Lia McLean, 2 Xiao-yi Sun, 1 Margaret Stanley, 2 Nell Almond 1 f and Geoffrey ... Present address: Sir William Dunn School of Pathology, Univer- sity of Oxford ...
Journal of General Virology (1990), 71, 2185-2190.
Printed in Great Britain
Increased antibody responses to human papiHomavirus type 16 L1 protein expressed by recombinant vaccinia virus lacking serine protease inhibitor genes Jian Zhou,1 Lionel Crawford, 1. Lia McLean, 2 Xiao-yi Sun, 1 Margaret Stanley, 2 Nell Almond 1f and Geoffrey L. Smith2:~ I l C R F Tumour Virus Group, Department of Pathology and 2Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 IQP, U.K.
The L1 gene of human papillomavirus type 16 (HPV16) driven by the vaccinia virus major late 4b gene promoter has been inserted into three different sites of the vaccinia virus genome. Insertion into the thymidine kinase (TK) gene was achieved by selection of TKmutants in BUdR on TK- cells. Insertion into two vaccinia virus serine protease inhibitor (serpin) genes was achieved by co-insertion of the Escherichia coli xanthine guanine phosphoribosyltransferase gene linked to the vaccinia virus 7-5K promoter and selection of mycophenolic acid-resistant recombinant
viruses. Each recombinant virus expressed a 57K L1 protein at similar levels and with similar kinetics. However, immunization of mice with these recombinant viruses induced different levels of antibody to the L1 protein. Viruses lacking serpin genes B13R and B24R induced significantly higher antibody levels than did viruses lacking the TK gene. The presence of functional B13R and B24R gene products is therefore somehow immunosuppressive at least for antibody responses to the L1 protein of HPV-16.
A major problem with research with human papillomaviruses (HPV), particularly type 16 (Diirst et al., 1985), is the lack of any system for producing virus particles in quantity. Virion proteins, such as the major coat protein L1, must therefore be produced in expression vectors. For immunological purposes eukaryotic vectors are preferable as they are more likely to generate proteins with correct post-translational modifications including phosphorylation and glycosylation and these may be important for the recognition and processing of the antigen. Vaccinia virus recombinants have been widely used to express eukaryotic proteins including HPV antigens (Mackett et al., 1984; Browne et al., 1988) and have the advantage over some other eukaryotic vectors in that infection of an animal with recombinant virus presents the native protein to the immune system over an extended period. Expression of the HPV-16 L1 gene in vaccinia virus poses conflicting requirements. Firstly, this gene con-
tains the sequence TTTTTNT twice within its coding region (Seedorf et al., 1985). This sequence functions as a termination signal for the vaccinia virus DNA-dependent RNA polymerase early during infection (Rohrmann et al., 1986; Yuen & Moss, 1987) so that full-length L1 transcripts would not be produced from exclusively early vaccinia virus promoters. Consistent with this, much greater levels of L1 expression were obtained from the 4b (major late) rather than 7-5K (early and late) vaccinia virus promoter (Browne et al., 1988). Secondly, stimulation of T cell responses may be better if the antigen is expressed from early rather than from late vaccinia virus promoters, whereas B cell responses may result from the use of either class of promoter (Coupar et al., 1986). Early promoters may therefore be preferable if stimulation of both humoral and cell-mediated immunity is required. The vaccinia virus thymidine kinase (TK) gene has been a widely used site for insertion of foreign D N A since this is both non-essential for virus replication and enables genetic selection of T K - recombinant viruses. However, T K - recombinants are severely attenuated (Buller et al., 1985) so that the immune response evoked against a foreign antigen may be reduced. We have
i" Present address: National Institute of Biological Standards and Control, Blanche Lane, Potters Bar, Hertfordshire EN6 2QG, U.K. :~ Present address: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, U.K. 0000-9550 © 1990 SGM
investigated the consequence of insertion of HPV-16 L1 into other sites in the virus genome and compared the immunogenicity of recombinant viruses expressing this gene. The insertion sites chosen are the serine protease inhibitor (serpin) genes B13R and B24R (Kotwal & Moss, 1989; Smith et al., 1989) (Fig. 1). These genes are expressed early during infection (Smith et al., 1989) and in cowpox virus the homologue of the vaccinia virus B13R gene is responsible for a haemorrhagic pock phenotype (Pickup et al., 1986) and influences the inflammatory response (Palumbo et al., 1989; Chua et al., 1990). Recombinant vaccinia viruses which express the HPV-16 L1 gene and which are defective for TK or serpin genes B13R or B24R were constructed as follows. The serpin coding regions were disrupted by the HPV L 1 gene linked to the vaccinia virus strong late 4b promoter (Rosel & Moss, 1985; Kent, 1988) and the Escherichia coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene driven by the constitutively active 7.5K promoter (Boyle & Coupar, 1988) (Fig. 1). Insertion of HPV-16 L1 into the TK gene was similar to that described (Browne et al., 1988). The 2.5kb SfaNI-AvalI D N A fragment from HPV-16-pAT153 (Dfirst et al., 1983), which starts 10 nucleotides upstream of the first ATG of the L1 open reading frame and terminates 865 nucleotides downstream of the L1 TAA stop codon, was modified by addition of BglII linkers and cloned into the BamHI site of pRK19 (Kent, 1988) downstream of the 4b promoter to form plasmid pRK19/16 L1. Plasmid pSX5 was constructed by the insertion of a 1479 bp PvuII-SalI fragment containing serpin B24R (derived from the vaccinia virus Sail fragment) into SmaI- and SalIcut pUC9. The Ecogpt gene joined to the vaccinia virus 7-5K promoter was isolated as "an EcoRI fragment from plasmid pGpt07/14 (Boyle & Coupar, 1988), treated with Klenow enzyme to create blunt ends and cloned into the coding region of B24R at the unique KpnI site, which had been treated with T4 DNA polymerase, to form plasmid pSX3. An XbaI-SmaI fragment containing the 4b promoter linked to HPV L1 was then isolated from a modified version of pRK19/16 L1, treated with Klenow polymerase to create blunt ends and cloned into pSX3, which had been cleaved with BamHI and treated with Klenow enzyme, to form pSX5. Plasmid pLC19 was constructed by cloning a 5652 bp EcoRI-SalI fragment (derived from the vaccinia virus SalI G fragment) into EcoRI- and Sa/I-cut pUC13. This plasmid, pSTH1, contains most of serpin B 13R. The complete serpin gene was re-formed by cleavage of the vaccinia virus SalI I fragment with BglII, treatment with Klenow enzyme to form blunt ends, cleavage with SalI, isolation of a 1221 bp fragment and insertion of this into pSTH1 that had been cleaved with HindIII, treated with Klenow enzyme
~ ' N,,
/ B 13R\ FB~4R
• Virus DNA • Virus promoter [ ] gpt gene
E. coligpt HPV-16 L1 • HPV ORF P7-5 P4b B24R
E. coli gpt HPV-16 L1 P7.5 P4b B 13R
I ~' F
P4b HPV-16 L1
Fig. 1. Upper panel, location of the serpin genes B13R and B24R and the TK gene on the vaccinia virus genome. The serpin genes and TK gene are in the HindIIl B and J fragments, respectively. The I-IindlII map is shown on the top line and the SalI sites are indicated below. The arrows indicate the direction of transcription. Lower panel, plasmid vectors used to construct recombinant vaccinia viruses. Abbreviation: ORF, open reading frame.
and digested with SalI. The plasmid formed, pGS124, was digested with EcoRI and BglII, treated with Klenow enzyme and the large fragment was isolated and selfligated to form pYC 15. pYC 15 was digested with HincII and ligated with a 2-1 kb fragment containing the Ecogpt gene joined to the vaccinia virus 7.5K promoter (isolated as described above) to form pYC16, pYC16 was digested with PstI, treated with T4 DNA polymerase and ligated with the XbaI-SmaI fragment containing the HPV L1 gene joined to the 4b promoter (above) rendered bluntended with Klenow enzyme. Plasmids pLC19 and pSX5 were transfected into cells infected with wild-type (wt) vaccinia virus strain WR (Mackett et al., 1984). Recombinant viruses were selected by plaque assay in the presence of mycophenolic acid, xanthine and hypoxanthine (Falkner & Moss, 1988; Boyle & Coupar, 1988). For cloning of HPV L1 into the TK gene, plasmid pRK19/16 L1 was transfected into wt-infected cells and T K - recombinant virus was selected by plaque assay in the presence of BUdR on
human T K - 143 cells and screened for the presence of HPV L1 D N A (Mackett et al., 1984). The recombinant vaccinia viruses containing the HPV L1 gene inserted into the TK, B 13R or B24R genes were termed p R K 19i 16 L1 vv, pLC19 vv and pSX5 vv, respectively. Stocks of recombinant viruses were grown and titrated in CV-1 cells as described (Mackett et al., 1985a). The genomes of these recombinant viruses were analysed by restriction enzyme digestion, agarose gel electrophoresis and Southern blotting (data not shown). In the case of pRKI9/16 L1 vv, the 5 kb HindlII J fragment containing the T K gene had increased in size to 7.7 kb (the predicted size with addition of the HPV L1 gene) and this band also hybridized with HPV L1 DNA. For pSX5 vv, the Sail I fragment, containing serpin B24R, had increased in size consistent with addition of both the HPV L1 and Ecogpt genes. Serpin B13R crosses the Sail G and Sail I junction so that after SalI digestion of pLC19 vv the Sail G fragment had increased in size due to insertion of L1, and the Sail I fragment had increased in size due to insertion of the Ecogpt gene. In summary, the viruses all had genome structures consistent with the insertion of foreign D N A as predicted. Expression of the L1 gene was analysed by Western blot analysis using an anti-HPV L1 monoclonal antibody Camvir 1 (McLean et al., 1990; Fig. 2). Lysates from cells infected with wt or recombinant vaccinia viruses were separated by electrophoresis on 10~ polyacrylamide gels and the proteins blotted onto nitrocellulose filters. The filters were incubated in 3~o bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at 37 °C for 1 h and then with Camvir 1. After a 30 rain wash in PBS containing 1 ~ NP40, the filter was incubated with antimouse ~25I-labelled IgG, washed again, dried and autoradiographed. In lysates from cells infected with each of the three recombinant viruses there was a polypeptide of 57000 Mr that was absent from lysates of cells infected with wt virus. This polypeptide was of the predicted size for HPV L1 and was synthesized at the same level and with the same kinetics in each case. The expression and nuclear location of the L1 protein in recombinant virus-infected cells was also demonstrated by immunofluorescence (data not shown). The immunogenicity of the three recombinant viruses was compared by immunization of groups of 10 female BALB/c mice with 1 x 107 p.f.u, of virus by intraperitoneal (i.p.) or subcutaneous (s.c.) injection (2 x 106 p.f.u. s.c., 8 x 106 p.f.u.i.p.). Inoculations were repeated after 2 weeks and sera collected 7 days later and tested for antibodies to H P V L1 by ELISA (Fig. 3). HPV16 LI protein was produced in E. coli by expression vector pKK223-3 (Pharmacia) and was kindly provided by Dr D. H. Davis. One-hundred gl samples of diluted L1 protein (containing approximately 1 gg of protein per ml)
Fig. 2. Western blot analysis of HPV-16 L1 expressedby recombinant vaccinia viruses. CV-1 cellswere infectedat 10 p.f.u./cellwith wt (lane 1) or recombinantvaccinia viruses pRK19/16 L1 vv (lane 2), pSX5 vv (lane 3) or pLCI9 vv (lane 4) and harvestedat 48 h p.i. The positionsof Mr markers are shown on the left. were placed in fiat-bottomed microtitre plate wells and left overnight at 4 °C. After washing three times with borate-buffered saline and blocking with 1 ~ BSA, the antigen-fixed wells were reacted with test antiserum, followed by peroxidase-linked sheep anti-mouse IgG. The wells were then reacted with o-phenylenediamine (800 gg/ml) plus H 2 0 : (0.025~o) and the absorbance of the supernatants was measured by an ELISA Kinetic Microplate Reader (Molecular Devices) at 490 nm. The data are expressed as kinetic rates (mOD/min) corrected for the control serum values. All mice immunized with recombinant virus expressing HPV L1 showed antibody responses against this antigen; sera from mice immunized with wt virus remained at background level. However, the antibody titres in the pLC19 vv and pSX5 vv groups were higher than in those receiving pRK19/16 LI vv (Fig. 3) and could be detected at 1:500 dilution. These differences could have been due to serpin gene inactivation. Alternatively, these different titres may be due to virus attenuation resulting from loss of the T K gene in virus pRK19/16 L1 vv. To address the latter possibility, T K - virus (derived from wt virus by passage in BUdR) was used for construction of additional serpinnegative viruses. These T K - and serpin-negative viruses induced similar levels of anti-HPV L1 antibody to their T K + counterparts (Fig. 3) demonstrating that the increased levels of antibodies are due to serpin gene inactivation. Interestingly, antiserum induced by pLC 19
Fig. 3. ELISA m e a s u r e m e n t of anti-HPV LI antibody levels from vaccinated mice. From left to right the data are the m e a n of sera from 10 mice immunized with pRK19/16 L1 vv, pSX5 vv (B24R), pSX5)TK- (B24R), pLC19 vv (B13R) or p L C 1 9 / T K - (B13R). The bar on each column indicates the standard deviation of the m e a n value.
vv (B13R insertion) and pRK19/16 L1 vv (TK insertion) recognized different epitopes of LI (D. H. Davies & M. Wishart, personal communication). Many recombinant vaccinia viruses have been used to immunize animals and generate good immune responses against a foreign gene product, for example the influenza virus haemagglutinin (Smith et al., 1983b), hepatitis B virus surface antigen (Smith et al., 1983a), herpes simplex virus glycoprotein D (Paoletti et al., 1984; Cremer et al., 1985) and the Epstein-Barr virus gp340 (Mackett & Arrand, 1985). In these experiments neutralizing antibodies were produced and experimental animals have subsequently been protected against challenge with the appropriate viruses (Moss et al., 1984). Besides the humoral immune response, cell-mediated reactions were also found against the foreign antigens (Bennink et al., 1984, 1986; Wiktor et al., 1984; McMichael et al., 1986). The ability of recombinant vaccinia viruses to stimulate both humoral and cell-mediated responses against the expressed foreign antigens greatly enhances the potential uses of the vaccinia viruses to prevent these virus infections. To attempt to increase the immunogenicity of vaccinia
virus recombinants we have inserted a foreign gene, in this case the HPV LI gene, into different sites in the vaccinia virus genome and compared the immune response of mice immunized with these different viruses. Serpin genes B13R and B24R which are located between 10 and 17 kb from the right end of the genome (Kotwal & Moss, 1989; Smith et al., 1989) were chosen for insertion sites. A third serpin gene is located in the H i n d l I I K fragment near the opposite end of the virus genome (Boursnell et al., 1988). The function and essentiality of these genes in vaccinia virus was unknown, but in cowpox virus which has homologues of the B13R and B24R genes (Pickup et al., 1986; Kotwal & Moss, 1989), the BI3R gene i'~ non-essential for virus growth and is responsible for a haemorrhagic pock phenotype (Pickup e t a / . , 1986). Recently, it has been suggested that this haemorrhagic phenotype is attributable to the serpin gene product directly or indirectly inhibiting the infiltration of the infected area by white cells, and it is the presence of these cells in pocks formed by serpindeficient virus that prevents haemorrhage (Palumbo et al., 1989). Extracts from cells infected with serpinpositive cowpox virus also prevent the migration of lymphocytes in vitro (Chua et al., 1990). Whether the serpin causes haemorrhage by direct inhibition of blood clotting is unknown. However, our observation that antibody responses to a foreign antigen are reduced by serpin genes B13R or B24R is consistent with a model in which the serpin gene product prevents the infiltration of the infected area by inflammatory cells, resulting in a diminished immune response. Another proposed function for the serpin genes is the inhibition of cellular proteases which are involved in the proteolytic degradation of intracellular antigens (Smith et al., 1989). The observation that vaccinia virus may block the presentation of some antigens to class I major histocompatibility complex-restricted cytotoxic T cells (Coupar et al., 1986) and that this blockage may be overcome by expressing unstable proteins (Townsend et al., 1988) or peptide fragments (Gould et al., 1989) is consistent with this proposal. The potential for successful immunoprophylaxis of HPV infections is encouraged by the fact that bovine papillomavirus type 1 L1 expressed in E. coli protects cattle from development of warts due to virus infection (Pilacinski et al., 1985). Vaccinia virus recombinants might offer some advantages for immunization against HPV due to the vaccine's stability, low cost, ease of administration and ability to stimulate T cell and antibody responses. Although the HPV L1 protein is nuclear, other nuclear virus antigens expressed by vaccinia virus have induced good immune responses in vaccinated animals (Mackett et al., 1985b; Yewdell et al., 1985). Moreover, immunization of animals with
recombinant viruses expressing nuclear antigens of polyoma virus have caused the prevention or regression of polyoma virus-induced tumours (Lathe et al., 1987). It remains to be determined Whether deletion of the serpin genes can influence the immune responses to other foreign proteins expressed by vaccinia virus but it is possible that immune responses (antibody and T cell) may be generally increased by the deletion of these genes. We are indebted to Professor H. zur Hausen for the HPV-16 plasmid, to Dr D. H. Davies for the L1 protein produced by the pKK223-3 expression vector, to Dr D. B. Boyle for the Ecogpt gene and to Mrs Helen Wilson for preparing the manuscript. G. L. S is a Lister InstituteJenner Research Fellow.
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(Received 28 February 1990; Accepted 24 May 1990)