Generation of a Transgenic Plasmodium cynomolgi Parasite Expressing Plasmodium vivax Circumsporozoite Protein for Testing P. vivax CSP-Based Malaria Vaccines in Non-Human Primates

Abstract

Background/Objectives: Malaria, caused by infection with Plasmodium parasites, exacts a heavy toll worldwide. There are two licensed vaccines for malaria as well as two monoclonal antibodies that have shown promising efficacy in field trials. The vaccines and monoclonal antibodies target the major surface protein (circumsporozoite protein, CSP) of Plasmodium falciparum. Yet P. falciparum is only one of the four major species of Plasmodium that infect humans. Plasmodium vivax is the second leading cause of malaria, but the P. vivax vaccine and monoclonal development lags far behind that for P. falciparum owing to the lack of basic preclinical tools such as in vitro culture or mouse models that replicate the key biological features of P. vivax. Notably among these features is the ability to form dormant liver stages (hypnozoites) that reactivate and drive the majority of the P. vivax malaria burden. Plasmodium cynomolgi is a simian parasite which is genotypically very close and phenotypically similar to P. vivax; it can infect non-human primates commonly used in research and replicates many features of P. vivax, including relapsing hypnozoites. Methods: Recently, a strain of P. cynomolgi has been adapted to in vitro cultures allowing parasite transgenesis. Here, we created a transgenic P. cynomolgi parasite in which the endogenous P. cynomolgi CSP has been replaced with P. vivax CSP, with the goal of enabling the preclinical study of anti-P. vivax CSP interventions to protect against primary and relapse infections. Results: We show that the in vitro-generated transgenic Pcy[PvCSP] parasite expresses both serotypes of P. vivax CSP and retains full functionality in vivo, including the ability to transmit to laboratory-reared Anopheles mosquitoes and cause relapsing infections in rhesus macaques. To our knowledge, this is the first gene replacement in a relapsing Plasmodium species. Conclusions: This work can directly enable the in vivo development of anti-P. vivax CSP interventions and provide a blueprint for the study of relapsing malaria through reverse genetics.

Keywords: Plasmodium cynomolgi; Plasmodium vivax; circumsporozoite protein; hypnozoite; pre-erythrocytic malaria vaccines.

Figure 1

The construction and validation of a P. vivax-expressing P. cynomolgi parasite. (A) The plasmid map and CSP sequence used for the Cas9-mediated generation of the Pcy[PvCSP] transgenic parasite. (B) PCR results confirming the successful integration of the Pv csp gene replacing the native Pcy csp after the in vitro expansion of the transfected parasites. The top graphic indicates the primers and expected amplicon size with genotyping primer pairs. Bottom images show PCR gels of transfected cultures The T1–T3 using the primers indicated above. Culture T1 is shown to have the wild-type Pcy CSP (band at ~1200 pb on right image) as well as the Pv CSP (bands at ~750 pb on left image for both 3′ and 5′ integration sites), indicating a mixed population. Culture T2 has only wild-type parasites, evidenced by the lack of a band at ~750 bp for either transgene integration site (left gel). Culture T3 is a pure population of transgenic parasites, evidenced by the band at ~750 bp for both 3′ and 5′ Pv-transgene integration sites and the lack of a band at ~1200 bp for the Pcy wild-type CSP detection.

Figure 2

A Pcy[PvCSP] transgenic parasite completes the parasite life cycle between rhesus macaques and A. stephensi mosquitoes including relapse. (A) An overview of the NHP infections and mosquito feeds, with the purpose of each stage indicated above. (B) Parasitemia curves demonstrating the infectivity of the transgenic parasite at both blood and sporozoite stages. The parasitemia presented was quantified by thin-smear microscopy, and parasite-negativity for animals infected with sporozoites (center panel) was confirmed by a qRT-PCR (see Supplemental Figure S1). Pcy[PvCSP] = Plasmodium cynomolgi parasite with an endogenous circumsporozoite gene replaced with a P. vivax sequence. RM = rhesus macaque. Rx = antimalarial treatment specific to clearing blood-stage parasites (no action against hypnozoites). RC = radical cure with Tafenoquine to clear hypnozoites.

Figure 3

Confirmation of PvCSP integration and protein expression following infection of NHPs and cycling in A. stephensi mosquitoes. (A) PCR for integration of Pv csp gene replacing native Pcy csp after cycling in three NHPs and Anopheles mosquitoes using infected blood samples from relapsed parasites, wild-type parental non-transgenic parasites (WT), Culture T3 transgenic parasites (T3), and no template sample using primer sets described in Figure 1. (B) Immunofluorescent imaging comparing expression of two major Pv CSP variants in parasite field isolates from Thailand and Peru, both of which predominantly express VK210, to our Pcy[PvCSP] transgenic parasite that expresses both VK210 and VK247. Peruvian strain stained with anti-Pv CSP 247 exhibits one location of autofluorescent debris in upper left quadrant and is distinct in size and focus-plane from true sporozoites as no defined sporozoites could be found. Secondary only control lacks any CSP-specific antibody.