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Asian zoonotic schistosomiasis caused by S. japonicum is still considered to be a major health problem in China, the Philippines and some parts of Indonesia. One of the major factors that is delaying the elimination of this disease in locations with low prevalence is the low sensitivity of the currently used microscopic diagnostic method. To facilitate the efforts undertaken in the disease endemic countries to control and eliminate the disease, the development of more sensitive and specific diagnostic tools is required. Therefore, multi-epitope recombinant proteins were applied to improve the sensitivity and specificity of ELISA antigens to be used in the diagnosis of S. japonicum in humans. The eight Schistosoma proteins with potential diagnostic performance as ELISA antigens, SjTPx-1, Sj7TR, SjPCS, SjLP40, SjPrx-4, SjSAP4, SjSAP5 and Sj23-LHD, were subjected to epitope prediction analyses to extract their “epitope” sequences. Based on the results, 13 “epitope” sequences were extracted and by combining 4 to 10 of them together, three multi-epitope proteins, namely SjChimeric 1, SjChimeric 2, and SjChimeric 3, were designated and constructed. Recombinant proteins of all eight of the single proteins and the three multi-epitope proteins were produced, and their diagnostic performance as ELISA antigens was evaluated with a panel of archived serum samples collected from 30 stool-positive patients, 30 endemic negative individuals and 30 non-endemic individuals. The ELISA results showed better and improved diagnostic performance of SjChimeric 2 (93.3% sensitivity and 96.7% specificity) and SjChimeric 3 (90% sensitivity and 93.3 specificity) as compared with those of the single proteins. This study suggests that the introduction of multi-epitope proteins can improve the diagnostic performance of the ELISA antigen and promote the development of more sensitive and specific diagnostic tools to detect human schistosomiasis.
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Table 4. Primers used in this study for molecular cloning
Target gene Forward (5'-3') Reverse (5'-3')
SjTPx-1 TTA GGA TCC ATG GTA CTG ATT CCA AAT TTA AAG CTT TAA TCA GTG ATT CAC TTT Sj7TR GCC ATA TGC CTG CTG AAC CAG TTG AAT CGA GCT CGA ATT CCT ATA TTT CCT C SjPCS GCC ATA TGG AGA GAG AAC TTG T GCG TCG ACT TAG TGG TTT ATA CAA SjLP40 TTA GTG GTT TAT ACA ACG TGT ACA C CGG AGC TCT TAA TGA CTG ACTG SjPrx-4 GCG GAT CCA TGC TGT TAC CAA CG GCC TCG AGT CAT GTA GAT GAA GAG SjSAP5 CCG GAA TTC ATG TCA ACA TTG AAC CGG TCG ACT TAT GCA AAA GGA T SjSAP4 CGC ATA TGG CAA ACC ACA CTG AG GCG TCG ACT TAT AAT GGA CAC AAC Sj23LHD GCG AAT TCG CAT GAC TGG TGC GCG TCG ACC TAG TTG CGT TTT AAG SjChimeric 1 GCG AAT TCA TGG ACA AGA TCG ACG CGC TCG AGT TAC TTC GGC AGT TGA A SjChimeric 2 CGC ATA TGA AAC AGC TGT GC GCG TCG ACT TAG CTT TTC GCT TC SjChimeric 3 GCC ATA TGA TGT GCA TCC ACA TTA CGG TCG ACT TAG ATA AAG CTT TTC
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Table 5. List of selected peptides Protein Predicted
epitope Selected peptide Start
position
SjTPx-1 SjTPx-1_E1 YSHLAWTKQDRKSGGLG 78
SjTPx-1_E2 ACSTDSKYSHLAWTKQDRKSGGLGDMRIPLLADPTKSIARA YGVLDEEEGNAFRGLFIIDPKGILRQITVNDKPVGRSVDETL
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Sj7TR Sj7TR_E LTHDEQTDDAPLSEDIEQKET 30
SjPCS SjPCS_E MNQVDSDTQLPRGFVVLRK 279
SjLP40 SjLP40_E1 PDYESITFTDNRQLGIHPKSANQ 221
SjLP40_E2 GPTILDDGANGKRLHLEVPVD 260
SjPrx-4 SjPrx-4_E IIAFGERCEEFEKKNCQV 51
SjSAP4 SjSAP4_E1 LRTMLTWCQPSLKTANES 100
SjSAP4_E2 CIHIMASETEMILKDFIDFVEINKLRTMLTWCQPSLKTAN ESNSTFVCSTCETIVTFMKTFTQSEEAKS
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SjSAP5 SjSAP5_E1 QACNSTTNSLTHRTGFGDNILCEE 113
SjSAP5_E2 KQLCESIPPGSLRNTCLTYVNEYLLLALKVMANSVRPESI CKALQACNSTTNSLTHRTGFGDNILCEECRSGFILLKSFI
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Sj23LHD Sj23LHD_E1 DKIDDEINTLMTGALENP 110
Sj23LHD_E2 IQTSFHCCGVKGPDDYKGNVP 139
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Table 6. ELISA results of single proteins and multi-epitope recombinant proteins with human samples
Protein Sensitivity (%) Specificity (%) PPV NPV
SjTPx-1 83.3 93.3 92.6 84.9
Sj7TR 80.0 93.3 92.3 82.4
SjPCS 73.3 83.3 81.5 75.8
SjLP40 83.3 90.0 89.3 84.4
SjPrx-4 83.3 86.7 86.1 83.8
SjSAP4 86.7 90.0 89.7 87.1
SjSAP5 83.3 86.7 86.2 83.8
Sj23LHD 76.7 100.0 100.0 81.1
SjChimeric 1 80.0 100.0 100.0 83.3
SjChimeric 2 93.3 96.7 96.6 93.6
SjChimeric 3 90.0 93.3 93.1 90.3
PPV, positive predictive value; NPV, negative predictive value.
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Figure 13. Constructs of multi-epitope recombinant proteins.
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Figure 14. Amplification of target genes by PCR. (A) SjTPx-1; (B) Sj7TR; (C) SjPCS; (D) SjLP40;
(E) SjPrx-4; (F) Sj23LHD, SjSAP5 and SjSAP4: Lane 1: Sj23LHD; Lane 2: SjSAP5; Lane 3: SjSAP4;
Lane M: marker 100 bp.
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Figure 15. Expression and purification analysis of single recombinant proteins. (A) SjTPx-1;
(B) Sj7TR; (C) SjPCS; (D) SjLP40; (E) SjPrx-4; (F) SjSAP4; (G) SjSAP5; (H) Sj23LHD: Lane M: molecular marker; Lane 1: Bacterial lysate before induction; Lane 2: Bacterial lysate after IPTG induction; Lane 3: purified proteins.
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Figure 16. Expression and purification analysis of multi-epitope recombinant proteins.
(A) SjChimeric 1; (B) SjChimeric 2; (C) SjChimeric 3. Lane M: molecular marker; Lane 1:
Bacterial lysate before induction; Lane 2: Bacterial lysate after IPTG induction; Lane 3:
purified proteins.
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Figure 17. ELISA results of single recombinant proteins and multi-epitope recombinant proteins against human serum samples. Red dashed lines correspond to the cut-off values.
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GENERAL DISCUSSION
The WHO has targeted the elimination of schistosomiasis by 2025 in all 78 of the countries in which it remains endemic. Thus, drug development and strengthened disease surveillance are needed to ensure the success of the control programs in these areas. Therefore, the aims of this study were to develop and test tools that can provide a better understanding of schistosomiasis control in terms of the assessment of therapeutic efficacy in an animal model for drug development and to improve the sensitivity and specificity of serodiagnosis for S. japonicum infection.
In the first chapter of this study, the establishment of real-time PCR was done to assess the egg burden in infected organs using an experimental mouse model. As one of the pathological analyses, assessment of egg burden in the affected organs has been done by histopathological examination, a time-consuming and laborious method. Taking advantage of the real-time PCR protocol targeting the S. japonicum mitochondrial NADH dehydrogenase I region for detecting the parasite DNA from stool samples (23), the method was applied to evaluate the egg burden in organ samples in a mouse model of S. japonicum infection. The results suggested that real-time PCR would improve the accuracy of evaluating egg burden in a mouse model experimentally infected with S. japonicum. Real-time PCR has been widely used to detect S.
japonicum DNA in stool samples (24,106), but this is the first time, to my knowledge, that the method has been applied to detect S. japonicum DNA in the organs of infected mice. Real-time PCR can complement traditional histopathological examinations.
In the second chapter, the study aimed to characterize the immunohistochemical properties of S. japonicum peroxiredoxin 4 and evaluate the serodiagnostic potential of this protein as an antigen in ELISA. In the era of whole genome sequencing, the S. japonicum genome has been successfully assembled (107-109), which has led to opportunities for continued development and improvement of serological assays by analyzing many more antigens such as
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excretory/secretory (110) and transmembrane proteins (101) from the online database. Three types of S. japonicum thioredoxin peroxidase (TPx), also called peroxiredoxin (Prx), have been cloned and characterized (72). Among them, SjTPx-2 and SjTPx-3 were evaluated as vaccine candidates against schistosome infection (81,82), and only SjTPx-1 was evaluated as a diagnostic antigen candidate in human (31), water buffalo (76) and dog (111). A new type of Prx family in S. japonicum as S. japonicum Prx 4 (SjPrx-4) was identified from the database.
SjPrx-4 was characterized for both its antioxidant activity and immunolocalization and evaluated for its diagnostic potential in archived S. japonicum-infected patient serum samples.
The results of antioxidant activity and immunohistochemistry showed that SjPrx-4 was an antioxidant protein and it localized in the tegument of the parasite. These findings suggested that SjPrx-4 was exposed to host tissues, and it might help the pathogen not only to evade host immune defenses and protect itself from oxidative stress (112) but also to modulate host immune responses. The results of ELISA with patient sera have confirmed the diagnostic potential of SjPrx-4 with a sensitivity of 83.3% and specificity of 87.8%. However, when comparing the results of SjPrx-4 with those of SjTPx-1, different kinetics of infected human antibodies response to two antigens were found. Therefore, it was surmised that the two antigens might complement each other to offer a better synergistic diagnostic potential. The protocol of mixing multiple antigens in a cocktail format has also been used to improve the diagnostic capabilities of the tests of other parasitic diseases (63,65,113,114). Therefore, SjPrx-4 and SjTPx-1 were combined, and the diagnostic performance of the combined antigen in ELISA was evaluated. The results confirmed that the combined antigens improved the sensitivity and specificity of the ELISA to 90% and 92.7%, respectively, as compared with those of the single antigens alone. The use of combined antigens in ELISA not only helps to improve the sensitivity of the diagnostic test but also suggests a possible way to construct multi-epitope fusion antigens to enhance the accuracy of the diagnosis of human schistosomiasis.
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Thus, the objective of the third chapter of this study, is to develop more sensitive and specific means to diagnose human schistosomiasis using the multi-epitope fusion protein, SjPrx-4 from the previous chapter and other antigens with reported diagnostic potentials, such as SjTPx-1, Sj7TR (31), SjPCS, Sj40LP, SjSAP4, SjSAP5 and Sj23LHD (33). Based on B-cell epitope prediction from these eight proteins, three multi-epitope fusion proteins were designed, and their recombinant proteins were produced as SjChimeric 1, SjChimeric 2 and SjChimeric 3. The diagnostic performances of the eight recombinant individual proteins were reevaluated as well as the three fusion proteins using ELISA. The ELISA results showed that all of the single antigens had sensitivities and specificities of more than 70%. On the other hand, the fusion proteins SjChimeric 2 and SjChimeric 3 showed high sensitivities of 93% and 90%
respectively. Their specificities were also high at 96.7% and 93.3%, respectively. SjChimeric 1, however, did not fare so well in terms of sensitivity as compared to that of the single antigens.
Generally, multi-epitope fusion proteins improve the sensitivity of the test as compared to that with the individual proteins. SjChimeric 2 showed the best diagnostic potential among the three multi-epitope recombinant proteins being evaluated. Therefore, SjChimeric 2 might be a good diagnostic antigen that can be applied in the diagnosis of human schistosomiasis in areas with a low prevalence of the disease.
On other hand, one of the major factors hindering the control of S. japonicum infection in the endemic areas is the neglected contribution of animals to the transmission of this parasitic disease (115,116). Animals known to be important in the transmission include dogs, pigs, cattle and water buffaloes (117,118). Therefore, the development of a sensitive and specific diagnostic test for the surveillance of infection in multiple host species is necessary. Further study to evaluate the antigens such as SjChimeric 2 for their diagnostic performance in ELISA with animal samples should be conducted.
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The development of more sensitive and reliable tests will help to identify the true status of the infection and allow the selection of appropriate control measures in endemic areas in which the disease prevalence has already reached a level of near elimination. Furthermore, the antigens offering improved performance can help in developing point-of-care testing with high sensitivity. Such testing is needed for field application to detect the infection in a short time to avoid delayed or unnecessary treatment and to confirm parasite clearance on site. Attainment of these goals will help to minimize economic losses and lead to more efficient drug administration in the treatment of schistosomiasis.
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GENERAL CONCLUSION
In chapter 1, the application of real-time PCR targeting of the NADH dehydrogenase I gene of S. japonicum was done for the assessment of the egg burden in organ tissue samples collected from a mouse model experimentally infected with S. japonicum. The Ct value determined by real-time PCR for the liver sample correlated well with its egg burden as estimated by microscopic examination of the tissue sections. Real-time PCR could detect the parasite DNA in brain and spleen whereas the egg was not observed by microscopic examination of the affected tissue sections. Real-time PCR has proved to be a powerful tool in providing an acute estimation of the egg burden in the infected organs collected from an animal model and may lead to improved drug development and provide additional insights into the pathobiology of schistosomiasis.
In chapter 2, biochemical properties of S. japonicum peroxiredoxin-4 (SjPrx-4) and its diagnostic performance as an ELISA antigen were reported. S. japonicum may use SjPrx-4 as an antioxidant enzyme to protect itself from oxidative stress derived from the host immune system and the parasite metabolism. Recombinant protein of SjPrx-4 was also a potential antigen in ELISA that could detect S. japonicum infection in humans. Moreover, combination of the antigens SjPrx-4 and SjTPx-1 improved the sensitivity of the test to 90%, suggesting the possibility of fusing antigens to improve the sensitivity of ELISA in the diagnosis of human schistosomiasis.
In chapter 3, three multi-epitope fusion antigens were constructed using B-cell epitope sequences extracted from eight S. japonicum proteins previously reported as potential antigens to develop a more sensitive ELISA. Two multi-epitope recombinant proteins, SjChimeric 2 and SjChimeric 3, showed higher sensitivity in the ELISA than those of the eight single recombinant proteins alone. Among them, SjChimeric 2 showed the highest sensitivity of 93.3% and high specificity of 96.7%. SjChimeric 2 could potentially be used as a diagnostic antigen to develop
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a robust diagnostic test that can strengthen disease surveillance to ensure the success of the control programs in the endemic areas already showing low prevalence of the disease.
In conclusion, this study has proposed two useful tools for schistosomiasis control. First, real-time PCR can be applied to the evaluation of egg burden in the organs collected from parasite-infected animal models for use in novel drug development to promote better disease control. Second, multi-epitope fusion proteins might be useful in developing reliable and accurate diagnostic tests for disease surveillance and assessment of the efficacy of an MDA program directed at the elimination of schistosomiasis.
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ACKNOWLEDGEMENTS
This thesis would have not been completed without the great help and support of my supervisors, colleagues, friends and family.
First and foremost, I would like to express my special and sincere gratitude to my supervisor, Prof. Shin-ichiro Kawazu, for his great help and excellent mentoring all throughout my studies. Thank you, Sensei, for giving me this opportunity to pursue my doctoral degree in Japan. I have learned a lot from you, and I consider this knowledge to be a precious gift in my life. Your unending support and guidance in my research work enabled me to proceed to its final completion.
I would also like to thank my co-supervisors, Prof. Hiroshi Suzuki at NRCPD, and Prof.
Tadashi Itagaki at Iwate University, for their useful advice and comments to improve my research work.
My special thanks to all of the other professors at the NRCPD for their comments and suggestions during my progress report seminars at the NRCPD.
My sincere appreciation also goes to Dr. Jose Ma. Angeles, my mentor, and my brother.
This work would not have been possible without your help. I greatly value your support and guidance in my study. It will take a lifetime for me to express how thankful I am to you.
I greatly appreciate my seniors Dr. Thu Thuy Nguyen, Dr. Kharleezelle J. Moendegand Dr.
Adrian Miki C. Macalanda for their sincere friendship, beneficial discussions and help.
I would like to thank Assoc. Prof. Yasuyuki Goto of the University of Tokyo for his advice, comments and suggestions to improve my research work.
I would like to express my sincerest gratitude to Assoc. Prof. Masashi Kirinoki of the Department of Tropical Medicine and Parasitology, Dokkyo Medical University School of Medicine, Japan for providing me the samples used in my experiments and for his guidance on animal infection.
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I would like to acknowledge the financial support from MEXT and the academic support of both the Obihiro University of Agriculture and Veterinary Medicine (OUAVM) and the United Graduate School of Veterinary Sciences, Gifu University, for my doctoral study. My special thanks also go to the International Student Affairs Section of OUAVM, including Ms. Mizuki Chiba, Ms. Yuki Hara and other staff members, for their support and assistance throughout my doctoral study.
I am really thankful to the current and former international students and to all individuals that I met during my stay in Obihiro; I am glad to have enjoyed their friendship.
I would also like to extend my heartfelt gratitude to my current and former lab mates Assist.
Prof. Keisuke Suganuma, Dr. Daiki Mizushima, Dr. Ehab Mossaad, Dr. Batdorj Davaasuren, Dr. Nthatisi I. Molefe, Dr. Yuma Ohari, Ms. Atcharaphan Wanlop, Ms. Afraa Elata, Ms. Luna Higuchi, Ms. Chiho Oto and other students for helping and giving me some insights in my experiments, and to friends in Obihiro for their help and friendship during my doctoral studies.
To Onishi san, I can’t thank you enough for all of the help that you have extended to me during my stay here in Obihiro.
I would like to thank current and former Vietnamese at Obihiro, Mr. Dong Van Hieu, Ms.
Tran Thi Huong Giang, Mr. Bui Van Dung, Ms. Nguyen Thi Minh Huong, Dr. Dao Duy Tung, Ms. Nguyen Tra Vy and Ms. Mong Thi Ho Diep, for their kind help given to me in ordinary life.
I am deeply grateful to Dr. Van Cao, my supervisor at the Pasteur Institute in Ho Chi Minh City, Viet Nam, for allowing and supporting me to pursue my doctoral degree in Japan.
Finally, I express my deepest gratitude to my parents Dang Dinh Ba and Trinh Thi Hong and my younger sister Dang Trinh Bich Diep for the everlasting love of family. I am eternally grateful to my relatives and friends in Viet Nam for their constant support, understanding, love and guidance all throughout my study.
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