Characterization of the immune response elicited by the vaccinia virus L3 protein delivered as naked DNA

Abstract

Poxviruses are complex dsDNA viruses with over 200 genes, many of them with unknown role in the stimulation of immune responses. Among these, the vaccinia virus (VACV) L3L ORF encodes an essential protein for the transcription of the VACV early genes. To the best of our knowledge, the immune response elicited by L3 has not been characterized. In this regard, our data describes a DNA L3-coding plasmid (pL3L) that stimulates both, humoral- and cell-mediated immune responses in a mouse model. Cell-mediated immune responses were measured by IFN-γ and IL-4 ELISPOT assays. We performed CD8⁺ cells depletion and flow cytometry analysis to account for the contribution of cytotoxic T lymphocytes in the IFN-γ production. Moreover, results from ELISPOT were confirmed by measuring the concentration of IL-4 and IFN-γ in supernatant of antigen-stimulated splenocytes by cytokine ELISA. Additionally, dominant antigenic regions of L3 protein were identified by epitope mapping analysis. Humoral immune responses were assessed by ELISA. Specifically, the production of total IgG, IgG1 (TH-2) and IgG2a (TH-1) were determined one week after the final immunization. Our ELISPOT data shows pL3L-immunized animals to produce significantly higher frequencies of IFN-γ Spot-Forming Cells (SFC) versus controls. IL-4 levels remained unchanged in all three groups, demonstrating the increase in antigen-specific IFN-γ releasing cells. Flow cytometry assay results showed that CD8⁺ T cells are a major contributor to the production of IFN-γ. Moreover, our formulation enhances the production of total IgG, predominantly IgG2a isotype. Immunization with pL3L promotes a robust cytotoxic immune response, crucial against viral pathogens. In addition, our vaccine candidate promotes an increase in IgG levels, especially IgG2a (TH-1 type). Our data encourages further studies of L3 as a novel antigen in vaccine development against poxviruses.

Keywords: Bioterror agents; DNA-vaccine; L3L; Novel antigen; Smallpox; Vaccine.

Fig. 1.

VACV L3-specific cell-mediated immune response determined by ELISPOT analysis. Each group consisted of four 4–6-week-old female Balb/c mice immunized three times, two weeks apart via intramuscular (i.m.) injection as follows: Naïve (negative control), pVax1 (backbone control), and pL3L. (A) Antigen-specific IFN–γ response to VVWR L3 peptides after overnight incubation. (B) Antigen-specific IL-4 response to VVWR L3 peptides after 48 h of incubation. Data are averages of at least three independent experiments, and the immune responses among groups of mice are presented as the mean ± standard error of the mean (SEM). A p value of <0.05 was considered significant. Con A data not shown as its values are out of scale.

Fig. 2.

Contribution of CD8⁺ T cells in IFN-γ production. (A) Contribution of CD8⁺ Cell Population to IFN-γ production: Pools of splenocytes were incubated with anti-mouseCD8⁺ antibodies and magnetically separated using the autoMACS. Magnetically retained CD8a⁺ cells are eluted as the positively selected cell fraction. (B) Flow cytometry analysis: an 83.9% decrease in the CD8⁺ T cells population was observed after magnetic separation and results were analyzed by FlowJo. Each experiment was conducted independently at least three times and the immune responses among groups are presented as the mean ± standard error of the mean (SEM). A p value of <0.05 was considered significant. Con A data not shown as its values are out of scale.

Fig. 3.

L3L-specific cytokine profile. Mice were immunized three times via i.m injection, two weeks apart from each dose. Splenocytes isolated from immunized mice were stimulated with VVWR L3 peptide pool for 5 days. Supernatants were collected, and cytokine ELISA was performed. Our data shows a significant increase in the production of IFN-γ in mice receiving pL3L when compared to non-immunized groups (p = 0.0215). Meanwhile, the production of IL-4 was not altered and all groups had similar values. The immune responses among groups are presented as the mean ± standard error of the mean (SEM) of at least three independent experiments. A p value of <0.05 was considered significant.

Fig. 4.

Total IgG Response Against L3 VVWR Antigen. Serum was isolated from mice one week after the final immunization. Total IgG responses were assessed by ELISA. Our data shows a significant increase in the production of total IgG in animals receiving pL3L when compared to both controls (ANOVA test, KWT = 33.31, p < 0.0001). Data is presented as the mean ± standard error of the mean (SEM) of at least three independent experiments.

Fig. 5.

Enhancement in the production of IgG isotypes. Serum was isolated from mice one week after the final immunization. IgG isotyping was assessed by ELISA using a secondary (A) murine anti-IgG2a or (B) murine anti-IgG1 antibodies. Our data demonstrates an enhancement of both isotypes, especially IgG2a (ANOVA test, KWT = 20.64, p< 0.0001). Immune responses among groups are presented as the mean ± standard error of the mean (SEM) of at least three independent experiments.

Fig. 6.

Protein localization of VACV L3 immunodominant peptides identified by ELISPOT analysis. Antigen-specific IFN–γ response to VVWR L3 individual 11-mer overlapped 15-mer -peptides was measured after overnight incubation. Data are averages of at least three independent experiments, and the immune responses among mouse groups are presented as the mean ± standard error of the mean (SEM). A p value of <0.05 was considered significant.