VH Gene Research Articles

Complement activation contributes to the anti-methicillin-resistant Staphylococcus aureus effect of natural anti-keratin antibody

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major public health problem worldwide because of its strong resistance to a variety of antibiotics. Natural immunoglobulin (Ig) M antibodies have been reported to protect against microbial infections. In the present study, the function of a monoclonal natural anti-keratin antibody IgM (named 3B4) in MRSA infection was evaluated. The binding of 3B4 to MRSA was studied using immunofluorescence assay and flow cytometry (FCM). The binding of 3B4 to mannose-binding lectin (MBL) and complement activation were detected by ELISA. For the in vivo study, transgenic mice for the VH gene from 3B4 (TgVH 3B4) were used. After infection, the bacterial burden was examined in the kidney, spleen and enterocelia. Inflammatory cytokine levels and the neutrophil ratio in peritoneal lavage fluid (PLF) were assessed by ELISA and FCM, respectively. Additionally, the total serum hemolytic activity (CH50) in the early stage of infection was detected by ELISA. The results showed that 3B4 bound directly to MRSA and MBL, and the interaction between 3B4 and MRSA/MBL led to the activation of the classic and the MBL pathway in vitro. After 48 h of MRSA infection, the bacterial load in the kidney, spleen and enterocelia was significantly decreased in TgVH 3B4 mice (P < 0.05) compared with wild-type mice. Levels of IL-6, TNF-α, and IFN-γ were increased after MRSA infection. The levels of IL-6 and TNF-α in TgVH 3B4 mice were decreased by 49.1% and 59.4% compared to wild-type mice. Additionally, the neutrophil ratio in the PLF of TgVH 3B4 mice was decreased by 65.9%. The CH50 value was significantly higher in TgVH 3B4 mice than in wild-type mice, indicating that 3B4 promoted the activation of the complement system in MRSA infected mice. The results reveal an important role of 3B4 in the anti-MRSA immune response, and the complement activation contributes to this effect.

Functional and Molecular Characteristics of Novel and Conserved Cross-Clade HIV Envelope Specific Human Monoclonal Antibodies.

To define features of the B cell response to HIV that may be translated to vaccine development, we have isolated a panel of monoclonal antibodies (MAbs) from HIV-infected patients. These MAbs are all highly reactive to HIV envelope (Env) from multiple clades, and include gp120 and gp41 specificities. Three of the MAbs exhibit substantial homology to previously described VH1-69, VH3-30, and VH4-59 HIV broadly neutralizing antibody lineages. An inherently autoreactive VH4-34 encoded MAb was reactive to diverse Env despite its minimal mutation from germline. Its isolation is consistent with our previous observation of increased VH4-34+antibodies in HIV-infected patients. These results suggest that conserved developmental processes contribute to immunoglobulin repertoire usage and maturation in response to HIV Env and that intrinsically autoreactive VH genes, despite the absence of mutation, could serve as effective templates for maturation and development of protective antibodies. These results also bear significant implications for the development of immunogens.

The ϕ6 cystovirus protein P7 becomes accessible to antibodies in the transcribing nucleocapsid: a probe for viral structural elements.

Protein P7 is a component of the cystovirus viral polymerase complex. In the unpackaged procapsid, the protein is situated in close proximity to the viral directed RNA polymerase, P2. Cryo-electron microscopy difference maps from the species ϕ6 procapsid have demonstrated that P7 and P2 likely interact prior to viral RNA packaging. The location of P7 in the post-packaged nucleocapsid (NC) remains unknown. P7 may translocate closer to the five-fold axis of a filled procapsid but this has not been directly visualized. We propose that monoclonal antibodies (Mabs) can be selected that serve as probe- reagents for viral assembly and structure. A set of Mabs have been isolated that recognize and bind to the ϕ6 P7. The antibody set contains five unique Mabs, four of which recognize a linear epitope and one which recognizes a conformational epitope. The four unique Mabs that recognize a linear epitope display restricted utilization of Vκ and VH genes. The restricted genetic range among 4 of the 5 antibodies implies that the antibody repertoire is limited. The limitation could be the consequence of a paucity of exposed antigenic sites on the ϕ6 P7 surface. It is further demonstrated that within ϕ6 nucleocapsids that are primed for early-phase transcription, P7 is partially accessible to the Mabs, indicating that the nucleocapsid shell (protein P8) has undergone partial disassembly exposing the protein’s antigenic sites.

Construction of scFv library of human antibodies against tetanus toxin.

Over the last three decades, monoclonal antibodies have become powerful therapeutics and diagnostics tools. Progress in the antibody engineering, and the appearance of great selection technology such as phage display has made human antibodies production possible against antigens with high affinities. The purpose of this study was to construct an immune antibody library from a vaccinated donor against tetanus toxin. A blood sample was drawn from the donor who was vaccinated with tetanus toxoid. PBMC were isolated by using ficoll. After RNA extraction and cDNA synthesis. In order to amplify VH and VL regions, two uniplex PCRs were performed and put considering NcoI & HindIII, MluI & NotI restriction sites respectively for their cloning. These amplicons were cloned to pSEX81 vector and transformed to E. coli XL1blue strain. Eventually, recombinant plasmids were extracted and sequenced. The result showed acceptable similarity between antibody gene library nucleotide sequences and the antibody genes were deposited in this database. In this study, the VH and VL genes human antibody library was constructed and confirmed by using DNA sequencing and alignment of sequences with blast database.

Central nervous system infiltrates are characterized by features of ongoing B cell-related immune activity in MP4-induced experimental autoimmune encephalomyelitis

In multiple sclerosis (MS) lymphoid follicle-like aggregates have been reported in the meninges of patients. Here we investigated the functional relevance of B cell infiltration into the central nervous system (CNS) in MP4-induced experimental autoimmune encephalomyelitis (EAE), a B cell-dependent mouse model of MS. In chronic EAE, B cell aggregates were characterized by the presence of CXCL13+ and germinal center CD10+ B cells. Germline transcripts were expressed in the CNS and particularly related to TH17-associated isotypes. We also observed B cells with restricted VH gene usage that differed from clones found in the spleen. Finally, we detected CNS-restricted spreading of the antigen-specific B cell response towards a myelin and a neuronal autoantigen. These data imply the development of autonomous B cell-mediated autoimmunity in the CNS in EAE — a concept that might also apply to MS itself.

CD27-IgD- memory B cells are modulated by in vivo interleukin-6 receptor (IL-6R) blockade in rheumatoid arthritis.

IntroductionEnhanced B cell activity, particularly memory B cells have gained interest in evaluating response during therapies with biologics. CD27-IgD- double-negative (DN) B cells lacking the conventional memory marker CD27 are reported to be part of the memory compartment, however, only scarce data is available for rheumatoid arthritis (RA). We therefore focused on DN B cells in RA, studied their isotypes and modulation during interleukin-6 receptor (IL-6R) inhibition by tocilizumab (TCZ).MethodsDN B cells were phenotypically analyzed from 40 RA patients during TCZ at baseline week 12, week 24 and 1 year. A single B cell polymerase chain reaction (PCR) approach was used to study Ig receptors, VH gene rearrangements and specific isotypes.ResultsPhenotypic analysis showed a significantly expanded population of DN B cells in RA which contain a heterogeneous mixture of IgG-, IgA- and IgM-expressing cells with a clear dominance of IgG+ cells. DN B cells carry rearranged heavy chain gene sequences with a diversified mutational pattern consistent with memory B cells. In contrast to tumor necrosis factor alpha (TNF-α) inhibition, a significant reduction in mutational frequency of BCR gene rearrangements at week 12, 24 and 1 year (P <0.0001) was observed by in vivo IL-6R inhibition. These changes were observed for all BCR isotypes IgG, IgA and IgM at week 12, 24 and 1 year (P <0.0001). IgA-RF, IgA serum level and IgA+ DN B cells decreased significantly (P <0.05) at week 12 and week 24 during TCZ. Patients with a good European League Against Rheumatism (EULAR) response to TCZ had less DN B cells at baseline as compared to moderate responders (P = 0.006). Univariate logistic regression analysis revealed that the frequency of DN B cells at baseline is inversely correlated to a subsequent good EULAR response (P = 0.024) with an odds ratio of 1.48 (95% confidence interval as 1.05 to 2.06).ConclusionsIn RA, the heterogeneous DN B cell compartment is expanded and dominated by IgG isotype. TCZ can modulate the mutational status of DN Ig isotype receptors over 1 year. Interestingly, the frequency of DN B cells in RA may serve as a baseline predictor of subsequent EULAR response to TCZ.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0580-y) contains supplementary material, which is available to authorized users.

Correlation of immunoglobulin variable heavy chain gene mutation status with prognosis in patients with mantle cell lymphoma

To study the relationship between immunoglobulin variable heavy chain (IgVH) gene mutation status and clinical features, pathologic findings and biologic behavior of mantle cell lymphoma (MCL). IgVH gene was amplified in 60 cases of MCL with FR1-JH and FR2-JH primers in BIOMED-2. The sequence was determined by cloning. The IgVH somatic mutational status was analyzed using NCBI's Ig-Blast tool. The relationship between IgVH gene mutation status and clinicopathologic features was also analyzed. Forty percent (24 cases, 28 functional Ig genes) of the MCL cases displayed somatically mutated VH genes (defined as > 2% mutated), whereas 60.0% (36 cases, 40 functional Ig genes) showed unmutated VH genes. The most widely used genes were VH3-21 (27.9%) and VH4-34 (19.1%). The former were mainly used by unmutated cases, while the later mainly by mutated cases.Intraclonal heterogeneity was noted in 19 cases. There was no correlation of VH mutation status and specific VH gene with survival (P > 0.05). MCL comprises at least two subsets that do not correlate with morphology: one with unmutated VH genes and one with mutated VH genes. The biased use of VH3-21 and VH4-34 is noted. The nonrandom usage of IgVH segments suggests specific antigens may play a role in the pathogenesis and progression of MCL subsets. There is no correlation of IgVH mutation status and specific VH gene with survival.

Sterile DJH rearrangements reveal that distance between gene segments on the human Ig H chain locus influences their ability to rearrange.

Rearrangement of the Ig locus occurs in two steps. First, a JH gene is rearranged to a D gene followed by a VH gene rearranging to the DJH rearrangement. By next generation sequencing, we analyzed 9969 unique DJH rearrangements and 5919 unique VHDJH rearrangements obtained from peripheral blood B cells from 110 healthy adult donors. We found that DJH rearrangements and nonproductive VHDJH rearrangements share many features but differ significantly in their use of D genes and propensity for somatic hypermutation. In D to JH gene rearrangements, the D genes proximal to the JH locus are used more frequently than JH locus distal D genes, whereas VH locus proximal D genes were observed more frequently in nonproductive VHDJH rearrangements. We further demonstrate that the distance between VH, D, and JH gene segments influence their ability to rearrange within the human Ig locus.

VH replacement in primary immunoglobulin repertoire diversification

The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D)J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. VH replacement represents a RAG-mediated secondary rearrangement in which an upstream VH element recombines with a rearranged VHDHJH joint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original VH element and the conventional RSS of the invading VH gene, leaving behind a footprint of up to five base pairs (bps) of the original VH gene that is often further obscured by exonuclease activity and N-nucleotide addition. We have previously demonstrated that VH replacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive VHDHJH allele. Using this mouse model, we characterized the role of VH replacement in the diversification of the primary Ig repertoire through the modification of productive VHDHJH rearrangements. Our results indicate that VH replacement occurs before Ig light chain rearrangement and thus is not involved in the editing of self-reactive antibodies.

Antibody repertoire diversification through VH gene replacement in mice cloned from an IgA plasma cell

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 VHQ52(NT); Vκgr32(NT) Ig monoclonal mice reprogrammed from the nucleus of an intestinal IgA(+) plasma cell. In VHQ52(NT) mice, IgA replaced IgM to drive early B-cell development and peripheral B-cell maturation. In VHQ52(NT) 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.

Spatial Regulation of V-(D)J Recombination at Antigen Receptor Loci.

Lymphocytes express a diverse repertoire of antigen receptors, which are able to recognize a large variety of foreign pathogens. Functional antigen receptor genes are assembled by V(D)J recombination in immature B cells (Igh and Igk) and T cells (Tcr b and Tcra/d). V(D)J recombination takes place in the 3' proximal domain containing the D, J, and C gene segments, whereas 31 (Tcrb) to 200 (Igh) V genes are spread over a large region of 0.67 (Tcrb) to 3 (Igk) megabase pairs. The spatial regulation of V(D)J recombination has been best studied for the Igh locus, which undergoes reversible contraction by long-range looping in pro-B cells. This large-scale contraction brings distantly located VH genes into close proximity of the DJH-rearranged gene segment, which facilitates VH-DJH recombination. The B-cell-specific Pax5, ubiquitous YY1, and architectural CTCF/cohesin proteins regulate Igh locus contraction in pro-B cells by binding to multiple sites in the VH gene cluster. These regulators also control the pro-B-cell-specific activity of the distally located PAIR elements, which may be involved in the regulation of VH-DJH recombination by promoting locus contraction. Moreover, the large VH gene cluster of the Igh locus undergoes flexible long-range looping, which guarantees similar participation of all VH genes in VH-DJH recombination to generate a diverse antibody repertoire. Importantly, long-range looping is a more general regulatory principle, as other antigen receptor loci also undergo reversible contraction at the developmental stage, where they engage in V-(D)J recombination.

Development of a fluorescence-linked immunosorbent assay for detection of avermectins using a fluorescent single-domain antibody

A schematic diagram of the FLISA based on a fluobody for the determination of AVMs. Note: FLISA, fluorescence-linked immunosorbent assay; GFP, green fluorescent protein; fluobody, fluorescent single-domain antibody; ABM, abamectin; OVA, ovalbumin; and AVMs, avermectins.

Chromatin Interactions in the Control of Immunoglobulin Heavy Chain Gene Assembly.

Expression of antibody heavy chain occurs via precisely timed developmental activation of the immunoglobulin heavy chain (IgH) gene locus during B cell development. IgH locus activation permits coordinated gene rearrangements that assemble variable (VH), diversity (DH), and joining (JH) gene segments into functional genes. Chromosomal conformation changes and epigenetic mechanisms play critical roles in ensuring rearrangement fidelity while minimizing hazardous consequences of broken DNA ends generated during recombination. In this review, we summarize the current status of regulatory mechanisms that underpin effective IgH gene assembly. For this, the germline locus is divided into two parts: a 2.4Mb 5' part that contains all VH gene segments and a 300kb 3' domain that contains DH and JH gene segments, as well as exons that encode IgH isotypes. Structural features of each part are discussed individually, followed by consideration of how the two parts come together to complete IgH recombination. Throughout we emphasize current insights, propose plausible mechanisms, and highlight key questions for future studies.

Setd1a regulates progenitor B‐cell‐to‐precursor B‐cell development through histone H3 lysine 4 trimethylation and Ig heavy‐chain rearrangement

SETD1A is a member of trithorax-related histone methyltransferases that methylate lysine 4 at histone H3 (H3K4). We showed previously that Setd1a is required for mesoderm specification and hematopoietic lineage differentiation in vitro. However, it remains unknown whether or not Setd1a controls specific hematopoietic lineage commitment and differentiation during animal development. Here, we reported that homozygous Setd1a knockout (KO) mice are embryonic lethal. Loss of the Setd1a gene in the hematopoietic compartment resulted in a blockage of the progenitor B-cell-to-precursor B-cell development in bone marrow (BM) and B-cell maturation in spleen. The Setd1a-cKO (conditional knockout) mice exhibited an enlarged spleen with disrupted spleen architecture and leukocytopenia. Mechanistically, Setd1a deficiency in BM reduced the levels of H3K4me3 at critical B-cell gene loci, including Pax5 and Rag1/2, which are critical for the IgH (Ig heavy-chain) locus contractions and rearrangement. Subsequently, the differential long-range looped interactions of the enhancer Eμ with proximal 5' DH region and 3' regulatory regions as well as with Pax5-activated intergenic repeat elements and 5' distal VH genes were compromised by the Setd1a-cKO. Together, our findings revealed a critical role of Setd1a and its mediated epigenetic modifications in regulating the IgH rearrangement and B-cell development.

Accumulation of self-reactive naïve and memory B cell reveals sequential defects in B cell tolerance checkpoints in Sjögren's syndrome.

Sjögren’s syndrome (SS) is an autoimmune disease characterised by breach of self-tolerance towards nuclear antigens resulting in high affinity circulating autoantibodies. Although peripheral B cell disturbances have been described in SS, with predominance of naïve and reduction of memory B cells, the stage at which errors in B cell tolerance checkpoints accumulate in SS is unknown. Here we determined the frequency of self- and poly-reactive B cells in the circulating naïve and memory compartment of SS patients. Single CD27−IgD+ naïve, CD27+IgD+ memory unswitched and CD27+IgD− memory switched B cells were sorted by FACS from the peripheral blood of 7 SS patients. To detect the frequency of polyreactive and autoreactive clones, paired Ig VH and VL genes were amplified, cloned and expressed as recombinant monoclonal antibodies (rmAbs) displaying identical specificity of the original B cells. IgVH and VL gene usage and immunoreactivity of SS rmAbs were compared with those obtained from healthy donors (HD). From a total of 353 VH and 293 VL individual sequences, we obtained 114 rmAbs from circulating naïve (n = 66) and memory (n = 48) B cells of SS patients. Analysis of the Ig V gene repertoire did not show significant differences in SS vs. HD B cells. In SS patients, circulating naïve B cells (with germline VH and VL genes) displayed a significant accumulation of clones autoreactive against Hep-2 cells compared to HD (43.1% vs. 25%). Moreover, we demonstrated a progressive increase in the frequency of circulating anti-nuclear naïve (9.3%), memory unswitched (22.2%) and memory switched (27.3%) B cells in SS patients. Overall, these data provide novel evidence supporting the existence of both early and late defects in B cell tolerance checkpoints in patients with SS resulting in the accumulation of autoreactive naïve and memory B cells.

Detection of the Malignant B Cell Clone in Multiple Myeloma Via High Throughput Sequencing Is Robust to Significant Levels of Somatic Hypermutation

Background: High-throughput sequencing (HTS) of antibody gene rearrangements is an emerging tool for minimal residual disease (MRD) monitoring in B cell malignancies in which the malignant clone harbors a monoclonal Ig heavy chain (IgH) and/or light chain (κ or λ) rearrangement. This approach has shown promise in B-ALL and CLL, but experience with this technique applied to samples from multiple myeloma patients is limited.Approach: We conducted HTS of PCR-amplified IgH (VDJ and DJ) rearrangements from bone marrow aspirates of 21 patients with various plasma cell dyscrasias (MM, MGUS, LPL) and peripheral blood of a patient with plasma cell leukemia. In 17/21 samples, an aliquot was enriched for CD138+ cells by immunomagnetic separation and analyzed separately. Dominant clones from enriched and un-enriched aliquots were compared to verify the malignant clonotype sequence(s). Disease burden in un-enriched samples was also evaluated by microscopy of the bone marrow aspirate smear and ranged from 0 (hemodilute) to 37% plasma cells.Results: In 19 out of 21 samples, a clearly dominant IgH gene rearrangement (>2.5% of total sequences, range 2.9-99.9%) was identified with clear separation from background frequency (at least 2.7-fold higher frequency than next most common clone). In 17/17 cases with paired CD138-enriched samples, the dominant sequences in the enriched and un-enriched samples were identical, indicating successful identification of the malignant clonal Ig rearrangements in the un-enriched sample. More than one IgH rearrangement suitable for longitudinally tracking the malignant clone was identified in 8 of 21 cases. The two cases without an expected, productive IgH rearrangement were IgG-κ and IgG-λ. This suggested that somatic hypermutation (SHM) in the primer binding sites might interfere with some clonal amplifications, so we investigated the degree of SHM in the VH segment of the 19 cases with at least one detected dominant Ig rearrangement. A total of 18 productive VDJ rearrangements were identified, and had SHM frequencies ranging from 2% to 19% in the sequenced portions of the rearranged VH gene. 8 myeloma clones harbored an identifiable DJ rearrangement, none of which showed evidence of SHM. Finally, 3 myeloma clones harbored nonproductive VDJ rearrangements, two with no SHM, and one with 2 SHM in 84 bp of the sequenced VH gene.Conclusion: HTS of Ig heavy and light chain rearrangements can successfully identify the malignant plasma cell clone in clinical specimens, including those with low disease burden and significant SHM. Application of this technique to MRD evaluation in multiple myeloma warrants further development. DisclosuresCarlson:Adaptive Biotechnologies: Consultancy, Equity Ownership. Vogl:Celgene Corporation: Consultancy; Amgen: Consultancy; Millennium/Takeda: Research Funding; GSK: Research Funding; Acetylon: Research Funding. Stadtmauer:Janssen: Consultancy.

The splenic autoimmune response to ADAMTS13 in thrombotic thrombocytopenic purpura contains recurrent antigen-binding CDR3 motifs

AbstractAcquired thrombotic thrombocytopenic purpura (TTP) is the consequence of a severe ADAMTS13 deficiency resulting from autoantibodies inhibiting ADAMTS13 or accelerating its clearance. Despite the success of plasma exchange the risk of relapse is high. From 2 patients (A and B), splenectomized for recurrent episodes of acquired TTP, the splenic B-cell response against ADAMTS13 was characterized through generation of human monoclonal anti-ADAMTS13 autoantibodies (mAbs) by cloning an immunoglobulin G (IgG)4κ- and IgG4λ-Fab library using phage display technology and by Epstein-Barr virus transformation of switched memory B cells (CD19+/CD27+/IgG+). Sequence analysis of the anti-ADAMTS13 IgGs of both patients revealed that the VH gene use was limited in our patients to VH1-3 (55%), VH1-69 (17%), VH3-30 (7%), and VH4-28 (21%) and contained 8 unique and thus far not reported heavy-chain complementarity determining region 3 motifs, of which 4 were shared by the 2 patients. The discovery of several highly similar anti-ADAMTS13 autoantibodies in 2 unrelated TTP patients suggests that the autoimmune response is antigen driven, because the probability that such similar immunoglobulin rearrangements happen by chance is very low (<10−9).

Multiple factors influence the contribution of individual immunoglobulin light chain genes to the naïve antibody repertoire.

BackgroundThe naïve antibody repertoire is initially dependent upon the number of germline V(D)J genes and the ability of recombined heavy and light chains to pair. Individual VH and VL genes are not equally represented in naïve mature B cells, suggesting that positive and negative selection also shape the antibody repertoire. Among the three member murine Vκ10 L chain family, the Vκ10C gene is under-represented in the antibody repertoire. Although it is structurally functional and accessible to both transcriptional and recombination machinery, the Vκ10C promoter is inefficient in pre-B cell lines and productive Vκ10C rearrangements are lost as development progresses from pre-B cells through mature B cells. This study examined VH/Vκ10 pairing, promoter mutations, Vκ10 transcript levels and receptor editing as possible factors that are responsible for loss of productive Vκ10C rearrangements in developing B cells.ResultsWe demonstrate that the loss of Vκ10C expression is not due to an inability to pair with H chains, but is likely due to a combination of other factors. Levels of mRNA are low in sorted pre-B cells and undetectable in B cells. Mutation of a single base in the three prime region of the Vκ10C promoter increases Vκ10C promoter function in pre-B cell lines. Pre-B and B cells harbor disproportionate levels of receptor-edited productive Vκ10C rearrangements.ConclusionsOur findings suggest that the weak Vκ10C promoter initially limits the amount of available Vκ10C L chain for pairing with H chains, resulting in sub-threshold levels of cell surface B cell receptors, insufficient tonic signaling and subsequent receptor editing to limit the numbers of Vκ10C-expressing B cells emigrating from the bone marrow to the periphery.

Rapid development of broadly influenza neutralizing antibodies through redundant mutations.

The neutralizing antibody response to influenza virus is dominated by antibodies that bind to the globular head of haemagglutinin, which undergoes a continuous antigenic drift, necessitating the re-formulation of influenza vaccines on an annual basis. Recently, several laboratories have described a new class of rare influenza-neutralizing antibodies that target a conserved site in the haemagglutinin stem. Most of these antibodies use the heavy-chain variable region VH1-69 gene, and structural data demonstrate that they bind to the haemagglutinin stem through conserved heavy-chain complementarity determining region (HCDR) residues. However, the VH1-69 antibodies are highly mutated and are produced by some but not all individuals, suggesting that several somatic mutations may be required for their development. To address this, here we characterize 197 anti-stem antibodies from a single donor, reconstruct the developmental pathways of several VH1-69 clones and identify two key elements that are required for the initial development of most VH1-69 antibodies: a polymorphic germline-encoded phenylalanine at position 54 and a conserved tyrosine at position 98 in HCDR3. Strikingly, in most cases a single proline to alanine mutation at position 52a in HCDR2 is sufficient to confer high affinity binding to the selecting H1 antigen, consistent with rapid affinity maturation. Surprisingly, additional favourable mutations continue to accumulate, increasing the breadth of reactivity and making both the initial mutations and phenylalanine at position 54 functionally redundant. These results define VH1-69 allele polymorphism, rearrangement of the VDJ gene segments and single somatic mutations as the three requirements for generating broadly neutralizing VH1-69 antibodies and reveal an unexpected redundancy in the affinity maturation process.

Genetic Assessment of B-cell Responses Following Immunization with HIV Env Protein and Adjuvants or SHIV (AD8) Infection in Nonhuman Primates

OA16.04 Background: An effective preventive vaccine for HIV-1 should generate potent neutralizing antibody responses against the HIV envelope (Env). However, broadly-neutralizing antibodies from HIV+ individuals often have unique characteristics such as extensive somatic hypermutation (SHM), long CDRH3 regions and VH gene restriction. Therefore, we monitored the development of these characteristics in the preclinical setting. Methods: NHP were immunized with HIV Env clade C protein alone, or with a panel of clinical adjuvants or were infected with SHIV(AD8) virus. Env-specific B cells were sorted, barcoded, deep sequenced, and analyzed using a newly curated NHP Ig reference database. In total we analyzed VH gene usage, SHM, and CDRH3 length from over 58,000 unique Ig heavy chains. Results: The adjuvants tested here did not significantly increase germline divergence compared to unadjuvanted protein, nor did they give rise to B cells with long CDRH3 regions. However, animals and individual reads with higher SHM showed evidence of a narrowed VH gene repertoire and were enriched in the IGHV3Q gene. By comparison, antibodies from SHIV infected animals showed an increase in SHM in animals with better neutralization responses. Better neutralization responses further correlated with higher viral loads and viral Env diversity, indicating that chronic antigen stimulation and diversity is likely a driving force for higher SHM and the development of antibody potency and breadth. Finally, we correlated the presence of 3 VH genes: IGHV4A, 4D and 3J with better neutralization, indicating it may be possible to identify a VH gene signature of neutralization. Conclusions: This study is the first comprehensive comparison of antibody genetics using vaccine and infection models in NHP. Our data suggest that future vaccine regimens should be designed to mimic chronic viral infection, utilizing heterologous Env immunogens and prolonged innate and antigen stimulation to drive affinity maturation and neutralization breadth.