The development and establishment of a heat inactivated preparation of Mycobacterium tuberculosis (H37Rv) as the first international standard for nucleic acid amplification techniques.

A need to develop an inactivated Mycobacterium tuberculosis (Mtb) preparation, to be used as a DNA standard, as an important and urgent requirement for Tuberculosis (TB) diagnostics standardization was identified in 2018. A candidate material was generated using a heat inactivated culture of Mtb strain H37Rv. A lyophilised preparation was evaluated for its suitability as an International Standard for molecular detection of Mtb DNA in an international collaborative study. Together with the use of quantitative PCR assays and rapid diagnostic tests, this candidate standard was demonstrated to be fit-for-purpose. Based on the results from this collaborative study, it is proposed this lyophilised heat inactivated Mtb preparation (NIBSC code: 20/152) to be established by the World Health Organization Expert Committee on Biological Standardization, as the First WHO International Standard for Mycobacterium tuberculosis (H37Rv) for nucleic acid amplification techniques with an assigned unitage of 6.3 log10 or 2 × 106 International Units per vial. The intended uses of this IS are for calibration of secondary or in-house reference preparations used in the assays for the molecular detection of Mtb DNA. It may also be used for assay validation and monitoring the performance in terms of limit of detection of rapid diagnostic tests.

Efficacy and immunogenicity of different BCG doses in BALB/c and CB6F1 mice when challenged with H37Rv or Beijing HN878.

Two strains of mice (BALB/c and CB6F1) were vaccinated with a range of Bacille Calmette-Guérin (BCG) Danish doses from 3 × 105 to 30 CFU/mouse, followed by aerosol infection with Mtb (H37Rv or West-Beijing HN878 strain). The results indicated that both strains of mice when infected with HN878 exhibited significant protection in their lungs with BCG doses at 3 × 105-3000 CFU (BALB/c) and 3 × 105-300 CFU (CB6F1). Whereas, a significant protection was seen in both strains of mice with BCG doses at 3 × 105-300 CFU when infected with H37Rv. A significant increase in the frequencies of BCG-specific IFNγ+ IL2+ TNFα+ CD4 T cells in the BCG doses at 3 × 105-3000 CFU (BALB/c) and 3 × 105-300 CFU (CB6F1) was seen. The IFNγ+ IL2+ TNFα+ CD4 T cells correlated with the Mtb burden in the lungs of HN878 infected mice (BALB/c and CB6F1) whereas, IFNγ+ TNFα+ CD4 T cells correlated with the BALB/c mice infected with H37Rv or HN878. The BCG dose at 3000 CFU (an equivalent single human dose in the mice by body weight) is protective in both strains of mice infected with H37Rv or HN878 and may serve an interesting dose to test new TB vaccine in a preclinical animal model.

Three-dimensional in situ morphometrics of Mycobacterium tuberculosis infection within lesions by optical mesoscopy and novel acid-fast staining.

Tuberculosis (TB) preclinical testing relies on in vivo models including the mouse aerosol challenge model. The only method of determining colony morphometrics of TB infection in a tissue in situ is two-dimensional (2D) histopathology. 2D measurements consider heterogeneity within a single observable section but not above and below, which could contain critical information. Here we describe a novel approach, using optical clearing and a novel staining procedure with confocal microscopy and mesoscopy, for three-dimensional (3D) measurement of TB infection within lesions at sub-cellular resolution over a large field of view. We show TB morphometrics can be determined within lesion pathology, and differences in infection with different strains of Mycobacterium tuberculosis. Mesoscopy combined with the novel CUBIC Acid-Fast (CAF) staining procedure enables a quantitative approach to measure TB infection and allows 3D analysis of infection, providing a framework which could be used in the analysis of TB infection in situ.

Comparative fitness analysis of D-cycloserine resistant mutants reveals both fitness-neutral and high-fitness cost genotypes.

Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for six decades without significant appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We therefore investigated why Mycobacterium tuberculosis fails to become resistant to D-cycloserine. To address this question, we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low rate of emergence of D-cycloserine resistance mutations is the dominant biological factor delaying the appearance of clinical resistance to this antibiotic. Furthermore, we also identified potential compensatory mechanisms able to minimize the severe fitness costs of primary D-cycloserine resistance conferring mutations.

Comparison of pellicle and shake flask-grown BCG strains by quality control assays and protection studies.

A global BCG vaccine shortage began in 2013 which impacted availability for infant vaccinations, as well as preclinical studies and clinical trials of new TB vaccines. Stakeholders met in 2015 at McGill University in Montreal to discuss the shortage and potential mitigation strategies. Manufacturing BCG through a more tractable liquid fermentation process instead of the traditional pellicle growth method was considered a potentially viable strategy. This pilot program compared pellicle-grown and shake flask-grown BCG strains (as a first step towards modeling fermenter-produced BCG vaccine) in selected quality control assays, as well as mouse and guinea pig protection studies. Conventional pellicle-grown, lyophilized BCG WHO Reference Reagents (Danish, Moreau, Russian, Tokyo strains) were obtained from the National Institute for Biological Standards and Control (NIBSC), UK. Strains were grown in shake flasks and glycerol stocks prepared. Shake flask-grown BCG culture preparations generally met the requirements of quality control testing at NIBSC. In mouse and guinea pig protection studies there were no significant differences in lung colony forming units (CFUs) between shake flask-grown and pellicle-grown preparations, with the exception of BCG Russian, where the shake flask-grown preparation protected better in mice (P = 0.0042), but the pellicle-grown preparation protected better in guinea pigs (P = 0.0015). Producing BCG vaccines by a more tractable liquid growth process could be a viable solution to the global BCG shortage.

Immunogenicity and Protective Efficacy of the DAR-901 Booster Vaccine in a Murine Model of Tuberculosis.

BACKGROUND: The development of a novel tuberculosis vaccine is a leading global health priority. SRL172, an inactivated, whole-cell mycobacterial vaccine, was safe, immunogenic and reduced the incidence of culture-confirmed tuberculosis in a phase III trial in HIV-infected and BCG immunized adults in Tanzania. Here we describe the immunogenicity and protective efficacy of DAR-901, a booster vaccine against tuberculosis manufactured from the same seed strain using a new scalable method. METHODS: We evaluated IFN-γ responses by ELISpot and antibody responses by enzyme linked immunosorbent assay in C57BL/6 and BALB/c mice after three doses of DAR-901. In an aerosol challenge model, we evaluated the protective efficacy of the DAR-901 booster in C57BL/6 mice primed with BCG and boosted with two doses of DAR-901 at 4 dosage levels in comparison with homologous BCG boost. RESULTS: DAR-901 vaccination elicited IFN-γ responses to mycobacterial antigen preparations derived from both DAR-901 and Mycobacterium tuberculosis. DAR-901 immunization enhanced antibody responses to DAR-901 but not Mycobacterium tuberculosis lysate or purified protein derivative. Among animals primed with BCG, boosting with DAR-901 at 1 mg provided greater protection against aerosol challenge than a homologous BCG boost (lungs P = 0.036, spleen P = 0.028). CONCLUSIONS: DAR-901 induces cellular and humoral immunity and boosts protection from M. tuberculosis compared to a homologous BCG boost.

The establishment of sub-strain specific WHO Reference Reagents for BCG vaccine.

As the latest addition to the sub-strain specific WHO Reference Reagents of BCG vaccine, an international collaborative study was completed to evaluate the suitability of a candidate BCG Moreau-RJ sub-strain as a WHO Reference Reagent of BCG vaccine. This follows the recent replacement of the WHO 1st International Reference Preparation for BCG vaccine, by three sub-strain specific WHO Reference Reagents of BCG vaccine (Danish 1331, Tokyo 172-1 and Russian BCG-I) in order to complete the coverage of most predominant sub-strains used for BCG vaccine production and distribution for use worldwide. The study used cultural viable count and modified ATP assays to quantify the preparation and multiplex PCR to confirm the identity of the sub-strain. The establishment of this WHO Reference Reagent of BCG vaccine of Moreau-RJ sub-strain was approved by the WHO Expert Committee on Biological Standardization meeting in October 2012. This preparation is available for distribution by NIBSC-MHRA, UK. The data from real-time stability monitoring demonstrated that these Reference Reagents of BCG vaccine are very stable in storage condition at -20°C. They serve as the valuable source of BCG Reference Reagents for use as comparators (1) for viability assays (such as cultural viable count and modified ATP assays); (2) for in vivo assays (such as the absence of virulent mycobacteria, dermal reactivity and protection assays) in the evaluation of candidate TB vaccines in non-clinical models; (3) for identity assays using molecular biology techniques.

OX40 ligand fusion protein delivered simultaneously with the BCG vaccine provides superior protection against murine Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis infection claims approximately 2 million lives per year, and improved efficacy of the BCG vaccine remains a World Health Organization priority. Successful vaccination against M. tuberculosis requires the induction and maintenance of T cells. Targeting molecules that promote T-cell survival may therefore provide an alternative strategy to classic adjuvants. We show that the interaction between T-cell-expressed OX40 and OX40L on antigen-presenting cells is critical for effective immunity to BCG. However, because OX40L is lost rapidly from antigen-presenting cells following BCG vaccination, maintenance of OX40-expressing vaccine-activated T cells may not be optimal. Delivering an OX40L:Ig fusion protein simultaneously with BCG provided superior immunity to intravenous and aerosol M. tuberculosis challenge even 6 months after vaccination, an effect that depends on natural killer 1.1(+) cells. Attenuated vaccines may therefore lack sufficient innate stimulation to maintain vaccine-specific T cells, which can be replaced by reagents binding inducible T-cell costimulators.

Laboratory investigation of immune responses to acellular pertussis vaccines when used for boosting adolescents after primary immunisation with whole cell pertussis vaccines: a comparison with data from clinical study.

The lack of unequivocal immunological correlates of human protection and an absence of a validated animal model for acellular pertussis vaccines, compounded by limited opportunity to undertake efficacy studies in humans and laboratory evaluation side by side, has made it difficult to compare vaccines and formulations. In the present study, the effect on the booster response to pertussis in adolescents primed in infancy with whole cell pertussis vaccine, of three low dose acellular pertussis/diphtheria/tetanus toxoid (TdPa) formulations with or without inactivated poliomyelitis vaccine (IPV) components, was investigated. To assess the relationship between laboratory vaccine evaluation and clinical trial performance, parallel evaluation of the same TdPa vaccines were carried out in a mouse booster model with whole cell pertussis vaccine priming. Prior to boosting, the clinical subjects had low cell mediated immune responses (CMI) responses to pertussis vaccine components. After boosting, all TdPa formulations stimulated CMI responses to the pertussis vaccine components assessed. The booster responses to the pertussis antigens remained skewed towards Th1 type even though acellular pertussis vaccines were used. In general the antibody and CMI responses to pertussis antigens in the mouse model followed the trend seen in the human subjects. Protection against aerosol challenge with virulent Bordetella pertussis was related to the magnitude of the antibody and CMI responses in the mouse model. As in the human subjects, the responses remained skewed towards Th1 type.

Therapeutic efficacy of high-dose intravenous immunoglobulin in Mycobacterium tuberculosis infection in mice.

Intravenous immunoglobulin (IVIg) is used to treat patients with primary antibody deficiencies and, at high doses, to treat a range of autoimmune and inflammatory disorders. With high-dose IVIg (hdIVIg), immunomodulatory mechanisms act on a range of cells, including T cells, B cells, and dendritic cells. Here, we demonstrate that the treatment of M. tuberculosis-infected mice with a single cycle of hdIVIg resulted in substantially reduced bacterial loads in the spleen and lungs when administered at either an early or late stage of infection. Titration of the IVIg showed a clear dose-response effect. There was no reduction in bacterial load when mice were given equimolar doses of another human protein, human serum albumin, or maltose, the stabilizing agent in the IVIg preparation. HdIVIg in vitro had no inhibitory effect on the growth of M. tuberculosis in murine bone marrow-derived macrophages. In addition, the effect of hdIVIg on bacterial loads was not observed in nude mice, suggesting the involvement of conventional T cells. Analysis of T cells infiltrating the lungs revealed only small increases in CD8(+) but not CD4(+) T-cell numbers in hdIVIg-treated mice. The mechanism of action of hdIVIg against tuberculosis in mice remains to be determined. Nevertheless, since hdIVIg is already widely used clinically, the magnitude and long duration of the therapeutic effect seen here suggest that IVIg, or components of it, may find ready application as an adjunct to therapy of human tuberculosis.

The effect of pertussis whole cell and acellular vaccines on pulmonary immunology in an aerosol challenge model.

Recent clinical trials have shown that the new generation of acellular pertussis vaccines (Pa) can confer protection against whooping cough with negligible adverse reactions. We have compared the effects of pertussis whole cell and acellular vaccines on pulmonary immune responses after aerosol challenge in a murine model of infection. Mice were vaccinated with PBS, Pw or Pa and challenged with Bordetella pertussis by the aerosol route. Cytokine gene expression was analysed from lung tissue and cells; lung lymphocytes were re-stimulated in vitro and cytokines produced measured. The results obtained are consistent with the proposal that a strong Th-1 response is associated with bacterial clearance in both the non-vaccinated and Pw vaccinated mice. The acellular vaccine treated mice cleared the bacterial challenge (with an intermediate efficacy) in the presence of low levels of any of the cytokines assessed. This suggests that Pa protects via a Th-2 independent mechanism.