Repeated intravenous (IV) administration of radiation-attenuated sporozoite (RAS) vaccines induces Plasmodium-specific CD8+ liver-resident T (Trm) cells in mice and achieves sterile protection against challenge. Our heterologous “prime-and-trap” vaccine strategy was previously shown to simplify and improve upon RAS vaccination. Prime-and-trap vaccination combines epidermal priming by DNA-encoded circumsporozoite protein (CSP) antigen followed by a single IV dose of freshly dissected RAS (fresh-RAS) to direct and trap activated and expanding CD8+ T cells in the liver. Prime-and-trap vaccination protects mice against wild-type sporozoite (spz) challenge. Assessment of prime-and-trap vaccines in nonhuman primate (NHP) models and/or humans would be greatly enabled if fresh-RAS could be replaced by cryopreserved RAS (cryo-RAS). Here, we investigated if fresh-RAS could be replaced with cryo cryo-RAS for prime-and-trap vaccination in BALB/cj mice. Despite a reduction in spz vaccine liver burden following cryo-RAS administration compared with fresh-RAS, cryo-RAS induced a similar level of Plasmodium yoelii (Py) CSP-specific CD8+ liver Trm cells and completely protected mice against Pyspz challenge 112 days after vaccination. Additionally, when the glycolipid adjuvant 7DW8-5 was coadministered with cryo-RAS, 7DW8-5 permitted the dose of cryo-RAS to be reduced 4-fold while still achieving high rates of sterile protection. In summary, cryo-RAS with and without 7DW8-5 were compatible with prime-and-trap malaria vaccination in a mouse model, which may accelerate the pathway for this vaccine strategy to move to NHPs and humans.

Malaria is caused by Plasmodium parasites and was responsible for over 247 million infections and 619,000 deaths in 2021. Radiation-attenuated sporozoite (RAS) vaccines can completely prevent blood stage infection by inducing protective liver-resident memory CD8+ T cells. Such T cells can be induced by ‘prime-and-trap’ vaccination, which here combines DNA priming against the P. yoelii circumsporozoite protein (CSP) with a subsequent intravenous (IV) dose of liver-homing RAS to “trap” the activated and expanding T cells in the liver. Prime-and-trap confers durable protection in mice, and efforts are underway to translate this vaccine strategy to the clinic. However, it is unclear whether the RAS trapping dose must be strictly administered by the IV route. Here we show that intradermal (ID) RAS administration can be as effective as IV administration if RAS are co-administrated with the glycolipid adjuvant 7DW8-5 in an ultra-low inoculation volume. In mice, the co-administration of RAS and 7DW8-5 in ultra-low ID volumes (2.5 µL) was completely protective and dose sparing compared to standard volumes (10–50 µL) and induced protective levels of CSP-specific CD8+ T cells in the liver. Our finding that adjuvants and ultra-low volumes are required for ID RAS efficacy may explain why prior reports about higher volumes of unadjuvanted ID RAS proved less effective. The ID route may offer significant translational advantages over the IV route and could improve sporozoite vaccine development.

Generating liver resident-memory CD8+ T cells (TRM cells) is critical for effective liver-stage malaria vaccine. The role of biological sex in liver-stage vaccine protection is understudied. Here we report sex-specific immune responses and protection outcomes for the two-step heterologous ‘Prime-and-Trap’ liver-stage malaria vaccine designed to induce liver TRM cells. The Prime-and-Trap strategy combines sporozoite antigen DNA priming with a single intravenous dose of liver-homing radiation-attenuated sporozoites (RAS) to direct and “trap” activated and expanding T cells in the liver. This strategy induces robust liver CD8+ TRM cell responses and confers sterile protection in the P. yoelii (Py) rodent malaria model in female mice. However, when tested in male mice, it induces weaker liver CD8+ TRM cell responses and little to no protection from sporozoite challenge. We examined sex-divergent immune responses in the spleen and liver following each step of the Prime-and-Trap regimen. We found that sexually dimorphic activation of certain immune pathways drives attenuated downstream retention of TRM cells in the liver, leading male mice to generate a sub-optimal immune response that ultimately fails to protect from Py sporozoite challenge. Our findings emphasize the importance of incorporating sex as a variable for evaluation in the design of robust liver-stage malaria vaccines. Supported by grants from NIH (R01 AI141857).

7DW8-5 is a potent glycolipid adjuvant that improves malaria vaccine efficacy in mice by inducing IFN-γ and increasing protective CD8+ T cell responses. The addition of 7DW8-5 was previously shown to improve the efficacy of a CD8+ T cell-mediated heterologous ‘prime-and-trap’ malaria vaccine against Plasmodium yoelii sporozoite challenge in inbred female mice. Here, we report significant differential sex-specific responses to 7DW8-5 in inbred and outbred mice. Male mice express significantly less IFN-γ and IL-4 compared to females following intravenous 7DW8-5 administration. Additionally, unlike in female mice, 7DW8-5 did not improve the vaccine efficacy against sporozoite challenge in prime-and-trap vaccinated male mice. Our findings highlight the importance of including both female and male sexes in experimental adjuvant studies.

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