Germline VH Research Articles

Characteristics and Functions of Infection-enhancing Antibodies to the N-terminal Domain of SARS-CoV-2.

Fcγ-receptor (FcγR)-independent enhancement of SARS-CoV-2 infection mediated by N-terminal domain (NTD)-binding monoclonal antibodies (mAbs) has been observed in vitro, but the functional significance of these antibodies in vivo is less clear. We characterized 1,213 SARS-CoV-2 spike (S)-binding mAbs derived from COVID-19 convalescent patients for binding specificity to the SARS-CoV-2 S protein, VH germ-line usage, and affinity maturation. Infection enhancement in a vesicular stomatitis virus (VSV)-SARS-CoV-2 S pseudovirus (PV) assay was characterized in respiratory and intestinal epithelial cell lines, and against SARS-CoV-2 variants of concern (VOC). Proteomic deconvolution of the serum antibody repertoire was used to determine functional attributes of secreted NTD-binding mAbs. We identified 72/1213 (5.9%) mAbs that enhanced SARS-CoV-2 infection in a PV assay. The majority (68%) of these mAbs recognized the NTD, were identified in patients with mild and severe disease, and persisted for at least 5 months post-infection. Infection enhancement by NTD-binding mAbs was not observed in intestinal and respiratory epithelial cell lines and was diminished or lost against SARS-CoV-2 VOC. Proteomic deconvolution of the serum antibody repertoire from 2 of the convalescent patients identified, for the first time, NTD-binding, infection-enhancing mAbs among the circulating immunoglobulins directly isolated from serum. Functional analysis of these mAbs demonstrated robust activation of FcγRIIIa associated with antibody binding to recombinant S proteins. Functionally active NTD-specific mAbs arise frequently during natural infection and can last as major serum clonotypes during convalescence. These antibodies display functional attributes that include FcγR activation, and may be selected against by mutations in NTD associated with SARS-CoV-2 VOC.

VHH CDR-H3 conformation is determined by VH germline usage

VHHs or nanobodies are single antigen binding domains originating from camelid heavy-chain antibodies. They are used as diagnostic and research tools and in a variety of therapeutic molecules. Analyzing variable domain structures from llama and alpaca we found that VHHs can be classified into two large structural clusters based on their CDR-H3 conformation. Extended CDR-H3 loops protrude into the solvent, whereas kinked CDR-H3 loops fold back onto framework regions. Both major families have distinct properties in terms of their CDR-H3 secondary structure, how their CDR-H3 interacts with the framework region and how they bind to antigens. We show that the CDR-H3 conformation of VHHs correlates with the germline from which the antibodies are derived: IGHV3-3 derived antibodies almost exclusively adopt a kinked CDR-H3 conformation while the CDR-H3 adopts an extended structure in most IGHV3S53 derived antibodies. We do not observe any bias stemming from V(D)J recombination in llama immune repertoires, suggesting that the correlation is the result of selection processes during B-cell development. Our findings demonstrate a previously undescribed impact of germline usage on antigen interaction and contribute to a better understanding on how properties of the antibody framework shape the immune repertoire.

Signatures of VH1-69-derived hepatitis C virus neutralizing antibody precursors defined by binding to envelope glycoproteins

An effective preventive vaccine for hepatitis C virus (HCV) remains a major unmet need. Antigenic region 3 (AR3) on the E1E2 envelope glycoprotein complex overlaps with the CD81 receptor binding site and represents an important epitope for broadly neutralizing antibodies (bNAbs) and is therefore important for HCV vaccine design. Most AR3 bNAbs utilize the VH1-69 gene and share structural features that define the AR3C-class of HCV bNAbs. In this work, we identify recombinant HCV glycoproteins based on a permuted E2E1 trimer design that bind to the inferred VH1-69 germline precursors of AR3C-class bNAbs. When presented on nanoparticles, these recombinant E2E1 glycoproteins efficiently activate B cells expressing inferred germline AR3C-class bNAb precursors as B cell receptors. Furthermore, we identify critical signatures in three AR3C-class bNAbs that represent two subclasses of AR3C-class bNAbs that will allow refined protein design. These results provide a framework for germline-targeting vaccine design strategies against HCV.

SARS-CoV-2 Delta and Omicron variants evade population antibody response by mutations in a single spike epitope

Population antibody response is thought to be important in selection of virus variants. We report that SARS-CoV-2 infection elicits a population immune response that is mediated by a lineage of VH1-69 germline antibodies. A representative antibody R1-32 from this lineage was isolated. By cryo-EM, we show that it targets a semi-cryptic epitope in the spike receptor-binding domain. Binding to this non-ACE2 competing epitope results in spike destruction, thereby inhibiting virus entry. On the basis of epitope location, neutralization mechanism and analysis of antibody binding to spike variants, we propose that recurrent substitutions at 452 and 490 are associated with immune evasion of the identified population antibody response. These substitutions, including L452R (present in the Delta variant), disrupt interactions mediated by the VH1-69-specific hydrophobic HCDR2 to impair antibody-antigen association, enabling variants to escape. The first Omicron variants were sensitive to antibody R1-32 but subvariants that harbour L452R quickly emerged and spread. Our results provide insights into how SARS-CoV-2 variants emerge and evade host immune responses.

HIV gp120-specific memory develops from pre-existing memory and naive B cells following vaccination in humans

Abstract The most potent and broad HIV envelope (Env)-specific antibodies often when reverted to their inferred germline versions representing the naïve B cell receptor, fail to bind Env suggesting that the initial responding B cell population is not exclusively comprised of a naïve population, but also a pre-existing cross-reactive antigen-experienced B cell pool that expands following Env exposure. Previously we isolated gp120-reactive monoclonal antibodies (mAbs) from participants from HVTN105, a phase I trial testing AIDSVAX B/E (gp120 protein) combined with a DNA immunogen. Using MiSeq-based deep sequencing VH-lineage tracking we identified several of these mAb lineages in pre-immune peripheral blood. Additionally, we sought to characterize the gp120-specific B cell repertoire prior to immunization, and the features of those pre-immune B cell clonal lineages that ultimately respond to the vaccine. Several of these pre-immune lineages also persisted in the bone marrow, including CD138+ long-lived plasma cell compartment, ~7 months after the final vaccination. Greater gp120 binding activity was observed from the non-naïve pre-immune VH members than their germline VH versions. The majority of the pre-immune gp120-reactive lineages included IgM, however IgG and IgA members were also predominant prior to vaccination. Additionally, the majority of the pre-immune lineage members exhibited somatic hypermutation prior to vaccination, suggesting that the vaccine-induced gp120-specific antibody lineages originated from both naïve B cells and cross-reactive memory B cells. Supported by grants from the NIH (R01AI117787, R21AI116285).

Identification and Characterization of Differentially Expressed IgM Transcripts of Channel Catfish Vaccinated with Antigens of Virulent Aeromonas hydrophila

Channel catfish (Ictalurus punctatus) is the top species produced in US aquaculture and motile Aeromonas septicemia, caused by virulent Aeromonas hydrophila (vAh), is one of the most severe diseases that afflict catfish farms. Previously, vaccination of fish with extracellular proteins (ECP) of vAh was shown to produce a robust antibody-mediated immune response against vAh infection. In this study, we analyzed IgM transcripts that were differentially expressed in the head kidney and liver of ECP-immunized and mock-immunized (control) fish with emphasis on a variable domain of heavy chain. Quantitative PCR analysis indicated that immunized fish produced significantly more IgM transcripts than control fish. Full-length IgM heavy chain cDNA was cloned, which encoded typical IgM peptide, including signal peptide, variable domain (VH), constant domain (CH), and carboxyl terminal peptide. Great sequence diversity was revealed in a VH segment, with the third complementarity diversity region (CDR3) being most variable. Using germline VH gene grouping method, variants (clones) of VH characterized in this study belonged to nine VH families. The most unique variants (approximately 49%) were found in the VH2 family. Vaccinated fish apparently had more unique variants than in the control fish. There were 62% and 79% of unique variants in the head kidney and liver of vaccinated fish, respectively, while 44% and 27% unique variants in the head kidney and liver of control fish, respectively. Among the unique variants in VH2 family, approximately 87% of them were found in vaccinated fish. Two-dimensional gel electrophoresis of semi-purified IgM protein confirmed that matured IgM protein was as variable as IgM transcripts identified in this study, with isoelectric points crossing from 6 to 10. Results of this study provided insight into the molecular and genetic basis of antibody diversity and enriched our knowledge of the complex interplay between antigens and antibodies in Ictalurid catfish.

Ontogeny of the Alloimmune Anti-Canine Factor VIII Inhibitor Response in Severe Hemophilia Α Dogs

The development of inhibitors to Factor VIII (FVIII) is a major complication of hemophilia A (HA) treatment. Most preclinical studies in HA animals have been limited to a xenoprotein response, e.g., evaluating the murine immune response to human FVIII (hFVIII). Severe HA dogs are a naturally occurring outbred model that recapitulates the spontaneous bleeding phenotype of the disease; their severe HA is due to intron 22 inversion (INV22) of the canine F8 gene, analogous to the INV22 found in 45% of severe HA patients. Importantly, similar to HA INV22 patients, about 20% of these animals develop an anti-canine FVIII (cFVIII) inhibitor response. This ability of the HA dogs to develop inhibitors in a species-specific manner provides an opportunity to probe the molecular ontogeny of an alloimmune anti-FVIII response.Here, we use antibody phage display to capture the humoral anti-cFVIII IgG repertoire in a dog with severe HA. This dog developed and maintained high titers of cFVIII inhibitor (89 Bethesda Units, BU) after exposure to cFVIII protein. The dog then received AAV liver-directed gene therapy encoding a cFVIII transgene, and after an initial anamnestic response characterized by rapid increase of cFVIII inhibitor titer (peak titer of 182 BU), the dog exhibited successful inhibitor eradication and immune tolerance induction, similar to our previous report (Finn et al., Blood 2010).Canine peripheral blood mononuclear cells were used to construct a single-chain variable fragment (scFv) phage display library which was sequentially selected four times against immobilized recombinant B-domain deleted (BDD) cFVIII protein. The amount of enrichment per round of selection plateaued during the third round from which 55 scFv/phage clones were isolated for characterization. Individual phage clones were sequenced by Sanger sequencing and screened for binding to both canine and human BDD-FVIII by ELISA. ScFv/phage clones were tested for inhibitory activity in a modified Bethesda assay.We identified 26 distinct scFv clones binding cFVIII based on heavy chain/light chain composition comprising 16 distinct CDRH3 sequences and 23 distinct CDRL3 sequences. The heavy chains of the 26 clones were derived from six canine germline IGHV genes, namely IGHV3-2, IGHV3-5, IGHV3-9, IGHV3-19, IGHV3-38, and IGHV3-41. The 16 distinct CDRH3 sequences had a mean length of 10.1 ± 3.6 amino acids, shorter than the 13.5 ± 3.6 amino acids previously reported for total canine CDRH3 repertoires (Steiniger et al., Mol. Immunol. 2014). Only 10 of the 26 FVIII-specific clones contained a lambda light chain, despite canine IgG repertoires dominated by the use of lambda light chains (Steiniger et al.). Eight of the 26 clones exhibited inhibitory activity. Although this dog was never exposed to hFVIII, 12/26 (46%) clones bound both cFVIII and hFVIII, consistent with the high degree of homology between the two orthologues with 85% identity. For three clones, framework region mutations and different light chain pairings resulted in altered inhibitory activity and hFVIII binding. Interestingly, one clone in particular comprised the majority of randomly screened scFv from the third and fourth rounds of phage library selection.These results suggest that cFVIII-specific B cells arise from multiple germline VH genes and exhibit high CDRH3 diversity. While the IgG-FVIII interaction has previously been suggested to be mostly influenced by the IgG heavy chain, our data suggest that for some antibodies, the IgG light chain may influence both inhibitory activity and epitope reactivity. Future studies will assess B cell clonal evolution via deep sequencing and longitudinal epitope specificity during multi-year immune tolerance induction by continuous exposure to cBDD-FVIII gene therapy. This is the first in-depth assessment of FVIII immunogenicity in a large HA model that avoids the use of a non-species specific antigen. Direct comparison with ongoing studies in HA inhibitor patients will inform the immunodominant epitopes of FVIII across species and thus provide insights on FVIII immunogenicity. DisclosuresDoshi: Janssen: Consultancy; Spark Therapeutics: Speakers Bureau. Samelson-Jones: Spark: Research Funding; Pfizer: Consultancy, Research Funding. Siegel: Verismo Therapeutics, Inc: Other: Co-Founder and Equity Holder; Vetigenics, LLC: Other: Co-Founder and Equity Holder.

Access to ultra-long IgG CDRH3 bovine antibody sequences using short read sequencing technology

The advances in high-throughput DNA sequencing and recombinant antibody technologies has presented new methods for characterizing antibody repertoires and significantly increased our understanding on the functional role of antibodies in immunity and their use in diagnostics, vaccine antigen design and as biological therapeutics. A subset of Bos taurus antibodies possesses unique ultra-long third complementary-determining region of the heavy chain (CDRH3) and are of special interest because they are thought to have unique functional abilities of broadly neutralizing properties – a functional role that has not been fully explored in vaccine development. Next generation sequencing technologies that are widely used to profile immunoglobulin (Ig) repertoires are based on short-read methods such as the Illumina technology. Although this technology has worked well in sequencing Ig V-D-J regions of most jawed vertebrates, it has faced serious technical challenges with sequencing regions in bovine Ig bearing ultra-long CDRH3 sequences, which are longer than 120 bp. To overcome this limitation, we have developed a sequencing strategy based on nested PCR products that allows sequence assembly of full-length bovine Ig heavy-chain (IgH) V-D-J regions. We have used this strategy to sequence IgH V-D-J regions of two Bos indicus breeds, Ankole and Boran. We confirm the presence of ultra-long CDRH3 sequences in IgG transcripts in both African cattle breeds, and provide preliminary evidence for differences and preferences in germline VH, DH and JH allele gene usage as well as differences in the length of the VH region in the two bovine breeds. Our method provides tools that should allow more robust analyses of ultra-long CDRH3 sequences aiding antibody and epitope discovery in different cattle breeds and their role in mediating immunity.

A SARS-CoV-2 antibody broadly neutralizes SARS-related coronaviruses and variants by coordinated recognition of a virus-vulnerable site

Potent neutralizing SARS-CoV-2 antibodies often target the spike protein receptor-binding site (RBS), but the variability of RBS epitopes hampers broad neutralization of multiple sarbecoviruses and drifted viruses. Here, using humanized mice, we identified an RBS antibody with a germline VH gene that potently neutralized SARS-related coronaviruses, including SARS-CoV and SARS-CoV-2 variants. X-ray crystallography revealed coordinated recognition by the heavy chain of non-RBS conserved sites and the light chain of RBS with a binding angle mimicking the angiotensin-converting enzyme 2 (ACE2) receptor. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, which was enhanced by immunoglobulin G3 (IgG3) class switching. The coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Low-dose therapeutic antibody treatment in hamsters reduced the virus titers and morbidity during SARS-CoV-2 challenge. The structural basis for broad neutralizing activity may inform the design of a broad spectrum of therapeutics and vaccines.

Analysis of expressed immunoglobulin heavy chain genes in familial B-CLL

In this study, we wished to determine whether familial chronic lymphocytic leukemia of B-cell phenotype (CLL) shares with sporadic B-CLL the same immunoglobulin (Ig) heavy chain variable region (VH) gene usage and occurrence of somatic mutation, to gain insight into the pathogenetic relatedness of these epidemiologically distinct forms of CLL. We therefore analyzed the expressed Ig heavy chain genes in 23 cases (11 families) of familial CLL, and compared these results with data previously reported for sporadic CLL. In addition, we assessed the relationship of the occurrence of somatic mutation to several clinical and phenotypic features. The distribution of V genes among these cases was similar to that observed in sporadic CLL: VH3 > VH1 > VH4. Thirteen of the 23 cases (57%) showed germ line VH gene sequences, whereas somatic mutations were detected in 10 cases (43%). The average mutation frequency of these latter 10 cases of was 6.7% (ranging from 1.7% to 8.8%), and evidence of antigen selection was noted in 6. Intraclonal variation, followed by clonal evolution and the appearance of a second clone over a 20-year period was observed in 1 case, suggesting that mutations can continue to accumulate after neoplastic transformation. The presence of somatic mutations correlated with age at presentation, low white blood cell (WBC) count, and low fluorescence intensity of surface CD5, and the potential significance of these relationships is discussed. Our data indicate that familial and sporadic B-CLL display a similar pattern of immunoglobulin gene usage and frequency of somatic mutation, and are consistent with a common ontogeny and immunogenetic origin for these 2 epidemiologically distinct forms of CLL. (Blood. 2000;95:1413-1419)

HIV-1 VRC01 Germline-Targeting Immunogens Select Distinct Epitope-Specific B Cell Receptors

Activating precursor B cell receptors of HIV-1 broadly neutralizing antibodies requires specifically designed immunogens. Here, we compared the abilities of three such germline-targeting immunogens against the VRC01-class receptors to activate the targeted B cells in transgenic mice expressing the germline VH of the VRC01 antibody but diverse mouse light chains. Immunogen-specific VRC01-like B cells were isolated at different time points after immunization, their VH and VL genes were sequenced, and the corresponding antibodies characterized. VRC01 B cell sub-populations with distinct cross-reactivity properties were activated by each immunogen, and these differences correlated with distinct biophysical and biochemical features of the germline-targeting immunogens. Our study indicates that the design of effective immunogens to activate B cell receptors leading to protective HIV-1 antibodies will require a better understanding of how the biophysical properties of the epitope and its surrounding surface on the germline-targeting immunogen influence its interaction with the available receptor variants invivo.

A VH1-69 antibody lineage from an infected Chinese donor potently neutralizes HIV-1 by targeting the V3 glycan supersite.

An oligomannose patch around the V3 base of HIV-1 envelope glycoprotein (Env) is recognized by multiple classes of broadly neutralizing antibodies (bNAbs). Here, we investigated the bNAb response to the V3 glycan supersite in an HIV-1-infected Chinese donor by Env-specific single B cell sorting, structural and functional studies, and longitudinal analysis of antibody and virus repertoires. Monoclonal antibodies 438-B11 and 438-D5 were isolated that potently neutralize HIV-1 with moderate breadth, are encoded by the VH1-69 germline gene, and have a disulfide-linked long HCDR3 loop. Crystal structures of Env-bound and unbound antibodies revealed heavy chain-mediated recognition of the glycan supersite with a unique angle of approach and a critical role of the intra-HCDR3 disulfide. The mechanism of viral escape was examined via single-genome amplification/sequencing and glycan mutations around the N332 supersite. Our findings further emphasize the V3 glycan supersite as a prominent target for Env-based vaccine design.

Dynamics of heavy chain junctional length biases in antibody repertoires

Antibody variable domain sequence diversity is generated by recombination of germline segments. The third complementarity-determining region of the heavy chain (CDR H3) is the region of highest sequence diversity and is formed by the joining of heavy chain VH, DH and JH germline segments combined with random nucleotide trimming and additions between these segments. We show that CDR H3 and junctional segment length distributions are biased in human antibody repertoires as a function of VH, VL and JH germline segment utilization. Most length biases are apparent in the naive and antigen experienced B cell compartments but not in nonproductive recombination products, indicating B cell selection as a major driver of these biases. Our findings reveal biases in the antibody CDR H3 diversity landscape shaped by VH, VL, and JH germline segment use during naive and antigen-experienced repertoire selection.

Genetic removal of the CH1 exon leads to the production of hypofunctional heavy chain-only IgG2a in rats

Despite great values in many applications, heavy chain-only antibodies (HcAbs) are naturally only produced in camelids and sharks, which are not easy to access and handle. Production of the type of antibodies in small laboratory animals would remarkably facilitate their applications. We previously reported a mouse line in which the CH1 exon of mouse γ1 was deleted that could express heavy chain-only IgG1 antibodies. However, these mice showed an extremely weak IgG1 response to specific antigens when immunized, and we could only achieve single VH domains with low affinity to antigens using these mice. One possibility is that the mouse germline VH repertoire was not sufficient to support the expression of functional heavy chain-only antibodies. In this study, we report the generation of a rat line in which the CH1 exon of the γ2a gene was removed and the γ1 and γ2b genes were silenced. Although the genetically modified rats expressed heavy chain-only IgG2a, they also exhibited a very weak IgG2a response to antigen immunization. Panning of a phage library constructed using IgG2a VH segments amplified from immunized rats identified antigen-specific single VH antibodies, which also exhibited much lower affinity than that of commercial mAbs. Together with our previous report, this study suggests that the simple genetic removal of the CH1 exon does not guarantee the successful expression of functional heavy chain-only antibodies.

Public Endowment of B Cells

Using a transgenic mouse strain expressing the human VH1-69 germline gene used by many broadly neutralizing antibodies to influenza A virus, Sangesland et al. show that the VH1-69 gene segment provides the essentials for mounting antibody responses against the conserved hemagglutinin stem epitope.

A broadly neutralizing germline-like human monoclonal antibody against dengue virus envelope domain III.

Dengue is the most widespread vector-borne viral disease caused by dengue virus (DENV) for which there are no safe, effective drugs approved for clinical use. Here, by using sequential antigen panning of a yeast antibody library derived from healthy donors against the DENV envelop protein domain III (DIII) combined with depletion by an entry defective DIII mutant, we identified a cross-reactive human monoclonal antibody (mAb), m366.6, which bound with high affinity to DENV DIII from all four DENV serotypes. Immunogenetic analysis indicated that m366.6 is a germline-like mAb with very few somatic mutations from the closest VH and Vλ germline genes. Importantly, we demonstrated that it potently neutralized DENV both in vitro and in the mouse models of DENV infection without detectable antibody-dependent enhancement (ADE) effect. The epitope of m366.6 was mapped to the highly conserved regions on DIII, which may guide the design of effective dengue vaccine immunogens. Furthermore, as the first germline-like mAb derived from a naïve antibody library that could neutralize all four DENV serotypes, the m366.6 can be a tool for exploring mechanisms of DENV infection, and is a promising therapeutic candidate.

VH1-69 Utilizing Antibodies Are Capable of Mediating Non-neutralizing Fc-Mediated Effector Functions Against the Transmitted/Founder gp120.

Multiple antibody effector functions arise in HIV-1 infection that could be harnessed to protect against infection or clear the persistent reservoir. Here, we have investigated the genetic and functional memory B cell and antibody landscape present during early infection in six individuals infected with either subtype A, C, or an A/C recombinant HIV-1. These individuals demonstrated varying levels of plasma autologous neutralization (nAb) against the transmitted/founder envelope (T/F Env) pseudovirus and non-neutralizing Fc-mediated effector function (nnFc) antibody-dependent cell-mediated cytotoxicity (ADCC) against the T/F Env gp120 protein at ~7 months after infection. Genetic analysis of the immunoglobulin heavy (VH) and light (VL) chain variable domain gene segments from 352 autologous T/F Env gp120-specific single B cells recovered at this same 7-month time-point revealed an over-representation of the VH1-69 germline in five of six individuals. A defining feature of the VH1-69 utilizing gp120-specific antibodies was their significantly more hydrophobic complementarity-determining region-2 (CDRH2) regions compared to other VH CDRH2 sequences from each individual. While none of the VH1-69 antibodies possessed strong neutralizing activity against virions pseudotyped with the autologous T/F Env, almost a third were capable of mediating high ADCC activity, as assayed by intracellular granzyme B activity in CEM.NKr.CCR5 target cells coated with autologous T/F Env gp120. High ADCC mediating VH1-69 antibodies exhibited shorter complementarity-determining region-3 (CDRH3) lengths and a more neutral isoelectric point than antibodies lacking this function. In the individual that developed the highest autologous ADCC responses, the high granzyme B producing antibodies bound to surface expressed envelope in the absence of CD4 and were not enhanced by the addition of soluble CD4. Overall, VH1-69 utilizing antibodies are commonly induced against gp120 in diverse HIV-1 infections and a subset of these antibodies can mediate ADCC functions, serving as a bridge between the innate and adaptive immune response to HIV-1.

A primer set for comprehensive amplification of V-genes from rhesus macaque origin based on repertoire sequencing

Recombinant antibodies serve as therapeutic molecules for a broad range of applications. High affinity antibodies are typically isolated following an active and effective immunization. Human-like antibodies may be obtained from immunized nonhuman primates (NHP), such as rhesus macaque, when immunized human origin is not available. For the isolation of such antibodies, strategies like phage and yeast display, are employed. These strategies are primarily based on the amplification of the rearranged variable (V) regions coded by mRNA, obtained from lymphatic source of immunized animals. To amplify these genomic sequences, designated set of primers are required, ideally covering the immune animal V-gene repertoire. Such primer sets are commonly designed based on the germline repertoire of specific animals according to immunoglobulin databases. However, In case of rhesus macaque, however, the known immunoglobulin germline V-gene database is still limited. The emergence and continuous improvements in high-throughput sequencing (HTS) technologies now enable the profiling of an immune repertoire for both basic and applicative studies, among which is the identification and expression of novel alleles. We report here on the profiling of non-immunized rhesus macaque (Macaca mulatta) expressed antibody repertoire, using HTS and advanced tailored bioinformatics tools. This analysis resulted in 32,480 and 73,354 complete heavy and light variable gene (VH and VL) sequences, respectively. Further analysis of these sequences, using the IgDiscover tool, resulted in the identification of 102, 214 and 48 inferred VH, Vκ and Vλ germline sequences, respectively, of which over 50% are novel alleles. This dataset, together with other recently published datasets, enabled the design of a comprehensive primer set (v2018), which demonstrated the broadest coverage of rhesus macaque germline genes identified up to date. The newly designed primer set was confirmed for its extent of coverage of the V-genes in various datasets of rhesus macaque germlines as well as the expressed repertoire mapped in this study. Among other things, an improvement of 28% and 50% in the coverage of the VH and VL expressed repertoire was demonstrated in comparison to a primer set we have previously designed. This primer set can be further used for various applications that require the complete coverage of the NHP V-gene repertoire.

An innovative flow cytometry method to screen human scFv-phages selected by in vivo phage-display in an animal model of atherosclerosis

Atherosclerosis is a chronic, progressive inflammatory disease that may develop into vulnerable lesions leading to thrombosis. This pathology is characterized by the deposition of lipids within the arterial wall and infiltration of immune cells leading to amplification of inflammation. Nowadays there is a rising interest to assess directly the molecular and cellular components that underlie the clinical condition of stroke and myocardial infarction. Single chain fragment variable (scFv)-phages issuing from a human combinatorial library were selected on the lesions induced in a rabbit model of atherosclerosis after three rounds of in vivo phage display. We further implemented a high-throughput flow cytometry method on rabbit protein extracts to individually test one thousand of scFv-phages. Two hundred and nine clones were retrieved on the basis of their specificity for atherosclerotic proteins. Immunohistochemistry assays confirmed the robustness of the designed cytometry protocol. Sequencing of candidates demonstrated their high diversity in VH and VL germline usage. The large number of candidates and their diversity open the way in the discovery of new biomarkers. Here, we successfully showed the capacity of combining in vivo phage display and high-throughput cytometry strategies to give new insights in in vivo targetable up-regulated biomarkers in atherosclerosis.

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.