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

Center for Biologics Evaluation and Research, United States Food and Drug Administration, Bethesda, MD 20892, USA.
Clinical and vaccine Immunology: CVI (Impact Factor: 2.47). 06/2009; 16(7):1025-32. DOI: 10.1128/CVI.00067-09
Source: PubMed


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.

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Available from: Steven C Derrick, Aug 14, 2014
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    • "CD4 molecules are primarily located on T helper cells (Th cells) (Miceli & Parnes, 1993). Parra et al., (2009) proposed a new assay method for correlates of protection. The assay involves biomarker panels and growth inhibition bioassay of mycobacteria. "
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    ABSTRACT: The efficacy of every vaccine depends in part on appropriate vaccine handling from the manufacturer to its utilization. There is need for continuous monitoring of vaccine to ensure that cold-chain system is maintained throughout the product lifespan. This study aims to validate the BCG vaccines used in immunization in SouthEast , Nigeria. The potency of the vaccines was determined by viable count on Soybean-Casein-Digest Agarafter incubation of 50µl of 1: 40,000 dilutions of the vaccines at 37°C for 30 days. Ten replicate plates were used and result reported as mean ±SD. The viable counts in the vaccines were compared with the labeled potency on the vials. The immunogenicity test was done by Antibody Induction Method. This involves measuring the neutralizing antibodies in a control group (given physiological saline) and immunized group after 30 days using the enzyme-linked immunosorbent assay (ELISA). Anti-tuberculosis antibodies' concentration was determined by absorbance measurement at 450nm wavelength. Viable counts in the BCG vaccine samples were 54.6±11.79 > 51.91±11.35 > 48.18±15.33 > 44.91±16.29 > 44.55±15.69 CFU/50µl (dilution factor was 40,000). The immunogenicity test shows that the IgG titers for the BCG vaccines from control, Enugu/Ebonyi, Imo, Anambra and Abia were 0.645, 1.567, 1.507, 1.451 and 1.286 respectively while the IgMtitres were 0.689, 0.736, 0.805, 0.792 and 0.715 respectively. One way analysis of variance shows that there is statistical difference in the IgG antibody titer produced by the control compared to the vaccines (P value < 0.0001). The IgG antibody was enough to confer protection. Neither the control nor the vaccines from the states produced enough protective IgM. There is no statistical difference in the IgM antibody titer produced by the control compared to the vaccines (P value = 0.1058). The vaccines were all within their labeled potency and have good immunogenicity profile.
    African journal of pharmacy and pharmacology 01/2015; 8(47):1186-1191. DOI:10.5897/AJPP2014.4198 · 0.84 Impact Factor
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    • "To assess the impact of malaria infection on the effectiveness of novel TB vaccine candidates, C57BL/6 mice were vaccinated with three unique immunizing preparations using different vaccination strategies. In an initial experiment with a novel vaccine, mice were immunized with the E6-85 TB fusion protein (ESAT6-Antigen 85B) suspended in DDA/MPL adjuvant [26], [27]. As controls, other groups of mice were immunized with BCG. "
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    ABSTRACT: 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. 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. 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.
    PLoS ONE 12/2011; 6(12):e28164. DOI:10.1371/journal.pone.0028164 · 3.23 Impact Factor
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    ABSTRACT: New vaccines and novel immunization strategies are needed to improve the control of the global tuberculosis epidemic. To facilitate vaccine development, we have been creating in vitro mycobacterial intra-macrophage growth inhibition assays. Here we describe the development of an in vitro assay designed for BSL-2 laboratories which measures the capacity of vaccine-induced immune splenocytes to control the growth of isoniazid-resistant Mycobacterium bovis BCG (INH(r) BCG). The use of the INH(r) BCG as the infecting organism allows the discrimination of BCG bacilli used in murine vaccinations from BCG used in the in vitro assay. In this study, we showed that protective immune responses evoked by four different types of Mycobacterium tuberculosis vaccines [BCG, an ESAT6/Antigen 85B fusion protein formulated in DDA/MPL adjuvant, a DNA vaccine expressing the same fusion protein, and a TB Modified Vaccinia Ankara construct expressing four TB antigens (MVA-4TB)] were detected. Importantly, the levels of vaccine-induced protective immunity seen in the in vitro assay correlated with the results from in vivo protection studies in the mouse model of pulmonary tuberculosis. Furthermore, the growth inhibition data for the INH(r) BCG assay was similar to the previously reported results for a M. tuberculosis infection assay. The cytokine expression profiles at day 7 of the INH(r) BCG growth inhibition studies were also similar but not identical to the cytokine patterns detected in earlier M. tuberculosis co-culture assays. Overall, we have shown that a BSL-2 compatible in vitro growth inhibition assay using INH(r) BCG as the intra-macrophage target organism should be useful in developing and evaluating new TB immunization strategies.
    Vaccine 10/2009; 28(2):317-22. DOI:10.1016/j.vaccine.2009.10.047 · 3.62 Impact Factor
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