title: Evaluating the immunogenicity and transmission-blocking potential of Plasmodium falciparum whole parasite, liposomal malaria vaccine candidates
Creators: name: Du Toit-Thompson, Taymin F
Publisher: name: Griffith University
Year: 2021
Subjects: subject: Transmission of the malaria parasite
subject: sexual-stage parasite
subject: Liposomes
subject: immunomodulatory molecules

description: Malaria is a mosquito-borne infectious disease which resulted in 409,000 deaths and 229 million clinical cases across 87 malaria endemic countries in 2019. Anti-malarial drugs are used to prevent and treat malaria, and vector control is currently the most effective means to control malaria parasite transmission. However, the development of resistance by the mosquito to insecticides and resistance by the parasite to all current anti-malarial drugs, as well as the significant cost of anti-malarial drugs, has heightened the urgency for research into alternative strategies to control and eliminate malaria, such as a vaccine. There have been difficulties in developing an efficacious malaria vaccine, in part due to the Plasmodium parasite and its complex life cycle in both the Anopheles spp. mosquito vector and vertebrate host. The majority of malaria vaccine approaches target a specific Plasmodium life cycle stage, with sexual blood-stage vaccine candidates explored to a comparatively minor extent. By disrupting he parasite’s ability to infect and develop within the mosquito a transmission-blocking vaccine (TBV), targeting the sexual-stage parasite, does not aim to protect the vaccine but instead aims to provide a community-based protection against infection with the malaria parasite. This project focuses on vaccine candidates targeting the sexual-stage parasite, with the overall aim to interrupt transmission of the malaria parasite. Vaccine candidates explored here were formulated with liposomes and the immunomodulatory molecules: mannosylated core peptide (F3) and/or 3D(6-acyl) PHAD (PHAD). Liposomes have been previously investigated for the development of malaria vaccine candidates including TBV candidates, and a whole asexual blood-stage parasite vaccine was recently formulated with F3 liposomes and was shown to induce protective immunity against blood-stage challenge. Initially, vaccine candidates were examined as single component vaccine formulations, with P. falciparum NF54 gametocytes representing the sexual-stage parasite in the human host and the P. falciparum antigen Pfs25/Pfs25-derived peptides representing the sexual-stage parasites in the mosquito. Immunogenicity was evaluated by measuring antigen-specific antibody responses by enzymelinked immunosorbent assay (ELISA) and cellular responses by splenocyte proliferation assays and cytokine bead arrays (CBA). Based on the quality and quantity of the induced cellular and humoral immune responses, the optimal vaccine formulation combining gametocytes and the Pfs25/Pfs25-derived peptides was then examined as a multi-component formulation to facilitate the induction of immune responses that target the sexual-stage of the parasite in both the human and mosquito hosts. In addition to measuring immunogenicity, standard membrane feeding assays (SMFA) were used in the multi-component vaccine experiment to evaluate the functionality of the induced antibody responses. The induction of a cross-stage functional immune response that targeted the asexual blood-stage parasite was also examined in the single and multi-component experiments by challenging mice with heterologous P. yoelii 17X bloodstage parasites. In the single-component vaccine experiments, when examining the humoral immune responses, mice immunised with Pfs25-EPA and Pfs25-derived peptide-DT vaccine formulations had high Immunoglobulin G (IgG) titres against the vaccinating antigen and the presence of liposomes allowed faster antibody acquisition following immunisation. IgG1 was the predominant IgG subclass induced following immunisation, with lower levels of the other IgG subclasses observed. The level of Pfs25-specific IgG was much lower in sera from mice immunised with vaccine formulations containing Pfs25-derived peptide-DT compared to vaccine formulations containing Pfs25-EPA. Therefore, we did not consider a Pfs25-derived peptide-DT vaccine component for the multi-component vaccine formulation. Mice immunised with formulations containing gametocytes with liposomes and PHAD (or PHAD in combination with F3) had significantly higher P. falciparum NF54 asexual blood-stage parasite-specific IgG titres compared with the other gametocyte-vaccinated groups. Antibodies against whole gametocyte antigens were not able to be measured due to a lack of coating antigen for ELISAs. IgG specific for P. yoelii 17X asexual blood-stage parasites was only observed in the sera of mice immunised with vaccine formulations containing gametocytes with liposomes and PHAD (or PHAD in combination with F3), albeit at low levels. Interestingly, we also detected IgG specific for Pfs25 in sera from mice immunised with whole gametocytes, likely due to the presence of Pfs25-expressing gametes which may have exflagellated during purification of gametocytes for the vaccine formulations. In examining the cellular immune responses, all groups immunised with Pfs25-EPA, excluding Pfs25-EPA + PHAD liposomes, had Pfs25-specific proliferative responses significantly higher than background. Splenocytes from mice immunised with Pfs25-EPA + PHAD liposomes had significantly higher T helper 1 (Th1), Th2 and Th17-related cytokine production, with the exception of Interleukin-10 (IL-10), compared with all other groups. All vaccine formulations containing P. falciparum gametocytes induced strong proliferative responses to P. falciparum asexual parasitised red blood cells (pRBCs) and gametocytes, with no significant differences between the P. falciparum asexual pRBC (NF54 and 7G8 strains) and P. falciparum NF54 gametocyte stimulants indicating the induction of stage and strain-transcending cellular immune responses. Proliferative responses to P. yoelii pRBCs were comparatively lower in all groups, although this was only statistically significant in mice vaccinated with gametocytes + naked liposomes. In genral, Th1 and Th2-related cytokine responses were higher in groups immunised with vaccine formulations containing gametocytes with liposomes and PHAD (or PHAD in combination with F3) and Th17 responses were only detected in these groups. Comparatively, all groups had lower Th1, Th2 and Th17-related cytokine responses in response to P. yoelii pRBC stimulation. As the data suggest that the presence of PHAD in the vaccine formulation was critical for the induction of cellular immune responses, this was selected as the optimal liposome formulation for the multi-component vaccine experiment. For the multi-component experiment, Pfs25-EPA and P. falciparum gametocytes were formulated together with liposomes and PHAD and this multi-component vaccine was evaluated alongside vaccine formulations containing the individual antigen components to examine whether the inclusion of two vaccine components in the vaccine formulation would result in immune interference. Mice immunised with Pfs25-EPA + PHAD liposomes had significantly higher Pfs25-specific IgG titres compared with mice immunised with Pfs25-EPA + gametocytes + PHAD liposomes and gametocytes + PHAD liposomes. The lower Pfs25- specific IgG, following immunisation with the multi-component vaccine, may be due to immune interference. No significant differences were observed when comparing the IgG titres against the P. falciparum NF54 asexual blood-stage parasite in groups immunised with Pfs25- EPA + gametocytes + PHAD liposomes or gametocytes + PHAD liposomes. This indicates that there was no immune interference with the induction of these antibodies. IgG specific for Pfs25 was also detected in sera from mice immunised with gametocytes + PHAD liposomes in the multi-component vaccine experiment. Immunisation with vaccine formulations containing gametocytes induced strong P. falciparum whole parasite-specific splenocyte proliferative responses, with no differences observed in the proliferative response against any of the P. falciparum whole parasite antigen stimulants. Comparatively, immunisation with vaccine formulations containing gametocytes induced lower P. yoelii pRBC-specific splenocyte proliferative responses. Proliferative responses were significantly lower in splenocytes from mice immunised with gametocytes + PHAD liposomes following stimulation with P. yoelii pRBCs compared to stimulation with Pf NF54 gametocytes. Splenocytes from mice immunised with Pfs25-EPA + PHAD liposomes, Pfs25- EPA + gametocytes + PHAD liposomes and gametocytes + PHAD liposomes had significantly higher proliferative responses following stimulation with Pfs25 compared to stimulation with media. This was in contrast with the lack of a significantly higher proliferative response in the Pfs25-EPA + PHAD liposomes group in the single-component vaccine experiment. Splenocytes from mice immunised with gametocytes in the multi-component vaccine experiment produced Th1, Th2 and Th17-related cytokines in response to whole parasite antigen stimulation, with much lower responses observed against P. yoelii pRBCs. Pfs25- specific cytokine responses in the Pfs25-EPA + PHAD liposome immunised mice were inconsistent with the results from the single-component experiment, suggesting that the analysis for this multi-component experiment did not work and therefore it should be repeated in future experiments. Functionality of vaccine-induced immune responses was examined in both single and multicomponent vaccine experiments. The SMFA measured the functionality of antibodies induced by the vaccine formulations in the multi-component experiment. Pooled post-vaccination sera samples from the multi-component vaccine and the individual Pfs25-EPA or gametocyte vaccine formulations had antibodies with potent functional activity, reducing the prevalence of infected mosquitoes and the density of oocysts in infected mosquitoes by &gt;90%. Further analysis with different strains of Plasmodium gametocytes in the vaccine or the SMFA should be conducted in future experiments to evaluate strain-transcending inhibitory activity of antibodies and to enable assessment of the breadth of the vaccine-induced transmissionblocking immune response. To examine the induction of cross-stage protective immunity against asexual blood-stage parasites, mice were challenged with P. yoelii 17X pRBCs. Moderate protection was observed in mice immunised with gametocytes + PHAD liposomes in the single-component vaccine experiment, but this was not reproduced in the multicomponent vaccine experiment. The heterologous species blood-stage challenge using a rodent parasite does not appear to be a robust or reproducible model for assessing cross-stage immunity induced by a vaccine containing human parasites or parasite antigens. Future preclinical evaluation could be conducted in Aotus monkeys to evaluate the induction of crossstage protection against homologous challenge with P. falciparum asexual blood-stage parasites. Malaria remains a global public health issue which urgently requires more effective strategies to control the transmission of the malaria parasite. A multi-component vaccine which could target parasite antigens across different hosts could be critical for stopping parasite transmission. Through the experiments conducted in this project we have generated important data which supports further development of the TBV candidates examined here and provides further justification of a multi-component vaccine utilising multiple antigens to target the malaria parasite at different points in the Plasmodium life cycle. However, further studies are still needed to be able to progress the multi-component vaccine candidate into clinical evaluation.