1. Introduction

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), that has been included in the top 10 leading causes of death in the world today and is the current leading cause of death among infectious diseases ^[1][2][1,2]. This devastating title came at the expense of 1.4 million lives in 2019 alone [3]. According to the World Health Organization (WHO), a quarter of the world population is infected with Mtb ^[3] [3] and most of these cases are related to poverty and located in low and middle-income countries ^[2][3][2,3].

Although there is a licensed vaccine against TB, the Mycobacterium bovis bacilli Calmette–Guérin (BCG) vaccine, it has several limitations, namely its high variability of efficacy in the population and low protection against pulmonary tuberculosis [4]. This explains the number of Mtb infections worldwide, despite the existence of a vaccine and its availability.

Treatment for TB normally consists of a long antibiotic regimen that decreases the patients’ compliance [5]. Nonetheless, the efficacy of this treatment is now compromised by the emergence of multidrug-resistant Mycobacterium tuberculosis bacteria against which there is only 50% chance of cure with the available drug treatment [6]. The WHO has set the goal to reduce TB morbidity by 90% and mortality by 95% by 2035 [6].

While drug research and diagnostics need to evolve in order to achieve control over Mtb infections, the discovery of a new and effective vaccine takes a central stage for its cost-effectiveness and ability to prevent or help with the treatment of TB, even of multidrug resistant strains ^[4][6][7][4,6,7]. By using a vaccine, it is possible to decrease both the transmission of the pathogen and the use of antibiotics to treat it, which means that the vaccine controls the disease and overcomes the antimicrobial resistance (AMR) [7].

Currently, in the TB vaccine pipeline, there are different types of vaccines, including live attenuated, inactive and subunit vaccine candidates ^[4][8][4,8]. Subunit vaccines are not the most advanced at the moment, since the current phase III clinical trials only comprise whole-cell vaccines, however they are promising candidates [9]. This type of vaccines overcomes some of the safety concerns associated with live attenuated or inactive vaccines [10]. Furthermore, as they are designed using specific antigens, they can be targeting different aspects of the infection, optimized as the research progresses and provide additional immune responses for more complex pathogens, such as Mtb ^[8][11][8,11].

While being very safe, subunit vaccines cannot rely solely on the antigen to achieve a desired effect as they are generally poorly immunogenic [10]. Adjuvants are needed in their formulation in order to increase immunogenicity, decrease the antigen dose, ensure a targeted delivery and optimize the antigens interaction with the immune cells ^[10][11][10,11]. Undeniably, adjuvants are an important part of new subunit vaccines design and in general, adjuvants identification, design and characterization is essential to modern vaccines formulation [12]. Additionally, understanding how adjuvants work plays a determinant role in vaccine development and can be the key factor to make or break the vaccine’s chances to succeed [12].

3. Understanding the Adjuvant’s Immune Role by Understanding TB Immunity

After inhalation of infected aerosols, the phagocytosis of the Mtb pathogen by the alveolar macrophages takes place. The block of the lysosome-phagosome fusion leads to the survival of the bacterium within the macrophages with activation of a cell-mediated immune response, such as CD4+ and CD8+ T lymphocytes [17]. From there, the immune response leads to a very important and characteristic cell-mediated consequence which is the formation of a granuloma [17]. This structure composed by macrophages, lymphocytes, stem cells and epithelial cells has the ability to control bacterial replication and induce a latent stage of the disease [17]. Understanding this immune response and the mediators involved is essential for the development of a vaccine and for choosing an appropriate adjuvant. The main problem with a TB vaccine is that the host immune responses towards Mtb are not yet fully understood and, as said before, there is no defined correlate of protection [18].

Most research lines in the field of TB vaccination focus on T-helper 1 (Th1) type of response, namely polyfunctional CD4+ cells expressing simultaneously IFN-γ, TNF-α, and IL-2 cytokines ^[13][19][20][13,19,20]. While it seems that investigators are moving towards the discovery and establishment of a correlate of protection, the findings are not clear. Looking at the pathogen’s behaviour to understand if these cells are indicative of protection, it is possible to see some ambiguous results since CD4+ cells are related to protection against disease and with immune responses to successful treatments but a high expression of these cells are also related to an increase in the bacterial load during active infection [19]. Moreover, most of the studies already concentrate in finding these cells and thus it is possible that there is some other immune correlate that is not being investigated [19].

Nevertheless, studies on mice and on patients have shown a clear importance of polyfunctional Th1 cells in TB protection and, thus, Th1 is still a desired response in new vaccine candidates ^[12][19][12,19]. In fact, as will be shown in this review, most of these new formulations show protective results and a clear Th1 response.

Th17 cells have also been described as important for immunity against Mtb and they are an immune marker used in some studies ^[12][17][12,17]. It has been previously described, in appropriate mice models, that IL-17 does not play an important role on the early control of the bacteria in the lung, upon infection with Mtb. However, Th17 cells are important for neutrophil recruitment and they are induced by the Mtb infection [20]. The same authors describe that IL-17-producing T-cells may be important for vaccine-mediated protection due to their probable ability to populate the lung and other tissues and, upon infection, start a signal that leads to bacterial load control [20].

In the selection of adjuvants, it is important to categorize the type of response that they are capable to promote, especially when combined with antigens specific for Mtb [18]. It is clear that CD4 T cells are crucial for protection as well as the production of IFN-γ and TNF-α although not enough ^[18][21][18,21]. While other immune mediators are not yet established as essential, their role in Mtb immunity is worth exploring and adjuvant’s associated response in new vaccine candidates is an excellent resource for new information regarding these immune responses.

4. Adjuvants in New TB Vaccine Candidates

This section aims to give insights on adjuvants that are being used in innovative TB vaccine candidates. The available data on these new formulations are reported and discussed. The immune response that they trigger will be explored by analysing the available data, which can potentially contribute to the discussion on vaccine formulation. A brief overview of the mechanism of action of the adjuvants described in this review is presented in Figure 1.

Figure 1. Overview of Proposed Mechanisms of Action of Adjuvants in vaccines. (A) Receptors and Molecule Activation of adjuvants. The active principle of GLA-SE and AS0, the MPLA, activates TLR4 and downstream pathways, namely MyD88 and, after internalization of the CD14/TLR4/MD-2 complex, the “late” TRAM-TRIF pathway. Starch interacts with C-type DC-SIGN receptor. IC31, more specifically, CpG motifs, as well as pDNA present in TMC microparticles activate TLR9 inside the endosome. AS01 and Chitosan activate the inflammasome and promote the subsequent release of pro-inflammatory cytokines. CDNs activate STING receptors in the endoplasmic reticulum. (B) IC31′s KLK peptide increases the permeability of the adjuvant system in the membrane to deliver the antigen and CpG motifs. (C) Cationic Liposome adjuvants, such as CAF01, promote a depot effect on the injection site and release the antigen in a controlled way, stimulating innate immunity.

Figure 1

Overview of Proposed Mechanisms of Action of Adjuvants in vaccines. (A) Receptors and Molecule Activation of adjuvants. The active principle of GLA-SE and AS0, the MPLA, activates TLR4 and downstream pathways, namely MyD88 and, after internalization of the CD14/TLR4/MD-2 complex, the “late” TRAM-TRIF pathway. Starch interacts with C-type DC-SIGN receptor. IC31, more specifically, CpG motifs, as well as pDNA present in TMC microparticles activate TLR9 inside the endosome. AS01 and Chitosan activate the inflammasome and promote the subsequent release of pro-inflammatory cytokines. CDNs activate STING receptors in the endoplasmic reticulum. (B) IC31′s KLK peptide increases the permeability of the adjuvant system in the membrane to deliver the antigen and CpG motifs. (C) Cationic Liposome adjuvants, such as CAF01, promote a depot effect on the injection site and release the antigen in a controlled way, stimulating innate immunity.