Results from robustness experiments.

<p>Average fold difference between ID₅₀ values calculated from robustness experiments that varied the number of cells that were used in the assay. Assays were performed using the optimal cell number, twice the optimal cell number, four times the optimal cell number, one-half of the optimal cell number, and one-quarter of the optimal cell number. The ID₅₀ values for each condition were required to remain within 3-fold of the ID₅₀ values generated by the optimal cell number.</p

Example of a luminometer validation curve.

<p>Each month the values of a NIST-traceable-calibrated validation plate are plotted on a graph. The graphs are then superimposed and the values must remain within 10% of the established baseline (mean of the 20 initial runs).</p

Example of a DEAE-Dextran titration curve using two pseudoviruses.

<p>The optimal concentration of DEAE-Dextran (x-axis) to use in the TZM-bl NAb Assay is calculated by selecting a concentration lower than the concentration yielding the peak RLU values on both titration curves of two pseudoviruses (in this instance QHO692.42, CAAN5342.A2). By picking the concentration lower than the peak, one avoids potential cell toxicity that may result with the use of other pseudoviruses. The vertical dotted line in the graph represents the concentration of DEAE-Dextran that maximizes the infectivity of the pseudovirus without being toxic to the TZM-bl cells.</p

CAVD/CA-VIMC Regional Laboratories that completed the Assay Implementation Plan.

<p>CAVD/CA-VIMC Regional Laboratories that completed the Assay Implementation Plan.</p

Pictorial representation of the TZM bl NAb Assay.

<p>Briefly, pseudovirus infection of TZM-bl Cells stimulates expression of Luciferase Reporter gene thereby emitting luminescence (A). When the pseudovirus is neutralized prior to the infection of TZM-bl Cells, the Luciferase reporter gene is not expressed and no luminescence is emitted (B).</p

Results from precision experiments: inter-operator and inter-assay.

<p>(A) Results show the %CV between the ID₅₀ values generated by two operators carrying out the identical experiment on the same days. Each point represents one sample/pseudovirus combination. (B) Results show the %CV between ID₅₀ values generated from identical experiments conducted by the same operator on 3 different days. Each point represents one sample/pseudovirus combination.</p

Example of Neutralization Curve.

<p>The neutralization curve depicts the linear portion of the curve between 20% and 80% neutralization.</p

Maximum likelihood phylogenetic analysis of southern African clade C acute/early envelope nucleotide sequences (n = 200) (Table 1).

<p>Branches are colored according to country/region. Bootstrap values > 80% of 100 resampled replicates are illustrated as filled circles on nodes. South African samples from Soweto/Johannesburg (Gauteng province), Cape Town (Western Cape Province), and KwaZulu-Natal are highlighted in light green, red, and blue respectively, sequences from other locations in South Africa are shown in grey. Clades that were from the same geographic region are highlighted. Only one of these regional grouping (>2 sequence clusters) had strong bootstrap support (4 sequences from Tanzania, highlighted in orange).</p

Comparison of neutralization susceptibility and viral characteristics of pre- and post-seroconversion clade C panel viruses.

<p>Pseudotyped clade C viruses were partitioned into three infection stage groups according to their sequential gain of HIV-1 specific antibody responses as a marker of time from infection; pre-seroconversion (Ab-) (n = 58) indicated in blue, indeterminate (Ab-/+) (n = 26) indicated in grey and post-seroconversion (Ab+) (n = 55) indicated in red. Each point represents the geometric mean of viruses tested for neutralization sensitivity using a panel of 30 South African sera. (A) Serum neutralization potency as measured by GMT, over the 30 sera/plasma included in this study; sera below the threshold of detection (dilutions of 1:20 was the limit tested) were given the value of 10. The two subgroups evident among the pre-seroconversion viruses did not cluster phylogenetically. One-sided Wilcoxon rank sum test employed with median values shown in red and interquartile ranges in black (B) Neutralization breadth per infection stage against 30 clade C serum samples measured as the percentage of viruses neutralized at ID₅₀ > 1:20. Three very sensitive viruses, two tier 1A (SO032_A2.8–1, CH0505.w4.3) and one tier 1B, (6644.v2.c33), were excluded from the analysis. (C), (D) V1V2 loop amino acid length variation and glycan density per infection stage. Mann-Whitney two-sided tests used in panel B, C and D, with median values shown in red and interquartile ranges in black. Uncorrected p-values <0.05 are provided, however as four comparisons were made, only p-values <0.015 should be considered significant.</p

Characteristics of the acute/early clade C panel virus donors by infection stage and country/region of origin.

<p>Characteristics of the acute/early clade C panel virus donors by infection stage and country/region of origin.</p