Rajesh Ringe

Variations in autologous neutralization and CD4 dependence of b12 resistant HIV-1 clade C env clones obtained at different time points from antiretroviral naïve Indian patients with recent infection.

BackgroundLimited information is available on HIV-1 Indian clade C sensitivities to autologous antibodies during the course of natural infection. In the present study, a total of 37 complete envelope clones (Env) were amplified at different time points predominantly from the plasma of five Indian patients with recent HIV-1 infection and envelope-pseudotyped viruses were examined for their magnitude of sensitivity to autologous plasma antibodies during natural course of infection.ResultsVariable low levels of neutralization were consistently detected with contemporaneous autologous plasma. In contrast to clade B and African clade C HIV-1 envelopes, Env clones obtained from four patients were found to be resistant to IgG1b12. The majority of the Env clones were resistant to 2G12 and 2F5 due to the absence of the minimal motifs required for antibody recognition, but were sensitive to 4E10. Nonetheless, Env clones from one patient were found to be sensitive to 2G12, atypical for clade C, and one Env clone exhibited unusual sensitivity to 17b, suggesting spontaneous exposure of CD4i epitopes. Phylogenetic analysis revealed that Env clones were closely clustered within patients. Variation in the potential N-linked glycosylation pattern also appeared to be different in patients over the course of infection. Interestingly, we found that the sensitivity of Envs to contemporaneous autologous NAbs correlated positively with increased sensitivity to soluble CD4 and inversely with anti-CD4 antibody and Envs with increased NAb sensitivity were able to efficiently infect HeLa cells expressing low CD4.ConclusionOur data showed considerable variations in autologous neutralization of these early HIV-1 clade C Envs in each of these patients and indicate greater exposure to CD4 of Envs that showed increased autologous neutralization. Interestingly, Env clones obtained from a single patient at different time points were found to retain sensitivity to b12 antibody that binds to CD4 binding site in Env in contrast to Envs obtained from other patients. However, we did not find any association between increased b12 sensitivity of Envs obtained from this particular patient with their degree of exposure to CD4.

Subtle alteration of residues including N-linked glycans in V2 loop modulate HIV-1 neutralization by PG9 and PG16 monoclonal antibodies.

Recent discovery of several potent and broadly neutralizing monoclonal antibodies (MAbs) (such as PG9 and PG16) to HIV-1 provided clues on newer vaccine targets. In the present study, we found an env clone obtained from a slow progressor showing significant resistance to PG9 and PG16 MAbs in sharp contrast to other contemporaneous autologous env clones. By constructing chimeric envelopes and specific substitutions we found that both loop length and spatial orientation of glycan residues in addition to the net charge of the β sheet C region that directly binds to PG9 CDRH3 within V2 loop significantly modulated HIV-1 sensitivity to PG9 and PG16 MAbs. Similar observation were made with several other Envs which varied in length, glycan content and net charge in PG9 contacting complementary region in V2 loop. Our data indicated that subtle change within V2 loop alone modulates exposition of quaternary epitopes that are targets of PG9/PG16 MAbs.

Association of Enhanced HIV-1 Neutralization by a Single Y681H Substitution in gp41 with Increased gp120-CD4 Interaction and Macrophage Infectivity

HIV-1 variants that show unusual sensitivity to autologous antibodies due to presence of critical neutralization signatures would likely contribute towards rational envelope based HIV-1 vaccine design. In the present study, we found that presence of a naturally occurring H681 in gp41 membrane proximal external region (MPER) of a clade C envelope (Env) obtained from a recently infected Indian patient conferred increased sensitivity to autologous and heterologous plasma antibodies. Furthermore, Env-pseudotyped viruses expressing H681 showed increased sensitivity to soluble CD4, b12 and 4E10 monoclonal antibodies both in related and unrelated Envs and was corroborated with increased Env susceptibility and binding to cellular CD4 as well as with prolonged exposure of MPER epitopes. The increased gp120-CD4 interaction was further associated with relative exposure of CD4-induced epitopes and macrophage infectivity. In summary, our data indicate that Y681H substitution exposes neutralizing epitopes in CD4bs and MPER towards comprehensive interference in HIV-1 entry.

A single amino acid substitution in the C4 region in gp120 confers enhanced neutralization of HIV-1 by modulating CD4 binding sites and V3 loop.

Identification of vulnerability in the HIV-1 envelope (Env) will aid in Env-based vaccine design. We recently found an HIV-1 clade C Env clone (4-2.J45) amplified from a recently infected Indian patient showing exceptional neutralization sensitivity to autologous plasma in contrast to other autologous Envs obtained at the same time point. By constructing chimeric Envs and fine mapping between sensitive and resistant Env clones, we found that substitution of highly conserved isoleucine (I) with methionine (M) (ATA to ATG) at position 424 in the C4 domain conferred enhanced neutralization sensitivity of Env-pseudotyped viruses to autologous and heterologous plasma antibodies. When tested against monoclonal antibodies targeting different sites in gp120 and gp41, Envs expressing M424 showed significant sensitivity to anti-V3 monoclonal antibodies and modestly to sCD4 and b12. Substitution of I424M in unrelated Envs also showed similar neutralization phenotype, indicating that M424 in C4 region induces exposure of neutralizing epitopes particularly in CD4 binding sites and V3 loop.

HIV-1 clade C env clones obtained from an Indian patient exhibiting expanded coreceptor tropism are presented with naturally occurring unusual amino acid substitutions in V3 loop

HIV-1 subtype C is predominantly circulating in India and has been reported to be strictly CCR5 tropic irrespective of disease stages. In the present study, we examined env clones obtained from a late stage Indian patient with a history of multiple sexual partners and opportunistic infections for coreceptor usage and V3 loop sequence. The env clones were found to exploit several coreceptors in addition to CCR5 in a cell-associated and cell-free manner. Analysis of V3 loop sequence revealed that the NARI-VB105 env clones were presented with unique amino acid substitutions with GPGR motif, atypical of clade C envelope. Further genetic analysis showed the V3 sequences albeit belonging to subtype C; however clustered distinctly to that of other clade C envelopes originated in different geographical regions. Modelling data revealed that NARI-VB105 V3 loop contained several basic residues giving rise a high net positive charge of +8 to these envelopes.

Evidence of extended alternate coreceptor usage by HIV-1 clade C envelope obtained from an Indian patient

HIV-1 clade C tends to exclusively use CCR5 irrespective of disease stages. We previously reported envelopes (Envs) obtained from an Indian patient (VB105) that used CXCR4, CXCR6, CCR2b, CCR3, GPR15, and CX3CR1 as additional coreceptors besides CCR5 for entry. Here we show that the primary VB105 virus was able to replicate in peripheral blood mononuclear cells (PBMCs) in presence of inhibitors that antagonizes all the above seven coreceptors at excess doses. In addition, VB105 Envs were found to efficiently infect CCR5-defective T cells (MOLT-4) in presence of excess TAK-779, AMD3100, vMIP-1 and vMIP-2 further substantiated the usage of additional coreceptors beyond the seven coreceptors as reported earlier by VB105 Env. Interestingly, VB105 Envs showed spontaneous exposure of CD4-induced epitopes and found to be associated with increased infection of macrophages. Information on HIV-1 clade C using alternate coreceptors in primary cells to better understand their impact on pathogenesis and efficacy to future entry inhibitors.

HIV-1 clade C envelopes obtained from late stage symptomatic Indian patients varied in their ability towards relative CD4 usages and sensitivity to CCR5 antagonist TAK-779

The mechanism by which strictly CCR5 using HIV-1 clade C variants exacerbate disease progression in absence of coreceptor switch is not clearly known. We previously reported HIV-1 clade C envelopes (Env) obtained from late stage Indian patients with expanded coreceptor tropism. Here we compared such Envs (having expanded coreceptor tropism) with strictly CCR5 using Envs also obtained from late stage in their capacity to utilize CD4 and CCR5 for productive entry. We found that while envelopes with low CD4 dependence tend to infect primary CD4(+) T cells better than those required optimum CD4 for entry, no significant association was found between low CD4 usage and infectivity of primary CD4(+) T cells by Env-pseudotyped viruses and their sensitivity to CCR5 antagonist TAK-779. Interestingly, Envs that readily infected HeLa cells expressing low CD4 showed relative resistance to T20 indicating that conformational intermediates of these envelopes remained for a shorter period of time than is required for efficient inhibition by T20.

Unique C2V3 Sequence in HIV-1 Envelope Obtained from Broadly Neutralizing Plasma of a Slow Progressing Patient Conferred Enhanced Virus Neutralization

Broadly neutralizing antibodies to HIV-1 usually develops in chronic infections. Here, we examined the basis of enhanced sensitivity of an env clone amplified from cross neutralizing plasma of an antiretroviral naïve chronically infected Indian patient (ID50 >600-fold higher compared to other autologous env clones). The enhanced autologous neutralization of pseudotyped viruses expressing the sensitive envelope (Env) was associated with increased sensitivity to reagents and monoclonal antibodies targeting distinct sites in Env. Chimeric viruses constructed by swapping fragments of sensitive Env into resistant Env backbone revealed that the presence of unique residues within C2V3 region of gp120 governed increased neutralization. The enhanced virus neutralization was also associated with low CD4 dependence as well as increased binding of Env trimers to IgG1b12 and CD4-IgG2 and was independent of gp120 shedding. Our data highlighted vulnerabilities in the Env obtained from cross neutralizing plasma associated with the exposure of discontinuous neutralizing epitopes and enhanced autologous neutralization. Such information may aid in Env-based vaccine immunogen design.

Short communication: evidence of HIV type 1 clade C env clones containing low V3 loop charge obtained from an AIDS patient in India that uses CXCR6 and CCR8 for entry in addition to CCR5.

Abstract HIV-1 clade C is the major subtype circulating in India and preferentially uses CCR5 during the entire disease course. We have recently shown that env clones from an Indian patient; NARI-VB105 uses multiple coreceptors for entry and was presented with an unusual V3 loop sequence giving rise to high net V3 loop positive charges. Here we show that env clones belonging to subtype C obtained from an AIDS patient, NARI-VB52, use CXCR6 and CCR8 in addition to CCR5 for entry. However, unlike the NARI-105 patient, the env clones contained a low V3 loop net charge of +3 with a conserved GPGQ motif typical of CCR5 using subtype C strains, indicating that residues outside the V3 loop contributed to extended coreceptor use in this particular patient.

Determinants in V2C2 region of HIV-1 clade C primary envelopes conferred altered neutralization susceptibilities to IgG1b12 and PG9 monoclonal antibodies in a context-dependent manner.

In the present study by examining pseudoviruses expressing patient chimeric envelopes (Envs) made between an IgG1b12 (b12)-sensitive (2-5.J3) and a b12-resistant (4.J22) HIV-1 clade C envelope, we identified determinants in the V2C2 region that governed susceptibility to b12 monoclonal antibody, but not to other CD4 binding site antibodies. Interestingly, when the V2C2 sequence of the 2-5.J3 Env was transferred to other b12-resistant primary clade C Envs, their susceptibility to b12 varied, indicating that this effect was context dependent. In addition, we identified determinants within the V2 region in the b12-resistant envelope that significantly modulated the neutralization of Env-pseudotyped viruses to PG9/PG16 MAbs. The enhanced neutralization susceptibilities of Envs to b12 and PG9 MAbs were correlated with increased exposure of their corresponding epitopes highlighting vulnerabilities in the V2C2 region that altered Env conformation necessary for the efficient accessibility of b12 and PG9 antibodies.