Microbiology & Immunology - Theses
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ItemInterventions for the control of Zika virus transmission: Wolbachia and a single-dose Zika vaccine( 2021)ABSTRACT The versatility of flaviviruses such as Zika virus (ZIKV), dengue virus (DENV) and West Nile virus (WNV) that are capable of replicating in both invertebrate and vertebrate hosts, opens opportunities in the design of interventions aimed at their control. Control at the human level can arise from the generation of vaccines and antivirals, while control at the vector level can be achieved through the regulation and manipulation of mosquito populations. The uncontrollable spread of ZIKV in the Americas during 2015 to 2017 lead the WHO to declare it as a global public health emergency for most of 2016. Now, with a very reduced, but sustained number of cases every year in the Americas, ZIKV is still a treat that can re-emerge in other parts of the world. Currently, there is no licensed vaccines or antivirals against ZIKV that can prepare us for future outbreaks, for which it is crucial to generate and validate them now. Furthermore, since the Zika pandemic, a deeper understanding of viral infections in the context of pregnancy became an obligatory area in the study of all emerging viruses. On the other hand, the success of Wolbachia and specifically the wMel strain as a biological agent to control the spread of dengue virus from mosquito populations to humans, has expanded the options for vector control. Nonetheless, as an endosymbiont highly resistant to genetic manipulation, the identification and characterization of alternative Wolbachia strains is vital due to the unlikely, but always potential, emergence of arboviruses resistant to wMel. In a translational approach and to address some these important questions in the study of control measures against ZIKV, in this doctoral thesis we: a) generated a ZIKV virus-like particle (VLP) based vaccine candidate and assessed the immunogenicity of these particles in mice. To generate our vaccine candidate, we utilised the Adenovirus-vector system, and standardized a protocol for the generation and purification of highly immunogenic and antigenic VLPs. The ZIKV-VLPs were highly morphologically similar to ZIKV by electron microscopy and were antigenic against known anti-Flavivirus neutralizing antibodies. We observed that ZIKV-VLPs alone and inactivated ZIKV in a single dose generated an immune response that lasted over 6 months, but it did not neutralize the infection of cells virus in vitro. Thus, the ZIKV-VLPs needed to be in combination with an adjuvant to generate a neutralizing immune response. From the three adjuvants that we used, Aluminum oxyhydroxide (Alhydrogel) was found as the most effective in a single dose regime, since it not only neutralized the virus, but also it generated a greater number of specific B-memory cells. We additionally observed that the generation of the neutralizing antibodies persisted for up to 6 months, induced a strong B-cell population and importantly generated subpopulations of immunoglobulins that are less related to antibody-dependent enhancement and disease severity. Our results indicate that the produced ZIKV VLPs are robust for use as a single-dose approach as a vaccine candidate; b) identified potential next generation Wolbachia strains by screening a panel of Drosophila simulans flies trans-infected with 12 different Wolbachia strains. We found that 75% of these strains (8 out of 12) significantly reduced ZIKV RNA levels and exhibited a significant negative correlation between Wolbachia levels and virus replication. This D. simulans model system can support further research in the selection of new Wolbachia strains as a pre-screening method prior to the artificial transfer into Aedes mosquitoes aimed at preventing the transmission of arboviruses; and in a more basic fashion c) evaluated the replication kinetics of ZIKV and the related encephalitic flavivirus West Nile strain Kunjin virus (WNVKUN) in early-term placental cell lines. We have observed that WNVKUN in fact replicates with a greater rate and to higher titres that ZIKV in these cell lines. These results would indicate the potential for all flaviviruses to replicate in placental tissue, but it is the ability to cross the placenta itself that is the restrictive factor in the clinical progression and presentation of congenital Zika syndrome. This additionally, provided some insights into the particular ability of ZIKV in causing microcephaly when maternal infection happens during the first trimester of pregnancy. Overall, the findings presented in this doctoral thesis have provided important insights into infection of ZIKV in the placenta and into the development of preventative strategies to protect the human population from future ZIKV outbreaks.
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ItemUnderstanding human B cell and antibody responses against seasonal influenza viruses( 2020)Vaccination is the best available means to reduce the burden of seasonal influenza. However, current influenza vaccines need to be updated frequently to keep up with evolution among circulating viruses. Antigenic evolution, otherwise termed drift, is most rapid among A/H3N2 viruses, and the A/H3N2 component of vaccines is frequently updated. Despite this, influenza vaccine effectiveness against the A/H3N2 subtype has been poor in recent years, especially among previously vaccinated individuals. Protection induced by inactivated influenza vaccines is largely mediated by B cells and antibodies reactive against the head of the hemagglutinin (HA) protein, with help from T follicular helper cells. The cellular and molecular mechanisms that underlie the attenuating effects of prior vaccination and existing immunity are largely undefined. It has been suggested that existing antibodies clear or mask antigen, or that memory B cells induced by prior exposures competitively dominate responses so that B cells and antibodies become focused on epitopes that are shared between prior and prevailing vaccine strains. The aim of the work presented in this PhD thesis was to examine the impact of pre-existing immune responses induced by prior infection with different A/H3N2 strains on influenza vaccine immunogenicity. In depth antibody as well as B cell assessments were performed to understand the impact of existing antibodies and memory B cells following vaccination and provide insights into the design of new vaccine strategies. As a lead up to the ex vivo analysis of B cells from vaccinees, we first sought to understand how human naive versus memory B cells differentiate in vitro. Experiments were conducted in Chapter 3 to compare the stimuli required for their differentiation into plasmablasts, and subsequently understand how they change phenotypically once stimulated. Specifically, sorted human naive and memory B cells from healthy individuals were stimulated in vitro to induce differentiation into plasmablasts. Data obtained in this PhD thesis showed that stimulation with the Toll-like receptor (TLR) 7/8 agonist R848 in the presence of monocytes induced the highest activation of both naive and memory B cells. Conversely, stimulation with the TLR9 agonist CpG or with R848 in the absence of monocytes induced little to no differentiation of naive B cells but were able to stimulate memory B. cell differentiation. Despite robust differentiation into antibody secreting plasmablasts, naive-derived B cells remained phenotypically distinct from memory-derived B cells up to day 6 after in vitro activation, with differential expression of CD27, CD38 and CD20. This work resulted in a first-author publication in Clin Transl Immunol, 2019. The focus of Chapters 4 and 5 was to understand how prior influenza virus infection affects antibody and B cell responses to influenza vaccination. To address this question, vaccine responses were investigated in a unique influenza vaccine-naive cohort in Viet Nam, that had been monitored for both clinical and asymptomatic influenza virus infection for more than 9 years. In 2016, twenty-eight participants without documented A/H3N2 virus infection (since 2007) and 72 participants who had been infected with A/H3N2 viruses, belonging to a range of genetic clades, received an inactivated trivalent influenza vaccine containing an A/Hong Kong/4801/2014-like (H3N2) antigen. This work investigated whether influenza vaccination induced naive B cell responses specific for new epitopes or largely recalled B cells specific for conserved epitopes, common to the vaccine A/H3N2 component and prior infecting strains. Hemagglutination inhibition antibody titres were measured in pre- and serial post-vaccination sera against 40 A/H3N2 viruses spanning 1968-2018 to understand how the titre and cross-reactivity of antibodies against the HA head evolve. B cells were assessed by flow cytometry using a panel of phenotypic markers in addition to recombinant HA probes representing the vaccine and recently infecting strains (A/Perth/16/2009, A/Victoria/361/2011 and A/Switzerland/9715293/2013). Participants who had at least one pre-vaccination A/H3N2 virus infection had on average 2 to 3-fold higher vaccine-specific antibody titres, steeper titre rises in the weeks following vaccination (mean peak on day 14), and less titre decay by days 21 and 280 compared to participants without prior infection. Moreover, participants with prior infection exhibited greater and better-maintained titre rises against viruses that circulated a year after vaccination, indicating that prior infection extends the strain coverage of antibodies induced by vaccination. Notably, A/H3N2 viruses that circulated 275-340 days after vaccination caused illness in only 1.4% of participants with infection prior to vaccination and in 14% of participants without prior infection. This suggests that vaccine effectiveness can be enhanced by pre-existing immunity. However, it was also clear that the range of strains against which antibodies were induced was dictated by the strain with which participants were previously infected, indicating that vaccination may simply recall rather than update antibody-mediated immunity. HA-probe reactive B cell frequencies and activation status increased substantially after vaccination. The greatest increases in HA probe-reactive B cells were detected among participants who had recent prior infection, with the majority of B cells exhibiting cross-reactivity with prior strains. A modest but significant increase in the frequency of B cells that reacted with the HA of the vaccine strain, but not of past strains, could be detected in participants who lacked prior infection. The phenotype of vaccine HA single-positive B cells, including increased IgM expression, indicated that they may have been naive-derived B cells. Vaccination induced B cells that preferentially reacted with the HA of A/Perth/16/2009 and/or A/Victoria/361/2011 viruses, but not A/Switzerland/9715293/2013 viruses, among participants who had prior A/Perth/16/2009-like virus infection. However, B cells induced by vaccination in participants who had prior A/Switzerland/9715293/2013-like virus infection were equally cross-reactive with HA of all tested viruses. These results support the inference that immune responses to standard inactivated influenza vaccines are dominated and shaped by recalled memory B cells with limited activation of naive B cells to update immunity. Overall, this PhD thesis investigated how pre-existing immunity induced by documented influenza virus infection affected the humoral response to seasonal influenza vaccines in healthy adults. This work provides new insights into the capacity of influenza vaccines to stimulate naive B cells, which may be limited due to memory B cell dominance and to a lack of sufficient stimulation to activate naive B cells. This knowledge could be used to design new vaccine strategies and improve influenza vaccine-induced protection.
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ItemDeveloping vaccines to prevent mycobacterial infections( 2020)Tuberculosis (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.