Periodic Reporting for period 3 - EMI-TB (Eliciting Mucosal Immunity to Tuberculosis)
Reporting period: 2018-01-01 to 2019-06-30
Tuberculosis (TB) is a global health problem, killing 1.5 million of people every year. The only currently available vaccine, Mycobacterium bovis BCG, is effective against severe childhood forms, but it demonstrates a variable efficacy against the pulmonary form of TB in adults. Many of these adult TB cases result from the reactivation of an initially controlled, latent Mycobacterium tuberculosis (MTB) infection. Effective prophylactic vaccination remains the key long-term strategy for combating TB as a global health threat. Continued belief in reaching this goal requires unrelenting innovation in the formulation and delivery of candidate vaccines. This project focused on mucosal vaccination, which has been considered in the past, but not implemented efficiently. The innovation of the project was centred on several important aspects of vaccine development and testing, including the use of novel technologies for vaccine delivery, novel modes of targeting mucosal immune tissues and cells and application of novel tools for identifying early predictors and correlates of immune protection and/or disease progression. Our key objectives were:
1. To establish an optimal vaccine delivery system for inducing protective mucosal immunity against MTB
2. To identify novel, early-stage infection associated CD8+ T-cell epitopes for inducing protective CD8+ T-cell mediated immunity
3. To establish correlates of protective immune response in MTB-exposed humans and vaccinated animals and define predictors of vaccine efficacy
4. To test the most promising vaccine candidate in the NHP model of MTB infection
1. To establish an optimal vaccine delivery system for inducing protective mucosal immunity against MTB
2. To identify novel, early-stage infection associated CD8+ T-cell epitopes for inducing protective CD8+ T-cell mediated immunity
3. To establish correlates of protective immune response in MTB-exposed humans and vaccinated animals and define predictors of vaccine efficacy
4. To test the most promising vaccine candidate in the NHP model of MTB infection
To achieve the Project's objectives, the work was divided in 6 workpackages (WPs), all of which have contributed to the project’s successful outcome. The main achievements of the project in relation to the set objectives could be described as follows:
Objective 1: To establish an optimal vaccine delivery system for inducing protective mucosal immunity against MTB
This was established through a series of experiments performed in the first 3 years of the project using the mouse model and subsequently validated in guinea pigs and non-human primates. We identified B. subtilis spores, Yc-NaMA nanoparticles and apoptotic body like liposomes as the most suitable delivery systems for mucosal vaccination. Each of these 3 delivery systems was tested comprehensively in vivo and in vitro, in terms of their interaction with the immune cells and tissues, providing the mechanistic evidence for protective mucosal immunity.
Objective 2: To identify novel, early-stage infection associated CD8+ T-cell epitopes
We initially focused on HLA-A2 epitopes that have been identified by the bioinformatics approach for testing of immunogenicity and protective immunity. However, extensive studies in HLA-A2 transgenic mice failed to demonstrate their immunogenicity irrespective of the mode of delivery (with adjuvants, as DNA vaccines, adenoviral vectors etc). This necessitated change of focus on HLA-E epitopes which are not restrictive to the same level as A,B and C. Several HLA-E peptides were identified as immunogenic in human PBMC from TB patients and LTBI. These peptides merit further testing for potential inclusion into existing TB vaccine candidates.
Objective 3: To establish correlates of protective immune response in MTB-exposed humans
We have performed correlates of immunity/infection studies in two separate cohorts in Maputo (Mozambique) and Vigo (Spain). We applied the systems biology approach and compared the immunological, transcriptomic and proteomic signatures in TB patients, exposed (LTBI) contacts and negative controls. We identified several proteins expressed in human mucosal samples that are associated with the clinical status of the TB disease. Most importantly, we identified a transcriptomic signature that differentiates the LTBI individuals into those who resemble TB patients and those who are more similar to negative contacts, thus potentially having a predictive potential for reactivation of TB in LTBI.
Objective 4: To test the most promising vaccine candidate in the NHP model of MTB infection
We have performed an NHP study in which we compared BCG with the lead vaccine candidate Spore-FP1 as boost to BCG. Following three pilot studies, we opted for a vaccination regimen that included one intradermal and one aerosol boost with Spore-FP1. The results of the NHP trial indicated that the Spore-FP1 candidate was safe, amenable for aerosolization and immunogenic, both systemically and in the mucosa. However, boosting BCG with Spore-FP1 did not confer statistically significant increase in protection in this animal model, in terms of pathological, clinical and microbiological readouts. Further refinement of this vaccine candidate is therefore required.
Objective 1: To establish an optimal vaccine delivery system for inducing protective mucosal immunity against MTB
This was established through a series of experiments performed in the first 3 years of the project using the mouse model and subsequently validated in guinea pigs and non-human primates. We identified B. subtilis spores, Yc-NaMA nanoparticles and apoptotic body like liposomes as the most suitable delivery systems for mucosal vaccination. Each of these 3 delivery systems was tested comprehensively in vivo and in vitro, in terms of their interaction with the immune cells and tissues, providing the mechanistic evidence for protective mucosal immunity.
Objective 2: To identify novel, early-stage infection associated CD8+ T-cell epitopes
We initially focused on HLA-A2 epitopes that have been identified by the bioinformatics approach for testing of immunogenicity and protective immunity. However, extensive studies in HLA-A2 transgenic mice failed to demonstrate their immunogenicity irrespective of the mode of delivery (with adjuvants, as DNA vaccines, adenoviral vectors etc). This necessitated change of focus on HLA-E epitopes which are not restrictive to the same level as A,B and C. Several HLA-E peptides were identified as immunogenic in human PBMC from TB patients and LTBI. These peptides merit further testing for potential inclusion into existing TB vaccine candidates.
Objective 3: To establish correlates of protective immune response in MTB-exposed humans
We have performed correlates of immunity/infection studies in two separate cohorts in Maputo (Mozambique) and Vigo (Spain). We applied the systems biology approach and compared the immunological, transcriptomic and proteomic signatures in TB patients, exposed (LTBI) contacts and negative controls. We identified several proteins expressed in human mucosal samples that are associated with the clinical status of the TB disease. Most importantly, we identified a transcriptomic signature that differentiates the LTBI individuals into those who resemble TB patients and those who are more similar to negative contacts, thus potentially having a predictive potential for reactivation of TB in LTBI.
Objective 4: To test the most promising vaccine candidate in the NHP model of MTB infection
We have performed an NHP study in which we compared BCG with the lead vaccine candidate Spore-FP1 as boost to BCG. Following three pilot studies, we opted for a vaccination regimen that included one intradermal and one aerosol boost with Spore-FP1. The results of the NHP trial indicated that the Spore-FP1 candidate was safe, amenable for aerosolization and immunogenic, both systemically and in the mucosa. However, boosting BCG with Spore-FP1 did not confer statistically significant increase in protection in this animal model, in terms of pathological, clinical and microbiological readouts. Further refinement of this vaccine candidate is therefore required.
Our project achieved two major outputs which will have an immediate (Output 1) and a long-term, (Socio-economic; Output 2) impact in TB field. The first output will enable scientific progress in mucosal TB vaccine field and the second will enable better monitoring disease progression and control.
1. Aerosolised protein vaccine delivery: We produced strong evidence that protein subunit vaccine formulations based on particles and designed to partly mimic MTB itself are capable of inducing mucosal and systemic immunity and confer protection in mice and guinea pigs against aerosol MTB challenge. Our lead vaccine candidates, Spore-FP1, was optimised for aerosolized delivery and this is the first example of a successful aerosolised delivery of a protein subunit vaccine for TB. While aerosolised Spore-FP1 ultimately did not induce sufficient protection in NHP, it nevertheless opened the route for aerosolised delivery and testing of other types of non-live, protein-based vaccines in future studies.
2. Molecular signature of TB infection: we have performed transcriptomic and proteomic analysis of systemic (blood) and mucosal clinical samples (i.e. saliva and sputum) from TB patients, LTBI and uninfected contacts, and have uncovered unique molecular signatures associated with infection and/or protection. Such approaches had been employed in the past for blood only but are largely unexplored for mucosal samples. These exploratory studies have provided us with a unique LTBI transcriptomic and proteomic signature associated with ‘TB-like’ and ‘Not TB-like’ LTBI profiles, which could be further exploited to identify individuals at greatest risk of TB disease in this population.
1. Aerosolised protein vaccine delivery: We produced strong evidence that protein subunit vaccine formulations based on particles and designed to partly mimic MTB itself are capable of inducing mucosal and systemic immunity and confer protection in mice and guinea pigs against aerosol MTB challenge. Our lead vaccine candidates, Spore-FP1, was optimised for aerosolized delivery and this is the first example of a successful aerosolised delivery of a protein subunit vaccine for TB. While aerosolised Spore-FP1 ultimately did not induce sufficient protection in NHP, it nevertheless opened the route for aerosolised delivery and testing of other types of non-live, protein-based vaccines in future studies.
2. Molecular signature of TB infection: we have performed transcriptomic and proteomic analysis of systemic (blood) and mucosal clinical samples (i.e. saliva and sputum) from TB patients, LTBI and uninfected contacts, and have uncovered unique molecular signatures associated with infection and/or protection. Such approaches had been employed in the past for blood only but are largely unexplored for mucosal samples. These exploratory studies have provided us with a unique LTBI transcriptomic and proteomic signature associated with ‘TB-like’ and ‘Not TB-like’ LTBI profiles, which could be further exploited to identify individuals at greatest risk of TB disease in this population.