CD4-Binding Site Directed Cross-Neutralizing scFv Monoclonals from HIV-1 Subtype C Infected Indian Children

Abstract

Progression of human immunodeficiency virus type-1 (HIV-1) infection in children is faster than adults. HIV-1 subtype C is responsible for more than 50% of the infections globally and more than 90% infections in India. To date, there is no effective vaccine against HIV-1. Recent animal studies and human Phase I trials showed promising results of the protective effect of anti-HIV-1 broadly neutralizing antibodies (bnAbs). Interaction between CD4 binding site (CD4bs) on the HIV-1 envelope glycoprotein and CD4 receptor on the host immune cells is the primary event leading to HIV-1 infection. The CD4bs is a highly conserved region, comprised of a conformational epitope, and is a potential target of bnAbs such as VRC01 that is presently under human clinical trials. Recombinant scFvs can access masked epitopes due to their small size and have shown the potential to inhibit viral replication and neutralize a broad range of viruses. Pediatric viruses are resistant to many of the existing bnAbs isolated from adults. Therefore, in this study, pooled peripheral blood mononuclear cells from 9 chronically HIV-1 subtype C infected pediatric cross-neutralizers whose plasma antibodies exhibited potent and cross-neutralizing activity were used to construct a human anti-HIV-1 scFv phage library of 9 × 108 individual clones. Plasma mapping using CD4bs-specific probes identified the presence of CD4bs directed antibodies in 4 of these children. By extensive biopanning of the library with CD4bs-specific antigen RSC3 core protein, we identified two cross-neutralizing scFv monoclonals 2B10 and 2E4 demonstrating a neutralizing breadth and GMT of 77%, 17.9 µg/ml and 32%, 51.2 µg/ml, respectively, against a panel of 49 tier 1, 2 and 3 viruses. Both scFvs competed with anti-CD4bs bnAb VRC01 confirming their CD4bs epitope specificity. The 2B10 scFv was effective in neutralizing the 7 subtype C and subtype A pediatric viruses tested. Somatic hypermutations in the VH gene of scFvs (10.1–11.1%) is comparable with that of the adult antibodies. These cross-neutralizing CD4bs-directed scFvs can serve as potential reagents for passive immunotherapy. A combination of cross-neutralizing scFvs of diverse specificities with antiretroviral drugs may be effective in suppressing viremia at an early stage of HIV-1 infection and prevent disease progression.

An HIV-1 broadly neutralizing antibody from a clade C infected pediatric elite neutralizer potently neutralizes the contemporaneous and autologous evolving viruses

“…However, no reduction in ELISA binding of plasma antibodies with CD4bs probe RSC3 mutant protein Δ371I/P363N, as compared to its wild-type protein RSC3 core, was observed. In addition, the plasma antibodies did not bind to MPER peptides either (Figure 1D), suggesting the absence of CD4bs and MPER directed antibodies in the 2015 time point AIIMS_330 plasma, as was also seen in its earlier 2013 time point plasma sample (38). Based on the elite neutralizing activity exhibited by AIIMS_330 (2015) plasma, this donor was selected for isolating anti-HIV-1 bNAbs.…”

“…Further, the bNAbs present in the plasma of infected children demonstrated similar epitope specificities as that observed for the adult bNAbs (24). Recently, from pooled peripheral blood mononuclear cells (PBMCs) of select HIV-1 clade C infected pediatric long-term non-progressors (LTNPs), we constructed an anti-HIV-1 human single chain recombinant fragment (scFv) phage library, from which one of the CD4-binding site (CD4bs) targeted scFv 2B10 demonstrated broad neutralizing activity with a breadth of 78% (38).…”

An HIV-1 Broadly Neutralizing Antibody from a Clade C-Infected Pediatric Elite Neutralizer Potently Neutralizes the Contemporaneous and Autologous Evolving Viruses

“…In addition, the plasma antibodies also did not bind to peptides of the membraneproximal external region (MPER) ( Fig. 1F), suggesting the absence of CD4bs-and MPER-directed antibodies in AIIMS_330 plasma of the 2015 time point, as was also seen in its earlier 2013 time point plasma sample (38). Based on the elite neutralizing activity exhibited by AIIMS_330 (2015) plasma, this donor was selected for isolating anti-HIV-1 bNAbs.…”

“…Further, the bNAbs present in the plasma of infected children demonstrated epitope specificities similar to those observed for the adult bNAbs (24). Recently, from pooled peripheral blood mononuclear cells (PBMCs) of select HIV-1 clade C (HIV-1C)-infected pediatric long-term nonprogressors (LTNPs), we constructed an anti-HIV-1 human single-chain recombinant fragment (scFv) phage library, from which one of the CD4-binding site (CD4bs)-targeted scFvs, 2B10, demonstrated broad neutralizing activity with a breadth of 78% (38).…”

Viral Characteristics Associated with Maintenance of Elite Neutralizing Activity in Chronically HIV-1 Clade C-Infected Monozygotic Pediatric Twins

“…Chronically HIV-1-infected children have been shown to have potent plasma bnAbs with diverse epitope specificities (5,6). In our pediatric cohort of antiretroviral-naive HIC-1 clade C (HIV-1C)-infected children previously characterized for plasma antibody responses (6,(13)(14)(15)(16)(17)(18), we identified a pair of chronically infected antiretroviral-naive HIV-1C-infected children, AIIMS_329 and AIIMS_330, defined herein as genetically identical twins by their identical HLA phenotypes, who had acquired the infection at birth by vertical transmission. Both twins AIIMS_329 and AIIMS_330 developed potent plasma neutralizing antibodies (nAbs), with the latter showing elite neutralizing activity since the first sampling.…”