1. Isolate and characterize bnAbs. Currently isolated bnAbs do not account for all of the plasma neutralizing activity, suggesting the presence of new bnAbs yet to be discovered. Furthermore, most isolated bnAbs present unusual genetic features imposing potential elicitation barriers. Hence, we propose to isolate new classes of bnAbs that may be more easily generated. We have ongoing collaborations to obtain plasma and PBMC samples from SHIV-infected RMs from Cecilia Cheng-Mayer, from HIV-1 seroconverters from the ADARC acute infection cohort from Martin Markowitz, and from long-term non-progressors (LTNPs) from Andrés Finzi and Cécile Tremblay at the University of Montréal. We have screened these plasma samples and detected cross-reactive nAbs in 20-40% of the screened subjects. We will select the top 3-5 subjects and apply our previous strategy of antigenically probing memory B-cells by FACS, followed by single B-cell RT-PCR, to recover individual mAbs. To isolate Env trimer-dependent mAbs, we will display Env trimers on the cell surface and are currently testing methods to enrich Env trimer-binding B-cells. The isolated mAbs will be examined for neutralization activity and epitope mapping.
2. Determine bnAb precursors. To understand how bnAbs are developed, we face two fundamental immunological questions: 1) what are the naïve and/or founder (n/f) B-cells that have been immunologically selected to generate HIV-1-specific bnAbs, and 2) how do these selected n/f B-cells react with HIV-1 Env? We propose to address these questions by a longitudinal analysis to identify n/f B-cells that eventually develop into bnAb producing B-cells. For this study we have obtained longitudinal plasma and PBMC samples from SHIV-RMs and HIV-1 seroconverters. We will apply the Illumina 2x300bp pair-end MiSeq sequencing platform to analyze the subject's antibody repertoire over time. We are currently developing a bioinformatics pipeline to identify sequences that are clonally related to the isolated bnAbs based on sequence identity and VDJ gene composition. To track an isolated bnAb back in time, we will sequence longitudinally collected samples from time points prior to the mAb isolation. As we examine earlier samples, we expect to find increasingly lower hypermutation frequencies until the full VDJ and their joining regions can be linked to 1 – 5 top candidate progenitor sequences. We will initially focus on the IgG sequences, but eventually will also examine the IgM sequences. We will then repeat this process for light chain sequences, with the ultimate goal of finding complete heavy and light chain progenitor sequences for expression and functional tests. In parallel, autologous envs will be isolated by single genome amplification (SGA) from early time point plasma samples. Homologous env clones have already been isolated from SHIV inoculums. Representative env sequences will be cloned to generate Env-pseudotyped viruses for neutralization tests. The Env protein sequences will also be expressed in appropriate forms to assess n/f B-cell receptor binding.
3. Determine how neutralization breadth is developed. While only a fraction of infected individuals mount bnAb responses and such responses typically do not appear until 2-3 years after infection, almost all infected individuals develop nAbs against autologous or homologous viral strains during the first year of infection. Currently we do not understand how the early narrow autologous nAbs develop into bnAbs in some individuals. We hypothesize that bnAbs arise through two mechanisms that are not mutually exclusive. First, through n/f B-cells that precisely target a conserved neutralizing epitope. In this case, once the antibody obtains sufficient somatic hypermutation to neutralize autologous strains, it simultaneously acquires neutralization breadth against heterologous strains. Second, through epitope shifting. In this case, the n/f B-cells that originally target suboptimal epitopes with narrow autologous neutralizing activity shift to optimal epitopes (by additional somatic mutations) in response to autologous viral escape, thus acquiring neutralization breadth. For these studies, we will test longitudinal plasmas to determine the time period at which antibodies transition from autologous to heterologous neutralization and gain breadth. We will then isolate mAbs and perform antibody deep sequencing before and after the transition time. We will compare the isolated mAbs with and without neutralization breadth to determine key changes. Through the mAb sequences, we will determine their clonal relationships and track their development by deep sequencing of longitudinal samples. For longitudinal mAbs that belong to the same clonal lineage, we will compare mAb epitopes to determine temporal changes that correspond to differences in neutralization breadth. Comparisons among longitudinally acquired mAbs will reveal the requirements for bnAb development and hence test our hypothesis on the mechanisms of the broadening of antibody neutralization activity.
Our research will fill in knowledge gaps in our understanding of the process of an effective B-cell response. It is our view that this knowledge is required to induce anti-HIV-1 bnAbs through vaccination. Having access to precious samples from longitudinally followed SHIV-RMs and HIV-1 seroconverters, we have the opportunity to address two critical steps during this process: the initial activation of rare B-cell precursors and the later antibody somatic hypermutation and affinity maturation to gain broadly neutralizing function. Results from these studies will lead towards our ultimate goal of developing an effective HIV-1 vaccine.
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