VH Families Research Articles

The influence of variable-heavy chain families on IgG2, 3, 4, FcγRs and B-cell superantigens protein G and L binding using biolayer interferometry.

As the most abundant immunoglobulin in blood and the most common human isotype used for therapeutic monoclonal antibodies, the engagement and activation of its Fc receptors by IgGs are crucial for antibody function. Assumed to be relatively constant within subtypes, recent studies reveal that antibody variable regions exert distal effects of modulating antibody-receptor interactions on antibody isotypes. These variable (V)-region distal effects are also expected for the IgG subtypes. With an in-depth understanding of the V-region effects, researchers can make a more informed antibody engineering approach and antibody purification strategy accounting for the functions of microbial immune evasion . In this study, we created a panel of IgG2/IgG3/IgG4 antibodies by changing the VH family (VH1-7) frameworks while retaining the complementary determining regions of pertumuzab and measured their interactions with FcγRIa, FcγRIIaH167, FcγRIIaR167, FcγRIIb/c, FcγRIIIaF176, FcγRIIIaV176, FcγRIIIbNA1 and FcγRIIIbNA2 receptors alongside B-cell superantigens Protein L and G using biolayer interferometry. The panel of 21 IgGs demonstrated that the VH frameworks influenced receptor binding sites on the constant region in a non-canonical manner. However, there was minimal influence on the binding of bacterial B-cell superantigens Proteins L and Protein G on the IgGs, showing their robustness against V-region effects. These results demonstrate the role of V-regions during the humanization of therapeutic antibodies that can influence FcR-dependent immune responses while retaining binding by bacterial B-cell superantigens for antibody purification. These in vitro measurements provide a clue to detailed antibody engineering and understanding of antibody superantigen functions that would be relevant with in vivo validation.

Mutational features of immunoglobulin heavy chain variable region gene in patients with chronic lymphocytic leukemia

Objective: To investigate the mutational features of the immunoglobulin heavy chain variable region (IgHV) gene in patients with chronic lymphocytic leukemia (CLL) using immunophenotypic and molecular genetic methods. Methods: The laboratory results of 266 CLL patients who underwent IgHV gene examination at Sino-US diagnostics laboratory from February 2020 to February 2021 were analyzed for the IgVH mutational status and presence of specific IgVH fragments. In addition, their immunophenotypic, molecular, chromosomal karyotypic, and FISH profiles were investigated and correlated with the IgVH mutational status. Results: Among 266 patients, 172 were male and 94 were female, with a media age of 67 years (20-82 years).There were more patients with mutated IgHV (m-IgHV) than unmutated IgHV (un-IgHV) (69.2%∶30.8%). There was association of VH family and the presence of gene fragments: the overall incidence of VH families including VH3 family (142/266, 53.4%), VH4 family (75/266, 28.2%), and VH1 family (34/266, 12.8%) was about 95%, among which the proportion of VH4-34 (26/266, 9.8%), VH3-23 (25/266, 9.4%), VH3-7 (24/266, 9.0%), and VH4-39 (16/266, 6.0%) was about 35%. VH3-20 and VH3-49 only occurred in un-IgHV (P<0.05). In addition, the expression rates of CD38 (26.3% vs. 3.0%), CD79b (71.1%∶45.5%) and 11q deletion (25.5%∶5.3%) were higher in un-IgHV, and single trisomy 12 (37.9%∶5.6%) were more commonly found in m-IgHV (P<0.05). MYD88 was one of the major mutation genes in m-IgHV, while ATM had the highest mutation rate in un-IgHV. Conclusion: CLL patients have differential expression in terms of IgHV gene mutations, correlating to their immunophenotype and genetics characteristics.

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.

Lack of Bmf Facilitates the Selection of Highly Responsive B-Cell Receptor Clones in Chronic Lymphocytic Leukemia

Introduction: Chronic lymphocytic leukemia (CLL) is a disease of inhibited cell death, increased proliferation and high importance of interactions with the microenvironment e.g. with T-cells or stromal cells. A central effector in this concert is the activation of B-cell receptor (BCR) signalling pathway, associated with selection of specific BCR qualities (either autonomous signalling or reactivity to chronic (auto)-antigenic stimuli) and these signals play an essential role in CLL disease development and progression, also evidenced by the clinical success of kinase inbibitors directed at this signal. Mechanisms modulating selection of specific BCR types are ill understood, but since checkpoints during BCR selection in B cells are guarded by the Bcl2 family of proteins it is likely that cell death proteins are also influencing these decisions in CLL. To study the importance of microenvironmental interactions and antigen stimulation Tcl1 tg mice, which develop a murine CLL highly similar to the human disease 1,2, were frequently applied in the past, aiming to overcome the limitations of more or less artificial CLL in vitro culture systems. During clonal evolution of CLL, predominantly unmutated and stereotyped IgVH-11 and IgVH-12 BCRs are selected in Tcl1 tg mice which were shown to be specific for the autoantigen phosphatidylcholine (PtC) 3, however, the detailed mechanisms of clonal selection in CLL are still unclear. Here we propose a role of the BH3-only and pro-apoptotic protein Bmf in clonal selection by eliminating CLL cells expressing highly responsive BCRs.Methods: Tcl1 tg mice were crossed to Bmf knockout (KO) mice and CLL development monitored over time. After establishment of overt leukemia, mice were sacrificed and the overall survival time analysed. Additionally, we performed BCR sequencing, RNA-Seq and phosphoproteomics after 10µg/ml aIgM treatment using mass cytometry (Helios, CyTOF) and flow cytometry.Results: We found that Tcl1 tg Bmf KO mice developed phenotypically unchanged murine CLL but had an earlier onset of disease (Figure 1A) and an altered usage of BCR IgVH genes. While Tcl1 tg mice usually use PtC specific IgVH genes from the VH11 and V12 family, absence of Bmf led to a skewing to non PtC specific IgVH genes, mainly form the VH1 family (Figure 1B). We thus speculated that lack of Bmf favoured the selection of CLL clones with hyper responsive BCRs, when compared to Tcl1 tg mice with functional Bmf. Indeed we could show that after aIgM stimulation Tcl1 tg BmfKO CLL cells showed increased BCR signalling as measured by mass cytometry (Figure 1C), which was also confirmed by conventional flow cytometry (Figure 1D). Importantly, strong BCR stimulation induces cell death in B cells. Indeed, this can be observed in CLL cells from Tcl1 mice. In Tcl1 tumors that are deficient for BMF this cell death induction is not observed, suggesting that loss of Bmf protects from excessive cell death due to strong BCR stimulation.Conclusions: Here we report a novel role of Bmf in the selection of BCR clones in murine CLL. The higher BCR signalling capacity and the skewing of IgVH gene usage in the absence of Bmf indicates that highly responsive BCR clones are more often selected instead of eliminated during clonal selection.. Interestingly, a SNP in the Bmf gene has been reported to be susceptibility locud for CLL, suggesting that low expression of BMF may have similar effects in huma CLL. Indeed, in preliminary analyses, we observed a similar contribution of Bmf in clonal selection and BCR responsiveness in human CLL and decreased resistance to the BCR signalling inhibitor ibrutinib. Therefore our findings might also be important in a clinical context.

P-036: Implementation of IgH/k Next Generation Sequencing for Multiple Myeloma Minimal Residual Disease monitoring: advantages in patients’ management during daily clinical practice.

<h3>Background</h3> The introduction of novel agents into the therapeutic algorithm of Multiple Myeloma (MM) has led to remarkable improvements in the rate of Minimal Residual Disease (MRD) negative status. Sustained MRD negativity is a robust prognosticator and a driver of treatment strategies. Therefore, it is expected that MRD molecular tracking would become critical in the management of MM, and could be included in daily clinical practice. <h3>Aim</h3> To evaluate the performances of next generation sequencing (NGS), implemented as best practice in MM MRD daily evaluation. <h3>Methods</h3> A cohort of 166 newly diagnosed transplant-eligible and -ineligible MM pts were screened to define the ID clonotypes. MRD was firstly assessed at achievement of at least a very good partial response during maintenance, and thereafter once a year. In high-risk pts, MRD was also detected between 1° and 2° autologous stem-cell transplantation (ASCT). The ID clonotypes screening has been performed by NGS, using an assay covering IgH and Igk genes (Invivoscribe®). The MRD tracking has been done both by conventional ASO-qPCR and by NGS. Data were analyzed by Lymphotrack Dx and MRD software (Invivoscribe®). <h3>Results</h3> The ID clonotypes screening was successful in 158/166 pts (95%), even though a small proportion of pts resulted polyclonal (14/158). Overall, 63, 27 and 54 pts had IgH, Igk and IgH/k ID clonotypes, respectively, mainly restricted to the VH3 (47%) family genes. MRD assessment was performed by NGS in 124/144 pts (overall, 96 MRD samples for 64 pts monitored to date), with 10-5 sensitivity in most cases (75/96). 44 pts were assessed during maintenance and 23 achieved MRD-negativity. On the contrary, 20 pts were evaluated between 1° and 2° ASCT, resulting MRD-positive in all cases. Overall, few FU samples (13/96) were reported as "positive-not-quantifiable" (PNQ), since had residual disease levels of 10-7, much lower than the internal control. In a subset of 20 pts with trackable IgH/Igk sequences, pts-specific assays were designed, according to the EuroMRD guidelines. In these pts, MRD was tested both by ASO-qPCR (including a standard curve and healthy donor cells, to define the sensitivity and the specificity, respectively) and by NGS. Overall, ASO-qPCR allowed the tracking of a single MRD clone (with up to 10-4 sensitivity) in 12/20 pts (60%), whereas NGS was feasible in all of them, confirming the ASO-qPCR results, yet with higher sensitivity and >95% confidence. Moreover, in 2 cases, NGS allowed to disclose ASO-qPCR PNQ results due to nonspecific amplification, thus unveiling MRD negativity with 10-5 sensitivity. <h3>Conclusion</h3> NGS has been successfully implemented as best practice in the management of MM pts, showing the superiority of this method over ASO-qPCR conventional approach. The ID clonotype was defined in almost all pts included in the workflow, thus allowing their MRD molecular monitoring, according to the clinical requirements. <h3>Acknowledgment</h3> AIRC IG2018-22059

Distinction between peanut allergy and tolerance by characterization of B cell receptor repertoires.

BackgroundSpecific IgE against a peanut 2S albumin (Ara h 2 or 6) is the best predictor of clinically relevant peanut sensitization. However, sIgE levels of peanut allergic and those of peanut sensitized but tolerant patients partly overlap, highlighting the need for improved diagnostics to prevent incorrect diagnosis and consequently unnecessary food restrictions. Thus, we sought to explore differences in V(D)J gene transcripts coding for peanut 2S albumin‐specific monoclonal antibodies (mAbs) from allergic and sensitized but tolerant donors.Methods2S albumin‐binding B‐cells were single‐cell sorted from peripheral blood of peanut allergic (n=6) and tolerant (n=6) donors sensitized to Ara h2 and/or 6 (≥ 0.1 kU/l) and non‐atopic controls (n=5). h 2 and/or 6 (≥ 0.1 kU/l). Corresponding h heavy and light chain gene transcripts were heterologously expressed as mAbs and tested for specificity to native Ara h2 and 6. HCDR3 sequence motifs were identified by Levenshtein distances and hierarchically clustering.ResultsThe frequency of 2S albumin‐binding B cells was increased in allergic (median: 0.01%) compared to tolerant (median: 0.006%) and non‐atopic donors (median: 0.0015%, p = 0.008). The majority of mAbs (74%, 29/39) bound specifically to Ara h 2 and/or 6. Non‐specific mAbs (9/10) were mainly derived from non‐atopic controls. In allergic donors, 89% of heavy chain gene transcripts consisted of VH3 family genes, compared with only 54% in sensitized but tolerant and 63% of non‐atopic donors. Additionally, certain HCDR3 sequence motifs were associated with allergy (n = 4) or tolerance (n = 3) upon hierarchical clustering of their Levenshtein distances.ConclusionsPeanut allergy is associated with dominant VH3 family gene usage and certain public antibody sequences (HCDR3 motifs).

TLR-9 agonist and CD40-targeting vaccination induces HIV-1 envelope-specific B cells with a diversified immunoglobulin repertoire in humanized mice.

The development of HIV-1 vaccines is challenged by the lack of relevant models to accurately induce human B- and T-cell responses in lymphoid organs. In humanized mice reconstituted with human hematopoietic stem cells (hu-mice), human B cell-development and function are impaired and cells fail to efficiently transition from IgM B cells to IgG B cells. Here, we found that CD40-targeted vaccination combined with CpG-B adjuvant overcomes the usual defect of human B-cell switch and maturation in hu-mice. We further dissected hu-B cell responses directed against the HIV-1 Env protein elicited by targeting Env gp140 clade C to the CD40 receptor of antigen-presenting cells. The anti-CD40.Env gp140 vaccine was injected with CpG-B in a homologous prime/boost regimen or as a boost of a NYVAC-KC pox vector encoding Env gp140 clade C. Both regimens elicited Env-specific IgG-switched memory hu-B cells at a greater magnitude in hu-mice primed with NYVAC-KC. Single-cell RNA-seq analysis showed gp140-specific hu-B cells to express polyclonal IgG1 and IgG3 isotypes and a broad Ig VH/VL repertoire, with predominant VH3 family gene usage. These cells exhibited a higher rate of somatic hypermutation than the non-specific IgG+ hu-B-cell counterpart. Both vaccine regimens induced splenic GC-like structures containing hu-B and hu-Tfh-like cells expressing PD-1 and BCL-6. We confirmed in this model that circulating ICOS+ memory hu-Tfh cells correlated with the magnitude of gp140-specific B-cell responses. Finally, the NYVAC-KC heterologous prime led to a more diverse clonal expansion of specific hu-B cells. Thus, this study shows that CD40-targeted vaccination induces human IgG production in hu-mice and provides insights for the development of a CD40-targeting vaccine to prevent HIV-1 infection in humans.

Reshaping nanobodies for affinity purification on protein a

Nanobodies (Nbs) are 15 kDa recombinant, single-domain, antigen-specific fragments derived from heavy-chain only antibodies (HCAbs) occurring naturally in species of Camelidae. The beneficial properties of Nbs make them suitable tracers for diagnostic and therapeutic purposes. Whereas Nbs with a terminal hexa-histidine tag (His-tag) are easily purified via immobilized metal affinity chromatography, previous studies revealed a negative impact of the His-tag on the biodistribution of Nb-based tracers. Thus, it is important to develop alternative purification methods for Nbs without a His-tag. Protein A (SpA), a surface protein of Staphylococcus aureus, binds the Fc-region of IgG molecules and also to a lesser extent human heavy chain family-3 variable (VH) regions. Nbs also belong to this VH family, although many fail to be recognized by SpA. Here it is demonstrated that non-SpA binding Nbs can be mutagenized for purification by SpA affinity chromatography and that these Nb variants retain their thermostability and antigen affinity, while biodistribution remains unaffected.

Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments

Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.

Generation and characterization of a specific single-chain antibody against DSPP as a prostate cancer biomarker: Involvement of bioinformatics-based design of novel epitopes

Isolation of specific single chain antibodies (scFvs) against key epitopes of cancer markers are applied for cancer immunotherapy and diagnosis. In this study following the prediction of the 3D structure of the DSP part of Dentin sialophosphoprotein (DSPP), the epitope was chosen using in silico programs. Panning process was applied to isolate specific human scFv against the epitope. PCR and DNA fingerprinting differentiated the specific clones, which were evaluated by phage ELISA. Following DNA sequencing, the 3D structure of isolated scFv was modeled and Docked on DSP. Results demonstrated the selection of a specific anti-DSPP scFv with 40% frequency, which reacted significantly with the predicted epitope and PCa patients' urines in ELISA tests (P-value < 0.05). The VH and VL of the isolated scFv were from VH1 and VL3 gene families with several amino acid changes in CDRs and FRs domains. The scFv tightly bound to the DSP epitope with the lowest energy level by hydrogen bonds, cation-pi, hydrophobic and ionic interactions demonstrating the specificity of Ag-Ab interactions. The anti-DSPP scFv selected in this study with significant specificity to DSPP antigen offers a promising new agent for both PCa early detection and treatment of cancers with DSPP expression.

The Dual Method Approach (DMA) Resolves Measurement Range Limitations of Heat Pulse Velocity Sap Flow Sensors

Sap flow, the movement of fluid in the xylem of plants, is commonly measured with the heat pulse velocity (Vh) family of methods. The observable range of Vh in plants is ~−10 to ~+270 cm/h. However, most Vh methods only measure a limited portion of this range, which restricts their utility. Previous research attempted to extend the range of Vh methods, yet these approaches were analytically intensive or impractical to implement. The Dual Method Approach (DMA), which is derived from the optimal measurement ranges of two Vh methods, the Tmax and the heat ratio method (HRM), also known as the “slow rates of flow” method (SRFM), is proposed to measure the full range of sap flow observable in plants. The DMA adopts an algorithm to dynamically choose the optimal Vh measurement via the Tmax or HRM/SRFM. The DMA was tested by measuring sap flux density (Js) on Tecoma capensis (Thunb.) Lindl., stems and comparing the results against Js measured gravimetrically. The DMA successfully measured the entire range of Vh observed in the experiment from 0.020 to 168.578 cm/h, whereas the HRM/SRFM range was between 0.020 and 45.063 cm/h, and the Tmax range was between 2.049 cm/h and 168.578 cm/h. A linear regression of DMA Js against gravimetric Js found an R2 of 0.918 and error of 1.2%, whereas the HRM had an R2 of 0.458 and an error of 49.1%, and the Tmax had an R2 of 0.826 and an error of 0.5%. Different methods to calculate sapwood thermal diffusivity (k) were also compared with the kVand method showing better accuracy. This study demonstrates that the DMA can measure the entire range of Vh in plants and improve the accuracy of sap flow measurements.

Detection of Clinically Relevant Molecular Alterations in Chronic Lymphocytic Leukemia (CLL) By Nanopore Sequencing

IntroductionChronic Lymphocytic Leukaemia (CLL) is the most prevalent leukaemia in the Western world and characterised by clinical heterogeneity. IgHV mutation status, mutations in the TP53 gene and deletions of the p-arm of chromosome 17 are currently used to predict an individual patient's response to therapy and give an indication as to their long-term prognosis. Current clinical guidelines recommend screening patients prior to initial, and any subsequent, treatment. Routine clinical laboratory practices for CLL involve three separate assays, each of which are time-consuming and require significant investment in equipment. Nanopore sequencing offers a rapid, low-cost alternative, generating a full prognostic dataset on a single platform. In addition, Nanopore sequencing also promises low failure rates on degraded material such as FFPE and excellent detection of structural variants due to long read length of sequencing. Importantly, Nanopore technology does not require expensive equipment, is low-maintenance and ideal for patient-near testing, making it an attractive DNA sequencing device for low-to-middle-income countries.MethodsEleven untreated CLL samples were selected for the analysis, harbouring both mutated (n=5) and unmutated (n=6) IgHV genes, seven TP53 mutations (five missense, one stop gain and one frameshift) and two del(17p) events. Primers were designed to amplify all exons of TP53, along with the IgHV locus, and each primer included universal tails for individual sample barcoding. The resulting PCR amplicons were prepared for sequencing using a ligation sequencing kit (SQK-LSK108, Oxford Nanopore Technologies, Oxford, UK). All IgHV libraries were pooled and sequenced on one R9.4 flowcell, with the TP53 libraries pooled and sequenced on a second R9.4 flowcell. Whole genome libraries were prepared from 400ng genomic DNA for each sample using a rapid sequencing kit (SQK-RAD004, Oxford Nanopore Technologies, Oxford, UK), and each sample sequenced on individual flowcells on a MinION mk1b instrument (Oxford Nanopore Technologies, Oxford, UK). We developed a bespoke bioinformatics pipeline to detect copy-number changes, TP53 mutations and IgHV mutation status from the Nanopore sequencing data. Results were compared to short-read sequencing data obtained earlier by targeted deep sequencing (MiSeq, Illumina Inc, San Diego, CA, USA) and whole genome sequencing (HiSeq 2500, Illumina Inc, San Diego CA, USA).ResultsFollowing basecalling and adaptor trimming, the raw data were submitted to the IMGT database. In the absence of error correction, it was possible to identify the correct VH family for each sample; however the germline homology was not sufficient to differentiate between IgHVmut and IgHVunmut CLL cases. Following bio-informatic error correction and consensus building, the percentage to germline homology was the same as that obtained from short-read sequencing and nanopore sequencing also called the same productive rearrangements in all cases.A total of 77 TP53 variants were identified, including 68 in non-coding regions, and three synonymous SNVs. The remaining 6 were predicted to be functional variants (eight missense and two stop-gains) and had all been identified in early MiSeq targeted sequencing. However, the frameshift mutation was not called by the analysis pipeline, although it is present in the aligned reads. Using the low-coverage WGS data, we were able to identify del(17p) events, of 19Mb and 20Mb length, in both patients with high confidence.ConclusionsHere we demonstrate that characterization of the IgHV locus in CLL cases is possible using the MinION platform, provided sufficient downstream analysis, including error correction, is applied. Furthermore, somatic SNVs in TP53 can be identified, although similar to second generation sequencing, variant calling of small insertions and deletions is more problematic. Identification of del(17p) is possible from low-coverage WGS on the MinION and is inexpensive. Our data demonstrates that Nanopore sequencing can be a viable, patient-near, low-cost alternative to established screening methods, with the potential of diagnostic implementation in resource-poor regions of the world. DisclosuresSchuh:Giles, Roche, Janssen, AbbVie: Honoraria.

The proliferation and clonal migration of B cells in the systemic and mucosal tissues of channel catfish suggests there is an interconnected mucosal immune system

IgM transcripts from different mucosal and systemic tissues from a single adult channel catfish have been evaluated. Arrayed heavy chain cDNA libraries from each of these different mucosal and systemic tissues were separately constructed, hybridized with VH family specific probes and a variety of approaches were used to define their structural relationships. Baseline hybridization studies indicated that the tissue libraries had different VH expression patterns, and sequencing studies indicated this was not simply due to varying proportions of the same B cell population. In the systemic tissues of PBL, spleen, and anterior kidney >95% of the sequenced clones in the arrayed libraries represented different heavy chain rearrangements. Diversity was also found in the mucosal libraries of skin, gill lamellae, and two non-adjoining regions of the intestine, but additional populations were identified which indicated localized clonal expansion. Various clonal sets were characterized in detail, and their genealogies indicated somatic mutation accompanied localized clonal expansion with some members undergoing additional mutations and expansion after migration to different mucosal sites. PCR analyses indicated these mucosal clonal sets were more abundant within different mucosal tissues rather than in the systemic tissues. These studies indicate that the mucosal immune system in fish can express B cell transcripts differently from those found systemically. These studies further indicate that the mucosal immune system is interconnected with clonal B cells migrating between different mucosal tissues, results which yield new insight into immune diversity in early vertebrate phylogeny.

Antibody Epitope Specificity for dsDNA Phosphate Backbone Is an Intrinsic Property of the Heavy Chain Variable Germline Gene Segment Used.

Analysis of protein sequences by the informational spectrum method (ISM) enables characterization of their specificity according to encoded information represented with defined frequency (F). Our previous data showed that F(0.367) is characteristic for variable heavy chain (VH) domains (a combination of variable (V), diversity (D) and joining (J) gene segments) of the anti-phosphocholine (PC) T15 antibodies and mostly dependent on the CDR2 region, a site for PC phosphate group binding. Because the T15 dsDNA-reactive U4 mutant also encodes F(0.367), we hypothesized that the same frequency may also be characteristic for anti-DNA antibodies. Data obtained from an analysis of 60 spontaneously produced anti-DNA antibody VH domain sequences supported our hypothesis only for antibodies, which use V gene segment in germline configuration, such as S57(VH31), MRL-DNA22, and VH11, members of the VH1 (J558) and VH7 (S107) gene families. The important finding is that out of seven V gene segments used by spontaneous anti-DNA antibodies, F(0.367) is only expressed by the germline configuration of these three V gene segments. The data suggest that antibody specificity for the phosphate group moiety delineated as F(0.367) is the intrinsic property of the V germline gene segments used, whereas paratope/epitope interaction with antigens bearing this epitope, such as PC or dsDNA, requires corresponding antibody VH conformation that is susceptible to somatic mutation(s).

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.

Effect of VH-VL Families in Pertuzumab and Trastuzumab Recombinant Production, Her2 and FcγIIA Binding.

Many therapeutic antibodies are humanized from animal sources. In the humanization process, complementarity determining region grafting is tedious and highly prone to failure. With seven known VH families, and up to six known κ VL families, there are choices aplenty. However, the functions of these families remain largely enigmatic. To study the role of these V-region families, we made 84 recombinant combinations of the various VH and VL family whole IgG1 variants of both Trastuzumab and Pertuzumab. We managed to purify 66 of these to investigate the biophysical characteristics: recombinant protein production, and both Her2 and FcγIIA binding. Our findings revealed combinations that showed improved recombinant antibody production and both antigen and receptor binding kinetics. These findings show the need to rethink antibodies as a whole protein, relooking of the functions of the antibody domains, and the need to include immunoglobulin receptor investigations for effective antibody therapeutics development.

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.

Human IgG repertoire of malaria antigen-immunized human immune system (HIS) mice

Humanized mouse models present an important tool for preclinical evaluation of new vaccines and therapeutics. Here we show the human variable repertoire of antibody sequences cloned from a previously described human immune system (HIS) mouse model that possesses functional human CD4+ T cells and B cells, namely HIS-CD4/B mice. We sequenced variable IgG genes from single memory B-cell and plasma-cell sorted from splenocytes or whole blood lymphocytes of HIS-CD4/B mice that were vaccinated with a human plasmodial antigen, a recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP). We demonstrate that rPfCSP immunization triggers a diverse B-cell IgG repertoire composed of various human VH family genes and distinct V(D)J recombinations that constitute diverse CDR3 sequences similar to humans, although low hypermutated sequences were generated. These results demonstrate the substantial genetic diversity of responding human B cells of HIS-CD4/B mice and their capacity to mount human IgG class-switched antibody response upon vaccination.

Detection of naive B cells expressing potentially protective antibodies

Abstract Most effective vaccines work through the induction of protective antibodies able to neutralize or eradicate infection. Unfortunately, the induction of protective antibodies has proved difficult for a number of infections, such as HIV/AIDS, or the protective antibodies that are elicited through vaccination do not bind all strains of the virus, such as with influenza. Broadly neutralizing antibodies against these viruses, displaying protective potentials in experimental animal models, have been isolated from infected individuals. The detailed characterization of these antibodies revealed that they are derived from specific, but infrequent, VH/VL gene pairing. To better characterize the rare naïve B cells that express the germline (gl) forms of these antibodies (gl B cell receptors, glBCRs) we developed unique anti-idiotype antibodies, that specifically recognize the gl form of HIV-1 and influenza broadly neutralizing antibodies, b12 and FI6, respectively. The abilities of anti-idiotypic antibodies against gl-b12 and -FI6 IgG to specifically identify the rare naïve human B cells that express gl-b12 or -FI6 were evaluated. In the case of FI6, we generated an anti-idiotype that bound to 5 closely related alleles within the VH3 family. Using this anti-idiotype in conjugation with a Flu protein that can bind to gl-FI6, we have begun to characterize a family of gl-FI6-like antibodies. For b12, one anti-idiotype displayed superior ability to target naïve B cells expressing gl-b12-like BCRs. The availability of this novel reagent allows for the high-throughput, in-depth, characterization of the rare B cells that are the precursors of broadly neutralizing antibodies, and will assist efforts to elicit such antibodies through vaccination

Immunogenetics of chronic lymphocytic leukemia.

Cytogenetic aberrations as well as presence of IGVH mutations are the underlying reason for clinical heterogeneity in Chronic Lymphocytic Leukemia (CLL). The presence of IGVH mutations as well as the predominant gene usage shows geographical variations. However, there is no study from India addressing immunogenetics of CLL. In a first Indian study we document the immunogenetics of CLL in a large tertiary hospital. We analyzed IGVH mutation status, VH gene usage, cytogenetic abnormalities using FISH, immunophenotyping data and correlated them with standard clinical variables in 84 patients of CLL. Advanced Rai stage (Stage 3/4) was seen in 45% of our patients, where as 13q deletion was the commonest clonal cytogenetic abnormality detected in 48.4% of the cases. IGVH unmutated cases (55.2%) showed higher proportion expressing CD38 and CD49d, a preferential usage for VH1 and VH3 families (55.2%), presentation at an advanced Rai stage (52.8%) as well as more frequent presence of p53 deletions. As compared to the IGVH mutated cases greater proportion of IGVH unmutated patients (70%) required treatment. However, there was no significant difference in the time to treatment between mutated and unmutated cases which can be attributed to relatively short median follow up of 10 months. To summarize, we have seen a higher proportion of IGVH unmutated patients in our cohort (55.2%). The commonly used VH genes in the Indian population are IGVH 2-5, IGVH 1-2 and IGVH 1-69. Longer clinical follow up and a larger cohort is necessary to confirm the prognostic value of IGVH mutation analysis in Indian Patients with CLL.