The RT and protease genes were amplified in BP from 20 patients. Mutations conferring resistance to at least one antiretroviral drug were observed in all but one of the patients (VN 18). The RT and protease genes were amplified in PBMC from all patients, except for the RT gene in patient VN 07. All patients harboured viruses archived in PBMC which bore at least one resistance mutation in the RT and/or protease gene.
HIV-1 was amplified from the SP of 15 patients, 14 of whom harboured resistant strains. The RT and/or protease gene could not be amplified in SP from five patients with low SP HIV RNA load (below 500 copies/ml). HIV DNA was amplified from NSC of seven patients, five of whom harboured resistant archived strains. Among the 13 patients in whom amplification was unsuccessful, the HIV DNA load in NSC was below the detection limit in 10, and between 40 and 60 copies/106 cells in the other three.
The genotypic resistance patterns differed between HIV RNA in BP and HIV DNA in PBMC in 16 of 20 patients (80%) (VN 01, VN 03–06, VN 08, VN 10–15, VN 17–20). Only patients VN 05 and VN 18 were off treatment. Overall, the blood compartment and the genital compartment exhibited different genotypic resistance patterns in six of 20 patients (30%) (VN 07, VN 09, VN 11, VN 14, VN 15, and VN 18). In four patients (VN 07, VN 09, VN 11, VN 18), viral strains present in the genital compartment harboured more resistance mutations than those in the blood compartment (in either HIV RNA or proviral DNA) (Table 4).
To explore the inter- and intra-individual genetic diversity of HIV-1, we analysed all available sequences [55 RT (Fig. 1a) and 57 protease gene sequences (Fig. 1b)]. All sequences showed the expected patient-specific clustering. Nineteen patients were infected by subtype B strains, while patient VN 12 was infected by a CRF02 strain (data not shown). In most cases, we observed genetic diversity not only between the blood compartment and the genital compartment, but also between the cell-free viruses and the cell-associated archived proviruses in a given compartment. However, strains present in SP were more likely to be closely related to BP viruses.
In order to determine the origin of viral strains in the male genital compartment, we analyzed the sequences of protease gene clones in three patients: VN 09, VN 14, and VN 15 (Fig. 2). In all three cases, the most striking finding was the homogeneity of viral quasi-species in BP and SP, and the greater genetic diversity of archived proviruses. The homology of the SP and BP viral quasi-species of patients VN 09 and VN 14 suggested that the cell-free viral quasi-species in the genital compartment of these two patients probably arose by passive diffusion from BP.
Further analysis of resistance mutations showed the persistence of wild-type HIV-1 archived in PBMC and NSC. Some primary major resistance mutations were only detected in the clones, and not by direct sequence analysis (mutations 30N and 90M for PBMC1 and PBMC7, respectively, from patient VN 09). Moreover, the clones from the genital compartment bore some resistance mutations that were only present in this compartment (mutation 30N in clone SC5 from patient VN 15).
Understanding HIV-1 compartmentalization and the nature of archived proviruses has important implications in patient management. In this study, we compared the genetic diversity and the mutational resistance patterns of the cell-free HIV quasi-species and cell-associated archived proviruses, in the blood and genital compartments of HIV-infected men in whom several antiretroviral regimens had failed.
We have confirmed the compartmentalization between blood and the male genital tract. We also found evidence of local viral production, a phylogenetically distinct viral population and a distinct mutational pattern in the male genital tract.
Local virus production is suggested by the significantly higher HIV RNA load in the SP of patient VN 20 relative to his HIV RNA load in BP, and particularly by the correlation between HIV RNA load in SP and HIV DNA load in NSC. Only 55% patients had detectable levels of HIV proviral DNA in NSC, which limits any definitive conclusion to rule out between local viral production versus phramacological compartmentalization. However, the homology between clone NSC4 and clones from the SP of patient VN 15 is suggestive of local virus production in the male genital tract. Given the low levels of HIV RNA and HIV DNA in SP and NSC respectively, we are conscious that variants identified by our cloning method might not be representative of all quasi-species present in the genital compartment.
Differences in the resistance patterns between blood and genital tract viruses observed in six patients also point to HIV compartmentalization. Moreover, we observed some primary mutations only in the genital tract, emphasizing the fact that the storage of archived proviruses differ according to the anatomic reservoir. Moreover, it is particularly interesting that M184V persisted in the genital tract in three (VN 07, VN 09, VN 18) out of the five patients off treatment despite a presumable reversion in the blood compartment, suggesting a different dynamics of the reversion of resistance mutations according to the compartment. The most striking feature was the intra-individual diversity of archived proviruses in blood and male genital tract, with the persistence of both wild-type and multi-resistant viruses, confirming findings from Ruff et al. . The presence of wild-type viruses was particularly noteworthy in these 20 extensively pre-treated patients, 15 of whom were on a failing antiretroviral combination at the time of the study, and harboured resistant viruses in BP. The trend towards a correlation between HIV DNA load in PBMC and the nadir CD4 cell count suggests that the archived viral pool increases with the progression of HIV disease. These findings indicate that wild-type viral sequences archived in the two reservoirs early in infection had not been completely replaced by the dynamic processes affecting the pool of latently infected cells, despite of the presence of resistant circulating virions in BP and SP. In other words, the production of resistant circulating viral particles in BP and SP appears to reflect the predominant viral population emerging under drug-selection pressure.
The resistance patterns of clones of archived HIV DNA exhibited wide intra-individual diversity, and revealed the existence of some resistance mutations that were not seen during routine genotypic resistance testing of BP, suggesting that all previously circulating wild-type and drug-resistant forms of the virus in a given patient can be archived in PBMC as well as in NSC. Archived viruses in latently infected seminal cells are likely to be replication-competent  and may re-emerge in favourable conditions. Such conditions could be created by exposure of productive cells in the male genital tract to suboptimal antiretroviral concentrations, permitting ongoing HIV-1 production and infectiousness [25,38]. Indeed, although all NRTI and NNRTI are known to reach suppressive concentrations in the male genital tract [39–43], PI diffusion in this compartment is variable [27,28,38,43–48]. Thus, with the widespread administration of combined antiretroviral regimens, it will be then critical to deliver adequate drug concentrations to all compartments in which the virus can replicate.
As regards sexual transmission of resistant HIV strains, we show that, at a given time, routine HIV RNA quantification and genotypic resistance tests applied to BP can only partially predict this risk, as recently confirmed by mathematical models . We also show, with regard to sub-compartmentalization in the male genital tract, that semen cells  and free viral particles present in seminal plasma might be differently involved in the spread of resistant HIV-1 strains. As sexual intercourse is the main route of HIV transmission, our findings suggest that people living with HIV should themselves be a target of prevention campaigns. Given the increasing prevalence of HIV-1 strains with reduced drug susceptibility, further studies are needed to monitor the role of sexual transmission in the spread of drug-resistant virus.
We thank all the patients who agreed to participate in this study. We would also like to thank Dr Agnès Mogenet and Professor Jean-Louis Bresson for the clinical monitoring. This work was supported by a scholarship from the Fondation pour la Recherche Médicale, and by grants from the French National AIDS Research Agency (Agence Nationale de Recherche sur le Sida).
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