Tools for Assessing the Protective Efficacy of TB Vaccines in Humans: in vitro Mycobacterial Growth Inhibition Predicts Outcome of in vivo Mycobacterial Infection

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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

F1000Research ◽

10.12688/f1000research.51640.2 ◽

2021 ◽

Vol 10 ◽

pp. 257

Author(s):

Rachel Tanner ◽

Emily Hoogkamer ◽

Julia Bitencourt ◽

Andrew White ◽

Charelle Boot ◽

...

Keyword(s):

Growth Inhibition ◽

Protective Immunity ◽

Vaccine Development ◽

Autologous Serum ◽

Inhibition Assay ◽

Growth Inhibition Assay ◽

Vaccine Candidates ◽

Mycobacterial Growth

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.

Download Full-text

Comparative studies on tuberculin hypersensitivity in vivo and in vitro in patients with probable mycobacterial infection

Immunology Letters ◽

10.1016/s0165-2478(97)88641-5 ◽

1997 ◽

Vol 56 (1-3) ◽

pp. 443

Author(s):

E Sapundjieva

Keyword(s):

Comparative Studies ◽

Mycobacterial Infection

Download Full-text

Stage-dependent effect of deferoxamine on growth of Plasmodium falciparum in vitro

Blood ◽

10.1182/blood.v76.6.1250.1250 ◽

1990 ◽

Vol 76 (6) ◽

pp. 1250-1255 ◽

Cited By ~ 36

Author(s):

S Whitehead ◽

TE Peto

Keyword(s):

Plasmodium Falciparum ◽

Growth Inhibition ◽

Antimalarial Activity ◽

Clinical Malaria ◽

Inhibitory Effect ◽

Parasite Development ◽

And Control ◽

Speed Of Onset

Abstract Deferoxamine (DF) has antimalarial activity that can be demonstrated in vitro and in vivo. This study is designed to examine the speed of onset and stage dependency of growth inhibition by DF and to determine whether its antimalarial activity is cytostatic or cytocidal. Growth inhibition was assessed by suppression of hypoxanthine incorporation and differences in morphologic appearance between treated and control parasites. Using synchronized in vitro cultures of Plasmodium falciparum, growth inhibition by DF was detected within a single parasite cycle. Ring and nonpigmented trophozoite stages were sensitive to the inhibitory effect of DF but cytostatic antimalarial activity was suggested by evidence of parasite recovery in later cycles. However, profound growth inhibition, with no evidence of subsequent recovery, occurred when pigmented trophozoites and early schizonts were exposed to DF. At this stage in parasite development, the activity of DF was cytocidal and furthermore, the critical period of exposure may be as short as 6 hours. These observations suggest that iron chelators may have a role in the treatment of clinical malaria.

Download Full-text

Regeneration linked miRNA modify tumor phenotype and can enforce multi-lineage growth arrest in vivo

Scientific Reports ◽

10.1038/s41598-021-90009-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Siamak Salehi ◽

Oliver D. Tavabie ◽

Augusto Villanueva ◽

Julie Watson ◽

David Darling ◽

...

Keyword(s):

Cell Proliferation ◽

Growth Inhibition ◽

Tumor Aggressiveness ◽

Novel Treatment ◽

Tumor Phenotype ◽

Aggressive Tumor ◽

In Vivo Growth ◽

Regulation Of Regeneration

AbstractRegulated cell proliferation is an effector mechanism of regeneration, whilst dysregulated cell proliferation is a feature of cancer. We have previously identified microRNA (miRNA) that regulate successful and failed human liver regeneration. We hypothesized that these regulators may directly modify tumor behavior. Here we show that inhibition of miRNAs -503 and -23a, alone or in combination, enhances tumor proliferation in hepatocyte and non-hepatocyte derived cancers in vitro, driving more aggressive tumor behavior in vivo. Inhibition of miRNA-152 caused induction of DNMT1, site-specific methylation with associated changes in gene expression and in vitro and in vivo growth inhibition. Enforced changes in expression of two miRNA recapitulating changes observed in failed regeneration led to complete growth inhibition of multi-lineage cancers in vivo. Our results indicate that regulation of regeneration and tumor aggressiveness are concordant and that miRNA-based inhibitors of regeneration may constitute a novel treatment strategy for human cancers.

Download Full-text

β-elemene alleviates airway stenosis via the ILK/Akt pathway modulated by MIR143HG sponging miR-1275

Cellular & Molecular Biology Letters ◽

10.1186/s11658-021-00261-0 ◽

2021 ◽

Vol 26 (1) ◽

Author(s):

Guoying Zhang ◽

Cheng Xue ◽

Yiming Zeng

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Cell Model ◽

Protective Efficacy ◽

Rabbit Model ◽

Akt Pathway ◽

Loss Of Function ◽

Airway Stenosis

Abstract Background We have previously found that β-elemene could inhibit the viability of airway granulation fibroblasts and prevent airway hyperplastic stenosis. This study aimed to elucidate the underlying mechanism and protective efficacy of β-elemene in vitro and in vivo. Methods Microarray and bioinformatic analysis were used to identify altered pathways related to cell viability in a β-elemene-treated primary cell model and to construct a β-elemene-altered ceRNA network modulating the target pathway. Loss of function and gain of function approaches were performed to examine the role of the ceRNA axis in β-elemene's regulation of the target pathway and cell viability. Additionally, in a β-elemene-treated rabbit model of airway stenosis, endoscopic and histological examinations were used to evaluate its therapeutic efficacy and further verify its mechanism of action. Results The hyperactive ILK/Akt pathway and dysregulated LncRNA-MIR143HG, which acted as a miR-1275 ceRNA to modulate ILK expression, were suppressed in β-elemene-treated airway granulation fibroblasts; β-elemene suppressed the ILK/Akt pathway via the MIR143HG/miR-1275/ILK axis. Additionally, the cell cycle and apoptotic phenotypes of granulation fibroblasts were altered, consistent with ILK/Akt pathway activity. In vivo application of β-elemene attenuated airway granulation hyperplasia and alleviated scar stricture, and histological detections suggested that β-elemene's effects on the MIR143HG/miR-1275/ILK axis and ILK/Akt pathway were in line with in vitro findings. Conclusions MIR143HG and ILK may act as ceRNA to sponge miR-1275. The MIR143HG/miR-1275/ILK axis mediates β-elemene-induced cell cycle arrest and apoptosis of airway granulation fibroblasts by modulating the ILK/Akt pathway, thereby inhibiting airway granulation proliferation and ultimately alleviating airway stenosis.

Download Full-text

Chemical and biological control of Fusarium species involved in garlic dry rot at early crop stages

European Journal of Plant Pathology ◽

10.1007/s10658-021-02265-0 ◽

2021 ◽

Author(s):

Letizia Mondani ◽

Giorgio Chiusa ◽

Paola Battilani

Keyword(s):

Growth Inhibition ◽

Fusarium Species ◽

Maximum Growth ◽

Severity Index ◽

Field Application ◽

Dry Rot ◽

Great Capacity ◽

Chemical Products

AbstractThe aim of the study was to test in vitro and in vivo the efficacy of triazoles and biocontrol agents (BCAs) against Fusarium proliferatum and F. oxysporum, the former signaled as the main causal agent of garlic dry rot and the latter also involved. In vitro trials were organized using potato dextrose agar with added chemicals or BCAs inoculated with selected F. proliferatum and F. oxysporum. Garlic cloves were dipped before sowing in suspensions prepared with the fungicides showing the best performances in vitro; then they were dipped in Fusaria suspension before sowing. In in vitro trials, the maximum Fusaria growth inhibition was performed by Propiconazole + Prochloraz (100%), followed by Tebuconazole (88.9%). BCAs showed great capacity to control Fusaria, with a maximum growth inhibition of 80% (Trichoderma harzianum + T. gamsii). In vivo bacterial BCAs showed a similar capacity to control F. proliferatum and F. oxysporum compared to chemical products (mean of severity index 18.6% and 11.7%, respectively). In vivo results confirmed the in vitro performances, except for Trichoderma, which had the worst performances in vivo. Therefore, the results are preliminary but promising for future field application.

Download Full-text

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.

Download Full-text