猪流行性腹泻病毒全病毒灭活疫苗量效关系研究

doi:10.11843/j.issn.0366-6964.2022.05.021

摘要/Abstract

摘要： 本研究旨在探索猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)灭活疫苗不同抗原含量对仔猪感染的保护作用。测定不同浓缩倍数PEDV感染性病毒粒子数和病毒粒子总数，用不同浓缩倍数抗原分别制备灭活疫苗免疫小鼠，利用ELISA抗体检测方法、中和试验、ELISPOT方法测定体液免疫与细胞免疫产生情况，筛选合适抗原含量疫苗进行仔猪免疫并测定抗体，利用攻毒试验研究浓缩疫苗与保护之间的关系。结果显示，当抗原含量达8×10⁶ pfu·mL^-1以上时，灭活疫苗能有效刺激小鼠产生体液免疫及细胞免疫。以2 mL含8×10⁶ pfu·mL^-1抗原的灭活PEDV疫苗免疫仔猪可抵抗PEDV攻毒引起的腹泻及连续排毒,相较于总病毒粒子数，感染性病毒粒子数与抗体产生呈显著相关(r = 0.998 1)，中和效价与仔猪保护呈显著相关性(r=0.974 7)。PEDV灭活疫苗可以提供良好的免疫保护，其中感染性病毒数量是提升抗体水平的关键因子，抗体滴度作为一项体液免疫的指标是判断免疫保护的重要参考。

关键词: 猪流行性腹泻病毒, 灭活疫苗, 量效关系

Abstract: This study aims to investigate the protective effect of infected piglets which were immunized with different dose of inactivated porcine epidemic diarrhea virus (PEDV) vaccines. The number of infective virus particles and total virus particles of PEDV with different concentrations were determined, and the mice were immunized with different concentration vaccine prepared as antigen, respectively. The humoral and cellular immune production were determined by ELISA antibody detection method, neutralization test and ELISPOT method. Vaccine with appropriate antigen content was selected to immunize piglets, then the antibody was determined. The relationship between concentrated vaccine and protective effect was studied by challenge experiment. The results showed that, when the antigen dose was equal or greater than 8×10⁶ pfu·mL^-1, the inactive vaccine could effectively stimulate mice to produce humoral and cellular immunity. The piglets immunized with 2 mL inactivated PEDV vaccine containing 8×10⁶ pfu·mL^-1 antigen could resist diarrhea and continuous viral shedding caused by PEDV challenge. Compared with the total number of virus particles, the number of infectious virus particles was significantly correlated with antibody production (r=0.998 1), and neutralization titer was significantly correlated with piglet protection (r=0.974 7). PEDV inactivated vaccine can provide good immune protection, in which the number of infectious virus particles is the key factor to improve the antibody level. Antibody titer, as an index of humoral immunity, is an important reference for judging immune protection.

Key words: porcine epidemic diarrhea virus, inactivated vaccine, dose-titration

中图分类号:

S855.3

郝建伟, 薛春宜, 曹永长. 猪流行性腹泻病毒全病毒灭活疫苗量效关系研究[J]. 畜牧兽医学报, 2022, 53(5): 1536-1543.

HAO Jianwei, XUE Chunyi, CAO Yongchang. The Dose-titration Study of Inactivated Vaccine of Porcine Epidemic Diarrhea Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1536-1543.

参考文献 23

[1]	MASTERS P S,PERLMAN S.Coronaviridae[M]//KNIPE D M,HOWLEY P M.Fields Virology.Philadelphia:Wolters Kluwer,2013:825.
[2]	WOOD E N.An apparently new syndrome of porcine epidemic diarrhoea[J].Vet Rec,1977,100(12):243-244.
[3]	SONG D,PARK B.Porcine epidemic diarrhoea virus:a comprehensive review of molecular epidemiology,diagnosis,and vaccines[J].Virus Genes,2012,44(2):167-175.
[4]	WON H,LIM J,NOH Y H,et al.Efficacy of porcine epidemic diarrhea vaccines:a systematic review and meta-analysis[J].Vaccines,2020,8(4):642.
[5]	SONG D,MOON H,KANG B.Porcine epidemic diarrhea:a review of current epidemiology and available vaccines[J].Clin Exp Vaccine Res,2015,4(2):166-176.
[6]	SATO T,TAKEYAMA N,KATSUMATA A,et al.Mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo[J].Virus Genes,2011,43(1):72-78.
[7]	THAN V T,CHOE S E,VU T T H,et al.Genetic characterization of the spike gene of porcine epidemic diarrhea viruses (PEDVs) circulating in Vietnam from 2015 to 2016[J].Vet Med Sci,2020,6(3):535-542.
[8]	COLLIN E A,ANBALAGAN S,OKDA F,et al.An inactivated vaccine made from a U.S. field isolate of porcine epidemic disease virus is immunogenic in pigs as demonstrated by a dose-titration[J].BMC Vet Res,2015,11(1):62.
[9]	CAVANAGH D.Severe acute respiratory syndrome vaccine development:experiences of vaccination against avian infectious bronchitis coronavirus[J].Avian Pathol,2003,32(6):567-582.
[10]	FLECK R.疫苗诱导对猪肺炎支原体的免疫防卫[J].汪秉刚,译.国外畜牧学(猪与禽),2002(5):44-46.FLECK R.Vaccination-induced immune defense against M. Hyo[J].WANG B G,trans.Animal Sci Abroad-Pigs Poultry,2002(5):44-46.(in Chinese)
[11]	HAO J W,XUE C Y,HE L L,et al.Bioinformatics insight into the spike glycoprotein gene of field porcine epidemic diarrhea strains during 2011-2013 in Guangdong,China[J].Virus Genes,2014,49(1):58-67.
[12]	ZHANG X B,HAO J W,ZHEN J,et al.Rapid quantitation of porcine epidemic diarrhea virus (PEDV) by Virus Counter[J].J Virol Methods,2015,223:1-4.
[13]	HAO J W,ZHANG Y,FANG S K,et al.Evaluation of purified recombinant spike fragments for assessment of the presence of serum neutralizing antibodies against a variant strain of porcine epidemic diarrhea virus[J].Virol Sin,2017,32(4):307-316.
[14]	GANGES L,CROOKE H R,BOHóRQUEZ J A,et al.Classical swine fever virus:the past,present and future[J].Virus Res,2020,289:198151.
[15]	WANG J,CUI X,WANG X B,et al.Efficacy of the Bartha-K61 vaccine and a gE-/gI-/TK- prototype vaccine against variant porcine pseudorabies virus (vPRV) in piglets with sublethal challenge of vPRV[J].Res Vet Sci,2020,128:16-23.
[16]	WANG D,FANG L R,XIAO S B.Porcine epidemic diarrhea in China[J].Virus Res,2016,226:7-13.
[17]	GERDTS V,ZAKHARTCHOUK A.Vaccines for porcine epidemic diarrhea virus and other swine coronaviruses[J].Vet Microbiol,2017,206:45-51.
[18]	MOGLER M A,GANDER J,HARRIS D L.Development of an alphavirus RNA particle vaccine against porcine epidemic diarrhea virus[J].Ann Proc Amer Assn Swine Vets,2014,63-64.
[19]	GOEDE D,MURTAUGH M P,NEREM J,et al.Previous infection of sows with a "mild" strain of porcine epidemic diarrhea virus confers protection against infection with a "severe" strain[J].Vet Microbiol,2015,176(1-2):161-164.
[20]	WU P,RODRIGUEZ Y Y,HERSHEY B J,et al.Validation of a binary ethylenimine (BEI) inactivation procedure for biosafety treatment of foot-and-mouth disease viruses (FMDV),vesicular stomatitis viruses (VSV),and swine vesicular disease virus (SVDV)[J].Vet Microbiol,2021,252:108928.
[21]	SONG D S,OH J S,KANG B K,et al.Oral efficacy of Vero cell attenuated porcine epidemic diarrhea virus DR13 strain[J].Res Vet Sci,2007,82(1):134-140.
[22]	BANDRICK M,ARIZA-NIETO C,BAIDOO S K,et al.Colostral antibody-mediated and cell-mediated immunity contributes to innate and antigen-specific immunity in piglets[J].Dev Comp Immunol,2014,43(1):114-120.
[23]	JUNG K,SAIF L J,WANG Q H.Porcine epidemic diarrhea virus (PEDV):an update on etiology,transmission,pathogenesis,and prevention and control[J].Virus Res,2020,286:198045.