Mutant/mutation
Three mutant are described that express CSP from P. falciparum (PF3D7_0304600) fused to Ovalbumin (OVA).
No detailed description is provided of the generation of DNA constructs and the generation of the mutants. It seems likely that the PfCSP-OVA transgenes are introduced in mutants PfCSP-OVA(surface) and PfCSP-OVA(fusion) as an additional copy under the control of the promoter of the sporozoite/liver stage specific  UIS4 gene

We generated three lines of transgenic parasites co-expressing PfCSP (PF3D7_0304600) and the OVA323-339 (OT-II epitope) in different relative spatial locations. First, PfCSP-OVAcytosol contained PfCSP on the surface of the SPZ and OVA (whole protein, fused with mCherry) expressed cytosolically under the control of a strong constitutive promoter Hsp70.
Second, PfCSP-OVAsurface carried intact PfCSP, and PbCSP molecule with OVA260-386 inserted between the repeat and C terminus in order to target OVA to the sporozoite surface but on a separate protein.
Third, PfCSP-OVAfusion carried OVA260-386 inserted between the repeat and C-terminus of PfCSP. Since the insertion of the OVA260-386 rendered PfCSP non-functional it was also necessary to insert PbCSP to ensure SPZs can still emerge from mosquitoes

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2024.08.10.607257

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.

Phenotype
In previous experiments, different transgenic T cells might have intrinsically different capacity to help B cells (e.g. different TCR affinity). To address this limitation, we generated three lines of transgenic parasites co-expressing PfCSP (PF3D7_0304600) and the OVA323-339 (OT-II epitope) in different relative spatial locations. First, PfCSP-OVAcytosol contained PfCSP on the surface of the SPZ and OVA (whole protein, fused with mCherry) expressed cytosolically under the control of a strong constitutive promoter Hsp70.
Second, PfCSP-OVAsurface carried intact PfCSP, and PbCSP molecule with OVA260-386 inserted between the repeat and C terminus in order to target OVA to the sporozoite surface but on a separate protein.
Third, PfCSP-OVAfusion carried OVA260-386 inserted between the repeat and C-terminus of PfCSP. Since the insertion of the OVA260-386 rendered PfCSP non-functional it was also necessary to insert PbCSP to ensure SPZs can still emerge from mosquitoes.

Mice were immunizated by intravenous injection with 3-6 ×104 irradiated (15kRad) sporozoites of the three different mutant parasites.
Analysis of immunized mice with these mutant parasites showed that Ighg2A10 B cells can receive help from CD4+ T cells that are specific for bystander surface, but not cytosolic SPZ antigens.

Additional information

Other mutants