AU699547B2 - Papillomavirus polyprotein constructs - Google Patents

Description

WO 97/05164 PCT/AU96/00473 -1- "PAPILLOMAVIRUS POLYPROTEIN CONSTRUCTS" FIELD OF THE INVENTION This invention relates to polyprotein constructs and in particular polyprotein constructs comprising a plurality of papillomavirus (PV) amino acid sequences which may be used in compositions for eliciting an immune response against PV, and particularly human papillomavirus (HPV), in a host animal.

BACKGROUND OF THE INVENTION Papillomaviruses induce benign hyperproliferative lesions in humans and in many animal species, some of which undergo malignant conversion. The biology of papillomavirus infection is summarised in a review by J.P. Sundberg, entitled "Papillomavirus Infections in Animals" In "Papillomaviruses and Human Disease" edited by K. Syrjanen, L. Gissmann and L.G. Koss, Springer Verlag (1987).

Papillomaviruses are a family of small DNA viruses encoding up to eight early (El, E2, E3, E4, E5, E6, E7 and E8) and two late genes (L1 and L2). These viruses have been classified in several distinct groups such as HPV which are differentiated into types 1 to depending upon DNA sequence homology. A clinicopathological grouping of HPV and the malignant potential of the lesions with which they are most frequently associated are summarised in "Papillomaviruses and Human Cancer" by H. Pfister, CRC Press, Inc.

(1990). For example, HPV type 1 (HPV-1) is present in plantar warts, HPV-6 or HPV-11 are associated with condylomata acuminata (anogenital warts), and HPV-16 or HPV-18 are common in pre-malignant and malignant lesions of the cervical squamous epithelium.

The immunological approach to the prevention of HPV disease requires a thorough analysis of the viral proteins against which humoral and cellular immune responses are mounted during and after infection. However, despite recent limited T WO 97/05164 PCT/AU96/00473 -2success (Kreider et al., 1986, j. Virol., 59, 369; Sterling et al., 1990, Virol., 64, 6305; Meyers et al., 1992, Science, 257, 971; Dollard et al., 1992, Genes and Development, 6, 1131), papillomaviruses are notoriously refractory to growth in cultured cells (Teichaman and LaPorta, 1987 In "The Papovaviridae", Vol 2 edited by N.P. Salzman and P.M. Howley, p.109). As a consequence, the lack of viral reagents has delayed the analysis of the immune response to PV infection.

The recent advent of recombinant expression systems in vitro has allowed the production of viral proteins encoded by both early and late genes in relatively large amounts and in a purified form (Tindle et al., 1990, J. Gen. Virol., 71, 1347; Jarrett et al., 1991, Virology, 184, 33; Ghim et al., 1992, Virology, 190, 548; Stacey et al., 1991, Gen. Virol., 73, 2337). These systems have, for the first time, allowed the analysis of the host immune response to these viral proteins.

Interest in immune responses to the non-structural early open reading frame (ORF) proteins of HPV has centred on HPV-16 E7 because of an apparent association between serum antibodies to this protein and cervical cancer (for a review, see "Immune Response to Human Papillomaviruses and the Prospects of Human Papillomavirus-Specific Immunisation" by Tindle and Frazer In "Human Pathogenic Papillomaviruses" edited by H. zur Hausen, Current Topics in Microbiology Immunology, 186, Springer-Verlag, Berlin, 1994).

The immune responses to other HPV early ORF proteins have also been investigated including HPV-16 E6 (Stacey et al., 1992, J. Gen. Virol., 73, 2337; Bleul et al., 1991, J. Clin. Microbiol., 29, 1579; Dillner, 1990, Int. J. Cancer, 46, 703; and MOller et al., 1992, Virology, 187, 508), HPV-16 E2 (Dillner et al., 1989 Proc.Natl. Acad.

Sci.USA, 86, 3838; Dillner, 1990, supra; Lehtinen et al., 1992, J. Med. Virol., 37, 180; Mann et al., 1990, Cancer Res., 50, 7815; and Jenison et al., 1990, J. Infect. Dis., 162, i 60) and HPV-16 E4 (Kichel et al., 1991, Int. i. Cancer, 48, 682; Jochmus-Kudielka et 30 al., 1989, JNCI, 81, 1698; and Barber et al., 1992, Cancer Immunol. Immunother., rma~ I- WO 97/05164 PCT/AU96/00473 -3- 33). However, comparison of these studies reveals a lack of correlation between the results of the various assays which have been used in assessing HPV early ORF protein reactivity in serum (Tindle and Frazer, 1994, supra).

In addition, antibodies to other HPV early ORF proteins have not yet been sought with sufficient rigour in large enough numbers of patients to determine their utility as disease markers or as indicators of HPV protein immunogenicity following HPV infection.

A problem associated with immunising animals with preparations of individual PV proteins is that most of these proteins are comparatively small and might therefore not comprise many reactive epitopes. In addition, immunodominance of particular B or T cell epitopes within a single PV protein would vary presumably between animals of different major histocompatibility (MHC) backgrounds. To this end, the efficacy of such immunogens, in respect of eliciting an immune response against PV, might be expected to differ between animals of diverse MHC background.

In addition, there is surprisingly little knowledge regarding which PV proteins are expressed by infected cells at various stages of differentiation, and hence it is not possible to predict which proteins will be responsible for defining appropriate immunological targets.

The present invention provides a polyprotein construct comprising a plurality of PV early ORF proteins in one fused or linked construct to improve the efficacy of immune stimulation against PV infection and to avoid the need to define specific immunological targets.

SUMMARY OF THE INVENTION In one aspect, the present invention provides as an isolated product, a polyprotein construct comprising at least two amino acid sequences fused directly or indirectly WO 97/05164 PCT/AU96/00473 -4together, each of said sequences being the sequence of an early open reading frame (ORF) protein of papillomavirus (PV) or an immunogenic variant or fragment thereof, and at least one of said sequences being other than the E6 or E7 protein sequence or an immunogenic variant or fragment thereof.

In yet another aspect, the present invention provides a composition for eliciting a humoral and/or cellular immune response against PV in a host animal, said composition comprising an immunologically effective amount of a construct as described above, together with a pharmaceutically acceptable carrier and/or diluent.

In yet another aspect, this invention provides a method for eliciting a humoral and/or cellular response against PV in a host animal, which method comprises administering to the host animal an immunologically effective amount of a polyprotein construct as described above. In a related aspect, the invention also extends to use of such a polyprotein construct in eliciting an immune response against PV in a host animal.

Preferably, the host animal is a human, however the host animal may also be a nonhuman mammal.

II The present invention also extends to a nucleic acid molecule which encodes a polypeptide construct as broadly described above. Such a nucleic acid molecule may be delivered to a host animal in a nucleic acid vaccine composition with a pharmaceutically acceptable carrier and/or diluent, for expression of the encoded polyprotein construct in vivo in a host animal. Alternatively, the nucleic acid molecule may be included in a recombinant DNA molecule comprising an expression control sequence operatively linked to the nucleic acid molecule.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers." WO 97/05164 PCT/AU96/00473 DETAILED DESCRIPTION OF THE INVENTION The term "polyprotein construct" as used herein is used to describe a protein construct made up of individual proteins that have been joined together in a sequence whereby they retain their original relevant biological activities.

The term "isolated" as used herein denotes that the polyprotein construct has undergone at least one purification or isolation step, and preferably is in a form suitable for administration to a host animal.

By use of the term "immunologically effective amount" herein in the context of treatment of PV infection, it is meant that the administration of that amount to an individual PV infected host, either in a single dose or as part of a series, that is effective for treatment of PV infection. By the use of the term "immunologically effective amount" herein in the context of prevention of PV infection, it is meant that the administration of that amount to an individual host, either in a single dose or as part of a series, that is effective to delay, inhibit, treat or prevent PV infection or disease. The effective amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the immunogen, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

Preferably, the amino acid sequences in the polyprotein construct substantially correspond to the sequences of wild-type early ORF proteins of PV, including allelic or other variants thereof. Suitable variants include variants having single or multiple amino acid substitutions or additions to the wild-type sequences, and may have at least 50-60%, more preferably at least 70-80%, and most preferably at least 90%, similarity to the wildtype amino acid sequences, provided the variant is capable of eliciting an immune 1 WO 97/05164 PCT/AU96/00473 -6response against PV in a host animal. The amino acid sequences may also be immunogenic fragments of the wild-type early ORF proteins, that is fragments of the proteins which are capable of eliciting an immune response in a host animal. Suitably, the immunogenic fragment will comprise at least five, and more preferably at least ten, contiguous amino acid residues of the particular protein. Such immunogenic fragments may also be recognised by PV-specific antibodies, particularly antibodies which have a protective or therapeutic effect in relation to PV infection. Preferably, the immunogenic fragment is a non-full length fragment of a wild-type amino acid sequence, which may for example comprise a deletion mutant of an early ORF protein corresponding to at least 50%, more preferably 60-70%, and even 80-90% of the full length wild-type amino acid sequence.

The amino acid sequences in the polyprotein construct of the present invention may be selected from the group consisting of the El, E2, E3, E4, E5 (E5a, E5b), E6, E7 and E8 proteins of PV, and may be included in the construct in any desired order. By way of example, the construct may be selected from the group consisting of: E6/E4 E6/E5a/E4 E6/E7/E4 E6/E7/E5a/E4 E6/E7/E1/E4 E6/E7/E5a/E1/E4 E6/E7/E5a/El/E2/E4 E6/E7/E5a/E5b/E1/E2/E4 E2/E1/E5b E2/E5a/E5b E2/E1/E5a/E5b E2/E4/E5alE5b/E6/E7/El E2/E3/E4/E5/E8/E6/E7/E1.

P:\OPERUMS\65102-96,7- 4/9/98 -7- As described above, at least one of the early ORF proteins is other than the E6 or E7 proteins. Preferably one of the early ORF proteins in the construct is the E4 protein.

The polyprotein constructs of this invention preferably comprise at least three, and more preferably three, four or five early ORF protein sequences. In addition, two or more different polyprotein constructs based on different combinations of early ORF proteins and/or different PV genotypes may be included in a single composition for prophylactic or therapeutic use.

Cc 10 In the polyprotein constructs of this invention, the amino acid sequences may be fused or linked directly together. Alternatively, they may be linked with a linker sequence of from 1 to 50, preferably 1 to 20, and more preferably 1 to 5, amino acid residues between the separate amino acid sequences. By way of example, such a linker sequence may be an amino acid sequence encoded by the nucleotide sequence comprising a 15 restriction endonuclease site. Linker sequences as described above may also be provided before and/or after the amino acid sequences in the polyprotein constructs.

The polyprotein constructs of this invention may also comprise a tag protein or peptide moiety fused or otherwise coupled thereto to assist in purification of the 20 polyprotein construct. Suitable tag moieties include, for example, glutathione-Stransferase (GST) and FLAG (International Biotechnologies), with the (His), tag moiety being preferred. The constructs may further comprise a component to enhance the immunogenicity of the polyprotein. The component may be an adjuvant such as diphtheria or cholera toxin or E. coli heat labile toxin or a non-toxic derivative thereof such as the holotoxoid or B subunit of cholera toxin or LT. In addition, the polyprotein construct of the invention may comprise a lipid binding region to facilitate incorporation into ISCOMs. A preferred lipid binding region is an influenza haemagglutinin tail.

WO 97/05164 PCT/AU96/00473 -8- The present invention also provides a nucleic acid molecule comprising a sequence of nucleotides which encodes a polyprotein construct as broadly described above.

The nucleic acid molecule may be RNA or DNA, single stranded or double stranded, in linear or covalently closed circular form. It will be appreciated that the sequence of nucleotides of this aspect of the invention may be obtained from natural, synthetic or semi-synthetic sources; furthermore, this nucleotide sequence may be a naturally-occurring sequence, or it may be related by mutation, including single or multiple base substitutions, deletions, insertions and inversions, to such a naturallyoccurring sequence, provided always that the nucleic acid molecule comprising such a sequence is capable of being expressed as a polyprotein construct as described herein.

The nucleotide sequence may have expression control sequences positioned adjacent to it, such control sequences being derived from either a homologous or a heterologous source.

Since nucleic acid molecules may be delivered directly as "naked DNA" to a host animal, (see, for example, Wolfe et al., 1990, Science 247:1465 and Fynan et al., 1993, Proc.Natl. Acad. Sci. USA, 90:11478), the present invention also includes a nucleic acid vaccine composition comprising a nucleic acid molecule as described above, together with a pharmaceutically acceptable carrier and/or diluent.

Immunisation with an isolated nucleic acid molecule allows in vivo synthesis of the encoded polyprotein construct by the host animal in a manner similar to the manner in which PV proteins are expressed during infection by PV. In this aspect, the present invention also extends to a method for eliciting an immune response against PV in a host animal, which method comprises administering to the host animal an immunologically effective amount of a nucleic acid molecule as described above. The invention also P:\OPERUMS\65102-96,9 -4/9/98 -9extends to use of such a nucleic acid molecule in eliciting an immune response against PV in a host animal.

This invention also provides a recombinant DNA molecule comprising an expression control sequence having promoter and initiator sequences, the nucleotide sequence encoding the polyprotein construct being located 3' to the promoter and initiator sequences and a terminator sequence located 3' to this sequence of nucleotides.

In yet another aspect' the invention provides a recombinant DNA cloning vehicle such as a plasmid capable of expressing the polyprotein construct, as well as a host cell 10 containing a recombinant DNA cloning vehicle and/or a recombinant DNA molecule as described above.

Suitable expression control sequences and host cell/cloning vehicle combinations are well known in the art, and are described by way of example, in Sambrook et al. (1989) 15 Molecular Cloning A Laboratory Manual, 2nd ed. Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press. Thus, the nucleotide sequence may be ligated into any o suitable expression vector, which may be either a prokaryotic or eukaryotic expression vector. Preferably, the vector is a prokaryotic expression vector such as pTrcHisA or ir.. pGEX-STOP (a pGEX expression vector (Amrad/Pharmacia Biotech) which has been 20 manipulated so as to result in truncation of the GST moiety). Whilst the host cell is preferably a prokaryotic cell, more preferably a bacterium such as E. coli, it will be understood that the host cell may alternatively be a yeast or other eukaryotic cell, or insect cells infected with baculovirus or the like.

Once recombinant DNA cloning vehicles and/or host cells expressing a polyprotein construct of this invention have been identified, the expressed polypeptides synthesised by the host cells, for example, as a fusion protein, can be isolated substantially free of contaminating host cell components by techniques well known to those skilled in the art.

WO 97/05164 PCT/AU96/00473 The polyprotein construct-encoding DNA sequence is formed by linking or "fusing" sequences encoding each of the individual protein moieties. The first sequence in the polyprotein DNA construction has a promoter element and a ribosome binding site. These elements assure that transcription of the polyprotein DNA into mRNA begins at a defined site and that the signal, the ribosome binding site, needed for translation of mRNA into protein is present. Synthesis of the polyprotein is made continuous from one protein component to the next by removing or altering any initiation or binding signals and stop codons from the subsequent protein-encoding sequences. The stop codon, normally a signal for the ribosome to stop translation and to end the polypeptide, is not altered or removed from the last DNA sequence. The individual protein encoding sequences are jointed such that a proper phasing is made of the mRNA reading frames for translation of the sequence into the desired amino acids. Once a DNA sequence encoding a polyprotein construct or a "polyprotein gene" is made, it is necessary to demonstrate that the construction leads to production of a stable polyprotein construct.

If the resulting protein is not stable, for example because the junctions between the proteins are vulnerable to proteolytic digestion, then the junction regions are modified.

This can be done by inserting different amino acids at or near the junction or by building spacers of amino acids between the individual proteins. Linkers or spacers can also be I introduced to modify the overall activity of the polyprotein. By adjusting the space between and orientation of the individual proteins it is possible to modify the total activity of the polyprotein construct. Further details of the preparation of polyprotein constructs of the present invention by recombinant DNA techniques are disclosed, by way of example, in US Patent No. 4774180, the disclosure of which is incorporated herein by reference.

Preferably, the polymerase chain reaction (PCR) is used to amplify the nucleotide sequences encoding each of the individual PV early ORF proteins. The nucleotide sequences which are amplified may be full length or non full-length fragments thereof.

.estriction endonuclease sites may be incorporated in the oligonucleotide primers used for PCR to furnish directional ligation of the amplification products in the same WO 97/05164 PCT/AU96/00473 -11translational frame and to enable directional cloning into a suitable expression vector.

The primers may encode an artificial initiator codon or a termination codon.

The first nucleotide sequence has an initiator codon. This initiator codon may either be the normal wild-type initiator codon of the first sequence or may be inserted artificially at another chosen position of this sequence. Synthesis of the polyprotein construct is made continuous from one protein component to the next by removing or altering any initiation or binding signals and termination codons. The termination codon must be present in the last nucleotide sequence. This is effected normally by not altering or removing the termination codon of the last nucleotide sequence. However, this termination codon may be inserted artificially, by methods known to persons skilled in the art, by first removing the normal, wild-type termination codon of the last nucleotide sequence and inserting another, in the correct reading frame, at another position of this sequence.

The polyprotein construct-encoding DNA sequence may incorporate restriction sites at the flanking ends to facilitate insertion of the DNA sequence into a suitable expression vector.

The PV can be a human or an animal PV, and is preferably HPV. The HPV may be of any genotype, and may for example be selected from the group consisting of HPV- 6, HPV-11, HPV-16, HPV-18, HPV-33, HPV-35, HPV-31 and HPV-45. Preferably, the HPV is HPV-6 or HPV-11.

The present invention is particularly, but not exclusively, directed to polyprotein constructs comprising early ORF proteins of the HPV-6 and HPV-11 genotypes which are causative agents of condylomata acuminata, however it will be appreciated that the invention extends to variants of the corresponding proteins in other HPV genotypes, particularly the HPV-16 and HPV-18 genotypes, and other genotypes which have oncogenic potential of a type similar to HPV-16 and HPV-18.

WO 97/05164 PCT/AU96/00473 -12- The polyprotein constructs of the present invention may comprise early ORF proteins of a single HPV genotype, or alternatively they may comprise early ORF proteins from more than one HPV genotype. In addition, a combination of more than one polyprotein construct may be used in cases where not all early ORF proteins are represented in the one polyprotein construct, or where immune responses to more than one HPV genotype are desired.

The polyprotein constructs of the present invention are provided as isolated proteins, that is they are substantially free of other PV proteins, and find particular utility for the treatment of genital warts, cervical cancer or other conditions caused by HPV in man. The polyprotein constructs can be included in pharmaceutical compositions for the treatment or prevention of diseases involving HPV as well as the other conditions discussed above.

The polyprotein constructs of the invention may be used to raise antibodies and/or induce cellular immune responses, either in subjects for which protection against infection by PV is desired, i.e. as prophylactic vaccines, or to heighten the immune response to an PV infection already present, i.e. as therapeutic vaccines. They also can be injected into production species to obtain antisera. In lieu of the polyclonal antisera obtained in the production species, monoclonal antibodies may be produced using the standard methods or by more recent modifications thereof by immortalising spleen or other antibody-producing cells for injection into animals to obtain antibody-producing clones. The polyclonal or monoclonal antibodies obtained, corrected if necessary for species variations, can also be used as therapeutic agents.

Direct administration of the polyprotein constructs to a host animal such as a human can confer either protective immunity against PV or, if the subject is already infected, a boost to the subject's own immune response to more effectively combat the progress of the PV induced disease.

WO 97/05164 PCT/AU96/00473 -13- The magnitude of the prophylactic or therapeutic dose of a polyprotein constructs of this invention will, of course, vary with the group of patients (age, sex, etc.), the nature or the severity of the condition to be treated and with the particular polyprotein construct and its route of administration. In general, the weekly dose range for use lies within the range of from about 0.1 to about 5 pg per kg body weight of a mammal.

Any suitable route of administration may be employed for providing a mammal, especially a human, with an effective dosage of a polyprotein construct of this invention.

For example, oral, rectal, vaginal, topical, parenteral, ocular, nasal, sublingual, buccal, intravenous and the like may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, suppositories, aerosols and the like. Said dosage forms also include injected or implanted slow releasing devices specifically designed for this purpose or other forms of implants modified to additionally act in this fashion.

If the polyprotein constructs are to be administered as vaccines, they are formulated according to conventional methods for such administration to the subject to be protected. The polyprotein constructs may be delivered in accordance with this invention in ISCOMSTM (immune stimulating complexes), liposomes or encapsulated in compounds such as acrylates or poly(DL-lactide-co-glycoside) to form microspheres.

They may also be incorporated into oily emulsions and delivered orally.

SOther adjuvants, as well as conventional pharmaceutically acceptable carriers, excipients, buffers or diluents, may also be included in vaccine compositions of this invention. Generally, a vaccine composition in accordance with the present invention will comprise an immunologically effective amount of the polyprotein construct, and 'optionally an adjuvant, in conjunction with one or more conventional pharmaceutically acceptable carriers and/or diluents. An extensive though not exhaustive list of adjuvants can be found in Coulter and Cox, "Advances in Adjuvant Technology and Application", in Animal Parasite Control Utilizing Biotechnology, Chapter 4, Ed. Young, CRC WO 97/05164 PCT/AU96/00473 -14- Press, 1992. As used herein "pharmaceutically acceptable carriers and/or diluents" include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art and is described by way of example in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, U.S.A.

In practical use, a polyprotein construct of this invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. oral or parenteral (including intravenous and intra-arterial). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water glycols, oils, alcohols, flavouring agents, preservatives, colouring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets.

Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.

In addition to the common dosage forms set out above, the polyprotein constructs of this invention may also be administered by controlled release means and/or delivery devices, including by way of example, the controlled release preparations disclosed in International Patent Specification No. PCT/AU93/00677 (Publication No. WO 94/15636).

SPharmaceutical compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units such as capsules, cachets or

I

WO 97/05164 97/05164 PCT/AU96/00473 tablets each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

Further features of the present invention are more fully described in the following Example(s). It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the present invention, and should not be understood in any way as a restriction on the broad description of the invention as set out above.

EXAMPLES

Example 1 Amplification and cloning of early open reading frames (ORFs) of HPV6b A clone containing the entire genome of HPV6b in pBR322 (de Villiers, 1981, 1.

Virol, 40:932) was used as the template for separate PCR amplifications of E6, E7, El, E2 and E4 open reading frame (ORF) sequences.

Appropriate restriction enzyme recognition sequences were included in the oligonucleotides used for amplification (Table I; 1-7) to allow sequential assembly of these amplified early gene sequences into a 'polyprotein' sequence as depicted in Figure 1A.

'td i WO 97/05164 PCT/AU96/00473 -16- In this scheme, E6 was amplified with oligonucleotides containing a Smal site at the 5' end and Hindlll, Ncol and Xbal sites at the 3' end. As well, E4 was amplified with oligonucleotides containing Xbal, Sad, Kpnl and Spel sites 5' and a Bgill site 3'.

These amplified fragments were cloned as Smal/Xbal (E6) and XballBglll (E4) (Figure 1B) in the vector pSP70 (Promega Corporation) which had been modified by the removal of an EcoRV/EcoRI fragment to contain a portion of the pGEM3Zf (Promega Corporation) polylinker Hindll through EcoRI. As well, unwanted sites upstream of the Smal site were removed by cleaving with Smal/Xhol and insertion of a Smal/Sall/Xhol linker to create the vector pSP70 (MOD).

The E6/E4 cassette was able to be removed by cleavage with Smal/Bglll and this was then cloned for expression into the pGEX-STOP vector which produces a non-fusion protein with a C-terminal six-histidine sequence for purification purposes.

Using the introduced restriction enzyme recognition sequences, other early ORF sequences were incorporated into the E6/E4 cassette cloned into pSP70 (MOD) and then the newly created cassette cloned as a Smal/Bglll fragment into pGEX-STOP.

In this manner polyprotein constructs containing E6/E5a/E4, E6/E7/E4, E6/E7/E5a/E4, E6/E7/E1/E4 and E6/E7/E5a/E1/E4 were assembled. Complete DNA sequence data for the first three constructs is included and sequence data across the junctions of El is included for the latter two. DNA sequencing revealed the Spel site was inactivated by a single base change which occurred either during oligonucleotide synthesis, PCR or cloning.

As well the tetrafusion construct of E6/E7/E5a/E4 was cloned for expression into pET23b (Novagen) by firstly subcloning the tetramer as a Smal/Bgill fragment into the Smal/BamHI sites of the vector pRIT2T (AMRAD Pharmacia Biotech). The tetramer was WO 97/05164 PCT/AU96/00473 -17then removed by restriction with Smal and Sall and cloned into the Hincll/Xhol sites of the vector pET23b.

A further construct containing E2 and E5b, but which could also accommodate the addition of El and E5a, was created by amplifying E2 with oligonucleotides containing a Smal site at the 5' end and Xbal, Ncol, Kpnl and Sad, sites at the 3' end (Table 1; 8) and with E5b amplified using oligonucleotides with an Xbal site 5' and Xhol, Bgll sites 3' (Table 1; These amplified fragments were then cloned into pSP70 (MOD) as depicted in Figure 1C.

WO 97/05164 PCT/AU96/00473 18 Table 1

I'

ii Oligonucleotidles used for PCR Early Forward Reverse 1 E6 5 'GCGCCCCGGGATGGAAAGTGC 5

GCGCTCTAGACCATGGAAGCT

AAATGCCTC

3

TGGGTAACATGTCTTCCATGC

3 (SEQ ID No. 1) (SEQ ID. No.2) 2 E4 "GCGCTCTAGAGAGCTCGGTACC "GCGCAGATCTTAGGCGTAGCT ACTAGTG GAG CACCAAACATTG G GAACTGTTAC 3 GAAG 3 (SEQ ID No. 3) (SEQ ID No. 4) 3 E~a 5 GCGCCCATGGGAAGTGGTGCCT "GCGCTCTAGATTGCTGTGTGG

GTACAAATAGC

3 TAACAATATAG 3 (SEQ ID No. 5) (SEQ ID No. 6) 4 E7 5 GCGCAAGC11fCATGGAAGACAT "GCGCCCATGGGGTCTTCGGT GTTACCCTAAAG 3 GCGCAGATGG 3 (SEQ ID No. 7) (SEQ ID No. 8) El 5 GCGCGAGCTCGCGGACGATTCA "GCGCGGTACCTAAAGTTCTAA GGTACAGAAAATG 3 CAACTGTTCCTG 3 (SEQ ID No. 9) (SEQ ID No. 6 E2 5 GCGCG GTACCGAAGCAATAGCC 5 'GCGCACTAGTCAATAG GTG CA AAGCGTUAG 3

GTGACATAAATC

3 (SEQ ID No. 11) (SEQ ID No. 12) 7 ES b 5 GCGCTCTAGACTAACATGTCAAT 5 'GCG CGAG CTCA-FTCATATATA TTAATGATG 3

TATAATCACC

3 (SEQ ID No. 13) (SEQ ID No. 14) 8 E2 YGCGCCCCGG GATGGAAGCAATA 5 'GCGCTCTAGACC-ATGG GGTAC GCCAAGCG 3

CGAGCTCCAATAGGTGCAGTG

ACATAAATC

3 (SEQ ID No. 15) (SEQ ID No. 16) 9 E5b 5 GCGCTCTAGACTAACATGTCAAT 5

GCGCAGATCTCTCGAGAT[CA

TTAATGATG TATATrATATAATCAC 3 (SEQ ID No. 17) J(SEQ ID No. 18)J WO 97/05164 PCT/AU96/00473 -19- Example 2 Expression of different polyprotein constructs The following constructs in pGEX-STOP were expressed in E. coli strain BL21 and protein production was assayed by PAGE followed by Western blotting:

I

i) E6/E4 ii) E6/E5a/E4 iii) E6/E7/E4 iv) E6/E7/E5a/E4 Construct (iv) in pET23b, expressed in E. coli strains BL21(DE3)pLysS and AD494(DE3)pLysS (Novagen), was also assayed for protein production by Western blotting and also by Coomassie Blue staining for the latter strain.

Cultures of 200mL were grown in Terrific broth (Tartoff and Hobbs, Focus, 9: 12, 1987) in the presence of 100 pg/mL ampicillin (BL21) and 34pg/ml cloramphenicol [BL21(DE3)pLysS] and 15pg/mL kanamycin [AD494( DE3)pLysS]. At OD 6 oo 1 protein expression was induced by the addition of IPTG to 0.4mM. Following induction samples were taken at 1, 2, 3, 4 and 5 hours and in some cases after overnight culture.

Figure 2 shows a Western blot result for the E6/E4 construct. This was probed with a polyclonal rabbit anti-E4 antibody (MWE4 raised to the peptide LGNEHEESNSPLATPCVWPT conjugated to ovalbumin). An immunoreactive band of kDa was present in the 4 hour-induced sample (lanes 2 4, arrow) which was not present in the uninduced sample (lane 3).

The same 30kDa band can also be seen in the induced sample in Figure 3, lane 3, arrow (lane 2-uninduced) while the E6/E5a/E4 trimer construct of 40kDa was poorly represented after a 4 hour induction period (lane 5, arrow; uninduced sample-lane 4) using the same anti-E4 antibody.

WO 97/05164 PCT/AU96/00473 In contrast however, a trimer construct of E6/E7/E4 41 kDa) could be easily detected after 5 hours induction using an anti-hexahistidine monoclonal antibody (Dianova) [Figure 4, lane 4, arrow; uninduced sample lane 3].

The same trimer construct was again easily visualised after 5 hours induction using the anti-E4 antibody MWE4 (Figure 5, lane TRI, arrow; control sample lane C) and the tetramer consisting of E6/E7/E5a/E4 (-51 kDa) could also be detected (lane TET, arrow). Although this band is weak, it must be noted that a considerable amount of high molecular weight material is also immunoreactive, indicating the tetramer is reasonably well expressed but possibly prone to aggregation.

Figure 6 indicates that an anti-E6 antibody (prepared as described below) was able to detect E6/E7/E4 after 5 hours induction (lane TRI, arrow) but not E6/E7/E5a/E4 (lane TET; lane C uninduced). However, an anti-E7 antibody (prepared as described below) was able to detect after 5 hours induction both the trimer (Figure 7, lane TRI, arrow; lane C uninduced) and the tetramer (lane TET, arrow; lane C uninduced), with the latter again showing indications of aggregation. A monoclonal antibody raised to an E4 peptide also recognised the trimer.

The phenomenon of aggregation was clearly apparent when the E6/E7/E5a/E4 tetramer was expressed in the pET23b plasmid in BL21 (DE3)pLysS (Figure 8 a Western blot probed with MWE4). Lanes 2-5 are 1 hour, 2 hour, 3 hour and overnight uninduced samples and lanes 6-9 represent 1 hour, 2 hour, 3 hour and overnight induced samples.

After 1 hour induction a band of E6/E7/E5a/E4 can clearly be seen (arrow), but with increased times of induction this seems to decrease and aggregated forms are increased.

In contrast, when strain AD494(DE3)pLysS was used to express the tetramer, a substantial signal was obtained at the -50kDa position on a Western blot of the insoluble fraction (Figure 9, arrow) following 2 hours induction, which still persisted at 3 hours. This immunoreactive band was not present in control samples and no protein was detected in the samples from the soluble fractions.

U~i~PIIP~_ WO 97/05164 PCT/AU96/00473 -21- Figure 10 shows the Coomassie stained profile of an identical gel, indicating that the immunoreactive bands present after 2 and 3 hours induction (Figure 9) can clearly be visualised as stained bands (arrow) which are not present in the control samples.

Example 3 DNA sequencing of polyprotein constructs Polyprotein constructs were sequenced in both directions by the dideoxy method using primers that generated overlapping sequence information. The TSequencingTM Kit (Pharmacia was used to generate "S-labelled chain-terminated fragments which were analysed on a Sequi-GenTM (Biorad) electrophoretic gel apparatus. The DNA and corresponding amino acid sequences for E6/E5a/E4 (CSL690.SEQ), E6/E7/E4 (CSL760.SEQ) and E6/E7/E5a/E4 (CSL673.SEQ) are shown below. (SEQ ID Nos: 19 and 21 and 22, and 23 and 24, respectively).

For constructs E6/E7/E1/E4 (CSL 791) and E6/E7/E5a/E1/E4 (CSL 762), which were created from E6/E7/E4 and E6/E7/E5a/E4, respectively, DNA sequence analysis across the junctions of El with its neighbours is shown below (SEQ ID Nos. 25 and 26, 27 and 28, and 29 and 30, respectively).

WO 97/05164 WO 9705164PCT/AU96/00473 File CSL69O.SEQ Range: 1 Codon Table Univers 22 11'' Mode Normal E6/E5a/E4 SEQ ID Nos, 1 9 (DNA) and 20 (amino acid) ATG GAA AGT GCA AAT GCC TCC ACG TCT G-CA ACG ACC ATA GAC CAG TTG TGC AAG Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gin Leu Cys Lys 63 72 81 90 99 108 ACG TTT AAT CTA TCT ATG CAT ACG TTG CAA ATT AAT TGT GTG TTT TGC AAG PAT Thr Phe Asn Leu Ser Met His Thr Leu Gin Ile Asn Cys Val Phe Cys Lys Asn 117 126 135 144 153 162 GCA CTG ACC ACA GCA GAG ATT TAT TCA TAT GCA TAT AAA CAC CTA AAG GTC CTG Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr Lys His Leu Lys Val L-eu 171 180 189 198 207 216 TTT CGA GGC GGC TAT CCA TAT GCA GCC TGC GCG TGC TGC CTA GAA TTT CAT GGA Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys Ala Cys Cys Leu Glu Phe His Gly 225 234 243 252 261 270 AAA ATA PAC CPA TAT AGA CAC TTT GAT TAT GCT GGA TAT GCA ACA ACA GTT GA Lys Ile Asn Gin Tyr Arg His Phe Asp Tyr Ala Giy Tyr Ala Thr Thr Val Glu 279 288 297 306 315 324 GAA GPA ACT AAA CPA GAC ATC TTA GAC GTG CTA ATT CGG TGC TAC CTG TGT CAC Glu Giu Thr Lys Gin Asp Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His 333 342 351 360 369 378 PAA CCG CTG TGT GAA GTA GPA AAG GTA AAA CAT ATA CTA ACC AAG GCG CGG TTC Lys Pro Leu Cys Giu Val Giu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe ATA AAG Ile Lys CTA AAT TGT ACG TGG AAG GGT CrC TGC CTA CAC TGC TGG ACA ACA TGC.

Leu Asn Cys Thr Trp Lys Giy Arg Cys Leu His Cys Trp Thr Thr Cys ATG GPA GAC Met Giu Asp 495 GCA GGA ACA Ala Giy Thr ATG TTA CCC AAG CTT CCA TGG GAA GTG GTG CCT GTA CAA ATA GCT Met Leu Pro Lys Leu Pro Trp Glu Val Val Pro Val Gin le Ala

ACC

Thr AGC ACA. TTC ATA CTG CCT GTT ATA ATT GCA TTT GTT GTA TGT Ser Thr Phe Ile Leu Pro Vai Ile Ile Ala Phe Vai Vai Cys 567 576 585 WO 97/05164 PTA9/07 PCT/AU96/00473 23 TTT GTT AGC ATC ATA CTT ATT GTA TGG ATA TCT GAG TTT ATT GTG TAC ACA TCT Phe Val Ser Ile Ile Leu Ile Val Trp Ile Ser Giu Phe Ile Val Tyr Thr Ser GTG CTA GTA Val Leu Vai CTA ACA CTG CTT TTA TAT TTA CTA TTG TGG CTG CTA TTA ACA ACC Leu Thr Leu Leu Leu Tyr Leu Leu Leu Trp Leu Leu Leu Thr Thr CCC TTG CAA TTT TTC CTA CTA ACT CTA CTT GTG TGT TAG TGT CCC GCA TTG TAT Pro Leu Gin Phe Phe Leu Leu Thr Leu Leu Vai Cys Tyr Cys Pro Ala Leu Tyr 711 720 729 738 747 756 ATA CAC TAG TAT ATT GTT ACC ACA GAG GAA TGT AGA GAG GTG GGT ACG ACT PAT Ile His Tyr Tyr Ile Vai Thr Thr Gin Gin Ser Arg Giu Leu Giy Thr Thr Asn 765 774 783 792 801 810 GGA GCA CCA PAC ATT GGG AAG TAT GTT ATG GCA GCA GAG TTA TAT GTT CTC CTG Giy Ala Pro Asn Ile Giy Lys Tyr Val Met Ala Ala Gin Leu Tyr Val Leu Leu 819 828 837 846 855 864 CAT CTG TAT GTA GCA CTA GAG PAG, PAG TAT CCA TTC CTG PAT GTA CTA CAT ACA His Leu Tyr Leu Ala Leu His Lys Lys Tyr Pro Phe L e Asn Leu Leu His Thr 873 882 891 900 909 918 CCC CCG GAG AGA CCT CCA CCC TTG TGT CCT CPA GCA CCA AGG PAG ACG GAG TGC Pro Pro His Arg Pro Pro Pro Leu Cys Pro Gin Ala Pro Arg Lys Thr Gin Cys 927 936 945 954 963 972 AA.A CGG CGC CTA GGA AAC GAG GAG GAG GAG TCC AAC AGT CCC CTT GCA ACG GCT Lys Arg Arg Leu Gly Asn Glu His Giu Giu Ser Asn Ser Pro Leu Ala Thr Pro 1008 1017 1026 TGT GTG TGG CCC. ACA TTG Cys Vai Trp Pro Thr Leu GAG CCG TGG Asp Pro Trp ACA GTG GAA Thr Val Giu ACC ACA ACG, Thr Thr Thr TCA TGA CTA Ser Set Let 1080 1035 1044 1053 1062 1071 ACJA ATC ACG Thr Ile Thr ACC AGC ACC Thr Ser Thr AAA GAG GGA Lys Asp Gly ACA ACA GTA Thr Thr Val ACA GTTI GAG CTA Thr Val Gin Leu 'Cr' CTAp Arg Leu 1089 1098 1107 AGA TCT CAT Arg Ser His CAG CAT CAG His His His CAT GAG TAA 3' His His

I

WO 97/05164 PCT/AU96/00473 -24- File CSL760.SEQ Range: 1- Codon Table Univers 1128 Mode Normal E6/E7/E4 SEQ ID Nos. 21 (DNA) and 22 (amino acid) ATG GAA AGT GCA AAT GCC Met Glu Ser Ala Asn Ala TCC ACG TCT GCA ACG ACC Ser Thr Ser Ala Thr Thr ATA GAC Ile Asp CAG TTG TGC AAG Gin Leu Cys Lys ACG TTT AAT CTA TCT ATG CAT ACG TTG CAA ATT AAT TGT GTG TTT TGC AAG AAT Thr Phe Asn Leu Ser Met His Thr Leu Gin Ile Asn Cys Val Phe Cys Lys Asn 117 126 135 144 153 162 GCA CTG ACC ACA GCA GAG ATT TAT TCA TAT GCA TAT AAA CAC CTA AAG GTC CTG Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr Lys His Leu Lys Val Leu 171 180 189 198 207 216 TTT CGA GGC GGC TAT CCA TAT GCA GCC TGC GCG TGC TGC CTA GAA TTT CAT GGA Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys Ala Cys Cys Leu Glu Phe His Gly 225 234 243 252 261 270 AAA ATA AAC CAA TAT AGA CAC TTT GAT TAT GCT GGA TAT GCA ACA ACA GTT GAA Lys Ile Asn Gin Tyr Arg His Phe Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu 279 288 297 306 315 324 GAA GAA ACT AAA CAA GAC ATC TTA GAC GTG CTA ATT CGG TGC TAC CTG TGT CAC Glu Glu Thr Lys Gin Asp Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His 333 342 351 360 369 378 AAA CCG CTG TGT GAA GTA GAA AAG GTA AAA CAT ATA CTA ACC AAG GCG CGG TTC Lys Pro Leu Cys Glu Val Glu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe 387 396 405 414 423 432 ATA AAG CTA AAT TGT ACG TGG AAG GGT CGC TGC CTA CAC TGC TGG ACA ACA TGC Ile Lys Leu Asn Cys Thr Trp Lys Gly Arg Cys Leu His Cys Trp Thr Thr Cys 459 ATG GAA GAC ATG TTA CCC AAG CTT CAT GGA AGA CAT GTT ACC CTA AAG GAT ATT Met Glu Asp Met Leu Pro Lys Leu His Gly Arg His Val Thr Leu Lys Asp Ile 495 504 513 522 531 540 GTA TTA GAC CTG CAA CCT CCA GAC CCT GTA GGG TTA CAT TGC TAT GAG CAA TTA Val Leu Asp Leu Gin Pro Pro Asp Pro Val Gly Leu His Cys Tyr Glu Gin Leu 558 567 576 585 594 WO 97/05164 WO 9705164PCT/AU96/00473 GTA GAC AGC TCA GAA GAT GAG Vai Asp Ser Ser Giu Asp Giu 25 GTG GAC GAA GTG GAC GGA CAA Val Asp Giu Val Asp Giy Gin GAT TCA CAA CCT Asp Ser Gin Pro TTA AAA CAA CAT TTC CAA ATA GTG ACC TGT TGC TGT GGA TGT GAC AGC AAC GTT Leu Lys Gin His Phe Gin Ile Val Thr Cys Cys Cys Giy Cys Asp Ser Asn Vai CGA CTG GTT GTG CAG TGT ACA Arg Leu Vai Val Gin Cys Thr GAA ACA GAC ATC AGA GAA GTG CAA CAG CTT CTG Giu Thr Asp Ile Arg Giu Vai Gin Gin Leu Leu TTG GGA ACA CTA AAC ATA GTG TGT CCC ATC TGC GCA CCG AAG ACC CCA TGG TCT Leu Giy Thr Leu Asn Ile Vai Cys Pro Ile Cys Ala Pro Lys Thr Pro Trp Ser 765 774 783 792 801 810 AGA GAG CTC GGT ACC ACT AAT GGA GCA CCA AAC ATT GGG AAG TAT GTT ATG GCA Arg Giu Leu Giy Thr Thr Asn Giy Ala Pro Asn Ile Giy Lys Tyr Vai Met Ala 819 828 837 846 855 864 GCA CAG TTA TAT GTT CTC CTG CAT CTG TAT CTA GCA CTA CAC AAG, AAG TAT CCA Ala Gin Leu Tyr Vai Leu Leu His Leu Tyr Leu Ala Leu His Lys Lys Tyr Pro 873 882 891 900 909 918 TTC CTG AAT CTA CTA CAT ACA CCC CCG CAC AGA CCT CCA CCC TTG TGT CCT CAA Phe Leu Asn Leu Leu His Thr Pro Pro His Arg Pro Pro Pro Leu Cys Pro Gin GCA CCA AGG AAG ACG CAG TGC Ala Pro Arg Lys Thr Gin Cys AAA CGC CGC CTA GGA AAC Lys Arg Arg Leu Giy Asn GAG CAC GAG GAG TCC Giu His Giu Giu Ser 1008 1017 1026 AAC AGT CCC Asn Ser Pro CTT GCA ACG CCT TGT GTG TGG CCC ACA Leu Ala Thr Pro Cys Val Trp Pro Thr TTG GAC CCG Leu Asp Pro TGG ACA GTG Trp Thr Vai 1035 1044 1053 1062 1071i 1080 GAA ACC ACA ACC Giu Thr Thr Thr TCA TCA Ser Ser CTA ACA ATC ACG Leu Thr Ile Thr ACC AGC ACC AAA GAC GGA ACA ACA Thr Ser Thr Lys Asp Giy Thr Thr 1089 1098 1107 1116 1125 GTA ACA GTT Val Thr Vai CAG CTA CGC Gin Leu Arg CTA AGA TCT CAT Leu Arg Set His CAC CAT CAC CAT CAC TAA 3' His His His His His WO 97/05164 WO 9705164PCT/AU96/00473 26 File CSL673.DNA Range: 1 Codon Table Univers 13 m-de Normal al F6/E7/E5aIE4 SEQ ID Nos. 23 (DNA) and 24 (amino acid) ATG GAA AGT GCA AAT GCC Met Giu Ser Ala Asn Ala TCC ACG TCT GCA Ser Thr Ser Ala ACG ACC ATA GAC CAG TTG TGC AAG Thr Thr Ile Asp Gin Leu Cys Lys ACG TTT AAT CTA TCT ATG CAT ACG TTG CAA ATT AAT TGT GTG TTT TGC AAG AAT Thr Phe Asn Leu Ser Met His Thr Leu Gin Ile Asn Cys Val Phe Cys Lys Asn 117 126 135 144 153 162 GCA CTG ACC ACA GCA GAG ATT TAT TCA TAT GCA TAT AAA CAC CTA AAG GTC CTG Ala Leu Thr Thr Ala Giu Ile Tyr Ser Tyr Ala Tyr Lys His Leu Lys Val Leu 171 180 189 198 207 216 TTT CGA GGC GGC TAT CCA TAT GCA GCC TGC GCG TGC TGC CTA GAA TTT CAT GGA Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys Ala Cys Cys Leu Glu Phe His Gly 234 AAA ATA AAC CAA TAT AGA CAC TTT GAT TAT GCT GGA TAT GCA ACA ACA GTT GAA Lys Ile Asn Gin Tyr Arg His Phe Asp Tyr Ala Giy Tyr Ala Thr Thr Val Giu 279 288 297 306 315 324 GAA GAA ACT AAA CAA GAC ATC TTA GAC GTG CTA ATT CGG TGC TAC CTG TGT CAC Giu Giu Thr Lys Gin Asp Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His 333 342 351 360 369 378 AAA CCG CTG TGT GAA GTA GAA AAG GTA AAA CAT ATA CTA ACC AAG GCG CGG TTC Lys Pro Leu Cys GiU Val Giu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe 396 ATA AAG CTA AAT TGT ACG TGG AAG GGT CGC TGC CTA CAC TGC TGG ACA ACA TGC Ile Lys Leu Asn Cys Thr Trp Lys Gly Arg Cys Leu His Cys Trp Thr Thr Cys 441 450 459 468 477 486 ATG GAA GAC ATG TTA CCC AAG, CTT CAT GGA AGA CAT GTT ACC CTA AAG GAT ATT Met Glu Asp Met Leu Pro Lys Leu His Gly Arg His Val Thr Leu Lys Asp Ile 495 GTA TTA GAC Val Leu Asp 504 CTG CAA CCT CCA GAC Leu Gin Pro Pro Asp 513 CCT GTA GGG TTA CAT TGC TAT GAG CAA TTA Pro Val Gly Leu His Cys Tyr Glu Gin Leu CAn R'7 r. C; Q 4 ~II II WO 97/05164 PCT/AU96/00473 -27 GTG GAC GAA GTG GAC GGA CAA GAT TCA CAA CCT Val Asp Glu Val Asp Gly Gin Asp Ser Gin Pro GTA GAC AGC TCA GAA GAT GAG Val Asp Ser Ser Glu Asp Glu TTA AAA CAA CAT TTC CAA ATA GTG ACC TGT TGC TGT GGA TGT GAC AGC AAC GTT Leu Lys Gin His Phe Gin Ile Val Thr Cys Cys Cys Gly Cys Asp Ser Asn Val 702 CGA CTG GTT GTG CAG TGT ACA GAA ACA GAC ATC AGA GAA GTG CAA CAG CTT CTG Arg Leu Val Val Gin Cys Thr Glu Thr Asp Ile Arg Glu Val Gin Gin Leu Leu 711 720 729 738 747 756 TTG GGA ACA CTA AAC ATA GTG TGT CCC ATC TGC GCA CCG AAG ACC CCA TGG GAA Leu Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro Lys Thr Pro Trp Glu 765 774 783 792 801 810 GTG GTG CCT GTA CAA ATA GCT GCA GGA ACA ACC AGC ACA TTC ATA CTG CCT GTT Val Val Pro Val Gin Ile Ala Ala Gly Thr Thr Ser Thr Phe Ile Leu Pro Val 819 828 837 846 855 864 ATA ATT GCA TTT GTT GTA TGT TTT GTT AGC ATC ATA CTT ATT GTA TGG ATA TCT Ile Ile Ala Phe Val Val Cys Phe Val Ser Ile Ile Leu Ile Val Trp Ile Ser GAG TTT ATT GTG TAC ACA Glu Phe Ile Val Tyr Thr TCT GTG CTA Ser Val Leu GTA CTA ACA CTG CTT TTA TAT TTA CTA Val Leu Thr Leu Leu Leu Tyr Leu Leu 954 963 972 CAA TTT TTC CTA CTA ACT CTA CTT GTG Gin Phe Phe Leu Leu Thr Leu Leu Val TTG TGG CTG CTA TTA ACA ACC CCC TTG Leu Trp Leu Leu Leu Thr Thr Pro Leu 981 990 999 TGT TAC TGT CCC GCA TTG TAT ATA CAC Cys Tyr Cys Pro Ala Leu Tyr Ile His 1008 1017 1026 TAC TAT ATT Tyr Tyr Ile GTT ACC ACA CAG Val Thr Thr Gin 1071 CAA TCT Gin Ser 1080 1035 1044 1053 1062 AGA GAG CTC Arg Glu Leu GGT ACC ACT Gly Thr Thr AAT GGA GGA Asn Gly Ala CCA AAC ATT Pro Asn Ile GGG AAG TAT Gly Lys Tyr GTT ATG GCA Val Met Ala 1134 1089 1098 1107 1116 1125 GCA CAG TTA Ala Gin Leu TAT GTT CTC Tyr Val Leu CTG CAT CTG Leu His Leu TAT CTA GCA Tyr Leu Ala CTA CAC AAG AAG TAT CCA Leu His Lys Lys Tyr Pro 11 4-q 1152 1161 1170 1179 1188 WO 97/05164 PTA9/07 PCT/AU96/00473 28 TTC CTG AAT Phe Leu Asn CTA CTA CAT Leu Leu His ACA CCC CCG Thr Pro Pro CAC AGA CCT CCA CCC TTG TGT CCT CAA His Arg Pro Pro Pro Leu Cys Pro Gin 1197 1206 1215 1224 1233 1242 GCA CCA AGG AAG ACG CAG Ala Pro Arg Lys Thr Gin TGC AAA. CGC Cys Lys Arg CGC CTA GGA AAC GAG CAC GAG GAG TCC Arg Leu Gly Asn Giu His Glu Giu Ser 1251 1260 1269 1278 1287 1296 AAC AGT CCC CTT GCA ACG CCT Asn Ser Pro Leu Ala Thr Pro TGT GTG TGG Cys Val Trp 1323 CCC ACA TTG GAC CCG TGG ACA GTG Pro Thr Leu Asp Pro Trp Thr Val 1305 1314 1332 1341 1350 GAA ACC ACA ACC TCA TCA CTA ACA ATC ACG ACC AGC ACC AAA GAC GGA ACA ACA Glu Thr Thr Thr Ser Ser Leu Thr Ile Thr Thr Ser Thr Lys Asp Gly Thr Thr 1359 1368 1377 1386 1395 GTA ACA GTT CAG CTA CGC CTA AGA TCT CAT CAC CAT CAC CAT CAC TAA 3' Val Thr Val Gin Leu Arg Leu Arg Ser His His His His His His Yunction of El and E4 OJRFz for CSL791 and CSL762 SEQ ID Nos. 25(DN'A)and 26(amino acid) Modified I4nl $pal itGAG GAA GAT GGA AGC MAT AGC CAA GCG TTT AGA TGC GTG CCA GGA ACA GTT GTT AGA ACT TTA GGT ACC ACT MAT GGA GCA CCA MAC ATT GGG MAG TAT GTT ATG GCA 3' Glu Glu Asp Gly Ser Asn Ser Gin Ala Phe Arg Cys Val Pro Gly Thr Val Val Arg Thr Leu Gly Thr Thr Asn Gly Ala Pro Asn Ile Gly Lys Tyr Val Met Ala Yunction of E5a and El for CSL762 SEQ ID Nos. 27(DNA)and 28 (amino acid) Thai Sadl 0' TGT CCC GCA TTG TAT ATA CAC TAC TAT AT? GTT ACC ACA CAG CMA TCT AGA GAG CTC GCG GAC GAT TCA GGT ACA GMA MT GAG GGG TCT GGG TGT ACA GGA 3' Cys Pro Ala Leu Tyr Ile His Tyr Tyr Ile Val Thr Thr Gin Gin Ser Arg Glu Leu Ala Asp Asp Ser Gly Thr Glu Asn Glu Gly Ser Gly Cys Thr Gly E~a runction of E7 and El for CSL791 SEQ ID Nos. 29(DNA) and 30(amino acid) mcol Ahal Saad TTG GGA ACA CTA MAC ATA GTG TGT CCC ATC TGC GCA CCG MAG ACC CCA TGG TCT AGA GAG CTC GCG GAC GAT TCA GGT ACA GMA MT GAG GGG TCT GGG TGT ACA GGA 3' Leu Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro Lys Thr Pro Trp Ser Arg Giu Leu Ala Asp Asp Ser Gly Thr Glu Asn GlU Gly Ser Giy Cys Thr Gly r I--~s zIQz.WQ_ lmt "Wt WO 97/05164 PCT/AU96/00473 Example 4 Preparation of antibodies to HPV6b early ORF protein products The following peptides corresponding to portions of the sequence of the relevant E proteins, were synthesised and conjugated to diphtheria toxoid: E6 dip. tox-C-QYRHFDYAQYATTVEEETKQDILD E7 MHGRHVTLKDIVLDLQPPD-C-dip. tox For the E6 peptide two rabbits (following pre-bleeding) were each inoculated with approximately 54pg peptide/104pg diphtheria toxoid in Freund's complete adjuvant followed at 3-weekly intervals by a similar dose of peptide conjugate in Freund's incomplete adjuvant. Bleeds were taken one week after the second dose and one week following the third dose. The same regime was used for the E7 peptide using peptide/103 pg diphtheria toxoid.

Serum derived from the bleeds were tested for specific antibody in a solid phase EIA against biotin-conjugated peptide which had been bound to plates coated with strepavidin.

Example 5 Purification of polyprotein E6/E7/E4 The trimer polyprotein E6/E7/E4 was expressed in E. coli BL21 cells by induction of cells at ODoo 1 using 0.4mM IPTG. The cells were harvested by centrifugation (4,000g, 20 minutes), resuspended in 30mM Tris pH8.0, disrupted by sonication (MSE, amplitude 18/m, 4 x 30 seconds) and inclusion bodies pelleted by centrifugation (12,000g, 30 minutes). The pellet containing the trimer was solubilized in 8M Urea, Tris pH8.0 for 16 hours in the presence of protease inhibitors (Boehringer Cat. No.

1697498) and then centrifuged at 12,000g for 30 minutes and the supernatant collected.

To this, Tris-(2-carboxyethyl)phosphine (TCEP) [Pierce] was added to 1.2mM final concentration. The supernatant was applied to Q-sepharose HP (Pharmacia) and the WO 97/05164 PCT/AU96/00473 -31column washed with one column volume of 8M Urea, 1.2mM TCEP, 30 mM Tris Fractions were then eluted using a gradient containing 0 to 1M NaCI in 10 column volumes of the washing buffer. The fractions obtained were examined by Western blot from 4 to 20% SDS-PAGE probed with the anti-E4 antibody MWE4.

Figure 11 shows a Western blot of material obtained from Q-sepharose. An immunoreactive band of 41kDa is evident in supernatant material from the urea solubilisation lane 3, and in fractions corresponding to 120 to 150 mM NaCI (lanes 8 and 9, arrow).

Supernatant from the urea solublisation was also applied to a column containing Chelating Sepharose Fast Flow (Pharmacia) to take advantage of the C-terminal six histidine sequence. Relatively poor binding of the trimer to the Nickel column was observed under the conditions described. The trimer was eluted from the column using a 0 to 500 mM imidazole gradient.

Example 6 In a further example of the present invention, a DNA sequence coding for a single polyprotein (Fig. 12) is formed by fusion of DNA fragments encoding HPV-6 early ORF proteins wherein the order of the ORFs is E2, E4, E5a, E5b, E6, E7 and El.

The DNA sequences encoding the early ORF proteins are amplified individually by PCR using HPV-6 genomic DNA using the primers set out in Table 2.

WO 97/05164 PCT/AU96/00473 -32- Table 2 Gene Oligonucleotides E2 5'-GTG TGT GAG CTC ATG GAA GCA ATA GCC AAG-3' (SEQ ID No. 31) and 5'-GTG TGT GTC GAC CAA TAG GTG CAG TGA CAT-3' (SEQ ID No. 32) E4 5'-GTG TGT GTC GAC ATG GGA GCA CCA AAC ATT-3' (SEQ ID No. 33) and TGT AGA TCT TAG GCG TAG CTG AAC TGT-3' (SEQ ID No. 34) 5'-GTG TGT AGA TCT ATG GAA GTG GTG CCT GTA-3' (SEQ ID No. 35) and 5'-GTG TGT CTT AAG TTG CTG TGT GGT AAC AAT-3' (SEQ ID No. 36) 5'-GTG TGT CTT AAG ATG ATG CTA ACA TGT CAA-3' (SEQ ID No. 37) and 5'-GTG TGT CCG CGG ATT CAT ATA TAT ATA ATC-3' (SEQ ID No. 38) E6 5'-GTG TGT CCG CGG ATG GAA AGT GCA AAT GCC-3' (SEQ ID No. 39) and 5'-GTG TGT GCT AGC GGG TAA CAT GTC TTC CTA-3' (SEQ ID No. E7 5'-GTG TGT GCT AGC ATG CAT GGA AGA CAT GTT-3' (SEQ ID No. 41) and 5'-GTG TGT CGA TCG GGT CTT CGG TGC GCA GAT-3' (SEQ ID No. 42) El 5'-GTG TGT CGA TCG ATG GCG GAC GAT TCA GGT-3' (SEQ ID No. 43) and 5'-GTG TGT GGT ACC TCA TAA AGT TCT AAC AAC-3' (SEQ ID No. 44) The primers are synthesised to incorporate artificial restriction enzyme sites at the and 3' termini of the amplification products. These restriction enzyme sites are used to facilitate the fusion of PCR products encoding the appropriate early ORF proteins in the desired order and in the correct translational frame. The restriction enzyme sites are also used to aid the cloning of the PCR products into the expression vector pTrcHisA.

When cloned into this vector, the polyprotein construct is expressed as an N-terminal s WO 97/05164 PCT/AU96/00473 -33 hexaHis fusion. The nucleotide sequence and deduced amino acid sequence of this fusion are shown below (SEQ ID Nos. 45 and 46, respectively).

WO 97/05164 PCT/AU96/00473 -34- INFORMATIION FOR HEXLAHIS-POLYPROTEIN FUSION SEQUENCE: SEQUENCE CHARACTERISTICS: LENGTH: 4770 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genornic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: HUMAN PAPILLOMAVIRUS TYPE 6 (ix) FEATURE: NAME/KEY: CDS LOCATION:1. .4761 OTHER INFORMATION: /codon-start= 1 /product= "HPV-6 Polyprotein" (ix) FE.ATURE: NAME/KEY: misc RNA LOCATION:1. .108 OTHER INFORMATION: /function= "Tag used for protein purification" /product= "hexaHis leader sequence from p~zcHisA" (ix) FEATURE: NAME/KEY: misc feature LOCATION:109. .114 OTHER INFORMATION: /label= SadI (ix) FEATURE: NAME/KEY: mRNA LOCATION:115. .1218 OTHER INFORMATION: /gene= "HPV-6 E2" (ix) FEATURE: NAME/KEY: maisc feature LOCATION:1219. .1224 OTHER INFORMATION: /label= Sall (ix) FEATURE: NAME/KEY: mRNA LOCATION:1225. .1551 OTHER INFORMATION: /gene= "HPV-6 E4" (ix) FEATURE: NAME/KEY: misc feature LOC?3TION:1552..1557 OTHER INFORMATION: /label= BglII (ix) FEATURE: NAME/KEY: znINA LOCATION:1558. .1830 OTHER INFORMATION: /gene= "HPV-6 EWa (ix) FEATURE: NAME/KEY: misc feature LOCATION:1831. .1836 OTHER INFORMATION: /label= BfrI (ix) FEATURE: NAME/KEY: mRNA LOC.ATION:1837. .2052 OTHER INFORMATION: /gene= "HPV-6 ~Crnwwrr WO 97/05164 35 (ix) FEATURE: NAME/KEY: misc feature LOCATION:2053.:2058 OTHER INFOR~MATION: /abe= SacI (ix) FEATURE: NAME/KEY: mRNA LOCATION:2059. .2508 OTHER INFORMATION: /gene= "HPV-6 E6" (ix) FEATURE: NAME/KEY: misc-feature LOCATION:2509. .2514 OTHER INFORMATION: /label= NheI (ix) FEATURE: NAME/KEY: mRNA.

LOC.ATION:2515. .2808 OTHER INFORMATION: /gene= "HPV-6 E7" (ix) FEATURE: NAME/KEY: misc-feature LOCATION':2B09. .2814 OTHER INFORMATION: /label= PvuI (ix) FEATURE: NAME/KEY: xnRNA LOCATION:2815. .4764 OTHER INFORMATION: /gene= HPV-6 El" (ix) FEATURE: NAME/KEY: misc-feature LOCATION:4765. .47'70 OTHER INFORMATION:/abei= KpnI GGG GGT TCT CAT CAT CAT CAT CAT CAT GGT ATG GCT AGC ATG ACT Giy Gly Scr His His His His His His Gly Met Ala Ser Met Thr 5 10 GGA CAG CAA MTG GGT CGG GAT CTG TAC GAC GAT GAC GAT AAG GAT Gly Gin Gin Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 25 TGG GGA TCC GAG CTC ATG GAA GCA MTA GCC AAG CGT TTA GAT GCG Trp Gly Sex Giu Leu Met Giu Ala Ile Ala Lys Axg Leu Asp Ala 40 CAG GAA CAG TTG TTA GAA CTT TAT GAA GAA AAC AGT ACT GAC CTA Gin Glu Gin Leu Leu Glu Leu Tyr Giu Glu Asn Ser Thr Asp Leu 55 AAA CAT GTA TTG CAT TGG AAA TGC ATG AGA CAT GAA AGT GTA TTA Lys His Val Leu His Trp Lys Cys Met Axg His Giu Ser Val Leu 70 75 TAT AAA GCA AAA CAA MTG GGC CTA AGC CAC MTA GGA. MG CAA GTA Tyr Lys Ala Lys Gin Met Gly Leu Ser His Ile Giy Met Gin Vai 90 9 CCA CCA TTA AAG GTG TCC GAA GCA. AAA GGA CAT AAT GCC MTT GAA Pro Pro Leu Lys Val Ser Giu Ala Lys Giy His Asn Ala Ile Giu 100 105 110 CAA AMG CAT TTA GAA TCA TTA TTA AGG ACT GAG TAT AGT ATG GAA Gin Met His Leu Giu Ser Leu Leu Arg Thr Giu Tyr Ser Met Giu 115 120 125 PCTIAU96/00473 48 96 144 192 240 288 336 384

ATG

Met 1

GGT

Gly

CGA

Axrg

TGC

Cys

CAC

His

TTA

Leu

GTG

Vai

ATG

Met CCG TGG ACA Pro Trp Thr 130 AAA CGC TGT Lys Arg Cys TTA CAA GAA ACA AGT TMT Leu Gin Giu Thr Ser Tyr 135 TTT AAA AAiA CGG GGC AAA Phe Lys Lys Arg Gly Lys GAA ATG TGG CAA ACA CCA CCT Gi~u Met Trp Gin Thr Pro Pro 140 ACT GTA GAA GTT AAA TTT GAT Thr Val Giu Val Lys Phe Asp 432 480 WO 97/05164 PCTIAU96/00473 36 GGC TGT GCA AAC AAJ Gly Cys Ala Asn Asn 165 GTG CAG GAC AAT GAC.

Val Gin Asp Asn Asp 180 AAG GGT ATA TAT TAC Lys Gay Ile Tyr Tyr 195 TTT GTA AAA GAG GCA Phe Val Lys Giu Ala 210 TGT TAT GGC AGC ACA Cys Tyr Gay Sex Thr 225 ACA CAA GAA GTA TCC Thr Gin Giu Val Set 245 ACC TCC ACC GTT GTG Thr Sex Thx Leu Vai 260 CCG CCT AGG AAA CGA Pro Pro Arg Lys Arg 275 TTG TGT GTG GCC CAC Leu Cys Val Ala His 290 ATC ACT AAC AAT CAC Ile Thr Asn Asn His 305 TCA GCT ACG CCT ATA Sex Aia Thr Pro Ile 325 TGT TTT AGA TAT AGG Cys Phe Arg Tyr Arg 340 ATA TCA TCA ACG TGG Ile Sex Sex Thr Trp 355 GCC ATT GTA ACT GTA Ala Ile Val Thr Val 370 TTA GAT GTT GTA AAA Leu Asp Vai Val Lys 385 ATG TCA CTG CAC CTA Met Sex Leu His Leu 405 TAT GTT ATG GCA GCA Tyr Val Met Ala Ala 420 CTA CAC AAG AAG TAT Leu His Lys Lys Tyr 435 ACA ATG GAT TAT Thr Met Asp Tyr ACC TGG GTA AAG Thr Txp Vai Lys 185 ACA TGT GGA CAA Thr Cys Giy Gin 200 GAA AAG TAT GGG Giu Lys Tyr Giy 215 GTT ATA TGT TCT Vai Ile Cys Sex 230 ATT CCT GAA TCT Ile Pro Giu Sex TCC TCA AGC ACC Ser Sex Sex Thr GTA TGG ACA Vai Trp Thr CAT AGT ATG His Sex Met AAA ACA TAT Lys Thr Tyr 205 ACC AAA CAT Thr Lys His 220 GCA TCT GTA Ala Sex Vai 235 ACA TAC ACC Thx Tyr Thr GAA GAC GCA Giu Asp Aia CAG TCC CCT Gin Sex Pro 285 AGT GGA AAC Sex Giy Asn 300 GAT GTG TAT Asp Vai Tyr 175 GTA GAT GCT Vai Asp Ala 190 TAT GTA AAC Tyx Vai Asn TGG GAA GTA Trp Giu Vai TCT AGC ACT Sex Sex Thx 240 CCC GCA CAG Pro Ala Gin 255 GTG CAA ACG Val Gin Thx 270 TGC AAC GCC Cys Asn Ala CAC AAC CTC His Asn Leu

ACT

Thx 250

AAG

Lys GCA CGA GGA Ala Axg Gly 280 A=T GGA CCC Ile Gly Pro 295 GAG GAG GAG Asp Gin His 310 GTG CAA ITT Vai Gin Phe CTA AAT GAG Leu Asn Asp 265 GTG CAA Vai Gin GIG GAC Val Asp CAA AGA Gin Arg CAA GGT Gin Giy 330 AGA GAG Axg His 345 528 576 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 1344 CAC TGG His Trp ACA TAT Thx Tyr 375 ATA CCC Ile Pro 390 TTG GTG Leu Vai GAG TTA Gin Leu GCC ICC TGA Ala Sex Sex 360 GAT AGT GAG Asp Sex Giu CCT ACC AlT Pro Th Ile GAG ATG GGA Asp Met Giy 410 TAT GTT GTG Tyr Vai Leu 425 GGG AAC AAC AGT Axg Asn Asn Sex 315 GAA ICC AAT IGI Giu Sex Asn Gys AGA, CAT TTA ITT Arg His Leu Phe 350 AAG GCA GCA CAT Lys Ala Pro His 365 GAA CAA AGG CAA Giu Gin Arg Gin 380 AGC GAG AAA CTG Sex His Lys Leu 395 GCA GGA AAG All Ala Pro Asn Ile CTG CAT CTG TAT Leu His Leu Tyx 430 AAG AGT Asn Set 320 TTA AAG Leu Lys 335 GAT TTA Asp Leu AAA CAT Lys His GAG TT Gin Phe GGA TT Giy Phe 400 GGG AAG Giy Lys 415 GTA G-CA Leu Ala CGA TIC GIG AAT Pro Phe Leu Asn 440 CI CIA CAT ACA CCC GGG GAG Leu Leu His Thx Pro Pro His 445 WO 97/05164 -37 AGA CCT CCA CCC TTG TGT CCT CAA GCA CCA AGG AA( Arg Pro Pro Pro Leu Cys Pro Gin Ala pro Arg Ly 450 455 46( CGC CGC CTA GGA AAC GAG CAC GAG GAG TCC AAC AG~ Arg Arg Leu Gly Asn Giu His Giu Giu Sex Asn Se~ 465 470 475 CCT TGT GTG TGG CCC ACA TTG GAC CCG TGG ACA GT( Pro Cys Val Trp Pro Thr Leu Asp Pro Trp Thr Va2 485 490 TCA TCA CTA ACA ATC ACG ACC AGC ACC AAA GAC GG2 Ser Ser Leu Thr Ile Thr Thr Ser Thr Lys Asp G1l 500 505 GTT CAG CTA CGC CTA AGA TCT ATG GAA GTG GTG CCI Vai Gin Leu Arg Leu Arg Sex Met Giu Val Val Prc 515 520 G4CA GGA ACA ACC AGC ACA TTC ATA CTG CCT GTT AT) Ala Giy Thr Thr Ser Thr Phe Ile Leu Pro Vai Ile PCT/AU96/00473 G ACG CAG TGC AAA 1392 s Thr Gin Cys Lys r' CCC CTT GCA ACG 1440 r Pro Leu Ala Thr 480 3GAA ACC ACA ACC 1488 L Giu Thr Thr Thr 495 SACA ACA GTA ACA 1536 TThr Thr Vai Thr 510 GTA CAA ATA GCT 1584 D Val Gin Ile Aia 525 SATT GCA TTT GTT 1632 Ile Alda Phe Valt 530 535 540 TGT TTT GTT AGC Cys Phe Vai Ser ATC ATA Ile Ile 550 CTT ATT GTA TGG Leu Ile Vai Trp 555 ATA TCT GAG TTT Ile Ser Giu Phe GTG TAC ACA TCT Val Tyr Thr Ser CTA GTA CTA ACA Leu Vai Leut Thr CTG CTT Leu Leu 570 TTA TAT TTA Leu Tyr Leu CTA TTG Leu Leu 575 TGG CTG CTA TTA ACA ACC CCC TTG CAA TTT TTC CTA CTA ACT CTA CTT Trp Leu Leu Leu Thr Thr Pro Leu Gin Phe Phe Leu Leu Thr Leu Leu 580 585 590 GTG TGT TAC-TGT Vai Cys Tyr Cys 595 CAG CAA CTT AAG Gin Gin Leu Lys 610 CCC GCA TTG Pro Ala Leu ATG ATG CTA Met Met Leu 615 ATA CAC TAC TAT Ile his Tyr Tyr GTT ACC ACA Vai Thr Thr GGA GAT ACC Gly Asp Thr ACA TGT CAA TTT Thr Cys Gin Phe AAa' GAT Asn Asp 620 TGG CTG GGT TTG TGG TTG TITA TGT GCC Trp Leu Giy Leu Trp, Leu Leu Cys Ala 625 630 TTT ATT Phe Ile 635 GTA GGG ATG TTG Val Giy Met Leu TTA TTA TTG ATG Leu Leu Leu Met CAC TAT AGA GCT GTA CAA His Tyr Arg Ala Val Gin 645 650 GGG GAT AAA CAC Gly Asp Lys His ACC AAA Thr Lys 655 1680 1728 1776 1824 1872 1920 1968 2016 2064 2112 2160 2208 2256 TGrT AAG AAG TGT AAC AAA CAC AAC TGT Cys Lys Lys Cys Asn Lys His Asn Cys 660 665 AATI GAT GAT TAT Asn Asp Asp Tyr GTA ACT ATG Val Thr Met 670 CAT TAT ACT His Tyr Thr 675 AGT GCA AAT Sex Ala Asn 690 ACT GAT GGT GAT TAT ATA TAT ATG ANT CCG CGG ATG GAA Thr Asp Giy Asp Tyr Ile Tyr Met A1n. Pro Axg Met Giu 680 685 GCC TCC ACG Ala Ser Thr TCT GCA ACG ACC ATA GAC Ser Ala Thr Thr Ile Asp 695 700 CAG TTG TGC AAG Gin Leu Cys Lys TGT GTG TTT TGC Cys Vai Phe Cys 720 ACG' "ITT Thr Phe 705 AAT CTA TCT Asn Leu Sex ATG CAT ACG TTG CAA ATT AAT Met His Thr Leu Gin Ile As-n 710 715 AAG AAT GCA CTG ACC ACA GCA GAG ATT TAT Lys Asn Ala Leu Thr Thr Ala Giu Ile Ty4: 725 730 TCA TAT GCA TAT AAA CAC Sex Tyr Ala Tyr Lys His .735 CTA AAG GTC CTG TTT CGA GGC GGC TAT CCA TAT GCA GCC TGC GCG TGC Leu Lys Val Leu Phe Arg Giy Gly Tyr Pro Tyr Ala Ala Cys Ala Cys 1' WO 97/05164 PCT/AU96/00473 38 TGC CTA GAA TTT CAT GGA AAA Cys Leu Giu Phe His Giy Lys 755 GCT GGA TMT GCA ACA ACA GTT Ala Gly Tyr Ala Thr Thr Vai 770 775 ATA AAC.

Ile Asn 760 CAA TAT AGA Gin Tyr Arg TTT GAT TAT Phe Asp Tyr GAA GAA GAA ACT Giu Giu Giu Thx CTG TGT CAC AAA Leu Cys His Lys 795 CAA GAC ATC TTA Gin Asp Ile Leu GAC GTG Asp Val 785 CTA ATT CGG TGC TAC Leu Ile Arg Cys Tyr 790 CCG CTG TGT GAA GTA Pro Leu Cys Giu Val 800 GAA AhG GTA AAA Giu Lys Val Lys CAT MTA CTA ACC His Il.e Leu Thr 805 GGT CGC TGC CTA Gly Arg Cys Leu AAG GCG CGG Lys Ala Axg 810 TTC MTA AAG Phe Ile Lys CTA AAT Leu Asn 815 TGT ACG TGG Cys Thr Trp GAC ATG TTA Asp Met Leu 835 MTT GTA TTA Ile Vai Leu 850 CAC TGC His Cys 825 TGG ACA ACA Trp Thr Thm TGC ATG GAA Cys Met Giu 830 CTA AAG GAT Leu Lys Asp CCC GCT AGC ATG Pro Ala Set Met GGA AGA CAT Giy Arg His GTT ACC Val Thr 845 GAC CTG CAA CCT CCA GAC CCT GTA GGG TTA CAT TGC TAT Asp Leu Gin Pro Pro Asp Pro Val Giy Leu His Cys Tyr 855 860 GAG CAA Giu Gin 865 TTA GTA GAC Leu Vai Asp AGC TCA Set Sex 870 GAA GAT GAG Giu Asp GiU GAC GAA GTG GAC Asp Giu Vai Asp CAA GAT TCA CAA Gin Asp Ser Gin CCT TTA AAA CAA CAT TTC CAA ATA GTG ACC TGT TGC Pro Leu Lys Gin His Phe Gin Ile Vai Thr Cys Cys 885 890 895 2304 2352 24~00 2448 2496 2544 2592 2640 2688 2736 2'78 4 2832 2880 2928 2976 3024 3 07 2 31i20 TGT GGA TGT Cys Gly Cys AGC AAC GTT CGA Ser Asn Val Arg CTG GTT Leu Vai 905 GTG CAG TGT Vai Gin Cys ACA GAA ACA Thr Giu Thr 910 GAC ATC AGA GAA GTG CAA CAG CTT CTG TTG GGA ACA CTA AAC ATA GTG Asp Ile Arg Giu Vai Gin Gin Leu Leu Leu Giy Thr Leu Asn Ile Vai 915 920 925 TGT CCC Cys Pro 930 ATC TGC GCA Ile Cys Ala CCG AAG Pro Lys 935 ACC CGA TCG ATG Thr Axg Sex Met GAC GAT TCA GGT Asp Asp Set Giy ACA GAA AAT GAG GGG TCT Thr Giu Asn Giu Giy Sex 945 950 MTA GTG CAA CAC CCA ACA Ile Vai Gin His Pro Thr 965 GGG TGT ACA Giy Cys Thr GGA TGG Giy Trp 955 TTT ATG GTA GAA Phe Met Vai Giu GGT ACA CAA ATA TCA GAC GAT GAG GAT GAG Giy Thx Gin Ile Sex Asp Asp G23u Asp Giu 970 97 GAG GTG GAG GAC Giu Vai Giu Asp 980 AAT MTT ACA CAC Asn Ile Thr His 995 AGT GGG TAT GAC Sex Giy Tyr Asp GTG GAC TTT Vai Asp Phe MTT GMT GAC AGC Ile Asp Asp Sex 990 AAT TCA CTG Asn Set Leu GAA GCA CAG Giu Ala Gin 1000 GCA TTG TTT AAC AGG CAG Ala leu Phe Asn Axg Gin 1005 GAG GCG GAC Glu Ala Asp 1010 ACC CMT TAT GCG ACT GTG Thx His Tyx Ala Thr Vai 1015 CAG GAC CIA AAA, CGA AAG TAT Gin Asp Leu Lys Arg Lys Tyr 1020 TTA GGT AGT CCA TMT GTT AGT CCT ATA AAC ACT ATA GCC GAG GCA GTG Leu Giy Sex Pro Tyr Vai Ser Pro Ile Asn Th Ile Ala Giu Ala Vai 1025 1030 1035 1040 I r WO 97/05164 PCT/AU96/00473 GAA AGT GAA ATA AGT CCA Giu Ser Giu Ile Ser Pro 1045 CC~A AAA AAG GTA AAG CGA Pro Lys Lys Val Lys Axg 1060 39 CGA TTG GAC GCC ATT AAA CTT ACA AGA CAG Arg Leu Asp Ala Ile Lys Leu Thr Arg Gin 1050 1055 CGG CTG TTT CAA ACC AGG GAA CTA ACG GAC A rg Leu Phe Gin Thr Axg Giu Leu Thr Asp 1065 10"70 AG? GGA TAT GGC Ser Gly Tyr Giy 1075 TAT TCT GAA Tyr Sex Giu GTG GAA GCT Val Giu Ala 1080 GGA ACG GGA ACG CAG GTA Gly Thr Giy Thr Gin Val 1085 3168 3216 3264 3312 3360 GAG AAA CAT Giu Lys His 1090 GGC GTA CCG Gly Vai Pro GAA AAT Giu Asn 1095 GGG GGA GAT Giy Giy Asp GGT CAG Gly Gin 1100 GAA AAG GAC Giu Lys Asp

ACA

Thr 1105 GGA AGG GAC ATA GAG GGG GAG GAA CAT ACA GAG GCG GAA GCG CCC Giy Axg Asp Ile Giu Giy Giu Giu His Thr Giu Ala Giu Ala Pro 1110 11l5 1120 ACZA AAC AGT Thr Asn Sex TTA AAA TGT Leu Lys Cys GTA CGG GAG Val Arg Giu 1125 CAT GCA GGC ACA GCA His Ala Gly Thr Al1a 1130 AAA GAT Lys Asp 1140 TTA CGG GCA Leu Arg Ala GCA TTA CTT Ala Leu Leu 1145 GGA ATA TTG GAA TTG Gly Ile Leu Giu Leu 1135 GGT AAG TTT AAA GAA Giy Lys Phe Lys Giu 1150 CCA TTT AAA AGT GAT Pro Phe Lys Sex Asp 1165 TTT GGT ATA CAT CAT Phe Gly Ile His His 1180 TGC TTT GGG CTG TCT TTT ATA GAT TTA AT? AGG Cys Phe Gly Leu Sex Phe Ile Asp Leu Ile Axg 1155 1160 AAA ACA ACA TGT TTA GAT TGG GTG GTA GCA GGG Lys Thr Thr Cys Leu Asp Tip Vai Val Ala Giy 1170 1175 AGC MAA Sex Ile 1185 TCA GAG GCA Ser Giu Ala TTT CAA AAA Phe Gin Lys 1190 TTA ATT GAG CCA Leu Ile Giu Pro 1195 TTA AGT TTA TAT Leu Sex Leu Tyr 1200 GTA TTG TTA GTA Val Leu Leu Val 1215 G-CA CAT ATA CAA Ala His Ile Gin TGG CTA ACA Tip Leu Thx 1205 AAT GCA TGG GGA ATG Asn Ala Tip Gly Met 1210 TTA TTA AGA TTT AAA GTA AAT Leu Leu Arg Phe Lys Vai Asn 1220 CTT GC-A ACG CTA TTA AAT ATA Leu Ala Thr Leu Leu Asn Ile 1235 AAA AGT AGA Lys Ser Axg 1225 CCT GAA AAC Pro Giu Asn 1240 AGT ACC GTT GCA CGT ACA Ser Thr Val Aia Arg Thr 1230 CAA ATG TTA ATA GAG CCA Gin Met Leu Ile Giu Pro 1245 3456 3504 3552 3600 3648 3696 3-744 3792 3840 3888 3936 CCA AAA ATA CAA AG? GGT GTT GCA GCC Pro Lys Ile Gin Sex Gly Val Ala Ala 1250 1255 CTG TM' TGG CGT ACA GGT Leu Tyr Tip Phe Axg Thr Gly 1260 A'A TCA AAT Ile Ser Asn 1265 GCC AGT ACA GTT1 ATA Ala Sex Thr Val Ile 1270 GGG GAA GCA CCA GAA TGG ATA ACA Gly Giu Ala Pro Giu Tip Ile Thr 127 5 1280 CGt CAA ACA GTT Arg Gin Thx Vai ATT GAA Ile Giu 1285 CAC GGG TTG His Giy Leu GCA GAC AGT Ala Asp Sex 12 90 CAG TTT AAA TTA Gin Phe Lys Leu 1295 ACA GAA ATG GTG CAG TGG Thx Glu Met Val Gin Tip 1300 GAA ATT GCA 'ITT GAA TAT Giu Ile Ala Phe Giu Tyx 1315 GCG TAT GAT AAT Ala Tyr Asp Asn 1305 GCA CAA AGG GGA Ala Gin Arg Gly 1320 GAC ATA TGC Asp Ile Cys GAG GAG AG? Glu Giu Sex 1310 GA? TTT GAT TCT AAT- GCA Asp Phe Asp Sex Asn Ala -1325 3984- CGA GCA TTT TTA AAT AGC AAT ATG CAG GCA AAA TAT GTG AAA GAT TGT Mxg A.1g Rhe Leu Asn Sex Asn Met Gin Ala Lys Tyr Vai Lys Asp Cys 4032 WO 97105164 PCT/AU96/00473 40 1340 1330 1335 GCA ACT ATG Ala Thr Met 1345 ATA AAA CAA Ile Lys Gin AAT TGG AAA Asn Trp Lys TGT AGA CAT TAT AAA Cys Arg His Tyr Lys 1350 TGG ATA AAA CAT AGG Trp Ile Lys His Arg 1365 CCA ATT GTA CAA TTC Pro Ile Val Gin Phe 1380 CAT GCA GAA ATG His Ala Giu Met 1355 AGG AAG ATG TCT Axg Lys Met Sex 1360 GGT TCT AAA ATA GAA GGC ACA GGA Giy Ser Lys Ile Giu Gly Thr Gly 1370 1375 CTA CGA CAT Leu Arg His 1385 CAA AAT ATA GAA TTC Gin Asn Ile Giu Phe 1390 CAC GGT ACG CCA AAA His Gly Thr Pro Lys 1405 4080 4128 4176 4224 4272 4320 ATT CCT TTT TTA ACT AAA TTT AAA TTA TGG CTG Ile Pro Phe Leu Thr Lys Phe Lys Leu Trp, Leu 1395 1400 AAA AAC TGC ATA GCC ATA GTA GGC Lys Asn Cys Ile Ala Ile Vai Giy 1410 1415 CCT CCA GAT Pro Pro Asp ACT GGG Thr Giy 1420 TTT TGT Phe Cys 1425 ATG AGT TTA ATA AGC Met Sex Leu Ile Sex 1430 TTT CTA GGA Phe Leu Gly GGT ACA GTT Gly Thr Val 1435 AAA TCG TAC Lys Ser Tyr ATT AGT CAT Ile Ser His 1440 GAT GCT AAG Asp Ala Lys 1455 GTA AAT TCC AGC AGC CAT TTT Val Asn 5cr Sex Sex His Phe 1445 GTA GCA TTG TTA GAT GAT GCA Vai Ala Leu Leu Asp Asp Ala 1460 ACA TAT ATG AGA AAT TTG TTA Thx Tyr Met Axg Asn Leu Leu 147S TGG TTG CAA CCG TTA GTA Trp Leu Gin Pro Leu Val 1450 ACA CAG CCA Thx Gin Pro 1465 GAT GGT AAT Asp Giy Asn 1480 TGT TGG ATA TAT ATG GAT Cys Trp Ile Tyr Met Asp 14,70 CCT ATG AGT ATT GAC AGA Pro Met Se Ile Asp Axg 1485 AAG CAT AAA GCA TTG ACA TTA ATT AAA TGT CCA CCT CTG CTA GTA ACG Lys His Lys Ala Leu Thr Leu Ile Lys Cys Pro Pro Leu Leu Val Thr 1490 1495 1500 TCC AAC: ATA GAT ATT ACT AAA GAA GAT AAA TAT AAG TAT TTA CAT ACT Sex Asn Ile Asp Ile Thr Lys Giu Asp, Lys Tyr Lys Tyr Leu His Thr 1505 1510 1515 1520 AGA GTA ACA ACA TTT ACA TTT CCA AAT CCA TTC CCT TTT GAC AGA MAT Arg Val Thr Thr Phe Thx Phe Pro Asn Pro Phe Pro Phe Asp Arg Asn 1525 1530 1535 GGG AAT GCA GTG TAT GAA CTG TCA AAT ACA AAC TGG AAA Gly Asn Al1a Val Tyx Giu Leu Sex Asn Thr Asn Trp Lys 1540 1545 TGT TTT TTT Cys Phe Phe 1550 4512 4560 4608 4656 4704 4752 47'70 GAA AGA CTG TCG TCA AGC CTA GAC ATT CAG Giu Arg Leu Sex Sex Sex Leu Asp Ile Gin 1555 1560 GAT TCT GAG GAC GAG GAA Asp Sex Giu Asp Giiu Giu 1565 GAT GGA AGC AAT Asp Giy Sex Asn 1570 AGC CAA GCG TTT AGA TGC GTG Sex Gin Ala Phe Axrg Cys Val 1575 CCA GGA ACA GTT GTT Pro Giy Thr Val Vai 1580 AGA ACT TTA TGAGGTACC Axg Thx Leu 1585

Claims (16)

1. A composition for eliciting a humoral and/or cellular immune response against papillomavirus in a host animal, said composition comprising an immunologically effective amount of a polyprotein construct comprising at least two amino acid sequences fused directly or indirectly together, each of said sequences being the sequence of an early ORF protein of papillomavirus (PV) or an immunogenic variant or fragment thereof, and at least one of said sequences being other than the E6 or E7 protein sequence or an immunogenic variant or fragment thereof, together with a pharmaceutically acceptable carrier and/or diluent.

2. A composition according to claim 1, wherein said construct comprises sequences of early ORF proteins of human PV, or immunogenic variants or fragments thereof.

3. A composition according to claim 2, wherein said early ORF proteins are selected from the group consisting of the El, E2, E3, E4, E5 (E5a, E5b), E6, E7 and E8 proteins of PV.

4. A composition according to any of claims 1 to 3, wherein said construct is selected from the group consisting of: E6/E4 E6/E5a/E4 E6/E7/E4 E6/E7/E5a/E4 E6/E7/E1/E4 E6/E7/E5a/E1/E4 E6/E7/E5a/E1/E2/E4 E6/E7/E5a/E5b/E1/E2/E4 E2/E1/E5b i ~RRAI g- i Ce rr V. ^l P:\OPER\]MS\65102-96,42 2011098 -42- E2/E5a/E5b E2/E1/E5a/E5b E2/E4/E5a/E5b/E6/E7/E1 E2/E3/E4/E5/E8/E6/E7/E1. rt C* C. SI. 4 8 8D 4* 8* 8 88 A composition according to claim 1, wherein said construct further comprises one or more linker sequences between and/or before and/or after said amino acid sequences.

6. A composition according to claim 5, wherein said linker sequence(s) comprise from 1 to 5 amino acid residues.

7. A composition according to claim 1, wherein said construct further comprises a tag protein or peptide moiety fused or otherwise coupled thereto.

8. A composition according to claim 7, wherein said tag moiety is selected from the group consisting of glutathione-S-transferase (GST) and FLAG.

9. A composition according to claim 1, wherein said construct further comprises an adjuvant moiety fused or otherwise coupled thereto. A composition according to claim 9, wherein said adjuvant moiety is selected from diphtheria toxin, cholera toxin and E. coli heat labile toxin (LT) and non-toxic derivatives thereof such as the holotoxoid or B sub-unit of cholera toxin or LT.

11. A composition according to claim 1, wherein said construct further comprisesa lipid binding region.

12. A composition according to claim 11, wherein said lipid binding region is an influenza haemagglutinin tail. R r I ;LP_ P:\OPER\MS\65102-96,43 20/10/98 -43

13. A composition according to any of claims 1 to 12, further comprising an adjuvant.

14. A method for eliciting a humoral and/or cellular immune response against papillomavirus in a host animal, which method comprises administering to the host animal an immunologically effective amount of a polyprotein construct comprising at least two amino acid sequences fused directly or indirectly together, each of said sequences being the sequence of an early ORF protein of papillomavirus (PV) or an immunogenic variant or fragment thereof, and at least one of said sequences being other than the E6 or E7 protein sequence or an -I immunogenic variant or fragment thereof. A method according to claim 14, wherein said polyprotein construct is administered in a composition together with a pharmaceutically acceptable carrier Sand/or diluent.

16. A method according to claim 15, wherein said composition further comprises an adjuvant.

17. A method according to any of claims 14 to 16, wherein said host animal is a human.

18. Use of a polyprotein construct comprising at least two amino acid sequences fused directly or indirectly together, each of said sequences being the sequence of an early ORF protein of papillomavirus (PV) or an immunogenic variant or fragment thereof, and at least one of said sequences being other than the E6 or E7 protein sequence or an immunogenic variant or fragment thereof, for the preparation of a composition for eliciting an immune response against papillomavirus in a host animal. r RAZ/ L.

102-96,44 -20/10/98 44- 19. A composition for eliciting an immune response against papillomavirus in a host animal comprising a nucleic acid molecule which encodes a polyprotein construct j comprising at least two amino acid sequences fused directly or indirectly together, each of said sequences being the sequence of an early ORF protein of papillomavirus (PV) or an immunogenic variant or fragment thereof, and at least one of said sequences being other than the E6 or E7 protein sequence or an immunogenic variant or fragment thereof, together with a pharmaceutically acceptable carrier and/or diluent. I. 20. A composition according to claim 19, further comprising an adjuvant. 21. A method for eliciting an immune response against papillomavirus in a host ao. a j animal, which method comprises administering to the host animal an immunologically effective amount of a nucleic acid molecule which encodes a apolyprotein construct comprising at least two amino acid sequences fused directly .or indirectly together, each of said sequences being the sequence of an early ORF protein of papillomavirus (PV) or an immunogenic variant or fragment thereof, and at least one of said sequences being other than the E6 or E7 protein sequence or an immunogenic variant or fragment thereof. o co 22. A method according to claim 21, wherein said nucleic acid molecule is administered in a composition together with a pharmaceutically acceptable carrier and/or diluent. 23. A method according to claim 22, wherein said composition further comprises an adjuvant. 24. A method according to any of claims 21 to 23, wherein said host animal is a human. RA- P:\OPER\JMS\65102-96,45 20110/98 Use of a nucleic acid molecule which encodes a polyprotein construct comprising at least two amino acid sequences fused directly or indirectly together, each of said sequences being the sequence of an early ORF protein of papillomavirus (PV) or an immunogenic variant or fragment thereof, and at least one of said sequences being other than the E6 or E7 protein sequence or an immunogenic variant or fragment thereof, for the preparation of a composition for eliciting an immune response against papillomavirus in a host animal. Dated this 20th day of October 1998. o D q 9 CSL Limited AND The University of Queensland oo000 By their Patent Attorneys D o SDav Davies Collison Cave I 04 *000 a 099 o9 *o 0 *a *4 oo *oa lsi 0 4 9~ e c r r f/ 'F ie r