Repertoire diversity and maturation of HIV-1 vaccine-induced B cell responses

In the work presented in this thesis, I characterized B cell responses in rhesus macaques inoculated with HIV-1 envelope glycoprotein (Env)-based vaccines to define genetic and functional properties of the elicited antibodies for an improved understanding of Env immunogenicity.

In Paper I, we investigated antibody heavy chain V gene segment expression in the overall B cell repertoire and in response to vaccination by using 2 approaches, 454-pyrosequencing and single-cell RT-PCR of bulk and sorted memory B cells, respectively. Both methods gave highly concordant results and identified differential expression of the Ig gene segments. We further investigated the VH repertoire of antigen-specific memory B cells induced by an HIV-1 Env trimer immunogen. We found that the antibody response against Env was highly polyclonal consisting of most of the expressed VH segments in immunized subject. In Paper II, we isolated and comprehensively characterized genetic properties of a panel of vaccine-induced MAbs from Env immunized subjects. We demonstrated that the MAbs were genetically diverse with no bias in their V gene usage towards the distinct epitope regions they were mapped to, consistent with a highly polyclonal response. MAbs targeting two sub-regions, the CD4 binding site (CD4bs) and variable region 3 (V3), were neutralizing. The combined activity of these MAbs recapitulated the neutralizing activity observed in unfractionated plasma samples of the animals from which they were isolated, consistent with that these were the predominant specificities elicited.

In Paper III, we used Illumina high-throughput sequencing (HTS) and developed a novel computational tool, IgDiscover, for germline Ig V allele identification. Previous work using targeted genomic PCR of germline V genes from Chinese rhesus macaques revealed a striking allelic diversity between animals and the presence of many novel alleles, highlighting the limitations of existing macaque V gene databases. By using IgDiscover to construct individualized Ig germline gene databases, we can improve the accuracy of Ab repertoire analyses in outbred populations considerably. In Paper IV, we investigated B cell responses induced by a new generation, well-ordered Env trimers. We used HTS sequencing to investigate how the response evolved over time and whether it disseminated into different immune compartments. We developed an IgDiscover extension module, Clonoquery, to trace hundreds of B cell clonal lineages targeting distinct sub-regions of Env in the peripheral blood, bone marrow, spleen, draining lymph node and gut. We detected broad dissemination of Env-specific B cell clones in all compartments measured, except gut. We showed that some lineages were greatly expanded and that the mean level of somatic hypermutation of the variants within each lineage increased with boosting, replacing previous variants from the same lineage in both blood and bone marrow.

In conclusion, this thesis offers a new information about the genetic and functional composition of Env-specific B cell responses in immunized rhesus macaques, a highly relevant model for understanding vaccine-induced response in humans. It also offers valuable information about the rhesus macaque Ig repertoire in general as well as broadly applicable tools for repertoire sequencing.

List of scientific papers

I. Christopher Sundling, Zhenhai Zhang, Ganesh E. Phad, Zizhang Sheng, Yimeng Wang, John R. Mascola, Yuxing Li, Richard T. Wyatt, Lawrence Shapiro and Gunilla B. Karlsson Hedestam. Single-cell and deep sequencing of IgG-switched macaque B cells reveal a diverse Ig repertoire following immunization. Journal of Immunology. 2014, 192(8):3637-44.
https://doi.org/10.4049/jimmunol.1303334

II. Ganesh E. Phad, Néstor Vázquez Bernat, Yu Feng, Jidnyasa Ingale, Paola Andrea Martinez Murillo, Sijy O'Dell, Yuxing Li, John R. Mascola, Christopher Sundling, Richard T. Wyatt and Gunilla B. Karlsson Hedestam. Diverse antibody genetic and recognition properties revealed following HIV-1 envelope glycoprotein immunization. Journal of Immunology. 2015, 194(12):5903-14.
https://doi.org/10.4049/jimmunol.1500122

III. Martin M. Corcoran, Ganesh E. Phad, Néstor Vázquez Bernat, Christiane Stahl-Hennig, Noriyuki Sumida, Mats A. A. Persson, Marcel Martin, Gunilla B. Karlsson Hedestam. Production of individualized V gene databases reveals high levels of immunoglobulin genetic diversity. Nature Communications. 2016, 7:13642.
https://doi.org/10.1038/ncomms13642

IV. Ganesh E. Phad, Pradeepa Pushparaj, Karen Tran, Paola Martinez-Murillo, Monika Adori, Viktoriya Dubrovskaya, Néstor Vázquez Bernat, Komal Bhullar, Sanjana Narang, Chiara Sorini, Eduardo Villablanca, Sijy O’Dell, John Mascola, Christopher Sundling, Marcel Martin, Ben Murrell, Martin Corcoran, Richard Wyatt and Gunilla B. Karlsson Hedestam. Clonal lineage tracing reveals dissemination and maturation of vaccine-induced B cell responses in multiple immune compartments. [Manuscript]