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WO1996039178A1 - Recombinant humain du type 5 de l'adenovirus a deficience de replication, utilise comme porteur de vaccins - Google Patents

Recombinant humain du type 5 de l'adenovirus a deficience de replication, utilise comme porteur de vaccins Download PDF

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Publication number
WO1996039178A1
WO1996039178A1 PCT/US1996/009495 US9609495W WO9639178A1 WO 1996039178 A1 WO1996039178 A1 WO 1996039178A1 US 9609495 W US9609495 W US 9609495W WO 9639178 A1 WO9639178 A1 WO 9639178A1
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Prior art keywords
virus
recombinant
protein
adenovirus
rabies
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PCT/US1996/009495
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English (en)
Inventor
Hildegund C. J. Ertl
James M. Wilson
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The Wistar Institute Of Anatomy And Biology
Trustees Of The University Of Pennsylvania
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Publication date
Priority claimed from US08/461,837 external-priority patent/US5698202A/en
Application filed by The Wistar Institute Of Anatomy And Biology, Trustees Of The University Of Pennsylvania filed Critical The Wistar Institute Of Anatomy And Biology
Priority to US08/973,223 priority Critical patent/US6019978A/en
Priority to AU62616/96A priority patent/AU6261696A/en
Publication of WO1996039178A1 publication Critical patent/WO1996039178A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20111—Lyssavirus, e.g. rabies virus
    • C12N2760/20134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention relates generally to recombinant adenoviruses as vaccine components, and more particularly, to the use of replication deficient adenoviruses as vaccine carriers, which induce protective immune responses in mammalian hosts.
  • a replication competent, recombinant adenovirus is an adenovirus with intact or functional essential genes, (i.e., Ela, Elb, E2a, E2b and E4) .
  • Such recombinant viruses containing a variety of inserted genes have been used as vaccine compositions with some success [see, e.g. Davis, U.S. Patent No. 4,920,309].
  • One of these recombinant adenoviruses expressing the rabies G protein was shown to induce protective immunity in animals upon challenge with rabies virus [L. Prevac, J. Infect. Dis.. 161:27-30 (1990) ⁇ .
  • Ad with pseudorabies glycoprotein 50 inserted into the El deletion site under the control of a homologous Ad promoter In rabbits and mice, after immunization and challenge, only partial protection was obtained (i.e., about one-third).
  • marmosets ta arins
  • intramuscular immunizations and viral challenge full protection was obtained.
  • a vaccine should be effective at a low dosage to control the occurrence of side effects or to enable sufficient amounts of vaccine to be introduced into animals in the wild.
  • a vaccinia rabies glycoprotein (VRG) vaccine is being used for oral wild- life immunization [B. Brochier et al. Vaccine. 12:1368-
  • compositions and methods of vaccinating a human and/or animal against a disease using an adenovirus defective vaccine composition which produces a high level of protection upon administration of a low vaccine dose.
  • vaccination with a vaccine composition described herein, which is directed against rabies has been found to require as little as a single dose of 10 4 pfu of rabies vaccine vector to induce complete protection. This effect is also accomplished by administration routes other than the oral route.
  • the invention provides a replication-defective recombinant adenovirus (rAd) vaccine containing DNA encoding a selected heterologous protein from a disease-causing agent, which elicits a protective immune response against the agent.
  • This recombinant adenovirus of the invention contains at least a partial, but functional, deletion of the Ad E3 gene.
  • the recombinant virus contains a sequence comprising a non-adenovirus promoter directing the replication and expression of the DNA encoding the heterologous protein.
  • an rAd is Adrab.gp, which contains a rabies gp gene and is useful in a method for treating or preventing rabies.
  • the invention provides pharmaceutical and veterinary compositions which contain the rAd of the invention.
  • the invention provides for the use of the rAd in the manufacture of the compositions described above.
  • the invention provides a method of vaccinating a human or animal against disease comprising administering to said human or animal an effective amount of a replication-defective recombinant adenovirus vaccine containing DNA encoding a selected heterologous protein which elicits a protective immune response against an agent causing the disease.
  • This adenovirus of the invention contains at least a partial, but functional, deletion of the Ad E3 gene.
  • the recombinant virus contains a sequence comprising a non-adenovirus promoter directing the replication and expression of the DNA encoding the heterologous protein.
  • the present invention provides a method of preventing rabies infection in an animal comprising administering to the animal an effective amount of a recombinant replication-defective Adrab.gp adenovirus containing DNA encoding a rabies virus glycoprotein.
  • Fig. 1A is a schematic representation of the
  • Fig. IB is a schematic map of the pAd.CMVlacZ
  • plasmid also known as H5.020CMVlacZ plasmid, which contains adenovirus map units ( .u.) 0-1 as represented by the black bar at the top of the circular plasmid, followed by a cytomegalovirus enhancer/promoter (CMV enh/prom) represented by the striped arrow to the right of the black bar, a human betagalactosidase gene represented by the dark gray bar at the righthand side of the circular plasmid; a polyadenylation signal represented by the short white bar at the bottom of the circular plasmid, adenovirus m.u.
  • CMV enh/prom cytomegalovirus enhancer/promoter
  • Fig. 1C is a schematic map of the plasmid pAdCMV.rabgp which results from blunt end cloning of the Bglll fragment of pSG ⁇ .ragp to the larger NotI fragment of pAdCMV.lacZ.
  • pAdCMV.rapgp is substantially similar to the pAd.CMVlacZ plasmid, but which contains the rabies glycoprotein sequence in place of the lacZ gene.
  • pAdCMV.rapgp [SEQ ID NO: 1] contains adenovirus m.u.
  • 0-1 as represented by the black bar at the top of the circular plasmid (nucleotides 12 to 364 of SEQ ID NO: 1) ; followed by a cytomegalovirus enhancer/promoter (CMV enh/prom) represented by the striped arrow to the right of the black bar [nucleotides 382 to 863 of SEQ ID NO: 1] ; a rabies glycoprotein gene represented by the dotted bar at the righthand side of the circular plasmid (nucleotides 1178 to 2827 of SEQ ID NO: 1) ; a polyadenylation signal represented by the short white bar at the lower righthand portion of the circular plasmid [nucleotides 2836-3034 of SEQ ID NO: 1] ; adenovirus m.u.
  • CMV enh/prom cytomegalovirus enhancer/promoter
  • SEQ ID NO: 9-16 represented by the long black bar at the lower portion of the circular plasmid (nucleotides 3061 to 5524 of SEQ ID NO: 1) ; and plasmid sequences from plasmid pAT153 including an origin of replication and ampicillin resistance gene represented by the light gray bar at the upper lefthand portion of the circular plasmid (nucleotides 5525 to 8236 of SEQ ID NO: 1) .
  • Restriction endonuclease enzymes are represented by conventional designations.
  • SEQ ID NO: ' 2 provides the rabies protein sequence encoded by the nucleotide sequence within pAdCMV.rabgp.
  • Fig. ID is a schematic map of recombinant adenovirus Adrab.gp (also known as H5.020CMV.rab) , which results from homologous recombination between pAdCMV.rabgp and Ad strain dl7001.
  • Ad dl7001 is an Ad5 variant that carries an approximately 3 kb deletion of the Ad5 sequence (GenBank Accession No. M73260) between m.u. 78.4 through 86.
  • the CMV/rabies glycoprotein/pA minicassette of pAd.CMVrab is inserted between deleted adenovirus m.u.l and 9, with the remaining Ad5 m.u. 9-100 having the above-mentioned E3 gene deletion.
  • Fig. 2 is a bar graph plotting 3 H-thymidine ([3H]TdR) incorporation, measured at counts per minute ⁇ standard deviation (cpm + SD) , for irradiated splenocytes plated at 5 x 10 5 cells per well of a round bottom microtiter plate and incubated with 5 (diagonally striped), 1 (cross-hatched) or 0.2 (solid) ⁇ g/ml of betapropionolactone-inactivated Evelyn Rockitniki Abelseth rabies strain (ERA-BPL) or approximately 1 (diagonally striped), 0.1 (cross-hatched), and 0.01 (solid) pfu of Adrab.gp per cell or medium only as a negative control for 60 minutes at 37°C.
  • [3H]TdR 3 H-thymidine
  • Fig. 3A is a graph plotting % specific lysis
  • Fig. 3B is a graph of an experiment similar to Fig. 3A, but in which the activated lymphocytes were tested at different E:T ratios on H-2 compatible L929 cells stably transfected with a neomycin-expressing vector (t.L929.neo) in the 51 Cr-release assay, as a control.
  • a neomycin-expressing vector t.L929.neo
  • Fig. 4A is a graph plotting number of cells vs. intensity of fluorescence for L929 fibroblasts plated in 24-well Costar plates in medium supplemented with 2% fetal bovine serum (FBS) following infection with 1 pfu/cell of VRG, as described in Example 5 below.
  • FBS fetal bovine serum
  • Cells harvested 12 hours after infection and stained by indirect immunofluorescence with monoclonal antibody (MAb) 509-6 were analyzed by fluorescence activated cell sorting (FACS) .
  • the line on the graph labeled "B” is the threshold below which 99% of the population are negative.
  • Line “C” represents the region that encompasses all events on the histogram.
  • Fig. 4B is a graph similar to Fig. 4A above, except the cells were harvested 36 hours after infection.
  • Fig. 4C is a graph similar to Fig. 4A above, except the cells were harvested 60 hours after infection.
  • Fig. 4D is a graph similar to Fig. 4A above, except the cells, harvested 12 hours after infection, were stained using cells treated only with the fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin (Ig) as a control.
  • FITC fluorescein isothiocyanate
  • Ig goat anti-mouse immunoglobulin
  • Fig. 4E is a graph similar to Fig. 4D above, except the cells were harvested 36 hours after infection.
  • Fig. 4F is a graph similar to Fig. 4D above, except the cells were harvested 60 hours after infection.
  • Fig. 4G is a graph similar to Fig. 4A above, except the cells were infected with 1 pfu Adrab.gp virus, and cells were harvested 12 hours after infection.
  • Fig. 4H is a graph similar to Fig. 4G, except the cells were harvested 36 hours after infection.
  • Fig. 41 is a graph similar to Fig. 4G, except the cells were harvested 60 hours after infection.
  • Fig. 4J is a graph similar to Fig. 4G above, except the cells were stained by indirect immunofluorescence using cells treated only with FITC- labeled goat anti-mouse Ig as a control.
  • Fig. 4K is a graph similar to Fig. 4J above, except the cells were harvested 36 hours after infection.
  • Fig. 4L is a graph similar to Fig. 4J above, except the cells were harvested 60 hours after infection.
  • Fig. 5A is a graph plotting optical density at 405 nm vs. serum dilution for duplicate samples ⁇ SD, as described in Example 6B below for mice immunized with a replication-competent E3 deleted adenovirus (open box) or Adrab.gp (solid box) . Native age-matched control mice were used as controls (X) . Mice were bled 10 days after immunization and serum antibody titers to adenoviral antigens were determined by an ELISA on plates coated with 1 ⁇ g/mL of purified H5.020CMVlacZ virus.
  • Fig. 5B is a graph similar to that of Fig. 5A for mice immunized as described in Fig. 6A below, and bled at 16 days.
  • Fig. 6A is a graph plotting mean percentage (%) specific lysis of triplicates ⁇ SD vs. E:T cell ratio for C3H/He mice inoculated with 10 6 pfu of replication competent E3 deleted adenovirus and boosted 3 weeks later with Adrab.gp (open box) . Control mice were inoculated with Adrab.gp only (solid box) .
  • Fig. 6B is a graph similar to Fig. 6A, except the L929 cells were transfected with pSV2neo.
  • Fig. 7 is a graph plotting % survival of vaccinated mice vs. days after challenge with rabies virus. Mice were challenged 3 days (open triangle) , 7 days (open square) , and 10 days (solid square) after vaccination. X represents naive mice controls. See, Example 7.
  • the present invention provides compositions and methods of effectively inducing a protective immune response to a disease agent.
  • the compositions include a recombinant replication-defective adenovirus, and pharmaceutical and veterinary compositions containing the rAd.
  • the rAd backbone was previously used for gene therapy.
  • the inventors have surprisingly found that use of such a recombinant Ad, described in detail below, provides substantially complete immune protection in vaccinates.
  • the recombinant adenovirus presents an immunogenic protein in such a manner that a protective immune response is observed in substantially all vaccinates after a single administration.
  • substantially all is meant greater than 90% of the vaccinates.
  • the recombinant vaccine permits successful vaccination with very few booster administrations. Also unexpectedly, the recombinant vaccine permits vaccination at an unexpectedly lower dosage than is normally used in similar vaccines in which the same protein is present in another recombinant virus.
  • this recombinant, replication defective Ad vaccine is advantageous over, e.g., the vaccinia vaccine, because it permits lower doses of antigen to be expressed for an extended period of time by a non-lytic virus.
  • vaccinia expresses higher doses of antigen, e.g., a rabies antigen, it is a lytic virus which causes a rapid demise of infected cells.
  • the finding that the recombinant replication-defective Ad, e.g., Adrab.gp virus, used in the method of the present invention is more efficacious than the currently used vaccinia rabies (VRG) vaccine is unexpected and incompatible with current thinking that the antigenic dose governs the magnitude of the immune response.
  • the use of the recombinant replication defective adenovirus also confers safety and efficacy advantages over other vaccine carriers, such as vaccinia.
  • the adenovirus construct results in slow accumulation of the rabies virus G protein on the surface of infected cells without causing visible cell damage (data not shown) . In contrast, cells infected with VRG recombinant rapidly express substantial amounts of the rabies virus G protein on the cell surface but then die shortly after infection.
  • the adenoviral construct persists for at least seven days in immunocompetent mice.
  • the present invention provides a recombinant replication-defective Ad which is thus highly unlikely to spread within a host or among individuals, particularly in view of the fact that the recombinant.
  • El-deleted dl7001 Ad virus which is the backbone of the exemplary replication defective recombinant Ad used in the examples below has already been approved for use in humans for gene therapy, i.e., for the replacement of faulty or missing genes.
  • the recombinant virus lacks oncogenic potential because the El gene that can function as an oncogene in some adenovirus strains has been deleted. Further, cells infected with the recombinant, replication defective adenovirus are completely eliminated by CD8 T cells within 21 days in immunocompetent hosts.
  • the recombinant, replication defective Ad of this invention is highly efficacious at inducing cytolytic T cells and antibodies to the inserted heterologous protein expressed by the virus. This has been demonstrated with a recombinant, replication defective Ad containing a sequence encoding the rabies virus glycoprotein as the heterologous gene, which Ad has been administered to animals by other than the oral route.
  • the recombinant virus of this invention is also surprisingly more effective as a vaccine than other, previously reported, replication defective adenovirus vaccines. See, for example, Ragot et al, Eliot et al, and Jacobs et al, all cited above.
  • the vaccine composition useful in the present invention can be used at lower doses. This vaccine can also be administered in a single inoculation to obtain substantially complete protection.
  • the recombinant replication- defective adenovirus of the invention and particularly the preferred embodiment which makes use of the pAdCMV.lacZ (or H5.020CMVlacZ) Ad vector described below can be used as a prophylactic or therapeutic vaccine against any pathogen for which the antigen(s) crucial for induction of an immune response able to limit the spread of the pathogen has been identified and for which the cDNA is available.
  • the Recombinant Adenovirus can be used as a prophylactic or therapeutic vaccine against any pathogen for which the antigen(s) crucial for induction of an immune response able to limit the spread of the pathogen has been identified and for which the cDNA is available.
  • minicassette refers to the nucleotide sequence comprised of (a) a non-Ad promoter, which directs the replication and expression of (b) the following nucleotide sequence which encodes a heterologous protein immunogen, which is followed by (c) a polyA nucleotide sequence.
  • vector or plasmid is meant the construct comprised of 5' sequences of the Ad virus (usually Ad m.u. 0-1) deleted of the El gene (which occurs between Ad m.u. 1-9) , which may contain a heterologous nucleotide sequence, but which does not contain the 3• end of the Ad virus (generally between about Ad m.u.
  • recombinant, replication defective Ad is meant the infectious recombinant virus, deleted of its El gene, into which location is inserted the minicassette, and which contains all of the 3• sequences essential to an infectious virus except for a functional deletion in the E3 gene region.
  • the recombinant virus of the method of the invention is a replication-defective recombinant adenovirus containing a deletion of its El gene and at least a partial, functional deletion of its E3 gene.
  • a minicassette is inserted, which comprises a nucleotide sequence encoding a heterologous protein immunogen and a non-adenovirus promoter directing the replication and expression of the nucleotide sequence encoding the heterologous protein.
  • Any Ad that infects the target cells is appropriate for use in this invention.
  • Desirable adenoviruses are human type C adenoviruses, including serotypes Ad2 and Ad5.
  • the DNA sequences of a number of adenovirus types, including type Ad5 are available from GenBank [Accession No. M73260] .
  • the adenovirus sequences may be obtained from any known adenovirus type, including the presently identified 41 human types [Horwitz et al. Virology. 2d ed. , B. N. Fields, Raven Press, Ltd., New York (1990) ] . Similarly, adenoviruses known to infect other animals may also be employed in this invention. The selection of the adenovirus type and strain is not anticipated to limit the following invention. A variety of adenovirus strains are available from the American Type Culture Collection, Rockville, Maryland, or available by request from a variety of commercial and institutional sources. In the following exemplary embodiment, an adenovirus type 5 (Ad5) sequence obtained from GenBank [Ace. No. M73260] is used for convenience.
  • Adenoviruses of the present invention are replication defective, i.e., intact adenoviruses which have been rendered replication defective by deleting the early gene locus that encodes Ela and Elb. See, K.F. Kozarsky and J. M. Wilson, Curr. Opin. Genet. Dev.. 2:499-503 (1993). Similarly, a replication defective adenovirus may be designed by deleting less than the entire Ela and Elb locus, but enough to functionally disable the El genes.
  • Ad useful in this invention is that the E3 gene is deleted, i.e., from about m.u. 78.5 to about m.u. 84.3 of Ad5. While the presently preferred embodiment contains a complete deletion of that sequence, it may be possible to partially delete the E3 sequence to disable the functional abilities of the E3 gene.
  • a preferred recombinant Ad virus may be produced by using a plasmid vector pAd.CMVlacZ as described in Fig. IB.
  • This plasmid contains adenovirus sequences Ad m.u. 0-1 (i.e., it is fully deleted of Ela and Elb genes) , after which a selected minigene may be inserted, e.g., the rabies glycoprotein under control of a heterologous promoter and other regulatory sequences, if desired, followed by the sequence Ad m.u.9 to 16 and plasmid sequences.
  • the resulting recombinant adenovirus is capable of functioning as a rabies vaccine.
  • This recombinant virus called Adrab.gp or H5020.CMVrab, is described in detail in Example 1 and in flow chart form in Figs. 1A through ID.
  • the preferred recombinant Ad of this invention contains a minicassette which uses the cytomegalovirus (CMV) promoter [see, e.g., Boshart et al, Cell.
  • CMV cytomegalovirus
  • the promoter is inserted in the site of the El deletion and directs the replication and expression of the protein encoded by the selected heterologous gene.
  • this invention is not limited by the selection of the promoter, except that the promoter should be heterologous to the Ad virus, i.e., the El Ad promoter is replaced using techniques known to those of skill in the art.
  • Other desirable promoters include the Rous sarcoma virus LTR promoter/enhancer, the SV40 promoter, and the chicken cytoplasmic ⁇ -actin promoter [T. A. Kost et al, Nucl. Acids Res.. 11(23) :8287 (1983)]. Still other promoter/enhancer sequences may be readily selected by one of skill in the art.
  • a nucleic acid sequence preferably in the form of DNA, encoding a protein heterologous to the Ad is inserted using techniques known to those of skill in the art.
  • the heterologous nucleic acid encodes a protein which is desirably capable of inducing an immune response to a pathogen.
  • a protein may be a protein from rabies virus, human papilloma virus, human immunodeficiency virus (HIV) , respiratory syncytial virus (RSV) .
  • the vaccine method of the present invention may also be employed with a tumor-associated protein specific for a selected malignancy. These tumor antigens include viral oncogenes, such as E6 and E7 of human papilloma virus or cellular oncogenes such as mutated ras or p53.
  • the protein is preferably HIV glycoprotein 120 for which sequences are available from GenBank.
  • the condition is human papilloma virus infection
  • the protein is selected from the group consisting of E6, E7 and/or LI [Seedorf, K. et al, Virol.. 145:181-185 (1985)].
  • the condition is respiratory syncytial virus infection
  • the protein is selected from the group consisting of the glyco- (G) protein and the fusion (F) protein, for which sequences are available from GenBank.
  • G glyco-
  • F fusion protein
  • the condition is rabies and the protein is the rabies glycoprotein [see, U.S. Patent No. 4,393,201].
  • rabies strains are well known and available from academic and commercial sources, including depositaries such as the American Type Culture Collection, or may be isolated using known techniques.
  • the strain used in the examples below is the Evelyn Rockitniki Abelseth (ERA) strain.
  • ERA Evelyn Rockitniki Abelseth
  • cDNA encoding the rabies virus glycoprotein is inserted under control of a CMV promoter into the pAdCMV.lacZ (or H5.020CMVlacZ) Ad vector and supplied with the essential genes for infectivity and viral formation in a helper cell line using standard techniques, as described in detail in Example 1. Immunization studies revealed that a single administration of the resulting recombinant replciation defective virus conferred complete protection at a relatively low dose following challenge with rabies virus. II. Formulation of Vaccine
  • a recombinant replication defective Ad bearing a gene encoding an immunogenic protein may be administered to a human or veterinary patient, preferably suspended in a biologically compatible solution or pharmaceutically acceptable delivery vehicle.
  • a suitable vehicle is sterile saline.
  • Other aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non- aqueous sterile suspensions known to be pharmaceutically acceptable carriers and well known to those of skill in the art may be employed for this purpose.
  • a vaccinal composition of the invention may be formulated to contain other components, including, e.g. adjuvants, stabilizers, pH adjusters, preservatives and the like. Such components are well known to those of skill in the vaccine art.
  • the recombinant, replication defective viruses are administered in an "effective amount", that is, an amount of recombinant virus that is effective in a route of administration to transfect the desired cells and provide sufficient levels of expression of the selected gene to provide a vaccinal benefit, i.e., protective immunity.
  • an effective amount that is, an amount of recombinant virus that is effective in a route of administration to transfect the desired cells and provide sufficient levels of expression of the selected gene to provide a vaccinal benefit, i.e., protective immunity.
  • Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, rectal, oral and other parental routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the immunogen or the disease.
  • a prophylactically effective amount or dose of the Ad vaccine is generally in the range of from about 100 ⁇ l to about 10 ml of saline solution containing concentrations of from about 1 x 10 4 to 1 x 10 7 plaque forming units (pfu) virus/ml.
  • a preferred dose is from about 1 to about 10 ml saline solution at the above concentrations.
  • the preferred dose is about 10 4 pfu of the recombinant virus per mouse, preferably suspended in about 0.1 L saline.
  • a larger animal would preferably be administered about a 1 mL dose containing about 1 x 10 5 Adrab.gp pfu suspended in saline.
  • optional booster immunizations may be desired.
  • the following examples illustrate the preferred methods for preparing the vectors and the recombinant viruses used in the vaccine and method of the invention. These examples are illustrative only and do not limit the scope of the invention.
  • Adrab.gp A recombinant, replication defective adenovirus expressing the rabies virus G protein of the Evelyn Rockitniki Abelseth (ERA) strain of rabies virus [ATCC VR-332; U. S. Patent No. 3,423,505] (ERA) was constructed as follows. See the flowchart of Figs. 1A to ID.
  • the 1650 bp rabies virus G cDNA (nucleotides 1178 to 2827 of SEQ ID NO: 1) was purified from the pSG ⁇ rab.gp plasmid [S.R. Burger et al, J. Gen. Virol.. 22:359-367 (1991)] upon digestion with Bglll, and blunt-ended with Klenow to supply the G gene. See also United States Patent No. 4,393,201, issued July 12, 1983.
  • the pAd.CMVlacZ vector [J. Wilson et al, Hum. Gene Ther.. 5_:501-519 (1994); K. Kozarsky et al, J. Biol. Chem.. 269:13695-13702 (1994)], which contains Ad5 m.u. 0-1, followed by the cytomegalovirus (CMV) enhancer/promoter, the beta galactosidase (lacZ) gene, a polyadenylation signal (pA) , adenovirus m.u. 9-16 and plasmid sequences from plasmid pAT153 including an origin of replication and ampicillin resistance gene, was completely digested with NotI to remove the lacZ gene and provide an "5.6 kb backbone.
  • CMV cytomegalovirus
  • lacZ beta galactosidase
  • pA polyadenylation signal
  • pA adenovirus m.u. 9-16
  • pAdCMV.rabgp contains adenovirus m.u.
  • nucleotides 12 to 364 of SEQ ID NO: 1 followed by a cytomegalovirus enhancer/promoter (nucleotides 382 to 863 of SEQ ID NO: 1) ; the rabies glycoprotein gene (nucleotides 1178 to 2827 of SEQ ID NO: 1) ; a polyadenylation signal (nucleotides 2836-3034 of
  • SEQ ID NO: 1 SEQ ID NO: 1 ; adenovirus m.u. 9-16 (nucleotides 3061 to 5524 of SEQ ID NO: 1) ; and plasmid sequences from plasmid pAT153 (nucleotides 5525 to 8236 of SEQ ID NO: 1) .
  • the remaining nucleotides of SEQ ID NO: 1 are the result of cloning and plasmid construction.
  • the 3' end of the adenovirus sequence was needed to replace the pAT153 plasmid sequences of pAdCMV.rabgp.
  • the plasmid pAdCMV.rabgp was linearized with Nhel.
  • the linearized plasmid was co-transfected into 293 packaging cells [ATCC CRL 1573] which contain and express the transforming genes of human adenovirus type 5 to allow replication of the adenovirus [F. L. Graham et al, J. Gen. Virol.. 3_6.59-72 (1977)].
  • the transfected packaging cells were grown in DMEM with 10%
  • Ad5dl7001 a variant that carries a 3 kb deletion between m.u. 78.4 through 86 in the nonessential E3 region (provided by Dr. William Wold, Washington, University, St. Louis, MO)].
  • This Ad5dl7001 had been digested with a restriction enzyme Clal to remove the left end, i.e., 917 bp from the 5' end of the adenovirus sequence, rendering the DNA non-infectious.
  • Adrab.gp replication defective clone
  • the recombinant, replication defective Ad Adrab.gp contains Ad5 m.u. 0-1, followed by the CMV enhancer/promoter, the rabies G gene, a pA site, and Ad5 m.u. 9-78.4 and 86-100.
  • Adrab.gp contains Ad5 m.u. 0-1, followed by the CMV enhancer/promoter, the rabies G gene, a pA site, and Ad5 m.u. 9-78.4 and 86-100.
  • the recombinant replciation defective Ad is substantially identical to Adrab.gp, except that this virus contains E. coli lacZ in place of the rabies G protein and only a partial deletion of E3.
  • the plasmid pAd.CMVlacZ described above was linearized with Nhel and co-transfected into 293 cells with a partially E3 deleted Ad5 DNA (sub 360 DNA, H5sub360) , which had been digested with Clal to eliminate the sequence of m.u. 83.5 to 85.
  • homologous recombination, followed by plaqing and harvesting produced the resulting recombinant adenovirus, designated H5.010CMVlacZ.
  • This virus contains the sequence from Ad5 m.u. 0-1, followed by the CMV enhancer/promoter, the Escherichia coli lacZ gene, a pA site, and Ad5 m.u. 9- 83.5 and 85-100.
  • adenoviral recombinants Adrab.gp H5.010CMVlacZ, and Ad5dl7001, a replication competent adenovirus, on 293 cells for 72 hours. Virus was recovered on the third round of freeze-thawing. Cell- free supematants were either used directly or they were further purified by CsCl density centrifugation. Viral stocks were titrated on 293 cells using a plaque assay.
  • Example 2 Immunofluorescence and T Cell Studies To confirm that the Adrab.gp recombinant virus expresses the rabies virus G protein on infected cells in a form recognized by antibodies and cytolytic T cells directed against rabies virus, a series of in vitro experiments were performed initially.
  • HeLa cells which had been maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10% FBS, HEPES buffer and antibiotics in a 10% C0 2 incubator] were infected for 48 hours with 1 pfu of Adrab.gp virus per cell or as a control with H5.020CMVlacZ. Cells were stained 24 hours later by an indirect immunofluorescence assay using three MAbs (designated 523-11, 509-6, and
  • the B cell hybridoma cells 509-6, 1112-1, and 523-11 secrete antibodies to different antigenic sites of the rabies virus G protein (509-6 to site I, 1112-1 to site II, and 523-11 to site III [T.J. Wiktor et al, Proc. Natl. Acad. Sci. USA. 25:3938-3945 (1978)].
  • These hybridoma cells were grown in DMEM supplemented with 10% FBS.
  • Ascetic fluid was prepared in BALB/c mice. The assay was performed as follows.
  • the HeLa cells were infected for various times with 1 pfu of recombinant adenovirus or with 1 pfu of the vaccinia VRG virus described above per cell in 24- well Costar plates seeded with 5 x 10 5 cells per well. Cells were harvested at varied times after infection by treatment with trypsin and incubated for 60 minutes on ice with the MAbs identified above. Cells were washed once with phosphate-buffered saline (PBS) and then incubated with a FITC-labeled goat anti-mouse immunoglobulin (Ig) antibody. Cells were washed and analyzed by a fluorescence activated cell sorter (FACS) .
  • FACS fluorescence activated cell sorter
  • a rabies virus-specific helper T cell clone obtained from splenocytes of VRG immune C3H/He mice in the inventors' laboratory, was cultured (2 x 10 4 cells/well) in 96-well round-bottom microtiter plate with 5 x 10 5 irradiated syngeneic C3H/He splenocytes pretreated with different antigen preparations (1, 0.1 and 0.01 pfu Adrab.gp per cell) in DMEM supplemented with 2% FBS and 10 ⁇ 6 M 2-mercaptoethanol and 10% rat
  • Adrab.gp causes expression of the rabies virus G protein in a form that is readily recognized by both rabies virus-specific antibodies and T cells of the helper and the cytolytic subset.
  • Fig. 2 illustrates that Adrab.gp induces proliferation of a rabies virus G protein T helper cell clone in the presence of antigen presenting cells.
  • mice were immunized with the Adrab.gp recombinant virus at several doses using different routes of immunization as follows. Groups of eight to twelve week old outbred ICR [Harlan Sprague-Dawley (Indianapolis, IN)] or C3H/He mice [The Jackson Laboratories (Bar Harbor, ME) ] were injected subcutaneously (s.c), orally (per os) , intranasally (i.n.)f or upon anesthesia and surgical exposure of the trachea intratracheally (i.t.), with the recombinant adenoviruses of the previous examples diluted in 100 to
  • VRG which had been propagated on HeLa cells as described in T. J. Wiktor et al, Proc. Natl. Acad. Sci. USA r £1-7194-7198 (1984)] was given s.c. Mice were bled by retro-orbital puncture in regular intervals after immunization to assess serum antibody titers.
  • the challenge virus standard (CVS)-24 strain of rabies virus that is antigenically closely related to the ERA strain but shows higher virulence in mice, was derived from brain suspensions of infected newborn ICR mice [T.J. Wiktor et al, J. Virol.. 21:626-633 (1977].
  • mice were challenged with 10 mean lethal doses (LD 50 ) of CVS-24 virus given intramuscularly (i.m.) into the masseter muscle; they were observed for the following 3 weeks for symptoms indicative of a rabies virus infection. Mice that developed complete bilateral hind leg paralysis (proceeding death by 24 to 48 hours) were euthanized for humanitarian reasons.
  • LD 50 mean lethal doses
  • CVS-24 virus given intramuscularly (i.m.) into the masseter muscle; they were observed for the following 3 weeks for symptoms indicative of a rabies virus infection. Mice that developed complete bilateral hind leg paralysis (proceeding death by 24 to 48 hours) were euthanized for humanitarian reasons.
  • VNA virus neutralizing antibody
  • Table 1 illustrates the data expressed as neutralization titers which are the reciprocal of the serum dilution resulting in a 50% reduction in the number of infected cells. Samples were assayed in duplicate in serial 3-fold dilutions starting with a dilution of 1:5. Standard deviations were within 10% for any given experiment. As illustrated by the results in Table 1, virus given s.c, i.t., or i.n. induced a potent neutralizing antibody response if given at 10 6 pfu. Oral immunization with Adrab.gp or systemic immunization with H5.020CMVlacZ failed to induce a measurable antibody response to rabies virus.
  • Adrab.gp Recombinant Induces Neutralizing Antibodies to Rabies Virus
  • mice were vaccinated with an equal dose of PFUs of unpurified and gradient purified recombinant adenovirus. Both groups of mice developed identical virus neutralizing antibody titers.
  • mice inoculated s.c. with Adrab.gp virus developed rabies virus G protein-specific cytolytic T cells able to kill H-2 compatible L929 target cells stably transfected with a plasmid vector expressing the rabies virus G protein under the control of the SV40 early promoter [Z. Q. Xiang et al, J. Virol. Meth.. 47:103-116 (1994)].
  • L929 mouse fibroblasts were maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) , HEPES buffer and antibiotics in a 10% C0 2 incubator.
  • DMEM Dulbecco's minimal essential medium
  • FBS fetal bovine serum
  • L929 cells stably transfected with pSG ⁇ rab.gp [S.R. Burger et al, cited above] , expressing the rabies virus G protein as well as L929 cells transfected with pSV2neo [ATCC Accession No. 37149] were maintained in 10% DMEM supplemented with 10% FBS.
  • splenocytes were harvested from immunized C3H/He mice. Single cells were prepared and incubated at 6 x 10 6 cells per well with 1 pfu per cell of the Adrab.gp recombinant virus in 1.6 ml of DMEM supplemented with 10 ⁇ 6 M 2-mercaptoethanol and 2% FBS for 5 days in a humidified 10% C0 2 incubator. The effector cells were then co-cultured with 51 Cr-labeled L929 cells expressing the rabies virus G protein upon stable transfection with the pSG5rab.gp vector at varied effector-to-target cells ratios.
  • Fig. 3A which illustrates that the Adrab.gp construct induces cytolytic T cells to the rabies virus G protein. See, also the results of Fig. 3B, in which lymphocytes were tested at different E:T ratios on an L929 cell line transfected with Adrab.gp or a neomycin expressing control.
  • mice immunized as described in Example 3A above, were challenged with 10 LD 50 of rabies virus. Briefly, mice immunized with the Adrab.gp or the VRG recombinant virus were challenged 2 to 5 weeks after immunization with 10 LD 50 of the virulent CVS-24 strain of rabies virus given i.m. into the masseter muscle. Mice that subsequently developed complete bilateral hind leg paralysis indicative of a terminal rabies virus infection were euthanized for humanitarian reasons. Survivors were observed for a total of 21 days.
  • mice immunized with Adrab.gp i.m., i.t., or i.n. using doses ranging from 10 4 to 2 x 10 6 pfu were fully protected against infection; 87% of mice inoculated with 10 3 pfu were protected.
  • mice vaccinated with VRG showed partial protection; the group receiving the highest dose, i.e., 2 x 10 6 pfu, had a mortality rate above 50% raising to ⁇ 90% in mice inoculated with 2 x 10 4 pfu of VRG.
  • Table 2
  • the same cell line infected with 1 pfu of Adrab.gp per cell shows low expression of the rabies virus G protein on day 1.
  • the level of expression increases until days 3 to 4 after infection and then reaches plateau levels (data shown for days 1 to 3 in Fig. 4A through Fig. 4L) .
  • the replication-defective recombinant adenoviruses are non-lytic and maintain stable infection and expression of virus-encoded proteins for extended periods of time. In tissue culture, expression has been shown for 7 days in vivo; using the H5.010CMVlacZ recombinant virus, stable levels of expression were demonstrated in immunocompromised mice for 10 months.
  • a non-lytic virus e.g., the recombinant replication defective adenovirus, that expresses antigens for prolonged periods of time might thus be more immunogenic compared to a replicating agent that causes death of the infected cells within 24 to 48 hours, e.g., vaccinia.
  • the inventors compared the immune response to rabies proteins upon immunization of mice with a replication-defective El deleted adenovirus and a replication-competent adenovirus. Both adenoviruses were of the human strain 5 and both were deleted in E3. These recombinant viruses were tested by enzyme linked immunoadsorbent assay (ELISA) (Figs. 5A and 5B) .
  • ELISA enzyme linked immunoadsorbent assay
  • the ELISAs were conducted in 96-well microtiter plates coated with 0.1 to 0.2 ⁇ g per well of ERA-BPL virus or 1-2 ⁇ g per well of purified H5.010CMVlacZ virus, using an alkaline phosphatase conjugated goat anti-mouse Ig as second antibody as described in detail in Xiang and Ertl, Virus Res.. 24.J297-314 (1992).
  • the antibody response to the El deleted Adrab.gp virus solid box
  • was superior to that of a replication competent Ad virus open box
  • Figs. 5A and 5B illustrate that expression of El causes a reduction in the antibody response to adenovirus.
  • Example 6 Further Comparative Studies The following study was performed to test if pre-existing immunity to adenoviral proteins interferes with stimulation of a rabies G protein-specific immune response to the Adrab.gp construct.
  • Groups of C3H/He mice were immunized with 10 5 or 10 6 pfu of a replication- competent adenovirus human serotype 5 that had been deleted of the E3 gene.
  • Mice were injected 4 weeks later with 10 6 pfu of Adrab.gp.
  • Control mice were only injected with Adrab.gp (10 6 pfu) .
  • Mice were bled 12 days later and neutralizing antibody titers were determined (Table 3) .
  • Table 3 Table 3
  • mice pre-immunized with adenovirus developed VNA to rabies virus upon booster immunization with the Adrab.gp construct. Titers were equivalent, or marginally lower, when compared to those in control mice that had only received Adrab.gp, indicating that antibodies to adenoviruses only marginally inhibit the B cell response to proteins expressed by adenovirus recombinants.
  • sera from pre-immune (both doses of adenovirus) or naive mice were shown to have titers of 40 IU to rabies virus. Protection to rabies virus is correlated to antibody titers and 2 IU are considered sufficient to protect against a severe challenge.
  • Pre-immunity to adenovirus does, thus, not impair the ability of the Adrab.gp vaccine to elicit protective immunity.
  • FIG. 6A and 6B illustrate that the cytolytic T cell response to rabies virus G protein expressing target cells upon immunization with Adrab.gp is only slightly reduced in animals immune to adenovirus. Nevertheless, adenovirus-immune mice still developed significant T cell responses to the rabies virus G protein upon immunization with Adrab.gp.
  • Example 7 Additional Challenge Studies In this experiment the kinetic of the induction of protective immunity upon vaccination was tested with the Adrab.gp virus. Vaccination to rabies virus is in general given post-exposure, hence it is crucial for the vaccine to induce a rapid immune response before the rabies virus has reached the central nervous system.
  • mice were immunized with 10 6 PFU of Adrab.gp s.c. Immunized mice were challenged 3 (0), 7 (D) , and 10 ( ⁇ ) days after vaccination with 10 LD 50 of rabies virus given i.m. Naive mice (X) served as controls. Mice were observed for four weeks to record mortality. As shown in Fig. 7, mice vaccinated with Adrab.gp virus 10 days previously were completely protected; while more than half of the animals were protected as early as seven days after a single injection. Mice vaccinated three days before challenge succumbed to the infection.
  • GGTGTACACA GGAAGTGACA ATTTTCGCGC GGTTTTAGGC GGATGTTGTA 250
  • GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC 700
  • CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG 800
  • AAGCATGTCC CTGACTCGCA TGTTTTCCCT GACCAAATCC GCCAGAAGGC 4796
  • GTCGGTGCTC GTCCAGACGG GCCAGGGTCA TGTCTTTCCA CGGGCGCAGG 5046
  • GTCCTCGTCA GCGTAGTCTG GGTCACGGTG AAGGGGTGCG CTCCGGGCTG 5096 CGCGCTGGCC AGGGTGCGCT TGAGGCTGGT CCTGCTGGTG CTGAAGCGCT 5146

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Abstract

L'invention concerne un adénovirus de recombinaison à déficience de réplication, qui contient une délétion complète de son gène E1 et au moins une délétion partielle de son gène E3, ledit virus contenant dans le site de la délétion d'E1 une séquence comprenant un promoteur de non-adénovirus dirigeant la réplication et l'expression de l'ADN codant une protéine hétérologue provenant d'un agent pathogène, qui, lorsqu'il est administré à un mammifère dans ledit virus de recombinaison, induit une réponse immunitaire de protection sensiblement complète contre ledit agent. Les produits pharmaceutiques et vétérinaires contenant ledit adénovirus de recombinaison sont également décrits.
PCT/US1996/009495 1995-06-05 1996-06-05 Recombinant humain du type 5 de l'adenovirus a deficience de replication, utilise comme porteur de vaccins WO1996039178A1 (fr)

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WO1997012981A1 (fr) * 1995-10-05 1997-04-10 Microbix Biosystems Inc. Adenovirus recombine de la rage
WO1999003885A1 (fr) * 1997-07-18 1999-01-28 Transgene S.A. Composition antitumorale a base de polypeptide immunogene de localisation cellulaire modifiee
WO2001014416A3 (fr) * 1999-08-25 2001-12-06 Merck & Co Inc Genes synthetiques humains du virus de papillome
AU2001294562B8 (en) * 2000-09-15 2002-03-26 Merck & Co., Inc. Enhanced First Generation Adenovirus Vaccines Expressing Codon Optimized HIV1-Gag, Pol, Nef and Modifications
WO2003018055A1 (fr) * 2001-08-23 2003-03-06 Merck & Co., Inc. Vaccin mettant en oeuvre des proteines e du virus du papillome administrees par vecteur viral
US6733993B2 (en) 2000-09-15 2004-05-11 Merck & Co., Inc. Enhanced first generation adenovirus vaccines expressing codon optimized HIV1-gag, pol, nef and modifications
US6787351B2 (en) 1999-07-06 2004-09-07 Merck & Co., Inc. Adenovirus carrying gag gene HIV vaccine
WO2006002594A1 (fr) * 2004-07-07 2006-01-12 Institute Of Military Veterinary, Academy Of Military Medical Sciences, Pla Adenovirus canin de type 2 recombinant, procede d'elaboration et utilisation
US7001995B1 (en) 1999-08-25 2006-02-21 Merck & Co., Inc. Synthetic human papillomavirus genes
US7300657B2 (en) 2002-12-17 2007-11-27 Crucell Holland B.V. Recombinant viral-based malaria vaccines
US7947822B2 (en) 2003-09-15 2011-05-24 The United States Of America As Represented By The Department Of Health And Human Services HIV vaccines based on Env of multiple clades of HIV
US20110159034A1 (en) * 1997-06-09 2011-06-30 Mcmichael Andrew Methods and reagents for vaccination which generate a cd8 t cell immune response
US8454972B2 (en) 2004-07-16 2013-06-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Method for inducing a multiclade immune response against HIV utilizing a multigene and multiclade immunogen
WO2022068247A1 (fr) * 2020-09-29 2022-04-07 广州恩宝生物医药科技有限公司 Vaccin quadrivalent à adénovirus

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JOURNAL OF GENERAL VIROLOGY, 1990, Vol. 71, ELOIT et al., "Construction of a Defective Adenovirus Vector Expressing the Pseudorabies Virus Glycoprotein gp50 and its use as a Live Vaccine", pages 2425-2431. *
JOURNAL OF VIROLOGY, January 1989, Vol. 63, No. 1, DEWAR et al., "Synthesis and Processing of Human Immunodeficiency Virus Type 1 Envelope Proteins Encoded by a Recombinant Human Adenovirus", pages 129-136. *
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WO1997012981A1 (fr) * 1995-10-05 1997-04-10 Microbix Biosystems Inc. Adenovirus recombine de la rage
US20110159034A1 (en) * 1997-06-09 2011-06-30 Mcmichael Andrew Methods and reagents for vaccination which generate a cd8 t cell immune response
WO1999003885A1 (fr) * 1997-07-18 1999-01-28 Transgene S.A. Composition antitumorale a base de polypeptide immunogene de localisation cellulaire modifiee
US7332478B2 (en) 1997-07-18 2008-02-19 Transgene Antitumoral composition based on immunogenic polypeptide with modified cell location
US6884786B1 (en) 1997-07-18 2005-04-26 Transgene S.A. Antitumoral composition based on immunogenic polypeptide with modified cell location
US6787351B2 (en) 1999-07-06 2004-09-07 Merck & Co., Inc. Adenovirus carrying gag gene HIV vaccine
US7001995B1 (en) 1999-08-25 2006-02-21 Merck & Co., Inc. Synthetic human papillomavirus genes
WO2001014416A3 (fr) * 1999-08-25 2001-12-06 Merck & Co Inc Genes synthetiques humains du virus de papillome
JP4799789B2 (ja) * 1999-08-25 2011-10-26 メルク・シャープ・エンド・ドーム・コーポレイション ヒト細胞中での発現用に最適化された合成ヒトパピローマウイルス遺伝子
JP2003511010A (ja) * 1999-08-25 2003-03-25 メルク エンド カムパニー インコーポレーテッド ヒト細胞中での発現用に最適化された合成ヒトパピローマウイルス遺伝子
AU772611B2 (en) * 1999-08-25 2004-05-06 Merck Sharp & Dohme Corp. Synthetic human papillomavirus genes
US7211569B2 (en) 1999-08-25 2007-05-01 Merck & Co., Inc. Synthetic human papilloma virus genes
AU2001294562B2 (en) * 2000-09-15 2007-05-24 Merck & Co., Inc. Enhanced First Generation Adenovirus Vaccines Expressing Codon Optimized HIV1-Gag, Pol, Nef and Modifications
WO2002022080A3 (fr) * 2000-09-15 2002-05-02 Merck & Co Inc Vaccins adenoviraux de premiere generation evolues, exprimant les proteines gag, pol et nef du vih-1 a optimisation des codons et leurs modifications
US6733993B2 (en) 2000-09-15 2004-05-11 Merck & Co., Inc. Enhanced first generation adenovirus vaccines expressing codon optimized HIV1-gag, pol, nef and modifications
AU2001294562B8 (en) * 2000-09-15 2002-03-26 Merck & Co., Inc. Enhanced First Generation Adenovirus Vaccines Expressing Codon Optimized HIV1-Gag, Pol, Nef and Modifications
WO2003018055A1 (fr) * 2001-08-23 2003-03-06 Merck & Co., Inc. Vaccin mettant en oeuvre des proteines e du virus du papillome administrees par vecteur viral
US7867764B2 (en) 2002-12-17 2011-01-11 Crucell Holland B.V. Recombinant viral-based malaria vaccines
US7521229B2 (en) 2002-12-17 2009-04-21 Crucell Holland B.V. Recombinant viral-based malaria vaccines
US7524947B2 (en) 2002-12-17 2009-04-28 Crucell Holland B.V. Recombinant viral-based malaria vaccines
EP2258850A1 (fr) 2002-12-17 2010-12-08 Crucell Holland B.V. Vaccins contre la malaria basés sur un virus recombinant
US7387894B2 (en) 2002-12-17 2008-06-17 Crucell Holland B.V. Recombinant viral-based malaria vaccines
US7300657B2 (en) 2002-12-17 2007-11-27 Crucell Holland B.V. Recombinant viral-based malaria vaccines
US8097453B2 (en) 2002-12-17 2012-01-17 Crucell Holland B.V. Recombinant viral-based malaria vaccines
US8361478B2 (en) 2002-12-17 2013-01-29 Crucell Holland B.V. Recombinant viral-based malaria vaccines
US7947822B2 (en) 2003-09-15 2011-05-24 The United States Of America As Represented By The Department Of Health And Human Services HIV vaccines based on Env of multiple clades of HIV
US8323961B2 (en) 2003-09-15 2012-12-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services HIV vaccines based on adenoviral vectors encoding Env from multiple clades of HIV
WO2006002594A1 (fr) * 2004-07-07 2006-01-12 Institute Of Military Veterinary, Academy Of Military Medical Sciences, Pla Adenovirus canin de type 2 recombinant, procede d'elaboration et utilisation
US8454972B2 (en) 2004-07-16 2013-06-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Method for inducing a multiclade immune response against HIV utilizing a multigene and multiclade immunogen
WO2022068247A1 (fr) * 2020-09-29 2022-04-07 广州恩宝生物医药科技有限公司 Vaccin quadrivalent à adénovirus

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