WO1994017810A1 - Vaccin recombine contre le cytomegalovirus - Google Patents

Vaccin recombine contre le cytomegalovirus Download PDF

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WO1994017810A1
WO1994017810A1 PCT/US1994/002107 US9402107W WO9417810A1 WO 1994017810 A1 WO1994017810 A1 WO 1994017810A1 US 9402107 W US9402107 W US 9402107W WO 9417810 A1 WO9417810 A1 WO 9417810A1
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exon
adenovirus
glu
strain
hcmv
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Stanley A. Plotkin
Robert P. Ricciardi
Eva Gonczol
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Wistar Institute of Anatomy and Biology
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
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    • 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
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • 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
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention refers generally to a recombinant human cytomegalovirus vaccine, and more specifically to a subunit vaccine.
  • Cytomegalovirus is one of a group of highly host specific herpes viruses that produce unique large cells bearing intranuclear inclusions.
  • the envelope of the human cytomegalovirus (HCMV) is characterized by a major glycoprotein complex recently termed gB or gCI, which was previously referred to as gA.
  • HCMV causes cyto gratisic inclusion disease and has been associated with a syndrome resembling infectious mononucleosis in adults. It also induces complications in immunocompromised individuals.
  • CMV infection in utero is an important cause of central nervous system damage in newborns. Although the virus is widely distributed in the population, about 40% of women enter pregnancy without antibodies and thus are susceptible to infection. About 1% of these women undergo primary infection in utero. Classical cytomegalic inclusion disease is rare; however, a proportion of the infected infants, including those who were symptom free, are subsequently found to be mentally retarded.
  • HCMV vaccines have been developed or are in the process of development. Vaccines based on live attenuated strains of HCMV have been described. [See, e.g., S. A. Plotkin et al, Lancet, .1:528-30 (1984); S. A. Plotkin et al, J. Infect. Pis.. 134:470-75 (1976); S. A. Plotkin et al, "Prevention of Cytomegalovirus Pisease by Towne Strain Live Attenuated Vaccine", in birth Pefects, Original Article Series, £0.(1) :271-287 (1984); J. P. Glazer et al, Ann. Intern. Med..
  • Adenoviruses have been developed previously as efficient heterologous gene expression vectors.
  • an adenovirus vector has been employed to express herpes simplex virus glycoprotein gB [P. C. Johnson et al, Virol.. 164:1-14 (1988)]; human immunodeficiency virus type 1 envelope protein [R. L. Pewar et al, J. Virol.. (52:129-136 (1988)]; and hepatitis B surface antigen [A. R. Pavis et al, Proc. Natl. Acad. Sci.. U.S.A.. 82:7560-7564 (1985); J. E. Morin et al, Proc. Natl. Acad. Sci..
  • Adenoviruses have also been found to be non-toxic as vaccine components in humans [See, e.g., E. T. Takajuji et al, J. Infect. Pis.. 140:48-53 (1970); P. B. Collis et al, J. Inf. Pis.. 128:74-750 (1973); and R. B. Couch et al, Am. Rev. Respir. Pis. f iB_8 . :394-403 (1963)].
  • the present invention provides a non-defective recombinant adenovirus containing an immediate-early exon-4 (IE-exon-4) subunit of the HCMV free from association with any additional " human proteinaceous material.
  • IE-exon-4 immediate-early exon-4
  • the HCMV subunit is under the control of regulatory sequences capable of expressing the IE-exon 4 subunit in vitro and in vivo.
  • Another aspect of the present invention is a vaccine composition comprising a non-defective recombinant adenovirus, as described above.
  • the invention provides a method of using the recombinant adenovirus containing the subunit gene encoding IE-exon-4, in the manufacture of a vaccine composition useful against HCMV infection.
  • the inventors have found that presenting these HCMV subunit proteins expressed by in vivo transcription of the gene to a vaccinate is particularly capable of eliciting a protective immune response.
  • the invention provides an adenovirus-produced HCMV IE-exon-4 subunit, which subunit may also form vaccine compositions to protect humans against HCMV.
  • the present invention provides a novel murine model useful for demonstrating cytotoxic T lymphocyte (CTL) response to individual HCMV proteins.
  • CTL cytotoxic T lymphocyte
  • the present invention provides novel immunogens and vaccine components for HCMV which comprise an adenovirus expression system capable of expressing a selected HCMV subunit gene in vivo.
  • an immunogenic composition i.e., a composition which elicits a humoral and/or a cell-mediated immune response, may be expressed in, and isolated from, the recombinant adenovirus expression system.
  • an immunogenic composition may be used in a vaccine for protecting against human CMV infection.
  • any adenovirus strain capable of replicating in mammalian ceils in vitro may be used to construct an expression vector for the selected HCMV subunit.
  • a preferred expression system involves a non-defective adenovirus strain, including, but not limited to, adenovirus type 5.
  • adenovirus type 5 adenovirus type 5
  • other desirable adenovirus strains may be employed which are capable of being orally administered, for use in expressing the CMV subunit in vivo .
  • Such strains useful for in vivo production of the subunit in addition to adenovirus-5 strains include adenovirus type 4, 7, and 21 strains. [See, e.g., Takajuji et al, cited above].
  • Appropriate strains of adenovirus including those identified above and those employed in the examples below are publicly available from sources such as the American Type Culture Collection, Rockville, Maryland.
  • the presently preferred subunit protein for use in the present invention is the HCMV IE-exon 4 subunit.
  • the full length IE1 gene was reported by Stenberg et al, J. Virol.. 41:190-199 (1984).
  • An Xbal E fragment containing the exon 4 subunit of the IE (or IE1) gene of the Towne strain of HCMV was reported to GenBank, Los Alamos, New Mexico in September 15, 1989 by Stenberg et al.
  • the nucleic acid sequences of the coding region of the IE-exon-4 are provided in SEQ IP NO:l, in which the native TC nucleotides which precede the lysine codon have been modified to the ATG initiation codon.
  • SEQ IP NO:2 provides amino acid sequences of the IE-exon 4 protein.
  • the HCMV IE-exon 4 subunit may be produced in vitro by recombinant techniques in large quantities sufficient for use in a subunit vaccine.
  • more than one HCMV subunit may be employed in a vaccine according to the teachings of the present invention.
  • the recombinant adenovirus containing the subunit may itself be employed as an immunogen or vaccine component, capable of expressing the subunit in vivo .
  • One embodiment of the invention provides a replication competent adenovirus-5 vector carrying the HCMV IE-exon 4 gene.
  • the desired subunit may be isolated from an available strain of HCMV for insertion into the selected adenovirus.
  • a number of strains of human CMV have been isolated.
  • the Towne strain of CMV a preferred strain for use in preparation of a vaccine of this invention because of its broad antigenic spectrum and its attenuation, was isolated from the urine of a two month old male infant with cytomegalic inclusion disease (symptoms - central nervous system damage and hepatosplenomegaly) .
  • This strain of CMV was isolated by Stanley A. Plotkin, M.P. of The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania, and is described in J. Virol.. 11 (6) : 991 (1973) .
  • This strain is freely available from The Wistar Institute or from the American Type Culture Collection (ATCC) , 12301 Parklawn Prive, Rockville, Maryland, USA, under accession number VR-977.
  • ATCC American Type Culture Collection
  • other strains of CMV useful in the practice of this invention may be obtained from depositories like the ATCC or from other institutes or universities.
  • the subunit sequence can be chemically synthesized by resort to conventional methods known to one of skill in the art and, e.g., SEQ IP NOS: l and 2. Alternatively, the sequence may be purchased from commercial sources.
  • the recombinant adenovirus of the present invention may also contain multiple copies of the HCMV subunit.
  • the recombinant virus may contain more than one HCMV subunit type, so that the virus may express two or more HCMV subunits or immediate early antigens and subunits together.
  • the sequences of other HCMV subunits of two HCMV strains have been published [See, e.g., Mach et al, J. Gen. Virol..
  • the CMV subunit sequence is preferably inserted in an adenovirus strain under the control of an expression control sequence in the virus itself.
  • the adenovirus vector of the present invention preferably contains other sequences of interest in addition to the HCMV subunit. Such sequences may include regulatory sequences, enhancers, suitable promoters, secretory signal sequences and the like.
  • sequences may include regulatory sequences, enhancers, suitable promoters, secretory signal sequences and the like.
  • the techniques employed to insert the subunit sequence into the adenovirus vector and make other alterations in the viral PNA, e.g., to insert linker sequences and the like, are known to one of skill in the art. See, e.g., T. Maniatis et al, "Molecular Cloning.
  • adenovirus expression vectors for expression of an HCMV IE-exon 4 subunit protein is within the skill of the art.
  • Example 1 below describes in detail the construction of a non-defective adenovirus containing the HCMV IE-exon-4 subunit.
  • the recombinant adenovirus itself may be used directly as an immunogen or a vaccine component.
  • the recombinant adenovirus containing the HCMV subunit, e.g., the IE-exon-4 subunit, is introduced directly into the patient by vaccination.
  • the recombinant virus when introduced into a patient directly, infects the patient's cells and produces the CMV subunit in the patient's cells.
  • the inventors have found that this method of presenting these HCMV genes to a vaccinate is particularly capable of eliciting a protective immune response.
  • Examples 2 and 3 below demonstrate the ability of the adenovirus recombinant of this invention, Ad-IE, containing subunit IE-exon-4 to elicit a CTL response from immunized mice.
  • the recombinant viral vector containing the CMV subunit protein e.g., the IE-exon 4 subunit
  • it may be infected into a suitable host cell for in vitro expression.
  • the infection of the recombinant viral vector is performed in a conventional manner.
  • Suitable host cells include mammalian cells or cell lines, e.g., A549 (human lung carcinoma) or 293 (transformed human embryonic kidney) cells.
  • the host cell once infected with the recombinant virus of the present invention, is then cultured in a suitable medium, such as Minimal Essential Medium (MEM) for mammalian cells.
  • MEM Minimal Essential Medium
  • the culture conditions are conventional for the host cell and allow the subunit, e.g., IE-exon4, to be produced either intracellularly, or secreted extracellularly into the medium.
  • Conventional protein isolation techniques are employed to isolate the expressed subunit from the selected host cell or medium.
  • the subunit When expressed in vitro and isolated from culture, the subunit, e.g., IE-exon4, may then be formulated into an appropriate vaccine composition.
  • Such compositions may generally contain one or more of the recombinant CMV subunits.
  • a pharmaceutically acceptable vaccine composition having appropriate pH, isotonicity, stability and other conventional characteristics is within the skill of the art.
  • such vaccines may optionally contain other components, such as adjuvants and/or carriers, e.g., aqueous suspensions of aluminum and magnesium hydroxides.
  • the present invention also includes a method of vaccinating humans against human CMV infection with the recombinant adenovirus vaccine composition.
  • This vaccine composition is preferably orally administered, because adenoviruses are known to replicate in cells of the stomach. Previous studies with adenoviruses have shown them to be safe when administered orally [see, e.g., Collis et al, cited above].
  • the present invention is not limited by the route of administration selected for the vaccine.
  • a dosage of between 10 5 and 10 8 plaque forming units may be used. Additional doses of the vaccines of this invention may also be administered where considered desirable by the physician.
  • the dosage regimen involved in the method for vaccination against CMV infection with the recombinant virus of the present invention can be determined considering various clinical and environmental factors known to affect vaccine administration.
  • the vaccine composition may comprise one or more recombinantly-produced human CMV subunit proteins, preferably including the IE-exon-4 subunit.
  • the in vitro produced subunit proteins may be introduced into the patient in a vaccine composition as described above, preferably employing the oral, nasal or subcutaneous routes of administration.
  • the dosage for all routes of administration of the in vitro vaccine containing one or more of the CMV subunit proteins is generally greater than 20 micrograms of protein per kg of patient body weight, and preferably between 40 and 80 micrograms of protein per kilogram.
  • adenovirus The utility of the recombinant adenoviruses of the present invention is demonstrated through the use of a novel mouse experimental model which characterizes cytotoxic T lymphocyte (CTL) responses to individual proteins of strictly human-restricted viruses.
  • CTL cytotoxic T lymphocyte
  • the model as used herein is based on the use of two types of recombinant viruses, an adenovirus and a canarypox virus, both expressing a gene of the same HCMV protein.
  • This model is useful in identifying immunodominant HCMV proteins and immunodominant epitopes of individual proteins to incorporate into an appropriate immunizing vector, analysis of proteins of various HCMV strains, immunization protocols and the longevity of cell-mediated immunity to individual proteins or epitopes; and investigation of the optimal vector for effective introduction of a certain antigen or epitope to the host immune system.
  • mice are immunized with one recombinant, such as that of the invention, and CTL activity tested in target cells infected with the other recombinant.
  • Example 2 provides a murine model of the cytotoxic T lymphocyte (CTL) response to the glycoprotein B (gB) gene of human cytomegalovirus (HCMV) based on the use of gB-expressing adenovirus (Ad-gB) and several poxvirus recombinants.
  • CTL cytotoxic T lymphocyte
  • gB glycoprotein B gene of human cytomegalovirus
  • the protection of humans from CMV infection or virus-induced diseases is based on antibody dependent and/or T-cell dependent immune responses.
  • the following experimental data demonstrates that an adenovirus recombinant containing the major immediate early (IE) gene of HCMV elicits a protective immune response in mice.
  • the nucleic acid sequences of the coding region of the IE-exon-4 are provided in SEQ IP NO:l, in which the native TC nucleotides which precede the lysine codon have been modified to the ATG initiation codon.
  • PCR polymerase chain reaction
  • the PCR primers were synthesized so as to incorporate the proper restriction endonuclease cleavage site, Xbal, (underlined in SEQ IP NOS: 3 and 4 below) for insertion into the Xbal site of the adenovirus vector.
  • Xbal restriction endonuclease cleavage site
  • the 5' primer was also modified so that an ATG start translation codon was inserted at the first amino acid position of exon 4.
  • the oligonucleotides used as primers were the following: 5' IE-exon 4: SEQ IP NO:3:
  • the 5' oligonucleotide corresponds to nucleotide positions 1 to 27 (sense orientation) and the 3' oligonucleotide corresponds to nucleotide positions 1251 to 1222 (anti-sense orientation) of an Xbal E fragment of the HCMV IE1 gene (Towne strain) available from GenBank, Los Alamos, New Mexico (Accession #M11630, Code #8SMIE4) . This fragment was used as an Exon 4 gene template for the PCR reaction. The full length IE1 gene was reported by Stenberg et al, J. Virol.. 4 ⁇ :190-199 (1984).
  • the 5' and 3* primers 400 ng each were mixed with 0.1 ⁇ g of purified HCMV genomic PNA and the PNA was amplified using the Perkin-Elmer amplitaq kit.
  • the final reaction volume was 100 ⁇ l and the thermocycling conditions were 94°C, l min; 52°C, 1 min; 72°C, 1 min, repeated for a total of 35 cycles.
  • Amplified PNA was purified by cutting the proper size PNA fragment out of a 1.2% agarose gel, digested with Xbal.
  • the E3 coding region (between map units 78.5 and 84.0) of the adenovirus is replaced by the exon-4 fragment.
  • the correct orientation allows for the proper transcription of the gene fragment (in the sense orientation) from the adenovirus E3 promoter.
  • the exon-4 product of the HCMV-IE gene was shown to be a target for CP8 cytotoxic and CP4 lymphopr ⁇ liferative T cell responses in humans.
  • the Ad- IE-exon-4 construct is non-defective in replication (i.e., capable of replicating normally) in tissue culture cells.
  • This Ad-IE-exon-4 recombinant was used in the in vitro cytotoxic T lymphocyte (CTL) assay and mouse model described below.
  • This CTL assay is a system in which two types of viral expression vectors, poxvirus and adenovirus, carrying the same HCMV IE-exon 4 subunit gene, are alternately used for immunization of animal or for infection of target cells to show that HCMV IE-exon 4 subunit is an inducer of CTL in mice.
  • This model system the relative immunogenicities of both a gB antigen expressed by different recombinant viruses and the IE exon 4 subunit antigen has been evaluated.
  • A. Recombinant Viruses Used in CTL Assays The following recombinant viruses were used in the CTL assay of Example 3 below to demonstrate the vaccine utility of the recombinant adenoviruses of the present invention.
  • Wild-type human adenovirus type 5 WT-Ad
  • Ad-gB non-defective adenovirus-gB recombinant
  • E3-deleted adenovirus type 5 mutant lacking the Xbal P fragment of adenovirus PNA was constructed by overlap recombination, using plasmid pAd-5 mu 59.5-100, which was deleted in E3 sequences (mu 78.5-84) using the techniques described in EP No. 389,286 and Marshall et al, cited above, and pAd-5 mu 0-75.9.
  • the vaccinia WR strain [obtained from Pr. Enzo Paoletti, Virogenetics Corp, Troy, NY] was used to develop a recombinant expressing HCMV-gB ( (VacW)-gB) .
  • This recombinant was derived using a strategy similar to that described for the VacC-gB recombinant (Gonczol et al. , cited above) .
  • a canarypox recombinant [ALVAC-CMV (VCP139) which is subsequently referred to as Cp-gB] expressing the HCMV-gB gene was constructed using a strategy similar to that described for a canarypox-rabies recombinant in Taylor et al.. Vaccine. :190-193 (1991) [also obtained from Or. Enzo Paoletti] .
  • HCMV Downe strain glycoprotein B glycoprotein B
  • canarypox donor plasmid consisting of a polylinker flanked by genomic sequence from which a nonessential gene was specifically deleted (at a unique EcoRI site within a 3.3 kbp PvuII subgenomic fragment of canarypox PNA) .
  • Expression of the gB protein gene was placed under the transcriptional control of an early/late vaccinia virus promoter (H6) previously described [Percus et al., J. Virol. , .62:3829-3835 (1989)].
  • H6 early/late vaccinia virus promoter
  • the Cp-gB recombinant and parental canarypox virus (WT-Cp) were propagated on primary chick embryo fibroblasts (CEF) cells [ATCC CRL 1590].
  • Ad-gB and WT-Ad were purified by CsCl gradient centrifugation.
  • VacC-gB, VacW-gB and WT-Vac were purified by sucrose gradient centrifugation, and Cp-gB and WT-Cp were concentrated on sucrose cushion.
  • spleens were aseptically removed and cell suspensions were prepared by gently pressing the spleens through a stainless steel mesh.
  • Cells were suspended at 2.5 x 10 6 viable cells/ml in RPMI 1640 medium containing 5% FBS (Gibco) , 2 x 10 ⁇ 5 M 2-mercaptoethanol, 14 mM HEPES buffer, glutamine and 50 ⁇ g/ml gentamicin.
  • CP4 or CP8 cells For in vitro depletion of CP4 or CP8 cells, 3 x 10 6 spleen cells were incubated with anti-mouse CP4 monoclonal antibody (MAb) [Pharmingen; Cat.3:01061 P; 20 ⁇ g/3xl0 6 cells] or CP8 MAb [Accurate; Cat.#:CL-8921; diluted 1:4] for 60 minutes at 4°C, and further incubated in the presence of rabbit complement [Accurate; Low-tox M; diluted 1:10] for 30 minutes at 37°C. The cells were washed twice and used as effector cells in a 51 Cr-release test.
  • MAb monoclonal antibody
  • H-2 d mouse MC57 (H-2 ) cells [also termed MC-57G, P.P. Aden et al, Immunoqenetics. 2:209-221 (1976)] and mouse NCTC clone 929 (H-2 k ) cells [ATCC CCL 1] were used as target cells.
  • the HCMV neutralization titer of mouse sera was determined on MRC-5 cells [ATCC CCL 171] by the microneutralization method as described in Gonczol et al., J. Virol. Methods. 14:37-41 (1986).
  • the target cells were infected with Ad-gB or
  • Target cells were washed in the modified RPMI 1640 medium described above and 2 x 10 6 cells were labeled with 100 ⁇ Ci of [ 51 Cr]NaCr04 [Amersham, specific activity 250-500 mCi/mg] for 1 hour.
  • the labeled target cells were washed 3 times in phosphate-buffered saline (PBS) and then mixed with the effector cells at various effector:target ratios in triplicate using 96-well U-bottomed microtiter plates and incubated for 4 hours.
  • PBS phosphate-buffered saline
  • Percentage specific 51 Cr release was calculated as: [(cpm experimental release - cpm spontaneous release) / (cpm maximal release - cpm spontaneous release)] x 100. Standard deviation of the mean of triplicate cultures was less than 10%, and spontaneous release was always less than 25%.
  • mice were immunized with Ad-IE-exon-4 recombinant virus and target cells were infected with Vac(WR strain)-IE recombinant virus or parental vaccinia virus. Briefly, mice were immunized i.p. with Ad-IE-exon-4 at 1-2 x 10 8 plaque forming units (p.f.u).
  • MHC class-I matched and mismatched target cells were infected with Ad-IE-exon-4, or parental adenovirus, or with Vac-IE- exon-4 or parental vaccinia virus. Percentage specific 31 chromium release was calculated as : [ (cpm experimental release-cpm spontaneous release)/(cpm maximal release-cpm spontaneous release)] x 100.
  • the CBA mice immunized with the Ad-IE-exon-4 recombinant developed a HCMV-IE-exon-4 specific cytotoxic T cell response.
  • Example 3 Protection Study using Ad-IE exon-4 HCMV-protein-specific protection was demonstrated in Ad-HCMV immunized mice from a vaccinia- HCMV recombinant-induced encephalitis/meningitis and death, as follows. The model is described above.
  • CBA mice were immunized i.p. with 2 x 10 8 p.f.u. of the Ad-HCMV subunit protein recombinant virus, e.g. Ad-IE-exon 4 of Example 1, and 5- 18 days later were challenged intracerebrally (i.e.) with a lethal dose of a vaccinia(WR strain)-HCMV recombinant virus (e.g. Vac(WR)-gB).
  • Vaccinia(WR strain)-IE or vaccinia(WR strain)-gB recombinant viruses were obtained from Or. Paoletti, Virogenetics Corporation, Troy, NY.
  • the WR-strain of vaccinia is neurovirulent for mice.
  • CBA mice immunized with the Ad-IE-exon-4 recombinant were protected against a lethal dose of vaccinia WR-IE recombinant virus.
  • the protection was HCMV-IE protein specific.
  • Ninety percent of CBA mice, immunized i.p. with Ad-IE-exon-4 recombinant virus were protected against a lethal dose of Vac(WR-strain)-IE recombinant virus, inoculated intracerebrally.
  • MOLECULE TYPE protein
  • Glu Met Lys Cys lie Gly Leu Thr Met Gin Ser Met Tyr Glu 60 65 70
  • Gin Cys Ser Pro Asp Glu lie Met Ala Tyr Ala Gin Lys lie 155 160 165
  • Phe Lys lie Leu Asp Glu Glu Arg Asp Lys Val Leu Thr His 170 175 180 lie Asp His lie Phe Met Asp lie Leu Thr Thr Cys Val Glu 185 190 195
  • MOLECULE TYPE CPNA
  • SEQUENCE DESCRIPTION SEQ ID NO:3: TTATCCTCCT CTAGAATGAA ACAGATTAAG 30

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Abstract

L'invention concerne un système d'expression recombiné de l'adénovirus non défectif pour l'expression de protéines exon 4 immédiates-précoces, ledit adénovirus recombiné exprimant le HCMV étant utilisé en tant que compositon immunogène et vaccin.
PCT/US1994/002107 1993-02-12 1994-02-10 Vaccin recombine contre le cytomegalovirus Ceased WO1994017810A1 (fr)

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US5591439A (en) * 1989-03-24 1997-01-07 The Wistar Institute Of Anatomy And Biology Recombinant cytomegalovirus vaccine
US6086876A (en) * 1997-02-07 2000-07-11 The Wistar Insitute Methods and compositions for the inhibition of interleukin-12 production
US6448389B1 (en) 1996-04-23 2002-09-10 The Wistar Institute Of Anatomy And Biology Human cytomegalovirus DNA constructs and uses therefor
US6586192B1 (en) 1998-05-29 2003-07-01 Thomas Jefferson University Compositions and methods for use in affecting hematopoietic stem cell populations in mammals
WO2004020405A2 (fr) 2002-08-30 2004-03-11 Biorexis Pharmaceutical Corporation Proteines de fusion de transferrine modifiees
US6846486B1 (en) 2000-02-24 2005-01-25 Advanced Biotherapy Concepts, Inc. Method of treating allergy by administering an anti-histamine antibody
US7094569B2 (en) 2001-05-24 2006-08-22 Soogyun Kim Hair follicle growth factor proteins
WO2007059513A2 (fr) 2005-11-15 2007-05-24 The Children's Hospital Of Philadelphia Compositions et procedes de modulation d’hemostase
WO2007068803A1 (fr) 2005-12-14 2007-06-21 Licentia Ltd Nouveau facteur neurotrophique et utilisations de celui-ci
US7314618B2 (en) 2000-07-19 2008-01-01 Advanced Research And Technology Institute Antibodies to fibroblast growth factor (FGF23)
US7371846B2 (en) 1999-05-24 2008-05-13 The Trustees Of The University Of Pennsylvania CD4-independent HIV envelope proteins as vaccines and therapeutics
US7608433B2 (en) 2005-02-09 2009-10-27 Idexx Laboratories Method of detection of classical swine fever
WO2009133247A1 (fr) 2008-04-30 2009-11-05 Licentia Oy Facteur neurotrophique manf et ses utilisations
WO2010060035A1 (fr) 2008-11-21 2010-05-27 The Children's Hospital Of Philadelphia Facteur v de serpent et procédés d'utilisation en tant que procoagulant
US7754482B2 (en) 2004-05-27 2010-07-13 The Trustees Of The University Of Pennsylvania Artificial antigen presenting cells and uses therefor
EP2384764A2 (fr) 2006-02-23 2011-11-09 The Children's Hospital of Philadelphia Compositions et méthodes pour moduler une hémostase au moyen de différentes formes de facteur V activé
WO2012074868A2 (fr) 2010-12-03 2012-06-07 Ms Technologies, Llc Expression optimisée de molécules d'acide nucléique codant pour la résistance au glyphosate dans cellules végétales
EP2479189A1 (fr) 2006-12-12 2012-07-25 Biorexis Pharmaceutical Corporation Bibliothèques de protéines hybrides de transferrine
EP2551281A1 (fr) 2007-10-25 2013-01-30 Nevalaita, Lina Variants d'épissage de GDNF et leurs utilisations
WO2013049804A1 (fr) 2011-09-30 2013-04-04 The Children's Hospital Of Philadelphia Compositions et procédés pour moduler l'homéostasie
US8617560B2 (en) * 1999-06-04 2013-12-31 Florian Kern Peptides for vaccination against human CMV
WO2014191630A2 (fr) 2013-05-28 2014-12-04 Helsingin Yliopisto Modèle animal non humain codant pour un gène manf non fonctionnel
US9345677B2 (en) 2003-04-04 2016-05-24 Incyte Corporation Compositions, methods and kits relating to HER-2 cleavage
WO2017075619A1 (fr) 2015-10-30 2017-05-04 Spark Therapeutics, Inc. Variants du facteur viii à cpg réduit, compositions, procédés et utilisations pour le traitement d'affections hémostatiques
US9783817B2 (en) 2013-03-04 2017-10-10 Arkansas State University Methods of expressing and detecting activity of expansin in plant cells
WO2018226887A1 (fr) 2017-06-07 2018-12-13 Spark Therapeutics, Inc. Agents d'activation destinés à la transfection de cellules et/ou la production de vecteur raav améliorées
WO2019152957A1 (fr) 2018-02-02 2019-08-08 Arizona Board Of Regents On Behalf Of Arizona State University Lymphocytes t de type adn-récepteur antigénique chimérique pour immunothérapie
US10611800B2 (en) 2016-03-11 2020-04-07 Pfizer Inc. Human cytomegalovirus gB polypeptide
US10842885B2 (en) 2018-08-20 2020-11-24 Ucl Business Ltd Factor IX encoding nucleotides
WO2022079083A1 (fr) 2020-10-15 2022-04-21 F. Hoffmann-La Roche Ag Constructions d'acide nucléique pour transcription de va-arn
WO2022079082A1 (fr) 2020-10-15 2022-04-21 F. Hoffmann-La Roche Ag Constructions d'acides nucléiques améliorées pour activation de gènes simultanée
US11629172B2 (en) 2018-12-21 2023-04-18 Pfizer Inc. Human cytomegalovirus gB polypeptide
WO2023198685A1 (fr) 2022-04-13 2023-10-19 F. Hoffmann-La Roche Ag Procédé de détermination de génomes d'aav
WO2023227438A1 (fr) 2022-05-23 2023-11-30 F. Hoffmann-La Roche Ag Procédé raman de différenciation d'un sérotype de particules aav et d'un état de chargement de particules aav
WO2023232922A1 (fr) 2022-06-03 2023-12-07 F. Hoffmann-La Roche Ag Procédé de production de particules d'aav recombinées
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WO2024013239A1 (fr) 2022-07-14 2024-01-18 F. Hoffmann-La Roche Ag Procédé de production de particules de virus adéno-associé recombinant
WO2024056561A1 (fr) 2022-09-12 2024-03-21 F. Hoffmann-La Roche Ag Procédé de séparation de particules de vaa pleines et vides
WO2024165456A1 (fr) 2023-02-07 2024-08-15 F. Hoffmann-La Roche Ag Procédé de détection d'anticorps anti-particules d'aav
WO2024194280A1 (fr) 2023-03-21 2024-09-26 F. Hoffmann-La Roche Ag Méthode destinée à produire des préparations de particules aav recombinantes
US12209262B2 (en) 2018-08-20 2025-01-28 Ucl Business Ltd Factor IX encoding nucleotides
WO2025168663A1 (fr) 2024-02-09 2025-08-14 F. Hoffmann-La Roche Ag Procédé de production de particules virales adéno-associées recombinées
WO2025252480A1 (fr) 2024-06-07 2025-12-11 F. Hoffmann-La Roche Ag Procédé de purification d'adn plasmidique
WO2026022064A1 (fr) 2024-07-22 2026-01-29 F. Hoffmann-La Roche Ag Nouveaux polypeptides rep et rep orf de vaa
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US6448389B1 (en) 1996-04-23 2002-09-10 The Wistar Institute Of Anatomy And Biology Human cytomegalovirus DNA constructs and uses therefor
US6086876A (en) * 1997-02-07 2000-07-11 The Wistar Insitute Methods and compositions for the inhibition of interleukin-12 production
US6586192B1 (en) 1998-05-29 2003-07-01 Thomas Jefferson University Compositions and methods for use in affecting hematopoietic stem cell populations in mammals
US7371846B2 (en) 1999-05-24 2008-05-13 The Trustees Of The University Of Pennsylvania CD4-independent HIV envelope proteins as vaccines and therapeutics
US8617560B2 (en) * 1999-06-04 2013-12-31 Florian Kern Peptides for vaccination against human CMV
US6846486B1 (en) 2000-02-24 2005-01-25 Advanced Biotherapy Concepts, Inc. Method of treating allergy by administering an anti-histamine antibody
US8586317B2 (en) 2000-07-19 2013-11-19 Advanced Research & Technology Institute Methods of diagnosing hypophosphatemic disorders
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US7314618B2 (en) 2000-07-19 2008-01-01 Advanced Research And Technology Institute Antibodies to fibroblast growth factor (FGF23)
US7335641B2 (en) 2001-05-24 2008-02-26 Soogyun Kim Method for stimulating hair follicle cell proliferation
US7094569B2 (en) 2001-05-24 2006-08-22 Soogyun Kim Hair follicle growth factor proteins
WO2004020405A2 (fr) 2002-08-30 2004-03-11 Biorexis Pharmaceutical Corporation Proteines de fusion de transferrine modifiees
US9345677B2 (en) 2003-04-04 2016-05-24 Incyte Corporation Compositions, methods and kits relating to HER-2 cleavage
EP3363907A1 (fr) 2004-05-27 2018-08-22 The Trustees of the University of Pennsylvania Nouvel antigène artificiel présentant des cellules et utilisations associées
US8722400B2 (en) 2004-05-27 2014-05-13 The Trustees Of The University Of Pennsylvania Artificial antigen presenting cells and uses therefor
US10286066B2 (en) 2004-05-27 2019-05-14 The Trustees Of The University Of Pennsylvania Artificial antigen presenting cells and uses thereof
US7754482B2 (en) 2004-05-27 2010-07-13 The Trustees Of The University Of Pennsylvania Artificial antigen presenting cells and uses therefor
EP2295588A1 (fr) 2004-05-27 2011-03-16 The Trustees Of The University Of Pennsylvania Nouvel antigène artificiel présentant des cellules et utilisations associées
US9555105B2 (en) 2004-05-27 2017-01-31 The Trustees Of The University Of Pennsylvania Artificial antigen presenting cells and uses thereof
US7608433B2 (en) 2005-02-09 2009-10-27 Idexx Laboratories Method of detection of classical swine fever
WO2007059513A2 (fr) 2005-11-15 2007-05-24 The Children's Hospital Of Philadelphia Compositions et procedes de modulation d’hemostase
EP2431390A1 (fr) 2005-11-15 2012-03-21 The Children's Hospital of Philadelphia Compositions et procédés de modulation de l'hémostase
EP2431389A1 (fr) 2005-11-15 2012-03-21 The Children's Hospital of Philadelphia Compositions et procédés de modulation de l'hémostase
EP2423224A1 (fr) 2005-11-15 2012-02-29 The Children's Hospital of Philadelphia Compositions et procédés de modulation de l'hémostase
WO2007068803A1 (fr) 2005-12-14 2007-06-21 Licentia Ltd Nouveau facteur neurotrophique et utilisations de celui-ci
EP2384764A2 (fr) 2006-02-23 2011-11-09 The Children's Hospital of Philadelphia Compositions et méthodes pour moduler une hémostase au moyen de différentes formes de facteur V activé
EP2479189A1 (fr) 2006-12-12 2012-07-25 Biorexis Pharmaceutical Corporation Bibliothèques de protéines hybrides de transferrine
EP2551281A1 (fr) 2007-10-25 2013-01-30 Nevalaita, Lina Variants d'épissage de GDNF et leurs utilisations
WO2009133247A1 (fr) 2008-04-30 2009-11-05 Licentia Oy Facteur neurotrophique manf et ses utilisations
WO2010060035A1 (fr) 2008-11-21 2010-05-27 The Children's Hospital Of Philadelphia Facteur v de serpent et procédés d'utilisation en tant que procoagulant
WO2012074868A2 (fr) 2010-12-03 2012-06-07 Ms Technologies, Llc Expression optimisée de molécules d'acide nucléique codant pour la résistance au glyphosate dans cellules végétales
WO2013049804A1 (fr) 2011-09-30 2013-04-04 The Children's Hospital Of Philadelphia Compositions et procédés pour moduler l'homéostasie
US9783817B2 (en) 2013-03-04 2017-10-10 Arkansas State University Methods of expressing and detecting activity of expansin in plant cells
WO2014191630A2 (fr) 2013-05-28 2014-12-04 Helsingin Yliopisto Modèle animal non humain codant pour un gène manf non fonctionnel
WO2017075619A1 (fr) 2015-10-30 2017-05-04 Spark Therapeutics, Inc. Variants du facteur viii à cpg réduit, compositions, procédés et utilisations pour le traitement d'affections hémostatiques
US10611800B2 (en) 2016-03-11 2020-04-07 Pfizer Inc. Human cytomegalovirus gB polypeptide
WO2018226887A1 (fr) 2017-06-07 2018-12-13 Spark Therapeutics, Inc. Agents d'activation destinés à la transfection de cellules et/ou la production de vecteur raav améliorées
WO2019152957A1 (fr) 2018-02-02 2019-08-08 Arizona Board Of Regents On Behalf Of Arizona State University Lymphocytes t de type adn-récepteur antigénique chimérique pour immunothérapie
US12338459B2 (en) 2018-02-02 2025-06-24 Arizona Board Of Regents On Behalf Of Arizona State University DNA-chimeric antigen receptor T cells for immunotherapy
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US11629172B2 (en) 2018-12-21 2023-04-18 Pfizer Inc. Human cytomegalovirus gB polypeptide
US11857622B2 (en) 2020-06-21 2024-01-02 Pfizer Inc. Human cytomegalovirus GB polypeptide
WO2022079083A1 (fr) 2020-10-15 2022-04-21 F. Hoffmann-La Roche Ag Constructions d'acide nucléique pour transcription de va-arn
WO2022079082A1 (fr) 2020-10-15 2022-04-21 F. Hoffmann-La Roche Ag Constructions d'acides nucléiques améliorées pour activation de gènes simultanée
US12312606B2 (en) 2020-10-15 2025-05-27 Hoffmann-La Roche Inc. Nucleic acid constructs for VA RNA transcription
WO2023198685A1 (fr) 2022-04-13 2023-10-19 F. Hoffmann-La Roche Ag Procédé de détermination de génomes d'aav
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