1 Department of Pathology, UniversiO, of Cambridge, Tennis Court Road, Cambridge ..... Ts-iao Ch-hia Research Studentship (Downing College, University of.
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Journal of General Virology (1994), 75, 165 169. Printed in Great Brita#t
Delayed-type hypersensitivity response to human papillomavirus type 16 E6 protein in a mouse model M a r k A. Chambers, ~ Simon N. Staeey, 2 John R. Arrand 2 and Margaret A. Stanley ~* 1Department of Pathology, UniversiO, of Cambridge, Tennis Court Road, Cambridge CB2 1QP and 2 Cancer Research Campaign Department of Molecular Biology, Paterson blstitute for Cancer Research, Christie Hospital, Manchester M20 9BX, U.K.
A mouse model incorporating the epitheliotropic nature of human papillomavirus (HPV) infections has been used to study an immune response to HPV type 16 (HPV-16) E6 protein in vivo. Using a transplantation technique, a novel immortal keratinocyte cell line expressing the E6 protein has been grafted onto syngeneic mice to re-form a differentiated epithelium overlying a granulation tissue bed. By this approach the
presentation of viral antigens to the immune system can be modelled in a way analogous to the natural infection. Here we report a delayed-type hypersensitivity (DTH) reaction in grafted mice challenged intradermally with a recombinant vaccinia virus expressing the HPV-16 E6 protein. The specificity of the response was confirmed by the absence o f a DTH reaction to challenge with virus expressing either HPV-16 E7 or LI protein.
More than 65 different types of human papillomavirus (HPV) have been described, classified on the basis of nucleotide homology. At least 20 genotypes are associated with lesions of the genital tract. Benign genital warts (condylomata acuminata of low malignant potential) are most frequently associated with types 6 and 11, whereas types 16 and 18 are found predominantly in anogenital dysplasias and carcinomas. Our understanding of the immunobiology of papillomaviruses, although limited, is central to any strategy for prophylactic or therapeutic intervention. The evidence from immunosuppressed patients (Alloub et al., 1989; Kent et al., 1987; Sillman et al., 1984; Lutzner et al., 1983; Mullen et al., 1976) and regressing warts (Fierlbeck et al., 1989; Aiba et al., 1986) suggests an important role for the immune system in papillomavirus infections. Humoral responses to the transforming proteins E6 and E7 in benign genital disease are low (Jenison et al., 1990) in contrast to the levels of antibody response demonstrated by the serum of patients with cervical carcinoma (Muller et al., 1992; Mann et al., 1990; Diltner, 1990; Jochmus-Kudielka et at., 1989). Despite this, doubt still exists as to whether seroconversion is an inevitable consequence of HPV infection, and furthermore no apparent benefit has been attributed to the presence of HPV-16- or -18-specific antibodies in the serum. In contrast to humoral immunity there is good evidence that cell-mediated immune responses are important in the pathogenesis of HPV infections. The
spontaneous regression of HPV-associated lesions is accompanied by a pronounced local infiltration of inflammatory cells including cytotoxic T lymphocytes (CTLs), macrophages and natural killer cells (Fierlbeck et al., 1989; Aiba et al., 1986), and a histological appearance characteristic of a delayed-type hypersensitivity (DTH) response. Recently, an animal model has been described that enables immune responses to HPV-16 early gene proteins within the BALB/c mouse to be studied (McLean et al., 1993). The model incorporates the exclusively epithelial nature of HPV-16 by the presentation of antigen to the animal as a differentiated syngeneic epithelial graft expressing viral protein. Mice primed in this way with HPV-16 E7-expressing cell lines respond with a DTH reaction on subsequent challenge with recombinant E7 protein (McLean et al., 1993; M. A. Chambers, Z. Wei, N. Coleman, A. A. Nash & M. A. Stanley, unpublished results). The reaction is specific to the E7 protein and is dependent on the presence of CD4 + T cells. Using this animal model we have demonstrated a DTH reaction to recombinant E6 protein specifically in mice primed by the grafting of E6-expressing syngeneic keratinocytes. The materials and methods used in this report have been described previously (McLean et al., 1993), with the following exceptions. BALB/MK cells were obtained from S. A. Aaronson, Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Md., U.S.A. and are described in Weissman & Aaronson (1983). They were cultured in keratinocyte serum-free
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Fig. 1. Expressionof E6 protein by VACE6 early (a) and late (b) in infection. CV-1 cells were infected with 10 p.f.u./cell VACE6 (oddnumbered lanes) or parental virus WR (even-numberedlanes) for (a) 4 h or (b) 12 h. Cells were metabolicallylabelled with [35S]cysteine from 2 h p.i.. then tysed in RIPA buffer and immunoprecipitated as described previously (Stacey et al., 1992). Sera used for immunoprecipitation were: (a) polyclonal anti-E, coli-derived fl-galactosidas~E6 fusion protein (lanes 1 and 2); cervical carcinoma patient A (lanes 3 and 4); normal human control serum (lanes 5 and 6); polyclonal anti-vaccinia virus (lanes 7 and 8); (b) cervical carcinoma patient A (lanes 1 and 2); cervical carcinoma patient B (lanes 3 and 4): anti-E6 monoclonal antibody C1P5 (lanes 5 and 6); polyclonal anti-MS2-E6 fusion protein (lanes 7 and 8). Mr markers are shown on the right of each panel (lanes M).
medium (SFM) (Gibco-BRL) supplemented with the bovine pituitary extract and h u m a n epidermal growth factor supplied with the medium in a 31 °C humidified atmosphere containing 5 % CO,,. Recombinant vaccinia virus expressing HPV-16 E6 protein was constructed as follows. The HPV-16 E6 open reading frame (ORF) was isolated as a 5 4 2 b p Eco0109(112) to DdeI(654) fragment, blunt-ended with the Klenow fragment and B a m H I ( C G G A T C C G ) linkers were attached. After digestion with B a m H I the fragment was ligated into the B a m H I site of vaccinia virus insertion vector p1109, which contains the 7-5K promoter and an in-frame N c o I start codon (M. Mackett, unpublished results). This resulted in amino acids 2 and 3 of the E6 O R F (F and Q) being replaced with two vector-encoded amino acids (D and P). The insertion vector also contained the dominant selectable gpt marker gene. The vector was used to insert the E 6 0 R F into the thymidine kinase (TK) locus of the W R strain of vaccinia virus, by selection for the gpt marker. The recombinant virus was produced by transfection of the insertion vector into WR-infected CV-1 cells. Following at least three rounds of plaque purification in medium containing 25 g g / m l mycophenolic acid (Sigma M-5255), 250 t.tg/ml xanthine (Sigma X-0250) and 15 g g / m l hypoxanthine (Sigma H9377) a single clone was selected, designated VACE6. The presence and location of the E 6 0 R F was confirmed
by Southern blotting and by testing for the T K phenotype using B U d R selection (data not shown). Expression of the E6 protein was investigated using the radioimmunoprecipitation assay (RIPA) (Fig. 1). This figure shows that E6 was expressed by VACE6 but not by W R at both early and late times following infection. The E6 protein could be detected by only some antisera raised against Escherichia coli-derived fusion proteins, in line with previous observations (Stacey eta[., 1992). This suggests that the dominant epitopes recognized by these antisera are masked or buried in the structure of the E6 protein when it is in its native conformation. Sera from E6-seropositive cervical carcinoma patients readily recognized the vaccinia virus E6 protein in RIPAs, demonstrating the antigenic authenticity of the recombinant protein. To determine whether mice would m o u n t a D T H response to HPV-16 E6 [as reported by McLean et al. (1993) for HPV-16 E7] two new cell lines were derived by infection of B A L B / M K cells with either the recombinant retrovirus L X S N or LXSN16E6 (obtained from C. L. Halbert, Fred Hutchinson Cancer Research Center, Seattle, Wash., U.S.A. ; described in Halbert et al., 1991). Infection of cells was carried out in 60 m m dishes seeded with 5 x 105 cells. Virus was added to 3 ml of SFM containing 4 lag/ml polybrene (hexadimethrine bromide; Sigma). The cells were incubated for 8 h prior to
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Fig. 2. Immunoprecipitation of HPV-16 radiolabelled E6 protein. Samples were precipitated with rabbit polyclonal E6 antiserum and run on a 17.5% polyacrylamide gel prior to autoradiography. Lane 1, MKLE6SN cells: lane 2, M K L X S N cells; lane 3, CaSki cells (human cervical squamous cell carcinoma cell line containing integrated HPV16). The positions of size markers and the E6 protein are given.
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24 48 Time after challenge with recombinant vaccinia virus (h) Fig. 3. DTH response to specificrecombinant vaccinia virus challenge. Mice were grafted with or without 107 MKLE6SN cells and challenged 7 days later in the left ear with 107 p.f.u, of the vaccinia virus recombinant shown. The graph shows the difference in thickness between the left and right (unchallenged control) ears measured 24 and 48 h after intradermal inoculation. Each group contained five mice: error bars represent the S.D. of the group. Production of VACE6 is described in the text. VACE7 and VACL1 have been described previously (McLean et al., 1993). A--A, MKLE6SN+VACE6; I1--11, MKLE6SN+VACL1; O O, No graft+VACE7; [] D, No graft +VACL1 ; /~ A, No graft +VACE6. refeeding with fresh SFM. Clones of infected cells were established by selection in medium containing 200 g g / m l G418 (Sigma). Infection of cells with L X S N resulted in the monoclonal line M K L X S N , containing no H P V sequences. The infection of ceils with LXSN16E6 resulted in the monoclonal line M K L E 6 S N which by R I P A was shown to express the HPV-16 E6 protein (see Fig. 2). We examined the ability of 107 M K L E 6 S N cells to
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form an epithelium when grafted onto syngeneic mice (methodology as in McLean et al., 1993). Seven days after grafting the cells formed an epithelium exhibiting full thickness atypia with very limited differentiation potential. The graft had a highly irregular basal layer and in some levels there was early invasion of the granulation tissue bed (data not shown). This is not a consequence of E6 expression since a similar phenomenon has been seen with the control line M K L X S N , containing no H P V sequences (data not shown). To assay for D T H reactivity to HPV-16 E6, mice were grafted with 107 M K L E 6 S N cells and challenged intradermally 7 days later in the left ear with 107 p.f.u. recombinant VACE6 or V A C L 1. Similarly, three control groups of mice were sham-grafted with 200 gl PBS and subsequently challenged with 10 v p.f.u. VACE7, VACL1 or VACE6. A significant ear swelling response (P < 0.002, Student's t-test) was seen in the group grafted with 107 M K L E 6 S N cells and challenged with VACE6 compared with each of the control groups (see Fig. 3). We conclude that HPV-16 E6 protein produced in the epithelium can prime immunocompetent mice in an antigen-specific manner. The immunogenicity of HPV-16 E6 has been demonstrated before. Serum antibody responses from cervical carcinoma patients have been demonstrated using baculovirus-derived recombinant E6 protein as the target antigen (Stacey et al., 1992) and to specific E6 peptides (Muller et al., 1992). However, for humoral responses it appears that reactivity to HPV-16 E6 is associated with HPV-16-containing cervical cancer but not to HPVnegative cervical cancer or interestingly to HPV-16containing cervical intraepithelial neoplasia (CIN) (Ghosh et al., 1993). For some time it has been known that the ability to m o u n t a D T H reaction to specific viral or non-specific antigens is a factor in wart immunity (Thivolet et al., 1977). For example, positive skin tests have been reported in patients with regressing warts or with a history of past warts challenged with purified papillomavirus capsid antigen (Viac et al., 1977), in C I N patients on challenge with HPV-16 L 1 peptides expressed as recombinant bacterial fusion proteins (Hopfl et al., 1991), whereas contact dermatitis induced by dinitrochlorobenzene at the site of recalcitrant verrucae provokes their rejection (Greenberg et al., 1973). To date, the bulk of ceil-mediated immunity studies on HPV-16 have focused on responses to the E7 protein with a few notable exceptions. Stauss et al. (1992) have demonstrated three murine class I (H-2K b or D b) binding peptides within HPV-16 E6 capable of stimulating primary C T L responses in vitro, and have underlined the importance of peptide length for C T L induction. Extrapolation to the h u m a n situation has proved less successful, however. In a typical study, only one of 29
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p a t i e n t s w i t h C I N w a s p o s i t i v e in a l y m p h o c y t e p r o l i f e r a t i o n a s s a y u s i n g p u r i f i e d H P V - 1 6 E6 p r o t e i n ( C u b i c et al., 1989) a n d o n l y o n e T cell d e t e r m i n a n t in H P V - 1 6 E6 ( D R 7 D w 7 - r e s t r i c t e d ) has b e e n d e f i n e d u s i n g s y n t h e t i c p e p t i d e s to s t i m u l a t e p e r i p h e r a l b l o o d m o n o n u c l e a r cells f r o m n o r m a l i n d i v i d u a l s ( S t r a n g et al., 1990). T h e a b i l i t y o f H P V - 1 6 E6 to act as a t u m o u r r e j e c t i o n a n t i g e n has b e e n d e m o n s t r a t e d f o l l o w i n g i m m u n i z a t i o n with recombinant E 6 v a c c i n i a virus, in the r a t ( M e n e g u z z i et al., 1991), a n d w i t h cells e x p r e s s i n g t h e E 6 p r o t e i n , in t h e m o u s e ( C h e n et al., 1992). I n t h e l a t t e r m o d e l , EG-specific, C D 8 + C T L s c o u l d be g e n e r a t e d in vitro f r o m s p l e e n cell p o p u l a t i o n s d e r i v e d f r o m t h e EG-immunized mice. T o o u r k n o w l e d g e , t h e d a t a p r e s e n t e d h e r e are t h e first d e m o n s t r a t i o n o f a n in vivo C D 4 - m e d i a t e d r e s p o n s e to t h e H P V - 1 6 E6 p r o t e i n in t h e m o u s e . T a k e n t o g e t h e r with the demonstration of a DR-restricted determinant in H P V - 1 6 E6 ( S t r a n g et al., 1990), this m a y i n d i c a t e a r o l e f o r C D 4 + cells in t h e r e s p o n s e to H P V a d d i t i o n a l to t h a t i m p l i e d f o r C D 8 + C T L s . I n v i e w o f these d a t a a n d t h o s e o f C h e n et al. (1992) t h e E 6 p r o t e i n o f H P V - 1 6 is a n a t t r a c t i v e c a n d i d a t e as a t a r g e t a n t i g e n in a n y v a c c i n e strategy. A l t h o u g h t h e W R - b a s e d E6 r e c o m b i n a n t v a c c i n i a v i r u s d e s c r i b e d h e r e is t o o v i r u l e n t f o r use in humans, the model system described here provides an ideal opportunity for testing the antigenicity of poxvirus r e c o m b i n a n t s b a s e d o n m o r e a t t e n u a t e d f o r m s o f the virus. The authors would like to thank Esther Choolun and Debs Jordan for their technical assistance, and Dr Nicholas Coleman for his help with histology. Mark A. Chambers was supported by the Nita King Studentship (Department of Pathology, University of Cambridge), the Ts-iao Ch-hia Research Studentship (Downing College, University of Cambridge), and the Harnett Fund (Clinical School, University of Cambridge). Simon N. Stacey and John R. Arrand are supported by the Cancer Research Campaign.
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(Received 16 June 1993; Accepted 30 August 1993)
