Immunoglobulin gene usage and affinity maturation in antiviral antibodies
Author: Pushparaj, Pradeepa
Date: 2023-05-12
Location: Eva and George Klein Hall, Solnavägen 9, Karolinska Institutet, Solna
Time: 09.00
Department: Inst för mikrobiologi, tumör- och cellbiologi / Dept of Microbiology, Tumor and Cell Biology
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Thesis (3.379Mb)
Abstract
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.
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.
List of papers:
I. Phad GE, Pushparaj P, Tran K, Dubrovskaya V, Àdori M, Martinez Murillo P, Vázquez Bernat N, Singh S, Dionne G, O'Dell S, Bhullar K, Narang S, Sorini C, Villablanca EJ, Sundling C, Murrell B, Mascola JR, Shapiro L, Pancera M, Martin M, Corcoran M, Wyatt RT, Karlsson Hedestam GB. Extensive dissemination and intraclonal maturation of HIV Env vaccine-induced B cell responses. Journal of Experimental Medicine. February 2020; 217(2):e20191155.
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II. Sheward DJ*, Pushparaj P*, Das H*, Greaney AJ, Kim C, Kim S, Hanke L, Dyrdak R, Lee J, Peacock TP, McInerney G, Albert J, Corcoran M, Bloom JD, Murrell B#, Karlsson Hedestam GB#, Hällberg BM.# Structural basis of broad SARS-CoV-2 cross-neutralization by affinity-matured public antibodies. *These authors contributed equally. [Submitted]
III. Pushparaj P, Nicoletto A*, Sheward DJ*, Das H, Castro Dopico X, Perez Vidakovics L, Hanke L, Chernyshev M, Narang S, Kim S, Fischbach J, Ekström S, McInerney G, Hällberg BM, Murrell B, Corcoran M, Karlsson Hedestam GB. Immunoglobulin germline gene polymorphisms influence the function of SARS-CoV-2 neutralizing antibodies. Immunity. January 2023; 56(1):193-206. *These authors contributed equally.
Fulltext (DOI)
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IV. Pushparaj P, Nicoletto A, Castro Dopico X, Sheward DJ, Sungyong Kim, Ekström S, Murrell B, Corcoran M and Karlsson Hedestam GB. Frequent usage of IGHV3-30-3 in SARS-CoV-2 neutralizing antibody responses. Frontiers in Virology. March 2023, Vol 3.
Fulltext (DOI)
I. Phad GE, Pushparaj P, Tran K, Dubrovskaya V, Àdori M, Martinez Murillo P, Vázquez Bernat N, Singh S, Dionne G, O'Dell S, Bhullar K, Narang S, Sorini C, Villablanca EJ, Sundling C, Murrell B, Mascola JR, Shapiro L, Pancera M, Martin M, Corcoran M, Wyatt RT, Karlsson Hedestam GB. Extensive dissemination and intraclonal maturation of HIV Env vaccine-induced B cell responses. Journal of Experimental Medicine. February 2020; 217(2):e20191155.
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Sheward DJ*, Pushparaj P*, Das H*, Greaney AJ, Kim C, Kim S, Hanke L, Dyrdak R, Lee J, Peacock TP, McInerney G, Albert J, Corcoran M, Bloom JD, Murrell B#, Karlsson Hedestam GB#, Hällberg BM.# Structural basis of broad SARS-CoV-2 cross-neutralization by affinity-matured public antibodies. *These authors contributed equally. [Submitted]
III. Pushparaj P, Nicoletto A*, Sheward DJ*, Das H, Castro Dopico X, Perez Vidakovics L, Hanke L, Chernyshev M, Narang S, Kim S, Fischbach J, Ekström S, McInerney G, Hällberg BM, Murrell B, Corcoran M, Karlsson Hedestam GB. Immunoglobulin germline gene polymorphisms influence the function of SARS-CoV-2 neutralizing antibodies. Immunity. January 2023; 56(1):193-206. *These authors contributed equally.
Fulltext (DOI)
Pubmed
View record in Web of Science®
IV. Pushparaj P, Nicoletto A, Castro Dopico X, Sheward DJ, Sungyong Kim, Ekström S, Murrell B, Corcoran M and Karlsson Hedestam GB. Frequent usage of IGHV3-30-3 in SARS-CoV-2 neutralizing antibody responses. Frontiers in Virology. March 2023, Vol 3.
Fulltext (DOI)
Institution: Karolinska Institutet
Supervisor: Karlsson Hedestam, Gunilla
Co-supervisor: Loré, Karin
Issue date: 2023-04-18
Rights:
Publication year: 2023
ISBN: 978-91-8017-003-1
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