scholarly journals Application of a whole blood mycobacterial growth inhibition assay to study immunity against Mycobacterium tuberculosis in a high tuberculosis burden population

PLoS ONE ◽  
2017 ◽  
Vol 12 (9) ◽  
pp. e0184563 ◽  
Author(s):  
Richard Baguma ◽  
Adam Penn-Nicholson ◽  
Erica Smit ◽  
Mzwandile Erasmus ◽  
Jonathan Day ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Growth Inhibition ◽  
Whole Blood ◽  
Inhibition Assay ◽  
Growth Inhibition Assay ◽  
Mycobacterial Growth

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

2009 ◽  
Vol 16 (7) ◽  
pp. 1025-1032 ◽  
Author(s):  
Marcela Parra ◽  
Amy L. Yang ◽  
JaeHyun Lim ◽  
Kristopher Kolibab ◽  
Steven Derrick ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Immune Responses ◽  
Growth Inhibition ◽  
In Vitro Assays ◽  
Functional Assay ◽  
Inhibition Assay ◽  
Growth Inhibition Assay ◽  
Vitro Assay ◽  
Mycobacterial Growth

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.

scholarly journals Evaluation of Tuberculosis Vaccine Candidate, pcDNA3.1-rpfD using Mycobacterial Growth Inhibition Assay (MGIA)

2021 ◽  
Vol 29 (1) ◽  
pp. 1-8
Author(s):  
Mifa Nurfadilah ◽  
Andriansjah Rukmana ◽  
Fithriyah Sjatha
Keyword(s):  
Growth Inhibition ◽  
Vaccine Candidate ◽  
Negative Control ◽  
Automated System ◽  
Control Group ◽  
Functional Assay ◽  
Inhibition Assay ◽  
Peripheral Blood Mononuclear ◽  
Growth Inhibition Assay ◽  
Mycobacterial Growth

Resuscitation-promoting factor D (RpfD) is a protein involved in the resuscitation of dormant bacteria. A new tuberculosis vaccine carrying the rpfD gene has been successfully constructed, pcDNA3.1-rpfD. It was demonstrated that this vaccine exhibits cellular and humoral immune responses. Therefore, within this study, the efficacy of this new vaccine candidate was evaluated using mycobacterial growth inhibition assay (MGIA). MGIA is a functional assay that measures the complex host immune response, peripheral blood mononuclear cell (PBMC) and splenocyte from BALB/c mice against mycobacteria. With BACTECTM MGITTM 960 automated system, the effect of vaccination on bacterial growth was reported as a time to positivity (TTP) in hours. The mean of TTP from the vaccinated group (both pcDNA3.1-rpfD and BCG) was higher than the negative control group. These results suggest that pcDNA3.1-rpfD may be effective in controlling tuberculosis growth and may provide a clue for the development of the tuberculosis vaccine. In addition, despite previous evidence that IFNγ was essential for tuberculosis immunity, IFNγ (interferon gamma) production was found not to be correlated with mycobacterial inhibition. Therefore, these findings offer an alternative method to evaluate vaccine candidates than the assessment using IFNγ only.

scholarly journals Ex vivo mycobacterial growth inhibition assay (MGIA) for tuberculosis vaccine testing - a protocol for mouse splenocytes

2015 ◽  
Author(s):  
Andrea Andrea Zelmer ◽  
Rachel Tanner ◽  
Elena Stylianou ◽  
Sheldon Morris ◽  
Angelo Izzo ◽  
...  
Keyword(s):  
Immune Response ◽  
Clinical Trials ◽  
Growth Inhibition ◽  
Ex Vivo ◽  
Inhibition Assay ◽  
Growth Inhibition Assay ◽  
Mouse Splenocytes ◽  
Vaccine Testing ◽  
Mycobacterial Growth ◽  
Detailed Protocol

The testing of vaccines for tuberculosis is costly and time-consuming, and dependent on preclinical animal challenge models and clinical trials. We have recently developed a mycobacterial growth inhibition assay (MGIA) to test vaccine efficacy ex vivo. This assay measures the summative effect of the host immune response and may serve as a novel tool to facilitate vaccine testing. It has generated much interest recently, and to facilitate technology transfer and reproducibility between laboratories, we here describe a detailed protocol for an ex vivo MGIA in mouse splenocytes.

scholarly journals A mycobacterial growth inhibition assay (MGIA) for bovine TB vaccine development

Tuberculosis ◽  
2017 ◽  
Vol 106 ◽  
pp. 118-122 ◽  
Author(s):  
Ilaria Pepponi ◽  
Bhagwati Khatri ◽  
Rachel Tanner ◽  
Bernardo Villarreal-Ramos ◽  
Martin Vordermeier ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Vaccine Development ◽  
Inhibition Assay ◽  
Growth Inhibition Assay ◽  
Mycobacterial Growth

scholarly journals The in vitro direct mycobacterial growth inhibition assay (MGIA) for the early evaluation of TB vaccine candidates and assessment of protective immunity: a protocol for non-human primate cells

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 257
Author(s):  
Rachel Tanner ◽  
Emily Hoogkamer ◽  
Julia Bitencourt ◽  
Andrew White ◽  
Charelle Boot ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Protective Immunity ◽  
Vaccine Development ◽  
Autologous Serum ◽  
Inhibition Assay ◽  
Growth Inhibition Assay ◽  
Vaccine Candidates ◽  
Mycobacterial Growth

The only currently available approach to early efficacy testing of tuberculosis (TB) vaccine candidates is in vivo preclinical challenge models. These typically include mice, guinea pigs and non-human primates (NHPs), which must be exposed to virulent M.tb in a ‘challenge’ experiment following vaccination in order to evaluate protective efficacy. This procedure results in disease development and is classified as ‘Moderate’ in severity under EU legislation and UK ASPA licensure. Furthermore, experiments are relatively long and animals must be maintained in high containment level facilities, making them relatively costly. We describe an in vitro protocol for the direct mycobacterial growth inhibition assay (MGIA) for use in the macaque model of TB vaccine development with the aim of overcoming some of these limitations. Importantly, using an in vitro assay in place of in vivo M.tb challenge represents a significant refinement to the existing procedure for early vaccine efficacy testing. Peripheral blood mononuclear cell and autologous serum samples collected from vaccinated and unvaccinated control animals are co-cultured with mycobacteria in a 48-well plate format for 96 hours. Adherent monocytes are then lysed to release intracellular mycobacteria which is quantified using the BACTEC MGIT system and colony-forming units determined relative to an inoculum control and stock standard curve. We discuss related optimisation and characterisation experiments, and review evidence that the direct NHP MGIA provides a biologically relevant model of vaccine-induced protection. The potential end-users of the NHP MGIA are academic and industry organisations that conduct the assessment of TB vaccine candidates and associated protective immunity using the NHP model. This approach aims to provide a method for high-throughput down-selection of vaccine candidates going forward to in vivo efficacy testing, thus expediting the development of a more efficacious TB vaccine and offering potential refinement and reduction to the use of NHPs for this purpose.

scholarly journals Adaption of the ex vivo mycobacterial growth inhibition assay for use with murine lung cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hannah Painter ◽  
Satria A. Prabowo ◽  
Felipe Cia ◽  
Lisa Stockdale ◽  
Rachel Tanner ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Ex Vivo ◽  
Lung Cells ◽  
Inhibition Assay ◽  
Growth Inhibition Assay ◽  
Murine Lung ◽  
Mycobacterial Growth
Sign in / Sign up

Export Citation Format

Share Document