Identification of CD4-Binding Site Dependent Plasma Neutralizing Antibodies in an HIV-1 Infected Indian Individual

Dissecting antibody specificities in the plasma of HIV-1 infected individuals that develop broadly neutralizing antibodies (bNAbs) is likely to provide useful information for refining target epitopes for vaccine design. Several studies have reported CD4-binding site (CD4bs) antibodies as neutralization determinants in the plasma of subtype B-infected individuals; however there is little information on the prevalence of CD4bs specificities in HIV-infected individuals in India. Here, we report on the presence of CD4bs antibodies and their contribution to virus neutralization in the plasma from a cohort of HIV-1 infected Indian individuals. Plasma from 11 of the 140 HIV-1 infected individuals (7.9%) studied here exhibited cross-neutralization activity against a panel of subtype B and C viruses. Analyses of these 11 plasma samples for the presence of CD4bs antibodies using two CD4bs-selective probes (antigenically resurfaced HXB2gp120 core protein RSC3 and hyperglycosylated JRFLgp120 mutant ΔN2mCHO) revealed that five (AIIMS 617, 619, 627, 642, 660) contained RSC3-reactive plasma antibodies and only one (AIIMS 660) contained ΔN2mCHO-reactive antibodies. Plasma antibody depletion and competition experiments confirmed that the neutralizing activity in the AIIMS 660 plasma was dependent on CD4bs antibodies. To the best of our knowledge, this is the first study to report specifically on the presence of CD4bs antibodies in the plasma of a cohort of HIV-1 infected Indian donors. The identification of CD4bs dependent neutralizing antibodies in an HIV-1 infected Indian donor is a salient finding of this study and is supportive of ongoing efforts to induce similar antibodies by immunization

Isolation and characterization of cross-neutralizing human anti-V3 single-chain variable fragments (scFvs) against HIV-1 from an antigen preselected phage library

Recently conducted human phase- I trials showed protective effect of anti-HIV-1 broadly neutralizing antibodies (bnAbs). The V3 region of the HIV-1 envelope is highly conserved as it is the co-receptor binding site, and is highly immunogenic. Recombinant single-chain antibody fragments (scFvs) can serve as potential tools for construction of chimeric/bispecific antibodies that can target different epitopes on the HIV-1 envelope. Previously, we have constructed a V3 specific human scFv phage recombinant library by a combinational approach of Epstein–Barr virus (EBV) transformation and antigen (V3) preselection, using peripheral blood mononuclear cells (PBMCs), from a subtype C HIV-1 infected antiretroviral naive donor. In the present study, by biopanning this recombinant scFv phage library with V3B (subtype B) and V3C (subtype C) peptides, we identified unique cross reactive anti-V3 scFv monoclonals. These scFvs demonstrated cross-neutralizing activity when tested against subtype A, subtype B, and subtype C viruses. Further, molecular modeling of the anti-V3 scFvs with V3C and V3B peptides predicted their sites of interaction with the scFvs, providing insights for future immunogen design studies. A large collection of such monoclonal antibody fragments with diverse epitope specificities can be useful immunotherapeutic reagents along with antiretroviral drugs to prevent HIV-1 infection and disease progression

A novel strategy for efficient production of anti-V3 human scFvs against HIV-1 clade C

<p>Abstract</p> <p>Background</p> <p>Production of human monoclonal antibodies that exhibit broadly neutralizing activity is needed for preventing HIV-1 infection, however only a few such antibodies have been generated till date. Isolation of antibodies by the hybridoma technology is a cumbersome process with fewer yields. Further, the loss of unstable or slowly growing clones which may have unique binding specificities often occurs during cloning and propagation and the strongly positive clones are often lost. This has been avoided by the process described in this paper, wherein, by combining the strategy of EBV transformation and recombinant DNA technology, we constructed human single chain variable fragments (scFvs) against the third variable region (V3) of the clade C HIV-1 envelope.</p> <p>Results</p> <p>An antigen specific phage library of 7000 clones was constructed from the enriched V3- positive antibody secreting EBV transformed cells. By ligation of the digested scFv DNA into phagemid vector and bio panning against the HIV-1 consensus C and B V3 peptides followed by random selection of 40 clones, we identified 15 clones that showed V3 reactivity in phage ELISA. DNA fingerprinting analysis and sequencing showed that 13 out of the 15 clones were distinct. Expression of the positive clones was tested by SDS-PAGE and Western blot. All the 13 anti-V3 scFvs showed cross-reactivity against both the clade C and B V3 peptides and did not show any reactivity against other unrelated peptides in ELISA. Preliminary neutralization assays indicated varying degrees of neutralization of clade C and B viruses. EBV transformation, followed by antigen selection of lines to identify specific binders, enabled the selection of phage from un-cloned lines for scFv generation, thus avoiding the problems of hybridoma technology. Moreover, as the clones were pretested for antigen binding, a comparatively small library sufficed for the selection of a considerable number of unique antigen binding phage. After selection, the phage clones were propagated in a clonal manner.</p> <p>Conclusions</p> <p>This strategy can be efficiently used and is cost effective for the generation of diverse recombinant antibodies. This is the first study to generate anti-V3 scFvs against HIV-1 Clade C.</p

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

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

Alterations in B Cell Compartment Correlate with Poor Neutralization Response and Disease Progression in HIV-1 Infected Children

Several B cell defects are reported in HIV-1 infected individuals including variation in B cell subsets, polyclonal B cell activation and exhaustion, with broadly neutralizing antibodies elicited in less than 10–20% of the infected population. HIV-1 disease progression is faster in children than adults. B Lymphocyte Stimulator (BLyS), expressed on dendritic cells (DCs), is a key regulator of B cell homeostasis. Understanding how DCs influence B cell phenotype and functionality (viral neutralization), thereby HIV-1 disease outcome in infected children, is important to develop interventional strategies for restoration of B cell function. In this study, a total of 38 vertically transmitted HIV-1 infected antiretroviral therapy (ART) naïve children and 25 seronegative controls were recruited. Based on the CD4 counts and years post-infection, infected children were categorized as long-term non-progressors (LTNPs) (n = 20) and progressors (n = 18). Eight of these progressors were followed up at 6–12 months post-ART. Percentages (%) of DCs, B cell subsets, and expression of BLyS on DCs were analyzed by flow-cytometry. Plasma levels of B cell growth factors were measured by ELISA and viral neutralization activity was determined using TZM-bl assay. Lower (%) of myeloid DCs (mDCs), plasmacytoid DCs, and high expression of BLyS on mDCs were observed in HIV-1 infected progressors than seronegative controls. Progressors showed lower % of naive B cells, resting memory B cells and higher % of mature activated, tissue-like memory B cells as compared to seronegative controls. Higher plasma levels of IL-4, IL-6, IL-10, and IgA were observed in progressors vs. seronegative controls. Plasma levels of IgG were high in progressors and in LTNPs than seronegative controls, suggesting persistence of hypergammaglobulinemia at all stages of disease. High plasma levels of BLyS in progressors positively correlated with poor viral neutralizing activity. Interestingly on follow up, treatment naïve progressors, post-ART showed increase in resting memory B cells along with reduction in plasma BLyS levels that correlated with improvement in viral neutralization. This is the first study to demonstrate that reduction in plasma BLyS levels correlates with restoration of B cell function, in terms of viral neutralization in HIV-1-infected children

Cross-neutralization activity of six select plasma samples from HIV-1 infected Indian individuals against a panel of subtype B and subtype C pseudoviruses.

<p>The neutralization breadth of plasma antibodies from 6 plasma samples obtained from HIV-1 infected ART-naive individuals from India were assessed against a panel of 22 subtype B and C viruses. The AIIMS ID of the plasma samples is provided at the top of the table. Plasma neutralization is shown as the reciprocal value of the ID50, which is the plasma dilution at which virus infectivity is inhibited to 50%. ID50>1000 (Bold), ID50 = 61–1000 (Italic) and 60, where ID50 was not reached. Each experiment was performed at least twice, independently.</p><p>Cross-neutralization activity of six select plasma samples from HIV-1 infected Indian individuals against a panel of subtype B and subtype C pseudoviruses.</p

% depletion of gp120 specific antibodies from select CNP samples using CD4bs probes.

<p>The CNP samples that exhibited CD4bs specificities (AIIMS 660 and AIIMS 619) were depleted at 1:30 dilution in three rounds of incubation with the CD4bs probes (HXB2gp120, HXB2 gp120-D368R, RSC3, ΔN2mCHO and BSA) coupled magnetic beads. The depletion was verified by reduction in the ELISA binding of the depleted (flow-through) as compared to the undepleted plasma, with the corresponding wild type gp120. The plasma antibodies, before and after depletion, were tested for binding to the respective wild-type gp120 (HXB2 gp120 for both RSC3 and D368R depleted plasma and JRFL gp120 for ΔN2mCHO depleted plasma. Percent depletion of CD4bs plasma antibodies by each probe was calculated as: % depletion = 100-[100× (OD405 after 3 rounds of adsorption /OD405 before adsorption)] for RSC3 and ΔN2mCHO. For HXB2 gp120- D368R depleted plasma sample: % depletion = [100× (OD405 after 3 rounds of adsorption /OD405 before adsorption)]. BSA coupled beads were used as control.</p

Clinical profile of 8 CNP of HIV-1 infected antiretroviral naive individuals.

<p>Clinical profile of 8 CNP of HIV-1 infected antiretroviral naive individuals.</p