Chief Investigator: Dr Danny Wilson

Funding Amount: $74,662

Recipient: University of Adelaide

Overview:

P. vivax causes ~80 million malaria cases annually, with children in Australia’s neighbouring countries badly affected. In a major collaboration between the Menzies Institute and University of Adelaide, we will develop a unique model of P. vivax vaccine candidate PvMSP3. Using this model, we will detail the unknown function of PvMSP3 and quantify the immune response targeting PvMSP3 that protects children from disease. This study will assess PvMSP3 vaccine candidacy and provide innovative tools to shorten the timeframe of vaccine development against this debilitating childhood pathogen.

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Research Outcomes:

Researchers: Dr Danny Wilson, Dr Michelle Boyle

Research Completed: 2020

Research Findings: Plasmodium falciparum and Plasmodium vivax together account for the majority of sickness and deaths caused by malaria, with P. vivax representing a significant disease burden in Australia’s neighbouring southeast Asia region. Merozoite Surface Proteins (MSPs) are a broad range of functionally distinct proteins localized on the surface of the parasite form which invades and established disease-causing infection in human red blood cells, the merozoite. Despite being diverse many MSPs are essential for asexual blood stage growth and are potential vaccine candidates. While many of the P. falciparum MSPs are well studied, investigation of P. vivax MSPs has been severely hindered by the lack of suitable reagents to study protein immunogenicity and the inability to grow P. vivax in long-term in vitro culture.  Plasmodium knowlesi a primate Plasmodium related to P. vivax and an emerging zoonotic pathogen in its own right, has been adapted to long-term in vitro culture in human red blood cells (Lim et al., 2013; Moon et al., 2013). Efficient CRISPR-Cas9 gene editing of human adapted P. knowelsi has been developed and in this study, we have used this gene editing technique for thestudy of P. vivax MSPs on a background of P. knowlesi malaria, a phylogenetically similar model to P. vivax. We have achieved in this study:

1. Quantification and functional characterisation of protective antibodies targeting PvMSP3a.

Using recombinantly expressed PvMSP3a, we assessed antibody levels and the types of antibodies targeting this protein in malaria exposed children and adult sera. The key findings of this study were:

• Seroprevalence of complement-fixing antibodies was highest against the central region of PvMSP3α (77.6%).
• IgG1, IgG3, and IgM antibody subclasses were significantly correlated with complement mediated immune responses to PvMSP3α.
• Complement-fixing antibody levels were similar between age groups, but IgM was predominant in children and IgG3 more prevalent in adults.
• Complement fixing functional antibodies increased following acute infection to 7 days after treatment, however rapidly waned by day 28.
• Complement fixing IgM antibodies were more predominant in children and IgG3 more prevalent in adults

This study demonstrates PvMSP3α antibodies acquired during P. vivax infection can mediate complement-fixation and shows the important influence of age in shaping these specific antibody responses. This study was published in 2019 with the Channel 7 Research Foundations significant contribution cited.

Oyong, D. A., Wilson, D. W., Barber, B. E., William, T., Jiang, J., Galinski, M. R., Fowkes, F. J. I., Grigg, M. J., Beeson, J. G., Anstey, N. M., and Boyle, M. J. (2019) Induction and Kinetics of Complement-Fixing Antibodies Against Plasmodium vivax Merozoite Surface Protein 3alpha and Relationship With Immunoglobulin G Subclasses and Immunoglobulin M. J Infect Dis 220, 1950-1961

• Gene-editing and functional characterisation of PvMSP3a

Malaria transfection generally takes several months longer than most other cell based systems. In this study, we are seeking to not just modify one gene, but all four PkMSP3s and ultimately replace these with a single PvMSP3 (PvMSP3a). Using the latest CRISPR-Cas9 gene editing techniques, we have:

• Sequentially removed all 4 endogenous PkMSP3s and have begun functionally characterizing growth defects with single, double or triple PkMSP3 knockouts.
• Knocked out 3 of 4 PkMSPs on chromosome 10 in one transfection. This required the CRISPR-Cas9 mediated removal of 8000 bp of DNA coding sequence, a difficult task in malaria biology. It is necessary to remove these antigens before inserting PvMSP3a to replace the remaining PkMSP3 on chromosome 14.
• Integrated PvMSP3a into the PkMSP3 locus on chromosome 14 in preparation for CRISPR-Cas9 mediated removal of the remaining PkMSP3s. This will create the final parasite line expressing only the single PvMSP3a.

As part of this project, we obtained Australian Society for Parasitology funding for Ms Isabelle Henshall to travel to the Laboratory of Assoc Prof Robert Moon (London School of Tropical Medicine and Hygiene), the world leader in P. knowlesi transfection technologies, where she spent 3 weeks training in his laboratory which has improved our throughput and will fast track final development of a PvMSP3a expressing P. knowlesi parasite line.

We have obtained P. vivax exposed sera from PNG donors and have all the ethics approvals in place to use this sera set to assess naturally acquired antibody levels against PvMSP3a using functional assays available in the laboratory.

This arm of the study is likely to result in one to two initial papers (Paper 1: characterisation of MSP3 Knock-out lines; Paper 2: assessment of naturally acquired antibodies in malaria exposed sera to PvMSP3a) in the next 18 months.

Combined, Ch7RF funding is on track to deliver 2-3 papers within 3.5 years of starting the study, with one paper already published and delivering insights into immunity to P. vivax malaria.

Key Outcomes: Malaria is caused by mosquito borne Plasmodium parasites that infect and replicate inside human red blood cells (RBCs). The stage that enters the RBC, the merozoite, is a target of vaccine development as blocking host cell entry prevents disease. Despite P. vivax being a leading cause of childhood morbidity globally, research into suitable vaccine candidates to prevent disease caused by this parasite is limited. P. vivax merozoite surface protein 3a (PvMSP3a) is a protein on the surface of the P. vivax merozoite that has been identified as a promising vaccine candidate. With the help of the Channel 7 Children’s Research Foundation, we have quantified the specific function of antibodies targeting PvMSP3a that protect children from malaria and developed gene-edited parasites to characterize the location, function and vaccine potential of this leading P. vivax vaccine candidate.

Research Papers: Oyong, D. A., Wilson, D. W., Barber, B. E., William, T., Jiang, J., Galinski, M. R., Fowkes, F. J. I., Grigg, M. J., Beeson, J. G., Anstey, N. M., and Boyle, M. J. (2019) Induction and Kinetics of Complement-Fixing Antibodies Against Plasmodium vivax Merozoite Surface Protein 3alpha and Relationship With Immunoglobulin G Subclasses and Immunoglobulin M. J Infect Dis220, 1950-1961

Related Publications:

Future Outcomes: