Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), an infectious disease that led to the death of 1.4 million individuals in 2019 (1). The current licensed vaccine against TB, a live attenuated strain of Mycobacterium bovis known as Bacillus Calmette-Guérin (BCG), is able to provide protection against disseminated forms of disease but is ineffective at providing protection against pulmonary TB in children and adults. Therefore, in order to lessen TB burden worldwide, a more efficacious vaccine and improved vaccine delivery strategies are urgently needed.

The development of more robust TB vaccines has unique requirements that have made progress challenging. Vaccines traditionally provide durable protection by prophylactically inducing neutralizing antibodies that can serve as a first line of defense against pathogens (2). Although numerous pathogens require antibodies for protection, this is less clear in the case of TB. While research on the role of antibodies in TB is ongoing and is of interest due to new data (3), no evidence has yet to suggest that neutralizing antibodies are required for protection against Mtb. Therefore, TB vaccine research has focused on identifying antigens and delivery strategies that maximize the generation of T cell responses. In particular, research has shown that CD4⁺ T cells are a critical component of protective immunity. Mouse studies, however, have demonstrated that T cells are not recruited to the lungs until weeks after infection is established (4). Mtb is able to suppress T cell recruitment and responses by utilizing several immune evasion mechanisms (IEM) to impede antigen-presenting cell (APC) function, thereby dampening adaptive responses. Researchers have attempted to bypass the delayed T cell response by targeting specific APCs such as dendritic cells (DCs), but there is limited evidence that this can preclude the immunosuppressive effects of Mtb upon challenge (5, 6).

Despite these challenges, recent developments have reinvigorated interest in TB vaccine research. A study in humans found that BCG revaccinated adults have increased protection compared to control groups (7). Moreover, while BCG is administered through the intradermal route, a recent study found that administering BCG intravenously can induce robust T cell responses and afford non-human primates (NHPs) protection against Mtb challenge (8, 9). Recent clinical trials of novel vaccine candidates have also yielded promising results. The Phase IIB clinical trial results of the M72/AS01_E subunit adjuvanted vaccine demonstrated 49.7% protection against Mtb (10). M72 is a recombinant fusion protein consisting of antigens Mtb32A and Mtb39A and AS01_E is an adjuvant that combines monophosphoryl lipid (MPL) with QS-21 (a purified saponin fraction). The AS01 adjuvant system success extends to other infectious disease vaccines as it is used in the FDA approved shingles vaccine “SHINGRIX” and is currently in clinical development for use in malaria (11). These successes set an important threshold for the clinical development of future TB vaccines.

To develop effective antigen-specific T cell responses, host immunity requires non-specific innate cell activation. In the case of a subunit vaccine, priming is accomplished with adjuvants. Modern adjuvants prime host immunity through binding of receptors that recognize pathogen-associated and damage-associated molecular patterns, such as toll like receptors (TLRs), C-type lectin receptors (CLRs), and NOD like receptor (NLRs) on the surface of APCs. Adjuvants are able to activate these receptors and induce downstream signaling pathways such as NFκB signaling, which in turn enables the activation of adaptive immune responses. Given the expense and time-consuming nature of vaccine development, adjuvant compounds and formulations that have been shown to be safe and effective are often repurposed for testing novel vaccine candidates. The majority of current TB vaccine candidates contain adjuvants ( Table 1 ). However, a TB-specific adjuvant that is able to induce strong immune responses in the lung but minimize corresponding tissue damage is required. Adjuvanted vaccines delivered directly to the upper or lower respiratory tract may have increased efficacy compared with parenterally administration against respiratory pathogens, such as Mtb. Therefore, development of new adjuvants with defined modes of action will be necessary in order to generate improved vaccination strategies that can provide protective immunity to TB.

To address the needs of TB vaccine development and lack of TB-targeted adjuvants, the National Institute of Allergy and Infectious Diseases (NIAID) held a workshop in July 2020 titled “Advancing Vaccine Adjuvants for Mycobacterium tuberculosis Therapeutics.” This workshop brought together vaccine and adjuvant developers from industry and academia and researchers in fields beyond TB. It is here that we identified research in four areas that will be essential for development of optimal adjuvants for TB vaccines: 1) correlates of protective immunity, 2) targeting specific immune cells, 3) immune evasion mechanisms, and 4) animal models. In this review article, we discuss these four areas in detail and highlight priority areas which the broader TB research community can address in order to develop efficacious adjuvants and vaccination strategies.

Active or passive targeting of specific immune cells through vaccination is an important step in the development of a safe and effective vaccine for TB. Mtb is primarily transmitted via inhalation and establishes infection in the lung through phagocytic uptake of the bacilli by tissue-resident macrophages. The initial recognition and immune activation by innate immune subsets sets the stage for either clearance or persistent containment within a granuloma or active disease through suppression of the immune system (89). Cells involved in lung-specific innate and adaptive immune responses are important frontline targets for Mtb vaccination approaches. The majority of Mtb vaccines are delivered via the intramuscular (such as M72/AS01_E) or intradermal (BCG) route, creating additional challenges in the recruitment of tissue resident memory cells to the site of initial infection. Targeting vaccines to specific cell types could help to overcome some of the shortfalls of current vaccine approaches while improving both safety and efficacy. Several groups have worked to overcome these challenges through targeting specific immune cells via passive (adjuvants, delivery systems) or active (mucosal vaccination, prime-pull, receptor targeting) immunization strategies with great success in pre-clinical and early clinical investigations. Coordination of the innate and adaptive immune response is important for resolution of Mtb infection and targeting of specific immune cells that orchestrate this response at the site of infection is critical in the development of an effective vaccine.

The context in which antigens are presented to the immune system controls the immunological outcome of antigenic exposure (90). The innate immune system uses pathogen recognition receptors (PRRs) to decode the nature of the antigen (e.g., viral, bacterial, fungal) and to translate this into an appropriate adaptive immune response. Central to the idea of cell-targeting is activation of the innate immune response by pathogen associated molecular patterns (PAMPs) and the specific cellular targets they encounter are crucial for early control of infection and for the subsequent development of protective long-term adaptive immunity. The use of passive and active cell targeting strategies opens the door towards the rational design of vaccines for Mtb that could lead to more durable and protective mediated immune responses.

An important reason for the poor efficacy of natural immunity to Mtb or BCG vaccine-induced immunity is immune evasion mechanisms that lead to ineffective crosstalk between innate and adaptive immunity (95, 132). By identifying critical Mtb factors that prevent optimal innate immune responses and elucidating the molecular basis for how host pathways are subverted by Mtb, we can engineer vaccines that target specific pathogen and host pathways to improve the immunogenicity and efficacy.

Mtb has evolved multiple strategies to evade innate immunity and impede T cell responses. By impairing DC functions (i.e., co-stimulation, cytokine production and antigen presentation) during early stages of infection in the lung, Mtb induces delayed and suboptimal antigen-specific T cell responses that fail to eradicate infection or provide lasting protection (4, 133–136). Vaccine strategies need to augment the induction of additional Mtb-specific T cell subsets, such as Th₁₇ cells, that will work in concert with Th₁ responses to enhance protective immunity (137). Mtb also inhibits macrophage microbicidal functions and dampens production of early proinflammatory cytokines and chemokines critical for shaping the nature and magnitude of T cell subsets that home to the site of infection (95, 138–140). Moreover, emerging evidence that myeloid-derived suppressor cells (MDSC) (141) and/or neutrophils (142) suppress T cell responses to Mtb infection suggests that targeting these cell types may improve vaccine-induced immunity.

Several Mtb genes have been implicated in evading DC and macrophage responses and, when deleted in BCG or Mtb, show enhanced vaccine-induced protection against TB in animal models. Thus, removing immune evasion genes shared by Mtb and BCG is an important approach for improving live attenuated vaccination strategies. The following studies are examples of applying knowledge gained from mechanistic studies of Mtb immune evasion genes and the host pathways that they modulate towards designing better vaccines for TB.

Inhibition of apoptosis as an immune evasion strategy is exemplified by the nuoG gene in Mtb, which encodes a subunit of NADH dehydrogenase and inhibits macrophage apoptosis (143, 144). Infection of mice with MtbΔnuoG led to an increase in apoptosis along with earlier activation of T cells compared to WT, suggesting that nuoG dampens the ability of innate cells to initiate T cell responses (145). Moreover, deleting nuoG in BCGΔureC::hly, a recombinant vaccine strain that is more protective than BCG in animal models, led to increased apoptosis following vaccination and significantly lowered bacterial burdens in Mtb-challenged mice (146). Deletion of Mtb sigH, which regulates multiple stress-induced proteins in Mtb, also led to increased apoptosis and chemokine responses in infected macrophages relative to wild type (147, 148). Mucosal vaccination with MtbΔsigH in NHPs resulted in increased survival and reduced lung pathology following challenge compared to BCG, with higher central and effector memory T cells in the lung (9).

Cell surface proteins are well positioned to modulate innate immune functions. The Mtb serine protease Hip1 is present in the cell wall of Mtb and dampens macrophage and DC functions via proteolytically cleaving its substrate, Mtb GroEL2 (138, 149). Hip1 inhibits TLR2 and inflammasome-dependent macrophage proinflammatory responses, impairs CD40-mediated costimulatory responses in DCs, and restricts Th₁₇ polarisation during infection (91, 136, 138). Both an Mtb hip1 mutant and BCGΔhip1 strain augmented CD40 expression on DCs, enhanced macrophage and DC functions and led to higher lung Th₁₇ responses (150). In a mucosal DC vaccination model, BCGΔhip1 induced immune responses that significantly reduced Mtb burden following challenge (150). These studies suggest that impeding CD40-CD40L interactions allows Mtb to induce suboptimal immunity, and that adjuvants that augment CD40 during vaccination are likely to improve efficacy. Another cell surface protein implicated in dampening innate immunity is LprG, a lipoprotein that binds to TLR2 on macrophages and has been implicated in inhibiting MHC class II antigen presentation and phagosome/lysosome fusion (151–153). Deleting lprG in BCG led to higher levels of pro-inflammatory cytokines, lower bacterial burdens and higher Th₁₇ responses following vaccination compared to BCG in murine models (154).

Many Mtb secreted proteins have also been implicated in evading host immune responses. SapM is secreted extracellularly via the SecA2 pathway and is involved in preventing phagosome maturation in macrophages (155, 156). Vaccination of mice with BCGΔsapM increased protection following challenge and led to increased activation and recruitment of DCs (157, 158). Together, these studies illustrate that deleting immune evasion genes in the context of live attenuated vaccine strains can significantly improve efficacy and suggest that sequential deletion of multiple immune evasion genes in a single vaccine strain is likely to lead to synergistic effects that improve vaccine efficacy.

Studies focused on innate immune pathways modulated by Mtb may also provide key insights into strategies for enhancing innate immunity during vaccination. Adjuvants that trigger and engage specific DC responses during Mtb infection may be of particular interest as a way to combat Mtb induced delays in DC activation and suppression of antigen presentation to CD4⁺ T cells. Approaches to enhancing Th₁₇ responses include adjuvants that engage CD40 on DCs (91) and MPL/chitosan formulations that induce Th₁₇ polarising cytokines in DCs (114). Ligands that stimulate TLR7 and TLR9 on DCs have also been shown to upregulate MHCII and reduce anti-inflammatory IL-10 responses following BCG vaccination (159). Other receptors to target include inflammasome components and other cytosolic recognition receptors known to play a role in protective immunity to Mtb infection (160). Developing adjuvants that target additional immune evasion pathways in DCs, such as autophagy, can enhance antigen presentation (93). Targeting costimulatory and coinhibitory molecules on DCs has the potential to be beneficial in improving the immunogenicity of candidate vaccine by fine-tuning the pro- and anti-inflammatory pathways necessary for optimal immunity (91, 161–163). Additionally, adjuvants that limit early induction of IL-10 and T-regulatory cell expansion may be effective given the role of these responses in dampening immune responses during infection (164–166). Finally, we need a deeper understanding of the suppressive functions of neutrophils and MDSCs in order to develop strategies that target these cell types using adjuvant-like approaches or by targeted depletion.

In summary, studies on Mtb immune evasion mechanisms have given us valuable insights, not only on how Mtb manipulates host immunity, but have also identified pathogen and host targets for designing live attenuated vaccine strains and novel vaccine adjuvants that enhance vaccine efficacy.

TB is a complex disease and no specific animal model perfectly mimics or recapitulates Mtb infection in humans. However, harnessing the strength of different animal models available will prove useful in developing and testing new adjuvants for TB.

NHPs have played a significant role in TB research and have been increasingly used for vaccine and adjuvant development. The strength of this NHP model lies in its ability to recapitulate disease similar to that in humans. NHPs can develop a latent version of Mtb infection, produce granulomatous lesions, and be used as a model to study HIV/TB co-infection. The use of primates has provided key insights such as the enhanced protective effect of intravenous BCG vaccination and the safety and efficacy of trial TB vaccines including M72/AS01_E and ID93 + GLA-SE (8, 167). Different routes of vaccination can be tested in NHPs, as the intravenous BCG study also included comparisons to intradermal, aerosol, and intradermal & aerosol routes of administration (8). NHPs have been used to test different vaccination strategies such as the prime-boost approach in which macaques are primed with BCG and then boosted with experimental vaccines and adjuvants 3-4 months after priming (168, 169). Additionally, NHP studies on protective vaccines can help inform immune correlates of protection, such as the study which observed protection using pulmonary-delivered BCG and the intravenous BCG study (8, 51). Given their similarity to humans, the NHP model is best equipped to study the priority areas outlined in this review. However, due to the prohibitive costs and challenges of working with primates (along with limited number of animals available for research), the use of small animal models should continue to be used to perform initial pre-clinical studies of potential adjuvants.

Historically, small animal models (mice and guinea pigs) have been used by developers to test adjuvant suitability for inducing protective immunity under rigorous in vivo conditions. The mouse model represents an excellent screen for first time in vivo assessment of a test vaccine. Using mice affords greater feasibility in testing of different variables such as the effect of different doses, alternative routes of inoculation and different inoculation times post-vaccination in a relatively short time frame. Additionally, the ability to use knockout and transgenic mice provides an additional level of interrogation for vaccine formulations and a better understanding of mechanisms involved in inducing protective immune responses (37). For instance, a recent analysis of the effect of intranasal vaccination using ODN as a molecular adjuvant showed that immunity could be achieved in a type I interferon-dependent manner (170). Another study demonstrated that intranasal vaccination was the best route for inducing protective immunity compared to intraperitoneal, subcutaneous, or intragastric routes (170). The cost of mouse models also allows for long-term immune profiling in vaccination studies. Extended timepoints have been previously been used to identify improved protective efficacy of subunit vaccine candidates compared to BCG (171). New approaches are also being used that allow mouse models to better mimic the heterogenous immune responses observed in humans. The diversity outbred (DO) mice, where initial breeding is done with inbred and wild-caught strains, have been developed with a level of genetic diversity similar to humans and NHPs. In a BCG vaccine study with DO mice, a diverse response was observed in which some vaccinated mice were protected and others were highly susceptible to Mtb challenge (172). Another attempt to improve the mouse model is the recent development of the ultra-low dose model. Infecting with 1-2 CFU of Mtb via aerosol, instead of the conventional ~100 CFU dose, causes mice to produce a heterogenous immune response and granulomatous structure in the lung similar to humans (173). The use of these and other mouse models will therefore continue to be important for testing novel adjuvants and vaccination strategies against TB.

Guinea pigs serve as an additional model and is traditionally employed after a vaccine formulation has demonstrated success in mice. Guinea pigs were originally used in TB research to understand disease pathogenesis due to their susceptibility to Mtb infection as well as their use in distinguishing “mammalian” bacilli from “avian type” bacilli due to their resistance to the latter (174). Subsequent studies by Smith and colleagues (175, 176) demonstrated that aerosol infection of guinea pigs with virulent Mtb resulted in pulmonary pathology similar to that observed in humans as well as a lethal course of disease (177). This model therefore allow researchers to test the ability of a vaccine formulation to limit disease, reduce Mtb burden, and prolong survival. The success of the guinea pig to identify vaccine candidates that have the potential to succeed in humans was demonstrated by a study which found the M72 vaccine candidate was able to boost BCG responses in the model (178). The long-term guinea pig model was used until recently to determine the efficacy of a vaccine as a low dose aerosol infection with virulent Mtb able to induce progressive pulmonary and extra-pulmonary disease similar to acute TB in humans (179). Vaccination with BCG resulted in the significant reduction of pathology and prolonged survival in guinea pigs, therefore validating the use of this model to test vaccines that can be efficacious in humans. Guinea pigs can therefore provide valuable insight into the ability of different formulations to significantly limit disease progression.

Together, the use of both mouse and guinea pig models of TB have contributed to a better understanding of adjuvant mechanisms in vaccines. The NIH/NIAID TB Vaccine testing program (180) has used both the C57BL/6 short-term mouse model and the Hartley guinea pig as a short- and long-term model to test vaccines and novel adjuvants. Both models have similar experimental strategies; animals are vaccinated with the test vaccine formulation, rested, and then infection is established by aerosol challenge with a low dose of Mtb (approximately 100 CFU for the mouse and 10 CFU for the guinea pig). The transition of testing adjuvants from the mouse to the guinea pig model has resulted in down selection of candidate vaccines under the NIH testing contracts and few have been able to reach the clinical testing stage. Of note has been the M72/AS01_E vaccine, which demonstrated activity in the mouse and guinea pig models (178). Using both models also reconcile differences observed experimentally. Recent investigations with ODN adjuvants have highlighted these discrepancies; when the same formulation and inoculation route were used in both models, the adjuvant proved less effective in guinea pigs. These results have been observed with other adjuvants and emphasize the need to test vaccines in both the mouse and guinea pig models While adjuvanted subunit vaccines have in general been less effective at limiting disease in the guinea pig model, it is not clear whether this is a limitation of the model or whether guinea pigs are selecting out formulations that may perform poorly in clinical trials. Unfortunately, there are only a small number of adjuvanted vaccines currently in clinical trials, some of which have been successfully tested in the guinea pig model (181, 182). An increased understanding of immune mechanisms in the guinea pig would be useful to determine if adjuvants were providing the appropriate signals to generate protective immunity. With the limited number of immunological tools available for the guinea pig to perform in-depth analyses (compared to mice), it is difficult to determine why such differences occur. Developing novel reagents for the guinea pig is an area that must be supported to achieve a better understanding of immunological responses to infection and vaccination. Additionally, a systematic analysis of cell phenotypes, expression of PRRs and other innate immune factors will be required to determine why adjuvants have provided limited protective immunity in the guinea pig model. This will help strengthen the use of this model for use in adjuvant studies for TB.

In summary, while NHP models more closely mimic human disease, small animal models provide feasability in testing vaccine adjuvants in a pre-clinical setting. With the rapid increase in the development of new molecular adjuvants, the mouse and guinea pig models provide the capacity to test them rigorously in an infectious disease setting. Harnessing the strength of all three of these animal models will therefore be crucial for developing new and improved adjuvants for TB vaccines.

Recent advances in vaccine development, including an adjuvanted TB vaccine providing nearly 50% protection and observations on improving BCG, have reinvigorated the field. As our understanding of the complex interplay between Mtb and the host immune system is improved, new strategies for adjuvant development may be employed to further boost efficacy and develop a protective vaccine against TB.

In this review, we have highlighted four areas of importance for researchers in the TB field to address in order to generate more efficacious adjuvants and vaccination strategies. Defining correlates of protection is necessary to dictate which pathways should be targeted in order to induce protective immune responses. Similarly, learning how to target cells that are responsible for inducing those protective immune responses serves as a direct access to immune responses that are often dampened during infection. This is critical as Mtb is able to encode immune evasion proteins that serve to actively impede key immune cell subsets. Further characterizing these proteins give us direct insight into pathways that can be activated to overcome pathogen inhibition. Additionally, having appropriate animal models is necessary to test whether adjuvants and vaccines can be beneficial to humans while at the same time serving as a tool to further study infection and disease. While research into these topics can contribute directly to the generation of new adjuvants and vaccines, current adjuvants and vaccines are instructive in determining the requirements for protective immunity to TB. While intravenous delivery of BCG in humans is controversial, the study in NHPs (8) served as a model for identifying correlates of protection that can be targeted through safer, more practical means. Similarly, the M72/AS01_E vaccine study (10) demonstrated the benefit of adjuvants in TB vaccine strategies and contributed potential insights into mechanisms of protection. Cross-talk between basic TB science research and TB translational research should be encouraged as each of these inform the other. Additionally, insights from within the field are able to inform other fields that require generation of similar immune responses for their own models of protection. Therefore, advancing research in these four areas identified at the workshop has the potential to improve immunology in general while working to reduce TB burden worldwide.

ABE, AI, SMM, ELS, RNM, DJF, JTE, JR and JAT developed, wrote, and edited sections of the review as well as edited the entire manuscript. ABE provided significant revisions to the manuscript. All authors contributed to the article and approved the submitted version.

The workshop and publication have been supported by the Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under the contract HHSN272201600001G.

Author RNM was employed by company Columbus Technologies & Services Inc. JE and SM are employees of and own shares in Inimmune Corporation.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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