Oral vaccination with subunit vaccines protects animals against aerosol infection with Mycobacterium tuberculosis

Abstract

Immunity against Mycobacterium tuberculosis depends largely on activation of cell-mediated responses, and gamma interferon has been shown to play a crucial role in this process in both humans and animal models. Since the lung is normally the organ in which infection is initiated and is the major site of pathology, immune responses in the lung play a significant role in restricting initial infection with M. tuberculosis. The aim of the present study was to stimulate efficient immunity in the lung by targeting the gut mucosa. Detoxified monophosphoryl lipid A (MPL) has been shown to be a relatively nontoxic adjuvant which efficiently promotes the induction of type 1 responses when it is given by the traditional subcutaneous route. We have therefore compared subcutaneous immunization of mice to oral immunization by using a model subunit vaccine carrying two immunodominant proteins from M. tuberculosis, in combination with MPL-based adjuvants. While less effective when used to prime a response, a heterologous priming and boosting vaccination strategy employing oral boosting induced significant systemic type 1 responses which equaled and surpassed those attained by subcutaneous immunization protocols. Moreover, the increased immune responses observed correlated with the induction of substantial protection against subsequent aerosol infection with virulent M. tuberculosis at levels comparable to, or better than, those obtained by multiple subcutaneous vaccinations. These results demonstrate that booster vaccinations via mucosal surfaces, by combining efficient subunit vaccines with the potent adjuvant MPL, may be an effective method of addressing some of the shortcomings of current vaccination strategies.

FIG. 1.

Oral vaccination with MPL-based adjuvants does not prime effective immune responses. (A) Systemic immune responses as assessed from IFN-γ levels after in vitro restimulation of spleen cells from mice harvested 2 weeks after the booster vaccination. Results are IFN-γ levels (in picograms per milliliter) from four mice per experimental group ± standard deviations. (B) Protective efficacy as assessed by determining reductions in the numbers of CFU in the lungs of mice 6 weeks after aerosol infection. Results are mean log₁₀ numbers of CFU ± standard deviations from four mice per experimental group. Protection expressed as the log reduction in numbers of CFU in vaccinated versus naïve animals is shown above each group. sc, subcutaneous.

FIG. 2.

The failure of MPL to prime effective immune responses by the oral route is shared by classic oral adjuvants. (A) Systemic immune responses as assessed from IFN-γ levels after in vitro restimulation of spleen cells from mice harvested 2 weeks after the booster vaccination. Results are IFN-γ levels (in picograms per milliliter) from four mice per experimental group ± standard deviations. (B) Protective efficacy as assessed by the log reduction in numbers of CFU from the lungs of mice 6 weeks after aerosol infection. Results are mean log₁₀ numbers of CFU ± standard deviations from four mice per experimental group. Protection expressed as the log reduction in numbers of CFU in vaccinated compared to naïve animals is shown above each group. sc, subcutaneous.

FIG. 3.

Oral delivery of MPL-based adjuvants effectively boosts immune responses in a dose-dependent manner. (A) Systemic immune responses as assessed from IFN-γ levels after in vitro restimulation of spleen cells from mice harvested 2 weeks after the booster vaccination. Results are IFN-γ levels (in picograms per milliliter) from four mice per experimental group ± standard deviations. (B) Protective efficacy as assessed by the log reduction in numbers of CFU from the lungs of mice 6 weeks after aerosol infection. Results are mean log₁₀ numbers of CFU ± standard deviations from four mice per experimental group. Protection expressed as the log reduction in numbers of CFU in vaccinated versus naïve animals is shown above each group. sc, subcutaneous; Ag, antigen.

FIG. 4.

(A) Percentages of total lung lymphocytes positive for both CD4 and IFN-γ isolated from the lungs of perfused mice after aerosol infection. Results are from six mice per experimental group. (B) Results of staining cells from mice which had received 0, 1, or 10 μg of the hybrid antigen as a booster vaccine 4 days postinfection for CD4 and IFN-γ.

FIG. 5.

(A) Systemic immune responses as assessed from IFN-γ levels after in vitro restimulation of spleen cells from mice harvested 2 weeks after the booster vaccination. Results are IFN-γ levels (in picograms per milliliter) from four mice per experimental group ± standard deviations. (B) Protective efficacy as assessed by measuring the numbers of CFU from the lungs of mice 6 weeks after aerosol infection. Results are mean log₁₀ numbers of CFU ± standard deviations from four mice per experimental group. Protection expressed as the log reduction in the numbers of CFU in vaccinated versus naïve animals is shown above each group. sc, subcutaneous.