WO1991002804A1 - Recombinant adenovirus sequence expressing rhabdovirus antigen and use as a vaccine - Google Patents

Recombinant adenovirus sequence expressing rhabdovirus antigen and use as a vaccine Download PDF

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Publication number
WO1991002804A1
WO1991002804A1 PCT/GB1990/001217 GB9001217W WO9102804A1 WO 1991002804 A1 WO1991002804 A1 WO 1991002804A1 GB 9001217 W GB9001217 W GB 9001217W WO 9102804 A1 WO9102804 A1 WO 9102804A1
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dna sequence
virus
recombinant
adenovirus
recombinant adenovirus
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PCT/GB1990/001217
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French (fr)
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Chil Yong Kang
Lud Prevec
Frank Graham
Jim Campbell
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Ov Limited
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C12N2760/20122—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
    • 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 to a recombinant adenovirus DNA sequence, a recombinant adenovirus expressing the DNA sequence and a rhabdovirus vaccine including the
  • Rabies is caused by a virus of the Lyssavirus genera of the Rhabdoviridae family of viruses. It is estimated that over 20000 people still die
  • recombinant virus vaccines in which a gene encoding a rabies antigen is carried and is expressed m an
  • VSV vesicular stomatitis virus
  • Adenoviruses have a number of properties such a restricted host range, stability, and ability to infect by the oral route, that make them particularly useful for the development of rabies vaccine.
  • a recombinant adenovirus DNA sequence comprising an inserted DNA sequence which codes for a lyssavirus antigen.
  • the adenovirus DNA sequence may be derived from a human adenovirus.
  • the human adenovirus is human adenovirus type 5.
  • Expression of the inserted DNA sequence may be controlled by an adenovirus promoter.
  • the adenovirus promoter may be the E3 promoter or major late promoter of human adenovirus.
  • the inserted DNA sequence may replace an adenovirus DNA sequence.
  • the inserted DNA sequence may replace an early adenovirus DNA sequence.
  • Preferably the inserted sequence replaces at least a portion of the E3 adenovirus gene.
  • the inserted DNA may be contained within a cassette of DNA from another virus.
  • the other virus may be a Simian virus.
  • the Simian virus is SV40.
  • the inserted DNA may be introduced between the promoter and poly-A addition site of the earlier region of SV40.
  • the inserted DNA sequence may encode a rabies virus antigen.
  • the rabies virus antigen may be a glycoprotein.
  • a recombinant virus comprising a recombinant adenovirus DNA sequence as defined in any of the five immediately preceding paragraphs.
  • the recombinant virus may be Ad5RGILP as deposited with the ATCC under
  • a vaccine comprising an effective amount of a recombinant virus as defined in the immediately preceding paragraph and a pharmaceutically acceptable excipient.
  • the effective amount of virus may be at least 10 4 PFU per
  • a method of immunizing a mammal against a lyssavirus-mduced disease comprising administering a recombinant virus or vaccine according to any of the two immediately preceding paragraphs to the mammal.
  • the vaccine may be administered orally or nassally.
  • a recombinant adenovirus expressing the DNA sequence and a rhabdovirus vaccine including the recombinant adenovirus in accordance with the invention will now be described, by way of example only, with reference to the accompanying drawings in which:
  • Fig. 1 illustrates the construction of a recombinant adenovirus
  • Fig. 2 illustrates the results of tests of the efficacy of the vaccine of the invention.
  • the strategy used to produce a recombinant human adenovirus 5 containing a rabies virus glycoprotein is shown m Fig. 5.
  • Plasmid SV2neo (Southern, P.J. and Berg, P.
  • the synthetic polylinker sequence into which a desired gene can be inserted into which a desired gene can be inserted.
  • the SV40 promoter, polylinker and SV40 poly-A addition signal form a "cassette" which is bounded by XbaI sites.
  • pSV2X3RG had the rabies virus DNA in the correct orientation to allow transcription from the SV40 promoter.
  • the cassette containing the inserted rabies glycoprotein was removed by XbaI digestion and isolated by agarose separation.
  • Plasmid pFGDXl contains the sequence of the human adenovirus type 5 genome, including the E3 promoter, from the BamHI site at 59.5% to 100%, except for the XbaI D fragment from 78.5% to 84.3%.
  • Plasmid pBCRG was selected for growth and purification. 293 cells (Graham, F.L.,
  • a recombinant adenovirus (A5RGILP) having the desired rabies gene insert was identified by restriction enzyme analysis, using HindIII, XbaI, and EcoRI, and purified.
  • the recombinant adenovirus A5RGILP was twice plaque purified in 293 cells, and the final plaque isolate expanded and grown in KB cell (described in a paper by H. Eagle (Proc. Soc. Exp. Biol. and Med. (1955) 89, 362.) Human, oral, epidermoid carcmema, HeLa markers. (ATCC CCL 17 KB)) suspension cultures.
  • A5GRILP was extracted from the infective cells, and purified by twice banding on CsCl density gradients. The recombinant virus was dialyzed and titrated prior to use. The rabies gene insert is in the same orientation as the adenovirus E3 genes which it replaced to form the recombinant virus. The rabies gene m A5RGILP is expressed from the transcription origins at either the adenovirus E3 promoter or major late promoter.
  • FIMT fluoresence inhibition microtest
  • Ad5RGILP virus may also serve as a very effective immunogen in a wide range of animal species.
  • mice received 0.1 ml inoculum. Oral adminstration was performed under light Fluothane anaethesia using micro tubing fitted over a 23 gauge needle attached to a tuberculin syringe. Tubing was inserted approximately 1 cm past their pharynx. The animals were bled via the tail 4 weeks later, and sera was assayed individually. Antibody titers in mice are presented in Table 1 as the means of seropositivity results within individual groups; figures in brackets represent numbers of seropositive animals total number in group. Antibody titers are expressed in FIMT units, one unit being the highest two-fold dilution of serum inhibiting replication of ERA rabies challenge virus by at least 50%, as measured by fluorescent focus formation. All sera were heat
  • mice Following this initial success, we conducted a more extensive investigation of the efficacy of the Ad5RGILP recombinant virus vaccine in mice. In these studies, groups of 5 week old mice, housed five to a cage and fed Purina rodent food ad lib were given a single
  • recombinant virus with titers ranging from 10 4 to 10 7 plaque forming units (PFU).
  • Control groups received 0.1 ml virus diluent (PBS/2% foetal cal serum alone).
  • mice were bled via the tail vein (0.2 to 0.3 ml per animal), sera were collected individually, heat inactivated (56oC for 30 minutes), and assayed for rabies virus neutralizing antibodies by FIMT.
  • the animal were challenged mtracerebrally with 0.3 ml of ERA strain of rabies virus
  • the vaccine of this invention has proven effective in oral administration to skunks and foxes in inducing high levels of neutralizing antibody and in protecting the animals from death after challenge with lethal doses of rabies virus.
  • Ad5GRILP virus A sample of recombinant Ad5GRILP virus was deposited on March 4, 1988, under the Budapest Treaty on the

Abstract

This invention provides a recombinant adenovirus DNA sequence comprising an inserted DNA sequence which codes for a lyssavirus antigen; and a recombinant adenovirus incorporating this DNA sequence. The invention also provides a vaccine comprising an effective amount of the recombinant adenovirus virus and a pharmaceutically-acceptable excipient; and a method of immunising a mammal against a lyssavirus-induced disease comprising administering the virus or vaccine to the mammal.

Description

RECOMBINANT ADENOVIRUS SEQUENCE EXPRESSING RHABDOVIRUS ANTIGEN AND USE AS A VACCINE
This invention relates to a recombinant adenovirus DNA sequence, a recombinant adenovirus expressing the DNA sequence and a rhabdovirus vaccine including the
recombinant adenovirus.
Although countless lives have been saved by
vaccination against rabies since the time of Pasteur, this disease nonetheless continues to be a serious
worldwide problem. Rabies is caused by a virus of the Lyssavirus genera of the Rhabdoviridae family of viruses. It is estimated that over 20000 people still die
annually from rabies virus infection, mainly in southeast Asia. Even in those parts of the world where the
instance of human death is negligible, distress is caused to hundreds of thousands of individuals vaccinated, and considerable economic loss results from the large number of domestic animals succumbing to the disease. Infected wild life constitutes a major reservoir of rabies, and while currently available vaccines for humans and
domestic animals have proven safe and effective, there are economic and practical limitations to the use of only these vaccines for larger scale disease control
programmes. As an alternative to known vaccines,
recombinant virus vaccines, in which a gene encoding a rabies antigen is carried and is expressed m an
infectious vector virus, would seem to hold considerable potential. Recombinant vaccinia virus vectors expressing the rabies glycoprotein have proved effective in inducing an immune response and in protecting animals of a number of species against a lethal rabies challenge. Human adenoviruses have also been investigated as potential non-lyssavirus rhabdovirus glycoprotem vectors, and have been shown effective for the induction of neutralizing antibodies to vesicular stomatitis virus (VSV)
glycoprotein in mice, dogs, pigs and cows. Adenoviruses have a number of properties such a restricted host range, stability, and ability to infect by the oral route, that make them particularly useful for the development of rabies vaccine.
According to one aspect of the invention there is provided a recombinant adenovirus DNA sequence comprising an inserted DNA sequence which codes for a lyssavirus antigen. The adenovirus DNA sequence may be derived from a human adenovirus. Preferably the human adenovirus is human adenovirus type 5.
Expression of the inserted DNA sequence may be controlled by an adenovirus promoter. The adenovirus promoter may be the E3 promoter or major late promoter of human adenovirus.
The inserted DNA sequence may replace an adenovirus DNA sequence. The inserted DNA sequence may replace an early adenovirus DNA sequence. Preferably the inserted sequence replaces at least a portion of the E3 adenovirus gene.
The inserted DNA may be contained within a cassette of DNA from another virus. The other virus may be a Simian virus. Preferably the Simian virus is SV40. The inserted DNA may be introduced between the promoter and poly-A addition site of the earlier region of SV40.
The inserted DNA sequence may encode a rabies virus antigen. The rabies virus antigen may be a glycoprotein.
According to another aspect of the invention there is provided a recombinant virus comprising a recombinant adenovirus DNA sequence as defined in any of the five immediately preceding paragraphs. The recombinant virus may be Ad5RGILP as deposited with the ATCC under
accession no. VR2204.
According to another aspect of the invention there is provided a vaccine comprising an effective amount of a recombinant virus as defined in the immediately preceding paragraph and a pharmaceutically acceptable excipient. The effective amount of virus may be at least 104 PFU per
0.1 ml of vaccine.
According to a further aspect of the invention there is provided a method of immunizing a mammal against a lyssavirus-mduced disease comprising administering a recombinant virus or vaccine according to any of the two immediately preceding paragraphs to the mammal.
The vaccine may be administered orally or nassally. A recombinant adenovirus expressing the DNA sequence and a rhabdovirus vaccine including the recombinant adenovirus in accordance with the invention will now be described, by way of example only, with reference to the accompanying drawings in which:
Fig. 1 illustrates the construction of a recombinant adenovirus;
Fig. 2 illustrates the results of tests of the efficacy of the vaccine of the invention.
1. Construction of the human adenovirus type 5 rabies glycoprotein gene recombinant.
The strategy used to produce a recombinant human adenovirus 5 containing a rabies virus glycoprotein is shown m Fig. 5.
Plasmid SV2neo (Southern, P.J. and Berg, P.
Transformation of mammalian cells to antibiotic
resistance with a bacterial gene under control of the SV 40 early region promoter, J. Molecular Applied Genetics, (1982) 1, 327-334) was modified through conversion of the single NdeI (N) site to an XbaI (X) site, replacement of the BamI (B) to EcoRI (E) fragment by an XbaI linker, and by deletion of all neo gene and SV40 sequences by
replacing the HindIII to HpaI plasmid sequences with a synthetic polylinker containing HindIII, BamHI, EcoRI, SmaI (S) and XhoI (Xh) restriction sites. In the
resultant plasmid, pSV2X3, the SV40 early promoter sequence (represented by a filled box in Fig. 1) is
separated from the SV40 poly-A addition signal
(represented by an unfilled box in Fig. 1) by the
synthetic polylinker sequence into which a desired gene can be inserted. The SV40 promoter, polylinker and SV40 poly-A addition signal form a "cassette" which is bounded by XbaI sites.
A complete cDNA copy of the glycoprotein gene, obtained from Connaught Medical Research Laboratories, for the ERA strain of rabies virus in pBR322 was used as the source of the rabies glycoprotein gene to be inserted into pSV2X3. The cloned rabies virus DNA is described in Malek, L.T., Soostmeyer, G., Garvin, R.T. and James, E. The rabies glycoprotein gene is expressed in E. coli as a denatured polypeptide. In: Chanock, R. Lerner, R. eds. Modern Approaches to Vaccines. Cold Spring Harbor
Laboratory, 1984, 203-208. The gene was isolated by digestion of the plasmid with EcoRI and BamHI and, after blunt end filling using Klenow polymerase was then
inserted into the SmaI site in the polylinker in pSV2X3.
One of the resulting plasmids, pSV2X3RG, had the rabies virus DNA in the correct orientation to allow transcription from the SV40 promoter. The cassette containing the inserted rabies glycoprotein was removed by XbaI digestion and isolated by agarose separation.
The cassette was then inserted into the XbaI site of plasmid pFGDXl (Haj-Ahmad, Y. and Graham, F.L. Development of a helper-independent human adenovirus vector and its use in the transfer of the herpes simplex virus thymidine kmase gene, J. of Virology (1986) 57, 267-274). Plasmid pFGDXl contains the sequence of the human adenovirus type 5 genome, including the E3 promoter, from the BamHI site at 59.5% to 100%, except for the XbaI D fragment from 78.5% to 84.3%.
In one of the plasmids thus formed, pBCRG, the rabies glycoprotem gene-containing cassette was
inserted in the correct orientation so that the SV40 transcription direction was in the same direction as the adenovirus E3 promoter. Plasmid pBCRG, was selected for growth and purification. 293 cells (Graham, F.L.,
Smiley, J., Russell, W.C. and Nairn, R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. of General Virology (1977) 36, 59-72) were transfected with pBCRG, along with EcoRI digested adenovirus type 5 DNA using the calcium
phosphate precipitations techniques. A recombinant adenovirus (A5RGILP) having the desired rabies gene insert was identified by restriction enzyme analysis, using HindIII, XbaI, and EcoRI, and purified. The recombinant adenovirus A5RGILP was twice plaque purified in 293 cells, and the final plaque isolate expanded and grown in KB cell (described in a paper by H. Eagle (Proc. Soc. Exp. Biol. and Med. (1955) 89, 362.) Human, oral, epidermoid carcmema, HeLa markers. (ATCC CCL 17 KB)) suspension cultures. A5GRILP was extracted from the infective cells, and purified by twice banding on CsCl density gradients. The recombinant virus was dialyzed and titrated prior to use. The rabies gene insert is in the same orientation as the adenovirus E3 genes which it replaced to form the recombinant virus. The rabies gene m A5RGILP is expressed from the transcription origins at either the adenovirus E3 promoter or major late promoter.
2. Antibody responses following adminstration of Ad5RGILP virus.
The initial animal experiment involved the
introduction of the recombinant Ad5RGILP virus into mice and dogs. Mice were inoculated orally or parentally while dogs were given virus by either the subcutaneous or mtranasal route. Pre- and post-inoculation serum samples were taken and coded prior to analysis and rabies virus neutralizing antibody titers were determined by the fluoresence inhibition microtest (FIMT). As seen in
Table 1, both species responded well to the parentally administered vaccine. In mice responding to the oral vaccine route, it is uncertain whether seroconversion was affected by the buccal or intestinal routes since some of the inoculum in individual animals may have been
regurgitated or may have been deposited in the buccal cavity during withdrawal of the tubing. Particularly significant is the observation that virus given to dogs by the intranasal route was highly effective in inducing neutralizing antibodies. This is in keeping with our previous observations using the adenovirus vector
expressing the VSV glycoprotem. Since the latter recombinant was also effective in inducing VSV antibodies in calves and pigs, it suggests that Ad5RGILP virus may also serve as a very effective immunogen in a wide range of animal species.
Dogs received vaccine in 0.5 ml doses, with the second dose, were given, 14 days later. Serum antibody titers were measured at 2 and 4 weeks post-primary vaccination, mice (3 week old females from Charles
River) received 0.1 ml inoculum. Oral adminstration was performed under light Fluothane anaethesia using micro tubing fitted over a 23 gauge needle attached to a tuberculin syringe. Tubing was inserted approximately 1 cm past their pharynx. The animals were bled via the tail 4 weeks later, and sera was assayed individually. Antibody titers in mice are presented in Table 1 as the means of seropositivity results within individual groups; figures in brackets represent numbers of seropositive animals total number in group. Antibody titers are expressed in FIMT units, one unit being the highest two-fold dilution of serum inhibiting replication of ERA rabies challenge virus by at least 50%, as measured by fluorescent focus formation. All sera were heat
inactivated at 56º for 30 minutes before assay. Table 1: Antibody responses following administration of Ad5RGILP virus
Animals Dose Vaccination route Antibody titer
(PFU) 1st 2nd 0 wk 2 wk 4 wk - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Dog 1 5×5105 SC SC Neg. 256 512
Dog 2 SC SC Neg. 1024 1024
Dog 3 IN SC Neg. 256 512
Dog 4 IN IN Neg. 256 4096
Mice 1×107 Oral Oral Neg. - 1124 (4/10) Mice 1×106 Oral Oral Neg. - 424 (4/10) Mice 1×107 IM IM Neg. - 2925 (10/10) Mice 1×105 IM IM Neg. - 1312 (8/10) - - - - -- - - - -- -- - - - - - -- - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
SC : Subcutaneous
IN : Intra-Nasal
IM : Int ra-Muscular
3. Protection of mice against rabies virus after minimization with Ad5RGILP virus vaccine.
Following this initial success, we conducted a more extensive investigation of the efficacy of the Ad5RGILP recombinant virus vaccine in mice. In these studies, groups of 5 week old mice, housed five to a cage and fed Purina rodent food ad lib were given a single
mtraperitoneal dose of 0.1 ml gradient purified
recombinant virus, with titers ranging from 10 4 to 107 plaque forming units (PFU). Control groups received 0.1 ml virus diluent (PBS/2% foetal cal serum alone). After
3 weeks, animals were bled via the tail vein (0.2 to 0.3 ml per animal), sera were collected individually, heat inactivated (56ºC for 30 minutes), and assayed for rabies virus neutralizing antibodies by FIMT. Three days following collection of serum, the animal were challenged mtracerebrally with 0.3 ml of ERA strain of rabies virus
(20 TCID 50/dose) and observed for 21 days. Death occurred mainly between days 10 and 14. All animals developing rabies exhibited typical pathological signs including ruffled hair, cradled posture, weight loss, progressing to spasms and/or paralysis of extremities and death. The result of this challenge study is presented in Figure 2. As seen in Figure 2, injection of Ad5GRILP virus at 10 PFU/mouse induced a high titer of
neutralizing antibody in all animals. Lower doses in 10-fold dilutions produced a response in decreasing proportions of animals, and also induced correspondingly reduced titers of antibodies and responding animals.
Although 100% of control (ummmunized) mice died
following the rabies virus challenge, all animals that had developed even a minimal detectable antibody response following vaccine administration showed no sign of rabies infection during a 21 day observation period. Three animals that had been given lower doses of Ad5GRILP virus, and in which no neutralizing antibodies were detected, also survived the rabies challenge, suggesting that sub-detectable levels of antibodies provide
protection or that the protection has been caused by stimulation of some other defense mechanιsm(s). This protect ive st imulat ion of the immune systems by Ad5GRILP virus was rabies-specific since the group of 15 animals, inoculated with adenovirus type 5 alone, remained
susceptible to the challenge with rabies virus two weeks later (data not shown).
The vaccine of this invention has proven effective in oral administration to skunks and foxes in inducing high levels of neutralizing antibody and in protecting the animals from death after challenge with lethal doses of rabies virus.
Deposits
A sample of recombinant Ad5GRILP virus was deposited on March 4, 1988, under the Budapest Treaty on the
International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure in American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, U.S.A. under ATCC accession number VR2204 issued on March 25, 1988.

Claims

1. A recombinant adenovirus DNA sequence comprising an inserted DNA sequence which codes for a lyssavirus antigen.
2. A recombinant adenovirus DNA sequence according to claim 1 in which the adenovirus DNA sequence is derived from a human adenovirus.
3. A recombinant adenovirus DNA sequence according to claim 2 in which the human adenovirus is human adenovirus type 5.
4. A recombinant adenovirus DNA sequence according to any preceding claim in which expression of the inserted DNA sequence is controlled by an adenovirus promoter.
5. A recombinant adenovirus DNA sequence according to claim 4 in which the adenovirus promoter is the E3 promoter or major late promoter of human adenovirus.
6. A recombinant adenovirus DNA sequence according to any preceding claim in which the inserted DNA sequence replaces an adenovirus DNA sequence.
7. A recombinant adenovirus DNA sequence according to claim 6 in which the inserted DNA sequence replaces an early adenovirus DNA sequence.
8. A recombinant adenovirus DNA sequence according to claim 7 in which the inserted DNA sequence replaces at least a portion of the E3 gene.
9. A recombinant adenovirus DNA sequence according to any preceding claim in which the inserted DNA is
contained within a cassette of DNA from another virus.
10. A recombinant adenovirus DNA sequence according to claim 9 in which the other virus is a Simian virus.
11. A recombinant adenovirus DNA sequence according to claim 10 in which the Simian virus is SV40.
12. A recombinant adenovirus DNA sequence according to claim 11 in which the inserted DNA is introduced between the promoter and poly-A addition site of the early region of SV40.
13. A recombinant virus comprising a recombinant
adenovirus DNA sequence according to any preceding claim.
14. A vaccine comprising an effective amount of a recombinant virus as defined in claim 13 and a
pharmaceutically-acceptable excipient.
15. A method of immunising a mammal against a iyssavirus-induced disease comprising administering a virus or vaccine according to claim 13 or 14 to the mammal.
16. A method according to claim 15 in which the virus or vaccine is administered by the oral or nasal route.
17. A method of preparing a recombinant adenovirus DNA sequence comprising inserting a DNA sequence coding for a lyssavirus antigen into an adenovirus DNA sequence.
18. A method of preparing a vaccine comprising mixing a recombinant virus comprising a recombinant adenovirus DNA sequence with a pharmaceutically- acceptable carrier.
PCT/GB1990/001217 1989-08-22 1990-08-03 Recombinant adenovirus sequence expressing rhabdovirus antigen and use as a vaccine WO1991002804A1 (en)

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GB8919102.7 1989-08-22
GB898919102A GB8919102D0 (en) 1989-08-22 1989-08-22 A recombinant adenovirus dna sequence,a recombinant adenovirus expressing the dna sequence and a rhabdovirus vaccine including the recombinant adenovirus

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EP3307313A1 (en) * 2015-06-12 2018-04-18 GlaxoSmithKline Biologicals SA Adenovirus polynucleotides and polypeptides

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Title
Technological Advances in Vaccine Development, 1988, Alan R. Liss, Inc., F.L. GRAHAM et al.: "Cloning and Expression of Glycoprotein Genes in Human Adenovirus Vectors1", pages 243-250 see the whole article, especially the Abstract *
The Journal of Infectious Diseases, Volume 161, No. 1, 1 January 1990, The University of Chicago Press, L. PREVEC et al.: "A Recombinant Human Adenovirus Vaccine against Rabies", pages 27-30 see the whole article *

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EP0586076A2 (en) * 1992-08-07 1994-03-09 American Home Products Corporation Recombinant adenovirus vaccines
EP0586076A3 (en) * 1992-08-07 1994-04-20 American Home Products Corporation Recombinant adenovirus vaccines
AU680826B2 (en) * 1992-08-07 1997-08-14 Wyeth Recombinant adenovirus vaccines
KR100347219B1 (en) * 1992-08-07 2003-02-25 와이어쓰 Recombinant Adenovirus Vaccine
WO1997012981A1 (en) * 1995-10-05 1997-04-10 Microbix Biosystems Inc. Rabies recombinant adenovirus
US7037716B2 (en) * 1998-04-24 2006-05-02 Crucell Holland B.V. Packaging systems for human recombinant adenovirus to be used in gene therapy
WO2006002594A1 (en) * 2004-07-07 2006-01-12 Institute Of Military Veterinary, Academy Of Military Medical Sciences, Pla A recombinant canine adenovirus type-2 and the preparation method and usage thereof

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