Memory B cells.

Recent advances in studies of immune memory in mice and humans have reinforced the concept that memory B cells play a critical role in protection against repeated infections, particularly from variant viruses. Hence, insights into the development of high-quality memory B cells that can generate broadly neutralizing antibodies that bind such variants are key for successful vaccine development. Here, we review the cellular and molecular mechanisms by which memory B cells are generated and how these processes shape the antibody diversity and breadth of memory B cells. Then, we discuss the mechanisms of memory B cell reactivation in the context of established immune memory; the contribution of antibody feedback to this process has now begun to be reappreciated.

B cell diversification in gut-associated lymphoid tissues: From birds to humans.

Several species generate their preimmune repertoire in gut-associated lymphoid tissues (GALT), compensating a reduced germline V gene repertoire by post-rearrangement diversification mechanisms (gene conversion and/or somatic hypermutation) in these environments that act as primary lymphoid organs. We summarize here these processes for three different species (chickens, sheep, and rabbits) and further discuss the analogous process that T-independent B cell responses in humans represent: we indeed recently showed that response against bacterial polysaccharides mobilize marginal zone B cells that prediversified against gut antigens. While the initial diversification strategy differs in these two cases, i.e., repertoire formation driven by gut-derived mitotic signals vs. response against gut antigens, the common feature of these two processes is the mobilization of a B cell compartment prediversified in GALT for immune responses against distinct systemic antigens.

SARS-CoV-2 intra-host diversity, antibody response, and disease severity after reinfection by the variant of concern Gamma in Brazil.

The rapid spread of the SARS-CoV-2 Variant of Concern (VOC) Gamma in Amazonas during early 2021 fueled a second large COVID-19 epidemic wave and raised concern about the potential role of reinfections. Very few cases of reinfection associated with the VOC Gamma have been reported to date, and their potential impact on clinical, immunological, and virological parameters remains largely unexplored. Here we describe 25 cases of SARS-CoV-2 reinfection in Brazil. SARS-CoV-2 genomic analysis confirmed that individuals were primo-infected with distinct viral lineages between March and December 2020 (B.1.1, B.1.1.28, B.1.1.33, B.1.195, and P.2) and reinfected with the VOC Gamma between 3 to 12 months after primo-infection. We found a similar mean cycle threshold (Ct) value and limited intra-host viral diversity in both primo-infection and reinfection samples. Sera of 14 patients tested 10-75 days after reinfection displayed detectable neutralizing antibodies (NAb) titers against SARS-CoV-2 variants that circulated before (B.1.*), during (Gamma), and after (Delta and Omicron) the second epidemic wave in Brazil. All individuals had milder or no symptoms after reinfection, and none required hospitalization. These findings demonstrate that individuals reinfected with the VOC Gamma may display relatively high RNA viral loads at the upper respiratory tract after reinfection, thus contributing to onward viral transmissions. Despite this, our study points to a low overall risk of severe Gamma reinfections, supporting that the abrupt increase in hospital admissions and deaths observed in Amazonas and other Brazilian states during the Gamma wave was mostly driven by primary infections. Our findings also indicate that most individuals analyzed developed a high anti-SARS-CoV-2 NAb response after reinfection that may provide some protection against reinfection or disease by different SARS-CoV-2 variants.

Antibody diversity in IVIG: Therapeutic opportunities for novel immunotherapeutic drugs.

Significant progress has been made in the elucidation of human antibody repertoires. Furthermore, non-canonical functions of antibodies have been identified that reach beyond classical functions linked to protection from pathogens. Polyclonal immunoglobulin preparations such as IVIG and SCIG represent the IgG repertoire of the donor population and will likely remain the cornerstone of antibody replacement therapy in immunodeficiencies. However, novel evidence suggests that pooled IgA might promote orthobiotic microbial colonization in gut dysbiosis linked to mucosal IgA immunodeficiency. Plasma-derived polyclonal IgG and IgA exhibit immunoregulatory effects by a diversity of different mechanisms, which have inspired the development of novel drugs. Here we highlight recent insights into IgG and IgA repertoires and discuss potential implications for polyclonal immunoglobulin therapy and inspired drugs.

Locus architecture and RAG scanning determine antibody diversity.

Diverse mammalian antibody repertoires are produced via distant genomic contacts involving immunoglobulin Igh variable (V), diversity (D), and joining (J) gene segments and result in V(D)J recombination. How such interactions determine V gene usage remains unclear. The recombination-activating gene (RAG) chromatin scanning model posits that RAG recombinase bound to the recombination center (RC) linearly tracks along chromatin by means of cohesin-mediated loop extrusion; a proposition supported by cohesin depletion studies. A mechanistic role for chromatin loop extrusion has also been implicated for Igh locus contraction. In this opinion, we provide perspective on how loop extrusion interfaces with the 3D conformation of the Igh locus and newly identified enhancers that regionally regulate VH gene usage during V(D)J recombination, shaping the preselected repertoire.

Chickens, more than humans, focus the diversity of their immunoglobulin genes on the complementarity-determining region but utilise amino acids, indicative of a more cross-reactive antibody repertoire.

The mechanisms of B-cell diversification differ greatly between aves and mammals, but both produce B cells and antibodies capable of supporting an effective immune response. To see how differences in the generation of diversity might affect overall repertoire diversity, we have compared the diversity characteristics of immunoglobulin genes from domestic chickens to those from humans. Both use V(D)J gene rearrangement and somatic hypermutation, but only chickens use somatic gene conversion. A range of diversity analysis tools were used to investigate multiple aspects of amino acid diversity at both the germline and repertoire levels. The effect of differing amino acid usages on antibody characteristics was assessed. At both the germline and repertoire levels, chickens exhibited lower amino acid diversity in comparison to the human immunoglobulin genes, especially outside of the complementarity-determining region (CDR). Chickens were also found to possess much larger and more hydrophilic CDR3s with a higher predicted protein binding potential, suggesting that the antigen-binding site in chicken antibodies is more flexible and more polyreactive than that seen in human antibodies.

The major role of junctional diversity in the horse antibody repertoire.

The antibody repertoire (Rep-seq) sequencing revolutionized the diversity of antigen B cell receptor studies, allowing deep and quantitative analysis to decipher the role of adaptive immunity in health and disease. Particularly, horse (Equus caballus) polyclonal antibodies have been produced and used since the century XIX to treat and prophylaxis diphtheria, tuberculosis, tetanus, pneumonia, and, more recently, COVID-19. However, our knowledge about the horse B cell receptors repertories is minimal. We present a deep horse antibody heavy chain repertoire (IGH) characterization of non-infected horses using NGS (Next generation sequencing). This study obtained a mean of 248,169 unique IgM clones and 66,141 unique IgG clones from four domestic adult horses. Rarefaction analysis showed sequence coverage was between 52 % and 82 % in IgM and IgG isotypes. We observed that besides horses antibody can use all functional IGHV genes, around 80 % of their antibodies use only three IGHV gene segments, and around 55 % use only one IGHJ gene segment. This limited VJ diversity seems to be compensated by the junctional diversity of these antibodies. We observed that the junctional diversity in horse antibodies is widespread, present in more than 90 % of horse antibodies. Besides this, the length of this region seems to be higher in horse antibodies than in other species. N1 and N2 nucleotides addition range from 0 to 111 nucleotides. In addition, around 45 % of the antibody clones have more than ten nucleotides in both the N1 and N2 junction regions. This diversity mechanism may be one of the most important in providing variability to the equine antibody repertoire. This study provides new insights regarding horse antibody composition, diversity generation, and particularities compared to other species, such as the frequency and length of N nucleotide addition. This study also points out the urgent need to better characterize TdT in horses and other species to better understand antibody repertoire characteristics.

Different classes of genomic inserts contribute to human antibody diversity.

Recombination of antibody genes in B cells can involve distant genomic loci and contribute a foreign antigen-binding element to form hybrid antibodies with broad reactivity for Plasmodium falciparum. So far, antibodies containing the extracellular domain of the LAIR1 and LILRB1 receptors represent unique examples of cross-chromosomal antibody diversification. Here, we devise a technique to profile non-VDJ elements from distant genes in antibody transcripts. Independent of the preexposure of donors to malaria parasites, non-VDJ inserts were detected in 80% of individuals at frequencies of 1 in 104 to 105 B cells. We detected insertions in heavy, but not in light chain or T cell receptor transcripts. We classify the insertions into four types depending on the insert origin and destination: 1) mitochondrial and 2) nuclear DNA inserts integrated at VDJ junctions; 3) inserts originating from telomere proximal genes; and 4) fragile sites incorporated between J-to-constant junctions. The latter class of inserts was exclusively found in memory and in in vitro activated B cells, while all other classes were already detected in naïve B cells. More than 10% of inserts preserved the reading frame, including transcripts with signs of antigen-driven affinity maturation. Collectively, our study unravels a mechanism of antibody diversification that is layered on the classical V(D)J and switch recombination.

Greater Breadth of Vaccine-Induced Immunity in Females than Males Is Mediated by Increased Antibody Diversity in Germinal Center B Cells.

Inactivated influenza vaccines induce greater antibody responses in females than males among both humans and mice. To test the breadth of protection, we used recombinant mouse-adapted A/California/2009 (maA/Cal/09) H1N1 viruses containing mutations at one (1M), two (2M), or three (3M) antigenic sites, in addition to a virus containing the 1M mutation and a substitution of the Ca2 antigenic site (Sub) with one derived from an H5 hemagglutinin (HA) to challenge mice of both sexes. Following maA/Cal/09 vaccination, females produced greater virus-specific, class-switched total IgG and IgG2c antibodies against the vaccine and all mutant viruses, and antibodies from females recognized a greater number of unique, linear HA epitopes than did antibodies from males. While females had greater neutralizing antibody titers against the vaccine virus, both sexes showed a lower neutralization capacity against mutant viruses. After virus challenge, vaccinated females had lower pulmonary virus titers and reduced morbidity than males for the 1M and 2M viruses, but not the Sub virus. Females generated greater numbers of germinal center (GC) B cells containing superior somatic hypermutation (SHM) frequencies than vaccinated males. Deletion of activation-induced cytidine deaminase (Aicda) eliminated female-biased immunity and protection against the 2M virus. Harnessing methods to improve GC B cell responses and frequencies of SHM, especially in males, should be considered in the development of universal influenza vaccines. IMPORTANCE Adult females develop greater antibody responses to influenza vaccines than males. We hypothesized that female-biased immunity and protection would be dependent on the extent of virus diversity as well as molecular mechanisms in B cells which constrain the breadth of epitope recognition. We developed a panel of mouse-adapted (ma) A/Cal/09 viruses that had mutations in the immunodominant hemagglutinin. Following vaccination against maA/Cal/09, females were better able to neutralize maA/Cal/09 than males, but neutralization of mutant maA/Cal/09 viruses was equally poor in both sexes, despite vaccinated females being better protected against these viruses. Vaccinated females benefited from the greater production of class-switched, somatically hypermutated antibodies generated in germinal center B cells, which increased recognition of more diverse maA/Cal/09 hemagglutinin antigen epitopes. Female-biased protection against influenza infection and disease after vaccination is driven by differential mechanisms in males versus females and should be considered in the design of novel vaccine platforms.

Gene prediction in the immunoglobulin loci.

The V(D)J recombination process rearranges the variable (V), diversity (D), and joining (J) genes in the immunoglobulin (IG) loci to generate antibody repertoires. Annotation of these loci across various species and predicting the V, D, and J genes (IG genes) are critical for studies of the adaptive immune system. However, because the standard gene finding algorithms are not suitable for predicting IG genes, they have been semimanually annotated in very few species. We developed the IGDetective algorithm for predicting IG genes and applied it to species with the assembled IG loci. IGDetective generated the first large collection of IG genes across many species and enabled their evolutionary analysis, including the analysis of the "bat IG diversity" hypothesis. This analysis revealed extremely conserved V genes in evolutionary distant species, indicating that these genes may be subjected to the same selective pressure, for example, pressure driven by common pathogens. IGDetective also revealed extremely diverged V genes and a new family of evolutionary conserved V genes in bats with unusual noncanonical cysteines. Moreover, unlike all other previously reported antibodies, these cysteines are located within complementarity-determining regions. Because cysteines form disulfide bonds, we hypothesize that these cysteine-rich V genes might generate antibodies with noncanonical conformations and could potentially form a unique part of the immune repertoire in bats. We also analyzed the diversity landscape of the recombination signal sequences and revealed their features that trigger the high/low usage of the IG genes.

Helios expressing regulatory T cells are correlated with decreased IL-2 producing CD8 T cells and antibody diversity in Mozambican individuals living chronically with HIV-1.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) causes impairment of T and B cell responses, which begins during the acute phase of infection and is not completely restored by antiretroviral treatment. Regulatory T cell (Tregs) can improve overall disease outcome by controlling chronic inflammation but may also suppress beneficial HIV-1 specific immune responses. We aimed to analyze the profile of Tregs and their correlation with the status of T cells activation, the expression of IL-2 and IFNγ and the profile of HIV-1 specific antibodies response in Mozambican people living chronically with HIV-1 (PLWH-C). RESULTS: In PLWH-C, the proportion of total Tregs was positively correlated with the proportion of IL-2+CD4 T cells (r = 0.647; p = 0.032) and IL-2+IFNγ+CD8 T cells (r = 0.551; p = 0.014), while the proportions of Helios+Tregs correlated inversely with levels of IL-2+CD8 T cells (r = - 0.541; p = 0.017). Overall, PLWH-C, with (82%) or without virologic suppression (64%), were seronegative for at least HIV-1 p31, gp160 or p24, and the breadth of antibody responses was positively correlated with proportions of CD38+HLA-DR+CD8 T cells (r = 0.620; p = 0.012), viral load (r = 0.452; p = 0.040) and inversely with absolute CD4 T cells count (r = - 0.481; p = 0.027). Analysis of all individuals living HIV-1 showed that the breadth of HIV-1 antibody responses was inversely correlated with the proportion of Helios+Tregs (r = - 0.45; p = 0.02). CONCLUSION: Among Mozambican people living with HIV-1, seronegativity to some HIV-1 proteins is common, particularly in virologically suppressed individuals. Furthermore, lower diversity of HIV-specific antibodies is correlated to lower immune activation, lower viral replication and higher CD4 counts, in PLWH-C. Elevation in the proportion of Helios+Tregs is related to a reduction of CD8 T expressing intracellular IL-2, in PLWH-C, but may contribute to impairment of B cell function.

Extensive age-dependent loss of antibody diversity in naturally short-lived turquoise killifish.

Aging individuals exhibit a pervasive decline in adaptive immune function, with important implications for health and lifespan. Previous studies have found a pervasive loss of immune-repertoire diversity in human peripheral blood during aging; however, little is known about repertoire aging in other immune compartments, or in species other than humans. Here, we perform the first study of immune-repertoire aging in an emerging model of vertebrate aging, the African turquoise killifish (Nothobranchius furzeri). Despite their extremely short lifespans, these killifish exhibit complex and individualized heavy-chain repertoires, with a generative process capable of producing millions of distinct productive sequences. Whole-body killifish repertoires decline rapidly in within-individual diversity with age, while between-individual variability increases. Large, expanded B-cell clones exhibit far greater diversity loss with age than small clones, suggesting important differences in how age affects different B-cell populations. The immune repertoires of isolated intestinal samples exhibit especially dramatic age-related diversity loss, related to an elevated prevalence of expanded clones. Lower intestinal repertoire diversity was also associated with transcriptomic signatures of reduced B-cell activity, supporting a functional role for diversity changes in killifish immunosenescence. Our results highlight important differences in systemic vs. organ-specific aging dynamics in the adaptive immune system.

Ig Enhancers Increase RNA Polymerase II Stalling at Somatic Hypermutation Target Sequences.

Somatic hypermutation (SHM) drives the genetic diversity of Ig genes in activated B cells and supports the generation of Abs with increased affinity for Ag. SHM is targeted to Ig genes by their enhancers (diversification activators [DIVACs]), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase II (Pol II) and Pol II occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol II or production of full-length transcripts, indicating accumulation of stalled Pol II. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of ssDNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol II stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.

Anticuerpos irregulares en donantes de sangre / Anticuerpos irregulares en donantes de sangre Irregular antibodies in blood donor

Introducción: El sistema inmunológico puede reconocer una gran cantidad de antígenos cuando está expuesto a ellos. Los linfocitos B producen gran variedad de anticuerpos, con el fin de generar la especificidad de los receptores para el reconocimiento de dichos antígenos. La presencia de anticuerpos irregulares, es una de las causas de reacciones adversas transfusionales por incompatibilidad entre donante y receptor. Objetivo: Describir la genética, estructura y función de los anticuerpos irregulares en los donantes de sangre. Métodos: Se llevó a cabo una revisión de la literatura, en idioma inglés y español, a través de bases de datos como Pubmed, ScienceDirect, NCBI, Redalyc y SciElo de artículos publicados en los últimos 10 años. Análisis y síntesis de la información: El sistema inmunológico genera una gran diversidad de anticuerpos mediante el proceso de recombinación somática entre los segmentos Variables (V), de diversidad (D) y de unión (J) de la línea germinal de las inmunoglobulinas, como mecanismo de defensa del organismo frente a sustancias o antígenos extraños. Los anticuerpos irregulares son aquellos diferentes al sistema sanguíneo ABO y los más comúnmente encontrados en los donantes de sangre son anti-D, anti-E, anti-K y anti-M. Conclusiones: La importancia clínica de los anticuerpos irregulares en donantes se basa en su asociación con las reacciones hemolíticas, dada la capacidad que tienen los antígenos de algunos grupos sanguíneos para generar anticuerpos de tipo IgG que causan lisis prematura de los eritrocitos(AU)

Introduction: The immune system can recognize a large number of antigens when it is exposed to them; B Lymphocytes produces a great variety of antibodies, in order to generate the specificity of the receivers for the recognition of said antigens. The presence to irregular antibodies is one of the causes to the adverse reactions to the transfusion when for blood incompatibility between donor and receptor. Objective: To describe the genetics, structure and function of irregular antibodies in blood donors. Methods: A literature review was carried out, in English and Spanish, through databases such as Pubmed, ScienceDirect, NCBI, Redalyc and Scielo of articles published in the last 10 years. Analysis and synthesis of information: The immune system generates a great diversity of antibodies through the somatic recombination process between the Variable (V), diversity (D) and joining (J) segments of the germ line of immunoglobulins, as a defense mechanism of the organism against foreign substances or antigens. Irregular antibodies are those other than the ABO blood system and those most commonly found in blood donors are anti-D, anti-E, anti-K, and anti-M. Conclusions: The clinical significance of irregular antibodies in donors is based on their association with hemolytic reactions, due to the ability of antigens in some blood groups to generate IgG-type antibodies that cause premature erythrocyte lysis(AU)

An Analysis of the Effects of Spaceflight and Vaccination on Antibody Repertoire Diversity.

Ab repertoire diversity plays a critical role in the host's ability to fight pathogens. CDR3 is partially responsible for Ab-Ag binding and is a significant source of diversity in the repertoire. CDR3 diversity is generated during VDJ rearrangement because of gene segment selection, gene segment trimming and splicing, and the addition of nucleotides. We analyzed the Ab repertoire diversity across multiple experiments examining the effects of spaceflight on the Ab repertoire after vaccination. Five datasets from four experiments were analyzed using rank-abundance curves and Shannon indices as measures of diversity. We discovered a trend toward lower diversity as a result of spaceflight but did not find the same decrease in our physiological model of microgravity in either the spleen or bone marrow. However, the bone marrow repertoire showed a reduction in diversity after vaccination. We also detected differences in Shannon indices between experiments and tissues. We did not detect a pattern of CDR3 usage across the experiments. Overall, we were able to find differences in the Ab repertoire diversity across experimental groups and tissues.

Antibody Repertoire Analysis of Tumor-Infiltrating B Cells Reveals Distinct Signatures and Distributions Across Tissues.

The role of B cells in the tumor microenvironment (TME) has largely been under investigated, and data regarding the antibody repertoire encoded by B cells in the TME and the adjacent lymphoid organs are scarce. Here, we utilized B cell receptor high-throughput sequencing (BCR-Seq) to profile the antibody repertoire signature of tumor-infiltrating lymphocyte B cells (TIL-Bs) in comparison to B cells from three anatomic compartments in a mouse model of triple-negative breast cancer. We found that TIL-Bs exhibit distinct antibody repertoire measures, including high clonal polarization and elevated somatic hypermutation rates, suggesting a local antigen-driven B-cell response. Importantly, TIL-Bs were highly mutated but non-class switched, suggesting that class-switch recombination may be inhibited in the TME. Tracing the distribution of TIL-B clones across various compartments indicated that they migrate to and from the TME. The data thus suggests that antibody repertoire signatures can serve as indicators for identifying tumor-reactive B cells.

IL-7R signaling activates widespread VH and DH gene usage to drive antibody diversity in bone marrow B cells.

Generation of the primary antibody repertoire requires V(D)J recombination of hundreds of gene segments in the immunoglobulin heavy chain (Igh) locus. The role of interleukin-7 receptor (IL-7R) signaling in Igh recombination has been difficult to partition from its role in B cell survival and proliferation. With a detailed description of the Igh repertoire in murine IL-7Rα-/- bone marrow B cells, we demonstrate that IL-7R signaling profoundly influences VH gene selection during VH-to-DJH recombination. We find skewing toward 3' VH genes during de novo VH-to-DJH recombination more severe than the fetal liver (FL) repertoire and uncover a role for IL-7R signaling in DH-to-JH recombination. Transcriptome and accessibility analyses suggest reduced expression of B lineage transcription factors (TFs) and targets and loss of DH and VH antisense transcription in IL-7Rα-/- B cells. Thus, in addition to its roles in survival and proliferation, IL-7R signaling shapes the Igh repertoire by activating underpinning mechanisms.

Impact of interleukin-32 germ-line rs28372698 and intronic rs12934561 polymorphisms on cancer development: A systematic review and meta-analysis.

OBJECTIVE: The pro-inflammatory cytokine IL-32 has high susceptibility to develop cancer. But no previous meta-analysis was done to provide firm evidence. This systematic review and meta-analysis was designed to evaluate the association of IL-32 gene polymorphisms (rs28372698 and rs12934561) with cancer. METHOD: Eligible studies were selected using authentic databases searching from January 2013 to January 2021. Demographic data and genotypic information were extracted and organized from the selected studies. Review Manager (RevMan) version 5.4 was used to perform data analysis and data arrangement for meta-analysis. RESULTS: A total of seven studies with 3395 patients and 3781 controls were included in this study. IL-32 rs28372698 polymorphism implied that mutant allele (TT) carriers had a significantly higher risk of cancer (OR = 1.43, p = 0.032). Codominant 3, recessive and allele models also showed 1.36-, 1.38- and 1.11-fold increased risk, respectively (p < 0.05). Besides, the Asian population showed a significantly increased risk in codominant 2 (OR = 1.74), codominant 3 (OR = 1.78), recessive (OR = 1.76) and allele model (OR = 1.16). IL-32 rs12934561 showed significantly reduced cancer risk in codominant 1 (OR = 0.66. p = 0.035), codominant 2 (OR = 0.76, p = 0.007), and dominant model (OR = 0.72, p = 0.012). After subgroup analysis, an association of rs12934561 was found in Asians (codominant 1: OR = 0.54, p = 7.28 × 10-8; codominant 2: OR = 1.40, p = 0.019; codominant 3: OR = 0.76, p = 0.0006; dominant model: OR = 0.64, p = 1.12 × 10-5; overdominant model: OR = 0.64, p = 3.92 × 10-7) but not in Caucasians. After stratifying with the control source, a significant (p < 0.05) association of rs28372698 and rs12934561 was found with cancer in population-based controls. No publication bias was found, and the outcome of this meta-analysis was not influenced by any individual study confirmed from sensitivity analysis. Moreover, trial sequential analysis (TSA) established a link between rs28372698 and rs12934561 polymorphisms and cancer. CONCLUSION: The outcome of this meta-analysis revealed that IL-32 rs28372698 and rs12934561 polymorphisms are associated with cancer. Moreover, the Asian dynasty had a significant association compared to Caucasians.

Amplification of a minimally biased antibody repertoire for in vitro display using a universal primer-based amplification method.

Immune hosts are valuable sources for antibody discovery. To construct in vitro display antibody libraries from immune repertoires, singleplex or multiplex PCR amplification were employed using primers targeting multiple immunoglobulin genes. However, during this process, the B cell receptor repertoire is distorted due to interactions between multiple target genes and primers. To minimize this alternation, we devised a new method for harvesting immunoglobulin genes and tested its performance in rabbit variable heavy chain (VH) and variable kappa light chain (VK) genes. Double-stranded cDNA was synthesized using primers containing V/J gene-specific regions and universal sequence parts for in vitro display. VH and VK gene libraries were obtained through subsequent PCR amplification using primers with universal sequences. Next-generation sequencing analysis confirmed that universal PCR libraries had more diverse VH and VK clonotypes, and a less biased clonal distribution, than conventional singleplex or multiplex gene-specific PCR libraries.