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.

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Molecular diagnostic tests for Plasmodium falciparum parasites are increasingly used to enable ultrasensitive detection of infection in clinical trials and field surveillance studies. Ribonucleic acid (RNA)-based assays targeting 18S rRNA are particularly sensitive with limits of detection reported to comprise a single infected red blood cell (RBC) in a relatively large volume of blood. However, the validation testing at such limiting concentrations is hampered by the so-called Poisson distribution of such rare events, which can lead laboratorians to inaccurately set the limit of detection higher (i.e., less sensitive) than the assay can actually detect. Here we set out to formally demonstrate the analytical sensitivity of the Plasmodium 18S rRNA quantitative reverse transcription PCR (qRT-PCR). Fluorescence-activated cell sorting (FACS) was used on synchronous P. falciparum cultures doubly stained for DNA and RNA and was followed by qRT-PCR on the individual sorted cells spiked with negative whole blood. Over 95% of individual single-ring infected RBCs were detected by qRT-PCR. The formally measured median 18S rRNA content per individual ring-stage P. falciparum parasite was 9,550 copies (interquartile range 8,130–12,300). Thus, one can confidently rely on Plasmodium 18S rRNA qRT-PCR to detect one parasite per 50-µL blood sample.

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