THIS INVENTION relates to papillomaviruses and in particular antigens and vaccines that may be effective in treatment of infections caused by such viruses.
Papillomavirus infections are known not only in humans but also in animals such as sheep, dogs, cattle, coyotes, wolves, possums, deer, antelope, beaver, turtles, bears, lizards, monkeys, chimpanzees, giraffes, impala, elephants, whales, cats, pigs, gerbils, elks, yaks, dolphins, parrots, goats, rhinoceros, camels, lemmings, chamois, skunks, Tasmanian devils, badgers, lemurs, caribou, armadillo, newts and snakes (see for example xe2x80x9cPapillomavirus Infections in Animalsxe2x80x9d by J P Sundberg which is described in Papillomaviruses and Human Disease, edited by K Syrjanen, L Gissman and L G Ross, Springer Verlag 1987).
It is also known (eg. In Papillomaviruses and Human Cancer edited by H Pfister and published by CRC Press Inc 1990) that papillomaviruses are included in several. distinct groups such as human papillomaviruses (HPV) which are differentiated into types 1-56 depending upon DNA sequence homology. A clinicopathological grouping of HPV and the malignant potential of the lesions with which they are most frequently associated may be separated as follows.
In a first group may be listed types 1 and 4 which cause benign plantar warts, types 2, 26, 28 and 29 which cause benign common warts, Types 3, 10 and 27 which cause benign flat warts and Type 7 which causes butcher""s warts. This first group of infections occur in normal or immunocompetent individuals.
In a second group which refer to immunocompromised individuals there may be listed Types 5 and 8 which cause highly malignant macular lesions, Types 9, 12, 14, 15, 17, 19-25, 36 and 46-50 which cause macular or flat lesions which are benign or rarely malignant. These macular lesions are otherwise known as epidermodyplasia verruci formis (EV).
In a third group which infect particularly the genital tract there may be listed Types 6, 11, 34 and 39, 41-44, and 51-55 which cause condylomata which are rarely malignant, Types 13 and 32 which cause benign focal epithelial hyperplasia, Types 16 and 18 which cause epithelial dysplasia and other lesions considerable potential including bowenoid papulosis, and Types 30, 31, 33, 35, 45 and 56 which cause condylomata with intermediate malignant potential. The condylomata appear mostly in the anogenital tract and in particular the cervix. Types 16 and 18 are associated with the majority of in situ and invasive carcinomas of the cervix, vagina, vulva and anal canal. The condylomata may also occur in the aerodigestive tract.
In particular HPV16 is associated with premalignant and malignant diseases of the genito-urinary tract, and in particular with carcinoma of the cervix (Durst et al., PNAS 80 3812-3815, 1983; Gissmann et al., J. Invest. Dermatol 83 265-285, 1984). Presently, there is no information on the role of humoral responses in the neutralization of HPV16.
The detection of antibodies against HPV16 fusion proteins (Jenison et al., J Virol 65 1208-1218, 1990; Rachel et al, Int. J Cancer 48 682-688, 1991) and synthetic HPV16L1 peptides (Dillner et al. Int. J Cancer 45 529-535, 1990) in the serum of patients with HPV16 infection confirms that there are B epitopes within the capsid proteins of HPV, though few patients have HPV16 L1-specific antibodies identified by these techniques. There is no system for HPV16 propagation in vitro, and human genital lesions produce few HPV16 virions; therefore HPV16 particles have not been available for immunological studies.
The animal papillomaviruses may also include bovine papillomavirus (BPV) and in particular types BPV1, BPV2, BPV3, BPV4, BPV5 and BPV6 which are also differentiated by DNA sequence homology. In general the other animal papillomaviruses infect deer, horses, rabbits, dogs, rodents and birds. Papillomaviruses are small DNA viruses encoding for up to eight early and two late genes. (for review see Lancaster and Jenson 1987 Cancer Metast. Rev. p6653-6664; and Pfister 1987 Adv. Cancer Res 48, 113-147). The organisation of the late genes is simpler than the early genes. The late genes L1 and L2 slightly overlap each other in most cases. The putative L2 proteins are highly conserved among different papillomaviruses particularly the sequence of 10 basic amino acids at the C-terminal end. The broad domain in the middle reveals only small clustered similarities. The L1 ORF however appears monotonously conserved in all known cases. (See Syrjanen et al above) The amino acid sequence homology reaches 50% with the comparison between HPV1a, HPV6b, BPV1 and CRPV (Cotton tail rabbit papillomavirus).
In regard to immunotherapy concerning papillomavirus infections prior methods of treatment of warts and epithelial skin lesions have involved the use of surgery which can be painful and traumatic with scarring often a result with the risk that reinfection can occur. Treatment with chemicals has also been used. A common treatment agent is salicylic acid which is the main ingredient in strengths ranging from 10% to 40% in tinctures and plasters. Formalin in strengths of 38-20% has also been proposed. Cryotherapy has been used for treatment of skin warts. Gluteraldehyde as a treatment agent has also been used. Podophyllin has also been used with varying success for both skin warts and anogenital condylomata. The types of surgery that has been used on anogenital condylomata has included surgical excision, cryosurgery and laser surgery. The use of interferons has also been proposed (see Syrjanen et al above).
Antibodies to the L1 protein of bovine papillomavirus (BPV) have virus-neutralization activity (Pilacinski et a., 1986) and HPV11 virions can be inactivated in an in vitro model by specific antisera (Christensen and Kreider, J. Virol 64 3151-3156, 1990).
There is also some evidence that spontaneous regression of HPV1-induced cutaneous warts is associated with increased humoral immune responses to wart protein (Kirchner, Prog. Med. Virol 33 1-41, 1986).
Vaccines have also been proposed with indifferent success. It has been proposed to use vaccines containing autogenous tumor homogenates [Abcarian et al J. Surg Res 22: 231-236 (1977) Dis Colon Rectum 25:648-51 (1982) Dis Colon Rectum 19: 237-244 (1976)]. However it has recently been advocated that patients should no longer be treated with autogenous vaccines because of the potential oncogenic effect of the viral DNA (Bunney 1986 Br Med J 293 1045-1047).
In relation to production of genetically engineered vaccines this matter has been discussed in Pfister (1990) above and it seems that difficulty has been experienced in obtaining an effective vaccine because of the plethora of different papillomavirus types. Pfister however points out that attention should be directed to the so called early proteins (ie. E1, E2, E3, E4, E5, E6, E7 or E8) because these proteins are most likely synthesised in the proliferating basal cells of a wart infection in contrast to the structural proteins which are expressed in the upper epidermal layers. Therefore according to Pfister (1990) virus capsid protein appears to be limited in relation to use in a vaccine. The use of recombinant vaccinia viruses in in vitro test systems for papillomavirus early proteins in eukaryotic cells has been discussed also in Pfister (1990). This may take the form of a live vaccine consisting of genetically modified vaccina virus expressing papillomavirus proteins or on the surface of paraformaldehyde fixed autologous cells infected in vitro with vaccinia recombinants or transfected with other expression vectors. Another strategy for vaccine development as discussed in Pfister (1990) is to use an immune stimulating complex of the glycoside Quil A.
Data on successful proplylactic vaccination exist only for bovine fibropapillomas homogenised homogenate of bovine fibropapillomas and has been shown to provide limited immunity (Olson et al J Am Vet Med Assoc 135, 499 (1959) Cancer Res 22 463 (1962)). A vaccine including an engineered L1 fusion protein (Pilacinski et al. UCLA Symp. Molecular and Cellular Biology New Series Vol 32 papillomaviruses Molecular and Clinical Aspects Alan R Liss New York 1985 257) has also been used in calves but proved unsuccessful in humans (Barthold et al J. Am Vet Med Assoc. 165, 276, 1974). In Pfister (1990) it is stated that there is presently no evidence for a possible prevention of HPV infection by the use of a capsid protein vaccine, but induction of an antitumor cell immunity appears to be feasible.
The L1 and L2 genes have been the basis of vaccines for the prevention and treatment of papillomavirus infections and immunogens used in the diagnosis and detection of papillomaviruses (International Patent Specifications WO8605816 and WO830623). However, it appears that no commercial usage of these vaccines have taken place.
Therefore it is an object of the invention to provide virus like particles (VLPs) which may be useful as diagnostic agents as well as forming a component of a vaccine for use with papillomavirus infections.
The invention therefore in one aspect includes a method for production of papillomavirus like particles (VLPs) including the steps of:
(i) constructing one or more recombinant DNA molecules which; each encode papillomavirus L1 protein or a combination of papillomavirus L1 protein and papillomavirus L2 protein; and
(ii) transfecting a suitable host cell with said one or more recombinant DNA molecules so that virus like particles (VLPs) are produced within the cell after expression of the L1 or combination of L1 and L2 proteins.
The invention in another aspect includes a vaccine containing the papillomavirus VLPs in combination with a suitable adjuvant.
In relation to step (i) only papillomavirus L1 protein is required to form VLPs of some papillomaviruses. Suitably only the L1 protein is required to form VLPs of BVP1, HPV11 and HPV6 including HPV6b. However VLPs may also be formed in relation to BPV1, HPV11 or HPV6b containing both L1 and L2 proteins. For the formation of VLPs of other papillomaviruses such as HPV16, both the L1 and L2 proteins are required. This situation is also believed applicable to HPV18 which has similar pathological symptoms to HPV16 and also similar DNA sequence homology. Further it will be appreciated that the L1 and L2 genes may be included in the same DNA recombinant molecule or in different DNA recombinant molecules.
Preferably the recombinant DNA molecules are contained in recombinant virus which may transfect the host cell. Suitable viruses that may be used for this purpose include baculovirus, vaccinia, sindbis virus, SV40, Sendai virus, adenovirus, retrovirus or poxviruses. Suitable host cells may include host cells that are compatible with the above viruses and these include insect cells such as Spodoptera frugiperda, CHO cells, chicken embryo fibroblasts, BHK cells, human SW13 cells, drosophila, mosquito cells derived from Aedes albopictus or monkey epithelial cells. It will also be appreciated that other eukaryote cells may comprise yeast cells or other mammalian cells.
The DNA recombinant molecule is suitable obtained from a source of papillomavirus genome whereby L1 protein or L2 protein may be amplified by PCR amplification using suitably designed primers discussed hereinafter. Preferably a gene encoding L1 protein is inserted in a plasmid containing a suitable promoter and a DNA fragment containing the L1 protein and promoter is incorporated in a primary plasmid which may constitute the recombinant DNA molecule which may be inserted into a recombinant virus vector as described above.
A gene encoding the L2 protein may also be linked to a suitable promoter and preferably a DNA fragment incorporating the L2 gene and promoter is inserted into the primary plasmid to provide a doubly recombinant plasmid or secondary plasmid which plasmid may also be inserted in a recombinant virus vector as described above to form a doubly recombinant virus vector.
However the invention also includes the embodiment wherein the primary plasmid and/or the secondary plasmid may infect a suitable host cell to produce VLPs containing L1 protein or VLPs containing L1 and L2 protein under appropriate experimental conditions. The latter VLPs are the ideal immunogen for a papillomavirus specific vaccine, as the L2 protein is immunodominant in natural infection.
Other suitable DNA recombinant molecules include cosmids as well as recombinant viruses. Suitable expression systems include prokaryotic expression systems including E coli and any plasmid or cosmid expression vector or eukaryotic systems including host cells described above in combination with a recombinant virus vector or alternatively yeast cells and yeast plasmids.
In the situation where plasmids are used which incorporate genes encoding L1 or both L1 and L2 and wherein such plasmids may infect a suitable host cell for production of VLPs such plasmids should also include a suitable promoter to enhance expression of the VLP structural proteins and a polymerase may also be utilised which is associated with the relevant promoter. However in this situation VLPs may only be obtained under specific experimental conditions.
The L1 and L2 genes may be driven off any mammalian or viral promoter with a mammalian or viral polyadenylation signal. Preferably the L1 and L2 genes are transcribed from any vaccinia virus promoter which may be an early promoter or late promoter as considered appropriate. A list of such promoters is given in Davision and Moss (1989) J. Mol. Biol 210 749-769 and (1989) J. Mol. Biol 210 771-784.
In the experimental work that has taken place the L1 gene is located downstream of a vaccinia 4b promoter and the L2 gene is located downstream of a synthetic vaccinia 28 k late promoter. The host cell is monkey epithelial cells.
The VLPs may be obtained from the transfected cells by any suitable means of purification. The VLPs may be combined with any suitable adjuvant such as ISCOMS, alum, Freunds Incomplete or Complete Adjuvant, Quil A and other saponins or any other adjuvant as described for example in Vanselow (1987) S. Vet. Bull. 57 881-896.
Reference may now be made to various preferred embodiments of the invention as illustrated in the attached drawings. In these preferred embodiments it should be noted that the specific papillomaviruses, VLPs and specific constructs of DNA recombinant molecules are given by way of example.