2026_Assessment of Vaccination With Recombinant Protein Ama1 Plasmodium Knowlesi Expressed in Pichia Pastoris Against Chimeric Rodent Malaria Parasite
| Format: | General Document |
|---|
| _version_ | 1860798363742502912 |
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| building | INTELEK Repository |
| collection | Online Access |
| collectionurl | https://intelek.unisza.edu.my/intelek/pages/search.php?search=!collection8804 |
| copyright | Copyright©PWB2026 |
| country | Malaysia |
| date | 2025-09-07 |
| format | General Document |
| id | 17471 |
| institution | UniSZA |
| originalfilename | ASSESSMENT OF VACCINATION WITH RECOMBINANT PROTEIN AMA1 Plasmodium knowlesi EXPRESSED IN Pichia pastoris AGAINST CHIMERIC RODENT MALARIA PARASITE (MASTER_2025).pdf |
| person | Musa Ahmad Aminu |
| recordtype | oai_dc |
| resourceurl | https://intelek.unisza.edu.my/intelek/pages/view.php?ref=17471 |
| sourcemedia | Server storage Scanned document |
| spelling | 17471 https://intelek.unisza.edu.my/intelek/pages/view.php?ref=17471 https://intelek.unisza.edu.my/intelek/pages/search.php?search=!collection8804 General Document Malaysia Library Staff (Top Management) Library Staff (Management) Library Staff (Support) Terengganu Faculty of Health Sciences English application/pdf 1.4 148 Public Access Server storage Scanned document Universiti Sultan Zainal Abidin Universiti Sultan Zainal Abidin Dissertations, Academic Copyright©PWB2026 Thesis Musa Ahmad Aminu Plasmodium Knowlesi Apical Membrane Antigen 1 (AMA1) Recombinant Protein Vaccine Pichia Pastoris Expression System Chimeric Rodent Malaria Parasite Blood-stage Malaria Vaccine Murine Immunization-challenge Model IgG Antibody Response Pre-clinical Vaccine Evaluation Zoonotic Malaria Malaria—Vaccination Zoonoses 2026_Assessment of Vaccination With Recombinant Protein Ama1 Plasmodium Knowlesi Expressed in Pichia Pastoris Against Chimeric Rodent Malaria Parasite 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 potentially fatal zoonotic parasite that has, since 2018, been the sole cause of all indigenous malaria infections and deaths 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. Recombinant PkAMA1 (DI-II and DI-II-III) was strategically expressed in P. pastoris, 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 twice in three experimental groups. Under aseptic conditions, 50 μg of purified rPkAMA1 (domains 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 the 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 × 10³ blood-stage parasites expressing the 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 were analyzed using the GraphPad Prism software version 10.1.1 (207). The biostatistical tests used in this study were ANOVA, Tukey’s HSD test, Kaplan–Meier survival plot, and log-rank (Mantel–Cox) test. Protein characterization and quantification demonstrated that the rPkAMA1 was properly expressed and suitable for downstream application. rPkAMA1 DI-II was best expressed at 72 hours of 1% methanol induction and OD600 3–4, while rPkAMA1 DI-II-III was best expressed at 48 hours of 2% methanol induction and OD600 4–5. Immunization with rPkAMA1 (DI-II and DI-II-III) protected mice against the chimeric rodent malaria parasite expressing PkAMA1 (PkAMA1-CRMP), a novel finding for the mouse model. For the protective efficacy experiment of the antigen, mice in the immunized groups exhibited reduced blood-stage parasitemia levels, remaining prepatent (number of days within ≤ 2% parasitemia after challenge). In contrast, all unimmunized mice became patent before or on day 5. For the assessment of the 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), providing a novel finding on the potency of the DI-II domain. 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. 2025-09-07 uuid:744d6557-0731-4dad-807f-82c259510c30 macOS Version 15.6.1 (Build 24G90) Quartz PDFContext ASSESSMENT OF VACCINATION WITH RECOMBINANT PROTEIN AMA1 Plasmodium knowlesi EXPRESSED IN Pichia pastoris AGAINST CHIMERIC RODENT MALARIA PARASITE (MASTER_2025).pdf Malaria—Molecular aspects Malaria—prevention & control |
| spellingShingle | 2026_Assessment of Vaccination With Recombinant Protein Ama1 Plasmodium Knowlesi Expressed in Pichia Pastoris Against Chimeric Rodent Malaria Parasite |
| state | Terengganu |
| subject | Dissertations, Academic Malaria—Vaccination Zoonoses Malaria—Molecular aspects Malaria—prevention & control |
| summary | 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 potentially fatal zoonotic parasite that has, since 2018, been the sole cause of all indigenous malaria infections and deaths 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. Recombinant PkAMA1 (DI-II and DI-II-III) was strategically expressed in P. pastoris, 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 twice in three experimental groups. Under aseptic conditions, 50 μg of purified rPkAMA1 (domains 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 the 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 × 10³ blood-stage parasites expressing the 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 were analyzed using the GraphPad Prism software version 10.1.1 (207). The biostatistical tests used in this study were ANOVA, Tukey’s HSD test, Kaplan–Meier survival plot, and log-rank (Mantel–Cox) test. Protein characterization and quantification demonstrated that the rPkAMA1 was properly expressed and suitable for downstream application. rPkAMA1 DI-II was best expressed at 72 hours of 1% methanol induction and OD600 3–4, while rPkAMA1 DI-II-III was best expressed at 48 hours of 2% methanol induction and OD600 4–5. Immunization with rPkAMA1 (DI-II and DI-II-III) protected mice against the chimeric rodent malaria parasite expressing PkAMA1 (PkAMA1-CRMP), a novel finding for the mouse model. For the protective efficacy experiment of the antigen, mice in the immunized groups exhibited reduced blood-stage parasitemia levels, remaining prepatent (number of days within ≤ 2% parasitemia after challenge). In contrast, all unimmunized mice became patent before or on day 5. For the assessment of the 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), providing a novel finding on the potency of the DI-II domain. 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. |
| title | 2026_Assessment of Vaccination With Recombinant Protein Ama1 Plasmodium Knowlesi Expressed in Pichia Pastoris Against Chimeric Rodent Malaria Parasite |
| title_full | 2026_Assessment of Vaccination With Recombinant Protein Ama1 Plasmodium Knowlesi Expressed in Pichia Pastoris Against Chimeric Rodent Malaria Parasite |
| title_fullStr | 2026_Assessment of Vaccination With Recombinant Protein Ama1 Plasmodium Knowlesi Expressed in Pichia Pastoris Against Chimeric Rodent Malaria Parasite |
| title_full_unstemmed | 2026_Assessment of Vaccination With Recombinant Protein Ama1 Plasmodium Knowlesi Expressed in Pichia Pastoris Against Chimeric Rodent Malaria Parasite |
| title_short | 2026_Assessment of Vaccination With Recombinant Protein Ama1 Plasmodium Knowlesi Expressed in Pichia Pastoris Against Chimeric Rodent Malaria Parasite |
| title_sort | 2026_assessment of vaccination with recombinant protein ama1 plasmodium knowlesi expressed in pichia pastoris against chimeric rodent malaria parasite |