Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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HTGTS-Rep-Seq for Humanized Antibodies that Broadly Neutralize Omicron Sub-variants

ABSTRACT: During V(D)J recombination antibody diversity is enhanced by non-templated junctional modifications that generate immensely diverse heavy chain (HC) and light chain (LC) complementarity-determining 3 antigen-contact regions (CDR3s). We previously developed a mouse model that generates diverse antibody repertoires by rearranging a single human VH1-2 and Vk1-33, associated with highly diverse CDR3s generated by V(D)J recombination with mouse Ds and/or Js. Immunization of this model with SARS-CoV-2 D614G spike elicited an antibody that potently neutralized SARS-CoV-2 variants through Omicron BA.2.754. Here, we report a next generation mouse model in which a single VH1-2 rearranges to human D3-3 and JH6, generating diverse HC-CDR3s much longer on average than those of our prior model. Omicron BA.4/.5 spike ferritin nanoparticle-immunization of the new model elicited four highly-related humanized antibodies that potently neutralize downstream Omicron sub-variants. All four antibodies had 12 AA HC-CDR3s with two aromatic amino acids that engage an epitope comprising a hydrophobic patch opened-up by early omicron lineage mutations and conserved in subsequent variants. Immunization of our prior, shorter CDR3-based model, elicited slightly less potent neutralizing antibodies that bound the same Omicron epitope, and were similar in all other aspects to those from the long, fully-human CDR3 model. One tested antibody from each set reduced lung viral titers in a mouse-adapted BQ1.1 challenge. The antibodies we describe are highly related to, but more potent than, recently described antibodies from omicron-infected humans. These studies validate the utility of single human VH- and Vk-rearranging mice for discovering humanized antibodies that neutralize emerging pathogens.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Mus musculus

SUBMITTER: Himanshu Batra

PROVIDER: E-MTAB-16106 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Developing B lymphocytes undergo V(D)J recombination to assemble germline V, D, and J gene segments into exons that encode the antigen-binding variable region of immunoglobulin (Ig) heavy (H) and light (L) chains. IgH and IgL chains associate to form the B cell receptor (BCR), which upon antigen binding activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. Ability of V(D)J recombination to generate vast primary B cell repertoires results from combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as HTGTS repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or non-productive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions and also for following antibody affinity maturation processes. We employed high-throughput genome-wide translocation sequencing adapted repertoire sequencing (HTGTS-Rep-seq) to study antibody repertoires. For HTGTS-Rep-seq libraries, we utilize bait coding ends of J segments to identify, in unbiased fashion, mouse IgH DJH repertoires [processed tlx files] along with both productive and non-productive IgH V(D)J repertoires from both pro-B and peripheral B cells [processed xls files of samples 1-18, 21-51]. Similarly, we also identify mouse productive and non-productive Igk repertoires from peripheral B cells [processed xls files of samples 19,20,52-57].

Project description:We previously reported the establishment of a rabbit (Oryctolagus cuniculus) model of Systemic Lupus Erythematosus (SLE) in which peptide immunization led to lupus-like autoantibody production including anti-Sm, -RNP, -SS-A, -SS-B and -dsDNA. Some neurological symptoms in form of seizures and nystagmus were observed. The animals used in the previous and in the present study were from a National Institute of Allergy and Infectious Diseases colony of rabbits that were pedigreed, immunoglobulin allotype-defined but not inbred. Their genetic heterogeneity may correspond to that found among patients of a given ethnicity. We extended the information about this rabbit model of SLE by microarray based expression profiling. We first demonstrated that human expression arrays could be used with rabbit RNA to yield information on molecular pathways. We then designed a study evaluating gene expression profiles in 8 groups of control and SLE rabbits (46 rabbits in total). Genes significantly upregulated in SLE rabbits were associated with NK cytotoxicity, antigen presentation, leukocyte migration, cytokine activity, protein kinases, RNA spliceosomal ribonucleoproteins, intracellular signaling cascades, and glutamate receptor activity. These results link increased immune activation with up-regulation of components associated with neurological and anti-RNP responses, demonstrating the utility of the rabbit SLE model to uncover biological pathways related to SLE-induced clinical symptoms, including Neuropsychiatric Lupus. Our finding of distinct gene expression patterns in rabbits that made anti-dsDNA compared to those that only made other anti-nuclear antibodies should be further investigated in subsets of SLE patients with different autoantibody profiles. An important goal of biomedical research is to translate basic findings into clinical applications. Models in inbred mice that spontaneously develop SLE, along with various mutant, transgenic and knockout models have documented a variety of genetic defects leading to SLE, but from the clinical perspective, the degree to which these findings using the inbred or homogeneous artificially mutated strains apply to individuals in heterogeneous outbred human populations is open to question. Given that there is still no cure available for SLE, it is important that we continue to explore possibilities using new animal models of SLE including neuropsychiatric lupus (NPSLE). The gene expression study reported here was conducted to expand our understanding of SLE and in particular NPSLE using our rabbit model. We reported earlier that immunization of rabbits with the SM- or GR-MAP peptides led to development of anti-nuclear autoantibodies, including anti-dsDNA, as well as neurological symptoms in the form of seizures and nystagmus in some rabbits. After establishing that it was possible to use the Affymetrix U95 human microarray for the rabbit gene expression studies, through comparative hybridization of identically prepared cRNA from human and rabbit PWBC, the human microarray was used due to lack of rabbit-specific microarrays. We describe unique gene expression changes associated with lupus like serological patterns in immunized rabbits. Our results also demonstrate that caution must be applied when choosing the structure of the carrier Multiple Antigen Peptide (MAP-peptide) for immunization. We discovered that using MAP-4 rather than MAP-8 significantly altered patterns of immune response and gene expression. Currently, microarrays specific for study of gene expression profiles are not available for rabbits. Therefore, we first conducted studies that compared identically prepared rabbit and human cRNA binding to the Affymetrix U95 microarray available for human gene expression analyses. It was determined that the human microarray could be used with rabbit cRNA to yield information on genetic pathways activated and/or suppressed in autoantibody-producing immunized rabbits. In the current report, gene expression profiles of a total of 46 rabbits, from 4 generations within a pedigreed group of control and immunized rabbits, were obtained and analyzed.

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HTGTS-Rep-Seq for Humanized Antibodies that Broadly Neutralize Omicron Sub-variants

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