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.
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 . 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.
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.
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