WO1989005855A1 - Proteine de cytomegalovirus humain - Google Patents

Proteine de cytomegalovirus humain Download PDF

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Publication number
WO1989005855A1
WO1989005855A1 PCT/GB1988/001112 GB8801112W WO8905855A1 WO 1989005855 A1 WO1989005855 A1 WO 1989005855A1 GB 8801112 W GB8801112 W GB 8801112W WO 8905855 A1 WO8905855 A1 WO 8905855A1
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Prior art keywords
hcmv
polypeptide
antibodies
recombinant
antibody
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PCT/GB1988/001112
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English (en)
Inventor
Barclay George Barrell
Stephan Beck
Anthony C. Minson
Geoffrey Lilley Smith
Martin Patrick Cranage
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Cogent Limited
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Publication of WO1989005855A1 publication Critical patent/WO1989005855A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • 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
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to HCMV proteins, and particularly to those that may be useful, directly or indirectly, in diagnostic procedures, prophylaxis and therapy.
  • MHC- loci Major histocompatibility complexes, MHC- loci, have been identified so far only in vertebrates including mammals, birds, amphibians and probably reptiles and fish.
  • the MHC encoded proteins, the MHC-antigens are known to play a vital role in the self versus non-self recognition in the immune response. They are divided into class I and class II molecules, each consisting of a set of cell-surface glycoproteins .
  • a polypeptide which comprises at least part of the a ino acid sequence encoded by the nucleotide sequence 1* ⁇ 3 to 12-47 as depicted in Fig. 2 hereof, or an allele or variant thereof, and having at least one epitope characteristic of HCMV.
  • the invention also includes sub- genomic DNA sequences encoding such a polypeptide, recombinant cloning and expression vectors containing such DNA, recombinant microorganisms and cell cultures capable of producing such a polypeptide, and vaccines against HCMV incorporating such a polypeptide or a recombinant viral vector capable of expressing the polypeptide in a mammal immunized therewith.
  • the invention further includes monoclonal and polyclonal antibody binding specifically to such polypeptide.
  • the polypeptide can be used in diagnostic kits and procedures to detect HCMV specific antibodies in a clinical sample.
  • the antibodies hereof can be used in diagnostic kits and procedures to detect HCMV antigens in a clinical sample, and they can also be used therapeutically or prophylactically for administration by way of passive immunisation against HCMV infection.
  • Figure 1 shows a physical map of HCMV strain ADI69.
  • the linear double stranded DNA genome of HCMV consists of a long unique (U- Q ) and a short unique (Ug) region both of which are flanked by repeat sequences (boxed) which allow the virus to exist in four equimolar isomers (Oram, et al, J.Gen. Virol. 59, 111-129 (1982)).
  • the scale shows the genome size in kilobases (kb). Below the scale the Hindlll map of the generally accepted prototype orientation is shown. The Hindlll O-fragment is indicated by shadowing and the approximate position and of the predicted HCMV-H301 gene is indicated by an arrow.
  • Figure 2 shows the nucleotide sequence of the HCMV- H301 gene in 5' ⁇ 3' direction.
  • the predicted coding sequence starting at position 1 3 (start codon, ATG) to position 1247 (stop codon, TGA) has been translated into the corresponding amino acid sequence which is written above the nucleotide sequence in the one letter code.
  • the potential TATA-box (position 100) at -42 upstream of the start codon and the potential polyadenylation site AATAAA (position 1722) at +473 downstream of the stop codon are indicated by boxing.
  • a second polyadenylation site (position 132) of an upstream open reading frame, ending at position 3 (stop codon, TAA) is also indicated by boxing.
  • HLA complex HLA complex in human
  • H-2 complex H-2 complex in mouse
  • RT1 complex RT1 complex in rat
  • RLA complex RLA complex in rabbit and many more (Klein et al supra) .
  • the structure of class I genes has been studied in detail (Klein et al , supra, and Hood, et al , Ann.Rev. Immunol. , 29-568 (1983)) .
  • each gene is between 3,000 and 12,000 bp long, depending upon the variable length of their introns .
  • class I genes show a very conserved gene structure.
  • the coding sequence of each gene is about 1,100 bp long and is divided into 8 exons, separated by f introns.
  • Exon 1 encodes the leader sequence (LD)
  • exons 2, 3 and 4 encode the ⁇ l , a.2 and ⁇ 3 domain respectively
  • exon 5 encodes the transmembrane region (TM)
  • exons 6, 7 and 8 encode the cytoplasmic domain (CY) and the 3' untranslated region.
  • the structure of the HCMV- H301 gene is quite different. There are no indications of suitable GT/AG splicing signals for the typical upstream and downstream intron boundaries in class I genes (Hood et al , supra)
  • the predicted coding sequence of 1 , 104 bp (position 142 - 1246, Fig. 2) , however, coincides perfectly with the average length of the coding sequence of known class I genes. We therefore assume, that the HCMV-H301 gene is not spliced.
  • the predicted gene shows all characteristic elements of a complete gene.
  • TATA-box potential promotor
  • AATAAA potential polyadenylation site
  • MHC class I glycoproteins are cell surface molecules and accordingly their ⁇ chain can be divided into an extracellular region, a hydrophobic anchor or transmembrane region and an intracellular or cytoplasmic region. Like other membrane proteins class I precursors also have a short, hydrophobic leader sequence at the NH2 ⁇ terminus which becomes cleaved off before the molecules are expressed on the cell surface.
  • HCMV-H301 amino acid sequence was aligned to some ⁇ chains of class I antigens from mouse (H- 2) , rat (RT1), human (HLA) and rabbit (RLA) (Klein et al, supra, Klein, et al , Immunol. Today 7, 41-44 (1986) , Claverie et al , Proteins 1, 60-65 (1986)).
  • Fig. 2 homologous amino acids are marked by ( : ) for 50% - 100% ho ology and (.) for 25 - 50 homology.
  • C cysteines in the a.2 and ⁇ 3 domains are highlighted by asterisks (*).
  • N_-X-T/S N-linked " glycosylation sites
  • TM transmembrane region
  • LD HCMV-H301 leader sequence
  • the bulk of the class I ⁇ chain consists of the extracellular region which is divided into three domains , ⁇ l , ⁇ 2 and ⁇ 3, respectively. Each domain consists of about 90 amino acids.
  • the main structural features of the extracellular region are four highly conserved cysteines (C), two in the ⁇ 2 domain and two in the ⁇ 3 domain.
  • C highly conserved cysteines
  • the ⁇ l domain normally contains no cysteine. It is presumed, that the two cysteines in both the ⁇ 2 and ⁇ 3 domain, form a covalent disulfide bridge by looping out the intervening amino acid sequence similar to Ig molecules. This creates a loop of 62 amino acids in the ⁇ 2 domain and a loop of 55 amino acids in the c_3 domain.
  • HCMV-H301 ⁇ chain HCMV-H301 ⁇ chain. It also can be divided into the corresponding ⁇ l , ⁇ 2 and ⁇ 3 domain.
  • the ⁇ l domain also contains no cysteine and shows an average homology of 30% in the amino acid sequence to various ⁇ l domains compared.
  • the ⁇ 2 domain shares the two highly conserved cysteines and shows an average homology of 26% in the amino acid sequence. However, the corresponding loop between the two cysteines consists of 72 amino acids rather than 62 amino acids found in all the other ⁇ 2 domains compared.
  • the average homology of 27% in the amino acid sequence of the HCMV-H301 ⁇ 3 domain seems to be clustered in mainly 3 distinct regions (overlined in Fig. 2) .
  • the HCMV- H301 ⁇ chain shows a potential TM sequence (boxed in Fig. 2), consisting of 23 uncharged, mainly hydrophobic amino acids.
  • the cytoplasmic terminal region of HCMV-H301 contains no potential phosphorylation site and shows no significant homology in the amino acid sequence. Assuming that the predicted HCMV-H301 gene is transcribed, translated and expressed on the surface of the infected cell, the absence of any phosphorylation site in the HCMV- H301 cytoplasmic region could have a signal significance in the viral budding process in order to distinguish between membrane patches expressing host or viral encoded proteins, and in a similar way, the 13 potential glycosylation sites of the HCMV-H301 ⁇ chain could also present a signal significance on the surface of an infected cell by increasing the negative charge on the cell surface.
  • HCMV 'HLA' Gene H301 Uses of HCMV 'HLA' Gene H301 and its Expression Polypeptide Both the HCMV 'HLA' gene H301 and its expression polypeptide having most characteristic features of an MHC class I antigen have a variety of commercial uses.
  • the expression polypeptide may be produced either in a recombinant expression system using the HCMV 'HLA' gene H301 , or by any other preparative process which utilises the gene sequence information as provided herein and with reference to Fig. 1.
  • the main commercial uses can be summarised as follows : i) use of the HCMV 'HLA' gene H301 and of its expression polypeptide in vaccination systems; ii) use of the HCMV 'HLA' gene H301 and of its expression polypeptide (in crude preparations, as a purified product or as a recombinant) to raise antibodies (monoclonal, polyclonal and engineered) suitable for use in diagnostic tests and as therapeutic and prophylactic agents; and iii) use of the expression polypeptide as reagents in diagnostic kits.
  • the present invention provides an alternative approach to the production of a vaccine against HCMV by providing: a) a sequence of HCMV 'HLA 1 gene H301 which encodes b) an expression polypeptide having most characteristic features of an MHC class I antigen and acting as a receptor for 2 .
  • the provision of both the gene sequence and the expression polypeptide sequence provides for two alternative vaccination routes .
  • HCMV 'HLA' gene H301 sequence is isolated and introduced into a suitable mammalian virus vector by conventional genetic engineering techniques (see for example the detailed description below) and transferring the plasmid into the host e.g. the human for vaccination.
  • suitable virus vectors are the poxviruses such as vaccinia virus.
  • the receptor protein itself may be utilised as a vaccine according to techniques well known in the art. These techniques comprise compounding the protein with a suitable adjuvant or excipient of the kind conventionally employed in vaccine formulations . This form of vaccination might be more appropriate than the recombinant vaccine for example in immunosuppressed individuals . ii) Antibody Preparation
  • the vaccination techniques described above are simply techniques used to stimulate a given hosts immune response and thereby prime the immune system to a foreign antigen. These same techniques may be applied to experimental animals, from which antibodies produced against the antigen (in this case the HCMV 'HLA' gene expression polypeptide) may be harvested.
  • the host animal is immunised with either a recombinant virus vector carrying the HCMV 'HLA' gene H301 or with the expression polypeptide itself.
  • Antibodies specific to the expression polypeptide are then extracted from the host animals antiserum, using standard techniques well known in the art. Monoclonal antibodies may also be prepared from the cells of the immunised animals using standard techniques well known in the art.
  • Antibodies are prepared to a high degree of specificity by contacting them with the expression polypeptide immobilised on a suitable support such as an affinity column gel and then separating bound antibody from the expression polypeptide by eluting the affinity column with a reagent which destroys the polypeptide-antibody binding. iii) Use of Antibodies for Diagnosis- Therapy and
  • Both the antibodies produced as described above and the expression polypeptide itself, can be used in methods for, and incorporated as parts of kits for, detecting infection with HCMV.
  • HCMV is detected in a biological sample (usually sera) taken from a patient.
  • a biological sample usually sera
  • the expression polypeptide is used as a reagent to detect antibodies to HCMV the biological sample.
  • the presence of serum antibodies to HCMV indicates past or present infection with HCMV.
  • the methods for detecting infection using both the expression polypeptide and antibodies thereto comprise a variety of conventional assay procedures, based for example on ELISA, RIA or immunofluorescence.
  • the expression polypeptide could be immobilised on a support (such as an ELISA plate or a dip-stick) and then contacted with the clinical sample. After washing, the support is contacted with labelled anti-human i munoglobulin which binds to any HCMV antibody which has been found by immobilised HCMV protein.
  • the label may be an enzyme and binding of the labelled anti-human immunoglobulin is detected by applying a suitably chromogenic substrate.
  • Alternative labelling/detection systems are also well known in the art. Expression of HCMV 'HLA' gene H301 and production of antibody to this protein 1. Expression of the HLA gene in bacteria
  • the BamHI-Pstl fragment was cloned into plasmid pEX-3 that had been cut with BamHI and Pstl so that the HLA gene was in frame, as a fusion protein downstream, of the body of ⁇ - galactosidase.
  • the resulting plasmid was called CXI.
  • the pEX-3 plasmid and E_. coli strain POPS are designed so that high levels of ⁇ -galactosidase fused to other protein coding sequences may be synthesized in an inducible manner (Stanley, et al, (1984) . EMBO J. 3_ l429 ⁇ 34) .
  • CXI was introduced into E_.
  • the nucleotide sequence of the HLA gene showed that an additional ATG codon was present upstream of the ATG used to initiate translation of the HLA gene and before the first convenient upstream restriction enzyme site. Consequently, before this gene could be adequately expressed in vaccinia virus it was necessary to remove this additional ATG. This was done as follows. First, the HLA gene was isolated from a plasmid pAT 153/HCMV Hindlll 0 which contained the HCMV genomic Hindlll 0 fragment (Oram et al, (1982) J. Gen. Virol., 59, 111-129).
  • pAT153/HCMV Hindlll 0 was cut with Hindlll, the 8683bp HCMV Hindlll 0 fragment isolated and cut with Stul into fragments of 6728 and 1955 bp.
  • the 6728 bp Hindlll-Stul fragment was further digested with BamHI-EcoRI and a 1370 bp BamHI-EcoRI fragment isolated and cloned into plasmids pUCl ⁇ and pUC19 (Yanisch-Perron et al, (1985), Gene 33, 103-119) that had been cut with BamHI and EcoRI, to form plasmids pSB-l8/02 and pSB-19/02, respectively.
  • Plasmid pSB-19/02 was used for the removal of the additional ATG upstream of the HLA gene by Bal31 exonuclease trimming.
  • BamHI cut pSB-19/02 was digested with Bal31 for 18, 22, 26 or 30 minutes, the ragged ends of DNA repaired with Klenow DNA polymerase and then digested with EcoRI. Isolated DNA fragments were cloned into Smal and EcoRI out pUCl ⁇ . Thirty six clones were isolated and sequenced and one identified that had lost the first two nucleotides of the upstream ATG codon. This clone was called pSBH301-26.
  • the modified HLA gene was transferred from pSBH301- 26 into vaccinia virus by first cloning it into plasmid vectors pGS62 (Smith et al (1987). Virology, 160, 336-345) and pRK19- (pRK19 is constructed in a similar way to pGS62 (Smith et al, supra) but instead of the 7» promoter it has the vaccinia late 4b gene promoter (Rose et al , (1985). J. Virol. , 56, 830-836).
  • a 1261 bp BamHI-EcoRI fragment was purified from pSBH301-26 and inserted downstream of vaccinia promoters p7.5K (Smith et al , supra) or p4b (Rose et al , supra) in plasmids pGS62 and pRK19, respectively.
  • Resultant plasmids pl02 and pl03 were used to generate TK " recombinant vaccinia viruses V102 and V103 using previously established methods (Mackett, et al (1984). J. Virol. 4_9_, 857-64) .
  • HLA gene by recombinant vaccinia viruses V102 and V103 was demonstrated by infecting CV-1 cells with these viruses, radiolabelling the cells with 35s-methionine and analysing extracts of the infected cells by polyacrylamide gel electrophoresis and autoradiography.
  • Cells infected with recombinant vaccinia virus V102 or V103 but not HCMV gB-VAC (Cranage, et al, (1986). EMBO J. 5_, 3057-63) or WT vaccinia virus or uninfected cells synthesise a diffuse protein of M r 71K. This represents the product of the HLA gene and Is considerably larger than the primary amino acid chain of 39-5 kD predicts. The difference is most likely attributable to the 13 potential N-linked glycosylation sites. The diffuse nature of the band is consistent with the presence of glycosylation.
  • nucletide sequence of Fig. 2 could be varied, within the constraints of the genetic code, to encode the same polypeptide.
  • polypeptide itself may be varied from that shown in Fig. 2, for example by way of amino acid addition, deletion, substitution, insertion or inversion, while retaining the essential antigenicity characteristic of HCMV.
  • variants may arise as natural alleles , or may be produced synthetically by methods well known in the art.
  • Antibodies of the present invention include not only entire immunoglobulins, but also fragments thereof having the characteristic complementarity determining regions (see eg. WO 86/01533).
  • the monoclonal antibodies hereof may be those raised by immunization and produced from hybridomas , or alternatively they can be expressed from recombinant host cells transformed with DNA encoding the immunoglobulin.
  • the antibody may, furthermore, be a hybrid having different binding characteristics in its variable regions, and/or a hybrid comprising the variable or complementarity determining regions produced by immunizing a non-human animal with the polypeptide hereof and constant regions obtained from a different antibody such as a human immunoglobulin (see eg. GB 2188638A and EP 173494A) .

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Abstract

On a identifié un polypeptide de CMVH ayant les traits caractéristiques d'un antigène de classe I de MHC vertébré. La protéine est codée par une séquence de nucléotides commençant à la position 143 et terminant à la position 1247 du gène HCMV-H301 comme le montre la figure 2. La séquence polypeptidique ou génétique dans un vecteur viral pourrait être utile à la vaccination contre le CMHV ou dans la production et la purification d'anticorps développés contre le CMVH. Le polypeptide et les anticorps développés contre celui-ci trouvent également une utilisation dans des kits de diagnostic permettant de détecter une infection provoquée par le CMVH.
PCT/GB1988/001112 1987-12-15 1988-12-15 Proteine de cytomegalovirus humain WO1989005855A1 (fr)

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GB878729251A GB8729251D0 (en) 1987-12-15 1987-12-15 Human cytomegalovirus protein

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992018630A1 (fr) * 1991-04-18 1992-10-29 Fondation Nationale De Transfusion Sanguine Compose comprenant une sequence peptidique neutralisant l'infection par le cytomegalovirus humain (hcmv) et composition le comportant
WO1995006717A2 (fr) * 1993-09-03 1995-03-09 Viagene, Inc. Procedes de suppression des rejets de greffes
EP0601585A3 (fr) * 1992-12-09 1995-04-26 Enzo Therapeutics Inc Acide nucléique pour l'inhibition ou la régulation des fonctions d'un gène de la réponse immunitaire, méthode employant le-dit acide nucléique et système immuno-compatible contenant le-dit acide nucléique.
US5698390A (en) * 1987-11-18 1997-12-16 Chiron Corporation Hepatitis C immunoassays
US5712088A (en) * 1987-11-18 1998-01-27 Chiron Corporation Methods for detecting Hepatitis C virus using polynucleotides specific for same
US5714596A (en) * 1987-11-18 1998-02-03 Chiron Corporation NANBV diagnostics: polynucleotides useful for screening for hepatitis C virus
US6027729A (en) * 1989-04-20 2000-02-22 Chiron Corporation NANBV Diagnostics and vaccines
US6171782B1 (en) 1987-11-18 2001-01-09 Chiron Corporation Antibody compositions to HCV and uses thereof
US6861212B1 (en) 1987-11-18 2005-03-01 Chiron Corporation NANBV diagnostics and vaccines
US9346874B2 (en) 2009-12-23 2016-05-24 4-Antibody Ag Binding members for human cytomegalovirus

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
J. Gen. Virol. vol. 68, 1987 (GB) J.E. Grundy et al.: "B2 Microglobulin enhances the infectivity of cytomegalovirus and when bound to the virus enables class I HLA molecules to be used as a virus receptor", pages 793-803 *
J. Gen. Virol., vol. 68, 1987 (GB) J.A. McKeating et al.: "Cytomegalovirus in urine specimens has host B2 microglobulin bound to the viral envelope: a machanism of evading the host immune response", pages 785-792 *
J. Gen. Virol., vol. 68, 1987 (GB) J.E. Grundy et al.: "Cytomegalovirus strain AD169 binds B2 microglobulin in vitro after release from cells", pages 777-784 *
Nature, vol. 331, 21 January 1988, S. Beck et al.: "Human cytomegalovirus encodes a glycoprotein homologous to MHC class-I antigens", pages 269-272 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6096541A (en) * 1987-11-18 2000-08-01 Chiron Corporation Cell culture systems for HCV
US5714596A (en) * 1987-11-18 1998-02-03 Chiron Corporation NANBV diagnostics: polynucleotides useful for screening for hepatitis C virus
US7790366B1 (en) 1987-11-18 2010-09-07 Novartis Vaccines And Diagnostics, Inc. NANBV diagnostics and vaccines
US6861212B1 (en) 1987-11-18 2005-03-01 Chiron Corporation NANBV diagnostics and vaccines
US5698390A (en) * 1987-11-18 1997-12-16 Chiron Corporation Hepatitis C immunoassays
US5712088A (en) * 1987-11-18 1998-01-27 Chiron Corporation Methods for detecting Hepatitis C virus using polynucleotides specific for same
US6074816A (en) * 1987-11-18 2000-06-13 Chiron Corporation NANBV diagnostics: polynucleotides useful for screening for hepatitis C virus
US6171782B1 (en) 1987-11-18 2001-01-09 Chiron Corporation Antibody compositions to HCV and uses thereof
US6027729A (en) * 1989-04-20 2000-02-22 Chiron Corporation NANBV Diagnostics and vaccines
WO1992018630A1 (fr) * 1991-04-18 1992-10-29 Fondation Nationale De Transfusion Sanguine Compose comprenant une sequence peptidique neutralisant l'infection par le cytomegalovirus humain (hcmv) et composition le comportant
EP0601585A3 (fr) * 1992-12-09 1995-04-26 Enzo Therapeutics Inc Acide nucléique pour l'inhibition ou la régulation des fonctions d'un gène de la réponse immunitaire, méthode employant le-dit acide nucléique et système immuno-compatible contenant le-dit acide nucléique.
WO1995006717A2 (fr) * 1993-09-03 1995-03-09 Viagene, Inc. Procedes de suppression des rejets de greffes
WO1995006717A3 (fr) * 1993-09-03 1995-04-06 Viagene Inc Procedes de suppression des rejets de greffes
US9346874B2 (en) 2009-12-23 2016-05-24 4-Antibody Ag Binding members for human cytomegalovirus

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