WO1994023744A1 - Recombinant cytomegalovirus vaccine - Google Patents

Recombinant cytomegalovirus vaccine Download PDF

Info

Publication number
WO1994023744A1
WO1994023744A1 PCT/US1994/004180 US9404180W WO9423744A1 WO 1994023744 A1 WO1994023744 A1 WO 1994023744A1 US 9404180 W US9404180 W US 9404180W WO 9423744 A1 WO9423744 A1 WO 9423744A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
fragment
ser
val
leu
Prior art date
Application number
PCT/US1994/004180
Other languages
French (fr)
Inventor
Stanley A. Plotkin
Robert P. Ricciardi
Eva Gonczol
Klara Berencsi
Robert F. Rando
Original Assignee
The Wistar Institute Of Anatomy And Biology
THE GOVERNMENT OF THE UNITED STATES OF AMERICA on behalf of THE DEPARTMENT OF HEALTH AND HUMAN SERVICES
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 The Wistar Institute Of Anatomy And Biology, THE GOVERNMENT OF THE UNITED STATES OF AMERICA on behalf of THE DEPARTMENT OF HEALTH AND HUMAN SERVICES filed Critical The Wistar Institute Of Anatomy And Biology
Priority to EP94915386A priority Critical patent/EP0708658A4/en
Publication of WO1994023744A1 publication Critical patent/WO1994023744A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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
    • 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
    • C12N2710/10011—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 containing fragments of a HCMV major glycoprotein complex subunit gB gene.
  • 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 cytomegalic 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.
  • Preliminary estimates based on surveys of approximately 4,000 newborns from several geographical areas indicate that the virus causes significant damage of the central nervous system leading to mental deficiency in at least 10%, and perhaps as high as 25%, of infected infants.
  • HCMV in humans has also been observed to cause serious complications and infections in the course of organ transplantations, especially with kidney and liver transplants.
  • 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, 20(1) :271-287 (1984); J. P. Glazer et al, Ann. Intern. Med..
  • a proposed HCMV vaccine using a recombinant vaccinia virus expressing HCMV glycoprotein B has also been described. [See, e.g., Cranage, M. P. et al, EMBO J.. 5:3057-3063 (1986).] However, vaccinia models for vaccine delivery are believed to cause local reactions. Additionally, vaccinia vaccines are considered possible causes of encephalitis.
  • Adenoviruses have been developed previously as efficient heterologous gene expression vectors. For example, an adenovirus vector has been employed to express herpes simplex virus glycoprotein gB [P. C. Johnson et al, Virol..
  • Adenoviruses have also been found to be non-toxic as vaccine components in humans [See, e.g., E. T. Takajuji et al, J.
  • the present invention provides a non-defective recombinant adenovirus containing a fragment of a gB subunit of the HCMV free from association with any additional human proteinaceous material.
  • the HCMV subunit is under the control of regulatory sequences capable of expressing the HCMV gB subunit fragment 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 vaccinating a human against HCMV comprising administering to the patient the recombinant adenovirus containing the subunit gene encoding a gB protein fragment in a vaccine composition.
  • This method of presenting these HCMV gene fragments to a vaccinate is particularly capable of eliciting an immune response.
  • the invention provides an adenovirus-produced gB subunit fragment, which fragment may also form vaccine compositions to protect humans against HCMV.
  • the preferred fragment comprises about amino acids 1 to about 303 of the gB protein SEQ IP NO:2, gB-.. 3(a .
  • Fig. IA illustrates diagrammatically the cloning of the gB gene into the early region 3 (E3) transcription unit of Ad5. Represented are the 3.1kb fragment containing the gB gene by the open box; the adenovirus sequences extending from 59.5 to 100 mu (except for the deletion of the 78.5 to 84.7 mu length) by the filled portion of the circle: the large BamHI fragment of the pBR322 by the thin line of the circle.
  • restriction enzymes are identified as follows: X is Xbal, B is BamHI.
  • Fig. IB illustrates diagrammatically the construction of the recombinant adenovirus virus Ad5/gB, containing the gB gene of the Towne strain of HCMV described in Example 1.
  • This figure shows the 59.5 mu to 76 mu region where homologous recombination occurs (as indicated by the crossed lines) between wild type Ad5 viral sequence and the adenovirus sequences present on the pAd5 plasmid containing the gB gene.
  • the plaque purified recombinant virus retains the cloning Xbal sites and'the direction of transcription of the gB gene from the E3 promoter is indicated by the bent arrow. Restriction enzymes are as identified above.
  • the present invention provides novel immunogenic components for HCMV which comprise an adenovirus expression system capable of expressing a selected HCMV subunit gene fragment in vivo.
  • the selected subunit fragment for use in an immunogenic composition may be expressed in, and isolated from, the recombinant adenovirus expression system.
  • any adenovirus strain capable of replicating in mammalian cells 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.
  • 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.
  • a number of strains of isolated human CMV may be employed from which a desired gB subunit is derived.
  • 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. and is described in J. Virol.. 11 (6) : 991 (1973) .
  • This strain is freely available from The istar Institute or from the ATCC under accession number VR-977.
  • 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 HCMV subunit may be produced in vitro by recombinant techniques in large quantities sufficient for use in an immunogenic composition or subunit vaccine.
  • the recombinant adenovirus containing the subunit may itself be employed as an immunogenic or vaccine component, capable of expressing the subunit in vivo .
  • the presently preferred subunit proteins for use in the present invention are the HCMV gB subunit fragments.
  • One embodiment of the present invention provides a replication competent (non-defective) adenovirus vector carrying a fragment of the HCMV gB gene which contains a CTL epitope and/or B cell epitope.
  • a preferred gene fragment encodes about amino acid 1 to about amino acid 303 of the gB subunit protein SEQ IP NO:2.
  • Another suitable fragment of gB SEQ IP NO:2 is the fragment spanning about amino acid 1 to about amino acid 700 of SEQ IP NO:2.
  • Still another suitable gB fragment spans about amino acid 1 to about amino acid 465 of SEQ IP NO:2.
  • HCMV subunit may be employed in a vaccine according to the teachings of the present invention.
  • sequences of the subunits of two HCMV strains have been published [See, e.g., Mach et al, J. Gen. Virol.. 67_ ⁇ 1461-1467 (1986); Cranage et al, (1986) cited above; and Spaete et al, Virol.. 167:207-225 (1987) .
  • These subunit sequences can therefore be chemically synthesized by conventional methods known to one of skill in the art, or the sequences 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, subunit fragments, or immediate early antigens and subunits together.
  • 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 subunit protein is within the skill of the art.
  • Example 3 below provides construction details for the non-defective adenovirus containing these gB fragments.
  • 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 gB subunit fragment, 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 an immune response.
  • Examples 5 and 6 demonstrate the ability of a recombinant adenovirus containing the gB fragment, amino acid 1-303 of SEQ IP NO:2, to induce a gB-specific, protective CTL response in mice.
  • adenovirus recombinants as immunogens capable of inducing a CTL response is surprising in view of the results obtained in the same assays of the examples with other known virus types, which have been used in vaccines previously.
  • the recombinant viral vector containing the CMV subunit protein e.g., the gB ⁇ r ⁇ subunit fragment
  • 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, without limitation, mammalian cells and 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., gB W( ⁇ subunit fragment, 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., gB ⁇ , may then be formulated into an appropriate vaccine composition.
  • Such compositions may generally contain one or more of the recombinant CMV subunits.
  • 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 a fragment of a gB 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.
  • Such an immune response is capable of providing protection against exposure to the whole human CMV microorganism.
  • 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.
  • mice are immunized with one recombinant of the invention, and CTL activity is tested in target cells infected with the other recombinant.
  • Examples 4-6 below provide a murine model of the cytotoxic T lymphocyte (CTL) response to the amino acid 1-303 fragment of the glycoprotein B (gB) gene [SEQ IP NO:2] 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
  • Ad-gB gB-expressing adenovirus
  • MHC major histocompatibility complex
  • the gB gene was cloned from the Towne strain of HCMV [Wistar Institute] as follows. The gB gene was first mapped to the 20.5 kb Hind III P fragment of HCMV using oligonucleotides that corresponded to the 5' and 3' termini of the published AP-169 gB sequence [See, Cranage et al (1986), cited above]. The Hind III fragment was cut with Xbal to generate a 9.8 kb fragment. This fragment was then cut with X alll to generate a 3.1 kb fragment. The 3.1 kb Xmalll fragment which contained the gB gene, had Xbal linkers attached to its 5' and 3' termini.
  • pAd5 Bam-B An adenovirus type 5 plasmid, pAd5 Bam-B, which contains the 59.5 - 100 mu region of the Ad5 adenovirus genome cloned into the BamHI site of pBR322 [See, R. L. Berkner et al, Nucl. Acids Res.. 1 ⁇ :6003-6020 (1983) and M. E. Morin et al, cited above] was digested with Xbal to remove the 78.5 mu - 84.7 mu sequences of the Ad5 genome. The 78.5 to 84.7 mu deletion removes most of the coding region of the E3 transcription unit of Ad5 but leaves the E3 promoter intact.
  • Fig. IA provides a diagrammatic illustration of the above description.
  • the 0-76 mu fragment of wild type Ad5 virus was isolated by digesting the viral PNA with EcoRI [See, U. Petterson et al, J. Mol. Biol. , 72:125-130 (1973)]. This fragment was co- transfected with the 59.5 to 100 mu BamHI fragment of pAd5 Bam-B containing the gB gene as described above into human embryonic kidney 293 cells, available from the American Type Culture Collection.
  • the Ad-gB recombinant was generated by overlap recombination between the viral sequences as illustrated in Fig. IB.
  • the gB recombinant virus was plaque purified on human lung carcinoma A549 cells [ATCC CCL185] using standard procedures. Viruses containing both orientations of the gB gene, as determined by Southern blotting, were isolated.
  • the recombinant containing the gB gene in the same 5• to 3' direction as the adenovirus E3 promoter of the adenovirus type 5 strain is under the transcriptional control of the E3 promoter.
  • the plaque purified recombinant virus retains the cloning Xbal sites.
  • the above-described cloned gB gene is devoid of its natural promoter according to the PNA sequence of gB identified in Spaete et al, (1987) , cited above.
  • Example 2 Production of the Full-Length gB Subunit
  • the adenovirus gB plasmid construct and the Ad5 mu 0-76 PNA of Example 1 were cotransfected into 293 cells, human cells transformed by adenovirus 5 early genes [See, Graham et al, J. Gen. Virol. ,36:59-72 (1977); and ATCC CRL1573] employing conventional procedures.
  • This transfection generated a functional recombinant virus by homologous overlap recombination as shown in
  • Southern blot analysis confirmed the presence of an adenovirus, type 5, containing the HCMV gB subunit (referred to as either Ad-5/gB or Ad-gB) recombinant virus which was subsequently purified by plaque purification using standard procedures.
  • the recombinant virus AP-5/gB expresses gB subunit protein as determined by conventional assays, i.e., immunofluorescence on fixed cells and by Western blot using monospecific guinea pig antiserum and monoclonal antibodies to gB protein [See, e.g., T. Maniatis et al, cited above].
  • the Ad-5/gB recombinant also referred to as Ad-gB, is also described in applicant's publication [Marshall et al., J. Infect. Pis.. 162:1177-1181 (1990)] published after the filing date of the original parent application from which this application claims priority.
  • Ad-gBi. 303 and Ad-gB M55 recombinant viruses were constructed by overlap recombination as described for Ad- gB in Example 2 above. Briefly, in order to clone the subfragments of the gB gene, five oligonucleotide primers for polymerase chain reactions (PCR) were synthesized.
  • PCR polymerase chain reactions
  • the primers were designed to anneal with various portions of the gB PNA sequence and promote amplification of the gene.
  • all of the oligonucleotide primers were engineered to contain an Xba I site so that the PCR product could be digested with this enzyme in order to facilitate cloning into the pAd-5 vector.
  • 5' gB primer SEQ IP NO:3:
  • oligonucleotides correspond to the following nucleotide positions of the HCMV gB gene (Towne strain) as reported by Spaete et al, Virology. 167:207-225 (1988).
  • SEQ IP N0:3 corresponds to nucleotide positions 895 to 922 in the sense orientation; SEQ IP NO:4 to nucleotide positions 3090 to 3067 anti-sense; SEQ IP NO:5 to nucleotide positions 2375 to 2350 anti-sense; SEQ IP NO:6 to nucleotide positions 1877 to 1847 anti-sense; and SEQ IP NO:7 to nucleotide positions 1432 to 1400 anti- sense.
  • the specific segments or fragments of the gB gene were amplified using the Perkin-Elmer AmplitaqTM kit by mixing 400 ng of the 5' gB primer with each of the 3' primers separately (400 ng of each) and 0.1 ⁇ g of purified HCMV genomic PNA or 0.1 ⁇ g of previously cloned intact gB gene (see Example 2) .
  • the final reaction mixture was 100 / xL and the thermocycling conditions were 94°C, 1 minute; 52°C, 1 minute; 72°C, 1 minute, repeated for a total of 35 cycles.
  • Amplified PNA was purified by cutting the proper PNA fragment out of a 1.2% agarose gel, digested with Xbal.
  • Example 4 CTL Assays A. Recombinant Viruses Used
  • Wild-type human adenovirus type 5 WT-Ad
  • Ad-gB Ad-gB
  • 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 Example 1, and pAd-5 mu 0-75.9 [G. S. Marshall et al, J. Infect. Pis.. 162:1177-1181 (1990), hereby incorporated by reference].
  • 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. 9.:190-193 (1991) [also obtained from Pr. 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
  • Cp-gB recombinant and parental canarypox virus were propagated on primary chick embryo fibroblasts.
  • B. Expression of the gB protein in Cp-gB recombinant virus Chicken embryo fibroblast (CEF) cells [ATCC CRL 1590] infected with either Cp-gB or with the parental wild-type canarypox (WT-Cp) virus preparations were analyzed by Western blot assay using the 4A guinea-pig serum directed against the gB protein.
  • a diffuse band at the 140 kPa position and a double band of 55 and 58 kPa were detected in both Cp-gB-infected CEF cells and in HCMV-infected MRC-5 cells.
  • the presence of these gB-specific proteins presumably representing the glycosylated 140 kPa precursor and the differentially glycosylated cleavage products (55 and 58 kPa) indicates that the Cp-gB recombinant expresses the inserted gB gene.
  • the slight difference between the mobility of 55 and 58 kPa cleavage products of control and recombinant gB may reflect different glycosylation patterns.
  • 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.
  • mice Six- to 8-week-old female BALB/c and CBA mice (from Harlan Sprague-Pawley and Jackson) and 12-week-old male BALB/k mice (from The Wistar Institute Animal Facility) were immunized intraperitoneally (i.p.) with the recombinant viruses described above at 1-5 x IO 8 pfu unless otherwise stated.
  • spleens were aseptically removed and cell suspensions were prepared by gently pressing the spleens through a stainless steel mesh.
  • 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
  • mice MC57 (H- 2 b ) cells [also termed MC-57G, P.P. Aden et al, Im unogenetics, 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).
  • 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%.
  • This CTL assay is a system in which two types of viral expression vectors, poxvirus and adenovirus, carrying the same fragment of the HCMV-gB gene, are alternately used for immunization of animal or for infection of target cells to show that HCMV-gB fragment is an inducer of CTL in mice.
  • poxvirus and adenovirus carrying the same fragment of the HCMV-gB gene
  • Ad-gB j . 303 and Ad-gB WJ5 recombinant viruses were constructed as described in Example 3 above.
  • CBA mice were immunized i.p. with IO 8 pfu of the Ad-gB, Two weeks later spleen cells were restimulated in vitro with Ad-gB infected autologous spleen cells and tested for ability to lyse Wt-Ad, Vac-gB or Wt-Vac infected L929 (MHC-class I matched) cells.
  • CBA mice were immunized with 1 x 10 8 pfu of Wt-Ad, Ad5 ⁇ 3 (an E3 deleted mutant virus, the parental strain of the recombinant viruses) , Ad-gB, Ad-gB ⁇ or Ad-gB ⁇ _ 155 .
  • Ad-gB an E3 deleted mutant virus, the parental strain of the recombinant viruses
  • Ad-gB ⁇ an E3 deleted mutant virus, the parental strain of the recombinant viruses
  • Ad-gB Ad-gB ⁇
  • Ad-gB ⁇ _ 155 Ad-gB
  • VacWR-gB a neurovirulent vaccinia strain expressing the HCMV-gB protein
  • Ad-gB and Ad-gB ⁇ -immunized mice survived (92% and 95% survival, respectively) , while all of the Ad-gB j .i jj -i munized mice died, indicating a protection epitope on the N-terminal part of the gB protein between amino acid 155 and 303.
  • HCMV major glycoprotein complexes e.g., gcll or gcIII, or immediate-early antigens
  • gcll or gcIII e.g., immediate-early antigens
  • immediate-early antigens may be expressed in a non- defective adenovirus recombinant in the same manner as described above for subunit gB fragment.
  • GGT ACG GAT CTT ATT CGC TTT GAA CGT AAT ATC GTC TGC ACC 336 Gly Thr Asp Leu lie Arg Phe Glu Arg Asn lie Val Cys Thr 100 105 110
  • TAC ATC CAC ACC ACT TAT CTG CTG GGC AGC AAC ACG GAA TAC 504 Tyr lie His Thr Thr Tyr Leu Leu Gly Ser Asn Thr Glu Tyr 155 160 165
  • AAG ATC TTC ATC GCC GGC AAC TCG GCC TAC GAG TAC GTG GAC 1890 Lys lie Phe lie Ala Gly Asn Ser Ala Tyr Glu Tyr Val Asp 620 625 630
  • GCC ATT GGG GCC GTG GGT GGC GCG GTG GCC TCC GTG GTC GAA 2226 Ala lie Gly Ala Val Gly Gly Ala Val Ala Ser Val Val Glu 730 735 740
  • ATC ATC CTC GTG GCC ATA GCC GTC GTC ATT ATC ATT TAT TTG 2310 lie lie Leu Val Ala lie Ala Val Val lie lie lie Tyr Leu 760 765 770
  • Val Val Asp lie Ser Pro Phe Tyr Asn Gly Thr Asn Arg Asn Ala Ser 275 280 285
  • Ser lie Ser Thr Val Asp Ser Met lie Ala Leu Asp lie Asp Pro Leu

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention provides a non-defective adenovirus recombinant expression system for the expression of an immunogenic fragment of the HCMV gB subunit, said recombinant HCMV-expressing adenovirus being useful as a vaccine.

Description

RECOMBINANT CYTOMEGALOVIRUS VACCINE
This work was performed with government support under National Institutes of Health grants AI-07278 and HD-18957. The U.S. government has certain rights in this invention.
Field of the Invention The present invention refers generally to a recombinant human cytomegalovirus vaccine, and more specifically to a subunit vaccine containing fragments of a HCMV major glycoprotein complex subunit gB gene.
Background of the Invention
Cytomegalovirus (CMV) 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 cytomegalic 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. Preliminary estimates based on surveys of approximately 4,000 newborns from several geographical areas indicate that the virus causes significant damage of the central nervous system leading to mental deficiency in at least 10%, and perhaps as high as 25%, of infected infants. Assuming that about 1% of newborn infants per year excrete CMV and that about one fourth of those develop mental deficiency, in the United States this means approximately 10,000 brain-damaged children born per year. This is a formidable number, particularly in view of the ability of these children to survive [J. Infect. Pis.. 123 (5) :555 (1971)].
HCMV in humans has also been observed to cause serious complications and infections in the course of organ transplantations, especially with kidney and liver transplants.
Several 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, 20(1) :271-287 (1984); J. P. Glazer et al, Ann. Intern. Med.. 91:676-83 (1979); and U. S. Patent 3,959,466.] A proposed HCMV vaccine using a recombinant vaccinia virus expressing HCMV glycoprotein B has also been described. [See, e.g., Cranage, M. P. et al, EMBO J.. 5:3057-3063 (1986).] However, vaccinia models for vaccine delivery are believed to cause local reactions. Additionally, vaccinia vaccines are considered possible causes of encephalitis. Adenoviruses have been developed previously as efficient heterologous gene expression vectors. For example, 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.. 63.:129-136 (1988)]; and hepatitis B surface antigen [A. R. Pavis et al, Proc. Nat1. Acad. Sci.. U.S.A.. 82.:7560-7564 (1985); J. E. Morin et al, Proc. Natl. Acad. Sci.. U.S.A.. 84:4626-4630 (1987)]. 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.. J38_:394-403 (1963)].
There remains a need in the art for additional vaccines capable of preventing CMV infection by generating neutralizing antibody and cellular responses to CMV in the human immune system.
Summary of the Invention
In one aspect, the present invention provides a non-defective recombinant adenovirus containing a fragment of a gB subunit of the HCMV free from association with any additional human proteinaceous material. In this recombinant adenovirus, the HCMV subunit is under the control of regulatory sequences capable of expressing the HCMV gB subunit fragment in vitro and in vivo. Another aspect of the present invention is a vaccine composition comprising a non-defective recombinant adenovirus, as described above.
In a further aspect, the invention provides a method of vaccinating a human against HCMV comprising administering to the patient the recombinant adenovirus containing the subunit gene encoding a gB protein fragment in a vaccine composition. The inventors have found that this method of presenting these HCMV gene fragments to a vaccinate is particularly capable of eliciting an immune response.
In still a further aspect the invention provides an adenovirus-produced gB subunit fragment, which fragment may also form vaccine compositions to protect humans against HCMV. Currently, the preferred fragment comprises about amino acids 1 to about 303 of the gB protein SEQ IP NO:2, gB-..3(a .
Other aspects and advantages of the present invention are described further in the following detailed description of preferred embodiments of the present invention.
Brief Pescription of the Prawings
Fig. IA illustrates diagrammatically the cloning of the gB gene into the early region 3 (E3) transcription unit of Ad5. Represented are the 3.1kb fragment containing the gB gene by the open box; the adenovirus sequences extending from 59.5 to 100 mu (except for the deletion of the 78.5 to 84.7 mu length) by the filled portion of the circle: the large BamHI fragment of the pBR322 by the thin line of the circle.
In the figure, the restriction enzymes are identified as follows: X is Xbal, B is BamHI.
Fig. IB illustrates diagrammatically the construction of the recombinant adenovirus virus Ad5/gB, containing the gB gene of the Towne strain of HCMV described in Example 1. This figure shows the 59.5 mu to 76 mu region where homologous recombination occurs (as indicated by the crossed lines) between wild type Ad5 viral sequence and the adenovirus sequences present on the pAd5 plasmid containing the gB gene. The plaque purified recombinant virus retains the cloning Xbal sites and'the direction of transcription of the gB gene from the E3 promoter is indicated by the bent arrow. Restriction enzymes are as identified above.
Petailed Pescrintion of the Invention
The present invention provides novel immunogenic components for HCMV which comprise an adenovirus expression system capable of expressing a selected HCMV subunit gene fragment in vivo.
Alternatively the selected subunit fragment for use in an immunogenic composition, such as a vaccine, may be expressed in, and isolated from, the recombinant adenovirus expression system. As provided by the present invention, any adenovirus strain capable of replicating in mammalian cells in vitro may be used to construct an expression vector for the selected HCMV subunit. However, a preferred expression system involves a non-defective adenovirus strain, including, but not limited to, adenovirus type 5. Alternatively, 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.
Similarly, a number of strains of isolated human CMV may be employed from which a desired gB subunit is derived. For example, 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. and is described in J. Virol.. 11 (6) : 991 (1973) . This strain is freely available from The istar Institute or from the ATCC under accession number VR-977. However, 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.
In the practice of one embodiment of this invention the HCMV subunit may be produced in vitro by recombinant techniques in large quantities sufficient for use in an immunogenic composition or subunit vaccine. Alternatively, the recombinant adenovirus containing the subunit may itself be employed as an immunogenic or vaccine component, capable of expressing the subunit in vivo .
The presently preferred subunit proteins for use in the present invention are the HCMV gB subunit fragments. One embodiment of the present invention provides a replication competent (non-defective) adenovirus vector carrying a fragment of the HCMV gB gene which contains a CTL epitope and/or B cell epitope. A preferred gene fragment encodes about amino acid 1 to about amino acid 303 of the gB subunit protein SEQ IP NO:2. Another suitable fragment of gB SEQ IP NO:2 is the fragment spanning about amino acid 1 to about amino acid 700 of SEQ IP NO:2. Still another suitable gB fragment spans about amino acid 1 to about amino acid 465 of SEQ IP NO:2.
More particularly, it is anticipated that smaller fragments containing all or a portion of the gB fragment spanning amino acids about 155 to about 303 will also be desirable for vaccine use. This region is suspected of containing at least a CTL epitope (see Examples 5 and 6 below) . It is anticipated that in the construction of the adenovirus vectors of this invention, any of the subunits of the HCMV envelope protein may be employed. In a manner similar to the use of the gB fragment in this vaccine, other subunits of CMV which may be employed in the production of a vaccine according to the invention may be selected from the gcll, gcIII, or immediate early subunits of the human virus. Alternatively, more than one HCMV subunit may be employed in a vaccine according to the teachings of the present invention. In addition to isolating the desired subunit from an available strain of HCMV for insertion into the selected adenovirus, the sequences of the subunits of two HCMV strains have been published [See, e.g., Mach et al, J. Gen. Virol.. 67_ι1461-1467 (1986); Cranage et al, (1986) cited above; and Spaete et al, Virol.. 167:207-225 (1987) . These subunit sequences can therefore be chemically synthesized by conventional methods known to one of skill in the art, or the sequences purchased from commercial sources. The recombinant adenovirus of the present invention may also contain multiple copies of the HCMV subunit. Alternatively, the recombinant virus may contain more than one HCMV subunit type, so that the virus may express two or more HCMV subunits, subunit fragments, or immediate early antigens and subunits together.
In the construction of the adenovirus vector of the present invention, 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. 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. A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982) . Thus, given the disclosures contained herein the construction of suitable adenovirus expression vectors for expression of an HCMV subunit protein is within the skill of the art. Example 3 below provides construction details for the non-defective adenovirus containing these gB fragments.
The recombinant adenovirus itself, constructed as described above, may be used directly as an immunogen or a vaccine component. According to this embodiment of the invention, the recombinant adenovirus, containing the HCMV subunit, e.g., the gB subunit fragment, 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 an immune response. Examples 5 and 6 demonstrate the ability of a recombinant adenovirus containing the gB fragment, amino acid 1-303 of SEQ IP NO:2, to induce a gB-specific, protective CTL response in mice.
The use of these adenovirus recombinants as immunogens capable of inducing a CTL response is surprising in view of the results obtained in the same assays of the examples with other known virus types, which have been used in vaccines previously. According to another embodiment of this invention, once the recombinant viral vector containing the CMV subunit protein, e.g., the gB^rø subunit fragment, is constructed, 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. [See, Maniatis et al, supra-] Suitable host cells include, without limitation, mammalian cells and 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. The culture conditions are conventional for the host cell and allow the subunit, e.g., gBW(β subunit fragment, 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.
When expressed in vitro and isolated from culture, the subunit, e.g., gB^^, may then be formulated into an appropriate vaccine composition. Such compositions may generally contain one or more of the recombinant CMV subunits.
The preparation of a pharmaceutically acceptable vaccine composition, having appropriate pH, isotonicity, stability and other conventional characteristics is within the skill of the art. Thus such vaccines may optionally contain other components, such as adjuvants and/or carriers, e.g., aqueous suspensions of aluminum and magnesium hydroxides. Thus, 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]. However, the present invention is not limited by the route of administration selected for the vaccine.
When the recombinant adenovirus is administered as the vaccine, a dosage of between 105 and 108 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.
Alternatively, the vaccine composition may comprise one or more recombinantly-produced human CMV subunit proteins, preferably a fragment of a gB 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 presence of the subunit produced either in vivo or as part of an in vitro expressed subunit administered with a carrier, stimulates an immune response in the patient. Such an immune response is capable of providing protection against exposure to the whole human CMV microorganism. 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.
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. For example, 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. According to this model, mice are immunized with one recombinant of the invention, and CTL activity is tested in target cells infected with the other recombinant. Specifically, Examples 4-6 below provide a murine model of the cytotoxic T lymphocyte (CTL) response to the amino acid 1-303 fragment of the glycoprotein B (gB) gene [SEQ IP NO:2] of human cytomegalovirus (HCMV) based on the use of gB-expressing adenovirus (Ad-gB) and several poxvirus recombinants. Using this model, it has been demonstrated that the human CMV subunit gB (HCMV-gB) amino acid 1-303 fragment can elicit a major histocompatibility complex (MHC) class I-restricted HCMV-gB-specific CTL response in mice.
The following examples illustrate the construction of a non-defective adenovirus strain capable of expressing the HCMV major envelope glycoprotein gBj^ fragment and the efficacy of these compositions as an HCMV vaccine. These examples are illustrative only and do not limit the scope of the present invention.
Example 1 - Construction of a Non-defective Adenovirus - αB fAd-αB) Recombinant
The gB gene was cloned from the Towne strain of HCMV [Wistar Institute] as follows. The gB gene was first mapped to the 20.5 kb Hind III P fragment of HCMV using oligonucleotides that corresponded to the 5' and 3' termini of the published AP-169 gB sequence [See, Cranage et al (1986), cited above]. The Hind III fragment was cut with Xbal to generate a 9.8 kb fragment. This fragment was then cut with X alll to generate a 3.1 kb fragment. The 3.1 kb Xmalll fragment which contained the gB gene, had Xbal linkers attached to its 5' and 3' termini.
An adenovirus type 5 plasmid, pAd5 Bam-B, which contains the 59.5 - 100 mu region of the Ad5 adenovirus genome cloned into the BamHI site of pBR322 [See, R. L. Berkner et al, Nucl. Acids Res.. 1^:6003-6020 (1983) and M. E. Morin et al, cited above] was digested with Xbal to remove the 78.5 mu - 84.7 mu sequences of the Ad5 genome. The 78.5 to 84.7 mu deletion removes most of the coding region of the E3 transcription unit of Ad5 but leaves the E3 promoter intact. The Xbal-linked 3.1 kb fragment of CMV containing the gB gene was inserted into this Xbal site of pAd5 Bam-B. Fig. IA provides a diagrammatic illustration of the above description. To generate recombinant virus, the 0-76 mu fragment of wild type Ad5 virus was isolated by digesting the viral PNA with EcoRI [See, U. Petterson et al, J. Mol. Biol. , 72:125-130 (1973)]. This fragment was co- transfected with the 59.5 to 100 mu BamHI fragment of pAd5 Bam-B containing the gB gene as described above into human embryonic kidney 293 cells, available from the American Type Culture Collection. The Ad-gB recombinant was generated by overlap recombination between the viral sequences as illustrated in Fig. IB. The gB recombinant virus was plaque purified on human lung carcinoma A549 cells [ATCC CCL185] using standard procedures. Viruses containing both orientations of the gB gene, as determined by Southern blotting, were isolated. The recombinant containing the gB gene in the same 5• to 3' direction as the adenovirus E3 promoter of the adenovirus type 5 strain is under the transcriptional control of the E3 promoter. The plaque purified recombinant virus retains the cloning Xbal sites. The above-described cloned gB gene is devoid of its natural promoter according to the PNA sequence of gB identified in Spaete et al, (1987) , cited above.
Example 2 - Production of the Full-Length gB Subunit The adenovirus gB plasmid construct and the Ad5 mu 0-76 PNA of Example 1 were cotransfected into 293 cells, human cells transformed by adenovirus 5 early genes [See, Graham et al, J. Gen. Virol. ,36:59-72 (1977); and ATCC CRL1573] employing conventional procedures. This transfection generated a functional recombinant virus by homologous overlap recombination as shown in
Fig. IB.
Southern blot analysis confirmed the presence of an adenovirus, type 5, containing the HCMV gB subunit (referred to as either Ad-5/gB or Ad-gB) recombinant virus which was subsequently purified by plaque purification using standard procedures.
The recombinant virus AP-5/gB, expresses gB subunit protein as determined by conventional assays, i.e., immunofluorescence on fixed cells and by Western blot using monospecific guinea pig antiserum and monoclonal antibodies to gB protein [See, e.g., T. Maniatis et al, cited above]. The Ad-5/gB recombinant, also referred to as Ad-gB, is also described in applicant's publication [Marshall et al., J. Infect. Pis.. 162:1177-1181 (1990)] published after the filing date of the original parent application from which this application claims priority.
Example 3 - Construction of the gB gene fragments
Ad-gBi.303 and Ad-gBM55 recombinant viruses were constructed by overlap recombination as described for Ad- gB in Example 2 above. Briefly, in order to clone the subfragments of the gB gene, five oligonucleotide primers for polymerase chain reactions (PCR) were synthesized.
The primers were designed to anneal with various portions of the gB PNA sequence and promote amplification of the gene. In addition, all of the oligonucleotide primers were engineered to contain an Xba I site so that the PCR product could be digested with this enzyme in order to facilitate cloning into the pAd-5 vector. 5' gB primer : SEQ IP NO:3:
4889: 5'-ACACGCAAGAGA TCTAGA CGCGCCTCAT 3' primer at amino acid 700 of gB protein: SEQ IP N0:4: 5'-TCGTCCAGAC TCTAGA GGTAGGGC
3' primer at aa 465: SEQ IP NO:5:
5'-CGACTCCAT TCTAGA TTAATGAGTTGCATT 3' primer at aa 303: SEQ IP NO:6:
5'-CAAAGTCGGAG TCTAGAG TCTAGTTCGGAAA 3' primer at aa 155: SEQ IP NO:7:
5'-CAGATAAGTGG TCTAGA TCTAAGCGTAGCTACG The above oligonucleotides correspond to the following nucleotide positions of the HCMV gB gene (Towne strain) as reported by Spaete et al, Virology. 167:207-225 (1988). SEQ IP N0:3 corresponds to nucleotide positions 895 to 922 in the sense orientation; SEQ IP NO:4 to nucleotide positions 3090 to 3067 anti-sense; SEQ IP NO:5 to nucleotide positions 2375 to 2350 anti-sense; SEQ IP NO:6 to nucleotide positions 1877 to 1847 anti-sense; and SEQ IP NO:7 to nucleotide positions 1432 to 1400 anti- sense. These immediately preceding nucleotide numbers are not identical to those of SEQ IP NO: 1 because the Spaete et al sequence, to which these numbers correspond, contains additional 5' non-coding sequence while SEQ IP NO: 1 reports only the PNA sequence corresponding to the coding region of the gB protein [SEQ IP NO: 2].
The specific segments or fragments of the gB gene were amplified using the Perkin-Elmer Amplitaq™ kit by mixing 400 ng of the 5' gB primer with each of the 3' primers separately (400 ng of each) and 0.1 μg of purified HCMV genomic PNA or 0.1 μg of previously cloned intact gB gene (see Example 2) . The final reaction mixture was 100 /xL and the thermocycling conditions were 94°C, 1 minute; 52°C, 1 minute; 72°C, 1 minute, repeated for a total of 35 cycles. Amplified PNA was purified by cutting the proper PNA fragment out of a 1.2% agarose gel, digested with Xbal. repurified by cutting the digested fragments out of a 1.2% agarose gel and then ligated into the Xbal site of the cloning vector pAd-5. Positive recombinants were verified by PNA sequence analysis and sequence analysis confirmed the orientation of the clones.
Example 4 - CTL Assays A. Recombinant Viruses Used
The following recombinant viruses were used in the CTL assays of Examples 5-6 below to demonstrate the immunogenicity and vaccine utility of the recombinant adenoviruses of the present invention. Wild-type human adenovirus type 5 (WT-Ad) and the Ad-gB recombinant were propagated in human lung carcinoma A549 cells [ATCC CCL185], as described in Example 1. An E3-deleted adenovirus type 5 mutant lacking the Xbal P fragment of adenovirus PNA (Ad5ΔE3) 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 Example 1, and pAd-5 mu 0-75.9 [G. S. Marshall et al, J. Infect. Pis.. 162:1177-1181 (1990), hereby incorporated by reference]. A vaccinia virus recombinant containing the gB subunits (VacC-gB) described previously in Gonczol et al, Vaccine. :631-637 (1991) and the parental Copenhagen strain of vaccinia, VC-2 (also known as wild-type vaccinia (WT-Vac) ) were grown in Vero cells [E. Gonczol et al, Vaccine. J3:130-136 (1990); J. Tartaglia et al, Crit. Rev. Immunol.. i :13-30 (1990)].
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. 9.:190-193 (1991) [also obtained from Pr. Enzo Paoletti] . Briefly, the gene encoding the HCMV (Towne strain) glycoprotein B was inserted into a 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)]. Cp-gB was derived and plaque-purified by standard methods [Panicali and Paoletti, Proc. Natl. Acad. Sci. USA. 29:4927-4931 (1982)]. The Cp-gB recombinant and parental canarypox virus (WT-Cp) were propagated on primary chick embryo fibroblasts. B. Expression of the gB protein in Cp-gB recombinant virus Chicken embryo fibroblast (CEF) cells [ATCC CRL 1590] infected with either Cp-gB or with the parental wild-type canarypox (WT-Cp) virus preparations were analyzed by Western blot assay using the 4A guinea-pig serum directed against the gB protein. Western blot assays and the 4A guinea-pig serum, used as gB-specific antibody, were described previously in Gonczol et al., J. Virol.. 58:661-664 (1986). Uninfected and HCMV-infected MRC-5 cell [ATCC CCL 171] lysates were included as controls.
A diffuse band at the 140 kPa position and a double band of 55 and 58 kPa were detected in both Cp-gB-infected CEF cells and in HCMV-infected MRC-5 cells. The presence of these gB-specific proteins presumably representing the glycosylated 140 kPa precursor and the differentially glycosylated cleavage products (55 and 58 kPa) indicates that the Cp-gB recombinant expresses the inserted gB gene. The slight difference between the mobility of 55 and 58 kPa cleavage products of control and recombinant gB may reflect different glycosylation patterns.
C. Murine Model and CTL Assay
For immunization of mice, 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.
Six- to 8-week-old female BALB/c and CBA mice (from Harlan Sprague-Pawley and Jackson) and 12-week-old male BALB/k mice (from The Wistar Institute Animal Facility) were immunized intraperitoneally (i.p.) with the recombinant viruses described above at 1-5 x IO8 pfu unless otherwise stated. One to 12 weeks later, 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 106 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 μg/ml gentamicin. Spleen cell cultures were restimulated in vitro with Ad-gB (multiplicity of infection (m.o.i.) = 10) or VacC-gB (m.o.i. = 0.5 ) infected autologous spleen cells for 5 days in 24-well plates. Cytolytic activity of nonadherent spleen cells was tested in a chromium release assay which was performed as follows.
1. T-cell subset depletion
For in vitro depletion of CP4 or CP8 cells, 3 x 106 spleen cells were incubated with anti-mouse CP4 monoclonal antibody (MAb) [Pharmingen; Cat.3:01061 P; 20 μg/3xl06 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 51Cr-release test.
2. Chromium release assay
P815 (H-2d) [ATCC TIB 64], mouse MC57 (H- 2b) cells [also termed MC-57G, P.P. Aden et al, Im unogenetics, 2:209-221 (1976)] and mouse NCTC clone 929 (H-2k) 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 Ad-5ΔE3 (multiplicity of infection (m.o.i.) = 40-80, 40 hours) or with Vac-gB or WT-Vac (m.o.i. = 5-10, 4 hours). Target cells were washed in the modified RPMI 1640 medium described above and 2 x 106 cells were labeled with 100 μCi of [51Cr]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.
Percentage specific 51Cr 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%.
This CTL assay is a system in which two types of viral expression vectors, poxvirus and adenovirus, carrying the same fragment of the HCMV-gB gene, are alternately used for immunization of animal or for infection of target cells to show that HCMV-gB fragment is an inducer of CTL in mice. Using this model system, the relative immunogenicity of the gB fragment expressed by different recombinant viruses has been evaluated.
Example 5 - CTL Responses to Adenovirus Containing gB Fragments
Ad-gBj.303 and Ad-gBWJ5 recombinant viruses were constructed as described in Example 3 above. In CTL experiments performed as described in Example 4, CBA mice were immunized i.p. with IO8 pfu of the Ad-gB,
Figure imgf000022_0001
Two weeks later spleen cells were restimulated in vitro with Ad-gB infected autologous spleen cells and tested for ability to lyse Wt-Ad, Vac-gB or Wt-Vac infected L929 (MHC-class I matched) cells.
All recombinants showed an Ad virus-specific CTL response, but only Ad-gB (containing the complete gB coding sequence) and Ad-gB^^ exerted gB-specific CTL, indicating the presence of a CTL-epitope on the N- terminal part of the gB protein between amino acid 155 and 303.
Example 6 - Protection Studies with Adenovirus Containing gB Fragments
Using the murine model described in Example 4, CBA mice were immunized with 1 x 108 pfu of Wt-Ad, Ad5Δ3 (an E3 deleted mutant virus, the parental strain of the recombinant viruses) , Ad-gB, Ad-gB^^ or Ad-gBι_155. Five to ten days later the immunized mice were challenged i.c. with VacWR-gB (a neurovirulent vaccinia strain expressing the HCMV-gB protein) . Control mice, immunized with the Wt-Ad or Ad5Δ3 virus died within 4-7 days after the challenge.
Ad-gB and Ad-gB^^-immunized mice survived (92% and 95% survival, respectively) , while all of the Ad-gBj.ijj-i munized mice died, indicating a protection epitope on the N-terminal part of the gB protein between amino acid 155 and 303.
Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. For example, use of other appropriate non-defective adenovirus strains for construction of analogous expression systems to express the HCMV gB fragment may be constructed according to the disclosure of the present invention.
Additionally, the other subunits of HCMV major glycoprotein complexes, e.g., gcll or gcIII, or immediate-early antigens, may be expressed in a non- defective adenovirus recombinant in the same manner as described above for subunit gB fragment. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Wistar Institute of Anatomy, Biology Government of USA Pept. Health and Human Services
(ii) TITLE OF INVENTION: Recombinant Cytomegalovirus
Vaccine
(iii) NUMBER OF SEQUENCES: 7
(iv) CORRESPONPENCE APPRESS:
(A) APPRESSEE: Howson and Howson
(B) STREET: Spring House Corporate Center, PO Box 457
(C) CITY: Spring House (P) STATE: Pennsylvania
(E) COUNTRY: USA
(F) ZIP: 19477
(V) COMPUTER REAPABLE FORM:
(A) MEPIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-POS/MS-POS
(P) SOFTWARE: Patentln Release #1.0, Version #1.25
(Vi) CURRENT APPLICATION PATA:
(A) APPLICATION NUMBER:
(B) FILING PATE:
(C) CLASSIFICATION:
(Vϋ) PRIOR APPLICATION PATA:
(A) APPLICATION NUMBER: US 08/048,978
(B) FILING PATE: 16-APR-1993
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Bak, Mary E.
(B) REGISTRATION NUMBER: 31,215
(C) REFERENCE/POCKET NUMBER: WST6CPCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 215-540-9200
(B) TELEFAX: 215-540-5818 (2) INFORMATION FOR SEQ IP NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2724 base pairs
(B) TYPE: nucleic acid
(C) STRANPEPNESS: double (P) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cPNA
(ix) FEATURE:
(A) NAME/KEY: CPS
(B) LOCATION: 1..2721
(Xi) SEQUENCE PESCRIPTION: SEQ IP NO:l:
ATG GAA TCC AGG ATC TGG TGC CTG GTA GTC TGC GTT AAC TTG 42 Met Glu Ser Arg lie Trp Cys Leu Val Val Cys Val Asn Leu 1 5 10
TGT ATC GTC TGT CTG GGT GCT GCG GTT TCC TCA TCT TCT ACT 84 Cys lie Val Cys Leu Gly Ala Ala Val Ser Ser Ser Ser Thr 15 20 25
CGT GGA ACT TCT GCT ACT CAC AGT CAC CAT TCC TCT CAT ACG 126 Arg Gly Thr Ser Ala Thr His Ser His His Ser Ser His Thr 30 35 40
ACG TCT GCT GCT CAT TCT CGA TCC GGT TCA GTC TCT CAA CGC 168 Thr Ser Ala Ala His Ser Arg Ser Gly Ser Val Ser Gin Arg 45 50 55
GTA ACT TCT TCC CAA ACG GTC AGC CAT GGT GTT AAC GAG ACC 210 Val Thr Ser Ser Gin Thr Val Ser His Gly Val Asn Glu Thr 60 65 70
ATC TAC AAC ACT ACC CTC AAG TAC GGA GAT GTG GTG GGG GTC 252 lie Tyr Asn Thr Thr Leu Lys Tyr Gly Asp Val Val Gly Val
75 80
AAC ACC ACC AAG TAC CCC TAT CGC GTG TGT TCT ATG GCA CAG 294 Asn Thr Thr Lys Tyr Pro Tyr Arg Val Cys Ser Met Ala Gin 85 90 95
GGT ACG GAT CTT ATT CGC TTT GAA CGT AAT ATC GTC TGC ACC 336 Gly Thr Asp Leu lie Arg Phe Glu Arg Asn lie Val Cys Thr 100 105 110
TCG ATG AAG CCC ATC AAT GAA GAC CTG GAC GAG GGC ATC ATG 378 Ser Met Lys Pro lie Asn Glu Asp Leu Asp Glu Gly lie Met 115 120 125 GTG GTC TAC AAA CGC AAC ATC GTC GCG CAC ACC TTT AAG GTA 420 Val Val Tyr Lys Arg Asn lie Val Ala His Thr Phe Lys Val 130 135 140
CGA GTC TAC CAG AAG GTT TTG ACG TTT CGT CGT AGC TAC GCT 462 Arg Val Tyr Gin Lys Val Leu Thr Phe Arg Arg Ser Tyr Ala
145 150
TAC ATC CAC ACC ACT TAT CTG CTG GGC AGC AAC ACG GAA TAC 504 Tyr lie His Thr Thr Tyr Leu Leu Gly Ser Asn Thr Glu Tyr 155 160 165
GTG GCG CCT CCT ATG TGG GAG ATT CAT CAT ATC AAC AGT CAC 546 Val Ala Pro Pro Met Trp Glu lie His His lie Asn Ser His 170 175 180
AGT CAG TGC TAC AGT TCC TAC AGC CGC GTT ATA GCA GGC ACG 588 Ser Gin Cys Tyr Ser Ser Tyr Ser Arg Val lie Ala Gly Thr 185 190 195
GTT TTC GTG GCT TAT CAT AGG GAC AGC TAT GAA AAC AAA ACC 630 Val Phe Val Ala Tyr His Arg Asp Ser Tyr Glu Asn Lys Thr 200 205 210
ATG CAA TTA ATG CCC GAC GAT TAT TCC AAC ACC CAC AGT ACC 672 Met Gin Leu Met Pro Asp Asp Tyr Ser Asn Thr His Ser Thr
215 220
CGT TAC GTG ACG GTC AAG GAT CAA TGG CAC AGC CGC GGC AGC 714 Arg Tyr Val Thr Val Lys Asp Gin Trp His Ser Arg Gly Ser 225 230 235
ACC TGG CTC TAT CGT GAG ACC TGT AAT CTG AAT TGT ATG GTG 756 Thr Trp Leu Tyr Arg Glu Thr Cys Asn Leu Asn Cys Met Val 240 245 250
ACC ATC ACT ACT GCG CGC TCC AAG TAT CCC TAT CAT TTT TTC 798 Thr lie Thr Thr Ala Arg Ser Lys Tyr Pro Tyr His Phe Phe 255 260 265
GCA ACT TCC ACG GGT GAT GTG GTT GAC ATT TCT CCT TTC TAC 840 Ala Thr Ser Thr Gly Asp Val Val Asp lie Ser Pro Phe Tyr 270 275 280
AAC GGA ACT AAT CGC AAT GCC AGC TAT TTT GGA GAA AAC GCC 882 Asn Gly Thr Asn Arg Asn Ala Ser Tyr Phe Gly Glu Asn Ala
285 290
GAC AAG TTT TTC ATT TTT CCG AAC TAC ACT ATC GTC TCC GAC 924 Asp Lys Phe Phe lie Phe Pro Asn Tyr Thr lie Val Ser Asp 295 300 305 TTT GGA AGA CCG AAT TCT GCG TTA GAG ACC CAC AGG TTG GTG 966 Phe Gly Arg Pro Asn Ser Ala Leu Glu Thr His Arg Leu Val 310 315 320
GCT TTT CTT GAA CGT GCG GAC TCA GTG ATC TCC TGG GAT ATA 1008 Ala Phe Leu Glu Arg Ala Asp Ser Val lie Ser Trp Asp lie 325 330 335
CAG GAC GAG AAG AAT GTT ACT TGT CAA CTC ACT TTC TGG GAA 1050 Gin Asp Glu Lys Asn Val Thr Cys Gin Leu Thr Phe Trp Glu 340 345 350
GCC TCG GAA CGC ACC ATT CGT TCC GAA GCC GAG GAC TCG TAT 1092 Ala Ser Glu Arg Thr lie Arg Ser Glu Ala Glu Asp Ser Tyr
355 360
CAC TTT TCT TCT GCC AAA ATG ACC GCC ACT TTC TTA TCT AAG 1134 His Phe Ser Ser Ala Lys Met Thr Ala Thr Phe Leu Ser Lys 365 370 375
AAG CAA GAG GTG AAC ATG TCC GAC TCT GCG CTG GAC TGT GTA 1176 Lys Gin Glu Val Asn Met Ser Asp Ser Ala Leu Asp Cys Val 380 385 390
CGT GAT GAG GCC ATA AAT AAG TTA CAG CAG ATT TTC AAT ACT 1218 Arg Asp Glu Ala lie Asn Lys Leu Gin Gin lie Phe Asn Thr 395 400 405
TCA TAC AAT CAA ACA TAT GAA AAA TAT GGA AAC GTG TCC GTC 1260 Ser Tyr Asn Gin Thr Tyr Glu Lys Tyr Gly Asn Val Ser Val 410 415 420
TTT GAA ACC ACT GGT GGT TTG GTG GTG TTC TGG CAA GGT ATC 1302 Phe Glu Thr Thr Gly Gly Leu Val Val Phe Trp Gin Gly lie
425 430
AAG CAA AAA TCT CTG GTG GAA CTC GAA CGT TTG GCC AAC CGC 1344 Lys Gin Lys Ser Leu Val Glu Leu Glu Arg Leu Ala Asn Arg 435 440 445
TCC AGT CTG AAT CTT ACT CAT AAT AGA ACC AAA AGA AGT ACA 1386 Ser Ser Leu Asn Leu Thr His Asn Arg Thr Lys Arg Ser Thr 450 455 460
GAT GGC AAC AAT GCA ACT CAT TTA TCC AAC ATG GAG TCG GTG 1428 Asp Gly Asn Asn Ala Thr His Leu Ser Asn Met Glu Ser Val 465 470 475
CAC AAT CTG GTC TAC GCC CAG CTG CAG TTC ACC TAT GAC ACG 1470 His Asn Leu Val Tyr Ala Gin Leu Gin Phe Thr Tyr Asp Thr 480 485 490 TTG CGC GGT TAC ATC AAC CGG GCG CTG GCG CAA ATC GCA GAA 1512 Leu Arg Gly Tyr lie Asn Arg Ala Leu Ala Gin lie Ala Glu
495 500
GCC TGG TGT GTG GAT CAA CGG CGC ACC CTA GAG GTC TTC AAG 1554 Ala Trp Cys Val Asp Gin Arg Arg Thr Leu Glu Val Phe Lys 505 510 515
GAA CTT AGC AAG ATC AAC CCG TCA GCT ATT CTC TCG GCC ATC 1596 Glu Leu Ser Lys lie Asn Pro Ser Ala lie Leu Ser Ala lie 520 525 530
TAC AAC AAA CCG ATT GCC GCG CGT TTC ATG GGT GAT GTC CTG 1638 Tyr Asn Lys Pro lie Ala Ala Arg Phe Met Gly Asp Val Leu 535 540 545
GGT CTG GCC AGC TGC GTG ACC ATT AAC CAA ACC AGC GTC AAG 1680 Gly Leu Ala Ser Cys Val Thr lie Asn Gin Thr Ser Val Lys 550 555 560
GTG CTG CGT GAT ATG AAT GTG AAG GAA TCG CCA GGA CGC TGC 1722 Val Leu Arg Asp Met Asn Val Lys Glu Ser Pro Gly Arg Cys
565 570
TAC TCA CGA CCA GTG GTC ATC TTT AAT TTC GCC AAC AGC TCG 1764 Tyr Ser Arg Pro Val Val lie Phe Asn Phe Ala Asn Ser Ser 575 580 585
TAC GTG CAG TAC GGT CAA CTG GGC GAG GAT AAC GAA ATC CTG 1806 Tyr Val Gin Tyr Gly Gin Leu Gly Glu Asp Asn Glu lie Leu 590 595 600
TTG GGC AAC CAC CGC ACT GAG GAA TGT CAG CTT CCC AGC CTC 1848 Leu Gly Asn His Arg Thr Glu Glu Cys Gin Leu Pro Ser Leu 605 610 615
AAG ATC TTC ATC GCC GGC AAC TCG GCC TAC GAG TAC GTG GAC 1890 Lys lie Phe lie Ala Gly Asn Ser Ala Tyr Glu Tyr Val Asp 620 625 630
TAC CTC TTC AAA CGC ATG ATT GAC CTC AGC AGC ATC TCC ACC 1932 Tyr Leu Phe Lys Arg Met lie Asp Leu Ser Ser lie Ser Thr
635 640
GTC GAC AGC ATG ATC GCC CTA GAC ATC GAC CCG CTG GAA AAC 1974 Val Asp Ser Met lie Ala Leu Asp lie Asp Pro Leu Glu Asn 645 650 655
ACC GAC TTC AGG GTA CTG GAA CTT TAC TCG CAG AAA GAA TTG 2016 Thr Asp Phe Arg Val Leu Glu Leu Tyr Ser Gin Lys Glu Leu 660 665 670 CGT TCC AGC AAC GTT TTT GAT CTC GAG GAG ATC ATG CGC GAG 2058 Arg Ser Ser Asn Val Phe Asp Leu Glu Glu lie Met Arg Glu 675 680 685
TTC AAT TCG TAT AAG CAG CGG GTA AAG TAC GTG GAG GAC AAG 2100 Phe Asn Ser Tyr Lys Gin Arg Val Lys Tyr Val Glu Asp Lys 690 695 700
GTA GTC GAC CCG CTG CCG CCC TAC CTC AAG GGT CTG GAC GAC 2142 Val Val Asp Pro Leu Pro Pro Tyr Leu Lys Gly Leu Asp Asp
705 710
CTC ATG AGC GGC CTG GGC GCC GCG GGA AAG GCC GTT GGC GTA 2184 Leu Met Ser Gly Leu Gly Ala Ala Gly Lys Ala Val Gly Val 715 720 725
GCC ATT GGG GCC GTG GGT GGC GCG GTG GCC TCC GTG GTC GAA 2226 Ala lie Gly Ala Val Gly Gly Ala Val Ala Ser Val Val Glu 730 735 740
GGC GTT GCC ACC TTC CTC AAA AAC CCC TTC GGA GCC TTC ACC 2268 Gly Val Ala Thr Phe Leu Lys Asn Pro Phe Gly Ala Phe Thr 745 750 755
ATC ATC CTC GTG GCC ATA GCC GTC GTC ATT ATC ATT TAT TTG 2310 lie lie Leu Val Ala lie Ala Val Val lie lie lie Tyr Leu 760 765 770
ATC TAT ACT CGA CAG CGG CGT CTC TGC ATG CAG CCG CTG CAG 2352 lie Tyr Thr Arg Gin Arg Arg Leu Cys Met Gin Pro Leu Gin
775 780
AAC CTC TTT CCC TAT CTG GTG TCC GCC GAC GGG ACC ACC GTG 2394 Asn Leu Phe Pro Tyr Leu Val Ser Ala Asp Gly Thr Thr Val 785 790 795
ACG TCG GGC AAC ACC AAA GAC ACG TCG TTA CAG GCT CCG CCT 2436 Thr Ser Gly Asn Thr Lys Asp Thr Ser Leu Gin Ala Pro Pro 800 805 810
TCC TAC GAG GAA AGT GTT TAT AAT TCT GGT CGC AAA GGA CCG 2478 Ser Tyr Glu Glu Ser Val Tyr Asn Ser Gly Arg Lys Gly Pro 815 820 825
GGA CCA CCG TCG TCT GAT GCA TCC ACG GCG GCT CCG CCT TAC 2520 Gly Pro Pro Ser Ser Asp Ala Ser Thr Ala Ala Pro Pro Tyr 830 835 840
ACC AAC GAG CAG GCT TAC CAG ATG CTT CTG GCC CTG GTC CGT 2562 Thr Asn Glu Gin Ala Tyr Gin Met Leu Leu Ala Leu Val Arg
845 850 CTG GAC GCA GAG CAG CGA GCG CAG CAG AAC GGT ACA GAT TCT 2604 Leu Asp Ala Glu Gin Arg Ala Gin Gin Asn Gly Thr Asp Ser 855 860 865
TTG GAC GGA CAG ACT GGC ACG CAG GAC AAG GGA CAG AAG CCC 2646 Leu Asp Gly Gin Thr Gly Thr Gin Asp Lys Gly Gin Lys Pro 870 875 880
AAC CTG CTA GAC CGA CTG CGA CAC CGC AAA AAC GGC TAC CGA 2688 Asn Leu Leu Asp Arg Leu Arg His Arg Lys Asn Gly Tyr Arg 885 890 895
CAC TTG AAA GAC TCC GAC GAA GAA GAG AAC GTC TGA 2724
His Leu Lys Asp Ser Asp Glu Glu Glu Asn Val 900 905
(2) INFORMATION FOR SEQ IP NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 907 amino acids
(B) TYPE: amino acid (0) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
( i) SEQUENCE OESCRIPTION: SEQ IP NO:2:
Met Glu Ser Arg lie Trp Cys Leu Val Val Cys Val Asn Leu Cys lie 1 5 10 15
Val Cys Leu Gly Ala Ala Val Ser Ser Ser Ser Thr Arg Gly Thr Ser 20 25 30
Ala Thr His Ser His His Ser Ser His Thr Thr Ser Ala Ala His Ser 35 40 45
Arg Ser Gly Ser Val Ser Gin Arg Val Thr Ser Ser Gin Thr Val Ser 50 55 60
His Gly Val Asn Glu Thr lie Tyr Asn Thr Thr Leu Lys Tyr Gly Asp 65 70 75 80
Val Val Gly Val Asn Thr Thr Lys Tyr Pro Tyr Arg Val Cys Ser Met
85 90 95
Ala Gin Gly Thr Asp Leu lie Arg Phe Glu Arg Asn lie Val Cys Thr 100 105 110
Ser Met Lys Pro lie Asn Glu Asp Leu Asp Glu Gly lie Met Val Val 115 120 125 Tyr Lys Arg Asn lie Val Ala His Thr Phe Lys Val Arg Val Tyr Gin 130 135 140
Lys Val Leu Thr Phe Arg Arg Ser Tyr Ala Tyr lie His Thr Thr Tyr 145 150 155 160
Leu Leu Gly Ser Asn Thr Glu Tyr Val Ala Pro Pro Met Trp Glu lie
165 170 175
His His lie Asn Ser His Ser Gin Cys Tyr Ser Ser Tyr Ser Arg Val 180 185 190 lie Ala Gly Thr Val Phe Val Ala Tyr His Arg Asp Ser Tyr Glu Asn 195 200 205
Lys Thr Met Gin Leu Met Pro Asp Asp Tyr Ser Asn Thr His Ser Thr 210 215 220
Arg Tyr Val Thr Val Lys Asp Gin Trp His Ser Arg Gly Ser Thr Trp 225 230 235 240
Leu Tyr Arg Glu Thr Cys Asn Leu Asn Cys Met Val Thr lie Thr Thr
245 250 255
Ala Arg Ser Lys Tyr Pro Tyr His Phe Phe Ala Thr Ser Thr Gly Asp 260 265 270
Val Val Asp lie Ser Pro Phe Tyr Asn Gly Thr Asn Arg Asn Ala Ser 275 280 285
Tyr Phe Gly Glu Asn Ala Asp Lys Phe Phe lie Phe Pro Asn Tyr Thr 290 295 300 lie Val Ser Asp Phe Gly Arg Pro Asn Ser Ala Leu Glu Thr His Arg 305 310 315 320
Leu Val Ala Phe Leu Glu Arg Ala Asp Ser Val lie Ser Trp Asp lie
325 330 335
Gin Asp Glu Lys Asn Val Thr Cys Gin Leu Thr Phe Trp Glu Ala Ser 340 345 350
Glu Arg Thr lie Arg Ser Glu Ala Glu Asp Ser Tyr His Phe Ser Ser 355 360 365
Ala Lys Met Thr Ala Thr Phe Leu Ser Lys Lys Gin Glu Val Asn Met 370 375 380
Ser Asp Ser Ala Leu Asp Cys Val Arg Asp Glu Ala lie Asn Lys Leu 385 390 395 400 Gin Gin lie Phe Asn Thr Ser Tyr Asn Gin Thr Tyr Glu Lys Tyr Gly
405 410 415
Asn Val Ser Val Phe Glu Thr Thr Gly Gly Leu Val Val Phe Trp Gin 420 425 430
Gly lie Lys Gin Lys Ser Leu Val Glu Leu Glu Arg Leu Ala Asn Arg 435 440 445
Ser Ser Leu Asn Leu Thr His Asn Arg Thr Lys Arg Ser Thr Asp Gly 450 455 460
Asn Asn Ala Thr His Leu Ser Asn Met Glu Ser Val His Asn Leu Val 465 470 475 480
Tyr Ala Gin Leu Gin Phe Thr Tyr Asp Thr Leu Arg Gly Tyr lie Asn
485 490 495
Arg Ala Leu Ala Gin lie Ala Glu Ala Trp Cys Val Asp Gin Arg Arg 500 505 510
Thr Leu Glu Val Phe Lys Glu Leu Ser Lys lie Asn Pro Ser Ala lie 515 520 525
Leu Ser Ala lie Tyr Asn Lys Pro lie Ala Ala Arg Phe Met Gly Asp 530 535 540
Val Leu Gly Leu Ala Ser Cys Val Thr lie Asn Gin Thr Ser Val Lys 545 550 555 560
Val Leu Arg Asp Met Asn Val Lys Glu Ser Pro Gly Arg Cys Tyr Ser
565 570 575
Arg Pro Val Val lie Phe Asn Phe Ala Asn Ser Ser Tyr Val Gin Tyr 580 585 590
Gly Gin Leu Gly Glu Asp Asn Glu lie Leu Leu Gly Asn His Arg Thr 595 600 605
Glu Glu Cys Gin Leu Pro Ser Leu Lys lie Phe lie Ala Gly Asn Ser 610 615 620
Ala Tyr Glu Tyr Val Asp Tyr Leu Phe Lys Arg Met lie Asp Leu Ser 625 630 635 640
Ser lie Ser Thr Val Asp Ser Met lie Ala Leu Asp lie Asp Pro Leu
645 650 655
Glu Asn Thr Asp Phe Arg Val Leu Glu Leu Tyr Ser Gin Lys Glu Leu 660 665 670 Arg Ser Ser Asn Val Phe Asp Leu Glu Glu lie Met Arg Glu Phe Asn 675 680 685
Ser Tyr Lys Gin Arg Val Lys Tyr Val Glu Asp Lys Val Val Asp Pro 690 695 700
Leu Pro Pro Tyr Leu Lys Gly Leu Asp Asp Leu Met Ser Gly Leu Gly 705 710 715 720
Ala Ala Gly Lys Ala Val Gly Val Ala lie Gly Ala Val Gly Gly Ala
725 730 735
Val Ala Ser Val Val Glu Gly Val Ala Thr Phe Leu Lys Asn Pro Phe 740 745 750
Gly Ala Phe Thr lie lie Leu Val Ala lie Ala Val Val lie lie lie 755 760 765
Tyr Leu lie Tyr Thr Arg Gin Arg Arg Leu Cys Met Gin Pro Leu Gin 770 775 780
Asn Leu Phe Pro Tyr Leu Val Ser Ala Asp Gly Thr Thr Val Thr Ser 785 790 795 800
Gly Asn Thr Lys Asp Thr Ser Leu Gin Ala Pro Pro Ser Tyr Glu Glu
805 810 815
Ser Val Tyr Asn Ser Gly Arg Lys Gly Pro Gly Pro Pro Ser Ser Asp 820 825 830
Ala Ser Thr Ala Ala Pro Pro Tyr Thr Asn Glu Gin Ala Tyr Gin Met 835 840 845
Leu Leu Ala Leu Val Arg Leu Asp Ala Glu Gin Arg Ala Gin Gin Asn 850 855 860
Gly Thr Asp Ser Leu Asp Gly Gin Thr Gly Thr Gin Asp Lys Gly Gin 865 870 875 880
Lys Pro Asn Leu Leu Asp Arg Leu Arg His Arg Lys Asn Gly Tyr Arg
885 890 895
His Leu Lys Asp Ser Asp Glu Glu Glu Asn Val 900 905 (2) INFORMATION FOR SEQ IP NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANOEPNESS: single (P) TOPOLOGY: unknown
(ii) MOLECULE TYPE: PNA (genomic)
(xi) SEQUENCE PESCRIPTION: SEQ IO NO:3:
ACACGCAAGA GATCTAGACG CGCCTCAT 28
(2) INFORMATION FOR SEQ IO NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANOEPNESS: single (P) TOPOLOGY: unknown
(ii) MOLECULE TYPE: PNA (genomic)
(xi) SEQUENCE PESCRIPTION: SEQ IO NO:4:
TCGTCCAGAC TCTAGAGGTA GGGC 24
(2) INFORMATION FOR SEQ IO NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANPEPNESS: single (P) TOPOLOGY: unknown
(ii) MOLECULE TYPE: PNA (genomic)
(xi) SEQUENCE PESCRIPTION: SEQ IO NO:5:
CGACTCCATT CTAGATTAAT GAGTTGCATT 30 (2) INFORMATION FOR SEQ IO NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANOEPNESS: single (P) TOPOLOGY: unknown
(ii) MOLECULE TYPE: PNA (genomic)
(xi) SEQUENCE PESCRIPTION: SEQ IP NO:6:
CAAAGTCGGA GTCTAGAGTC TAGTTCGGAA A 31
(2) INFORMATION FOR SEQ IO NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANOEPNESS: single (P) TOPOLOGY: unknown
(ii) MOLECULE TYPE: PNA (genomic)
(xi) SEQUENCE PESCRIPTION: SEQ IO NO:7:
CAGATAAGTG GTCTAGATCT AAGCGTAGCT ACG 33

Claims

WHAT IS CLAIMEP IS:
1. A non-defective recombinant adenovirus containing a human cytomegalovirus protein gene encoding a cytomegalovirus protein gB subunit fragment containing at least one CTL epitope, said gene being under the control of an expression control sequence, said virus capable of expressing said subunit protein.
2. The adenovirus according to claim 1 wherein said fragment is selected from the group consisting of:
(a) the fragment spanning about amino acid 1 to about amino acid 303,
(b) the fragment spanning about amino acid 1 to about amino acid 700,
(c) the fragment spanning about amino acid 1 to about amino acid 465,
(d) fragments spanning about amino acid 155 to about amino acid 303, and
(e) smaller fragments of (a) through (d) of SEQ IP NO:2.
3. An immunogenic composition comprising a a non-defective recombinant adenovirus and a suitable pharmaceutical carrier, wherein said recombinant adenovirus comprises a human cytomegalovirus protein gene encoding a cytomegalovirus protein gB subunit fragment containing at least one CTL epitope, said gene being under the control of an expression control sequence and said virus is capable of expressing said subunit protein in vivo in an animal.
4. The composition according to claim 3 wherein said fragment is selected from the group consisting of:
(a) the fragment spanning about amino acid 1 to about amino acid 303,
(b) the fragment spanning about amino acid 1 to about amino acid 700,
(c) the fragment spanning about amino acid 1 to about amino acid 465,
(d) fragments spanning about amino acid 155 to about amino acid 303, and
(e) smaller fragments of (a) through (d) of SEQ 10 NO:2.
5. The composition according to claim 3 wherein said protein gene encodes an additional cytomegalovirus subunit protein fragment or a selected cytomegalovirus subunit protein.
6. The composition according to claim 3 wherein said gB subunit fragment is about amino acid 1 to about amino acid 303 of SEQ 10 NO:2.
7. The composition according to claim 3 wherein said adenovirus is selected from the group consisting of an adenovirus type 5, adenovirus type 4 and adenovirus type 7 strain.
8. The composition according to claim 7 wherein said gB subunit fragment is obtained from the Towne strain cytomegalovirus, and the adenovirus is type 5.
9. The use of a non-defective recombinant adenovirus comprising a human cytomegalovirus protein gene encoding a cytomegalovirus protein gB subunit fragment containing at least one CTL epitope, said gene being under the control of an expression control sequence and said virus being capable of expressing said subunit protein in vivo in an animal, in the preparation of a CMV vaccine.
10. The use according to claim 9 wherein said fragment is selected from the group consisting of:
(a) the fragment spanning about amino acid 1 to about amino acid 303,
(b) the fragment spanning about amino acid 1 to about amino acid 700,
(c) the fragment spanning about amino acid 1 to about amino acid 465,
(d) fragments spanning about amino acid 155 to about amino acid 303, and
(e) smaller fragments of (a) through (d) of SEQ 10 NO:2.
11. The use according to claim 9 wherein said adenovirus is present in an effective amount of between 10s to 108 plaque forming units.
12. An immunogenic composition comprising a gB subunit protein fragment containing at least one CTL epitope expressed in a recombinant adenovirus vector.
13. The composition according to claim 12 wherein said fragment is selected from the group consisting of:
(a) the fragment spanning about amino acid 1 to about amino acid 303,
(b) the fragment spanning about amino acid 1 to about amino acid 700,
(c) the fragment spanning about amino acid 1 to about amino acid 465,
(d) fragments spanning about amino acid 155 to about amino acid 303, and
(e) smaller fragments of (a) through (d) of SEQ 10 NO:2.
PCT/US1994/004180 1993-04-16 1994-04-15 Recombinant cytomegalovirus vaccine WO1994023744A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP94915386A EP0708658A4 (en) 1993-04-16 1994-04-15 Recombinant cytomegalovirus vaccine

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US4897893A 1993-04-16 1993-04-16
US08/048,978 1993-04-16

Publications (1)

Publication Number Publication Date
WO1994023744A1 true WO1994023744A1 (en) 1994-10-27

Family

ID=21957451

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/004180 WO1994023744A1 (en) 1993-04-16 1994-04-15 Recombinant cytomegalovirus vaccine

Country Status (3)

Country Link
EP (1) EP0708658A4 (en)
CA (1) CA2160583A1 (en)
WO (1) WO1994023744A1 (en)

Cited By (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5591439A (en) * 1989-03-24 1997-01-07 The Wistar Institute Of Anatomy And Biology Recombinant cytomegalovirus vaccine
EP0837928A1 (en) * 1995-06-06 1998-04-29 Virogenetics Corporation Recombinant poxvirus - cytomegalovirus compositions and uses
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
WO2003000720A1 (en) * 2001-06-26 2003-01-03 The Council Of The Queensland Institute Of Medical Research Novel human cytomegalovirus (hcmv) cytotoxic t cell epitopes, polyepitopes, compositions comprising same and diagnostic and prophylactic and therapeutic 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 (en) 2002-08-30 2004-03-11 Biorexis Pharmaceutical Corporation Modified transferrin fusion proteins
WO2004020454A2 (en) 2002-08-30 2004-03-11 Biorexis Pharmaceutical Corporation Modified transferrin fusion proteins comprising duplicate transferrin amino or carboxy terminal domains
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 (en) 2005-11-15 2007-05-24 The Children's Hospital Of Philadelphia Compositions and methods for modulating hemostasis
WO2007068803A1 (en) 2005-12-14 2007-06-21 Licentia Ltd Novel neurotrophic factor protein and uses thereof
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 (en) 2008-04-30 2009-11-05 Licentia Oy Neurotrophic factor manf and uses thereof
WO2010060035A1 (en) 2008-11-21 2010-05-27 The Children's Hospital Of Philadelphia Snake factor v and methods of use as a procoagulant
US7754482B2 (en) 2004-05-27 2010-07-13 The Trustees Of The University Of Pennsylvania Artificial antigen presenting cells and uses therefor
EP2384764A2 (en) 2006-02-23 2011-11-09 The Children's Hospital of Philadelphia Compositions and methods for modulating hemostasis using variant forms of activated factor V
WO2012074868A2 (en) 2010-12-03 2012-06-07 Ms Technologies, Llc Optimized expression of glyphosate resistance encoding nucleic acid molecules in plant cells
EP2479189A1 (en) 2006-12-12 2012-07-25 Biorexis Pharmaceutical Corporation Transferrin fusion protein libraries
EP2551281A1 (en) 2007-10-25 2013-01-30 Nevalaita, Lina Splice variants of GDNF and uses thereof
WO2013049804A1 (en) 2011-09-30 2013-04-04 The Children's Hospital Of Philadelphia Compositions and methods for modulating hemostasis
WO2014191630A2 (en) 2013-05-28 2014-12-04 Helsingin Yliopisto Non-human animal model encoding a non-functional manf gene
US9345677B2 (en) 2003-04-04 2016-05-24 Incyte Corporation Compositions, methods and kits relating to HER-2 cleavage
US9783817B2 (en) 2013-03-04 2017-10-10 Arkansas State University Methods of expressing and detecting activity of expansin in plant cells
WO2019152957A1 (en) 2018-02-02 2019-08-08 Arizona Board Of Regents On Behalf Of Arizona State University Dna-chimeric antigen receptor t cells for immunotherapy
WO2022079083A1 (en) 2020-10-15 2022-04-21 F. Hoffmann-La Roche Ag Nucleic acid constructs for va rna transcription
WO2022079082A1 (en) 2020-10-15 2022-04-21 F. Hoffmann-La Roche Ag Nucleic acid constructs for simultaneous gene activation
WO2023198685A1 (en) 2022-04-13 2023-10-19 F. Hoffmann-La Roche Ag Method for determining aav genomes
WO2023227438A1 (en) 2022-05-23 2023-11-30 F. Hoffmann-La Roche Ag Raman-based method for the differentiation of aav particle serotype and aav particle loading status
WO2023232922A1 (en) 2022-06-03 2023-12-07 F. Hoffmann-La Roche Ag Method for producing recombinant aav particles
WO2024013239A1 (en) 2022-07-14 2024-01-18 F. Hoffmann-La Roche Ag Method for producing recombinant aav particles
WO2024056561A1 (en) 2022-09-12 2024-03-21 F. Hoffmann-La Roche Ag Method for separating full and empty aav particles

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4920209A (en) * 1984-11-01 1990-04-24 American Home Products Corporation Oral vaccines

Family Cites Families (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
WO1990000062A1 (en) * 1988-07-01 1990-01-11 The Children's Hospital, Incorporated Antibody recognition of fragments from cytomegalovirus
WO1990006771A1 (en) * 1988-12-12 1990-06-28 The Children's Hospital, Incorporated Proteolytic fragments obtained from human cytomegalovirus glycoprotein complex i
ES2091221T3 (en) * 1989-03-24 1996-11-01 Wistar Inst RECOMBINANT CYTOMEGALOVIRUS VACCINE.

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4920209A (en) * 1984-11-01 1990-04-24 American Home Products Corporation Oral vaccines

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP0708658A4 *
THE EMBO JOURNAL, Volume 5, No. 11, issued November 1986, CRANAGE et al.: "Identification of the Human Cytomegalovirus Glycoprotein B Gene and Induction of Neutralizing Antibodies via Its Expression in Recombinant Vaccinia Virus", pages 3057-3063, see entire document. *

Cited By (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5591439A (en) * 1989-03-24 1997-01-07 The Wistar Institute Of Anatomy And Biology Recombinant cytomegalovirus vaccine
EP0837928A4 (en) * 1995-06-06 2003-01-22 Virogenetics Corp Recombinant poxvirus - cytomegalovirus compositions and uses
EP0837928A1 (en) * 1995-06-06 1998-04-29 Virogenetics Corporation Recombinant poxvirus - cytomegalovirus compositions and uses
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
US6846486B1 (en) 2000-02-24 2005-01-25 Advanced Biotherapy Concepts, Inc. Method of treating allergy by administering an anti-histamine antibody
US7314618B2 (en) 2000-07-19 2008-01-01 Advanced Research And Technology Institute Antibodies to fibroblast growth factor (FGF23)
EP2184296A1 (en) 2000-07-19 2010-05-12 Advanced Research And Technology Institute, Inc. Fibroblast growth factor (fgf23) and methods for use
US8586317B2 (en) 2000-07-19 2013-11-19 Advanced Research & Technology Institute Methods of diagnosing hypophosphatemic disorders
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
WO2003000720A1 (en) * 2001-06-26 2003-01-03 The Council Of The Queensland Institute Of Medical Research Novel human cytomegalovirus (hcmv) cytotoxic t cell epitopes, polyepitopes, compositions comprising same and diagnostic and prophylactic and therapeutic uses therefor
US7524503B2 (en) 2001-06-26 2009-04-28 The Council Of The Queensland Institute Of Medical Research Human cytomegalovirus (HCMV) cytotoxic T cell epitopes, polyepitopes compositions comprising same and diagnostic and prophylactic and therapeutic uses therefor
WO2004020454A2 (en) 2002-08-30 2004-03-11 Biorexis Pharmaceutical Corporation Modified transferrin fusion proteins comprising duplicate transferrin amino or carboxy terminal domains
WO2004020405A2 (en) 2002-08-30 2004-03-11 Biorexis Pharmaceutical Corporation Modified transferrin fusion proteins
US9345677B2 (en) 2003-04-04 2016-05-24 Incyte Corporation Compositions, methods and kits relating to HER-2 cleavage
US10286066B2 (en) 2004-05-27 2019-05-14 The Trustees Of The University Of Pennsylvania Artificial antigen presenting cells and uses thereof
EP3363907A1 (en) 2004-05-27 2018-08-22 The Trustees of the University of Pennsylvania Novel artificial antigen presenting cells and uses therefor
US9555105B2 (en) 2004-05-27 2017-01-31 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 (en) 2004-05-27 2011-03-16 The Trustees Of The University Of Pennsylvania Novel artificial antigen presenting cells and uses thefor
US8722400B2 (en) 2004-05-27 2014-05-13 The Trustees Of The University Of Pennsylvania Artificial antigen presenting cells and uses therefor
US7608433B2 (en) 2005-02-09 2009-10-27 Idexx Laboratories Method of detection of classical swine fever
WO2007059513A2 (en) 2005-11-15 2007-05-24 The Children's Hospital Of Philadelphia Compositions and methods for modulating hemostasis
EP2431389A1 (en) 2005-11-15 2012-03-21 The Children's Hospital of Philadelphia Compositions and methods for modulating hemostasis
EP2423224A1 (en) 2005-11-15 2012-02-29 The Children's Hospital of Philadelphia Compositions and methods for modulating hemostasis
EP2431390A1 (en) 2005-11-15 2012-03-21 The Children's Hospital of Philadelphia Compositions and methods for modulating hemostasis
WO2007068803A1 (en) 2005-12-14 2007-06-21 Licentia Ltd Novel neurotrophic factor protein and uses thereof
EP2384764A2 (en) 2006-02-23 2011-11-09 The Children's Hospital of Philadelphia Compositions and methods for modulating hemostasis using variant forms of activated factor V
EP2479189A1 (en) 2006-12-12 2012-07-25 Biorexis Pharmaceutical Corporation Transferrin fusion protein libraries
EP2551281A1 (en) 2007-10-25 2013-01-30 Nevalaita, Lina Splice variants of GDNF and uses thereof
WO2009133247A1 (en) 2008-04-30 2009-11-05 Licentia Oy Neurotrophic factor manf and uses thereof
WO2010060035A1 (en) 2008-11-21 2010-05-27 The Children's Hospital Of Philadelphia Snake factor v and methods of use as a procoagulant
WO2012074868A2 (en) 2010-12-03 2012-06-07 Ms Technologies, Llc Optimized expression of glyphosate resistance encoding nucleic acid molecules in plant cells
WO2013049804A1 (en) 2011-09-30 2013-04-04 The Children's Hospital Of Philadelphia Compositions and methods for modulating hemostasis
US9783817B2 (en) 2013-03-04 2017-10-10 Arkansas State University Methods of expressing and detecting activity of expansin in plant cells
WO2014191630A2 (en) 2013-05-28 2014-12-04 Helsingin Yliopisto Non-human animal model encoding a non-functional manf gene
WO2019152957A1 (en) 2018-02-02 2019-08-08 Arizona Board Of Regents On Behalf Of Arizona State University Dna-chimeric antigen receptor t cells for immunotherapy
WO2022079083A1 (en) 2020-10-15 2022-04-21 F. Hoffmann-La Roche Ag Nucleic acid constructs for va rna transcription
WO2022079082A1 (en) 2020-10-15 2022-04-21 F. Hoffmann-La Roche Ag Nucleic acid constructs for simultaneous gene activation
WO2023198685A1 (en) 2022-04-13 2023-10-19 F. Hoffmann-La Roche Ag Method for determining aav genomes
WO2023227438A1 (en) 2022-05-23 2023-11-30 F. Hoffmann-La Roche Ag Raman-based method for the differentiation of aav particle serotype and aav particle loading status
WO2023232922A1 (en) 2022-06-03 2023-12-07 F. Hoffmann-La Roche Ag Method for producing recombinant aav particles
WO2024013239A1 (en) 2022-07-14 2024-01-18 F. Hoffmann-La Roche Ag Method for producing recombinant aav particles
WO2024056561A1 (en) 2022-09-12 2024-03-21 F. Hoffmann-La Roche Ag Method for separating full and empty aav particles

Also Published As

Publication number Publication date
EP0708658A1 (en) 1996-05-01
EP0708658A4 (en) 1997-05-21
CA2160583A1 (en) 1994-10-27

Similar Documents

Publication Publication Date Title
US5552143A (en) Recombinant cytomegalovirus vaccine
US5591439A (en) Recombinant cytomegalovirus vaccine
EP0708658A1 (en) Recombinant cytomegalovirus vaccine
WO1994017810A1 (en) Recombinant cytomegalovirus vaccine
US6723695B1 (en) CTL epitopes from EBV
US6998252B1 (en) Recombinant poxviruses having foreign DNA expressed under the control of poxvirus regulatory sequences
US6448389B1 (en) Human cytomegalovirus DNA constructs and uses therefor
Volkmer et al. Cytolytic T lymphocyte recognition of the murine cytomegalovirus nonstructural immediate-early protein pp89 expressed by recombinant vaccinia virus.
JP2001520507A (en) Recombinant poxvirus-cytomegalovirus compositions and uses
US20020081316A1 (en) Novel avian herpes virus and uses thereof
JP2007254489A (en) Immunogenic composition containing recombinant attenuated poxvirus expressing htlv antigen
EP0538299A1 (en) Equine herpesvirus-4 tk?- vaccine
US5369025A (en) Recombinant fowlpox vaccine for protection against Marek's disease
CA2337965C (en) Attenuated equine herpesvirus
WO1994003595A1 (en) Non-shedding live herpesvirus vaccine
AU708460B2 (en) A polynucleotide herpes virus vaccine
CA2012895C (en) Recombinant cytomegalovirus vaccine
EP0606452B1 (en) Vector vaccines of recombinant feline herpesvirus
WO1990001546A1 (en) Equine herpesvirus-1 vaccine
US5674499A (en) Equine herpesvirus gene 15 mutants
GB2282601A (en) Coronavirus vaccines
EP0668355A1 (en) Vaccine for the protection of horses against equine herpesvirus infection
JP2003235588A (en) Vaccine for use in therapeutic treatment of hsv
US6025181A (en) Equine herpesvirus-4TK mutant
Epstein Recent progress with vaccines against Epstein-Barr virus infection

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2160583

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 1994915386

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1994915386

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1994915386

Country of ref document: EP