Chimeric Plasmodium falciparum parasites expressing Plasmodium vivax circumsporozoite protein fail to produce salivary gland sporozoites

Abstract

BackgroundRodent malaria parasites where the gene encoding circumsporozoite protein (CSP) has been replaced with csp genes from the human malaria parasites, Plasmodium falciparum or Plasmodium vivax, are used as pre-clinical tools to evaluate CSP vaccines in vivo. These chimeric rodent parasites produce sporozoites in Anopheles stephensi mosquitoes that are capable of infecting rodent and human hepatocytes. The availability of chimeric P. falciparum parasites where the pfcsp gene has been replaced by the pvcsp would open up possibilities to test P. vivax CSP vaccines in small scale clinical trials using controlled human malaria infection studies.MethodsUsing CRISPR/Cas9 gene editing two chimeric P. falciparum parasites, were generated, where the pfcsp gene has been replaced by either one of the two major pvcsp alleles, VK210 or VK247. In addition, a P. falciparum parasite line that lacks CSP expression was also generated. These parasite lines have been analysed for sporozoite production in An. stephensi mosquitoes.ResultsThe two chimeric Pf-PvCSP lines exhibit normal asexual and sexual blood stage development in vitro and produce sporozoite-containing oocysts in An. stephensi mosquitoes. Expression of the corresponding PvCSP was confirmed in oocyst-derived Pf-PvCSP sporozoites. However, most oocysts degenerate before sporozoite formation and sporozoites were not found in either the mosquito haemocoel or salivary glands. Unlike the chimeric Pf-PvCSP parasites, oocysts of P. falciparum parasites lacking CSP expression do not produce sporozoites.ConclusionsChimeric P. falciparum parasites expressing P. vivax circumsporozoite protein fail to produce salivary gland sporozoites. Combined, these studies show that while PvCSP can partially complement the function of PfCSP, species-specific features of CSP govern full sporozoite maturation and development in the two human malaria parasites.Electronic supplementary materialThe online version of this article (10.1186/s12936-018-2431-1) contains supplementary material, which is available to authorized users.

Generation of a Genetically Modified Chimeric Plasmodium falciparum Parasite Expressing Plasmodium vivax Circumsporozoite Protein for Malaria Vaccine Development

“…This Pvcspchi expression cassette contains a codon-optimized Pvcsp open reading frame (ORF) containing the N-and C-terminal regions of the Pvcsp VK210 allele that flank a chimeric repeat sequence comprising repeats of both the VK210 allele (three times the repeat GDRADGQPA/GDRAAGQPA) and the VK247 allele (three times the repeat ANGAGNQPG/ANGAGDQPG). The ORF of Pvcsp-chi is flanked by EcoRV/EcoRI restriction sites and cloned into pLf0040 (cut EcoRV/EcoRI) (Marin- Mogollon et al, 2018) which contains 967bp of the promoter regions of Pfcsp (PF3D7_0304600) and 917bp of 3'UTR of Pfcsp resulting in intermediate plasmid T24 (Figure S1). This plasmid was digested with ApaI/SacII to obtain the complete Pvcsp-chi expression cassette containing the Pfcsp promoter, the Pvcsp-chi ORF with the chimeric VK210/VK247 repeats, and the Pfcsp 3'UTR.…”

“…After the negative selection, iRBC were harvested from cultures for genotyping by diagnostic PCR and Southern blot analysis. Subsequently, selected parasites were cloned by limiting dilution as previously described (Marin-Mogollon et al, 2018). Briefly, serial dilutions were performed with uninfected RBC in complete medium (1% hematocrit and 20% serum) and cultured in a total volume of 100 µl incubated in 96-well plates, resulting in 8 rows with the following numbers of iRBC per well: 100, 10, 5, 2.5, 1.25, 0.6, 0.3, 0.15.…”

“…The availability of comparable chimeric P. falciparum parasites expressing PvCSP would permit the analysis of protective efficacy of PvCSP-based vaccines in clinical studies using P. falciparum-based CHMI, bypassing the need for P. vivax sporozoite production and hypnozoite elimination. However, in contrast to efficient PvCSP expression and sporozoite production in chimeric rodent parasites, we found that chimeric P. falciparum lines with the PfCSP gene replaced by PvCSP, failed to produce salivary gland sporozoites, indicating that PvCSP cannot fully complement the role of PfCSP in P. falciparum sporozoite formation (Marin-Mogollon et al, 2018). To develop chimeric P. falciparum sporozoites expressing PvCSP, we therefore aimed at generating a chimeric P. falciparum line expressing both the endogenous PfCSP and an engineered PvCSP.…”

Potential and Limitations of Cross-Protective Vaccine against Malaria by Blood-Stage Naturally Attenuated Parasite

“…This indicates that it is possible to develop a cross-species protective vaccine. Then, Marin-Mogollon et al tried to make a recombinant Pf that expressed P. vivax CSP [ 44 ], to develop a cross-protective human malaria vaccine. They successfully generated the blood-stage recombinant parasite (asexual and sexual); however, unfortunately this recombinant parasite did not produce hemocoel or salivary gland sporozoites in the mosquito stage.…”

Investigating immune responses to parasites using transgenesis

“…In order to investigate the potential of the circumsporozoite protein (CSP) as a vaccine antigen in vivo , the csp gene of rodent malaria parasites was replaced with the csp genes from human parasites such as P. falciparum and P. vivax . The chimeric rodent parasites could produce sporozoites in Anopheles stephensi mosquitoes, capable of infecting human and rodent hepatocytes [40]. Thus, by expressing recombinant antigens from a human parasite into a mouse parasite, it is possible to take advantage of a mouse model to identify neutralising immune responses in a mouse model and hence discover potential vaccine candidates for human malaria.…”