Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2002-06-17
2004-04-27
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C435S252300, C435S252330, C530S350000, C536S023720
Reexamination Certificate
active
06727092
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The subject invention relates to methods for the simultaneous detection of Hepatitis C Virus (HCV) antigens as well as antibodies produced in response to HCV antigens. Furthermore, the subject invention allows one to detect antigens in the early, acute stage of infection, even prior to the development of antibodies, thereby allowing for early detection of infected blood and blood products, and thus improving the safety of the blood supply.
2. Background Information
Recent epidemiological studies indicate that HCV infects more than 170 million people worldwide and that, in more than 50% of the cases, the infection is chronic. In the United States, there are approximately 4 million people infected, and 30,000 new infections are estimated to occur annually (NIH Conference,
Hepatology Suppl
1:2S (1997)). In addition, HCV is responsible for 8,000-10,000 deaths annually in the United States and is the leading indicator for liver transplantation.
The HCV genome is a single-stranded RNA molecule of positive polarity that is approximately 9400-9500 nucleotides in length. The organization of the coding regions resembles that of other flaviviruses [Major et al.,
Hepatology
25:1527 (1997)] as well as the more recently discovered GB viruses [Muerhoff A S, et al.,
J Virol
69:5621 (1995)]. The HCV genome possesses a large open reading frame (ORF) encoding a polyprotein precursor of 3010 to 3033 amino acids depending on the particular isolate [Choo et al., Proc Natl Acad Sci USA 88:2451 (1991); Grakoui et al., J Virol 67:1385 (1993)]. HCV structural genes (core and envelope) are encoded near the 5′-end of the genome, followed by the proteases and helicase, the helicase cofactor and the replicase. Noncoding regions (NCR), thought to be important in replication, are found at each end of the genome.
HCV infection occurs primarily through parenteral exposure, i.e., through shared needles, by tattooing, or through transfusion of contaminated blood or blood products. Following exposure, the virus enters a susceptible hepatocyte and viral replication occurs. There is an eclipse phase period of approximately 10 days during which time there is no evidence of viral presence (i.e., viral RNA cannot be detected), serum transaminase levels are within normal limits, and there is no evidence of an immune response to HCV [Busch et al.,
Transfusion
40:143 (2000)]. Typically, about 10 days following exposure, HCV RNA can be detected, often with viral loads between 100,000-120,000,000 HCV RNA copies per ml of serum. Several weeks later, there is typically an increase in ALT levels indicating inflammation of the liver; antibodies are detected an average of about 70 days after exposure.
One of the preventive measures employed to limit the spread of HCV infections is to screen blood for exposure to HCV, either by the detection of antibodies to HCV or by the detection of viral-specific molecules (e.g., HCV RNA or HCV core proteins) in serum/plasma. Blood or blood products derived from individuals identified as having been exposed to HCV, by these tests, are removed from the blood supply and are not utilized for distribution to recipients of blood products (see, e.g., U.S. Pat. No. 6,172,189). These tests may also be utilized in the clinical setting to diagnose liver disease attributable to HCV infection.
Due to the unavailability of native, intact HCV virions, serologic antibody tests have relied on recombinant antigens or synthetic peptides, representing selected fragments of the viral polyprotein. The first generation anti-HCV screening tests were based on detection of antibodies directed against a recombinant protein (HCV genotype 1a) originating from sequences located in the nonstructural NS-4 protein (C100-3) [Choo et al.,
Science
244:359 (1989); Kuo et al.,
Science
244:362 (1989)]. The first generation assays failed to detect antibodies in approximately 10% of individuals having chronic HCV infection and up to 10-30% of individuals presenting with acute HCV infection. The second generation anti-HCV assays have incorporated recombinant proteins from three different regions of the HCV genome (HCV genotype 1a), including amino acid sequences from the core, NS3, and NS4 protein [Mimms et al.,
Lancet
336:1590 (1990); Bresters et al.,
Vox Sang
62:213 (1992)], allowing a marked improvement over the first generation tests in identifying HCV infected blood donors [Aach et al.,
N Engl J Med
325:1325 (1991); Kleinman et al.,
Transfusion
32:805 (1992)]. The second generation assays detect antibodies in close to 100% of chronic HCV cases [Hino K.,
Intervirology
37:77 (1994)] and in nearly 100% of the acute cases by 12 weeks post infection [Alter et al.,
N Engl J Med
327:1899 (1992); Bresters et al.,
Vox Sang
62:213 (1992)]. The third generation test includes a recombinant protein expressing amino acid sequences from the NS5 region, as well as antigens from the core, NS3 and NS4. Some studies have indicated a slight improvement in sensitivity in comparing the third generation tests to second generation tests [Lee et al.,
Transfusion
35:845 (1995); Courouce et al.
Transfusion
34:790-795 (1994)], but this improvement is largely attributed to changes in the NS3 protein rather than the inclusion of NS5 [Courouce et al.,
Lancet
343:853 (1994)].
In general, the second and third generation HCV antibody tests detect exposure to HCV about 70 days after exposure. Since HCV establishes persistent, and in many cases lifelong infection, the detection of antibodies to HCV represents a very efficient method for determining exposure to HCV. However, antibody testing alone will frequently fail to detect HCV infected individuals during the first 70 days after exposure.
The existing HCV antigen tests rely on detecting the presence of the HCV core antigen in serum or plasma. The core (or nucleocapsid) protein comprises the first 191 amino acids of the polyprotein. Two different types of serologic assays have been developed which permit detection of HCV core antigens in serum. One assay format detects HCV core antigens in subjects prior to seroconversion and is utilized in screening blood donors, while the other assay format detects core antigens only in hepatitis C patients, regardless of their HCV antibody status and is utilized in clinical laboratories to diagnose exposure to HCV or to monitor antiviral therapy.
Recent data on samples obtained during the pre-seroconversion period indicate that the HCV antigen test detects exposure to HCV significantly earlier than antibody testing [Aoyagi et al.,
J Clin Microbiol
37:1802 (1999); Peterson et al.,
Vox Sang
78:80(2000); Dawson et al.,
Transfusion, SD
161, 40(2000); Muerhoff et al., 7
th
International Meeting on Hepatitis C virus and related viruses
, Dec. 3-7, 2000], and represents an alternative to nucleic acid testing for detecting exposure to HCV during the pre-seroconversion period. The advantages of HCV antigen detection are that the test is rapid, simple, may not require sample extraction or other pretreatment, and is not as prone to handling errors (e.g., contamination) as may occur in the HCV RNA tests.
In clinical laboratories, the HCV antigen test has comparable sensitivity to the HCV DNA tests in detecting exposure to HCV in patients infected with different HCV genotypes [Dickson et al.,
Transplantation
68:1512 (1999)] and in monitoring antiviral therapy [Tanaka et al.,
Hepatology
32:388 (2000); Tanaka et al.,
J Hepatol
23:742 (1995)]. Thus, HCV core antigen tests present a practical alternative to HCV RNA for screening blood donors or for monitoring antiviral therapy.
The uniqueness of the current invention lies in its ability to detect HCV antibodies and HCV antigens simultaneously (see also International Application No. PCT/JP99/04129). This combination test or “combo” assay utilizes antigen detection to identify exposure to HCV during the pre-se
Dawson George
Desai Suresh
Guetierrez Robin A.
Jiang Lily
Leary Thomas P.
Abbott Laboratories
Becker Cheryl L.
Wortman Donna C.
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