MTBVAC: A Tuberculosis Vaccine Candidate Advancing Towards Clinical Efﬁcacy Trials in TB Prevention

Tuberculosis (TB) remains a major global health burden, causing more than 10 million new cases and 1.6 million deaths each year. Currently, the only approved TB vaccine in use in humans, is the one hundred years old vaccine, BCG, an attenuated vaccine derived from an isolate of Mycobacterium bovis that causes TB in cattle. BCG shows a variable efﬁcacy in preventing pulmonary forms of the disease in humans, so new vaccines are needed to help stop TB transmission. Among the 15 diverse TB vaccine candidates in clinical trials, MTBVAC is the only one based on rational attenuation of a human clinical isolate of Mycobacterium tuberculosis , which contains the largest number of antigens of the TB vaccine candidates in the pipeline. MTBVAC was designed and constructed as a response to the need to confer a better TB protection in terms of pulmonary disease prevention in newborns, adolescents


Introduction
Tuberculosis (TB) is an infectious disease that ranks among the leading causes of death worldwide.Its causative agent is Mycobacterium tuberculosis, also known as the Koch bacillus, discovered by Robert Koch in 1882. 1 Scientific community has extensively studied TB, revealing the early-stage relationship between the development of the disease and the immune status of patients. 2 significant part of the bacillus' infectivity is due to its personto-person transmission through the respiratory route.Transmitted through aerosols, these deposit in the pulmonary alveoli, where they infect alveolar macrophages. 3The lung is the primary target organ of M. tuberculosis, although 10-20% of immunocompetent patients develop extrapulmonary forms of the disease, a figure that increases to 60% in immunocompromised patients. 4efore the arrival of the pandemic caused by the SARS-CoV-2 coronavirus (COVID- 19), TB was the leading cause of death caused by a single infectious pathogen, surpassing HIV/AIDS, and malaria.It is estimated that a quarter of the global population (more than two billion people worldwide) is infected with M. tuberculosis, although only 5-10% will develop the disease throughout their life. 5urthermore, due to its high prevalence in developing countries, transmission appears to be related to migratory movements, as well as contexts of poverty and overcrowding. 6,7ne of the most pressing aspects is the increasing resistance cases of TB, reflected in the emergence of multidrug-resistant (MDR-TB) TB that does not respond to at least isoniazid and rifampicin, the 2 most powerful anti-TB drugs and extensively drug-resistant (XDR-TB) MDR TB that is resistant to isoniazid and rifampin, plus any fluoroquinolone and at least one of three injectable second-line drugs (i.e., amikacin, kanamycin, or capreomycin) M. tuberculosis strains.This has led to the need to find new ways to combat the disease with better diagnosis of TB, increased discovery of new drugs, shortening treatment time and decreasing transmission, particularly through preventive measures. 8,9urrently, vaccination is considered the most cost-effective measure in preventing a disease and the development of resistance to its treatment. 9The only licensed vaccine for TB prevention is Bacillus Calmette-Guérin (BCG) vaccine, which is based on the attenuation of Mycobacterium bovis originally isolated from cattle. 10First administered over a century ago, the World Health Organisation (WHO) recommends its use in countries with incidence higher than 10 TB cases per 100,000 population per year. 11CG provides protection with an efficacy rate of 60-80% against meningeal and miliary TB, which are disseminated and aggressive forms of the disease that occur during childhood. 12However, clinical trials have shown variable efficacy preventing pulmonary forms, the transmissible form of the disease, especially in adolescents and adults. 9,13BCG has been shown to provide protection against TB for up to 10-15 years post-vaccination. 14Several hypotheses have been proposed to explain this variation in efficacy, including the variability between different BCG strains, dosage, route of administration, 15 patient's genetic profile, nutritional status, 16,17 viral or helminthic infections, 16 and exposure to environmental mycobacteria 17,18 or geographical variation. 12uring the subcultivation process of BCG attenuation between 1908 and 1921, more than 100 additional genes were lost from BCG relative to M. tuberculosis.Among them, deletion of Region of Difference 1 (RD1).Many authors attribute the limited efficacy of BCG to its antigenic composition, insufficiently related to that of M. tuberculosis.BCG lacks 23-28% of the CD4 epitopes present in the pathogen, 15 including ESAT6 and CFP10 coded in RD1, which is present in M. tuberculosis and absent in BCG, being mainly responsible for the attenuation of BCG. 19urrently, there are 15 different candidates in clinical trials in the global TB vaccine pipeline, which are included in Fig. 1 of this article. 5,20tudies on subunit vaccine candidate M72/AS01 E have shown promising results, demonstrating its ability to reduce the risk of developing active disease in individuals previously infected with M. tuberculosis, achieving disease prevention (POD) in approximately 50% of the volunteers studied. 21herefore, the need for new vaccines to improve BCG protection led to the development of several candidates, including (Mycobacterium Tuberculosis Vaccine (MTBVAC), based on the rational attenuation of M. tuberculosis.Developed by the University of Zaragoza (Spain) in collaboration with the Pasteur Institute (France), MTBVAC has demonstrated the ability to induce a comprehensive, pathogen-specific immune response superior to the current BCG vaccine in different animal models (Martín et al.,  2021).Since 2008, the Spanish biopharmaceutical company Biofabri has been the industrial partner of the University of Zaragoza and responsible for the clinical and industrial development of the vaccine in collaboration with TuBerculosis Vaccine Initiative (TBVI) and US International AIDDS Vaccine Initiative (IAVI).Currently, MTBVAC is progressing with its Phase 3 efficacy trial in newborns in Sub-Saharan Africa. 9

Methodology
A scoping review of international publications over the last 20 years was conducted to cover the complete development of MTB-VAC.A literature search was performed in the PubMed database using the key terms "Tuberculosis", "Prevention and control", "Vaccines", "Attenuated vaccines" and "MTBVAC".After selecting the articles and removing duplicates, a first screening phase was conducted by reading the abstract, followed by a second phase of reading the full text to exclude irrelevant articles.During the review process, a couple of articles were added based on citation identification.
Additionally, multiple searches were conducted in the Clin-icalTrials.govdatabase to determine the status of clinical trials associated with each referenced vaccine.

Mycobacterium tuberculosis vaccine: MTBVAC
MTBVAC has been designed and constructed with the aim of providing better protection than the current BCG against respiratory forms of the disease.In its conception we have considered the experience of 100 years of BCG use, both being live attenuated vaccines, can be administered intradermally in a single dose and the same distribution and storage methods can be used.MTBVAC aims to be a universally accessible and affordable vaccine for countries with the highest TB burden that protects better than BCG against pulmonary TB.MTBVAC is postulated as a preventive vaccine with better protection than BCG at birth, and as a revaccination strategy in adolescents and adults previously vaccinated with BCG in TB-endemic countries. 22TBVAC was constructed by rationally attenuating a clinical isolate of M. tuberculosis Mt103, belonging to Lineage 4 (Euro-American), one of the most globally widespread modern lineages of M. tuberculosis. 23According to the first Geneva consensus published in 2005, 24 it was designed based on the construction of a strain of M. tuberculosis with two stable and independent genetic deletions without antibiotic resistance markers, located in the phoP and fadD26 genes, conferring a PhoP-/PDIM-phenotype to MTBVAC, and maintaining the rest of the antigens present in the bacillus and absent in the current BCG vaccine. 25Today MTBVAC is the only vaccine based on the attenuation of M. tuberculosis itself. 26,27hoPR is a two-component signal transduction system that is highly relevant to the pathogenesis of M. tuberculosis.PhoP acts as a transcriptional regulator of a wide variety of genes related to functions involved in the pathogenicity of the bacillus, controlling up to 2% of its genome. 28,29PhoPR was identified as a major factor of virulence in TB while studying an MDR-TB outbreak caused by a M. bovis strain that showed an increased expression of phoP gene due to an insertion sequence IS6110.This MDR-TB outbreak resulted in more than a hundred deaths in Spain during the 1990s, primarily among AIDS patients before triple therapy against HIV was implemented. 26hoPR controls the biosynthesis of several lipid components of the cell wall, such as diacyltrehalose (DAT), polyacyltrehalose (PAT) and sulfolipids (SL), responsible for inhibiting the host's innate response and inducing a productive cough that facilitates the transmission of the pathogen. 30In turn, it regulates the expression of ESX-1, responsible for the secretion of the virulence factor ESAT6, 29 which when secreted inhibits autophagy, a mechanism responsible for the destruction of the intracellular pathogens, and induces apoptosis of infected cells, facilitating the spread of the bacillus between cells. 31Finally, PhoPR acts on a small RNA strand (mcr7), which represses the translation of tatC, a protein belonging to the TAT (Twin Arginine Translocation) system, responsible for antigen secretion, thus reducing the secretion of Ag85A and Ag85C.MTB-VAC, as a phoP deletion mutant, shows the absence of cell wall components such as DAT, PAT, and SL.Furthermore, phoP deletion inhibits ESAT6 secretion and increased secretion of Ag85 major tuberculous antigens 30,31 and increases production and secretion of the second messenger c-di-AMP, which could be relevant for enhancing the innate immune response of the host. 30he fadD26 gene is responsible for the biosynthesis of lipid components of the M. tuberculosis cell wall called phthiocerol dimycocerosates (PDIMs) a major virulence factor in M. tuberculosis strains.PDIMs function lies in the disruption of the phagosome, in conjunction with the action of ESAT6, making them crucial for the survival of the pathogen within the host.The absence of PDIMs because of the deletion of fadD26 in MTBVAC increases the susceptibility of the pathogen to the innate immune response, as it is unable to prevent the maturation of the phagosome, which leads to death of the bacterium. 29,31eclinical studies on prototype vaccine SO2 and MTBVAC The phoP gene was inactivated in a M. tuberculosis clinical isolate by insertion of a kanamycin resistance marker into it.The resulting strain, called SO2, also contained a single mutation in the PDIM locus, conferring it a PhoP-and PDIM-deficient double phenotype.SO2 was used as a prototype TB vaccine candidate (PDIM-deficient phoP mutant) in preclinical animal models to study attenuation and protection against TB challenge. 25,32,33SO2 preclinical studies confirmed a superior attenuation profile to BCG, 34 as well as better protection in guinea pigs 35 (Table 1).SO2 did not meet the Geneva Consensus criteria required to enter clinical trials, due to the inactivation of PhoP through the insertion of an antibiotic resistance marker and a single mutation in the fadD26 gene.
MTBVAC was constructed by two independent stable deletions in phoP and fadD26 genes allowing, thereby enabling its progression into clinical development. 25,27Since then, MTBVAC has demonstrated safety, protection, and immunogenicity comparable to prototype SO2 in studies in mice, guinea pigs 25 and rhesus macaques. 36,37In addition, MTBVAC confers superior protection to BCG in adult and neonatal mouse models 38 and guinea pigs 22 (Table 2).
In 2012, MTBVAC vaccine candidate was approved for entry into human clinical trials.

PHASE 1a. Dose-escalation study to assess the safety and immunogenicity of MTBVAC vaccine compared to BCG vaccine
Between 2012 and 2014, a randomised, double-blind, BCGcontrolled clinical trial was conducted in Lausanne (Switzerland) to assess the safety and local tolerability of MTBVAC in healthy adults aged 18-45 years. 39Thirty-six (36) participants were randomised  29 Comparative Metabolomics between M. tuberculosis and the MTBVAC To compare the differences between metabolites produced by MTBVAC and M. tuberculosis.
34 metabolites were identified, of which 25 were increased in MTBVAC and 9 in M. tuberculosis.Pérez I et al. (2020) 23 Study MTBVAC representing the three modern M. tuberculosis lineages reveal that the Euro-American genetic background.
To evaluate the efficacy of the new variants of MTBVAC (L2 and L3 strains) compared to the original (L4 strain) and determine whether MTBVAC can protect against three modern strains.
All three variants of MTBVAC confer protection against the disease irrespective of the phylogenetic lineage used as the basis for their construction.Greater protection is observed for the variant based on the L4 lineage.White AD et al.
(2021) 37 Protection study of intradermal vaccination with MTBVAC in rhesus macaques against aerosol challenge with M. tuberculosis To determine the protection of a single intradermal dose of MTBVAC in rhesus macaques and to characterise the immune response for comparison with that defined in both preclinical studies in macaques and in human clinical trials.
MTBVAC vaccination conferred significantly improved protection against the BCG vaccinated group and the non-vaccinated group.Immunological profiles demonstrated a predominantly Th1-type response that correlated with results in clinical and preclinical trials.Pérez I et al. (2022) 30 Metabolomic study of MTBVAC To analyse the expression of the second messenger c-di-AMP in MTBVAC and its impact on vaccine efficacy.
Deletion of phoP in MTBVAC induces increased production of c-di-AMP, which influences attenuation and efficacy, leading to an increased IL-1␤-mediated immune response to BCG.
into three cohorts, verifying its safety by progressively increasing the MTBVAC dose [low-dose (5 × 10 3 CFU); intermediate-dose (5 × 10 4 CFU); and high-dose (5 × 10 5 CFU)], in a 3:1 ratio.As primary objective of the study, MTBVAC showed a safety profile comparable to BCG, with no serious adverse effects.MTBAVC was shown to be at least as immunogenic as BCG with evidence of dose-dependent induction of specific CD4+ T cells (IFN-␥+, TNF-␣+, IL-2+) in both groups, although the study sample did not allow for statistically significant differences.The results obtained suggested that both BCG and MTBVAC predisposed to the development of a central memory T-cell-mediated immune response, rather than an effector memory T-cell-mediated response, the former being essential for the maintenance of long-term immunological memory with a greater tendency in MTBVAC 40 (Fig. 2).

PHASE 1b. Dose-escalation study and immunogenicity study to compare MTBVAC with BCG in neonates with a safety arm in adults
Between 2015 and 2018, a new trial was conducted in Worcester, near Cape Town (South Africa) with the aim of evaluating  26 the safety and immunogenicity of MTBVAC in newborns in a TB endemic area. 41Prior to this, a safety arm of 18 adult participants was developed in which participants were assigned to the MTBVAC and BCG groups in a 1:1 ratio.Once the safety results of this cohort were obtained, the study was conducted by distributing 36 newborns in a 3:1 ratio in three cohorts of progressive doses of MTBVAC [low-dose (2.5 × 10 3 CFU); intermediate-dose (2.5 × 10 4 CFU); and high-dose (2.5 × 10 5 CFU)].
The study showed that MTBVAC had an acceptable reactogenicity profile, inducing as in the phase 1a trial, a dose-dependent Th1 response mediated by polyfunctional CD4+ T cell.Thus, significantly reduced responses were observed in the low-dose group (2.5 × 10 3 CFU) and therefore this dose was discontinued in future studies.On the other hand, statistically significant responses were observed between the high-dose group (2.5 × 10 5 CFU) and the BCG group.In addition, reduced frequencies of CD4+ Th17, Th22 and CD8+ T lymphocytes were observed.High IFN-␥ conversion frequencies were observed for the first time in the IGRA test, due to cross-reactivity with CFP10 antigens 42 that correspond with the protection in animal models 43 (Fig. 2).

PHASE 1b/2a. MTBVAC study in adults with and without latent TB infection in South-Africa (A-050)
In 2018, MTBVAC initiated a Phase 1a/2b trial in South Africa with the aim of defining the appropriate dose of inoculation, as well as evaluating the safety and immunogenicity of MTBVAC in adults with or without latent TB disease previously vaccinated with BCG in childhood.For this purpose, 144 participants were selected and distributed into 8 cohorts 1-4 those without TB disease and 5-8 those with TB disease.Progressive doses of MTBVAC were used: 5 × 10 3 CFU in cohorts 1 and 5, 5 × 10 4 CFU in cohorts 2 and 6, 5 × 10 5 CFU in cohorts 3 and 7, 5 × 10 6 CFU in cohorts 4 and 8.The study has been completed and the results are pending publication 44 (Fig. 2).

PHASE 2a. Safety and immunogenicity study to define the dose of MTBVAC in South African neonates
Finally, a concurrent Phase 2a trial was initiated in South Africa in 2019 to define the appropriate dose of administration in newborns, evaluate the safety and immunogenicity of MTBVAC, as well as to assess the frequency of IGRA test conversion, following the results of the Phase 1b trial.99 HIV-unexposed infants without BCG vaccination were randomised into three cohorts in a 3:1 ratio [25 participants were assigned to each MTBVAC progressive dose group (2.5 × 10 4 CFU, 2.5 × 10 5 CFU and 2.5 × 10 6 CFU) and 24 to the BCG group].o study MTBVAC trained immunity and protection against experimental lethal pneumonia compared to BCG in mouse model.
To determine the ability of MTBVAC to induce "trained immunity" and offer protection against a heterologous model of pneumococcal pneumonia in mice.
MTBVAC is able to generate metabolic and immunomodulatory effects and epigenomic modifications through mechanisms similar to BCG, providing protection against heterologous infections unrelated to TB, such as Streptococcus pneumoniae.Dijkman K et al. (2021) 64 Pulmonary MTBVAC vaccination in non-human primates To assess the impact of using MTBVAC inoculated through the respiratory mucosa in primates to determine their ability to induce adaptative immunity.
Pulmonary vaccination with MTBVAC resulted in a local antigen-specific response that has previously been correlated with protection against TB.Both T lymphocytes and antibodies generated by the vaccine showed an enhanced capacity to respond to M. tuberculosis.trial in newborn babies.This is a randomised, double-blind, BGGcontrolled, multi-centre clinical trial, with the evaluation being conducted in six different sites, four of them in South Africa for efficacy study, one in Senegal and one in Madagascar for safety and immunogenicity compared to BCG.The objective of this study is to evaluate the safety, immunogenicity and, most importantly, the efficacy of MTBVAC in healthy HIV-negative infants born to HIVpositive or HIV-negative mothers.A total of 7120 participants are expected to be recruited and randomised 1:1 to either the MTBVAC or BCG group.Recruitment is currently underway, and the trial is expected to end in September 2029 46 (Fig. 2).

Comparison of MTBVAC with other candidate vaccines in PHASE 3
In addition to MTBVAC, other vaccine candidates are in Phase 3 of clinical development, such as VPM1002, GamTBvac, Immuvac (MIP), and revaccination with BCG 5 (Fig. 1).
VPM1002 is a live attenuated vaccine based on recombinant BCG that has demonstrated safety and immunogenicity in Phase 1 and 2a clinical trials.Its potential application as a post-exposure immunisation technique in adults and as a preventive method for TB in newborns is being studied. 47It was designed to stimulate the CD8+ T cell-mediated response, resulting in an increase in IL-17 production. 48amTBvac is a subunit-based vaccine containing ESAT6-CFP10 antigens.GamTBvac has shown an acceptable safety profile while inducing a specific and durable Th1 and humoral immune response. 49This specific Th1 cellular response is mediated by IFN-␥, with a better profile at medium doses, showing a progressive increase in IFN-␥ production without a significant subsequent decline in cytokine production over time. 50he main difference between MTBVAC and the other candidate TB vaccines in Phase 3 is its larger antigen composition.MTBVAC is the first and only vaccine candidate based on the attenuation of M. tuberculosis in clinical trials. 31BCG was developed over a century ago through repeated subculture attenuation of M. bovis. 51On the other hand, VPM1002 is a recombinant BCG vaccine (BCG ureC::hly) in which the urease C gene has been replaced by the coding gene (hly) of listeriolysin O from Listeria monocytogenes. 47Immuvac (MIP) is a vaccine based on heat-killed Mycobacterium indicus pranii. 9Finally, GamTBvac is a recombinant subunit vaccine formed by the fusion of two mycobacterial antigens (Ag85A and ESAT6-CFP10) with the dextran-binding domain immobilised on dextran, mixed with an adjuvant core consisting of DEAE-dextran with CpG oligodeoxynucleotides (TLR9 antagonists). 52urrently, the most reliable criterion for efficacy is the demonstration of disease prevention.4][55] Additionally, revaccination with BCG is conducting a study to demonstrate prevention of M. tuberculosis infection, which is its primary indication. 56Furthermore, VPM1002 is studying the prevention of M. tuberculosis infection 57 and the recurrence of active TB disease (both pulmonary and extrapulmonary). 58nother aspect to consider is the design of these studies in relation to their comparative efficacy with BCG, which is used as a control in the trials as it is the only approved vaccine for human use and therefore considered the gold standard.It is worth noting that the studies of MTBVAC have been designed with the goal of demonstrating superior efficacy to BCG, while other vaccines like VPM1002 aim for non-inferiority, meaning the validation of possible alternatives without compromising the protection provided by the reference vaccine.However, given the wide range of BCG formulations, data should be interpreted carefully, avoiding extrapolation of results from one formulation to other existing formulations. 9dditionally, both MTBVAC and GamTBvac express ESAT6-CF10 antigens, which can result in cross reactivity with the IGRA diagnostic test.Consequently, there is a need to develop new diagnostic techniques that reduce the number of false positives after vaccination. 26,52inally, it is important to note that the administration of live attenuated vaccines, such as MTBVAC, VPM1002, or BCG, poses a risk in individuals infected with HIV.Therefore, trials are being planned to study the safety of theses vaccines in this population. 20 new study whose purpose is to evaluate the safety and immunogenicity of MTBVAC in adolescents and adults living with and without HIV in South Africa (HVTN605A5421) is scheduled to begin in early 2024.59

Research priorities in other MTBVAC uses
After more than two decades of development, MTBVAC is starting Phase 3 efficacy trial in newborns against TB disease.However, its effects go beyond achieving TB prevention. 26In addition to MTBVAC protection against TB, other uses that take advantage of the non-specific effect of live attenuated vaccines are under investigation in pre-clinical studies.Its potential as a treatment for BCG-refractory non-muscle-invasive bladder cancer has been demonstrated, stimulating a tumour-specific immune response mediated by CD4+ and CD8+ T lymphocytes. 60Similarly, its ability to induce a Th1-type immune response, to the detriment of an IgE-producing Th2 response, has demonstrated MTBVAC's ability to reverse established asthma in mice. 61Furthermore, its capacity to induce "trained immunity" 62,63 has been studied, which refers to epigenetic changes in primary myeloid cells, enhancing the immune response against non-bacterial stimuli and providing heterologous protection against Streptococcus pneumoniae 62 (Table 3).
On the other hand, its respiratory administration has been studied as an alternative route to the intradermal inoculation used in most studies.In these cases, MTBVAC was able to induce a local antigen-specific response that extended beyond the site of inoculation and has been previously correlated with protection against TB. 64The respiratory administration of MTBVAC could facilitate universal vaccination in the future (Table 3).

Conclusions
MTBVAC emerged as a response to the need for a better preventive strategy than BCG vaccination, given its variable efficacy.Based on the attenuation of M. tuberculosis, MTBVAC showed an optimal safety profile, as well as an adequate degree of reactogenicity and immunogenicity in preclinical animal studies, which supported its entry into human clinical trials in 2012.After 20 years of development, MTBVAC has started Phase 3 clinical trial to evaluate its efficacy in newborns, with plans for a further trial in adolescents and adults in the future.
While it is not the only candidate to have reached this point, its properties and proven potential over the years make it a very promising candidate.

Conflict of interest
C.M. is co-inventor on a patent on Tuberculosis Vaccine held by the University of Zaragoza and Biofabri.Biofabri is an industrial partner of University of Zaragoza and exclusive licensee and industrial and clinical developer of MTBVAC.The authors declare that they have no other known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

PHASE 3 .
Efficacy, safety and immunogenicity evaluation of MTBVAC in newborns in sub-Saharan Africa Currently, as of September 2022, MTBVAC received partial funding through the European & Developing Countries Clinical Trials Partnership (EDCTP2) to conduct a multi-centre Phase 3 efficacy

Table 1
Preclinical studies conducted with the SO2 vaccine prototype.

Table 2
Preclinical studies conducted with the MTBVAC vaccine.

Table 3
Studies of other indications and routes of administration of MTBAVC vaccine.