Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2002-01-03
2004-01-27
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C536S024300, C536S024320, C536S024330
Reexamination Certificate
active
06682884
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of Invention
The present invention relates to methods and devices for the diagnosis of infections. In particular, the present invention relates to methods and kits for detection of
Hepatitis B
Virus Viremia (HBV).
2. Description of the Prior Art
Hepatitis B
virus (HBV) is an enveloped hepatotropic DNA virus. Acute and chronic HBV infection causes significant liver diseases and it is estimated that more than 300 million individuals world wide are chronically infected with HBV. The HBV genome is unique in the world of viruses due to its compact nature, use of overlapping reading frames, and dependence on a reverse-transcriptional step, though the virion contains primarily DNA. The human
hepatitis B
virus is a member of the
Hepadna Viridae
family which includes duck hepatitis virus (DHBV), Ground squirrel hepatitis virus (GSHV), snow goose
hepatitis B
virus (sgHBV), woodchuck hepatitis virus (WHV) and wooly monkey hepatitis virus.
Furthermore, persistent viral infection leads to chronic active hepatitis, liver cirrhosis and the development of
hepatocellular carcinoma.
It has recently been appreciated that individuals who recover from HBV infection have a broad based cellular immune response to HBV structural proteins. Indeed, cytotoxic lymphocyte activity (CTL) may be critical for promoting viral clearance from the liver and CTL activity has been detected many years after resolution of acute infection. The presence of CTL activity may be due to persistence of low level HBV infection in the liver that can be identified only by molecular techniques such as PCR. Thus, the concept has arisen that even if individuals serologically recover from HBV infection, the virus, in most instances is never completely irradicated from the liver.
Hepatitis B
is of great medical importance because
Hepatocellular carcinomas
(HCC), one of the most common cancers afflicting humans, is primarily caused by chronic HBV infection. In the last few decades, the correlation between HBV and the development of HCC has been well established. However, the mechanism by which HBV transforms hepatocytes remains elusive. It is noticed that before HBV can transform a cell, the virus first infects it. However, the mechanism through which HBV enters hepatocyte has not been resolved despite further understanding of the viral protein involved. Much more research is needed before it is fully understood by the scientist and the spread of this infectious agent is controlled.
It is noticed that in individuals who become persistently infected with HBV is due to lack of a broad based cellular immune response for unclear reasons. In this context, there are often deletions and mutations within the envelope and core genes that may allow for persistent viral infection to occur. Alternatively, these mutant viral strains may evolve as the result of immune selection pressure by the host. In specific instances, mutations in the viral genome can lead to or contribute to the generation of latent viral infection. Furthermore, specific mutations in the precore region that includes the regulatory elements may lead to more severe disease such as fulminant hepatitis.
Cellular and humoral immune responses to HBV antigens are believed to play an essential role in the elimination of virus by the infected host. The activity of a broad-based cellular immune response to different HBV antigens has been demonstrated to be one of the most important factors contributing to virus clearance from infected hepatocytes. One hypothesis to explain the development of persistent viral infection is that HBV-specific CTLs are unable to clear virus from the liver because of substantially decreased intrahepatic levels or qualitative changes in CTL activity. Cellular immune responses against HBV may therefore play an important role in the pathogenesis of viral hepatitis as well as determine the development, severity and outcome of chronic liver disease. The cellular immune response to HBV is strong and multispecific in acutely infected patients, and these T lymphocytes typically secrete TH1-like antiviral cytokines such as interferon-a (IFN-a) and tumor necrosis factor-a (TNF-a) upon antigen stimulation. In contrast, the cellular immune response in patients persistently infected with HBV is weak and narrowly focused.
However, it is known that some chronically infected individuals spontaneously clear HBV from serum, and this phenomenon is often accompanied by increased CD4+ T lymphocyte responses and acute exacerbation of liver disease as manifested by increased serum alanine aminotransferase levels. The observation of spontaneous HBV clearance in some patients implies that the suboptimal cellular immune response may be reversible. Therefore, strategies to enhance the HBV-specific immune response or to alter the balance between certain components of the response may be able to terminate persistent infection.
Early and rapid diagnosis of HBV infection is of great importance. Yet, conventional methods for detection of HBV from serum, plasma are inaccurate and/or slow. Serologic markers are commonly used as diagnostic and/or prognostic indicators of acute or chronic HBV infection. The most common marker of HBV infection is the presence of HBV surface antigen (HBs Ag). Although carriers may clear HBs Ag and develop antibody to HBs Ag, there appears to still be a risk of serious liver complications later in life. HBs Ag is generally used as a secondary marker to indicate active HBV replication associated with progressive live disease. Failure to clear HBs Ag appears to increase the risk of end stage liver disease.
Various strains of HBV can either produce HBs Ag that is not detectable in serum or the strain can lose the ability to make HBs Ag even when an active infection is present. The ability to detect HBV DNA in serum has been reported to have prognostic value for the outcome of acute and chronic HBV infections. The methodology can allow the detection of HBV DNA after HBs Ag clearance or detection of HBV lacking serologic makers.
The following requirements need to be fulfilled for an optimal assay for HBV diagnosis.
High sensitivity and specificity;
Rapid results; and
High reproducibility.
Kits for detection of HBV are commercially available. One such kit is produced by Hoffmann-La Roche and sold under the tradename Amplicor. This kit makes use of amplification by Polymerase Chain Reaction (PCR) to create amplicons specific to HBV followed by Enzyme Linked Immunosorbent Assay (ELISA) to detect the amplicons. Furthermore, the test is an in vitro amplification test for the quantification of
Hepatitis B
Viral DNA in human serum or plasma. The test is not intended for use in screening blood or blood products for the presence of
Hepatitis B
Virus.
In light of the foregoing, there is a need for a more sensitive and specific detection protocol for clinical samples. The inventors of the present invention have been successful in developing a kit and a method for detecting HBV in a more sensitive, specific and rapid manner. The present invention obviates the problems associated with the conventional kits.
Several terms used in the invention are defined as follows.
The term “primer” refers to a synthetic oligonucleotide sequence synthesized for annealing to a specific nucleotide sequence of interest. The primer initiates DNA synthesis to occur using thermostable DNA dependent DNA polymerase. Selecting the proper primer is one of the most important steps in designing a PCR kit. The primer set must hybridize to the target sequence with little or no hybridization to other sequences that are also present in the sample.
The term “probe” refers to a synthetic oligonucleotide sequence which lies internal to the + strand of the amplified product resulting from a PCR reaction.
The term “hybridization” refers to annealing of nucleotide sequences to each other under optimal conditions. Typically, a nucleotide A binds to nucleotide T and nucleotide G binds to nucleotide C.
The term “biological samples” refers to the samples
Kondiboyina Venkata Ramana
Sharma Vijay
Lackenbach & Siegel LLP
Nissen J. Harold
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