Chemistry: molecular biology and microbiology – Vector – per se
Patent
1998-01-26
2000-12-12
Degen, Nancy
Chemistry: molecular biology and microbiology
Vector, per se
536 2372, C12N 1511, C12N 1581
Patent
active
061597295
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention is directed to a synthetic DNA molecule encoding purified human papillomavirus type 11 L1 protein and derivatives thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic of the construction of the HPV6/11 hybrid L1 gene, using synthetic oligonucleotides.
FIGS. 2A-2D show the nucleotide sequence of the HPV6/11 hybrid (SEQ ID NO:38), published HPV6a (SEQ ID NO:36) and published HPV11 L1 (SEQ ID NO:37) genes.
FIG. 3 shows the bidirectional yeast expression vector pGAL1-10 used to express papillomavirus L1 capsid proteins.
FIG. 4 is a Northern analysis of HPV11 L1 mRNA from yeast.
FIG. 5 shows expression of HPV11 L1 protein in yeast by Western analysis (immunoblot).
FIG. 6 shows ELISA reactivities of HPV11 L1 VLPs expressed from wild-type (wt) HPV11 compared to HPV6/11 hybrid DNA.
FIG. 7 is an electron micrograph of HPV11 L1 VLPs expressed in yeast.
FIGS. 8A-8B shows the nucleotide sequence of the HPV6/11 hybrid gene (SEQ ID NO:34).
BACKGROUND OF THE INVENTION
Papillomavirus (PV) infections occur in a variety of animals, including humans, sheep, dogs, cats, rabbits, monkeys, snakes and cows. Papillomaviruses infect epithelial cells, generally inducing benign epithelial or fibroepithelial tumors at the site of infection. PV are species specific infective agents; a human papillomavirus cannot infect a nonhuman animal.
Papillomaviruses may be classified into distinct groups based on the host that they infect. Human papillomaviruses (HPV) are further classified into more than 70 types based on DNA sequence homology. PV types appear to be type-specific immunogens in that a neutralizing immunity to infection by one type of papillomavirus does not confer immunity against another type of papillomavirus.
In humans, different HPV types cause distinct diseases. HPV types 1, 2, 3, 4, 7, 10 and 26-29 cause benign warts in both normal and immunocompromised individuals. HPV types 5, 8, 9, 12, 14, 15, 17, 19-25, 36 and 46-50 cause flat lesions in immunocompromised individuals. HPV types 6, 11, 34, 39, 41-44 and 51-55 cause nonmalignant condylomata of the genital or respiratory mucosa. HPV types 16, 18, 31, 33, 35, 45, and 58 cause epithelial dysplasia of the genital mucosa and are associated with the majority of in situ and invasive carcinomas of the cervix, vagina, vulva and anal canal.
Papillomaviruses are small (50-60 nm), nonenveloped, icosahedral DNA viruses that encode for up to eight early and two late genes. The open reading frames (ORFs) of the virus genomes are designated E1 to E8 and L1 and L2, where "E" denotes early and "L" denotes late. L1 and L2 code for virus capsid proteins. The early (E) genes are associated with functions such as viral replication, transcriptional regulation and cellular transformation.
The L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa. L2 protein is a minor capsid protein which has a predicted molecular weight of 55-60 kDa and an apparent molecular weight of 75-100 kDa as determined by polyacrylamide gel electrophoresis. Immunological data suggest that most of the L2 protein is internal to the L1 protein within the viral capsomere. The L1 ORF is highly conserved among different papillomaviruses. The L2 proteins are less conserved among different papillomaviruses.
The L1 and L2 genes have been identified as good targets for immunoprophylaxis. Some of the early genes have also been demonstrated to be potential targets of vaccine development. Studies in the cottontail rabbit papillomavirus (CRPV) and bovine papillomavirus (BPV) systems have shown that immunizations with recombinant L1 and/or L2 proteins (produced in bacteria or by using vaccinia vectors) protected animals from viral infection. Expression of papillomavirus L1 genes in baculovirus expression systems or using vaccinia vectors resulted in the assembly of virus-like particles (VLP) which have been used to induce high-titer virus-neutralizing antibody responses that correlate with protection from viral challenge. Furthermore, the L1 and L2
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George Hugh A.
Hofmann Kathryn J.
Jansen Kathrin U.
Joyce Joseph G.
Lehman E. Dale
Degen Nancy
Giesser Joanne M.
Merck & Co., Ltd.
Schwartzman Robert
Tribble Jack L.
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