Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2001-05-09
2002-11-12
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C435S005000, C435S069100, C435S071100, C530S388300
Reexamination Certificate
active
06479282
ABSTRACT:
BACKGROUND OF THE INVENTION
Hepatitis B virus (HBV) is the most significant of the hepatotrophic viruses in terms of the number of people chronically infected and the severity of the complications of infection. It is a major cause of human liver disease which can lead to chronic infection, cirrhosis and hepatocellular carcinoma, resulting in over a million deaths worldwide each year.
HBV is a double stranded DNA virus that may be carried by as much as 20% or more of the apparently healthy population in certain parts of the world, such as Africa, Asia and the Pacific Region (Principles and Practice of Clinical Virology, 3rd Edition, Chapter 2: Hepatitis Viruses, pp. 162-180). The reservoir of carriers worldwide is estimated at a number over 300 million. HBV was originally thought to be spread exclusively by blood and blood products, although it now appears that HBV can also be transmitted by intimate contact, such as sexual contact, and other routes may also be possible. Thus, transmission of infection may result from accidental inoculation of minute amounts of blood, or fluid contaminated with blood, during medical, surgical and dental procedures; immunization with inadequately sterilized syringes and needles; intravenous or percutaneous drug abuse; tatooing; ear, nose and other piercing; acupuncture; laboratory accidents; and, accidental inoculation with razors and similar objects that have been contaminated with blood.
The genomes of a variety of isolates of HBV have been cloned and the complete nucleotide sequence thereof determined. Although there is some variation in sequence (up to about 12% of nucleotides) between these isolates, the genetic organization and other essential features are conserved. The genome is around 3200 base pairs in length and analysis of the protein coding potential reveals four conserved ORFs. The four ORFs are located on the same DNA strand and the strands of the genome have accordingly been designated the plus (incomplete strand) and minus (complete strand).
HBV belongs to the hepadnaviridae family and consists of an outer envelope of host-derived lipids containing a virion-encoded surface antigen (HBsAg). This 42 nm lipoprotein shell encloses an icosahedral nucleocapsid assembled from the core antigen (HBcAg) that contains the viral genomic DNA and the viral polymerase (for review, see Nassal and Schaller, 1993). The core protein is the cytoplasmic product of the C-gene, composed of 183 or 185 amino acid residues (21 kDa) depending on sero-subtypes, which can be divided into an N-terminal assembly domain (residues 1-149) and a C-terminal very basic protamine-like domain (residues 150-185), respectively responsible for polymerization into particles and RNA packaging. The HBc protein has the ability to form disulfide-linked homodimers which spontaneously assemble into particles (Zhou and Standring, 1992).
HBcAg is a very powerful immunogen inducing strong humoral, T helper (Th) and cytotoxic T cell (CTL) responses and functioning as both T-cell-dependent and T-cell-independent antigen (Milich et al., 1997a, b). Anti-HBc arise in virtually all infected individuals. They are produced very early after infection and may be detected a few days after the detection of HBsAg in the blood of infected subjects. Moreover, in acute infections, HBsAg declines over a period of several weeks and is replaced by detectable levels of HBsAg antibody (anti-HBs). During a “window” period, when neither HBsAg nor its homologous antibody is detectable, anti-HBc may be the only detectable serological marker of HBV infection. In addition, anti-HBc usually persists longer than any other HBV marker. Anti-HBc is therefore the most useful marker for the diagnosis of an ongoing or past HBV infection and for epidemiological purposes.
Intensive studies have shown that HBcAg can be produced in a variety of heterologous expression systems including
E. coli
and undergoes correct folding and self-assembly to form core particles similar to native capsids (Pasek et al., 1979; Cohen and Richmond, 1982; Naito et al., 1997; Wizeman and von Brunn, 1999). Bacterially expressed HBc molecules assemble into particles of two sizes arranged respectively with a triangulation number T=3 (90 dimers) or T=4 (120 dimers) icosahedral symmetry (Crowther et al., 1994; Wingfield et al., 1995). The physiological implications of this dimorphic switch are not clear, although the T=4 form is reported to outnumber the T=3 form by ~13 to 1 in capsids isolated from the human liver (Kenney et al., 1995). The crystal structure of the T=4 capsid of the bacterially expressed truncated protein (aa .1-149) has been solved by X-ray crystallography to 3.3 Å resolution (Wynne et al., 1999). The monomer fold is characterized by four &agr;-helices and the absence of &bgr;-sheets. In agreement with previous biochemical analyses, the structural data revealed two regions required for the dimerization of core monomers and for the subsequent assembly of the dimers into core particles (Wynne et al., 1999).
Many different procedures have been described to purify HBcAg. Common purification procedures are based on sedimentation of the core particles on sucrose gradients which do not allow the removal of all contaminating
E. coli
material and are often associated with a low yield. As a consequence, most of the commercially available anti-HBc detection systems are immunoassays based on inhibition or competition where human anti-HBc inhibit a labeled anti-HBc from binding to an immobilized recombinant HBcAg (Pujol et al., 1994). Consequently, the presence of anti-HBc in the sample generates a low signal value, whereas its absence results in a high signal value. This conventional test format circumvents the need for highly purified HBcAg, but has potential drawbacks which include poor specificity and poor reproducibility especially near assay cutoff (Dodd and Popovsky, 1991). False-positive anti-HBc reactivity has been attributed to cross-reactive antibodies or interfering substances in human serum (Robertson et al., 1991). It has been shown that the pretreatment of serum samples with reducing agents could significantly improve the specificity of anti-HBc determination in competitive assay (Robertson et al., 1991; Spronk et al., 1991; Weare et al., 1991). However in order to avoid cost-intensive remeasurements in routine diagnosis and discarding of blood donations due to (false)-positive anti-HBc results, there is still a need for increasing the test's specificity.
SUMMARY OF THE INVENTION
In an attempt to improve the production of a high-quality, highly purified recombinant antigen for use in diagnosis of anti-HBc antibodies in biological samples, HBcAg was expressed in yeast. The highly purified recombinant protein was characterized and its suitability as a diagnostic antigen was evaluated, by way of example, in a new sandwich enzyme immunoassay (EIA) in which anti-HBc antibodies were captured by binding to recombinant HBcAg on a solid phase and then detected by using recombinant HBcAg labeled with an appropriate marker.
As explained hereabove, assays for the diagnosis of the potential presence of HBV in a patient, as indicated by the presence of anti-HBc antibodies, are known in the art. However, it is believed that such assays are not sufficiently sensitive and specific and a need exists for the production of a high-quality recombinant HBcAg allowing the development of reliable tests, in particular sandwich assays.
Consequently, the subject of the present invention is a new expression system or cassette which is functional in a cell derived from a yeast selected from the group consisting of strain Pichia and Schizosaccharomyces, especially selected from the group consisting of
Pichia pastoris, Pichia methanolica
and
Schizosaccharomyces pombe
and allowing the expression of HBc DNA or fragments thereof encoding HBcAg or fragments thereof, placed under the control of the elements necessary for its expression. A large number of these cells are commercially available in collection
Letourneur Odile
Watelet Bénédicte
Bio Merieux
Oliff & Berridg,e PLC
Park Hankyel T.
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