bioinformatics tools. Results Here we present CaPSID (Computational Pathogen Sequence IDentification, a comprehensive bioinformatics platform for identifying, querying and visualizing both exogenous and endogenous pathogen nucleotide sequences in tumor genomes and transcriptomes. CaPSID includes a scalable, high performance database for data storage and a web application that integrates the genome browser JBrowse. CaPSID also provides useful metrics for sequence analysis of pre-aligned BAM files, such as gene and genome coverage, and is optimized to run efficiently on multiprocessor computers with low memory usage. Conclusions To demonstrate the usefulness and efficiency of CaPSID, we carried out a comprehensive analysis of both a simulated dataset and transcriptome samples from ovarian cancer. CaPSID correctly identified all of the human and pathogen sequences in the simulated dataset, while in the ovarian dataset CaPSID’s predictions were successfully validated in vitro.

  3. X-Ray Structures of the Hexameric Building Block of the HIV Capsid

    Energy Technology Data Exchange (ETDEWEB)

    Pornillos, Owen; Ganser-Pornillos, Barbie K.; Kelly, Brian N.; Hua, Yuanzi; Whitby, Frank G.; Stout, C. David; Sundquist, Wesley I.; Hill, Christopher P.; Yeager, Mark; (Scripps); (Utah)

    2009-09-11

    The mature capsids of HIV and other retroviruses organize and package the viral genome and its associated enzymes for delivery into host cells. The HIV capsid is a fullerene cone: a variably curved, closed shell composed of approximately 250 hexamers and exactly 12 pentamers of the viral CA protein. We devised methods for isolating soluble, assembly-competent CA hexamers and derived four crystallographically independent models that define the structure of this capsid assembly unit at atomic resolution. A ring of six CA N-terminal domains form an apparently rigid core, surrounded by an outer ring of C-terminal domains. Mobility of the outer ring appears to be an underlying mechanism for generating the variably curved lattice in authentic capsids. Hexamer-stabilizing interfaces are highly hydrated, and this property may be key to the formation of quasi-equivalent interactions within hexamers and pentamers. The structures also clarify the molecular basis for capsid assembly inhibition and should facilitate structure-based drug design strategies.

  4. Cyclophilin A stabilizes the HIV-1 capsid through a novel non-canonical binding site

    Science.gov (United States)

    Liu, Chuang; Perilla, Juan R.; Ning, Jiying; Lu, Manman; Hou, Guangjin; Ramalho, Ruben; Himes, Benjamin A.; Zhao, Gongpu; Bedwell, Gregory J.; Byeon, In-Ja; Ahn, Jinwoo; Gronenborn, Angela M.; Prevelige, Peter E.; Rousso, Itay; Aiken, Christopher; Polenova, Tatyana; Schulten, Klaus; Zhang, Peijun

    2016-03-01

    The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection.

  5. A molecular thermodynamic model for the stability of hepatitis B capsids

    Science.gov (United States)

    Kim, Jehoon; Wu, Jianzhong

    2014-06-01

    Self-assembly of capsid proteins and genome encapsidation are two critical steps in the life cycle of most plant and animal viruses. A theoretical description of such processes from a physiochemical perspective may help better understand viral replication and morphogenesis thus provide fresh insights into the experimental studies of antiviral strategies. In this work, we propose a molecular thermodynamic model for predicting the stability of Hepatitis B virus (HBV) capsids either with or without loading nucleic materials. With the key components represented by coarse-grained thermodynamic models, the theoretical predictions are in excellent agreement with experimental data for the formation free energies of empty T4 capsids over a broad range of temperature and ion concentrations. The theoretical model predicts T3/T4 dimorphism also in good agreement with the capsid formation at in vivo and in vitro conditions. In addition, we have studied the stability of the viral particles in response to physiological cellular conditions with the explicit consideration of the hydrophobic association of capsid subunits, electrostatic interactions, molecular excluded volume effects, entropy of mixing, and conformational changes of the biomolecular species. The course-grained model captures the essential features of the HBV nucleocapsid stability revealed by recent experiments.

  6. Sequence analysis and structural implications of rotavirus capsid proteins.

    Science.gov (United States)

    Parbhoo, N; Dewar, J B; Gildenhuys, S

    Rotavirus is the major cause of severe virus-associated gastroenteritis worldwide in children aged 5 and younger. Many children lose their lives annually due to this infection and the impact is particularly pronounced in developing countries. The mature rotavirus is a non-enveloped triple-layered nucleocapsid containing 11 double stranded RNA segments. Here a global view on the sequence and structure of the three main capsid proteins, VP2, VP6 and VP7 is shown by generating a consensus sequence for each of these rotavirus proteins, for each species obtained from published data of representative rotavirus genotypes from across the world and across species. Degree of conservation between species was represented on homology models for each of the proteins. VP7 shows the highest level of variation with 14-45 amino acids showing conservation of less than 60%. These changes are localised to the outer surface alluding to a possible mechanism in evading the immune system. The middle layer, VP6 shows lower variability with only 14-32 sites having lower than 70% conservation. The inner structural layer made up of VP2 showed the lowest variability with only 1-16 sites having less than 70% conservation across species. The results correlate with each protein's multiple structural roles in the infection cycle. Thus, although the nucleotide sequences vary due to the error-prone nature of replication and lack of proof reading, the corresponding amino acid sequence of VP2, 6 and 7 remain relatively conserved. Benefits of this knowledge about the conservation include the ability to target proteins at sites that cannot undergo mutational changes without influencing viral fitness; as well as possibility to study systems that are highly evolved for structure and function in order to determine how to generate and manipulate such systems for use in various biotechnological applications.

  7. Quasispecies dynamics in main core epitopes of hepatitis B virus by ultra-deep-pyrosequencing

    Institute of Scientific and Technical Information of China (English)

    Maria Homs; Maria Buti; David Tabernero; Josep Quer; Alex Sanchez; Noelia Corral; Rafael Esteban

    2012-01-01

    AIM:To investigate the variability of the main immunodominant motifs of hepatitis B virus (HBV) core gene by ultra-deep-pyrosequencing (UDPS).METHODS:Four samples (2 genotype A and 2 genotype D) from 4 treatment-naive patients were assessed for baseline variability.Two additional samples from one patient (patient 4,genotype D) were selected for analysis:one sample corresponded to a 36-mo treatment-free period from baseline and the other to the time of viral breakthrough after 18 mo of lamivudine treatment.The HBV region analyzed covered amino acids 40 to 95 of the core gene,and included the two main epitopic regions,Th50-69 and B74-84.UDPS was carried out in the Genome Sequencer FLX system (454 Life Sciences,Roche).After computer filtering of UDPS data based on a Poisson statistical model,122 813 sequences were analyzed.The most conserved position detected by UDPS was analyzed by site-directed mutagenesis and evaluated in cell culture.RESULTS:Positions with highest variability rates were mainly located in the main core epitopes,confirming their role as immune-stimulating regions.In addition,the distribution of variability showed a relationship with HBV genotype.Patient 1 (genotype A) presented the lowest variability rates and patient 2 (genotype A) had 3 codons with variability higher than 1%.Patient 3 and 4 (both genotype D) presented 5 and 8 codons with variability higher than 1%,respectively.The median baseline frequencies showed that genotype A samples had higher variability in epitopic positions than in the other positions analyzed,approaching significance (P =0.07,sample 1 and P =0.05,sample 2).In contrast,there were no significant differences in variability between the epitopic and other positions in genotype D cases.Interestingly,patient 1 presented a completely mutated motif from amino acid 64 to 67 (E64LMT67),which is commonly recognized by T helper cells.Additionally,the variability observed in all 4 patients was particularly associated with the E64

  8. Peptide dot immunoassay and immunoblotting: electroblotting from aluminum thin-layer chromatography plates and isoelectric focusing gels to activated nitrocellulose

    DEFF Research Database (Denmark)

    Bjerrum, O.J.; Holm, A.; Lauritzen, Edgar;

    1993-01-01

    Peptide dot immunoassay, electroblotting, activated nitrocellulose, dot blot, membranes, peptides and proteins......Peptide dot immunoassay, electroblotting, activated nitrocellulose, dot blot, membranes, peptides and proteins...

  9. Rapid detection of fungal alpha-amylase in the work environment with a lateral flow immunoassay

    NARCIS (Netherlands)

    Bogdanovic, J.; Koets, M.; Sander, I.; Wouters, I.; Meijster, T.; Heederik, D.J.J.; Amerongen, van A.; Doekes, G.

    2006-01-01

    Background Occupational allergen exposure assessment usually requires airborne dust sampling at the worksite followed by dust extraction and enzyme immunoassay (EIA) analysis at the laboratory. Use of semiquantitative lateral flow immunoassays (LFIAs) may allow a more rapid detection procedure with

  10. The Effects of Sample Matrices on Immunoassays to Detect Microcystin-LR in Water

    Science.gov (United States)

    Abstract: Immunoassays are widely used biochemical techniques to detect microcystins in environmental samples. The use of immunoassays for the detection of microcystins is vulnerable to matrix components and other interferents. This study is an evaluation of the effects of interf...

  11. Identification of Autoantigen Epitopes in Alopecia Areata.

    Science.gov (United States)

    Wang, Eddy H C; Yu, Mei; Breitkopf, Trisia; Akhoundsadegh, Noushin; Wang, Xiaojie; Shi, Feng-Tao; Leung, Gigi; Dutz, Jan P; Shapiro, Jerry; McElwee, Kevin J

    2016-08-01

    Alopecia areata (AA) is believed to be a cell-mediated autoimmune hair loss disease. Both CD4 and cytotoxic CD8 T cells (CTLs) are important for the onset and progression of AA. Hair follicle (HF) keratinocyte and/or melanocyte antigen epitopes are suspected potential targets of autoreactive CTLs, but the specific epitopes have not yet been identified. We investigated the potential for a panel of known epitopes, expressed by HF keratinocytes and melanocytes, to induce activation of CTL populations in peripheral blood mononuclear cells. Specific synthetic epitopes derived from HF antigens trichohyalin and tyrosinase-related protein-2 induced significantly higher frequencies of response in AA CTLs compared with healthy controls (IFN-gamma secretion). Apoptosis assays revealed conditioned media from AA peripheral blood mononuclear cells stimulated with trichohyalin peptides elevated the expression of apoptosis markers in primary HF keratinocytes. A cytokine array revealed higher expression of IL-13 and chemokine ligand 5 (CCL5, RANTES) from AA peripheral blood mononuclear cells stimulated with trichohyalin peptides compared with controls. The data indicate that AA affected subjects present with an increased frequency of CTLs responsive to epitopes originating from keratinocytes and melanocytes; the activated CTLs secreted soluble factors that induced apoptosis in HF keratinocytes. Potentially, CTL response to self-antigen epitopes, particularly trichohyalin epitopes, could be a prognostic marker for human AA.

  12. The three-dimensional structure of Infectious flacherie virus capsid determined by cryo-electron microscopy

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Cryo-electron microscopy and image reconstruction were used to determine the three-dimensional structure of Infectious flacherie virus (IFV). 5047 particles were selected for the final reconstruction. The FSC curve showed that the resolution of this capsid structure was 18 ·. The structure is a psuedo T=3 (P=3) icosahedral capsid with a diameter of 302.4 · and a single shell thickness of 15 ·. The density map showed that IFV has a smooth surface without any prominent protrude or depression. Comparison of the IFV structure with those of the insect picorna-like virus-Cricket paralysis virus (CrPV)and human picornavirus-Human rhinovirus 14 (HRV 14) revealed that the IFV structure resembles the CrPV structure. The "Rossmann canyon" is absent in both IFV and CrPV particles. The polypeptide topology of IFV VP2, IFV VP3 was predicted and the subunit location at the capsid surface was further analyzed.

  13. A Ginzburg-Landau model for the expansion of a dodecahedral viral capsid

    Science.gov (United States)

    Zappa, E.; Indelicato, G.; Albano, A.; Cermelli, P.

    2013-11-01

    We propose a Ginzburg-Landau model for the expansion of a dodecahedral viral capsid during infection or maturation. The capsid is described as a dodecahedron whose faces, meant to model rigid capsomers, are free to move independent of each other, and has therefore twelve degrees of freedom. We assume that the energy of the system is a function of the twelve variables with icosahedral symmetry. Using techniques of the theory of invariants, we expand the energy as the sum of invariant polynomials up to fourth order, and classify its minima in dependence of the coefficients of the Ginzburg-Landau expansion. Possible conformational changes of the capsid correspond to symmetry breaking of the equilibrium closed form. The results suggest that the only generic transition from the closed state leads to icosahedral expanded form. Our approach does not allow to study the expansion pathway, which is likely to be non-icosahedral.

  14. Automated liquid operation method for microfluidic heterogeneous immunoassay.

    Science.gov (United States)

    Yi, Hui; Pan, Jian-Zhang; Shi, Xiao-Tong; Fang, Qun

    2013-02-15

    In this work, an automated liquid operation method for multistep heterogeneous immunoassay toward point of care testing (POCT) was proposed. A miniaturized peristaltic pump was developed to control the flow direction, flow time and flow rate in the microliter range according to a program. The peristaltic pump has the advantages of simple structure, small size, low cost, and easy to build and use. By coupling the peristaltic pump with an antibody-coated capillary and a reagent-preloaded cartridge, the complicated liquid handling operation for heterogeneous immunoassay, including sample metering and introduction, multistep reagent introduction and rinsing, could be triggered by an action and accomplished automatically in 12 min. The analytical performance of the present immunoassay system was demonstrated in the measurement of human IgG with fluorescence detection. A detection limit of 0.68 μg/mL IgG and a dynamic range of 2-300 μg/mL were obtained.

  15. Potential applications of immunoassays in studies of flatfish recruitment

    Science.gov (United States)

    Feller, Robert J.

    The fisheries recruitment-stock problem, a lack of correlation between measures of reproductive output of the parent stock and recruitment to the fishery, has several potential biotic and abiotic causes. Immunoassays may be useful in examining several aspects of this and several other problems in flatfish ecology: stock identification, parasitism and disease, and trophic interactions. Given stage-specific antisera capable of recognozing antigenic moieties of, for instance, eggs, larvae, or newly-settled juveniles, it is possible to screen stomach contents of many putative predators ( e.g., shrimp or crabs) rapidly for the presence and amounts of platfish prey. This trophic application of immunological methods has great promise for measuring loss of potential recruits to predation. All immunoassays are limited by the quality of antisera used and the researcher's ability to interpret quantitative data in an ecologically meaningful way. Key references for applications of immunoassays in fish-related questions are provided with recommendations for their utilization.

  16. Structural Comparison of Different Antibodies Interacting with Parvovirus Capsids

    Energy Technology Data Exchange (ETDEWEB)

    Hafenstein, Susan; Bowman, Valorie D.; Sun, Tao; Nelson, Christian D.S.; Palermo, Laura M.; Chipman, Paul R.; Battisti, Anthony J.; Parrish, Colin R.; Rossmann, Michael G.; Cornell; Purdue

    2009-05-13

    The structures of canine parvovirus (CPV) and feline parvovirus (FPV) complexed with antibody fragments from eight different neutralizing monoclonal antibodies were determined by cryo-electron microscopy (cryoEM) reconstruction to resolutions varying from 8.5 to 18 {angstrom}. The crystal structure of one of the Fab molecules and the sequence of the variable domain for each of the Fab molecules have been determined. The structures of Fab fragments not determined crystallographically were predicted by homology modeling according to the amino acid sequence. Fitting of the Fab and virus structures into the cryoEM densities identified the footprints of each antibody on the viral surface. As anticipated from earlier analyses, the Fab binding sites are directed to two epitopes, A and B. The A site is on an exposed part of the surface near an icosahedral threefold axis, whereas the B site is about equidistant from the surrounding five-, three-, and twofold axes. One antibody directed to the A site binds CPV but not FPV. Two of the antibodies directed to the B site neutralize the virus as Fab fragments. The differences in antibody properties have been linked to the amino acids within the antibody footprints, the position of the binding site relative to the icosahedral symmetry elements, and the orientation of the Fab structure relative to the surface of the virus. Most of the exposed surface area was antigenic, although each of the antibodies had a common area of overlap that coincided with the positions of the previously mapped escape mutations.

  17. Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain.

    Science.gov (United States)

    Selzer, Lisa; Kant, Ravi; Wang, Joseph C-Y; Bothner, Brian; Zlotnick, Adam

    2015-11-20

    Hepatitis B virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty hepatitis B virus (HBV) T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage when compared with CTDs of Cp183-WT capsids. Cryo-electron microscopy studies of trypsin-digested capsids show that CTDs at five-fold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at five-fold, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the hepatitis B virus lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle.

  18. Assembly and characterization of foot-and-mouth disease virus empty capsid particles expressed within mammalian cells

    DEFF Research Database (Denmark)

    Gullberg, Maria; Muszynski, Bartosz; Organtini, Lindsey J.;

    2013-01-01

    The foot-and-mouth disease virus (FMDV) structural protein precursor, P1-2A, is cleaved by the virus-encoded 3C protease (3Cpro) into the capsid proteins VP0, VP1 and VP3 (and 2A). In some systems, it is difficult to produce large amounts of these processed capsid proteins since 3Cpro can be toxic...

  19. Poliovirus-associated protein kinase: Destabilization of the virus capsid and stimulation of the phosphorylation reaction by Zn sup 2+

    Energy Technology Data Exchange (ETDEWEB)

    Ratka, M.; Lackmann, M.; Ueckermann, C.; Karlins, U.; Koch, G. (Univ. of Hamburg (West Germany))

    1989-09-01

    The previously described poliovirus-associated protein kinase activity phosphorylates viral proteins VP0 and VP2 as well as exogenous proteins in the presence of Mg{sup 2+}. In this paper, the effect of Zn{sup 2+} on the phosphorylation reaction and the stability of the poliovirus capsid has been studied in detail and compared to that of Mg{sup 2+}. In the presence of Zn{sup 2+}, phosphorylation of capsid proteins VP2 and VP4 is significantly higher while phosphorylation of VP0 and exogenous phosphate acceptor proteins is not detected. The results indicate the activation of more than one virus-associated protein kinase by Zn{sup 2+}. The ion-dependent behavior of the enzyme activities is observed independently of whether the virus was obtained from HeLa or green monkey kidney cells. The poliovirus capsid is destabilized by Zn{sup 2+}. This alteration of the poliovirus capsid structure is a prerequisite for effective phosphorylation of viral capsid proteins. The increased level of phosphorylation of viral capsid proteins results in further destabilization of the viral capsid. As a result of the conformational changes, poliovirus-associated protein kinase activities dissociate from the virus particle. The authors suggest that the destabilizing effect of phosphorylation on the viral capsid plays a role in uncoating of poliovirus.

  20. Structural Transitions and Energy Landscape for Cowpea Chlorotic Mottle Virus Capsid Mechanics from Nanomanipulation in Vitro and in Silico

    NARCIS (Netherlands)

    Kononova, Olga; Snijder, Joost; Brasch, Melanie; Cornelissen, Jeroen; Dima, Ruxandra I.; Marx, Kenneth A.; Wuite, Gijs J. L.; Roos, Wouter H.; Barsegov, Valeri

    2013-01-01

    Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Combined AFM experiments and computational modeling on subsecond timescales of the indentation nanomechanics of

  1. Structural Transitions and Energy Landscape for Cowpea Chlorotic Mottle Virus Capsid Mechanics from Nanomanipulation in Vitro and in Silico

    Science.gov (United States)

    Kononova, Olga; Snijder, Joost; Brasch, Melanie; Cornelissen, Jeroen; Dima, Ruxandra I.; Marx, Kenneth A.; Wuite, Gijs J. L.; Roos, Wouter H.; Barsegov, Valeri

    2013-10-01

    Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Virus shells can have applications as nanocontainers and delivery vehicles in biotechnology and medicine. Combined AFM experiments and computational modeling on sub-second timescales of the indentation nanomechanics of Cowpea Chlorotic Mottle Virus (CCMV) capsid show that the capsid's physical properties are dynamic and local characteristics of the structure, which depend on the magnitude and geometry of mechanical input. Surprisingly, under large deformations the CCMV capsid transitions to the collapsed state without substantial local structural alterations. The enthalpy change in this deformation state dH = 11.5 - 12.8 MJ/mol is mostly due to large-amplitude out-of-plane excitations, which contribute to the capsid bending, and the entropy change TdS = 5.1 - 5.8 MJ/mol is mostly due to coherent in-plane rearrangements of protein chains, which result in the capsid stiffening. Dynamic coupling of these modes defines the extent of elasticity and reversibility of capsid mechanical deformation. This emerging picture illuminates how unique physico-chemical properties of protein nanoshells help define their structure and morphology, and determine their viruses' biological function.

  2. B Cell Epitope-Based Vaccination Therapy

    Directory of Open Access Journals (Sweden)

    Yoshie Kametani

    2015-08-01

    Full Text Available Currently, many peptide vaccines are undergoing clinical studies. Most of these vaccines were developed to activate cytotoxic T cells; however, the response is not robust. Unlike vaccines, anti-cancer antibodies based on passive immunity have been approved as a standard treatment. Since passive immunity is more effective in tumor treatment, the evidence suggests that limited B cell epitope-based peptide vaccines may have similar activity. Nevertheless, such peptide vaccines have not been intensively developed primarily because humoral immunity is thought to be preferable to cancer progression. B cells secrete cytokines, which suppress immune functions. This review discusses the possibility of therapeutic antibody induction by a peptide vaccine and the role of active and passive B cell immunity in cancer patients. We also discuss the use of humanized mice as a pre-clinical model. The necessity of a better understanding of the activity of B cells in cancer is also discussed.

  3. Breaking a virus: Identifying molecular level failure modes of a viral capsid by multiscale modeling

    Science.gov (United States)

    Krishnamani, V.; Globisch, C.; Peter, C.; Deserno, M.

    2016-07-01

    We use coarse-grained (CG) simulations to study the deformation of empty Cowpea Chlorotic Mottle Virus (CCMV) capsids under uniaxial compression, from the initial elastic response up to capsid breakage. Our CG model is based on the MARTINI force field and has been amended by a stabilizing elastic network, acting only within individual proteins, that was tuned to capture the fluctuation spectrum of capsid protein dimers, obtained from all atom simulations. We have previously shown that this model predicts force-compression curves that match AFM indentation experiments on empty CCMV capsids. Here we investigate details of the actual breaking events when the CCMV capsid finally fails. We present a symmetry classification of all relevant protein contacts and show that they differ significantly in terms of stability. Specifically, we show that interfaces which break readily are precisely those which are believed to form last during assembly, even though some of them might share the same contacts as other non-breaking interfaces. In particular, the interfaces that form pentamers of dimers never break, while the virtually identical interfaces within hexamers of dimers readily do. Since these units differ in the large-scale geometry and, most noticeably, the cone-angle at the center of the 5- or 6-fold vertex, we propose that the hexameric unit fails because it is pre-stressed. This not only suggests that hexamers of dimers form less frequently during the early stages of assembly; it also offers a natural explanation for the well-known β-barrel motif at the hexameric center as a post-aggregation stabilization mechanism. Finally, we identify those amino acid contacts within all key protein interfaces that are most persistent during compressive deformation of the capsid, thereby providing potential targets for mutation studies aiming to elucidate the key contacts upon which overall stability rests.

  4. Breaking a virus: Identifying molecular level failure modes of a viral capsid by multiscale modeling

    Science.gov (United States)

    Krishnamani, V.; Globisch, C.; Peter, C.; Deserno, M.

    2016-10-01

    We use coarse-grained (CG) simulations to study the deformation of empty Cowpea Chlorotic Mottle Virus (CCMV) capsids under uniaxial compression, from the initial elastic response up to capsid breakage. Our CG model is based on the MARTINI force field and has been amended by a stabilizing elastic network, acting only within individual proteins, that was tuned to capture the fluctuation spectrum of capsid protein dimers, obtained from all atom simulations. We have previously shown that this model predicts force-compression curves that match AFM indentation experiments on empty CCMV capsids. Here we investigate details of the actual breaking events when the CCMV capsid finally fails. We present a symmetry classification of all relevant protein contacts and show that they differ significantly in terms of stability. Specifically, we show that interfaces which break readily are precisely those which are believed to form last during assembly, even though some of them might share the same contacts as other non-breaking interfaces. In particular, the interfaces that form pentamers of dimers never break, while the virtually identical interfaces within hexamers of dimers readily do. Since these units differ in the large-scale geometry and, most noticeably, the cone-angle at the center of the 5- or 6-fold vertex, we propose that the hexameric unit fails because it is pre-stressed. This not only suggests that hexamers of dimers form less frequently during the early stages of assembly; it also offers a natural explanation for the well-known β-barrel motif at the hexameric center as a post-aggregation stabilization mechanism. Finally, we identify those amino acid contacts within all key protein interfaces that are most persistent during compressive deformation of the capsid, thereby providing potential targets for mutation studies aiming to elucidate the key contacts upon which overall stability rests.

  5. Internal Proteins of the Procapsid and Mature Capsids of Herpes Simplex Virus 1 Mapped by Bubblegram Imaging

    Science.gov (United States)

    Wu, Weimin; Newcomb, William W.; Cheng, Naiqian; Aksyuk, Anastasia; Winkler, Dennis C.

    2016-01-01

    ABSTRACT The herpes simplex virus 1 (HSV-1) capsid is a huge assembly, ∼1,250 Å in diameter, and is composed of thousands of protein subunits with a combined mass of ∼200 MDa, housing a 100-MDa genome. First, a procapsid is formed through coassembly of the surface shell with an inner scaffolding shell; then the procapsid matures via a major structural transformation, triggered by limited proteolysis of the scaffolding proteins. Three mature capsids are found in the nuclei of infected cells. A capsids are empty, B capsids retain a shrunken scaffolding shell, and C capsids—which develop into infectious virions—are filled with DNA and ostensibly have expelled the scaffolding shell. The possible presence of other internal proteins in C capsids has been moot as, in cryo-electron microscopy (cryo-EM), they would be camouflaged by the surrounding DNA. We have used bubblegram imaging to map internal proteins in all four capsids, aided by the discovery that the scaffolding protein is exceptionally prone to radiation-induced bubbling. We confirmed that this protein forms thick-walled inner shells in the procapsid and the B capsid. C capsids generate two classes of bubbles: one occupies positions beneath the vertices of the icosahedral surface shell, and the other is distributed throughout its interior. A likely candidate is the viral protease. A subpopulation of C capsids bubbles particularly profusely and may represent particles in which expulsion of scaffold and DNA packaging are incomplete. Based on the procapsid structure, we propose that the axial channels of hexameric capsomers afford the pathway via which the scaffolding protein is expelled. IMPORTANCE In addition to DNA, capsids of tailed bacteriophages and their distant relatives, herpesviruses, contain internal proteins. These proteins are often essential for infectivity but are difficult to locate within the virion. A novel adaptation of cryo-EM based on detecting gas bubbles generated by radiation

  6. Research Progress of Ig Y Application in Immunoassay

    Institute of Scientific and Technical Information of China (English)

    Zhang Jingjing

    2015-01-01

    Immunoglobulin of yolk( IgY) is the major antibody in birds,reptiles and amphibian,which is generally extracted from yolk of immunized hen,with a biological role similar to mammal Ig G. Compared with IgG,IgY has many significant characters. It is extensively applied in production of polyclonal antibodies against various antigens,immunoassay and immunotherapy,and many medical areas in both animals and human. This article summarized research progress of IgY application in immunoassay.

  7. Prediction of epitopes using neural network based methods

    DEFF Research Database (Denmark)

    Lundegaard, Claus; Lund, Ole; Nielsen, Morten

    2011-01-01

    been evaluated to be among the very best performing MHC:peptide binding predictors available. Here we describe the background for these methods, and the rationale behind the different optimization steps implemented in the methods. We go through the practical use of the methods, which are publicly...... available in the form of relatively fast and simple web interfaces. Furthermore, we will review results obtained in actual epitope discovery projects where previous implementations of the described methods have been used in the initial selection of potential epitopes. Selected potential epitopes were all...

  8. Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen.

    OpenAIRE

    Lin, S F; Sun, R; Heston, L; Gradoville, L; Shedd, D; Haglund, K; Rigsby, M; Miller, G.

    1997-01-01

    We describe a recombinant antigen for use in serologic tests for antibodies to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV). The cDNA for a small viral capsid antigen (sVCA) was identified by immunoscreening of a library prepared from the BC-1 body cavity lymphoma cell line induced into KSHV lytic gene expression by sodium butyrate. The cDNA specified a 170-amino-acid peptide with homology to small viral capsid proteins encoded by the BFRF3 gene of Epstein-Barr virus and the ORF65 gene...

  9. Capsid coding sequences of foot-and-mouth disease viruses are determinants of pathogenicity in pigs

    DEFF Research Database (Denmark)

    Lohse, Louise; Jackson, Terry; Bøtner, Anette;

    2012-01-01

    The surface exposed capsid proteins, VP1, VP2 and VP3, of foot-and-mouth disease virus (FMDV) determine its antigenicity and the ability of the virus to interact with host-cell receptors. Hence, modification of these structural proteins may alter the properties of the virus. In the present study we...... B64 virus and the two chimeric viruses are identical to each other except for the capsid coding region. Animals exposed to O1K B64 did not exhibit signs of disease, while pigs exposed to each of the other viruses showed typical clinical signs of foot-and-mouth disease (FMD). All pigs infected...

  10. Solid-State NMR Studies of HIV-1 Capsid Protein Assemblies

    OpenAIRE

    HAN, YUN; Ahn, Jinwoo; Concel, Jason; Byeon, In-Ja L.; Gronenborn, Angela M.; YANG, Jun; Polenova, Tatyana

    2010-01-01

    In mature HIV-1 virions, a 26.6 kDa CA protein is assembled into a characteristic cone shaped core (capsid) that encloses the RNA viral genome. The assembled capsid structure is best described by a fullerene cone model that is made up from a hexameric lattice containing a variable number of CA pentamers, thus allowing for closure of tubular or conical structures. In this report, we present a solid-state NMR analysis of the wild type HIV-1 CA protein, prepared as conical and spherical assembli...

  11. Transient expression of Human papillomavirus type 16 L2 epitope fused to N- and C-terminus of coat protein of Potato virus X in plants

    Indian Academy of Sciences (India)

    Noemi Cerovska; Hana Hoffmeisterova; Tomas Moravec; Helena Plchova; Jitka Folwarczna; Helena Synkova; Helena Ryslava; Viera Ludvikova; Michal Smahel

    2012-03-01

    Transient expression of foreign genes based on plant viral vectors is a suitable system for the production of relevant immunogens that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study the epitope derived from HPV-16 L2 minor capsid protein (amino acids 108–120) was expressed from Potato virus X (PVX)-based vector pGR106 as N- or C-terminal fusion with the PVX coat protein (PVX CP) in transgenic Nicotiana benthamiana plants. The fusion protein L2108-120-PVX CP was successfully expressed in plants at a level of 170 mg/kg of fresh leaf tissue. The C-terminal fusion protein PVX CP- L2108-120 was expressed using mutated vector sequence to avoid homologous recombination at a level of 8 mg/kg of fresh leaf tissue. Immunogenicity of L2108-120-PVX CP virus-like particles was tested after immunization of mice by subcutaneous injection or tattoo administration. In animal sera the antibodies against the PVX CP and the L2108-120 epitope were found after both methods of vaccine delivery.

  12. Immunogenicity of papaya mosaic virus-like particles fused to a hepatitis C virus epitope: evidence for the critical function of multimerization.

    Science.gov (United States)

    Denis, Jérôme; Majeau, Nathalie; Acosta-Ramirez, Elizabeth; Savard, Christian; Bedard, Marie-Claude; Simard, Sabrina; Lecours, Katia; Bolduc, Marilène; Pare, Christine; Willems, Bernard; Shoukry, Naglaa; Tessier, Philippe; Lacasse, Patrick; Lamarre, Alain; Lapointe, Réjean; Lopez Macias, Constantino; Leclerc, Denis

    2007-06-20

    Plant-virus-based vaccines have emerged as a promising avenue in vaccine development. This report describes the engineering of an innovative vaccine platform using the papaya mosaic virus (PapMV) capsid protein (CP) as a carrier protein and a C-terminal fused hepatitis C virus (HCV) E2 epitope as the immunogenic target. Two antigen organizations of the PapMV-based vaccines were tested: a virus-like-particle (VLP; PapMVCP-E2) and a monomeric form (PapMVCP(27-215)-E2). While the two forms of the vaccine were both shown to be actively internalized in vitro in bone-marrow-derived antigen presenting cells (APCs), immunogenicity was demonstrated to be strongly dependent on antigen organization. Indeed, C3H/HeJ mice injected twice with the multimeric VLP vaccine showed a long-lasting humoral response (more than 120 days) against both the CP and the fused HCV E2 epitope. The antibody profile (production of IgG1, IgG2a, IgG2b, IgG3) suggests a Th1/Th2 response. Immunogenicity of the PapMV vaccine platform was not observed when the monomer PapMVCP-E2 was injected. These results demonstrate for the first time the potential of the PapMV vaccine platform and the critical function of multimerization in its immunogenicity.

  13. Sensitive enzyme immunoassay for hepatitis B virus core-related antigens and their correlation to virus load.

    Science.gov (United States)

    Kimura, Tatsuji; Rokuhara, Akinori; Sakamoto, Yoko; Yagi, Shintaro; Tanaka, Eiji; Kiyosawa, Kendo; Maki, Noboru

    2002-02-01

    A sensitive enzyme immunoassay (EIA) specific for hepatitis B virus core antigen (HBcAg) and hepatitis B e antigen (HBeAg) was developed. We designated the precore/core gene products as hepatitis B virus (HBV) core-related antigens (HBcrAg). In order to detect HBcrAg even in anti-HBc/e antibody-positive specimens, the specimens were pretreated in detergents. The antibodies are inactivated by this pretreatment and, simultaneously, the antigens are released and the epitopes are exposed. The assay demonstrated 71 to 112% recovery using HBcrAg-positive sera. We observed no interference from the tested anticoagulants or blood components. When the cutoff value was tentatively set at 10(3) U/ml, all healthy control (HBsAg/HBV-DNA negative; n = 108) and anti-HCV antibody-positive (n = 59) sera were identified as negative. The assay showed a detection limit of 4 x 10(2) U/ml using recombinant antigen. Detection limits were compared in four serially diluted HBV high-titer sera. The HBcrAg assay demonstrated higher sensitivity than HBV-DNA transcription-mediated amplification (TMA) or HBeAg radio immunoassay (RIA) in the dilution test. HBcrAg concentrations correlated well with HBV-DNA TMA (r = 0.91, n = 29) and in-house real-time detection-PCR (r = 0.93, n = 47) in hepatitis B patients. On HBeAg/anti-HBe antibody seroconversion panels, the HBcrAg concentration changed in accordance with HBV-DNA levels. HBcrAg concentration provides a reflection of HBV virus load equivalent to HBV-DNA level, and the assay therefore offers a simple method for monitoring hepatitis B patients.

  14. Design and Characterization of Epitope-Scaffold Immunogens That Present the Motavizumab Epitope from Respiratory Syncytial Virus

    Energy Technology Data Exchange (ETDEWEB)

    McLellan, Jason S.; Correia, Bruno E.; Chen, Man; Yang, Yongping; Graham, Barney S.; Schief, William R.; Kwong, Peter D. (UWASH); (NIH)

    2012-06-28

    Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, but an effective vaccine has not yet been developed. An ideal vaccine would elicit protective antibodies while avoiding virus-specific T-cell responses, which have been implicated in vaccine-enhanced disease with previous RSV vaccines. We propose that heterologous proteins designed to present RSV-neutralizing antibody epitopes and to elicit cognate antibodies have the potential to fulfill these vaccine requirements, as they can be fashioned to be free of viral T-cell epitopes. Here we present the design and characterization of three epitope-scaffolds that present the epitope of motavizumab, a potent neutralizing antibody that binds to a helix-loop-helix motif in the RSV fusion glycoprotein. Two of the epitope-scaffolds could be purified, and one epitope-scaffold based on a Staphylococcus aureus protein A domain bound motavizumab with kinetic and thermodynamic properties consistent with the free epitope-scaffold being stabilized in a conformation that closely resembled the motavizumab-bound state. This epitope-scaffold was well folded as assessed by circular dichroism and isothermal titration calorimetry, and its crystal structure (determined in complex with motavizumab to 1.9 {angstrom} resolution) was similar to the computationally designed model, with all hydrogen-bond interactions critical for binding to motavizumab preserved. Immunization of mice with this epitope-scaffold failed to elicit neutralizing antibodies but did elicit sera with F binding activity. The elicitation of F binding antibodies suggests that some of the design criteria for eliciting protective antibodies without virus-specific T-cell responses are being met, but additional optimization of these novel immunogens is required.

  15. Development of a widely applicable immunoassay for insulin in marine teleosts that regulates cross-reactivity using biotinylation and inhibits interference by plasma components.

    Science.gov (United States)

    Andoh, Tadashi

    2016-01-15

    Amino acids are important insulinotropins in fish, and their effects vary between amino acids and fish species. Insulin levels are indicative of growth efficiency and stress levels in fish; however, interspecies comparisons of insulin levels are hampered by the difficulty of measuring insulin concentration in each fish. We developed a widely applicable competitive immunoassay using biotinylated yellowtail (Seriola quinqueradiata) insulin for measuring insulin in marine teleosts, including yellowtail and red seabream (Pagrus major), which are the most common species raised by aquaculture in Japan. Amino acid sequence substitution was limited at the ninth residue of the A-chain (A9) between these two species, and analysis of the primary structures of insulins from six phylogenetically far teleosts suggested that the sequences of yellowtail and red seabream insulins are identical to those of many teleosts, except the A9 residue. However, A9 is known to be an epitope that confers cross-reactive differences on insulin. We solved this problem through immunoreactive invalidation of this residue by biotinylation. The binding-inhibition curves of yellowtail and red seabream insulins were identical following the use of this technique. However, yellowtail and red seabream plasma was found to contain components that interfere with immunoassays. This problem was solved by the extraction of plasma using equal volume of acid-ethanol in yellowtail and by cooling at 0°C during the cross-reaction between the ligand and antibody in red seabream. Serially diluted plasma samples from both species exhibited linearity after these treatments. In a recovery test using plasma with added yellowtail insulin, the average recovery varied from 96.2% to 109.4%. A post-feeding rise in insulin was confirmed by this immunoassay in yellowtail, and peak of the rise was 39.8±7ng/ml at 1h postfeeding from 3.9±1.1ng/ml at 0h. This indicates that this assay is sufficient for measuring the baseline

  16. Polyglutamine- and temperature-dependent conformational rigidity in mutant huntingtin revealed by immunoassays and circular dichroism spectroscopy.

    Directory of Open Access Journals (Sweden)

    Valentina Fodale

    Full Text Available BACKGROUND: In Huntington's disease, expansion of a CAG triplet repeat occurs in exon 1 of the huntingtin gene (HTT, resulting in a protein bearing>35 polyglutamine residues whose N-terminal fragments display a high propensity to misfold and aggregate. Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT, which likely influence its biological and biophysical properties. Developing assays to characterize and measure these conformational changes in isolated proteins and biological samples would advance the testing of novel therapeutic approaches aimed at correcting mutant HTT misfolding. Time-resolved Förster energy transfer (TR-FRET-based assays represent high-throughput, homogeneous, sensitive immunoassays widely employed for the quantification of proteins of interest. TR-FRET is extremely sensitive to small distances and can therefore provide conformational information based on detection of exposure and relative position of epitopes present on the target protein as recognized by selective antibodies. We have previously reported TR-FRET assays to quantify HTT proteins based on the use of antibodies specific for different amino-terminal HTT epitopes. Here, we investigate the possibility of interrogating HTT protein conformation using these assays. METHODOLOGY/PRINCIPAL FINDINGS: By performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein at different temperatures, we have discovered a temperature-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins. Circular dichroism spectroscopy confirms the temperature and polyglutamine-dependent change in HTT structure, revealing an effect of polyglutamine length and of temperature on the alpha-helical content of the protein. CONCLUSIONS/SIGNIFICANCE: The temperature- and polyglutamine

  17. An assessment on epitope prediction methods for protozoa genomes

    Directory of Open Access Journals (Sweden)

    Resende Daniela M

    2012-11-01

    Full Text Available Abstract Background Epitope prediction using computational methods represents one of the most promising approaches to vaccine development. Reduction of time, cost, and the availability of completely sequenced genomes are key points and highly motivating regarding the use of reverse vaccinology. Parasites of genus Leishmania are widely spread and they are the etiologic agents of leishmaniasis. Currently, there is no efficient vaccine against this pathogen and the drug treatment is highly toxic. The lack of sufficiently large datasets of experimentally validated parasites epitopes represents a serious limitation, especially for trypanomatids genomes. In this work we highlight the predictive performances of several algorithms that were evaluated through the development of a MySQL database built with the purpose of: a evaluating individual algorithms prediction performances and their combination for CD8+ T cell epitopes, B-cell epitopes and subcellular localization by means of AUC (Area Under Curve performance and a threshold dependent method that employs a confusion matrix; b integrating data from experimentally validated and in silico predicted epitopes; and c integrating the subcellular localization predictions and experimental data. NetCTL, NetMHC, BepiPred, BCPred12, and AAP12 algorithms were used for in silico epitope prediction and WoLF PSORT, Sigcleave and TargetP for in silico subcellular localization prediction against trypanosomatid genomes. Results A database-driven epitope prediction method was developed with built-in functions that were capable of: a removing experimental data redundancy; b parsing algorithms predictions and storage experimental validated and predict data; and c evaluating algorithm performances. Results show that a better performance is achieved when the combined prediction is considered. This is particularly true for B cell epitope predictors, where the combined prediction of AAP12 and BCPred12 reached an AUC value

  18. A Compact Immunoassay Platform Based on a Multicapillary Glass Plate

    Directory of Open Access Journals (Sweden)

    Shuhua Xue

    2014-05-01

    Full Text Available A highly sensitive, rapid immunoassay performed in the multi-channels of a micro-well array consisting of a multicapillary glass plate (MCP and a polydimethylsiloxane (PDMS slide is described. The micro-dimensions and large surface area of the MCP permitted the diffusion distance to be decreased and the reaction efficiency to be increased. To confirm the concept of the method, human immunoglobulin A (h-IgA was measured using both the proposed immunoassay system and the traditional 96-well plate method. The proposed method resulted in a 1/5-fold decrease of immunoassay time, and a 1/56-fold cut in reagent consumption with a 0.05 ng/mL of limit of detection (LOD for IgA. The method was also applied to saliva samples obtained from healthy volunteers. The results correlated well to those obtained by the 96-well plate method. The method has the potential for use in disease diagnostic or on-site immunoassays.

  19. Detection of cyclopiazonic acid (CPA) in maize by immunoassay

    Science.gov (United States)

    Cyclopiazonic acid (a-CPA) is a tremorgenic mycotoxin that is commonly produced by certain of the Aspergilli, in particular A. flavus, which is more widely known for production of the aflatoxins. Despite the fact that a-CPA may co-occur with aflatoxins, immunoassay-based methods for monitoring for C...

  20. A compact immunoassay platform based on a multicapillary glass plate.

    Science.gov (United States)

    Xue, Shuhua; Zeng, Hulie; Yang, Jianmin; Nakajima, Hizuru; Uchiyama, Katsumi

    2014-05-23

    A highly sensitive, rapid immunoassay performed in the multi-channels of a micro-well array consisting of a multicapillary glass plate (MCP) and a polydimethylsiloxane (PDMS) slide is described. The micro-dimensions and large surface area of the MCP permitted the diffusion distance to be decreased and the reaction efficiency to be increased. To confirm the concept of the method, human immunoglobulin A (h-IgA) was measured using both the proposed immunoassay system and the traditional 96-well plate method. The proposed method resulted in a 1/5-fold decrease of immunoassay time, and a 1/56-fold cut in reagent consumption with a 0.05 ng/mL of limit of detection (LOD) for IgA. The method was also applied to saliva samples obtained from healthy volunteers. The results correlated well to those obtained by the 96-well plate method. The method has the potential for use in disease diagnostic or on-site immunoassays.

  1. Biosensor immunoassay for flumequine in broiler serum and muscle

    NARCIS (Netherlands)

    Haasnoot, W.; Gercek, H.; Cazemier, G.; Nielen, M.W.F.

    2007-01-01

    Flumequine (Flu) is one of the fluoroquinolones most frequently applied for the treatment of broilers in The Netherlands. For the detection of residues of Flu in blood serum of broilers, a biosensor immunoassay (BIA) was developed which was fast (7.5 min per sample) and specific (no cross-reactivity

  2. Flow cytometric immunoassay for sulfonamides in raw milk

    NARCIS (Netherlands)

    Keizer, de W.; Bienenmann-Ploum, M.; Bergwerff, A.A.; Haasnoot, W.

    2008-01-01

    Sulfonamide antibiotics are applied in veterinary medicine for the treatment of microbial infections. For the detection of residues of sulfonamides in milk, a multi-sulfonamide flow cytometric immunoassay (FCI) was developed using the Luminex MultiAnalyte Profiling (xMAP) technology. In this automat

  3. Dimethylamylamine: a drug causing positive immunoassay results for amphetamines.

    Science.gov (United States)

    Vorce, Shawn P; Holler, Justin M; Cawrse, Brian M; Magluilo, Joseph

    2011-04-01

    The Department of Defense (DoD) operates six forensic urine drug-testing laboratories that screen close to 5 million urine samples for amphetamines yearly. Recently, the DoD laboratories have observed a significant decrease in the confirmation rates for amphetamines because of specimens screening positive by two separate immunoassays and confirming negative by gas chromatography-mass spectrometry (GC-MS). Previous studies conducted by the Division of Forensic Toxicology, Armed Force Institute of Pathology (AFIP) utilizing a GC-MS basic drug screen and a designer drug screen revealed no common compound or compound classes as to the cause of the immunoassay-positive results. Additional information obtained from an immunoassay vendor suggested the anorectic compound dimethylamylamine (DMAA) may be the cause of the false-positive screens. An additional 134 false-positive samples were received and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS-MS) for DMAA. LC-MS-MS analysis revealed the presence of DMAA in 92.3% of the false-positive samples at a concentration of approximately 6.0 mg/L DMAA, causing a positive screen on both immunoassay kits.

  4. Multiplex biosensor immunoassays for antibiotics in the food chain

    NARCIS (Netherlands)

    Haasnoot, W.

    2009-01-01

    The use of antibiotics in food-producing animals may result in unwanted residues in food products. The main objective of the present research was to study the development and application of fast and automated multiplex surface plasmon resonance (SPR)-based biosensor immunoassays (BIAs), based on mul

  5. An improved competitive inhibition enzymatic immunoassay method for tetrodotoxin quantification

    OpenAIRE

    Stokes Amber N; Williams Becky L; French Susannah S

    2012-01-01

    Abstract Quantifying tetrodotoxin (TTX) has been a challenge in both ecological and medical research due to the cost, time and training required of most quantification techniques. Here we present a modified Competitive Inhibition Enzymatic Immunoassay for the quantification of TTX, and to aid researchers in the optimization of this technique for widespread use with a high degree of accuracy and repeatability.

  6. What Can a Urine Drug Screening Immunoassay Really Tell Us?

    Science.gov (United States)

    Nelson, Zachary J; Stellpflug, Samuel J; Engebretsen, Kristin M

    2016-10-01

    Urine drug screening has become standard of care in many medical practice settings to assess compliance, detect misuse, and/or to provide basis for medical or legal action. The antibody-based enzymatic immunoassays used for qualitative analysis of urine have significant drawbacks that clinicians are often not aware of. Recent literature suggests that there is a lack of understanding of the shortcomings of these assays by clinicians who are ordering and/or interpreting them. This article addresses the state of each of the individual immunoassays that are most commonly used today in order to help the reader become proficient in the interpretation and application of the results. Some literature already exists regarding sources of "false positives" and "false negatives," but none seem to present the material with the practicing clinician in mind. This review aims to avoid overwhelming the reader with structures and analytical chemistry. The reader will be presented relevant clinical knowledge that will facilitate appropriate interpretation of immunoassays regardless of practice settings. Using this review as a learning tool and a reference, clinicians will be able to interpret the results of commonly used immunoassays in an evidence-based, informed manner and minimize the negative impact that misinterpretation has on patient care.

  7. Galactomannan enzymatic immunoassay cross-reactivity caused by Prototheca species.

    Science.gov (United States)

    Van den Bossche, D; De Bel, A; Hendrickx, M; De Becker, A; Jacobs, R; Naessens, A; Piérard, D

    2012-10-01

    We report a reactive Aspergillus galactomannan enzymatic immunoassay against the serum of a patient with invasive Prototheca zopfii infection. Analysis of the supernatants of suspensions of P. zopfii and other Prototheca isolates revealed positive results as well. These data suggest cross-reactivity with the serum Aspergillus galactomannan assay in invasive protothecosis.

  8. An improved competitive inhibition enzymatic immunoassay method for tetrodotoxin quantification

    Directory of Open Access Journals (Sweden)

    Stokes Amber N

    2012-03-01

    Full Text Available Abstract Quantifying tetrodotoxin (TTX has been a challenge in both ecological and medical research due to the cost, time and training required of most quantification techniques. Here we present a modified Competitive Inhibition Enzymatic Immunoassay for the quantification of TTX, and to aid researchers in the optimization of this technique for widespread use with a high degree of accuracy and repeatability.

  9. Galactomannan Enzymatic Immunoassay Cross-Reactivity Caused by Prototheca Species

    OpenAIRE

    Van den Bossche, D.; De Bel, A.; Hendrickx, M.; Becker, A.; Jacobs, R.; Naessens, A; Piérard, D.

    2012-01-01

    We report a reactive Aspergillus galactomannan enzymatic immunoassay against the serum of a patient with invasive Prototheca zopfii infection. Analysis of the supernatants of suspensions of P. zopfii and other Prototheca isolates revealed positive results as well. These data suggest cross-reactivity with the serum Aspergillus galactomannan assay in invasive protothecosis.

  10. An improved competitive inhibition enzymatic immunoassay method for tetrodotoxin quantification.

    Science.gov (United States)

    Stokes, Amber N; Williams, Becky L; French, Susannah S

    2012-01-01

    Quantifying tetrodotoxin (TTX) has been a challenge in both ecological and medical research due to the cost, time and training required of most quantification techniques. Here we present a modified Competitive Inhibition Enzymatic Immunoassay for the quantification of TTX, and to aid researchers in the optimization of this technique for widespread use with a high degree of accuracy and repeatability.

  11. Kinase Activity Studied in Living Cells Using an Immunoassay

    Science.gov (United States)

    Bavec, Aljos?a

    2014-01-01

    This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

  12. Toward a dry reagent immunoassay of progesterone in bovine milk

    NARCIS (Netherlands)

    Posthuma-Trumpie, Geertruida Afina

    2008-01-01

    This thesis is aimed at the development of a dry reagent immunoassay of progesterone in cow's milk. Progesterone is a steroid hormone and regulates ovulation in female mammals. The concentration of progesterone in blood and in milk is in accordance with the reproductive cycle of the individual femal

  13. Is the turbidimetric immunoassay of haptoglobin phenotype-dependent?

    NARCIS (Netherlands)

    Rijn, H.J.M. van; Wilt, W. van der; Stroes, J.W.; Schrijver, J.

    1987-01-01

    Comparison of the turbidimetric immunoassay of haptoglobin with a reference method (the RID technique with appropriate correction for phenotype) clearly showed the turbidimetric assay to be phenotype-dependent. Correction factors for the three main phenotypes were calculated and reference values det

  14. A rapid dot immunoassay for detecting the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius with a "flow through" device.

    Science.gov (United States)

    Ajello, G W; Matar, G M; Swaminathan, B; Bibb, W F; Helsel, L O; Perkins, B A

    1995-06-01

    Brazilian purpuric fever (BPF) is a highly fatal pediatric disease that may follow an episode of purulent conjunctivitis caused by a virulent clone of Haemophilus influenzae biogroup aegyptius (Hae). Oral rifampin prophylaxis, by eliminating carriage of the BPF clone in children with conjunctivitis, may prevent onset of the systemic disease. A test to detect the BPF clone directly from eye swabs could identify those in need of prophylaxis. This is a preliminary report of a rapid dot immunoassay performed on a "flow-through" cartridge that was developed for use under field conditions. The test is based upon recognition of a unique epitope of the 25-kDa pilin protein on the surface of BPF clone cells by a monoclonal antibody. With 36 laboratory-maintained cultures of Hae (15 clone isolates and 21 others), sensitivity of the assay was 67% and specificity was 95%. When fimbrial-enriched (25-kDa+) phenotypes of five false-negative clone strains were prepared for use as test antigens, sensitivity rose to 100%. Evaluation of the immunoassay under field conditions is necessary to prove its efficacy.

  15. Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Warner, Marvin G.; Grate, Jay W.; Tyler, Abby J.; Ozanich, Richard M.; Miller, Keith D.; Lou, Jianlong; Marks, James D.; Bruckner-Lea, Cindy J.

    2009-09-01

    A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum toxin serotype A (BoNT/A). For the development of the assay, a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) has been used as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader. Detection down to 31 pM of the BoNT/Hc-fragment was demonstrated with a total incubation time of 3 hours, using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the immunochemical reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. These beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell as part of a sequential injection fluidic system. This flow cell was equipped with a fiber optic system for fluorescence measurements. In PBS buffer solution matrix, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.

  16. Strategic Use of Epitope Matching to Improve Outcomes.

    Science.gov (United States)

    Wiebe, Chris; Nickerson, Peter

    2016-10-01

    Understanding the events leading to allorecognition and the subsequent effector pathways engaged is key for the development of strategies to prolong graft survival. Optimizing patient outcomes will require 2 major advancements: (1) minimizing premature death with a functioning graft in the patients with stable graft function, and (2) maximizing graft survival by avoiding the aforementioned allorecognition. This necessitates personalized immunosuppression to avoid known metabolic side effects, risk for infection, and malignancy, while holding the alloimmune system in check. Since the beginning of transplant a key strategy to achieve this goal is to minimize HLA mismatching between donor and recipient. What has not evolved is any refinement in our evaluation of HLA relatedness between donor and recipient when HLA mismatch exists. Donor-recipient HLA mismatch at the amino acid level can now be determined. These mismatches serve as potential epitopes for de novo donor specific antibody development and correlate with late rejection and graft loss. It is in this context that HLA epitope analysis is considered as a strategy to permit safe immunosuppression minimization to improve patient outcomes through: (1) improved allocation schemes that favor donor-recipient pairs with a low HLA epitope mismatch load (especially at the class II loci) or avoiding specific epitope mismatches known to be highly immunogenic and (2) immunosuppressive minimization in patients with low epitope mismatch loads or without highly immunogenic epitope mismatches.

  17. Computational elucidation of potential antigenic CTL epitopes in Ebola virus.

    Science.gov (United States)

    Dikhit, Manas R; Kumar, Santosh; Vijaymahantesh; Sahoo, Bikash R; Mansuri, Rani; Amit, Ajay; Yousuf Ansari, Md; Sahoo, Ganesh C; Bimal, Sanjiva; Das, Pradeep

    2015-12-01

    Cell-mediated immunity is important for the control of Ebola virus infection. We hypothesized that those HLA A0201 and HLA B40 restricted epitopes derived from Ebola virus proteins, would mount a good antigenic response. Here we employed an immunoinformatics approach to identify specific 9mer amino acid which may be capable of inducing a robust cell-mediated immune response in humans. We identified a set of 28 epitopes that had no homologs in humans. Specifically, the epitopes derived from NP, RdRp, GP and VP40 share population coverage of 93.40%, 84.15%, 74.94% and 77.12%, respectively. Based on the other HLA binding specificity and population coverage, seven novel promiscuous epitopes were identified. These 7 promiscuous epitopes from NP, RdRp and GP were found to have world-wide population coverage of more than 95% indicating their potential significance as useful candidates for vaccine design. Epitope conservancy analysis also suggested that most of the peptides are highly conserved (100%) in other virulent Ebola strain (Mayinga-76, Kikwit-95 and Makona-G3816- 2014) and can therefore be further investigated for their immunological relevance and usefulness as vaccine candidates.

  18. Kinetics of antigen expression and epitope presentation during virus infection.

    Directory of Open Access Journals (Sweden)

    Nathan P Croft

    2013-01-01

    Full Text Available Current knowledge about the dynamics of antigen presentation to T cells during viral infection is very poor despite being of fundamental importance to our understanding of anti-viral immunity. Here we use an advanced mass spectrometry method to simultaneously quantify the presentation of eight vaccinia virus peptide-MHC complexes (epitopes on infected cells and the amounts of their source antigens at multiple times after infection. The results show a startling 1000-fold range in abundance as well as strikingly different kinetics across the epitopes monitored. The tight correlation between onset of protein expression and epitope display for most antigens provides the strongest support to date that antigen presentation is largely linked to translation and not later degradation of antigens. Finally, we show a complete disconnect between the epitope abundance and immunodominance hierarchy of these eight epitopes. This study highlights the complexity of viral antigen presentation by the host and demonstrates the weakness of simple models that assume total protein levels are directly linked to epitope presentation and immunogenicity.

  19. Display of the Viral Epitopes on Lactococcus lactis: A Model for Food Grade Vaccine against EV71.

    Science.gov (United States)

    Varma, Nadimpalli Ravi S; Toosa, Haryanti; Foo, Hooi Ling; Alitheen, Noorjahan Banu Mohamed; Nor Shamsudin, Mariana; Arbab, Ali S; Yusoff, Khatijah; Abdul Rahim, Raha

    2013-01-01

    In this study, we have developed a system for display of antigens of Enterovirus type 71 (EV71) on the cell surface of L. lactis. The viral capsid protein (VP1) gene from a local viral isolate was utilized as the candidate vaccine for the development of oral live vaccines against EV71 using L. lactis as a carrier. We expressed fusion proteins in E. coli and purified fusion proteins were incubated with L. lactis. We confirmed that mice orally fed with L. lactis displaying these fusion proteins on its surface were able to mount an immune response against the epitopes of EV71. This is the first example of an EV71 antigen displayed on the surface of a food grade organism and opens a new perspective for alternative vaccine strategies against the EV71. We believe that the method of protein docking utilized in this study will allow for more flexible presentations of short peptides and proteins on the surface of L. lactis to be useful as a delivery vehicle.

  20. Display of the Viral Epitopes on Lactococcus lactis: A Model for Food Grade Vaccine against EV71

    Directory of Open Access Journals (Sweden)

    Nadimpalli Ravi S. Varma

    2013-01-01

    Full Text Available In this study, we have developed a system for display of antigens of Enterovirus type 71 (EV71 on the cell surface of L. lactis. The viral capsid protein (VP1 gene from a local viral isolate was utilized as the candidate vaccine for the development of oral live vaccines against EV71 using L. lactis as a carrier. We expressed fusion proteins in E. coli and purified fusion proteins were incubated with L. lactis. We confirmed that mice orally fed with L. lactis displaying these fusion proteins on its surface were able to mount an immune response against the epitopes of EV71. This is the first example of an EV71 antigen displayed on the surface of a food grade organism and opens a new perspective for alternative vaccine strategies against the EV71. We believe that the method of protein docking utilized in this study will allow for more flexible presentations of short peptides and proteins on the surface of L. lactis to be useful as a delivery vehicle.

  1. Nanofluidic Devices with Two Pores in Series for Resistive-Pulse Sensing of Single Virus Capsids

    DEFF Research Database (Denmark)

    Harms, Zachary D.; Mogensen, Klaus Bo; Rodrigues de Sousa Nunes, Pedro André;

    2011-01-01

    We report fabrication and characterization of nanochannel devices with two nanopores in series for resistive-pulse sensing of hepatitis B virus (HBV) capsids. The nanochannel and two pores are patterned by electron beam lithography between two microchannels and etched by reactive ion etching. The...

  2. Essential C-Terminal region of the baculovirus minor capsid protein VP80 binds DNA

    NARCIS (Netherlands)

    Marek, M.; Merten, O.W.; Francis-Devaraj, F.; Oers, van M.M.

    2012-01-01

    The essential Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) minor capsid protein VP80 has been recently shown to interact with the virus-triggered, nuclear F-actin cytoskeleton. A role for VP80 in virus morphogenesis has been proposed in the maturation of progeny nucleocapsids and

  3. The conformation of double-stranded DNA inside bacteriophages depends on capsid size and shape.

    Science.gov (United States)

    Petrov, Anton S; Boz, Mustafa Burak; Harvey, Stephen C

    2007-11-01

    The packaging of double-stranded DNA into bacteriophages leads to the arrangement of the genetic material into highly-packed and ordered structures. Although modern experimental techniques reveal the most probable location of DNA inside viral capsids, the individual conformations of DNA are yet to be determined. In the current study we present the results of molecular dynamics simulations of the DNA packaging into several bacteriophages performed within the framework of a coarse-grained model. The final DNA conformations depend on the size and shape of the capsid, as well as the size of the protein portal, if any. In particular, isometric capsids with small or absent portals tend to form concentric spools, whereas the presence of a large portal favors coaxial spooling; slightly and highly elongated capsids result in folded and twisted toroidal conformations, respectively. The results of the simulations also suggest that the predominant factor in defining the global DNA arrangement inside bacteriophages is the minimization of the bending stress upon packaging.

  4. Immobilization and One-Dimensional Arrangement of Virus Capsids With Nanoscale Precision Using DNA Origami

    Science.gov (United States)

    Stephanopoulos, Nicholas; Liu, Minghui; Tong, Gary J.; Li, Zhe; Liu, Yan; Yan, Hao; Francis, Matthew B.

    2011-01-01

    Self-assembly has proven to be one of the most effective ways to arrange matter at the nanometer level. Biology, in particular, makes extensive use of self-assembly to position molecules over several length scales with a high degree of spatial control over structure. In recent years, one promising approach that takes advantage of biological self-assembly in order to build synthetic materials employs virus capsids, the protein shells that encapsulate the genetic material of viruses.1 Capsids are composed of multiple protein subunits that can assemble (either spontaneously or under an external stimulus) into a monodisperse structure with different geometries depending on the virus. By appropriately functionalizing the proteins that comprise the capsid, multiple copies of a molecule or other entity can be positioned with a predictable arrangement. A wide variety of components have been attached to and arranged by virus capsids, including chromophores,2 catalysts,3 nanoparticles and quantum dots,4 polymers,5 drug molecules,6 and imaging agents.7 PMID:20575574

  5. Porcine circovirus-2 capsid protein induces cell death in PK15 cells

    Energy Technology Data Exchange (ETDEWEB)

    Walia, Rupali; Dardari, Rkia, E-mail: rdardari@ucalgary.ca; Chaiyakul, Mark; Czub, Markus

    2014-11-15

    Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathways involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis. - Highlights: • IFN-γ enhances PCV2 replication that leads to cell death in PK15 cells. • IFN-γ enhances nuclear localization of the PCV2 Capsid protein. • Transient PCV2a and 2b-Capsid protein expression induces cell death. • Cell death is not dictated by specific Capsid protein sub-localization.

  6. Functional dissection of the alphavirus capsid protease: sequence requirements for activity

    Directory of Open Access Journals (Sweden)

    Pützer Brigitte M

    2010-11-01

    Full Text Available Abstract Background The alphavirus capsid is multifunctional and plays a key role in the viral life cycle. The nucleocapsid domain is released by the self-cleavage activity of the serine protease domain within the capsid. All alphaviruses analyzed to date show this autocatalytic cleavage. Here we have analyzed the sequence requirements for the cleavage activity of Chikungunya virus capsid protease of genus alphavirus. Results Amongst alphaviruses, the C-terminal amino acid tryptophan (W261 is conserved and found to be important for the cleavage. Mutating tryptophan to alanine (W261A completely inactivated the protease. Other amino acids near W261 were not having any effect on the activity of this protease. However, serine protease inhibitor AEBSF did not inhibit the activity. Through error-prone PCR we found that isoleucine 227 is important for the effective activity. The loss of activity was analyzed further by molecular modelling and comparison of WT and mutant structures. It was found that lysine introduced at position 227 is spatially very close to the catalytic triad and may disrupt electrostatic interactions in the catalytic site and thus inactivate the enzyme. We are also examining other sequence requirements for this protease activity. Conclusions We analyzed various amino acid sequence requirements for the activity of ChikV capsid protease and found that amino acids outside the catalytic triads are important for the activity.

  7. Cloning and Sequence Analysis of Capsid Protein Gene of Iridovirus Indonesian Isolates

    Directory of Open Access Journals (Sweden)

    Murwantoko .

    2015-11-01

    Full Text Available generated by an Adobe application 11.5606 Iridovirus was known as agents that caused serious systemic disease in freshwater and marine fishes. The mortality up to 100% of orange-spotted grouper (Epinephelus coioides due to iridovirus infection has been reported in Indonesia. The gene encoding capsid protein of iridovirus is supposed to be conserved and has the potency for the development of control methods. The objectives of this study are to clone the gene encoding capsid protein iridovirus and to analyze their sequences. The   spleen tissues of orange-spotted grouper were collected and extracted their DNA. The DNA fragment of capsid protein of iridovirus genes were amplified by PCR using designed primers with the extraction DNA as templates. The amplified DNA fragments were cloned in pBSKSII and sequenced.  The genes encoding capsid protein of iridovirus from Jepara and Bali were successfully amplified and cloned. The Jepara clone (IJP03 contained complete open reading frame (ORF of the gene composed by 1362 bp nucleotides which encoded 453 amino acids. Those Jepara and Bali (IGD01 clones shared 99.8% similarity in nucleotide level and 99.4% at amino acid level. Based on those sequences, Indonesian iridovirus was belonged to genus Megalocystivirus and shared 99,6-99,9% similarity on nucleotide level with DGIV, ISKNV, MCIV, and ALIV Normal 0 36 false false false

  8. Disassociation of the SV40 Genome from Capsid Proteins Prior to Nuclear Entry

    Directory of Open Access Journals (Sweden)

    Kuksin Dmitry

    2012-08-01

    Full Text Available Abstract Background Previously, we demonstrated that input SV40 particles undergo a partial disassembly in the endoplasmic reticulum, which exposes internal capsid proteins VP2 and VP3 to immunostaining. Then, in the cytoplasm, disassembly progresses further to also make the genomic DNA accessible to immune detection, as well as to detection by an ethynyl-2-deoxyuridine (EdU-based chemical reaction. The cytoplasmic partially disassembled SV40 particles retain some of the SV40 capsid proteins, VP1, VP2, and VP3, in addition to the viral genome. Findings In the current study, we asked where in the cell the SV40 genome might disassociate from capsid components. We observed partially disassembled input SV40 particles around the nucleus and, beginning at 12 hours post-infection, 5-Bromo-2-deoxyuridine (BrdU-labeled parental SV40 DNA in the nucleus, as detected using anti-BrdU antibodies. However, among the more than 1500 cells examined, we never detected input VP2/VP3 in the nucleus. Upon translocation of the BrdU-labeled SV40 genomes into nuclei, they were transcribed and, thus, are representative of productive infection. Conclusions Our findings imply that the SV40 genome disassociates from the capsid proteins before or at the point of entry into the nucleus, and then enters the nucleus devoid of VP2/3.

  9. The Herpes Simplex Virus 1 UL17 Protein Is the Second Constituent of the Capsid Vertex-Specific Component Required for DNA Packaging and Retention▿

    OpenAIRE

    Toropova, Katerina; Huffman, Jamie B.; Homa, Fred L.; James F Conway

    2011-01-01

    The herpes simplex virus (HSV) UL17 and UL25 minor capsid proteins are essential for DNA packaging. They are thought to comprise a molecule arrayed in five copies around each of the capsid vertices. This molecule was initially termed the “C-capsid-specific component” (CCSC) (B. L. Trus et al., Mol. Cell 26:479-489, 2007), but as we have subsequently observed this feature on reconstructions of A, B, and C capsids, we now refer to it more generally as the “capsid vertex-specific component” (CVS...

  10. Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform.

    Science.gov (United States)

    Hanson, Cynthia; Israelsen, Nathan D; Sieverts, Michael; Vargis, Elizabeth

    2016-11-10

    Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available. Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags(1). Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

  11. Epitope Mapping of Avian Influenza M2e Protein: Different Species Recognise Various Epitopes.

    Directory of Open Access Journals (Sweden)

    Noor Haliza Hasan

    Full Text Available A common approach for developing diagnostic tests for influenza virus detection is the use of mouse or rabbit monoclonal and/or polyclonal antibodies against a target antigen of the virus. However, comparative mapping of the target antigen using antibodies from different animal sources has not been evaluated before. This is important because identification of antigenic determinants of the target antigen in different species plays a central role to ensure the efficiency of a diagnostic test, such as competitive ELISA or immunohistochemistry-based tests. Interest in the matrix 2 ectodomain (M2e protein of avian influenza virus (AIV as a candidate for a universal vaccine and also as a marker for detection of virus infection in vaccinated animals (DIVA is the rationale for the selection of this protein for comparative mapping evaluation. This study aimed to map the epitopes of the M2e protein of avian influenza virus H5N1 using chicken, mouse and rabbit monoclonal or monospecific antibodies. Our findings revealed that rabbit antibodies (rAbs recognized epitope 6EVETPTRN13 of the M2e, located at the N-terminal of the protein, while mouse (mAb and chicken antibodies (cAbs recognized epitope 10PTRNEWECK18, located at the centre region of the protein. The findings highlighted the difference between the M2e antigenic determinants recognized by different species that emphasized the importance of comparative mapping of antibody reactivity from different animals to the same antigen, especially in the case of multi-host infectious agents such as influenza. The findings are of importance for antigenic mapping, as well as diagnostic test and vaccine development.

  12. Hidden symmetry of small spherical viruses and organization principles in "anomalous" and double-shelled capsid nanoassemblies.

    Science.gov (United States)

    Rochal, S B; Konevtsova, O V; Myasnikova, A E; Lorman, V L

    2016-09-29

    We propose the principles of structural organization in spherical nanoassemblies with icosahedral symmetry constituted by asymmetric protein molecules. The approach modifies the paradigmatic geometrical Caspar and Klug (CK) model of icosahedral viral capsids and demonstrates the common origin of both the "anomalous" and conventional capsid structures. In contrast to all previous models of "anomalous" viral capsids the proposed modified model conserves the basic structural principles of the CK approach and reveals the common hidden symmetry underlying all small viral shells. We demonstrate the common genesis of the "anomalous" and conventional capsids and explain their structures in the same frame. The organization principles are derived from the group theory analysis of the positional order on the spherical surface. The relationship between the modified CK geometrical model and the theory of two-dimensional spherical crystallization is discussed. We also apply the proposed approach to complex double-shelled capsids and capsids with protruding knob-like proteins. The introduced notion of commensurability for the concentric nanoshells explains the peculiarities of their organization and helps to predict analogous, but yet undiscovered, double-shelled viral capsid nanostructures.

  13. Mutation of the N-Terminal Region of Chikungunya Virus Capsid Protein: Implications for Vaccine Design

    Science.gov (United States)

    Liu, Xiang; Zaid, Ali; Goh, Lucas Y. H.; Hobson-Peters, Jody; Hall, Roy A.; Merits, Andres

    2017-01-01

    ABSTRACT Mosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of the Togaviridae family responsible for frequent outbreaks of arthritic disease in humans. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleolus. In encephalitic alphaviruses, nuclear translocation induces host cell transcriptional shutoff; however, the role of capsid protein nucleolar localization in arthritogenic alphaviruses remains unclear. Using recombinant enhanced green fluorescent protein (EGFP)-tagged expression constructs and CHIKV infectious clones, we describe a nucleolar localization sequence (NoLS) in the N-terminal region of capsid protein, previously uncharacterized in CHIKV. Mutation of the NoLS by site-directed mutagenesis reduced efficiency of nuclear import of CHIKV capsid protein. In the virus, mutation of the capsid protein NoLS (CHIKV-NoLS) attenuated replication in mammalian and mosquito cells, producing a small-plaque phenotype. Attenuation of CHIKV-NoLS is likely due to disruption of the viral replication cycle downstream of viral RNA synthesis. In mice, CHIKV-NoLS infection caused no disease signs compared to wild-type CHIKV (CHIKV-WT)-infected mice; lack of disease signs correlated with significantly reduced viremia and decreased expression of proinflammatory factors. Mice immunized with CHIKV-NoLS, challenged with CHIKV-WT at 30 days postimmunization, develop no disease signs and no detectable viremia. Serum from CHIKV-NoLS-immunized mice is able to efficiently neutralize CHIKV infection in vitro. Additionally, CHIKV-NoLS-immunized mice challenged with the related alphavirus Ross River virus showed reduced early and peak viremia postchallenge, indicating a cross-protective effect. The high degree of CHIKV-NoLS attenuation may improve CHIKV antiviral and rational vaccine design. PMID:28223458

  14. Mutation of the N-Terminal Region of Chikungunya Virus Capsid Protein: Implications for Vaccine Design

    Directory of Open Access Journals (Sweden)

    Adam Taylor

    2017-02-01

    Full Text Available Mosquito-transmitted chikungunya virus (CHIKV is an arthritogenic alphavirus of the Togaviridae family responsible for frequent outbreaks of arthritic disease in humans. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleolus. In encephalitic alphaviruses, nuclear translocation induces host cell transcriptional shutoff; however, the role of capsid protein nucleolar localization in arthritogenic alphaviruses remains unclear. Using recombinant enhanced green fluorescent protein (EGFP-tagged expression constructs and CHIKV infectious clones, we describe a nucleolar localization sequence (NoLS in the N-terminal region of capsid protein, previously uncharacterized in CHIKV. Mutation of the NoLS by site-directed mutagenesis reduced efficiency of nuclear import of CHIKV capsid protein. In the virus, mutation of the capsid protein NoLS (CHIKV-NoLS attenuated replication in mammalian and mosquito cells, producing a small-plaque phenotype. Attenuation of CHIKV-NoLS is likely due to disruption of the viral replication cycle downstream of viral RNA synthesis. In mice, CHIKV-NoLS infection caused no disease signs compared to wild-type CHIKV (CHIKV-WT-infected mice; lack of disease signs correlated with significantly reduced viremia and decreased expression of proinflammatory factors. Mice immunized with CHIKV-NoLS, challenged with CHIKV-WT at 30 days postimmunization, develop no disease signs and no detectable viremia. Serum from CHIKV-NoLS-immunized mice is able to efficiently neutralize CHIKV infection in vitro. Additionally, CHIKV-NoLS-immunized mice challenged with the related alphavirus Ross River virus showed reduced early and peak viremia postchallenge, indicating a cross-protective effect. The high degree of CHIKV-NoLS attenuation may improve CHIKV antiviral and rational vaccine design.

  15. Electrostatic potential of human immunodeficiency virus type 2 and rhesus macaque simian immunodeficiency virus capsid proteins

    Directory of Open Access Journals (Sweden)

    Katarzyna eBozek

    2012-06-01

    Full Text Available Human immunodeficiency virus type 2 (HIV-2 and simian immunodeficiency virus isolated from a macaque monkey (SIVmac are assumed to have originated from simian immunodeficiency virus isolated from sooty mangabey (SIVsm. Despite their close similarity in genome structure, HIV-2 and SIVmac show different sensitivities to TRIM5α, a host restriction factor against retroviruses. The replication of HIV-2 strains is potently restricted by rhesus (Rh monkey TRIM5α, while that of SIVmac strain 239 (SIVmac239 is not. Viral capsid protein is the determinant of this differential sensitivity to TRIM5α, as the HIV-2 mutant carrying SIVmac239 capsid protein evaded Rh TRIM5α-mediated restriction. However, the molecular determinants of this restriction mechanism are unknown. Electrostatic potential on the protein-binding site is one of the properties regulating protein-protein interactions. In this study, we investigated the electrostatic potential on the interaction surface of capsid protein of HIV-2 strain GH123 and SIVmac239. Although HIV-2 GH123 and SIVmac239 capsid proteins share more than 87% amino acid identity, we observed a large difference between the two molecules with the HIV-2 GH123 molecule having predominantly positive and SIVmac239 predominantly negative electrostatic potential on the surface of the loop between α-helices 4 and 5 (L4/5. As L4/5 is one of the major determinants of Rh TRIM5α sensitivity of these viruses, the present results suggest that the binding site of the Rh TRIM5α may show complementarity to the HIV-2 GH123 capsid surface charge distribution.

  16. T cell epitope-based allergy vaccines.

    Science.gov (United States)

    Larché, Mark

    2011-01-01

    Specific immunotherapy (SIT) with extracts containing intact allergen molecules is clinically efficacious, but associated with frequent adverse events related to the allergic sensitization of the patient. As a result, treatment is initiated in an incremental dose fashion which ultimately achieves a plateau (maintenance dose) that may be continued for several years. Reduction of allergic adverse events may allow safer and more rapid treatment Thus, many groups have developed and evaluated strategies to reduce allergenicity whilst maintaining immunogenicity, the latter being required to achieve specific modulation of the immune response. Peptide immunotherapy can be used to target T and/or B cells in an antigen-specific manner. To date, only approaches that target T cells have been clinically evaluated. Short, synthetic peptides representing immunodominant T cell epitopes of major allergens are able to modulate allergen-specific T cell responses in the absence of IgE cross linking and activation of effector cells. Here we review clinical and mechanistic studies associated with peptide immunotherapy targeting allergy to cats or to bee venom. 

  17. Comparison of an enzyme immunoassay to an indirect fluorescent immunoassay for the detection of antinuclear antibodies.

    Science.gov (United States)

    Reisner, B S; DiBlasi, J; Goel, N

    1999-04-01

    The standard method for detecting antinuclear antibodies (ANAs) is by immunofluorescence assay (IFA), a method that is labor intensive and subjective. In an attempt to overcome these limitations, several commercial enzyme immunoassays (EIAs) have been developed. We report the results of our evaluation of the ANA Microplate EIA (Sanofi Diagnostics Pasteur, Chaska, MN). For the evaluation, 808 serum samples were tested by EIA and IFA; 52 specimens were positive by both assays, 561 were negative by both assays, 91 were positive by EIA only, and 3 were positive by IFA only. Borderline results (not positive or negative) were obtained for 101 specimens, which were excluded when calculating the sensitivity, specificity, and positive and negative predictive values of this assay, which were 94.6%, 86.0%, 36.4%, and 99.5%, respectively. Because of its high negative predictive value, this assay can be used reliably to detect ANA-negative samples; however, the low positive predictive value indicates that EIA-positive specimens should be retested by an IFA to determine the final result.

  18. Species-specific and cross-reactive IgG1 antibody binding to viral capsid protein 1 (VP1 antigens of human rhinovirus species A, B and C.

    Directory of Open Access Journals (Sweden)

    Jua Iwasaki

    Full Text Available BACKGROUND: Human rhinoviruses (HRV are associated with upper and lower respiratory illnesses, including severe infections causing hospitalization in both children and adults. Although the clinical significance of HRV infections is now well established, no detailed investigation of the immune response against HRV has been performed. The purpose of this study was to assess the IgG1 antibody response to the three known HRV species, HRV-A, -B and -C in healthy subjects. METHODS: Recombinant polypeptides of viral capsid protein 1 (VP1 from two genotypes of HRV-A, -B and -C were expressed as glutathione S-transferase (GST fusion proteins and purified by affinity and then size exclusion chromatography. The presence of secondary structures similar to the natural antigens was verified by circular dichroism analysis. Total and species-specific IgG1 measurements were quantitated by immunoassays and immunoabsorption using sera from 63 healthy adults. RESULTS: Most adult sera reacted with the HRV VP1 antigens, at high titres. As expected, strong cross-reactivity between HRV genotypes of the same species was found. A high degree of cross-reactivity between different HRV species was also evident, particularly between HRV-A and HRV-C. Immunoabsorption studies revealed HRV-C specific titres were markedly and significantly lower than the HRV-A and HRV-B specific titres (P<0.0001. A truncated construct of HRV-C VP1 showed greater specificity in detecting anti-HRV-C antibodies. CONCLUSIONS: High titres of IgG1 antibody were bound by the VP1 capsid proteins of HRV-A, -B and -C, but for the majority of people, a large proportion of the antibody to HRV-C was cross-reactive, especially to HRV-A. The improved specificity found for the truncated HRV-C VP1 indicates species-specific and cross-reactive regions could be defined.

  19. Renewable Surface Fluorescence Sandwich Immunoassay Biosensor for Rapid Sensitive Botulinum Toxin Detection in an Automated Fluidic Format

    Energy Technology Data Exchange (ETDEWEB)

    Grate, Jay W.; Warner, Marvin G.; Ozanich, Richard M.; Miller, Keith D.; Colburn, Heather A.; Dockendorff, Brian P.; Antolick, Kathryn C.; Anheier, Norman C.; Lind, Michael A.; Lou, Jianlong; Marks, James D.; Bruckner-Lea, Cindy J.

    2009-03-05

    A renewable surface biosensor for rapid detection of botulinum toxin is described based on fluidic automation of a fluorescence sandwich immunoassay, using a recombinant fragment of the toxin heavy chain as a structurally valid simulant. Monoclonal antibodies AR4 and RAZ1 bind to separate epitopes of both this fragment and the holotoxin. The AR4 antibody was covalently bound to Sepharose beads and used as the capture antibody. A rotating rod flow cell was used to capture these beads delivered as a suspension by the sequential injection flow system, creating a 3.6 microliter column. After perfusing the bead column with sample and washing away the matrix, the column was perfused with Alexa 647 dye-labeled RAZ1 antibody as the reporter. Optical fibers coupled to the rotating rod flow cell at a 90 degree angle to one another delivered excitation light from a HeNe laser and collected fluorescent emission light for detection. After each measurement, the used sepharose beads are released and replaced with fresh beads. In a rapid screening approach to sample analysis, the toxin simulant was detected to concentrations of 10 pM in less than 20 minutes.

  20. Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA

    Science.gov (United States)

    TakYu, Zeta; Guan, Huijiao; Ki Cheung, Mei; McHugh, Walker M.; Cornell, Timothy T.; Shanley, Thomas P.; Kurabayashi, Katsuo; Fu, Jianping

    2015-06-01

    Immunoassays represent one of the most popular analytical methods for detection and quantification of biomolecules. However, conventional immunoassays such as ELISA and flow cytometry, even though providing high sensitivity and specificity and multiplexing capability, can be labor-intensive and prone to human error, making them unsuitable for standardized clinical diagnoses. Using a commercialized no-wash, homogeneous immunoassay technology (‘AlphaLISA’) in conjunction with integrated microfluidics, herein we developed a microfluidic immunoassay chip capable of rapid, automated, parallel immunoassays of microliter quantities of samples. Operation of the microfluidic immunoassay chip entailed rapid mixing and conjugation of AlphaLISA components with target analytes before quantitative imaging for analyte detections in up to eight samples simultaneously. Aspects such as fluid handling and operation, surface passivation, imaging uniformity, and detection sensitivity of the microfluidic immunoassay chip using AlphaLISA were investigated. The microfluidic immunoassay chip could detect one target analyte simultaneously for up to eight samples in 45 min with a limit of detection down to 10 pg mL-1. The microfluidic immunoassay chip was further utilized for functional immunophenotyping to examine cytokine secretion from human immune cells stimulated ex vivo. Together, the microfluidic immunoassay chip provides a promising high-throughput, high-content platform for rapid, automated, parallel quantitative immunosensing applications.

  1. High Throughput T Epitope Mapping and Vaccine Development

    Directory of Open Access Journals (Sweden)

    Giuseppina Li Pira

    2010-01-01

    Full Text Available Mapping of antigenic peptide sequences from proteins of relevant pathogens recognized by T helper (Th and by cytolytic T lymphocytes (CTL is crucial for vaccine development. In fact, mapping of T-cell epitopes provides useful information for the design of peptide-based vaccines and of peptide libraries to monitor specific cellular immunity in protected individuals, patients and vaccinees. Nevertheless, epitope mapping is a challenging task. In fact, large panels of overlapping peptides need to be tested with lymphocytes to identify the sequences that induce a T-cell response. Since numerous peptide panels from antigenic proteins are to be screened, lymphocytes available from human subjects are a limiting factor. To overcome this limitation, high throughput (HTP approaches based on miniaturization and automation of T-cell assays are needed. Here we consider the most recent applications of the HTP approach to T epitope mapping. The alternative or complementary use of in silico prediction and experimental epitope definition is discussed in the context of the recent literature. The currently used methods are described with special reference to the possibility of applying the HTP concept to make epitope mapping an easier procedure in terms of time, workload, reagents, cells and overall cost.

  2. Different Biotinylation Strategies for Competitive Immunoassay of Estradiol

    Institute of Scientific and Technical Information of China (English)

    ZHAO,Jin-Fu(赵金富); WANG,Yong-Cheng(王永成); LI,Yuan-Zong(李元宗); CHANG,Wen-Bao(常文保)

    2004-01-01

    Study on biotinylation strategies for competitive immunoassay of estradiol (E2) was carried out. Two types of competitive enzyme immunoassay (EIA) with Biotin-Avidin amplification system were established and optimized.The E2-Biotin conjugate was used as a tracer in one assay, and biotinylated antibody was used as a tracer in the other. In both of EIAs, horseradish peroxidase-labelled Avidin (Avidin-HRP) was used with a spectrometric determination of enzyme activity. The precision, sensitivity and specificity were measured and compared. The results showed that although both were satisfactory in specificity, the EIA with hapten-Biotin showed to be superior to the EIA with biotinylated antibody in sensitivity and precision. The limit of detection of serum E2 was 8 and 50 pg/mL with E2-Biotin and biotinylated antibody as tracer, respectively.

  3. Design and Fabrication of a PDMS Microchip Based Immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Shao, Guocheng; Wang, Wanjun; Wang, Jun; Lin, Yuehe

    2010-07-01

    In this paper, we describe the design and fabrication process of a polydimethylsiloxane (PDMS) microchip for on-chip multiplex immunoassay application. The microchip consists of a PDMS microfluidic channel layer and a micro pneumatic valve control layer. By selectively pressurizing the pneumatic microvalves, immuno reagents were controlled to flow and react in certain fluidic channel sites. Cross contamination was prevented by tightly closed valves. Our design was proposed to utilize PDMS micro channel surface as the solid phase immunoassay substrate and simultaneously detect four targets antigens on chip. Experiment result shows that 20psi valve pressure is sufficient to tightly close a 200µm wide micro channel with flow rate up to 20µl/min.

  4. Bacteriophage P23-77 capsid protein structures reveal the archetype of an ancient branch from a major virus lineage.

    Science.gov (United States)

    Rissanen, Ilona; Grimes, Jonathan M; Pawlowski, Alice; Mäntynen, Sari; Harlos, Karl; Bamford, Jaana K H; Stuart, David I

    2013-05-07

    It has proved difficult to classify viruses unless they are closely related since their rapid evolution hinders detection of remote evolutionary relationships in their genetic sequences. However, structure varies more slowly than sequence, allowing deeper evolutionary relationships to be detected. Bacteriophage P23-77 is an example of a newly identified viral lineage, with members inhabiting extreme environments. We have solved multiple crystal structures of the major capsid proteins VP16 and VP17 of bacteriophage P23-77. They fit the 14 Å resolution cryo-electron microscopy reconstruction of the entire virus exquisitely well, allowing us to propose a model for both the capsid architecture and viral assembly, quite different from previously published models. The structures of the capsid proteins and their mode of association to form the viral capsid suggest that the P23-77-like and adeno-PRD1 lineages of viruses share an extremely ancient common ancestor.

  5. Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing.

    Science.gov (United States)

    Adachi, Kei; Enoki, Tatsuji; Kawano, Yasuhiro; Veraz, Michael; Nakai, Hiroyuki

    2014-01-01

    Adeno-associated virus (AAV) capsid engineering is an emerging approach to advance gene therapy. However, a systematic analysis on how each capsid amino acid contributes to multiple functions remains challenging. Here we show proof-of-principle and successful application of a novel approach, termed AAV Barcode-Seq, that allows us to characterize phenotypes of hundreds of different AAV strains in a high-throughput manner and therefore overcomes technical difficulties in the systematic analysis. In this approach, we generate DNA barcode-tagged AAV libraries and determine a spectrum of phenotypes of each AAV strain by Illumina barcode sequencing. By applying this method to AAV capsid mutant libraries tagged with DNA barcodes, we can draw a high-resolution map of AAV capsid amino acids important for the structural integrity and functions including receptor binding, tropism, neutralization and blood clearance. Thus, Barcode-Seq provides a new tool to generate a valuable resource for virus and gene therapy research.

  6. DETERMINATION OF AMINOGLYCOSIDES IN FOOD BY FLUORESCENCE POLARIZATION IMMUNOASSAY

    Directory of Open Access Journals (Sweden)

    FARAFONOVA O.V.

    2015-01-01

    Full Text Available The methodic for quantitative determination of aminoglycoside antibiotics (gentamicin, kanamycin, streptomycin, amikacin, neomycin in food by polarization fluorescent immunoassay (FPIA is developed. The size and structure influence of a fluorescent molecule on a fluorescence polarization degree is analyzed. Affinity constants of antibodies to compounds and tracers were estimated, optimized working concentration of tracers and antibodies that provide the maximum value of analytical signal. Methods were tested in the antibiotics identification in milk, eggs and chicken.

  7. Epitope characterization and variable region sequence of f1-40, a high-affinity monoclonal antibody to botulinum neurotoxin type a (Hall strain.

    Directory of Open Access Journals (Sweden)

    Miles C Scotcher

    Full Text Available BACKGROUND: Botulism, an often fatal neuroparalytic disease, is caused by botulinum neurotoxins (BoNT which consist of a family of seven serotypes (A-H produced by the anaerobic bacterium Clostridium botulinum. BoNT, considered the most potent biological toxin known, is a 150 kDa protein consisting of a 100 kDa heavy-chain (Hc and a 50 kDa light-chain (Lc. F1-40 is a mouse-derived, IgG1 monoclonal antibody that binds the light chain of BoNT serotype A (BoNT/A and is used in a sensitive immunoassay for toxin detection. We report the fine epitope mapping of F1-40 and the deduced amino acid sequence of the variable regions of the heavy and light chains of the antibody. METHODS AND FINDINGS: To characterize the binding epitope of F1-40, three complementary experimental approaches were selected. Firstly, recombinant peptide fragments of BoNT/A light-chain were used in Western blots to identify the epitope domains. Secondly, a peptide phage-display library was used to identify the specific amino acid sequences. Thirdly, the three-dimensional structure of BoNT/A was examined in silico, and the amino acid sequences determined from the phage-display studies were mapped onto the three-dimensional structure in order to visualize the epitope. F1-40 was found to bind a peptide fragment of BoNT/A, designated L1-3, which spans from T125 to L200. The motif QPDRS was identified by phage-display, and was mapped to a region within L1-3. When the three amino acids Q138, P139 and D140 were all mutated to glycine, binding of F1-40 to the recombinant BoNT/A light chain peptide was abolished. Q-138, P-139 and D-140 form a loop on the external surface of BoNT/A, exposed to solvent and accessible to F1-40 binding. CONCLUSIONS: The epitope of F1-40 was localized to a single exposed loop (ss4, ss5 on the Lc of BoNT. Furthermore amino acids Q138, P139 and D140 forming the tip of the loop appear critical for binding.

  8. Targeted quantitative mass spectrometric immunoassay for human protein variants

    Directory of Open Access Journals (Sweden)

    Nedelkov Dobrin

    2011-04-01

    Full Text Available Abstract Background Post-translational modifications and genetic variations give rise to protein variants that significantly increase the complexity of the human proteome. Modified proteins also play an important role in biological processes. While sandwich immunoassays are routinely used to determine protein concentrations, they are oblivious to protein variants that may serve as biomarkers with better sensitivity and specificity than their wild-type proteins. Mass spectrometry, coupled to immunoaffinity separations, can provide an efficient mean for simultaneous detection and quantification of protein variants. Results Presented here is a mass spectrometric immunoassay method for targeted quantitative proteomics analysis of protein modifications. Cystatin C, a cysteine proteinase inhibitor and a potential marker for several pathological processes, was used as a target analyte. An internal reference standard was incorporated into the assay, serving as a normalization point for cystatin C quantification. The precision, linearity, and recovery characteristics of the assay were established. The new assay was also benchmarked against existing cystatin C ELISA. In application, the assay was utilized to determine the individual concentration of several cystatin C variants across a cohort of samples, demonstrating the ability to fully quantify individual forms of post-translationally modified proteins. Conclusions The mass spectrometric immunoassays can find use in quantifying specific protein modifications, either as a part of a specific protein biomarker discovery/rediscovery effort to delineate the role of these variants in the onset of the disease, progression, and response to therapy, or in a more systematic study to delineate and understand human protein diversity.

  9. Specific Noncompetitive Immunoassay for HT-2 Mycotoxin Detection.

    Science.gov (United States)

    Arola, Henri O; Tullila, Antti; Kiljunen, Harri; Campbell, Katrina; Siitari, Harri; Nevanen, Tarja K

    2016-02-16

    Here we demonstrate a novel homogeneous one-step immunoassay, utilizing a pair of recombinant antibody antigen-binding fragments (Fab), that is specific for HT-2 toxin and has a positive readout. Advantages over the conventional competitive immunoassay formats such as enzyme-linked immunosorbent assay (ELISA) are the specificity, speed, and simplicity of the assay. Recombinant antibody HT2-10 Fab recognizing both HT-2 and T-2 toxins was developed from a phage display antibody library containing 6 × 10(7) different antibody clones. Specificity of the immunoassay was introduced by an anti-immune complex (IC) antibody binding the primary antibody-HT-2 toxin complex. When the noncompetitive immune complex assay was compared to the traditional competitive assay, an over 10-fold improvement in sensitivity was observed. Although the HT2-10 antibody has 100% cross-reactivity for HT-2 and T-2 toxins, the immune complex assay is highly specific for HT-2 alone. The assay performance with real samples was evaluated using naturally contaminated wheat reference material. The half-maximal effective concentration (EC50) value of the time-resolved fluorescence resonance energy transfer (TR-FRET) assay was 9.6 ng/mL, and the limit of detection (LOD) was 0.38 ng/mL (19 μg/kg). The labeled antibodies can be predried to the assay vials, e.g., microtiter plate wells, and readout is ready in 10 min after the sample application.

  10. Single Step Nanoplasmonic Immunoassay for the Measurement of Protein Biomarkers

    Directory of Open Access Journals (Sweden)

    Shradha Prabhulkar

    2013-02-01

    Full Text Available A nanoplasmonic biosensor for highly-sensitive, single-step detection of protein biomarkers is presented. The principle is based on the utilization of the optical scattering properties of gold nanorods (GNRs conjugated to bio-recognition molecules. The nanoplasmonic properties of the GNRs were utilized to detect proteins using near-infrared light interferometry. We show that the antibody-conjugated GNRs can specifically bind to our model analyte, Glucose Transporter-1 (Glut-1. The signal intensity of back-scattered light from the GNRs bound after incubation, correlated well to the Glut-1 concentration as per the calibration curve. The detection range using this nanoplasmonic immunoassay ranges from 10 ng/mL to 1 ug/mL for Glut-1. The minimal detectable concentration based on the lowest discernable concentration from zero is 10 ng/mL. This nanoplasmonic immunoassay can act as a simple, selective, sensitive strategy for effective disease diagnosis. It offers advantages such as wide detection range, increased speed of analysis (due to fewer incubation/washing steps, and no label development as compared to traditional immunoassay techniques. Our future goal is to incorporate this detection strategy onto a microfluidic platform to be used as a point-of-care diagnostic tool.

  11. Graphene-based immunoassay for human lipocalin-2.

    Science.gov (United States)

    Vashist, Sandeep Kumar

    2014-02-01

    We have developed a highly sensitive immunoassay using graphene nano platelets (GNPs) for the rapid detection of human lipocalin-2 (LCN2) in plasma, serum, and whole blood. It has the dynamic range, linear range, limit of detection, and analytical sensitivity of 0.6 to 5120, 80 to 2560, 0.7, and 1pg/ml, respectively. It is the most sensitive assay for the detection of LCN2, which has 80-fold higher analytical sensitivity and 3-fold lesser immunoassay duration than the commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit. The functionalization of microtiter plate (MTP) with GNPs, dispersed in 3-aminopropyltriethoxysilane (APTES), provided the increased surface area that leads to higher immobilization density of capture antibodies. Moreover, the generation of free amino groups on MTP and GNPs by APTES enables the leach-proof covalent crosslinking of anti-human LCN2 capture antibody by its carboxyl groups using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) as the heterobifunctional crosslinker. The anti-LCN2 antibody-bound MTPs were highly stable given that they did not show any significant decrease in their functional activity when stored at 4°C in 0.1M phosphate-buffered saline (PBS) for 8weeks. The developed immunoassay correlated well with the conventional ELISA, thereby demonstrating its high precision and potential utility for highly sensitive analyte detection in industrial and clinical settings.

  12. Targeting of herpesvirus capsid transport in axons is coupled to association with specific sets of tegument proteins

    OpenAIRE

    Luxton, G.W. Gant; Haverlock, Sarah; Coller, Kelly Elizabeth; Antinone, Sarah Elizabeth; Pincetic, Andrew; Smith, Gregory Allan

    2005-01-01

    The capsids of neurotropic herpesviruses have the remarkable ability to move in specific directions within axons. By modulating bidirectional capsid transport to favor either retrograde (minus-end) or anterograde (plus-end) motion, these viruses travel to sensory ganglia or peripheral tissue at specific stages of infection. By using correlative motion analysis to simultaneously monitor the trafficking of distinct viral proteins in living neurons, we demonstrate that viral “tegument” proteins ...

  13. Location of the Bacteriophage P22 Coat Protein C-terminus Provides Opportunities for the Design of Capsid Based Materials

    OpenAIRE

    Servid, Amy; Jordan, Paul; O’Neil, Alison; Prevelige, Peter; Douglas, Trevor

    2013-01-01

    Rational design of modifications to the interior and exterior surfaces of virus-like particles (VLPs) for future therapeutic and materials applications is based on structural information about the capsid. Existing cryo-electron microscopy based models suggest that the C-terminus of the bacteriophage P22 coat protein (CP) extends towards the capsid exterior. Our biochemical analysis through genetic manipulations of the C-terminus supports the model where the CP C-terminus is exposed on the ext...

  14. Rationally Designed Interfacial Peptides Are Efficient In Vitro Inhibitors of HIV-1 Capsid Assembly with Antiviral Activity

    OpenAIRE

    Rebeca Bocanegra; María Nevot; Rosa Doménech; Inmaculada López; Olga Abián; Alicia Rodríguez-Huete; Cavasotto, Claudio N.; Adrián Velázquez-Campoy; Javier Gómez; Miguel Ángel Martínez; José Luis Neira; Mateu, Mauricio G.

    2011-01-01

    Virus capsid assembly constitutes an attractive target for the development of antiviral therapies; a few experimental inhibitors of this process for HIV-1 and other viruses have been identified by screening compounds or by selection from chemical libraries. As a different, novel approach we have undertaken the rational design of peptides that could act as competitive assembly inhibitors by mimicking capsid structural elements involved in intersubunit interfaces. Several discrete interfaces in...

  15. Cloning of the rhesus lymphocryptovirus viral capsid antigen and Epstein-Barr virus-encoded small RNA homologues and use in diagnosis of acute and persistent infections.

    Science.gov (United States)

    Rao, P; Jiang, H; Wang, F

    2000-09-01

    Epstein-Barr virus (EBV) is the most common cause of infectious mononucleosis and is associated with the development of several human malignancies. A closely related herpesvirus in the same lymphocryptovirus (LCV) genera as EBV naturally infects rhesus monkeys and provides an important animal model for studying EBV pathogenesis. We cloned the small viral capsid antigen (sVCA) homologue from the rhesus LCV and developed a peptide enzyme-linked immunosorbent assay (ELISA) to determine whether epitopes in the rhesus LCV sVCA are a reliable indicator of rhesus LCV infection. In order to define a "gold standard" for rhesus LCV infection, we also cloned the EBV-encoded small RNA 1 (EBER1) and EBER2 homologues from rhesus LCV and developed a reverse transcription (RT)-PCR assay to detect persistent LCV infection in rhesus monkey peripheral blood lymphocytes. Animals from a conventional and a hand-reared colony were studied to compare the prevalence of rhesus LCV infection in the two groups. There was a 100% correlation between the peptide ELISA and EBER RT-PCR results for rhesus LCV infection. In addition, specificity for LCV infection and exclusion of potential cross-reactivity to the rhesus rhadinovirus sVCA homologue could be demonstrated using sera from experimentally infected animals. These studies establish two novel assays for reliable diagnosis of acute and persistent rhesus LCV infections. The rhesus LCV sVCA peptide ELISA provides a sensitive and reliable assay for routine screening, and these studies of the hand-reared colony confirm the feasibility of raising rhesus LCV-naive animals.

  16. Whole-Chain Tick Saliva Proteins Presented on Hepatitis B Virus Capsid-Like Particles Induce High-Titered Antibodies with Neutralizing Potential.

    Directory of Open Access Journals (Sweden)

    Philipp Kolb

    Full Text Available Ticks are vectors for various, including pathogenic, microbes. Tick saliva contains multiple anti-host defense factors that enable ticks their bloodmeals yet also facilitate microbe transmission. Lyme disease-causing borreliae profit specifically from the broadly conserved tick histamine release factor (tHRF, and from cysteine-rich glycoproteins represented by Salp15 from Ixodes scapularis and Iric-1 from Ixodes ricinus ticks which they recruit to their outer surface protein C (OspC. Hence these tick proteins are attractive targets for anti-tick vaccines that simultaneously impair borrelia transmission. Main obstacles are the tick proteins´ immunosuppressive activities, and for Salp15 orthologs, the lack of efficient recombinant expression systems. Here, we exploited the immune-enhancing properties of hepatitis B virus core protein (HBc derived capsid-like particles (CLPs to generate, in E. coli, nanoparticulate vaccines presenting tHRF and, as surrogates for the barely soluble wild-type proteins, cysteine-free Salp15 and Iric-1 variants. The latter CLPs were exclusively accessible in the less sterically constrained SplitCore system. Mice immunized with tHRF CLPs mounted a strong anti-tHRF antibody response. CLPs presenting cysteine-free Salp15 and Iric-1 induced antibodies to wild-type, including glycosylated, Salp15 and Iric-1. The broadly distributed epitopes included the OspC interaction sites. In vitro, the anti-Salp15 antibodies interfered with OspC binding and enhanced human complement-mediated killing of Salp15 decorated borreliae. A mixture of all three CLPs induced high titered antibodies against all three targets, suggesting the feasibility of combination vaccines. These data warrant in vivo validation of the new candidate vaccines´ protective potential against tick infestation and Borrelia transmission.

  17. Phage displaying epitope of Candida albicans HSP90 and serodiagnosis

    Institute of Scientific and Technical Information of China (English)

    杨琼; 王丽; 卢大宁; 邢沈阳; 尹东; 朱筱娟

    2004-01-01

    @@ Recently, the frequent use of immunosuppressants and chemotherapeutic drugs for cancers has caused an increase in the frequency of life-threatening systemic candidiasis.1 Studies by Matthews et al2 indicated HSP90 fragments are major targets for the immune system in infection due to C. albicans, and anti-epitope LKVIRK of HSP90 antibody is a serological marker for diagnosis of invasive candidiasis. Cloning and sequencing HSP90 antigen revealed that the linear epitope LKVIRK, localized near the C-terminus of the 47 kDa protein which circulates in the sera of patients with invasive candidiasis, as a heat-stable breakdown product of large more heat-labile antigen HSP90.2 In this study, epitope LKVIRK was displayed on the surface of phage fd to develop a new serological test for systemic candidiasis.

  18. Antibody specific epitope prediction-emergence of a new paradigm.

    Science.gov (United States)

    Sela-Culang, Inbal; Ofran, Yanay; Peters, Bjoern

    2015-04-01

    The development of accurate tools for predicting B-cell epitopes is important but difficult. Traditional methods have examined which regions in an antigen are likely binding sites of an antibody. However, it is becoming increasingly clear that most antigen surface residues will be able to bind one or more of the myriad of possible antibodies. In recent years, new approaches have emerged for predicting an epitope for a specific antibody, utilizing information encoded in antibody sequence or structure. Applying such antibody-specific predictions to groups of antibodies in combination with easily obtainable experimental data improves the performance of epitope predictions. We expect that further advances of such tools will be possible with the integration of immunoglobulin repertoire sequencing data.

  19. Common food allergens and their IgE-binding epitopes.

    Science.gov (United States)

    Matsuo, Hiroaki; Yokooji, Tomoharu; Taogoshi, Takanori

    2015-10-01

    Food allergy is an adverse immune response to certain kinds of food. Although any food can cause allergic reactions, chicken egg, cow's milk, wheat, shellfish, fruit, and buckwheat account for 75% of food allergies in Japan. Allergen-specific immunoglobulin E (IgE) antibodies play a pivotal role in the development of food allergy. Recent advances in molecular biological techniques have enabled the efficient analysis of food allergens. As a result, many food allergens have been identified, and their molecular structure and IgE-binding epitopes have also been identified. Studies of allergens have demonstrated that IgE antibodies specific to allergen components and/or the peptide epitopes are good indicators for the identification of patients with food allergy, prediction of clinical severity and development of tolerance. In this review, we summarize our current knowledge regarding the allergens and IgE epitopes in the well-researched allergies to chicken egg, cow's milk, wheat, shrimp, and peanut.

  20. Mature Epitope Density - A strategy for target selection based on immunoinformatics and exported prokaryotic proteins

    DEFF Research Database (Denmark)

    Santos, Anderson R; Pereira, Vanessa Bastos; Barbosa, Eudes;

    2013-01-01

    BACKGROUND: Current immunological bioinformatic approaches focus on the prediction of allele-specific epitopes capable of triggering immunogenic activity. The prediction of major histocompatibility complex (MHC) class I epitopes is well studied, and various software solutions exist for this purpo...

  1. Branched peptide amphiphiles, related epitope compounds and self assembled structures thereof

    Science.gov (United States)

    Stupp, Samuel I.; Guler, Mustafa O.

    2008-11-18

    Branched peptide amphiphilic compounds incorporating one or residues providing a pendant amino group for coupling one or more epitope sequences thereto, such compounds and related compositions for enhanced epitope presentation.

  2. Structural transitions and energy landscape for Cowpea Chlorotic Mottle Virus capsid mechanics from nanomanipulation in vitro and in silico

    CERN Document Server

    Kononova, Olga; Brasch, Melanie; Cornelissen, Jeroen; Dima, Ruxandra I; Marx, Kenneth A; Wuite, Gijs J L; Roos, Wouter H; Barsegov, Valeri

    2015-01-01

    Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Virus shells can have applications as nanocontainers and delivery vehicles in biotechnology and medicine. Combined AFM experiments and computational modeling on sub-second timescales of the indentation nanomechanics of Cowpea Chlorotic Mottle Virus (CCMV) capsid show that the capsid's physical properties are dynamic and local characteristics of the structure, which depend on the magnitude and geometry of mechanical input. Surprisingly, under large deformations the CCMV capsid transitions to the collapsed state without substantial local structural alterations. The enthalpy change in this deformation state dH = 11.5 - 12.8 MJ/mol is mostly due to large-amplitude out-of-plane excitations, which contribute to the capsid bending, and the entropy change TdS = 5.1 - 5.8 MJ/mol is mostly due to coherent in-plane rearrangements of pr...

  3. Selective Inhibitor of Nuclear Export (SINE) Compounds Alter New World Alphavirus Capsid Localization and Reduce Viral Replication in Mammalian Cells.

    Science.gov (United States)

    Lundberg, Lindsay; Pinkham, Chelsea; de la Fuente, Cynthia; Brahms, Ashwini; Shafagati, Nazly; Wagstaff, Kylie M; Jans, David A; Tamir, Sharon; Kehn-Hall, Kylene

    2016-11-01

    The capsid structural protein of the New World alphavirus, Venezuelan equine encephalitis virus (VEEV), interacts with the host nuclear transport proteins importin α/β1 and CRM1. Novel selective inhibitor of nuclear export (SINE) compounds, KPT-185, KPT-335 (verdinexor), and KPT-350, target the host's primary nuclear export protein, CRM1, in a manner similar to the archetypical inhibitor Leptomycin B. One major limitation of Leptomycin B is its irreversible binding to CRM1; which SINE compounds alleviate because they are slowly reversible. Chemically inhibiting CRM1 with these compounds enhanced capsid localization to the nucleus compared to the inactive compound KPT-301, as indicated by immunofluorescent confocal microscopy. Differences in extracellular versus intracellular viral RNA, as well as decreased capsid in cell free supernatants, indicated the inhibitors affected viral assembly, which led to a decrease in viral titers. The decrease in viral replication was confirmed using a luciferase-tagged virus and through plaque assays. SINE compounds had no effect on VEEV TC83_Cm, which encodes a mutated form of capsid that is unable to enter the nucleus. Serially passaging VEEV in the presence of KPT-185 resulted in mutations within the nuclear localization and nuclear export signals of capsid. Finally, SINE compound treatment also reduced the viral titers of the related eastern and western equine encephalitis viruses, suggesting that CRM1 maintains a common interaction with capsid proteins across the New World alphavirus genus.

  4. Microplate-based assay for identifying small molecules that bind a specific intersubunit interface within the assembled HIV-1 capsid.

    Science.gov (United States)

    Halambage, Upul D; Wong, Jason P; Melancon, Bruce J; Lindsley, Craig W; Aiken, Christopher

    2015-09-01

    Despite the availability of >30 effective drugs for managing HIV-1 infection, no current therapy is curative, and long-term management is challenging owing to the emergence and spread of drug-resistant mutants. Identification of drugs against novel HIV-1 targets would expand the current treatment options and help to control resistance. The highly conserved HIV-1 capsid protein represents an attractive target because of its multiple roles in replication of the virus. However, the low antiviral potencies of the reported HIV-1 capsid-targeting inhibitors render them unattractive for therapeutic development. To facilitate the identification of more-potent HIV-1 capsid inhibitors, we developed a scintillation proximity assay to screen for small molecules that target a biologically active and specific intersubunit interface in the HIV-1 capsid. The assay, which is based on competitive displacement of a known capsid-binding small-molecule inhibitor, exhibited a signal-to-noise ratio of >9 and a Z factor of >0.8. In a pilot screen of a chemical library containing 2,400 druglike compounds, we obtained a hit rate of 1.8%. This assay has properties that are suitable for screening large compound libraries to identify novel HIV-1 capsid ligands with antiviral activity.

  5. Two-Dimensional Phase Transition of Viral Capsid Gives Insights into Subunit Interactions

    Science.gov (United States)

    Tresset, Guillaume; Chen, Jingzhi; Chevreuil, Maelenn; Nhiri, Naïma; Jacquet, Eric; Lansac, Yves

    2017-01-01

    We show that the thermal dissociation of icosahedral viral capsids can be interpreted in terms of a two-dimensional phase transition. The approach provides a useful framework to get direct insights into the interactions at work between viral components. We devise a mean-field lattice model that relates the melting temperature to subunit attractive energy, effective charge, and chemical potential. Through fluorescence thermal shift assay on a plant viral system, we illustrate how the model gives access to the interaction parameters for empty and loaded viral capsids in various ionic conditions. This work should help construct physical models accounting for the assembly and disassembly mechanisms of viruses, with possible fallout in the development of therapeutic inhibitors.

  6. Expression and subcellular targeting of canine parvovirus capsid proteins in baculovirus-transduced NLFK cells.

    Science.gov (United States)

    Gilbert, Leona; Välilehto, Outi; Kirjavainen, Sanna; Tikka, Päivi J; Mellett, Mark; Käpylä, Pirjo; Oker-Blom, Christian; Vuento, Matti

    2005-01-17

    A mammalian baculovirus delivery system was developed to study targeting in Norden Laboratories feline kidney (NLFK) cells of the capsid proteins of canine parvovirus (CPV), VP1 and VP2, or corresponding counterparts fused to EGFP. VP1 and VP2, when expressed alone, both had equal nuclear and cytoplasmic distribution. However, assembled form of VP2 had a predominantly cytoplasmic localization. When VP1 and VP2 were simultaneously present in cells, their nuclear localization increased. Thus, confocal immunofluorescence analysis of cells transduced with the different baculovirus constructs or combinations thereof in the absence or presence of infecting CPV revealed that the VP1 protein is a prerequisite for efficient targeting of VP2 to the nucleus. The baculovirus vectors were functional and the genes of interest efficiently introduced to this CPV susceptible mammalian cell line. Thus, we show evidence that the system could be utilized to study targeting of the CPV capsid proteins.

  7. Theory of morphological transformation of viral capsid shells during maturation process

    CERN Document Server

    Konevtsova, O V; Rochal, S B

    2015-01-01

    In the frame of the Landau-Ginzburg formalism we propose a minimal phenomenological model for a morphological transformation in viral capsid shells. The transformation takes place during virus maturation process which renders virus infectious. The theory is illustrated on the example of the HK97 bacteriophage and viruses with similar morphological changes in the protective protein shell. The transformation is shown to be a structural phase transition driven by two order parameters. The first order parameter describes the isotropic expansion of the protein shell while the second one is responsible for the shape symmetry breaking and the resulting shell faceting. The group theory analysis and the resulting thermodynamic model make it possible to choose the parameter which discriminates between the icosahedral shell faceting often observed in viral capsids and the dodecahedral one observed in viruses of the Parvovirus family. Calculated phase diagram illustrates the discontinuous character of the virus morpholog...

  8. DNA condensates organized by the capsid protein VP15 in White Spot Syndrome Virus.

    Science.gov (United States)

    Liu, Yingjie; Wu, Jinlu; Chen, Hu; Hew, Choy Leong; Yan, Jie

    2010-12-20

    The White Spot Syndrome Virus (WSSV) has a large circular double-stranded DNA genome of around 300kb and it replicates in the nucleus of the host cells. The machinery of how the viral DNA is packaged has been remained unclear. VP15, a highly basic protein, is one of the major capsid proteins found in the virus. Previously, it was shown to be a DNA binding protein and was hypothesized to participate in the viral DNA packaging process. Using Atomic Force Microscopy imaging, we show that the viral DNA is associated with a (or more) capsid proteins. The organized viral DNA qualitatively resembles the conformations of VP15 induced DNA condensates in vitro. Furthermore, single-DNA manipulation experiments revealed that VP15 is able to condense single DNA against forces of a few pico Newtons. Our results suggest that VP15 may aid in the viral DNA packaging process by directly condensing DNA.

  9. Antibody protection reveals extended epitopes on the human TSH receptor.

    Directory of Open Access Journals (Sweden)

    Rauf Latif

    Full Text Available Stimulating, and some blocking, antibodies to the TSH receptor (TSHR have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD. However, successful crystallization of TSHR residues 22-260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1-412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity.

  10. Interactions of the HSV-1 UL25 Capsid Protein with Cellular Microtubule-associated Protein

    Institute of Scientific and Technical Information of China (English)

    Lei GUO; Ying ZHANG; Yan-chun CHE; Wen-juan WU; Wei-zhong LI; Li-chun WANG; Yun LIAO; Long-ding LIU; Qi-han LI

    2008-01-01

    An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β-D-galactosidase activity assays. Immunofluorescence microscopy of the UL25 protein demonstrated its co-localization with cellular microtubule-associated protein in the plasma membrane. Further investigations with deletion mutants suggest that UL25 is likely to have a function in the nucleus.

  11. High-throughput epitope identification for snakebite antivenom

    DEFF Research Database (Denmark)

    Engmark, Mikael; De Masi, Federico; Laustsen, Andreas Hougaard

    Insight into the epitopic recognition pattern for polyclonal antivenoms is a strong tool for accurate prediction of antivenom cross-reactivity and provides a basis for design of novel antivenoms. In this work, a high-throughput approach was applied to characterize linear epitopes in 966 individua...... toxins from pit vipers (Crotalidae) using the ICP Crotalidae antivenom. Due to an abundance of snake venom metalloproteinases and phospholipase A2s in the venoms used for production of the investigated antivenom, this study focuses on these toxin families....

  12. Transient Bluetongue virus serotype 8 capsid protein expression in Nicotiana benthamiana

    Directory of Open Access Journals (Sweden)

    Albertha R. van Zyl

    2016-03-01

    Full Text Available Bluetongue virus (BTV causes severe disease in domestic and wild ruminants, and has recently caused several outbreaks in Europe. Current vaccines include live-attenuated and inactivated viruses; while these are effective, there is risk of reversion to virulence by mutation or reassortment with wild type viruses. Subunit or virus-like particle (VLP vaccines are safer options: VLP vaccines produced in insect cells by expression of the four BTV capsid proteins are protective against challenge; however, this is a costly production method. We investigated production of BTV VLPs in plants via Agrobacterium-mediated transient expression, an inexpensive production system very well suited to developing country use. Leaves infiltrated with recombinant pEAQ-HT vectors separately encoding the four BTV-8 capsid proteins produced more proteins than recombinant pTRA vectors. Plant expression using the pEAQ-HT vector resulted in both BTV-8 core-like particles (CLPs and VLPs; differentially controlling the concentration of infiltrated bacteria significantly influenced yield of the VLPs. In situ localisation of assembled particles was investigated by using transmission electron microscopy (TEM and it was shown that a mixed population of core-like particles (CLPs, consisting of VP3 and VP7 and VLPs were present as paracrystalline arrays in the cytoplasm of plant cells co-expressing all four capsid proteins.

  13. Modeling of the human rhinovirus C capsid suggests possible causes for antiviral drug resistance.

    Science.gov (United States)

    Basta, Holly A; Ashraf, Shamaila; Sgro, Jean-Yves; Bochkov, Yury A; Gern, James E; Palmenberg, Ann C

    2014-01-05

    Human rhinoviruses of the RV-C species are recently discovered pathogens with greater clinical significance than isolates in the RV-A+B species. The RV-C cannot be propagated in typical culture systems; so much of the virology is necessarily derivative, relying on comparative genomics, relative to the better studied RV-A+B. We developed a bioinformatics-based structural model for a C15 isolate. The model showed the VP1-3 capsid proteins retain their fundamental cores relative to the RV-A+B, but conserved, internal RV-C residues affect the shape and charge of the VP1 hydrophobic pocket that confers antiviral drug susceptibility. When predictions of the model were tested in organ cultures or ALI systems with recombinant C15 virus, there was a resistance to capsid-binding drugs, including pleconaril, BTA-188, WIN56291, WIN52035 and WIN52084. Unique to all RV-C, the model predicts conserved amino acids within the pocket and capsid surface pore leading to the pocket may correlate with this activity.

  14. Motion of an antiviral compound in a rhinovirus capsid under rotational symmetry boundary conditions.

    Science.gov (United States)

    Yoneda, Shigetaka; Yoneda, Teruyo; Kurihara, Youji; Umeyama, Hideaki

    2002-08-01

    A molecular dynamics (MD) simulation of a complex of a rhinovirus protein shell referred to as a "capsid" and an anti-rhinovirus drug, WIN52084s, was performed under the rotational symmetry boundary conditions. For the simulation, the energy parameters of WIN52084s in all-atom approximations were determined by ab initio calculations using a 6-31G* basis set and the two-conformational two-stage restricted electrostatic potential fit method. The motion of WIN52084s and the capsid was focused on in the analysis of the trajectory of the simulation. The root mean square deviations of WIN52084s from the X-ray structure were decomposed to conformational, translational, and rotational components. The translation was further decomposed to radial, longitudinal, and lateral components. The conformation of WIN52084s was rigid, but moving in the pocket. The easiest path of motion for WlN52084s was on the longitudinal line, providing a track for the binding process required of the anti-rhinovirus drug to enter the pocket. The conformation of the pocket was also preserved in the simulation, although the position of the pocket in the capsid fluctuated in the lateral and radial directions.

  15. Enhancing the clinical potential of AAV vectors by capsid engineering to evade pre-existing immunity

    Directory of Open Access Journals (Sweden)

    Melissa eBartel

    2011-10-01

    Full Text Available Vectors based on adeno-associated viruses have shown considerable promise in both preclinical models and increasingly in clinical trials. However, one formidable challenge is pre-existing immunity due to widespread exposure to numerous AAV variants and serotypes within the human population, which affect efficacy of clinical trials due to the accompanying high levels of anti-capsid neutralizing antibodies. Transient immunosuppression has promise in mitigating cellular and humoral responses induced by vector application in naïve hosts, but cannot overcome the problem that pre-existing neutralizing antibodies pose towards the goal of safe and efficient gene delivery. Shielding of AAV from antibodies, however, may be possible by covalent attachment of polymers to the viral capsid or by encapsulation of vectors inside biomaterials. In addition, there has been considerable progress in using rational mutagenesis, combinatorial libraries, and directed evolution approaches to engineer capsid variants that are not recognized by anti-AAV antibodies generally present in the human population. While additional progress must be made, such strategies, alone or in combination with immunosuppression to avoid de novo induction of antibodies, have strong potential to significantly enhance the clinical efficacy of AAV vectors.

  16. Hierarchical Assembly of Plasmonic Nanostructures using Virus Capsid Scaffolds on DNA Origami Tiles

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Debin; Capehart, Stacy L.; Pal, Suchetan; Liu, Minghui; Zhang, Lei; Schuck, P. J.; Liu, Yan; Yan, Hao; Francis, Matthew B.; De Yoreo, James J.

    2014-07-07

    Plasmonic nanoarchitectures using biological scaffolds have shown the potential to attain controllable plasmonic fluorescence via precise spatial arrangement of fluorophores and plasmonic antennae. However, previous studies report a predominance of fluorescence quenching for small metal nanoparticles (less than ~60 nm) due to their small scattering cross-sections. In this work, we report the design and performance of fluorescent plasmonic structures composed of fluorophore-modified virus capsids and gold nanoparticles (AuNPs) assembled on DNA origami tiles. The virus capsid creates a scaffold for control over the three dimensional arrangement of the fluorophores, whereas the DNA origami tile provides precise control over the distance between the capsid and the AuNP. Using finite-difference time-domain (FDTD) numerical simulations and multimodal single-particle imaging measurements, we show that the judicial design of these structures places the dye molecules near the hot spot of the AuNP. This effectively increases the fluorescence intensity in the quenching regime of the AuNP, with an enhancement factor that increases with increasing AuNP size. This strategy of using biological scaffolds to control fluorescence paves the way for exploring the parameters that determine plasmonic fluorescence. It may lead to a better understanding of the antenna effects of photon absorption and emission, enabling the construction of multicomponent plasmonic systems.

  17. In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway

    Science.gov (United States)

    Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L.; Wagner, Jef; Himes, Benjamin A.; Zhao, Gongpu; Aiken, Christopher; Zandi, Roya; Zhang, Peijun

    2016-12-01

    HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

  18. Characterization of a nuclear localization signal of canine parvovirus capsid proteins.

    Science.gov (United States)

    Vihinen-Ranta, M; Kakkola, L; Kalela, A; Vilja, P; Vuento, M

    1997-12-01

    We investigated the abilities of synthetic peptides mimicking the potential nuclear localization signal of canine parvovirus (CPV) capsid proteins to translocate a carrier protein to the nucleus following microinjection into the cytoplasm of A72 cells. Possible nuclear localization sequences were chosen for synthesis from CPV capsid protein sequences (VP1, VP2) on the basis of the presence of clustered basic residues, which is a common theme in most of the previously identified targeting peptides. Nuclear targeting activity was found within the N-terminal residues 4-13 (PAKRARRGYK) of the VP1 capsid protein. While replacement of Arg10 with glycine did not affect the activity, replacement of Lys6, Arg7, or Arg9 with glycine abolished it. The targeting activity was found to residue in a cluster of basic residues, Lys5, Arg7, and Arg9. Nuclear import was saturated by excess of unlabelled peptide conjugates (showing that it was a receptor-mediated process). Transport into the nucleus was an energy-dependent and temperature-dependent process actively mediated by the nuclear pores and inhibited by wheat germ agglutinin.

  19. Viral capsid is a pathogen-associated molecular pattern in adenovirus keratitis.

    Directory of Open Access Journals (Sweden)

    Ashish V Chintakuntlawar

    2010-04-01

    Full Text Available Human adenovirus (HAdV infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9 signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9(-/- mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD. These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins.

  20. Detection of major capsid protein of infectious myonecrosis virus in shrimps using monoclonal antibodies.

    Science.gov (United States)

    Seibert, Caroline H; Borsa, Mariana; Rosa, Rafael D; Cargnin-Ferreira, Eduardo; Pereira, Alitiene M L; Grisard, Edmundo C; Zanetti, Carlos R; Pinto, Aguinaldo R

    2010-10-01

    Infectious myonecrosis virus (IMNV) has been causing a progressive disease in farm-reared shrimps in Brazil and Indonesia. Immunodiagnostic methods for IMNV detection, although reliable, are not employed currently because monoclonal antibodies (MAbs) against this virus are not available. In this study, a fragment of the IMNV major capsid protein gene, comprising amino acids 300-527 (IMNV(300-527)), was cloned and expressed in Escherichia coli. The nucleotide sequence of the recombinant IMNV(300-527) fragment displayed a high degree of identity to the major capsid protein of IMNV isolates from Brazil (99%) and Indonesia (98%). Ten MAbs were generated against the expressed fragment, and eight of these, mostly IgG(2a) or IgG(2b), were able to bind to IMNV in tissue extracts from shrimps infected naturally in immunodot-blot assays. Six of these MAbs recognized a approximately 100 kDa protein in a Western-blot, which is the predicted mass of IMNV major capsid protein, and also bound to viral inclusions present in muscle fibroses and in coagulative myonecrosis, as demonstrated by immunohistochemistry. Among all those MAbs created, four did not cross-react with non-infected shrimp tissues; this observation supports their applicability as a sensitive and specific immunodiagnosis of IMNV infection in shrimps.

  1. Epitope mapping by epitope excision, hydrogen/deuterium exchange, and peptide-panning techniques combined with in silico analysis.

    Science.gov (United States)

    Clementi, Nicola; Mancini, Nicasio; Criscuolo, Elena; Cappelletti, Francesca; Clementi, Massimo; Burioni, Roberto

    2014-01-01

    The fine characterization of protective B cell epitopes plays a pivotal role in the development of novel vaccines. The development of epitope-based vaccines, in fact, cannot be possible without a clear definition of the antigenic regions involved in the binding between the protective antibody (Ab) and its molecular target. To achieve this result, different epitope-mapping approaches have been widely described (Clementi et al. Drug Discov Today 18(9-10):464-471, 2013). Nowadays, the best way to characterize an Ab bound region is still the resolution of Ab-antigen (Ag) co-crystal structure. Unfortunately, the crystallization approaches are not always feasible. However, different experimental strategies aimed to predict Ab-Ag interaction and followed by in silico analysis of the results may be good surrogate approaches to achieve this result. Here, we review few experimental techniques followed by the use of "basic" informatics tools for the analysis of the results.

  2. File list: Oth.Epd.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Epd.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Epidermis SRX71842...0,SRX512368,SRX512366,SRX807621,SRX512367,SRX512372,SRX512373,SRX807620 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Epd.50.Epitope_tags.AllCell.bed ...

  3. File list: Oth.Unc.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.20.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags Unclassified ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.20.Epitope_tags.AllCell.bed ...

  4. File list: Oth.CeL.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CeL.20.Epitope_tags.AllCell dm3 TFs and others Epitope tags Cell line SRX099638...099636 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.CeL.20.Epitope_tags.AllCell.bed ...

  5. File list: Oth.Unc.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.50.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags Unclassified ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.50.Epitope_tags.AllCell.bed ...

  6. File list: Oth.Adl.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adl.10.Epitope_tags.AllCell dm3 TFs and others Epitope tags Adult SRX181427,SRX...ttp://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Adl.10.Epitope_tags.AllCell.bed ...

  7. File list: Oth.Prs.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Prs.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Prostate SRX084528...,SRX084527,SRX084524 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Prs.05.Epitope_tags.AllCell.bed ...

  8. File list: Oth.PSC.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Pluripotent stem c...ell SRX555489 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.PSC.20.Epitope_tags.AllCell.bed ...

  9. File list: Oth.Kid.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Kid.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Kidney SRX065541,S...RX644719,SRX170375,SRX644723 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Kid.10.Epitope_tags.AllCell.bed ...

  10. File list: Oth.Brs.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Brs.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Breast SRX667411,S...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Brs.10.Epitope_tags.AllCell.bed ...

  11. File list: Oth.EmF.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Embryonic fibroblas...RX255460,SRX204644,SRX542102,SRX204643,SRX204642 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.50.Epitope_tags.AllCell.bed ...

  12. File list: Oth.ALL.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.50.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags All cell types ...493939 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.50.Epitope_tags.AllCell.bed ...

  13. File list: Oth.Bld.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Blood SRX718015,SRX...,SRX180155,SRX695808 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Bld.50.Epitope_tags.AllCell.bed ...

  14. File list: Oth.Emb.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.05.Epitope_tags.AllCell dm3 TFs and others Epitope tags Embryo SRX066244,SR...X815533,SRX066245,SRX815531,SRX066247 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Emb.05.Epitope_tags.AllCell.bed ...

  15. File list: Oth.CDV.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CDV.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Cardiovascular SRX1...304813 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.CDV.20.Epitope_tags.AllCell.bed ...

  16. File list: Oth.Emb.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.10.Epitope_tags.AllCell dm3 TFs and others Epitope tags Embryo SRX066244,SR...X815533,SRX066245,SRX815531,SRX066247 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Emb.10.Epitope_tags.AllCell.bed ...

  17. File list: Oth.Neu.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Neural SRX275807,SR...SRX691799,SRX691794,SRX759286,SRX691798,SRX691797,SRX275809,SRX275811,SRX691795,SRX022866 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Neu.20.Epitope_tags.AllCell.bed ...

  18. File list: Oth.Epd.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Epd.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Epidermis SRX51236...8,SRX512367,SRX718420,SRX512372,SRX512366,SRX512373,SRX807621,SRX807620 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Epd.05.Epitope_tags.AllCell.bed ...

  19. File list: Oth.NoD.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.50.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags No description ...http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.50.Epitope_tags.AllCell.bed ...

  20. File list: Oth.Utr.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Utr.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Uterus SRX248763,S...,SRX735140,SRX735139,SRX210703,SRX210702,SRX095386,SRX968127 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Utr.05.Epitope_tags.AllCell.bed ...

  1. File list: Oth.ALL.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.10.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags All cell types ...211370,SRX493939,SRX211371 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.10.Epitope_tags.AllCell.bed ...

  2. File list: Oth.Emb.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Embryo SRX485264,SR...SRX663358,SRX967653 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.50.Epitope_tags.AllCell.bed ...

  3. File list: Oth.Myo.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Muscle SRX1470542,...SRX1470544 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Myo.10.Epitope_tags.AllCell.bed ...

  4. File list: Oth.Myo.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Muscle SRX1470542,...SRX1470544 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Myo.20.Epitope_tags.AllCell.bed ...

  5. File list: Oth.Neu.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Neural SRX367452,S...RX367451 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Neu.20.Epitope_tags.AllCell.bed ...

  6. File list: Oth.PSC.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Pluripotent stem ce...708,ERX320411,SRX647912,SRX204802,SRX352995 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.50.Epitope_tags.AllCell.bed ...

  7. File list: Oth.Emb.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.20.Epitope_tags.AllCell dm3 TFs and others Epitope tags Embryo SRX815533,SR...X066244,SRX066245,SRX815531,SRX066247 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Emb.20.Epitope_tags.AllCell.bed ...

  8. File list: Oth.Prs.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Prs.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Prostate SRX084527...,SRX084528,SRX084524 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Prs.20.Epitope_tags.AllCell.bed ...

  9. File list: Oth.Brs.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Brs.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Breast SRX667411,S...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Brs.05.Epitope_tags.AllCell.bed ...

  10. File list: Oth.NoD.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags No description htt...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.NoD.50.Epitope_tags.AllCell.bed ...

  11. File list: Oth.ALL.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags All cell types SRX1...460,ERX320411,SRX695808 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.ALL.05.Epitope_tags.AllCell.bed ...

  12. File list: Oth.NoD.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.10.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags No description ...http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.10.Epitope_tags.AllCell.bed ...

  13. File list: Oth.Myo.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Muscle SRX1470542,...SRX1470544 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Myo.05.Epitope_tags.AllCell.bed ...

  14. File list: Oth.Unc.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Unclassified SRX88...9798 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Unc.10.Epitope_tags.AllCell.bed ...

  15. File list: Oth.Bon.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bon.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Bone SRX065557,SRX...096356,SRX096358,SRX316960,SRX065556 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Bon.05.Epitope_tags.AllCell.bed ...

  16. File list: Oth.Liv.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Liv.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Liver SRX1165095,S...RX1165103,SRX1165096,SRX1165104,SRX1165100,SRX1165101,SRX1165102,SRX1165090,SRX1165091 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.05.Epitope_tags.AllCell.bed ...

  17. File list: Oth.Neu.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Neural SRX367452,S...RX367451 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Neu.10.Epitope_tags.AllCell.bed ...

  18. File list: Oth.ALL.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags All cell types SRX...322539,SRX170374,SRX644727,SRX644719,SRX644723 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.ALL.20.Epitope_tags.AllCell.bed ...

  19. File list: Oth.Neu.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Neural SRX275807,SR...SRX759284,SRX691794,SRX759286,SRX691798,SRX691797,SRX691795,SRX022866,SRX275809,SRX275811 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Neu.50.Epitope_tags.AllCell.bed ...

  20. File list: Oth.Pan.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Pan.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Pancreas SRX747491,...SRX747492 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Pan.50.Epitope_tags.AllCell.bed ...

  1. File list: Oth.Unc.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.10.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags Unclassified ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.10.Epitope_tags.AllCell.bed ...

  2. File list: Oth.Lng.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Lng.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Lung SRX119639,SRX...119641,SRX119640,SRX119642,SRX119638,SRX119637 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Lng.20.Epitope_tags.AllCell.bed ...

  3. File list: Oth.Emb.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.50.Epitope_tags.AllCell dm3 TFs and others Epitope tags Embryo SRX066244,SR...X815533,SRX066247,SRX066245,SRX815531 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Emb.50.Epitope_tags.AllCell.bed ...

  4. File list: Oth.Bon.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bon.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Bone SRX065557,SRX...096356,SRX096358,SRX316960,SRX065556 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Bon.50.Epitope_tags.AllCell.bed ...

  5. File list: Oth.Epd.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Epd.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Epidermis SRX71842...0,SRX512368,SRX512366,SRX807621,SRX512367,SRX512372,SRX512373,SRX807620 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Epd.20.Epitope_tags.AllCell.bed ...

  6. File list: Oth.CDV.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CDV.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Cardiovascular SRX...096360,SRX096362 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.CDV.20.Epitope_tags.AllCell.bed ...

  7. File list: Oth.Gon.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Gonad SRX153153,SRX...153152,SRX153151 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Gon.20.Epitope_tags.AllCell.bed ...

  8. File list: Oth.Neu.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Neural SRX367452,S...RX367451 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Neu.05.Epitope_tags.AllCell.bed ...

  9. In silico-accelerated identification of conserved and immunogenic variola/vaccinia T-cell epitopes

    DEFF Research Database (Denmark)

    Moise, Leonard; McMurry, Julie A; Buus, Søren;

    2009-01-01

    Epitopes shared by the vaccinia and variola viruses underlie the protective effect of vaccinia immunization against variola infection. We set out to identify a subset of cross-reactive epitopes using bioinformatics and immunological methods. Putative T-cell epitopes were computationally predicted...

  10. File list: Oth.Prs.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Prs.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Prostate SRX084528...,SRX084527,SRX084524 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Prs.10.Epitope_tags.AllCell.bed ...

  11. File list: Oth.EmF.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Embryonic fibroblas...RX255459,SRX255462,SRX255460,SRX204643,SRX204642 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.20.Epitope_tags.AllCell.bed ...

  12. File list: Oth.CDV.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CDV.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Cardiovascular SRX...096360,SRX096362 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.CDV.50.Epitope_tags.AllCell.bed ...

  13. File list: Oth.Gon.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Gonad SRX204898,SR...X204899 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Gon.50.Epitope_tags.AllCell.bed ...

  14. File list: Oth.CeL.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CeL.10.Epitope_tags.AllCell dm3 TFs and others Epitope tags Cell line SRX099635...099636 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.CeL.10.Epitope_tags.AllCell.bed ...

  15. File list: Oth.Myo.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Muscle SRX344965,SR...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Myo.20.Epitope_tags.AllCell.bed ...

  16. File list: Oth.Bld.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Blood SRX180156,SRX...,SRX180155,SRX695808 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Bld.05.Epitope_tags.AllCell.bed ...

  17. File list: Oth.Neu.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Neural SRX367452,S...RX367451 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Neu.50.Epitope_tags.AllCell.bed ...

  18. File list: Oth.Liv.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Liv.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Liver SRX1165103,S...RX1165095,SRX1165100,SRX1165101,SRX1165090,SRX1165104,SRX1165102,SRX1165096,SRX1165091 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.20.Epitope_tags.AllCell.bed ...

  19. File list: Oth.Kid.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Kid.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Kidney SRX065541,S...RX644719,SRX527876,SRX644723 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Kid.05.Epitope_tags.AllCell.bed ...

  20. File list: Oth.NoD.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags No description htt...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.NoD.20.Epitope_tags.AllCell.bed ...

  1. File list: Oth.ALL.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.20.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags All cell types ...211371,SRX493939,SRX381289 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.20.Epitope_tags.AllCell.bed ...

  2. File list: Oth.Unc.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Unclassified SRX88...9798 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Unc.50.Epitope_tags.AllCell.bed ...

  3. File list: Oth.Brs.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Brs.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Breast SRX667411,S...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Brs.20.Epitope_tags.AllCell.bed ...

  4. File list: Oth.Utr.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Utr.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Uterus SRX188854,S...,SRX210703,SRX679119,SRX095385,SRX210702,SRX968127,SRX095386 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Utr.20.Epitope_tags.AllCell.bed ...

  5. File list: Oth.Gon.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Gonad SRX204899,SR...X204898 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Gon.05.Epitope_tags.AllCell.bed ...

  6. File list: Oth.Gon.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Gonad SRX204898,SR...X204899 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Gon.10.Epitope_tags.AllCell.bed ...

  7. File list: Oth.PSC.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Pluripotent stem ce...822,SRX266828,SRX352996,ERX320411,SRX204802 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.20.Epitope_tags.AllCell.bed ...

  8. File list: Oth.Prs.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Prs.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Prostate SRX084527...,SRX084528,SRX084524 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Prs.50.Epitope_tags.AllCell.bed ...

  9. File list: Oth.Adl.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adl.05.Epitope_tags.AllCell dm3 TFs and others Epitope tags Adult SRX181427,SRX...ttp://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Adl.05.Epitope_tags.AllCell.bed ...

  10. File list: Oth.ALL.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags All cell types SRX1...995,SRX275809,SRX275811 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.ALL.50.Epitope_tags.AllCell.bed ...

  11. File list: Oth.CDV.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CDV.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Cardiovascular SRX...096360,SRX096362 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.CDV.10.Epitope_tags.AllCell.bed ...

  12. File list: Oth.Pan.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Pan.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Pancreas SRX747491,...SRX747492 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Pan.05.Epitope_tags.AllCell.bed ...

  13. File list: Oth.Gon.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Gonad SRX153153,SRX...153152,SRX153151 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Gon.50.Epitope_tags.AllCell.bed ...

  14. File list: Oth.YSt.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.YSt.10.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags Yeast strain SR...1370,SRX493939,SRX211371 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.10.Epitope_tags.AllCell.bed ...

  15. File list: Oth.YSt.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.YSt.50.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags Yeast strain SR...3939 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.50.Epitope_tags.AllCell.bed ...

  16. File list: Oth.NoD.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.20.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags No description ...http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.20.Epitope_tags.AllCell.bed ...

  17. File list: Oth.CDV.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CDV.10.Epitope_tags.AllCell mm9 TFs and others Epitope tags Cardiovascular SRX1...304813 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.CDV.10.Epitope_tags.AllCell.bed ...

  18. File list: Oth.Kid.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Kid.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Kidney SRX065541,S...RX170376,SRX065542,SRX065543 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Kid.50.Epitope_tags.AllCell.bed ...

  19. File list: Oth.Unc.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Unclassified SRX88...9798 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Unc.05.Epitope_tags.AllCell.bed ...

  20. File list: Oth.CDV.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CDV.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Cardiovascular SRX1...304813 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.CDV.50.Epitope_tags.AllCell.bed ...

  1. File list: Oth.Oth.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Oth.10.Epitope_tags.AllCell mm9 TFs and others Epitope tags Others SRX228677,SR...X228676,SRX228679,SRX228678 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Oth.10.Epitope_tags.AllCell.bed ...

  2. File list: Oth.Kid.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Kid.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Kidney SRX065541,S...RX644727,SRX644719,SRX644723 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Kid.20.Epitope_tags.AllCell.bed ...

  3. File list: Oth.Myo.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Muscle SRX1470542,...SRX1470544 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Myo.50.Epitope_tags.AllCell.bed ...

  4. File list: Oth.PSC.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Pluripotent stem c...ell SRX555489 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.PSC.10.Epitope_tags.AllCell.bed ...

  5. File list: Oth.Adl.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adl.50.Epitope_tags.AllCell dm3 TFs and others Epitope tags Adult SRX181419,SRX...ttp://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Adl.50.Epitope_tags.AllCell.bed ...

  6. File list: Oth.Liv.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Liv.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Liver SRX1165095,S...RX1165103,SRX1165100,SRX1165096,SRX1165104,SRX1165101,SRX1165090,SRX1165102,SRX1165091 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.10.Epitope_tags.AllCell.bed ...

  7. File list: Oth.CeL.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CeL.05.Epitope_tags.AllCell dm3 TFs and others Epitope tags Cell line SRX330995...099636 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.CeL.05.Epitope_tags.AllCell.bed ...

  8. File list: Oth.NoD.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.05.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags No description ...http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.05.Epitope_tags.AllCell.bed ...

  9. File list: Oth.YSt.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.YSt.05.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags Yeast strain SR...1370,SRX211371,SRX493939 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.05.Epitope_tags.AllCell.bed ...

  10. File list: Oth.Oth.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Oth.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Others SRX228677,SR...X228679,SRX228676,SRX228678 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Oth.50.Epitope_tags.AllCell.bed ...

  11. File list: Oth.Utr.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Utr.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Uterus SRX188854,S...,SRX210703,SRX968127,SRX610673,SRX610674,SRX610672,SRX095386 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Utr.50.Epitope_tags.AllCell.bed ...

  12. File list: Oth.Adl.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adl.20.Epitope_tags.AllCell dm3 TFs and others Epitope tags Adult SRX181427,SRX...ttp://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Adl.20.Epitope_tags.AllCell.bed ...

  13. File list: Oth.Oth.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Oth.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Others SRX228677,SR...X228676,SRX228679,SRX228678 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Oth.05.Epitope_tags.AllCell.bed ...

  14. File list: Oth.Dig.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Dig.10.Epitope_tags.AllCell mm9 TFs and others Epitope tags Digestive tract SRX...365692 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Dig.10.Epitope_tags.AllCell.bed ...

  15. File list: Oth.Liv.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Liv.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Liver SRX1165103,S...RX1165095,SRX1165100,SRX1165101,SRX1165104,SRX1165102,SRX1165090,SRX1165091,SRX1165096 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.50.Epitope_tags.AllCell.bed ...

  16. File list: Oth.PSC.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Pluripotent stem ce...821,ERX320410,SRX266822,SRX352996,ERX320411 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.05.Epitope_tags.AllCell.bed ...

  17. File list: Oth.Dig.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Dig.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Digestive tract SRX...365692 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Dig.20.Epitope_tags.AllCell.bed ...

  18. File list: Oth.Gon.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Gonad SRX153152,SRX...153153,SRX153151 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Gon.05.Epitope_tags.AllCell.bed ...

  19. File list: Oth.ALL.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags All cell types SRX...644715,SRX555489,SRX644719,SRX527876,SRX644723 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.ALL.05.Epitope_tags.AllCell.bed ...

  20. File list: Oth.Myo.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Muscle SRX344965,SR...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Myo.50.Epitope_tags.AllCell.bed ...

  1. File list: Oth.Myo.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.10.Epitope_tags.AllCell mm9 TFs and others Epitope tags Muscle SRX039346,SR...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Myo.10.Epitope_tags.AllCell.bed ...

  2. File list: Oth.Myo.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Muscle SRX039346,SR...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Myo.05.Epitope_tags.AllCell.bed ...

  3. File list: Oth.ALL.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.05.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags All cell types ...211370,SRX211371,SRX493939 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.05.Epitope_tags.AllCell.bed ...

  4. File list: Oth.Bon.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bon.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Bone SRX065557,SRX...096356,SRX096358,SRX316960,SRX065556 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Bon.10.Epitope_tags.AllCell.bed ...

  5. File list: Oth.ALL.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags All cell types SRX1...802,SRX204643,SRX204642 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.ALL.20.Epitope_tags.AllCell.bed ...

  6. File list: Oth.Brs.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Brs.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Breast SRX667411,S...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Brs.50.Epitope_tags.AllCell.bed ...

  7. File list: Oth.Gon.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.10.Epitope_tags.AllCell mm9 TFs and others Epitope tags Gonad SRX153153,SRX...153151,SRX153152 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Gon.10.Epitope_tags.AllCell.bed ...

  8. Generation and molecular characterization of a monoclonal antibody reactive with conserved epitope in sphingomyelinases D from Loxosceles spider venoms.

    Science.gov (United States)

    Dias-Lopes, C; Felicori, L; Rubrecht, L; Cobo, S; Molina, L; Nguyen, C; Galéa, P; Granier, C; Molina, F; Chávez-Olortegui, C

    2014-04-11

    We report the production of a neutralizing monoclonal antibody able to recognize the venoms of three major medically important species of Loxosceles spiders in Brazil. The mAb was produced by immunization of mice with a toxic recombinant L. intermedia sphingomyelinase D {SMases D isoform (rLiD1)} [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho venoms as antigens. One clone (LiD1mAb16) out of seventeen anti-rLiD1 hybridomas was cross-reactive with the three whole Loxosceles venoms. 2D Western blot analysis indicated that LiD1mAb16 was capable of interacting with 34 proteins of 29-36kDa in L. intermedia, 33 in L. gaucho and 27 in L. laeta venoms. The results of immunoassays with cellulose-bound peptides revealed that the LiD1mAb16 recognizes a highly conserved linear epitope localized in the catalytic region of SMases D toxins. The selected mAb displayed in vivo protective activity in rabbits after challenge with rLiD1. These results show the potential usefulness of monoclonal antibodies for future therapeutic approaches and also opens up the perspective of utilization of these antibodies for immunodiagnostic assays in loxoscelism.

  9. Mutational Analysis of the Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Construction of AAV2 Vectors with Altered Tropism

    OpenAIRE

    Wu, Pei; Xiao, Wu; Conlon, Thomas; Hughes, Jeffrey; Agbandje-McKenna, Mavis; FERKOL, THOMAS; Flotte, Terence; Muzyczka, Nicholas

    2000-01-01

    Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mut...

  10. Mast Cells Produce a Unique Chondroitin Sulfate Epitope.

    Science.gov (United States)

    Farrugia, Brooke L; Whitelock, John M; O'Grady, Robert; Caterson, Bruce; Lord, Megan S

    2016-02-01

    The granules of mast cells contain a myriad of mediators that are stored and protected by the sulfated glycosaminoglycan (GAG) chains that decorate proteoglycans. Whereas heparin is the GAG predominantly associated with mast cells, mast cell proteoglycans are also decorated with heparan sulfate and chondroitin sulfate (CS). This study investigated a unique CS structure produced by mast cells that was detected with the antibody clone 2B6 in the absence of chondroitinase ABC digestion. Mast cells in rodent tissue sections were characterized using toluidine blue, Leder stain and the presence of mast cell tryptase. The novel CS epitope was identified in rodent tissue sections and localized to cells that were morphologically similar to cells chemically identified as mast cells. The rodent mast cell-like line RBL-2H3 was also shown to express the novel CS epitope. This epitope co-localized with multiple CS proteoglycans in both rodent tissue and RBL-2H3 cultured cells. These findings suggest that the novel CS epitope that decorates mast cell proteoglycans may play a role in the way these chains are structured in mast cells.

  11. An epitope delivery system for use with recombinant mycobacteria

    NARCIS (Netherlands)

    Hetzel, C.; Janssen, R.; Ely, S.J.; Kristensen, N.M.; Bunting, K.; Cooper, J.B.; Lamb, J.R.; Young, D.B.; Thole, J.E.R.

    1998-01-01

    We have developed a novel epitope delivery system based on the insertion of peptides within a permissive loop of a bacterial superoxide dismutase molecule. This system allowed high-level expression of heterologous peptides in two mycobacterial vaccine strains, Mycobacterium bovis bacille Calmette- G

  12. High-Throughput Tools for Characterization of Antibody Epitopes

    DEFF Research Database (Denmark)

    Christiansen, Anders

    multiple years. Taken together, the presented studies demonstrated new applications for the investigated techniques focusing on their utilization in epitope mapping. In the process, new insights were obtained into how antibodies recognize their targets in a major disease, i.e. food allergy....

  13. High-throughput epitope profiling of snake venom toxins

    DEFF Research Database (Denmark)

    Engmark, Mikael; Andersen, Mikael Rørdam; Laustsen, Andreas Hougaard

    Insight into the molecular details of polyclonal antivenom antibody specificity is a prerequisite for accurate prediction of cross-reactivity and can provide a basis for design of novel antivenoms. In this work, a highthroughput approach was applied to characterize linear elements in epitopes in ...... toxins from four African mamba and three neurotoxic cobra snakes obtained from public databases....

  14. Development of a human papillomavirus competitive luminex immunoassay for 9 HPV types.

    Science.gov (United States)

    Roberts, Christine; Green, Tina; Hess, Erica; Matys, Katie; Brown, Martha J; Haupt, Richard M; Luxembourg, Alain; Vuocolo, Scott; Saah, Alfred; Antonello, Joseph

    2014-01-01

    In the clinical trials of the quadrivalent human papillomavirus (qHPV) vaccine, antibodies were measured by a competitive Luminex immunoassay (HPV-4 cLIA). A nine-valent HPV (9vHPV) vaccine targeting the types in the qHPV vaccine (HPV6/11/16/18), as well as 5 of the next most frequent HPV types found in cervical cancers worldwide (HPV31/33/45/52/58) is under development. To support the 9vHPV vaccine program, a nine-multiplexed cLIA (HPV-9 cLIA) was developed. Antibody titers were determined in a competitive format, where type-specific phycoerythrin (PE)-labeled, neutralizing mAbs (mAbs-PE) compete with an individual's serum antibodies for binding to conformationally sensitive, neutralizing epitopes on the VLPs. Neutralizing antibody levels were quantitated against a reference standard - a pool of sera from 6 Rhesus macaques that were immunized with the 9vHPV vaccine. Specificity of the mAbs was assessed by measuring their individual binding capacities to the type-specific and non-type-specific VLPs at alternative concentrations of the mAbs. Antibody assignments to the HPV-9 cLIA reference standard for HPV6/11/16/18 were determined to provide for a measure of consistency in serostatus assignment between the HPV-4 and HPV-9 cLIAs. Antibody assignments to the HPV-9 reference standard for HPV31/33/45/52/58 were obtained by calibration to HPV11 using a direct binding IgG assay. For each HPV VLP type, the cross-reactivity of the mAb-PEs in the HPV-9 cLIA was <1% (i.e., the mAb-PEs result in <1% non-specific binding). The antibody concentrations assigned to the HPV-9 cLIA reference standard for types 6/11/16/18/31/33/45/52/58 were 3,817, 2,889, 23,061, 5,271, 3,942, 2,672, 1,489, 1274, and 2263 mMU/mL, respectively.

  15. Micromotor-based lab-on-chip immunoassays

    Science.gov (United States)

    García, Miguel; Orozco, Jahir; Guix, Maria; Gao, Wei; Sattayasamitsathit, Sirilak; Escarpa, Alberto; Merkoçi, Arben; Wang, Joseph

    2013-01-01

    Here we describe the first example of using self-propelled antibody-functionalized synthetic catalytic microengines for capturing and transporting target proteins between the different reservoirs of a lab-on-a-chip (LOC) device. A new catalytic polymer/Ni/Pt microtube engine, containing carboxy moieties on its mixed poly(3,4-ethylenedioxythiophene) (PEDOT)/COOH-PEDOT polymeric outermost layer, is further functionalized with the antibody receptor to selectively recognize and capture the target protein. The new motor-based microchip immunoassay operations are carried out without any bulk fluid flow, replacing the common washing steps in antibody-based protein bioassays with the active transport of the captured protein throughout the different reservoirs, where each step of the immunoassay takes place. A first microchip format involving an `on-the-fly' double-antibody sandwich assay (DASA) is used for demonstrating the selective capture of the target protein, in the presence of excess of non-target proteins. A secondary antibody tagged with a polymeric-sphere tracer allows the direct visualization of the binding events. In a second approach the immuno-nanomotor captures and transports the microsphere-tagged antigen through a microchannel network. An anti-protein-A modified microengine is finally used to demonstrate the selective capture, transport and convenient label-free optical detection of a Staphylococcus aureus target bacteria (containing proteinA in its cell wall) in the presence of a large excess of non-target (Saccharomyces cerevisiae) cells. The resulting nanomotor-based microchip immunoassay offers considerable potential for diverse applications in clinical diagnostics, environmental and security monitoring fields.Here we describe the first example of using self-propelled antibody-functionalized synthetic catalytic microengines for capturing and transporting target proteins between the different reservoirs of a lab-on-a-chip (LOC) device. A new catalytic

  16. Two highly similar LAEDDTNAQKT and LTDKIGTEI epitopes in G glycoprotein may be useful for effective epitope based vaccine design against pathogenic Henipavirus.

    Science.gov (United States)

    Parvege, Md Masud; Rahman, Monzilur; Nibir, Yead Morshed; Hossain, Mohammad Shahnoor

    2016-04-01

    Nipah virus and Hendra virus, two members of the genus Henipavirus, are newly emerging zoonotic pathogens which cause acute respiratory illness and severe encephalitis in human. Lack of the effective antiviral therapy endorses the urgency for the development of vaccine against these deadly viruses. In this study, we employed various computational approaches to identify epitopes which has the potential for vaccine development. By analyzing the immune parameters of the conserved sequences of G glycoprotein using various databases and bioinformatics tools, we identified two potential epitopes which may be used as peptide vaccines. Using different B cell epitope prediction servers, four highly similar B cell epitopes were identified. Immunoinformatics analyses revealed that LAEDDTNAQKT is a highly flexible and accessible B-cell epitope to antibody. Highly similar putative CTL epitopes were analyzed for their binding with the HLA-C 12*03 molecule. Docking simulation assay revealed that LTDKIGTEI has significantly lower binding energy, which bolstered its potential as epitope-based vaccine design. Finally, cytotoxicity analysis has also justified their potential as promising epitope-based vaccine candidate. In sum, our computational analysis indicates that either LAEDDTNAQKT or LTDKIGTEI epitope holds a promise for the development of universal vaccine against all kinds of pathogenic Henipavirus. Further in vivo and in vitro studies are necessary to validate the obtained findings.

  17. MIMOX: a web tool for phage display based epitope mapping

    Directory of Open Access Journals (Sweden)

    Honda Wataru

    2006-10-01

    Full Text Available Abstract Background Phage display is widely used in basic research such as the exploration of protein-protein interaction sites and networks, and applied research such as the development of new drugs, vaccines, and diagnostics. It has also become a promising method for epitope mapping. Research on new algorithms that assist and automate phage display based epitope mapping has attracted many groups. Most of the existing tools have not been implemented as an online service until now however, making it less convenient for the community to access, utilize, and evaluate them. Results We present MIMOX, a free web tool that helps to map the native epitope of an antibody based on one or more user supplied mimotopes and the antigen structure. MIMOX was coded in Perl using modules from the Bioperl project. It has two sections. In the first section, MIMOX provides a simple interface for ClustalW to align a set of mimotopes. It also provides a simple statistical method to derive the consensus sequence and embeds JalView as a Java applet to view and manage the alignment. In the second section, MIMOX can map a single mimotope or a consensus sequence of a set of mimotopes, on to the corresponding antigen structure and search for all of the clusters of residues that could represent the native epitope. NACCESS is used to evaluate the surface accessibility of the candidate clusters; and Jmol is embedded to view them interactively in their 3D context. Initial case studies show that MIMOX can reproduce mappings from existing tools such as FINDMAP and 3DEX, as well as providing novel, rational results. Conclusion A web-based tool called MIMOX has been developed for phage display based epitope mapping. As a publicly available online service in this area, it is convenient for the community to access, utilize, and evaluate, complementing other existing programs. MIMOX is freely available at http://web.kuicr.kyoto-u.ac.jp/~hjian/mimox.

  18. Fluorescence polarization immunoassay for salinomycin based on monoclonal antibodies

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A fluorescence polarization immunoassay(FPIA) for the determination of salinomycin(SAL) was developed by using anti-SAL monoclonal antibodies(mAb).Fluorescein labeled SAL(tracer) was synthesized by the N-hydroxysuccinimide active ester method and purified using thin layer chromatography(TLC).The developed FPIA for SAL had a dynamic range from 0.60 to 2193 ng/mL with an IC50 value of 33.2 ng/mL and a detection limit(LOD) of 0.08 ng/mL.No significant cross-reactivities were observed with other drugs but 67.6% with narasin.

  19. Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

    Science.gov (United States)

    Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

    1999-04-01

    Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

  20. Bupropion interference with immunoassays for amphetamines and LSD.

    Science.gov (United States)

    Vidal, Christian; Skripuletz, Thomas

    2007-06-01

    A 50-year-old male patient suddenly had lost consciousness, although he had previously been healthy. On arrival at hospital seizures arose. The authors investigated a urine sample of the patient, and performed toxicological drug screening with immunochemical Cloned Enzyme Donor Immunoassay (CEDIA) assays. Positive findings for amphetamines and LSD could not be confirmed. Using gas chromatography/mass spectrometry (GC/MS), and liquid chromatography/mass spectrometry (LC/MS), the authors identified bupropion, a drug used to aid in smoking cessation, as the interfering compound, which may cause false-positive results for amphetamines and LSD using the CEDIA assays.

  1. Pholcodine interference in the immunoassay for opiates in urine.

    Science.gov (United States)

    Svenneby, G; Wedege, E; Karlsen, R L

    1983-01-01

    The excretion in urine after single oral therapeutic doses of morphine derivatives was analysed with radioimmunoassay (RIA) and homogeneous enzyme immunoassay (EMIT) for opiates. In contrast to the rapid excretion of ethylmorphine and codeine, pholcodine showed positive results for opiates 2-6 weeks after intake when the urines were analysed with the RIA-method. When analysed with the EMIT-method, positive results were obtained for pholcodine for approximately 10 days. As pholcodine is a common component in cough mixtures, its prolonged excretion could represent a hazard in interpreting the results from drug analyses of urines.

  2. Screening of cannabinoids, benzoylecgonine and opiates in whole blood and urine using emit II plus immunoassay and konelab 30

    DEFF Research Database (Denmark)

    Simonsen, Kirsten Wiese; Christiansen, Nobuko; Müller, Irene Breum

    2004-01-01

    Screening,cannabinoids,benzoylecgonine,opiates in whole blood and urine, emit II, immunoassay,konelab 30......Screening,cannabinoids,benzoylecgonine,opiates in whole blood and urine, emit II, immunoassay,konelab 30...

  3. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine

    Science.gov (United States)

    Poh, Chit Laa; Kirk, Kristin; McBride, William John Hannan; Aaskov, John; Grollo, Lara

    2016-01-01

    Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine. PMID:27223692

  4. Epitope-specific antibody levels in tuberculosis: biomarkers of protection, disease and response to treatment.

    Directory of Open Access Journals (Sweden)

    Graham H Bothamley

    2014-06-01

    Full Text Available Monoclonal antibodies restricted to Mycobacterium tuberculosis can measure epitope-specific antibody levels in a competition assay. Immunodominant epitopes were defined from clinical samples and related to the clinical spectrum of disease. Antibody to the immunodominant epitopes was associated with HLA-DR15. Occupational exposure showed a different response and was consistent with recognition of dormancy related proteins and protection despite exposure to tuberculosis. Studies in leprosy revealed the importance of immune deviation and the relationships between T and B cell epitopes. During treatment, antibody levels increased, epitope spreading occurred, but the affinity constants remained the same after further antigen exposure, suggesting constraints on the process of epitope selection. Epitope-specific antibody levels have a potential role as biomarkers for new vaccines which might prevent the progression of latent to active tuberculosis and as tools to measure treatment effects on subpopulations of tubercle bacilli.

  5. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine.

    Directory of Open Access Journals (Sweden)

    Babu Ramanathan

    Full Text Available Dengue virus (DENV is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine.

  6. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine.

    Science.gov (United States)

    Ramanathan, Babu; Poh, Chit Laa; Kirk, Kristin; McBride, William John Hannan; Aaskov, John; Grollo, Lara

    2016-01-01

    Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine.

  7. A multiplexed immunoassay for detection of antibodies to Actinobacillus pleuropneumoniae (App) in pigs

    DEFF Research Database (Denmark)

    Berger, Sanne Schou; Boas, Ulrik; Andresen, Lars Ole

    2014-01-01

    our diagnostic tools, we are currently developing a novel indirect fluorescent microsphere immunoassay that can facilitate simultaneous detection of antibodies towards multiple App serovars within a single serum sample volume. The multiplex immunoassay is based on Luminex technology (8) and has...

  8. Determination of hepatitis B surface antigen using magnetic immunoassays in a thin channel.

    Science.gov (United States)

    Tsai, H Y; Chan, J R; Li, Y C; Cheng, F C; Fuh, C Bor

    2010-08-15

    We report novel methods for detection of hepatitis B surface antigen (HBsAg) based on competitive and sandwiched magnetic immunoassays using functional magnetic nanoparticles in a thin channel. Magnetic nanoparticles labeled with hepatitis B antibody are flowed through a thin channel to form a predeposition layer for capturing HBsAg. Competitive and sandwiched magnetic immunoassays were studied and detection limit, linear range, and sample selectivity were compared. The detection limits of competitive and sandwiched magnetic immunoassays were found to be 0.26 and 0.25 pg/ml, respectively. The linear range of HBsAg concentration was 0.26 pg/ml-2.6 ng/ml for competitive magnetic immunoassay and was 0.89 pg/ml-8.9 ng/ml for sandwiched magnetic immunoassay. The advantages of these methods over ELISA and other methods for HBsAg detection are lower detection limits and wider linear ranges. The running time was less than 30 min. Competitive magnetic immunoassay was faster than sandwiched magnetic immunoassay for detection of HBsAg. The measurements of HBsAg in serum samples from these methods differed by about 10% from those of ELISA. These methods can provide simple, fast, and sensitive detections of biomarkers and other immunoassay-related samples.

  9. Supramolecular Au Nanoparticle Assemblies as Optical Probes for Enzyme-Linked Immunoassays

    NARCIS (Netherlands)

    Rica, de la Roberto; Velders, Aldrik H.

    2011-01-01

    A novel enzyme-linked immunoassay with nanoparticle assemblies as optical probes is presented. The macrocycle cucurbit[7]uril assembles gold nanoparticles, and the enzymatic generation of ammonium by urease as the label of the immunoassay disperses the probes to yield variations of plasmon absorbanc

  10. Evaluation of an immunoassay for determination of plasma efavirenz concentrations in resource-limited settings

    DEFF Research Database (Denmark)

    Abdissa, Alemseged; Wiesner, Lubbe; McIlleron, Helen

    2014-01-01

    to be implemented in resource-limited settings. This study evaluated a commercially available immunoassay for measurement of plasma efavirenz. Methods: The immunoassay-based method was applied to measure efavirenz using a readily available Humastar 80 chemistry analyzer. We compared plasma efavirenz concentrations...

  11. Rationally designed interfacial peptides are efficient in vitro inhibitors of HIV-1 capsid assembly with antiviral activity.

    Directory of Open Access Journals (Sweden)

    Rebeca Bocanegra

    Full Text Available Virus capsid assembly constitutes an attractive target for the development of antiviral therapies; a few experimental inhibitors of this process for HIV-1 and other viruses have been identified by screening compounds or by selection from chemical libraries. As a different, novel approach we have undertaken the rational design of peptides that could act as competitive assembly inhibitors by mimicking capsid structural elements involved in intersubunit interfaces. Several discrete interfaces involved in formation of the mature HIV-1 capsid through polymerization of the capsid protein CA were targeted. We had previously designed a peptide, CAC1, that represents CA helix 9 (a major part of the dimerization interface and binds the CA C-terminal domain in solution. Here we have mapped the binding site of CAC1, and shown that it substantially overlaps with the CA dimerization interface. We have also rationally modified CAC1 to increase its solubility and CA-binding affinity, and designed four additional peptides that represent CA helical segments involved in other CA interfaces. We found that peptides CAC1, its derivative CAC1M, and H8 (representing CA helix 8 were able to efficiently inhibit the in vitro assembly of the mature HIV-1 capsid. Cocktails of several peptides, including CAC1 or CAC1M plus H8 or CAI (a previously discovered inhibitor of CA polymerization, or CAC1M+H8+CAI, also abolished capsid assembly, even when every peptide was used at lower, sub-inhibitory doses. To provide a preliminary proof that these designed capsid assembly inhibitors could eventually serve as lead compounds for development of anti-HIV-1 agents, they were transported into cultured cells using a cell-penetrating peptide, and tested for antiviral activity. Peptide cocktails that drastically inhibited capsid assembly in vitro were also able to efficiently inhibit HIV-1 infection ex vivo. This study validates a novel, entirely rational approach for the design of capsid

  12. Species Specific Bacterial Spore Detection Using Lateral-Flow Immunoassay with DPA-Triggered Tb Luminescence

    Science.gov (United States)

    Ponce, Adrian

    2003-01-01

    A method of detecting bacterial spores incorporates (1) A method of lateral-flow immunoassay in combination with (2) A method based on the luminescence of Tb3+ ions to which molecules of dipicolinic acid (DPA) released from the spores have become bound. The present combination of lateral-flow immunoassay and DPA-triggered Tb luminescence was developed as a superior alternative to a prior lateral-flow immunoassay method in which detection involves the visual observation and/or measurement of red light scattered from colloidal gold nanoparticles. The advantage of the present combination method is that it affords both (1) High selectivity for spores of the species of bacteria that one seeks to detect (a characteristic of lateral-flow immunoassay in general) and (2) Detection sensitivity much greater (by virtue of the use of DPA-triggered Tb luminescence instead of gold nanoparticles) than that of the prior lateral-flow immunoassay method

  13. Immunoassay Methods and their Applications in Pharmaceutical Analysis: Basic Methodology and Recent Advances.

    Science.gov (United States)

    Darwish, Ibrahim A

    2006-09-01

    Immunoassays are bioanalytical methods in which the quantitation of the analyte depends on the reaction of an antigen (analyte) and an antibody. Immunoassays have been widely used in many important areas of pharmaceutical analysis such as diagnosis of diseases, therapeutic drug monitoring, clinical pharmacokinetic and bioequivalence studies in drug discovery and pharmaceutical industries. The importance and widespread of immunoassay methods in pharmaceutical analysis are attributed to their inherent specificity, high-throughput, and high sensitivity for the analysis of wide range of analytes in biological samples. Recently, marked improvements were achieved in the field of immunoassay development for the purposes of pharmaceutical analysis. These improvements involved the preparation of the unique immunoanalytical reagents, analysis of new categories of compounds, methodology, and instrumentation. The basic methodologies and recent advances in immunoassay methods applied in different fields of pharmaceutical analysis have been reviewed.

  14. AN ENVIRONMENTAL TECHNOLOGY VERIFICATION (ETV) TESTING OF THREE IMMUNOASSAY TEST KITS FOR ANTHRAX, BOTULINUM TOXIN AND RICIN

    Science.gov (United States)

    Immunoassay test kits are based on immunoassay methods, where specific antibodies are used to detect and measure the contaminants of interest. Immunoassay test kits rely on the reaction of a contaminant or antigen with a selective antibody to give a product that can be measures....

  15. Sensitive Immunoassays of Nitrated Fibrinogen in Human Biofluids

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Zhiwen; Wu, Hong; Du, Dan; Wang, Jun; Wang, Hua; Qian, Weijun; Bigelow, Diana J.; Pounds, Joel G.; Smith, Richard D.; Lin, Yuehe

    2010-05-05

    Three new sandwich immunoassays for detection of nitrated biomarker have been established with potential applications in biomedical studies and clinical practice. In this study, nitrated human fibrinogen, a potential oxidative stress biomarker for several pathologies, was chosen as the target. To improve the sensitivity and overcome the interference caused by the complexity of human biofluids, we developed three sandwich strategies using various combinations of primary antibody and secondary antibody. All three strategies demonstrated high sensitivity and selectivity towards nitrated forms of fibrinogen in buffer, but their performances were dramatically reduced when tested with human plasma and serum samples. Systematically optimizations were carried out to investigate the effects of numerous factors, including sampling, coating, blocking, and immunoreactions. Our final optimization results indicate that two of these strategies retain sufficient sensitivity and selectivity for use as assays in human physiological samples. Specifically, detection limits reached the pM level and the linear response ranges were up to nM level with a correlation coefficient > 0.99. To our best knowledge, this is the first example of using an electrochemical immunoassay for a nitrated biomarker in a physiological fluid. This novel approach provides a rapid, sensitive, selective, cost efficient and robust bioassay for detection of oxidative stress in pathology and for clinical applications. Moreover, the sandwich strategies developed in this paper can be readily used to establish effective methods targeting other nitration biomarkers.

  16. Development of competitive immunoassays to hydroxyl containing fungicide metabolites.

    Science.gov (United States)

    Gough, Kevin C; Jarvis, Shila; Maddison, Ben C

    2011-01-01

    This paper describes the isolation of monoclonal antibodies and the development of competitive immunoassays to pesticide metabolites of the fungicides imazalil, carbendazim and thiabendazole. The metabolite specific hydroxyl residues were used as the reactive group with which to link the metabolite to the carrier proteins Keyhole Limpet Haemocyanin (KLH) and Bovine Serum Albumin (BSA). In each case immune responses in mice were raised and monoclonal antibodies were produced. Antibodies were developed into competitive ELISAs to the appropriate metabolite. The antibody raised to a metabolite of imazalil was optimised into a competitive ELISA format which had an assay IC50 of 7.5 μg/L and a limit of detection (LOD) of 1.1 μg/L. A single antibody isolated against the metabolite of carbendazim had assay IC50s of 3.2 and 2.7 μg/L for the metabolites of carbendazim and thiabendazole respectively with an LOD of 0.38 μg/L for both. These sensitive immunoassays may have application in the monitoring of human exposure to these fungicide residues either by occupational or non-occupational routes.

  17. Application of photonic crystal enhanced fluorescence to a cytokine immunoassay.

    Science.gov (United States)

    Mathias, Patrick C; Ganesh, Nikhil; Cunningham, Brian T

    2008-12-01

    Photonic crystal surfaces are demonstrated as a means for enhancing the detection sensitivity and resolution for assays that use a fluorescent tag to quantify the concentration of an analyte protein molecule in a liquid test sample. Computer modeling of the spatial distribution of resonantly coupled electromagnetic fields on the photonic crystal surface are used to estimate the magnitude of enhancement factor compared to performing the same fluorescent assay on a plain glass surface, and the photonic crystal structure is fabricated and tested to experimentally verify the performance using a sandwich immunoassay for the protein tumor necrosis factor-alpha (TNFalpha). The demonstrated photonic crystal fabrication method utilizes a nanoreplica molding technique that allows for large-area inexpensive fabrication of the structure in a format that is compatible with confocal microarray laser scanners. The signal-to-noise ratio for fluorescent spots on the photonic crystal is increased by at least 5-fold relative to the glass slide, allowing a TNF-alpha concentration of 1.6 pg/mL to be distinguished from noise on a photonic crystal surface. In addition, the minimum quantitative limit of detection on the photonic crystal surface is one-third the limit on the glass slide--a decrease from 18 to 6 pg/mL. The increased performance of the immunoassay allows for more accurate quantitation of physiologically relevant concentrations of TNF-alpha in a protein microarray format that can be expanded to multiple cytokines.

  18. The development of immunoassays for detection of chemical warfare agents

    Energy Technology Data Exchange (ETDEWEB)

    Lenz, D.E. [Army Medical Research Inst. of Chemical Defense, Aberdeen Proving Ground, MD (United States)

    1995-06-01

    With the advent of enzyme linked immunoabsorbant assays (ELISA) and monoclonal antibodies in the last two decades, there has been considerable effort devoted to the development of antibodies to detect and quantify low molecular weight toxic substances in environmental or biological fluids. Polyclonal antibodies against paraoxon (the toxic metabolite of parathion) were reported as capable of detecting paraoxon in body fluids at a level of 10{sup -9} M ({approximately}260 pg/mL) when used in a competitive inhibition enzyme immunoassay (CIEIA). Monoclonal antibodies developed against a structural analogue of the chemical warfare agent soman were capable of detecting soman in buffer solutions at a level of 10{sup -6} M ({approximately}180 ng/mL). In addition, these antibodies were highly specific for soman even in the presence of its major hydrolysis product. Subsequent studies with antisoman monoclonal antibodies reported an extension of the level of sensitivity to -80 ng/mL. Furthermore these antibodies did not cross react with other chemical warfare nerve agents such as sarin or tabun. In all cases, the time for a confirmatory test was two hours or less. Immunoassays for T-2 micotoxins have also been reported with a minimal detection range of 2 pg/assay to 50 ng/assay for the polyclonal and monoclonal T-2 antibodies respectively. These antibodies offer a sensitive, rapid and low cost approach to the diagnosis or detection of the presence of toxic chemical substances.

  19. Capsid structure and dynamics of a human rhinovirus probed by hydrogen exchange mass spectrometry.

    Science.gov (United States)

    Wang, Lintao; Smith, David L

    2005-06-01

    Viral capsids are dynamic protein assemblies surrounding viral genomes. Despite the high-resolution structures determined by X-ray crystallography and cryo-electron microscopy, their in-solution structure and dynamics can be probed by hydrogen exchange. We report here using hydrogen exchange combined with protein enzymatic fragmentation and mass spectrometry to determine the capsid structure and dynamics of a human rhinovirus, HRV14. Capsid proteins (VP1-4) were labeled with deuterium by incubating intact virus in D(2)O buffer at neutral pH. The labeled proteins were digested by immobilized pepsin to give peptides analyzed by capillary reverse-phase HPLC coupled with nano-electrospray mass spectrometry. Deuterium levels incorporated at amide linkages in peptic fragments were measured for different exchange times from 12 sec to 30 h to assess the amide hydrogen exchange rates along each of the four protein backbones. Exchange results generally agree with the crystal structure of VP1-4,with extended, flexible terminal and surface-loop regions in fast exchange and folded helical and sheet structures in slow exchange. In addition, three alpha-helices, one from each of VP1-3, exhibited very slow exchange, indicating high stability of the protomeric interface. The beta-strands at VP3 N terminus also had very slow exchange, suggesting stable pentamer contacts. It was noted, however, that the interface around the fivefold axis had fast and intermediate exchange, indicating relatively more flexibility. Even faster exchange rates were found in the N terminus of VP1 and most segments of VP4, suggesting high flexibilities, which may correspond to their potential roles in virus uncoating.

  20. Exacerbated Leishmaniasis Caused by a Viral Endosymbiont can be Prevented by Immunization with Its Viral Capsid

    Science.gov (United States)

    Castiglioni, Patrik; Hartley, Mary-Anne; Rossi, Matteo; Prevel, Florence; Desponds, Chantal; Utzschneider, Daniel T.; Eren, Remzi-Onur; Zangger, Haroun; Brunner, Livia; Collin, Nicolas; Zehn, Dietmar; Kuhlmann, F. Matthew; Beverley, Stephen M.; Ronet, Catherine

    2017-01-01

    Recent studies have shown that a cytoplasmic virus called Leishmaniavirus (LRV) is present in some Leishmania species and acts as a potent innate immunogen, aggravating lesional inflammation and development in mice. In humans, the presence of LRV in Leishmania guyanensis and in L. braziliensis was significantly correlated with poor treatment response and symptomatic relapse. So far, no clinical effort has used LRV for prophylactic purposes. In this context, we designed an original vaccine strategy that targeted LRV nested in Leishmania parasites to prevent virus-related complications. To this end, C57BL/6 mice were immunized with a recombinant LRV1 Leishmania guyanensis viral capsid polypeptide formulated with a T helper 1-polarizing adjuvant. LRV1-vaccinated mice had significant reduction in lesion size and parasite load when subsequently challenged with LRV1+ Leishmania guyanensis parasites. The protection conferred by this immunization could be reproduced in naïve mice via T-cell transfer from vaccinated mice but not by serum transfer. The induction of LRV1 specific T cells secreting IFN-γ was confirmed in vaccinated mice and provided strong evidence that LRV1-specific protection arose via a cell mediated immune response against the LRV1 capsid. Our studies suggest that immunization with LRV1 capsid could be of a preventive benefit in mitigating the elevated pathology associated with LRV1 bearing Leishmania infections and possibly avoiding symptomatic relapses after an initial treatment. This novel anti-endosymbiotic vaccine strategy could be exploited to control other infectious diseases, as similar viral infections are largely prevalent across pathogenic pathogens and could consequently open new vaccine opportunities. PMID:28099431

  1. Optimization of Substitution Matrix for Sequence Alignment of Major Capsid Proteins of Human Herpes Simplex Virus

    Directory of Open Access Journals (Sweden)

    Vipan Kumar Sohpal

    2011-12-01

    Full Text Available Protein sequence alignment has become an informative tool in modern molecular biology research. A number of substitution matrices have been readily available for sequence alignments, but it is challenging task to compute optimal matrices for alignment accuracy. Here, we used the parameter optimization procedure to select the optimal Q of substitution matrices for major viral capsid protein of human herpes simplex virus. Results predict that Blosum matrix is most accurate on alignment benchmarks, and Blosum 60 provides the optimal Q in all substitution matrices. PAM 200 matrices results slightly below than Blosum 60, while VTML matrices are intermediate of PAM and VT matrices under dynamic programming.

  2. Foot-and-mouth disease virus capsid proteins; analysis of protein processing, assembly and utility as vaccines

    DEFF Research Database (Denmark)

    Belsham, Graham

    precursor enhances the yield of processed capsid proteins and their assembly into empty capsid particles within mammalian cells. Such particles can potentially form the basis of a vaccine but they may only have the same properties as the current inactivated vaccines. We have expressed the FMDV P1-2A alone...... or with FMDV 3Cpro using a “single cycle” alphavirus vector based on Semliki Forest virus (SFV). Cattle vaccinated with these rSFV-FMDV vectors alone, produced anti-FMDV antibodies but the immune response was insufficient to give protection against FMDV challenge. However, vaccination with these vectors primed...... a much stronger immune response against FMDV post-challenge. In subsequent experiments, cattle were sequentially vaccinated with a rSFV-FMDV followed by recombinant FMDV empty capsid particles, or vice versa, prior to challenge. Animals given a primary vaccination with the rSFV-FMDV vector...

  3. Development and validation of a monoclonal based immunoassay for the measurement of fungal alpha-amylase: focus on peak exposures.

    Science.gov (United States)

    Elms, J; Denniss, S; Smith, M; Evans, P G; Wiley, K; Griffin, P; Curran, A D

    2001-03-01

    The inhalation of flour dust has been implicated in the induction of sensitisation and elicitation of respiratory symptoms, such as asthma in bakers. In addition to the cereal allergens present in wheat flour, enzymes in flour improvers, in particular fungal alpha-amylase, are now known to be a significant cause of respiratory allergy in the baking industry.A monoclonal antibody based enzyme-linked immunoassay (ELISA) was developed using two monoclonal antibodies that recognised two distinct epitopes of the fungal alpha-amylase enzyme. The ELISA had an inter-assay variation of 12.0% at 1360 pg/ml and 12.8% at 564 pg/ml and intra-assay variation of 4.9% at 1340 pg/ml and 6.1% at 504 pg/ml. The assay had a sensitivity of 200 pg/ml. Competitive inhibition assays confirmed that the monoclonal antibodies had no cross reactivity with other enzymes used in the baking industry and could distinguish added fungal alpha-amylase from cereal amylase. We assessed the levels of exposure to dust, total protein and fungal alpha-amylase in four UK bakeries ranging in size and technical capabilities. Within the bakeries we surveyed, workers were exposed to variable levels of inhalable dust (0.8-39.8 mg/m3), total protein (0-5.7 mg/m3) and fungal alpha-amylase (0-29.8 ng/m3). Consecutive 15 min personal samples taken over a 1 h period demonstrated that the ELISA could measure fungal alpha-amylase exposure in such a 15 min period. Short term peak exposures to fungal alpha-amylase could be identified which may contribute to the sensitisation in individuals who appear to have low exposure levels if measured over a full shift period.

  4. Epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp2 fragment of the replicase polyprotein contains a cluster of B-cell epitopes

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Bøtner, Anette; Toft, P.;

    2001-01-01

    We screened phage display libraries of porcine reproductive and respiratory syndrome virus (PRRSV) protein fragments with sera from experimentally infected pigs to identify linear B-cell epitopes that are commonly recognized during infection in vivo. We identified 10 linear epitope sites (ES) 11...

  5. Construction and immunogenicity prediction of Plasmodium falciparum CTL epitope minigene vaccine

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The minigenes encoding Plasmodium falciparum CTL epitopesrestricted to human MHC class I molecular HLA-A2 and HLA-B51, which were both at high frequency among Chinese population, were constructed as mono-epitope CTL vaccines named pcDNA3.1/tr and pcDNA3.1/ sh. The minigenes of the two epitopes were then tandem linked to form a dimeric CTL epitope minigene recombinant vaccine. After DNA transfection, the epitope minigenes were expressed respectively in two human cell lines, each bearing one MHC class I molecule named CIR/HLA-A2.1 and K562/HLA-B51. The intracellular expression of the CTL epitope minigenes not only enhanced the stability of HLA-A2.1 and HLA-B51 molecules but also increased the assemblage of MHC class I molecules on cell surfaces, which testified the specific process and presentation of those endogenous expressed epitopes. For the cells transfected with the dimeric minigene encoding two tandem linked epitopes, the expression and presentation of each epitope were also detected on cell membranes that bore different MHC class I molecules. It meant that the adjacency of the two CTL epitopes did not interfere with the specific process and presentation of each epitope. Compared with the ordinary CTL studies that inoculated synthesized epitope peptides with peripheral blood cells, this work aimed to process the epitopes directly inside HLA class I allele specific human cells, and thus theoretically imitated the same procedure in vivo. It was also an economical way to predict the immunogenicity of CTL epitopes at an early stage especially in laboratories with limited financial resource.

  6. Three Candidate Epitope-Vaccines in Combination Inducing High Levels of Multiantibodies Against HIV-1

    Institute of Scientific and Technical Information of China (English)

    刘祖强; 田海军; 王颖; 陈应华

    2003-01-01

    HIV-1 mutation results in immune evasion, which presents a serious challenge for conventional strategies for developing effective vaccines.So far, much experimental evidence indicates that HIV-1 particles in the blood of patients can be cleaned principally by neutralizing antibodies.Based on these facts, we prepared triple combination of epitope-vaccines with the objective of inducing antibodies with predefined multi-epitope-specificity against HIV-1.According to the sequences of three neutralizing epitopes (RILAVERYLKD, ELDKWA and GPGRAFY, designated E1, E2, and E3, respectively) on HIV-1 envelope proteins, three epitope-peptides ((E1)2: C-(RILAVERYLKDG)2; (E2)4: C-(ELDKWAG)4; and (E3)2: C-(GPGRAFY)2) were synthesized and then conjugated with carrier protein keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA), and used for immunizing rabbits.After the vaccine course, the triple combination of epitope-vaccines induced high levels of predefined multi-epitope-specific antibodies.An immunoblotting-analysis demonstrated that the antibodies could recognize the native epitopes on both gp41 protein and V3 loop peptide.Furthermore, we compared the immune responses of three doses of epitope-peptides in the candidate epitope-vaccine.Strong antibody responses to three epitopes were observed in a dose dependent manner, with increasing dose raising the immune response.This result indicated that immunotolerance did not occur using an epitope vaccine dose of 80 μg.Thus, our results demonstrate that epitope-vaccines in combination can synchronously induce high levels of antibodies with predefined multi-epitope-specificity against HIV-1, and may be used to develop effective vaccines against HIV as a new strategy.

  7. Anti-epitope antibody,a novel site-directed antibody against human acetylcholinesterase

    Institute of Scientific and Technical Information of China (English)

    Xing-mei ZHANG; Gang LIU; Man-ji SUN

    2004-01-01

    AIM: To construct synthetic antigens using the epitope of human brain acetylcholinesterase (hbAChE) for induction and detection of the specific antibody against the epitope, and to analyse the immunogenicity of the antibody.METHODS: The epitope (RTVLVSMNYR, amino acids 143-152) of hbAChE was chemically synthesized, coupled with the carrier protein keyhole limpet hemocyanin (KLH) to construct an artificial immunogen (KLH-epitope), and injected into rabbits to raise antibody. The epitope conjugated with bovine serum albumin (BSA) was used as the detection antigen. The specificity of the antibody was tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The immunoreaction between the anti-recombinant human butyrylcholinesterase (rhBChE)polyclonal antibody and the biotinylated-epitope was examined by indirect ELISA. RESULTS: The erythrocyte AChE, the hbAChE, rhBChE and the BSA-epitope all immunoreacted with the anti-epitope antibody against the epitope (143-152) of hbAChE, whereas the torpedo AChE did not. CONCLUSION: The hbAChE, the human erythrocyte AChE and hBChE share the conservative antigenic epitope RTVLVSMNYR, hence they can all immunoreact with the anti-epitope antibody. Since the epitope of hbAChE is less similar with the aligned amino acid sequences of AChE of Torpedo californica or Torpedo marmorata, there is not any immunoreactivity between them. The R, M, and N residues in the epitope seem to be necessary radicals for the conservation of antigenicity.

  8. Sizing up large protein complexes by electrospray ionisation-based electrophoretic mobility and native mass spectrometry : morphology selective binding of Fabs to hepatitis B virus capsids

    NARCIS (Netherlands)

    Bereszczak, Jessica Z; Havlik, Marlene; Weiss, Victor U; Marchetti-Deschmann, Martina; van Duijn, Esther; Watts, Norman R; Wingfield, Paul T; Allmaier, Guenter; Steven, Alasdair C; Heck, Albert J R

    2014-01-01

    The capsid of hepatitis B virus (HBV) is a major viral antigen and important diagnostic indicator. HBV capsids have prominent protrusions ('spikes') on their surface and are unique in having either T = 3 or T = 4 icosahedral symmetry. Mouse monoclonal and also human polyclonal antibodies bind either

  9. Preparation and epitope characterization of monoclonal antibodies against firefly luciferase

    Institute of Scientific and Technical Information of China (English)

    徐沁; 丁建芳; 胡红雨; 许根俊

    1999-01-01

    The 6-His tagged firefly luciferase was highly expressed in E. coli and purified to homogeneity by affinity chromatography and gel filtration. After immunizing Balb/c mice with the antigen, 6 hybridomas clones were found to secrete monoelonal antibodies (mAbs) and the mAbs were also purified separately. The competitive binding experiments show that 2 mAbs can bind heat-denatured antigen or its proteolytic fragments but not the native lueiferase, suggesting that their epitopes might be accommodated in the internal segments of the protein. On the other hand, the other 4 mAbs are capable of binding both native and denatured antigens. It infers that their epitopes locate in the segments on the protein surface. The results also suggest that the six mAbs are all sequence-specific.

  10. Identification of an epitope of SARS-coronavirus nucleocapsid protein

    Institute of Scientific and Technical Information of China (English)

    YING LIN; JIN WANG; HONG XIA WANG; HUA LIANG JIANG; JIAN HUA SHEN; YOU HUA XIE; YUAN WANG; GANG PEI; BEI FEN SHEN; JIA RUI WU; BING SUN; XU SHEN; RUI FU YANG; YI XUE LI; YONG YONG JI; YOU YU HE; MUDE SHI; WEI LU; TIE LIU SHI

    2003-01-01

    The nucleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a majorvirion structural protein. In this study, two epitopes (N1 and N2) of the N protein of SARS-CoV werepredicted by bioinformatics analysis. After immunization with two peptides, the peptides-specific antibodieswere isolated from the immunized rabbits. The further experiments demonstrated that N1 peptide-inducedpolyclonal antibodies had a high affinity to bind to E. coli expressed N protein of SARS-CoV. Furthermore, itwas confirmed that N1 peptide-specific IgG antibodies were detectable in the sera of severe acute respiratorysyndrome (SARS) patients. The results indicated that an epitope of the N protein has been identified andN protein specific Abs were produced by peptide immunization, which will be useful for the study of SARS-CoV.

  11. 'Multi-epitope-targeted' immune-specific therapy for a multiple sclerosis-like disease via engineered multi-epitope protein is superior to peptides.

    Directory of Open Access Journals (Sweden)

    Nathali Kaushansky

    Full Text Available Antigen-induced peripheral tolerance is potentially one of the most efficient and specific therapeutic approaches for autoimmune diseases. Although highly effective in animal models, antigen-based strategies have not yet been translated into practicable human therapy, and several clinical trials using a single antigen or peptidic-epitope in multiple sclerosis (MS yielded disappointing results. In these clinical trials, however, the apparent complexity and dynamics of the pathogenic autoimmunity associated with MS, which result from the multiplicity of potential target antigens and "epitope spread", have not been sufficiently considered. Thus, targeting pathogenic T-cells reactive against a single antigen/epitope is unlikely to be sufficient; to be effective, immunospecific therapy to MS should logically neutralize concomitantly T-cells reactive against as many major target antigens/epitopes as possible. We investigated such "multi-epitope-targeting" approach in murine experimental autoimmune encephalomyelitis (EAE associated with a single ("classical" or multiple ("complex" anti-myelin autoreactivities, using cocktail of different encephalitogenic peptides vis-a-vis artificial multi-epitope-protein (designated Y-MSPc encompassing rationally selected MS-relevant epitopes of five major myelin antigens, as "multi-epitope-targeting" agents. Y-MSPc was superior to peptide(s in concomitantly downregulating pathogenic T-cells reactive against multiple myelin antigens/epitopes, via inducing more effective, longer lasting peripheral regulatory mechanisms (cytokine shift, anergy, and Foxp3+ CTLA4+ regulatory T-cells. Y-MSPc was also consistently more effective than the disease-inducing single peptide or peptide cocktail, not only in suppressing the development of "classical" or "complex EAE" or ameliorating ongoing disease, but most importantly, in reversing chronic EAE. Overall, our data emphasize that a "multi-epitope-targeting" strategy is required for

  12. A General Method to Discover Epitopes from Sera.

    Directory of Open Access Journals (Sweden)

    Kurt Whittemore

    Full Text Available Antigen-antibody complexes are central players in an effective immune response. However, finding those interactions relevant to a particular disease state can be arduous. Nonetheless many paths to discovery have been explored since deciphering these interactions can greatly facilitate the development of new diagnostics, therapeutics, and vaccines. In silico B cell epitope mapping approaches have been widely pursued, though success has not been consistent. Antibody mixtures in immune sera have been used as handles for biologically relevant antigens, but these and other experimental approaches have proven resource intensive and time consuming. In addition, these methods are often tailored to individual diseases or a specific proteome, rather than providing a universal platform. Most of these methods are not able to identify the specific antibody's epitopes from unknown antigens, such as un-annotated neo antigens in cancer. Alternatively, a peptide library comprised of sequences unrestricted by naturally-found protein space provides for a universal search for mimotopes of an antibody's epitope. Here we present the utility of such a non-natural random sequence library of 10,000 peptides physically addressed on a microarray for mimotope discovery without sequence information of the specific antigen. The peptide arrays were probed with serum from an antigen-immunized rabbit, or alternatively probed with serum pre-absorbed with the same immunizing antigen. With this positive and negative screening scheme, we identified the library-peptides as the mimotopes of the antigen. The unique library peptides were successfully used to isolate antigen-specific antibodies from complete immune serum. Sequence analysis of these peptides revealed the epitopes in the immunized antigen. We present this method as an inexpensive, efficient method for identifying mimotopes of any antibody's targets. These mimotopes should be useful in defining both components of the

  13. Elicitation of structure-specific antibodies by epitope scaffolds

    OpenAIRE

    2010-01-01

    Elicitation of antibodies against targets that are immunorecessive, cryptic, or transient in their native context has been a challenge for vaccine design. Here we demonstrate the elicitation of structure-specific antibodies against the HIV-1 gp41 epitope of the broadly neutralizing antibody 2F5. This conformationally flexible region of gp41 assumes mostly helical conformations but adopts a kinked, extended structure when bound by antibody 2F5. Computational techniques were employed to transpl...

  14. Combinatorial Contextualization of Peptidic Epitopes for Enhanced Cellular Immunity

    Science.gov (United States)

    Ito, Masaki; Hayashi, Kazumi; Adachi, Eru; Minamisawa, Tamiko; Homma, Sadamu; Koido, Shigeo; Shiba, Kiyotaka

    2014-01-01

    Invocation of cellular immunity by epitopic peptides remains largely dependent on empirically developed protocols, such as interfusion of aluminum salts or emulsification using terpenoids and surfactants. To explore novel vaccine formulation, epitopic peptide motifs were co-programmed with structural motifs to produce artificial antigens using our “motif-programming” approach. As a proof of concept, we used an ovalbumin (OVA) system and prepared an artificial protein library by combinatorially polymerizing MHC class I and II sequences from OVA along with a sequence that tends to form secondary structures. The purified endotoxin-free proteins were then examined for their ability to activate OVA-specific T-cell hybridoma cells after being processed within dendritic cells. One clone, F37A (containing three MHC I and two MHC II OVA epitopes), possessed a greater ability to evoke cellular immunity than the native OVA or the other artificial antigens. The sensitivity profiles of drugs that interfered with the F37A uptake differed from those of the other artificial proteins and OVA, suggesting that alteration of the cross-presentation pathway is responsible for the enhanced immunogenicity. Moreover, F37A, but not an epitopic peptide, invoked cellular immunity when injected together with monophosphoryl lipid A (MPL), and retarded tumor growth in mice. Thus, an artificially synthesized protein antigen induced cellular immunity in vivo in the absence of incomplete Freund's adjuvant or aluminum salts. The method described here could be potentially used for developing vaccines for such intractable ailments as AIDS, malaria and cancer, ailments in which cellular immunity likely play a crucial role in prevention and treatment. PMID:25343355

  15. Common antiviral cytotoxic t-lymphocyte epitope for diverse arenaviruses.

    Science.gov (United States)

    Oldstone, M B; Lewicki, H; Homann, D; Nguyen, C; Julien, S; Gairin, J E

    2001-07-01

    Members of the Arenaviridae family have been isolated from mammalian hosts in disparate geographic locations, leading to their grouping as Old World types (i.e., lymphocytic choriomeningitis virus [LCMV], Lassa fever virus [LFV], Mopeia virus, and Mobala virus) and New World types (i.e., Junin, Machupo, Tacaribe, and Sabia viruses) (C. J. Peters, M. J. Buchmeier, P. E. Rollin, and T. G. Ksiazek, p. 1521-1551, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996; P. J. Southern, p. 1505-1519, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996). Several types in both groups-LFV, Junin, Machupo, and Sabia viruses-cause severe and often lethal human diseases. By sequence comparison, we noted that eight Old World and New World arenaviruses share several amino acids with the nucleoprotein (NP) that consists of amino acids (aa) 118 to 126 (NP 118-126) (RPQASGVYM) of LCMV that comprise the immunodominant cytotoxic T-lymphocyte (CTL) epitope for H-2(d) mice (32). This L(d)-restricted epitope constituted >97% of the total bulk CTLs produced in the specific antiviral or clonal responses of H-2(d) BALB mice. NP 118-126 of the Old World arenaviruses LFV, Mopeia virus, and LCMV and the New World arenavirus Sabia virus bound at high affinity to L(d). The primary H-2(d) CTL anti-LCMV response as well as that of a CTL clone responsive to LCMV NP 118-126 recognized target cells coated with NP 118-126 peptides derived from LCMV, LFV, and Mopeia virus but not Sabia virus, indicating that a common functional NP epitope exists among Old World arenaviruses. Use of site-specific amino acid exchanges in the NP CTL epitope among these arenaviruses identified amino acids involved in major histocompatibility complex binding and CTL recognition.

  16. Structural Basis for the Development of Avian Virus Capsids That Display Influenza Virus Proteins and Induce Protective Immunity

    OpenAIRE

    Pascual, Elena; Mata, Carlos P.; Gómez-Blanco, Josué; Moreno, Noelia; Bárcena, Juan; Blanco, Esther; Rodríguez-Frandsen, Ariel; Nieto, Amelia; Carrascosa, José L.; Castón, José R.

    2014-01-01

    Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ∼70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an am...

  17. Analogs of LDL Receptor Ligand Motifs in Dengue Envelope and Capsid Proteins as Potential Codes for Cell Entry

    OpenAIRE

    Juan Guevara; Jaime Romo; Troy McWhorter; Natalia Valentinova Guevara

    2015-01-01

    It is established that cell entry of low density lipoprotein particles (LLPs) containing Apo B100 and Apo E is mediated by receptors and GAGs. Receptor ligand motifs, XBBBXXBX, XBBXBX, and ΨBΨXB, and mono- and bipartite NLS sequences are abundant in Apo E and Apo B100 as well as in envelope and capsid proteins of Dengue viruses 1–4 (DENV1–4). Synthetic, fluorescence-labeled peptides of sequences in DENV2 envelope protein, and DENV3 capsid that include these motifs were used to conduct a quali...

  18. Optimal selection of epitopes for TXP-immunoaffinity mass spectrometry

    Directory of Open Access Journals (Sweden)

    Joos Thomas

    2010-06-01

    Full Text Available Abstract Background Mass spectrometry (MS based protein profiling has become one of the key technologies in biomedical research and biomarker discovery. One bottleneck in MS-based protein analysis is sample preparation and an efficient fractionation step to reduce the complexity of the biological samples, which are too complex to be analyzed directly with MS. Sample preparation strategies that reduce the complexity of tryptic digests by using immunoaffinity based methods have shown to lead to a substantial increase in throughput and sensitivity in the proteomic mass spectrometry approach. The limitation of using such immunoaffinity-based approaches is the availability of the appropriate peptide specific capture antibodies. Recent developments in these approaches, where subsets of peptides with short identical terminal sequences can be enriched using antibodies directed against short terminal epitopes, promise a significant gain in efficiency. Results We show that the minimal set of terminal epitopes for the coverage of a target protein list can be found by the formulation as a set cover problem, preceded by a filtering pipeline for the exclusion of peptides and target epitopes with undesirable properties. Conclusions For small datasets (a few hundred proteins it is possible to solve the problem to optimality with moderate computational effort using commercial or free solvers. Larger datasets, like full proteomes require the use of heuristics.

  19. Common food allergens and their IgE-binding epitopes

    Directory of Open Access Journals (Sweden)

    Hiroaki Matsuo

    2015-10-01

    Full Text Available Food allergy is an adverse immune response to certain kinds of food. Although any food can cause allergic reactions, chicken egg, cow's milk, wheat, shellfish, fruit, and buckwheat account for 75% of food allergies in Japan. Allergen-specific immunoglobulin E (IgE antibodies play a pivotal role in the development of food allergy. Recent advances in molecular biological techniques have enabled the efficient analysis of food allergens. As a result, many food allergens have been identified, and their molecular structure and IgE-binding epitopes have also been identified. Studies of allergens have demonstrated that IgE antibodies specific to allergen components and/or the peptide epitopes are good indicators for the identification of patients with food allergy, prediction of clinical severity and development of tolerance. In this review, we summarize our current knowledge regarding the allergens and IgE epitopes in the well-researched allergies to chicken egg, cow's milk, wheat, shrimp, and peanut.

  20. 赭曲霉毒素A模拟表位的筛选及免疫学鉴定%SCREENING AND IMMUNOLOGICAL CHARACTERIZATION OF THE MIMICKING EPITOPE OF OCHRATOXIN A

    Institute of Scientific and Technical Information of China (English)

    王耀; 胡骁飞; 裴亚峰; 赵东; 刘运超; 张改平; 李志西; 邓瑞广

    2012-01-01

    To screen the mimicking epitope of Ochratoxin A from Ph. D.-7TM and establish the immunoassay for detecting OTA with it. An anti-Ochratoxin A monoclonal antibody was used as ligand, biopanning was done to screen the mimicking epitope from a Ph.D. -7TM, which was displayed as a fusion protein with the coat protein 11I of filamentous phage M13, the positive clones was identified by ELISA, and established a immunoassay for detecting OTA with the positive phages. After four rounds of panning, 22 positive clones could bind to the OTA mAb and the binding could be blocked by the free ochratoxin A. With the DNA sequencing and peptide core sequence analysis, the amino sequence of the mimicking epitope is MPLWXDL(X is any amino acid residues). A competitive ELISA immunoassay was established with the positive clones, the linear range of the inhibition curves is between 250pg/ml and 8000pg/ml. Based on the OTA mAb, the phage display technique can be successfully applied to screen the mimicking epitope of Ochratoxin A, the obtain phage can replaced Ochratoxin A to establish the immunoassay.%从噬菌体随机七肽库中筛选得到赭曲霉毒素A的模拟表位,并以其替代毒素标品建立赭曲霉毒素A免疫学快速检测方法。以纯化的抗赭曲霉毒素A单克隆抗体(OTAmAb)为配基,亲和筛选融合表达在丝状M13噬菌体次要衣壳蛋白(PⅢ)上的随机七肽库,以ELISA方法鉴定阳性克隆,并以筛选的模拟表位建立赭曲霉毒素A的ELISA检测方法。经过4轮亲和筛选,共得到22株与OTAmAb特异结合的阳性噬菌体克隆,且该结合都能够被OTA标品阻断,DNA测序及多肽核心序列分析后,得到赭曲霉毒素A的模拟表位的主要序列为:MPLWXDL,X为任意氨基酸,以阳性噬菌体克隆建立的竞争ELISA检测方法,线性范围为250~8000pg/ml。基于制备的OTAmAb,利用噬菌体随机七肽库可成功筛选到赭曲霉毒素A的模拟表位,并能够替