Summary Immunization with radiation‐attenuated Plasmodium sporozoites (RAS) induces sterile and long‐lasting protective immunity. Although intravenous (IV) route of RAS immunization is reported to induce superior immunity compared to intradermal (ID) injection, its role in the maintenance of sterile immunity is yet to be understood. We investigated whether the route of homologous sporozoite challenge of Plasmodium berghei (Pb) RAS‐immunized mice would influence the longevity of protection. C57BL/6 mice immunized with Pb‐RAS by IV were 100% protected upon primary IV/ID sporozoite challenge. In contrast, ID immunization resulted in 80% protection, regardless of primary challenge route. Interestingly, the route of primary challenge was found to bring difference in the maintenance of sterile protection. While IV Pb RAS‐immunized mice remained protected at all challenges regardless of the route of primary challenge, ID Pb‐RAS‐immunized mice receiving ID primary challenge became parasitaemic upon secondary IV challenge. Significantly, primary IV challenge of Pb RAS ID‐immunized mice resulted in 80% and 50% survival at secondary and tertiary challenges, respectively. According to phenotypically diverse liver CD8+ T cells, the percentages and the numbers of both CD8+ T effector memory and resident memory cells were significantly higher in IV than in ID Pb RAS‐immunized mice. IFN‐γ‐producing CD8+ T cells specific to Pb TRAP130 and MIP‐4‐Kb‐17 were also found significantly higher in IV mice than in ID mice. The enhanced T‐cell generation and the longevity of protection appear to be dependent on the parasite load during challenge when infection is tolerated under suboptimal CD8+ T‐cell response.

Pre-erythrocytic vaccines prevent malaria by targeting parasites in the clinically silent sporozoite and liver stages and preventing progression to the virulent blood stages. The leading pre-erythrocytic vaccine RTS,S/AS01E (Mosquirix®) entered implementation programs in 2019 and targets the major sporozoite surface antigen called circumsporozoite protein or CSP. However, in phase III clinical trials, RTS,S conferred partial protection with limited durability, indicating a need to improve CSP-based vaccination. Previously, we identified highly expressed liver stage proteins that could potentially be used in combination with CSP and are referred to as pre-erythrocytic vaccine antigens (PEVA). Here, we developed heterologous prime-boost CSP vaccination models to confer partial sterilizing immunity against Plasmodium yoelii (Py)(protein prime/adenovirus 5 (Ad5) boost) and P. berghei (Pb) (DNA prime/Ad5 boost) in mice. When combined as individual antigens with PyCSP, 3 of 8 PyPEVA significantly enhanced sterile protection against sporozoite challenge, compared to PyCSP alone. Similar results were obtained when 3 PbPEVA and PbCSP were combined in a single vaccine regimen. In general, PyCSP antibody responses were similar after CSP alone versus CSP+PEVA vaccinations. Both Py and Pb CSP+PEVA combination vaccines induced robust CD8 + T cell responses including signature IFN-γ increases. In the Pb model system, IFN-γ responses were significantly higher in hepatic than splenic CD8 + T cells. The addition of novel antigens may enhance the degree and duration of sterile protective immunity conferred by a human vaccine such as RTS,S.

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Exposure to Plasmodium sporozoites (spz), the causative agent of malaria, induces anti-disease immunity, but fails to protect against repeated Plasmodium infections. In contrast, multiple immunizations with radiation-attenuated Plasmodium spz (RAS) induce sterile lasting protection in humans, non-human primates, and murine models. The mechanisms of protective immunity are multifactorial and CD8 T cells are considered sine qua none of protection. The Plasmodium berghei (Pb) RAS-induced protection in C57Bl/6 mice correlates with a network of liver stage antigens (LS Ags)-activated liver CD8 T cell subsets including resident memory (RM) CD8 TRM cells. A protective Pb LS Ag, termed MIF-4G, induced liver CD8 T cells that recognize H-2Kb-restricted epitope, Kb17. One of the current efforts to improve malaria vaccines focuses on maximizing the induction of liver CD8 TRM cells by Prime-Trap immunization regimen. Here we asked if priming with DNA-MIF-4G followed by trapping with Adenovirus serotype 5 (Ad5) vectored-Kb17 would induce Kb17-specific liver CD8 TRM cells as well as protection against Pb spz challenge. Liver Kb17-specific CD8 TRM cells (CD69+CXCR6+) exceeded the number of these cells in the spleens, 27% vs 2%, respectively. Protection measured by liver parasite burden was CD8 T cell dependent. In contrast, prime:boost immunization with DNA/Ad5 MIF-4G induced only 8% liver CD8 TRM cells, as did control immunization with DNA/Ad5 empty vector. Utilizing LS Ags and the corresponding CD8 T cell-inducing epitopes in Prime-Trap immunization lends itself for further explorations as an ancillary approach to malaria vaccine regimens where liver CD8 TRM may be crucial in preventing virulent blood stages by targeting LS parasites.