Study of Immunity at the Genital Mucosa of HIV-1 Infected and Healthy Women

Eighty-five percent of new cases of HIV infection involve the South and the mode of transmission is predominantly heterosexual. In northern countries, sexual transmission is also majority. These epidemiological considerations clearly indicate that HIV is mainly transmitted mucosally.

Clinical observations show that some people are "resistant" to HIV, ie they do not become infected despite repeated sexual exposure to the virus. The "resistance" to HIV exposed but uninfected partners may be due to the absence of infection of target cells in the mucosa of the host, or to elimination of HIV or of infected cell during effective infection. Biological factors that govern individual resistance to HIV remain poorly understood. An immune response directed against cervicovaginal HIV antigens, including viral envelope glycoproteins has been described in a small number of HIV-negative women sexually exposed to the virus and resistant to infection. These observations demonstrate that there is a compartmentalization of the mucosal immune response of women, which can be induced against HIV antigens independently of systemic humoral immunity. In addition, a cytotoxic cervicovaginal immunity against HIV has been shown in some women, suggesting that viral antigens are presented to the immune system in a HLA-I, and therefore that HIV antigens were able to cross the mucosal barrier. A second hypothesis, which does not exclude the first, is that an immune response can be induced by repeated deposition of viral antigens on the cervicovaginal mucosa. It has been shown in HEPS women (Highly sexually exposed to HIV-1 persistently IgG seronegative purpose) the existence of IgA antibodies directed against gp41 can inhibit both the transcytosis of the virus through a monolayer epithelial cells and to neutralize the infection of CD4 T cells in vitro.

Soluble factors also play an important role in mucosal transmission of HIV. Indeed, lymphokines RANTES, MIP-1a and MIP-1b, SDF-1 natural ligands of HIV co-receptors are found in varying concentrations in vaginal secretions. More recently, it has been shown that the CCL20/MIP3alpha, which is produced by epithelial cells genital HIV could interact with X4-and R5-tropic to inhibit infection.

The most common techniques used to study the immunological and microbiological factors of female genital tract are cervicovaginal lavage (CVL) and swabbing the cervix. As part of a comprehensive approach to different aspects of mucosal immunity, it is necessary to use techniques for the acquisition of a large number of information while using small amounts of samples. Thus, techniques such as transcriptomic analysis on microarrays, which can tell us about changes in thousands of genes, and the use of techniques for multiplex quantification of soluble factors (cytokines and soluble factors of immunity innate mucosal) must be adapted, tested and validated before the start of a vaccine trial so that the samples needed for immunological assessment be exploited optimally.

A first phase I trial (ANRS VAC14) evaluating the safety and immunogenicity in mucosal and systemic recombinant gp160 protein oligomeric administered nasally or vaginally with or without adjuvant (DC-Chol), was performed in women seronegative for HIV. The goal was to induce secretory IgA (SIgA) level specific genital mucosa. In this study, the storage conditions of the supernatants of cervicovaginal secretions and cells were not optimal for a study of the transcriptome. However, on a few samples we have shown the feasibility of transcriptomic analysis using Illumina technology. We were able on these samples to determine the profiles of mRNA expression and cytokine profiles. These preliminary results led us to propose for the new study, to preserve RNA, cell pellets treated with buffer RLT+ Qiagen before freezing at -80 °C. In addition, protease inhibitors are added directly into the washing liquid to preserve cervicovaginal immunoglobulins and cytokines degradation.

We propose here a feasibility study of the immunological and transcriptomic analysis of cervicovaginal samples of women infected or not infected with HIV-1. Secondarily we assess tolerance samples taken by cytobrush and cervicovaginal washings, efficiency and reproducibility of the sample by cytobrush and cervicovaginal lavage for transcriptomic analysis, measurement of cytokines by Luminex technology, quantification of IgG and IgA. In blood we will determine the phenotype of B cells and Tfh cell frequency (T follicular helper) and quantification of serum immunoglobulins and will perform a transcriptomic analysis of blood cells. Finally we will make correlations with the observed responses at the genital mucosa.

This pathophysiological exploratory study will be performed in 20 women infected with HIV-1 and 20 healthy women recruited from two centers in France and will include a screening visit and two visits M0 and M1 during which mucous and blood samples will be performed.

The results of the study will capitalize skills in biology mucosa, using powerful tools to assess mucosal immunological parameters. These standardized tools can be used both for the evaluation of mucosal immune response induced by prototype vaccines, and cohort studies to search for correlates of protection. These tools can also be used in the evaluation of the local tolerance to the application of molecules with microbicides.