EP1005543A2 - Menschliches herpesvirus 7, diagnostische und immunologische reagentien und deren verwendungen - Google Patents

Menschliches herpesvirus 7, diagnostische und immunologische reagentien und deren verwendungen

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Publication number
EP1005543A2
EP1005543A2 EP98935607A EP98935607A EP1005543A2 EP 1005543 A2 EP1005543 A2 EP 1005543A2 EP 98935607 A EP98935607 A EP 98935607A EP 98935607 A EP98935607 A EP 98935607A EP 1005543 A2 EP1005543 A2 EP 1005543A2
Authority
EP
European Patent Office
Prior art keywords
human
protein
åesvirus
hhv
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98935607A
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English (en)
French (fr)
Inventor
Paola Secchiero
Gabriella Campadelli-Fiume
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universita di Bologna
University of Maryland Biotechnology Institute UMBI
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Universita di Bologna
University of Maryland Biotechnology Institute UMBI
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Application filed by Universita di Bologna, University of Maryland Biotechnology Institute UMBI filed Critical Universita di Bologna
Publication of EP1005543A2 publication Critical patent/EP1005543A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16511—Roseolovirus, e.g. human herpesvirus 6, 7
    • C12N2710/16522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Human herpesvirus 7 is a DNA-containing beta-herpesvirus isolated from peripheral blood mononuclear cells (PBMC's), from saliva of healthy individuals, or from individuals affected by chronic fatigue syndrome.
  • PBMC's peripheral blood mononuclear cells
  • the natural history of HHV- 7 infection in the human host is not well understood.
  • An association of primary infection with exanthema subitum or febrile illness has been established, in some cases with central nervous system complications such as generalized convulsions and hemiplegia.
  • the virus persists in the organism, is shed in the saliva from where it can be isolated, and can be reactivated by culturing PBMC's.
  • Cells susceptible to HHV-7 infection include CD4+ T-lymphocytes in vivo and in vitro, and CD68+ cells of the monocyte/macrophage lineage in Kaposi's sarcoma lesions.
  • CD4 is an essential component of the cellular receptor for virus entry.
  • HHV-7 is widely diffused in the general population and that seroconversion for HHV-7 occurs in most individuals during the first four years of life.
  • HHV-7 persists in the host in a latent state after primary infection and, therefore, is an opportunistic agent in immunocompromised hosts, such as transplant recipients. Once reactivated, HHV-7 may be pathogenic and would further worsen a state of immunodeficiency due to HHV-7 's selective tropism for CD4+ T-lymphocytes.
  • HHV-7 U14 gene product The gene encodes a protein of 648 amino acid residues, with a predicted molecular weight of about 74 kDa. HHV-7 genome organization overlaps with that of human herpesvirus-6 (HHV-6) and homologies at the encoded amino acid level vary from about 44% to about 85%.
  • Patent 5,230,997 to Frenkel relates to HHV-7 as a newly characterized herpesvirus
  • U.S. Patent 5,514,577 to Draper et al. relates to oligonucleotide therapies for modulating the effects of herpesviruses.
  • PCT publication WO 94/03605 relates to compositions and methods for detecting human herpesvirus-6 strain Z29. None of these documents suggest reagents for serological detection of HHV-7 as provided by the present disclosure.
  • MAbs 5E1 and 3B1 recognize a family of five antigenically related proteins (87, 85, 70, 61 and 57 kDa, respectively). The 85 kDa species is phosphorylated, and the family of proteins is designated as the pp85 complex.
  • MAb 5E1 is specific for HHV-7-infected cells as measured by lack of reactivity to uninfected cells and lack of cross-reactivity to HHV-6-infected cells.
  • Pp85 is the immunodominant target of the human response to HHV-7 infection.
  • the gene that encodes pp85 and the source of pp85 were not known prior to the present disclosure.
  • HHV-7 serodiagnosis does not exist. Serological diagnoses of HHV-7 infections are currently carried out using HHV- 7 infected cells as antigen. An important issue related to interpretation of serologic assays is specificity, especially the need for distinguishing between HHV-6 and HHV-7. Because of genetic similarity between HHV-6 and HHV-7, cross-reaction can affect results obtained in most assays based on whole cell lysates. Therefore, absorption steps with infected cell antigens from the other virus are required to increase specificity of the assays.
  • the present invention seeks to overcome these and other drawbacks inherent in the prior art by providing reagents heretofore unavailable for use in specific serological detection of HHV-7.
  • a serological assay uses antigen for the detection of specifically binding antibodies and, therefore, the success of the assay depends upon availability and purity of antigen.
  • the present inventors have mapped the gene encoding pp85, the immunodominant protein of HHV-7 infection, and have demonstrated that pp85 is encoded by the U14 gene of HHV-7.
  • the inventors have also defined a functional role for pp85 as a viral tegument protein.
  • the invention thereby also provides reagents for recombinantly expressing protein pp85, and reagents for serological detection of HHV- 7.
  • serological detection means detection of antibodies having binding specificity for pp85 protein, or for epitopic fragments thereof in a test sample.
  • the invention provides a purified nucleic acid molecule having a nucleotide sequence encoding human herpesvirus-7 pp85 protein, or an epitopic fragment thereof, the protein having an amino acid sequence essentially as set forth in SEQ ID NO:3 or SEQ ID NO:4.
  • the nucleic acid molecule may have a nucleotide sequence as set forth in SEQ ID NO: 1 or 2.
  • the nucleic acid molecule may be a fragment of SEQ ID NO: 1 or 2 that encodes an HHV-7-specific epitope of HHV-7 pp85 protein. HHV-7 specific epitopes are provided as SEQ ID NOS.:6, 7, 8, 9, " 10, or 11.
  • a further aspect of the invention is a purified nucleic acid molecule complementary to any of the above- described nucleic acid molecules.
  • Table 1 lists the identity of sequences of the present disclosure having sequence identifiers.
  • Another aspect of the invention is a purified human herpesvirus-7 pp85 protein having an amino acid sequence essentially as set forth in SEQ ID NO:3, or SEQ ID NO:4.
  • the peptide having no cross-reactivity means that antibodies having binding specificity for the peptide have no significant binding specificity for HHV-6 protein products.
  • a method for serological detection of human herpesvirus-7 in a biological sample is an important embodiment of the present invention.
  • the method provides substantially no cross reactivity with human herpesvirus-6 and comprises contacting the sample with a human herpesvirus-7-specific epitope of human herpesvirus-7 p ⁇ 85 protein; and determining binding of the epitope to the sample. Binding indicates presence of antibodies having binding specificity for human herpesvirus-7 in the biological sample.
  • the human herpesvirus-7 specific epitope may be pp85 protein.
  • Determining binding is by a method whereby binding of the pp85 protein or fragment thereof to the sample is detected, including immunofluorescence assay, enzyme-linked immunosorbent assay, radioimmunoassay, agglutination, hemagglutination, or immunoblotting, for example.
  • a further embodiment of the invention is a method for diagnosis of human herpesvirus-7 infection in a subject. The method comprises contacting a biological sample from the subject with a human herpesvirus-7-specific epitope of human herpesvirus-7 pp85 protein; and determining binding of the epitope to the sample. Binding indicates diagnosis of human herpesvirus-7 infection in the subject.
  • Another embodiment of the invention is a method for diagnosis of human herpesvirus-7 infection in a subject comprising contacting a first biological sample and a second biological sample obtained later in time than the first sample from the subject with a human herpesvirus-7-specific epitope of human herpesvirus-7 pp85 protein; and determining binding of the epitope to the first sample and the second sample. Greater binding of the epitope to the second sample compared to binding of the epitope in the first sample indicates diagnosis of human herpesvirus-7 infection in the subject.
  • the level of binding in the first sample may be considered a background level of antibody present in the subject. In this way, the subject is his or her own control.
  • a method of preparing recombinant human herpesvirus-7 pp85 protein encoded by the human herpesvirus-7 U14 gene is an aspect of the present invention.
  • the method comprises preparing a recombinant host cell bearing the U14 gene, the host being capable of expressing the encoded protein, culturing the recombinant host cell to produce pp85 protein; and collecting the pp85 protein.
  • the pp85 protein has an amino acid sequence essentially as set forth in SEQ ID NO:3, or SEQ ID NO:4.
  • a method of preparing an epitopic fragment of pp85 using a recombinant system is a further aspect of the invention.
  • the recombinant host cell may bear a fragment of the U14 gene encoding the epitopic fragment.
  • kits for serological detection of human herpesvirus-7 in a human biological sample comprises in packaged combination a carrier means adapted to receive a plurality of container means in close confinement therewith; a first container means including pp85 protein or an epitopic fragment of pp85 protein; and a second container means including a detectable antibody having specific binding affinity for human immunoglobulin.
  • the kit may further comprise a detectable antibody having specific binding affinity for pp85 protein or for an epitopic fragment thereof for use as a control.
  • a method for conferring protection to human herpesvirus-7 infection or to reactivation of HHV-7 in a subject comprising administering an epitope of pp85 protein to the subject, wherein the subject has an immune reaction to the epitope, thereby providing protection to human herpesvirus-7 infection or reactivation is a further embodiment of the present invention.
  • the pp85 protein, or epitopic fragment thereof is produced by recombinant means and is , therefore, more pure than protein isolated from nonrecombinant sources.
  • the i munodominant protein was virally encoded or cellularly encoded and induced by viral infection, or, if the protein was virally encoded, which open reading frame of the HHV-7 genome encoded the immunodominant protein.
  • a homologue of the HHV-7 U14 gene was predicted to be encoded in the HHV-6 genome.
  • the immunodominant protein for HHV-6 is not the U14 gene product, but protein pi 00, the product of the Ul l gene.
  • the immunodominant protein in HCMV is ppl50 encoded by the UL32 gene, and pp65, encoded by the UL 38 gene.
  • the U14 gene product has not previously been shown to be a major immunogenic protein. No immune response in humans to homologues of the U14 gene product has been reported prior to the present disclosure.
  • the HHV-7 cDNA expression library constructed by the present inventors as described in Example 1 herein was the first to be successfully screened using monoclonal antibody 5E1.
  • FIG. 1 shows the arrangement of clones pBLUESCRIPTTM-2c and -8a identified by immunoscreening of a HHV-7-infected cell cDNA library with MAb 5E1.
  • the cDNA from U14 ORF is cloned and expressed in fusion with the prokaryotic ⁇ -galactosidase gene.
  • Clone ⁇ BLUESCRIPTTM-8a lacks 153 bp at the N- terminus and the EcoRI cloning site is absent.
  • the HHV-7 genomic coordinates of the inserted sequence are shown below each arrangment, and the locations of the start and stop codons are indicated. Non-coding region of the cDNA is shaded. MCS, multiple cloning site.
  • the present invention provides compositions and methods for serological detection of human herpesvirus-7 where substantially no cross reactivity with human herpesvirus-6 occurs.
  • Human herpesvirus-7 U14 recombinant nucleic acid molecules are provided for generation or synthesis of protein pp85 or epitopes thereof for serodiagnostic assays.
  • Such protein or peptide epitopes are further provided for vaccines for conferring protection to herpesvirus-7 infection or reactivation.
  • the present invention results from the identification of the gene that encodes the major determinant of immunoresponse to HHV-7 infection, i.e., protein pp85.
  • the gene is viral as opposed to cellular and is the U14 gene of HHV-7. This information provides the basis for development of specific reagents for serological detection, diagnosis, immunization, and for studies of pathogenesis of HHV-7.
  • the present inventors By immunoscreening a cDNA library from HHV-7-infected cells with MAb 5E1 that is directed to the proteins of the pp85 complex, the present inventors mapped the gene encoding pp85 to the U14 open reading frame of the HHV-7 genome.
  • a prokaryotically-expressed fusion protein containing the U14 open reading frame reacted with MAb 5E1 in immunoblot assay.
  • pp85 the product of the U14 gene, is a component of the HHV-7 tegument, and suggests that the HHV-7 tegument is not a homogenous structure but rather is composed of substructures including an outermost layer containing pp85.
  • the identified gene is U14 and the open reading frame (ORF) of U14 is provided for generating viral recombinant protein p ⁇ 85, or epitopic fragments thereof, and for serodiagnostic immunoassay tests such as Western blotting or ELISA assays, for example.
  • ORF open reading frame
  • Use of recombinantly expressed pp85 protein, or epitopic fragments thereof, provides standardization and increases the sensitivity of HHV-7 serodiagnostic methods.
  • Nucleic acid embodiments of the present invention include deoxyribonucleic acid and ribonucleic acid molecules.
  • the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:l, or SEQ ID NO:2.
  • the term "essentially as set forth” means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:l, or SEQ ID NO:2, and has relatively few codons that are not identical, or functionally equivalent, to the codons of said sequences.
  • the term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, for example, and also refers to codons that encode biologically equivalent amino acids.
  • nucleic acid molecule includes nucleic acid segments and smaller fragments of such segments; and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
  • vectors including, for example, plasmids, cosmids, phage, viruses, and the like.
  • gene is used for simplicity to refer to a functional protein-, polypeptide- or peptide-encoding unit. As will be understood by those in the art in light of this disclosure, this functional term includes genomic sequences, cDNA sequences or combinations thereof.
  • “Purified” or “isolated substantially away from other coding sequences” means that the gene of interest forms the significant part of the coding region of the nucleic acid molecule, and that the nucleic acid molecule does not contain large portions of naturally-occurring coding nucleic acid, such as large genomic fragments or other functional genes or cDNA coding regions. This refers to the nucleic acid molecule as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
  • the amino acid sequence of the proteins and peptides of the present invention is also that encoded by messenger RNA.
  • Phosphoprotein pp85 is phosphorylated in in vitro infection of mammalian cells, however, the present inventors have produced the protein recombinantly in E. coli where phosphorylation is not expected to occur as described in Example 2.
  • the recombinantly-produced protein binds to Mab 5E1 and to human seropositive serum, therefore, phosphorylation is not required for use of pp85 or epitopes thereof in the immunoassays provided herein. Binding to human seropositive serum demonstrates the utility of the present reagents for diagnosis in human serum.
  • "Pp85" means either the phosphorylated or nonphosphorylated forms of the protein.
  • amino acid sequence essentially as set forth in SEQ ID NO:3, or SEQ
  • biologically functional equivalent is well understood in the art and is further defined as a protein having a sequence essentially as set forth in SEQ ID NO:3, or SEQ ID NO:4 capable of binding human seropositive serum. Modifications and changes may be made in the sequence of pp85, or an epitopic fragment thereof, and still obtain a peptide or protein having like or otherwise desirable characteristics.
  • amino acids may be substituted for other amino acids in a peptide without appreciable loss of function. Since it is the interactive capacity and nature of an amino acid sequence that defines the peptide's functional activity, certain amino acid sequences may be chosen (or its underlying DNA coding sequence) and nevertheless obtain a peptide with like properties. It is thus contemplated by the inventors that certain changes may be made in the sequence of pp85 protein, or epitopic fragments thereof, (or underlying DNA) without appreciable loss of its ability to function.
  • amino acid may be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent peptide.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those which are within ⁇ 1 are more preferred, and those within ⁇ 0.5 are most preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Epitopic regions of protein pp85 are shown herein to be immunogenic or are predicted to be immunogenic, i.e., each region may be a substantial part of an epitope that induces an immune response in a host subject.
  • An epitope is a relatively short sequence of amino acids that is "complementary" to and therefore will bind antigen binding sites on antibodies directed to that antigen.
  • complementary as related to protein embodiments, is meant the amino acids or peptides exhibit an attractive force towards each other.
  • epitopes of pp85 of the present invention have specific binding affinity for human antibodies having binding specificity for HHV-7 and do not bind antibodies having specificity for HHV-6.
  • Such epitopic sequences are useful for detection of antibodies that bind said epitopes, and for administering to a subject for conferring protection to HHV-7 infection or reactivation.
  • a peptide region of pp85 from amino acids 471-586, 484 to 509, 484-502, 504-516, or 489-498 of human herpes virus-7 pp85 protein as set forth in SEQ ID NO.: 6, 7, 8, 9, 10, or 11, has been demonstrated to include an epitope of pp85.
  • pp85 may include amino acids 464-469, 489-498, 505-512, 545-550, 560- 563, 566-572, 581-585, 610-616, or 633-640 of SEQ ID NO:3 or SEQ ID NO:4.
  • the size of the epitope is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core amino acids.
  • the smallest useful epitope sequence contemplated by the present inventors is about 4-8 amino acids in length. This size generally corresponds to the smallest peptide antigens prepared in accordance with the invention. However, the size of the antigen may be larger where desired, as long as it contains an epitopic core sequence.
  • nucleic acid embodiments of the present invention include a recombinant vector comprising a purified nucleic acid molecule having a nucleotide sequence encoding a pp85 protein or epitopic peptide, the protein having an amino acid sequence essentially as set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, or SEQ ID NO.: 11.
  • the term "recombinant vector” refers to a vector that has been modified to contain a nucleic acid segment that encodes a pp85 protein or epitopic peptide, or fragment of interest thereof.
  • the recombinant vector may be further defined as an expression vector comprising a promoter operatively linked to said pp85 protein or peptide encoding nucleic acid molecule.
  • the recombinant vector may also comprise other sequences such as expression control sequences, markers, amplifying genes, signal sequences, and the like.
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' nucleotides, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth herein, including the maintenance of biological protein activity where protein expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences which may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region, or may include various coding or noncoding internal sequences, i.e., amino acids that form the junction between a protein fused to pp85 protein, for example.
  • nucleic acid segments of the present invention may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably.
  • the pp85 protein has been successfully expressed in a prokaryotic expression system by the present inventors, in particular, using the pBLUESCRIPTTM vector in E. coli cells.
  • Other expression systems contemplated by the present inventors include, yeast-based, insect-based, or mammalian cell-based systems.
  • For expression one would position the coding sequences adjacent to and under the control of the promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame of the protein between about 1 and about 50 nucleotides "downstream" of (i.e., 3' of) the chosen promoter.
  • polyadenylation site is placed about 30 to 2000 nucleotides "downstream" of the termination site of the protein at a position prior to transcription termination.
  • promoters examples include the polyhedrin promoter for baculovirus transfer vectors, ⁇ -galactosidase, lac, or trc, for bacterial vectors; or RSV LTR, CMV, SV40, MMTV-LTR (dexamethasone-inducible), HSV-TK (thymidine kinase) for mammaliam vectors, or the like.
  • Preferred promoters are the polyhedrin or bacterial promoters. Of the mammalian promoters, SV40 and CMV are preferred.
  • vectors may be further defined as eukaryotic plasmids such as SV40, baculovirus, or yeast vectors; prokaryotic vectors such as bacterial plasmids, e.g., pUC and derivatives thereof; or viral vectors such as a herpesvirus, pCMV, adenoviral, lambdaphage Ml 3 or derivatives thereof; or retroviral vectors such as HIV-, HHV-6- or HHV-7-based vectors.
  • Preferred vectors are plasmid or baculovirus vectors, and most preferred vectors are plasmid vectors.
  • the recombinant vector may comprise a further DNA sequence encoding at least one polypeptide for co-expression with pp85, or an epitopic fragment thereof.
  • This DNA sequence is operatively attached to the recombinant vector in reading frame so it is expressed in the host.
  • the different structural DNA sequences carried by the vector may be separated by termination and start sequences so that the proteins will be expressed separately or they may be part of a single reading frame and therefore be produced as a fusion protein by methods known in the art.
  • a fusion protein containing pp85 protein or epitopic peptide thereof where the fusion does not interfere with the immunogenic function of the protein or peptide is an aspect of the present invention.
  • Example 2 provides a pp85 protein fused with ⁇ galactosidase sequences.
  • a further aspect of the present invention is a host cell, made recombinant with a recombinant vector comprising a nucleic acid encoding pp85 protein or an epitopic peptide thereof.
  • the recombinant host cell may be a prokaryotic or a eukaryotic cell. In a more preferred embodiment, the recombinant host cell is a eukaryotic cell.
  • the term "engineered” or "recombinant” cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding pp85 protein or epitopic peptide thereof, has been introduced.
  • engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced gene.
  • engineered cells are cells having a gene or genes introduced through the hand of man.
  • Recombinantly introduced genes will either be in the form of a cDNA gene (i.e., they will not contain introns), a copy of a genomic gene, or will include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene, or combinations thereof.
  • Exemplary host cells may be further defined as an insect cell line such as Spodoptera frugiperda (Sf9); mammalian cells such as human fibroblast cell lines, Vero cells, CHO cells, or COS cells; Saccharomyces cerevisiae, Escherichia coli cells, or the like.
  • Sf9 Spodoptera frugiperda
  • mammalian cells such as human fibroblast cell lines, Vero cells, CHO cells, or COS cells
  • Saccharomyces cerevisiae Escherichia coli cells, or the like.
  • Preferred host cells are Sf9 or CHO cells, and most preferred host cells are CHO cells.
  • GST glutathione -S-transferase
  • MalBP maltose binding protein
  • fusion between the protein of interest and the tag occurs through a sequence recognized by specific proteases, e.g. enterokinase, thrombin, or factor Xa that allow removal of the proteic tag sequence from the purified fusion protein.
  • peptides including an antigenic epitope of pp85 may be synthesized synthetically. Synthesis of epitopic sequences, or peptides that include an antigenic epitope within their sequence is achieved using conventional synthetic techniques such as the solid phase method using an automatic peptide synthesizer (such as an Applied Biosystems Model430A Peptide Synthesizer) with standard t- butoxycarbonyl (t-Boc) chemistry that is well known to one skilled in this art in light of this disclosure. Peptide antigens synthesized in this manner may be aliquotted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, preferably, in a powder or lyophilized state pending use.
  • an automatic peptide synthesizer such as an Applied Biosystems Model430A Peptide Synthesizer
  • t-Boc t- butoxycarbonyl
  • the amino acid composition of the synthesized peptides may be determined by amino acid analysis with an automated amino acid analyzer to confirm that they correspond to the expected compositions.
  • the purity of the peptides may be determined by sequence analysis or HPLC.
  • HHV-7-specific reagents are provided to substantially eliminate any potential cross-reactivity to the homolog gene product of genetically-related herpesvirus such as HHV-6 and HCMV. The specificity of the reagent, therefore, is significantly increased.
  • a further embodiment of the invention is use of pp85 in assembly of a subunit vaccine for conferring protection to HHV-7 infection or reactivation of HHV-7 in a subject.
  • “Subunit vaccine,” as used herein, means an assembly of at least one epitope of the pp85 protein together with an HHV-7 immunogenic protein or peptide other than pp85 protein or peptide, or with an immunogenic protein from a closely related herpesvirus, such as a beta herpesvirus, as material for immunization of a human subject.
  • an immunogenic protein from a closely related herpesvirus include, but are not limited to, the UI 1 gene product of HHV-6 or HHV-7, or the UL32 or UL38 gene products of HCMV.
  • An amount of pp85, or an epitopic fragment thereof, effective to induce disease protection or reactivation in the subject is administered to the subject in order to confer protection to HHV-7 infection.
  • the vaccine may be administered once to afford a certain degree of protection against HHV-7 or it may be repeated at preset intervals.
  • the vaccine may suitably be readministered at anytime.
  • a typical interval for revaccination is about 1 day to 6 months, and more preferably about 10 days to 4 months.
  • the vaccine may be administered as a booster at other times as well.
  • the protein pp85 or epitopic fragment thereof may be administered to a subject by administering an expression vector containing the U14 gene or fragment encoding an epitopic fragment of pp85.
  • the expression vector is expressed in vivo thereby producing the pp85 protein or epitopic fragment thereof.
  • a nucleic acid vaccine is provided that in vivo provides a protein vaccine.
  • the immunoassay comprises the steps of contacting pp85 or an epitopic fragment thereof with the biological sample for a sufficient time to permit binding between antibody that may be present in the sample and pp85 protein, or epitopic fragment thereof, to form an incubate, and determining the presence of bound pp85 or epitopic fragment thereof in the incubate. Determining antibody-bound pp85 or epitopic fragment thereof may be by a number of detecting means.
  • One detecting means uses a detectable label attached to pp85 or epitopic fragment thereof.
  • Another detecting means is by contacting the incubate with a detectably labelled antibody specific for human immunoglobulin such as IgG, IgA, or IgM.
  • a detectably labelled antibody may be an enzyme-linked antibody, a fluorescent- tagged antibody or a radiolabeled antibody, for example. Where the antigen is labeled, a secondary antibody having binding specificity for human immunoglobulin need not be labeled. The presence of antibodies that bind pp85 or an epitopic fragment thereof in the human biological sample is detectable as a measure of the detectable label and demonstrates detection of HHV-7.
  • Antibodies fall into a number of different immunoglobulin classes and, generally, as used herein, antibodies mean immunoglobulins IgG, IgA, or IgM.
  • IgG antibodies are the common circulating type of antibodies and a primary target of serological detection herein. Detection of IgM antibody is also important herein especially where HHV-7 reactivation is suspected or where a newly acquired infection is suspected such as may occur in a transplant recipient of an infected organ.
  • a biological sample is a fluid sample from a human subject suspected of having
  • HHV-7 infection or reactivation If the human subject has HHV-7 infection or reactivation, then the sample would contain antibodies to HHV-7, in particular, antibodies of various immunoglobulin classes having binding specificity for pp85 or an epitopic fragment thereof.
  • samples include blood, plasma, serum, cerebrospinal fluid, semen, pleural fluid, B-cell secretions, saliva, urine, sputum, mucus, or the like.
  • immunoassays capable of using reagents of the present invention to serologically detect HHV-7.
  • Such immunoassays are intended or fall within the scope of the present invention and include competition assays where MAb 5E1, for example, would compete with antibody present in a test sample for binding pp85 or an epitope thereof; or indirect assays (sandwiched); for example.
  • a detectable antigen may be added to a test sample, and a second antibody having binding specificity for human immunoglobulin may be used to detect the antigen-primary antibody complex.
  • appropriate means such as a label
  • appropriate means for determining binding of pp85 or epitope thereof to the sample.
  • appropriate indicator means include fluorescent, radioactive, enzymatic, colorimetric, bioluminescent, or other ligands, such as avidin/biotin, fluorescein, or rhodamine, which are capable of giving a detectable signal.
  • fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents.
  • enzyme tags colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific binding.
  • kits for vaccination of a human comprises in packaged combination a carrier means adapted to receive a container means in close confinement therewith and a first container means including pp85 protein or epitopic peptide thereof, or a recombinant molecule capable of expressing pp85 protein or an epitopic peptide thereof.
  • the first container means includes recombinantly produced pp85, or epitopic fragments thereof.
  • the kit further comprises an HHV-7 immunogenic protein or peptide other than pp85 protein or peptide, or an immunogenic protein from a closely related herpesvirus, such as a beta herpesvirus.
  • a further aspect of the present invention is an HHV-7 vaccine comprising a human subject-protecting amount of pp85 protein or epitopic peptide thereof.
  • the vaccine may comprise a recombinant vector, including viral vectors other than HHV-7 itself, capable of expressing pp85 protein or an epitopic peptide thereof in a host.
  • the vaccine may comprise a physiologically acceptable carrier.
  • the vaccine according to the invention is administered in amounts sufficient to stimulate the immune system and confer protection to HHV-7 infection or reactivation.
  • the vaccine may be provided in powder form as a unit form, or in about 1-1000 doses of vaccine per sealed container, and more preferably about 10 to 100 doses.
  • Physiologically acceptable carriers for vaccination must not interfere with the immunological response elicited by the vaccine and/or with the expression of its protein or peptide product.
  • exemplary carriers are water, saline, dextrose, glycerol, ethanol, or the like.
  • the carrier or diluent may be compatible with administration methods such as injection, eye drops, nose drops, and the like.
  • adjuvants and stabilizers may also be contained in the vaccine.
  • adjuvants such as aluminum hydroxide, aluminum phosphate, plant and animal oils, and the like, are administered with the vaccine in amounts sufficient to enhance the immune response to pp85 immunogenic protein or peptide.
  • the amount of adjuvant added to the vaccine will vary depending on the nature of the adjuvant, generally ranging from about 0.1 . o about 100 times the weight of the active ingredient, preferably from about 1 to about 10 times the weight of the active ingredient.
  • the vaccine of the present invention may also contain various stabilizers.
  • Any suitable stabilizer can be used including carbohydrates such as sorbitol, mannitol, starch, sucrose, dextrin, or glucose; proteins such as albumin or casein; and buffers such as alkaline metal phosphate and the like.
  • a stabilizer is particularly advantageous when a dry vaccine preparation is prepared by lyophilization.
  • the vaccine may be administered by any suitable known method of inoculating including nasally, ophthalmically, by injection, in drinking water, in food, by exposure, and the like.
  • a vaccine according to the present invention can be administered by injection.
  • the vaccines are preferably administered parenterally.
  • Parenteral administration as used herein means administration by intravenous, subcutaneous, intramuscular, or intraperitoneal injection.
  • the vaccine may be provided in a sterile container in unit form or in other amounts. It is preferably stored frozen, below -20° C, and more preferably below -70° C. It is thawed prior to use, and may be refrozen immediately thereafter.
  • the present example provides data demonstrating that the HHV-7 gene encoding pp85 is U14.
  • Cells and viruses Primary cord blood mononuclear cells (CBMCs) were purified from heparinized blood by Ficoll-Hypaque (Pharmacia) gradient centrifugation. SupTl T lymphoblastoid CD4+ T cells were cultured in RPMI 1640, 90%; and fetal bovine serum, 10%.
  • CBMCs were grown for two days with phytohemagglutinin (PHA-P) (Sigma Biochimici, Milano, Italy), 5U/ml, and two further days with PHA-P and recombinant interleukin 2 (5U/ml) (Boehringer, Mannheim, Germany) prior to infection.
  • PHA-P phytohemagglutinin
  • 5U/ml recombinant interleukin 2
  • the HHV-7 strains were RK and AL. Strain RK was obtained from Dr. N. Frenkel, National Institutes of Health) and strain AL was isolated by the present inventors.
  • CBMCs and SupTl were infected with HHV-7 (RK or AL). Progress of infection was monitored by indirect immunofluorescence assay (IF A) microscopy with MAb 5E1 (Dr. Gabriella Campadelli-Fiume, Univ. of Bologna, Italy).
  • a cDNA library from HHV-7 -infected cells SupTl cells infected with HHV-7 (AL) were harvested 6 days after infection, when approximately 70% of the cells were infected (as evaluated by the appearance of cytopathic effect (CPE) and by IF A). RNA was extracted and polyadenylated RNA was selected by oligo(dT)-cellulose column chromatography using standard techniques. cDNA was retrotranscribed with an oligo(dT)-XhoI primer and using 5 ⁇ g of purified polyadenylated RNA as template. A directional cDNA library was constructed based on the ZAPII kit (EcoRI-Xhol) (Stratagene, La Jolla, CA).
  • BLUESCRIPTTM phagemids were rescued by excision from lambda ZAPIITM by superinfection with the EXASSISTTM helper phage (Stratagene). Rescued phagemids were then used to infect E. coli SOLRTM cells (Stratagene), and ampicillin-resistant colonies were selected. Plasmid DNA was prepared by using the QIAPREPTM Plasmid kit (Qiagen, Chatsworth, CA) and the presence of inserts was verified by restriction enzyme digestion and agarose gel electrophoresis. Nucleotide sequence of the insert was then determined with a Taq DYEDEOXYTM cycle sequencing kit (Applied Biosystems Inc. [ABI], Foster City, CA), according to the manufacturer's instructions, using an ABI automated DNA sequencer. DNA sequence analysis was performed with PC/GENETM (IntelliGenetics, Mountain View, CA). Protein alignments were performed with BESTFITTM program.
  • FIG. 1 The arrangement of the HHV-7 sequence in pBLUESCRIPTTM-2c and -8a is schematically depicted in FIG. 1.
  • the insert contains the entire U14 ORF and additional 402 bp at the 3' terminus.
  • the initiating methionine is inserted in frame with Escherichia coli ⁇ -galactosidase sequences, giving a fusion protein of ⁇ -gal:U14 that stops at the U14 stop codon at the 21.831 map unit in the genome (FIG. 1). No other significant open reading frame is apparent in this insert.
  • the insert also contained the HHV-7 U14 ORF in frame with the ⁇ -galactosidase sequence, although it lacked 153 bp at the 5' end of the ORF.
  • mapping experiments taken together with the specificity of Mab 5E1 for pp85 demonstrate that the HHV-7 gene encoding pp85 is U14.
  • the mapping studies were unequivocally confirmed by the ability of MAb 5E1 to react with the prokaryotically-expressed fusion protein ⁇ -gal:U14 as provided in Example 2.
  • Alignment of HHV-7 and HHV-6 U14 protein sequences as deduced from the nucleic acid sequences shows an overall degree of identity of 48.9%. This figure is lower than that obtained with the proteins that display the highest degree of conservation, such as the major capsid protein (68.8% identity and 82.6% similarity) and helicase (74.9% identity and 85.7% similarity).
  • a closer insight into the alignment of HHV-7 and HHV-6 U14 homologues shows that the similarity is not uniformly distributed throughout the entire sequence.
  • clone pBLUESCRIPTTM-2c that contained the entire U14 ORF in frame with ⁇ -galactosidase sequences was used to verify if the prokaryotically- expressed U14 maintains reactivity with MAb 5E1.
  • the DNA of pBLUESCRIPTTM clone 2c from Example 1 was transfected into BL21 (protease deficient) and XL-1BLUE bacterial cells (Stratagene). Bacteria transfected with pBLUESCRIPTTM alone without any insert served as a control. Expression from the transfected cells was either uninduced or induced with 1 mM IPTG and incubated at 30°C for 2 h. Cells were pelletted, lysed with 1% NP40, 1% DOC in PBS in the presence of protease inhibitors and centrifuged.
  • the proteins were separated by denaturing electrophoresis, transferred to nitrocellulose sheet, and reacted with MAb 5E1, after blocking non-specific binding sites with 5% skimmed milk in PBS.
  • the antibody reacted with a major band of apparent molecular weight of about
  • the antibody also reacted with a number of minor species of lower apparent molecular weight, some of which were present also in the immunoblot with pBLUESCRIPTTM alone.
  • the apparent molecular weight of the prokaryotically-expressed protein is higher than the predicted molecular weight of U14 gene product (73,893 Da), and is consistent with the apparent molecular weight of the major protein species (85 kDa) detected by MAb 5E1 in lysates of HHV-7-infected CBMCs.
  • EXAMPLE 3 pp85 is a Viral Structural Protein Positioned at the Outer Periphery of the Tegument
  • pp85 is a viral structural protein and define a functional role for pp85.
  • HHV-7(RK)-infected and uninfected CBMCs were fixed five days after infection, or mock infection, with a freshly-made solution of 3% paraformaldehyde in PBS for 30 min at room temperature, washed and further processed for cryomicrotomy and immunolabeling. An aliquot of cells was post-fixed with 3% glutaraldehyde and 2% osmium tetroxide for standard embedding and thin- sectioning. Cultures employed for cryomicrotomy contained more than 10% of cells displaying virus particles. Frozen-thawed sections were indirectly labelled with MAb
  • HHV-7(RK)-infected or uninfected CBMCs were harvested at day 6 after infection, fixed with 3% paraformaldehyde in PBS and processed for cryomicrotomy and immunolabelling.
  • concentrations of MAb 5E1 and of gold-conjugated anti-mouse antibodies were adjusted to produce no detectable background in control uninfected cell samples.
  • Cultures of CBMCs infected with HHV-7 include a mixture of cells exhibiting various degrees of structural preservation, depending on the cytopathic effect of the infection and the age of the cells. As expected, virus particles were most frequently associated with cells displaying advanced cytopathic effect, and included all forms of morphogenetic maturation typical of members of the Herpesviridae family. Cryosectioned HHV-7 particles typically included a core surrounded by the electron- opaque substance of the tegument and, in the more mature forms, of the outer envelope membrane. The immunogold reactivity of MAb 5E1 with virus-infected cells was specifically directed to the virus particles and to electron-dense amorphous cytoplasmic areas.
  • Cytoplasmic membranes or other compartments of the host cell were not labeled. Most immunogold granules were precisely positioned at the outer periphery of the virion tegument. In particular, this reaction took place irrespective of the presence of the viral envelope membrane.
  • clone pBLUESCRIPT-2c that contained the entire U14 ORF in frame with ⁇ -galactosidase sequences was used to verify if human sera known to carry antibodies to HHV-7, including antibodies to pp85 made in HHV-7-infected cord blood mononuclear cells (CBMCs), react to prokaryotically expressed pp85.
  • CBMCs cord blood mononuclear cells
  • BL21 or XL- 1 BLUE cells carrying pBLUESCRIPT-2c were induced to express U14 gene product by induction with lmM IPTG and incubated at 30°C for 2 h, as in Example 2.
  • Cells were pelleted, lysed in 1% NP40, l%DOC in PBS in the presence of protease inhibitors and pelleted.
  • the proteins present in the supernatant were separated by SDS-PAGE, transferred to nitrocellulose and reacted with a number of human sera.
  • the human sera employed had previously been tested for reactivity to pp85 made in HHV-7-infected CBMCs.
  • HHV-7 U14 cDNA was excised from the pBLUESCRIPT-2c construct by digestion with Knpl and Notl restriction enzymes.
  • the purified insert was ligated into pcDNA3.1 vector (Invitrogen), via sites Kpnl and Notl, under the control of CMV promoter.
  • Expression of U14 was analyzed in transient transfection experiments, by using the human 293 cell line. Positive reactivity to Mab 4E1 was specifically observed in the cells transfected with the expression vector containing the U14 cDNA, and not in the control cells transfected with the expression vector without insert.
  • Epitopes were further determined by overlapping peptides. Twelve overlapping peptides were synthesized covering the region between amino acid positions 471 to 586 of SEQ ID NO: 4. Reactivity between each peptide and monoclonal antibody 5E1 was measured in ELISA tests. HHV-7 specific epitopes were thus mapped to peptide 484- 509 (SEQ ID NO: 8), to peptide 484-502 (SEQ ID NO:9), and to peptide 504-516 (SEQ ID NO: 10). In particular, amino acids in the region of residues 489-498 (SEQ ID NO: 11) are absent from the HHV-6 herpesvirus.
EP98935607A 1997-07-10 1998-07-10 Menschliches herpesvirus 7, diagnostische und immunologische reagentien und deren verwendungen Withdrawn EP1005543A2 (de)

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