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.

[1]  H. McShane,et al.  Induction of specific antibodies, IgG-secreting plasmablasts and memory B cells following BCG vaccination , 2021, bioRxiv.

[2]  H. McShane,et al.  A non-human primate in vitro functional assay for the early evaluation of TB vaccine candidates , 2021, NPJ vaccines.

[3]  F. Giannoni,et al.  Drug-Resistant Tuberculosis 2020: Where We Stand , 2020, Applied Sciences.

[4]  R. Reljic,et al.  Adaption of the ex vivo mycobacterial growth inhibition assay for use with murine lung cells , 2020, Scientific Reports.

[5]  H. McShane,et al.  Tools for Assessing the Protective Efficacy of TB Vaccines in Humans: in vitro Mycobacterial Growth Inhibition Predicts Outcome of in vivo Mycobacterial Infection , 2020, Frontiers in Immunology.

[6]  T. Ottenhoff,et al.  Disparate Tuberculosis Disease Development in Macaque Species Is Associated With Innate Immunity , 2019, Front. Immunol..

[7]  Sang-Nae Cho,et al.  In vitro Mycobacterial Growth Inhibition in South Korean Adults With Latent TB Infection , 2019, Front. Immunol..

[8]  H. Dockrell,et al.  Optimisation, harmonisation and standardisation of the direct mycobacterial growth inhibition assay using cryopreserved human peripheral blood mononuclear cells , 2019, Journal of immunological methods.

[9]  T. Ottenhoff,et al.  Prevention of tuberculosis infection and disease by local BCG in repeatedly exposed rhesus macaques , 2019, Nature Medicine.

[10]  N. Beeching,et al.  Human Hookworm Infection Enhances Mycobacterial Growth Inhibition and Associates With Reduced Risk of Tuberculosis Infection , 2018, Front. Immunol..

[11]  P. Bettencourt,et al.  Regulation of mycobacterial infection by macrophage Gch1 and tetrahydrobiopterin , 2018, Nature Communications.

[12]  T. Fletcher,et al.  Immunological correlates of mycobacterial growth inhibition describe a spectrum of tuberculosis infection , 2018, Scientific Reports.

[13]  M. Netea,et al.  Mycobacterial growth inhibition is associated with trained innate immunity , 2018, The Journal of clinical investigation.

[14]  F. Cia,et al.  High monocyte to lymphocyte ratio is associated with impaired protection after subcutaneous administration of BCG in a mouse model of tuberculosis , 2018, F1000Research.

[15]  M. Roederer,et al.  Toward Tuberculosis Vaccine Development: Recommendations for Nonhuman Primate Study Design , 2017, Infection and Immunity.

[16]  H. Dockrell,et al.  Assay optimisation and technology transfer for multi-site immuno-monitoring in vaccine trials , 2017, PloS one.

[17]  M. Hatherill,et al.  Application of a whole blood mycobacterial growth inhibition assay to study immunity against Mycobacterium tuberculosis in a high tuberculosis burden population , 2017, PloS one.

[18]  H. McShane,et al.  A mycobacterial growth inhibition assay (MGIA) for bovine TB vaccine development , 2017, Tuberculosis.

[19]  T. Scriba,et al.  The Cross-Species Mycobacterial Growth Inhibition Assay (MGIA) Project, 2010–2014 , 2017, Clinical and Vaccine Immunology.

[20]  C. Aagaard,et al.  Optimisation of a murine splenocyte mycobacterial growth inhibition assay using virulent Mycobacterium tuberculosis , 2017, Scientific Reports.

[21]  A. Lescano,et al.  A malaria vaccine protects Aotus monkeys against virulent Plasmodium falciparum infection , 2017, npj Vaccines.

[22]  A. Thomas,et al.  Variable BCG efficacy in rhesus populations: Pulmonary BCG provides protection where standard intra-dermal vaccination fails. , 2017, Tuberculosis.

[23]  P. Bettencourt,et al.  The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays , 2017, Scientific Reports.

[24]  A. Benedetti,et al.  Treatment of isoniazid-resistant tuberculosis with first-line drugs: a systematic review and meta-analysis. , 2017, The Lancet. Infectious diseases.

[25]  H. McShane,et al.  Replacing, reducing and refining the use of animals in tuberculosis vaccine research. , 2016, ALTEX.

[26]  S. Stibitz,et al.  A simplified mycobacterial growth inhibition assay (MGIA) using direct infection of mouse splenocytes and the MGIT system. , 2016, Journal of microbiological methods.

[27]  F. Gleeson,et al.  Alternative BCG delivery strategies improve protection against Mycobacterium tuberculosis in non-human primates: Protection associated with mycobacterial antigen-specific CD4 effector memory T-cell populations , 2016, Tuberculosis.

[28]  H. Dockrell,et al.  Polyfunctional CD4 T-cells correlate with in vitro mycobacterial growth inhibition following Mycobacterium bovis BCG-vaccination of infants. , 2016, Vaccine.

[29]  H. McShane,et al.  In vitro mycobacterial growth inhibition assays: A tool for the assessment of protective immunity and evaluation of tuberculosis vaccine efficacy. , 2016, Vaccine.

[30]  Ann Williams,et al.  A new tool for tuberculosis vaccine screening: Ex vivo Mycobacterial Growth Inhibition Assay indicates BCG-mediated protection in a murine model of tuberculosis , 2016, BMC Infectious Diseases.

[31]  H. Dockrell,et al.  Human biomarkers: can they help us to develop a new tuberculosis vaccine? , 2016, Future microbiology.

[32]  Ryung S. Kim,et al.  Association of Human Antibodies to Arabinomannan With Enhanced Mycobacterial Opsonophagocytosis and Intracellular Growth Reduction , 2016, The Journal of infectious diseases.

[33]  Fergus Gleeson,et al.  Ultra low dose aerosol challenge with Mycobacterium tuberculosis leads to divergent outcomes in rhesus and cynomolgus macaques. , 2016, Tuberculosis.

[34]  J. Knight,et al.  Distinct Transcriptional and Anti-Mycobacterial Profiles of Peripheral Blood Monocytes Dependent on the Ratio of Monocytes: Lymphocytes , 2015, EBioMedicine.

[35]  T. Nathaniel,et al.  ANTI-TUBERCULAR ACTIVITY OF EDTA AND HOUSEHOLD CHEMICALS AGAINST MYCOBACTERIUM SMEGMATIS, A SURROGATE FOR MULTI-DRUG RESISTANT TUBERCULOSIS , 2015 .

[36]  H. McShane,et al.  Ex vivo mycobacterial growth inhibition assay (MGIA) for tuberculosis vaccine testing - a protocol for mouse splenocytes , 2015, bioRxiv.

[37]  S. Wolfensohn,et al.  Refinement of welfare through development of a quantitative system for assessment of lifetime experience , 2015 .

[38]  J. Flynn,et al.  Immunology studies in non‐human primate models of tuberculosis , 2015, Immunological reviews.

[39]  Andrew Carkeet,et al.  Exact Parametric Confidence Intervals for Bland-Altman Limits of Agreement , 2015, Optometry and vision science : official publication of the American Academy of Optometry.

[40]  Cécile Crosnier,et al.  A PfRH5-Based Vaccine Is Efficacious against Heterologous Strain Blood-Stage Plasmodium falciparum Infection in Aotus Monkeys , 2015, Cell host & microbe.

[41]  W. Ho,et al.  Monkey Models of Tuberculosis: Lessons Learned , 2014, Infection and Immunity.

[42]  J. Kappes,et al.  Development of a luciferase based viral inhibition assay to evaluate vaccine induced CD8 T-cell responses. , 2014, Journal of immunological methods.

[43]  R. Borrow,et al.  Development and Use of a Serum Bactericidal Assay Using Pooled Human Complement To Assess Responses to a Meningococcal Group A Conjugate Vaccine in African Toddlers , 2014, Clinical and Vaccine Immunology.

[44]  J. Sterne,et al.  Protection by BCG vaccine against tuberculosis: a systematic review of randomized controlled trials. , 2014, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[45]  H. McShane,et al.  Evaluation of a Human BCG Challenge Model to Assess Antimycobacterial Immunity Induced by BCG and a Candidate Tuberculosis Vaccine, MVA85A, Alone and in Combination , 2013, The Journal of infectious diseases.

[46]  S. Morris,et al.  Time to Detection of Mycobacterium tuberculosis Using the MGIT 320 System Correlates with Colony Counting in Preclinical Testing of New Vaccines , 2013, Clinical and Vaccine Immunology.

[47]  H. McShane,et al.  Mycobacterial growth inhibition in murine splenocytes as a surrogate for protection against Mycobacterium tuberculosis (M. tb). , 2013, Tuberculosis.

[48]  H. Dockrell,et al.  Inhibition of Mycobacterial Growth In Vitro following Primary but Not Secondary Vaccination with Mycobacterium bovis BCG , 2013, Clinical and Vaccine Immunology.

[49]  G. Gstraunthaler,et al.  A plea to reduce or replace fetal bovine serum in cell culture media , 2013, Cytotechnology.

[50]  A. Hill,et al.  Can growth inhibition assays (GIA) predict blood-stage malaria vaccine efficacy? , 2012, Human vaccines & immunotherapeutics.

[51]  A. Martineau,et al.  A functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged BCG or M.Tb (BCGlux/M.Tb lux). , 2011, Journal of visualized experiments : JoVE.

[52]  A. Thomas,et al.  Vaccination with Plasmodium knowlesi AMA1 Formulated in the Novel Adjuvant Co-Vaccine HT™ Protects against Blood-Stage Challenge in Rhesus Macaques , 2011, PloS one.

[53]  M. Niederweis,et al.  Mycobacterium tuberculosis Can Utilize Heme as an Iron Source , 2011, Journal of bacteriology.

[54]  Adrian V. S. Hill,et al.  Establishment of an Aerosol Challenge Model of Tuberculosis in Rhesus Macaques and an Evaluation of Endpoints for Vaccine Testing , 2010, Clinical and Vaccine Immunology.

[55]  S. Morris,et al.  A practical in vitro growth inhibition assay for the evaluation of TB vaccines. , 2009, Vaccine.

[56]  W. Jacobs,et al.  Development of a Murine Mycobacterial Growth Inhibition Assay for Evaluating Vaccines against Mycobacterium tuberculosis , 2009, Clinical and Vaccine Immunology.

[57]  A. Thomas,et al.  MVA.85A Boosting of BCG and an Attenuated, phoP Deficient M. tuberculosis Vaccine Both Show Protective Efficacy Against Tuberculosis in Rhesus Macaques , 2009, PloS one.

[58]  T. Cohen,et al.  Modeling the effects of strain diversity and mechanisms of strain competition on the potential performance of new tuberculosis vaccines , 2008, Proceedings of the National Academy of Sciences.

[59]  D. Cohen,et al.  TRPV4 enhances the cellular uptake of aminoglycoside antibiotics , 2008, Journal of Cell Science.

[60]  G. Bjune,et al.  The emergence of Beijing family genotypes of Mycobacterium tuberculosis and low‐level protection by bacille Calmette–Guérin (BCG) vaccines: is there a link? , 2006, Clinical and experimental immunology.

[61]  Hong Zhou,et al.  Immunity to Recombinant Plasmodium falciparum Merozoite Surface Protein 1 (MSP1): Protection in Aotus nancymai Monkeys Strongly Correlates with Anti-MSP1 Antibody Titer and In Vitro Parasite-Inhibitory Activity , 2006, Infection and Immunity.

[62]  I. Stănescu,et al.  Validation overview of bio-analytical methods , 2005, Gene Therapy.

[63]  M. Denis,et al.  Ability of T cell subsets and their soluble mediators to modulate the replication of Mycobacterium bovis in bovine macrophages. , 2004, Cellular immunology.

[64]  J. Ellner,et al.  Investigation of the relationships between immune-mediated inhibition of mycobacterial growth and other potential surrogate markers of protective Mycobacterium tuberculosis immunity. , 2002, The Journal of infectious diseases.

[65]  J. Ellner,et al.  A whole blood bactericidal assay for tuberculosis. , 2001, The Journal of infectious diseases.

[66]  M. Levin,et al.  Evaluation of human antimycobacterial immunity using recombinant reporter mycobacteria. , 2000, The Journal of infectious diseases.

[67]  J. Ellner,et al.  Lymphocyte-dependent inhibition of growth of virulent Mycobacterium tuberculosis H37Rv within human monocytes: requirement for CD4+ T cells in purified protein derivative-positive, but not in purified protein derivative-negative subjects. , 1998, Journal of immunology.

[68]  E. Gormley,et al.  Cellular responses and Mycobacterium bovis BCG growth inhibition by bovine lymphocytes , 1997, Immunology and cell biology.

[69]  P. E. M. Fine,et al.  Variation in protection by BCG: implications of and for heterologous immunity , 1995, The Lancet.

[70]  M. Horwitz,et al.  Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3. , 1990, Journal of immunology.

[71]  J. O. Irwin,et al.  The estimation of the bactericidal power of the blood , 1938, Epidemiology and Infection.