Vaccine-induced plasmablast responses in rhesus macaques: Phenotypic characterization and a source for generating antigen-specific monoclonal antibodies

Abstract

Over 100 broadly neutralizing antibodies have been isolated from a minority of HIV infected patients, but the steps leading to the selection of plasmacells producing such antibodies remain incompletely understood, hampering the development of vaccines able to elicit them. Rhesus macaques have become a preferred animal model system used to study SIV/HIV, for the characterization and development of novel therapeutics and vaccines as well as to understand pathogenesis. However, most of our knowledge about the dynamics of antibody responses is limited to the analysis of serum antibodies or monoclonal antibodies generated from memory B cells. In a vaccine setting, relatively little is known about the early cellular responses that elicit long-lived plasma cells and memory B cells and the tools to dissect plasmablast responses are not available in macaques. In the current study, we show that the majority (>80%) of the vaccine-induced plasmablast response are antigen-specific by functional ELISPOT assays. While plasmablasts are easily defined and isolated in humans, those same phenotypic markers have not been useful for identifying macaque plasmablasts. Here we describe an approach that allows for the isolation and single cell sorting of vaccine-induced plasmablasts. Finally, we show that isolated plasmablasts can be used to efficiently recover antigen-specific monoclonal antibodies through single cell expression cloning. This will allow detailed studies of the early plasmablast responses in rhesus macaques, enabling the characterization of both their repertoire breadth as well as the epitope specificity and functional qualities of the antibodies they produce, not only in the context of SIV/HIV vaccines but for many other pathogens/vaccines as well.

Influence of Plasma Cell Niche Factors on the Recruitment and Maintenance of IRF4 hi Plasma Cells and Plasmablasts in Vaccinated, Simian Immunodeficiency Virus-Infected Rhesus Macaques with Low and High Viremia

“…1D) (29,31,39). Expression of CD27 was low in both subsets, in agreement with previous findings (34,35) (data not shown).…”

“…High expression of the transcription factor IRF4 is characteristic of both PB and PC (24)(25)(26), while expression of CD138 is most often associated with PC differentiation (27,28). Consistent with other reports and through backgating analysis of IRF4 hi populations of interest, these PC/PB subsets were identified as lacking expression of CD20 (29)(30)(31)(32) but as CD19 ϩ/Ϫ (31,33,34). Thus, for all sites examined, PB were identified as CD20 Ϫ IRF4 hi CD138 Ϫ and PC as CD20 Ϫ IRF4 hi CD138 ϩ (Fig.…”

“…Many studies have found evidence for the protective effect of SIV-and HIV-specific antibodies early postchallenge (62)(63)(64)(65)(66)(67)(68)), yet few have examined how the generation, recruitment, and maintenance of antibody-secreting cells themselves impact viral control in the chronic phase of infection. Unlike memory B cells, which express their receptor (BCR) on the cell surface, the majority of PC lack surface expression of their secreted immunoglobulin (33,34), making consistent identification of SIV-specific PC a challenge. Furthermore, chronic HIV and SIV infection induces a number of B cell abnormalities affecting PB/PC, including hypergammaglobulinemia, reductions in memory B cells and antibody responses to recall antigens, progressive loss of lymphoid tissue architecture, and an altered isotype ratio in the gut, favoring IgG over IgA (69).…”

Identification of Vaccine-Altered Circulating B Cell Phenotypes Using Mass Cytometry and a Two-Step Clustering Analysis

“…To further elucidate germinal center cell commitment to memory B cells or PB/PCs, future studies should incorporate markers that can characterize additional B cell subsets through their intracellular Ig levels, capacity to secrete Ab, or proliferate (56), as well as analyze additional tissues where most Absecreting cells preferentially reside (27,47). In addition, reagents to detect specific BCRs as described for rotavirus, influenza, papillomavirus, and other Ags (27,(76)(77)(78)(79) could be used to reveal SPADE clusters that are enriched with vaccine-specific cells.…”

“…PB/PCs were defined as CD3 (56). The cynomolgus macaque (Macaca fascicularis) model presented in this study could include phenotypes of PB/PCs that differ from other primates, and we expect that our Ab panel may identify a portion of PBs when gating HLA-DR + CD20 low cells.…”