Veterinary Immunology and Immunopathology, 35 ( 1992 ) 191-197

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Elsevier Science Publishers B.V., Amsterdam

Enhancement after feline immunodeficiency virus vaccination Margaret J. Hosie, Robert Osborne, George Reid, James C. Neil and Oswald Jarrett Universityof Glasgow, Department of VeterinaryPathology, Bearsden, GlasgowG61 1QH, UK

ABSTRACT Hosie, M.J., Osborne, R., Reid, G., Neil, J.C. and Jarrett, O., 1992. Enhancement after feline immunodeficiency virus vaccination. Vet.Immunol. Immunopathol., 35:191-197. Cats were vaccinated with one of the three preparations: purified feline immunodeficiency virus (FIV) incorporated into immune stimulating complexes (ISCOMs), recombinant FIV p24 ISCOMs, or a fixed, inactivated cell vaccine in quil A. Cats inoculated with the FIV ISCOMs or the recombinant p24 ISCOMs developed high titres of antibodies against the core protein p24 but had no detectable antibodies against the env protein gp 120 or virus neutralising antibodies. In contrast, all of the cats inoculated with the fixed, inactivated cell vaccine developed anti-env antibodies and four of five had detectable levels of neutralising antibody. However, none of the vaccinated cats were protected from infection after intraperitoneal challenge with 20 infectious units of FIV. Indeed there appeared to be enhancement of infection after vaccination as the vaccinated cats become viraemic sooner than the unvaccinated controls, and 100% of the vaccinated cats became viraemic compared with 78% of the controls. The mechanism responsible for this enhancement remains unknown.

ABBREVIATIONS FIV, feline immunodeficiency virus; HIV, human immunodeficiency virus; ISCOM, immune stimulating complex; PBT, peripheral blood T-cells; SPF, specific pathogen free. INTRODUCTION

The development of a safe, effective vaccine against feline immunodeficiency virus (FIV) infection is of importance for two reasons. First, FIV is a valuable model for the study of human immunodeficiencyvirus (HIV) infection and therefore successful vaccination against FIV would provide encouragement that an effective vaccine against HIV might be developed using simCorrespondence to: Margaret J. Hosie, Department of Veterinary Pathology, University of Glasgow, Bearsden, Glasgow G61 1QH, UK.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00

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ilar strategies. In this respect, FIV has a significant advantage over other animal model systems since it is a naturally occurring infection which is transmissible and causes disease in its natural host. Second, FIV is an important pathogen in the cat, causing immunodeficiency with a poor prognosis. Hence an effective vaccine would also be of great veterinary importance. The aims of this study were to compare the relative efficacies of three vaccine preparations in mounting humoral responses to FIV core and env proteins and in responding to challenge with live homologous virus. MATERIALS AND METHODS

Virus FIV/Glasgow-8 was grown in feline peripheral blood T-cells (PBT). Approximately 6 X 106 PBT were resuspended in FIV-infected tissue culture supernatant overnight and then were grown in normal T-cell medium containing RPMI-1640 with 10% foetal bovine serum, 2 mM glutamine (Gibco, Paisley, U K ) , 100 units penicillin m1-1 (Gibco), 10 #g streptomycin ml -~ ( Gibco ), 2 X 10- 5 mol 2-mercaptoethanol ( Sigma, Poole, UK) and 100 units recombinant human interleukin-2 m l - 1 (kindly provided by J. Nunberg, Cetus, Emeryville, CA).

Preparation of lSCOMs To prepare FIV ISCOMs, 1.5 ml of virus purified from 400 ml of tissue culture supernatant by ammonium sulphate precipitation was added to 1.5 ml of 4% decanoyl N-methylglucamide and incubated for 1 h at 37°C. Lyophilised quil A was added to a final concentration of 0.1% together with 150 /tl of lipid mix containing 10 mg phosphatidylcholine and l 0 mg cholesterol ml-1 in 20% decanoyl N-methylglucamide in Tris-saline. The preparation was then dialysed over 2 days against frequent changes of 50 mM ammonium acetate. Recombinant p24 ISCOMs were prepared in the same way using recombinant p24 expressed in Escherichia coli as described previously (Reid et al., 1991 ).

Analysis of lSCOMs The FIV and recombinant p24 ISCOMs were analysed by electron microscopy and by density gradient centrifugation on a 10-40% sucrose gradient at 35K for 12 h using an SW40.1 rotor. Electron microscopy revealed the typical regular cage-like structures, approximately 40 nm in diameter. The ISCOM preparations banded at a density of 1.087 g 1-1. The protein content of the FIV ISCOMs was determined by immunoblot analysis. These contained predominantly p24, as well as p17 and small

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19 3

amounts of p55 and gp 120. The relative concentrations of the proteins were determined by Coomassie blue staining of a sodium dodecyl sulphate polyacrylamide gel using ovalbumin as a standard. Using this method the protein content of the FIV ISCOMs was estimated to be 10/lg of p24 and p 17 per dose. There was negligible gp 120 by Coomassie blue staining. The recombinant p24 ISCOMs contained 50 #g of p24 per dose.

Preparation offixed, inactivated cell vaccine FIV/Glasgow-8 was grown in PBT with uninfected cells being added at weekly intervals. The cells were harvested when the culture was producing large amounts of virus as determined by ELISA for p24 production (FIV antigen detection kit: IDEXX, Slough, U K ) . The cells were treated with glutaraldehyde using a modification of procedures described previously (Zaia and Oxman, 1977; Stott et al., 1984). The infected culture was spun at 800 × g for 5 rain, then 6 × 107 cells were resuspended in ice cold Earle's balanced salt solution and 6 ml of 0.15% glutaraldehyde were added. The cells were left on ice for 5 rain and 12 ml of 0.1 M glycine were added on ice. The cells were centrifuged at 800 X g for 5 rain; the supernatant was discarded and the cells were resuspended in 6 ml of normal T-cell medium with 10% dimethylsulphoxide, 600 #1 of Tris pH 8.0 and 13.2 pl offl-propiolactone. The cells were incubated at 37°C in the dark for 2 h, dispensed into 200 #1 volumes and stored at - 70°C. One aliquot was thawed, washed and co-cultivated with uninfected cells; the culture was maintained for 1 month. The culture was examined daily for the appearance of cytopathic effect and samples of tissue culture supernatant were taken at weekly intervals to be tested by ELISA for p24 production. There was no evidence of cytopathic effect in the culture and the results of the weekly tests for antigen production were consistently negative. For each dose of vaccine, 2 X 106 cells were thawed and washed twice in phosphate buffered saline (PBS). The cells were resuspended in 1 ml of PBS containing 50 pg of quil A and kept on ice during the short period prior to inoculation. RESULTS

Vaccination with FIV ISCOMs Four 5-month-old specific pathogen free (SPF) cats were inoculated subcutaneously with FIV ISCOMs at 0, 5 and 18 weeks. These four cats, together with four age-matched unvaccinated controls, were challenged intraperitoneally at 20 weeks with 20 infectious units of FIV. On the day of challenge, all four vaccinated cats were seropositive by ELISA (FIV antibody detection kit;

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IDEXX) and had antibodies to p55, p24 and pl 7 by immunoblot analysis. No anti-env antibodies could be detected and there were no neutralising antibodies.

Vaccination with recombinant p24 ISCOMs Four 10-week-old SPF kittens were inoculated subcutaneously with ISCOMs containing 50 #g of recombinant p24 per dose at 0, 3, 5 and 7 weeks. These five cats were challenged intraperitoneally at 9 weeks with 20 infectious units of FIV, as were the cats vaccinated with fixed, inactivated cells and their age-matched unvaccinated controls. On the day of challenge, all four vaccinated cats were seropositive by ELISA (FIV antibody detection kit; IDEXX) and had high titres of antibodies to p24 by immunoblot analysis. No neutralising antibodies were detected.

Vaccination with fixed, inactivated cells Five 10-week-old kittens were inoculated subcutaneously at 0, 3, 6, 9, 12 and 15 weeks with 2 × 106 cells per dose and challenged on Week 21. On the day of challenge all five vaccinated cats were seropositive by ELISA (FIV antibody detection kit, IDEXX) and had antibodies to p55, p24 and pl 7 by immunoblot analysis. Anti-env antibodies were detected by radioimmunoprecipitation of [~4C]glucosamine-labelled FIV-infected PBT, using the method previously described by Hosie and Jarrett (1990). Antibodies against both gpl20 and gp41 were detected from Week 6 (data not shown). Four of the five vaccinated cats had detectable neutralising antibody titres, three of 25 and one of 100. TABLEI Proportion of viraemic cats at intervals after challenge Weeks post challenge 0

3

5

FIV ISCOMS Controls

0/4 0/4

3/4 1/4

4/4 3/4

p24 ISCOMS Cell vaccine Controls

0/4 0/5 0/5

4/4 4/5 0/5

6

8

9

4/4 3/4 4/4 5/5 4/5

4/4 5/5 4/5

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Enhancement of FIV infection after vaccination The development of viraemia in the cats vaccinated with FIV ISCOMs, recombinant p24 ISCOMs and fixed, inactivated cells (cell vaccine) and the unvaccinated controls is shown in Table 1. In both challenge experiments the unvaccinated controls became viraemic later than the vaccinated cats. Furthermore, whereas 13 of 13 vaccinated cats became viraemic, only seven of nine unvaccinated controls became infected following challenge. DISCUSSION

Protection against FIV infection has been demonstrated recently by others using inactivated whole virus or inactivated virus-infected cells (Yamamoto et al., 1991 ). In these experiments only those cats with very high levels of neutralising antibodies were protected (J. Yamamoto, personal communication, 1991 ). Other cats with lower levels of neutralising antibodies became viraemic. However, in contrast with the findings of this study, cats that developed low titres of neutralising antibodies did not become viraemic earlier than the controls. In this study one cat in each of the unvaccinated control group did not become infected after challenge, demonstrating that the challenge dose was close to the minimum infectious dose. A similarly low dose of challenge virus was used in the study which demonstrated protection. Enhancement of infection after vaccination has been demonstrated in other viral systems. For example, it has been shown with West Nile virus (Fagbami et al., 1988) and Dengue virus (Halstead, 1988) that the binding of antibody-antigen complexes to cellular Fc receptors facilitates the attachment of virus particles to target cells. Furthermore, in vitro studies with HIV demonstrated that Fc receptors induced by cytomegalovirus allowed immune complexes of HIV to infect fibroblasts which were otherwise not permissive to HIV infection (McKeating et al., 1990). This result suggested that antibodymediated HIV infection ofmonocytes, macrophages and fibroblasts could occur in vivo. Enhancement of retroviral infection after vaccination was also recorded in cats inoculated with purified feline leukaemia virus envelope protein (Pedersen et al., 1986 ). The cats developed antibodies against the envelope protein, but these did not neutralise the virus in vitro and the cats were more susceptible to infection with feline leukaemia virus compared with unvaccinated cats. In this study cats in only one of the three vaccine groups produced anti-env antibodies although there was enhancement of infection in all three groups. It would appear that enhancement was not mediated via opsonisation of the challenge virus by anti-env antibodies followed by increased uptake via Fc receptors. It was not possible to demonstrate enhancing antibodies in any of the cats in the three vaccine groups (data not shown). Furthermore, the cats

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inoculated with recombinant p24 ISCOMs produced antibodies to only the core protein 1924. Such antibodies should not be expected to enhance infection by this mechanism. Therefore we would suggest that the e n h a n c e m e n t seen in this study arose by a different mechanism. Inoculation with any FIV protein m a y activate the feline i m m u n e system, thereby enhancing infection by the challenge virus. We have recently d e m o n s t r a t e d that the receptor for FIV is present on activated T-cells (Hosie et al., 1992 ); hence any procedure activating the i m m u n e system and increasing the expression o f the FIV receptor m a y enhance infection after challenge. This e n h a n c e m e n t m a y have been overcome in the presence o f very high levels o f neutralising a n t i b o d y in the study which demonstrated protection. To test this hypothesis future vaccine experiments will test imm u n i s a t i o n with ISCOMs containing a non-FIV antigen. It will also be desirable to identify which viral components are unnecessary for protection or which elicit e n h a n c e m e n t and to omit these from future vaccine preparations.

ACKNOWLEDGEMENTS The authors t h a n k Jan Cole for technical assistance. This work was supported by a grant from the Medical Research Council AIDS Directed Programme. REFERENCES Fagbami, A., Halstead, S.B., Marchette, N. and Larsen, K., 1988. Heterologousflavivirus infection-enhancingantibodies in sera of Nigerians. Am. J. Trop. Med. Hyg., 38:2055-2070. Halstead, S.B., 1988. Pathogenesis of dengue: challenges to molecular biology. Science, 239: 476-480. Hosie, M.J. and Jarrett, O., 1990. Serologicalresponses to feline immunodeficiencyvirus. AIDS, 4:215-220. Hosie, M.J., Willett, B.W., Dunsford, T.H., Neil, J.C. and Jarrett, O., 1992. A monoclonal antibody which blocks infection with feline immunodeficiencyvirus. Abstract MoA 0042, VIII. Int. Conf. on AIDS, 19-24 July, Amsterdam, Netherlands. McKeating, J.A., Griffith, P.D. and Weiss, R.A., 1990. HIV susceptibility conferred to human fibroblasts by cytomegalovirus-inducedFc receptor. Nature, 343:659-661. Pedersen, N.C., Johnson, L., Birch, D. and Theilen, G.H., 1986. Possible immunoenhancement of persistent viremia by feline leukaemia virus envelope glycoproteinvaccine in challengeexposure conditions where whole inactivated virus vaccines were protective. Vet. Immunol. Immunopathol., 11: 123-148. Reid, G., Rigby, M., McDonald, M., Hosie, M.J., Neil, J.C. and Jarrett, O., 1991. Immunodiagnosis of feline immunodeficiency virus infection using recombinant viral p 17 and p24. AIDS, 5: 1477-1483. Stott, E.J., Thomas, L.H., Taylor, G., Collins, A.P., Jebbett, J. and Crouch, S., 1984. A comparison of three vaccines against respiratory syncytialvirus in calves. J. Hyg., 93:251-261. Yamamoto, J.K., Okuda, T., Ackley,C.D., Louie, H., Pembroke, E., Zochlinski,H., Munn, R.J.

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and Gardner, M.B., 1991. Experimental vaccine protection against feline immunodeficiency virus. AIDS Res. Hum. Retroviruses, 7:911-922. Zaia, A. and Oxman, M.N., 1977. Antibody to Varicella-Zoster virus-induced membrane antigen: immunofluorescence assay using monodisperse glutaraldehyde-fixed target cells. J. Infect. Dis., 136: 519-530.

Enhancement after feline immunodeficiency virus vaccination.

Cats were vaccinated with one of the three preparations: purified feline immunodeficiency virus (FIV) incorporated into immune stimulating complexes (...
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