Vaccine compositions for human papillomavirus

The present application claims priority under 35 U.S.C. §119(e) to U.S. patent application Ser. Nos. 60/125,208, filed Mar. 18, 1999 and 60/148,544, filed Aug. 12, 1999, each of which is incorporated herein in its entirety.

The invention was made using U.S. Government funds, and therefore the U.S. Government has rights in the invention.

The invention relates to preparation and use of viral coat proteins. In particular, the invention relates to preparations of human papillomavirus L1 protein.

Human papillomaviruses are involved in a variety of disease states, including benign warts and cancer. There has been considerable effort to produce vaccines against human papillomaviruses, especially against types 16 and 18, which are associated with cervical cancer. Papillomaviruses contain two structural proteins that encapsidate the viral minichromosome, L1 and L2. In the virus particle, 72 pentamers of L1 form an outer shell; L2, probably one copy per L1 pentamer, is located on the inside of the L1 shell. The L1 and L2 proteins are therefore important candidates to use as immunogens. Because papillomaviruses cannot be propagated in cell culture, however, recombinant methods must be used to produce papillomavirus proteins.

Bacterial expression systems are generally effective and inexpensive ways to produce large quantities of recombinant proteins, but it has been difficult to produce large quantities of papillomavirus L1 protein in bacterial expression systems. Thus, there is a need in the art for methods of obtaining preparations of human papillomavirus L1 proteins which can be used as immunogens and as diagnostic reagents from bacterial expression systems.

It is an object of the present invention to provide stable preparations of human papillomavirus L1 protein for use as immunogens and as diagnostic tools.

One embodiment of the invention is a composition which comprises a multimer of a human papillomavirus L1 protein and a physiologically compatible carrier. As used herein, the term “L1 protein” refers to a truncated L1 polypeptide that does not include amino terminal residues 1-8 of HPV16 L1 , or the corresponding structural residues of the L1 protein of other HPV subtypes.

As used herein, the term “multimer” refers to more than one L1 monomers; however, the invention is most advantageous where “multimer” refers to 5 L1 monomers (i.e., a pentamer) up to and including 60 monomers (12 pentamers).

As used herein, the term “stable” means that the preparation of L1 protein is not proteolytically degraded or denatured by proteases when placed in 10 mM salt, 0.1 mM EDTA at 10° C. for 24 hours.

Preferably, the preparation of human papillomavirus L1 protein is also soluble.

As used herein, “soluble” means that the preparation of L1 protein remains in the supernatant after centrifugation in a table-top centrifuge at 10,000 rpm for 5 minutes.

These and other objects of the invention are provided by one or more of the embodiments which are described below.

Another embodiment of the invention is a method of immunizing a human against a human papillomavirus. A composition which comprises a multimer of a human papillomavirus L1 protein is administered to a human at a dose effective to induce an immune response against the L1 protein in the human. As used herein, “effective to induce an immune response” refers to the ability to induce an antibody response to the L1 protein or to induce a CTL response to L1 .

Yet another embodiment of the invention is a method of detecting the presence of antibodies against a human papillomavirus in a biological sample. A biological sample is contacted with a multimer of a human papillomavirus L1 protein. Antibodies which bind to the multimer are detected. Detection of antibodies which are bound to the multimer identifies the presence of antibodies against the human papillomavirus in the biological sample.

A further embodiment of the invention is a method of detecting a specific subtype of human papillomavirus in a biological sample. A biological sample is contacted with an antibody which specifically binds to an L1 protein of a specific subtype of human papillomavirus. L1 protein which is bound to the antibody is detected. Detection of L1 protein which is bound to the antibody identifies the presence of the specific subtype of human papillomavirus in the biological sample.

Even another embodiment of the invention is a solid support comprising a multimer of a human papillomavirus L1 protein.

Another embodiment of the invention is a solid support comprising an antibody which specifically binds to an L1 protein of a specific type of human papillomavirus.

Still another embodiment of the invention is a method of producing a soluble multimer of a human papillomavirus L1 protein which is truncated at its amino terminus. A recombinant human papillomavirus L1 protein is expressed in a bacterial host cell. The recombinant human papillomavirus L1 protein comprises amino acids 9-505 as shown in SEQ ID NO: 1. Preferably the L1 protein comprises amino acids 10-505, 11-505, 12-505, and even 13-505 as shown in SEQ ID NO:1. The recombinant human papillomavirus L1 protein is treated to remove bacterial host cell proteins. A soluble multimer of the human papillomavirus L1 protein is thereby formed.

The present invention thus provides the art with a simple and effective method of producing large quantities of human papillomavirus L1 protein. Preparations of human papillomavirus protein can be used, inter alia, as immunogens and diagnostic tools.

FIG. 1 . Two views of the HPV 16 L1 subunit. The outward facing loops are at the top of the figure.

FIG. 2 . Interaction between L1 monomers in a pentameric capsomere.

FIG. 3. A pentameric capsomere of HPV16 L1 , viewed normal to the five-fold axis.

FIG. 4 . View of pentameric HPV16 L1 capsomere from a direction corresponding to the outside of the icosahedral particle.

FIG. 5 . The HPV16 L1 T=1 icosahedral particle.

FIG. 6 . Interactions between pentamers in an L1 T=1 icosahedral particle.

FIGS. 7A-7B. FIG. 7A, HPV16 L1 pentamer, with polypeptide chains in a worm-like representation. Positions of residues that vary significantly among HPV types are indicated by arrows. FIG. 7B, surface representation of outer (left) and inner (right) views of L1 pentamer.

It is a discovery of the present invention that L1 coat proteins of human papillomaviruses can be expressed in large quantities from bacterial host cells, particularly E. coli cells. The expressed L1 proteins associate to form soluble multimers. L1 multimers can be used, for example, as immunogens, to induce an immune response against human papillomavirus infection, as diagnostic tools for detecting the presence of human papillomavirus in biological samples, and as tools for mapping receptor interactions.

Multimers of Human Papillomavirus L1 Protein

An L1 multimer of the invention can comprise 2 or more, 3, 4, preferably 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 60 or more molecules of human papillomavirus L1 protein . Pentamers of L1 protein are preferred. A 60-subunit (12-pentamer) T=1 icosahedral particle, as described below, is particularly preferred. Preferably, the multimers are soluble in an aqueous solution. Multimers of L1 protein from any human papillomavirus can be produced including, but not limited to, HPV1, HPV2, HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV11, HPV12, HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV25, HPV26, HPV27, HPV28, HPV29, HPV31, HPV33, HPV34, HPV35, HPV36, HPV37, HPV38, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV66. Optionally, an L1 multimer can comprise a molecule of human papillomavirus L2 protein from the same or from a different human papillomavirus type.

In one embodiment, the L1 molecules in the multimers are less than full-length. For example, truncated forms of human papillomavirus 16 L1 protein which lack 9, 10, 11, or 12 consecutive amino acids beginning at the amino terminus can be used to form L1 multimers. In order to be soluble, however, the expressed human papillomavirus 16 L1 proteins must comprise at least amino acids 13-505 as shown in SEQ ID NO: 1. L1 proteins from other human papillomavirus types, such as HPV 18, are also expected to be soluble with amino terminus truncations. The precise extent of the amino terminal amino acids which can be deleted will depend upon the particular amino acid sequence of the L1 protein and can be predicted by structurally aligning the L1 protein with residues of the L1 protein of HPV16. Amino acid sequences of other human papillomaviruses can be obtained, for example, from databases such as Genbank, Swiss-prot, PIR protein Data Base, PDB Protein Data Bank, or EMBL, or from the HPV sequence database, which can be accessed at <http://hpv-web.lanl.gov>. The amino acid sequences of the HPV16 and HPV18 L1 proteins are provided herein as SEQ ID NOS: 1 and 3, respectively.

L1 molecules in the multimers can comprise the natural carboxy terminus of the protein or can be truncated at the carboxy terminus by up to 30 amino acids. Again, the precise extent of the truncation will depend on the particular amino acid sequence of the L1 protein.

Icosahedral Particles

L1 multimers comprising L1 protein which is truncated at its amino terminus associate to form multimeric particles. For example, pentamers of L1 protein can associate to form 12-pentamer (60-subunit) particles with icosahedral symmetry (T=I icosahedral particles). T=1 icosahedral particles are L1 multimers which form naturally at a pH of 4-5 and a concentration of at least 0.2, 0.25, 0.3, 0.35, 0.4, 0.5, 1, 5, 10, or 20 mg/ml. Preferably, however, the T=1 icosahedral particles are stable at pH values above 4.5, such as pH 5, 5.5, 6, 6.5, or 7. Most preferably, the T=1 icosahedral particles are stable at neutral pH.

Any means known in the art which will maintain immunogenicity of the particles can be used to stabilize the particles. For example, L1 pentamers can be cross-linked to stabilize the structure of a T=1 icosahedral particle. In one embodiment, cysteine residues are introduced at particular positions in the L1 protein, to generate inter-pentamer disulfide bonds that will keep the particles assembled at higher pH values. Methods of substituting or adding codons for one or more amino acids to a coding sequence for a polypeptide are well known in the art. The positions at which cysteine residues can be introduced to result in the placement of two cysteine residues at the correct distance for formation of a disulfide bond can readily be determined by inspection of the crystal structure of the assembled L1 pentamers. Alternatively, mutations can be introduced into the L1 amino acid sequence which would improve non-covalent interactions between L1 pentamers, such as the formation of salt bridges between oppositely charged amino acids. Chemical cross-linking can also be used to stabilize the T=1 icosahedral particles.

Pentamers of L1 proteins contain outward-facing “loops” of amino acids which are clearly responsible for antigenic differences among human papillomavirus types and may also confer specificity for particular cell-surface receptors (see Example 4, below). The discovery that the outward facing loops contain most of the sequence variability (and hence type variation) and that other surfaces, as well as the hydrophobic core, are conserved can be exploited to produce antibodies and vaccines which are specific for particular types of human papillomavirus. The term “outward facing loop” refers to residues in the BC, BE, FG, and HI loops of the L1 protein. The ability of the L1 pentamers to associate to form T=1 icosahedral particles is also important for the generation of effective vaccines against human papillomavirus. The T=1 icosahedral particles are highly polyvalent and are therefore effective immunogens. Moreover, because of their polyvalency, the T=1 icosahedral particles will bind tightly to cells with the appropriate receptor. Thus, they might penetrate those cells and produce an even broader immune response, including, for example, CTL immunity as well as antibody production.

Expression of L1 Multimers in Bacterial Host Cells

Polynucleotide segments encoding human papillomavirus L1 proteins can be inserted into expression constructs using standard molecular biology techniques and used to express large quantities of L1 protein which is capable of associating to form soluble multimers. Amino acid sequences of human papillomavirus proteins, as well as nucleotide sequences which encode the proteins, can be obtained from databases such as Genbank, Swiss-prot, EMBL, PIR Protein Data Base, PDB Protein Data Bank, or from the HPV sequence database, found at <http://hpv-web.lanl.gov/>.

An L1 expression construct comprises a promoter which is functional in a chosen bacterial host cell. The skilled artisan can readily select an appropriate promoter from the large number of cell type-specific promoters known and used in the art. The expression construct can also contain a transcription terminator which is functional in the bacterial host cell. The expression construct comprises a polynucleotide segment which encodes the desired portion of the L1 protein. The polynucleotide segment is located downstream from the promoter. Transcription of the polynucleotide segment initiates at the promoter. The expression construct can be linear or circular and can contain sequences, if desired, for autonomous replication.

A variety of bacterial host cells are available and can be used to express or to propagate L1 expression constructs. Suitable bacterial host cells include strains of E. coli and Bacillus. Preferably, L1 protein is produced in E. coli cells. Expression constructs can be introduced into bacterial host cells using any standard transfer technique for bacterial cells known in the art.

To facilitate purification, the L1 protein can be expressed as a fusion protein, such as a GST fusion protein. After expression of the recombinant L1 protein from the bacterial host cells, the recombinant L1 protein is treated to remove bacterial host cell proteins. This can be accomplished using any method known in the art. When L1 protein is expressed in E. coli cells, the expressed protein must be treated to remove the E. coli protein GroEL. Treatment with 2 mM ATP and 3.5 M urea is especially effective at removing GroEL protein from the expressed L1 protein (see Example 1, below).

If inclusion of human papillomavirus L2 protein in an L1 multimer is desired, expression constructs encoding L2 can be introduced into the bacterial host cell, as described above, and used to co-express L2 protein in the cell. The amino acid sequences of L2 proteins from HPV16 and HPV18 are shown in SEQ ID NOS: 2 and 4, respectively. Amino acid sequences of other human papillomavirus L2 proteins can be found in databases such as Genbank, Swiss-prot, EMBL, PIR Protein Data Base, PDB Protein Data Bank, and the HPV sequence database at <http://hpv-web.lanl.gov/>. Alternatively, L1 and L2 proteins can be expressed from the same expression construct.

Compositions of L1 Multimers

L1 multimers of the invention, including T=1 icosahedral particles, can be used in a composition. L1 multimer compositions are useful, for example, in immunoassays, as vaccine compositions, and as tools for use in characterizing cell-surface receptors which bind to L1 proteins.

In one embodiment of the invention, the composition can consist essentially of L1 multimers without the presence of L2 molecules. Alternatively, an L1 multimer composition can consist of L1 multimers alone.

Compositions of the invention can be used as vaccine compositions, for example, to enhance or induce an immune response of a human to a human papillomavirus. Immune responses which can be enhanced or induced include both humoral and cell-mediated responses. Compositions comprising particular preparations of L1 multimers can be tested for the ability to induce an immune response using assays well known in the art. For example, a composition can be injected into a laboratory animal, such as a rat or mouse, and the animal can be monitored for the appearance of immune reactants, such as antibodies, directed against L1 protein. Alternatively, assays such as cytotoxic T lymphocyte assays can be performed to determine whether a particular composition of L1 multimers is immunogenic.

For use as a vaccine, a composition of the invention preferably comprises a multimer of a human papillomavirus L1 protein, preferably a pentamer or a T=1 icosahedral particle comprising twelve L1 pentamers which is stable at neutral pH. Stable L1 multimers are resistant to proteolytic degradation or denaturation. Optionally, the L1 multimer can include a molecule of an human papillomavirus L2 protein.

Vaccine compositions of the invention typically comprise a physiologically compatible carrier. Physiologically compatible carriers are well known to those in the art. Such carriers include, but are not limited to, a simple low salt solution which permits preservation of the integrity of the L1 protein, e.g., 10 mM NaCl, 0.1 mM EDTA, or to large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Pharmaceutically acceptable salts can also be used in L1 multimer compositions, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as salts of organic acids such as acetates, proprionates, malonates, or benzoates. Compositions of the invention can also contain liquids, such as water, saline, glycerol, and ethanol, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents.

Administration of L1 Multimer Compositions as Vaccines

Various methods can be used to administer an L1 multimer vaccine composition to a human, including intravenous, intramuscular, or subcutaneous injection. Vaccine compositions of the invention are administered at a dose effective to induce an immune response against the L1 protein. The particular dosages of the composition used to enhance an immune response will vary, for example, according to the L1 composition being used and the mammal to which the composition is administered. Generally, about 5 μg to about 50 μg of L1 multimers per kg of patient body weight, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg, or about 200 to about 250 μg/kg will be administered to an adult human.

Such ranges by no means preclude use of a higher or lower amount of a component, as might be warranted in a particular application. For example, the actual dose and schedule may vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on individual differences in pharmacokinetics, drug disposition, and metabolism.

Diagnostic Assays

Because of the polyvalency of L1 multimers, particularly the T=1 icosahedral particles, L1 multimers of the invention can be used to produce antibodies which specifically bind to human papillomavirus L1 proteins. Taking advantage of the high variability of the outward-facing loops of L1 pentamers, for example, antibodies can be generated which can distinguish among L1 proteins of different human papillomaviruses. These antibodies can be used, for example, in diagnostic assays, to detect directly the presence of human papillomavirus or specific types of human papillomavirus associated with a particular disease state in a biological sample. Similarly, L1 multimers themselves can be used in immunoassays, to detect the presence of antibodies to human papillomavirus in a biological sample. Use of type-specific antibodies or L1 multimers represents a significant improvement over immunoassays which use polyclonal antisera directed against a prototype human papillomavirus.

Polyclonal or monoclonal antibodies, including single-chain antibodies, which specifically bind to L1 proteins can be constructed using techniques well known in the art. Preferably, antibodies which specifically bind to human papillomavirus L1 proteins do not bind to human papillomavirus L2 proteins or to human proteins. Most preferably, the antibodies specifically bind to a specific type of human papillomavirus, such as HPV 16, HPV 18, or any of the other specific types of human papillomavirus disclosed above. Antibodies which specifically bind to L1 proteins typically do not detect other proteins in immunochemical assays and can immunoprecipitate an L1 protein or multimer from solution.

Immunoassays which can be used to detect binding of L1 proteins and antibodies include, but are not limited to, Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Typically, such assays provide a detection signal at least 5-, 10-, or 20-fold higher than a background signal. Optionally, either the antibody or the L1 multimer can comprise a detectable label, such as a fluorescent, chemiluminescent, or enzymatic tag or a radiolabel.

An antibody or an L1 multimer can be bound to a solid support, such as a glass or plastic slide, tissue culture plate, microtiter well, tube, column, column matrix, protein, or particle such as a bead, including but not limited to a latex, polystyrene, or glass bead, or a flexible membrane, such as a nitrocellulose or nylon membrane. Means of coupling protein moieties to such surfaces are well known in the art, and any coupling means which does not destroy the ability of an antibody and an L1 protein to bind to each other can be used.

A biological sample to be tested is contacted with the antibody or L1 multimer. The biological sample can be any sample suspected of comprising a human papillomavirus, such as a biopsy, smear, or tissue section of a tissue such as skin, cervix, anogenital epithelium, larynx, upper respiratory tract, conjunctiva, or tissues of the oral cavity.

The following examples are provided for exemplification purposes only and are not intended to limit the scope of the invention.

Human papillomavirus type 16

1

Met Gln Val Thr Phe Ile Tyr Ile Leu Val Ile Thr Cys Tyr Glu Asn

1 5 10 15

Asp Val Asn Val Tyr His Ile Phe Phe Gln Met Ser Leu Trp Leu Pro

20 25 30

Ser Glu Ala Thr Val Tyr Leu Pro Pro Val Pro Val Ser Lys Val Val

35 40 45

Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn Ile Tyr Tyr His Ala Gly

50 55 60

Thr Ser Arg Leu Leu Ala Val Gly His Pro Tyr Phe Pro Ile Lys Lys

65 70 75 80

Pro Asn Asn Asn Lys Ile Leu Val Pro Lys Val Ser Gly Leu Gln Tyr

85 90 95

Arg Val Phe Arg Ile His Leu Pro Asp Pro Asn Lys Phe Gly Phe Pro

100 105 110

Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp Ala Cys

115 120 125

Val Gly Val Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly Ile Ser

130 135 140

Gly His Pro Leu Leu Asn Lys Leu Asp Asp Thr Glu Asn Ala Ser Ala

145 150 155 160

Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg Glu Cys Ile Ser Met Asp

165 170 175

Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys Pro Pro Ile Gly

180 185 190

Glu His Trp Gly Lys Gly Ser Pro Cys Thr Asn Val Ala Val Asn Pro

195 200 205

Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn Thr Val Ile Gln Asp Gly

210 215 220

Asp Met Val His Thr Gly Phe Gly Ala Met Asp Phe Thr Thr Leu Gln

225 230 235 240

Ala Asn Lys Ser Glu Val Pro Leu Asp Ile Cys Thr Ser Ile Cys Lys

245 250 255

Tyr Pro Asp Tyr Ile Lys Met Val Ser Glu Pro Tyr Gly Asp Ser Leu

260 265 270

Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe Val Arg His Leu Phe Asn

275 280 285

Arg Ala Gly Thr Val Gly Glu Asn Val Pro Asp Asp Leu Tyr Ile Lys

290 295 300

Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser Ser Asn Tyr Phe Pro Thr

305 310 315 320

Pro Ser Gly Ser Met Val Thr Ser Asp Ala Gln Ile Phe Asn Lys Pro

325 330 335

Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp Gly

340 345 350

Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met

355 360 365

Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu Thr Thr Tyr Lys Asn Thr

370 375 380

Asn Phe Lys Glu Tyr Leu Arg His Gly Glu Glu Tyr Asp Leu Gln Phe

385 390 395 400

Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Asp Val Met Thr Tyr

405 410 415

Ile His Ser Met Asn Ser Thr Ile Leu Glu Asp Trp Asn Phe Gly Leu

420 425 430

Gln Pro Pro Pro Gly Gly Thr Leu Glu Asp Thr Tyr Arg Phe Val Thr

435 440 445

Gln Ala Ile Ala Cys Gln Lys His Thr Pro Pro Ala Pro Lys Glu Asp

450 455 460

Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys

465 470 475 480

Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu

485 490 495

Gln Ala Gly Leu Lys Ala Lys Pro Lys Phe Thr Leu Gly Lys Arg Lys

500 505 510

Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr Thr Ala Lys Arg Lys Lys

515 520 525

Arg Lys Leu

530

2

473

Human papillomavirus type 16

2

Met Arg His Lys Arg Ser Ala Lys Arg Thr Lys Arg Ala Ser Ala Thr

1 5 10 15

Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile

20 25 30

Ile Pro Lys Val Glu Gly Lys Thr Ile Ala Glu Gln Ile Leu Gln Tyr

35 40 45

Gly Ser Met Gly Val Phe Phe Gly Gly Leu Gly Ile Gly Thr Gly Ser

50 55 60

Gly Thr Gly Gly Arg Thr Gly Tyr Ile Pro Leu Gly Thr Arg Pro Pro

65 70 75 80

Thr Ala Thr Asp Thr Leu Ala Pro Val Arg Pro Pro Leu Thr Val Asp

85 90 95

Pro Val Gly Pro Ser Asp Pro Ser Ile Val Ser Leu Val Glu Glu Thr

100 105 110

Ser Phe Ile Asp Ala Gly Ala Pro Thr Ser Val Pro Ser Ile Pro Pro

115 120 125

Asp Val Ser Gly Phe Ser Ile Thr Thr Ser Thr Asp Thr Thr Pro Ala

130 135 140

Ile Leu Asp Ile Asn Asn Thr Val Thr Thr Val Thr Thr His Asn Asn

145 150 155 160

Pro Thr Phe Thr Asp Pro Ser Val Leu Gln Pro Pro Thr Pro Ala Glu

165 170 175

Thr Gly Gly His Phe Thr Leu Ser Ser Ser Thr Ile Ser Thr His Asn

180 185 190

Tyr Glu Glu Ile Pro Met Asp Thr Phe Ile Val Ser Thr Asn Pro Asn

195 200 205

Thr Val Thr Ser Ser Thr Pro Ile Pro Gly Ser Arg Pro Val Ala Arg

210 215 220

Leu Gly Leu Tyr Ser Arg Thr Thr Gln Gln Val Lys Val Val Asp Pro

225 230 235 240

Ala Phe Val Thr Thr Pro Thr Lys Leu Ile Thr Tyr Asp Asn Pro Ala

245 250 255

Tyr Glu Gly Ile Asp Val Asp Asn Thr Leu Tyr Phe Ser Ser Asn Asp

260 265 270

Asn Ser Ile Asn Ile Ala Pro Asp Pro Asp Phe Leu Asp Ile Val Ala

275 280 285

Leu His Arg Pro Ala Leu Thr Ser Arg Arg Thr Gly Ile Arg Tyr Ser

290 295 300

Arg Ile Gly Asn Lys Gln Thr Leu Arg Thr Arg Ser Gly Lys Ser Ile

305 310 315 320

Gly Ala Lys Val His Tyr Tyr Tyr Asp Leu Ser Thr Ile Asp Pro Ala

325 330 335

Glu Glu Ile Glu Leu Gln Thr Ile Thr Pro Ser Thr Tyr Thr Thr Thr

340 345 350

Ser His Ala Ala Ser Pro Thr Ser Ile Asn Asn Gly Leu Tyr Asp Ile

355 360 365

Tyr Ala Asp Asp Phe Ile Thr Asp Thr Ser Thr Thr Pro Val Pro Ser

370 375 380

Val Pro Ser Thr Ser Leu Ser Gly Tyr Ile Pro Ala Asn Thr Thr Ile

385 390 395 400

Pro Phe Gly Gly Ala Tyr Asn Ile Pro Leu Val Ser Gly Pro Asp Ile

405 410 415

Pro Ile Asn Ile Thr Asp Gln Ala Pro Ser Leu Ile Pro Ile Val Pro

420 425 430

Gly Ser Pro Gln Tyr Thr Ile Ile Ala Asp Ala Gly Asp Phe Tyr Leu

435 440 445

His Pro Ser Tyr Tyr Met Leu Arg Lys Arg Arg Lys Arg Leu Pro Tyr

450 455 460

Phe Phe Ser Asp Val Ser Leu Ala Ala

465 470

3

568

Human papillomavirus type 18

3

Met Cys Leu Tyr Thr Arg Val Leu Ile Leu His Tyr His Leu Leu Pro

1 5 10 15

Leu Tyr Gly Pro Leu Tyr His Pro Arg Pro Leu Pro Leu His Ser Ile

20 25 30

Leu Val Tyr Met Val His Ile Ile Ile Cys Gly His Tyr Ile Ile Leu

35 40 45

Phe Leu Arg Asn Val Asn Val Phe Pro Ile Phe Leu Gln Met Ala Leu

50 55 60

Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro Ser Val Ala

65 70 75 80

Arg Val Val Asn Thr Asp Asp Tyr Val Thr Pro Thr Ser Ile Phe Tyr

85 90 95

His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro Tyr Phe Arg

100 105 110

Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys Val Ser Ala

115 120 125

Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro Asn Lys Phe

130 135 140

Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln Arg Leu Val

145 150 155 160

Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro Leu Gly Val

165 170 175

Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp Thr Glu Ser

180 185 190

Ser His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg Asp Asn Val

195 200 205

Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly Cys Ala Pro

210 215 220

Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys Ser Arg Pro

225 230 235 240

Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn Thr Val Leu

245 250 255

Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met Asp Phe Ser

260 265 270

Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile Cys Gln Ser

275 280 285

Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp Pro Tyr Gly

290 295 300

Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe Ala Arg His

305 310 315 320

Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro Gln Ser Leu

325 330 335

Tyr Ile Lys Gly Thr Gly Met Pro Ala Ser Pro Gly Ser Cys Val Tyr

340 345 350

Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser Asp Ser Gln Leu Phe

355 360 365

Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn Asn Gly Val

370 375 380

Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Pro Ser

385 390 395 400

Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Pro Val Pro Gly Gln

405 410 415

Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val Glu Glu Tyr

420 425 430

Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu Thr Ala Asp

435 440 445

Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu Glu Asp Trp

450 455 460

Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val Asp Thr Tyr

465 470 475 480

Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp Ala Ala Pro

485 490 495

Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp Asn Val Asp

500 505 510

Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro Leu Gly Arg

515 520 525

Lys Phe Leu Val Gln Ala Gly Leu Arg Arg Lys Pro Thr Ile Gly Pro

530 535 540

Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr Ser Ser Lys Pro Ala Lys

545 550 555 560

Arg Val Arg Val Arg Ala Arg Lys

565

4

462

Human papillomavirus type 18

4

Met Val Ser His Arg Ala Ala Arg Arg Lys Arg Ala Ser Val Thr Asp

1 5 10 15

Leu Tyr Lys Thr Cys Lys Gln Ser Gly Thr Cys Pro Pro Asp Val Val

20 25 30

Pro Lys Val Glu Gly Thr Thr Leu Ala Asp Lys Ile Leu Gln Trp Ser

35 40 45

Ser Leu Gly Ile Phe Leu Gly Gly Leu Gly Ile Gly Thr Gly Ser Gly

50 55 60

Thr Gly Gly Arg Thr Gly Tyr Ile Pro Leu Gly Gly Arg Ser Asn Thr

65 70 75 80

Val Val Asp Val Gly Pro Thr Arg Pro Pro Val Val Ile Glu Pro Val

85 90 95

Gly Pro Thr Asp Pro Ser Ile Val Thr Leu Ile Glu Asp Ser Ser Val

100 105 110

Val Thr Ser Gly Ala Pro Arg Pro Thr Phe Thr Gly Thr Ser Gly Phe

115 120 125

Asp Ile Thr Ser Ala Gly Thr Thr Thr Pro Ala Val Leu Asp Ile Thr

130 135 140

Pro Ser Ser Thr Ser Val Ser Ile Ser Thr Thr Asn Phe Thr Asn Pro

145 150 155 160

Ala Phe Ser Asp Pro Ser Ile Ile Glu Val Pro Gln Thr Gly Glu Val

165 170 175

Ala Gly Asn Val Phe Val Gly Thr Pro Thr Ser Gly Thr His Gly Tyr

180 185 190

Glu Glu Ile Pro Leu Gln Thr Phe Ala Ser Ser Gly Thr Gly Glu Glu

195 200 205

Pro Ile Ser Ser Thr Pro Leu Pro Thr Val Arg Arg Val Ala Gly Pro

210 215 220

Arg Leu Tyr Ser Arg Ala Tyr Gln Gln Val Ser Val Ala Asn Pro Glu

225 230 235 240

Phe Leu Thr Arg Pro Ser Ser Leu Ile Thr Tyr Asp Asn Pro Ala Phe

245 250 255

Glu Pro Val Asp Thr Thr Leu Thr Phe Asp Pro Arg Ser Asp Val Pro

260 265 270

Asp Ser Asp Phe Met Asp Ile Ile Arg Leu His Arg Pro Ala Leu Thr

275 280 285

Ser Arg Arg Gly Thr Val Arg Phe Ser Arg Leu Gly Gln Arg Ala Thr

290 295 300

Met Phe Thr Arg Ser Gly Thr Gln Ile Gly Ala Arg Val His Phe Tyr

305 310 315 320

His Asp Ile Ser Pro Ile Ala Pro Ser Pro Glu Tyr Ile Glu Leu Gln

325 330 335

Pro Leu Val Ser Ala Thr Glu Asp Asn Asp Leu Phe Asp Ile Tyr Ala

340 345 350

Asp Asp Met Asp Pro Ala Val Pro Val Pro Ser Arg Ser Thr Thr Ser

355 360 365

Phe Ala Phe Phe Lys Tyr Ser Pro Thr Ile Ser Ser Ala Ser Ser Tyr

370 375 380

Ser Asn Val Thr Val Pro Leu Thr Ser Ser Trp Asp Val Pro Val Tyr

385 390 395 400

Thr Gly Pro Asp Ile Thr Leu Pro Ser Thr Thr Ser Val Trp Pro Ile

405 410 415

Val Ser Pro Thr Ala Pro Ala Ser Thr Gln Tyr Ile Gly Ile His Gly

420 425 430

Thr His Tyr Tyr Leu Trp Pro Leu Tyr Tyr Phe Ile Pro Lys Lys Arg

435 440 445

Lys Arg Val Pro Tyr Phe Phe Ala Asp Gly Phe Val Ala Ala

450 455 460