Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-11-16
2001-01-09
Wortman, Donna C. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S826000
Reexamination Certificate
active
06172193
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an escape mutant of the surface antigen of the hepatitis B virus.
BACKGROUND OF THE INVENTION
Serum from patients infected with hepatitis B virus (HBV) commonly have three distinct structures that contain the hepatitis B surface antigen (HBsAg): Dane particles, spherical particles, and filamentous particles. Dane particles are spheres that are 42 nm in diameter with a core that is 28 nm in diameter. The spherical particles have a diameter of about 22 nm. Filamentous particles have a diameter of about 22 nm and a length from about 50 nm to about 230 nm.
The particles contain three glycoprotein designated the major, middle, and large proteins. The hepatitis B surface antigen open reading frame of HBV-DNA is divided into three regions, pre-S 1, pre-S2, and S. This open reading frame encodes the major, middle, and large proteins. The complete amino acid sequence for the major protein is given in Valenzuela et al.,
Nature
, 280:815-819 (1979). The amino acid sequence in Valenzuela et al. is 226 amino acids long and the amino acid positions referred to in this application refer to the amino acid sequence disclosed in Valenzuela et al. HBsAg contains several antigenic determinant,;, the most important of these are the a determinant, the d/y determinant, and the w/r determinant.
Hepatitis B vaccines have been used (extensively in humans in recent years. A typical vaccine, e.g., RECOMBIVAX HB vaccine, available from Merck, contains HBsAg of the adw subtype that has been produced recombinantly in yeast. As the human population becomes vaccinated, the virus is put under pressure to evolve around the vaccine. Mutants that evolve in response to the vaccine are termed “escape mutants.” Current vaccines may not be effective against these (escape mutants. Also, some current immunodiagnostic tests may not detect these escape mutants.
Certain HBV escape mutants have been reported previously. A mutation at position 145 from glycine to arginine was reported by Carman et al.,
Lancet
, 336:325-329 (1990). Also, a mutation where additional amino acids were inserted after amino acid 122 was reported in WO 95/21189. Other mutants are described in McMahon et al.,
Hepatology,
15(5):757-766 (1992). In addition, studies have been conducted on artificial mutant proteins. An example of this type of study is Mazngold et al.,
Virology
, 211:535-543 (1995). Mangolet et al. made various mutations at positions 121, 124, 137, 139, 147, and 149.
The invention provides isolated mutant hepatitis B surface antigen proteins, fragments thereof, and particles containing these proteins. These mutant proteins, fragments, and particles can be used in improved vaccines and these mutant proteins, fragments, and particles and specific binding agents to them can be used in improved immunoassays.
SUMMARY OF THE INVENTION
The invention provides an isolated mutant hepatitis B surface antigen protein which comprises an amino acid sequence of a surface antigen protein of hepatitis B virus which infects humans, in which the amino acid at position 121 is not cysteine and at least one of the amino acids at positions 120, 122, 123, 147, or 149 is not a conserved amino acid for its position. The invention also provides an isolated mutant hepatitis B surface antigen protein which comprises an amino acid sequence of a surface antigen protein of hepatitis B virus which infects humans, in which the amino acid at position 122 is not a conserved amino acid for its position.
The invention also provides a vaccine which comprises an immunogenic amount of a mutant hepatitis B surface antigen protein, a fragment thereof, or a particle containing the protein, in a pharmaceutically acceptable carrier. The invention also provides specific binding agents which specifically recognize a mutant hepatitis B surface antigen protein, a fragment thereof, or a particle containing the protein.
The invention also provides a method for detecting in a sample a mutant hepatitis B surface antigen protein or a particle containing the protein comprising: (a) contacting a sample with a specific binding agent which specifically recognizes a mutant surface antigen protein of the invention under conditions suitable for binding to occur; and (b) measuring the extent of the binding of the specific binding agent, wherein the extent of the binding correlates to the presence or amount of mutant hepatitis B surface antigen protein or a particle containing the protein in the sample; to determine the presence or amount of mutant hepatitis B surface antigen protein or a particle containing the protein in the sample. Method; for calibrating a method for determining the amount of a mutant hepatitis B surface antigen protein or a particle containing the protein, in a sample and for testing the binding affinity of a specific binding agent a mutant hepatitis B surface antigen protein, a fragment thereof, or a particle containing the protein, are also provided.
The invention provides a method of detecting in a sample a nucleic acid sequence coding for a mutant hepatitis B surface antigen protein comprising: (a) providing a sample suspected. of containing a nucleic acid sequence coding for a mutant hepatitis B surface antigen protein of the invention; and (b) detecting the nucleic acid sequence coding for the mutant hepatitis B surface antigen protein.
Additional features and advantages of the invention are set forth in the description which follows and in part will be apparent from the description. The objectives and other advantages of the invention will be realized and attained by the isolated mutant hepatitis B surface antigen proteins, fragments thereof, and particles containing these proteins, specific binding agents thereto, and immunoassays and vaccines using these proteins, fragments thereof, and particles containing these proteins and/or specific binding agents as particularly pointed out in the written description, claims, and appended drawings. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.
REFERENCES:
patent: 5286452 (1994-02-01), Hansen
patent: 5589401 (1996-12-01), Hansen et al.
patent: 5639637 (1997-06-01), Thomas et al.
patent: 0 511 855 A1 (1992-11-01), None
patent: 0 522 030 B1 (1993-01-01), None
patent: WO 87/06594 (1987-11-01), None
patent: WO 94/21812 (1994-09-01), None
patent: WO 94/25486 (1994-11-01), None
patent: WO 94/26904 (1994-11-01), None
patent: WO 95/21189 (1995-08-01), None
patent: WO 97/39029 (1997-10-01), None
patent: WO 98/45421 (1998-10-01), None
European Search Report for Application No. 97830635.5, dated Apr. 8, 1999.
Antoni et al., “Site-directed Mutagenesis of Cysteine Residues of Hepatitis B Surface Antigen,”Eur. J. Biochem., 222(1):121-127 (May 1994).
Ashton-Rickardt et al., “Mutants of the Hepatitis B Virus Surface Antigen that Define Some Antigenically Essential Residues in the Immunodominant a Region,”J. Med. Virol., 29(3):196-203 (Nov. 1989). (abstract only).
Ashton-Rickardt et al., “Mutations that Change the Immunological Subtype of Hepatitis B Virus Surface Antigen and Distinguish Between Antigenic and Immunogenic Determination,”J. Med. Virol., 29(3):204-214 (Nov. 1989). (abstract only).
Bruce et al., “Mutations of Some Critical Amino Acid Residues in the Hepatitis B Virus Surface Antigen,”J. Med. Virol., 46(2):157-161 (Jun. 1995).
Cariani et al., “Emergence of Hepatitis B Virus S Geene Mutant in a Liver Transplant Recipient,”J. Med. Virol., 47:410-415 (1995).
Carman et al., “Vaccine-induced Escape Mutant of Hepatitis B Virus,”The Lancet, 336:325-329 (Aug. 11, 1990).
Grethe et al., “Characterization of Unusual Escape Variants of Hepatitis B Virus Isolated From a Hepatitis B Surface Antigen-Negative Subject,”J. Virol., 72(9):7692-7696 (Sep. 1998).
Howard et al., “Hepatitis B Surface Antigen Variation and Protective Immunity,”Intervirology, 38:35-40 (1995).
Mangold et al., “Secretion and Antigenicity of Hepatitits
Fiordalisi Gianfranco
Palla Mario
Primi Daniele
Brumback Brenda G.
DiaSorin International Inc.
Popovich & Wiles, P.A.
Wortman Donna C.
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