Safety and immunogenicity of an FP9-vectored candidate tuberculosis vaccine (FP85A), alone and with candidate vaccine MVA85A in BCG-vaccinated healthy adults: a phase I clinical trial.

The safety and immunogenicity of a new candidate tuberculosis (TB) vaccine, FP85A was evaluated alone and in heterologous prime-boost regimes with another candidate TB vaccine, MVA85A. This was an open label, non-controlled, non-randomized Phase I clinical trial. Healthy previously BCG-vaccinated adult subjects were enrolled sequentially into three groups and vaccinated with FP85A alone, or both FP85A and MVA85A, with a four week interval between vaccinations. Passive and active data on adverse events were collected. Immunogenicity was evaluated by Enzyme Linked Immunospot (ELISpot), flow cytometry and Enzyme Linked Immunosorbent assay (ELISA). Most adverse events were mild and there were no vaccine-related serious adverse events. FP85A vaccination did not enhance antigen 85A-specific cellular immunity. When MVA85A vaccination was preceded by FP85A vaccination, cellular immune responses were lower compared with when MVA85A vaccination was the first immunisation. MVA85A vaccination, but not FP85A vaccination, induced anti-MVA IgG antibodies. Both MVA85A and FP85A vaccinations induced anti-FP9 IgG antibodies. In conclusion, FP85A vaccination was well tolerated but did not induce antigen-specific cellular immune responses. We hypothesize that FP85A induced anti-FP9 IgG antibodies with cross-reactivity for MVA85A, which may have mediated inhibition of the immune response to subsequent MVA85A. ClinicalTrials.gov identification number: NCT00653770

A Phase I study evaluating the safety and immunogenicity of MVA85A, a candidate TB vaccine, in HIV-infected adults

Objectives Control of the tuberculosis (TB) epidemic is a global health priority and one that is likely to be achieved only through vaccination. The critical overlap with the HIV epidemic requires any effective TB vaccine regimen to be safe in individuals who are infected with HIV. The objectives of this clinical trial were to evaluate the safety and immunogenicity of a leading candidate TB vaccine, MVA85A, in healthy, HIV-infected adults. Design This was an open-label Phase I trial, performed in 20 healthy HIV-infected, antiretroviral-naïve subjects. Two different doses of MVA85A were each evaluated as a single immunisation in 10 subjects, with 24 weeks of follow-up. The safety of MVA85A was assessed by clinical and laboratory markers, including regular CD4 counts and HIV RNA load measurements. Vaccine immunogenicity was assessed by ex vivo interferon γ (IFN-γ) ELISpot assays and flow-cytometric analysis. Results MVA85A was safe in subjects with HIV infection, with an adverse-event profile comparable with historical data from previous trials in HIV-uninfected subjects. There were no clinically significant vaccine-related changes in CD4 count or HIV RNA load in any subjects, and no evidence from qPCR analyses to indicate that MVA85A vaccination leads to widespread preferential infection of vaccine-induced CD4 T cell populations. Both doses of MVA85A induced an antigen-specific IFN-γ response that was durable for 24 weeks, although of a lesser magnitude compared with historical data from HIV-uninfected subjects. The functional quality of the vaccine-induced T cell response in HIV-infected subjects was remarkably comparable with that observed in healthy HIV-uninfected controls, but less durable. Conclusion MVA85A is safe and immunogenic in healthy adults infected with HIV. Further safety and efficacy evaluation of this candidate vaccine in TB- and HIV-endemic areas is merited

Preclinical Development of an In Vivo BCG Challenge Model for Testing Candidate TB Vaccine Efficacy

There is an urgent need for an immunological correlate of protection against tuberculosis (TB) with which to evaluate candidate TB vaccines in clinical trials. Development of a human challenge model of Mycobacterium tuberculosis (M.tb) could facilitate the detection of such correlate(s). Here we propose a novel in vivo Bacille Calmette-Guérin (BCG) challenge model using BCG immunization as a surrogate for M.tb infection. Culture and quantitative PCR methods have been developed to quantify BCG in the skin, using the mouse ear as a surrogate for human skin. Candidate TB vaccines have been evaluated for their ability to protect against a BCG skin challenge, using this model, and the results indicate that protection against a BCG skin challenge is predictive of BCG vaccine efficacy against aerosol M.tb challenge. Translation of these findings to a human BCG challenge model could enable more rapid assessment and down selection of candidate TB vaccines and ultimately the identification of an immune correlate of protection

Immunogenicity and protective efficacy of BCG + CT – MVA85A.

<p>Balb/c mice received BCG±CT <i>i.n.</i> followed by 1×10⁶ CFU MVA85A 10 weeks later. Lungs (<b>A</b>) and spleen (<b>B</b>) were taken at 10 (black circles), 11 (dark grey circles) and 14 (light grey circles) weeks post-BCG and cytokine-producing cells responding to an Ag85A peptide pool quantified using ICS. Responses from animals receiving BCG – MVA85A (closed circles) were compared with those receiving BCG + CT followed by MVA85A (open circles). Statistical analysis was performed using a Mann Whitney test. n = 10, five each from two experiments. (<b>C</b>) Balb/c mice were vaccinated as above. Control groups included unvaccinated and BCG <i>i.d.</i> A group receiving BCG <i>i.n.</i> was included to compare BCG – MVA85A <i>i.n.</i> to BCG <i>i.n</i>. Animals were exposed to ∼100 CFU <i>M.tb</i> via aerosol four weeks post-MVA85A. Four weeks post-challenge, lungs and spleen were homogenised and plated for CFU quantitation. (<b>D</b>) Balb/c mice were vaccinated and challenged as described above. Groups receiving BCG – MVA85A and BCG + CT – MVA85A received an anti-IL-17 blocking antibody (MAB421; R&D Systems) administered <i>i.p.</i> every three days post-challenge. One group receiving BCG – MVA85A received an IgG2a isotype control antibody (MAB006; R&D Systems) on the same regimen. Mice were culled four weeks post-challenge and lung CFU quantitated as described above. Statistical analysis was performed using a one way ANOVA and post-hoc tests on the vaccinated groups (n = 8–16).</p

Effect of cholera toxin on BCG immunogenicity and <i>M.tb</i> challenge outcome.

<p>(<b>A</b>) Cytokine gating in spleens and lungs, unstimulated vs. Ag85A peptide pool-stimulated. (<b>B</b>) Balb/c mice received 4×10⁵ CFU BCG <i>i.n.</i> or <i>i.d.</i> followed 10 weeks later by 1×10⁶ PFU MVA85A <i>i.d.</i> At weeks 10, 11 and 14 post-BCG, lungs were examined for cytokine production by ICS following Ag85A peptide pool stimulation in the presence of Brefeldin A and GolgiStop. (<b>C</b> & <b>D</b>) Balb/c mice received 4×10⁵ CFU BCG±2 µg CT <i>i.n</i>. After 10 weeks, lungs and spleen were dissected and stimulated with PPD in the presence of Brefeldin A and GolgiStop. Percentages of CD4⁺ T cells producing IFN-γ and IL-17 were calculated following ICS on lung cells (<b>C</b>) and splenocytes (<b>D</b>). Mice receiving BCG only are plotted with closed circles and those receiving CT have open circles. P values were calculated using a Mann Whitney test (n = 5). (<b>E</b>) Ten weeks post-BCG, mice received ∼100 CFU <i>M.tb</i> via aerosol and four weeks later lungs and spleen were homogenised and plated out for CFU quantitation. Statistical analysis was performed using a one way ANOVA and post-hoc tests on the vaccinated groups (n = 8).</p

Histological analysis of <i>M.tb</i>-exposed lungs.

<p>Four Balb/c mice from each group shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078312#pone-0078312-g002" target="_blank">Figure 2C</a> had one lung lobe inflated with 10% NBF and dissected. Lungs were embedded in paraffin, sectioned, mounted onto slides and stained with haematoxylin and eosin. Images were cropped so that no external white space was visible (<b>A</b>). Analysis was performed using a k means clustering algorithm in MATLAB (<b>B</b>). (<b>C</b>) Representative sections from each group. (<b>D</b>) Percentages of the image represented by one of three colours (purple, red or white) were calculated and plotted, n = 2-4.</p

Genetic regulation of antibody responsiveness to immunization in substrains of BALB/c mice.

Antibody-mediated immunity is highly protective against disease. The majority of current vaccines confer protection through humoral immunity, but there is high variability in responsiveness across populations. Identifying immune mechanisms that mediate low antibody responsiveness may provide potential strategies to boost vaccine efficacy. Here, we report diverse antibody responsiveness to unadjuvanted as well as adjuvanted immunization in substrains of BALB/c mice, resulting in high and low antibody response phenotypes. Furthermore, these antibody phenotypes were not affected by changes in environmental factors such as the gut microbiota composition. Antigen-specific B cells following immunization had a marked difference in capability to class switch, resulting in perturbed IgG isotype antibody production. In vitro, a B-cell intrinsic defect in the regulation of class-switch recombination was identified in mice with low IgG antibody production. Whole genome sequencing identified polymorphisms associated with the magnitude of antibody produced, and we propose candidate genes that may regulate isotype class-switching capability. This study highlights that mice sourced from different vendors can have significantly altered humoral immune response profiles, and provides a resource to interrogate genetic regulators of antibody responsiveness. Together these results further our understanding of immune heterogeneity and suggest additional research on the genetic influences of adjuvanted vaccine strategies is warranted for enhancing vaccine efficacy