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

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

Broadly neutralizing plasma antibodies effective against autologous circulating viruses in infants with multivariant HIV-1 infection

Broadly neutralizing antibodies (bnAbs) develop in a subset of HIV-1 infected individuals over 2-3 years of infection. Infected infants develop plasma bnAbs frequently and as early as 1-year post-infection suggesting factors governing bnAb induction in infants are distinct from adults. Understanding viral characteristics in infected infants with early bnAb responses will provide key information about antigenic triggers driving B cell maturation pathways towards induction of bnAbs. Herein, we evaluate the presence of plasma bnAbs in a cohort of 51 HIV-1 clade-C infected infants and identify viral factors associated with early bnAb responses. Plasma bnAbs targeting V2-apex on the env are predominant in infant elite and broad neutralizers. Circulating viral variants in infant elite neutralizers are susceptible to V2-apex bnAbs. In infant elite neutralizers, multivariant infection is associated with plasma bnAbs targeting diverse autologous viruses. Our data provides information supportive of polyvalent vaccination approaches capable of inducing V2-apex bnAbs against HIV-1.</p

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

Broadly neutralizing antibodies (bNAbs) have demonstrated protective effects against HIV-1 in primate studies and recent human clinical trials. Elite neutralizers are potential candidates for isolation of HIV-1 bNAbs. The coexistence of bNAbs such as BG18 with neutralization-susceptible autologous viruses in an HIV-1-infected adult elite controller has been suggested to control viremia. Disease progression is faster in HIV-1-infected children than in adults. Plasma bNAbs with multiple epitope specificities are developed in HIV-1 chronically infected children with more potency and breadth than in adults. Therefore, we evaluated the specificity of plasma neutralizing antibodies of an antiretroviral-naive HIV-1 clade C chronically infected pediatric elite neutralizer, AIIMS_330. The plasma antibodies showed broad and potent HIV-1 neutralizing activity with >87% (29/33) breadth, a median inhibitory dilution (ID50) value of 1,246, and presence of N160 and N332 supersite-dependent HIV-1 bNAbs. The sorting of BG505.SOSIP.664.C2 T332N gp140 HIV-1 antigen-specific single B cells of AIIMS_330 resulted in the isolation of an HIV-1 N332 supersite-dependent bNAb, AIIMS-P01. The AIIMS-P01 neutralized 67% of HIV-1 cross-clade viruses, exhibited substantial indels despite limited somatic hypermutations, interacted with native-like HIV-1 trimer as observed in negative stain electron microscopy, and demonstrated high binding affinity. In addition, AIIMS-P01 neutralized the coexisting and evolving autologous viruses, suggesting the coexistence of vulnerable autologous viruses and HIV-1 bNAbs in the AIIMS_330 pediatric elite neutralizer. Such pediatric elite neutralizers can serve as potential candidates for isolation of novel HIV-1 pediatric bNAbs and for understanding the coevolution of virus and host immune response. IMPORTANCE More than 50% of the HIV-1 infections globally are caused by clade C viruses. To date, there is no effective vaccine to prevent HIV-1 infection. Based on the structural information of the currently available HIV-1 bNAbs, attempts are under way to design immunogens that can elicit correlates of protection upon vaccination. Here, we report the isolation and characterization of an HIV-1 N332 supersite-dependent bNAb, AIIMS-P01, from a clade C chronically infected pediatric elite neutralizer. The N332 supersite is an important epitope and is one of the current HIV-1 vaccine targets. AIIMS-P01 potently neutralized the contemporaneous and autologous evolving viruses and exhibited substantial indels despite low somatic hypermutations. Taken together with the information on infant bNAbs, further isolation and characterization of bNAbs contributing to the plasma breadth in HIV-1 chronically infected children may help provide a better understanding of their role in controlling HIV-1 infection

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

ELISA binding of CD4bs-selective probes with known bNAbs.

<p>To assess functionality of the CD4bs probes, binding of the recombinant proteins was checked with different concentrations of known bNAbs. Binding of the RSC3 and its RSC3Δ371I mutant was assessed with VRC01 and b12 (A), of HXB2 gp120 and its D368R mutant with b12 and 447-52D (B), JRFLgp120 and its hyperglycosylated mutant ΔN2mCHO with b12 and 447-52D (C). The selectivity of mutant ΔN2mCHO was confirmed using two characterized plasma samples (D). Human mAb 1418 to parvovirus B19 was used as negative control in all assays.</p

Cross-neutralizing anti-HIV-1 human single chain variable fragments(scFvs) against CD4 binding site and N332 glycan identified from a recombinant phage library

More than 50% of HIV-1 infection globally is caused by subtype_C viruses. Majority of the broadly neutralizing antibodies (bnAbs) targeting HIV-1 have been isolated from non-subtype_C infected donors. Mapping the epitope specificities of bnAbs provides useful information for vaccine design. Recombinant antibody technology enables generation of a large repertoire of monoclonals with diverse specificities. We constructed a phage recombinant single chain variable fragment (scFv) library with a diversity of 7.8 x 10(8) clones, using a novel strategy of pooling peripheral blood mononuclear cells (PBMCs) of six select HIV-1 chronically infected Indian donors whose plasma antibodies exhibited potent cross neutralization efficiency. The library was panned and screened by phage ELISA using trimeric recombinant proteins to identify viral envelope specific clones. Three scFv monoclonals D11, C11 and 1F6 selected from the library cross neutralized subtypes A, B and C viruses at concentrations ranging from 0.09 mu g/mL to 100 mu g/mL. The D11 and 1F6 scFvs competed with mAbs b12 and VRC01 demonstrating CD4bs specificity, while C11 demonstrated N332 specificity. This is the first study to identify cross neutralizing scFv monoclonals with CD4bs and N332 glycan specificities from India. Cross neutralizing anti-HIV-1 human scFv monoclonals can be potential candidates for passive immunotherapy and for guiding immunogen design

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