Antibodies and other binding molecules specific for...

Details Antibodies and other binding molecules specific for... Antibodies and other binding molecules specific for...

2000-01-13

2003-04-01

Wortman, Donna C. (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S339000, C530S388300, C436S820000

Reexamination Certificate

active

06541198

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to antibodies land other binding molecules specific for hepatitis B viral antigens (HBV), peptides comprising epitopes recognised by such molecules, and cell lines capable of producing antibodies. The invention is further concerned with the use of such molecules in diagnosis of hepatitis B virus (HBV).
BACKGROUND OF THE INVENTION
The virus that causes hepatitis B or serum hepatitis appears to infect only man and chimpanzees. Hepatitis B virus (HBV) infection in humans is widespread.
The hepatitis infection is transmitted by three general mechanisms: (1) by parenteral inoculation of infected blood or body fluids, either in large amounts as in blood transfusions or in minute amounts as through an accidental skin prick; (2) by close family or sexual contact; and (3) by some mothers, who infected during pregnancy, transmit the virus to their new-born children. Under natural conditions, HBV is not highly contagious. Transmission by inhalation occurs rarely, if ever.
The transmission route through contaminated blood or blood products is a major threat to the human health.
Infection with HBV often results in subclinical or acute self-limited liver disease or can result in chronic long-term infection. Chronic HBV infection elicits a spectrum of disease entities ranging from the most severe form of chronic active hepatitis (CAH) to less severe chronic persistent hepatitis (CPH) to the asymptomatic carrier (ASC) state. An array of diagnostic assays have recently been developed to aid the clinician in differentiating hepatitis B virus infections from other forms of viral hepatitis (i.e., HAV, HEV, HCV). However, the ability to distinguish between an acute hepatitis B (AH-B) infection and symptomatic chronic hepatitis B (CH-B) infection is still problematic. This is especially true since CAH and CPH patients often demonstrate a cyclic pattern of hepatitis characterised by acute exacerbations (A.E.) of liver injury alternating with normal liver function.
After infection with HBV, large quantities of the virus and associated particles are present in the serum. During symptomatic phases of infection, both acute and chronic HBV patients have elevated liver enzyme levels, possess the hepatitis B surface antigen (HBsAg) in their serum, and produce antibodies to the nucleocapsid antigen (HBcAg). Antibodies specific for the HBsAg or the hepatitis B e antigen (HBeAg) are not detected. The appearance of antibody to HBsAg is usually not observed until approximately two months following disappearance, of circulating HBsAg. The viral particles present in the serum are known to shed their surface coat exposing the nucleocapsid, known as the core antigen (HBcAg). Antibody production of HBcAg occurs early in the course of the acute phase of HBV infection and can persist for many years, and chronically infected patients can produce high titers of anti-HBc antibodies.
The HBsAg is established as the most important marker of acute or chronic hepatitis B infection, detectable in serum of infected individuals. HBsAg screening of donor blood for example, is essential to avoid transmission of hepatitis B. It is clear that sensitivity is of utmost importance in diagnostic HBV assays.
HBV Surface Antigens (IBsAg)
The HBV surface antigens (HBsAg) are the translational products of a large open reading frame (ORF) that is demarcated into three domains; each of these domains begins with an in-frame ATG codon that is capable of functioning as a translational initiation site. These domains are referred to as Pre-S1, Pre-S2, and S in their respective 5′ to 3′ order in the gene. Thus, these domains define three polypeptides referred to as S or HBsAg (226 amino acids), Pre-S2+S (281 amino acids), and Pre-S1+Pre-S2+S (289-400 amino acids), also referred to, respectively, as major protein (S-protein), middle protein (M-protein), and large protein (L-protein) (Toillais et al., 1985, Nature, 317, 489-495).
Definition of an HBsAg Subtype
The HBsAg in the viral envelope part of HBV has one well-characterised group specific determinant “a” and two sets of mutually exclusive subtype determinants d/y and w/r. Thus four major subtypes of HBsAg—adw, ayw; adr, and ayr—denote the phenotypes of the virion (Le Bouvier et al., 1975, Amer. J. Med. Sci. 270, 165). Subdivision of “a” specificity into al, a2, a3, and other intermediate specificities which are later redefined as subdeterminants of w (w1-w4) at an international workshop in Paris in 1975 (Courouce et al., 1976, BibI Hematol, Basel, Karger, vol. 42), the issue of HBsAg subtypes acquired a considerable degree of complexity. These subtypes were ayw1, ayw2, ayw3, ayw4, ayr, adw2, adw4, and adr. With the identification of the q determinant (Magnius et al., 1975, Acta Pathol Micr Scand, 83B, 295-297) the number of subtypes increased from eight to nine, due to the subdivision of the adr subtype into a q-positive and a q-negative category (Courouce-Pauty et al., 1978, Vox Sang, 35, 304-308). Sequencing of complete genomes encoding adw2 and ayw3 subtypes revealed numerous substitutions throughout the genome (Valenzuela et al., 1980, ICN-UCLA, Symposia on Animal Virus Genetics, NY, Ac. Press, pp57-70). A number of these substitutions in the S-gene were claimed to be associated with the expression of d and y specificity (Okamoto et al., 1986, J Gen Virol, 67, 2305-2314). Analysis of reactivity patterns with monoclonal antibodies after chemical modification of HBsAg revealed the importance of Lys 122 for the expression of the d determinant (Peterson et al., 1984, J Immun, 132, 920-927). Later studies on two blood donors carrying surface antigens of compound subtypes adyr and adwr respectively, showed that amino acid substitutions at position 122 and 160 alone explained the expression of d/y and w/r specificity, respectively (Okamoto et al., 1987, J Virol, 61, 3030-3034). Both the d to y and w to r changes were mediated by a shift from Lys to Arg at the corresponding positions. Therefore, major subtypic variations of HBsAg exist.
Definition of an HBsAg Genotype
Sequencing of viral genomes, and comparison has defined four genomic groups of HBV on a divergence of 8% or more of the complete genome, and were designated with A-D (Okamoto et al., 1988, J Gen Virol, 69, 2575-2583). Genomes encoding the subtype adw were found in genomic groups A-C, while the genomes encoding ayw were all found in group D and group B (Sastrosoewignjo et al., 1991, J Gastroenterol Hepatol, 6, 491-498). Genomes encoding both the adr and ayr subtype occurred in genomic group C alongside with adw.
Also two new genotypes of HBV designated with E and F were recently identified (Norder et al., 1994, Virology, 198, 489-503).
Immune Escape Mutants in Relation to Genotypes
Apart from the genetic variability of HBV based on the divergence of HBV strains over long periods of time resulting in geographically related subtypes and genotypes, considerable interest has recently also been focused on two kinds of immune escape mutants. The first of these to be described was a mutation from Trp 28 to a stop codon in the precore sequence, that specifically prevented the expression of HBeAg although leaving that of HBcAg unaffected (Carman et al., 1989, Lancet, ii, 588-591; Brunetto et al., 1991, Proc Natl Acad Sci USA, 88, 4186-4190).
Vaccine escape mutants are described involving the “a” determinant of HBsAg, an important part of which is formed by a loop encompassing amino acid residues 139-147 stabilised by a disulphide bridge between two cysteinic residues at these positions (Waters et al., 1991, Virua Res, 22, 1-12; Stirk et al., 1992, Intervirology, 33, 148-158). One mutation from Gly to Arg at residue 145 of HBsAg was revealed in several vaccinees in Italy (Carman et al., 1990, Lancet, ii, 325-329) and Singapore (Harrison et al., 1991, J Hepatol 13 (suppl 4), S105-107). Another presumed vaccine escape mutation from Lys to Glu at position 141 has only been reported from West Africa (Allison et al., 1993, Abstr. Ixth Int Congr of Virology, Glasgow, pp1

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