ICP/CDS/Oll
8 July 1986
ENGLISH ONLY
I
, REPORT WORKING GROUP ON VACCINE DEVELOPMENT AND VACCINE STRATEGIES FOR JAPANESE ENCEPHALITIS
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Convened by the
REGIONAL OFFICE FOR THE WESTERN PACIFIC OF THE WORLD HEALTH ORGANIZATION Tokyo, Japan, 10-12 December 1985
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Not for Sale Printed and distributed by the
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Regional Office for the Western Pacific of the World Health Organization Manila, Philippines July 1986
NOTES
The views expressed in this report are those of the members of the Working Group on Vaccine Development and Vaccination Strategies for Japanese Encephalitis and do not necessarily reflect the policies of the World Health Organization.
This report has been prepared by the Regional Office for the Western Pacific of the World Health Organization for governments of Member States in the Region and for members of the Working Group on Vaccine Development and Vaccination Strategies for Japanese Encephalitis, Tokyo, Japan, 10-12 December 1985.
CONTENTS
1.
INTRODUCTION.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
1
2. 3. 4. 5.
JAPANESE ENCEPHALITIS ••••••••••••••••••••••••••••••••.••.• CURRENTLY USED JAPANESE ENCEPHALITIS VACCINES •••••••.••••. FIELD TRIAL OF MONOVALENT AND BIVALENT INACTIVATED JAPANESE ENCEPHALITIS VACCINES IN THAII~ND •••••••••••••••• RECENT PROGRESS IN VACCINE DEVELOPMENT 5.1 5.2 5.3 Inactivated Japanese encephalitis vaccines ••••••••••• Live attenuated Japanese encephalitis vaccines ••..••• Molecular approaches to vaccine development ••••••••••
1 2 2 3
3 4 4 4 4
6.
GUIDELINES ON STRATEGIES FOR JAPANESE ENCEPHALITIS VACCINES AND FOR VACCINATION OF HUMANS •••••••••••••••••••. 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8
Rationale for use of vaccines in human beings •••••••• Type of vaCC1ne •••••••••••••••••••••••••••••••••••••• Vaccine strains .......................................................................... .. Potency 0 f vacc ine .................................................................... ..
4 4 5 5 5 5 6 6 6 6 6
Purity of vaccine ••••••••••••••••••••••••••••••..•••• Safety of vaccine ...................................................................... .. Storage of vaccine .••••.•••••••.•.••••...•••••••..•.. Contra-indications ................................................................... ..
6.9 Quality control •••••••••••••••••••••••••••••••••••••• 6.10 Subjects of immunization •••••••••••.••••••••••••••••• 6.11 Adequate time of vaccination ••••••••••••.•••••••••••• 7.
RECOMMENDATIONS ...................................................................................... ANNEXES ANNEX 1 - AGENDA •••••••••••••...••••••••••••••••••••..•••• ANNEX 2 - LIST OF MEMBERS, CONSULTANTS, OBSERVERS AND SECRE TARIAT ••.•••...••••••••••••••••••.••••..••• ANNEX 3 - A REVIEW ON VACCINE AND VACCINATION FOR JAPANESE ENCEPHALITIS BY DR AKIRA OYA •••••••...•
8/9
10/11
15
1.
INTRODUCTION
The Working Group on Vaccine Development and Vaccination Strategies for Japanese Encephalitis met in Tokyo, Japan, from 10 to 12 December 1985. Dr H. Nakajima, Director, WHO Regional Office for the Western ~a~ific, welcomed the Group. In view of the importance of Japanese encepha11t1s (JE), the Regional Office for the Western Pacific organized meeting~ in Tokyo in 1983 and Sendai in 1984. The working groups at these prev10us meetings recommended ways to improve surveillance and control of Japanese encephalitis, including improved methods of production and ut~lizati?n of vaccine. It was considered opportune therefore to convene th1s meet1ng to review progress and make new recommendations to WHO. The objectives of the meeting were: (1) to discuss the effectiveness of current vaccines and to review progress in the development and utilization of new Japanese encephalitis vaccines; to formulate guidelines for Japanese encephalitis vaccines; to recommend means to strengthen regional and interregional cooperation in production, quality control and utilization of monovalent and polyvalent vaccines.
(2) (3)
The Group elected Dr Akira Oya, Chairman, Dr Hoang Thuy Nguyen, Vice Chairman, and Dr Robert Shope, Rapporteur. The agenda and list of participants are attached as Annexes 1 and 2.
2.
JAPANESE ENCEPHALITIS
Japanese encephalitis continues to be an important public health problem in the South-East Asia and Western Pacific Regions. Countries affected include Burma, China, India, Indonesia, Japan, Republic of Korea, Malaysia, Nepal, Philippines, Singapore, Sri Lanka, Thailand and Viet Nam. Pigs, birds and other animals are involved in amplification of transmission. Major human epidemics occur during the summer or monsoon season when there are large numbers of Culex tritaeniorrhynchus mosquitos breeding in areas of rice cultivation. Japanese encephalitis was once a major disease in Japan, but in the last twenty years the prevalence has decreased drastically, partly in association with the systematic use of inactivated mouse brain vaccine. Major epidemics continue however to be recorded in neighbouring countries. There were 1 179 confirmed cases in 1982 in the Republic of Korea where the fatality rate is approximately 10% and more than 2 000 cases in Viet Nam in 1985. There are regular seasonal epidemics of encephalitis in China, Thailand and Viet Nam. Clinical attack rates in Viet Nam are as high as 6.9 per 100 000.
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3.
CURRENTLY USED JAPANESE ENCEPHALITIS VACCINES
Two inactivated vaccines are currently in use~ The first is a mouse brain preparation of the Nakayama strain, which is manufactured in Japan and the Republic of Korea. Its cost of over UStl per dose has restricted its use in mass immunization in less developed nations. In addition tests of human vaccinees and cross-challenge experiments in mice indicated'that there are at least two closely-related antigenic groups of isolates of JE virus. These are represented by the Nakayama and Beijing-l strains. The second is a primary hamster kidney preparation, made in China from the P3 strain. Recent studies in China have shown that its efficacy, not yet ideal, can be improved by physical concentration methods.
4.
FIELD TRIAL OF MONOVALENT AND BIVALENT INACTIVATED JAPANESE ENCEPHALITIS VACCINES IN THAILAND
In Japan a bivalent vaccine was experimentally manufactured because of the existence of two closely related antigenic variants of Japanese encephalitis virus. Experimental lots of Beijing-l vaccine were manufactured by the same technique as that used for inactivated mouse brain vaccine of the Nakayama strain. In humans the antibody levels overall were relatively high to both viruses. A vaccine consisting of a mixture of equal antigenic masses of Nakayama and Beijing-l strains induced in mice neutralizing antibody of higher titre to both strains than did either vaccine alone. Thus it was postulated that bivalent vaccine would be more efficacious in humans than the currently used Nakayama monovalent vaccine. A vaccine field trial in 73 000 children aged 1-14 years was carrierl out by the Thai Ministry of Public Health and the Armed Forces Research Institute of Medical Sciences (AFRIMS). Vaccine was administered in Kampanphet Province, Northern Thailand, before the start of the encephalitis season in 1985. Volunteers gave informal consent and were randomizad into three groups, receiving subcutaneous inoculations of (a) Nakayama monovalent vaccine, (b) Nakayama-Beijing bivalent vaccine, or (c) tetanus toxoid. Vaccines from the Biken Foundation of Japan were used. Ninety per cent of enrolled participants received two doses of vaccine and ten per cent received one dose. Subjects vaccinated at the beginning of the study were bled and sera were tested for neutralizing antibody. Serological responses of about equal magnitude to Nakayama virus were found in subjects receiving monovalent and bivalent vaccine. Serological responses, however, to Beijing-l virus were not improved in recipients of the bivalent vaccine as compared with recipients of the monovalent vaccine.
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The finding of low titre Beijing-l responses in subjects receiving. the bivalent vaccine was unexpected. The explanation is not known. Tests ~n mice indicated that both components of the bivalent vaccine were sta~le for at least 384 days so lack of stability does not appear to be respons~ble. Potency tests of vaccine handled under field conditions are planned. Analysis of morbidity and mortality of the vaccinees during the 1985 encephalitis season is not yet complete. Initial results indicate that there were 11 JE cases among vaccines confirmed by the IgM capture ELISA of cerebrospinal fluid. Nine cases were diagnosed in persons receiving the tetanus toxoid and two cases were in recipients of the bivalent vaccine, however, one of those had received only one dose of JE vaccine. There were no cases in the monovalent Nakayama vaccine recipients.
5.
RECENT PROGRESS IN VACCINE DEVELOPMENT
5.1
Inactivated Japanese encephalitis vaccine
The inactivated Nakayama mouse brain vaccine has recently been produced in both India and Thailand (in addition to Japan and the Republic of Korea). The vaccines from both countries met the standards for manufacture in Japan. Experimental inactivated tissue culture vaccines were developed in both China and India in an effort to improve on the available hamster kidney product. Three JE virus strains isolated in 1983 in China were shown to give average yields in primary hamster kidney cells of between 8.1 and 8.7 log ID50ml in contrast to an average yield of 6.8 for the P3 strain currently incorporated into the vaccine. Vaccines prepared from two of these strains protected mice from homologous virus challenge more effectively than homologous P3 vaccine. These vaccines were not tried in humans. Concentrated inactivated hamster kidney vaccine was prepared by hollow fiber ultrafiltration in an effort to improve efficiency. Forty-four children in Shanghai received a single inoculation with minimal side effects. An additional 59 children 7-10 years old from Heilongjiang were vaccinated with one dose. PRNT seroconversion rate after one month was 90.6%, a rate comparable with that observed after two inoculations of commercial inactivated mouse brain vaccine and significantly higher than after two inoculations of standard hamster kidney cell vaccine. The concentrated hamster kidney cell vaccine has been used only experimentally. JE viruses were adapted to chicken embryo fibrablasts in India and formalin-inactivated experimental vaccines were prepared. The protective index in mice of vaccines prepared from the Nakayama strain and two Indian isolates was equal or superior to that of commercial inactivated mouse brain vaccine used as a standard.
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5.2
Live attenuated Japanese encephalitis vaccines
Live attenuated vaccines are effectively used in horses in China and in pigs in Japan and other nations. Trials in humans in China were carried out with two experimental vaccines but seroconversion rates and duration of antibody were not superior to those of inactivated hamster kidney vaccine. No further development of attenuated vaccines is currently reported. 5.3 Molecular approaches to vaccine development
Japanese encephalitis virus contains three structural proteins: M(VI), C(V2) and E(V3). The major surface glycoprotein E plays an important role in neutralization and in stimulation of cytotoxic T/cells. The E protein contains at least eight domains of antigenic determinants. These have been characterized using monoclonal antibody studies. Some domains are associated with induction of neutralizing antibody, and monoclonal antibody to one (group 8) was efficient in protecting mice from challenge with JE virus. At least three institutions have now reported cloning portions of the JE virus genome. Sequence analysis was obtained for significant portions, including the region coding for the E glycoprotein, which has protective potential. With this encouraging progress, there is now expectation that a genetically engineered or synthetic polypeptide vaccine can be produced.
6.
GUIDELINES ON STRATEGIES FOR JAPANESE ENCEPHALITIS VACCINES AND FOR VACCINATION OF HUMANS
6.1
Rationale for use of vaccines in human beings
Control of Japanese encephalitis may be performed by (1) control of vector mosquitos, (2) control of amplifying hosts such as pigs, and (3) immunization of humans. Although vector control and immunization of pigs are valuable anti-JE methods, these may involve some practical problems or may lead to further complications, including pesticide resistance. Immunization of humans is broadly applicable, logistically feasible, relatively effective, and is successfully used in some countries. 6.2 Type of vaccine
Inactivated vaccines, using JE V1rUS grown in adult m1ce and in hamster kidney cell culture, are currently available for human use. Fluid type and freeze dried type are provided as the final product for mouse brain origin vaccine and the fluid type is provided for hamster kidney origin vaccine. 6.3 Vaccine strains
Since minor antigenic differences are noted among virus isolates from nature, vaccine strains should be selected to cover as wide an antigenic
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spectrum as possible. The strain selected should be stable, able to grow to a high titre and also be similar antigenically to those viruses found in natural infections in the area where the vaccine will be used. Nakayama-NIH, Beijing-l and P3 may be candidate strains; other antigenically similar strains are acceptable. In some areas bivalent vaccines may be used if field trials indicate their efficacy. 6.4 Potency of vaccine
Several techniques are 1n use 1n different laboratories to determine vaccine potency. These include antibody production in mice measured by plaque reduction neutralization and the mouse vaccination-challenge test. A comparative assessment of these techniques should be carried out. There is evidence that neutralizing antibody in the circulating blood plays the main role in protection against encephalitis. Therefore it is rational to assess potency by determining the vaccine's ability to induce neutralizing antibody in man and animals using the plaque reduction neutralization test (PRNT). The PRNT should incorporate a WHO mouse antibody standard when it becomes available. The vaccination-challenge test is relatively strain-specific; the challenge viruses should therefore include the homologous as well as at least one current strain. 6.5 Purity of vaccine
In order to remove non-viral substances and especially the encephalitogenic factor, JE antigen must be purified when prepared from infected mouse brain. The level of purity may be assessed by the specific activity per unit amount of protein in the vaccine. Eighty microgram per ml is the maximum total protein recommended for the mouse brain vaccine 1n Japan. Hamster kidney cell culture vaccine should contain less than 1 microgram calf serum protein per mI. 6.6 Safety of vaccine
Both purified brain vaccine and cell culture vaccine have a long history of development and improvement in quality, and severe adverse reactions such as anaphylaxis and encephalitogenic reactivity are very rare. Mild systemic reactions including fever are reported in less than I per cent. Some allergic reactions such as exanthem occur in low frequencey (less than 0.02 per cent). The vaccines are tested for toxicity in guinea pigs and for presence of live JE virus or other adventitious agents by intracerebral inoculation of mice with blind passage. 6.7 Storage of vaccine
The fluid vaccine should be stored at 4 0 to lOoC in the dark. The fluid vaccine frozen even once may lose considerable potency. Its potency is not guaranteed when it is kept in an environment of higher temperature. The fluid vaccine may be used for one year after passage of national assay. The potency of freeze-dried vaccine with moisture content less than 3% is stable for five years when stored at temperatures beloW 100C. Freeze-dried vaccine is particularly recommended where a cold chain system is not available.
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6.8
Contra-indications
There are no specific contra-indications to the administration of JE vaccine. Pregnant women or people suffering from chronic diseases like tuberculosis may be vaccinated under the direction of a physician. Persons with known prior allergic reactions to the vaccine should be re-vaccinated only under the supervision of a physician. 6.9 Quality control
No international requirements for JE vaccine are available. Therefore only national laboratories are responsible for certification. The quality of the final product of mouse brain type vaccine is controlled by the National Institute of Health, Tokyo, upon request, and that of the cell culture type by the National Control Institute for Pharmaceutical and Biological Products, Beijing. Items and procedures for national certification are described in the Minimum Requirements issued by these Governments (see Annex 3). 6.10 Subjects of immunization The object of immunization with Japanese encephalitis is the establishment and continued maintenance of individual immunity in the population potentially exposed to the virus. People of any ag~ can be immunized if indicated. This includes travellers to endemic areas from non-endemic areas. If they are residents of an endemic area of Japanese encephalitis, most of the adults may already be immune to Japanese encephalitis. Therefore, it is reasonable to select younger age groups and other high risk groups as the main targets of immunization. Morbidity statistics must be considered when a mass immunization programme is attempted. It is safe to immunize children under 6 months of age, but immunization in this age group is usually avoided if most of the mothers are considered to be already immune to Japanese encephalitis. 6.11 Adequate time of vaccination To provide effective immunization, two inoculations of JE vacc~ne are given at 7 to 14-day intervals followed by one booster I to 12 months later. It is estimated that at least one month is needed to obtain immunity after the initial administration of JE vaccine in non-immune individuals. Vaccination must be completed therefore at least one month ahead of the JE epidemic season. Vaccination may be given at anytime. In non-epidemic areas re-vaccination is recommended every three years to maintain a protective level of antibody.
7.
RECOMMENDATIONS
(1) WHO should coordinate a collaborative study to determine the most appropriate method for potency testing of JE inactivated vaccines.
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(2) WHO should develop m1n1mum requirements for JE inactivated vaccines. In formulating the requirements, the following should be taken into consideration: monitoring for murine viruses, and methods for determination of purity. (3) WHO should establish an international antibody standard for the potency testing of JE vaccine. (4) WHO should encourage longitudinal field studies under different ecological conditions to determine the duration of immunity following the initial vaccination series. (5) WHO should support and encourage the development and improvement of inactivated tissue culture Japanese encephalitis vaccines. (6) WHO should encourage field studies on the efficacy of improved inactivated vaccines and molecularly derived vaccines as they are developed. (7) The Working Group endorses the recommendations made by the Working Groups in December 1983 and August 1984, including recommendations that WHO should encourage research on the application of molecular technology to the development of second generation Japanese encephalitis vaccines; that WHO should continue to develop and distribute the IgM capture ELISA, and to train health workers in its preparation and use; and that the "Technical guidelines on Japanese encephalitis" should be revised.
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ANNEX 1 AGENDA
1.
Japanese encephalitis vaccine and vaccination Overview on JE vaccine New JE (Beijing) vaccine Field trial of Beijing vaccine JE vaccine and vaccination in the Republic of Korea JE vaccine and vaccination in the People's Republic of China JE vaccine and vaccination in Thailand Review of JE epidemic in Viet Nam Dr S. Nadhirat Prof. Hoang Thuy Nguyen Dr Chen Bo Quan Dr S.B. Paik Dr A. Oya Dr K. Fukai Dr Kitano
2.
Development of new JE vaccine Overview Bivalent vaCClne Live attenuated vaccine 1n China Molecular aspects of JE virus in relation to vaccine developments Dr K. Yasui Dr A. Igarashi Dr R. Shope Dr K. Fukai Dr He Shimin
3.
Discussion and formulation of guidelines for JE vaccination Dr A. Oya
4.
Recornrnendat ions
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ANNEX 2
LIST OF MEMBERS, CONSULTANT, SECRETARIAT AND OBSERVERS
1.
MEMBERS
Dr Janet Blok Virologist Queensland Institute of Medical Research Herston, Brisbane Queensland 4006 Australia Dr Chen Bo Quan Institute of Virology Chinese Academy of Medical Science Beijing People's Republic of China Dr He Shimin Shanghai Institute of Biological Products Shanghai People's Republic of China Dr Akira Igarashi Institute for Tropical Medicine Nagasaki University 12-4 Sakamoto-machi Nagasaki Japan 852 Dr N. Ishida President Tohoku University 2-1 Seiryomachi Sendai 980 Japan
... /
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Annex 2
Dr K.H. Kim Director Korea Green Cross Research Institute 729. Seocho-Dong. KangNam-Ku Seoul T-l35 Republic of Korea Dr T. Kitano Department of Virology and Rickettsiology National Institute of Health 10-35 Kamiosaki. 2-chome Sh inagawa-ku Tokyo Japan Professor Hoang Thuy Nguyen Director National Institute of Health 1. Yersin Street Hanoi Socialist Republic of Viet Nam Dr Akira Oya Director Department of Virology and Rickettsiology National Institute of Health 10-35 Kamiosaki. 2-chome Shinagawa-ku Tokyo Japan Dr Sung Bok Paik Director Department of Virology National Institute of Health Seoul 122 Republic of Korea Dr K. Pavri Director National Institute of Virology Pune India
... /
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Annex 2
Dr Nadhirat Sangkawibha Director-General Department of Medical Sciences Yod-se, Bangkok 10100 Thailand Dr Robert E. Shope Professor of Epidemiology Yale Arbovirus Research Unit Yale University School of Medicine Box 3333 New Haven, Connecticut 06510 United States of America Dr Kotaro Yasui Department of Microbiology Tokyo Metropolitan Institute of Neurosciences 2-6 Musashidai, Fuchu Tokyo Japan 2. CONSULTANT Dr Konosuke Fukai Chairman, Board of Directors Research Foundation for Microbial Diseases Osaka University 3-1 Yamada-Oka Suita, Osaka Japan 3. OBSERVERS Dr C.H. Hoke Chief Department of Virology Armed Forces Research Institute of Medical Sciences Bangkok Thailand
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Annex 2
Dr Thomas O'Rourke Virology Department United States Naval Medical Research Unit No. 2 Bureau of Research and Laboratories San Lazaro Compound Riza1 Avenue, Sta. Cruz Manila
4.
SECRETARIAT Dr T. Umenai Regional Adviser in Communicable Diseases WHO Regional Office for the Western Pacific Manila Dr T. Bektimirov Chief Medical Officer Virus Diseases Unit WHO Headquarters Geneva Dr C.K. Sanyakorn Director Disease Prevention and Control WHO Regional Office for South-East Asia New Delhi
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ANNEX 3
VACCINE AND VACCINATION FOR JAPANESE ENCEPHALITIS A REVIEW by Dr Akira Oya
1.
Vaccine for human use
Japanese encephalitis (JE) vaccine for human use is produced and used in Japan, Republic of Korea and China, including Taiwan (Province of China). Inactivated vaccine of mouse brain origin is used in Japan, Republic of Korea and Taiwan Province. Inactivated vaccine employing primary hamster kidney cell culture is used in China.
1.1
Purified inactivated vaccine from infected mouse brain (1)
Production
Purified inactivated vaccine is produced in Japan either by application of ultracentrifuge or by alcohol precipitation. Manufacturers in Republic of Korea and Taiwan Province principally follow the alcohol precipitation method of Japan. Formalin is used for the inactivation of JE virus. Approximately, 12 million dOses are produced annually in Japan. In the Republic of Korea, the production of JE vaccine has increased rapidly during the recent decade and 6 million doses were produced in 1983. Two types of final products are available - the fluid type and the freeze-dried type. The former is routinely used in Japan and the latter is for use outside of Japan, mainly in tropical areas in view of its greater stability at a higher environmental temperature. (2) Quality control
JE vaccine in Japan is prepared in accordance with the m1n1mum requirement for JE vaccine, issued by the Ministry of Health and Welfare. Essentially the same requirement with certain modifications has been adopted in the Republic of Korea and Taiwan Province. Fluid-type JE vaccine as a final product is sterile, colourless and transparent with a pH range 6.8 7.4. Residual formaldehyde is within 0.01 w/v per cent. Thimerosal can be added less than 0.012 w/v per cent. As an indicator of purity, the nitrogen content of protein is controlled not to exceed 10 ug/ml. Great consideration has been given to making JE vaccine free from either by the antibody production test in mice or by the intracerebral challenge test. However, the direct challenge method using mice in general has the disadvantage of always using a special strain of challenge virus and in addition shows a wider error range of results. Instead, the antibody
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Annex 3
production method could be applied in any virus challenge system. The flexible nature of the potency test may be more applicable to testing the potency of JE vaccine against a variety of JE strains. There is as yet no international standard of potency of JE vaccine. The deterioration of potency will occur in 10% at 4 0 C for four months and in 50% at 25 0 C for ten days. The potency of lyophilized vaccine is extremely stable compared with the fluid type and has remained unchanged for five years at 4 oC. The moisture content of lyophilized JE vaccine in Japan is required not to exceed 3%. (3)
Efficacy
A large-scale field trial was carried out involving 400 000 children in Taiwan Province in 1965 by Hsu, et al. More than 130 000 children aged 3-11 years were vaccinated with JE vaccine produced in Japan and an equal number of children were kept as a placebo group receiving tetanus toxoid. After the immunization, they were exposed to natural infection. Morbidity rates for JE were 3.58 per 100 000 among children receiving two shots of vaccine and 18.20 among the placebo group. The efficacy of JE vaccine was calculated as 80%. Antibody production in the vaccine was examined by many investigators. Sero-conversion rate was observed in 90% to 100% against homologous strain to the vaccine strain. However, the rate decreased to 60 - 90% when challenged with the heterologous strain of JE V1rus. Antibody titres produced by two doses of primary vaccination fell rapidly one year after vaccination. When one dose of booster vaccination was given, antibody titre increased rapidly and fell slowly. It was observed that the neutralizing antibody titre was kept well at levels of more than 1:10 three years after a booster. (4) Side reaction
Since the use of JE vaccine of the mouse brain type started in Japan, special concern has been expressed about the possible incidence of a severe adverse reaction in the central nervous tissue after vaccination. In 1965, a special survey team was organized by clinical doctors under the Ministry of Health and Welfare, who conducted an extensive survey on the side reactions among 21 396 vaccinees; fever, general malaise and abdominal symptoms were noted in 1.2% of vaccinees. With the cooperation of the countrywide network of hospitals the survey team searched for any severe neurological diseases occurring within one month after vaccination. They found 26 cases during the ten years from 1957 to 1966. Reported diseases were meningitis type (9), convulsion type (10), demyelinization type (2) and polyneuritis type (5). The incidence was thought to be very small, considering the millions of vaccinations made every year in Japan. No etiological relation between those diseases and JE vaccination was confirmed.
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Annex 3
1.2 Inactivated vaccine from cell culture (1) Production in China Formalin-inactivated JE vaccine employing primary hamster kidney (HK) cells has been produced on a large scale and used since 1967 in China. In 1983, approximately 100 million doses were produced by six institutes of biological products in China. HI cells were cultivated in large roller bottles in a growth medium containing calf serum. After the growth medium is discarded, HK cells are seeded with the P3 strain of JE virus and maintenance medium containing human albumin is added. The culture fluid is harvested after an appropriate time of incubation, clarified by filtration, inactivated by adding 1:2000 formalin, and used for vaccine. Quality control is done according to national requirements, mainly by the control department of the Institute. The National Control Institute independently checks samples, mainly for potency and for residual amount of calf serum. The method used for potency test is the challenge test similar to that described by Otani. Potency is expressed as 50% immune dose and the minimum level of potency of JE vaccine is required to have 0.00024 ml IDSO in mice. The residual amount of calf serum in JE vaccine is required not to exceed 1 ug/ml determined by passive haemagglutination using human erythrocytes coated with antibody against calf serum. (2) Efficacy and side reaction
Children under 10 years were given three initial doses in 1973, a booster dose in 1974 and left for the following three years. Thirteen JE cases were found in approximately 71 000 vaccinated children during five years and 11 out of approximately 3 500 were reported as JE cases in the control group. The protective rate of the vaccine was estimated as around 95% (Huang, 1982). The sero-conversion rate was examined in children in a non-epidemic area, Yianji City, Jilin Province. Primary school children received two doses of JE vaccine in 1973. A month later the sero-conversion rate was 60% which declined to 10.3% one year later (1974). The antibody positive rate increased to 93.1% when a booster injection was made in 1974. At the beginning of the mass vaccination using tissue culture vaccine, certain lots were found to produce some allergic reaction such as skin rashes and shock. Such a reaction was found to correlate with the residual amount of calf serum in the vaccines. Thereafter, the residual amount of calf serum was strictly regulated and the incidence of the side reaction markedly decreased. The incidence of systemic and local reaction with the current vaccine was reported to be mild and found in 0.02% of vaccinees out of 1.6 million. Cases of high fever of over 390 C were found in 0.002%
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Annex 3
2.
Research on live vaccine
Hammon and his co-workers have attempted to develop live virus vaccine by passages of JE virus in hamster kidney cells. They obtained a highly attenuated mutant which does not kill the weanling mice by inoculation. However, it was found at the same time to be essentially non-infective to man. Takehara, et al. (1969), Inoue (1964), Sazawa, et al. (1969) and Yoshida, et al. (1981) obtained independently attenuated strains of JE virus in cell cultures. They did not produce an appreciable titre of viraemia and produced no adverse reaction in pigs. They are now used for immunization of pigs in Japan, mainly to prevent stillbirth. Chinese researchers have devoted a considerable amount of time to developing attenuated JE virus vaccine independently of other countries for use in humans as well as animals in recent decades. The 2-8 strain was administered subcutaneously to 500 000 horses without any severe adverse reaction and positive conversion of the neutralizing antibody was observed in 80 - 90%. Epidemiological evaluation showed a protection rate of 86.7% (1974). Subsequently, the 2-8 strain was administered to 8 000 children and sero-conversion was found in approximately 50%. Another attenuated strain 5-3 was administered to 205 359 children aged 2 - 10 years in 1973 and the incidence of JE cases was observed for five successive years. The protection rate of the vaccine was estimated as 85 - 96% for each of the five years referring case incidence in 26 182 controls. No particular observation was made about the presence of an adverse reaction. 3. Vaccines for animal use and applied for public health purposes
Minimizing the susceptible population of animals which are responsible for transmitting the virus to vector mosquitos in nature seemS to be a reasonable way to control a JE epidemic. Pigs are well established for playing this amplifying role of infected mosquitos in nature. The effective immunization of pigs could thus prevent JE epidemic among humans. Vaccinated pigs indeed showed no viraemia after challenge and did not the transmit virus to mosquitos. Large-scale field experiments have been conducted in Nagasaki (Takahashi, et al., 1971) and in Kyoto (Tsuchiya, et al., 1970) in Japan. However, as the reports revealed, great difficulty has been encountered in putting this idea into practice. One of the difficulties encountered was finding a suitable time for vaccinating pigs. Since newborn piglets carry the maternal antibody for approximately four months after birth and are sacrificed 6 - 8 months after birth in Japan, effective vaccination should be done during the very limited period when pigs have no antibody. However, the attempt to immunize pigs in order to prevent an epidemic could be promising if the trial is carefully designed.
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Annex 3
4.
Effective immunization with JE vaccine (1) Adequate time of vaccination
It is estimated that at least one month is needed for man to obtain immunity after the initial administration of JE vaccine, provided that he has no immunological memory of JE virus antigen before. Vaccination must therefore, be completed at least one month ahead of the start of the JE epidemic if the period of the epidemic can be roughly presumed. In the area where the epidemic is rather incidental and hard to predict, it is recommended to give the people an immune memory by two shots of JE vaccine at 7 to 14 days interval anytime, to be followed by one booster shot one month later to give an immune memory of JE virus antigen. A booster vaccination is given anytime when the epidemic starts. It was evidenced that the effect of the booster vaccination was expected as early as seven days after vaccination. (2) Subjects of immunization
People of any age can be included among the subjects of JE vaccination if necessary. If they are residents in endemic areas of JE, most of the adults may already be immune to JE. Thus, it is reasonable to select the younger age groups as the main target of immunization. Morbidity statistics must be referred when mass vaccination programme is attempted. Children under 6 months after birth can be excluded if most mothers are considered to be already immune to JE. (3) Contraindication
There seems no contraindication specially considered for the administration of current JE vaccine. On the contrary, pregnant women or people who have some chronic diseases such as tuberculosis may be vaccinated if necessary. In case some allergic reaction apparently related to the JE vaccination is noted, it is better to be relieved from further JE vaccination.