EP0429470A1 - Antibody recognition of fragments from cytomegalovirus - Google Patents

Antibody recognition of fragments from cytomegalovirus

Info

Publication number
EP0429470A1
EP0429470A1 EP89907929A EP89907929A EP0429470A1 EP 0429470 A1 EP0429470 A1 EP 0429470A1 EP 89907929 A EP89907929 A EP 89907929A EP 89907929 A EP89907929 A EP 89907929A EP 0429470 A1 EP0429470 A1 EP 0429470A1
Authority
EP
European Patent Office
Prior art keywords
fragments
chymotrypsin
molecular weight
peptides
monoclonal antibodies
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.)
Ceased
Application number
EP89907929A
Other languages
German (de)
English (en)
French (fr)
Inventor
Bruce E. Kari
Richard C. Gehrz
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.)
Childrens Hospital Inc
Original Assignee
Childrens Hospital Inc
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Childrens Hospital Inc filed Critical Childrens Hospital Inc
Publication of EP0429470A1 publication Critical patent/EP0429470A1/en
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
    • C07K16/088—Varicella-zoster virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
    • C07K16/089—Cytomegalovirus
    • 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

  • Human cytomegalovirus contains several disulfide-linked glycoprotein complexes (Britt, 1984,
  • glycoproteins are held together by disulfide bonds and contain largely N-linked oligosaccharides (Rasmussen et al., 1988).
  • Complex gC-I can be immunoprecipitated by human serum positive for HCMV and will also stimulate human T cells (Liu et al., 1988).
  • Antibody 11B4 was also observed to be non-neutralizing and had the ability to inhibit the neutralizing activity of other antibodies in a plaque-reduction assay. These gC-I specific murine monoclonal antibodies were found to recognize several clinical isolates of HCMV as determined by immunofluoresence. This suggested that the epitopes recognized by these antibodies are conserved (Lussenhop et al., 1988).
  • proteolysis has been one approach used to determine if epitopes are physically close or distant on viral glycoproteins. For example, proteolysis has been used to determine the location of anti- genic determinants on glycoproteins from tick-borne encephalitis virus (Heinz et al., 1983), murine leukemia virus (Pinter et al., 1982) and glycoprotein D from HSV (Eisenberg et al., 1982).
  • the present invention provides immunogenic proteolytic fragments of gC-I which contained all three immunoreactive domains of gC-I.
  • trypsin or chymotrypsin a set of fragments could be generated which were recognized by antibodies from all three domains. These fragments were also recognized by several human sera positive for HCMV. Trypsin and chymotrypsin fragments were also examined for T cell reactivity. Lymphocyte proliferative responses were detected with fragments made with either enzyme in the case of individuals who reacted strongly with whole gC-I, but higher responses were consistently obtained with trypsin fragments. Cells obtained from an individual who had low responses to whole gC-I had low responses to the fragments as well. But a T cell clone from this individual was observed to react with trypsin fragments, but not chymotrypsin fragments.
  • the HCMV envelope glycoprotein complex gC-I was digested with chymotrypsin and fragments immunoaffinity purified. Two major glycosylated peptides were obtained which had molecular weights (MWs) of 34,000 and 43,000 under non-re ⁇ ucing conditions. After reduction, one major glycosylated fragment, with a MW of 34,000, was observed in addition to at least two other peptides with MWs of 30,000 and 28,000. Under non-reducing conditions, monoclonal antibodies (MoAbs) which bind to all three domains of gC-I immunoprecipitated the 34,000 and 43,000 MW fragments and reacte ⁇ with them in Western blot. After reduction, the MoAb assigned to domain III and one MoAb in domain I were non-reactive while all other MoAbs reacted with the 34,000, 30,000 and 28,000 MW peptides in Western blot.
  • MoAbs monoclonal antibodies
  • Proteolvsis Either purified Towne strain HCMV or whole cells at 7 to 14 days post infection were solubilized with 1.0% NP-40 in 50 mM Tris buffer (pH 7.4) containing 150 mM NaCl. Insoluble material was removed by centrifugation at 16,000 ⁇ g for 30 min. The extract was collected and protein content determined with the BCA protein assay (Pierce). The extract was digested with either TPCK-Trypsin (Worthington) or TLCK-chymotrypsin (Sigma) at an enzyme-to-protein ratio of 1:50 for 24 to 48 hours at room temperature.
  • Proteolysis was terminated with either enzyme by addition of phenylmethyl-sulfonyl fluoride (PMSF). Extracts were also digested with pronase (Sigma) using the same conditions. Pronase digestion was stopped by addition of BSA. Purification of whole gC-I or its proteolytic fragments. Whole gC-I or its fragments were isolated by a modification of an immunoaffinity method using biotinylated monoclonal antibodies and streptavidin agarose (Gretch et al., 1987). Briefly, a biotinylated monoclonal antibody was added to 1.0% NP-40 extracts containing whole gC-I or its fragments.
  • PMSF phenylmethyl-sulfonyl fluoride
  • the antibody was incubated with the extracts for 30 min. before adding streptavidin agarose. This mixture was allowed to react for an additional 45 min. with constant mixing.
  • the agarose beads were pelleted by centrifugation and then washed twice with PBS containing 0.1% NP-40 and twice with PBS. Proteins and peptides were eluted from the bound antibody by heating at 100oC in a Tris buffer (0.2 M Tris, pH 6.8, containing 4% SDS) for 3 min.
  • Chymotrypsin fragments were digested with t7e enzyme N-Glycanase
  • Eluted fragments were reduced with beta-mercaptoethanol and diluted witn phosonate-buffered saline (pH 8.6) containing 1.0% NP-40 so that the final SDS concentration was 0.1%.
  • the protease inhibitor 1, 10-phenanthroline hydrate was added according to the manufacturer's instructions. The reaction was done at room temperature for 24 hours with constant mixing. At the end of this time, SDS was added to bring the SDS concentration back to 1.0% and the reaction mixture was dialyzed against 0.1% SDS overnight. Samples were concentrated prior to SDS-PAGE.
  • Lymphocyte proliferation assays Lymphocyte proliferation assays. Lymphocyte proliferation assays were performed as previously described (Liu et al., 1988). Briefly, proteolytic fragments used for T cell analysis were extensively dialyzed against PBS to remove toxic substances. Dialysis membrane with a molecular weight cut out of 10-12,000 was used.
  • proteolysis was stopped at 0.5, 2, and 24 hours by addition of PMSF and gC-I fragments immunoprecipitated with 41C2. In addition, an aliquot was allowed to remain at room temperature for 24 hours without exposure to proteolysis. Proteins and glycoproteins immunoprecipitated with 41C2 were examined by SDS-PAGE with and without reduction of disulfide bonds.
  • the gC-I comDlexes immunoprecipitated by 41C2 from the initial extract or from the same extract after 24 hours at room temperature were similar if not identical regardless of the label used. This demonstrated that gC-I in the absence of trypsin or chymotrypsin was stable in the extract for at least 24 hours. In the absence of proteolysis, complexes typical of gC-I were obtained which had molecular weights from 130,000 to greater than 200,000. The most abundant glycoproteins obtaine ⁇ from these complexes after reduction had molecular weights of 130,000, 93 , 000 and 50-52,000 regardless of the radioactive label used.
  • the chymotrypsin and trypsin glycopeptide fragments obtained from gC-I isolated from extracellular virus were the same as those immunoprecipitated when gC-I was obtained from infected cells.
  • antibodies from all three domains were capable of immunoprecipitating the same fragments, suggesting that they contained the three domains previously described (Lussenhop et al., 1988).
  • pronase a non-specific protease
  • CMV titers were determined by a latex aggultination assay (LA) or immunofluoresences (IF). The first number represents the LA titer and the second the IF titer.
  • LA latex aggultination assay
  • IF immunofluoresences
  • gC-I contains glycoproteins which have homology with gB from HSV
  • these serum were tested for their reactivity with HSV by immunofluoresence. Of those tested, only one (A, 3) was positive for HSV
  • the negative murine antibody control was not reactive with the peptides recognized by the monoclonal antibodies.
  • the pattern obtained with all positive human serum was identical to the monoclonal antibodies and the negative human sera (A,1) was non-reactive.
  • monoclonal antibodies 1184 and 26B11 were not reactive with the chymotrypsin fragments of gC-I. All other monoclonal antibodies reacted strongly with the 34,000 and 30,000 molecular weight peptides and, with the exception of 41C2, reacted strongly with a 28,000 molecular weight peptide. Of the human positive serum tested, four (A,2, A,3, I,1 and I,2) reacted strongly with the
  • gC-I from HCMV strain AD169 was also digested with chymotrypsin, fragments of immunoaffinity purified and examined by Western blot under non-reducing conditions to determine whether or not all three domains would be present.
  • all monoclonal antibodies reacted with the fragments in Western blot, demonstrating the presence of these domains in AD169.
  • the pattern was slightly different. A weak band was detected at 63, 000 molecular weight, but the lower molecular weight peptides appeared more diffuse than they did with peptide from Towne strain HCMV. A broad band covering molecular weights from 40,000 to 35,000 was detected along with a band at 31,000 and another diffuse band with a molecular weight of 23,000.
  • PBMC Peripheral blood mononuclear cells
  • MNC mononuclear cells
  • the chymotrypsin fragments also contained T helper cell epitopes; however, more of these appear to remain in the trypsin fragments. That T cell epitopes were deleted by chymotrypsin was clearly demonstrated by the positive reactivity of a T cell clone with the trypsin fragments, but not with chymotrypsin fragments. Furthermore, the individual from which the T cell clone was obtained showed a very low response to either trypsin or chymotrypsin fragments when mononuclear cell cultures were used. Thus, the population of T cells present in the mononuclear cell cultures from this individual recognizing the T cell epitope present in the trypsin fragments must have been low.
  • the chymotrypsin fragments from gC-I also contained the epitope recognized by antibody 11B4 which was placed in domain III. This was established by the ability of 11B4 to immunoprecipitate the chymotrypsin fragments, and its reactivity in Western blot under non-reducing conditions.
  • Antibody 11B4 is a non-neutralizing antibody which blocks binding of neutralizing antibodies (Lussennop et al., 1938). From this perspective, anti bodies directed toward this epitope would have benefit for the virus, but not the host. It would seem that the chymotrypsin fragments could be used as part of a synthetic vaccine, although the epitope recognized by 11B4 should be avoided in any attempt to make a synthetic vaccine.
  • 11B4 appears to recognize a conformational epitope since its reactivity with the chymotrypsin fragments was lost after reduction of disulfide bonds. Unlike 11B4, those antibodies which neutralized Towne strain HCMV and the human serum were reactive after reduction of disulfide bonds. Furthermore, after reduction, three peptides were detected by our monoclonal antibodies and human serum. One of the major differences between these three peptides was the extent of glycosylation. The 34,000 molecular weight peptide was most heavily glycosylated, but could be reduced to at least 30,000 molecular weight by action of N-glycanase.
  • a synthetic vaccine might include only the linear amino acid sequences of the peptides in the chymotrypsin fragments. Such subunit vaccines are within the scene of this invention.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
EP89907929A 1988-07-01 1989-06-15 Antibody recognition of fragments from cytomegalovirus Ceased EP0429470A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US21430288A 1988-07-01 1988-07-01
US214302 1988-07-01

Publications (1)

Publication Number Publication Date
EP0429470A1 true EP0429470A1 (en) 1991-06-05

Family

ID=22798566

Family Applications (1)

Application Number Title Priority Date Filing Date
EP89907929A Ceased EP0429470A1 (en) 1988-07-01 1989-06-15 Antibody recognition of fragments from cytomegalovirus

Country Status (5)

Country Link
EP (1) EP0429470A1 (ja)
JP (1) JPH03505457A (ja)
AU (1) AU3849989A (ja)
IL (1) IL90833A0 (ja)
WO (1) WO1990000062A1 (ja)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5124440A (en) * 1986-11-24 1992-06-23 The Childrens Hospital, Inc. Antibody and T cell recognition sites on glycoproteins comprising the GCI complex of human cytomegalovirus
WO1991004277A1 (en) * 1989-09-14 1991-04-04 Children's Biomedical Research Institute Monoclonal antibodies specific to cytomegalovirus glycoprotein
WO1994023744A1 (en) * 1993-04-16 1994-10-27 The Wistar Institute Of Anatomy And Biology Recombinant cytomegalovirus vaccine
EP0914441A2 (en) 1996-04-23 1999-05-12 The Wistar Institute Of Anatomy And Biology Novel human cytomegalovirus dna constructs and uses therefor

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4689225A (en) * 1984-11-02 1987-08-25 Institut Merieux Vaccine for cytomegalovirus
DE3619720A1 (de) * 1986-06-12 1987-12-17 Behringwerke Ag Hauptglykoprotein des menschlichen cytomegalovirus, seine herstellung und verwendung
US5126130A (en) * 1986-11-24 1992-06-30 The Childrens Hospital Incorporated Monoclonal antibodies reactive with specific antigenic sites on human cytomegalovirus glycoprotein a

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9000062A1 *

Also Published As

Publication number Publication date
JPH03505457A (ja) 1991-11-28
IL90833A0 (en) 1990-01-18
AU3849989A (en) 1990-01-23
WO1990000062A1 (en) 1990-01-11

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