Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1995-06-06
2001-01-16
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007920, C530S350000, C530S389400
Reexamination Certificate
active
06174685
ABSTRACT:
The present invention relates to the human herpesvirus type 6 protein p100 and parts thereof having its specific immunological properties. It further relates to antibodies specifically reacting with the protein or parts thereof and to DNA sequences encoding said protein or parts thereof, to recombinant vectors containing these DNA sequences and to host organisms transformed with these vectors. Furthermore, it relates to the preparation of the proteins and DNA sequences and their use in pharmaceutical or diagnostic compositions.
The human herpesvirus type 6 (HHV-6) has recently been shown to be closely related to human cytomegalovirus (HCMV) on the basis of amino acid sequence homology (Littler et al., 1990; Lawrence et al., 1990; Chang and Balachandran, 1991; Neipel et al., 1991), genomic position and orientation of conserved herpesvirus genes (Neipel et al., 1991), and antigenic properties (Larcher et al., 1988; Yamamoto et al., 1990; Littler et al., 1990). Until today, only two proteins of HHV-6 and their genes have been described in more detail: the major capsid protein (MCP) (Littler et al., 1990) with a molecular weight of 135 kda, and a phosphoprotein of 41 kda termed HHV-6 p41 (Chang and Balachandran, 1991). The latter one is homologous to UL44 of HCMV.
In order to be able to distinguish infections caused by HHV6 and HCMV it is desirable to have a reagent which is specific for the human herpesvirus type 6.
Thus, the technical problem underlying the present invention essentially is to provide a protein having immunogenic properties and the capability to induce the formation of antibodies lacking crossreactivity with HCMV and other human herpesviruses. Furthermore, it is a technical problem to provide the corresponding DNA sequences.
The solution to this technical problem is achieved by providing the embodiments characterized in the claims.
The present invention therefore relates to a DNA sequence encoding the HHV-6 (human herpesvirus type 6) protein p100 having the amino acid sequence given in FIGS.
3
A-
3
E (SEQ ID NO:1) starting from the position corresponding to nucleotide 639 to the position corresponding to nucleotide 3248.
The protein p100 is a structural protein from human herpesvirus type 6 with a molecular weight of about 100 kda that is in part homologous to pp150 of HCMV. It can be obtained by expression of the gene which is located in the region of the EcoRI fragments 6/7 of HHV-6 strain U1102 (distance to the left end of the HHV-6 genome 21-25 kb). The protein p100 has immunogenic properties and lacks crossreactivity with human cytomegalovirus and other human herpesviruses (Yamamoto et al., 1990). It can, therefore, be used as a reagent for detecting HHV-6 antibodies and for the differential diagnosis of HHV-6 infection versus CMV-infection.
The present invention further relates to the corresponding DNA sequence given in FIGS.
3
A-
3
E (SEQ ID NO:2) from position 639 to position 3248.
A DNA sequence encoding p100 can be isolated from an HHV-6 genome as disclosed herein. If the obtained DNA sequence differs from the DNA sequence given in
FIG. 3
, the above DNA can be derived therefrom by conventional in vitro mutagenesis techniques. Furthermore, the person skilled in the art equipped with the technical teaching disclosed herein will be able to obtain the DNA sequences of the present invention by conventional DNA synthesis techniques.
In a further embodiment, the present invention relates to a DNA sequence hybridizing to the above DNA sequence and encoding a protein having the specific immunological properties of the HHV-6 protein p100. In this context, the term “hybridization” refers to conventional hybridization conditions, preferably to hybridization conditions under which the T
m
value is between T
m
=−20 to T
m
=−27° C. Most preferably, the term “hybridization” refers to stringent hybridization conditions. The term “having the specific immunological properties” characterizes the entire protein defined by the amino acid sequence in
FIG. 3
as well as parts of this protein which react with antibodies specific for the protein and substantially without crossreactivity to components of human cytomegalovirus and other herpesviruses. Examples of such immunogenic parts or epitopes of the protein are the amino acid sequences encoded by the nucleotide sequence given in
FIG. 3
from position 2960 to position 3141 (SEQ ID NO:3) or the nucleotide sequence given in
FIG. 3
from position 2408 to position 2959 (SEQ ID NO:4). These epitomes may also be used in the diagnostic composition described below.
The present invention further relates to recombinant vectors containing the above DNA sequences whereby the DNA sequences may be under the control of a homologous or heterologous promoter allowing its expression in a desired host cell.
A further embodiment of the present invention is a host organism transformed with one of the recombinant vectors of the present invention wherein the host organism is a bacterium, preferably of the genus Escherichia, a yeast, preferably of the genus Saccharomyces, a plant cell or an animal cell, preferably a mammalian cell.
The present invention also relates to the preparation of the HHV-6 protein p100 which comprises the steps of cultivating a transformed host organism and recovering said protein from the culture.
A further object of the present invention is to provide antibodies specifically reacting with the HHV-6 protein p100 or parts thereof having its specific immunological properties but not with components of human cytomegalovirus and other herpesviruses. The person skilled in the art provided with the proteins and fragments thereof of the present invention can produce these antibodies according to conventional methods. In a preferred embodiment of the antibodies of the present invention, the antibodies are monoclonal antibodies.
Another object of the invention is to provide pharmaceutical compositions containing the HHV-6 protein p100 or parts thereof having its specific immunological properties and/or antibodies directed to them, wherein the pharmaceutical compositions are suitable for the prophylaxis or treatment of HHV-6 infections.
A further object of the invention is to provide a composition containing the HHV-6 protein p100 or parts thereof having its specific immunological properties or the corresponding DNA sequences or antibodies of the invention.
These compositions may additionally contain parts of the major capsid protein gene of HHV-6, especially the DNA sequences given in
FIG. 1
(SEQ ID NOS:5 and 6) and/or the polypeptide being encoded by these DNA sequences or parts of the gene encoding the phosphorylated HHV-6 protein of 41 kda, especially the DNA sequence given in
FIG. 2
(SEQ ID NO:7) and/or the polypeptide being encoded by these DNA sequences. Since the HHV-6 protein p100 has the capability to induce the formation of antibodies lacking crossreactivity with human cytomegalovirus or human herpesviruses, it may be used in the differential diagnosis for distinguishing whether an infection is caused by HHV-6 or human cytomegalovirus or other herpesviruses.
REFERENCES:
Littler et al., Identification, Cloning, and Expression of the Major Capsid Protein Gene of Human Herpesvirus 6, Journal of Virology, vol. 64, No. 2, Feb. 1990, p. 714-772.
Larcher et al., Serological Crossreaction of Human Herpesvirus 6 with Cytomegalovirus, The Lancet, Oct. 22, 1988, p. 963-964.
Neipel et al., The Unique Region of the Human Herpesvirus 6 Genome is Essentially Collinear With the ULSegment of Human Cytomegalovirus, Journal of General Virology (1991), 72, p. 2293-3397.
Chang et al., Identification, Characterization, and Sequence Analysis of a cDNA Encoding a Phosphoprotein of Human Herpesvirus 6, Journal of Virology, vol. 65, No. 6, Jun. 1991, p. 288-2894.
Lawrence et al., Human Herpesvirus 6 Is Closely Related to Human Cytomegalovirus, Journal of Virology, vol. 64, No. 1, Jan. 1990, p. 287-299.
Josephs et al., Genomic Analysis of the Human B-Lymphotropic Virus (HBLV), Science, vol. 234, (Oct. 31, 1986
Fleckenstein Bernhard
Neipel Frank
Behring Diagnostics GmbH
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Ketter James
Yucel Irem
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