Malaria Infections Do Not Compromise Vaccine-Induced Immunity against Tuberculosis in Mice

BACKGROUND: Given the considerable geographic overlap in the endemic regions for malaria and tuberculosis, it is probable that co-infections with Mycobacterium tuberculosis and Plasmodium species are prevalent. Thus, it is quite likely that both malaria and TB vaccines may be used in the same populations in endemic areas. While novel vaccines are currently being developed and tested individually against each of these pathogens, the efficacy of these vaccines has not been evaluated in co-infection models. To further assess the effectiveness of these new immunization strategies, we investigated whether co-infection with malaria would impact the anti-tuberculosis protection induced by four different types of TB vaccines in a mouse model of pulmonary tuberculosis. PRINCIPAL FINDINGS: Here we show that the anti-tuberculosis protective immunity induced by four different tuberculosis vaccines was not impacted by a concurrent infection with Plasmodium yoelii NL, a nonlethal form of murine malaria. After an aerogenic challenge with virulent M. tuberculosis, the lung bacterial burdens of vaccinated animals were not statistically different in malaria infected and malaria naïve mice. Multi-parameter flow cytometric analysis showed that the frequency and the median fluorescence intensities (MFI) for specific multifunctional T (MFT) cells expressing IFN-γ, TNF-α, and/or IL-2 were suppressed by the presence of malaria parasites at 2 weeks following the malaria infection but was not affected after parasite clearance at 7 and 10 weeks post-challenge with P. yoelii NL. CONCLUSIONS: Our data indicate that the effectiveness of novel TB vaccines in protecting against tuberculosis was unaffected by a primary malaria co-infection in a mouse model of pulmonary tuberculosis. While the activities of specific MFT cell subsets were reduced at elevated levels of malaria parasitemia, the T cell suppression was short-lived. Our findings have important relevance in developing strategies for the deployment of new TB vaccines in malaria endemic areas

Early Pulmonary Cytokine and Chemokine Responses in Mice Immunized with Three Different Vaccines against Mycobacterium tuberculosis Determined by PCR Array▿ †

In this study, the early pulmonary cytokine and chemokine responses in mice immunized with either BCG vaccine, a ΔsecA2 mutant of Mycobacterium tuberculosis, or a DNA vaccine expressing an ESAT6-antigen 85B fusion protein and then aerogenically challenged with a low dose of M. tuberculosis were evaluated by PCR array. The cellular immune responses at day 10 postchallenge were essentially equivalent in the lungs of mice immunized with either the highly immunogenic BCG vaccine or the ΔsecA2 M. tuberculosis mutant strain. Specifically, 12 immune biomolecules (including gamma interferon [IFN-γ], interleukin-21 [IL-21], IL-27, IL-17f, CXCL9, CXCL10, and CXCL11) were differentially regulated, relative to the levels for naïve controls, in the lungs of vaccinated mice at this time point. Although the vaccine-related immune responses evoked in mice immunized with the DNA vaccine were relatively limited at 10 days postinfection, upregulation of IFN-γ RNA synthesis as well as increased expression levels of CXCL9, CXCL10, and CXCL11 chemokines were detected

Mycobacterium bovis BCG Immunization Induces Protective Immunity against Nine Different Mycobacterium tuberculosis Strains in Mice▿ †

Recent preclinical and epidemiologic studies have suggested that certain Mycobacterium tuberculosis genotypes (in particular, Beijing lineage strains) may be resistant to Mycobacterium bovis BCG vaccine-induced antituberculosis protective immunity. To investigate the strain specificity of BCG-induced protective responses in a murine model of pulmonary tuberculosis, C57BL/6 mice were vaccinated with BCG vaccine and then challenged 2 months later with one of nine M. tuberculosis isolates. Four of these strains were from the W-Beijing lineage (HN878, N4, NHN5, and ChS) while four were non-Beijing-type isolates (C913, CDC1551, NY669, and NY920). As a control, the WHO standard M. tuberculosis Erdman strain was evaluated in these vaccination/challenge experiments. To assess the protective responses evoked by BCG immunization, organ bacterial burdens and lung pathology were assessed in vaccinated and naïve mice at 4, 12, and 20 weeks postchallenge as well as during the day of infection. At 4 weeks after the aerosol challenge with each of these strains, significantly reduced bacterial growth in the lungs and spleens and significantly improved lung pathology were seen in all vaccinated animals compared to naïve controls. After 12 weeks, reduced organ bacterial burdens were detected in vaccinated animals infected with six of nine challenge strains. Although lung CFU values were lower in vaccinated mice for only three of nine groups at 20 weeks postchallenge, significantly decreased lung inflammation was seen in all immunized animals relative to controls at 20 weeks postchallenge. Taken together, these data demonstrate that BCG vaccination protects against infection with diverse M. tuberculosis strains in the mouse model of pulmonary tuberculosis and suggest that strain-specific resistance to BCG-induced protective immunity may be uncommon

Development of a Murine Mycobacterial Growth Inhibition Assay for Evaluating Vaccines against Mycobacterium tuberculosis▿ †

The development and characterization of new tuberculosis (TB) vaccines has been impeded by the lack of reproducible and reliable in vitro assays for measuring vaccine activity. In this study, we developed a murine in vitro mycobacterial growth inhibition assay for evaluating TB vaccines that directly assesses the capacity of immune splenocytes to control the growth of Mycobacterium tuberculosis within infected macrophages. Using this in vitro assay, protective immune responses induced by immunization with five different types of TB vaccine preparations (Mycobacterium bovis BCG, an attenuated M. tuberculosis mutant strain, a DNA vaccine, a modified vaccinia virus strain Ankara [MVA] construct expressing four TB antigens, and a TB fusion protein formulated in adjuvant) can be detected. Importantly, the levels of vaccine-induced mycobacterial growth-inhibitory responses seen in vitro after 1 week of coculture correlated with the protective immune responses detected in vivo at 28 days postchallenge in a mouse model of pulmonary tuberculosis. In addition, similar patterns of cytokine expression were evoked at day 7 of the in vitro culture by immune splenocytes taken from animals immunized with the different TB vaccines. Among the consistently upregulated cytokines detected in the immune cocultures are gamma interferon, growth differentiation factor 15, interleukin-21 (IL-21), IL-27, and tumor necrosis factor alpha. Overall, we have developed an in vitro functional assay that may be useful for screening and comparing new TB vaccine preparations, investigating vaccine-induced protective mechanisms, and assessing manufacturing issues, including product potency and stability

Relative gene expression in mice infected with <i>P. yoelii</i> and naïve controls at days 1, 5 and 9 after the malaria infection.

a<p>Gene expression in mice infected with <i>P. yoelii</i> relative to naïve controls.</p>b<p>Mean±the standard error of the mean (SEM) for 4–5 mice. For these immune mediators, significant differences were detected by unpaired t test analysis (p<0.05) in the levels of gene expression for PyNL infected and naïve mice.</p>c<p>(–) Significant differences in gene expression were not detected between malaria infected mice and controls.</p

BCG vaccination confers partial protection against <i>P. yoelii</i> 17XNL infections in mice.

<p>Two months after immunization with BCG, vaccinated (closed squares) and naïve mice (closed circles) were challenged with an erythrocytic stage <i>P. yoelii</i> infection. The post-infection parasitemia levels were determined by blood smear microscopy. These data are representative results from 3 experiments. In each study, 10 mice were used for each experimental group. * Significant difference in parasitemia levels (p<0.05) as determined by unpaired t test analysis.</p

Relative gene expression in BCG vaccinated, <i>P. yoelii</i> infected mice compared to non–immunized controls.

a<p>Gene expression in BCG vaccinated mice compared to naïve controls.</p>b<p>Gene expression in BCG vaccinated <i>P. yoelii</i> infected mice compared to non–immunized, malaria infected controls at days 1, 5, and 9 post–infection.</p>c<p>Mean±SEM for 4–5 mice. For these immune mediators, significant differences in gene expression were detected by unpaired t test analysis (p<0.05) between BCG vaccinated and naïve mice or BCG vaccinated, PyNL infected mice and non–infected PyNL infected controls.</p>d<p>(–) Significant differences were not detected between vaccinated mice and controls.</p