Final Report Summary - GENINVADE (Parasite population genomics and functional studies towards development of a blood stage malaria vaccine)
The research programme was conducted continuously over a five year period as planned. This involved experiments and analysis on human malaria parasites at the molecular, functional, and population levels:
(i) Population genetic analyses of P. falciparum in sites of contrasting endemicity in West Africa, to finely determine signatures of selection with high-resolution throughout the genome, and help refine hypotheses on mechanisms used by merozoites to invade erythrocytes and evade acquired immune responses.
Multi-population genomic analyses of samples from West Africa were completed as planned, with results indicating loci under recent selection and under locally differing selection. Most of the results are published (Mobegi et al. Mol. Biol. Evol. 2014; Nwakanma et al. J. Infect. Dis. 2014; Duffy et al. BMC Genomics 2015; Duffy et al. Molecular Ecology 2017), and further final results are currently submitted for publication. The research approaches and insights also enabled supplementary analyses to contribute to related studies of variation in P. falciparum and the related parasite species P. reichenowi (Otto et al. Nature Communications 2014), and to population genetics and genomics of P. knowlesi (Divis et al. PLOS Pathogens 2015; Assefa et al. Proc. Natl. Acad. Sci. USA 2015; Divis et al. Emerg. Infect. Dis. 2017; Divis et al. Molecular Ecology 2018). Although our aim was initially to focus on selection on genes expressed at the merozoite stage, results also indicated interesting genomic loci under selection that was independent of this life-cycle stage, and leads to additional hypotheses for future experimental investigation.
(ii) Experimental culture analysis of merozoite invasion into erythrocytes to identify the receptor-ligand interactions used by different parasite populations ex vivo. Novel receptor knockdown assays on cultured erythrocytes will be employed, and parasite adaptation experiments conducted to identify constraints on the use of alternative invasion pathways.
The spectrum of alternative invasion phenotypes was compared across sites in three different West African countries (Senegal, Guinea and Ghana), as published (Bowyer et al. Infection and Immunity 2015). Parasite culture adaptation experiments did not show significant shifts in invasion phenotypes, but genome sequencing allowed us to identify genes affected by loss-of-function mutations that are selected in culture (Claessens et al. Sci. Rep. 2017). These latter results are particularly interesting and have led to ideas for studies on parasite adaptation and development that go beyond the scope of the defined project. Continued analysis of an expanded panel of Ghanaian cultured isolates, to identify additional emerging mutants and convergent evolution, will soon be submitted for publication.
(iii) Innovative approaches to select individual parasites and characterise cell tropism, transcript profiles, and genome sequences. This is aimed to validate population level findings and revolutionise approaches to genetics and phenotyping of parasites in the future. Candidate molecule discoveries will be taken forwards to receptor-ligand interaction assays, antibody inhibition and immuno-epidemiological studies.
The single-cell sorting and sequencing approaches yielded preliminary results that are useful for design of further proof-of-principle studies, which require further work as this is a pioneering area in which methods development is still needed. We developed a new assay for detecting variation in the asexual multiplication rates of different P. falciparum lines, and identified a spectrum of rates among clinical isoles from West Africa in an initial study we have published (Murray et al. Sci. Rep. 2017), with further results from an expanded panel of isolates to be submitted for publication. Analysis of transcriptome variation in schizonts of laboratory-adapted and clinical isolates has identified new variant genes, in a report being prepared for publication.
(i) Population genetic analyses of P. falciparum in sites of contrasting endemicity in West Africa, to finely determine signatures of selection with high-resolution throughout the genome, and help refine hypotheses on mechanisms used by merozoites to invade erythrocytes and evade acquired immune responses.
Multi-population genomic analyses of samples from West Africa were completed as planned, with results indicating loci under recent selection and under locally differing selection. Most of the results are published (Mobegi et al. Mol. Biol. Evol. 2014; Nwakanma et al. J. Infect. Dis. 2014; Duffy et al. BMC Genomics 2015; Duffy et al. Molecular Ecology 2017), and further final results are currently submitted for publication. The research approaches and insights also enabled supplementary analyses to contribute to related studies of variation in P. falciparum and the related parasite species P. reichenowi (Otto et al. Nature Communications 2014), and to population genetics and genomics of P. knowlesi (Divis et al. PLOS Pathogens 2015; Assefa et al. Proc. Natl. Acad. Sci. USA 2015; Divis et al. Emerg. Infect. Dis. 2017; Divis et al. Molecular Ecology 2018). Although our aim was initially to focus on selection on genes expressed at the merozoite stage, results also indicated interesting genomic loci under selection that was independent of this life-cycle stage, and leads to additional hypotheses for future experimental investigation.
(ii) Experimental culture analysis of merozoite invasion into erythrocytes to identify the receptor-ligand interactions used by different parasite populations ex vivo. Novel receptor knockdown assays on cultured erythrocytes will be employed, and parasite adaptation experiments conducted to identify constraints on the use of alternative invasion pathways.
The spectrum of alternative invasion phenotypes was compared across sites in three different West African countries (Senegal, Guinea and Ghana), as published (Bowyer et al. Infection and Immunity 2015). Parasite culture adaptation experiments did not show significant shifts in invasion phenotypes, but genome sequencing allowed us to identify genes affected by loss-of-function mutations that are selected in culture (Claessens et al. Sci. Rep. 2017). These latter results are particularly interesting and have led to ideas for studies on parasite adaptation and development that go beyond the scope of the defined project. Continued analysis of an expanded panel of Ghanaian cultured isolates, to identify additional emerging mutants and convergent evolution, will soon be submitted for publication.
(iii) Innovative approaches to select individual parasites and characterise cell tropism, transcript profiles, and genome sequences. This is aimed to validate population level findings and revolutionise approaches to genetics and phenotyping of parasites in the future. Candidate molecule discoveries will be taken forwards to receptor-ligand interaction assays, antibody inhibition and immuno-epidemiological studies.
The single-cell sorting and sequencing approaches yielded preliminary results that are useful for design of further proof-of-principle studies, which require further work as this is a pioneering area in which methods development is still needed. We developed a new assay for detecting variation in the asexual multiplication rates of different P. falciparum lines, and identified a spectrum of rates among clinical isoles from West Africa in an initial study we have published (Murray et al. Sci. Rep. 2017), with further results from an expanded panel of isolates to be submitted for publication. Analysis of transcriptome variation in schizonts of laboratory-adapted and clinical isolates has identified new variant genes, in a report being prepared for publication.