Evidence of genotypic resistance diversity of archived and circulating viral strains in blood and semen of pre-treated HIV-infected men
Ghosn, Jadea,c; Viard, Jean-Paulb; Katlama, Christinec; de Almeida, Martad; Tubiana, Rolandc; Letourneur, Francke; Aaron, Laurentb; Goujard, Cécilef; Salmon, Dominiqueg; Leruez-Ville, Mariannea; Rouzioux, Christinea; Chaix, Marie-Laurea
From the aLaboratoire de Virologie, EA MRT 3620 Université R. Descartes, CHU Necker, Paris, the bService d'Immunologie Clinique, CHU Necker, Paris, the cService de Maladies Infectieuses, CHU Pitié-Salpêtrière, Paris, the dCECOS, CHU Cochin, Paris, the eInstitut Cochin, Paris, the fService de Médecine Interne, CHU Bicêtre, Le Kremlin-Bicêtre and the gService de Médecine Interne, CHU Cochin, Paris, France.
Correspondence to Jade Ghosn, MD, Laboratoire de Virologie, CHU Necker Enfants Malades, 149, rue de Sèvres, 75015 Paris, France.
Tel: + 33 1 44 49 49 61; fax: +33 1 44 49 49 60; e-mail: email@example.com
Received: 8 May 2003; revised: 10 July 2003; accepted: 12 August 2003.
Objective: To study the genetic diversity of drug-resistant HIV strains present in blood and in semen, especially those archived in peripheral blood mononuclear cells (PBMC) and non-sperm cells (NSC).
Methods: Paired blood and semen samples were collected from twenty heavily pre-treated HIV-infected men. HIV RNA in blood plasma (BP) and seminal plasma (SP), as well as proviral DNA in PBMC and NSC were quantified and used for resistance genotyping. Phylogenetic analysis of protease gene clones was used to explore the diversity of the viral quasi-species.
Results: Median BP HIV RNA, PBMC proviral DNA, SP HIV RNA and non-sperm cell proviral DNA loads were respectively: 4.77, 3.65, 3.16 and 1.77 log10 copies per ml or per 106 cells. Resistant HIV strains were found in the BP and PBMC of all the patients, in the SP of 14 patients, and in the NSC of five patients. Overall, the blood and genital compartments exhibited different genotypic resistance patterns in six patients (30%), with additional resistance mutations in the semen of four patients. Phylogenetic analysis of clones of HIV protease gene showed that viral strains in SP originated not only from passive diffusion from BP, but also from local production in semen. The storage of archived proviruses differed according to the anatomic reservoir.
Conclusion: HIV resistant strains are frequent (70%) in the semen of heavily pre-treated men, and the diversity of genotypic resistance pattern confirms HIV compartmentalization. Thus, the risk of sexual transmission of resistant strains can only be partly predicted by standard tests applied to BP.
Although human immunodeficiency virus (HIV) infection leads to a systemic disease, several lines of evidence suggest HIV compartmentalization both at the cellular level [1–6] and in anatomic compartments, such as the central nervous system [7–9] and the kidney [10–12]. The male genital tract is a potential reservoir for HIV partly because of its unique vascular features (e.g. the blood–testes barrier), and also because T lymphocytes and macrophages isolated from semen of HIV-infected men harbour provirus [13,14]. Several comparative studies of viral populations in blood and semen indicate that the male genital tract may constitute a distinct compartment in some patients, based on the isolation of phylogenetically distinct viral quasi-species from semen and blood [15–17]. Although antiretroviral combination therapy appears to reduce viral shedding in semen , the rate and pattern of emergence of resistance may differ between the blood compartment and the male genital tract [19–22]. Furthermore, as HIV may evolve in a compartment-specific manner, and given the role of semen in sexual transmission, it is important to study drug-resistant viruses in the male genital tract. Distinct resistance patterns may arise from the compartmentalization of viral replication [18,21, 23,24], a phenomenon possibly enhanced by suboptimal drug concentrations in semen [25–28]. The presence of resistant strains of HIV-1 in the male genital tract increases the risk of sexual transmission of resistant strains [19,21,22,25]. Recent data suggesting the spread of sexually transmitted drug-resistant HIV-1 strains in Europe and the United States underline the major public health implications of this issue [29–32].
Increasing numbers of HIV-1-infected patients have a history of multiple treatment failure. Yet, little is known about viral resistance in the male genital tract of patients who have resistant HIV-1 strains in their blood compartment. We therefore evaluated the frequency of HIV-1 resistant strains in the genital compartment of heavily pre-treated men with a history of therapeutic failure. We first quantified HIV RNA in blood plasma (BP) and seminal plasma (SP), and HIV DNA in peripheral blood mononuclear cells (PBMC) and non-sperm cells (NSC). We then used the genotypic viral mutational pattern as a marker to study and compare cell-free and archived cell-associated strains in blood and semen.
Patients and methods
Patients and study design
HIV-1 infected men were eligible for this cross-sectional multicentre study if they met the following criteria: age > 18 years, blood plasma HIV RNA > 1000 copies/ml (3 log10), previous failure of at least three antiretroviral regimens, no clinical acute genital infection, willingness to participate to the study and to sign a written informed consent. The study was approved by the Cochin Hospital ethics committee.
Sample collection and processing
Single paired samples of blood and semen were collected on the same day for each patient. Semen was obtained by masturbation at the Necker Clinical Investigations Center after a recommended 2-day period of sexual abstinence. Samples were collected in sterile containers and processed within 1 h after collection according to WHO recommendations. Spermatozoa and NSC were counted and spermatozoa motility was evaluated using standard methods. Two-millilitre aliquots diluted 1 : 1 in RPMI culture medium (Gibco-BRL, Cergy-Pontoise, France) containing 5 mg/ml of bromeline (Sigma-Aldrich Chimie, Saint-Quentin, France) were then centrifuged for 20 min at 300 g on a two-layer discontinuous gradient of 47.5 and 95% Percoll (Sigma-Aldrich Chimie). SP, NSC and spermatozoa were recovered separately. Selected spermatozoa and NSC were washed twice in RPMI medium at 600 g and 200 g respectively, then counted and kept as dry pellets at −80°C. SP fractions were centrifuged for 10 min at 6000 g and supernatants were aliquoted and stored at −80°C.
PBMC were isolated from whole blood by centrifugation on a one-layer Ficoll Hypaque gradient. The PBMC were washed three times in RPMI medium, then counted and kept as dry pellets at −80°C.
HIV RNA in blood plasma
Free HIV virions (HIV RNA) in BP were quantified by using the HIV-1 Monitor 1.5 HIV RNA assay kit (Roche SA, Meylan, France) according to the manufacturer's instructions: the detection limit was 200 copies/ml.
HIV RNA in seminal plasma
Free HIV virions were extracted from 250 μl of SP by the Nuclisens kit (Organon Teknika, Fresnes, France). HIV RNA was amplified with the HIV-1 Monitor 1.5 assay (Roche SA). The internal control provided with the kit was routinely added to the Nuclisens lysis buffer prior to extraction in order to validate both the extraction and amplification steps. Each batch included one positive and one negative control, consisting of seminal plasma from HIV-seronegative subjects, spiked or unspiked with a predetermined number of HIV RNA copies. The detection limit was 200 copies/ml.
HIV DNA assay in non-sperm cells and in peripheral blood mononuclear cells
HIV proviral DNA was quantified in 5 × 106 PBMC and 0.2 to 3 × 106 NSC. Four hundred microlitres of lysis buffer from the Whole Blood Specimen Preparation Kit (Roche SA) were added to the cellular fractions. DNA was extracted using the QIAamp DNA Blood Mini kit (Qiagen, Courtaboeuf, France) and quantified by spectrophotometry. Five hundred nanograms of extracted DNA was amplified with an ‘in-house’ real-time polymerase chain reaction (PCR) method on the ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystem, Courtaboeuf, France), targeting a conserved region of the HIV-1 LTR gene, with a sensitivity of 10 copies per reaction .
Genotypic resistance tests and sequences alignment
The HIV-1 reverse transcriptase (RT) and protease genes were amplified from cell-associated proviral DNA and from cell-free HIV RNA by a first-round PCR followed by nested PCR using published primers . PCR final products were visualized on gels then purified using the QIAquick PCR Purification HIV kit (Qiagen, Valencia, California, USA). After purification, PCR products were sequenced using the fluorescent dideoxy-terminator method (Big Dye Terminator kit; Applied Biosystem, Perkin Elmer, Foster City, California, USA) on an Applied Biosystem 377 automated DNA sequencer (Applied Biosystem, Perkin Elmer). Sequences were aligned using Sequence Navigator software. The amino acids at codons associated with resistance to nucleoside analogue reverse transcriptase inhibitors (NRTI), non-nucleoside analogue reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI) are reported according to the 2002 IAS list except for mutation 63P in the protease gene (http://www.iasusa.org). HIV drug resistance was defined according to the HIV-1 genotypic resistance interpretation algorithm of the French National Agency for Research on AIDS (http://www.hivfrenchresistance.org).
Clones of the HIV-1 protease gene
Protease gene PCR products from three patients were cloned into the pCR Topo 2-1 plasmid (TOPO TA Cloning kits; Invitrogen BV, The Netherlands) as recommended by the manufacturer. DNA was purified with the Mini-Prep kit (Qiagen) and then sequenced as described above.
Direct sequences of the RT and protease genes from the twenty patients, and sequences of protease gene clones from three patients were aligned with Clustal W 1.6 software. Pairwise evolutionary distances were estimated using Kimura's two-parameter method, then the trees were constructed by a neighbour-joining method (neighbour program implemented in the Phylip package) . The reliability of each tree topology was estimated from 100 bootstrap replicates . Trees were also inferred by using the maximum likelihood model.
Spearman's test was used to calculate correlations. Viral loads were compared using the Mann–Whitney test.
Patients’ characteristics at inclusion
Twenty HIV-infected men were enrolled between December 2001 and May 2002 (Table 1). Their median age was 42.5 years (range, 36–51 years), the median CD4 cell count at inclusion was 258 × 106/l (range, 72–560), the median nadir CD4 cell count was 40 × 106/l (range, 1–218). Thirteen patients (65%) had a history of AIDS-defining events (1993 CDC definition). The median time since the initiation of antiretroviral treatment was 84 months (range, 68–170). All patients had been heavily pre-treated, with a median of seven previous antiretroviral regimens (range, 3–15). At the time of sampling, five patients had been in a ‘wash-out’ phase for a median of 6 weeks (range, 4–12 weeks), while the other 15 were receiving a failing antiretroviral regimen containing a median of four drugs (range, 3–6).
The semen characteristics of the 20 patients were as follows: median ejaculate volume 2 ml (range, 0.1–6; normal value > 2 ml), median spermatozoa count 44 million/ml (range, 0–380; normal value > 30 × 106/ml), median percentage of spermatozoa with rapid motility 10% (range, 0–35%; normal value > 25%), median NSC count 8.5 million/ml (range, 0–32; normal value < 1 × 106/ml).
HIV RNA values in blood plasma and seminal plasma
HIV RNA was detectable in the BP of all 20 patients, with a median of 4.77 log10 or 59 150 copies/ml (range, 1680–474 000 copies/ml) (Table 1). The median HIV RNA value in the BP of patients without treatment (n = 5; 113 000 copies/ml, 5.05 log10) did not differ from that of patients on treatment (n = 15; 36 000 copies/ml, 4.55 log10) (P = 0.17, Mann–Whitney test).
The median HIV RNA value in SP was 1450 copies/ml or 3.16 log10 (range, < 200–260 000 copies/ml). The median HIV RNA value in the SP of men without treatment (n = 5; 13 750 copies/ml, 4.13 log10) was higher than that of men on treatment (n = 15; 590 copies/ml, 2.77 log10) (P = 0.07, Mann–Whitney test).
The median HIV RNA value was significantly higher in BP (60 000 copies/ml, 4.77 log10) than in SP (1450 copies/ml, 3.16 log10) (P < 0.05, Mann–Whitney test). In one of the patients off treatment (VN 20), HIV RNA load was higher in SP (4.92 log10) than in BP (4.19 log10).
HIV DNA values in PBMC and non-sperm cells
HIV DNA was detected in PBMC of all the patients, with a median value of 3.65 log10 or 4500 copies/106 PBMC (range, 173–100 000 copies/106 cells) (Table 1). A trend towards a correlation was found between HIV DNA load in PBMC and the nadir CD4 cell count (P = 0.0787; r2 = −0.4; Spearman's test). HIV DNA was detected in NSC of nine of 20 patients (45%). The median HIV DNA value was 1.77 log10 or 60 copies/106 NSC (range, 32–416 copies/106 cells) in these nine patients. The other 11 patients had undetectable HIV DNA in NSC. Patients with undetectable HIV RNA in SP had undetectable HIV DNA in NSC. All patients with detectable HIV DNA in NSC had detectable HIV RNA in SP. HIV RNA load in SP correlated strongly with HIV DNA load in NSC (P = 0.0016; r2 = 0.724, Spearman's test).
Mutations at positions conferring resistance to antiretroviral drugs are reported in Tables 2 and 3.
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).
Phylogenetic analysis of direct sequences in the 20 patients
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.
Phylogenetic analysis of HIV protease gene clones in three patients
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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