Mutant/mutation
The mutant (PbviVac) expresses P. vivax CSP (see below). The Pvcsp gene is introduced as an 'additional' copy in the neutral p230p locus and is under control of the uis4 promoter and 3'UTR regions. The mutant is drug-selectable marker free.

Protein (function)
The CS protein is the major protein on the surface of sporozoites and is critical for development of sporozoites within the oocysts and is involved in motility and invasion of both the salivary gland of the mosquito and the liver cells. The protein is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. Specific motifs in CS are involved in sporozoite binding to mosquito salivary glands and in sporozoite attachment to heparan sulfate proteoglycans in the liver of the mammalian host. During substrate-dependent locomotion of sporozoites, CS is secreted at the sporozoite anterior pole, translocated along the sporozoite axis and released on the substrate at the sporozoite posterior pole. Following sporozoite invasion of hepatocytes, the CS is released in the host cell cytoplasm.

P. vivax CSP used in this study:
The PvCSP coding sequence of a Pv field isolate from Thailand was initially amplified and compared to reference PvCSP sequences, including those of Pv strains P01 and Sal-1. 
As expected, the coding regions of both the N- and C-termini were highly conserved among the different PvCSP sequences, with a single non-synonymous polymorphic site at position 38, resulting in a transition from an asparagine to a glycine in the Pv Thailand isolate, while most variability occurred in the protein’s central repeat region. Our results indicate that the sequence of the PvCSP gene present in the Thailand field isolate employed in our study is similar to that of the most common and well-adapted variant of the PvCSP protein, VK210. This isolate presents 16 repeats of the VK210 variant’s most common peptide repeat motifs, GDRA(D/A)GQPA24, as well as a single occurrence of two other repeat motifs, GARADGQPA and GNGAGQAA, the latter of which is also found in Pv strain Sal-1, as well as in Sri Lanka’s and Brazil’s Pv populations.

Phenotype
The mutant expresses CSP from both P. berghei and P. vivax in sporozoites and liver stages. Evidence is presented for a surface localization of PvCSP in sporozoites. Wild type sporozoite production and sporozoites show wild type infectivity to hepatocytes.

Additional information
From the Abstract of the paper:
'Whole-sporozoite (WSp) vaccination, targeting pre-erythrocytic (PE) parasite stages, is  a promising strategy for immunization against malaria and several PfWSp-based vaccine candidates are currently undergoing clinical evaluation. In contrast, no WSp candidates have been developed for Pv, mainly due to constraints in the production of Pv sporozoites in the laboratory. Recently, we developed a novel approach for WSp vaccination against Pf based on the use of transgenic rodent P. berghei (Pb) sporozoites expressing immunogens of this human-infective parasite. We showed that this platform can be used to deliver PE Pf antigens, eliciting both targeted humoral responses and cross-species cellular immune responses against Pf. Here we explored this WSp platform for the delivery of Pv antigens. As the Pv circumsporozoite protein (CSP) is a leading vaccine candidate antigen, we generated a transgenic Pb parasite, PbviVac, that, in addition to its endogenous PbCSP, expresses PvCSP under the control of a strictly PE promoter. Immunofluorescence microscopy analyses confirmed that both the PbCSP and the PvCSP antigens are expressed in PbviVac sporozoites and liver stages and that PbviVac sporozoite infectivity of hepatic cells is similar to that of its wild-type Pb counterpart. Immunization of mice with PbviVac sporozoites elicits the production of anti-PvCSP antibodies that efficiently recognize and bind to Pv sporozoites.

Other mutants