Immunoglobulin gene usage and affinity maturation in antiviral antibodies

The ability of antibodies to block infections makes them highly relevant for successful vaccine development. Through the papers described in this thesis, I attempt to characterize the functional and genetic aspects of antiviral antibodies induced by infection and vaccination. In Paper I, we characterized the distribution and maturation of HIV-1 envelope glycoproteins (Env)-specific antibody lineages post-vaccination in different immune compartments of rhesus macaques. Vaccine-induced Env-specific antibody lineages were disseminated across the periphery, lymph node, spleen, and bone marrow (BM) but not in gut tissue. We observed a consistent increase in the somatic hypermutation (SHM) levels of Env-specific antibody sequences after each boost and the SHM levels strongly correlated with the affinity of members from a potent neutralizing antibody lineage. In Paper II, we set out to understand the role of SHM in a broad, potent, public class of antibodies isolated from a healthcare worker who was previously infected with SARS-CoV2. I selected a potent neutralizing antibody and reverted the heavy chain (HC) to the germline sequence. I then sequentially introduced individual or combinations of SHM so that we could test the functional impact of this. We found a substantial gain of antibody potency and breadth when certain SHM mutations were reintroduced, and we identified two key mutations that largely contributed to the breadth of this lineage. Furthermore, we showed that the mature antibody retained neutralizing activity against potential future viral variants by deep mutational scanning (DMS) experiments. A high-resolution structure of this antibody obtained by cryo-electron microscopy (cryo-EM) confirmed important interactions made by the identified SHMs with the SARS-CoV-2 spike (S). In Papers III and IV, we investigated the effect of immunoglobulin heavy chain variable (IGHV) gene polymorphisms on the function of human SARS-CoV-2 antibodies isolated post-infection. We genotyped a cohort of previously infected healthcare workers and evaluated the neutralization activity of germline-reverted and allele-swapped S-specific IGHV1-69*20-using antibodies from two independent donors carrying this allele. Neutralization was retained when reverting the IGHV region to the germline IGHV1- 69∗20 allele but lost when reverting to other IGHV1-69 alleles demonstrating a strong allele-dependence in these antibodies. A high resolution cryo-EM structure of one of the antibodies revealed significant contacts made by two IGHV1-69*20-germline encoded amino acid residues with the S, illustrating the impact of IGHV polymorphisms on antibody functions. We next focused on the IGHV3-30 group of genes, which are frequently used by S-specific antibodies. By IGHV genotype and haplotype analysis we observed that IGHV3-30-3 gene was deleted in many individuals, and the IGHV3-30 alleles were heterogeneously distributed in our cohort. When the IGHV region of an IGHV3-30-3*01 neutralizing antibody was swapped with IGHV3-30 alleles, the neutralization remained unaffected demonstrating functional redundancy within this gene group, at least for this antibody lineage. The results from my doctoral research provide insight into functional and genetic properties of antibodies induced by viral antigens, which have important clinical relevance both for guided-vaccine design and monoclonal antibody therapeutics, and for our general understanding of antibody responses in the population

Frequent use of IGHV3-30-3 in SARS-CoV-2 neutralizing antibody responses

The antibody response to SARS-CoV-2 shows biased immunoglobulin heavy chain variable (IGHV) gene usage, allowing definition of genetic signatures for some classes of neutralizing antibodies. We investigated IGHV gene usage frequencies by sorting spike-specific single memory B cells from individuals infected with SARS-CoV-2 early in the pandemic. From two study participants and 703 spike-specific B cells, the most used genes were IGHV1-69, IGHV3-30-3, and IGHV3-30. Here, we focused on the IGHV3-30 group of genes and an IGHV3-30-3-using ultrapotent neutralizing monoclonal antibody, CAB-F52, which displayed broad neutralizing activity also in its germline-reverted form. IGHV3-30-3 is encoded by a region of the IGH locus that is highly variable at both the allelic and structural levels. Using personalized IG genotyping, we found that 4 of 14 study participants lacked the IGHV3-30-3 gene on both chromosomes, raising the question if other, highly similar IGHV genes could substitute for IGHV3-30-3 in persons lacking this gene. In the context of CAB-F52, we found that none of the tested IGHV3-33 alleles, but several IGHV3-30 alleles could substitute for IGHV3-30-3, suggesting functional redundancy between the highly homologous IGHV3-30 and IGHV3-30-3 genes for this antibody

Frequent use of IGHV3-30-3 in SARS-CoV-2 neutralizing antibody responses

The antibody response to SARS-CoV-2 shows biased immunoglobulin heavy chain variable (IGHV) gene usage, allowing definition of genetic signatures for some classes of neutralizing antibodies. We investigated IGHV gene usage frequencies by sorting spike-specific single memory B cells from individuals infected with SARS-CoV-2 early in the pandemic. From two study participants and 703 spike-specific B cells, the most used genes were IGHV1-69, IGHV3-30-3, and IGHV3-30. Here, we focused on the IGHV3-30 group of genes and an IGHV3-30-3-using ultrapotent neutralizing monoclonal antibody, CAB-F52, which displayed broad neutralizing activity also in its germline-reverted form. IGHV3-30-3 is encoded by a region of the IGH locus that is highly variable at both the allelic and structural levels. Using personalized IG genotyping, we found that 4 of 14 study participants lacked the IGHV3-30-3 gene on both chromosomes, raising the question if other, highly similar IGHV genes could substitute for IGHV3-30-3 in persons lacking this gene. In the context of CAB-F52, we found that none of the tested IGHV3-33 alleles, but several IGHV3-30 alleles could substitute for IGHV3-30-3, suggesting functional redundancy between the highly homologous IGHV3-30 and IGHV3-30-3 genes for this antibody

Immunoglobulin germline gene polymorphisms influence the function of SARS-CoV-2 neutralizing antibodies

The human immunoglobulin heavy-chain (IGH) locus is exceptionally polymorphic, with high levels of allelic and structural variation. Thus, germline IGH genotypes are personal, which may influence responses to infection and vaccination. For an improved understanding of inter-individual differences in antibody responses, we isolated SARS-CoV-2 spike-specific monoclonal antibodies from convalescent health care workers, focusing on the IGHV1-69 gene, which has the highest level of allelic variation of all IGHV genes. The IGHV1-69∗20-using CAB-I47 antibody and two similar antibodies isolated from an independent donor were critically dependent on allele usage. Neutralization was retained when reverting the V region to the germline IGHV1-69∗20 allele but lost when reverting to other IGHV1-69 alleles. Structural data confirmed that two germline-encoded polymorphisms, R50 and F55, in the IGHV1-69 gene were required for high-affinity receptor-binding domain interaction. These results demonstrate that polymorphisms in IGH genes can influence the function of SARS-CoV-2 neutralizing antibodies

Immunoglobulin germline gene polymorphisms influence the function of SARS-CoV-2 neutralizing antibodies

The human immunoglobulin heavy-chain (IGH) locus is exceptionally polymorphic, with high levels of allelic and structural variation. Thus, germline IGH genotypes are personal, which may influence responses to infection and vaccination. For an improved understanding of inter-individual differences in antibody responses, we isolated SARS-CoV-2 spike-specific monoclonal antibodies from convalescent health care workers, focusing on the IGHV1-69 gene, which has the highest level of allelic variation of all IGHV genes. The IGHV1-69∗20-using CAB-I47 antibody and two similar antibodies isolated from an independent donor were critically dependent on allele usage. Neutralization was retained when reverting the V region to the germline IGHV1-69∗20 allele but lost when reverting to other IGHV1-69 alleles. Structural data confirmed that two germline-encoded polymorphisms, R50 and F55, in the IGHV1-69 gene were required for high-affinity receptor-binding domain interaction. These results demonstrate that polymorphisms in IGH genes can influence the function of SARS-CoV-2 neutralizing antibodies