A modified mycobacterial growth inhibition assay for the functional assessment of vaccine-mediated immunity.

The Mycobacterial growth inhibition assay (MGIA) is an ex-vivo assay used to measure the overall functional immune response elicited by infection or vaccination. In tuberculosis (TB) vaccine development, MGIA is a potentially important tool for preclinical evaluation of early-stage vaccine candidates to complement existing assays, and to potentially reduce the need for lengthy and costly pathogenic Mycobacterium tuberculosis (Mtb) animal challenge experiments. The conventional method of MGIA in mice entails directly infecting mixed cell cultures, most commonly splenocytes, from immunised mice with mycobacteria. However, this direct infection of mixed cell populations may yield unreliable results and lacks sufficient sensitivity to discriminate well between different vaccines due to the low number of mycobacteria-permissive cells. Here, we modified the assay by inclusion of mycobacteria-infected congenic murine macrophage cell lines as the target cells, and by measuring the total number of killed cells rather than the relative reduction between different groups. Thus, using splenocytes from Mycobacterium bovis BCG immunised mice, and J774 and MH-S (BALB/c background) or BL/6-M (C57Bl/6 background) macrophage cell lines, we demonstrated that the modified assay resulted in at least 26-fold greater mycobacterial killing per set quantity of splenocytes as compared to the conventional method. This increased sensitivity of measuring mycobacterial killing was confirmed using both the standard culture forming unit (CFU) assay and luminescence readings of luciferase-tagged virulent and avirulent mycobacteria. We propose that the modified MGIA can be used as a highly calibrated tool for quantitating the killing capacity of immune cells in preclinical evaluation of vaccine candidates for TB.

Immunogenicity of PE18, PE31, and PPE26 proteins from Mycobacterium tuberculosis in humans and mice.

Introduction: The large family of PE and PPE proteins accounts for as much as 10% of the genome of Mycobacterium tuberculosis. In this study, we explored the immunogenicity of three proteins from this family, PE18, PE31, and PPE26, in humans and mice. Methods: The investigation involved analyzing the immunoreactivity of the selected proteins using sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy donors from the TB endemic country Mozambique. Antigen-recall responses were examined in PBMC from these groups, including the evaluation of cellular responses in healthy unexposed individuals. Moreover, systemic priming and intranasal boosting with each protein, combined with the Quil-A adjuvant, were conducted in mice. Results: We found that all three proteins are immunoreactive with sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy controls. Likewise, antigen-recall responses were induced in PBMC from all groups, and the proteins stimulated proliferation of peripheral blood mononuclear cells from healthy unexposed individuals. In mice, all three antigens induced IgG antibody responses in sera and predominantly IgG, rather than IgA, responses in bronchoalveolar lavage. Additionally, CD4+ and CD8+ effector memory T cell responses were observed in the spleen, with PE18 demonstrating the ability to induce tissue-resident memory T cells in the lungs. Discussion: Having demonstrated immunogenicity in both humans and mice, the protective capacity of these antigens was evaluated by challenging immunized mice with low-dose aerosol of Mycobacterium tuberculosis H37Rv. The in vitro Mycobacterial Growth Inhibition Assay (MGIA) and assessment of viable bacteria in the lung did not demonstrate any ability of the vaccination protocol to reduce bacterial growth. We therefore concluded that these three specific PE/PPE proteins, while immunogenic in both humans and mice, were unable to confer protective immunity under these conditions.

Mucosal and systemic immune responses after a single intranasal dose of nanoparticle and spore-based subunit vaccines in mice with pre-existing lung mycobacterial immunity.

Tuberculosis (TB) is a major global health threat that claims more than one million lives annually. With a quarter of the global population harbouring latent TB, post-exposure vaccination aimed at high-risk populations that could develop active TB disease would be of great public health benefit. Mucosal vaccination is an attractive approach for a predominantly lung disease like TB because it elicits both local and systemic immunity. However, the immunological consequence of mucosal immunisation in the presence of existing lung immunity remains largely unexplored. Using a mycobacterial pre-exposure mouse model, we assessed whether pre-existing mucosal and systemic immune responses can be boosted and/or qualitatively altered by intranasal administration of spore- and nanoparticle-based subunit vaccines. Analysis of lung T cell responses revealed an increasing trend in the frequency of important CD4 and CD8 T cell subsets, and T effector memory cells with a Th1 cytokine (IFNγ and TNFα) signature among immunised mice. Additionally, significantly greater antigen specific Th1, Th17 and IL-10 responses, and antigen-induced T cell proliferation were seen from the spleens of immunised mice. Measurement of antigen-specific IgG and IgA from blood and bronchoalveolar lavage fluid also revealed enhanced systemic and local humoral immune responses among immunised animals. Lastly, peripheral blood mononuclear cells (PBMCs) obtained from the TB-endemic country of Mozambique show that individuals with LTBI showed significantly greater CD4 T cell reactivity to the vaccine candidate as compared to healthy controls. These results support further testing of Spore-FP1 and Nano-FP1 as post-exposure TB vaccines.

Serological analysis reveals differential antibody responses between TB patients and latently infected individuals from the TB endemic country of Mozambique.

Serological antibody profiling of tuberculosis (TB) patients and household contacts with latent TB infection (LTBI) could identify risk indicators of disease progression, and potentially also serve as an easily accessible diagnostic tool to discriminate between these two stages of Mycobacterium tuberculosis (Mtb) infection. Yet, despite significant efforts over many decades, neither application has yet fully materialised, and this is at least in part due to inconsistent and varying antibody profiles from different TB endemic regions. In this study, we conducted a retrospective exploratory analysis of serum antibodies in a cohort of active TB patients (ATB) and their interferon-gamma release assay (IGRA) positive household contacts (LTBI), as well as healthy controls (HC) from Mozambique, a country with a high TB burden from the Sub-Saharan region. Using several Mtb antigens as well as crude preparations of culture filtrate proteins (CFP) from Mtb and Bacille Calmette Guérin (BCG), we report that the most discriminatory response for TB and LTBI was observed for serum IgA antibodies to the MPT64 antigen, followed by IgG antibodies to Ag85B and CFP, with ATB patients having significantly higher levels than LTBI or BCG-vaccinated healthy controls. Conversely, sera from LTBI individuals had higher levels of IgG antibodies to the HBHA antigen than ATB. While our sample size (n = 21 for ATB, 18 for LTBI and 17 for HC) was too small to fully evaluate the diagnostic potential of these differing serological profiles, our study however preliminarily indicated high level of sensitivity (95%) and specificity (97%) of an ELISA MPT64-IgA test for discriminating TB from LTBI and healthy controls, supporting the notion that it alone, or possibly in combination with other antigens such as Ag85B or CFP could lead to development of an easily accessible diagnostic tool for TB.

Glehnia littoralis Root Extract Induces G0/G1 Phase Cell Cycle Arrest in the MCF-7 Human Breast Cancer Cell Line.

Glehnia littoralis (GL) is widely used as an oriental medicine for cough, fever, stroke and other disease conditions. However, the anti-cancer properties of GL on MCF-7 human breast cancer cells have not been investigated. In order to elucidate anti-cancer properties and underlying cell death mechanisms, MCF-7cells (5 X 104/well) were treated with Glehnia littoralis root extract at 0-400 ug/ml. A hot water extract of GL root inhibited the proliferation of MCF-7 cells in a dose-dependent manner. Analysis of the cell cycle after treatment of MCF-7 cells with increasing concentrations of GL root extract for 24 hours showed significant cell cycle arrest in the G1 phase. RT-PCR and Western blot analysis both revealed that GL root extract significantly increased the expression of p21 and p27 with an accompanyingdecrease in both CDK4 and cyclin D1. Our reuslts indicated that GL root extract arrested the proliferation of MCF-7 cells in G1 phase through inhibition of CDK4 and cyclin D1 via increased induction of p21 and p27. In summary, the current study showed that GL could serve as a potential source of chemotherapeutic or chemopreventative agents against human breast cancer.