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: 2.37). 06/2009; 16(7):1025-32. DOI: 10.1128/CVI.00067-09
Source: PubMed

ABSTRACT 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.


Available from: Steven C Derrick, Aug 14, 2014
  • [Show abstract] [Hide abstract]
    ABSTRACT: Tuberculosis (TB) continues being one of the diseases having the greatest mortality rates around the world, 8.7 million cases having been reported in 2011. An efficient vaccine against TB having a great impact on public health is an urgent need. Usually, selecting antigens for vaccines has been based on proteins having immunogenic properties for patients suffering TB and having had promising results in mice and non-human primates. Our approach has been based on a functional approach involving the pathogen–host interaction in the search for antigens to be included in designing an efficient, minimal, subunit-based anti-TB vaccine. This means that Mycobacterium tuberculosis has mainly been involved in studies and that lipoproteins represent an important kind of protein on the cell envelope which can also contribute towards this pathogen's virulence. This study has assessed the expression of four lipoproteins from M. tuberculosis H37Rv, that is, Rv1411c (LprG), Rv1911c (LppC), Rv2270 (LppN) and Rv3763 (LpqH), and the possible biological activity of peptides derived from these. Five peptides were found for these proteins which had high specific binding to both alveolar A549 epithelial cells and U937 monocyte-derived macrophages which were able to significantly inhibit mycobacterial entry to these cells in vitro.
    Chemical Biology &amp Drug Design 07/2014; DOI:10.1111/cbdd.12365 · 2.51 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A hundred and five years ago, Albert Calmette and Camille Guerin began a daunting task, which is unmatched even today, that led to the most widely used vaccine in human history. Despite a century of scientific advances, BCG (an acronym for Bacillus Calmette-Guerin) remains the only vaccine for prevention of tuberculosis. Due to the fact that the use of BCG vaccines will continue, either as a stand-alone or as a prime vaccine in prime-boost immunization strategies, the World Health Organization (WHO) has underlined the necessity for further work toward better characterization, evaluation and quality control of the BCG vaccine, taking into account recent advances in genetics and molecular biology. The potential benefit of such improved characterization could be addressed to better and easier differentiation between sub-strains used by different manufacturers. It may help to ensure consistency of production in terms of genetic stability and it may also help the clinical evaluation of new antituberculosis vaccines. Last but not least, the state-of-the-art technologies could facilitate the quality control performed by the manufacturers and by National Control Authorities as well.
    Biotechnology & Biotechnological Equipment 07/2014; 28(3):387-391. DOI:10.1080/13102818.2014.927200 · 0.38 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: While formulating Mycobacterium bovis BCG in lipid-based adjuvants has been shown to increase the vaccine's protective immunity, the biological mechanisms responsible for the enhanced potency of lipid encapsulated BCG are unknown. To assess whether mixing BCG in adjuvant increases its immunogenicity by altering post-vaccination organ distribution and persistence, mice were immunized subcutaneously with conventional BCG Pasteur or BCG formulated in DDA/TDB adjuvant and the bio-distribution of BCG bacilli was evaluated in mouse lungs, spleens, lymph nodes, and livers for up to 1 year. Although BCG was rarely detected in mouse livers, mycobacteria were found in mouse lungs, spleens, and lymph nodes for at least 1 year post-vaccination. However, at various time points during the 1 year study, the frequency of lung and spleen infections and the number of mycobacteria in infected organs of individual mice were highly variable. In contrast, mycobacteria were nearly always detected in the lymph nodes of vaccinated mice. While the frequency and extent of lymph node infections generally were not significantly different between mice vaccinated with adjuvanted or nonadjuvanted BCG preparations, multiparameter flow cytometry analysis of lymph node cells showed significantly higher frequencies of CD4+ and CD8+ T cells expressing IFN-γ and IFN-γ/TNF-α in mice immunized with adjuvanted BCG. Overall, our data suggest that the relationship between lymph node infection and the generation of anti-tuberculosis protective responses following BCG vaccination should be further investigated.
    Vaccine 11/2014; 33(1). DOI:10.1016/j.vaccine.2014.11.004 · 3.49 Impact Factor