The CCR5 antagonist maraviroc, used in combination with antiretroviral drugs in HIV-infected patients, is only active against CCR5-using viruses. However, some patients were given maraviroc although they were infected by R5X4 dual-mixed viruses. This was to assess the immunological benefit of maraviroc therapy. In the MARIMUNO study, patients with undetectable plasma HIV RNA received a 24-week maraviroc supplement to an efficient antiretroviral therapy. Since the positive selection of CXCR4-using viruses in cell reservoirs may influence any response to later treatment, we investigated how the frequency of CXCR4-using variants in R5X4 dual-mixed virus populations responded to maraviroc selection pressure using ultra-deep sequencing (UDS).
We explored 22 patients from the MARIMUNO study infected with R5X4 dual-mixed viruses according to the recombinant virus assay Toulouse Tropism Test. The frequency of CXCR4-using variants was determined in peripheral blood mononuclear cells (PBMCs) before maraviroc intensification (week 0) and after 24 weeks of maraviroc (week 24); both samples were tested in the same run. UDS was performed on a 454 GS Junior system. The sequence reads of the V3 env regions were analysed with PyroVir software (Inserm-Transfert) developed to provide a fast and automated position-specific process for inferring HIV-1 tropism from V3 env 454 ultra-deep pyrosequencing data.
The mean total HIV-1 DNA before maraviroc intensification was 2.4 log copies/106 cells; it was 2.5 log copies/106 cells 24 weeks later (Wilcoxon rank test, P=0.3). UDS with the PyroVir genotypic algorithm detected CXCR4-using viruses in the 22 R5X4 infected patients at week 0 with a mean frequency of 59% (range: 3-100%). CXCR4-using viruses were detected in 21/22 patients at week 24 with a mean frequency of 52% (range: 10-92%). We found no correlation between the HIV DNA concentration in PBMCs and the number of CXCR4-using variants or their frequency. The frequency of CXCR4-using variants did not increase between weeks 0 and 24 except in patient 4 whose increase was 32%. The mean numbers of unique amino acid variants and X4 amino acid variants were similar at both stages.
A 24-week course of a CCR5 antagonist does not select CXCR4-using viruses in the PBMCs of patients on suppressive therapy infected with R5X4 dual-mixed viruses. These results indicate little or no residual HIV replication that could be subjected to selection pressure.