title: A new tool for tuberculosis vaccine screening: Ex vivo Mycobacterial Growth Inhibition Assay indicates BCG-mediated protection in a murine model of tuberculosis.
abstract: In the absence of a validated animal model and/or an immune correlate which predict vaccine-mediated protection, large-scale clinical trials are currently the only option to prove efficacy of new tuberculosis candidate vaccines. Tools to facilitate testing of new tuberculosis (TB) vaccines are therefore urgently needed.
  
  
  
  We present here an optimized ex vivo mycobacterial growth inhibition assay (MGIA) using a murine Mycobacterium tuberculosis infection model. This assay assesses the combined ability of host immune cells to inhibit mycobacterial growth in response to vaccination. C57BL/6 mice were immunized with Bacillus Calmette-Guérin (BCG) and growth inhibition of mycobacteria by splenocytes was assessed. Mice were also challenged with Mycobacterium tuberculosis Erdman, and bacterial burden was assessed in lungs and spleen.
  
  
  
  Using the growth inhibition assay, we find a reduction in BCG CFU of 0.3-0.8 log10 after co-culture with murine splenocytes from BCG vaccinated versus naïve C57BL/6 mice. BCG vaccination in our hands led to a reduction in bacterial burden after challenge with Mycobacterium tuberculosis of approx. 0.7 log10 CFU in lung and approx. 1 log10 CFU in spleen. This effect was also seen when using Mycobacterium smegmatis as the target of growth inhibition. An increase in mycobacterial numbers was found when splenocytes from interferon gamma-deficient mice were used, compared to wild type controls, indicating that immune mechanisms may also be investigated using this assay.
  
  
  
  We believe that the ex vivo mycobacterial growth inhibition assay could be a useful tool to help assess vaccine efficacy in future, alongside other established methods. It could also be a valuable tool for determination of underlying immune mechanisms.
authors: London School of Hygiene and Tropical Medicine, London, UK. andrea.zelmer@lshtm.ac.uk.
authors: Andrea
authors: 0000-0001-9621-4406
authors: A
authors: Zelmer
authors: The Jenner Institute, Oxford University, Oxford, UK.
authors: Rachel
authors: R
authors: Tanner
authors: The Jenner Institute, Oxford University, Oxford, UK.
authors: Elena
authors: E
authors: Stylianou
authors: London School of Hygiene and Tropical Medicine, London, UK.
authors: Timon
authors: T
authors: Damelang
authors: Center for Biologics Evaluation and Research, Silver Spring, MD, USA.
authors: Sheldon
authors: S
authors: Morris
authors: Colorado State University, Fort Collins, CO, USA.
authors: Angelo
authors: A
authors: Izzo
authors: Public Health England, Porton Down, UK.
authors: Ann
authors: A
authors: Williams
authors: Public Health England, Porton Down, UK.
authors: Sally
authors: S
authors: Sharpe
authors: The Jenner Institute, Oxford University, Oxford, UK.|Animal and Plant Health Agency, Weybridge, UK.|Translational Research Unit, National Institute for Infectious Diseasess "Lazzaro Spallanzani", Rome, Italy.
authors: Ilaria
authors: I
authors: Pepponi
authors: Aeras, Rockville, MD, USA.
authors: Barry
authors: B
authors: Walker
authors: Aeras, Rockville, MD, USA.
authors: David A
authors: DA
authors: Hokey
authors: The Jenner Institute, Oxford University, Oxford, UK.
authors: Helen
authors: H
authors: McShane
authors: Aeras, Rockville, MD, USA.
authors: Michael
authors: M
authors: Brennan
authors: London School of Hygiene and Tropical Medicine, London, UK.
authors: Helen
authors: H
authors: Fletcher
mesh_terms: D000818:Animals; D001500:BCG Vaccine / Q000276:immunology* / Q000494:pharmacology; D018920:Coculture Techniques; D015169:Colony Count, Microbial / Q000379:methods*; D004195:Disease Models, Animal; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005260:Female; D007371:Interferon-gamma / Q000235:genetics / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D009163:Mycobacterium bovis / Q000276:immunology; D020102:Mycobacterium smegmatis / Q000254:growth & development / Q000276:immunology; D009169:Mycobacterium tuberculosis / Q000276:immunology / Q000472:pathogenicity; D013154:Spleen / Q000166:cytology / Q000276:immunology / Q000382:microbiology; D014376:Tuberculosis / Q000276:immunology / Q000517:prevention & control*; D032581:Tuberculosis Vaccines / Q000276:immunology / Q000494:pharmacology*; D014611:Vaccination
chemical_list: D001500:BCG Vaccine; D032581:Tuberculosis Vaccines; D007371:Interferon-gamma
keywords: Growth inhibition assay; Mycobacteria; Tuberculosis; Vaccines