New initiatives for the control of Japanese encephalitis by vaccination:

Vaccine 18 (2000) 1±25

www.elsevier.com/locate/vaccine

New initiatives for the control of Japanese encephalitis by vaccination: Minutes of a WHO/CVI meeting, Bangkok, Thailand, 13±15 October 1998 Theodore F. Tsai* Centers for Disease Control and Prevention, National Center for Infectious Diseases, Fort Collins, CO 80522, USA

Abstract Japanese encephalitis (JE) is a leading cause of viral encephalitis in Asia that, in several countries, has been controlled e€ectively through national vaccine programs. However, in recent years, transmission has been recognized or has intensi®ed in new locations where the available vaccines are either una€ordable or unlicensed. In addition, the near-eradication of poliomyelitis from Asia has elevated JE in the public health agenda of preventable childhood diseases, and surveillance of acute neurological infections to con®rm polio eradication, simultaneously, has led to a greater awareness of the disease burden attributable to JE. The only internationally licensed JE vaccine, an inactivated mouse-brain derived vaccine, is ecacious but is problematic from the perspectives of reactogenicity, requirement for numerous doses, cost and reliance on a neurological tissue substrate. A live-attenuated vaccine distributed only in China also is ecacious and requires fewer doses; however, production and regulatory standards are unresolved. Several approaches toward developing novel JE vaccines that could ®ll the gap in JE vaccine need are under pursuit. The minutes and recommendations of a meeting of experts to discuss these issues, jointly sponsored by the World Health Organization and the Children's Vaccine Initiative in Bangkok, Thailand, 13±15 October, 1998, are presented. Published by Elsevier Science Ltd.

1. Introduction

Abbreviations: AE, (vaccine-related) adverse events; ATCC, American Type Culture Collection; cDNA, copy deoxyribonucleic acid; CPE, cytopathic e€ect; CSF, cerebrospinal ¯uid; DALY, disability adjusted life year; DT, diphtheria±tetanus; DTP, diphtheria± tetanus±pertussis; EPI, Expanding Immunization; GMP, good manufacturing practice; GMT, geometric mean titre; HI, hemagglutination inhibition; Hib, Haemophilus in¯uenza Type B; HIV, human immunode®ciency virus; Ic, ip; intracerebral, intraperitoneal; IgE, IgA; IgM, IgG; classes of immunoglobulins; JE, Japanese encephalitis; MBP, myelin basic protein; NS1, nonstructural protein 1; pfu, plaque-forming unit; PHK, primary hamster kidney cells; PIV, puri®ed inactivated vaccine; YF, yellow fever. * Current aliation: Clinical Research, Wyeth Lederle Vaccines, 401 N. Middletown Rd, Pearl River, NY 10965, USA; Tel.: 914 7324053; fax: 914 732 5517. E-mail address: [email protected] (T.F. Tsai). 0264-410X/00/$ - see front matter Published by Elsevier Science Ltd. PII: S 0 2 6 4 - 4 1 0 X ( 0 0 ) 0 0 0 3 7 - 2

Japanese encephalitis (JE), a mosquitoborne ¯aviviral infection transmitted in Asia, is a grave disease of children in the region, accounting for more than 16 000 reported cases and 5000 deaths annually. In the last 25 years, transmission has intensi®ed in certain countries and the disease has extended its geographical range to previously una€ected areas of Asia and to northern Australia. The availability of e€ective vaccines that have eliminated or reduced signi®cantly disease rates in Japan, Republic of Korea, Taiwan and China provides a means to control the disease elsewhere in the region (Fig. 1). But the only internationally approved vaccine (an inactivated mouse brain-derived vaccine) is relatively costly and the other proven vaccine (made from the live attenuated SA14-14-2 strain)

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is distributed only in China. Furthermore, in many countries, disease surveillance to con®rm the burden of disease and to identify high-risk areas has been inadequate to guide vaccine policy. With the near eradication of poliomyelitis, JE has risen in public health priority as a vaccine-preventable disease, so that regional and international e€orts to delineate disease burden, vaccine need, and to compare attributes of the available vaccines are timely. Under the auspices of the WHO Steering Committee on ¯aviviruses and Working Group on dengue and Japanese encephalitis and the WHO South-East Asia Regional Oce, a group of international experts and national public health authorities met in Bangkok in October 1998 to discuss the following issues: 1. Needs and requirements for JE vaccine by country and in the region. 2. The current status of quali®cation and development of live-attenuated SA14-14-2 and candidate JE vaccines, respectively. 3. Global supply of inactivated mouse brain-derived and SA14-14-2 vaccines and their suciency to meet regional demands to control JE. Dr Scott Halstead delivered a generous tribute to Dr Yu Yong Xin for his seminal role in developing the attenuated SA14-14-2 vaccine, an accomplishment he likened to Theiler's derivation of the 17D yellow fever strain, which was honored by a Nobel Prize. Dr Halstead underscored the largely empirical process to develop, and to prove in humans, the robust attenuation and protective ecacy of the vaccine, a feat that is the more remarkable when viewed in the context of the few successful live viral vaccines that ever have been licensed. Dr Yu has been rewarded by the knowledge that thousands of childhood illnesses and deaths have been averted through his e€orts, but among the international public health and scienti®c communities, his achievement largely has been unheralded, in part

Fig. 1. Cases of Japanese encephalitis reported to the World Health Organization, 1966±1996.

because of his humility and self-e€acing personality. Dr Halstead invited the group to redress this oversight by saluting Dr Yu, which his colleagues endorsed, in a standing ovation. 2. Burden of disease 2.1. Incidence Although JE is a WHO reportable disease, ocial noti®cations signi®cantly underestimate true incidence. Although many countries report all cases of clinical encephalitis without laboratory con®rmation, resulting in an overestimation of cases, in general, this bias is far outweighed by underreporting. In numerous countries where viral transmission has been proven and where sporadic cases have been recognized, no surveillance systems are in place and, where surveillance exists, sensitivity is variable. For example, in Indonesia, Philippines, and Malaysia where few or no cases have ever been reported, hospital-based studies disclosed JE to be the etiology in 17±50% of hospitalized encephalitis cases, indicating a high level of endemic transmission not re¯ected in ocial noti®cations (Table 1) [1±6]. Further contributing to underreporting is the failure to recognize cases presenting with atypical clinical presentations (e.g. milder febrile illnesses without clinical signs of encephalitis, Guillian Barre syndrome, and acute psychosis) and patients dying outside of hospital. To estimate JE incidence in the region, a representative incidence rate was inferred from recent investigations in which incidence was measured by active case-®nding with laboratory con®rmation of the diagnosis in a de®ned population at risk (Table 2) [7±12]. These rates were similar to those in placebo and unvaccinated children in vaccine trials in Taiwan and in ThailandÐranging from 1.8 to 2.5/10 000 in their respective control groups [10, 13±15]. JE attack rates in immunologically naõÈ ve adult populations provide surrogate estimates of disease rates for children in areas with endemic transmission, and with one exception, these rates also fell within the 95% con®dence interval of the rates in placebo study groups above. Incidence rates in unvaccinated children in Chinese vaccine ®eld trials, however, have been considerably higher, ranging upwards to 5.7±64/10 000 (Table 3) [16]. This di€erence is unexplained but may re¯ect a higher risk of acquiring the disease in those populations due to di€erent standards of living or other factors. The total population at risk in the region can be estimated from crude assumptions that it comprises (a) principally residents of rural areas, who number approximately two thirds of the Asia population of 3 billion, and (b) children <15 years of age, since nearly

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Table 1 Proportion of hospitalized encephalitis cases due to Japanese encephalitis, various locations and yearsa Age

Study location

JE/clinical encephalitis (%)

7 months±13 years 6 months±15 years 18 months±12 years 10 months±10 years 6 months±12 years Unselected Unselected

1991±1994, Beijing, China 1993, Sichuan province, China 1990±1995, Denpasar, Indonesia 1990±1992, Pennang, Malaysia 1986±1989, Lucknow, India 1986, Koshi zone, Nepal 1985, Manila, Philippines

5/97 99/158 40/77 5/13 92/394 119/124 22/129

(5%) (63%)b (52%) (38%) (23%) (96%)c (17%)

a

Proportions can vary widely, depending on factors such as age of subjects under surveillance, laboratory diagnostic sensitivity and speci®city, hospital referral patterns, seasonal and secular patterns, as well as true disease incidence. b 4 epidemic months. c 2 epidemic months.

all cases are in children of this age. Using 1994 population estimates, the segment of the rural population of Asia under 15 years is 700 million children. Applying a representative rate of 2.5/10 000 to that population at risk, in the absence of any intervention, the annual incidence of JE in the region is estimated to be 175 000 cases. Assuming that 25% of cases are fatal and that 45% of surviving patients retain a neurological de®cit, 43 750 fatal cases and 78 750 cases with disabilities are estimated annually (see below). The gap between the calculated number of cases and ocial noti®cations is explained partially by vaccination in some countries but it re¯ects principally the lack of reporting from populous countries where no surveillance currently exists. An estimate of the expected number of cases in individual countries was calculated from the incidence rate above and the estimated rural population under 15 years of age, corrected for estimated vaccine coverage. A comparison of reported and expected number of cases by country indicates the shortfall in reports and suggests locations where surveillance should be intensi®ed to assess disease burden (Table 4). In addition, with the absolute decline in numbers of cases from China, the relative

contribution of cases from other countries in the region has gained importance. 2.2. Disability The spectrum and extent of disabilities in recovered JE patients is poorly studied, although it is clear that some disabilities improve or resolve with time, underscoring the importance of long-term follow-up studies. Such studies are dicult to perform and few have been reported [17±29]. A review of clinical reports in which cases were laboratory-con®rmed, and in which disability also was assessed after hospital discharge, disclosed a wide range of outcomes. A placebo-controlled study of dexamethasone therapy in Thailand, that represented a high standard of supportive care, reported a fatal outcome in 25% of cases and neurological sequelae in an additional 45% of cases 3 months later [17]. A follow-up study of 59 cases in Taiwan found neurological or psychological de®cits in 38% of patients followed for 1 year but in only 10% of those followed for 4 years; however, individual cases were not observed longitudinally [18]. In contrast, among 55 childhood cases in a series from Luck-

Table 2 Incidence of Japanese encephalitis among children in locations with endemic transmission (including surveillance of placebo recipients in vaccine ®eld trials in Taiwan, 1965±71 and in Thailand, 1984±85) or in immunologically naõÈ ve populations (including adults) exposed to a novel viral introduction or entering an endemic zone (bold); selected studies Study site

Cases/Population

Annual rate/10,000

Comment

1995, Torres Strait, Australia 1991, Okinawa, Japan 1990, Saipan, Northern Mariana Islands 1984±85, Kampangphet, Thailand 1972, Nham Phong, Thailand 1970, Chiangmai Valley, Thailand 1968±71, Taiwan (except Taipei)

3/8000 3/20,000 10/40,000 11/21,516 9/2101 100/680,000 120/ > 12  106 240/ > 12  106 35/140,514 24/131,865

3.8 1.5 2.5 2.5 42.8 1.5 0.1 0.2 2.5 1.8

Cx. annulirostris-borne outbreak Heterogeneous population at risk; island highly urbanized Cx. annulirostris-borne outbreak Placebo recipients in vaccine trial, 1±14 year old US soldiers Transmission extended over 8 months Consecutive years after mass vaccination initiated in 1967 Consecutive years after mass vaccination initiated in 1967 Unvaccinated Placebo recipients in vaccine trial, 3±7 year old

1965, Taiwan (except Taipei)

(1.3±11.0) (0.5±4.4) (1.4±4.6) (1.4±4.6) (22.6±81.2) (1.2±1.8) (0.08±0.13) (0.18±0.23) (1.8±3.5) (1.2±2.7)

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Table 3 Ecacy of SA14-14-2 live-attenuated Japanese encephalitis (JE) vaccine in ®eld trials, China, 1988±92a Province

Year

Study group

No. of subjects

JE cases

Incidence/100,000

Ecacy % (95% CI)

Guizhou

1988

Vaccinatedb Nonvaccinated Vaccinatedc Nonvaccinated Vaccinatedb Nonvaccinated Vaccinatedc Nonvaccinated Vaccinated Nonvaccinated Vaccinatedd Nonvaccinated Vaccinatede Nonvaccinated

86,132 21,149 86,933 16,869 64,027 4546 63,927 5784 65,941 7262 29,639 29,006 79,599 6587

1 12 0 12 2 9 1 37 0 24 (3 yr) 2 46 2 4

1.16 56.7 2.30 71.1 3.12 198.0 1.56 639.6

98.0 (96±100)

1989 Jiang-Xi

1989 1990 1991±3

Yunnan

1991

Anhui

1992

109.6 6.75 158.6 2.5 60.7

100 98.4 (97±100) 99.8 (98±100) 100 95.7 (94±99) 95.8 (94±99)

a JE incidence rates in nonvaccinated children that are much higher than in trials of inactivated JE vaccine are unexplained but could re¯ect di€erences in human exposure and viral transmission patterns in China. b Children 1±10 immunized with single primary dose. c Combination of 1±10 year old children immunized in previous years(s), 1 year-old children given primary dose, and 2 year-old children given booster dose. d Children 1±7 years old immunized with single primary dose only. e Children 1±6 years.

now, India, 71% had signi®cant neurological or psychomotor retardation 12±18 months after discharge and, at a 2 year follow up, few of the de®cits had resolved. Furthermore, in the best documented study of long-term disability, 10 years after the 1947 Guam outbreak, neurological sequelae were present in 40% of surviving patients, 11% of them considered severe [23]. A 5-year follow-up of cases in Thailand also found serious sequelae, such as limb paralysis, in more than one-third of the patients and ®ne motor de®cits in 72% [29]. Psychological and behavioral disorders were present in almost three-quarters of the children. None of the above studies were controlled, although, to evaluate the proportion of cases with psychomotor retardation, ®ne motor de®cits, and behavioral disorders, comparative observations of a control group

are essential. Dr Xu Zhi Yi reported preliminary results of a neurological and psychological study of patients who had recovered from JE or encephalitis due to other causes, 6 to >20 years after their acute illness. The original patient population was comprised of 468 encephalitis patients hospitalized between 1973 and 1997 at two pediatric hospitals in Shanghai. After arduous e€orts to trace the discharged patients, 29 JE and 43 control patients were interviewed and examined. Severe neurological de®cits (paralysis, convulsions, and optic atrophy) were present in 32% of the JE cases but in none of the controls. Of considerable interest, optic atrophy and convulsions in three cases did not develop until 7 or more years after recovery from the acute illness, indicating the potential for considerable latency in the onset of JE-associated adverse

Table 4 Japanese encephalitis (JE) surveillance gaps by country; expected cases calculated from typical JE incidence rate applied to rural population of children under 15 years Country

Cases without interventiona

1±(vaccine coverage  0.9)

Expected cases

Reported cases

%

Bangladesh China India Indonesia Myanmar Philippines Thailand Viet Nam Total

10,566 57,975 59,787 11,245 3134 2876 3467 5567 154,618

1 1±(0.5  0.9) 1 1 1 1 1±(0.5  0.9) 1±(0.2  0.9)

10,566 31,886 59,787 11,245 3134 2876 1907 4565 125,966

0 18,472 1243 0 0 0 752 3447 23,914

0 58 2 0 0 0 39 76 19

a

Based on rural population <15 years and incidence rate of 2.5/10,000.

T.F. Tsai / Vaccine 18 (2000) 1±25

outcomes. This study also illuminated the additive burdens of various neurological and psychological de®cits on activities of daily living, which were impaired in a greater proportion of JE survivors than in control patients. These ®ndings, and the previously mentioned Thai study demonstrating psychomotor or behavioral disorders in the majority of JE survivors, underscore the critical importance of subtle de®cits impeding cerebral function, especially in developing children, which more than deaths, contributes to the disease burden of JE. While the focus of JE surveillance has emphasized encephalitis cases, in related infections such as St Louis encephalitis, milder clinical presentations Ð classi®ed under a syndrome of febrile headache Ð far outnumber cases with overt encephalitis [30]. Undoubtedly, some proportion of these cases also re¯ect neurological infection with the possibility of permanent sequelae. E€orts to de®ne more fully the spectrum of symptomatic JE infections may disclose a far greater burden of disease than has been appreciated from surveillance of encephalitis alone. 2.3. Comparison of Japanese encephalitis and Hemophilus in¯uenza b meningitis incidence A comparison of JE and Hemophilus in¯uenza b (Hib) meningitis incidence in Asia is appropriate since both are vaccine-preventable diseases resulting in neurological infections of children [31]. Hib incidence has been reported to vary in middle and low income Asian countries, from 3 to 10/100 000 in children under 5 years of age in Hong Kong, Malaysia and in Caucasian residents of New Caledonia, to 25±163/ 100 000 in Fiji, among the Melanesian population of New Caledonia, the Vietnamese population of Hong Kong and Vanuatu. The latter rate was exceptional and the highest among any of twenty-one populations in Africa, Asia, or South America. The consensus range of 3±70/100 000, encompassing Asian countries of all income levels and used in a model of Hib disease burden in Asia, is in the range of JE rates in China and elsewhere in the region noted above. 3. Cost±e€ectiveness of immunization against JE A prevention±e€ectiveness analysis for a regional JE vaccination program has not been attempted until recently because of gaps in data on the burden of disease and the outcome of cases, particularly with respect to morbidity and disability in survivors of the illness. A cost±e€ectiveness analysis of alternative JE vaccination programs in Thailand showed that a vaccination program at 18 months of age, in which two doses were administered at the local cost of $1.75,

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results in a cost saving of $15 000 per case averted or $60 000 per life saved [32]. The approximate savings in terms of disability-life-years (DALY), $1200/DALY, would be considered cost±e€ective by World Bank standards that specify the per DALY cost of a prevention program should be less than the per capita GNP, for Thailand, $2000±3000. By comparison, the cost savings of an averted death due to HIb disease is $5000±30 000 and the cost per DALY of childhood Hib vaccination ranges from $150 to 800/DALY. Dr Xu Zhi Yi presented a conservative analysis of the cost±e€ectiveness of the JE vaccination program in Shanghai, based on cases averted in a post-vaccination period between 1989±1998. Extrapolating from observations during the prevaccination era, 11 946 expected cases were prevented by vaccination. Focusing only on direct costs of treatment (c. 129 million yuan for the expected number of cases) and work loss in disabled survivors (i.e. without considering the productivity losses due to a premature death or the medical care costs and loss of family productivity in disabled survivor households), if 20.7% of the expected cases were fully disabled, at a rate of productivity of 20 000 yuan/ year and for 40 years of lost productivity, the cost attributed to work loss was nearly 2.3 trillion yuan. The cost of vaccination of 100 000 newborns per year in this 10 year period (at a rate of 4 yuan per dose and for ®ve doses) was 20 million yuan, for a cost±bene®t ratio exceeding 100. The expenditure per case prevented was 1674 yuan and the per resident expenditure to maintain the vaccination program was 200 yuan. While these studies are important steps toward calculating the cost±e€ectiveness of JE vaccination, further analyses are needed, employing a more comprehensive quanti®cation of disease burden outcomes. The demonstration of a vaccination program's cost± e€ectiveness, although essential in arguments for uptake of a given vaccine into a national program, is only one of many factors that in¯uence such decisions. An analysis of predictors of hepatitis B vaccine acceptance into national schedules found that vaccine cost as a percentage of GDP was the single most important variable, after which the presence of an e€ective EPI infrastructure was an important secondary factor; high disease burden was a relatively poor predictor. This analysis underscores potential obstacles to acceptance of the only internationally distributed JE vaccine into national programs because of the vaccine's cost and its recommended age of administration beyond the usual EPI interval. 4. Control of JE by means other than human vaccination Although JE viral transmission, in principle, can be

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modulated by interventions aimed at the mosquito vector and vertebrate amplifying hosts, prevention of human disease by vector control or pig immunization is impractical and cannot reduce risk to the same extent as human immunization [33±34]. In the typical rural areas at risk, it is infeasible to treat the vast expanses and ubiquitous breeding sites exploited by vector mosquitoes. Adulticides may be indicated in emergencies but such interventions are costly and of unproven ecacy, and the perennial risk of transmission favors an ongoing preventive program. Pig vaccination is indicated to reduce economic losses from spontaneous abortions but, it cannot be relied upon to prevent human cases because alternate vertebrate hosts can amplify the virus, and because pigs bear young throughout the year, hindering compliance. Because adult pigs do not become ill with infection, the incentives for farmers to vaccinate their animals are indirect. Environmental modi®cations associated with economic development also have yielded unintended e€ects resulting in decreased viral transmission. For example, diminished land area under rice cultivation and applications of agricultural pesticides have reduced vector populations in rural areas, and even in developing countries, the rural population at greatest risk has declined. In many areas, zoning ordinances, changing life styles and economies of scale associated with centralized pig rearing, have led to a physical separation of the principal viral amplifying host from humans, reducing their risk of exposure. Urbanization of an entire country in the case of Singapore has been associated with an interruption of viral transmission to the local population, as was demonstrated in a recent serosurvey indicating no JE immunity in children under 12 years [35]. In Thailand and Vietnam, where JE vaccine has been incorporated into national immunization programs, the most signi®cant reductions in disease incidence preceded the establishment of these programs. Although these observations are of academic interest, the ultimate control and prevention of JE necessitates active interventions aimed at protecting humans. 5. JE vaccine manufacturing capacity and country vaccine requirements: country reports 5.1. India Endemic transmission of JE had been recognized in Southern India for several decades before 1973, when a series of outbreaks emerged, ®rst in West Bengal and subsequently in disparate areas of several northern and central states. In response to the size and number of outbreaks, in 1987, the Central Research Institute in

Kasauli, with technical assistance from Japan, began production of inactivated mouse brain-derived vaccine. Attempts to develop a vaccine with local strains were unsuccessful in producing any as potent as the conventional Nakayama strain vaccine, and human studies indicated that the Nakayama strain vaccine elicited neutralizing antibodies to local JE strains. Vaccine implementation in areas with outbreaks has been associated with a decline in cases nationwide from 6489 in 1989, to no more than 3000 cases annually since 1992. Vaccine requirements for India are dicult to estimate but, on the basis of demand from local health authorities, no more than 1.65 million doses have been required, although demand has ¯uctuated greatly to as low as 25 000 doses. When the Kasauli facility was established, an annual capacity of 2 million doses was planned; should future demand exceed that capacity, an additional production unit would be required which could be sited elsewhere in the country. The cost of inactivated JE vaccine is considered high compared to other vaccines included in the national immunization programme and, its cost makes largescale vaccination impractical. Demand for JE vaccine could increase several fold over current usage should a less expensive vaccine become available. Local research e€orts to develop a JE vaccine from the SA14-14-2 strain and from immunostimulatory complexes have been reported. 5.2. Japan Between 1948 and 1966, JE epidemics in Japan produced 1000±5000 cases annually, 50% of them fatal. By 1972, the annual number of cases had been reduced markedly, to fewer than 100, and after 1992, to below 10. It is generally accepted that the introduction of mass vaccination, beginning in 1955, led to this remarkable diminution in human cases. National requirements for formalin inactivated JE vaccine, issued in 1954, speci®ed that the supernatant of a 5% infected mouse brain homogenate should contain total solid materials of <20 mg/ml [36]. To reduce brain matter that might be associated with allergic myelitis, the requirements were revised in 1957, 1962, 1965, 1971 and in 1989 to improve purity and also to provide broader antigenic reactivity. Currently, JE vaccine made from the Beijing-1 strain contains <0.01 mg/ml protein nitrogen. The Beijing-1 vaccine is given in half the volume of the previous Nakayama strain vaccine and it is administered in a 0.5 ml dose for adults and half that volume for children. The quantity of JE vaccine produced in Japan increased from 376 000 doses in 1955 to 7.7 million doses in 1964. After improved puri®cation procedures employing alcohol±protamine precipitation or ultra-

T.F. Tsai / Vaccine 18 (2000) 1±25

centrifugation were introduced, vaccine production increased signi®cantly to 50 million doses by 1968. Annual production declined gradually to 9±10 million doses between 1984 and 1988. The switch in vaccine strain from Nakayama to Beijing-1 in 1989, reduced by half the volume of vaccine needed without a signi®cant reduction in the number of doses produced, around 9.4 million per year. The standard vaccination schedule of two doses within a 1 week interval, followed by a booster in a year, produces 100% seroconversion with a log GMT of 3.64 to the homologous Beijing-1 strain, and only a slightly lower 97.2% seroconversion to the Nakayama strain, with a corresponding log GMT of 2.56. The vaccine is administered principally to primary school and junior high school children with coverage rates, between 1977 and 1995, ranging from 42 to 66% overall, but reaching 80% in high risk areas. Exportations of liquid JE vaccine between 1967 and 1984 ranged from 4800 to 304 000 doses per year but the introduction of freeze-dried vaccine for export in 1968 led to a signi®cant increase in export volume to 1.5 million doses in 1995. Japanese manufacturers recently have been concerned that genotypically distinct strains isolated in northern Thailand were poorly neutralized by hyperimmune mouse sera raised against the two conventional vaccine strains (see below), and by the general issue of protection extended by Nakayama and Beijing-1 strain based-vaccines against strains from tropical Asia. 5.3. Thailand After the decision to introduce JE vaccine to the national immunization program was made in 1985, the Government Pharmaceutical Organization (GPO), the country's national vaccine manufacturer, obtained technical assistance from Biken and the Japanese Government to establish local production of inactivated mouse brain-derived vaccine. Production of a liquid vaccine using the Nakayama strain began in 1988. In ®eld trials comparing its immunogenicity with Bikenmanufactured vaccine, the local vaccine was equally immunogenic, producing antibody conversions in 99.4% of seronegative 5±9 year old vaccinees after two doses. The vaccine was added in a stepwise manner into the routine EPI schedules of high-risk provinces with additional catch-up campaigns aimed at 5±6 year old children [37]. Currently, the vaccine is given routinely in 34 of 76 provinces, accounting for one-third of the birth cohort, while the catch-up program has been discontinued. The GPO has been able to produce only 40% of the national vaccine need and the shortfall has been ®lled by imported vaccine that is relatively costly. To

7

increase capacity, in 1998, the GPO with Biken assistance has begun to change over production to the Beijing-1 strain, with the expectation that clinical trials can be undertaken in 1999±2001 and full scale production by 2002. Because the Beijing-1 vaccine is administered in half the volume of the Nakayma strain vaccine, a doubling of yield is expected using the current manufacturing facilities. The GPO expects that it can fully meet the national demand of c. 2.7 million doses by 2002±2003. In conjunction with the change of viral strain, improved vaccine stability has been sought by freezedrying. Vaccine stability is an important issue because production consists principally of 5 ml multidose vials. While the relative potency of liquid vaccine stored at 48C declines by c. 50% by 18 months, in experimentally produced lots of freeze-dried vaccine, relative potency was unchanged through 30 months. After storage at 378C for 1 week, the freeze-dried vaccine lots retained almost double the relative potency of liquid vaccine. Reconstituted lyophylized vaccine exhibits stability characteristics of the liquid vaccine. 5.4. Viet Nam JE occurs throughout the country with incidence rates of 5±15/100 000 in the provinces at greatest risk in the North [38±39]. The fatality rate among cases ranges from 14 to 20% with neurological sequelae in 60±70% of patients recovering from the acute illness. Approximately 2000±3000 cases of acute encephalitis are reported annually with epidemiological characteristics, including seasonality (with a June±July peak) and an age distribution (mainly in children from 1 to 15 years with peak between 3±5 years) that are highly suggestive of JE. From population estimates that 1.5 million children 1±5 years old live in high risk areas, at least 3 million vaccine doses are needed. In 1989, the WHO regional oce in collaboration with the Japanese BikenKanonji Institute assisted the National Institute of Hygeine and Epidemiology (NIHE) to establish production of Nakayama strain inactivated vaccine. Between 1990 and 1998, all 146 lots produced by NIHE passed the minimum WHO and Japanese requirements for physical, chemical and biological characteristics. Field trials showed that locally produced vaccine was as immunogenic as the Biken vaccine, producing 100% seroconversion in subjects given two doses, with a geometric mean neutralizing antibody titer that was only slightly lower, log 3.48 vs log 3.71. A ®eld study between 1993±1997 in Gialouong district, Bacninh province, comparing JE rates over a 5 year interval, in 33 552 1±5 year old children vaccinated with two to three doses and 38 448 unvaccinated children disclosed a protective ecacy of 98.6%.

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Although vaccine coverage increased from 12.6% to only 46.6% in those years, the vaccine's e€ectiveness was demonstrated by a reduction of JE incidence rates from 21.8/100 000 in 1995 to 6.8/100 000 in 1997. It is of interest that a much greater reduction in encephalitis cases occurred between 1988 and 1992, before vaccination was established, when incidence declined from 99.5/100 000 to 21.7/100 000. In 1997, the NIHE produced 800 000 doses and in 1998, production is projected to achieve 1.5 million doses. However, the national vaccine need is c. 6 million doses and currently, only a portion of that shortfall, c. 330±400 000 doses, is met by vaccine imported from other countries. With a limited supply, the vaccine is being introduced progressively into the national immunization program, focusing initially on geographic areas with the highest risk. 5.5. Republic of Korea An outbreak in 1949 leading to 5616 cases, 2729 fatal, marked the beginning of a period in which outbreaks of thousands of cases recurred every 2±3 years. In the largest outbreak on record, 6897 cases were reported in 1958. Starting in 1967, JE vaccination was introduced, using small quantities of the Nakayama strain vaccine imported from Japan. Although domestic vaccine production was initiated in 1971, the annual number of doses distributed in that decade remained under 500 000, leading to vaccine coverage rates under 5%. After outbreaks of 1197 and 139 cases occurred in 1982 and 1983, national health authorities initiated mass immunizations of 3±15 year old children at state public health centers and, simultaneously, encouraged vaccinations in the private sector. In 1984, 9.8 million doses were distributed, resulting in a coverage rate of 89.1%, and between 1985±1992, coverage was maintained consistently above 97.4%. In the last 15 years, fewer than ten cases have been reported annually, nearly all in adults. Four Korean manufacturers annually produce 3.6 million vaccine doses that are distributed through six pharmaceutical companies. Currently, an additional 400 000 doses are imported in bulk from Biken and distributed by another company but annual domestic capacity potentially could be increased to 9.5 million doses. As the incidence of disease in the Republic of Korea has declined, vaccine-associated adverse events have attracted concern from the public and medical community. In 1994, four highly publicized deaths temporally associated with JE vaccination produced strong antivaccine sentiment among the public and concern in the medical community that was re¯ected in an acute reduction of vaccine coverage to 79% (see below). The Korean FDA is now placing greater emphasis

on vaccine safety issues in the approval of new vaccines, which re¯ects a major change from past policies that expedited vaccine distribution to control important infectious diseases. 5.6. China Although a shift in the age of JE cases toward adults and the elderly has been reported from Japan, Republic of Korea and Taiwan, as childhood cases in China have been controlled by immunization, this same trend was not been seen. In an analysis of cases from 1991 to 1997 in Shanghai, where vaccine coverage also is high, a slightly higher age-speci®c incidence in the elderly was seen in birth cohorts before 1931± 1935, but in subsequent cohorts, that peak has disappeared. These observations indicate no need for a change in vaccination policy to target adults. To the contrary, several observations suggest that the age of primary vaccination should be reduced to cover infants. In 1996±1997 the peak incidence of cases reported in Shanghai has been in infants under 1 year of age. Serological studies indicate a decline in antibody prevalence from 70% in cord blood samples to 13% in 6±7 month old infants, underscoring the increased susceptibility of infants with a decline of maternal immunity. Nationally, the volume of vaccine needed to immunize the birth cohort in 2005 is estimated to be smaller than the 1997 requirement because of a projected decline in the birth rate from 17/1000 to 14/1000: the estimated 2005 birth cohort is 17.7 million infants while the 1997 birth cohort was 20 million. JE vaccine is administered only during 3 month campaigns each spring to 1 year old children, and in high risk areas to infants as young as 6 months. As many as six doses of the inactivated primary hamster kidney cell-derived vaccine are needed to fully immunize a child with two primary, and ®ve booster, doses for a total national need of roughly 110 million doses annually. However, if one or two doses of live attenuated vaccine was sucient for protection, only 18±36 million doses of the latter vaccine would be needed, re¯ecting a tremendous savings in vaccine and administration costs. Accordingly, domestic vaccine institutes have gradually shifted production from the inactivated to attenuated vaccine with a projected annual capacity of 110 million doses by the end of the century, which is ample for domestic demand. 5.7. Democratic People's Republic of Korea JE was transmitted in an endemic pattern until the beginning of the 1970s, when vaccination and vector control activities brought the disease under control to an extent that almost no cases have been reported in

T.F. Tsai / Vaccine 18 (2000) 1±25

recent years. The Ministry of Public Health maintains an active program of mosquito, pig and goat surveillance from May to October, and human case surveillance through county and provincial levels during the transmission season, from August to October. Surveillance is focused on areas below 800 m elevation including coastal and piedmont areas. Human vaccination is targeted at children between 1 and 15 years of age but, since 1994, a series of natural disasters that led to shortages in vaccine production materials and the destruction of vaccine production and control facilities resulted in a decline in coverage to 53% in 1997. Requests have been made to WHO and other international organizations for sucient doses to immunize 3.5 million people. Vector-control programs have been organized to supplement vaccination. From January to March, e€orts are directed at exterminating overwintering mosquitoes. March±April and September±October are designated ``Hygienic Campaign Months'', when antiepidemic stations organize the elimination of mosquito breeding habitats and overwintering hibernacula respectively. From July to September, adult mosquitoes are driven away by smoke and all families are instructed to use bednets. 5.8. Australia The disease emerged in Australia as recently as 1995 when an outbreak of three cases on Badu island in the Torres Strait islands was shown by mosquito collections and animal and human serosurveys to have resulted from local viral transmission by Cx annulirostris mosquitoes [7]. Nucleotide sequences of viral isolates recovered from the outbreak and from Papua New Guinea, whose southern coast is only a few kilometers from the northernmost of the islands, were nearly identical, indicating Papua New Guinea as the likely source of the introduction. Serosurveys indicated widespread transmission in the Strait and a mass vaccination campaign was initiated. By January 1996, 3440 persons, principally outer island residents, were immunized without incident, with vaccine purchased from Canada in a 3-dose schedule [40]. Continued local transmission on the outer islands in the next 2 years was documented by sentinel animal surveillance but no human cases recurred until 1998, when a case occurred in a Badu Island child unimmunized for religious reasons. Pig infections were documented on six other islands and for the ®rst time, also among sentinels at the northern tip of the Cape York Peninsula. In March 1998, the ®rst human case acquired on the Australia mainland was documented in a ®sherman who was exposed near the mouth of the Mitchell River on the western aspect of the Peninsula [41]. Pigs located nearby seroconverted over an interval of several

9

weeks, suggesting sustained local transmission. Isolates recovered from viremic pigs were similar genetically to strains recovered in 1995 and from Papua New Guinea, mimicking the pattern of introduction 3 years earlier. Animal, mosquito and human surveillance have been organized to monitor the resumption of viral transmission that would indicate the establishment of local enzootic transmission on the mainland. JE vaccine has been incorporated into the routine immunization schedule of infants in the Torres Strait outer islands but no decision has been made on mass or routine vaccination of Cape York Peninsula residents. 5.9. Other countries in South-East Region The SEARO representative delivered reports on behalf of other countries and gave a regional perspective. The 10 countries in the region perceive the relative public health importance of JE di€erently, consequently their approaches to the disease are heterogeneous. Only the DPRK, Thailand, Sri Lanka incorporate JE vaccine into routine EPI schedules while India distributes the vaccine on an ad hoc basis to control epidemics. While JE is recognized to be epidemic in Nepal, resources have been unavailable to purchase vaccine. Indonesia, Myanmar, and Bangladesh have no plans to initiate vaccination and have focused their e€orts on surveillance. JE is not recognized in the Maldives or Bhutan. JE surveillance and vaccination programs in Sri Lanka, Myanmar and Nepal illustrate distinct approaches of three countries in the region. Outbreaks have occurred regularly in the west central region of the Sri Lanka since at least 1985, leading most recently to 306 cases in 1996 and 138 cases in 1997, with a case±fatality ratio of 15% [42]. Mass campaigns fully funded by the national government have been conducted during inter-epidemic intervals in high-risk areas, targetting children 1±10 years of age. Three doses are given for primary immunization followed by boosters every 3±4 years. Coverage was 61% in the seven divisions vaccinated in 1997, using Beijing-1 strain vaccine imported from Seiken. The estimated annual need is 1 million doses. A total of 188 JE cases were reported in a series of outbreaks between 1974 and 1979 in areas of Myanmar bordering Thailand. The outbreaks were seasonal, with a peak between September and November, and a€ected principally children and adults under 20 years. Although the disease was severe with a case fatality ratio of 47%, recurrences have not been recognized and the Ministry of Health has not considered JE to be a public health problem. No vaccination program is planned. The ®rst serologically con®rmed JE cases in Nepal were reported in 1978, followed by cyclical outbreaks

10

T.F. Tsai / Vaccine 18 (2000) 1±25

in the southern Terai from July to November every 2± 3 years until 1982, when the epidemic pattern became less regular [6]. In the largest recorded outbreak, 2336 cases were reported in 1997. Between 1978 and 1997, 14 920 cases with a case±fatality ratio of 24% were reported. The government has been unable to a€ord mass vaccination but preparatory trials are planned in 1999. By one estimate, the birth cohort within areas at risk in the SEA region exceeds 11 million, thus 22 million doses of inactivated vaccine would be needed annually to provide sucient doses for primary immunization, with an additional 45 million doses annually for booster doses (assuming boosters every 4 years). In addition, catch-up immunization would be required for the >225 million children under 15 years in the SEA region. These needs present challenges to the traditional EPI approach to childhood vaccination. Unlike the universal and age-based schedules of other EPI vaccines, JE vaccination potentially could be targeted geographically within high-risk areas and the seasonal occurrence of disease potentially favors a campaign approach to vaccination. But perhaps most importantly, as currently recommended, JE vaccination is recommended at a later age than other EPI vaccines, necessitating additional visits beyond 9±12 months and increased administration costs (see below). In developing countries, national authorities need assistance to prioritize JE among other vaccine-preventable diseases in formulating vaccination policies. In countries where the disease burden of JE is uncertain, improved surveillance is needed to de®ne disease incidence and geographic areas at risk. 6. International vaccines Ð inactivated mouse brainderived vaccine 6.1. Immunogenicity and ecacy of inactivated mouse brain-derived vaccine [36] The basis of the current vaccine potency assay and the choice of strains of the current formulation were reviewed. Experience from intraperitoneal viral challenge experiments in passively immunized weanling mice showed that protection correlated with levels of passively transferred antibody, accordingly, the procedure for potency testing was simpli®ed by merely measuring antibody production in vaccinated mice. In this model, minute antibody levels are protective because passively immunized animals without demonstrable neutralizing antibody titers are still protected to a degree. How closely this mouse model re¯ects human susceptibility and protection is uncertain but, very likely, it is a conservative standard because the viral challenge dose of 103±104 mouse LD50 is con-

siderably greater than the dose introduced by a mosquito bite, estimated to be <100 mouse LD50. Moreover, human experimental studies, in which intradermally-inoculated inactivated viral antigen in a dose equivalent to that introduced by a mosquito, was protective suggested that very small quantities of antigen are immunogenic and can induce protection. Presumably the small quantity of immune complexes taken up by follicular dendritic cells was sucient to stimulate a protective humoral response. Sterilizing immunity may not be needed, since some viral replication with production of antibody against NS1 is found in naturally exposed recipients of inactivated vaccine. In the monkey intracerebral challenge model, an 8 day incubation period elapses before symptom onset, allowing ample time for a memory T-cell response even in previously immunized animals without demonstrable antibodies. In humans, an incubation period of several days to weeks between mosquito bite exposure and neuroinvasion allows low levels of neutralizing antibody to provide adequate defense in the interval before a secondary antibody response is mounted. Furthermore, humans in areas with endemic transmission are frequently boosted naturally by exposure to infected mosquitoes. The number and intervals for booster doses beyond childhood have not been well de®ned while the need for these data has increased, as the age distribution of cases has shifted to adults and with anti-vaccine sentiment towards minimizing booster doses because of vaccine side-e€ects. In developing countries, the required number of booster doses in late childhood is an economic issue. The persistence of neutralizing antibodies after primary immunization with two doses and booster immunization 1 year later was reported in Japanese studies that indicated 90±100% of children retained adequate titers for at least 3 years [36]. A 1996 study found that avarage neutralizing antibodies titers declined tenfold to levels of log 2,89, 3±5 years after primary immunization. In a study carried out in Republic of Korea in 1996, neutralizing antibody in primary school children were analysed by interval since the last booster dose. Antibody prevalences and GMTs at 6, 18, and 30 months were 98% [152/155] (239); 99% [103/104] (188); and 96% [43/45] (134); at 40 months, only 5 of 7 children [71%] were seropositive. Studies of antibody persistence in subjects from endemic areas may be confounded by natural boosting by bites of infected mosquitoes, however, a small study of unexposed US soldiers also found that high neutralizing antibody titers persisted for 3 years after primary immunization with three doses [43]. Additional data are needed to establish the interval for booster immunization after the initial series. The absence of data is re¯ected in the diverse national schedules recommended in various

T.F. Tsai / Vaccine 18 (2000) 1±25

countries, with Republic of Korea recommending, until recently, a total of 12 annual booster doses through age 15 years (Fig. 2). T-cell memory induced by vaccination has been studied in few subjects but, in persons immunized >10 years previously, JE virus speci®c CD4 and T memory cell responses were stimulated by revaccination and lasted up to 7 months, although none were detected in prebooster samples, despite the possibility of prior natural reexposures. Memory T-cells responded chie¯y to JE virus but also to other ¯aviviruses, e.g. West Nile and dengue viruses, with a heterotypic speci®city similar to that induced by live dengue vaccine. T-cell clones were shown to recognize viral E protein but not prM [44]. It is unknown if CD8 cytotoxic T-cells are elicited in vaccinees. Studies describing anamestic antibody responses 3 years after a previous immunization indicate that memory T-cells probably persist for that interval but in most individuals, they are unlikely to persist beyond 10±15 years. T-cell immunity is relatively weaker in infants and in the elderly, and JE cases in the elderly may be explained by a decline in memory cells or a weaker response of existing memory cells. The latter is suggested by the poorer humoral response of elderly persons to booster immunization and in recovered cases, by high levels of viral antibody indicating an anamestic response. How T-cell memory is maintained, how it correlates with protection, and whether memory T-cells can persist without periodic boosts is unknown. The mouse brain-derived vaccine initially was produced from the Nakayama strain, isolated in 1935, but subsequent laboratory studies disclosed antigenic di€erences of ®eld strains assignable to four distinct groups, one including the Nakayama strain, and three others. Although no clinical evidence of a reduced protective ecacy of the Nakayama formulated vaccine has been shown, candidate vaccines produced from a series of other strains were examined in order to address the theoretical concern of antigenic variation.

11

The Beijing-1 strain, isolated in 1948, was exceptional in retaining a high degree of potency through the extensive puri®cation procedures from native infected mouse brain. In addition, the Beijing-1 strain demonstrated a closer antigenic relationship to recent Japanese ®eld isolates than the Nakayama strain, and immunized mice more rapidly produced higher neutralizing antibody titers to newer strains. While lots of vaccine formulated with the Nakayama strain occasionally failed potency testing at the Japanese National Institute of Health, after switching to the Beijing strain in 1989, such failures have become rare. Several human immunogenicity studies have shown a higher response rate (e.g. 98% to Beijing-1 vaccine vs 83±89% to Nakayama vaccine) and higher GMTs (e.g. 286 vs 240, respectively) after primary two-dose immunization. The longevity of immunity after two doses is known to be brief but the recommended third dose 1 year later produces high neutralizing antibody titers in 100% of boosted vaccinees, lasting 3±4 years. Recent genetic comparisons of ®eld strains have resulted in the classi®cation of strains based on nucleotide sequence similarities into four genotypes [45±51]. The relevance of these distinctions to human disease and protection, however, is unproven. Both Nakayama and Beijing-1 viruses are members of the same genotype, and in a ®eld trial, the ecacy of Nakayama vaccine in Thailand, where two other genotypes also are transmitted, was 91%. Moreover, a specially formulated bivalent vaccine containing both Nakayama and Beijing-1 antigens was no more ecacious in this ®eld trial than the monovalent Nakayama vaccine. These observations notwithstanding, concern remains over the degree of protection extended to strains not well neutralized by immune serum from vaccinated mice [52]. The neutralizing antibody titers of mice immunized with commercial Beijing-1 vaccine ranged from only dex 1.37 to 1.65 against several Thai ®eld strains, represented by Th44/92 and Th67/93, while the serum exhibited titers of dex 2.16 and 3.13 against Nakayama and Beijing-1 viruses, respectively. In view of the important protective role of antibody, these distinctions are of concern, and comparative potency testing of vaccines against new or local strains as a regulatory requirement has been discussed. However, as stated above, the unproven relevance of these observations to vaccine ecacy in humans should be underscored. 6.2. Current knowledge of adverse events

Fig. 2. Vaccination schedules for mouse brain-derived inactivated Japanese encephalitis vaccine, by county.

6.2.1. Europe Since 1982, more than 400 000 doses of inactivated JE vaccine have been distributed in Denmark (population 5.2 million), mainly to adult travellers. Routine vaccine-related adverse events (AE) surveillance in Denmark is sensitive, especially for JE vaccine because

12

T.F. Tsai / Vaccine 18 (2000) 1±25

it is distributed only through the State Serum Institute. Between 1982±1995, 350 000 doses were distributed and 101 AEs were registered, including 73 cases with allergic mucocutaneous reactions [53,54]. Allergic reactions and serious reactions requiring hospitalization were reported most frequently between 1989±1992. More than 70% of these reactions were reported after the second or later dose, with a median duration from vaccination to reaction of 2 days (range 0±12 days). Ten of these patients (15%) were hospitalized and medical treatment was required in 67%. Other reactions such as fever, in¯uenza-like symptoms, local reactions, and headache were registered in 28 patients. In addition, three cases of demyelinating disease were reported in 1983, 1992, and 1995. Symptoms in the latter patients began 6 days after revaccination, and 18 days after the ®rst dose, respectively. MRI scans showed changes consistent with acute disseminated encephalomyelitis. A case-control study comparing vaccinees with allergic reactions (principally urticaria and/or angioedema) in 1992±1993 to vaccinees without reported side-e€ects found that a history of other hypersensitivities was a risk factor, with an odds ratio of 5.0. Young age and female gender also were associated with increased risk of vaccine-related hypersensitivity. These observations led to a change in vaccine recommendations to further restrict vaccine use, especially in young persons with an allergic history [54±58]. 6.2.2. Republic of Korea Since 1994, two deaths from acute anaphylaxis and four cases of acute encephalopathy or acute disseminated encephalomyelitis (two fatal) temporally related to JE vaccination were reported. One of the fatal cases was in a 15 year old girl who received her ninth dose of JE vaccine and her third dose of hantaviral vaccine (also made in mouse brain) 4 and 2 weeks, respectively, before the onset of stupor, seizures and acute encephalopathy. In an investigation of the timing of the events, their biological plausibility and the elimination of other etiologies, a causal association with the vaccine could not be ruled out. These cases aroused public concern over the vaccine's safety and parental refusals to accept the vaccine led to a decline in national coverage to 79% in 1994. In view of the undocumented need for annual boosters and to minimize risk of vaccine-related side e€ects, in 1995, the National Immunization Program Advisory Committee temporarily changed the national vaccination schedule to a biannual interval for JE vaccine booster administration. However, this decision was followed by a price increase of the vaccine, creating a burden among some sectors of the public. Governmental authorities recently initiated a re-evaluation of the safety and quality of licensed domestically-produced vaccines and

a general assessment of the national JE vaccination program. 6.2.3. Other regions Reports of JE vaccine-related AEs in Japan and in the United States were reviewed. The two principal safety concerns are hypersensitivity reactions due to gelatin and other allergens (such as mouse serum protein) and neurological reactions. All inactivated mouse brain derived-JE vaccines manufactured in Japan contain gelatin of bovine or swine origin, ranging in concentration from 0.01% to 0.02% w/v; JE vaccine distributed in the US contains 0.05% w/v of swine-derived gelatin. Sakaguchi recently reported two JE vaccine-related syndromes, with clinical manifestations of urticaria and wheezing in vaccinees with anti-gelatin IgE or, hypotension and cyanosis in vaccinees without gelatin reaginic antibodies [59]. The second safety concern is the triggering of acute disseminated encephalomyelitis or other neurological disorders by traces of mouse neural proteins remaining after puri®cation [60± 64]. Passive reporting in Japan in 1966 disclosed neurological side e€ects in 1±2 per million vaccinees and current risk is considered to be lower with the higher purity of contemporary JE vaccines. The Japanese National Adverse Reactions Reporting System (NARRS) speci®es prede®ned criteria for JE vaccine adverse events, including anaphylaxis within 24 h after vaccination, encephalopathy or other central nervous system disorder within 7 days, systemic skin eruption or fever >398C within 2 days, and any other atypical (unexpected) AE. From April 1996 to March 1998, c. 4827 litres of JE vaccine were distributed in Japan; in 1997, the seven manufacturers produced 2500 litres for the domestic market. Vaccine coverage is estimated to be 87.2% and 83.1%, respectively, for the two 0.25 ml primary doses, 66.7% for the ®rst 0.5 ml booster dose, 57.6% for the second booster dose and 30.5% for the third. During this interval, 186 JE vaccine AEs were reported to NARRS with rates for individual manufacturers ranging from 0 to 5.4 cases/100 000 ml. Among the reported cases were 14 cases of anaphylactic shock, 25 anaphylactoid reactions and 13 irreversible neurological disorders, including one death. From Ministry of Health and Welfare data on the number of doses distributed, the calculated rate per million doses of all AEs was 25.7; for anaphylactic shock, 1.9; and for irreversible neurological disorders, 1.8. In the United States 170 000 doses were distributed annually between 1993±1997 to travellers and military personnel, for a total of 717000 doses. During this interval, 141 JE vaccine AEs Ð 80 involving JE vaccine administered alone, were reported to the national Vaccine Adverse Events Reporting System (VAERS). A higher proportion of reports were in female (53%)

T.F. Tsai / Vaccine 18 (2000) 1±25

vaccinees than in males (43%) and in adults 20±24 years old than in other age groups. The latter may re¯ect the age distribution of travellers and military personnel but because the underlying age and sex composition of vaccinees is unknown, rates cannot be calculated. A fatal illness with brain oedema and pneumonia was reported in a 6 year old child whose symptoms began 13 days after immunization. Six other cases were hospitalized, ®ve in males. Among the 80 reactions following administration of JE vaccine alone, 31 were characterized by urticaria/pruritus and/or angioedema, for an estimated rate of 1/22 500 doses; there were no cases of anaphylactic shock. One-third of these reactions occurred on the vaccination day and the remainder, between 1±7 days after vaccination. Adverse events surveillance systems in Japan and the USA are limited by their passive design, although it is likely that they would detect serious and life-threatening reactions. Comparisons of JE vaccine related AEs in the countries also is confounded by di€erences in vaccine formulation, including vaccine strain, and content of gelatin and other additives. Although hypersensitivity events in Japan were rare, well documented reactions to gelatin indicate a need for further studies and, potentially, e€orts to reduce or eliminate gelatin from the vaccine. 7. National vaccines Ð live-attenuated SA14-14-2 vaccine 7.1. History of development and viral strain characteristics Although routine childhood immunization with inactivated JE vaccine produced in primary hamster

Fig. 3. Derivation of the live-attenuated SA14-14-2 strain of Japanese encephalitis virus (Yu Yong Xin Ð see reference [16]).

13

kidney (PHK) cells had greatly reduced JE incidence in China, the vaccine's limited ecacy and need for numerous doses stimulated development of an improved vaccine [16]. The eventual derivation of the SA14-14-2 strain through an empirical process of serial passage, principally in PHK cells, struck a ®ne balance between safety through stable neuroattenuation, and immunogenicity, with sucient viral replication to stimulate immunity. Properties of intermediate viral clones from which the vaccine strain eventually was derived illustrate the inherent subtleties of JE viral attenuation (Fig. 3). The early 12-1-7 clone exhibited a low degree of pathogenicity, however, it was unstable and reverted to neurovirulence after one mouse brain passage or several PHK cell passages. The 9-7 strain derived from it was neuroattenuated in mice and monkeys and did not revert with mouse or PHK cell passage, but it produced seroconversions in only 20% of vaccinated children. To increase immunogenicity, the strain was passaged orally in hamsters and plaque puri®ed to derive the 5-3 strain. This clone retained neuroattenuation characteristics but its immunogenicity in children improved only marginally, to 65%. Further passages in mice and plaque puri®cations led to the 14-14-2 clone which was stably neuroattenuated yet was immunogenic in 85±100% of vaccinated children. Vaccine produced in PHK cells was highly ecacious and has been distributed since 1989 to more than 100 million Chinese children without evidence of signi®cant adverse events. This success attracted international attention and attempts to adapt the strain to a cell culture substrate more acceptable internationally. However, as shown by previous experience, this process may lead to changes in attenuation. An initial attempt to adapt the strain to primary dog kidney cells found that only six additional passages led to further attenuation and a reduction of antibody responses to only 40% in vaccinated children. The SA14-14-2 strain grows to a titer of >107 in PHK cells, and produces CPE and small plaques under overlay. In contrast to its parent, the strain is avirulent by i.c. or i.p. routes in weanling mice, Syrian hamsters, and in cytoxan immunosuppressed mice. The virus is virulent for nu/nu mice only by i.c. inoculation. Monkeys inoculated by a combination of intrathalamic and intraspinal routes develop asymptomatic infections and on neuropathological examination, exhibit a minor degree of in¯ammatory reaction only along the needle track, with minimal neuronal infection or neuronal death. Small plaque morphology and neuroattenuation in mice is retained through at least twenty-three further PHK cell passages, using conditions of infection (e.g. m.o.i. and incubation temperature) identical to those employed in production. In guinea pigs, a single dose of SA14-14-2 virus elicits immunity that signi®cantly reduces viremia levels

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T.F. Tsai / Vaccine 18 (2000) 1±25

produced by viral challenge. Immunity could be passively transferred by either spleen cells or serum. Evidence that the live vaccine elicited a stronger cellular immune response than inactivated vaccine was seen in challenge experiments comparing mice immunized with the respective vaccines. Despite equal titers of circulating neutralizing antibody, survival after i.c. challenge was signi®cantly higher in animals previously immunized with live vaccine. Cytoxan immunosuppresion of SA14-14-2 vaccinated mice did not alter their resistance to lethal viral challenge in contrast to mice vaccinated with inactivated vaccine, in whom survival was reduced by 90% after immunosuppresion. In vaccination/challenge studies, survival was signi®cantly greater in mice vaccinated with one dose of live vaccine compared with two doses of inactivated mouse brain or PHK cell derived vaccines, and challenge with the P3, Nakayama or 12 ®eld strains isolated in China. However, experiments to demonstrate neutralization of, or protection, against strains representing other genotypes have not been undertaken. Although the growth of SA14-14-2 virus itself in Cx tritaeniorhychus never has been evaluated, the attenuated 2-8 clone, derived from the same pedigree, was not transmitted in experimental studies [65]. A chimeric virus constructed from SA14-14-2 and yellow fever 17D viruses (ChimeriVax, see below), however, was not infectious for Cx tritaeniorhychus by intrathoracic inoculation although, remarkably, the virus was infectious for Ae aegypti by this route, suggesting a more important role for YF virus derived nonstructural proteins than JE viral derived structural proteins for mosquito infectivity in this experimental system. SA-14-14-2 viremia levels have not been studied in vaccinees and it is unknown whether circulating infectivity titers are suciently high or are maintained for a sucient interval to pose a practical risk of mosquitoborne transmission after vaccination. Attenuated JE swine vaccine used to prevent pig stillbirths in Japan was shown not to grow in mosquitoes.

expected if they were associated with a speci®c incubation period. Temperature elevations were limited to a single day in most cases [66]. In uncontrolled observations of 1946 children (1±6 years) vaccinated with the Wuhan Institute-produced vaccine local reactions, occurring in 6.2% of vaccinees overall, were more common in older children who had had previous doses of inactivated JE vaccine. A controlled study, conducted under the auspices of the Rockefeller Foundation in the city of Chengdu in southwestern China, compared hospitalizations and speci®c illnesses and symptoms in block randomized cohorts of 13 266 vaccinated children and 12 951 children in whom routine JE vaccination was deferred for 1 month [2]. The 1±2 year old children were followed prospectively for 1 month after this initial visit and on their return, parents were questioned about hospitalizations and the occurrence of speci®c illnesses in the intervening month, including encephalitis, meningitis, and the new onset of seizures. No subject had a central nervous system illness, and the proportion of vaccinated and unvaccinated children with the new onset of seizures, hospitalizations, fever lasting >3 days, and various other illnesses such as diarrhoea, respiratory infections and reactions consistent with anaphylaxis were similar. A subset of 266 vaccinated children was examined prospectively for post-vaccination side e€ects 1, 2, 3 and 7 days after the vaccination visit. Low rates of various minor local and systemic symptoms were observed and fever occurred in 5% of vaccinees. This study provides convincing evidence of the short-term safety of SA14-14-2 vaccine during the ®rst 30 days after immunization. Concern that a live vaccine derived from an encephalitogenic virus might lead to vaccine-associated encephalitis could not be addressed satisfactorily even with a study of 26 000 children. From the observation of no cases in the month after immunization, an interval expected to encompass the incubation period of

7.2. Safety

Table 5 Japanese encephalitis cases in children immunized with SA14-14-2 vaccine in ®eld trialsa

Vaccine safety has been evaluated in several smallscale studies and in two large-scale post-marketing studies. Studies of 588 512 children 1±15 years vaccinated with the Chengdu Institute-manufactured vaccine and of 60 000 children given Wuhan Institute-vaccine reported no temporally associated encephalitis cases and, among other vaccine-associated adverse events, fever was reported in fewer than 1/500, with lower rates for rash and other systemic symptoms [16]. Daily examination for fever among 867 vaccinated children disclosed low rates of fever (e388C) with onsets distributed evenly over the 21 day observation period, without clustering, as might have been

Study

Cases/vaccinated

Rate/100/000b

Guizhou, 1988 Jiang-Xi Yunnan, 1991 Anhui, 1992

1/86,132 2/64,027 2/29,639 2/145,758

1.16 3.12 6.75 1.37

a Rates of JE among vaccinated subjects could re¯ect primary vaccine failure; errors in diagnosis; and cases due to the vaccine itself. The incidence rates were combined into a weighted estimate, as indicated by Cochran's test of homogeneity. Weighted combined incidence estimate=1.59/100,000 (95%CI, 0.22±2.95). Estimate=awipÃi/ awi, where wi=ni/pÃi(1-pÃi ) and SE (combined estimate=1/awi. b Cochran's test of homogeneity not signi®cant, p = 0.61.

T.F. Tsai / Vaccine 18 (2000) 1±25

infectious encephalitis, the upper 95% con®dence interval indicates that encephalitis is unlikely to complicate SA14-14-2 vaccination at a rate higher than 2.3/10 000. Encephalitis rates in children vaccinated with SA1414-2 in ecacy trials provide an additional source of data. Although the studies were conducted in di€erent geographic areas of China and in di€erent years, the observations were combinable in a weighted average because they were reasonably homogeneous, as shown by Cochran's test (Table 5). In studies that were conducted over 2 or more years, only observations in the ®rst year were analysed to focus on risk from the ®rst vaccine dose. The weighted average encephalitis risk in the four studies was 1.59/100 000 with 95% con®dence intervals of 0.22±2.95. These cases potentially represent naturally acquired JE associated with primary vaccine failure; encephalitis due to other causes, misclassi®ed as JE; or potentially, cases of vaccine-associated encephalitis. The onset of the cases several months after vaccination strongly suggests that they were not vaccine-associated. 7.3. Immunogenicity Immunogenicity in children follows a dose±response gradient with antibody responses in >92% of vaccinees receiving 106.0 viral pfu from vaccines produced after licensure in 1988 [16]. Antibody responses are seen in all subjects given a second dose. Among responders to primary immmunization, all retained measurable neutralizing antibodies for 3 years. Response rates and antibody persistence were signi®cantly higher than in a comparison group of children receiving inactivated PHK cell derived vaccine. Although a single dose is highly immunogenic, national policy recommends two doses to insure a high degree of protection and to avoid the unmeasured interference to primary immune responses caused by maternal antibodies. Vaccine is o€ered only during annual springtime campaigns, which is problematic, because children who miss that annual opportunity must wait a full year before they can avail themselves of the vaccine. Inclusion of JE vaccine into an age-based primary immunization schedule with other pediatric vaccines undoubtedly would improve coverage. A study of 331 children found that 92% developed neutralizing antibodies after the ®rst dose and 99% seroconverted after receiving two doses either in a short one month schedule or longer 2.5 month interval, although GMT s were higher with the long schedule [67]. The study was limited by the age of subjects who were middle school aged children but, in a Korean study of 68 children with a mean age of 3 years who were vaccinated with a single 106 pfu dose, 65 (96%) seroconverted with a geometric mean neutralizing antibody titer of 184 [68].

15

With high levels of compliance in infant vaccination programs, these data indicate that booster vaccination at school entry lacks a strong rationale. 7.4. Ecacy Protective ecacy was evaluated in four ®eld trials of over 300 000 children. The ecacy of one dose was >95% in every trial and, in one location where the vaccinated 1±10 year old children were followed through ®ve transmission seasons, >98% protective ecacy was maintained through this interval [16]. The above trials were conducted in such a way that may have introduced biases in risk of exposure in vaccinated and unvaccinated subjects. However, when a fully e€ective vaccine is licensed and in actual use, it is not ethically permissible to restudy vaccine ecacy using a placebo controlled ®eld trial. A reliable, sensitive and accurate alternative that takes advantage of inadvertent vaccine failures, usually due to missed immunizations, is a case-control study in which the vaccination histories of cases and matched controls are compared. A study conducted in Sichuan province in which all village children of the same age as serologically con®rmed JE cases were selected as controls, found an e€ectiveness of 98% with two doses and 80% after one dose (95% CI 44±93%) [69]. The broad con®dence interval of the latter estimate re¯ects the small number of subjects in that category. The results of this study support the previously cited ecacy studies while extending those observations, by measuring e€ectiveness of JE vaccine distributed under the actual ®eld conditions of vaccine delivery, storage and administration. 7.5. Production and regulatory issues International expectations of safety and ecacy of live attenuated virus vaccines were discussed. Prevention of transmission of adventitious infections from the virus seed, the cell substrate, and the serum or trypsin used in the manufacturing process, is a general concern with all live virus vaccines. For the SA14-14-2 vaccine, the lack of precedence for a PHK cell substrate in production of an internationally accepted vaccine was a particular concern. Current controls assure the absence of a broad range of known rodent viruses but these need to be constantly reviewed and state-ofart developments introduced, as appropriate. Validation issues with respect to the neutralization of SA14-14-2 virus in assays to detect other infectious agents, and the choice of indicator cell cultures should be presented. In addition, the principle of reducing the risk of adventitious agents entering the manufacturing process should be emphasized by using healthy animals, preferably from a closed SPF colony that is

16

T.F. Tsai / Vaccine 18 (2000) 1±25

monitored regularly, as a source material for preparation of PHK cells. In common with all live virus vaccines, steps to exclude potential contaminants of serum and trypsin employed in manufacturing, including speci®c bovine and porcine viruses and transmissible spongiform encephalopathy agents, would be expected. The demonstration of consistency of vaccine production in PHK cell culture can be approached by providing information on the consistency of primary cell production and virus production. The former includes aspects of cell growth such as morphological characteristics and days to plating con¯uency. Consistency of viral titers are demonstrated by precise and reproducible titration procedures and, possibly, by monitoring genotypic and phenotypic markers. It is desirable to demonstrate the consistency measures under conditions of scale-up and large-scale production. Regulatory perspectives on the extent of testing required to characterize a cell substrate have changed in the last 10 years. Recently published WHO requirements provide guidance on international expectations for the quality of cell substrates used for production of vaccines. The creation of a well characterized master PHK cell bank at the secondary or tertiary level was discussed as a potential way to improve lot to lot consistency and to simplify quality control. Although experience of the Chengdu Institute showed that plating eciency declined by 40% after primary cells were frozen and thawed, this does not exclude possibility of future improvements that would make this approach practicable. Attempts to adapt the strain to other cell cultures would be expected to result in fundamental changes in biological characteristics, and attempts using MRC-5, primary chick and duck embryo cells, and PDK cells were unsuccessful (see above). Early research indicates the strain can be adapted to Vero cells with the preservation of neuroattenuation in mice but it is unclear how equivalence with the PHK produced SA14-14-2 vaccine should be proven (see below). WHO guidelines that address many of the above issues concerning the manufacture and control of live attenuated JE vaccine will be an important step towards the future internationalization of vaccines beyond the SA14-14-2 vaccine [70]. The group discussed several scenarios to approach internationalization of a locally produced vaccine, including international collaboration to assure the production and regulatory process, contract production in another country with release by the manufacturer and regulatory oversight in the original country, or licensing technology to a third country that would take responsibility for the production and regulatory process. Each approach has advantages and disadvantages, such as the need for bridging trials when the production site is changed and expensive facility invest-

ment. All would depend on the assurance of strong regulatory infrastructure in the country of manufacture. 8. Alternative JE vaccines in near-term development [71] 8.1. Vero cell-derived inactivated vaccines Applying its extensive experience in producing inactivated rabies and polio vaccines in Vero cells, Pasteur±Merieux±Connaught Inc. (PMC) has manufactured a formalin inactivated JE vaccine in ATCC CCL81 Vero cells, using the P-3 vaccine strain. The strain, obtained at its 88th passage level from the Chinese National Vaccine and Serum Institute, is similar to the Beijing-1 strain (known as P-1 in China). Cells are propagated with gamma-radiated calf serum from New Zealand, the USA or Canada but they are fed with protein-free 483 medium. Inactivated viral antigen is concentrated and puri®ed by dia®ltration and chromatographic steps so that the ®nished vaccine contains no animal proteins and <100 pcg/dose of residual DNA. Inactivation is controlled by inoculating susceptible cell cultures and mice. Both lyophylized and liquid preparations will be developed. Potency of GMP-produced lots in mice is equivalent to that of the reference mouse brain-derived standard and Phase I clinical trials in young adults have been initiated in Sweden. Phase II dose-ranging studies in adults and children and large-scale safety and immunogencity trials in Asian and western countries are planned by 2000, but the manufacturer intends to submit serological correlates of protection in lieu of ecacy data from a ®eld trial. Large scale GMP manufacturing in 2000± 4500 litre containers using microcarrier technology is planned to meet global demand for a childhood and adult vaccine in Asia, and for travellers and military elsewhere. Issues that will be addressed in clinical development include immune responses in ¯avivirus naõÈ ve and previously infected persons, booster interval, alterations of viral epitopes by formalin inactivation and immune response to various viral topotypes. Vaccine produced by a similar process in Beijing has entered a Phase II trial. Initial studies disclosed 95% seroconversion with a neutalizing GMT of 30 in subjects given two doses. Chinese manufacturers estimate that domestically produced Vero cell-derived vaccine will cost $1/dose. The National Institute of Preventive Medicine, Taiwan has taken a parallel approach and produced pilot lots of inactivated vaccine produced from Vero cell microcarrier cultures and adsorbed to aluminum hydroxide. Preclinical studies in mice indicate equivalent potency to the licensed mouse brain vaccine and non-human primate studies have shown the immunogenicity of a single dose. Biken also has completed pre-clinical evaluation of

T.F. Tsai / Vaccine 18 (2000) 1±25

an inactivated Vero cell derived-vaccine, produced from ATCC Vero cells, established at passage levels 126 for the master cell bank and 132 for the working cell bank. For production purposes, the cells are expanded by six passages and grown on Cytodex 1 carriers. Master and working seeds of Beijing-1 virus prepared in Vero cells were shown by nucleotide sequencing and phenotypically to be identical to virus obtained after ®ve further cell culture passages. Infected cell culture ¯uid harvested 4 days after growth at 378C was concentrated by ultra-®ltration and inactivated with formalin (1:1500 v/v). The inactivated preparation was further concentrated twice by sucrose density gradient centrifugation, resulting in a viral protein content of 7.8 mg/ml, which is similar to the 7.5 mg/ ml content of commercial mouse brain-derived vaccine. Residual calf serum protein and residual Vero cell DNA were less than the WHO requirement. Electron microscopic examination of the candidate and commercial mouse brain vaccines disclosed similar concentrations of whole virions with a minor component of slow-sedimenting hemagglutinins. Mice immunized with the trial vaccine were protected against i.p. or i.v. challenge with a lethal dose of Beijing-1 virus. Neutralizing antibody levels in immunized animals were two to three-fold higher than titers in animals immunized with corresponding amounts of mouse-brain derivedvaccine. Scaled-up production in a 50 litre fermenter is underway and GMP production of a pilot lot for clinical testing is planned. A second generation inactivated JE vaccine, adapting the SA14-14-2 PDK strain to Vero cells, was developed in the Republic of Korea. Selection of an attenuated virus for the vaccine strain provides an extra margin of safety in production and for vaccinees, and because the attenuated virus produces less CPE, puri®cation is simpli®ed. Each dose contains <5 ng of Vero cell protein and <10 pcg of DNA. The ®nal product contains no gelatin or bovine-derived protein, 0.1% thimerosol as a preservative, and will be adsorbed to aluminum hydroxide. A master seed was established at the fourth Vero cell passage level and working and production seeds, at the 5th and 6th passage levels, respectively. Attenuation after Vero cell passage was veri®ed by comparing pfu/ LD50 ratios in mice inoculated with the puri®ed inactivated vaccine (PIV), wild-type SA14, and SA14-14-2 PHK strains. PIV was attenuated with a pfu/LD50 of 106. A cGMP lot of the PIV vaccine is undergoing preclinical studies prior to the initiation of Phase I human testing. Mice immunized with bulk lot PIV vaccine developed tenfold higher neutralizing antibody titers than animals immunized with similar quantities (on a weight basis) of commercial mouse brain-derived vaccine. Vaccine stability was shown by the equivalence of antibody titers of mice immunized with bulk lot vaccine that had been stored for 8 months or with the

17

commercial vaccine. The bulk lot PIV protected mice against lethal viral challenge at a lower dose (ED50 0.9 ng) than the commercial vaccine (ED50, 1.5 ng). Groups of ®ve human volunteers, screened for absence of JE and other ¯aviviral antibodies, will be vaccinated with doses of 2.5, 1.0, 0.2 and 0.04 mg of JE PIV adsorbed to aluminum hydroxide. As a control, commercial inactivated mouse brain derived JE vaccine (JE-VAX, Biken-PMC) will be administered as recommended at 0, 1 and 2 months. JE PIV will be administered at 0, 1 and 6 months and neutralizing antibodies measured from blood drawn at frequent intervals after vaccination, up to 7 months. In addition to establishing safety of the vaccine, the dose range study should allow estimation of a median e€ective immunizing dose for the JE PIV. 8.2. Genetically engineered vaccine Chambers et al. developed a chimeric attenuated JE 1 vaccine (ChimeriVax OraVax) in which the prM and envelope protein genes of attenuated SA14-14-2 JE virus replace corresponding sequences in a 17D yellow fever (YF) virus cDNA clone [72]. The approach harnesses the replicative apparatus of the attenuated 17D virus to vector the principal structural proteins of the attenuated JE virus. Chimeric DNA is transcribed to RNA and transfection yields a virus that is more neuroattenuated in monkeys than the licensed 17D vaccine. Monkeys infected by i.c. inoculation exhibit no illness and are viremic for three days. Using the histopathological scoring system standardized for YF-17D neurovirulence testing, discriminatory and target areas in chimeric virus inoculated animals were scored lower than 17D vaccine inoculated animals. YF/SA14-14-2 chimeric virus-infected animals produced JE neutralizing antibody titers >6400. Monkeys immunized with the chimeric vaccine containing 4.3 or 5.3 dex pfu developed low levels of viremia (<2 dex pfu) for several days and antibody responses peaking to titers of 2560 at 30 days. Intracerebral challenge with wild type JE virus 60 days later produced 1 death in six animals and no detectable viremias and an anamnestic antibody response whereas 6 of 6 unimmunized controls died. The chimeric virus is YF-17D virus-like in the patterns of viremia produced after infection but neurovirulence is dictated by its JE sequence. The expectation is that a single dose of the chimeric virus will safely provide life-long immunity to JE, paralleling those attributes of YF-17D vaccine. 9. Expanded Japanese encephalitis immunization through national immunization program National programs to control JE in Thailand were

18

T.F. Tsai / Vaccine 18 (2000) 1±25

®rst organized in 1973 and followed a succession of approaches, beginning with vector control and health education, to the eventual introduction of routine JE vaccination into the national immunization program and catch-up immunization in 28 provinces of the country. The Thai experience of staged provincial vaccination by incidence rate is a model for other countries in the region contemplating routine JE vaccination. JE has been recognized as a signi®cant cause of childhood morbidity in Thailand, particularly in north and northeastern provinces, since the early 1970s, when annual outbreaks produced up to 2400 cases and 400 deaths. A preliminary vaccine immunogenicity and safety study had been conducted in Chiangmai as early as 1973, while JE control in the north was initiated through the addition of vector control and health education programs emphasizing personal protection, through an existing dengue and vector control program. These e€orts had little impact on disease incidence and they were reduced while a vaccine ecacy trial was conducted in Kamphangphet. That trial showed a protective ecacy of 91%, and its success was followed by the introduction of childhood vaccination into local programs, and by pilot studies assessing the feasibility of vaccine delivery in the EPI with measles or with DT, at school entry [73,74]. The discouraging experience of vector control and the positive outcome of the vaccine ecacy trial motivated the National Advisory Committee on Immunization to expand existing local vaccine programs and to add JE vaccine to the EPI schedule, with administration of two doses at 18 months and a booster at 24±36 months. The policy evolved with the stepwise extension of JE vaccination from eight provinces in 1990, to 17 provinces in 1991, and 21 provinces in 1994. Currently, the vaccine is administered in 34 provinces with a national coverage rate for two doses of 79% in a 1996 survey. Parallel catch-up school-based immunization with two doses given to ®rst graders was launched in 10 provinces in 1994, and in 28 provinces in 1997. The catch-up program was discontinued in 1998, as school entrants now have been immunized as infants. Since the national vaccine program was launched, JE incidence has declined to approx. 500 cases a year with 50 deaths. Public response to JE vaccination has been enthusiastic in northern provinces where the disease is endemic, as evidenced by the willingness of parents to pay for the vaccine through private channels. In locations with lower transmission rates, public response has not been evaluated, although judging from media coverage, introduction of the vaccine seems to have been welcomed. A critical factor in the decision to provide routine JE immunization was the availability of vaccine produced locally (by the Thai GPO) at reduced cost;

albeit as a state enterprise, that cost re¯ects a public subsidy. Currently, half of the vaccine used in national programs is produced locally and the remainder is imported. The decision to place JE control under the EPI was also important to maintain budgetary security and continuity because under dengue and malaria control programs, the vaccination program had to compete for resources and personnel and was given a lower Ð and, often uncertain priority. The introduction of JE vaccine has added a burden of three extra visits to the EPI schedule at 18 and 24±36 months and an attendant need for a cold chain for those visits. Administration at an earlier age, with DTP and, or hepatitis B vaccine would be preferable. A 1984 study in Chiangmai showed no unfavourable interactions in school children given JE and DT vaccines concurrently and, other studies disclosed no interference after simultaneous administration with OPV, measles, and BCG. With the near term introduction of numerous vaccines and medications (e.g. hepatitis B, vitamin A), their interactions with JE vaccine should be studied to maximize ¯exibility in the design of local schedules. Additional studies of vaccine safety and immune response in HIV infected infants also are needed. 10. Prospects for increasing vaccine supply 10.1. Inactivated vaccine Current regional production of inactivated JE vaccine from infected mouse brain is in the range of 12 million doses annually and at maximum capacity, c. 30 million doses could be produced. Since China maintains an independent capacity for JE vaccine production, the estimated maximum capacity probably is sucient to meet regional needs. However, vaccine cost and side-e€ects are issues that could mitigate against scaled-up production and reliance on this vaccine for expanded JE vaccination. Biken, the largest Japanese manufacturer, currently produces 5±6 million doses annually, with a FOB price of $1.50±3.50 per dose. The per dose price can be reduced by providing liquid instead of lyophylized vaccine, with multi-dose vials, and with bulk vaccine provided to other countries for local ®lling. Scaled-up production would not necessarily lead to a reduction in vaccine cost because the manufacturing process from mouse brains does not lend itself to economies of scale. In addition to the actual costs of vaccine production, other costs associated with the exportation of JE vaccine include the need for a cold chain, loss of potency of frozen liquid vaccine, the costs of diluent to reconstitute lyophylized vaccine, quality of packaging materials, transportation costs and even the costs of the package insert. An unresolved issue is the need for

T.F. Tsai / Vaccine 18 (2000) 1±25

potency testing against local strains or even localityspeci®c formulations. A second objection to the mouse brain derived vaccine has been risk for AEs, arising from mouse or neural antigens remaining after puri®cation, or to additives, such as gelatin. Since 1965, considerable e€orts have been made to purify the mouse brain vaccine and a ®nal product of 96±99% purity has been achieved. Proteins of neural origin have been a speci®c concern and early studies proved that 1 mg was the minimal quantity of myelin basic protein (MBP) (with complete Freund's adjuvant) needed to produce allergic encephalomyelitis in guinea pigs, while 0.05 mg was insucient to induce the disease. The ®nished vaccine now contains <2 ng/ml of MBP. In clinical trials of other tissue-derived products, prion contamination has been a public concern, and with increased awareness, risk of transmissible spongiform encephalopathy (TSE) from mouse brain may be raised as an issue. Several factors mitigate against such a risk, including the absence of a naturally occurring TSE in mice, the young age of mice used in vaccine production, and the species barrier. On the other hand, mice can be infected experimentally with these agents and the system by which mice are supplied to the manufacturer, from multiple small suppliers, introduces a potential for contamination. Furthermore, formalin used to inactivate JE virus stabilizes prions. In regards to vaccine-related hypersensitivity, allergy to mouse proteins is not uncommon among animal caretakers and, even among the general population, an age-related increase in antibodies to mouse IgA has been shown. Although hypersensitivity to gelatin apparently underlies some cases, no attempts have been made to rule out sensitivity to murine proteins in others. Public objection to the use of animals in vaccine production and testing, and to animal product vaccine constituents are a growing trend that will in¯uence manufacturers in their decisions to expand capacity. 10.2. Live attenuated vaccine Annual production of all JE vaccine from the six Chinese vaccine institutes currently stands at 100 million doses, of which 35±40 million doses are SA1414-2 vaccine manufactured in Wuhan and Chengdu. In principle, this output is sucient to immunize the entire birth cohort of China. However, vaccine coverage is estimated to be in the range of 50±75% and some provincial public health authorities cite a shortage. A contributing factor is the failure of local administrators to comply with scienti®cally validated immunization schedules. For example, despite the promulgation of a nationally recommended vaccination schedule of two total doses (given at 12 and 24 months), administration of SA14-14-2 vaccine varies

19

by province, with Szechuan province recommending an additional dose at seven years. Vaccine supply e€ectively would be increased by one third if local authorities adhered to the proven two-dose schedule. The current annual production of SA14-14-2 vaccine at the Chengdu facility is 20±30 lots (100±200 bulks) of 1 million doses each. Lot size is limited by the availability of hamsters and personnel to harvest their kidneys. Cells from one 10±12 day old hamster yield 300 doses Ð accordingly, each lot (bulk) requires 3300 (500) hamsters. Requirements for increased lyophilization capacity is a critical limiting factor. With renovations of the Wuhan plant to meet GMP standards and the completion of a new GMP facility in Langzhou, total SA14-14-2 vaccine production in the range of 110 million doses is expected as early as 1999. Con®rmation that a single dose of SA14-14-2 vaccine was ecacious would signi®cantly reduce total vaccination cost and the required vaccine supply (see above). Live SA14-14-2 vaccine also could be of value in reducing the number of booster doses currently required in children previously immunized with inactivated vaccine. In the Republic of Korea study cited previously, 10 children who had been immunized with two to three doses of inactivated vaccine but who had no measurable neutralizing antibodies were boosted with a single dose of SA14-14-2 vaccine. All developed anamnestic neutralizing antibody responses with a GMT of 3778. Although more data are required, these observations suggest the potential utility and cost savings of live vaccine boosters to maintain immunity in populations previously immunized with inactivated vaccine. The future possibility of SA14-14-2 vaccine production in a GMP facility and under GMP principles in a country other than China could make the vaccine available internationally. Boran Pharmaceuticals holds an exclusive agreement with the Chinese government to produce and distribute the vaccine internationally and has developed plans for production outside of China. The company described the quali®cation of a new master seed and more inclusive lot release and quality control testing, including state of the art retrovirus testing. However, the equivalence of vaccine produced in a new location, possibly from hamsters of a di€erent origin and under di€erent manufacturing conditions, is an issue that might be raised by regulatory authorities where production is planned. 10.3. Improved surveillance Improved surveillance is needed to assess disease burden in countries where it is poorly characterized. The Thai experience was reviewed as a model. At the village level, clinical cases with fever and alterations in

20

T.F. Tsai / Vaccine 18 (2000) 1±25

consciousness are referred for lumbar puncture at a district or provincial hospital. Patients with a pre-dominantly lymphocytic pleocytosis are considered probable cases of viral encephalitis and CSF and serum are referred for IgM determinations. Despite active e€orts to solicit diagnostic samples, however, adequate clinical specimens are submitted for laboratory con®rmation in less than one-third of cases and ocial noti®cations are based on clinically diagnosed cases. Reports from provincial health ocers are used locally to improve disease control and are forwarded to the Epidemiology Division in the Ministry of Health and National Institute of Health for con®rmation. Between 1968 and 1977, reported JE morbidity and mortality both were high. With increased reporting between 1978 and 1987, case-fatality rates declined as more (and milder) cases were recognized and since 1988, morbidity has declined with the implementation of vaccination. Reported incidence rates in the prevaccination era were as high as 8.5/100 000 in some northern provinces, but with the introduction of routine vaccination to those areas, the highest rates, c. 2/ 100 000, now are in southern provinces. To measure the impact of vaccination on reported incidence, e€orts should be made to obtain a laboratory diagnosis in a greater proportion of cases. Development of simple diagnostic kits that can be used in the ®eld would increase the speci®city of reporting and provide a truer picture of disease incidence and vaccine e€ectiveness. 11. Funding perspectives JE vaccine production in China is sucient to satisfy vaccine needs of the entire region if a single dose of the live vaccine was shown to be e€ective. Data presented above suggest that this may be the case and additional data to con®rm the e€ectiveness of single-dose immunization could be accumulated relatively simply in a case-control study (as was done in Szechuan province) by expanding the number of subjects investigated. JE vaccine coverage in the region can be expanded without additional vaccine production by eliminating unnecessary booster immunizations and by redirecting them where they are needed. Millions of doses are wasted annually in these misguided e€orts. Additional studies are needed to de®ne minimum e€ective schedules and doses. To aid the development and evaluation of higher quality vaccines, it is in the interest of all interested parties to support focused research on surrogate measures of protection, since placebo controlled trials will not be acceptable. Reasoning from evidence of clinical protection in humans, the gold standard must

refer to two doses of live vaccine or multiple doses of killed vaccine. The burden of supporting additional research and evaluation should be shared by manufacturers, the Children's Vaccine Initiative, World Bank and other entities with an interest in preventing the disease. 12. Summary and recommendations 12.1. Consensus statement 1. Virtually all persons residing in the WHO SouthEast Asia and Western Paci®c Regions are at risk from Japanese Encephalitis. 2. JE disease burden estimates in the region vary in their completeness. There are three principal reasons for reporting an absence or low numbers of JE cases Ð little or no disease, incomplete surveillance, or successful vaccination in progress. Incomplete case detection is a leading explanation for the apparent absence of disease in countries not currently undertaking vaccination. 3. Human vaccination is the only e€ective long-term control measure against JE. All at-risk residents should receive a safe and ecacious vaccine as part of their national immunization programme. 4. Approximately 3.2 billion people live in these countries, with an annual birth cohort of c. 70 million. Current vaccine production, from whatever source, is unable to meet the requirements to vaccinate this population with the routine and catch-up vaccinations that would be needed to bring the disease under control. 5. Only mouse brain-derived JE vaccines currently are licensed for international use. There is a shortfall between global requirements and current maximum potential production of 30 million doses per year (equivalent to immunizing c. 10 million individuals). 6. The large-scale deployment of a live attenuated vaccine in China has resulted in the successful control of the disease in that country. Accordingly, every e€ort should be expended to ensure that the production and regulation of this vaccine conforms to international standards. 7. Although presently unavailable, promising second generation JE candidate vaccines are under development, but marketing approval is unlikely in less than four years.

12.2. Recommendations for countries in a€ected areas 1. Countries should strengthen surveillance of JE to better assess the burden of disease, including the

T.F. Tsai / Vaccine 18 (2000) 1±25

number of cases and deaths, psychiatric and other long-term sequelae and their economic implications. 2. Countries should adopt JE vaccine as part of their national immunization schedules and monitor its impact. 3. Many countries where JE is a problem also are experiencing ®nancial diculties. In view of the e€ectiveness of the vaccine and the ongoing burden of disease, countries are urged to maintain, as a minimum, their current level of administration of JE vaccine. 4. Countries should reduce JE vaccine doses in the national immunization programme to the lowest prudent number.

12.3. Recommendations for all countries 1. Countries should harmonize registration requirements and indications for use of JE vaccines, ensuring the requirements are based on scienti®c evidence. 2. Through continuous training and consultation, countries should increase their Awareness of Good Manufacturing Practice (GMP) to enable them to produce vaccines that meet international standards.

12.4. Recommendations for WHO/CVI 1. Strengthen national capabilities for disease burden assessment. 2. Promote development of a simple laboratory diagnostic test for JE. 3. Establish production and control guidelines for live JE vaccines and international standards for JE antibodies and vaccines. 4. Develop criteria for assessing JE vaccine ecacy and guidelines that de®ne optimal vaccine presentations and administration schedules. 5. Continue supporting National Regulatory Agencies to evaluate JE vaccines. 6. Collect additional data on JE vaccines including adverse events in HIV-infected individuals and a potential role of vitamin A supplementation with respect to ecacy of vaccination [75]. 7. Inform potential donors of the value of supporting JE-endemic countries to administer the vaccine.

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13. List of participants 13.1. Temporary advisers Dr Sang Ja Ban, Korea Food and Drug Administration & Welfare (KFDA), 5-Nokbun-Dong, Eeunpyuong-Ku, Seoul, Republic of Korea, Tel.: +82 2 380 1860; fax: +82 2 388 0125. Dr Natth Bhamarapravati, Department of Pathology, Mahidol University at Salaya, Center for Vaccine Development, Institute of Sciences & Technology, for Development, 25/25 Phutthamonthon 4, Nakhonpathom 73170, Thailand, Tel.: +66 2 441 97 44/9336; fax: +66 2 441 9744/9336, e-mail: [email protected] Dr Virender Gupta, Deputy Assistant Director, Central Research Institute, Kasauli 173204 (Shimla Hills), Himachal Pradesh, India, Tel.: +01 792 72739; fax: +01 792 72049. Dr Scott B. Halstead, Medical Science & Technology Division, 800 North Quincy Street, Arlington, VA 22217-5660, USA, Tel.: +1 703 696 4257; fax: +1 703 696 1212 e-mail: [email protected] Dr A. Igarashi, Director, WHO Collaborative Center for Ref. & Research, Nagasaki University, 1-12-4 Sakamoto, Japan 852, Nagasaki, Japan, Tel.: +81 95 849 7827; fax: +81 95 949 7830. Col. Bruce L. Innis, Walter Reed Army Institute of Research, (WRAIR), Department of Virus Diseases, 14th Street and Dahlia, N.W., Washington, DC 203075100, USA, Tel.: +1 202 782 7019/1 301 295 7757; fax: +1 202 782 0442/1 301 295 7767. Dr Ichiro Kurane, Director, National Institute of Infectious Diseases, Department of Virology 1, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan, Tel.: +81 3 5285 1169; fax: +81 3 5285 1169; e-mail: [email protected] Dr Chung Keel Lee, Chief Ocer for Technical Cooperation, International Vaccine Institute, Seoul National University Campus, Shillim-Dong, Kwanakku, Seoul, Korea 151-742, Tel.: +82 2 872 2801; fax: +82 2 872 2803; e-mail: [email protected] Dr Huynh Phuong Lien, Head, National Institute of Health and Epidemiology, Department of Virology, 1 Yersin Street, Hanoi, Viet Nam, Tel.: +84 4 971 3310; fax: +84 4 971 3310. Dr Akira Oya, 1-18-5, O-Okayama, Maguro, Tokyo 152, Japan, Tel.: +81 3 371 7716; fax: +81 3 371 9102; e-mail: [email protected] Dr Tove Ronne, Head, Statens Serum Institute, Department of Epidemiology, Copenhagen 2300 S, Denmark, Tel: +45 32 68 32 68/45 33 12 19 49; fax: +45 32 68 38 74; e-mail: [email protected] Mr Kwon Sun Yon, Ministry of Public Health, Democratic People's Republic of Korea, Pyongyang,

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T.F. Tsai / Vaccine 18 (2000) 1±25

Tel.: +850 2 44 126/42 635; fax:+ 850 238 17639/850 2 81 76 03. Dr Supamit Chunsuttiwat, Division of General Communicable Diseases, Ministry of Public Health, Tiwanon Road, Montaburi 11000, Thailand, Tel.: +66 2 591 8432; fax: +66 2 591 8432. Dr Yoksan Sutee, Department of Pathology, Mahidol University at Salaya, Center for Vaccine Development, Institute of Sciences & Technology, for Development 25/25 Phutthamonthon 4, Thailand, Tel.: +66 2 441 9744/9336; fax: +66 2 441 9744/9336; e-mail: [email protected] Dr Hiroshi Takahashi, Communicable Disease Epidemiology, WA-DOH, 1610 N.E. 150th Street, Seattle, WA 98155, USA, Tel.: +1 206 361 2914; fax: +1 206 361 2930; e-mail: [email protected] Dr Theodore F. Tsai, Assistant Director for Medical Science, CDC, National Center for Infectious Diseases, Division of Vector-Bourne Infectious Diseases P.O. Box 2087, Fort Collins, CO 80522, USA, Tel.: +1 914-732-4053; fax: +1 914-732-5517; email: [email protected] Dr Somsak Watanasri, Director, Ministry of Public Health (MOPH), Oce of the Permanent Secretary, Nontaburi 11000, Thailand, Tel.: +662 591 8199; fax: +662 591 8579. Dr David Wood, Principal Scientist, NIBSC, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK, Tel.: +441 1707 654 753; fax: +441 1707 646 730; e-mail: [email protected] Dr Xu Zhi-Yi, Professor & Chairman, Shanghai Medical University, Department of Epidemiology, School of Public Health, 138 Yi Xue Yuan Road, Shanghai, China, Tel.: +86 6404 1900 ext. 2216; fax: +82 2 872 2803; e-mail: [email protected] Dr Han Yong Sik, Ministry of Public Health, Democratic People's Republic of Korea Phongyang, Tel.: +850 2 44 126/42 635; fax: +850 238 17 639/850 2 81 76 03. Dr Yu Yong Xin, National Institute for Control of Pharmaceutical and Biological Products Temple of Heaven, Beijing, 100050, Peoples Republic of China, Tel.: +86 10 67 01 755 ext. 436; fax: +86 10 67 017 683; e-mail: [email protected] Dr Cao Liam Zhi, Director, Division of Biological Product, State Drug Administration, Ministry of Public Health, 44 Houhai Beiyan, Beijing 100725, PRC, Tel.: +86 10 640 123 76; fax: +86 10 640 331 24. 13.2. Representatives from industry Mr Hyn Soo Kim, Boran Pharmaceuticals, 3 F, Koryo Academytel, 437-3, Ahyun-dong, apo-gu, Seoul 121-010, Republic of Korea, Tel.: +82 2 365 7000;

fax: +82 2 365 7009; e-mail: [email protected] Dr Yoichiro Kino, Manager, Division One, Second Research Department, Katetsuken, Kyokushi, Kikuchi, Kumamoto 896-1298, Japan, Tel.: +81 968 37 4059; fax: +81 968 37 4350. Dr Jean Lang, Pasteur MeÂrieux Connaught, 1541 Avenue Marcel MeÂrieux, 69280 Marcy L'Etoile, France, Tel.: +33 4 72 73 70 36; fax: +33 4 72 73 78 13. Dr Thomas P. Monath, Vice President, Research and Medical A€airs, OraVax Inc., 38 Sidney Street, Cambridge MA 02139, USA, Tel.: +1 617 494 1339; fax: +1 617 494 8872; e-mail: [email protected] Dr Soo Ok Kim, Research Scientist, R&D Center, Cheil Jedang Corporation, 522-1 dokpyong-Ri, Majang-Myon, Ichon-Si, Kyonggi-Do 467-810, Korea, Tel.: +82 336 639 4373; fax: +82 336 632 2784; email: [email protected] 13.3. Participants without WHO/CVI funds Dr Kenneth Eckels, Walter Reed Army Institute of Research (WRAIR), Department of Biologics Research, Building 501, Forest Glen Section, Washington DC 20307-5100, USA, Tel.: +1 301 295 7757/ 7758; fax: +1 301 295 7767. Dr J. Hanna, Public Health Physician, Tropical Public Health Unit, P.O. Box 1103, Cairns QLD 4870, Australia, Tel.: +61 740 503 600; fax: 61 740 311 440; e-mail: [email protected] Dr Toyokazu Ishikawa, Manager of Research and Development Division, Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Japan, Tel.: +81 875 25 4171; fax: +81 875 23 1660; e-mail: [email protected] Dr Li De Fu, Deputy Director, National Institute for Control of Pharmaceutical and Biological Products, Temple of Heaven, 2 Tiantan Xii, Beijing 1000, PRC, Fax: +86 1 6701 7683. Dr Insu P. Lee, Consultant to the Commissioner, Korea Food & Administration & Welfare (KFDA), 5 Nokbun-Dong, Eunpyong-Ku, Seoul, Republic of Korea, Tel.: +82 2 380 1860; fax: +82 2 388 0125. Dr Krisda Manoonvong, Director, Government Pharmaceutical Organization, Bangkok, Thailand. Dr Seung-il Shin, Project Leader, International Vaccine Institute, Seoul National University Campus, Shillim Don, Kwana-Ku, Seoul 151742, Republic of Korea, Tel.: +82 2 872 2801; fax: +82 2 872 2803; email: [email protected] Dr Young Mo Sohn, Director, Yonsei University College of Medicine, Department of Pediatrics, Youngdon Severance Hospital, 146-92 Dogok-dong, Kangnam-gu, Seoul, Republic of Korea, Tel.: +82 2

T.F. Tsai / Vaccine 18 (2000) 1±25

3461 3354; fax: +82 2 3461 9473; e-mail: [email protected]

[10]

13.4. WHO secretariat Dr John Clements, Medical Ocer, Expanded Programme on Immunization, Global Programme for Vaccines and Immunization, CH-1211 Geneva 27, Switzerland, Tel.: +41 22 791 4402; fax: +41 22 791 4193; e-mail: [email protected] Dr Julie Milstien, Scientist, Vaccine Supply and Quality, Global Programme for Vaccines And Immunization, CH-1211 Geneva 27, Switzerland, Tel.: +41 22 791 3564; fax: 41 22 791 4193; e-mail: [email protected] Dr Yuri Pervikov, Medical Ocer, Vaccine Research and Development, Global Programme for Vaccines and Immunization, CH-1211 Geneva 27, Switzerland, Tel: +41 22 791 2601; fax: +41 22 791 4860; e-mail: [email protected] Dr Jos Vandelaer, Regional Oce for South-EastAsia (SEARO), World Health Organization, Indraprastha Estate, Mahatma Gandhi Road, New Delhi 110002, India Tel.: +91 11 331 7804; fax: +91 11 331 8607. 13.5. CVI Secretariat Dr Mark Miller, Medical Ocer, Children's Vaccine Initiative, GPV, CH-1211 Geneva 27, Switzerland, Tel.: +41 22 791 2368; fax: +41 22 791 4888; e-mail: [email protected] References [1] Xu YH, Zhaori GT, Vene S, et al. Viral etiology of acute childhood encephalitis in Beijing diagnosed by analysis of single samples. Pediatr Infect Dis J 1996;15:1018±24. [2] Liu ZL, Hennessy S, Strom BL, Tsai TF, et al. Short-term safety of live-attenuated Japanese encephalitis vaccine (SA14-142): Results of a 26,239-subject randomized trial. J Infect Dis 1997;176:1366±9. [3] Kari K. personal communication. [4] Cardosa MJ, Hooi TP, Kaur P. Japanese encephalitis virus is an important cause of encephalitis among children in Penang. Southeast Asia J Trop Med Publ Health 1995;26:272±5. [5] Kumar R, Mathur A, Kumar A, Sharma S, Chakraborty S, Chaturvedi UC. Clinical features and prognostic indicators of Japanese encephalitis in children in Lucknow (India). Indian J Med Res 1990;91:321±7. [6] Joshi DD. Japanese encephalitis in Nepal. Southeast Asian J Trop Med Publ Health 1995;26:34±40. [7] Hanna JN, Ritchie SA, Phillips DA, et al. An outbreak of Japanese encephalitis in the Torres Strait, Australia, 1995. Med J Aust 1996;165:256±60. [8] Berg SW, Mitchell BS, Hanson RK, et al. Systemic reactions in US Marine Corps personnel who received Japanese encephalitis vaccine. J Infect Dis 1997;24:265±6. [9] Paul WS, Moore PS, Karabatsos N, Flood SP, Yamada S,

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