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Humoral immunity and correlation between ELISA, hemagglutination inhibition, and neutralization tests after vaccination against tick-borne encephalitis virus in children
Journal of Virological Methods 134 (2006) 136–139
Humoral immunity and correlation between ELISA, hemagglutination inhibition, and neutralization tests after vaccination against tick-borne encephalitis virus in children Giulietta Venturi a , Rosanna Mel b , Antonella Marchi a , Sebastiano Mancuso b , Floriana Russino b , Giusi Da Pra b , Nunziato Papa c , Gianni Bertiato c , Cristiano Fiorentini a , Maria Grazia Ciufolini a,∗ a
Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanit`a, Viale Regina Elena 299, Rome 00161, Italy b Department of Public Health, Via S. Andrea 8, Belluno 32100, Italy c Department of Laboratory Medicine, S. Martino Hospital, Viale Europa, Belluno 32100, Italy
Received 30 May 2005; received in revised form 15 December 2005; accepted 15 December 2005 Available online 3 February 2006
Abstract Vaccination against tick-borne encephalitis (TBE) virus is the measure of choice for disease control in endemic areas, as no treatment is available. In Italy, the province of Belluno is one of the most active TBE virus infection foci. In this study sera were examined from vaccinated children living in areas around Belluno in order to monitor the immune response after anti-TBE vaccination. For the assessment of neutralizing antibodies, a plaque reduction neutralization test (PRNT) was optimized and the correlation between enzyme-linked immunosorbent assay (ELISA), hemaglutination inhibition (HI), and neutralizing antibodies titers was evaluated. All children had high serum levels of TBE IgG in ELISA test after the vaccination, in agreement with previous studies. HI and PRNT titers ranged between very low and high levels. A good correlation between HI and PRNT titers, and with IgG ELISA titers, was observed. PRNT is an useful assay for monitoring protective immunity after the completion of anti-TBE vaccination. This type-specific assay is an important tool for differential diagnosis in cases where the presence of cross-reactive antibodies due to other flavivirus infections or vaccinations cannot be excluded. © 2005 Elsevier B.V. All rights reserved. Keywords: Antibodies; Vaccine; Flavivirus; Diagnosis
1. Introduction Tick-borne encephalitis (TBE) virus is a member of the genus flavivirus within the family Flaviviridae, which comprises of 70 distinct but antigenically related viruses. Several flaviviruses are pathogenic to man, e.g. yellow fever (YF) virus, dengue viruses, West Nile (WN) virus, and Japanese encephalitis (JE) virus (Monath, 1990). These are small spherical enveloped viruses, with single strand RNA positive genome, a capsid composed of a single basic protein, and two membrane-associated proteins (membrane and envelope – E proteins). The E protein presents hemagglutination activity, and is the major viral antigen that
∗
Corresponding author. Tel.: +39 0649903235; fax: +39 0649902082. E-mail address: [email protected] (M.G. Ciufolini).
0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.12.010
induces the formation of neutralizing antibodies and a protective immune response. All flavivirus E proteins share common antigenic sites as shown originally by cross-reactivity studies using hemagglutination inhibition (HI) tests (Casals, 1957). Different flavivirus serocomplexes have been established with virus neutralization tests (NT) (Calisher et al., 1989). TBE virus is prevalent over a wide area on the Eurasian continent, in many European countries, Russia, and countries of Far-east Asia, including Japan (Dumpis et al., 1999; Takashima et al., 1997). Presently, a total of 10,000–12,000 hospitalized cases are registered annually, including about 3000 in Europe (Suss, 2003). The virus is transmitted by tick bite. In central Europe approximately 10% of infected people develop neurological symptoms that may include meningitis, meningoencephalitis, meningoencephalomyelitis, or paralysis, with mortality of 1–2% (Kaiser,
G. Venturi et al. / Journal of Virological Methods 134 (2006) 136–139
1999). Active immunization can successfully be used for containing TBE (Kunz, 2003). For testing immunity after TBE virus infection or for monitoring the immune response after TBE vaccination, the IgG enzyme-linked immunosorbent assay (ELISA) is normally used, due to its simplicity and ease of automation (Holzmann, 2003). An excellent correlation between ELISA IgG units and the antibody titers obtained by HI or NT has been demonstrated provided that there was no other exposure to flavivirus antigens except TBE virus/vaccination (Holzmann et al., 1996). In Italy, the province of Belluno is one of the most active TBE virus infection foci (Caruso et al., 1999), and immunization with anti-TBE vaccine of volunteers at risk of disease is being recommended until 1996. In this study, sera were examined from children residing in areas around Belluno in order to monitor the immune response after anti-TBE vaccination, and the correlation between ELISA, HI, and neutralizing antibodies was evaluated. For the assessment of neutralizing antibodies we optimized a plaque reduction neutralization test (PRNT), a simple method that has been used rarely in the past because of the difficulties of reproducibility with TBE virus. 2. Materials and methods 2.1. Patient sera Thirty-six healthy male and female children living in areas around Belluno, aged between 6 and 12 years, completed vaccination with three half-adult doses (0.25 ml) of FSME-Immuno vaccine, in the period June 2001–August 2002. Serum samples were collected 1–14 months (mean: 123 days) after the last vaccine dose, and sent to Belluno Hospital for the ELISA test, and to Virology Laboratory of the Italian Ministry of Health (Istituto Superiore di sanit`a) for HI and NT tests. 2.2. ELISA
137
strain ir968 (Verani et al., 1981), diluted 1:10 in serum-free maintenance medium. After 4 days of incubation, a cytopathic effect was observed and supernatant was collected, clarified and frozen in aliquots. Infectivity titration was performed by plaque assay using PS cells. 2.4. Plaque reduction neutralization test The assay was carried out in a six-well tissue culture plates with subconfluent PS cell monolayers (approximately 40–50% confluence). Sera were diluted 1:10 in serum-free maintenance medium, heat-inactivated, and titrated in duplicate in two-fold dilution steps (100 ul/well). Equal volumes (100 ul) of TBE virus dilution containing 80 PFU, and serum dilutions, were mixed, and incubated over night at 4 ◦ C. PS cells were counted and seeded, kept 2 h to attach in six-well tissue culture plates, and then infected with 200 ul of the virus–serum mixtures in duplicate. After 1 h incubation at 37 ◦ C and 5% CO2 , the inocula were aspirated and the wells were overlayed with a mixture of one part 2% Gum Tragacanth and one part 2× MEM supplemented with 2.5% inactivated FCS and 2% 1M HEPES. The plates were incubated at 37 ◦ C and 5% CO2 for 4 days, and then were stained with 1.5% crystal violet. A titration of TBE virus with three dilutions in duplicate (the working dilution, 1:2 and 1:8 dilutions) was performed in each assay and used as a control for the assay: those performing too low or too high virus doses were not accepted. A mouse ascitic fluid obtained after inoculation with TBE virus strain ir968 has been used as a positive control only when performing PRNT optimization experiments, together with an anti TBE-negative human serum as assayed in the HI test, as the negative control. Neutralizing antibody titers were calculated as the reciprocal of the serum dilution that gave an 80% reduction of the number of plaques (NT80 ), as compared to the virus control. Titers ≥10 were considered positive.
The IgG and IgM antibodies against TBE virus were detected in serum using a commercially available ELISA system (FSME IgG and IgM Immunozym, Progen Biotech GMBH, Heidelberg). Standard calibrators are supplied by the kit to obtain a reference curve, which allows a quantitative evaluation of anti-TBE IgG antibodies. Results are expressed in Vienna units per milliliter (VIEU/ml). Absorbance values <63 VIEU/ml are considered negative, values between 63 and 126 VIEU/ml are considered borderline, and values >126 VIEU/ml are considered positive, according to manufacturer’s instructions. Samples with an absorbance exceeding that of the reference curve were prediluted (1 + 1) with the FSME incubation buffer. The concentrations thus obtained had to be multiplied by the factor two. High level positive and low level positive control sera are supplied by the manufacture and added to each test.
2.5. Hemagglutination-inhibition test
2.3. Virus
In order to evaluate the correlation between NT and HI titers, and between ELISA IgG titers and NT or HI titers, Spearman and Kendall ranks correlation coefficients were computed. The analysis was performed by the statistical package Stata 8.
PS (porcine kidney) culture cells were infected with a 10% (w/v) suckling mouse brain suspension of the italian TBE virus
A standard HI test was employed, according to Clarke and Casals (1958). Briefly, sera were pre-absorbed with kaolin and goose erythrocytes, and tested subsequently at two-fold dilutions, starting at 1:10. Arcton antigen extracted from mouse brain infected with TBE virus (strain ir968) was used at eight hemagglutinating units. The antigen’s calibration was controlled within each assay. To assess the specificity of the reaction, sera were titrated in parallel against at least one different flavivirus that cross-reacts at lower titer (WN virus), and against Arbia virus antigen, as negative control. HI tests were carried out at pH 6.6 for TBE. 2.6. Statistical analysis
138
G. Venturi et al. / Journal of Virological Methods 134 (2006) 136–139
3. Results 3.1. ELISA test results All serum samples but one (patient 26) were negative by the IgM ELISA test. All serum samples were positive by the IgG ELISA test with titers ranging from 737 to 12006 VIEU/ml (mean: 6786.86 VIEU/ml). 3.2. Optimization of plaque reduction neutralization test Fig. 2. Correlation between ELISA anti-TBE IgG and HI antibody titers.
PS cells monolayers at different degrees of confluence were assayed for the formation of visible plaques when inoculated with TBE virus (ir968). Different incubation times were also tested. Visible plaques of 2–3 mm diameter, were reproducibly obtained with PS cells monolayers at low degree of confluence (40–50%), and 4 days of incubation. Eleven PRNTs were carried out for the titration of neutralizing antibodies: in all assays visible plaques were obtained. Ten PRNT assays performed virus doses ranging from 75 to 118 PFU (mean 93, standard deviation 15); one assay performing 145 PFU was not accepted. The mouse ascitic fluid obtained after inoculation with TBE virus strain ir968, used as positive control, had an NT antibody titer of 80; the human serum anti TBE-negative as assayed in the HI test, used as the negative control, showed an NT antibody titer <10.
Fig. 3. Correlation between ELISA anti-TBE IgG and neutralizing (NT80 ) antibody titers.
4. Discussion 3.3. Correlation between hemagglutination inhibition and plaque reduction neutralization test, and correlation between ELISA IgG antibody titers and HI or neutralizing antibody titers HI and neutralizing antibody titers between 10 and >1280 have been detected (Fig. 1). The correlation between NT80 and HI titers is consistently high (Spearman’s coefficient = 0.79 and Kendall’s tau = 0.67). The results obtained with the two methods are shown in Fig. 1. A good correlation was also observed between ELISA IgG antibody titers and both HI and neutralizing antibody titers (Spearman’s coefficient 0.9564 and 0.8422, respectively; Kendall’s tau 0.8554 and 0.6987, respectively), as shown in Figs. 2 and 3.
Fig. 1. Hemagglutination inhibition (HI) and neutralizing (NT80 : reciprocal of the serum dilution that gave an 80% reduction of the number of plaques in neutralization test) antibody titers.
As no treatment is available, immunization is the measure of choice for disease control of TBE in endemic areas. Individuals of all ages are thought to be equally susceptible to infection (Logar et al., 2000), and immunization against TBE is recommended for all individuals, including infants and children, who live in TBE endemic areas, or travel to countries that have a high risk of infection. In this study, serum samples of 36 children were examined. Anti-TBE vaccination had been required voluntarily for children mainly because of their recreational activities outdoors in TBE endemic areas. They thus received three half-adult doses of FSME-Immuno vaccine, as recommended by the Italian Ministry of Health (Circ. Min.Sanit`a n.10, 13-07-2000). The pediatric formulation of the vaccine was not available. In this study, children showed serum levels of anti-TBE IgG in ELISA test after the vaccination ranging from 737 to 12006 VIEU/ml (mean: 6786,86 VIEU/ml). These results are in good agreement with those reported in other studies (Eder and Kollaritsch, 2003; Kunz, 2003), in particular they confirm the efficacy of using half-adult doses in children. In one case a positive anti-TBE IgM ELISA result was obtained, but clinical evidence of TBE disease was not observed. The production and titers of HI and neutralizing antibodies in this population were studied. Most of the available data concerning the neutralizing antibody response after vaccination were obtained by Chiron Vaccines. One thousand TCID50 (50% tissue culture infectious doses) of TBE virus were used for the NT, and cell cultures microscopic screening for cytopathic effects was performed after 4–5 days of incubation with
G. Venturi et al. / Journal of Virological Methods 134 (2006) 136–139
serum–virus mixtures (Klockmann et al., 1991). Other methods have been described for the detection of neutralizing antibodies: Vene et al. (1998) developed a rapid fluorescent focus inhibition test (RFFIT), using 50 FFD50 (50% focus forming dose) of virus and visualization of virus foci in immunofluorescence after 24 h of incubation of cells with serum–virus mixtures. Neutralizing titers obtained with RFFIT were almost identical to those obtained with a standard PRNT, which was less reproducible. A good correlation with HI results was also reported. Holzmann et al. (1996) performed the NT with 1000 PFU/ml of virus, and on day 4 post infection the supernatants were tested for the presence or absence of viral antigen in a four-layer ELISA system. They reported a good correlation between ELISA IgG units and the antibody titers obtained by HI or NT, provided that there was no other exposure to flavivirus antigens except TBE virus/vaccination. In this study, visible plaques with TBE virus were obtained using subconfluent PS cell monolayers, and thus a simple PRNT was optimized and validated in the population of vaccinated children. Visible plaques were not obtained with Vero cells, which have been used for PRNTs in other studies (Calisher et al., 1989; Vene et al., 1998). In the present study HI and NT80 titers ranged between very low (10) and high (>1280) levels. A very low NT80 and HI titer of 10–20 was observed in 5/36 and 3/36 serum samples, respectively (Fig. 1). A good correlation between HI and NT80 titers (Fig. 1), and between ELISA IgG antibody titers and HI or NT80 titers (Figs. 2 and 3), was observed. A statistically significant negative correlation between antibody titers and days elapsed from last vaccine dose could not be demonstrated in this small population (data not shown). The kinetic curve of persistence of neutralizing TBE antibodies was investigated recently in order to assess if the 3 years booster intervals recommended currently should be extended. The results obtained in cross-sectional studies show that following four immunization doses protective antibodies can be detected far beyond a period of 3 years, thus supporting strongly the reconsideration of currently recommended booster intervals (Rendi-Wagner et al., 2004a,b; Zent et al., 2004). The availability of a NT will allow us to monitor the persistence of immunity after the completion of the vaccination cycle. Moreover, the NT, which is undertaken in very few laboratories in Europe is very important because of the increased contacts with tropical and subtropical countries which has augmented the risk of acquiring other flavivirus infections or of being vaccinated against YF or JE (Monath, 1994). Acknowledgment The authors thank Dr. Giada Minelli for kind help with statistical analysis. References Calisher, C.H., Karabatsos, N., Dalrymple, J.M., Shope, R.E., Porterfield, J.S., Westaway, E.G., Brandt, W.E., 1989. Antigenic relationships between
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flaviviruses as determined by cross-neutralization tests with polyclonal antisera. J. Gen. Virol. 70 (Pt. 1), 37–43. Caruso, G., Mondardini, V., Granata, C., Guzzo, E., Papa, N., Ciufolini, M.G., 1999. Recenti acquisizioni sulla encefalite da zecca. Giornale Italiano di Malattie Infettive, supplemento al volume 5, no. 2, S226–S228. Casals, J., 1957. Viruses: the versatile parasites; the arthropod-borne group of animal viruses. Trans. N. Y. Acad. Sci. 19 (3), 219–235. Circolare del Ministero della Sanit`a. n.10/13.07.2000, 2000. Malattie trasmesse da zecche: cenni di epidemiologia-misure di prevenzione. Clarke, D.H., Casals, J., 1958. Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses. Am. J. Trop. Med. Hyg. 7 (5), 561–573. Dumpis, U., Crook, D., Oksi, J., 1999. Tick-borne encephalitis. Clin. Infect. Dis. 28 (4), 882–890. Eder, G., Kollaritsch, H., 2003. Antigen dependent adverse reactions and seroconversion of a tick-borne encephalitis vaccine in children. Vaccine 21 (September (25–26)), 3575–3583. Holzmann, H., Kundi, M., Stiasny, K., Clement, J., McKenna, P., Kunz, C., Heinz, F.X., 1996. Correlation between ELISA, hemagglutination inhibition, and neutralization tests after vaccination against tick-borne encephalitis. J. Med. Virol. 48 (1), 102–107. Holzmann, H., 2003. Diagnosis of tick-borne encephalitis. Vaccine 21 (Suppl. 1), S36–S40. Kaiser, R., 1999. The clinical and epidemiological profile of tick-borne encephalitis in southern Germany 1994–98: a prospective study of 656 patients. Brain 122 (Pt. 11), 2067–2078. Klockmann, U., Bock, H.L., Kwasny, H., Praus, M., Cihlova, V., Tomkova, E., Krivanec, K., 1991. Humoral immunity against tick-borne encephalitis virus following manifest disease and active immunization. Vaccine 9 (January (1)), 42–46. Kunz, C., 2003. TBE vaccination and the Austrian experience. Vaccine 21 (Suppl. 1), S50–S55. Logar, M., Arnez, M., Kolbl, J., Avsic-Zupanc, T., Strle, F., 2000. Comparison of the epidemiological and clinical features of tick-borne encephalitis in children and adults. Infection 28 (2), 74–77. Monath, T.P., 1990. Flaviviruses. In: Fields, B.N., Knipe, D.M. (Eds.), Virology, second ed. Raven Press, New York, pp. 763–814. Monath, T.P., 1994. Dengue: the risk to developed and developing countries. Proc. Natl. Acad. Sci. U.S.A. 91 (7), 2395–2400. Rendi-Wagner, P., Kundi, M., Zent, O., Dvorak, G., Jaehnig, P., Holzmann, H., Mikolasek, A., Kollaritsch, H., 2004a. Persistence of protective immunity following vaccination against tick-borne encephalitis-longer than expected? Vaccine 22 (21–22), 2743–2749. Rendi-Wagner, P., Kundi, M., Zent, O., Banzhoff, A., Jaehnig, P., Stemberger, R., Dvorak, G., Grumbeck, E., Laaber, B., Kollaritsch, H., 2004b. Immunogenicity and safety of a booster vaccination against tick-borne encephalitis more than 3 years following the last immunisation. Vaccine 23 (4), 427–434. Suss, J., 2003. Epidemiology and ecology of TBE relevant to the production of effective vaccines. Vaccine 21 (Suppl. 1), S19–S35. Takashima, I., Morita, K., Chiba, M., Hayasaka, D., Sato, T., Takezawa, C., Igarashi, A., Kariwa, H., Yoshimatsu, K., Arikawa, J., Hashimoto, N., 1997. A case of tick-borne encephalitis in Japan and isolation of the virus. J. Clin. Microbiol. 35 (8), 1943–1947. Verani, P., Ciufolini, M.G., Nicoletti, L., Lopes, M.C., Amaducci, L., Fratiglioni, L., Paci, P., Leoncini, F., Calducci, M., 1981. Circulation of TBE virus in Italy: seroepidemiological and ecovirological studies. In: Kunz, C. (Ed.), Tick-borne Encephalitis. Facultas Verlag, Wien, pp. 265–272. Vene, S., Haglund, M., Vapalahti, O., Lundkvist, A., 1998. A rapid fluorescent focus inhibition test for detection of neutralizing antibodies to tick-borne encephalitis virus. J. Virol. Methods 73 (1), 71–75. Zent, O., Plentz, A., Schwarz, T.F., Fruhwein, N., Kuhr, H.B., Broeker, M., Banzhoff, A., Jilg, W., 2004. TBE booster immunization according to the rapid immunization schedule: are 3-year booster intervals really necessary? Vaccine 23 (3), 312–315.
Humoral immunity and correlation between ELISA, hemagglutination inhibition, and neutralization tests after vaccination against tick-borne encephalitis virus in children Giulietta Venturi a , Rosanna Mel b , Antonella Marchi a , Sebastiano Mancuso b , Floriana Russino b , Giusi Da Pra b , Nunziato Papa c , Gianni Bertiato c , Cristiano Fiorentini a , Maria Grazia Ciufolini a,∗ a
Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanit`a, Viale Regina Elena 299, Rome 00161, Italy b Department of Public Health, Via S. Andrea 8, Belluno 32100, Italy c Department of Laboratory Medicine, S. Martino Hospital, Viale Europa, Belluno 32100, Italy
Received 30 May 2005; received in revised form 15 December 2005; accepted 15 December 2005 Available online 3 February 2006
Abstract Vaccination against tick-borne encephalitis (TBE) virus is the measure of choice for disease control in endemic areas, as no treatment is available. In Italy, the province of Belluno is one of the most active TBE virus infection foci. In this study sera were examined from vaccinated children living in areas around Belluno in order to monitor the immune response after anti-TBE vaccination. For the assessment of neutralizing antibodies, a plaque reduction neutralization test (PRNT) was optimized and the correlation between enzyme-linked immunosorbent assay (ELISA), hemaglutination inhibition (HI), and neutralizing antibodies titers was evaluated. All children had high serum levels of TBE IgG in ELISA test after the vaccination, in agreement with previous studies. HI and PRNT titers ranged between very low and high levels. A good correlation between HI and PRNT titers, and with IgG ELISA titers, was observed. PRNT is an useful assay for monitoring protective immunity after the completion of anti-TBE vaccination. This type-specific assay is an important tool for differential diagnosis in cases where the presence of cross-reactive antibodies due to other flavivirus infections or vaccinations cannot be excluded. © 2005 Elsevier B.V. All rights reserved. Keywords: Antibodies; Vaccine; Flavivirus; Diagnosis
1. Introduction Tick-borne encephalitis (TBE) virus is a member of the genus flavivirus within the family Flaviviridae, which comprises of 70 distinct but antigenically related viruses. Several flaviviruses are pathogenic to man, e.g. yellow fever (YF) virus, dengue viruses, West Nile (WN) virus, and Japanese encephalitis (JE) virus (Monath, 1990). These are small spherical enveloped viruses, with single strand RNA positive genome, a capsid composed of a single basic protein, and two membrane-associated proteins (membrane and envelope – E proteins). The E protein presents hemagglutination activity, and is the major viral antigen that
∗
Corresponding author. Tel.: +39 0649903235; fax: +39 0649902082. E-mail address: [email protected] (M.G. Ciufolini).
0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.12.010
induces the formation of neutralizing antibodies and a protective immune response. All flavivirus E proteins share common antigenic sites as shown originally by cross-reactivity studies using hemagglutination inhibition (HI) tests (Casals, 1957). Different flavivirus serocomplexes have been established with virus neutralization tests (NT) (Calisher et al., 1989). TBE virus is prevalent over a wide area on the Eurasian continent, in many European countries, Russia, and countries of Far-east Asia, including Japan (Dumpis et al., 1999; Takashima et al., 1997). Presently, a total of 10,000–12,000 hospitalized cases are registered annually, including about 3000 in Europe (Suss, 2003). The virus is transmitted by tick bite. In central Europe approximately 10% of infected people develop neurological symptoms that may include meningitis, meningoencephalitis, meningoencephalomyelitis, or paralysis, with mortality of 1–2% (Kaiser,
G. Venturi et al. / Journal of Virological Methods 134 (2006) 136–139
1999). Active immunization can successfully be used for containing TBE (Kunz, 2003). For testing immunity after TBE virus infection or for monitoring the immune response after TBE vaccination, the IgG enzyme-linked immunosorbent assay (ELISA) is normally used, due to its simplicity and ease of automation (Holzmann, 2003). An excellent correlation between ELISA IgG units and the antibody titers obtained by HI or NT has been demonstrated provided that there was no other exposure to flavivirus antigens except TBE virus/vaccination (Holzmann et al., 1996). In Italy, the province of Belluno is one of the most active TBE virus infection foci (Caruso et al., 1999), and immunization with anti-TBE vaccine of volunteers at risk of disease is being recommended until 1996. In this study, sera were examined from children residing in areas around Belluno in order to monitor the immune response after anti-TBE vaccination, and the correlation between ELISA, HI, and neutralizing antibodies was evaluated. For the assessment of neutralizing antibodies we optimized a plaque reduction neutralization test (PRNT), a simple method that has been used rarely in the past because of the difficulties of reproducibility with TBE virus. 2. Materials and methods 2.1. Patient sera Thirty-six healthy male and female children living in areas around Belluno, aged between 6 and 12 years, completed vaccination with three half-adult doses (0.25 ml) of FSME-Immuno vaccine, in the period June 2001–August 2002. Serum samples were collected 1–14 months (mean: 123 days) after the last vaccine dose, and sent to Belluno Hospital for the ELISA test, and to Virology Laboratory of the Italian Ministry of Health (Istituto Superiore di sanit`a) for HI and NT tests. 2.2. ELISA
137
strain ir968 (Verani et al., 1981), diluted 1:10 in serum-free maintenance medium. After 4 days of incubation, a cytopathic effect was observed and supernatant was collected, clarified and frozen in aliquots. Infectivity titration was performed by plaque assay using PS cells. 2.4. Plaque reduction neutralization test The assay was carried out in a six-well tissue culture plates with subconfluent PS cell monolayers (approximately 40–50% confluence). Sera were diluted 1:10 in serum-free maintenance medium, heat-inactivated, and titrated in duplicate in two-fold dilution steps (100 ul/well). Equal volumes (100 ul) of TBE virus dilution containing 80 PFU, and serum dilutions, were mixed, and incubated over night at 4 ◦ C. PS cells were counted and seeded, kept 2 h to attach in six-well tissue culture plates, and then infected with 200 ul of the virus–serum mixtures in duplicate. After 1 h incubation at 37 ◦ C and 5% CO2 , the inocula were aspirated and the wells were overlayed with a mixture of one part 2% Gum Tragacanth and one part 2× MEM supplemented with 2.5% inactivated FCS and 2% 1M HEPES. The plates were incubated at 37 ◦ C and 5% CO2 for 4 days, and then were stained with 1.5% crystal violet. A titration of TBE virus with three dilutions in duplicate (the working dilution, 1:2 and 1:8 dilutions) was performed in each assay and used as a control for the assay: those performing too low or too high virus doses were not accepted. A mouse ascitic fluid obtained after inoculation with TBE virus strain ir968 has been used as a positive control only when performing PRNT optimization experiments, together with an anti TBE-negative human serum as assayed in the HI test, as the negative control. Neutralizing antibody titers were calculated as the reciprocal of the serum dilution that gave an 80% reduction of the number of plaques (NT80 ), as compared to the virus control. Titers ≥10 were considered positive.
The IgG and IgM antibodies against TBE virus were detected in serum using a commercially available ELISA system (FSME IgG and IgM Immunozym, Progen Biotech GMBH, Heidelberg). Standard calibrators are supplied by the kit to obtain a reference curve, which allows a quantitative evaluation of anti-TBE IgG antibodies. Results are expressed in Vienna units per milliliter (VIEU/ml). Absorbance values <63 VIEU/ml are considered negative, values between 63 and 126 VIEU/ml are considered borderline, and values >126 VIEU/ml are considered positive, according to manufacturer’s instructions. Samples with an absorbance exceeding that of the reference curve were prediluted (1 + 1) with the FSME incubation buffer. The concentrations thus obtained had to be multiplied by the factor two. High level positive and low level positive control sera are supplied by the manufacture and added to each test.
2.5. Hemagglutination-inhibition test
2.3. Virus
In order to evaluate the correlation between NT and HI titers, and between ELISA IgG titers and NT or HI titers, Spearman and Kendall ranks correlation coefficients were computed. The analysis was performed by the statistical package Stata 8.
PS (porcine kidney) culture cells were infected with a 10% (w/v) suckling mouse brain suspension of the italian TBE virus
A standard HI test was employed, according to Clarke and Casals (1958). Briefly, sera were pre-absorbed with kaolin and goose erythrocytes, and tested subsequently at two-fold dilutions, starting at 1:10. Arcton antigen extracted from mouse brain infected with TBE virus (strain ir968) was used at eight hemagglutinating units. The antigen’s calibration was controlled within each assay. To assess the specificity of the reaction, sera were titrated in parallel against at least one different flavivirus that cross-reacts at lower titer (WN virus), and against Arbia virus antigen, as negative control. HI tests were carried out at pH 6.6 for TBE. 2.6. Statistical analysis
138
G. Venturi et al. / Journal of Virological Methods 134 (2006) 136–139
3. Results 3.1. ELISA test results All serum samples but one (patient 26) were negative by the IgM ELISA test. All serum samples were positive by the IgG ELISA test with titers ranging from 737 to 12006 VIEU/ml (mean: 6786.86 VIEU/ml). 3.2. Optimization of plaque reduction neutralization test Fig. 2. Correlation between ELISA anti-TBE IgG and HI antibody titers.
PS cells monolayers at different degrees of confluence were assayed for the formation of visible plaques when inoculated with TBE virus (ir968). Different incubation times were also tested. Visible plaques of 2–3 mm diameter, were reproducibly obtained with PS cells monolayers at low degree of confluence (40–50%), and 4 days of incubation. Eleven PRNTs were carried out for the titration of neutralizing antibodies: in all assays visible plaques were obtained. Ten PRNT assays performed virus doses ranging from 75 to 118 PFU (mean 93, standard deviation 15); one assay performing 145 PFU was not accepted. The mouse ascitic fluid obtained after inoculation with TBE virus strain ir968, used as positive control, had an NT antibody titer of 80; the human serum anti TBE-negative as assayed in the HI test, used as the negative control, showed an NT antibody titer <10.
Fig. 3. Correlation between ELISA anti-TBE IgG and neutralizing (NT80 ) antibody titers.
4. Discussion 3.3. Correlation between hemagglutination inhibition and plaque reduction neutralization test, and correlation between ELISA IgG antibody titers and HI or neutralizing antibody titers HI and neutralizing antibody titers between 10 and >1280 have been detected (Fig. 1). The correlation between NT80 and HI titers is consistently high (Spearman’s coefficient = 0.79 and Kendall’s tau = 0.67). The results obtained with the two methods are shown in Fig. 1. A good correlation was also observed between ELISA IgG antibody titers and both HI and neutralizing antibody titers (Spearman’s coefficient 0.9564 and 0.8422, respectively; Kendall’s tau 0.8554 and 0.6987, respectively), as shown in Figs. 2 and 3.
Fig. 1. Hemagglutination inhibition (HI) and neutralizing (NT80 : reciprocal of the serum dilution that gave an 80% reduction of the number of plaques in neutralization test) antibody titers.
As no treatment is available, immunization is the measure of choice for disease control of TBE in endemic areas. Individuals of all ages are thought to be equally susceptible to infection (Logar et al., 2000), and immunization against TBE is recommended for all individuals, including infants and children, who live in TBE endemic areas, or travel to countries that have a high risk of infection. In this study, serum samples of 36 children were examined. Anti-TBE vaccination had been required voluntarily for children mainly because of their recreational activities outdoors in TBE endemic areas. They thus received three half-adult doses of FSME-Immuno vaccine, as recommended by the Italian Ministry of Health (Circ. Min.Sanit`a n.10, 13-07-2000). The pediatric formulation of the vaccine was not available. In this study, children showed serum levels of anti-TBE IgG in ELISA test after the vaccination ranging from 737 to 12006 VIEU/ml (mean: 6786,86 VIEU/ml). These results are in good agreement with those reported in other studies (Eder and Kollaritsch, 2003; Kunz, 2003), in particular they confirm the efficacy of using half-adult doses in children. In one case a positive anti-TBE IgM ELISA result was obtained, but clinical evidence of TBE disease was not observed. The production and titers of HI and neutralizing antibodies in this population were studied. Most of the available data concerning the neutralizing antibody response after vaccination were obtained by Chiron Vaccines. One thousand TCID50 (50% tissue culture infectious doses) of TBE virus were used for the NT, and cell cultures microscopic screening for cytopathic effects was performed after 4–5 days of incubation with
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serum–virus mixtures (Klockmann et al., 1991). Other methods have been described for the detection of neutralizing antibodies: Vene et al. (1998) developed a rapid fluorescent focus inhibition test (RFFIT), using 50 FFD50 (50% focus forming dose) of virus and visualization of virus foci in immunofluorescence after 24 h of incubation of cells with serum–virus mixtures. Neutralizing titers obtained with RFFIT were almost identical to those obtained with a standard PRNT, which was less reproducible. A good correlation with HI results was also reported. Holzmann et al. (1996) performed the NT with 1000 PFU/ml of virus, and on day 4 post infection the supernatants were tested for the presence or absence of viral antigen in a four-layer ELISA system. They reported a good correlation between ELISA IgG units and the antibody titers obtained by HI or NT, provided that there was no other exposure to flavivirus antigens except TBE virus/vaccination. In this study, visible plaques with TBE virus were obtained using subconfluent PS cell monolayers, and thus a simple PRNT was optimized and validated in the population of vaccinated children. Visible plaques were not obtained with Vero cells, which have been used for PRNTs in other studies (Calisher et al., 1989; Vene et al., 1998). In the present study HI and NT80 titers ranged between very low (10) and high (>1280) levels. A very low NT80 and HI titer of 10–20 was observed in 5/36 and 3/36 serum samples, respectively (Fig. 1). A good correlation between HI and NT80 titers (Fig. 1), and between ELISA IgG antibody titers and HI or NT80 titers (Figs. 2 and 3), was observed. A statistically significant negative correlation between antibody titers and days elapsed from last vaccine dose could not be demonstrated in this small population (data not shown). The kinetic curve of persistence of neutralizing TBE antibodies was investigated recently in order to assess if the 3 years booster intervals recommended currently should be extended. The results obtained in cross-sectional studies show that following four immunization doses protective antibodies can be detected far beyond a period of 3 years, thus supporting strongly the reconsideration of currently recommended booster intervals (Rendi-Wagner et al., 2004a,b; Zent et al., 2004). The availability of a NT will allow us to monitor the persistence of immunity after the completion of the vaccination cycle. Moreover, the NT, which is undertaken in very few laboratories in Europe is very important because of the increased contacts with tropical and subtropical countries which has augmented the risk of acquiring other flavivirus infections or of being vaccinated against YF or JE (Monath, 1994). Acknowledgment The authors thank Dr. Giada Minelli for kind help with statistical analysis. References Calisher, C.H., Karabatsos, N., Dalrymple, J.M., Shope, R.E., Porterfield, J.S., Westaway, E.G., Brandt, W.E., 1989. Antigenic relationships between
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