In Silico Identification of Immunogenic ERv 53-63 Epitope; A Potential Candidate for Tuberculosis Vaccine Design

Immunization With a Bivalent Adenovirus-vectored Tuberculosis Vaccine Provides Markedly Improved Protection Over Its Monovalent Counterpart Against Pulmonary Tuberculosis

Novel vaccines against M. tuberculosis

Background: Preclinical animal experiments measuring vaccine immunogenicity and safety are essential, not only to establish if the vaccine should progress further, but to generate information on how the vaccine should be administered in humans. Animal models that represent human vaccine responses well are vital to translate information about vaccine dose to clinical phases. Vaccine dose is a key aspect in creating an effective vaccine. However, if the wrong dose is chosen, vaccine candidates may be mistakenly discarded and considerable resources wasted. Current methods of finding optimal vaccine dose are mostly empirically based, which may be leading to sub-optimal doses progressing into later clinical trials. A current example of this is in the tuberculosis (TB) vaccine developmental pipeline, where a series of adjuvanted subunit vaccines, the H-series, have progressed through to later stages of clinical development with a high dose that has been shown to less immunogenic than lower doses. In drug development, mathematical model-based methods are routinely used alongside empirical evaluations, to inform dose-finding. I hypothesised that vaccine development may benefit from the application of similar quantitative methods. As such, I launched the new field of vaccine immunostimulation/immunodynamic (IS/ID) mathematical modelling. My aims for this thesis were 1) to establish differences in Bacillus Calmette–Guerin (BCG) Interferon-Gamma (IFN-γ) response by human subpopulation, then develop a IS/ID model to represent these response dynamics and identify the most representative macaque subpopulation for human BCG responses. Aim 2) was to predict human H-series vaccine IFN-γ response using IS/ID model calibrated to mouse multi-dose IFN-γ data and allometric scaling. Methods: For aim 1, longitudinal data on IFN-γ emitting CD4+ T cells following vaccination BCG were available in humans and macaques. Human (sub)population covariates were: baseline BCG vaccination status, time since BCG vaccination, gender and monocyte/lymphocyte cell count ratio. The macaque (sub)population covariate was colony of origin. I developed a two-compartmental mathematical model describing the post-BCG IFN-γ immune response dynamics. The model was calibrated to the human and macaque data using 4 Nonlinear Mixed Effects Modelling (NLMEM) to establish if there were differences in IFN-γ dynamics for both species subpopulations. I then established which macaque subpopulation best described human data. For aim 2, longitudinal data on IFN-γ emitting CD4+ T cells following two vaccinations with five doses of novel TB vaccine H56+IC31 in mice were generated. I then assessed the shape of the dose response curve at early and late time points. I calibrated the T cell model to the mouse data and established the change in key model parameters across dose. Using the change in model parameters across dose found in the mice, I predicted the immune response dynamics in humans for different doses and which dose was most immunogenic. Results: In aim 1, I found that BCG status in humans (baseline BCG-naive or baseline BCG-vaccinated) was associated with differences in the peak and end IFN-γ response after vaccination with BCG. When the mathematical model was calibrated to the BCG data for both macaques and humans, significant differences (p<0.05) in key model parameters were found after stratification by macaque colony and human baseline-BCG status. Indonesian cynomolgus macaques had the closest immune response dynamics to the baseline BCG-naive humans. In aim 2, a peaked curve was the best description of the mouse H56+IC31 dose response curve for early and late time points. Calibrating a revaccination model to the data and mapping changes in the estimated mouse model parameters across dose group to the estimated human model parameters, I found at day 224 (a latest time point), the model-predicted median number of human IFN-γ secreting CD4+ T cells were the highest for the dose group in the range 1-10μg H56/H1+500 nmol IC31. This suggests a dose of 1-10μg may be the most immunogenic in humans. Discussion: Finding the most predictive animal model and optimal vaccine dose is essential for efficiently accelerating the development of new, effective, TB vaccines. I demonstrated that mathematical modelling was a useful tool to quantify BCG immune response dynamics in macaques and humans. I established which macaque subpopulation should be used to represent a human BCG (or potentially new TB vaccine) induced IFN-γ response in future clinical trials. Using IFN-γ as marker of vaccine immunogenicity, mathematical modelling predictions using preclinical data suggested that doses in current novel TB vaccines clinical 5 trials on healthy BCG-vaccinated participants should be between 1-10μg H56/H1+500 nmol IC31, a result which has been recently corroborated in an empirical H56+IC31 dose-ranging trial. This project has demonstrated the potential utility of mathematical modelling in vaccine development. I believe future work on IS/ID modelling should include data on more complex immune response networks and different animal and human subpopulations. This future work is entirely feasible and would establish IS/ID modelling as a legitimate tool to accelerate vaccine development.

Pre-print submitted to BioRxiv.Abstract: Eradication of tuberculosis (TB), the world's leading cause of death due to infectious disease, requires a highly efficacious TB vaccine. Many TB vaccine candidates are in pre-clinical and clinical development but only a few can be advanced to large - scale efficacy trials due to limited global resources. We aimed to perform a statistically rigorous comparison of the antigen - specific T cell responses induced by six novel TB vaccine candidates and the only licensed TB vaccine, Bacillus Calmette - Guerin (BCG). We propose that the antigen - specific immune response induced by such vaccines provides an objective, data - driven basis for prioritisation of vaccine candidates for efficacy testing. We analyze d frequencies of antigen - specific CD4 and CD8 T cells expressing IFN γ, IL - 2, TNF and/or IL - 17 from adolescents or adults, with or without Mycobacterium tuberculosis ( M.tb ) infection, who received MVA85A, A ERAS - 402, H1:IC31, H56:IC31, M72/AS01 E, ID93 + GLA - SE or BCG. Two key response characteristics were analyzed, namely response magnitude and cytokine co - expression profile of the memory T cell response that persisted above the pre-vaccination response to the final study visit in each trial. All vaccines preferentially induced antigen - specific CD4 T cell responses expressing Th1 cytokines; levels of IL - 17 - expressing cells were low or not detected. In M.tb - uninfected and - infected individuals, M72/AS01 E induced higher memory Th1 cytokine - expressing CD4 T cell response s than other novel vaccine candidates. Cytokine co - expression profile s of memory CD4 T cells induced by different novel vaccine candidates were alike. Our study suggests that the T cell response feature which most differentiated between the TB vaccine candidates was response magnitude, whilst functional profiles suggested a lack of response diversity. Since M72/AS01 E induced the highest memory CD4 T cell response it demonstrated the best vaccine take. In the absence of immunological correlates of protection the likelihood of finding a protective vaccine by empirical testing of candidates may be increased by the addition of candidates that induce distinct immune characteristics.

Tuberculosis: Poor Awareness Leads to Poor Control

Development of Mycobacterium tuberculosis attenuated strains as live vaccine candidates for tuberculosis

Mycobacterium kansasii is a bacterium included in non-tuberculous mycobacteria (NTM) that can cause lung disease. It shares a significant number of antigens with Mycobacterium tuberculosis (Mtb), suggesting that it has the potential to be used as a tuberculosis (TB) vaccine. Therefore, we subcutaneously vaccinated mice with reference strain, M. kansasii-ATCC12478 [M. kansasii-American Type Culture Collection (ATCC)], and clinically isolated strain, M. kansasii-SM-1 to evaluate potential as a TB vaccine by comparing with bacille Calmette-Guerin (BCG) vaccine. Ten weeks after vaccination, we evaluated immunogenicity of M. kansasii-ATCC and M. kansasii-SM-1, and M. kansasii-SM-1 immunization induces potent Mtb antigen-specific IFN-γ-producing CD4+ T cells than M. kansasii-ATCC. Upon Mtb infection, M. kansasii-SM-1 provided better protection than M. kansasii-ATCC, which was comparable to the efficacy of BCG. These results showed that the clinical strain M. kansasii-SM-1, which exhibits an enhanced Mtb antigen-specific Th1 response, shows greater vaccine efficacy compared to M. kansasii-ATCC. In this study, we demonstrated that vaccine efficacy can vary depending on the strain of M. kansasii and that its efficacy can be comparable to BCG. This suggests that M. kansasii has the potential to be a live TB vaccine candidate.IMPORTANCEMycobacterium kansasii, a non-tuberculous mycobacteria (NTM) species causing lung disease, shares key antigens with Mycobacterium tuberculosis (Mtb), indicating its potential for TB vaccine development. Subcutaneous vaccination of mice with M. kansasii strains reference strain M. kansasii-ATCC12478 [(M. kansasii-American Type Culture Collection (ATCC)] and clinically isolated strain M. kansasii-SM-1 revealed differences in immunogenicity. M. kansasii-SM-1 induced a robust Mtb antigen-specific IFN-γ-producing CD4+ T cell response compared to M. kansasii-ATCC. Additionally, M. kansasii-SM-1 conferred better protection against Mtb infection than M. kansasii-ATCC, which is comparable to bacille Calmette-Guerin (BCG). These findings underscore the variable vaccine efficacy among M. kansasii strains, with M. kansasii-SM-1 exhibiting promising potential as a live TB vaccine candidate, suggesting its comparative effectiveness to BCG.

Tuberculosis(TB), malaria and helminthiasis are a major challenge for the global public health in the 21st century. The HIV-associated TB epidemic, occurrence of drug resistant strains of Mycobacterium tuberculosis (M.tb) and the limited efficacy of the Bacille Calmette Guerin (BCG) vaccine are important obstacles of reducing TB morbidity and mortality. An estimated 1.5 million people died from TB in 2013, of these approximately one quarter were HIV positive. A new TB vaccine should be safe and efficacious in all populations, including HIV positives. In Sub-Saharan Africa, there is substantial geographical overlap of malaria tropica and soil-transmitted helminth infections and co-infections are common. Intervention strategies mostly neglect co-morbidity, although there is evidence that helminths impact on clinical malaria. The human gut microbiota has an extensive role in nutrition and host health. Gastrointestinal helminths and the gut microbiota share the same niche and close interactions are likely. Chapter 2 documents the clinical trial testing the safety and immunogenicity of the TB vaccine H1/IC31 in HIV infected volunteers. The trial was designed as a phase II, multi-centre, double-blind, placebo-controlled trial and volunteers with a CD4+ lymphocyte count above 350/mm3 and no evidence of active TB were included. H1/IC31 consists of a fusion protein of Ag85B and ESAT-6, both secreted, immuno-dominant proteins isolated from M.tb culture supernatants. Safety was assessed based on medical history, clinical examinations, and blood and urine testing. Immunogenicity was tested using whole blood stimulation followed by intracellular cytokine staining and flow cytometry. The vaccine was safe and well tolerated in HIV infected individuals and CD4+ lymphocyte counts and viral loads remained constant. H1/IC31 was observed to be immunogenic and induced specific Th1 responses with bi-functional CD4+ T cells expressing IL-2 and TNF-a and polyfunctional CD4+T cells expressing IFN-g ,IL-2 and TNF-a. The ancillary study in chapter 3 investigates the induction and maintenance of CD4+ memory T cells following vaccination with H1/IC31. Induction of vaccine specific central (TCM) and effector (TEM) memory CD4+ T cells was detected. The magnitude was highly heterogeneous and the volunteers were grouped into non-, intermediate and high responder based on maintenance of vaccine specific TCM or TEM until 6 months after initial vaccination. Amplicon based transcript quantification of peripheral whole blood using next generation sequencing was performed to identify differentially expressed genes either induced by vaccination or present at baseline. Higher expression of genes implicated in resolution of inflammation were detected in high responder three days after the first vaccination. At baseline, high expression of genes involved in antiviral innate immunity was observed in non-responders and correlated with impaired vaccine specific maintenance of TCM and TEM. A functional variant of TLR-8 was present in a subgroup of TEM high responder, that was previously reported to result in slower disease progression in HIV infected individuals. Summarizing, HIV infected individuals with high expression levels of genes involved in antiviral innate immunity were found to have an affected long-term maintenance of H1/IC31 induced cellular memory response. In chapter 4 co-infections of Plasmodium falciparum (P.falciparum) and soil-transmitted helminths and the effect on clinical presentation of malaria are investigated in children aged 2 months to 9 years from the coastal region of Tanzania. Opposite to Hookworm infections, children co-infected with P. falciparum and Enterobius vermicularis (E. vermicularis) showed a significant reduction of clinical malaria cases. Expression of IL-6 and TNF-a by monocytes and conventional dendritic cells from peripheral blood after stimulation of toll-like receptors with known agonists was reduced in children infected with E. vermicularis. Transcriptome analysis of whole blood revealed lower expression of genes implicated in Th1 responses, pro-inflammation and IFN inducible genes in children with E. vermicularis. The gut microbiome from children with E. vermicularis showed a higher diversity and a function towards an anti-inflammatory enterotype. For the first time it was demonstrated, that E. vermicularis reduces the risk of clinical malaria by suppression of pro-inflammatory cytokine expression at the level of the systemic innate immune system.

The only licensed tuberculosis (TB) vaccine, Bacillus Calmette Guerin (BCG), fails to reliably protect adolescents and adults from pulmonary TB, resulting in ~1.6 million deaths annually. Protein subunit vaccines have shown promise against TB in clinical studies. Unfortunately, most subunit vaccines require multiple administrations, which increases the risk of loss to follow-up and necessitates more complex and costly logistics. Given the well-documented adjuvant effect of BCG, we hypothesized that BCG co-administration could compensate for a reduced number of subunit vaccinations. To explore this, we developed an expression-optimized version of our H107 vaccine candidate (H107e), which does not cross-react with BCG. In the CAF®01 adjuvant, a single dose of H107e induced inferior protection compared to three H107e/CAF®01 administrations. However, co-administering a single dose of H107e/CAF®01 with BCG significantly improved protection, which was equal to BCG co-administered with three H107e/CAF®01 doses. Importantly, combining BCG with a single H107e/CAF®01 dose also increased protection in previously BCG-primed animals. Overall, a single dose of H107e/CAF®01 with BCG induced long-lived immunity and triggered BCG-specific Th17 responses. These data support co-administration of BCG and subunit vaccines in both BCG naïve and BCG-primed individuals as an improved TB vaccine strategy with reduced number of vaccination visits.

A recombinant adenovirus expressing immunodominant TB antigens can significantly enhance BCG-induced human immunity

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. As a result of the coronavirus disease 2019 (COVID-19) pandemic, the global TB mortality rate in 2020 is rising, making TB prevention and control more challenging. Vaccination has been considered the best approach to reduce the TB burden. Unfortunately, BCG, the only TB vaccine currently approved for use, offers some protection against childhood TB but is less effective in adults. Therefore, it is urgent to develop new TB vaccines that are more effective than BCG. Accumulating data indicated that peptides or epitopes play essential roles in bridging innate and adaptive immunity and triggering adaptive immunity. Furthermore, innovations in bioinformatics, immunoinformatics, synthetic technologies, new materials, and transgenic animal models have put wings on the research of peptide-based vaccines for TB. Hence, this review seeks to give an overview of current tools that can be used to design a peptide-based vaccine, the research status of peptide-based vaccines for TB, protein-based bacterial vaccine delivery systems, and animal models for the peptide-based vaccines. These explorations will provide approaches and strategies for developing safer and more effective peptide-based vaccines and contribute to achieving the WHO’s End TB Strategy.

Background: ​Mycobacterium Tuberculosis (TB) is a bacterial organism responsible for causing TB in various parts of the human body, but it mainly affects the lung parenchyma. TB is one of the leading causes of morbidity and mortality globally. In the year 2015, 10.4 million adults suffered from TB and 1800,000 died while 1000,000 paediatric population suffered from TB and 170,000 passed away due to this illness. Objective: ​To find an association between pulmonary tuberculosis and BCG vaccination in the paediatric population. Methodology:​ ​It was an observational case-control study. ​The study was carried out in the departments of Pulmonology of The Children's Hospital and the Institute of Child Health, Lahore. The duration​ of the study was six months. A total of 122 patients fulfilling the inclusion criteria were enrolled in the study. BCG vaccination status was determined. Results: ​The mean age of the cases was 7.67±4.30 years that of controls was 7.79±4.13 years. Among cases, there were 32(52.2%) males and 29(47.5%) females while among controls there were 39(63.9%) males and 22(36.1%) females. In the cases group there were 27(44.3%) children with BCG Vaccination and 34(55.7%) without BCG vaccination on the other side among controls there were 33(54.1%) children with BCG vaccination and 28(45.9%) without BCG vaccination. There was no significant association between BCG vaccination and study groups (r=0.67, p-value=0.27). Conclusion: ​There was no association between pulmonary TB and BCG vaccination in the paediatric population.

At present, the epidemic of tuberculosis (TB) is severe, and the only available vaccine,Bacillus Calmette-Guerin (BCG), is not high efficacious.Thus, there is a need for the development of novel TB vaccines which are more safe, effective, practical and inexpensive.This review summarizes the advance in the development of TB vaccines, including recombinant BCG vaccine, live attenuated vaccine, subunit vaccine, DNA vaccine and viral vector vaccine. Key words: Mycobacterium tuberculosis; BCG vaccines; Vaccines, subunit; Vaccines, DNA

The development of a new tuberculosis (TB) vaccine has become one of the main objectives of the scientific community. Protein antigens have been widely explored as subunit TB vaccines, however lipid antigens could be equally important to be used or included in such a vaccine. The aim of this study was to demonstrate the potential of a liposome formulation composed of an extract of lipids from Mycobacterium smegmatis (Ms) as a TB vaccine candidate. We evaluated the immunogenicity of this formulation as well as the cross reactive response against antigens from Mycobacterium tuberculosis (MTb) in BALB/c mice. We determined the anti-liposome IgG response in sera from TB patients and from healthy subjects who displayed a positive (PPD+) or negative (PPD-) tuberculin skin test. A significant increase in anti-liposome IgG (p<0.05) was detected in animals immunized with Bacille Calmette-Guérin (BCG) compared with all groups, and in the group immunized with liposomes from Ms (LMs) compared to animals immunized with either LMs adjuvanted with aluminium (LMs-A) or the negative control group (phosphate buffered saline, PBS) respectively. With respect to the cross reactive response against a cocktail of cell wall antigens (CWA) from MTb, significantly higher IgG levels were observed in animals immunized with BCG and LMs compared to negative controls and either, aluminium-adjuvanted liposomes (LMs-A) or montanide (LMs-M) (p<0.05). Furthermore, the anti-liposome IgG response was significantly superior in sera from pulmonary TB patients compared to PPD+ and PPD- healthy subjects (p<0.001) suggesting the expression of these antigens in vivo during active MTb infection. The results obtained provide some evidence for the potential use of liposomes containing total lipid extracts of Ms as a TB vaccine candidate.

Various strategies have been implemented to prevent tuberculosis. Vaccination with Bacille Calmette Guerin (BCG) vaccine is still used around the world. Generally, most people in have gained BCG vaccine as an infant, but the effectiveness of these vaccines do not survive to adulthood. Therefore, the necessary replacement BCG vaccine more effective to eliminate tuberculosis. Mycobacterium tuberculosis is an intracellular pathogen and it was inside the macrophage, which is considered to be the most important component of the immune system. M. tuberculosis has two sets of genes are highly polymorphic referred to as PE and PPE families. These unique families of proteins account for about 10% of the mycobacterial genome and have attracted great interest from a variety of different studies around the world. One member of the as a vaccine candidate is Rv 2430c. It is known that the sera of all patients infected with TB showed strong antibody responses against Rv 2430c compared to healthy individuals. The existence of these antibodies indicates that this protein is found in vivo during infection and is a native immunogenic molecule. The purpose of this study was to clone the Open Reading Frame (ORF) Rv 2430c M. tuberculosis Indonesian isolates to host cells Esherichia coli JM 109. The method used is by isolating -chromosomal DNA from clinical isolates from Indonesia, amplifying the ORF Rv 2430c with , ligating into cloning vectors pGEM-T and transform to E.coli JM 109. Characterization of clones do with migration analysis, restriction analysis and . The results obtained are recombinant clones that carry insertion was Rv 2430cKeywords: Mycobacterium tuberculosis, Rv 2430c, ORF, , JM 109.