Virology Division, Department of Infectious Diseases and Immunology, Veterinary Faculty, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands1
Institute of Virology, Erasmus University Rotterdam, Rotterdam, The Netherlands2
Virbac Laboratories Inc., 06511 Carros Cedex, France3
Author for correspondence: Harrie Glansbeek. Fax +31 30 2536723. e-mail H.Glansbeek{at}vet.uu.nl
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Abstract |
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Introduction |
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Attempts to vaccinate cats against FIP have been largely unsuccessful. Vaccination with an avirulent FCoV (Pedersen & Black, 1983 ) or a recombinant vaccinia virus expressing the S protein (Vennema et al., 1990
) failed to induce protection and even exacerbated the disease. Administration of closely related human, canine or porcine coronaviruses also failed to protect cats (Barlough et al., 1984
, 1985
; Stoddart et al., 1988
; Woods & Pedersen, 1979
). Currently, a temperature-sensitive strain of FIPV is marketed as a vaccine (Christianson et al., 1989
). Although its ability to protect cats against FIPV was demonstrated (Gerber et al., 1990
; Gerber, 1995
), the efficacy of this vaccine is a matter of debate (Fehr et al., 1997
; McArdle et al., 1995
; Scott et al., 1995
).
A major obstacle for vaccine development is the fact that coronavirus antibodies are not protective rather, they enhance disease progression, as demonstrated by passive immunization of kittens with anti-FIPV antibodies (Weiss & Scott, 1981 ). This effect is due to anti-S antibodies: vaccination of kittens with a recombinant vaccinia virus expressing the S protein (Vennema et al., 1990
), but not with M or N protein recombinants (Vennema et al., 1991
), resulted in early death. Antibodies against the S protein were also shown to induce antibody-dependent enhancement of infection of macrophages in vitro (Corapi et al., 1992
; Hohdatsu et al., 1998
; Olsen et al., 1992
). On the other hand, cell-mediated immunity appears to play a protective role. Cats that have recovered from FIP exhibit strong blastogenic and delayed hypersensitivity responses (Pedersen & Floyd, 1985
). In addition, the N protein was found to induce protective immunity in a vaccination protocol where kittens were primed with a recombinant raccoon poxvirus and boosted with an avirulent FCoV (Wasmoen et al., 1995
). In view of its internal position in the virion, it is unlikely that antibodies played a role in the protective immunity induced by this vaccination protocol.
A recent approach to induce cell-mediated immunity against infectious agents utilizes plasmids encoding the protection-relevant antigen(s) and their endogenous expression in the host organism. These DNA vaccines often efficiently prime antigen-specific CD4+ T helper cells as well as CD8+ cytotoxic T cells (CTLs). DNA vaccines have been shown to induce protective immunity against herpes simplex virus (Manickan et al., 1995 ), pseudorabies virus (PRV) (Gerdts et al., 1997
; Haagmans et al., 1999
; van Rooij et al., 2000
), influenza A virus (Yokoyama et al., 1997
) and lymphocytic choriomeningitis virus (LCMV) infection (Yokoyama et al., 1997
).
Immunity induced by DNA vaccination is enhanced when the immune system is boosted with another vaccine formulation, e.g. recombinant vaccinia virus. This was elegantly shown in studies with the malaria parasite Plasmodium berghei. Repeated application of plasmid DNA encoding pre-erythrocyte antigens conferred only limited protection to mice. However, priming with DNA followed by a single boost with a recombinant vaccinia virus expressing the same antigen resulted in complete protection and high levels of CD8+ T cells (Schneider et al., 1998 ). A similar DNA/vaccinia virus protocol was found to elicit the highest CTL responses in a human immunodeficiency virus vaccination study (Hanke et al., 1998
).
Another improvement of DNA vaccine efficacy has been attained by the co-injection of cytokine-encoding plasmids. In this respect, plasmids encoding IL-12 are particularly promising in that they stimulate T helper 1 (Th1) responses (Chow et al., 1998 ; Sin et al., 1999a
, b
; Tsuji et al., 1997
) and enhance the induction of antigen-specific CD8+ CTLs (Hamajima et al., 1997
; Kim et al., 1997
; Okada et al., 1997
; Tan et al., 1999
; Tsuji et al., 1997
). In several studies, co-delivery of IL-12-encoding plasmids with DNA vaccines resulted in enhanced protection against virus infections and tumours (Boretti et al., 2000
; Chow et al., 1998
; Sin et al., 1999a
, b
; Tan et al., 1999
).
In view of these considerations, we have investigated the potential of DNA vaccination against FIP, adopting a primeboost protocol as well as co-delivery of IL-12-encoding plasmids.
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Methods |
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To construct a vector encoding both FIPV-M and FIPV-N, the entire expression cassette of VR1012-M (containing the CMV promoter, FIPV-M cDNA and BGH polyA) was purified after ApaLI digestion and cloned into DraI-digested VR1012-N to yield VRMVRN. To introduce the stimulatory CpG sequences present in the ampicillin resistance (Ampr) gene (Roman et al., 1997 ; Sato et al., 1996
), this gene was excised from pcDNA-3 (Invitrogen) by BspHI digestion, blunt-ended using Klenow and ligated into DraIII-digested VRMVRN to yield VRMVRN-CpG.
IL-12 is a heterodimeric protein which is composed of disulfide-bonded 35 kDa (p35) and 40 kDa (p40) subunits. The genes encoding the subunits of feline IL-12 have been cloned in our laboratory (Schijns et al., 1997 ). Each cDNA was initially cloned separately into EcoRV-digested VR1012, yielding VR1012-p35 and -p40. To obtain a vector that encodes both the p35 and the p40 chain, the entire expression cassette from VR1012-p35 was excised by ApaLI digestion. The 3' recessive ends were filled in using Klenow and the fragment was cloned into DraI-digested VR1012-p40 to yield VR1012-fIL12.
Plasmids were grown in the PC2495 strain of Escherichia coli and purified on columns (Qiagen), according to the manufacturers directions.
Radioimmunoprecipitation of expressed FIPV-M and -N proteins.
COS-7 cells were seeded in 35 mm diameter dishes at 5x105 cells per dish. After a culture period of 16 h, cells were transfected with 1 µg of plasmid DNA using Lipofectamin Plus (Gibco BRL), according to the manufacturers instructions. Cells were washed with PBS 24 h after transfection and kept for 30 min in cysteine- and methionine-free Dulbeccos minimal essential media (DMEM) containing 10 mM HEPES (pH 7·2) and 5% dialysed foetal calf serum (FCS). [35S]Methionine (Amersham) was added to a final concentration of 11·1 MBq/ml and incubation was continued for 1 h at 37 °C. Subsequently, cells were lysed by a 10 min incubation on ice with lysis buffer (20 mM TrisHCl pH 7·5, 1 mM EDTA, 100 mM NaCl, 1% Triton X-100, 1 µg/ml pepstatin A, 1 µg/ml aprotinin, 1 µg/ml leupeptin) and centrifuged for 15 min at 10000 g and 4 °C. For immunoprecipitation, 100 µl of the supernatant was diluted with 1 ml detergent solution (50 mM TrisHCl pH 8·0, 62·5 mM EDTA, 0·4 % deoxycholate, 1% NP-40, 0·7% SDS, 0·1 mg/ml BSA), whereafter 3 µl ascites fluid obtained from an experimentally FIPV-infected kitten was added. After overnight incubation at 4 °C, 50 µl of a 10% (w/v) suspension of formalin-fixed Staphylococcus aureus cells (Pansorbin) (Calbiochem) was added and the incubation was continued for 30 min at 4 °C. The bacteria were spun down, washed three times with RIPA buffer (10 mM TrisHCl pH 7·4, 150 mM NaCl, 0·1% SDS, 1% deoxycholate, 1% NP-40) and resuspended in 30 µl Laemmlis sample buffer containing 5% -mercaptoethanol. Samples were heated for 1 min at 95 °C and analysed by SDSPAGE in 15% gels, followed by fluorography.
Radioimmunoprecipitation assay for the analysis of antibodies in cat sera.
Felis catus whole foetus (fcwf-D) cells were infected with FIPV strain 79-1146 at an m.o.i. of 10. After an incubation of 4·5 h, cells were washed with PBS and cultured for 30 min in cysteine- and methionine-free DMEM containing 10 mM HEPES (pH 7·2) and 5% dialysed FCS. [35S]Methionine was added to a final concentration of 11·1 MBq/ml and the incubation was continued for 2 h at 37 °C. Subsequently, cells were lysed by a 10 min incubation on ice with lysis buffer and centrifuged for 15 min at 10000 g and 4 °C. For precipitation, 25 µl of the supernatant was diluted with 1 ml TESV (20 mM TrisHCl pH 7·3, 1 mM EDTA, 100 mM NaCl) containing 1% Triton X-100, whereafter 25 µl cat serum was added. After overnight incubation at 4 °C, 50 µl of a 10% (w/v) suspension of formalin-fixed S. aureus cells was added and the incubation was continued for 30 min at 4 °C. The bacteria were spun down, washed three times with RIPA buffer and resuspended in 30 µl Laemmlis sample buffer containing 5% -mercaptoethanol. Samples were heated for 1 min at 95 °C and analysed by SDSPAGE in 10% gels, followed by fluorography.
In vitro expression of recombinant feline IL-12.
COS-7 cells were seeded in 35 mm diameter dishes at 5x105 cells per well. After a culture period of 16 h, cells were transfected with 1 µg of plasmid DNA using Lipofectamin Plus, according to the manufacturers instructions. Culture media were collected 72 h after transfection. Cytokine activity released into the culture medium was analysed using a bioassay, described previously by Gately et al. (1997) . In short, human peripheral blood lymphocytes (PBLs), isolated using Lymphoprep (Nycomed), were cultured for 2 days in Iscoves medium containing 5 µg/ml concanavalin A. To stimulate the formation of blasts, recombinant human IL-2 was added (50 units/ml) and cells were cultured for an additional 3 days. Cells were washed, seeded in 96-well plates (2x104 cells per well) and cultured in the presence of the culture media for transfected cells. Recombinant human IL-12 (Genzyme) was used as a positive control. After 48 h, [3H]thymidine (Amersham) was added and the incubation was continued for 4 h, whereafter the cells were harvested by an automated cell harvester. The incorporated radioactivity was quantified by liquid scintillation counting.
Production of recombinant vaccinia virus stocks.
Construction of the recombinant vaccinia viruses vSC, vFN and vFM has been described previously (Vennema et al., 1991 ). To produce new virus stocks, RK-13 cells were infected with recombinant virus at an m.o.i. of 0·1. After a culture period of 34 days, cells were harvested and disrupted in 10 mM Tris (pH>9). The homogenate was centrifuged for 10 min at 1100 r.p.m. and the supernatant was collected. Virus stocks were titrated on RK-13 cells.
Virus neutralization assay.
FIPV strain 79-1146 (50 µl of 1x106·5 TCID50/ml) or PRV strain NIA-3 (50 µl of 2x105 p.f.u./ml) were incubated overnight at 37 °C with twofold dilutions of heat-inactivated plasma from kittens (50 µl), whereafter the viruses were added to fcwf-D cells (16000 cells per well in 96-well plates). After an incubation period of 18 h, cells were stained with crystal violet to visualize plaques.
Design of vaccination/challenge trials.
To evaluate the efficacy of DNA vaccines, two vaccination/challenge experiments were performed using female, specific-pathogen-free HsdCpb:CADS(BR) kittens (Harlan). At the start of the experiments, the kittens were 1416 weeks of age.
In the first experiment, three groups (AC) of kittens (n=5) were injected with different plasmids in 1 ml PBS. Kittens in group A received 200 µg of plasmid DNA encoding the PRV glycoprotein D (VR1012-gD) (Haagmans et al., 1999 ). Group B kittens each received 200 µg VR1012-M and 200 µg VR1012-N. Group C kittens were injected with 200 µg VR1012-M, 200 µg VR1012-N, 200 µg VR1012-p35 and 200 µg VR1012-p40. Vaccinations were done four times at intervals of 3 weeks. Each vaccine dose was distributed equally over four sites by two intradermal injections and two intramuscular injections (upper hind limbs). Four weeks after the fourth vaccination, all kittens were challenged oronasally with 1000 TCID50 FIPV 79-1146.
In the second experiment, three groups (AC) of four kittens each were vaccinated with the following plasmids in 0·8 ml PBS. Group A kittens (control) were inoculated with 400 µg VR1012-gD, group B with 400 µg plasmid DNA encoding both FIPV-M and FIPV-N (VRNVRM-CpG) and group C with 400 µg VRMVRN-CpG and 400 µg of plasmid DNA encoding both subunits of feline IL-12 (VR1012-fIL12). Cats were vaccinated twice (3-week-interval) by intradermal injection. At 3 weeks after the second DNA vaccination, the kittens of group A received a subcutaneous injection of 1x108 p.f.u. of recombinant vaccinia virus vSC, while the kittens in groups B and C were boosted by a similar injection of a mixture containing 1x108 p.f.u. of recombinant vaccinia virus expressing FIPV-N (vFN) and 1x108 p.f.u. of recombinant vaccinia virus expressing FIPV-M (vFM). Kittens were challenged oronasally with 50 TCID50 FIPV 79-1146 at week 3 after the last vaccination.
To avoid unnecessary suffering, kittens were euthanased once they had entered the irreversible terminal phase of FIP, as judged by the veterinary experts of the animal facility.
For both vaccination/challenge experiments, the approval of the Ethical Committee of Utrecht University was obtained.
Statistical analysis.
The significance of the differences in the numbers of PBLs was analysed using Students t-test. Evaluation of statistical differences in survival after FIPV challenge was performed using Coxs proportional hazard model.
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Results |
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As shown in the results of a bioassay (Fig. 2), proliferation of human PBLs was stimulated by culture media both of COS-7 cells co-transfected with VR1012-p35 and -p40 (lane 4) and of cells transfected with VR1012-fIL12 (lane 5); no biological activity was found in culture media of cells transfected with VR1012-p35 (lane 2) or -p40 (lane 3) alone. These results demonstrate that biologically active feline IL-12 was produced as predicted.
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As shown in Fig. 3, VR1012-gD induced neutralizing antibodies in all kittens from group A. No PRV-neutralizing antibodies were detected in kittens from the other groups (data not shown).
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As shown in Fig. 5, the co-delivery of VR1012-p35 and -p40 did not improve protection rather, it seemed to enhance the susceptibility to FIPV; all kittens died within 30 days after challenge, while in each of the two other groups, two of five kittens survived for more than 40 days. All kittens from group C had high titres of FIPV-neutralizing antibodies at day 14 after challenge. These titres were not significantly different from those of kittens from groups A or B (data not shown).
Vaccination of kittens using a primeboost protocol
In a second trial, kittens were vaccinated twice with the plasmid encoding both FIPV-M and -N (VRMVRN-CpG) and boosted with recombinant vaccinia viruses expressing FIPV-N (vFN) and -M (vFM). Control kittens were primed with the expression vector encoding the PRV glycoprotein D (VR1012-gD) and boosted using the control recombinant vaccinia virus vSC. All animals were challenged with FIPV 3 weeks after the poxviruses had been administered.
No antibodies against FIPV-M or -N could be demonstrated in the sera of kittens vaccinated with VRMVRN-CpG and boosted with vFN and vFM (data not shown). However, analysis of sera taken 1 week after the oronasal challenge with 50 TCID50 FIPV 79-1146 demonstrated clearly that the immune system had been primed. Unlike the control cats (group A), all kittens from groups B and C had significant antibody levels against the M protein. Due to the known non-specific precipitation of the N protein by cat sera (see group A), the interpretation of the responses to the N protein is less clear-cut. Yet, the observations of Fig. 6 show that at least one cat had seroconverted, while the same appeared likely for the other animals from groups B and C.
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Discussion |
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These observations leave us with the question as to why protection was not achieved. Obviously the quality and/or degree of the immune responses induced by our vaccinations were insufficient. Because assays to measure CTL responses are not established in our laboratory for the feline species yet, we had no opportunity to directly evaluate this parameter. However, the protective effects described following vaccinations with poxviruses expressing the FIPV M or N protein (Vennema et al., 1991 ; Wasmoen et al., 1995
) suggest the existence of CTL epitopes on these antigens and we may assume that FIPV-specific cellular immune responses were induced but that their levels may have been just too low. In order to allow the quantitative analysis of these T cell responses in the future, we are presently establishing assays for feline CTLs.
While antibodies to the N protein obviously do not neutralize virus infectivity, those recognizing the exposed amino-terminal domain of the M protein potentially do. Indeed, a monoclonal antibody against the M protein can inhibit infection of feline macrophages in vitro (Kida et al., 2000 ). Anti-M antibodies might induce complement-mediated neutralization of FIPV, since those directed against the homologous protein of transmissible gastroenteritis virus, a related coronavirus, do neutralize in the presence of complement (Laviada et al., 1990
; Woods et al., 1988
). However, our results indicate that anti-M antibodies are not important for protecting cats against FIP. At day 7 after challenge, all vaccinated kittens had high titres of these antibodies, while titres of (infection-enhancing) S-specific antibodies were still low.
Cytokines play a critical role in orchestrating immune responses and there is much interest in the use of plasmids encoding cytokines as genetic adjuvants. Co-delivery of plasmids encoding IL-12 along with DNA vaccine formulations has been shown to augment antigen-specific CD4+ Th1 (Chow et al., 1998 ; Sin et al., 1999a
, b
; Tsuji et al., 1997
) and CD8+ CTL responses (Chow et al., 1998
; Hamajima et al., 1997
; Kim et al., 1997
; Okada et al., 1997
; Tsuji et al., 1997
). In cats, IL-12 co-delivery improved protection against feline immunodeficiency virus (Boretti et al., 2000
; Leutenegger et al., 2000
). Contrary to our expectation, co-injection of IL-12-encoding plasmids did not contribute to protection on the contrary, it clearly enhanced the susceptibility of the animal to FIPV challenge. The adverse effects of IL-12 were also demonstrated by the lower numbers of PBLs after challenge.
We can only speculate about how IL-12 may have caused the increase in susceptibility to FIPV. Factors known to diminish resistance of cats to FIPV are changes in the humoral (enhanced production of antibodies against the S protein) or cellular (inhibited activity) immune responses (Hayashi et al., 1983 ; Pedersen & Floyd, 1985
; Vennema et al., 1990
). We have no evidence from our experiments that IL-12 caused an enhanced production of S antibodies, which are known to exacerbate FIPV infection of macrophages through the binding of FIPV/antibody complexes to Fc receptors (Corapi et al., 1992
; Olsen et al., 1992
). The titres of neutralizing antibodies measured after challenge did not differ significantly between the groups. Enhanced infection of macrophages could also have occurred through Fc receptor upregulation via induction of interferon (IFN)-
(Horvath et al., 1996
; Mortola et al., 1998
; Puddu et al., 1997
). A similar mechanism has been described for IFN-
-mediated enhancement of dengue virus infection (Kontny et al., 1988
).
More likely, our co-administration of IL-12 may have led to a suppression of cell-mediated immunity. The observation that the PBL counts during the recovery phase were lower in IL-12-treated cats than in the controls supports this idea. Moreover, in the first trial injections of IL-12 DNA diminished the induction of antibodies during vaccination, possibly through effects on specific helper responses. Although IL-12 is a potent adjuvant for the induction of cell-mediated immunity, several studies have shown dose-dependent effects, with high cytokine concentrations sometimes leading to a suppression of the immune response. For instance, induction of cell-mediated immunity after vaccination with a recombinant adenovirus expressing hepatitis C virus antigens was potentiated by co-administration of a recombinant adenovirus expressing IL-12; high-dose co-administration of this vector, however, inhibited the immune response (Lasarte et al., 1999 ). Immunosuppression was accompanied by increased apoptosis in the spleen (Lasarte et al., 1999
).
Also, dose effects of IL-12 have been observed after DNA vaccination. Repeated injections with a low dose of IL-12 protein were found to enhance CTL induction, whereas a high dose suppressed generation of antigen-specific CTL responses (Lee et al., 2000 ). Similarly, antigen-specific T cell responses were enhanced by co-delivery of a low dose of IL-12 DNA during priming, while high IL-12 expression during priming or during the boost with recombinant vaccinia viruses was strongly suppressive (Gherardi et al., 2000
). The immunosuppressive effects seem to result from nitric oxide (NO), since the effect could be overcome by specific inhibitors of inducible NO synthase (Lasarte et al., 1999
; Gherardi et al., 2000
). In addition to the suppressive effect on the induction of cell-mediated immunity after vaccination, Orange et al. (1994)
also found that a high concentration of IL-12 can enhance susceptibility to viruses. They showed that treatment with a low dose of IL-12 enhanced immunity to LCMV infection, while the mice treated with high doses showed a dramatic decrease in CTL induction and a 2-log increase in LCMV titres in both spleen and kidneys. In view of the observations discussed above, we hypothesize that the adverse effects of IL-12 co-expression during our DNA vaccination against FIPV was caused by overexpression of the cytokine; lower levels might still enhance immunity against FIPV.
In summary, we show that DNA vaccination with vectors encoding the M and N proteins did not protect cats against FIP. Co-delivery of vectors encoding feline IL-12 also failed to induce protective immunity and even gave rise to adverse effects. Our study demonstrates that plasmids encoding IL-12 are no panacea for adjuvanting genetic vaccines.
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Acknowledgments |
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References |
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Received 17 May 2001;
accepted 30 August 2001.