title: Immunity to Babesiosis and Discovery of Next Generation Vaccines
Creators: name: Al-Nazal, Hanan A
Publisher: name: Griffith University
Year: 2020
Subjects: subject: Babesia
subject: babesiosis disease
subject: Babesia microti
subject: parasite vaccine

description: The genus Babesia comprises intraerythrocytic protozoa that cause babesiosis disease with flu-like symptoms in young immunocompetent individuals that can develop into anaemia, organ failure, neurological complications, and even death in older or immunocompromised patients. Babesia spp. are known to infect both humans and a wide range of wild and domestic animals. More than 100 species of Babesia have been identified but only a few species are known to cause disease in humans. Babesiosis causes huge economic losses in the cattle industry, with 1.2 billion cattle at risk of Babesia infection worldwide. In Australia alone, this disease costs the cattle industry millions of dollars, not just as a result of the mortality and abortion in infected cattle, but also due to the costs of control measures such as acaricidal baths and drug administration. Current control measures for bovine babesiosis include chemotherapeutics, acaricides, and live vaccines, which have inherent limitations and are non-sustainable, while no human vaccine is currently available to protect against human babesiosis. Effective, safe and sustainable vaccines are needed. In this thesis, novel approaches were taken to develop whole Babesia microti (King strain) parasite vaccines. In Chapter 3 the B. microti parasitised red blood cell (pRBCs) dose was characterised in a rodent model using two strains of mice (BALB/c and C56BL/6) and the dose of B. microti pRBCs was optimised for two different inoculation routes. We noted that BALB/c mice suffered from recrudescence, while C56BL/6 cleared parasites and no recrudescent parasitemia peak was detected. The findings in Chapter 3 were used in whole blood-stage parasite vaccine studies in the subsequent chapters. We subsequently examined whether a chemically attenuated B. microti pRBCs vaccine, where the parasite is treated in vitro with the chemical agent, tafuramycin-A, that previously induced protective immunity in pre-clinical vaccine studies of the related malaria parasite, could protect the vaccinee. We showed that mice intravenously administered B. microti pRBCs that had been treated with 2μM tafuramycin-A in vitro, were protected against homologous challenge. We then immunised mice with a threedose regimen of a chemically attenuated whole blood stage B. microti vaccine and showed protection with prolonged immunological memory at 9 months after the last immunisation. Protection was shown to be CD4+ T-cell-dependent. Analysis of the protective efficacy is described in Chapter 4. A live attenuated B. microti vaccine has limitations associated with storage, and delivery considerations. An alternative strategy is a liposome-based antigen delivery platform, whereby liposomes are used to deliver antigens to target cells. In Chapter 5, we produced freshly prepared mannosylated/ unmannosylated liposomes containing parasite extract of B. microti pRBCs using the thin-film hydration method as described in Chapter 2 and examined in a three-dose regimen their protective efficacy against homologous challenge with B. microti pRBCs compared with the chemically attenuated B. microti pRBCs vaccine in a rodent model. We then examined freshly prepared mannosylated liposomes containing parasite extract of B. divergens pRBCs, and studied induced immune responses and protective efficacy against heterologous challenge. Furthermore, we prepared lyophilised mannoyslated liposomes containing B. microti as described in Chapter 2 and examined their protective efficacy and induced immune responses to those freshly prepared containing B. microti against homologous challenge in order to address some of the difficulties associated with the storage and delivery of current whole blood-stage parasite vaccine approaches. We then compared in the protective efficacy and immune responses between lyophilised mannoyslated liposomes containing B. microti or B. divergens pRBCs against homologous or heterologous challenge. Stability of lyophilised vaccine stored at 4°C for a month prior to initiation of the immunisation regimen was evaluated and found to protect against homologous challenge. Lyophilised mannosylated liposomes containing B. microti pRBCs induced immunity with immunological memory at 3 months after the last immunisation. This protection was again determined to be CD4+ T-cell-dependent. Analysis of the protective efficacy is described in Chapter 5. We present a preclinical study employing a novel strategy of live whole organism (chemical attenuated B. microti vaccine) and non-live mannosylated liposomal vaccines to combat babesiosis. The data obtained further our understanding of the immune responses elicited following immunisation with both chemically attenuated B. microti and mannosylated liposome vaccines containing whole parasite antigens. Vaccination with freshly prepared mannosylated liposomes and lyophilised vaccines containing B. divergens antigens induces cross-species protection against B. microti, and as such this strategy could be employed for producing a vaccine from cultured B. divergens parasites. Lyophilised mannosylated liposomes provide a feasible strategy towards practical vaccine storage for use in the field. These data provide strong grounds towards further investigation of an effective babesial vaccine in a clinical setting.