According to the calculations described herein, V3 loop neutralization epitopes recognized by either mAbs 447-52D or 2219 are conserved in 37% of HIV-1 viruses infecting patients worldwide despite the sequence variation observed in the V3 loop. This implies that a vaccine capable of generating both the 447-52D- and 2219-like antibodies in humans would potentially be capable of neutralizing 37% of viruses worldwide, across all subtypes. However, realizing this potential would mean that all HIV-1 isolates worldwide would present the V3 loop to the human antibody response with accessibility that is comparable to SF162. In reality, the accessibility of the V3 loop in worldwide isolates is highly variable and, unfortunately, most of the time V3 is less available (more masked) than it is in the SF162 envelope, at least partly due to glycosylation of the envelope. Thus, the actual percentage of worldwide isolates that could be neutralized by these antibodies is likely to be much lower than that represented by these calculations. It remains to be seen to what extent these masking effects can be overcome by the induction of relatively high levels of these antibodies and/or the induction of antibodies with higher affinities. Nevertheless, we cannot neutralize an epitope that isn't there, so this study establishes a rational baseline for the comparative utility of these antibodies, and the method described can be applied to other epitopes recognized by neutralizing mAbs.
Thus, using data generated from bioinformatics, crystallography, epidemiology, and viral neutralization studies, we developed an approach for measuring the degree of conservation of neutralization epitopes in a variable region of the gp120 envelope glycoprotein of diverse, globally relevant strains of HIV-1. This first version of the method depends on the fidelity of several techniques. The percentages we have calculated are only first estimates that may very well underestimate or overestimate the breadth of 447 and 2219, and the accuracy of the method may be improved in subsequent versions.
First, although a major advantage of this method is that it reduces a complex structural interaction to a sequence motif, V3 loop crown structures may contain a sequence motif, but due to backbone folding, may not fit the combining site of some antibodies, and vice versa (ones that do not contain the sequence motif but fold into a perfect shape for binding through weaker contacts). Indeed, some of the outlier sequences in have been determined to be resistant to neutralization due to folding effects despite the presence of the sequence motif (data not shown). Methods to incorporate backbone effects may improve the estimates.
Second, the method depends on neutralization assays and correlates 3D structural observations directly with neutralization patterns without using potentially noisy V3 loop-antibody binding data as an intermediary. However, some Q315 V3 loop peptides have been observed to bind 447-52D,7
and a few are neutralized relatively well compared to the average for Q315 viruses. A better understanding of the relationship between V3 loop-antibody binding observations and V3 loop-mediated neutralization may provide further refinement of the motif to include some Q315 viruses in the definition of the 447-52D epitope. In addition, although the SF162 chimeric pseudovirus system used partitioned the data sufficiently in this case, global interactions between non-V3 and V3 positions in the gp120 monomer and trimer may still have biased these results. A better understanding of these effects may improve the results.
Third, the LANL database is a biased representation of the worldwide distribution of HIV-1 isolates. Improvements in this database to reduce sampling bias may increase the precision of the estimates derived by our method.
Fourth, the method depends on the accuracy of epidemiologic estimates of the global distribution of subtypes. As these estimates become more precise and/or change over time due to virus evolution,12
the epidemiologic relevance of these calculations may improve. Indeed, greater detail may result from calculating the distribution in every defined subtype or from the subtype distribution in specific geographic regions instead of extrapolating from just the three subtypes (A/AG, B, and C) that currently make up 86% of the worldwide pandemic as we did here.
Fifth, the method depends on our statistical and phylogenetic techniques to precisely assess how well all the LANL sequences corresponding to the derived 3D motif fit the mAb under study. Novel methods to make this assessment may improve the precision of the calculation.
Finally, the method also depends on the quality of the crystal structures and psV neutralization assay data. With additional structural and viral data, the precision and relevance of the calculations may be improved.
The method described here is applicable to any crystallographically resolved mAb/peptide epitope complex—including those in sequence variable, surface exposed regions on any pathogen—and it clearly clusters known HIV-1 viruses into vaccine-relevant groups that bear little or no relationship to the subtype designations of viral groups based on genotyping (). This in silico serotyping is relevant to vaccine design because it rapidly allows comparison of promising neutralizing antibodies that can be studied for the rational engineering of protective antibody responses. Upon application to epitopes located in several regions of gp120, this method may serve as a tool for the rational design of multivalent neutralizing antibody-based vaccines, which will protect against the maximum proportion of HIV-1 strains while targeting the minimum number of epitopes.
FIG. 4. Worldwide HIV-1 clade distribution. This diagram depicts the estimated global distribution of HIV-1 genetic subtypes in the year 2000.25 Hatched shape overlays illustrate the theoretical coverage of the two mAbs (447-52D and 2219) calculated in this work. (more ...)