A recombinant novirhabdovirus presenting at the surface the E Glycoprotein from West Nile Virus (WNV) is immunogenic and provides partial protection against lethal WNV challenge in BALB/c mice

Abstract

West Nile Virus (WNV) is a zoonotic mosquito-transmitted flavivirus that can infect and cause disease in mammals including humans. Our study aimed at developing a WNV vectored vaccine based on a fish Novirhabdovirus, the Viral Hemorrhagic Septicemia virus (VHSV). VHSV replicates at temperatures lower than 20°C and is naturally inactivated at higher temperatures. A reverse genetics system has recently been developed in our laboratory for VHSV allowing the addition of genes in the viral genome and the recovery of the respective recombinant viruses (rVHSV). In this study, we have generated rVHSV vectors bearing the complete WNV envelope gene (EWNV) (rVHSV-EWNV) or fragments encoding E subdomains (either domain III alone or domain III fused to domain II) (rVHSV-DIIIWNV and rVHSV-DII-DIIIWNV, respectively) in the VHSV genome between the N and P cistrons. With the objective to enhance the targeting of the EWNV protein or EWNV-derived domains to the surface of VHSV virions, Novirhadovirus G-derived signal peptide and transmembrane domain (SPG and TMG) were fused to EWNV at its amino and carboxy termini, respectively. By Western-blot analysis, electron microscopy observations or inoculation experiments in mice, we demonstrated that both the EWNV and the DIIIWNV could be expressed at the viral surface of rVHSV upon addition of SPG. Every constructs expressing EWNV fused to SPG protected 40 to 50% of BALB/cJ mice against WNV lethal challenge and specifically rVHSV-SPGEWNV induced a neutralizing antibody response that correlated with protection. Surprisingly, rVHSV expressing EWNV-derived domain III or II and III were unable to protect mice against WNV challenge, although these domains were highly incorporated in the virion and expressed at the viral surface. In this study we demonstrated that a heterologous glycoprotein and non membrane-anchored protein, can be efficiently expressed at the surface of rVHSV making this approach attractive to develop new vaccines against various pathogens.

Figure 1. Recovery of six rVHSV expressing WNV antigens by reverse genetics.

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.

Figure 2. Schematic diagram of immunization and challenge schedule.

5-week old BALB/c mice were immunized three times with different antigens at two-week interval. Serum samples from immunized mice were collected every two weeks in order to assess antibody production. One month after the last immunization, the mice were challenged intraperitoneally with a lethal dose (1,000 PFU) of WNV (Israël-98 strain). Blood samples were collected on days 3 and 7 post-challenge to quantify the viremia by qRT-PCR. Mice were sacrificed on day 21 post-challenge, after a last serum sample was harvested.

Figure 3. Expression of E_WNV antigens in rVHSV-infected cells.

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).

Figure 4. Analysis of virion incorporation of E_WNV antigens.

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).

Figure 5. Detection of DIII_WNV at the virus surface of rVHSV-SP_GE_WNV and rVHSV-SP_GDIII_WNVTM_G by immunogold.

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.

Figure 6. Purified rVHSV-SP_GE_WNV is immunogenic and induces the production of antibodies against E_WNV in BALB/c mice.

(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 t test. (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).

Figure 7. Individual production of total IgG after immunization with rVHSV-SP_GE_VWN and rVHSV-SP_GE_VWNΔTMTM_G.

5-week-old BALB/c mice in groups of 12 individuals were immunized three times with different antigens as indicated (see fig.2 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).

Figure 8. Survival Curves of mice immunized with rVHSV carrying WNV E antigens and infected with a lethal dose of WNV.

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).