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Protection against European isolates of tick-borne encephalitis virus after vaccination with a new tick-borne encephalitis vaccine
Protection against European isolates of tick-borne encephalitis virus after vaccination with a new tick-borne encephalitis vaccine U . K l o c k m a n n *,~, K . K f i v a n e c * , J . R . S t e p h e n s o n ++ a n d J. H i l f e n h a u s *
A highly pur!fi'ed, &activated tick-borne encephalitis (TBE) virus particle vaccine has been developed. In this stud)' we report on the efficacy of this new vaccine to protect against TBE virus isolates from different geographical areas q[ Europe and the Asian part oj the USSR. Keywords: Tick-borne encephalitis: virus particle vaccine: mice
INTRODUCTION
MATERIALS
The viruses of the tick-borne encephalitis (TBE) complex of flaviviruses cause the most clinically important arthropod-transmitted disease in Europe and in the USSR and has raised a significant health problem in several countries, especially Austria and Czechoslovakia 1'2. Virus strains of TBE can be assigned to a Western subtype which occurs in every European country with the exception of the UK, Spain, Portugal and the Benelux countries and is mainly transmitted by 1xodes ricinus. A Far Eastern subtype is prevalent in the eastern part of the USSR, with lxodes persulcatus as its main vector a. A closely related virus has been shown to be the causative agent of louping ill, the animal form of the disease which is widespread in the UK and is found in several domestic and wild animals including birds 4 s. Although louping ill virus does not normally cause severe infections of man, several human infections have been described with some proving fatal 9. Recently, a new highly purified, inactivated TBE virus particle vaccine was developed 1° which has been shown in a phase I/II clinical trial to offer an effective and well-tolerated prophylactic means against the disease ~. The efficacy of this vaccine has been well documented against the virus from which it was derived ~o, ~~ but until now we have not reported on its potency to protect against virus isolates from different geographical regions. In order to further assess the quality of this vaccine we evaluated its protective capacity against different isolates of the virus.
Viruses
*Behringwerke AG, Department of Microbiology Research, PO Box 11 40, D-3550 Marburg 1, Germany. tCzechoslovak Academy of Sciences, Department of Parasitology, Branisovska 31, 37005 Ceske Budejovice, Czechoslovakia. *PHLS Centre for Applied Microbiology and Research, Division of Biologics, Porton Down, Salisbury, Wiltshire SP4 0JG, UK. ~To whom correspondence should be addressed. (Received 18 June 1990; revised and accepted 13 September 1990) 0264-410x/91/030210433 1991 Butterworth-HeinemannLtd 210 Vaccine, Vol. 9, March 1991
AND
METHODS
Isolates of TBE virus (TBEV) used in this study are listed in Table I. TBEV isolate K 23 representing the vaccine strain was obtained from infected ticks caught in the municipal forest of Karlsruhe, Germany, and was provided by R. Ackermann of the Neurological Clinic, University of Cologne, Germany. J.S. Porterfield from the Sir William Dunn School of Pathology, University of Oxford, UK, generously supplied the Austrian isolate IX 10 which is closely related to the Austrian prototype strain Neud6rfl ~2. The Yugoslavian isolate H 60 was kindly provided by Emoke Ferenci, National Institute of Hygiene, Budapest, Hungary. The Scottish isolate of louping ill virus (ADRA/3) was obtained from infected lamb brain and was provided by H.W. Reid, Moredun Institute, Edinburgh, UK. The other TBEV isolates form part of a strain collection of one of us (K.K.). The Russian isolates Absettarov (Western subtype) and Sofyn (prototype strain of the Far Eastern subtype) were both isolated from infected man, the first one from the blood of a sick child and the second one from the brain of a patient who died of encephalitis in the Primorye Territory. The Czechoslovakian prototype strain Hypr was derived from the blood of a patient bitten by a tick in Brno, Moravia, and was originally provided by the Institute of Virology, Slovak Academy of Sciences, Bratislava. Isolate Petracova was obtained from a 17-year-old woman at Tachov, West Bohemia. Cg 1 was isolated from the stomach of a bank vale (Clethrionomys glareolus) at Orlik, Bohemia, by J. Kolman, Czechoslovak Academy of Sciences, Prague, CSFR. Field isolate 274/II (Ceske Budejovice) was obtained from infected ticks by K.K. and does not exhibit an extended mouse brain passage history. Further characteristics of the isolates can be taken from the references cited. TBE vaccine and immunizations TBE vaccines were prepared
as described
pre-
A new TBE vaccine: U. Klockmann et al. Table 1
Origins of the TBEV isolates analysed
Isolate designation
Geographic origin
Source of isolation
Year of isolation
Reference
K 23 IX 10 H 60 Hypr Trpisovsky Cg 1 Petracova Gbelce 274/11 Klodobok Dobrostan Absettarov (Western subtype) Sofyn (Far Eastern subtype) ADRA/3 (Iouping ill)
Germany Austria Yugoslavia CSFR CSFR CSFR CSFR CSFR CSFR Poland Bulgaria USSR USSR UK
Ixodes ricinus Ixodes ricinus Ixodes ricinus Human blood Human blood Clethrionomys glareolus Human blood Ixodes ricinus Ixodes ricinus Ixodes ricinus Ixodes ricinus Human blood Human brain Lamb brain
1975 1971 1981 1953 1954 1957 1959 1980 1987 1953 1967 1951 1937 1979
17
Table 2
18 19 20 21 22 23 24, 25 16
Surviving mice after being vaccinated and challenged with different TBEV isolates Protection (% survivors) after immunization with TBE vaccines containing: 1.5
0.15
TBEI isolates K 23 IX 10 H 60 Gbelce Petracova Klodobok Dobrostan Absettarov Sofyn Louping ill
0.015
0.0015
EDso (rig)
50 70 70 50 70 30 80 20 30 60
10 0 0 0 20 10 30 0 10 0
2.3 1.8 1.8 2.8 1.7 4.5 0.8 7.5 3.4 2.3
#g m1-1 TBE antigen 100 100 90a 100 100 100 100 100 100 100
100 100 100 100 80 90 90 100 100 100
Groups of ten mice were immunized and challenged as described in Materials and methods. The ED~ (effective dose~) indicates the amount of antigen (in nanograms) which is sufficient to protect 50% of vaccinated animals aOne mouse in this group died immediately after inoculation, probably from shock
viouslylO,11. Immunizations of 17 healthy male volunteers aged 20-50 years consisted of two vaccinations given at an interval of 4 weeks and a third one 10 months later. Blood samples of vaccinees were taken 4 weeks after the third immunization. Immunizations of mice were carried out in groups of ten animals by administering 0.2 mi per mouse of serial ten-fold dilutions of TBE vaccines containing 0.2% AI(OH)3 twice at an interval of 14 days. Blood samples were taken 1 week after the second vaccination. Neutralization test The neutralization test (NT) was essentially carried out as described previously ~3. Briefly, dilutions of serum were mixed with an equal volume of each TBEV isolate adjusted to contain 100-1000 50% tissue culture infectious doses. Serum-virus mixtures were tested in quintuple in the human cell line A 549 for infectious TBEV. Serum-neutralizing antibodies were determined as the reciprocal of the highest serum dilution that inhibited the cytopathic effect in 50% of the cells.
RESULTS Table 2 shows that vaccinated mice can be protected against a challenge-infection of 100 mouse LDso of different TBEV isolates of the Western subtype, an isolate of the Far Eastern subtype (Sofyn), and a TBEV related flavivirus causing animal diseases (louping ill virus). It can be seen that the protection rates are dependent on the amount of antigen administered. Table 3 shows that sera of human vaccinees are capable of neutralizing all the TBEV isolates tested including louping ill virus. The highest neutralization titres were obtained against TBEV isolate K 23 representing the vaccine virus. The neutralizing capacity of sera against the isolates from Austria and Czechoslovakia move in the same range and is above that against isolates from the more eastern parts of Europe (isolate Dobrostan from Bulgaria; isolate Absettarov from the European part of the USSR). Surprisingly, neutralization titres against the isolate from the Far Eastern part of the USSR (Sofyn) were not lower than against the isolate from the European part of this country.
Mouse challenge potency test Mice (strain: N M R I outbred; sex: female; age: 22 days; body weight: ~ :20 g) were immunized as described above. Eight days after the second vaccination the mice were challenged by intraperitoneal inoculation of 100 mouse LDso of each TBEV isolate. The isolates were additionally titrated in mice to determine the exact challenge virus dose administered. Percentages of surviving animals were determined 20 days postinfection.
DISCUSSION Previously, we have shown that our new TBE vaccine raises a protective immune response in laboratory animals 1° and induces high levels of neutralizing antibodies in human vaccinees 11,~3. In both studies, however, immune responses elicited by this vaccine were determined against the homologous TBEV isolate. As
Vaccine, Vol. 9, M a r c h 1991
211
A n e w TBE vaccine: U. K l o c k m a n n et al. Table 3
Neutralizing antibody titres in human vaccinees against different TBEV isolates
TBEV isolates
Minimum
Maximum
Median
GMT
SF
K 23 IX 10 Hypr Trpisovsky Pet racova 274/11 Gbelce Cg 1 Dobrostan Absettarov Sofyn Louping ill
129 74 28 43 43 49 37 21 21 21 21 37
1023 295 295 295 447 195 295 170 56 112 97 97
295 112 129 112 129 112 112 49 25 49 65 74
307 134 110 114 129 110 98 55 31 46 56 66
2.05 1.62 2.27 1.83 2.04 1.78 2.03 2.18 1.43 1.68 1.69 1.39
Seventeen healthy male volunteers were immunized three times with TBE vaccines as described in Materials and methods. Sera taken 4 weeks after the third vaccination were assayed for neutralizing antibodies against the TBEV isolates listed above G M T = g e o m e t r i c mean titre; S F - s c a t t e r factor (a measure for the variation of the GMT)
antigenic variations of TBEV isolates from different geographic areas may occur we investigated whether this new vaccine is also protective against other TBEV isolates obtained. We were able to demonstrate that with this vaccine mice were protected from a lethal challenge with several TBEV isolates obtained over a period of more than 30 years from all over Europe and the Asian part of the USSR. In addition, we could show that antibodies induced by vaccination of human volunteers neutralized all isolates tested. Thus this TBE vaccine proved to be efficacious against various TBEV isolates including louping ill virus, a natural pathogen of sheep. In principle this result is not unexpected because Heinz and Kunz 14, Heinz et al. 15 and Stephenson et al. 1~ demonstrated that the envelope glycoprotein of European isolates of TBEV, the major and most likely single determinant for the induction of protective immunity, exhibits a remarkable homogeneity. It is not subjected to a high selective pressure in nature as is characteristic for the glycoproteins of influenza viruses, paramyxoviruses and ~-viruses. However, as the neutralizing antibody titres of our human vaccinees were found to be lower against the heterologous TBEV isolates than against the homologous isolate minor differences between neutralizing epitopes of various isolates must exist.
8 9 10
11
12
13
14
15
16
17
ACKNOWLEDGEMENTS
18
The authors thank S. Jacobs, H. Eife, B. H6cker and A. Kliebe for their skilful technical assistance.
19 20
REFERENCES 1 2
3 4 5
6 7
212
Heinz, F.X. Epitope mapping of flavivirus glycoproteins. Adv. Virus Res. 1986, 31, 103 Kunz, C. Die Fr0hsommer-Meningoencephalitis. In Klinische Virologie (Eds Gsell, O. et al.) Urban & Schwarzenberg, MLinchen, 1986, pp. 275-285 Clarke, D.H. Further studies on antigenic relationship among the viruses of the group B tick-borne complex. Bull. WHO 1964, 31,45 Bannatyne, C.L., Wilson, R.L., Reid, H.W., Buxton, D. and Pow, I. Louping ill virus infection of pigs. Vet. Rec. 1980, 106, 13 Doherty, P.C., Vantsis, J.T. and Hart, R. Louping-ill encephalomyelitis in the sheep. VI. Infection of the 120-day foetus. J. Comp. Path. 1972, 82, 385 Mackenzie, C.P. Recovery of a dog from Iouping-ill. J. Small Anita. Pract. 1982, 23, 233 Reid, H.W., Dungan, J.S., Phillips, J.D.P., Moss, R. and Watson, A. Studies on Iouping ill virus (flavivirus group) in wild red grouse (Lagopus lagopus scoticus). J. Hyg. 1978, 81,321
V a c c i n e , Vol. 9, M a r c h 1991
21 22
23
24
25
Timoney, P.J. Susceptibility of the horse to experimental inoculation with Iouping ill virus. J. Comp. Path. 1980, 98, 73 Williams H. and Thorburn, H. Serum antibodies to Iouping ill virus. Scott. Med. J. 1962, 7, 353 Klockmann, U., Bock, H.L., Franke, V., Hein, B., Reiner, G. and Hilfenhaus, J. Preclinical investigations of the safety, immunogenicity and efficacy of a purified, inactivated tick-borne encephalitis vaccine. J. Biol. Stand. 1989, 17, 331 Bock, H.L., Klockmann, U., J0ngst, C., SchindeI-KiJnzel, F., Theobald, K. and Zerban, R. A new vaccine against tick-borne encephalitis: Initial trial in man including a dose-response study. Vaccine 1990, 8, 22 Heinz, F.X. and Kunz, C. Protease treatment and chemical crosslinking of a flavivirus: tick-borne encephalitis virus. Arch. Virol. 1979, 68, 207 Klockmann, U., Bock, H.L., Kwasny, H., Praus, M., Cihlova, V., Tomkova, E. and Krivanec, K. Humoral immunity against tick-borne encephalitis virus following manifest disease and active immunization. Vaccine 1991, 9, 42 Heinz, F.X. and Kunz, C. Homogeneity of the structural gtycoprotein from European isolates of tick-borne encephalitis virus: Comparison with other flaviviruses. J. Gen. Virol. 1981, 57, 263 Heinz, F.X., Berger, R., Tuma, W. and Kunz, C. A topological and functional model of epitopes on the structural glycoprotein of tick-borne encephalitis virus defined by monoclonal antibodies. Virology 1983, 126, 525 Stephenson, J.R, Lee, J.M. and Wilton-Smith, P.D. Antigenic variation among members of the tick-borne encephalitis complex. J. Gen. Virol. 1984, 65, 81 Rhese-KLipper, B., Danielova, V., Klenk, W., Abar, B. and Ackermann, R. The isolation of central European encephalitis (tick-borne encephalitis) virus from Ixodes ricinus (L.) ticks in Southern Germany. Zbl. Bakt. Hyg. I. Abt. Orig. 1978, .,I),242, 148 Pospisil, L., Jandasek, L. and Pesek, J. The isolation of new strains of meningoencephalitis virus in the Brno region in the summer of 1953. Lek. Listy 1954, 9, 3 Strauss, J. and Kolman, J. Isolation and serological identification of 16 strains of Czechoslovak TBE virus. C. Epidem. 1954, 3, 67 Kozuch, O., Chunikhin, S.P., Gresikova, M., Nosek, J., Kurenkov, V.B. and Lysy, J. The ecological marker 'Cg' of tick-borne encephalitis virus in different rodent species. Acta Virologica 1981, 25, 219 Kozuch, O., Nosek, J. and Lysy, J. Natural focus of tick-borne encephalitis in Soutfiern Slovakia. Biologica (Bratisl.) 1980, 37 Przesmycki, F., Taytsch, Z., Semkov, R. and WalentynoviczStanczyk, R. Investigation of tick-borne encephalitis. I. Biology of tick-borne encephalitis viral strains isolated in Poland. Przegl. epidem. 1954, 8, 204 Ilyenko, V.I. A contribution to the methods of producing a tissue-culture formolized vaccine against tick-borne encephalitis. Acta Virologica 1959, 4, 37 Pletnev, A.G., Yamshchikov, V.F. and Blinov, V.M. Nucleotide sequence of the genome and complete amino acid sequence of the polyprotein of tick-borne encephalitis virus. Virology 1990,174, 250 Smorodintseff, A.A., Kagan, N.W., Levkovitsch, E.N. and Dankovskij, N.L. Experimenteller und epidemiologischer Beitrag zur aktiven Immunisierung gegen die FrLihling-Sommer-Zechenencephalitis. Arch. ges. Virusforsch. 1941, 2, 1
A highly pur!fi'ed, &activated tick-borne encephalitis (TBE) virus particle vaccine has been developed. In this stud)' we report on the efficacy of this new vaccine to protect against TBE virus isolates from different geographical areas q[ Europe and the Asian part oj the USSR. Keywords: Tick-borne encephalitis: virus particle vaccine: mice
INTRODUCTION
MATERIALS
The viruses of the tick-borne encephalitis (TBE) complex of flaviviruses cause the most clinically important arthropod-transmitted disease in Europe and in the USSR and has raised a significant health problem in several countries, especially Austria and Czechoslovakia 1'2. Virus strains of TBE can be assigned to a Western subtype which occurs in every European country with the exception of the UK, Spain, Portugal and the Benelux countries and is mainly transmitted by 1xodes ricinus. A Far Eastern subtype is prevalent in the eastern part of the USSR, with lxodes persulcatus as its main vector a. A closely related virus has been shown to be the causative agent of louping ill, the animal form of the disease which is widespread in the UK and is found in several domestic and wild animals including birds 4 s. Although louping ill virus does not normally cause severe infections of man, several human infections have been described with some proving fatal 9. Recently, a new highly purified, inactivated TBE virus particle vaccine was developed 1° which has been shown in a phase I/II clinical trial to offer an effective and well-tolerated prophylactic means against the disease ~. The efficacy of this vaccine has been well documented against the virus from which it was derived ~o, ~~ but until now we have not reported on its potency to protect against virus isolates from different geographical regions. In order to further assess the quality of this vaccine we evaluated its protective capacity against different isolates of the virus.
Viruses
*Behringwerke AG, Department of Microbiology Research, PO Box 11 40, D-3550 Marburg 1, Germany. tCzechoslovak Academy of Sciences, Department of Parasitology, Branisovska 31, 37005 Ceske Budejovice, Czechoslovakia. *PHLS Centre for Applied Microbiology and Research, Division of Biologics, Porton Down, Salisbury, Wiltshire SP4 0JG, UK. ~To whom correspondence should be addressed. (Received 18 June 1990; revised and accepted 13 September 1990) 0264-410x/91/030210433 1991 Butterworth-HeinemannLtd 210 Vaccine, Vol. 9, March 1991
AND
METHODS
Isolates of TBE virus (TBEV) used in this study are listed in Table I. TBEV isolate K 23 representing the vaccine strain was obtained from infected ticks caught in the municipal forest of Karlsruhe, Germany, and was provided by R. Ackermann of the Neurological Clinic, University of Cologne, Germany. J.S. Porterfield from the Sir William Dunn School of Pathology, University of Oxford, UK, generously supplied the Austrian isolate IX 10 which is closely related to the Austrian prototype strain Neud6rfl ~2. The Yugoslavian isolate H 60 was kindly provided by Emoke Ferenci, National Institute of Hygiene, Budapest, Hungary. The Scottish isolate of louping ill virus (ADRA/3) was obtained from infected lamb brain and was provided by H.W. Reid, Moredun Institute, Edinburgh, UK. The other TBEV isolates form part of a strain collection of one of us (K.K.). The Russian isolates Absettarov (Western subtype) and Sofyn (prototype strain of the Far Eastern subtype) were both isolated from infected man, the first one from the blood of a sick child and the second one from the brain of a patient who died of encephalitis in the Primorye Territory. The Czechoslovakian prototype strain Hypr was derived from the blood of a patient bitten by a tick in Brno, Moravia, and was originally provided by the Institute of Virology, Slovak Academy of Sciences, Bratislava. Isolate Petracova was obtained from a 17-year-old woman at Tachov, West Bohemia. Cg 1 was isolated from the stomach of a bank vale (Clethrionomys glareolus) at Orlik, Bohemia, by J. Kolman, Czechoslovak Academy of Sciences, Prague, CSFR. Field isolate 274/II (Ceske Budejovice) was obtained from infected ticks by K.K. and does not exhibit an extended mouse brain passage history. Further characteristics of the isolates can be taken from the references cited. TBE vaccine and immunizations TBE vaccines were prepared
as described
pre-
A new TBE vaccine: U. Klockmann et al. Table 1
Origins of the TBEV isolates analysed
Isolate designation
Geographic origin
Source of isolation
Year of isolation
Reference
K 23 IX 10 H 60 Hypr Trpisovsky Cg 1 Petracova Gbelce 274/11 Klodobok Dobrostan Absettarov (Western subtype) Sofyn (Far Eastern subtype) ADRA/3 (Iouping ill)
Germany Austria Yugoslavia CSFR CSFR CSFR CSFR CSFR CSFR Poland Bulgaria USSR USSR UK
Ixodes ricinus Ixodes ricinus Ixodes ricinus Human blood Human blood Clethrionomys glareolus Human blood Ixodes ricinus Ixodes ricinus Ixodes ricinus Ixodes ricinus Human blood Human brain Lamb brain
1975 1971 1981 1953 1954 1957 1959 1980 1987 1953 1967 1951 1937 1979
17
Table 2
18 19 20 21 22 23 24, 25 16
Surviving mice after being vaccinated and challenged with different TBEV isolates Protection (% survivors) after immunization with TBE vaccines containing: 1.5
0.15
TBEI isolates K 23 IX 10 H 60 Gbelce Petracova Klodobok Dobrostan Absettarov Sofyn Louping ill
0.015
0.0015
EDso (rig)
50 70 70 50 70 30 80 20 30 60
10 0 0 0 20 10 30 0 10 0
2.3 1.8 1.8 2.8 1.7 4.5 0.8 7.5 3.4 2.3
#g m1-1 TBE antigen 100 100 90a 100 100 100 100 100 100 100
100 100 100 100 80 90 90 100 100 100
Groups of ten mice were immunized and challenged as described in Materials and methods. The ED~ (effective dose~) indicates the amount of antigen (in nanograms) which is sufficient to protect 50% of vaccinated animals aOne mouse in this group died immediately after inoculation, probably from shock
viouslylO,11. Immunizations of 17 healthy male volunteers aged 20-50 years consisted of two vaccinations given at an interval of 4 weeks and a third one 10 months later. Blood samples of vaccinees were taken 4 weeks after the third immunization. Immunizations of mice were carried out in groups of ten animals by administering 0.2 mi per mouse of serial ten-fold dilutions of TBE vaccines containing 0.2% AI(OH)3 twice at an interval of 14 days. Blood samples were taken 1 week after the second vaccination. Neutralization test The neutralization test (NT) was essentially carried out as described previously ~3. Briefly, dilutions of serum were mixed with an equal volume of each TBEV isolate adjusted to contain 100-1000 50% tissue culture infectious doses. Serum-virus mixtures were tested in quintuple in the human cell line A 549 for infectious TBEV. Serum-neutralizing antibodies were determined as the reciprocal of the highest serum dilution that inhibited the cytopathic effect in 50% of the cells.
RESULTS Table 2 shows that vaccinated mice can be protected against a challenge-infection of 100 mouse LDso of different TBEV isolates of the Western subtype, an isolate of the Far Eastern subtype (Sofyn), and a TBEV related flavivirus causing animal diseases (louping ill virus). It can be seen that the protection rates are dependent on the amount of antigen administered. Table 3 shows that sera of human vaccinees are capable of neutralizing all the TBEV isolates tested including louping ill virus. The highest neutralization titres were obtained against TBEV isolate K 23 representing the vaccine virus. The neutralizing capacity of sera against the isolates from Austria and Czechoslovakia move in the same range and is above that against isolates from the more eastern parts of Europe (isolate Dobrostan from Bulgaria; isolate Absettarov from the European part of the USSR). Surprisingly, neutralization titres against the isolate from the Far Eastern part of the USSR (Sofyn) were not lower than against the isolate from the European part of this country.
Mouse challenge potency test Mice (strain: N M R I outbred; sex: female; age: 22 days; body weight: ~ :20 g) were immunized as described above. Eight days after the second vaccination the mice were challenged by intraperitoneal inoculation of 100 mouse LDso of each TBEV isolate. The isolates were additionally titrated in mice to determine the exact challenge virus dose administered. Percentages of surviving animals were determined 20 days postinfection.
DISCUSSION Previously, we have shown that our new TBE vaccine raises a protective immune response in laboratory animals 1° and induces high levels of neutralizing antibodies in human vaccinees 11,~3. In both studies, however, immune responses elicited by this vaccine were determined against the homologous TBEV isolate. As
Vaccine, Vol. 9, M a r c h 1991
211
A n e w TBE vaccine: U. K l o c k m a n n et al. Table 3
Neutralizing antibody titres in human vaccinees against different TBEV isolates
TBEV isolates
Minimum
Maximum
Median
GMT
SF
K 23 IX 10 Hypr Trpisovsky Pet racova 274/11 Gbelce Cg 1 Dobrostan Absettarov Sofyn Louping ill
129 74 28 43 43 49 37 21 21 21 21 37
1023 295 295 295 447 195 295 170 56 112 97 97
295 112 129 112 129 112 112 49 25 49 65 74
307 134 110 114 129 110 98 55 31 46 56 66
2.05 1.62 2.27 1.83 2.04 1.78 2.03 2.18 1.43 1.68 1.69 1.39
Seventeen healthy male volunteers were immunized three times with TBE vaccines as described in Materials and methods. Sera taken 4 weeks after the third vaccination were assayed for neutralizing antibodies against the TBEV isolates listed above G M T = g e o m e t r i c mean titre; S F - s c a t t e r factor (a measure for the variation of the GMT)
antigenic variations of TBEV isolates from different geographic areas may occur we investigated whether this new vaccine is also protective against other TBEV isolates obtained. We were able to demonstrate that with this vaccine mice were protected from a lethal challenge with several TBEV isolates obtained over a period of more than 30 years from all over Europe and the Asian part of the USSR. In addition, we could show that antibodies induced by vaccination of human volunteers neutralized all isolates tested. Thus this TBE vaccine proved to be efficacious against various TBEV isolates including louping ill virus, a natural pathogen of sheep. In principle this result is not unexpected because Heinz and Kunz 14, Heinz et al. 15 and Stephenson et al. 1~ demonstrated that the envelope glycoprotein of European isolates of TBEV, the major and most likely single determinant for the induction of protective immunity, exhibits a remarkable homogeneity. It is not subjected to a high selective pressure in nature as is characteristic for the glycoproteins of influenza viruses, paramyxoviruses and ~-viruses. However, as the neutralizing antibody titres of our human vaccinees were found to be lower against the heterologous TBEV isolates than against the homologous isolate minor differences between neutralizing epitopes of various isolates must exist.
8 9 10
11
12
13
14
15
16
17
ACKNOWLEDGEMENTS
18
The authors thank S. Jacobs, H. Eife, B. H6cker and A. Kliebe for their skilful technical assistance.
19 20
REFERENCES 1 2
3 4 5
6 7
212
Heinz, F.X. Epitope mapping of flavivirus glycoproteins. Adv. Virus Res. 1986, 31, 103 Kunz, C. Die Fr0hsommer-Meningoencephalitis. In Klinische Virologie (Eds Gsell, O. et al.) Urban & Schwarzenberg, MLinchen, 1986, pp. 275-285 Clarke, D.H. Further studies on antigenic relationship among the viruses of the group B tick-borne complex. Bull. WHO 1964, 31,45 Bannatyne, C.L., Wilson, R.L., Reid, H.W., Buxton, D. and Pow, I. Louping ill virus infection of pigs. Vet. Rec. 1980, 106, 13 Doherty, P.C., Vantsis, J.T. and Hart, R. Louping-ill encephalomyelitis in the sheep. VI. Infection of the 120-day foetus. J. Comp. Path. 1972, 82, 385 Mackenzie, C.P. Recovery of a dog from Iouping-ill. J. Small Anita. Pract. 1982, 23, 233 Reid, H.W., Dungan, J.S., Phillips, J.D.P., Moss, R. and Watson, A. Studies on Iouping ill virus (flavivirus group) in wild red grouse (Lagopus lagopus scoticus). J. Hyg. 1978, 81,321
V a c c i n e , Vol. 9, M a r c h 1991
21 22
23
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