title: Novel Use of Delayed Death Agents in the Development of a Chemically Attenuated Whole Malaria Vaccine
Creators: name: Low, Leanne
Publisher: name: Griffith University
Year: 2018
Subjects: subject: Delayed death agents
subject: Malaria vaccine
subject: Apicoplast
subject: Plasmodium falciparum

description: Malaria vaccine research has generally focused upon single parasite derived proteins for the development of a sub-unit vaccine. However, many of these proteins are highly variable and are unable to elicit protective responses to multiple strains. A vaccine approach utilizing the whole parasite is advantageous as it contains multiple parasite antigens, including those that are conserved between parasite strains. The discovery of the apicoplast, a structure found in all malaria parasites, offers an alternative means to current vaccine approaches for crippling the malaria parasite. Studies have demonstrated that specific targeting of critical metabolic pathways harboured by the apicoplast causes parasite death. Interestingly, some antibiotics have been observed to cause a ‘delayed death’ phenomenon, in which the effects of treatment are observed in the progeny rather than the treated parasite itself. Given the importance of the apicoplast to the malaria parasite and the ability to pharmacologically target it, we investigated the possibility of developing a whole bloodstage parasite vaccine candidate by specifically targeting the apicoplast. We initially generated apicoplast-minus parasites based on previously published methodology; Plasmodium falciparum can be chemically attenuated with a delayed death drug (doxycycline) and subsequently rescued with a crucial product produced by the apicoplast, isopentenyl pyrophosphate (IPP). This method renders parasites dependent on IPP supplementation, following loss of their apicoplast. We successfully produced apicoplast-minus parasites in vitro, optimising concentrations of doxycycline and IPP. Furthermore, we determined that doxycycline was required for at least two cycles of replication for complete attenuation. Despite success in vitro, our attempts at translating these studies to an in vivo rodent model was hindered by the lack of a long-term culturing system for rodent malaria parasites and the inability to sustain IPP levels in blood to allow for in vivo generation of apicoplast-minus parasites. We, therefore, decided upon a different approach; current investigation of chemoprophylactic immunization involving administration of infective sporozoites under chloroquine treatment (termed CPS) has shown promising results in human studies. Human studies employing the CPS model have observed sterile protection to homologous challenge, yet little to no blood-stage protection was noted. Similar investigation with blood-stage parasites showed protective immunity could be elicited in human and rodent studies; however, these studies were impacted by the requirement for delayed administration of treatment. We hypothesised that the use of a delayed death-causing drug could eliminate the lag time between infection and treatment commencement while also allowing for the parasite to persist long enough to produce a protective response. To investigate this theory, a controlled infection immunization (CII) model was established and studied in two mouse strains. Mice were given P. chabaudi AS or P. yoelii YM infection with immediate doxycycline chemoprophylaxis. We demonstrate that the delayed death effect caused by doxycycline allowed the persistence of the parasite. In turn, this persistence allowed the induction of strong protection from homologous challenge in groups receiving CIIs. Heterologous protection was also observed in mice immunized by P. chabaudi CII. Elucidation of the mechanisms of protective immunity found that different types of immune responses could be elicited by the various rodent/parasite combinations: CD4+ T cells were predominant mediators of immunity in P. chabaudi CII mice, whereas immunity was B-cell dependent for P. yoelii CII. Th1 and/or Th2 cytokine responses were also detected in various host/parasite groupings. Refinement of the CII protocol to use a longer-acting drug, azithromycin, demonstrated the possibility of reducing repeated drug treatment to a single dose. Given the encouraging outcome of our pre-clinical studies, we conducted a pilot clinical study investigating the persistence and immunogenicity of P. falciparum trophozoite parasites under doxycycline chemoprophylaxis. Findings from the clinical study show that the CII model was tolerable in a clinical setting as well as garnering results which corroborate our pre-clinical data. These findings will inform future clinical investigations of the CII approach. Malaria has a significant impact on public health worldwide. There have been several measures implemented to alleviate the burden of disease; however, the development of a vaccine is vital for substantial control of malarial infections. The discovery of the apicoplast, identification of its key synthetic processes and the revelation that delayed death-causing drugs specifically target this organelle offer an Achilles’ heel to an otherwise resilient organism. The exploitation of the apicoplast in vaccine development is a novel approach, and our studies indicate that the use of delayed deathcausing drugs in in vivo attenuation of blood-stage malaria is effective in inducing protection.