Production of human papillomavirus type 45 virus-like particles in insect cells using a recombinant baculovirus

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1 ELSEVIER FEMS Microbiology Letters 141 (1996) MICROBIOLOGY LETTERS Production of human papillomavirus type 45 virus-like particles in insect cells using a recombinant baculovirus Abstract Antoine Touzk, Catherine Dupuy, Martine Chabaud, Pierre Le Cann, Pierre Coursaget * Laboratoire d lmmunologie des Maladies Infectieuses, Facultk de Pharmacie, 31 avenue Mange, Tours, France Received 15 January 1996; revised version received 20 May 1996; accepted 20 May 1996 The Ll major capsid protein of human papillomavirus type 45 was expressed in insect cells using recombinant baculovirus technology. Human papillomavirus type 45 Ll major capsid protein self-assembled into empty virus-like particles (VLPs). These SO-60 nm diameter particles were present in the nuclei of recombinant baculovirus-infected insect cells and had a density of g/cm3 in c&urn chloride. The expressed human papillomavirus type 45 Ll protein sequence is identical to the reference human papillomavirus type 45 strain except for one amino acid located at position 49 of the human papillomavirus type 45 Ll protein. It must be noted that the quantity of purified human papillomavirus type 45 virus-like particles is at a lower level than that previously observed with human papillomavirus type 16. Nevertheless, the ability to generate preparative amounts of human papillomavirus type 45 virus-like particles is of great importance for the production of an anti-human papillomavirus vaccine. Keyworrls: Papillomavirus; Human papillomavirus type 45; Ll protein; Virus-like particles; Baculovirus 1. Introduction Genital infection with human papillomavirus (HPV) is one of the most common sexually transmitted diseases. Moreover, HPV are now considered to be the most important risk factor in the development of cervical cancer, the second most important cancer in women worldwide [1,2]. About 43 of the 74 distinct HPV types so far identified are known to infect the genital tract. Among them, HPV 16, 18, 45 and 31 account for about 80% of the HPV infections associated with cervical cancer [1,3]. A recent * Corresponding author. Tel.: ; Fax: worldwide study of the prevalence of HPV detection in cervical cancer showed that HPV 16 was present in 50% of specimens, HPV 18 in 14%, HPV 45 in 8% and HPV 31 in 5% [l]. However, there were significant geographical variations, with a higher prevalence of HPV 45 observed in tropical African countries, where this genotype was detected in lo-33% of cervical cancer specimens tested [l]. A growing body of evidence indicates that HPV virus-like particles (VLPs) may be valuable immunogens in the prevention of HPV infections and related malignant lesions. (i) Immunization with HPV 11 virions has elicited antibodies in animals which inhibit the infectivity of this virus in a xenograft experiment [4]. These neutralizing antibodies recognized conformational /96/$ Published by Elsevier Science B.V. PHSO (96)

2 112 A. TouzP et ul. I FEMS Microbiology Letters 141 (1996) epitopes of the viral capsid protein [5,6]. (ii) In a canine model, immunization with formalin-inactivated canine oral papillomavirus (COPV) protected dogs against COPV challenge [7]. (iii) Immunization of cottontail rabbits with VLPs composed of the cottontail rabbit papillomavirus (CRPV) Ll major capsid protein expressed in the baculovirus expression system has recently been shown to protect rabbits against CRPV challenge [8,9]. Because of the prevalence of different HPV types in cervical cancer, a vaccine against HPV genital infection and cervical cancer must contain HPV types 16, 18, 45 VLPs and perhaps those of less frequent types such as HPV 31 and 33 [l]. Because HPV virions cannot be obtained by cell culture, HPV Ll major capsid protein has been expressed in eukaryotic expression systems such as recombinant vaccinia virus, recombinant baculovirus and more recently in yeast [ Expressed Ll protein was shown to self-assemble into VLPs which resembled empty papillomavirus virions. Thus far, HPV 16, 18 and 33 VLPs have been produced [lo, In order to develop an anti- HPV vaccine with a broad activity against types associated with cervical cancer, we have cloned and expressed HPV 45 Ll gene in the baculovirus expression system. 2. Materials and methods 2.1. Molecular cloning of HP V 45 Ll gene HPV 45 Ll open reading frame (1620 bp) was amplified by PCR from cells obtained by cervical scraping from a HPV 45 infected Senegalese woman (isolate 58/T). The following synthetic oligonucleotide primers were used: 5 -ATATAGATCTT- CATGGCACACAATATT-3 (nt ) as forward primer and 5 -ATGAAGCTTCATATTAT- TTCTTACTAC-3 (nt ) as reverse primer [17]. Upstream of the ATG and downstream of the stop codon, BgZII and Hind111 restriction enzyme sites were added respectively (bold-faced letters). PCR was carried out with Taq DNA polymerase (Eurobio, Les Ullis, France) in a Perkin-Elmer Thermocycler. The PCR product was cloned into the pcri1 vector (TA cloning kit, Invitrogen, San Die- go, CA, USA) and then sub-cloned into the pblue- BacIII vector (Invitrogen) digested with BgflI and Hind111 restriction enzymes in order to obtain pblue- BacIII-Ll HPV 45. HPV 45 Ll gene was then sequenced with the dideoxynucleotides method (Genome Express, Grenoble, France). Spodoptera frugiperda cells (Sfp) were co-transfected with Autographa californica nuclear polyhedrosis virus (AcMNPV) linearized genomic DNA (Linear AcMNPV Transfection Module, Invitrogen) and pbluebaciii-ll HPV 45 DNA. A recombinant baculovirus encoding the Ll major capsid protein HPV 45 (AC Ll 45) gene was selected in one round of plaque purification by visual examination and confirmed by PCR Production and characterization of HPV 4.5 LI protein 39 cells, maintained in supplemented Grace s insect medium with 10% fetal calf serum (Life Technologies, Eragny, France), were infected at a multiplicity of infection of 20 with AC Ll 45. Four days post-infection, cells were harvested and fractionated into cytoplasmic and nuclear fractions by Nonidet P40 treatment (0.5%) followed by centrifugation (10000 Xg, 15 min). The nuclear pellet was resuspended in 8 M urea. These fractions were separated on 12% SDS-PAGE, blotted onto BA83 nitrocellulose (Schleicher and Schuell, Dassel, Germany), probed with papillomavirus common epitope rabbit antiserum (Biogenex Laboratories, San Ramon, CA, USA) and proteins were revealed by chemiluminescence with the ECL Western blotting kit (Amersham, Buckinghamshire, UK). Nuclear extract of AC Ll 16 infected Sf9 cells was also used as positive control [ Purzjication of HPV 45 VLPs HPV 45 VLPs were purified essentially as previously described for HPV 16 VLPs [IS]. Sf9 cells were grown in spinner culture ( ml, 2 X lo6 cells/ml). Four days post-infection with AC Ll 45, cells were collected and resuspended in ice-cold PBS containing 0.5% Nonidet P40. After incubation on ice for 15 min, the cell lysate was centrifuged ( X g, 15 min). The nuclear pellet was resus-

3 A. To& et al. IFEMS Microbiology Letters 141 (1996) pended in PBS and sonicated by three bursts of 15 s each at 60% maximal power (Vibra-cell, Bioblock Scientific, Strasbourg, France). The nuclear lysate was loaded onto a 40% (w/v) sucrose cushion and centrifuged in a Beckman SW28 rotor (150 min, rpm, 4 C). The pellet was resuspended in PBS, loaded onto CsCl and centrifuged to equilibrium in the same rotor (20 h, rpm, 4 C). CsCl gradient fractions were collected and investigated for density by refractometry, for HPV reactivity with anti-hpv 16 VLP mouse sera in an ELISA (detailed below) and by electron microscopy for the presence of VLPs. Microplates (CML, Nemours, France) were coated with each gradient fraction diluted by two-fold dilution from l/250 to l/32000 in 0.1 M carbonate buffer (ph 9.6) and incubated overnight at 4 C. After 4 washes with PBS-0.1% Tween 20 (washing buffer), non-specific binding sites were blocked by incubation for 30 min at 37 C with 200 ~1 PBS-l% newborn calf serum. After removing blocking solution, 100 pl of HPV 16 VLP-immunized mouse sera diluted l/2000 in dilution buffer (PBS 5 X, 10% newborn calf serum, 2% Tween 20) was incubated for 90 min at 45 C. After 4 washes, 100 pl of horseradish peroxidase conjugated to goat antimouse immunoglobulins (Dako, Glostrup, Denmark), l/1000 in dilution buffer, was added and incubated at 45 C. After 90 min and 4 washes, 100 ul of substrate solution containing o-phenylene diamine and Hz02 was added and incubated at room temperature for 30 min. Optical densities (492 mn) were determined and the antigenic titer was determined using a cut-off value of 0.1 (mean of controls plus two standard deviations). Mouse anti-hpv 16 VLP sera were obtained after subcutaneous immunization of BALBlc mice at days 0, 7, 14 and 28 with 5 pg of HPV 16 VLPs adsorbed on aluminum hydroxide (0.2%) Electron microscopy Positive samples detected with anti-hpv 16 Ll VLPs mouse sera (antigenic titer > 16000) and with density of were applied to glow discharged carbon-coated grids, negatively stained with 1.5% uranyl acetate and observed at x magnification in a JEOL 1010 electron microscope. In order to compare the amount of HPV 45 VLPs produced to that observed previously with HPV 16, each HPV VLP preparation was mixed with an equal volume of baculovirus-expressed recombinant hepatitis B core particles (HBc particles) and then examined by electron microscopy as previously described. HBc particles have a diameter of 30 nm, making them easily distinguishable from papillomavirus VLPs. We counted the total number of HPV VLPs and HBc Ag particles in five fields at X magnification randomly. 3. Results The Ll gene of HPV 45 was cloned by PCR amplification using DNA derived from cervical scraping of a HPV 45 infected woman. The amplification of the full-length Ll gene was successful in one sample (58/T) out of three HPV 45 positive samples investigated. Nucleotide sequence analysis of the cloned gene revealed 99.9% homology to the HPV 45 Ll prototype sequence [17]. Three point mutations were detected at positions 5559, 5675 and 5796 on the HPV 45 genome (Table 1). The alignment of HPV 45 Ll amino acid sequences derived from 58/ T isolate to HPV 45 Ll prototype revealed that only the point mutation at nucleotide 5675 altered the primary structure of the protein: Ser-49 was replaced by Asn-49 (Table 1). This point mutation did not affect the three potential N-linked glycosylation sites (Asn-X-Ser or Asn-X-Thr) located at residues 173, 370, and 425 of the HPV 45 Ll protein. The HPV 45 Ll gene was cloned into the baculovirus transfer vector under the control of the polyhedrin promoter using pbluebacii1 vector. After co-transfection of the plasmid with AcMNPV linear DNA into St9 Table 1 Sequence comparison of the HPV 45 Ll coding region (nt 553& 7149) HPV 45 isolate Sequence aa 49 nt 5559 nt 5675 nt 5796 Prototype GGT AGC GTT Ser 58/T GGC AK GTC Asn

4 114 A. To& et al. IFEMS Microbiology Letters 141 (1996) Fig. 1. Western blot analysis of HPV 45 and HPV 16 Ll proteins expression and sub-cellular localization. Recombinant Ll proteins were detected with papillomavirus common epitope rabbit antiserum. Lane 1: nuclear extract of St9 cells infected with AC Ll 16. Lane 2: cytoplasmic extract of St9 cells infected with AC Ll 45. Lane 3: nuclear extract of St9 cells infected with AC Ll 45. cells, recombinant baculovirus AC Ll 45 was purified using a plaque purification assay. The synthesis of the HPV 45 Ll protein in AC Ll 45 infected Sf9 cells was analyzed by Western blot. HPV 45 Ll protein was detected as a 60 kda band Fig. 3. Electron micrographs of purified HPV 45 virus-like particles. VLPs were negatively stained with uranyl acetate and ob- served under X magnification. in the nuclei of AC Ll 45 infected cells (lane 3 in Fig. 1). As a control, HPV 16 Ll protein appeared as a 56 kda band (lane 1 in Fig. 1). The size of the band obtained for HPV 45 Ll protein was consistent with the predicted coding capacities of the HPV 45 Ll open reading frame. This protein was not detected in the cytoplasm of AC Ll 45 infected Sf9 cells (lane 2 in Fig. 1). In addition, the 60 kda protein was detected with CamVir-1 (anti-hpv 16 Ll protein monoclonal antibody, Pharmingen, San Diego, CA, USA) and HPV 16 Ll VLP mouse antisera (data not shown). Antigenic activity was detected with HPV 16 VLP mouse anti-sera in the CsCl fractions corresponding to densities of (Fig. 2). These fractions were analyzed by electron microscopy, and VLPs were observed (Fig. 3). These particles were spherical particles with diameters of nm. The morphology and density in CsCl of these particles were similar to other HPV VLPs produced [ To compare the yield of VLPs from HPV 45 and HPV 16 [15], each of these VLPs was mixed with HBc particles and the relative proportion of HPV VLPs was assessed by electron microscopy. The amount of HPV 45 VLPs produced was determined to be four times less than that observed for HPV 16 VLPS. 6 II 10 CsCl gradient fractions Fig. 2. Detection of HPV 45 VLPs by ELISA in CsCl gradient fractions with anti-hpv 16 VLP mouse sera. 4. Discussion Our results indicate that the Ll protein of HPV 45

5 A. To& et al. I FEMS Microbiology Letters 141 (1996) has the same intrinsic ability to form virus-like particles as Ll of other high risk types when expressed in insect cells using the recombinant baculovirus system, and that L2 or other papillomavirus viral proteins are not required for assembly. These VLPs are nm in diameter and have a CsCl density of g/cm3, similar to HPV 16 VLPs [lo,ls]. Kirnbauer et al. have pointed out the essential role of Asp at position 202 for self-assembly of HPV 16 Ll protein [lo]. It has been shown that the production of VLPs is reduced 122-fold if this Asp-202 is replaced by His [13]. The alignment of the Ll protein sequence of isolate 58/T (HPV 45) with isolate 114/K (HPV 16) [lo] shows that Asp-202 at position 229 of the HPV 45 Ll protein corresponds to Asp at position 202 of the HPV 16 Ll protein. Moreover, the region surrounding this position is relatively well conserved. However, with the same level of Ll protein expression, determined by Coomassie blue staining (data not shown), the amount of HPV 45 VLPs produced was four times less compared to HPV 16 VLPs obtained in a previous study [15]. Sasagawa et al. [13] have also observed variations in the amount of HPV 16 VLPs produced in relation to different isolates of HPV 16. We can therefore speculate that the same variations in the self-assembly of the Ll protein of different HPV 45 strains could exist. Thus, it would be interesting to amplify and express the Ll gene from other strains in order to increase the yield of HPV 45 VLPs. Nevertheless, the ability to generate preparative amounts of HPV 45 VLPs is of great importance for the production of an anti-hpv vaccine. Acknowledgments We thank Pierre-Yves Sizaret, Facultt de MCdetine de Tours, for electron microscopy. This work was supported by the Minis&e de 1 Enseignement Suptrieur et de la Recherche, the Ligue contre le Cancer and the Association Paul Metadier (Tours). References [l] Bosch, F.X., Manos, M.M., Munoz, N., Scherman, M., Jansen, A., Peto, J., Schiffman, M.H., Moreno, V., Kurman, R. and Shah, K.V. (1995) Prevalence of papillomavirus in cervi- cal cancer: a worldwide perspective. J. Natl. Cancer Inst. 87, [2] Zur Hausen, H. (1989) Papillomaviruses in anogenital cancer as a model to understand the role of viruses in human cancer. Cancer Res. 49, [3] Chan, S.Y., Delius, H., Halpem, A.L. and Bernard, H.U. (1995) Analysis of genomic sequences of 95 papillomavirus types: uniting typing, phylogeny, and taxonomy. J. Virol. 69, [4] Ghim, S.J., Christensen, N.D., Kreider, J.W. and Jenson, A.B. (1991) Comparison of neutralization of BPV-1 infection of Cl27 cells and bovine fetal skin xenograft. Int. J. Cancer 49, [5] Christensen, N.D. and Kreider, J.W. (1990) Antibodymediated neutralization in vivo of infectious papillomaviruses. J. Virol. 64, [6] Christensen, N.D., Kreider, J.W., Cladel, N.M., Patrick, S.D. and Welsh, P.A. (1990) Monoclonal antibody-mediated neutralization of infectious papillomavirus type 11. J. Virol. 64, [7] Bell, J.A., Sundberg, J.P., Ghim, S.J., Newsome, J., Jenson, A.B. and Schlegel, R. (1994) A formalin-inactivated vaccine protects against mucosal papillomavirus infection: a canine model. Pathobiol. 62, [8] Breitburd, F., Kirnbauer, R., Hubbert, N.L., Nonnenmacher, B., Trin-Dinh-Desmarquet, C., Orth, G., Schiller, J.T. and Lowy, D.R. (1995) Immunization with virus-like particles from cottontail rabbit papillomavirus (CRPV) can protect against experimental CRPV infection. J. Virol. 69, [9] Jansen, K.U., Rosolowsky, M., Schultz, L.D., Markus, H.Z., Cook, J.C., Donelly, J.J., Martinez, D., Ellis, R.W. and Shaw, A.R. (1995) Vaccination with yeast-expressed Cottontail rabbit papillomavirus (CRPV) virus-like particles protects rabbits from CRPV-induced papilloma formation. Vaccine 13, [lo] Kirnbauer, R., Taub, J., Greenstone, H., Roden, R., Dtirst, M., Gissmann, L., Lowy, D.R. and Schiller, J.T. (1993) Efficient self-assembly of human papillomavirus type 16 Ll and Ll-L2 into virus-like particles. J. Virol. 67, [ll] Rose, R., Bonnez, W., Reichman, R.C. and Garcea, R.L. (1993) Expression of human papillomavirus type 11 Ll protein in insect cells: in vivo and in vitro self-assembly of viruslike particles, J. Virol. 63, [12] Rose, R., Bonnez, W., Da Rin, C., McCance, D.J. and Reichman, R.C. (1994) Serological differentiation of human papillomavirus types 11, 16 and 18 using recombinant virus-like particles. J. Gen. Virol. 75, [13] Sasagawa, T., Pushko, P., Steers, G., Gscbmeissner, SE., Nasser Hajibagher, M.A., Finch, J., Crawford, L. and Tommasino, M. (1995) Synthesis and assembly of virus-like particles of human papillomaviruses type 6 and type 16 in fission yeast Schizosaccharomyces pombe. Virology 206, [14] Volpers, C., Schumacher, P., Streek, R.E. and Sapp, M. (1994) Assembly of the major and the minor capsid protein of human papillomavirus type 33 into virus-like particles and tubular structures in insect cells. Virology 200,

6 116 A. TouzP et al. IFEMS Microbiology Letters 141 (1996) Ill-116 [15] Le Cann, P., Coursaget, P., lochmann, S. and To&, A. (1994) Self-assembly of human papillomavirus type 16 capsids by expression of the Ll protein in insect cells. FEMS Microbiol. Lett. 117, [16] Schiller, J.T. and Roden, R.B.S. (1995) Papillomavirus-like particles. Papillomavirus report 6, [I 71 Delius, H. and Hofmann, B. (1994) Primer-directed sequencing of human papillomavirus types. Cur. Top. Microbial. Immunol. 186,