We characterized a large and diverse panel of HIV-1 Env pseudoviruses to better define the relative susceptibility of each isolate to NAbs and to identify subgroups of viruses that represent distinct categories of neutralization sensitivity. This panel represents a broad diversity of HIV-1 isolates in terms of genetic subtype, geographic distribution, and stage of infection, including viruses derived from acute HIV-1 infection. Importantly, this virus panel includes previously described reference panels of viruses, as well as isolates representative of clades and regions of the world that are epicenters of the AIDS epidemic. While some specific clades and CRFs are not well represented in this panel (i.e., clades D, G, and F and CRF01_AE), efforts are under way to clone and characterize HIV-1 Env pseudoviruses of these subtypes for utilization in NAb assays. Nonetheless, the existing panel provides a solid diversity of molecularly cloned viruses for characterizing the range of neutralization sensitivities of HIV-1 primary isolates and for defining subgroups of viruses for tiered NAb assessment. Furthermore, the known gp160 sequence of these viruses will facilitate efforts to map the epitope specificities of broadly neutralizing MAbs and sera from vaccinated or infected individuals and will serve as a foundation for future studies to delineate potential neutralization serotypes. Additional detailed information on these isolates and corresponding gp160 sequence data is being made available for reference on the Los Alamos National Laboratories website (www.hiv.lanl.gov/content/nab-reference-strains
), and designated reference clones will be made available to other investigators through the NIH ARRRP.
Our initial rank-ordering analysis demonstrates that many isolates in this panel exhibit a similar range of average NAb sensitivities, with a smaller proportion of viruses demonstrating a distinctly more sensitive or resistant phenotype. Most viruses were neutralized by each of the HIV-1+
plasma pools tested, although the degree of sensitivity to individual pools varied among isolates. The epitope specificities of NAbs represented in the majority of these plasma pools are unknown, although we have previously investigated the constituent samples of the clade B-Zepto and clade C-SA pools (4
). These studies found evidence of NAbs targeting the CD4bs of gp120 and, to a lesser extent, the membrane-proximal external region (MPER) of gp41; however, the majority of neutralizing activity could not be effectively mapped. While we did not detect any pronounced synergy or hindrance associated with the pooling of these individual plasma samples in the studies for this report, it should be noted that these experiments were not optimally designed to thoroughly investigate these questions.
Given the range of neutralization sensitivities observed for individual isolates within this panel, we employed clustering analysis as a method to distinguish four subsets of viruses representative of distinct tiers of neutralization sensitivity. The three isolates demonstrating the greatest sensitivity to neutralization (MW965.26, SF162.LS, and MN) form their own unique cluster (tier 1A) and have historically been utilized for tier 1 screening of candidate vaccine sera for minimal NAb activity. Importantly, our analysis has defined an additional subset of viruses with above-average sensitivities that we have termed tier 1B. These isolates will provide an important bridge in the tiered algorithm of testing between the hypersensitive tier 1A isolates and the much larger subgroup of tier 2 HIV-1 isolates that exhibit moderate sensitivity to antibody-mediated neutralization. To date, many candidate HIV-1 vaccine immunogens have been shown to elicit antibody responses that can neutralize tier 1A viruses, yet none have demonstrated the capacity to elicit neutralizing activity against most tier 2 reference viruses (8
). Thus, we recommend the inclusion of tier 1B isolates in the screening of candidate vaccine sera as a means to better detect and identify incremental advances in immunogen design that result in NAbs with increasing breadth and potency. The majority of viruses in our panel clustered into the tier 2 category and thus may be a fair representation of the average neutralization sensitivity of circulating strains of HIV-1. Utilization of these viruses in the second tier of testing will provide important insight into the potency and cross-reactive breadth of NAbs elicited by candidate vaccine immunogens. We also identified a subgroup of tier 3 viruses that were unusually resistant to neutralization by one or more of the HIV-1+
plasma pools. These isolates will provide an additional level of stringency for assessing the potency of candidate vaccine sera which demonstrate NAb activity against tier 2 isolates.
We investigated assay parameters in addition to the ID50
titer for the rank-ordering and clustering analysis of our HIV-1 Env pseudovirus panel. In particular, pAUC was found to be an informative measure of NAb activity that provided rank order and clustering results that were highly concordant with those of our analyses based on NAb ID50
titer. Furthermore, we investigated the robustness of our hierarchical clustering assignments by introducing measures of laboratory assay variance and random resampling with replacement of results for bootstrap comparisons. These analyses demonstrate that the large majority of viruses cluster into their designated tier category with ≥80% probability. While particular viruses were ambiguous in tier assignment with the introduction of these variables, the results support the general categorization of these isolates into four tiered subgroups based on neutralization sensitivity. Alternate approaches for identifying the number of clusters (e.g., the method of Calinski and Harabasz [10
]) and for assigning viruses to clusters (e.g., k
-medoids, agglomerative hierarchical clustering, and divisive hierarchical clustering) show some variability in both the number of clusters identified and virus cluster assignments depending on the methods used for analysis. We report the results using k
-means, a partitional clustering method, but other partitional clustering methods produced very similar results.
The previous studies which led to the recommended standard reference panels of clade B and clade C HIV-1 isolates incorporated NAb phenotyping using four broadly neutralizing MAbs and a limited number of HIV-1+
serum samples (22
). While these data were useful in selecting viruses which fit the general criteria of not being overly sensitive or resistant to NAbs, it was not clear that these viruses reflected the range of neutralization sensitivities observed in the larger context of globally diverse HIV-1 isolates. In this study, we have applied a more rigorous analytical assessment of the neutralization sensitivities of these commonly used reference viruses. Our results demonstrate that these standard reference panels, as well as several clade A and transmitted/founder clade B viruses, represent a broad distribution in neutralization rank order. While the majority of isolates in these panels were categorized as tier 2 viruses, each panel had one or two viruses that were categorized as tier 1B. The standard clade B reference panel also contains two viruses that were categorized as tier 3 isolates (PVO.4 and TRJO4551.58). Thus, our data support the continued use of these standard reference panels for the testing of candidate vaccine sera. Having defined the relative neutralization sensitivity rank order of viruses within each reference panel will also facilitate the interpretation of results when these panels are employed.
We have also investigated whether specific virus characteristics are associated with neutralization sensitivity. No associations were found with the stage of infection or the source of material from which the gp160 Env gene was cloned. Notably, there was no evidence that the transmitted viruses were different than other viruses in overall neutralization sensitivity. We did observe a significant trend toward increased neutralization sensitivity when the virus and HIV-1+
plasma pool were of the same clade, supportive of previous such observations (7
). However, it should be noted that this was determined using a limited number of matched virus isolates and plasma pools. To investigate whether this clade match effect influenced our rank ordering and clustering results, these analyses were repeated after the matching clade log10
titers were removed from the average. In fact, we found that exclusion of these data did not greatly affect our results (data not shown).
Some HIV-1-specific MAbs can only neutralize viruses known to have a highly neutralization-sensitive phenotype (5
). We therefore evaluated two such MAbs, MAb 1.5E directed to the CD4bs and MAb 17b directed to the coreceptor binding site. Both MAbs could neutralize viruses within the tier 1A and 1B categories but were ineffective against viruses in the tier 2 and 3 categories. Since our virus categorization was done with HIV-1+
plasma and not MAbs, these results confirm that the neutralization capacity of some MAbs is restricted to a small subset of particularly neutralization-sensitive viruses. Our data were similar for the anti-V3 MAb 447, which neutralized primarily within the tier 1A and 1B categories of viruses. These data are consistent with a model suggesting that, for most virus isolates, the conformation of the native Env trimer acts to restrict access to coreceptor and CD4 receptor epitopes on gp120 (20
) and that the V3 loop region is not well exposed on most primary isolates of HIV-1 (5
). Thus, it is possible that the heightened susceptibility of tier 1A and the more sensitive tier 1B viruses to these MAbs and to NAbs in general may be related to an enhanced open configuration of the Env trimer on the virion surface which makes these epitopes more accessible for antibody binding.
In summary, this study provides the first analytically derived definition of tiered neutralization phenotypes of HIV-1 isolates. We used a set of varied plasma pool reagents to characterize the overall neutralization sensitivities of a diverse panel of more than 100 HIV-1 Env pseudoviruses. Viruses were collected from diverse geographic regions and stages of infection and represented most of the major circulating genetic subtypes of HIV-1 worldwide. Viruses could be categorized into four groups, designated tiers 1A, 1B, 2, and 3, with the majority of viruses belonging to tier 2. This viral panel will facilitate the full characterization of novel MAbs and will allow a careful and systematic assessment of the potency and breadth of neutralization by vaccine sera. The known Env sequences and clonal nature of these Env pseudoviruses will also allow investigation of the viral epitopes that contribute to neutralization by HIV+ plasma, vaccine sera, and MAbs.