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Early archives of genetically-restricted proviral DNA in the female genital tract after heterosexual transmission of HIV-1

Chomont, Nicolasa; Hocini, Hakima; Grésenguet, Gérardb; Brochier, Célinec; Bouhlal, Hichama; Andréoletti, Laurentd; Becquart, Pierree; Charpentier, Charlottea; de Dieu Longo, Jeanb; Si-Mohamed, Alia; Kazatchkine, Michel Da; Bélec, Laurenta

doi: 10.1097/QAD.0b013e328011f94b
Basic Science

Objectives and method: In order to characterize human immunodeficiency virus type 1 (HIV-1) variants that are transmitted in women via heterosexual intercourse, the env V1-V3 sequences of HIV-1 provirus (DNA) and free virus (RNA) in paired samples of blood and cervicovaginal secretions of untreated chronically and primary infected African women were compared.

Results: Env RNA sequences retrieved from plasma and genital compartments formed a single cluster in primary infection. In contrast, env RNA sequences from these two compartments were distinct in chronically infected women. Analysis of proviral DNA of primary infected women showed that most HIV-1 sequences derived from the genital epithelia form independent clusters from HIV-1 sequences of DNA from peripheral blood mononuclear cells and RNA recovered from plasma and genital secretions. Similarly, the analysis of proviral DNA in the genital compartment of chronically infected women showed the persistence of genetically-restricted cluster of HIV-1.

Conclusions: These observations indicate that a viral subpopulation is archived as proviral DNA in the female genital tract early in primary infection, and suggest that HIV-1 variants from the male donor are selected in the female mucosal site during male to female transmission of HIV-1.

From the aUniversité Paris V, Unité INSERM Internationale d'Immunologie Humaine U743, Equipe « Immunité et Biothérapie Muqueuse », Centre de Recherches Biomédicales des Cordeliers, and Laboratoire de Virologie, Hôpital Européen Georges Pompidou, Paris, France

bUnité de Recherche et d'Intervention sur les Maladies Sexuellement Transmissibles et le SIDA, Faculté des Sciences de la Santé, & Centre National de Référence des Maladies Sexuellement Transmissibles et du SIDA, Bangui, Central African Republic

cUniversité Aix-Marseille I, Laboratoire Evolution, Génomique, Environnement, Marseille

dLaboratoire de Virologie, Centre Hospitalier Universitaire, Reims

eUnité 36, Institut de Recherche et Développement, Montpellier, France.

Received 5 October, 2005

Revised 25 September, 2006

Accepted 13 October, 2006

Correspondence to Laurent Bélec, MD, PhD, Université Paris V, Unité INSERM Internationale Immunologie Humaine, Equipe « Immunité et Biothérapie Muqueuse », Centre de Recherches Biomédicales des Cordeliers, 15, rue de l'Ecole de Médecine, 75006 Paris, France. e-mail: laurent.belec@u430.bhdc.jussieu.fr

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Introduction

Male-to-female heterosexual transmission of the human immunodeficiency virus type 1 (HIV-1) is mainly characterized by the selection of HIV variants between the transmitter and the newly infected female recipient [1], the homogeneous nature of circulating HIV early in infection [2], and a subsequent compartmentalization of HIV variants between female genital secretions and blood in chronic infection [3–5]. Thus, homogeneous HIV-1 variants are present in blood at early infection as assessed by genetically-restricted env gene sequences [2,6–8]. In addition, recent evidence indicates that the recipient viruses in plasma are monophyletic, encoding compact, glycan-restricted envelope glycoprotein [9]. In chronic infection, the female genital tract and the blood harbor genetically distinct populations of replicating HIV-1 [3,4,10]. Genetic compartmentalization may be the consequence of the tissular, cellular and cytokinic microenvironment and of the HIV-specific immune response, within the mucosal and systemic compartments [4]. In acutely HIV-1-infected women, provirus populations were genetically distinct between the cellular fraction of cervical secretions and peripheral blood mononuclear cells [10], suggesting that compartmentalization with the female genital tract probably occurs early during virus mucosal transmission. In the present study, we investigated the genetic diversity of HIV-1 variants in the female genital compartment at the time of primary infection.

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Material and methods

Study population

Consecutive women attending the ‘Centre National de Référence des Maladies Sexuellement Transmissibles et du SIDA’ in Bangui, Central African Republic (CAR), were enrolled in a prospective study cohort. We followed the ethical recommendations of the CAR Ministry of Health, and oral informed consent was obtained from all participants. Paired samples of cervicovaginal secretions (CVS) and peripheral blood were collected. All women were asymptomatic with regard to HIV disease. No woman was treated with antiretroviral therapy at the time of sampling. The characteristics of the study population have been reported previously [11].

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Sample processing and clinical monitoring

Plasma and serum were separated from ethylenediamine-tetraacetic acid-anticoagulated and clotted blood. CVS were collected by vaginal washing with 3.0 ml of phosphate-buffered saline, as previously described [12]. The cellular and the cell-free fractions of CVS were separated by centrifugation at 1000 g for 10 min. Samples containing hemoglobin were excluded by second-derivative spectrophotometry (sensitivity threshold, 10 μg/ml), as described [13], and/or traces of contaminating semen, as determined by means of a Y chromosome polymerase chain reaction (PCR) in DNA extracted from the cellular pellet of CVS [14]. HIV-1 seropositivity was diagnosed by enzyme-linked immunosorbent assay followed by western blotting. HIV seronegative sera, and sera showing incomplete patterns upon western blotting were subjected to a gag-specific reverse transcriptase (RT)-PCR that is sensitive for all the subtypes found in our study population [15]. In sera exhibiting complete western blot patterns, a potassium thiocyanate (KSCN)-based dissociation assay of gp160-anti-gp160 complexes was used to distinguish between acute and chronic infections, as described [16,17].

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Polymerase chain reaction amplification and cloning of HIV-1 env gene

RNA was extracted from plasma and from the acellular fraction of CVS using the Qiamp Viral RNA minikit (Qiagen AG, Basel, Switzerland). DNA was extracted from peripheral blood mononuclear cells (PBMC) and from the pellet of centrifuged CVS using the Qiagen DNA minikit (Qiagen AG). RNA was reverse transcribed and amplified by means of the Superscript one step RT-PCR kit for long templates (Invitrogen, Groningen, The Netherlands). RT-PCR was carried out with E00 and ES8b as primers which allow amplification of a region of gp120 including V1, V2 and V3. Products of the first PCR were then amplified in a nested PCR assay using the primers E20 and E115. In order to minimize possible virus population sampling bias, a minimum of four independent PCR products obtained from each sample were used for cloning. In addition, for all PCR-positive samples, multiple PCRs were performed with serial 10-fold dilutions of DNA extracted from PBMC and cellular pellet of CVS. cDNA obtained after reverse transcription of RNA extracted from plasma and acellular fraction of CVS were similarly diluted. In all cases, V1-V3 envelope gene could not be amplified when DNA template or cDNA were diluted.

Cloning was carried out by means of the TOPO TA Cloning kit for sequencing (Invitrogen). Plasmids were extracted using the REAL prep 96 plasmid kit (Qiagen AG). The insert size was evaluated following digestion of the plasmid with EcoRI (Invitrogen). Plasmids containing inserts of the expected size were further sequenced.

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Molecular analyses

Nucleotide sequences were analyzed using Sequence Navigator and were aligned by using the Clustal X program [18] followed by visual inspection and manual adjustment. Pair-wise distances among sequences were estimated by the DNADist program in the Phylip package [19]. Average genetic distances were calculated as the mean of pair-wise distances in a given population. Phylogenetic trees were built using Phylip. Distance-matrix-based trees were estimated with the neighbor-joining method [20] using the Kimura's two-parameter model of nucleotide substitution [21] excluding positions where gaps were present in any sequence. The reliability of the branching order was estimated by performing 1000 bootstraps replicates.

Assessment of the compartmentalization of HIV variants was performed using the cladistic approach, as described by Slatkin and Maddison [22] and applied by Poss et al. [23]. In brief, for each woman, phylogenetic trees were constructed by parsimony using DNApars from the phylip package [24] for sequences coming from each couple of compartments. Each sequence was further recoded as a two-states character according to the compartment where it comes from. The number of evolutionary steps required to fit these character-states to the most parsimonious trees found previously was estimated using DNApars [24]. The observed mean number of compartment changes, s, was then estimated from 1000 bootstrap trees inferred from the nucleotide sequences with seqboot from the Phylip package. To test if s was smaller than expected from an equivalent HIV variants population with no compartmentalization (that corresponds to the null hypothesis of no compartmentalization of HIV variants), 109 random phylogenetic trees were generated, and the calculated mean number of compartment changes under the null hypothesis, s′, was estimated using the random trees search option of the software PAUP 3.1 (Phylogenetic Analysis Using Parsimony and other methods [20,25]). Finally, the observed s and calculated s′ distributions were compared.

The number of potential N-linked glycosylation sites on V1-V3 of gp120 was determined by using the N-glycosite tool from the Los Alamos HIV sequence database. Amino-acid sequences of the V3 loop were used to predict coreceptor usage, as previously described [26], using the net charge of the V3 loop and the nature of the amino acid in positions 11 and 25 [27–29].

Nucleotide sequences were further aligned to reference sequences and submitted to the Los Alamos HIV sequence database for HIV subtype identification.

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Statistics

Average genetic distances were compared using the non parametric Mann–Whitney U test. Numbers of N-linked glycosylation sites in each compartment were compared using the Student t-test. The proportions of viruses using CCR5 as coreceptor were compared by the binomial proportions test.

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Nucleotide sequence accession numbers

All reported sequences have been submitted to GenBank (accession numbers AY502140 to AY502591).

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Results

Study population

Paired samples of genital secretions and blood were obtained from 274 consecutive women. In the study samples, 135 CVS from menstruating women or containing hemoglobin (60/274, 22%) and/or traces of contaminating semen (100/274, 36%) were excluded. Among 139 women for whom vaginal secretions were devoid of hemoglobin and Y chromosome, 36 (26 %) were seropositive for HIV-1. Among these latter women, four showed indeterminate patterns of western blot suggesting HIV seroconversion with positive gag-specific RT-PCR; and two showed complete patterns of western blot with low KSCN avidity indexes reflecting recent infection (less than 3 months) (Table 1). Furthermore, two of the 103 HIV-1-seronegative women were found to be positive upon screening by gag-specific RT-PCR used to diagnose early acute infection. Thus, eight women were diagnosed as early infected by HIV. Among the population of 30 HIV-1-infected women with typical complete western blot pattern and high KSCN avidity indexes, seven women were randomly selected as controls (Table 1).

Table 1

Table 1

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Variability of the HIV-1 env gene in acutely and in chronically infected women

A total of 452 env sequences were obtained from free virions and cell-associated-viruses in the systemic compartment and the genital tract of the 15 women in the study population. Sequences of viruses from each of the 15 women clustered together demonstrating no contamination between patients (data not shown). To assess the variability of the env gene of viruses cloned from the genital tract and from peripheral blood, we calculated the average genetic diversity of V1-V3 sequences in samples from both compartments when the biological sample was available and successfully amplified by PCR. HIV-1 RNA was successfully amplified from all available plasma samples (14 out of 15 women, Table 1) and from the CVS samples of five among eight acutely and five among seven chronically HIV-1-infected women (Fig. 1). The average genetic diversity of plasma HIV-1 RNA was significantly lower in samples from acutely infected women (mean of 0.27%) than in samples from chronically infected women (mean of 1.98%) (P = 0.0006). This statistical difference was not observed when comparing the average genetic diversities of CVS HIV-1 RNA between the two groups (1.55 and 0.64%, respectively; P = 0.42). Finally, in acutely infected women, as in the chronically infected ones, the mean average genetic diversities of HIV-1 RNA in plasma and in paired CVS were not statistically different (P = 0.37 and P = 0.06, respectively).

Fig. 1

Fig. 1

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Compartmentalization of HIV-1 RNA in systemic and genital compartments

We further compared the V1-V3 env sequences of HIV-1 RNA variants in systemic and genital compartments of four acutely infected (#36, #44, #57 and #270) and five chronically infected (#29, #104, #128, #149 and #214) women (Table 1).

Phylogenetic trees of variants in samples obtained from acutely-infected women (#36, #44 and #57) demonstrated that viral populations were homogeneous between the plasma and genital compartments (Fig. 2, upper panel), suggesting the absence of viral compartmentalization between systemic and genital compartments at the early stage of infection. Thus, in women #36 and #57, all HIV-1 RNA sequences derived from the systemic and genital compartments were closely related. In woman #44, only three of 29 clones obtained from CVS and plasma were found to be genetically distant. Altogether, our results indicate that these three acutely-infected women did not exhibit partitioning of CVS from plasma V1-V3 HIV-1 RNA sequences, as depicted in Table 2, confirming no significant compartmentalization of HIV-1 RNA variants between blood and genital compartments early in infection. In contrast, statistical analysis demonstrated significant compartmentalization between V1-V3 HIV-1 RNA sequences detected in genital and systemic compartments of woman #270 (Table 2; P value < 0.001), although the limited number of positive RT-PCR (n = 2) and clones (n = 8) obtained from her genital secretions could have biased the result. When analyzing phylogenetic trees from the three chronically HIV-1-infected women #29, #104 and #128, distinct patterns were observed (Fig. 2, lower panel). Env sequences of HIV-1 RNA variants in plasma were highly heterogeneous whereas sequences derived from CVS were highly homogeneous. In women #104 and #128, the HIV-1 RNA populations from the systemic and genital compartments formed two distinct clusters supported by high bootstrap values (999 and 1000, respectively). In woman #29, three among 16 HIV-1 variants from the systemic compartment were more closely related to variants detected in the genital compartment than to variants from plasma. The apparent compartmentalization of the HIV-1 variants between the systemic and compartments observed in these three women was confirmed by the cladistic statistical analysis (Table 2; all P values < 0.001). Interestingly, the statistical analyses for the two remaining chronically-infected women #149 and #214, who were infected more recently than women #29, #104 and #128 (as indicated by the KSCN index in Table 1), showed no significant compartmentalization of HIV-1 variants between the systemic and genital compartments (Table 2; P values = 0.25 and 0.09, respectively). Taken together, these observations indicate that the compartmentalization of HIV-1 RNA populations is much stronger in chronically HIV-1-infected women than in acutely HIV-1-infected women.

Fig. 2

Fig. 2

Table 2

Table 2

We then determined the number of potential N-linked glycosylation sites in the 452 clones derived from the study population (Fig. 2). The mean number of glycosylation sites on V1-V3 sequences derived from plasma was compared to that of sequences derived from CVS. No difference was found between samples obtained from acutely-infected women, whereas three of the five paired samples from chronically-infected women showed significant differences in the number of N-linked glycosylation sites between the two compartments, further demonstrating compartmentalization of HIV-1 in these women. We further determined the predicted tropism of each sequenced clone, in both compartments. All viruses were predicted to use CCR5 (data not shown), which did not allow to evidence viral compartmentalization with regard to coreceptor usage.

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Compartmentalization of genital and systemic HIV-1 RNA and DNA variants

HIV-1 RNA and DNA variants obtained from the four compartments were analyzed for two acutely (#36 and #57) and two chronically (#128 and #149) HIV-1-infected women for whom sufficient material was available (Table 1). In the case of acutely-infected women, the phylogenetic trees showed that HIV-1 RNA and DNA sequences from the systemic compartment and HIV-1 RNA sequences from the genital compartment clustered together (Fig. 3a); in contrast, HIV-1 env DNA clones from the genital compartment constituted a distinct cluster in both women. Statistical analyses confirmed in both women the lack of compartmentalization between the HIV-1 RNA and DNA sequences from the systemic compartment and the HIV-1 RNA variants from the genital compartment, as well as the compartmentalization between the HIV-1 DNA variants from the genital compartment and the HIV-1 RNA or DNA variants from blood and the HIV-1 RNA variants from genital secretions (Table 2). The HIV-1 variants obtained from chronically-infected women (#128 and #149) showed higher variability and overall higher compartmentalization than those obtained from acutely-infected women (Fig. 3a), and this feature was confirmed by the statistical analyses (Table 2).

Fig. 3

Fig. 3

We also analyzed neighbor-joining trees corresponding to HIV-1 RNA and DNA variants retrieved from the genital tract. As depicted in Fig. 3b, HIV-1 DNA and RNA variants from the chronically-infected woman #128 formed two distinct and well-supported clusters (bootstrap values of 1000). Statistical analysis rejected the hypothesis of no compartmentalization in this woman (Table 2; all P values < 0.001). In woman #149, four distinct (i.e. two clusters of HIV-1 DNA sequences and two clusters of HIV-1 RNA sequences) and well-supported (bootstrap values of 1000) clusters were observed. Statistical analysis confirmed the compartmentalization between HIV-1 RNA and DNA variants from genital tract in this woman (Table 2; P values < 0.05). Similarly, statistical analyses demonstrated the compartmentalization between HIV-1 RNA and DNA variants from genital tract in acutely-infected women #36 and #57 (Table 2; P values < 0.05), although the genetic distances between the HIV-1 DNA and RNA genital variants appeared lower than in the case of chronically-infected women (Fig. 3b). In both acutely infected women #36 and #57, but not in chronically infected women #128 and #149, few HIV-1 DNA genital variants seemed more closely related to HIV-1 RNA genital variants, suggesting recent migration events between HIV-1 DNA and HIV-1 RNA genital compartments or a still-in progress compartmentalization. Taken together, these observations indicate that HIV-1 DNA sequences from the genital compartment constitute an independent cluster of viral variants in both acutely and chronically HIV-1-infected women.

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Discussion

In the present study, a viral subpopulation is archived as proviral HIV-1 DNA in the female genital tract early in primary infection, suggesting that HIV-1 variants from the male donor may be selected at the female mucosal level during male-to-female HIV-1 transmission.

All acutely-infected women displayed homogeneous HIV-1 populations in plasma, indicating that viral selection occurs during the early phase of sexual transmission of HIV-1, as previously suggested by Zhu et al. [1]. Variants in plasma were closely related to those retrieved from the genital compartment of acutely-infected women, suggesting that viral selection resulting in an oligoclonal distribution of variants early in infection occurs within the genital compartment and prior to viral dissemination to the systemic compartment. In addition, HIV-1 RNA variants were homogeneous in plasma and genital compartments upon phylogenetic analysis, reflecting the absence of genetic diversity early in infection. Our observations are consistent with previous studies showing that homogeneous HIV strains are present in blood early during primary infection, including in women [2,6–8], but differ from those reporting frequent heterogeneity of HIV-1 variants in blood during primary infection in women living in Kenya [30] or in Rwanda [31]. Thus, Long et al. reported that a heterogeneous virus was present in 18 out of 28 (64%) Kenyan women before seroconversion [30]. One major hypothesis proposed by Long et al. is that there could be a gender-specific, subtype-related transmission of HIV-1, resulting in blood heterogeneity of clades A or D HIV-1 variants during male-to-female, but not female-to-male, heterosexual transmission of the virus. According to that latter hypothesis, the possibility exists that the CRF 11cpx variants of HIV-1 mainly present in our study women may be homogeneously transmitted during male-to-female heterosexual transmission, as previously hypothesized for clade C HIV-1 variants during both female-to-male and male-to-female transmissions [30]. However, other hypotheses on virus diversity in blood during primary infection should be envisioned, including sexual behaviour (sex workers or married women), possible ‘inoculum’ effect of HIV-1 load in semen which is highly variable between men [32], or other yet unknown cofactor of heterosexual HIV transmission.

The mean genetic diversity of HIV-1 V1-V3 RNA sequences in the plasma of acutely infected women was significantly lower than that in the plasma of chronically infected women, as previously reported [1,2,6,33]. When analyzing the phylogenetic trees of variants from chronically infected women, it was observed that env sequences of HIV RNA variants in plasma were highly heterogeneous whereas sequences derived from CVS were highly homogeneous. Furthermore, the number of potential N-linked glycosylation sites was similar in genital and systemic variants from acutely infected women, and limited as previously observed for plasma viruses in acute infection [9], whereas significant differences in the number of N-linked glycosylation sites between variants from the two compartments could be observed in chronically infected women. These observations confirmed that HIV-1 RNA populations are compartmentalized in chronically-infected women, in agreement with previous studies in chronic infection [1,3,4,10,23,34], and demonstrate that compartmentalization is acquired at late stages of the natural history of HIV disease, likely resulting from differential immune selective pressure between mucosal and systemic compartments [35].

We next addressed viral reservoirs in the genital and systemic compartments by molecular and cladistic analyses. In the case of acutely-infected women, HIV-1 RNA and DNA sequences derived from the systemic compartment clustered together. By contrast, most of HIV-1 env DNA clones obtained from CVS constituted a distinct population from that derived from RNA variants. In chronically HIV-1-infected women, four distinct RNA and DNA HIV-1 clusters were observed in blood and cervicovaginal secretions. The finding that DNA and RNA V1-V3 sequences derived from HIV-1 variants in the genital tract do not cluster together in the case of acutely-infected women, suggests that genetically-restricted variants are archived as cell-associated DNA in the genital tract early during primary infection. We can hypothesize that such archived HIV-1 DNA may correspond to the restricted virus subpopulation originating from the male donor that had been selected for transmission. Semen-originating HIV-1 variants may have poor replicating capacity within the female genital tract, thus remaining dominant only in the cell-associated fraction of the cervicovaginal secretions. These latter variants will be at the origin of new HIV-1 variants adapted to replicate within the female genital tissue, and to spread and replicate efficiently within the systemic environment. Thus, the female genital environment may act as a ‘viral filter’ by selecting variants harboring phenotypic advantage to replicate in both the genital and further the systemic compartments. Recently, Miller et al. showed that the mucosal barrier of female rhesus macaque greatly limits the infection of cervicovaginal tissues [36]. It is likely that HIV variants capable of replication in the specific micro-environment of the genital tract of the recipient host then derive from these original clones early during primary infection, in keeping with the hypothesis of viral selection occurring at the mucosal level during heterosexual transmission of HIV-1. Genetically-restricted HIV-1 DNA was also present in genital secretions of chronically-infected women, suggesting long-lasting persistence of archived HIV-1 DNA possibly corresponding to non-replication competent viruses. HIV-1 DNA sequences retrieved from genital secretions may thus represent a genetically-restricted cluster of variants that is archived in the female genital compartment at a very early stage during primary infection. This hypothesis is supported by the presence of genetically distinct HIV-1 DNA and RNA sequences in the genital compartment: the HIV-1 RNA sequences likely reflect the viral evolution in the newly infected host whereas the HIV-1 DNA sequences may represent the archive of the originally transmitted virus. In chronically-infected individuals, distinct immune selective pressure would then lead to divergent evolving genetic patterns of HIV variants in blood and in the genital compartment. The restricted nature of male-to-female transmitted HIV-1 variants provides new perspectives for the development of effective preventive HIV vaccines.

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Acknowledgements

N.C. was the recipient of a scholarship of the Ministry of Research, and of the Agence Nationale de Recherches sur le SIDA et les Hépatites Virales (ANRS), France.

Sponsorship: This work was supported by ANRS, and by the Association pour la Recherche en Infectiologie, Paris.

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Keywords:

HIV; genital tract; mucosal immunity; compartmentalization

© 2007 Lippincott Williams & Wilkins, Inc.