Gene expression and cytokine profile correlate with mycobacterial growth in a human BCG challenge model.

BACKGROUND: Bacillus Calmette-Guerin (BCG) vaccine is the most widely administered vaccine in the world, yet its mechanism of action remains unclear. We hypothesize that certain immune pathways are associated with reduced mycobacterial growth following BCG challenge in human volunteers. METHODS: We used samples from a mycobacterial challenge in which previously BCG-vaccinated or BCG-naive adults in the United Kingdom were challenged intradermally with a standard dose of BCG. Any remaining BCG was quantified in a skin biopsy specimen obtained 2 weeks after challenge and used as a measure of BCG growth and functional antimycobacterial immunity. We measured the immune response over the 2-week challenge, using DNA microarrays and flow cytometry, and correlated this with mycobacterial growth. RESULTS: The magnitude of the immune response to BCG is greater in previously vaccinated volunteers, and this correlates with reduced mycobacterial growth but increased scarring at the vaccination site. In particular, the interferon γ and interleukin 17 pathways are strongly induced in previously vaccinated volunteers and correlate with reduced mycobacterial growth in this population. CONCLUSION: This study identifies pathways associated with control of mycobacterial growth in vivo in human volunteers and supports the use of BCG challenge as a tool for evaluating vaccine efficacy and identifying mechanisms of antimycobacterial immunity

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

Tuberculosis (TB) remains a leading global cause of morbidity and mortality and an effective new vaccine is urgently needed. A major barrier to the rational development of novel TB vaccines is the lack of a validated immune correlate or biomarker of protection. Mycobacterial Growth Inhibition Assays (MGIAs) provide an unbiased measure of ability to control mycobacterial growth in vitro, and may represent a functional correlate of protection. However, the biological relevance of any potential correlate can only be assessed by determining the association with in vivo protection from either a controlled mycobacterial infection or natural development of TB disease. Our data demonstrate that the direct MGIA using peripheral blood mononuclear cells (PBMC) is measuring a biologically relevant response that correlates with protection from in vivo human BCG infection across two independent cohorts. This is the first report of an MGIA correlating with in vivo protection in the species-of-interest, humans, and furthermore on a per-individual as well as per-group basis. Control of mycobacterial growth in the MGIA is associated with a range of immune parameters measured post-BCG infection in vivo including the IFN-γ ELISpot response, frequency of PPD-specific IFN-γ or TNF-α producing CD4+ T cells and frequency of specific sub-populations of polyfunctional CD4+ T cells. Distinct transcriptomic profiles are associated with good vs. poor mycobacterial control in the MGIA, with good controllers showing enrichment for gene sets associated with antigen processing/presentation and the IL-23 pathway, and poor controllers showing enrichment for hypoxia-related pathways. This study represents an important step toward biologically validating the direct PBMC MGIA for use in TB vaccine development and furthermore demonstrates the utility of this assay in determining relevant immune mechanisms and pathways of protection

Development of a non-human primate BCG infection model for the evaluation of candidate tuberculosis vaccines.

The lack of validated immunological correlates of protection makes tuberculosis vaccine development difficult and expensive. Using intradermal bacille Calmette-Guréin (BCG) as a surrogate for aerosol Mycobacterium tuberculosis (M.tb) in a controlled human infection model could facilitate vaccine development, but such a model requires preclinical validation. Non-human primates (NHPs) may provide the best model in which to do this. Cynomolgus and rhesus macaques were infected with BCG by intradermal injection. BCG was quantified from a skin biopsy of the infection site and from draining axillary lymph nodes, by culture on solid agar and quantitative polymerase chain reaction. BCG was detected up to 28 days post-infection, with higher amounts of BCG detected in lymph nodes after high dose compared to standard dose infection. Quantifying BCG from lymph nodes of cynomolgus macaques 14 days post-high dose infection showed a significant reduction in the amount of BCG detected in the BCG-vaccinated compared to BCG-naïve animals. Demonstrating a detectable vaccine effect in the lymph nodes of cynomolgus macaques, which is similar in magnitude to that seen in an aerosol M.tb infection model, provides support for proof-of-concept of an intradermal BCG infection model and evidence to support the further evaluation of a human BCG infection model

Process of assay selection and optimization for the study of case and control samples from a phase IIb efficacy trial of a candidate tuberculosis vaccine, MVA85A.

The first phase IIb safety and efficacy trial of a new tuberculosis vaccine since that for BCG was completed in October 2012. BCG-vaccinated South African infants were randomized to receive modified vaccinia virus Ankara, expressing the Mycobacterium tuberculosis antigen 85A (MVA85A), or placebo. MVA85A did not significantly boost the protective effect of BCG. Cryopreserved samples provide a unique opportunity for investigating the correlates of the risk of tuberculosis disease in this population. Due to the limited amount of sample available from each infant, preliminary work was necessary to determine which assays and conditions give the most useful information. Peripheral blood mononuclear cells (PBMC) were stimulated with antigen 85A (Ag85A) and purified protein derivative from M. tuberculosis in an ex vivo gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISpot) and a Ki67 proliferation assay. The effects of a 2-h or overnight rest of thawed PBMC on ELISpot responses and cell populations were determined. Both the ELISpot and Ki67 assays detected differences between the MVA85A and placebo groups, and the results correlated well. The cell numbers and ELISpot responses decreased significantly after an overnight rest, and surface flow cytometry showed a significant loss of CD4(+) and CD8(+) T cells. Of the infants tested, 50% had a positive ELISpot response to a single pool of flu, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) (FEC) peptides. This pilot work has been essential in determining the assays and conditions to be used in the correlate study. Moving forward, PBMC will be rested for 2 h before assay setup. The ELISpot assay, performed in duplicate, will be selected over the Ki67 assay, and further work is needed to evaluate the effect of high FEC responses on vaccine-induced immunity and susceptibility to tuberculosis disease

Lessons from the first clinical trial of a non-licensed vaccine among Ugandan adolescents: a phase II field trial of the tuberculosis candidate vaccine, MVA85A

Background: A more effective vaccine for tuberculosis (TB) is a global public health priority. Vaccines under development will always need evaluation in endemic settings, most of which have limited resources. Adolescents are an important target population for a new TB vaccine and for other vaccines which are relevant at school-age. However, in most endemic settings there is limited experience of trials of investigational products among adolescents, and adolescents are not routinely vaccinated. Methods: We used Modified vaccinia Ankara-expressing Ag85A (MVA85A), a well-tolerated candidate vaccine for tuberculosis, to assess the effect of Schistosoma mansoni infection on vaccine immunogenicity among Ugandan adolescents in primary school. We describe here the challenges and lessons learned in designing and implementing this first clinical trial among Ugandan adolescents using a non-licensed vaccine. Results: The school based immunization study was feasible and adhered to Good Clinical Practice principles.  Engagement with the community and all stakeholders was critical for successful implementation of the trial. Creative and adaptable strategies were used to address protocol-specific, operational and logistical challenges. This study provided lessons and solutions that can be applied to other trials among adolescents in similar settings elsewhere, and to school-based immunization programs. Conclusion: Sufficient time and resources should be planned for community preparation and sensitization to ensure buy in and acceptance of a project of this kind. This trial shows that challenges to implementing early field trials in Africa are not insurmountable and that necessary well-planned high-quality ethical trials are feasible and should be encouraged. Trial Registration: ClinicalTrials.gov NCT02178748 03/06/201

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

Safety and immunogenicity of ChAdOx1 85A prime followed by MVA85A boost compared with BCG revaccination among Ugandan adolescents who received BCG at birth: a randomised, open-label trial

Background BCG confers reduced, variable protection against pulmonary tuberculosis. A more effective vaccine is needed. We evaluated the safety and immunogenicity of candidate regimen ChAdOx1 85A–MVA85A compared with BCG revaccination among Ugandan adolescents. Methods After ChAdOx1 85A dose escalation and age de-escalation, we did a randomised open-label phase 2a trial among healthy adolescents aged 12–17 years, who were BCG vaccinated at birth, without evident tuberculosis exposure, in Entebbe, Uganda. Participants were randomly assigned (1:1) using a block size of 6, to ChAdOx1 85A followed by MVA85A (on day 56) or BCG (Moscow strain). Laboratory staff were masked to group assignment. Primary outcomes were solicited and unsolicited adverse events (AEs) up to day 28 and serious adverse events (SAEs) throughout the trial; and IFN-γ ELISpot response to antigen 85A (day 63 [geometric mean] and days 0–224 [area under the curve; AUC). Findings Six adults (group 1, n=3; group 2, n=3) and six adolescents (group 3, n=3; group 4, n=3) were enrolled in the ChAdOx1 85A-only dose-escalation and age de-escalation studies (July to August, 2019). In the phase 2a trial, 60 adolescents were randomly assigned to ChAdOx1 85A–MVA85A (group 5, n=30) or BCG (group 6, n=30; December, 2019, to October, 2020). All 60 participants from groups 5 and 6 were included in the safety analysis, with 28 of 30 from group 5 (ChAdOx1 85A–MVA85A) and 29 of 30 from group 6 (BCG revaccination) analysed for immunogenicity outcomes. In the randomised trial, 60 AEs were reported among 23 (77%) of 30 participants following ChAdOx1 85A–MVA85A, 31 were systemic, with one severe event that occurred after the MVA85A boost that was rapidly self-limiting. All 30 participants in the BCG revaccination group reported at least one mild to moderate solicited AE; most were local reactions. There were no SAEs in either group. Ag85A-specific IFN-γ ELISpot responses peaked on day 63 in the ChAdOx1 85A–MVA85A group and were higher in the ChAdOx1 85A-MVA85A group compared with the BCG revaccination group (geometric mean ratio 30·59 [95% CI 17·46–53·59], p<0·0001, day 63; AUC mean difference 57 091 [95% CI 40 524–73 658], p<0·0001, days 0–224). Interpretation The ChAdOx1 85A–MVA85A regimen was safe and induced stronger Ag85A-specific responses than BCG revaccination. Our findings support further development of booster tuberculosis vaccines. Funding UK Research and Innovations and Medical Research Council. Translations For the Swahili and Luganda translations of the abstract see Supplementary Materials section

Safety of a controlled human infection model of tuberculosis with aerosolised, live-attenuated Mycobacterium bovis BCG versus intradermal BCG in BCG-naive adults in the UK: a dose-escalation, randomised, controlled, phase 1 trial

Background: Mycobacterium tuberculosis is the main causative agent of tuberculosis. BCG, the only licensed vaccine, provides inadequate protection against pulmonary tuberculosis. Controlled human infection models are useful tools for vaccine development. We aimed to determine a safe dose of aerosol-inhaled live-attenuated Mycobacterium bovis BCG as a surrogate for M tuberculosis infection, then compare the safety and tolerability of infection models established using aerosol-inhaled and intradermally administered BCG. Methods: This phase 1 controlled human infection trial was conducted at two clinical research facilities in the UK. Healthy, immunocompetent adults aged 18–50 years, who were both M tuberculosis-naive and BCG-naive and had no history of asthma or other respiratory diseases, were eligible for the trial. Participants were initially enrolled into group 1 (receiving the BCG Danish strain); the trial was subsequently paused because of a worldwide shortage of BCG Danish and, after protocol amendment, was restarted using the BCG Bulgaria strain (group 2). After a dose-escalation study, during which participants were sequentially allocated to receive either 1 × 103, 1 × 104, 1 × 105, 1 × 106, or 1 × 107 colony-forming units (CFU) of aerosol BCG, the maximum tolerated dose was selected for the randomised controlled trial. Participants in this trial were randomly assigned (9:12), by variable block randomisation and using sequentially numbered sealed envelopes, to receive aerosol BCG (1 × 107 CFU) and intradermal saline or intradermal BCG (1 × 106 CFU) and aerosol saline. Participants were masked to treatment allocation until day 14. The primary outcome was to compare the safety of a controlled human infection model based on aerosol-inhaled BCG versus one based on intradermally administered BCG, and the secondary outcome was to evaluate BCG recovery in the airways of participants who received aerosol BCG or skin biopsies of participants who received intradermal BCG. BCG was detected by culture and by PCR. The trial is registered at ClinicalTrials.gov, NCT02709278, and is complete. Findings: Participants were assessed for eligibility between April 7, 2016, and Sept 29, 2018. For group 1, 15 participants were screened, of whom 13 were enrolled and ten completed the study; for group 2, 60 were screened and 33 enrolled, all of whom completed the study. Doses up to 1 × 107 CFU aerosol-inhaled BCG were sufficiently well tolerated. No significant difference was observed in the frequency of adverse events between aerosol and intradermal groups (median percentage of solicited adverse events per participant, post-aerosol vs post-intradermal BCG: systemic 7% [IQR 2–11] vs 4% [1–13], p=0·62; respiratory 7% [1–19] vs 4% [1–9], p=0·56). More severe systemic adverse events occurred in the 2 weeks after aerosol BCG (15 [12%] of 122 reported systemic adverse events) than after intradermal BCG (one [1%] of 94; difference 11% [95% CI 5–17]; p=0·0013), but no difference was observed in the severity of respiratory adverse events (two [1%] of 144 vs zero [0%] of 97; 1% [−1 to 3]; p=0·52). All adverse events after aerosol BCG resolved spontaneously. One serious adverse event was reported—a participant in group 2 was admitted to hospital to receive analgesia for a pre-existing ovarian cyst, which was deemed unrelated to BCG infection. On day 14, BCG was cultured from bronchoalveolar lavage samples after aerosol infection and from skin biopsy samples after intradermal infection. Interpretation: This first-in-human aerosol BCG controlled human infection model was sufficiently well tolerated. Further work will evaluate the utility of this model in assessing vaccine efficacy and identifying potential correlates of protection

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