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Preparation and immune protective efficacy analysis of an inactivated vaccine against infectious hematopoietic necrosis (IHN)

  • Corresponding author: LU Tongyan, lutongyan@hrfri.ac.cn
  • Received Date: 2021-05-11
    Accepted Date: 2021-08-23
    Available Online: 2021-10-14
  • Infectious hematopoietic necrosis disease (IHN) is an acute infectious viral disease that can cause sudden death of salmon. Vaccine immunization is the most effective way to prevent and control the disease. At present, there is no commercial vaccine to prevent the disease in China. The objective of the present study was to prepare an inactivated vaccine against IHN and evaluate its protective immunity in Oncorhynchus mykiss. In this study, infectious hematopoietic necrosis viruses (IHNV) were successively cultured on Epithelioma papulosum cyprinid (EPC) cells with different multiplicity of infection (MOI). The optimal proliferation pattern of IHNV on EPC cells was determined by measuring the titer of IHNV in each passage combined with the virus harvest time. IHNV was inactivated by β-propanolactone (BPL) at different final concentrations at 24 °C, and the inactivity was then verified in vitro and in vivo to determine the optimal inactivation condition. Inactivated IHNV prepared with the optimal inactivation protocol was intraperitoneally injected into O. mykiss [(10±2) g)] with different doses, and the protective effect of the inactivated vaccine was analyzed by detecting relative percent survival (RPS) after challenge, expression levels of immune-related factors and serum neutralizing antibody titers at different time post vaccination. It was shown that different proliferation patterns had some effects on the proliferation of IHNV on EPC. We chose MOI of 0.0001 as the best inoculation dose on EPC cells, and the virus was harvested on the 3rd day post inoculation at 15 °C. The in vivo and in vitro safety tests showed that the best inactivation condition was to inactivate IHNV at 24 °C for 24 h with the final concentration of 3.0 mmol/L BPL. 10 μL per fish was chosen as the optimal immunization dose, and more O. mykiss were immunized. The RPS was 91.37%, 84.28%, 84.15% and 47.5% at 7, 21, 45 and 60 d post immunization (d.p.i), respectively, and significant difference was observed on RPS between 60 d.p.i and other time points. Compared with the negative group, the expression levels of Mx-1 and IFN-γ were significantly up-regulated in spleen and head-kidney at 7, 15 and 30 d.p.i, and reached the maximum at 7 d.p.i (5 folds). The expressions of CD4 and IgM genes were significantly up-regulated in spleen and head-kidney at 15 d.p.i. In the detection of neutralizing antibody titer, the average neutralizing antibody titer in O. mykiss serum was 67.25, 43.40 and 29.78 at 30, 45 and 60 d.p.i respectively, with a decreasing trend and significant differences among different groups. The results indicated that the IHN-BPL inactivated vaccine developed in this study could induce specific and non-specific immune response in O. mykiss, and could provide significant immunoprotection, which will provide references for the development of inactivated vaccines against IHN.
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Preparation and immune protective efficacy analysis of an inactivated vaccine against infectious hematopoietic necrosis (IHN)

    Corresponding author: LU Tongyan, lutongyan@hrfri.ac.cn
  • 1. Key Laboratory of Aquatic Animal Diseases and Immune Technology of Heilongjiang Province, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin    150070, China
  • 2. National Demonstration Centre for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China

Abstract: Infectious hematopoietic necrosis disease (IHN) is an acute infectious viral disease that can cause sudden death of salmon. Vaccine immunization is the most effective way to prevent and control the disease. At present, there is no commercial vaccine to prevent the disease in China. The objective of the present study was to prepare an inactivated vaccine against IHN and evaluate its protective immunity in Oncorhynchus mykiss. In this study, infectious hematopoietic necrosis viruses (IHNV) were successively cultured on Epithelioma papulosum cyprinid (EPC) cells with different multiplicity of infection (MOI). The optimal proliferation pattern of IHNV on EPC cells was determined by measuring the titer of IHNV in each passage combined with the virus harvest time. IHNV was inactivated by β-propanolactone (BPL) at different final concentrations at 24 °C, and the inactivity was then verified in vitro and in vivo to determine the optimal inactivation condition. Inactivated IHNV prepared with the optimal inactivation protocol was intraperitoneally injected into O. mykiss [(10±2) g)] with different doses, and the protective effect of the inactivated vaccine was analyzed by detecting relative percent survival (RPS) after challenge, expression levels of immune-related factors and serum neutralizing antibody titers at different time post vaccination. It was shown that different proliferation patterns had some effects on the proliferation of IHNV on EPC. We chose MOI of 0.0001 as the best inoculation dose on EPC cells, and the virus was harvested on the 3rd day post inoculation at 15 °C. The in vivo and in vitro safety tests showed that the best inactivation condition was to inactivate IHNV at 24 °C for 24 h with the final concentration of 3.0 mmol/L BPL. 10 μL per fish was chosen as the optimal immunization dose, and more O. mykiss were immunized. The RPS was 91.37%, 84.28%, 84.15% and 47.5% at 7, 21, 45 and 60 d post immunization (d.p.i), respectively, and significant difference was observed on RPS between 60 d.p.i and other time points. Compared with the negative group, the expression levels of Mx-1 and IFN-γ were significantly up-regulated in spleen and head-kidney at 7, 15 and 30 d.p.i, and reached the maximum at 7 d.p.i (5 folds). The expressions of CD4 and IgM genes were significantly up-regulated in spleen and head-kidney at 15 d.p.i. In the detection of neutralizing antibody titer, the average neutralizing antibody titer in O. mykiss serum was 67.25, 43.40 and 29.78 at 30, 45 and 60 d.p.i respectively, with a decreasing trend and significant differences among different groups. The results indicated that the IHN-BPL inactivated vaccine developed in this study could induce specific and non-specific immune response in O. mykiss, and could provide significant immunoprotection, which will provide references for the development of inactivated vaccines against IHN.

  • 传染性造血器官坏死病(infectious hematopoietic necrosis,IHN)是一种致死性极强的病毒病,多发生于鲑鳟幼鱼,死亡率最高达100%[1-2]。我国最早于20世纪80年代在辽宁本溪暴发IHN疫情,从2006年开始,我国虹鳟(Oncorhynchus mykiss)主要养殖省份开始陆续大规模暴发IHN,养殖虹鳟幼鱼死亡率高达90%以上[3-4],严重威胁了我国虹鳟养殖业的健康可持续发展。传染性造血器官坏死病毒(infectious hematopoietic necrosis virus,IHNV)基因组从3′端至5′端依次为3′N-P-M-G-NV-L 5′[5],对G基因全部或部分基因序列进行系统发育分析,发现IHNV共分为5个基因型,北美国家主要为U、M和L型[6],欧洲等国家主要为E型[7-8],亚洲等国家主要为J型[9-10]。中国目前主要流行IHNV毒株基因型主要为J基因型[11-12],但2012年辽宁省首次监测到M基因型MN基因亚型IHNV(LN12-17)[12],于2013年分离的BjLL株属于U基因型[13]。与此同时,研究发现不同基因型的IHNV毒株对虹鳟具有不同的致死性,J基因型对虹鳟的致死率大于U基因型和M基因型[14]。为了有效防控IHN的暴发,国内外学者开展了大量关于IHN疫苗的研究,主要包括灭活疫苗、减毒活疫苗、亚单位疫苗和DNA疫苗[15-16],但大多数疫苗研究仍停留于实验室水平,仅加拿大具有允许商业化生产的IHN DNA疫苗(Apex-IHN)[17],而我国目前尚无防治IHN的商业化疫苗和药物。

    灭活疫苗因抗原含量高、质量稳定、安全性高、生产成本低等优点成为现今获批的商业化疫苗中的主流疫苗产品[18]。细胞培养灭活疫苗在制备时首要决定条件是稳定、高效体外增殖方案和简单、安全的灭活工艺,不同毒株需建立最合适的体外增殖方法和灭活工艺。前期研究发现,采用β-丙内酯(β-propanolactone,BPL)灭活IHNV制备疫苗的免疫保护效果比甲醛和二元乙烯亚胺(BEI)好[19-20],但目前尚无IHNV体外增殖方案系统性研究的相关报道。因此,本实验开展了关于J基因型IHNV毒株在胖头鱥上皮细胞(Epithelioma papulosum cyprinid,EPC)的增殖方案、BPL灭活IHNV制备灭活疫苗并对其进行免疫保护效力分析等一系列研究,以期为今后IHN灭活疫苗的研制提供技术支撑,加速我国自主IHN疫苗的研究进程。

    • MEM培养基(minimum essential medium,MEM)、双抗(Penicillin Streptomycin)和胎牛血清(Fetal bovine serum,FBS)购自Gibco生物公司,BPL购自德国Serva公司,硫代硫酸钠(Sodium thiosulfate)购自Sigma-Aldrich®化学试剂公司。

      IHNV毒株由本课题组分离保存[11],EPC细胞由中国水产科学研究院长江水产研究所曾令兵教授惠赠,用细胞培养液(MEM培养基+10%胎牛血清+1%双抗)将其静置培养于25 °C二氧化碳细胞培养箱;健康虹鳟[(10±1) g]购自辽宁本溪艾格莫林实业有限公司,实验用鱼饲养于温度为(13±0.5) °C的室内循环水系统中。

    • 将IHNV分离株病毒原液用细胞维持液(MEM培养基+2%胎牛血清+1%双抗)分别稀释为1 mL的感染复数(multiplicity of infection,MOI)为0.1、0.01、0.001、0.000 1和0.000 01的病毒培养液,接种到6孔单层EPC细胞上,于15 °C CO2恒温培养箱内孵育1 h后弃去含有病毒的培养液,每孔更换为2 mL细胞维持液,于15 °C静置培养,当细胞病变达80%以上时,收获病毒于−80 °C冰箱反复冻融后,以12 000 r/min离心10 min (4 °C),取上清液为第1代IHNV。将第1代IHNV作为感染液以同样的方法传代,传至第10代时结束病毒连续传代。将每次传代收获的上清液分装后保存于−80 °C冰箱,并对其进行病毒滴度的测定。

    • 取出以MOI为0.01、0.001、0.000 1和0.000 01在EPC细胞中传代时获得的第2代、第5代和第10代病毒液,分别按照与原病毒液相对应的MOI接种于6孔细胞培养板的单层EPC细胞。分别在接种后第1、2、3、4和5天收获病毒,冻存于−80 °C冰箱。按照已有文献[21]的方法对收获的病毒进行滴度测定,绘制各组IHNV生长曲线。

    • 将BPL溶液用细胞维持液稀释后,在IHNV病毒液中分别加入终浓度为3.5、3.0和2.5 mmol/L的BPL溶液,加入后迅速混匀。将加入BPL的病毒液放置于摇床,于24 °C (室温)、100 r/min条件下分别在灭活1、6、12、24和48 h后加入终浓度为20 mmol/L的硫代硫酸钠中和BPL,以此终止灭活。将上述灭活液保存于−80 °C冰箱备用。

    • 将灭活液用细胞维持液稀释10倍后接种于6孔单层EPC细胞,于15 °C CO2培养箱孵育1 h,弃去含有灭活液的培养基,补加2 mL细胞维持液。连续7 d观察是否出现IHNV典型的细胞病变,若出现细胞病变,说明该灭活条件不可以使IHNV完全灭活;若未出现病变,需将其置于−80 °C冰箱反复冻融2次后,于4 °C、12 000 r/min离心10 min,取上清液,将细胞上清液进行盲传3代,若盲传3次均未出现细胞病变,说明该灭活条件可使IHNV完全灭活。

    • 将健康虹鳟分为5组,每组20尾,第1~4组为上述确定完全灭活的IHNV灭活液,第5组为磷酸盐缓冲溶液(PBS)对照组,每尾腹腔注射100 μL灭活液或PBS,连续21 d观察虹鳟摄食是否正常、有无IHN临床症状以及有无因接种疫苗引起的毒性反应。若灭活疫苗注射免疫虹鳟后一切正常,结合生产实际筛选灭活时间短、灭活剂量小的灭活条件作为最佳灭活IHNV的灭活工艺。

    • 将健康虹鳟随机分成4组,第1~3组分别每尾腹腔注射5、10和20 μL IHN BPL灭活疫苗,作为免疫组;第4组腹腔注射PBS,作为阴性对照组。在免疫后第7、14天,从各免疫组和对照组分别随机各取60尾虹鳟,每20尾1个平行,每尾腹腔注射50 μL 100 TCID50 IHNV[22],连续观察21 d,统计各组虹鳟21 d累积死亡数,并计算相对免疫保护率 (RPS,%),计算公式:

      相对免疫保护率= [1−(免疫组死亡率/对照组死亡率)]×100%。

    • 将680尾健康虹鳟分为2组,每组340尾,免疫组腹腔注射IHN-BPL灭活疫苗,免疫剂量为上述得出的最佳免疫剂量;对照组腹腔注射等剂量的PBS。在免疫后3、7、15、30 d分别从各组中随机挑选5尾虹鳟采集其头肾和脾脏组织,冻存于−80 °C冰箱用于免疫相关因子表达分析;在免疫后30、45和60 d分别从各组中随机挑选10尾虹鳟进行尾静脉采血,血样于4 °C过夜,500 r/min离心10 min,取上清液制备血清,用于血清中和抗体效价测定。

    • 在免疫后7、21、45和60 d,分别从免疫组和对照组随机取60尾虹鳟,每20尾1个平行,每尾腹腔注射50 μL 100 TCID50 IHNV原液进行攻毒实验[21],连续观察21 d,统计各组虹鳟21 d累积死亡数,并计算RPS。

    • 将血清样品利用细胞维持液进行连续2倍系列稀释(1∶4~1∶256)后,与100 TCID50的IHNV病毒液以1∶1 (体积比) 的比例混合,置于15 °C CO2培养箱中孵育1 h,接种于96孔单层EPC细胞,于15 °C CO2培养箱孵育1 h后弃去含有血清和IHNV的细胞培养液,更换为每孔100 μL的细胞维持液。将细胞培养板置于15 °C静置培养,连续观察7 d,记录病变及未病变孔数,以抑制50%细胞病变的血清最高稀释度作为血清中和效价,按Reed-Muench氏法计算血清中和抗体效价[23]

    • 利用TRIzol法提取虹鳟头肾和脾脏组织中RNA,将β-actin作为内参基因,利用One Step SYBR Prime Script Plus RT-PCR试剂盒检测各个组织RNA中IFN-γ[24]Mx-1[25]CD8、CD4[26]lgM[27]表达量变化,并通过2−△△Ct计算各基因的相对表达水平。以PBS对照组的头肾和脾脏组织RNA作为阴性对照。

    2.   结果
    • 将IHNV毒株以不同的MOI在EPC细胞中连续传代,待细胞病变达80%以上时收获病毒,测定其TCID50。结果显示,当MOI为0.1时,随着传代次数的增加,TCID50逐渐下降,传至第6代时再无任何细胞病变,TCID50降为0 (图1-a);而当MOI为0.01、0.001、0.000 1和0.000 01时,在传代的过程中病毒滴度均有少量下降,但随着传代次数的增多逐渐趋于稳定,稳定时IHNV的TCID50依次为107.42、107.42、107.50和107.40。结果表明,当以MOI为0.1进行连续传代时,IHNV无法持续在EPC细胞中进行增殖;当以MOI为0.01、0.001、0.000 1和0.000 01进行连续传代时,IHNV滴度均能稳定地保持在较高水平,但是MOI为0.000 1时,获得的IHNV滴度均高于其余MOI组 (图1-b)。综上可知,MOI 0.1的接种浓度不适合用于IHNV在EPC细胞上的体外连续传代,MOI 0.000 1的接种浓度更适合用于IHNV在EPC细胞中的体外连续传代。

      Figure 1.  Virus titers of IHNV inoculated with different MOIs

    • 将以不同MOI接种EPC细胞后获得的第2、第5及第10代IHNV接种于单层EPC细胞,接种后5 d内,每天收获病毒液进行滴度测定,并绘制生长曲线。结果发现,当MOI为0.01、0.001和0.000 1时,IHNV的滴度在接种后3 d即趋于稳定,总体上表现为在接种后第1~3天呈对数增长,第3~4天缓慢增长并达到峰值,第3~5天的TCID50依次分别为107.42、107.50和107.52(MOI=0.01);107.34、107.49和107.49(MOI=0.001);107.44、107.55和107.55(MOI=0.000 1)。当MOI为0.000 01时,IHNV滴度在接种后5 d才到达峰值,第3~5天的TCID50依次为106.95、107.28和107.42 (图2)。该结果表明,以MOI为0.01、0.001和0.000 1接种IHNV时,病毒增殖达到平台期需要的时间相对较短,在接种后3 d即可达到107.00以上,更适合用于IHNV的大规模培养。

      Figure 2.  Growth curve of IHNV at MOIs

      综合考虑病毒滴度、传代稳定性及病毒收获时间的长短等因素,本研究推荐病毒收获时间最短、病毒滴度最高、传代最稳定的最小接种剂量——MOI 0.000 1为IHNV在EPC细胞上连续传代的最佳接种剂量。即IHNV在EPC细胞中的最佳增殖方案为接种浓度MOI 0.000 1,15 °C培养3 d。

    • 在24 °C下,分别利用终浓度为3.5、3.0和2.5 mmol/L的BPL溶液于摇床(100 r/min)灭活IHNV病毒液,在不同时间点用20 mmol/L硫代硫酸钠终止灭活。将处理后的病毒灭活液接种于单层EPC细胞以确定灭活病毒的感染性(表1),同时腹腔注射于健康虹鳟来验证其是否对鱼体产生毒副作用。在24 °C下,采用终浓度分别为3.5和3.0 mmol/L的BPL在灭活24和48 h后均可完全灭活IHNV。将其腹腔注射免疫21 d,免疫虹鳟均摄食正常、未出现毒副作用、未出现IHN临床症状及死亡。综合灭活时间短、灭活剂量小等实际生产因素,最终选择以终浓度为3.0 mmol/L的BPL在24 °C下灭活24 h来制备IHN灭活疫苗。

      BPL终浓度/(mmol/L)
      BPL final concentration
      灭活时间/h
      inactivation time
      16122448
      2.5+++++++++++++
      3.0+++++++++------
      3.5+++++++++------
      注:+++. 盲传3代均发生了细胞病变;++. 盲传时第1代未病变,其余2代均病变了;---. 盲传3代时均未发生细胞病变
      Notes: +++. the cytopathic effect occurred in all 3 generations of blind transmission; ++. only the first generation has no cytopathic effect occurred in 3 generations of blind transmission; ---. no cytopathic effect occurred in each of the 3 generations of blind transmission

      Table 1.  Inactivation effect of IHNV by different inactivation conditions

    • 将以最佳灭活条件制备的IHN-BPL灭活疫苗,分别以每尾5、10和20 μL免疫剂量免疫虹鳟,并在免疫后7、14 d进行攻毒,RPS结果表明,当免疫剂量为10 μL/尾时,RPS在各个免疫时间都最高,依次为76.67%和73.33%,且与其余2个免疫剂量组间差异显著(P<0.05)(图3)。因此,该灭活疫苗对虹鳟的最佳免疫剂量为10 μL/尾。

      Figure 3.  Relative percent survival of O. mykiss immunized with inactivated vaccine

    • 将以最佳灭活条件制备的IHN-BPL灭活疫苗,腹腔注射免疫虹鳟(10 μL/尾),分别在免疫后7、21、45和60 d进行攻毒,分别计算21 d累积死亡率和RPS。疫苗组在不同免疫时间段的21 d累积死亡率均低于对照组,且差异极显著(P<0.05)(图4-a);疫苗组的相对免疫保护率从免疫后7 d至60 d依次为91.37%、84.28%、84.15%和47.50%,第60天时相对免疫保护率最低,与其他时间点差异显著(P<0.05)(图4-b)。

      Figure 4.  Cumulative mortality and relative percent survival of O. mykiss immunized with inactivated vaccine

    • 于免疫后30、45和60 d,尾静脉采血,分别测定各组10尾虹鳟血清IHNV中和抗体效价(表2),平均中和抗体效价依次为62.25、43.40和29.78。免疫组中和抗体效价均与对照组间差异极显著(P<0.01),免疫组中和抗体效价从第30天至第60天呈下降趋势,且彼此间差异极显著(P<0.01)(图5)。

      免疫
      时间/d
      immune
      time
      中和抗体效价
      neutralizing antibody titer
      免疫组(n=10)
      immune group
      对照组(n=10)
      control group
      30 41.00,117.41,44.92,50.81,62.75,
      39.61,102.81,36.00,44.44,132.75
      4, 2, 2, 2, 2,
      2, 8, 4, 4, 8
      45 38.03, 31.98, 39.46, 47.05, 32.00,
      109.54, 32.00, 40.29, 31.65, 32.00
      4, 2, 2, 4, 4,
      4, 4, 2, 2, 2
      60 29.62, 36.54, 26.03, 31.03, 31.18,
      55.08, 26.08, 20.00, 36.10, 6.10
      4, 2, 4, 4, 2,
      2, 4, 4, 2, 2

      Table 2.  Neutralizing antibody titer of O. mykiss immunized with IHN BPL inactivated vaccine

      Figure 5.  Neutralizing antibody titers of serum

    • 在免疫后3、7、15和21 d分别测定虹鳟头肾和脾脏中Mx-1和IFN-γ相对表达量,并在免疫后15和30 d测定IgMCD4和CD8相对表达量。以PBS注射虹鳟组为阴性对照。在免疫组虹鳟脾脏中,Mx-1在第3、7、15、30天时均显著上调(P<0.05),第7天时表达倍数最高(5倍),IFN-γ在第7天时极显著上调(P<0.01),且表达量最高;免疫的虹鳟头肾和脾脏中,Mx-1和IFN-γ均在免疫后第7天显著上调,此时表达量倍数最大分别为5倍和10倍 (图6)。表明BPL灭活疫苗在免疫后第3天时,即可诱导鱼体产生非特异性免疫应答。在免疫后第15和第30天虹鳟脾脏中,CD4、CD8和IgM均显著上调(P<0.05),这3种基因间差异不显著;在头肾中,CD4和IgM分别在第15天显著上调(P<0.05) (图7)。综上表明,该疫苗免疫虹鳟后可使CD4、CD8和IgM表达上调,激发机体产生免疫应答反应。

      Figure 6.  Relative expression level of Mx-1 (a) and IFN-γ (b) genes in spleen and head kidney of O. mykiss

      Figure 7.  Relative expression of CD4, CD8 and IgM genes in O. mykiss spleen (a) and head kidney (b)

    3.   讨论
    • 自我国于1985年首次分离获得IHNV毒株以来,已公布的我国IHNV有上百余株,主要来源于我国甘肃、辽宁、河北、北京、云南、四川、新疆、青海、山东及黑龙江等地区。对其G基因全部或部分基因序列进行系统发育分析发现,我国现行IHNV毒株大部分属于J基因型[11-12],病毒滴度测定为106.15~108.36 TCID50/0.1 mL[21, 28-29]。本课题组对2012年至今流行病学调查分离到的百余株IHNV的生物学特性、免疫原性、毒力等进行综合分析,筛选出了一株免疫原性好、毒力强的IHNV分离株。Xu等[22]利用该分离株的G基因构建IHN DNA疫苗免疫虹鳟后,利用我国不同省份IHNV分离株进行攻毒,该疫苗均能有效抵抗IHNV的感染,RPS高达90%以上,进一步证明该毒株可被选为IHN灭活疫苗候选毒株。因此,本研究也选择该毒株用于制备IHN灭活疫苗。

    • 稳定高效的病毒体外增殖方案是灭活疫苗产业化的首要条件。IHNV病毒滴度的差异性主要取决于不同分离毒株和不同体外增殖方案,而同一IHNV分离株病毒滴度稳定性主要取决于其体外增殖方案。因此,本研究就IHNV体外增殖方案开展了一系列实验,筛选出了一种病毒滴度稳定、病毒收获时间短、接种剂量小的最佳增殖方案,旨在为IHNV灭活疫苗或减毒活疫苗的大规模制备提供保障。

      病毒体外增殖方案主要决定因素有细胞系、温度、接种病毒剂量、血清浓度和pH。自IHN首次暴发后,国内外学者对其病原IHNV进行了大量研究,研究发现IHNV对EPC细胞、胖头鱥肌肉细胞(muscle cells of fathead minnow,FHM)和蓝鳃太阳鱼成纤维细胞(bluegill fry,BF-2)最敏感[30],世界动物卫生组织(World Organization for Animal Health,OIE)推荐使用EPC作为IHNV体外增殖检测细胞系[31],故本研究选用EPC细胞为IHNV的体外增殖细胞系。EPC细胞是鱼类病毒学研究中常用的细胞系,且其培养条件简单,对血清含量要求低,常用含有10% FBS和1%双抗的MEM基础培养基,于25 °C培养。研究表明,在适当的pH和体积比为2%的血清浓度下,病毒的增殖效果几乎不受影响[32],故本研究未进行关于血清浓度和pH值对IHNV增殖影响的研究,直接采用含2%胎牛血清的MEM培养基作为IHNV培养液。IHNV在4~20 °C均可在细胞上增殖,最适增殖温度为15 °C[33],故本研究选择将接种IHNV的EPC细胞置于15 °C CO2培养箱进行病毒扩增。病毒接种剂量的大小不仅对宿主细胞的生长状态有影响,还对病毒自身的增殖有影响。前期研究发现,在IHNV传代培养过程中,当接种剂量过大时,会出现错误包装形成大量的缺陷病毒,而缺陷病毒不具备繁殖能力,随着传代次数的增加,缺陷病毒会快速累积,最终导致IHNV无法连续进行体外传代;当接种剂量过小时,病毒繁殖所需时间较长,延长了生产周期,增加了污染概率,提高了疫苗生产成本。本研究结果同样发现,IHNV在EPC中的最佳接种剂量为MOI 0.000 1,MOI过大或过小均会影响IHNV滴度的稳定性和细胞病变的快慢程度。综合生产实际考虑,IHNV在EPC细胞中的最佳增殖方案:接种浓度为MOI 0.000 1,15 °C培养3 d即可收获病毒。该病毒增殖方案可为大规模培养IHNV提供技术支持,有利于促进IHN灭活疫苗和减毒活疫苗规模化制备。

      Anderson等[20]分别应用MOI 10同步接毒法和MOI 0.01异步接毒法扩增IHNV,制备灭活疫苗,发现以MOI 0.01扩增病毒后制备的灭活疫苗免疫保护率显著高于MOI 10,而MOI 0.1制备的疫苗分别在免疫56和14 d后攻毒,RPS均大于90%[28],综上可知,用不同接种剂量扩增的IHNV制备灭活疫苗,免疫保护率不同,本研究采用的接种剂量为MOI=0.000 1,制备灭活疫苗免疫虹鳟7 d后,相对免疫保护率最高达90%以上,在制备灭活疫苗时大大减少了疫苗候选毒株病毒原液的消耗,降低了生产成本。

    • 近20年来,关于IHN灭活疫苗的研究越来越多。IHN灭活疫苗是指将细胞培养获得的IHNV经物理或化学方法灭活制成的疫苗。至今应用于制备IHN灭活疫苗的化学灭活剂主要有BPL、甲醛和二乙烯亚胺(BEI),其中甲醛虽然便宜且易获得,但其相较于BPL和BEI更容易使病毒蛋白质发生变性,使病毒免疫原性在一定程度上减弱[18, 34]。Anderson等[20]和Tang等[19]分别用上述3种灭活剂灭活IHNV细胞培养液,并用灭活液免疫虹鳟,发现BPL相较于其他2种灭活剂免疫效果最好,高达90%;故本研究选择BPL作为灭活IHNV的灭活剂。目前已有的IHN灭活疫苗大多在4~6 °C下灭活[19, 28, 35],仅Anderson等[20]在室温(22 °C)下灭活IHNV,而在实际生产中,灭活温度为室温时,可有效减少耗能,降低生产成本,故本研究选择在室温(24 °C)下灭活IHNV。Anderson等[20]应用终浓度为2.7 mmol/L的BPL在22 °C下灭活48 h可完全灭活IHNV,而本研究应用终浓度为3 mmol/L的BPL在24 °C下灭活24 h完全灭活IHNV,相较而言,本研究筛选出的IHNV最佳灭活条件耗时更短,更贴合实际生产,有利于推进IHN灭活疫苗产业化的进程。

    • 现有IHN-BPL灭活疫苗研究中未见相关最佳免疫剂量的报道,本研究在开展最佳免疫剂量筛选实验时,发现大免疫剂量(20 μL)在免疫后第7、14天的相对免疫保护率均显著低于小免疫剂量(10 μL),这可能是IHNV灭活液中抗原含量不均一导致的,这一现象曾在IHN DNA疫苗[21]免疫实验中也出现过。以最佳免疫剂量免疫虹鳟后,随着免疫时间的延长,免疫保护率呈下降趋势,平均相对免疫保护率从最初91.37% (第7天)降为47.5% (第60天),Anderson等[20]应用IHN-BPL灭活疫苗免疫虹鳟后,免疫保护率变化趋势与本研究一致,但其添加弗氏完全佐剂(FCA)后制备的IHN-BPL灭活疫苗免疫虹鳟后第28至第56天,免疫保护率均显著提高。因此,下一步可通过筛选合适的免疫佐剂提高该疫苗的免疫保护率。

      虹鳟免疫接种含G蛋白的重组疫苗后,可诱发虹鳟产生IFN介导的先天性免疫反应,诱导Mx的表达上调,并且在一定程度上可以抵抗机体感染病毒[21, 36-37],CD4+和CD8+T细胞和IgM免疫球蛋白在免疫保护反应中发挥着重要的作用[26-27]。本研究发现,在免疫后第7天时Mx-1和IFN-γ在免疫虹鳟脾脏和头肾中均显著上调;在第15天时,CD4和IgM在免疫虹鳟脾脏和头肾中均显著上调,表明该疫苗免疫虹鳟后第7天可诱导机体产生非特异性免疫应答反应,可持续到第15天,并有效抵御IHNV的感染。免疫后第30、45、60天免疫虹鳟血清中和抗体效价与相对免疫保护率趋势相对应,均逐渐降低,但攻毒后累积死亡率与对照组差异显著(P<0.05),Tang等[19]测定血清中特异性IgM抗体效价于免疫后第5周显著上调达最大值,故该疫苗可在免疫后第30天刺激机体产生一定的特异性免疫反应。

    4.   结论
    • 本实验就IHNV体外增殖方案和IHNV灭活疫苗制备及免疫保护效力分析开展了研究,制定了一种接种剂量小、收毒时间短、病毒滴度稳定的IHNV体外增殖方案,成功制备了具有较好保护效果的IHN灭活疫苗实验室产品。

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