Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1997-03-25
2001-10-23
Scheiner, Laurie (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069300, C435S071100, C435S254110, C435S254210, C435S254200, C435S254230, C424S227100
Reexamination Certificate
active
06306625
ABSTRACT:
FIELD OF THE INVENTION
This invention refers generally to the construction of yeast strains synthesizing simultaneously different heterologous proteins or polypeptides. More specifically the invention refers to the production of hepatitis B surface antigen particles by introducing two or more vectors, each carrying an individual polypeptide expression cassette into a single yeast cell and allowing assembly of the mixed (composite) particle.
BACKGROUND OF THE INVENTION
Hepatitis B virus (HBV) infection in humans is associated with the occurrence in the serum of various structures carrying the hepatitis B surface antigen (HBsAg) [Tiollais et al,
Nature,
17:489 (1985)]. In addition to infectious virions, filamentous and spherical particles of 22 nm in diameter (containing about 100 envelope proteins) are present which are formed by association of host-derived lipids with the three hepatitis surface proteins: the major (S), middle (M) and large (L) proteins. These proteins share the same sequence of 226 amino acids on the HBV genome, known as the S protein coding sequence. The entire 163 amino acid coding sequence which immediately precedes the S protein coding sequence on the HBV genome is referred to herein as the preS coding sequence. The middle protein includes the additional 55 amino acid amino-terminal region (preS2 region) which immediately precedes the S protein (M protein: 55 plus 226 amino acids). The large protein includes the remaining 108 amino acid region (preS1 region) of the preS coding sequence (L protein: 108 plus 55 plus 226 amino acids). See, Heermann et al,
J. Virol.,
52:396 (1984).
Because the promoter for the S and M specific transcripts is embedded within the open reading frame of the L protein, transformation of mammalian cells with DNA encoding the complete open reading frame for the L protein may result in synthesis of all three surface proteins. In mammalian cells, HBsAg particles are secreted [reviewed by Tiollais et al,
Nature,
17:489 (1985)]. However, an overproduction of L protein relative to S protein leads to an inhibition of secretion of HBsAg particles [See, e.g., Ou et al,
J. Virol.,
61:782 (1987)].
In yeast, transcription initiation is directed by the 5′ yeast promoter sequence and viral transcription initiation signals within the HBV genes are not functional. For that reason, expression of the genes encoding the S, M or L protein in yeast leads in each case to only one primary translation product. The S protein, the M protein and the L protein have been expressed independently by several laboratories [See, e.g., Valenzuela et al,
Nature,
298:347 (1982); Valenzuela et al,
Biotech.,
3:317 (1985); Itoh et al,
Biochem. Biophys. Res. Comm.,
138:268 (1986) and others].
Glycosylation of the surface proteins produced in
S. cerevisiae
differs from glycosylation observed in mammalian cells. The major protein is not glycosylated, whereas the middle and the large protein are produced in N- and O-linked glycosylated and non-glycosylated forms. N-linked chains of the high mannose type were identified, as well as O-linked oligosaccharide chain(s) [See, Itoh, et al, cited above; Langley et al,
Gene,
67:229 (1988)]. When extracted from yeast, both the S and M protein are recovered as lipoproteic particles closely resembling the 22 nm particle present in serum of human HBV patients. The L protein was also recovered in lipoproteic structures, but their exact nature was not elucidated [See, Rutgers et al, “Viral Hepatitis and Liver Disease”, ed. A. J. Zuckerman, A. R. Liss, New York, pp. 304-308 (1988)].
HBsAg particles have been used as carrier matrices for presentation of foreign epitopes by fusing heterologous sequences within the preS2 region, leading to particles with the heterologous epitopes exposed at their surface. In one instance, it was shown that the presentation resulted in improved immunogenicity. [Valenzuela et al,
Biotech.,
3:323 (1985); Rutgers et al,
Biotech.,
6:1065 (1988)].
S. M. Kingsman et al,
Biotech. Gen. Eng. Rev.,
3:377 (1985) refers to expression of heterologous genes in
S. cerevisiae
, including HBsAg in unglycosylated form. This document also refers to the
S. cerevisiae
Ty element for use in multicopy chromosomal integrative vectors.
Rutter et al, U.S. Pat. No. 4,769,238 refers to the synthesis of HBsAg in yeast and the construction of an expression vector containing the S protein but excluding the 163 amino acid presequence.
Valenzuela et al, U.S. Pat. No. 4,722,840 refers to hybrid polypeptides formed of an HBsAg fragment fused to a heterologous amino acid sequence defining an epitopic site of a pathogen or toxin. The presurface polypeptide of HBsAg links the particle forming and heterologous sequences.
A. A. Mohamad et al, Abstracts, Meeting on Hepatitis B. Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 27 (1987) refers to expression of HBsAg particles in a baculovirus expression system. The particles are believed to be of the S or M HBV proteins in glycosylated and unglycosylated form, the L protein in both forms, or particles having all three proteins.
R. E. Streeck et al,
J. Cell Biochem.,
12B Suppl., 6 (Abstract F010) (1988) refers to insertion of sequences, e.g. for poliovirus type 1, into the S gene of HBV and expression in mammalian cells of mixed particles containing HBs-Polio-Ag and HBsAg.
European Patent Application 0,198,474 refers to expression in bacterial cells of plasmids carrying the PreS
1
-PreS
2
-S protein coding region of HBsAg.
PCT Application WO88/01646 refers to a recombinant DNA containing a transposase, a control sequence and transposable element enabling the insertion of exogenous DNA into prokaryotic and eukaryotic cells.
Although vaccines presently described and in use have great efficacy, a certain percentage of persons receiving such vaccines, particularly immunocompromised persons, e.g., hemodialysis patients, are non- or slow responders [See, e.g., Hadler et al,
New Engl. J. Med.,
315:209-215 (1986); and Bruguera et al,
PostGrad. Med. J.,
63:155-158 (1987)].
Thus, there remains a need in the art for methods and compositions useful in preparing additional effective vaccines to HBV.
REFERENCES:
patent: 4722840 (1988-02-01), Valenzuela et al.
patent: 4769238 (1988-09-01), Rutter et al.
patent: 4818527 (1989-04-01), Thornton et al.
patent: 4963483 (1990-10-01), Ellis et al.
patent: 0 198 474 (1986-10-01), None
patent: WO 88/01646 (1988-03-01), None
Tiollais et al., “The hepatitis B virus”,Nature, 317, pp. 489-495 (1985).
Heermann et al., “Large Surface Proteins of Hepatitis B Virus Containing the Pre-s Sequence”,J. Virol., 52, No. 2 pp. 396-402 (1984).
Eble et al., “Hepatitis B Surface Antigen: an Unusual Secreted Protein Initially Synthesized as a Transmembrane Polypeptide”,Mol. Cell. Biol., 6, No. 5, pp. 1454-1463 (1986).
Ou et al., “Regulation of Secretion of the Hepatitis B Virus Major Surface Antigen by the PreS-1 Protein”J. Virol., 61, No. 3.: pp. 782-786 (1987).
Valenzuela et al., “Construction of plasmids and expression of HBsAg in Yeast”Nature, 298, pp. 348-350 (1982).
Valenzuela et al., “Synthesis and Assembly in Yeast of Hepatitis B Surface Antigen Particles Containing The Polyalbumin Receptor”,Biotech, 3, pp. 317-320 (1985).
Itoh et al., “Express of Hepatitis B Virus Surface Antigen P31 Gene in Yeast,”Biochem. and Biophys. Res. Comm., 138, No. 1 pp. 268-274 (1986).
Langley et al., “Characterization of purified hepatitis B surface antigen containing pre-S(2) epitopes expressed inSaccharomyces cerevisiae”,Gene, 67 pp. 229-245 (1988).
Rutgers et al., “Expression of Different Forms of Hepatitis B Virus Envelope Proteins in Yeast”,Viral Hepatitis and Liver Disease, ed. A. J. Zuckerman, A. R., Liss, New York, pp. 304-308 (1988).
Rutgers et al., “Hepatitis B Surface Antigen as Carrier Matrix for the Repetitive Epitope of the Circumsporozoite Protein ofPlasmodium falciparum”, Biotech., 6, pp. 1065-1070 (1988).
Kingsman et al., “Heterologous Gene Expression i
Jacobs Eric
Rutgers Apolonia
Kerekes Zoltan
Kinzig Charles M.
Scheiner Laurie
SmithKline Beecham Biologicals, sa
Venetianer Stephen
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