Plasma viral loads following SHIV_SF162P3 vaginal challenge of 2G12-treated and control macaques.

<p>A total of nine female Indian rhesus macaques were divided into treatment groups of five animals for <i>i.v.</i> administration of 2G12, two animals to receive the isotype control (Dengue anti-NS1, DEN3), and two additional controls were challenged prior to the beginning of the protection study to confirm viral fitness, but were not treated with antibody. In (A) two 2G12-treated (40 mg/kg) animals became infected: 90154 reached peak viremia of 2×10⁷ on day 21 similar to controls; 95113 showed a one-week delay of infection onset and peak viremia was lower at 5×10⁶. The remaining three 2G12-treated animals were protected against infection and showed no measurable viremia. In (B) all 4 control animals experienced peak viremia between 1×10⁷ and 4×10⁷ on day 21. The quantity of SIV viral RNA genomic copy equivalents (vRNA copy Eq/ml) in EDTA-anticoagulated plasma was determined using quantitative RT PCR <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000433#ppat.1000433-Friedrich1" target="_blank">[52]</a>. The assay minimum detection is 150 copies of vRNA Eq/ml (2.1 log) with a 99% confidence level.</p

Half-life of transferred 2G12 in macaque serum.

<p>The data represents the half-life (t_1/2) of serum 2G12 determined from data in three different ELISA formats over a period of three weeks following <i>i.v</i>. transfer of 40 mg/kg of 2G12. The half-life of transferred 2G12 ranged between 7.2 and 15.6 days in the 5 macaques with a somewhat shorter half-life observed in animal 01038. The average half-life of all animals as measured in the three ELISA formats is about 11 days. The half-life of 2G12 in rhesus macaques has previously been noted as about 13 days <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000433#ppat.1000433-Mascola1" target="_blank">[5]</a>.</p

Comparison of SHIV neutralization by b12 and 2G12 in rhesus PBMC-based and pseudovirus luciferase reporter gene assays.

<p>The selection of SHIV_SF162P3 for the protection study was based on the results of 2G12 neutralization of rhesus PBMCs and pseudovirus assays against the panel shown.</p

Comparison of b12 and 2G12 transudated to the vagina following intravenous administration.

<p>Each antibody treatment group consisted of three female Indian Rhesus macaques which were <i>i.v.</i>-administered 5 mg/kg of either b12 or 2G12 following Depo-provera treatment. Vaginal secretions from each animal were absorbed to cellulose wicks. A set of 3 samples per animal was taken at 6 hours, 12 hours, 24 hours, 4 days, and 7 days post <i>i.v.</i> antibody administration. The concentration of antibody in mucosal secretions was determined by ELISA from the clarified supernatant extracted from the wicks. Resulting data were compared to the corresponding antibody standard curve using nonlinear regression. Arithmetic means and standard deviations were calculated for each set of triplicate samples per animal. Data points were calculated from all animals at each timepoint and error bars represent the standard error of means. The typical time for viral challenge in protection experiments is indicated. The differences in the mean concentrations of b12 and 2G12 at each timepoint were evaluated in a student's t test and determined to be non-significant. Analyses performed in GraphPad Prism Software for Mac, Version 5.0a.</p

Comparison of antibody-dependent cell-mediated viral inhibition (ADCVI) by 2G12 and b12.

<p>Target cells (CEM.NKR-CCR5) were infected with SHIV_SF162P3 and incubated for 48 hours, washed to remove cell-free virus and combined with Rhesus PBMC effector cells and serially diluted antibody. Viral inhibition was measured after incubation for 7 days. 2G12 is somewhat less effective than b12 in mediating ADCVI for a strict concentration comparison. An unpaired Two-tailed t test (P = 0.3285) of b12 and 2G12 ADCVI with an F test comparison of variance reveals no significant difference (P = 0.4154). Analysis performed in GraphPad Prism Software for Mac, Version 5.0a.</p

MHC genotyping of macaques against MHC Class Ι alleles.

<p>Macaque samples were tested against a panel of nine MHC class Ι alleles that have previously been shown to be important in SIV epitope presentation or increased resistance to SIV infection <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000433#ppat.1000433-Yant1" target="_blank">[24]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000433#ppat.1000433-Kaizu1" target="_blank">[25]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000433#ppat.1000433-Loffredo1" target="_blank">[26]</a>. The alleles are: <i>Mamu-A*01</i>, <i>Mamu-A*02</i>, <i>Mamu-A*08</i>, <i>Mamu-A*11, Mamu-B*01</i>, <i>Mamu-B*03</i>, <i>Mamu-B*04, Mamu-B*08, and Mamu-B*17</i>. Animal 90140 is positive for <i>Mamu-A*01</i> and animal 95066 was determined to carry the <i>Mamu-B*01</i> allele. <i>Mamu-A*01</i> has been associated with moderate control of SIVmac239 replication <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000433#ppat.1000433-Zhang1" target="_blank">[29]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000433#ppat.1000433-Mothe1" target="_blank">[30]</a>. <i>Mamu-B*01</i> remains on the panel based on early reports of SIV-derived epitopes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000433#ppat.1000433-Yasutomi1" target="_blank">[31]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000433#ppat.1000433-Su1" target="_blank">[32]</a>, but subsequent studies show that <i>Mamu-B*01</i> does not bind SIV-derived epitopes and has no effect on SIV disease progression <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000433#ppat.1000433-Loffredo1" target="_blank">[26]</a>. <b>^{P, N, I, C}</b> denotes protected, non-protected, isotype control, and non-antibody treated control animals, respectively.</p

2G12 serum antibody concentrations following passive administration.

<p>The concentrations of transferred 2G12 in the serum of all experimental animals on the day of challenge (day 0) and during the following three weeks were determined by ELISA using three different formats. For each animal, the results from the different ELISA formats are shown in separate columns. The concentrations of 2G12 in the macaque sera were determined from the measurement of binding to monomeric JR-FL (gp120), to an immobilized synthetic oligomannose dendron conjugated to BSA <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000433#ppat.1000433-Wang1" target="_blank">[21]</a> (Man4D), and to a highly specific anti-idiotype-2G12 antibody (MIgG1 L13) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000433#ppat.1000433-Roux2" target="_blank">[23]</a> (anti-id). In all formats, a dilution series of serum was compared to a 2G12 standard curve and the concentration determined using a nonlinear regression curve fit analysis performed in GraphPad Prism Software for Mac, Version 5.0a.</p

Pre-existing neutralizing antibody mitigates B cell dysregulation and enhances the Env-specific antibody response in SHIV-infected rhesus macaques

<div><p>Our central hypothesis is that protection against HIV infection will be powerfully influenced by the magnitude and quality of the B cell response. Although sterilizing immunity, mediated by pre-formed abundant and potent antibodies is the ultimate goal for B cell-targeted HIV vaccine strategies, scenarios that fall short of this may still confer beneficial defenses against viremia and disease progression. We evaluated the impact of sub-sterilizing pre-existing neutralizing antibody on the B cell response to SHIV infection. Adult male rhesus macaques received passive transfer of a sub-sterilizing amount of polyclonal neutralizing immunoglobulin (Ig) purified from previously infected animals (SHIVIG) or control Ig prior to intra-rectal challenge with SHIV_SF162P4 and extensive longitudinal sampling was performed. SHIVIG treated animals exhibited significantly reduced viral load and increased <i>de novo</i> Env-specific plasma antibody. Dysregulation of the B cell profile was grossly apparent soon after infection in untreated animals; exemplified by a ≈50% decrease in total B cells in the blood evident 2–3 weeks post-infection which was not apparent in SHIVIG treated animals. IgD+CD5+CD21+ B cells phenotypically similar to marginal zone-like B cells were highly sensitive to SHIV infection, becoming significantly decreased as early as 3 days post-infection in control animals, while being maintained in SHIVIG treated animals, and were highly correlated with the induction of Env-specific plasma antibody. These results suggest that B cell dysregulation during the early stages of infection likely contributes to suboptimal Env-specific B cell and antibody responses, and strategies that limit this dysregulation may enhance the host’s ability to eliminate HIV.</p></div

High-resolution longitudinal B cell phenotypic profile.

<p>Heatmap shows data corresponding to the change (log2) from baseline value for each subset frequency at each time point. Paired two tailed t-test for all post-baseline time points vs. baseline was computed independently for NIgG and SHIVIG groups; populations shown had at least one significant time point p<0.01 compared to baseline. Subsets were clustered hierarchically based on Euclidean distance and complete linkage. Magenta subset label indicates a significant difference of at least one time point for the log2 change from baseline between NIgG and SHIVIG groups at p<0.01 as determined by two-tailed unpaired t-test. [X] indicates subset is measured as the frequency of X parent population, otherwise parent population is total CD19+CD20+ B cells. Rectangle outline is used to emphasize select subsets.</p

Effect of passively transferred neutralizing IgG on plasma viral load and CD4⁺ T cells in SHIV_SF162P4 infected macaques.

<p>Male rhesus macaques were treated with NIgG (n = 4) or SHIVIG (n = 5) and blood samples collected at regular intervals after viral exposure. (<b>A</b>) RNA was isolated from plasma, and viral SHIV RNA was quantified by RT-PCR. (<b>B</b>) CD4⁺ T cell count was determined by flow cytometry and normalized to percentage of baseline value. Baseline value was defined as the average value of the -1 w.p.i and either -1 day p.i. (NIgG group) or 0 day p.i. (SHIVIG group). Symbols represent group mean±SEM. * indicates significant difference (p<0.05) between groups at indicated time point as determined by two-tailed t-test.</p