Vero-cell rabies vaccine produced using serum-free medium

A new rabies vaccine was developed from Vero cells adhered to microcarriers, cultivated in a bioreactor in serum-free medium and infected with the PV/VERO-Paris rabies virus strain. The viral suspensions were concentrated by tangential filtration, purified by chromatography and inactivated with beta-propiolactone. In immunogenicity studies performed in mice immunized with three doses of the new vaccine (seven batches) and the commercial Verorab and HDCV, mean titers of neutralizing antibodies of 10.3-34.6, 6.54 and 9.36 IU/ml were found, respectively. The vaccine presented stability during 14 months at 2-8 degrees C, 30 days at 37 degrees C and 8 h at 45 degrees C. The use of serum-free medium facilitated the downstream process leading to residual cellular DNA values <22.8 pg per dose of vaccine in all produced batches. The effective immunogenicity induced in mice by this vaccine, the degree of purity of the product, the high antigen yield and the reduction of the cost of the product due to the virus production and purification processes, makes this technology very important for countries where rabies presents a great public health problem.     

Immunogenicity and safety study of Indirab: A Vero cell based chromatographically purified human rabies vaccine

A chromatographically purified Vero cell rabies vaccine, Indirab manufactured by Bharat Biotech International Limited, Hyderabad, India was subjected to safety and immunogenicity studies by both intramuscular and intradermal routes of administration in parallel with a reference vaccine, Verorab. A Pre-exposure study was undertaken in 239 subjects by intramuscular (IM) route (Study I), Post-exposure study in 188 patients by intramuscular route (Study II) and Simulated post-exposure study in 134 subjects by intradermal (ID) route (Study III). All subjects in these studies were administered with either the test or the reference vaccine as per WHO approved intramuscular and intradermal regimens. The blood samples were collected on days 0, 14 and 35 in case of Study 1, and days 0, 14, 28 and 90 in case of studies II and III. In all studies both vaccine groups had adequate antibody titers (>0.5 IU/mL) on all days tested post-vaccination and there was no significant difference in the titers observed (p > 0.05). Some side effects like pain, induration, itching and fever were noted in both vaccine groups in all studies. Both vaccines were well tolerated. Hence it can be concluded that Indirab is as safe and immunogenic as Verorab when administered by both intramuscular and intradermal routes.     

Long-term stability of Vero cell-derived inactivated Japanese encephalitis vaccine prepared using serum-free medium

We established a method of producing a Vero cell-derived Japanese encephalitis vaccine using serum-free medium, and tested its stability using various stabilizers during the inactivation process and storage at 4 degrees C and 28 degrees C. Similar to previously reported results of cell culture in serum-containing medium, Vero cells were cultured in a serum-free medium multiplied well, and the viral yield was successfully increased to about 10(9)PFU/ml. Following formalin-inactivation and purification via ethanol precipitation and sucrose density ultracentrifugation of the virus solution, the vaccine had the same quality as, and higher immunogenicity, the mouse brain-derived vaccine in current use. Testing of several stabilizers showed that the addition of 0.5% glycine during the virus inactivation process facilitated the maintenance of immunogenicity for a long period of time. Furthermore, the addition of 0.5% glycine and 1.0% sorbitol as vaccine stabilizers after purification led to the maintenance of immunogenicity for 1 year, not dependent on the storage temperature (4 degrees C or 28 degrees C). These results indicate that, in contrast to the current mouse brain-derived vaccine, the Vero cell-derived vaccine can be prepared using serum-free medium containing no animal-derived components, and that the vaccine can be stored at room temperature by adding stabilizers, suggesting the possibility of producing room temperature-stable vaccines.     

Evaluation of novel HIV vaccine candidates using recombinant vesicular stomatitis virus vector produced in serum-free Vero cell cultures

Acquired Immune Deficiency Syndrome (AIDS) in humans is a result of the destruction of the immune system caused by Human Immunodeficiency Virus (HIV) infection. This serious epidemic is still progressing world-wide. Despite advances in treatment, a safe and effective preventive HIV vaccine is desired to combat this disease, and to save millions of lives. However, such a vaccine is not available yet although extensive amounts of resources in research and development have been invested over three decades. In light of the recently approved Ebola virus disease vaccine based on a recombinant vesicular stomatitis virus (rVSV-ZEBOV), we present the results of our work on three novel VSV-vectored HIV vaccine candidates. We describe the design, rescue, production and purification method and evaluate their immunogenicity in mice prior to preclinical studies that will be performed in non-human primates. The production of each of the three candidate vaccines (rVSV-B6-NL4.3Env/SIVtm, rVSV-B6-NL4.3Env/Ebtm and rVSV-B6-A74Env(PN6)/SIVtm) was evaluated in small scale in Vero cells and it was found that production kinetics on Vero cells vary depending on the HIV gp surface protein used. Purified virus preparations complied with the WHO restrictions for the residual DNA and host cell protein contents. Finally, when administered to mice, all three rVSV-HIV vaccine candidates induced an HIV gp140-specific antibody response.     

Immunogenicity, safety and lot consistency in adults of a chromatographically purified Vero-cell rabies vaccine: a randomized, double-blind trial with human diploid cell rabies vaccine

The immunogenicity and safety of a chromatographically purified rabies vaccine (CPRV) was evaluated using US veterinary medical students. In the first study, 242 healthy adults were enrolled in a randomized, modified double-blind, multicenter trial and received five doses of either CPRV or human diploid cell vaccine (HDCV) by intramuscular injection on days 0, 3, 7, 14, and 28 concurrently with human rabies immunoglobulin in a simulated post-exposure prophylaxis regimen. Post-immunization titers in the CPRV and HDCV groups reached 0.5 IU/ml (the WHO-recommended minimally acceptable titer) or greater in all subjects in both vaccine groups by day 14 and remained above that level through day 90. In the second study, 438 healthy adults were enrolled in a randomized, double-blind, multicenter trial and assigned to receive five doses from one of three lots of CPRV by intramuscular injection on days 0, 3, 7, 14, and 28 in a simulated post-exposure prophylaxis regimen to evaluate lot consistency. Post-immunization titers rapidly increased to over 0.5 IU/ml by day 14 for all subjects and remained above that level through day 42 when the study was terminated. The three lots were considered equivalent. The percentage of subjects with at least one local reaction during the five-dose regimen was slightly lower in the CPRV group than in the HDCV group (P=0.06). The most frequently reported local reaction for all doses of vaccine was pain at the injection site. Headache, myalgia, and malaise were the most frequently reported systemic events. The percentage of subjects with at least one systemic event was significantly lower for CPRV (P=0.0084). No vaccine-related serious adverse reaction was reported in these studies. The results of these studies indicate that CPRV administered intramuscularly to healthy adults is immunogenic and is associated with fewer local and systemic reactions than HDCV.     

Purification of rabies virus produced in Vero cells grown in serum free medium

Rabies is a viral zoonosis caused by negative-stranded RNA viruses of the Lyssavirus genus. It can affect all mammals including humans. Dogs are the main source of human rabies deaths, contributing up to 99% of all rabies transmissions to humans. Vaccination against rabies is still the sole efficient way to fight against the disease.Cell culture vaccines are recommended by World Health Organization (WHO) for pre and post exposure prophylaxis; among them Vero cell rabies vaccines which are used worldwide. In this work we studied the purification of inactivated rabies virus produced in Vero cells grown in animal component free conditions, using different methods. Cells were grown in VP-SFM medium in stirred bioreactor, then infected at an MOI of 0.05 with the LP2061 rabies virus strain. Collected harvests were purified by zonal centrifugation, and by chromatography supports, namely the Capto Core 700 and the monolithic CIM-QA column. Generated data were compared in terms of residual DNA level, host cell proteins (HCP) level and the overall recovery yield.Rabies virus purification using the monolithic column resulted in the highest antigen recovery yield, equal to 94%. Capto Core 700 showed a lower yield, about 84%; whereas the purification yield by zonal centrifugation was equal to 60%. In terms of host cell residual DNA removal, zonal centrifugation was the most efficient method; the removal yield was equal to 88.5%; elimination of host cell DNA was slightly lower when using the monolithic CIM-QA (equal to 73%). Whereas Capto Core 700 showed the lowest level (49.2%). Host cell protein removal varied between 92.6% for the monolithic column and 78.6% for the zonal centrifugation. Capto Core 700 eliminated 86.5% of HCP.     

Increasing Vero viable cell densities for yellow fever virus production in stirred-tank bioreactors using serum-free medium

In this work, changes in Vero cell cultivation methods have been employed in order to improve cell growth conditions to obtain higher viable cell densities and to increase viral titers. The propagation of the 17DD yellow fever virus (YFV) in Vero cells grown on Cytodex I microcarriers was evaluated in 3-L bioreactor vessels. Prior to the current changes, Vero cells were repeatedly displaying insufficient microcarrier colonization. A modified cultivation process with four changes has resulted in higher cell densities and higher virus titers than previously observed for 17DD YFV.     

53例人用狂犬病无佐剂纯化疫苗接种后抗体检测结果分析

目的观察暴露后人群接种国产人用狂犬病无佐剂纯化疫苗(Vero cell)的免疫效果。方法对53例研究对象按照0、3、7、14和28 d程序进行暴露后免疫,采用WHO认可的RFFIT法检测免疫14 d,45 d的血清抗体。观察每针次接种后72 h内局部和全身反应。结果所有接种对象均未出现严重副反应。首剂免疫14 d,45 d ELISA法检测抗体阳性率分别为52.83%,94.34%,RFFIT法检测抗体阳性率分别为100%,100%。几何平均滴度(GMT)为17.74 IU/ml,15.50 IU/ml。结论国产人用狂犬病无佐剂纯化疫苗(Vero cell)具有良好的安全性和免疫原性。     

Safety and immunogenicity of a serum-free purified Vero rabies vaccine in healthy adults: A randomised phase II pre-exposure prophylaxis study

A serum-free, highly purified Vero cell rabies vaccine (PVRV-NG) is under development. We previously demonstrated that pre-exposure prophylaxis (PrEP) with PVRV-NG had a satisfactory safety profile and was immunogenically non-inferior to the licensed purified Vero cell rabies vaccine in adults. Here, we evaluated the safety and immunogenic non-inferiority of PrEP with PVRV-NG compared to the licensed human diploid cell vaccine (HDCV) in healthy adults (NCT01784874). Participants received three vaccinations (days 0, 7, and 28) as PrEP with or without a booster injection after 12 months. Rabies virus neutralising antibodies (RVNA) were evaluated on days 0, 28 (subgroup only), and 42, and Months 6, 12, and 12 + 14 days (booster group only). Non-inferiority (first primary objective) was based on the proportion of participants with RVNA titres ≥ 0.5 IU/mL (World Health Organization criteria for seroconversion) on day 42, expected to be ≥ 99% (second primary objective). Safety was evaluated after each dose and monitored throughout the study. At day 42, PVRV-NG was non-inferior to HDCV and the first primary objective was met; seroconversion was observed for 98.3% of PVRV-NG recipients and 99.1% of HDCV recipients. As < 99% of participants in the PVRV-NG group had RVNA titres ≥ 0.5 IU/mL, the second primary objective was not met. Booster vaccination produced a strong increase in RVNA titres for all groups, primed with PVRV-NG or HDCV. RVNA geometric mean titres tended to be higher for HDCV than PVRV-NG primary vaccine recipients. In a complementary evaluation using alternative criteria for seroconversion (complete virus neutralization at 1:5 serum dilution), 99.6% and 100% of participants in the PVRV-NG and HDCV groups, respectively, achieved seroconversion across the vaccine groups. No major safety concerns were observed during the study. PVRV-NG was well tolerated, with a similar safety profile to HDCV in terms of incidence, duration, and severity of adverse events after primary and booster vaccinations.ClinicalTrials.gov number: NCT01784874.     

人用狂犬病无佐剂纯化疫苗的安全性与免疫原性的初步观察

目的评价国产人用狂犬病无佐剂纯化疫苗(Vero细胞微载体)的安全性和免疫原性。方法对502人(A组)接种人用狂犬病无佐剂纯化疫苗(Vero细胞微载体)另100人(B组)作为对照接种巴斯德公司生产的狂犬病纯化疫苗。采用0、3、7、14和28天程序,观察每针次接种后72小时内局部和全身反应及14天、45天的免疫应答水平。结果所有接种对象均未出现严重局部和全身副反应。首剂免疫14天,A、B组抗体阳性率均达到100%,几何平均滴度为5.2IU/ml和5.6IU/ml。第45天,A组抗体几何平均滴度上升至9.5IU/mll,与B组相似(9.8IU/ml)。结论人用狂犬病无佐剂纯化疫苗(Vero细胞微载体)具有良好安全性和免疫原性。     

One-year study of the 2-1-1 intramuscular postexposure rabies vaccine regimen in 100 severely exposed Thai patients using rabies immune globulin and Vero cell rabies vaccine.

The 2-1-1 rabies postexposure treatment schedule is an abbreviated regimen in which a tissue culture rabies vaccine is administered intramuscularly at two sites on day 0, and at one site on days 7 and 21. Compared to the standard five-dose intramuscular regimen, the 2-1-1 schedule reduces the number of clinic visits from five to three and the amount of vaccine used by 20%. One hundred Thai patients, who were severely exposed to rabies, were treated with rabies immune globulin and the 2-1-1 regimen using purified Vero cell rabies vaccine. They were followed for 1 year. Rabies antibody titres were measured in 10% of this group. All patients survived and adverse reactions were mild. A satisfactory antibody response (a titre greater than 0.5 IU ml-1) occurred in all ten patients studied at day 14, but persisted for 90 days in 80% and for 360 days in only 50%. The authors therefore do not recommend use of the 2-1-1 schedule in severely exposed patients who also need to receive rabies immune globulin.     

Antibody response of patients after postexposure rabies vaccination with small intradermal doses of purified chick embryo cell vaccine or purified Vero cell rabies vaccine

Although the introduction of tissue culture vaccines for rabies has dramatically improved the immunogenicity and safety of rabies vaccines, they are often prohibitively expensive for developing countries. To examine whether smaller doses of these vaccines could be used, we tested the safety and immunogenicity of purified chick embryo cell vaccine (PCECV) on 211 patients in Thailand with World Health Organization (WHO) category II and III exposures to rabies. The patients presented at two Thai hospitals and were randomized into three groups. Patients in Group 1 received 0.1 ml PCECV intradermally at two sites on days 0, 3, 7, and at one site on days 30 and 90. Group 2 was treated similarly, except that purified Vero cell rabies vaccine (PVRV) was used instead of PCECV. Group 3 received 1.0 ml PCECV intramuscularly on days 0, 3, 7, 14, 30 and 90. After 0, 3, 7, 14, 30 and 90 days serum was collected from the subjects and the geometric mean titres (GMTs) of rabies virus neutralizing antibody determined. After 14 days the GMT of 59 patients vaccinated intradermally with PCECV was equivalent to that of patients who received PVRV. Adverse reactions were more frequent in patients who received vaccines intradermally, indicating the reactions were associated with the route of injection, rather than the vaccine per se. We conclude that PCECV is a safe and highly immunogenic vaccine for postexposure rabies vaccination when administered intradermally in 0.1-ml doses using the two-site method ("2,2,2,0,1,1") recommended by WHO.     

Purified Vero cell rabies vaccine and human diploid cell strain vaccine: comparison of neutralizing antibody responses to post-exposure regimens

Neutralizing antibody responses to conventional rabies post-exposure regimens of human diploid cell strain vaccine (HDCSV) and the new purified Vero cell rabies vaccine (PVRV) were compared in 58 healthy Thai veterinary students. The geometric mean titres (GMTs) of the group given HDCSV were slightly higher than those given PVRV, but on day 28 the peak GMTs of the two groups were statistically similar. The early antibody response to PVRV was unaffected by the addition of passive immunization, whereas the level of HDCSV response was reduced on day 14, so that there was no difference on that day between the GMTs of the two vaccine groups given HRIG. However, by day 91 the GMT of those given PVRV and HRIG was lower than in those given HDCSV alone or with HRIG. The appearance of antibody was less rapid than was observed in previous studies using multiple-site intradermal vaccination. Side effects were trivial. Our results confirm the promise of this new, potentially more economical tissue culture vaccine, but they suggest that the regimen could be improved.     

Immunogenicity, safety and consistency of new trivalent inactivated influenza vaccine

To augment the available influenza vaccine supply, a phase III study was conducted to evaluate the immunogenicity, safety, and consistency of a new trivalent inactivated influenza vaccine manufactured by CSL Limited. Healthy adults (ages 18-64) were randomized to receive either a single dose of TIV from multi-dose vials with thimerosal, TIV from pre-filled syringes without thimerosal, or placebo. Of the TIV recipients, 97.8% achieved a post-vaccination titer > or =40 against H1N1, 99.9% against H3N2 component, and 94.2% against influenza B. Few local or systemic adverse events were noted after vaccination with either TIV presentation. TIV was well tolerated and immunogenic.     

国产流感裂解疫苗的免疫效果及安全性观察

我国是流感的高发区,每年均有不同程度流行,严重威胁人民健康.接种流感疫苗是预防流感的最佳方法.安全性、免疫原性较好的疫苗将成为未来流感疫苗的发展方向.为了解国产流感病毒裂解疫苗在人群中接种的安全性和免疫效果,并与同类进口流感疫苗做对比,于2006年11月至2007年4月在陕西省屑县进行临床研究,结果报告如下.     

High immunogenic enterovirus 71 strain and its production using serum-free microcarrier Vero cell culture

Developing an effective vaccine against enterovirus 71 (EV71) infection provides the best means to control the disease. We have previously reported that large-scale preparation of a low immunogenic EV71 strain can be achieved using serum free microcarrier Vero cell culture in a 2-l bioreactor [Wu SC, Liu CC, Lian WC. Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development. Vaccine 2004;22:3858-64]. This present work further investigated the virus growth and the immunogenicity of two high immunogenic strains (EV71-075 and EV71-117) prepared in serum-free microcarrier cell cultures. Our results showed that serum free culture increased cell death rate after infection, reduced the virus specific productivity, but resulted in elicitation of higher neutralizing titers in immunized mice as compared to that parallel obtained in serum-containing cultures. Therefore, serum-free microcarrier culture is a valuable process for developing inactivated EV71 vaccines.     

An immunogenicity study of a newly introduced purified Vero cell rabies vaccine (Abhayrab) manufactured in India

Purified Vero cell culture rabies vaccine "Abhayrab" manufactured by Human Biologicals Institute, Ooty, India was subjected for immunogenicity studies. Pre-exposure study was undertaken on 60 healthy volunteers (Group I) with vaccination on days 0, 7 and 21. A group of 75 patients of category II (Group II), 67 of category III (Group III) were given post-exposure prophylaxis and 88 patients of category III were administered with rabies immunoglobulins (Group IV) along with post-exposure prophylaxis as per World Health Organization (WHO) recommendations with a booster on day 90. The volunteers and patients vaccinated showed very few adverse side effects. The blood samples collected from volunteers (Group I) on days 14, 35 and 365 and patients (Group II-IV) on days 14, 30, 90 and 365 showed geometric mean titres (GMT) of >0.5 IU/ml. The study indicated new rabies vaccine manufactured in India was found to be safe and immunogenic.     

脊髓灰质炎灭活疫苗加强免疫的免疫原性和安全性观察

目的 评价完成脊髓灰质炎灭活疫苗(inactivated polio vaccine,IPV)基础免疫的儿童,在18月龄进行IPV加强免疫的安全性和免疫原性.方法 2011至2012年选择在2、3、4月龄各接种1剂IPV完成基础免疫,并且在18月龄完成1剂IPV加强免疫接种的儿童,共有97名研究对象入组,检测加强免疫前、后的血清中脊髓灰质炎中和抗体,计算抗体几何平均滴度(GMT)和保护率.同时观察接种后30 d内发生的不良反应,包括局部疼痛、红肿、硬结,发热、呕吐、异常哭闹、嗜睡、食欲下降、易激惹,以及其他身体不适的所有症状和用药情况,对疫苗安全性进行描述性分析.结果 最终有84名调查对象完成了免疫前后的血清采样.加强免疫前,脊髓灰质炎Ⅰ型、Ⅱ型、Ⅲ型的中和抗体阳性率均为100％(84/84),GMT(95％CI)分别为1∶148.5(116.49 ～ 189.29)、1∶237.68(178.39～316.67)、1∶231.87(181.27 ～ 296.58);加强免疫后,3个型别中和抗体阳性率均为100％(84/84),GMT(95％CI)分别为1∶1612.14(1470.57 ～ 1767.34)、1∶1854.92(1715.83 ～ 2005.29)、1∶1625.50(1452.12～ 1819.58).加强免疫前,脊髓灰质炎Ⅰ型、Ⅱ型、Ⅲ型的中和抗体滴度分布集中于1∶128～1∶512范围内,分别占各型的65％ (55/84)、55％ (46/84)、74％ (62/84).加强免疫后,3个型别中和抗体滴度升高,抗体≥1∶1024者分别占93％ (78/84)、95％(80/84)、92％ (77/84).共有96名研究对象完成了安全性观察,16名出现不良反应,总发生率17％.观察到的局部反应主要为压痛3％ (3/96),无红肿和硬结;全身反应分别为食欲下降8％(8/96)、易激惹8％(8/96)、发热7％(7/96)、异常哭闹6％(6/96)、嗜睡6％(6/96)、呕吐1％ (1/96).所有反应均为轻度和中度;局部反应全部发生在接种当日,持续1 ～2d;全身反应多在接种后2d内发生,持续时间多小于3d.结论 IPV加强免疫具有良好的免疫原性和安全性,可提供有效的保护水平.