AU683220C - Self-assembling recombinant papillomavirus capsid proteins - Google Patents

Description

SELF-ASSEMBLING RECOMBINANT PAPILLOMA VIRUS CAPSID PROTEINS

Field of the Invention This invention relates to recombinant viral proteins. It relates particularly to recombinant viral proteins that are suitable for use in the diagnosis, prophylaxis and therapy of viral infections.

Background of the Invention

Papillomaviruses infect the epithelia of a wide variety of species of animals, including humans, generally inducing benign epithelial and fibro-epithelial tumors, or warts, at the site of infection. Each species of vertebrate is infected by a distinct group of papillomaviruses, each papiUomavirus group comprising several papiUomavirus types. For example, more than

60 different human papiUomavirus (HPV) genotypes have been isolated. Papillomaviruses are highly species specific infective agents; for example, a bovine papiUomavirus cannot induce papiUomas in a heterologous species, such as humans. PapiUomavirus types ALSO appear to be highly specific as immunogens in that a neutralizing immunity to infection against one papiUomavirus type does not usuaUy confer immunity against another type, even when the types infect an homologous species.

In humans, genital warts, which are caused by human papillomaviruses, represent a sexuaUy transmitted disease. Genital warts are very common, and subcUnical, or inapparent

HPV infection is even more common than clinical infection. Some benign lesions in humans, particularly those arising from certain papiUomavirus types, undergo malignant progression. For that reason, infection by one of the malignancy associated papiUoma virus types is considered one of the most significant risk factors in the development of cervical cancer, the second most common cancer of women worldwide (zur Hausen, H., 1991; Schiffman, M.

1992). Several different HPV genotypes have been found in cervical cancer, with HPV16 being the most common type that is isolated from 50% of cervical cancers.

Immunological studies demonstrating the production of neutralizing antibodies to papiUomavirus antigens indicate that papiUomavirus infections and malignancies associated with these infections in vertebrate animals could be prevented through immunization; however the development of effective papiUomavirus vaccines has been impeded by a number of difficulties.

First, it has not been possible to generate in vitro the large stocks of infectious virus required to determine the structural and immunogenic features of papiUomavirus that are fundamental to the development of effective vaccines. Cultured cells express papiUomavirus oncoproteins and other non-structural proteins and these have been extensively studied in vitro; but expression of the structural viral proteins, LI and L2 (and the subsequent assembly of infectious virus) occurs only in terminally differentiated layers of infected epithelial tissues. Therefore, the characterization of viral genes, proteins, and structure has necessarUy been assembled from studies of virus harvested from papiUomas. In particular, papiUomavirus structure and related immunity have been carried out in the bovine papiUomavirus system because large amounts of infectious virus particles can be isolated from bovine papUlomavirus (BPV) warts.

The information derived from studies of papUlomavirus structure to date indicates that aU papiUomaviruses are non-enveloped 50-60 nm icosahedral structures (Crawford, L., et al., 1963) which are comprised of conserved LI major capsid protein and less weU conserved L2 minor capsid protein (Baker, C, 1987). There is no sequence relationship between the two proteins. The function and location of L2 in the capsid is unclear; however immunologic data suggests that most of L2 is internal to LI. Recently, high resolution cryoelectron microscopic analysis of BPV1 and HPV1 virions has determined that the two viruses have a very simUar structure, with 72 pentameric capsomers, each capsomer presumably composed of five LI molecules, forming a virion sheU with T=7 symmetry (Baker, T., 1991). The location of the minor L2 capsid protein in the virion has not been determined, and it is not certain whether L2 or other viral proteins are needed for capsid assembly. SuperficiaUy, papUlomavirus structure resembles that of the polyoma 45 nm virion, which has the same symmetry and capsomere number (Liddington, R., et al., 1991); however, the systems of intracapsomer contact for polyomavirus and papiUomavirus species are different, and the major and minor capsid proteins of polyomavirus are not geneticaUy related to LI and L2. Bovine papiUomavirus studies are facUitated by a quantitative focal transformation infectivity assay developed for BPV that is not avaUable for HPV (Dvoretzky, I., et al, 1980), and an understanding of immunity to papUlomavirus has therefore also been derived from the bovine papUlomavirus system. Limited studies using intact bovine papUlomavirus demonstrated that the non-cutaneous inoculation of infectious or formalin-inactivated BPV virus was effective as a vaccine to prevent experimental BPV infection in calves (Olson. C, et al., 1960; Jarrett, W., et al., 1990). Unfortunately, BPV virions cannot be used to develop vaccines against papUlomavirus which infects other species, or even vaccines against other bovine types, because of the great specificity of these viruses, as weU as concern for the oncogenic potential of intact viral particles. A significant conclusion of studies of papUlomavirus immunity is that the abilitv of antibodies to neutralize papiUoma virus appears to be related to their ability to react with type-specific, conformationaUy dependent epitopes on the virion surface. For example. rabbit antisera raised against infectious BPVl virions inhibits focal transformation of C127 ceUs (Doretzky, I., et al., 1980), as weU as the transformation of fetal bovine skin grafts; whereas antisera raised against denatured virions does not (Ghim, S., et al., 1991).

In contrast, neutralizing sera generated against bacteriaUy derived BPV LI and L2

(PUacinski, W. et al., 1984; Jin, X., et al., 1989) and against in vitro synthesized cottontaU rabbit papUlomavirus (CRPV) LI and L2 (Christensen, N., et al., 1991; Lin, Y-L, et al., 1992), neither of which has the structural features of native virions, had low titers, and the use of recombinant HPV LI fusion peptides expressed in E. coli to detect ceUular immune reactivity has had only limited success (Hδpfl, R. et al, 1991). The results in the BPV system are consistent with those of the HPV system, in which monoclonal antibodies that neutralized HPV11 infection in a mouse xenograft assay recognized native, but not denatured, HPV11 virions (Christensen, N., et al., 1990).

There have been isolated attempts to produce papiUomavirus capsids in vitro. Zhou. J. et al. (1991) and (1992) produced virus-like particles by cloning HPV LI and L2 genes. and HPV LI and L2 genes in combination with HPV E3/E4 genes into a vaccinia virus vector and infecting CV-1 mammalian ceUs with the recombinant vaccinia virus. These studies were interpreted by Zhou to establish that expression of HPV16 LI and L2 proteins in epithelial ceUs is necessary and sufficient to aUow assembly of virion type particles. CeUs infected with doubly recombinant vaccinia virus which expressed LI and L2 proteins showed smaU (40 nm) virus-like particles in the nucleus that appeared to be incompletely assembled arrays of HPV capsomers. Expressing LI protein alone, or L2 protein alone, was expressed did not produce virus-like particles; ceUs doubly infected with singly recombinant vaccinia virus containing LI and L2 genes also did not produce particles. No neutralizing activity was reported.

Ghim et al., (1992) reported that when LI from HPV1, a non-genital virus type associated mainly with warts on the hands and feet, was expressed in mammalian ceUs, the LI protein contained conformational epitopes found on intact virions. Ghim did not determine if particles were produced, nor was it evaluated if the LI protein might induce neutralizing antibodies. Even more recently, Hagansee. et al. ( 1993) reported that when LI from HPV1 was expressed in human ceUs, it self-assembled into virus-like particles. No neutralizing antibody studies were performed. Studies in other virus systems, for example, parvovirus, indicate that capsid assembly alone may not confer immunogenicity. Parvovirus VP2, by itself, was able to self-assemble when expressed in insect ceUs, but only particles containing both VPl and VP2 were able to induce neutralizing antibodies (Kajigaya, S., et al., 1991). It would be advantageous to develop methods for producing renewable papiUomavirus reagents of any selected species and type in ceU culture. It would also be beneficial to produce such papUlomavirus reagents having the immunity conferring properties of the conformed native virus particles that could be used as a subunit vaccine.

It is therefore the object of the invention to provide these recombinant conformed papiUomavirus proteins, as weU as methods for their production and use.

Summary of the Invention The invention is directed to the diagnosis and prevention of papiUomavirus infections and their benign and malignant sequelae by providing recombinant papiUomavirus capsid proteins that self assemble to form capsomer structures comprising conformational epitopes that are highly specific and highly immunogenic. Therefore, according to the invention there is provided a genetic construct, comprising a papUlomavirus LI conformational coding sequence, inserted into a baculovirus transfer vector, and operatively expressed by a promoter of that vector. The papiUomavirus LI conformational coding sequence can be isolated from a bovine, monkey, or human gene. In a preferred embodiment, the papiUomavirus LI conformational coding sequence is isolated from a wUd type HPV 16 gene.

In a particularly preferred embodiment, the papUlomavirus Ll conformational coding sequence is SEQ ID NO:6. The genetic construct can further comprise a papiUomavirus L2 coding sequence.

According to another aspect of the invention there is provided a non-mammalian eukaryotic host ceU transformed by the genetic constructs of the invention.

According to yet another aspect of the invention there is provided a method for producing a recombinant papUlomavirus capsid protein, assembled into a capsomer structure or a portion thereof, comprising the steps of (1) cloning a papUlomavirus gene that codes for an Ll conformational capsid protein into a transfer vector wherein the open reading frame of said gene is under the control of the promoter of said vector; (2) transferring the recombinant vector into a host ceU, wherein the cloned papUlomavirus gene expresses the papUlomavirus capsid protein; and (3) isolating capsomer structures, comprising the papiUomavirus capsid protein, from the host ceU. In a preferred embodiment, the cloned papiUomavirus gene consists essentially of the conformational Ll coding sequence, and the expressed protein assembles into capsomer structures consisting essentiaUy of Ll capsid protein. In another preferred embodiment, the cloning step of the method further comprises the cloning of a papUlomavirus gene coding for L2 capsid protein, whereby said Ll and L2 proteins are coexpressed in the host ceU, and wherein the isolated capsomer structures comprise Ll and

L2 capsid proteins; provided that said transfer vector is not a vaccinia virus when said host ceU is a mammalian ceU. The conformational Ll coding sequence can be cloned from a bovine, monkey, or human papiUomavirus. According to a preferred embodiment, the conformational Ll coding sequence is cloned from a wUd type HPV16 papiUomavirus. In a particularly preferred embodiment, the conformational Ll coding sequence is SEQ ID NO:6. Also in a preferred embodiment, the host ceU into which the genetic construct is transfected is an insect ceU. Also preferred are embodiments wherein the transfer vector is a baculovirus based transfer vector, and the papUlomavirus gene is under the control of a promoter that is active in insect ceUs. Accordingly in this embodiment, the recombinant baculovirus DNA is transfected into Sf-9 insect ceUs, preferably co-transfected with wUd-type baculovirus DNA into Sf-9 insect ceUs.

In an alternative embodiment of the method of the invention, the transfer vector is a yeast transfer vector, and the recombinant vector is transfected into yeast ceUs. According to yet another aspect of the invention there is provided a virus capsomer structure, or a portion thereof, consisting essentiaUy of papUlomavirus Ll capsid protein, produced by the method the invention. Alternatively, the virus capsomer structure can consist essentiaUy of papUlomavirus Ll and L2 capsid proteins, produced by the method of the invention. In a particularly preferred embodiment, the virus capsomer structure comprises papUlomavirus Ll capsid protein that is the expression product of an HPV16 Ll

DNA cloned from a wUd type virus.

The virus capsids or capsomer structures of the invention, or portions or fragments thereof. can consist essentiaUy of papUlomavirus Ll capsid protein. Alternatively, these capsids or capsomer structures or their fragments can consist essentially of wild type HPV 16 papiUomavirus Ll capsid protein.

The virus capsid structures according to any of the methods of the invention comprise capsid proteins having immunogenic conformational epitopes capable of inducing neutralizing antibodies against native papUlomavirus. The capsid proteins can be bovine, monkey or human papiUomavirus Ll proteins. In a preferred embodiment, the papUlomavirus Ll capsid protein is the expression product of a wild type HPV 16 L l gene.

In a particularly preferred embodiment, the HPV16 Ll gene comprises the sequence of SEQ

ID NO:6.

According to yet another aspect of the invention there is provided a unit dose of a vaccine, comprising a peptide having conformational epitopes of a papiUomavirus Ll capsid protein, or Ll protein and L2 capsid proteins, in an effective immunogenic concentration sufficient to induce a papUlomavirus neutralizing antibody titer of at least about 10³ when administered according to an immunizing dosage schedule. In a preferred embodiment, the vaccine comprises an Ll capsid protein which is an HPV16 capsid protein. In a particularly preferred embodiment, the vaccine comprises an Ll capsid protein that is a wUd type

HPV16 Ll protein.

Use of the Ll open reading frame (ORF) from a wUd type HPV16 papUlomavirus genome, according to the methods of the invention, particularly facUitates the production of preparative amounts of virus-like particles on a scale suitable for vaccine use. According to yet another aspect of the invention, there is provided a method of preventing or treating papiUomavirus infection in a vertebrate, comprising the administration of a papUlomavirus capsomer structure or a fragment thereof according to the invention to a vertebrate, according to an immunity-producing regimen. In a preferred embodiment, the papiUomavirus capsomer structure comprises wUd type HPV16 Ll capsid protein. The invention further provides a method of preventing or treating papUlomavirus infection in a vertebrate, comprising the administration of the papUlomavirus capsomer structure of the invention, or a vaccine product comprising the capsomer structure to a vertebrate, according to an immunity-producing regimen. In a preferred embodiment, the papUlomavirus vaccine comprises wUd type HPV 16 Ll capsid protein. Also within the scope of the invention is a method for immunizing a vertebrate against papiUomavirus infection, comprising administering to the vertebrate a recombinant genetic construct of the invention comprising a conformational pap lomavirus Ll coding sequence, and aUowing said coding sequence to be expressed in the ceUs or tissues of said vertebrate, whereby an effective, neutralizing, immune response to papUlomavirus is induced. In a preferred embodiment, the conformational papiUomavirus Ll coding sequence is derived from human papUlomavirus HPV 16. In a particularly preferred embodiment, the human papUlomavirus HPV16 is a wild type papUlomavirus.

According to yet another aspect of the invention, there is provided a method of detecting humoral immunity to papiUomavirus infection in a vertebrate comprising the steps of: (a) providing an effective antibody-detecting amount of a papUlomavirus capsid peptide having at least one conformational epitope of a papUlomavirus capsomer structure;

(b) contacting the peptide of step (a) with a sample of bodUy fluid from a vertebrate to be examined for papUlomavirus infection, and aUowing papUlomavirus antibodies contained in said sample to bind thereto, forming antigen-antibody complexes; (c) separating said complexes from unbound substances; (d) contacting the complexes of step (c) with a detectably labeUed immunoglobulin-binding agent; and (e) detecting anti-papiUomavirus antibodies in said sample by means of the labeUed immunoglobulin-binding agent that binds to said complexes. In a preferred embodiment of this aspect of the invention, the peptide consists essentiaUy of papUlomavirus Ll capsid protein. According to an alternative embodiment, the peptide consists essentiaUy of the expression product of a human papUlomavirus HPV 16. In a particularly preferred embodiment, the peptide consists essentiaUy of the expression product of a wUd type human papUlomavirus HPV16 gene, for example, the peptide can consist essentiaUy of the expression product of SEQ ID NO:6.

According to yet another aspect of the invention, there is provided a method of detecting papUlomavirus in a specimen from an animal suspected of being infected with said virus, comprising contacting the specimen with antibodies having a specificity to one or more conformational epitopes of the capsid of said papUlomavirus, wherein the antibodies have a detectable signal producing label, or are attached to a detectably labeUed reagent; aUowing the antibodies to bind to the papUlomavirus; and determining the presence of papUlomavirus present in the specimen by means of the detectable label.

According to yet another aspect of the invention, there is provided a method of determining a ceUular immune response to papUlomavirus in an animal suspected of being infected with the virus, comprising contacting immunocompetent ceUs of said animal with a recombinant wUd type papiUomavirus Ll capsid protein, or combined recombinant Ll and L2 capsid proteins according to the invention; and assessing cellular immunity to papUlomavirus by means of the proliferative response of said ceUs to the capsid protein. In a preferred embodiment of this aspect of the invention, the recombinant papUlomavirus protein is introduced into the skin of the animal.

According to yet another aspect of the invention there is provided a papUlomavirus infection diagnostic kit, comprising capsomer structures consisting essentially of papUlomavirus Ll capsid protein, or capsomer structures comprising papUlomavirus Ll protein and L2 capsid proteins, or antibodies to either of these capsomer structures, singly or in combination, together with materials for carrying out an assay for humoral or cellular immunity against papiUomavirus, in a unit package container.

DetaUed Description of the Invention

We have discovered that the gene coding for the Ll major capsid protein of BPV or HPV16, foUowing introduction into host ceUs by means of an appropriate transfer vector, can express Ll at high levels, and that the recombinant Ll has the intrinsic capacity to self-assemble into empty capsomer structures that closely resemble those of an intact virion.

Further, the self-assembled recombinant Ll capsid protein of the invention, in contrast to Ll protein extracted from recombinant bacteria, or denatured virions, has the efficacy of intact papiUomavirus particles in the abUity to induce high levels of neutralizing antiserum that can protect against papUlomavirus infection. The high level of immunogenicity of the capsid proteins of the invention implies strong antibody binding properties that make them sensitive agents in serological screening tests to detect and measure antibodies to conformational virion epitopes. Their immunogenicity also indicates that the capsid proteins of the invention can also be used as highly effective vaccines or immunogens to elicit neutralizing antibodies to protect a host animal against infection by papiUomavirus. These observations were recently pubUshed in Kirnbauer, et al., (1992), and formed the basis of U.S. application Serial No. 07/941,371.

We have now discovered that the capsid protein Ll expressed by wUd type HPV16 genomes isolated from benign papiUomavirus lesions, when expressed in the baculovirus system described, wUl self-assemble with an efficiency heretofore unknown and comparable to that of bovine papiUovirus Ll capsid protein.

The HPV 16 Ll Gene Sequence

The source of HPV16 Ll DNA, as disclosed in published studies, for example, by Zhou, et al.(1991) was the prototype clone, GenBank Accession No. K02718, that had been isolated from a cervical carcinoma (Seedorf, et al., 1985). We have found that Ll from wUd type HPV16 genome, which differs from the prototype genome by a single point mutation, wUl self-assemble into virus-like particles with an efficiency simUar to that seen with BPV

Ll or BPV L1/L2. Compared with the self-assembly seen when Ll from the prototype HPV genome is used with L2, Ll from a wUd-type genome self-assembles at least 100 times more efficiently.

To provide genetic insight into the self-assembly efficiency of different HPV 16 Ll expression products, the open reading frames from HPV 16 Ll genes isolated from both benign lesions and lesions associated with dysplasia or carcinoma were sequenced. The analysis detected two errors in the published sequence of the published Ll sequence of the prototype strain, as foUows:

(1) there should be an insertion of three nucleotides (ATC) between nt 6902 and 6903, which results in the insertion of a serine in the Ll protein; and (2) there should be a deletion in the published prototype sequence of three nucleotides (GAT), consisting of nt 6952-6954, which deletes an aspartate from the Ll protein sequence. The corrected nucleotide sequence of the prototype HPV16 Ll genome, consisting of nt 5637-7155, is that of SEQ ID NO:5, listed herein. The numbering of the nucleotide bases in Sequence ID Nos. 5 and 6 is indexed to

1, and the numbering of nucleotide bases of the published HPV sequence, that is from nt

5638-7156, corresponds to those of the sequence listing from 1-1518. The sites referred to in the original sequence can be thus readUy identified by one skUled in the art.

Three other HPV16 Ll genomes, clone 16PAT; and clones 114/16/2 and 114/16/11, were sequenced and those sequences compared to that of the corrected prototype.

Clone 16PAT, kindly provided by Dennis McCance at the University of Rochester

School of Medicine, and cloned from a dysplastic (pre-malignant) lesion of the cervix, expresses an Ll that does not self-assemble efficiently.

Clones 114/16/2 and 114/16/11, kindly provided by Matthias Durst of the German Cancer Research Center in Heidelburg, were both cloned from non-malignant lesions, and both expressed Ll protein that self-assembled efficiently.

Comparison of Genetic Characteristics of HPV16 Ll associated with Dysplasia, Malignant Progression and Benign Lesions Clone 16PAT, isolated from papUlomavirus infected dysplastic lesions and the prototype HPV16, isolated from malignant cervical carcinoma, both encode Histidine at nt 6242-6244, whUe clones 2 and 11, isolated from benign papiUomavirus infected lesions (like isolates of many other papUlomavirus) encode Aspartate at this site.

It appears that this single amino acid difference between the prototype, malignancy- associated HPV16 species, and the HPV 16 species from benign lesions accounts for the difference in self-assembly efficiency. It is likely that among closely related HPV types, Aspartate at this locus may be necessary for efficient self-assembly, and that the substitution of Histidine for Aspartate impairs this ability in the capsid protein. The impairment in capsid assembly in malignancy-associated viruses, associated with loss of the conformational epitopes required for the production of neutralizing antibodies, may also be linked to a lowered immunogenicity which would aUow the papUlomavirus to escape immune control.

Accordingly, HPV 16 Ll genes that express capsid protein that self-assembles efficiently can be obtained by (1) isolation of the wUd type HPV16 Ll open reading frame from benign lesions of papUlomavirus infection; or

(2) carrying out a site specific mutation in the prototype sequence at nt 6242-6244 to encode

Aspartate.

Recombinant Capsid Protein The method of the invention provides a means of preparing recombinant capsid particles for any papiUomavirus. Particles consisting of either Ll or L2 capsid protein alone, or consisting of both Ll and L2 capsid proteins together can be prepared. L1/L2 capsid protein particles are more closely related to the composition of native papUlomavirus virions, but L2 does not appear to be as significant as Ll in conferring immunity, probably because most of L2 is internal to Ll in the capsid structure. Although Ll can self-assemble by itself, in the absence of L2, self-assembled L1/L2 capsid protein particles are more closely related to the composition of native papUlomavirus virions. Accordingly, particles comprising Ll alone are simpler, whUe those comprising L1/L2 may have an even more authentic structure. Both self-assembled Ll and L1/L2 particles induce high-titer neutralizing antibodies and may therefore be suitable for vaccine production. Particles comprising Ll capsid protein expressed by a wUd type HPV genome, either as Ll alone or L1/L2 together, are particularly preferred.

Production of the recombinant Ll, or combined L1/L2, capsid particles is carried out by cloning the Ll (or Ll and L2) gene(s) into a suitable vector and expressing the corresponding conformational coding sequences for these proteins in a eukaryotic ceU transformed by the vector. It is believed that the abUity to form a capsid-like structure is intimately related to the abUity of the capsid protein to generate high-titer neutralizing antibody, and that in order to produce a capsid protein that is capable of self-assembling into capsid structures having conformational epitopes, substantiaUy aU of the capsid protein coding sequence must be expressed. Accordingly, substantiaUy aU of the capsid protein coding sequence is cloned. The gene is preferably expressed in a eukaryotic ceU system. Insect ceUs are preferred host ceUs; however, yeast cells are also suitable as host cells if appropriate yeast expression vectors are used. Mammalian cells simUarly transfected using appropriate mammalian expression vectors can also be used to produce assembled capsid protein, however, cultured mammalian cells are less advantageous because they are more likely than non-mammalian ceUs to harbor occult viruses which might be infectious for mammals.

According to a preferred protocol, a baculovirus system is used. The gene to be cloned, substantiaUy aU of the coding sequence for bovine papUlomavirus (BPVl) or human papUlomavirus (HPV16) Ll capsid protein, or human papUlomavirus HPV 16 Ll and L2, is inserted into a baculovirus transfer vector containing flanking baculovirus sequences to form a gene construct, and the recombinant DNA is co-transfected with wUd type baculovirus

DNA into Sf-9 insect ceUs as described in Example 1, to generate recombinant virus which, on infection, can express the inserted gene at high levels. The actual production of protein is made by infecting fresh insect ceUs with the recombinant baculovirus; accordingly, the Ll capsid protein and the Ll and L2 capsid proteins are expressed in insect ceUs that have been infected with recombinant baculovirus as described in Example 2.

In the procedure of Example 1, the complete Ll gene of BPVl was amplified by polymerase chain reaction (PCR; Saiki, R., et al., 1987) and cloned into AcMNPV

(Autographa califomica nuclear polyhedrosis virus) based baculovirus vector (Summers, M. et al., 1987). The Ll open reading frame was put under the control of the baculovirus polyhedrin promoter. After co-transfection of the Ll clone with the wUd type (wt) baculovirus DNA into Sf-9 insect ceUs (ATCC Accession No. CRL 1711) and plaque purification of recombinant clones, high titer recombinant virus was generated. Extracts from ceUs infected with wt AcMNPV or BPVl Ll recombinant viruses (AcBPV-Ll) (Example 2) were analyzed by polyacrylamide gel electrophoresis. After Coomassie blue staining, a unique protein of the predicted size, 55 kilodaltons, was detected in extracts from the cultures infected with the AcBPVl-Ll virus. The identity of this protein as BPV Ll was verified by immunoblotting, using a BPV Ll specific monoclonal antibody (Nakai, Y., et al.,

1986). Thus, the expression of BPV Ll by means of recombinant virus were demonstrated by SDS-PAGE analysis of lysates from infected insect cells.

To test the hypothesis that papiUomavirus Ll has the ability to self-assemble into virus-like particles when overexpressed in et TcΛoepus ceλls,, sections from AcBPV-Ll infected ceUs were examined for the presence of papUlomavirus- like structures. CeUs infected with the BPV recombinant virus contained many circular structures of approximately 50 nm which were preferentiaUy localized in the nucleus; these structures were absent from wUd type baculovirus infected cells. These results suggested that self assembly of Ll into virus-like particles had occurred, since in vivo papiUomavirus virion assembly takes place in the nucleus and the diameter of the virions has been reported as 55 nm.

FoUowing expression of the conformed capsid protein in the host ceU, virus particles are purified from lysates of infected ceUs as described in Example 4. To obtain further evidence that the Ll protein had self-assembled, virus-like particles were isolated from the infected insect ceUs by means of gradient centrifugation. We demonstrated the conformation of purified recombinant BPV Ll and HPV16 Ll capsid proteins by electron microscopy, compared with authentic BPV virions.

High molecular mass structures were separated from lysates of Ll recombinant or wUd type infected ceUs by centrifugation through a 40% sucrose cushion and the peUeted material was subjected to CsCl density gradient centrifugation. Fractions were coUected and tested for reactivity to the BPV Ll specific monoclonal antibody by immunoblotting.

Ll positive fractions from the gradient were adsorbed onto carbon fUm grids, stained with 1% uranyl acetate and examined by transmission electron microscopy. In electron microscopy, the positive fractions contained numerous circular structures that exhibited a regular array of capsomers. Consistent with previous reports of the density of empty BPV virions (Larsen, P., et al., 1987), the density of the CsCl fraction containing the peak of the virus-like particles was approximately 1.30 gm/ml. Most were approximately 50 nm in diameter, although smaUer circles and partiaUy assembled structures were also seen. In electron microscopy, the larger particles were very simUar in size and subunit structure to infectious BPV virions that had been stained and photographed concurrently. These particles were not observed in preparations from mock infected or wt AcMNPV infected ceUs. These results indicate that BPV Ll has the intrinsic capacity to assemble into virus¬ like particles in the absence of L2 or other papUlomavirus proteins. In addition, specific factors limited to differentiating epithelia or mammalian ceUs are not required for papUlomavirus capsid assembly.

To determine if the abUity to self-assemble in insect ceUs is a general feature of papUlomavirus Ll, we also expressed the Ll of HPV16, the HPV type most often detected in human genital cancers, via an analogous recombinant baculovirus. A protein of the expected 58 kd size was expressed at high levels in the insect ceUs infected with the HPV16-

Ll recombinant virus, as demonstrated by SDS-PAGE. This protein reacted strongly with an HPV16 Ll monoclonal antibody upon immunoblotting. The monoclonal antibody also lightly stained five other bands ranging in apparent molecular weight from approximately 28 kd to approximately 48 kd. The antibody also reacted weakly with BPV Ll, thus this antibody lightly stained the 55 kd protein of BPV Ll on the same immunoblot. After CsCl gradient purification, immunoreactive fractions were examined by electron microscopy and found to contain 50 nm papiUomavirus-like particles upon electron microscopy. Although somewhat fewer completely assembled particles were seen in the human system in comparison to the BPV Ll preparations, possibly due to the lower levels of expression or greater extent of HPV16 Ll degradation seen in SDS-PAGE, the results conclusively indicate that the Ll of the HPV16 and presumably the Ll proteins of other types, have the intrinsic capacity to assemble into virion-type structures. Preparations of recombinant papiUomavirus capsid particles for Rhesus monkey PV have also been carried out as described in the Examples.

Recombinant Conformed Capsid Proteins as Immunogens

Subunit vaccines, based on self-assembled major capsid proteins synthesized in heterologous ceUs, have been proved effective in preventing infections by several pathogenic viruses, including human hepatitis B (Stevens, C, et al., 1987). Studies demonstrating that infectious or formalin inactivated BPV is effective as a vaccine, whUe BPV transformed ceUs are ineffective, suggest that viral capsid proteins, rather than early gene products, elicit the immune response. Other data in the scientific literature indicates that Ll protein extracted from bacteria was partiaUy successful in eliciting an immune response despite the low titers of neutralizing antibodies. Accordingly, the BPV Ll that was expressed and assembled into virus-like particles in insect ceUs was studied for its abUity to induce neutralizing antisera in rabbits. Two types of preparations were tested: whole ceU extracts of Ll recombinant or wUd type infected Sf-9 ceUs and partiaUy purified particles isolated by differential centrifugation and ammonium sulfate precipitation. FoUowing a primary inoculation, the rabbits received two biweekly booster inoculations.

The rabbit sera were tested for the abUity to inhibit BPV infection of mouse C127 ceUs, as measured by a reduction in the number of foci induced by a standard amount of BPV virus. A representative assay was conducted in which the titers of neutralizing antisera induced in animals inoculated with recombinant BPV Ll was compared to antisera against intact and denatured BPV virions. The immune sera generated by inoculation with baculovirus derived Ll were able to reduce the infectivity of the BPV virus by 50% at a dilution of at least 1:11,000 (a titer of 11,000; Table 1 ), whereas the preimmune sera from the same rabbits did not inhibit focal transformation at a dUution of 1:20, the lowest dUution tested. Both the crude preparations and partially purified particles were effective in inducing high titer neutralizing antisera, with 290,000 being the highest titer measured. This was the same as the neutralizing titer of the positive control antiserum raised against infectious BPV virions. In comparison, the highest titer generated in a previous study using bacteriaUy derived Ll was 36 (PUancinski, W., et al., 1984). The serum from the rabbit inoculated with the extract from the wUd type baculovirus infected ceUs was unable to inhibit infectivity at a dUution of 1:20, indicating that the neutralizing activity was Ll specific.

Disruption of the partiaUy purified Ll particles, by boUing in 1% SDS, abolished the abUity of the preparation to induce neutralizing antibodies (Table 1). The demonstration that Ll can self-assemble into virion-like particles that elicit neutralizing antisera titers at least three orders of magnitude higher than previous in vitro-produced antigens suggests the recombinant Ll capsid proteins has the potential to induce effective long term protection against naturaUy transmitted papUlomavirus. In view of these results, it appears that the Ll particles assembled in insect ceUs mimic infectious virus in the presentation of conformationaUy dependent immunodominant epitopes. These results also establish that L2 is not required for the generation of high titer neutralizing antibodies. The reported weak neutralizing immunogenicity of bacteriaUy derived Ll may occur because it does not assume an appropriate conformation or has not assembled into virion like structures. Also, multiple electrophoretic variants of Ll have been detected in virions (Larsen, P., et al., 1987). Some of these modified species, which are probably absent in the bacteriaUy derived Ll, may facUitate the generation of neutralizing antibodies.

The abUity of recombinant Ll (or L2) papiUomavirus capsid proteins such as those disclosed herein to induce high titer neutralizing antiserum makes them suitable for use as vaccines for prophylaxis against communicable papillomatosis. Examples of populations at risk that could benefit from immunization are bovine herds, which are susceptible to papiUoma warts; aU humans for non-genital types of HPV infection: and sexually active humans for genital HPV types of infection.

Therapeutic vaccination can be useful for productive papiUomavirus lesions, which usuaUy express Ll (and L2) capsid proteins. Such lesions are most likely to occur in benign infections, such as warts or laryngeal papiUomatosis. Laryngeal pap lomatosis in newborns is usuaUy contracted by the infant during passage through the birth canal where infectious papiUomavirus is present in vaginal secretions. Therapeutic vaccination of infected pregnant women against the papUlomavirus can induce neutralizing IgG antibody capable of passing through the placental barrier and into the circulation of the fetus to provide prophylactic passive immunity in the infant against this type of papiUomavirus infection. Additional infant-protecting mechanisms are provided by maternal IgA which is secreted into the vaginal fluid and into breast mUk. Jarrett (1991) demonstrates some therapeutic efficacy for L2 in treating BPV-induced warts. Malignant tumors typicaUy do not express Ll or L2, and the efficacy of vaccination with recombinant Ll or L2 in conditions such as cervical cancer, is uncertain.

Protective immunity against both benign and malignant papUlomavirus disease can be induced by administering an effective amount of recombinant Ll capsid protein to an individual at risk for papUlomavirus infection. A vaccine comprising the capsid protein can be directly administered, either parenteraUy or locaUy, according to conventional immunization protocols. In an alternative embodiment, the conformational coding sequence of Ll can be cloned into a transfer vector, for example, a semliki forest virus vector (which produces a mUd transient infection), the recombinant virus introduced into the ceUs or tissues of the recipient where the immunizing capsid protein is then expressed. Vaccinia virus can also be used as a vehicle for the gene. Recombinant Conformed Capsid Proteins as Serological Screening Agents

Published serologic studies of human immune response to papUlomavirus virion proteins have principaUy utUized bacteriaUy derived Ll and L2 capsid proteins, and the results have not correlated weU with other measures of HPV infection (Jenison, S., et al., 1990). BPV papiUomavirus immunity studies described above indicate that papUlomavirus virion proteins extracted from bacteria do not present the conformationaUy dependent epitopes that appear to be type-specific and recognized by most neutralizing antibodies. Compared with such assays that primarUy recognize linear epitopes, a serological test using self-assembled Ll particles is likely to be a more accurate measure of the extent of anti- HPV virion immunity in the human population. The recombinant Ll capsid proteins disclosed herein, presenting conformational epitopes, can therefore be used as highly specific diagnostic reagents to detect immunity conferring neutralizing antibody to papiUoma virus in binding assays of several types. The procedures can be carried out generaUy as either solid phase or solution assays that provide a means to detect antibodies in bodily fluids that specificaUy bind to the capsid protein in antigen-antibody pairs. Examples of procedures known to those skilled in the art for evaluating circulating antibodies are solution phase assays, such as double-antibody radioimmunoassays or enzyme immunoassays, or solid phase assays such as strip radioimmunoassay based on Western blotting or an enzyme-linked immunoabsorbent assay (ELISA) as disclosed in U.S. Patent No. 4,520,113 to Gallo et al., or immunochromatographic assays as disclosed in U.S. Patent No. 5.039,607 to Skold et al

A preferred ELISA method for the detection of antibodies is that disclosed in Harlow. E.. and Lane, D. in Antibodies: A Laboratory Manual Cold Spring Harbor, NY, 1988, pp. 563-

578. The recombinant Ll or L1/L2 capsid proteins disclosed herein can also be used to measure ceUular immunity to papUlomavirus by means of in vivo or in vitro assays, for example, antigen-induced T-ceU proliferative responses as described by Bradley, L., 1980, and particularly ceUular responses to viral antigens, as described in U.S. Patent No.

5,081,029 to Starling. CeUular immunity to papiUomavirus can also be determined by the classical in vivo delayed hypersensitivity skin test as described by Stites, D., 1980; or in a preferred method, according to Hopfl, R., et al., 1991, by the intradermal injection of recombinant HPV Ll fusion proteins.

The capsid proteins of the invention can also be used as immunogens to raise polyclonal or monoclonal antibodies, according to methods weU known in the art. These papiUomavirus-specific antibodies, particularly in combination with labeUed second antibodies, specific for a class or species of antibodies, can be used diagnosticaUy according to various conventional assay procedures, such as immunohistochemistry, to detect the presence of capsid proteins in samples of body tissue or bodUy fluids.

The genetic manipulations described below are disclosed in terms of their general application to the preparation of elements of the genetic regulatory unit of the invention.

OccasionaUy, the procedure may not be applicable as described to each recombinant molecule included within the disclosed scope. The situations for which this occurs wiU be readUy recognized by those skiUed in the art. In all such cases, either the operations can be successfuUy performed by conventional modifications known to those skUled in the art, e.g. by choice of an appropriate alternative restriction enzyme, by changing to alternative conventional reagents, or by routine modification of reaction conditions. Alternatively, other procedures disclosed herein or otherwise conventional wUl be applicable to the preparation of the corresponding recombinant molecules of the invention. In all preparative methods, aU starting materials are known or readUy preparable from known starting materials. In the foUowing examples, aU temperatures are set forth in degrees Celsius; unless otherwise indicated, aU parts and percentages are by weight.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utUize the invention to its fullest extent. The foUowing preferred embodiments are therefore to be construed as merely illustrative and not limiting the remainder of the disclosure in any way whatsoever.

EXAMPLE 1 FuU length Ll, or Ll and L2 open reading frames (ORF) were amplified by PCR using the cloned prototypes of BPVl DNA (Chen, E., et al., 1982), GenBank Accession No.

X02346 or HPV16 DNA (Seedorf, K., et al., 1985), GenBank Accession No. K02718; or wUd type HPV16 DNA SEQ ID NO:2) as templates. Unique restriction sites were incorporated into the oligonucleotide primers (underlined). BPV1-L1 primer sequence SEQ ID NO:3):

5^,-CCGCTGAATTCAATATGGCGTTGTGGCAACAAGGCCAGAAGCTGTAT-3^, (sense) and SEQ ID NO:4):

5'-GCGGTGGTACCGTGCAGTTGACTTACCTTCTGTTTTACATTTACAGA-3'

(antisense); HPV16-L1 primer sequence SEQ ID NO:5):

5'-CCGCTAGATCTAATATGTCTCTTTGGCTGCCTAGTGAGGCC-3' (sense); and

SEQ ID NO:6):

5'-GCGGTAG ^TClACACTAATTCAACATACATACAATACTTACAGC-3'(antisense).

Ll coding sequences begin at the 1st methionine codon (bold) for BPVl and the 2nd methionine for HPV16. BPV1-L1 was cloned as a 5'-EcoRI to 3'-KpnI fragment and

HPV16-L1 as a 5'-BglII to 3'-BglII fragment into the multiple cloning site downstream of the polyhedrin promoter of the AcMNPV based baculovirus transfer vector pEV mod

(Wang, X., et al. 1991) and verified by sequencing through the AcMNPV/Ll junction. A quantity of 2 μg of CsCl-purified recombinant plasmid was cotransfected with 1 μg wUd type AcMNPV DNA (Invitrogen, San Diego, California) into Sf-9 cells (ATCC) using lipofectin

(Gibco/BRL, Gaithersburg, Maryland) (Hartig, P., et al., 1991) and the recombinant baculoviruses plaque-purified as described (Summers, M., et al., 1987).

EXAMPLE 2 Expression of Ll Proteins or L1/L2 proteins in Insect Cells

Sf-9 ceUs were either mock infected (mock) or infected at a multiplicity of infection of 10 with either wt AcMNPV (wt) or AcBPV-Ll (B-Ll ), AcHPV16-Ll ( 16-Ll), or AcHPV16-Ll (16-Ll) and AcHPVl6-L2 (16-L2) recombinant virus. After 72 hours, ceUs were lysed by boUing in Laemmli buffer and the lysates subjected to SDS-PAGE in 10% gels. Proteins were either stained with 0.25% Coomassie blue or immunoblotted and probed with BPV Ll mAb AU-1 (Nakai, Y., et al., 1986), or HPV16L1 mAb CAMVIR-1 (McLean,

C, et al., 1990) and ¹²⁵I-labeled Fab anti-mouse IgG (Amersham). P designates polyhedrin protein. The anti BPV Ll mAb recognized the expected 55 kd protein. The anti-HPV16Ll mAb strongly stained the expected 58 kd protein, as weU as lightly staining five lower molecular weight bands, as discussed above. As also discussed above, this anti-HPV16Ll lightly cross-reacted with the BPV Ll protein.

EXAMPLE 3 Production of antisera

Rabbits were immunized by subcutaneous injection either with whole ceU Sf-9 lysates (3xl0⁷ ceUs) prepared by one freeze/thaw cycle and 20x dounce homogenization (rabbit #1,2, and 8) or with 200 μg of Ll protein partiaUy purified by differential centrifugation and 35% ammonium sulfate precipitation (#3,4,6, and 7), in complete Freund's adjuvant, and then boosted twice at two week intervals, using the same preparations in incomplete

Freund's adjuvant.

EXAMPLE 4

Purification of Particles and Transmission Electron Microscopic (EMK) Analysis

500 ml of Sf-9 ceUs (2xl0⁶/ml) were infected with AcBPV-Ll or AcHPV 16-Ll or AcHPV16-Ll/L2 (16-L1/L2) recombinant baculoviruses. After 72 hr, the harvested ceUs were sonicated in PBS for 60 sec. After low speed clarification, the lysates were subjected to centrifugation at 110,000g for 2.5 hr through a 40% (wt/vol) sucrose/PBS cushion

(SW-28). The resuspended peUets were centrifuged to equUibrium at 141,000g for 20 hr at room temperature in a 10-40% (wt/wt) CsCl /PBS gradient. Fractions were harvested from the bottom and analyzed by SDS-PAGE. Immunoreactive fractions were dialyzed against PBS, concentrated by Centricon 30 (MUlipore) ultrafUtration, and (for HPV16-L1) peUeted by centrifugation for 10 min at 30 psi in a A-100 rotor in an airfuge (Beckman). BPVl virions (Fig. 2B) were purified from a bovine wart (generously provided by A.B. Jenson) as described (Cowsert, L., et al., 1987). Purified particles were adsorbed to carbon coated TEM grids, stained with 1% uranyl acetate and examined with a PhUips electron microscope EM 400T at 36,000x magnification. Results were obtained by electron microscopy, and are discussed above. EXAMPLE 5

BPVl neutralization assay

Serial dUutions of sera obtained 3 wk after the second boost were incubated with approximately 500 focus forming units of BPVl virus for 30 min, the virus absorbed to C127 ceUs for 1 hr and the ceUs cultured for 3 weeks (Dvoretzky, I., et al, 1980). The foci were stained with 0.5% methylene blue/0.25% carbol fuchsin/methanol. The results were obtained by evaluating the number of foci; these results are discussed below. Anti-AcBPV-Ll was obtained from rabbit #1 and anti-wt AcMNPV from rabbit #8 (Table 1). Preimmune sera at 1:400 dUution was used as a standard. Anti-AcBPV-Ll at either 1:400 or 1:600 dUution substantiaUy eliminated foci, whereas anti-wt AcMNPV at either

1:400 or 1:600 dUution appeared to produce an increase in the number of foci. The normal rabbit serum negative control designated "nrs" at 1:00 dUution was used as a standard for the anti-BPV-1 virion, which appeared to substantiaUy eliminate foci at either 1:400 or 1:600 dUution. The anti-BPV-1 virion was raised against native BPV virions in a previous study (Nakai, Y., et al., 1986). FinaUy, Dako is the commerciaUy avaUable (Dako Corp., Santa

Barbara, CaUfornia) rabbit antiserum raised against denatured BPV virions. This serum produced a large number of foci, apparently greater than a no Ab control. As a negative control, a no virus test produced substantiaUy no foci.

EXAMPLE 6

Serum Neutralizing Titer against BPVl

Assays were carried out as in Example 5. Rabbits #1, 2, and 8 were inoculated with crude whole ceU Sf-9 lysates, and rabbits # 3,4,6, and 7 with partiaUy purified Ll protein (Table 1). Rabbits #6 and 7 were immunized with Ll protein preparations that had been denatured by boUing in 1% SDS. At least two bleeds, taken 3-6 weeks after the second boost, were tested for each rabbit and found to have the same titer. The titer of the preimmune sera from each of the rabbits was less than 20, the lowest dUution tested.

-2 ID- TABLE

recproca o uton t at cause ocusre uct r tprovided by A.B. Jenson (Nakai, Y., et al., 1986).

BIBLIOGRAPHY

U.S. Patent No. 5,081,029 to Starling et al. U.S. Patent No. 5,039,607 to Skold et al.

U.S. Patent No. 4,520,113 to GaUo et al.

Baker, C. in The Papovaviridae: Vol.2. The PapiUomaviruses (N. Salzman et al., eds.) Plenum Press, New York, 1987. p.321.

Baker, T., et al. Biophys. J. 60:1445 (1991).

Bradley, L. et al. in Selected Methods in CeUular Immunology. B. MisheU and S. Shiigi, eds. San Francisco: W.H. Freeman and Co., 1980. pp. 164-166.

Christensen, N., et al. Virology 64:5678 (1990).

Christensen, , et al. Virology 181:572 (1991).

Crawford, L., et al. Virology 21:258 (1963).

Dvoretzky, I., et al. Virology 103:369 (1980). Ghim, S., et al. Comparison of neutralization of BPV-1 infection of C127 ceUs and bovine fetal skin xenografts. Int. J. Cancer 49: 285 (1991).

Ghim, S., et al. HPV1-L1 protein expressed in cos ceUs displays conformational epitopes found on intact virions. Virology 190:548-552 (1992).

Hagensee, M., et al. Self-assembly of human papUlomavirus type 1 capsids by expression of the Ll protein alone or by coexpression of the Ll and L2 capsid proteins. J. of Virology 67(l):315-322. Hopfl, R., et al. Skin test for HPV type 16 proteins in cervical intraepithelial neoplasia.

Lancet 337:373 (1991).

Jarrett, W., et al. Veterinary Record 126:449 (1990). Jarrett, W., et al. Studies on vaccination against papUlomaviruses: prophylactic and therapeutic vaccination with recombinant structural proteins. Virology 184:33 (1991).

Jenison, S., et al. J. Infectious Dis. 162:60 (1990). Jenson, A., et al. Identification of linear epitopes BPV-1 Ll protein recognized by sera of infected or immunized animals. Pathobiology 59:396 (1991)

Jin, X., et al. J. Gen. Virology 70:1133 (1989). Kajigaya, S., et al. Proc. Natl. Acad. Sci. USA 88:4646 (1991). Kirnbauer, R., et al. PapiUomavirus Ll major capsid protein self-assembles into virus-like particles that are highly immunogenic. Proc. Natl. Acad. Sci. USA 89: 12180-12184 (1992).

Larsen, P., et al. J. Virology 61:3596 (1987).

Liddington, R., et al. Nature 354:278 (1991).

Lin, Y-L., et al. Effective vaccination against papiUoma development by immunization with Ll or L2 structural protein of cottontaU rabbit papUlovirus. Virology 187:612 (1992).

McLean, C, et al. Production and characterization of a monoclonal antibody to human papiUomavirus type 16 using recombinant vaccinia virus. J. Clin. Pathol 43:488 (1990).

Nakai, Y. Intervirol. 25:30 (1986). Olson, C, et al. Amer. J. Vet. Res. 21:233 (1960). PUacinski, W., et al. Biotechnology 2:356 (1984). Saiki, R. K., et al. Science 239:487 (1987).

Seedorf, et al. Human papUlomavirus type 16 DNA seqeunce. Virology 145: 181-185 (1985) Shiffman, M. J. National Cancer Inst. 84:394 (1992). Stevens, C, et al. JAMA 257:2612 (1987).

Stites, D. Chapter 27 in Basic and Clinical Immunology 3d Ed. H. Fudenberg et al., eds. Los Altos: Lange Medical Publications, 1980.

Summers, M., et al. Texas Agricultural Experiment Station, CoUege Station, Texas. A Manual of Methods for Baculovirus Vectors and Insect CeU Culture Procedures ( 1987). BuUetin No. 1555. Zhou, J., et al. Expression of vaccinia recombinant HPV 16 Ll and L2 ORF proteins in epithelial ceUs is sufficient for assembly of HPV virion-like particles. J. Virology 185:251 (1991). zur Hausen, H. Science 254:1167 (1991).

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: The Government of the United States, as represented by the Secretary of Health and Human Services

(ii) TITLE OF INVENTION: SELF- ASSEMBLING RECOMBINANT PAPILLOMAVIRUS CAPSID PROTEINS

(iii) NUMBER OF SEQUENCES: 6

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: KNOBBE, MARTENS, OLSON & BEAR

(B) STREET: 620 Newport Center Drive, Sixteenth Floor

(C) CITY: Newport Beach

(D) STATE: CA

(E) COUNTRY: USA

(F) ZIP: 92660

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release =1.0, Version #1.25

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/941,371

(B) FILING DATE: 03-SEP-1992

(A) APPLICATION NUMBER: US 08/032,869

(B) FILING DATE: 16-MAR-1993

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 714-760-0404

(B) TELEFAX: 714-760-9502

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1517 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Human papiUomavirus

■:3ι STRAIN: HPV16 ( ix) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 1..1518

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATG TCT CTT TGG CTG CCT AGT GAG GCC ACT GTC TAC TTG CCT CCT GTC 48

Met Ser Leu Trp Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val 1 5 10 15

CCA GTA TCT AAG GTT GTA AGC ACG GAT GAA TAT GTT GCA CGC ACA AAC 96

Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn 20 25 30

ATA TAT TAT CAT GCA GGA ACA TCC AGA CTA CTT GCA GTT GGA CAT CCC 144 lie Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro 35 40 45

TAT TTT CCT ATT AAA AAA CCT AAC AAT AAC AAA ATA TTA GTT CCT AAA 192 Tyr Phe Pro lie Lys Lys Pro Asn Asn Asn Lys lie Leu Val Pro Lys 50 55 60

GTA TCA GGA TTA CAA TAC AGG GTA TTT AGA ATA CAT TTA CCT GAC CCC 240 Val Ser Gly Leu Gin Tyr Arg Val Phe Arg lie His Leu Pro Asp Pro 65 70 75 80

AAT AAG TTT GGT TTT CCT GAC ACC TCA TTT TAT AAT CCA GAT ACA CAG 288 Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gin 85 90 95

CGG CTG GTT TGG GCC TGT GTA GGT GTT GAG GTA GGT CGT GGT CAG CCA 336 Arg Leu Val Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gin Pro 100 105 110

TTA GGT GTG GGC ATT AGT GGC CAT CCT TTA TTA AAT AAA TTG GAT GAC 384 Leu Gly Val Gly lie Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp 115 120 125

ACA GAA AAT GCT AGT GCT TAT GCA GCA AAT GCA GGT GTG GAT AAT AGA 432 Thr Glu Asn Ala Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg 130 135 140

GAA TGT ATA TCT ATG GAT TAC AAA CAA ACA CAA TTG TGT TTA ATT GGT 480 Glu Cys lie Ser Met Asp Tyr Lys Gin Thr Gin Leu Cys Leu lie Gly 145 150 155 160

TGC AAA CCA CCT ATA GGG GAA CAC TGG GGC AAA GGA TCC CCA TGT ACC 528 Cys Lys Pro Pro lie Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr 165 170 175

AAT GTT GCA GTA AAT CCA GGT GAT TGT CCA CCA TTA GAG TTA ATA AAC 576 Asn Val Ala Val Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu lie Asn 180 185 190

ACA GTT ATT CAG GAT GGT GAT ATG GTT CAT ACT GGC TTT GGT GCT ATG 624 Thr Val lie Gin Asp Gly Asp Met Val His Thr Gly Phe Gly Ala Met 195 200 205 GAC TTT ACT ACA TTA CAG GCT AAC AAA AGT GAA GTT CCA CTG GAT ATT 672 Asp Phe Thr Thr Leu Gin Ala Asn Lys Ser Glu Val Pro Leu Asp lie 210 215 220

TGT ACA TCT ATT TGC AAA TAT CCA GAT TAT ATT AAA ATG GTG TCA GAA 720 Cys Thr Ser lie Cys Lys Tyr Pro Asp Tyr lie Lys Met Val Ser Glu 225 230 235 240

CCA TAT GGC GAC AGC TTA TTT TTT TAT TTA CGA AGG GAA CAA ATG TTT 768 Pro Tyr Gly Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gin Met Phe 245 250 255

GTT AGA CAT TTA TTT AAT AGG GCT GGT ACT GTT GGT GAA AAT GTA CCA 816 Val Arg His Leu Phe Asn Arg Ala Gly Thr Val Gly Glu Asn Val Pro 260 265 270

GAC GAT TTA TAC ATT AAA GGC TCT GGG TCT ACT GCA AAT TTA GCC AGT 864 Asp Asp Leu Tyr lie Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser 275 280 285

TCA AAT TAT TTT CCT ACA CCT AGT GGT TCT ATG GTT ACC TCT GAT GCC 912 Ser Asn Tyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala 290 295 300

CAA ATA TTC AAT AAA CCT TAT TGG TTA CAA CGA GCA CAG GGC CAC AAT 960 Gin lie Phe Asn Lys Pro Tyr Trp Leu Gin Arg Ala Gin Gly His Asn 305 310 315 320

AAT GGC ATT TGT TGG GGT AAC CAA CTA TTT GTT ACT GTT GTT GAT ACT 1008 Asn Gly lie Cys Trp Gly Asn Gin Leu Phe Val Thr Val Val Asp Thr 325 330 335

ACA CGC AGT ACA AAT ATG TCA TTA TGT GCT GCC ATA TCT ACT TCA GAA 1056 Thr Arg Ser Thr Asn Met Ser Leu Cys Ala Ala lie Ser Thr Ser Glu 340 345 350

ACT ACA TAT AAA AAT ACT AAC TTT AAG GAG TAC CTA CGA CAT GGG GAG 1104 Thr Thr Tyr Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu 355 360 365

GAA TAT GAT TTA CAG TTT ATT TTT CAA CTG TGC AAA ATA ACC TTA ACT 1152 Glu Tyr Asp Leu Gin Phe lie Phe Gin Leu Cys Lys lie Thr Leu Thr 370 375 380

GCA GAC GTT ATG ACA TAC ATA CAT TCT ATG AAT TCC ACT ATT TTG GAG 1200 Ala Asp Val Met Thr Tyr lie His Ser Met Asn Ser Thr lie Leu Glu 385 390 395 400

GAC TGG AAT TTT GGT CTA CAA CCT CCC CCA GGA GGC ACA CTA GAA GAT 1248 Asp Trp Asn Phe Gly Leu Gin Pro Pro Pro Gly Gly Thr Leu Glu Asp 405 410 415

ACT TAT AGG TTT GTA ACA TCC CAG GCA ATT GCT TGT CAA AAA CAT ACA 1296 Thr Tyr Arg Phe Val Thr Ser Gin Ala lie Ala Cys Gin Lys His Thr 420 425 430

CCT CCA GCA CCT AAA GAA GAT CCC CTT AAA AAA TAC ACT TTT TGG GAA 1344 Pro Pro Ala Pro Lys Glu Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu 435 440 445 GTA AAT TTA AAG GAA AAG TTT TCT GCA GAC CTA GAT CAG TTT CCT TTA 1392 Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gin Phe Pro Leu 450 455 460

GGA CGC AAA TTT TTA CTA CAA GCA GGA TTG AAG GCC AAA CCA AAA TTT 1440 Gly Arg Lys Phe Leu Leu Gin Ala Gly Leu Lys Ala Lys Pro Lys Phe 465 470 475 480

ACA TTA GGA AAA CGA AAA GCT ACA CCC ACC ACC TCA TCT ACC TCT ACA 1488 Thr Leu Gly Lys Arg Lys Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr 485 490 495

ACT GCT AAA CGC AAA AAA CGT AAG CTG TA 1518

Thr Ala Lys Arg Lys Lys Arg Lys Leu 500 505

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1518 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1518

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

ATG TCT CTT TGG CTG CCT AGT GAG GCC ACT GTC TAC TTG CCT CCT GTC 48

Met Ser Leu Trp Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val 1 5 10 15

CCA GTA TCT AAG GTT GTA AGC ACG GAT GAA TAT GTT GCA CGC ACA AAC 96

Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn 20 25 30

ATA TAT TAT CAT GCA GGA ACA TCC AGA CTA CTT GCA GTT GGA CAT CCC 144 lie Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro 35 40 45

TAT TTT CCT ATT AAA AAA CCT AAC AAT AAC AAA ATA TTA GTT CCT AAA 192 Tyr Phe Pro lie Lys Lys Pro Asn Asn Asn Lys lie Leu Val Pro Lys 50 55 60

GTA TCA GGA TTA CAA TAC AGG GTA TTT AGA ATA CAT TTA CCT GAC CCC 240 Val Ser Gly Leu Gin Tyr Arg Val Phe Arg lie His Leu Pro Asp Pro 65 70 75 ^" 80 AAT AAG TTT GGT TTT CCT GAC ACC TCA TTT TAT AAT CCA GAT ACA CAG 288 Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gin 85 90 95

CGG CTG GTT TGG GCC TGT GTA GGT GTT GAG GTA GGT CGT GGT CAG CCA 336 Arg Leu Val Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gin Pro 100 105 110

TTA GGT GTG GGC ATT AGT GGC CAT CCT TTA TTA AAT AAA TTG GAT GAC 384 Leu Gly Val Gly lie Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp 115 120 125

ACA GAA AAT GCT AGT GCT TAT GCA GCA AAT GCA GGT GTG GAT AAT AGA 432 Thr Glu Asn Ala Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg 130 135 140

GAA TGT ATA TCT ATG GAT TAC AAA CAA ACA CAA TTG TGT TTA ATT GGT 480 Glu Cys lie Ser Met Asp Tyr Lys Gin Thr Gin Leu Cys Leu lie Gly 145 150 155 160

TGC AAA CCA CCT ATA GGG GAA CAC TGG GGC AAA GGA TCC CCA TGT ACC 528 Cys Lys Pro Pro lie Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr 165 170 175

AAT GTT GCA GTA AAT CCA GGT GAT TGT CCA CCA TTA GAG TTA ATA AAC 576 Asn Val Ala Val Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu lie Asn 180 185 190

ACA GTT ATT CAG GAT GGT GAT ATG GTT GAT ACT GGC TTT GGT GCT ATG 624 Thr Val He Gin Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met 195 200 205

GAC TTT ACT ACA TTA CAG GCT AAC AAA AGT GAA GTT CCA CTG GAT ATT 672 Asp Phe Thr Thr Leu Gin Ala Asn Lys Ser Glu Val Pro Leu Asp He 210 215 220

TGT ACA TCT ATT TGC AAA TAT CCA GAT TAT ATT AAA ATG GTG TCA GAA 720 Cys Thr Ser He Cys Lys Tyr Pro Asp Tyr He Lys Met Val Ser Glu 225 230 235 240

CCA TAT GGC GAC AGC TTA TTT TTT TAT TTA CGA AGG GAA CAA ATG TTT 768 Pro Tyr Gly Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gin Met Phe 245 250 255

GTT AGA CAT TTA TTT AAT AGG GCT GGT ACT GTT GGT GAA AAT GTA CCA 816 Val Arg His Leu Phe Asn Arg Ala Gly Thr Val Gly Glu Asn Val Pro 260 265 270

GAC GAT TTA TAC ATT AAA GGC TCT GGG TCT ACT GCA AAT TTA GCC AGT 864 Asp Asp Leu Tyr He Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser 275 280 285

TCA AAT TAT TTT CCT ACA CCT AGT GGT TCT ATG GTT ACC TCT GAT GCC 912 Ser Asn Tyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala 290 295 300

CAA ATA TTC AAT AAA CCT TAT TGG TTA CAA CGA GCA CAG GGC CAC AAT 960 Gin He Phe Asn Lys Pro Tyr Trp Leu Gin Arg Ala Gin Gly His Asn 305 310 315 320 AAT GGC ATT TGT TGG GGT AAC CAA CTA TTT GTT ACT GTT GTT GAT ACT 1008 Asn Gly He Cys Trp Gly Asn Gin Leu Phe Val Thr Val Val Asp Thr 325 330 335

ACA CGC AGT ACA AAT ATG TCA TTA TGT GCT GCC ATA TCT ACT TCA GAA 1056 Thr Arg Ser Thr Asn Met Ser Leu Cys Ala Ala He Ser Thr Ser Glu 340 345 350

ACT ACA TAT AAA AAT ACT AAC TTT AAG GAG TAC CTA CGA CAT GGG GAG 1104 Thr Thr Tyr Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu 355 360 365

GAA TAT GAT TTA CAG TTT ATT TTT CAA CTG TGC AAA ATA ACC TTA ACT 1152 Glu Tyr Asp Leu Gin Phe He Phe Gin Leu Cys Lys He Thr Leu Thr 370 375 380

GCA GAC GTT ATG ACA TAC ATA CAT TCT ATG AAT TCC ACT ATT TTG GAG 1200 Ala Asp Val Met Thr Tyr He His Ser Met Asn Ser Thr He Leu Glu 385 390 395 400

GAC TGG AAT TTT GGT CTA CAA CCT CCC CCA GGA GGC ACA CTA GAA GAT 1248 Asp Trp Asn Phe Gly Leu Gin Pro Pro Pro Gly Gly Thr Leu Glu Asp 405 410 415

ACT TAT AGG TTT GTA ACC CAG GCA ATT GCT TGT CAA AAA CAT ACA CCT 1296 Thr Tyr Arg Phe Val Thr Gin Ala He Ala Cys Gin Lys His Thr Pro 420 425 430

CCA GCA CCT AAA GAA GAT GAT CCC CTT AAA AAA TAC ACT TTT TGG GAA 1344 Pro Ala Pro Lys Glu Asp Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu 435 440 445

GTA AAT TTA AAG GAA AAG TTT TCT GCA GAC CTA GAT CAG TTT CCT TTA 1392 Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gin Phe Pro Leu 450 455 460

GGA CGC AAA TTT TTA CTA CAA GCA GGA TTG AAG GCC AAA CCA AAA TTT 1440 Gly Arg Lys Phe Leu Leu Gin Ala Gly Leu Lys Ala Lys Pro Lys Phe 465 470 475 480

ACA TTA GGA AAA CGA AAA GCT ACA CCC ACC ACC TCA TCT ACC TCT ACA 1488 Thr Leu Gly Lys Arg Lys Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr 485 490 495

ACT GCT AAA CGC AAA AAA CGT AAG CTG TA 1517

Thr Ala Lys Arg Lys Lys Arg Lys Leu 500 505

(2) INFORMATION FOR SEQ ID NO: 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bovine papiUomavirus

(vii) IMMEDIATE SOURCE:

(B) CLONE: BPVl N

( i) SEQUENCE DESCRIPTION: SEQ ID NO:3 : CCGCTGAATT CAATATGGCG TTGTGGCAAC AAGGCCAGAA GCTGTAT 47

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(vii) IMMEDIATE SOURCE: (B) CLONE: BPVl Y

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GCGGTGGTAC CGTGCAGTTG ACTTACCTTC TGTTTTACAT TTACAGA 47

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vii) IMMEDIATE SOURCE:

(B) CLONE: HPV16 N

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: ^"30

CCGCTAGATC TAATATGTCT CTTTGGCTGC CTAGTGAGGC C 41

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(vii) IMMEDIATE SOURCE:

(B) CLONE: HPV16 Y

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GCGGTAGATC TACACTAATT CAACATACAT ACAATACTTA CAGC 44

Claims (46)

WHAT IS CLAIMED IS:

1. A genetic construct, comprising a papUlomavirus Ll conformational coding sequence, inserted into a baculovirus transfer vector, and operatively expressed by a promoter of that vector.

2. The genetic construct of Claim 1, wherein said papUlomavirus Ll conformational coding sequence is isolated from a bovine, monkey, or human gene.

3. The genetic construct of Claim 2, wherein said papUlomavirus Ll conformational coding sequence is isolated from a wUd type HPV16 gene.

4. The genetic construct of Claim 3, wherein said papUlomavirus Ll conformational coding sequence is SEQ ID NO:6.

5. The genetic construct of Claim 3, further comprising a papUlomavirus L2 coding sequence.

6. A non-mammalian eukaryotic host ceU transformed by the genetic construct of any one of Claims 1-5.

7. A method for producing a recombinant papUlomavirus capsid protein, assembled into a capsomer structure or a portion thereof, comprising the steps of: cloning a papiUomavirus gene that codes for an Ll conformational capsid protein into a transfer vector wherein the open reading frame of said gene is under the control of the promoter of said vector; transferring the recombinant vector into a host ceU, wherein the cloned papUlomavirus gene expresses said papUlomavirus capsid protein; and isolating capsomer structures, comprising said papUlomavirus capsid protein, from said ceU.

8. The method of Claim 7, wherein the cloned papUlomavirus gene consists essentiaUy of the conformational Ll coding sequence, and the expressed protein assembles into capsomer structures consisting essentiaUy of Ll capsid protein.

9. The method of Claim 7, wherein the cloning step further comprises the cloning of a papUlomavirus gene coding for L2 capsid protein, whereby said Ll and L2 proteins are coexpressed, and wherein the isolated capsomer structures comprise Ll and L2 capsid proteins; provided that said transfer vector is not a vaccinia virus when said host ceU is a mammalian cell.

10. The method of Claim 7 or 9 wherein the conformational Ll coding sequence is cloned from a bovine, monkey, or human papUlomavirus.

11. The method of Claim 7 or 9, wherein the conformational Ll coding sequence is cloned from a wUd type HPV 16 papUlomavirus.

12. The method of Claim 11, wherein said conformational Ll coding sequence is SEQ ID NO:6.

13. The method of Claim 7, wherein said host ceU is an insect ceU.

14. The method of Claim 7, wherein said vector is a baculovirus based transfer vector, and the papUlomavirus gene is under the control of a promoter that is active in insect ceUs.

15. The method of Claim 7, wherein said recombinant baculovirus DNA is transfected into Sf-9 insect ceUs.

16. The method of Claim 15, wherein said recombinant baculovirus DNA is co-transfected with wUd-type baculovirus DNA into Sf-9 insect ceUs.

17. The method of Claim 7, wherein said vector is a yeast transfer vector, and the recombinant vector is transfected into yeast ceUs.

18. A virus capsomer structure, or a portion thereof, consisting essentiaUy of papUlomavirus Ll capsid protein, produced by the method of Claim 7.

19. A virus capsomer structure, consisting essentiaUy of papUlomavirus Ll and L2 capsid proteins, produced by the method of Claim 9.

20. A virus capsomer structure according to Claim 18 or 19 wherein said papUlomavirus Ll capsid protein is the expression product of an HPV16 Ll DNA cloned from a wUd type virus.

21. A virus capsid or a capsomer structure, or a portion thereof, consisting essentially of papUlomavirus Ll capsid protein.

22. A virus structure according to Claim 21, consisting essentially of wUd type HPV16 papUlomavirus Ll capsid protein.

23. A virus structure according to any one of Claims 18, 19. 21, or 22, wherein said capsid protein includes an immunogenic conformational epitope capable of inducing neutralizing antibodies against native papUlomavirus.

24. A virus structure according to Claim 23. wherein said papUlomavirus Ll capsid protein is selected from the group consisting of bovine, monkey, or human papiUomavirus Ll proteins.

25. A virus structure according to Claim 24, wherein said papiUomavirus Ll capsid protein is the expression product of a wUd type HPV16 Ll gene.

26. A virus structure according to Claim 25, wherein said HPV16 Ll gene comprises the sequence of SEQ ID NO:6.

27. A unit dose of a vaccine, comprising a peptide having conformational epitopes of a papiUomavirus Ll capsid protein, or Ll protein and L2 capsid proteins, in an effective immunogenic concentration sufficient to induce a papUlomavirus neutralizing antibody titer of at least about 10³ when administered according to an immunizing dosage schedule.

28. The vaccine of Claim 27, wherein said Ll capsid protein is an HPV16 capsid protein.

29. The vaccine of Claim 28, wherein said Ll capsid protein is a wUd type

HPV16 Ll protein.

30. A method of preventing or treating papUlomavirus infection in a vertebrate, comprising the administration of a papUlomavirus structure according to Claim 18 or 19 to said vertebrate, according to an immunity-producing regimen.

31. The method of Claim 30 wherein said papUlomavirus structure comprises wUd type HPV 16 Ll capsid protein.

32. A method of preventing or treating papUlomavirus infection in a vertebrate, comprising the administration of the papUlomavirus structure according to Claim 30 to said vertebrate, according to an immunity-producing regimen.

33. A method of preventing or treating papUlomavirus infection in a vertebrate, comprising the administration of the papUlomavirus vaccine of Claim 27 to said vertebrate, according to an immunity-producing regimen.

34. The method of Claim 33 wherein said papUlomavirus vaccine comprises wUd type HPV16 Ll capsid protein.

35. A method for immunizing a vertebrate against papUlomavirus infection, comprising administering to said vertebrate a recombinant genetic construct comprising a conformational papUlomavirus Ll coding sequence, and aUowing said coding sequence to be expressed in the ceUs or tissues of said vertebrate, whereby an effective, neutralizing, immune response to papUlomavirus is induced.

36. A method according to Claim 35, wherein said conformational papUlomavirus

Ll coding sequence is derived from human papUlomavirus HPV 16.

37. The method of Claim 36, wherein said human papUlomavirus HPV 16 is a wUd type papUlomavirus.

38. A method of detecting humoral immunity to papUlomavirus infection in a vertebrate comprising the steps of:

(a) providing an effective antibody-detecting amount of a papUlomavirus capsid peptide having at least one conformational epitope of a papUlomavirus capsomer structure;

(b) contacting the peptide of step (a) with a sample of bodUy fluid from a vertebrate to be examined for papUlomavirus infection, and aUowing papUlomavirus antibodies contained in said sample to bind thereto, forming antigen- antibody complexes; (c) separating said complexes from unbound substances;

(d) contacting the complexes of step (c) with a detectably labeUed immunoglobulin-binding agent; and

(e) detecting anti-papiUomavirus antibodies in said sample by means of the labeUed immunoglobulin-binding agent that binds to said complexes.

39. The method of Claim 37, wherein said peptide consists essentiaUy of papUlomavirus Ll capsid protein.

40. The method of Claim 38, wherein said peptide consists essentiaUy of the expression product of a human papUlomavirus HPV 16.

41. The method of Claim 39, wherein said peptide consists essentiaUy of the expression product of a wUd type human papUlomavirus HPV 16 gene.

42. The method of Claim 40, wherein said peptide consists essentiaUy of the expression product of SEQ ID NO:6.

43. A method of detecting papUlomavirus in a specimen from an animal suspected of being infected with said virus, comprising contacting said specimen with antibodies having a specificity to one or more conformational epitopes of the capsid of said papiUomavirus, said antibodies having a detectable signal producing label, or being attached to a detectably labeUed reagent; aUowing said antibodies to bind to said papUlomavirus; and determining the presence of papiUomavirus present in said specimen by means of said detectable label.

44. A method of determining a ceUular immune response to papUlomavirus in an animal suspected of being infected with said virus, comprising; contacting immunocompetent cells of said animal with a recombinant wild type papUlomavirus Ll capsid protein, or combined Ll and L2 capsid protein according to Claim 18 or 19; and assessing ceUular immunity to said papUlomavirus by means of the proUferative response of said ceUs to said capsid protein.

45. The method of Claim 44, wherein said recombinant papUlomavirus protein is introduced into the skin of said animal.

46. A papUlomavirus infection diagnostic kit, comprising capsomer structures consisting essentiaUy of papUlomavirus Ll capsid protein, or capsomer structures comprising papUlomavirus Ll protein and L2 capsid proteins, or antibodies to either of said capsomers structures, singly or in combination, together with materials for carrying out an assay for humoral or ceUular immunity against papUlomavirus, in a unit package container.