Protection of CD4-IgG2-treated rhesus macaques in a high-dose SIVmac239 challenge experiment.

<p>To maintain serum concentrations, CD4-IgG2 (or control human polyclonal IgG) was administered subcutaneously over a two-week period by an ALZET osmotic pump. Animals were challenged intrarectally with a single high dose inoculum (3–5×10³ TCID₅₀) of SIVmac239 3-days after initiation of CD4-IgG2 administration. (<b>A</b>) Viral loads for animals treated with 200 mg of control polyclonal human IgG as a function of time following SIVmac239 challenge. All control animals became infected. (<b>B</b>) Viral loads for animals administered 20 mg CD4-IgG2 as a function of time following SIVmac239 challenge. Three out of 6 animals were fully protected and one infected animal showed delayed primary viremia. Due to a technical problem with the ALZET osmotic pump, one of the protected animals (98045) did not receive the full dose of 20 mg but this animal did not become infected. (<b>C</b>) Viral loads for animals administered 200 mg CD4-IgG2 as a function of time following SIVmac239 challenge. Five out of 7 animals were protected and showed no sign of infection at any time point. The minimum detection level was 125 SIV RNA copies/ml with a 95% confidence level. Open symbol indicates protected animal, closed symbol indicates infected animal.</p

Plasma concentration of CD4-IgG2 in treated animals.

<p>Animals administered 200 mg of CD4-IgG2 showed plasma concentrations in the range of 500 to 1400 ng/ml at the time of challenge. No apparent correlation between plasma concentration and protection was observed. The CD4-IgG2 concentration at the time of challenge in animals administered 20 mg was 100 ng/ml for 1 animal and below the limit of detection (8 ng/ml) for 2 animals. Serum samples from the remaining 3 animals administered 20 mg were unavailable for this analysis.</p

Anti-human CD4 response in animals treated with CD4-IgG2.

<p>Animal sera were tested in a human CD4-specific ELISA to detect macaque antibody responses against CD4-IgG2. Serum samples were tested up to 23 days post-viral challenge and no responses were detected before day 15, indicating that the animal protection outcome was independent of a response against human CD4. Serum samples from 3 animals administered 20 mg CD4-IgG2 were unavailable for this analysis.</p

IDEPI: Rapid Prediction of HIV-1 Antibody Epitopes and Other Phenotypic Features from Sequence Data Using a Flexible Machine Learning Platform

<div><p>Since its identification in 1983, HIV-1 has been the focus of a research effort unprecedented in scope and difficulty, whose ultimate goals — a cure and a vaccine – remain elusive. One of the fundamental challenges in accomplishing these goals is the tremendous genetic variability of the virus, with some genes differing at as many as 40% of nucleotide positions among circulating strains. Because of this, the genetic bases of many viral phenotypes, most notably the susceptibility to neutralization by a particular antibody, are difficult to identify computationally. Drawing upon open-source general-purpose machine learning algorithms and libraries, we have developed a software package IDEPI (IDentify EPItopes) for learning genotype-to-phenotype predictive models from sequences with known phenotypes. IDEPI can apply learned models to classify sequences of unknown phenotypes, and also identify specific sequence features which contribute to a particular phenotype. We demonstrate that IDEPI achieves performance similar to or better than that of previously published approaches on four well-studied problems: finding the epitopes of broadly neutralizing antibodies (bNab), determining coreceptor tropism of the virus, identifying compartment-specific genetic signatures of the virus, and deducing drug-resistance associated mutations. The cross-platform Python source code (released under the GPL 3.0 license), documentation, issue tracking, and a pre-configured virtual machine for IDEPI can be found at <a href="https://github.com/veg/idepi" target="_blank">https://github.com/veg/idepi</a>.</p></div

IDEPI performance, measured by MCC, as a function of the number of model features.

<p>(A): on a representative of each of the four classification problems, (B): on predicting resistance to a particular broadly neutralizing monoclonal antibody. Abbreviations: NVP - Nevirapine; DRAM - drug resistance associated mutations; HAD - HIV associated dementia; bNab - broadly neutralizing antibody. The optimal number of features is highlighted with a filled circle for each line plot.</p

IDEPI workflow.

<p>Abbreviations: MSA - multiple sequence analysis; mRMR - minimum redundancy maximum relevance; SVM - support vector machine.</p

IDEPI performance in predicting phenotype and recovering features from simulated data.

<p>Forward feature selection (to optimize MCC), and 10-fold nested cross-validation were used to learn the models. <b>L</b>: the number of sites in an epitope; <b>M</b>: how many escape mutations are needed to confer resistance; epitope recover classes are based on simulated evolutionary rates; <b>FP</b>: mean (per replicate) number of selected features not in a simulated epitope; a feature was counted as recovered if it were selected in 50% or more of cross-validation replicates.</p><p>IDEPI performance in predicting phenotype and recovering features from simulated data.</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

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

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