Accelerating the development of effective vaccines for protection against Mycobacterium tuberculosis
Lead Research Organisation:
London School of Hygiene & Tropical Medicine
Department Name: Infectious and Tropical Diseases
Abstract
The aim of this project is to accelerate and improve the preclinical development of effective vaccines for protection against tuberculosis (TB). TB is a primarily airborne infection caused by members of the Mycobacterium tuberculosis complex (MTBC). The BCG vaccine is the only clinically approved vaccine against TB. However, it has variations in protection (0-80%) globally [2]. In addition, variations in protection against different TB strains (representative of different lineages of MTBC) have been reported in animal models [3]. Increasing our understanding of these variations may have important implications on vaccine design and testing. An important step involves inclusion of TB strains when preclinically validating vaccines. During preclinical vaccine development, specific biological indictors are analysed to help find candidates that induce a strong immune response. Within the TB field, indicators of a strong immune response (which may indicate protection) remain unknown.
The mycobacterial growth inhibition assay (MGIA) is able to quantify mycobacteria, based on the principle that a reduction of bacterial growth will be observed following co-culture of mycobacteria with cells from immunised individuals versus unimmunised. In this project, the MGIA is used as a read-out of the ability of cells from mice to inhibit MTB growth. Since initial bacterial input is the same, the bacterial growth inhibitory effect of cells isolated from vaccinated mice is compared with unvaccinated, as a potential surrogate of vaccine efficacy. Further, this assay is being utlilised to investigate variations in the ability of BCG-vaccinated mice to inhibit growth of different TB strains, with the later aim to set up a system in which novel vaccines can also be investigated. To date, an MGIA has been established in mouse spleen and lung cells. Growth inhibition of an MTB laboratory strain has been shown in lung and spleen cells from mice vaccinated with BCG, as well as a novel TB vaccine, compared with unvaccinated mice. In order to understand the differences in the ability of cells from vaccinated and unvaccinated animals to control bacterial growth, RNA seq was performed to determine changes in gene expression which may account for these variations. Flow cytometric analysis was also performed to determine changes in cell populations over the assay culture period. A further RNA seq experiment has been performed to understand variations observed in the ability of cells from vaccinated animals to control growth of different lineages of the MTBC.
To carry out this analysis, I have obtained a Home Office licence (whole organ/organism physiology). I have attended two quantitative immunology courses in preparation for transcriptomic analysis (quantitative). I completed a placement at SATVI (Cape Town, South Africa) focusing on host signatures and machine learning (quantitative). I am taking part in an industrial placement in Seattle in January 2020 where I will be involved in mouse MTB challenge studies (whole organ/organism physiology) and performing bioinformatic analysis on samples obtained from the challenge study (quantitative). During the studentship I expect to increase my understanding of vaccine research and development, and the molecular mechanisms of MTB infection and disease progression. I hope to gain skills in experimentation in animal models, bacterial culture and quantitative analysis of transcription.
1. Sharma A, Bloss E, Heilig CM, Click ES. Tuberculosis caused by Mycobacterium africanum, United States, 2004-2013. Emerg Infect Dis 2016:22,396-403.
2. Colditz GA, Brewer TF, Berkey CS et al. Efficacy of BCG vaccine in the prevention of tuberculosis. Metaanalysis of the published literature. JAMA 1994;271:698-702.
3. Henao-Tamayo M et al. The efficacy of the BCG vaccine against newly emerging clinical strains of mycobacterium tuberculosis. PLoS One 2015:10,1-15.
The mycobacterial growth inhibition assay (MGIA) is able to quantify mycobacteria, based on the principle that a reduction of bacterial growth will be observed following co-culture of mycobacteria with cells from immunised individuals versus unimmunised. In this project, the MGIA is used as a read-out of the ability of cells from mice to inhibit MTB growth. Since initial bacterial input is the same, the bacterial growth inhibitory effect of cells isolated from vaccinated mice is compared with unvaccinated, as a potential surrogate of vaccine efficacy. Further, this assay is being utlilised to investigate variations in the ability of BCG-vaccinated mice to inhibit growth of different TB strains, with the later aim to set up a system in which novel vaccines can also be investigated. To date, an MGIA has been established in mouse spleen and lung cells. Growth inhibition of an MTB laboratory strain has been shown in lung and spleen cells from mice vaccinated with BCG, as well as a novel TB vaccine, compared with unvaccinated mice. In order to understand the differences in the ability of cells from vaccinated and unvaccinated animals to control bacterial growth, RNA seq was performed to determine changes in gene expression which may account for these variations. Flow cytometric analysis was also performed to determine changes in cell populations over the assay culture period. A further RNA seq experiment has been performed to understand variations observed in the ability of cells from vaccinated animals to control growth of different lineages of the MTBC.
To carry out this analysis, I have obtained a Home Office licence (whole organ/organism physiology). I have attended two quantitative immunology courses in preparation for transcriptomic analysis (quantitative). I completed a placement at SATVI (Cape Town, South Africa) focusing on host signatures and machine learning (quantitative). I am taking part in an industrial placement in Seattle in January 2020 where I will be involved in mouse MTB challenge studies (whole organ/organism physiology) and performing bioinformatic analysis on samples obtained from the challenge study (quantitative). During the studentship I expect to increase my understanding of vaccine research and development, and the molecular mechanisms of MTB infection and disease progression. I hope to gain skills in experimentation in animal models, bacterial culture and quantitative analysis of transcription.
1. Sharma A, Bloss E, Heilig CM, Click ES. Tuberculosis caused by Mycobacterium africanum, United States, 2004-2013. Emerg Infect Dis 2016:22,396-403.
2. Colditz GA, Brewer TF, Berkey CS et al. Efficacy of BCG vaccine in the prevention of tuberculosis. Metaanalysis of the published literature. JAMA 1994;271:698-702.
3. Henao-Tamayo M et al. The efficacy of the BCG vaccine against newly emerging clinical strains of mycobacterium tuberculosis. PLoS One 2015:10,1-15.
People |
ORCID iD |
Helen Fletcher (Primary Supervisor) | |
Hannah Painter (Student) |
Publications
Painter H
(2020)
Adaption of the ex vivo mycobacterial growth inhibition assay for use with murine lung cells.
in Scientific reports
Studentship Projects
Project Reference | Relationship | Related To | Start | End | Student Name |
---|---|---|---|---|---|
MR/N013638/1 | 30/09/2016 | 29/09/2025 | |||
1784589 | Studentship | MR/N013638/1 | 30/09/2016 | 03/01/2021 | Hannah Painter |
MR/R502273/1 | 30/09/2017 | 29/09/2021 | |||
1784589 | Studentship | MR/R502273/1 | 30/09/2016 | 03/01/2021 | Hannah Painter |
Description | Keystone Scholarship |
Amount | $1,200 (USD) |
Organisation | National Institutes of Health (NIH) |
Sector | Public |
Country | United States |
Start | 01/2019 |
End | 01/2019 |
Description | MRC National Productivity Investment Fund |
Amount | £9,376 (GBP) |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2020 |
End | 03/2020 |
Title | Murine lung mycobacterial growth inhibition assay |
Description | The splenocyte mycobacterial growth inhibition assay (MGIA) [1] has been optimised and adapted for use in murine lung as a potential 'in vitro challenge model' to analyse tuberculosis (TB) vaccine efficacy in mice. The MGIA has been developed as a tool to enable existing and preclinical tuberculosis vaccine to be evaluated in animals models in tandem with TB challenge studies. 1. Zelmer, A. et al. Ex vivo mycobacterial growth inhibition assay (MGIA) for tuberculosis vaccine testing - a protocol for mouse splenocytes. bioRxiv 1-10 (2015). doi:http://dx.doi.org/10.1101/020560 |
Type Of Material | Technology assay or reagent |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | The assay does not require the animals to be challenged with Mycobacterium tuberculosis, reducing both the length of study and severity level assigned to it. In addition, since multiple cell inputs for the assay can be harvested from a single animal, less animals are required to design a experiment with sufficient power. The assay incorporates both the refinement and reduction principles of the 3Rs of animals in research. |
URL | https://www.nature.com/articles/s41598-020-60223-y |
Description | MRC National Productivity Investment Fund - PhD Innovation Placements |
Organisation | Seattle Children's Hospital |
Country | United States |
Sector | Hospitals |
PI Contribution | refer to Validate pump prime NPF awarded to Hannah Painter covered flights, living expenses, visa, insurance and a lab consumables fund for project. |
Collaborator Contribution | refer to Validate pump prime Dr Rhea Coler to host Hannah Painter for three-month placement at Seattle Children's. |
Impact | Following NFP award to Hannah Painter, Helen Fletcher and Rhea Coler applies for further funding from Validate (see record). |
Start Year | 2019 |
Description | Validate Pump Prime |
Organisation | Seattle Children's Hospital |
Country | United States |
Sector | Hospitals |
PI Contribution | Validate pump prime funded awarded to Dr Rhea Coler and Professor Helen Fletcher. Project will use the murine lung MGIA (developed by Painter, Fletcher and Zelmer at LSHTM) to test candidate TB vaccines developed under funding awarded to Dr Coler. MTB laboratory and clinical strains for MGIA grown by Hannah Painter and shipped to Coler Group. Transcriptional analysis over an MTB challenge time course performed by RNA seq. RNA extraction protocols developed by Hannah Painter at LSHTM and transferred to Coler Group in person during placement (funded by MRC National Productivity Fund). RNA seq data analysis to be performed by Hannah Painter, using training and pipeline developed at LSHTM. HPC at LSHTM used for analysis. |
Collaborator Contribution | In vivo murine studies, MTB challenge and downstream CFU, ICS and ELISA. |
Impact | in progress |
Start Year | 2019 |