Microbiology and Tumorbiology Center, Karolinska Institute, S-171 77 Stockholm, Sweden1
Department of Vaccine Research, Swedish Institute for Infectious Disease Control, S-105 21 Stockholm, Sweden2
Department of Veterinary Pathology, Faculty of Veterinary Medicine, University College Dublin, Dublin 4, Ireland3
Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland4
Author for correspondence: Gregory Atkins. Fax +353 1 6799294. e-mail gatkins{at}tcd.ie
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study we have tested four different types of vaccine in a setting where immunized mice were challenged with three LIV strains of graded virulence to reveal differences in the efficacy of protection. Two of the vaccines were prototype Semliki Forest virus (SFV)-based vaccines consisting of recombinant particles or a layered naked DNA/RNA replicon vaccine (expressing SFV RNA from a cytomegalovirus promotor; Berglund et al.,1998b ). These were compared to a commercially available formaldehyde-inactivated virus preparation, which is available for animals but has not been licensed for human use. The fourth vaccine used was a standard naked DNA vaccine. Recombinant vaccines expressed the LIV prME spike protein complex, which contains neutralizing antibody epitopes (Gao et al.,1994
), and/or the nonstructural NS1 protein. Both antigens have previously been shown to induce strong humoral and cellular immune responses, which can be protective in mice (Schlesinger et al.,1986
; Jacobs et al.,1992
; Konishi et al.,1992
; Heinz et al.,1995
; Pugachev et al.,1995
; Schmaljohn et al.,1997
; Colombage et al.,1998
; Lin et al.,1998
; Timofeev et al., 1998
; Fleeton et al.,1999
).
In the standard naked DNA vaccine, the antigens are expressed from a cytomegalovirus immediate early promoter (Koprowski & Weiner, 1998 ); such naked DNA vaccines have previously been shown to be protective against flavivirus infection (Schmaljohn et al.,1997
; Colombage et al.,1998
; Lin et al.,1998
).
In the SFV vectors, the subgenomic RNA encoding the virus structural proteins, which are expressed at high level during infection, has been substituted with the antigen-encoding gene (Liljeström & Garoff, 1991 ; Berglund et al., 1993
; Tubulekas et al.,1997
; Atkins et al.,1999
; Berglund et al.,1999
). Replication of SFV RNA leads to cell death by apoptosis (Glasgow et al.,1997
, 1998
; Kohno et al.,1998
), which may enhance cross-priming of dendritic cells (Albert et al., 1998a
, b
) and may also constitute a safety feature that removes the vaccine from tissue.
Two versions of the SFV vector were used. In the layered DNA/RNA system, the SFV vector replicon (encoding replicase and antigen) is placed under the control of a cytomegalovirus immediate early promoter. Injection of purified preparations of such naked plasmid DNA has been shown to induce strong immune responses (Berglund et al., 1998a , b
; Smerdou & Liljeström, 1999a
). Recombinant SFV particles, on the other hand, are prepared by packaging in vitromade recombinant RNAs into virions, using a helper system which provides the SFV structural proteins (Smerdou & Liljeström, 1999b
). A number of studies have shown that vaccination with such particles leads to strong and protective immune responses (Zhou et al.,1994
, 1995
; Mossman et al.,1996
; Berglund et al.,1997
, 1999
; Fleeton et al.,1999
). Both types of vaccine will result in the same pattern of replication and antigen expression upon entry into a cell.
In this study we have used the stringent LIV challenge model to test the comparative protective efficacy of different vaccine constructs expressing the LIV prME and NS1 antigens. This model indicates that vaccines based on recombinant particles induce better protective responses than the other vaccines tested.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction and expression of LIV antigens from plasmid vaccines.
Plasmids pSFV-prME, pSFV-NS1 (Fleeton et al., 1999 ) and pSCII-LiprME, containing cloned genes derived from the LI/369 strain of LIV which is closely related to LI/31 (Shiu et al., 1992
), have been described previously. All plasmids for immunization were based on the pBK-CMV vector backbone (Stratagene). Construction of pBK-LacZ and pBK-SFV-LacZ has been described earlier (Berglund et al., 1998b
). pBK-SFV-prME and pBK-SFV-NS1 were constructed as follows: the AgeISpeI fragment from pBK-SFV-LacZ was removed and replaced with an AgeISpeI fragment containing prME or NS1 sequences (including 5'and 3' flanking pSFV vector sequences) from pSFV-prME or pSFV-NS1 respectively. To construct pBK-prME and pBK-NS1 plasmids, the laci region of pBK-CMV was first removed by digestion with SpeI and NheI followed by religation. A BamHIXmaI fragment from pSFV-NS1 was inserted into BamHI/XmaI-cut pBK-CMV vector. A SmaIStuI fragment from pBK-SFV-NS1 was subsequently ligated into the SmaI site of pBK-NS1 in order to generate the same sequence at the 3' end of the NS1 coding sequence as pBK-SFV-NS1. A BglII fragment from pSCII-LIprME was ligated into the BamHI site of pBK-CMV to generate pBK-prME. All plasmid preparations for immunization of mice and transfection of BHK cells were prepared using an endotoxin-free plasmid purification kit (Qiagen).
Metabolic labelling of transfected cells with [35S]methionine was done essentially as previously described (Berglund et al., 1998b ) using the Effectene lipofection kit (Qiagen). Briefly, transfected cells were incubated for 20 h before radiolabelling for 30 min. Fresh medium containing 10x the normal amount of methionine was added and cells were incubated further for various times. Cell lysates and medium samples were analysed by immunoprecipation followed by SDSPAGE (Fleeton et al., 1999
). At each time-point the number of transfected cells expressing either prME or NS1 was monitored by indirect immunofluorescence staining.
Production of recombinant virus.
Packaging of recombinant RNA encoding either prME or NS1 (Fleeton et al., 1999 ) into rSFV particles was done using a two-helper RNA (Smerdou & Liljeström, 1999b
). Briefly, BHK cells were co-transfected with recombinant RNA and two helper RNAs, one of which encodes the SFV capsid protein, the other for the SFV envelope proteins. After 24 h incubation, medium containing recombinant virus stocks was harvested. Indirect immunofluorescence of infected BHK cells was performed to determine the titre of the recombinant virus stocks (Salminen et al., 1992
).
Antibody assays.
Antibodies (total IgG) in serum from immunized mice were detected by whole cell ELISA as previously described (Fleeton et al., 1999 ). Briefly, BHK cells were transfected with rSFV-prME or rSFV-NS1 RNA, plated onto 96-well plates and incubated overnight at 37 °C, 5% CO2. Cells were then fixed with methanolacetone (1:1) and incubated in blocking buffer before addition of twofold serial dilutions of mouse serum samples. The secondary antibody was alkaline phosphatase-conjugated anti-IgG. Cut-off titres were calculated as the reciprocal of the final dilution giving an absorbance (A) exceeding 5 standard deviations above the mean A (mean+5SD) from a panel of 1020 mock-immunized mice.
A fluorescent focus inhibition test described previously (Vene et al., 1998 ) was used to detect LIV-neutralizing antibodies. Sera were first heated to 56 °C for 30 min to inactivate complement. Duplicate serum samples (including positive and negative controls) were diluted serially twofold in Eagles MEM (3% FBS, 10 mM HEPES, 2 mM glutamine) in 50 µl aliquots in 96-well plates. LIV (LI/I, LI/31 or MA54) was added at approximately 100 focus forming units (f.f.u.)/50 µl to wells containing serum dilutions. In addition, a virus only control was added to each plate containing approximately 100, 10 and 1 f.f.u. per well. Plates were incubated for 90 min at 37 °C. Following this incubation, approximately 1x105 BHK cells in complete BHK medium were added to each well and plates were further incubated for 20 h at 37 °C. Cells were then washed with PBS and fixed at -20 °C with methanolacetone (1:1). A rabbit anti-LIV hyperimmune serum was added to each well and plates were incubated for 1 h at 37 °C. An FITC-conjugated anti-rabbit IgG was used to detect LIV infected cells. Neutralizing antibody titres were calculated as the reciprocal of the last serum dilution that showed 80% reduction in the number of fluorescent cells as compared to the virus control.
Challenge experiments.
In challenge experiments, male BALB/c or C57Bl/6 mice were used, purchased as specific pathogen-free animals aged 68 weeks from Harlan UK. For particle vaccination, 106 infectious particles were given in 0·5 ml PBS for the intraperitoneal (i.p.) route or 100 µl (50 µl into each hind-limb muscle) for the intramuscular (i.m.) route. Unless stated otherwise DNA vaccines were given as 100 µl containing 50 µg of DNA was given i.m. (50 µl into each hind limb muscle). These doses have been found to stimulate effective immunity for recombinant particles (Berglund et al.,1999 ; Fleeton et al.,1999
) and DNA vaccines (Koprowski & Weiner, 1998
; Berglund et al.,1998b
; Lin et al.,1998
) in previous studies. Mice inoculated with LacZ-encoding plasmids or PBS were used as controls. The commercial vaccine was purchased from Schering-Plough Animal Health and consists of formalin-inactivated particles in a mineral oil emulsion. It was stored at 4 °C, and warmed at 37 °C for 30 min and mixed thoroughly prior to inoculation as instructed by the manufacturer. It was given subcutaneously (s.c.) as 0·2 ml of emulsion. Vaccinated animals were boosted at 3 weeks after immunization, unless stated otherwise, and challenged 3 weeks after the final vaccination. All animals were monitored daily for 3 weeks after challenge.
Stocks of the LI/I, LI/31 and MA54 strains of LIV were prepared as clarified brain homogenates from intracerebrally infected neonatal mice, as previously described (Fleeton et al.,1999 ), and stored in aliquots at -70 °C. All strains were obtained from E. A. Gould, Institute of Microbiology and Environmental Microbiology, Oxford, UK, and were handled under level 3 containment. In all cases, groups of 10 vaccinated or control mice were infected with 100 LD50 of challenge virus given s.c., and infected mice were housed under veterinary supervision in a containment area and carcasses disposed of by incineration. Male mice were used in challenge experiments rather than the female mice used previously (Fleeton et al.,1999
), because preliminary experiments indicated that a higher proportion died following challenge with the LI/I strain of LIV. Significance of survival results was assessed (at the 5% probability level) using the Fisher exact test for numbers of dead and standard error of the mean for survival time.
Pathological examination of mice surviving challenge.
Three mice from each group showing statistically significant survival at 3 weeks after challenge were anaesthetized with halothane and perfused via the left ventricle with 10% formal saline for 5 min. Brains and spinal cords from a total of 91 mice were processed for histological examination as previously described (Sheahan et al.,1996 ).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Humoral immunity
Since the commercial vaccine is produced in BHK cells and as a result induced high levels of antibodies to the BHK cells used in the assays (data not shown), studies of the immune response to this vaccine were not taken further.
BALB/c and C57Bl/6 mice were immunized with three different recombinant vaccines: rSFV particles, pBK-SFV DNA and pBK plasmid DNA encoding LIV antigens (Table 1). All vaccines induced humoral responses specific for the encoded antigens following one immunization of either 106 particles or 50 µg of plasmid. A second immunization 3 weeks after priming resulted in seroconversion of all vaccinated mice, with the exception of two mice immunized with pBK-SFV-NS1. In addition, a significant elevation of the antibody in serum from vaccinated mice was detected. Vaccination of mouse strain C57Bl/6 produced slightly higher titres than those in BALB/c mice.
|
Since the LI/31 and MA54 strains differ from LI/I in two neutralization epitopes (Gao et al.,1994 ), we analysed serum from mice immunized twice with recombinant vaccines for neutralizing activity against all three strains. Mice vaccinated with prME showed the presence of neutralizing antibodies against LI/I and LI/31, but not against the MA54 strain (Table 2
).
|
|
|
|
Pathological examination of challenged mice
Neuronal degeneration, gliosis and leukocytic infiltration characteristic of LIV infection were present in one BALB/c mouse vaccinated with pBK-SFV-prME and NS1 which survived to 3 weeks following challenge with LI/I. Most other mice appeared normal; a minority showed occasional small perivascular aggregates of lymphocytes in the leptomeninges (Fig. 2).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have previously shown that SFV recombinant particles expressing the prME and/or NS1 antigens of LIV efficiently induced protective immunity in mice against challenge by the less virulent LI/I antibody escape variant and the virulent LI/31 prototype strain of LIV (Fleeton et al.,1999 ). In this study we have also included the highly virulent MA54 isolate as a challenge virus, and have tested vaccine constructs based on recombinant SFV particles, a standard naked DNA vector, a layered DNA/RNA SFV vector, and also a commercially available inactivated virus vaccine. The commercial LIV vaccine used in this study is a formaldehyde-inactivated particle vaccine with oil adjuvant. However, the particles are concentrated by ultrafiltration during vaccine preparation, and it may be that cellular debris is included in the preparation, which gives rise to the anti-BHK cell antibodies detected in this study. In mice, this vaccine appears to have intermediate efficacy in inducing protection against challenge; it is not as effective as recombinant particles, but is more effective than the two DNA vaccines tested.
It is clear that all recombinant vaccines tested induced at least partial immunity against challenge in some combinations of antigen/challenge virus. As found in our previous study (Fleeton et al.,1999 ), the NS1 antigen was generally less effective at inducing protective immunity than the prME antigen. However, in order to limit the spread of possible antibody escape mutants, an ideal vaccine should probably include both NS1 and prME, a combination that also induces efficient protective immunity. From previous studies, it is clear that the LI/31 strain of LIV differs from the less virulent LI/I antibody escape variant in two neutralization epitopes, located in the E protein. The virulent MA54 strain has the same epitopes as LI/31 at this location, as do all vaccines used in this study (Gao et al.,1994
). However, we have shown that there is no significant difference in neutralization of LI/I or LI/31 by the polyclonal antisera induced by vaccination with prME, but neutralization of the virulent MA54 strain could not be detected. That immunity to MA54 can be induced by vaccination with prME is, however, shown by the partial protection elicited by double vaccination with recombinant particles expressing prME. One explanation of these results could be that several epitopes are important in neutralization of LIV by polyclonal sera, and that MA54 differs from LI/31 and LI/I in one or more epitopes that are important for neutralization. These may be separate from the two well-characterized epitopes which differ in LI/31 and LI/I, but such a possibility remains to be investigated. It is also clear that MA54 is divergent from LI/31 and LI/I in sequence evolution, since it is an Irish isolate derived from cattle, whereas LI/31 and LI/I are Scottish and Welsh isolates respectively, derived from sheep (Gao et al.,1994
; McGuire et al.,1998
). It is possible therefore that the virulence of MA54 may also be determined by other regions of the genome.
Serum from a small number of mice immunized with recombinant vaccines that survived challenge with LI/I or LI/31 showed significantly increased serum neutralization antibody titres (320) against LI/I and LI/31, and low levels of neutralizing activity against MA54 (1020) at 3 weeks post-challenge. This result is in agreement with a recent study which showed that anamnestic neutralizing antibody responses after challenge correlate with protection in vaccinated mice (Konishi et al., 1999 ).
It has recently been shown that low doses (0·110 µg) of pBK-SFV-LacZ (Berglund et al., 1998b ), or an equivalent Sindbis-based DNA vaccine plasmid expressing the gD protein of herpesvirus (Driver et al.,1998
), induced higher antigen-specific humoral immune responses in mice than conventional DNA vaccines expressing the same antigens. In this study we found the same trend when the NS1 antigen was expressed, where the antibody response was absent when a conventional DNA vaccine was used at low doses. In contrast, this difference was not observed when the prME spike complex antigen was expressed, suggesting that the outcome of vaccination may be due to the nature of the antigen expressed.
pBK-SFV vaccines in general did not protect immunized mice as well as pBK vaccines from challenge. However, similar levels of protection were obtained where pre-challenge antigen specific IgG was equivalent. Thus, for both plasmid based vaccines pre-challenge antibody levels correlated with protection.
Immunization with recombinant particles is clearly the most efficient way to induce protective immunity. The reason for this is not obvious at present. Upon entry into a cell both SFV DNA and rSFV particle vaccines induce the same pattern of RNA replication and antigen expression. However, it is possible that different cell populations are targeted by recombinant particles compared to DNA in vivo. This might have an effect on antigen expression and presentation and, ultimately, in protection from challenge.
The results of the challenge experiments were substantiated by extensive pathology studies. In all cases (except for a single mouse), pathological studies showed that in mice surviving challenge, neurons, the target cells for LIV in the central nervous system, appeared normal. Isolated aggregates of lymphocytes in the leptomeninges of some survivors were not accompanied by any evidence of neuronal degeneration or gliosis, and probably resulted from immune stimulation caused by the challenge virus.
This study and a previous one (Fleeton et al.,1999 ) have established LIV infection of mice as an efficient and stringent model to test vaccine technology. A triple challenge model is employed using strains of graded virulence, and two antigens that differ in their protective potency. This LIV model is currently being extended to sheep, the natural host of LIV. It should be possible to test biosafety parameters of new recombinant vaccines using both mice and sheep, as well as efficacy of protection, and to field test new vaccines in areas where louping ill is prevalent.
In conclusion, we have established LIV as a new model for testing vaccine vector constructs. Our initial results in mice indicate that recombinant SFV particles induce better protective immunity than DNA vaccines or the currently available commercial vaccine.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Albert, M. L., Sauter, B. & Bhardwaj, N. (1998b). Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392, 86-89.[Medline]
Atkins, G. J. , Sheahan, B. J. & Liljeström, P. (1999). The molecular pathogenesis of Semliki Forest virus: a model virus made useful? Journal of General Virology 80, 2287-2297.
Berglund, P., Sjöberg, M., Atkins, G. J., Sheahan, B. J., Garoff, H. & Liljeström, P. (1993). Semliki Forest virus expression system: production of conditionally infectious recombinant particles. Bio/Technology 11, 916-920.[Medline]
Berglund, P., Quesada-Rolander, M., Putkonen, P., Biberfeld, G., Thorstensson, R. & Liljeström, P. (1997). Outcome of immunization of cynomolgus monkeys with recombinant Semliki Forest virus encoding human immunodeficiency virus type 1 envelope protein and challenged with a high dose of SHIV-4 virus. AIDS Research and Human Retroviruses 13, 1487-1495.[Medline]
Berglund, P., Fleeton, M., Smerdou, C., Tubulekas, I., Sheahan, B. J., Atkins, G. J. & Liljeström, P. (1998a). Vaccination with recombinant suicidal DNA/RNA. In New Developments and New Applications in Animal Cell Technology. Edited by O. Merten, P. Perrin & B. Griffiths. Dordrecht: Kluwer Academic Publishers.
Berglund, P., Smerdou, C., Fleeton, M. N., Tubulekas, I. & Liljeström, P. (1998b). Enhancing immune responses using suicidal DNA vaccines. Nature Biotechnology 16, 562-565.[Medline]
Berglund, P., Fleeton, M. N., Smerdou, C. & Liljeström, P. (1999). Immunization with recombinant Semliki Forest virus induces protection against influenza challenge in mice. Vaccine 17, 497-507.[Medline]
Colombage, G., Hall, R., Pavy, M. & Lobigs, M. (1998). DNA-based and alphavirus-vectored immunization with prM and E proteins elicits long-lived and protective immunity against the flavivirus, Murray Valley encephalitis virus. Virology 250, 151-163.[Medline]
Davidson, M. M., Williams, H. & Macleod, A. J. (1991). Louping ill in man: a forgotten disease. Journal of Infection 23, 241-249.[Medline]
Driver, D. A., Townsend, K., Brumm, D., Polo, J. M., Belli, B. A., Catton, D. J., Hsu, D., Mittelstaedt, D., McCormack, J. E., Karavodin, L., Dubensky, T. W., Chang, S. M. W. & Banks, T. A. (1998). DNA immunization against Herpes Simplex virus: enhanced efficacy using a Sindbis virus-based vector. Journal of Virology 72, 950-958.
Fleeton, M. N., Sheahan, B. J., Gould, E. A., Atkins, G. J. & Liljeström, P. (1999). Recombinant Semliki Forest virus particles encoding the prME or NS1 proteins of louping ill virus protect mice from lethal challenge. Journal of General Virology 80, 1189-1198.[Abstract]
Gao, G. F., Hussain, M. H., Reid, H. W. & Gould, E. A. (1994). Identification of naturally occurring monoclonal antibody escape variants of louping ill virus.Journal of General Virology 75, 609-614.[Abstract]
Glasgow, G. M., McGee, M. M., Mooney, D. A., Sheahan, B. J. & Atkins, G. J. (1997). Death mechanisms in cultured cells infected by Semliki Forest virus. Journal of General Virology 78, 1559-1563.[Abstract]
Glasgow, G. M., McGee, M. M., Tarbatt, C. J., Mooney, D. A., Sheahan, B. J. & Atkins, G. J. (1998). The Semliki Forest virus vector induces p53-independent apoptosis. Journal of General Virology 79, 2405-2410.[Abstract]
Heinz, F. X., Allison, S. L., Stiasny, K., Schalich, J., Holzman, H., Mandl, C. W. & Künz, C. (1995). Recombinant and virion-derived soluble and particulate immunogens for vaccination against tick-borne encephalitis. Vaccine 13, 1636-1642.[Medline]
Jacobs, S. C., Stephenson, J. R. & Wilkinson, G. W. G. (1992). High level expression of the tick-borne encephalitis virus NS1 protein by using an adenovirus-based vector: protection elicited in a murine model. Journal of Virology 66, 2086-2095.[Abstract]
Kohno, A., Emi, N., Kasai, M., Tanimoto, M & Saito, H. (1998). Semliki Forest virus-based DNA expression vector transient protein production followed by cell death. Gene Therapy 5, 415-418.[Medline]
Konishi, E., Pincus, S., Paoletti, E., Shope, R. E., Burrage, T. & Mason, P. W. (1992). Mice immunized with a subviral particle containing the Japanese encephalitis virus prM/M and E proteins are protected from lethal JEV infection. Virology 188, 714-720.[Medline]
Konishi, E., Yamaoka, M., Khin-Sane-Win, Kurane, I., Takada, K. & Mason, P. W. (1999). The anamnestic neutralising antibody response is critical for protection of mice from challenge following vaccination with a plasmid encoding the Japanese encephalitis virus premembrane and envelope genes. Journal of Virology 73, 5527-5534.
Koprowski, H. & Weiner, D. B. (1998). DNA vaccination/genetic vaccination. Current Topics in Microbiology and Immunology 226, 5-13.
Liljeström, P. & Garoff, H. (1991). A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Bio/Technology 9, 1356-1361.[Medline]
Lin, Y. L., Chen, L. K., Liao, C. L., Yeh, C. T., Ma, S. H., Chen, J. L., Huang, Y. L., Chen, S. S. & Chiang, H. Y. (1998). DNA immunization with Japanese encephalitis virus nonstructural protein NS1 elicits protective immunity in mice.Journal of Virology 72, 191-200.
McGuire, K., Holmes, E. C., Gao, G. F., Reid, H. W. & Gould, E. A. (1998). Tracing the origins of louping ill virus by molecular phylogenetic analysis. Journal of General Virology 79, 981-988.[Abstract]
Monath, T. P. & Heinz, F. X. (1996). Flaviviruses. In Fields Virology, pp. 961-1034. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: LippincottRaven.
Mossman, S., Bex, F., Berglund, P., Arthos, J., ONeill, S. P., Riley, D., Maul, D. H., Bruck, C., Momin, P., Burny, A., Fultz, P. N., Mullins, J. I., Liljeström, P. & Hoover, E. A. (1996). Protection against lethal SIVsmmPBj14 disease by a recombinant Semliki Forest virus gp160 vaccine and by gp120 subunit vaccine. Journal of Virology 70, 1953-1960.[Abstract]
Pugachev, K. V., Mason, P. W., Shope, R. E. & Frey, T. K. (1995). Double-subgenomic Sindbis virus recombinants expressing immunogenic proteins of Japanese encephalitis virus induce significant protection in mice against lethal JEV infection. Virology 212, 587-594.[Medline]
Salminen, A., Wahlberg, J. M., Lobigs, M., Liljeström, P. & Garoff, H. (1992). Membrane fusion process of Semliki Forest virus. II. Cleavage-dependent reorganisation of the spike protein complex controls virus entry. Journal of Cell Biology 116, 349-357.[Abstract]
Schlesinger, J. J., Brandriss, M. W., Cropp, C. P. & Monath, T. P. (1986). Protection against yellow fever in monkeys by immunization with yellow fever nonstructural protein NS1. Journal of Virology 60, 1153-1155.[Medline]
Schmaljohn, C., Vanderzanden, L., Bray, M., Custer, D., Meyer, B., Li, D., Rossi, C., Fuller, D., Fuller, J., Haynes, J. & Huggins, J. (1997). Naked DNA vaccines expressing the prM and E genes of Russian spring summer encephalitis virus and Central European encephalitis virus protect mice from homologous and heterologous challenge. Journal of Virology 71, 9563-9569.[Abstract]
Sheahan, B. J., Ibrahim, M. A. & Atkins, G. J. (1996). Demyelination of olfactory pathways in mice following intranasal infection with the avirulent A7 strain of Semliki Forest virus. European Journal of Veterinary Pathology 2, 117-125.
Shiu, S. Y. W., Reid, H. W. & Gould, E. A. (1992). Louping ill virus envelope protein expressed by recombinant baculovirus and vaccinia virus fails to stimulate protective immunity. Virus Research 26, 213-229.[Medline]
Smerdou, C. & Liljeström, P. (1999a). Non-viral amplification systems for gene transfer: vectors based on alphaviruses. Current Opinion in Molecular Therapy (in press).
Smerdou, C. & Liljeström, P. (1999b). Two-helper RNA system for production of recombinant Semliki Forest virus particles. Journal of Virology 73, 1092-1098.
Timofeev, A. V., Ozherelkov, S. V., Pronin, A. V., Deeva, A. V., Karganova, G. G., Elbert, L. B. & Stephenson, J. R. (1998). Immunological basis for protection in a murine model of tick-borne encephalitis by a recombinant adenovirus carrying the gene encoding the NS1 non-structural protein. Journal of General Virology 79, 689-695.[Abstract]
Tubulekas, I., Berglund, P., Fleeton, M. & Liljeström, P. (1997). Alphavirus expression vectors and their use as recombinant vaccines a minireview. Gene 190, 191-195.[Medline]
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. Journal of Virological Methods 73, 71-75.[Medline]
Zhou, X. P., Berglund, G., Rhodes, G., Parker, S. E., Jondal, M. & Liljeström, P. (1994). Self-replicating Semliki Forest virus RNA as recombinant vaccine. Vaccine 12, 1510-1514.[Medline]
Zhou, X. P., Berglund, G., Zhao, H., Liljeström, P. & Jondal, M. (1995). Generation of cytotoxic and humoral immune responses by nonreplicative recombinant Semliki Forest virus. Proceedings of the National Academy of Sciences, USA 92, 3009-3013.[Abstract]
Received 2 September 1999;
accepted 10 November 1999.