Nonstructural protein 2 (nsP2) of Chikungunya virus (CHIKV) enhances protective immunity mediated by a CHIKV envelope protein expressing DNA Vaccine

Abstract

Chikungunya virus (CHIKV) is an important emerging mosquito-borne alphavirus, indigenous to tropical Africa and Asia. It can cause epidemic fever and acute illness characterized by fever and arthralgias. The epidemic cycle of this infection is similar to dengue and urban yellow fever viral infections. The generation of an efficient vaccine against CHIKV is necessary to prevent and/or control the disease manifestations of the infection. In this report, we studied immune response against a CHIKV-envelope DNA vaccine (pEnv) and the role of the CHIKV nonstructural gene 2 (nsP2) as an adjuvant for the induction of protective immune responses in a relevant mouse challenge model. When injected with the CHIKV pEnv alone, 70% of the immunized mice survived CHIKV challenge, whereas when co-injected with pEnv+pnsP2, 90% of the mice survived viral challenge. Mice also exhibited a delayed onset signs of illness, and a marked decrease in morbidity, suggesting a nsP2 mediated adjuvant effect. Co-injection of the pnsP2 adjuvant with pEnv also qualitatively and quantitatively increased antigen specific neutralizing antibody responses compared to vaccination with pEnv alone. In sum, these novel data imply that the addition of nsP2 to the pEnv vaccine enhances anti-CHIKV-Env immune responses and maybe useful to include in future CHIKV clinical vaccination strategies.

FIG. 1.

Plasmid CHIKV-nsP2 construction and in vitro expression. (a) Schematic diagram of the CHIKV-nsP2 gene Including the Kozak consensus sequence and immunoglobulin E (IgE) leader; (b) pVax1 backbone vector indicating the inclusion of the nsP2 gene insert; (c) indirect immunofluorescence assay of transfected HeLa cells expressing pVax1 or pnsP2. Images indicate transfected cells viewed under Zeiss LSM510 confocal microscope, following the addition of anti-nsP2 antisera, followed by staining with a FITC-conjugated mouse secondary antibody (upper and lower left panel) or DAPI stain (upper and lower middle panels), followed by an overlayed image (FITC+DAPI shown in the upper right and lower panels); (d) immunoprecipitation of synthesized CHIKV non-structural protein 2 (nsP2), from the transfection/expression reaction mixtures, using a His-tag antibody. The image indicates that the protein product from the gene construct pnsP2 is at its predicted molecular weight.

FIG. 2.

Immunization schema and post-vaccination antibody responses. (a) Vaccination and blood/tissue sampling time course. (b) Anti-CHIKV antibody measurement by ELISA. Sera were collected a week after the third immunization and the total IgG produced was measured in each group. Values represent the mean (±SD) of duplicate wells; p values (p<0.0085) indicate that the data are statistically significant between pEnv and pEnv+pnsP2 (i.e., the titer for pEnv + pnsP2 vaccinated animals is significantly higher. (c) IgG subtype ELISA analysis. The graph shows CHIKV-Env specific IgG1, IgG2a, IgG2b, and IgG3 antibody levels in mice that were vaccinated with pEnv or pEnv+pnsP2. Error bars represent standard deviation (±S.D) of duplicate wells. (d) IgG1 to IgG2a level binding ratios are shown for mice (n=5) immunized with pEnv or pEnv+pnsP2 intramuscularly, delivered through MID-EP. The values shown represent the mean (±S.D). The ratios were not significantly different.

FIG. 3.

Immunogenicity of CHIKV-nsP2. As indicated in the Materials and Methods section, C57BL/6 mice were immunized three times with 50 μg of pEnv, pEnv+pnsP2, pnsP2, or control pVax1 constructs and sacrificed 1 week following last immunization. Splenocytes were harvested, stimulated overnight with the specific peptide pools from proteins (a) Env-E1 or (b) Env-E2 and (c) Nsp2 and plates were processed and the spot forming units (SFU) were quantified by an automated ELISpot reader. For the NsP2 we used only the pools from the overlapping peptide of the protein. The raw data was normalized to SFU per million splenocytes. Values represent the mean of duplicate wells.

FIG. 3.

Immunogenicity of CHIKV-nsP2. As indicated in the Materials and Methods section, C57BL/6 mice were immunized three times with 50 μg of pEnv, pEnv+pnsP2, pnsP2, or control pVax1 constructs and sacrificed 1 week following last immunization. Splenocytes were harvested, stimulated overnight with the specific peptide pools from proteins (a) Env-E1 or (b) Env-E2 and (c) Nsp2 and plates were processed and the spot forming units (SFU) were quantified by an automated ELISpot reader. For the NsP2 we used only the pools from the overlapping peptide of the protein. The raw data was normalized to SFU per million splenocytes. Values represent the mean of duplicate wells.

FIG. 4.

CHIKV viremia and pathogenesis in vaccinated mice. Ten mice from the control or the experimental vaccination groups were inoculated with 7log₁₀ PFU (plaque forming units) of CHIKV by the intranasal route and subsequently examined for viremia, disease phenotype, and survival, and sacrificed for pathologic analysis. In this viral challenge model, (a) pVax1 and NsP2 immunized mice exhibited robust replication, but Env+nsP2 co-immunized mice which exhibited a 2 to 3 log reduction in mean viral tier. 50% of the mice in the pVax1 group were euthanized on day 12 post-infection, as they lost 30% of the original body weight. Significantly lower viremia (p<0.0146) in pEnv+pnsP2 vaccination group compared to pVax1 alone, pnsP2 alone groups. (b) Long-term survival of CHIKV challenged mice, demonstrating increased survival, coupled with a disease-free state, of mice in the pEnv+pnsP2 groups compared to the pVax1 or pEnv alone groups. (c and d) Proinflammatory cytokines of TNF-a and IL-6 in mouse sera after CHIKV challenge. Sera samples were evaluated on day 12-post challenge with naïve mouse sera samples used as controls. Cytokines in serum levels were determined by specific ELISAs, and results shown are geometric mean values obtained from five mice at time point.

FIG. 5.

DNA vaccination using CHIKV-nsP2 with EP is completely protective against lethal challenge: Immunohistopathology of brain tissue from immunized mice after CHIK viral challenge experiment. Brain tissues were collected from day 10 through 14 and were sectioned and prepared as described in the Materials and Methods section. Sections from pVax1 vaccination group: (a) infiltration of cells into the arteriole and blood capillary; (d) large focal accumulation of glial cells and formation of glial nodule. Sections from pEnv vaccination group: (b) hyperemia and mild perivascular edema; (e) mild gliosis. Sections from pEnv+pnsP2 vaccination group: (c) normal brain pathology; (f) low levels of gliosis.