Our Affinofile system provides a more comprehensive method to characterize the CD4 and CCR5 usage pattern of extant R5 isolates; in essence, their efficiency of CD4 and CCR5 usage generates a signature 3-D surface plot which can then be correlated with the clinical or pathogenic characteristics of the virus. We wish to emphasize that each virus always gave its signatory surface curve in multiple independent experiments and that its vector metrics, derived from the function F(x, y) that describes the topology of the surface plot, can be used for quantitative comparisons of CD4 and CCR5 usage patterns. The robustness of the assay system and the mathematical transformation that results in the sensitivity vectors is underscored by the clustering of the θ values for each of the viral isolates examined (Fig. and ). We propose to call our system 293-Affinofile cells to reflect their ability to profile the receptor affinities of viral isolates.
Primary biological isolates from well-defined cohorts can be subjected to the same analysis, and their patterns of CD4 and CCR5 usage, reduced to the metrics whose results are shown in Fig. and , may be used to correlate the efficiency of CD4 and CCR5 usage with various aspects of viral pathogenicity. For example, the increased pathogenicity and/or fusogenicity of AIDS versus pre-AIDS R5 strains has been attributed to their increased efficiency of CD4 and/or CCR5 usage, as demonstrated by increased resistance to various entry inhibitors (31
). Conversely, acutely transmitted “founder” viruses, though R5 tropic, can grow in activated PBMCs but not primary macrophages (42
). Indeed, increased macrophage tropism has been used as a surrogate marker for the ability to use low levels of CCR5 (7
). However, Goodenow and Collman (7
) have cautioned that coreceptor preference can be distinct from target cell tropism. Thus, not all R5-tropic strains can use CCR5 on macrophages, and dually X4R5-tropic strains may use one coreceptor on one cell type and not the other. 293-Affinofile cells present a unique opportunity to directly examine the efficiency of CD4 and CCR5 usage of viral isolates over a wide range of expression levels; the ability to ascribe a quantitative metric that defines an isolate's overall CD4/CCR5 usage efficiency may illuminate the complexities that underlie the biological phenotypes mentioned above.
Recently, entry inhibitors, such as enfurtivide (T-20) and maraviroc (a CCR5 antagonist), have emerged as a new class of drugs to treat HIV. Not surprisingly, viral resistance mechanisms have already been identified (40
). Current CDC HIV treatment guidelines recommend the use of coreceptor tropism assays (Trofile) prior to starting maraviroc. The Trofile assay is also proscribed in the case of treatment failure while on a regimen including an entry inhibitor (32
). The use of 293-Affinofile cells may complement the Trofile assay to better predict the success of regimens that include entry inhibitors and may also provide better insights into mechanisms underlying CCR5 inhibitor resistance. For example, for CCR5 inhibitor-resistant isolates that have been shown to use the inhibitor-bound form of CCR5 (37
), Moore and colleagues used our 293-Affinofile cells to show that the degree of resistance is inversely proportional to the level of CCR5 cell surface expression (38
). In addition, the extent of inhibitor resistance, as measured by the plateau inhibitory effect (and represented by the maximal percent inhibition value) in the presence of a saturating amount of inhibitor (vicriviroc or maraviroc), can vary significantly between two different inhibitor-resistant isolates, especially when CCR5 levels are limiting (38
). This nuance may not be appreciated when using the commercial Trofile assay to generate resistance profiles, as the Trofile assay relies on U87-CD4/CCR5 cells (50
) that express very high levels of CCR5. It would be of interest to determine how inhibitor resistance phenotypes would be reflected in changes in sensitivity vector metrics.
In the case of the V3 RT isolates, the angular metric suggests that their infection efficiency is more sensitive to changes in CCR5 levels (θ
shifts closer to 90°) and, thus, would be more sensitive to CCR5 inhibitors and, by extension, fusion peptide inhibitors. We have confirmed this experimentally (23
and data not shown). The decreased efficiency of CCR5 usage likely prolongs the target window of opportunity for CCR5 inhibitors or peptide inhibitors of six-helix bundle formation to work. However, sensitivity angles should only be interpreted in the context of their mean induction levels (M
). For the V3 crown mutants, the YA-to-RT mutations also led to a decrease in M
, indicating an overall decrease in infection efficiency for the RT mutants due to their dependence on higher levels of CCR5 for efficient infection. On the other hand, for the comparison of the CP versus ES clones, the increase in both sensitivity vector angles and mean induction levels (M
) for the CP clones (Fig. ) indicates that their overall increased infection efficiency is likely due to their more efficient usage of CCR5 (Fig. ). Conversely, it implies that ES clones have reduced entry efficiencies and a reduced ability to use low levels of CCR5.
There is a growing body of evidence that the ability to use low levels of CD4 and coreceptor contributes to expanded cellular tropism and disease pathogenesis. Until now, studies have been limited by using surrogate or indirect assays for the efficiency of CD4 and CCR5 usage. Our 293-Affinofile cells can be used to directly measure and profile the CD4/CCR5 usage efficiency of any given viral isolate and provide novel quantitative metrics that can be used for multiple comparisons. We have automated the complex computational analysis required to derive the sensitivity vector metrics on a Web-based platform (http://versa.biomath.ucla.edu
) so that anyone using our Affinofile cells can obtain the vector metrics by imputing the raw infectivity data as described above. In an accompanying study, Doms and colleagues used our Affinofile cells and VERSA, in part, to show that a V3 loop-truncated R5 virus envelope compensated for its inefficient usage of CCR5 by increasing its ability to use low levels of CD4 (1
). Not only is our system a valuable tool for better understanding the relationship between viral pathogenesis and the efficiency of CD4/CCR5 usage, but the application of sensitivity vector analysis to clinical isolates may have implications for guiding entry inhibitor use.