Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1997-09-22
2001-01-23
Wortman, Donna (Department: 1643)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007600, C435S007920, C435S069300, C435S071100, C435S252330, C435S254230, C435S320100, C530S350000, C436S513000, C436S518000, C436S531000, C436S534000, C436S548000
Reexamination Certificate
active
06177241
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the use of peptides derived from herpesvirus proteins to improve the specificity of enzyme immunoassays for the detection of herpesvirus-specific IgM antibody.
2. Discussion of the Art
Diagnosis of herpesvirus infection is typically accomplished by serological methods, i.e., demonstration of the appearance of antibodies to herpesvirus antigens in a previously seronegative subject. Herpesvirus IgM antibodies can be a sensitive and specific indicator of primary herpesvirus infection in immunocompetent subjects. However, its detection varies widely because of low levels of IgM antibodies produced in some patients.
A problem with assays utilizing herpesvirus antigens for detecting herpesvirus-specific IgM antibodies is that the assays are so sensitive that low levels of herpesvirus-specific IgM antibodies that are present in individuals of low risk are frequently detected. In other words, these assays indicate too many false positive results. The diagnosis of recent herpesvirus infection, e.g., 0 to 8 months, is questionable because the low level of IgM antibodies present in individuals experiencing an asymptomatic reactivation from a previous herpesvirus infection may be detected. If the level of herpesvirus antigen in the reagent is reduced to improve the specificity of the assay, the sensitivity of the assay is also reduced. In other words, it is undesirable to obtain a positive result in asymptomatic patients who have not had an infection within the previous eight months. Accordingly, it would be desirable to provide an assay for herpesvirus that not only detects IgM antibodies indicative of recent infection, but that also fails to detect IgM antibodies that are not indicative of recent infection.
Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus in human beings. It is rarely pathogenic in healthy adults but is associated with several diseases in immunocompromised individuals (such as persons infected with HIV and transplant recipients). Furthermore, HCMV is the most common cause of congenital infection in humans. Intrauterine primary infections are second only to Down's syndrome as a known cause of mental retardation. Less severe complications result from secondary infections. Because infections are either asymptomatic or accompanied by symptoms that are not specific of HCMV (such as fever and leukopenia), laboratory techniques are the sole means of diagnosing acute HCMV infection. Diagnosis of HCMV infection can be obtained by direct demonstration of the virus or virus components in pathological materials or indirectly through serology. Diagnosis of primary HCMV infection is accomplished exclusively by serological methods, i.e. demonstration of the appearance of antibodies to HCMV in a previously seronegative subject. HCMV-specific IgM is a sensitive and specific indicator of primary HCMV infection in immunocompetent subjects, and it is very often produced during active viral reactivation in transplant recipients. However its detection varies widely and a very poor agreement has been found among the results obtained with different commercial kits.
European Patent Application 262531 discloses immunogenic portions of HCMV structural phosphoprotein of 150 kD, encoded by the gene localized in the Hind III-Y/N fragment of the viral genome; according to that European Patent Application, such immunogenic portions of pp150 are encoded, in particular, by an EcoRI-PstI fragment of approximately 1.5 kb, localized inside the region of EcoRI-Y fragment from HCMV genome of AD169 strain. Subsequent and more exhaustive studies have however shown the disclosure of European Patent Application 262531 to be incorrect, in that protein pp150 is shown to be encoded by UL32, is shown to have a length much greater than 1.5 kb and, finally, is shown to be quite outside any such EcoRI-PstI fragment, as may be defined within the EcoRI-Y fragment of the AD169 strain. Furthermore, the EcoRI-Y fragment is wholly outside of Hind III-Y/N fragment.
According to U.S. Ser. No. 08/765,856, filed Dec. 27, 1996 (hereinafter “U.S. Ser. No. 08/765,856”), there is a correlation between the immunogenic properties shown by the proteic material referred to in the aforementioned European patent application (which, however, providing an incorrect identification of the polypeptide, causes it to remain substantially undetermined) and the inclusion into the peptide of epitope A1C2, encoded bag UL32 nucleotides 1783 to 1842, running 5′→43′, i.e., of the region corresponding to amino acids 595 to 614 of ppUL32, whose identification is posterior (Novak J. P. et al. 1991. Mapping of serologically relevant regions of human cytomegalovirus phosphoprotein pp150 using synthetic peptides. J. Gen. Virol. 72; 1409-1413).
U.S. Ser. No. 08/765,856 discloses a recombinant proteic material, in particular, a fusion protein, capable of binding by immunoreaction with antibodies produced against human Cytomegalovirus, in particular, both IgM and IgG, capable of being used as an antigen for detecting such antibodies in the relevant serologic tests, with substantially the same efficacy as the virus and/or of infected cell lyses. U.S. Ser. No. 08/765,856 further discloses diagnostic reagents and kits derived from that proteic material.
U.S. Ser. No. 08/765,856 discloses recombinant antigens, in particular fusion proteins, to detect Cytomegalovirus-specific IgM in human sera by enzyme immunoassay. U.S. Ser. No. 08/765,856 further discloses diagnostic, reagents and kits derived from those proteic materials.
According to U.S. Ser. No. 08/765,856, there is provided a recombinant proteic material, capable of binding with antibodies against human Cytomegalovirus (HCMV), characterized in that it consists of a fusion protein comprising a first region, carrying at least part of the sequence between aa 202 and aa 434, inclusive, read in 5′→3′ direction, of protein pp52, a second region, carrying at least a part of the sequence between aa 1006 and aa 1048, inclusive, read in 5′→3′ direction, of pp150, and a third region, carrying at least a part of the sequence between aa 595 and 614 inclusive, in 5′→3′ direction, of pp150. These regions are capable of being variably arranged with respect to one another within the fusion protein.
These regions preferably comprise the sequences of pp52 and pp150 in full, and, according to a preferred embodiment, a fusion protein according to U.S. Ser. No. 08/765,856 comprises in a sequential manner, running 5′→3′: the first region, immediately downstream wherefrom there is placed the third region, and then the second region, downstream from the third region, a bridge sequence being inserted between the third and the second regions. The bridge sequence consists, running 5′→3′, of the following series of aminoacids: lys leut gln glu phe. See SEQ ID NO: 7.
U.S. Ser. No. 08/765,856 further provides for a reagent for diagnosing HCMV infection by serological methods comprising a fusion protein as defined previously. In particular, the diagnostic reagent comprises a fusion protein, whereof at least part of the amino acids is encoded by the nucleotide sequence shown in the sequence listing as SEQ ID NO: 1, read from nucleotide 001 to nucleotide 900.
U.S. Ser. No. 08/765,856 also relates to diagnostic reagents for direct detection of HCMV through serology, comprising at least one monospecific polyclonal serum, or monoclonal antibodies (MaB), directed against the fusion protein.
According to another aspect of U.S. Ser. No. 081765,856, there is provided a mixture of recombinant antigens to detect Cytomegalovirus-specific IgM in human sera by enzyme immunoassay, the mixture being characterized in that it includes, in combination:
(i) a first fusion protein comprising: a first region, carrying an amino acid sequence (H10) corresponding to at least part of the sequence between a2L 202 and aa 434, inclusive, read in 5′→3′ directio
Abbott Laboratories
Weinstein David L.
Wortman Donna
Zeman Mary K.
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