Selection of therapeutic H5N1 monoclonal antibodies following IgVH repertoire analysis in mice

Abstract

The rapid rate of influenza virus mutation drives the emergence of new strains that inflict serious seasonal epidemics and less frequent, but more deadly, pandemics. While vaccination provides the best protection against influenza, its utility is often diminished by the unpredictability of new pathogenic strains. Consequently, efforts are underway to identify new antiviral drugs and monoclonal antibodies that can be used to treat recently infected individuals and prevent disease in vulnerable populations. Next Generation Sequencing (NGS) and the analysis of antibody gene repertoires is a valuable tool for Ab discovery. Here, we describe a technology platform for isolating therapeutic monoclonal antibodies (MAbs) by analyzing the IgVH repertoires of mice immunized with recombinant H5N1 hemagglutinin (rH5). As an initial proof of concept, 35 IgVH genes selected using a CDRH3 search algorithm, co-expressed in a murine IgG2a expression vector with a panel of germline murine kappa genes, and culture supernatants screened for antigen binding. Seventeen of the 35 IgVH MAbs (49%) bound rH5VN1203 in preliminary screens and 8 of 9 purified MAbs inhibited 3 heterosubtypic strains of H5N1 virus when assayed by HI, and 2 MAbs demonstrated prophylactic and therapeutic activity in virus-challenged mice. This is the first example in which an NGS discovery platform has been used to isolate anti-influenza MAbs with relevant therapeutic activity.

Characterization of the naive murine antibody repertoire using unamplified high-throughput sequencing

“…It provides a comparative foundation when looking at host response to antigen [ 54 ] and has been used to isolated therapeutic antibodies. Antibodies for influenza in a mouse model and were a valuable tool in the detection of antigen specific responses [ 16 , 34 ].…”

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library

“…Finally, future work that integrates FACS data with computational sequence analysis may decrease the rate of false positives. Prior computational work has focused on choosing the most frequent binders, 4 , 24-25 , 27 or identifying antibodies shared between replicate mice. 28 Other approaches for future study include computational structural analysis of putative binders and computational methods for detection of affinity maturation.…”

A combination of TLR-4 agonist and saponin adjuvants increases antibody diversity and protective efficacy of a recombinant West Nile Virus antigen

“…At 21 days following intramuscular immunization, RNA was prepared from splenocytes of immunized mice ( n = 5/group), and antibody repertoire diversity assessed by deep-sequencing of antibody immunoglobulin heavy chain (IgH) variable (V) regions as previously described. 41 The number of specific IgH V gene sequences induced by each adjuvant was determined across a range of significance scores, defined as the negative log of the e-score cutoff value, which indicate the probability of a given sequence being specific to that adjuvant group. SLA-LSQ induced a larger number of total IgH V-gene sequences (Fig.…”

“…Antibody repertoire in splenocytes was assessed by HiSeq analysis of amplified CDR3 sequences as described previously. 41 , 45 Sequences were quantified by identification of unique molecular identifiers (UMI), with specific sequences for each adjuvant determined by calculation of a significance score. The number of specific sequence counts a as well as the number of specific CDR3 sequences b was determined across a range of negative log e-score cutoff values.…”

“…Preparation, sequencing and data analysis were performed as described with the following modifications. 41 In addition to the combined barcode UMI sequence in the cDNA primer, an additional six base barcode was added to the 3′ end of the upstream PCR primer, allowing samples from each individual to be tagged with a unique barcode pair. Sequencing of pooled barcoded samples was performed with a 2 × 250 HISEQ analysis, yielding 101,692,094 total paired end reads of which a total of 41,287,384 had designated barcode pairs.…”