Human herpesvirus type 6 protein p100, the corresponding DNA sequences, their preparation and use

Abstract: The invention relates to the human herpesvirus type 6 protein p100 and parts thereof having its specific immunological properties. It further relates to antibodies directed to them and to the corresponding DNA sequences. They can be used in pharmaceutical or diagnostic compositions, optionally together with other HHV-6 proteins or the corresponding DNA sequences.

Description: The present invention relates to the human herpesvirus type 6 protein p100 and parts thereof having its specific immunological properties as provided hereinbelow. It further relates 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 41kda 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 an isolated DNA sequence encoding the HHV-6 (human herpesvirus type 6) protein p100 having the amino acid sequence given in Tab. 3 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 100kda 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, therefcre, 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 Tab. 3 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 Tab. 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 Tab. 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 Tab. 3 from position 2960 to position 3141 or the nucleotide sequence given in Tab. 3 from position 2408 to position 2959. These epitopes 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. Another object of the invention is to provide pharmaceutical compositions containing the HHV-6 protein p100 or parts thereof having its specific immunological properties as described above , 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 as described above the corresponding DNA Sequences of the invention. These compositions may additionally contain parts of the major capsid protein gene of HHV-6, especially the DNA sequences given in Tab. 1 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 Tab. 2 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. The present invention is explained in more detail in the following description and the figures: Tab. 1 shows the DNA sequences of the viral inserts of clones pMF94 and pMF295. Both sequences are part of the major capsid protein gene of HHV-6 as published in Littler et al., 1990. Tab. 2 shows the DNA sequence of the viral insert of clone pMF90. The sequence is identical with nucleotides 117-194 of the sequence published in Chang and Balachandran, 1991. Tab. 3 shows the complete DNA sequence of the HHV-6 EcoRI fragments numbered 6 and 7 (starting from the left end). These fragments contain the entire p100 gene of HHV-6. Furthermore, the amino acid sequence of p100 is shown. Fig. 1 shows a Western blot analysis wherein antiserum of rabbits immunized with HHV-6 infected HSB-2 cells and antibodies against the HHV-6 protein p100 purified from this antiserum are reacted with viral proteins. Fig. 2 shows the restriction map of the HHV-6 genome. Fig. 3 shows the results of the expression of HHV-6 proteins in the expression vector pROS in a Western blot with rabbit serum and a PAGE Coomassie staining. Fig. 4 shows the reactivity of the serum of four patients with HHV-6 epitopes. The DNA sequences encoding the immunogenic proteins and parts thereof were identified in a genomic HHV-6 gene bank with mono- and polyspecific rabbit antisera against HHV-6 proteins. Rabbits were immunized with whole HHV-6 infected HSB-2 cells. The obtained antiserum reacted with at least 7 viral proteins (Fig. 4). Antibodies against a 100kda protein of HHV-6 were purified from this serum. For this purpose, entire viral protein was subjected to preparative SDS polyacrylamide electrophoresis. Viral protein with a molecular weight of 100kda was transferred to nitrocellulose membranes and incubated with the diluted rabbit serum. Antibodies that were specifically bound on the nitrocellulose sheets were eluted with 100mM glycin at pH 2.7. The obtained antibodies reacted specifically with an HHV-6 virion protein of about 100kda (Fig. 4). Both serum preparations were used to screen the genomic library. The construct of a genomic library DNA from cosmids containing the entire HHV-6 genome in overlapping fragments was sheared by sonication. After addition of EcoRI linkers, EcoRI digestions and size fractionation it was ligated into the commercially available vector lambda zapII (Stratagene Inc., La Jolla, USA). After in vitro packaging a gene bank of 3 x 10 5 independent recombinants was obtained. Positive clones were identified by immunological screening using the sera mentioned and a commercially available detection system ('Pico blue', Stratagene Inc., La Jolla, USA). The identified lambda clones were then subcloned into the Bluescript SK- vector by 'in vivo excision' following the supplier's instructions (Stratagene Inc.). , Four clones that were especially reactive in Western blots (pMF101, pMF90, pMF94, pMF295) were chosen for further characterization. The inserts of these clones were sequenced by Sanger's chain termination method. Data were analyzed by the Genetics Computer Group (GCG, Madison, Wisconsin, USA) sequence analysis package. The predicted amino acid sequences were used for homology searches with the computer program FASTA (Pearson & Lipman, 1988) in a library containing all of the published herpesvirus sequences. The clones pMF94 and pMF295 were found to contain parts of the published Major Capsid Protein gene of HHV-6 (Tab. 1) (Littler et al., 1990), while pMF90 contains part of an open reading frame homologous to UL44 of HCMV (Tab. 2). The corresponding HHV-6 gene has recently been identified using monoclonal antibodies against a phosphorylated HHV-6 protein of 41kda (chang and Balachandran, 1991). However, the epitope identified by Chang et al. is located after amino acid 227 of their sequence, while pMF90 covers amino acids 119-187 only. No homologous gene could be found for the predicted amino acid sequence of clone pMF101. The insert of pMF101 was used to locate the gene within the virus genome. By hybridization with 7 cosmid clones that encompass the entire HHV-6 genome (Neipel et al., 1991) it could be located within an 1.4kb EcoRI fragment close to the left terminal repeat (Fig. 2). Further sequencing in this area revealed an open reading frame coding. for a protein of 870 amino acids with a predicted molecular weight of 97kda (termed p100 hereinafter). Five fragments of p100, comprising almost the complete protein (pDF446-4, pDF446-3, pD2Hae, pD2Hind, pMF101R), were prokaryotically expressed as β-galactosidase fusion protein in the vector pROS (Ellinger et al). In Western blot assays only the carboxyterminal clones reacted with both rabbit human HHV-6 positive sera (Fig. 3, Fig. 4). Fusion protein expressed from pMF101R was used to purify antibodies from rabbit serum as described above. The antibodies were used to carry out Western blot analyses with HHV-6 infected and uninfected HSB-2 cells. A protein of 100kda was detected in infected cells only. Of all expression clones investigated so far the carboxyterminal parts of p100 were most reliably recognized by human HHV-6 positive sera in Western blot analyses. Since it would be possible only with great technical elaboration to isolate virion proteins in the amounts necessary for diagnostic aids, the manner of preparation by gene manipulation according to the invention is especially advantageous. In Western blot analyses using HHV-6 infected cells a protein of 100kda is recognized most reliably by human sera. It could not have been expected that prokaryotically expressed p100 or parts thereof are invariably recognized by human sera, as the homologous gene of HCMV codes for a much larger protein, and the immunogenic parts of the HHV-6 gene did not show any homology to HCMV pp150. It is also surprising that the. prokaryotically expressed part. of an phosphorylated HHV-6 protein homologous to HCMV UL44 (pMF90) is recognized by most HHV-6 positive human sera. It is possible according to the invention to use p100 and/or the fragment of the UL44 homologue of HHV-6 (pMF90) and/or the phosphorylated HHV-6 protein of 41 kD, or immunogenic parts thereof, which have been prepared, in prokaryotic or eukaryotic cells, for example yeast cells, human or animal cells, as a reagent for detecting HHV-6 antibodies, for example in an ELISA assay. EXAMPLE A fragment of 182 bp from the carboxyterminal part of HHV-6 p100 (nucleotides 2960-3141 in Tab. 3) was ligated in the expression vector pROS (Ellinger,S. et al., 1989). The clone is termed pMF101R. The BamHI-HindIII fragments from plasmid pMF90, pMF94, and pMF295 were also ligated into pROS. They are termed pD2MF90, pD2MF94, and pD2MF295, respectively. Transformation of the resulting hybrid plasmid into E. coli JK50 was followed by isolation of clones whose plasmid DNA had the expected restriction pattern. After induction of the lac promoter with isopropyl-β-D-thiogalacto-pyranoside (IPTG) the clones expressed large amounts of a fusion protein having a viral fraction. The fusion proteins were isolated from the bacterial cells and used in Western blotting experiments. All human sera that were HHV-6 positive in a standard immunofluorescence assay using HHV-6 infected HSB-2 cells recognized at least one of the fusion proteins (Fig. 3). Human sera that were found to be HHV-6 negative using the immunofluorescence did react weakly or not at all. Thus, prokaryotically expressed parts of p100 or the UL44 homologue of HHV-6 can be used in a diagnostic assay that is more sensitive and specific than the immunofluorescence used so far. References: Chang, C.K. and Balachandran, N. (1991) Identification, Characterization, and Sequence Analysis of a cDNA Encoding a phosphoprotein of Human Herpesvirus 6. J. Virol., 65:2884-2894 Larcher, C., Huemer, H.P., Margreiter, R., and Dierich, M.P. (1988) Serological crossreaction of human herpesvirus-6 with cytomegalovirus (letter). Lancet, 2:963-964 Lawrence, G.L., Chee, M., Craxton, M.A., Gompels, U.A, Honess, R.W., and Barrell, B.G. (1990) Human herpesvirus 6 is closely related to human cytomegalovirus. J. Virol., 64:287-299 Littler, E., Lawrence, G., Liu, M.Y., Barrell, B.G., and Arrand, J.R. (1990) Identification, cloning, and expression of the major capsid protein gene of human herpesvirus 6. J. Virol., 64:714-722 Neipel, F., Ellinger, K., and Fleckenstein, B. (1991) The unique region of the human herpesvirus type 6 genome is essentially colinear to the UL segment of human cytomegalovirus. J. Gen. Virol. Yamamoto, M., Black, J.B., Stewart, J.A., Lopez, C., and Pellett, P.E. (1990) Identification of a nucleocapsid protein as a specific serological marker of human herpesvirus 6 infection. J. Clin. Microbiol., 28:1957-1962 Ellinger, S., Glockshuber, R., Jahn, G., and Plückthun, A. (1989) Cleavage of procaryotically expressed human immunodeficiency virus fusion proteins by factor Xa and application in western blot (immunoblot) assays; J. Clin. Microbiol. 27 (5): 971-976 Pearson, W.R., and Lipman, D. J. (1988) Improved tools for biological sequence comparison; Proc. Natl. Acad. Sci. U.S.A., 85 (8):2444-2448; INFORMATION FOR SEQ ID NO: 2 SEQUENCE CHARACTERISTICS: TYPE: nucleotide LENGTH: 441 base pairs STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: virus DNA ORGANISM: HHV-6 PROPERTIES: coding for a part of HHV-6 major capsid protein INFORMATION FOR SEQ ID NO: 3 SEQUENCE CHARACTERISTICS: TYPE: nucleotide LENGTH: 219 base pairs STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: virus. DNA ORGANISM: HHV-6 PROPERTIES: coding for a part of HHV-6 major capsid protein INFORMATION FOR SEQ ID NO: 4 SEQUENCE CHARACTERISTICS: TYPE: nucleotide LENGTH: 212 base pairs STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: virus DNA ORGANISM: HHV-6 PROPERTIES: coding for a part of phosphorylated HHV-6 protein of 41 KD

Claims: Claims for the following Contracting States : AT, BE, CH, DE, DK, FR, GB, IT, LI, LU, NL, PT, SE An isolated DNA sequence encoding the HHV-6 (human herpesvirus type 6) protein p100 having the amino acid sequence given in Tab. 3 starting from the position corresponding to nucleotide 639 to the position corresponding to nucleotide 3248. The DNA sequence according to claim 1 having the nucleotide sequence from position 639 to the position corresponding to nucleotide 3248. A DNA sequence hybridizing to a DNA sequence according to claim 1 or 2 and encoding the entire protein defined by the amino acid sequence in Tab. 3 or parts of said protein which react with antibodies specific for said protein and substantially without crossreactivity to components of human cytomegalovirus and other herpesviruses. The DNA sequence according to claim 3 having the nucleotide sequence given in Tab. 3 from position 2960 to position 3141. The DNA sequence according to claim 3 having the nucleotide sequence given in Tab. 3 from position 2408 to position 2959. An HHV-6 protein p100 or a part thereof , which reacts with antibodies specific for the protein having the amino acid sequence given in Tab. 3 and substantially without crossreactivity to components of human cytomegalovirus and other herpesviruses, being encoded by a DNA sequence according to any one of claims 1 to 5. A recombinant vector comprising the DNA according to any one of claims 1 to 5. The recombinant vector according to claim 7 in which said DNA sequence is under the control of a homologous or heterologous promoter allowing its expression in a desired host cell. A transformed host organism obtainable by transformation with the vector according to claim 7 or 8. The transformed host organism according to claim 9 which 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. A process for the production of the HHV-6 protein p100 or a part thereof as claimed in claim 6 comprising the steps of cultivating the transformed host organism as claimed in claim 9 or 10 and recovering said protein or said part thereof from the culture. A pharmaceutical composition containing a protein according to claim 6 optionally in combination with a pharmaceutically acceptable carrier and/or diluent. The pharmaceutical composition according to claim 12 for the prophylaxis or treatment of HHV-6 infections. A composition comprising a part of the HHV-6 protein p100 as claimed in claim 6 and/or the DNA encoding said part as claimed in anyone of the claims 2 to 5. The composition according to claim 14, additionally containing parts of the major capsid protein gene of HHV-6, especially the DNA sequences given in Tab. 1 and/or the polypeptide being encoded by these DNA sequences. The composition according to claim 14, additionally containing parts of the gene encoding the phosphorylated HHV-6 protein of 41 kD, especially the DNA sequences given in Tab. 2 and/or the polypeptide being encoded by these DNA sequences. The composition according to any one of the claims 14 to 16 for carrying out an ELISA assay. The composition according to any one of the claims 14 to 17 for the differential diagnosis of HHV-6. Claims for the following Contracting State : ES A process for the production of a HHV-6 protein p100 or a part thereof, which reacts with antibodies specific for said protein and substantially without crossreactivity to components of human cytomegalovirus and other herpesviruses, being encoded by a DNA sequence encoding the HHV-6 (human herpesvirus type 6) protein p100, having the amino acid sequence given in Tab. 3 starting from the position corresponding to nucleotide 639 to the position corresponding to nucleotide 3248, comprising the steps of cultivating a transformed host organism containing a vector with said DNA sequence and recovering said protein from the culture. A process according to claim 1, wherein said DNA sequence has the nucleotide sequence from position 639 to position 3248. A process according to claim 1 or 2, wherein said DNA sequence hybridises to a DNA sequence according to claim 1 or 2 and encoding a protein having the specific immunological properties of the HHV-6 protein p100. A process according to claim 3, wherein said DNA sequence has the nucleotide sequence given in Tab. 3 from position 2960 to position 3141. A process according to claim 3, wherein said DNA sequence has the nucleotide sequence given in Tab. 3 from position 2408 to position 2959. A process according to any of claims 1 - 5, wherein said DNA sequence is under the control of a homologous or heterologous promoter allowing its expression in a desired host cell. A process according to any of claims 1 - 6, wherein the transformed 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. A process for producing a pharmaceutical composition by mixing a protein produced according to any of claims 1 - 7 optionally with a pharmaceutically acceptable carrier and/or diluent. A process according to claim 8, wherein said pharmaceutical composition is for the prophylaxis or treatment of HHV-6 infections. A process for producing a composition containing a DNA sequence encoding the HHV-6 (human herpesvirus type 6) protein p100 having the amino acid sequence given in Tab. 3 starting from the position corresponding to nucleotide 639 to the position corresponding to nucleotide 3248 and/or a protein produced according to any of claims 1 - 7. A process according to claim 10, wherein said DNA sequence has the nucleotide sequence from position 639 to position 3248. A process according to claim 10 or 11, wherein said DNA sequence hybridises to a DNA sequence according to claim 10 or 11 and encoding a protein which reacts with antibodies specific for the HHV-6 (human herpesvirus type 6) protein p100 and substantially without crossreactivity to components of human cytomegalovirus and other herpesviruses. A process according to any of claims 10-12, wherein said composition additionally contains parts of the major capsid protein gene of HHV-6, especially the DNA sequences given in Tab. 1 and/or the polypeptide being encoded by these DNA sequences.

Inventors: FLECKENSTEIN BERNHARD, NEIPEL FRANK, FLECKENSTEIN, BERNHARD, NEIPEL, FRANK
Applicants: DADE BEHRING MARBURG GMBH
Application year: 1992
Main classification: C12N15
IPC codes: C12N15, A61K38, A61K39, C12N5, C07K14, G01N33, A61P31, C12P21, C12N1, C12R1, C07K16