鸭病毒性肝炎弱毒疫苗（A66株）的研制
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摘要
鸭病毒性肝炎(Duck Viral Hepatitis, DVH)是雏鸭的一种高度致死性急性传染病,以肝炎为其主要特征,病原是鸭肝炎病毒(Duck Hepatitis Virus, DHV)。我国流行的鸭肝炎以1型DHV (DHV1)为主。疫苗接种是预防本病最有效的措施,我国目前尚无商品化疫苗。本研究以1型DHV A66弱毒株第60代次(E60)鸡胚毒为始发毒,经鸡胚克隆化选育获得DHV A66株,在对A66株的安全性、免疫原性、纯净性和遗传稳定性检测的基础上,进一步开展鸭病毒性肝炎活疫苗(A66株)的安全性和效力试验、生产工艺等研究及中试生产工作,并在农业部行政审批许可后进行了疫苗临床试验,最终完成了鸭病毒性肝炎活疫苗(A66株)研制。
1、DHV A66弱毒株的克隆筛选
以1型DHV A66弱毒株第60代次(E60)鸡胚毒为始发毒,采用有限稀释法接种SPF鸡胚进行3次克隆化筛选、2次纯培养和1次扩大培养获得66代次(E66)种毒,通过毒价、毒力、免疫原性、特异性、纯净性等测定,表明克隆纯化的E66代种毒符合疫苗研制毒种要求,可作为鸭病毒性肝炎活疫苗研发的原始毒种,并命名为DHV A66株。
2、DHV A66株种毒的安全性、免疫原性、遗传稳定性和纯净性研究
以DHV A66株E66代次种毒,分别经皮下注射和口服途径大剂量接种1日龄敏感雏鸭各10只,接种雏鸭临床观察正常,剖检观察肝脏等内脏组织无肉眼可见病理变化,肝脏等组织切片检查无异常。再以该种毒经皮下注射途径大剂量接种1日龄敏感雏鸭20只,于接种后不同时间采集血液进行细胞计数、血沉测定、血红蛋白测定等血常规检查,同时取血样分离血清进行反映肝脏机能状态的谷丙转氨酶(ALT)、谷草转氨酶(AST)、谷氨酰胺转移酶(GGT)、碱性磷酸酶(ALP)等血清酶和胆红素测定。结果显示：试验鸭的血常规及四种血清酶和总胆红素(TBIL)测定值与健康对照鸭均无明显差异,但强毒对照鸭于接毒后24-36h的测定值与健康鸭有显著(P<0.05)或极显著差异(P<0.01)。
毒株遗传稳定性方面,以DHV A66株E66代次的种毒,每次用1～3日龄敏感雏鸭10只,连续传代回归10次进行毒力返强试验。结果各回归代次雏鸭肝组织能稳定分离到DHV,且组织含毒量相对稳定在103.5-4.8ELD50/ml范围；各代次回归试验鸭健康,剖检观察正常,病理组织学检查正常；各代次试验鸭的血液常规指标、四种主要血清酶和总胆红素(TBIL)值均正常。对DHV的A66-F10与F0全基因组序列比对和分析表明,全基因组核苷酸序列没有发生插入或缺失,两者仅有6个核苷酸差异,同源性达99.9%以上,ORF氨基酸序列未发生改变。充分证明DHV A66株具有良好的安全性和遗传稳定性。
以DHV A66株种毒经9～10日龄SPF鸡胚连续传代至E80代,通过对不同代次种毒的病毒含量及对1日龄雏鸭的毒力和免疫力的测定,结果表明：不同代次种毒毒价在108.10-8.50ELD50/ml之间,皮下注射免疫的半数免疫量(IMD50在106.10-6.40IMD50/ml范围,表明A66株免疫原性稳定而良好。因此,确定E66为原始种子代次,基础种子代次为E67～72,种子继代培养不超过3代,即生产用抗原最高代次为E75。通过对A66株E66、E70和E75代次鸡胚混合毒的最小免疫量测定,并取A66株鸡胚传代较高代次E75以不同倍数最小免疫量进行免疫试验,确定A66株的最小免疫量为103.3ELD50；根据免疫试验结果及其它因素综合考虑,确定A66株的免疫剂量(即使用剂量)应≥104.3ELD50。
按照现行《中华人民共和国兽药典》[16]规定方法,对DHV A66株种毒E66、E70代毒种进行无菌检验和外源病毒检验,结果细菌和支原体检验阴性,12种现行《中华人民共和国兽药典》[16]规定检测的外源病毒和鸡传染性贫血病毒(CIAV)也全部为阴性,表明DHV A66株毒种纯净。
3、检验用鸭肝炎强毒株(W株)的系统鉴定
通过对DHV W强毒株接种雏鸭复壮,进行病毒传代、毒力及其稳定性测定、血清学特异性及与DHV R85952强毒参考株进行毒力比较试验等,认定DHV W毒株可作为检验用强毒。对W毒株的攻毒剂量和保存期试验表明：W株的攻毒剂量1～7日龄选用103.0LD50/鸭,7～10日龄选用104.0LD50/鸭；W株的肝组织毒冻干后在-20℃和-70℃中保存期分别可达5年和8年。
4、鸭病毒性肝炎活疫苗(A66株)的安全性、效力和保存期研究
对5批实验室制备的DHV(A66株)活疫苗,分别通过皮下注射、口服途径对1日龄敏感雏鸭进行一次单剂量接种、单剂量重复接种和一次大剂量接种的安全性试验。临床观察试验雏鸭的精神、采食、饮水、生长、增重等均正常,肝功能检查、解剖观察肝脏等内脏组织器官及肝脏组织切片检查无异常。试验结果表明：A66活疫苗对雏鸭使用安全性好、无副作用。
用A66株最高代次(E75代)生产用毒种制备的三批疫苗,每批疫苗分别经皮下注射、肌肉注射、饮水和口服途径免疫易感雏鸭各10只。试验结果表明：经皮下和肌肉注射途径免疫雏鸭,2-3天就能产生免疫保护力；经口服、滴鼻和饮水途径免疫雏鸭,5天能产生良好的保护效果。以该三批疫苗1/10使用剂量免疫1日龄易感雏鸭5天后(120h)用强毒株进行攻毒,免疫组雏鸭全部保护,表明用A66株最高代次毒种制备的疫苗仍具有良好的免疫效力。将疫苗按免疫剂量接种1日龄易感雏鸭,雏鸭的免疫期至少达60天以上,即一次免疫就能有效保护雏鸭安全度过易感期(6周龄以内雏鸭)。
采用10倍免疫剂量的DHV(A66株)活疫苗,分别经皮下注射和口服途径接种1日龄易感雏鸭各20只,5日后随机取出其中10只与20只1日龄易感雏鸭进行第1代同居饲养5日,再取10只同居感染鸭与20只1日龄易感雏鸭进行第2代同居感染试验,如此连续进行5代次的同居感染试验。通过定时采集接种鸭及同居鸭肛拭子样品进行PCR检测,测定DHV A66株的感染和排毒情况；对接种或同居5日的试验鸭进行攻毒保护试验,测定同居感染的保护情况。结果表明：A66株活疫苗经皮下注射和口服途径接种雏鸭,均可以通过粪便向体外排泄病毒；同居易感雏鸭可经粪—口途径接触感染,感染雏鸭可获得鸭肝炎的免疫保护；同居感染现象随着同居代次的增加,同居感染的排毒时间逐渐延迟、感染率逐步下降,至同居5代时这种同居感染现象基本终止。
选择经鸭肝炎疫苗免疫的种鸭所产种蛋孵化的雏鸭,采用鸡胚中和试验测定其鸭肝炎母源抗体水平；对不同母源抗体水平的雏鸭于1日龄时用1羽份疫苗进行免疫,分别于免疫后2天和5天用1型DHV W株强毒攻击,结果表明,母源抗体水平对鸭肝炎活疫苗免疫效果无明显影响。
以制备的3批A66株活疫苗分别经过不同保存温度、不同保存期后,测定其鸡胚半数致死量(ELD50)的变化情况,判定疫苗的稳定性；通过对疫苗的免疫攻毒保护试验、物理性状观察、水分及真空度测定,以确定疫苗的保存期。试验结果表明DHV (A66株)活疫苗在-15℃条件下可保存2年以上,2-8℃条件下可保存1年以上；疫苗用生理盐水稀释后在室温(25)℃条件下存放8h毒价下降不明显。
5、鸭病毒性肝炎活疫苗(A66株)的生产工艺研究和中试生产
选择蔗糖、明胶、蛋白(氨基酸)等组份,与真空度、升温速度等因素,通过水平正交设计试验进行冻干保护剂筛选和冻干曲线的优化研究。根据试验结果确定A66株活疫苗生产工艺为：种毒以100～1000倍稀释、经尿囊腔途径接种9～11日龄SPF鸡胚,0.2ml/胚,收获接种后36～72h死亡鸡胚组织(胚体+胚膜+胚液),用组织捣碎机匀浆3～5分钟后保存；优化的保护剂配方和冻干条件为：5%蔗糖、1.5%明胶、6%甘氨酸的保护剂,真空度0.02mbar,升温速度为1.5℃/min。抗原与保护剂1：1比例,在冻干曲线：-40℃3h;-25℃13h;-10℃4h;25℃4h时,疫苗冻干效果最佳。按照确定的保护剂和冻干条件验证3批产品,其物理性状、真空度、残余水分含量均符合《中华人民共和国兽药典》[16]规定,3批疫苗冻干前后毒价损失≤100.6滴度。
按照本研究制定的《鸭病毒性肝炎活疫苗中间试制规程及质量标准》(草案),在南京天邦生物科技有限公司进行中试生产了5批鸭肝炎活疫苗(A66株),共计605万羽份。5批疫苗按照中间试制规程和质量标准进行检验,结果物理性状、无菌检验、支原体检验、病毒含量、安全检验、效力检验、剩余水分测定、真空度测定均符合标准。表明该疫苗生产工艺合理,产品质量稳定,适合规模化生产。
6、鸭病毒性肝炎活疫苗(A66株)临床试验
2008年12月27日获得农业部兽用生物制品临床试验批件(批件号：200844)后,自2009年2月至6月在安徽省5个鸭场(企业)应用5个批次的中间试制产品进行临床试验,试验共计免疫雏鸭5.9万羽。经临床观察、攻毒试验、抗体检测及生产性能的统计分析,结果表明：鸭病毒性肝炎活疫苗(A66株)对免疫雏鸭安全、有效、无不良反应。
Duck viral hepatitis (DVH) is a highly lethal acute infectious disease of ducklings; its main characteristics is hepatitis and the pathogen is Duck hepatitis virus (DHV). DHV1is the mainly epidemic in China. Vaccination is the most effective measure to prevent the disease, and there is no commercial vaccine in China. In the study, In the study, we selected the60passages virus (E60) of DHV1A66attenuated strain by chicken embryo passage as the origin virus, by cloning selection on SPF chicken embryo acquired DHV A66strain. On the basis of the detection of the safety, immunogenicity, purity and the genetic stability of A66strain, we do further research of the safety, production technology and trial production of the vaccine, and completed the clinical test research of the vaccine after the permission of the Ministry of Agriculture, finally, we developed DHV living vaccine (A66strain).
1. Cloning selection and purification of DHV1A66attenuated strain
Treating the E60of DHV1A66attenuated strain by chicken embryo passage as the origin virus, cloning selection of inoculation on SPF chicken embryos by the limited dilution for3times, purified cultivation for2times and1times expanding cultivation till66passages(E66), detecting the virus titre,virulence,immunogenicity, purity and exogenous virus, the results showed that the E66has met the requirements of producing vaccine. Therefore, we determined the chicken embryo virus E66of DHV as the origin virus for the development of DHV living vaccine, which we named DHV A66strain.
2. The safety, genetic stability, immunogenicity and purity of DHV A66strain
1-day-old ducklings were inoculated by subcutaneous injection and oral route with the E66passage virus of DHV A66; And the clinical observation of the ducklings was normal; there were no visible pathological changes on liver and other visceral organs by necropsy observation; histological examination of liver and other tissues was normal. Then,201-day-old ducklings inoculated by subcutaneous injection with a large dose of this virus were collated blood to do cell count, erythrocyte sedimentation rate, to determine the hemoglobin and other blood tests at different time after inoculation, at the same time, to detect alanine aminotransferase(ALT), aspartate aminotransferase(AST), glutamine transferase (GGT), alkaline phosphatase (ALP) and other serum enzymes and bilirubin to reflect the liver function by separated serum. The results showed:the measured values of blood routine, four serum enzymes and total bilirubin (TBIL). of the test ducklings have no obvious difference with healthy control ducklings, but there were significant (P<0.05) or extremely significant difference (P<0.01) between the measured values of the virulent control ducklings after24-36h inoculation and healthy10ducklings.
In terms of genetic stability of strains, using101-3days old ducklings infected by the E66passage virus of DHV1A66each time, we did the test of virulence rejuvenation by the continuous passage for10times, the duckling liver tissues of each regressive passage can be detected DHV, the virus titre of which is relatively stable at the range of103.5-4.8ELD50/ml; the ducklings of each regressive generation are healthy, pathological observation and pathological examination are all normal; the measured values of blood routine, four serum enzymes and total bilirubin (TBIL) of ducklings for each regressive generation are all normal. Compared and analyzed the the whole genome sequence between A66-F10and F0of DHV1, the results show that there is not insertion or deletion in whole genome sequence, with the difference of only6nucleotides, more than99.9%homology, and the sequences of ORF amino acid don't change. The above prove DHV1A66attenuated strain has a good safety and genetic stability.
Continuous passage on9-10days old SPF chicken embryos inoculated with the virus of DHV1A66till E80generation, we determined the content of each generation virus and the immune ability of1-day-old ducklings, which showed that:the virus titre of different generations is in10ELD50/lml to10ELD50/lml, the IMD50of subcutaneous immunization is in106.10IMD50/lml to10640IMD50/1ml, and indicated that the immunity of strain A66is stable and good. Accordingly E66is the original seed passage, E67-72are basic seed passages, the sub-cultivation of the seeds is not more that3generations, that is E75is the highest passage generation of the antigen used in production. By the determination of the minimum immune amount of the mix virus of E66, E70and E75chick embryo passages of A66strain, and the immune test with the minimum immune dose of different times taken by the high chicken embryo passages E75of A66strain, we determined that the minimum immune of A66strain is103.3ELD5o; we determined that the immune dose of A66strain should be higher than104.3ELD50, according to the results of immune tests and integrated consideration of other factors.
Under the current provisions of "China Veterinary Pharmacopoeia", to determine the bacteria and the exogenous virus, the result was negative for bacteria, mycolasma,12types of exogenous virus need to determine by the current provisions of "China Veterinary Pharmacopoeia" and chicken infectious anemia virus (CIAV), which indicated that the virus seed of DHV A66strain is purified.
3. System identification of duck hepatitis virulent strain (W strain) used for detection
After the rejuvenation of DHV1W virulent strain inoculated ducklings, we did the virus passage, detected the virulence, stability and serological specificity, and compared the virulence with the virulent DHV1R85952, etc, in order to determine DHV1W suitable for detection. The text of the dose of attacking virus and retention period of W strain show that1-7day-old ducklings immunized10LD50/duck and7-10day-old ducklings immunized104LD50/duck; the retention period of the liver tissue freeze-dried of W strain, stored at-20℃and-70℃,are respectively for5and8years.
4. The safety, effectiveness and retention period of the duck virual hepatitis living vaccine (A66strain)
5batches A66vaccine made in laboratory, respectively, by subcutaneous injection and oral route on1day-old ducklings, are done the safety texts of a single dose of vaccination, a single dose of repeat vaccination and a large dose of vaccination, the spirit, feeding, drinking, growth and weight gain of the ducklings were all normal by clinical observation; the results of liver function test, anatomical observation of liver and other organs, and the liver histological examination were normal. The results showed that:A66live vaccine is safe and has no side effects for young duckling.
Using the virus seeds of the highest passages of attenuated strain A66(E75) for the production of vaccine to prepare three batches, each batch of vaccine was treated with subcutaneous injection, intramuscular injection, drinking water and oral route on10susceptible ducklings. The results showed that:the ducklings immunized by subcutaneous and intramuscular injection can generate protective immunity in2to3days; the ducklings immunized by oral, intranasal and drinking water means, produce good protective effect in5days. Using1/10dose of the three batches vaccines to immune susceptible1-day ducklings, which are attacked with a virulent virus after5days (120h), immuned ducklings are all protected, and this indicates that the vaccines produced by the highest passages of A66have good immune efficacy. The immune period of the1-day-old ducklings inoculated the immune dose of vaccine is more than60days, that means once immunization can effectively protect the duckling to live through susceptible period (less than6-weeks-old ducklings.)
Each201-day-old ducklings immunized with with10-time dose of A66vaccine, respectively by the oral route and subcutaneous injection, after5days,10of which selected randomly cohabit and feed for5days with201-day-old susceptible ducklings for the1st generation;10of1st generation cohabitation infected ducklings cohabit with201-day-old susceptible ducklings for the2st generation; in this way,5consecutive passages of cohabitation infection experiments are done. The anal swab samples of inoculated ducks and cohabited ducks collected regularly are done PCR, to determine the infection and detoxification of DHV1A66inoculated ducks or5-day cohabited ducks are done the attacking-virus protection text to determine the conservation status of cohabitation infection. The results showed that:the ducklings immunized A66vaccine by subcutaneous injection and oral route can both excrete the virus with the feces; the cohabited susceptible ducklings can be infected by the fecal-oral route, infected ducklings can acquire the immune protection; with the increase of cohabited generations, the excretion time of virus is delayed gradually, the infectious rate is declined gradually, the phenomenon of cohabited infection ends basically till5st generational cohabitation. immune protection; with the increase of cohabited generations, the excretion time of virus is delayed gradually, the infectious rate is declined gradually, the phenomenon of cohabited infection ends basically till5st generational cohabitation.
The ducklings from hatching eggs produced by ducklings immunized by DHV vaccine were detected the level of maternal antibody of DHV using chick embryo neutralization test; the ducklings with different levels of maternal antibodies were immunized with1plume dose at1day old, and then were attacked the virulent DHV1W on second day and5st day after immunization; the results showed that the level of maternal antibodies has no obvious effect on immune effect of the vaccine.
3batches A66strain live vaccine prepared, saved at different temperatures and period, were detected the changes of the half chicken embryo lethal dose(ELD50) to judge the stability of vaccine; the retention period of vaccines was determined by attacking-virus immune protection test, observation of physical properties, the measurement of water and vacuum.The results showed that:A66live vaccine can be stored at-15℃in2years,2～8℃in1year; the titre of vaccine diluted with saline and stored for8h at25℃isn't declined obviously.
5. Production technology and intermediate test production of the duck virual hepatitis living vaccine (A66strain)
The test of level orthogonal design is to select the freeze-dried protectant and to optimize the freeze-dried curve, by selecting sucrose, gelatin, protein (amino acids) and other components, and by controlling the degree of cacuum, heating rate and other factors. According to the results of the tests, the productive technology of A66strain live vaccine is that, Dilution of strain virus by100-1000times, inoculation of9-11days SPF chicken embryos by urine cysts,0.2ml/embryo, harvest of dead chicken organization (body+membrane+liquid) after36-72h inoculation, homogenate of the tissue for3-5min and save; The formula of optimal protectant and freeze-dried condition is:5%sucrose,1.5%gelatin,6%glycine protection agents, vacuum0.02mbar, heating rate of1.5℃/min.1:1ratio of antigen and protective agents, in the freeze-drying curve:-40℃3h;-25℃13h;-10℃4h;25℃4h, which is the best freeze-dried vaccine. To verify the three batches by the identified protectant and the freeze-dried condition, its physical properties, vacuum, residual water content are in line with the "China Veterinary Pharmacopoeia" and the loss of virus titre is low to100.6.
According to this study, we developed a draft "Duck hepatitis vaccine trial procedures and quality standards ", and produced5batches of vaccine A66, a total of6.05million doses in Nanjing Tianbang Biotechnology Co., Ltd. To detect5batched of vaccine by trial procedures and quality standards, the results of physical traits, sterility test, Mycoplasma test, virus content, security testing, effectiveness testing, residual moisture content, determination of the vacuum are all in line with the standards. Which indicated that the production technology is reasonable; the production is stable and is suitable for large-scale production.
6. The duck virual hepatitis living vaccine (A66strain) in clinical trials
After the acquirement of the approval document of clinical trials of veterinary biological products of the Ministry Agriculture (number:200844) on27December2008, we had applied5batch products for clinical trials to test a total of59,000young ducklings in5duck farms of Anhui Province since February to June2009.
1、DHV A66弱毒株的克隆筛选
以1型DHV A66弱毒株第60代次(E60)鸡胚毒为始发毒,采用有限稀释法接种SPF鸡胚进行3次克隆化筛选、2次纯培养和1次扩大培养获得66代次(E66)种毒,通过毒价、毒力、免疫原性、特异性、纯净性等测定,表明克隆纯化的E66代种毒符合疫苗研制毒种要求,可作为鸭病毒性肝炎活疫苗研发的原始毒种,并命名为DHV A66株。
2、DHV A66株种毒的安全性、免疫原性、遗传稳定性和纯净性研究
以DHV A66株E66代次种毒,分别经皮下注射和口服途径大剂量接种1日龄敏感雏鸭各10只,接种雏鸭临床观察正常,剖检观察肝脏等内脏组织无肉眼可见病理变化,肝脏等组织切片检查无异常。再以该种毒经皮下注射途径大剂量接种1日龄敏感雏鸭20只,于接种后不同时间采集血液进行细胞计数、血沉测定、血红蛋白测定等血常规检查,同时取血样分离血清进行反映肝脏机能状态的谷丙转氨酶(ALT)、谷草转氨酶(AST)、谷氨酰胺转移酶(GGT)、碱性磷酸酶(ALP)等血清酶和胆红素测定。结果显示：试验鸭的血常规及四种血清酶和总胆红素(TBIL)测定值与健康对照鸭均无明显差异,但强毒对照鸭于接毒后24-36h的测定值与健康鸭有显著(P<0.05)或极显著差异(P<0.01)。
毒株遗传稳定性方面,以DHV A66株E66代次的种毒,每次用1～3日龄敏感雏鸭10只,连续传代回归10次进行毒力返强试验。结果各回归代次雏鸭肝组织能稳定分离到DHV,且组织含毒量相对稳定在103.5-4.8ELD50/ml范围；各代次回归试验鸭健康,剖检观察正常,病理组织学检查正常；各代次试验鸭的血液常规指标、四种主要血清酶和总胆红素(TBIL)值均正常。对DHV的A66-F10与F0全基因组序列比对和分析表明,全基因组核苷酸序列没有发生插入或缺失,两者仅有6个核苷酸差异,同源性达99.9%以上,ORF氨基酸序列未发生改变。充分证明DHV A66株具有良好的安全性和遗传稳定性。
以DHV A66株种毒经9～10日龄SPF鸡胚连续传代至E80代,通过对不同代次种毒的病毒含量及对1日龄雏鸭的毒力和免疫力的测定,结果表明：不同代次种毒毒价在108.10-8.50ELD50/ml之间,皮下注射免疫的半数免疫量(IMD50在106.10-6.40IMD50/ml范围,表明A66株免疫原性稳定而良好。因此,确定E66为原始种子代次,基础种子代次为E67～72,种子继代培养不超过3代,即生产用抗原最高代次为E75。通过对A66株E66、E70和E75代次鸡胚混合毒的最小免疫量测定,并取A66株鸡胚传代较高代次E75以不同倍数最小免疫量进行免疫试验,确定A66株的最小免疫量为103.3ELD50；根据免疫试验结果及其它因素综合考虑,确定A66株的免疫剂量(即使用剂量)应≥104.3ELD50。
按照现行《中华人民共和国兽药典》[16]规定方法,对DHV A66株种毒E66、E70代毒种进行无菌检验和外源病毒检验,结果细菌和支原体检验阴性,12种现行《中华人民共和国兽药典》[16]规定检测的外源病毒和鸡传染性贫血病毒(CIAV)也全部为阴性,表明DHV A66株毒种纯净。
3、检验用鸭肝炎强毒株(W株)的系统鉴定
通过对DHV W强毒株接种雏鸭复壮,进行病毒传代、毒力及其稳定性测定、血清学特异性及与DHV R85952强毒参考株进行毒力比较试验等,认定DHV W毒株可作为检验用强毒。对W毒株的攻毒剂量和保存期试验表明：W株的攻毒剂量1～7日龄选用103.0LD50/鸭,7～10日龄选用104.0LD50/鸭；W株的肝组织毒冻干后在-20℃和-70℃中保存期分别可达5年和8年。
4、鸭病毒性肝炎活疫苗(A66株)的安全性、效力和保存期研究
对5批实验室制备的DHV(A66株)活疫苗,分别通过皮下注射、口服途径对1日龄敏感雏鸭进行一次单剂量接种、单剂量重复接种和一次大剂量接种的安全性试验。临床观察试验雏鸭的精神、采食、饮水、生长、增重等均正常,肝功能检查、解剖观察肝脏等内脏组织器官及肝脏组织切片检查无异常。试验结果表明：A66活疫苗对雏鸭使用安全性好、无副作用。
用A66株最高代次(E75代)生产用毒种制备的三批疫苗,每批疫苗分别经皮下注射、肌肉注射、饮水和口服途径免疫易感雏鸭各10只。试验结果表明：经皮下和肌肉注射途径免疫雏鸭,2-3天就能产生免疫保护力；经口服、滴鼻和饮水途径免疫雏鸭,5天能产生良好的保护效果。以该三批疫苗1/10使用剂量免疫1日龄易感雏鸭5天后(120h)用强毒株进行攻毒,免疫组雏鸭全部保护,表明用A66株最高代次毒种制备的疫苗仍具有良好的免疫效力。将疫苗按免疫剂量接种1日龄易感雏鸭,雏鸭的免疫期至少达60天以上,即一次免疫就能有效保护雏鸭安全度过易感期(6周龄以内雏鸭)。
采用10倍免疫剂量的DHV(A66株)活疫苗,分别经皮下注射和口服途径接种1日龄易感雏鸭各20只,5日后随机取出其中10只与20只1日龄易感雏鸭进行第1代同居饲养5日,再取10只同居感染鸭与20只1日龄易感雏鸭进行第2代同居感染试验,如此连续进行5代次的同居感染试验。通过定时采集接种鸭及同居鸭肛拭子样品进行PCR检测,测定DHV A66株的感染和排毒情况；对接种或同居5日的试验鸭进行攻毒保护试验,测定同居感染的保护情况。结果表明：A66株活疫苗经皮下注射和口服途径接种雏鸭,均可以通过粪便向体外排泄病毒；同居易感雏鸭可经粪—口途径接触感染,感染雏鸭可获得鸭肝炎的免疫保护；同居感染现象随着同居代次的增加,同居感染的排毒时间逐渐延迟、感染率逐步下降,至同居5代时这种同居感染现象基本终止。
选择经鸭肝炎疫苗免疫的种鸭所产种蛋孵化的雏鸭,采用鸡胚中和试验测定其鸭肝炎母源抗体水平；对不同母源抗体水平的雏鸭于1日龄时用1羽份疫苗进行免疫,分别于免疫后2天和5天用1型DHV W株强毒攻击,结果表明,母源抗体水平对鸭肝炎活疫苗免疫效果无明显影响。
以制备的3批A66株活疫苗分别经过不同保存温度、不同保存期后,测定其鸡胚半数致死量(ELD50)的变化情况,判定疫苗的稳定性；通过对疫苗的免疫攻毒保护试验、物理性状观察、水分及真空度测定,以确定疫苗的保存期。试验结果表明DHV (A66株)活疫苗在-15℃条件下可保存2年以上,2-8℃条件下可保存1年以上；疫苗用生理盐水稀释后在室温(25)℃条件下存放8h毒价下降不明显。
5、鸭病毒性肝炎活疫苗(A66株)的生产工艺研究和中试生产
选择蔗糖、明胶、蛋白(氨基酸)等组份,与真空度、升温速度等因素,通过水平正交设计试验进行冻干保护剂筛选和冻干曲线的优化研究。根据试验结果确定A66株活疫苗生产工艺为：种毒以100～1000倍稀释、经尿囊腔途径接种9～11日龄SPF鸡胚,0.2ml/胚,收获接种后36～72h死亡鸡胚组织(胚体+胚膜+胚液),用组织捣碎机匀浆3～5分钟后保存；优化的保护剂配方和冻干条件为：5%蔗糖、1.5%明胶、6%甘氨酸的保护剂,真空度0.02mbar,升温速度为1.5℃/min。抗原与保护剂1：1比例,在冻干曲线：-40℃3h;-25℃13h;-10℃4h;25℃4h时,疫苗冻干效果最佳。按照确定的保护剂和冻干条件验证3批产品,其物理性状、真空度、残余水分含量均符合《中华人民共和国兽药典》[16]规定,3批疫苗冻干前后毒价损失≤100.6滴度。
按照本研究制定的《鸭病毒性肝炎活疫苗中间试制规程及质量标准》(草案),在南京天邦生物科技有限公司进行中试生产了5批鸭肝炎活疫苗(A66株),共计605万羽份。5批疫苗按照中间试制规程和质量标准进行检验,结果物理性状、无菌检验、支原体检验、病毒含量、安全检验、效力检验、剩余水分测定、真空度测定均符合标准。表明该疫苗生产工艺合理,产品质量稳定,适合规模化生产。
6、鸭病毒性肝炎活疫苗(A66株)临床试验
2008年12月27日获得农业部兽用生物制品临床试验批件(批件号：200844)后,自2009年2月至6月在安徽省5个鸭场(企业)应用5个批次的中间试制产品进行临床试验,试验共计免疫雏鸭5.9万羽。经临床观察、攻毒试验、抗体检测及生产性能的统计分析,结果表明：鸭病毒性肝炎活疫苗(A66株)对免疫雏鸭安全、有效、无不良反应。
Duck viral hepatitis (DVH) is a highly lethal acute infectious disease of ducklings; its main characteristics is hepatitis and the pathogen is Duck hepatitis virus (DHV). DHV1is the mainly epidemic in China. Vaccination is the most effective measure to prevent the disease, and there is no commercial vaccine in China. In the study, In the study, we selected the60passages virus (E60) of DHV1A66attenuated strain by chicken embryo passage as the origin virus, by cloning selection on SPF chicken embryo acquired DHV A66strain. On the basis of the detection of the safety, immunogenicity, purity and the genetic stability of A66strain, we do further research of the safety, production technology and trial production of the vaccine, and completed the clinical test research of the vaccine after the permission of the Ministry of Agriculture, finally, we developed DHV living vaccine (A66strain).
1. Cloning selection and purification of DHV1A66attenuated strain
Treating the E60of DHV1A66attenuated strain by chicken embryo passage as the origin virus, cloning selection of inoculation on SPF chicken embryos by the limited dilution for3times, purified cultivation for2times and1times expanding cultivation till66passages(E66), detecting the virus titre,virulence,immunogenicity, purity and exogenous virus, the results showed that the E66has met the requirements of producing vaccine. Therefore, we determined the chicken embryo virus E66of DHV as the origin virus for the development of DHV living vaccine, which we named DHV A66strain.
2. The safety, genetic stability, immunogenicity and purity of DHV A66strain
1-day-old ducklings were inoculated by subcutaneous injection and oral route with the E66passage virus of DHV A66; And the clinical observation of the ducklings was normal; there were no visible pathological changes on liver and other visceral organs by necropsy observation; histological examination of liver and other tissues was normal. Then,201-day-old ducklings inoculated by subcutaneous injection with a large dose of this virus were collated blood to do cell count, erythrocyte sedimentation rate, to determine the hemoglobin and other blood tests at different time after inoculation, at the same time, to detect alanine aminotransferase(ALT), aspartate aminotransferase(AST), glutamine transferase (GGT), alkaline phosphatase (ALP) and other serum enzymes and bilirubin to reflect the liver function by separated serum. The results showed:the measured values of blood routine, four serum enzymes and total bilirubin (TBIL). of the test ducklings have no obvious difference with healthy control ducklings, but there were significant (P<0.05) or extremely significant difference (P<0.01) between the measured values of the virulent control ducklings after24-36h inoculation and healthy10ducklings.
In terms of genetic stability of strains, using101-3days old ducklings infected by the E66passage virus of DHV1A66each time, we did the test of virulence rejuvenation by the continuous passage for10times, the duckling liver tissues of each regressive passage can be detected DHV, the virus titre of which is relatively stable at the range of103.5-4.8ELD50/ml; the ducklings of each regressive generation are healthy, pathological observation and pathological examination are all normal; the measured values of blood routine, four serum enzymes and total bilirubin (TBIL) of ducklings for each regressive generation are all normal. Compared and analyzed the the whole genome sequence between A66-F10and F0of DHV1, the results show that there is not insertion or deletion in whole genome sequence, with the difference of only6nucleotides, more than99.9%homology, and the sequences of ORF amino acid don't change. The above prove DHV1A66attenuated strain has a good safety and genetic stability.
Continuous passage on9-10days old SPF chicken embryos inoculated with the virus of DHV1A66till E80generation, we determined the content of each generation virus and the immune ability of1-day-old ducklings, which showed that:the virus titre of different generations is in10ELD50/lml to10ELD50/lml, the IMD50of subcutaneous immunization is in106.10IMD50/lml to10640IMD50/1ml, and indicated that the immunity of strain A66is stable and good. Accordingly E66is the original seed passage, E67-72are basic seed passages, the sub-cultivation of the seeds is not more that3generations, that is E75is the highest passage generation of the antigen used in production. By the determination of the minimum immune amount of the mix virus of E66, E70and E75chick embryo passages of A66strain, and the immune test with the minimum immune dose of different times taken by the high chicken embryo passages E75of A66strain, we determined that the minimum immune of A66strain is103.3ELD5o; we determined that the immune dose of A66strain should be higher than104.3ELD50, according to the results of immune tests and integrated consideration of other factors.
Under the current provisions of "China Veterinary Pharmacopoeia", to determine the bacteria and the exogenous virus, the result was negative for bacteria, mycolasma,12types of exogenous virus need to determine by the current provisions of "China Veterinary Pharmacopoeia" and chicken infectious anemia virus (CIAV), which indicated that the virus seed of DHV A66strain is purified.
3. System identification of duck hepatitis virulent strain (W strain) used for detection
After the rejuvenation of DHV1W virulent strain inoculated ducklings, we did the virus passage, detected the virulence, stability and serological specificity, and compared the virulence with the virulent DHV1R85952, etc, in order to determine DHV1W suitable for detection. The text of the dose of attacking virus and retention period of W strain show that1-7day-old ducklings immunized10LD50/duck and7-10day-old ducklings immunized104LD50/duck; the retention period of the liver tissue freeze-dried of W strain, stored at-20℃and-70℃,are respectively for5and8years.
4. The safety, effectiveness and retention period of the duck virual hepatitis living vaccine (A66strain)
5batches A66vaccine made in laboratory, respectively, by subcutaneous injection and oral route on1day-old ducklings, are done the safety texts of a single dose of vaccination, a single dose of repeat vaccination and a large dose of vaccination, the spirit, feeding, drinking, growth and weight gain of the ducklings were all normal by clinical observation; the results of liver function test, anatomical observation of liver and other organs, and the liver histological examination were normal. The results showed that:A66live vaccine is safe and has no side effects for young duckling.
Using the virus seeds of the highest passages of attenuated strain A66(E75) for the production of vaccine to prepare three batches, each batch of vaccine was treated with subcutaneous injection, intramuscular injection, drinking water and oral route on10susceptible ducklings. The results showed that:the ducklings immunized by subcutaneous and intramuscular injection can generate protective immunity in2to3days; the ducklings immunized by oral, intranasal and drinking water means, produce good protective effect in5days. Using1/10dose of the three batches vaccines to immune susceptible1-day ducklings, which are attacked with a virulent virus after5days (120h), immuned ducklings are all protected, and this indicates that the vaccines produced by the highest passages of A66have good immune efficacy. The immune period of the1-day-old ducklings inoculated the immune dose of vaccine is more than60days, that means once immunization can effectively protect the duckling to live through susceptible period (less than6-weeks-old ducklings.)
Each201-day-old ducklings immunized with with10-time dose of A66vaccine, respectively by the oral route and subcutaneous injection, after5days,10of which selected randomly cohabit and feed for5days with201-day-old susceptible ducklings for the1st generation;10of1st generation cohabitation infected ducklings cohabit with201-day-old susceptible ducklings for the2st generation; in this way,5consecutive passages of cohabitation infection experiments are done. The anal swab samples of inoculated ducks and cohabited ducks collected regularly are done PCR, to determine the infection and detoxification of DHV1A66inoculated ducks or5-day cohabited ducks are done the attacking-virus protection text to determine the conservation status of cohabitation infection. The results showed that:the ducklings immunized A66vaccine by subcutaneous injection and oral route can both excrete the virus with the feces; the cohabited susceptible ducklings can be infected by the fecal-oral route, infected ducklings can acquire the immune protection; with the increase of cohabited generations, the excretion time of virus is delayed gradually, the infectious rate is declined gradually, the phenomenon of cohabited infection ends basically till5st generational cohabitation. immune protection; with the increase of cohabited generations, the excretion time of virus is delayed gradually, the infectious rate is declined gradually, the phenomenon of cohabited infection ends basically till5st generational cohabitation.
The ducklings from hatching eggs produced by ducklings immunized by DHV vaccine were detected the level of maternal antibody of DHV using chick embryo neutralization test; the ducklings with different levels of maternal antibodies were immunized with1plume dose at1day old, and then were attacked the virulent DHV1W on second day and5st day after immunization; the results showed that the level of maternal antibodies has no obvious effect on immune effect of the vaccine.
3batches A66strain live vaccine prepared, saved at different temperatures and period, were detected the changes of the half chicken embryo lethal dose(ELD50) to judge the stability of vaccine; the retention period of vaccines was determined by attacking-virus immune protection test, observation of physical properties, the measurement of water and vacuum.The results showed that:A66live vaccine can be stored at-15℃in2years,2～8℃in1year; the titre of vaccine diluted with saline and stored for8h at25℃isn't declined obviously.
5. Production technology and intermediate test production of the duck virual hepatitis living vaccine (A66strain)
The test of level orthogonal design is to select the freeze-dried protectant and to optimize the freeze-dried curve, by selecting sucrose, gelatin, protein (amino acids) and other components, and by controlling the degree of cacuum, heating rate and other factors. According to the results of the tests, the productive technology of A66strain live vaccine is that, Dilution of strain virus by100-1000times, inoculation of9-11days SPF chicken embryos by urine cysts,0.2ml/embryo, harvest of dead chicken organization (body+membrane+liquid) after36-72h inoculation, homogenate of the tissue for3-5min and save; The formula of optimal protectant and freeze-dried condition is:5%sucrose,1.5%gelatin,6%glycine protection agents, vacuum0.02mbar, heating rate of1.5℃/min.1:1ratio of antigen and protective agents, in the freeze-drying curve:-40℃3h;-25℃13h;-10℃4h;25℃4h, which is the best freeze-dried vaccine. To verify the three batches by the identified protectant and the freeze-dried condition, its physical properties, vacuum, residual water content are in line with the "China Veterinary Pharmacopoeia" and the loss of virus titre is low to100.6.
According to this study, we developed a draft "Duck hepatitis vaccine trial procedures and quality standards ", and produced5batches of vaccine A66, a total of6.05million doses in Nanjing Tianbang Biotechnology Co., Ltd. To detect5batched of vaccine by trial procedures and quality standards, the results of physical traits, sterility test, Mycoplasma test, virus content, security testing, effectiveness testing, residual moisture content, determination of the vacuum are all in line with the standards. Which indicated that the production technology is reasonable; the production is stable and is suitable for large-scale production.
6. The duck virual hepatitis living vaccine (A66strain) in clinical trials
After the acquirement of the approval document of clinical trials of veterinary biological products of the Ministry Agriculture (number:200844) on27December2008, we had applied5batch products for clinical trials to test a total of59,000young ducklings in5duck farms of Anhui Province since February to June2009.
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