Development and characterization of a live varicella vaccine (Oka strain)

A live varicella vaccine (Oka strain) was developed by serial passage of the Oka strain isolated in our laboratory, in human embryonic lung cells (11 times at 34 C) and guinea pig embryo cells (12 times at 37 C). It is slightly temperature sensitive at 39 C and shows a higher ratio of infectivity in guinea pig embryo cells to infectivity in human embryo cells than wild-type strains. The DNA digest with Hpa I enzyme of the Oka strain contained one unique fragment (K), although its mobility differed only slightly from that of the corresponding fragment of wild-type strains. Studies with clinical varicella zoster virus (VZV) isolates from vaccinees indicated that tests on the ratio of infectivity in guinea pig embryo fibroblasts (GPEF) to that in human embryo fibroblasts (HuEF) and the profile of the DNA digest with Hpa I are useful for differentiation of the vaccine strain from wild-type strains. The vaccine virus showed stable immunogenicity during at least 15 further repeated passages in human diploid cells, a character which seems helpful for production of a large quantity of vaccine virus for practical use.     

Immunization of acute leukemic children with a live varicella vaccine (Oka strain)

A total of 52 acute leukemic children have been safely and effectively vaccinated with live varicella (Oka strain) vaccine given under close clinical and immunological observation. The incidence of zoster in the vaccinated children group was slightly less than that in the group that had experienced natural varicella.     

Clinical application of a live varicella vaccine (Oka strain) in a hospital

A total of 239 children, including 22 high-risk children and 55 non-high risk diseased children have been immunized with a live varicella vaccine (Oka strain) since June, 1978. No clinical reaction attributable to the vaccine has been observed. Of these children, 87 received emergency vaccination. Of 47 children receiving emergency vaccination because they had been in contact with varicella patients either in hospital, school or a playground, only 5 developed varicella and their symptoms were mild. Of 40 children receiving emergency vaccination because of exposure to varicella in their home, 10 developed mild varicella and 30 were protected. Clinical symptoms of varicella when seen seemed to be due to incomplete protection because the vaccine was given too late rather than to clinical reactions to the vaccine. During follow-up period of 6 to 66 months after vaccination, 8 children showed very mild rashes without fever as the result of exogenous varicella infection.     

Clinical trial of the Oka strain of live attenuated varicella vaccine on healthy children

Clinical and serological follow-up studies were made on 257 healthy children who had received live varicella vaccine (strain Oka) in Showa Hospital. Good antibody responses were shown with a seroconversion rate of 98.4% (253/257) by the immune adherence hemagglutination test. Mild adverse reactions were observed in 11 of the vaccinated children. During observation periods of 6 months to 4 years, 6 of the 253 children who were successfully vaccinated contracted mild varicella, while all 4 vaccinees who showed no primary immune response contracted mild to moderate clinical varicella. It is concluded that this vaccine is highly immunogenic and causes few clinical reactions in normal children.     

二代测序法深度分析水痘疫苗Oka株病毒基因特征

采用二代测序(next-generation sequencing,NGS)技术,对上海生物制品研究所有限责任公司(简称SIBP)2015-2017这3个年度生产的Oka株水痘疫苗进行深度测序,从分子水平分析疫苗质量的一致性,并与不同厂家疫苗/毒种进行比较,分析它们之间的遗传特征相关性。结果显示,3个年度生产的水痘疫苗中平均有77个位点的疫苗型位点检出频率(proportion of vaccine-type allele,Pv)≥10%,至少在两批疫苗中出现Pv≥10%的位点数共76个,两两之间Pv最大差值<10%,与平均值的最大差值为<5%。共有40个位点的Pv均值≥30%,其中19个位点导致氨基酸残基变异;15个位于ORF62区域。筛选出35个位点,比较5个公司水痘疫苗/毒种的Oka株病毒基因序列,结果显示它们之间存在相关性,同时存在一定的差异。研究表明,SIBP不同年度生产的水痘疫苗Oka株病毒具有良好的分子水平上的一致性,为疫苗质量管理与市场监督提供了方法和基础数据。     

A clinical trial of live attenuated varicella vaccine (Biken) in children with malignant diseases

The live attenuated varicella vaccine (Biken) derived from the Oka strain was used for immunization of childhood cancer patients in remission but on chemotherapy. Thirty nine patients were immunized without any severe adverse effects but 6 recipients had a small number of vesicles. Seroresponses were observed in 90% of recipients examined. During the follow-up period 6 leukemia recipients were suffered from natural varicella in family or community contacts. No vaccinee developed herpes-zoster during this study period.     

Application of a live attenuated varicella vaccine to hospitalized children and its protective effect on spread of varicella infection.

Twenty-three hospitalized children with no history of varicella or no detectable complement fixing (CF) antibody, were vaccinated with a live attenuated varicella vaccine (Oka strain) immediately after the occurrence of a case of varicella in a children's ward of hospital. These children suffered from the nephrotic syndrome, nephritis, purulent meningitis, hepatitis etc., and 12 of them were receiving steroid therapy. An antibody response was noticed in all the vaccinated children, with mild fever in 6 and a mild rash in 2 of 6. It was uncertain whether these reactions were due to vaccinatin or to naturally acquired infection modified by vaccination. No other clinical reactions or abnormalities of the blood or urine were detected. Thus the spread of varicella infection was prevented, with the exception of one severe case in an unvaccinated patient. In another trial, 16 children with renal diseases were also vaccinated. All the children showed an immune response with no clinical reactions and no abnormalities in blood and urine examinations. Thus live varicella vaccine (Oka strain) can be used safely and effectively for hospitalized children, and its effectiveness in preventing spread of varicella infection was confirmed.     

Development of a live attenuated varicella vaccine.

The Oka strain of varicella virus, isolated in our labolatory, was serially cultivated in guinea-pig embryo cultures (GPEC), and a considerable amount of cell-free virus was obtained from infected cell. GPEC passage virus at the 6th passage level was used in a small scale field trial. Susceptible children of 1 to 10 years old were injected subcutaneously with 100 to 1,000 PFU of virus. No clinical reactions due to the vaccination were observed in any children, and a high rate of antibody response was obtained with viral doses of more than 200 PFU. Attenuated virus obtained by passage in GPEC was propagated in human diploid (WI-38) cells, and it was also effective in inducing an immune response without clinical reactions. The results show that the Oka strain of varicella virus passaged in GPEC and human diploid (WI-38) cells may be used safely and effectively as a live attenuated vaccine.     

T lymphocyte cytotoxicity with natural varicella-zoster virus infection and after immunization with live attenuated varicella vaccine

Varicella-zoster virus (VZV) specific cytotoxicity was investigated during acute primary VZV infection, in naturally immune subjects and after vaccination with the live attenuated varicella vaccine by using T cell cultures (TCC) generated by stimulating PBMC with VZV Ag and autologous VZV-superinfected lymphoblastoid cell lines as targets. Lysis of VZV-infected lymphoblastoid cell lines was observed by TCC from acutely infected subjects, naturally immune subjects, and recipients of the varicella vaccine. VZV glycoprotein I induced cytotoxic T cells but killing was less efficient than killing by TCC stimulated with VZV Ag. The TCC were primarily CD4+ (mean 86.6%) T lymphocytes with 15.2% of the cells coexpressing Leu-19. TCC were predominantly restricted by HLA class II as demonstrated by lack of any blocking using class I mAb and blocking of 15 to 71% by L243, a mAb to class II. Unrestricted killing as measured by killing of K562 cells occurred in all TCC but was minimally greater than that observed against uninfected autologous targets. Phenotypes of PBMC during acute infection had an initial increase in CD4+ cells and an overall decrease in the percentage of circulating Leu-11+ (CD16). No enhanced K562 killing was demonstrated in PBMC from subjects with acute infection compared to subjects without infection. CD4+ CTL may function as an important primary host response in acute varicella. Immunization with live attenuated varicella vaccine induced VZV-specific, memory CTL responses comparable to those of naturally immune subjects. The demonstration of their persistence long after primary VZV infection may indicate a role for CTL in restriction of viral replication during episodes of VZV reactivation from latency.     

儿童2剂水痘疫苗不同间隔免疫程序的比较

目的了解水痘疫苗2剂不同间隔时间免疫程序的安全性及免疫原性。方法按照随机、双盲的原则,选择符合条件的1~3岁、4~6岁、7~12岁儿童各200名,每个年龄组按照2剂间隔3个月和6个月的免疫程序,分别接种2剂冻干水痘减毒活疫苗。观察各组接种后的不良反应率,用膜抗原荧光抗体法(FAMA)检测疫苗接种前、后血清抗体阳转率、GMT水平和平均增长倍数。结果接种2剂水痘疫苗后,总体不良反应率在4%~13%;同年龄组不同间隔时间接种2剂疫苗后,不良反应发生率差异无统计学意义;不同年龄组间不良反应发生率差异无统计学意义。同年龄组不同间隔时间免前及接种2剂后,血清GMT水平差异均无统计学意义;同年龄组接种1剂与2剂水痘疫苗的血清GMT水平,两者间差异有显著性统计学意义。接种2剂水痘疫苗的GMT增长倍数高于接种1剂。不同间隔时间接种第2剂水痘疫苗后抗体阳转率差异无统计学意义;同年龄组接种2剂后抗体阳转率高于接种1剂,两者差异有统计学意义。结论间隔3个月和6个月接种第2剂水痘疫苗,在安全性及免后GMT水平、增长倍数及抗体阳转率等4个方面,两者差异均无统计学意义。     

Immune response induced by live attenuated varicella vaccine and short term follow-up on immunity of the vaccinated children

Live attenuated varicella vaccine (Oka strain, 500 to 750 PFU) was inoculated into 234 children with various underlying diseases. Varicella-zoster virus (VZV) specific immune adherence hemagglutination antibody developed in 95% of initially seronegative subjects. VZV specific cellular immune responses were detected in 91% of subjects within 6 weeks after vaccination. A preliminary survey of 46 vaccinated normal-risk subjects during the first and the third year revealed excellent protection with the exception of one case who had not shown any antibody response at the fourth week.     

Antibody response in school children to live rubella vaccine (Cendehill strain)

The efficacy of an attenuated rubella virus vaccine, Cendevax, was tested on 65 school children. Forty-nine of them (75%) had pre-existing antibodies and in these there was no increase in the HAI antibody titres after administration of the vaccine. Sixteen children (25%) had no demonstrable rubella HAI antibody prior to vaccination. From the latter group, postvaccination serum samples were available from only 11, and 10 of these seronegative children showed seroconversion after vaccination. The geometric mean HAI titre was 1:180. Seven of the 10 postvaccination serum samples had complement-fixing antibodies and specific IgM antibodies were detected by the immunofluorescence test in 8. No correlation was observed between the CF and the IgM antibodies.     

Comparison of the complete DNA sequences of the Oka varicella vaccine and its parental virus

The DNA sequences of the Oka varicella vaccine virus (V-Oka) and its parental virus (P-Oka) were completed. Comparison of the sequences revealed 42 base substitutions, which led to 20 amino acid conversions and length differences in tandem repeat regions (R1, R3, and R4) and in an origin of DNA replication. Amino acid substitutions existed in open reading frames (ORFs) 6, 9A, 10, 21, 31, 39, 50, 52, 55, 59, 62, and 64. Of these, 15 base substitutions, leading to eight amino acid substitutions, were in the gene 62 region alone. Further DNA sequence analysis showed that these substitutions were specific for V-Oka and were not present in nine clinical isolates. The immediate-early gene 62 product (IE62) of P-Oka had stronger transactivational activity than the mutant IE62 contained in V-Oka in 293 and CV-1 cells. An infectious center assay of a plaque-purified clone (S7-01) from the V-Oka with 8 amino acid substitutions in ORF 62 showed smaller plaque formation and less-efficient virus-spreading activity than did P-Oka in human embryonic lung cells. Another clone (S-13) with only five substitutions in ORF 62 spread slightly faster than S7-01 but not as effectively as P-Oka. Moreover, transient luciferase assay in 293 cells showed that transactivational activities of IE62s of S7-01 and S7-13 were lower than that of P-Oka. Based on these results, it appears that amino acid substitutions in ORF 62 are responsible for virus growth and spreading from infected to uninfected cells. Furthermore, the Oka vaccine virus was completely distinguishable from P-Oka and 54 clinical isolates by seven restriction-enzyme fragment length polymorphisms that detected differences in the DNA sequence.     

Use of live varicella vaccine in children with acute leukemia and malignant lymphoma

Live varicella vaccine was given to 7 children with acute leukemia and 1 child with non-Hodgkin's lymphoma. Seroconversion without any clinical reactions was observed in 4 children in remission from acute leukemia without interruption of anticancer medication. These children have been free from varicella for 5 to 11 months after vaccination. Mild to severe clinical reactions developed in 3 of 4 children who were receiving remission induction chemotherapy. It is concluded that live varicella vaccine is effective in children with acute leukemia without interrupting anticancer medication, but that it is not suitable for children with leukemia and lymphoma who are receiving remission induction chemotherapy with antileukemic agents.     

A five-year immunological follow-up study of the institutionalized handicapped children vaccinated with live varicella vaccine or infected with natural varicella

Twenty-two institutionalized handicapped children who were susceptible to varicella were vaccinated with live varicella vaccine of the Oka strain and their immune status was followed for 5 years under conditions without exposure to natural varicella. Simultaneously, 7 children infected with natural varicella were followed. Of the 22 vaccinees, 16 showed sero-positive conversion by the fluorescent antibody to membrane antigen (FAMA) test, the other 6 remaining seronegative during 5 years of observation period. All the 16 cases showing seroconversion had detectable antibody for 5 years after vaccination, and 14 of them gave a positive reaction in the varicella skin test. All the 7 cases after natural varicella gave positive reactions in both the FAMA and skin test. These results suggest that immunity conferred by the vaccination would persist long even in the absence of exposure to natural varicella, though further follow-up studies are needed.     

冻干水痘减毒活疫苗转瓶生产工艺的建立

应用旋转培养的方法,建立冻干水痘减毒活疫苗的生产工艺。选择长成致密单层的2BS细胞,接种带状疱疹病毒Oka株,待细胞病变达75％以上时,收获病毒液,经超声破碎、离心、澄清,冻干后,按常规检定,疫苗各项检定符合《WHO水痘活疫苗规程》及《冻干水痘减毒活疫苗制造及检定试行规程》要求。与克氏瓶相比,应用旋转培养,不但提高了疫苗单产,降低了牛血清蛋白残留量,而且疫苗质量也保持稳定。     

Indicators of immunity in children vaccinated with live poliomyelitis vaccine]

Serological examination of 1057 children, residents of Leningrad, vaccinated with poliomyelitis vaccine at the appropriate calendar dates according to the scheme, showed the presence of antibodies to the polioviruses in 81.5-99.1% of the cases. There were more serologically negative children against the virus type III, and much less--against the virus type II. The value of the mean geometrical titres somewhat decreased with the advance of the children's age and the time lapse after the vaccination and revaccination. The greatest antibody titres determined were against the poliovirus type II, and the least--against type III. No antibodies against the viruses of types I and III were revealed in case of deficiency against the poliovirus type II. The number of children with the absence of antibodies against the poliovirus of all the types was insignificant.