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

2013 ◽  
Vol 20 (11) ◽  
pp. 1683-1689 ◽  
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.

scholarly journals Mycobacterial Growth Inhibition Assay (MGIA) as a Host Directed Diagnostic Tool for the Evaluation of the Immune Response in Subjects Living With Type 2 Diabetes Mellitus

2021 ◽  
Vol 11 ◽  
Author(s):  
Miriam Bobadilla-del-Valle ◽  
Francisco Leal-Vega ◽  
Pedro Torres-Gonzalez ◽  
Anabel Ordaz-Vazquez ◽  
Maria de Lourdes Garcia-Garcia ◽  
...  
Keyword(s):  
Immune Response ◽  
Glycemic Control ◽  
Growth Inhibition ◽  
Healthy Subjects ◽  
Control Group ◽  
Inhibition Assay ◽  
Poor Control ◽  
Growth Inhibition Assay ◽  
Mycobacterial Growth

The lack of efficient and cost-effective diagnostic tools contributes to poor control of tuberculosis in endemic countries. Moreover, host biological processes influence susceptibility, and infection resolution. It is well known that comorbidities such as type 2 diabetes mellitus (DM2) affect the host immune response, making individuals more susceptible to Mycobacterium tuberculosis infection. Currently, there are no laboratory tools that can identify those subjects who have a higher risk of developing the disease. In this study, we used a whole blood mycobacterial growth inhibition assay to assess the immune response capacity to inhibit mycobacterial growth between healthy subjects and those living with DM2 with optimal and poor glycemic control. We also measured cytokine levels in the culture supernatant by cytokine bead arrays. We included 89 patients with DM2: 54 patients with optimal control (mean age 56.2 ± 11.75 years) and 35 patients with poor control (mean age 52.05 ± 9.94 years). We also included 44 healthy subjects as controls (mean age 42.12 ± 11.75 years). We compared the Δlog UFC (a value that represents the difference between mycobacterial growth in the control tube versus the subject’s blood) between each group. Our results demonstrate that patients with DM2 had a lower capacity to inhibit M. tuberculosis growth (Δlog UFC DM2 subjects 0.9581 (-0.3897 to 2.495) vs Δlog UFC healthy subjects 0.7190 (-0.2678 to 2.098); p=0.013). Comparing subjects living with DM2 (optimal and poor glycemic control) vs healthy subjects, we found only significant differences between healthy subjects and patients poorly controlled (Δlog UFC optimal control group 0.876 (-0.3897 to 2.495); Δlog UFC poor control group 1.078 (0.068 to 2.33); Δlog UFC healthy subjects 0.7190 (-0.2678 to 2.098); p= 0.022). Therefore, glycemic control assessed by glycosylated hemoglobin values influences the capacity of the host to control the infection. Our results confirm that the whole blood mycobacterial growth inhibition assay has potential utility as an in vitro marker of M. tuberculosis immunological control in vivo in subjects living with DM2. This assay can be used to evaluate the immune response of each individual against M. tuberculosis, allowing clinicians to choose a more specific host-directed therapy.

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

npj Vaccines ◽  
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.

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 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
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