Title : Assessment of vaccination with AMA1 of plasmodium knowlesi expressed in pichia pastoris against chimeric rodent malaria parasite
Abstract:
Introduction: Malaria continues to be one of the most significant infectious diseases globally. Its existence in various species complicates the development of an efficient broad-spectrum solution. One species that is gaining increased attention in Southeast Asia is Plasmodium knowlesi, a zoonotic parasite that has since 2018 been the sole cause of all indigenous malaria infections in Malaysia. To address the burden, this research was designed to evaluate the P. knowlesi Apical Membrane Antigen 1 (PkAMA1) as a malaria vaccine candidate.
Methodology: Recombinant PkAMA1 (DI-II and DI-II-III) was strategically expressed in P. pastoris (culture-induction-purification-characterization), followed by a pre-clinical study using a murine immunization-challenge model to assess the immunological efficacy of the protein on the chimeric rodent malaria parasite. This was elucidated in duplicate in 3 experimental groups. Under aseptic conditions, 50 µg of purified rPkAMA1 (domain DI-II and DI-II-III) was administered to groups 1 and 2, respectively, via the intramuscular route (prime immunization), whereas group 3 was unimmunized (naïve). Fourteen days later, mice in immunized groups were boosted with the same antigen concentration. On day 14 after boost immunization, half of each group was sacrificed for comparative immunoassay, while the remaining half was challenged with 1.0 x103 blood-stage parasite expressing PkAMA1 antigen via the intraperitoneal route. After the challenge, parasitemia monitoring (patency = >2%) and survival time analysis were evaluated daily for 14 days. All data was analyzed using the GraphPad Prism software version 10.1.1(207). The biostatistical tests used in this study were ANOVA, Kaplan–Meier survival plot and log-rank (Mantel-Cox) test.
Result: Protein characterization and quantification demonstrated that the rPkAMA1 was rightly expressed and suitable for downstream application. rPkAMA1 DI-II was best expressed at 72 hours of 1% methanol induction and OD600 3, while rPkAMA1 DI-II-III was best expressed at 48 hours of 2% methanol induction and OD600. Immunization with both rPkAMA1 protects against the chimeric rodent malaria parasite expressing PkAMA1. For the protective efficacy experiment of the antigen, mice in the immunized groups exhibited reduced blood-stage parasitemia levels, remaining prepatent (number of days to reach a 0.5-2% parasitemia after challenge), whereas all unimmunized mice become patent before and on day 5. For the assessment of protection experiment, ELISA quantified parasite-specific IgG produced against the PkAMA1 antigen and the titers were statistically significant (P<0.0001). There was also a significant difference in survival rates observed between both immunized groups compared to the naïve group. Furthermore, the study demonstrated that the immune response generated by rPkAMA1 (DI-II) was equivalent in terms of antibody responses to that elicited by the complete rPkAMA1 protein (DI-II-III), highlighting the potency of the DI-II domain.
Conclusion: The immunological insights revealed by this study can aid the utilization of PkAMA1 as an efficacious blood-stage vaccine for P. knowlesi. Future research should focus on the assessment of cellular immunity and memory responses to further validate the antigen's potential. These would facilitate pre-clinical trials and the antigen’s prospective role in malaria eradication.
