Identification of New Regions in HIV-1 gp120 Variable 2 and 3 Loops that Bind to α4β7 Integrin Receptor

<div><p>Background</p><p>The gut mucosal homing integrin receptor α4β7 present on activated CD4⁺ T cells interacts with the HIV-1 gp120 second variable loop (V2). Case control analysis of the RV144 phase III vaccine trial demonstrated that plasma IgG binding antibodies specific to scaffolded proteins expressing the first and second variable regions (V1V2) of HIV envelope protein gp120 containing the α4β7 binding motif correlated inversely with risk of infection. Subsequently antibodies to the V3 region were also shown to correlate with protection. The integrin receptor α4β7 was shown to interact with the LDI/V motif on V2 loop but recent studies suggest that additional regions of V2 loop could interact with the α4β7. Thus, there may be several regions on the V2 and possibly V3 loops that may be involved in this binding. Using a cell line, that constitutively expressed α4β7 receptors but lacked CD4, we examined the contribution of V2 and V3 loops and the ability of V2 peptide-, V2 integrin-, V3-specific monoclonal antibodies (mAbs), and purified IgG from RV144 vaccinees to block the V2/V3-α4β7 interaction.</p><p>Results</p><p>We demonstrate that α4β7 on RPMI8866 cells bound specifically to its natural ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) as well as to cyclic-V2 and cyclic-V3 peptides. This binding was inhibited by anti-α4β7-specific monoclonal antibody (mAb) ACT-1, mAbs specific to either V2 or V3 loops, and by purified primary virions or infectious molecular clones expressing envelopes from acute or chronic subtypes A, C, and CRF01_AE viruses. Plasma from HIV-1 infected Thai individuals as well as purified IgG from uninfected RV144 vaccinees inhibited (0–50%) the binding of V2 and V3 peptides to α4β7.</p><p>Conclusion</p><p>Our results indicate that in addition to the tripeptide LDI/V motif, other regions of the V2 and V3 loops of gp120 were involved in binding to α4β7 receptors and this interaction was blocked by anti-V2 peptide, anti-V2 integrin, and anti-V3 antibodies. The ability of purified IgG from some of the uninfected RV144 vaccinees to inhibit α4β7 raises the hypothesis that anti-V2 and anti-V3 antibodies may play a role in blocking the gp120-α4β7 interaction after vaccination and thus prevent HIV-1 acquisition.</p></div

Demographic Characteristics of RV144 Participants with Clinical Dengue Episodes and Non-Dengue Serious Adverse Events (Controls).

<p>AE: Adverse event; SAE: Serious Adverse Event; SD: Standard Deviation</p><p>Demographic Characteristics of RV144 Participants with Clinical Dengue Episodes and Non-Dengue Serious Adverse Events (Controls).</p

Distribution of the Adverse Events (AEs) and Serious Adverse Events (SAEs) for Clinical Dengue Episodes and Other Infectious SAEs During RV144.

<p>*One subject had two episodes of dengue classified as SAEs.</p><p>Distribution of the Adverse Events (AEs) and Serious Adverse Events (SAEs) for Clinical Dengue Episodes and Other Infectious SAEs During RV144.</p

Geometric Mean Titer (GMT) ratio (Post/Pre) and 95% confidence interval (CI) for clinical dengue cases (both SAEs and AEs) and subjects with other infectious Serious Adverse Events (SAEs) displayed by serology dengue status.

<p>* Higher GMT ratios in clinical dengue subjects p<0.001; higher GMT ratios in subjects with clinical dengue cases and positive serology</p><p>** p = 0.037.</p><p>Geometric Mean Titer (GMT) ratio (Post/Pre) and 95% confidence interval (CI) for clinical dengue cases (both SAEs and AEs) and subjects with other infectious Serious Adverse Events (SAEs) displayed by serology dengue status.</p

Distribution of the number of clinical dengue episodes over the months of the year during the RV144 trial between July 2004 and November 2008.

<p>Distribution of the number of clinical dengue episodes over the months of the year during the RV144 trial between July 2004 and November 2008.</p

Regional distribution of HIV-1 subtypes and recombinants.

<p>The relative proportion of each subtype/recombinant is shown in the pie chart for each country. The data on Figure 1 are from the former studies in the region and estimates made by AVAN Task Force members of the manuscript based on the unpublished data of their own research projects.</p

Frequency of symptoms and biological parameters evoking dengue and recorded as clinical dengue episodes with a positive dengue serology.

<p>Frequency of symptoms and biological parameters evoking dengue and recorded as clinical dengue episodes with a positive dengue serology.</p

AIDS Vaccine Trials across Asia to Date.

<p>CDC, Center for Disease Control and Prevention; DoD, Department of Defense; EU, European Union.</p

Amino Acid Sequences for V2 and V3 Peptides.

<p>Amino Acid Sequences for V2 and V3 Peptides.</p

RPMI8866 cell titration curves and inhibition of binding of α4β7 integrin receptor to MAdCAM-1.

<p>(<b>A</b>) The top of the Fig shows a schematic representation of the experiment. Varying numbers of RPMI8866 cells in media were added to 96-well U bottom plates followed by the addition of AlamarBlue^® dye. Cell numbers ranged from 800–400,000 cells per well. The plate was incubated at 37°C in a CO₂ incubator. Fluorescence was measured for 8 hours at 1 hour intervals using a M2 plate reader (excitation 560 nm; emission 590 nm). The data are plotted as relative fluorescence units as a function of time. The average ± S.D. of six replicates for each time point are shown. (<b>B</b>) Schematic of the assay set up. RMPI8866 cells were incubated with media only (solid circles) or with ACT-1 (0.5 μg/well; open circles, left panel) and then added to MAdCAM-1 (natural ligand of α4β7 integrin; 0.2 μg/well) coated plates. The plates were washed and 100 μl of media and 10 μl of AlamarBlue^® dye were added to each well. Fluorescence was measured immediately after the addition of the dye (time 0) and then at 2 hours intervals for 8 hours. The percent inhibition at the 8 hour time point is shown. The data are plotted as relative fluorescence units as a function of time and represent the average ± SEM of 12 experiments done in triplicate.</p