Identification and characterization of epitopes on the merozoite surface protein-??? (Msp-142) of plasmodium knowlesi / Cheong Fei Wen
Malaria is one of the infections that causes high global mortality and morbidity annually. Plasmodium knowlesi has been recognised as the fifth human Plasmodium species that can cause human malaria and it can be potentially life threatening. The merozoite surface protein 1 (MSP-1) undergoes two...
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my.um.stud.116312021-01-03T20:29:22Z Identification and characterization of epitopes on the merozoite surface protein-??? (Msp-142) of plasmodium knowlesi / Cheong Fei Wen Cheong, Fei Wen R Medicine (General) Malaria is one of the infections that causes high global mortality and morbidity annually. Plasmodium knowlesi has been recognised as the fifth human Plasmodium species that can cause human malaria and it can be potentially life threatening. The merozoite surface protein 1 (MSP-1) undergoes two proteolytic steps during maturation of merozoites and invasion of merozoites into erythrocytes. During the first process, the MSP-1 precursor polypeptide is cleaved into four major fragments including MSP-142. The secondary process further cleaves the MSP-142 into two fragments, MSP-133 and MSP-119. In the present study, the ~28 kDa recombinant protein MSP-133 and ~42 kDa recombinant protein MSP-142 of P. knowlesi (pkMSP-133 and pkMSP-142 respectively) were expressed using Escherichia coli system. The purified proteins were evaluated with malaria and non-malaria human patient sera using Western Blot and ELISA assays. In the Western Blot assays, pkMSP-133 showed sensitivity of 98.3% and specificity of 97.4% in detecting malaria antibodies. In ELISA, the sensitivity for pkMSP-133 was 76.3%, with the specificity 94.9%. The pkMSP-142 had a sensitivity of 91.0% for detection of human malaria in both assays. Specificity of pkMSP-142 was 97.5% and 92.6% in Western blots and ELISA, respectively. High sensitivity and specificity of pkMSP-133 and pkMSP-142 in immunoassays reveals that these two recombinant proteins could be useful in general sero-epidemiological screening. Sensitivity and specificity obtained for pkMSP-142 in Western Blot and ELISA assays were consistently higher (>90%) as compared to pkMSP-133 which had lower sensitivity in ELISA (<80%). Besides, the MSP-142, which is made up of the MSP-133 and MSP-119 regions, has immunodominant T cell and B cell epitopes. Hence, this study aimed to evaluate the immunogenicity of pkMSP-142 using mouse model and to identify iv the potential epitopes. Mice immunized with pkMSP-142 had increased levels of cytokine interferon-gamma, interleukin-2, interleukin-4, and interleukin-10 significantly as compared to the levels in negative control mice. Furthermore, the endpoint titres of pkMSP-142-raised antibody were high, with the following IgG isotype distribution: IgG1 > IgG2b > IgG3 > IgG2a. Potential epitopes on P. knowlesi MSP-142 were identified using synthetic peptide library and phage display library approaches. In these approaches, pkMSP-142- immunized mice sera were used for screening of the potential epitopes. Nine potential epitopes were identified using synthetic peptide library, and 14 using the phage display library. Two regions (residues 37-95 and residues 240-289) were identified to be the potential dominant epitope regions. Two peptides from the peptide library, P10 (TAKDGMEYYNKMGELYKQ) and P31 (RCLLGFKEVGGKCVPASI), were selected and evaluated using mouse model. P10 and P31-immunized mice sera reacted with recombinant P. knowlesi MSP-142, and the IgG isotype distribution was IgG2b > IgG1 > IgG2a > IgG3. Antibodies raised against P10 and P31 recognised P. knowlesi blood stage parasites, indicating that both peptides were immunogenic and might correspond to the epitopes that serve as the binding sites for antibodies on the parasites. Furthermore, interferon-gamma and interleukin-2 levels were significantly higher in P31-immunized mice. With further evaluations, P10 and P31 can potentially be used in the development of malaria vaccine and therapeutic agents 2014 Thesis NonPeerReviewed application/pdf http://studentsrepo.um.edu.my/11631/4/cheong.pdf Cheong, Fei Wen (2014) Identification and characterization of epitopes on the merozoite surface protein-??? (Msp-142) of plasmodium knowlesi / Cheong Fei Wen. PhD thesis, University of Malaya. http://studentsrepo.um.edu.my/11631/ |
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R Medicine (General) Cheong, Fei Wen Identification and characterization of epitopes on the merozoite surface protein-??? (Msp-142) of plasmodium knowlesi / Cheong Fei Wen |
description |
Malaria is one of the infections that causes high global mortality and morbidity
annually. Plasmodium knowlesi has been recognised as the fifth human
Plasmodium species that can cause human malaria and it can be potentially life
threatening.
The merozoite surface protein 1 (MSP-1) undergoes two proteolytic steps during
maturation of merozoites and invasion of merozoites into erythrocytes. During the first
process, the MSP-1 precursor polypeptide is cleaved into four major fragments
including MSP-142. The secondary process further cleaves the MSP-142 into two
fragments, MSP-133 and MSP-119.
In the present study, the ~28 kDa recombinant protein MSP-133 and ~42 kDa
recombinant protein MSP-142 of P. knowlesi (pkMSP-133 and pkMSP-142 respectively)
were expressed using Escherichia coli system. The purified proteins were evaluated
with malaria and non-malaria human patient sera using Western Blot and ELISA assays.
In the Western Blot assays, pkMSP-133 showed sensitivity of 98.3% and specificity of
97.4% in detecting malaria antibodies. In ELISA, the sensitivity for pkMSP-133 was
76.3%, with the specificity 94.9%. The pkMSP-142 had a sensitivity of 91.0% for
detection of human malaria in both assays. Specificity of pkMSP-142 was 97.5% and
92.6% in Western blots and ELISA, respectively. High sensitivity and specificity of
pkMSP-133 and pkMSP-142 in immunoassays reveals that these two recombinant
proteins could be useful in general sero-epidemiological screening.
Sensitivity and specificity obtained for pkMSP-142 in Western Blot and ELISA
assays were consistently higher (>90%) as compared to pkMSP-133 which had lower
sensitivity in ELISA (<80%). Besides, the MSP-142, which is made up of the MSP-133
and MSP-119 regions, has immunodominant T cell and B cell epitopes. Hence, this study
aimed to evaluate the immunogenicity of pkMSP-142 using mouse model and to identify
iv
the potential epitopes. Mice immunized with pkMSP-142 had increased levels of
cytokine interferon-gamma, interleukin-2, interleukin-4, and interleukin-10 significantly
as compared to the levels in negative control mice. Furthermore, the endpoint titres of
pkMSP-142-raised antibody were high, with the following IgG isotype distribution:
IgG1 > IgG2b > IgG3 > IgG2a.
Potential epitopes on P. knowlesi MSP-142 were identified using synthetic
peptide library and phage display library approaches. In these approaches, pkMSP-142-
immunized mice sera were used for screening of the potential epitopes. Nine potential
epitopes were identified using synthetic peptide library, and 14 using the phage display
library. Two regions (residues 37-95 and residues 240-289) were identified to be the
potential dominant epitope regions. Two peptides from the peptide library, P10
(TAKDGMEYYNKMGELYKQ) and P31 (RCLLGFKEVGGKCVPASI), were
selected and evaluated using mouse model. P10 and P31-immunized mice sera reacted
with recombinant P. knowlesi MSP-142, and the IgG isotype distribution was IgG2b >
IgG1 > IgG2a > IgG3. Antibodies raised against P10 and P31 recognised P. knowlesi
blood stage parasites, indicating that both peptides were immunogenic and might
correspond to the epitopes that serve as the binding sites for antibodies on the parasites.
Furthermore, interferon-gamma and interleukin-2 levels were significantly higher in
P31-immunized mice. With further evaluations, P10 and P31 can potentially be used in
the development of malaria vaccine and therapeutic agents |
format |
Thesis |
author |
Cheong, Fei Wen |
author_facet |
Cheong, Fei Wen |
author_sort |
Cheong, Fei Wen |
title |
Identification and characterization of epitopes on the merozoite surface protein-??? (Msp-142) of plasmodium knowlesi / Cheong Fei Wen |
title_short |
Identification and characterization of epitopes on the merozoite surface protein-??? (Msp-142) of plasmodium knowlesi / Cheong Fei Wen |
title_full |
Identification and characterization of epitopes on the merozoite surface protein-??? (Msp-142) of plasmodium knowlesi / Cheong Fei Wen |
title_fullStr |
Identification and characterization of epitopes on the merozoite surface protein-??? (Msp-142) of plasmodium knowlesi / Cheong Fei Wen |
title_full_unstemmed |
Identification and characterization of epitopes on the merozoite surface protein-??? (Msp-142) of plasmodium knowlesi / Cheong Fei Wen |
title_sort |
identification and characterization of epitopes on the merozoite surface protein-??? (msp-142) of plasmodium knowlesi / cheong fei wen |
publishDate |
2014 |
url |
http://studentsrepo.um.edu.my/11631/4/cheong.pdf http://studentsrepo.um.edu.my/11631/ |
_version_ |
1738506507914313728 |
score |
13.188404 |