Immunogenicity of Schistosoma japonicum Sj26gst mRNA vaccine candidate

doi:10.12140/j.issn.1000-7423.2023.05.004

Abstract

Abstract:

Objective To evaluate glutathione S-transferase subunit of relative molecular mass (M_r) 26 000 (Sj26gst) mRNA of Schistosoma japonicum and assess its immunogenicity. Methods The Sj26gst coding sequence was optimized by the commonly used human codons according to the Sj26gst coding region sequence (NC_002544.1). The gene was synthesized by adding the 3' and 5' non-coding regions (UTR) of beta-globulin at both ends, and then connected to pcDNA3.1(+) to construct the linear pcDNA3.1/Sj26gst plasmid which was used for transient transcription of Sj26gst mRNA in vitro. The Sj26gst mRNA was transfected into HeLa cells by using liposomes. The HeLa cells were collected to detect the relative expression level of Sj26gst mRNA by quantitative immunofluorescence PCR (qRT-PCR) and examine the expression level of Sj26GST protein by Western blotting at 2, 6, 12, 24, 48 hours after the transfection. Female BALB/c mice were randomly divided into four groups: Sj26gst-mRNA group, pcDNA3.1/Sj26gst group, pcDNA3.1 group and PBS group (6 mice in each group). Mice were immunized three times at two-week interval with 100 μg of Sj26gst mRNA, pcDNA3.1/Sj26gst plasmid, pcDNA3.1 plasmid, or PBS (100 μl), respectively. Blood samples were collected from the tail vein before immunization (0 week) and at 2, 4, 6 and 8 weeks after the first immunization for serum preparation which was used to detect SJ26GST specific IgG antibody levels by ELISA. At 8 weeks post-immunization, the mice spleen tissue was collected aseptically to prepare spleen lymphocyte suspension, which was cultured at 37 ℃ for 48 h, subsequently, 10 μl CCK8 reagent (cell counting kit-8) was added for culturing additional 4 h to determine the spleen cell stimulation index. The levels of cytokines Th1 (TNF-α, INF-γ), Th2 (IL-10, IL-4) and Th17 (IL-17) in the lymphocyte supernatant were assayed by ELISA. One-way analysis of variance was used for inter-group comparisons. Results The optimized coding sequence was 858 bp in length, the GC content was 63.2%, and the free energy change of the secondary structure was -485.6 kcal/mol. The in vitro transcribed Sj26gst mRNA band was a single and non-dispersive band. The RT-PCR results showed that the relative expression level of Sj26gst mRNA in HeLa cells is (6.21 ± 0.83) at 2 h after Sj26gst mRNA transfection, and its peak level was (14.26 ± 1.23) at 6 h, followed by a decrease. The western blotting results showed that Sj26GST protein bands of M_r 26 000 were detected at 6, 12, 24 and 48 hours after Sj26gst mRNA transfection. The ELISA results showed that the A₄₅₀ value of Sj26gst mRNA group, pcDNA3.1/Sj26gst group, pcDNA3.1 group and PBS group were 0.85 ± 0.16, 0.42 ± 0.21, 0.14 ± 0.03 and 0.11 ± 0.02, at eight weeks after the first immunization, respectively. There were statistically significant differences between Sj26gst mRNA group and pcDNA3.1/Sj26gst group, pcDNA3.1/Sj26gst group and pcDNA3.1/Sj26gst group (t = 16.46, 12.35; all P < 0.05). The ELISA results showed that the TNF-α, INF-γ, IL-10, and IL-17 expression levels in the supernatants of splenocytes from Sj26gst mRNA-immunized mice were (756.23 ± 134.35), (598.46 ± 50.47), (713.42 ± 118.25), and (301.45 ± 48.23) pg/ml, respectively. In the PcDNA3.1/Sj26gst group, the corresponding levels were (587.26 ± 32.48), (401.45 ± 46.25), (386.27 ± 23.75) and (175.24 ± 41.23) pg/ml. These levels were higher than those in the pcDNA3.1 group [(19.34 ± 2.01), (35.14 ± 5.25), (32.11 ± 15.20), (19.75 ± 7.21) pg/ml] and the PBS group [(18.75 ± 1.98), (34.12 ± 4.78), (29.36 ± 8.72), (15.34 ± 2.51) pg/ml] (t = 54.59, 19.57, 52.36, 26.47; all P < 0.05). Conclusion The Sj26gst mRNA plasmid with stable expression of Sj26GST was constructed. The Sj26gst mRNA could induce high level of specific antibodies against Sj26GST and stimulate Th1 cellular immune response.

Key words: Schistosoma japonicum, Sj26gst, mRNA vaccine, Sequence optimization, Cellular immune responses

CLC Number:

R383.24

TAN Xiao, ZHU Qi, LIU Zhongqi, LI Jia, PENG Dingjin. Immunogenicity of Schistosoma japonicum Sj26gst mRNA vaccine candidate[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2023, 41(5): 546-551.

Figures/Tables 4

References 20

[1]	McManus DP, Dunne DW, Sacko M, et al. Schistosomiasis[J]. Nat Rev Dis Primers, 2018, 4: 13.
[2]	Lin JJ. Endemic status and control of animal schistosomiasis in China[J]. Chin J Schisto Control, 2019, 31(1): 40-46. (in Chinese)
	(林矫矫. 我国家畜血吸虫病流行情况及防控进展[J]. 中国血吸虫病防治杂志, 2019, 31(1): 40-46.)
[3]	Feng JX, Gong YF, Luo ZW, et al. Scientific basis of strategies for schistosomiasis control and prospect of the 14th Five-Year Plan in China[J]. Chin J Parasitol Parasit Dis, 2022, 40(4): 428-435. (in Chinese)
	(冯家鑫, 公衍峰, 罗卓韦, 等. 我国血吸虫病防治策略的科学基础与“十四五”展望[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(4): 428-435.)
[4]	Lu S, Wang SX, Grimes-Serrano JM. Current progress of DNA vaccine studies in humans[J]. Expert Rev Vaccines, 2008, 7(2): 175-191.
[5]	Cai Y, Rodriguez S, Hebel H. DNA vaccine manufacture: scale and quality[J]. Expert Rev Vaccines, 2009, 8(9): 1277-1291.
[6]	Ledwith BJ, Manam S, Troilo PJ, et al. Plasmid DNA vaccines: investigation of integration into host cellular DNA following intramuscular injection in mice[J]. Intervirology, 2000, 43(4/5/6): 258-272.
[7]	Li MY, Wang ZN, Xie CY, et al. Advances in mRNA vaccines[J]. Int Rev Cell Mol Biol, 2022, 372: 295-316.
[8]	De Beuckelaer A, Grooten J, De Koker S. Type I interferons modulate CD8⁺ T cell immunity to mRNA vaccines[J]. Trends Mol Med, 2017, 23(3): 216-226.
[9]	McManus DP, Loukas A. Current status of vaccines for schistosomiasis[J]. Clin Microbiol Rev, 2008, 21(1): 225-242.
[10]	Tang CL, Pan Q, Dai WQ, et al. Administration of anti-CTLA-4 monoclonal antibody augments protective immunity induced by Schistosoma japonicum glutathione-S-transferase[J]. Parasite Immunol, 2019, 41(8): e12657.
[11]	Arnon R, Tarrab-Hazdai R, Steward M. A mimotope peptide-based vaccine against Schistosoma mansoni: synthesis and characterization[J]. Immunology, 2000, 101(4): 555-562.
[12]	Chen B, Zhang GL, Zhang GH. Research progress on clinical trials of schistosomiasis vaccine candidates[J]. Chin J Parasitol Parasit Dis, 2022, 40(4): 511-515. (in Chinese)
	(陈兵, 张国莉, 张高红. 血吸虫病候选疫苗临床研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(4): 511-515.)
[13]	Pourseif MM, Masoudi-Sobhanzadeh Y, Azari E, et al. Self-amplifying mRNA vaccines: mode of action, design, development and optimization[J]. Drug Discov Today, 2022, 27(11): 103341.
[14]	Li L, Petrovsky N. Molecular mechanisms for enhanced DNA vaccine immunogenicity[J]. Expert Rev Vaccines, 2016, 15(3): 313-329.
[15]	Tan X, Xiao CL, Xiao F, et al. Immunoprotective effect of DNA vaccine against Schistosoma japonicum surface membrane protein SjOST48[J]. Chin J Parasitol Parasit Dis, 2021, 37(7): 791-795. (in Chinese)
	(谭潇, 肖楚丽, 肖非, 等. 日本血吸虫表膜蛋白SjOST48 DNA疫苗的免疫保护作用研究[J]. 中国免疫学杂志, 2021, 37(7): 791-795.)
[16]	Wadman M. The overlooked superpower of mRNA vaccines[J]. Science, 2021, 373(6554): 479.
[17]	Pardi N, Carre?o JM, O’dell G, et al. Development of a pentavalent broadly protective nucleoside-modified mRNA vaccine against influenza B viruses[J]. Nat Commun, 2022, 13(1): 4677.
[18]	Rutitzky LI, Stadecker MJ. Exacerbated egg-induced immunopathology in murine Schistosoma mansoni infection is primarily mediated by IL-17 and restrained by IFN-γ[J]. Eur J Immunol, 2011, 41(9): 2677-2687.
[19]	VanBlargan LA, Himansu S, Foreman BM, et al. An mRNA vaccine protects mice against multiple tick-transmitted flavivirus infections[J]. Cell Rep, 2018, 25(12): 3382-3392.e3.
[20]	Pardi N, Hogan MJ, Pelc RS, et al. Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination[J]. Nature, 2017, 543(7644): 248-251.