Recombinant Semliki Forest virus particles expressing louping ill virus antigens induce a better protective response than plasmid-based DNA vaccines or an inactivated whole particle vaccine

Marina N. Fleeton1, Peter Liljeström1,2, Brian J. Sheahan3 and Gregory J. Atkins4

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
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Abstract
Introduction
Methods
Results
Discussion
References
 
Louping ill virus (LIV) infection of mice was used as a model to evaluate the protective efficacy of Semliki Forest virus (SFV)-based vaccines in comparison to a standard DNA vaccine and a commercial chemically inactivated vaccine. The recombinant SFV-based vaccines consisted of suicidal particles and a naked layered DNA/RNA construct. The nucleic acid vaccines expressed the spike precursor prME and the nonstructural protein 1 (NS1) antigens of LIV. Three LIV strains of graded virulence for mice were used for challenge. One of these was a naturally occurring antibody escape variant. All vaccines tested induced humoral immunity but gave varying levels of protection against lethal challenge. Only recombinant SFV particles administered twice gave full protection against neuronal degeneration and encephalitis induced by two of the three challenge strains, and partial protection against the highly virulent strain, whereas the other vaccines tested gave lower levels of partial protection.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
We are using louping ill virus (LIV) infection as a model for testing the efficacy and biosafety of different types of recombinant vaccines. LIV is a member of the tick-borne complex of flaviviruses, prevalent in tick-infested upland pastures in Britain and Ireland. It is primarily known as a disease of sheep, but has also been shown to infect and cause disease in deer, cattle, goats, grouse and occasionally man (Davidson et al.,1991 ; Monath & Heinz, 1996 ). LIV is a good model to test vaccine technology since it produces fatal encephalitis in mice which is similar to the disease in sheep; pathological examination of the brains of surviving vaccinated animals to determine efficiency of protection is straightforward. Sheep represent a large animal model for experimentation, which is cheaper and easier to manipulate than other models, and it should be possible to conduct field trials of any recombinant vaccine produced for this virus, since outbreaks of louping ill occur in predictable isolated areas with sparse human populations. Antibody escape variants, which are less efficiently neutralized by monoclonal antibodies active against the vaccine strain, occur naturally and it is possible that such variants could be selected by the use of inactivated vaccine. Such variants have been sequenced and the neutralization epitopes identified (Gao et al.,1994 ). One such variant (LI/I) was used in this study, which also included the prototype LI/31 strain and a highly virulent strain, MA54. Challenge with these virus strains represents a gradation of virulence in outbred mice, where the LI/I strain is least virulent, LI/31 of intermediate virulence and MA54 most virulent (Gao et al.,1994 ).

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 vitro–made 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
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cells and antibodies.
BHK-21 cells were maintained as previously described (Liljeström & Garoff, 1991 ). For immunoprecipitation a rabbit hyperimmune antiserum was used; for immunofluorescence, anti-E MAb 4.2 and anti-NS1 MAb 7.3 were used (Gao et al., 1994 ).

{blacksquare} 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 AgeI–SpeI fragment from pBK-SFV-LacZ was removed and replaced with an AgeI–SpeI 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 BamHI–XmaI fragment from pSFV-NS1 was inserted into BamHI/XmaI-cut pBK-CMV vector. A SmaI–StuI 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 SDS–PAGE (Fleeton et al., 1999 ). At each time-point the number of transfected cells expressing either prME or NS1 was monitored by indirect immunofluorescence staining.

{blacksquare} 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 ).

{blacksquare} 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 methanol–acetone (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 10–20 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 Eagle’s 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 methanol–acetone (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.

{blacksquare} Challenge experiments.
In challenge experiments, male BALB/c or C57Bl/6 mice were used, purchased as specific pathogen-free animals aged 6–8 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.

{blacksquare} 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
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Abstract
Introduction
Methods
Results
Discussion
References
 
Expression of LIV antigens from recombinant vaccines
LIV sequences encoding the envelope proteins prME and the nonstructural protein NS1 had previously been cloned into rSFV (Fleeton et al., 1999 ). For this study the same LIV sequences were inserted into two different DNA expression vectors: a CMV-based vector, pBK, and an SFV-based plasmid vector, pBK-SFV (Fig. 1a). In cells transfected with pBK vector, the CMV promoter drives the expression of the antigen-coding genes. However, for pBK-SFV, the role of the promoter is to direct the synthesis of a recombinant SFV RNA replicon. Translation of this RNA molecule in the cytoplasm of transfected cells produces the SFV replicase complex, which results in self-amplification of the recombinant RNA encoding the antigen.



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Fig. 1. (a) Schematic diagram of recombinant sequences inserted into plasmids used in this study (not to scale). First is the pBK plasmid, second pBK-SFV and lastly is pSFV, which was used to make recombinant particles. The CMV immediate early promoter is shown in dark grey, the SFV replicase genes in light grey and LIV antigen (either prME or NS1) in white. Expression of LIV genes in the SFV-based plasmids is controlled by the SFV 26S subgenomic promoter (arrow). (b) Analysis of [35S]methionine-labelled envelope proteins expressed by pBK-SFV-prME or pBK-prME in transfected BHK cells. Lysate and medium samples were taken at the times indicated after radiolabelling, and immunoprecipitated with a rabbit anti-LIV hyperimmune serum. The positions of E and prM proteins are indicated by arrows. (c)Analysis of radiolabelled NS1 protein from cells transfected with pBK-SFV-NS1 or pBK-NS1. Lysate and medium samples were taken at the times indicated and immunoprecipitated with rabbit anti-LIV hyperimmune serum. Samples were divided in two prior to running on SDS–PAGE: one half was heated to 94 °C (+), the other to 37 °C (-), to demonstrate dimer formation. The positions of molecular mass markers ranging from 97·4 to 21·5 kDa are indicated on the left.

 
We have previously shown that the LIV antigens, prME and NS1, are expressed and correctly processed in BHK cells transfected with rSFV-prME and rSFV-NS1 (Fleeton et al., 1999 ). In this study we analysed antigen expression from the plasmid constructs pBK-SFV and pBK. BHK cells were transfected by lipofection with plasmids encoding either prME or NS1. Correct processing of LIV antigens expressed from pBK-SFV or pBK vectors was confirmed by pulse–chase analysis (Fig. 1b, c). Lysates from cells transfected with pBK-SFV-prME or pBK-prME showed the presence of three distinct bands with apparent molecular masses corresponding to the envelope precursor prME, and processed E and prM proteins. Later time-points showed the release of the E protein from transfected cells into the medium. Immunoprecipitation of lysates from cells transfected with pBK-SFV-NS1 or pBK-NS1 indicated the presence of monomeric and heat-labile dimeric forms of NS1. Five hours after pulse-labelling, the dimeric form could be detected in the medium of transfected cells.

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.


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Table 1. Geometric mean antibody titre of sera from BALB/c and C57Bl/6 mice following immunization with recombinant vaccines

 
We analysed effects of dose by immunizing BALB/c mice with 50, 5 or 1 µg of naked DNA (Table 1). The standard DNA vaccine pBK-prME showed higher responses than pBK-SFV-prME at the 50 µg dose whereas at the 5 and 1 µg doses the responses were similar. In contrast, when NS1 was the antigen, the pBK-SFV-NS1 vaccine induced a high response at the 50 µg dose, but at 5 and 1 µg the pBK-NS1 vaccine did not induce a response.

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).


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Table 2. Neutralizing antibody titres detected in serum from mice immunized with recombinant vaccines

 
Protection against challenge
As shown in Table 3, partial protection was obtained by a single inoculation with recombinant particles expressing the prME and NS1 LIV antigens against the two least virulent LIV strains, i.e. LI/I and LI/31. Partial protection was also obtained for the standard DNA vaccine against the LI/31 strain. No protection was obtained following single inoculation for the layered DNA/RNA vaccine (pBK-SFV) or following challenge with the virulent MA54 strain for any vaccine tested.


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Table 3. Challenge after single immunization with vaccines expressing prME and NS1

 
The results of challenge following double inoculation with vaccine are shown in Table 4. Partial protection at best was obtained with the two DNA vaccines, although only against the less virulent LI/I and LI/31 strains. No protection was obtained with any DNA vaccine against the virulent MA54 strain. In general, vaccines expressing prME gave better protection than those expressing NS1 alone. Recombinant particles expressing prME gave complete protection against LI/I and LI/31, and partial protection against the virulent MA54 strain; protection was obtained against MA54 only with recombinant particles and the commercial vaccine. Protection against challenge was obtained with recombinant particles after vaccination by both the i.m. and i.p. routes. The commercial vaccine gave partial protection against all three challenge viruses.


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Table 4. Challenge after double inoculation with vaccines expressing prME and/or NS1, and commercial vaccine

 
Table 5 shows the results of challenge in C57Bl/6 mice following double inoculation with vaccine. The outcome of vaccination and challenge in this mouse strain was generally similar to BALB/c mice, i.e. recombinant particles protected efficiently, the standard DNA vaccine partially and the layered DNA/RNA vaccine inefficiently.


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Table 5. Challenge with LI/31 after double inoculation of C57Bl/6 mice with vaccines expressing prME and/or NS1

 
We also tested the ability of double administration of low dose (5 µg) plasmid vaccines encoding prME and NS1 to protect BALB/c mice from LI/I challenge. Although the standard DNA vaccine gave better protection (3/10 mice dead) than the layered DNA/RNA vaccine (7/10 mice dead), there was no statistical significance in this result (P=0·08).

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).



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Fig. 2. Examples of the central nervous system pathology of mice following LIV challenge. (a) Frontal cerebral cortex, unvaccinated BALB/c mouse, 7 days following infection with MA54. Many neurons appear shrunken and show nuclear condensation and fragmentation. Haematoxylin and eosin, x400. (b) Pons, BALB/c mouse, rSFV-NSI vaccinated, 3 weeks following i.p. challenge with LI/31. Small perivascular aggregate of lymphocytes in the subarachnoid space. Haematoxylin and eosin, x400.

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Previous studies have shown that the flavivirus NS1 or prME proteins can elicit immune responses when expressed by standard naked DNA vaccines from the cytomegalovirus promoter (Schmaljohn et al.,1997 ; Lin et al.,1998 ; Colombage et al.,1998 ), by adenovirus (Jacobs et al.,1992 ; Timofeev et al., 1998 ) or alphavirus (Pugachev et al., 1995 ; Colombage et al.,1998 ; Fleeton et al.,1999 ) vectors. However, so far no comparative study has been undertaken of the relative efficiency of these expression systems in inducing protective immunity; one problem in making such comparisons has been the lack of a suitable stringent challenge model. Here we propose such a test model that makes comparisons possible. We have expressed two antigens from a heterologous virus (LIV) using CMV and SFV vectors, and shown that the protective efficacy of these antigens and vector constructs can be demonstrated using challenge virus strains of graded virulence.

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 (10–20) 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 (0·1–10 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
 
We thank Peter Nowlan, Cormac O’Carroll and Caroline Woods, of the BioResources Unit, TCD, for help with animal experiments, and Marie Moore and Sheila Worrell for technical assistance with the histopathological studies. This work was supported by the Swedish Medical Research Council, the Swedish Council for Engineering Sciences, the EU Biotechnology Programme, the Wellcome Trust and BioResearch Ireland.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 2 September 1999; accepted 10 November 1999.