Complex adenovirus-mediated expression of West Nile virus C, PreM, E, and NS1 proteins induces both humoral and cellular immune responses

Abstract

West Nile Virus (WNV), a member of the family Flaviviridae, was first identified in Africa in 1937. In recent years, it has spread into Europe and North America. The clinical manifestations of WNV infection range from mild febrile symptoms to fatal encephalitis. Two genetic lineages (lineages I and II) are recognized; lineage II is associated with mild disease, while lineage I has been associated with severe disease, including encephalitis. WNV has now spread across North America, significantly affecting both public and veterinary health. In the efforts to develop an effective vaccine against all genetic variants of WNV, we have studied the feasibility of inducing both neutralizing and cellular immune responses by de novo synthesis of WNV antigens using a complex adenoviral vaccine (CAdVax) vector. By expressing multiple WNV proteins from a single vaccine vector, we were able to induce both humoral and cellular immune responses in vaccinated mice. Neutralization assays demonstrated that the antibodies were broadly neutralizing against both lineages of WNV, with a significant preference for the homologous lineage II virus. The results from this study show that multiple antigens synthesized de novo from a CAdVax vector are capable of inducing both humoral and cellular immune responses against WNV and that a multiantigen approach may provide broad protection against multiple genetic variants of WNV.

FIG. 1.

WNV and CAdVax vector genomes. (A) Schematic representation of the structure of a single positive-stranded RNA genome of WNV. Open boxes, ORFs of WNV proteins that are synthesized as a polyprotein precursor, which is processed into each individual protein, as labeled (NC, noncoding region). (B) Genome structure of CAdVax-WNVII that expresses the C, preM, E, and NS1 proteins of WNVII. Large arrows, promoter regions; boxes with lines, poly(A) site, as labeled. The expression cassette containing the C and NS1 proteins is inserted at the left end of the vector within the E1 region that has been deleted from the Ad genome, and the cassette expressing the E and preM proteins is inserted in the right end of the vector into the E4 region that has also been deleted, except for orf6 (ITR, inverted terminal repeat; hCMVie, human cytomegalovirus intermediate-early promoter; BGH-poly(A), bovine growth hormone polyadenylation site; ψ, Ad packaging signal.

FIG. 2.

Western blot analysis of E-protein expression. Whole-cell lysates of Vero cells transduced with CAdVax-WNVII and controls were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane, as described in Materials and Methods. Lysates from uninfected cells and cells transduced with an Ad vector expressing the Marburg virus nucleoprotein antigen (CAdVax-M11) were used as negative controls. MAb 7H2 was used to probe for the WNV E protein (∼52 kDa), and a polyclonal β-actin antibody was used as an internal control for total protein loading.

FIG. 3.

Immunochemical staining of WNV proteins in CAdVax-WNVII-transduced cells. Vero cells transduced at an MOI of 30 with CAdVax-WNVII (right column) or the negative control vaccine, CAdVax-M11 (left column), are shown. At 2 days posttransduction, the cells were fixed and probed with primary antibodies specific for E, preM (PrM), M, or NS1 and stained with FITC-conjugated secondary antibodies. The fluorescence in positively stained cells was visualized with Axiovert-25 UV-microscope with FITC filters. Magnification, ×300.

FIG. 4.

CAdVax-WNVII vaccination of mice induces humoral immune response. ELISAs were conducted to determine the antibody titers in sera taken from groups of mice vaccinated with CAdVax-WNVII, the CAdVax-M11 control, or PBS. An MAb against the WNV E protein was used as a positive control. The arrows indicate vaccination time points. Titers were defined as the dilution of serum that produced a positive signal two times that of the background. Each point represents the mean titer for five individual animals, with standard deviations indicated by the bars above and below each point. Statistical analysis was performed by using an ANOVA model (**, P < 0.01; *, P < 0.05).

FIG. 5.

CAdVax-WNVII vaccination of mice induces neutralizing antibody responses. Sera from mice vaccinated with CAdVax-WNVII, CAdVax-M11, or PBS were collected 2 weeks after the booster immunizations and were used for plaque reduction neutralization tests. Serial dilutions of each serum sample were tested for neutralizing activity against a lineage I virus (strain NY99) and a lineage II virus (strain Uganda37), as described in Materials and Methods. Serial dilutions of sera were incubated with each virus strain, followed by infection of Vero cell monolayers under an agar medium overlay. Each point represents the mean number of plaques for five individual animals, with standard deviations indicated by the bars above and below each point. Statistical analysis was performed by using an ANOVA model (**, P < 0.01; *, P < 0.05).

FIG. 6.

Vaccination with CAdVax-WNVII induces cell-mediated immune responses against multiple WNV antigens. Four C57BL/6 mice from each vaccine group were euthanized 10 weeks after the primary vaccinations, and splenocytes from each animal were collected and analyzed individually for WNV-specific CTL activity by using a murine IFN-γ ELISPOT assay. Splenocytes were incubated with syngeneic target cells expressing either the preM and E proteins or the C and NS1 proteins. As negative and positive controls, splenocytes were incubated with media only or concanavalin A, respectively. Following incubation, the numbers of IFN-γ SFU were detected by ELISPOT assay and counted on an AID ELISPOT assay plate reader. The results are presented as the number of IFN-γ SFU per 2 × 10⁶ cells. Statistical analysis was performed by using an ANOVA model (**, P < 0.01; *, P < 0.05); bars, standard deviations; ConA, concanavalin A; RV, retrovirus.