Department of Medical Microbiology, University of Cape Town, Faculty of Health Sciences, Anzio Road, Observatory 7925, Cape Town, South Africa1
Departments of Medicine and Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA2
Author for correspondence: Anna-Lise Williamson.Fax +27 21 4484110. e-mail annalise{at}medmicro.uct.ac.za
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Abstract |
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HPV vaccine development has been further impaired by the inability to infect laboratory animals with HPV. This has prevented the demonstration of protection against HPV challenge. The present study describes the development and use of a recombinant HPV-16 L1vaccinia virus (VV) challenge model in mice to demonstrate protection in VLP-16-vaccinated mice and to investigate the importance of cell-mediated immunity induced by VLP-16. The use of recombinant VV expressing the nucleoprotein of vesicular stomatitis virus as a challenge system was first described by Bachmann et al. (1994) to investigate the efficacy of a potential vesicular stomatitis virus vaccine. This technique has also been applied to assess human immunodeficiency virus type 1 vaccine regimes in mice (Belyakov et al., 1998
; Kent et al., 1998
). The present study shows that immunization with VLP-16 elicits T cell proliferative responses characterized by the production of both Th1 and Th2 cytokines and is capable of protecting mice against challenge with recombinant HPV-16 L1VV (VVL1R-16).
VLP-16 were produced in a baculovirus expression system (Rose et al., 1994 b ). To examine the T cell response to VLP-16, groups of 68-week-old BALB/c mice (Animal Unit, University of Cape Town) were immunized intraperitoneally (i.p.) either with a single dose of 20 µg VLP-16 or with three consecutive weekly doses of 10 µg VLP-16. Two weeks after the last inoculation, splenocytes were isolated by passage through a steel mesh (Sigma) to obtain a single-cell suspension. Contaminating red blood cells were removed by centrifugation over Ficoll-Hypaque density gradients as described previously (Boyum, 1968
). Viable cells were resuspended at 2x106 cells/ml in RPMI-1640 medium supplemented with 10% foetal calf serum. For the lymphoproliferation assay, splenocytes (2x105 cells per well) were seeded in triplicate into round-bottomed 96-well culture plates (Nunc). Splenocytes were incubated for 6 days at 37 °C in a humidified 5% CO2 atmosphere in the presence of purified VLP-16 (15 µg/ml) or baculovirusinsect cell extract (10% v/v). [3H]Thymidine (1 µCi per well) was added to each well for the last 18 h of the assay. The cells were harvested by using an automated cell harvester (PHD, Cambridge Technology) and the radioactivity was measured by using a liquid scintillation counter (Tricarb-4640). For generation of cytokine-containing supernatants, splenocytes (2x105 cells per well) were seeded in quadruplicate into round-bottomed 96-well culture plates (Nunc) in the presence or absence of purified VLP-16 (15 µg/ml). Culture supernatants were collected at 4, 5 and 6 days and stored at -20 °C. Interferon (IFN)-
(Th1-type) and interleukin (IL)-4 (Th2-type) were assessed by a sandwich ELISA according to the manufacturer's recommendations (Biotrak, Amersham).
A single i.p. immunization of 20 µg VLP-16 resulted in detectable T cell proliferation after only a single cycle of in vitro stimulation (Fig. 1a). When mice were given three consecutive weekly booster doses, proliferative responses to VLP-16 were increased 12-fold (Fig. 1b
). Because the VLP were purified from a baculovirus expression system, insect cell extract was used as a control for non-specific proliferation to residual insect cell proteins. Lymphocytes from VLP-16-primed mice did not proliferate in response to these insect cell proteins, indicating that the response to VLP-16 was antigen-specific. Lymphocytes from control mice, given placebo inoculations with PBS, did not proliferate to HPV antigen or insect cell proteins.
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Two groups of four female BALB/c mice (812 weeks old) were compared. Group A was vaccinated with 10 µg VLP-16 (Bachmann et al., 1994 ) and group B was not vaccinated. After 13 days, groups A and B were challenged with VVL1R-16. Five days after challenge, the ovaries were harvested. The ovaries were chopped finely, placed in McIlvain's buffer (4 mM citric acid, 0·2 M Na2HPO4.12H2O) (100 mg ovary/ml) and homogenized by using a ten Broeck grinder (30 strokes). This was followed by three cycles of freezethawing and then preparations were centrifuged at 2000 r.p.m. for 5 min. The supernatant fluid was titrated in CV-1 cells and the VVL1R-16 titre per mouse was calculated.
VLP-16-immunized BALB/c mice were better able to control VVL1R-16 infection following challenge than the unvaccinated control mice (4·6 log10 protection, P<0·05; Fig. 2a). Protection was VLP-16-specific, as challenge with wild-type VV only resulted in a 1 log10 reduction in vaccinated mouse ovarian virus titre (2·4x107 p.f.u.) compared with unvaccinated mice (2·3x108 p.f.u.). X-Gal staining of titration plates containing virus from mouse ovaries confirmed that uncleared virus in vaccinated mice was recombinant VVL1R-16. A similar experiment with VLP-16-immunized C57 BL/6 mice confirmed that they were also protected from VVL1R-16 challenge compared with their unvaccinated littermates (2·3 log10 protection) but the level of protection was lower compared with BALB/c mice (Fig. 2b
). This finding is consistent with previous reports that have shown that C57 BL/6 mice are genetically more resistant to pox virus infection than the innately susceptible BALB/c strain (O'Neill & Brenan, 1987
). These results indicate that BALB/c mice may be a better strain for the VVL1R-16 challenge model.
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References |
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Received 30 March 1999;
accepted 19 May 1999.