Sample records for human antibody immune

  1. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library. (United States)

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei


    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  2. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library (United States)

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei


    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  3. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

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    Han Wang


    Full Text Available Tetanus neurotoxin (TeNT produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  4. Generation of mouse anti-human urate anion exchanger antibody by genetic immunization and its identification

    Institute of Scientific and Technical Information of China (English)

    XU Guo-shuang; WU Di; CHEN Xiang-mei; SHI Suo-zhu; HONG Quan; ZHANG Ping; LU Yang


    Background Human urate anion exchanger (hURAT1) as a major urate transporter expressed on renal tubular epithelial cells regulates blood urate level by reabsorbing uric acid. Antibody is an important tool to study hURAT1. This study aimed, by genetic immunization, to produce mouse anti-hURAT1 polyclonal antibody with high throughput and high specificity and to detect the location of hURAT1 in human kidney.Methods Human renal total RNA was isolated and the entire cDNA of hURAT1 was amplified by RT-PCR. The sequence of intracellular high antigenicity fragment (A280 to R349) was chosen by prediction software of protein antigenicity, and its cDNA was amplified from cDNA of hURAT1, and then cloned into pBQAP-TT vector to construct recombinant plasmid pBQAP-TT-hURAT1-210 for genetic immunization. Mice were inoculated with this recombinant plasmid and two other adjuvant plasmids, pCMVi-GMCSF and pCMVi-Flt3L, which helped to enhance the antibody’s generation. After four weeks, the mice were sacrificed to obtain the anti-hURAT1 antibody from serum. The antibody was identified by western blot analysis and immunohistochemistry. At the same time, rabbit anti-hURAT1 antibody was produced by protein immunization. The specificity and efficiency between the rabbit and mouse anti-hURAT1 antibody were compared by western blot analysis and immunohistochemistry.Results The entire cDNA of hURAT1 and cDNA of its intracellular high immunogenic fragment were amplified successfully. Recombinant plasmid pBQAP-TT-hURAT1-210 for genetic immunization was confirmed by restriction digestion and sequencing. Both the mouse anti-hURAT1 antibody and rabbit anti-hURAT1 antibody recognized 58kD hURAT1 and 64kD glycosylated hURAT1 protein bands in western blot. Immunohistochemically, hURAT1 was located at the brush border membrane of renal proximal tubular cells. In addition, the throughput and specificity of the mouse anti-hURAT1 antibody were higher than those of the rabbit anti-hURAT1 antibody

  5. Human immune system mice: current potential and limitations for translational research on human antibody responses. (United States)

    Vuyyuru, Raja; Patton, John; Manser, Tim


    It has recently become possible to generate chimeric mice durably engrafted with many components of the human immune system (HIS mice). We have characterized the maturation and function of the B cell compartment of HIS mice. The antibody response of HIS mice to T cell-dependent B cell antigens is limited, and contributing factors may be the general immaturity of the B cell compartment, infrequent helper T cells selected on human MHC class II antigens, and incomplete reconstitution of secondary lymphoid organs and their microenvironments. In contrast, HIS mice generate protective antibody responses to the bacterium Borrelia hermsii, which acts as a T cell-independent antigen in mice, but do not respond to purified polysaccharide antigens (PPS). We speculate that the anti-B. hermsii response of HIS mice is derived from an abundant B cell subset that may be analogous to B1 B cells in mice. We suggest that failure of HIS mice to respond to PPS is due to the lack of a B cell subset that may originate from adult bone marrow and is highly dependent on human interleukin-7 for development.

  6. Harnessing the immune system's arsenal: producing human monoclonal antibodies for therapeutics and investigating immune responses (United States)

    Sullivan, Meghan; Kaur, Kaval; Pauli, Noel


    Monoclonal antibody technology has undergone rapid and innovative reinvention over the last 30 years. Application of these technologies to human samples revealed valuable therapeutic and experimental insights. These technologies, each with their own benefits and flaws, have proven indispensable for immunological research and in our fight to provide new treatments and improved vaccines for infectious disease. PMID:21876728

  7. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

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    Tal Noy-Porat


    Full Text Available Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1 that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  8. Natural and Immune Human Antibodies Reactive with Antigens of Virulent Neisseria gonorrhoeae: Immunoglobulins G, M, and A (United States)

    Cohen, Irun R.


    Natural and immune human antibodies reactive with heat-labile and heat-stable antigens of virulent Neisseria gonorrhoeae were studied by use of an indirect fluorescent-antibody (IFA) procedure. The immunoglobulin class of the reactive antibodies was identified by using fluorescein-conjugated antisera specific for human IgG, IgA, or IgM in the IFA procedure. The effects of heat and mercaptoethanol on IFA reactivities were also studied. It appeared that antibodies of the IgG, IgM, and IgA classes present in the sera of both infected persons (immune antibodies) and normal persons with no history of gonococcal infection (natural antibodies) react with heat-stable somatic antigens. Immune IgG antibodies, however, were distinguishable from natural IgG antibodies by their ability to recognize heat-labile surface antigens. The distinction between natural and immune IgM antibodies was less obvious. IgM antibodies from both infected and normal persons appeared to react with heat-labile antigens. Some, but not all, infected persons had immune IgA antibodies to heat-labile as well as to heat-stable antigens. Treatment of sera with mercaptoethanol had no effect on IgG antibodies. The IFA activity of IgM antibodies was decreased, but not abolished. The effects of mercaptoethanol on IgA antibodies were variable. Some sera showed a decrease in IgA titer, and others showed an increase in IgA activity to certain antigens. Immune IgG antibodies were more resistant to heating than were natural IgG antibodies. Natural and immune IgM antibodies appeared equally sensitive to heating. IgA activity, on the other hand, was increased by heating sera at 60 C, but was decreased at higher temperatures. Thus, it appears that natural and immune human IgG antibodies to N. gonorrhoeae may be distinguished by their interactions with heat-labile antigens and by their resistance to heating. Images PMID:4961630

  9. Generation of human antigen-specific monoclonal IgM antibodies using vaccinated "human immune system" mice.

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    Pablo D Becker

    Full Text Available BACKGROUND: Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the 'humanization' of murine monoclonal antibodies (mAbs is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique. METHODOLOGY/PRINCIPAL FINDINGS: After transplantation with CD34+CD38⁻ human hematopoietic progenitor cells, BALB/c Rag2⁻/⁻IL-2Rγc⁻/⁻ mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. "Human Immune System" mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19+CD27+ B cells were retrovirally transduced with the human B cell lymphoma (BCL-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively. CONCLUSION/SIGNIFICANCE: This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens.

  10. Passive immunization against dental caries and periodontal disease: development of recombinant and human monoclonal antibodies. (United States)

    Abiko, Y


    Indigenous micro-organisms in the oral cavity can cause two major diseases, dental caries and periodontal diseases. There is neither agreement nor consensus as to the actual mechanisms of pathogenesis of the specific virulence factors of these micro-organisms. The complexity of the bacterial community in dental plaque has made it difficult for the single bacterial agent of dental caries to be determined. However, there is considerable evidence that Streptococcus mutans is implicated as the primary causative organism of dental caries, and the cell-surface protein antigen (SA I/II) as well as glucosyltransferases (GTFs) produced by S. mutans appear to be major colonization factors. Various forms of periodontal diseases are closely associated with specific subgingival bacteria. Porphyromonas gingivalis has been implicated as an important etiological agent of adult periodontitis. Adherence of bacteria to host tissues is a prerequisite for colonization and one of the important steps in the disease process. Bacterial coaggregation factors and hemagglutinins likely play major roles in colonization in the subgingival area. Emerging evidence suggests that inhibition of these virulence factors may protect the host against caries and periodontal disease. Active and passive immunization approaches have been developed for immunotherapy of these diseases. Recent advances in mucosal immunology and the introduction of novel strategies for inducing mucosal immune responses now raise the possibility that effective and safe vaccines can be constructed. In this regard, some successful results have been reported in animal experimental models. Nevertheless, since the public at large might be skeptical about the seriousness of oral diseases, immunotherapy must be carried out with absolute safety. For this goal to be achieved, the development of safe antibodies for passive immunization is significant and important. In this review, salient advances in passive immunization against caries

  11. Dengue viruses are enhanced by distinct populations of serotype cross-reactive antibodies in human immune sera.

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    Ruklanthi de Alwis


    Full Text Available Dengue viruses (DENV are mosquito-borne flaviviruses of global importance. DENV exist as four serotypes, DENV1-DENV4. Following a primary infection, individuals produce DENV-specific antibodies that bind only to the serotype of infection and other antibodies that cross-react with two or more serotypes. People exposed to a secondary DENV infection with another serotype are at greater risk of developing more severe forms of dengue disease. The increased risk of severe dengue in people experiencing repeat DENV infections appear to be due, at least in part, to the ability of pre-existing serotype cross-reactive antibodies to form virus-antibody complexes that can productively infect Fcγ receptor-bearing target cells. While the theory of antibody-dependent enhancement (ADE is supported by several human and small animal model studies, the specific viral antigens and epitopes recognized by enhancing human antibodies after natural infections have not been fully defined. We used antibody-depletion techniques to remove DENV-specific antibody sub-populations from primary DENV-immune human sera. The effects of removing specific antibody populations on ADE were tested both in vitro using K562 cells and in vivo using the AG129 mouse model. Removal of serotype cross-reactive antibodies ablated enhancement of heterotypic virus infection in vitro and antibody-enhanced mortality in vivo. Further depletion studies using recombinant viral antigens showed that although the removal of DENV E-specific antibodies using recombinant E (rE protein resulted in a partial reduction in DENV enhancement, there was a significant residual enhancement remaining. Competition ADE studies using prM-specific Fab fragments in human immune sera showed that both rE-specific and prM-specific antibodies in primary DENV-immune sera significantly contribute to enhancement of heterotypic DENV infection in vitro. Identification of the targets of DENV-enhancing antibodies should contribute to

  12. Monoclonal antibodies in human organ transplantation and auto-immune diseases. (United States)

    Wijdenes, J; Roy, C; Morel-Fourrier, B; Racadot, E


    The usefulness of monoclonal antibodies (mAbs) in the transplantation field has become evident over the last couple of years. Different mAbs have been used as a prophylactic treatment after transplantation, in a therapeutic way against acute organ rejection and new diagnostic tools to predict clinical rejection immerge. One can even hope that with humanised mAbs or human mAbs obtained by repertoire cloning the formation of human anti-mouse antibodies will be solved although on the one hand this appeared not to be a big problem and on the other hand anti-idiotypic antibodies can still be expected. However, the most puzzling question is how the mAbs modulates the immuno-response and this not only in organ rejection but also in auto-immune diseases. Only one out of many CD25 mAbs with seemingly similar epitope recognition can be used in therapeutical treatment of acute Graft versus Host Disease. The same mAb is not, however, very efficient in the prophylactic treatment of kidney transplantation without association of suboptimal doses of cyclosporin A. Another example is a CD4 mAb which is efficient in the treatment of polyarthritis with no side effects but which provokes transient but clear side effects when used in psoriasis or multiple sclerosis patients. A second CD4 mAb with high inhibitory activity in several bioassays compared to the first CD4 mAb has no beneficial effect at all on polyarthritis. Also the question why there is a percentage of "no response" patients among apparently identical "good response" patients remains unanswered. However it becomes clear from these experiences that: 1) mAbs recognizing a similar epitope and being of the same isotype will not automatically have the same effect in therapy. 2) side effects may be depending of the disease treated. 3) the activities of mAbs in bioassays and even animal models very often do not reflect the in vivo situation in human. 4) efficiency of the treatment with mAbs can be increased by a better

  13. Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

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    Britton, W.J.; Hellqvist, L.; Basten, A.; Raison, R.L.


    Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae.

  14. Toll-like receptor activation enhances cell-mediated immunity induced by an antibody vaccine targeting human dendritic cells

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    Berger Marc A


    Full Text Available Abstract Previously, we have successfully targeted the mannose receptor (MR expressed on monocyte-derived dendritic cells (DCs using a fully human MR-specific antibody, B11, as a vehicle to deliver whole protein tumor antigens such as the human chorionic gonadotropin hormone (hCGβ. Since MRs play a role in bridging innate immunity with adaptive immunity we have explored several toll-like receptor (TLR-specific ligands that may synergize with MR targeting and be applicable as adjuvants in the clinic. We demonstrate that antigen-specific helper and cytolytic T cells from both healthy donors and cancer patients were effectively primed with B11-hCGβ-treated autologous DCs when a combination of one or several TLR ligands is used. Specifically, concomitant signaling of DCs via TLR3 with dsRNA (poly I:C and DC TLR 7/8 with Resiquimod (R-848, respectively, elicited efficient antigen presentation-mediated by MR-targeting. We demonstrate that MR and TLRs contribute towards maturation and activation of DCs by a mechanism that may be driven by a combination of adjuvant and antibody vaccines that specifically deliver antigenic targets to DCs.

  15. Electroporation-aided DNA immunization generates polyclonal antibodies against the native conformation of human endothelin B receptor. (United States)

    Allard, Bertrand; Priam, Fabienne; Deshayes, Frédérique; Ducancel, Frédéric; Boquet, Didier; Wijkhuisen, Anne; Couraud, Jean-Yves


    Endothelin B receptor (ET(B)R) is a G protein-coupled receptor (GPCR) specific for endothelin peptides (including endothelin-1, ET1), which mediates a variety of key physiological functions in normal tissues, such as modulation of vasomotor tone, tissue differentiation, or cell proliferation. Moreover, ET(B)R, overexpressed in various cancer cells including melanoma, has been implicated in the growth and progression of tumors, as well as in controlling T cell homing to tumors. To gather information on receptor structure and function, antibodies are generally considered choice molecular probes, but generation of such reagents against the native conformation of GPCRs is a real technical challenge. Here, we show that electroporation-aided genetic immunization, coupled to cardiotoxin pretreatment, is a simple and very efficient method to raise large amounts of polyclonal antibodies highly specific for native human ET(B)R (hET(B)R), as assessed by both flow cytometry analysis of different stably transfected cell lines and a new and rapid cell-based enzyme-linked immunosorbent assay that we also describe. The antibodies recognized two major epitopes on hET(B)R, mapped within the N-terminal extracellular domain. They were used to reveal hET(B)R on membranes of three different human melanoma cell lines, by flow cytometry and confocal microscopy, a method that we show is more relevant than mRNA polymerase chain reaction in assessing receptor expression. In addition, ET-1 partially competed with antibodies for receptor binding. The strategy described here, thus, efficiently generated new immunological tools to further analyze the role of ET(B)R under both normal and pathological conditions, including cancers. Above all, it can now be used to raise monoclonal antibodies against hET(B)R and, more generally, against GPCRs that constitute, by far, the largest reservoir of potential pharmacological targets.

  16. How does the recombinant human interferon beta induce antibodies in immune tolerant mice?

    NARCIS (Netherlands)

    Kijanka, G.M.


    Therapeutic proteins revolutionized the treatment of severe diseases like multiple sclerosis, diabetes, haemophilia and many more. Unfortunately, their usage is often limited due to the formation of anti drug antibodies (ADAs), which may block the activity of these protein drugs and may lead to

  17. Reliability of the nanopheres-DNA immunization technology to produce polyclonal antibodies directed against human neogenic proteins. (United States)

    Arnaoty, Ahmed; Gouilleux-Gruart, Valérie; Casteret, Sophie; Pitard, Bruno; Bigot, Yves; Lecomte, Thierry


    The molecular domestication of several DNA transposons that occurred during the evolution of the mammalian lineage, has led to the emergence of at least 43 genes, known as neogenes. To date, the limited availability of efficient commercial antibodies directed against most of their protein isoforms hampers investigation of their expression in vitro and in situ. Since immunization protocols using peptides or recombinant proteins have revealed that it is difficult to recover antibodies, we planned to produce antisera in mice using a new technique of nanopheres/DNA immunization, the ICANtibodies™ technology. Here, we investigate the possibilities of obtaining polyclonal antibodies for 24 proteins or protein domains using this immunization strategy. We successfully obtained 13 antisera that were able to detect neogenic proteins by Western blotting and ELISA in protein extracts of transiently-transfected cells and various cancer cell lines, plus another two that only detected the in ELISA and in in situ hybridizations. The features required for the production of these antibodies are analyzed and discussed, and examples are given of the advantages they offer for the study of neogenic proteins.

  18. Lipoxin A4 decreases human memory B cell antibody production via an ALX/FPR2-dependent mechanism: A link between resolution signals and adaptive immunity (United States)

    Ramon, Sesquile; Bancos, Simona; Serhan, Charles N.; Phipps, Richard P.


    Summary Specialized proresolving mediators (SPMs) are endogenous bioactive lipid molecules that play a fundamental role in the regulation of inflammation and its resolution. SPMs are classified into lipoxins, resolvins, protectins and maresins. Lipoxins and other SPMs have been identified in important immunological tissues including bone marrow, spleen and blood. Lipoxins regulate functions of the innate immune system including the promotion of monocyte recruitment and increase macrophage phagocytosis of apoptotic neutrophils. A major knowledge gap is whether lipoxins influence adaptive immune cells. Here, we analyzed the actions of lipoxin A4 (LXA4) and its receptor ALX/FPR2 on human B cells. LXA4 decreased IgM and IgG production on activated B cells through ALX/FPR2-dependent signaling, which downregulated NF-κB p65 nuclear translocation. LXA4 also inhibited human memory B cell antibody production and proliferation, but not naïve B cell function. Lastly, LXA4 decreased antigen-specific antibody production in vivo. To our knowledge, this is the first description of the actions of lipoxins on human B cells, which shows a link between resolution signals and adaptive immunity. Regulating antibody production is crucial to prevent unwanted inflammation. Harnessing the ability of lipoxins to decrease memory B cell antibody production can be beneficial to threat inflammatory and autoimmune disorders. PMID:24166736

  19. Human recombinant antibodies against Plasmodium falciparum merozoite surface protein 3 cloned from peripheral blood leukocytes of individuals with immunity to malaria demonstrate antiparasitic properties

    DEFF Research Database (Denmark)

    Lundquist, Rasmus; Nielsen, Leif Kofoed; Jafarshad, Ali


    Immunoglobulins from individuals with immunity to malaria have a strong antiparasitic effect when transferred to Plasmodium falciparum malaria infected patients. One prominent target of antiparasitic antibodies is the merozoite surface antigen 3 (MSP-3). We have investigated the antibody response...

  20. A monoclonal antibody against the human SUMO-1 protein obtained by immunization with recombinant protein and CpG-DNA-liposome complex. (United States)

    Kim, Dongbum; Lee, Joo Young; Song, Dae-Geun; Kwon, Sanghoon; Lee, Younghee; Pan, Cheol-Ho; Kwon, Hyung-Joo


    Post-translational modification regulated by conjugation of a small ubiquitin-like modifier (SUMO) is involved in various cellular processes. In this study, we expressed and purified recombinant human SUMO-1 (hSUMO-1). BALB/c mice were immunized with a complex of hSUMO-1 protein and Lipoplex(O) to produce hSUMO-1-specific antibodies. Using conventional hybridoma technology, we obtained four hybridoma clones derived from the mouse with the highest antibody titer against hSUMO-1. Based on Western blot analysis, our hSUMO-1 monoclonal antibody specifically recognizes hSUMO-1, but not other SUMO proteins. These results support that the anti-hSUMO-1 monoclonal antibody produced with the aid of Lipoplex(O) adjuvant is specific and that Lipoplex(O) is useful for development of monoclonal antibodies against recombinant protein. In addition, we analyzed human tissues to examine the distribution of hSUMO-1. Higher expression of hSUMO-1 was detected in normal adrenal gland, esophagus, pancreas, liver, stomach, kidney, and uterus than in corresponding cancer tissues, suggesting a tumor suppressive function of hSUMO-1.

  1. Humoral Immunity to Commensal Oral Bacteria in Human Infants: Salivary Antibodies Reactive with Actinomyces naeslundii Genospecies 1 and 2 during Colonization (United States)

    Cole, Michael F.; Bryan, Stacey; Evans, Mishell K.; Pearce, Cheryl L.; Sheridan, Michael J.; Sura, Patricia A.; Wientzen, Raoul; Bowden, George H. W.


    The secretory immune response in saliva to colonization by Actinomyces naeslundii genospecies 1 and 2 was studied in 10 human infants from birth to 2 years of age. Actinomyces species were not recovered from the mouths of the infants until approximately 4 months after the eruption of teeth. However, low levels of secretory immunoglobulin A1 (SIgA1) and SIgA2 antibodies reactive with whole cells of A. naeslundii genospecies 1 and 2 were detected within the first month after birth. Although there was a fivefold increase in the concentration of SIgA between birth and age 2 years, there were no differences between the concentrations of SIgA1 and SIgA2 antibodies reactive with A. naeslundii genospecies 1 and 2 over this period. When the concentrations of SIgA1 and SIgA2 antibodies reactive with whole cells of A. naeslundii genospecies 1 and 2 were normalized to the concentrations of SIgA1 and SIgA2 in saliva, the A. naeslundii genospecies 1- and 2-reactive SIgA1 and SIgA2 antibodies showed a significant decrease from birth to 2 years of age. The fine specificities of A. naeslundii genospecies 1- and 2-reactive SIgA1 and SIgA2 antibodies were examined by Western blotting of envelope proteins. Similarities in the molecular masses of proteins recognized by SIgA1 and SIgA2 antibodies, both within and between subjects over time, were examined by cluster analysis and showed considerable variability. Taken overall, our data suggest that among the mechanisms Actinomyces species employ to persist in the oral cavity are the induction of a limited immune response and clonal replacement with strains differing in their antigen profiles. PMID:9712779

  2. Colostrum from cows immunized with a vaccine associated with bovine neonatal pancytopenia contains allo-antibodies that cross-react with human MHC-I molecules. (United States)

    Kasonta, Rahel; Holsteg, Mark; Duchow, Karin; Dekker, James W; Cussler, Klaus; Bendall, Justin G; Bastian, Max


    In 2006, a new haemorrhagic syndrome affecting newborn calves, Bovine Neonatal Pancytopenia (BNP), was reported in southern Germany. It is characterized by severe bleeding, destruction of the red bone marrow, and a high case fatality rate. The syndrome is caused by alloreactive, maternal antibodies that are ingested by the calf with colostrum and result from a dam vaccination with one particular vaccine against Bovine-Viral-Diarrhoea-Virus. Because bovine colostrum is increasingly gaining interest as a dietary supplement for human consumption, the current study was initiated to elucidate whether BNP alloantibodies from BNP dams (i.e. animals that gave birth to a BNP-affected calf) cross-react with human cells, which could pose a health hazard for human consumers of colostral products. The present study clearly demonstrates that BNP alloantibodies cross-react with human lymphocytes in vitro. In agreement with previous reports on BNP, the cross-reactive antibodies are specific for MHC-I molecules, and sensitize opsonised human cells for in vitro complement lysis. Cross-reactive antibodies are present in serum and colostrum of individual BNP dams. They can be traced in commercial colostrum powder manufactured from cows immunized with the vaccine associated with BNP, but are absent from commercial powder manufactured from colostrum excluding such vaccinated cows. In humans alloreactive, MHC-I specific antibodies are generally not believed to cause severe symptoms. However, to minimize any theoretical risk for human consumers, manufacturers of bovine colostrum for human consumption should consider using only colostrum from animals that have not been exposed to the vaccine associated with BNP.

  3. Colostrum from cows immunized with a vaccine associated with bovine neonatal pancytopenia contains allo-antibodies that cross-react with human MHC-I molecules.

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    Rahel Kasonta

    Full Text Available In 2006, a new haemorrhagic syndrome affecting newborn calves, Bovine Neonatal Pancytopenia (BNP, was reported in southern Germany. It is characterized by severe bleeding, destruction of the red bone marrow, and a high case fatality rate. The syndrome is caused by alloreactive, maternal antibodies that are ingested by the calf with colostrum and result from a dam vaccination with one particular vaccine against Bovine-Viral-Diarrhoea-Virus. Because bovine colostrum is increasingly gaining interest as a dietary supplement for human consumption, the current study was initiated to elucidate whether BNP alloantibodies from BNP dams (i.e. animals that gave birth to a BNP-affected calf cross-react with human cells, which could pose a health hazard for human consumers of colostral products. The present study clearly demonstrates that BNP alloantibodies cross-react with human lymphocytes in vitro. In agreement with previous reports on BNP, the cross-reactive antibodies are specific for MHC-I molecules, and sensitize opsonised human cells for in vitro complement lysis. Cross-reactive antibodies are present in serum and colostrum of individual BNP dams. They can be traced in commercial colostrum powder manufactured from cows immunized with the vaccine associated with BNP, but are absent from commercial powder manufactured from colostrum excluding such vaccinated cows. In humans alloreactive, MHC-I specific antibodies are generally not believed to cause severe symptoms. However, to minimize any theoretical risk for human consumers, manufacturers of bovine colostrum for human consumption should consider using only colostrum from animals that have not been exposed to the vaccine associated with BNP.

  4. Prophylactic antibody treatment and intramuscular immunization reduce infectious human rhinovirus 16 load in the lower respiratory tract of challenged cotton rats

    Directory of Open Access Journals (Sweden)

    Jorge C.G. Blanco


    Full Text Available Human rhinoviruses (HRV represent the single most important etiological agents of the common cold and are the most frequent cause of acute respiratory infections in humans. Currently the performance of available animal models for immunization studies using HRV challenge is very limited. The cotton rat (Sigmodon hispidus is a well-recognized model for the study of human respiratory viral infections. In this work we show that, without requiring any genetic modification of either the host or the virus, intranasal infection of cotton rats with HRV16 resulted in measurable isolation of infective virus, lower respiratory tract pathology, mucus production, and expression of interferon-activated genes. Intramuscular immunization with live HRV16 generated robust protective immunity that correlated with high serum levels of neutralizing antibodies. In addition, cotton rats treated prophylactically with hyperimmune anti-HRV16 serum were protected against HRV16 intranasal challenge. Finally, protection by immunization was efficiently transferred from mothers to newborn animals resulting in a substantial reduction of infectious virus loads in the lung following intranasal challenge. Overall, our results demonstrate that the cotton rat provides valuable additional model development options for testing vaccines and prophylactic therapies against rhinovirus infection.

  5. Characterization of serum antibodies from women immunized with Gardasil: A study of HPV-18 infection of primary human keratinocytes. (United States)

    Wang, Hsu-Kun; Wei, Qing; Moldoveanu, Zina; Huh, Warner K; Vu, Huong Lan; Broker, Thomas R; Mestecky, Jiri; Chow, Louise T


    The prevalent human papillomaviruses (HPVs) infect human epithelial tissues. Infections by the mucosotropic HPV genotypes cause hyperproliferative ano-genital lesions. Persistent infections by high-risk (HR) HPVs such as HPV-16, HPV-18 and related types can progress to high grade intraepithelial neoplasias and cancers. Prophylactic HPV vaccines are based on DNA-free virus-like particles (VLPs) composed of the major capsid protein L1 of HPV-16, -18, -6 and -11 (Gardasil) or HPV-16 and -18 (Cervarix). Sera from vaccinated animals effectively prevent HPV pseudovirions to infect cell lines and mouse cervical epithelia. Both vaccines have proven to be highly protective in people. HPV pseudovirions are assembled in HEK293TT cells from matched L1 and L2 capsid proteins to encapsidate a reporter gene. Pseudovirions and genuine virions have structural differences and they infect cell lines or primary human keratinocytes (PHKs) with different efficiencies. In this study, we show that sera and isolated IgG from women immunized with Gardasil prevent authentic HPV-18 virions from infecting PHKs, whereas non-immune sera and purified IgG thereof are uniformly ineffective. Using early passage PHKs, neutralization is achieved only if immune sera are added within 2-4h of infection. We attribute the timing effect to a conformational change in HPV virions, thought to occur upon initial binding to heparan sulfate proteoglycans (HSPG) on the cell surface. This interpretation is consistent with the inability of immune IgG bound to or taken up by PHKs to neutralize the virus. Interestingly, the window of neutralization increases to 12-16h in slow growing, late passage PHKs, suggestive of altered cell surface molecules. In vivo, this window might be further lengthened by the time required to activate the normally quiescent basal cells to become susceptible to infection. Our observations help explain the high efficacy of HPV vaccines.

  6. Human Antibodies Can Cross Guinea Pig Placenta and Bind Its Neonatal Fc Receptor: Implications for Studying Immune Prophylaxis and Therapy during Pregnancy

    Directory of Open Access Journals (Sweden)

    Evi Budo Struble


    Full Text Available Despite increased use of monoclonal and polyclonal antibody therapies, including during pregnancy, there is little data on appropriate animal models that could humanely be used to understand determinants of protection and to evaluate safety of these biologics in the mother and the developing fetus. Here, we demonstrate that pregnant guinea pigs can transport human IgG transplacentally at the end of pregnancy. We also observe that human IgG binds to an engineered soluble variant of the guinea pig neonatal Fc receptor in vitro in a manner similar to that demonstrated for the human variant, suggesting that this transplacental transport mirrors the receptor-based mechanism seen in humans. Using an intravenous antihepatitis B-specific immune globulin preparation as an example, we show that this transport results in neutralizing activity in the mother and the newborn that would potentially be prophylactic against hepatitis B viral infection. These preliminary data lay the groundwork for introducing pregnant guinea pigs as an appropriate model for the evaluation of antibody therapies and advancing the health of women and neonates.

  7. Broad blockade antibody responses in human volunteers after immunization with a multivalent norovirus VLP candidate vaccine: immunological analyses from a phase I clinical trial.

    Directory of Open Access Journals (Sweden)

    Lisa C Lindesmith


    Full Text Available Human noroviruses (NoVs are the primary cause of acute gastroenteritis and are characterized by antigenic variation between genogroups and genotypes and antigenic drift of strains within the predominant GII.4 genotype. In the context of this diversity, an effective NoV vaccine must elicit broadly protective immunity. We used an antibody (Ab binding blockade assay to measure the potential cross-strain protection provided by a multivalent NoV virus-like particle (VLP candidate vaccine in human volunteers.Sera from ten human volunteers immunized with a multivalent NoV VLP vaccine (genotypes GI.1/GII.4 were analyzed for IgG and Ab blockade of VLP interaction with carbohydrate ligand, a potential correlate of protective immunity to NoV infection and illness. Immunization resulted in rapid rises in IgG and blockade Ab titers against both vaccine components and additional VLPs representing diverse strains and genotypes not represented in the vaccine. Importantly, vaccination induced blockade Ab to two novel GII.4 strains not in circulation at the time of vaccination or sample collection. GII.4 cross-reactive blockade Ab titers were more potent than responses against non-GII.4 VLPs, suggesting that previous exposure history to this dominant circulating genotype may impact the vaccine Ab response. Further, antigenic cartography indicated that vaccination preferentially activated preexisting Ab responses to epitopes associated with GII.4.1997. Study interpretations may be limited by the relevance of the surrogate neutralization assay and the number of immunized participants evaluated.Vaccination with a multivalent NoV VLP vaccine induces a broadly blocking Ab response to multiple epitopes within vaccine and non-vaccine NoV strains and to novel antigenic variants not yet circulating at the time of vaccination. These data reveal new information about complex NoV immune responses to both natural exposure and to vaccination, and support the potential

  8. Probiotic Lactobacillus rhamnosus GG enhanced Th1 cellular immunity but did not affect antibody responses in a human gut microbiota transplanted neonatal gnotobiotic pig model.

    Directory of Open Access Journals (Sweden)

    Ke Wen

    Full Text Available This study aims to establish a human gut microbiota (HGM transplanted gnotobiotic (Gn pig model of human rotavirus (HRV infection and diarrhea, and to verify the dose-effects of probiotics on HRV vaccine-induced immune responses. Our previous studies using the Gn pig model found that probiotics dose-dependently regulated both T cell and B cell immune responses induced by rotavirus vaccines. We generated the HGM transplanted neonatal Gn pigs through daily feeding of neonatal human fecal suspension to germ-free pigs for 3 days starting at 12 hours after birth. We found that attenuated HRV (AttHRV vaccination conferred similar overall protection against rotavirus diarrhea and virus shedding in Gn pigs and HGM transplanted Gn pigs. HGM promoted the development of the neonatal immune system, as evidenced by the significantly enhanced IFN-γ producing T cell responses and reduction of regulatory T cells and their cytokine production in the AttHRV-vaccinated pigs. The higher dose Lactobacillus rhamnosus GG (LGG feeding (14 doses, up to 109 colony-forming-unit [CFU]/dose effectively increased the LGG counts in the HGM Gn pig intestinal contents and significantly enhanced HRV-specific IFN-γ producing T cell responses to the AttHRV vaccine. Lower dose LGG (9 doses, up to 106 CFU/dose was ineffective. Neither doses of LGG significantly improved the protection rate, HRV-specific IgA and IgG antibody titers in serum, or IgA antibody titers in intestinal contents compared to the AttHRV vaccine alone, suggesting that an even higher dose of LGG is needed to overcome the influence of the microbiota to achieve the immunostimulatory effect in the HGM pigs. This study demonstrated that HGM Gn pig is an applicable animal model for studying immune responses to rotavirus vaccines and can be used for studying interventions (i.e., probiotics and prebiotics that may enhance the immunogenicity and protective efficacy of vaccines through improving the gut microbiota.

  9. Human immune system variation. (United States)

    Brodin, Petter; Davis, Mark M


    The human immune system is highly variable between individuals but relatively stable over time within a given person. Recent conceptual and technological advances have enabled systems immunology analyses, which reveal the composition of immune cells and proteins in populations of healthy individuals. The range of variation and some specific influences that shape an individual's immune system is now becoming clearer. Human immune systems vary as a consequence of heritable and non-heritable influences, but symbiotic and pathogenic microbes and other non-heritable influences explain most of this variation. Understanding when and how such influences shape the human immune system is key for defining metrics of immunological health and understanding the risk of immune-mediated and infectious diseases.

  10. Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions. (United States)

    Schlothauer, Tilman; Herter, Sylvia; Koller, Claudia Ferrara; Grau-Richards, Sandra; Steinhart, Virginie; Spick, Christian; Kubbies, Manfred; Klein, Christian; Umaña, Pablo; Mössner, Ekkehard


    Recombinant human IgG antibodies (hIgGs) completely devoid of binding to Fcγ receptors (FcγRs) and complement protein C1q, and thus with abolished immune effector functions, are of use for various therapeutic applications in order to reduce FcγR activation and Fc-mediated toxicity. Fc engineering approaches described to date only partially achieve this goal or employ a large number of mutations, which may increase the risk of anti-drug antibody generation. We describe here two new, engineered hIgG Fc domains, hIgG1-P329G LALA and hIgG4-P329G SPLE, with completely abolished FcγR and C1q interactions, containing a limited number of mutations and with unaffected FcRn interactions and Fc stability. Both 'effector-silent' Fc variants are based on a novel Fc mutation, P329G that disrupts the formation of a proline sandwich motif with the FcγRs. As this motif is present in the interface of all IgG Fc/FcγR complexes, its disruption can be applied to all human and most of the other mammalian IgG subclasses in order to create effector silent IgG molecules.

  11. A rapid immunization strategy with a live attenuated tetravalent dengue vaccine elicits protective neutralizing antibody responses in non-human primates

    Directory of Open Access Journals (Sweden)

    Yuping eAmbuel


    Full Text Available Dengue viruses (DENVs cause approximately 390 million cases of DENV infections annually and over 3 billion people worldwide are at risk of infection. No dengue vaccine is currently available nor is there an antiviral therapy for DENV infections. We have developed a tetravalent live-attenuated DENV vaccine (TDV that consists of a molecularly characterized attenuated DENV-2 strain (TDV-2 and three chimeric viruses containing the pre-membrane and envelope genes of DENV-1, -3 and -4 expressed in the context of the TDV-2 genome. To impact dengue vaccine delivery in endemic areas and immunize travelers, a simple and rapid immunization strategy (RIS is preferred. We investigated RIS consisting of two full vaccine doses being administered subcutaneously or intradermally on the initial vaccination visit (day 0 at two different anatomical locations with a needle-free disposable syringe jet injection (DSJI delivery devices (PharmaJet in non-human primates (NHP. This vaccination strategy resulted in efficient priming and induction of neutralizing antibody responses to all four DENV serotypes comparable to those elicited by the traditional prime and boost (two months later vaccination schedule. In addition, the vaccine induced CD4+ and CD8+ T cells producing IFN-γ, IL-2, and TNF-α, and targeting the DENV-2 NS1, NS3 and NS5 proteins. Moreover, vaccine-specific T cells were cross-reactive with the non-structural NS3 and NS5 proteins of DENV-4. When animals were challenged with DENV-2 they were protected with no detectable viremia, and exhibited sterilizing immunity (no increase of neutralizing titers post- challenge. RIS could decrease vaccination visits and provide quick immune response to all four DENV serotypes. This strategy could increase vaccination compliance and would be especially advantageous for travelers into endemic areas.

  12. Enhancement of anamnestic immunospecific antibody response in orally immunized chickens

    DEFF Research Database (Denmark)

    Mayo, Susan; Carlsson, Hans-Erik; Zagon, Andrea


    of the immunization in week 18, demonstrating the presence of memory cells following the two initial oral immunizations. Considering that oral immunization results in approximately ten times lower concentrations of immunospecific antibodies in the egg yolk, compared to traditional subcutaneous immunization schemes......Production of immunospecific egg yolk antibodies (IgY antibodies) in egg laying hens through oral immunization is an attractive alternative to conventional antibody production in mammals for economic reasons as well as for animal welfare reasons. Oral immunization results in a systemic humoral...... response, but oral booster immunizations lack efficiency. The aim of the present study was to develop immunization schemes in which the concentration of immunospecific IgY would increase following oral booster immunizations. Two groups of egg laying hens (5 in each group) were immunized orally (each...


    NARCIS (Netherlands)



    Immune inhibition of release of the DNA virues, herpes simplex virus types 1 and 2 and pseudorabies virus by anti-viral and anti-host cell sera occurred while two RNA viruses, influenza and encephalomyocarditis, were inhibited only by anti-viral sera (not anti-host cell sera). Simian virus 40 and su


    NARCIS (Netherlands)



    Immune inhibition of release of the DNA virues, herpes simplex virus types 1 and 2 and pseudorabies virus by anti-viral and anti-host cell sera occurred while two RNA viruses, influenza and encephalomyocarditis, were inhibited only by anti-viral sera (not anti-host cell sera). Simian virus 40 and

  15. Human monoclonal antibodies against anthrax lethal factor and protective antigen act independently to protect against Bacillus anthracis infection and enhance endogenous immunity to anthrax

    NARCIS (Netherlands)

    Albrecht, Mark T.; Li, Han; Williamson, E. Diane; LeButt, Chris S.; Flick-Smith, Helen C.; Quinn, Conrad P.; Westra, Hans; Galloway, Darrell; Mateczun, Alfred; Goldman, Stanley; Groen, Herman; Baillie, Les W. J.


    The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action

  16. Human monoclonal antibodies against anthrax lethal factor and protective antigen act independently to protect against Bacillus anthracis infection and enhance endogenous immunity to anthrax

    NARCIS (Netherlands)

    Albrecht, Mark T.; Li, Han; Williamson, E. Diane; LeButt, Chris S.; Flick-Smith, Helen C.; Quinn, Conrad P.; Westra, Hans; Galloway, Darrell; Mateczun, Alfred; Goldman, Stanley; Groen, Herman; Baillie, Les W. J.


    The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action

  17. Tabhu: tools for antibody humanization

    DEFF Research Database (Denmark)

    Olimpieri, Pier Paolo; Marcatili, Paolo; Tramontano, Anna


    Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can...... into a suitable human template. Unfortunately, this procedure may results in a partial or complete loss of affinity of the grafted molecule that can be restored by back-mutating some of the residues of human origin to the corresponding murine ones. This trial-and-error procedure is hard and involves expensive...... and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps...

  18. Passive transfer of maternal immunity in the dromedary (Camelus dromedarius), involvement of heavy chain antibodies. (United States)

    Salhi, Imed; Bessalah, Salma; Mbarek, Sonia Ben; Chniter, Mohamed; Seddik, Mabrouk-Mouldi; Khorchani, Touhami; Hammadi, Mohamed


    In many mammalian species, newborns are agammaglobulinemic; thus, colostrum and milk are the main sources of early protective antibodies. These antibodies are produced in the mother's serum and transferred to mammalian glands a few days before parturition. Here, we have studied the transfer of immunity from a she-camel immunized with human serum albumin (HSA) to her calf via colostrum and milk. Our results show that HSA-specific antibodies are produced in the mother's serum and are subsequently transferred to her colostrum. These specific antibodies are then transferred by suckling to the calf. The calf serum did not contain HSA-reactive antibodies at parturition and before the first feed, after suckling, a rise in reactivity was observed peaking at 24 h postpartum. The involvement of heavy chain antibodies (HCAbs) in the process of immunity transfer was also examined, and it was found that they were also transferred from the colostrum to the calf serum like conventional antibodies.

  19. Human anti-mouse antibodies. (United States)

    Klee, G G


    Human anti-mouse antibodies (HAMA) are human immunoglobulins with specificity for mouse immunoglobulins. This topic currently is of interest because of the increased use of monoclonal mouse antibodies as diagnostic reagents both for in vitro laboratory measurements and for in vivo imaging studies. Monoclonal mouse antibodies also are being used therapeutically. This short article reviews the production of HAMA in patients receiving monoclonal antibodies and illustrates the potential ways that HAMA can interfere with immunoassay measurements. Methods for measuring and neutralizing HAMA also are discussed.

  20. Study of Humoral Immunity to Commensal Oral Bacteria in Human Infants Demonstrates the Presence of Secretory Immunoglobulin A Antibodies Reactive with Actinomyces naeslundii Genospecies 1 and 2 Ribotypes (United States)

    Cole, Michael F.; Evans, Mishell K.; Kirchherr, Jennifer L.; Sheridan, Michael J.; Bowden, G. H. W.


    The mouths of three human infants were examined from birth to age 2 years to detect colonization of Actinomyces naeslundii genospecies 1 and 2. These bacteria did not colonize until after tooth eruption. The diversity of posteruption isolates was determined by ribotyping. Using immunoblotting and enzyme-linked immunosorbent assay, we determined the reactivity of secretory immunoglobulin A (SIgA) antibodies in saliva samples collected from each infant before and after colonization against cell wall proteins from their own A. naeslundii strains and carbohydrates from standard A. naeslundii genospecies 1 and 2 strains. A. naeslundii genospecies 1 and 2 carbohydrate-reactive SIgA antibodies were not detected in any saliva sample. However, SIgA antibodies reactive with cell wall proteins were present in saliva before these bacteria colonized the mouth. These antibodies could be almost completely removed by absorption with A. odontolyticus, a species known to colonize the human mouth shortly after birth. However, after colonization by A. naeslundii genospecies 1 and 2, specific antibodies were induced that could not be removed by absorption with A. odontolyticus. Cluster analysis of the patterns of reactivity of postcolonization salivary antibodies from each infant with antigens from their own strains showed that not only could these antibodies discriminate among strains but antibodies in saliva samples collected at different times showed different reactivity patterns. Overall, these data suggest that, although much of the salivary SIgA antibodies reactive with A. naeslundii genospecies 1 and 2 are directed against genus-specific or more broadly cross-reactive antigens, species, genospecies, and possibly strain-specific antibodies are induced in response to colonization. PMID:15138172

  1. [Cellular immunity in human periapical granuloma]. (United States)

    Terrié, B; Grégoire, G


    Numerous authors have produced different types of immunoglobulins in analyses of the human periapical granuloma. The present study examines the cellular immunity of the human periapical granuloma, and in particular the distribution of the lymphocyte sub-populations and the macrophage population. The technique used was that of cell surface marking, using monoclonal antibodies on frozen sections. The results reveal equal proportions of inductor T lymphocytes and suppressor T lymphocytes (whereas healthy tissue shows a ratio of 2/1), which explains the chronic nature of the lesion as far as the immune reaction is concerned. The presence of numerous macrophage cells shows that there are important local immune reactions.

  2. Tabhu: tools for antibody humanization.

    KAUST Repository

    Olimpieri, Pier Paolo


    SUMMARY: Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity into a suitable human template. Unfortunately, this procedure may results in a partial or complete loss of affinity of the grafted molecule that can be restored by back-mutating some of the residues of human origin to the corresponding murine ones. This trial-and-error procedure is hard and involves expensive and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps of the humanization experiment protocol. AVAILABILITY: CONTACT:, SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

  3. Antibody-Mediated Immunity against Tuberculosis: Implications for Vaccine Development


    Achkar, Jacqueline M; Casadevall, Arturo


    There is an urgent need for new and better vaccines against tuberculosis (TB). Current vaccine design strategies are generally focused on the enhancement of cell-mediated immunity. Antibody-based approaches are not being considered, mostly due to the paradigm that humoral immunity plays little role in the protection against intracellular pathogens. Here, we reappraise and update the increasing evidence for antibody-mediated immunity against Mycobacterium tuberculosis, discuss the complexity o...

  4. Passive Immunization against HIV/AIDS by Antibody Gene Transfer

    Directory of Open Access Journals (Sweden)

    Lili Yang


    Full Text Available Despite tremendous efforts over the course of many years, the quest for an effective HIV vaccine by the classical method of active immunization remains largely elusive. However, two recent studies in mice and macaques have now demonstrated a new strategy designated as Vectored ImmunoProphylaxis (VIP, which involves passive immunization by viral vector-mediated delivery of genes encoding broadly neutralizing antibodies (bnAbs for in vivo expression. Robust protection against virus infection was observed in preclinical settings when animals were given VIP to express monoclonal neutralizing antibodies. This unorthodox approach raises new promise for combating the ongoing global HIV pandemic. In this article, we survey the status of antibody gene transfer, review the revolutionary progress on isolation of extremely bnAbs, detail VIP experiments against HIV and its related virus conduced in humanized mice and macaque monkeys, and discuss the pros and cons of VIP and its opportunities and challenges towards clinical applications to control HIV/AIDS endemics.

  5. A novel H6N1 virus-like particle vaccine induces long-lasting cross-clade antibody immunity against human and avian H6N1 viruses. (United States)

    Yang, Ji-Rong; Chen, Chih-Yuan; Kuo, Chuan-Yi; Cheng, Chieh-Yu; Lee, Min-Shiuh; Cheng, Ming-Chu; Yang, Yu-Chih; Wu, Chia-Ying; Wu, Ho-Sheng; Liu, Ming-Tsan; Hsiao, Pei-Wen


    Avian influenza A(H6N1) virus is one of the most common viruses isolated from migrating birds and domestic poultry in many countries. The first and only known case of human infection by H6N1 virus in the world was reported in Taiwan in 2013. This led to concern that H6N1 virus may cause a threat to public health. In this study, we engineered a recombinant H6N1 virus-like particle (VLP) and investigated its vaccine effectiveness compared to the traditional egg-based whole inactivated virus (WIV) vaccine. The H6N1-VLPs exhibited similar morphology and functional characteristics to influenza viruses. Prime-boost intramuscular immunization in mice with unadjuvanted H6N1-VLPs were highly immunogenic and induced long-lasting antibody immunity. The functional activity of the VLP-elicited IgG antibodies was proved by in vitro seroprotective hemagglutination inhibition and microneutralization titers against the homologous human H6N1 virus, as well as in vivo viral challenge analyses which showed H6N1-VLP immunization significantly reduced viral load in the lung, and protected against human H6N1 virus infection. Of particular note, the H6N1-VLPs but not the H6N1-WIVs were able to confer cross-reactive humoral immunity; antibodies induced by H6N1-VLP vaccine robustly inhibited the hemagglutination activities and in vitro replication of distantly-related heterologous avian H6N1 viruses. Furthermore, the H6N1-VLPs were found to elicit significantly greater anti-HA2 antibody responses in immunized mice than H6N1-WIVs. Collectively, we demonstrated for the first time a novel H6N1-VLP vaccine that effectively provides broadly protective immunity against both human and avian H6N1 viruses. These results, which uncover the underlying mechanisms for induction of wide-range immunity against influenza viruses, may be useful for future influenza vaccine development.

  6. Antibody-mediated protection against mucosal simian-human immunodeficiency virus challenge of macaques immunized with alphavirus replicon particles and boosted with trimeric envelope glycoprotein in MF59 adjuvant. (United States)

    Barnett, Susan W; Burke, Brian; Sun, Yide; Kan, Elaine; Legg, Harold; Lian, Ying; Bost, Kristen; Zhou, Fengmin; Goodsell, Amanda; Zur Megede, Jan; Polo, John; Donnelly, John; Ulmer, Jeffrey; Otten, Gillis R; Miller, Christopher J; Vajdy, Michael; Srivastava, Indresh K


    We have previously shown that rhesus macaques were partially protected against high-dose intravenous challenge with simian-human immunodeficiency virus SHIV(SF162P4) following sequential immunization with alphavirus replicon particles (VRP) of a chimeric recombinant VEE/SIN alphavirus (derived from Venezuelan equine encephalitis virus [VEE] and the Sindbis virus [SIN]) encoding human immunodeficiency virus type 1 HIV-1(SF162) gp140DeltaV2 envelope (Env) and trimeric Env protein in MF59 adjuvant (R. Xu, I. K. Srivastava, C. E. Greer, I. Zarkikh, Z. Kraft, L. Kuller, J. M. Polo, S. W. Barnett, and L. Stamatatos, AIDS Res. Hum. Retroviruses 22:1022-1030, 2006). The protection did not require T-cell immune responses directed toward simian immunodeficiency virus (SIV) Gag. We extend those findings here to demonstrate antibody-mediated protection against mucosal challenge in macaques using prime-boost regimens incorporating both intramuscular and mucosal routes of delivery. The macaques in the vaccination groups were primed with VRP and then boosted with Env protein in MF59 adjuvant, or they were given VRP intramuscular immunizations alone and then challenged with SHIV(SF162P4) (intrarectal challenge). The results demonstrated that these vaccines were able to effectively protect the macaques to different degrees against subsequent mucosal SHIV challenge, but most noteworthy, all macaques that received the intramuscular VRP prime plus Env protein boost were completely protected. A statistically significant association was observed between the titer of virus neutralizing and binding antibodies as well as the avidity of anti-Env antibodies measured prechallenge and protection from infection. These results highlight the merit of the alphavirus replicon vector prime plus Env protein boost vaccine approach for the induction of protective antibody responses and are of particular relevance to advancing our understanding of the potential correlates of immune protection against

  7. Construction of human antibody gene libraries and selection of antibodies by phage display. (United States)

    Frenzel, André; Kügler, Jonas; Wilke, Sonja; Schirrmann, Thomas; Hust, Michael


    Antibody phage display is the most commonly used in vitro selection technology and has yielded thousands of useful antibodies for research, diagnostics, and therapy.The prerequisite for successful generation and development of human recombinant antibodies using phage display is the construction of a high-quality antibody gene library. Here, we describe the methods for the construction of human immune and naive scFv gene libraries.The success also depends on the panning strategy for the selection of binders from these libraries. In this article, we describe a panning strategy that is high-throughput compatible and allows parallel selection in microtiter plates.

  8. Early Rise of Blood T Follicular Helper Cell Subsets and Baseline Immunity as Predictors of Persisting Late Functional Antibody Responses to Vaccination in Humans.

    Directory of Open Access Journals (Sweden)

    Fabiana Spensieri

    Full Text Available CD4+ T follicular helper cells (T(FH have been identified as the T-cell subset specialized in providing help to B cells for optimal activation and production of high affinity antibody. We recently demonstrated that the expansion of peripheral blood influenza-specific CD4(+IL-21(+ICOS1(+ T helper (T(H cells, three weeks after vaccination, associated with and predicted the rise of protective neutralizing antibodies to avian H5N1. In this study, healthy adults were vaccinated with plain seasonal trivalent inactivated influenza vaccine (TIIV, MF59(®-adjuvanted TIIV (ATIIV, or saline placebo. Frequencies of circulating CD4(+ T(FH1 ICOS(+ T(FH cells and H1N1-specific CD4(+-IL-21(+ICOS(+ CXCR5(+ T(FH and CXCR5(- T(H cell subsets were determined at various time points after vaccination and were then correlated with hemagglutination inhibition (HI titers. All three CD4(+ T cell subsets expanded in response to TIIV and ATIIV, and peaked 7 days after vaccination. To demonstrate that these T(FH cell subsets correlated with functional antibody titers, we defined an alternative endpoint metric, decorrelated HI (DHI, which removed any correlation between day 28/day 168 and day 0 HI titers, to control for the effect of preexisting immunity to influenza vaccine strains. The numbers of total circulating CD4(+T(FH1 ICOS(+ cells and of H1N1-specific CD4(+IL-21(+ICOS(+ CXCR5(+, measured at day 7, were significantly associated with day 28, and day 28 and 168 DHI titers, respectively. Altogether, our results show that CD4(+ T(FH subsets may represent valuable biomarkers of vaccine-induced long-term functional immunity.

  9. Early Rise of Blood T Follicular Helper Cell Subsets and Baseline Immunity as Predictors of Persisting Late Functional Antibody Responses to Vaccination in Humans (United States)

    Borgogni, Erica; Zedda, Luisanna; Cantisani, Rocco; Chiappini, Nico; Schiavetti, Francesca; Rosa, Domenico; Castellino, Flora; Montomoli, Emanuele; Bodinham, Caroline L.; Lewis, David J.; Medini, Duccio; Bertholet, Sylvie; Del Giudice, Giuseppe


    CD4+ T follicular helper cells (TFH) have been identified as the T-cell subset specialized in providing help to B cells for optimal activation and production of high affinity antibody. We recently demonstrated that the expansion of peripheral blood influenza-specific CD4+IL-21+ICOS1+ T helper (TH) cells, three weeks after vaccination, associated with and predicted the rise of protective neutralizing antibodies to avian H5N1. In this study, healthy adults were vaccinated with plain seasonal trivalent inactivated influenza vaccine (TIIV), MF59®-adjuvanted TIIV (ATIIV), or saline placebo. Frequencies of circulating CD4+ TFH1 ICOS+ TFH cells and H1N1-specific CD4+IL-21+ICOS+ CXCR5+ TFH and CXCR5- TH cell subsets were determined at various time points after vaccination and were then correlated with hemagglutination inhibition (HI) titers. All three CD4+ T cell subsets expanded in response to TIIV and ATIIV, and peaked 7 days after vaccination. To demonstrate that these TFH cell subsets correlated with functional antibody titers, we defined an alternative endpoint metric, decorrelated HI (DHI), which removed any correlation between day 28/day 168 and day 0 HI titers, to control for the effect of preexisting immunity to influenza vaccine strains. The numbers of total circulating CD4+ TFH1 ICOS+ cells and of H1N1-specific CD4+IL-21+ICOS+ CXCR5+, measured at day 7, were significantly associated with day 28, and day 28 and 168 DHI titers, respectively. Altogether, our results show that CD4+ TFH subsets may represent valuable biomarkers of vaccine-induced long-term functional immunity. Trial Registration NCT01771367 PMID:27336786

  10. Role of antibody in immunity and control of chicken coccidiosis. (United States)

    Wallach, Michael


    Research has been carried out worldwide to try to elucidate the mechanism of protective immunity against coccidiosis. It was concluded from early studies that cellular immunity is the key to protection against Eimeria, whereas humoral immunity plays a very minor role in resistance against infection. By contrast, other studies have pointed towards the ability of antibody to block parasite invasion, development and transmission and to provide passive and maternal immunity against challenge infection. Herein, recent results demonstrate the ability of antibodies (raised by live immunization or against purified stage-specific Eimeria antigens) to inhibit parasite development in vitro and in vivo and readdress the question of the role of antibody in protection against coccidiosis. Copyright 2010 Elsevier Ltd. All rights reserved.

  11. Humoral Immunity to Commensal Oral Bacteria in Human Infants: Salivary Secretory Immunoglobulin A Antibodies Reactive with Streptococcus mitis biovar 1, Streptococcus oralis, Streptococcus mutans, and Enterococcus faecalis during the First Two Years of Life (United States)

    Cole, Michael F.; Bryan, Stacey; Evans, Mishell K.; Pearce, Cheryl L.; Sheridan, Michael J.; Sura, Patricia A.; Wientzen, Raoul L.; Bowden, George H. W.


    Secretory immunoglobulin A (SIgA) antibodies reactive with the pioneer oral streptococci Streptococcus mitis biovar 1 and Streptococcus oralis, the late oral colonizer Streptococcus mutans, and the pioneer enteric bacterium Enterococcus faecalis in saliva samples from 10 human infants from birth to age 2 years were analyzed. Low levels of salivary SIgA1 and SIgA2 antibodies reactive with whole cells of all four species were detected within the first month after birth, even though S. mutans and E. faecalis were not recovered from the mouths of the infants during the study period. Although there was a fivefold increase in the concentration of SIgA between birth and age 2 years, there were no differences between the concentrations of SIgA1 and SIgA2 antibodies reactive with the four species over this time period. When the concentrations of SIgA1 and SIgA2 antibodies reactive with all four species were normalized to the concentrations of SIgA1 and SIgA2 in saliva, SIgA1 and SIgA2 antibodies reactive with these bacteria showed a significant decrease from birth to 2 years of age. Adsorption of each infant’s saliva with cells of one species produced a dramatic reduction of antibodies recognizing the other three species. Sequential adsorption of saliva samples removed all SIgA antibody to the bacteria, indicating that the SIgA antibodies were directed to antigens shared by all four species. The induction by the host of a limited immune response to common antigens that are likely not involved in adherence may be among the mechanisms that commensal streptococci employ to persist in the oral cavity. PMID:10085031

  12. Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization. (United States)

    Hansen, Debra T; Robida, Mark D; Craciunescu, Felicia M; Loskutov, Andrey V; Dörner, Katerina; Rodenberry, John-Charles; Wang, Xiao; Olson, Tien L; Patel, Hetal; Fromme, Petra; Sykes, Kathryn F


    Antibodies are essential for structural determinations and functional studies of membrane proteins, but antibody generation is limited by the availability of properly-folded and purified antigen. We describe the first application of genetic immunization to a structurally diverse set of membrane proteins to show that immunization of mice with DNA alone produced antibodies against 71% (n = 17) of the bacterial and viral targets. Antibody production correlated with prior reports of target immunogenicity in host organisms, underscoring the efficiency of this DNA-gold micronanoplex approach. To generate each antigen for antibody characterization, we also developed a simple in vitro membrane protein expression and capture method. Antibody specificity was demonstrated upon identifying, for the first time, membrane-directed heterologous expression of the native sequences of the FopA and FTT1525 virulence determinants from the select agent Francisella tularensis SCHU S4. These approaches will accelerate future structural and functional investigations of therapeutically-relevant membrane proteins.

  13. Antibody response to measles immunization in India*


    Job, J. S.; John, T J; Joseph, A.


    Antibody response to measles vaccine was measured in 238 subjects aged 6-15 months. Seroconversion rates ranged from 74% at 6 months of age to 100% at 13-15 months; the differences in age-specific rates were not statistically significant. The postimmunization antibody titres increased with increasing age of the vaccinee. Seroconversion rates and antibody titres in 49 subjects with grades I and II malnutrition were not significantly different from those in the 189 normal subjects.

  14. Exceptional Antibodies Produced by Successive Immunizations.

    Directory of Open Access Journals (Sweden)

    Patricia J Gearhart


    Full Text Available Antibodies stand between us and pathogens. Viruses mutate quickly to avoid detection, and antibodies mutate at similar rates to hunt them down. This death spiral is fueled by specialized proteins and error-prone polymerases that change DNA sequences. Here, we explore how B lymphocytes stay in the race by expressing activation-induced deaminase, which unleashes a tsunami of mutations in the immunoglobulin loci. This produces random DNA substitutions, followed by selection for the highest affinity antibodies. We may be able to manipulate the process to produce better antibodies by expanding the repertoire of specific B cells through successive vaccinations.

  15. Immune responses to chlamydial antigens in humans. (United States)

    Hanna, L; Kerlan, R; Senyk, G; Stites, D P; Juster, R P; Jawetz, E


    Antibody titer, lymphocyte stimulation and leukocyte migration inhibition with chlamydial antigens were determined repeatedly over many months on human subjects. The volunteers were retrospectively placed into four groups on the basis of clinical, laboratory and epidemiologic criteria. Group A consisted of persons with proven or probable chlamydial infection, including an illness confirmed by chlamydial isolation or seroconversion, or a clinically compatible illness with positive serologic results. Group B were sexual partners or close contacts of group A individuals. Group C were laboratory workers with prolonged exposure to viable chlamydiae or their antigens. Group D included persons of comparable age as those in groups A and B, but lacking a history of symptomatic chlamydial infection or of contact with chlamydiae. Individual cases illustrated the rise of antibody and some cell mediated immunity reactions (CMI) with active chlamydial infection. By contrast, laboratory exposure resulted in elevation of CMI but not of antibody. Statistical analysis of the results in 46 volunteers tested repeatedly indicated a strong association of specific antibody with lymphocyte stimulation, but not with leukocyte migration inhibition. Regression analysis suggested that the type of exposure markedly influenced the relationship between antibody and lymphocyte stimulation. Measurement of immunotype-specific antibody titer by microimmunofluorescence (or an equally sensitive method) remains the best laboratory indicator of past chlamydial infection. Neither antibody nor CMI can, as yet, be definitely related to resistance to re-infection in humans.

  16. Vault nanocapsules as adjuvants favor cell-mediated over antibody-mediated immune responses following immunization of mice.

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    Upendra K Kar

    Full Text Available BACKGROUND: Modifications of adjuvants that induce cell-mediated over antibody-mediated immunity is desired for development of vaccines. Nanocapsules have been found to be viable adjuvants and are amenable to engineering for desired immune responses. We previously showed that natural nanocapsules called vaults can be genetically engineered to elicit Th1 immunity and protection from a mucosal bacterial infection. The purpose of our study was to characterize immunity produced in response to OVA within vault nanoparticles and compare it to another nanocarrier. METHODOLOGY AND PRINCIPAL FINDINGS: We characterized immunity resulting from immunization with the model antigen, ovalbumin (OVA encased in vault nanocapsules and liposomes. We measured OVA responsive CD8(+ and CD4(+ memory T cell responses, cytokine production and antibody titers in vitro and in vivo. We found that immunization with OVA contain in vaults induced a greater number of anti-OVA CD8(+ memory T cells and production of IFNγ plus CD4(+ memory T cells. Also, modification of the vault body could change the immune response compared to OVA encased in liposomes. CONCLUSIONS/SIGNIFICANCE: These experiments show that vault nanocapsules induced strong anti-OVA CD8(+ and CD4(+ T cell memory responses and modest antibody production, which markedly differed from the immune response induced by liposomes. We also found that the vault nanocapsule could be modified to change antibody isotypes in vivo. Thus it is possible to create a vault nanocapsule vaccine that can result in the unique combination of immunogen-responsive CD8(+ and CD4(+ T cell immunity coupled with an IgG1 response for future development of vault nanocapsule-based vaccines against antigens for human pathogens and cancer.

  17. Rabbits immunized with thyroid-stimulating hormone produce autoantiidiotypic thyroid-stimulating antibodies. (United States)

    Beall, G N; Rapoport, B; Chopra, I J; Kruger, S R


    We immunized rabbits with thyroid-stimulating hormone (TSH) to investigate the hypothesis that such immunization could result in production of thyroid-stimulating autoantiidiotypic antibodies to anti-TSH. Thyroid-stimulating immunoglobulin (TSI) appeared in the serum of several rabbits after immunization. At 160 d, TSI equivalent to 6-18 microU TSH/1.5 mg IgG was present in two of six human (h)TSH-, two of six hTSH beta chain-, and two of the four surviving bovine (b)TSH-immunized animals. Control (human serum albumin-immunized rabbits) serum TSI was 4.3 +/- 0.4 (mean +/- SD) at this time. Antiidiotypic antibodies that could bind to monoclonal anti-hTSH were found in the sera of the bTSH-immunized rabbits. The peak TSI activity occurred 3 mo after a TSH booster immunization and declined gradually during subsequent weeks. Evidence that antiidiotypic antibodies to anti-TSH can cause thyroid stimulation strengthens the notion that such antibodies may be the cause of Graves' hyperthyroidism.

  18. Maternal antibodies: clinical significance, mechanism of interference with immune responses, and possible vaccination strategies

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    Stefan eNiewiesk


    Full Text Available Neonates have an immature immune system which cannot adequately protect against infectious diseases. Early in life, immune protection is accomplished by maternal antibodies transferred from mother to offspring. However, decaying maternal antibodies inhibit vaccination as is examplified by the inhibition of seroconversion after measles vaccination. This phenomenon has been described in both human and veterinary medicine and is independent of the type of vaccine being used. This review will discuss the use of animal models for vaccine research. I will review clinical solutions for inhibition of vaccination by maternal antibodies, and the testing and development of potentially effective vaccines. These are based on new mechanistic insight about the inhibitory mechanism of maternal antibodies. Maternal antibodies inhibit the generation of antibodies whereas the T cell response is usually unaffected. B cell inhibition is mediated through a cross-link between B-cell receptor (BCR with the Fcg receptor IIB (FcgRIIB by a vaccine-antibody complex. In animal experiments, this inhibition can be partially overcome by injection of a vaccine-specific monoclonal IgM antibody. IgM stimulates the B-cell directly through cross-linking the BCR via complement protein C3d and antigen to the complement receptor 2 (CR2 signaling complex. In addition, it was shown that interferon alpha binds to the CD21 chain of CR2 as well as the interferon receptor and that this dual receptor usage drives B cell responses in the presence of maternal antibodies. In lieu of immunizing the infant the concept of maternal immunization as a strategy to protect neonates has been proposed. This approach would still not solve the question of how to immunize in the presence of maternal antibodies but would defer the time of infection to an age where infection might not have such a detrimental outcome as in neonates. I will review successful examples and potential challenges of implementing

  19. Antibody (United States)

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...


    Directory of Open Access Journals (Sweden)

    S. A. Donina


    Full Text Available Abstract. At present, immunogenicity evaluation of influenza vaccines is performed by quantitative assessment of increased serum antibodies. It was, however, shown that the degree of human defense against influenza is mostly related to their qualitative characteristics, i.e., avidity (functional activity. Leading role of local immunity is demonstrated in protection against influenza. Such immunity is mediated by IgA antibodies from mucosal airways. Meanwhile, the avidity issues for local antibodies still remain open.In present study, an attempt was undertaken to evaluate post-vaccination local immunological memory for influenza A virus, according to IgA antibodies from upper respiratory secretions. Two techniques were used to evaluate antibody avidity, that were previously applied for studying this phenomenon with serum imunoglobulins, i.e., a dynamic test (measurement of antigen-antibody reaction rates, and a test with urea, a chaotropic agent (avidity is determined as a strength of antigen-antibody complex. A total of 202 persons (18 to 20 years old were enrolled into the study.With both tests, a broad range of individual avidity values was observed for the antibodies. A significant cohort (up to 30 per cent of persons immunized with live influenza vaccine, showed sharply increased avidity of secretory IgA antibodies by both methods, along with accumulation of these immunoglobulins after vaccination. A reverse relationship is revealed between avidity levels of these antibodies before vaccination, and increase of this parameter post-immunization. The data present convincing arguments for specific renewal of local humoral immunological memory, as induced by live influenza vaccine. The study substantiates a necessity for application of the both tests in parallel, when determining avidity of secretory IgA antibodies. (Med. Immunol., vol. 10, N 4-5, pp 423-430.

  1. Rabbits immunized with thyroid-stimulating hormone produce autoantiidiotypic thyroid-stimulating antibodies.


    Beall, G N; Rapoport, B; Chopra, I J; Kruger, S R


    We immunized rabbits with thyroid-stimulating hormone (TSH) to investigate the hypothesis that such immunization could result in production of thyroid-stimulating autoantiidiotypic antibodies to anti-TSH. Thyroid-stimulating immunoglobulin (TSI) appeared in the serum of several rabbits after immunization. At 160 d, TSI equivalent to 6-18 microU TSH/1.5 mg IgG was present in two of six human (h)TSH-, two of six hTSH beta chain-, and two of the four surviving bovine (b)TSH-immunized animals. Co...

  2. Parasite glycans and antibody-mediated immune responses in Schistosoma infection. (United States)

    van Diepen, Angela; Van der Velden, Niels S J; Smit, Cornelis H; Meevissen, Moniek H J; Hokke, Cornelis H


    Schistosome infections in humans are characterized by the development of chronic disease and high re-infection rates after treatment due to the slow development of immunity. It appears that anti-schistosome antibodies are at least partially mediating protective mechanisms. Efforts to develop a vaccine based on immunization with surface-exposed or secreted larval or worm proteins are ongoing. Schistosomes also express a large number of glycans as part of their glycoprotein and glycolipid repertoire, and antibody responses to those glycans are mounted by the infected host. This observation raises the question if glycans might also form novel vaccine targets for immune intervention in schistosomiasis. This review summarizes current knowledge of antibody responses and immunity in experimental and natural infections with Schistosoma, the expression profiles of schistosome glycans (the glycome), and antibody responses to individual antigenic glycan motifs. Future directions to study anti-glycan responses in schistosomiasis in more detail in order to address more precisely the possible role of glycans in antibody-mediated immunity are discussed.

  3. A human-mouse hybridoma producing monoclonal antibody against human sperm coating antigen. (United States)

    Kyurkchiev, S D; Shigeta, M; Koyama, K; Isojima, S


    Since anti-sperm antibodies were first discovered in the sera of women, the relationship of these antibodies to sterility has been studied by many investigators. In order to determine the antigens of spermatozoa responsible for raising antibodies to spermatozoa in humans, many studies have been carried out by purifying human spermatozoa cell membrane and seminal plasma components. Since it was found that the purification was difficult by physiochemical procedures, the immunoaffinity chromatography bound monoclonal antibody (Mab) to spermatozoa antigens was attempted for this purpose. The establishment of hybridomas producing Mabs to human seminal plasma and human spermatozoa was reported by Shigeta et al. (1980), Isojima, Koyoma & Fujiwara (1982), Lee et al. (1982) and Isahakia & Alexander (1984). The ordinary approaches to obtain the Mabs consisted of xenogenic immunization with human semen and cell fusion of immunized spleen cells with mouse myeloma cells. However, the antigenic epitopes of human spermatozoa, which induced antibody production, are xenogenic for the mouse, and therefore there is a possibility that there is a difference in recognized antigenic epitopes in humans as isotypic and in mice as xenogenic. In order to study these antigenic epitopes, which correspond to antibodies against spermatozoa in women, the establishment of human-mouse hybridomas, which produced anti-semen antibodies as produced in sterile women, became essential. In these studies, we used recently developed cell fusion techniques to fuse immunized human peripheral lymphocytes with mouse myeloma cells. PMID:3456978

  4. Antidotes, antibody-mediated immunity and the future of pharmaceutical product development. (United States)

    Caoili, Salvador Eugenio C


    If new scientific knowledge is to be more efficiently generated and applied toward the advancement of health, human safety must be more effectively addressed in the conduct of research. Given the present difficulties of accurately predicting biological outcomes of novel interventions in vivo, the imperative of human safety suggests the development of novel pharmaceutical products in tandem with their prospective antidotes in anticipation of possible adverse events, to render the risks of initial clinical trials more acceptable from a regulatory standpoint. Antibody-mediated immunity provides a generally applicable mechanistic basis for developing antidotes to both biologicals and small-molecule drugs (such that antibodies may serve as antidotes to pharmaceutical agents as a class including other antibodies) and also for the control and prevention of both infectious and noninfectious diseases via passive or active immunization. Accordingly, the development of prophylactic or therapeutic passive-immunization strategies using antipeptide antibodies is a plausible prelude to the development of corresponding active-immunization strategies using peptide-based vaccines. In line with this scheme, global proliferation of antibody- and vaccine-production technologies, especially those that obviate dependence on the cold chain for storage and transport of finished products, could provide geographically distributed breakout capability against emerging and future health challenges.

  5. Malaria vaccines: immunity, models and monoclonal antibodies

    DEFF Research Database (Denmark)

    Hviid, Lars; Barfod, Lea


    Although experts in the field have agreed on the malaria vaccine technology roadmap that should be followed (, the path towards an effective malaria vaccine remains littered with intellectual and practical pot-holes. The animal models that are currently...... available are problematic, and current understanding of the exact mechanisms and targets of protective immune responses is incomplete. However, recent technological advances might help overcome some of these hurdles....

  6. Production of Monoclonal Antibody against Human Nestin. (United States)

    Hadavi, Reza; Zarnani, Amir Hassan; Ahmadvand, Negah; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Sadeghi, Mohammad-Reza; Soltanghoraee, Haleh; Akhondi, Mohammad Mehdi; Tarahomi, Majid; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah


    We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140-250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays.

  7. A Cayley Tree Immune Network Model with Antibody Dynamics

    CERN Document Server

    Anderson, R W; Perelson, A S; Anderson, Russell W.; Neumann, Avidan U.; Perelson, Alan S.


    Abstract: A Cayley tree model of idiotypic networks that includes both B cell and antibody dynamics is formulated and analyzed. As in models with B cells only, localized states exist in the network with limited numbers of activated clones surrounded by virgin or near-virgin clones. The existence and stability of these localized network states are explored as a function of model parameters. As in previous models that have included antibody, the stability of immune and tolerant localized states are shown to depend on the ratio of antibody to B cell lifetimes as well as the rate of antibody complex removal. As model parameters are varied, localized steady-states can break down via two routes: dynamically, into chaotic attractors, or structurally into percolation attractors. For a given set of parameters, percolation and chaotic attractors can coexist with localized attractors, and thus there do not exist clear cut boundaries in parameter space that separate regions of localized attractors from regions of percola...

  8. Sequential Immunization Elicits Broadly Neutralizing Anti-HIV-1 Antibodies in Ig Knockin Mice. (United States)

    Escolano, Amelia; Steichen, Jon M; Dosenovic, Pia; Kulp, Daniel W; Golijanin, Jovana; Sok, Devin; Freund, Natalia T; Gitlin, Alexander D; Oliveira, Thiago; Araki, Tatsuya; Lowe, Sarina; Chen, Spencer T; Heinemann, Jennifer; Yao, Kai-Hui; Georgeson, Erik; Saye-Francisco, Karen L; Gazumyan, Anna; Adachi, Yumiko; Kubitz, Michael; Burton, Dennis R; Schief, William R; Nussenzweig, Michel C


    A vaccine that elicits broadly neutralizing antibodies (bNAbs) against HIV-1 is likely to be protective, but this has not been achieved. To explore immunization regimens that might elicit bNAbs, we produced and immunized mice expressing the predicted germline PGT121, a bNAb specific for the V3-loop and surrounding glycans on the HIV-1 spike. Priming with an epitope-modified immunogen designed to activate germline antibody-expressing B cells, followed by ELISA-guided boosting with a sequence of directional immunogens, native-like trimers with decreasing epitope modification, elicited heterologous tier-2-neutralizing responses. In contrast, repeated immunization with the priming immunogen did not. Antibody cloning confirmed elicitation of high levels of somatic mutation and tier-2-neutralizing antibodies resembling the authentic human bNAb. Our data establish that sequential immunization with specifically designed immunogens can induce high levels of somatic mutation and shepherd antibody maturation to produce bNAbs from their inferred germline precursors.

  9. Increased titers of neutralizing antibodies after immunization with both envelope proteins of the porcine endogenous retroviruses (PERVs

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    Denner Joachim


    Full Text Available Abstract Despite enormous difficulties to induce antibodies neutralizing HIV-1, especially broadly neutralizing antibodies directed against the conserved membrane proximal external region (MPER of the transmembrane envelope protein, such antibodies can be easily induced in the case of gammaretroviruses, among them the porcine endogenous retroviruses (PERVs. In addition to neutralizing antibodies directed against the transmembrane envelope protein p15E, neutralizing antibodies were also induced by immunization with the surface envelope protein gp70. PERVs represent a special risk for xenotransplantation using pig tissues or organs since they are integrated in the genome of all pigs and infect human cells and a vaccine may protect from transmission to the recipient. To investigate the effect of simultaneous immunization with both proteins in detail, a study was performed in hamsters. Gp70 and p15E of PERV were produced in E. coli, purified and used for immunization. All animals developed binding antibodies against the antigens used for immunization. Sera from animals immunized with p15E recognized epitopes in the MPER and the fusion peptide proximal region (FPPR of p15E. One MPER epitope showed a sequence homology to an epitope in the MPER of gp41 of HIV-1 recognized by broadly neutralizing antibodies found in HIV infected individuals. Neutralizing antibodies were detected in all sera. Most importantly, sera from animals immunized with gp70 had a higher neutralizing activity when compared with the sera from animals immunized with p15E and sera from animals immunized with gp70 together with p15E had a higher neutralizing activity compared with sera from animals immunized with each antigen alone. These immunization studies are important for the development of vaccines against other retroviruses including the human immunodeficiency virus HIV-1.

  10. DNA immunization as a technology platform for monoclonal antibody induction. (United States)

    Liu, Shuying; Wang, Shixia; Lu, Shan


    To combat the threat of many emerging infectious diseases, DNA immunization offers a unique and powerful approach to the production of high-quality monoclonal antibodies (mAbs) against various pathogens. Compared with traditional protein-based immunization approaches, DNA immunization is efficient for testing novel immunogen designs, does not require the production or purification of proteins from a pathogen or the use of recombinant protein technology and is effective at generating mAbs against conformation-sensitive targets. Although significant progress in the use of DNA immunization to generate mAbs has been made over the last two decades, the literature does not contain an updated summary of this experience. The current review provides a comprehensive analysis of the literature, including our own work, describing the use of DNA immunization to produce highly functional mAbs, in particular, those against emerging infectious diseases. Critical factors such as immunogen design, delivery approach, immunization schedule, use of immune modulators and the role of final boost immunization are discussed in detail.

  11. Immune TB Antibody Phage Display Library as a Tool To Study B Cell Immunity in TB Infections. (United States)

    Hamidon, Nurul Hamizah; Suraiya, Siti; Sarmiento, Maria E; Acosta, Armando; Norazmi, Mohd Nor; Lim, Theam Soon


    B cells and in particular antibodies has always played second fiddle to cellular immunity in regard to tuberculosis (TB). However, recent studies has helped position humoral immunity especially antibodies back into the foray in relation to TB immunity. Therefore, the ability to correlate the natural antibody responses of infected individuals toward TB antigens would help strengthen this concept. Phage display is an intriguing approach that can be utilized to study antibody-mediated responses against a particular infection via harvesting the B cell repertoire from infected individuals. The development of disease-specific antibody libraries or immune libraries is useful to better understand antibody-mediated immune responses against specific disease antigens. This study describes the generation of an immune single-chain variable fragment (scFv) library derived from TB-infected individuals. The immune library with an estimated diversity of 10(9) independent clones was then applied for the identification of monoclonal antibodies against Mycobacterium tuberculosis α-crystalline as a model antigen. Biopanning of the library isolated three monoclonal antibodies with unique gene usage. This strengthens the role of antibodies in TB immunity in addition to the role played by cellular immunity. The developed library can be applied against other TB antigens and aid antibody-derived TB immunity studies in the future.

  12. Novel mutations in Marburg virus glycoprotein associated with viral evasion from antibody mediated immune pressure. (United States)

    Kajihara, Masahiro; Nakayama, Eri; Marzi, Andrea; Igarashi, Manabu; Feldmann, Heinz; Takada, Ayato


    Marburg virus (MARV) and Ebola virus, members of the family Filoviridae, cause lethal haemorrhagic fever in humans and non-human primates. Although the outbreaks are concentrated mainly in Central Africa, these viruses are potential agents of imported infectious diseases and bioterrorism in non-African countries. Recent studies demonstrated that non-human primates passively immunized with virus-specific antibodies were successfully protected against fatal filovirus infection, highlighting the important role of antibodies in protective immunity for this disease. However, the mechanisms underlying potential evasion from antibody mediated immune pressure are not well understood. To analyse possible mutations involved in immune evasion in the MARV envelope glycoprotein (GP) which is the major target of protective antibodies, we selected escape mutants of recombinant vesicular stomatitis virus (rVSV) expressing MARV GP (rVSVΔG/MARVGP) by using two GP-specific mAbs, AGP127-8 and MGP72-17, which have been previously shown to inhibit MARV budding. Interestingly, several rVSVΔG/MARVGP variants escaping from the mAb pressure-acquired amino acid substitutions in the furin-cleavage site rather than in the mAb-specific epitopes, suggesting that these epitopes are recessed, not exposed on the uncleaved GP molecule, and therefore inaccessible to the mAbs. More surprisingly, some variants escaping mAb MGP72-17 lacked a large proportion of the mucin-like region of GP, indicating that these mutants efficiently escaped the selective pressure by deleting the mucin-like region including the mAb-specific epitope. Our data demonstrate that MARV GP possesses the potential to evade antibody mediated immune pressure due to extraordinary structural flexibility and variability.

  13. Sperm-immobilizing monoclonal antibody to human seminal plasma antigens. (United States)

    Shigeta, M; Watanabe, T; Maruyama, S; Koyama, K; Isojima, S


    Rat spleen cells immunized to human azoospermic semen (a mixture of seminal plasma components) and mouse myeloma cells (P3/X63 Ag8U1; P3U1) (Marguilies et al., 1976) were successfully fused with polyethylene glycol (PEG 1500) and 19 of 89 fused cell cultures were found to produce sperm-immobilizing antibody. The cells that produced antibody indicating the highest sperm-immobilizing activity were distributed into wells for further recloning and 10 clones producing sperm-immobilizing antibody were established. The clone (1C4) producing the highest antibody titre was found to produce a large amount of IgG in culture supernatants and to contain a mixture of rat and mouse chromosomes. It was proved by immunodiffusion test that the monoclonal antibody was produced to the human seminal plasma antigen No. 7 which is common to human milk protein. Using this hybridoma which produced a large amount of monoclonal sperm-immobilizing antibody, a new method could be developed for purifying human seminal plasma antigen by immunoaffinity chromatography with bound antibody from the hybridoma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6783353

  14. Differing rates of antibody acquisition to merozoite antigens in malaria: implications for immunity and surveillance. (United States)

    McCallum, Fiona J; Persson, Kristina E M; Fowkes, Freya J I; Reiling, Linda; Mugyenyi, Cleopatra K; Richards, Jack S; Simpson, Julie A; Williams, Thomas N; Gilson, Paul R; Hodder, Anthony N; Sanders, Paul R; Anders, Robin F; Narum, David L; Chitnis, Chetan; Crabb, Brendan S; Marsh, Kevin; Beeson, James G


    Antibodies play a key role in acquired human immunity to Plasmodium falciparum (Pf) malaria and target merozoites to reduce or prevent blood-stage replication and the development of disease. Merozoites present a complex array of antigens to the immune system, and currently, there is only a partial understanding of the targets of protective antibodies and how responses to different antigens are acquired and boosted. We hypothesized that there would be differences in the rate of acquisition of antibodies to different antigens and how well they are boosted by infection, which impacts the acquisition of immunity. We examined responses to a range of merozoite antigens in 2 different cohorts of children and adults with different age structures and levels of malaria exposure. Overall, antibodies were associated with age, exposure, and active infection, and the repertoire of responses increased with age and active infection. However, rates of antibody acquisition varied between antigens and different regions within an antigen following exposure to malaria, supporting our hypothesis. Antigen-specific responses could be broadly classified into early response types in which antibodies were acquired early in childhood exposure and late response types that appear to require substantially more exposure for the development of substantial levels. We identified antigen-specific responses that were effectively boosted after recent infection, whereas other responses were not. These findings advance our understanding of the acquisition of human immunity to malaria and are relevant to the development of malaria vaccines targeting merozoite antigens and the selection of antigens for use in malaria surveillance. © Society for Leukocyte Biology.

  15. CD44 antibodies and immune thrombocytopenia in the amelioration of murine inflammatory arthritis.

    Directory of Open Access Journals (Sweden)

    Patrick J Mott

    Full Text Available Antibodies to CD44 have been used to successfully ameliorate murine models of autoimmune disease. The most often studied disease model has been murine inflammatory arthritis, where a clear mechanism for the efficacy of CD44 antibodies has not been established. We have recently shown in a murine passive-model of the autoimmune disease immune thrombocytopenia (ITP that some CD44 antibodies themselves can induce thrombocytopenia in mice, and the CD44 antibody causing the most severe thrombocytopenia (IM7, also is known to be highly effective in ameliorating murine models of arthritis. Recent work in the K/BxN serum-induced model of arthritis demonstrated that antibody-induced thrombocytopenia reduced arthritis, causing us to question whether CD44 antibodies might primarily ameliorate arthritis through their thrombocytopenic effect. We evaluated IM7, IRAWB14.4, 5035-41.1D, KM201, KM114, and KM81, and found that while all could induce thrombocytopenia, the degree of protection against serum-induced arthritis was not closely related to the length or severity of the thrombocytopenia. CD44 antibody treatment was also able to reverse established inflammation, while thrombocytopenia induced by an anti-platelet antibody targeting the GPIIbIIIa platelet antigen, could not mediate this effect. While CD44 antibody-induced thrombocytopenia may contribute to some of its therapeutic effect against the initiation of arthritis, for established disease there are likely other mechanisms contributing to its efficacy. Humans are not known to express CD44 on platelets, and are therefore unlikely to develop thrombocytopenia after CD44 antibody treatment. An understanding of the relationship between arthritis, thrombocytopenia, and CD44 antibody treatment remains critical for continued development of CD44 antibody therapeutics.

  16. Molecular Pathways: Immune Checkpoint Antibodies and their Toxicities. (United States)

    Cousin, Sophie; Italiano, Antoine


    The emergence of immune checkpoint inhibitors for solid tumor treatments represents a major oncologic advance. Since the approval of ipilimumab, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) antibody, for the treatment of metastatic melanoma, many drugs, especially those targeting PD-1/PD-L1, have demonstrated promising antitumor effects in many types of cancer. By reactivating the immune system, these immunotherapies have led to the development of new toxicity profiles, also called immune-related adverse events (irAE). IrAEs can involve many organ systems, and their management is radically different from that of cytotoxic drugs; irAEs require immunosuppressive treatments, such as corticoids or TNFα antibody. In addition, the occurrence of irAEs has raised significant questions. Here, we summarize progress that has been made toward answering these questions, focusing on (i) the impact of immunotherapy dose on irAE occurrence, (ii) the correlation between irAE and patient outcome, (iii) the safety of immune checkpoint inhibitors in patients already treated for autoimmune disease, and (iv) the suspected effect on tumor growth of steroids used for the management of irAEs. Clin Cancer Res; 22(18); 4550-5. ©2016 AACR.

  17. Immunity to rhabdoviruses in rainbow trout: the antibody response

    DEFF Research Database (Denmark)

    Lorenzen, Niels; Lapatra, S.E.


    Interactions between host and pathogen, as in the case of fish pathogenic viruses, represent interesting models for analyses of the relationships between structure and function of the teleost immune system. Two salmonid rhabdoviruses, IHNV and VHSV, have received special attention due to their oc......Interactions between host and pathogen, as in the case of fish pathogenic viruses, represent interesting models for analyses of the relationships between structure and function of the teleost immune system. Two salmonid rhabdoviruses, IHNV and VHSV, have received special attention due...... to their occasional detrimental effect on rainbow trout farming. Research efforts have been focused on understanding the mechanisms involved in protective immunity. Several specific and nonspecific cellular and humoral parameters are believed to be involved, but only the antibody response has been characterised...

  18. Lactogenic immunity in transgenic mice producing recombinant antibodies neutralizing coronavirus. (United States)

    Castilla, J; Sola, I; Pintado, B; Sánchez-Morgado, J M; Enjuanes, L


    Protection against coronavirus infections can be provided by the oral administration of virus neutralizing antibodies. To provide lactogenic immunity, eighteen lines of transgenic mice secreting a recombinant IgG1 monoclonal antibody (rIgG1) and ten lines of transgenic mice secreting recombinant IgA monoclonal antibodies (rIgA) neutralizing transmissible gastroenteritis coronavirus (TGEV) into the milk were generated. Genes encoding the light and heavy chains of monoclonal antibody (MAb) 6A.C3 were expressed under the control of regulatory sequences derived from the mouse genomic DNA encoding the whey acidic protein (WAP) and beta-lactoglobulin (BLG), which are highly abundant milk proteins. The MAb 6A.C3 binds to a highly conserved epitope present in coronaviruses of several species. This MAb does not allow the selection of neutralization escaping virus mutants. The antibody was expressed in the milk of transgenic mice with titers of one million as determined by RIA, and neutralized TGEV infectivity by one million fold corresponding to immunoglobulin concentrations of 5 to 6 mg per ml. Matrix attachment regions (MAR) sequences were not essential for rIgG1 transgene expression, but co-microinjection of MAR and antibody genes led to a twenty to ten thousand-fold increase in the antibody titer in 50% of the rIgG1 transgenic animals generated. Co-microinjection of the genomic BLG gene with rIgA light and heavy chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and BLG genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of BLG co-integration. Antibody expression levels were transgene copy number independent and integration site dependent. The generation of transgenic animals producing virus neutralizing antibodies in the milk could be a general approach to provide protection

  19. A Potential of an Anti-HTLV-I gp46 Neutralizing Monoclonal Antibody (LAT-27 for Passive Immunization against Both Horizontal and Mother-to-Child Vertical Infection with Human T Cell Leukemia Virus Type-I

    Directory of Open Access Journals (Sweden)

    Hideki Fujii


    Full Text Available Although the number of human T-cell leukemia virus type-I (HTLV-I-infected individuals in the world has been estimated at over 10 million, no prophylaxis vaccines against HTLV-I infection are available. In this study, we took a new approach for establishing the basis of protective vaccines against HTLV-I. We show here the potential of a passively administered HTLV-I neutralizing monoclonal antibody of rat origin (LAT-27 that recognizes epitopes consisting of the HTLV-I gp46 amino acids 191–196. LAT-27 completely blocked HTLV-I infection in vitro at a minimum concentration of 5 μg/mL. Neonatal rats born to mother rats pre-infused with LAT-27 were shown to have acquired a large quantity of LAT-27, and these newborns showed complete resistance against intraperitoneal infection with HTLV-I. On the other hand, when humanized immunodeficient mice were pre-infused intravenously with humanized LAT-27 (hu-LAT-27, all the mice completely resisted HTLV-I infection. These results indicate that hu-LAT-27 may have a potential for passive immunization against both horizontal and mother-to-child vertical infection with HTLV-I.

  20. Amelioration of murine immune thrombocytopenia by CD44 antibodies: a potential therapy for ITP? (United States)

    Crow, Andrew R; Song, Seng; Suppa, Sara J; Ma, Shuhua; Reilly, Michael P; Andre, Pierrette; McKenzie, Steven E; Lazarus, Alan H


    To explore the potential for monoclonal antibodies as a treatment for immune thrombocytopenia (ITP) and to further explore their mechanisms of action, we tested 8 monoclonal CD44 antibodies in murine ITP and found 4 antibodies that could successfully ameliorate ITP; 2 of these antibodies function at a full 3-log fold lower dosage compared with IVIg. Further characterization of the 2 most successful antibodies (5035-41.1D and KM114) demonstrated that, similar to IVIg: (1) the presence of the inhibitory IgG receptor FcγRIIB was required for their ameliorative function, (2) complement-deficient mice responded to anti-CD44 treatment, and (3) human transgenic FcγRIIA-expressing mice also responded to the CD44 therapeutic modality. Dissimilar to IVIg, the Fc portion of the CD44 antibody was not required. These data demonstrate that CD44 antibodies can function therapeutically in murine ITP and that they could potentially provide a very-low-dose recombinant therapy for the amelioration of human ITP.

  1. Immunity to rhabdoviruses in rainbow trout: the antibody response

    DEFF Research Database (Denmark)

    Lorenzen, Niels; Lapatra, S.E.


    Interactions between host and pathogen, as in the case of fish pathogenic viruses, represent interesting models for analyses of the relationships between structure and function of the teleost immune system. Two salmonid rhabdoviruses, IHNV and VHSV, have received special attention due to their oc......Interactions between host and pathogen, as in the case of fish pathogenic viruses, represent interesting models for analyses of the relationships between structure and function of the teleost immune system. Two salmonid rhabdoviruses, IHNV and VHSV, have received special attention due...... in detail so far. Analysis of the specificity of anti-virus trout antibodies has been complicated by a generally insufficient ability of the antibodies to bind the viral proteins in assays such as immunoblotting. However, other assays, specifically designed for detection of fish anti IHNV/VHSV antibodies...... by different kinds of epitopes being differently immunogenic in fish and in mammals. Also, it may be assumed that the requirements for the assay-antigens in terms of antigenicity, may differ for mammals and fish. The present text includes an initial presentation of the pathogens, followed by basic and applied...

  2. A cross-reactive monoclonal antibody to nematode haemoglobin enhances protective immune responses to Nippostrongylus brasiliensis.

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    Natalie E Nieuwenhuizen

    Full Text Available BACKGROUND: Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection. METHODOLOGY/PRINCIPAL FINDINGS: Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four -HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens. CONCLUSION: The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity

  3. Dromedary immune response and specific Kv2.1 antibody generation using a specific immunization approach. (United States)

    Hassiki, Rym; Labro, Alain J; Benlasfar, Zakaria; Vincke, Cécile; Somia, Mahmoud; El Ayeb, Mohamed; Muyldermans, Serge; Snyders, Dirk J; Bouhaouala-Zahar, Balkiss


    Voltage-gated potassium (Kv) channels form cells repolarizing power and are commonly expressed in excitable cells. In non-excitable cells, Kv channels such as Kv2.1 are involved in cell differentiation and growth. Due to the involvement of Kv2.1 in several physiological processes, these channels are promising therapeutic targets. To develop Kv2.1 specific antibody-based channel modulators, we applied a novel approach and immunized a dromedary with heterologous Ltk- cells that overexpress the mouse Kv2.1 channel instead of immunizing with channel protein fragments. The advantage of this approach is that the channel is presented in its native tetrameric configuration. Using a Cell-ELISA, we demonstrated the ability of the immune serum to detect Kv2.1 channels on the surface of cells that express the channel. Then, using a Patch Clamp electrophysiology assay we explored the capability of the dromedary serum in modulating Kv2.1 currents. Cells that were incubated for 3h with serum taken at Day 51 from the start of the immunization displayed a statistically significant 2-fold reduction in current density compared to control conditions as well as cells incubated with serum from Day 0. Here we show that an immunization approach with cells overexpressing the Kv2.1 channel yields immune serum with Kv2.1 specific antibodies.

  4. Pathogenic role of modified LDL antibodies and immune complexes in atherosclerosis. (United States)

    Lopes-Virella, Maria F; Virella, Gabriel


    There is strong evidence supporting a key role of the adaptive immune response in atherosclerosis, given that both activated Th cells producing predominantly interferon-γ and oxidized LDL (oxLDL) and the corresponding antibodies have been isolated from atheromatous plaques. Studies carried out using immune complexes (IC) prepared with human LDL and rabbit antibodies have demonstrated proatherogenic and pro-inflammatory properties, mostly dependent on the engagement of Fcγ receptors Ⅰ and Ⅱ in macrophages and macrophage-like cell lines. Following the development of a methodology for isolating modified LDL (mLDL) antibodies from serum and isolated IC, it was confirmed that antibodies reacting with oxLDL and advanced glycation end product-modified LDL are predominantly IgG of subtypes 1 and 3 and that mLDL IC prepared with human reagents possesses pro-inflammatory and proatherogenic properties. In previous studies, LDL separated from isolated IC has been analyzed for its modifications, and the reactivity of antibodies isolated from the same IC with different LDL modifications has been tested. Recently, we obtained strong evidence suggesting that the effects of mLDL IC on phagocytic cells are modulated by the composition of the mLDL. Clinical studies have shown that the level of mLDL in circulating IC is a strong predictor of cardiovascular disease (CVD) and, in diabetic patients, other significant complications, such as nephropathy and retinopathy. In conclusion, there is convincing ex vivo and clinical data supporting the hypothesis that, in humans, the humoral immune response to mLDL is pathogenic rather than protective.

  5. Intranasal Immunization of Mice to Avoid Interference of Maternal Antibody against H5N1 Infection.

    Directory of Open Access Journals (Sweden)

    Fenghua Zhang

    Full Text Available Maternally-derived antibodies (MDAs can protect offspring against influenza virus infection but may also inhibit active immune responses. To overcome MDA- mediated inhibition, active immunization of offspring with an inactivated H5N1 whole-virion vaccine under the influence of MDAs was explored in mice. Female mice were vaccinated twice via the intraperitoneal (IP or intranasal (IN route with the vaccine prior to mating. One week after birth, the offspring were immunized twice via the IP or IN route with the same vaccine and then challenged with a lethal dose of a highly homologous virus strain. The results showed that, no matter which immunization route (IP or IN was used for mothers, the presence of MDAs severely interfered with the active immune response of the offspring when the offspring were immunized via the IP route. Only via the IN immunization route did the offspring overcome the MDA interference. These results suggest that intranasal immunization could be a suitable inoculation route for offspring to overcome MDA interference in the defense against highly pathogenic H5N1 virus infection. This study may provide references for human and animal vaccination to overcome MDA-induced inhibition.

  6. Antibody and immune memory persistence post infant hepatitis B vaccination

    Directory of Open Access Journals (Sweden)

    Hudu SA


    Full Text Available Shuaibu A Hudu,1,2 Yasmin A Malik,3 Mohd Taib Niazlin,1 Nabil S Harmal,1,4 Ariza Adnan,5 Ahmed S Alshrari,1 Zamberi Sekawi1 1Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Malaysia; 2Department of Pathology and Medical Microbiology, College of Health Sciences, Usmanu Danfodiyo University Sokoto, Sokoto State, Nigeria; 3Department of Clinical Science, Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Selangor, Malaysia; 4Department of Medical Microbiology, Faculty of Medicine and Health Sciences, Sana'a University, Sana'a, Yemen; 5Cluster of Laboratory Medical Sciences, Faculty of Medicine Universiti Teknologi MARA, Sungai Buloh, Selangor, Malaysia Objectives: This study aimed to evaluate the level of hepatitis B immunity among undergraduate students 23 years after commencement of the nationwide hepatitis B childhood immunization program in Malaysia. Methods: A total of 402 serum samples obtained from volunteer undergraduate students were screened for the presence of hepatitis B surface antibodies using qualitative ELISA. Results: Results showed that 62.7% of volunteers had protective anti-hepatitis B surface antigens (≥10 IU/L, of whom 67.9% received three doses of the vaccine. The estimated post-vaccination immunity was found to be at least 20 years, indicating persistent immunity against hepatitis B and a significant association (P < 0.05 with duration of vaccination. Anamnestic response 1 month post-hepatitis B booster was 94.0% and highly significant (P < 0.01. Isolated anti-hepatitis B core antigen (anti-HBc prevalence was found to be 5.0%, all having had a positive anamnestic response. Conclusion: Immunity after primary vaccination with hepatitis B recombinant vaccine persists for at least 20 years post-vaccination, with significant association with the number of vaccinations. Furthermore, the presence of anamnestic response to

  7. Generation of human antibody fragments against Streptococcus mutans using a phage display chain shuffling approach

    Directory of Open Access Journals (Sweden)

    Barth Stefan


    Full Text Available Abstract Background Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials. Results The antibody derivatives were generated using a chain-shuffling approach based on human antibody variable gene phage-display libraries. Like the parent antibody, these derivatives bound specifically to SAI/II, the surface adhesin of the oral pathogen S. mutans. Conclusions Humanization of murine antibodies can be easily achieved using phage display libraries. The human antibody fragments bind the antigen as well as the causative agent of dental caries. In addition the human diabody derivative is capable of aggregating S. mutans in vitro, making it a useful candidate passive immunotherapeutic agent for oral diseases.


    Institute of Scientific and Technical Information of China (English)

    MALan; CAOXiao-Mei; BENKun-Long; CHENYun-Liang


    Clinical and experimental investigations have shown that secretory IgA antibodies to human sperm may bc one of the most important factors in immunological infertility. Studies on the effects of these antibodies on sperm function arc helpful to understand the role of

  9. Identification of anti-CD98 antibody mimotopes for inducing antibodies with antitumor activity by mimotope immunization. (United States)

    Saito, Misa; Kondo, Masahiro; Ohshima, Motohiro; Deguchi, Kazuki; Hayashi, Hideki; Inoue, Kazuyuki; Tsuji, Daiki; Masuko, Takashi; Itoh, Kunihiko


    A mimotope is an antibody-epitope-mimicking peptide retrieved from a phage display random peptide library. Immunization with antitumor antibody-derived mimotopes is promising for inducing antitumor immunity in hosts. In this study, we isolated linear and constrained mimotopes from HBJ127, a tumor-suppressing anti-CD98 heavy chain mAb, and determined their abilities for induction of antitumor activity equal to that of the parent antibody. We detected elevated levels of antipeptide responses, but failed to detect reactivity against native CD98-expressing HeLa cells in sera of immunized mice. Phage display panning and selection of mimotope-immunized mouse spleen-derived antibody Fab library showed that HeLa cell-reactive Fabs were successfully retrieved from the library. This finding indicates that native antigen-reactive Fab clones represented an undetectable minor population in mimotope-induced antibody repertoire. Functional and structural analysis of retrieved Fab clones revealed that they were almost identical to the parent antibody. From these results, we confirmed that mimotope immunization was promising for retrieving antitumor antibodies equivalent to the parent antibody, although the co-administration of adjuvant compounds such as T-cell epitope peptides and Toll-like receptor 4 agonist peptides is likely to be necessary for inducing stronger antitumor immunity than mimotope injection alone.

  10. Nasal Immunization Confers High Avidity Neutralizing Antibody Response and Immunity to Primary and Recurrent Genital Herpes in Guinea Pigs (United States)

    Persson, Josefine; Zhang, Yuan; Olafsdottir, Thorunn A.; Thörn, Karolina; Cairns, Tina M.; Wegmann, Frank; Sattentau, Quentin J.; Eisenberg, Roselyn J.; Cohen, Gary H.; Harandi, Ali M.


    Genital herpes is one of the most prevalent sexually transmitted infections in both the developing and developed world. Following infection, individuals experience life-long latency associated with sporadic ulcerative outbreaks. Despite many efforts, no vaccine has yet been licensed for human use. Herein, we demonstrated that nasal immunization with an adjuvanted HSV-2 gD envelope protein mounts significant protection to primary infection as well as the establishment of latency and recurrent genital herpes in guinea pigs. Nasal immunization was shown to elicit specific T cell proliferative and IFN-γ responses as well as systemic and vaginal gD-specific IgG antibody (Ab) responses. Furthermore, systemic IgG Abs displayed potent HSV-2 neutralizing properties and high avidity. By employing a competitive surface plasmon resonance (SPR) analysis combined with a battery of known gD-specific neutralizing monoclonal Abs (MAbs), we showed that nasal immunization generated IgG Abs directed to two major discontinuous neutralizing epitopes of gD. These results highlight the potential of nasal immunization with an adjuvanted HSV-2 envelope protein for induction of protective immunity to primary and recurrent genital herpes. PMID:28082979

  11. Influence of the oncolytic parvovirus H-1, CTLA-4 antibody tremelimumab and cytostatic drugs on the human immune system in a human in vitro model of colorectal cancer cells

    Directory of Open Access Journals (Sweden)

    Heinrich B


    Full Text Available Bernd Heinrich,* Katrin Goepfert,* Maike Delic, Peter R Galle, Markus MoehlerUniversity Medical Center of the Johannes Gutenberg University Mainz, 1st Department of Internal Medicine, Langenbeckstrasse, Mainz, Germany *These authors contributed equally to this workIntroduction: Tumor-directed and immune-system-stimulating therapies are of special interest in cancer treatment. Here, we demonstrate the potential of parvovirus H-1 (H-1PV to efficiently kill colorectal cancer cells and induce immunogenicity of colorectal tumors by inducing maturation of dendritic cells (DCs alone and also in combination with cytostatic drugs in vitro. Using our cell culture model, we have additionally investigated the effects of anti-CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4 receptor antibody tremelimumab on this process.Materials and methods: Colon carcinoma cell lines were treated with different concentrations of cytostatic drugs or tremelimumab or were infected with H-1PV in different multiplicities of infection (MOIs, and viability was determined using MTT assays. Expression of CTLA-4 in colon carcinoma cell lines was measured by FACScan™. For the coculture model, we isolated monocytes using adherence, and differentiation into immature DCs (iDCs was stimulated using interleukin-4 and granulocyte-macrophage colony-stimulating factor. Maturation of iDCs into mature DCs (mDCs was induced by a cytokine cocktail. SW480 colon carcinoma cells were infected with H-1PV or treated with cytostatic drugs. Drug treated and H-1PV-infected SW480 colon carcinoma cells were cocultured with iDCs and expression of maturation markers was measured using FACScan™. Cytokine measurements were performed using enzyme-linked immunosorbent assay.Results: Colon carcinoma cells SW480 were potently infected and killed by H-1PV. CTLA-4 expression in SW480 cells increased after infection with H-1PV and also after treatment with cytostatic drugs. Tremelimumab had no influence on

  12. Loss of Rubella Antibody from Immune Globulin Treated with Kaolin (United States)

    Cabasso, V. J.; Louie, R. E.; Hok, K. A.; Robinson, C. T.; Davis, P. C.; Miner, R. C.


    Sera and immune globulin (IG) preparations are customarily treated with kaolin before titration of their rubella hemagglutination-inhibiting (HI) antibody in order to rid them of nonspecific inhibitors of hemagglutination. The treatment was shown in this investigation to have no adverse effect on the antibody level of the sera but was found to remove considerable amounts of gamma-globulin from IG preparations. Evidence of this removal was obtained by serological tests, by spectrophotometric determination of protein concentration and by disc electrophoresis. In contrast to kaolin, heparin-manganese chloride (MnCl2) treatment of IG preparations had essentially no effect on the level of antibody globulin by all the criteria used. Heparin-MnCl2-treated IG lots were in these respects similar, if not identical, to their untreated counterparts. Since nonspecific inhibitors associated with the β-lipoprotein fraction of serum are removed by the method employed to fractionate the IG samples, it seems unnecessary to treat the latter in any way for the HI test. No difficulty was encountered in this investigation with several untreated IG lots. Images PMID:4190525

  13. Consumption of purple sweet potato leaves modulates human immune response: T-lymphocyte functions, lytic activity of natural killer cell and antibody production

    Institute of Scientific and Technical Information of China (English)

    Chiao-Ming Chen; Sing-Chung Li; Ya-Ling Lin; Ching-Yun Hsu; Ming-Jer Shieh; Jen-Fang Liu


    AIM: To study the immunological effects of physiological doses of purple sweet potato leaves (PSPL).METHODS: The randomized crossover study (two periods,each lasting for 2 wk) involved 16 healthy non-smoking adults of normal weight. The 6-wk study consisted of a run-in (wk 1) PSPL diet (daily consumption of 200 g PSPL) or a control diet (low polyphenols, with the amount of carotenoids adjusted to the same level as that of PSPL) (wk 2-3), washout diet (wk 4), and switched diet (wk 5-6). Fasting blood was collected weekly in the morning. T-lymphocyte function was assessed via the proliferation and secretion of immunoreactive cytokines.Salivary IgA secretion and the specific cytotoxic activities of cytotoxic T lymphocytes and natural killer (NK) cells were determined.RESULTS: The plasma β-carotene level increased with time in both groups, while the plasma polyphenol level decreased in the control group, and no significant difference was detected between the two groups.Although plasma polyphenol levels did not significantly increase in the PSPL group at the end of the study, they were significantly elevated in urine. PSPL consumption produced a significant increase in proliferation responsiveness of peripheral blood mononuclear cells (PBMC) and their secretion of immunoreactive IL-2 and IL-4. As well, lytic activity in NK cells was elevated in a time-dependent fashion. Salivary TgA secretion significantly decreased in control group after 2 wk, and returned to baseline following dietary switch to PSPL.CONCLUSION: Consumption of PSPL modulates various immune functions including increased proliferation responsiveness of PBMC, secretion of cytokines IL-2 and IL-4, and the lytic activity of NK cells. The responsible determinants of PSPL remain to be elucidated, as does the biological significance of the present observations.

  14. Antisperm antibodies and human reproduction. (United States)

    Check, J H


    To present strategies in diagnosing and treating infertility related to antisperm antibodies. Antisperm antibodies (ASA) were detected on sperm using the direct immunobead (IBD) test. Treatments included intrauterine insemination (IUI) with pretreatment with chymotrypsin/galactose vs. in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI). Intrauterine insemination with protein digestive enzyme treatment was much more effective than IUI without enzymatic therapy. However IVF with ICSI provided three times the pregnancy rate for males with sperm coated with ASA than IUI with chymotrypsin treated sperm. It is advisable to include measurement for ASA on the initial semen analysis. However, another option is to perform it initially only with an abnormal post-coital test. The decision for IUI with chymotrypsin pretreatment of the sperm vs. IVF with ICSI may depend on insurance and financial issues.

  15. Synthetic immunology: modulating the human immune system. (United States)

    Geering, Barbara; Fussenegger, Martin


    Humans have manipulated the immune system to dampen or boost the immune response for thousands of years. As our understanding of fundamental immunology and biotechnological methodology accumulates, we can capitalize on this combined knowledge to engineer biological devices with the aim of rationally manipulating the immune response. We address therapeutic approaches based on the principles of synthetic immunology that either ameliorate disorders of the immune system by interfering with the immune response, or improve diverse pathogenic conditions by exploiting immune cell effector functions. We specifically highlight synthetic proteins investigated in preclinical and clinical trials, summarize studies that have used engineered immune cells, and finish with a discussion of possible future therapeutic concepts.

  16. Immunotherapy of melanoma with the immune costimulatory monoclonal antibodies targeting CD137

    Directory of Open Access Journals (Sweden)

    Li SY


    Full Text Available Shi-Yan Li, Yizhen Liu Cancer Research Institute, Scott and White Healthcare, Temple, TX, USA Abstract: Knowledge of how the immune system recognizes and attempts to control cancer growth and development has improved dramatically. The advent of immunotherapies for cancer has resulted in robust clinical responses and confirmed that the immune system can significantly inhibit tumor progression. Until recently, metastatic melanoma was a disease with limited treatment options and a poor prognosis. CD137 (also known as 4-1BB a member of the tumor necrosis factor (TNF receptor superfamily, is an activation-induced T cell costimulator molecule. Growing evidence indicates that anti-CD137 monoclonal antibodies possess strong antitumor properties, the result of their powerful capability to activate CD8+ T cells, to produce interferon (IFN-γ, and to induce cytolytic markers. Combination therapy of anti-CD137 with other anticancer agents, such as radiation, has robust tumor-regressing abilities against nonimmunogenic or poorly immunogenic tumors. Of importance, targeting CD137 eliminates established tumors, and the fact that anti-CD137 therapy acts in concert with other anticancer agents and/or radiation therapy to eradicate nonimmunogenic and weakly immunogenic tumors is an additional benefit. Currently, BMS-663513, a humanized anti-CD137 antibody, is in clinical trials in patients with solid tumors, including melanoma, renal carcinoma, ovarian cancer, and B-cell malignancies. In this review, we discuss the basis of the therapeutic potential of targeting CD137 in cancer treatment, focusing in particular, on BMS-663513 as an immune costimulatory monoclonal antibody for melanoma immunotherapy. Keywords: anti-CD137 monoclonal antibodies, immune costimulator molecule, BMS-663513

  17. V(H)3 antibody response to immunization with pneumococcal polysaccharide vaccine in middle-aged and elderly persons. (United States)

    Serpa, Jose A; Valayam, Josemon; Musher, Daniel M; Rossen, Roger D; Pirofski, Liise-anne; Rodriguez-Barradas, Maria C


    Pneumococcal disease continues to cause substantial morbidity and mortality among the elderly. Older adults may have high levels of anticapsular antibody after vaccination, but their antibodies show decreased functional activity. In addition, the protective effect of the pneumococcal polysaccharide vaccine (PPV) seems to cease as early as 3 to 5 years postvaccination. Recently, it was suggested that PPV elicits human antibodies that use predominantly V(H)3 gene segments and induce a repertoire shift with increased V(H)3 expression in peripheral B cells. Here we compared V(H)3-idiotypic antibody responses in middle-aged and elderly subjects receiving PPV as initial immunization or revaccination. We studied pre- and postvaccination sera from 36 (18 vaccine-naïve and 18 previously immunized subjects) middle-aged and 40 (22 vaccine-naïve and 18 previously immunized subjects) elderly adults who received 23-valent PPV. Concentrations of IgGs to four individual serotypes (6B, 14, 19F, and 23F) and of V(H)3-idiotypic antibodies (detected by the monoclonal antibody D12) to the whole pneumococcal vaccine were determined by enzyme-linked immunosorbent assay (ELISA). PPV elicited significant IgG and V(H)3-idiotypic antibody responses in middle-aged and elderly subjects, regardless of whether they were vaccine naïve or undergoing revaccination. Age did not influence the magnitude of the antibody responses, as evidenced by similar postvaccination IgG and V(H)3 antibody levels in both groups, even after stratifying by prior vaccine status. Furthermore, we found similar proportions (around 50%) of elderly and middle-aged subjects experiencing 2-fold increases in V(H)3 antibody titers after vaccination. Age or repeated immunization does not appear to affect the V(H)3-idiotypic immunogenicity of PPV among middle-aged and elderly adults.

  18. Broad epitope coverage of a human in vitro antibody library (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


    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

  19. Probing cocaine-antibody interactions in buffer and human serum.

    Directory of Open Access Journals (Sweden)

    Muthu Ramakrishnan

    Full Text Available BACKGROUND: Despite progress in cocaine immunotherapy, the kinetic and thermodynamic properties of antibodies which bind to cocaine and its metabolites are not well understood. It is also not clear how the interactions between them differ in a complex matrix such as the serum present in the human body. In the present study, we have used microscale thermophoresis (MST, isothermal titration calorimetry (ITC, and surface plasmon resonance (SPR we have evaluated the affinity properties of a representative mouse monoclonal (mAb08 as well as those of polyclonal antibodies purified from vaccinated mouse and human patient serum. RESULTS: MST analysis of fluorescently tagged mAb08 binding to cocaine reveals an approximately 15 fold decrease in its equilibrium dissociation constant in 20-50% human serum compared with that in saline buffer. A similar trend was also found using enriched polyclonal antibodies purified from vaccinated mice and patient serum, for which we have used fluorescently tagged bovine serum albumin conjugated to succinyl norcocaine (BSA-SNC. This conjugate closely mimics both cocaine and the hapten used to raise these antibodies. The ITC data also revealed that cocaine has a moderate affinity of about 2 µM to 20% human serum and very little interaction with human serum albumin or nonspecific human IgG at that concentration range. In a SPR inhibition experiment, the binding of mAb08 to immobilized BSA-SNC was inhibited by cocaine and benzoylecgonine in a highly competitive manner, whereas the purified polyclonal antibodies from vaccinated humans and mice, revealed preferential selectivity to pharmacologically active cocaine but not to the inactive metabolite benzoylecgonine. We have also developed a simple binding model to simulate the challenges associated with cocaine immunotherapy using the variable quantitative and kinetic properties of the antibodies. CONCLUSIONS: High sensitivity calorimetric determination of antibody binding to

  20. [Neutralizing Monoclonal and Chimeric Antibodies to Human IFN-γ]. (United States)

    Larina, M V; Aliev, T K; Solopova, O N; Pozdnyakova, L P; Korobova, S V; Yakimov, S A; Sveshnikov, P G; Dolgikh, D A; Kirpichnikov, M P


    Autoiminune disorders are chronic diseases characterized by abnormal immune response directed against self-antigens that leads to tissue damage and violation of its normal functioning. Such diseases often result in disability or even death of patients. Nowadays a number of monoclonal antibodies to pro-inflammatory cytokines and their receptors are successfully used for the targeted treatment of autoimmune diseases. One of the perspective targets in autoimmune disease therapy is interferon gamma, a key cytokine in Th1 cells differentiation, activation of macrophages, and inflammation. In the present work, 5 monoclonal antibodies to human IFN-γ were obtained. For the development of potential therapeutic agent, we have performed neutralizing activity and affinity analysis of the antibodies. Based on the data obtained, the monoclonal antibody F1 was selected. This antibody has a dissociation constant 1.7 x 10(-9) M and IC90 = 8.9 ± 2.0 nM measured upon antibody inhibition of the IFN-γ-induced HLA-DR expression on the surface of U937 cells. We have constructed a bicistronic vector for the production of recombinant chimeric Fab fragment F1 chim in E. coli cells. The recombinant chimeric Fab fragment Fl chim neutralizes IFN-γ activity in vitro and has a dissociation constant 1.8 x 10(-9) M.

  1. Secondary Mechanisms of Affinity Maturation in the Human Antibody Repertoire

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    Bryan S. Briney


    Full Text Available V(DJ recombination and somatic hypermutation (SHM are the primary mechanisms for diversification of the human antibody repertoire. These mechanisms allow for rapid humoral immune responses to a wide range of pathogenic challenges. V(DJ recombination efficiently generate a virtually limitless diversity through random recombination of variable (V, diversity (D and joining (J genes with diverse nontemplated junctions between the selected gene segments. Following antigen stimulation, affinity maturation by SHM produces antibodies with refined specificity mediated by mutations typically focused in complementarity determining regions (CDRs, which form the bulk of the antigen recognition site. While V(DJ recombination and SHM are responsible for much of the diversity of the antibody repertoire, there are several secondary mechanisms that, while less frequent, make substantial contributions to antibody diversity including V(DDJ recombination (or D-D fusion, somatic-hypermutation-associated insertions and deletions, and affinity maturation and antigen contact by non-CDR regions of the antibody. In addition to enhanced diversity, these mechanisms allow the production of antibodies that are critical to response to a variety of viral and bacterial pathogens but that would be difficult to generate using only the primary mechanisms of diversification.

  2. Addressing the Immunogenicity of the Cargo and of the Targeting Antibodies with a Focus on Demmunized Bacterial Toxins and on Antibody-Targeted Human Effector Proteins. (United States)

    Grinberg, Yehudit; Benhar, Itai


    Third-generation immunotoxins are composed of a human, or humanized, targeting moiety, usually a monoclonal antibody or an antibody fragment, and a non-human effector molecule. Due to the non-human origin of the cytotoxic domain, these molecules stimulate potent anti-drug immune responses, which limit treatment options. Efforts are made to deimmunize such immunotoxins or to combine treatment with immunosuppression. An alternative approach is using the so-called "human cytotoxic fusion proteins", in which antibodies are used to target human effector proteins. Here, we present three relevant approaches for reducing the immunogenicity of antibody-targeted protein therapeutics: (1) reducing the immunogenicity of the bacterial toxin, (2) fusing human cytokines to antibodies to generate immunocytokines and (3) addressing the immunogenicity of the targeting antibodies.

  3. Activation of human complement by immunoglobulin G antigranulocyte antibody. (United States)

    Rustagi, P K; Currie, M S; Logue, G L


    The ability of antigranulocyte antibody to fix the third component of complement (C3) to the granulocyte surface was investigated by an assay that quantitates the binding of monoclonal anti-C3 antibody to paraformaldehyde-fixed cells preincubated with Felty's syndrome serum in the presence of human complement. The sera from 7 of 13 patients with Felty's syndrome bound two to three times as much C3 to granulocytes as sera from patients with uncomplicated rheumatoid arthritis. The complement-activating ability of Felty's syndrome serum seemed to reside in the monomeric IgG-containing serum fraction. For those sera capable of activating complement, the amount of C3 fixed to granulocytes was proportional to the amount of granulocyte-binding IgG present in the serum. Thus, complement fixation appeared to be a consequence of the binding of antigranulocyte antibody to the cell surface. These studies suggest a role for complement-mediated injury in the pathophysiology of immune granulocytopenia, as has been demonstrated for immune hemolytic anemia and immune thrombocytopenia. PMID:7174786

  4. Broadly Neutralizing Antibodies against HIV-1 As a Novel Aspect of the Immune Response. (United States)

    Shcherbakov, D N; Bakulina, A Y; Karpenko, L I; Ilyichev, A A


    The human immunodeficiency virus-1 (HIV-1) has the ability to evade the adaptive immune response due to high mutation rates. Soon after the discovery of HIV-1, it was originally proposed that neutralizing of antibodies to the virus occurs rarely or cannot be elicited at all. In the 1990s, there appeared reports that sera of select HIV-1-infected individuals contained antibodies capable of neutralizing different virus subtypes. Such antibodies were named broadly neutralizing antibodies (bNAbs). Since 2009, the development of new cell technologies has intensified research efforts directed at identifying new bNAbs with a neutralization potency of over 90% of primary HIV-1 isolates. These antibodies have unique characteristics which include high levels of somatic mutations and unusually long variable loops that penetrate through the glycan shield of HIV-1 Env to contact the protein surface. In this review, we will attempt to summarize the latest data on bNAbs against HIV-1 in terms of their interactions with the sites of vulnerability on HIV-1 glycoproteins.

  5. Human cysticercosis: antigens, antibodies and non-responders. (United States)

    Flisser, A; Woodhouse, E; Larralde, C


    Immunoelectrophoresis of sera from patients with brain cysticercosis against a crude antigenic extract from Cysticercus cellulosae indicates that nearly 50% of the patients do not make sufficient antibodies to ostensively precipitate. The other 50% of the patients who do make precipitating antibodies show a very heterogeneous response in the number of antigens they recognize as well as in the type of antigen--as classified by their electrophoretic mobilities. The most favoured, called antigen B, is recognized by 84% of positive sera and corresponds to one or a limited number of antigens isoelectric at pH 8.6. Indirect immunofluorescence with monospecific anti-human immunoglobulins, performed upon the immunoelectrophoretic preparations, reveal that all cysticercus antigens induced the synthesis of antibodies in the immunoglobulin classes in the order G greater than M greater than E greater than A greater than D. Finally, antigen H (an anodic component) seems to favour IgE relative to its ability to induce IgG. Thus, although in natural infection a good proportion of cysticercotic patients do not seem to mount an energetic antibody response against the parasite, giving rise to some speculations about immunosuppression, the fact that 50% do synthesize antibodies allows for some optimistic expectations from vaccination of humans--in view of the good results of vaccination in experimental animals mediated by IgG antibodies. A likely prospect for a human vaccine would be antigen B because it is the most frequently detected by humans, although its immunizing and toxic properties remain to be properly studied. Images FIG. 1 FIG. 3 FIG. 6 PMID:7389197

  6. Fully human antagonistic antibodies against CCR4 potently inhibit cell signaling and chemotaxis.

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    Urs B Hagemann

    Full Text Available CC chemokine receptor 4 (CCR4 represents a potentially important target for cancer immunotherapy due to its expression on tumor infiltrating immune cells including regulatory T cells (Tregs and on tumor cells in several cancer types and its role in metastasis.Using phage display, human antibody library, affinity maturation and a cell-based antibody selection strategy, the antibody variants against human CCR4 were generated. These antibodies effectively competed with ligand binding, were able to block ligand-induced signaling and cell migration, and demonstrated efficient killing of CCR4-positive tumor cells via ADCC and phagocytosis. In a mouse model of human T-cell lymphoma, significant survival benefit was demonstrated for animals treated with the newly selected anti-CCR4 antibodies.For the first time, successful generation of anti- G-protein coupled chemokine receptor (GPCR antibodies using human non-immune library and phage display on GPCR-expressing cells was demonstrated. The generated anti-CCR4 antibodies possess a dual mode of action (inhibition of ligand-induced signaling and antibody-directed tumor cell killing. The data demonstrate that the anti-tumor activity in vivo is mediated, at least in part, through Fc-receptor dependent effector mechanisms, such as ADCC and phagocytosis. Anti-CC chemokine receptor 4 antibodies inhibiting receptor signaling have potential as immunomodulatory antibodies for cancer.

  7. Antibody level of New Zealand children immunized with the triple vaccine DTP (diphtheria-tetanus-pertussis).


    Lau, R. C.


    Enzyme-linked immunosorbent assay (ELISA) tests were used to measure IgG antibody levels in 2638 New Zealand children who had been immunized with the triple vaccine DTP. The percentage of children immune to diphtheria decreased with age. The percentage of children immune to tetanus varied from 67.1 to 55.0%. The percentage of children with measurable antibody to pertussis increased with age. The mean percentages of children with measurable antibody or immunity to one or more DTP components we...

  8. Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

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    M Jedi-Tehrani


    Full Text Available Introduction: As monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, use of these proteins has always been considered in infertility and reproduction research. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. Material and Methods: To produce specific clones against human sperm surface antigens, proteins were extracted using solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete Freund’s adjuvant in the first injection and incomplete Adjuvant in the following booster injections. Hybridoma cells producing ASA were cloned by limiting dilution. Results: Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with rat and mice spermatozoa was examined but they did not have any cross reactivity. Conclusion: The produced antibodies can be used in further studies to characterize and evaluate each of the antigens present on human sperm surface and determining their role in fertilization.

  9. Efficient generation of human IgA monoclonal antibodies. (United States)

    Lorin, Valérie; Mouquet, Hugo


    Immunoglobulin A (IgA) is the most abundant antibody isotype produced in humans. IgA antibodies primarily ensure immune protection of mucosal surfaces against invading pathogens, but also circulate and are present in large quantities in blood. IgAs are heterogeneous at a molecular level, with two IgA subtypes and the capacity to form multimers by interacting with the joining (J) chain. Here, we have developed an efficient strategy to rapidly generate human IgA1 and IgA2 monoclonal antibodies in their monomeric and dimeric forms. Recombinant monomeric and dimeric IgA1/IgA2 counterparts of a prototypical IgG1 monoclonal antibody, 10-1074, targeting the HIV-1 envelope protein, were produced in large amounts after expression cloning and transient transfection of 293-F cells. 10-1074 IgAs were FPLC-purified using a novel affinity-based resin engrafted with anti-IgA chimeric Fabs, followed by a monomers/multimers separation using size exclusion-based FPLC. ELISA binding experiments confirmed that the artificial IgA class switching of 10-1074 did not alter its antigen recognition. In summary, our technical approach allows the very efficient production of various forms of purified recombinant human IgA molecules, which are precious tools in dissecting IgA B-cell responses in physiological and pathophysiological conditions, and studying the biology, function and therapeutic potential of IgAs.

  10. Radioimmunoassay for zearalenone and zearalanol in human serum: production, properties, and use of porcine antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Thouvenot, D.; Morfin, R.F.


    To produce antigens susceptible to raise antibodies for resorcylic acid lactones, the 6'-carboxymethyloxime derivatives of zearalenone and zearalanone were bound to bovine serum albumin. Pigs could be immunized by using these antigens, the best titer in antibodies being obtained with the zearalenone antigen. The procine antibodies were specific for the resorcylic acid lactones of structural resemblance with zearalenone. This specificity made the antibodies usable for a radioimmunoassay of zearalenone and zearalanol, which may be found in human and animal sera. The range of the assay was between 0.25 and 10 ng. The limit of detection was 5 ppb (5 ng/ml) in human serum.

  11. RA8, A human anti-CD25 antibody against human treg cells

    Energy Technology Data Exchange (ETDEWEB)

    Arias, Robyn; Flanagan, Meg; Miller, Keith D.; Nien, Yu-Chih; Hu, Peisheng; Gray, Dixon; Khawli, Leslie A.; Epstein, Alan L.


    Although anti-CD25 antibodies exist for clinical use in patients, there is a need for the development of a human Treg antibody that will abrogate the immunosuppressive function of this small but critical T cell subtype. Based upon mounting evidence that the level of Treg cells in the tumor microenvironment correlates with clinical prognosis and stage in man, it appears that Treg cells play an important role in the tumor's ability to overcome host immune responses. In mice, the rat anti-mouse CD25 antibody PC61 causes depletion of CD25-bearing Treg cells both peripherally in lymphatic tissues and in the tumor microenvironment, without inducing symptoms of autoimmunity. A similar antibody, though with the ability to delete Treg cells specifically, would be an important new tool for reversing tumor escape associated with Treg immunosuppression in man. To begin to generate such a reagent, we now describe the development of a human anti-CD25 antibody using a novel yeast display library. The target antigen CD25-Fc was constructed and used for five rounds of selection using a non-immune yeast display library that contained as many as 109 single chain variable fragments (scFv). Two unique clones with low KD values (RA4 and RA8) were then selected to construct fully human anti-CD25 antibodies (IgG1/kappa) for stable expression. One antibody, RA8, showed excellent binding to human CD25+ cell lines and to human Treg cells and appears to be an excellent candidate for the generation of a human reagent that may be used in man for the immunotherapy of cancer.

  12. Changes in the repertoire of natural antibodies caused by immunization with bacterial antigens

    DEFF Research Database (Denmark)

    Shilova, N V; Navakouski, M J; Huflejt, M


    The repertoire of natural anti-glycan antibodies in naïve chickens and in chickens immunized with bacteria Burkholderia mallei, Burkholderia pseudomallei, and Francisella tularensis as well as with peptides from an outer membrane protein of B. pseudomallei was studied. A relatively restricted...... pattern of natural antibodies (first of all IgY against bacterial cell wall peptidoglycan fragments, L-Rha, and core N-acetyllactosamine) shrank and, moreover, the level of detectable antibodies decreased as a result of immunization....

  13. Belimumab: anti-BLyS human monoclonal antibody, anti-BLyS monoclonal antibody, BmAb, human monoclonal antibody to B-lymphocyte stimulator. (United States)


    Belimumab is a fully human monoclonal antibody that specifically recognizes and inhibits the biological activity of B-lymphocyte stimulator, or BLyS. Belimumab is in phase III trials for the treatment of systemic lupus erythematosus (SLE) and has completed a phase II trial in rheumatoid arthritis (RA); the product may also have potential in the treatment of other autoimmune disorders. In May 2001, Cambridge Antibody Technology (now MedImmune) completed its discovery programme and Human Genome Sciences identified belimumab as a candidate for clinical development. More than 1000 distinct human antibodies specific to BLyS were characterized by the collaboration.B-lymphocyte stimulator is a naturally occurring protein discovered by Human Genome Sciences that stimulates B-lymphocytes to develop into mature B cells. Laboratory studies have indicated that higher than normal levels of B-lymphocyte stimulator may contribute to the pathogenesis of autoimmune diseases, such as SLE and RA. Human Genome Sciences (HGS) and Cambridge Antibody Technology signed a collaborative agreement in August 1999 to study the B-lymphocyte stimulator as a human protein target. HGS is also developing other BLyS products. In March 2000, HGS and Cambridge Antibody Technology expanded their agreement into a 10-year collaboration and product development alliance, providing Human Genome Sciences with the right to use the antibody technology of Cambridge Antibody Technology to fully develop human antibodies for therapeutic and diagnostic purposes. Cambridge Antibody Technology will receive royalty payments on product sales from HGS, as well as the development and milestone payments it has already received. Belimumab will be manufactured in Human Genome Sciences' manufacturing facility, located in Rockville, MD, USA. HGS holds commercial rights to the drug. In July 2005, GlaxoSmithKline (GSK) exercised its co-development and co-promotion option to belimumab. In an agreement made in June 1996, HGS had

  14. Immunization routes in cattle impact the levels and neutralizing capacity of antibodies induced against S. aureus immune evasion proteins. (United States)

    Boerhout, Eveline; Vrieling, Manouk; Benedictus, Lindert; Daemen, Ineke; Ravesloot, Lars; Rutten, Victor; Nuijten, Piet; van Strijp, Jos; Koets, Ad; Eisenberg, Susanne


    Vaccines against S. aureus bovine mastitis are scarce and show limited protection only. All currently available vaccines are applied via the parenteral (usually intramuscular) route. It is unknown, however, whether this route is the most suitable to specifically increase intramammary immunity to combat S. aureus at the site of infection. Hence, in the present study, immunization via mucosal (intranasal; IN), intramuscular (triangle of the neck; IM), intramammary (IMM) and subcutaneous (suspensory ligament; SC) routes were analyzed for their effects on the quantity of the antibody responses in serum and milk as well as the neutralizing capacity of the antibodies within serum. The experimental vaccine comprised the recombinant S. aureus immune evasion proteins extracellular fibrinogen-binding protein (Efb) and the leukotoxin subunit LukM in an oil-in-water adjuvant combined with a hydrogel and alginate. The highest titer increases for both Efb and LukM specific IgG1 and IgG2 antibody levels in serum and milk were observed following SC/SC immunizations. Furthermore, the harmful effects of Efb and leukotoxin LukMF' on host-defense were neutralized by serum antibodies in a route-dependent manner. SC/SC immunization resulted in a significant increase in the neutralizing capacity of serum antibodies towards Efb and LukMF', shown by increased phagocytosis of S. aureus and increased viability of bovine leukocytes. Therefore, a SC immunization route should be considered when aiming to optimize humoral immunity against S. aureus mastitis in cattle.

  15. Design and construction of immune phage antibody library against Tetanus neurotoxin: Production of single chain antibody fragments. (United States)

    Sadreddini, Sanam; Seifi-Najmi, Mehrnosh; Ghasemi, Babollah; Kafil, Hossein Samadi; Alinejad, Vahideh; Sadreddini, Sevil; Younesi, Vahid; Jadidi-Niaragh, Farhad; Yousefi, Mehdi


    Tetanus neurotoxin (TeNT) is composed of a light (LC) and heavy chain (HC) polypeptides, released by anaerobic bacterium Clostridium tetani and can cause fatal life-threatening infectious disease. Toxin HC and LC modules represents receptor binding and zinc metalloprotease activity, respectively. The passive administration of animal-derived antibodies against tetanus toxin has been considered as the mainstay therapy for years. However, this treatment is associated with several adverse effects due to the presence of anti-isotype antibodies. In the present study, we have produced the fully human single chain antibody fragments (HuScFv) from two human antibody phage display libraries. Twenty-four different HuscFvs were isolated from two anti TeNT immune libraries. Our produced human ScFv (HuScFv) were converted to IgG platform and analyzed regarding their specific reactivity to TeNT. All of the selected scFvs have the same VL but different VH. Three HuscFvs from the first library (TTX15, 51, 75) and two HuscFvs from the second library (TTX16, 20) were chosen to convert to IgG1 using pOptiVEC and pcDNA3.3 systems. Production of IgG1 from transfected DG44 and binding capacity of them to tetanus toxin and toxoid were measured by ELISA. ELISA results showed no detectable production of TTX16 and TTX20 IgG1. Although, TTX51 and TTX75 were converted and produced as IgG1, no reactivity to tetanus toxin and toxoid was observed. However, TTX15 was successfully produced as whole IgG1 platform with reactivity to both tetanus toxin and toxoid. The latter would be an appropriate replacement for conventional polyclonal antibodies if would meet the further characterization including specificity determination, affinity measurement and toxin neutralizing assays. Our results demonstrated production of functional IgG1 derived from TTX15 scFv and might be an appropriate replacement for polyclonal Tetabulin but it needs further characterization.

  16. Antibodies and immune profiles of individuals occupationally exposed to formaldehyde: six case reports. (United States)

    Thrasher, J D; Broughton, A; Micevich, P


    Six patients with multiple subjective health complaints, which have been correlated with chronic exposure to formaldehyde during the course of their education and occupations, were tested for the existence of antibodies (IgE, IgM, and IgG) to formaldehyde (F) conjugated to human serum albumin (F-HSA). In addition, the percentage and absolute numbers of peripheral lymphocyte subpopulations as determined by surface markers were investigated. Antibody titers to F-HSA were present as follows: IgE (2 patients), IgM (3 of 4 tested patients), and IgG (5 patients). Analysis of lymphocyte subpopulations showed T-helper/suppressor (H/S) ratios ranging from 0.8 to 3.3. All 6 patients had elevated Tal cells (antigen memory cells), whereas interleuken 2 receptor positive cells were within expected values. Following formaldehyde exposure, 5 of the patients complained of an initial flulike illness from which they have not completely recovered. The sixth individual had a history of recurrent respiratory infections and surgical removal of hyperplastic ethmoid sinus tissue. The common occurrence of anti-F-HSA antibodies, flulike illness, and Tal cells are interpreted as suggestive of a chronic antigenic stimulation of the immune system in these 6 patients. Further immunological work-up of additional subjects and immune parameters with similar history of formaldehyde exposure and subjective health complaints is warranted.

  17. Early eradication of persistent Salmonella infection primes antibody-mediated protective immunity to recurrent infection. (United States)

    Johanns, Tanner M; Law, Calvin Y; Kalekar, Lokeshchandra A; O'Donnell, Hope; Ertelt, James M; Rowe, Jared H; Way, Sing Sing


    Typhoid fever is a systemic, persistent infection caused by host-specific strains of Salmonella. Although the use of antibiotics has reduced the complications associated with primary infection, recurrent infection remains an important cause of ongoing human morbidity and mortality. Herein, we investigated the impacts of antibiotic eradication of primary infection on protection against secondary recurrent infection. Using a murine model of persistent Salmonella infection, we demonstrate protection against recurrent infection is sustained despite early eradication of primary infection. In this model, protection is not mediated by CD4(+) or CD8(+) T cells because depletion of these cells either alone or in combination prior to rechallenge does not abrogate protection. Instead, infection followed by antibiotic-mediated clearance primes robust levels of Salmonella-specific antibody that can adoptively transfer protection to naïve mice. Thus, eradication of persistent Salmonella infection primes antibody-mediated protective immunity to recurrent infection.

  18. Human immune cell targeting of protein nanoparticles - caveospheres (United States)

    Glass, Joshua J.; Yuen, Daniel; Rae, James; Johnston, Angus P. R.; Parton, Robert G.; Kent, Stephen J.; de Rose, Robert


    Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells--an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines.

  19. Differential Kinetics and Distribution of Antibodies in Serum and Nasal and Vaginal Secretions after Nasal and Oral Vaccination of Humans


    Rudin, Anna; Johansson, Eva-Liz; Bergquist, Charlotta; Holmgren, Jan


    Although nasal vaccination has emerged as an interesting alternative to systemic or oral vaccination, knowledge is scarce about the immune responses after such immunization in humans. In the present study, we have compared the kinetics and organ distribution of the antibody responses after nasal and oral vaccination. We immunized female volunteers nasally or orally with cholera toxin B subunit (CTB) and determined the specific antibody levels in serum and nasal and vaginal secretions, as well...

  20. Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice. (United States)

    Murphy, Andrew J; Macdonald, Lynn E; Stevens, Sean; Karow, Margaret; Dore, Anthony T; Pobursky, Kevin; Huang, Tammy T; Poueymirou, William T; Esau, Lakeisha; Meola, Melissa; Mikulka, Warren; Krueger, Pamela; Fairhurst, Jeanette; Valenzuela, David M; Papadopoulos, Nicholas; Yancopoulos, George D


    Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.

  1. Antibody complementarity-determining regions (CDRs: a bridge between adaptive and innate immunity.

    Directory of Open Access Journals (Sweden)

    Elena Gabrielli

    Full Text Available BACKGROUND: It has been documented that, independently from the specificity of the native antibody (Ab for a given antigen (Ag, complementarity determining regions (CDR-related peptides may display differential antimicrobial, antiviral and antitumor activities. METHODOLOGY/PRINCIPAL FINDINGS: In this study we demonstrate that a synthetic peptide with sequence identical to V(HCDR3 of a mouse monoclonal Ab (mAb specific for difucosyl human blood group A is easily taken up by macrophages with subsequent stimulation of: i proinflammatory cytokine production; ii PI3K-Akt pathway and iii TLR-4 expression. Significantly, V(HCDR3 exerts therapeutic effect against systemic candidiasis without possessing direct candidacidal properties. CONCLUSIONS/SIGNIFICANCE: These results open a new scenario about the possibility that, beyond the half life of immunoglobulins, Ab fragments may effectively influence the antiinfective cellular immune response in a way reminiscent of regulatory peptides of innate immunity.

  2. Oral antibiotics enhance antibody responses to keyhole limpet hemocyanin in orally but not muscularly immunized chickens. (United States)

    Murai, Atsushi; Kitahara, Kazuki; Okumura, Shouta; Kobayashi, Misato; Horio, Fumihiko


    Recent studies have emphasized the crucial role of gut microbiota in triggering and modulating immune response. We aimed to determine whether the modification of gut microbiota by oral co-administration of two antibiotics, ampicillin and neomycin, would lead to changes in the antibody response to antigens in chickens. Neonatal chickens were given or not given ampicillin and neomycin (0.25 and 0.5 g/L, respectively) in drinking water. At 2 weeks of age, the chicks were muscularly or orally immunized with antigenic keyhole limpet hemocyanin (KLH), and then serum anti-KLH antibody levels were examined by ELISA. In orally immunized chicks, oral antibiotics treatment enhanced antibody responses (IgM, IgA, IgY) by 2-3-fold compared with the antibiotics-free control, while the antibiotics did not enhance antibody responses in the muscularly immunized chicks. Concomitant with their enhancement of antibody responses, the oral antibiotics also lowered the Lactobacillus species in feces. Low doses of antibiotics (10-fold and 100-fold lower than the initial trial), which failed to change the fecal Lactobacillus population, did not modify any antibody responses when chicks were orally immunized with KLH. In conclusion, oral antibiotics treatment enhanced the antibody response to orally exposed antigens in chickens. This enhancement of antibody response was associated with a modification of the fecal Lactobacillus content, suggesting a possible link between gut microbiota and antibody response in chickens. © 2015 Japanese Society of Animal Science.

  3. Induction of antibodies reactive to cardiac myosin and development of heart alterations in cruzipain-immunized mice and their offspring. (United States)

    Giordanengo, L; Maldonado, C; Rivarola, H W; Iosa, D; Girones, N; Fresno, M; Gea, S


    Human and murine infection with Trypanosoma cruzi parasite is usually accompanied by strong humoral and cellular immune response to cruzipain, a parasite immunodominant antigen. In the present study we report that the immunization of mice with cruzipain devoid of enzymatic activity, was able to induce antibodies which bind to a 223-kDa antigen from a mouse heart extract. We identified this protein as the mouse cardiac myosin heavy chain by sequencing analysis. The study of IgG isotype profile revealed the occurrence of all IgG isotypes against cruzipain and myosin. IgG1 showed the strongest reactivity against cruzipain, whereas IgG2a was the main isotype against myosin. Anti-cruzipain antibodies purified by immunoabsorption recognized the cardiac myosin heavy chain, suggesting cross-reactive epitopes between cruzipain and myosin. Autoimmune response in mice immunized with cruzipain was associated to heart conduction disturbances. In addition, ultrastructural findings revealed severe alterations of cardiomyocytes and IgG deposit on heart tissue of immunized mice. We investigated whether antibodies induced by cruzipain transferred from immunized mothers to their offsprings could alter the heart function in the pups. All IgG isotypes against cruzipain derived from transplacental crossing were detected in pups' sera. Electrocardiographic studies performed in the offsprings born to immunized mothers revealed conduction abnormalities. These results provide strong evidence for a pathogenic role of autoimmune response induced by a purified T. cruzi antigen in the development of experimental Chagas' disease.

  4. Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Bao-Ping Wu; Bing Xiao; Tian-Mo Wan; Ya-Li Zhang; Zhen-Shu Zhang; Dian-Yuan Zhou; Zhuo-Sheng Lai; Chun-Fang Gao


    AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer. METHODS: Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer.RT-PCR was used to amplify the heavy chain Fd and light chain к and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected. RESULTS: The amplified fragments of Fd and к gained by RT-PCR were about 650bp. Fd and к PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 x 106. The libraries were enriched about 120-fold by 3 cycles of adsorption-elution- multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5clones of Fab phage antibodies which had binding activities with antigens of colorectal cancer. CONCLUSION: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.

  5. Hypogammaglobulinemia in BLT humanized mice--an animal model of primary antibody deficiency.

    Directory of Open Access Journals (Sweden)

    Francisco Martinez-Torres

    Full Text Available Primary antibody deficiencies present clinically as reduced or absent plasma antibodies without another identified disorder that could explain the low immunoglobulin levels. Bone marrow-liver-thymus (BLT humanized mice also exhibit primary antibody deficiency or hypogammaglobulinemia. Comprehensive characterization of B cell development and differentiation in BLT mice revealed other key parallels with primary immunodeficiency patients. We found that B cell ontogeny was normal in the bone marrow of BLT mice but observed an absence of switched memory B cells in the periphery. PC-KLH immunizations led to the presence of switched memory B cells in immunized BLT mice although plasma cells producing PC- or KLH- specific IgG were not detected in tissues. Overall, we have identified the following parallels between the humoral immune systems of primary antibody deficiency patients and those in BLT mice that make this in vivo model a robust and translational experimental platform for gaining a greater understanding of this heterogeneous array of humoral immunodeficiency disorders in humans: (i hypogammaglobulinemia; (ii normal B cell ontogeny in bone marrow; and (iii poor antigen-specific IgG response to immunization. Furthermore, the development of strategies to overcome these humoral immune aberrations in BLT mice may in turn provide insights into the pathogenesis of some primary antibody deficiency patients which could lead to novel clinical interventions for improved humoral immune function.

  6. [Immunization experiments for producing antibody-like substances in caterpillars of Mamestra brassicae L. (Insecta, Lepid., Noct.)]. (United States)

    Luther, P; Otto, D; Köhler, W; Fischer, G


    The agglutinins against human blood cells described in caterpillars of Mamestra brassicae L. were not demonstrable when feeding the animals with a semisynthetic food. After injection or oral intake of certain bacteria (E. coli or streptococci of group C) or even Pope's broth the "antibody-like substances" known from feeding with natural food are being formed, and they agglutinated all human blood cells. The individual animals showed differences regarding the strength of agglutinin formation. The immune reactions observed possibly indicate the existence of a primitive immune system in these species (arthropods).

  7. Human germline antibody gene segments encode polyspecific antibodies. (United States)

    Willis, Jordan R; Briney, Bryan S; DeLuca, Samuel L; Crowe, James E; Meiler, Jens


    Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.

  8. Phase I study of anticolon cancer humanized antibody A33. (United States)

    Welt, Sydney; Ritter, Gerd; Williams, Clarence; Cohen, Leonard S; John, Mary; Jungbluth, Achim; Richards, Elizabeth A; Old, Lloyd J; Kemeny, Nancy E


    Humanized A33 (huA33; IgG1) monoclonal antibody detects a determinant expressed by 95% of colorectal cancers and can activate immune cytolytic mechanisms. The present study was designed to (a) define the toxicities and maximum tolerated dose of huA33 and (b) determine huA33 immunogenicity. Patients (n = 11) with advanced chemotherapy-resistant colorectal cancer received 4-week cycles of huA33 at 10, 25, or 50 mg/m(2)/week. Serum samples were analyzed using biosensor technology for evidence of human antihuman antibody (HAHA) response. Eight of 11 patients developed a HAHA response. Significant toxicity was limited to four patients who developed high HAHA titers. In two of these cases, infusion-related reactions such as fevers, rigors, facial flushing, and changes in blood pressure were observed, whereas in the other two cases, toxicity consisted of skin rash, fever, or myalgia. Of three patients who remained HAHA negative, one achieved a radiographic partial response, with reduction of serum carcinoembryonic antigen from 80 to 3 ng/ml. Four patients had radiographic evidence of stable disease (2, 4, 6, and 12 months), with significant reductions (>25%) in serum carcinoembryonic antigen levels in two cases. The complementarity-determining region-grafted huA33 antibody is immunogenic in the majority of colon cancer patients (73%). HAHA activity can be measured reproducibly and quantitatively by BIACORE analysis. Whereas the huA33 construct tested here may be too immunogenic for further clinical development, the antitumor effects observed in the absence of antibody-mediated toxicity and in this heavily pretreated patient population warrant clinical testing of other IgG1 humanized versions of A33 antibody.

  9. Antibody blockade of IL-17 family cytokines in immunity to acute murine oral mucosal candidiasis. (United States)

    Whibley, Natasha; Tritto, Elaine; Traggiai, Elisabetta; Kolbinger, Frank; Moulin, Pierre; Brees, Dominique; Coleman, Bianca M; Mamo, Anna J; Garg, Abhishek V; Jaycox, Jillian R; Siebenlist, Ulrich; Kammüller, Michael; Gaffen, Sarah L


    Antibodies targeting IL-17A or its receptor, IL-17RA, are approved to treat psoriasis and are being evaluated for other autoimmune conditions. Conversely, IL-17 signaling is critical for immunity to opportunistic mucosal infections caused by the commensal fungus Candida albicans, as mice and humans lacking the IL-17R experience chronic mucosal candidiasis. IL-17A, IL-17F, and IL-17AF bind the IL-17RA-IL-17RC heterodimeric complex and deliver qualitatively similar signals through the adaptor Act1. Here, we used a mouse model of acute oropharyngeal candidiasis to assess the impact of blocking IL-17 family cytokines compared with specific IL-17 cytokine gene knockout mice. Anti-IL-17A antibodies, which neutralize IL-17A and IL-17AF, caused elevated oral fungal loads, whereas anti-IL-17AF and anti-IL-17F antibodies did not. Notably, there was a cooperative effect of blocking IL-17A, IL-17AF, and IL-17F together. Termination of anti-IL-17A treatment was associated with rapid C. albicans clearance. IL-17F-deficient mice were fully resistant to oropharyngeal candidiasis, consistent with antibody blockade. However, IL-17A-deficient mice had lower fungal burdens than anti-IL-17A-treated mice. Act1-deficient mice were much more susceptible to oropharyngeal candidiasis than anti-IL-17A antibody-treated mice, yet anti-IL-17A and anti-IL-17RA treatment caused equivalent susceptibilities. Based on microarray analyses of the oral mucosa during infection, only a limited number of genes were associated with oropharyngeal candidiasis susceptibility. In sum, we conclude that IL-17A is the main cytokine mediator of immunity in murine oropharyngeal candidiasis, but a cooperative relationship among IL-17A, IL-17AF, and IL-17F exists in vivo. Susceptibility displays the following hierarchy: IL-17RA- or Act1-deficiency > anti-IL-17A + anti-IL-17F antibodies > anti-IL-17A or anti-IL-17RA antibodies > IL-17A deficiency.

  10. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus (United States)

    Marcobal, Angela; Liu, Xiaowen; Zhang, Wenlei; Dimitrov, Antony S.; Jia, Letong; Lee, Peter P.; Fouts, Timothy R.; Parks, Thomas P.


    Abstract Eradication of human immunodeficiency virus type 1 (HIV-1) by vaccination with epitopes that produce broadly neutralizing antibodies is the ultimate goal for HIV prevention. However, generating appropriate immune responses has proven difficult. Expression of broadly neutralizing antibodies by vaginal colonizing lactobacilli provides an approach to passively target these antibodies to the mucosa. We tested the feasibility of expressing single-chain and single-domain antibodies (dAbs) in Lactobacillus to be used as a topical microbicide/live biotherapeutic. Lactobacilli provide an excellent platform to express anti-HIV proteins. Broadly neutralizing antibodies have been identified against epitopes on the HIV-1 envelope and have been made into active antibody fragments. We tested single-chain variable fragment m9 and dAb-m36 and its derivative m36.4 as prototype antibodies. We cloned and expressed the antibody fragments m9, m36, and m36.4 in Lactobacillus jensenii-1153 and tested the expression levels and functionality. We made a recombinant L. jensenii 1153-1128 that expresses dAb-m36.4. All antibody fragments m9, m36, and m36.4 were expressed by lactobacilli. However, we noted the smaller m36/m36.4 were expressed to higher levels, ≥3 μg/ml. All L. jensenii-expressed antibody fragments bound to gp120/CD4 complex; Lactobacillus-produced m36.4 inhibited HIV-1BaL in a neutralization assay. Using a TZM-bl assay, we characterized the breadth of neutralization of the m36.4. Delivery of dAbs by Lactobacillus could provide passive transfer of these antibodies to the mucosa and longevity at the site of HIV-1 transmission. PMID:26950606

  11. Immunoepidemiology of Wuchereria bancrofti infection: parasite transmission intensity, filaria-specific antibodies, and host immunity in two East African communities. (United States)

    Jaoko, Walter G; Michael, Edwin; Meyrowitsch, Dan W; Estambale, Benson B A; Malecela, Mwele N; Simonsen, Paul E


    We compared the age profiles of infection and specific antibody intensities in two communities with different transmission levels in East Africa to examine the contribution of humoral responses to human immunity to the vector-borne helminth Wuchereria bancrofti. The worm intensities were higher and exhibited a nonlinear age pattern in a high-transmission community, Masaika, in contrast to the low but linearly increasing age infection profile observed for a low-transmission community, Kingwede. The mean levels of specific immunoglobulin G1 (IgG1), IgG2, IgG4, and IgE were also higher in Masaika, but intriguingly, the IgG3 response was higher in Kingwede. The age-antibody patterns differed in the two communities but in a manner apparently contrary to a role in acquired immunity when the data were assessed using simple correlation methods. By contrast, multivariate analyses showed that the antibody response to infection may be classified into three types and that two of these types, a IgG3-type response and a response measuring a trade-off in host production of IgG4 and IgG3 versus production of IgG1, IgG2, and IgE, had a negative effect on Wuchereria circulating antigen levels in a manner that supported a role for these responses in the generation of acquired immunity to infection. Mathematical modeling supported the conclusions drawn from empirical data analyses that variations in both transmission and worm intensity can explain community differences in the age profiles and impacts of these antibody response types. This study showed that parasite-specific antibody responses may be associated with the generation of acquired immunity to human filarial infection but in a form which is dependent on worm transmission intensity and interactions between immune components.

  12. Generation and Characterization of Novel Human IRAS Monoclonal Antibodies

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    Bo Wang


    Full Text Available Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS, has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10–120aa through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10–120aa. Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.

  13. Capsid protein: evidences about the partial protective role of neutralizing antibody-independent immunity against dengue in monkeys. (United States)

    Gil, Lázaro; Izquierdo, Alienys; Lazo, Laura; Valdés, Iris; Ambala, Peris; Ochola, Lucy; Marcos, Ernesto; Suzarte, Edith; Kariuki, Thomas; Guzmán, Guadalupe; Guillén, Gerardo; Hermida, Lisset


    The role of cellular immune response in dengue virus infection is not yet fully understood. Only few studies in murine models propose that CD8(+) T-cells are associated with protection from infection and disease. At the light of recent reports about the protective role of CD8(+) T-cells in humans and the no correlation between neutralizing antibodies and protection observed in several studies, a vaccine based on cell-mediated immunity constitute an attractive approach. Our group has developed a capsid-based vaccine as nucleocpasid-like particles from dengue-2 virus, which induced a protective CD4(+) and CD8(+) cell-mediated immunity in mice, without the contribution of neutralizing antibodies. Herein we evaluated the immunogenicity and protective efficacy of this molecule in monkeys. Neither IgG antibodies against the whole virus nor neutralizing antibodies were elicited after the antigen inoculation. However, animals developed a cell-mediated immunity, measured by gamma interferon secretion and cytotoxic capacity. Although only one out of three vaccinated animals was fully protected against viral challenge, a viral load reduction was observed in this group compared with the placebo one, suggesting that capsid could be the base on an attractive vaccine against dengue.

  14. Cooperativity between CD8+ T cells, non-neutralizing antibodies, and alveolar macrophages is important for heterosubtypic influenza virus immunity.

    Directory of Open Access Journals (Sweden)

    Brian J Laidlaw


    Full Text Available Seasonal epidemics of influenza virus result in ∼36,000 deaths annually in the United States. Current vaccines against influenza virus elicit an antibody response specific for the envelope glycoproteins. However, high mutation rates result in the emergence of new viral serotypes, which elude neutralization by preexisting antibodies. T lymphocytes have been reported to be capable of mediating heterosubtypic protection through recognition of internal, more conserved, influenza virus proteins. Here, we demonstrate using a recombinant influenza virus expressing the LCMV GP33-41 epitope that influenza virus-specific CD8+ T cells and virus-specific non-neutralizing antibodies each are relatively ineffective at conferring heterosubtypic protective immunity alone. However, when combined virus-specific CD8 T cells and non-neutralizing antibodies cooperatively elicit robust protective immunity. This synergistic improvement in protective immunity is dependent, at least in part, on alveolar macrophages and/or other lung phagocytes. Overall, our studies suggest that an influenza vaccine capable of eliciting both CD8+ T cells and antibodies specific for highly conserved influenza proteins may be able to provide heterosubtypic protection in humans, and act as the basis for a potential "universal" vaccine.

  15. Human papillomavirus targets crossroads in immune signaling

    NARCIS (Netherlands)

    Tummers, Bart


    Persistent infections with high-risk type human papillomaviruses (hrHPVs) can progress to cancer. HrHPVs infect keratinocytes (KCs) and successfully suppress host immunity for up to two years despite the fact that KCs are well equipped to detect and initiate immune responses to invading pathogens.

  16. Factors of Innate and Adaptive Local Immunity in Children with Primary Deficiencies of Antibody Formation

    Directory of Open Access Journals (Sweden)

    L.I. Chernyshova


    Full Text Available In 40 children with various types of primary immunodeficiencies (PID of antibody formation we examined factors of local immunity in saliva. It is found that in the saliva of children with PID of antibody formation in comparison with immunocompetent children the concentration of factors of adaptive immunity is significantly reduced. Lack of adaptive immunity in the PID of antibody formation to some extent is compensated by increased concentrations of innate immune factors on the mucous membranes — the free Sc, as well as lactoferrin in selective immunodeficiency of IgA. At PID of antibody formation we observed increased TNF-α level in the saliva, which may indicate the persistence of local inflammation on the membranes of the respiratory tract.

  17. DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 Human IgG Antibody Produced by AnaptysBio, Inc. (United States)


    ECBC-TR-1339 DARPA ANTIBODY TECHNOLOGY PROGRAM STANDARDIZED TEST BED FOR ANTIBODY...CHARACTERIZATION: CHARACTERIZATION OF AN MS2 HUMAN IGG ANTIBODY PRODUCED BY ANAPTYSBIO, INC. DARPA ATP Standardized Test Bed for Antibody...Characterization: Characterization of an MS2 human IgG antibody produced by AnaptysBio DARPA ATP Standardized Test Bed for Antibody

  18. Human Lung Immunity against Mycobacterium tuberculosis (United States)

    Schwander, Stephan; Dheda, Keertan


    The study of human pulmonary immunity against Mycobacterium tuberculosis (M.tb) provides a unique window into the biological interactions between the human host and M.tb within the broncho-alveolar microenvironment, the site of natural infection. Studies of bronchoalveolar cells (BACs) and lung tissue evaluate innate, adaptive, and regulatory immune mechanisms that collectively contribute to immunological protection or its failure. In aerogenically M.tb–exposed healthy persons lung immune responses reflect early host pathogen interactions that may contribute to sterilization, the development of latent M.tb infection, or progression to active disease. Studies in these persons may allow the identification of biomarkers of protective immunity before the initiation of inflammatory and disease-associated immunopathological changes. In healthy close contacts of patients with tuberculosis (TB) and during active pulmonary TB, immune responses are compartmentalized to the lungs and characterized by an exuberant helper T-cell type 1 response, which as suggested by recent evidence is counteracted by local suppressive immune mechanisms. Here we discuss how exploring human lung immunity may provide insights into disease progression and mechanisms of failure of immunological protection at the site of the initial host–pathogen interaction. These findings may also aid in the identification of new biomarkers of protective immunity that are urgently needed for the development of new and the improvement of current TB vaccines, adjuvant immunotherapies, and diagnostic technologies. To facilitate further work in this area, methodological and procedural approaches for bronchoalveolar lavage studies and their limitations are also discussed. PMID:21075901

  19. Production of High Affinity Human Single-chain Antibody Against PreS1 of Hepatitis B Virus:Comparison of Large Na(i)ve and In vitro Immune Phage Displayed Antibody Library%高亲和力抗乙型肝炎病毒PreS1的人源单链抗体的获得:天然及免疫抗体库的对比研究

    Institute of Scientific and Technical Information of China (English)

    张志超; 胡学军; 包永明; 杨青; 张红梅; 安利佳


    A large nave phage displayed human single-chain variable fragments antibody (scFv) library and an in vitro immune library were constructed in parallel conditions, based on the PBLs from healthy and sero-negative blood donors, part of which were in vitro immunized by peptide PreS1 conjugated to BSA. After 3 rounds of panning against PreS1, measurement of antibody-antigen reaction revealed: a scFv specific to PreS1 from the immune library was obtained, which affinity (k=10-7~10-8 M) was higher than that from the nave one (k=10-6~10-7 M). Sequencing of the two scFv showed they were human antibodies, which may be of interest in therapy of Hepatitis B. This investigation also illustrated that the method of in vitro immunization results in antibody library more satisfied even than the large nave one.%以健康人的外周血淋巴细胞为来源,以偶联BSA的乙型肝炎病毒PreS1肽体外免疫.分别从免疫和未经免疫的淋巴细胞提取RNA,扩增抗体基因,构建大容量天然单链抗体(scFv)噬菌体展示文库和体外免疫scFv抗体库.以PreS1肽进行3轮淘选后,抗原抗体反应结果显示,从免疫库中获得了亲和力10-7~10-8 M的抗乙型肝炎病毒PreS1的单链抗体,高于天然库的结果(10-6~10-7 M).测序结果表明两株抗体均为人抗体.为基因工程抗体用于临床治疗乙型肝炎奠定基础.同时证明淋巴细胞体外免疫方法构建的免疫抗体库优于大容量天然抗体库.

  20. Antibody therapies for melanoma: new and emerging opportunities to activate immunity (Review). (United States)

    Malas, Sadek; Harrasser, Micaela; Lacy, Katie E; Karagiannis, Sophia N


    The interface between malignant melanoma and patient immunity has long been recognised and efforts to treat this most lethal form of skin cancer by activating immune responses with cytokine, vaccine and also antibody immunotherapies have demonstrated promise in limited subsets of patients. In the present study, we discuss different antibody immunotherapy approaches evaluated in the context of melanoma, each designed to act on distinct targets and to employ different mechanisms to restrict tumour growth and spread. Monoclonal antibodies recognising melanoma-associated antigens such as CSPG4/MCSP and targeting elements of tumour-associated vasculature (VEGF) have constituted long-standing translational approaches aimed at reducing melanoma growth and metastasis. Recent insights into mechanisms of immune regulation and tumour-immune cell interactions have helped to identify checkpoint molecules on immune (CTLA4, PD-1) and tumour (PD-L1) cells as promising therapeutic targets. Checkpoint blockade with antibodies to activate immune responses and perhaps to counteract melanoma-associated immunomodulatory mechanisms led to the first clinical breakthrough in the form of an anti-CTLA4 monoclonal antibody. Novel modalities to target key mechanisms of immune suppression and to redirect potent effector cell subsets against tumours are expected to improve clinical outcomes and to provide previously unexplored avenues for therapeutic interventions.

  1. Maternal antibody transfer can lead to suppression of humoral immunity in developing zebra finches (Taeniopygia guttata). (United States)

    Merrill, Loren; Grindstaff, Jennifer L


    Maternally transferred antibodies have been documented in a wide range of taxa and are thought to adaptively provide protection against parasites and pathogens while the offspring immune system is developing. In most birds, transfer occurs when females deposit immunoglobulin Y into the egg yolk, and it is proportional to the amount in the female's plasma. Maternal antibodies can provide short-term passive protection as well as specific and nonspecific immunological priming, but high levels of maternal antibody can result in suppression of the offspring's humoral immune response. We injected adult female zebra finches (Taeniopygia guttata) with one of two antigens (lipopolysaccharide [LPS] or keyhole limpet hemocyanin [KLH]) or a control and then injected offspring with LPS, KLH, or a control on days 5 and 28 posthatch to examine the impact of maternally transferred antibodies on the ontogeny of the offspring's humoral immune system. We found that offspring of females exposed to KLH had elevated levels of KLH-reactive antibody over the first 17-28 days posthatch but reduced KLH-specific antibody production between days 28 and 36. We also found that offspring exposed to either LPS or KLH exhibited reduced total antibody levels, compared to offspring that received a control injection. These results indicate that high levels of maternal antibodies or antigen exposure during development can have negative repercussions on short-term antibody production and may have long-term fitness repercussions for the offspring.

  2. Monoclonal antibodies to intermediate filament proteins of human cells: unique and cross-reacting antibodies. (United States)

    Gown, A M; Vogel, A M


    Monoclonal antibodies were generated against the intermediate filament proteins of different human cells. The reactivity of these antibodies with the different classes of intermediate filament proteins was determined by indirect immunofluorescence on cultured cells, immunologic indentification on SDS polyacrylamide gels ("wester blot" experiments), and immunoperoxidase assays on intact tissues. The following four antibodies are described: (a) an antivimentin antibody generated against human fibroblast cytoskeleton; (b), (c) two antibodies that recognize a 54-kdalton protein in human hepatocellular carcinoma cells; and (d) an antikeratin antibody made to stratum corneum that recognizes proteins of molecular weight 66 kdaltons and 57 kdaltons. The antivimentin antibody reacts with vimentin (58 kdaltons), glial fibrillary acidic protein (GFAP), and keratins from stratum corneum, but does not recognize hepatoma intermediate filaments. In immunofluorescence assays, the antibody reacts with mesenchymal cells and cultured epithelial cells that express vimentin. This antibody decorates the media of blood vessels in tissue sections. One antihepatoma filament antibody reacts only with the 54 kdalton protein of these cells and, in immunofluorescence and immunoperoxidase assays, only recognizes epithelial cells. It reacts with almost all nonsquamous epithelium. The other antihepatoma filament antibody is much less selective, reacting with vimentin, GFAP, and keratin from stratum corneum. This antibody decorates intermediate filaments of both mesenchymal and epithelial cells. The antikeratin antibody recognizes 66-kdalton and 57-kdalton proteins in extracts of stratum corneum and also identifies proteins of similar molecular weights in all cells tested. However, by immunofluorescence, this antibody decorates only the intermediate filaments of epidermoid carcinoma cells. When assayed on tissue sections, the antibody reacts with squamous epithelium and some, but not all

  3. Polyclonal and Specific Antibodies Mediate Protective Immunity against Enteric Helminth Infection

    NARCIS (Netherlands)

    McCoy, Kathy D.; Stoel, Maaike; Stettler, Rebecca; Merky, Patrick; Fink, Katja; Senn, Beatrice M.; Schaer, Corinne; Massacand, Joanna; Oderrnatt, Bernhard; Oettgen, Hans C.; Zinkernagel, Rolf M.; Bos, Nicolaas A.; Hengartner, Hans; Macpherson, Andrew J.; Harris, Nicola L.


    Anti-helminth immunity involves CD4(+) T cells, yet the precise effector mechanisms responsible for parasite killing or expulsion remain elusive. We now report an essential role for antibodies in mediating immunity against the enteric helminth Heligmosomoides polygyrus (Hp), a natural murine parasit

  4. Multi-Dimensional Measurement of Antibody-Mediated Heterosubtypic Immunity to Influenza.

    Directory of Open Access Journals (Sweden)

    Jiong Wang

    Full Text Available The human immune response to influenza vaccination depends in part on preexisting cross-reactive (heterosubtypic immunity from previous infection by, and/or vaccination with, influenza strains that share antigenic determinants with the vaccine strains. However, current methods for assessing heterosubtypic antibody responses against influenza, including the hemagglutination-inhibition (HAI assay and ELISA, are time and labor intensive, and require moderate amounts of serum and reagents. To address these issues we have developed a fluorescent multiplex assay, mPlex-Flu, that rapidly and simultaneously measures strain specific IgG, IgA, and IgM antibodies against influenza hemagglutinin (HA from multiple viral strains. We cloned, expressed and purified HA proteins from 12 influenza strains, and coupled them to multiplex beads. Assay validation showed that minimal sample volumes (<5 μl of serum were needed, and the assay had a linear response over a four Log10 range. The assay detected nanogram levels of anti-influenza specific antibodies, had high accuracy and reproducibility, with an average percentage coefficient of variation (%CV of 9.06 for intra-assay and 12.94 for inter-assay variability. Pre- and post-intramuscular trivalent influenza vaccination levels of virus specific Ig were consistent with HAI titer and ELISA measurements. A significant advantage of the mPLEX-Flu assay over the HAI assay is the ability to perform antigenic cartography, determining the antigenic distances between influenza HA's, without mathematical correction for HAI data issues. For validation we performed antigenic cartography on 14 different post-influenza infection ferret sera assayed against 12 different influenza HA's. Results were in good agreement with a phylogenetic tree generated from hierarchical clustering of the genomic HA sequences. This is the first report of the use of a multiplex method for antigenic cartography using ferret sera. Overall, the m

  5. Modulation of antibody-mediated immune response by probiotics in chickens. (United States)

    Haghighi, Hamid R; Gong, Jianhua; Gyles, Carlton L; Hayes, M Anthony; Sanei, Babak; Parvizi, Payvand; Gisavi, Haris; Chambers, James R; Sharif, Shayan


    Probiotic bacteria, including Lactobacillus acidophilus and Bifidobacterium bifidum, have been shown to enhance antibody responses in mammals. The objective of this study was to examine the effects of a probiotic product containing the above bacteria in addition to Streptococcus faecalis on the induction of the chicken antibody response to various antigens, both systemically and in the gut. The birds received probiotics via oral gavage and subsequently were immunized with sheep red blood cells (SRBC) and bovine serum albumin (BSA) to evaluate antibody responses in serum or with tetanus toxoid (TT) to measure the mucosal antibody response in gut contents. Control groups received phosphate-buffered saline. Overall, BSA and SRBC induced a detectable antibody response as early as week 1 postimmunization (p.i.), which lasted until week 3 p.i. Probiotic-treated birds had significantly (P probiotics. However, treatment with probiotics did not enhance the serum IgM and IgG antibody responses to BSA. Immunization with TT resulted in the presence of specific IgA and IgG antibody responses in the gut. Again, treatment with probiotics did not change the level or duration of the antibody response in the gut. In conclusion, probiotics enhance the systemic antibody response to some antigens in chickens, but it remains to be seen whether probiotics have an effect on the generation of the mucosal antibody response.

  6. Longitudinal analysis of antibody response to immunization in paediatric survivors after allogeneic haematopoietic stem cell transplantation (United States)

    Inaba, Hiroto; Hartford, Christine M.; Pei, Deqing; Posner, Meredith J.; Yang, Jie; Hayden, Randall T.; Srinivasan, Ashok; Triplett, Brandon M.; McCulllers, Jon A.; Pui, Ching-Hon; Leung, Wing


    Summary The long-term antibody responses to re-immunization in recipients of allogeneic haematopoietic stem cell transplantation (allo-HSCT) have not been well studied. We prospectively and longitudinally evaluated the antibody responses to 8 vaccine antigens (diphtheria, tetanus, pertussis, measles, mumps, rubella, hepatitis B, and poliovirus) and assessed the factors associated with negative titres in 210 allo-HSCT recipients at St. Jude Children’s Research Hospital. Antibody responses lasting for more than 5 years after immunization were observed in most patients for tetanus (95.7%), rubella (92.3%), poliovirus (97.9%), and, in diphtheria-tetanus-acellular pertussis (DTaP) recipients, diphtheria (100%). However, responses to pertussis (25.0%), measles (66.7%), mumps (61.5%), hepatitis B (72.9%), and diphtheria in tetanus-diphtheria (Td) recipients (48.6%) were less favourable, with either only transient antibody responses or persistently negative titres. Factors associated with vaccine failure were older age at immunization; lower CD3, CD4 or CD19 counts; higher IgM concentrations; positive recipient cytomegalovirus serology; negative titres before immunization; acute or chronic graft-versus-host disease; and radiation during preconditioning. These response patterns and clinical factors can be used to formulate re-immunization and monitoring strategies. Patients at risk for vaccine failure should have long-term follow-up; those with loss of antibody response or no seroconversion should receive booster immunizations. PMID:22017512

  7. Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein.

    Directory of Open Access Journals (Sweden)

    Zhifeng Chen

    Full Text Available Respiratory syncytial virus (RSV is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction.

  8. [Immune response genes products in human physiology]. (United States)

    Khaitov, R M; Alekseev, L P


    Current data on physiological role of human immune response genes' proteomic products (antigens) are discussed. The antigens are specified by a very high level of diversity that mediates a wide specter ofphysiological functions. They actually provide integrity and biological stability of human as species. These data reveal new ideas on many pathological processes as well as drafts new approaches for prophylaxis and treatment.

  9. Human Monoclonal Antibodies as a Countermeasure Against Botulinum Toxins (United States)


    REPORT Human monoclonal antibodies as a countermeasure against Botulinum toxins 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: In this report, we...Prescribed by ANSI Std. Z39.18 - 31-Aug-2012 Human monoclonal antibodies as a countermeasure against Botulinum toxins Report Title ABSTRACT In this report...DTRA Final Report: Human monoclonal antibodies as a countermeasure against Botulinum toxins   Page 1 of 22 DTRA Final Report: Human monoclonal

  10. A novel polymorphism of human complement component C3 detected by means of a monoclonal antibody

    DEFF Research Database (Denmark)

    Koch, C; Behrendt, N


    A mouse monoclonal antibody, HAV 4-1, obtained after immunization of a BALB/c mouse with purified C3F, detected a novel genetic polymorphism of human complement component C3 in a simple immunoblotting system. The frequency of HAV 4-1-positive genes was 20.1%. Reactivity of HAV 4-1 was closely rel...

  11. [Immune system evolution. (From cells to humans)]. (United States)

    Belek, A S


    The great variety of cells and molecules observed in the mammalian immune system can be explained by stepwise acquisition of them during phylogeny. Self/nonself discrimination and cell-mediated immunity have been present since the early stages of evolution. Although some inducible antimicrobial molecules have been demonstrated in invertebrates, immunoglobulins appear in vertebrates. T and B cell diversity, development of the lymphoid organs, MHC molecules, complement and cytokines are the characteristics that appear through the evolution of vertebrates. Further knowledge that will be obtained from phylogenetic studies will improve our understanding of the immune system of human.

  12. A high-affinity human monoclonal IgM antibody reacting with multiple strains of Mycoplasma hominis

    DEFF Research Database (Denmark)

    Moller, SA; Birkelund, Svend; Borrebaeck, CA


    Human monoclonal antibodies were produced against Mycoplasma hominis by in vitro immunization of peripheral blood lymphocytes from a healthy seropositive donor using low amounts of antigen (5 ng/ml). The immune B lymphocytes were subsequently immortalized by Epstein-Barr virus transformation...

  13. Impact of immunization technology and assay application on antibody performance--a systematic comparative evaluation.

    Directory of Open Access Journals (Sweden)

    Michael C Brown

    Full Text Available Antibodies are quintessential affinity reagents for the investigation and determination of a protein's expression patterns, localization, quantitation, modifications, purification, and functional understanding. Antibodies are typically used in techniques such as Western blot, immunohistochemistry (IHC, and enzyme-linked immunosorbent assays (ELISA, among others. The methods employed to generate antibodies can have a profound impact on their success in any of these applications. We raised antibodies against 10 serum proteins using 3 immunization methods: peptide antigens (3 per protein, DNA prime/protein fragment-boost ("DNA immunization"; 3 per protein, and full length protein. Antibodies thus generated were systematically evaluated using several different assay technologies (ELISA, IHC, and Western blot. Antibodies raised against peptides worked predominantly in applications where the target protein was denatured (57% success in Western blot, 66% success in immunohistochemistry, although 37% of the antibodies thus generated did not work in any of these applications. In contrast, antibodies produced by DNA immunization performed well against both denatured and native targets with a high level of success: 93% success in Western blots, 100% success in immunohistochemistry, and 79% success in ELISA. Importantly, success in one assay method was not predictive of success in another. Immunization with full length protein consistently yielded the best results; however, this method is not typically available for new targets, due to the difficulty of generating full length protein. We conclude that DNA immunization strategies which are not encumbered by the limitations of efficacy (peptides or requirements for full length proteins can be quite successful, particularly when multiple constructs for each protein are used.

  14. Influenza vaccine induces intracellular immune memory of human NK cells.

    Directory of Open Access Journals (Sweden)

    Yaling Dou

    Full Text Available Influenza vaccines elicit antigen-specific antibodies and immune memory to protect humans from infection with drift variants. However, what supports or limits vaccine efficacy and duration is unclear. Here, we vaccinated healthy volunteers with annual vaccine formulations and investigated the dynamics of T cell, natural killer (NK cell and antibody responses upon restimulation with heterologous or homologous influenza virus strains. Influenza vaccines induced potential memory NK cells with increased antigen-specific recall IFN-γ responses during the first 6 months. In the absence of significant changes in other NK cell markers (CD45RO, NKp44, CXCR6, CD57, NKG2C, CCR7, CD62L and CD27, influenza vaccines induced memory NK cells with the distinct feature of intracellular NKp46 expression. Indeed, surface NKp46 was internalized, and the dynamic increase in NKp46(intracellular+CD56dim NK cells positively correlated with increased IFN-γ production to influenza virus restimulation after vaccination. In addition, anti-NKp46 antibodies blocked IFN-γ responses. These findings provide insights into a novel mechanism underlying vaccine-induced immunity and NK-related diseases, which may help to design persisting and universal vaccines in the future.

  15. Influenza vaccine induces intracellular immune memory of human NK cells. (United States)

    Dou, Yaling; Fu, Binqing; Sun, Rui; Li, Wenting; Hu, Wanfu; Tian, Zhigang; Wei, Haiming


    Influenza vaccines elicit antigen-specific antibodies and immune memory to protect humans from infection with drift variants. However, what supports or limits vaccine efficacy and duration is unclear. Here, we vaccinated healthy volunteers with annual vaccine formulations and investigated the dynamics of T cell, natural killer (NK) cell and antibody responses upon restimulation with heterologous or homologous influenza virus strains. Influenza vaccines induced potential memory NK cells with increased antigen-specific recall IFN-γ responses during the first 6 months. In the absence of significant changes in other NK cell markers (CD45RO, NKp44, CXCR6, CD57, NKG2C, CCR7, CD62L and CD27), influenza vaccines induced memory NK cells with the distinct feature of intracellular NKp46 expression. Indeed, surface NKp46 was internalized, and the dynamic increase in NKp46(intracellular)+CD56dim NK cells positively correlated with increased IFN-γ production to influenza virus restimulation after vaccination. In addition, anti-NKp46 antibodies blocked IFN-γ responses. These findings provide insights into a novel mechanism underlying vaccine-induced immunity and NK-related diseases, which may help to design persisting and universal vaccines in the future.

  16. Delayed adaptive immunity is related to higher MMR vaccine-induced antibody titers in children. (United States)

    Strömbeck, Anna; Lundell, Anna-Carin; Nordström, Inger; Andersson, Kerstin; Adlerberth, Ingegerd; Wold, Agnes E; Rudin, Anna


    There are notable inter-individual variations in vaccine-specific antibody responses in vaccinated children. The aim of our study was to investigate whether early-life environmental factors and adaptive immune maturation prior and close to measles-mumps-rubella (MMR) immunization relate to magnitudes of vaccine-specific antibody titers. In the FARMFLORA birth cohort, including both farming and non-farming families, children were immunized with the MMR vaccine at 18 months of age. MMR vaccine-induced antibody titers were measured in plasma samples obtained at 36 months of age. Infants' blood samples obtained at birth, 3-5 days and at 4 and 18 months of age were analyzed for T- and B-cell numbers, proportions of naive and memory T and B cells, and fractions of putative regulatory T cells. Multivariate factor analyses show that higher anti-MMR antibody titers were associated with a lower degree of adaptive immune maturation, that is, lower proportions of memory T cells and a lower capacity of mononuclear cells to produce cytokines, but with higher proportions of putative regulatory T cells. Further, children born by cesarean section (CS) had significantly higher anti-measles titers than vaginally-born children; and CS was found to be associated with delayed adaptive immunity. Also, girls presented with significantly higher anti-mumps and anti-rubella antibody levels than boys at 36 months of age. These results indicate that delayed adaptive immune maturation before and in close proximity to immunization seems to be advantageous for the ability of children to respond with higher anti-MMR antibody levels after vaccination.

  17. Microbiota-driven immune cellular maturation is essential for antibody-mediated adaptive immunity to Staphylococcus aureus infection in the eye. (United States)

    Zaidi, Tanweer; Zaidi, Tauqeer; Cywes-Bentley, Colette; Lu, Roger; Priebe, Gregory P; Pier, Gerald B


    As an immune-privileged site, the eye, and particularly the outer corneal surface, lacks resident mature immune effector cells. Physical barriers and innate mediators are the best-described effectors of immunity in the cornea. When the barriers are breached, infection can result in rapid tissue destruction, leading to loss of visual acuity and frank blindness. To determine the cellular and molecular components needed for effective adaptive immunity on the corneal surface, we investigated which immune system effectors were required for protection against Staphylococcus aureus corneal infections in mice, which are a serious cause of human eye infections. Both systemically injected and topically applied antibodies to the conserved cell surface polysaccharide poly-N-acetylglucosamine (PNAG) were effective at mediating reductions in corneal pathology and bacterial levels. Additional host factors impacting protection included intercellular adhesion molecule 1 (ICAM-1)-dependent polymorphonuclear leukocyte (PMN) recruitment, functional CD4(+) T cells, signaling via the interleukin-17 (IL-17) receptor, and IL-22 production. In germfree mice, there was no protective efficacy of antibody to PNAG due to the lack of LY6G(+) inflammatory cell coeffector recruitment to the cornea. Protection was manifest after 3 weeks of exposure to conventional mice and acquisition of a resident microbiota. We conclude that in the anterior eye, ICAM-1-mediated PMN recruitment to the infected cornea along with endogenous microbiota-matured CD4(+) T cells producing both IL-17 and IL-22 is required for antibody to PNAG to protect against S. aureus infection. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  18. Human Immune Responses to Dengue Viruses. (United States)


    FA titer of these antisera. We found using these hyper- immunized murine ascitis fluids that the homologous antiserum was most active in augmenting...statistically significant (pɘ.05). CHyperimmune mouse ascitis fluid was used as a source of anti-dengue 2 anti- body at a 1:20 dilution. PBL without anti-dengue 2 antibody. *statistically significant (pɘ.05), l1not significant. bHyperimmune mouse ascitis fluid was used as a source

  19. Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries

    Directory of Open Access Journals (Sweden)

    Antti Tullila


    Full Text Available Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab library.

  20. Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries (United States)

    Tullila, Antti; Nevanen, Tarja K.


    Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library. PMID:28561803

  1. General in vitro method to analyze the interactions of synthetic polymers with human antibody repertoires. (United States)

    Soshee, Anandakumar; Zürcher, Stefan; Spencer, Nicholas D; Halperin, Avraham; Nizak, Clément


    Recent reports on the hitherto underestimated antigenicity of poly(ethylene glycol) (PEG), which is widely used for pharmaceutical applications, highlight the need for efficient testing of polymer antigenicity and for a better understanding of its molecular origins. With this goal in mind, we have used the phage-display technique to screen large, recombinant antibody repertoires of human origin in vitro for antibodies that bind poly(vinylpyrrolidone) (PVP). PVP is a neutral synthetic polymer of industrial and clinical interest that is also a well-known model antigen in animal studies, thus allowing the comparison of in vitro and in vivo responses. We have identified 44 distinct antibodies that bind specifically to PVP. Competitive binding assays show that the PVP-antibody binding constant is proportional to the polymerization degree of PVP and that specific binding is detected down to the vinylpyrrolidone (VP) monomer level. Statistical analysis of anti-PVP antibody sequences identifies an amino-acid motif that is shared by many phage-display-selected anti-PVP antibodies that are similar to a previously described natural anti-PVP antibody. This suggests a role for this motif in specific antibody/PVP interactions. Interestingly, sequence analysis also suggests that only a single antibody chain containing this shared motif is responsible for antibody binding to PVP, as confirmed upon systematic deletion of either antibody chain for 90% of selected anti-PVP antibodies. Overall, a large number of antibodies in the human repertoires we have screened bind specifically to PVP through a small number of shared amino acid motifs, and preliminary comparison points to significant correlations between the sequences of phage-display-selected anti-PVP antibodies and their natural counterparts isolated from immunized mice in previous studies. This study pioneers the use of antibody phage-display to explore the antigenicity of biotechnologically relevant polymers. It also paves the

  2. Plasmodium falciparum synthetic LbL microparticle vaccine elicits protective neutralizing antibody and parasite-specific cellular immune responses. (United States)

    Powell, Thomas J; Tang, Jie; Derome, Mary E; Mitchell, Robert A; Jacobs, Andrea; Deng, Yanhong; Palath, Naveen; Cardenas, Edwin; Boyd, James G; Nardin, Elizabeth


    Epitopes of the circumsporozoite (CS) protein of Plasmodium falciparum, the most pathogenic species of the malaria parasite, have been shown to elicit protective immunity in experimental animals and human volunteers. The mechanisms of immunity include parasite-neutralizing antibodies that can inhibit parasite motility in the skin at the site of infection and in the bloodstream during transit to the hepatocyte host cell and also block interaction with host cell receptors on hepatocytes. In addition, specific CD4+ and CD8+ cellular mechanisms target the intracellular hepatic forms, thus preventing release of erythrocytic stage parasites from the infected hepatocyte and the ensuing blood stage cycle responsible for clinical disease. An innovative method for producing particle vaccines, layer-by-layer (LbL) fabrication of polypeptide films on solid CaCO3 cores, was used to produce synthetic malaria vaccines containing a tri-epitope CS peptide T1BT comprising the antibody epitope of the CS repeat region (B) and two T-cell epitopes, the highly conserved T1 epitope and the universal epitope T. Mice immunized with microparticles loaded with T1BT peptide developed parasite-neutralizing antibodies and malaria-specific T-cell responses including cytotoxic effector T-cells. Protection from liver stage infection following challenge with live sporozoites from infected mosquitoes correlated with neutralizing antibody levels. Although some immunized mice with low or undetectable neutralizing antibodies were also protected, depletion of T-cells prior to challenge resulted in the majority of mice remaining resistant to challenge. In addition, mice immunized with microparticles bearing only T-cell epitopes were not protected, demonstrating that cellular immunity alone was not sufficient for protective immunity. Although the microparticles without adjuvant were immunogenic and protective, a simple modification with the lipopeptide TLR2 agonist Pam3Cys increased the potency and

  3. A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells (United States)

    Colcher, D.; Horan Hand, P.; Nuti, M.; Schlom, J.


    Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts of apparently normal human liver. As a result of further screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a ``pancarcinoma'' reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.

  4. The ABCs (Antibody, B cells, and Carbohydrate epitopes) of cholera immunity: considerations for an improved vaccine. (United States)

    Provenzano, Daniele; Kovác, Pavol; Wade, William F


    Cholera, a diarrheal disease, is known for explosive epidemics that can quickly kill thousands. Endemic cholera is a seasonal torment that also has a significant mortality. Not all nations with extensive rural communities can achieve the required infrastructure or behavioral changes to prevent epidemic or endemic cholera. For some communities, a single-dose cholera vaccine that protects those at risk is the most efficacious means to reduce morbidity and mortality. It is clear that our understanding of what a protective cholera immune response is has not progressed at the rate our understanding of the pathogenesis and molecular biology of cholera infection has. This review addresses V. cholerae lipopolysaccharide (LPS)-based immunogens because LPS is the only immunogen proven to induce protective antibody in humans. We discuss the role of anti-LPS antibodies in protection from cholera, the importance and the potential role of B cell subsets in protection that is based on their anatomical location and the intrinsic antigen-receptor specificity of various subsets is introduced.

  5. [Human single chain antibodies directed to tumor necrosis factor]. (United States)

    Vikhrova, M A; Batanova, T A; Lebedev, L R; Shingarova, L N; Frank, L A; Kirpichnikov, M P; Tikunova, N V


    Six unique phage antibodies to human TNF have been selected from a combinatorial library of human single chain fragment variable. ELISA and Western-blotting was used to study selected phage antibodies binding with TNF. The specificity of selected antibodies was determined by binding with interferon alpha and gamma, bovine serum albumin, ovalbumin and ubiquitin. Two antibodies, sA1 and sB3, were converted into a soluble single-chain antibody form and their affinity was 2.5 and 13.7 nM respectively.


    NARCIS (Netherlands)



    Pulmonary infections occur so frequently in recipients of lung transplants as well as of combined heart and lung transplants that it has been suggested that the function of the defense system in lung transplants is impaired. Therefore, we investigated in rats whether antibody responses against intra

  7. The schistosoma-specific antibody response after treatment in non-immune travellers

    DEFF Research Database (Denmark)

    Duus, Liv Marie; Christensen, Anders Vittrup; Navntoft, Dorte;


    ) and Membrane Bound Antigen (MBA) assays in assessment of treatment efficacy and number of treated non-immune individuals with signs of treatment failure. In a retrospective study, residents in Denmark diagnosed with positive Schistosoma antibodies in the period 1987 - 2004 were offered follow-up including...... analyses for GAA, MBA, IgE and eosinophil count. Among 98 patients with positive antibody at time of diagnosis, 73 were examined for eggs and 27% had detectable eggs. 15% still had detectable living eggs after 1 course of treatment. At follow-up it was demonstrated that antibodies continued to increase...... for up to 6 months after treatment and average duration of positive GAA antibody was approximately 10 y. The study confirms that the GAA- and MBA-IFAT are not suitable in monitoring results of therapy. Treatment failure in 15% of non-immune patients indicates that studies are needed to define the correct...

  8. A novel human-derived antibody against NY-ESO-1 improves the efficacy of chemotherapy. (United States)

    Gupta, Anurag; Nuber, Natko; Esslinger, Christoph; Wittenbrink, Mareike; Treder, Martin; Landshammer, Alexandro; Noguchi, Takuro; Kelly, Marcus; Gnjatic, Sacha; Ritter, Erika; von Boehmer, Lotta; Nishikawa, Hiroyoshi; Shiku, Hiroshi; Old, Lloyd; Ritter, Gerd; Knuth, Alexander; van den Broek, Maries


    We investigated whether antibodies against intracellular tumor-associated antigens support tumor-specific immunity when administered together with a treatment that destroys the tumor. We propose that released antigens form immune complexes with the antibodies, which are then efficiently taken up by dendritic cells. We cloned the first human monoclonal antibodies against the Cancer/Testis (CT) antigen, NY-ESO-1. We tested whether the monoclonal anti-NY-ESO-1 antibody (12D7) facilitates cross-presentation of a NY-ESO-1-derived epitope by dendritic cells to human CD8+ T cells, and whether this results in the maturation of dendritic cells in vitro. We investigated the efficacy of 12D7 in combination with chemotherapy using BALB/c mice bearing syngeneic CT26 tumors that express intracellular NY-ESO-1. Human dendritic cells that were incubated with NY-ESO-1:12D7 immune complexes efficiently stimulated NY-ESO-1(157-165)/HLA-A2-specific human CD8+ T cells to produce interferon-γ, whereas NY-ESO-1 alone did not. Furthermore, the incubation of dendritic cells with NY-ESO-1:12D7 immune complexes resulted in the maturation of dendritic cells. Treatment of BALB/c mice that bear CT26/NY-ESO-1 tumors with 5-fluorouracil (5-FU) plus 12D7 was significantly more effective than chemotherapy alone. We propose systemic injection of monoclonal antibodies (mAbs) against tumor-associated antigens plus a treatment that promotes the local release of those antigens resulting in immune complex formation as a novel therapeutic modality for cancer.

  9. Exploring the antigenic response to multiplexed immunizations in a chicken model of antibody production

    DEFF Research Database (Denmark)

    Kousted, Tina Mostrup; Kalliokoski, Otto; Christensen, Sofie Kjellerup


    . With an upper limit of 1 mg protein material recommended for chicken immunizations, we found that the maximum number of immunogens that can be reliably used is most likely in the low double digits. The limiting factor for a response to an immunogen could not be related to the number of splenic plasma cells...... plasma cell involvement of less than 5%. Two breeds of egg-layers were compared with respect to antibody production in an initial experiment, but differences in antibody productivity were negligible. Although our findings support the use of multiplexed immunizations in the hen, we find that the number...

  10. A malaria vaccine that elicits in humans antibodies able to kill Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)


    Full Text Available BACKGROUND: Plasmodium falciparum merozoite surface protein 3 is a malaria vaccine candidate that was identified, characterised, and developed based on a unique immuno-clinical approach. The vaccine construct was derived from regions fully conserved among various strains and containing B cell epitopes targeted by human antibodies (from malaria-immune adults that are able to mediate a monocyte-dependent parasite killing effect. The corresponding long synthetic peptide was administered to 36 volunteers, with either alum or Montanide ISA720 as adjuvant. METHODS AND FINDINGS: Both formulations induced cellular and humoral immune responses. With alum, the responses lasted up to 12 mo. The vaccine-induced antibodies were predominantly of cytophilic classes, i.e., able to cooperate with effector cells. In vitro, the antibodies induced an inhibition of the P. falciparum erythrocytic growth in a monocyte-dependent manner, which was in most instances as high as or greater than that induced by natural antibodies from immune African adults. In vivo transfer of the volunteers' sera into P. falciparum-infected humanized SCID mice profoundly reduced or abrogated parasitaemia. These inhibitory effects were related to the antibody reactivity with the parasite native protein, which was seen in 60% of the volunteers, and remained in samples taken 12 mo postimmunisation. CONCLUSION: This is the first malaria vaccine clinical trial to clearly demonstrate antiparasitic activity by vaccine-induced antibodies by both in vitro and in vivo methods. The results, showing the induction of long-lasting antibodies directed to a fully conserved polypeptide, also challenge current concepts about malaria vaccines, such as unavoidable polymorphism, low antigenicity, and poor induction of immune memory.

  11. A malaria vaccine that elicits in humans antibodies able to kill Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Pierre Druilhe


    Full Text Available Plasmodium falciparum merozoite surface protein 3 is a malaria vaccine candidate that was identified, characterised, and developed based on a unique immuno-clinical approach. The vaccine construct was derived from regions fully conserved among various strains and containing B cell epitopes targeted by human antibodies (from malaria-immune adults that are able to mediate a monocyte-dependent parasite killing effect. The corresponding long synthetic peptide was administered to 36 volunteers, with either alum or Montanide ISA720 as adjuvant.Both formulations induced cellular and humoral immune responses. With alum, the responses lasted up to 12 mo. The vaccine-induced antibodies were predominantly of cytophilic classes, i.e., able to cooperate with effector cells. In vitro, the antibodies induced an inhibition of the P. falciparum erythrocytic growth in a monocyte-dependent manner, which was in most instances as high as or greater than that induced by natural antibodies from immune African adults. In vivo transfer of the volunteers' sera into P. falciparum-infected humanized SCID mice profoundly reduced or abrogated parasitaemia. These inhibitory effects were related to the antibody reactivity with the parasite native protein, which was seen in 60% of the volunteers, and remained in samples taken 12 mo postimmunisation.This is the first malaria vaccine clinical trial to clearly demonstrate antiparasitic activity by vaccine-induced antibodies by both in vitro and in vivo methods. The results, showing the induction of long-lasting antibodies directed to a fully conserved polypeptide, also challenge current concepts about malaria vaccines, such as unavoidable polymorphism, low antigenicity, and poor induction of immune memory.

  12. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity. (United States)

    Asti, Lorenzo; Uguzzoni, Guido; Marcatili, Paolo; Pagnani, Andrea


    The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10-6), outperforming other sequence- and structure-based models.

  13. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity.

    Directory of Open Access Journals (Sweden)

    Lorenzo Asti


    Full Text Available The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10-6, outperforming other sequence- and structure-based models.

  14. A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words

    Energy Technology Data Exchange (ETDEWEB)

    Mirick, G. R.; Bradt, B. M.; Denardo, S. J.; Denardo, G. L. [Calfornia Univ., Sacramento (United States). Davis Medical Center


    The United States Food and Drugs Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ({sup 9}0yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ({sup 1}31I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) antiCD20 MAns for use in radioimmunotherapy (RIT) of non-Hodgikin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, essays were developed to determine HAGE (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to humanize MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades.

  15. A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words. (United States)

    Mirick, G R; Bradt, B M; Denardo, S J; Denardo, G L


    The United States Food and Drug Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ((90)yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ((131)I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) anti-CD20 MAbs for use in radioimmunotherapy (RIT) of non-Hodgkin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, assays were developed to determine HAGA (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to ''humanize'' MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades.

  16. A human PrM antibody that recognizes a novel cryptic epitope on dengue E glycoprotein. (United States)

    Chan, Annie Hoi Yi; Tan, Hwee Cheng; Chow, Angelia Yee; Lim, Angeline Pei Chiew; Lok, Shee Mei; Moreland, Nicole J; Vasudevan, Subhash G; MacAry, Paul A; Ooi, Eng Eong; Hanson, Brendon J


    Dengue virus (DENV) is a major mosquito-borne pathogen infecting up to 100 million people each year; so far no effective treatment or vaccines are available. Recently, highly cross-reactive and infection-enhancing pre-membrane (prM)-specific antibodies were found to dominate the anti-DENV immune response in humans, raising concern over vaccine candidates that contain native dengue prM sequences. In this study, we have isolated a broadly cross-reactive prM-specific antibody, D29, during a screen with a non-immunized human Fab-phage library against the four serotypes of DENV. The antibody is capable of restoring the infectivity of virtually non-infectious immature DENV (imDENV) in FcγR-bearing K562 cells. Remarkably, D29 also cross-reacted with a cryptic epitope on the envelope (E) protein located to the DI/DII junction as evidenced by site-directed mutagenesis. This cryptic epitope, while inaccessible to antibody binding in a native virus particle, may become exposed if E is not properly folded. These findings suggest that generation of anti-prM antibodies that enhance DENV infection may not be completely avoided even with immunization strategies employing E protein alone or subunits of E proteins.

  17. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity

    DEFF Research Database (Denmark)

    Asti, Lorenzo; Uguzzoni, Guido; Marcatili, Paolo


    The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high...... of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10-6), outperforming other sequence- and structure-based models....

  18. Human immune system mice immunized with Plasmodium falciparum circumsporozoite protein induce protective human humoral immunity against malaria. (United States)

    Huang, Jing; Li, Xiangming; Coelho-dos-Reis, Jordana G A; Zhang, Min; Mitchell, Robert; Nogueira, Raquel Tayar; Tsao, Tiffany; Noe, Amy R; Ayala, Ramses; Sahi, Vincent; Gutierrez, Gabriel M; Nussenzweig, Victor; Wilson, James M; Nardin, Elizabeth H; Nussenzweig, Ruth S; Tsuji, Moriya


    In this study, we developed human immune system (HIS) mice that possess functional human CD4+ T cells and B cells, named HIS-CD4/B mice. HIS-CD4/B mice were generated by first introducing HLA class II genes, including DR1 and DR4, along with genes encoding various human cytokines and human B cell activation factor (BAFF) to NSG mice by adeno-associated virus serotype 9 (AAV9) vectors, followed by engrafting human hematopoietic stem cells (HSCs). HIS-CD4/B mice, in which the reconstitution of human CD4+ T and B cells resembles to that of humans, produced a significant level of human IgG against Plasmodium falciparum circumsporozoite (PfCS) protein upon immunization. CD4+ T cells in HIS-CD4/B mice, which possess central and effector memory phenotypes like those in humans, are functional, since PfCS protein-specific human CD4+ T cells secreting IFN-γ and IL-2 were detected in immunized HIS-CD4/B mice. Lastly, PfCS protein-immunized HIS-CD4/B mice were protected from in vivo challenge with transgenic P. berghei sporozoites expressing the PfCS protein. The immune sera collected from protected HIS-CD4/B mice reacted against transgenic P. berghei sporozoites expressing the PfCS protein and also inhibited the parasite invasion into hepatocytes in vitro. Taken together, these studies show that our HIS-CD4/B mice could mount protective human anti-malaria immunity, consisting of human IgG and human CD4+ T cell responses both specific for a human malaria antigen.

  19. Antibody Profiling in Naive and Semi-immune Individuals Experimentally Challenged with Plasmodium vivax Sporozoites.

    Directory of Open Access Journals (Sweden)

    Myriam Arévalo-Herrera


    Full Text Available Acquisition of malaria immunity in low transmission areas usually occurs after relatively few exposures to the parasite. A recent Plasmodium vivax experimental challenge trial in malaria naïve and semi-immune volunteers from Colombia showed that all naïve individuals developed malaria symptoms, whereas semi-immune subjects were asymptomatic or displayed attenuated symptoms. Sera from these individuals were analyzed by protein microarray to identify antibodies associated with clinical protection.Serum samples from naïve (n = 7 and semi-immune (n = 9 volunteers exposed to P. vivax sporozoite-infected mosquito bites were probed against a custom protein microarray displaying 515 P. vivax antigens. The array revealed higher serological responses in semi-immune individuals before the challenge, although malaria naïve individuals also had pre-existing antibodies, which were higher in Colombians than US adults (control group. In both experimental groups the response to the P. vivax challenge peaked at day 45 and returned to near baseline at day 145. Additional analysis indicated that semi-immune volunteers without fever displayed a lower response to the challenge, but recognized new antigens afterwards.Clinical protection against experimental challenge in volunteers with previous P. vivax exposure was associated with elevated pre-existing antibodies, an attenuated serological response to the challenge and reactivity to new antigens.

  20. The natural immunity to evolutionary atavistic endotoxin for human cancer. (United States)

    Moncevičiūtė-Eringienė, Elena; Rotkevič, Kristina; Grikienis, Ruta Grikienyte


    We propose a new theory of the immunological control of cancer corresponding to the hypothesis that the specific natural immunity to evolutionary atavistic endotoxin has a potential role in human cancer prevention. The results of our studies have shown that IgMNAE, i.e. endogenous or spontaneous IgM class antibodies to enterobacterial lipopolysaccharide molecules (lipid A), control the immune mechanisms responsible for the internal medium stability not only against the damaging impact of the carcinogenic factors, but also against the malignant transformation of its own degenerated cells. Among people who in 1979 and 1982 had IgMNAE in their blood serum, after 15-30years fell ill with cancer 10%, versus 15% among people who had no IgMNAE (pimmunity to endotoxin it is possible to see the formation of the respective evolutionary protective reactions which protect the damaged cells from acquiring resistance to damaging factors and thus from becoming an independent new parasitic population. Thereby the presented theory of the immunological control of cancer has a causal connection with our evolutionary resistance theory of the origin of cancer. Collectively, these data suggest that activation of natural immunity to endotoxin and production of vaccines against evolutionary atavistic endotoxin or gram-negative bacterial endotoxin can be helpful when applied in cancer prophylaxis for persons with a low level of natural immunity to endotoxin and perhaps in creating immunotherapeutic methods for stopping the endogenous parasitism of tumour cells by binding IgMNAE to atavistic endotoxin in cancer patients.

  1. Analysis of Antibodies Directed against Merozoite Surface Protein 1 of the Human Malaria Parasite Plasmodium falciparum (United States)

    Woehlbier, Ute; Epp, Christian; Kauth, Christian W.; Lutz, Rolf; Long, Carole A.; Coulibaly, Boubacar; Kouyaté, Bocar; Arevalo-Herrera, Myriam; Herrera, Sócrates; Bujard, Hermann


    The 190-kDa merozoite surface protein 1 (MSP-1) of Plasmodium falciparum, an essential component in the parasite's life cycle, is a primary candidate for a malaria vaccine. Rabbit antibodies elicited by the heterologously produced MSP-1 processing products p83, p30, p38, and p42, derived from strain 3D7, were analyzed for the potential to inhibit in vitro erythrocyte invasion by the parasite and parasite growth. Our data show that (i) epitopes recognized by antibodies, which inhibit parasite replication, are distributed throughout the entire MSP-1 molecule; (ii) when combined, antibodies specific for different regions of MSP-1 inhibit in a strictly additive manner; (iii) anti-MSP-1 antibodies interfere with erythrocyte invasion as well as with the intraerythrocytic growth of the parasite; and (iv) antibodies raised against MSP-1 of strain 3D7 strongly cross-inhibit replication of the heterologous strain FCB-1. Accordingly, anti-MSP-1 antibodies appear to be capable of interfering with parasite multiplication at more than one level. Since the overall immunogenicity profile of MSP-1 in rabbits closely resembles that found in sera of Aotus monkeys immunized with parasite-derived MSP-1 and of humans semi-immune to malaria from whom highly inhibiting antigen-specific antibodies were recovered, we consider the findings reported here to be relevant for the development of MSP-1-based vaccines against malaria. PMID:16428781

  2. Immune antibodies and helminth products drive CXCR2-dependent macrophage-myofibroblast crosstalk to promote intestinal repair.

    Directory of Open Access Journals (Sweden)

    Julia Esser-von Bieren


    Full Text Available Helminth parasites can cause considerable damage when migrating through host tissues, thus making rapid tissue repair imperative to prevent bleeding and bacterial dissemination particularly during enteric infection. However, how protective type 2 responses targeted against these tissue-disruptive multicellular parasites might contribute to homeostatic wound healing in the intestine has remained unclear. Here, we observed that mice lacking antibodies (Aid-/- or activating Fc receptors (Fcrg-/- displayed impaired intestinal repair following infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb, whilst transfer of immune serum could partially restore chemokine production and rescue wound healing in Aid-/- mice. Impaired healing was associated with a reduced expression of CXCR2 ligands (CXCL2/3 by macrophages (MΦ and myofibroblasts (MF within intestinal lesions. Whilst antibodies and helminths together triggered CXCL2 production by MΦ in vitro via surface FcR engagement, chemokine secretion by intestinal MF was elicited by helminths directly via Fcrg-chain/dectin2 signaling. Blockade of CXCR2 during Hpb challenge infection reproduced the delayed wound repair observed in helminth infected Aid-/- and Fcrg-/- mice. Finally, conditioned media from human MΦ stimulated with infective larvae of the helminth Ascaris suum together with immune serum, promoted CXCR2-dependent scratch wound closure by human MF in vitro. Collectively our findings suggest that helminths and antibodies instruct a chemokine driven MΦ-MF crosstalk to promote intestinal repair, a capacity that may be harnessed in clinical settings of impaired wound healing.

  3. Immune antibodies and helminth products drive CXCR2-dependent macrophage-myofibroblast crosstalk to promote intestinal repair. (United States)

    Esser-von Bieren, Julia; Volpe, Beatrice; Sutherland, Duncan B; Bürgi, Jérôme; Verbeek, J Sjef; Marsland, Benjamin J; Urban, Joseph F; Harris, Nicola L


    Helminth parasites can cause considerable damage when migrating through host tissues, thus making rapid tissue repair imperative to prevent bleeding and bacterial dissemination particularly during enteric infection. However, how protective type 2 responses targeted against these tissue-disruptive multicellular parasites might contribute to homeostatic wound healing in the intestine has remained unclear. Here, we observed that mice lacking antibodies (Aid-/-) or activating Fc receptors (Fcrg-/-) displayed impaired intestinal repair following infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb), whilst transfer of immune serum could partially restore chemokine production and rescue wound healing in Aid-/- mice. Impaired healing was associated with a reduced expression of CXCR2 ligands (CXCL2/3) by macrophages (MΦ) and myofibroblasts (MF) within intestinal lesions. Whilst antibodies and helminths together triggered CXCL2 production by MΦ in vitro via surface FcR engagement, chemokine secretion by intestinal MF was elicited by helminths directly via Fcrg-chain/dectin2 signaling. Blockade of CXCR2 during Hpb challenge infection reproduced the delayed wound repair observed in helminth infected Aid-/- and Fcrg-/- mice. Finally, conditioned media from human MΦ stimulated with infective larvae of the helminth Ascaris suum together with immune serum, promoted CXCR2-dependent scratch wound closure by human MF in vitro. Collectively our findings suggest that helminths and antibodies instruct a chemokine driven MΦ-MF crosstalk to promote intestinal repair, a capacity that may be harnessed in clinical settings of impaired wound healing.

  4. The Fc and not CD4 Receptor Mediates Antibody Enhancement of HIV Infection in Human Cells (United States)

    Homsy, Jacques; Meyer, Mia; Tateno, Masatoshi; Clarkson, Sarah; Levy, Jay A.


    Antibodies that enhance human immunodeficiency virus (HIV) infectivity have been found in the blood of infected individuals and in infected or immunized animals. These findings raise serious concern for the development of a safe vaccine against acquired immunodeficiency syndrome. To address the in vivo relevance and mechanism of this phenomenon, antibody-dependent enhancement of HIV infectivity in peripheral blood macrophages, lymphocytes, and human fibroblastoid cells was studied. Neither Leu3a, a monoclonal antibody directed against the CD4 receptor, nor soluble recombinant CD4 even at high concentrations prevented this enhancement. The addition of monoclonal antibody to the Fc receptor III (anti-FcRIII), but not of antibodies that react with FcRI or FcRII, inhibited HIV type 1 and HIV type 2 enhancement in peripheral blood macrophages. Although enhancement of HIV infection in CD4+ lymphocytes could not be blocked by anti-FcRIII, it was inhibited by the addition of human immunoglobulin G aggregates. The results indicate that the FcRIII receptor on human macrophages and possibly another Fc receptor on human CD4+ lymphocytes mediate antibody-dependent enhancement of HIV infectivity and that this phenomenon proceeds through a mechanism independent of the CD4 protein.

  5. A monoclonal antibody against human MUDENG protein. (United States)

    Wagley, Yadav; Choi, Jun-Ha; Wickramanayake, Dimuthu Dhammika; Choi, Geun-Yeol; Kim, Chang-Kyu; Kim, Tae-Hyoung; Oh, Jae-Wook


    MUDENG (mu-2-related death-inducing gene, MuD) encodes a predicted ∼54-kDa protein in humans, considered to be involved in trafficking proteins from endosomes toward other membranous compartments as well as in inducing cell death. Here we report on the generation of a mouse monoclonal antibody (MAb) against the middle domain of human (h) MuD. This IgG sub 1 MAb, named M3H9, recognizes residues 244-326 in the middle domain of the MuD protein. Thus, the MuD proteins expressed in an astroglioma cell line and primary astrocytes can be detected by the M3H9 MAb. We showed that M3H9 MAb can be useful in enzyme-linked immunosorbent assay (ELISA) and immunoblot experiments. In addition, M3H9 MAb can detect the expression of the MuD protein in formalin-fixed, paraffin-embedded mouse ovary and uterus tissues. These results indicate that the MuD MAb M3H9 could be useful as a new biomarker of hereditary spastic paraplegia and other related diseases.

  6. SSB peptide and DNA co-immunization induces inhibition of anti-dsDNA antibody production in rabbits

    Institute of Scientific and Technical Information of China (English)


    Background Patients with systemic lupus erythematosus often have various autoantibodies.The relationship between these antibodies is still poorly understood.The aim of the present study was to observe the anti-SSB antibody and anti-dsDNA antibody production profiles following immunization with synthetic SSB peptide alone,DNA alone or co-immunization with these two antigens.Methods SSB 214-225 aa peptide was synthesized by organic chemistry solid-phase peptide synthesis.Rabbits were immunized with the foliowing antigens:synthetic SSB peptide linked with keyhole limpet hemocyanin (KLH),DNA,SSB plus dsDNA,KLH and PBS.Antibodies were measured by ELISA.Histopathology and direct immufluorescence assays were also applied.Results Ainit-SSB and anti-dsDNA antibodies were produced following immunization with SSB peptide and DNA respectively.The level of SSB antibody in the co-immunization group was higher than that of the SSB peptide immunization group.The level of anti-dsDNA antibody in the co-immunization group was,however,lower than that in the DNA immunization group.Meanwhile,the level of anti-SSB antibody was higher than that of anti-DNA antibody in the co-immunization group.No morphological or immunological abnormalities were found in the heart,liver,kidney,spleen or skin tissues.Conclusion Inhibition of anti-dsDNA-antibody was induced by co-immunization with synthesized SSB peptide and DNA,which might explain,at least partly,the mild disease in some LE subsets associated with SSB antibody.


    Institute of Scientific and Technical Information of China (English)

    刘秉慈; MelvinSpira; 许增禄


    Injectable bovine collagen has been used clinically for years.But both the necessity of repeated injections to maintain corrections and the question of adverse allergic reactions developing from the use of a xenogenic collagen have been an area of serious concern.To overoome these adyerse effects,we have developed injectable collagen preparations from human placenta.Gamma irradiation was used for sterilization and crosslinking of the collagen.We observed the mouse immune respose to gamma-irradiated human placenta soluble and insoluble collagen follow-ing multiple injections.After six injections of these materials,no total IgG level increase was found,nor was anti-body specifically directed against human collagen found.Mouse antibody levels were also observed following Zyderm Ⅱ and Zyplast repetitive injections and follow-ing repetitive implantations of coated vicryl and chromic gut.No humoral immune response was found in this het-erologous type system.

  8. The antibody response to Plasmodium falciparum Merozoite Surface Protein 4: comparative assessment of specificity and growth inhibitory antibody activity to infection-acquired and immunization-induced epitopes

    Directory of Open Access Journals (Sweden)

    Black Casilda G


    Full Text Available Abstract Background Malaria remains a global public health challenge. It is widely believed that an effective vaccine against malaria will need to incorporate multiple antigens from the various stages of the parasite's complex life cycle. Plasmodium falciparum Merozoite Surface Protein 4 (MSP4 is a vaccine candidate that has been selected for development for inclusion in an asexual stage subunit vaccine against malaria. Methods Nine monoclonal antibodies (Mabs were produced against Escherichia coli-expressed recombinant MSP4 protein and characterized. These Mabs were used to develop an MSP4-specific competition ELISA to test the binding specificity of antibodies present in sera from naturally P. falciparum-infected individuals from a malaria endemic region of Vietnam. The Mabs were also tested for their capacity to induce P. falciparum growth inhibition in vitro and compared against polyclonal rabbit serum raised against recombinant MSP4 Results All Mabs reacted with native parasite protein and collectively recognized at least six epitopes. Four of these Mabs recognize reduction-sensitive epitopes within the epidermal growth factor-like domain found near the C-terminus of MSP4. These sera were shown to contain antibodies capable of inhibiting the binding of the six Mabs indicating infection-acquired responses to the six different epitopes of MSP4. All of the six epitopes were readily recognized by human immune sera. Competition ELISA titres varied from 20 to 640, reflecting heterogeneity in the intensity of the humoral response against the protein among different individuals. The IgG responses during acute and convalescent phases of infection were higher to epitopes in the central region than to other parts of MSP4. Immunization with full length MSP4 in Freund's adjuvant induced rabbit polyclonal antisera able to inhibit parasite growth in vitro in a manner proportionate to the antibody titre. By contrast, polyclonal antisera raised to

  9. The immune system in the aging human. (United States)

    Rymkiewicz, Paulina Dominika; Heng, Yi Xiong; Vasudev, Anusha; Larbi, Anis


    With the improvement of medical care and hygienic conditions, there has been a tremendous increment in human lifespan. However, many of the elderly (>65 years) display chronic illnesses, and a majority requires frequent and longer hospitalization. The robustness of the immune system to eliminate or control infections is often eroded with advancing age. Nevertheless, some elderly individuals do cope better than others. The origin of these inter-individual differences may come from genetic, lifestyle conditions (nutrition, socio-economic parameters), as well as the type, number and recurrence of pathogens encountered during life. The theory we are supporting is that chronic infections, through life, will induce profound changes in the immune system probably due to unbalanced inflammatory profiles. Persistent viruses such a cytomegalovirus are not eliminated and are a driven force to immune exhaustion. Because of their age, elderly individuals may have seen more of these chronic stimulators and have experienced more reactivation episodes ultimately leading to shrinkage of their repertoire and overall immune robustness. This review integrates updates on immunity with advancing age and its impact on associated clinical conditions.

  10. Identification of human nonpancreatic-type ribonuclease by antibodies obtained against a synthetic peptide. (United States)

    Bravo, M I; Cuchillo, C M; Nogués, M V


    An antibody that recognizes human nonpancreatic-type ribonuclease was obtained by immunizing a rabbit with a 14-residue synthetic peptide corresponding to the N-terminal sequence of eosinophil-derived neurotoxin which is identical to human liver ribonuclease. This amino acid sequence is unique to this protein. The anti N-peptide antibody was purified by protein A-Sepharose and by using ELISA and SDS-PAGE immunoblot techniques, the antibody reactivity against EDN and partially purified nonpancreatic-type ribonucleases from human plasma and urine was observed. Cross-reactivity with bovine pancreatic ribonuclease A and other proteins was not detected. In addition, the activity of the nonpancreatic-type ribonuclease was not affected by the antibody. The immune response was elicited without the need for a carrier protein showing that the N-terminal sequence of nonpancreatic ribonuclease contains a specific epitope. This antibody can be used for the immunological identification of both the native and denatured forms of this type of enzyme.

  11. Prophylactic and therapeutic activity of fully human monoclonal antibodies directed against Influenza A M2 protein

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    Gwerder Myriam


    Full Text Available Abstract Influenza virus infection is a prevalent disease in humans. Antibodies against hemagglutinin have been shown to prevent infection and hence hemagglutinin is the major constituent of current vaccines. Antibodies directed against the highly conserved extracellular domain of M2 have also been shown to mediate protection against Influenza A infection in various animal models. Active vaccination is generally considered the best approach to combat viral diseases. However, passive immunization is an attractive alternative, particularly in acutely exposed or immune compromized individuals, young children and the elderly. We recently described a novel method for the rapid isolation of natural human antibodies by mammalian cell display. Here we used this approach to isolate human monoclonal antibodies directed against the highly conserved extracellular domain of the Influenza A M2 protein. The identified antibodies bound M2 peptide with high affinities, recognized native cell-surface expressed M2 and protected mice from a lethal influenza virus challenge. Moreover, therapeutic treatment up to 2 days after infection was effective, suggesting that M2-specific monoclonals have a great potential as immunotherapeutic agents against Influenza infection.

  12. A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

    NARCIS (Netherlands)

    Huls, GA; Heijnen, IAFM; Cuomo, ME; Koningsberger, JC; Boel, E; de Vries, ARV; Loyson, SAJ; Helfrich, W; Henegouwen, GPV; van Meijer, M; de Kruif, J; Logtenberg, T


    A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab), The purified huMab had an affinity of 5 nM and effectively mediated tumor cell kil

  13. Urine antibody against human cancer antigen NY-ESO-1


    Jäger, Dirk; Stockert, Elisabeth; Karbach, Julia; Herrlinger, Kristina; Atmaca, Akin; Arand, Michael; Chen, Yao-Tseng; Gnjatic, Sacha; Old, Lloyd J.; Knuth, Alexander; Jäger, Elke


    NY-ESO-1 is one of the most immunogenic tumor antigens known to date. Spontaneous humoral and cellular immune responses against NY-ESO-1 are detected in a substantial proportion of patients with NY-ESO-1 positive cancers. NY-ESO-1 serum antibody is dependent on the presence of NY-ESO-1+ cancer cells, and antibody titers correlate with the clinical development of the disease. NY-ESO-1 serum antibody is associated with detectable NY-ESO-1-specific CD8+ T cell reactivity. High titers of NY-ESO-1...

  14. Immune response in mice to ingested soya protein: antibody production, oral tolerance and maternal transfer

    DEFF Research Database (Denmark)

    Christensen, Hanne Risager; Pedersen, Susanne Brix; Frøkiær, Hanne


    While allergic reactions to soya are increasingly investigated, the normal immune response to ingested soya is scarcely described. In the present study, we wanted to characterise the soya-specific immune response in healthy mice ingesting soya protein. Mice fed a soya-containing diet (F0) and mice...... of the first (F1) and second (F2) offspring generation bred on a soya protein-free diet were used either directly or were transferred between the soya-containing and soya protein-free diet during pregnancy or neonatal life. The mice were compared as to levels of naturally occurring specific antibodies analysed...... by ELISA, and to the presence of oral tolerance detected as a suppressed antibody and cell-proliferation response upon immunisation with soya protein. F0 mice generated soya-specific antibodies, while oral tolerance to the same soya proteins was also clearly induced. When F0 dams were transferred to soya...

  15. Simultaneous Raising of Rabbit Monoclonal Antibodies to Fluoroquinolones with Diverse Recognition Functionalities via Single Mixture Immunization. (United States)

    Liu, Na; Zhao, Zhiyong; Tan, Yanglan; Lu, Lei; Wang, Lin; Liao, Yucai; Beloglazova, Natalia; De Saeger, Sarah; Zheng, Xiaodong; Wu, Aibo


    Highly specific monoclonal and polyclonal antibodies are the key components in a diverse set of immunoassay applications, from research work to routine monitoring and analysis. In the current manuscript, combinatorial strategies for a single mixture immunization, screening and rabbit hybridoma cell technology were described. Fluoroquinolones (FQs) drugs were chosen as representative analytes. Six FQs were conjugated with bovine serum albumin and used as immunogens for subsequent immunization, while a mixture of all was injected for coimmunization. The hybridomas obtained against the individual and multiple FQs were used for the production of diverse varieties of rabbit monoclonal antibodies (RabMAbs) against the target analytes. As was proven by indirect competitive ELISA and quantitative lateral flow immunoassay, this approach opens a new way for simultaneously obtaining functional monoclonal antibodies which are capable of recognizing both individual and multiple analytes in a single preparation circle. This addresses various needs of different monitoring regulations as analytical methodology advances.

  16. Production and Characterization of a Murine Monoclonal Antibody Against Human Ferritin (United States)

    Bayat, Ali Ahmad; Yeganeh, Omid; Ghods, Roya; Zarnani, Amir Hassan; Ardekani, Reza Bahjati; Mahmoudi, Ahmad Reza; Mahmoudian, Jafar; Haghighat-Noutash, Farzaneh; Jeddi-Tehrani, Mahmood


    Background Ferritin is an iron storage protein, which plays a key role in iron metabolism. Measurement of ferritin level in serum is one of the most useful indicators of iron status and also a sensitive measurement of iron deficiency. Monoclonal antibodies may be useful as a tool in various aspects of ferritin investigations. In this paper, the production of a murine monoclonal antibody (mAb) against human ferritin was reported. Methods Balb/c mice were immunized with purified human ferritin and splenocytes of hyper immunized mice were fused with Sp2/0 myeloma cells. After four times of cloning by limiting dilution, a positive hybridoma (clone: 2F9-C9) was selected by ELISA using human ferritin. Anti-ferritin mAb was purified from culture supernatants by affinity chromatography. Results Determination of the antibody affinity for ferritin by ELISA revealed a relatively high affinity (2.34×109 M -1) and the isotype was determined to be IgG2a. The anti-ferritin mAb 2F9-C9 reacted with 79.4% of Hela cells in flow cytometry. The antibody detected a band of 20 kDa in K562 cells, murine and human liver lysates, purified ferritin in Western blot and also ferritin in human serum. Conclusion This mAb can specifically recognize ferritin and may serve as a component of ferritin diagnostic kit if other requirements of the kit are met. PMID:24285995

  17. Induction of neutralizing antibodies in mice immunized with scorpion toxins detoxified by liposomal entrapment

    Directory of Open Access Journals (Sweden)

    S.G. Fonseca


    Full Text Available The possibility of producing neutralizing antibodies against the lethal effects of scorpion toxins was evaluated in the mouse model by immunization with an immunogen devoid of toxicity. A toxic fraction (5 mg from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes. The liposomes were treated for 1 h at 37oC with a 1% (w/w trypsin solution in 0.2 M sodium carbonate buffer, pH 8.3. This treatment led to a strong reduction in venom toxicity. Immunization was performed as follows: mice were injected sc with 20 µg of the liposome-entrapped toxic fraction on days 1 and 21 and a final injection (20 µg was administered ip on day 36. After injection of the immunogen, all mice developed an IgG response which was shown to be specific for the toxic antigen. The antibodies were measured 10 days after the end of the immunization protocol. In an in vitro neutralization assay we observed that pre-incubation of a lethal dose of the toxic fraction with immune serum strongly reduced its toxicity. In vivo protection assays showed that mice with anti-toxin antibodies could resist the challenge with the toxic fraction, which killed, 30 min after injection, all non-immune control mice

  18. Changes in antibody profile after treatment of human onchocerciasis. (United States)

    Lee, S J; Francis, H L; Awadzi, K; Ottesen, E A; Nutman, T B


    To define the changes in antibody response to Onchocerca volvulus antigens after treatment of patients with onchocerciasis, IgG and IgE antibodies were examined quantitatively and qualitatively in 21 patients and 3 control individuals before and sequentially for 14 days after treatment with diethylcarbamazine. The quantitative levels of IgE and IgG responses (both polyclonal and O. volvulus-specific) remained essentially unchanged for all patients, but 9 of the 21 patients showed intensified responses to one or more parasite-specific antigens, and 8 of 21 developed antibodies to previously undetected antigens. There was a significant correlation between the intensities of infection and the development of newly recognized anti-O. volvulus antibodies. These studies demonstrate that O. volvulus-specific IgE and IgG antibody responses are, at least transiently, enhanced by treatment with diethylcarbamazine and that after treatment, parasites possibly release antigens previously hidden from the host's immune response.

  19. Preserved antiviral adaptive immunity following polyclonal antibody immunotherapy for severe murine influenza infection. (United States)

    Stevens, Natalie E; Hatjopolous, Antoinette; Fraser, Cara K; Alsharifi, Mohammed; Diener, Kerrilyn R; Hayball, John D


    Passive immunotherapy may have particular benefits for the treatment of severe influenza infection in at-risk populations, however little is known of the impact of passive immunotherapy on the formation of memory responses to the virus. Ideally, passive immunotherapy should attenuate the severity of infection while still allowing the formation of adaptive responses to confer protection from future exposure. In this study, we sought to determine if administration of influenza-specific ovine polyclonal antibodies could inhibit adaptive immune responses in a murine model of lethal influenza infection. Ovine polyclonal antibodies generated against recombinant PR8 (H1N1) hemagglutinin exhibited potent prophylactic capacity and reduced lethality in an established influenza infection, particularly when administered intranasally. Surviving mice were also protected against reinfection and generated normal antibody and cytotoxic T lymphocyte responses to the virus. The longevity of ovine polyclonal antibodies was explored with a half-life of over two weeks following a single antibody administration. These findings support the development of an ovine passive polyclonal antibody therapy for treatment of severe influenza infection which does not affect the formation of subsequent acquired immunity to the virus.

  20. Mathematical modeling provides kinetic details of the human immune response to vaccination

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    Dustin eLe


    Full Text Available With major advances in experimental techniques to track antigen-specific immune responses many basic questions on the kinetics of virus-specific immunity in humans remain unanswered. To gain insights into kinetics of T and B cell responses in human volunteers we combine mathematical models and experimental data from recent studies employing vaccines against yellow fever and smallpox. Yellow fever virus-specific CD8 T cell population expanded slowly with the average doubling time of 2 days peaking 2.5 weeks post immunization. Interestingly, we found that the peak of the yellow fever-specific CD8 T cell response is determined by the rate of T cell proliferation and not by the precursor frequency of antigen-specific cells as has been suggested in several studies in mice. We also found that while the frequency of virus-specific T cells increases slowly, the slow increase can still accurately explain clearance of yellow fever virus in the blood. Our additional mathematical model describes well the kinetics of virus-specific antibody-secreting cell and antibody response to vaccinia virus in vaccinated individuals suggesting that most of antibodies in 3 months post immunization are derived from the population of circulating antibody-secreting cells. Taken together, our analysis provides novel insights into mechanisms by which live vaccines induce immunity to viral infections and highlight challenges of applying methods of mathematical modeling to the current, state-of-the-art yet limited immunological data.


    Institute of Scientific and Technical Information of China (English)

    白银; 王琰; 周丽君; 俞莉章


    To construct and express a human-mouse chimeric antibody against human bladder cancer. Method: The variable region genes of anti-human bladder cancer monoclonal antibody BDI-1 were cloned by RT-PCR. A human-mouse chimeric antibody expression vector was constructed and transfected into CHO cells. The chimeric antibody against bladder cancer was expressed and characterized. Result: Eukaryotic expression vector of the chimeric antibody against human bladder carcinoma was successfully constructed, and was expressed in eukaryotic cells; the expressed chimeric antibody ch-BDI showed same specificity as its parent McAb against human bladder cancer cells. Conclusion: The constructed chimeric antibody was expressed successfully in eukaryotic cells, and the chimeric antibody had desired affinity against human bladder cancer cells.

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

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



    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.

  3. Human CD8+ T cells mediate protective immunity induced by a human malaria vaccine in human immune system mice. (United States)

    Li, Xiangming; Huang, Jing; Zhang, Min; Funakoshi, Ryota; Sheetij, Dutta; Spaccapelo, Roberta; Crisanti, Andrea; Nussenzweig, Victor; Nussenzweig, Ruth S; Tsuji, Moriya


    A number of studies have shown that CD8+ T cells mediate protective anti-malaria immunity in a mouse model. However, whether human CD8+ T cells play a role in protection against malaria remains unknown. We recently established human immune system (HIS) mice harboring functional human CD8+ T cells (HIS-CD8 mice) by transduction with HLA-A∗0201 and certain human cytokines using recombinant adeno-associated virus-based gene transfer technologies. These HIS-CD8 mice mount a potent, antigen-specific HLA-A∗0201-restricted human CD8+ T-cell response upon immunization with a recombinant adenovirus expressing a human malaria antigen, the Plasmodium falciparum circumsporozoite protein (PfCSP), termed AdPfCSP. In the present study, we challenged AdPfCSP-immunized HIS-CD8 mice with transgenic Plasmodium berghei sporozoites expressing full-length PfCSP and found that AdPfCSP-immunized (but not naïve) mice were protected against subsequent malaria challenge. The level of the HLA-A∗0201-restricted, PfCSP-specific human CD8+ T-cell response was closely correlated with the level of malaria protection. Furthermore, depletion of human CD8+ T cells from AdPfCSP-immunized HIS-CD8 mice almost completely abolished the anti-malaria immune response. Taken together, our data show that human CD8+ T cells mediate protective anti-malaria immunity in vivo.

  4. From network-to-antibody robustness in a bio-inspired immune system. (United States)

    Fernandez-Leon, Jose A; Acosta, Gerardo G; Mayosky, Miguel A


    Behavioural robustness at antibody and immune network level is discussed. The robustness of the immune response that drives an autonomous mobile robot is examined with two computational experiments in the autonomous mobile robots trajectory generation context in unknown environments. The immune response is met based on the immune network metaphor for different low-level behaviours coordination. These behaviours are activated when a robot sense the appropriate conditions in the environment in relation to the network current state. Results are obtained over a case study in computer simulation as well as in laboratory experiments with a Khepera II microrobot. In this work, we develop a set of tests where such an immune response is externally perturbed at network or low-level behavioural modules to analyse the robust capacity of the system to unexpected perturbations. Emergence of robust behaviour and high-level immune response relates to the coupling between behavioural modules that are selectively engaged with the environment based on immune response. Experimental evidence leads discussions on a dynamical systems perspective of behavioural robustness in artificial immune systems that goes beyond the isolated immune network response.

  5. Mechanism of human antibody-mediated neutralization of Marburg virus. (United States)

    Flyak, Andrew I; Ilinykh, Philipp A; Murin, Charles D; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L; Hashiguchi, Takao; Bornholdt, Zachary A; Slaughter, James C; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G; Ward, Andrew B; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E


    The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition.

  6. Generation and Characterization of Specific Antibodies to the Murine and Human Ectonucleotidase NTPDase8. (United States)

    Pelletier, Julie; Salem, Mabrouka; Lecka, Joanna; Fausther, Michel; Bigonnesse, François; Sévigny, Jean


    The ectonucleotidase nucleoside triphosphate diphosphohydrolase-8 (NTPDase8) is the last member of the Ecto-NTPDase family to be discovered and characterized. It is a transmembrane protein which regulates the concentration of the agonists of P1 and P2 receptors at the cell surface. The functions of the enzyme are still not known partly due to the lack of specific tools such as antibodies. In this work, guinea pig polyclonal antibodies against mouse NTPDase8 and mouse monoclonal antibodies against human NTPDase8 have been generated and characterized. For the production of antibodies against mouse NTPDase8 several techniques have been tried. Several peptide antigens in several hosts (rabbit, rat, hamster, and guinea pig) failed to give a positive reaction suggesting that NTPDase8 is poorly immunogenic. In this study, we describe the successful process that led to anti-mouse NTPDase8, namely the cDNA immunization technique. Monoclonal antibodies to human NTPDase8 were also obtained by cDNA immunization followed by a final injection with transfected human embryonic kidney (HEK 293T) cells expressing human NTPDase8. The specificity of these antibodies was evaluated by Western blot, immunocytochemistry, immunohistochemistry and flow cytometry. In contrast, all commercial antibodies to NTPDase8 peptides that we have tested failed to give a specific positive signal against the expressed NTPDase8 protein when used to probe Western blots. In addition, immunohistochemistry experiments confirmed the presence of NTPDase8 in mouse liver canaliculi. The tools generated in this work will help characterize NTPDase8 localization and function in future studies and its contribution to the modulation of P1 and P2 receptor activation.

  7. Discovery of diverse and functional antibodies from large human repertoire antibody libraries. (United States)

    Schwimmer, Lauren J; Huang, Betty; Giang, Hoa; Cotter, Robyn L; Chemla-Vogel, David S; Dy, Francis V; Tam, Eric M; Zhang, Fangjiu; Toy, Pamela; Bohmann, David J; Watson, Susan R; Beaber, John W; Reddy, Nithin; Kuan, Hua-Feng; Bedinger, Daniel H; Rondon, Isaac J


    Phage display antibody libraries have a proven track record for the discovery of therapeutic human antibodies, increasing the demand for large and diverse phage antibody libraries for the discovery of new therapeutics. We have constructed naïve antibody phage display libraries in both Fab and scFv formats, with each library having more than 250 billion clones that encompass the human antibody repertoire. These libraries show high fidelity in open reading frame and expression percentages, and their V-gene family distribution, VH-CDR3 length and amino acid usage mirror the natural diversity of human antibodies. Both the Fab and scFv libraries show robust sequence diversity in target-specific binders and differential V-gene usage for each target tested, supporting the use of libraries that utilize multiple display formats and V-gene utilization to maximize antibody-binding diversity. For each of the targets, clones with picomolar affinities were identified from at least one of the libraries and for the two targets assessed for activity, functional antibodies were identified from both libraries.

  8. Exquisite specificity and peptide epitope recognition promiscuity, properties shared by antibodies from sharks to humans. (United States)

    Marchalonis, J J; Adelman, M K; Robey, I F; Schluter, S F; Edmundson, A B


    This review considers definitions of the specificity of antibodies including the development of recent concepts of recognition polyspecificity and epitope promiscuity. Using sets of homologous and unrelated peptides derived from the sequences of immunoglobulin and T cell receptor chains we offer operational definitions of cross-reactivity by investigating correlations of either identities in amino acid sequence, or in hydrophobicity/hydrophilicity profiles with degree of binding in enzyme-linked immunosorbent assays. Polyreactivity, or polyspecificity, are terms used to denote binding of a monoclonal antibody or purified antibody preparation to large complex molecules that are structurally unrelated, such as thyroglobulin and DNA. As a first approximation, there is a linear correlation between degree of sequence identity or hydrophobicity/hydrophilicity and antigenic cross-binding. However, catastrophic interchanges of amino acids can occur where changing of one amino acid out of 16 in a synthetic peptide essentially eliminates binding to certain antibodies. An operational definition of epitope promiscuity for peptides is the case where two peptides show little or no identity in amino acid sequence but bind strongly to the same antibody as shown by either direct binding or competitive inhibition. Analysis of antibodies of humans and sharks, the two most divergent species in evolution to express antibodies and the combinatorial immune response, indicates that the capacity for both exquisite specificity and epitope recognition promiscuity are essential conserved features of individual vertebrate antibodies.

  9. The use of ammonium sulphate precipitation method for the determination of antigen-binding capacity and affinity of anti-tetanus antibodies in human serum. (United States)

    Alausa, O K


    Radioimmunoassay of anti-tetanus antibodies produced in human serum in response to tetanus toxoid immunization was carried out by employing the non-specific precipitation effect of ammonium sulphate described by Farr (1958). The results showed that the method was sensitive and was able to differentiate between immune and non-immune persons. The effects of booster dose injection on the quantity and quality of antibodies produced during immunization were discussed, and the possible use of the method to predict the amount of immunogen, the timing and number of injections required for optimal host response in immunization schedules was suggested.

  10. Increased levels of IgG antibodies against human HSP60 in patients with spondyloarthritis.

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    Astrid Hjelholt

    Full Text Available Spondyloarthritis (SpA comprises a heterogeneous group of inflammatory diseases, with strong association to human leukocyte antigen (HLA-B27. A triggering bacterial infection has been considered as the cause of SpA, and bacterial heat shock protein (HSP seems to be a strong T cell antigen. Since bacterial and human HSP60, also named HSPD1, are highly homologous, cross-reactivity has been suggested in disease initiation. In this study, levels of antibodies against bacterial and human HSP60 were analysed in SpA patients and healthy controls, and the association between such antibodies and disease severity in relation to HLA-B27 was evaluated.Serum samples from 82 patients and 50 controls were analysed by enzyme-linked immunosorbent assay (ELISA for immunoglobulin (IgG1, IgG2, IgG3 and IgG4 antibodies against human HSP60 and HSP60 from Chlamydia trachomatis, Salmonella enteritidis and Campylobacter jejuni. Disease severity was assessed by the clinical scorings Bath Ankylosing Spondylitis Disease Activity Index (BASDAI, Bath Ankylosing Spondylitis Functional Index (BASFI and Bath Ankylosing Spondylitis Metrology Index (BASMI. Levels of IgG1 and IgG3 antibodies against human HSP60, but not antibodies against bacterial HSP60, were elevated in the SpA group compared with the control group. Association between IgG3 antibodies against human HSP60 and BASMI was shown in HLA-B27⁺ patients. Only weak correlation between antibodies against bacterial and human HSP60 was seen, and there was no indication of cross-reaction. These results suggest that antibodies against human HSP60 is associated with SpA, however, the theory that antibodies against human HSP60 is a specific part of the aetiology, through cross-reaction to bacterial HSP60, cannot be supported by results from this study. We suggest that the association between elevated levels of antibodies against human HSP60 and disease may reflect a general activation of the immune system and an increased

  11. Origin, diversity and maturation of human antiviral antibodies analyzed by high-throughput sequencing

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    Ponraj ePrabakaran


    Full Text Available Our understanding of how antibodies are generated and function could help develop effective vaccines and antibody-based therapeutics against viruses such as HIV-1, SARS Coronavirus (CoV, and Hendra and Nipah viruses (henipaviruses. Although broadly neutralizing antibodies (bnAbs against the HIV-1 were observed in patients, elicitation of such bnAbs remains a major challenge when compared to other viral targets. We previously hypothesized that HIV-1 could have evolved a strategy to evade the immune system due to absent or very weak binding of germline antibodies to the conserved epitopes that may not be sufficient to initiate and/or maintain an effective immune response. To further explore our hypothesis, we used the 454 sequence analysis of a large naïve library of human IgM antibodies which had been used for selecting antibodies against SARS Coronavirus (CoV receptor-binding domain (RBD, and soluble G proteins (sG of Hendra and Nipah viruses (henipaviruses. We found that the human IgM repertoires from the 454 sequencing have diverse germline usages, recombination patterns, junction diversity and a lower extent of somatic mutation. In this study, we identified germline intermediates of antibodies specific to HIV-1 and other viruses as observed in normal individuals, and compared their genetic diversity and somatic mutation level along with available structural and functional data. Further computational analysis will provide framework for understanding the underlying genetic and molecular determinants related to maturation pathways of antiviral bnAbs that could be useful for applying novel approaches to the design of effective vaccine immunogens and antibody-based therapeutics.

  12. Molluskan Hemocyanins Activate the Classical Pathway of the Human Complement System through Natural Antibodies (United States)

    Pizarro-Bauerle, Javier; Maldonado, Ismael; Sosoniuk-Roche, Eduardo; Vallejos, Gerardo; López, Mercedes N.; Salazar-Onfray, Flavio; Aguilar-Guzmán, Lorena; Valck, Carolina; Ferreira, Arturo; Becker, María Inés


    Molluskan hemocyanins are enormous oxygen-carrier glycoproteins that show remarkable immunostimulatory properties when inoculated in mammals, such as the generation of high levels of antibodies, a strong cellular reaction, and generation of non-specific antitumor immune responses in some types of cancer, particularly for superficial bladder cancer. These proteins have the ability to bias the immune response toward a Th1 phenotype. However, despite all their current uses with beneficial clinical outcomes, a clear mechanism explaining these properties is not available. Taking into account reports of natural antibodies against the hemocyanin of the gastropod Megathura crenulata [keyhole limpet hemocyanin (KLH)] in humans as well as other vertebrate species, we report here for the first time, the presence, in sera from unimmunized healthy donors, of antibodies recognizing, in addition to KLH, two other hemocyanins from gastropods with documented immunomodulatory capacities: Fisurella latimarginata hemocyanin (FLH) and Concholepas concholepas hemocyanin (CCH). Through an ELISA screening, we found IgM and IgG antibodies reactive with these hemocyanins. When the capacity of these antibodies to bind deglycosylated hemocyanins was studied, no decreased interaction was detected. Moreover, in the case of FLH, deglycosylation increased antibody binding. We evaluated through an in vitro complement deposition assay whether these antibodies activated the classical pathway of the human complement system. The results showed that all three hemocyanins and their deglycosylated counterparts elicited this activation, mediated by C1 binding to immunoglobulins. Thus, this work contributes to the understanding on how the complement system could participate in the immunostimulatory properties of hemocyanins, through natural, complement-activating antibodies reacting with these proteins. Although a role for carbohydrates cannot be completely ruled out, in our experimental setting

  13. Preferential germline usage and VH/VL pairing observed in human antibodies selected by mRNA display. (United States)

    Chen, Lei; Kutskova, Yuliya A; Hong, Feng; Memmott, John E; Zhong, Suju; Jenkinson, Megan D; Hsieh, Chung-Ming


    Since the invention of phage display, in vitro antibody display technologies have revolutionized the field of antibody discovery. In combination with antibody libraries constructed with sequences of human origin, such technologies enable accelerated therapeutic antibody discovery while bypassing the laborious animal immunization and hybridoma generation processes. Many in vitro display technologies developed since aim to differentiate from phage display by displaying full-length IgG proteins, utilizing eukaryotic translation system and codons, increasing library size or real-time kinetic selection by fluorescent activated cell sorting. We report here the development of an mRNA display technology and an accompanying HCDR3 size spectratyping monitor for human antibody discovery. Importantly, the mRNA display technology maintains a monovalent linkage between the mRNA (genotype) and display binding protein (phenotype), which minimizes avidity effect common in other display systems and allows for a stringent affinity and off-rate selection. The mRNA display technology successfully identified 100 human antibodies in 15 different selections against various targets from naïve human antibody libraries. These antibodies in general have high affinity and diversity. By analyzing the germline usage and combination of antibodies selected by the mRNA display technology, we identified trends and determined the productivity of each germline subgroup in the libraries that could serve as the knowledge base for constructing fully synthetic, next generation antibody libraries.

  14. A Rapid and Simple Approach to Preparation of Monoclonal Antibody Based on DNA Immunization

    Institute of Scientific and Technical Information of China (English)

    YiNi; KeMa; JingNi; XiujuanZheng; YingWang; SidongXiong


    Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb). But it is quite difficult to obtain pure proteins especially with natural structures. Here we attempt to replace the protein inoculation with DNA immunization in the preparation of mAb. The eukaryotic expression vectors pcDNA3-PreS2/S and pVAX-PreS2/S encoding the HBV M protein were constructed and prepared for DNA immunization. Female BALB/c mice developed a well antibody response to the target antigen after muscle injection with corresponding plasmids. The mice with effective antibodies induced were used for preparation of mAb. We found the mice immunized with three administrations of pcDNA3-PreS2/S and boosted by intrasplenic injection with the same plasmid could be exploited for preparation of mAb. And positive hybridoma cell 2D3 that can secrete specific mAb was cloned and analyzed. Our studies demonstrate that gene immunization may provide a convenient and efficient way to prepare mAbs. Cellular & Molecular Immunology.

  15. A Rapid and Simple Approach to Preparation of Monoclonal Antibody Based on DNA Immunization

    Institute of Scientific and Technical Information of China (English)

    Yi Ni; Ke Ma; Jing Ni; Xiujuan Zheng; Ying Wang; Sidong Xiong


    Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb).But it is quite difficult to obtain pure proteins especially with natural structures. Here we attempt to replace the protein inoculation with DNA immunization in the preparation of mAb. The eukaryotic expression vectors pcDNA3-PreS2/S and pVAX-PreS2/S encoding the HBV M protein were constructed and prepared for DNA immunization. Female BALB/c mice developed a well antibody response to the target antigen after muscle injection with corresponding plasmids. The mice with effective antibodies induced were used for preparation of mAb. We found the mice immunized with three administrations of pcDNA3-PreS2/S and boosted by intrasplenic injection with the same plasmid could be exploited for preparation of mAb. And positive hybridoma cell 2D3 that can secrete specific mAb was cloned and analyzed. Our studies demonstrate that gene immunization may provide a convenient and efficient way to prepare mAbs.

  16. The schistosoma-specific antibody response after treatment in non-immune travellers. (United States)

    Duus, Liv Marie; Christensen, Anders Vittrup; Navntoft, Dorte; Tarp, Britta; Nielsen, Henrik V; Petersen, Eskild


    Egg detection is the gold standard in diagnosing and controlling treatment in schistosomiasis, but sensitivity is poor in lightly infected individuals, whereas Schistosoma-specific antibodies are more sensitive. The purpose of the study was to evaluate use of Gut Associated Antigen (GAA) and Membrane Bound Antigen (MBA) assays in assessment of treatment efficacy and number of treated non-immune individuals with signs of treatment failure. In a retrospective study, residents in Denmark diagnosed with positive Schistosoma antibodies in the period 1987 - 2004 were offered follow-up including analyses for GAA, MBA, IgE and eosinophil count. Among 98 patients with positive antibody at time of diagnosis, 73 were examined for eggs and 27% had detectable eggs. 15% still had detectable living eggs after 1 course of treatment. At follow-up it was demonstrated that antibodies continued to increase for up to 6 months after treatment and average duration of positive GAA antibody was approximately 10 y. The study confirms that the GAA- and MBA-IFAT are not suitable in monitoring results of therapy. Treatment failure in 15% of non-immune patients indicates that studies are needed to define the correct dose of praziquantel in those individuals or to evaluate if resistance to praziquantel is a growing problem.

  17. On the Meaning of Affinity Limits in B-Cell Epitope Prediction for Antipeptide Antibody-Mediated Immunity

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    Salvador Eugenio C. Caoili


    Full Text Available B-cell epitope prediction aims to aid the design of peptide-based immunogens (e.g., vaccines for eliciting antipeptide antibodies that protect against disease, but such antibodies fail to confer protection and even promote disease if they bind with low affinity. Hence, the Immune Epitope Database (IEDB was searched to obtain published thermodynamic and kinetic data on binding interactions of antipeptide antibodies. The data suggest that the affinity of the antibodies for their immunizing peptides appears to be limited in a manner consistent with previously proposed kinetic constraints on affinity maturation in vivo and that cross-reaction of the antibodies with proteins tends to occur with lower affinity than the corresponding reaction of the antibodies with their immunizing peptides. These observations better inform B-cell epitope prediction to avoid overestimating the affinity for both active and passive immunization; whereas active immunization is subject to limitations of affinity maturation in vivo and of the capacity to accumulate endogenous antibodies, passive immunization may transcend such limitations, possibly with the aid of artificial affinity-selection processes and of protein engineering. Additionally, protein disorder warrants further investigation as a possible supplementary criterion for B-cell epitope prediction, where such disorder obviates thermodynamically unfavorable protein structural adjustments in cross-reactions between antipeptide antibodies and proteins.

  18. Two new monoclonal antibodies for biochemical and flow cytometric analyses of human interferon regulatory factor-3 activation, turnover, and depletion. (United States)

    Rustagi, Arjun; Doehle, Brian P; McElrath, M Juliana; Gale, Michael


    Interferon regulatory factor-3 (IRF-3) is a master transcription factor that drives the host intracellular innate immune response to virus infection. The importance of IRF-3 in innate immune responses is highlighted by the fact that pathogenic viruses have developed strategies for antagonism of IRF-3. Several tools exist for evaluation of viral regulation of IRF-3 activation and function, but high-quality monoclonal antibodies that mark the differential activation states of human IRF-3 are lacking. To study IRF-3 activation, turnover, and depletion in a high-throughput manner in the context of virus infection, we have developed two new monoclonal antibodies to human IRF-3. These antibodies detect IRF-3 in virus-infected cells in a wide variety of assays and provide a new tool to study virus-host interactions and innate immune signaling.

  19. 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.

  20. Limited impact of passive non-neutralizing antibody immunization in acute SIV infection on viremia control in rhesus macaques.

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    Taku Nakane

    Full Text Available BACKGROUND: Antiviral antibodies, especially those with neutralizing activity against the incoming strain, are potentially important immunological effectors to control human immunodeficiency virus (HIV infection. While neutralizing activity appears to be central in sterile protection against HIV infection, the entity of inhibitory mechanisms via HIV and simian immunodeficiency virus (SIV-specific antibodies remains elusive. The recent HIV vaccine trial RV144 and studies in nonhuman primate models have indicated controversial protective efficacy of HIV/SIV-specific non-neutralizing binding antibodies (non-NAbs. While reports on HIV-specific non-NAbs have demonstrated virus inhibitory activity in vitro, whether non-NAbs could also alter the pathogenic course of established SIV replication in vivo, likewise via neutralizing antibody (NAb administration, has been unclear. Here, we performed post-infection passive immunization of SIV-infected rhesus macaques with polyclonal SIV-specific, antibody-dependent cell-mediated viral inhibition (ADCVI-competent non-NAbs. METHODS AND FINDINGS: Ten lots of polyclonal immunoglobulin G (IgG were prepared from plasma of ten chronically SIVmac239-infected, NAb-negative rhesus macaques, respectively. Their binding capacity to whole SIVmac239 virions showed a propensity similar to ADCVI activity. A cocktail of three non-NAb lots showing high virion-binding capacity and ADCVI activity was administered to rhesus macaques at day 7 post-SIVmac239 challenge. This resulted in an infection course comparable with control animals, with no significant difference in set point plasma viral loads or immune parameters. CONCLUSIONS: Despite virus-specific suppressive activity of the non-NAbs having been observed in vitro, their passive immunization post-infection did not result in SIV control in vivo. Virion binding and ADCVI activity with lack of virus neutralizing activity were indicated to be insufficient for antibody

  1. Comparative Analysis of Immune Repertoires between Bactrian Camel's Conventional and Heavy-Chain Antibodies (United States)

    Yang, Kai; Zhang, Wei; Zhang, Changjiang; Fu, Longfei; Ren, Zhe; Wang, Changxi; Wu, Jinghua; Lu, Ruxue; Ye, Yanrui; He, Mengying; Nie, Chao; Yang, Naibo; Wang, Jian; Yang, Huanming; Liu, Xiao


    Compared to classical antibodies, camel heavy chain antibodies (HCAbs) are smaller in size due to lack of the light chain and the first constant domain of the heavy chain (CH1 region). The variable regions of HCAbs (VHHs) are more soluble and stable than that of conventional antibodies (VHs). Even with such simple structure, they are still functional in antigen binding. Although HCAbs have been extensively investigated over the past two decades, most efforts have been based upon low throughput sequence analysis, and there are only limited reports trying to analyze and describe the complete immune repertoire (IR) of camel HCAbs. Here we leveraged the high-throughput data generated by Next Generation Sequencing (NGS) of the variable domains of the antibody heavy chains from three Bactrian camels to conduct in-depth comparative analyses of the immunoglobulin repertoire. These include analyses of the complementary determining region 3 (CDR3) length and distribution, mutation rate, antibody characteristic amino acids, the distribution of the cysteine (Cys) codons, and the non-classical VHHs. We found that there is higher diversity in the CDR2 than in the other sub-regions, and there is a higher mutation rate in the VHHs than in the VHs (P VH and VHH clones, we also observed other substitutions at the positions NO.40/54/57/96/101 that could lead to additional structural alterations. We also found that VH-derived VHH clones, referred to as non-classical VHH clones in this study, accounted for about 8% of all clones. Further, only 5%-10% clones had the Trp > Arg AA substitution at the first position of framework 4 for all types of clones. We present, for the first time, a relatively complete picture of the Bactrian camel antibody immune repertoire, including conventional antibody (Ab) and HCAbs, using PCR and in silico analysis based on high-throughput NGS data. PMID:27588755

  2. Complement-mediated virus infectivity neutralisation by HLA antibodies is associated with sterilising immunity to SIV challenge in the macaque model for HIV/AIDS.

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    Mark Page

    Full Text Available Sterilising immunity is a desired outcome for vaccination against human immunodeficiency virus (HIV and has been observed in the macaque model using inactivated simian immunodeficiency virus (SIV. This protection was attributed to antibodies specific for cell proteins including human leucocyte antigens (HLA class I and II incorporated into virions during vaccine and challenge virus preparation. We show here, using HLA bead arrays, that vaccinated macaques protected from virus challenge had higher serum antibody reactivity compared with non-protected animals. Moreover, reactivity was shown to be directed against HLA framework determinants. Previous studies failed to correlate serum antibody mediated virus neutralisation with protection and were confounded by cytotoxic effects. Using a virus entry assay based on TZM-bl cells we now report that, in the presence of complement, serum antibody titres that neutralise virus infectivity were higher in protected animals. We propose that complement-augmented virus neutralisation is a key factor in inducing sterilising immunity and may be difficult to achieve with HIV/SIV Env-based vaccines. Understanding how to overcome the apparent block of inactivated SIV vaccines to elicit anti-envelope protein antibodies that effectively engage the complement system could enable novel anti-HIV antibody vaccines that induce potent, virolytic serological response to be developed.

  3. Human Metapneumovirus Antagonism of Innate Immune Responses

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    Xiaoyong Bao


    Full Text Available  Human metapneumovirus (hMPV is a recently identified RNA virus belonging to the Paramyxoviridae family, which includes several major human and animal pathogens. Epidemiological studies indicate that hMPV is a significant human respiratory pathogen with worldwide distribution. It is associated with respiratory illnesses in children, adults, and immunocompromised patients, ranging from upper respiratory tract infections to severe bronchiolitis and pneumonia. Interferon (IFN represents a major line of defense against virus infection, and in response, viruses have evolved countermeasures to inhibit IFN production as well as IFN signaling. Although the strategies of IFN evasion are similar, the specific mechanisms by which paramyxoviruses inhibit IFN responses are quite diverse. In this review, we will present an overview of the strategies that hMPV uses to subvert cellular signaling in airway epithelial cells, the major target of infection, as well as in primary immune cells.

  4. Defensins: natural component of human innate immunity. (United States)

    Jarczak, Justyna; Kościuczuk, Ewa M; Lisowski, Paweł; Strzałkowska, Nina; Jóźwik, Artur; Horbańczuk, Jarosław; Krzyżewski, Józef; Zwierzchowski, Lech; Bagnicka, Emilia


    The widespread use of antibiotics has contributed to a huge increase in the number of resistant bacteria. New classes of drugs are therefore being developed of which defensins are a potential source. Defensins are a group of antimicrobial peptides found in different living organisms, involved in the first line of defense in their innate immune response against pathogens. This review summarizes the results of studies of this family of human antimicrobial peptides (AMPs). There is a special emphasis on describing the entire group and individual peptides, history of their discovery, their functions and expression sites. The results of the recent studies on the use of the biologically active peptides in human medicine are also presented. The pharmaceutical potential of human defensins cannot be ignored, especially considering their strong antimicrobial activity and properties such as low molecular weight, reduced immunogenicity, broad activity spectrum and resistance to proteolysis, but there are still many challenges and questions regarding the possibilities of their practical application.

  5. Human Monoclonal Antibodies Broadly Neutralizing against Influenza B Virus (United States)

    Yasugi, Mayo; Kubota-Koketsu, Ritsuko; Yamashita, Akifumi; Kawashita, Norihito; Du, Anariwa; Sasaki, Tadahiro; Nishimura, Mitsuhiro; Misaki, Ryo; Kuhara, Motoki; Boonsathorn, Naphatsawan; Fujiyama, Kazuhito; Okuno, Yoshinobu; Nakaya, Takaaki; Ikuta, Kazuyoshi


    Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus. PMID:23408886

  6. Immune System Regulation in the Induction of Broadly Neutralizing HIV-1 Antibodies

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    Garnett Kelsoe


    Full Text Available In this brief review, we discuss immune tolerance as a factor that determines the magnitude and quality of serum antibody responses to HIV-1 infection and vaccination in the context of recent work. We propose that many conserved, neutralizing epitopes of HIV-1 are weakly immunogenic because they mimic host antigens. In consequence, B cells that strongly bind these determinants are removed by the physiological process of immune tolerance. This structural mimicry may represent a significant impediment to designing protective HIV-1 vaccines, but we note that several vaccine strategies may be able to mitigate this evolutionary adaptation of HIV and other microbial pathogens.

  7. Chronic pediatric pulmonary disease and primary humoral antibody based immune disease. (United States)

    Dosanjh, A


    Chronic inflammation of the larger airways is a common occurrence in children. A number of factors such as younger age, premature birth, male gender, exposure to environmental smoke or pollution, and crowded housing can increase a child's susceptibility to chronic lung disease. Chronic bronchitis may be caused by an underlying humoral immunodeficiency if the clinical course is recurrent or prolonged. Primary humoral immunodeficiency accounts for approximately 70% of all immunodeficiencies. The differential of chronic bronchitis also includes Cystic Fibrosis, ciliary defects and immune cellular and phagocytic defects. This review will summarize the most common humoral antibody based immune based deficiencies associated with chronic pulmonary disease.

  8. CTA1-DD adjuvant promotes strong immunity against human immunodeficiency virus type 1 envelope glycoproteins following mucosal immunization. (United States)

    Sundling, Christopher; Schön, Karin; Mörner, Andreas; Forsell, Mattias N E; Wyatt, Richard T; Thorstensson, Rigmor; Karlsson Hedestam, Gunilla B; Lycke, Nils Y


    Strategies to induce potent and broad antibody responses against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) at both systemic and mucosal sites represent a central goal for HIV-1 vaccine development. Here, we show that the non-toxic CTA1-DD adjuvant promoted mucosal and systemic humoral and cell-mediated immune responses following intranasal (i.n.) immunizations with trimeric or monomeric forms of HIV-1 Env in mice and in non-human primates. Env-specific IgG subclasses in the serum of immunized mice reflected a balanced Th1/Th2 type of response. Strikingly, i.n. immunizations with Env and the CTA1-DD adjuvant induced substantial levels of mucosal anti-Env IgA in bronchial alveolar lavage and also detectable levels in vaginal secretions. By contrast, parenteral immunizations of Env formulated in Ribi did not stimulate mucosal IgA responses, while the two adjuvants induced a similar distribution of Env-specific IgG-subclasses in serum. A single parenteral boost with Env in Ribi adjuvant into mice previously primed i.n. with Env and CTA1-DD, augmented the serum anti-Env IgG levels to similar magnitudes as those observed after three intraperitoneal immunizations with Env in Ribi. The augmenting potency of CTA1-DD was similar to that of LTK63 or CpG oligodeoxynucleotides (ODN). However, in contrast to CpG ODN, the effect of CTA1-DD and LTK63 appeared to be independent of MyD88 and toll-like receptor signalling. This is the first demonstration that CTA1-DD augments specific immune responses also in non-human primates, suggesting that this adjuvant could be explored further as a clinically safe mucosal vaccine adjuvant for humoral and cell-mediated immunity against HIV-1 Env.

  9. Production and characterization of murine monoclonal anti-human DNase II antibodies, and their use for immunoaffinity purification of DNase II from human liver and urine. (United States)

    Nakajima, Tamiko; Yasuda, Toshihiro; Takeshita, Haruo; Mori, Shinjiro; Mogi, Kouichi; Kaneko, Yasushi; Nakazato, Emiko; Kishi, Koichiro


    Four murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion (SRED) and DNA-cast polyacrylamide gel electrophoresis (DNA-cast PAGE) were very useful for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining of the 32-kDa enzyme band, which is one of the three non-identical subunits of human DNase II molecule separated by sodium dodecyl sulfate (SDS)-PAGE followed by blotting on a transfer membrane. A formyl-cellulofine resin conjugated with each antibody specifically adsorbed and efficiently desorbed the active DNase II enzyme. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.

  10. Rheumatoid factor interference in immunogenicity assays for human monoclonal antibody therapeutics. (United States)

    Tatarewicz, Suzanna; Miller, Jill M; Swanson, Steven J; Moxness, Michael S


    Rheumatoid factors (RFs) are endogenous human antibodies that bind to human gamma globulins. RFs demonstrate preferential binding to aggregated gamma globulins and are involved in the clearing mechanism of immune complexes. Immunoassays designed to measure human anti-human antibodies (HAHA) after administration of monoclonal antibody therapeutics are thus vulnerable to interference from RFs. When using a sensitive electrochemiluminescent (ECL) bridging immunoassay, samples from subjects with rheumatoid arthritis demonstrated much higher baseline reactivity than healthy subjects. Interference was found to be dependent on the aggregation state of the therapeutic antibody that had been conjugated with the detection reagent (ruthenium). Size exclusion high performance liquid chromatography (SE-HPLC) demonstrated that of the total integrated peaks, as little as 0.55% high molecular weight aggregates (>600kDa) were sufficient to cause increased reactivity. Stability studies of the ruthenium and biotin conjugated therapeutic antibody indicated that storage time, temperature and buffer formulation were critical in maintaining the integrity of the reagents. Through careful SE-HPLC monitoring we were able to choose appropriate storage and buffer conditions which led to a reduction in the false reactivity rate in therapeutic-naïve serum from a rheumatoid arthritis population.


    Institute of Scientific and Technical Information of China (English)

    Ma Yunguo; Yuan Mei; Fei Lihua; Li Li


    Objective: To investigate the immune responses induced by apomucin which is a mixture of mucin core peptide, in mice for elucidating the role of mucin core peptide in the modulation of cancers. Methods:Apomucin was isolated from human pancreatic cancer cell line SW1990. The mice were immunized with this apomucin (10μg/time×6) plus DETOX. Results: When immunized, all mice developed delayed-type hypersensitivity (DTH) after challenged with apomucin or synthetic peptide MUC-2 or MUC-3, while the mice immunized with apomucin alone did not develop DTH.No antibodies were detected by ELISA after immunization. When the spleen cells of vaccinated mice were cocultured with this apomucin (10-50μg/ml) and rhIL-2(50U/ml) in vitro, the proliferated lymphocytes showed cytotoxicity against human cancer cells, including colon cancer, gastric cancer, pancreatic cancer and leukemia as measured by Cr-51 release assay. Antibodies against MUC-2 and MUC-3 could block the cytotoxicity. Conclusion: It was identified that a vaccine combined of apomucin and immune adjuvant DETOX can induce cellular immune response and anti-tumor cytotoxicity in mice.

  12. Immunoproteomic analysis of human serological antibody responses to vaccination with whole-cell pertussis vaccine (WCV.

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    Yong-Zhang Zhu

    Full Text Available BACKGROUND: Pertussis (whooping cough caused by Bordetella pertussis (B.p, continues to be a serious public health threat. Vaccination is the most economical and effective strategy for preventing and controlling pertussis. However, few systematic investigations of actual human immune responses to pertussis vaccines have been performed. Therefore, we utilized a combination of two-dimensional electrophoresis (2-DE, immunoblotting, and mass spectrometry to reveal the entire antigenic proteome of whole-cell pertussis vaccine (WCV targeted by the human immune system as a first step toward evaluating the repertoire of human humoral immune responses against WCV. METHODOLOGY/PRINCIPAL FINDINGS: Immunoproteomic profiling of total membrane enriched proteins and extracellular proteins of Chinese WCV strain 58003 identified a total of 30 immunoreactive proteins. Seven are known pertussis antigens including Pertactin, Serum resistance protein, chaperonin GroEL and two OMP porins. Sixteen have been documented to be immunogenic in other pathogens but not in B.p, and the immunogenicity of the last seven proteins was found for the first time. Furthermore, by comparison of the human and murine immunoproteomes of B.p, with the exception of four human immunoreactive proteins that were also reactive with mouse immune sera, a unique group of antigens including more than 20 novel immunoreactive proteins that uniquely reacted with human immune serum was confirmed. CONCLUSIONS/SIGNIFICANCE: This study is the first time that the repertoire of human serum antibody responses against WCV was comprehensively investigated, and a small number of previously unidentified antigens of WCV were also found by means of the classic immunoproteomic strategy. Further research on these newly identified predominant antigens of B.p exclusively against humans will not only remarkably accelerate the development of diagnostic biomarkers and subunit vaccines but also provide detailed insight

  13. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. (United States)

    Wesolowski, Janusz; Alzogaray, Vanina; Reyelt, Jan; Unger, Mandy; Juarez, Karla; Urrutia, Mariela; Cauerhff, Ana; Danquah, Welbeck; Rissiek, Björn; Scheuplein, Felix; Schwarz, Nicole; Adriouch, Sahil; Boyer, Olivier; Seman, Michel; Licea, Alexei; Serreze, David V; Goldbaum, Fernando A; Haag, Friedrich; Koch-Nolte, Friedrich


    Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.

  14. Role of B Cells and Antibodies in Acquired Immunity against Mycobacterium tuberculosis (United States)

    Achkar, Jacqueline M.; Chan, John; Casadevall, Arturo


    Accumulating evidence has documented a role for B cells and antibodies (Abs) in the immunity against Mycobacterium tuberculosis (Mtb). Passive transfer studies with monoclonal antibodies (mAbs) against mycobacterial antigens have shown protection against the tubercle bacillus. B cells and Abs are believed to contribute to an enhanced immune response against Mtb by modulating various immunological components in the infected host including the T-cell compartment. Nevertheless, the extent and contribution of B cells and Abs to protection against Mtb remains uncertain. In this article we summarize the most relevant findings supporting the role of B cells and Abs in the defense against Mtb and discuss the potential mechanisms of protection. PMID:25301934

  15. Evaluation of human antibody responses to diphtheria toxin subunits A and B in various age groups. (United States)

    Karakus, R; Caglar, K; Aybay, C


    This study aimed to evaluate human antibody responses to diphtheria toxin subunits in various age groups. Antibodies against the intact diphtheria toxin and the diphtheria toxin subunits A and B were evaluated in 1319 individuals using a double-antigen ELISA. Although high levels of protection (83.6%, 95% CI 79.2-87.4) were found in children and adolescents, the middle-aged adult population was less protected (28.8%, 95% CI 24.3-33.6). An increase in age was associated with a decrease in the frequency of protected individuals in the 0-39-year age group (p antibodies against the intact toxin. In children aged antibodies were observed were found to correlate with the ages at which booster doses are administered. Overall, males appeared to be more protected than females (OR 1.67, 95% CI 1.34-2.08, p antibody levels of > or =0.1 IU/mL against the intact toxin, but did not have protective antibody against subunit B. Determination of anti-subunit B antibody levels should help in evaluating the effectiveness of diphtheria boosters and other aspects of diphtheria immunity.

  16. 21 CFR 640.100 - Immune Globulin (Human). (United States)


    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Immune Globulin (Human). 640.100 Section 640.100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Immune Globulin (Human) § 640.100...

  17. Lineage Structure of the Human Antibody Repertoire in Response to Influenza Vaccination (United States)

    Jiang, Ning; He, Jiankui; Weinstein, Joshua A.; Penland, Lolita; Sasaki, Sanae; He, Xiao-Song; Dekker, Cornelia L.; Zheng, Nai-ying; Huang, Min; Sullivan, Meghan; Wilson, Patrick C.; Greenberg, Harry B.; Davis, Mark M.; Fisher, Daniel S.; Quake, Stephen R.


    The human antibody repertoire is one of the most important defenses against infectious disease, and the development of vaccines has enabled the conferral of targeted protection to specific pathogens. However, there are many challenges to measuring and analyzing the immunoglobulin sequence repertoire, such as the fact that each B cell contains a distinct antibody sequence encoded in its genome, that the antibody repertoire is not constant but changes over time, and the high similarity between antibody sequences. We have addressed this challenge by using high-throughput long read sequencing to perform immunogenomic characterization of expressed human antibody repertoires in the context of influenza vaccination. Informatic analysis of 5 million antibody heavy chain sequences from healthy individuals allowed us to perform global characterizations of isotype distributions, determine the lineage structure of the repertoire and measure age and antigen related mutational activity. Our analysis of the clonal structure and mutational distribution of individuals’ repertoires shows that elderly subjects have a decreased number of lineages but an increased pre-vaccination mutation load in their repertoire and that some of these subjects have an oligoclonal character to their repertoire in which the diversity of the lineages is greatly reduced relative to younger subjects. We have thus shown that global analysis of the immune system’s clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects. PMID:23390249

  18. Food availability and maternal immunization affect transfer and persistence of maternal antibodies in nestling pigeons.

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    Ahmad Ismail

    Full Text Available The ability of mothers to transfer antibodies (Abs to their young and the temporal persistence of maternal Abs in offspring constitute important life-history traits that can impact the evolution of host-parasite interactions. Here, we examined the effects of food availability and parental immunization on the transfer and persistence of maternal antibodies in nestling pigeons (Columba livia. This species can transmit maternal Abs to offspring before hatching through the egg yolk and potentially after hatching through crop milk. However, the role of this postnatal substance in immunity remains elusive. We used a full cross-fostering design to disentangle the effects of food limitation and parental immunization both before and after hatching on the levels and persistence of maternal Abs in chicks. Parents were immunized via injection with keyhole limpet hemocyanin antigens. Using an immunoassay that specifically detected the IgY antibodies that are known to be transmitted via the yolk, we found that the levels of anti-KLH Abs in newly hatched chicks were positively correlated with the levels of anti-KLH Abs in the blood of their biological mothers. However, this correlation was not present between chicks and their foster parents, suggesting limited IgY transfer via crop milk to the chick's bloodstream. Interestingly, biological mothers subjected to food limitation during egg laying transferred significantly fewer specific maternal Abs, which suggests that the transfer of antibodies might be costly for them. In addition, the persistence of maternal Abs in a chick's bloodstream was not affected by food limitation or the foster parents' anti-KLH Ab levels; it was only affected by the initial level of maternal anti-KLH Abs that were present in newly hatched chicks. These results suggest that the maternal transfer of Abs could be costly but that their persistence in an offspring's bloodstream may not necessarily be affected by environmental conditions.

  19. Production and Characterization of Monoclonal Antibody Against Recombinant Human Erythropoietin

    Institute of Scientific and Technical Information of China (English)



    Objective To produce specific monoclonal antibody(mAb)against recombinant human erythropoietin(rHuEPO)for development of higmy efficient methods for erythropoietin detection in biological fluids.Methods rHuEPO was covalently coupled with bovine serum albumin(BSA)and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology.The obtained F3-mAb was characterized by enzyme-linked immunosorbent assay (ELISA),SDS-PAGE and Western blot.Results The isotype of F3-mAb Was found to be IgM with an affinity constant of 2.1x108 L/mol.The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work.Conclusions The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.

  20. MEMS reagent and sample handling procedure: Feasibility of viral antibody detection by passive immune agglutination (United States)

    Bailey, G. D.; Tenoso, H. J.


    An attempt was made to develop a test requiring no preadsorption steps for the assessment of antibodies to rubella and mumps viruses using the passive immune agglutination (PIA) method. Both rubella and mumps antigens and antibodies were prepared. Direct PIA tests, using rubella antigen-coated beads, and indirect PIA tests, using rubella antibody-coated beads, were investigated. Attempts, using either method, were unsuccessful. Serum interference along with nonspecific agglutination of beads by the rubella antigen resulted in no specific response under the test conditions investigated. A new, highly sensitive approach, the enzyme immunoassay (EIA) test system, is recommended to overcome the nonspecificity. This system is a logical outgrowth of some of the solid phase work done on MEMS and represents the next generation tests system that can be directly applied to early disease detection and monitoring.

  1. Detection of opsonic antibodies against Enterococcus faecalis cell wall carbohydrates in immune globulin preparations. (United States)

    Hufnagel, M; Sixel, K; Hammer, F; Kropec, A; Sava, I G; Theilacker, C; Berner, R; Huebner, J


    Three different commercially available polyvalent immune globulins (IG) were investigated for the existence of antibodies against cell wall carbohydrates of four different E. faecalis serotypes (using a cell wall carbohydrate-enzyme-linked immunosorbent assay), and whether these antibodies mediated opsonic killing (using an opsonic-killing assay). All three IG preparations contained antibodies against all four serotypes (CPS-A to CPS-D). However, only one of the three IG preparations showed opsonic killing against all four serotypes. Average killing was higher against serotypes A and B (72 and 79 %, respectively) than against serotypes C and D (30 and 37 %, respectively). Such IG preparations could play a role as an adjuvant therapeutic option in life-threatening infections with E. faecalis, particularly when resistant strains are involved.

  2. Dermatologic complications of anti-PD-1/PD-L1 immune checkpoint antibodies. (United States)

    Sibaud, Vincent; Meyer, Nicolas; Lamant, Laurence; Vigarios, Emmanuelle; Mazieres, Julien; Delord, Jean Pierre


    The therapeutic use of anti-PD-1/PD-L1 antibodies (nivolumab, pembrolizumab) is rapidly increasing. Given their mechanism of action that triggers T-cell activation, these immune checkpoint inhibitors induce specific adverse events that are mostly of immunologic origin. In this way, cutaneous toxicities represent the most frequent immune-related adverse events (irAEs). The purpose of this review is to summarize the most prevalent dermatologic complications induced by PD-1/PD-L1 immune checkpoint-blocking antibodies and to compare their dermatologic safety profile with anti-CTLA-4 ipilimumab. More than 40% of melanoma patients treated with anti-PD-1 therapy are faced with dermatologic irAEs. However, these cutaneous complications usually remain self-limiting and readily manageable. Nonspecific macular papular rash and pruritus represent the most common manifestations. More characteristic lichenoid dermatitis or psoriasis may also develop. Vitiligo is also frequent in patients with melanoma but has not been reported in other types of solid cancers. Mucosal involvement may also occur, including xerostomia and lichenoid reactions. Although available data remain scarce, anti-PD-L1 antibodies present a similar dermatologic safety profile. Dermatologic irAEs induced by PD-1 or PD-L1 blockade therapy rarely result in significant morbidity or permanent discontinuation of treatment. However, early recognition and appropriate management are crucial for restricting dose-limiting toxicities.

  3. Kinetics of antibody responses after primary immunization with meningococcal serogroup C conjugate vaccine or secondary immunization with either conjugate or polysaccharide vaccine in adults

    NARCIS (Netherlands)

    de Voer, Richarda M.; van der Klis, Fiona R. M.; Engels, Carla W. A. M.; Schepp, Rutger M.; van de Kassteele, Jan; Sanders, Elisabeth A. M.; Rijkers, Ger T.; Berbers, Guy A. M.


    In the Netherlands the meningococcal serogroup C conjugate (MenCC) vaccine is administered as a single dose at 14 months. We evaluated the kinetics of isotype-specific antibodies in adults (n = 21) after primary immunization with MenCC or secondary immunization with MenCC or plain MenC polysaccharid

  4. Opsonising antibodies to P. falciparum merozoites associated with immunity to clinical malaria.

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    Danika L Hill

    Full Text Available Naturally acquired humoral immunity to the malarial parasite Plasmodium falciparum can protect against disease, although the precise mechanisms remain unclear. Although antibody levels can be measured by ELISA, few studies have investigated functional antibody assays in relation to clinical outcomes. In this study we applied a recently developed functional assay of antibody-mediated opsonisation of merozoites, to plasma samples from a longitudinal cohort study conducted in a malaria endemic region of Papua New Guinea (PNG. Phagocytic activity was quantified by flow cytometry using a standardized and high-throughput protocol, and was subsequently evaluated for association with protection from clinical malaria and high-density parasitemia. Opsonising antibody responses were found to: i increase with age, ii be enhanced by concurrent infection, and iii correlate with protection from clinical episodes and high-density parasitemia. Stronger protective associations were observed in individuals with no detectable parasitemia at baseline. This study presents the first evidence for merozoite phagocytosis as a correlate of acquired immunity and clinical protection against P. falciparum malaria.

  5. Humoral immune response to the entire human immunodeficiency virus envelope glycoprotein made in insect cells

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    Rusche, J.R.; Lynn, D.L.; Robert-Guroff, M.; Langlois, A.J.; Lyerly, H.K.; Carson, H.; Krohn, K.; Ranki, A.; Gallo, R.C.; Bolognesi, D.P.; Putney, S.D.


    The human immunodeficiency virus envelope gene was expressed in insect cells by using a Baculovirus expression vector. The protein has an apparent molecular mass of 160 kDa, appears on the surface of infected insect cells, and does not appear to be cleaved to glycoproteins gp120 and gp41. Goats immunized with the 160-kDa protein have high titers of antibody that neutralizes virus infection as measured by viral gene expression or cell cytolysis. In addition, immune sera can block fusion of human immunodeficiency virus-infected cells in culture. Both neutralization and fusion-blocking activities are bound to and eluted from immobilized gp120.

  6. The Plasmodium falciparum erythrocyte invasion ligand Pfrh4 as a target of functional and protective human antibodies against malaria.

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    Linda Reiling

    Full Text Available BACKGROUND: Acquired antibodies are important in human immunity to malaria, but key targets remain largely unknown. Plasmodium falciparum reticulocyte-binding-homologue-4 (PfRh4 is important for invasion of human erythrocytes and may therefore be a target of protective immunity. METHODS: IgG and IgG subclass-specific responses against different regions of PfRh4 were determined in a longitudinal cohort of 206 children in Papua New Guinea (PNG. Human PfRh4 antibodies were tested for functional invasion-inhibitory activity, and expression of PfRh4 by P. falciparum isolates and sequence polymorphisms were determined. RESULTS: Antibodies to PfRh4 were acquired by children exposed to P. falciparum malaria, were predominantly comprised of IgG1 and IgG3 subclasses, and were associated with increasing age and active parasitemia. High levels of antibodies, particularly IgG3, were strongly predictive of protection against clinical malaria and high-density parasitemia. Human affinity-purified antibodies to the binding region of PfRh4 effectively inhibited erythrocyte invasion by P. falciparum merozoites and antibody levels in protected children were at functionally-active concentrations. Although expression of PfRh4 can vary, PfRh4 protein was expressed by most isolates derived from the cohort and showed limited sequence polymorphism. CONCLUSIONS: Evidence suggests that PfRh4 is a target of antibodies that contribute to protective immunity to malaria by inhibiting erythrocyte invasion and preventing high density parasitemia. These findings advance our understanding of the targets and mechanisms of human immunity and evaluating the potential of PfRh4 as a component of candidate malaria vaccines.

  7. Assessment of antibody titers and immunity to Hepatitis B in children receiving chemotherapy (United States)

    Shams Shahemabadi, A; Salehi, F; Hashemi, A; Vakili, M; Zare, F; Esphandyari, N; Kashanian, S


    Background There is a decrease in vaccine-specific antibody to certain vaccine-preventable diseases in children after chemotherapy, but the frequency of non-immune patients is not clear. In the present case-control study, was taken under investigation protection level to Hepatitis B infection in children 6 months after completing chemotherapy. Materials and Methods In this study 68 patients with cancer and 68 healthy children were enrolled. Patients were 1.5 -12 years old with completed standard chemotherapy at least for 6 months. All the patients and healthy children were negative for HBsAg and HBeAg and had received Hepatitis B vaccination. IgG antibody concentrations against Hepatitis B Virus (HBV) were determined in the patients receiving chemotrapy and healthy subjects serum by ELISA method. IgG antibody titer > 10 mIU/ml was considered as baseline protective titer for preventing HBV infection. Results Anti-HBs antibody titer in 19.12% of patients was less than 10 mIU/ml and 11.76% of the patients had borderline antibody titer (10-20 mIU/ml). In healthy subjects, 2.94% and 5.88% had antibody titer < 10 mIU/ml and 10-20 mIU/ml, respectively. According to statistical analysis, frequency of non immune subjects in children with cancer was significantly higher than those in healthy children (P-value=0.024). Conclusion HBV vaccination post-intensive chemotherapy in the children with cancer is strongly recommended. PMID:24575253

  8. Cytokines and immune surveillance in humans (United States)

    Sonnenfeld, Gerald


    Evidence from both human and rodent studies has indicated that alterations in immunological parameters occur after space flight. Among the parameters shown, by us and others, to be affected is the production of interferons. Interferons are a family of cytokines that are antiviral and play a major role in regulating immune responses that control resistance to infection. Alterations in interferon and other cytokine production and activity could result in changes in immunity and a possible compromise of host defenses against both opportunistic and external infections. The purpose of the present study is to explore further the effects of space flight on cyotokines and cytokine-directed immunological function. Among the tests carried out are interferon-alpha production, interferon-gamma production, interleukin-1 and -2 production, signal transduction in neutrophils, signal transduction in monocytes, and monocyte phagocytic activity. The experiments will be performed using peripheral blood obtained from human subjects. It is our intent to eventually carry out these experiments using astronauts as subjects to determine the effects of space flight on cytokine production and activity. However, these subjects are not currently available. Until they become available, we will carry out these experiments using subjects maintained in the bed-rest model for microgravity.

  9. Intranasal administration of HIV-DNA vaccine formulated with a polymer, carboxymethylcellulose, augments mucosal antibody production and cell-mediated immune response. (United States)

    Hamajima, K; Sasaki, S; Fukushima, J; Kaneko, T; Xin, K Q; Kudoh, I; Okuda, K


    We previously reported that intramuscular (i.m.) immunization of DNA vaccine encoding human immunodeficiency virus type 1 (HIV-1)IIIB env and rev genes alone or in combination with appropriate adjuvant induces substantial and enhanced immune response against HIV-1. In the present study, we examined whether a polymer, low-viscosity carboxymethylcellulose sodium salt (CMCS-L), has an adjuvant effect on immune response induced by DNA vaccination. BALB/c mice were immunized with HIV-DNA vaccine formulated with CMCS-L via the intranasal (i.n.) and i.m. routes. The combination with the polymer elicited higher levels of antigen-specific serum IgG and fecal IgA antibodies than DNA vaccine alone. For cell-mediated immunity, HIV-specific delayed-type hypersensitivity response and cytotoxic T lymphocyte activity were measured by the footpad-swelling test and the 51Cr-release assay, respectively. Both were enhanced by the combination with CMCS-L via i.n. and i.m. inoculation. Cytokine analysis in culture media of bulk splenocytes harvested from immunized animals showed higher levels of IL-4 production in i.n. -immunized mice compared with i.m.-immunized mice. Nevertheless, the increased IFN-gamma production resulting from the combination with CMCS-L was observed only in i.n.-immunized mice. These data indicate that i.n. immunization of HIV-DNA vaccine formulated with CMCS-L enhances HIV-specific mucosal antibody (Ab) and systemic Ab and cell-mediated immune response.

  10. The natural antibody repertoire of sharks and humans recognizes the potential universe of antigens. (United States)

    Adelman, Miranda K; Schluter, Samuel F; Marchalonis, John J


    In ancestral sharks, a rapid emergence in the evolution of the immune system occurred, giving jawed-vertebrates the necessary components for the combinatorial immune response (CIR). To compare the natural antibody (NAb) repertoires of the most divergent vertebrates with the capacity to produce antibodies, we isolated NAbs to the same set of antigens by affinity chromatography from two species of Carcharhine sharks and from human polyclonal IgG and IgM antibody preparations. The activities of the affinity-purified anti-T-cell receptor (anti-TCR) NAbs were compared with those of monoclonal anti-TCR NAbs that were generated from a systemic lupus erythematosus patient. We report that sharks and humans, representing the evolutionary extremes of vertebrate species sharing the CIR, have NAbs to human TCRs, Igs, the human senescent cell antigen, and to numerous retroviral antigens, indicating that essential features of the combinatorial repertoire and the capacity to recognize the potential universe of antigens is shared among all jawed-vertebrates.

  11. Anti-BlyS antibody reduces the immune reaction against enzyme and enhances the efficacy of enzyme replacement therapy in Fabry disease model mice. (United States)

    Sato, Yohei; Ida, Hiroyuki; Ohashi, Toya


    Formation of antibodies against a therapeutic enzyme is an important complication during enzyme replacement therapy (ERT) for lysosomal storage diseases. Fabry disease (FD) is caused by a deficiency of alpha-galactosidase (GLA), which results in the accumulation of globotriaosylceramide (GL-3). We have shown immune tolerance induction (ITI) during ERT in FD model mice by using an anti-B lymphocyte stimulator (anti-BlyS) antibody (belimumab). A single dose of the anti-BlyS antibody temporarily lowered the percentage of B cells and IgG antibody titer against recombinant human GLA. Administration of a low maintenance dose of the anti-BlyS antibody suppressed the B cell population and immunotolerance was induced in 20% of mice, but antibody formation could not be prevented. We then increased the maintenance dose of the anti-BlyS antibody and immunotolerance was induced in 50% of mice. Therapeutic enzyme distribution and clearance of GL-3 were also enhanced by a high maintenance dose of the anti-BlyS antibody.

  12. Uptake of antigen-antibody complexes by human dendritic cells. (United States)

    Fanger, N A; Guyre, P M; Graziano, R F


    Fc receptors specific for IgG (FcγR) potentiate the immune response by facilitating the interaction between myeloid cells and antibody-coated targets (1-3). Monocyte and neutrophil FcyR engagement can lead to the induction of lytic-type mechanisms associated with innate responses. FcyR triggering can also play a key role in adaptive immune responses. For example, FcyR-directed capture and uptake of antigens (Ag) by dendritic cells (DC) results in processing and presentation to naive Ag-specific T cells, leading to their expansion and maturation into effector T-cell populations. This chapter describes methodology currently in use to explore and manipulate antigen-antibody (Ag-Ab) uptake by FcyR expressed on DC.

  13. A novel monoclonal anti-CD81 antibody produced by genetic immunization efficiently inhibits Hepatitis C virus cell-cell transmission.

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    Isabel Fofana

    Full Text Available BACKGROUND AND AIMS: Hepatitis C virus (HCV infection is a challenge to prevent and treat because of the rapid development of drug resistance and escape. Viral entry is required for initiation, spread, and maintenance of infection, making it an attractive target for antiviral strategies. METHODS: Using genetic immunization, we produced four monoclonal antibodies (mAbs against the HCV host entry factor CD81. The effects of antibodies on inhibition of HCV infection and dissemination were analyzed in HCV permissive human liver cell lines. RESULTS: The anti-CD81 mAbs efficiently inhibited infection by HCV of different genotypes as well as a HCV escape variant selected during liver transplantation and re-infecting the liver graft. Kinetic studies indicated that anti-CD81 mAbs target a post-binding step during HCV entry. In addition to inhibiting cell-free HCV infection, one antibody was also able to block neutralizing antibody-resistant HCV cell-cell transmission and viral dissemination without displaying any detectable toxicity. CONCLUSION: A novel anti-CD81 mAb generated by genetic immunization efficiently blocks HCV spread and dissemination. This antibody will be useful to further unravel the role of virus-host interactions during HCV entry and cell-cell transmission. Furthermore, this antibody may be of interest for the development of antivirals for prevention and treatment of HCV infection.


    Institute of Scientific and Technical Information of China (English)


    Objective: Preparation of anti-human androgen receptor(hAR) monoclonal antibody (McAb). Methods: Four cells lines of hybridoma secreting specific monoclonal antibodies against AR were first established by fusion SP2/0 cell with spleen cell from BALB/c mice immunized with the coupling complex of hAR-KLH. Results: Paraffin-embedded sections of 45 prostate cancers were detected. There was an overall concordance of 91% using Immunohistochemistry between AR polyclonal antibody from Zymed and hAR-N McAb selfmade. Conclusion: The results show that the McAb obtained in this study would be a useful tool to detect the AR status in prostate cancer.

  15. Generation and selection of immunized Fab phage display library against human B cell lymphoma

    Institute of Scientific and Technical Information of China (English)

    Yongmei Shen; Xiaochun Yang; Ningzheng Dong; Xiaofang Xie; Xia Bai; Yizhen Shi


    The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production of monoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody (HAMA) response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the K light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94×107. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains (VH) and variable light domains (VL) were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.

  16. Advances in alloimmune thrombocytopenia: perspectives on current concepts of human platelet antigens, antibody detection strategies, and genotyping. (United States)

    Hayashi, Tomoya; Hirayama, Fumiya


    Alloimmunisation to platelets leads to the production of antibodies against platelet antigens and consequently to thrombocytopenia. Numerous molecules located on the platelet surface are antigenic and induce immune-mediated platelet destruction with symptoms that can be serious. Human platelet antigens (HPA) cause thrombocytopenias, such as neonatal alloimmune thrombocytopenia, post-transfusion purpura, and platelet transfusion refractoriness. Thirty-four HPA are classified into 28 systems. Assays to identify HPA and anti-HPA antibodies are critically important for preventing and treating thrombocytopenia caused by anti-HPA antibodies. Significant progress in furthering our understanding of HPA has been made in the last decade: new HPA have been discovered, antibody-detection methods have improved, and new genotyping methods have been developed. We review these advances and discuss issues that remain to be resolved as well as future prospects for preventing and treating immune thrombocytopenia.

  17. Monitoring human parvovirus B19 virus-like particles and antibody complexes in solution by fluorescence correlation spectroscopy. (United States)

    Toivola, Jouni; Michel, Patrik O; Gilbert, Leona; Lahtinen, Tomi; Marjomäki, Varpu; Hedman, Klaus; Vuento, Matti; Oker-Blom, Christian


    Fluorescence correlation spectroscopy (FCS) was used in monitoring human parvovirus B19 virus-like particle (VLP) antibody complexes from acute phase and past-immunity serum samples. The Oregon Green 488-labeled VLPs gave an average diffusion coefficient of 1.7 x 10(-7) cm2 s(-1) with an apparent hydrodynamic radius of 14 nm. After incubation of the fluorescent VLPs with an acute phase serum sample, the mobility information obtained from the fluorescence intensity fluctuation by autocorrelation analysis showed an average diffusion coefficient of 1.5 x 10(-8) cm2 s(-1), corresponding to an average radius of 157 nm. In contrast, incubation of the fluorescent VLPs with a past-immunity serum sample gave an average diffusion coefficient of 3.5 x 10(-8) cm2 s(-1) and a radius of 69 nm. A control serum devoid of B19 antibodies caused a change in the diffusion coefficient from 1.7 x 10(-7) to 1.6 x 10(-7) cm2 s(-1), which is much smaller than that observed with acute phase or past-immunity sera. Thus, VLP-antibody complexes with different diffusion coefficients could be identified for the acute phase and past-immunity sera. FCS measurement of VLP-immune complexes could be useful in distinguishing between antibodies present in acute phase or past-immunity sera as well as in titration of the VLPs.

  18. Recombinant anti-human melanoma antibodies are versatile molecules. (United States)

    Neri, D; Natali, P G; Petrul, H; Soldani, P; Nicotra, M R; Vola, R; Rivella, A; Creighton, A M; Neri, P; Mariani, M


    The low cost, high versatility, and reliable production of bacterially produced recombinant antibody fragments speeds up the development of tumor-targeting agents. High-quality recombinant anti-melanoma antibodies are much sought after in the scientific community. We cloned the murine antibody 225.28S, currently used in radioimmunoimaging of human melanoma lesions, in single-chain Fv configuration (scFv) for soluble expression in bacteria. The recombinant antibody fragment conserved the binding specificity of the parental antibody. In order to arm the scFv(225.28S) with biologically useful effector functions, we developed vectors for soluble expression of scFv(225.28S) in bacteria that allow both covalent and noncovalent chemical antibody modification at positions that do not interfere with antigen binding. An expression vector was developed that appends a cysteine residue at the C-terminal extremity of the recombinant antibody, thus allowing reaction with thiol-specific reagents, including 99mTc labeling, at a position that does not interfere with antigen binding. The scFv(225.28S) was also successfully expressed with a casein kinase II substrate tag that enables efficient and stable 32P labeling. For noncovalent antibody modification, we developed an expression vector that appends the human calmodulin gene at the C-terminal extremity of scFv(225.28S). The calmodulin domain is poorly immunogenic and can be targeted with chemically modified high-affinity calmodulin ligands. The recombinant anti-human melanoma antibodies described in this article should prove useful "building blocks" for the development of anti-melanoma diagnostic and therapeutic strategies.

  19. Antibody response to Salmonella typhi lw human Schistosomiasis mansoni

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    Maria Imaculada Muniz-Junqueira


    Full Text Available Antibody response to Salmonella typhi O and H antigens was evaluated in 24 individuals with either hepatointestinal or hepatosplenic schistosomiasis mansoni before and after typhoid vaccination, and compared with that of non-infected controls. Before vaccination, Schistosoma-infected patients showed a higher frequency of positive antibody to O antigen and the same frequency to H antigen when compared with that of healthy individuals. However, those with hepatosplenic schistosomiasis showed higher titres of antibody to H antigen than those with hepatointestinal disease or healthy individuals. Infected subjects, particularly those with hepatointestinal disease, showed a decreased response after typhoid vaccine. Tins diminished ability to mount an immune response towards typhoid antigens dining schistosomiasis may interfere ivith the clearance of the bacteria from blood stream and, therefore, play a role in the prolonged survival of salmonella as obsewed in some patients with chronic salmonellosis associated with schistosomiasis.

  20. Antibodies against high-risk human papillomavirus proteins as markers for invasive cervical cancer. (United States)

    Combes, Jean-Damien; Pawlita, Michael; Waterboer, Tim; Hammouda, Doudja; Rajkumar, Thangarajan; Vanhems, Philippe; Snijders, Peter; Herrero, Rolando; Franceschi, Silvia; Clifford, Gary


    Different human papillomavirus (HPV) genes are expressed during the various phases of the HPV life cycle and may elicit immune responses in the process towards malignancy. To evaluate their association with cervical cancer, antibodies against proteins from HPV16 (L1, E1, E2, E4, E6 and E7) and HPV18/31/33/35/45/52/58 (L1, E6 and E7) were measured in serum of 307 invasive cervical cancer cases and 327 controls from Algeria and India. Antibody response was evaluated using a glutathione S-transferase-based multiplex serology assay and HPV DNA detected from exfoliated cervical cells using a GP5+/6+-mediated PCR assay. Among HPV16 DNA-positive cases, seroprevalence of HPV16 antibodies ranged from 16% for HPV16 E1 to 50% for HPV16 E6 and all were significantly higher than controls. Seroprevalence of E6, E7 and L1 antibodies for HPV18 and for at least one of HPV31/33/35/45/52/58 were also higher in cases positive for DNA of the corresponding type (50% and 30% for E6 of HPV18 and HPV31/33/35/45/52/58 combined, respectively). E6 and E7 antibodies were rarely found in controls, but cross-reactivity was evident among cancer cases positive for DNA of closely phylogenetically-related HPV types. E6 or E7 antibodies against any of the eight HPV types were detected in 66.1% of all cervical cancer cases, as compared to 10.1% of controls. E6, and to a lesser extent E7, antibodies appear to be specific markers of HPV-related malignancy. However, even among cases positive for the same type of HPV DNA, approximately one-third of cervical cancer cases show no detectable immune response to either E6 or E7.

  1. Altered immune response of immature dendritic cells upon dengue virus infection in the presence of specific antibodies

    NARCIS (Netherlands)

    Torres, Silvia; Flipse, Jacky; Upasani, Vinit C; van der Ende-Metselaar, Heidi; Urcuqui-Inchima, Silvio; Smit, Jolanda M; Rodenhuis-Zybert, Izabela A


    Dengue virus (DENV) replication is known to prevent maturation of infected DCs thereby impeding the development of adequate immunity. During secondary DENV infection, dengue-specific antibodies can suppress DENV replication in immature DCs (immDCs), however how dengue-antibody complexes (DENV-IC) in


    Institute of Scientific and Technical Information of China (English)

    王俊梅; 张波; 杨邵敏; 韩继生; 李冰思; 侯琳


    Objective. To develop monoclonal antibodies against the catalytic subunit of human telomerase reverse transcriptase (hTERT) for its expression detection of human tumors. Methods. A dominant epitope in hTERT (peptide hTERT7)was automatically synthesized based on Fmoc method, and was used to immunize Balb/c mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, and the characterization was performed by Western blotting and immunohistochemical staining. The heavy chain variable region of antibody was cloned by RT-PCR and sequenced. Results. Antigenic peptide hTERT7 was synthesized and confirmed by MALDI-TOF-MS and HPLC analysis. One hybridoma cell line secreting anti-hTERT7 antibodies designated as M2 was established after primary screening and consequent 3 rounds of limited dilution. M2 was IgG1 in isotyping. The competi tive assay showed that the M2 antibody was hTERT7 -specific, and the affinity constant was about 1×106 mol-1. The antibody reacted with cell extracts from HeLa cancer cells but not with those from normal 2BS cells in ELISA assay. For in situ staining of immunohistochemistry, the positive staining presented in the nuclear compartment of HeLa, while 2BS was negative. The heavy chain variable region from M2 re vealed that the monoclonal antibody was mouse origin. Conclusions. The developed mouse monoclonal antibody is hTERT-specific and able to recognize native cellular hTERT in ELISA and immunohistochemistry, which makes the immuno-detection of telom erase hTERT expression in cancer cells or tissues possible.

  3. Human C-C chemokine receptor 3 monoclonal antibody inhibits pulmonary inflammation in allergic mice

    Institute of Scientific and Technical Information of China (English)

    Kai WANG; Hua-hao SHEN; Wen LI; Hua-qiong HUANG


    Aim:To evaluate the effect of C-C chemokine receptor 3 (CCR3) blockade on pulmonary inflammation and mucus production in allergic mice. Methods:We used the synthetic peptide of the CCR3 NH2-terminal as the immunizing antigen and generated murine monoclonal antibody against the human CCR3. In addition,the generated antibody was administered to mice sensitized and challenged with ovalbumin. The inflammatory cells in bronchoalveolar lavage,cytokine levels,pulmonary histopathology,and mucus secretion were examined. Results:The Western blotting analysis indicated that the generated antibody bound to CCR3 specifically. The allergic mice treated with the antihuman CCR3 antibody exhibited a significant reduction of pulmonary inflammation accompanied with the alteration of cytokine. Conclusion:The antibody we generated was specific to CCR3. The inhibition of airway inflammation and mucus overproduction by the antibody suggested that the blockade of CCR3 is an appealing therapeutical target for asthma. The present research may provide an experimental basis for the further study of this agent.

  4. 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


    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.

  5. Sex hormones and the immune response in humans

    NARCIS (Netherlands)

    Bouman, Annechien; Heineman, Maas Jan; Faas, Marijke M.


    In addition to their effects on sexual differentiation and reproduction, sex hormones appear to influence the immune system. This results in a sexual dimorphism in the immune response in humans: for instance, females produce more vigorous cellular and more vigorous humoral immune reactions, are more

  6. Sporozoite neutralizing antibodies elicited in mice and rhesus macaques immunized with a Plasmodium falciparum repeat peptide conjugated to meningococcal outer membrane protein complex

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    Craig ePrzysiecki


    Full Text Available Antibodies that neutralize infectivity of malaria sporozoites target the central repeat region of the circumsporozoite (CS protein, which in Plasmodium falciparum is comprised primarily of 30-40 tandem NANP tetramer repeats. We evaluated immunogenicity of an alum-adsorbed (NANP6 peptide conjugated to an outer membrane protein complex (OMPC derived from Neisseria meningitidis, a carrier protein used in a licensed H. influenzae pediatric vaccine. Mice immunized with alum-adsorbed (NANP6-OMPC, with or without Iscomatrix© as co-adjuvant, developed high levels of anti-repeat peptide antibody that inhibited in vitro invasion of human hepatoma cells by transgenic P. berghei sporozoites that express P. falciparum CS repeats (PfPb. Inhibition of sporozoite invasion in vitro correlated with in vivo resistance to challenge by the bites of PfPb infected mosquitoes. Challenged mice had > 90% reduction of hepatic stage parasites as measured by real-time PCR, and either sterile immunity, i.e. no detectable blood stage parasites, or delayed prepatent periods which indicate neutralization of a majority, but not all, sporozoites. Rhesus macaques immunized with two doses of (NANP6-OMPC/MAA formulated with Iscomatrix© developed anti-repeat antibodies that persisted for ~2 years. A third dose of (NANP6-OMPC/MAA+ Iscomatrix© at that time elicited strong anamnestic antibody responses. Rhesus macaque immune sera obtained post second and third dose of vaccine displayed high levels of sporozoite neutralizing activity in vitro that correlated with presence of high anti-repeat antibody titers. These preclinical studies in mice of different MHC haplotypes and a non-human primate support use of CS peptide-OMPC conjugates as a highly immunogenic platform to evaluate CS protective epitopes. Potential pre-erythrocytic vaccines can be combined with sexual blood stage vaccines as a multi-antigen malaria vaccine to block invasion and transmission of Plasmodium parasites

  7. Activated human nasal epithelial cells modulate specific antibody response against bacterial or viral antigens.

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    Chiou-Yueh Yeh

    Full Text Available Nasal mucosa is an immune responsive organ evidenced by eliciting both specific local secretory IgA and systemic IgG antibody responses with intra-nasal administration of antigens. Nevertheless, the role of nasal epithelial cells in modulating such responses is unclear. Human nasal epithelial cells (hNECs obtained from sinus mucosa of patients with chronic rhinosinusitis were cultured in vitro and firstly were stimulated by Lactococcus lactis bacterium-like particles (BLPs in order to examine their role on antibody production. Secondly, both antigens of immunodominant protein IDG60 from oral Streptococcus mutans and hemagglutinin (HA from influenza virus were tested to evaluate the specific antibody response. Stimulated hNECs by BLPs exhibited a significant increase in the production of interleukin-6 (IL-6, and thymic stromal lymphopoietin (TSLP. Conditioned medium of stimulated hNECs has effects on enhancing the proliferation of CD4+ T cells together with interferon-γ and IL-5 production, increasing the costimulatory molecules on dendritic cells and augmenting the production of IDG60 specific IgA, HA specific IgG, IgA by human peripheral blood lymphocytes. Such production of antigen specific IgG and IgA is significantly counteracted in the presence of IL-6 and TSLP neutralizing antibodies. In conclusion, properly stimulated hNECs may impart immuno-modulatory effects on the antigen-specific antibody response at least through the production of IL-6 and TSLP.

  8. Characterization of a novel inhibitory human monoclonal antibody directed against Plasmodium falciparum Apical Membrane Antigen 1. (United States)

    Maskus, Dominika J; Królik, Michał; Bethke, Susanne; Spiegel, Holger; Kapelski, Stephanie; Seidel, Melanie; Addai-Mensah, Otchere; Reimann, Andreas; Klockenbring, Torsten; Barth, Stefan; Fischer, Rainer; Fendel, Rolf


    Malaria remains a major challenge to global health causing extensive morbidity and mortality. Yet, there is no efficient vaccine and the immune response remains incompletely understood. Apical Membrane Antigen 1 (AMA1), a leading vaccine candidate, plays a key role during merozoite invasion into erythrocytes by interacting with Rhoptry Neck Protein 2 (RON2). We generated a human anti-AMA1-antibody (humAbAMA1) by EBV-transformation of sorted B-lymphocytes from a Ghanaian donor and subsequent rescue of antibody variable regions. The antibody was expressed in Nicotiana benthamiana and in HEK239-6E, characterized for binding specificity and epitope, and analyzed for its inhibitory effect on Plasmodium falciparum. The generated humAbAMA1 shows an affinity of 106-135 pM. It inhibits the parasite strain 3D7A growth in vitro with an expression system-independent IC50-value of 35 μg/ml (95% confidence interval: 33 μg/ml-37 μg/ml), which is three to eight times lower than the IC50-values of inhibitory antibodies 4G2 and 1F9. The epitope was mapped to the close proximity of the RON2-peptide binding groove. Competition for binding between the RON2-peptide and humAbAMA1 was confirmed by surface plasmon resonance spectroscopy measurements. The particularly advantageous inhibitory activity of this fully human antibody might provide a basis for future therapeutic applications.

  9. Adjuvants and immunization strategies to induce influenza virus hemagglutinin stalk antibodies.

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    Peter H Goff

    Full Text Available The global population remains vulnerable in the face of the next pandemic influenza virus outbreak, and reformulated vaccinations are administered annually to manage seasonal epidemics. Therefore, development of a new generation of vaccines is needed to generate broad and persistent immunity to influenza viruses. Here, we describe three adjuvants that enhance the induction of stalk-directed antibodies against heterologous and heterosubtypic influenza viruses when administered with chimeric HA proteins. Addavax, an MF59-like nanoemulsion, poly(I:C, and an RNA hairpin derived from Sendai virus (SeV Cantell were efficacious intramuscularly. The SeV RNA and poly(I:C also proved to be effective respiratory mucosal adjuvants. Although the quantity and quality of antibodies induced by the adjuvants varied, immunized mice demonstrated comparable levels of protection against challenge with influenza A viruses on the basis of HA stalk reactivity. Finally, we present that intranasally, but not intramuscularly, administered chimeric HA proteins induce mucosal IgA antibodies directed at the HA stalk.

  10. Innate immune defences in the human endometrium

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    Kelly Rodney W


    Full Text Available Abstract The human endometrium is an important site of innate immune defence, giving protection against uterine infection. Such protection is critical to successful implantation and pregnancy. Infection is a major cause of preterm birth and can also cause infertility and ectopic pregnancy. Natural anti-microbial peptides are key mediators of the innate immune system. These peptides, between them, have anti-bacterial, anti-fungal and anti-viral activity and are expressed at epithelial surfaces throughout the female genital tract. Two families of natural anti-microbials, the defensins and the whey acidic protein (WAP motif proteins, appear to be prominent in endometrium. The human endometrial epithelium expresses beta-defensins 1–4 and the WAP motif protein, secretory leukocyte protease inhibitor. Each beta-defensin has a different expression profile in relation to the stage of the menstrual cycle, providing potential protection throughout the cycle. Secretory leukocyte protease inhibitor is expressed during the secretory phase of the cycle and has a range of possible roles including anti-protease and anti-microbial activity as well as having effects on epithelial cell growth. The leukocyte populations in the endometrium are also a source of anti-microbial production. Neutrophils are a particularly rich source of alpha-defensins, lactoferrin, lysozyme and the WAP motif protein, elafin. The presence of neutrophils during menstruation will enhance anti-microbial protection at a time when the epithelial barrier is disrupted. Several other anti-microbials including the natural killer cell product, granulysin, are likely to have a role in endometrium. The sequential production of natural anti-microbial peptides by the endometrium throughout the menstrual cycle and at other sites in the female genital tract will offer protection from many pathogens, including those that are sexually transmitted.

  11. Human antibody and antigen response to IncA antibody of Chlamydia trachomatis. (United States)

    Tsai, P Y; Hsu, M C; Huang, C T; Li, S Y


    The high prevalence of C. trachomatis worldwide has underscored the importance of identifying specific immunogenic antigens in facilitating diagnosis as well as vaccine development. The aim of this study is to evaluate IncA antibody and antigen production in natural human infections. Our temporal expression study showed that IncA transcription and protein expression could be detected as early as 4 hours after the start of infection. Antibody responses could be detected in urine and genital swab samples from C. trachomatis-positive patients. It is especially interesting to note that the IncA antigen could be detected in urine. In conclusion, we have identified IncA as an important antigen in human. The potential applicability of the IncA antibody or antigen in the diagnosis as well as to vaccine development for C. trachomatis is also discussed.


    Directory of Open Access Journals (Sweden)

    I. V. Losev


    Full Text Available There exists a real hazard of transferring zoonotic influenza A viruses, either swine, or avian, into human population. In such case, severity of such pandemics depends on the pathogen-specific immunity in the population. Virtual absence of such immunity in humans was declared in the literature. In this work, we assessed systemic, local, and T-cell immunity to potentially pandemic H3N2sw, H5N1, H5N2, H7N3, H7N9 and H2N2 influenza A viruses in a group of healthy adults of different age. Our results indicate that these subjects develop the following immune reactions: (i local (i.e., nasal IgA and cellular (CD4+ and CD8v memory T cells heterosubtypic immunity, in absence of detectable virus-specific serum antibodies to avian influenza A viruses; (ii Local immune responses (as nasal IgA to human A (H2N2 virus which circulated in 1957-1968 were detected both in subjects who could be primed at that time, but also in subjects born after 1968; (iii full-scale systemic and local immunity to potentially pandemic А (H3N2sw swine virus was found in the group. Conclusion. In order of proper epidemiological forecasts and planning appropriate preventive measures for potentially pandemic Influenza A viruses, a regular monitoring of collective immunity should be performed using different adaptive markers. In this respect, any conclusion based on molecular analysis only could lead to considerable mistakes, and should be accomplished by the mentioned immunological studies.

  13. Preventive immunization of aged and juvenile non-human primates to beta-amyloid

    Directory of Open Access Journals (Sweden)

    Kofler Julia


    Full Text Available Abstract Background Immunization against beta-amyloid (Aβ is a promising approach for the treatment of Alzheimer’s disease, but the optimal timing for the vaccination remains to be determined. Preventive immunization approaches may be more efficacious and associated with fewer side-effects; however, there is only limited information available from primate models about the effects of preclinical vaccination on brain amyloid composition and the neuroinflammatory milieu. Methods Ten non-human primates (NHP of advanced age (18–26 years and eight 2-year-old juvenile NHPs were immunized at 0, 2, 6, 10 and 14 weeks with aggregated Aβ42 admixed with monophosphoryl lipid A as adjuvant, and monitored for up to 6 months. Anti-Aβ antibody levels and immune activation markers were assessed in plasma and cerebrospinal fluid samples before and at several time-points after immunization. Microglial activity was determined by [11C]PK11195 PET scans acquired before and after immunization, and by post-mortem immunohistochemical and real-time PCR evaluation. Aβ oligomer composition was assessed by immunoblot analysis in the frontal cortex of aged immunized and non-immunized control animals. Results All juvenile animals developed a strong and sustained serum anti-Aβ IgG antibody response, whereas only 80 % of aged animals developed detectable antibodies. The immune response in aged monkeys was more delayed and significantly weaker, and was also more variable between animals. Pre- and post-immunization [11C]PK11195 PET scans showed no evidence of vaccine-related microglial activation. Post-mortem brain tissue analysis indicated a low overall amyloid burden, but revealed a significant shift in oligomer size with an increase in the dimer:pentamer ratio in aged immunized animals compared with non-immunized controls (P  Conclusions Our results indicate that preventive Aβ immunization is a safe therapeutic approach lacking adverse CNS immune system

  14. [New antibodies in cancer treatment]. (United States)

    Pestalozzi, B C; Knuth, A


    Since the development of hybridoma technology in 1975 monoclonal antibodies with pre-defined specificity can be produced. Only twenty years later did it become possible to make therapeutic use of monoclonal antibodies in oncology. To this end it was necessary to attach the antigen-binding site of a mouse antibody onto the scaffold of a human antibody molecule. Such chimeric or "humanized" antibodies may be used in passive immunotherapy without eliciting an immune response. Rituximab and trastuzumab are such humanized antibodies. They are used today routinely in the treatment of malignant lymphoma and breast cancer, respectively. These antibodies are usually used in combination with conventional cytostatic anticancer drugs.

  15. Epitope Mapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients▿


    Song, Ruijiang; Franco, David; Kao, Chia-Ying; Yu, Faye; Huang, Yaoxing; Ho, David D.


    Ibalizumab is a humanized monoclonal antibody that binds human CD4, the primary receptor for human immunodeficiency virus type 1 (HIV-1). With its unique specificity for domain 2 of CD4, this antibody potently and broadly blocks HIV-1 infection in vitro by inhibiting a postbinding step required for viral entry but without interfering with major histocompatibility complex class II (MHC-II)-mediated immune function. In clinical trials, ibalizumab has demonstrated anti-HIV-1 activity in patients...

  16. Monitoring the systemic human memory B cell compartment of melanoma patients for anti-tumor IgG antibodies.

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    Amy E Gilbert

    Full Text Available Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10 to primary and metastatic melanoma cells compared to healthy volunteers (n = 10 (P<0.0001. Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21 (P<0.0001. Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800 compared to 2% of cultures from healthy controls (n = 600 produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer.

  17. Local and Systemic Antibody Responses in Humans with Helicobacter pylori Infection

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    Thomas G Blanchard


    Full Text Available Immunization can prevent or cure an otherwise chronic helicobacter infection in several animal models despite the chronic nature of natural helicobacter infections. Differences in the antigenic specificity of the antibodies may contribute to the protection observed in these experimental animals. The goal of the present study was to compare the local and systemic antibody responses of humans with chronic Helicobacter pylori infection with those of an individual with spontaneous resolution of infection to find an immunological correlate of protection. Spontaneous resolution of infection was accompanied by a change in immunoblot profiles. Whereas a broad range of H pylori antigens was recognized in chronically infected patients (including the patient who ultimately cleared the infection spontaneously, resolution of infection in the absence of therapeutic agents resulted in the recognition of only several immunodominant antigens. The most dominant antigen was approximately 66 kDa in molecular mass. Immunoblot analysis demonstrated that these antibodies were specific for the structural subunits of the urease enzyme. These studies suggest that the success of antihelicobacter immunization may be due to the ability of vaccination to induce an immune response against antigens that are normally not immunodominant during the course of infection.

  18. Standardization of the antibody-dependent respiratory burst assay with human neutrophils and Plasmodium falciparum malaria. (United States)

    Llewellyn, David; Miura, Kazutoyo; Fay, Michael P; Williams, Andrew R; Murungi, Linda M; Shi, Jianguo; Hodgson, Susanne H; Douglas, Alexander D; Osier, Faith H; Fairhurst, Rick M; Diakite, Mahamadou; Pleass, Richard J; Long, Carole A; Draper, Simon J


    The assessment of naturally-acquired and vaccine-induced immunity to blood-stage Plasmodium falciparum malaria is of long-standing interest. However, the field has suffered from a paucity of in vitro assays that reproducibly measure the anti-parasitic activity induced by antibodies in conjunction with immune cells. Here we optimize the antibody-dependent respiratory burst (ADRB) assay, which assesses the ability of antibodies to activate the release of reactive oxygen species from human neutrophils in response to P. falciparum blood-stage parasites. We focus particularly on assay parameters affecting serum preparation and concentration, and importantly assess reproducibility. Our standardized protocol involves testing each serum sample in singlicate with three independent neutrophil donors, and indexing responses against a standard positive control of pooled hyper-immune Kenyan sera. The protocol can be used to quickly screen large cohorts of samples from individuals enrolled in immuno-epidemiological studies or clinical vaccine trials, and requires only 6 μL of serum per sample. Using a cohort of 86 samples, we show that malaria-exposed individuals induce higher ADRB activity than malaria-naïve individuals. The development of the ADRB assay complements the use of cell-independent assays in blood-stage malaria, such as the assay of growth inhibitory activity, and provides an important standardized cell-based assay in the field.

  19. Secretion of respiratory syncytial virus inhibitors and antibody in human milk throughout lactation. (United States)

    Toms, G L; Gardner, P S; Pullan, C R; Scott, M; Taylor, C


    Neutralising inhibitors to respiratory syncytial (RS) virus have been demonstrated in the whey of most samples of human milk tested. Although high titres were secreted in colostra of some mothers (1/10-1/2,560; median 1/40) inhibitor levels in milk collected after the first week of lactation were uniformly low (median 1/10). High neutralising titres correlated with high colostral levels of specific antiviral IgA but, unlike neutralising activity, IgA antiviral antibody persisted in the milk of only four of 18 mothers. Similarly, antiviral IgG and IgM antibodies were not generally detected after the first post-partum week. Differences in antibody secretion among mothers did not correlate with differences in total protein or total immunoglobulin secretion, and appeared to reflect maternal immune status. In one mother a marked rise in specific antiviral IgA and IgG secretions during the second and third months of lactation suggested a response to virus infection. The relevance of maternal immunity and colostral and milk antiviral antibody to protection of breast-fed babies from RS-virus bronchiolitis is discussed.

  20. Identification and characterization of human eosinophil cationic protein by an epitope-specific antibody. (United States)

    Boix, E; Carreras, E; Nikolovski, Z; Cuchillo, C M; Nogués, M V


    The eosinophil cationic protein (ECP) is a basic secretion protein involved in the immune response system. ECP levels in biological fluids are an indicator of eosinophil-specific activation and degranulation and are currently used for the clinical monitoring and diagnosis of inflammatory disorders. A polyclonal epitope-specific antibody has been obtained by immunizing rabbits with a conjugated synthetic peptide. A sequence corresponding to a large exposed loop in the human ECP three-dimensional structure (D115-Y122) was selected as a putative antigenic epitope. The antibody was purified on an affinity column using recombinant ECP (rECP) as antigen. The antibody (D112-P123 Ab) specifically recognizes rECP and its native glycosylated and nonglycosylated forms in plasma, granulocytes, and sputum. The antibody detects as little as 1 ng of rECP, can be used both in reducing and nonreducing conditions, and does not cross-react with the highly homologous eosinophil-derived neurotoxin or other proteins of the pancreatic ribonuclease superfamily.

  1. Serological characterization of guinea pigs infected with H3N2 human influenza or immunized with hemagglutinin protein

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    Bushnell Ruth V


    Full Text Available Abstract Background Recent and previous studies have shown that guinea pigs can be infected with, and transmit, human influenza viruses. Therefore guinea pig may be a useful animal model for better understanding influenza infection and assessing vaccine strategies. To more fully characterize the model, antibody responses following either infection/re-infection with human influenza A/Wyoming/03/2003 H3N2 or immunization with its homologous recombinant hemagglutinin (HA protein were studied. Results Serological samples were collected and tested for anti-HA immunoglobulin by ELISA, antiviral antibodies by hemagglutination inhibition (HI, and recognition of linear epitopes by peptide scanning (PepScan. Animals inoculated with infectious virus demonstrated pronounced viral replication and subsequent serological conversion. Animals either immunized with the homologous HA antigen or infected, showed a relatively rapid rise in antibody titers to the HA glycoprotein in ELISA assays. Antiviral antibodies, measured by HI assay, were detectable after the second inoculation. PepScan data identified both previously recognized and newly defined linear epitopes. Conclusions Infection and/or recombinant HA immunization of guinea pigs with H3N2 Wyoming influenza virus resulted in a relatively rapid production of viral-specific antibody thus demonstrating the strong immunogenicity of the major viral structural proteins in this animal model for influenza infection. The sensitivity of the immune response supports the utility of the guinea pig as a useful animal model of influenza infection and immunization.

  2. Antigen-specific acquired immunity in human brucellosis: implications for diagnosis, prognosis, and vaccine development

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    Anthony P Cannella


    Full Text Available Brucella spp. are facultative intracellular Gram negative bacteria with specific tropism for monocytes/macrophages. Clinical manifestations of brucellosis are primarily immune-mediated and not thought to be due to bacterial virulence factors. Acquired immunity to brucellosis has been studied through observations of naturally infected hosts (cattle, goats, laboratory mouse models, and human infection. Cell-mediated immunity drives the clinical manifestations of human disease after exposure to Brucella species but high antibody responses are not associated with protective immunity. The precise mechanisms by which cell-mediated immune responses confer protection or lead to disease manifestations remain poorly understood. Descriptive studies of immune responses in human brucellosis show that TH1 (interferon-gamma are associated with dominant immune responses, findings consistent with animal studies. Whether these T cell responses are protective, or determine the different clinical responses associated with brucellosis is unknown, especially with regard to undulant fever manifestations, relapsing disease, or are associated with responses to distinct sets of Brucella spp. antigens are unknown. Few data regarding T cell responses in terms of specific recognition of Brucella spp. protein antigens and peptidic epitopes, either by CD4+ or CD8+ T cells, have been identified in human brucellosis patients. Additionally because current attenuated Brucella vaccines used in animals cause human disease, there is a true need for a recombinant protein subunit vaccine for human brucellosis, as well as for improved diagnostics in terms of prognosis and identification of unusual forms of brucellosis. This review will focus on current understandings of antigen-specific immune responses induced by Brucella protein antigens that has promise for yielding new insights into vaccine and diagnostics development, and for understanding pathogenetic mechanisms of human

  3. Current status of cancer immunodetection with radiolabeled human monoclonal antibodies. (United States)

    De Jager, R; Abdel-Nabi, H; Serafini, A; Pecking, A; Klein, J L; Hanna, M G


    The use of radiolabeled murine monoclonal antibodies (MoAbs) for cancer immunodetection has been limited by the development of human antimouse antibodies (HAMA). Human monoclonal antibodies do not elicit a significant human antihuman (HAHA) response. The generation and production of human monoclonal antibodies met with technical difficulties that resulted in delaying their clinical testing. Human monoclonal antibodies of all isotypes have been obtained. Most were immunoglobulin (Ig) M directed against intracellular antigens. Two antibodies, 16.88 (IgM) and 88BV59 (IgG3k), recognize different epitopes on a tumor-associated antigen, CTA 16.88, homologous to cytokeratins 8, 18, and 19. CTA 16.88 is expressed by most epithelial-derived tumors including carcinomas of the colon, pancreas, breast, ovary, and lung. The in vivo targeting by these antibodies is related to their localization in nonnecrotic areas of tumors. Repeated administration of 16.88 over 5 weeks to a cumulative dose of 1,000 mg did not elicit a HAHA response. Two of 53 patients developed a low titer of HAHA 1 to 3 months after a single administration of 88BV59. Planar imaging of colorectal cancer with Iodine-131 (131I)-16.88 was positive in two studies in 9 of 12 and 16 of 20 patients preselected by immunohistochemistry. Tumors less than 2 cm in diameter are usually not detected. The lack of immunogenicity and long tumor residence time (average = 17 days) makes 16.88 a good candidate for therapy. Radioimmunlymphoscintigraphy with indium-111 (111In)-LiLo-16.88 administered by an intramammary route was used in the presurgical staging of primary breast cancer. The negative predictive value of lymph node metastases for tumors less than 3 cm was 90.5%. Planar and single photon emission computed tomography imaging of colorectal carcinoma with technetium-99m (99mTc) 88BV59 was compared with computed tomography (CT) scan in 36 surgical patients. The antibody scan was more sensitive than the CT scan in detecting

  4. Modulation of innate immune responses by influenza-specific ovine polyclonal antibodies used for prophylaxis.

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    Catherine Rinaldi

    Full Text Available In the event of a novel influenza A virus pandemic, prophylaxis mediated by antibodies provides an adjunct control option to vaccines and antivirals. This strategy is particularly pertinent to unvaccinated populations at risk during the lag time to produce and distribute an effective vaccine. Therefore, development of effective prophylactic therapies is of high importance. Although previous approaches have used systemic delivery of monoclonal antibodies or convalescent sera, available supply is a serious limitation. Here, we have investigated intranasal delivery of influenza-specific ovine polyclonal IgG antibodies for their efficacy against homologous influenza virus challenge in a mouse model. Both influenza-specific IgG and F(ab'2 reduced clinical scores, body weight loss and lung viral loads in mice treated 1 hour before virus exposure. Full protection from disease was also observed when antibody was delivered up to 3 days prior to virus infection. Furthermore, effective prophylaxis was independent of a strong innate immune response. This strategy presents a further option for prophylactic intervention against influenza A virus using ruminants to generate a bulk supply that could potentially be used in a pandemic setting, to slow virus transmission and reduce morbidity associated with a high cytokine phenotype.

  5. Beyond immediate hypersensitivity: evolving roles for IgE antibodies in immune homeostasis and allergic diseases. (United States)

    Burton, Oliver T; Oettgen, Hans C


    Immunoglobulin E (IgE) antibodies have long been recognized as the antigen-specific triggers of allergic reactions. This review briefly introduces the established functions of IgE in immediate hypersensitivity and then focuses on emerging evidence from our own investigations as well as those of others that IgE plays important roles in protective immunity against parasites and exerts regulatory influences in the expression of its own receptors, FcεRI and CD23, as well as controlling mast cell homeostasis. We provide an overview of the multifaceted ways in which IgE antibodies contribute to the pathology of food allergy and speculate regarding potential mechanisms of action of IgE blockade.

  6. Immunization of chickens with quail and turkey perivitelline membrane proteins: production of antibodies and their effects on fertility. (United States)

    Kapoor, P; Compton, M M; Howarth, B


    The cross-reactivity of antibodies developed against zona pellucida proteins and their subsequent deleterious effect on fertility in heterologous species is well documented. However, similar investigations have not been undertaken in avian species. In Experiment 1, White Leghorn hens were immunized with intact germinal discs (GD) of quail and chickens. Chicken GD proteins did not elicit an immune response in chicken hens, whereas quail GD proteins were highly immunogenic. Anti-quail GD antibodies did not bind with chicken inner perivitelline membrane (IPM) proteins as determined by immunoblot analysis. To examine the fertility of immunized hens, artificial insemination was performed at weekly intervals for 4 wk following the booster immunization. No significant differences were detected in fertility or hatchability of immunized hens when compared with unimmunized control hens. In Experiment 2, White Leghorn hens were immunized with intact turkey GD, solubilized turkey perivitelline membrane (PM) modified with dinitrophenol (DNP), and solubilized chicken IPM modified with DNP. High titers of antibodies were detected against the turkey GD and the DNP-modified turkey PM proteins. A weak immune response was observed in hens immunized with modified chicken IPM proteins. The fertility and hatchability of eggs laid by immunized hens, however, were not significantly different from those of unimmunized hens. Antibodies from immunized hens were further analyzed using an in vitro assay that assesses sperm penetration of intact IPM. Sperm penetration of intact IPM was inhibited to the same extent when IPM was preincubated with preimmune as well as anti-PM immunoglobulins. Collectively, these results suggested that the antibodies developed in these hens did not cross-react with the chicken IPM proteins involved in sperm-egg interaction and thus did not influence the fertility.

  7. Immune status following alemtuzumab treatment in human CD52 transgenic mice. (United States)

    Turner, Michael J; Lamorte, Michael J; Chretien, Nathalie; Havari, Evis; Roberts, Bruce L; Kaplan, Johanne M; Siders, William M


    Alemtuzumab is a monoclonal antibody against the CD52 antigen present at high levels on the surface of lymphocytes. While treatment of multiple sclerosis patients with alemtuzumab results in marked depletion of lymphocytes from the circulation, it has not been associated with a high incidence of serious infections. In a human CD52 transgenic mouse, alemtuzumab treatment showed minimal impact on the number and function of innate immune cells. A transient decrease in primary adaptive immune responses was observed post-alemtuzumab but there was little effect on memory responses. These results potentially help explain the level of immunocompetence observed in alemtuzumab-treated MS patients.

  8. Production of neutralizing monoclonal antibody against human vascular endothelial growth factor receptor Ⅱ

    Institute of Scientific and Technical Information of China (English)

    Rong LI; Dong-sheng XIONG; Xiao-feng SHAO; Jia LIU; Yuan-fu XU; Yuan-sheng XU; Han-zhi LIU; Zhen-ping ZHU; Chun-zheng YANG


    AIM: To prepare neutralizing monoclonal antibody (mAb) against extracellular immunoglobulin (Ig)-like domainⅢ of vascular endothelial growth factor receptor KDR and study its biological activity. METHODS: Soluble KDR Ig domain Ⅲ (KDR-Ⅲ) fusion protein was expressed in E Coli and purified from the bacterial periplasmic extracts via an affinity chromatography. Monoclonal antibodies against KDR-Ⅲ were prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [3H]-thymidine incorporation assay were also used to detect the activity of anti-KDR mAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on vascular endothelial growth factor-induced mitogenesis of human endothelial ceils.RESULTS: A monoclonal antibody, Ycom1D3 (IgG1), was generated from a mouse immunized with the recombinant KDR-Ⅲ protein. Ycom1D3 bound specifically to both the soluble KDR-Ⅲ and the cell-surface expressed KDR. Ycom1D3 effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated KDR activation in human endothelial cells. Furthermore, the antibody efficiently neutralized VEGF-induced mitogenesis of human endothelial cells. CONCLUSION: Our results suggest that the anti-KDR mAb, Ycom1D3, has potential applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.


    NARCIS (Netherlands)



    The selectivity of anticancer agents may be improved by antibody-directed enzyme prodrug therapy (ADEPT), The immunogenicity of antibody-enzyme conjugates and the low tumor to normal tissue ratio calls for the use of a human enzyme and the development of a monoclonal antibody (MAb) against that enzy

  10. Induction of neutralizing antibodies in mice immunized with scorpion toxins detoxified by liposomal entrapment



    The possibility of producing neutralizing antibodies against the lethal effects of scorpion toxins was evaluated in the mouse model by immunization with an immunogen devoid of toxicity. A toxic fraction (5 mg) from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes. The liposomes were treated for 1 h at 37oC with a 1% (w/w) trypsin solution in 0.2 M sodium carbonate buffer, pH 8.3. This treatment led to a strong reduction in venom toxicity. Immuni...

  11. Neutralization Interfering Antibodies: A “Novel” Example of Humoral Immune Dysfunction Facilitating Viral Escape?

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    Roberto Burioni


    Full Text Available The immune response against some viral pathogens, in particular those causing chronic infections, is often ineffective notwithstanding a robust humoral neutralizing response. Several evasion mechanisms capable of subverting the activity of neutralizing antibodies (nAbs have been described. Among them, the elicitation of non-neutralizing and interfering Abs has been hypothesized. Recently, this evasion mechanism has acquired an increasing interest given its possible impact on novel nAb-based antiviral therapeutic and prophylactic approaches. In this review, we illustrate the mechanisms of Ab-mediated interference and the viral pathogens described in literature as able to adopt this “novel” evasion strategy.

  12. Identification of a common immune signature in murine and human systemic Salmonellosis (United States)

    Lee, Seung-Joo; Liang, Li; Juarez, Silvia; Nanton, Minelva R.; Gondwe, Esther N.; Msefula, Chisomo L.; Kayala, Matthew A.; Necchi, Francesca; Heath, Jennifer N.; Hart, Peter; Tsolis, Renée M.; Heyderman, Robert S.; MacLennan, Calman A.; Felgner, Philip L.; Davies, D. Huw; McSorley, Stephen J.


    Despite the importance of Salmonella infections in human and animal health, the target antigens of Salmonella-specific immunity remain poorly defined. We have previously shown evidence for antibody-mediating protection against invasive Salmonellosis in mice and African children. To generate an overview of antibody targeting in systemic Salmonellosis, a Salmonella proteomic array containing over 2,700 proteins was constructed and probed with immune sera from Salmonella-infected mice and humans. Analysis of multiple inbred mouse strains identified 117 antigens recognized by systemic antibody responses in murine Salmonellosis. Importantly, many of these antigens were independently identified as target antigens using sera from Malawian children with Salmonella bacteremia, validating the study of the murine model. Furthermore, vaccination with SseB, the most prominent antigenic target in Malawian children, provided mice with significant protection against Salmonella infection. Together, these data uncover an overlapping immune signature of disseminated Salmonellosis in mice and humans and provide a foundation for the generation of a protective subunit vaccine. PMID:22331879

  13. Passive immunization with anti-ActA and anti-listeriolysin O antibodies protects against Listeria monocytogenes infection in mice (United States)

    Asano, Krisana; Sashinami, Hiroshi; Osanai, Arihiro; Hirose, Shouhei; Ono, Hisaya K.; Narita, Kouji; Hu, Dong-Liang; Nakane, Akio


    Listeria monocytogenes is an intracellular pathogen that causes listeriosis. Due to its intracellular niche, L. monocytogenes has evolved to limit immune recognition and response to infection. Antibodies that are slightly induced by listerial infection are completely unable to protect re-infection of L. monocytogenes. Thus, a role of antibody on the protective effect against L. monocytogenes infection has been neglected for a long time. In the present study, we reported that passive immunization with an excessive amount of antibodies against ActA and listeriolysin O (LLO) attenuates severity of L. monocytogenes infection. Combination of these antibodies improved survival of L. monocytogenes infected mice. Bacterial load in spleen and liver of listerial infected mice and infected RAW264.7 cells were significantly reduced by administration of anti-ActA and anti-LLO antibodies. In addition, anti-LLO antibody neutralized LLO activity and inhibited the bacterial escape from the lysosomal compartments. Moreover, anti-ActA antibody neutralized ActA activity and suppressed actin tail formation and cell-to-cell spread. Thus, our studies reveal that passive immunization with the excessive amount of anti-ActA and -LLO antibodies has potential to provide the protective effect against listerial infection. PMID:28004800

  14. Traumatic spinal cord injury in mice with human immune systems. (United States)

    Carpenter, Randall S; Kigerl, Kristina A; Marbourg, Jessica M; Gaudet, Andrew D; Huey, Devra; Niewiesk, Stefan; Popovich, Phillip G


    Mouse models have provided key insight into the cellular and molecular control of human immune system function. However, recent data indicate that extrapolating the functional capabilities of the murine immune system into humans can be misleading. Since immune cells significantly affect neuron survival and axon growth and also are required to defend the body against infection, it is important to determine the pathophysiological significance of spinal cord injury (SCI)-induced changes in human immune system function. Research projects using monkeys or humans would be ideal; however, logistical and ethical barriers preclude detailed mechanistic studies in either species. Humanized mice, i.e., immunocompromised mice reconstituted with human immune cells, can help overcome these barriers and can be applied in various experimental conditions that are of interest to the SCI community. Specifically, newborn NOD-SCID-IL2rg(null) (NSG) mice engrafted with human CD34(+) hematopoietic stem cells develop normally without neurological impairment. In this report, new data show that when mice with human immune systems receive a clinically-relevant spinal contusion injury, spontaneous functional recovery is indistinguishable from that achieved after SCI using conventional inbred mouse strains. Moreover, using routine immunohistochemical and flow cytometry techniques, one can easily phenotype circulating human immune cells and document the composition and distribution of these cells in the injured spinal cord. Lesion pathology in humanized mice is typical of mouse contusion injuries, producing a centralized lesion epicenter that becomes occupied by phagocytic macrophages and lymphocytes and enclosed by a dense astrocytic scar. Specific human immune cell types, including three distinct subsets of human monocytes, were readily detected in the blood, spleen and liver. Future studies that aim to understand the functional consequences of manipulating the neuro-immune axis after SCI

  15. Generation and isolation of target-specific single-domain antibodies from shark immune repertoires. (United States)

    Müller, Mischa Roland; O'Dwyer, Ronan; Kovaleva, Marina; Rudkin, Fiona; Dooley, Helen; Barelle, Caroline Jane


    The drive to exploit novel targets and biological pathways has lead to the expansion of classical antibody research into innovative fragment adaptations and novel scaffolds. The hope being that alternative or cryptic epitopes may be targeted, tissue inaccessibility may be overcome, and easier engineering options will facilitate multivalent, multi-targeting approaches. To this end, we have been isolating shark single domains to gain a greater understanding of their potential as therapeutic agents. Their unique shape, small size, inherent stability, and simple molecular architecture make them attractive candidates from a drug discovery perspective. Here we describe protocols to capture the immune repertoire of an immunized shark species and to build and select via phage-display target-specific IgNAR variable domains (VNARs).

  16. Kinetics of epsilon antitoxin antibodies in different strategies for active immunization of lambs against enterotoxaemia

    Directory of Open Access Journals (Sweden)

    Heni F. Costa


    Full Text Available Enterotoxaemia, a common disease that affects domestic small ruminants, is mainly caused by the epsilon toxin of Clostridium perfringens type D. The present study tested four distinct immunization protocols to evaluate humoral response in lambs, a progeny of non-vaccinated sheep during gestation. Twenty-four lambs were randomly allocated into four groups according to age (7, 15, 30 and 45 days, receiving the first dose of epsilon toxoid commercial vaccine against clostridiosis with booster after 30 days post vaccination. Indirect ELISA was performed after the first vaccine dose and booster to evaluate the immune response of the lambs. Results showed that for the four protocols tested all lambs presented serum title considered protective (≥0.2UI/ml epsilon antitoxin antibodies and also showed that the anticipation of primovaccination of lambs against enterotoxaemia conferred serum title considered protective allowing the optimization of mass vaccination of lambs.

  17. Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire. (United States)

    Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei


    High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies.

  18. Characterization of human antiviral adaptive immune responses during hepatotropic virus infection in HLA-transgenic human immune system mice. (United States)

    Billerbeck, Eva; Horwitz, Joshua A; Labitt, Rachael N; Donovan, Bridget M; Vega, Kevin; Budell, William C; Koo, Gloria C; Rice, Charles M; Ploss, Alexander


    Humanized mice have emerged as a promising model to study human immunity in vivo. Although they are susceptible to many pathogens exhibiting an almost exclusive human tropism, human immune responses to infection remain functionally impaired. It has recently been demonstrated that the expression of HLA molecules improves human immunity to lymphotropic virus infections in humanized mice. However, little is known about the extent of functional human immune responses in nonlymphoid tissues, such as in the liver, and the role of HLA expression in this context. Therefore, we analyzed human antiviral immunity in humanized mice during a hepatotropic adenovirus infection. We compared immune responses of conventional humanized NOD SCID IL-2Rγ-deficient (NSG) mice to those of a novel NOD SCID IL-2Rγ-deficient strain transgenic for both HLA-A*0201 and a chimeric HLA-DR*0101 molecule. Using a firefly luciferase-expressing adenovirus and in vivo bioluminescence imaging, we demonstrate a human T cell-dependent partial clearance of adenovirus-infected cells from the liver of HLA-transgenic humanized mice. This correlated with liver infiltration and activation of T cells, as well as the detection of Ag-specific humoral and cellular immune responses. When infected with a hepatitis C virus NS3-expressing adenovirus, HLA-transgenic humanized mice mounted an HLA-A*0201-restricted hepatitis C virus NS3-specific CD8(+) T cell response. In conclusion, our study provides evidence for the generation of partial functional antiviral immune responses against a hepatotropic pathogen in humanized HLA-transgenic mice. The adenovirus reporter system used in our study may serve as simple in vivo method to evaluate future strategies for improving human intrahepatic immune responses in humanized mice.

  19. Discovery of human antibodies against black cobra toxins

    DEFF Research Database (Denmark)

    Øhlenschlæger, Mia; Andersen, Mikael Rørdam; Lohse, Brian

    Snakebite envenoming represents a major health threat intropical parts of the developing world1. Animal-derivedantisera currently constitute the only effective treatment option,but are associated with severe side effects due toincompatibility with the human immune system. We aim atdiscovering human...

  20. IgA antibodies to Toxoplasma gondii in human tears

    NARCIS (Netherlands)

    Meek, B.; Klaren, V.N.A.; Haeringen, van N.J.; Kijlstra, A.; Peek, R.


    PURPOSE. To investigate whether mucosal immune responses directed against the ubiquitous parasite Toxoplasma gondii can be detected in tears of healthy humans. METHODS. Nonstimulated tears and blood were obtained from 62 healthy humans (mean age, 35 ± 10 [SD] years). Serum anti-T. gondii immunoglobu

  1. Differential expression of anti-glycan antibodies in schistosome-infected humans, rhesus monkeys and mice (United States)

    Luyai, Anthony E; Heimburg-Molinaro, Jamie; Prasanphanich, Nina Salinger; Mickum, Megan L; Lasanajak, Yi; Song, Xuezheng; Nyame, A Kwame; Wilkins, Patricia; Rivera-Marrero, Carlos A; Smith, David F; Van Die, Irma; Secor, W Evan; Cummings, Richard D


    Schistosomiasis is a debilitating parasitic disease of humans, endemic in tropical areas, for which no vaccine is available. Evidence points to glycan antigens as being important in immune responses to infection. Here we describe our studies on the comparative humoral immune responses to defined schistosome-type glycan epitopes in Schistosoma mansoni-infected humans, rhesus monkeys and mice. Rhesus anti-glycan responses over the course of infection were screened on a defined glycan microarray comprising semi-synthetic glycopeptides terminating with schistosome-associated or control mammalian-type glycan epitopes, as well as a defined glycan microarray of mammalian-type glycans representing over 400 glycan structures. Infected rhesus monkeys generated a high immunoglobulin G (IgG) antibody response to the core xylose/core α3 fucose epitope of N-glycans, which peaked at 8–11 weeks post infection, coinciding with maximal ability to kill schistosomula in vitro. By contrast, infected humans generated low antibody levels to this epitope. At 18 months following praziquantel therapy to eliminate the parasite, antibody levels were negligible. Mice chronically infected with S. mansoni generated high levels of anti-fucosylated LacdiNAc (GalNAcβ1, 4(Fucα1, 3)GlcNAc) IgM antibodies, but lacked a robust response to the core xylose/core α3 fucose N-glycan antigens compared with other species studied, and their sera demonstrated an intermediate level of schistosomula killing in vitro. These differential responses to parasite glycan antigens may be related to the ability of rhesus monkeys to self-cure in contrast to the chronic infection seen in humans and mice. Our results validate defined glycan microarrays as a useful technology to evaluate diagnostic and vaccine antigens for schistosomiasis and perhaps other infections. PMID:24727442

  2. Antibody responses to bacteriophage phi X-174 in human subjects exposed to the antarctic winter-over model of spaceflight (United States)

    Shearer, W. T.; Lugg, D. J.; Rosenblatt, H. M.; Nickolls, P. M.; Sharp, R. M.; Reuben, J. M.; Ochs, H. D.


    BACKGROUND: It has been proposed that exposure to long-term spaceflight conditions (stress, isolation, sleep disruption, containment, microbial contamination, and solar radiation) or to ground-based models of spaceflight will alter human immune responses, but specific antibody responses have not been fully evaluated. OBJECTIVE: We sought to determine whether exposure to the 8-month Antarctic winter-over model of spaceflight would alter human antibody responses. METHODS: During the 1999 Australian National Antarctic Research Expeditions, 11 adult study subjects at Casey, Antarctica, and 7 control subjects at Macquarie Island, sub-Antarctica, received primary and secondary immunizations with the T cell-dependent neoantigen bacteriophage phi X-174. Periodic plasma samples were analyzed for specific antibody function. RESULTS: All of the subjects from Casey, Antarctica, cleared bacteriophage phi X-174 normally by 1 week after primary immunization, and all had normal primary and secondary antibody responses, including immunologic memory amplification and switch from IgM to IgG antibody production. One subject showed a high normal pattern, and one subject had a low normal pattern. The control subjects from Macquarie Island also had normal immune responses to bacteriophage phi X-174. CONCLUSIONS: These data do not support the hypothesis that de novo specific antibody responses of subjects become defective during the conditions of the Antarctic winter-over. Because the Antarctic winter-over model of spaceflight lacks the important factors of microgravity and solar radiation, caution must be used in interpreting these data to anticipate normal antibody responses in long-term spaceflight.

  3. Rescue and expression of human immunoglobulin genes to generate functional human monoclonal antibodies. (United States)

    Lewis, A P; Parry, N; Peakman, T C; Crowe, J S


    Human monoclonal antibody production has been hampered for many years by the instability of cell lines and low levels of expression of the antibodies. We describe here the rescue of human immunoglobulin genes utilizing micro-mRNA preparation from a small number of human hybridoma cells and conventional cDNA cloning. This allows cloning and immediate high-level expression from full-length human heavy and light chain cDNA molecules and provides a mechanism to rescue whole human monoclonal antibodies of proven efficacy.

  4. The crossroads between cancer immunity and autoimmunity: antibodies to self antigens. (United States)

    Benvenuto, Monica; Mattera, Rosanna; Masuelli, Laura; Tresoldi, Ilaria; Giganti, Maria Gabriella; Frajese, Giovanni Vanni; Manzari, Vittorio; Modesti, Andrea; Bei, Roberto


    The production of autoantibodies to self antigens is dependent on the failure of immune tolerance. Cancer cells express antigens which elicit a spontaneous immune response in cancer patients. The repertoire of autoantibodies found in cancer patients partly covers that of patients with autoimmune diseases. Biological activities of autoantibodies to self antigens may induce paraneoplastic syndromes which reflect the attempt of cancer patients to counteract tumor growth. Autoantibodies with similar specificities may have different effects in cancer and autoimmune disease patients due to different immunological microenvironments. Tregs dysfunction has been observed in patients with paraneoplastic syndromes and/or with autoimmune diseases, while the increase of Tregs has been associated with poor cancer patients prognosis. Novel therapies have employed antibodies against Tregs immune-checkpoint receptors with the aim to boost immune response in cancer patients. The presence of autoantibodies to tumors antigens has also been investigated as a marker for cancer detection and cancer patients prognosis. This report reviews the current knowledge on the analysis and meaning of autoantibodies to self antigens detected in cancer and autoimmune disease patients.

  5. A study of the human immune response to Lolium perenne (rye) pollen and its components, Lol p I and Lol p II (rye I and rye II). I. Prevalence of reactivity to the allergens and correlations among skin test, IgE antibody, and IgG antibody data. (United States)

    Freidhoff, L R; Ehrlich-Kautzky, E; Grant, J H; Meyers, D A; Marsh, D G


    In a stratified random sample of 320 white adults, the prevalence of puncture skin test positivity (ST +) to Lolium perenne (rye grass)-pollen extract (LPE) was 16%. Fifteen percent of all subjects (or 84% of subjects classified LPE IgE antibody positive [Ab +]) was classified IgE Ab + to highly purified Lol p I (Rye I), and 4% of all subjects (or 26% of subjects classified LPE IgE Ab +) was classified IgE Ab + to highly purified Lol p II (Rye II). These data and similar results obtained in an allergy-enriched group of 361 subjects are consistent with previous studies that Lol I is a major allergen and Lol II is a minor allergen of LPE. Whether we studied LPE, Lol I, or Lol II, responder subjects were younger than nonresponder subjects and more male than female subjects were responders. We then investigated the quantitative interrelationships among ST, IgE, and IgG Ab responsiveness to LPE, Lol I, and Lol II in the allergy-enriched group. For each allergen, log-log correlations were strong and significant for ST versus IgE Ab and for IgE Ab versus IgG Ab. All subjects IgE Ab + to Lol I or Lol II were IgG Ab + to that allergen, supporting other evidence for a commonality in the genetic control influencing the production of IgE and IgG Abs to a given allergen. Log-log correlations among ST end points, IgE Ab levels, or IgG Ab levels were strong for LPE versus either Lol I or Lol II but weak between Lol I and Lol II, consistent with the reported lack of cross-reactivity between Lol I and Lol II. Despite these findings, almost all Lol II + subjects were Lol I + by ST (98%), IgE Ab (91%), and IgG Ab (83%), suggesting that the Ia-restricted immune recognition of both these molecules is at least in part under a common genetic control.

  6. Human antibody fragments specific for Bothrops jararacussu venom reduce the toxicity of other Bothrops sp. venoms. (United States)

    Roncolato, Eduardo Crosara; Pucca, Manuela Berto; Funayama, Jaqueline Carlos; Bertolini, Thaís Barboza; Campos, Lucas Benício; Barbosa, José Elpidio


    Approximately 20,000 snakebites are registered each year in Brazil. The classical treatment for venomous snakebite involves the administration of sera obtained from immunized horses. Moreover, the production and care of horses is costly, and the use of heterologous sera can cause hypersensitivity reactions. The production of human antibody fragments by phage display technology is seen as a means of overcoming some of these disadvantages. The studies here attempted to test human monoclonal antibodies specific to Bothrops jararacussu against other Bothrops sp. venoms, using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Using the Griffin.1 phage antibody library, this laboratory previously produced scFvs capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom. The structural and functional similarities of the various forms of phospholipase A2 (PLA₂) in Bothrops venom served as the basis for the present study wherein the effectiveness of those same scFvs were evaluated against B. jararaca, B. neuwiedi, and B. moojeni venoms. Each clone was found to recognize all three Bothrops venoms, and purified scFvs partially inhibited their in vitro phospholipase activity. In vivo assays demonstrated that the scFv clone P2B7 reduced myotoxicity and increased the survival of animals that received the test venoms. The results here indicate that the scFv P2B7 is a candidate for inclusion in a mixture of specific antibodies to produce a human anti-bothropic sera. This data demonstrates that the human scFv P2B7 represents an alternative therapeutic approach to heterologous anti-bothropic sera available today.

  7. Antiidiotypic antibody related to the 84 kD human sperm membrane protein

    Institute of Scientific and Technical Information of China (English)



    Wistar rats were inoculated with purified YWK-I antibody.The anti-idiotypic antibodies were isolated from rat sera by successive passage over affinity chromatography columns of YWK-I mAb and normal mouse Igs.Specificity of anti-Id antibody was established by ELISA.The 84kD protein inhibited the binding of anti-Id to YWK-I mAb,but failed to repress antibody against normal mouse Ig binding to YWK-I mAb.In competitive inhibition assay,84kD protein had shown the ability to compete with anti-Id binding to YWK-I mAb in a dose-dependent manner.Crude sperm extract showed a lower competitive ability.No effect was found with the irrelevant 36kD sperm protein.The antisera from the Balb/C micr immunized with AId contained Ab3 that reacted with 84kD sperm protein.The binding of anti-Id to YWK-I mAb was inhibited by Ab3 in a dose-dependent fashion and Ab3 was shown to be able to induce human sperm agglutination.These results indicate that anti-Id which may mimic an epitope of the 84kD protein could be exploited as an antigen to raise antibodies against sperm protein.

  8. Expression and purification of human ARP1 protein and rapid preparation of polyclonal antibody. (United States)

    Sun, Mingjuan; Zou, Rongjiang; Dong, Xiaoyi; Zong, Ying; Gao, Yun; Wang, Lianghua; Jiao, Binghua


    Angiopoietin-related protein 1 (ARP1) is one of the antiangiogenic factors and plays an important role in endothelial cell proliferation, migration, and blood vessel network formation. Here a rapid method to prepare ARP1 polyclonal antibody in 1 month was developed. The gene of fibrinogen homology domain (FD) for ARP1 was cloned and the protein was expressed in a soluble form of MBP-FD fused protein. The MBP-FD protein was purified using amylose affinity chromatography of maltose-binding protein. Polyclonal antibodies against MBP-FD were obtained through immunization in BALB/c mice. The titer was determined by indirect enzyme-linked immunosorbent assay (ELISA), and the antibody specificity was assessed by Western blot. The full-length ARP1 protein in stable form expressed in transfected human large lung cancer cell lines NCI-H460 was detected by immunocytochemistry (ICC) analysis using ARP1 polyclonal antibodies. The result shows that the antibody possesses good specificity and sensitivity. This work provides a substantial base for the further studies of ARP1 function and associated mechanisms. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.

  9. Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1. (United States)

    Douthwaite, Julie A; Sridharan, Sudharsan; Huntington, Catherine; Hammersley, Jayne; Marwood, Rose; Hakulinen, Jonna K; Ek, Margareta; Sjögren, Tove; Rider, David; Privezentzev, Cyril; Seaman, Jonathan C; Cariuk, Peter; Knights, Vikki; Young, Joyce; Wilkinson, Trevor; Sleeman, Matthew; Finch, Donna K; Lowe, David C; Vaughan, Tristan J


    Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding VH CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long VH CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long VH CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs.

  10. Critical epitopes in the nucleocapsid protein of SFTS virus recognized by a panel of SFTS patients derived human monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Li Yu

    Full Text Available BACKGROUND: SFTS virus (SFTSV is a newly discovered pathogen to cause severe fever with thrombocytopenia syndrome (SFTS in human. Successful control of SFTSV epidemic requires better understanding of the antigen target in humoral immune responses to the new bunyavirus infection. METHODOLOGY/PRINCIPAL FINDINGS: We have generated a combinatorial Fab antibody phage library from two SFTS patients recovered from SFTSV infection. To date, 94 unique human antibodies have been generated and characterized from over 1200 Fab antibody clones obtained by screening the library with SFTS purified virions. All those monoclonal antibodies (MAbs recognized the nucleocapsid (N protein of SFTSV while none of them were reactive to the viral glycoproteins Gn or Gc. Furthermore, over screening 1000 mouse monoclonal antibody clones derived from SFTSV virions immunization, 462 clones reacted with N protein, while only 16 clones were reactive to glycoprotein. Furthermore, epitope mapping of SFTSV N protein was performed through molecular simulation, site mutation and competitive ELISA, and we found that at least 4 distinct antigenic epitopes within N protein were recognized by those human and mouse MAbs, in particular mutation of Glu10 to Ala10 abolished or significantly reduced the binding activity of nearly most SFTS patients derived MAbs. CONCLUSIONS/SIGNIFICANCE: The large number of human recombinant MAbs derived from SFTS patients recognized the viral N protein indicated the important role of the N protein in humoral responses to SFTSV infection, and the critical epitopes we defined in this study provided molecular basis for detection and diagnosis of SFTSV infection.

  11. The immune landscape of human tumors (United States)

    Bindea, Gabriela; Mlecnik, Bernhard; Angell, Helen K; Galon, Jérôme


    Understanding the spontaneous immune response of cancer patients is critical for the design of efficient anticancer immunotherapies. The power of integrative tumor immunology approaches allowed for a comprehensive view of the immune system evolution in the course of tumor progression and recurrence. We have demonstrated that tumor-infiltrating immune cells are spatiotemporally regulated, a finding that has profound implications for the development of efficient anticancer immunotherapies. PMID:24800163

  12. The Efficacy of Humanized Antibody against the Sporothrix Antigen, gp70, in Promoting Phagocytosis and Reducing Disease Burden (United States)

    de Almeida, José R. F.; Santiago, Karla L.; Kaihami, Gilberto H.; Maranhão, Andrea Q.; de Macedo Brígido, Marcelo; de Almeida, Sandro R.


    Sporotrichosis is a subcutaneous mycosis distributed worldwide and is frequently reported in countries with tropical climates, as Latin America countries. We previously demonstrated that mice with sporotrichosis produce specific antibodies against a 70-kDa fungal protein, indicating that specific antibodies against this molecule may help to control the sporotrichosis. IgG1 monoclonal antibody was generated, and called mAbP6E7, in mice against a 70-kDa glycoprotein (gp70) of S. schenckii. The mAbP6E7 showed prophylactic and therapeutic activity against sporotrichosis. However, this antibody has a murine origin, and this can generate an immune response when administered to humans, precluding its use for a prolonged time. For its possible use in the treatment of human sporotrichosis, we humanized the mAbP6E7 by genetic engineering. Once expressed, the humanized antibodies had good stability and were able to bind to the 70-kDa cell wall antigens of Sporothrix schenckii and S. brasiliensis. The humanized P6E7 were able to opsonize S. schenckii yeasts, thus increasing the phagocytic index in human monocyte-derived macrophages. The treatment with humanized P6E7 decreased fungal burden in vivo. These data suggest that humanized P6E7 may have a therapeutic role in sporotrichosis.

  13. Passive Immunization in JNPL3 Transgenic Mice Using an Array of Phospho-Tau Specific Antibodies.

    Directory of Open Access Journals (Sweden)

    Cristina d'Abramo

    Full Text Available Recent work from our lab and few others have strongly suggested that immunotherapy could be an effective means of preventing the development of tau accumulation in JNPL3 transgenic mice, carrying the human P301L mutation. The aim of this study was to test the efficacy of a variety of specific tau monoclonal antibodies in JNPL3. Starting at 3 months of age, mice were treated for 4 months with weekly intraperitoneal injections of saline or purified tau monoclonal antibodies (10 mg/Kg different in specificity for pathological tau: CP13 (pSer202, RZ3 (pThr231 and PG5 (pSer409. As expected, not all the antibodies tested showed efficacy at preventing the development of tau pathology at the described dose, with some of them even worsening the pathological scenario. Only by targeting the pSer202 epitope with CP13 was a conspicuous reduction of insoluble or soluble tau in cortex and hindbrain obtained. Here we report about the importance of screening in vivo multiple tau antibodies in order to select the antibodies to direct into future clinical studies.

  14. Protection of gnotobiotic rats against dental caries by passive immunization with bovine milk antibodies to Streptococcus mutans. (United States)

    Michalek, S M; Gregory, R L; Harmon, C C; Katz, J; Richardson, G J; Hilton, T; Filler, S J; McGhee, J R


    A multivalent vaccine consisting of whole cell antigens of seven strains, representing four serotypes (b, c, d and g), of mutans streptococci was used to hyperimmunize a group of cows. Serum samples from these animals contained immunoglobulin G1 (IgG1) antibody activity to seven serotypes (a to g) of mutans streptococci. Whey obtained from the animal with the highest serum antibody activity, which also contained high levels of IgG1 antibody, was used in passive caries immunity studies. Gnotobiotic rats monoinfected with Streptococcus mutans MT8148 serotype c or Streptococcus sobrinus OMZ176 (d) or 6715 (g) and provided a caries-promoting diet containing immune whey had lower plaque scores, numbers of streptococci in plaque, and degree of caries activity than similarly infected animals given a diet containing control whey obtained from nonimmunized cows. To establish the nature of the protective component(s) present in the immune whey, an ultrafiltrate fraction of the whey was prepared. This preparation contained higher levels of IgG1 anti-S. mutans antibody activity than the immune whey. Rats monoinfected with S. mutans MT8148 and provided with a diet supplemented with 0.1% of this fraction exhibited a degree of caries protection similar to that seen in animals provided a diet containing 100% immune whey. In fact, a diet containing as little as 0.01% of the ultrafiltrate fraction gave some degree of protection against oral S. mutans infection. The active component in the immune whey was the IgG1 anti-S. mutans antibody, since rats monoinfected with S. mutans MT8148 and provided a diet supplemented with purified immune whey IgG1 had significantly reduced plaque scores, numbers of S. mutans in plaque, and caries activity compared with control animals. Prior adsorption of the IgG fraction with killed S. mutans MT8148 whole cells removed antibody activity and abrogated caries protection.

  15. Circulating levels of chromatin fragments are inversely correlated with anti-dsDNA antibody levels in human and murine systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Jørgensen, Mariann H; Rekvig, Ole Petter; Jacobsen, Rasmus S


    Anti-dsDNA antibodies represent a central pathogenic factor in Lupus nephritis. Together with nucleosomes they deposit as immune complexes in the mesangial matrix and along basement membranes within the glomeruli. The origin of the nucleosomes and when they appear e.g. in circulation is not known...... an inverse correlation between anti-dsDNA antibodies and the DNA concentration in the circulation in both murine and human serum samples. High titer of anti-DNA antibodies in human sera correlated with reduced levels of circulating chromatin, and in lupus prone mice with deposition within glomeruli....... The inverse correlation between DNA concentration and anti-dsDNA antibodies may reflect antibody-dependent deposition of immune complexes during the development of lupus nephritis in autoimmune lupus prone mice. The measurement of circulating DNA in SLE sera by using qPCR may indicate and detect...

  16. Bispecific antibodies and trispecific immunocytokines for targeting the immune system against cancer: preparing for the future. (United States)

    Fournier, Philippe; Schirrmacher, Volker


    Monoclonal anti-tumor antibodies (mAbs) that are clinically effective usually recruit, via their constant fragment (Fc) domain, Fc receptor (FcR)-positive accessory cells of the immune system and engage these additionally against the tumor. Since T cells are FcR negative, these important cells are not getting involved. In contrast to mAbs, bispecific antibodies (bsAbs) can be designed in such a way that they involve T cells. bsAbs are artificially designed molecules that bind simultaneously to two different antigens, one on the tumor cell, the other one on an immune effector cell such as CD3 on T cells. Such dual antibody constructs can cross-link tumor cells and T cells. Many such bsAb molecules at the surface of tumor cells can thus build a bridge to T cells and aggregate their CD3 molecules, thereby activating them for cytotoxic activity. BsAbs can also contain a third binding site, for instance a Fc domain or a cytokine that would bind to its respective cytokine receptor. The present review discusses the pros and cons for the use of the Fc fragment during the development of bsAbs using either cell-fusion or recombinant DNA technologies. The recombinant antibody technology allows the generation of very efficient bsAbs containing no Fc domain such as the bi-specific T-cell engager (BiTE). The strong antitumor activity of these molecules makes them very interesting new cancer therapeutics. Over the last decade, we have developed another concept, namely to combine bsAbs and multivalent immunocytokines with a tumor cell vaccine. The latter are patient-derived tumor cells modified by infection with a virus. The virus-Newcastle Disease Virus (NDV)-introduces, at the surface of the tumor cells, viral molecules that can serve as general anchors for the bsAbs. Our strategy aims at redirecting, in an Fc-independent fashion, activities of T cells and accessory cells against autologous tumor antigens. It creates very promising perspectives for a new generation of efficient

  17. A role for plasma cell targeting agents in immune tolerance induction in autoimmune disease and antibody responses to therapeutic proteins. (United States)

    Rosenberg, A S; Pariser, A R; Diamond, B; Yao, L; Turka, L A; Lacana, E; Kishnani, P S


    Antibody responses to life saving therapeutic protein products, such as enzyme replacement therapies (ERT) in the setting of lysosomal storage diseases, have nullified product efficacy and caused clinical deterioration and death despite treatment with immune-suppressive therapies. Moreover, in some autoimmune diseases, pathology is mediated by a robust antibody response to endogenous proteins such as is the case in pulmonary alveolar proteinosis, mediated by antibodies to Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF). In this work, we make the case that in such settings, when the antibody response is high titered, sustained, and refractory to immune suppressive treatments, the antibody response is mediated by long-lived plasma cells which are relatively unperturbed by immune suppressants including rituximab. However, long-lived plasma cells can be targeted by proteasome inhibitors such as bortezomib. Recent reports of successful reversal of antibody responses with bortezomib in the settings of ERT and Thrombotic Thrombocytopenic Purpura (TTP) argue that the safety and efficacy of such plasma cell targeting agents should be evaluated in larger scale clinical trials to delineate the risks and benefits of such therapies in the settings of antibody-mediated adverse effects to therapeutic proteins and autoantibody mediated pathology.

  18. The immune response of the human brain to abdominal surgery

    DEFF Research Database (Denmark)

    Forsberg, Anton; Cervenka, Simon; Jonsson Fagerlund, Malin


    OBJECTIVE: Surgery launches a systemic inflammatory reaction that reaches the brain and associates with immune activation and cognitive decline. Although preclinical studies have in part described this systemic-to-brain signaling pathway, we lack information on how these changes appear in humans....... This study examines the short- and long-term impact of abdominal surgery on the human brain immune system by positron emission tomography (PET) in relation to blood immune reactivity, plasma inflammatory biomarkers, and cognitive function. METHODS: Eight males undergoing prostatectomy under general...... to change in [(11) C]PBR28 binding (p = 0.027). INTERPRETATION: This study translates preclinical data on changes in the brain immune system after surgery to humans, and suggests an interplay between the human brain and the inflammatory response of the peripheral innate immune system. These findings may...

  19. Assessment of humoral immune responses to blood-stage malaria antigens following ChAd63-MVA immunization, controlled human malaria infection and natural exposure.

    Directory of Open Access Journals (Sweden)

    Sumi Biswas

    Full Text Available The development of protective vaccines against many difficult infectious pathogens will necessitate the induction of effective antibody responses. Here we assess humoral immune responses against two antigens from the blood-stage merozoite of the Plasmodium falciparum human malaria parasite--MSP1 and AMA1. These antigens were delivered to healthy malaria-naïve adult volunteers in Phase Ia clinical trials using recombinant replication-deficient viral vectors--ChAd63 to prime the immune response and MVA to boost. In subsequent Phase IIa clinical trials, immunized volunteers underwent controlled human malaria infection (CHMI with P. falciparum to assess vaccine efficacy, whereby all but one volunteer developed low-density blood-stage parasitemia. Here we assess serum antibody responses against both the MSP1 and AMA1 antigens following i ChAd63-MVA immunization, ii immunization and CHMI, and iii primary malaria exposure in the context of CHMI in unimmunized control volunteers. Responses were also assessed in a cohort of naturally-immune Kenyan adults to provide comparison with those induced by a lifetime of natural malaria exposure. Serum antibody responses against MSP1 and AMA1 were characterized in terms of i total IgG responses before and after CHMI, ii responses to allelic variants of MSP1 and AMA1, iii functional growth inhibitory activity (GIA, iv IgG avidity, and v isotype responses (IgG1-4, IgA and IgM. These data provide the first in-depth assessment of the quality of adenovirus-MVA vaccine-induced antibody responses in humans, along with assessment of how these responses are modulated by subsequent low-density parasite exposure. Notable differences were observed in qualitative aspects of the human antibody responses against these malaria antigens depending on the means of their induction and/or exposure of the host to the malaria parasite. Given the continued clinical development of viral vectored vaccines for malaria and a range of other

  20. Preexisting human antibodies neutralize recently emerged H7N9 influenza strains (United States)

    Henry Dunand, Carole J.; Leon, Paul E.; Kaur, Kaval; Tan, Gene S.; Zheng, Nai-Ying; Andrews, Sarah; Huang, Min; Qu, Xinyan; Huang, Yunping; Salgado-Ferrer, Marlene; Ho, Irvin Y.; Taylor, William; Hai, Rong; Wrammert, Jens; Ahmed, Rafi; García-Sastre, Adolfo; Palese, Peter; Krammer, Florian; Wilson, Patrick C.


    The emergence and seasonal persistence of pathogenic H7N9 influenza viruses in China have raised concerns about the pandemic potential of this strain, which, if realized, would have a substantial effect on global health and economies. H7N9 viruses are able to bind to human sialic acid receptors and are also able to develop resistance to neuraminidase inhibitors without a loss in fitness. It is not clear whether prior exposure to circulating human influenza viruses or influenza vaccination confers immunity to H7N9 strains. Here, we demonstrate that 3 of 83 H3 HA-reactive monoclonal antibodies generated by individuals that had previously undergone influenza A virus vaccination were able to neutralize H7N9 viruses and protect mice against homologous challenge. The H7N9-neutralizing antibodies bound to the HA stalk domain but exhibited a difference in their breadth of reactivity to different H7 influenza subtypes. Mapping viral escape mutations suggested that these antibodies bind at least two different epitopes on the stalk region. Together, these results indicate that these broadly neutralizing antibodies may contribute to the development of therapies against H7N9 strains and may also be effective against pathogenic H7 strains that emerge in the future. PMID:25689254

  1. CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes (United States)

    Yeap, Wei Hseun; Wong, Kok Loon; Shimasaki, Noriko; Teo, Esmeralda Chi Yuan; Quek, Jeffrey Kim Siang; Yong, Hao Xiang; Diong, Colin Phipps; Bertoletti, Antonio; Linn, Yeh Ching; Wong, Siew Cheng


    Antibody-dependent cellular cytotoxicity (ADCC) is exerted by immune cells expressing surface Fcγ receptors (FcγRs) against cells coated with antibody, such as virus-infected or transformed cells. CD16, the FcγRIIIA, is essential for ADCC by NK cells, and is also expressed by a subset of human blood monocytes. We found that human CD16− expressing monocytes have a broad spectrum of ADCC capacities and can kill cancer cell lines, primary leukemic cells and hepatitis B virus-infected cells in the presence of specific antibodies. Engagement of CD16 on monocytes by antibody bound to target cells activated β2-integrins and induced TNFα secretion. In turn, this induced TNFR expression on the target cells, making them susceptible to TNFα-mediated cell death. Treatment with TLR agonists, DAMPs or cytokines, such as IFNγ, further enhanced ADCC. Monocytes lacking CD16 did not exert ADCC but acquired this property after CD16 expression was induced by either cytokine stimulation or transient transfection. Notably, CD16+ monocytes from patients with leukemia also exerted potent ADCC. Hence, CD16+ monocytes are important effectors of ADCC, suggesting further developments of this property in the context of cellular therapies for cancer and infectious diseases. PMID:27670158

  2. In Vitro Characterization of Human Cytomegalovirus-Targeting Therapeutic Monoclonal Antibodies LJP538 and LJP539 (United States)

    Patel, Hetalkumar D.; Nikitin, Pavel; Gesner, Thomas; Lin, James J.; Barkan, David T.; Ciferri, Claudio; Carfi, Andrea; Akbarnejad Yazdi, Tahmineh; Skewes-Cox, Peter; Wiedmann, Brigitte; Jarousse, Nadine; Zhong, Weidong; Feire, Adam


    Human cytomegalovirus (HCMV) infection is usually benign in healthy individuals but can cause life-threatening disease in those with compromised immune systems. Approved drugs available to treat HCMV disease, including ganciclovir, cidofovir, and foscarnet, have significant toxicities that limit their use in certain patient populations. LJP538 and LJP539 are human monoclonal antibodies that are being evaluated as immunoglobulin therapeutics. The antibodies target glycoproteins gB and the gH/gL/UL128/UL130/UL131a pentameric complex, respectively. Here we present an in vitro characterization of these antibodies. We show that LJP538 and LJP539 are more potent than a marketed immunoglobulin at inhibiting HCMV infection of various cell lines relevant to pathogenesis. We find that LJP538 and LJP539 are active against a panel of clinical isolates in vitro and demonstrate minor-to-moderate synergy in combination. Passage of HCMV in the presence of LJP538 or LJP539 alone resulted in resistance-associated mutations that mapped to the target genes. However, no loss of susceptibility to the combination of antibodies was observed for >400 days in culture. Finally, the binding regions of LJP538 and LJP539 are conserved among clinical isolates. Taken together, these data support the use of LJP538 and LJP539 in combination for clinical trials in HCMV patients. PMID:27270290

  3. Generation and characterization of a polyclonal antibody against human high mobility group box 4. (United States)

    Yang, Fen; Li, Runsheng; Hong, Aizhen; Duan, Fei; Li, Yuhua


    A human high mobility group box 4 (hHMGB4) expression construct (pET‑28a/hHMGB4) was generated by cloning the hHMGB4 full‑length cDNA in the expression vector pET‑28a(+). The hHMGB4 fusion protein with His6‑Tag was prepared using E.coli BL21 (DE3) transformed with pET‑28a/hHMGB4 and purified via preparative SDS‑PAGE plus electroelution. Immunization of rabbits with the purified hHMGB4 generated polyclonal antibodies. The titer of the antiserum was determined to be 1:102,400 by ELISA analysis. Western blotting analysis showed that the antibody specifically recognized the recombinant hHMGB4 protein and also the endogenous hHMGB4 protein in prostate cancer cells. In addition, immunohistochemical staining analysis using the prepared antibody revealed marked hHMGB4 staining in the nuclei of the human prostate tissue. These data demonstrate that the anti‑hHMGB4 polyclonal antibody may be a useful reagent for the functional study of hHMGB4.

  4. Design and construction of a new human naïve single-chain fragment variable antibody library, IORISS1. (United States)

    Pasello, Michela; Zamboni, Silvia; Mallano, Alessandra; Flego, Michela; Picci, Piero; Cianfriglia, Maurizio; Scotlandi, Katia


    Human monoclonal antibodies are a powerful tool with increasingly successful exploitations and the single chain fragment variable format can be considered the building block for the implementation of more complex and effective antibody-based constructs. Phage display is one of the best and most efficient methods to isolate human antibodies selected from an efficient and variable phage display library. We report a method for the construction of a human naïve single-chain variable fragment library, termed IORISS1. Many different sets of oligonucleotide primers as well as optimized electroporation and ligation reactions were used to generate this library of 1.2×10(9) individual clones. The key difference is the diversity of variable gene templates, which was derived from only 15 non-immunized human donors. The method described here, was used to make a new human naïve single-chain fragment variable phage display library that represents a valuable source of diverse antibodies that can be used as research reagents or as a starting point for the development of therapeutics. Using biopanning, we determined the ability of IORISS1 to yield antibodies. The results we obtained suggest that, by using an optimized protocol, an efficient phage antibody library can be generated.

  5. Studying the immune response to human viral infections using zebrafish. (United States)

    Goody, Michelle F; Sullivan, Con; Kim, Carol H


    Humans and viruses have a long co-evolutionary history. Viral illnesses have and will continue to shape human history: from smallpox, to influenza, to HIV, and beyond. Animal models of human viral illnesses are needed in order to generate safe and effective antiviral medicines, adjuvant therapies, and vaccines. These animal models must support the replication of human viruses, recapitulate aspects of human viral illnesses, and respond with conserved immune signaling cascades. The zebrafish is perhaps the simplest, most commonly used laboratory model organism in which innate and/or adaptive immunity can be studied. Herein, we will discuss the current zebrafish models of human viral illnesses and the insights they have provided. We will highlight advantages of early life stage zebrafish and the importance of innate immunity in human viral illnesses. We will also discuss viral characteristics to consider before infecting zebrafish with human viruses as well as predict other human viruses that may be able to infect zebrafish.

  6. An antibody against the surfactant protein A (SP-A)-binding domain of the SP-A receptor inhibits T cell-mediated immune responses to Mycobacterium tuberculosis. (United States)

    Samten, Buka; Townsend, James C; Sever-Chroneos, Zvjezdana; Pasquinelli, Virginia; Barnes, Peter F; Chroneos, Zissis C


    Surfactant protein A (SP-A) suppresses lymphocyte proliferation and IL-2 secretion, in part, by binding to its receptor, SP-R210. However, the mechanisms underlying this effect are not well understood. Here, we studied the effect of antibodies against the SP-A-binding (neck) domain (alpha-SP-R210n) or nonbinding C-terminal domain (alpha-SP-R210ct) of SP-R210 on human peripheral blood T cell immune responses against Mycobacterium tuberculosis. We demonstrated that both antibodies bind to more than 90% of monocytes and 5-10% of CD3+ T cells in freshly isolated PBMC. Stimulation of PBMC from healthy tuberculin reactors [purified protein derivative-positive (PPD+)] with heat-killed M. tuberculosis induced increased antibody binding to CD3+ cells. Increased antibody binding suggested enhanced expression of SP-R210, and this was confirmed by Western blotting. The antibodies (alpha-SP-R210n) cross-linking the SP-R210 through the SP-A-binding domain markedly inhibited cell proliferation and IFN-gamma secretion by PBMC from PPD+ donors in response to heat-killed M. tuberculosis, whereas preimmune IgG and antibodies (alpha-SP-R210ct) cross-linking SP-R210 through the non-SP-A-binding, C-terminal domain had no effect. Anti-SP-R210n also decreased M. tuberculosis-induced production of TNF-alpha but increased production of IL-10. Inhibition of IFN-gamma production by alpha-SP-R210n was abrogated by the combination of neutralizing antibodies to IL-10 and TGF-beta1. Together, these findings support the hypothesis that SP-A, via SP-R210, suppresses cell-mediated immunity against M. tuberculosis via a mechanism that up-regulates secretion of IL-10 and TGF-beta1.

  7. Antibody-mediated immunity induced by engineered Escherichia coli OMVs carrying heterologous antigens in their lumen

    Directory of Open Access Journals (Sweden)

    Laura Fantappiè


    Full Text Available Background: Outer membrane vesicles (OMVs from Gram-negative bacteria are gaining increasing attention as vaccine platform for their built-in adjuvanticity and for their potential use as carriers of heterologous antigens. These 2 properties offer the opportunity to make highly effective, easy to produce multi-valent vaccines. OMVs can be loaded with foreign antigens by targeting protein expression either to the outer membrane or to the periplasm of the OMV-producing strain. Periplasmic expression is simple and relatively efficient but leads to the accumulation of recombinant antigens in the lumen of OMVs and the ability of OMVs carrying internalized antigens to induce antigen-specific antibody responses has been only marginally investigated and is considered to be sub-optimal. Methods: We have systematically analyzed in qualitative and quantitative terms antibody responses induced by OMVs carrying different heterologous antigens in their lumen. Group A Streptococcus (GAS Slo, SpyCEP, Spy0269 and Group B Streptococcus (GBS SAM_1372 were fused to the OmpA leader sequence for secretion and expressed in Escherichia coli. OMVs from the recombinant strains were purified and tested for immunogenicity and protective activity. Results: All proteins were incorporated into the OMVs lumen in their native conformation. Upon mice immunization, OMVs induced high functional antibody titers against the recombinant proteins. Furthermore, immunization with Slo-OMVs and SpyCEP-OMVs protected mice against GAS lethal challenge. Conclusions: The efficiency of antigen delivery to the vesicular lumen via periplasmic expression, and the surprisingly high immunogenicity and protective activity of OMVs carrying internalized recombinant antigens further strengthens the potential of OMVs as vaccine platform.

  8. Extending mass spectrometry contribution to therapeutic monoclonal antibody lead optimization: characterization of immune complexes using noncovalent ESI-MS. (United States)

    Atmanene, Cédric; Wagner-Rousset, Elsa; Malissard, Martine; Chol, Bertrand; Robert, Alain; Corvaïa, Nathalie; Van Dorsselaer, Alain; Beck, Alain; Sanglier-Cianférani, Sarah


    Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases including cancers, immunological disorders, and other pathologies. These large biomolecules display specific structural features, which affect their efficiency and need, therefore, to be extensively characterized using sensitive and orthogonal analytical techniques. Among them, mass spectrometry (MS) has become the method of choice to study mAb amino acid sequences as well as their post-translational modifications. In the present work, recent noncovalent MS-technologies including automated chip-based nanoelectrospray MS and traveling wave ion mobility MS were used for the first time to characterize immune complexes involving both murine and humanized mAb 6F4 directed against human JAM-A, a newly identified antigenic protein (Ag) overexpressed in tumor cells. MS-based structural insights evidenced that heterogeneous disulfide bridge pairings of recombinant JAM-A alter neither its native structure nor mAbs 6F4 recognition properties. Investigations focused on mAb:Ag complexes revealed that, similarly to murine mAb, humanized mAb 6F4 binds selectively up to four antigen molecules with a similar affinity, confirming in this way the reliability of the humanization process. Noncovalent MS appears as an additional supporting technique for therapeutic mAbs lead characterization and development.

  9. Therapeutic monoclonal antibodies in human breast milk: a case study. (United States)

    Ross, Elle; Robinson, Steven E; Amato, Carol; McMillan, Colette; Westcott, Jay; Wolf, Tiffany; Robinson, William A


    Recently, therapeutic monoclonal antibodies have been introduced for the treatment of advanced melanoma and other diseases. It remains unclear whether these drugs can be safely administered to women who are breast feeding because of the potential hazardous side effects for nursing infants. One such therapy for metastatic melanoma is ipilimumab, a human monoclonal antibody that blocks cytotoxic T-lymphocyte-antigen-4, and is the preferred treatment for patients with metastatic melanoma when other molecular therapies are not viable. This study measured ipilimumab levels in the breast milk of a patient undergoing treatment that were enough to raise concerns for a nursing infant exposed to ipilimumab.

  10. Antibodies specific for a segment of human membrane IgE deplete IgE-producing B cells in humanized mice. (United States)

    Brightbill, Hans D; Jeet, Surinder; Lin, Zhonghua; Yan, Donghong; Zhou, Meijuan; Tan, Martha; Nguyen, Allen; Yeh, Sherry; Delarosa, Donnie; Leong, Steven R; Wong, Terence; Chen, Yvonne; Ultsch, Mark; Luis, Elizabeth; Ramani, Sree Ranjani; Jackman, Janet; Gonzalez, Lino; Dennis, Mark S; Chuntharapai, Anan; DeForge, Laura; Meng, Y Gloria; Xu, Min; Eigenbrot, Charles; Lee, Wyne P; Refino, Canio J; Balazs, Mercedesz; Wu, Lawren C


    IgE-mediated hypersensitivity is central to the pathogenesis of asthma and other allergic diseases. Although neutralization of serum IgE with IgE-specific antibodies is in general an efficacious treatment for allergic asthma, one limitation of this approach is its lack of effect on IgE production. Here, we have developed a strategy to disrupt IgE production by generating monoclonal antibodies that target a segment of membrane IgE on human IgE-switched B cells that is not present in serum IgE. This segment is known as the M1' domain, and using genetically modified mice that contain the human M1' domain inserted into the mouse IgE locus, we demonstrated that M1'-specific antibodies reduced serum IgE and IgE-producing plasma cells in vivo, without affecting other immunoglobulin isotypes. M1'-specific antibodies were effective when delivered prophylactically and therapeutically in mouse models of immunization, allergic asthma, and Nippostrongylus brasiliensis infection, likely by inducing apoptosis of IgE-producing B cells. In addition, we generated a humanized M1'-specific antibody that was active on primary human cells in vivo, as determined by its reduction of serum IgE levels and IgE plasma cell numbers in a human PBMC-SCID mouse model. Thus, targeting of human IgE-producing B cells with apoptosis-inducing M1'-specific antibodies may be a novel treatment for asthma and allergy.

  11. Human immunodeficiency virus/acquired immune deficiency syndrome: Using drug from mathematical perceptive. (United States)

    Chatterjee, Amar Nath; Saha, Shubhankar; Roy, Priti Kumar


    Entry of acquired immune deficiency syndrome virus into the host immune cell involves the participation of various components of host and viral cell unit. These components may be categorized as attachment of the viral surface envelope protein subunit, gp120, to the CD4(+) receptor and chemokine coreceptors, CCR5 and CXCR4, present on T cell surface. The viral fusion protein, gp41, the second cleaved subunit of Env undergoes reconfiguration and the membrane fusion reaction itself. Since the CD4(+) T cell population is actively involved; the ultimate outcome of human immunodeficiency virus infection is total collapse of the host immune system. Mathematical modeling of the stages in viral membrane protein-host cell receptor-coreceptor interaction and the effect of antibody vaccine on the viral entry into the susceptible host cell has been carried out using as impulsive differential equations. We have studied the effect of antibody vaccination and determined analytically the threshold value of drug dosage and dosing interval for optimum levels of infection. We have also investigated the effect of perfect adherence of drug dose on the immune cell count in extreme cases and observed that systematic drug dosage of the immune cells leads to longer and improved lives.

  12. Formation of infectious dengue virus-antibody immune complex in vivo in marmosets (Callithrix jacchus) after passive transfer of anti-dengue virus monoclonal antibodies and infection with dengue virus. (United States)

    Moi, Meng Ling; Ami, Yasushi; Shirai, Kenji; Lim, Chang-Kweng; Suzaki, Yuriko; Saito, Yuka; Kitaura, Kazutaka; Saijo, Masayuki; Suzuki, Ryuji; Kurane, Ichiro; Takasaki, Tomohiko


    Infection with a dengue virus (DENV) serotype induces cross-reactive, weakly neutralizing antibodies to different dengue serotypes. It has been postulated that cross-reactive antibodies form a virus-antibody immune complex and enhance DENV infection of Fc gamma receptor (FcγR)-bearing cells. We determined whether infectious DENV-antibody immune complex is formed in vivo in marmosets after passive transfer of DENV-specific monoclonal antibody (mAb) and DENV inoculation and whether infectious DENV-antibody immune complex is detectable using FcγR-expressing cells. Marmosets showed that DENV-antibody immune complex was exclusively infectious to FcγR-expressing cells on days 2, 4, and 7 after passive transfer of each of the mAbs (mAb 4G2 and mAb 6B6C) and DENV inoculation. Although DENV-antibody immune complex was detected, contribution of the passively transferred antibody to overall viremia levels was limited in this study. The results indicate that DENV cross-reactive antibodies form DENV-antibody immune complex in vivo, which is infectious to FcγR-bearing cells but not FcγR-negative cells.

  13. Monoclonal antibodies against human granulocytes and myeloid differentiation antigens. (United States)

    Mannoni, P; Janowska-Wieczorek, A; Turner, A R; McGann, L; Turc, J M


    Monoclonal antibodies (MCA) were obtained by immunizing BALB/c mice with 99% pure granulocytes from normal donors or with a whole leukocyte suspension obtained from a chronic myelogenous leukemia (CML) patient, and then fusing the mouse spleen cells with a 315-43 myeloma cell clone. Four MCA were selected and studied using ELISA, immunofluorescence, cytotoxicity assays, and FACS analysis. Antibodies 80H.1, 80H.3, and 80H.5 (from normals) and 81H.1 (from CML) detected antigens expressed on neutrophils. Antibodies 80H.1 and 80H.3 (IgG) also reacted with monocytes but not with other blood cell subsets. Antibodies 80H.5 and 81H.1 (IgM) were cytotoxic and reacted strongly with most of the cells of the neutrophil maturation sequence, i.e., myeloblasts, promyelocytes, myelocytes, and mature granulocytes. Antibodies 80H.5 and 81H.1 also inhibited CFU-GM growth stimulated by leukocyte feeder layers or placental conditioned media, but did not inhibit BFU-E and CFU-E. Antigens recognized by 80H.3, 80H.5, and 81H.1 were expressed both on a proportion of cells from HL.60, KG.1, ML.1, and K562 myeloid cell lines, and on a proportion of blast cells isolated from patients with acute myelogenous leukemia. They were not found on lymphoid cell lines or lymphoid leukemia cells. These MCA recognize either late differentiation antigens expressed on mature neutrophils and monocytes (80H.1 and 80H.3) or early differentiation antigens (80H.5 and 81H.1) specific to the granulocytic lineage. They may be useful for a better definition of those antigens specific to hematopoietic stem cells and their relationship with normal or neoplastic hematopoiesis.

  14. Multivalent porous silicon nanoparticles enhance the immune activation potency of agonistic CD40 antibody. (United States)

    Gu, Luo; Ruff, Laura E; Qin, Zhengtao; Corr, Maripat; Hedrick, Stephen M; Sailor, Michael J


    One of the fundamental paradigms in the use of nanoparticles to treat disease is to evade or suppress the immune system in order to minimize systemic side effects and deliver sufficient nanoparticle quantities to the intended tissues. However, the immune system is the body's most important and effective defense against diseases. It protects the host by identifying and eliminating foreign pathogens as well as self-malignancies. Here we report a nanoparticle engineered to work with the immune system, enhancing the intended activation of antigen presenting cells (APCs). We show that luminescent porous silicon nanoparticles (LPSiNPs), each containing multiple copies of an agonistic antibody (FGK45) to the APC receptor CD40, greatly enhance activation of B cells. The cellular response to the nanoparticle-based stimulators is equivalent to a 30-40 fold larger concentration of free FGK45. The intrinsic near-infrared photoluminescence of LPSiNPs is used to monitor degradation and track the nanoparticles inside APCs.

  15. Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

    Energy Technology Data Exchange (ETDEWEB)

    McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.


    A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.

  16. Can we find a solution to the human immunodeficiency virus/acquired immune deficiency syndrome controversy? Is acquired immune deficiency syndrome the consequence of continuous excessive stressing of the body? (United States)

    Hässig, A; Wen-Xi, L; Stampfli, K


    The time of re-evaluation of the role of human immunodeficiency viruses in the pathogenesis of acquired immune deficiency syndrome has now come, now that methods are available for the direct detection of human immunodeficiency viruses and for the detection of cellular anti-human immunodeficiency virus immune reactions. It has been shown that human immunodeficiency virus infections are common among anti-human immunodeficiency virus antibody negative high-risk individuals. The disease is brought under control by cellular immune reactions and the anti-human immunodeficiency virus antibody test remains negative. Apart from proof that infection with human immunodeficiency viruses has occurred, a positive result in an anti-human immunodeficiency virus-antibody test is also an indication of an independent immunosuppression state. According to the definition of the Centers of Disease Control classical acquired immune deficiency syndrome is the consequence of infection with human immunodeficiency virus in association with continuous excessive stress, such as observed in the known risk groups. At the center of the pathogenetic process is hypercortisolism-determined damage of T lymphocytes, in which insufficiency of thymus is prominent. For this reason, in our view, there are indications for shifting efforts from the prophylaxis of infection with human immunodeficiency viruses to the prophylaxis of acquired immune deficiency syndrome by reducing stress factors.

  17. Immune-mediated steroid-responsive epileptic spasms and epileptic encephalopathy associated with VGKC-complex antibodies. (United States)

    Suleiman, Jehan; Brenner, Tanja; Gill, Deepak; Troedson, Christopher; Sinclair, Adriane J; Brilot, Fabienne; Vincent, Angela; Lang, Bethan; Dale, Russell C


    Autoantibodies that bind to voltage-gated potassium-channel complex proteins (VGKC-complex antibodies) occur frequently in adults with limbic encephalitis presenting with cognitive impairment and seizures. Recently, VGKC-complex antibodies have been described in a few children with limbic encephalitis, and children with unexplained encephalitis presenting with status epilepticus. We report a case of infantile-onset epileptic spasms and developmental delay compatible with epileptic encephalopathy. Our patient was a female infant, aged 4 months at presentation. She had evidence of immune activation in the central nervous system with elevated cerebrospinal fluid neopterin and mirrored oligoclonal bands, which prompted testing for autoantibodies. VGKC-complex antibodies were elevated (201 pmol/L, normalVGKC-complex antibodies might represent a marker of immune therapy responsiveness in a subgroup of patients with infantile epileptic encephalopathy.

  18. Correlation of antinuclear antibody immunofluorescence patterns with immune profile using line immunoassay in the Indian scenario

    Directory of Open Access Journals (Sweden)

    Sebastian Wendy


    Full Text Available Background: Immunity status, individual response to disease and types of antibodies produced are well known to vary from person to person, place to place and probably from population to population. A broad spectrum of specific auto antibodies that have so far been associated with specific rheumatic diseases, as noted in Western literature, has been well taken as a reference standard all over the world. There is neither research work nor any data correlating the auto antibodies and their antinuclear antibody (ANA patterns with the immunoprofile in the Indian population to date. Aims: To understand a definite association between ANA patterns and specific antibodies in the serum in the Indian study population and to document similarities / differences with the West. Settings and Design: This prospective and retrospective double blind study was undertaken on the South Indian population referred for ANA testing by Indirect Immunofluorescence method and by immunoline methods. Materials and Methods: Serum samples of patients from a random South Indian population who sought medical help for rheumatic disease were subjected for ANA testing by indirect immunofluorescence (IIF method and line immunoassay during the study period of 27 months. Serum samples were processed in dilution of 1:100 using HEp - 2010 / liver biochip (Monkey (EUROIMMUN AG. The serum samples which were further processed for line immunoassay were treated in 1:100 dilution on nylon strips coated with recombinant and purified antigens as discrete lines with plastic backing (EUROIMMUN AG coated with antigens nRNP / Sm, Sm, SSA, Ro-52, SSB, Scl-70, PM-Scl, PCNA, Jo-1, CENP-B, dsDNA, nucleosomes, histones, ribosomal protein-P, anti-mitochondrial antibodies (AMA-M2 along with a control band. The analysis was done by comparing the intensity of the reaction with positive control line by image analysis. Results: The antinuclear antibody indirect immunofluorescence (ANA - IIF patterns obtained

  19. Detection of human κ-opioid antibody using microresonators with integrated optical readout. (United States)

    Timurdogan, Erman; Ozber, Natali; Nargul, Sezin; Yavuz, Serhat; Kilic, M Salih; Kavakli, I Halil; Urey, Hakan; Alaca, B Erdem


    Label-free detection of the interaction between hexahistidine-tagged human κ-opioid receptor membrane protein and anti-His antibody is demonstrated in liquid by an optical microelectromechanical system utilizing electromagnetically actuated microresonators. Shift in resonance frequency due to accretion of mass on the sensitive surface of microresonators is monitored via an integrated optical readout. A frequency resolution of 2Hz is obtained. Together with a sensitivity of 7 ppm/(ng/ml) this leads to a minimum detectable antibody concentration of 5.7 ng/ml for a 50-kHz device. The measurement principle is shown to impart immunity to environmental noise, facilitate operation in liquid media and bring about the prospect for further miniaturization of actuator and readout leading to a portable biochemical sensor. Copyright 2010 Elsevier B.V. All rights reserved.

  20. Stereospecific antibodies to methadone. I. Radioimmunoassay of d,l-methadone in human serum. (United States)

    Bartos, F; Olsen, G D; Leger, R N; Bartos, D


    Anti-d,l-methadone antibodies were produced in rabbits immunized with d,l-methadol-hemisuccinate thyroglobulin conjugate. Using the antiserum, a radioimmunoasay (RIA) for determination of d,l-methadone in human serum has been developed and is described. Concentration of d,l-methadone of 1.4 pmol in a native serum sample (volume 0.1 ml or less) could be measured directly by RIA. The antibodies crossreact 100% with d,l-methadone, 50% with d-methadone, 50% with l-methadone and 100% with alpha-d-methadol. No crossreactivity was found with alpha 1-methadol, morphine, meperidine, dextropropoxyphene, 2-ethyl-5-methyl-3,3-diphenyl-l-pyrroline and 2-ethylidene-l, 5-dimethyl-3,3-diphenylpyrrolidene. High sensitivity and small sample requirements make this method suitable for future monitoring of patients on methadone maintenance and for studies where other procedures have lack of sensitivity.

  1. Production of the Polyclonal Anti-human Metallothionein 2A Antibody with Recombinant Protein Technology

    Institute of Scientific and Technical Information of China (English)

    Faiz M.M.T.MARIKAR; Qi-Ming SUN; Zi-Chun HUA


    Metallothionein 2A (MT2A) is a small stress response protein that can be induced by exposure to toxic metals. It is highly expressed in breast cancer cells. In this study, the eDNA encoding the human MT2A protein was expressed as glutathione S-transferase (GST) fusion protein in Escherichia coli.Recombinant MT2A proteins were loaded onto 12% sodium dodecylsulfate-polyacrylamide gel and separated by electrophoresis, the recombinant protein was visualized by Coomassie blue staining and the 33 kDa recombinant GST-MT2A fusion protein band was cut out from the gel. The gel slice was minced and used to generate polyclonal antisera. Immunization of rabbit against MT2A protein allowed the production of high titer polyclonal antiserum. This new polyclonal antibody recognized recombinant MT2A protein in Western blot analysis. This low-cost antibody will be useful for detection in various immuno-assays.

  2. Early infections by myxoma virus of young rabbits (Oryctolagus cuniculus) protected by maternal antibodies activate their immune system and enhance herd immunity in wild populations. (United States)

    Marchandeau, Stéphane; Pontier, Dominique; Guitton, Jean-Sébastien; Letty, Jérôme; Fouchet, David; Aubineau, Jacky; Berger, Francis; Léonard, Yves; Roobrouck, Alain; Gelfi, Jacqueline; Peralta, Brigitte; Bertagnoli, Stéphane


    The role of maternal antibodies is to protect newborns against acute early infection by pathogens. This can be achieved either by preventing any infection or by allowing attenuated infections associated with activation of the immune system, the two strategies being based on different cost/benefit ratios. We carried out an epidemiological survey of myxomatosis, which is a highly lethal infectious disease, in two distant wild populations of rabbits to describe the epidemiological pattern of the disease. Detection of specific IgM and IgG enabled us to describe the pattern of immunity. We show that maternal immunity attenuates early infection of juveniles and enables activation of their immune system. This mechanism associated with steady circulation of the myxoma virus in both populations, which induces frequent reinfections of immune rabbits, leads to the maintenance of high immunity levels within populations. Thus, myxomatosis has a low impact, with most infections being asymptomatic. This work shows that infection of young rabbits protected by maternal antibodies induces attenuated disease and activates their immune system. This may play a major role in reducing the impact of a highly lethal disease when ecological conditions enable permanent circulation of the pathogen.

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

    NARCIS (Netherlands)

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


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

  4. Concerted activity of IgG1 antibodies and IL-4/IL-25-dependent effector cells trap helminth larvae in the tissues following vaccination with defined secreted antigens, providing sterile immunity to challenge infection.

    Directory of Open Access Journals (Sweden)

    James P Hewitson


    Full Text Available Over 25% of the world's population are infected with helminth parasites, the majority of which colonise the gastrointestinal tract. However, no vaccine is yet available for human use, and mechanisms of protective immunity remain unclear. In the mouse model of Heligmosomoides polygyrus infection, vaccination with excretory-secretory (HES antigens from adult parasites elicits sterilising immunity. Notably, three purified HES antigens (VAL-1, -2 and -3 are sufficient for effective vaccination. Protection is fully dependent upon specific IgG1 antibodies, but passive transfer confers only partial immunity to infection, indicating that cellular components are also required. Moreover, immune mice show greater cellular infiltration associated with trapping of larvae in the gut wall prior to their maturation. Intra-vital imaging of infected intestinal tissue revealed a four-fold increase in extravasation by LysM+GFP+ myeloid cells in vaccinated mice, and the massing of these cells around immature larvae. Mice deficient in FcRγ chain or C3 complement component remain fully immune, suggesting that in the presence of antibodies that directly neutralise parasite molecules, the myeloid compartment may attack larvae more quickly and effectively. Immunity to challenge infection was compromised in IL-4Rα- and IL-25-deficient mice, despite levels of specific antibody comparable to immune wild-type controls, while deficiencies in basophils, eosinophils or mast cells or CCR2-dependent inflammatory monocytes did not diminish immunity. Finally, we identify a suite of previously uncharacterised heat-labile vaccine antigens with homologs in human and veterinary parasites that together promote full immunity. Taken together, these data indicate that vaccine-induced immunity to intestinal helminths involves IgG1 antibodies directed against secreted proteins acting in concert with IL-25-dependent Type 2 myeloid effector populations.

  5. Concerted Activity of IgG1 Antibodies and IL-4/IL-25-Dependent Effector Cells Trap Helminth Larvae in the Tissues following Vaccination with Defined Secreted Antigens, Providing Sterile Immunity to Challenge Infection (United States)

    Hewitson, James P.; Filbey, Kara J.; Esser-von Bieren, Julia; Camberis, Mali; Schwartz, Christian; Murray, Janice; Reynolds, Lisa A.; Blair, Natalie; Robertson, Elaine; Harcus, Yvonne; Boon, Louis; Huang, Stanley Ching-Cheng; Yang, Lihua; Tu, Yizheng; Miller, Mark J.; Voehringer, David; Le Gros, Graham; Harris, Nicola; Maizels, Rick M.


    Over 25% of the world's population are infected with helminth parasites, the majority of which colonise the gastrointestinal tract. However, no vaccine is yet available for human use, and mechanisms of protective immunity remain unclear. In the mouse model of Heligmosomoides polygyrus infection, vaccination with excretory-secretory (HES) antigens from adult parasites elicits sterilising immunity. Notably, three purified HES antigens (VAL-1, -2 and -3) are sufficient for effective vaccination. Protection is fully dependent upon specific IgG1 antibodies, but passive transfer confers only partial immunity to infection, indicating that cellular components are also required. Moreover, immune mice show greater cellular infiltration associated with trapping of larvae in the gut wall prior to their maturation. Intra-vital imaging of infected intestinal tissue revealed a four-fold increase in extravasation by LysM+GFP+ myeloid cells in vaccinated mice, and the massing of these cells around immature larvae. Mice deficient in FcRγ chain or C3 complement component remain fully immune, suggesting that in the presence of antibodies that directly neutralise parasite molecules, the myeloid compartment may attack larvae more quickly and effectively. Immunity to challenge infection was compromised in IL-4Rα- and IL-25-deficient mice, despite levels of specific antibody comparable to immune wild-type controls, while deficiencies in basophils, eosinophils or mast cells or CCR2-dependent inflammatory monocytes did not diminish immunity. Finally, we identify a suite of previously uncharacterised heat-labile vaccine antigens with homologs in human and veterinary parasites that together promote full immunity. Taken together, these data indicate that vaccine-induced immunity to intestinal helminths involves IgG1 antibodies directed against secreted proteins acting in concert with IL-25-dependent Type 2 myeloid effector populations. PMID:25816012

  6. An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of a Human Antibody

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    Krause, Jens C.; Ekiert, Damian C.; Tumpey, Terrence M.; Smith, Patricia B.; Wilson, Ian A.; Crowe, Jr., James E. (Vanderbilt); (Scripps); (CDC)


    The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in a predicted hot-spot motif, introduction of a new hot-spot motif, a molecular duplication due to polymerase slippage, a deletion due to misalignment, and additional somatic point mutations. Atomic resolution structures of the wild-type antibody and a variant in which the insertion was removed revealed that the three-amino-acid insertion near the base of heavy-chain complementarity-determining region (CDR) H2 resulted in a bulge in that loop. This enlarged CDR H2 loop impinges on adjacent regions, causing distortion of the CDR H1 architecture and its displacement away from the antigen-combining site. Removal of the insertion restores the canonical structure of CDR H1 and CDR H2, but binding, neutralization activity, and in vivo activity were reduced markedly because of steric conflict of CDR H1 with the hemagglutinin antigen.

  7. EBV Infection of Mice with Reconstituted Human Immune System Components. (United States)

    Münz, Christian


    Epstein-Barr virus (EBV) was discovered 50 years ago as the first candidate human tumor virus. Since then, we have realized that this human γ-herpesvirus establishes persistent infection in the majority of adult humans, but fortunately causes EBV-associated diseases only in few individuals. This is an incredible success story of the human immune system, which controls EBV infection and its transforming capacity for decades. A better understanding of this immune control would not only benefit patients with EBV-associated malignancies, but could also provide clues how to establish such a potent, mostly cell-mediated immune control against other pathogens and tumors. However, the functional relevance of EBV-specific immune responses can only be addressed in vivo, and mice with reconstituted human immune system components (huMice) constitute a small animal model to interrogate the protective value of immune compartments during EBV infection, but also might provide a platform to test EBV-specific vaccines. This chapter will summarize the insights into EBV immunobiology that have already been gained in these models and provide an outlook into promising future avenues to develop this in vivo model of EBV infection and human immune responses further.

  8. Cationic lipid/DNA complexes (JVRS-100) combined with influenza vaccine (Fluzone) increases antibody response, cellular immunity, and antigenically drifted protection. (United States)

    Lay, Marla; Callejo, Bernadette; Chang, Stella; Hong, David K; Lewis, David B; Carroll, Timothy D; Matzinger, Shannon; Fritts, Linda; Miller, Christopher J; Warner, John F; Liang, Lily; Fairman, Jeffery


    Safe and effective adjuvants for influenza vaccines that could increase both the levels of neutralizing antibody, including against drifted viral subtypes, and T-cell immunity would be a major advance in vaccine design. The JVRS-100 adjuvant, consisting of DOTIM/cholesterol cationic liposome-DNA complexes, is particularly promising for vaccines that require induction of high levels of antibody and T-cell immunity, including CD8(+) cytotoxic T lymphocytes (CTL). Inclusion of protein antigens with JVRS-100 results in the induction of enhanced humoral and cell-mediated (i.e., CD4(+) and CD8(+) T cells) immune responses. The JVRS-100 adjuvant combined with a split trivalent influenza vaccine (Fluzone-sanofi pasteur) elicited increased antibody and T-cell responses in mice and non-human primates compared to vaccination with Fluzone alone. Mice vaccinated with JVRS-100-Fluzone and challenged with antigenically drifted strains of H1N1 (PR/8/34) and influenza B (B/Lee/40) viruses had higher grade protection, as measured by attenuation of weight loss and increased survival, compared to recipients of unadjuvanted vaccine. The results indicate that the JVRS-100 adjuvant substantially increases immunogenicity and protection from drifted-strain challenge using an existing influenza vaccine.

  9. Intranasal immunization of mice with recombinant Streptococcus gordonii expressing NadA of Neisseria meningitidis induces systemic bactericidal antibodies and local IgA. (United States)

    Ciabattini, Annalisa; Giomarelli, Barbara; Parigi, Riccardo; Chiavolini, Damiana; Pettini, Elena; Aricò, Beatrice; Giuliani, Marzia M; Santini, Laura; Medaglini, Donata; Pozzi, Gianni


    NadA and NhhA, two surface proteins of serogroup B Neisseria meningitidis identified as candidate vaccine antigens, were expressed on the surface of the human oral commensal bacterium Streptococcus gordonii. Recombinant strains were used to immunize BALB/c mice by the intranasal route and the local and systemic immune response was assessed. Mice were inoculated with recombinant bacteria administered alone or with LTR72, a partially inactivated mutant of Escherichia coli heat-labile enterotoxin, as a mucosal adjuvant. Intranasal immunization with live bacteria expressing NadA induced a significant serum antibody response, with a prevalence of the IgG2a subclass, bactericidal activity in the sera of 71% of animals, and a NadA-specific IgA response in nasal and bronchoalveolar lavages. A formalin-inactivated recombinant strain of S. gordonii expressing NadA was also administered intranasally, inducing a systemic and mucosal humoral response comparable to that of live bacteria. The administration of recombinant bacteria with the mucosal adjuvant LTR72 stimulated a stronger systemic antibody response, protective in 85% of sera, while did not increase the local IgA response. Recombinant S. gordonii expressing NhhA induced a systemic but not mucosal antibody response. These data support the role of NadA as vaccine candidate against serogroup B meningococci, and the use of S. gordonii as vector for intranasal vaccination.

  10. Glycan masking of hemagglutinin for adenovirus vector and recombinant protein immunizations elicits broadly neutralizing antibodies against H5N1 avian influenza viruses.

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    Shih-Chang Lin

    Full Text Available The highly pathogenic avian influenza (HPAI H5N1 virus, a known trigger of diseases in poultry and humans, is perceived as a serious threat to public health. There is a clear need for a broadly protective H5N1 vaccine or vaccines for inducing neutralizing antibodies against multiple clades/subclades. We constructed single, double, and triple mutants of glycan-masked hemagglutiinin (HA antigens at residues 83, 127 and 138 (i.e., g83, g127, g138, g83+g127, g127+g138, g83+g138 and g83+g127+g138, and then obtained their corresponding HA-expressing adenovirus vectors and recombinant HA proteins using a prime-boost immunization strategy. Our results indicate that the glycan-masked g127+g138 double mutant induced more potent HA-inhibition, virus neutralization antibodies, cross-clade protection against heterologous H5N1 clades, correlated with the enhanced bindings to the receptor binding sites and the highly conserved stem region of HA. The immune refocusing stem-specific antibodies elicited by the glycan-masked H5HA g127+g138 and g83+g127+g138 mutants overlapped with broadly neutralizing epitopes of the CR6261 monoclonal antibody that neutralizes most group 1 subtypes. These findings may provide useful information in the development of a broadly protective H5N1 influenza vaccine.

  11. Serologic analysis of anti-porcine endogenous retroviruses immune responses in humans after ex vivo transgenic pig liver perfusion. (United States)

    Xu, Hui; Sharma, Ajay; Okabe, Jeannine; Cui, Cunqi; Huang, Liping; Wei, Yuan Yuan; Wan, Hua; Lei, Ying; Logan, John S; Levy, Marlon F; Byrne, Guerard W


    Improvements in xenotransplantation may significantly increase the availability of organs for human transplantation. The use of porcine organs, however, has raised concern about possible transmission of porcine endogenous retroviruses (PERV) to the recipients. The authors developed monoclonal antibodies specific to the PERV Gag viral product and show that these antibodies can detect PERV antigen under a variety of assay conditions, including enzyme linked immunosorbent assay (ELISA), Western blot, and immunofluorescence staining methods. Two patients in fulminant hepatic failure were treated by extracorporeal perfusion using transgenic porcine livers before receiving orthotopic liver transplants. Despite the use of immune suppression that allowed survival of the allograft, these patients both showed a strong immune response to the xenograft suggesting a largely intact capability to mount a humoral immune response. However, analysis of patient serum samples over a 3 to 4 year period has showed no evidence of an immune response to PERV antigens, suggesting a lack of PERV infection.

  12. Conserved natural IgM antibodies mediate innate and adaptive immunity against the opportunistic fungus Pneumocystis murina. (United States)

    Rapaka, Rekha R; Ricks, David M; Alcorn, John F; Chen, Kong; Khader, Shabaana A; Zheng, Mingquan; Plevy, Scott; Bengtén, Eva; Kolls, Jay K


    Host defense against opportunistic fungi requires coordination between innate and adaptive immunity for resolution of infection. Antibodies generated in mice vaccinated with the fungus Pneumocystis prevent growth of Pneumocystis organisms within the lungs, but the mechanisms whereby antibodies enhance antifungal host defense are poorly defined. Nearly all species of fungi contain the conserved carbohydrates β-glucan and chitin within their cell walls, which may be targets of innate and adaptive immunity. In this study, we show that natural IgM antibodies targeting these fungal cell wall carbohydrates are conserved across many species, including fish and mammals. Natural antibodies bind fungal organisms and enhance host defense against Pneumocystis in early stages of infection. IgM antibodies influence recognition of fungal antigen by dendritic cells, increasing their migration to draining pulmonary lymph nodes. IgM antibodies are required for adaptive T helper type 2 (Th2) and Th17 cell differentiation and guide B cell isotype class-switch recombination during host defense against Pneumocystis. These experiments suggest a novel role for the IgM isotype in shaping the earliest steps in recognition and clearance of this fungus. We outline a mechanism whereby serum IgM, containing ancient specificities against conserved fungal antigens, bridges innate and adaptive immunity against fungal organisms.

  13. The human antibody response to the surface of Mycobacterium tuberculosis.

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    Casey C Perley

    Full Text Available Vaccine-induced human antibodies to surface components of Haemophilus influenzae and Streptococcus pneumonia are correlated with protection. Monoclonal antibodies to surface components of Mycobacterium tuberculosis are also protective in animal models. We have characterized human antibodies that bind to the surface of live M. tuberculosis.Plasma from humans with latent tuberculosis (TB infection (n = 23, active TB disease (n = 40, and uninfected controls (n = 9 were assayed by ELISA for reactivity to the live M. tuberculosis surface and to inactivated M. tuberculosis fractions (whole cell lysate, lipoarabinomannan, cell wall, and secreted proteins.When compared to uninfected controls, patients with active TB disease had higher antibody titers to the surface of live M. tuberculosis (Δ = 0.72 log10, whole cell lysate (Δ = 0.82 log10, and secreted proteins (Δ = 0.62 log10, though there was substantial overlap between the two groups. Individuals with active disease had higher relative IgG avidity (Δ = 1.4 to 2.6 to all inactivated fractions. Surprisingly, the relative IgG avidity to the live M. tuberculosis surface was lower in the active disease group than in uninfected controls (Δ =  -1.53, p = 0.004. Patients with active disease had higher IgG than IgM titers for all inactivated fractions (ratios, 2.8 to 10.1, but equal IgG and IgM titers to the live M. tuberculosis surface (ratio, 1.1. Higher antibody titers to the M. tuberculosis surface were observed in active disease patients who were BCG-vaccinated (Δ = 0.55 log10, p = 0.008, foreign-born (Δ = 0.61 log10, p = 0.004, or HIV-seronegative (Δ = 0.60 log10, p = 0.04. Higher relative IgG avidity scores to the M. tuberculosis surface were also observed in active disease patients who were BCG-vaccinated (Δ = 1.12, p < 0.001 and foreign-born (Δ = 0.87, p = 0.01.Humans with active TB disease produce antibodies to the surface of M. tuberculosis with low avidity and with a low IgG/IgM ratio

  14. The Human Antibody Response to the Surface of Mycobacterium tuberculosis (United States)

    Perley, Casey C.; Frahm, Marc; Click, Eva M.; Dobos, Karen M.; Ferrari, Guido; Stout, Jason E.; Frothingham, Richard


    Background Vaccine-induced human antibodies to surface components of Haemophilus influenzae and Streptococcus pneumonia are correlated with protection. Monoclonal antibodies to surface components of Mycobacterium tuberculosis are also protective in animal models. We have characterized human antibodies that bind to the surface of live M. tuberculosis. Methods Plasma from humans with latent tuberculosis (TB) infection (n = 23), active TB disease (n = 40), and uninfected controls (n = 9) were assayed by ELISA for reactivity to the live M. tuberculosis surface and to inactivated M. tuberculosis fractions (whole cell lysate, lipoarabinomannan, cell wall, and secreted proteins). Results When compared to uninfected controls, patients with active TB disease had higher antibody titers to the surface of live M. tuberculosis (Δ = 0.72 log10), whole cell lysate (Δ = 0.82 log10), and secreted proteins (Δ = 0.62 log10), though there was substantial overlap between the two groups. Individuals with active disease had higher relative IgG avidity (Δ = 1.4 to 2.6) to all inactivated fractions. Surprisingly, the relative IgG avidity to the live M. tuberculosis surface was lower in the active disease group than in uninfected controls (Δ = –1.53, p = 0.004). Patients with active disease had higher IgG than IgM titers for all inactivated fractions (ratios, 2.8 to 10.1), but equal IgG and IgM titers to the live M. tuberculosis surface (ratio, 1.1). Higher antibody titers to the M. tuberculosis surface were observed in active disease patients who were BCG-vaccinated (Δ = 0.55 log10, p = 0.008), foreign-born (Δ = 0.61 log10, p = 0.004), or HIV-seronegative (Δ = 0.60 log10, p = 0.04). Higher relative IgG avidity scores to the M. tuberculosis surface were also observed in active disease patients who were BCG-vaccinated (Δ = 1.12, p<0.001) and foreign-born (Δ = 0.87, p = 0.01). Conclusions/Significance Humans

  15. Isolation of ScFv antibodies of rP27Kip1 from phage display libraries constructed from immunized and non-immunized repertoires

    Institute of Scientific and Technical Information of China (English)

    曹跃琼; 乔守怡; 袁有忠; 黄建生; 赵寿元


    Through mRNA extract, RT and a series of PCR, phage antibody libraries were constructed from rP27Kiplimmunized and non-immunized mice. After only one round of selection with rP27Kipl, clones from each library were chosen randomly and digested by Taq I and Hinf I. 11 of 64 clones from the immunized animal had consistent restriction pattern, while none of the 64 clones from the non-immunized animal had, except that one had the same fragments pattern as that of the 11 clones. The 12 fragments were expressed in E. coli BL21(DE3)/pET-28b(+) system. ELISA showed that some of the fragments could bind to rP27Kipl specifically. All these results implied that specific antibody can be obtained by genetic engineering without hybridoma or many rounds of growth and panning selection.

  16. Recognition of influenza H3N2 variant virus by human neutralizing antibodies (United States)

    Bangaru, Sandhya; Nieusma, Travis; Kose, Nurgun; Thornburg, Natalie J.; Kaplan, Bryan S.; King, Hannah G.; Singh, Vidisha; Lampley, Rebecca M.; Cisneros, Alberto; Edwards, Kathryn M.; Edupuganti, Srilatha; Lai, Lilin; Richt, Juergen A.; Webby, Richard J.; Ward, Andrew B.; Crowe, James E.


    Since 2011, over 300 human cases of infection, especially in exposed children, with the influenza A H3N2 variant (H3N2v) virus that circulates in swine in the US have been reported. The structural and genetic basis for the lack of protection against H3N2v induced by vaccines containing seasonal H3N2 antigens is poorly understood. We isolated 17 human monoclonal antibodies (mAbs) that neutralized H3N2v virus from subjects experimentally immunized with an H3N2v candidate vaccine. Six mAbs exhibited very potent neutralizing activity (IC50 < 200 ng/ml) against the H3N2v virus but not against current human H3N2 circulating strains. Fine epitope mapping and structural characterization of antigen-antibody complexes revealed that H3N2v specificity was attributable to amino acid polymorphisms in the 150-loop and the 190-helix antigenic sites on the hemagglutinin protein. H3N2v-specific antibodies also neutralized human H3N2 influenza strains naturally circulating between 1995 and 2005. These results reveal a high level of antigenic relatedness between the swine H3N2v virus and previously circulating human strains, consistent with the fact that early human H3 seasonal strains entered the porcine population in the 1990s and reentered the human population, where they had not been circulating, as H3N2v about a decade later. The data also explain the increased susceptibility to H3N2v viruses in young children, who lack prior exposure to human seasonal strains from the 1990s. PMID:27482543

  17. Induction of anti-tumor immunity by trifunctional antibodies in patients with peritoneal carcinomatosis

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    Lindhofer Horst


    Full Text Available Abstract Peritoneal carcinomatosis (PC from epithelial tumors is a fatal diagnosis without efficient treatment. Trifunctional antibodies (trAb are novel therapeutic approaches leading to a concerted anti-tumor activity resulting in tumor cell destruction. In addition, preclinical data in mouse tumor models demonstrated the induction of long lasting tumor immunity after treatment with trAb. We describe the induction of anti-tumor specific T-lymphocytes after intraperitoneal administration of trAb in patients with PC. 9 patients with progressive PC from gastric (n = 6 and ovarian cancer (n = 2, and cancer of unknown primary (n = 1 received 3 escalating doses of trAb after surgery and/or ineffective chemotherapy. The trAb EpCAM × CD3 (10, 20, 40 μg or HER2/neu × CD3 (10, 40, 80 μg were applicated by intraperitoneal infusion. Four weeks after the last trAb application, all patients were restimulated by subdermal injection of trAb + autologous PBMC + irradiated autologous tumor cells. Immunological reactivity was tested by analyzing PBMC for specific tumor reactive CD4+/CD8+ T lymphocytes using an IFN-γ secretion assay. In 5 of 9 patients, tumor reactive CD4+/CD8+ T-lymphocytes increased significantly, indicating specific anti-tumor immunity. A clinical response (stable disease, partial regression has been observed in 5 of 9 patients, with a mean time to progression of 3.6 months. Follow-up showed a mean survival of 11.8 months (median 8.0 months after trAb therapy. TrAb are able to induce anti-tumor immunity after intraperitoneal application and restimulation. The induction of long-lasting anti-tumor immunity may provide an additional benefit of the intraperitoneal therapy with trAb and should be further elevated in larger clinical trials.

  18. Recognition determinants of broadly neutralizing human antibodies against dengue viruses. (United States)

    Rouvinski, Alexander; Guardado-Calvo, Pablo; Barba-Spaeth, Giovanna; Duquerroy, Stéphane; Vaney, Marie-Christine; Kikuti, Carlos M; Navarro Sanchez, M Erika; Dejnirattisai, Wanwisa; Wongwiwat, Wiyada; Haouz, Ahmed; Girard-Blanc, Christine; Petres, Stéphane; Shepard, William E; Desprès, Philippe; Arenzana-Seisdedos, Fernando; Dussart, Philippe; Mongkolsapaya, Juthathip; Screaton, Gavin R; Rey, Félix A


    Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.

  19. An improved and robust DNA immunization method to develop antibodies against extracellular loops of multi-transmembrane proteins. (United States)

    Hazen, Meredith; Bhakta, Sunil; Vij, Rajesh; Randle, Steven; Kallop, Dara; Chiang, Vicki; Hötzel, Isidro; Jaiswal, Bijay S; Ervin, Karen E; Li, Bing; Weimer, Robby M; Polakis, Paul; Scheller, Richard H; Junutula, Jagath R; Hongo, Jo-Anne S


    Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry. As expected, the use of the immune modulators fetal liver tyrosine kinase 3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor positively enhanced the immune response against MRP4. Imaging studies using CMV-based plasmids expressing luciferase showed that the in vivo half-life of the target antigen was less than 48 h using CMV-based plasmids, thus necessitating frequent boosting with DNA to achieve an adequate immune response. We also describe a comparison of plasmids, which contained MRP4 cDNA with either the CMV or CAG promoters, used for immunizations. The observed luciferase activity in this comparison demonstrated that the CAG promoter-containing plasmid pCAGGS induced prolonged constitutive expression of MRP4 and an increased anti-MRP4 specific immune response even when the plasmid was injected less frequently. The method described here is one that can be broadly applicable as a general immunization strategy to develop antibodies against multi-transmembrane proteins, as well as target antigens that are difficult to express or purify in native and functionally active conformation.

  20. Chikungunya virus neutralization antigens and direct cell-to-cell transmission are revealed by human antibody-escape mutants.

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    Chia Yin Lee


    Full Text Available Chikungunya virus (CHIKV is an alphavirus responsible for numerous epidemics throughout Africa and Asia, causing infectious arthritis and reportedly linked with fatal infections in newborns and elderly. Previous studies in animal models indicate that humoral immunity can protect against CHIKV infection, but despite the potential efficacy of B-cell-driven intervention strategies, there are no virus-specific vaccines or therapies currently available. In addition, CHIKV has been reported to elicit long-lasting virus-specific IgM in humans, and to establish long-term persistence in non-human primates, suggesting that the virus might evade immune defenses to establish chronic infections in man. However, the mechanisms of immune evasion potentially employed by CHIKV remain uncharacterized. We previously described two human monoclonal antibodies that potently neutralize CHIKV infection. In the current report, we have characterized CHIKV mutants that escape antibody-dependent neutralization to identify the CHIKV E2 domain B and fusion loop "groove" as the primary determinants of CHIKV interaction with these antibodies. Furthermore, for the first time, we have also demonstrated direct CHIKV cell-to-cell transmission, as a mechanism that involves the E2 domain A and that is associated with viral resistance to antibody-dependent neutralization. Identification of CHIKV sub-domains that are associated with human protective immunity, will pave the way for the development of CHIKV-specific sub-domain vaccination strategies. Moreover, the clear demonstration of CHIKV cell-to-cell transmission and its possible role in the establishment of CHIKV persistence, will also inform the development of future anti-viral interventions. These data shed new light on CHIKV-host interactions that will help to combat human CHIKV infection and inform future studies of CHIKV pathogenesis.

  1. Protection of gnotobiotic rats against dental caries by passive immunization with bovine milk antibodies to Streptococcus mutans.


    Michalek, S. M.; Gregory, R L; Harmon, C C; Katz, J; Richardson, G J; Hilton, T.; Filler, S J; McGhee, J R


    A multivalent vaccine consisting of whole cell antigens of seven strains, representing four serotypes (b, c, d and g), of mutans streptococci was used to hyperimmunize a group of cows. Serum samples from these animals contained immunoglobulin G1 (IgG1) antibody activity to seven serotypes (a to g) of mutans streptococci. Whey obtained from the animal with the highest serum antibody activity, which also contained high levels of IgG1 antibody, was used in passive caries immunity studies. Gnotob...

  2. Seasonal changes in human immune responses to malaria

    DEFF Research Database (Denmark)

    Hviid, L; Theander, T G


    Cellular as well as humorol immune responses to malaria antigens fluctuate in time in individuals living in molono-endemic areas, particularly where malaria transmission is seasonal. The most pronounced changes are seen in association with clinical attacks, but osymptomatic infection can also lead...... to apparent immune depression. However, recent data have shown that seasonal variation in cellular immune responses may occur even in the absence of detectable porositaemia. Here, Lars Hviid and Thor G. Theonder review the seasonal variation in human immune responses to malaria, and discuss its possible...

  3. Amelioration of murine passive immune thrombocytopenia by IVIg and a therapeutic monoclonal CD44 antibody does not require the Myd88 signaling pathway.

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    Andrew R Crow

    Full Text Available Immune thrombocytopenia (ITP is an autoimmune bleeding disorder characterized by a low platelet count and the production of anti-platelet antibodies. The majority of ITP patients have antibodies to platelet integrin α(IIbβ₃ (GPIIbIIIa which can direct platelet phagocytosis by macrophages. One effective treatment for patients with ITP is intravenous immunoglobulin (IVIg which rapidly reverses thrombocytopenia. The exact mechanism of IVIg action in human patients is unclear, although in mouse models of passive ITP, IVIg can rapidly increase platelet counts in the absence of adaptive immunity. Another antibody therapeutic that can similarly increase platelet counts independent of adaptive immunity are CD44 antibodies. Toll-like receptors (TLRs are pattern recognition receptors which play a central role in helping direct the innate immune system. Dendritic cells, which are notable for their expression of TLRs, have been directly implicated in IVIg function as an initiator cell, while CD44 can associate with TLR2 and TLR4. We therefore questioned whether IVIg, or the therapeutic CD44 antibody KM114, mediate their ameliorative effects in a manner dependent upon normal TLR function. Here, we demonstrate that the TLR4 agonist LPS does not inhibit IVIg or KM114 amelioration of antibody-induced thrombocytopenia, and that these therapeutics do not ameliorate LPS-induced thrombocytopenia. IVIg was able to significantly ameliorate murine ITP in C3H/HeJ mice which have defective TLR4. All known murine TLRs except TLR3 utilize the Myd88 adapter protein to drive TLR signaling. Employing Myd88 deficient mice, we found that both IVIg and KM114 ameliorate murine ITP in Myd88 deficient mice to the same extent as normal mice. Thus both IVIg and anti-CD44 antibody can mediate their ameliorative effects in murine passive ITP independent of the Myd88 signaling pathway. These data help shed light on the mechanism of action of IVIg and KM114 in the amelioration of

  4. Detection of anti-liver cell membrane antibody using a human hepatocellular carcinoma cell line

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    Lobo-Yeo, A.; McSorley, C.; McFarlane, B.M.; Mieli-Vergani, G.; Mowat, A.P.; Vergani, D.


    A radioimmunometric technique for the detection of autoantibodies to liver membrane antigens has been developed using Alexander cells, a human hepatocellular carcinoma cell line. After incubation of Alexander cells with serum, antimembrane antibodies were detected by addition of /sup 125/I-labeled Protein A. Binding ratios in 15 children with uncontrolled autoimmune chronic active hepatitis and in seven children with primary sclerosing cholangitis were significantly higher than in 18 age-matched normal controls. Nine patients with inactive autoimmune chronic active hepatitis, 13 with alpha 1-antitrypsin deficiency and five with fulminant hepatic failure had ratios similar to controls. In nine patients with Wilson's disease, there was a modest but significant increase in binding ratio. In four children with autoimmune chronic active hepatitis, binding ratios fell during effective immunosuppressive therapy. Sera from patients with systemic lupus erythematosus or rheumatoid arthritis gave normal results, excluding that binding derives from Fc-mediated immune complex capture. A positive correlation was found between Alexander cell binding values and anti-liver-specific protein antibody titers, suggesting that the two assays detect antibodies against shared antigenic determinants. The Alexander cell assay is a simple, rapid and sensitive technique to detect antibody to liver cell membrane antigens.

  5. Antigen capture ELISA system for henipaviruses using polyclonal antibodies obtained by DNA immunization. (United States)

    Kaku, Yoshihiro; Noguchi, Akira; Marsh, Glenn A; Barr, Jennifer A; Okutani, Akiko; Hotta, Kozue; Bazartseren, Boldbaatar; Broder, Christopher C; Yamada, Akio; Inoue, Satoshi; Wang, Lin-Fa


    A novel antigen-capture sandwich ELISA system targeting the glycoproteins of the henipaviruses Nipah virus (NiV) and Hendra virus (HeV) was developed. Utilizing purified polyclonal antibodies derived from NiV glycoprotein-encoding DNA-immunized rabbits, we established a system that can detect the native antigenic structures of the henipavirus surface glycoproteins using simplified and inexpensive methods. The lowest detection limit against live viruses was achieved for NiV Bangladesh strain, 2.5 × 10(4) TCID(50). Considering the recent emergence of genetic variants of henipaviruses and the resultant problems that arise for PCR-based detection, this system could serve as an alternative rapid diagnostic and detection assay.

  6. Human Immune Responses to Dengue Viruses. (United States)


    between the PBL of non- inmune donors and dengue virus-infected cells, which results in IFN production. We use dengue virus-infected B lymphoblastoid cell...or, J.J., M.W. Brandriss, and E.E. Walsh. 1985. Protection against 17D yellow fever encephalitis in mice by passive transfer of monoclonal antibodies

  7. Studies of neutralising antibodies to SV40 in human sera. (United States)

    Minor, P; Pipkin, P; Jarzebek, Z; Knowles, W


    It has been suggested that the low levels of antibody to the simian polyoma virus SV40 found in human sera may be linked to the use of polio vaccines. Panels of sera from areas of the world with different vaccination histories were examined to see if consistent differences could be identified. In a total of 2,054 sera from the United Kingdom, 692 from Africa and 923 from Poland taken between 1985 and 1997, the seroprevalence was generally between 3 and 5%, although exceptionally one collection from Morocco had a prevalence of 100%, and one from Poland of 0.4%. The seroprevalence showed no obvious age-dependent increase and titres were low compared to post infection animal sera. The results are consistent with previous studies and reveal no general geographically based differences related to possible differences in vaccination history, but the origin of the SV40 antibody in human sera remains to be established.

  8. Identification of skin immune cells in non-human primates. (United States)

    Adam, Lucille; Rosenbaum, Pierre; Cosma, Antonio; Le Grand, Roger; Martinon, Frédéric


    The skin is a valuable target for vaccine delivery because it contains many immune cell populations, notably antigen presenting cells. Skin immune cells have been extensively described in mice and humans but not in non-human primates, which are pertinent models for immunological research in vaccination. The aim of this work was to describe immune cell populations in the epidermis, dermis and skin draining lymph nodes in cynomolgus macaques by a single 12-parameter flow cytometry protocol. Given that skin cells share several markers, we defined a gating strategy to identify accurately immune cells and to limit contamination of one immune cell population by another. The epidermis contained CD1a(+)CD1c(-) Langerhans cells (LCs), CD3(+) T cells and putative NK cells. The dermis contained CD1a(+)CD1c(-) cells, which were similar to LCs, CD1a(+)CD1c(+) dermal dendritic cells (DDCs), CD163(high)CD11b(+) resident macrophages, CD3(+) T cells and putative NK cells. The skin also contained CD66(+) polymorphonuclear cells in some animals. Thus, immune cell populations in the macaque are similar to those in humans despite some differences in phenotype. In skin draining lymph nodes, we identified migratory LCs, CD1a(+)CD1c(+) DDCs and macrophages. The simultaneous identification of these different immune cells with one panel of markers avoids the use of large amounts of precious sample and may improve the understanding of immune mechanisms in the skin after treatment or vaccination.

  9. Humoral immune responses induced by anti-idiotypic antibody fusion protein of 6B11scFv/hGM-CSF in BALB/c mice

    Institute of Scientific and Technical Information of China (English)


    Background We have previously developed and characterized a monoclonal anti-idiotype antibody, designated 6B11, which mimics an ovarian carcinoma associated antigen OC166-9 and whose corresponding monoclonal antibody is COC166-9 (Ab1). In this study, we evaluate the humoral immune responses induced by the fusion protein 6B11 single-chain variable fragment (scFv)/human granulocyte macrophage colony-stimulating factor (hGM-CSF) and 6B11scFv in BALB/c mice. Methods The fusion protein 6B11scFv/hGM-CSF was constructed by fusing a recombinant single-chain variable fragment of 6B11scFv to GM-CSF. BALB/c mice were administrated by 6B11scFv/hGM-CSF and 6B11scFv, respectively. Results The fusion protein 6B11scFv/hGM-CSF retained binding to the anti-mouse F(ab)2' and was also biologically active as measured by proliferation of human GM-CSF dependent cell TF1 in vitro. After immunization with the 6B11scFv/hGM-CSF and 6B11ScFv, BALB/c mice showed significantly enhanced Ab3 antibody responses to 6B11scFv/hGM-CSF compared with the 6B11scFv alone. The level of Ab3 was the highest after the first week and maintained for five weeks after the last immunization. Another booster was given when the Ab3 titer descended, and it would reach to the high level in a week. Conclusion The fusion protein 6B11scFv/hGM-CSF can induce humoral immunity against ovarian carcinoma in vivo. We also provide the theoretical foundation for the application of the fusion protein 6B11scFv/hGM-CSF for active immunotherapy of ovarian cancer.

  10. Discovery Of Human Antibodies Against Spitting Cobra Toxins

    DEFF Research Database (Denmark)

    Bojsen-Møller, Laura; Lohse, Brian; Harrison, Robert

    spitting cobras are among the most medically important snakes in sub-Saharan regions due to the severity of the clinical outcomes caused by their cytotoxic venom, which is derived from cytotoxins of the 3FTx toxin family and PLA2. Here we report the results of our progress in identifying human antibodies...... targeting relevant toxins from the venom of the black necked spitting cobra (Naja nigricolis)....

  11. Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model.

    Directory of Open Access Journals (Sweden)

    Xiaodong Xiao

    Full Text Available Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252 and two anti-V-specific human mAb (m253, m254 by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.

  12. Platelet antibodies, activated platelets and serum leptin in childhood immune thrombocytopenic purpura. (United States)

    Badrawy, Hosny; Elsayh, Khalid I; Zahran, Asmaa M; El-Ghazali, Mohamad Hamdy


    The aim of this study was to evaluate the levels of platelet-associated antibodies (PAIgG and PAIgM), activated platelets and serum leptin in children with acute immune thrombocytopenic purpura (ITP). The study included 40 patients with ITP and 40 healthy age- and sex-matched controls. PAIgG, PAIgM and activated platelet levels were estimated by flow cytometry, and serum leptin levels were estimated by ELISA. Activated platelets and serum leptin were significantly higher in the ITP patients than in the controls. The percentage and mean fluorescence intensity of PAIgG and PAIgM staining were significantly higher in the patients than in the controls. Serum leptin and activated platelet levels in patients with thrombocytopenia of brief duration were significantly lower than those in patients with thrombocytopenia of prolonged duration. The levels of activated platelets, serum leptin and PAIgG were positively correlated, and the levels of serum leptin, activated platelets and platelet counts were negatively correlated. The increased levels of activated platelets, serum leptin and platelet-associated antibodies in children with acute ITP suggest that these factors could play a role in ITP pathogenesis. Additionally, activated platelets and serum leptin could have prognostic significance in paediatric acute ITP. Copyright © 2013 S. Karger AG, Basel.

  13. Modulation of Human Immune Response by Fungal Biocontrol Agents (United States)

    Konstantinovas, Cibele; de Oliveira Mendes, Tiago A.; Vannier-Santos, Marcos A.; Lima-Santos, Jane


    Although the vast majority of biological control agents is generally regarded as safe for humans and environment, the increased exposure of agriculture workers, and consumer population to fungal substances may affect the immune system. Those compounds may be associated with both intense stimulation, resulting in IgE-mediated allergy and immune downmodulation induced by molecules such as cyclosporin A and mycotoxins. This review discusses the potential effects of biocontrol fungal components on human immune responses, possibly associated to infectious, inflammatory diseases, and defective defenses. PMID:28217107

  14. A murine monoclonal anti-idiotypic antibody detects a common idiotope on human, mouse and rabbit antibodies to allergen Lol p IV. (United States)

    Zhou, E M; Dzuba-Fischer, J M; Rector, E S; Sehon, A H; Kisil, F T


    A syngeneic mouse monoclonal anti-idiotypic antibody (anti-Id), designated as B1/1, was generated against a monoclonal antibody (MoAb 91) specific for Ryegrass pollen allergen Lol p IV. This anti-Id recognized an idiotope (Id) that was also present on other monoclonal antibodies with the same specificity as MoAb 91. Observations that (i) the anti-Id inhibited the binding of MoAb 91 to Lol p IV and (ii) the Id-anti-Id interaction could be inhibited by Lol p IV indicated that the Id was located within or near the antigen combining site. These properties served to characterize B1/1 as an internal image anti-Id. Evidence that an immune response in different species to Lol p IV elicits the formation of antibodies which express a common Id was provided by the observations that (i) the Id-anti-Id interactions could be inhibited by mouse, human and rabbit antisera to Lol p IV and (ii) the binding of these antisera to Lol p IV could be inhibited by the anti-Id. Interestingly, the internal image anti-Id B1/1 also recognized an Id on a monoclonal antibody which was directed to an epitope of Lol p IV, different from that recognized by MoAb 91.

  15. Acquired immune heterogeneity and its sources in human helminth infection. (United States)

    Bourke, C D; Maizels, R M; Mutapi, F


    Similarities in the immunobiology of different parasitic worm infections indicate that co-evolution of humans and helminths has shaped a common anti-helminth immune response. However, recent in vitro and immuno-epidemiological studies highlight fundamental differences and plasticity within host-helminth interactions. The 'trade-off' between immunity and immunopathology inherent in host immune responses occurs on a background of genetic polymorphism, variable exposure patterns and infection history. For the parasite, variation in life-cycle and antigen expression can influence the effector responses directed against them. This is particularly apparent when comparing gastrointestinal and tissue-dwelling helminths. Furthermore, insights into the impact of anti-helminthic treatment and co-infection on acquired immunity suggest that immune heterogeneity arises not from hosts and parasites in isolation, but also from the environment in which immune responses develop. Large-scale differences observed in the epidemiology of human helminthiases are a product of complex host-parasite-environment interactions which, given potential for exposure to parasite antigens in utero, can arise even before a parasite interacts with its human host. This review summarizes key differences identified in human acquired immune responses to nematode and trematode infections of public health importance and explores the factors contributing to these variations.

  16. Antibodies against human BLyS and APRIL attenuate EAE development in marmoset monkeys. (United States)

    Jagessar, S Anwar; Heijmans, Nicole; Oh, Luke; Bauer, Jan; Blezer, Erwin L A; Laman, Jon D; Migone, Thi-Sau; Devalaraja, Matt N; 't Hart, Bert A


    B lymphocyte stimulator (BLyS, also indicated as BAFF (B-cell activating factor) and CD257), and A Proliferation Inducing Ligand (APRIL, CD256) are two members of the TNF superfamily with a central role in B cell survival. Antibodies against these factors have potential therapeutic relevance in autoimmune inflammatory disorders with a proven pathogenic contribution of B cells, such as multiple sclerosis (MS). In the current study we performed a multi-parameter efficacy comparison of monoclonal antibodies against human anti-BLyS and anti-APRIL in a common marmoset (Callithrix jacchus) model of experimental autoimmune encephalomyelitis (EAE). A MS-like disease was induced by immunization with recombinant human myelin/oligodendrocyte glycoprotein (rhMOG) in complete Freund's adjuvant. The results show that the anti-BLyS and anti-APRIL antibody cause significant depletion of circulating CD20+ B cells, but a small subset of CD20 + CD40(high) B cells was not depleted. Induction of CD20+ B cell depletion from lymph nodes was only observed in the anti-BLyS treated monkeys. Both antibodies had a significant inhibitory effect on disease development, but all monkeys developed clinically evident EAE. Anti-BLyS treated monkeys were sacrificed with the same clinical signs as saline-treated monkeys, but nevertheless displayed significantly reduced spinal cord demyelination. This effect was not observed in the anti-APRIL treated monkeys. The two antibodies had a different effect on T cell subset activation and the profiles of ex vivo released cytokines. In conclusion, treatment with anti-BLyS and anti-APRIL delays the development of neurological disease in a relevant preclinical model of MS. The two mAbs achieve this effect via different mechanisms.

  17. A review of the equine age-related changes in the immune system: comparisons between human and equine aging, with focus on lung-specific immune-aging. (United States)

    Hansen, S; Baptiste, K E; Fjeldborg, J; Horohov, D W


    The equine aging process involves many changes to the immune system that may be related to genetics, the level of nutrition, the environment and/or an underlying subclinical disease. Geriatric horses defined as horses above the age of 20, exhibit a decline in body condition, muscle tone and general well-being. It is not known whether these changes contribute to decreased immune function or are the result of declining immune function. Geriatric years are characterized by increased susceptibility to infections and a reduced antibody response to vaccination as a result of changes in the immune system. Humans and horses share many of these age-related changes, with only a few differences. Thus, inflamm-aging and immunosenescence are well-described phenomena in both human and equine research, particularly in relation to the peripheral blood and especially the T-cell compartment. However, the lung is faced with unique challenges because of its constant interaction with the external environment and thus may not share similarities to peripheral blood when considering age-related changes in immune function. Indeed, recent studies have shown discrepancies in cytokine mRNA and protein expression between the peripheral blood and bronchoalveolar lavage immune cells. These results provide important evidence that age-related immune changes or 'dys-functions' are organ-specific.

  18. Reshaped Human Monoclonal Antibodies for Therapy and Passive Immunization (United States)


    0C. The annealed DNA was extended with Klenow fragment of DNA polymerase I in a reaction mixture containing T4 DNA ligase and 0.5 mM of each of dATP...and T4 DNA ligase . The DNA was transformed into E. coli TG1 made competent by the method of Simanis (7). Single stranded DNA was prepared from...introduced. The initial synthesis of the mutant strand was with Klenow fragment of DNA polymerase I and DNA ligase as described above or with T7 DNA

  19. A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies. (United States)

    Chan, Jo-Anne; Howell, Katherine B; Langer, Christine; Maier, Alexander G; Hasang, Wina; Rogerson, Stephen J; Petter, Michaela; Chesson, Joanne; Stanisic, Danielle I; Duffy, Michael F; Cooke, Brian M; Siba, Peter M; Mueller, Ivo; Bull, Peter C; Marsh, Kevin; Fowkes, Freya J I; Beeson, James G


    Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

  20. Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system.

    LENUS (Irish Health Repository)

    Little, Mark A


    Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3) antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener\\'s granulomatosis). Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ⁻\\/⁻ mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17%) more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.

  1. Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system.

    Directory of Open Access Journals (Sweden)

    Mark A Little

    Full Text Available Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3 antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener's granulomatosis. Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ⁻/⁻ mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17% more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.

  2. Innate immune functions of microglia isolated from human glioma patients

    Directory of Open Access Journals (Sweden)

    Grimm Elizabeth


    Full Text Available Abstract Background Innate immunity is considered the first line of host defense and microglia presumably play a critical role in mediating potent innate immune responses to traumatic and infectious challenges in the human brain. Fundamental impairments of the adaptive immune system in glioma patients have been investigated; however, it is unknown whether microglia are capable of innate immunity and subsequent adaptive anti-tumor immune responses within the immunosuppressive tumor micro-environment of human glioma patients. We therefore undertook a novel characterization of the innate immune phenotype and function of freshly isolated human glioma-infiltrating microglia (GIM. Methods GIM were isolated by sequential Percoll purification from patient tumors immediately after surgical resection. Flow cytometry, phagocytosis and tumor cytotoxicity assays were used to analyze the phenotype and function of these cells. Results GIM expressed significant levels of Toll-like receptors (TLRs, however they do not secrete any of the cytokines (IL-1β, IL-6, TNF-α critical in developing effective innate immune responses. Similar to innate macrophage functions, GIM can mediate phagocytosis and non-MHC restricted cytotoxicity. However, they were statistically less able to mediate tumor cytotoxicity compared to microglia isolated from normal brain. In addition, the expression of Fas ligand (FasL was low to absent, indicating that apoptosis of the incoming lymphocyte population may not be a predominant mode of immunosuppression by microglia. Conclusion We show for the first time that despite the immunosuppressive environment of human gliomas, GIM are capable of innate immune responses such as phagocytosis, cytotoxicity and TLR expression but yet are not competent in secreting key cytokines. Further understanding of these innate immune functions could play a critical role in understanding and developing effective immunotherapies to malignant human gliomas.

  3. Characterization of serum and mucosal antibody responses in white sturgeon (Acipenser transmontanus Richardson) following immunization with WSIV and a protein hapten antigen. (United States)

    Drennan, John D; Lapatra, Scott E; Swan, Christine M; Ireland, Sue; Cain, Kenneth D


    Serum and cutaneous mucus antibodies were monitored in white sturgeon for 15 weeks following intraperitoneal immunization. Ten fish were immunized (50 microg) with white sturgeon iridovirus (WSIV) or white sturgeon gonad (WSGO) tissue culture cells emulsified with or without FCA. An additional group was immunized with FITC:KLH+FCA. Fish were booster immunized at 6 weeks. Fish immunized with FITC:KLH+FCA produced significant serum antibodies to FITC by 6 weeks and this response peaked at 12 weeks (average titer 31,000). Mucosal antibodies to FITC were first detected at 12 weeks and significantly elevated by 15 weeks (average titer 18). Anti-WSIV antibody titers were detected in the serum by 9 weeks in fish immunized with WSIV and WSIV+FCA, but only a small number responded to immunization. At 15 weeks, four fish immunized with WSIV produced serum antibodies (average titer 838) and one fish immunized with WSIV+FCA had a serum titer of 1600. Mucosal anti-WSIV antibody titers of 8 and 16 were observed in two fish from the WSIV group at 12 weeks while four different fish from this group responded at 15 weeks (average titer 4). Western Blot using a monoclonal antibody confirmed immunoglobulin in mucus, and specificity to WSIV was further demonstrated by immunocytochemistry using serum from fish immunized with WSIV. Specific antibody was not detected in mucus of fish immunized with WSIV+FCA, WSGO, or WSGO+FCA. Collectively, these experiments demonstrate that white sturgeon can generate a specific antibody response following immunization, and is the first report showing mucosal immunoglobulin is present in this species.

  4. Immunization with Protein D from Non-Typeable Haemophilus influenzae (NTHi) Induced Cytokine Responses and Bioactive Antibody Production (United States)

    Davoudi Vijeh Motlagh, Atefeh; Siadat, Seyed Davar; Abedian Kenari, Saeid; Mahdavi, Mehdi; Behrouzi, Ava; Asgarian-Omran, Hossein


    Background Outer membrane protein D (PD) is a highly conserved and stable protein in the outer membrane of both encapsulated (typeable) and non-capsulated (non-typeable) strains of Haemophilus influenzae. As an immunogen, PD is a potential candidate vaccine against non-typeable H. influenzae (NTHi) strains. Objectives The aim of this study was to determine the cytokine pattern and the opsonic antibody response in a BALB/c mouse model versus PD from NTHi as a vaccine candidate. Methods Protein D was formulated with Freund’s and outer membrane vesicle (OMV) adjuvants and injected into experimental mice. Sera from all groups were collected. The bioactivity of the anti-PD antibody was determined by opsonophagocytic killing test. To evaluate the cytokine responses, the spleens were assembled, suspension of splenocytes was recalled with antigen, and culture supernatants were analyzed by ELISA for IL-4, IL-10, and IFN-γ cytokines. Results Anti-PD antibodies promoted phagocytosis of NTHi in both immunized mice groups (those administered PD + Freund’s and those administered PD + OMV adjuvants, 92.8% and 83.5%, respectively, compared to the control group). In addition, the concentrations of three cytokines were increased markedly in immunized mice. Conclusions We conclude that immunization with PD protects mice against NTHi. It is associated with improvements in both cellular and humoral immune responses and opsonic antibody activity. PMID:27942362

  5. Native and recombinant proteins to analyze auto-antibodies to myeloperoxidase in pauci-immune crescentic glomerulonephritis

    NARCIS (Netherlands)

    Boomsma, MM; Stegeman, CA; Oost-Kort, WW; Kallenberg, CGM; Moguilevsky, N; Limburg, PC; Tervaert, JWC


    The prevalence of Anti-Neutrophil Cytoplasmic Antibodies (ANCA) directed against myeloperoxidase (MPO) in pauci-immune necrotizing crescentic glomerulonephritis (NCGN) is dependent on the assay(s) used, We investigated the frequency of MPO-ANCA as detected by different assays for MPO-ANCA in a large

  6. The Use of Monoclonal Antibodies in Human Prion Disease (United States)

    Bodemer, Walter

    Detection of PrP and its pathological isoform(s) is the key to understanding the etiology and pathogenesis of transmissible spongiform encephalopathy. There is ample evidence that PrP isoforms constitute a major component of an unknown and perhaps unconventional infectious agent. An etiological relationship between human and zoonotic transmissible spongiform encephalopathies may be revealed with monoclonal antibodies. Knowledge of the conformational transition rendering a nonpathogenic, almost ubiquitous cellular protein into a pathogenic one is crucial to defining pathomechanisms. The stepwise or even continuous formation of pathogenic molecules can be monitored. Any improvement in the early diagnosis could help to conceive new therapeutic measures which are not currently available. Determination of PrP isoforms in tissue, cells, or body fluids may be of prognostic value. Many experimental approaches in molecular medicine and molecular biology of the prion protein already rely on monoclonal antibodies. Recombinant antibodies such as the single-chain Fv may soon replace traditional hybridoma techniques. Binding affinity can easily be manipulated by a number of techniques, including in vitro mutagenesis - a step which could never be carried out using the traditional hybridoma technology. Monoclonal antibodies are and will remain an essential support for ongoing research on the prion protein in general and on the unconventional infectious prions.

  7. The immunomodulatory effects of shark cartilage on the mouse and human immune system

    Directory of Open Access Journals (Sweden)

    ali Sheikhian


    Materials and methods: In an experimental study, the effects of different doses of shark cartilage on humoral (antibody titer immune response against sheep red blood cells (SRBC, were measured in mouse. In addition, we evaluated the modulatory effects of the shark cartilage on the natural killer (NK activity of the peritoneal cells of mouse against a tumor cell line called K562, according to the standard methods. The proliferative response of the human peripheral blood mononuclear cells was measured under the influence of shark cartilage. Results: Pure shark cartilage enhanced antibody response against SRBC in vivo. The hemagglutination titer which was 1/147 in the control group (injected with hen cartilage, increased to 1/1355 in the test group. The optimal dose was 100 mg/ml. both type of cartilage had blastogenic effect on peripheral blood mononuclear cells (the blastogenic index was 6.7 and 4.9 for impure shark cartilage and hen cartilage, respectively. NK activity was inhibited completely by pure shark cartilage (the amount of the killing activity of the effector peritoneal cells for the control and test groups against target cells was 25.9% and 5.5% respectively. Conclusion: Shark cartilage has a potent immunomodulatory effect on the specific immune mechanisms and some inhibitory effects on the innate immune mechanisms such as NC activity. Since the specific immunity has a more pivotal role against tumor formation, shark cartilage can be used as a cancer immunotherapeutic.

  8. Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin

    Energy Technology Data Exchange (ETDEWEB)

    Whittle, James R.R.; Zhang, Ruijun; Khurana, Surender; King, Lisa R.; Manischewitz, Jody; Golding, Hana; Dormitzer, Philip R.; Haynes, Barton F.; Walter, Emmanuel B.; Moody, M. Anthony; Kepler, Thomas B.; Liao, Hua-Xin; Harrison, Stephen C. (Harvard-Med); (Novartis); (US-FDA); (Duke)


    Seasonal antigenic drift of circulating influenza virus leads to a requirement for frequent changes in vaccine composition, because exposure or vaccination elicits human antibodies with limited cross-neutralization of drifted strains. We describe a human monoclonal antibody, CH65, obtained by isolating rearranged heavy- and light-chain genes from sorted single plasma cells, coming from a subject immunized with the 2007 trivalent influenza vaccine. The crystal structure of a complex of the hemagglutinin (HA) from H1N1 strain A/Solomon Islands/3/2006 with the Fab of CH65 shows that the tip of the CH65 heavy-chain complementarity determining region 3 (CDR3) inserts into the receptor binding pocket on HA1, mimicking in many respects the interaction of the physiological receptor, sialic acid. CH65 neutralizes infectivity of 30 out of 36 H1N1 strains tested. The resistant strains have a single-residue insertion near the rim of the sialic-acid pocket. We conclude that broad neutralization of influenza virus can be achieved by antibodies with contacts that mimic those of the receptor.

  9. Production and characterization of antibodies against irradiated human erythrocytes membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Amancio, Francisco F. [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil)]|[Pernambuco Univ., Recife, PE (Brazil). Dept. de Biofisica e Radiobiologia; Andrade Junior, Heitor F. [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina. Inst. de Medicina Tropical


    Gamma irradiation affects people in several situations, with few if any sensitive biological assay of its action. Nucleic acids and proteins are affected by radiation, but only the former was used in most dosimetric techniques. The irradiation of proteins promotes structural modifications attributed to free radicals from water radiolysis. Theoretically, antibodies induced by irradiated proteins could recognize these radical-related new epitopes, allowing their use as a probe. Human erythrocyte membrane proteins (HEMP), few and well defined molecules, are certainly exposed to radiation, being the ideal target. With this rationale, we study the production of antibodies in mice immunized with {sup 60} Co irradiated HEMPs. Menbranes from hypotonic lysis with differential centrifugation of A+ erythrocytes, were irradiated in a Gammacell 220 with 400, 800 and 1600 Gy, and used as immunogen for Balb/c mice, after SDS-PAGE. Irradiated HEMP induced antibodies recognize only irradiated human erthrocytes in an intact cell indirect immunofluorescence assay (ICIIFA). When used in Wester-blot against non-irradiated HEMPs, those sera recognize most proteins, suggesting a pool of abs directed both to native, as detected by Western Blot, or irradiated, as detected by ICIFA, HEMPs. Those data confirmed our assumptions, allowing the use of those abs in the search for a method of biological dosimetry. (author). 18 refs., 3 figs.

  10. Monoclonal antibodies to human colorectal tumor-associated antigens: improved elicitation and subclass restriction. (United States)

    Morgan, A C; Woodhouse, C S; Knost, J A; Abrams, P G; Clarke, G C; Arthur, L O; McIntyre, R; Ochs, J J; Foon, K A; Oldham, R K


    Monoclonal antibodies to tumor-associated antigens (TAA) of human colorectal cancer were elicited using immunosorbents of lectins combined with peripheral protein extracts of xenografted colon adenocarcinoma. This method of immunization was compared with whole cells from surgical specimens and to crude membranes from xenografted tumors. The immunosorbent immunogens were superior to the other immunogens in three ways: (1) the number of hybrids reactive with colon tumor cells or extracts, but not with lymphoid cells or extracts, (2) the number of stable hybrids after cloning, and (3) the number of hybridoma clones reactive with tissue sections of colon tumors, but not normal colonic mucosa. In addition, lectin immunosorbents elicited primarily IgG antibodies, especially IgG3, with almost 50% of the clones of interest reacting to seemingly less immunogenic glycoproteins. The improved elicitation of monoclonal antibodies to TAA by the use of lectin immunosorbents and peripheral protein extracts has considerable potential for generating reagents useful in diagnosis and therapy of human tumors.

  11. Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals. (United States)

    Wang, Bo; Lee, Chang-Han; Johnson, Erik L; Kluwe, Christien A; Cunningham, Josephine C; Tanno, Hidetaka; Crooks, Richard M; Georgiou, George; Ellington, Andrew D


    Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.

  12. The intestinal flora is required to support antibody responses to systemic immunization in infant and germ free mice. (United States)

    Lamousé-Smith, Esi S; Tzeng, Alice; Starnbach, Michael N


    The presence of a complex and diverse intestinal flora is functionally important for regulating intestinal mucosal immune responses. However, the extent to which a balanced intestinal flora regulates systemic immune responses is still being defined. In order to specifically examine whether the acquisition of a less complex flora influences responses to immunization in the pre-weaning stages of life, we utilize a model in which infant mice acquire an intestinal flora from their mothers that has been altered by broad-spectrum antibiotics. In this model, pregnant dams are treated with a cocktail of antibiotics that alters both the density and microbial diversity of the intestinal flora. After challenge with a subcutaneous immunization, the antibiotic altered flora infant mice have lower antigen specific antibody titers compared to control age-matched mice. In a second model, we examined germ free (GF) mice to analyze how the complete lack of flora influences the ability to mount normal antibody responses following subcutaneous immunization. GF mice do not respond well to immunization and introduction of a normal flora into GF mice restores the capacity of these mice to respond. These results indicate that a gastrointestinal flora reduced in density and complexity at critical time points during development adversely impacts immune responses to systemic antigens.

  13. The intestinal flora is required to support antibody responses to systemic immunization in infant and germ free mice.

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    Esi S Lamousé-Smith

    Full Text Available The presence of a complex and diverse intestinal flora is functionally important for regulating intestinal mucosal immune responses. However, the extent to which a balanced intestinal flora regulates systemic immune responses is still being defined. In order to specifically examine whether the acquisition of a less complex flora influences responses to immunization in the pre-weaning stages of life, we utilize a model in which infant mice acquire an intestinal flora from their mothers that has been altered by broad-spectrum antibiotics. In this model, pregnant dams are treated with a cocktail of antibiotics that alters both the density and microbial diversity of the intestinal flora. After challenge with a subcutaneous immunization, the antibiotic altered flora infant mice have lower antigen specific antibody titers compared to control age-matched mice. In a second model, we examined germ free (GF mice to analyze how the complete lack of flora influences the ability to mount normal antibody responses following subcutaneous immunization. GF mice do not respond well to immunization and introduction of a normal flora into GF mice restores the capacity of these mice to respond. These results indicate that a gastrointestinal flora reduced in density and complexity at critical time points during development adversely impacts immune responses to systemic antigens.

  14. Breadth of humoral response and antigenic targets of sporozoite-inhibitory antibodies associated with sterile protection induced by controlled human malaria infection (United States)

    Peng, Kaitian; Goh, Yun Shan; Siau, Anthony; Franetich, Jean-François; Chia, Wan Ni; Ong, Alice Soh Meoy; Malleret, Benoit; Wu, Ying Ying; Snounou, Georges; Hermsen, Cornelus C.; Adams, John H.; Mazier, Dominique; Preiser, Peter R.; Sauerwein, Robert W.; Grüner, Anne-Charlotte; Rénia, Laurent


    The development of an effective malaria vaccine has remained elusive even until today. This is due to our incomplete understanding of the immune mechanisms that confer and/or correlate with protection. Human volunteers have been protected experimentally from a subsequent challenge by immunization with Plasmodium falciparum sporozoites under drug cover. Here, we demonstrate that sera from the protected individuals contain neutralizing antibodies against the pre erythrocytic stage. To identify the antigen(s) recognized by these antibodies, a newly developed library of P. falciparum antigens was screened with the neutralizing sera. Antibodies from protected individuals recognized a broad antigenic repertoire of which three antigens, PfMAEBL, PfTRAP and PfSEA1 were recognized by most protected individuals. As a proof of principle, we demonstrated that anti-PfMAEBL antibodies block liver stage development in human hepatocytes. Thus, these antigens identified are promising targets for vaccine development against malaria. PMID:27130708

  15. Antibody titers against vaccine and contemporary wild poliovirus type 1 in children immunized with IPV+OPV and young adults immunized with OPV. (United States)

    Lukashev, Alexander N; Yarmolskaya, Maria S; Shumilina, Elena Yu; Sychev, Daniil A; Kozlovskaya, Liubov I


    In 2010, a type 1 poliovirus outbreak in Congo with 445 lethal cases was caused by a virus that was neutralized by sera of German adults vaccinated with inactivated polio vaccine with a reduced efficiency. This seroprevalence study was done in two cohorts immunized with other vaccination schedules. Russian children aged 3-6 years immunized with a combination of inactivated and live polio vaccines were reasonably well protected against any wild type poliovirus 1, including the Congolese isolate. Adults aged 20-29 years immunized only with live vaccine were apparently protected against the vaccine strain (92% seropositive), but only 50% had detectable antibodies against the Congo-2010 isolate. Both waning immunity and serological divergence of the Congolese virus could contribute to this result.

  16. Antibody response to recombinant human coagulation factor VIII in a new rat model of severe hemophilia A. (United States)

    Lövgren, K M; Søndergaard, H; Skov, S; Weldingh, K N; Tranholm, M; Wiinberg, B


    Neutralizing antibodies toward FVIII replacement therapy (inhibitors) are the most serious treatment-related complication in hemophilia A (HA). A rat model of severe HA (F8(-/-) ) has recently been developed, but an immunological characterization is needed to determine the value of using the model for research into inhibitor development. Characterize the antibody response towards recombinant human coagulation factor VIII (rhFVIII) in the HA rat, following a human prophylactic dosing regimen. Two identical studies were performed, which included a total of 17 homozygous HA rats (F8(-/-) , 0% FVIII activity), 12 heterozygous rats (F8(+/-) ), and 12 wild-type (F8(+/+) ) rats. All rats received intravenous injections of rhFVIII at 50 IU kg(-1) twice weekly for 4 weeks. Predosing blood samples were analyzed for binding and neutralizing anti-rhFVIII antibodies at weeks 1-7. In both studies, antibodies developed after 4-6 administrations of rhFVIII, and neutralizing antibodies reached levels similar to human patients (range 1-111 BU, median 6.0 BU) at the end of the study. There was no significant difference between the two studies or between genotypes in time to response or levels reached for binding and neutralizing antibodies. Interestingly, early spontaneous bleeds were associated with a faster antibody response. Following intravenous administration of human FVIII, according to a clinical prophylaxis regimen, a robust and reproducible antibody response is seen in this HA rat model, suggesting that the model is useful for intervention studies with the aim of suppressing, delaying, or preventing the inhibitor response. Also, bleeds seem to have an adjuvant effect on the immune response. © 2016 International Society on Thrombosis and Haemostasis.

  17. Delineation of BmSXP antibody V-gene usage from a lymphatic filariasis based immune scFv antibody library. (United States)

    Rahumatullah, Anizah; Ahmad, Azimah; Noordin, Rahmah; Lim, Theam Soon


    Phage display technology is an important tool for antibody generation or selection. This study describes the development of a scFv library and the subsequent analysis of identified monoclonal antibodies against BmSXP, a recombinant antigen for lymphatic filariasis. The immune library was generated from blood of lymphatic filariasis infected individuals. A TA based intermediary cloning approach was used to increase cloning efficiency for the library construction process. A diverse immune scFv library of 10(8) was generated. Six unique monoclonal antibodies were identified from the 50 isolated clones against BmSXP. Analysis of the clones showed a bias for the IgHV3 and Vκ1 (45.5%) and IgHV2 and Vκ3 (27.3%) gene family. The most favored J segment for light chain is IgKJ1 (45.5%). The most favored D and J segment for heavy chain are IgHD6-13 (75%) and IgHJ3 (47.7%). The information may suggest a predisposition of certain V genes in antibody responses against lymphatic filariasis.

  18. Active immunizations with peptide-DC vaccines and passive transfer with antibodies protect neutropenic mice against disseminated candidiasis. (United States)

    Xin, Hong


    We previously report that peptide-pulsed dendritic cell (DC) vaccination, which targeting two peptides (Fba and Met6) expressed on the cell surface of Candida albicans, can induce high degree of protection against disseminated candidiasis in immunocompetent mice. Passive transfer of immune sera from the peptide immunized mice or peptide-related monoclonal antibodies demonstrated that protection was medicated by peptide-specific antibodies. In this study the efficacy of active and passive immunization against disseminated candidiasis was tested in mice with cyclophosphamide-induced neutropenia. Peptide-DC vaccines were given to mice prior to induction of neutropenia. We show active immunization with either Fba or Met6 peptide-DC vaccine significantly improved the survival and reduced the fungal burden of disseminated candidiasis in those immunocompromised mice. Importantly, we show that administration of two protective monoclonal antibodies also protect neutropenic mice against the disease, implying possibility of developing a successful passive immunotherapy strategy to treat the disease and protect against disseminated candidiasis. The results of this study are crucial as they address the fundamental questions as to whether the synthetic peptide vaccine induced immunity protects the host during a neutropenic episode. We anticipate that this peptide-vaccine study will serve as the foundation of future investigations into new peptide vaccines comprised of cell surface peptides from other medically important Candida species, as well as other fungi.

  19. Serum and mucosal antibody responses to inactivated polio vaccine after sublingual immunization using a thermoresponsive gel delivery system. (United States)

    White, Jessica A; Blum, Jeremy S; Hosken, Nancy A; Marshak, Joshua O; Duncan, Lauren; Zhu, Changcheng; Norton, Elizabeth B; Clements, John D; Koelle, David M; Chen, Dexiang; Weldon, William C; Oberste, M Steven; Lal, Manjari


    Administering vaccines directly to mucosal surfaces can induce both serum and mucosal immune responses. Mucosal responses may prevent establishment of initial infection at the port of entry and subsequent dissemination to other sites. The sublingual route is attractive for mucosal vaccination, but both a safe, potent adjuvant and a novel formulation are needed to achieve an adequate immune response. We report the use of a thermoresponsive gel (TRG) combined with a double mutant of a bacterial heat-labile toxin (dmLT) for sublingual immunization with a trivalent inactivated poliovirus vaccine (IPV) in mice. This TRG delivery system, which changes from aqueous solution to viscous gel upon contact with the mucosa at body temperature, helps to retain the formulation at the site of delivery and has functional adjuvant activity from the inclusion of dmLT. IPV was administered to mice either sublingually in the TRG delivery system or intramuscularly in phosphate-buffered saline. We measured poliovirus type-specific serum neutralizing antibodies as well as polio-specific serum Ig and IgA antibodies in serum, saliva, and fecal samples using enzyme-linked immunosorbent assays. Mice receiving sublingual vaccination via the TRG delivery system produced both mucosal and serum antibodies, including IgA. Intramuscularly immunized animals produced only serum neutralizing and binding Ig but no detectable IgA. This study provides proof of concept for sublingual immunization using the TRG delivery system, comprising a thermoresponsive gel and dmLT adjuvant.

  20. Mass Cytometry of the Human Mucosal Immune System Identifies Tissue- and Disease-Associated Immune Subsets. (United States)

    van Unen, Vincent; Li, Na; Molendijk, Ilse; Temurhan, Mine; Höllt, Thomas; van der Meulen-de Jong, Andrea E; Verspaget, Hein W; Mearin, M Luisa; Mulder, Chris J; van Bergen, Jeroen; Lelieveldt, Boudewijn P F; Koning, Frits


    Inflammatory intestinal diseases are characterized by abnormal immune responses and affect distinct locations of the gastrointestinal tract. Although the role of several immune subsets in driving intestinal pathology has been studied, a system-wide approach that simultaneously interrogates all major lineages on a single-cell basis is lacking. We used high-dimensional mass cytometry to generate a system-wide view of the human mucosal immune system in health and disease. We distinguished 142 immune subsets and through computational applications found distinct immune subsets in peripheral blood mononuclear cells and intestinal biopsies that distinguished patients from controls. In addition, mucosal lymphoid malignancies were readily detected as well as precursors from which these likely derived. These findings indicate that an integrated high-dimensional analysis of the entire immune system can identify immune subsets associated with the pathogenesis of complex intestinal disorders. This might have implications for diagnostic procedures, immune-monitoring, and treatment of intestinal diseases and mucosal malignancies.

  1. Could an experimental dengue virus infection fail to induce solid immunity against homologous viral challenge in non-human primates? (United States)

    Valdés, Iris; Gil, Lázaro; Lazo, Laura; Marcos, Ernesto; Martín, Jorge; Suzarte, Edith; Castro, Jorge; Romero, Yaremis; Guillén, Gerardo; Hermida, Lisset


    There are several dengue vaccine candidates at advanced stages of development, but none of them are licensed. Despite the reactogenicity and immunogenicity profile in humans of the tetravalent ChimeriVax™ dengue vaccine candidate, in efficacy trials, it has failed to confer complete protection against dengue virus (DENV)-1 and DENV-2. However, full protection against the four serotypes had been observed previously in monkeys immunized with this vaccine candidate. Some authors have tried to explain this contradiction by hypothesizing that protection rates in non-human primates (NHPs) are associated with a lack of post-challenge anamnestic immune responses. Here, we studied the protection and anamnestic response patterns after homologous challenge in NHPs previously infected with DENV-2. Two immunization schemes were used, varying the viral doses and the intervals between them. Animals developed immunity against DENV-2 that provided full protection against reinfection with a homologous virus. However, all monkeys showed a significant increase in antiviral and neutralizing antibody titers after challenge. Our results suggest that sterilizing immunity could not be induced by infection with the virus despite the lack of detectable viremia in some animals in which an increase in antibody titer was observed. For this reason, we propose that the lack of an anamnestic neutralizing antibody response after challenge, as suggested by some authors, should be carefully reviewed as a criterion for evaluating the functionality of vaccine candidates.

  2. Systemic and mucosal immune responses to sublingual or intramuscular human papilloma virus antigens in healthy female volunteers.

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    Zhiming Huo

    Full Text Available The sublingual route has been proposed as a needle-free option to induce systemic and mucosal immune protection against viral infections. In a translational study of systemic and mucosal humoral immune responses to sublingual or systemically administered viral antigens, eighteen healthy female volunteers aged 19-31 years received three immunizations with a quadravalent Human Papilloma Virus vaccine at 0, 4 and 16 weeks as sublingual drops (SL, n = 12 or intramuscular injection (IM, n = 6. IM antigen delivery induced or boosted HPV-specific serum IgG and pseudovirus-neutralizing antibodies, HPV-specific cervical and vaginal IgG, and elicited circulating IgG and IgA antibody secreting cells. SL antigens induced ~38-fold lower serum and ~2-fold lower cervical/vaginal IgG than IM delivery, and induced or boosted serum virus neutralizing antibody in only 3/12 subjects. Neither route reproducibly induced HPV-specific mucosal IgA. Alternative delivery systems and adjuvants will be required to enhance and evaluate immune responses following sublingual immunization in humans.ClinicalTrials.govNCT00949572.

  3. Identification of a human monoclonal antibody to replace equine diphtheria antitoxin for treatment of diphtheria intoxication. (United States)

    Sevigny, Leila M; Booth, Brian J; Rowley, Kirk J; Leav, Brett A; Cheslock, Peter S; Garrity, Kerry A; Sloan, Susan E; Thomas, William; Babcock, Gregory J; Wang, Yang


    Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 μg of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic.

  4. Human anti-rhinosporidial antibody does not cause metabolic inactivation or morphological damage in endospores of Rhinosporidium seeberi, in vitro

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    Arseculeratne S


    Full Text Available This report describes the use of the MTT-reduction and Evan′s blue-staining tests for the assessment of the viability and morphological integrity, respectively, of rhinosporidial endospores after exposure to sera from rhinosporidial patients with high titres of anti-rhinosporidial antibody. Sera from three patients, with nasal, ocular and disseminated rhinosporidiosis respectively were used, with human serum without anti-rhinosporidial antibody for comparison, with or without added fresh guinea pig serum as a source of complement. All four sera tested, with or without guinea-pig serum, had no effect on the morphological integrity or the viability of the endospores and it is suggested that anti-rhinosporidial antibody has no direct protective role against the endospores, the infective stage, in rhinosporidiosis. This finding is compatible with the occurrence of chronicity, recurrence and dissemination that are characteristic of rhinosporidiosis despite the presence of high titres of anti-rhinosporidial antibody in patients with these clinical characteristics. The possible occurrence of humoral mechanisms of immunity that involve anti-rhinosporidial antibody with cells such as leucocytes and NK cells, in vivo, cannot yet be discounted, although the presence of high titres of anti-rhinosporidial antibody in patients with chronic, recurrent and disseminated lesions might indicate that such antibody is non-protective in vivo.

  5. A variety of human monoclonal antibodies against epidermal growth factor receptor isolated from a phage antibody library. (United States)

    Kurosawa, Gene; Kondo, Mariko; Kurosawa, Yoshikazu


    When the technology for constructing human antibody (Ab) libraries using a phage-display system was developed, many researchers in Ab-related fields anticipated that it would be widely applied to the development of pharmaceutical drugs against various diseases, including cancers. However, successful examples of such applications are very limited. Moreover, researchers who utilize phage-display technology now show divergent ways of thinking about phage Ab libraries. For example, there is debate about what should be the source of VH and VL genes for the construction of libraries to cover the whole repertoire of Abs present in the human body. In the immune system, the introduction of mutations into V genes followed by selection based on binding activity, termed Ab maturation, is required for the production of Abs exhibiting high affinity to the antigen (Ag). Therefore, introduction of mutations and selection are required for isolation of Abs with high affinity after isolation of clones from phage Ab libraries. We constructed a large human Ab library termed AIMS, developed a screening method termed ICOS, and succeeded in isolating many human monoclonal Abs (mAbs) that specifically and strongly bind to various tumor-associated Ags. Eight anti-EGFR mAbs were included, which we characterized. These mAbs showed various different activities against EGFR-expressing cancer cells. In this paper, we describe these data and discuss the possibility and necessity that the mAbs isolated from the AIMS library might be developed as therapeutic drugs against cancers without introduction of mutations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Human immunodeficiency virus antibodies and the vaccine problem. (United States)

    Chiodi, F; Weiss, R A


    Despite the great advances made in controlling human immunodeficiency virus type 1 (HIV-1) infection with antiretroviral drug treatment, a safe and efficacious HIV vaccine has yet to be developed. Here, we discuss why clinical trials and vaccine development for HIV have so far been disappointing, with an emphasis on the lack of protective antibodies. We review approaches for developing appropriate HIV immunogens and the stimulation of long-lasting B-cell responses with antibody maturation. We conclude that candidate reagents in the pipeline for HIV vaccine development are unlikely to be particularly effective. Although the major funders of HIV vaccine research and development are placing increasing emphasis on clinical product development, a genuine breakthrough in preventing HIV infection through vaccines is more likely to come from novel immunogen research.

  7. DNA vaccine molecular adjuvants SP-D-BAFF and SP-D-APRIL enhance anti-gp120 immune response and increase HIV-1 neutralizing antibody titers. (United States)

    Gupta, Sachin; Clark, Emily S; Termini, James M; Boucher, Justin; Kanagavelu, Saravana; LeBranche, Celia C; Abraham, Sakhi; Montefiori, David C; Khan, Wasif N; Stone, Geoffrey W


    the gp120 trimer, the inaccessibility of the conserved sequences, highly variable protein sequences, and the loss of HIV-1-specific antibody-producing cells during development. We have shown previously that tumor necrosis factor (TNF) superfamily ligands, including BAFF and APRIL, can be multitrimerized using the lung protein SP-D (surfactant protein D), enhancing immune responses. Here we show that DNA or DNA-protein vaccines encoding BAFF or APRIL multitrimers, IL-12p70, and membrane-bound HIV-1 Env gp140 induced tier 1 and tier 2 neutralizing antibodies in a mouse model. BAFF and APRIL enhanced the immune reaction, improved antibody binding, and increased the numbers of anti-HIV-1 antibody-secreting cells. Adaptation of this vaccine design may prove useful in designing preventive HIV-1 vaccines for humans. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. Biological effects and use of PrPSc- and PrP-specific antibodies generated by immunization with purified full-length native mouse prions. (United States)

    Petsch, Benjamin; Müller-Schiffmann, Andreas; Lehle, Anna; Zirdum, Elizabeta; Prikulis, Ingrid; Kuhn, Franziska; Raeber, Alex J; Ironside, James W; Korth, Carsten; Stitz, Lothar


    The prion agent is the infectious particle causing spongiform encephalopathies in animals and humans and is thought to consist of an altered conformation (PrP(Sc)) of the normal and ubiquitous prion protein PrP(C). The interaction of the prion agent with the immune system, particularly the humoral immune response, has remained unresolved. Here we investigated the immunogenicity of full-length native and infectious prions, as well as the specific biological effects of the resulting monoclonal antibodies (MAbs) on the binding and clearance of prions in cell culture and in in vivo therapy. Immunization of prion knockout (Prnp(0/0)) mice with phosphotungstic acid-purified mouse prions resulted in PrP-specific monoclonal antibodies with binding specificities selective for PrP(Sc) or for both PrP(C) and PrP(Sc). PrP(Sc)-specific MAb W261, of the IgG1 isotype, reacted with prions from mice, sheep with scrapie, deer with chronic wasting disease (CWD), and humans with sporadic and variant Creutzfeldt-Jakob disease (CJD) in assays including a capture enzyme-linked immunosorbent assay (ELISA) system. This PrP(Sc)-specific antibody was unable to clear prions from mouse neuroblastoma cells (ScN2a) permanently infected with scrapie, whereas the high-affinity MAb W226, recognizing both isoforms, PrP(Sc) and PrP(C), did clear prions from ScN2a cells, as determined by a bioassay. However, an attempt to treat intraperitoneally prion infected mice with full-length W226 or with a recombinant variable-chain fragment (scFv) from W226 could only slightly delay the incubation time. We conclude that (i) native, full-length PrP(Sc) elicits a prion-specific antibody response in PrP knockout mice, (ii) a PrP(Sc)-specific antibody had no prion-clearing effect, and (iii) even a high-affinity MAb that clears prions in vitro (W226) may not necessarily protect against prion infection, contrary to previous reports using different antibodies.

  9. Serum or breast milk immunoglobulins mask the self-reactivity of human natural IgG antibodies. (United States)

    Djoumerska-Alexieva, Iglika; Manoylov, Iliyan; Dimitrov, Jordan D; Tchorbanov, Andrey


    B cells producing IgG antibodies specific to a variety of self- or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein-destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab')2 fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self-reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients.

  10. Humans Have Antibodies against a Plant Virus: Evidence from Tobacco Mosaic Virus (United States)

    Liu, Ruolan; Vaishnav, Radhika A.; Roberts, Andrew M.; Friedland, Robert P.


    Tobacco mosaic virus (TMV), a widespread plant pathogen, is found in tobacco (including cigarettes and smokeless tobacco) as well as in many other plants. Plant viruses do not replicate or cause infection in humans or other mammals. This study was done to determine whether exposure to tobacco products induces an immune response to TMV in humans. Using a sandwich ELISA assay, we detected serum anti-TMV antibodies (IgG, IgG1, IgG3, IgG4, IgA, and IgM) in all subjects enrolled in the study (20 healthy smokers, 20 smokeless-tobacco users, and 20 non-smokers). Smokers had a higher level of serum anti-TMV IgG antibodies than non-smokers, while the serum level of anti-TMV IgA from smokeless tobacco users was lower than smokers and non-smokers. Using bioinformatics, we also found that the human protein TOMM40L (an outer mitochondrial membrane 40 homolog – like translocase) contains a strong homology of six contiguous amino acids to the TMV coat protein, and TOMM40L peptide exhibited cross-reactivity with anti-TMV antibodies. People who smoke cigarettes or other tobacco products experience a lower risk of developing Parkinson’s disease, but the mechanism by which this occurs is unclear. Our results showing molecular mimicry between TMV and human TOMM40L raise the question as to whether TMV has a potential role in smokers against Parkinson’s disease development. The potential mechanisms of molecular mimicry between plant viruses and human disease should be further explored. PMID:23573274

  11. Correlated effects of selection for immunity in White Leghorn chicken lines on natural antibodies and specific antibody responses to KLH and M. butyricum

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    Gourichon David


    Full Text Available Abstract Background The effect of selection for three general immune response traits on primary antibody responses (Ab to Mycobacterium butyricum or keyhole limpet hemocyanin (KLH was studied in four experimental lines of White Leghorn chicken. Birds underwent 12 generations of selection for one of three different general immune criteria; high antibody response to Newcastle disease virus 3 weeks after vaccination (ND3, high cell-mediated immune response, using the wing web response to phytohemglutinin (PHA and high phagocytic activity, measured as carbone clearance (CC. Line ND3-L was selected on ND3, line PHA-L was selected on PHA, and line CC-L on CC, but all lines were measured for all three traits. The fourth line was a contemporary random bred control maintained throughout the selection experiment. Principal component analysis was used to distinguish clusters based on the overall set of immune measures. Results In the KLH immunised group, no differences were present between lines for natural antibodies binding to KLH and LPS, and, lines ND3-L and PHA-L had higher titers to LTA and anti-Gal titers measured before the immunisation protocol. The measure of ND3 was correlated positively with LPS titers measured post KLH immunisation and with the difference between LPS titers measured at day 0 and 7 post immunisation. In the M. butyricum immunised group, Line ND3-L showed significantly higher specific antibody response to M. butyricum, and this result agrees well with the hypothesis that the Th-1 pathway was expected to be selected for in this line. Conclusion This study has shown that the two different antigens KLH and M. butyricum gave rise to different responses in the set of selected lines, and that the response was only enhanced for the antigen associated with the same response mechanism as that for the trait (ND3, PHA or CC for which the line was selected. Interactions between innate and acquired immunity have been observed mainly for the

  12. Production of monoclonal antibodies to human glomerular basement membrane.

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    Full Text Available Using the technique of somatic cell fusion, we produced monoclonal antibodies to collagenase-digested human glomerular basement membrane (GBM. Fourteen monoclonal antibodies which reacted with normal human kidney in indirect immunofluorescence (IIF studies were produced. An analysis of the binding patterns indicated that the antigens recognized could be divided into six broad groups. Monoclonal antibody B3-H10 (Group 1 reacted with only GBM in a fine granular pattern. A5-B12 and B5-C2 (Group 2 reacted with GBM and peritubular capillary in a linear pattern. B2-A12 (Group 3 reacted with only epithelial cells. Al-C9 and A4-E2 (Group 4 showed a mesangial pattern in glomerulus and a lineal pattern in tubular basement membrane (TBM, Bowman's capsule and peritubular capillary. A1-E1, A1-E11, A2-E6, A3-B6, A4-F8 and B5-H2 (Group 5 recognized determinants common to GBM, TBM, Bowman's capsule and/or peritubular capillary. A3-F1 and B5-E10 (Group 6 reacted with TBM and Bowman's capsule. The staining pattern of B3-H10 (Group 1 was characteristic because it was not linear, but finely granular along the GBM. The staining pattern of B2-A12 (Group 3 was also characteristic because only epithelial cells were stained, and processes of epithelial cells were observed as fine fibrils. To the best of our knowledge, these two types of monoclonal antibodies have not been reported previously.

  13. Anthrax lethal factor as an immune target in humans and transgenic mice and the impact of HLA polymorphism on CD4+ T cell immunity. (United States)

    Ascough, Stephanie; Ingram, Rebecca J; Chu, Karen K; Reynolds, Catherine J; Musson, Julie A; Doganay, Mehmet; Metan, Gökhan; Ozkul, Yusuf; Baillie, Les; Sriskandan, Shiranee; Moore, Stephen J; Gallagher, Theresa B; Dyson, Hugh; Williamson, E Diane; Robinson, John H; Maillere, Bernard; Boyton, Rosemary J; Altmann, Daniel M


    Bacillus anthracis produces a binary toxin composed of protective antigen (PA) and one of two subunits, lethal factor (LF) or edema factor (EF). Most studies have concentrated on induction of toxin-specific antibodies as the correlate of protective immunity, in contrast to which understanding of cellular immunity to these toxins and its impact on infection is limited. We characterized CD4+ T cell immunity to LF in a panel of humanized HLA-DR and DQ transgenic mice and in naturally exposed patients. As the variation in antigen presentation governed by HLA polymorphism has a major impact on protective immunity to specific epitopes, we examined relative binding affinities of LF peptides to purified HLA class II molecules, identifying those regions likely to be of broad applicability to human immune studies through their ability to bind multiple alleles. Transgenics differing only in their expression of human HLA class II alleles showed a marked hierarchy of immunity to LF. Immunogenicity in HLA transgenics was primarily restricted to epitopes from domains II and IV of LF and promiscuous, dominant epitopes, common to all HLA types, were identified in domain II. The relevance of this model was further demonstrated by the fact that a number of the immunodominant epitopes identified in mice were recognized by T cells from humans previously infected with cutaneous anthrax and from vaccinated individuals. The ability of the identified epitopes to confer protective immunity was demonstrated by lethal anthrax challenge of HLA transgenic mice immunized with a peptide subunit vaccine comprising the immunodominant epitopes that we identified.

  14. Anthrax lethal factor as an immune target in humans and transgenic mice and the impact of HLA polymorphism on CD4+ T cell immunity.

    Directory of Open Access Journals (Sweden)

    Stephanie Ascough


    Full Text Available Bacillus anthracis produces a binary toxin composed of protective antigen (PA and one of two subunits, lethal factor (LF or edema factor (EF. Most studies have concentrated on induction of toxin-specific antibodies as the correlate of protective immunity, in contrast to which understanding of cellular immunity to these toxins and its impact on infection is limited. We characterized CD4+ T cell immunity to LF in a panel of humanized HLA-DR and DQ transgenic mice and in naturally exposed patients. As the variation in antigen presentation governed by HLA polymorphism has a major impact on protective immunity to specific epitopes, we examined relative binding affinities of LF peptides to purified HLA class II molecules, identifying those regions likely to be of broad applicability to human immune studies through their ability to bind multiple alleles. Transgenics differing only in their expression of human HLA class II alleles showed a marked hierarchy of immunity to LF. Immunogenicity in HLA transgenics was primarily restricted to epitopes from domains II and IV of LF and promiscuous, dominant epitopes, common to all HLA types, were identified in domain II. The relevance of this model was further demonstrated by the fact that a number of the immunodominant epitopes identified in mice were recognized by T cells from humans previously infected with cutaneous anthrax and from vaccinated individuals. The ability of the identified epitopes to confer protective immunity was demonstrated by lethal anthrax challenge of HLA transgenic mice immunized with a peptide subunit vaccine comprising the immunodominant epitopes that we identified.

  15. Anthrax Lethal Factor as an Immune Target in Humans and Transgenic Mice and the Impact of HLA Polymorphism on CD4+ T Cell Immunity (United States)

    Ascough, Stephanie; Ingram, Rebecca J.; Chu, Karen K.; Reynolds, Catherine J.; Musson, Julie A.; Doganay, Mehmet; Metan, Gökhan; Ozkul, Yusuf; Baillie, Les; Sriskandan, Shiranee; Moore, Stephen J.; Gallagher, Theresa B.; Dyson, Hugh; Williamson, E. Diane; Robinson, John H.; Maillere, Bernard; Boyton, Rosemary J.; Altmann, Daniel M.


    Bacillus anthracis produces a binary toxin composed of protective antigen (PA) and one of two subunits, lethal factor (LF) or edema factor (EF). Most studies have concentrated on induction of toxin-specific antibodies as the correlate of protective immunity, in contrast to which understanding of cellular immunity to these toxins and its impact on infection is limited. We characterized CD4+ T cell immunity to LF in a panel of humanized HLA-DR and DQ transgenic mice and in naturally exposed patients. As the variation in antigen presentation governed by HLA polymorphism has a major impact on protective immunity to specific epitopes, we examined relative binding affinities of LF peptides to purified HLA class II molecules, identifying those regions likely to be of broad applicability to human immune studies through their ability to bind multiple alleles. Transgenics differing only in their expression of human HLA class II alleles showed a marked hierarchy of immunity to LF. Immunogenicity in HLA transgenics was primarily restricted to epitopes from domains II and IV of LF and promiscuous, dominant epitopes, common to all HLA types, were identified in domain II. The relevance of this model was further demonstrated by the fact that a number of the immunodominant epitopes identified in mice were recognized by T cells from humans previously infected with cutaneous anthrax and from vaccinated individuals. The ability of the identified epitopes to confer protective immunity was demonstrated by lethal anthrax challenge of HLA transgenic mice immunized with a peptide subunit vaccine comprising the immunodominant epitopes that we identified. PMID:24788397

  16. Large scale immune profiling of infected humans and goats reveals differential recognition of Brucella melitensis antigens. (United States)

    Liang, Li; Leng, Diana; Burk, Chad; Nakajima-Sasaki, Rie; Kayala, Matthew A; Atluri, Vidya L; Pablo, Jozelyn; Unal, Berkay; Ficht, Thomas A; Gotuzzo, Eduardo; Saito, Mayuko; Morrow, W John W; Liang, Xiaowu; Baldi, Pierre; Gilman, Robert H; Vinetz, Joseph M; Tsolis, Renée M; Felgner, Philip L


    Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host.

  17. Pathogens and host immunity in the ancient human oral cavity

    DEFF Research Database (Denmark)

    Warinner, Christina; Rodrigues, João F Matias; Vyas, Rounak


    cavity has long served as a reservoir for bacteria implicated in both local and systemic disease. We characterize (i) the ancient oral microbiome in a diseased state, (ii) 40 opportunistic pathogens, (iii) ancient human-associated putative antibiotic resistance genes, (iv) a genome reconstruction...... of the periodontal pathogen Tannerella forsythia, (v) 239 bacterial and 43 human proteins, allowing confirmation of a long-term association between host immune factors, 'red complex' pathogens and periodontal disease, and (vi) DNA sequences matching dietary sources. Directly datable and nearly ubiquitous, dental...... calculus permits the simultaneous investigation of pathogen activity, host immunity and diet, thereby extending direct investigation of common diseases into the human evolutionary past....

  18. Hepatitis A and B immunizations of individuals infected with human immunodeficiency virus. (United States)

    Laurence, Jeffrey C


    All persons at risk for infection with human immunodeficiency virus (HIV) types 1 and 2, including men who have sex with men, those with multiple heterosexual contacts, abusers of illegal injection drugs, and persons frequently exposed to blood and blood products, are also at high risk for hepatitis A virus (HAV) and acute and chronic hepatitis B virus (HBV) infections. HIV can prolong the duration and increase the level of HAV viremia and augment HAV-related liver abnormalities. HIV also magnifies HBV viremia and the risk of HBV reactivation, chronic active HBV infection, cirrhosis, and death. Because of these concerns, hepatitis A vaccination is recommended for all HIV-positive/HAV seronegative persons, with 2 standard doses given 6 to 12 months apart. Immune response to hepatitis A vaccines is excellent, even in moderately immune-suppressed individuals. Hepatitis B vaccination is also recommended for all HIV-positive persons lacking prior immunity. However, immune reactivity to hepatitis B vaccines is frequently suboptimal in terms of patients' rate of response, antibody titer, and durability. Relatively high CD4+ T-cell counts (> or =500/mm3) and low levels of HIV viremia (hepatitis B vaccine response. Higher hepatitis B vaccine doses, prolongation of the vaccination schedule, or both, as prescribed for many patients with non-HIV-related immune deficiencies, may be considered initially. Revaccination should be instituted if postvaccination titers of antibodies to hepatitis B surface antigen are <10 mIU/mL (<10 IU/L). Nonresponders may also react to a subsequent vaccine course if CD4+ T-cell counts rise to 500/mm3 following institution of highly active antiretroviral therapy; vaccine adjuvant trials are under way. Universal, age-based immunization of all young and middle-aged adults appears to be the most comprehensive way of protecting all populations who are at high risk.

  19. 21 CFR 640.102 - Manufacture of Immune Globulin (Human). (United States)


    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Manufacture of Immune Globulin (Human). 640.102 Section 640.102 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... microorganisms. Preservatives to inhibit growth of microorganisms shall not be used during processing. (c)...

  20. The human spleen. Development and role in the immune system.

    NARCIS (Netherlands)

    Timens, Willem


    In the present thesis an extensive in situ characterization is given of cellular constituents of the human spleen, that play a role in the human immune system. The development of immunocompetent cells in their micro-environment was studied in early embryonic life, fetal life, infancy and childhood,

  1. Senescence of T Lymphocytes: Implications for Enhancing Human Immunity. (United States)

    Akbar, Arne N; Henson, Sian M; Lanna, Alessio


    As humans live longer, a central concern is to find ways to maintain their health as they age. Immunity declines during ageing, as shown by the increased susceptibility to infection by both previously encountered and new pathogens and by the decreased efficacy of vaccination. It is therefore crucial to understand the mechanisms responsible for this decrease in immunity and to develop new strategies to enhance immune function in older humans. We discuss here how the induction of senescence alters leukocyte, and specifically T cell, function. An emerging concept is that senescence and nutrient sensing-signalling pathways within T cells converge to regulate functional responses, and the manipulation of these pathways may offer new ways to enhance immunity during ageing. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Antibodies are not required to a protective immune response against dengue virus elicited in a mouse encephalitis model. (United States)

    Amorim, Jaime Henrique; dos Santos Alves, Rúbens Prince; Bizerra, Raíza; Araújo Pereira, Sara; Ramos Pereira, Lennon; Nascimento Fabris, Denicar Lina; Santos, Robert Andreata; Romano, Camila Malta; de Souza Ferreira, Luís Carlos


    Generating neutralizing antibodies have been considered a prerequisite to control dengue virus (DENV) infection. However, T lymphocytes have also been shown to be important in a protective immune state. In order to investigate the contribution of both humoral and cellular immune responses in DENV immunity, we used an experimental model in which a non-lethal DENV2 strain (ACS46) is used to intracranially prime Balb/C mice which develop protective immunity against a lethal DENV2 strain (JHA1). Primed mice generated envelope-specific antibodies and CD8(+) T cell responses targeting mainly non-structural proteins. Immune sera from protected mice did not confer passive protection to naïve mice challenged with the JHA1 strain. In contrast, depletion of CD4(+) and CD8(+) T lymphocytes significantly reduced survival of ACS46-primed mice challenged with the JHA1 strain. Collectively, results presented in this study show that a cellular immune response targeting non-structural proteins are a promising way in vaccine development against dengue.

  3. The modulatory influence of some Echinacea-based remedies on antibody production and cellular immunity in mice (United States)

    Sokolnicka, Irena; Skopińska-Różewska, Ewa; Skopiński, Piotr


    Echinacea purpurea-containing remedies are herbal medicines used in respiratory tract infections and several inflammatory conditions as enhancers of non-specific and modulators of specific cellular immunity. They also exert anti-inflammatory, anti-viral, and anti-microbial activity. The aim of the present study was to compare the in vivo influence of orally administered three Echinacea purpurea-based remedies (IMMUNAL drops, ECHINACEA FORTE drops, IMMUNAL FORTE tablets) on some parameters of cellular and humoral immunity in mice. Results Feeding mice for seven days with IMMUNAL drops resulted in enhanced anti-SRBC antibody production and modulatory effect on proliferative response to PHA of their splenic lymphocytes. No stimulatory effect was observed on splenocytes chemokinesis. Mice fed with ECHINACEA FORTE drops presented enhanced response to PHA of their splenocytes. However, contrary to the previous group, no enhancement of antibody production was observed. In this group, lymphocyte-induced immunological angiogenesis (LIA) and chemokinesis (spontaneous migration – SM) of spleen lymphocytes was diminished after feeding mice with both doses (LIA) or with a higher dose (SM) of remedy. Lymphocyte-induced immunological angiogenesis activity of splenocytes collected from animals fed with prophylactic and therapeutic IMMUNAL FORTE tablet doses did not differ from the controls. PMID:27095917

  4. Autoimmune dermatologic toxicities from immune checkpoint blockade with anti-PD-1 antibody therapy: a report on bullous skin eruptions. (United States)

    Jour, George; Glitza, Isabella C; Ellis, Rachel M; Torres-Cabala, Carlos A; Tetzlaff, Michael T; Li, Janet Y; Nagarajan, Priyadharsini; Huen, Auris; Aung, Phyu P; Ivan, Doina; Drucker, Carol R; Prieto, Victor G; Rapini, Ronald P; Patel, Anisha; Curry, Jonathan L


    Monoclonal antibodies against the immune checkpoint programmed cell death receptor 1 (PD-1) improve the hosts' antitumor immune response and have showed tremendous promise in the treatment of advanced solid tumors and hematologic malignancies. Reports of serious autoimmune dermatologic toxicities from immune checkpoint blockade therapy, however, are emerging. We report our experience with five patients who presented with pruritic vesicles and blisters on the skin while treated with anti-PD-1 antibody immunotherapy with either nivolumab or pembrolizumab. Four of the patients' skin biopsies revealed subepidermal bullae with immunohistochemical study for type IV collagen labeling the floor of the blister cavity and direct immunofluorescence studies (in three of the four patients tested) decorated linear IgG and C3 immune deposits on the blister roof, diagnostic of bullous pemphigoid. One patient developed bullous erythema multiforme. All patients had partial or complete resolution of skin lesions following treatment with systemic corticosteroid and cessation of checkpoint blockade. Recognition and treatment of rare immune-related bullous dermatologic toxicities will become increasingly important as more patients are treated with effective and newer immune checkpoint blockade therapy. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. HAHA--nothing to laugh about. Measuring the immunogenicity (human anti-human antibody response) induced by humanized monoclonal antibodies applying ELISA and SPR technology. (United States)

    Nechansky, Andreas


    Immunogenicity induced by passively applied proteins is a serious issue because it is directly related to the patient's safety. The out-come of an immune reaction to a therapeutic protein can range from transient appearance of antibodies without any clinical significance to severe life threatening conditions. Within this article, enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) methodology to measure immunogenicity are compared and the pros and cons are discussed.

  6. Differences in the composition of the human antibody repertoire by B cell subsets in the blood

    Directory of Open Access Journals (Sweden)

    Eva Szymanska eMroczek


    Full Text Available The vast initial diversity of the antibody repertoire is generated centrally by means of a complex series of V (D J gene rearrangement events, variation in the site of gene segment joining, and TdT catalyzed N- region addition. Although the diversity is great, close inspection has revealed distinct and unique characteristics in the antibody repertoires expressed by different B cell developmental subsets. In order to illustrate our approach to repertoire analysis, we present an in-depth comparison of V (D J gene usage, hydrophobicity, length, DH reading frame, and amino acid usage between heavy chain repertoires expressed by immature, transitional, mature, memory IgD+, memory IgD-, and plasmacytes isolated from the blood of a single individual. Our results support the view that in both human and mouse the H chain repertoires expressed by individual, developmental B cell subsets appear to differ in sequence content. Sequencing of unsorted B cells from the blood is thus likely to yield an incomplete or compressed view of what is actually happening in the immune response of the individual. Our findings support the view that studies designed to correlate repertoire expression with diseases of immune function will likely require deep sequencing of B cells sorted by subset.

  7. Human intestinal dendritic cells as controllers of mucosal immunity

    Directory of Open Access Journals (Sweden)

    David Bernardo


    Full Text Available Dendritic cells are the most potent, professional antigen-presenting cells in the body; following antigen presentation they control the type (proinflammatory/regulatory of immune response that will take place, as well as its location. Given their high plasticity and maturation ability in response to local danger signals derived from innate immunity, dendritic cells are key actors in the connection between innate immunity and adaptive immunity responses. In the gut dendritic cells control immune tolerance mechanisms against food and/or commensal flora antigens, and are also capable of initiating an active immune response in the presence of invading pathogens. Dendritic cells are thus highly efficient in controlling the delicate balance between tolerance and immunity in an environment so rich in antigens as the gut, and any factor involving these cells may impact their function, ultimately leading to the development of bowel conditions such as celiac disease or inflammatory bowel disease. In this review we shall summarize our understanding of human intestinal dendritic cells, their ability to express and induce migration markers, the various environmental factors modulating their properties, their subsets in the gut, and the problems entailed by their study, including identification strategies, differences between humans and murine models, and phenotypical variations along the gastrointestinal tract.

  8. Evasion of the human innate immune system by dengue virus


    Pagni, Sarah; Fernandez-Sesma, Ana


    Dengue virus is a worldwide health problem, with billions of people at risk annually. Dengue virus causes a spectrum of diseases, namely dengue fever, dengue hemorrhagic fever and dengue shock syndrome with the latter two being linked to death. Understanding how dengue is able to evade the immune system and cause enhanced severity of disease is the main topics of interest in the Fernandez-Sesma laboratory at Mount Sinai School of Medicine. Using primary human immune cells, our group investiga...

  9. [Towards an industrial control of the cloning of lymphocytes B human for the manufacturing of monoclonal antibodies stemming from the human repertoire]. (United States)

    Guillot-Chene, P; Lebecque, S; Rigal, D


    Monoclonal antibodies (mAbs) are efficient drugs for treating infectious, inflammatory and cancer diseases. Antibodies secreted by human lymphocytes that have been isolated from either peripheral blood or tissues present the definite interest of being part of the physiological or disease-related response to antigens present in the human body. However, attempts to generate hybridomas with human B cells have been largely unsuccessful, and cloning of human B cells has been achieved only via their inefficient immortalization with Epstein Barr Virus (EBV). However, recent progress in our understanding of the molecular mechanisms of polyclonal B cell activation has dramatically increased the capacity to clone human B cells. In particular, activation of human naïve and memory B cells through CD40 or memory B cells only through TLR9 was shown to greatly facilitate their immortalization by EBV. Industrial development based on these observations will soon provide large collections of high affinity human mAbs of every isotype directly selected by the human immune system directed to recognize epitopes relevant for individual patients. Moreover, after CD40 activation, these mAbs will cover the full human repertoire, including the natural auto-immune repertoire. Full characterization of the biological activity of these mAbs will in turn bring useful information for selecting vaccine epitopes. This breakthrough in human B cell cloning opens the way into new areas for therapeutic use of mAbs.

  10. Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals

    DEFF Research Database (Denmark)

    Aasted, Bent; Blixenkrone-Møller, Merete; Larsen, Else Bang


    Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four...... antibodies bound to lymphocytes from some of the animals. These were the antibodies against CD8 and CD4 antigen, the antibody to C3b-receptor, and the antibody to the HLA-DR antigen. The CD8 antigen-reactive antibody reacted with lymphocytes from mink, cat, dog, and sheep, while the CD4 antigen...

  11. Expression cloning and production of human heavy-chain-only antibodies from murine transgenic plasma cells

    NARCIS (Netherlands)

    D.D. Drabek (Dubravka); R. Janssens (Rick); Boer, E. (Ernie de); Rademaker, R. (Rik); Kloess, J. (Johannes); J.J. Skehel (John ); Grosveld, F. (Frank)


    textabstractSeveral technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and

  12. Complex Adaptive Immunity to enteric fevers in humans: Lessons learned and the path forward

    Directory of Open Access Journals (Sweden)

    Marcelo B. Sztein


    Full Text Available Salmonella enterica serovar Typhi (S. Typhi, the causative agent of typhoid fever, and S. Paratyphi A and B, causative agents of paratyphoid fever, are major public health threats throughout the world. Although two licensed typhoid vaccines are currently available, they are only moderately protective and immunogenic necessitating the development of novel vaccines. A major obstacle in the development of improved typhoid, as well as paratyphoid vaccines is the lack of known immunological correlates of protection in humans. Considerable progress has been made in recent years in understanding the complex adaptive host responses against S. Typhi. Although the induction of S. Typhi-specific antibodies (including their functional properties and memory B cells, as well as their cross-reactivity with S. Paratyphi A and S. Paratyphi B has been shown, the role of humoral immunity in protection remains undefined. Cell mediated immunity (CMI is likely to play a dominant role in protection against enteric fever pathogens. Detailed measurements of CMI performed in volunteers immunized with attenuated strains of S. Typhi have shown, among others, the induction of lymphoproliferation, multifunctional type 1 cytokine production and CD8+ cytotoxic T cell responses. In addition to systemic responses, the local microenvironment of the gut is likely to be of paramount importance in protection from these infections. In this review we will critically assess current knowledge regarding the role of CMI and humoral immunity following natural S. Typhi and S. Paratyphi infections, experimental challenge, and immunization in humans. We will also address recent advances regarding cross-talk between the host’s gut microbiota and immunization with attenuated S. Typhi, mechanisms of systemic immune responses, and the homing potential of S. Typhi-specific B and T cells to the gut and other tissues.

  13. [Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment]. (United States)

    Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I


    Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

  14. Phage display-derived human antibodies in clinical development and therapy. (United States)

    Frenzel, André; Schirrmann, Thomas; Hust, Michael


    Over the last 3 decades, monoclonal antibodies have become the most important class of therapeutic biologicals on the market. Development of therapeutic antibodies was accelerated by recombinant DNA technologies, which allowed the humanization of murine monoclonal antibodies to make them more similar to those of the human body and suitable for a broad range of chronic diseases like cancer and autoimmune diseases. In the early 1990s in vitro antibody selection technologies were developed that enabled the discovery of "fully" human antibodies with potentially superior clinical efficacy and lowest immunogenicity. Antibody phage display is the first and most widely used of the in vitro selection technologies. It has proven to be a robust, versatile platform technology for the discovery of human antibodies and a powerful engineering tool to improve antibody properties. As of the beginning of 2016, 6 human antibodies discovered or further developed by phage display were approved for therapy. In 2002, adalimumab (Humira®) became the first phage display-derived antibody granted a marketing approval. Humira® was also the first approved human antibody, and it is currently the best-selling antibody drug on the market. Numerous phage display-derived antibodies are currently under advanced clinical investigation, and, despite the availability of other technologies such as human antibody-producing transgenic mice, phage display has not lost its importance for the discovery and engineering of therapeutic antibodies. Here, we provide a comprehensive overview about phage display-derived antibodies that are approved for therapy or in clinical development. A selection of these antibodies is described in more detail to demonstrate different aspects of the phage display technology and its development over the last 25 years.

  15. Recombinant human C1-inhibitor prevents acute antibody-mediated rejection in alloimmunized baboons

    NARCIS (Netherlands)

    Tillou, Xavier; Poirier, Nicolas; Le Bas-Bernardet, Stephanie; Hervouet, Jeremy; Minault, David; Renaudin, Karine; Vistoli, Fabio; Karam, Georges; Daha, Mohamed; Soulillou, Jean Paul; Blancho, Gilles


    Acute antibody-mediated rejection is an unsolved issue in transplantation, especially in the context of pretransplant immunization. The deleterious effect of preformed cytotoxic anti-HLA antibodies through complement activation is well proven, but very little is known concerning complement blockade

  16. Antibody quality and protection from lethal Ebola virus challenge in nonhuman primates immunized with rabies virus based bivalent vaccine.

    Directory of Open Access Journals (Sweden)

    Joseph E Blaney

    Full Text Available We have previously described the generation of a novel Ebola virus (EBOV vaccine platform based on (a replication-competent rabies virus (RABV, (b replication-deficient RABV, or (c chemically inactivated RABV expressing EBOV glycoprotein (GP. Mouse studies demonstrated safety, immunogenicity, and protective efficacy of these live or inactivated RABV/EBOV vaccines. Here, we evaluated these vaccines in nonhuman primates. Our results indicate that all three vaccines do induce potent immune responses against both RABV and EBOV, while the protection of immunized animals against EBOV was largely dependent on the quality of humoral immune response against EBOV GP. We also determined if the induced antibodies against EBOV GP differ in their target, affinity, or the isotype. Our results show that IgG1-biased humoral responses as well as high levels of GP-specific antibodies were beneficial for the control of EBOV infection after immunization. These results further support the concept that a successful EBOV vaccine needs to induce strong antibodies against EBOV. We also showed that a dual vaccine against RABV and filoviruses is achievable; therefore addressing concerns for the marketability of this urgently needed vaccine.

  17. Seroepidemiology of Human Papillomavirus 16 (HPV16) L2 and Generation of L2-Specific Human Chimeric Monoclonal Antibodies

    National Research Council Canada - National Science Library

    Wang, Joshua W; Jagu, Subhashini; Wu, Wai-Hong; Viscidi, Raphael P; Macgregor-Das, Anne; Fogel, Jessica M; Kwak, Kihyuck; Daayana, Sai; Kitchener, Henry; Stern, Peter L; Gravitt, Patti E; Trimble, Cornelia L; Roden, Richard B S


    Presently, the seroprevalence of human papillomavirus (HPV) minor capsid antigen L2-reactive antibody is not well understood, and no serologic standard exists for L2-specific neutralizing antibodies...

  18. Detection of antibodies to bacterial cell wall peptidoglycan in human sera. [/sup 125/I tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Heymer, B.; Schleifer, K.H.; Read, S.; Zabriskie, J.B.; Krause, R.M.


    A radioimmunoassay has been developed for the measurement of antibodies to peptidoglycan in human sera including patients with rheumatic feaver and juvenile rheumatoid arthritis. The assay is based on the percentage of binding of the hapten /sup 125/I-L-Ala-..gamma..-D-Glu-L-Lys-D-Ala-D-Ala, the major peptide determinant of peptidoglycan. Because of differences in the avidity of the antibodies in different sera, the amount of antibody was expressed as pentapeptide hapten-binding capacity (pentapeptide-HBC in ng/ml of serum). Fourteen out of 105 normal blood donors had a pentapeptide-HBC value greater than or equal to 75 ng/ml serum. Values in healthy children 5 to 18 years of age were less than or equal to 50 ng/ml. Sixty-eight percent of the individuals with rheumatic fever had values greater than or equal to 75 ng/ml, an indication that streptococcal infections can stimulate an immune response to peptidoglycan. Thirty-five percent of the patients with juvenile rheumatoid arthritis had values greater than or equal to 75 ng/ml. Such a finding points to a possible association between bacterial infections and juvenile rheumatoid arthritis.

  19. Inhibition of Entamoeba histolytica proteolytic activity by human salivary IgA antibodies. (United States)

    Guerrero-Manríquez, G G; Sánchez-Ibarra, F; Avila, E E


    Entamoeba histolytica is a protozoan parasite that causes amoebiasis in humans; as the infection occurs mainly in the intestinal epithelium, the secretory immune response of the host could have an influence on the outcome. Secretory IgA antibodies against E. histolytica have been detected in asymptomatic and symptomatic patients, but little is known about their protective role. E. histolytica cysteine proteases seem to be involved in the pathogenesis of amoebiasis; therefore, it is important to evaluate the human IgA response against these proteases and its effect on their enzymatic activity. When human saliva samples with and without antibodies against E. histolytica were tested by Western blot against one purified 70 kDa amoebic cysteine protease, 84% of anti-amoeba-positive samples recognized it. The secretory IgA purified from a pool of anti-protease-positive samples had a strong in vitro inhibitory effect on the E. histolytica proteolytic activity. These results suggest that this effect, if it occurs in vivo, could be an important protective factor against this parasite.

  20. New monoclonal antibodies directed against human renin. Powerful tools for the investigation of the renin system.


    Galen, F X; Devaux, C.; Atlas, S; Guyenne, T; Menard, J; Corvol, P; Simon, D.; Cazaubon, C; Richer, P; Badouaille, G


    Monoclonal antibodies directed against human renin were obtained by the fusing of myeloma cells with spleen cells from Balb/c or high-responder Biozzi mice injected with pure tumoral or highly purified renal renin. These procedures resulted in the production of seven stable monoclonal antibodies to human renin. Antibodies in the hybridoma culture medium were screened by binding to pure iodinated renin or insolubilized renin in a solid phase assay. The concentration of purified antibodies that...

  1. Treatment with a human recombinant monoclonal IgG antibody against oxidized LDL in atherosclerosis-prone pigs reduces cathepsin S in coronary lesions

    DEFF Research Database (Denmark)

    Poulsen, Christian Bo; Al-Mashhadi, Ahmed Ludvigsen; von Wachenfeldt, Karin;


    BACKGROUND: Immunization with oxidized LDL (oxLDL) reduces atherosclerosis in rodents. We tested the hypothesis that treatment with a human recombinant monoclonal antibody against oxLDL will reduce the burden or composition of atherosclerotic lesions in hypercholesterolemic minipigs. METHODS AND ...

  2. Detection of auto-anti-idiotypic antibodies to Lol p I (rye I) IgE antibodies in human sera by the use of murine idiotypes: levels in atopic and non-atopic subjects and effects of immunotherapy. (United States)

    Hébert, J; Bernier, D; Mourad, W


    Anti-idiotypic antibodies (anti-Id Abs) are involved in the regulation of a number of immune responses including the IgE antibody production. In atopic patients, the increased synthesis of IgE antibodies could be related to a defective production of regulatory anti-Id Abs. In the present study, we first developed a sensitive assay for measuring the levels of anti-Id Abs directed against antibodies specific for Lol p I, the major allergenic determinant of Lolium perenne (rye grass). In this assay, we used previously described murine monoclonal anti-Lol p I antibodies that were shown to share epitopic specificities with human anti-Lol p I IgE and IgG antibodies, thus short-cutting the need for purification of F(ab')2 fragments of human IgG Abs and insuring optimal specificity and sensitivity. Levels of anti-Id Abs against two anti-Lol p I monoclonal antibodies (290A-167, 348A-6) were higher in normal volunteers than in untreated atopic patients. Specific immunotherapy increased the levels of anti-Id Abs to those of normal volunteers. These observations suggest a role for the Id-anti-Id network in the regulation of IgE antibody production.

  3. Identification and purification of human erythroid progenitor cells by monoclonal antibody to the transferrin receptor (TU 67). (United States)

    Herrmann, F; Griffin, J D; Sabbath, K D; Oster, W; Wernet, P; Mertelsmann, R


    Anti-TU 67 is a murine monoclonal antibody that recognizes the transferrin receptor. With respect to hematopoietic cells TU 67 is expressed by human multipotent colony-forming cells (CFU-Mix), erythroid progenitor cells (BFU-E and CFU-E) and a fraction of granulocyte/monocyte colony forming cells, but is not expressed by mature hematopoietic cells including erythrocytes, platelets, lymphocytes, and peripheral blood myeloid cells. The TU 67-positive fraction of normal bone marrow, separated by fluorescence-activated cell sorting (FACS) or immune rosettes, contained 87% of the erythroid progenitor cells. Erythroid progenitor cells were enriched up to 50-fold by using a combination of monoclonal antibodies to deplete mature hematopoietic cells, followed by positive selection of BFU-E and CFU-E by TU 67 antibody.

  4. A murine monoclonal antibody that binds N-terminal extracellular segment of human protease-activated receptor-4. (United States)

    Sangawa, Takeshi; Nogi, Terukazu; Takagi, Junichi


    Abstract A monoclonal antibody that recognizes native G protein coupled receptors (GPCR) is generally difficult to obtain. Protease-activated receptor-4 (PAR4) is a GPCR that plays an important role in platelet activation as a low-affinity thrombin receptor. By immunizing peptide corresponding to the N-terminal segment of human PAR4, we obtained a monoclonal antibody that recognizes cell surface expressed PAR4. Epitope mapping using a series of artificial fusion proteins that carry PAR4-derived peptide revealed that the recognition motif is fully contained within the 6-residue portion adjacent to the thrombin cleavage site. The antibody blocked PAR4 peptide cleavage by thrombin, suggesting its utility in the functional study of PAR4 signaling.

  5. Antecedent avian immunity limits tangential transmission of West Nile virus to humans.

    Directory of Open Access Journals (Sweden)

    Jennifer L Kwan

    Full Text Available BACKGROUND: West Nile virus (WNV is a mosquito-borne flavivirus maintained and amplified among birds and tangentially transmitted to humans and horses which may develop terminal neuroinvasive disease. Outbreaks typically have a three-year pattern of silent introduction, rapid amplification and subsidence, followed by intermittent recrudescence. Our hypothesis that amplification to outbreak levels is contingent upon antecedent seroprevalence within maintenance host populations was tested by tracking WNV transmission in Los Angeles, California from 2003 through 2011. METHODS: Prevalence of antibodies against WNV was monitored weekly in House Finches and House Sparrows. Tangential or spillover transmission was measured by seroconversions in sentinel chickens and by the number of West Nile neuroinvasive disease (WNND cases reported to the Los Angeles County Department of Public Health. RESULTS: Elevated seroprevalence in these avian populations was associated with the subsidence of outbreaks and in the antecedent dampening of amplification during succeeding years. Dilution of seroprevalence by recruitment resulted in the progressive loss of herd immunity following the 2004 outbreak, leading to recrudescence during 2008 and 2011. WNV appeared to be a significant cause of death in these avian species, because the survivorship of antibody positive birds significantly exceeded that of antibody negative birds. Cross-correlation analysis showed that seroprevalence was negatively correlated prior to the onset of human cases and then positively correlated, peaking at 4-6 weeks after the onset of tangential transmission. Antecedent seroprevalence during winter (Jan - Mar was negatively correlated with the number of WNND cases during the succeeding summer (Jul-Sep. CONCLUSIONS: Herd immunity levels within after hatching year avian maintenance host populations <10% during the antecedent late winter and spring period were followed on three occasions by

  6. Effective Depletion of Pre-existing Anti-AAV Antibodies Requires Broad Immune Targeting. (United States)

    Velazquez, Victoria M; Meadows, Aaron S; Pineda, Ricardo J; Camboni, Marybeth; McCarty, Douglas M; Fu, Haiyan


    Pre-existing antibodies (Abs) to AAV pose a critical challenge for the translation of gene therapies. No effective approach is available to overcome pre-existing Abs. Given the complexity of Ab production, overcoming pre-existing Abs will require broad immune targeting. We generated a mouse model of pre-existing AAV9 Abs to test multiple immunosuppressants, including bortezomib, rapamycin, and prednisolone, individually or in combination. We identified an effective approach combining rapamycin and prednisolone, reducing serum AAV9 Abs by 70%-80% at 4 weeks and 85%-93% at 8 weeks of treatment. The rapamycin plus prednisolone treatment resulted in significant decreases in the frequency of B cells, plasma cells, and IgG-secreting and AAV9-specific Ab-producing plasma cells in bone marrow. The rapamycin plus prednisolone treatment also significantly reduced frequencies of IgD(-)IgG(+) class-switched/FAS(+)CL7(+) germinal center B cells, and of activated CD4(+) T cells expressing PD1 and GL7, in spleen. These data suggest that rapamycin plus prednisolone has selective inhibitory effects on both T helper type 2 support of B cell activation in spleen and on bone marrow plasma cell survival, leading to effective AAV9 Abs depletion. This promising immunomodulation approach is highly translatable, and it poses minimal risk in the context of therapeutic benefits promised by gene therapy for severe monogenetic diseases, with a single or possibly a few treatments over a lifetime.

  7. Effective Depletion of Pre-existing Anti-AAV Antibodies Requires Broad Immune Targeting

    Directory of Open Access Journals (Sweden)

    Victoria M. Velazquez


    Full Text Available Pre-existing antibodies (Abs to AAV pose a critical challenge for the translation of gene therapies. No effective approach is available t