Interactions of protein antigens with antibodies.

There are now several crystal structures of antibody Fab fragments complexed to their protein antigens. These include Fab complexes with lysozyme, two Fab complexes with influenza virus neuraminidase, and three Fab complexes with their anti-idiotype Fabs. The pattern of binding that emerges is similar to that found with other protein-protein interactions, with good shape complementarity between the interacting surfaces and reasonable juxtapositions of polar residues so as to permit hydrogen-bond formation. Water molecules have been observed in cavities within the interface and on the periphery, where they often form bridging hydrogen bonds between antibody and antigen. For the most part the antigen is bound in the middle of the antibody combining site with most of the six complementarity-determining residues involved in binding. For the most studied antigen, lysozyme, the epitopes for four antibodies occupy approximately 45% of the accessible surface area. Some conformational changes have been observed to accompany binding in both the antibody and the antigen, although most of the information on conformational change in the latter comes from studies of complexes with small antigens.

Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens.

The presentation of antigenic peptides by major histocompatibility complex (MHC) class II molecules to CD4+ T cells is critical to the function of the immune system. In this study, we have utilized the sorting signal of the lysosomal-associated membrane protein LAMP-1 to target a model antigen, human papillomavirus 16 E7 (HPV-16 E7), into the endosomal and lysosomal compartments. The LAMP-1 sorting signal reroutes the antigen into the MHC class II processing pathway, resulting in enhanced presentation to CD4+ cells in vitro. In vivo immunization experiments in mice demonstrated that vaccinia containing the chimeric E7/LAMP-1 gene generated greater E7-specific lymphoproliferative activity, antibody titers, and cytotoxic T-lymphocyte activities than vaccinia containing the wild-type HPV-16 E7 gene. These results suggest that specific targeting of an antigen to the endosomal and lysosomal compartments enhances MHC class II presentation and vaccine potency.

CD27-CD70 interactions regulate B-cell activation by T cells.

CD27, a member of the tumor necrosis factor (TNF) receptor family, binds to its ligand CD70, a member of the TNF family, and subsequently induces T-cell costimulation and B-cell activation. CD27 is expressed on resting T and B cells, whereas CD70 is expressed on activated T and B cells. Utilizing transfected murine pre-B-cell lines expressing human CD27 or CD70, we have examined the effect of such transfectant cells on human B-cell IgG production and B-cell proliferation. We show that the addition of CD27-transfected cells to a T-cell-dependent, pokeweed mitogen-driven B-cell IgG synthesis system resulted in marked inhibition of IgG production, whereas the addition of CD70-transfected cells enhanced IgG production. The inhibition and enhancement of pokeweed mitogen-driven IgG production by CD27 and CD70 transfectants were abrogated by pretreatment with anti-CD27 and anti-CD70 monoclonal antibodies, respectively. In contrast, little or no inhibition of IgG production and B-cell proliferation was noted with CD27-transfected cells or either anti-CD27 or CD70 monoclonal antibody in a T-cell-independent Staphylococcus aureus/interleukin 2-driven B-cell activation system. In this same system CD70-transfected cells enhanced B-cell IgG production and B-cell proliferation, and this enhancement could be gradually abrogated by addition of increasing numbers of CD27-transfected cells. These results clearly demonstrate that interactions among subsets of T cells expressing CD27 and CD70 play a key role in regulating B-cell activation and immunoglobulin synthesis.

Changes in expression of putative antigens encoded by pigment genes in mouse melanomas at different stages of malignant progression.

Cutaneous melanomas of Tyr-SV40E transgenic mice (mice whose transgene consists of the tyrosinase promoter fused to the coding regions of simian virus 40 early genes) strikingly resemble human melanomas in their development and progression. Unlike human melanomas, the mouse tumors all arise in genetically identical individuals, thereby better enabling expression of specific genes to be characterized in relation to advancing malignancy. The products of pigment genes are of particular interest because peptides derived from these proteins have been reported to function as autoantigens with immunotherapeutic potential in some melanoma patients. However, the diminished pigmentation characteristic of many advanced melanomas raises the possibility that some of the relevant products may no longer be expressed in the most malignant cells. We have therefore investigated the contributions of several pigment genes in melanotic vs. relatively amelanotic components of primary and metastatic mouse melanomas. The analyses reveal marked differences within and among tumors in levels of mRNAs and proteins encoded by the wild-type alleles at the albino, brown, slaty, and silver loci. Tyrosinase (the protein encoded by the albino locus) was most often either absent or undetectable as melanization declined. The protein encoded by the slaty locus (tyrosinase-related protein 2) was the only one of those tested that was clearly present in all the tumor samples. These results suggest that sole reliance on targeting tyrosinase-based antigens might selectively favor survival of more malignant cells, whereas targeting the ensemble of the antigens tested might contribute toward a more inclusive and effective antimelanoma strategy.

Upregulation of class I major histocompatibility complex antigens by interferon gamma is necessary for T-cell-mediated elimination of recombinant adenovirus-infected hepatocytes in vivo.

Recombinant adenoviruses are attractive vehicles for liver-directed gene therapy because of the high efficiency with which they transfer genes to hepatocytes in vivo. First generation recombinant adenoviruses deleted of E1 sequences also express recombinant and early and late viral genes, which lead to development of destructive cellular immune responses. Previous studies indicated that class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTLs) play a major role in eliminating virus-infected cells. The present studies utilize mouse models to evaluate the role of T-helper cells in the primary response to adenovirus-mediated gene transfer to the liver. In vivo ablation of CD4+ cells or interferon gamma (IFN-gamma) was sufficient to prevent the elimination of adenovirus-transduced hepatocytes, despite the induction of a measurable CTL response. Mobilization of an effective TH1 response as measured by in vitro proliferation assays was associated with substantial upregulation of MHC class I expression, an effect that was prevented in IFN-gamma-deficient animals. These results suggest that elimination of virus-infected hepatocytes in a primary exposure to recombinant adenovirus requires both induction of antigen-specific CTLs as well as sensitization of the target cell by TH1-mediated activation of MHC class I expression.

Two Drosophila nervous system antigens, Nervana 1 and 2, are homologous to the beta subunit of Na+,K(+)-ATPase.

A nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana (Nrv), has been purified on the basis of reactivity of its carbohydrate epitope(s) with anti-horseradish peroxidase (HRP) antibodies that are specific markers for Drosophila neurons. Anti-Nrv monoclonal antibodies (mAbs), specific for the protein moiety of Nrv, were used to screen a Drosophila embryo cDNA expression library. Three cDNA clones (designated Nrv1, Nrv2.1, and Nrv2.2) were isolated that code for proteins recognized by anti-Nrv mAbs on Western blots. DNA sequencing and Southern blot analyses established that the cDNA clones are derived from two different genes. In situ hybridization to Drosophila polytene chromosomes showed that the cDNA clones map to the third chromosome near 92C-D. Nrv1 and Nrv2.1/2.2 have open reading frames of 309 and 322/323 amino acids, respectively, and they are 43.4% identical at the amino acid level. The proteins deduced from these clones exhibit significant homology in both primary sequence and predicted topology to the beta subunit of Na+,K(+)-ATPase. Immunoaffinity-purified Nrv is associated with a protein (M(r) 100,000) recognized on Western blots by anti-ATPase alpha-subunit mAb. Our results suggest that the Drosophila nervous system-specific antigens Nrv1 and -2 are neuronal forms of the beta subunit of Na+,K(+)-ATPase.

Invariance and restriction toward a limited set of self-antigens characterize neonatal IgM antibody repertoires and prevail in autoreactive repertoires of healthy adults.

Analysis of the reactivity of IgM with self-antigens in tissues by a quantitative immunoblotting technique showed striking invariance among newborns in the human and in the mouse. The self-reactive repertoire of IgM of adults was also markedly conserved; it comprised most anti-self reactivities that prevailed among neonates. Multivariate analysis confirmed the homogeneity of IgM repertoires of neonates toward self- and non-self-antigens. Multivariate analysis discriminated between newborn and adult repertoires for reactivity with two of five sources of self-proteins and with non-self-antigens. Our observations support the concept that naturally activated B lymphocytes are selected early in development and throughout life for reactivity with a restricted set of self-antigens.

Characterization of B cells in healthy pregnant women from late pregnancy to post-partum: a prospective observational study

BACKGROUND: B cells play a role in pregnancy due to their humoral and regulatory activities. To our knowledge, different maturational stages (from transitional to memory) of circulating B cell subsets have not yet been characterized (cell quantification and phenotype identification) in healthy pregnant women. Thus, the objective of our study was to characterize these subsets (as well as regulatory B cells) from late pregnancy to post-partum and to compare them with the circulating B cells of non-pregnant women. METHODS: In all of the enrolled women, flow cytometry was used to characterize the circulating B cell subsets according to the expression of IgD and CD38 (Bm1-Bm5 classification system). Regulatory B cells were characterized based on the expression of surface antigens (CD24, CD27, and CD38) and the production of IL-10 after lipopolysaccharide stimulation. RESULTS: Compared to the absolute counts of B cells in the non-pregnant women (n = 35), those in the pregnant women (n = 43) were significantly lower (p < 0.05) during the 3rd trimester of pregnancy and on delivery day (immediately after delivery). The percentages of these cells on delivery day and at post-partum were significantly lower than those in the non-pregnant women. In general, the absolute counts and percentages of the majority of the B cell subsets were significantly lower in the 3rd trimester of pregnancy and on delivery day than in the non-pregnant women. However, these counts and percentages did not differ significantly between the post-partum and the non-pregnant women. The most notable exceptions to the above were the percentages of naïve B cells (which were significantly higher in the 3rd trimester and on delivery day than in the non-pregnant women) and of CD24(hi)CD38(hi) regulatory B cells (which were significantly higher in the post-partum than in the non-pregnant women). CONCLUSION: According to our study, the peripheral B cell compartment undergoes quantitative changes during normal late pregnancy and post-partum. Such findings may allow us to better understand immunomodulation during human pregnancy and provide evidence that could aid in the development of new strategies to diagnose and treat pregnancy-associated disturbances. Our findings could also be useful for studies of the mechanisms of maternal responses to vaccination and infection.

Characterization of B cells in healthy pregnant women from late pregnancy to post-partum: a prospective observational study

Abstract BACKGROUND: B cells play a role in pregnancy due to their humoral and regulatory activities. To our knowledge, different maturational stages (from transitional to memory) of circulating B cell subsets have not yet been characterized (cell quantification and phenotype identification) in healthy pregnant women. Thus, the objective of our study was to characterize these subsets (as well as regulatory B cells) from late pregnancy to post-partum and to compare them with the circulating B cells of non-pregnant women. METHODS: In all of the enrolled women, flow cytometry was used to characterize the circulating B cell subsets according to the expression of IgD and CD38 (Bm1-Bm5 classification system). Regulatory B cells were characterized based on the expression of surface antigens (CD24, CD27, and CD38) and the production of IL-10 after lipopolysaccharide stimulation. RESULTS: Compared to the absolute counts of B cells in the non-pregnant women (n = 35), those in the pregnant women (n = 43) were significantly lower (p < 0.05) during the 3rd trimester of pregnancy and on delivery day (immediately after delivery). The percentages of these cells on delivery day and at post-partum were significantly lower than those in the non-pregnant women. In general, the absolute counts and percentages of the majority of the B cell subsets were significantly lower in the 3rd trimester of pregnancy and on delivery day than in the non-pregnant women. However, these counts and percentages did not differ significantly between the post-partum and the non-pregnant women. The most notable exceptions to the above were the percentages of naïve B cells (which were significantly higher in the 3rd trimester and on delivery day than in the non-pregnant women) and of CD24(hi)CD38(hi) regulatory B cells (which were significantly higher in the post-partum than in the non-pregnant women). CONCLUSION: According to our study, the peripheral B cell compartment undergoes quantitative changes during normal late pregnancy and post-partum. Such findings may allow us to better understand immunomodulation during human pregnancy and provide evidence that could aid in the development of new strategies to diagnose and treat pregnancy-associated disturbances. Our findings could also be useful for studies of the mechanisms of maternal responses to vaccination and infection.

Serological diagnosis of echinococcosis: the diagnostic potential of native antigens

PURPOSE: Human alveolar (AE) and cystic echinococcosis (CE) caused by the metacestode stages of Echinococcus multilocularis and E. granulosus, respectively, lack pathognomonic clinical signs. Diagnosis therefore relies on the results of imaging and serological studies. The primary goal of this study was to evaluate the efficacy of several easy-to-produce crude or partially purified E. granulosus and E. multilocularis metacestode-derived antigens as tools for the serological diagnosis and differential diagnosis of patients suspicious for AE or CE. METHODS: The sera of 51 treatment-naïve AE and 32 CE patients, 98 Swiss blood donors and 38 patients who were initially suspicious for echinococcosis but suffering from various other liver diseases (e.g., liver neoplasia, etc.) were analysed. RESULTS: According to the results of enzyme-linked immunosorbent assays (ELISA), metacestode-derived antigens of E. granulosus had sensitivities varying from 81 to 97% and >99.9% for the diagnosis of CE and AE, respectively. Antigens derived from E. multilocularis metacestodes had sensitivities ranging from 84 to 91% and >99.9% for the diagnosis of CE and AE, respectively. Specificities ranged from 92 to >99.9%. Post-test probabilities for the differential diagnosis of AE from liver neoplasias, CE from cystic liver lesions, and screening for AE in Switzerland were around 95, 86 and 2.2%, respectively. Cross-reactions with antibodies in sera of patients with other parasitic affections (fasciolosis, schistosomosis, amebosis, cysticercosis, and filarioses) did occur at variable frequencies, but could be eliminated through the use of confirmatory testing. CONCLUSIONS: Different metacestode-derived antigens of E. granulosus and E. multilocularis are valuable, widely accessible, and cost-efficient tools for the serological diagnosis of echinococcosis. However, confirmatory testing is necessary, due to the lack of species specificity and the occurrence of cross-reactions to other helminthic diseases.

Blood group antigens & antibodies as applied to blood transfusion.

Includes bibliographical references.

The Antigenic response to a booster injection of a multiple antigen preparation containing diphtheria, pertussis, and tetanus antigens given seven years or more after primary immunization /

Mode of access: Internet.

Cardiolipin antigens;

Mode of access: Internet.

Fusion of interleukin-2 to subunit antigens increase their antigenicity in vitro due to an interleukin-2 receptor beta-mediated antigen uptake mechanism

Subunit vaccines, based on one or more epitopes, offer advantages over whole vaccines in terms of safety but are less antigenic. We investigated whether fusion of the cytokine interleukin-2 (IL-2) to influenza-derived subunit antigens could increase their antigenicity. The fusion of IL-2 to the subunit antigens increased their antigenicity in vitro. Encapsulation of the subunit antigen in liposomes also increased its antigenicity in vitro, yet encapsulation of the subunit IL-2 fusion did not. The use of anti-IL-2 receptor beta (IL-2Rbeta) antibody to block the receptor subunit on macrophages suggested that the adjuvancy exerted by IL-2 in our in vitro system is due to, at least in part, a previously unreported IL-2Rbeta-mediated antigen uptake mechanism.

Quantitative Association Tests of Immune Responses to Antigens of Mycobacterium Tuberculosis: A Study of Twins in West Africa

There is now considerable evidence that host genetic factors are important in determining the outcome of infection with Mycobacterium tuberculosis (MTB). The aim of this study was to assess the role of several candidate genes in the variation observed in the immune responses to MTB antigens. In-vitro assays of T-cell proliferation, an in-vivo intradermal delayed hypersensitivity response; cytokine and antibody secretions to several mycobacterial peptide antigens were assessed in healthy, but exposed, West African twins. Candidate gene polymorphisms were typed in the NRAMP1, Vitamin D receptor, IL10, IL4, IL4 receptor and CTLA-4 genes. Variants of the loci IL10 (-1082 G/A), CTLA-4 (49 A/G) and the IL4 receptor (128 A/G) showed significant associations with immune responses to several antigens. T-cell proliferative responses and antibody responses were reduced, TNF-alpha responses were increased for subjects with the CTLA-4 G allele. The T-cell proliferative responses of subjects with IL10 GA and GG genotypes differed significantly. IL4 receptor AG and GG genotypes also showed significant differences in their T-cell proliferative responses to MTB antigens. These results yield a greater understanding of the genetic mechanisms that underlie the immune responses in tuberculosis and have implications for the design of therapeutic interventions.