5.1.2. Inactivated Whole-Cell Mycobacteria

Most of inactivated whole cell mycobacterial TB vaccine candidates have been designed as "therapeutic vaccines" seeking to reduce treatment length in people infected with latent TB, or reduce the likelihood of recurrence after the end of a treatment [48].

*M. vaccae*™ vaccine is a lysate comprised of inactivated *Mycobacterium vaccae* (non-tuberculous mycobacteria) developed as an immunotherapeutic agent to help shorten TB treatment for patients with drug-susceptible TB and licensed by the China Food and Drug Administration (FDA). A Phase III study of efficacy has been recently published [2,62] (Figures 2 and 3).

MIP Immuvac is a heat-killed *Mycobacterium indicus pranii* vaccine, approved by the drug controller general of India and FDA as an immune-therapeutic and immunoprophylactic strategy for use in multibacillary leprosy patients (as an adjunct to standard multidrug therapy), and for preventing the development of leprosy among close contacts of leprosy patients. A Phase III efficacy and safety trial for preventing pulmonary TB among healthy house-hold contacts of sputum smear-positive TB patients is underway in India [2] (Figure 2).

DAR-901 is based on a heat-inactivated non-tuberculous bacterium *M. vaccae* renamed as *Mycobacterium obuense* which was produced by former Aeras (Rockville, MD, USA) [63] for use as booster vaccine in BCG vaccines (Figure 3). It is the scalable manufacturing version of candidate vaccine SRL172, which showed efficacy in a Phase III trial among HIV-infected adults in Tanzania. DAR-901 is in a Phase IIb prevention of infection trial among BCG-primed adolescents, also in the United Republic of Tanzania. The trial is scheduled for completion in 2020 [2] (Figure 2).

RUTI®is a liposomal formulation containing fragmented, detoxified *Mtb* grown under stress conceived conditions, as a potential therapeutic vaccine (Figure 3). RUTI is sponsored by the Archivel Farma (Spain), and is currently in a Phase IIa of clinical trials [64,65] (Figure 2). When administered one month after isoniazid treatment, RUTI showed safety and immunogenicity in individuals with latent TB infection (LTBI). Plans for evaluation in a POD trial among HIV-infected and uninfected patients with LTBI are underway [47].

## *5.2. Subunit Vaccines*

## 5.2.1. Subunit Viral Vectored Vaccines

There are three subunit vaccines that use attenuated viral vectors by different routes of administration.

Ad5HuAg85A vaccine, developed by the University of McMaster in Canada, consists of a human adenovirus serotype 5 vector that expresses Ag85A and is administered intramuscularly (Figure 3). AdHu5Ag85A has been evaluated in a Phase 1 safety and immunogenicity study in BCG-naїve and BCG-immunized healthy adults by the intramuscular route showing adequate safety and tolerability [47,66] (Figure 2).

ChAdOx85A/MVA85A vaccine strategy, developed by the University of Oxford, like the recombinant pox vaccine MVA85A (mentioned above), ChAdOx85A is a simian adenovirus which also expresses Ag85A (Figure 3). Both vaccine candidates are tested together in a joint heterologous prime-boost regimen delivered through both systemic and mucosal routes in BCG-vaccinated individuals. A Phase I trial of intramuscular administration of ChAdOx85A in BCG vaccinated adults in the United Kingdom, tested alone and as part of a prime-boost strategy with MVA85A, has been completed. Aerosol administration of ChAdOx185A is currently evaluated in a Phase I trial among BCG-vaccinated adults in Switzerland (Figure 2). Safety and immunogenicity of aerosol administration of MVA85A in BCG-vaccinated individuals and in people with a latent TB infection have been evaluated. In 2019, plans for a Phase IIa safety and immunogenicity trial among adults and adolescents in Uganda were underway with the aim of evaluating intramuscular administration of ChAdOx185A and MVA85A [2].

TB/FLU-04L is based on an attenuated replication-deficient influenza virus vector expressing antigens Ag85A and ESAT-6 (Figure 3). It was designed as a preventive booster vaccine in BCG-vaccinated infants, adolescents and adults. It is about to start a Phase IIa study in individuals with latent TB [2] (Figure 2).

#### 5.2.2. Adjuvanted Subunit Vaccines

D93 + GLA-SE vaccine was developed by the Infectious Disease Research Institute (IDRI) in the United States. ID93+GLA-SE comprises four *Mtb* antigens, of which three are associated with virulence (Rv2608, Rv3619 and Rv3620) and one (Rv1813), with latency) formulated with the adjuvant GLA-SE for delivery [67] (Figure 3). It has been evaluated Phase IIa trial in South Africa among HIV-negative TB patients who have recently completed TB treatment for active pulmonary disease [68] (Figure 2). This is in preparation for Phase II safety and immunogenicity trials among adults undergoing active TB therapy. A POI Phase IIa trial in BCG-vaccinated healthy health care workers is also underway [2].

H56:IC31 is a fusion protein of three *Mtb* antigens (Ag85B, ESAT-6 and Rv2660c) and delivered in the adjuvant IC31© (Valneva Austria GmBH, Vienna, Austria) (Figure 3). H56:IC31 was discovered and developed by the Statem Serum Institut (SSI) of Copenhagen. It has been tested in three Phase I or I/IIa trials for safety and immunogenicity in BCG-vaccinated adults (Figure 2) showing acceptable safety and immunogenicity. It has completed a Phase Ib safety and immunogenicity trial in adolescents. A POR Phase IIb trial funded by EDCTP and coordinated by IAVI is underway in South Africa and the United Republic of Tanzania [2].

M72/AS01E is also a subunit candidate vaccine comprising two *Mtb* antigens (32A and 39A) formulated in the AS01 adjuvant for delivery (Figure 3), also used in the formulation of the malaria vaccine (RTS, S/AS01, GlaxoSmithKline) and the recombinant zoster vaccine Shingrix, GlaxoSmithKline [69]. It was evaluated in a IIb efficacy trial in in Kenya, South Africa and Zambia among *Mtb*-infected HIV-negative adults whose data showed 54.0% protective efficacy in *Mtb*–infected young adult women [69] (Figure 2). The immunogenicity analysis after end of the three-year follow-up showed that M72/AS01 elicited an immune response and provided protection against progression to pulmonary TB disease for at least three years [70]. This is the first time a proof-of-principle trial demonstrates vaccine-induced protection against clinical TB disease. However, whether M72/AS01 could provide protection against TB among *Mtb*-uninfected and HIV-negative individuals and in people from other geographical areas remain key questions to be answered. M72/AS01E has been exclusively licensed to the Medical Research Institute of Bill and Melinda Gates Foundation for further development.

GamTBvac is a fusion protein comprising *Mtb* antigens Ag85A and ESAT6–CFP-10 with the dextran-binding domain immobilized on dextran. It is formulated with an adjuvant consisting of a DEAE-dextran core and CpG oligodeoxynucleotides (TLR9 agonist) (Figure 3). GamTBvac is undergoing a Phase IIa safety and immunogenicity evaluation in healthy BCG-vaccinated adults, following a successful Phase 1 safety and immunogenicity trial in Russia [47] (Figure 2).

**Figure 3.** The diversity of TB vaccine candidates in clinical trials. Schematic table showing the main characteristics of the vaccine candidates. The table is coloured according to vaccine strategies indicated in Figure 2 and contains representative information for each candidate, including the mycobacterial origin from each vaccine and their antigenic content.

#### **6. Target Population for a New TB Vaccine**

In its Preferred Product Characteristics (PPC) document for TB vaccines, WHO defines two target populations for which a new TB vaccine could play a very important role in the fight against TB including resistant forms of the disease [3,4]. The WHO prioritizes two types of strategies one, safe, effective and affordable TB vaccines for adolescents and adults and the other, a TB vaccine for neonates and infants with improved safety and efficacy as compared to BCG.

Following the negative results of the first efficacy study which was conducted with MVA5A in infants, the priority population was changed to mainly adults. Modelling studies suggest that new effective vaccines for adolescents and adults, who are responsible for disease transmission, would have the greatest impact in halting TB incidence [71,72]. These studies suggest that although disease incidence in children under five years of age is considerable, TB transmission among this age-group is not common [73].

Prophylactic prime vaccines should be compared to BCG, since whole-cell vaccines seek to protect better than BCG. The immune responses elicited by live mycobacterial vaccines are considered to be specific and long-lasting, and these responses are not obtained with subunit vaccines. Vaccine persistence or restricted replication in vivo could account for the differences of immune responses, as observed for other live human vaccines, such as polio, measles, and yellow fever [48]. Considering the variability in BCG formulations, new TB vaccine trials which use BCG as reference comparator, should be interpreted cautiously with reference to a specific BCG formulation avoiding generalization of data to all BCGs [13].

Since the majority of the adolescent and adult population in countries endemic of TB has been previously vaccinated with BCG at birth, what is sought with subunit candidates comprising specific *Mtb* antigens is to potentiate the pre-existing immunity induced by BCG [2,46,62]. Recent studies indicate that new TB vaccines that are compared to BCG should be interpreted cautiously with reference to a specific BCG formulation and not presumed to generalize to all BCGs [13]. In addition, testing the new concepts of vaccines in relevant animal models such as NHP, should be key before advancing into expensive clinical trials of efficacy [74,75]. Something that has been questioned in the MVA85A efficacy study [38] was that the efficacy experiments in NHP, which showed lack of efficacy by the tested clinical route and dose of administration [76,77]. We should be very careful not to repeat mistakes with new candidates. Recently, it was published in NHP, that boosting BCG with M72/ASO1E or H56/CAF01or rAd5 failed to enhance BCG-induced protection against TB [78]. Thus, care should be taken with the advanced clinical development of such candidates.

Healthy newborns represent the most sensitive population without pre-existing immunity to BCG or environmental mycobacteria, which in older groups can lead to possible effects of masking and blocking vaccination [79,80]. We, therefore, think that the efficacy should be studied in newborns as first step, and once the efficacy of a new vaccine has been established, then such new candidates should be tested for efficacy in adolescents and adults where the impact on TB will be greater given that the pulmonary forms are responsible for this transmission [80].

**Author Contributions:** Writing—review and editing, C.M., N.A., D.M. and J.G.-A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work has been funded by the Ministry of Science (RTI2018-097625-B-I00) and the European and Developing Countries Clinical Trials Partnership (EDCTP) RIA2016V-1637.

**Acknowledgments:** In this section, you can acknowledge any support given which is not covered by the author contribution or funding sections. This may include administrative and technical support, or donations in kind (e.g., materials used for experiments).

**Conflicts of Interest:** C.M., D.M., N.A. and J.G.-A. are inventors of patents related to the tuberculosis vaccine, the owner of which is the University of Zaragoza and of which the Spanish biotechnology company Biofabri is responsible for industrial and clinical development of MTBVAC.
