欧亚类禽型H1N1猪流感病毒HA蛋白的表达及免疫原性评估

doi:10.3864/j.issn.0578-1752.2019.05.014

Abstract

Abstract:

【Objective】This study aimed to construct the recombinant plasmid expressing HA gene of Eurasian avian-like H1N1 (EA H1N1) swine influenza virus (SIV) and then evaluate its immunogenicity in mice. 【Method】HA gene of A/swine/Zhejiang/ 245/2013(H1N1) (ZJ245) was amplified by RT-PCR, and inserted into an eukaryotic expression vector pCAGGS. The recombinant plasmid, designated as pCAGGS-HA(ZJ245), was transfected into 293T cells, and the expressed HA protein was identified by indirect fluorescence assay (IFA) and Western blot. In order to evaluate the immunogenicity of the recombinant plasmid pCAGGS- HA(ZJ245), sixteen six-week-old female BALB/c mice were immunized with 100 μg of the recombinant plasmid by intramuscular injection, and then were boosted once with a 3-week interval. Another group of sixteen mice received 100 μL of phosphate-buffered saline (PBS) were used as unvaccinated control. Serum samples were collected every week after prime and boost immunization in order to detect the hemagglutinin inhibition (HI) antibodies, virus neutralization (VN) antibodies, respectively. Two weeks after the boost immunization, pCAGGS-HA(ZJ245)-immunized and PBS-inoculated mice were intranasally challenged with 50 μL(10 ^6.0EID₅₀) of the homologous ZJ245 and heterologous A/swine/Heilongjiang/44/2009(H1N1) (HLJ44), respectively. All mice per group were monitored daily for clinical signs of infection and body weight changes for two weeks. The mice that lost more than 25% of their initial body weight were euthanized on humane ground. On day 3 post-challenge three mice per group were euthanized and their organs including brain, nasal turbinate, lung, spleen and kidney were collected for virus titration in eggs. Immune efficacy of the recombinant plasmid pCAGGS-HA(ZJ245) was evaluated by body weight loss and virus replication titer in mice, respectively. 【Result】The recombinant plasmid pCAGGS-HA(ZJ245) was constructed by inserting HA gene of ZJ245 virus and verified by restriction endonuclease analysis and plasmid sequencing. IFA and Western blot analysis confirmed that the HA protein could be correctly expressed by the recombinant plasmid pCAGGS-HA(ZJ245) and had a good biological activity in vitro. Immunization and challenge trial indicated that low levels of HI and VN antibody against the homologous ZJ245 virus were initially detected one week after the first immunization, and significantly increased after the second immunization, with the HI titer of 76.88 and the VN titer of 152.5, respectively. Meanwhile, low levels of HI and VN antibodies against the heterologous HLJ44 virus were also detected. Compared with PBS-inoculated mice, the weight loss and viral replication rate of the pCAGGS-HA(ZJ245)-vaccinated mice when challenged with 10 ^6.0EID₅₀of the homologous ZJ245 virus were completely inhibited. When challenged with 10 ^6.0EID₅₀of the heterologous HLJ44 virus, the extent of weight loss and viral titer of the challenge virus detected in the pCAGGS-HA(ZJ245)- vaccinated mice were significantly lower than those in the PBS-inoculated mice (P<0.0001, P<0.001, P<0.05).【Conclusion】The recombinant plasmid pCAGGS-HA(ZJ245) could efficiently express HA protein, and provided complete protection for the immunized mice against the homologous ZJ245 virus infection and partial cross-protection against the heterologous HLJ44 virus infection, which indicated that the recombinant plasmid pCAGGS-HA(ZJ245) had good immunogenicity.

Key words: Eurasian avian-like H1N1 swine influenza virus, hemagglutinin protein, recombinant plasmid, immunogenicity, DNA vaccine

JIA YunHui,XU ChengZhi,SUI JinYu,WU YunPu,XU BangFeng,CHEN Yan,YANG HuanLiang,QIAO ChuanLing,CHEN HuaLan. Immunogenicity Evaluation of Eukaryotic Expressing Plasmids Encoding HA Protein of Eurasian Avian-Like H1N1 Swine Influenza Virus[J].Scientia Agricultura Sinica, 2019, 52(5): 930-938.

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References 32

[1]	BROWN I . The epidemiology and evolution of influenza viruses in pigs. Veterinary Microbiology, 2000,74(1/2):29-46. doi: 10.1016/S0378-1135(00)00164-4 pmid: 10799776
[2]	WEBBY R J, WEBSTER R G . Emergence of influenza A viruses. Philosophical Transactions of the Royal Society B Biological Sciences, 2001,356(1416):1817-1828. doi: 10.1098/rstb.2001.0997. doi: 10.1098/rstb.2001.0997 pmid: 1088557
[3]	BAUDON E, PEYRE M, PEIRIS M, COWLING B J . Epidemiological features of influenza circulation in swine populations: A systematic review and meta-analysis. PLoS ONE, 2017,12(6):e179044. doi: 10.1371/journal.pone.0179044. doi: 10.1371/journal.pone.0179044 pmid: 28591202
[4]	NEUMANN G, NODA T, KAWAOKA Y . Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature, 2009,459(7249):931-939. doi: 10.1038/nature08157. doi: 10.1038/nature08157 pmid: 19525932
[5]	SCHULTZ U, FITCH W M, LUDWIG S, MANDLER J, SCHOLTISSEK C . Evolution of pig influenza viruses. Virology, 1991,183(1):61-73. doi: 10.1016/0042-6822(91)90118-U pmid: 2053297
[6]	LIU J, BI Y, QIN K, FU G, YANG J, PENG J, MA G, LIU Q, PU J, TIAN F . Emergence of European avian influenza virus-like H1N1 swine influenza A viruses in China. Journal of Clinical Microbiology, 2009,47(8):2643-2646. doi: 10.1128/JCM.00262-09. doi: 10.1128/JCM.00262-09 pmid: 1460195
[7]	VINCENT A, AWADA L, BROWN I, CHEN H, CLAES F, DAUPHIN G, DONIS R, CULHANE M, HAMILTON K, LEWIS N, MUMFORD E, NGUYEN T, PARCHARIYANON S, PASICK J, PAVADE G, PEREDA A, PEIRIS M, SAITO T, SWENSON S, VAN REETH K, WEBBY R, WONG F, CIACCI-ZANELLA J . Review of influenza A virus in swine worldwide: a call for increased surveillance and research. Zoonoses Public Health, 2014,61(1):4-17. doi: 10.1111/zph.12049. doi: 10.1111/zph.12049 pmid: 23556412
[8]	GUAN Y, SHORTRIDGE K F, KRAUSS S, LI P H, KAWAOKA Y, WEBSTER R G . Emergence of avian H1N1 influenza viruses in pigs in China. Journal of Virology, 1996,70(11):8041-8046. doi: 10.1111/j.1365-2893.1996.tb00107.x pmid: 190877
[9]	YANG H, CHEN Y, QIAO C, HE X, ZHOU H, SUN Y, YIN H, MENG S, LIU L, ZHANG Q, KONG H, GU C, LI C, BU Z, KAWAOKA Y, CHEN H . Prevalence, genetics, and transmissibility in ferrets of Eurasian avian-like H1N1 swine influenza viruses. Proceedings of the National Academy of Sciences of the USA, 2016,113(2):392-397. doi: 10.1073/pnas.1522643113. doi: 10.1073/pnas.1522643113 pmid: 26711995
[10]	CAMPITELLI L, DONATELLI I, FONI E, CASTRUCCI M R, FABIANI C, KAWAOKA Y, KRAUSS S, WEBSTER R G . Continued evolution of H1N1 and H3N2 influenza viruses in pigs in Italy. Virology, 1997,232(2):310-318. doi: 10.1006/viro.1997.8514. doi: 10.1006/viro.1997.8514 pmid: 9191844
[11]	DE JONG J C, PACCAUD M F, DE RONDE-VERLOOP F M, HUFFELS N H, VERWEI C, WEIJERS T F, BANGMA P J, VAN KREGTEN E, KERCKHAERT J A, WICKI F, WUNDERLI W . Isolation of swine-like influenza A (H1N1) viruses from man in Switzerland and the Netherlands. Annales de l'Institut Pasteur/ Virologie, 1988,139(4):429-437. doi: 10.1016/S0769-2617(88)80078-9 pmid: 3214596
[12]	WANG D Y, QI S X, LI X Y, GUO J F, TAN M J, HAN G Y, LIU Y F, LAN Y, YANG L, HUANG W J, CHENG Y H, ZHAO X, BAI T, WANG Z, WEI H J, XIAO N, SHU Y L . Human infection with Eurasian avian-like influenza A(H1N1) virus, China. Emerging Infectious Diseases, 2013,19(10):1709-1711. doi: 10.3201/eid1910. 130420. doi: 10.3201/eid1910.130420 pmid: 3810748
[13]	YANG H, QIAO C, TANG X, CHEN Y, XIN X, CHEN H . Human infection from avian-like influenza A (H1N1) viruses in pigs, China. Emerging Infectious Diseases, 2012,18(7):1144-1146. doi: 10.3201/ eid1807.120009. doi: 10.3201/eid1807.120009 pmid: 3376805
[14]	SUI J, YANG D, QIAO C, XU H, XU B, WU Y, YANG H, CHEN Y, CHEN H . Protective efficacy of an inactivated Eurasian avian-like H1N1 swine influenza vaccine against homologous H1N1 and heterologous H1N1 and H1N2 viruses in mice. Vaccine, 2016,34(33):3757-3763. doi: 10.1016/j.vaccine.2016.06.009. doi: 10.1016/j.vaccine.2016.06.009 pmid: 27321744
[15]	RUAN B, WEN F, GONG X, LIU X M, WANG Q, YU L X, WANG S Y, ZHANG P, YANG H M, SHAN T L, ZHENG H, ZHOU Y J, TONG W, GAO F, TONG G Z, YU H . Protective efficacy of a high-growth reassortant H1N1 influenza virus vaccine against the European avian-like H1N1 swine influenza virus in mice and pigs. Veterinary Microbiology, 2018,222:75-84. doi: 10.1016/j.vetmic. 2018.07.003. doi: 10.1016/j.vetmic.2018.07.003
[16]	LIU L, LU J, ZHOU J, LI Z, ZHANG H, WANG D, SHU Y . Construction and comparison of different source neuraminidase candidate vaccine strains for human infection with Eurasian avian-like influenza H1N1 virus. Microbes and Infection, 2017,19(12):635-640. doi: 10.1016/j.micinf.2017.08.004. doi: 10.1016/j.micinf.2017.08.004 pmid: 28859895
[17]	WU Y, YANG D, XU B, LIANG W, SUI J, CHEN Y, YANG H, CHEN H, WEI P, QIAO C . Immune efficacy of an adenoviral vector-based swine influenza vaccine against antigenically distinct H1N1 strains in mice. Antiviral Research, 2017,147:29-36. doi: 10.1016/j.antiviral.2017.09.009. doi: 10.1016/j.antiviral.2017.09.009 pmid: 28941982
[18]	SHVARTSMAN D E, KOTLER M, TALL R D, ROTH M G, HENIS Y I . Differently anchored influenza hemagglutinin mutants display distinct interaction dynamics with mutual rafts. Journal of Cell Biology, 2003,163(4):879-888. doi: 10.1083/jcb.200308142. doi: 10.1083/jcb.200308142 pmid: 14623870
[19]	TREGONING J S, KINNEAR E . Using plasmids as DNA vaccines for infectious diseases. Microbiology Spectrum, 2014,2(6):1-16. doi: 10.1128/microbiolspec.PLAS-0028-2014.
[20]	徐汇洋, 许榜丰, 陈艳, 隋金钰, 杨焕良, 尹航, 杨大为, 乔传玲, 陈化兰 .一株H1N1猪流感病毒的进化分析与分子特征[J]. 中国农业科学,2015(15):3071-3078. doi: 10.3864/j.issn.0578-1752. 2015.15.018. doi: 10.3864/j.issn.0578-1752.2015.15.018
	XU H Y, XU B F, CHEN Y, SUI J Y, YANG H L, YIN H, YANG D W, QIAO C L, CHEN H L .Phylogenetic analysis and molecular characteristics of an H1N1subtype swine influenza virus.Scientia Agricultura Sinica, 2015(15):3071-3078. (in Chinese). doi: 10.3864/ j.issn.0578-1752.2015.15.018. doi: 10.3864/j.issn.0578-1752.2015.15.018
[21]	CHEN Y, ZHANG J, QIAO C, YANG H, ZHANG Y, XIN X , CHEN H. Co-circulation of pandemic 2009 H1N1, classical swine H1N1 and avian-like swine H1N1 influenza viruses in pigs in China. Infection, Genetics and Evolution, 2013, 13:331-338. doi: 10.1016/j.meegid. 2012.09.021. doi: 10.1016/j.meegid.2012.09.021 pmid: 23146831
[22]	PENSAERT M, OTTIS K, VANDEPUTTE J, KAPLAN M M, BACHMANN P A . Evidence for the natural transmission of influenza A virus from wild ducts to swine and its potential importance for man. Bulletin of the World Health Organ, 1981,59(1):75-78.
[23]	DUCATEZ M F, HAUSE B, STIGGER-ROSSER E, DARNELL D, CORZO C, JULEEN K, SIMONSON R, BROCKWELL-STAATS C, RUBRUM A, WANG D, WEBB A, CRUMPTON J C, LOWE J, GRAMER M, WEBBY R J . Multiple reassortment between pandemic (H1N1) 2009 and endemic influenza viruses in pigs, United States. Emerging Infectious Diseases, 2011,17(9):1624-1629. doi: 10.3201/ eid1709.110338. doi: 10.3201/eid1709.110338 pmid: 21892996
[24]	QIAO C, LIU L, YANG H, CHEN Y, XU H, CHEN H . Novel triple reassortant H1N2 influenza viruses bearing six internal genes of the pandemic 2009/H1N1 influenza virus were detected in pigs in China. Journal of Clinical Virology, 2014,61(4):529-534. doi: 10.1016/j.jcv. 2014.10.014. doi: 10.1016/j.jcv.2014.10.014 pmid: 25467861
[25]	WATSON S J, LANGAT P, REID S M, LAM TT, COTTEN M, KELLY M, VAN REETH K, QIU Y, SIMON G, BONIN E, FONI E, CHIAPPONI C, LARSEN L, HJULSAGER C, MARKOWSKA- DANIEL I, URBANIAK K DÜRRWALD R,SCHLEGEL M,HUOVILAINEN A,DAVIDSON I,DÁN Á,LOEFFEN W,EDWARDS S,BUBLOT M,VILA T,MALDONADO J,VALLS L,ESNIP3 CONSORTIUM,BROWN IH,PYBUS OG,KELLAM P,. Molecular epidemiology and evolution of influenza viruses circulating within European swine between 2009 and 2013. Journal of Virology, 2015,89(19):9920-9931. doi: 10.1128/JVI.00840-15. doi: 10.1128/JVI.00840-15 pmid: 4577897
[26]	RAJÃO D S, CHEN H, PEREZ D R, SANDBULTE M R, GAUGER P C, LOVING C L, SHANKS G D, VINCENT A . Vaccine-associated enhanced respiratory disease is influenced by Haemagglutinin and Neuraminidase in whole inactivated influenza virus vaccines. Journal of General Virology, 2016,97(7):1489-1499. doi: 10.1099/jgv.0. 000468.
[27]	PILLET S, KOBASA D, MEUNIER I, GRAY M, LADDY D, WEINER D B, VON MESSLING V, KOBINGER G P . Cellular immune response in the presence of protective antibody levels correlates with protection against 1918 influenza in ferrets. Vaccine, 2011,29(39):6793-6801. doi: 10.1016/j.vaccine.2010.12.059. doi: 10.1016/j.vaccine.2010.12.059 pmid: 21211587
[28]	JIANG Y, YU K, ZHANG H, ZHANG P, LI C, TIAN G, LI Y, WANG X, GE J, BU Z, CHEN H . Enhanced protective efficacy of H5 subtype avian influenza DNA vaccine with codon optimized HA gene in a pCAGGS plasmid vector. Antiviral Research, 2007,75(3):234-241. doi: 10.1016/j.antiviral.2007.03.009. doi: 10.1016/j.antiviral.2007.03.009
[29]	PING X, HU W, XIONG R, ZHANG X, TENG Z, DING M, LI L, CHANG C, XU K . Generation of a broadly reactive influenza H1 antigen using a consensus HA sequence. Vaccine, 2018,36(32 Pt B):4837-4845. doi: 10.1016/j.vaccine.2018.06.048. doi: 10.1016/j.vaccine.2018.06.048
[30]	MURPHY B R, CLEMENTS M L . The systemic and mucosal immune response of humans to influenza A virus. Current Topics in Microbiology and Immunology,1989, 146:107-116.
[31]	DE VLEESCHAUWER A R, VAN POUCKE S G, KARASIN A I, OLSEN CW, VAN REETH K . Cross-protection between antigenically distinct H1N1 swine influenza viruses from Europe and North America. Influenza Other Respiratory Viruses, 2011,5(2):115-122. doi: 10.1111/j.1750-2659.2010.00164.x. doi: 10.1111/j.1750-2659.2010.00164.x pmid: 21306575
[32]	BRAUCHER D R, HENNINGSON J N, LOVING C L, VINCENT A L, KIM E, STEITZ J, GAMBOTTO A A KEHRLI ME J R,. Intranasal vaccination with replication-defective adenovirus type 5 encoding influenza virus hemagglutinin elicits protective immunity to homologous challenge and partial protection to heterologous challenge in pigs. Clinical and Vaccine Immunology, 2012,19(11):1722-1729. doi: 10.1128/CVI.00315-12. doi: 10.1128/CVI.00315-12 pmid: 22933397

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Immunogenicity Evaluation of Eukaryotic Expressing Plasmids Encoding HA Protein of Eurasian Avian-Like H1N1 Swine Influenza Virus

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