Vaccination with plasmid DNA encoding TSA/LmSTI1 leishmanial fusion proteins confers protection against Leishmania major infection in susceptible BALB/c mice

Abstract

We have recently shown that a cocktail containing two leishmanial recombinant antigens (LmSTI1 and TSA) and interleukin-12 (IL-12) as an adjuvant induces solid protection in both a murine and a nonhuman primate model of cutaneous leishmaniasis. However, because IL-12 is difficult to prepare, is expensive, and does not have the stability required for a vaccine product, we have investigated the possibility of using DNA as an alternative means of inducing protective immunity. Here, we present evidence that the antigens TSA and LmSTI1 delivered in a plasmid DNA format either as single genes or in a tandem digene construct induce equally solid protection against Leishmania major infection in susceptible BALB/c mice. Immunization of mice with either TSA DNA or LmSTI1 DNA induced specific CD4(+)-T-cell responses of the Th1 phenotype without a requirement for specific adjuvant. CD8 responses, as measured by cytotoxic-T-lymphocyte activity, were generated after immunization with TSA DNA but not LmSTI1 DNA. Interestingly, vaccination of mice with TSA DNA consistently induced protection to a much greater extent than LmSTI1 DNA, thus supporting the notion that CD8 responses might be an important accessory arm of the immune response for acquired resistance against leishmaniasis. Moreover, the protection induced by DNA immunization was specific for infection with Leishmania, i.e., the immunization had no effect on the course of infection of the mice challenged with an unrelated intracellular pathogen such as Mycobacterium tuberculosis. Conversely, immunization of BALB/c mice with a plasmid DNA that is protective against challenge with M. tuberculosis had no effect on the course of infection of these mice with L. major. Together, these results indicate that the protection observed with the leishmanial DNA is mediated by acquired specific immune response rather than by the activation of nonspecific innate immune mechanisms. In addition, a plasmid DNA containing a fusion construct of the two genes was also tested. Similarly to the plasmids encoding individual proteins, the fusion construct induced both specific immune responses to the individual antigens and protection against challenge with L. major. These results confirm previous observations about the possibility of DNA immunization against leishmaniasis and lend support to the idea of using a single polygenic plasmid DNA construct to achieve polyspecific immune responses to several distinct parasite antigens.

FIG. 1.

Expression of TSA and LmSTI1 in HEK 293T cells. HEK 293T cells growing in six-well plates were transfected with DNA constructs (1 μg/well) encoding the L. major antigen TSA or LmSTI1 under the control of a constitutive CMV promoter. Seventy-two hours posttransfection, the cells were harvested, lysed, and analyzed by Western blotting using anti-TSA or anti-LmSTI1 polyclonal antiserum to determine the levels of protein expression. Lysates prepared from untransfected HEK 293T cells and bacterially expressed recombinant protein containing an N-terminal six-His tag were used as negative and positive controls, respectively, for antibody specificity. Both constructs directed high-level expression of proteins of the correct size, although TSA expression tended to be stronger than that of LmSTI1 (arrows).

FIG. 2.

IgG isotype antibody response to the recombinant proteins TSA and LmST1 in BALB/c mice immunized with TSA DNA and LmSTI1 DNA. Mice (three per group) were immunized three times i.m. (1-month intervals) with 100 μg of either TSA DNA or LmSTI1 DNA. One month after the last immunization, the animals were bled. Sera were obtained and tested by ELISA for specific anti-TSA (A) and -LmSTI1 (B) antibody responses of both IgG1 and IgG2a isotypes. This is one representative experiment of four separate experiments with virtually the same results.

FIG. 3.

Cytokine production by spleen cells of BALB/c mice immunized with TSA DNA and LmSTI1 DNA. Mice (three per group) were immunized three times i.m. (1-month intervals) with 100 μg of either TSA DNA or LmSTI1 DNA. One month after the last immunization, the animals were sacrificed, and their spleen cells were obtained and cultured in vitro for 3 days in the presence of medium or TSA or LmSTI1 protein. The supernatants were harvested and assayed by ELISA for both IFN-γ and IL-4. No IL-4 could be detected in any supernatant (not shown). This is one representative experiment of two separate experiments with virtually the same results.

FIG. 4.

Induction of CTL activity in BALB/c mice immunized with TSA DNA and LmSTI1 DNA. Mice (three per group) were immunized three times i.m. (1-month intervals) with 100 μg of either TSA DNA or LmSTI1 DNA. One month after the last immunization, the animals were sacrificed, and their spleen cells were obtained and stimulated for 6 days with either TSA DNA- or LmSTI1 DNA-transduced P815 cells. The stimulated cells were washed and tested for cytotoxicity in a 4-h ⁵¹Cr release assay against control-transduced P815 cells (EGPF), against TSA-transduced P815 cells, and against LmSTI1-transduced P815 cells. The results are expressed as percent (with standard deviation) specific cytotoxicity against the respective transduced cell targets. This is one representative experiment of two separate experiments with virtually the same results.

FIG. 5.

Vaccination of BALB/c mice against L. major infection with TSA DNA and LmSTI1 DNA. Mice (five per group) were immunized three times i.m. (1-month intervals) with saline; 100 μg of control DNA (pcDNA3) (Empty vector), TSA DNA, or LmSTI1 DNA; or a mixture containing 100 μg of TSA DNA and LmSTI1 DNA (100 μg of each). One month after the last immunization, the animals were infected in the right footpad with either 10⁴ amastigote forms (A) or 2 × 10⁵ promastigote (metacyclic) forms (B) of L. major, and footpad swelling was measured weekly thereafter. Standard errors for all points in A and B were less than 15%. This is one representative experiment of five separate experiments with virtually the same results.

FIG. 6.

Specificity of protection induced by DNA vaccination. Mice (five per group) were immunized three times i.m. (1-month intervals) with either saline or 100 μg of control DNA (pcDNA3) (Empty vector), TSA DNA, or Mtb8.4 DNA. One month after the last immunization, one set of mice was infected in the right footpad with 10⁴ amastigote forms of L. major, and footpad swelling was measured weekly thereafter (A). The other set of mice was challenged intravenously with 2 × 10⁵ viable M. tuberculosis organisms, and CFU in the spleen and lungs were enumerated 3 weeks later (B). This is one representative experiment of three separate experiments with virtually the same results. The bars indicate standard deviations.

FIG. 7.

Expression of a TSA/LmSTI1 fusion protein in HEK 293T cells. HEK 293T cells growing in six-well plates were transfected with DNA constructs (1 μg/well) encoding the L. major antigen TSA or LmSTI1 or a TSA/LmSTI1 fusion protein under the control of a constitutive CMV promoter. As described in the legend to Fig 1, lysates from transfected and untransfected cells were analyzed by Western blotting using anti-TSA or anti-LmSTI1 polyclonal antiserum to determine levels of protein expression. Lysates prepared from untransfected HEK 293T cells and bacterially expressed recombinant protein containing an N-terminal six-His tag were used as negative and positive controls, respectively, for antibody specificity. The fusion construct directed high-level expression of a TSA/LmSTI1 fusion protein of the correct size that was reactive with both the anti-TSA and anti-LmSTI1 polyclonal antisera (arrows).

FIG. 8.

Vaccination of BALB/c mice against L. major infection with a digene plasmid DNA construct. Mice (five per group) were immunized three times i.m. (1-month intervals) with either saline or 100 μg of control DNA (pcDNA3) (Empty vector) or with a pcDNA3 construct containing in tandem the genes for TSA and LmSTI1 (Di-gene-DNA). One month after the last immunization, the animals were infected in the right footpad with 10⁴ amastigote forms of L. major, and footpad swelling was measured weekly thereafter. This is one representative experiment of five separate experiments with virtually the same results.