A DNA vaccine producing LcrV antigen in oligomers is effective in protecting mice from lethal mucosal challenge of plague

Abstract

There is an urgent need to develop effective vaccines against pneumonic plague, a highly lethal and contagious disease caused by the Gram-negative bacterium Yersinia pestis. Here we demonstrate that a novel DNA vaccine expressing a modified V antigen (LcrV) of Y. pestis, with a human tissue plasminogen activator (tPA) signal sequence, elicited strong V-specific antibody responses in BALB/c mice. This tPA-V DNA vaccine protected mice from intranasal challenge with lethal doses of Y. pestis. In comparison, a DNA vaccine expressing the wild type V antigen was much less effective. Only tPA-V formed oligomers spontaneously, and elicited a higher IgG2a anti-V antibody response in immunized mice, suggesting increased TH1 type cellular immune response. Our data indicate that antigen engineering is effective in inducing high quality protective immune responses against conformationally sensitive antigens. These results support that optimized DNA vaccines have the potential to protect against bacterial pathogens than is generally recognized.

Fig. 1

Schematic diagram of the Y. pestis antigen inserts used in the DNA vaccine constructs. The wild-type genes (wt) and the genes with an additional tissue plasminogen activator (tPA) leader sequence were individually cloned into the DNA vector pJW4303. The natural leader sequence or the hydrophobic regions at the N-terminal region of each gene is marked.

Fig. 2

Y. pestis antigen-specific IgG responses in sera of DNA vaccine immunized BALB/c mice as measured by ELISA. Same amounts of transiently expressed Y. pestis proteins were used as the coating antigens. (a) Results are shown as the geometric mean titers with pooled mouse sera in duplicates from each group (6 mice per group) as indicated after the third DNA immunization. (b) Temporal mouse sera anti-V IgG responses measured by ELISA OD value in the pre-bleed and 4 weeks following each DNA immunization as indicated by arrows. Pooled sera from each group of 6 mice were used. Sera dilution was at 1:500. Solid square: mouse group immunized with the tPA-V DNA vaccine; open square: mouse group immunized with the wt-V DNA vaccine; and open circle: mouse group received empty vector DNA.

Fig. 3

Protection of mice immunized with different plague DNA vaccines expressing V, F1 or Pla antigens. The BALB/c mice received four DNA immunizations (Weeks 0, 4, 8 and 32) prior to the intranasal challenge at Week 34 with a dose of 5000 cfu (15 LD₅₀) Y. pestis Kim strain. There were 6 mice per group in this first challenge study. Animals were followed for 2 weeks after the challenge. Accumulated survivals were plotted to compare the efficacy of each vaccine pair (the wild type and tPA-) to the vector alone as indicated.

Fig. 4

Protection of the tPA-V or wt-V DNA vaccine immunized mice against intranasal challenge of Y. pestis Kim strain at one of the three escalating doses: 5000 cfu (15 LD₅₀), 20,000 cfu (60 LD₅₀) or 80,000 cfu (240 LD₅₀). There were 10 mice per group in this expanded challenge study. Mice received 4 DNA immunizations at Weeks 0, 4, 8, 16 and the lethal challenge was delivered at Week 18.

Fig. 5

Comparison of the wt-V and tPA-V antigen expression in vitro. The 293T cells were transiently transfected with either wt-V or tPA-V DNA vaccines. The cells were harvested 72 h later and the expressions of V antigen were examined by Western blot analysis. For each lane, 10 ng of samples were loaded. The mouse sera immunized with the tPA-V DNA vaccine were used to detect Y. pestis V antigen. (a) The conventional Western blot results; L: cell lysates and S: the supernatant of transfected 293T cells. (b) Urea (4M at final concentration) was added to the transiently expressed tPA-V samples from supernatant of transfected 293T cells and then the samples were either heat treated (Lane 2) or untreated (Lane 1) before subjected to Western blot analysis.

Fig. 6

The tPA-V antigen was not glycosylated. Fully denatured tPA-V protein transiently expressed from 293T cells was subjected to the treatment of PNGase F and then was analyzed by Western blot. Untreated or mock treated tPA-V samples were included as the negative controls. PNGase F treatment of S1.1 fragment of SARS-CoV spike protein was used as the positive control.

Fig. 7

Subtypes of anti-V IgG in mouse sera raised by the wt-V or tPA-V DNA vaccines as measured by the quantitative ELISA. The assay is similar to the regular ELISA as in Fig. 2 except the second antibodies used in this assay were purified anti-IgG1 or anti-IgG2a antibodies. The ELISA OD values were then converted to the concentration for each IgG subtypes in mouse sera by using the standard curves established with known concentration of IgG1 or IgGa2.