Ratios of subclinical to clinical Japanese encephalitis (JE) virus infections in vaccinated populations: evaluation of an inactivated JE vaccine by comparing the ratios with those in unvaccinated populations

Vaccine 21 (2002) 98–107

Ratios of subclinical to clinical Japanese encephalitis (JE) virus infections in vaccinated populations: evaluation of an inactivated JE vaccine by comparing the ratios with those in unvaccinated populations Eiji Konishi∗ , Tomoyuki Suzuki Department of Health Sciences, Kobe University School of Medicine, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142, Japan Received 9 May 2002; received in revised form 22 August 2002; accepted 26 August 2002

Abstract Japanese encephalitis (JE) virus is characterized as a virus that produces a large number of subclinical infections. In this report, we estimated a ratio of subclinical to clinical infections in vaccinated human populations who acquired natural infection with JE virus, and evaluated protective capacity of the currently approved inactivated JE vaccine by comparing the ratio with those reported for unvaccinated populations. We developed a sensitive immunostaining method for detecting nonstructural 1 (NS1) antibody to demonstrate JE virus infection in vaccinated individuals. Serum samples collected from human populations in western Japan showed NS1 antibody prevalences of approximately 10% in an urban area in 1981 and 1995 and 20% in a rural area from 1982 through 1983. Analysis of annual change in NS1 antibody titer using paired samples provided a mean duration of NS1 antibody responses of approximately 2 years, indicating that 5% of the urban population or 10% of the rural population acquired natural JE virus infection in 1 year. Based on the number of JE cases from 1982 through 1991 and the number of people acquiring natural infection, and on the assumption that annual infection rates obtained in the present study areas are representative of the infection rate in entire Japan except for non-endemic northern areas, the ratio of subclinical to clinical infections in vaccinated populations was estimated to be 2,000,000:1, which was 2000–80,000 times higher than the ratio previously reported for unvaccinated populations. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Japanese encephalitis; Inactivated vaccine; Subclinical infection

1. Introduction Japanese encephalitis (JE) virus is a mosquito-borne flavivirus distributing in Asia [1,2]. Although the majority of humans infected with JE virus are asymptomatic, some develop acute encephalitis at a ratio of subclinical to clinical infections in the range of 25:1–1000:1 [3]. A formalin-inactivated JE virion preparation is the only JE vaccine internationally accepted for human use [4,5]. The efficacy of the inactivated JE vaccine was evaluated in field studies by comparing numbers of patients in vaccinated and unvaccinated populations under a condition that both populations were exposed to JE virus infection at equivalent rates [6,7]. However, more exact evaluation can be achieved by determining how many vaccinated individuals who acquired natural JE virus infection are protected from disease (regarded as subclinical infections in this paper) and comparing ratios of subclinical to clinical infections in ∗

Corresponding author. Tel.: +81-78-796-4594; fax: +81-78-796-4594. E-mail address: [email protected] (E. Konishi).

vaccinated and unvaccinated populations. Natural JE virus infection has not been surveyed so far among vaccinated populations, since simple methods for differentiating infected from uninfected individuals in vaccinated populations have not been available. The JE virion contains a positive-strand genomic RNA with a long open reading frame coding for three structural proteins including capsid (C), premembrane (prM) and envelope (E) and seven nonstructural (NS) proteins including NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5 [8]. Since inactivated JE vaccine is composed of a purified virion fraction, antibodies induced in vaccinees are only those to structural proteins. On the other hand, humans who are infected with JE virus develop antibodies to both structural and nonstructural proteins. Therefore, vaccinated individuals who acquire natural infection can be distinguished from those uninfected, by demonstrating antibodies to nonstructural proteins. Antibody to NS1 is considered to be the best indicator for acquisition of natural JE virus infections among those to seven nonstructural proteins. NS1 is released from

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E. Konishi, T. Suzuki / Vaccine 21 (2002) 98–107

mammalian cells infected with JE virus in vitro [9], and immunization with NS1-expressing recombinant viruses [10] or plasmids [11] strongly elicits antibody to NS1 and can provide protection in mice. Protection by immunization with NS1 has been reported for other flaviviruses [12,13]. In humans, antibody to NS1 has been detected in JE patients and healthy individuals who may have acquired natural infection in Japan [14], indicating an ability of humans to induce NS1 antibody. Furthermore, Americans who were vaccinated with the inactivated JE vaccine and resided in the US, where JE virus is absent, induced antibodies to E, but not to NS1 [14], confirming that NS1 antibody is induced by infection but not vaccination. Recently, a capture enzyme-linked immunosorbent assay (ELISA) method to measure NS1 antibody has been reported [15]. This method clearly differentiates NS1 antibody levels in patient sera from those in sera of normal vaccinated individuals, but does not seem to be sensitive enough to detect low levels of NS1 antibody induced in vaccinated individuals who were asymptomatically infected. In Japan, a national JE vaccination program has been successfully carried out, reducing the annual number of confirmed human cases to less than 100 since 1972 and to less than 10 since 1992 [16]. On the other hand, JE virus continues to circulate in a mosquito–swine transmission cycle every summer excluding northern Japan, as demonstrated by seroconversion among swine, an amplifier of JE virus [16]. The JE virus activity suggests that humans are exposed to natural infection with JE virus. In the present study, we developed a sensitive immunostaining method for detecting NS1 antibody, and surveyed the presence of NS1 antibody among sera which had been collected from people residing in an urban or a rural area of western Japan in early 1980s and/or middle 1990s. A relatively high antibody prevalence was observed both in urban (approximately 10%) and rural (approximately 20%) areas. An estimated ratio of subclini-

99

cal to clinical JE virus infections in a vaccinated population was much higher than previously reported ratios in unvaccinated populations, indicating a protective capacity of the currently approved inactivated JE vaccine.

2. Materials and methods 2.1. Serum and plasma samples Sera from four JE patients were supplied by the Department of Virology, National Institute of Health, Korea, through Dr. Robert E. Shope, Yale Arbovirus Research Unit, Yale University School of Medicine, CT [17]. Plasmas from six JE patients were supplied by the Department of Virology, Armed Forces Research Institute of Medical Sciences, Thailand, through Dr. Ichiro Kurane, Department of Medicine, University of Massachusetts Medical Center, MA [14]. Sera from 30 healthy American volunteers used for evaluation of inactivated JE vaccine [18], who had no history of yellow fever vaccination and no detectable neutralizing antibodies against JE virus, were supplied by the Walter Reed Army Institute of Research through Dr. Robert E. Shope [17]. Serum specimens used in the present survey were collected in Kobe University Hospital in 1981 [19] and 1995 [20] and in Miki Health Center from 1982 through 1983 [21]. Age and gender compositions are listed in Table 1. Sera from individuals aged less than 6 months, which may have contained maternally-transferred antibodies, were excluded. Kobe and a neighboring city, Miki, are located in Hyogo Prefecture (west-central part of Japan) where more than 50% of swine population seroconverted (developed antibody to JE virus) every summer at least until 1999 as reported by the national JE surveillance program [16]. Kobe University Hospital is located in a residential area at central Kobe, whereas Miki populations reside in an area rich in rice

Table 1 Age and gender compositions of three human populations used in this survey Age (years)

Number of samples Urban 1995a

Urban 1981b

Male

Female

0–9 10–19 20–29 30–39 40–49 50–59 60–69 70–79 80–99

106 103 101 95 97 104 115 114 77

Total

912

a

Total

Male

108 108 102 104 101 104 112 78 92

214 211 203 199 198 208 227 192 169

0 23 60 58 48 54 54 68 0

909

1821

365

Samples collected from an urban population in 1995. Samples collected from an urban population in 1981. c Samples collected from a rural population from 1982 through 1983. b

Rural 1982–1983c Female

Total

Male

Female

Total

0 48 57 59 48 54 52 87 0

0 71 117 117 96 108 106 155 0

0 0 22 45 50 66 49 0 0

0 0 32 66 73 75 52 0 0

0 0 54 111 123 141 101 0 0

405

770

232

298

530

100

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fields which served as a habitat of Culex tritaeniorhynchus, a most important vector of JE virus. A significant number of adult mosquitoes were observed every summer from 1982 through 1985 as reported in a survey at several sites of Miki city [22]. Sera collected in Kobe and Miki were regarded as those from urban and rural populations, respectively. Although one of the periods when the urban samples were collected (1981) is different from the period when the rural samples were collected (1982–1983), we considered that these periods are equivalent, since the annual number of confirmed human JE cases in Japan was similar during these periods (23 cases in 1981, 21 cases in 1982 and 32 cases in 1983 [16]). A total of 314 pairs of sera collected at a 1-year interval from 137 individuals (43 males and 94 females) at Miki Health Center from 1982 through 1985 [23] were used for estimating duration of antibody responses. Ages of these individuals ranged from 20 to 80 years with most (77%) of the ages composed of 30–59 years. The use of all the human samples in the present study was approved by the Ethical Committee of the Kobe University School of Medicine (Ethical Committee Approval Number 81). The vaccination history of this survey population was based on the yearly amount of JE vaccine approved [5,24], improvement in vaccine potency [4], and vaccination rates [25]. Although immunization with inactivated JE vaccine was introduced into public health practice in 1954 with 400 thousand doses of less-refined vaccine preparations, production of a large number of doses (approximately 20 million doses or more) of well-refined vaccine preparations started in 1965 [4,5,24]. We therefore considered that almost all people of a certain age group received vaccination before 15 years of age (for instance, people below 32 years in 1982 and people below 45 years in 1995). In contrast to high vaccination rates before the age of 15 years, only a small percentage (18% in average) received booster immunizations in people aged 20 years or older [25], although duration of neutralizing antibody responses induced by inactivated JE vaccine is 3 years or longer [26–28]. 2.2. Plasmid The JE virus cDNA containing the NS1 signal sequence and the NS1 gene was amplified by the polymerase chain reactions with a template plasmid DNA, PM-6 (provided by Dr. Peter W. Mason of the Plum Island Animal Disease Center, United States Department of Agriculture, Greenport, NY [29]). The sense primer included an EcoRI site, an efficient eukaryotic initiation site [30] and a start codon, followed by the codons encoding Ala-Leu-Ala-Phe-Leu-Ala-Thr of the NS1 signal sequence. The anti-sense primer corresponded to the C-terminal six codons of NS1, a termination codon and an XbaI site. The amplified cDNA was inserted into the pcDNA3 vector (Invitrogen, San Diego, CA) at the EcoRI/XbaI site between the strong eukaryotic promoter derived from human cytomegalovirus and the polyadenylation

signal derived from the bovine growth hormone. The construct was designated pcJENS1. The proper insert of the gene cassette in pcJENS1 was confirmed by sequencing. DNA was purified using a Quantum Nucleic Acid Purification Kit (Bio-Rad Laboratories, Hercules, CA) following the manufacturer’s instruction. 2.3. Cells and transfection CHO-K1 cells [31] were grown in Eagle’s minimal essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum, non-essential amino acids, and kanamycin at 60 ␮g/ml, at 37 ◦ C in a humidified atmosphere of 5% CO2 –95% air. CHO-K1 cells were transfected with 1.0 ␮g of pcJENS1 by using lipopolyamine (Transfectam; Biosepra, Villeneuve-la-Garenne, France) according to the instructions supplied by the company. Following selection using medium supplemented with G418 (Life Technologies, Gaithersburg, MD) at 400 ␮g/ml, transfected cells displaying high-level NS1 protein expression were selected by limiting dilution cloning and immunostaining using a monoclonal antibody specific for NS1 (D2-7E11, see further) following the method we previously adopted for generating a cell line continuously expressing JE virus E antigen [31]. For preparation of cells used for immunochemical staining, equal numbers of NS1-expressing cells and non-expressing cells were mixed in growth medium and distributed into six-well microplates at 6 × 104 cells per well (3 × 104 cells per well for each cell clone). Cells were cultivated at 37 ◦ C in 5% CO2 –95% air for 2–3 days until the cells grew into colonies consisting of 16–64 cells. These cells were rinsed with phosphate-buffered saline (PBS), dried, and fixed with cold ethanol. 2.4. Immunochemical staining method to determine NS1 antibody titer Cells in wells of six-well microplates (used as antigens) were divided into 12 reaction areas by using a PAP (peroxidase–anti peroxidase) pen (Daido Inc., Japan). The cells were then rehydrated and blocked with the diluent consisting of PBS containing 0.05% Tween 20, 2% casein, 1% normal goat serum and 1% bovine serum albumin (BSA) at 37 ◦ C for 1 h. Subsequently, the cells were incubated with serial two-fold dilutions of test specimens starting from a 1:10 dilution in the diluent at 4 ◦ C for 1–2 days. Antigen–antibody reactions were detected by incubation with biotinylated anti-human IgG (heavy and light chain-specific: Vector Laboratories, Burlingame, CA) at 37 ◦ C for 30 min, the ABC (avidin–biotinylated peroxidase complex: Vector) reagents at 37 ◦ C for 30 min, and the VIP substrate (Vector) at 37 ◦ C for 10 min. When differences in stain intensity between NS1-expressing and non-expressing cell colonies were observed under a microscope, the reaction was determined to be positive. The NS1 antibody titer was expressed as the maximum serum or plasma dilution

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yielding a positive reaction. Serum or plasma samples showing NS1 antibody titers of 1:10 or more were considered to be positive for NS1 antibody. For immunochemical staining to detect antigens expressed in transfected cells, fixed cells were blocked with PBS containing 1% normal horse serum and incubated with monoclonal antibodies to JE virus E (J3-11B9), M (J2-2F1) or NS1 (D2-7E11, provided by Dr. Mary K. Gentry of Walter Reed Army Institute of Research, Washington, DC [10]). Cells were then incubated serially with biotinylated anti-mouse IgG, the ABC reagents and the VIP substrate. 2.5. Neutralization test Neutralizing antibodies in human sera were titrated using plaque reduction assays performed with the Nakayama strain of JE virus in the absence of complement as previously described [32]. The neutralization titer was expressed as the maximum serum dilution yielding a 90% reduction in plaque number. 2.6. Statistical analysis Significance of differences in antibody prevalences was evaluated by the chi-square test with the Yates’ correction factor. Significance of differences in mean antibody titers was evaluated by the Student’s t-test. The probability levels (P) of less than 0.05 were considered significant. 3. Results 3.1. Generation of a stable cell line expressing NS1 CHO cells transfected with pcJENS1 were selected in G418-containing medium and monitored for percentage of NS1-expressing cells in every passage using immunostaining with a monoclonal antibody D2-7E11. Although only 20% of NS1-expressing cells were observed following three passages in G418-containing medium, cloning of these cells by limiting dilution increased the percentage of NS1-expressing cells at 70–80% following the first cloning step and 80–90% following the third cloning step (data not shown). Among several clones containing NS1-expressing cells at high percentages, the clone ME4 showed the strongest reaction to JE patient sera in immunochemical staining. Although the percentage of NS1-expressing cells did not reach 100% in this clone, the percentage did not decrease at least for 10 passages and we decided to use ME4 clone for antigen of our immunostaining method to determine NS1 antibody titers. The ME4 cells were not stained with monoclonal antibodies to E (J3-11B9) and M (J2-2F1), confirming the specificity of expressed antigen (data not shown). During limiting-dilution cloning steps, we also selected a clone containing no NS1-expressing

Fig. 1. Micrograph showing a typical result of immunochemical staining method to determine NS1 antibody titers. NS1-expressing and non-expressing cells were incubated with a JE patient plasma at a 1:640 dilution and then serially with biotinylated anti-human IgG, the ABC reagents and the VIP substrate (see Section 2 for details). Dark staining is the result of specific immunoreactivity of NS1-expressing cells with NS1 antibody included in patient plasma. Note the difference in staining intensity between NS1-expressing and non-expressing cell colonies.

cells (ME11), which was used as a negative control for our immunostaining method. An example of staining results obtained with a patient sample is shown in Fig. 1. 3.2. Establishment of an immunochemical staining method to determine NS1 antibody titers Using NS1-expressing and non-expressing cell clones, we developed an immunostaining method to determine NS1 antibody titers (see Section 2 for detailed procedures). A pilot experiment using mice immunized with a plasmid encoding the prM and E genes of the Nakayama strain of JE virus indicated that NS1 antibody which were not detected prior to challenge were detected at titers of 1:80–1:1280 6–14 days following challenge with the Beijing P3 strain of JE virus (data not shown). NS1 antibody titers obtained by the present method were consistent with those obtained by a similar immunostaining method using cells infected with a recombinant vaccinia virus encoding the Nakayama strain NS1 and NS2a genes [32] as antigen. Better staining results were obtained using a cell line continuously expressing NS1 antigen than using a recombinant virus-infected cells transiently expressing NS1, since viral infection induced cytopathic effects in cells. This method was further evaluated by comparing NS1 antibody titers obtained with normal and patient samples. None of the sera from 30 healthy Americans negative for neutralizing antibody showed detectable levels of NS1 antibody (<1:10), whereas 10 serum/plasma samples from JE patients showed NS1 antibody titers ranging from 1:320 to 1:5120 (data not shown). Five repeated titrations using three patient plasma samples that showed NS1 antibody titers of 1:640–1:2560 provided variations of titers within a range of two-fold. These results indicate that the present

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immunostaining method to determine NS1 antibody titers is highly sensitive and reproducible. 3.3. NS1 antibody prevalence in urban and rural populations Serum samples collected in an urban area in 1995 and 1981 and in a rural area in 1982–1983 were titrated for NS1 antibody. Prevalences of NS1 antibody in male, female and total populations were 9.2, 9.6 and 9.4% in urban populations in 1995, 9.6, 10.1 and 9.9% in urban populations in 1981, and 17.2, 23.2 and 21.0% in rural populations in 1982–1983, respectively. No significant differences were observed between males and females and between urban populations in 1995 and 1981 (P > 0.05). However, NS1 antibody prevalences in rural populations were significantly higher than those in urban populations. Comparison of rural and urban populations of corresponding periods (early 1980s) provided differences in male (17.2% versus 9.6%, P < 0.01), female (23.2% versus 10.1%, P < 0.001) and total (21.0% versus 9.9%, P < 0.001) populations. Fig. 2 shows age-prevalence curves for male, female and total populations. Overall, the prevalence did not depend on ages and genders in both urban and rural populations, except for an urban population in 1995 which showed a higher prevalence at ages over 80 years (15.4%) than that of all ages (9.4%, P < 0.05) and a significant difference between males (3.0%) and females (12.7%, P < 0.05) at ages of 20–29 years. NS1 antibody was quantitatively analyzed using an urban population in 1995 in which sera were collected from people over a wide age range. Individual NS1 antibody titers ranged from <1:10 to 1:1280 in males and from <1:10 to 1:160 in females, and the frequency distributions of NS1 antibody titers of ≥1:10 are shown in Fig. 3. Although the majority of males (98.9%) and females (99.3%) had titers of 1:40 or less, a small population (0.5% in males) had titers equivalent to those obtained with JE patients (1:320 or more). These high levels of NS1 antibodies were shown in ages of 50 years or

Fig. 2. Age-dependent prevalences of NS1 antibody in male (䊊), female (䊐) and total (䉱) populations in an urban area in 1995 and 1981 and a rural area in 1982–1983. The numbers of samples are listed in Table 1. The asterisk indicates a significant difference between males and females (P < 0.05) and the dagger indicates a significant difference from the overall prevalence of 9.4% obtained with the urban population in 1995 (P < 0.05).

Fig. 3. Frequency distributions of NS1 antibody titers for a total of 84 males and 87 females positive for NS1 antibody. Frequencies were expressed as percentages of 912 males and 909 females of an urban population in 1995.

more. However, no statistically significant differences were detected in geometric mean NS1 antibody titers between males and females and among age groups (P > 0.05). 3.4. Relation between titers of NS1 antibody and neutralizing antibody An individual who acquired natural infection is considered to develop both NS1 antibody and neutralizing antibody. To obtain a quantitative relation between NS1 antibody and neutralizing antibody, we examined 1118 sera collected from an urban population in 1995 for neutralizing antibody titers. The overall prevalences of neutralizing antibody were 47.7% in males, 43.2% in females and 45.5% in total. Age-dependent prevalences of neutralizing antibody (Fig. 4) indicated relatively high percentages in age groups of 0–19 years, low percentages in age groups of 20–59 years and gradually increasing percentages in age groups over 60

Fig. 4. Age-dependent prevalences of neutralizing antibody in 583 males (䊊), 535 females (䊐) and totaling 1118 populations (䉱) of an urban area in 1995. The asterisk indicates a significant difference between males and females (P < 0.01).

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years. The prevalence in ages of 20–59 years (33.3%) was significantly lower than those of 0–19 years (56.0%, P < 0.001) and 60 years or older (55.2%, P < 0.001). Male and female populations had similar age-prevalence curves, except for an age group of 70–79 years in which males showed a higher prevalence than females (74.2% versus 40.0%, P < 0.01). In contrast to the age-dependent prevalence of neutralizing antibody, frequency distributions of neutralizing antibody titers were similar in all age groups (data not shown). To compare NS1 antibody titers with neutralizing antibody titers, we selected 918 samples of an urban population in 1995 on the basis of ages over 20 years. Selection of these ages is considered to minimize the effect of vaccination on neutralizing antibody titers, since most of the people receive vaccination before the age of 15 years but not after 20 years [25]. A quantitative comparison indicated that NS1 antibody titers were correlated with neutralizing antibody titers with correlation coefficients of 0.16 (P < 0.001) in these 918 samples and of 0.19 (P < 0.001) in 397 samples which were further selected according to the possession of neutralizing antibody (data not shown). These results indicate a quantitative relation between titers of NS1 antibody and neutralizing antibody, suggesting that increase in neutralizing antibody titers upon acquisition of natural JE virus infection is accompanied by the increase in NS1 antibody titers. Significant but low correlation coefficients in quantitative analyses suggest differences between immune responses to NS1 and viral surface proteins. 3.5. Duration of NS1 antibody responses To study how long humans who acquired natural infections and were asymptomatic remain positive for NS1 antibody, 314 paired sera collected at a 1-year interval were tested for NS1 antibody titers. In this population, 25.2 and 26.2% were positive for NS1 antibody in the first and second sera of these pairs, respectively. Fig. 5 shows a relation between NS1 antibody titers of the first sera and their differences from titers of the corresponding second sera. Most (192 of 234; 82%) of negative sera in the first year were negative in the second year and the remaining 42 (18%) seroconverted, and we considered that 18% of this population acquired natural JE virus infection within the past 1 year. The geometric mean difference between titers of the first and second sera in this seroconverting population was 1.69, indicating that the NS1 antibody titer increased to approximately 1:20 in average when an individual negative for NS1 antibody acquired JE virus infection within the past 1 year and was asymptomatic. Analysis using sera positive for NS1 antibody in the first year indicated that the first sample with higher NS1 antibody titers tended to decrease their titers to a larger extent in the second year. The geometric mean differences in sera with the first NS1 antibody titers of 1:40 and 1:20 were −2.18 and −0.80, respectively, indicating that these sera decreased their titers to approximately 1:10 in average 1 year after.

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Fig. 5. Relation between NS1 antibody titers of the first sera and the differences from titers of the second sera in 314 paired sera collected at an interval of 1 year. The difference was expressed as log2 ratio of the NS1 antibody titer of the second serum to that of the first serum, when samples below the limit of detection (<1:10) were assigned a value of 1:5. The numerals indicate geometric mean differences between titers of the first and second sera in pairs grouped according to the original NS1 antibody titer, excepting for pairs with the original NS1 antibody titer of <1:10 in which the pairs showing 1:10 or greater in the second sera were used for calculation.

Based on the pattern of annual change in NS1 antibody titers obtained in Fig. 5, we computed the mean duration of NS1 antibody responses by simulating serial changes of individual NS1 antibody titers which were increased at the time of seroconversion and subsequently decreased until seroreversion. Fig. 6A shows a frequency distribution of NS1 antibody titers in individuals who are considered to acquire natural JE virus infection within the past 1 year. Fig. 6B shows a frequency distribution of NS1 antibody titers in the same individuals as shown in Fig. 6A 1 year after,

Fig. 6. Simulation of annual changes in frequency distribution of NS1 antibody titers. This simulation was based on annual changes of individual NS1 antibody titers in 314 paired sera shown in Fig. 5. Frequency distribution for individuals who increased their NS1 antibody titers from <1:10 to 1:10–1:40 (seroconversion) within the past 1 year (A) was changed 1 (B), 2 (C) and 3 (D) years after seroconversion. Open bars and numerals above them indicate percentages of individuals whose NS1 antibody titers were decreased to <1:10 (seroreversion).

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Table 2 Simulation of percent seroreverting population to calculate the mean time between seroconversion and seroreversion Years to seroreversion (A)

Percentage of seroreverting populationa (B)

Cumulative percentage of negative population

1 2 3 4 5 6 7 8 9 10 11 12

52.02b 22.59b 12.09b 6.32 3.32 1.74 0.91 0.48 0.25 0.13 0.07 0.04

52.02 74.61 86.70 93.02 96.34 98.08 98.99 99.47 99.72 99.85 99.92 99.96

Total

99.96

A×B

52.02 45.18 36.27 25.29 16.59 10.44 6.39 3.83 2.26 1.32 0.76 0.43 200.77

a

Calculated by simulation based on annual changes of NS1 antibody titers shown in Fig. 5. b The same percentages as shown in Fig. 6.

indicating that 52% of the population seroreverted. Frequency distributions in two (Fig. 6C) and three (Fig. 6D) years after seroconversion indicated that percent negative populations were increased every year with a newly seroreverting population of 23 and 12%, respectively. In the same way, percent seroreverting populations, as well as cumulative percentages of the negative population, were simulated every year until the 12th year when almost 100% population became negative (Table 2). The time to seroreversion (designated A) was multiplied by the percent seroreverting population (designated B) every year, and the sum total of A × B (200.77) was divided by the sum total of percent seroreverting population (99.96), yielding a mean duration of NS1 antibody responses of 2.0 years. 3.6. Annual infection rates The antibody prevalence obtained in a cross-sectional study is based on the population who was infected in the past and maintains detectable levels of antibody. To show

what percentage of human populations acquired natural JE virus infection in 1 year (defined as annual infection rates), the NS1 antibody prevalences obtained in three human populations were divided by the mean duration of NS1 antibody responses. As shown in Table 3, the annual infection rates in urban and rural populations were calculated to be approximately 5 and 10%, respectively. In addition to the estimation using NS1 antibody, we analyzed annual change in neutralizing antibody titers using paired sera and estimated the mean duration of neutralizing antibody responses of 5.6 years (data not shown) by the same simulation method as described above. Based on this duration and the prevalence of neutralizing antibody in populations aged 20 years or more, we obtained an annual infection rate of 7.7% in one of the urban populations (in 1995) and 9.4% in the rural population (Table 3). Although annual infection rates obtained with neutralizing antibody titers are considered to be affected by vaccination, the effect of vaccination may not be so significant in a population over 20 years of age who have booster immunizations at a low frequency [25]. 4. Discussion Japanese encephalitis has been well controlled in recent 30 years in Japan [4,5,16]. Although current decline in JE cases has been explained basically by a national JE vaccination program, the reduced opportunity in human–mosquito contacts has been considered as another critical factor. The frequency of mosquito bites was reduced by use of insecticides and improved irrigation schemes for rice cultivation, and by the dissociation between the mosquito–swine transmission cycle and human dwellings [4,5]. Although the national JE surveillance program has reported infection of swine with JE virus every summer except for non-endemic northern areas [16], it has not been determined how frequent humans were exposed to bites by infected mosquitoes. Demonstration of natural infection with JE virus in human population is critical for assessing the national JE vaccination program. The present study reveals that people residing in an urban or a rural area of west Japan acquired natural JE

Table 3 Annual rate of natural JE virus infection in three human populations Population

Urban Urban Rural a

Year

1995 1981 1982–1983

NS1 antibody

Neutralizing antibody

Prevalence (%)a

Annual infection rate (%)b

Prevalence (%)

Annual infection rate (%)c

9.4 9.9 20.6

4.7 5.0 10.3

43.2d NDe 52.6f

7.7 ND 9.4

Prevalence obtained from data shown in Fig. 2. Prevalences were divided by the mean duration of NS1 antibody responses (2.0 years). c Prevalences were divided by the mean duration of neutralizing antibody responses (5.6 years). d Prevalence obtained with 918 urban people aged 20 years or older. e Not done. f Prevalences obtained with 188 rural people aged 20 years or older. b

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virus infection at relatively high percentages in early 1980s and middle 1990s. This result indicates that people still contact frequently with infected mosquitoes under the recent situation and that the inactivated JE vaccine contributes to protection of these people from the disease. Higher prevalence of NS1 antibody was obtained in the rural (20%) than urban (10%) area, suggesting that people in the rural area are more frequently exposed to infected mosquitoes. Similar NS1 antibody prevalences in different ages or genders are a characteristic feature for infection by a mosquito-transmitted virus. Several conditions were included in the immunostaining method to determine NS1 antibody titers in human sera, so that the method could be reliable and allow a large number of testings. Comparison of the stain intensity between NS1-expressing and non-expressing cell colonies in a single microscopic field contributed to increase in sensitivity. Also, use of a highly sensitive avidin–biotin complex method contributed to the exact decision for the reaction at the endpoint of serial two-fold dilutions of test samples. Another condition included in our present method is the use of 6-well microplates, since we experienced that the use of 96-well microplate sometimes provided negative results for patient sera. The failure to provide positive reactions is probably due to the surface tension of the reagent liquid in a relatively small space of the well. The use of six-well microplates contributed to the reproducibility of test results and the reduction in volume of reagents. Finally, we included several reagents in diluent of sera, such as Tween 20, BSA and casein [33] to decrease levels of nonspecific reactions. This contributed to observation at low serum dilutions. The present strategy to use NS1 antibody as a marker of infection cannot detect infection, when “sterile” immunity is achieved in the host in which JE virus present in mosquito saliva injected at the bite site is inactivated by neutralizing antibody before infecting the host cells and fails to produce NS1 protein. However, this “sterile” immune response seems to be induced only in a small population. A previous experiment using mice immunized with a recombinant vaccinia virus encoding the JE virus prM and E genes indicated that post-challenge NS1 antibody was detected even though the pre-challenge neutralizing antibody titer was as high as 1:2560 [10]. In our present study, age groups of 0–19 years which correspond to the period of vaccination (5–15 years) showed higher neutralizing antibody prevalences than, but equivalent NS1 antibody prevalences to those in age groups of 30–59 years, suggesting no reducing effect of vaccine-induced neutralizing antibody on development of NS1 antibody following infection. Furthermore, a half of the population used in the present study and one-fifth of the population used for surveys by the national JE surveillance program [16,34] do not have neutralizing antibody, indicating that infection can be established in these individuals without sterile immunity. Finally, an analysis of annual changes in NS1 antibody titers using 234 paired sera which were negative for NS1 antibody in the first year indicated that

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the percentage population positive for neutralizing antibody in the first year among 42 individuals who seroconverted (56%) was similar to that among 192 individuals who were also negative for NS1 antibody in the second year (58%). In the present study, we demonstrated infection by the presence of NS1 antibody and considered vaccine-induced protection as subclinical infection. When sterile immunity is achieved, this factor increases the annual infection rate obtained by NS1 antibody-based detection and also the estimated protective capacity of inactivated JE vaccine. The age-dependent prevalence of neutralizing antibody (Fig. 4) indicated that higher prevalences were observed in ages of 0–19 years and 60 years or older. This age prevalence pattern obtained in a location of west Japan was consistent with those obtained in different areas of Japan next year (1996) as reported by the national JE surveillance program [34]. The high percentage in young age groups are generally considered as a result of vaccination of people aged 15 years or younger and the low percentage in middle age groups as a result of the small population that received booster vaccinations [25]. The high prevalences in higher age groups may be explained by the fact that people aged 56 years or older did not receive vaccinations before the age of 15 years, since inactivated JE vaccine was authorized for practical use in 1954 [5]. Most of these populations are considered to be exposed to JE virus antigens initially by natural infection but not by vaccination, and the infection may induce stronger memory immune responses than those induced by vaccination. The difference in immune responses to the first exposure (infection or vaccination) may also be related to a high NS1 antibody prevalence of 15% in an age group of 80 years or older (Fig. 2) and high NS1 antibody titers of 1:320 or more in age groups of 50 years or older in urban populations in 1995 (Fig. 3). Duration of NS1 antibody responses was estimated by a simulation method, based on the pattern of annual change in NS1 antibody titer in a relatively large number of paired samples. Specifically, using seroconverting populations, the mean time between seroconversion and seroreversion was obtained on the basis of percent seroreverting population simulated every year. Although this study is, to our knowledge, the first example of using this type of simulation for estimating duration of antibody responses, we consider that this simulation would be an appropriate method alternative to the long-term monitoring of NS1 antibody titers. On the assumption that yearly 7.5% people in average acquired natural JE virus infection, we estimated the ratio of subclinical to clinical infections in vaccinated population during 10 years from 1982 from which information of vaccine history for JE patients was available using individual report cards [35]. The estimation was also based on the assumption that the annual infection rates obtained in the present study area would be similar to those in the other areas of Japan except northern areas. Since the national-scale production of well-refined JE vaccine started in 1965 [4,5,24], we selected people who received vaccination before 15 years

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of age (for instance, people below 32 years in 1982 and people below 41 years in 1991) for the estimation. Based on national population data [36,37], the cumulative number of vaccinated people from 1982 through 1991 in Japan except northern areas was 545,000,000, and the number of people who acquired natural JE virus infection was calculated to be 41,000,000 (545,000,000 × 7.5%). Since the total number of patients excluding those who did not receive vaccination was 22 during these 10 years [35], the ratio of subclinical to clinical infections in vaccinated population was calculated to be approximately 2,000,000:1 (41,000,000/22). This ratio was 2000–80,000 times higher than the ratio previously reported for unvaccinated populations (25:1–1000:1 [3]). More than half of the populations aged 20–59 years did not have detectable neutralizing antibody in our 90% plaque reduction assay, consistent with a survey report on vaccination rates showing that most people aged 20 years or older do not receive booster vaccinations [25]. In spite of these situations, annual numbers of confirmed JE cases have been less than 100 since 1972 [16]. It is considered that vaccinated people without detectable neutralizing antibody are protected from disease by an anamnestic neutralizing antibody response to the infected virus and the quickly-induced neutralizing antibody may block transportation of the virus from the peripheral replication sites to the brain. We have previously demonstrated using a mouse infection model that immunization with a plasmid encoding the JE virus prM and E genes induced only low or undetectable levels of neutralizing antibody, but produced a rapid rise of neutralizing antibody following an intraperitoneal challenge and provided protection from disease [32]. Thus, the role of inactivated JE vaccine seems to be the priming for immunologic memory in the vaccinated host. Periodic exposure to natural infection with JE virus serves as natural “boosting” and can maintain memory B cells in the vaccinated host for a long period. In conclusion, the present study reveals that relatively large populations acquired natural JE virus infection in west Japan and the majority of infected people were protected from disease by vaccination in childhood. This type of survey of natural JE virus infections would be important for elucidating a mechanism of protection by the current inactivated JE vaccine and for designing novel vaccination strategies.

Acknowledgements We thank Drs. Robert E Shope and Ichiro Kurane for patient samples, Dr. Peter W. Mason for plasmid PM6, Dr. Mary K. Gentry for monoclonal antibodies, and Drs. Ikuo Takashima, Kouichi Morita, Masaoki Yamaoka, Tomohiko Takasaki, Ken-ichiro Yamada, Mikio Nakayama, Satoru Arai, Yasuko Matsunaga, Nobuhiko Okabe, Akira Oya for valuable suggestions. We also thank Mss. Yoshimi Sakamori, Youko Matsumoto, Yumi Houki and Chiyoko Nukuzuma for technical assistance. This investigation received financial support from Research on Emerging and

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