title: Novel Approaches to Whole Parasite Blood-Stage Malaria Vaccine Development
Creators: name: Ssemaganda, Aloysious
Publisher: name: Griffith University
Year: 2018
Subjects: subject: Malaria vaccine
subject: Whole parasite
subject: Gametocyte densities
subject: Blood-stage vaccine
subject: Gold phosphines

description: The development of blood-stage vaccines capable of reducing severe disease remains a critical component of malaria eradication efforts. Such a vaccine will hypothetically reduce gametocyte densities which will in turn reduce transmission. An effective blood-stage vaccine will also bridge the gap between partially effective pre-erythrocytic vaccines such as RTS, S and control measures such as anti-malarial drugs and insecticide-treated bed nets that are threatened by resistance as well as inappropriate use. Herein, novel approaches to the development of whole parasite blood-stage malaria vaccines were examined. Firstly, clinical isolates of P. falciparum were successfully collected from volunteers with malaria living in Uganda. These clinical isolates are valuable reagents for preliminary evaluation of malaria vaccine candidate prior to deployment for further testing in large-scale clinical trials. The cryopreserved isolates were successfully revived in vitro, and culture-adapted. Preliminary anti-malarial drug susceptibility characterisation revealed that these parasites were sensitive to chloroquine and Riamet as well as gold(I) phosphine compounds auranofin and [Au(d2pype)2] Cl. However, further characterisation on a larger panel of established anti-malarial drugs will be required especially if these isolates are to be released for use in CHMI studies. Secondly, the utility of gold(I) phosphines as potential parasite attenuating agents for blood-stage malaria vaccine and drug development was examined. It was shown that gold(I) phosphines inhibited parasite growth following short-term in vitro culture. Furthermore, when mice were inoculated with completely attenuated parasites, no protection was observed following challenge with a wild-type malaria infection. However, protective immunity was attained in some experiments when mice were inoculated with partially attenuated parasites. Future studies will therefore be required to optimise the reproducibility of achieving partial attenuation in order to further develop this approach for an effective blood-stage malaria vaccine. In vivo, gold(I) phosphines were unable to inhibit parasite growth indicating that pharmacokinetic studies will be required to examine bioavailability following administration. Furthermore, these compounds need to be better designed to more efficiently target and selectively inhibit the parasite in vivo. The ultimate test of an effective malaria vaccine is the ability to protect against infection from multiple parasite species/strains. Therefore, a heterologous model was developed to assess the immunogenicity and protective efficacy of liposomes formulated with a mannose lipid core peptide, F3, (also referred to as mannosylated hereonin) containing human parasite, P. falciparum, antigens in a mouse model. To address one of the challenges to the development of whole parasite blood-stage vaccines, the induction of anti-red blood cell antibodies, it was shown that using a novel immunomagnetic method, red cell membranes were efficiently depleted from parasite antigen. When mice were immunised with liposomes containing red cell membrane-depleted parasite antigen, induction of antibodies against the surface of the red blood cell was significantly reduced compared with control mice that received injections of intact normal red blood cells. Furthermore, it was shown that depletion of red cell membranes from parasite antigen did not affect the immunogenicity of liposome formulations. These data strongly support the use of this method to minimize the induction of anti-red blood cell antibodies in vaccinees. To address storage and cold-chain maintenance limitations of vaccine products especially in malaria endemic regions, liposomes were lyophilized. Immunological assessments showed that lyophilization did not affect the induction of humoral immune responses but altered the cytokine response to a predominantly Th1 response compared to a balanced Th1/Th2 response observed following immunisation with freshly prepared liposomes. Therefore, optimisation of freeze-drying protocols will be required in order to achieve optimal cytokine responses consistently following immunisation with lyophilized liposomes. Vaccine efficacy studies revealed that freshly prepared mannosylated liposomes containing P. falciparum antigens, in some experiments, induced strong species-transcending protective immunity following challenge, with no direct correlate of immune protection. Future studies will thus be required to optimise formulations to attain reproducible protective efficacy. A whole parasite multi-stage liposome-based vaccine was then formulated with different combinations of F3, PHAD (a synthetic analogue of monophosphoryl Lipid A (MPLA), the circumsporozoite repeat peptide derived from the rodent parasite, P. yoelii (QGPGAP)4 and blood-stage Py17x antigens. Immunisation of mice resulted in low (QGPGAP)4-specific antibody titres and potent parasite-specific antibody responses which were associated with liposomes containing PHAD. Additionally, the parasite–specific antibody responses strongly correlated with protection against a homologous wild-type challenge infection. Collectively, the data presented in this thesis lays the foundation for future studies on the use of gold(I) phosphines as parasite attenuating agents as well as anti-malarial drugs. Additionally, these studies advance our understanding of the immune responses elicited following immunisation with liposomes containing inactivated whole parasite antigens and inform the design of future vaccine formulations suitable for human use. Importantly, the data strongly support transition of whole parasite liposome-based vaccines to human trials.