Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses

Fab and FcR binding.

<p>(<b>A</b>) Linear cross-clade epitope mapping of 7B2 IgG1_AAA by peptide microarray. FcR binding (response units), on-rate (ka) and off rate (kd) by Surface Plasmon Resonance (SPR) of 7B2 IgG1_AAA. (<b>B</b>) Fine mapping of the 7B2 epitope within the gp41 immunodominant loop. Top Graph shows the binding response at saturation (~140 seconds after starting injection of 7B2 Fab) of each Ala-substituted gp41_596-606 peptide normalized to wild type and the middle graph shows the normalized off-rate of the same peptides. Data are representative of at least two measurements on adjacent spots in the same sensor chip. Residues that are part of the 7B2 epitope are colored in orange. The Lys601Ala mutant peptide is highlighted in green since it gave a higher binding response and a decreased off-rate. Bottom graph is an example of sensogram showing 7B2 Fab binding to WT and select Alanine mutant gp41_596-606 peptides that were used to generate the top and middle graphs. (<b>C</b>) Binding between 7B2 and gp41 peptides in standard and reducing conditions. (<b>D</b>) The structure of the 7B2 Fab-gp41 peptide complex shows detailed polar interactions. Hydrogen bonds between functional groups in the peptide and the heavy chain of the Fab are indicated. (<b>E</b>) Comparison of the gp41 ID loop from our structure (far left) against its structure obtained from NMR (middle left) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005042#ppat.1005042.ref038" target="_blank">38</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005042#ppat.1005042.ref039" target="_blank">39</a>] and its conformation as shown in the BG505.SOSIP.664 structure (middle right) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005042#ppat.1005042.ref015" target="_blank">15</a>] superimposed against the 7B2 paratope. A superposition of all three ID conformations (far right) highlights the conformational variability of this region.</p

Data collection and refinement statistics for the 7B2 Fab-gp41 _596–606 structure.

<p>^a The crystal had two Fab-peptide complexes in the asymmetric unit. The dataset came from a single crystal.</p><p>^b Values in parentheses are for the highest resolution shells.</p><p>Data collection and refinement statistics for the 7B2 Fab-gp41 _596–606 structure.</p

7B2 IgG1_AAA, A32 IgG1_AAA and CH22 IgG1_AAA mAb concentrations in (A) plasma and (B) rectal secretions.

<p>Concentrations of mAb were measured by a binding assay with the infused antibody as a control for calculating concentration equivalents of Ab binding to Env protein (μg/ml). Visible red blood cells in the rectal weck elutions were observed at time points post infusion for some animals.</p

mAb concentrations at time of challenge.

<p>The average and range of antibody concentrations in plasma and rectal secretions from the PK study are indicated for the times that the subsequent infusion-challenge study was performed.</p><p>mAb concentrations at time of challenge.</p

Binding rate constants and affinities of rhesus FcγR3 binding to mAbs.

<p>Antibodies were measured for rhesus FcR binding by surface plasmon resonance (SPR). Mean and standard deviation from 3 independent assays are indicated where available.</p><p>Binding rate constants and affinities of rhesus FcγR3 binding to mAbs.</p

Viral loads and CD4 T cell counts following high dose SHIV BaL rectal challenge in rhesus macaques passively infused with 7B2 IgG_AAA, A32 IgG_AAA or CH22 IgG_AAA.

<p><b>(A)</b> Plasma viral RNA levels and (<b>B)</b> CD4 T cell counts in 7B2 IgG_AAA and palivizumab IgG treated rhesus monkeys following challenge with SHIV-BaL. (<b>C</b>) Plasma viral RNA levels and (<b>D</b>) CD4 counts in A32 IgG_AAA mAb and control palivizumab IgG mAb passively infused rhesus monkeys following challenge with SHIV-BaL. (<b>E)</b> Plasma viral RNA levels and (<b>F)</b> CD4 Counts in rhesus monkeys following challenge with SHIV-BaL after passive administration of CH22 or CH65 IgG mAbs.</p

7B2 mAb captures infectious SHIV BaL and SF162.

<p>SHIV BaL and SHIV SF162 virus capture by 7B2 and controls were measured by either <b>(A)</b> a plate–based capture assay (relative luciferase unit (RLU) on day 7 post infection shown) <b>(B)</b> or a column-based assay (% virus capture based on SIV <i>gag</i> viral RNA measurement for rVirion and RLU infectivity for iVirion percentages, respectively. The error bar is the SEM of three wells replicates. The dashed line is the positivity cutoff. The level of virion capture in the presence (red bars) and absence (blue bars) of soluble CD4 for HIV-1 SF162 <b>(C)</b> and HIV- BAL <b>(D)</b> are shown. Non-neutralizing mAb A32 and a neutralizing mAb 2G12 were used as negative and positive controls, respectively. Error bars show mean ± SEM from 3 separate experiments. <b>(E)</b> 7B2_AAA does not inhibit infection of rectal explants (gray) but does inhibit infectious transfer from migratory cells that emigrate from mucosal tissue (at the highest concentration (50 μg) (blue). This is reflective of CD4+ T cells being the primary targets of infection. Results are the average of two experiments.</p

Number of Transmitted/Founder (T/F) viruses.

<p>^AMinimum estimates of the number of T/F viruses for 7B2 IgG and palivizumab IgG treated rhesus macaques are shown. At a mean of 60 sequences per animal, there is 95% confidence that all variants that are >5% in prevalence have been enumerated.</p><p>*The difference in T/F variants between the 7B2 IgG_AAA treated animals and the negative control animals palivizumab IgG was statistically significant, p = 0.01; Mann-Whitney rank sum test, two tailed.</p><p>^B Minimum estimates of the number of T/F viruses for A32 IgG_AAA mAb and palivizumab IgG mAb treated rhesus macaques are shown. At a mean of 37 sequences per animal, there is 95% confidence that T/F variants with >8% prevalence in the population are represented.</p><p>*The difference in T/F variants between the A32 IgG_AAA treated animals and the negative control animals palivizumab IgG was statistically significant, p = 0.033; Mann-Whitney rank sum test, two tailed. ^C Minimum estimates of the number of T/F viruses for CH22 IgG_AAA mAb and CH65 IgG mAb treated rhesus macaques.</p><p>*The difference in T/F variants between the CH22 IgG_AAA treated animals and the negative control animals CH65 IgG was statistically significant, p = 0.011; Mann-Whitney rank sum test, two tailed.</p><p>Number of Transmitted/Founder (T/F) viruses.</p