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Establishment of an analyzing method for a Japanese encephalitis virus neutralization test in Vero cells
Vaccine 21 (2003) 1989–1994
Establishment of an analyzing method for a Japanese encephalitis virus neutralization test in Vero cells Motoharu Abe∗ , Syoji Kuzuhara, Yoichiro Kino The Chemo-Sero-Therapeutic Research Institute, Kikuchi Research Center, Kawabe Kyokushi Kikuchi, Kumamoto 869-1298, Japan Received 24 June 2002; received in revised form 22 November 2002; accepted 27 November 2002
Abstract We established a 50% plaque reduction analyzing method of neutralizing antibody for human serum to Japanese encephalitis virus (JEV) in Vero cells, called the ‘3 points least-squares regression method’ (3LSRM). Our method shows a high correlation with the chick embryo cell method (the current standard method for human serum), using the chart method established by the National Institute of Infectious Diseases of Japan, which is an equation made with retrospective data obtained with the 50% plaque reduction method as a standard measurement method for neutralizing antibody titers to JEV. Our new method is much simpler and more reliable than the current method in that it uses an established cell line, Vero cells, and its results are computed by the 3LSRM. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Japanese encephalitis virus; Neutralization test; Vero cells
1. Introduction Japanese encephalitis (JE) is an epidemic disease not only in Japan but also in many other Asian countries. Approximately 50,000 patients have been reported annually [1]; moreover, the JE epidemic area has been expanding [2,3]. The fatality rate is about 30%, and about 50% of those who survive develop neurological sequelae [4]. JE vaccine derived from mouse brain was licensed in Japan in 1954. Although a small number of patients have appeared since then in Japan, the infection rate in pigs, an intermediate host of JE virus (JEV), is very high even today. Therefore, vaccination and surveillance of JEV antibody in humans are considered to be very important as public health measures [5,6]. Methods of antibody measurement for JEV, hemagglutination inhibition tests (HI), complement-fixation tests (CF), and neutralization (NT) tests, etc. are known. Generally, compared with HI or CF, NT tests have the higher sensitivity, and since the functional antibody can be measured, considering protection against infection from viruses, the NT test is thought to be the most appropriate [7–10]. We are currently furthering the development of a Vero cell-derived inactivated JE vaccine [11]. In this clinical trial, we thought it important to measure the most suitable neutral∗
Corresponding author. Tel.: +81-968-37-4059; fax: +81-968-37-4350. E-mail address: [email protected] (M. Abe).
izing antibody (NTAb), considering protection against virus infection as a surrogate parameter. To date, NTAb testing using chick embryo (CE) cells developed for vaccine potency testing (CE cell method) has been adopted as an antibody assessment for human serum. In the testing, computations using the chart method, established by the National Institute of Infectious Diseases (NIID) of Japan, which is an equation made with retrospective data obtained with the 50% plaque reduction method using CE cells [12], are used as a standard analyzing method for NTAb titers to JEV. Because of the complicated nature of preparing CE cells, we thought it desirable to introduce the 50% plaque reduction method using an established cell line like Vero cells [12–15], conceivably a common method of the future. In fact, the potency testing of JE vaccines, in which mouse NTAb titers are measured, has already been shifted to a method using Vero cells (Vero cell method) from the CE cell method. However, there has been no report on an analyzing method for the measurement of human NTAb titer using Vero cells. In fact, the chart method is an equation made with retrospective data using the CE cell method, and we therefore judged that simply diverting to a Vero cell method could not be done. Moreover, since the chart method is a converting 50% plaque reduction method using only the plaque reduction rate of 1 dilution per sample, although it is simple compared to a serial dilution method, it is known that the margin of random error will be large and that the NTAb titers will tend
0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0264-410X(02)00772-7
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to fluctuate. Therefore, we were attracted by the necessity of establishing a new NTAb test method using Vero cells which has high reliability as a surrogate marker of the validity of a clinical trial.
of overlay medium (E-MEM supplemented with 2% FBS, 0.22% NaHCO3 , 0.03% l-glutamine, 1% methyl-cellulose) was added, and the plates were incubated for 5 days in an incubator (37 ◦ C, 5% CO2 ). 2.5. Fixation and staining
2. Materials and methods 2.1. Cells, virus, serum samples, and monoclonal antibodies Vero cells (CCL-81, ATCC) grown in six-well plates (Costar® six-well cell culture cluster, flat bottom, with lid, Catalog Number: 3506) were used for the NT test. Mouse brain-derived JEV Beijing-1 strain [16], which has been used for production of current inactivated JE vaccine in Japan since 1989 [17], was used as a challenge virus. All serum samples were taken from human volunteers after obtaining informed consent. However, the natures of antibodies, whether from vaccination or natural infection, were not known. Anti-JEV murine monoclonal antibodies (mAbs), used for investigating specificity of the method, were obtained from Dr. Yasui, Tokyo Metropolitan Institute for Neuroscience.
After 5 days of incubation, 1.5 ml per well of 10% formalin solution was added and the plates were left at room temperature for 1 h or more. After washing with water, 1.5 ml per well of 0.0375% methylene blue tetrahydrate solution was added to each well to stain virus plaque, and the plates were left at room temperature for about 1 h. After washing with water, the plates were dried and plaques were counted. If the average value of the number of plaques of 10 or more control wells was between 50 and 150, and when P-value was greater than 0.05 in a chi-square test for goodness of fit, the tests were qualified. 2.6. Analyzing method candidates We investigated three different 50% plaque reduction analyzing methods: the chart method, the 2 points plot method (2PM), and the 3 points least-squares regression method (3LSRM), as analyzing method candidates.
2.2. Production of cell plates Vero cells grown in a plastic flask were detached by 0.125% trypsin, 0.007 M ethylendiaminetetraacetic acid (EDTA), and were resuspended in cell growth medium (Eagle’s minimum essential medium (E-MEM) supplemented with 0.11% NaHCO3 , 10% fetal bovine serum (FBS), 100 units/ml penicillin (PC), 0.1 mg (titer)/ml streptomycin (SM), 0.03% l-glutamine). After adjustment of cell numbers to 2 × 105 cells per ml, 3 ml per well of cell suspension was dispensed to each well of six-well plates. The plates were left standing for 2 days in an incubator (37 ◦ C, 5% CO2 ) before the test was conducted. 2.3. Dilutions of serum samples and challenge virus Serial dilution of serum samples was carried out after heat inactivation (56 ◦ C, 30 min) using diluent medium (E-MEM supplemented with 0.15% NaHCO3 , 2% FBS, 100 units/ml PC, 0.1 mg (titer)/ml SM, 0.03% l-glutamine). Challenge virus was also diluted to give 100 plaques per well.
2.6.1. The chart method As in the case of the CE cell method, the 50% plaque reduction rate is computed using the equation made with retrospective data established by Dr. Oya et al. of the NIID. The equation, shown below, uses the 1 point (1 dilution) closest to 50% of the plaque reduction rate, according to the hypothesis that a relationship (near the 50% plaque reduction rate), between the plaque reduction and the serum dilution is effectively a straight line showing the same degree of slope. However, it is not necessary to calculate using the equation for each measurement, since the NIID has produced a conversion chart. Z = (Y − 50)/47.7622 + log X, Z: NTAb titer (log), Y: plaque reduction rate (%), Y = 10–90%, X: serum dilution.
2.4. Neutralization
2.6.2. 2 points plot method The simplest line is drawn using 2 points (2 dilutions) which interpose the 50% plaque reduction rate, and the serum dilution (which shows the 50% plaque reduction rate) is calculated by an equation using this simple line. This can be said to be the simplest 50% plaque reduction analyzing method using a regression line.
The same amounts of diluted serum sample and diluted challenge virus were mixed and NT was carried out at 37 ◦ C for 90 min. One-hundred micro liters of the neutralized liquid was inoculated into three wells of cell plate per each dilution and adsorption took place for 90 min in an incubator (37 ◦ C, 5% CO2 ). The control virus was inoculated into 12 wells (two plates). After adsorption, 3 ml per well
2.6.3. 3 points least-squares regression method A least-squares regression line is carried out using 3 points (3 dilutions), in which 2 points interpose the 50% plaque reduction rate and 1 point comparatively near 50%, and the serum dilution which shows the 50% plaque reduction rate is calculated. The correlation coefficient of the least-squares regression line indicates 3 points from which to choose the
M. Abe et al. / Vaccine 21 (2003) 1989–1994
most likely combination, which will have the highest correlation coefficient.
3. Results and discussion 3.1. Examination of the 50% plaque reduction analyzing method A typical relationship between the plaque reduction and the dilution of human sera is shown in Fig. 1 (serum sample 10 in Table 1). The slope is large, becoming a Sigmoid curve, arriving at a plateau immediately, and linearity is acquired in the width which is about 10 of a serum dilution. There are only about 3 points which show high linearity by serial four-fold dilution. When using Vero cells and human serum as a sample, it is necessary to analyze by efficiently choosing the data in a narrow domain which shows linearity. Moreover, as shown in Table 1, the slopes of the least-squares regression lines, using the 3LSRM, were not fixed, but varied independently of the NTAb titers, and the coefficient of variation (CV) of the slopes was large. We were apprehensive about the possibility of a heavy influence by individual differences in serum samples. Therefore, the creation of a simple equation common to the samples, as with the chart method, was considered to be difficult. A comparison of the 3LSRM and the chart method is shown in Fig. 2. The correlation coefficient is 0.977, and both 50% plaque reduction analyzing methods show a high correlation. Moreover, the least-squares regression line formula with 95% confidence intervals (CIs) becomes y = (0.93–1.16)x +(−0.49–0.20). As this formula includes y = 1x +0, both methods can be said to be correlated at 1:1, with
Fig. 1. A typical plaque reduction pattern corresponding to four-fold serum dilution and a typical least-squares regression line, using the 3LSRM, a 50% plaque reduction analyzing method using 3 points in which 2 points interpose the 50% plaque reduction rate and 1 point comparatively near 50%. The correlation coefficient of the least-squares regression line indicates 3 points from which to choose the most likely combination which will have the highest correlation coefficient. The NTAb titer, using the 3LSRM, of this serum sample is 102.39 .
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Table 1 The slopes and correlation coefficients, of the least-squares regression lines, using the 3LSRM, of 25 serum samples Sample no.
NTAb titer (log)
Slope
r
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
0.68 1.49 1.61 1.90 1.94 2.06 2.20 2.20 2.25 2.39 2.44 2.71 2.83 2.85 2.89 2.96 2.98 3.10 3.44 3.47 3.57 3.64 3.67 3.97 4.10
−34.47 −19.43 −29.13 −21.92 −23.47 −26.79 −34.62 −19.74 −14.98 −25.43 −25.42 −22.53 −14.29 −22.40 −30.48 −28.30 −27.89 −20.97 −28.17 −30.23 −28.17 −23.36 −23.22 −17.86 −25.36
1.000 0.962 0.976 1.000 0.994 0.987 0.998 0.983 0.993 1.000 0.993 0.955 1.000 0.967 0.993 0.978 0.999 1.000 1.000 0.999 0.980 0.990 0.994 0.996 0.996
Average S.D. CV (%)
− − −
−24.75 5.28 21.35
0.989 0.013 1.30
the level of significance of 5%. A comparison of the 3LSRM and the 2PM is shown in Fig. 3. Both 50% plaque reduction analyzing methods also show a high correlation. Moreover, the least-squares regression line formula with 95% CIs becomes y = (0.96–1.06)x + (−0.18–0.10). As this formula also includes y = 1x + 0, both methods can be said to be
Fig. 2. A comparison of the 3LSRM and the chart method using 20 serum samples. The regression line is determined by the least-squares method.
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Fig. 3. A comparison of the 3LSRM and the 2PM, using 20 serum samples. The regression line is determined by the least-squares method.
correlated at 1:1. Likewise, a comparison of the 2PM and the chart method produces similar results (data not shown). Therefore, there is no big difference in the NTAb titers calculated by the three above-mentioned analyzing methods. Although the chart method is an equation made with retrospective data using CE cells as mentioned above, and the slopes of the least-squares regression lines were not fixed against the hypothesis of the chart method, as mentioned above and shown in Table 1, the chart method can be used to compute NTAb titers in a Vero cell method with no serious problems. However, as for the general chart method, the total number of dilutions ranges from 1 to 3 per sample, and dilution multiples are fixed independent of a position of a 50% plaque reduction rate. On the other hand, as for the chart method employed in this study, the total number of dilutions was 6 per sample, and a point comparatively near the 50% plaque reduction rate was chosen. Therefore, it can be said that this chart method is more reliable than the general chart method. Moreover, the chart method is a 50% plaque reduction analyzing method which uses plaque reduction data for only 1 dilution, as mentioned above. Although the chart method is simple, a problem exists in terms of reliability. Likewise, the 2PM also uses data from only 2 dilutions. Therefore, there is no index distinguished though 1 dilution is an outlier. It is thought that the danger of overlooking this point is large. On the other hand, by the 3LSRM, a least-squares regression line using 3 points is used. For this reason, a correlation coefficient serves as a distinguishing index though 1 dilution is an outlier. It is considered possible to suppress, to a minimum, variation of measured values under the influence of an outlier. Moreover, the correlation coefficient was high, 0.95 or more, as shown in Table 1; therefore, it can be said that the 3LSRM is a technique which efficiently uses data in a narrow domain to show linearity. Accordingly, we decided to adopt the 3LSRM, as the analysing method for
Fig. 4. A typical plaque reduction pattern of a low-titered serum sample: (䊉) low dilution and (䊊) normal dilution (Table 2). The NTAb titer, using the low diluting method, of this serum sample is 100.68 . The regression line is determined by the least-squares method.
NTAb titers, using Vero cells, in our clinical study of a Vero cell-derived tissue culture inactivated JE vaccine. 3.2. Examination of a serum dilution The threshold of a serum dilution of NTAb assay for JEV is generally considered to be 1:10, from the influence of non-specific NT action of serum. However, the influence of non-specific NT action of serum in a low dilution, such as 1:1, 1:2, or 1:5, was not so big as to impede the measurement of NTAb titers (data not shown). Therefore, even in samples with an NTAb titer of less than 10, measurements performed with a low dilution showed that NTAb titer could be measured as shown in Fig. 4 (serum sample 1 in Table 1). Therefore, we adopted a technique in which the serum dilution is changed according to the range of NTAb titer as shown in Table 2; in addition, in principle, that data has to interpose 50% of the plaque reduction rate. Although NTAb titer is calculated even when it does not interpose, if measurement is conducted again, using a suitable diluting method, it is thought that a more exact NTAb titer will be calculated. Moreover, in the low diluting method (Table 2), when the plaque reduction rate becomes less than 50% in all serum dilutions, the NTAb titer becomes less than 1. In fact, we have several serum samples in which the NTAb titer is less Table 2 Diluting methods for the 3LSRM Diluting method
Serum dilution
Range of NTAb
High dilution Normal dilution Low dilution
1:640, 1:2560, 1:10240, 1:40960 1:10, 1:40, 1:160, 1:2560, 1:10240 (1:1)a , 1:2, 1:5, 1:10, 1:20
640–40960 10–10240 (1)a , 2–20
a The low diluting method requires a high volume of serum sample. Therefore, a serum dilution of 1:1 is performed if necessary.
M. Abe et al. / Vaccine 21 (2003) 1989–1994
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Fig. 5. As verification of continuity of diluting methods, four serum samples with NTAb titers of (䊉) (a): 104.10 , (䊊) (b): 103.97 , (䉱) (c): 102.40 , and () (d): 102.04 were diluted, and NTAb titers of each diluted sample were determined by the appropriate diluting method (Table 2). The regression line is determined by the least-squares method.
than 1. The high diluting method (Table 2) is used for the sample with the NTAb titer of 10240 or more. As verification of continuity of diluting methods, four serum samples of NTAb titer ranging from 102.04 to 104.10 (104.10 was the highest NTAb titer we have) were diluted, and NTAb titers of each diluted sample were determined by the appropriate diluting method (Table 2). As shown in Fig. 5, in all samples, a high correlation coefficient was shown and the range of all residuals was small, ±0.31 or less, with a suitable spread of data, independent of NTAb titer and dilution. 3.3. Examination of correlation of the CE cell method using the chart method, and the Vero cell method using the 3 points least-squares regression method A comparison of the Vero cell method using the 3LSRM and the CE cell method using the chart method is shown in Fig. 6. The correlation coefficient is 0.965, and both methods show a high correlation. Moreover, the least-squares
Fig. 6. A comparison of the Vero cell method using the 3LSRM and the CE cell method using the chart method, using 18 serum samples. The regression line is determined by the least-squares method.
regression line formula with 95% CIs becomes y = (0.92–1.23)x + (−0.15–0.73). As this formula includes y = 1x + 0, both methods can be said to be correlated at 1:1, with the level of significance of 5%. 3.4. Specificity and precision of the methods Specificity of the methods to the JEV was confirmed using anti-JEV mAbs [18,19] with or without neutralizing activities (data not shown). Precision was investigated in terms of two considerations: repeatability and intermediate precision. For repeatability, three serum samples were independently measured for NTAb titers, three times, under the same conditions (person, day, and passage number of Vero cells). For intermediate precision, four serum samples were measured for NTAb titers, 12 times, under random conditions. As results, intermediate precision and repeatability were almost equivalent. The standard deviation (S.D.) of NTAb titer was 0.127 or less, CV was 7.00% or less, and CIs of S.D. were 0.007–0.314, and it can be said that there is sufficient precision. In addition, it is known that the characteristics of Vero cells changed depending on the environment and the passage numbers, etc. [20–23]. It is thought that the suitable passage number of the cells used in the Vero cell method using the 3LSRM is between 138 and 161, which was determined by intermediate precision depending on passage numbers (data not shown). As mentioned above, we established a 50% plaque reduction method of analysis, using Vero cells, which shows a high correlation with the CE cell method using the chart method, high precision, and high specificity. Moreover, we established diluting methods for the 3LSRM, which can cover sera with a wide range of antibody titers. Generally, if there are NTAb titers of 10 or more by the CE cell method, it is thought that natural infection of a JEV can be prevented [24,25]. Therefore, from the results above,
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also in the Vero cell method using this 3LSRM, as with the CE method, if there are NTAb titers of 10 or more, it can be said that natural infection can be prevented. Moreover, information of NTAb titer of less than 10 is quite important for targeting a totally naive population in the case of a clinical trial or seroepidemiological study. Using the 3LSRM, with the correlation coefficient as an index, the distinction of an outlier becomes comparatively easy, and this analyzing method shows high reliability. Moreover, indefinite elements, such as individual differences in the judgment analysis were eliminated by defining the diluting method which set up a clear serum dilution according to each NTAb titer (Table 2), and by defining a clear standard of selection of the 3 points used for analysis. More complicated calculations are required for the 3LSRM than for the chart method. However, it is now possible to carry out such calculations by computer, very simply, even if the analysis is complicated. Accordingly, using spreadsheet software, we created a spreadsheet program which can easily calculate NTAb titers. The spreadsheet program allows the user to easily change to the modes most suitable for each diluting method (Table 2), and also allows for the free choice of the combination of three data used for the 3LSRM. Furthermore, the spreadsheet program can automatically perform a chi-square test for goodness of fit. Finally, the Vero cell method using the 3LSRM has already been used to achieve actual results in our clinical trial (Phase I, 2001). In the clinical trial, more than 500 samples were measured for NTAb titers, and the results showed high reliability.
Acknowledgements The authors are grateful to Ms. N. Sakuma, Ms. K. Hisama, and Ms. H. Izawa for their excellent technical assistance, to Ms. T. Ogata and Ms. M. Miura for their preparation of CE cells, and to Ms. H. Hagiwara for her preparation of the JEV bank. The authors also wish to thank to Mr. F. Matsuo for his helpful support. The authors are also indebted to Mr. Timothy Corrigan for his helpful discussions. References [1] Vaughn DW, Hoke Jr CH. The epidemiology of Japanese encephalitis: prospects for prevention. Epidemiol Rev 1992;14:197– 221. [2] Mackenzie JS, Poidinger M, Phillips D, et al. Emergence of Japanese encephalitis virus in the Australasian region. In: Saluzzo JF, Dodet B, editors. Factors in the emergence of arbovirus diseases. Paris: Elsevier; 1997. p. 191–201. [3] Mackenzie JS. The ecology Japanese encephalitis virus in the Australian region. Clin Virol 1999;27:1–17.
[4] Kitano T, Oya A. Japanese encephalitis vaccine. In: Arai Y, Asano T, Chino T, Katow S, Miyamura T, Nakamura R, et al., editors. Vaccine handbook. Tokyo: Maruzen; 1996. p. 103–13. [5] Hoke CH, Nisalak A, Sangawhipa N, et al. Protection against Japanese encephalitis by inactivated vaccines. N Engl J Med 1988;319(10):608–14. [6] Kurane I, Takasaki T. Immunogenicity and protective efficacy of the current inactivated Japanese encephalitis vaccine against different Japanese encephalitis virus strains. Vaccine 2000;18:33–5. [7] Okuno Y, Igarashi A, Fukai K. Neutralization tests for dengue and Japanese encephalitis viruses by the focus reduction method using peroxidase-anti-peroxidase staining. Biken J 1978;21(4):137–47. [8] Okuno Y, Fukunaga T, Srisupaluck S, Kasemsarn P, Dharakul C, Sangkawibha N. Serological and virological studies on patients with dengue hemorrhagic fever (DHF) in Chanthaburi province, Thailand. I. Serological studies on paired sera from DHF patients by neutralization (N), hemagglutination inhibition (HI) and staining tests. Biken J 1980;23(3):113–21. [9] Okuno Y, Fukunaga T, Tadano M, Okamoto Y, Ohnishi T, Takagi M. Rapid focus reduction neutralization test of Japanese encephalitis virus in microtiter system. Brief report. Arch Virol 1985;86(1– 2):129–35. [10] Markoff L. Points to consider in the development of a surrogate for efficacy of novel Japanese encephalitis virus vaccines. Vaccine 2000;18:26–32. [11] Sugawara K, Nishiyama K, Ishikawa Y, et al. Development of Vero cell-derived inactivated Japanese encephalitis vaccine. Biologicals 2002;30:303–14. [12] Yoshioka I, Aizawa C, Yamagishi H. Comparative study on neutralization tests of Japanese encephalitis. Kitasato Arch Exp Med 1966;39:47–55. [13] Horaud F. Absence of viral sequences in the WHO-Vero Cell Bank. A collaborative study. Dev Biol Stand 1992;76:43–6. [14] Montagnon BJ, Vincent-Falquet JC. Experience with the Vero cell line. Dev Biol Stand 1998;93:119–23. [15] Requirements for the use of animal cells as in vitro substrates for the production of biologicals (requirements for biological substances no. 50). WHO Tech. Report Ser.1998;878:19–52. [16] Okuno Y, Okamoto Y, Yamada A, Baba K, Yabuuchi H. Effect of current Japanese encephalitis vaccine on different strains of Japanese encephalitis virus. Vaccine 1987;5:128–32. [17] Tsai TF. New initiatives for the control of Japanese encephalitis by vaccination: minutes of a WHO/CVI meeting, Bangkok, Thailand, 13–15 October. Vaccine 2000;18:1–25. [18] Kimura-Kuroda J, Yasui K. Antigenic comparison of envelope protein E between Japanese encephalitis virus and some other flaviviruses using monoclonal antibodies. J Gen Virol 1986;67:2663–72. [19] Kimura-Kuroda J, Yasui K. Protection of mice against Japanese encephalitis virus by passive administration with monoclonal antibodies. J Immunol 1988;141(10):3606–10. [20] Montagnon B, Vincent-Falquet JC, Fanget B. Thousand litre scale microcarrier culture of Vero cells for killed polio virus vaccine. Promising results. Dev Biol Stand 1984;55:37–42. [21] Whitaker AM, Hayward CJ. The characterization of three monkey kidney cell lines. Dev Biol Stand 1985;60:125–31. [22] Montagnon BJ. Polio and rabies vaccines produced in continuous cell lines: a reality for Vero cell line. Dev Biol Stand 1989;70:27–47. [23] Vincent-Falquet JC, Peyron L, Souvras M, Moulin JC, Taktoff J, Patet J. Qualification of working cell banks for the Vero cell line to produce licensed human vaccines. Dev Biol Stand 1989;70:153–6. [24] Takahashi M. The effects of environmental and physiological conditions of Culex tritaeniorhynchus on the pattern of transmission of Japanese encephalitis virus. J Med Entomol 1976;13:275–84. [25] Oya A. Japanese encephalitis vaccine. Acta Paediatr Jpn 1988; 30:175–84.
Establishment of an analyzing method for a Japanese encephalitis virus neutralization test in Vero cells Motoharu Abe∗ , Syoji Kuzuhara, Yoichiro Kino The Chemo-Sero-Therapeutic Research Institute, Kikuchi Research Center, Kawabe Kyokushi Kikuchi, Kumamoto 869-1298, Japan Received 24 June 2002; received in revised form 22 November 2002; accepted 27 November 2002
Abstract We established a 50% plaque reduction analyzing method of neutralizing antibody for human serum to Japanese encephalitis virus (JEV) in Vero cells, called the ‘3 points least-squares regression method’ (3LSRM). Our method shows a high correlation with the chick embryo cell method (the current standard method for human serum), using the chart method established by the National Institute of Infectious Diseases of Japan, which is an equation made with retrospective data obtained with the 50% plaque reduction method as a standard measurement method for neutralizing antibody titers to JEV. Our new method is much simpler and more reliable than the current method in that it uses an established cell line, Vero cells, and its results are computed by the 3LSRM. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Japanese encephalitis virus; Neutralization test; Vero cells
1. Introduction Japanese encephalitis (JE) is an epidemic disease not only in Japan but also in many other Asian countries. Approximately 50,000 patients have been reported annually [1]; moreover, the JE epidemic area has been expanding [2,3]. The fatality rate is about 30%, and about 50% of those who survive develop neurological sequelae [4]. JE vaccine derived from mouse brain was licensed in Japan in 1954. Although a small number of patients have appeared since then in Japan, the infection rate in pigs, an intermediate host of JE virus (JEV), is very high even today. Therefore, vaccination and surveillance of JEV antibody in humans are considered to be very important as public health measures [5,6]. Methods of antibody measurement for JEV, hemagglutination inhibition tests (HI), complement-fixation tests (CF), and neutralization (NT) tests, etc. are known. Generally, compared with HI or CF, NT tests have the higher sensitivity, and since the functional antibody can be measured, considering protection against infection from viruses, the NT test is thought to be the most appropriate [7–10]. We are currently furthering the development of a Vero cell-derived inactivated JE vaccine [11]. In this clinical trial, we thought it important to measure the most suitable neutral∗
Corresponding author. Tel.: +81-968-37-4059; fax: +81-968-37-4350. E-mail address: [email protected] (M. Abe).
izing antibody (NTAb), considering protection against virus infection as a surrogate parameter. To date, NTAb testing using chick embryo (CE) cells developed for vaccine potency testing (CE cell method) has been adopted as an antibody assessment for human serum. In the testing, computations using the chart method, established by the National Institute of Infectious Diseases (NIID) of Japan, which is an equation made with retrospective data obtained with the 50% plaque reduction method using CE cells [12], are used as a standard analyzing method for NTAb titers to JEV. Because of the complicated nature of preparing CE cells, we thought it desirable to introduce the 50% plaque reduction method using an established cell line like Vero cells [12–15], conceivably a common method of the future. In fact, the potency testing of JE vaccines, in which mouse NTAb titers are measured, has already been shifted to a method using Vero cells (Vero cell method) from the CE cell method. However, there has been no report on an analyzing method for the measurement of human NTAb titer using Vero cells. In fact, the chart method is an equation made with retrospective data using the CE cell method, and we therefore judged that simply diverting to a Vero cell method could not be done. Moreover, since the chart method is a converting 50% plaque reduction method using only the plaque reduction rate of 1 dilution per sample, although it is simple compared to a serial dilution method, it is known that the margin of random error will be large and that the NTAb titers will tend
0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0264-410X(02)00772-7
1990
M. Abe et al. / Vaccine 21 (2003) 1989–1994
to fluctuate. Therefore, we were attracted by the necessity of establishing a new NTAb test method using Vero cells which has high reliability as a surrogate marker of the validity of a clinical trial.
of overlay medium (E-MEM supplemented with 2% FBS, 0.22% NaHCO3 , 0.03% l-glutamine, 1% methyl-cellulose) was added, and the plates were incubated for 5 days in an incubator (37 ◦ C, 5% CO2 ). 2.5. Fixation and staining
2. Materials and methods 2.1. Cells, virus, serum samples, and monoclonal antibodies Vero cells (CCL-81, ATCC) grown in six-well plates (Costar® six-well cell culture cluster, flat bottom, with lid, Catalog Number: 3506) were used for the NT test. Mouse brain-derived JEV Beijing-1 strain [16], which has been used for production of current inactivated JE vaccine in Japan since 1989 [17], was used as a challenge virus. All serum samples were taken from human volunteers after obtaining informed consent. However, the natures of antibodies, whether from vaccination or natural infection, were not known. Anti-JEV murine monoclonal antibodies (mAbs), used for investigating specificity of the method, were obtained from Dr. Yasui, Tokyo Metropolitan Institute for Neuroscience.
After 5 days of incubation, 1.5 ml per well of 10% formalin solution was added and the plates were left at room temperature for 1 h or more. After washing with water, 1.5 ml per well of 0.0375% methylene blue tetrahydrate solution was added to each well to stain virus plaque, and the plates were left at room temperature for about 1 h. After washing with water, the plates were dried and plaques were counted. If the average value of the number of plaques of 10 or more control wells was between 50 and 150, and when P-value was greater than 0.05 in a chi-square test for goodness of fit, the tests were qualified. 2.6. Analyzing method candidates We investigated three different 50% plaque reduction analyzing methods: the chart method, the 2 points plot method (2PM), and the 3 points least-squares regression method (3LSRM), as analyzing method candidates.
2.2. Production of cell plates Vero cells grown in a plastic flask were detached by 0.125% trypsin, 0.007 M ethylendiaminetetraacetic acid (EDTA), and were resuspended in cell growth medium (Eagle’s minimum essential medium (E-MEM) supplemented with 0.11% NaHCO3 , 10% fetal bovine serum (FBS), 100 units/ml penicillin (PC), 0.1 mg (titer)/ml streptomycin (SM), 0.03% l-glutamine). After adjustment of cell numbers to 2 × 105 cells per ml, 3 ml per well of cell suspension was dispensed to each well of six-well plates. The plates were left standing for 2 days in an incubator (37 ◦ C, 5% CO2 ) before the test was conducted. 2.3. Dilutions of serum samples and challenge virus Serial dilution of serum samples was carried out after heat inactivation (56 ◦ C, 30 min) using diluent medium (E-MEM supplemented with 0.15% NaHCO3 , 2% FBS, 100 units/ml PC, 0.1 mg (titer)/ml SM, 0.03% l-glutamine). Challenge virus was also diluted to give 100 plaques per well.
2.6.1. The chart method As in the case of the CE cell method, the 50% plaque reduction rate is computed using the equation made with retrospective data established by Dr. Oya et al. of the NIID. The equation, shown below, uses the 1 point (1 dilution) closest to 50% of the plaque reduction rate, according to the hypothesis that a relationship (near the 50% plaque reduction rate), between the plaque reduction and the serum dilution is effectively a straight line showing the same degree of slope. However, it is not necessary to calculate using the equation for each measurement, since the NIID has produced a conversion chart. Z = (Y − 50)/47.7622 + log X, Z: NTAb titer (log), Y: plaque reduction rate (%), Y = 10–90%, X: serum dilution.
2.4. Neutralization
2.6.2. 2 points plot method The simplest line is drawn using 2 points (2 dilutions) which interpose the 50% plaque reduction rate, and the serum dilution (which shows the 50% plaque reduction rate) is calculated by an equation using this simple line. This can be said to be the simplest 50% plaque reduction analyzing method using a regression line.
The same amounts of diluted serum sample and diluted challenge virus were mixed and NT was carried out at 37 ◦ C for 90 min. One-hundred micro liters of the neutralized liquid was inoculated into three wells of cell plate per each dilution and adsorption took place for 90 min in an incubator (37 ◦ C, 5% CO2 ). The control virus was inoculated into 12 wells (two plates). After adsorption, 3 ml per well
2.6.3. 3 points least-squares regression method A least-squares regression line is carried out using 3 points (3 dilutions), in which 2 points interpose the 50% plaque reduction rate and 1 point comparatively near 50%, and the serum dilution which shows the 50% plaque reduction rate is calculated. The correlation coefficient of the least-squares regression line indicates 3 points from which to choose the
M. Abe et al. / Vaccine 21 (2003) 1989–1994
most likely combination, which will have the highest correlation coefficient.
3. Results and discussion 3.1. Examination of the 50% plaque reduction analyzing method A typical relationship between the plaque reduction and the dilution of human sera is shown in Fig. 1 (serum sample 10 in Table 1). The slope is large, becoming a Sigmoid curve, arriving at a plateau immediately, and linearity is acquired in the width which is about 10 of a serum dilution. There are only about 3 points which show high linearity by serial four-fold dilution. When using Vero cells and human serum as a sample, it is necessary to analyze by efficiently choosing the data in a narrow domain which shows linearity. Moreover, as shown in Table 1, the slopes of the least-squares regression lines, using the 3LSRM, were not fixed, but varied independently of the NTAb titers, and the coefficient of variation (CV) of the slopes was large. We were apprehensive about the possibility of a heavy influence by individual differences in serum samples. Therefore, the creation of a simple equation common to the samples, as with the chart method, was considered to be difficult. A comparison of the 3LSRM and the chart method is shown in Fig. 2. The correlation coefficient is 0.977, and both 50% plaque reduction analyzing methods show a high correlation. Moreover, the least-squares regression line formula with 95% confidence intervals (CIs) becomes y = (0.93–1.16)x +(−0.49–0.20). As this formula includes y = 1x +0, both methods can be said to be correlated at 1:1, with
Fig. 1. A typical plaque reduction pattern corresponding to four-fold serum dilution and a typical least-squares regression line, using the 3LSRM, a 50% plaque reduction analyzing method using 3 points in which 2 points interpose the 50% plaque reduction rate and 1 point comparatively near 50%. The correlation coefficient of the least-squares regression line indicates 3 points from which to choose the most likely combination which will have the highest correlation coefficient. The NTAb titer, using the 3LSRM, of this serum sample is 102.39 .
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Table 1 The slopes and correlation coefficients, of the least-squares regression lines, using the 3LSRM, of 25 serum samples Sample no.
NTAb titer (log)
Slope
r
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
0.68 1.49 1.61 1.90 1.94 2.06 2.20 2.20 2.25 2.39 2.44 2.71 2.83 2.85 2.89 2.96 2.98 3.10 3.44 3.47 3.57 3.64 3.67 3.97 4.10
−34.47 −19.43 −29.13 −21.92 −23.47 −26.79 −34.62 −19.74 −14.98 −25.43 −25.42 −22.53 −14.29 −22.40 −30.48 −28.30 −27.89 −20.97 −28.17 −30.23 −28.17 −23.36 −23.22 −17.86 −25.36
1.000 0.962 0.976 1.000 0.994 0.987 0.998 0.983 0.993 1.000 0.993 0.955 1.000 0.967 0.993 0.978 0.999 1.000 1.000 0.999 0.980 0.990 0.994 0.996 0.996
Average S.D. CV (%)
− − −
−24.75 5.28 21.35
0.989 0.013 1.30
the level of significance of 5%. A comparison of the 3LSRM and the 2PM is shown in Fig. 3. Both 50% plaque reduction analyzing methods also show a high correlation. Moreover, the least-squares regression line formula with 95% CIs becomes y = (0.96–1.06)x + (−0.18–0.10). As this formula also includes y = 1x + 0, both methods can be said to be
Fig. 2. A comparison of the 3LSRM and the chart method using 20 serum samples. The regression line is determined by the least-squares method.
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Fig. 3. A comparison of the 3LSRM and the 2PM, using 20 serum samples. The regression line is determined by the least-squares method.
correlated at 1:1. Likewise, a comparison of the 2PM and the chart method produces similar results (data not shown). Therefore, there is no big difference in the NTAb titers calculated by the three above-mentioned analyzing methods. Although the chart method is an equation made with retrospective data using CE cells as mentioned above, and the slopes of the least-squares regression lines were not fixed against the hypothesis of the chart method, as mentioned above and shown in Table 1, the chart method can be used to compute NTAb titers in a Vero cell method with no serious problems. However, as for the general chart method, the total number of dilutions ranges from 1 to 3 per sample, and dilution multiples are fixed independent of a position of a 50% plaque reduction rate. On the other hand, as for the chart method employed in this study, the total number of dilutions was 6 per sample, and a point comparatively near the 50% plaque reduction rate was chosen. Therefore, it can be said that this chart method is more reliable than the general chart method. Moreover, the chart method is a 50% plaque reduction analyzing method which uses plaque reduction data for only 1 dilution, as mentioned above. Although the chart method is simple, a problem exists in terms of reliability. Likewise, the 2PM also uses data from only 2 dilutions. Therefore, there is no index distinguished though 1 dilution is an outlier. It is thought that the danger of overlooking this point is large. On the other hand, by the 3LSRM, a least-squares regression line using 3 points is used. For this reason, a correlation coefficient serves as a distinguishing index though 1 dilution is an outlier. It is considered possible to suppress, to a minimum, variation of measured values under the influence of an outlier. Moreover, the correlation coefficient was high, 0.95 or more, as shown in Table 1; therefore, it can be said that the 3LSRM is a technique which efficiently uses data in a narrow domain to show linearity. Accordingly, we decided to adopt the 3LSRM, as the analysing method for
Fig. 4. A typical plaque reduction pattern of a low-titered serum sample: (䊉) low dilution and (䊊) normal dilution (Table 2). The NTAb titer, using the low diluting method, of this serum sample is 100.68 . The regression line is determined by the least-squares method.
NTAb titers, using Vero cells, in our clinical study of a Vero cell-derived tissue culture inactivated JE vaccine. 3.2. Examination of a serum dilution The threshold of a serum dilution of NTAb assay for JEV is generally considered to be 1:10, from the influence of non-specific NT action of serum. However, the influence of non-specific NT action of serum in a low dilution, such as 1:1, 1:2, or 1:5, was not so big as to impede the measurement of NTAb titers (data not shown). Therefore, even in samples with an NTAb titer of less than 10, measurements performed with a low dilution showed that NTAb titer could be measured as shown in Fig. 4 (serum sample 1 in Table 1). Therefore, we adopted a technique in which the serum dilution is changed according to the range of NTAb titer as shown in Table 2; in addition, in principle, that data has to interpose 50% of the plaque reduction rate. Although NTAb titer is calculated even when it does not interpose, if measurement is conducted again, using a suitable diluting method, it is thought that a more exact NTAb titer will be calculated. Moreover, in the low diluting method (Table 2), when the plaque reduction rate becomes less than 50% in all serum dilutions, the NTAb titer becomes less than 1. In fact, we have several serum samples in which the NTAb titer is less Table 2 Diluting methods for the 3LSRM Diluting method
Serum dilution
Range of NTAb
High dilution Normal dilution Low dilution
1:640, 1:2560, 1:10240, 1:40960 1:10, 1:40, 1:160, 1:2560, 1:10240 (1:1)a , 1:2, 1:5, 1:10, 1:20
640–40960 10–10240 (1)a , 2–20
a The low diluting method requires a high volume of serum sample. Therefore, a serum dilution of 1:1 is performed if necessary.
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Fig. 5. As verification of continuity of diluting methods, four serum samples with NTAb titers of (䊉) (a): 104.10 , (䊊) (b): 103.97 , (䉱) (c): 102.40 , and () (d): 102.04 were diluted, and NTAb titers of each diluted sample were determined by the appropriate diluting method (Table 2). The regression line is determined by the least-squares method.
than 1. The high diluting method (Table 2) is used for the sample with the NTAb titer of 10240 or more. As verification of continuity of diluting methods, four serum samples of NTAb titer ranging from 102.04 to 104.10 (104.10 was the highest NTAb titer we have) were diluted, and NTAb titers of each diluted sample were determined by the appropriate diluting method (Table 2). As shown in Fig. 5, in all samples, a high correlation coefficient was shown and the range of all residuals was small, ±0.31 or less, with a suitable spread of data, independent of NTAb titer and dilution. 3.3. Examination of correlation of the CE cell method using the chart method, and the Vero cell method using the 3 points least-squares regression method A comparison of the Vero cell method using the 3LSRM and the CE cell method using the chart method is shown in Fig. 6. The correlation coefficient is 0.965, and both methods show a high correlation. Moreover, the least-squares
Fig. 6. A comparison of the Vero cell method using the 3LSRM and the CE cell method using the chart method, using 18 serum samples. The regression line is determined by the least-squares method.
regression line formula with 95% CIs becomes y = (0.92–1.23)x + (−0.15–0.73). As this formula includes y = 1x + 0, both methods can be said to be correlated at 1:1, with the level of significance of 5%. 3.4. Specificity and precision of the methods Specificity of the methods to the JEV was confirmed using anti-JEV mAbs [18,19] with or without neutralizing activities (data not shown). Precision was investigated in terms of two considerations: repeatability and intermediate precision. For repeatability, three serum samples were independently measured for NTAb titers, three times, under the same conditions (person, day, and passage number of Vero cells). For intermediate precision, four serum samples were measured for NTAb titers, 12 times, under random conditions. As results, intermediate precision and repeatability were almost equivalent. The standard deviation (S.D.) of NTAb titer was 0.127 or less, CV was 7.00% or less, and CIs of S.D. were 0.007–0.314, and it can be said that there is sufficient precision. In addition, it is known that the characteristics of Vero cells changed depending on the environment and the passage numbers, etc. [20–23]. It is thought that the suitable passage number of the cells used in the Vero cell method using the 3LSRM is between 138 and 161, which was determined by intermediate precision depending on passage numbers (data not shown). As mentioned above, we established a 50% plaque reduction method of analysis, using Vero cells, which shows a high correlation with the CE cell method using the chart method, high precision, and high specificity. Moreover, we established diluting methods for the 3LSRM, which can cover sera with a wide range of antibody titers. Generally, if there are NTAb titers of 10 or more by the CE cell method, it is thought that natural infection of a JEV can be prevented [24,25]. Therefore, from the results above,
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also in the Vero cell method using this 3LSRM, as with the CE method, if there are NTAb titers of 10 or more, it can be said that natural infection can be prevented. Moreover, information of NTAb titer of less than 10 is quite important for targeting a totally naive population in the case of a clinical trial or seroepidemiological study. Using the 3LSRM, with the correlation coefficient as an index, the distinction of an outlier becomes comparatively easy, and this analyzing method shows high reliability. Moreover, indefinite elements, such as individual differences in the judgment analysis were eliminated by defining the diluting method which set up a clear serum dilution according to each NTAb titer (Table 2), and by defining a clear standard of selection of the 3 points used for analysis. More complicated calculations are required for the 3LSRM than for the chart method. However, it is now possible to carry out such calculations by computer, very simply, even if the analysis is complicated. Accordingly, using spreadsheet software, we created a spreadsheet program which can easily calculate NTAb titers. The spreadsheet program allows the user to easily change to the modes most suitable for each diluting method (Table 2), and also allows for the free choice of the combination of three data used for the 3LSRM. Furthermore, the spreadsheet program can automatically perform a chi-square test for goodness of fit. Finally, the Vero cell method using the 3LSRM has already been used to achieve actual results in our clinical trial (Phase I, 2001). In the clinical trial, more than 500 samples were measured for NTAb titers, and the results showed high reliability.
Acknowledgements The authors are grateful to Ms. N. Sakuma, Ms. K. Hisama, and Ms. H. Izawa for their excellent technical assistance, to Ms. T. Ogata and Ms. M. Miura for their preparation of CE cells, and to Ms. H. Hagiwara for her preparation of the JEV bank. The authors also wish to thank to Mr. F. Matsuo for his helpful support. The authors are also indebted to Mr. Timothy Corrigan for his helpful discussions. References [1] Vaughn DW, Hoke Jr CH. The epidemiology of Japanese encephalitis: prospects for prevention. Epidemiol Rev 1992;14:197– 221. [2] Mackenzie JS, Poidinger M, Phillips D, et al. Emergence of Japanese encephalitis virus in the Australasian region. In: Saluzzo JF, Dodet B, editors. Factors in the emergence of arbovirus diseases. Paris: Elsevier; 1997. p. 191–201. [3] Mackenzie JS. The ecology Japanese encephalitis virus in the Australian region. Clin Virol 1999;27:1–17.
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