Link:  Pharm/Biotech Resources

Title:  Formalin-treated human papillomavirus L1 protein vaccine

United States Patent:  6,887,478

Issued:  May 3, 2005

Inventors:  Schlegel; C. Richard (Rockville, MD); Jenson; A. Bennett (Rockville, MD); Ghim; Shin-je (Washington, DC)

Assignee:  Georgetown University (Washington, DC)

Appl. No.:  665537

Filed:  September 22, 2003

Recombinantly produced L1 major capsid proteins which mimic conformational naturalizing epitopes on human and animal papilloma virions including canine and equine papilloma virions are provided. These recombinant proteins are useful as vaccines for conferring protection against papillomavirus infection. Antibodies to the recombinant protein are also provided. Such antibodies are useful in the diagnosis and treatment of viral infection.

FIELD OF THE INVENTION

The invention relates to the diagnosis, serotyping, prevention and treatment of viral diseases, particularly papillomavirus infections.

More particularly, the invention relates to the diagnosis, serotyping, prevention and treatment of human papillomavirus infections, equine papillomavirus infections and canine papillomavirus infections.

BACKGROUND OF THE INVENTION

Papillomaviruses (PV) are members of the papovavirus family and contain a double stranded circular DNA genome with a typical size of about 7900 base pairs (bp). Human papillomaviruses (HPV) are recognized as a cause of various epithelial lesions such as warts, condylomas and dysplasias. See, Gissman, L., Cancer Survey, 3:161 (1984); Boshart et al, EMBO J., 3:1151 (1984); Romanczuk et al, J. Virol., 65:2739-2744 (1991); Jenson et al, In "Papillomaviruses and human cancer" (H. Pfister. Ed.), pp. 11-43, CRC Press (1990); Schlegel, R., "Papillomaviruses and human cancer" In: Viral pathogenesis (ed. Fujinami, R.), Seminars in Virology 1:297-306 (1990); and Jenson et al, "Human Papillomaviruses" In Belshe, R. ed. Textbook of human virology, Second Edition: MASS:PSG, 1989:951.

HPVs are grouped into types based on the similarity of their DNA sequence. Two HPVs are taxonomically classified as being of the same type if their DNAs cross-hybridize to greater than 50% as measured by hybridization in solution under moderately stringent hybridization conditions.

A number of distinct papillomaviruses have been shown to infect humans. Papillomaviruses are highly species and tissue-specific, and are characterized by a specific mode of interaction with the squamous epithelia they infect. These small DNA tumor viruses colonize various stratified epithelia like skin and oral and genital mucosa, and induce the formation of self-limiting benign tumors known as papillomas (warts) or condylomas. These tumors are believed to arise from an initial event in the infectious cycle where the virus enhances the division rate of the infected stem cell in the epithelial basal layer, before it is replicated in the differentiating keratinocyte.

The term papillomavirus covers a large number of viruses which are considered responsible for several forms of viral infection ranging from relatively benign warts of the skin or mucous membranes to hyperplasias susceptible to progressing into dysplasias or intra-epithelial neoplasms, and malignant conversion to various forms of cancer, the most significant being that of the female uterine cervix.

A number of HPVs types have been identified. Furthermore, the preferential association of certain HPV types with anatomic location and distinct types of lesions gives support to the hypothesis that different HPV-induced lesions constitute distinct diseases, and that the clinical patterns of lesions express specific biological properties of distinct types of HPVs. Distinctive histological features have been associated with the infection of the skin or mucous membranes by different types of HPVs.

The genomes of different HPV types have been cloned and characterized. In particular, the genomes of two HPV types, HPV 16 and HPV 18, have been found to be associated with about 70% of invasive carcinomas of the uterine cervix.

Human papillomaviruses which infect the genital tract mucosa play a critical role in the development of cervical cancer. See, Lorincz et al, Obstetrics & Gynecology, 79:328-337 (1992); Beaudenon et al., Nature, 321:246-249 (1986); and Holloway et al, Gynecol. Onc., 41:123-128 (1991). For example, the majority of humans cervical carcinomas (95%) contain and express HPV DNA and it is the expression of two viral oncoproteins, E6 and E7, which appears to be critical for cellular transformation and maintenance of the transformed state. Despite the detailed knowledge concerning the molecular mechanism of action of these oncoproteins, there is little information available on the biology of papillomavirus infection, including the identity of viral receptors, the control of viral replication and assembly, and the host immune response to virus and virally-transformed cells. An effective vaccine against HPV infection could potentially reduce the incidence of human cervical dysplasia and carcinoma by 90-95%. However, there is no tissue culture system which permits sufficient keratinocyte differentiation to propagate the PV in-vitro. Because of the widespread occurrence of HPV infection, methods for detecting, preventing and treating viral infection are needed. Also, methods for detecting, preventing and treating papillomavirus infection in animals, e.g., equines and canines, are also needed.

Canine papillomas were one of the first animal systems studied when McFaydean and Hobday transmitted the oral papilloma in 1898. Today, dogs are commonly used as models for a variety of diseases and much is known about their physiology and immune system. Papillomas affect many anatomic locations in dogs, similar to the human diseases. Puppies may have marginal papillae on their tongues which are normal anatomic structures resembling oral papillomas. True papillomas can be found on the dorsal tongue and buccal mucosa, ocular mucous membranes, mucous membranes of the lower genital tracts of both males and females, and haired skin. The lesions are characterized by epithelial proliferation on thin fibrovascular stalks and there may be specific cytopathic effects in the stratum granulosum in which the cells swell, develop large keratohyalin-like granules, and may have intranuclear inclusions. Group-specific papillomavirus antigens can be detected by the cells exhibiting cytopathic effects.

The canine oral papillomavirus has been cloned and characterized (Sundberg et al, Amer. J. Vet. Res., 47(5), 1142-1177 (1986)). The COPV viral genome was cloned into pBR322, a restriction map constructed, with the completeness of the COPV genome confirmed by comparison of restriction fragment sizes derived from cloned and virion DNA. (Id.) It is known that COPV is antigenically similar to other papillomaviruses. For example, it has been reported that some of the antigenic and immunogenic epitopes of HPV16 and bovine, canine and avian papillomaviruses are shared. (Dillner et al, J. Virol., 65(12), 5862-6871, (1991)).

Strong evidence suggests that canine papillomaviruses play a role in squamous cell carcinoma development. For example, papillomavirus antigens are detected in penile and vulvar carcinomas. Also, it has been reported that intramuscular injection of canine oral papillomavirus results in the later development of cutaneous squamous cell carcinoma.

Papillomas are also prevalent in equines. In fact, papillomas are probably the most common equine tumor; however, few are ever submitted to diagnostic laboratories for histologic confirmation. Papillomas in equines generally affect the skin, mouth, lower genital tract and eyes. Papillomavirus which causes infection in equines is of the cutaneous type. Equine papillomaviruses have also been isolated and cloned. It is also known that equine papillomavirus infection causes millions of dollars in losses annually to the equine industry. Thus, based on the foregoing, it is clear that there exists a need for effective vaccines against papillomaviruses including HPV's and animal papillomaviruses such as COPV and equine papillomavirus.

SUMMARY OF THE INVENTION

Toward that end, a recombinantly produced L1 major capsid protein which mimics conformational neutralizing epitopes on human and animal papilloma virions is provided. The recombinant protein reproduces the antigenicity of the intact, infectious viral particle. The recombinant protein can be utilized to immunoprecipitate antibodies from the serum of patents infected or vaccinated with PV. Neutralizing antibodies to the recombinant protein are also provided. The antibodies are useful for the diagnosis and treatment of papilloma viral infection. The invention additionally provides subviral vaccines for the prevention of human and animal papillomavirus infection, e.g., for preventing equine and canine papillomavirus infection.

More specifically, recombinantly provided L1 major capsid proteins which mimic the conformational neutralizing epitopes on human, equine and canine papilloma virions are provided. These recombinant capsid proteins reproduce the antigenicity of the intact infectious human, canine or equine virus particle. The recombinant proteins can be utilized to immunoprecipitate antibodies from the serum of humans, equines or canines infected or vaccinated with the corresponding PV. Neutralizing antibodies to the human, canine or equine papillomavirus capsid protein are also provided. These antibodies are useful for the diagnosis and treatment of human, canine or equine papilloma viral infections. The invention further provides subviral vaccines for the prevention of human, canine and equine papillomavirus infection.

The invention further provides a unique and relevant canine animal model for the development of papillomavirus vaccines, in particular canine and human papillomavirus vaccines; which unlike the available rabbit and bovine papillomavirus models, utilizes the canine oral papillomavirus (COPV) which is tropic for mucous membranes and is assayable for infectivity under normal conditions of exposure.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Methods and compositions are provided for the prevention, detection and treatment of papillomavirus (PV) infection. The methods are based upon the production of a recombinant L1 major capsid protein which is capable of reproducing the conformational neutralizing epitopes on human and animal papillomavirus virions. The invention is further drawn to antigenic fragments of such recombinant L1 proteins.

Although papillomaviruses infect a wide variety of vertebrate species, they exhibit a remarkable conservation of genomic organization and capsid protein composition. Papillomaviruses consist of small (about 55 nm), non-enveloped virions which surround a genome of double-stranded, circular DNA. The genome is approximately 8,000 bp in length and can be divided into equal-length "early" and "late" regions. The "early" region encodes 7-8 genes involved in such processes as viral DNA replication (the E1 and E2 genes), RNA transcription (the E2 gene), and cell transformation (the E5, E6 and E7 genes). The "late" region encodes two structural proteins, L1 and L2, which represent the major and minor capsid proteins, respectively. All of the "early" and "late" genes are transcribed from the same strand of viral DNA.

There are a variety of PV types known in the art. Further, particular types of PVs are associated with particular infections such as flat warts, cutaneous warts, epidennodysplasia verruciformis, lesions and cervical cancer. Over 50 different HPV types have been identified in clinical lesions by viral nucleotide sequence homology studies. See, for example, Jenson et al, "Human papillomaviruses" In: Belshe, R. ed., Textbook of human virology, Second Edition, MASS: PSG, 1989:951 and Kremsdorf et al, J. Virol., 52:1013-1018 (1984). The HPV type determines, in part, the site of infection, the pathological features and clinical appearance as well as the clinical course of the respective lesion.

The L1 protein represents the most highly conserved protein of all the papillomavirus proteins. The nucleotide sequence of the L1 open reading frames of BPV-1, HPV-1A, and HPV-6B are given in U.S. Pat. No. 5,057,411, which disclosure is incorporated herein by reference. Furthermore, it is noted that L1 proteins and fusion proteins have been produced recombinantly. However, prior to the present invention, it was not known that L1 proteins with sufficient fidelity to maintain a conformation equivalent to that found in intact papillomavirus virions could be produced. Previously, recombinant L1 protein was produced as linear molecules which were incapable of protecting against papillomavirus infection. The present invention, in contrast, provides conformationally correct protein which is capable of inducing neutralizing antibodies which protect against animal and human papillomaviruses.

Because it is believed that there is little or no cross-immunity for PV types and immunity to infection is PV type-specific, it will be necessary to produce recombinant L1 protein for each specific PV type upon which protection or treatment is needed.

However, due to the homology between the L1 proteins and genes, hybridization techniques can be utilized to isolate the particular L1 gene of interest. Nucleotide probes selected from regions of the L1 protein which have been demonstrated to show sequence homology, can be utilized to isolate other L1 genes. Methods for hybridization are known in the art. See, for example, Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985); Molecular Cloning, A Laboratory Manual, Maniatis et al, eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982); and Molecular Cloning, A Laboratory Manual, Sambrook et al, eds., Cold Spring Harbor Laboratory, Second Edition, Cold Spring Harbor, N.Y. (1989). Alternatively, PCR methods can be utilized to amplify L1 genes or gene fragments. See, for example, U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,800,159.

Virus particles can also be isolated for a particular papillomavirus type, the DNA cloned, and the nucleic acid sequences encoding L1 proteins isolated. Methods for isolation of viral particles and cloning of virus DNAs have been reported. See, for example, Heilman et al., J. Virology, 36:395-407 (1980); Beaudenon et al, Nature, 321:246-249 (1986); Georges et al, J. Virolooy, 51:530-538 (1984); Kremsdorf et al, J. Virology, 52:1013-1018 (1984); Clad et al, Virology, 118:254-259 (1982); DeVilliers et al, J. Virology, 40:932-935 (1981); and European Patent Application 0133123.

Alternatively, the L1 protein for a particular papillomavirus can be isolated, the amino acid sequence determined and nucleic acid probes constructed based on the predicted DNA sequence. Such probes can be utilized in isolating the L1 gene from a library of the papillomavirus DNA. See, for example, Suggs et al, PNAS, 78(11):6613-6617 (1981). See also, Young and Davis, PNAS, 80:1194 (1983).

Since the recombinant L1 protein must be of suitable conformation to mimic that of the intact virus particle, the expression system is crucial to the invention. An expression system must be utilized which produces L1 protein in the correct conformation. That is, the recombinant L1 protein reproduces the antigenicity of intact infectious virus particles. Such expression systems should also produce high levels of capsid protein. Generally, the expression system will comprise a vector having the L1 protein of interest and the appropriate regulatory regions as well as a suitable host cell. Typically a suitable host will be one which provides eucaryotic mechanisms for processing of the proteins.

Ideally, a strong promoter is utilized for high expression of the recombinant protein. Of particular interest is the pSVL vector. The pSVL vector contains an SV40 origin of replication and when transfected in COS cells, which express Large T antigen, replicates to a high copy number.

Alternatively, baculovirus vectors can be utilized. A baculovirus system offers the advantage that a large percentage of cells can be induced to express protein due to the use of infection rather than transfection techniques. While baculovirus is an insect virus and grows in insect cells (Sf9), these cells retain many of the eucaryotic mechanisms for processing of proteins including glycosylation and phosphorylation which may be important for generating proteins of appropriate conformation. Baculovirus vector systems are known in the art. See, for example, Summers and Smith, Texas Agricultural Experimental Bulletin No. 1555 (1987); Smith et al, Mol. Cell Biol., 3:2156-2165 (1985); Posse, Virus Research, 5:4359 (1986); and Matsuura, J. Gen. Virol., 68:1233-1250 (1987).

In particular, this application exemplifies the expression of the canine oral papillomavirus (COPV) L1 protein in Sf9 cells using a baculovirus expression system and demonstrates that the resultant L1 proteins comprise conformational epitopes and confer protection when administered to naive dogs (beagles) upon challenge with live infectious COPV.

COPV was selected for several reasons. First, because of the high level of similarity between COPV and HPV's at the DNA and amino acid sequence level, genetic organizational level, as well as similar mucosal route of infection, COPV provides a highly suitable in vivo model for study of HPV vaccines. More specifically, dogs may be inoculated with COPV L1 proteins and challenged with live COPV in order to provide relevant in vivo evidence regarding the effectiveness of conformational PV L1 proteins to confer immunity against the corresponding papillomavirus.

While this is tremendously advantageous by itself, the COPV L1 protein is also important in its right. As discussed above, COPV is a mucosal papillomavirus which results in papillomas in canines that are found in the dorsal tongue and buccal mucosa; ocular mucous membranes, mucous membranes of the lower genital tracts of both males and females, and haired skin. Moreover, COPV is believed to play a role in squamous cell carcinoma. Therefore, a vaccine against COPV is highly desirable because it may be used to prevent papillomas in canines, and also squamous carcinoma caused by COPV.

Moreover, given the substantial similarities between COPV and HPVs, in particular those which cause cancer in humans, the COPV/beagle animal model has applicability in screening the effectiveness of potential antiviral agents for treating human papillomavirus infection. Essentially, this will involve administering an antiviral agent predicted to be useful for treating human papillomaviral infection to a beagle dog which has been infected with COPV and ascertaining the effects of this antiviral agent on the status of COPV infection. This may be effected, e.g., by observing the size and number of papillomas in the treated animal before and after treatment with the antiviral agent. Antiviral agents which inhibit papilloma development or result in their decrease in size and/or number in treated animals should possess similar activity in humans for treating HPV infection given the similarities between COPV and HPVs.

Another animal PV where L1 conformational proteins have application in the design of vaccines is equine papillomavirus. As noted, equine papillomavirus is probably the most common cause of equine tumor. Squamous cell carcinomas, which are believed to be caused by equine papillomaviral infection, are also common in horses. This is substantiated by the fact that such carcinomas have an anatomic distribution similar to papillomas. One of the most common locations of such carcinomas is the lower genital tract. Moreover, equine papillomavirus infection results in substantial expense (many millions of dollars yearly) to the equine industry. Therefore, an equine papillomavirus vaccine produced according to the invention should possess tremendous potential for protecting equines against equine papillomavirus infection and squamous cell carcinoma caused thereby. As with the afore-described papillomavirus vaccines, this vaccine will comprise a prophylactically effective amount of recombinant equine papillomavirus L1 proteins or fragments which exhibit the conformation of L1 proteins expressed by native equine papillomavirus virions. Based on the high level of sequence similarities between the L1 sequences of different papillomaviruses, the equine papillomavirus L1 sequence can readily be cloned and expressed in a suitable expression system, e.g., baculovirus.

For expression in an appropriate expression system, the L1 gene is operably linked into an expression vector and introduced into a host cell to enable the expression of the L1 protein by that cell. The gene with the appropriate regulatory regions will be provided in proper orientation and reading frame to allow for expression. Methods for gene construction are known in the art. See, in particular, Molecular Cloning, A Laboratory Manual, Sambrook et al, eds., Cold Spring Harbor Laboratory, Second Edition, Cold Spring Harbor, N.Y. (1989) and the references cited therein.

A wide variety of transcriptional and translational regulatory sequences may be employed. The signals may be derived from viral sources, where the regulatory signals are associated with a particular gene which has a high level of expression. That is, strong promoters, for example, of viral or mammalian sources, will be utilized. In this manner, the optimum conditions for carrying out the invention include the cloning of the L1 gene into an expression vector that will overexpress conformationally-dependent epitopes of the L1 protein in transfected or infected target cells.

The recombinant L1 protein is confirmed by reaction with antibodies or monoclonal antibodies which react or recognize conformational epitopes present on the intact virion. In this manner, the L1 protein can be verified as having the suitable conformation. Thus, other expression vectors and expression systems can be tested for use in the invention.

As discussed, it is essential that the expressed L1 protein be conformational, i.e., that it contain conformational epitopes that are necessary for a protective immunogenic response. This will typically be accomplished by expression of the entire L1 sequence of the particular papillomavirus, e.g., COPV or a human papillomavirus, e.g., HPV-6, HPV-11, HPV-16, HPV-18, among others. However, the invention also embraces expression of L1 DNA fragments, i.e., which do not comprise the entire L1 coding sequence but which upon expression still produce conformational L1 proteins, i.e., L1 proteins which contain conformational epitopes.

The specific L1 DNA fragments which results in the expression of conformational L1 proteins may be identified, e.g., by expressing different fragments of a particular L1 DNA, and ascertaining whether the resultant protein is conformational. This may be effected, e.g., by determining whether the particular L1 fragment reacts with or elicits the production of antibodies specific to conformational L1 epitopes.

To confirm that PV L1 DNA fragment encoding less than the entire L1 protein may be obtained which upon expression result in conformational L1 proteins, fragments of the COPV L1 open reading frame were expressed. In particular, fragments of the COPV L1 open reading frame were expressed which contained either a deletion in the amino-terminal or carboxy-terminal portion of the L1 sequence. It was found that the L1 protein containing the amino-terminal deletion expressed in a SV40 vector in COS cells apparently did not result in conformational L1 proteins (when tested with conformationally-dependent antibodies). By contrast, the L1 sequence which contained a deletion in the carboxy-terminal region when expressed in COS cells using the same SV40 vector system resulted in conformational L1 proteins (as demonstrated by binding to antibodies which recognize conformational epitopes). This carboxy-deletion consisted of deletion of the 26 amino acid fragment of the COPV L1 sequence, which was replaced by a 5 amino acid nuclear sequence of a nonstructural viral protein (large T protein) of SV40. The 26 amino acids of the COPV L1 sequence deleted include the nuclear signal sequences necessary for translocation of the native L1 protein into the cell nucleus. The particular nuclear signal sequence is not critical and is only necessary for transport into the nucleus. In this regard, many nuclear signal sequences are well known in the art. Thus, these results demonstrate that L1 fragments encompassing the carboxy terminus of the L1 protein are not necessary for reproducing conformational epitopes.

While only 26 amino acids of the carboxy-terminus were deleted, it is reasonable to assume that larger deletions will also result in conformational L1 proteins. As indicated, those deletions which are operable, i.e., which result in conformational L1 proteins, may be identified based on the reactivity of the resultant L1 fragments with conformational antibodies. This may be determined, e.g., by immunofluorescence or immunoprecipitation using conformational L1 antibodies.

Also, while only a carboxy-terminal deletion was demonstrated to yield conformational L1 proteins upon expression, it is believed that other deletions may also result in conformational L1 proteins. For example, internal deletions may also result in the formation of conformational L1 proteins. Also, it is expected that substitution mutations may be identified which result in conformational L1 proteins. Such substitutions may potentially be made in both the conserved and hypervariable regions of the L1 protein.

Moreover, while only COPV L1 fragments (containing deletion of 26 carboxy-terminal amino acids) were demonstrated to yield conformational L1 proteins, it is reasonable to expect that similar results will be obtained with other PV L1 sequences, given their high level of homology. It is especially reasonable to assume that similar results will be observed with carboxy-terminal deletions of HPV L1 sequences given the substantial similarities between HPVs and COPV.

COPV and HPVs associated with human malignancy are highly similar. They exhibit similar genetic organization, viral structure, capsid protein sequences, and selectively infect a mucosal site of infection. Based on these similarities, carboxy-deletions of HPV L1 sequences should also result in conformational L1 proteins when expressed according to the invention. This can be confirmed by testing with conformational antibodies specific to the particular HPV L1 fragment being expressed.

Once the L1 protein of suitable conformation has been expressed, antibodies can be raised against the recombinant protein or antigenic fragments thereof. The antibodies of the present invention may be prepared using known techniques.

Monoclonal antibodies are prepared using hybridoma technology as described by Kohler et al, Nature, 256:495 (1975); Kohler et al, Eur. J. Immunol., 6:511 (1976); Kohler et al, Eur. J. Immunol., 6:292 (1976); Hammerling et al, in: Monoclonal Antibodies and T-Cell Hybridomas, Elsavier, N.Y., pages 563-681 (1981). Such antibodies produced by the methods of the invention are capable of protecting against PV infection.

The term "antibody" includes both polyclonal and monoclonal antibodies, as well as fragments thereof, such as, for example, Fv, Fab and F(ab)₂ fragments which are capable of binding antigen or hapten. Such fragments are typically produced by proteolytic cleavage, such as papain, to produce Fab fragments or pepsin to produce F(ab)₂ fragments. Alternatively, hapten-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.

As indicated, both polyclonal and monoclonal antibodies may be employed in accordance with the present invention. Of special interest to the present invention are antibodies which are produced in humans or are "humanized" (i.e., non-immunogenic in a human) by recombinant or other technology. Humanized antibodies may be produced, for example, by placing an immunogenic portion of an antibody with a corresponding, but non-immunogenic portion, chimeric antibodies. See, for example, Robinson et al, International Patent Publication PCT/US86/02269; Akira et al, European Patent Application 184,187; Taniguchi, M. European Patent Application 171,496; Morrison et al, European Patent Application 173,494; Neuberger et al, PCT Application WO86/01533; Cabilly et al, European Patent Application 125,023; Better et al, Science, 240:1041-1043 (1988); Liu et al, PNAS, 84:3439-3443 (1987); Liu et al, J. Immunol., 139:3521-3526 (1987); Sun et al, PNAS, 84:214-218 (1987); Nishimura et al, Cancer Research, 47:999-1005 (1987); Wood et al, Nature, 314:446-449 (1985); and Shaw et al, J. National Cancer Inst., 80:1553-1559 (1988). General reviews of "humanized" chimeric antibodies are provided by Morrison, S. L., Science, 229:1202-1207 (1985) and by Oi et al, BioTechniques, 4:214 (1986).

The antibodies, or antibody fragments, of the present invention can be utilized to detect, diagnose, serotype, and treat papillomavirus infection. In this manner, the antibodies or antibody fragments are particularly suited for use in immunoassays.

Antibodies, or fragments thereof, may be labeled using any of a variety of labels and methods of labeling. Examples of types of labels which can be used in the present invention include, but are not limited to, enzyme labels, radioisotopic labels, non-radioactive isotopic labels, fluorescent labels, toxin labels, and chemiluminescent labels.

Examples of suitable enzyme labels include malate hydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase, etc.

Examples of suitable radioisotopic labels include ³H, ¹²⁵I, ¹³¹I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, and ¹⁰⁹Pd.

Examples of suitable fluorescent labels include a ¹⁵²Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, and allophycocyanin label, an o-phthaldehyde label, an fluorescamine label, etc.

Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin. Examples of chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, and imidazole label, and acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, an aequorin label, etc.

Those of ordinary skill in the art will know of other suitable labels which may be employed in accordance with the present invention. The binding of these labels to antibodies or fragments thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art. Typical techniques are described by Kennedy et al, Clin. Chim. Acta, 70:1-31 (1976), and Schurs et al, Clin. Chim. Acta, 81:1-40 (1977). Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method, all these methods incorporated by reference herein.

The detection of the antibodies (or fragments of antibodies) of the present invention may be improved through the use of carriers. Well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. Those skilled in the art will note many other suitable carriers for binding monoclonal antibody, or will be able to ascertain the same by use of routine experimentation.

By raising antibodies against L1 proteins which mimic the antigenicity of papillomavirus virions, the antibodies raised against such recombinant proteins are neutralizing and protective antibodies. The antibodies are able to prevent subsequent infection of the same type of papillomaviruses from which the L1 protein was derived.

That is, if a recombinant L1 protein from papillomavirus type 16 is utilized to raise antibodies, these antibodies will protect against subsequent infection of papillomavirus type 16. Thus, the method of the present invention provides for the prevention, treatment or detection of any HPV type.

The antibodies of the invention can be utilized to determine HPV types by serotyping as set forth in Jenson et al, J. Cutan. Pathol., 16:54-59 (1989). Determining the HPV type may be clinically important for determining the putative biological potential of some productively infected HPV-associated lesions, particularly benign and low-grade premalignant anogenital tract lesions. Thus, the present invention makes it possible to treat and prevent infection of any type of PV from which the L1 gene can be obtained and neutralizing antibodies obtained.

The invention also provides for pharmaceutical compositions as the antibodies can also be utilized to treat papillomavirus infections in mammals. The antibodies or monoclonal antibodies can be used in pharmaceutical compositions to target drug therapies to sites of PV infection. In this manner, the drugs or compounds of interest are linked to the antibody to allow for targeting of the drugs or compounds. Methods are available for linking antibodies to drugs or compounds. See, for example, EP 0,146,050; EP 0,187,658; and U.S. Pat. Nos. 4,673,573; 4,368,149; 4,671,958 and 4,545,988.

Such drug therapies include antiviral agents, toxic agents and photoactivatable compounds, such as coumarin, psoralen, phthalocyanimes, methylene blue, eosin, tetracycline, chlorophylls, porphyrins and the like. Such groups can be attached to the antibodies by appropriate linking groups. Antibody conjugates containing a photoactivatable compound are administered followed by irradiation of the target cells.

The antibody or antibody conjugates of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions such as by admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation are described, for example, in Remington's Pharmaceutical Sciences (16th Ed., Osol, A. Ed., Mack Easton Pa. (1980)). To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of antibody, either alone, or with a suitable amount of carrier vehicle.

The therapeutic or diagnostic compositions of the invention will be administered to an individual in therapeutically effective amounts. That is, in an amount sufficient to diagnose or treat PV infection. The effective amount will vary according to the weight, sex, age and medical history of the individual. Other factors include, the severity of the patient's condition, the type of PV, mode of administration, and the like. Generally, the compositions will be administered in dosages ranging from about 0.01 to about 2 picomoles/ml, more generally about 0.001 to about 20 picomoles/ml.

The pharmaceutically prepared compositions may be provided to a patient by any means known in the art including oral, intranasal, subcutaneous, intramuscular, intravenous, intraarterial, parenteral, etc.

Another aspect of the present invention involves the development of PV type-specific vaccines. The vaccines of the invention are those that contain the necessary antigenic determinants to induce formation of neutralizing antibodies in the host; possess high immunogenic potential; are safe enough to be administered without danger of clinical infection; devoid of toxic side-effects; suitable for administration by an effective route, for example, oral, intranasal, topical or parenteral; mimics the circumstances of natural infection; stable under conditions of long-term storage; and, compatible with the usual inert vaccine carriers.

The vaccines of the present invention include the conformationally correct recombinant L1 proteins or fragments thereof which provide the conformational epitopes present on the intact virions. Such amino acid sequences of the L1 protein comprise the antigenic component of the vaccine. It may be necessary or preferable to covalently link the antigen to an immunogenic carrier, i.e., bovine serum albumin or keyhole limpet hemocyanin. The vaccines of the invention may be administered to any mammal susceptible to infection with the papillomavirus. Human and non-animal mammals may benefit as hosts.

Administration of the vaccines may be parenteral, but preferably oral or intranasal, depending upon the natural route of infection. The dosage administered may be dependent upon the age, health, weight, kind of concurrent treatment, if any, and nature and type of the papillomavirus. The vaccine may be employed in dosage form such as capsules, liquid solutions, suspensions, or elixirs, for oral administration, or sterile liquid formulations such as solutions or suspensions for parenteral or intranasal use. An inert, immunologically acceptable carrier is preferably used, such as saline or phosphate-buffered saline.

The vaccines will be administered in therapeutically effective amounts. That is, in amounts sufficient to produce a protective immunological response. Generally, the vaccines will be administered in dosages ranging from about 0.1 mg protein to about 20 mg protein, more generally about 0.01 mg to about 100 mg protein. A single or multiple dosages can be administered.

The method of the present invention makes possible the preparation of subviral vaccines for preventing papillomavirus infection. Further, by following the methods of the invention, vaccines for any immunogenic type of specific papillomavirus can be made.

As more than one PV type may be associated with PV infections, the vaccines may comprise L1 antigenic amino acids from more than one type of PV. For example, as HPV 16 and 18 are associated with cervical carcinomas, a vaccine for cervical neoplasias may comprise L1 protein of HPV 16; of HPV 18; or both HPV 16 and 18.

In fact, a variety of neoplasias are known to be associated with PV infections. For example, HPVs 3a and 10 have been associated with flat warts. A number of HPV types have been reported to be associated with epidermodysplasia verruciformis (EV) including HPVs 3a, 5, 8, 9, 10, and 12. HPVs 1, 2, 4, and 7 have been reported to be associated with cutaneous warts and HPVs 6b, 11a, 13, and 16 are associated with lesions of the mucus membranes. See, for example, Kremsdorf et al, J. Virol., 52: 1013-1018 (1984); Beaudenon et al, Nature, 321:246-249 (1986); Heilman et al, J. Virol., 36:395-407 (1980); and DeVilliers et al, J. Virol., 40:932-935 (1981). Thus, vaccine formulations may comprise a mixture of L1 proteins from different PV types depending upon the desired protection.

In the same manner, the pharmaceutical compositions may contain a mixture of antibodies to different PV types.

As indicated, the L1 protein of the invention can be utilized for serotyping.

That is, monoclonal antibodies capable of reacting with conformationally correct L1 protein can be produced which can be used to serotype PV. In this manner, tissue or serum can be obtained from a patient and analyzed for the ability to immunoprecipitate such antibodies

In a broader sense, the antibodies can be used for serological screening. In this manner, populations of individuals can be tested for the ability to immunoprecipitate conformationally correct antibodies. Specific HPV type antibody responses can be determined.

The invention lends itself to the formulation of kits, particularly for the detection and serotyping of HPV. Such a kit would comprise a carrier being compartmentalized to receive in close confinement one or more containers, each container having antibodies for a particular HPV type or a mixture of antibodies for a variety of known HPV types. Other containers may contain means for detection such as enzyme substrates, labelled antigen/anti-antibody and the like.

For serological testing, the kits will comprise the conformationally correct recombinant L1 protein. Such a kit could also be utilized for vaccines.

While the present invention is generally directed to producing by recombinant method conformationally correct papillomavirus L1 proteins of any human or animal papillomavirus, as well the use of such proteins as vaccines, and or diagnosis and serotyping, in the preferred embodiments the recombinantly produced, conformationally correct L1 proteins will comprise human papillomavirus L1 proteins, canine oral papillomavirus (COPV) L1 proteins or equine papillomavirus L1 proteins.

As discussed supra, the canine oral papillomavirus (COPV) animal model offers a unique and highly relevant animal model for the development of human canine papillomavirus vaccines. Moreover, unlike the available rabbit and bovine papillomavirus models, COPV is tropic for mucous membranes and is assayable for infectivity under natural conditions of exposure. Using a beagle colony which exhibits a high, natural incidence of oral papillomas, the present inventors have demonstrated that these tumors express viral capsid proteins and contain intact viruses, which are preventable by immunization with virus-containing tumor extracts. Moreover, as described in greater detail infra, it has been demonstrated that administration of formalin-inactivated COPV or recombinant COPV conformational L1 proteins confers complete protection upon challenge with the virus.

As discussed, infection of the oral mucous by COPV results in the induction of well-differentiated, benign, squamous cell tumors (warts). These lesions contain episomal DNAs which have been cloned separately by two research groups (Sundberg et al, Amer. J. Vet. Res., 47:1142-1144 (1986); Bregman et al, Vet Patriol., 24:477-487, (1987). The COPV genome is slightly larger (8.2 Kb) than most other papillomavirus genomes (8.0 Kb) but the two isolates characterized to date exhibit identical restriction enzyme cleavage patterns. Inoculation of beagles with wart extracts, similar to the bovine and rabbit models, induces immunity to subsequent reinfection [unpublished results]. Unfortunately, in a small proportion of vaccinated animals, squamous cell carcinoma develops at the site of injection (Bregman et al, Vet Pathol., 24:477-487 (1987)). This presumably results from the neoplastic transformation of cutaneous keratinocytes by COPV which become entrapped in the needle during injection.

Sequencing results have demonstrated that the L1 gene of COPV is highly homologous to the L1 gene of HPV-1. Moreover, this virus possesses several critical characteristics which render it an ideal animal model for the "malignancy-associated" human papillomaviruses which distinguish it from the current rabbit and bovine models.

In particular, COPV, in contrast to CRPV, BPV-1 and BPV-2, infects and induces tumors at mucosal sites. This site mimics that for the mucosotrophic HPV-16 and HPV-18 which infect genital mucosa which are associated with cervical carcinoma. COPV has been isolated from genital mucous but not from cutaneous sites. Thus, COPV provides an ideal animal model for study of mucosotropic papillomaviruses which infect genital mucosa, and for screening and design of vaccines for providing immunity against such mucosotrophic papillomaviruses. This is extremely beneficial because of the fact that some mucosal HPVs, e.g., HPV-16 and HPV-18 are associated with cervical carcinoma.

Moreover, vaccines designed to prevent mucosal lesions may have specific requirements for generating IgA responses and for initiating an immune response in a specific subset of B lymphocytes.

Additionally, unlike the currently available CRPV and BPV models, COPV exhibits a high endogenous infection in a specific beagle colony. Thus, it is possible to escalate the efficacy of vaccines for preventing this naturally occurring infection. By contrast, the bovine and rabbit models require artificial means of infection (cutaneous abrasion) which may not necessarily reflect the natural mechanism of mucosal infections. Therefore, the beagle/COPV model should permit enhanced evaluation of the efficacy of putative vaccines against mucosal papillomaviruses such as COPV, HPV-16 and HPV-18 since it will better mimic in-vivo conditions than the CRPV and BPV models.

Further, carcinomas can develop at the site of benign tumors in a small percentage of animals as well as at the site of injection of crude "live" wart extracts. The limited conversion of benign lesions into carcinomas is also observed in human infected by mucosal papillomaviruses (HPV-16 and HPV-18) and represent the most serious consequence of HPV infection. Malignant conversion does not occur with cutaneous BPV, but does occur with CRPV in domestic rabbits.

This is highly significant because an effective vaccine against human papillomaviruses cell potentially reduce the incidence of human cervical dysplasia and carcinoma by 90-95%. However, due to the species specificity of these viruses, there are no animals into which HPV may be introduced to evaluate such vaccines. Moreover, because there are currently no tissue culture methods for propagating the virus, thereby eliminating the ability to assay viral neutralization in vitro. The only viable mechanisms for developing an HPV vaccine are to use prototype animal papillomaviruses which closely mimic the human disease process.

Thus, in light of the above, COPV should afford significant advantages over available rabbit and bovine papillomavirus animal models. Further, because the capsid proteins of COPV are closely related to HPV and since the biology of COPV closely mimics that of certain human papillomaviruses, e.g., HPV-16 and HPV-18, the identification of an effective COPV vaccine will yield direct benefits both because of potential veterinary applications, and more importantly to the development of vaccines against HPV's associated with cervical carcinoma.

Therefore, the present invention provides for the production of conformationally correct COPV L1 proteins, and the use of such COPV L1 proteins as vaccines against COPV, as well as the use thereof as an in vivo animal model for the development of human papillomavirus vaccines.

The present inventors studied the ability of conformationally correct COPV L1 proteins to afford immunity against COPV challenge in a beagle colony which exhibits a natural high incidence of oral warts formation as a consequence of viral infection. However, it is expected that the present invention will be applicable with any canine which is naturally susceptible to COPV infection.

Additionally, the present COPV/canine animal model further provides a means for delineating the role of antibodies against conformational epitopes of the COPV L1 proteins, as well as the L2 protein, in providing for resistance against COPV infection upon challenge with COPV.

This may be effected by injection of COPV wart extract (known to contain COPV viral particles) into susceptible animals. Also, the L1 and the L2 genes of COPV may be expressed in expression vectors which provide for the production of conformationally correct L1 and L2 proteins. As discussed supra, this will preferably be effected using eukaryotic expression vectors, e.g., mammalian, insect or yeast cells, e.g., Saccharomyces. Particularly preferred host cells for expression of COPV L1 proteins include by way of example COS cells and recombinant baculovirus infected Sf9 cells which produce L1 proteins which self-assemble into virus-like particles which antigenically mimic the intact COPV virion.

The conformationally correct COPV L1 and/or L2 proteins will be used to screen immune animal sera for the presence of L1 and L2 specific antibodies as well as to induce immunity in susceptible animals. The present invention further facilitates the identification of optimal conditions for inducing immunity in susceptible animals against COPV infection. Additionally, given the similarities between COPV and HPVs, the present invention further enables the identification of optimal conditions for inducing immunity against HPVs, in particular HPV-16 and HPV-18.

The ability of COPV L1 and L2 antibodies to inhibit COPV-induced tumors can be evaluated using virions purified from wart tissue or from other sources such as viral-producing tumors grown in nude mice.

Also, the conformationally correct COPV L1 and L2 proteins produced according to the invention can be used to generate monoclonal antibodies which may be used as therapeutics for providing passive immunization against COPV or as diagnostic agents. Further, monoclonal antibodies generated against intact virions may be used to define the molecular location of conformational, neutralizing epitopes on the COPV L1 and L2 proteins. Moreover, due to the structural and immunogenic similarity between COPV and HPVs, these antibodies may further have potential applicability in the development of human papillomavirus vaccines and diagnostic agents.

Immunization studies with COPV conformationally correct L1 and L2 proteins produced according to the present invention should enable the precise identification of specific dosages, carriers, adjuvants, frequency of administration and route of administration which provide for optimal immunity against COPV infection and possibly against HPV infection given the substantial similarities of COPV and HPV5. Immunity will be determined by studying vaccinated animals for the spontaneous appearance of oral warts.

Additionally, at selected times pre- and post-vaccination, animals will be evaluated for the presence of IgG, IgM and IgA antibodies which react with intact virus, or with L1 or L2 proteins. The temporal production of antibodies, as well as the ability of these antibodies to inhibit COPV infection will also be tested. In this regard, the present inventors have determined that injection of purified virus-like particles, with or without adjuvant, by systemic intradermal route of administration protects beagles from intraviral challenge with infectious COPV. Also, serum produced against intact virions were found to develop rapidly and to remain high in vaccinated beagles. By contrast, control, naive beagles were highly susceptible to challenge administered by the same route.

It has further been demonstrated by the present inventors that immunoglobulin fractions from vaccinated beagles which are passively transferred into weaning recipient beagles confer complete protection against COPV challenge. This provides additional evidence that the subject conformationally correct L1 and L2 proteins confer immunity against COPV by inducing a humoral (antibody) response against conformational epitopes contained on the L1 and L2 proteins.

Claim 1 of 8 Claims

1. A vaccine for conferring protection against human papillomavirus (HPV) infection in a human susceptible to human papillomavirus infection which comprises (i) a composition comprising a formalin-treated human papillomavirus L1 protein and (ii) a pharmaceutically-acceptable carrier.

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