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. 2012;7(10):e46713.
doi: 10.1371/journal.pone.0046713. Epub 2012 Oct 3.

Unique C2V3 sequence in HIV-1 envelope obtained from broadly neutralizing plasma of a slow progressing patient conferred enhanced virus neutralization

Rajesh Ringe  1 , Lipsa Das, Ipsita Choudhary, Deepak Sharma, Ramesh Paranjape, Virander Singh Chauhan, Jayanta Bhattacharya
Affiliations

Unique C2V3 sequence in HIV-1 envelope obtained from broadly neutralizing plasma of a slow progressing patient conferred enhanced virus neutralization

Rajesh Ringe et al. PLoS One. 2012.

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Neutralization sensitivity of pseudotyped viruses expressing NARI-LT5 envelopes to autologous and heterologous plasma antibodies.
Heterologous pooled HIV-1 positive plasma samples were prepared from ten Indian and ten South African (SA) donors.
Figure 2
Figure 2. Neutralization sensitivity of pseudotyped viruses expressing four different envelopes amplified from NARI-LT5 plasma to monoclonal antibodies targeting different sites in gp120 and gp41.
Figure 3
Figure 3. Neutralization properties of chimeric LT5 Envs.
A. Construction of chimeric LT5 envelopes. B. Neutralization sensitivity of pseudotyped viruses expressing wild type and chimeric LT5 envelopes to autologous plasma and human monoclonal antibodies targeting different sites in gp120 and gp41.
Figure 4
Figure 4. Effect of CCR5 antagonist, TAK-779 on entry inhibition of chimeric LT5 envelope in TZM-bl cells.
Pseudotyped viruses expressing different chimeric LT5 envelopes were titrated against different concentrations of TAK-779. The reduction in infection with the serially diluted TAK779 was assessed by measuring RLU and indicated as % neutralization on Y-axis. Note that comparable inhibition of CCR5 mediated entry of LT5.J4b and J12 were found despite differences in their sensitivity to 17b MAb.
Figure 5
Figure 5. Fine mapping of C2V3 region of LT5.J4b Env.
A. The alignment of C2V3 sequences of four LT5 Envs is shown. The secondary structure assignments are shown as arrows. Potential N-linked glycosylation sites are underlined. Residues of gp120 in direct contacts with CD4 are indicated by an asterisk. B. The panels of C2V3 mutant clones made to fine map the determinants in this region for neutralization sensitivity. All mutations were made in LT5.J12 Env backbone which acted as recipient Env to acquire corresponding amino acids present in LT5.J4b Envs. The mutant Envs were constructed by substitution of single amino acid or patches (combining more than one amino acid substitutions) from the sensitive LT5.J4b to LT5.J12 Env by blunt end ligation.
Figure 6
Figure 6. Effect of C2V3 on antibody binding and CD4 dependence (A) Binding of MAbs to envelope trimers expressed on the 293T cells.
The binding of ligands was expressed as RLU on the Y-axis for indicated Envs shown on X-axis. The RLU of only pSG3Δenv plasmid was taken as mock and the value was subtracted from the RLU of Env expressing cells. Each ligand was taken as 1 µg/ml for incubation with Env expressing cells for 45 min. The experiments were performed at two different time points (B) Infectivity in HeLa cells (RC49) expressing low CD4 on surface. RC49 cells were infected with equal TZM-bl infectious titer for indicated Envs with YU2 as positive control known to show higher infectivity in RC49. The infection in RC49 cells was measured as Focus Forming Units (FFU) and relative infectivity was in TZM-bl cells were measured and was expressed as FFUs per 5×105 RLU. (C) Inhibition by anti-CD4 (QS1420) MAb. The virus and anti-CD4 was added in the TZM-bl cells to allow them to compete for binding with CD4 on cells. The sensitivity of Envs to QS1420 is shown as percent neutralization of virus on Y-axis in a dose dependent manner.
Figure 7
Figure 7. Neutralizing antibody or sCD4 induced shedding of gp120 subunit from Env trimers.
Infectivity of the virus treated with a ligand was assessed in TZM-bl cells at different time points to probe the kinetics of MAb or sCD4 neutralization. Ligand concentrations were chosen to yield approximately 50% neutralization after 1 h of pre-incubation of virus with ligand at 37°C to allow for the monitoring of increases in neutralization activity over time. For LT5.J4b which is sensitive 0.02 µg/ml b12, 0.5 µg/ml 4E10, 0.3 µg/ml 2F5, 0.3 µg/ml 3906, 0.5 µg/ml sCD4 and 1∶5000 diluted LT5 plasma whereas for LT5.J12 resistant virus 10 µg/ml b12, 5 µg/ml 4E10, 4 µg/ml 2F5, 10 µg/ml 3906, 10 µg/ml sCD4 and 1∶20 diluted LT5 plasma were taken to treat respective viruses for the indicated time periods at 37°C. Percent neutralization of virus infectivity in TZM-bl cells was calculated with reference to the respective mock-treated virus control of each time point. The experiments were conducted in duplicate at two different time points and one is shown here.
Figure 8
Figure 8. Specificity of the LT5 plasma.
LT5 plasma (that showed 80% neutralization of indicated viruses) was incubated with gp120 outer domain (OD) construct, V3 peptide, 4E10 binding peptide and scrambled peptide at various concentrations for 1 hour and subsequently used to assess the neutralizing activity against LT5.J4b and heterologous Env pseudotyped viruses in TZM-bl cells. Assay was done in duplicate and twice. % infection of the viruses on Y-axis was measured as RLU.
Figure 9
Figure 9. Structure based prediction of the basis of enhanced LT5.J4b Env sensitivity to IgG1b12 (anti-CD4bs) and 3074 (anti-V3) MAbs.
A. Amino acid residues in C2V3 region of the LT5.J4b Env that modulated neutralization sensitivity. The model was prepared in UCSF Chimera in the background of JRFL crystal structure (RSCB PDB: 2B4C). The C2V3 amino acid residues that differed between sensitive (LT5.J4b) and other resistant Envs are highlighted. Residues in dark blue are in loop D that contacts CD4 which is in close proximity to major CD4 binding residues (SSGGDPE) (shown in yellow). B Predicated conformations of LT5.J4b and LT5.J12 Env structures in the background of JRFL gp120 crystal structure (2B4C) after energy minimization. C. Spatial orientation of loop D of LT5.J4b and LT5.J12 due to mutations in C2V3 region that showed closer proximity to IgG1b12 heavy chain. D. The interaction of heavy chain of 3074 (green) with V3 loops (LT5.J4b purple, LT5.J12 yellow.
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This study was supported by a research grant from the Department of Biotechnology, Ministry of Science and Technology, Government of India to JB (BT/PR12853/MED/29/141/2009). RR and DS are recipients of Senior Research Fellowships from the Council of Scientific and Industrial Research (CSIR), and the University Grants Commission (UGC), Government of India respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.