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

A Bacterial Lipooligosaccharide that Naturally Mimics the Epitope of the HIV-Neutralizing Antibody 2G12 as a Template for Vaccine Design

The broadly neutralizing antibody 2G12 binds a fairly conserved cluster of oligomannose sugars on the HIV surface glycoprotein gp120, which has led to the hypothesis that these sugars pose potential vaccine targets. Here, we present the chemical analysis, antigenicity, and immunogenicity of a bacterial lipooligosaccharide (LOS) comprised of a manno-oligosaccharide sequence analogous to the 2G12 epitope. Antigenic similarity of the LOS to oligomannose was evidenced by 2G12 binding to the LOS and the inability of sera elicited against synthetic oligomannosides, but incapable of binding natural oligomannose, to bind the LOS. Immunization with heat-killed bacteria yielded epitope-specific serum antibodies with the capacity to bind soluble gp120. Although these sera did not exhibit specific anti- HIV activity, our data suggest that this LOS may find utility as a template for the design of glycoconjugates to target HIV

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

ID50 values and percentage neutralization activity of plasma antibodies depleted using gp120 wild type and CD4bs-selective probes tested against different viruses.

<p>Plasma samples were adsorbed to BSA, HXB2gp120, HXB2gp120-D368R, RSC3, and ΔN2mCHO-coupled beads and then tested in TZM-bl neutralization assays. The final plasma dilution in the assay was 1:60. Reciprocal neutralization ID50 titres are shown. In cases where no neutralization was observed at 1:60, a value of 60 is noted.</p><p>^aID50 of untreated plasma, plasma adsorbed on BSA coupled beads; plasma absorbed on HXB2 gp120 coupled beads; plasma adsorbed on HXB2 gp120-D368R beads; plasma adsorbed on RSC3 coupled beads; plasma adsorbed on ΔN2mCHO coupled beads;</p><p>^b % reduction in ID50 by HXB2 gp120 adsorption relative to that of BSA coated beads; Percentage reduction in ID50 by HXB2 gp120-D368R relative to BSA-coupled beads; Percentage of gp120-directed neutralization contributed by CD4bs antibodies; Percentage reduction in ID50 by RSC3 adsorption relative to BSA coated beads; Percentage reduction in ID50 by ΔN2mCHO adsorption relative to BSA coated beads.</p><p>Values in bold italics are considered significant; i.e., cases where gp120 adsorbs >50% of the neutralizing activity and >25% of the activity is directed to the CD4bs. The calculations are shown in the Materials and Methods section.</p><p>ID50 values and percentage neutralization activity of plasma antibodies depleted using gp120 wild type and CD4bs-selective probes tested against different viruses.</p

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

ELISA binding assay to determine the presence of CD4bs antibodies in the CNP samples.

<p>Eleven (AIIMS 615, 616, 617, 619, 621, 642, 660, 206, 239, 249) CNP samples were tested for the presence of CD4bs antibodies using three sets of probes. HXB2 gp120 and its D368R mutant, RSC3 and CD4bs-defective mutant RSC3Δ371I, JRFLgp120 and hyperglycosylated ΔN2mCHO, which were used at 2μg/ml. Reciprocal Max50 binding titres were calculated using the least square regression method. Plasma of one healthy seronegative individual (D) was used as assay control. * denotes P values <0.05.</p

Cross-neutralization activity of two plasma samples from HIV-1 infected Indian individuals against a panel of 13 pseudoviruses (1 subtype A, 6 subtypes B and 6 subtype C viruses).

<p>The neutralization breadth of plasma antibodies from 2 samples of HIV-1 infected ART-naive individuals from India were assessed against a panel of 13 subtype B and C viruses. Plasma neutralization is shown as the reciprocal value of the ID50, which is the plasma dilution at which virus infectivity is inhibited to 50%. Data presentation is as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125575#pone.0125575.t001" target="_blank">Table 1</a>. Each experiment was performed at least twice, independently.</p><p>Cross-neutralization activity of two plasma samples from HIV-1 infected Indian individuals against a panel of 13 pseudoviruses (1 subtype A, 6 subtypes B and 6 subtype C viruses).</p