Utilisation d'un rhabdovirus de poisson pour le développement d'un vaccin vectorisé contre un virus zoonotique (le virus West Nile)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Chikungunya antibodies detected in non-human primates and rats in three Indian Ocean islands after the 2006 ChikV outbreak

International audienceThe role of terrestrial vertebrates in the epidemiology of chikungunya disease is poorly understood. We evaluated their exposure and amplification role during the 2006 chikungunya outbreak in the Indian Ocean. Blood samples were collected from 18 mammalian and reptile species from Reunion Island, Mauritius and Mayotte. Among the 1051 samples serologically tested for chikungunya virus (CHIKV), two crab-eating macaques (Macaca fascicularis) and two ship rats (Rattus rattus) proved to be exposed to CHIKV. CHIKV RNA was not detected in 791 analyzed sera. Our results confirm the preferential infection of simian primates and suggest that other vertebrates played a poor or no role in CHIKV transmission during the 2006 outbreak

Purified rVHSV-SP_GE_WNV is immunogenic and induces the production of antibodies against E_WNV in BALB/c mice.

<p>(A) Schematic diagram of immunization. Five 6-week-old BALB/c mice were injected subcutaneously with 10 μg of purified rVHSV-SP_GE_WNV three times at two-week interval. Mice sera were taken before the first immunization (day 0), one week (day 32) and three weeks (day 44) after the last immunization. (B) The presence of antibodies against WNV E glycoprotein in the serum of immunized mice was analyzed by ELISA on days 0 and 44 after the last immunization. Statistical significance was determined by <i>t test</i>. (C) Example of antibody specificity against WNV E and VHSV structural proteins tested by Western Blotting. Two μg of total viral proteins from sucrose purified-VHSV and 1 μg of rE_WNV were separated on a SDS-12% polyacrylamide gel and electrotransferred in a nitrocellulose membrane. The mouse sera, harvested 1 and 3 weeks after the last immunization (day 32 and 44, respectively), were used as primary antibodies to detect the five structural proteins of VHSV (left lane) and the rE_WNV (right lane).</p

Individual production of total IgG after immunization with rVHSV-SP_GE_VWN and rVHSV-SP_GE_VWNΔTMTM_G.

<p>5-week-old BALB/c mice in groups of 12 individuals were immunized three times with different antigens as indicated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091766#pone-0091766-g002" target="_blank">fig.2</a> for schedule details). Antibody production by each mouse was assessed by ELISA with individual sera collected on day 56 just before the WNV challenge. Each serum was diluted to 1∶100. OD values for each individual are represented directly after removal of the white (OD measured in the absence of serum).</p

Detection of DIII_WNV at the virus surface of rVHSV-SP_GE_WNV and rVHSV-SP_GDIII_WNVTM_G by immunogold.

<p>Sucrose purified-recombinant viral particles were adsorbed on electron microscopy nickel grids. After fixation, DIII_WNV and the glycoprotein G of VHSV (G_VHSV) were detected using specific mouse primary monoclonal antibodies. These mouse primary antibodies were detected by an anti-mouse secondary antibody coupled with a gold particle (black dots of 5 nm in diameter). After negative staining, recombinant viral particles were observed by transmission electron microscopy.</p

Recovery of six rVHSV expressing WNV antigens by reverse genetics.

<p>Six recombinant viruses containing an expression cassette between the N and P genes were recovered. These rVHSV expressing the entire WNV E glycoprotein (E_WNV; #2), or E_WNV fused to the signal peptide (SP_G) of the glycoprotein G of IHNV (SP_GE_WNV; #3), or E_WNV fused to the SP_G of the IHNV G and the transmembrane region (TM_G) of the VHSV G (SP_GE_WNVTM_G; #4), or fragments of E_WNV fused to SP_G and TM_G: the ectodomain part of E_WNV (SP_GE_WNVΔTMTM_G; #5), the domain III alone (SP_GDIII_WNVTM_G; #7) or associated with a portion of domain II (SP_GDIIDIII_WNVTM_G; #6). The titer of each rVHSV is indicated on the right.</p

rVSHV-SP_GE_WNV and rVSHV-SP_GE_WNVΔTMTM_G partially protect mice against a lethal WNV challenge.

<p>(a) The percentage of weight loss was calculated everyday post-challenge during 15 days. This percentage is based on the variation of weight observed everyday compared to that measured the day of the WNV challenge.</p><p>(b) Number of surviving mice on day 21 post-challenge.</p><p>(c) WNV genomic RNA was detected by quantitative RT-PCR in the blood of mice collected on days 3 and 7 post-challenge.</p

Expression of E_WNV antigens in rVHSV-infected cells.

<p>The expression of E_WNV antigens was assessed by indirect-immunofluorescence in BF-2 cells. The cells were infected or not infected (Mock) either with the empty vector (rVHSV) or the six recombinant viruses expressing E_WNV domains (as indicated). Cells were incubated for 48 h at 14°C. (A) At 48 h post-infection, cells were fixed and permeabilized with a mixture of alcohol/acetone, and protein expression was detected using a monoclonal antibody against E_WNV DIII (E24). (B) Detection of membrane expression of E_WNV antigens was performed on live cells using the E24 antibody, except for rVHSV-SP_GDIIDIII_WNVTM_G infected cells where E_WNV expression was achieved with mAb8150 (magnification X63).</p

Analysis of virion incorporation of E_WNV antigens.

<p>Sucrose gradient-purified viral proteins were separated on a SDS-12% polyacrylamide gel. (A) Ten μg of total viral proteins were visualized after Coomassie blue staining. (B) Four μg (lanes 1 to 6) and 2 μg (lane 7) of total viral proteins were loaded. The gel was electrotransferred onto a nitrocellulose membrane and incubated with a mixture of mAb8150 and E24 anti-E_WNV antibodies. (C) Thirty μg of total viral proteins were visualized after Coomassie blue staining. Lane 8 corresponds to rE_VWN (1 μg was loaded on each gel).</p

Survival Curves of mice immunized with rVHSV carrying WNV E antigens and infected with a lethal dose of WNV.

<p>At 28 days after the last immunization (day 56), immunized mice were challenged intraperitoneally with a lethal dose (1,000 PFU) of WNV (Israël-98 strain). Mice were observed daily for signs of morbidity. For statistical grouping, a comparison of survival between groups was performed with the log rank test on the Kaplan-Meier survival data using GraphPad Prism (GraphPad, San Diego, CA). Groups that were assigned to statistically similar groups (and, thus, share a letter) are not significantly different from each other (P>0.05), whereas those that were not assigned to statistically similar groups are significantly different (P<0.05).</p