1 The Jake Gittlen Cancer Research Institute, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
2 Department of Pathology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
3 Department of Comparative Medicine, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
4 Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
Correspondence
Neil D. Christensen
ndc1{at}psu.edu
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ABSTRACT |
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INTRODUCTION |
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Our previous studies have demonstrated that CRPV containing the regressive E6 gene plays a dominant role in controlling the spontaneous regression of CRPV-induced papillomas (Hu et al., 2002a). However, 100 % papilloma regression was not always achieved, even by the regressive-strain infections (Christensen, N. D., Hu, J. & Cladel, N. M., unpublished observations). Incomplete wart regressions on the same host by a particular virus infection may be due to incomplete host immunity. This phenomenon has been well-characterized in vaccination studies (Sundaram et al., 1998
; Han et al., 1999
; Hu et al., 2002b
). Different regression rates or phenotypes shown by the same virus infection in different hosts may result from inherent differences in host immunity (Salmon et al., 2000
; Hu et al., 2002a
). The link between papilloma regression and progression to the rabbit major histocompatibility complex (MHC) class II alleles DRA and DQA was reported in a previous study (Han et al., 1992
). Additional evidence demonstrated that rabbits homozygous for the DRA.DDQA.B haplotype were preferentially associated with early regression, whilst those homozygous for the DRA.CDQA.G haplotype were preferentially linked to wart persistence (Salmon et al., 2000
). An association between HLA type and human papillomavirus-induced cervical cancer has been reported (Bavinck et al., 1993
; Davidson et al., 2003
). In addition, malignant carcinomas associated with low-risk HPV types, such as HPV6 and HPV11, have been documented occasionally (Turazza et al., 1997
).
Studies using different viral antigens as immunogens have demonstrated that enhanced host immunity increased virion- or viral DNA-induced papilloma regression rates (Furumoto & Irahara, 2002). Other studies have demonstrated an increase of different types of HPV infection in renal-transplant recipients (Tieben et al., 1994
) and described the outgrowth of HPV-induced lesions in cyclosporin A (CsA)-treated transplant patients (Euvrard et al., 2003
). However, systematic documentation of the outcome of these HPV infections following cessation of CsA treatment is not presented in the literature. To investigate the impact of transient immune suppression on virus-induced papilloma evolution, we used CsA, an immunosuppressant that has been shown to inhibit lymphokine production by helper T cells in vitro and in vivo (Andrus & Lafferty, 1981
; Ali et al., 1982
; Dupont et al., 1985
). CsA has been widely used clinically to alleviate tissue allograft rejection (Jenkins et al., 1988
).
To determine the impact of genetic differences in host and virus during evolution of papillomavirus-induced tumours in transiently immunosuppressed animals, we used two rabbit strains [outbred and inbred New Zealand White (NZW)] and three CRPV variants (H.CRPVr, H.CRPVp-E6r and H.CRPVp-CE6rm). Outbred rabbits were purchased from a commercial supplier, whereas inbred rabbits were bred and maintained in our animal core facility. The three CRPV variants demonstrated a regressive phenotype in both outbred and inbred rabbits. These strains included H.CRPVr (Salmon et al., 2000), H.CRPVp-E6r (H.CRPVp containing the carboxyl-terminal portion of the H.CRPVr E6 gene; Hu et al., 2002a
) and H.CRPVp-CE6rm [H.CRPVp containing point mutations at three residues (E252G, G258D and S259P) in the E6 gene]. As this latter construct was not tested in our previous studies (Hu et al., 2002a
), but was highly regressive compared with other variants that we constructed, we chose to include the construct in the current study.
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METHODS |
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Inoculation of rabbit skin with plasmid viral DNA.
NZW outbred rabbits were purchased from Covance and EIII/JC inbred rabbits were bred and maintained in the animal facilities of the Pennsylvania State University College of Medicine. The Institutional Animal Care and Use committee of the Pennsylvania State University, College of Medicine, approved all animal-care and handling procedures. Viral DNA constructs (10 µg per site) were placed onto scarified sites in 50 µl volumes as described previously (Hu et al., 2002a). For inbred rabbits, four left-side sites and four right-side sites were challenged for H.CRPVr and H.CRPVp-E6r, respectively. For outbred rabbits, three of six CsA-treated rabbits were challenged at three sites each with H.CRPVr and H.CRPVp-E6r, whereas the remaining three rabbits were challenged at four additional sites with H.CRPVp-CE6rm.
Flow-cytometry analysis.
Peripheral blood lymphocytes (PBLs) were isolated from 10 ml blood. In brief, blood was diluted 1 : 2 with RPMI 1640 medium buffered with 10 mM HEPES and then underlaid with 10 ml Lympholyte-Rabbit (Cedarlane) and centrifuged at 1500 g at room temperature for 30 min. PBLs were collected at the interface and then diluted 1 : 2 with RPMI 1640 medium and centrifuged at 1500 g for 10 min. Contaminating red blood cells were lysed with ACK lysing buffer (Biofluids). PBLs were washed three times with RPMI 1640 medium and then counted. 1x107 PBLs were cultured in Eagle's medium [10 % fetal bovine serum (FBS), 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 0·1 mM non-essential amino acids, 50 µM 2-mercaptoethanol, 100 U penicillin ml1 and 100 µg streptomycin ml1] for about 3 h to allow time for optimal membrane-protein expression. After washing three times with PBS containing 2 % FBS, 1x106 PBLs were suspended in 30 µl PBS and incubated with 1 µl mAbs: mouse anti-rabbit CD4+ conjugated with fluorescein isothiocyanate (FITC) (RDI-CBL1400FT; Research Diagnostics) and mouse anti-rabbit CD8+ conjugated with FITC (RDI-CBL1402FT; Research Diagnostics) T-cell antibodies. The populations of cell-membrane markers on PBLs were determined by one-colour FSCAN flow-cytometry analysis (Hershey Medical Center Core Facility).
Confirmation of viral DNA in papillomas by DNA sequencing.
Biopsies of papillomas were collected monthly from rabbits. Total DNA was extracted by using a Qiagen DNeasy tissue kit. CRPV E6 plus E7 DNA fragments were amplified from each sample and partially purified by using a Qiagen PCR clean kit prior to sequencing. DNA sequencing was performed in the core facility of Pennsylvania State University College of Medicine. DNA alignment was analysed with DNAMAN software (version 5.2.9; Lynnon Biosoft).
Papilloma size determination and statistical analysis.
Papillomas were measured in three dimensions (lengthxwidthxheight) in mm, from which a geometric mean diameter (GMD) was calculated. Measurements were conducted weekly, beginning 3 weeks after initial viral DNA challenge. Data were represented as mean±SEM GMDs for papillomas per construct per group of animals. Statistical significance was determined by unpaired t-test comparisons. Regression rates occurring from H.CRPVr, H.CRPVp-E6r and H.CRPVp-CE6rm genomes on animals treated with CsA were compared with regression rates of papillomas from untreated animals by using Fisher's exact probability test for small samples.
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RESULTS |
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CsA decreased CD4+ and CD8+ T-cell levels in PBLs
CD4+ and CD8+ T cells are important for host defence against viral infections. The levels of CD4+ and CD8+ T cells in PBLs reflect levels of host immune response to pathogen invasion. CsA had been demonstrated to delay the maturation of thymus T cells. In our current study, significantly lower levels of CD4+ and CD8+ T cells were found in CsA-treated rabbits than in control rabbits (Fig. 2; P<0·05, t-test) 14 and 80 days after CsA injection. However, the levels of CD4+ and CD8+ T cells in CsA-treated rabbits returned to normal 2 months after CsA treatment was terminated (data not shown).
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Papilloma outgrowth in CsA-treated and non-treated EIII/JC inbred rabbits
To explore whether immunosuppression delayed regression of papillomas induced by H.CRPVr and H.CRPVp-E6r in EIII/JC inbred rabbits, eight rabbits were tested using the same protocol as described for the outbred rabbits. Four CsA-treated (17 challenge sites for each construct) and four control (16 challenge sites for each construct) rabbits were challenged with both H.CRPVr and H.CRPVp-E6r.
In the control group, papillomas induced by both H.CRPVr and H.CRPVp-E6r appeared around week 3 on all rabbits and began to regress and disappeared at all challenge sites around week 10. In contrast, papillomas on CsA-treated rabbits appeared at the same time and continued to grow. Although the growth rate was reduced after termination of CsA treatment, papillomas remained for several weeks. A significant difference was found for papilloma regression rates between CsA-treated rabbits and control rabbits induced by both constructs (Table 1; P<0·05, Fisher's exact test).
Papillomas induced by H.CRPVr were significantly smaller than those induced by H.CRPVp-E6r (Figs 6 and 7; P<0·05, t-test) at most time points. One of the CsA-treated rabbits (C0364) developed significantly smaller papillomas compared with the others. At week 21 for rabbit C0364, two papillomas induced by H.CRPVr and one induced by H.CRPVp-E6r regressed and the remaining ones were very small in size. However, papillomas on the remaining three rabbits continued to grow and were comparable in size to those that were formed during CsA treatment (Fig. 6
).
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Viral DNA stability in papillomas induced by different viral constructs
Papillomas induced by a regressive viral strain that becomes persistent might be a consequence of mutational changes in the viral genome, with selection of antigenic-escape variants. To determine whether any mutations had occurred in the persistent papillomas, we collected a biopsy of each papilloma from each CsA-treated rabbit (all the papillomas on three outbred and four inbred rabbits) weekly until week 10 and monthly from that time point on. The genetic sequence that we checked was E6 plus E7, which is the region that allows us to discriminate between the constructs that we used (H.CRPVr contains regressive E6 and E7, whereas H.CRPVp-E6r contains regressive E6 and progressive E7). All papillomas tested retained the original DNA sequences for the challenge constructs.
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DISCUSSION |
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We previously demonstrated that regressive E6 played a dominant role in triggering papilloma regression in both inbred and outbred rabbits (Hu et al., 2002a). The current study further supports this finding. The two regressive constructs that were tested in this study (H.CRPVr and H.CRPVp-E6r) did not show any differences in tumour outgrowth and regression in either inbred or outbred rabbits with intact host immunity. This implied that only the regressive E6 was strong enough to induce immune responses leading to the elimination of papillomas in these rabbits. However, in CsA-treated (immunocompromised) animals, remaining host immunity against viral DNA infection generated by H.CRPVr was much stronger than that induced by H.CRPVp-E6r, as manifested in earlier regressions and smaller papillomas (Table 1
, Figs 4 and 7
). Therefore, other genes or regions in the H.CRPVr genome must have played an additional role in triggering regression in these rabbits.
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In summary, short-term suppression of host immunity by CsA delayed papilloma regression in NWZ rabbits and changed the regression phenotype of regressive papillomavirus strains in EIII/JC inbred rabbits.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 15 July 2004;
accepted 14 October 2004.
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