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WO1991002004A1 - GLYCOPROTEINES IMMUNOGENIQUES DU CYTOMEGALOVIRUS HUMAIN gCII - Google Patents

GLYCOPROTEINES IMMUNOGENIQUES DU CYTOMEGALOVIRUS HUMAIN gCII Download PDF

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Publication number
WO1991002004A1
WO1991002004A1 PCT/US1990/004371 US9004371W WO9102004A1 WO 1991002004 A1 WO1991002004 A1 WO 1991002004A1 US 9004371 W US9004371 W US 9004371W WO 9102004 A1 WO9102004 A1 WO 9102004A1
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hcmv
glycoprotein
glycoproteins
gcii
group
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PCT/US1990/004371
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English (en)
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Richard C. Gehrz
Bruce E. Kari
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Children's Biomedical Research Center
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    • 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
    • C07K16/089—Cytomegalovirus
    • 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
    • C12N2710/16111—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

  • the present invention is directed to substantially pure, immunogenic glycoprotein complexes of HCMV which comprise at least two distinct groups of glycoproteins that are recognized by two different groups of monoclonal antibodies, to substantially pure component glycoproteins of these complexes, and to vaccines
  • HCMV cytomegalovirus
  • HCMV glycoproteins appear to be involved in human immune recognition of the virus. This has been demonstrated by the reactivity of HCMV glycoproteins with human convalescent sera (G.H. Farrar et al., J. Gen. Virol., 65, 1991 (1984); B. Nowak et al., Virology. 132, 325 (1984)) and peripheral blood mononuclear cells (Y.C. Liu et al., J. Virol., 62, 1066-1070 (March 1988)).
  • glycoproteins farrar et al., supra, were able to detect five polypeptides in the membranes of HCMV strain AD169 which were labeled by carbohydrate-specific methods.
  • glycoproteins had molecular weights ranging from 57,000 to 250,000.
  • gCIII Another complex, referred to hereinafter as gCIII, has been identified which contains a glycoprotein with homology to glycoprotein H from HSV (Cranage et al., J. Virol., 62, 1416-1422 (1988)). This glycoprotein was initially described by Rasmussen et al. P.N.A.S., 81, 876-880 (1984).
  • glycoproteins had molecular weights of 50,000-52,000 daltons. A very small amount of a glycoprotein with a molecular weight of 90,000 daltons was also detected.
  • glycoproteins comprising these glycoproteins as gCII.
  • the gCII glycoproteins recognized by 9E10 had a high content of O-linked oligosaccharides (Kari and Gehrz, Arch. Virol., 98, 171-188 (before May, 1988)), and were encoded for in the HXLF gene family of HCMV (Gretch et al., J. Virol., 62, 1956-1962 (June, 1988)).
  • HCMV infection can be a serious problem for a developing fetus, and therefore humoral immune responses to HCMV proteins in pregnant women and their infants have been studied (Ahlfors et al., Scand. J. Infect. Pis., 16, 129-137 (1984); Alford et al., J. Infect. Dis., 158, 917- 924 (1988)). Infant and adult cell-mediated immune responses to HCMV proteins have also been studied. Y.C. Liu et al., J. Virol., 62, 1066-1070 (March, 1988) studied the T-lymphocyte proliferation responses of adult and infant subjects to whole HCMV and to HCMV
  • PBMC peripheral blood mononuclear cells
  • glycoproteins glycoproteins
  • the present invention is directed to compositions of matter which are substantially pure glycoprotein complexes or glycoproteins which are
  • glycoproteins are associated via disulfide linkages into the glycopr ⁇ rtein complexes.
  • the glycoprotein complexes and their component glycoproteins are physically
  • the gCII complex comprises at least two distinct groups of
  • a preferred embodiment of the present invention is a substantially pure, immunogenic glycoprotein complex, which is on the membrane envelope of HCMV, and which contains
  • the complex which includes three glycoprotein
  • the Group 1 monoclonal antibody may be selected from the group consisting of monoclonal antibodies designated herein as 9E10, 8B4, and 26E2.
  • the Group 2 monoclonal antibody may be selected from the group consisting of monoclonal antibodies designated herein as 15F9, 12G9, 15G5, 23B10, 25C8, 27B4, and 40B7.
  • the gCII-200 complex was further analyzed in terms of human humoral immune response to the complex and its component glycoproteins. It was unexpectedly found that under reducing condition the component glycoproteins of gCII-200 are reactive with adult seropositive sera, but are not reactive with congenitally-infected infant sera. However, both the adult and the infant sera are reactive with the 52 kD and 158 kD glycoproteins of the gCI complex. Therefore, the present invention is further directed to the gCII-200 complex wherein the complex is reactive with HCMV-seropositive adult human sera, but is not substantially reactive with HCMV-seropositive, ⁇ ongenitally infected infant human sera. Preferably, the adult human sera and the infant human sera are both reactive with gCI glycoproteins having a molecular weight of about 52 kD or 158 kD.
  • glycoproteins of gCII-200 may be further distinguished by their reactive site within HCMV-infected cells.
  • the Group 1 monoclonal antibodies are N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the Group 1 MoAb 9E10 is preferably reactive with the nucleus of HCMV-infected fixed human cells.
  • the Group 2 MoAbs are preferably reactive with the cytoplasm of HCMV-infected fixed human cells, but are not reactive with the plasma membranes of HCMV-infected living human cells.
  • the present invention is also directed to a substantially pure, immunogenic glycoprotein having a molecular weight of about 50-52 kD, wherein the
  • glycoprotein is derivable from the membrane envelope of HCMV and can be associated with other envelope
  • glycoproteins by means of disulfide bonds, and wherein the about 50-52 kD glycoprotein reacts with a monoclonal antibody selected from the group consisting of 9E10 (IVI-10118), 8B4 and 26E2.
  • a monoclonal antibody selected from the group consisting of 9E10 (IVI-10118), 8B4 and 26E2.
  • 9E10 IVI-10118
  • 8B4 8B4
  • 26E2 a monoclonal antibody
  • the glycoprotein reacts with Group 1 MoAb 9E10 and does not substantially react with Group 2 MoAb 12G9.
  • the 50-52 kD glycoprotein reacts with both Group 1 and Group 2 MoAbs, including the monoclonal antibodies 9E10, 12G9, 26E2, 15F9, and 40B7.
  • non-reducing conditions means that glycoprotein complexes were isolated by detergent extraction from HCMV virion envelopes, and immunaffinity purified as described hereinbelow with gCII-specific monoclonal antibodies to obtain purified, unreduced gCII complexes
  • reducing conditions means that purified, unreduced gCII complexes were additionally solubilized, with subsequent alkylation, as described hereinbelow.
  • glycoproteins could then be separated by SDS-PAGE and analyzed in Western blot, as described hereinbelow.
  • the present invention is also directed to a substantially pure immunogenic glycoprotein having a molecular weight of about 39-48 kD, designated gp39-48(11), which can be detected in whole Towne HCMV in Western Blot using a Group 2 MoAb.
  • the present invention also includes two
  • substantially pure, immunogenic glycoproteins having molecular weights of about 90 kD or greater than about 200 kD, designated gp90(II) and gp200(II), respectively.
  • These glycoproteins are derivable from the membrane envelope of HCMV, can be associated with other envelope glycoproteins by means of disulfide bonds, and are reactive with the Group 2 MoAbs defined above.
  • the gp90(II) and gp200(II) glycoproteins can be obtained by reducing the gCII-200 complex described above. It has also been determined that these glycoproteins are
  • glycoproteins are detectable by HCMV-seropositive adult human sera in Western blot, but are not substantially detectable by HCMV-seropositive, congenitally infected infant human sera. Both adult and infant sera are detectable by gCI proteins of about 52 kD or about 158 kD molecular weight, however.
  • the gp90(II) and gp200(II) glycoproteins were also not substantially detectable by sera from HCMV sero-positive mothers of the congenitally infected infants, when the maternal sera was analyzed within 0-63 months post partum.
  • the present invention also provides a vaccine comprising an amount of one or more of the gp90(II) or gp200(II) glycoproteins which is effective to produce an immune response against HCMV in a mammal, when administered thereto, in combination with a
  • the present glycoprotein complexes and their component glycoproteins may be useful in the production of monoclonal antibodies, such as the gCII-specific monoclonal antibodies described above, which can in turn be used to diagnose HCMV, or to treat HCMV infections by blocking HCMV infectivity and/or toxicity.
  • the present complexes and glycoproteins are also useful to produce clonal populations of antigen-specific T-helper lymphocytes or antigen-specific T-cytotoxic lymphocytes , which in turn can be used for HCMV therapy.
  • the present invention also includes a method for inducing T helper cell (T h ) and T cytotoxic cell (T c ) response against HCMV, which includes the step of administering to a patient a vaccine against HCMV including an immunologically effective amount of gp90(II) or gp200(II) in combination with a pharmaceutically acceptable carrier.
  • glycoprotein complexes such as gCII-200 and their
  • gp93(I) the 93 kD glycoprotein of gCI is designated gp93(I) herein.
  • glycoproteins are designated gp39-48(II),
  • This complex is designated gCII-200 herein.
  • mice were immunized with 100 ug of whole Towne strain HCMV emulsified in complete Freund adjuvant and given a booster immunization with 100 ug whole Towne HCMV at 3 weeks. After several weeks a final boost was given using 20 ug of purified gCII which had been purified by anion exchange high performance liquid chromatography as previously reported by B.E. Kari et al., supra. Three days following the final boost with gCII, the mice were sacrificed and the spleen cells were fused with Sp2-O-Ag14 myeloma cells. Cloning was done as previously described by B.E. Kari et al., supra.
  • culture medium was assayed for antibody against gCII by enzyme linked immunosorbant assay (ELISA).
  • ELISA enzyme linked immunosorbant assay
  • purified gCII was fixed in 96 well dishes.
  • Hybridomas were subcloned twice by limiting dilution. Clonality was further assayed by agarose iso-electric focusing of purified MoAbs.
  • Glycoproteins were radiolabeled with [ 14 C]GlcN or [ 3 H]Arg (New England Nuclear) as previously described by B.E. Kari et al., supra. Labeled proteins were extracted from either extracellular virus or from
  • Tris buffer 50 mM Tris, pH 7.5, 150 mM NaCl, 2mM phenylmethylsulfonyl fluoride
  • solubilization buffer by heating at 100°C for 3 min. After SDS-PAGE, radioactive proteins were detected by fluorography using 3 H Enhance (New England Nuclear).
  • HCMV antigen was prepared by differential centrifugation of virions obtained from supernatants of roller bottle fibroblast cultures infected with Towne HCMV at a multiplicity of infection (MOI) of 1.
  • MOI multiplicity of infection
  • Glycoprotein complexes were obtained by detergent extraction from virion envelopes, and individual gCI and gCII complexes were immunoaffinity purified using gCI-specific and gCII-specific monoclonal antibodies designated as 41C2 and 15F9, respectively. After reduction, individual glycoproteins were separated by sodium dodecylsulfate-polyacrylamide gel
  • nitrocellulose paper as described below.
  • phenylmethylsulfonyl fluoride 2 mM, pH 7.5.
  • Strips of the blocked paper were incubated overnight with MoAbs in ascites diluted 1 to 500 or with human sera diluted 1 to 30 in TBS containing 1% gelatin. Strips were washed with TBS containing 0.05% Tween 20 and then with TBS before incubation with phosphatase labeled goat anti-mouse IgG (H + L) or goat anti-human IgG (H +
  • Cells infected with Towne strain HCMV were examined as living and fixed cells.
  • Cells in 6 well culture dishes were infected with Towne strain HCMV at a MOI of 3 ⁇ 10 -4 or 3.
  • Cultures of the cells infected at an MOI of 3 ⁇ 10 -4 were harvested at 12 days post infection, while cultures of the cells infected at an MOI of 3 were harvested at 4 and 7 days post infection.
  • Living cultures were then stained by indirect immunofluorescence using gCII MoAbs (Group 1 MoAbs 9E10, 26E2, and 8B4 and Group 2 MoAb 15F9) to detect gCII glycoproteins in the plasma membrane.
  • Duplicate cultures which had been fixed with acetone methanol were stained at the same time to detect internal gCII glycoproteins.
  • Example 7 sera were obtained from peripheral blood from infants with symptomatic congenital HCMV infection, mothers of HCMV-infected infants, and normal adult donors, and frozen at -70°C prior to
  • Example 7 serial dilutions of human sera were tested for antibody reactivity with viral proteins expressed on HCMV-infected fibroblasts in an indirect immunofluorescence assay, using polyvalent goat-anti-human immunoglobulin conjugated to fluorescein isothiocyanate (FITC).
  • FITC fluorescein isothiocyanate
  • glycoprotein complexes were obtained under non-reducing conditions by isolating glycoprotein complexes by detergent extraction from HCMV virion envelopes, and immunoaffinity purification of the complexes with gCII-specific monoclonal antibodies.
  • glycoprotein complexes having molecular weights of 93,000 and 450,000, which eluted from an anion exchange column at 0.3 and 0.9 molar NaCl, respectively.
  • the major glycoproteins of the 93,000 complex recognized by 9E10 in electrophoresis after reduction had molecular weights ranging from 50-52 kD.
  • a very small amount of a 90 kD glycoprotein was also detected by Kari et al. B.E. Kari and R.C. Gehrz, Arch. Virol., 98.
  • Group 2 MoAbs included five MoAbs (15F9, 12G9, 23B10, 27B4, and 40B7) which had an IgG2a subtype, MoAb 15G5 which had an IgGl subtype, and MoAb 25C8 which had an IgA subtype.
  • HCMV antibody was used as a negative control.
  • the mouse ascites did not react in Western blot.
  • MoAbs characterized as Group 1 MoAbs recognized gCII glycoproteins with molecular weights ranging from 47,000 to 63,000. These 47-63 kD glycoproteins are believed to include the 50-52 kD glycoprotein designated as gp52(II) by Kari and Gehrz, supra.
  • the MoAbs characterized as Group 2 MoAbs recognized proteins with a molecular weight ranging from 39,000 to 48,000, other proteins with a molecular weight of about 90,000, and other proteins with a molecular weight of greater than about 200,000.
  • the Group 2 MoAbs recognized proteins with a wider range of molecular weights than the Group 1 MoAbs.
  • the 90 kD and the greater than 200 kD proteins recognized by the Group 2 MoAbs were purified and repeatedly exposed to urea, SDS, and reducing reagents, without any further change in their molecular weights.
  • Group 1 and Group 2 gCII MoAbs were examined for their ability to immunoprecipitate gCII complexes from non-ionic detergent extracts of
  • the molecular weight range of the 47-52,000 glycoproteins recognized by MoAb 9E10 was similar to that of the 47-63,000 molecular weight glycoproteins recognized by MoAb 9E10 in Western blot, described above.
  • Kari and Gehrz, Arch. Virol., 98, 171-188 have previously shown that MoAb 9E10 can immunoprecipitate 50,000-52,000 molecular weight glycoproteins from gCII complexes independently of other gCII glycoproteins. Therefore, based on their molecular weights, the 47,000-52,000 molecular weight glycoproteins detected in the present 9E10
  • immunoprecipitates are uncomplexed glycoproteins, and include the 50-52 kD glycoprotein (gp52(II)) described by Kari and Gehrz, supra.
  • gp52(II) the 50-52 kD glycoprotein described by Kari and Gehrz, supra.
  • glycoprotein reacts with Group 1 MoAb 9E10 but not with Group 2 MoAb 12G9.
  • Disulfide-linked complexes ranging in molecular weight from 93,000 to greater than 200,000 were also detected. When these complexes were reduced, a major broad band was detected with molecular weights ranging from 47,000 to 63,000. A darker band within the 47-63 kD band could be detected which corresponds to the 50-52 kD glycoprotein (gp52(II)) described above.
  • glycoproteins 130,000 and of greater than about 200,000. After reduction, the most abundant glycoproteins had molecular weights of about 90,000 and greater than about 200,000.
  • glycoproteins were present in the extracts which are recognized by Group 1 MoAbs and not by Group 2 MoAbs, and other complexes and glycoproteins which are only
  • the greater than 200 kD complex was recognized by both the Group 2 MoAb 12G9 and the Group 1 MoAb 9E10, however. In any case, regardless of whether a Group 1 MoAb or a Group 2 MoAb was initially used in the serial
  • glycoproteins probably are not on the surface of infected cells.
  • fibroblasts were infected at an MOI of 3 and harvested at 4 days post infection. These cultures were fixed and examined by indirect immunofluorescence using gCII MoAbs. Only diffuse background staining was detected with fixed cells when no MoAb was used. With fixed infected cells, all Group 1 and 2 MoAbs showed cytoplasmic staining at either MOI. However, Group 1 MoAb 26E2 showed variable cytoplasmic staining, with some cells showing intense staining, and others very weak staining. This result was obtained with either MOI.
  • Staining of the nucleus could also be detected with Group 1 MoAb 9E10.
  • the nuclear staining appeared to be of two types. In some cells there was a diffuse staining of the whole nucleus, while in other cells intensely stained dots as well as diffuse staining were detected. The intense dots were also occasionally observed in the cytoplasm. No other Group 1 or 2 MoAb showed nuclear staining.
  • human skin fibroblasts were infected with Towne strain HCMV at an MOI of 1. Cells were scraped from culture dishes at 24, 48, 72, and 96 hours post infection. Culture extracts from these cells were immunoprecipitated using Group 2 MoAb 15F9. The purified gCII was reduced, subjected to SDS-PAGE, and examined by Western blot using MoAb 15F9.
  • An SP2
  • Proteins with molecular weights of about 90,000 and greater than about 200,000 were clearly detected at 72 hours post infection. This data indicated that proteins recognized by Group 2 MoAbs such as 15F9 are late
  • the human humoral immune response to gCII proteins was examined by Western blot using gCII which had been immunoaffinity purified with Group 2 MoAb 15F9. Purified gCII was reduced, the resulting proteins
  • nitrocellulose paper strips of nitrocellulose paper were probed with rabbit sera, adult and infant human sera, and monoclonal antibodies as described below.
  • gCII protein purified with Group 2 MoAb 15F9 was used for analysis with seropositive rabbit and adult convalescent HCMV sera.
  • each of the following was used as a probe of the purified gCII: rabbit anti-skin fibroblast serum (RSF); rabbit preimmune serum (R-); rabbit anti-Towne HCMV serum (R+); mouse ascites negative control (SP2); MoAb 35F10, which is specific for a 28,000 molecular weight HCMV protein;
  • Group 1 MoAb 9E10 Group 2 MoAb 12G9; Group 2 MoAb 15F9; six adult positive sera (A + ); and two adult HCMV negative sera (A-).
  • the gCII proteins are believed to be immunogenic in rabbits as well as in humans. However, no reactivity was detected with rabbit anti-skin
  • fibroblast serum RSF
  • R- rabbit preimmune serum
  • immunogenicity of gCII proteins may be specific to rabbit serum obtained from rabbits inoculated with Towne strain HCMV.
  • HCMV IMMUNOFLUORESCENCE TITERS FOR HUMAN SERA AND THEIR REACTIVITY WITH HCMV PROTEINS IN WESTERN BLOT
  • Immunofluorescence titers were determined in the clinical virology laboratory at the University of Minnesota and are expressed as the reciprocal of serum dilution. Indirect immunofluorescence titers of ⁇ 10 were considered negative.
  • CMV was isolated from the urine in the first week, but was not necessarily related to the patient's clinical symptom's. Ill did not have CMV infection in early infancy, but acquired CMV at six-months-of-age. Five of the adult positive sera showed strong reactivity with gCII proteins in Western Blot, again giving a pattern similar to that obtained with Group 2 MoAbs 12G9 and 15F9. Specifically, bands were detected which corresponded to glycoproteins having molecular weights of about 39-48 kD and other glycoproteins having molecular weights of about 90 kD to greater than 200 kD. One HCMV positive adult serum reacted with only a single protein having an apparent molecular weight of 100,000, which may have been non-specific since faint reactivity was detected with the negative sera.
  • HCMV MoAb 35F10
  • the 28,000 molecular weight protein was not associated with gCII by disulfide bonds, and is believed to be a contaminating HCMV protein.
  • HCMV antibody titers for the infant sera are also summarized in Table 2, above. Approximately 200 ug of gCII protein purified by Group 2 MoAb 15F9 was used in Western blot analysis with infant sera. The following were used as probes in Western Blot: HCMV MoAb 35F1; mouse ascites negative control (SP2); eleven infant sera positive for HCMV; two adult negative serum (A-) ; and seven adult positive serum (A+) .
  • the other 2 of 11 infant sera which contained gCII-specific antibodies were fraternal twins.
  • the first, 110 presented with anemia, hepatomegaly, and petecchiae due to abruptio placenta.
  • CMV was isolated from the urine during the first week but may not have been related to the patient's clinical symptoms.
  • a second twin, 111 did not have congenital CMV infection, but acquired CMV at six-months-of-age. Neither infant suffered any permanent injury to CMV.
  • HCMV protein 28,000 molecular weight HCMV protein; mouse negative ascites control (SP2); gCI-specific MoAb 39E11; eleven HCMV positive infant sera; two negative adult sera; and seven adult HCMV positive sera.
  • SP2 mouse negative ascites control
  • gCI-specific MoAb 39E11 eleven HCMV positive infant sera; two negative adult sera; and seven adult HCMV positive sera.
  • HCMV Human cytomegalovirus
  • CID cytomegalic inclusion disease
  • symptomatic congenital HCMV infection may have a deficit in the HCMV-specific humoral immune response.
  • Monoclonal antibodies were used to confirm the presence of individual gCI and gCII glycoproteins in the antigen preparations to be used for Western blot analysis of human sera.
  • Western blotting indicated that the purified gCI contained three glycoproteins.
  • gp52 glycoprotein having a molecular weight of about 52,000, designated gp52; and to a fully glycosylated precursor glycoprotein having a molecular weight of about 158,000, designated gpl58.
  • Western blotting with purified gCII indicated that gCII contained two antigenically distinct groups of glycoproteins, designated Group 1 and Group 2. Specifically, following Western blotting of purified gCII and blotting with gCII/Group 1-specific MoAb 9E10, bands were detected in the gel corresponding to glycoproteins having a molecular weight ranging from about 45,000- 63,000, designated gp45-63.
  • pp28 An unrelated non-glycosylated protein designated pp28 consistently co-precipitated with either gCI or gCII, and was recognized by MoAb 35F10. This monoclonal antibody recognizes a matrix protein of the virion.
  • HCMV-seropositive convalescent sera obtained from healthy adult donors, as well as in sera from congenitally infected infants.
  • Sera from 7 of 8 HCMV-positive adult donors contained antibodies reactive with all Group 1 and Group 2 gCII glycoproteins, as well as antibody reactive with the matrix protein pp28.
  • one apparently healthy seropositive adult lacked gCII-specific
  • HCMV-infected fibroblasts as determined by indirect immunofluorescence, expressed as reciprocal of serum dilution.
  • c Qualitative isolation of HCMV from urine and/or saliva in fibroblast cell culture (+/-) or quantitative excretion of HCMV In urine expressed as reciprocal dilution exhibiting TClD 50 .
  • the "i" symbol indicates equivocal reactivity with the lowest molecular weight glycoprotein in each group; i.e. gp45-63, gp39-48 or gp 28.
  • Patient 1 of Table 3 presented with symptoms of cytomegalic inclusion disease in the newborn period, including intrauterine growth retardation, microcephaly, hepatosplenomegaly, jaundice, and petechiae. After initial clinical improvement, this patient developed HCMV-associated interstitial pneumonia requiring prolonged hospitalization and supplemental oxygen at 10 weeks of age, associated with a quantitative increase in viral excretion and a rise in HCMV antibody titer.
  • Western blot of purified gCI and probing with sera from Patient 1 over a 32-month period indicated that this patient developed a quantitative increase in
  • gCII-specific MoAbs have been made using partially purified gCII as the immunizing antigen in mice.
  • Several MoAbs were obtained and characterized.
  • the MoAbs showed distinctions between high and low molecular weight glycoproteins found in gCII complexes. Accordingly, the MoAbs were characterized as belonging to either Group 1 or Group 2.
  • the gCII complex contains two distinct groups of proteins which are recognized by the two different groups of monoclonal antibodies.
  • glycoproteins having molecular weights of about 39-48 kD, about 90 kD, and greater than about 200 kD. These proteins were synthesized late in infection. (Proteins recognized by Group 1 MoAbs are also synthesized late in infection, but have much lower molecular weights, as reported by Gretch et al., J. Virol., 62, 1956-1962
  • Group 2 MoAbs showed strong immunofluorescence with fixed cells, but not with living infected cells, in contrast with the Group 1 MoAbs which stained the plasma membrane of the living infected cells. These results are further evidence that the proteins recognized by Group 2 MoAbs are different from those recognized by Group 1
  • the proteins recognized by Group 2 MoAbs may be products of different transcripts encoded by the HXLF gene family, or encoded by a different gene or genes.
  • One possibility is that the gCII complexes recognized on the surface. of infected cells are those which were immunoprecipitated by Group 1 MoAb 9E10, but were not immunoprecipitated by Group 2 MoAb 12G9. Accordingly, the gCII complexes recognized by both groups of MoAbs may only be expressed inside the cell. A less likely
  • epitopes recognized by Group 2 MoAbs may be on the surface of infected cells, but are not exposed enough for antibody binding.
  • Immunoaffinity purified gCII proteins were also recognized by antibody in adult convalescent sera.
  • the pattern obtained in Western blot of the human sera was very similar to that obtained with Group 2 MoAbs.
  • Some of the low molecular weight proteins recognized by the human sera may include proteins
  • glycoproteins and the 28,000 molecular weight protein in Western blot were also recognized by adult sera.
  • a major difference between adult and infant sera was the lack of reactivity with reduced gCII proteins.
  • the lack of reactivity by the infant sera may have resulted from the fact that the strain of HCMV which they were infected with did not express gCII.
  • the presence of antibody to gCII proteins in several adult sera suggested that wild type strains of HCMV do express these proteins.
  • infants who had gCII antibodies also had very low HCMV antibody titers to whole HCMV as detected by indirect immunofluorescence, as compared to infants who lacked gCII antibodies.
  • Liu et al., J. Virol., 62, 1066-1070 (1988) have previously reported finding no qualitative differences in the ability to immunoprecipitate gCI and gCII when comparing adult convalescent sera and congenitally infected infant sera. Therefore, these infants may have antibody which recognize conformational determinants on gCII complexes, but may lack significant amounts of antibodies which recognize continuous epitopes on gCII glycoproteins.
  • Example 7 the purpose of the study described therein was to characterize the antibodies to individual envelope glycoproteins of HCMV in sera from symptomatic congenital HCMV infants and their mothers, and to determine if susceptibility to clinical injury and/or viral persistence might be related to deficits in the antibody responses to these
  • glycoproteins or similarities in molecular weights among unrelated glycoproteins which make it difficult to discriminate the specificity of individual antibody responses.
  • purification of the individual glycoproteins prior to immunological analysis allowed the confirmation of their importance in the human antibody response in normal individuals, and the
  • HCMV-specific T h proliferation to immunodominant (glyco)proteins previously described by Gehrz et al., Lancet. 2, 844-847 (1977); and Gehrz et al., Clin. Exp. Immunol., 74, 333-338 (1988) may account for the lack of antibody response to gCII glycoproteins in congenitally infected infants, as described in Example 7 above. Furthermore, mothers of congenitally infected infants also appear to lack gCII-specific antibodies, indicating that susceptibility to the pathogenic effects of HCMV may be associated with maternal/fetal non-responsiveness to gCII and implying an essential role for gCII-specific antibodies in host defense.
  • gCII glycoproteins may be present to varying extents in different strains of HCMV, with virulent strains either not expressing gCII in sufficient quantity to stimulate the immune response, or containing gCII polymorphisms which do not expose or express immunodominant epitopes recognized by neutralizing gCII antibodies. Since these infants do appear to have antibodies recognizing conformation-dependent epitopes on unreduced gCII complexes (Liu et al., J. Virol., 62. 1066-1070 (1988)), it would seem that antigenic

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Abstract

Un complexe de glycoprotéines immunogéniques sensiblement pur du cytomégalovirus humain possède un poids moléculaire supérieur à environ 200 kD. Le complexe comprend au moins deux groupes distincts de glycoprotéines qui sont reconnus par deux groupes différents d'anticorps monoclonaux. Le complexe réagit avec des sérums séropositifs d'adultes mais n'est pas sensiblement réactif avec des sérums d'enfants infectés par voie congénitale.
PCT/US1990/004371 1989-08-07 1990-08-03 GLYCOPROTEINES IMMUNOGENIQUES DU CYTOMEGALOVIRUS HUMAIN gCII WO1991002004A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5552143A (en) * 1989-03-24 1996-09-03 The Wistar Institute Of Anatomy & Biology Recombinant cytomegalovirus vaccine
US5591439A (en) * 1989-03-24 1997-01-07 The Wistar Institute Of Anatomy And Biology Recombinant cytomegalovirus vaccine
AU681994B3 (en) * 1997-02-28 1997-09-11 Commonwealth Scientific And Industrial Research Organisation Moisture detection
US5807557A (en) * 1994-07-25 1998-09-15 The Trustees Of The University Of Pennsylvania Soluble herpesvirus glycoprotein complex
US6156319A (en) * 1994-07-25 2000-12-05 The Trustees Of The University Of Pennsylvania Soluble herpesvirus glycoprotein complex vaccine
US6448389B1 (en) 1996-04-23 2002-09-10 The Wistar Institute Of Anatomy And Biology Human cytomegalovirus DNA constructs and uses therefor
US9346874B2 (en) 2009-12-23 2016-05-24 4-Antibody Ag Binding members for human cytomegalovirus
CN112379088A (zh) * 2020-12-04 2021-02-19 福建农林大学 一种基于金纳米花技术检测铅残留的检测试纸

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. OF VIROLOGY, Volume 62, No. 3, issued March 1988, GRETCH et al., "Identification and Characterization of Three Distinct Families of Glycoprotein Complexes in the Envelopes of Human Cytomegalovirus", pages 875-881. *
J. OF VIROLOGY, Volume 62, No. 3, issued March 1988, LIU et al., "Human Immune Responses to Major Human Cytomegalovirus Glycoprotein Complexes", pages 1066-1070. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5552143A (en) * 1989-03-24 1996-09-03 The Wistar Institute Of Anatomy & Biology Recombinant cytomegalovirus vaccine
US5591439A (en) * 1989-03-24 1997-01-07 The Wistar Institute Of Anatomy And Biology Recombinant cytomegalovirus vaccine
US5807557A (en) * 1994-07-25 1998-09-15 The Trustees Of The University Of Pennsylvania Soluble herpesvirus glycoprotein complex
US6156319A (en) * 1994-07-25 2000-12-05 The Trustees Of The University Of Pennsylvania Soluble herpesvirus glycoprotein complex vaccine
US6541459B1 (en) 1994-07-25 2003-04-01 The Trustees Of The University Of Pennsylvania Soluble herpesvirus glycoprotein complex vaccine
US6448389B1 (en) 1996-04-23 2002-09-10 The Wistar Institute Of Anatomy And Biology Human cytomegalovirus DNA constructs and uses therefor
AU681994B3 (en) * 1997-02-28 1997-09-11 Commonwealth Scientific And Industrial Research Organisation Moisture detection
US9346874B2 (en) 2009-12-23 2016-05-24 4-Antibody Ag Binding members for human cytomegalovirus
CN112379088A (zh) * 2020-12-04 2021-02-19 福建农林大学 一种基于金纳米花技术检测铅残留的检测试纸
CN112379088B (zh) * 2020-12-04 2022-05-31 福建农林大学 一种基于金纳米花技术检测铅残留的检测试纸

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