Immune Response in Primates Vaccinated with Duck Embryo Cell Culture Rabies Vaccine

Adult rhesus monkeys (Macaca mulata) were vaccinated with four inactivated rabies vaccines, including two cell culture vaccines, one zonal purified cell culture vaccine, and a 10% extracted duck embryo vaccine. The vaccines were potency tested by both National Institutes of Health (NIH) and Habel methods and passed one or both tests. However, a vaccine having acceptable potency by one method frequently failed or was marginal by the other procedure. Groups of three monkeys were inoculated with each vaccine by one of two schedules. The first consisted of four weekly 1-ml doses followed by a 1-ml booster dose at 6 months, and the second consisted of seven daily 1-ml doses of vaccine with no booster. Both zonal purified and extracted duck embryo vaccines induced detectable neutralizing antibody by day 7 with either schedule, and antibody titers elicited by the cell culture vaccine remained high through 210 days. However, antibody titers produced by the 10% duck embryo vaccine dropped sharply after their 28-day peak. Duck embryo cell culture vaccines with low or marginal potency as measured by Habel or NIH tests still produced rapid, high levels of serum-neutralizing antibody in primates. LD(50) or NIH and Habel tests as measured in mice were not necessarily good indices of antibody response in the primate host. The need for a cell culture potency test that will yield a more predictable correlation with the definitive host's antibody response is discussed.     

Antibody Response to a Human Diploid Cell Rabies Vaccine

An experimentally killed rabies virus vaccine prepared in a human diploid cell strain (WI-38)—Wyeth rabies vaccine (WRV)—was used by various injection schedules in two separate studies to define more closely in human volunteer subjects an effective vaccination schedule for pre- or postexposure immunization, particularly for donors of rabies-hyperimmune plasma. To permit valid comparisons between our results and those of other workers, antibody levels achieved were expressed in terms of international units (IU) per milliliter of serum. Antibody response of previously nonvaccinated persons were only modest after a single dose of WRV, never reaching a level higher than 0.80 IU/ml over a 56-day testing period. Moreover, antibody was not detected at 0.16 IU/ml before the 14th day, either after a single dose or after two doses given 3 days apart. The best response followed four doses of WRV given within 4 weeks. This schedule resulted in the highest rate of seroconversion to the ≥6 IU/ml antibody level required of potential rabies-immune plasma donors. Giving the first vaccine dose in aluminum hydroxide diluent did not enhance the antibody response. There was a definite suggestion in the various injection schedules that higher and more sustained antibody levels were reached when the interval between the first and second vaccine doses was longest. The greater immunogenicity of WRV as compared with duck embryo vaccine was best demonstrated by the fact that a single booster dose of duck embryo vaccine to previously vaccinated individuals resulted in only a sevenfold antibody rise during the following 56 days, whereas a booster dose of WRV elicited a 69-fold rise. Al(OH)₃ in the first dose of WRV had no effect, but the enhancing effect of a longer interval between vaccine doses was noted once again; 20 of 20 subjects who received three doses of WRV with 4 weeks between doses developed good levels of rabies antibody, and 19 exceeded 6 IU/ml.     

柱层析法纯化人用地鼠肾细胞狂犬病疫苗

用Ｓｅｐｈａｒｏｓｅ－６Ｂ凝胶层析法纯化原代地鼠肾细胞狂犬病苗,可去除绝大部分杂蛋白,总蛋白含量减少９９．４％以上,纯化后抗原比活性提高２１０倍,单位疫苗剂量中牛血清含量降低１８．７倍以上。方法简单,价廉,适合于工业生产     

用CaG株毒种制备精制狂犬病疫苗的研究

通过对狂犬病毒aG株在金黄地鼠肾细胞上传代培养,获得一种新型狂犬病毒毒株,即地鼠肾细胞适应株(CaG株)[1].由于该毒种具有生产方法简单,成本低廉,且外源因子污染机率小等优点,因此试用该毒种生产精制狂犬病疫苗.在相同条件下,分别用豚鼠脑毒种和细胞毒种各生产3批病毒原液,经相同纯化工艺制备成精制狂犬病疫苗.经初步检定用细胞毒种制备的疫苗安全性良好,疫苗免疫效价与豚鼠脑毒种疫苗无明显差异.     

Purified Rabies Vaccine (Suckling Rat Brain Origin)

A 10% suckling rat brain rabies vaccine free from encephalitogenic activity was prepared and inactivated with 1:8,000 beta-propiolactone (BPL), or ultraviolet light, or a combination of ultraviolet light and BPL, or 1% phenol. Potency was excellent in all samples, with the exception of the phenolized product which was marginal. A purified suckling rat brain (SRB) vaccine prepared by zonal centrifugation and inactivated with 1:8,000 BPL contained about 0.01 the amount of protein nitrogen of the unpurified 10% SRB vaccine. This purified product passed the National Institutes of Health potency test for rabies vaccine after administration of a quantity equivalent to a standard 10% brain suspension.     

Vero细胞狂犬疫苗的研制

利用自建的狂犬病毒Vero细胞适应株和细胞反应器微载体系统培养Vero细胞和狂犬病毒,病毒滴度达到6．0～8．0log(10)LD(50)／ml。病毒原液纯化后制出的纯化狂犬疫苗质量基本上均能达到WHO对此类型疫苗的主要质控要求。表明疫苗小量试制工艺基本可行。     

Enhanced Growth and Plaquing of Rabies Virus in Static Chick Embryo Cell Culture

The 7-day egg passage line of HEP Flury strain of rabies virus was inoculated to primary chick embryo (CE) cells prepared in different ways to compare efficiencies of viral growth and plaquing. Special care to minimize cellular damage due to trypsin at the step of monodispersion and sowing a comparatively large number of cells for monolayer preparation were required for rabies plaquing, whereas such cares were not necessary for plaquing of vesicular stomatitis virus. Plaque number and size were increased by incorporation of a high concentration of thymidine into cell growth medium. Various other means to produce a static state of CE cells were tested, and a maximal plaquing efficiency was obtained when dishes receiving a massive number of dispersed cells in MEM plus 1% calf serum were incubated at 37 C for 1 day without any buffering for monolayer preparation and postinfection incubation was done at 32 C in a CO₂-incubator. Bottle cultures of CE cells prepared in a similar manner, when infected with HEP Flury virus, yielded a markedly higher titer of virus than CE cells prepared by our previous standard method.     

鸭呼肠孤病毒强毒株致鸭胚原代成纤维细胞凋亡的研究

通过光镜、电镜、DNA Ladder法、流式细胞术、荧光染色对鸭呼肠孤病毒(DRV)诱导鸭胚原代成纤维细胞(DEF)凋亡情况进行检测.结果显示,光镜可见细胞形态学上出现细胞皱缩,染色质浓染边移;电镜观察到细胞胞浆浓缩,细胞核染色质凝聚、部分形成凋亡小体;荧光染色结果显示,在感染后24h有激发绿色荧光的凋亡细胞出现,随着时间的推移,激发红色荧光的死亡细胞数量增多;DNA Ladder检测到感染后24～144h的DNA样品呈梯形条带;流式细胞术于感染后24h检测到凋亡细胞,其数量在72～96h达到高峰,144h开始下降.研究结果表明,DRV在DEF增殖的过程中具有诱导宿主细胞凋亡的作用.     

Immunogenicity and Safety in Adults of a New Chromatographically Purified Vero-cell Rabies Vaccine (CPRV): a Randomized, Double-blind Trial with Purified Vero-cell Rabies Vaccine (PVRV)

Recent improvements in chromatographic purification procedures have made it possible to develop a new chromatographically purified rabies vaccine (CPRV) by further purifying the current rabies vaccine prepared from Vero-cell culture (Verorab; Pasteur Mérieux Connaught). The immunogenicity and safety of primary immunization, followed by a booster at one year, with CPRV was compared to that of the purified Vero cell vaccine (PVRV) in a randomized, double-blind study carried out at four veterinary schools in France. A total of 330 healthy, male and female, first-year veterinary students, aged at least 18 years and who required pre-exposure rabies prophylaxis, were enrolled in this study. Included subjects were randomly assigned either CPRV (n = 163) or PVRV (n = 167) to be given as a primary immunization series of three intramuscular injections (D0, D7, D28), followed by a booster after 1 year (D365). Blood samples for serological analysis were taken at D0 (before first injection), D28, D42, D180, D365 (before booster) and D379. All subjects developed a strong immune response to the primary series, and at D42, all subjects had seroconverted for rabies neutralizing antibody (serum titre > or = 0.5 IU/ml). The rabies virus-neutralizing antibody GMT value at D42 in the CPRV group (23.0 IU/ml) was non-inferior to that in the PVRV group (29.6 IU/ml), according to a one-sided non-inferiority test. While antibody titres tended to decrease over the period of follow-up, at D365 (before booster), 97.5% subjects in the CPRV group and 98.8% of subjects in the PVRV group remained seroconverted. After booster, although the rabies antibody GMT value in the CPRV group was lower than that in the PVRV group, all subjects in both groups were seroconverted, and the difference is probably not clinically important. The incidence of local and systemic reactions tended to decrease with each dose during the primary immunization series, followed by a slight increase after booster (significant time-effect in an exploratory logistic regression analysis). Although mild or moderate local reactions tended to be more frequent after injection with CPRV compared to PVRV, systemic reactions were reported less often (significant group-effects in exploratory logistic regression analyses). One serious adverse event possibly related to vaccine occurred during this study (severe asthenia after the third dose of PVRV). This comparative study in healthy young adults demonstrates that the new chromatographically purified rabies vaccine is as immunogenic as PVRV, and seems to be associated with fewer systemic reactions.     

Correction: Neutralizing Antibody Response after Intramuscular Purified Vero Cell Rabies Vaccination (PVRV) in Iranian Patients with Specific Medical Conditions

Objective

Post exposure prophylaxis using one of the WHO-approved vaccines is the method of choice for preventing rabies. Abnormal immune function in patients with some specific medical conditions, such as pregnancy, chronic hepatitis B virus infection, different types of cancers like lymphoma, diabetes I and II, corticosteroid consumption by patients with rheumatoid arthritis and lupus erythematosus, could impair the immunologic response to various vaccines. The immune response to rabies vaccination has never been examined in patients with any of these described medical conditions. This study purposed to evaluate the neutralyzing antibody response after vaccination with purified Vero cell rabies vaccine (PVRV) according to the WHO-recommended Post–Exposure Prophylaxis (PEP) "ESSEN" regimen.

Methods

Thirty healthy volunteers and 50 volunteers with different medical conditions who were exposed to a suspected rabid animal in the 2nd or 3rd category of exposure received 5 doses of PVRV under the ESSEN protocol. Three blood samples were collected on days 0 (before the first dose), 14, and 35. The anti-rabies antibody titer was measured using the Rapid Fluorescent Foci Inhibition Test (RFFIT) and an ELISA Bio-Rad, Platelia, Rabies II kit.

Results

All subjects reached NAb titers above 0.5 IU/ml by day 14 after vaccination. On day 35 (1 week after receiving the last rabies vaccine), anti-rabies antibodies were in the protective level (>0.5 IU/ml) in both groups. There was no statistically significant difference in anti-rabies antibody response due to the type of exposure (category 2 or 3), and successful seroconversion was confirmed in both groups.

Conclusion

In conclusion, the ESSEN protocol using the PVRV vaccine is sufficient for rabies prophylaxis in patients with specific medical conditions.     

精制Vero细胞狂犬病疫苗的灭活和纯化

通过狂犬病病毒灭活和纯化试验,试验精制Vero细胞狂犬病疫苗。疫苗经检测残余小牛血清白蛋白含量,Vero细胞残余DNA含量,疫苗效价,安全试验均能达到WHO规程要求。初步建立了适合大规模生产精制Vero细胞狂犬病疫苗的灭活和纯化工艺。     

人用狂犬病疫苗浓缩前后内毒素含量变化的研究

本文采用显色鲎试验法测定人用狂犬病疫苗浓缩前后半成品中内毒素含量。经鲎试验抑制增强实验确定人用狂犬病疫苗半成品对鲎试验法测定内毒素无干扰作用。样品经系列稀释,测定光吸收值（５４５ｎｍ）,然后将其对数回归直线与标准品的反应直线比较,结果表明,两者间有平行关系,经对四个生物制品研究所的样品进行测定,结果显示,人用狂犬病疫苗经超滤浓缩后内毒素含量有所增加,但在被检３１批样品中,除一所二批浓苗外,其余２９批均低于以往测得的最低家兔致热剂量（３．３２ＥＵ／ｍｌ／Ｋｇ）。     

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines¹ support our understanding of invasion of the uterine wall² and remodeling of uterine spiral arteries^3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts^5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation^7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies^10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu¹³.We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues^7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models^10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.     

重组狂犬病病毒糖蛋白活酵母疫苗经小鼠口服给药后的免疫效果

目的:研究以活酵母为输送载体的狂犬病疫苗对小鼠的免疫保护能力和免疫疗程。方法:小鼠首先灌食高浓度空白活酵母INVSI,并于灌胃后8h和12h分别采集小鼠空肠和回肠组织并提取小肠浸出液培养,计算活酵母经肠胃环境后的存活率;分别取狂犬病糖蛋白(glycoprotein,G)分泌型表达菌株pYes-InG和胞内表达型菌株pYes-G灌胃小鼠,灌胃结束后12h采集小鼠血清和小肠组织,采用免疫组织化学方法检测抗原物质G在小肠上皮细胞的分布,采用ELISA检测小鼠血清中和性抗体的滴度。结果:活酵母经灌食消化8h后在小肠中的存活率最高达36.11%,12h后降至0.59%;口服分泌型pYes-InG重组酵母的小鼠小肠组织和血清中能检测到抗原物质G和低量的中和性抗体,ELISA分析显示,小鼠经过3~4次免疫接种,免疫效果基本恒定,而口服胞内表达型pYes-G重组酵母的小鼠小肠组织和血清中均未检测到目标物。结论:分泌型重组酵母pYes-InG经多次口服可对狂犬病起到一定的预防作用,但它诱导产生的中和性抗体浓度低,免疫应答慢,虽不适合用于控制突发性狂犬病的传染以及治疗狂犬病患者,但从免疫机制、免疫方式、安全性以及生产成本等因素考虑,仍具有良好的研究价值。     

微载体灌注培养制备Vero细胞狂犬病疫苗

本文研究在生物反应器中用微载体连续灌注培养Vero细胞生产狂犬病毒制备技术.在5L体积的生物反应器中,加入含10g/L微载体的199培养基,接种Vero细胞至细胞浓度达到1×105/mL,培养7d后细胞可生长至6～7×106/mL,然后以感染复数(MOI)为0.01接种狂犬病毒VaG株,接毒后24h开始收获,连续收获12d左右,收获的病毒滴度范围在6.0～8.5log LD50/mL,收获的病毒原液经浓缩、灭活和纯化等步骤制备成疫苗,各项质量指标均达到<中国生物制品规程>2000年版要求.实验表明,用生物反应器微载体灌注培养制备人用Vero细胞狂犬病疫苗小试工艺可行.     

微载体灌注培养制备Vero细胞狂犬病疫苗

本文研究在生物反应器中用微载体连续灌注培养Vero细胞生产狂犬病毒制备技术。在5L体积的生物反应器中,加入含10g/L微载体的199培养基,接种Vero细胞至细胞浓度达到1×105/mL,培养7d后细胞可生长至6~7×106/mL,然后以感染复数(MOI)为0.01接种狂犬病毒VaG株,接毒后24h开始收获,连续收获12d左右,收获的病毒滴度范围在6.0~8.5logLD50/mL,收获的病毒原液经浓缩、灭活和纯化等步骤制备成疫苗,各项质量指标均达到《中国生物制品规程》2000年版要求。实验表明,用生物反应器微载体灌注培养制备人用Vero细胞狂犬病疫苗小试工艺可行。     

Immunization of Cattle Against Rabies Using Inactivated Cell Culture Vaccines

Twenty-one heads of cattle were vaccinated with Madibovin, 31 with Rabdomun and 127 with Rabisin on 4 different farms. Rabies neutralizing antibody titre (≥0.5 IU/ml) was detected in 80% of 163 animals tested about 1 month and in 42% of 133 animals tested about 1 year after primary vaccination. On 3 of the farms 86 animals received booster vaccination about 1 year after primary vaccination. All these animals had antibody titre (≥0.5 IU/ml) about 1 month after booster and antibody levels were higher than after the primary vaccination. Rabies antibody titres (≥0.5 IU/ml) were detected in 96% of 50 animals tested 1 year after the booster. No significant differences (p>0.05) in antibody levels were detected between animals vaccinated with Madibovin or Rabisin (farm C) respectively with Rabisin or Rabdomun (farm D) at any collection time. Responses to rabies vaccines varied considerably between the farms. After primary vaccination of the animals on 2 farms with the same batch of Rabisin, the antibody levels clearly differed (p<0.0001) between the farms. Our results indicate that booster is always necessary after primary vaccination to ensure that all animals are protected.     

狂犬病病毒Vero细胞适应株的建立及其应用于疫苗生产的可行性探讨

将狂犬病病毒７ａＧ固定毒适应于Ｖｅｒｏ细胞,建立了稳定的ＶＲＧ适应株。病毒滴度可达７．６９ｌｏｇＬＤ５０／ｍｌ,病毒最适增殖时间５—７天,最适种毒比例１：１０￣３。应用转瓶培养Ｖｅｒｏ细胞增殖病毒,收获病毒液经β—丙内脂灭活,超离浓缩制备疫苗,效力试验结果（ＮＩＨ法）较原制疫苗成倍增高,且都大于２．５ＩＵ／２ｍｌ,表明ＶＲＧ适应株可应用于制备Ｖｅｒｏ细胞狂犬病疫苗。     

中国狂犬病疫苗生产株CTN-1全基因序列分析

本文首次对我国现行狂犬病疫苗生产用毒株CTN-1进行全长基因组序列测定和分析,为CTN-1疫苗在我国实际应用中的优势提供理论基础。利用RT-PCR方法分段扩增CTN-1全基因组序列,随后将PCR产物克隆到T载体、测序、拼接,用MegAlign软件比较CTN-1全基因组序列与国内外狂犬病疫苗株和街毒株全基因组序列的同源性;再以糖蛋白(G)基因为模板,用ClustalX和MEGA4软件进行系统进化分析。测序结果表明CTN-1全基因组序列的长度为11925nt(GenBank登录号FJ959397),序列分析表明CTN-1为基因Ⅰ型;CTN-1株全基因组序列与国内外狂犬病疫苗株和街毒株全基因组之间的同源性是81.5%～93.4%;与美国蝙蝠分离株SHBRV18的同源性最低,仅为81.5%;与新近从中国国内分离的野毒株HN10株的同源性最高,达93.4%。系统进化分析结果表明,CTN-1与国内不同地区大多数分离的狂犬病街毒株聚类于同一组内;而我国另一疫苗株aG株与国外疫苗株如Flury、PM、PV、ERA、RC-HL和个别中国街毒株分在另一组内。G基因氨基酸对比也显示CTN-1株与国内大多数街毒株的同源性高于其他疫苗株,结果说明CTN-1株较其他疫苗株病毒更适合制备用于预防中国狂犬病的灭活疫苗。