Mutant/mutation
The mutant expresses model T and B cell epitopes (OVA, HA, gB; 'polytope') fused to GFP under the control of the constitutive P. berghei elongation factor (EF)-1α promoter. The 'polytope' fused to GFP was introduced and maintained in the transgenic parasites as circular plasmids (episomes) by in vivo pyrimethamine selection.

Protein (function)
The transgene consists of T and B cell epitopes ('polytope') fused to GFP.
The T cell epitopes chosen were MHC I- and MHC II-restricted epitopes presented in C57BL/6 (B6) and BALB/c mice, which are differentially sensitive to P. berghei-mediated experimental cerebral malaria (ECM). For B6 mice, which are susceptible to ECM, MHC I- and II-restricted epitopes from chicken ovalbumin and an MHC I-restricted epitope from glycoprotein B of herpes simplex virus-1 were included. For ECM-resistant BALB/c mice, MHC I- and II-restricted epitopes from hemagglutinin of the influenza virus PR8 were included, whereas the MHC II-restricted OVA epitope can also be presented on I-Ad of BALB/c mice.

Sequences of overlapping oligonucleotides designed for PCR amplification of the selected CD4+ and CD8+ T cell epitopes (OVA257–264 [SIINFEKL], H-2Kb restricted; OVA323–339 [KISQAVHAAHAEINEAG], I-Ab and IAd restricted; gB498–505 [SSIEFARL], H-2Kb restricted; HA518–526 [IYSTVASSL], H-2Kd restricted; and HA126–138 [HNTNGVTAACSHE], I-Ad restricted), B cell epitopes FLAG (DYKDDDK) and c-myc (EQKLISEEDL), and restriction sites to facilitate cloning are illustrated in the Figure shown below.

Phenotype
The mutant expresses a variety of model T cell epitopes for which T cell receptor (TCR) transgenic mice are available. This approach of transgenic expression was necessary as MHC class I-restricted antigens to blood-stage infection have not been defined. It was demonstrated that the 'polytope' transgene was functionally expressed, correctly processed, and presented by the host immune system for specific stimulation of CD4+ and CD8+ T cells during blood-stage infection. Using these mutants it was demonstrated that antigens expressed by blood-stage P. berghei parasites are captured and cross-presented by CD8α DC to stimulate naive CD8+ T cell proliferation and lytic function  (see also 'Additional information').

Additional information
Expression of the 39-kDa polytope-GFP fusion protein by PbTG was confirmed by Western blot analysis using antibodies to GFP, FLAG, or c-myc

Evidence is presented indicating that bone marrow-derived DC were critical for the induction of T cell responses during blood-stage infection and data is presented that suggests that  CD8α DC are the major antigen-presenting cells for CD8+ T cells during blood-stage infection, whereas CD4 DC play a more extensive role in CD4+ T cell stimulation.

Other mutants
RMgm-5: mutant expressing GFP under the control of the constitutive promoter of the elongation factor 1 alpha (eef1a) gene of P. berghei.