A new member of the immunoglobulin superfamily-CTLA-4. eliminated by cytotoxic mechanisms. Finally, RT-PCR analysis of TCR-V repertoire usage showed that TCR-V12 (S)-Willardiine mRNA was never expressed during the infection. Taken together, these findings improve our understanding about immune response during human asymptomatic Ebola infection, and throw new light on protection against Ebola virus. and studies (S)-Willardiine have shown that filoviruses can infect and propagate in macrophagic and endothelial cells [9C12]. During recent Ebola outbreaks in Gabon, we found that fatal outcome was associated with a suboptimal humoral response, no specific IgG production, and early activation of peripheral blood mononuclear cells (PBMC), followed by extensive intravascular apoptosis . In contrast, survival was associated with early emergence of specific humoral responses, and tightly regulated activation of cytotoxic cells that coincided with clearance of viral antigens from blood. Recently, we identified several individuals with documented close contact with symptomatic EHF patients who nonetheless remained asymptomatic and mounted IgM and IgG responses to the NP and VP40 EBOV proteins . These asymptomatic individuals were infected by EBOV at a low Mouse monoclonal to CD8/CD38 (FITC/PE) level over several days. This first description of asymptomatic replicative Ebola infection raised questions about the mechanism of protection. As (S)-Willardiine symptomatic and asymptomatic subjects were infected by the same viral strain (as suggested by partial genotyping), the protective mechanism may have involved the transient and high circulating levels of proinflammatory cytokines (IL-1, TNF, IL-6, MCP and, MIP-1/) observed in asymptomatics early during the infection (5C7 days after the first putative infectious contact). In the present study, we investigated cellular immune responses (S)-Willardiine in seven asymptomatic individuals from 7 to 23 days after exposure to the virus. We showed that individuals asymptomatically infected with EBOV mounted a strong inflammatory response in temporal association with a counteract anti-inflammatory one. This inflammatory process is then followed by a well regulated T cell response leading to the generation of EBOV-specific IgG1 and IgG3 subclasses responses and to marked and sustained activation of cytotoxic cells involved in the elimination of infected cells from peripheral cirulation. Absence of TCR-V12 mRNA expression during the course of infection requires further investigation to fully understand immune responses. PATIENTS AND METHODS Asymptomatic individuals In a previous study, 7 of 24 exposed individuals were shown to be clearly infected by EBOV . These individuals were family members of symptomatic patients who cared for them with no physical protection such as gloves. They were directly exposed to infected materials from fatal and non fatal cases such as faeces, vomit, sweat and blood. These asymptomatic individuals were sampled 2, 3 or 4 4 times after the first exposure to a sick patient. Plasma was analysed in all 7 individuals, and RT-PCR was performed on samples from 3 of them. All samples (collected 7, 9, 16 and 23 days after the initial exposure) were obtained, treated and stored in exactly the same conditions. Detection of EBOV-specific IgG subclasses We used an ELISA IgG assay, developed at CIRMF, in which Ebo-Z antigens or lysates from normal cultured Vero E6 cells are applied to the microtiter plates overnight at 4C before washing with phosphate buffer saline (PBS) containing 01% Tween 20. The plates are saturated in PBS containing 3% bovine serum albumin (BSA) for 2 h at room temperature. Sera are diluted 1 : 25 in PBS containing 01% Tween 20, and 100 l of each serum is dispensed in duplicate wells and left overnight at 4C. After washing, the plates are incubated with biotin-conjugated mouse antibodies against human IgG subclasses (Sigma, l’Isle d’Abeau, France) for 2 h at room temperature. Binding is then revealed with streptavidin-peroxidase, and the TMB detector system is added (Dynex Technologies, Issy-les-Moulineaux, (S)-Willardiine France). Optical density is measured at 492 nm, on an ELISA plate reader (Diagnostics Pasteur,.