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Resistance of human immunodeficiency virus type 1 (HIV-1) to small-molecule CCR5 inhibitors is well demonstrated, but resistance to macromolecular CCR5 inhibitors (e.g., PSC-RANTES) that act by both CCR5 internalization and receptor blockade had not been reported until recently (3). The report of a single simian-human immunodeficiency virus SHIVSF162-p3 variant with one V3 and one gp41 sequence change in gp160 that conferred both altered replicative fitness and resistance to PSC-RANTES was therefore surprising. We introduced the same two mutations into both the parental HIV-1SF162 and the macaque-adapted SHIVSF162-p3 and found minor differences in entry fitness but no changes in sensitivity to inhibition by either PSC-RANTES or the small-molecule allosteric inhibitor TAK-779. We attribute the earlier finding to confounding fitness effects with inhibitor sensitivity.
A recent study by Dudley et al. (3) claimed to be “the first to describe the immediate selection and infection of a drug-resistant SHIV [simian-human immunodeficiency virus] variant in the face of a protective vaginal microbicide, PSC-RANTES.” The article further concluded, “This rhesus CCR5-specific/PSC-RANTES resistance selection is particularly alarming given the relative homogeneity of the SHIVSF162-p3 stock compared to the potential exposure to a heterogeneous HIV-1 [human immunodeficiency virus type 1] population in human transmission.” The study described a SHIVSF162-p3 variant with two amino acid substitutions, K315R in the V3 loop region (present as a minor component of the p3 challenge stock) and N640D in HR2 of gp41, that conferred greater replicative fitness and greater relative resistance to both the CCR5 inhibitor PSC-RANTES (to which the single macaque harboring this variant had been exposed prior to infection) and the small-molecule allosteric CCR5 inhibitor TAK-779 (1).
While the development of HIV-1 strains resistant to small-molecule CCR5 inhibitors has been observed (11, 14), this result was surprising for several reasons. First, the inhibitory mechanism of PSC-RANTES is different from those of the small-molecule allosteric inhibitors; the ability of the macromolecule to induce profound and prolonged intracellular coreceptor sequestration, together with its ability to sterically block coreceptor use, should provide additional barriers to the development of resistant viruses that retain use of CCR5 (10). Second, this interpretation is supported by the failure to generate PSC-RANTES-resistant strains in multiple long-term in vitro selection studies (R.N. and D.E.M., unpublished results). Finally, the development of escape mutants in an in vivo setting would be expected to require sustained inhibitory concentrations of the drug at sites of replication. The Dudley et al. study was based on a single-dose experiment under conditions in which even at the highest dose used, no detectable systemic exposure occurred (6).
The determination of resistance can be confounded by the fitness of a virus isolate (7), and the claim of resistance to PSC-RANTES was surprising given that infection with the parental HIV-1SF162 isolate with the consensus GPGR315 sequence is highly susceptible to PSC-RANTES inhibition (D.E.M., unpublished data) (Fig. (Fig.11 and Table Table1).1). These concerns prompted us to determine the impact of the K315R and N640D sequence variants on the entry fitness and sensitivity of both HIV-1SF162 and SHIVSF162-p3 to PSC-RANTES or TAK-779 in a single-round infection assay using either human or rhesus CCR5-expressing U87.CD4 target cells. We felt that it was important to extend the experiments of Dudley et al. (3) to HIV-1 since it is inhibition of HIV-1 infection of humans that is the intended application of a microbicide containing PSC-RANTES or related recombinant molecules (4, 12). We used site-directed mutagenesis to create three variants of the wild-type SF162 env sequence (R315, N640): K315, N640; R315, D640 (equivalent to the “resistant” SHIVSF162-p3 variant from macaque 584), and K315, D640. These four env genes were used to complement a luciferase reporter HIV-1 construct in a standard single-round infection assay that has the advantage of a dynamic range of up to 8 logs (9, 15). We found that D640 conferred a small but significant entry advantage over N640 in the single-cycle assay (Table (Table1),1), in agreement with the results reported by Dudley et al. (3). However, none of the SF162 mutations conferred any significant resistance to either PSC-RANTES or TAK-779 (Fig. (Fig.11 and Table Table1),1), whether or not we corrected for the modest difference in entry efficiency. We repeated these experiments using the SHIVSF162-p3 env clone that has the same sequence as that used in the experiments of Dudley et al. (3, 5) to determine if the finding of resistance was related to the other sequence differences between the macaque-adapted SHIV and SF162. Neither R315, D640, nor the combination of the two “resistance” mutations conferred resistance to either PSC-RANTES or TAK-779 on target cells expressing either human or rhesus CCR5 (Table (Table11 and Fig. 1C and D). The D640 substitution again conveyed a small entry advantage over N640 (data not shown). We thus conclude that the two mutations in SHIVSF162-p3 that were claimed to confer resistance to PSC-RANTES using either human or rhesus CCR5 for entry were selected by replicative fitness in macaque 584 and not by drug resistance. We find no evidence that the two mutations have any impact on the PSC-RANTES sensitivity of either HIV-1SF162 or SHIVSF162-p3 (Fig. (Fig.1),1), and we were unable to confirm the 5.5-fold increase in resistance to PSC-RANTES on target cells expressing human CCR5 or the 7-fold increase on target cells expressing rhesus CCR5 reported by Dudley et al. (3). We therefore attribute the conclusions of Dudley et al. (3) to confounding fitness effects with inhibitor sensitivity. Multiple rounds of replication in the assays employed by Dudley et al. (3) likely amplified the relatively minor differences in entry fitness that we (and they) observed and made the precise assessment of 50% inhibitory concentration (IC50) values more difficult, particularly given that 10-fold dilutions of inhibitors were used in their experiments.
We observed similar inhibitory activity of PSC-RANTES on entry of both HIV-1SF162 and SHIVSF162-p3 via both human and macaque CCR5, even though SHIVSF162-p3 has many mutations in env that occurred during multiple passages in macaques (5), and macaque CCR5 has 7 to 8 amino acid differences from human CCR5 (8, 13), including one polymorphic site that contributes to resistance to small-molecule CCR5 inhibitors (2). While it is possible that selection for either fitness or resistance to CCR5 inhibitors may show subtle differences between SHIV-infected macaques and HIV-1 infected humans, and may even vary between macaques with different CCR5 alleles, no such differences were observed in our experiments. Although we were unable to achieve the very high levels of macaque CCR5 expression reported by Dudley et al. (3), differences in coreceptor levels would be expected to affect all four SHIVSF162-p3 variants equally. Our results argue strongly that there is no simple mutational pathway that results in resistance to PSC-RANTES for either HIV-1SF162 or SHIVSF162-p3. We conclude that the results reported by Dudley et al. (3) give no cause for concern about the development of resistance to microbicides containing PSC-RANTES or similar compounds.
This work was supported by grants R01 AI052778 and U19 AI076981 from the National Institute of Allergy and Infectious Diseases (NIAID). Additional support for M.C. was provided by the La Jolla Foundation for Microbicide Research. O.H. acknowledges support from the Swiss National Science Foundation. R.E.O. acknowledges support from CONRAD, the World Health Organization, and the International Partnership for Microbicides.
We are grateful to Cecilia Cheng-Mayer for providing the SHIVSF162-p3 env molecular clone. Rhesus CCR5 (GenBank accession number U73740) was obtained from the NIH AIDS Research and Reference Reagent Program and was originally contributed by Preston Marx.
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Published ahead of print on 24 March 2010.