Induction of Transient Virus Replication Facilitates Antigen-Independent Isolation of SIV-Specific Monoclonal Antibodies

Abstract

Structural characterization of the HIV-1 Envelope (Env) glycoprotein has facilitated the development of Env probes to isolate HIV-specific monoclonal antibodies (mAbs). However, preclinical studies have largely evaluated these virus-specific mAbs against chimeric viruses, which do not naturally infect non-human primates, in contrast to the unconstrained simian immunodeficiency virus (SIV)mac239 clone. Given the paucity of native-like reagents for the isolation of SIV-specific B cells, we examined a method to isolate SIVmac239-specific mAbs without using Env probes. We first activated virus-specific B cells by inducing viral replication after the infusion of a CD8b-depleting mAb or withdrawal of antiretroviral therapy in SIVmac239-infected rhesus macaques. Following the rise in viremia, we observed 2-to 4-fold increases in the number of SIVmac239 Env-reactive plasmablasts in circulation. We then sorted these activated B cells and obtained 206 paired Ab sequences. After expressing 122 mAbs, we identified 14 Env-specific mAbs. While these Env-specific mAbs bound to both the SIVmac239 SOSIP.664 trimer and to infected primary rhesus CD4 + T cells, five also neutralized SIVmac316. Unfortunately, none of these mAbs neutralized SIVmac239. Our data show that this method can be used to isolate virus-specific mAbs without antigenic probes by inducing bursts of contemporary replicating viruses in vivo.

An Automated Fluorescence-Based Method to Isolate Bone Marrow-Derived Plasma Cells from Rhesus Macaques Using SIVmac239 SOSIP.664

“…We previously reported the serum neutralization titers of our cohort of 34 Indian RMs at the Wisconsin National Primate Research Center. 11 There we identified a conventional progressor, r10051, that developed SIVmac239-neutralizing titers of 1:2,157 80 weeks post-infection. After 2 additional years on antiretroviral therapy, its serum neutralization titers remained elevated (1:1,610).…”

“…To evaluate the Abs expressed by the plasma cells of r10051, we first determined their epitope specificity by ELISA with different Env subunits and mutants ( Table 1 ), as we described previously. 11 Of the 44 mAbs, 10 were SIVmac239 Env specific (22.7% of the total isolated mAbs; Table S1 ; GenBank: MT_678464–MT_678483). Four of these mAbs targeted the gp41 fusion protein, and four additional mAbs bound the variable (V) loops.…”

“…Picked bone marrow-derived plasma cells were immediately frozen on dry ice for subsequent amplification, cloning, expression, and purification of the VH and VL chains, as described previously. 11 , 34 Briefly, the VH and VL genes were produced by reverse-transcriptase PCR using random hexamers followed by two additional nested PCRs. The nested PCRs utilized 5′ V gene-specific primers and 3′ primers that bound to the heavy, kappa, or lambda constant regions and incorporated compatible ends with Gibson assembly subcloning.…”

“…We used replication-incompetent SIVmac239 and SIVmac316 pseudoviruses in a TZM-bl assay to evaluate the neutralization capabilities of our SIV-specific mAbs against SIVmac239 and SIVmac316, as described previously. 11 , 35 Briefly, we incubated the diluted SIV-specific mAbs mixed with pseudovirus at 37°C for 1 h before adding them onto TZM-bl cells. We tested mAb concentrations starting at 100 μg/mL, followed by six 4-fold dilutions.…”

“…To assess the ability of the isolated mAbs to bind native Env conformations present on SIVmac239-infected rhesus CD4+ T cells, we employed a flow cytometry-based binding assay, as described previously. 11 Briefly, we isolated CD4+ T cells from rhesus PBMCs using a nonhuman primate CD4+ T cell isolation kit (Miltenyi Biotec). These cells were later stimulated with phytohemagglutinin-P (Sigma-Aldrich), IL-2 (Roche), and anti-CD3/CD28/CD49d mAbs (NIH AIDS Reagent Program clone 6G12, Becton Dickinson [BD] clone L293, and BD clone 9F10, respectively) for 48 h in R10 medium (RPMI 1640, 10% heat-inactivated FBS, and antibiotic/antimycotic).…”