Efficient infection of non-human primates with purified, cryopreserved Plasmodium knowlesi sporozoites

Abstract

Background Plasmodium falciparum (Pf) sporozoite (SPZ) vaccines are the only candidate malaria vaccines that induce > 90% vaccine efficacy (VE) against controlled human malaria infection and the only malaria vaccines to have achieved reproducible VE against malaria in adults in Africa. The goal is to increase the impact and reduce the cost of PfSPZ vaccines by optimizing vaccine potency and manufacturing, which will benefit from identification of immunological responses contributing to protection in humans. Currently, there is no authentic animal challenge model for assessing P. falciparum malaria VE. Alternatively, Plasmodium knowlesi (Pk), which infects humans and non-human primates (NHPs) in nature, can be used to experimentally infect rhesus macaques (Macaca mulatta) to assess VE. Methods Sanaria has, therefore, produced purified, vialed, cryopreserved PkSPZ and conducted challenge studies in several naïve NHP cohorts. In the first cohort, groups of three rhesus macaques each received doses of 5 × 102, 2.5 × 103, 1.25 × 104 and 2.5 × 104 PkSPZ administered by direct venous inoculation. The infectivity of 1.5 × 103 PkSPZ cryopreserved with an altered method and of 1.5 × 103 PkSPZ cryopreserved for four years was tested in a second and third cohort of rhesus NHPs. The lastly, three pig-tailed macaques (Macaca nemestrina), a natural P. knowlesi host, were challenged with 2.5 × 103 PkSPZ cryopreserved six years earlier. Results In the first cohort, all 12 animals developed P. knowlesi parasitaemia by thick blood smear, and the time to positivity (prepatent period) followed a non-linear 4-parameter logistic sigmoidal model with a median of 11, 10, 8, and 7 days, respectively (r2 = 1). PkSPZ cryopreserved using a modified rapid-scalable method infected rhesus with a pre-patent period of 10 days, as did PkSPZ cryopreserved four years prior to infection, similar to the control group. Cryopreserved PkSPZ infected pig-tailed macaques with median time to positivity by thin smear, of 11 days. Conclusion This study establishes the capacity to consistently infect NHPs with purified, vialed, cryopreserved PkSPZ, providing a foundation for future studies to probe protective immunological mechanisms elicited by PfSPZ vaccines that cannot be established in humans.

Plasmodium knowlesi in pig-tailed macaques: a new model for malaria vaccine research

“…After a period of blood stage infection, cohort 1 animals were treated with antimalarial drugs and returned to the colony. Preliminary data describing the time to blood stage infection following PkSPZ challenge in this cohort was reported previously [32]. This data is again presented here along with substantial new data from this cohort.…”

“…1D). Thus, naïve PTM reliably develop patent blood stage infections after PkSPZ challenge, with a time to rst detection of blood stage parasites comparable to in rhesus macaques [32]. Then, upon re-challenge, all animals again reliably develop RT-PCR-detectable infections, but these typically remain below the limit of detection of thin blood smears.…”

“…The challenge dose for this study was 2.5x10 3 PkSPZ. This dose was selected because prior experiments in rhesus macaques demonstrated that 2.5x10 3 PkSPZ infects 100% of animals with highly reproducibile resulting blood stage parasite densities [32]. PkSPZ were vialled at 5x10 4 PkSPZ per vial, so a single vial was used for all animals challenged at a given time.…”

“…To evalute PTM as a model for malaria research, the study team therefore initiated a series of Pk challenge and infection studies at the Washington National Primate Research Center (WaNPRC). The team's earlier report described preliminary data con rming that PTM could be challenged with cryopreserved Pk sporozoites (PkSPZ), with an equivalent time to blood stage infection as in rhesus macaques [32]. Here, additional exploratory studies were performed to further evaluate infection outcomes in this proposed new research model.…”

Vaccination by single dose sporozoite injection of blood stage attenuated malaria parasites

“…In addition, mutant parasites with slow growing blood stages but still showing virulence characteristics could be used as a basis to generate double or triple gene-deletion mutants that might attenuate virulence. Finally, the human-infecting zoonotic P. knowlesi parasite could also be tested for slow growth as an intermediate pre-clinical model 51,52 as it can be cultured in vitro like P. falciparum but shows a higher transfection efficacy and a fast red blood stage cycle of just 24 hours 53 . Lastly, slow growing P. falciparum parasites were recently also identified by a screen using selection linked resistance marker integration, which could help to prescreen for gene-deletions resulting in an avirulent phenotype 54 .…”