Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Patent
1996-12-27
2000-06-13
Wortman, Donna
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
435 693, 435 712, 43525233, 43525423, 4353201, 436518, 436531, 436534, 436513, 436548, 530350, C12Q 170, G01N 33569
Patent
active
060748176
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to recombinant proteic materials useful as mono- and poly-epitope artificial antigens able to detect HCMV-specific immunoglobulines in Human sera, especially IgM, in place of purified virions. In particular, the invention relates to the joint use of three different fusion proteins obtained by recombinant methods, a first one including high immunogenic epitopes both from the non structural, DNA-binding viral protein of 52 kD (pp52) and from the structural phosphoprotein of 150 kD (pp150), a second one including an immunogenic epitope from the major matrix protein of the Human Cytomegalovirus (HCMV), namely the viral protein of 65 kD (pp65) encoded by the viral gene UL83, and a third one including an immunogenic epitope from the assembly protein of 38 kD (pp38) encoded by the viral gene UL80a. The invention further relates to a diagnostic kit and to diagnostic reagents derived therefrom and to plasmids expressing said recombinant antigens.
BACKGROUND ART
Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus in man. It is rarely pathogenic in healthy adults but is associated with several diseases in immunocompromised individuals (such as HIV-infected people 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 are the result of secondary infections. As 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 111. Diagnosis of primary HCMV infection is exclusively accomplished 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 while it is very often produced during active viral reactivation in transplant recipients [2-4]. However its detection varies widely and a very poor agreement has been found among the results obtained with different commercial kits [5].
From EP 262531 there are known immunogenic portions of HCVM structural phosphoprotein of 150 kD, encoded by the gene localized in the Hind III-Y/N fragment of the viral genome; according to said European patent, 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 afore deductions to be incorrect, in that (FIG. 1), protein pp150 is shown to be encoded by UL32, much longer than 1.5 kb and, anyway, 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 (in particular, to the side, towards the NH.sub.2 -terminus) of Hind III-Y/N fragment. According to the Applicants, therefore, there is a correlation between the immunogenic properties shown by the proteic material referred to in the afore European patent (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 by UL32 nucleotides 1783 to 1842, running 5'.fwdarw.3', 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).
Antigenic materials composed of single well characterized viral proteins, or portions of them, produced via molecular biology or peptide c
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Journal of Clinical Microbiolog
Flanders Richard T.
Landini Maria P.
Maine Gregory T.
Ripalti Alessandro
Abbott Laboratories
Weinstein David L.
Wortman Donna
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