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Journal of Clinical Microbiology, June 2007, p. 1838-1842, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.00113-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Genetic and Phenotypic Features of Blood and Genital Viral Populations of Clinically Asymptomatic and Antiretroviral-Treatment-Naive Clade A Human Immunodeficiency Virus Type 1-Infected Women

Laurent Andreoletti,^1* Katharina Skrabal,² Virginie Perrin,² Nicolas Chomont,³ Sentob Saragosti,² Gerard Gresenguet,⁴ Helene Moret,¹ Jerome Jacques,¹ Jean de Dieu Longo,⁴ Mathieu Matta,³ Fabrizio Mammano,² and Laurent Belec³

Laboratoire de Virologie Médicale, Hôpital Robert Debré, Centre Hospitalo-Universitaire de Reims, et Equipe d'Accueil 3798, Faculté de Médecine de Reims, France,¹ INSERM U552-Recherche Antivirale, Hôpital Bichat-Claude Bernard, Paris, France,² Unité Immunité et Biothérapie Muqueuse, Centre de Recherches Biomédicales des Cordeliers, Université René Descartes (Paris V), Paris, France,³ Unité de Recherche et d'Intervention sur les Maladies Sexuellement Transmissibles et le SIDA, Faculté des Sciences de la Santé, and Centre National de Référence des Maladies Sexuellement Transmissibles et du SIDA, Bangui, République Centrafricaine⁴

Received 16 January 2007/ Returned for modification 4 March 2007/ Accepted 16 April 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the present study, we assessed whether human immunodeficiency virus type 1 (HIV-1) genetic compartmentalization was associated with phenotypic CCR5 (R5) or CXCR4 (X4) coreceptor usage differences between the systemic and the genital viral populations. Four clinically asymptomatic and treatment-naïve clade A HIV-1-infected patients were selected from a cohort of 274 African women, because they were free of all the biological cofactors known to modify the kinetics of viral production in the genital tract. HIV RNA envelope sequences (V1 to V3) derived from plasma and cervicovaginal secretions (CVS) were amplified, subcloned, and sequenced. CCR5 or CXCR4 coreceptor usage was determined by production of recombinant viral particles, followed by single-cycle infection assays of indicator cell lines, using the tropism recombinant test. In these four selected patients, CVS-derived sequences appeared to be genetically distinct from blood-derived sequences (P 0.001). Two patients were found to harbor virus populations with only the R5 phenotype in both compartments, whereas viruses using CXCR4 in addition to CCR5 were detected in two other patients. In particular, one woman harbored genital virus populations with mixed R5 and X4 phenotypes associated with peripheral blood populations with only the R5 phenotype. These results demonstrate genetic compartmentalization of HIV between the plasma and genital secretions of clinically asymptomatic, treatment-naïve, clade A-infected women. Also, for one patient, we report phenotypic coreceptor usage differences between the systemic (R5) and genital (R5/X4) viral populations. These features may be critical for the development of further mucosal vaccines, therapies, or new preventive strategies to block heterosexual transmission.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Genital secretions are the source of most human immunodeficiency virus contamination, and more than 90% of new infections are transmitted heterosexually (5). Investigation of the genotypic and phenotypic features of human immunodeficiency virus type 1 (HIV-1) populations from the female genital tract is critical for the development of new therapeutic and preventive strategies to block heterosexual transmission (3, 12). Previous analyses have documented the existence of compartmentalized but phylogenetically related viral sequences between blood and the female genital tract (3, 6). Whether viral genetic compartmentalization is associated with phenotypic CCR5 (R5) or CXCR4 (X4) coreceptor usage differences between the viral populations from the blood and those from the female genital tract has not been directly addressed (3, 4, 6). Several genetic studies have predicted viral tropism in the female genital tract (4, 6) and have suggested that women infected by subtype B virus may harbor distinct CCR5-tropic or CXCR4-tropic viral populations between blood and the genital tract, whereas those infected with other HIV subtypes do not (4, 6). In a recent report, in which coreceptor usage was determined using a cell-cell fusion assay, R5-tropic variants were found in both systemic and genital compartments of five clade B HIV-1-infected women at the early stage of the disease (15). The usage of coreceptor CCR5 or CXCR4 by infectious variants present in the female genital tract are likely important determinants implicated in the mechanisms of heterosexual transmission from female to male (3). In the present study, we evaluated whether viral genetic compartmentalization between blood and the female genital tract was associated with phenotypic CCR5 or CXCR4 coreceptor usage differences between these two anatomic sites in clade A HIV-1 chronically infected women. Viral genomic sequences encoding the V1 to V3 loops of the env gene in cervicovaginal secretions (CVS) obtained by vaginal washing were compared to those obtained from matched blood samples. Moreover, virus coreceptor use was phenotypically determined for the first time for genital and blood viral populations by production of infectious recombinant viral particles carrying the tropism determinant region of primary viruses derived from plasma and CVS.

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Patients. We studied four patients who were part of a cohort of 274 women (mean age, 27 years; range, 15 to 49 years) who attended the Centre National de Référence des Maladies Sexuellement Transmissibles et du SIDA of Bangui (Central African Republic). The characteristics of this cohort of HIV-1-infected women and the results of basic laboratory investigations, including routine HIV-1 and syphilis testing and testing for sexually transmitted diseases (STDs), have been reported previously (10). The institutional review board in the Central African Republic approved this investigation, and each woman provided informed consent. Paired CVS and peripheral blood samples were obtained from all the patients at the time of inclusion. For the present study, we first selected 30 women because they were not pregnant, were in the third week of their menstrual cycles (and not taking any oral or parenteral contraception), were free of cervicitis and STDs, and their CVS were free of hemoglobin and semen trace contamination (3). Moreover, all of these women were clinically asymptomatic with regard to HIV disease at the time of sampling, and none of them had ever received antiretroviral therapy (Table 1). To study genetic and phenotypic diversity of HIV-1 variant populations with CVS and plasma, we finally focused our study on 4 of the 30 women because they demonstrated all of the required clinical and biological eligibility criteria and because they had HIV-1 RNA viral loads of 1,000 copies per ml in both blood and genital secretion samples (Table 1) (4).

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TABLE 1. Characteristics of the four clade A HIV-1-infected African women in the study

Sample collection and analysis. Plasma was separated from EDTA-anticoagulated blood and immediately stored in aliquots at –80°C until use. CVS were collected by a standardized nontraumatic 60-s vaginal washing procedure and then treated and stored as described previously (2). The absence of contaminating semen and hemoglobin in cervicovaginal secretions was determined, respectively, by a Y-chromosome PCR assay and by spectrophotometry, as described previously (2). We quantified HIV RNA in plasma and CVS by using an Amplicor HIV Monitor test 1.5 (Roche Diagnostic Systems, Branchburg, NJ) (1).

Nucleic acid extraction, reverse transcription-PCR, reverse transcription-PCR product subcloning, and sequence analysis. RNA was extracted from plasma and from the acellular fraction of CVS using a QIAamp viral RNA minikit (QIAGEN, Courtaboeuf, France) and a reverse transcription (RT)-PCR was carried out using E00 and ES8b primers that allowed amplification of the V1-V3 region of gp120 as described previously (17). To minimize sampling bias, the products obtained from at least two independent PCR amplifications were mixed and then cloned using a TOPO-TA cloning kit (Invitrogen, Groningen, The Netherlands) according to the manufacturer's instructions. Nucleotide sequencing of the V1-V3 region of the env gene was performed from each subcloned V1-V3 PCR product using an ABI model 3700 sequencer and an ABI Prism BigDye Terminator cycle sequencing Ready Reaction kit, version 2.0 (Applied Biosystems, Paris, France) (6). Sequences were aligned with Clustal W (16) and edited with MacClade software (9). Phylogenetic comparisons were performed using PAUP*4.0b10 software (Sinauer Associates, Inc., Sunderland, MA). After removing identical sequences, the total pairwise distances of sequences from plasma were compared to the total pairwise distances of sequences from vaginal secretions for each patient. P values were obtained by using the unpaired t test with Welch's correction (GraphPad Prism4 Software, Inc., San Diego, CA). Compartmentalization was evaluated by Slatkin and Maddison's method (14), which was adapted for HIV-1 populations (8, 11).

An HIV subtype was determined for each of the study women by submitting the sequencing data of the env V1-V3 region PCR products to the National Center for Biotechnology information (NCBI) sequence database for an HIV subtype identification process (http://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi) (1).

Tropism recombinant test. Tropism of the virus populations from different compartments was determined by the tropism recombinant test (TRT), as previously described (17). Briefly, the plasmid pNL4-3V, carrying a deletion of the principal tropism determinant domains (from variable loop V1 to V3 in the envelope gene), was linearized at the site of the deletion. 293-T cells were transfected using the calcium phosphate precipitation method with 8 µg of the linearized plasmid pNL4-3V and 0.8 µg of the PCR product obtained from different compartments, as described previously (17). Culture supernatants were collected 40 h after transfection and centrifuged for 10 min at 1,000 x g to remove cell debris. U373MG-CD4 target cells, stably transfected with an expression vector for the chemokine receptor CCR5 or CXCR4 and carrying an HIV-1 long terminal repeat-ß-galactosidase cassette, were seeded into 96-well plates 2 days before infection at a density of 2,000 cells/well (7). Infection with serial dilutions of viral supernatant was carried out in triplicate in complete medium with 2 µg/ml DEAE dextran. Forty hours after infection, cells were lysed using 100 µl/well of lysis buffer (5 mM MgCl₂ and 0.1% NP-40 in phosphate-buffered saline), and 100 µl/well of chromogenic substrate (6 mM chlorophenol red-ß-D-galactopyranoside [CPRG; Roche]) in lysis buffer was added (17). The optical density was measured at 570 nm, with the reference filter set at 690 nm, after 30 min to 6 h of incubation at 37°C. The coreceptor usage was determined by measurement of CPRG in the different target cells. The sensitivity values of the tropism assay for the detection of R5 and of X4 viruses in a mixed virus population were similar (13). A negative control (cells exposed to noninfectious supernatant produced by transfection in the absence of the PCR product) was included in each experiment. Optical density values greater than 1.5 times the negative control value were considered positive (17). At least two different nested PCR products derived from blood or from genital samples were assayed using this protocol.

Nucleotide sequence accession numbers. The V1-V3 region HIV RNA sequences corresponding to clones from the blood and CVS of the four patients have been deposited in the EMBL database under the following accession numbers: AJ893358 to AJ893507.

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Phylogenetic analyses of viral sequences from plasma and CVS. HIV RNA envelope sequences derived from plasma and CVS, representative of replicating viral populations in these two anatomic sites, were successively amplified, subcloned, and sequenced. Incomplete viral nucleotide sequences or amino acid sequences carrying frame shifts and stop amino acid codons were excluded from the present analyses. We compared 15 to 24 sequences spanning the V1 to V3 region of the viral envelope (nucleotides [nt] 806 to 876) for each anatomic site, resulting in a comparative analysis of 150 RNA viral sequences. All of the study HIV-1 RNA sequences were identified as HIV subtype A strains (not shown). Our phylogenetic study was performed for a comparative analysis of the HIV-1 RNA sequences from both anatomic sites, using the neighbor-joining method, after gap stripping (771 nt). The constructed phylogenetic trees clearly showed that the viral sequences from plasma and CVS clustered in two distinct groups of virus sequences between the blood and the genital tracts of the four study women (Fig. 1). Moreover, we observed the presence of four monophyletic groups corresponding to each study woman (Fig. 1).

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FIG. 1. Phylogenetic trees were built with PAUP*4.0b10 software (Sinauer Associates, Inc., Sunderland, MA). Maximum likelihood trees were constructed by using the HKY85 model of nucleotide substitution, with the transition/transversion ratio and alpha shape parameter for a gamma distribution estimated on a neighbor-joining tree. The resulting model was used in a heuristic search using subtree pruning regrafting (SPR) branch swapping to find the best maximum likelihood tree. Branch length distances calculated by PAUP* were used to calculate diversity by using the HKY85+G+I model of evolution in PAUP*. Sequences issued from the plasma (filled circles) and from the genital tract (open squares) of each patient are shown. Numbers at each branch point indicate the bootstrap values derived from 1,000 bootstrap resamplings. Bootstrap values greater than 95% are indicated.

Diversity analyses of viral sequences from plasma and CVS. We determined intracompartmental genetic diversity by measuring the mean pairwise distances of viral sequences within the peripheral blood and the genital tract of each study woman. The diversity of HIV-1 nucleotide sequences was found to be significantly different between plasma and vaginal secretions of the four selected women (Table 2). For patients 105, 231, and 259, the diversity was higher in plasma than in vaginal secretions. For patient 152, the diversity appeared to be higher in vaginal secretions than in plasma (Table 2). We then evaluated the HIV-1 compartmentalization between plasma and vaginal secretion sequences by using Slatkin and Madison's method for measuring the restriction of gene flow among viral populations (11, 14). Interestingly, this approach evidenced a highly significant compartmentalization between the peripheral blood and the genital tract HIV populations of the four study women (P < 0.001 for patients 152, 231, and 259; P = 0.001 for patient 105) (Table 2).

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TABLE 2. Comparison of genetic diversity, compartmentalization, and viral coreceptor usage of peripheral blood and genital secretions at the chronic phase of infection

Phenotypic determination of HIV coreceptor usage. Having determined the existence of a genetic compartmentalization of HIV envelope sequences between plasma and CVS, we asked whether this phenomenon could result in the presence of viral populations with different phenotypic traits encoded primarily by the HIV-1 V1-V3 region. Virus tropism was phenotypically determined, because genotypic prediction of virus tropism for non-B subtype HIV is generally not reliable. Indeed, three publicly available tropism prediction tools (Web sites http://ubik.microbiol.washington.edu/computing/pssm/, http://coreceptor.bioinf.mpi-sb.mpg.de/cgi-bin/coreceptor.pl, and http://genomiac2.ucsd.edu:8080/wetcat/index.html) indicated the exclusive use of CCR5 for all of our subtype A sequences, with the exception of three V3 env sequences from patient 105 (predicted only by the geno2pheno algorithm as CXCR4 using).

We therefore determined the phenotypic CCR5 or CXCR4 coreceptor usage of the different viral populations present in plasma and CVS of the four women by production of recombinant viral particles carrying patient-derived V1-V3 env domains (7, 17). Two patients (105 and 152) were found to harbor virus populations with only the R5 phenotype in both compartments (Table 2), whereas viruses using CXCR4 in addition to CCR5 were detected in two other patients. In particular, patient 259 displayed virus phenotypic compartmentalization, harboring a plasma virus population with only the R5 phenotype, while the viral population present in the CVS was able to use both coreceptors CCR5 and CXCR4 (Table 2). For patient 231, the plasma virus population was able to use both CCR5 and CXCR4 expressed on target cells, while we could not determine the tropism of the CVS virus population (Table 2) (17).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Our study aimed to examine whether the existence of a genetic compartmentalization might be associated with phenotypic coreceptor usage differences between the viral populations of blood and those of the genital tract in clade A HIV-1-infected African women. Virus tropism was phenotypically determined, because genotypic prediction of virus tropism for non-B subtype HIV is not fully reliable. We focused our study on four clade A HIV-1-infected patients who were free of all the biological cofactors known to modify the kinetics of viral production in the genital tract that, consequently, were capable of influencing the differential evolution of the viral populations in the female genital tract (1, 3). In addition to these stringent selective criteria of inclusion, the relatively small sample size of this study is due to the inherent difficulties in obtaining and preserving valuable biological material from field studies.

In each of the study women, the viral envelope V1-V3 nucleotide sequences derived from the cervicovaginal secretions clustered separately from those found in the matched plasma samples. The greater genetic distances between clones from the genital and the blood compartments than between clones from the same compartment were demonstrated to be statistically significant in all four cases (Fig. 1). In the four selected women, the viral genetic diversity between the blood and the genital tract appeared to be significantly different, which was highly suggestive of a compartmentalized genetic evolution of the variant populations between the female genital tract and the systemic compartments at the time of the chronic phase of HIV-1 infection (Table 2). Taken together, these findings supported the hypothesis that the female genital tract is an independent viral anatomic compartment that could harbor genetic variant populations that are distinctly different from those found in the peripheral blood of women recently infected or at the chronic phase of HIV-1 infection (3, 4, 6, 12, 15).

Our results also indicated that the level of genetic diversity of HIV RNA variant populations in genital secretions could be variable among women with a healthy genital tract, likely resulting from the variable dynamics of viral replication in the target cells of the female genital tract (1).

In the present study, virus tropism was phenotypically determined, because genotypic prediction of virus tropism for non-B subtype HIV is generally not reliable (http://ubik.microbiol.washington.edu/computing/pssm/; http://coreceptor.bioinf.mpi-sb.mpg.de/cgi-bin/coreceptor.pl; http://genomiac2.ucsd.edu:8080/wetcat/index.html). We therefore evaluated the coreceptor usage of viral populations from the female genital tract and from blood by producing recombinant virus particles expressing chimeric envelope proteins in which the tropism determinant region originated from patients' samples, followed by tropism assays, as described previously (17). In patients 105 and 152, only R5-tropic variants were identified, both in the peripheral blood and in the cervicovaginal secretions of these asymptomatic HIV-1 chronically infected women. In only one study woman (patient 231), this recombinant tropism assay did not allow us to determine the coreceptor use of the genital tract virus population, suggesting the possibility of an inefficient recombination or the production of a nonfunctional envelope protein complex. Alternatively, one cannot exclude the possibility that coreceptors other than CXCR4 or CCR5 could be used by these viruses to infect target cells located in the genital submucosa (3). Finally, in patient 259, we clearly identified the presence of mixed X4- and R5-tropic viral populations in the female genital tract, whereas we observed only R5-tropic viruses in their respective blood compartments. As suggested by the relatively low phylogenetic distances between the clones of the genital virus population of this patient, the presence of R5- and X4-tropic viruses does not seem to result from a local coinfection or superinfection by two independent viruses but could likely result from a differential genetic evolution between the blood and the genital compartment over time (3). Moreover, because of the negativity of the PCR Y chromosome in the genital sample, we can exclude the presence of seminal pollution by X4-tropic strains in the vaginal lumen of woman 259 (3). Taken together, these data demonstrate that the female genital tract can harbor both X4-tropic and R5-tropic variants at the chronic phase of infection and that female genital shedding is not limited to monotropic viruses. The biological characterization of X4-tropic viral variants in the genital fluids suggests the presence of permissive cells in the female genital tract, allowing a replicative local infection by such HIV variants (3, 4). Moreover, these X4-tropic viruses may be deposited onto the sexual male mucosal surfaces during heterosexual intercourse.

In conclusion, our study shows that the viral genetic compartmentalization between blood and the female genital tract can be associated with phenotypic CCR5 or CXCR4 coreceptor usage differences between these two compartments in clade A HIV-1 chronically infected women. Moreover, using a virus phenotypic assay, our data show that the female genital tract constitutes an independent viral compartment that can harbor both X4-tropic and R5-tropic HIV-1 variants at the chronic phase of infection. These findings may be of major interest for understanding mechanisms of HIV-1 transmission and in optimizing vaccine or therapeutic regimens.

 
We thank all the attendees of the Centre National de Référence des Maladies Sexuellement Transmissibles et du SIDA of Bangui.

K.S. is at present an employee of BioAlliance-Pharma. None of the other authors of the present work have a commercial or other association that might pose a conflict of interest. This work has not been previously presented in part in a recent meeting.

This work was supported by the Agence Nationale de Recherches sur le SIDA (ANRS, grant number FF-111-F, and doctoral fellowship to K.S.) and by SIDACTION, Paris, France.

 
* Corresponding author. Mailing address: Laboratoire de Virologie, Service de Microbiologie, Hôpital Robert Debré, Avenue du Général Koenig, 51092 Reims Cedex, France. Phone: 33 3 26 78 39 93. Fax: 33 3 26 78 41 34. E-mail: landreoletti{at}chu-reims.fr

Published ahead of print on 25 April 2007.

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Journal of Clinical Microbiology, June 2007, p. 1838-1842, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.00113-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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