**3. Nonspecific E**ff**ects of BCG**

One of the reasons why BCG is still used universally in middle and low income countries is because numerous studies indicate that similar to other live attenuated vaccines in use today, BCG have additional beneficial effects on the initially intended protection against TB [18–20]. Neonatal BCG is able to induce a strong Th1 cytokine response shown to enhance immune responses to other infant vaccines of the EPI [21]. In countries of low TB endemicity, BCG administration at birth has been related to reduction of childhood hospitalizations due to unrelated respiratory infections and sepsis [22,23]. In addition, there is emerging evidence that BCG may induce nonspecific resistance (T- and B-cell dependent) to other pathogens [20], which should be taken into consideration for AMR TB.

The non-specific beneficial effects ascribed to BCG have been attributed to the vaccine's ability to functionally and epigenetically reprogram innate immune cells, such as monocytes, macrophages, and NK (natural killer) cells, a process termed 'trained immunity' [24]. In human monocytes, BCG induced trained immunity has been attributed to the induction of metabolic pathways, which are regulated by epigenetic mechanisms at the level of chromatin organization [25]. In this context, future clinical trials could provide insight on the potential therapeutic role that modulation of these pathways may have during vaccination [26]. A recent large, multinational study conducted in sub-Saharan Africa suggests association of BCG vaccination with a reduced risk of malaria in children under the age of 5 years [27,28]. If these results are corroborated, they would denote that timely BCG vaccination could aid the global efforts to decrease malaria burden, including resistant forms of the disease.

To date, the preclinical and clinical down-selection process of new TB vaccine candidates has employed BCG as the reference gold standard comparator, because of its well-established safety profile [29,30]. Today, WHO also encourages incorporation of the nonspecific beneficial effects ascribed to BCG's ability to induce trained immunity in the design and development of novel TB vaccine candidates, especially those intended for BCG-replacement.

#### **4. New TB Vaccine Candidates from Discovery to Clinical Trials**

The most effective licensed vaccines against different infectious diseases confer protective immunity by /inducing neutralizing antibodies. Whereas, while for other diseases, such as HIV, malaria or TB, a strong response of cellular immunity is necessary, and we don't have correlated protection that could anticipate the efficacy of a new vaccine candidate [31]. The last 20 years have seen important breakthroughs in TB vaccinology, ranging from novel adjuvant systems or viral vectors for intracellular antigen presentation to advanced genetic engineering techniques for rational attenuation of *Mtb*. All these advances in vaccinology have led to the development of new TB vaccine candidates including novel prime and prime–boost regimes that could reach all age groups and TB population spectrums. The biology behind the host immune responses to *Mtb* are not yet understood [32,33]. Developing a protective vaccine requires not only finding the right antigens, but also activating the right ratio of protective and suppressive immune cells against these pathogens. In Douglas Young's words, "we need efficacious vaccines to understand immunology of TB" and we need "immunology studies to design a good TB vaccine".

Given the lack of BCG protection against respiratory forms of TB, in the last 20 years an enormous effort has been made in the research and development of new vaccines against TB. In the discovery phase, thousands of potential candidates were identified, of which hundreds have passed to preclinical evaluation in animal models and only a little more than a dozen have happened to be tested in early clinical studies in humans to date. There are different stages that each vaccine candidate needs to perform, including early first-in-human Phase I, then Phase II, and finally Phase III clinical trials, to reach marketing authorization. In the United States, for nearly two decades former Aeras (now International AIDS Vaccine Initiative, IAVI) [34], supported by the Bill and Melinda Gates Foundation, were dedicated to the discovery of new TB vaccine candidates. At the end of 2018, Aeras transferred its preclinical assets and clinical programs, biorepository, clinical staff, funding and other assets to International AIDS Vaccine Initiative (IAVI) [34]. In Europe, the research promoted by the different European Commission (EC) Framework Programs has made possible for hundreds of candidates to pass to preclinical evaluation of which several are currently in clinical trials [35]. In 2008, thanks to the EC funding programmes, the European TB Vaccine Initiative (TBVI) [36] was founded integrating at least 50 R&D consortium partners from the public (academia) and private (industry) sectors. TBVI is a non-profit product development partnership that facilitates the discovery and development of new, safe and effective TB vaccines and biomarkers that are accessible and affordable for global use. Clinical trials of a small number of selected TB vaccine candidates are supported by IAVI with funding sources from US NIH funding programmes and by the European and Developing Countries Clinical Trials Partnership (EDCTP) [37].

Given that the protection of BCG administered intradermally shows such limited results of efficacy, new studies changing the routes of administration of BCG have demonstrated encouraging results in non-human primates (NHP) applying BCG by the intravenous (IV) route against virulent *M. tuberculosis* challenge [38]. Although the difficulties of using the IV route for mass vaccination campaigns, the efficacy results demonstrating ability of IV BCG to substantially limit *Mtb* infection in highly susceptible rhesus macaques could have important implications in the preclinical evaluation of new candidates, as it could provide a prototype for identifying immune biomarkers and mechanisms of vaccine-induced protection against TB. The respiratory administration of BCG has demonstrate to be very promising by conferring very good immunity and protection in NHP [39]. If these results are confirmed in clinical studies, the aerosol route could be considered a possible universal vaccination route for BCG and new TB vaccine strategies.

Other live attenuated vaccines in preclinical testing fuelling the TB pipeline include the search for new candidates based on recombinant BCG has shown very promising results in preclinical animal models. On one hand, deletion of *zmp1* gene improves BCG-mediated protection in guinea pigs against TB [40] and on the other, inclusion of virulence genes, such as the RD1 region, genetically modified so as to not increase the virulence of BCG [41,42].
