猪圆环病毒2型合成肽疫苗佐剂筛选与免疫保护性研究

doi:10.16431/j.cnki.1671-7236.2023.03.022

摘要/Abstract

摘要： 【目的】筛选更安全高效的猪圆环病毒2型(Porcine circovirus type 2,PCV2)合成肽疫苗佐剂。【方法】将IMS 251C VG、GEL 02 PR、ISA 15A VG、ISA 206 VG、ISA 61 VG、台湾细菌鞭毛、卡波姆、多糖共8种佐剂与适量PCV2多肽抗原按照特定方法配制成不同疫苗,并验证其物理性状,采用小鼠试验初步检验疫苗安全性及免疫效果,筛选出较优的佐剂进行仔猪免疫攻毒保护试验,进一步评估各疫苗的免疫保护效果。【结果】物理性状显示,所有疫苗稳定性均较好,4 ℃保存期均超过12个月。ISA 61 VG油佐剂黏度较高且乳滴粒径较大,ISA 15A VG、ISA 206 VG双相佐剂次之,其余水佐剂均较低较小。小鼠试验结果显示,除台湾细菌鞭毛佐剂外其他佐剂安全性均良好;小鼠ELISA抗体水平检测结果显示,ISA 61 VG、GEL 02 PR、ISA 206 VG组免疫效果明显高于其余疫苗组;综合评定安全性和免疫效果,筛选ISA 61 VG、GEL 02 PR、卡波姆、多糖、ISA 206 VG共5个佐剂组进行仔猪免疫攻毒保护试验。仔猪免疫攻毒保护试验结果显示,免疫后,ISA 61 VG和GEL 02 PR组保护效果明显优于其他佐剂,其制备的疫苗产生的ELISA抗体和中和抗体水平均明显高于其他组,同时还可刺激产生更高水平的细胞因子γ-干扰素(IFN-γ);攻毒后,ISA 61 VG、GEL 02 PR、ISA 206 VG组疫苗的仔猪相对日增重显著高于其他佐剂组(P＜0.05),且实时荧光定量PCR和免疫组化结果显示,上述疫苗组可显著降低仔猪体内病毒载量(P＜0.05)。临床症状观察显示,ISA 61 VG和GEL 02 PR疫苗组无体温升高或被毛粗乱等现象,病理切片表明这2个疫苗组组织病变较轻。【结论】油佐剂ISA 61 VG佐剂和水佐剂GEL 02 PR制备的PCV2合成肽疫苗对预防PCV2感染具有较好的免疫保护作用,试验结果为PCV2合成肽疫苗的研究提供了数据基础。

关键词: 猪圆环病毒2型(PCV2); 合成肽疫苗; 佐剂筛选; 免疫保护

Abstract: 【Objective】 The purpose of this experiment was to screen safer and more efficient adjuvants for Porcine circovirus type 2 (PCV2) synthetic peptide vaccine.【Method】 The adjuvants of IMS 251C VG,GEL 02 PR,ISA 15A VG,ISA 206 VG,ISA 61 VG,Taiwan bacterial flagella,Carbomer,polysaccharide were used to make PCV2 synthetic peptide vaccine according to specific methods.The physical properties of each vaccine were verified,the safety and immune effect of the vaccine were preliminary tested by mouse test,and the better adjuvants were selected for immune challenge protection test of piglets to further evaluate the immune protection effect of each vaccine. 【Result】 The physical properties showed that all vaccines had good stability,and the storage period at 4 ℃ was more than 12 months.In terms of viscosity and emulsion droplet size,ISA 61 VG oil adjuvant was relatively higher and larger,and the two-phase adjuvant of ISA 15A VG and ISA 206 VG were second,the other water adjuvants were lower and smaller.The mouse test results showed that all adjuvants except Taiwan bacterial flagella adjuvant were safe.The results of mouse ELISA antibody level test showed that the immune effect of ISA 61 VG,GEL 02 PR and ISA 206 VG were better than other vaccines.Safety and immune effect were evaluated comprehensively,five adjuvant groups including ISA 61 VG,GEL 02 PR,Carbomer,polysaccharide and ISA 206 VG were selected for immune challenge protection test of piglets, and the results of immune challenge protection test of piglets showed that,the effects of ISA 61 VG and GEL 02 PR were obviously better than other adjuvants.The ELISA antibody and neutralizing antibody level produced by the vaccines prepared by them were significantly higher than those of other groups,and could also stimulate the production of higher levels of cytokine interferon-γ (IFN-γ).After challenge,the relative daily gain of piglets in ISA 61 VG,GEL 02 PR and ISA 206 VG groups was significantly higher than that in other adjuvant groups(P＜0.05),and the results of Real-time PCR and immunohistochemistry showed that the above vaccine groups could significantly reduce the viral load in piglets (P＜0.05).The observation of clinical symptoms showed that the two vaccine groups ISA 61 VG and GEL 02 PR had no temperature rise or roughened hair coats,and the pathological section showed that the pathological changes in the tissues of the two vaccine groups were mild.【Conclusion】 The results showed that the PCV2 synthetic peptide vaccine prepared by oil adjuvant ISA 61 VG and water adjuvant GEL 02 PR had better immune protection against PCV2 infection.This results provided a data basis for the study of PCV2 synthetic peptide vaccine.

Key words: Porcine circovirus type 2 (PCV2); synthetic peptide vaccine; adjuvant screening; immune protection

中图分类号:

S852.65^＋1

刘新月, 栗利芳, 王振豹, 张文亮, 张欣, 赵洪涛, 李鹏宇, 郑昊天, 肖进, 齐鹏. 猪圆环病毒2型合成肽疫苗佐剂筛选与免疫保护性研究[J]. 中国畜牧兽医, 2023, 50(3): 1068-1080.

LIU Xinyue, LI Lifang, WANG Zhenbao, ZHANG Wenliang, ZHANG Xin, ZHAO Hongtao, LI Pengyu, ZHENG Haotian, XIAO Jin, QI Peng. Adjuvants Screening and Immune Protection of Porcine Circovirus Type 2 Synthetic Peptide Vaccine[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1068-1080.

参考文献

[1] 欧阳海平,潘永飞,宋延华.猪圆环病毒2型疫苗研究进展[J].动物医学进展,2020,41(1):98-103. OUYANG H P,PAN Y F,SONG Y H.Progress on Porcine circovirus 2 vaccine[J].Progress in Veterinary Medicine,2020,41(1):98-103.(in Chinese)
[2] 张爱琼,梁海英,曾智勇,等.猪圆环病毒2型疫苗的研究进展[J].黑龙江畜牧兽医,2018,11:1-6. ZHANG A Q,LIANG H Y,ZENG Z Y,et al.Research progress of Porcine circovirus type 2 vaccine[J].Heilongjiang Animal Science and Veterinary Medicine,2018,11:1-6.(in Chinese)
[3] AFGHAH Z,WEBB B,MENG X J,et al.Ten years of PCV2 vaccines and vaccination:Is eradication a possibility?[J]. Veterinary Microbiology,2019,39(9):85-88.
[4] TRIBLE B R,ROWLAND R.Genetic variation of Porcine circovirus type 2(PCV2) and its relevance to vaccination,pathogenesis and diagnosis[J]. Virus Research,2012,164(1-2):68-77.
[5] FRANZO G,SEGALES J.Porcine circovirus 2 genotypes,immunity and vaccines:Multiple genotypes but one single serotype[J]. Pathogens,2020,9(12):1049.
[6] 王一平,郭龙军,唐青海,等.猪圆环病毒2型疫苗的研究进展[J].畜牧兽医学报,2012,43(9):1337-1345. WANG Y P,GUO L J,TANG Q H,et al.Research progress on vaccines of Porcine circovirus type 2[J].Acta Veterinaria et Zootechnica Sinica,2012,43(9):1337-1345.(in Chinese)
[7] LI W,MEDHA J,SMITA S,et al.Peptide vaccine:Progress and challenges[J]. Vaccines,2014,2(3):515-536.
[8] APOSTOLOPOULOS V,BOJARSKA J,CHAI T T,et al.A global review on short peptides:Frontiers and perspectives[J]. Molecules (Basel,Switzerland),2021,26(2):430.
[9] FOMER M,CAAS-ARRANZ R,DEFAUS S,et al.Peptide-based vaccines:Foot-and-mouth disease virus,a paradigm in animal health[J].Vaccines,2021,9(5):477.
[10] SALVADOR A,IGARYUA M,HEMANDEZ R M,et al.An overview on the field of micro-and nanotechnologies for synthetic peptide-based vaccines[J].Journal of Drug Delivery,2011,2011:181646.
[11] 李少华,唐康,张春梅,等.联合T、B细胞表位设计多肽疫苗的研究进展[J].中国免疫学杂志,2017,33(1):137-139. LI S H,TANG K,ZHANG C M,et al.Development of peptide vaccines designed with T and B cell epitopes[J].Chinese Journal of Immunology,2017,33(1):137-139.(in Chinese)
[12] 中国兽药典委员会.中华人民共和国兽药典(2020年版)三部[S].2020. COMMITTEE OF CHINA VETERINARY PHARMACOPOEIA.Veterinary Pharmacopoeia of People’s Republic of China,(2020 edition) Ⅲ[S].2020.(in Chinese)
[13] 吕慧,王先炜,李文良,等.猪圆环病毒2型重组Cap蛋白间接ELISA抗体检测方法的建立[J].畜牧与兽医,2009,41(11):6-11. LYU H,WANG X Y,LI W L,et al.Development of an indirect ELISA for detecting antibody to Porcine circovirus 2 using recombinant Cap protein[J].Animal Husbandry & Veterinary Medicine,2009,41(11):6-11.(in Chinese)
[14] 马涛,王先炜,李玉峰,等.不同佐剂对猪圆环病毒2型灭活抗原免疫效果的影响[J].中国兽医科学,2011,41(6):593-600. MA T,WANG X Y,LI Y F,et al.Effects of different adjuvants on immunogenicity of the inactivated Porcine cirovirus 2 antigen[J].Chinese Veterinary Science,2011,41(6):593-600.(in Chinese)
[15] 冯志新,姜平,王先炜,等.猪圆环病毒2型TaqMan实时PCR检测方法的建立[J].中国病毒学,2006,21(4):371-374. FENG Z X,JIANG P,WANG X W,et al.Development of a Real-time PCR method for detection of type 2 Porcine circovirus[J].Virologica Sinica,2006,21(4):371-374.(in Chinese)
[16] JEONG J,PARK C,KIM S,et al.Evaluation of the efficacy of a novel Porcine circovirus type 2 synthetic peptide vaccine[J].Canadian Journal of Veterinary Research,2018,82(2):146-153.
[17] 陈善真,赵焱,李中圣,等.含有Th细胞表位的合成PCV2 ORF2 Cap多肽抗原的表达及免疫原性分析[J].畜牧兽医学报,2015,46(12):2273-2281. CHEN S Z,ZHAO Y,LI Z S,et al.Expression and immunogenicity analysis of PCV2 Cap recombinant protein antigen which contains helper T cell epitope peptides[J].Acta Veterinaria et Zootechnica Sinica,2015,46(12):2273-2281.(in Chinese)
[18] 李学钊,洪炀,王治仓,等.免疫佐剂研究进展及前景展望[J].中国动物传染病学报,2014,22(4):81-86. LI X Z,HONG Y,WANG Z C,et al.Development and perspective of immune adjuvants[J].Chinese Journal of Animal Infectious Diseases,2014,22(4):81-86.(in Chinese)
[19] 裴世璇,宋吉健,韩忆侬,等.猪丹毒丝菌灭活疫苗免疫佐剂的筛选[J].畜牧兽医学报,2020,51(5):1083-1090. PEI S X,SONG J J,HAN Y N,et al.Comparison of adjuvant for inactivated swine erysipel as vaccine against Erysipelothrix rhusiopathiae[J].Acta Veterinaria et Zootechnica Sinica,2020,51(5):1083-1090.(in Chinese)
[20] MATHEW M K,VIRMANIJ N,BERA B C,et al.Protective efficacy of inactivated reverse genetics based equine influenza vaccine candidate adjuvanted with Montanide^TM Pet Gel in murine model[J].The Journal of Veterinary Medical Science,2019,81(12):1753-1762.
[21] 刘扶摇,王梦君,李雨轩,等.Montanide^TM Gel 01 ST水性佐剂对猪乙脑炎活疫苗免疫小鼠后的早期免疫保护效果的影响[J].中国预防兽医学报,2018,40(1):40-44. LIU F Y,WANG M J,LI Y X,et al.The effect of aqueous adjuvant Montanide^TM Gel 01 ST on early immune protection of swine Japanese encephalitis live-vaccine in mice[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(1):40-44.(in Chinese)
[22] 颜志斌,陈美静,唐栋,等.猪伪狂犬病病毒TK/gE/gI缺失株的构建及与Montanide Gel 01佐剂联合免疫效果评价[J].中国兽医学报,2021,41(9):1697-1703. YAN Z B,CHEN M J,TANG D,et al.Construction of Porcine pseudorabies virus with TK/gE/gI gene deletion and evaluation of its immune efficacy in combination with Montanide Gel 01 adjuvant[J].Chinese Journal of Veterinary Science,2021,41(9):1697-1703.(in Chinese)
[23] 秦守贤,孙涛.猪口蹄疫O型合成肽疫苗(多肽2600＋2700＋2800)新佐剂评估试验[J].中国畜牧兽医文摘,2018,34(2):65-66. QIN S X,SUN T.Evaluation of new adjuvant for synthetic peptide vaccine (peptide 2600＋2700＋2800) against swine foo-and-mouth disease[J]. Chinese Abstracts of Animal Husbandry and Veterinary Medicine,2018,34(2):65-66.(in Chinese)
[24] 侯博,王晨燕,彭瑶舜,等.PRV FB株gE/gI基因缺失株的构建及其灭活疫苗免疫保护效力评价[J].中国预防兽医学报,2021,43(9):965-971. HOU B,WANG C Y,PENG Y S,et al.Construction and efficacy evaluation of inactivated gE/gI gene deletion Pseudorabies virus FB vaccine candidates[J].Chinese Journal of Preventive Veterinary Medicine,2021,43(9):965-971.(in Chinese)
[25] 朱腾飞,孟凡增,姚浩,等.ISA 61 VG佐剂增强单核细胞增生李斯特氏菌灭活疫苗的保护性免疫应答[J].生物工程学报,2020,36(7):1378-1385. ZHU T F,MENG F Z,YAO H,et al.ISA 61 VG adjuvant enhances protective immune response of Listeria monocytogenes inactivated vaccine[J].Chinese Journal of Biotechnology,2020,36(7):1378-1385.(in Chinese)
[26] OBRADOVIC M R,CORSAUT L,DOLBEC D,et al.Experimental evaluation of protection and immunogenicity of Streptococcus suis bacterin-based vaccines formulated with different commercial adjuvants in weaned piglets[J]. Veterinary Research,2021,52(1):133.
[27] KHORASANI A,MADADGAR O,SOLEIMANJAHI H,et al.Evaluation of the efficacy of a new oil-based adjuvant ISA 61 VG FMD vaccine as a potential vaccine for cattle[J].Iranian Journal of Veterinary Research,2016,17(1):8-12.
[28] 庞胜美,吴文文,丁雪燕,等.细菌鞭毛蛋白免疫佐剂活性作用机制及应用研究进展[J].中国预防兽医学报,2020,42(8):850-855. PANG S M,WU W W,DING X Y,et al.Research progress on the mechanism and application of bacterial flagellin immunoadjuvant activity[J].Chinese Journal of Preventive Veterinary Medicine,2020,42(8):850-855.(in Chinese)
[29] 刘云霞,金宁一,秦宇豪,等.寨卡病毒DNA疫苗新型佐剂的筛选[J].中国病原生物学杂志,2019,14(3):253-256. LIU Y X,JIN N Y,QIN Y H,et al.Screening of new types of adjuvants for Zika DNA vaccines[J].Journal of Pathogen Biology,2019,14(3):253-256.(in Chinese)
[30] 解海东,熊祺琰,刘茂军,等.不同水性及水包油型佐剂对猪支原体肺炎灭活疫苗的免疫增强作用[J].畜牧兽医学报,2014,45(12):2028-2033. XIE H D,XIONG Q Y,LIU M J,et al.Study on immune enhancement effect of different aqueous and oil-in-water adjuvants to the Mycoplasma hyopneumoniae inactivated vaccine[J].Acta Veterinaria et Zootechnica Sinica,2014,45(12):2028-2033.(in Chinese)
[31] 史茜,徐新峰,李学文,等.多糖作为兽用疫苗佐剂作用机制的研究进展[J].中国畜牧兽医,2022,49(5):1895-1907. SHI Q,XU X F,LI X W,et al.Research progress on the mechanism of polysaccharideas adjuvant of veterinary vaccine[J].China Animal Husbandry & Veterinary Medicine,2022,49(5):1895-1907.(in Chinese)
[32] 赵思俊,孙晓亮,曲志娜,等.新型免疫佐剂研究进展[J].中国动物检疫,2016,33(7):58-61. ZHAO S J,SUN X L,QU Z N,et al.Study progress of new type of immunoadjuvant[J].China Animal Health Inspection,2016,33(7):58-61.(in Chinese)