**5. TB Vaccine Candidates in Clinical Trials**

As we previously mentioned, the lack of immune marker(s) for prediction of vaccine-elicited protection makes finding effective vaccines against TB extremely challenging, as it requires long and expensive efficacy trials with thousands of volunteers (Phase IIb proof-of-concept trials and Phase III efficacy trials) in endemic countries with a high incidence of TB after obtaining robust safety and immunogenicity data in previous trials with tens (Phase I) and then with hundreds (Phase II) of volunteers [32] (Figure 2).

**Figure 2.** Tuberculosis (TB) vaccine candidates in the pipeline of clinical trials. The diagram shows the advance in clinical trials of the 14 vaccine candidates coloured according to each vaccine strategy.

Unlike HIV and malaria, twenty years ago there was no new candidate for a vaccine against TB in clinical trials. After more than 10 years of previous clinical trials, in 2012 MVA85A was the first candidate since BCG in 1921 to enter efficacy evaluation as preventive TB vaccine in infants [43,44]. MVA85A vaccine was tested in a double-blind, placebo-control Phase IIb efficacy study for its ability to increase (or boost) the immunity in healthy infants HIV-uninfected (aged four to six months) recently vaccinated with BCG at birth, living in a highly endemic region for TB (Worcester, South Africa). A total of 2797 children were vaccinated (1399 with MVA85A and 1398 with a placebo) and followed up every three months for more than three years. Results showed that 32 children (2%) of the 1399 in the MVA85A arm, were diagnosed with TB and 39 children (3%) of the 1398 vaccinated with BCG + placebo. The difference between the two groups was not significant and the interpretation was absence of efficacy of MVA85A. For the TB vaccine scientific community, this study resulted highly informative, and it was a great step forward in the research of new vaccines paving the way for new TB vaccine efficacy studies. The Worcester study was coordinated by the South African TB Vaccine Initiative (SATVI) and the site is highly prepared for testing new promising TB vaccines. Years after this study, the scientific community continues learning and drawing conclusions about the immunology of the disease. After three years of follow-up of the children in the study, the QuantiFERON (QFT) and the risk of disease were studied by SATVI and Oxford University teams [45]. The extended immunogenicity analysis of the trial data showed that both children that remained QFT- (less than 0.35 IU/mL) or those who became QFT + (with less than 4 UI/mL) had a lower risk of developing TB as

compared to QFT + children with more than 4 IU/mL, whose risk of TB disease is high. This result may accelerate studies with other vaccines [45].

Current efficacy trials of new prophylactic TB vaccines attempt to measure either the prevention of infection against *Mtb* (POI), or the prevention of acquiring TB disease (POD) and the prevention of recurrent TB disease (POR) [46]. POR trials evaluate therapeutic vaccines administered as an adjunct to drug treatment to increase the effectiveness and shorten the duration of TB treatment in patients undergoing or completing TB treatment for active disease. The efficacy endpoints of POR trials are prevention of reactivation of existing infections and/or prevention of disease due to new infections. Considering lack of correlates of protection, POD trials provide the most reliable endpoint of vaccine efficacy and acceptance for Regulatory authorities.

In newborns, new TB vaccines should provide evidence of significant superiority over BCG. Global vaccination strategies targeting adolescents and adults would preferably include individuals with and without pre-existing *Mtb* infection, thus avoiding use of IGRAs which are expensive and can interfere with vaccine-induced immune results. In addition, testing candidates in both uninfected and infected individuals in future trials could avoid risks of excluding potentially efficacious candidates in the pipeline against infection but which lack efficacy in IGRA-positive individuals [47].

New TB vaccine candidates today in clinical trials could be divided into whole cell vaccines and subunit vaccines. Whole cell vaccines include live mycobacterial vaccines derived from live attenuated *Mtb* strains, *M. bovis* BCG or recombinant BCG and killed mycobacterial vaccines that could be formulated from other saprophytic mycobacterial species or *Mtb* [48] (Figure 3). Subunit vaccines contain *Mtb* antigens expressed as recombinant proteins that are formulated with different adjuvants or expressed by recombinant viral vectors that are used as vehicles for the administration of antigens [47]. Most of the current subunit vaccine candidates are vaccines with limited antigen diversity which are designed to enhance prior immunity mediated by T cells [49] (Figure 3).

There is a total of 14 TB vaccine candidates in clinical trials today, seven are based on subunits and seven consist on whole-cell mycobacteria [47,50] (Figure 3). Of the subunit candidates, four are mycobacterial fusion protein(s) in new adjuvant formulations (ID93: GLA-SE, H56.IC31, M72:ASO1E, GamTBVac) and three are based on recombinant live-attenuated or replication-deficient virus-vectored expressing one or more *Mtb* proteins (Ad5Ag85, ChadOx1.85/MVA85A, TB/FLU-04L). Of the whole-cell mycobacterial candidates, there are four candidates based on inactivated/extracts of mycobacteria (*M. vaccae*, MIP, DAR-901, RUTI) and three vaccine candidates are based on live attenuated mycobacteria, one is BCG revaccination, since a positive signal has been seen in prevention studies of *Mtb* infection [51], other is based on the use of recombinant BCG (VPM1002) and the third is derived from rationally attenuated *Mtb* (MTBVAC).
