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1
Raybould, Matthew I. J., Claire Marks, Aleksandr Kovaltsuk, Alan P. Lewis, Jiye Shi, and Charlotte M. Deane. "Public Baseline and shared response structures support the theory of antibody repertoire functional commonality." PLOS Computational Biology 17, no. 3 (2021): e1008781. http://dx.doi.org/10.1371/journal.pcbi.1008781.
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The naïve antibody/B-cell receptor (BCR) repertoires of different individuals ought to exhibit significant functional commonality, given that most pathogens trigger an effective antibody response to immunodominant epitopes. Sequence-based repertoire analysis has so far offered little evidence for this phenomenon. For example, a recent study estimated the number of shared (‘public’) antibody clonotypes in circulating baseline repertoires to be around 0.02% across ten unrelated individuals. However, to engage the same epitope, antibodies only require a similar binding site structure and the presence of key paratope interactions, which can occur even when their sequences are dissimilar. Here, we search for evidence of geometric similarity/convergence across human antibody repertoires. We first structurally profile naïve (‘baseline’) antibody diversity using snapshots from 41 unrelated individuals, predicting all modellable distinct structures within each repertoire. This analysis uncovers a high (much greater than random) degree of structural commonality. For instance, around 3% of distinct structures are common to the ten most diverse individual samples (‘Public Baseline’ structures). Our approach is the first computational method to find levels of BCR commonality commensurate with epitope immunodominance and could therefore be harnessed to find more genetically distant antibodies with same-epitope complementarity. We then apply the same structural profiling approach to repertoire snapshots from three individuals before and after flu vaccination, detecting a convergent structural drift indicative of recognising similar epitopes (‘Public Response’ structures). We show that Antibody Model Libraries derived from Public Baseline and Public Response structures represent a powerful geometric basis set of low-immunogenicity candidates exploitable for general or target-focused therapeutic antibody screening.
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Collins, Andrew M., Yan Wang, Krishna M. Roskin, Christopher P. Marquis, and Katherine J. L. Jackson. "The mouse antibody heavy chain repertoire is germline-focused and highly variable between inbred strains." Philosophical Transactions of the Royal Society B: Biological Sciences 370, no. 1676 (2015): 20140236. http://dx.doi.org/10.1098/rstb.2014.0236.
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The human and mouse antibody repertoires are formed by identical processes, but like all small animals, mice only have sufficient lymphocytes to express a small part of the potential antibody repertoire. In this study, we determined how the heavy chain repertoires of two mouse strains are generated. Analysis of IgM- and IgG-associated VDJ rearrangements generated by high-throughput sequencing confirmed the presence of 99 functional immunoglobulin heavy chain variable (IGHV) genes in the C57BL/6 genome, and inferred the presence of 164 IGHV genes in the BALB/c genome. Remarkably, only five IGHV sequences were common to both strains. Compared with humans, little N nucleotide addition was seen in the junctions of mouse VDJ genes. Germline human IgG-associated IGHV genes are rare, but many murine IgG-associated IGHV genes were unmutated. Together these results suggest that the expressed mouse repertoire is more germline-focused than the human repertoire. The apparently divergent germline repertoires of the mouse strains are discussed with reference to reports that inbred mouse strains carry blocks of genes derived from each of the three subspecies of the house mouse. We hypothesize that the germline genes of BALB/c and C57BL/6 mice may originally have evolved to generate distinct germline-focused antibody repertoires in the different mouse subspecies.
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Robinson, William H. "Sequencing the functional antibody repertoire—diagnostic and therapeutic discovery." Nature Reviews Rheumatology 11, no. 3 (2014): 171–82. http://dx.doi.org/10.1038/nrrheum.2014.220.
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Zheng, Tianqing, Jia Xie, Zhuo Yang, et al. "Antibody selection using clonal cocultivation of Escherichia coli and eukaryotic cells in miniecosystems." Proceedings of the National Academy of Sciences 115, no. 27 (2018): E6145—E6151. http://dx.doi.org/10.1073/pnas.1806718115.
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We describe a method for the rapid selection of functional antibodies. The method depends on the cocultivation of Escherichia coli that produce phage with target eukaryotic cells in very small volumes. The antibodies on phage induce selectable phenotypes in the target cells, and the nature of the antibody is determined by gene sequencing of the phage genome. To select functional antibodies from the diverse antibody repertoire, we devised a selection platform that contains millions of picoliter-sized droplet ecosystems. In each miniecosystem, the bacteria produce phage displaying unique members of the antibody repertoire. These phage interact only with eukaryotic cells in the same miniecosystem, making phage available directly for activity-based antibody selection in biological systems.
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de Bourcy, Charles F. A., Cesar J. Lopez Angel, Christopher Vollmers, Cornelia L. Dekker, Mark M. Davis, and Stephen R. Quake. "Phylogenetic analysis of the human antibody repertoire reveals quantitative signatures of immune senescence and aging." Proceedings of the National Academy of Sciences 114, no. 5 (2017): 1105–10. http://dx.doi.org/10.1073/pnas.1617959114.
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The elderly have reduced humoral immunity, as manifested by increased susceptibility to infections and impaired vaccine responses. To investigate the effects of aging on B-cell receptor (BCR) repertoire evolution during an immunological challenge, we used a phylogenetic distance metric to analyze Ig heavy-chain transcript sequences in both young and elderly individuals before and after influenza vaccination. We determined that BCR repertoires become increasingly specialized over a span of decades, but less plastic. In 50% of the elderly individuals, a large space in the repertoire was occupied by a small number of recall lineages that did not decline during vaccine response and contained hypermutated IgD+B cells. Relative to their younger counterparts, older subjects demonstrated a contracted naive repertoire and diminished intralineage diversification, signifying a reduced substrate for mounting novel responses and decreased fine-tuning of BCR specificities by somatic hypermutation. Furthermore, a larger proportion of the repertoire exhibited premature stop codons in some elderly subjects, indicating that aging may negatively affect the ability of B cells to discriminate between functional and nonfunctional receptors. Finally, we observed a decreased incidence of radical mutations compared with conservative mutations in elderly subjects’ vaccine responses, which suggests that accumulating original antigenic sin may be limiting the accessible space for paratope evolution. Our findings shed light on the complex interplay of environmental and gerontological factors affecting immune senescence, and provide direct molecular characterization of the effects of senescence on the immune repertoire.
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Schwimmer, Lauren J., Betty Huang, Hoa Giang, et al. "Discovery of diverse and functional antibodies from large human repertoire antibody libraries." Journal of Immunological Methods 391, no. 1-2 (2013): 60–71. http://dx.doi.org/10.1016/j.jim.2013.02.010.
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Nobrega, Alberto, Alf Grandien, Matthias Haury, Laura Hecker, Evelyne Malanchère, and Antonio Coutinho. "Functional diversity and clonal frequencies of reactivity in the available antibody repertoire." European Journal of Immunology 28, no. 4 (1998): 1204–15. http://dx.doi.org/10.1002/(sici)1521-4141(199804)28:04<1204::aid-immu1204>3.0.co;2-g.
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Kodangattil, Sreekumar, Christine Huard, Cindy Ross, et al. "The functional repertoire of rabbit antibodies and antibody discovery via next-generation sequencing." mAbs 6, no. 3 (2014): 628–36. http://dx.doi.org/10.4161/mabs.28059.
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Maleckar, J. R., and L. A. Sherman. "The composition of the T cell receptor repertoire in nude mice." Journal of Immunology 138, no. 11 (1987): 3873–76. http://dx.doi.org/10.4049/jimmunol.138.11.3873.
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Abstract Previous results from several laboratories have demonstrated the presence of functional T lymphocytes in congenitally athymic (nude) mice. The present study represents an analysis of the T cell receptor repertoire exhibited by such cells. Clones of H-2Kb-specific cytotoxic T lymphocytes (CTL) were generated under primary limiting dilution conditions by using spleen cells from nude mice. These clones were analyzed on a panel of Kb mutant target cells to assess the receptor specificity of each clone. Unlike thymic bearing mice the CTL repertoires of which are exceedingly diverse, it was found that in most cases the vast majority of clones from each individual exhibited the same reactivity pattern. The particular pattern varied from individual to individual. Clones from three animals that exhibited this phenomenon were additionally analyzed by using a monoclonal antibody that can detect the utilization of the gene products of the V beta 8 family. In one animal all clones were V beta 8 positive, whereas in the others, all clones were negative. We conclude that the T cell receptor repertoire in nude mice is extremely limited and represents in vivo expansion of a relatively small number of functional precursors.
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Kirchenbaum, Greg A., Giuseppe A. Sautto, Rodrigo B. Abreu, Paul V. Lehmann, and Ted M. Ross. "Assessment of Antibody Functional Affinity using FluoroSpot." Journal of Immunology 204, no. 1_Supplement (2020): 86.11. http://dx.doi.org/10.4049/jimmunol.204.supp.86.11.
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Abstract Studies of B cell immunity often rely upon serologic methodologies that primarily assess antibody specificity. However, functional affinity of the antigen-specific antibody repertoire is a key determinant of protective efficacy. Presently, detailed assessment of single B cell/antibody functional affinity is labor-intensive and low-throughput. Therefore, we sought to evaluate whether B cell FluoroSpot assays would enable distinction between B cells with differential functional affinity since fluorescent intensity is directly proportional to antibody abundance in this assay. To test our prediction, murine B cell hybridomas (P65C6-2, 36–65 and 36–71) secreting monoclonal antibodies (mAb) with differential affinity for the p-azophenylarsonate (Ars) hapten were utilized as model antibody-secreting cells (ASC). Additionally, usage of an anti-idiotypic mAb (17–63) specific for the high-affinity anti-Ars mAb (36–71) confirmed unambiguous segregation of Ars-specific ASC. Moreover, we also evaluated the capacity of a multiplexed FluoroSpot assay to simultaneously distinguish antibody functional affinity among influenza hemagglutinin-reactive murine B cell hybridomas, or in vivo differentiated ASC from immunized mice, secreting various IgG subclasses (IgG1/IgG2a/IgG2b) in parallel. Collectively, our data support the notion that FluoroSpot assays can be used to assess the functional affinity of antigen-specific B cells, and that this approach is well-suited for detailed assessment of humoral immunity.
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Reinhardt, Richard Lee, Hong-Erh Liang, and Richard M. Locksley. "Cytokine-secreting follicular T cells shape the antibody repertoire (34.19)." Journal of Immunology 182, no. 1_Supplement (2009): 34.19. http://dx.doi.org/10.4049/jimmunol.182.supp.34.19.
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Abstract High-affinity antibodies are critical for host protection against pathogens and toxins, and underlie successful vaccines. Generation of such antibodies requires T cell-dependent help, which mediates germinal center (GC) reactions where mutation and selection of B cells expressing receptors with high-affinity for antigen occurs. Follicular CD4 T (TFH) cells, which comprise only 5-10% of GC cells, have been imaged in stable conjugates with GC B cells, but no functional analysis of B/TFH cell interactions in GC has been performed. Using an IL-4 reporter system, we show that TFH cells comprise essentially all of the cytokine-secreting T cells in lymph nodes and are functionally distinct from T cells secreting the same cytokine in peripheral tissues. TFH cells with different cytokine profiles could be visualized in the same GC and could be isolated as conjugates with B cells undergoing cytokine-specific immunoglobulin isotype class switching, expressing high levels of AID, and evidence of somatic hypermutation. These findings support a model wherein GC B cells compete for cytokines produced by rare antigen-specific GC TFH cells that mediate highly localized effects and regulate the size, affinity and isotype of the B cell antibody response.
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Bai, Xuelian, Moonseon Jang, Nam Ju Lee, et al. "A Novel Synthetic Antibody Library with Complementarity-Determining Region Diversities Designed for an Improved Amplification Profile." International Journal of Molecular Sciences 23, no. 11 (2022): 6255. http://dx.doi.org/10.3390/ijms23116255.
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Antibody discovery by phage display consists of two phases, i.e., the binding phase and the amplification phase. Ideally, the selection process is dominated by the former, and all the retrieved clones are amplified equally during the latter. In reality, the amplification efficiency of antibody fragments varies widely among different sequences and, after a few rounds of phage display panning, the output repertoire often includes rapidly amplified sequences with low or no binding activity, significantly diminishing the efficiency of antibody isolation. In this work, a novel synthetic single-chain variable fragment (scFv) library with complementarity-determining region (CDR) diversities aimed at improved amplification efficiency was designed and constructed. A previously reported synthetic scFv library with low, non-combinatorial CDR diversities was panned against protein A superantigen, and the library repertoires before and after the panning were analyzed by next generation sequencing. The enrichment or depletion patterns of CDR sequences after panning served as the basis for the design of the new library. Especially for CDR-H3 with a higher and more random diversity, a machine learning method was applied to predict potential fast-amplified sequences among a simulated sequence repertoire. In a direct comparison with the previous generation library, the new library performed better against a panel of antigens in terms of the number of binders isolated, the number of unique sequences, and/or the speed of binder enrichment. Our results suggest that the amplification-centric design of sequence diversity is a valid strategy for the construction of highly functional phage display antibody libraries.
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Gould, Hannah J., and Yu-Chang Bryan Wu. "IgE repertoire and immunological memory: compartmental regulation and antibody function." International Immunology 30, no. 9 (2018): 403–12. http://dx.doi.org/10.1093/intimm/dxy048.
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Abstract It is now generally recognized that bone marrow is the survival niche for antigen-specific plasma cells with long-term immunological memory. These cells release antibodies into the circulation, needed to prime effector cells in the secondary immune response. These antibodies participate in the surveillance for antigen and afford immune defence against pathogens and toxins previously encountered in the primary immune response. IgE antibodies function together with their effector cells, mast cells, to exert ‘immediate hypersensitivity’ in mucosal tissues at the front line of immune defence. The constant supply of IgE antibodies from bone marrow plasma cells allows the rapid ‘recall response’ by mast cells upon re-exposure to antigen even after periods of antigen absence. The speed and sensitivity of the IgE recall response and potency of the effector cell functions are advantageous in the early detection and elimination of pathogens and toxins at the sites of attack. Local antigen provocation also stimulates de novo synthesis of IgE or its precursors of other isotypes that undergo IgE switching in the mucosa. This process, however, introduces a delay before mast cells can be sensitized and resume activity; this is terminated shortly after the antigen is eliminated. Recent results from adaptive immune receptor repertoire sequencing of immunoglobulin genes suggest that the mucosal IgE+ plasmablasts, which have undergone affinity maturation in the course of their evolution in vivo, are a source of long-lived IgE+ plasma cells in the bone marrow that are already fully functional.
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Gilchuk, Pavlo, Charles D. Murin, Jacob C. Milligan, et al. "Structural and functional analysis of cooperativity in a potent human antibody cocktail against Ebola virus." Journal of Immunology 204, no. 1_Supplement (2020): 167.19. http://dx.doi.org/10.4049/jimmunol.204.supp.167.19.
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Abstract Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus (EBOV). Systematic analysis of antibody repertoire in the human survivors identified a pair of potently neutralizing mAbs that cooperatively bind to the ebolavirus glycoprotein (GP). High-resolution structures revealed that in a two-antibody cocktail, molecular mimicry was a major feature of mAb/GP interactions. Broadly neutralizing mAb rEBOV-520 targeted a conserved epitope on the GP base region. MAb rEBOV-548 bound to a glycan cap epitope, possessed neutralizing and Fc-mediated effector function activities, and potentiated neutralization by rEBOV-520. Remodeling of the glycan cap structures by the cocktail enabled enhanced GP binding and virus neutralization. The cocktail demonstrated high levels of effectiveness against EBOV in nonhuman primates. These data illuminate structural principles of antibody cooperativity with implications for the design and development of antiviral immunotherapeutics.
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Kalled, S. L., and P. H. Brodeur. "Utilization of V kappa families and V kappa exons. Implications for the available B cell repertoire." Journal of Immunology 147, no. 9 (1991): 3194–200. http://dx.doi.org/10.4049/jimmunol.147.9.3194.
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Abstract A molecular cloning approach was used to determine the relative utilization of 2 individual V kappa 21 genes, 13 V kappa gene families, and the 4 functional J kappa gene segments among splenic B cells of nonimmunized BALB/c mice. Based on the observed frequency of individual V kappa gene expression, we estimate that the mouse genome encodes 150 to 180 functional V kappa genes, and we suggest that most functional V kappa exons are expressed at comparable frequencies in the preimmune antibody repertoire. In contrast, clear differences in J kappa segment utilization were observed, J kappa 4 being consistently underrepresented with respect to the other J kappa segments.
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Zwirner, J., W. Weissenhorn, L. Karlsson, et al. "Expression of a functional chimeric Ig-MHC class II protein." Journal of Immunology 148, no. 1 (1992): 272–76. http://dx.doi.org/10.4049/jimmunol.148.1.272.
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Abstract We have generated a chimeric protein molecule composed of the alpha- and beta-chains of the MHC class II I-E molecule fused to antibody V regions derived from anti-human CD4 mAb MT310. Expression vectors were constructed containing the functional, rearranged gene segments coding for the V region domains of the antibody H and L chains in place of the first domains of the complete structural genes of the I-E alpha- and beta-chains, respectively. Cells transfected with both hybrid genes expressed a stable protein product on the cell surface. The chimeric molecule exhibited the idiotype of the antibody MT310 as shown by binding to the anti-idiotypic mAb 20-46. A protein of the anticipated molecular mass was immunoprecipitated with anti-mouse IgG antiserum. Furthermore, human soluble CD4 did bind to the transfected cell line, demonstrating that the chimeric protein possessed the binding capacity of the original mAb. Thus, the hybrid molecule retained: 1) the properties of a MHC class II protein with regard to correct chain assembly and transport to the cell surface; as well as 2) the Ag binding capacity of the antibody genes used. The generation of hybrid MHC class II molecules with highly specific, non-MHC-restricted binding capacities will be useful for studying MHC class II-mediated effector functions such as selection of the T cell repertoire in thymus of transgenic mice.
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Hooijkaas, H., A. A. van der Linde-Preesman, W. M. Bitter, R. Benner, J. R. Pleasants, and B. S. Wostmann. "Frequency analysis of functional immunoglobulin C- and V-gene expression by mitogen-reactive B cells in germfree mice fed chemically defined ultra-filtered "antigen-free" diet." Journal of Immunology 134, no. 4 (1985): 2223–27. http://dx.doi.org/10.4049/jimmunol.134.4.2223.
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Abstract The frequencies of lipopolysaccharide (LPS)-reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of conventional (CV) and "antigen-free" C3H/HeCr mice of various ages. The antigen-free mice were germfree (GF)-raised and were fed an ultrafiltered solution of chemically defined (CD) low m.w. nutrients, and were thus devoid of exogenous antigenic stimulation. Spleen and BM cells were grown in a limiting dilution culture system that allows the growth and development of every newly formed LPS-reactive B cell into a clone of IgM-secreting cells which are capable of switching to other immunoglobulin (Ig) heavy chain isotypes (C-gene expression). The secretion of IgM and IgG1 was determined in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V-gene expression) were detected in plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The absolute frequency of LPS-reactive B cells and their capacity to switch to IgG1-secretion was not significantly different in 8- to 12-wk-old and 52-wk-old GF-CD mice and their age-matched CV controls. Moreover, no differences were observed in the frequencies of antigen-specific B cells within the pool of LPS reactive B cells. These frequencies ranged from 1 in 20 to 1 in 50 for NIP4-SRBC and NNP2-SRBC, from 1 in 100 to 1 in 150 for NIP0.4-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 2000 for SRBC and horse red blood cells. Within the limitations of having determined the switching capacity of IgM to IgG1 only and having assessed only a minor fraction of the total B cell antibody-specificity repertoire, the data indicate that young and old GF-CD mice, although devoid of exogenous antigenic and/or mitogenic stimulation, generate B cells with a similar switching capacity and a similar IgM antibody specificity repertoire as CV mice.
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Lacroix-Desmazes, Sébastien, Igor Resnick, Dorothea Stahl, et al. "Defective Self-Reactive Antibody Repertoire of Serum IgM in Patients with Hyper-IgM Syndrome." Journal of Immunology 162, no. 9 (1999): 5601–8. http://dx.doi.org/10.4049/jimmunol.162.9.5601.
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Abstract We have analyzed the self-reactive repertoires of IgM and IgG Abs in the serum of 19 patients with hyper-IgM syndrome (HIM) by means of a quantitative immunoblotting technique that allows for a quantitative comparison of Ab repertoires in health and disease by multiparametric statistical analysis. Normal tissue extracts of liver, lung, stomach, and kidney were used as sources of self Ags. Extracts of Pseudomonas aeruginosa and Staphylococcus epidermidis were used as sources of nonself Ags. We demonstrate a significant bias in repertoires of reactivities of IgM of patients with HIM with self Ags. Ab repertoires of IgM toward nonself Ags did not differ, however, between patients and controls. No difference was found between IgM repertoires of untreated patients and those of patients receiving substitutive treatment with i.v. IgG. IgG in the serum of HIM patients lacked reactivity with self Ags, although it exhibited a pattern of reactivity with nonself Ags that was similar to that of IgG of healthy controls. The data demonstrate that functional CD40-CD40 ligand interactions are essential for the selection of natural self-reactive B cell repertoires.
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Nielsen, Morten A., Lasse S. Vestergaard, John Lusingu, et al. "Geographical and Temporal Conservation of Antibody Recognition of Plasmodium falciparum Variant Surface Antigens." Infection and Immunity 72, no. 6 (2004): 3531–35. http://dx.doi.org/10.1128/iai.72.6.3531-3535.2004.
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ABSTRACT The slow acquisition of protection against Plasmodium falciparum malaria probably reflects the extensive diversity of important antigens. The variant surface antigens (VSA) that mediate parasite adhesion to a range of host molecules are regarded as important targets of acquired protective immunity, but their diversity makes them questionable vaccine candidates. We determined levels of VSA-specific immunoglobulin G (IgG) in human plasma collected at four geographically distant and epidemiologically distinct localities with specificity for VSA expressed by P. falciparum isolates from three African countries. Plasma levels of VSA-specific IgG recognizing individual parasite isolates depended on the transmission intensity at the site of plasma collection but were largely independent of the geographical origin of the parasites. The total repertoire of immunologically distinct VSA thus appears to be finite and geographically conserved, most likely due to functional constraints. Furthermore, plasma samples frequently had high IgG reactivity to VSA expressed by parasites isolated more than 10 years later, showing that the repertoire is also temporally stable. Parasites from patients with severe malaria expressed VSA (VSASM) that were better recognized by plasma IgG than VSA expressed by other parasites, but importantly, VSASM-type antigens also appeared to show substantial antigenic homogeneity. Our finding that the repertoire of immunologically distinct VSA in general, and in particular that of VSASM, is geographically and temporally conserved raises hopes for the feasibility of developing VSA-based vaccines specifically designed to accelerate naturally acquired immunity, thereby enhancing protection against severe and life-threatening P. falciparum malaria.
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Papp, Krisztián, Péter Végh, Kata Miklós, et al. "Detection of Complement Activation on Antigen Microarrays Generates Functional Antibody Profiles and Helps Characterization of Disease-Associated Changes of the Antibody Repertoire." Journal of Immunology 181, no. 11 (2008): 8162–69. http://dx.doi.org/10.4049/jimmunol.181.11.8162.
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Kumar, Rashmi, Martina P. Bach, Federica Mainoldi, et al. "Antibody repertoire diversification through VH gene replacement in mice cloned from an IgA plasma cell." Proceedings of the National Academy of Sciences 112, no. 5 (2015): E450—E457. http://dx.doi.org/10.1073/pnas.1417988112.
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In mammals, VDJ recombination is responsible for the establishment of a highly diversified preimmune antibody repertoire. Acquisition of a functional Ig heavy (H) chain variable (V) gene rearrangement is thought to prevent further recombination at the IgH locus. Here, we describe VHQ52NT; Vκgr32NT Ig monoclonal mice reprogrammed from the nucleus of an intestinal IgA+ plasma cell. In VHQ52NT mice, IgA replaced IgM to drive early B-cell development and peripheral B-cell maturation. In VHQ52NT animals, over 20% of mature B cells disrupted the single productive, nonautoimmune IgH rearrangement through VH replacement and exchanged it with a highly diversified pool of IgH specificities. VH replacement occurred in early pro-B cells, was independent of pre–B-cell receptor signaling, and involved predominantly one adjacent VH germ-line gene. VH replacement was also identified in 5% of peripheral B cells of mice inheriting a different productive VH rearrangement expressed in the form of an IgM H chain. In summary, editing of a productive IgH rearrangement through VH replacement can account for up to 20% of the IgH repertoire expressed by mature B cells.
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Perlmutter, R. M., B. Berson, J. A. Griffin, and L. Hood. "Diversity in the germline antibody repertoire. Molecular evolution of the T15 VN gene family." Journal of Experimental Medicine 162, no. 6 (1985): 1998–2016. http://dx.doi.org/10.1084/jem.162.6.1998.
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The T15 heavy chain variable region (VH) gene family in BALB/c mice includes four elements each greater than 88% homologous with the other. One of these elements, V1, encodes virtually all of the VH regions in BALB/c antiphosphorylcholine antibodies, while another element, V3, is a pseudogene and cannot be transcribed or translated. We have examined the structural features of this VH gene family in other mouse strains and, in particular, have cloned and sequenced the alleles of these gene segments present in B10.P mice. Each of the four B10.P sequences can be matched with its allelic counterpart in BALB/c mice. This represents the first successful analysis of allelism in antibody variable region gene segments. The V1B10.P allele, like its BALB/c counterpart, encodes most of the known phosphorylcholine binding heavy chains from C37BL/6 mice. Similarly, the V3B10.P gene segment is a pseudogene like V3BALB, although only two of four abnormalities present in the BALB/c allele are also present in the B10.P allele. Careful analysis of the specific substitutions observed in the T15 VH gene family suggests that environmental selection for functional combining regions contributes significantly to the pattern of variation in the germline antibody repertoire. In addition, evidence is presented supporting frequent gene conversion events in the divergence of antibody genes.
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Coutant, Frédéric, Jean-Jacques Pin, Florence Morfin-Sherpa, et al. "Impact of Host Immune Status on Discordant Anti-SARS-CoV-2 Circulating B Cell Frequencies and Antibody Levels." International Journal of Molecular Sciences 22, no. 20 (2021): 11095. http://dx.doi.org/10.3390/ijms222011095.
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Individuals with pre-existing chronic systemic low-grade inflammation are prone to develop severe COVID-19 and stronger anti-SARS-CoV-2 antibody responses. Whether this phenomenon reflects a differential expansion of antiviral B cells or a failure to regulate antibody synthesis remains unknown. Here, we compared the antiviral B cell repertoire of convalescent healthcare personnel to that of hospitalized patients with pre-existing comorbidities. Out of 277,500 immortalized B cell clones, antiviral B cell frequencies were determined by indirect immunofluorescence screening on SARS-CoV-2 infected cells. Surprisingly, frequencies of SARS-CoV-2 specific clones from the two groups were not statistically different, despite higher antibody levels in hospitalized patients. Moreover, functional analyses revealed that several B cell clones from healthcare personnel with low antibody levels had neutralizing properties. This study reveals for the first time a key qualitative defect of antibody synthesis in severe patients and calls for caution regarding estimated protective immunity based only on circulating antiviral antibodies.
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Carlson, Chris, Eline Luning Prak, Jeanne Dagloria, et al. "Functional IGH V polymorphism is frequent in humans: implications for immunity and vaccination (HUM8P.349)." Journal of Immunology 192, no. 1_Supplement (2014): 185.24. http://dx.doi.org/10.4049/jimmunol.192.supp.185.24.
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Abstract Antibody heavy chain variable region genes (VH) are important for antigen binding, as evidenced by the dominant usage of particular VHs, such as the VH3b subfamily, in the immune responses to the capsular polysaccharide of H. influenza b. The presence or absence of particular VH alleles may be important for immunity. To screen for polymorphic VH alleles in humans, we sequenced functionally rearranged VH from 500K circulating B cells in each of 100 healthy young adults. We focused on the naïve repertoire (0-1 mutations compared to the closest known germline allele), and analyzed the proportion of rearrangements that were in-frame without stop codons (IF). In mice, low IF VHs have not undergone robust selection and are often pseudogenes. We confirmed several polymorphically functional VH genes with variable IF fractions, including V3-20, V5-a, V3-f, V1-8, and V3-9. We also identified homozygous individuals carrying novel, non-functional alleles at V7-4-1, V6-1, V3-66 and V2-70. Comparison of VH usage patterns between individuals revealed significant variation in the number of functional VH contributing to the naïve repertoire, with between 36 and 43 functional VH segments observed in each individual. These data demonstrate a significant level of functional allelic diversity and VH copy number variation between individuals, which could contribute to differential disease susceptibility, as well as influence vaccine efficacy.
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Heeb, Silvan Rolf, Monica Schaller, and Johanna Anna Kremer Hovinga. "Anti-idiotypic network involved in restoring ADAMTS13 activity in immune-mediated thrombotic thrombocytopenic purpura (iTTP)." Journal of Immunology 204, no. 1_Supplement (2020): 224.29. http://dx.doi.org/10.4049/jimmunol.204.supp.224.29.
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Abstract The culprit of iTTP, a fatal autoimmune disease is a severe deficiency of the von Willebrand factor-cleaving protease ADAMTS13, caused by autoantibodies inhibiting its enzymatic activity. The pathophysiology upholding remission in certain patients but leading to relapse in others is unknown. We hypothesize that one mechanism keeping pathogenic autoantibodies in check is a network of anti-idiotypic antibodies, which we aimed to identify and characterize. From splenic mononuclear cells of two frequently relapsing iTTP patients (A and C), the anti-idiotypic IgG1 Fab κ/λ repertoire was amplified by phage display technology on previously generated selecting monoclonal anti-ADAMTS13 Fabs (Schaller et al., Blood 2014). Purified Fabs were pooled (pools A (n=4) and C (n=4)) and each pool was tested for its ability to neutralize anti-ADAMTS13 antibodies thereby restoring ADAMTS13 activity in plasma of 22 iTTP patients using the FRETS-vWF73 assay. Single anti-idiotypic clones revealed a highly diverse IGHV gene usage within and between the two iTTP patients, but four IGHV genes were shared among them. Partial neutralization of functional ADAMTS13 inhibitors of &gt;2BU/ml in 3/11 (27%) and 5/11 (45%) patients and full neutralization in 2/11 (18%) and 3/11 (27%) patients with lower inhibitor titer (1–2BU/ml) were shown for pools A and C, respectively. To summarize, we have identified functional anti-ADAMTS13 anti-idiotypes in the splenic IgG repertoire of two patients underlining the role of an anti-idiotypic response involved in remission of iTTP. The contribution of single anti-idiotypic Fabs to the neutralization potential and plasma mixing studies investigating acute and remission phase antibody repertoires are underway.
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Bende, Richard J., Wilhelmina M. Aarts, Robert G. Riedl, Daphne de Jong, Steven T. Pals, and Carel J. M. van Noesel. "Among B cell non-Hodgkin's lymphomas, MALT lymphomas express a unique antibody repertoire with frequent rheumatoid factor reactivity." Journal of Experimental Medicine 201, no. 8 (2005): 1229–41. http://dx.doi.org/10.1084/jem.20050068.
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We analyzed the structure of antigen receptors of a comprehensive panel of mature B non-Hodgkin's lymphomas (B-NHLs) by comparing, at the amino acid level, their immunoglobulin (Ig)VH-CDR3s with CDR3 sequences present in GenBank. Follicular lymphomas, diffuse large B cell lymphomas, Burkitt's lymphomas, and myelomas expressed a CDR3 repertoire comparable to that of normal B cells. Mantle cell lymphomas and B cell chronic lymphocytic leukemias (B-CLLs) expressed clearly restricted albeit different CDR3 repertoires. Lymphomas of mucosa-associated lymphoid tissues (MALTs) were unique as 8 out of 45 (18%) of gastric- and 13 out of 32 (41%) of salivary gland-MALT lymphomas expressed B cell antigen receptors with strong CDR3 homology to rheumatoid factors (RFs). Of note, the RF-CDR3 homology without exception included N-region–encoded residues in the hypermutated IgVH genes, indicating that they were stringently selected for reactivity with auto-IgG. By in vitro binding studies with 10 MALT lymphoma–derived antibodies, we showed that seven of these cases, of which four with RF-CDR3 homology, indeed possessed strong RF reactivity. Of one MALT lymphoma, functional proof for selection of subclones with high RF affinity was obtained. Interestingly, RF-CDR3 homology and t(11;18) appeared to be mutually exclusive features and RF-CDR3 homology was not encountered in any of the 19 pulmonary MALT lymphomas studied.
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VanBlargan, Laura A., Leslie Goo, and Theodore C. Pierson. "Deconstructing the Antiviral Neutralizing-Antibody Response: Implications for Vaccine Development and Immunity." Microbiology and Molecular Biology Reviews 80, no. 4 (2016): 989–1010. http://dx.doi.org/10.1128/mmbr.00024-15.
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SUMMARYThe antibody response plays a key role in protection against viral infections. While antiviral antibodies may reduce the viral burden via several mechanisms, the ability to directly inhibit (neutralize) infection of cells has been extensively studied. Eliciting a neutralizing-antibody response is a goal of many vaccine development programs and commonly correlates with protection from disease. Considerable insights into the mechanisms of neutralization have been gained from studies of monoclonal antibodies, yet the individual contributions and dynamics of the repertoire of circulating antibody specificities elicited by infection and vaccination are poorly understood on the functional and molecular levels. Neutralizing antibodies with the most protective functionalities may be a rare component of a polyclonal, pathogen-specific antibody response, further complicating efforts to identify the elements of a protective immune response. This review discusses advances in deconstructing polyclonal antibody responses to flavivirus infection or vaccination. Our discussions draw comparisons to HIV-1, a virus with a distinct structure and replication cycle for which the antibody response has been extensively investigated. Progress toward deconstructing and understanding the components of polyclonal antibody responses identifies new targets and challenges for vaccination strategies.
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Lucas, Alexander H., and Donald C. Reason. "Aging and the Immune Response to theHaemophilus influenzae Type b Capsular Polysaccharide: Retention of the Dominant Idiotype and Antibody Function in the Elderly." Infection and Immunity 66, no. 4 (1998): 1752–54. http://dx.doi.org/10.1128/iai.66.4.1752-1754.1998.
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ABSTRACT Anti-Haemophilus influenzae b polysaccharide (Hib PS) antibodies elicited in elderly subjects following conjugate vaccination expressed a light-chain variable-region (VL)-associated idiotype and had functional activities similar to those previously observed in children and younger adults. These findings indicate that advanced age is not accompanied by shifts in the major VLcomponent of the Hib PS-specific repertoire or by diminution of the protective function of antibodies.
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Sesterhenn, Fabian, Che Yang, Jaume Bonet, et al. "De novo protein design enables the precise induction of RSV-neutralizing antibodies." Science 368, no. 6492 (2020): eaay5051. http://dx.doi.org/10.1126/science.aay5051.
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De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines. Here, we present a protein design algorithm called TopoBuilder, with which we engineered epitope-focused immunogens displaying complex structural motifs. In both mice and nonhuman primates, cocktails of three de novo–designed immunogens induced robust neutralizing responses against the respiratory syncytial virus. Furthermore, the immunogens refocused preexisting antibody responses toward defined neutralization epitopes. Overall, our design approach opens the possibility of targeting specific epitopes for the development of vaccines and therapeutic antibodies and, more generally, will be applicable to the design of de novo proteins displaying complex functional motifs.
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Haynes, Joel, Virginia Perry, Evelyn Benson, et al. "In Depth Breadth Analyses of Human Blockade Responses to Norovirus and Response to Vaccination." Viruses 11, no. 5 (2019): 392. http://dx.doi.org/10.3390/v11050392.
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To evaluate and understand the efficacy of vaccine candidates, supportive immunological measures are needed. Critical attributes for a norovirus vaccine are the strength and breadth of antibody responses against the many different genotypes. In the absence of suitable neutralization assays to test samples from vaccine clinical trials, blockade assays offer a method that can measure functional antibodies specific for many of the different norovirus strains. This paper describes development and optimization of blockade assays for an extended panel of 20 different norovirus strains that can provide robust and reliable data needed for vaccine assessment. The blockade assays were used to test a panel of human clinical samples taken before and after vaccination with the Takeda TAK-214 norovirus vaccine. Great variability was evident in the repertoire of blocking antibody responses prevaccination and postvaccination among individuals. Following vaccination with TAK-214, blocking antibody levels were enhanced across a wide spectrum of different genotypes. The results indicate that adults may have multiple exposures to norovirus and that the magnitude and breadth of the complex preexisting antibody response can be boosted and expanded by vaccination.
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Gajula, Kiran S., Peter J. Huwe, Charlie Y. Mo, et al. "High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase." Nucleic Acids Research 42, no. 15 (2014): 9964–75. http://dx.doi.org/10.1093/nar/gku689.
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Abstract Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9–11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement. To this end, we developed a methodology (Sat-Sel-Seq) that couples saturation mutagenesis at each position across the targeting loop, with iterative functional selection and next-generation sequencing. This high-throughput mutational analysis revealed dominant characteristics for residues within the loop and additionally yielded enzymatic variants that enhance deaminase activity. To rationalize these functional requirements, we performed molecular dynamics simulations that suggest that AID and its hyperactive variants can engage DNA in multiple specific modes. These findings align with AID's competing requirements for specificity and flexibility to efficiently drive antibody maturation. Beyond insights into the AID-DNA interface, our Sat-Sel-Seq approach also serves to further expand the repertoire of techniques for deep positional scanning and may find general utility for high-throughput analysis of protein function.
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Ahmed, Rizwan, Zahra Omidian, Adebola Giwa, et al. "A newly discovered dual expresser lymphocyte that clonally expanded in Type 1 diabetes (T1D) patients secretes a public antibody that recognize public TCR in T1D." Journal of Immunology 204, no. 1_Supplement (2020): 142.10. http://dx.doi.org/10.4049/jimmunol.204.supp.142.10.
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Abstract PURPOSE We have recently discovered a new lymphocyte that co-express BCR and TCR (Ahmed et al, Cell, 2019: 177:11583) and referred as X cell to denote its crossover phenotype. Importantly, X cells express a public BCR that also encodes a potent autoantigen in its CDR3 sequence that is 10 fold more potent than native insulin peptide (InsB:9–23) in binding to DQ8 and activating autologous CD4 T cells. The x-autoantigen cross-activate insulin specific CD4 T cells as a peptide in the context of HLA-DQ8 molecules or as a soluble intact mAb (x-mAb). The goal of this study is to characterize autoreactive CD4 T cells that are responsive to x-mAb to determine their phenotype, cytokine profile and TCR repertoire and whether they express public TCRs. METHODS We used EBV-lymphoblastoid X cell clone as a source of x-mAb (IgM) and FACS based protocol to identify IgM reactive CD4 T cells (referred as IgMpos) and their functional properties. ImmunoSEQ assay used to characterize TCR repertoires. RESULTS Preliminary data show that frequency of IgMpos CD4 T cells is significantly higher in T1D as compared to Healthy subjects. In addition, IgMpos CD4 T cells exhibit an activated phenotype as compared to autologous IgMneg CD4 T cells, including expression of CD45RO, CD44, and CD69. Analysis of TCRVβ repertoire shows that IgMpos CD4 T cells are enriched for public clonally expanded TCRs as compared to IgMneg counterparts. CONCLUSIONS X cells in T1D patients are predominated by a single public BCR and that the secreted version of this BCR (x-mAb) is autoreactive against a specific subset of CD4 T cells that predominated by few clonotypes that express public TCRs. Our results are revealing previously unknown mechanism that appears to be a play critical role in pathogenesis of T1D.
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Yawata, Makoto, Nobuyo Yawata, Monia Draghi, Ann-Margaret Little, Fotini Partheniou, and Peter Parham. "Roles for HLA and KIR polymorphisms in natural killer cell repertoire selection and modulation of effector function." Journal of Experimental Medicine 203, no. 3 (2006): 633–45. http://dx.doi.org/10.1084/jem.20051884.
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Interactions between killer cell immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) class I ligands regulate the development and response of human natural killer (NK) cells. Natural selection drove an allele-level group A KIR haplotype and the HLA-C1 ligand to unusually high frequency in the Japanese, who provide a particularly informative population for investigating the mechanisms by which KIR and HLA polymorphism influence NK cell repertoire and function. HLA class I ligands increase the frequencies of NK cells expressing cognate KIR, an effect modified by gene dose, KIR polymorphism, and the presence of other cognate ligand–receptor pairs. The five common Japanese KIR3DLI allotypes have distinguishable inhibitory capacity, frequency of cellular expression, and level of cell surface expression as measured by antibody binding. Although KIR haplotypes encoding 3DL1*001 or 3DL1*005, the strongest inhibitors, have no activating KIR, the dominant haplotype encodes a moderate inhibitor, 3DL1*01502, plus functional forms of the activating receptors 2DL4 and 2DS4. In the population, certain combinations of KIR and HLA class I ligand are overrepresented or underrepresented in women, but not men, and thus influence female fitness and survival. These findings show how KIR–HLA interactions shape the genetic and phenotypic KIR repertoires for both individual humans and the population.
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Malkiel, Susan, Christopher Kuhlow, Patricio Mena, Gloria Monsalve, and Jorge Benach. "Mice with the Xid defect have impaired control against Borrelia burgdorferi infection but are more resistant to carditis (38.5)." Journal of Immunology 184, no. 1_Supplement (2010): 38.5. http://dx.doi.org/10.4049/jimmunol.184.supp.38.5.
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Abstract The antibody response is the main form of protection against Borrelia. Antibodies are functional in the elimination of the spirochetes and in resolution of disease. T cell-independent antibodies have been shown to have a protective role in defending the host against the Lyme disease spirochete B. burgdorferi. To determine the role of B1 cells in this antibody response, X-linked immunodeficient (Xid) CBA/N mice were used because they are severely deficient in B1 cells. Spirochete burdens were enhanced in the Xid mice compared to CBA/Ca controls at 3, 4 and 8 weeks postinfection, which may be explained by reduced levels of B. burgdorferi-specific IgM in the Xid mice. Meanwhile, the antibody repertoire of the IgM response to B.burgdorferi antigens appeared similar in both groups of mice by immunoblot. Despite impaired pathogen clearance, the Xids developed comparable arthritis but less carditis than controls. These results suggest that B1 cells play a dichotomous role in protecting the host against infection while promoting inflammation in the heart.
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Voss, William N., Yixuan J. Hou, Nicole V. Johnson, et al. "Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes." Science 372, no. 6546 (2021): 1108–12. http://dx.doi.org/10.1126/science.abg5268.
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The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an amino (N)-terminal domain (NTD)–directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multidonor class of “public” antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that “public” NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.
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Hughes, Darren L., Prachi Stafford, Samir W. Hamaia та ін. "Platelet integrin α2 I-domain specific antibodies produced via domain specific DNA vaccination combined with variable gene phage display". Thrombosis and Haemostasis 94, № 12 (2005): 1318–26. http://dx.doi.org/10.1160/th05-06-0410.
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SummaryAntibodies are a powerful tool for structure/function studies of platelet proteins. However, classic immunisation frequently elicits antibody responses against domains of minor functional interest. Robust strategies to generate antibodies against defined domains would be of significant interest in post-genome research. In this study, we report a new strategy using a combination of DNA vaccination and V gene phage display that allows the rapid generation of domain specific single-chain Fv antibodies (scFvs).This system was validated using the I-domain of α2 integrin as a model. The α2β1 integrin, which is expressed on many cell types, is the dominant collagen attachment receptor on platelets, functioning in close interplay with the collagen signalling receptor glycoproteinVI. A novel set of I-domain specific antibodies was obtained by a DNA vaccination/V gene repertoire cloning approach. Mice were first immunized with a DNA vaccine in which the α2 I-domain is expressed as a fusion protein with fragment C of tetanus toxoid (FrC-TT).Then the heavy and kappa light chain variable gene repertoires were rescued from immune splenocytes using antibody phage display. A total of four α2 I-domain specific scFvs were isolated by selection on recombinant I-domain or native platelet α2β1 integrin. Characterisation of the scFvs indicated that they recognised distinct epitopes that had profound differences in accessibility between native and recombinant I-domain. Our data suggest DNA immunisation and phage display represent versatile alternatives to protein immunisation and hybridoma-fusion techniques for the isolation of recombinant antibody reagents. This approach will be particularly useful for the generation of domain or splicevariant specific antibodies that recognise native protein.
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Zheng, Biao, Lei Fang, Qi Zhang, Wen Jiang, Shuhua Han, and Jun Qin. "Atg7 is critical for the functional maturation of germinal center B cells (LYM7P.625)." Journal of Immunology 194, no. 1_Supplement (2015): 200.17. http://dx.doi.org/10.4049/jimmunol.194.supp.200.17.
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Abstract The role of autophagy, especially individual components of the autophagy machinery, in antibody response has not been well established. Atg7 is an integral part in autophagesome formation and elongation. Using lineage- and stage-specific conditional knockout (CKO) mice, we have found that, in GC B cell-specific Atg7 CKO mice, although GC formation and structure were not affected by Atg7 deficiency, both primary and memory antibody responses were severely impaired in Atg7 CKO mice. Class-switch recombination (CSR) and Ig somatic hypermutation (SHM) were defective in Atg7-/- GC B-cells, leading to a severely inhibited production of class-switched high-affinity antibodies. Analysis of GC B-cell clones showed that Atg7-deficiency in GC B-cells causes a dramatic change in Ig gene composition in the responding B-cell repertoire, which is dominated by non-canonical Ig genes encoding lower affinity antibodies. Thus, our study demonstrated for the first time that a single gene mutation affects GC function without inhibiting GC formation. The underlying molecular mechanism for impaired GC function in Atg7 mutant mice is that Atg7 is critical in regulating activation-induced deaminase (AID). Atg7-deficiency in GC B cells led to delayed onset and reduced disease severity in collagen-induced arthritis (CIA), demonstrating that Atg7 is critical in the development and pathogenesis of autoimmune disease.
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Sherburn, Rebekah, William D. Tolbert, Suneetha Gottumukkala, Guillaume Beaudoin-Bussières, Andrés Finzi, and Marzena Pazgier. "Effects of gp120 Inner Domain (ID2) Immunogen Doses on Elicitation of Anti-HIV-1 Functional Fc-Effector Response to C1/C2 (Cluster A) Epitopes in Mice." Microorganisms 8, no. 10 (2020): 1490. http://dx.doi.org/10.3390/microorganisms8101490.
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Fc-mediated effector functions of antibodies, including antibody-dependent cytotoxicity (ADCC), have been shown to contribute to vaccine-induced protection from HIV-1 infection, especially those directed against non-neutralizing, CD4 inducible (CD4i) epitopes within the gp120 constant 1 and 2 regions (C1/C2 or Cluster A epitopes). However, recent passive immunization studies have not been able to definitively confirm roles for these antibodies in HIV-1 prevention mostly due to the complications of cross-species Fc–FcR interactions and suboptimal dosing strategies. Here, we use our stabilized gp120 Inner domain (ID2) immunogen that displays the Cluster A epitopes within a minimal structural unit of HIV-1 Env to investigate an immunization protocol that induces a fine-tuned antibody repertoire capable of an effective Fc-effector response. This includes the generation of isotypes and the enhanced antibody specificity known to be vital for maximal Fc-effector activities, while minimizing the induction of isotypes know to be detrimental for these functions. Although our studies were done in in BALB/c mice we conclude that when optimally titrated for the species of interest, ID2 with GLA-SE adjuvant will elicit high titers of antibodies targeting the Cluster A region with potent Fc-mediated effector functions, making it a valuable immunogen candidate for testing an exclusive role of non-neutralizing antibody response in HIV-1 protection in vaccine settings.
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Kretschmer, Karsten, Anke Jungebloud, Jana Stopkowicz, Britta Stoermann, Reinhard Hoffmann, and Siegfried Weiss. "Antibody Repertoire and Gene Expression Profile: Implications for Different Developmental and Functional Traits of Splenic and Peritoneal B-1 Lymphocytes." Journal of Immunology 171, no. 3 (2003): 1192–201. http://dx.doi.org/10.4049/jimmunol.171.3.1192.
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Dale, Gordon A., Caitlin D. Bohannon, Daniel J. Wilkins, et al. "Gene conversion contributes significantly to IgHV somatic hypermutation in mice and humans." Journal of Immunology 198, no. 1_Supplement (2017): 195.7. http://dx.doi.org/10.4049/jimmunol.198.supp.195.7.
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Abstract During antigen-driven affinity maturation germinal center B lymphocytes undergo multiple rounds of somatic mutagenesis in the rearranged IgHV gene segment. A direct consequence of this process is the generation of multiple antibody mutants that are, in general, specific for antigen. Survival of these mutants is contingent on preserving or improving antigen-specificity in the complementarity-determining regions (CDRs) as well as retaining a functional framework region (FWR). Broadly, somatic mutations in germinal center B cells can be classified as templated or untemplated. In animals such as rabbits and chickens, somatic mutations are mostly templated. Conversely, in mice and humans it is widely accepted that mutations are untemplated. Here, we argue in favor of the alternative, that a predominant number of somatic mutations in IgHV gene segments in mice and humans can be traced to germline IgHV genes and occur through gene conversion. Using the NP hapten model, we demonstrate that mutations in germinal center B cells and plasma cells are traceable to other germline IgHV genes. Subsequently we also demonstrate using the novel V-LAIR1-DJ antibodies described by Tan et al (Nature 2016) that mutations in human antibody IgHV rearrangements, including those in non-immunoglobulin sequences (LAIR1), are traceable to the human IgHV germline repertoire. Together, this suggests that the plethora of diversity generated in antigen specific B cells is primarily directed by the germline repertoire of IgHV genes.
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Pinschewer, Daniel D., Bénédict Fallet, Yi Hao, et al. "Chronic viral infection promotes efficient germinal center B cell responses." Journal of Immunology 204, no. 1_Supplement (2020): 247.4. http://dx.doi.org/10.4049/jimmunol.204.supp.247.4.
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Abstract Persistent viral infections subvert key elements of adaptive immunity. To compare germinal center (GC) B cell responses in chronic and acute lymphocytic choriomeningitis virus infection we exploit activation-induced deaminase (AID) fate-reporter mice and perform adoptive B cell transfer experiments. Chronic infection yields GC B cell responses of higher cellularity than acute infection, higher memory B cell and antibody secreting cell output for longer periods of time, a better representation of the late B cell repertoire in serum immunoglobulin and higher titers of protective neutralizing antibodies. GC B cells of chronically infected mice are similarly hypermutated as those emerging from acute infection. They efficiently adapt to viral escape variants and even in hypermutation-impaired AID-mutant mice, chronic infection selects for GC B cells with hypermutated BCRs and neutralizing antibody formation. These findings demonstrate that, unlike for CD8+ T cells, chronic viral infection drives a functional, productive and protective GC B cell response.
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Fassò, Marcella, Niroshana Anandasabapathy, Frances Crawford, John Kappler, C. Garrison Fathman та William M. Ridgway. "T Cell Receptor (Tcr)-Mediated Repertoire Selection and Loss of Tcr Vβ Diversity during the Initiation of a Cd4+ T Cell Response in Vivo". Journal of Experimental Medicine 192, № 12 (2000): 1719–30. http://dx.doi.org/10.1084/jem.192.12.1719.
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We recently described a novel way to isolate populations of antigen-reactive CD4+ T cells with a wide range of reactivity to a specific antigen, using immunization with a fixed dose of nominal antigen and FACS® sorting by CD4high expression. Phenotypic, FACS®, functional, antibody inhibition, and major histocompatibility complex–peptide tetramer analyses, as well as T cell receptor Vβ sequence analyses, of the antigen-specific CD4high T cell populations demonstrated that a diverse sperm whale myoglobin 110–121–reactive CD4+ T cell repertoire was activated at the beginning (day 3 after immunization) of the immune response. Within 6 d of immunization, lower affinity clones were lost from the responding population, leaving an expanded population of oligoclonal, intermediate affinity (and residual high affinity) T cells. This T cell subset persisted for at least 4 wk after immunization and dominated the secondary immune response. These data provide evidence that CD4+ T cell repertoire selection occurs early in the immune response in vivo and suggest that persistence and expansion of a population of oligoclonal, intermediate affinity T cells is involved in CD4+ T cell memory.
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Dzutsev, Amiran, Igor Belyakov, Dmitry Isakov, David Margulies, and Jay Berzofsky. "Avidity of CD8 T-cells sharpens immunodominance. (B9)." Journal of Immunology 178, no. 1_Supplement (2007): LB2. http://dx.doi.org/10.4049/jimmunol.178.supp.b9.
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Abstract In the course of viral infection, the immune system exploits only a fraction of the available CTL repertoire and focuses on a few of a myriad of potentially antigenic peptides. This phenomenon, known as immunodominance, depends on a number of factors, including antigen processing and transport, MHC binding, competition for antigen presenting cells, availability of the CD8 T-cell repertoire and other mechanisms that function largely by restricting the immune response. Here we elucidate a novel mechanism that increases the immunodominance of the epitope rather by enhancing the immune response. Using a peptide-specific MHC-restricted monoclonal antibody and functional assays of CTL activation, we show that T cells with high avidity for the immunodominant, H-2Dd-restricted, P18-I10 epitope expand rapidly following immunization, and this expansion in turn determines the level of the P18-I10 epitope immunodominance. This proliferation has little dependence on the number of MHC-peptide complexes. Since most self-reactive T-cells of high avidity are depleted in the thymus, the selection of immunodominant epitopes based on the expansion of high avidity T-cells in the periphery avoids the potential for autoimmunity.
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Haire, R. N., R. D. Buell, R. T. Litman, et al. "Diversification, not use, of the immunoglobulin VH gene repertoire is restricted in DiGeorge syndrome." Journal of Experimental Medicine 178, no. 3 (1993): 825–34. http://dx.doi.org/10.1084/jem.178.3.825.
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Immunoglobulin (Ig) genes were isolated from unamplified conventional as well as polymerase chain reaction-generated cDNA libraries constructed from the peripheral blood cells of a patient with complete DiGeorge syndrome. Comparison of the sequences of 36 heavy chain clones to the recently expanded database of human VH genes permitted identification of the germline VH genes that are expressed in this patient as well as placement of 19 of these genes in a partially resolved 0.8-mb region of the human VH locus. The pattern of VH gene use does not resemble the fetal (early) repertoire. However, as in the fetal repertoire, there are a number of cDNAs derived from germline genes that previously have been identified as autoantibodies. Two D mu sequences also were identified, as was another sequence resulting from a unique recombination event linking JH to an unidentified sequence containing a recombination signal sequence-like heptamer. All of the DiGeorge cDNAs are closely related to germline VH genes, showing little or no evidence of somatic mutation. In contrast, comparably selected IgM VH sequences derived from normal adult and age-matched human libraries, and from a second DiGeorge syndrome patient in whom the degree of thymic dysfunction is much less severe, exhibit considerable evidence of somatic mutation. The absence of somatic mutation is consistent with the atypical development of functional antibody responses associated with complete DiGeorge syndrome and implicates a role for T cells in the generation of diversity within the B cell repertoire.
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Benedict, Cindy L., Susan Gilfillan, and John F. Kearney. "The Long Isoform of Terminal Deoxynucleotidyl Transferase Enters the Nucleus And, Rather than Catalyzing Nontemplated Nucleotide Addition, Modulates the Catalytic Activity of the Short Isoform." Journal of Experimental Medicine 193, no. 1 (2001): 89–100. http://dx.doi.org/10.1084/jem.193.1.89.
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During variable/diversity/joining (V[D]J) recombination, the enzyme terminal deoxynucleotidyl transferase (Tdt) adds random nucleotides at the junctions of the rearranging gene segments, increasing diversity of the antibody (Ab) and T cell receptor repertoires. Two splice variants of Tdt have been described, but only one (short isoform of Tdt [TdtS]) has been convincingly demonstrated to catalyze nontemplated (N) addition in vitro. We have expressed each splice variant of Tdt in transgenic (Tg) mice and found that the TdtS transgene catalyzes N addition on the endogenous Tdt−/− background and in fetal liver, but that the long isoform of Tdt (TdtL) transgene does neither. In contrast to previous in vitro results, both TdtS and TdtL are translocated to the nucleus in our model. Furthermore, TdtL/TdtS double Tg mice exhibit less N addition in fetal liver than do TdtS Tg mice. Whereas the TdtS transgene was shown to have functional consequences on the antiphosphorylcholine (PC) B cell repertoire, TdtL Tg mice exhibit a normal PC response, and Tdt−/− mice actually exhibit an increase in the PC response and in TEPC 15 idiotype+ Ab production. We conclude that TdtL localizes to the nucleus in vivo where it serves to modulate TdtS function.
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Prechl, József. "Network Organization of Antibody Interactions in Sequence and Structure Space: the RADARS Model." Antibodies 9, no. 2 (2020): 13. http://dx.doi.org/10.3390/antib9020013.
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Adaptive immunity in vertebrates is a complex self-organizing network of molecular interactions. While deep sequencing of the immune-receptor repertoire may reveal clonal relationships, functional interpretation of such data is hampered by the inherent limitations of converting sequence to structure to function. In this paper, a novel model of antibody interaction space and network, termed radial adjustment of system resolution, RAdial ADjustment of System Resolution (RADARS), is proposed. The model is based on the radial growth of interaction affinity of antibodies towards an infinity of directions in structure space, each direction corresponding to particular shapes of antigen epitopes. Levels of interaction affinity appear as free energy shells of the system, where hierarchical B-cell development and differentiation takes place. Equilibrium in this immunological thermodynamic system can be described by a power law distribution of antibody-free energies with an ideal network degree exponent of phi square, representing a scale-free fractal network of antibody interactions. Plasma cells are network hubs, memory B cells are nodes with intermediate degrees, and B1 cells function as nodes with minimal degree. Overall, the RADARS model implies that a finite number of antibody structures can interact with an infinite number of antigens by immunologically controlled adjustment of interaction energy distribution. Understanding quantitative network properties of the system should help the organization of sequence-derived predicted structural data.
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Pasman, Yfke, and Azad Kaushik. "Elucidating the role of bovine heavy- and light-chain variable domains in polyspecific antigen-binding (P6104)." Journal of Immunology 190, no. 1_Supplement (2013): 141.15. http://dx.doi.org/10.4049/jimmunol.190.supp.141.15.
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Abstract The bovine antibody repertoire is generated from a limited germline sequence divergence as well as combinatorial diversity. Generation of an exceptionally long CDR3H (up to 61 amino acids) in the heavy chain variable region (VH) provides an additional mechanism to generate antibody diversity, not found in other species to date. These long CDR3H loops, present in polyspecific IgM, originate from VDJ recombination encoded by a specific VH gene, unusually long single DH-gene (capable of encoding up to 51 codons) and insertion of 15-18 base long conserved short nucleotide sequences (CSNS) specifically at VH-DH junction. In cattle, it is thought that antigen-binding is mainly a function of the variable-region of the heavy chain where light chain provides only structural support. To test this hypothesis, single chain variable fragment (scFv) and single domains (Fd) from polyspecific IgM were constructed. Both purified scFv as well as FdVH showed polyspecific binding to structurally dissimilar antigens in an ELISA. Whether scFv and FdVH bind to multiple epitopes on an antigen or if they recognize the same epitope can now be determined. The structural-functional complexities of these antibody fragments and the role of the heavy- and light-chains in antigen-binding will be discussed. [Supported by NSERC Canada]
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Serpa, Jose A., Josemon Valayam, Daniel M. Musher, Roger D. Rossen, Liise-anne Pirofski, and Maria C. Rodriguez-Barradas. "VH3 Antibody Response to Immunization with Pneumococcal Polysaccharide Vaccine in Middle-Aged and Elderly Persons." Clinical and Vaccine Immunology 18, no. 3 (2011): 362–66. http://dx.doi.org/10.1128/cvi.00408-10.
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ABSTRACTPneumococcal 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 VH3 gene segments and induce a repertoire shift with increased VH3 expression in peripheral B cells. Here we compared VH3-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 VH3-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 VH3-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 VH3 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 VH3 antibody titers after vaccination. Age or repeated immunization does not appear to affect the VH3-idiotypic immunogenicity of PPV among middle-aged and elderly adults.
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Basu, Uttiya, Andrew Franklin, and Frederick W. Alt. "Post-translational regulation of activation-induced cytidine deaminase." Philosophical Transactions of the Royal Society B: Biological Sciences 364, no. 1517 (2008): 667–73. http://dx.doi.org/10.1098/rstb.2008.0194.
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The assembled immunoglobulin genes in the B cells of mice and humans are altered by distinct processes known as class switch recombination (CSR) and somatic hypermutation, leading to diversification of the antibody repertoire. These two DNA modification processes are initiated by the B cell-specific protein factor activation-induced cytidine deaminase (AID). AID is post-translationally modified by phosphorylation at multiple sites, although functional significance during CSR has been implicated only for phosphorylation at serine-38 (S38). Although multiple laboratories have demonstrated that AID function is regulated via phosphorylation at S38, the precise biological role of S38 phosphorylation has been a topic of debate. Here, we discuss our interpretation of the significance of AID regulation via phosphorylation and also discuss how this form of AID regulation may have evolved in higher organisms.
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Panagides, Nadya, Lucia F. Zacchi, Mitchell J. De Souza, et al. "Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies." International Journal of Molecular Sciences 23, no. 15 (2022): 8470. http://dx.doi.org/10.3390/ijms23158470.
Full textAbstract:
Monoclonal antibodies (mAbs) are one of the most successful and versatile protein-based pharmaceutical products used to treat multiple pathological conditions. The remarkable specificity of mAbs and their affinity for biological targets has led to the implementation of mAbs in the therapeutic regime of oncogenic, chronic inflammatory, cardiovascular, and infectious diseases. Thus, the discovery of novel mAbs with defined functional activities is of crucial importance to expand our ability to address current and future clinical challenges. In vitro, antigen-driven affinity selection employing phage display biopanning is a commonly used technique to isolate mAbs. The success of biopanning is dependent on the quality and the presentation format of the antigen, which is critical when isolating mAbs against membrane protein targets. Here, we provide a comprehensive investigation of two established panning strategies, surface-tethering of a recombinant extracellular domain and cell-based biopanning, to examine the impact of antigen presentation on selection outcomes with regards to the isolation of positive mAbs with functional potential against a proof-of-concept type I cell surface receptor. Based on the higher sequence diversity of the resulting antibody repertoire, presentation of a type I membrane protein in soluble form was more advantageous over presentation in cell-based format. Our results will contribute to inform and guide future antibody discovery campaigns against cell surface proteins.