title: Trimeric gp120-specific bovine monoclonal antibodies require cysteine and aromatic residues in CDRH3 for high affinity binding to HIV Env.
abstract: We isolated HIV-1 Envelope (Env)-specific memory B cells from a cow that had developed high titer polyclonal immunoglobulin G (IgG) with broad neutralizing activity after a long duration vaccination with HIV-1AD8Env gp140 trimers. We cloned the bovine IgG matched heavy (H) and light (L) chain variable (V) genes from these memory B cells and constructed IgG monoclonal antibodies (mAbs) with either a human constant (C)-region/bovine V-region chimeric or fully bovine C and V regions. Among 42 selected Ig+ memory B cells, two mAbs (6A and 8C) showed high affinity binding to gp140 Env. Characterization of both the fully bovine and human chimeric isoforms of these two mAbs revealed them as highly type-specific and capable of binding only to soluble AD8 uncleaved gp140 trimers and covalently stabilized AD8 SOSIP gp140 cleaved trimers, but not monomeric gp120. Genomic sequence analysis of the V genes showed the third heavy complementarity-determining region (CDRH3) of 6A mAb was 21 amino acids in length while 8C CDRH3 was 14 amino acids long. The entire V heavy (VH) region was 27% and 25% diverged for 6A and 8C, respectively, from the best matched germline V genes available, and the CDRH3 regions of 6A and 8C were 47.62% and 78.57% somatically mutated, respectively, suggesting a high level of somatic hypermutation compared with CDRH3 of other species. Alanine mutagenesis of the VH genes of 6A and 8C, showed that CDRH3 cysteine and tryptophan amino acids were crucial for antigen binding. Therefore, these bovine vaccine-induced anti-HIV antibodies shared some of the notable structural features of elite human broadly neutralizing antibodies, such as CDRH3 size and somatic mutation during affinity-maturation. However, while the 6A and 8C mAbs inhibited soluble CD4 binding to gp140 Env, they did not recapitulate the neutralizing activity of the polyclonal antibodies against HIV infection.
authors.affiliation: a Department of Microbiology and Immunology , The University of Melbourne at The Peter Doherty Institute for Infection & Immunity , Melbourne , VIC , Australia.
authors.forename: Behnaz
authors.initials: B
authors.lastname: Heydarchi
authors.affiliation: a Department of Microbiology and Immunology , The University of Melbourne at The Peter Doherty Institute for Infection & Immunity , Melbourne , VIC , Australia.
authors.forename: Rob J
authors.initials: RJ
authors.lastname: Center
authors.affiliation: a Department of Microbiology and Immunology , The University of Melbourne at The Peter Doherty Institute for Infection & Immunity , Melbourne , VIC , Australia.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Bebbington
authors.affiliation: a Department of Microbiology and Immunology , The University of Melbourne at The Peter Doherty Institute for Infection & Immunity , Melbourne , VIC , Australia.
authors.forename: Jack
authors.initials: J
authors.lastname: Cuthbertson
authors.affiliation: a Department of Microbiology and Immunology , The University of Melbourne at The Peter Doherty Institute for Infection & Immunity , Melbourne , VIC , Australia.
authors.forename: Christopher
authors.initials: C
authors.lastname: Gonelli
authors.affiliation: a Department of Microbiology and Immunology , The University of Melbourne at The Peter Doherty Institute for Infection & Immunity , Melbourne , VIC , Australia.
authors.forename: Georges
authors.initials: G
authors.lastname: Khoury
authors.affiliation: a Department of Microbiology and Immunology , The University of Melbourne at The Peter Doherty Institute for Infection & Immunity , Melbourne , VIC , Australia.
authors.forename: Charlene
authors.initials: C
authors.lastname: Mackenzie
authors.affiliation: a Department of Microbiology and Immunology , The University of Melbourne at The Peter Doherty Institute for Infection & Immunity , Melbourne , VIC , Australia.
authors.forename: Marit
authors.initials: M
authors.lastname: Lichtfuss
authors.affiliation: a Department of Microbiology and Immunology , The University of Melbourne at The Peter Doherty Institute for Infection & Immunity , Melbourne , VIC , Australia.
authors.forename: Grant
authors.initials: G
authors.lastname: Rawlin
authors.affiliation: a Department of Microbiology and Immunology , The University of Melbourne at The Peter Doherty Institute for Infection & Immunity , Melbourne , VIC , Australia.
authors.forename: Brian
authors.initials: B
authors.lastname: Muller
authors.affiliation: a Department of Microbiology and Immunology , The University of Melbourne at The Peter Doherty Institute for Infection & Immunity , Melbourne , VIC , Australia.
authors.forename: Damian
authors.initials: D
authors.lastname: Purcell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D002417:Cattle; D022801:Complementarity Determining Regions / Q000276:immunology*; D003545:Cysteine; D015483:HIV Antibodies / Q000276:immunology*; D015699:HIV Envelope Protein gp120 / Q000276:immunology*; D015658:HIV Infections / Q000276:immunology / Q000662:veterinary; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology; D015202:Protein Engineering / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D007135:Immunoglobulin Variable Region; D003545:Cysteine
keywords: Aromatic residues; CDRH3; Cysteine; HIV; bovine; monoclonal antibodies; variable region