Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
2000-04-10
2004-11-16
Scheiner, Laurie (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S192100, C424S204100, C424S235100, C435S005000, C530S350000, C530S403000
Reexamination Certificate
active
06818219
ABSTRACT:
The present invention relates to a reagent for detecting and monitoring viral infections, such as those caused by the Epstein-Barr virus or EBV, which is in particular the causative agent of infectious mononucleosis, by the hepatitis C virus (HCV) or by the human immunodeficiency virus (HIV) and to its applications for the detection of a viral infection, in particular of an EBV infection, at any stage of the infection (primary infection, healthy carriers and induced tumours).
EBV is a herpesvirus which preferably infects the B lymphocytes and the epithelial cells.
This lymphocryptic virus escapes immunological surveillance. In general, the virus is carried by healthy carriers, with no special symptoms; however, under immunodeficient conditions, such as AIDS or chemotherapy following a transplant, as well as in endemic regions, such as in Africa or in Asia, the oncogenic potential of EBV is freed and leads to the emergence of various tumours, such as African Burkitt's lymphoma, undifferentiated carcinoma of the nasopharynx, certain lymphomas and Hodgkin's disease.
At present the serodiagnosis of EBV is carried out either by the Paul-Bunnell reaction (detection of heterophilic antibodies), a simple and rapid but nonspecific method (LINDERHOLM M. et al.,
J. Clin. Microbiol.,
1994, 32, 1, 259-261), by immunofluorescence (IF) tests which are specific for each class of antigens (VCA, EA and EBNA).
The development of a simpler and less expensive diagnostic test using an immunoenzymatic method such as the ELISA method is in this context particularly desirable and several tests have thus been proposed (M. GORGIEVSKY-HRISOHO et al.,
J. Clin. Microbiol.,
1990, 28, 2305-2311; J M. MIDDELDORP et al.,
J. Virol. Methods,
1988, 21, 133-159).
The immunoenzymatic methods proposed in the prior art exhibit, for the majority, the major disadvantage of lacking detection sensitivity (M. GORGIEVSKY-HRISOHO et al., cited above; J M. MIDDELDORP et al., cited above). To solve this problem of lack of sensitivity, some authors have proposed using various antigens, in combination; such combinations increase the sensitivity of the test in which these combinations are used, but not the specificity. The combinations of antigens which have been proposed in the prior art comprise, for example, the antigen p47-52 (major antigen EA-D, encoded by the open reading frame called BMRF1, SWISS-PROT accession No. P03191), the antigen p38 (encoded by the open reading frame called BALF2, SWISS-PROT accession No. P03227) and the antigen gp125 or gp110 (encoded by the open reading frame called BALF4, SWISS-PROT accession No. P03188) (W. M. J. Van GRUNSVEN et al.,
J. Virol.,
1993, 67, 3908-3916).
More recently, it has been shown that an 18 kDa capsid antigen (VCAp18), encoded by the open reading frame BFRF3 (SWISS-PROT accession No. P14348), is recognized by healthy carriers of EBV in immunoblot analysis and appears not to exhibit homologous sequences with the other human herpesviruses, unlike the major proteins, such as VCAp40 (sequence encoded by the open reading frame called BdRF1, SWISS-PROT accession No. P03219) and gp125/110 (R. BAER et al.,
Nature,
1984, 310, 207-211; M. S. CHEE et al.,
Curr. Topics Microbiol. Immunol.,
1990, 154, 125-170; A. J. DAVIDSON,
J. Gen. Virol.,
1986, 67, 1759-1816). The map of the antigenic domains of the antigen VCAp18 has been established and the major antigenic domain has been localized in the C-terminal region of the protein (W. M. J. Van GRUNSVEN et al.,
J. Infect. Dis.,
1994, 170, 13-18).
However, ELISA tests carried out with this capsid antigen VCAp18 have the disadvantage:
of not exhibiting sufficient sensitivity and specificity to detect all the anti-VCA Ig's produced (false-positives and/or false-negatives); in particular, as regards the false-negatives, they are essentially due to the small size of the synthetic peptide VCAp18/SEQ ID No. 2 (24 amino acids, SEQ ID No. 2 of the sequence listing), which may consequently not be recognized by all the VCA-positive individuals and/or because of the heterogeneity of the reactivity of the antibodies towards the synthetic peptide, compared with the same peptide, in its natural environment,
of not allowing differential diagnosis of the various stages of the EBV infection and/or of the pathologies induced by EBV, depending on the isotype profile of the Ig's produced (IgG, IgA and IgM), despite the use of the C-terminal fragment of the VCAp18 protein (SEQ ID No. 1 of the sequence listing), as reagent.
A similar situation is encountered with other viruses, such as HCV or HIV; indeed, in general, the use of one or more immunodominant fragments does not exhibit sufficient sensitivity to avoid false-negatives.
Accordingly, the Applicant company set itself the objective of providing a new reagent for the detection of viral infections, capable of being used in immunoenzymatic tests, which is both specific and sensitive and which makes it possible to obtain a gain in sensitivity of at least 15 to 30% compared with the prior art reagents.
Accordingly, the Applicant company also set itself the objective of providing a new reagent for the detection of EBV infections, capable of being used in immunoenzymatic tests, which is both specific and sensitive and which allows differential diagnosis of the stage of the infection and/or of the pathology, depending on the prevalent isotype; indeed, the presence of human anti-VCA IgMs is essentially the sign of a primary infection, the presence of human anti-VCA IgGs is essentially the sign of a past infection (healthy carriers, generally), whereas the presence of human anti-VCA IgAs suggests the emergence of a tumour. Such a reagent is more suitable for the requirements of practical use than the prior art reagents in the context of immunoenzymatic tests, in particular of the ELISA type.
The subject of the present invention is a reagent for the diagnosis of an infection caused by a virus, characterized in that it comprises essentially a mixture consisting of (1) an immunodominant fragment of a protein of the said virus comprising at most 60 amino acids, preferably between 20 and 30 amino acids and (2) a mixture (called mixotope) of convergent combinatory peptides derived from the said immunodominant fragment, which peptides are obtained by complete or partial artificial degeneration of the said immunodominant fragment by systematic or partial replacement of each amino acid with another according to a suitable replaceability matrix.
For the purposes of the invention, mixotope is understood to mean the mixture of all the combinatory peptides obtained from the selected immunodominant fragment by artificial or constructed degeneration; they are preferably obtained during a single synthesis and represent the peptide antigen and its variability in its antibody population recognition function; various mixotopes may be obtained from the same peptide; the factors which are involved in the constitution of a mixotope are:
on the one hand, the percentage degeneration of the native immunodominant fragment selected (total or partial degeneration); the conserved amino acids (isolated or forming a sequence), in the case of a partial degeneration, are preferably, as regards EBV, those which are involved in the structuring of the VCAp18 protein and
on the other hand, the mode of selection of the substitution of the amino acids of the said native immunodominant fragment; for each position of the sequence of the native immunodominant fragment chosen, the amino acid substitution is selected on the basis of the replacement matrix established by H. M. GEYSEN et al., (
J. Mol. Recog.,
1988, 1, 32-41), or modified, as illustrated in
FIG. 16
, taking into account the tolerance of the antibody recognition, depending on the amino acid substitution in the linear epitopes: for a given position, the amino acids exhibiting the highest percentage of “replaceability” are preferably chosen. However, it is preferable to take into account the conformation of the natural epitopes, before degeneration.
The mixotope
Auriault Claude
Bourez Brigitte
Diesis Eric
Gras-Masse Helene
Tartar Andre
Institut Pasteur de Lille
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Scheiner Laurie
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