狂犬病灭活疫苗免疫佐剂的比较试验

本文利用反向遗传操作系统拯救出狂犬病病毒的携带双G基因的HEP-dG株,选用铝佐剂、蜂胶和油乳剂制成3种狂犬病灭活疫苗,进行小鼠免疫试验.试验表明,油乳剂和蜂胶佐剂狂犬病灭活疫苗免疫组效价较高.蜂胶狂犬病灭活疫苗二次免疫产生的抗体水平明显高于首免,油乳剂狂犬病灭活疫苗首免抗体水平与蜂胶狂犬病灭活疫苗二免产生的抗体水平相当.携带双G基因的HEP-dG株具有良好的免疫原性,油乳剂是作为该狂犬病毒株的最适佐剂.     

铝胶佐剂对狂犬病灭活疫苗(Flury株)效力的影响

氢氧化铝胶是一种广泛应用于疫苗的佐剂,具有抗原吸附力强的特点,注入机体后可持续释放出抗原物质,刺激机体产生抗体,从而提高免疫效果,其主要用于细菌类灭活疫苗,在病毒类疫苗中也有应用,如狂犬病灭活疫苗等[1]。为了解不同浓度铝胶佐剂对狂犬病灭活疫苗(Flury株)效力的影响,进行了如下试验。     

狂犬病灭活疫苗CVS-11株对牛的免疫效果

野生狐和貉狂犬病流行于新疆、内蒙古和黑龙江地区,是当地牛、羊和骆驼等家畜狂犬病的主要传染源。在国内尚无野生动物口服疫苗的前提下,对家畜进行狂犬病灭活疫苗接种,具有重要的公共卫生学意义。本研究利用国产犬用狂犬病灭活疫苗(CVS-11株)对牛进行了免疫效果和安全性评价。以不同剂量疫苗肌内注射免疫成年牛350头,通过比较免疫后狂犬病病毒中和抗体水平和持续期,确定犬用狂犬病灭活疫苗(CVS-11株)在牛的最佳免疫剂量为1次性注射2剂量疫苗,免疫持续期为1年。结果表明,犬用狂犬病灭活疫苗(CVS-11株)免疫牛时,具有较高的免疫原性和安全性,适用于牛等大型动物的狂犬病注射免疫。     

嵌合犬瘟热病毒H基因的重组狂犬病病毒的构建及免疫原性研究

【目的】构建表达犬瘟热病毒（CDV）血凝素蛋白H基因的重组狂犬病病毒（RABV）,并研究其生物学特性及免疫原性。【方法】在RABV HEP-dG株基因组G与L基因之间插入CDV H基因,构建重组全长cDNA质粒pHEP-dG （H）。将pHEP-dG （H）与辅助质粒共同转染BHK细胞,拯救携带CDV H基因的重组RABV HEP-dG （H）。将重组病毒接种BHK细胞,分析病毒的扩散能力;将重组病毒接种小鼠,分析病毒的致病性和免疫原性。【结果】RT-PCR及直接免疫荧光显示,传至第3代的病毒仍能检测到H基因,表明CDV H基因已成功插入RABV基因组中,并在HEP-dG （H）中稳定遗传和正确表达。HEP-dG （H）在BHK细胞中的生长曲线与HEP-dG毒株相似,病毒滴度在96 h达到峰值,但HEP-dG （H）的滴度在每个时间点略低于HEP-dG。以感染复数（MOI）为0.005感染BHK细胞,HEP-dG （H）的扩散能力比HEP-dG毒株低。HEP-dG （H）与HEP-dG对6周龄成年小鼠均不致死,但HEP-dG （H）对成年小鼠体重的影响弱于HEP-dG。HEP-dG （H）与HEP-dG均能诱导小鼠产生抗RABV的中和抗体,免疫后7 d抗体已达到保护水平（0.5 EU/mL）;此外,HEP-dG （H）可诱导产生CDV中和抗体。HEP-dG （H）与HEP-dG免疫小鼠3周后,均能抵御RABV标准攻毒毒株CVS-24的攻击。【结论】本研究成功构建重组RABV HEP-dG （H）,其具有良好的免疫原性和安全性,可作为RABV-CDV新型二联基因工程候选疫苗。     

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

自2011年以来,新型伪狂犬病病毒(PRV)变异株在我国免疫猪场不断出现,导致目前商品化疫苗免疫效果不佳。为研究PRV FB株gE/gI基因缺失株疫苗防控新型PRV变异株感染的效果,本研究以PRV自然弱毒FB株作为亲本株,通过同源重组的方法构建了PRV g E/gI基因缺失株,将不同代次PRV FBΔgE/gI株与亲本PRV FB株分别接种BHK-21细胞,观察CPE和测定TCID50效价,分析缺失株的遗传稳定性,结果显示FBΔgE/gI株感染BHK-21细胞后产生与亲本株FB相似的CPE,且不同代FBΔgE/gI株的TCID50与亲本株相比均无明显变化。将FB株、FBΔgE/gI株、Bartha-K61株分别接种健康新西兰兔,比较三者的安全性,结果显示,接种BarthaK61株的家兔全部死亡(5/5),接种FB株的家兔死亡2只(2/5),而接种FBΔgE/gI株的家兔全部(5/5)健活,表明FBΔgE/gI株对新西兰兔的致病力较低,安全性更高。将FBΔgE/gI株分别与水相佐剂GEL02和两性佐剂ISA 206制成灭活疫苗免疫绵羊28 d后,采用ELISA法检测各组绵羊的gB和gE抗体,并且以PRV变异株FJ-2012攻毒,评估FBΔgE/gI株灭活疫苗对绵羊的保护效力,结果显示,接种GEL02和ISA 206为佐剂的FBΔgE/gI灭活疫苗绵羊在免疫后28 d,抗体均全部转阳;对照组绵羊在攻毒后6 d内全部死亡(5/5),以GEL02作为佐剂的灭活疫苗组绵羊死亡2只(2/5),而以ISA 206为佐剂的绵羊全部存活(5/5),保护率达到100%,表明以ISA 206作为佐剂的FBΔgE/gI株灭活疫苗能够完全保护绵羊抵御PRV变异株的攻击。本研究首次基于PRV自然弱毒FB株构建的FBΔgE/gI株,与Bartha-K61株相比具有更高的安全性,与佐剂ISA 206制成灭活疫苗免疫绵羊后能够完全抵抗新发变异PRV的攻击,具有完全保护作用。本研究为我国新发变异PRV疫苗的研发提供了新的参考依据。     

鸽新城疫(ND)蜂胶佐剂灭活疫苗免疫效果对比试验

采用鸽新城疫ND-GS 01株毒为种毒,分别研制出了尿囊液毒蜂胶佐剂灭活疫苗和四元材料(鸡胚尿囊液、羊水、尿囊膜、羊膜)混合病毒蜂胶佐剂灭活苗。对研制的两种灭活疫苗经安全性检验、急性毒性试验,免疫抗体消长水平与免疫持续期对比试验,结果表明,四元材料的疫苗与尿囊液疫苗性能一致。     

鸽新城疫rGX-mF株灭活疫苗的制备及免疫原性分析

为有效控制临床中鸽新城疫的发生，选取基因Ⅵ型鸽新城疫病毒弱毒株rGX-mF作为疫苗候选株，评价其生物学特性，并将rGX-mF灭活后制备成油佐剂灭活疫苗，接种雏鸽和SPF鸡进行rGX-mF株的免疫原性分析，为研制安全高效的鸽新城疫灭活疫苗奠定基础。结果表明，rGX-mF株能够在鸡胚中稳定传代，效价高，毒力弱；rGX-mF株灭活疫苗接种雏鸽后疫苗吸收良好，无局部和全身不良反应，安全性好，免疫持续期长达12个月；rGX-mF株灭活疫苗与LaSota株灭活疫苗相比，rGX-mF株灭活疫苗在鸽体内能产生更高水平的抗体；rGX-mF株灭活疫苗同时免疫SPF鸡和雏鸽，SPF鸡产生的抗体显著高于鸽产生的抗体，这可能与鸽和鸡的免疫系统存在差异有关。综上，利用基因Ⅵ型鸽新城疫病毒弱毒rGX-mF株制备的灭活疫苗可在鸽体内产生有效的抗体，免疫效果好，免疫持续期长。     

猪伪狂犬病活疫苗(Bartha-K61株)免疫产生期和免疫持续期的研究

为评价猪伪狂犬病活疫苗(Bartha-K61株,传代细胞源)的免疫保护效力,本研究对3批疫苗分别进行免疫产生期试验和免疫持续期试验。免疫产生期试验中将3批疫苗以单剂量免疫仔猪,在免疫后2、3、4、5、6 d连同对照组分别攻击伪狂犬病强毒,结果表明猪伪狂犬病活疫苗免后5 d即可产生坚强的免疫保护力。免疫持续期试验中将3批疫苗以单剂量免疫母猪,免疫后12、14个月连同对照猪分别攻击伪狂犬病强毒,结果显示免疫母猪及其所产仔猪均健康存活,表明猪伪狂犬病活疫苗(Bartha-K61株)的免疫持续期可长达14个月。     

密码子优化的RHDV2双VP60基因重组杆状病毒构建及表达蛋白免疫原性分析

【目的】试验旨在优化兔出血症病毒2型(Rabbit hemorrhagic disease virus 2,RHDV2)病毒样颗粒(virus-like particle, VLP)疫苗的制备策略,探究RHDV2 VLP疫苗对家兔的免疫原性,为低成本、高产量RHDV2新型疫苗研发提供新思路。【方法】根据昆虫细胞的密码子偏好性优化合成RHDV2 VP60全基因,将双VP60基因插入真核载体pFastBac^TM Dual,转化携带Bacmid质粒的大肠杆菌DH10Bac感受态细胞,构建含双VP60基因的重组杆粒Bacmid-VP60-VP60,转染Sf9昆虫细胞,通过Western blotting、间接免疫荧光试验(IFA)及透射电镜对重组杆状病毒Bacmid-VP60-VP60进行表达验证;将优化策略制备的重组蛋白抗原与氢氧化铝佐剂按照9∶1比例制备VLP灭活疫苗,通过安全性检验、最小免疫剂量、免疫持续期等评估优化策略制备的RHDV2 VLP疫苗的保护效果。【结果】试验成功构建重组杆粒Bacmid-VP60-VP60。Western blotting鉴定结果显示,...     

鸽新城疫灭活疫苗的研制及免疫效果研究

利用近年来分离到的鸽Ⅰ型副黏病毒ND32株进行鸽新城疫油乳剂灭活疫苗的研制,并对制备疫苗的性状、安全性、免疫效力等进行检验,结果表明制备疫苗的各项指标均符合生物制品通则的要求,对疫苗的最小免疫剂量、抗体产生及免疫持续期、与同类产品的交叉攻毒保护对比效果进行了进一步试验,结果表明疫苗的最小免疫剂量为0.15 mL/只,疫苗以0.3 mL/只免疫剂量进行一次免疫,免疫持续期为3个月,以0.3 mL/只免疫剂量进行二次免疫,免疫持续期可达6个月.交叉攻毒保护对比试验结果表明所制备的鸽新城疫灭活疫苗对鸽Ⅰ型副黏病毒ND32株和鸡新城疫病毒北京株(CVCC AV1611)的攻毒保护效果均优于鸡新城疫(La Sota株)灭活疫苗.     

Cell-mediated immune response to rabies virus in dogs following vaccination and challenge

Peripheral blood lymphocytes (PBL) from non-vaccinated dogs and from dogs either vaccinated intramuscularly (IM) or subcutaneously (SC) with an inactivated rabies virus vaccine (Rabguard-TC, Norden Laboratories, Lincoln, NE) or intramuscularly with an attenuated rabies virus vaccine (Endurall-R, Norden Laboratories, Lincoln, NE) were exposed in vitro to rabies virus. Blastogenesis of PBL was measured by incorporation of 3H-thymidine into the DNA of proliferating cells in the presence of a suboptimal concentration of phytohemagglutinin (PHA). Following the first vaccination, there was no difference in the blastogenic response of lymphocytes from dogs vaccinated IM with either the inactivated or attenuated rabies virus vaccines. The inactivated rabies vaccine stimulated as great or greater blastogenic response when it was given SC. The PBL from non-vaccinated control dogs were not stimulated by rabies virus. Dogs vaccinated with the inactivated vaccine developed a lymphocyte blastogenic response to rabies virus following challenge with virulent street rabies virus. Nonvaccinated control dogs did not develop a lymphocyte blastogenic response to rabies virus following challenge with virulent street rabies virus.     

Antibody response to vaccines for rhinotracheitis, caliciviral disease, panleukopenia, feline leukemia, and rabies in tigers (Panthera tigris) and lions (Panthera leo)

This article presents the results of a study of captive tigers (Panthera tigris) and lions (Panthera leo) vaccinated with a recombinant vaccine against feline leukemia virus; an inactivated adjuvanted vaccine against rabies virus; and a multivalent modified live vaccine against feline herpesvirus, calicivirus, and panleukopenia virus. The aim of the study was to assess the immune response and safety of the vaccines and to compare the effects of the administration of single (1 ml) and double (2 ml) doses. The animals were separated into two groups and received either single or double doses of vaccines, followed by blood collection for serologic response for 400 days. No serious adverse event was observed, with the exception of abortion in one lioness, potentially caused by the incorrect use of the feline panleukopenia virus modified live vaccine. There was no significant difference between single and double doses for all vaccines. The recombinant vaccine against feline leukemia virus did not induce any serologic response. The vaccines against rabies and feline herpesvirus induced a significant immune response in the tigers and lions. The vaccine against calicivirus did not induce a significant increase in antibody titers in either tigers or lions. The vaccine against feline panleukopenia virus induced a significant immune response in tigers but not in lions. This report demonstrates the value of antibody titer determination after vaccination of nondomestic felids.     

Results of vaccination of Asian elephants (Elephas maximus) with monovalent inactivated rabies vaccine

OBJECTIVE: To evaluate the humoral immune response of Asian elephants to a primary IM vaccination with either 1 or 2 doses of a commercially available inactivated rabies virus vaccine and evaluate the anamnestic response to a 1-dose booster vaccination. ANIMALS: 16 captive Asian elephants. PROCEDURES: Elephants with no known prior rabies vaccinations were assigned into 2 treatment groups of 8 elephants; 1 group received 1 dose of vaccine, and the other group received 2 doses of vaccine 9 days apart. All elephants received one or two 4-mL IM injections of a monovalent inactivated rabies virus vaccine. Blood was collected prior to vaccination (day 0) and on days 9, 35, 112, and 344. All elephants received 1 booster dose of vaccine on day 344, and a final blood sample was taken 40 days later (day 384). Serum was tested for rabies virus-neutralizing antibodies by use of the rapid fluorescent focus inhibition test. RESULTS: All elephants were seronegative prior to vaccination. There were significant differences in the rabies geometric mean titers between the 2 elephant groups at days 35, 112, and 202. Both groups had a strong anamnestic response 40 days after the booster given at day 344. CONCLUSIONS AND CLINICAL RELEVANCE: Results confirmed the ability of Asian elephants to develop a humoral immune response after vaccination with a commercially available monovalent inactivated rabies virus vaccine and the feasibility of instituting a rabies virus vaccination program for elephants that are in frequent contact with humans. A 2-dose series of rabies virus vaccine should provide an adequate antibody response in elephants, and annual boosters should maintain the antibody response in this species.     

The efficacy of a new single post-exposure treatment of rabies in mice without vaccination.

Local application of rabies immune serum and isoprinosine, an immunomodulator with antiviral activity was effective in mice infected with a sylvatic rabies virus. In this way, a single medical or veterinary treatment is only required, which is particularly important for developing but also for developed countries. The importance of using a post-exposure potency test to monitor rabies vaccines is emphasized. The same principle could be applied to other emerging viral infections of humans (for example, human immunodeficiency virus infection) and animals, for which no effective vaccines are available at this moment.     

Virology: The efficacy of a new single post‐exposure treatment of rabies in mice without vaccination

Summary

Local application of rabies immune serum and isoprinosine, an immunomodulator with antiviral activity was effective in mice infected with a sylvatic rabies virus. In this way, a single medical or veterinary treatment is only required, which is particularly important for developing but also for developed countries.

The importance of using a post‐exposure potency test to monitor rabies vaccines is emphasized. The same principle could be applied to other emerging viral infections of humans (for example, human immunodeficiency virus infection) and animals, for which no effective vaccines are available at this moment.     

Rabies – epidemiology,pathogenesis, public health concerns and advances in diagnosis and control: a comprehensive review

Rabies is a zoonotic, fatal and progressive neurological infection caused by rabies virus of the genus Lyssavirus and family Rhabdoviridae. It affects all warm-blooded animals and the disease is prevalent throughout the world and endemic in many countries except in Islands like Australia and Antarctica. Over 60,000 peoples die every year due to rabies, while approximately 15 million people receive rabies post-exposure prophylaxis (PEP) annually. Bite of rabid animals and saliva of infected host are mainly responsible for transmission and wildlife like raccoons, skunks, bats and foxes are main reservoirs for rabies. The incubation period is highly variable from 2 weeks to 6 years (avg. 2–3 months). Though severe neurologic signs and fatal outcome, neuropathological lesions are relatively mild. Rabies virus exploits various mechanisms to evade the host immune responses. Being a major zoonosis, precise and rapid diagnosis is important for early treatment and effective prevention and control measures. Traditional rapid Seller's staining and histopathological methods are still in use for diagnosis of rabies. Direct immunofluoroscent test (dFAT) is gold standard test and most commonly recommended for diagnosis of rabies in fresh brain tissues of dogs by both OIE and WHO. Mouse inoculation test (MIT) and polymerase chain reaction (PCR) are superior and used for routine diagnosis. Vaccination with live attenuated or inactivated viruses, DNA and recombinant vaccines can be done in endemic areas. This review describes in detail about epidemiology, transmission, pathogenesis, advances in diagnosis, vaccination and therapeutic approaches along with appropriate prevention and control strategies.     

Response of feral cats to vaccination at the time of neutering

OBJECTIVE: To determine whether administration of inactivated virus or modified-live virus (MLV) vaccines to feral cats at the time of neutering induces protective serum antiviral antibody titers. DESIGN: Prospective study. ANIMALS: 61 feral cats included in a trap-neuter-return program in Florida. PROCEDURES: Each cat received vaccines against feline panleukopenia virus (FPV), feline herpes virus (FHV), feline calicivirus (FCV), FeLV, and rabies virus (RV). Immediately on completion of surgery, vaccines that contained inactivated RV and FeLV antigens and either MLV or inactivated FPV, FHV, and FCV antigens were administered. Titers of antiviral antibodies (except those against FeLV) were assessed in serum samples obtained immediately prior to surgery and approximately 10 weeks later. RESULTS: Prior to vaccination, some of the cats had protective serum antibody titers against FPV (33%), FHV (21%), FCV (64%), and RV (3%). Following vaccination, the overall proportion of cats with protective serum antiviral antibody titers increased (FPV [90%], FHV [56%], FCV [93%], and RV [98%]). With the exception of the FHV vaccine, there were no differences in the proportions of cats protected with inactivated virus versus MLV vaccines. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that exposure to FPV, FHV, and FCV is common among feral cats and that a high proportion of cats are susceptible to RV infection. Feral cats appeared to have an excellent immune response following vaccination at the time of neutering. Incorporation of vaccination into trap-neuter-return programs is likely to protect the health of individual cats and possibly reduce the disease burden in the community.     

H9亚型禽流感油乳剂灭活苗免疫产生期内T淋巴细胞表型亚类的检测与研究

采用免疫荧光法、使用流式细胞检测仪对经H9亚型禽流感病毒人工感染SPF鸡、H9亚型禽流感油乳剂灭活苗免疫SPF鸡以及经免疫后使用H9亚型禽流感病毒攻毒后的SPF鸡外周血、脾脏、胸腺中T细胞表型亚类(CD4+、CD8+、TCR1+)的变化规律进行了监测,结果表明,H9亚型禽流感油乳剂灭活苗免疫后抗原的缓慢释放可在一定程度上激发机体的细胞免疫应答,使免疫活性T淋巴细胞得到活化,免疫后鸡体外周血中CD4+、CD8+和TCR1+T细胞的数量呈现出一明显升高的过程;同时,人工感染免疫鸡后,脾脏和胸腺TCR1+T细胞的数量上升,外周血CD4+、CD8+和TCR1+T细胞的数量少量降低或维持不变,随后短期即恢复正常;而人工感染SPF对照鸡后,外周血CD4+、CD8+和TCR1+T细胞的数量呈现下降趋势.     

A plaque-purified rabies virus (strains V-319) derived from a vampire bat (Desmodus rotundus) in Mexico: vaccine potential

A plaque-purified experimental rabies vaccine was developed from an isolate (strain V-319) made from a naturally infected vampire bat (Desmodus rotundus). Two different vaccines were prepared; one was live virus and the second was an inactivated rabies virus preparation. The live virus vaccine, as well as a betapropiolactone-inactivated vaccine, gave complete protection to challenge inoculation after 1 year. In contrast, greater than 80% of the non-vaccinated experimental control cattle died of rabies. The live virus vaccine could be given at doses as low as 10(5) PFU without loss of efficacy. It did not cause adverse reactions. More than 10,000 cattle have been vaccinated with the live virus vaccine under field conditions. No rabies deaths occurred in vaccinated cattle during a 2-year postvaccinal period. The serological responses of vaccinated cattle indicated protection that endured at least 1 year.