VH Gene Research Articles

Nucleotide Composition of Human Ig Nontemplated Regions Depends on Trimming of the Flanking Gene Segments, and Terminal Deoxynucleotidyl Transferase Favors Adding Cytosine, Not Guanosine, in Most VDJ Rearrangements.

The formation of nontemplated (N) regions during Ig gene rearrangement is a major contributor to Ab diversity. To gain insights into the mechanisms behind this, we studied the nucleotide composition of N regions within 29,962 unique human VHDJH rearrangements and 8728 unique human DJH rearrangements containing exactly one identifiable D gene segment and thus two N regions, N1 and N2. We found a distinct decreasing content of cytosine (C) and increasing content of guanine (G) across each N region, suggesting that N regions are typically generated by concatenation of two 3' overhangs synthesized by addition of nucleoside triphosphates with a preference for dCTP. This challenges the general assumption that the terminal deoxynucleotidyl transferase favors dGTP in vivo. Furthermore, we found that the G and C gradients depended strongly on whether the germline gene segments were trimmed or not. Our data show that C-enriched N addition preferentially happens at trimmed 3' ends of VH, D, and JH gene segments, indicating a dependency of the transferase mechanism upon the nuclease mechanism.

Belimumab promotes negative selection of activated autoreactive B cells in systemic lupus erythematosus patients

Belimumab has therapeutic benefit in active systemic lupus erythematosus (SLE), especially in patients with high-titer anti-dsDNA antibodies. We asked whether the profound B cell loss in belimumab-treated SLE patients is accompanied by shifts in the immunoglobulin repertoire. We enrolled 15 patients who had been continuously treated with belimumab for more than 7 years, 17 matched controls, and 5 patients who were studied before and after drug initiation. VH genes of sort-purified mature B cells and plasmablasts were subjected to next-generation sequencing. We found that B cell-activating factor (BAFF) regulates the transitional B cell checkpoint, with conservation of transitional 1 (T1) cells and approximately 90% loss of T3 and naive B cells after chronic belimumab treatment. Class-switched memory B cells, B1 B cells, and plasmablasts were also substantially depleted. Next-generation sequencing revealed no redistribution of VH, DH, or JH family usage and no effect of belimumab on representation of the autoreactive VH4-34 gene or CDR3 composition in unmutated IgM sequences, suggesting a minimal effect on selection of the naive B cell repertoire. Interestingly, a significantly greater loss of VH4-34 was observed among mutated IgM and plasmablast sequences in chronic belimumab-treated subjects than in controls, suggesting that belimumab promotes negative selection of activated autoreactive B cells.

IL-7 Enables Antibody Responses to Bacterial Polysaccharides by Promoting B Cell Receptor Diversity.

Polysaccharide vaccines such as the Vi polysaccharide (ViPS) of Salmonella enterica serovar Typhi induce efficient Ab responses in adults but not in young children. The reasons for this difference are not understood. IL-7 dependency in B cell development increases progressively with age. IL-7Rα-mediated signals are required for the expression of many VH gene segments that are distal to DH-JH in the IgH locus and for the complete diversification of the BCR repertoire. Therefore, we hypothesized that B cells generated in the absence of IL-7 do not recognize a wide range of Ags because of a restricted BCR repertoire. Compared with adult wildtype mice, young wildtype mice and IL-7-deficient adult mice generated a significantly reduced Ab response to ViPS. Additionally, ViPS-binding B cells in adult wildtype mice predominantly used distal VH gene segments. Transgenic expression of either IL-7 or a BCR encoded by a distal VH gene segment permitted young mice to respond efficiently to bacterial polysaccharides. These results indicate that restricted VH gene usage early in life results in a paucity of Ag-specific B cell precursors, thus limiting antipolysaccharide responses.

Vaccine Generation of Protective Ebola Antibodies and Identification of Conserved B-Cell Signatures.

We recently identified a single potently neutralizing monoclonal antibody (mAb), mAb114, isolated from a human survivor of natural Zaire ebolavirus (EBOV) infection, which fully protects nonhuman primates (NHPs) against lethal EBOV challenge. To evaluate the ability of vaccination to generate mAbs such as mAb114, we cloned antibodies from NHPs vaccinated with vectors encoding the EBOV glycoprotein (GP). We identified 14 unique mAbs with potent binding to GP, 4 of which were neutralized and had the functional characteristics of mAb114. These vaccine-induced macaque mAbs share many sequence similarities with mAb114 and use the same mAb114 VH gene (ie, IGHV3-13) when classified using the macaque IMGT database. The antigen-specific VH-gene repertoire present after each immunization indicated that IGHV3-13 mAbs populate an EBOV-specific B-cell repertoire that appears to become more prominent with subsequent boosting. These findings will support structure-based vaccine design aimed at enhanced induction of antibodies such as mAb114.

Isolation of human monoclonal antibodies that bind to two different antigens and are encoded by germline VH and VL genes

This paper reports isolation of two monoclonal antibodies (mAbs) that bind to both a membrane protein and a cytoplasmic protein. Most Abs established as markers for autoimmune disease bind to cytoplasmic or nuclear substances. However, it remains unknown how these Abs are produced. On the other hand, there were examples where clones originally isolated as Abs that bind to membrane proteins also showed binding activity to cytoplasmic or nuclear substances. Based on these results, the following hypothesis has been proposed. The Abs that had been originally produced against a membrane protein showed cross-reactivity against cytoplasmic or nuclear substances. In the present study we reported isolation of Abs that bound to both a membrane protein, CADM1, and a cytoplasmic protein, α-actinin-4. The method adopted in the present study could be generally applicable to isolation of Abs showing such dual specificity. Firstly, we constructed a huge human Ab library using various organs including naïve B-cell-rich organs such as bone marrow and umbilical cords. Then, we developed a comprehensive screening method for isolation of Abs that bound to cell surface antigens. Through extensive screenings with many kinds of cell we newly obtained a library composed of around 4000 independent clones that bind to membrane proteins. We screened this library with α-actinin-4 and succeeded in isolating two Abs. They bound to α-actinin-4 and a membrane protein CADM1. Furthermore, they are encoded by naïve heavy and light chain variable genes (VH & VL). These results suggested that cross-reactive Abs to both a membrane protein and a cytoplasmic protein could be present in germline repertoire of Ab in humans. This methodology adopted in the present study could be applied to isolation of cross-reactive Abs possibly involved in autoimmune diseases.

Immunoglobulin heavy chain variable region analysis in dairy goats

Based on the goat genome database, we have annotated the genomic organization of the goat immunoglobulin heavy chain variable region. The goat IgH locus is present on seven genome scaffolds, and contains ten VH, three DH and six JH segments. After the exclusion of three shorter segments, the VH genes were divided into two gene families based on sequence similarity. By analyzing the IgH cDNA sequences, we further identified that VH2 (54.2%), DH1 (61.7%) and JH1 (60.5%) segments were most frequently utilized in the expression of the immunoglobulin variable region, and that point mutations introduced by somatic hypermutation were the major mutation present in these expressed variable region. Compared with human and horses, DH-DH fusion occurred at a higher frequency in goat V(D)J recombination. These results provided variable insights into goat immunoglobulin heavy chain variable region genome loci and repertoire diversity.

Breadth and Functionality of Varicella-Zoster Virus Glycoprotein-Specific Antibodies Identified after Zostavax Vaccination in Humans.

Herpes zoster (HZ) (shingles) is the clinical manifestation of varicella-zoster virus (VZV) reactivation. HZ typically develops as people age, due to decreased cell-mediated immunity. However, the importance of antibodies for immunity against HZ prevention remains to be understood. The goal of this study was to examine the breadth and functionality of VZV-specific antibodies after vaccination with a live attenuated HZ vaccine (Zostavax). Direct enumeration of VZV-specific antibody-secreting cells (ASCs) via enzyme-linked immunosorbent spot assay (ELISPOT assay) showed that Zostavax can induce both IgG and IgA ASCs 7 days after vaccination but not IgM ASCs. The VZV-specific ASCs range from 33 to 55% of the total IgG ASCs. Twenty-five human VZV-specific monoclonal antibodies (MAbs) were cloned and characterized from single-cell-sorted ASCs of five subjects (>60 years old) who received Zostavax. These MAbs had an average of ∼20 somatic hypermutations per VH gene, similar to those seen after seasonal influenza vaccination. Fifteen of the 25 MAbs were gE specific, whereas the remaining MAbs were gB, gH, or gI specific. The most potent neutralizing antibodies were gH specific and were also able to inhibit cell-to-cell spread of the virus in vitro Most gE-specific MAbs were able to neutralize VZV, but they required the presence of complement and were unable to block cell-to-cell spread. These data indicate that Zostavax induces a memory B cell recall response characterized by anti-gE > anti-gI > anti-gB > anti-gH antibodies. While antibodies to gH could be involved in limiting the spread of VZV upon reactivation, the contribution of anti-gE antibodies toward protective immunity after Zostavax needs further evaluation.IMPORTANCE Varicella-zoster virus (VZV) is the causative agent of chickenpox and shingles. Following infection with VZV, the virus becomes latent and resides in nerve cells. Age-related declines in immunity/immunosuppression can result in reactivation of this latent virus, causing shingles. It has been shown that waning T cell immunity correlates with an increased incidence of VZV reactivation. Interestingly, serum with high levels of VZV-specific antibodies (VariZIG; IV immunoglobulin) has been administered to high-risk populations, e.g., immunocompromised children, newborns, and pregnant women, after exposure to VZV and has shown some protection against chickenpox. However, the relative contribution of antibodies against individual surface glycoproteins toward protection from shingles in elderly/immunocompromised individuals has not been established. Here, we examined the breadth and functionality of VZV-specific antibodies after vaccination with the live attenuated VZV vaccine Zostavax in humans. This study will add to our understanding of the role of antibodies in protection against shingles.

Induction and relationship of HIV Envelope-specific B cells in the peripheral blood and bone marrow following vaccination in humans.

Abstract Induction of long-lived plasma cells producing protective HIV-1 Envelope (Env)-specific antibody (Ab) is a primary goal of HIV vaccine strategies. However, vaccine-induced Env-specific human plasma cells have not yet been identified. In an effort to do so, samples were obtained from HVTN 105, a phase I trial in which participants were immunized with the same bivalent gp120 protein, AIDSVAX B/E, as used in RV144, although combined with a DNA immunogen (instead of ALVAC-HIV) in various prime/boost strategies. Participants who received a boosting regimen that included AIDSVAX B/E had robust peripheral blood plasmablast responses as measured by Env-specific ELISpot (n=21) and flow cytometry (n=20). The Env-specific immunoglobulin repertoire of the plasmablasts, assessed by monoclonal Ab generation (>110 mAbs total from 15 participants), was dominated by VH1 gene usage (~60%, VH1-2>VH1-46>VH1-24), and had modest mutation from germline (~5%). Ongoing epitope mapping has indicated V3 is a dominant target; however other regions including V1/V2 and C1/C5 were also targeted. The majority of the mAbs are high avidity, and a subset can mediate Ab-dependent cellular phagocytosis; additional functional analysis is ongoing. Bone marrow was evaluated ~7 months after immunization (n=3), and although ELISpot of CD138+ plasma cells could readily detect influenza specific IgG Ab secreting cells (ASCs), Env-specific IgG ASCs were not convincingly detected by ELISpot. However, when using VH deep sequencing of the bone marrow (n=4), multiple members of plasmablast-derived mAb clonal lineages were detected. Our results indicate that HIV-1 Env-specific B cells are present in human bone marrow after vaccination, but may persist at very low frequency.

Sequential Enhancer Sequestration Dysregulates Recombination Center Formation at the IgH Locus

Immunoglobulin heavy-chain (IgH) genes are assembled by DNA rearrangements that juxtapose a variable (VH), a diversity (DH), and a joining (JH) gene segment. Here, we report that in the absence of intergenic control region 1 (IGCR1), the intronic enhancer (Eμ) associates with the next available CTCF binding site located close to VH81X via putative heterotypic interactions involving YY1 and CTCF. The alternate Eμ/VH81X loop leads to formation of a distorted recombination center and altered DH rearrangements and disrupts chromosome conformation that favors distal VH recombination. Cumulatively, these features drive highly skewed, Eμ-dependent recombination of VH81X. Sequential deletion of CTCF binding regions on IGCR1-deleted alleles suggests that they influence recombination of single proximal VH gene segments. Our observations demonstrate that Eμ interacts differently with IGCR1- or VH-associated CTCF binding sites and thereby identify distinct roles for insulator-like elements in directing enhancer activity.

Age-associatedBcells expanded in autoimmune mice are memory cells sharing H-CDR3-selected repertoires.

Age-associated Bcells (ABCs) represent a distinct cell population expressing low levels of CD21 (CD21-/low ). The Ig repertoire expressed by ABCs in aged mice is diverse and exhibits signs of somatic hypermutation (SHM). A CD21-/low B-cell population is expanded in autoimmune diseases, e.g. systemic lupus erythematosus, as well as in lupus-prone NZB/W mice and in mice lacking a pre-Bcell receptor (SLC-/- ). However, the nature of the CD21-/low Bcells (hereafter ABCs) in autoimmunity is not well understood. Here we show that in young SLC-/- mice, the vast majority of the ABCs express memory B-cell (MBC) markers in contrast to wild-type controls. A similar population is present in lupus-prone MRL mice before and at disease onset. In SLC-/- mice, a majority of the ABCs are IgM+ , their VH genes have undergone SHM, show clonal diversification and clonal restriction at the H-CDR3 level. ABC hybridomas, established from SLC-/- mice, secrete typical lupus autoantibodies, e.g. anti-Smith antigen, and some of those that bind to DNA comprise a H-CDR3 that is identical to previously described IgM anti-DNA antibodies from lupus-prone mice. Together, these results reveal that ABCs in autoimmune mice are comprised of autoreactive MBCs expressing highly restricted H-CDR3 repertoires.

Primer Design and Inverse PCR on Yeast Display Antibody Selection Outputs.

The display of antibodies on the surface of Saccharomyces cerevisiae cells enables the high-throughput and precise selection of specific binders for the target antigen. The recent implementation of next-generation sequencing (NGS) to antibody display screening provides a complete picture of the entire selected polyclonal population. As such, NGS overcomes the limitations of random clones screening, but it comes with two main limitations: (1) depending upon the platform, the sequencing is usually restricted to the variable heavy chain domain complementary determining region 3 (HCDR3), or VH gene, and does not provide additional information on the rest of the antibody gene, including the VL; and (2) the sequence-identified clones are not physically available for validation. Here, we describe a rapid and effective protocol based on an inverse-PCR method to recover specific antibody clones based on their HCDR3 sequence from a yeast display selection output.

The Versatility of Framework Regions of Chicken VH and VL to Mutations.

To identify the interchangeability of VH and VL framework region (FR) residues, we artificially introduced random mutations at all residue positions in a chicken monoclonal antibody, which has only one functional VH and Vλ gene. When we classified the amino acids into 5 groups by their physicochemical properties, all FR residues could be replaced by another group except L23 (C), H36 (W), H86 (D), H104 (G), and H106 (G). Eighty-two (50.9%), 48 (29.8%), 17 (10.6%), and 9 FR residues (5.6%) could be replaced by 4, 3, 2, and 1 group(s), individually, without significant loss of reactivity. We also confirmed a similar level of versatility with 2 different chicken antibodies. This high level of versatility on FR residues has not been predicted because it has not been observed in the 150 chicken antibodies that we previously generated or in the 1,269 naïve chicken VH sequences publically available. In conclusion, chicken antibody FR residues are highly interchangeable and this property can be applied for improving the physicochemical property of antibody including thermal stability, solubility and viscosity.

Local Chromatin Features Including PU.1 and IKAROS Binding and H3K4 Methylation Shape the Repertoire of Immunoglobulin Kappa Genes Chosen for V(D)J Recombination.

V(D)J recombination is essential for the generation of diverse antigen receptor (AgR) repertoires. In B cells, immunoglobulin kappa (Igκ) light chain recombination follows immunoglobulin heavy chain (Igh) recombination. We recently developed the DNA-based VDJ-seq assay for the unbiased quantitation of Igh VH and DH repertoires. Integration of VDJ-seq data with genome-wide datasets revealed that two chromatin states at the recombination signal sequence (RSS) of VH genes are highly predictive of recombination in mouse pro-B cells. It is unknown whether local chromatin states contribute to Vκ gene choice during Igκ recombination. Here we adapt VDJ-seq to profile the Igκ VκJκ repertoire and present a comprehensive readout in mouse pre-B cells, revealing highly variable Vκ gene usage. Integration with genome-wide datasets for histone modifications, DNase hypersensitivity, transcription factor binding and germline transcription identified PU.1 binding at the RSS, which was unimportant for Igh, as highly predictive of whether a Vκ gene will recombine or not, suggesting that it plays a binary, all-or-nothing role, priming genes for recombination. Thereafter, the frequency with which these genes recombine was shaped both by the presence and level of enrichment of several other chromatin features, including H3K4 methylation and IKAROS binding. Moreover, in contrast to the Igh locus, the chromatin landscape of the promoter, as well as of the RSS, contributes to Vκ gene recombination. Thus, multiple facets of local chromatin features explain much of the variation in Vκ gene usage. Together, these findings reveal shared and divergent roles for epigenetic features and transcription factors in AgR V(D)J recombination and provide avenues for further investigation of chromatin signatures that may underpin V(D)J-mediated chromosomal translocations.

CD4-Binding Site Directed Cross-Neutralizing scFv Monoclonals from HIV-1 Subtype C Infected Indian Children.

Progression of human immunodeficiency virus type-1 (HIV-1) infection in children is faster than adults. HIV-1 subtype C is responsible for more than 50% of the infections globally and more than 90% infections in India. To date, there is no effective vaccine against HIV-1. Recent animal studies and human Phase I trials showed promising results of the protective effect of anti-HIV-1 broadly neutralizing antibodies (bnAbs). Interaction between CD4 binding site (CD4bs) on the HIV-1 envelope glycoprotein and CD4 receptor on the host immune cells is the primary event leading to HIV-1 infection. The CD4bs is a highly conserved region, comprised of a conformational epitope, and is a potential target of bnAbs such as VRC01 that is presently under human clinical trials. Recombinant scFvs can access masked epitopes due to their small size and have shown the potential to inhibit viral replication and neutralize a broad range of viruses. Pediatric viruses are resistant to many of the existing bnAbs isolated from adults. Therefore, in this study, pooled peripheral blood mononuclear cells from 9 chronically HIV-1 subtype C infected pediatric cross-neutralizers whose plasma antibodies exhibited potent and cross-neutralizing activity were used to construct a human anti-HIV-1 scFv phage library of 9 × 108 individual clones. Plasma mapping using CD4bs-specific probes identified the presence of CD4bs directed antibodies in 4 of these children. By extensive biopanning of the library with CD4bs-specific antigen RSC3 core protein, we identified two cross-neutralizing scFv monoclonals 2B10 and 2E4 demonstrating a neutralizing breadth and GMT of 77%, 17.9 µg/ml and 32%, 51.2 µg/ml, respectively, against a panel of 49 tier 1, 2 and 3 viruses. Both scFvs competed with anti-CD4bs bnAb VRC01 confirming their CD4bs epitope specificity. The 2B10 scFv was effective in neutralizing the 7 subtype C and subtype A pediatric viruses tested. Somatic hypermutations in the VH gene of scFvs (10.1–11.1%) is comparable with that of the adult antibodies. These cross-neutralizing CD4bs-directed scFvs can serve as potential reagents for passive immunotherapy. A combination of cross-neutralizing scFvs of diverse specificities with antiretroviral drugs may be effective in suppressing viremia at an early stage of HIV-1 infection and prevent disease progression.

Ecobody technology: rapid monoclonal antibody screening method from single B cells using cell-free protein synthesis for antigen-binding fragment formation

We report a rapid and cost-effective monoclonal antibody screening method from single animal B cells using reverse transcription (RT)-PCR and Escherichia coli cell-free protein synthesis (CFPS), which allows evaluation of antibodies within 2 working days. This process is named “Ecobody technology”. The method includes strategies to isolate B cells that specifically bind an antigen from the peripheral blood of immunised animals, and single-cell RT-PCR to generate DNA fragments of the VH and VL genes, followed by CFPS for production of fragments of antigen binding (Fab). In the CFPS step, we employed our techniques: 1) ‘Zipbody’ as a method for producing Fab, in which the association of heavy and light chains is facilitated by adhesive leucine zipper peptides fused at the C-termini of the Fab; and 2) an N-terminal SKIK peptide tag that can increase protein expression levels. Using Ecobody technology, we obtained highly-specific monoclonal antibodies for the antigens Vibrio parahaemolyticus and E. coli O26. The anti-V. parahaemolyticus Zipbody mAb was further produced in E. coli strain SHuffle T7 Express in inclusion bodies and refolded by a conventional method, resulting in significant antigen-binding activity (KD = 469 pM) and productivity of 8.5 mg purified antibody/L-culture.

HIV-1 Cross-Reactive Primary Virus Neutralizing Antibody Response Elicited by Immunization in Nonhuman Primates.

Elicitation of broadly neutralizing antibody (bNAb) responses is a major goal for the development of an HIV-1 vaccine. Current HIV-1 envelope glycoprotein (Env) vaccine candidates elicit predominantly tier 1 and/or autologous tier 2 virus neutralizing antibody (NAb) responses, as well as weak and/or sporadic cross-reactive tier 2 virus NAb responses with unknown specificity. To delineate the specificity of vaccine-elicited cross-reactive tier 2 virus NAb responses, we performed single memory B cell sorting from the peripheral blood of a rhesus macaque immunized with YU2gp140-F trimers in adjuvant, using JR-FL SOSIP.664, a native Env trimer mimetic, as a sorting probe to isolate monoclonal Abs (MAbs). We found striking genetic and functional convergence of the SOSIP-sorted Ig repertoire, with predominant VH4 or VH5 gene family usage and Env V3 specificity. Of these vaccine-elicited V3-specific MAbs, nearly 20% (6/33) displayed cross-reactive tier 2 virus neutralization, which recapitulated the serum neutralization capacity. Substantial similarities in binding specificity, neutralization breadth and potency, and sequence/structural homology were observed between selected macaque cross-reactive V3 NAbs elicited by vaccination and prototypic V3 NAbs derived from natural infections in humans, highlighting the convergence of this subset of primate V3-specific B cell repertories. Our study demonstrated that cross-reactive primary virus neutralizing B cell lineages could be elicited by vaccination as detected using a standardized panel of tier 2 viruses. Whether these lineages could be expanded to acquire increased breadth and potency of neutralization merits further investigation.IMPORTANCE Elicitation of antibody responses capable of neutralizing diverse HIV-1 primary virus isolates (designated broadly neutralizing antibodies [bNAbs]) remains a high priority for the vaccine field. bNAb responses were so far observed only in response to natural infection within a subset of individuals. To achieve this goal, an improved understanding of vaccine-elicited responses, including at the monoclonal Ab level, is essential. Here, we isolated and characterized a panel of vaccine-elicited cross-reactive neutralizing MAbs targeting the Env V3 loop that moderately neutralized several primary viruses and recapitulated the serum neutralizing antibody response. Striking similarities between the cross-reactive V3 NAbs elicited by vaccination in macaques and natural infections in humans illustrate commonalities between the vaccine- and infection-induced responses to V3 and support the feasibility of exploring the V3 epitope as a HIV-1 vaccine target in nonhuman primates.

Rescue Strategy for Nonviable Hybridomas: Amplifying Heavy- and Light-Chain Sequences from Genomic DNA by Polymerase Chain Reaction.

Many hybridoma researchers have lost an important hybridoma because of instability, mechanical breakdown of a freezer, or contamination. In many cases, however, it is possible to retrieve the heavy- and light-chain sequences from those hybridomas. Although most cloning methods associated with isolation of VH or VL gene sequences require the isolation of intact, nondegraded mRNA, alternative approaches based on genomic DNA have been reported. With genomic DNA, the DNA sample can remain relatively intact in cases in which the mRNA has been degraded. If intact DNA can be obtained from a nonviable hybridoma, the method described here can be used to recover the heavy- and light-chain sequences. The method uses PCR based on a combination of primers in the 5' untranslated region of VH and VL and in the JH and JL segments. Although a relatively large number of primers are required for this method, the ability to obtain a full-length V gene without having intact mRNA could be of general interest for rescuing lost hybridomas.

Sockeye salmon immunoglobulin VH usage and pathogen loads differ between spawning sites

The Immunological Imprinting Hypothesis proposes that juvenile anadromous fish respond to the pathogen fingerprint specific to their natal site by producing protective long lived plasma cells (LLPCs) that constitutively produce antibodies against those pathogens. Hence, fish returning to their natal streams have immunological protection from pathogens at that specific location. Here, we tested the hypothesis through analysis of antibody composition and usage in sockeye salmon populations in Alaska. Spleen and anterior kidney were sampled from salmon from six sites, and relative usage levels of six different Immunoglobulin VH gene families determined using RT-qPCR. Additionally, prevalence and pathogen loads were measured in each fish for Renibacterium salmoninarum, Flavobacterium psychrophilum, and Infectious Hematopoietic Necrosis Virus. Results revealed differences in VH usage, pathogen loads, and infection rates between spawning sites, while probability of infection was dependent on location for each pathogen analyzed. Further, several negative correlations between specific VH usage patterns and pathogen loads were uncovered. Greater understanding of site-dependent VH usage in spawning fish potentially suggests a method of natural immunization against common fish pathogens and thus protection of both farmed and wild populations.

Selection of nanobodies with broad neutralizing potential against primary HIV-1 strains using soluble subtype C gp140 envelope trimers

Broadly neutralizing antibodies (bnAbs) against HIV-1 protect from infection and reduce viral load upon therapeutic applications. However no vaccine was able so far to induce bnAbs demanding their expensive biotechnological production. For clinical applications, nanobodies (VHH) derived from heavy chain only antibodies from Camelidae, may be better suited due to their small size, high solubility/stability and extensive homology to human VH3 genes. Here we selected broadly neutralizing nanobodies by phage display after immunization of dromedaries with different soluble trimeric envelope proteins derived from HIV-1 subtype C. We identified 25 distinct VHH families binding trimeric Env, of which 6 neutralized heterologous primary isolates of various HIV-1 subtypes in a standardized in vitro neutralization assay. The complementary neutralization pattern of two selected VHHs in combination covers 19 out of 21 HIV-1 strains from a standardized panel of epidemiologically relevant HIV-1 subtypes. The CD4 binding site was preferentially targeted by the broadly neutralizing VHHs as determined by competition ELISAs and 3D models of VHH-Env complexes derived from negative stain electron microscopy. The nanobodies identified here are excellent candidates for further preclinical/clinical development for prophylactic and therapeutic applications due to their potency and their complementary neutralization patterns covering the majority of epidemiologically relevant HIV-1 subtypes.

Deacetylase activity of histone deacetylase 3 is required for productive VDJ recombination and B-cell development

Histone deacetylase 3 (HDAC3) is the catalytic component of NCoR/SMRT corepressor complexes that mediate the actions of transcription factors implicated in the regulation of B-cell development and function. We crossed Hdac3 conditional knockout mice with Mb1-Cre knockin animals to delete Hdac3 in early progenitor B cells. The spleens of Hdac3F/-Mb1-Cre+/- mice were virtually devoid of mature B cells, and B220+CD43+ B-cell progenitors accumulated within the bone marrow. Quantitative deep sequencing of the Ig heavy chain locus from B220+CD43+ populations identified a defect in VHDJH recombination with a severe reduction in productive rearrangements, which directly corresponded to the loss of pre-B cells from Hdac3Δ/- bone marrow. For Hdac3Δ/- B cells that did show productive VDJ rearrangement, there was significant skewing toward the incorporation of proximal VH gene segments and a corresponding reduction in distal VH gene segment use. Although transcriptional effects within these loci were modest, Hdac3Δ/- progenitor cells displayed global changes in chromatin structure that likely hindered effective distal V-DJ recombination. Reintroduction of wild-type Hdac3 restored normal B-cell development, whereas an Hdac3 point mutant lacking deacetylase activity failed to complement this defect. Thus, the deacetylase activity of Hdac3 is required for the generation of mature B cells.