Search

Although humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq (1) ) to the spike ectodomain (S-ECD (2) ) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing in vitro , we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified in vivo protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 Å (2) ). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning (3,4) our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.

Background PCV13 (conjugated polysaccharide) and PPSV23 (polysaccharide only) are two licensed vaccines targeting S. pneumoniae. The role of CD4 T-cell responses in pneumococcal vaccines among healthy participants and their impact on antibodies is not yet known. Methods Ten adults (5 old and 5 young) received PCV13 (prime) and a year later PPSV23 (boost). Blood samples were collected prior to and multiple time points after vaccination. CD4 T cells responding to CRM197, polysaccharide (PS), CRM197 conjugated polysaccharide (CPS), PCV13 and PPSV23 vaccines were measured by flow cytometry. Serum antibodies were analyzed via multiplex opsonophagocytosis (MOPA) and pneumococcal IgG assays. Results Vaccine-specific CD4 T cells were induced in all ten vaccinees post PCV13. Older vaccinees mounted higher peak responses and those specific for PCV13 and conjugated PS-1 were more polyfunctional compared to the younger group. Vaccine-elicited peripheral T follicular helper (Tfh) cells were only detected in the younger group who also exhibited a higher fold change in OPA titers post both vaccines. Importantly, Tfh cells following PCV13 correlated only with PCV13 serotype specific OPA titers after PPSV23 vaccination. Conclusions These findings demonstrate age related differences in immune response and the potential importance of Tfh in modulating functional antibody responses following pneumococcal vaccination.

Memory B cells (MBCs) and plasma antibodies against Plasmodium falciparum merozoite antigens are important components of the protective immune response against malaria. To gain understanding of how responses against P. falciparum develop in these two arms of the humoral immune system, we evaluated MBC and antibody responses against the most abundant merozoite antigen, merozoite surface protein 1 (MSP1), in individuals from a region in Uganda with high P. falciparum transmission. Our results showed that MSP1-specific B cells in adults with immunological protection against malaria were predominantly IgG+ classical MBCs, while children with incomplete protection mainly harbored IgM+ MSP1-specific classical MBCs. In contrast, anti-MSP1 plasma IgM reactivity was minimal in both children and adults. Instead, both groups showed high plasma IgG reactivity against MSP1 and whole merozoites, with broadening of the response against non-3D7 strains in adults. The antibodies encoded by MSP1-specific IgG+ MBCs carried high levels of amino acid substitutions and recognized relatively conserved epitopes on the highly variable MSP1 protein. Proteomics analysis of MSP119-specific IgG in plasma of an adult revealed a limited repertoire of anti-MSP1 antibodies, most of which were IgG1 or IgG3. Similar to MSP1- specific MBCs, anti-MSP1 IgGs had relatively high levels of amino acid substitutions and their sequences were predominantly found in classical MBCs, not atypical MBCs. Collectively, these results showed evolution of the MSP1-specific humoral immune response with cumulative P. falciparum exposure, with a shift from IgM+ to IgG+ B cell memory, diversification of B cells from germline, and stronger recognition of MSP1 variants by the plasma IgG repertoire.

Seasonal influenza vaccination elicits a diminished adaptive immune response in the elderly, and the mechanisms of immunosenescence are not fully understood. Using Ig-Seq, we found a marked increase with age in the prevalence of cross-reactive (CR) serum antibodies that recognize both the H1N1 (vaccine-H1) and H3N2 (vaccine-H3) components of an egg-produced split influenza vaccine. CR antibodies accounted for 73% ± 18% of the serum vaccine responses in a cohort of elderly donors, 65% ± 15% in late middle-aged donors, and only 13% ± 5% in persons under 35 years of age. The antibody response to non-HA antigens was boosted by vaccination. Recombinant expression of 19 vaccine-H1+H3 CR serum monoclonal antibodies (s-mAbs) revealed that they predominantly bound to non-HA influenza proteins. A sizable fraction of vaccine-H1+H3 CR s-mAbs recognized with high affinity the sulfated glycans, in particular sulfated type 2 N-acetyllactosamine (Galβ1-4GalNAcβ), which is found on egg-produced proteins and thus unlikely to contribute to protection against influenza infection in humans. Antibodies against sulfated glycans in egg-produced vaccine had been identified in animals but were not previously characterized in humans. Collectively, our results provide a quantitative basis for how repeated exposure to split influenza vaccine correlates with unintended focusing of serum antibody responses to non-HA antigens that may result in suboptimal immunity against influenza.

ObjectiveTo determine whether distinct aquaporin-4 (AQP4)-IgG lineages play a role in neuromyelitis optica spectrum disorder (NMOSD) pathogenesis, we profiled the AQP4-IgG polyclonal serum repertoire and identified, quantified, and functionally characterized distinct AQP4-IgG lineages circulating in 2 patients with NMOSD.MethodsWe combined high-throughput sequencing and quantitative immunoproteomics to simultaneously determine the constituents of both the B-cell receptor (BCR) and the serologic (IgG) anti-AQP4 antibody repertoires in the peripheral blood of patients with NMOSD. The monoclonal antibodies identified by this platform were recombinantly expressed and functionally characterized in vitro.ResultsMultiple antibody lineages comprise serum AQP4-IgG repertoires. Their distribution, however, can be strikingly different in polarization (polyclonal vs pauciclonal). Among the 4 serum AQP4-IgG monoclonal antibodies we identified in 2 patients, 3 induced complement-dependent cytotoxicity in a model mammalian cell line (p < 0.01).ConclusionsThe composition and polarization of AQP4-IgG antibody repertoires may play an important role in NMOSD pathogenesis and clinical presentation. Here, we present a means of coupling both cellular (BCR) and serologic (IgG) antibody repertoire analysis, which has not previously been performed in NMOSD. Our analysis could be applied in the future to clinical management of patients with NMOSD to monitor disease activity over time as well as applied to other autoimmune diseases to facilitate a deeper understanding of disease pathogenesis relative to autoantibody clones.

The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has sparked concern over the continued effectiveness of existing therapeutic antibodies and vaccines. Hence, together with increased genomic surveillance, methods to rapidly develop and assess effective interventions are critically needed. Here we report the discovery of SARS-CoV-2 neutralizing antibodies isolated from COVID-19 patients using a high-throughput platform. Antibodies were identified from unpaired donor B-cell and serum repertoires using yeast surface display, proteomics, and public light chain screening. Cryo-EM and functional characterization of the antibodies identified N3-1, an antibody that binds avidly (Kd,app = 68 pM) to the receptor binding domain (RBD) of the spike protein and robustly neutralizes the virus in vitro. This antibody likely binds all three RBDs of the trimeric spike protein with a single IgG. Importantly, N3-1 equivalently binds spike proteins from emerging SARS-CoV-2 variants of concern, neutralizes UK variant B.1.1.7, and binds SARS-CoV spike with nanomolar affinity. Taken together, the strategies described herein will prove broadly applicable in interrogating adaptive immunity and developing rapid response biological countermeasures to emerging pathogens.

SUMMARYAlthough humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq1) to the spike ectodomain (S-ECD2) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing in vitro, we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified in vivo protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 Å2). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning3,4 our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.

Newly emerged pathogens such as SARS-CoV-2 highlight the urgent need for assays that detect levels of neutralizing antibodies that may be protective. We studied the relationship between anti-spike ectodomain (ECD) and anti-receptor binding domain (RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by two different in vitro assays using convalescent plasma samples obtained from 68 COVID-19 patients, including 13 who donated plasma multiple times. Only 23% (16/68) of donors had been hospitalized. We also studied 16 samples from subjects found to have anti-spike protein IgG during surveillance screening of asymptomatic individuals. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers, and in vitro VN titer. Anti-RBD plasma IgG correlated slightly better than anti-ECD IgG titer with VN titer. The probability of a VN titer ≥160 was 80% or greater with anti-RBD or anti-ECD titers of ≥1:1350. Thirty-seven percent (25/68) of convalescent plasma donors lacked VN titers ≥160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease either VN or IgG titers. Analysis of 2,814 asymptomatic adults found 27 individuals with anti-RBD or anti-ECD IgG titers of ≥1:1350, and evidence of VN ≥1:160. Taken together, we conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titer of ≥1:1350 may provide critical information about protection against COVID-19 disease.

Newly emerged pathogens such as SARS-CoV-2 highlight the urgent need for assays that detect levels of neutralizing antibodies that may be protective. We studied the relationship between anti-spike ectodomain (ECD) and anti-receptor binding domain (RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by two differentin vitroassays using convalescent plasma samples obtained from 68 COVID-19 patients, including 13 who donated plasma multiple times. Only 23% (16/68) of donors had been hospitalized. We also studied 16 samples from subjects found to have anti-spike protein IgG during surveillance screening of asymptomatic individuals. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers, andin vitroVN titer. Anti-RBD plasma IgG correlated slightly better than anti-ECD IgG titer with VN titer. The probability of a VN titer ≥160 was 80% or greater with anti-RBD or anti-ECD titers of ≥1:1350. Thirty-seven percent (25/68) of convalescent plasma donors lacked VN titers ≥160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease either VN or IgG titers. Analysis of 2,814 asymptomatic adults found 27 individuals with anti-RBD or anti-ECD IgG titers of ≥1:1350, and evidence of VN ≥1:160. Taken together, we conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titer of ≥1:1350 may provide critical information about protection against COVID-19 disease.

International audience; Class-switched antibodies to double-stranded DNA (dsDNA) are prevalent and pathogenic in systemic lupus erythematosus (SLE), yet mechanisms of their development remain poorly understood. Humans and mice lacking secreted DNase DNASE1L3 develop rapid anti-dsDNA antibody responses and SLE-like disease. We report that anti-DNA responses in Dnase1l3-/- mice require CD40L-mediated T cell help, but proceed independently of germinal center formation via short-lived antibody-forming cells (AFCs) localized to extrafollicular regions. Type I interferon (IFN-I) signaling and IFN-I-producing plasmacytoid dendritic cells (pDCs) facilitate the differentiation of DNA-reactive AFCs in vivo and in vitro and are required for downstream manifestations of autoimmunity. Moreover, the endosomal DNA sensor TLR9 promotes anti-dsDNA responses and SLE-like disease in Dnase1l3-/- mice redundantly with another nucleic acid-sensing receptor, TLR7. These results establish extrafollicular B cell differentiation into short-lived AFCs as a key mechanism of anti-DNA autoreactivity and reveal a major contribution of pDCs, endosomal Toll-like receptors (TLRs), and IFN-I to this pathway.

Next-Generation Sequencing combined with bioinformatics is a powerful tool for analyzing the large number of DNA sequences present in the expressed antibody repertoire and these data sets can be used to advance a number of research areas including antibody discovery and engineering. The accurate measurement of the immune repertoire sequence composition, diversity and abundance is important for understanding the repertoire response in infections, vaccinations and cancer immunology and could also be useful for elucidating novel molecular targets. In this study 4 individual domestic cats (Felis catus) were subjected to antibody repertoire sequencing with total number of sequences generated 1079863 for VH for IgG, 1050824 VH for IgM, 569518 for VK and 450195 for VL. Our analysis suggests that a similar VDJ expression patterns exists across all cats. Similar to the canine repertoire, the feline repertoire is dominated by a single subgroup, namely VH3. The antibody paratope of felines showed similar amino acid variation when compared to human, mouse and canine counterparts. All animals show a similarly skewed VH CDR-H3 profile and, when compared to canine, human and mouse, distinct differences are observed. Our study represents the first attempt to characterize sequence diversity in the expressed feline antibody repertoire and this demonstrates the utility of using NGS to elucidate entire antibody repertoires from individual animals. These data provide significant insight into understanding the feline immune system function.

We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (T(m)) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher T(m) compared to the parental scFv and importantly, to retain full antigen binding activity after 2 hours of incubation at 70 °C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction.

Horse serum antibodies have been used for greater than a century for the treatment and prophylaxis of infectious diseases and envenomations. Little is known, however, about the immunogenetic diversity that produces horse serum antibodies. Here, we employed next-generation sequencing for a first-in-kind comprehensive analysis of the equine B-cell repertoire. Nearly 45,000 and 30,000 clonotypes were obtained for the heavy-chain (IGH) and lambda light-chain (IGL) loci, respectively. We observed skewed use of the common subgroups IGHV2 (92.49%) and IGLV8 (82.50%), consistent with previous reports, but also novel use of the rare genes IGHV6S1 and IGLV4S2. CDR-H3 amino acid composition revealed different amino acid patterns at positions 106 and 116 compared to human, rabbit, and mouse, suggesting that an extended conformation predominates among horse CDR-H3 loops. Our analysis provides new insights regarding the mechanisms employed to generate antibody diversity in the horse, and could be applicable to the optimized design of synthetic antibodies intended for future therapeutic use.

The developmental pathways of broadly neutralizing antibodies (bNAbs) against HIV are of great importance for the design of immunogens that can elicit protective responses. Here we show the maturation features of the HIV-neutralizing anti-V1V2 VRC26 lineage by simultaneously sequencing the exon together with the downstream intron of VRC26 members. Using the mutational landscapes of both segments and the selection-free nature of the intron region, we identify multiple events of amino acid mutational convergence in the complementarity-determining region 3 (CDR3) of VRC26 members, and determine potential intermediates with diverse CDR3s to a late stage bNAb from 2 years prior to its isolation. Moreover, we functionally characterize the earliest neutralizing intermediates with critical CDR3 mutations, with some emerging only 14 weeks after initial lineage detection and containing only ~6% V gene mutations. Our results thus underscore the utility of analyzing exons and introns simultaneously for studying antibody maturation and repertoire selection., Knowledge on how antibody responses have evolved is critical for the induction of protective immunity. Here the authors analyse, using high-throughput sequencing of both exon and intron regions, the mutation and lineage development of an HIV-neutralizing antibody to find an unexpected early emergence of broadly neutralizing species.

FCRL4, a low-affinity IgA Ab receptor with strong immunoregulatory potential, is an identifying feature of a tissue-based population of memory B cells (Bmem). We used two independent approaches to perform a comparative analysis of the Ag receptor repertoires of FCRL4+ and FCRL4− Bmem in human tonsils. We determined that FCRL4+ Bmem displayed lower levels of somatic mutations in their Ag receptors compared with FCRL4− Bmem but had similar frequencies of variable gene family usage. Importantly, Abs with reactivity to commensal microbiota were enriched in FCRL4+ cells, a phenotype not due to polyreactive binding characteristics. Our study links expression of the immunoregulatory FCRL4 molecule with increased recognition of commensal microbial Ags.

A better understanding of antitumor immune responses is key to advancing the field of cancer immunotherapy. Endogenous immunity in cancer patients, such as circulating anticancer antibodies or tumor-reactive B cells, has been historically yet incompletely described. Here, we demonstrate that tumor-draining (sentinel) lymph node (SN) is a rich source for tumor-reactive B cells that give rise to systemic IgG anticancer antibodies circulating in the bloodstream of breast cancer patients. Using a synergistic combination of high-throughput B-cell sequencing and quantitative immunoproteomics, we describe the prospective identification of tumor-reactive SN B cells (based on clonal frequency) and also demonstrate an unequivocal link between affinity-matured expanded B-cell clones in the SN and antitumor IgG in the blood. This technology could facilitate the discovery of antitumor antibody therapeutics and conceivably identify novel tumor antigens. Lastly, these findings highlight the unique and specialized niche the SN can fill in the advancement of cancer immunotherapy.SIGNIFICANCEUsing high-throughput molecular cloning and antibody proteomics to study coordinated antitumor immunity in breast cancer patients, we simultaneously demonstrate that the sentinel lymph node is a localized source of expanded antitumor B cells undergoing affinity maturation and that their secreted antibodies are abundant as systemic IgG circulating in blood.

The progression of antibody-mediated immunity can now be monitored at high-throughput on a single-cell level.

A major problem in autoimmunity has been identification of the earliest events that lead to breach of tolerance. Although there have been major advances in dissecting effector pathways and the multilineage immune responses to mitochondrial self-antigens in primary biliary cholangitis, the critical links between environmental factors and tolerance remain elusive. We hypothesized that environmental xenobiotic modification of the E2 subunit of the pyruvate dehydrogenase (PDC-E2) inner lipoyl domain can lead to loss of tolerance to genetically susceptible hosts. Previously we demonstrated that serum anti-PDC-E2 autoantibodies cross-react with the chemical xenobiotics 2-octynoic acid and 6,8-bis (acetylthio) octanoic acid and further that there is a high frequency of PDC-E2-specific peripheral plasmablasts. Herein we generated 104 recombinant monoclonal antibodies (mAbs) based on paired heavy-chain and light-chain variable regions of individual plasmablasts derived from primary biliary cholangitis patients. We identified 32 mAbs reactive with native PDC-E2, including 20 specific for PDC-E2 and 12 cross-reactive with both PDC-E2 and 2-octynoic acid and 6,8-bis (acetylthio) octanoic acid. A lower frequency of replacement somatic hypermutations, indicating a lower level of affinity maturation, was observed in the complementarity-determining regions of the cross-reactive mAbs in comparison to mAbs exclusively recognizing PDC-E2 or those for irrelevant antigens. In particular, when the highly mutated heavy-chain gene of a cross-reactive mAb was reverted to the germline sequence, the PDC-E2 reactivity was reduced dramatically, whereas the xenobiotic reactivity was retained. Importantly, cross-reactive mAbs also recognized lipoic acid, a mitochondrial fatty acid that is covalently bound to PDC-E2.ConclusionOur data reflect that chemically modified lipoic acid or lipoic acid itself, through molecular mimicry, is the initial target that leads to the development of primary biliary cholangitis. (Hepatology 2017;66:885-895).

In this study, we report that antigen-specific CD19+CD27+CD21lo (CD21lo) B cells are transiently induced 14 to 28 days after immunization, at the time germinal centers (GCs) peak. Although clonally related to memory B cells and plasmablasts, CD21lo cells form distinct clades within phylogenetic trees based on accumulated variable gene mutations, supporting exit from active GCs. CD21lo cells express a transcriptional program, suggesting that they are primed for plasma cell differentiation and are refractory to GC differentiation, although they do not spontaneously secrete antibody. In addition, CD21lo cells differentially express multiple cell surface markers and have elevated intracellular levels of Blimp-1 and T-bet protein compared with memory B cells. Together, these data support a model in which CD21lo cells are recent GC graduates that represent a distinct population from CD27+ classical memory cells, are refractory to GC reentry, and are predisposed to differentiate into long-lived plasma cells.

High-throughput immune repertoire sequencing has emerged as a critical step in the understanding of adaptive responses following infection or vaccination or in autoimmunity. However, determination of native antibody variable heavy-light pairs (VH-VL pairs) remains a major challenge, and no technologies exist to adequately interrogate the1 × 10(6) B cells in typical specimens. We developed a low-cost, single-cell, emulsion-based technology for sequencing antibody VH-VL repertoires from2 × 10(6) B cells per experiment with demonstrated pairing precision97%. A simple flow-focusing apparatus was used to sequester single B cells into emulsion droplets containing lysis buffer and magnetic beads for mRNA capture; subsequent emulsion RT-PCR generated VH-VL amplicons for next-generation sequencing. Massive VH-VL repertoire analyses of three human donors provided new immunological insights including (i) the identity, frequency and pairing propensity of shared, or 'public', VL genes, (ii) the detection of allelic inclusion (an implicated autoimmune mechanism) in healthy individuals and (iii) the occurrence of antibodies with features, in terms of gene usage and CDR3 length, associated with broadly neutralizing antibodies to rapidly evolving viruses such as HIV-1 and influenza.

Complementarity determining regions (CDR) are responsible for binding antigen and provide substantial diversity to the antibody repertoire, with VH CDR3 of the immunoglobulin variable heavy (VH) domain playing a dominant role. In this study, we examined 1200 unique canine VH and 500 unique variable light (VL) sequences of large and small canine breeds derived from peripheral B cells. Unlike the human and murine repertoire, the canine repertoire is heavily dominated by the Canis lupus familiaris IGHV1 subgroup, evolutionarily closest to the human IGHV3 subgroup. Our studies clearly show that the productive canine repertoire of all analyzed breeds shows similarities to both human and mouse; however, there are distinct differences in terms of VH CDR3 length and amino acid paratope composition. In comparison with the human and murine antibody repertoire, canine VH CDR3 regions are shorter in length than the human counterparts, but longer than the murine VH CDR3. Similar to corresponding human and mouse VH CDR3, the amino acids at the base of the VH CDR3 loop are strictly conserved. For identical CDR positions, there were significant changes in chemical paratope composition. Similar to human and mouse repertoires, the neutral amino acids tyrosine, glycine and serine dominate the canine VH CDR3 interval (comprising 35%) although the interval is nonetheless relatively depleted of tyrosine when compared to human and mouse. Furthermore, canine VH CDR3 displays an overrepresentation of the neutral amino acid threonine and the negatively charged aspartic acid while proline content is similar to that in the human repertoire. In general, the canine repertoire shows a bias towards small, negatively charged amino acids. Overall, this analysis suggests that functional canine therapeutic antibodies can be obtained from human and mouse sequences by methods of speciation and affinity maturation.

Most vaccines confer protection via the elicitation of serum antibodies, yet more than 100 y after the discovery of antibodies, the molecular composition of the human serum antibody repertoire to an antigen remains unknown. Using high-resolution liquid chromatography tandem MS proteomic analyses of serum antibodies coupled with next-generation sequencing of the V gene repertoire in peripheral B cells, we have delineated the human serum IgG and B-cell receptor repertoires following tetanus toxoid (TT) booster vaccination. We show that the TT(+) serum IgG repertoire comprises ∼100 antibody clonotypes, with three clonotypes accounting for40% of the response. All 13 recombinant IgGs examined bound to vaccine antigen with Kd ∼ 10(-8)-10(-10) M. Five of 13 IgGs recognized the same linear epitope on TT, occluding the binding site used by the toxin for cell entry, suggesting a possible explanation for the mechanism of protection conferred by the vaccine. Importantly, only a small fraction (5%) of peripheral blood plasmablast clonotypes (CD3(-)CD14(-)CD19(+)CD27(++)CD38(++)CD20(-)TT(+)) at the peak of the response (day 7), and an even smaller fraction of memory B cells, were found to encode antibodies that could be detected in the serological memory response 9 mo postvaccination. This suggests that only a small fraction of responding peripheral B cells give rise to the bone marrow long-lived plasma cells responsible for the production of biologically relevant amounts of vaccine-specific antibodies (near or above the Kd). Collectively, our results reveal the nature and dynamics of the serological response to vaccination with direct implications for vaccine design and evaluation.

Efforts to determine the antibody repertoire encoded by B cells in the blood or lymphoid organs using high-throughput DNA sequencing technologies have been advancing at an extremely rapid pace and are transforming our understanding of humoral immune responses. Information gained from high-throughput DNA sequencing of immunoglobulin genes (Ig-seq) can be applied to detect B-cell malignancies with high sensitivity, to discover antibodies specific for antigens of interest, to guide vaccine development and to understand autoimmunity. Rapid progress in the development of experimental protocols and informatics analysis tools is helping to reduce sequencing artifacts, to achieve more precise quantification of clonal diversity and to extract the most pertinent biological information. That said, broader application of Ig-seq, especially in clinical settings, will require the development of a standardized experimental design framework that will enable the sharing and meta-analysis of sequencing data generated by different laboratories.

Summary Humans are repeatedly exposed to influenza virus via infections and vaccinations. Understanding how multiple exposures and pre-existing immunity impact antibody responses is essential for vaccine development. Given the recent prevalence of influenza H1N1 A/California/7/2009 (CA09), we examined the clonal composition and dynamics of CA09 hemagglutinin (HA)-reactive IgG repertoire over 5 years in a donor with multiple influenza exposures. The anti-CA09 HA polyclonal response in this donor comprised 24 persistent antibody clonotypes, accounting for 72.6% ± 10.0% of the anti-CA09 HA repertoire over 5 years. These persistent antibodies displayed higher somatic hypermutation relative to transient serum antibodies detected at one time point. Additionally, persistent antibodies predominantly demonstrated cross-reactivity and potent neutralization toward a phylogenetically distant H5N1 A/Vietnam/1203/2004 (VT04) strain, a feature correlated with HA stem recognition. This analysis reveals how “serological imprinting” impacts responses to influenza and suggests that once elicited, cross-reactive antibodies targeting the HA stem can persist for years.

Antibodies that bind to both H1 and H3 influenza strains exist in the pre-vaccination serum repertoire of healthy adults; most vaccine-elicited clonotypes bind either H1 or H3 strains. Molecular understanding of serological immunity to influenza has been confounded by the complexity of the polyclonal antibody response in humans. Here we used high-resolution proteomics analysis of immunoglobulin (referred to as Ig-seq) coupled with high-throughput sequencing of transcripts encoding B cell receptors (BCR-seq) to quantitatively determine the antibody repertoire at the individual clonotype level in the sera of young adults before and after vaccination with trivalent seasonal influenza vaccine. The serum repertoire comprised between 40 and 147 clonotypes that were specific to each of the three monovalent components of the trivalent influenza vaccine, with boosted pre-existing clonotypes accounting for ∼60% of the response. An unexpectedly high fraction of serum antibodies recognized both the H1 and H3 monovalent vaccines. Recombinant versions of these H1 + H3 cross-reactive antibodies showed broad binding to hemagglutinins (HAs) from previously circulating virus strains; several of these antibodies, which were prevalent in the serum of multiple donors, recognized the same conserved epitope in the HA head domain. Although the HA-head-specific H1 + H3 antibodies did not show neutralization activity in vitro, they protected mice against infection with the H1N1 and H3N2 virus strains when administered before or after challenge. Collectively, our data reveal unanticipated insights regarding the serological response to influenza vaccination and raise questions about the added benefits of using a quadrivalent vaccine instead of a trivalent vaccine.

Plasma cells in human bone marrow (BM) are thought to be responsible for sustaining lifelong immunity, but its underlying basis is controversial. Here we use high-throughput sequence analysis of the same individual across 6.5 years to show that the BM plasma cell immunoglobulin heavy chain repertoire is remarkably stable over time. We find a nearly static bias in individual and combinatorial gene usage across time. Analysis of a second donor corroborates these observations. We also report the persistence of numerous BM plasma cell clonotypes (∼2%) identifiable at all points assayed across 6.5 years, supporting a model of serological memory based upon intrinsic longevity of human plasma cells. Donors were adolescents who completely recovered from neuroblastoma prior to the start of this study. Our work will facilitate differentiation between healthy and diseased antibody repertoires, by serving as a point of comparison with future deep-sequencing studies involving immune intervention., Longevity of antibody responses has been attributed to persistence of plasma cells in mice. Here the authors provide human data in support of this model by immunoglobulin sequencing bone marrow sections from two human donors over 6.5 years to show temporal stability of plasma cell clonotypes, but not other B cells.

The ensemble of antibodies found in serum and secretions represents the key adaptive component of B-cell mediated humoral immunity. The antibody repertoire is shaped by the historical record of exposure to exogenous factors such as pathogens and vaccines, as well as by endogenous host-intrinsic factors such as genetics, self-antigens, and age. Thanks to very recent technology advancements it is now becoming possible to identify and quantify the individual antibodies comprising the serological repertoire. In parallel, the advent of high throughput methods for antigen and immunosignature discovery opens up unprecedented opportunities to transform our understanding of numerous key questions in adaptive humoral immunity, including the nature and dynamics of serological memory, the role of polyspecific antibodies in health and disease and how protective responses to infections or vaccine challenge arise. Additionally, these technologies also hold great promise for therapeutic antibody and biomarker discovery in a variety of settings.

Variability in the developing antibody repertoire is focused on the third complementarity determining region of the H chain (CDR-H3), which lies at the center of the antigen binding site where it often plays a decisive role in antigen binding. The power of VDJ recombination and N nucleotide addition has led to the common conception that the sequence of CDR-H3 is unrestricted in its variability and random in its composition. Under this view, the immune response is solely controlled by somatic positive and negative clonal selection mechanisms that act on individual B cells to promote production of protective antibodies and prevent the production of self-reactive antibodies. This concept of a repertoire of random antigen binding sites is inconsistent with the observation that diversity (DH) gene segment sequence content by reading frame (RF) is evolutionarily conserved, creating biases in the prevalence and distribution of individual amino acids in CDR-H3. For example, arginine, which is often found in the CDR-H3 of dsDNA binding autoantibodies, is under-represented in the commonly used DH RFs rearranged by deletion, but is a frequent component of rarely used inverted RF1 (iRF1), which is rearranged by inversion. To determine the effect of altering this germline bias in DH gene segment sequence on autoantibody production, we generated mice that by genetic manipulation are forced to utilize an iRF1 sequence encoding two arginines. Over a one year period we collected serial serum samples from these unimmunized, specific pathogen-free mice and found that more than one-fifth of them contained elevated levels of dsDNA-binding IgG, but not IgM; whereas mice with a wild type DH sequence did not. Thus, germline bias against the use of arginine enriched DH sequence helps to reduce the likelihood of producing self-reactive antibodies.

Tyrosine and glycine constitute 40% of complementarity determining region 3 of the immunoglobulin heavy chain (CDR-H3), the center of the classic antigen-binding site. To assess the role of DH RF1-encoded tyrosine and glycine in regulating CDR-H3 content and potentially influencing B cell function, we created mice limited to a single DH encoding asparagine, histidine, and arginines in RF1. Tyrosine and glycine content in CDR-H3 was halved. Bone marrow and spleen mature B cell and peritoneal cavity B-1 cell numbers were also halved, whereas marginal zone B cell numbers increased. Serum immunoglobulin G subclass levels and antibody titers to T-dependent and T-independent antigens all declined. Thus, violation of the conserved preference for tyrosine and glycine in DH RF1 alters CDR-H3 content and impairs B cell development and antibody production.

Recent developments of high-throughput technologies are enabling the molecular-level analysis and bioinformatic mining of antibody-mediated (humoral) immunity in humans at an unprecedented level. These approaches explore either the sequence space of B-cell receptor repertoires using next-generation deep sequencing (BCR-seq), or the amino acid identities of antibody in blood using protein mass spectrometry (Ig-seq), or both. Generalizable principles about the molecular composition of the protective humoral immune response are being defined, and as such, the field could supersede traditional methods for the development of diagnostics, vaccines, and antibody therapeutics. Three key challenges remain and have driven recent advances: (1) incorporation of innovative techniques for paired BCR-seq to ascertain the complete antibody variable-domain VH:VL clonotype, (2) integration of proteomic Ig-seq with BCR-seq to reveal how the serum antibody repertoire compares with the antibody repertoire encoded by circulating B cells, and (3) a demand to link antibody sequence data to functional meaning (binding and protection).

The vast initial diversity of the antibody repertoire is generated centrally by means of a complex series of V (D) J gene rearrangement events, variation in the site of gene segment joining, and TdT catalyzed N- region addition. Although the diversity is great, close inspection has revealed distinct and unique characteristics in the antibody repertoires expressed by different B cell developmental subsets. In order to illustrate our approach to repertoire analysis, we present an in-depth comparison of V (D) J gene usage, hydrophobicity, length, DH reading frame, and amino acid usage between heavy chain repertoires expressed by immature, transitional, mature, memory IgD+, memory IgD-, and plasmacytes isolated from the blood of a single individual. Our results support the view that in both human and mouse the H chain repertoires expressed by individual, developmental B cell subsets appear to differ in sequence content. Sequencing of unsorted B cells from the blood is thus likely to yield an incomplete or compressed view of what is actually happening in the immune response of the individual. Our findings support the view that studies designed to correlate repertoire expression with diseases of immune function will likely require deep sequencing of B cells sorted by subset.

In man, as in mouse, diversification of the antibody repertoire appears to follow a strict developmental program whereby antigen specificities are serially acquired during ontogeny. When compared to the adult repertoire, the fetal antibody repertoire is highly enriched for polyreactive specificities of low affinity. Although the mechanisms governing the development of this fetal repertoire differ between human and mouse, the composition and structure of the fetal antibodies produced by both species are quite homologous. Specifically, both species use similar V gene segments and restrict the sequence and structure of the third complementarity determining region (HCDR3) of the antibody heavy chain. The precise role that this restriction of the HCDR3 might play in the development of immunocompetence in the human remains to be elucidated.

Sequence analysis of immunoglobulin (Ig) heavy and light chain transcripts can refine categorization of B cell subpopulations and can shed light on the selective forces that act during immune responses or immune dysregulation, such as autoimmunity, allergy, and B cell malignancy. High-throughput sequencing yields Ig transcript collections of unprecedented size. The authoritative web-based IMGT/HighV-QUEST program is capable of analyzing large collections of transcripts and provides annotated output files to describe many key properties of Ig transcripts. However, additional processing of these flat files is required to create figures, or to facilitate analysis of additional features and comparisons between sequence sets. We present an easy-to-use Microsoft® Excel® based software, named Immunoglobulin Analysis Tool (IgAT), for the summary, interrogation, and further processing of IMGT/HighV-QUEST output files. IgAT generates descriptive statistics and high-quality figures for collections of murine or human Ig heavy or light chain transcripts ranging from 1 to 150,000 sequences. In addition to traditionally studied properties of Ig transcripts—such as the usage of germline gene segments, or the length and composition of the CDR-3 region—IgAT also uses published algorithms to calculate the probability of antigen selection based on somatic mutational patterns, the average hydrophobicity of the antigen-binding sites, and predictable structural properties of the CDR-H3 loop according to Shirai’s H3-rules. These refined analyses provide in-depth information about the selective forces acting upon Ig repertoires and allow the statistical and graphical comparison of two or more sequence sets. IgAT is easy to use on any computer running Excel® 2003 or higher. Thus, IgAT is a useful tool to gain insights into the selective forces and functional properties of small to extremely large collections of Ig transcripts, thereby assisting a researcher to mine a data set to its fullest.

Immunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγnull engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model. Roche 454 sequencing was used to obtain over 685,000 reads from cDNA encoding immunoglobulin heavy (IGH) and light (IGK and IGL) genes isolated from immature, naïve, or total splenic B cells in engrafted NOD-scid-IL2Rγnull mice, and compared with over 940,000 reads from peripheral B cells of two healthy volunteers. We find that while naïve B-cell repertoires in humanized mice are chiefly indistinguishable from those in human blood B cells, and display highly correlated patterns of immunoglobulin gene segment use, the complementarity-determining region H3 (CDR-H3) repertoires are nevertheless extremely diverse and are specific for each individual. Despite this diversity, preferential DH-JH pairings repeatedly occur within the CDR-H3 interval that are strikingly similar across all repertoires examined, implying a genetic constraint imposed on repertoire generation. Moreover, CDR-H3 length, charged amino-acid content, and hydropathy are indistinguishable between humans and humanized mice, with no evidence of global autoimmune signatures. Importantly, however, a statistically greater usage of the inherently autoreactive IGHV4-34 and IGKV4-1 genes was observed in the newly formed immature B cells relative to naïve B or total splenic B cells in the humanized mice, a finding consistent with the deletion of autoreactive B cells in humans. Overall, our results provide evidence that key features of the primary repertoire are shaped by genetic factors intrinsic to human B cells and are principally unaltered by differences between mouse and human stromal microenvironments., PLoS ONE, 7 (4), ISSN:1932-6203

Anti-dsDNA antibodies tend to be enriched for heavy chain complementarity determining region 3 (CDR-H3) intervals of above average length that contain an increased frequency of charged amino acids. It is unclear whether these types of CDR-H3s are more common in the primary B-cell repertoire of auto-immune prone strains or whether their increased prevalence in affected individuals reflects positive selection and expansion of atypical CDR-H3s in the pathogenic response to self-antigen. Here, we present evidence that when compared to C3H, a MRL/MpJ(2+) parental strain, CDR-H3 intervals from pre-B cells of adult lupus-prone MRL/MpJ(2+) mice are longer on average and are enriched for charged amino acids. The predicted prevalence of deformed loops per Shirai H3 criteria is also higher. In contrast, the frequency of charge, the distribution of length, and the pattern of predicted deformed loop structures did not differ in sequences obtained from neonates of the same two strains. These observations suggest that the mechanisms that serve to shape the initial CDR-H3 repertoire in adults, but not neonates, are being regulated differently in C3H versus MRL/MpJ(2+). Dysregulation of the adult pre-B CDR-H3 antibody repertoire could be a contributing factor for the development of florid auto-immune disease in MRL/MpJ(2+) mice.