Dissection of HIV-1 Env-specific B cell responses in nonhuman primates

Abstract: Vaccine-induced protection is generally mediated by long-lived antigen-specific B cell responses. Most licensed vaccines target pathogens that display relatively low variability, but for highly variable pathogens, such as HIV-1, vaccine development is more challenging. This thesis is focused on understanding vaccine-induced B cell responses against the HIV-1 envelope glycoproteins (Env), a critical vaccine target. Information about the immunogenic properties of candidate Env immunogens remains limited and so far the elicitation of broadly neutralizing antibodies (bNAbs) were not reported for any vaccine regimen tested in primates. Thus, there is a need to investigate vaccine-induced B cell responses against Env in more detail and to identify means to improve upon current Env-based vaccine strategies. Here, I investigate B cell responses in nonhuman primates immunized with soluble HIV-1 Env trimers to address these questions, as well as to gain an enhanced understanding about B cell responses to complex protein antigens in general. In paper I we established several assays for the evaluation of B cell responses in macaques. Following immunization with soluble trimeric Env, we comprehensively analyzed the B cell responses in the periphery, bone marrow, and mucosal compartments and further evaluated the elicited Abs for neutralization activity and protection in a SHIV challenge model. We observed high levels of Env-specific B cell responses following immunizations, improved breadth of neutralization compared to responses elicited by a monomeric Env vaccine tested in humans and delayed acquisition of SHIV infection compared to in control immunized animals. In paper II we evaluated longitudinal B cell responses following immunization with soluble trimeric Env and influenza HA protein, the latter included for comparative purposes. We found that peripheral B cell responses declined rapidly following boost, while antigen-specific long-lived plasma cells were stable for >6 months following immunization, for both antigens. In paper III we established a system for highresolution evaluation of B cell responses in nonhuman primates. We first characterized the rhesus immunoglobulin loci to allow analyses of Ab gene usage and somatic hypermutation. We next isolated monoclonal antibodies (MAbs) targeting the HIV-1 primary receptor binding site (CD4bs) on Env and we examined the binding specificities of these Abs compared to infection-induced MAbs to unravel limitations of current vaccine-induced responses. In paper IV we optimized the RT-PCR method used in paper III for isolation of Ab V(D)J sequences from rhesus macaque B cells to facilitate future use of the macaque model for B cell studies. In conclusion, this thesis establishes several methods for the evaluation of B cell responses in nonhuman primates and it demonstrates that the soluble HIV-1 Env trimers induce potent, but relatively short-lived peripheral B cell responses. Additionally, we describe, for the first time, a set of vaccine-induced CD4bs-directed MAbs and we characterize their binding and neutralizing properties and discuss the implications of these results for improved Env vaccine design.