Abstract: The V1/V2 domain of the HIV-1 gp120 envelope protein has been shown to contribute to viral cell tropism during infection and also to viral recognition by neutralizing monoclonal antibodies. However, this domain has been poorly investigated. Carbohydrates have been demonstrated to dramatically influence immune reactivity of antisera to viral glycoprotein antigens. In this study, DNA sequences coding for V1/V2 domains from HIV-1 primary isolates of three subtypes (A, B, and C) were subcloned into a secretion vector and used to transfect CHO cells that are able to achieve the glycosylation of proteins. The structure of purified recombinant V1/V2 proteins was tested using two anti-V1/V2 monoclonal antibodies directed against either a linear or a conformational and glycosylation-dependent epitope (8.22.2 and 697-D). Serum or saliva of 14/82 seropositive patients with anti-V1/V2 reactivity demonstrated good recognition of the recombinant proteins. Deglycosylation of the recombinant proteins was found to increase the reactivity of the serum IgG to the clade A and C but not to clade B V1/V2 domain demonstrating that the recognition of glycosylation sites by serum IgG is clade dependent. When considering SIgA from parotid saliva, deglycosylation of all recombinant proteins tested decreased the reactivity, suggesting that glycosylation plays an important role in the recognition of V1/V2 domain target epitopes by this class of antibodies. In conclusion, these results suggest the influence of carbohydrate moieties on the specificity of the antibodies to the V1/V2 domain produced during HIV infection and the potential importance of viral glycans in vaccine responses after mucosal administration.

Abstract: BACKGROUND: Heterosexual human immunodeficiency virus (HIV) transmission implies the crossing of the vaginal mucosa by virions present in the semen, potentially using Langerhans cells as transporters. The recruitment of these cells in the mucosa is mediated by the chemokine macrophage inflammatory protein 3alpha (CCL20). The aim of this study was to evaluate the capacity of the semen to induce Langerhans cell recruitment via the production of CCL20 by vaginal epithelial cells. METHODS AND RESULTS: Using a vaginal epithelium model based on the SiHa cell line and human seminal plasma, we demonstrated that semen enhanced the production of CCL20. This secretion was regulated by the nuclear factor-kappaB intracellular signalling pathway. Fractionation of the seminal plasma indicated that the secretion of CCL20 was stimulated by high molecular weight compounds present in semen. Migration assays demonstrated that secreted CCL20 was able to promote the recruitment of Langerhans cell precursors (LCps), which remain permissive to X4 and R5 HIV infection. CONCLUSIONS: Our results demonstrate that epithelial cells respond to factors present in semen by secreting CCL20, leading to the enhancement of LCp recruitment. These data argue in favour of the implication of epithelial cells in the heterosexual transmission of HIV.

Abstract: BACKGROUND: Cytokines present in the sperm could influence the heterosexual transmission of HIV by modulating viral titers and influencing the early immune response in the vaginal mucosa. OBJECTIVES: To assess the relation between cytokine concentrations and HIV status in the seminal plasma. STUDY DESIGN: Twenty-five HIV positive subjects were tested for cytokine content and HIV-1 load in seminal and blood plasma through a cross-sectional study. RESULTS: HIV positive subjects exhibited a significantly higher amount of seminal IL-1beta as compared to a group of 33 HIV negative controls. In HIV positive subjects, amounts of IL-1beta and HIV-1 RNA in semen were significantly correlated and a trend for correlation was found between seminal IL-1beta and blood HIV-1 RNA. Amount of seminal IL-1beta was significantly lower in patients under HAART, according to the decrease of their viral loads in blood and semen. CONCLUSIONS: Considering that IL-1beta is known to enhance viral replication and to promote immune response, its dosage in semen could represent an interesting marker for identifying patients at high risk for HIV heterosexual transmission.

Abstract: Mucosa represents the main site of pathogen/cell interactions. The two main types of cells forming the epithelial structure [epithelial cells and Langerhans cells (LC)] coordinate the first defense responses to avoid infection. To evaluate the involvement of epithelial cells in the early steps leading to a specific adaptive immune response, we have studied the interactions between vaginal epithelial and LC through the establishment of a human vaginal epithelial mucosa. We demonstrate that normal human vaginal epithelial cells constitutively secrete the chemokine macrophage inflammatory protein 3alpha/CC chemokine ligand 20 (CCL20), known to recruit LC precursors (LCps) selectively via its cognate CC chemokine receptor 6 (CCR6). This secretion is up-regulated by the proinflammatory cytokine interleukin-1beta through the nuclear factor-kappaB pathway. Similar results were obtained with the human vaginal epithelial cell line SiHa, which displays numerous homologies with normal vaginal cells. The chemotactic activity of the secreted CCL20 was demonstrated by its ability to attract LCp CCR6+. Moreover, the use of neutralizing polyclonal antibodies directed against the CCL20 molecule abolished this migration completely, suggesting that CCL20 is the main attracting factor for LCps, which is produced by the vaginal cells. These data indicate that vaginal epithelial cells play an important role in the immunological defense by attracting immune cells to the site of epithelial/pathogen contact.

Abstract: X4 and R5 HIV strains are present in the semen of men infected with HIV but R5 isolates are transmitted preferentially. The role of human epithelial cells in this selection is addressed. Three human cervical cell lines-CaSki, SiHa, and HEC1A-and normal human vaginal cells from HIV-negative donors were characterized for HIV receptor expression and incubated with X4 and R5 laboratory-adapted strains or primary isolates. The infection was assessed by detection of intracellular HIV DNA. The three cell lines were shown to express on their surface the CXCR4 and GalCer molecules, but not the CD4 and CCR5 ones. The three cell lines and normal human vaginal cells were found to be selectively permissive to X4 HIV entry; the preincubation of the cell lines with rhSDF-1 inhibited this infection. The detection of the intracellular proviral DNA in the cell lines and in normal human vaginal cells demonstrated a selective integration of X4 strains. Additional experiments showed that no extracellular RNA was detected in the supernatants of HEC1A cells infected by X4 isolates either after 18 days of culture or after incubation with PHA-stimulated PBMCs and that no transmission occurred after co-culture between infected HEC1A cells and PHA-stimulated PBMCs. These results suggest specific sequestration of X4 strains by genital epithelial cells, which could explain, at least in part, the HIV tropism selection process during sexual intercourse.

Abstract: We have developed an in vitro reconstructed vaginal mucosa integrating Langerhans cells (LCs), obtained by differentiation of umbilical cord blood CD34(+) hematopoietic progenitor cells, and, in this model, we have investigated the infection of LCs by human immunodeficiency virus type 1 (HIV-1). Proviral DNA of X4 (LAI and NL4-3) and R5 (Ba-L) HIV-1 strains were detected in LCs integrated in the reconstituted mucosa. Infection of LCs could be specifically inhibited by the chemokines stromal cell-derived factor 1 (SDF-1) and RANTES (regulated on activation, normally T cell-expressed and -secreted), confirming the presence of functional coreceptors on LCs generated in vitro. A complete inhibition of LCs, by use of azidothymidine, a reverse-transcriptase inhibitor, was also observed. Moreover, HIV-1-infected LCs of the reconstructed mucosa were able to transmit R5 or X4 HIV-1 strains to activated peripheral blood mononuclear cells. Such a model could be a useful tool to study the mechanisms involved in transmission of HIV in the female genital tract.

Abstract: Enterovirus RNA has been found previously in specimens of muscle biopsy from patients with idiopathic dilated cardiomyopathy, chronic inflammatory muscle diseases, and fibromyalgia or chronic fatigue syndrome (fibromyalgia/chronic fatigue syndrome). These results suggest that skeletal muscle may host enteroviral persistent infection. To test this hypothesis, we investigated by reverse transcription-polymerase chain reaction (RT-PCR) assay the presence of enterovirus in skeletal muscle of patients with chronic inflammatory muscle diseases or fibromyalgia/chronic fatigue syndrome, and also of healthy subjects. Three of 15 (20%) patients with chronic inflammatory muscle diseases, 4 of 30 (13%) patients with fibromyalgia/chronic fatigue syndrome, and none of 29 healthy subjects was found positive. The presence of VP-1 enteroviral capsid protein was assessed by an immunostaining technique using the 5-D8/1 monoclonal antibody; no biopsy muscle from any patient or healthy subject was found positive. The presence of viral RNA in some muscle biopsies from patients exhibiting muscle disease, together with the absence of VP-1 protein, is in favor of a persistent infection involving defective viral replication.