Adaption of the ex vivo mycobacterial growth inhibition assay for use with murine lung cells.

In the absence of a correlate(s) of protection against human tuberculosis and a validated animal model of the disease, tools to facilitate vaccine development must be identified. We present an optimised ex vivo mycobacterial growth inhibition assay (MGIA) to assess the ability of host cells within the lung to inhibit mycobacterial growth, including Bacille Calmette-Guérin (BCG) and Mycobacterium tuberculosis (MTB) Erdman. Growth of BCG was reduced by 0.39, 0.96 and 0.73 log10 CFU following subcutaneous (s.c.) BCG, intranasal (i.n.) BCG, or BCG s.c. + mucosal boost, respectively, versus naïve mice. Comparatively, a 0.49 (s.c.), 0.60 (i.n.) and 0.81 (s.c. + mucosal boost) log10 reduction in MTB CFU was found. A BCG growth inhibitor, 2-thiophenecarboxylic acid hydrazide (TCH), was used to prevent quantification of residual BCG from i.n. immunisation and allow accurate MTB quantification. Using TCH, a further 0.58 log10 reduction in MTB CFU was revealed in the i.n. group. In combination with existing methods, the ex vivo lung MGIA may represent an important tool for analysis of vaccine efficacy and the immune mechanisms associated with vaccination in the organ primarily affected by MTB disease.

Phase III, placebo-controlled, randomized, double-blind trial of tableted, therapeutic TB vaccine (V7) containing heat-killed M. vaccae administered daily for one month.

OBJECTIVE: Immunotherapy of tuberculosis (TB) to shorten treatment duration represents an unmet medical need. Orally delivered, tableted TB vaccine (V7) containing heat-killed Mycobacterium vaccae (NCTC 11659) has been demonstrated in prior clinical studies to be safe and fast-acting immune adjunct. METHODS: The outcome of Phase III trial of V7 containing 10 µg of hydrolyzed M. vaccae was evaluated in 152 patients randomized at 2:1 ratio: V7 (N = 100), placebo (N = 52). Both arms received conventional 1st or 2nd line TB drugs co-administered with daily pill of V7 or placebo. RESULTS: After one month mycobacterial clearance was observed in 68% (P < 0.0001) and 23.1% (P = 0.04) of patients on V7 and placebo. Stratified conversion rates in V7 recipients with drug-sensitive and multidrug-resistant TB were 86.7% and 55.6% vs 27.2% and 15% in placebo. Patients on V7 gained on average 2.4 kg (P < 0.0001) vs 0.3 kg (P = 0.18) in placebo. Improvements in hemoglobin levels, erythrocyte sedimentation rate and leukocyte counts were significantly better than in controls. Liver function tests revealed that V7 can prevent chemotherapy-induced hepatic damage. CONCLUSION: Oral M. vaccae is safe, can overcome TB-associated weight loss and inflammation, reduce hepatotoxicity of TB drugs, improve sputum conversion three-fold OR 3.15; 95%CI (2.3,4.6), and cut treatment length by at least six-fold. Longer follow-up studies might be needed to further substantiate our findings (Clinicaltrials.gov: NCT01977768).

Impact of individual-level factors on Ex vivo mycobacterial growth inhibition: Associations of immune cell phenotype, cytomegalovirus-specific response and sex with immunity following BCG vaccination in humans.

Understanding factors associated with varying efficacy of Bacillus Calmette-Guérin (BCG) would aid the development of improved vaccines against tuberculosis (TB). In addition, investigation of individual-level factors affecting mycobacterial-specific immune responses could provide insight into confounders of vaccine efficacy in clinical trials. Mycobacterial growth inhibition assays (MGIA) have been developed to assess vaccine immunogenicity ex vivo and provide a measure of immune function against live mycobacteria. In this study, we assessed the impact of immune cell phenotype, cytomegalovirus (CMV)-specific response and sex on ex vivo growth inhibition following historical BCG vaccination in a cohort of healthy individuals (n = 100). A higher frequency of cytokine-producing NK cells in peripheral blood was associated with enhanced ex vivo mycobacterial growth inhibition following historical BCG vaccination. A CMV-specific response was associated with T-cell activation, a risk factor for TB disease and we also observed an association between T-cell activation and ex vivo mycobacterial growth. Interestingly, BCG-vaccinated females in our cohort controlled mycobacterial growth better than males. In summary, our present study has shown that individual-level factors influence capacity to control mycobacterial growth following BCG vaccination and the MGIA could be used as a tool to assess how vaccine candidates may perform in different populations.

RUTI Vaccination Enhances Inhibition of Mycobacterial Growth ex vivo and Induces a Shift of Monocyte Phenotype in Mice.

Tuberculosis (TB) is a major global health problem and there is a dire need for an improved treatment. A strategy to combine vaccination with drug treatment, termed therapeutic vaccination, is expected to provide benefit in shortening treatment duration and augmenting treatment success rate. RUTI candidate vaccine has been specifically developed as a therapeutic vaccine for TB. The vaccine is shown to reduce bacillary load when administered after chemotherapy in murine and guinea pig models, and is also immunogenic when given to healthy adults and individuals with latent TB. In the absence of a validated correlate of vaccine-induced protection for TB vaccine testing, mycobacterial growth inhibition assay (MGIA) has been developed as a comprehensive tool to evaluate vaccine potency ex vivo. In this study, we investigated the potential of RUTI vaccine to control mycobacterial growth ex vivo and demonstrated the capacity of MGIA to aid the identification of essential immune mechanism. We found an association between the peak response of vaccine-induced growth inhibition and a shift in monocyte phenotype following RUTI vaccination in healthy mice. The vaccination significantly increased the frequency of non-classical Ly6C- monocytes in the spleen after two doses of RUTI. Furthermore, mRNA expressions of Ly6C--related transcripts (Nr4a1, Itgax, Pparg, Bcl2) were upregulated at the peak vaccine response. This is the first time the impact of RUTI has been assessed on monocyte phenotype. Given that non-classical Ly6C- monocytes are considered to play an anti-inflammatory role, our findings in conjunction with previous studies have demonstrated that RUTI could induce a balanced immune response, promoting an effective cell-mediated response whilst at the same time limiting excessive inflammation. On the other hand, the impact of RUTI on non-classical monocytes could also reflect its impact on trained innate immunity which warrants further investigation. In summary, we have demonstrated a novel mechanism of action of the RUTI vaccine, which suggests the importance of a balanced M1/M2 monocyte function in controlling mycobacterial infection. The MGIA could be used as a screening tool for therapeutic TB vaccine candidates and may aid the development of therapeutic vaccination regimens for TB in the near future.

Historical BCG vaccination combined with drug treatment enhances inhibition of mycobacterial growth ex vivo in human peripheral blood cells.

Tuberculosis (TB) is a leading infectious cause of death globally. Drug treatment and vaccination, in particular with Bacillus Calmette-Guérin (BCG), remain the main strategies to control TB. With the emergence of drug resistance, it has been proposed that a combination of TB vaccination with pharmacological treatment may provide a greater therapeutic value. We implemented an ex vivo mycobacterial growth inhibition assay (MGIA) to discriminate vaccine responses in historically BCG-vaccinated human volunteers and to assess the contribution of vaccine-mediated immune response towards the killing effect of mycobacteria in the presence of the antibiotics isoniazid (INH) and rifampicin (RIF), in an attempt to develop the assay as a screening tool for therapeutic TB vaccines. BCG vaccination significantly enhanced the ability of INH to control mycobacterial growth ex vivo. The BCG-vaccinated group displayed a higher production of IFN-γ and IP-10 when peripheral blood mononuclear cells (PBMC) were co-cultured with INH, with a similar trend during co-culture with RIF. A higher frequency of IFN-γ+ and TNF-α+ CD3- CD4- CD8- cells was observed, suggesting the contribution of Natural Killer (NK) cells in the combined effect between BCG vaccination and INH. Taken together, our data indicate the efficacy of INH can be augmented following historical BCG vaccination, which support findings from previous observational and animal studies.

Glibenclamide Reduces Primary Human Monocyte Functions Against Tuberculosis Infection by Enhancing M2 Polarization.

Tuberculosis (TB) is a global public health problem, which is caused by Mycobacterium tuberculosis (Mtb). Type 2 diabetes mellitus (T2DM) is one of the leading predisposing factors for development of TB after HIV/AIDS. Glibenclamide is a widely used anti-diabetic drug in low and middle-income countries where the incidence of TB is very high. In a human macrophage cell line, glibenclamide, a K+ATP-channel blocker, promoted alternative activation of macrophages by enhancing expression of the M2 marker CD206 during M2 polarization. M2 macrophages are considered poorly microbicidal and associated with TB susceptibility. Here, we investigated the effect of glibenclamide on M1 and M2 phenotypes of primary human monocytes and further determined whether specific drug treatment for T2DM individuals influences the antibacterial function of monocytes in response to mycobacterial infection. We found that glibenclamide significantly reduced M1 (HLA-DR+ and CD86+) surface markers and TNF-α production on primary human monocytes against mycobacterial infection. In contrast, M2 (CD163+ and CD206+) surface markers and IL-10 production were enhanced by pretreatment with glibenclamide. Additionally, reduction of bactericidal activity also occurred when primary human monocytes from T2DM individuals who were being treated with glibenclamide were infected with Mtb in vitro, consistent with the cytokine responses. We conclude that glibenclamide reduces M1 and promotes M2 polarization leading to impaired bactericidal ability of primary human monocytes of T2DM individuals in response to Mtb and may lead to increased susceptibility of T2DM individuals to TB and other bacterial infectious diseases.

High monocyte to lymphocyte ratio is associated with impaired protection after subcutaneous administration of BCG in a mouse model of tuberculosis.

Background: The only available tuberculosis (TB) vaccine, Bacillus Calmette-Guérin (BCG), has variable efficacy. New vaccines are therefore urgently needed. Why BCG fails is incompletely understood, and the tools used for early assessment of new vaccine candidates do not account for BCG variability. Taking correlates of risk of TB disease observed in human studies and back-translating them into mice to create models of BCG variability should allow novel vaccine candidates to be tested early in animal models that are more representative of the human populations most at risk. Furthermore, this could help to elucidate the immunological mechanisms leading to BCG failure. We have chosen the monocyte to lymphocyte (ML) ratio as a correlate of risk of TB disease and have back-translated this into a mouse model. Methods: Four commercially available, inbred mouse strains were chosen. We investigated their baseline ML ratio by flow cytometry; extent of BCG-mediated protection from Mtb infection by experimental challenge; vaccine-induced interferon gamma (IFNγ) response by ELISPOT assay; and tissue distribution of BCG by plating tissue homogenates. Results: The ML ratio varied significantly between A/J, DBA/2, C57Bl/6 and 129S2 mice. A/J mice showed the highest BCG-mediated protection and lowest ML ratio, while 129S2 mice showed the lowest protection and higher ML ratio. We also found that A/J mice had a lower antigen specific IFNγ response than 129S2 mice. BCG tissue distribution appeared higher in A/J mice, although this was not statistically significant. Conclusions: These results suggest that the ML ratio has an impact on BCG-mediated protection in mice, in alignment with observations from clinical studies. A/J and 129S2 mice may therefore be useful models of BCG vaccine variability for early TB vaccine testing. We speculate that failure of BCG to protect from TB disease is linked to poor tissue distribution in a ML high immune environment.

Therapeutic vaccines for tuberculosis--a systematic review.

For eradication of tuberculosis (TB) by 2050, the declared aim of the Stop TB Partnership, novel treatment strategies are indispensable. The emerging epidemic of multi-drug resistant (MDR) TB has fuelled the debate about TB vaccines, as increasing numbers of patients can no longer be cured by pharmacotherapy. Of several proposed modalities, TB vaccines administered in therapeutic manner represents a promising alternative, despite the controversial history due to the occurrence of exacerbated immune response. A modified concept of immunotherapy is required in order to justify further exploration. In this paper we systematically reviewed the most advanced therapeutic vaccines for TB. We address the rationale of immunotherapeutic vaccination combined with optimized pharmacotherapy in active TB. We summarize preclinical and patient data regarding the five most advanced therapeutic vaccines currently in the pipeline. Of the five products that have been tested in animal models and in humans during active or latent TB, the quality of the published clinical reports of two of these products justify further studies in patients with active TB. This systematic review fuels further clinical evaluation eventually including head-to-head comparative studies.

Targeting multidrug-resistant tuberculosis (MDR-TB) by therapeutic vaccines.

Tuberculosis (TB) has scourged humankind for millennia, and latent infection affects nearly one-third of today's world population. The emergence of multidrug-resistant (MDR)-TB is a major global threat and reflects treatment failure of drug-sensitive disease. MDR-TB management is a burden for patients and society; success rates are unacceptably low with prolonged treatment duration. Mycobacterium tuberculosis (Mtb) possesses the ability to transform into a dormant state in which it can persist in the face of antimicrobial treatment and host defense. This sub-population of persisters is largely responsible for lengthy and difficult treatment. Targeting persistent bacilli could eventually improve the treatment success rate (currently 50-65 %) and shorten duration of treatment. A subset of therapies in the pipeline, termed therapeutic vaccines, use the host immune response to attack Mtb. The historical occurrence of an exacerbated host response has resulted in a negative perception of therapeutic vaccines. Thus, a renewed concept of immunotherapy is needed. We review current perspectives of immunotherapy in MDR-TB based on the knowledge of TB immunology and briefly discuss the profiles of several therapeutic vaccine products.