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

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.

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Inhibition of Mycobacterial GrowthIn Vitrofollowing Primary but Not Secondary Vaccination with Mycobacterium bovis BCG

Clinical and Vaccine Immunology ◽

10.1128/cvi.00427-13 ◽

2013 ◽

Vol 20 (11) ◽

pp. 1683-1689 ◽

Cited By ~ 52

Author(s):

Helen A. Fletcher ◽

Rachel Tanner ◽

Robert S. Wallis ◽

Joel Meyer ◽

Zita-Rose Manjaly ◽

...

Keyword(s):

Growth Inhibition ◽

Mycobacterium Bovis ◽

Primary Vaccination ◽

Inhibition Assay ◽

Peripheral Blood Mononuclear ◽

Growth Inhibition Assay ◽

Ifn Γ ◽

Mycobacterial Growth

ABSTRACTDespite the widespread use of theMycobacterium bovisBCG vaccine, there are more than 9 million new cases of tuberculosis (TB) every year, and there is an urgent need for better TB vaccines. TB vaccine candidates are selected for evaluation based in part on the detection of an antigen-specific gamma interferon (IFN-γ) response. The measurement of mycobacterial growth in blood specimens obtained from subjects immunized with investigational TB vaccines may be a betterin vitrocorrelate ofin vivovaccine efficacy. We performed a clinical study with 30 United Kingdom adults who were followed for 6 months to evaluate the abilities of both a whole-blood- and a novel peripheral blood mononuclear cell (PBMC)-based mycobacterial growth inhibition assay to measure a response to primary vaccination and revaccination with BCG. Using cryopreserved PBMCs, we observed a significant improvement in mycobacterial growth inhibition following primary vaccination but no improvement in growth inhibition following revaccination with BCG (P< 0.05). Mycobacterial growth inhibition following primary BCG vaccination was not correlated with purified protein derivative (PPD) antigen-specific IFN-γ enzyme-linked immunospot (ELISPOT) responses. We demonstrate that a mycobacterial growth inhibition assay can detect improved capacity to control growth following primary immunization, but not revaccination, with BCG. This is the first study to demonstrate that anin vitrogrowth inhibition assay can identify a difference in vaccine responses by comparing both primary and secondary BCG vaccinations, suggesting thatin vitrogrowth inhibition assays may serve as better surrogates of clinical efficacy than the assays currently used for the assessment of candidate TB vaccines.

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

Tuberculosis ◽

10.1016/j.tube.2017.07.008 ◽

2017 ◽

Vol 106 ◽

pp. 118-122 ◽

Cited By ~ 1

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

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

Clinical and Vaccine Immunology ◽

10.1128/cvi.00067-09 ◽

2009 ◽

Vol 16 (7) ◽

pp. 1025-1032 ◽

Cited By ~ 46

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.

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The Cross-Species Mycobacterial Growth Inhibition Assay (MGIA) Project, 2010–2014

Clinical and Vaccine Immunology ◽

10.1128/cvi.00142-17 ◽

2017 ◽

Vol 24 (9) ◽

Cited By ~ 17

Author(s):

Michael J. Brennan ◽

Rachel Tanner ◽

Sheldon Morris ◽

Thomas J. Scriba ◽

Jacqueline M. Achkar ◽

...

Keyword(s):

Growth Inhibition ◽

Functional Assay ◽

Major Advance ◽

Growth Inhibition Assay ◽

Content Type ◽

Vaccine Responses ◽

Vaccine Testing ◽

Mycobacterial Growth

ABSTRACT The development of a functional biomarker assay in the tuberculosis (TB) field would be widely recognized as a major advance in efforts to develop and to test novel TB vaccine candidates efficiently. We present preliminary studies using mycobacterial growth inhibition assays (MGIAs) to detect Mycobacterium bovis BCG vaccine responses across species, and we extend this work to determine whether a standardized MGIA can be applied in characterizing new TB vaccines. The comparative MGIA studies reviewed here aimed to evaluate robustness, reproducibility, and ability to reflect in vivo responses. In doing so, they have laid the foundation for the development of a MGIA that can be standardized and potentially qualified. A major challenge ahead lies in better understanding the relationships between in vivo protection, in vitro growth inhibition, and the immune mechanisms involved. The final outcome would be a MGIA that could be used with confidence in TB vaccine trials. We summarize data arising from this project, present a strategy to meet the goals of developing a functional assay for TB vaccine testing, and describe some of the challenges encountered in performing and transferring such assays.

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A non-human primate in vitro functional assay for the early evaluation of TB vaccine candidates

npj Vaccines ◽

10.1038/s41541-020-00263-7 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Rachel Tanner ◽

Andrew D. White ◽

Charelle Boot ◽

Claudia C. Sombroek ◽

Matthew K. O’Shea ◽

...

Keyword(s):

Growth Inhibition ◽

Mononuclear Cells ◽

Functional Assay ◽

Intermediate Precision ◽

Peripheral Blood Mononuclear ◽

Growth Inhibition Assay ◽

Early Evaluation ◽

Human Primate

AbstractWe present a non-human primate mycobacterial growth inhibition assay (MGIA) using in vitro blood or cell co-culture with the aim of refining and expediting early tuberculosis vaccine testing. We have taken steps to optimise the assay using cryopreserved peripheral blood mononuclear cells, transfer it to end-user institutes, and assess technical and biological validity. Increasing cell concentration or mycobacterial input and co-culturing in static 48-well plates compared with rotating tubes improved intra-assay repeatability and sensitivity. Standardisation and harmonisation efforts resulted in high consistency agreements, with repeatability and intermediate precision <10% coefficient of variation (CV) and inter-site reproducibility <20% CV; although some systematic differences were observed. As proof-of-concept, we demonstrated ability to detect a BCG vaccine-induced improvement in growth inhibition in macaque samples, and a correlation between MGIA outcome and measures of protection from in vivo disease development following challenge with either intradermal BCG or aerosol/endobronchial Mycobacterium tuberculosis (M.tb) at a group and individual animal level.

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In vitro mycobacterial growth inhibition assays: A tool for the assessment of protective immunity and evaluation of tuberculosis vaccine efficacy

Vaccine ◽

10.1016/j.vaccine.2016.07.058 ◽

2016 ◽

Vol 34 (39) ◽

pp. 4656-4665 ◽

Cited By ~ 35

Author(s):

Rachel Tanner ◽

Matthew K. O’Shea ◽

Helen A. Fletcher ◽

Helen McShane

Keyword(s):

Growth Inhibition ◽

Vaccine Efficacy ◽

Protective Immunity ◽

Mycobacterial Growth

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A novel mycobacterial growth inhibition assay employing live-cell imaging of virulent M. tuberculosis and monitoring of host cell viability

Tuberculosis ◽

10.1016/j.tube.2020.101977 ◽

2020 ◽

Vol 124 ◽

pp. 101977

Author(s):

Blanka Andersson ◽

Michaela Jonsson Nordvall ◽

Amanda Welin ◽

Maria Lerm ◽

Thomas Schön

Keyword(s):

Growth Inhibition ◽

Cell Viability ◽

Host Cell ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Inhibition Assay ◽

Growth Inhibition Assay ◽

Mycobacterial Growth

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

PLoS ONE ◽

10.1371/journal.pone.0184563 ◽

2017 ◽

Vol 12 (9) ◽

pp. e0184563 ◽

Cited By ~ 7

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

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

HAYATI Journal of Biosciences ◽

10.4308/hjb.29.1.1-8 ◽

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.

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