Developing vaccines to prevent mycobacterial infections

© 2020 Kirstie Maree MangasTuberculosis (TB) is a leading cause of disease by an infectious agent. It is the most common mycobacterial disease of humans, followed by leprosy and Buruli ulcer. TB is caused by infection with Mycobacterium tuberculosis (MTB). TB affects people in every part of the world, predominantly throughout Asia (especially India and China) and Africa. Roughly one-quarter of the world’s population is latently infected with MTB. Asymptomatically infected people carry an approximate 10% risk of developing active disease. In 2018, there were an estimated 10 million new cases of TB and 1.45 million associated deaths. TB is treatable however it requires six months of combination antibiotic therapy. Buruli ulcer (BU), is a neglected tropical disease and has been reported in more than 30 countries world-wide but the dominant endemic foci of this disease occur in rural regions of West Africa. In the past five years BU cases have increased dramatically in South-East Australia, near Melbourne. BU is caused by infection of subcutaneous tissue with Mycobacterium ulcerans, typically presenting as deep and extensively ulcerated skin lesions. MTB and M. ulcerans are closely related mycobacterial species, therefore a vaccine against one of these bacteria might induce cross-protection against the other. The Mycobacterium bovis ‘bacille Calmette-Guerin’ (BCG) vaccine, a live-attenuated whole cell vaccine against MTB, is widely used. It is most effective in children below two years of age and against disseminated TB but efficacy wanes after about 10-15 years. In adults, BCG is between 0-80% effective. There is no effective vaccine against any mycobacterial disease and immune correlates of protection for mycobacterial vaccines are not well defined. This thesis sought to address these knowledge gaps and explored the development of different vaccines to protect against TB and BU. The vaccines were tested in murine infection models and the types of immune responses induced by each vaccine were measured. Where a vaccine was able to elicit robust immune responses, the animals were then challenged with the mycobacterial pathogen to assess protective efficacy. The first chapter is an introduction to this thesis and includes a literature review of TB and BU, their respective causative agents, immune responses to infection, and recent vaccine developments. This chapter introduces the key concepts and motivations for this thesis. The second chapter describes the development of a protein-based vaccine against TB. The vaccines utilised MTB-specific proteins ESAT-6 and Ag85B in conjunction with lipopeptide adjuvant R4Pam2Cys in C57BL/6 mice. The vaccines were not capable of generating measurable interferon (IFN)-gamma responses from CD4+ T cells recovered from the spleen or from the lungs, which have been shown to be crucial to the control of TB. The vaccines were however able to induce high protein-specific antibody titres against Ag85B. These vaccines were then modified with M. ulcerans-specific proteins to try and develop a vaccine against M. ulcerans. The third chapter focusses on the development of two protein-based vaccines against two highly expressed M. ulcerans cell wall-associated proteins, MUL_3720 and Hsp18. These proteins were bound to lipopeptide adjuvant R4Pam2Cys and their ability to generate a strong antibody response was measured in BALB/c and C57BL/6 mice. M. ulcerans is predominantly an extracellular pathogen and a strong antibody response against M. ulcerans could play a role in prevention of infection. Both MUL_3720 and Hsp18 in conjunction with R4Pam2Cys were capable of generating strong protein-specific antibody responses in both mouse strains. These antibody responses remained augmented after subcutaneous challenge with M. ulcerans on the mouse tail, however strong antibody responses did not correlate to protection. All vaccinated mice succumbed to infection 40 days after M. ulcerans infection. This suggests that these proteins were not suitable vaccine candidates. There was also no difference in protection between vaccinated mice and mice vaccinated with BCG. The BCG vaccine is not wholly protective against M. ulcerans but previous studies have shown that the vaccine delays the onset of disease. The lack of difference in this study may be due to the high bacterial challenge dose and suggested the need for a different animal model of infection. The fourth chapter describes the development of a vaccine targeting the mycolactone biosynthesis pathway. Mycolactone is a polyketide toxin and is the main virulence factor of M. ulcerans. Mycolactone affects the host immune response, causing immune cells to display modulated or decreased cell function which enables bacteria to evade immune responses. Prior to the creation of a new vaccine formulation, a new murine model of infection was established to reflect a more realistic, lower pathogen challenge dose. In this new murine model, mouse tails were coated in engineered bioluminescent M. ulcerans and the contaminated skin was subcutaneously pierced with a sterile needle to replicate trauma-induced introduction of bacteria into the subcutaneous tissue. The bioluminescent bacteria enabled the visualisation and quantification of bacterial load over time using an in vivo imaging system (IVIS). Once a new murine challenge model was established, this chapter assessed the efficacy of a new vaccine formulation comprising a protein domain, enoyl reductase (ER). The ER functional domain is required for the biosynthesis of mycolactone. Recombinant ER protein was coupled to R4Pam2Cys and BALB/c mice were vaccinated and boosted. This vaccine provided comparable protection against BU compared to the BCG vaccine. Additionally, this vaccine was statistically more protective than no vaccination. Analysis of systemic cytokine responses suggest that control of disease correlates to the level of inflammatory cytokines found in the spleen compared to the draining lymph node (site of infection). The immune responses correlating to protection from a BCG vaccine differed to the responses generated by ER+R4Pam2Cys. This study indicates that protection against BU may be achievable by different immune responses. This study also suggests that the highly conserved mycolactone biosynthesis pathway may be an effective target for a vaccine. However, understanding the immune correlates of protection requires much further study. In conclusion, this thesis demonstrated that MTB proteins in conjunction with the chosen adjuvant (R4Pam2Cys) do not elicit immune responses, in particular IFN-gamma responses, that are typically required to protect against TB in a murine model. M. ulcerans proteins, Hsp18 and MUL_3720 also using the R4Pam2Cys adjuvant, did not induce protection against BU in a murine challenge model. However, vaccine-induced protection was observed by incorporating the M. ulcerans mycolactone ER functional domain with R4Pam2Cys and a murine model more reflective of a natural M. ulcerans infectious dose. These experiments highlighted the potential for an effective vaccine that targets the mycolactone biosynthesis pathway. This work also demonstrated that protection against M. ulcerans might be achieved via different combinations of immune responses. An effective vaccine against M. ulcerans will likely have useful lessons for developing vaccines protective against MTB (and vice-versa), whether through cross protection or by using vaccines as tools to probe and measure the host immune responses required for control or protection against infection