Panels of HIV-1 Subtype C Env Reference Strains for Standardized Neutralization Assessments

Abstract

In the search for effective immunologic interventions to prevent and treat HIV-1 infection, standardized reference reagents are a cost-effective way to maintain robustness and reproducibility among immunological assays. To support planned and ongoing studies where clade C predominates, here we describe three virus panels, chosen from 200 well-characterized clade C envelope (Env)-pseudotyped viruses from early infection. All 200 Envs were expressed as a single round of replication pseudoviruses and were tested to quantify neutralization titers by 16 broadly neutralizing antibodies (bnAbs) and sera from 30 subjects with chronic clade C infections. We selected large panels of 50 and 100 Envs either to characterize cross-reactive breadth for sera identified as having potent neutralization activity based on initial screening or to evaluate neutralization magnitude-breadth distributions of newly isolated antibodies. We identified these panels by downselection after hierarchical clustering of bnAb neutralization titers. The resulting panels represent the diversity of neutralization profiles throughout the range of virus sensitivities identified in the original panel of 200 viruses. A small 12-Env panel was chosen to screen sera from vaccine trials or natural-infection studies for neutralization responses. We considered panels selected by previously described methods but favored a computationally informed method that enabled selection of viruses representing diverse neutralization sensitivity patterns, given that we do not a priori know what the neutralization-response profile of vaccine sera will be relative to that of sera from infected individuals. The resulting 12-Env panel complements existing panels. Use of standardized panels enables direct comparisons of data from different trials and study sites testing HIV-1 clade C-specific products.IMPORTANCE HIV-1 group M includes nine clades and many recombinants. Clade C is the most common lineage, responsible for roughly half of current HIV-1 infections, and is a focus for vaccine design and testing. Standard reference reagents, particularly virus panels to study neutralization by antibodies, are crucial for developing cost-effective and yet rigorous and reproducible assays against diverse examples of this variable virus. We developed clade C-specific panels for use as standardized reagents to monitor complex polyclonal sera for neutralization activity and to characterize the potency and breadth of cross-reactive neutralization by monoclonal antibodies, whether engineered or isolated from infected individuals. We chose from 200 southern African, clade C envelope-pseudotyped viruses with neutralization titers against 16 broadly neutralizing antibodies and 30 sera from chronic clade C infections. We selected panels to represent the diversity of bnAb neutralization profiles and Env neutralization sensitivities. Use of standard virus panels can facilitate comparison of results across studies and sites.

Keywords: assay standardization; clinical trials; human immunodeficiency virus; immunoserology; neutralizing antibodies; vaccines.

FIG 1

Cumulative distributions of IC₅₀ neutralization titers from 16 bnAbs. Each line shows the proportion of 200 Envs, with IC₅₀s given by the value along the x axis. Gray lines at the lower and upper ranges of IC₅₀s indicate where censoring cutoffs differed among assays. Asterisks are intended to help locate the example of 3BNC117 censored at 20 and 50 μg/ml discussed in the text.

FIG 2

Heat maps of IC₅₀ neutralization titers from assaying 200 clade C envelopes against 16 bnAbs. (a) Hierarchically clustered heat map of IC₅₀ titers of 200 Envs against 16 bnAbs. The Env dendrogram is shown; the bnAb dendrogram is not shown. Leaf colors indicate 100 viruses included (red) or excluded (blue) by downselection. The histogram (black line) above the heat map summarizes the distribution of assay results, with histogram breakpoints at 10, 4.64, 2.15, 1.00, 0.464, 0.215, 0.10, 0.0464, 0.0215, and 0.01 μg/ml. Low IC₅₀s were censored at 0.01 μg/ml to standardize censoring thresholds across bnAbs. (b) 100-Env panel, downselected from alternating rows, i.e., red branches on the dendrogram. (c) 50-Env panel downselected by alternating over 100 Envs.

FIG 3

Magnitude-breadth curves (cumulative distribution functions) compare IC₅₀s from 100- and 50-Env panels (colored and gray lines, respectively) with those from all 200 Envs (black lines). Axes are scaled the same across all panels. Specificities for each bnAb are also listed. The curves are continuous distribution functions which indicate the proportion of viruses with IC₅₀ neutralization titers (expressed in micrograms per milliliter) no lower than the corresponding x axis value. Because the emphasis here is on comparisons within the same bnAb, the differences in censoring noted for Fig. 1 across different bnAbs are not relevant here. The differences are quantified in the supplemental material.

FIG 4

Comparison of Env sensitivity to neutralization by plasma samples and bnAbs. (a) Geometric mean plasma ID₅₀ per Env, stratified by the number of bnAbs with IC₅₀s below 10 μg/ml, from among the 16 tested. (b) Cumulative distribution of geometric mean ID₅₀s from 30 chronic plasma samples. Vertical lines indicate the 12 selected Envs. (c) Distribution of geometric mean ID₅₀s between 12-virus panel and nonpanel Envs. Five Envs with geometric mean ID₅₀s above 250 μg/ml are not shown. Colors in panels b and c summarize the number of neutralizing bnAbs shown in panel a.

FIG 5

Comparison of neutralization IC₅₀ titers between 12-Env panels. (a to c) Comparison of (a) clade C panel from 2006, (b) global panel (12), and (c) candidate clade C panel from the manuscript. As noted in the Fig. 1 legend, all assay results were censored above 10 and below 0.001 μg/ml to standardize dilution ranges across different experiments. NA, no data. Data for historical panels a and b were computed as geometric means from the CATNAP database, as detailed in the text. Env names in panels a and b are shortened per CATNAP, and panel c lists short names from Data Set S1.

FIG 6

Hierarchically clustered dendrogram of 200 tier 2 envelopes with heat map of neutralization ID₅₀s. The dendrogram was computed from squared Euclidean distance values using Ward's clustering method. Leaves (rows) were weighted by geometric mean neutralization titer for dendrogram layout. Colors indicate viruses selected for the candidate 12-Env panel (black). Panels defined by the automatic methods for lasso (red) and by k-medoids (blue) with k = 12 are also indicated. Other virus names are indicated in gray.