Protective killed Leptospira borgpetersenii vaccine induces potent Th1 immunity comprising responses by CD4 and gammadelta T lymphocytes

Abstract

Leptospira borgpetersenii serovar hardjo is the most common cause of bovine leptospirosis and also causes zoonotic infections of humans. A protective killed vaccine against serovar hardjo was shown to induce strong antigen-specific proliferative responses by peripheral blood mononuclear cells (PBMC) from vaccinated cattle by 2 months after the first dose of vaccine. This response was absent from nonvaccinated control cattle. The mean response peaked by 2 months after completion of the two-dose vaccination regimen, and substantial proliferation was measured in in vitro cultures throughout the 7 months of the study period. Variations in magnitude of the response occurred among the vaccinated animals, but by 7 months postvaccination there was a substantial antigen-specific response with PBMC from all vaccinated animals. Up to one-third of the PBMC from vaccinated animals produced gamma interferon (IFN-gamma) after 7 days in culture with antigen, as ascertained by flow cytometric analysis, and significant levels of IFN-gamma were measured in culture supernatants by enzyme-linked immunosorbent assay. Two-color immunofluorescence revealed that one-third of the IFN-gamma-producing cells were gammadelta T cells, with the remaining cells being CD4(+) T cells. The significance of this study is the very potent Th1-type immune response induced and sustained following vaccination with a killed bacterial vaccine adjuvanted with aluminum hydroxide and the involvement of gammadelta T cells in the response. Moreover, induction of this Th1-type cellular immune response is associated with the protection afforded by the bovine leptospiral vaccine against L. borgpetersenii serovar hardjo.

FIG. 1

Means and standard errors of proliferation by PBMC from vaccinated (A) or nonvaccinated (B) cattle, as determined by the incorporation of [³H]thymidine in response to either L. borgpetersenii serovar hardjo-bovis antigen, medium, or a suboptimal concentration of ConA. Cattle were vaccinated with the first dose administered at month 0 and the second dose at month 1.

FIG. 2

Proliferation by PBMC from individual vaccinated animals in response to culture with either L. borgpetersenii serovar hardjo-bovis antigen or medium at 3 months after the first dose of vaccine. The means of incorporated [³H]thymidine from quadruplicate wells are shown with the standard errors.

FIG. 3

The proliferation of PBMC from five vaccinated animals with low responses at 3 months after the first dose of the vaccine in response to L. borgpetersenii serovar hardjo-bovis antigen. The means for quadruplicate cultures are shown with the standard error. The dashed line indicates the mean values for the entire vaccinated group as a reference point.

FIG. 4

The mean and standard error of IFN-γ produced in PBMC cultures from vaccinated or nonvaccinated cattle after culture with leptospira antigen (Ag) or medium (Med). PBMC were evaluated at 3 months (A), 4 months (B), 6 months (C), or 7 months (D). The culture supernatants were assessed by ELISA, and the units shown are OD units from an ELISA plate reader using a wavelength of 450 nm. At months 3 and 4, 15 and 20% of the samples, respectively, were beyond the range of the assay. At months 6 and 7, 18 and 68% of the samples, respectively, were beyond the range when tested undiluted; however, dilutions of the supernatants were tested and the OD units were adjusted according to the dilution. At month 6 only vaccinated cattle were sampled. An asterisk indicates a response by the vaccinated group cultured with antigen which is significantly higher than the response by the nonvaccinated cattle at each time point (P < 0.01).

FIG. 5

Mean percentages of IFN-γ-positive cells from cultures of PBMC from vaccinated and nonvaccinated cattle. These cells were restimulated with PMA-ionomycin, permeabilized, and stained for intracellular IFN-γ using indirect immunofluorescence and flow cytometric analysis. The means and standard errors are shown. At month 6, only the vaccinated cattle were sampled. The asterisk indicates a significant difference in the response to antigen (P < 0.03).

FIG. 6

Percentages of IFN-γ-producing cells in PBMC from six vaccinated animals with high proliferative responses at month 5 after stimulation with L. borgpetersenii antigen are shown. The PBMC were cultured for 7 days and restimulated with PMA-ionomycin, and two-color immunofluorescence was performed and analyzed by flow cytometry. (A) An example of two-color flow cytometric analysis shown by dot plot. Detection of IFN-γ is shown on the y axis, and either CD4 or γδ-TCR is shown on the x axis. Double positives are in the upper right quadrant. (B) Means and standard errors of results with PBMC from six vaccinated animals. Shown are the total percentage of IFN-γ⁺ cells in the cultures and the percentage of cells double staining for CD4 and IFN-γ or for γδ-TCR and IFN-γ.