Source: http://www.google.de/patents/US5643579
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Patent US5643579 - Oral vaccines - Google PatenteSuche Bilder Maps Play YouTube News Gmail Drive Mehr » Erweiterte Patentsuche | Webprotokoll | Anmelden Erweiterte Patentsuche PatenteMethods and vaccines for the production of antibodies to infectious organisms are described. Live recombinant adenovirus containing a foreign gene coding for an antigen produced by another infectious organism is delivered to the intestine of a warm-blooded animal in an enteric-coated dosage form, whereupon...http://www.google.de/patents/US5643579?utm_source=gb-gplus-sharePatent US5643579 - Oral vaccines Ver�ffentlichungsnummerUS5643579 APublikationstypErteilung Anmeldenummer08/283,231 Ver�ffentlichungsdatum1. Juli 1997Eingetragen29. Juli 1994 Priorit�tsdatum1. Nov. 1984 ErfinderAlan R. DavisPaul P. HungUrspr�nglich Bevollm�chtigterAmerican Home Products CorporationWyeth US-Klassifikation424/227.1424/218.1424/233.1424/189.1424/205.1435/69.1Internationale KlassifikationA61K39/00C07K14/14C07K14/02C07K14/16 UnternehmensklassifikationC07K14/005C12N2720/12322C12N2740/16222A61K39/00C12N2730/10122 Europ�ische KlassifikationC07K 14/005ReferenzenPatentzitate (1)Nichtpatentzitate (25) Referenziert von (18)Externe LinksUSPTO USPTO-Zuordnung EspacenetOral vaccinesUS 5643579 A Zusammenfassung Methods and vaccines for the production of antibodies to infectious organisms are described. Live recombinant adenovirus containing a foreign gene coding for an antigen produced by another infectious organism is delivered to the intestine of a warm-blooded animal in an enteric-coated dosage form, whereupon the virus infects the gut wall and induces the production of antibodies or cell mediated immunity to both adenovirus and the other infectious organism.
What is claimed is: 1. A method for producing antibodies or cell mediated immunity to Hepatitis-B virus in a warm-blooded animal which comprises orally administering to said warm-blooded animal, in an enteric coated dosage form, live recombinant adenoviruses in which the virion structural protein is unchanged from that in the native adenovirus from which the recombinant adenovirus is produced, and which contain the gene coding for the hepatitis-B surface antigen which corresponds to said antibodies or induces said cell mediated immunity.
BACKGROUND OF THE INVENTION A major goal of biomedical research is to provide protection against viral disease through immunization. One approach has been to use killed vaccines. However, large quantities of material are required for killed vaccine in order to retain sufficient antigenic mass. In addition, killed vaccines are often contaminated with undesirable products during their preparation. Heterologous live vaccines, using appropriately engineered adenovirus, which is itself a vaccine, Chanock R. M. et al., JAMA, 195, 151 (1967), seem an excellent immunogen. Our invention concerns live oral vaccines using adenovirus as vector.
Adenoviruses contain a linear duplex DNA molecule (m.w. 20 these are incorporated into the viral particle which is morphologically complex and has a sophisticated assembly process. Previously SV40 T antigen has been expressed using an adenovirus recombinant (Solnick, D. Cell, 24, 135 (1981), Thummel, C. et at., Cell, 23, 825 (1981), Gluzman, Y. et al., in Eukaryotic viral Vectors, p. 187, Cold Spring Harbor (1982)). Also mouse dihydrofolate reductase has been expressed using an adenovirus recombinant (Berkner, K. and Sharp, P. A., Nucleic Acids Research, 12, 1925 (1984)).
DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1 Adenovirus Vectors Three adenovirus vectors (Gluzman, Y. et al., in Eukaryotic Viral Vectors p. 187, Cold Spring Harbor Laboratories, 1982) can easily be constructed. To maximize the length of foreign DNA that can be inserted, two expendable regions of the viral genome may be deleted, early regions 1 or early region 3 (E1 and E3), or both, of the adenovirus type 5 viral genome. ΔE1 is created by an in vivo recombinational event between a plasmid and a modified adenoviral DNA. (All plasmids described in this specification are propagated in E. coli). The plasmid is formed by insertion of adenoviral DNA sequences between 0 and 17 map units into pBR322 and subsequently, using restriction endonuclease digestion and ligation, deleting sequence between 1.4 and 9.1 map units and placing an Xbal restriction site at this junction. This plasmid is denoted in the art as pAC. The modified adenoviral DNA which contains a single Xbal restriction site at 4.0 map coordinates is formed as follows. Xbal cleaves wild type Ad5 DNA at four sites: 4, 29, 79, and 85 map. units. Modified DNA lacking sites at 29, 79, and 85 is isolated by cutting Ad5 DNA with Xbal, transfecting the DNA and isolating the modified adenovirus which lacks Xbal sites at positions 29 and/or 79. This procedure is repeated again and modified adenovirus is isolated containing only the Xbal site at position 4. Such modified adenoviruses can also be readily constructed using techniques of oligonucleotide-directed mutagenesis (Smith, M., and Gillam, S. (1981) in Genetic Engineering, Setlow, J. K. and Hollaender, A., Eds. Vol. 3, pp. 1-32, Plenum, New York). In this technique the Xbal restriction sites are destroyed using chemically synthesized oligonucleotides designed to produce silent changes in the amino acid coding regions defined by the respective Xbal restriction endonuclease sites. Vector ΔE3 is constructed by deletion of ΔE3 region sequences. Two modified adenoviruses (type 5) are formed by the procedures outlined above. One contains no Xbal sites, the other contains only the Xbal sites at map coordinates 29, 79, and 85. The left half of DNA of the mutant containing no Xbal sites is joined with the right half of DNA of the mutant containing Xbal sites at 79 and 85, forming a modified adenovirus containing Xbal sites at only 79 and 85 map coordinates. Cleavage of this DNA with Xbal followed by religation forms the ΔE3 viral DNA deleting the ΔE3 region between 79 and 85 map coordinates and placing a single Xbal cloning site at this junction. ΔE1 ΔE3 may be constructed by deletions in both regions in a similar manner.
EXAMPLE 2 6X Series Plasmids (For Hb.sub.S A.sub.g and rotavirus VP7) Plasmids that allow the placement of the adenovirus 2 late promoter upstream from DNA coding for hepatitis B surface antigen or rotavirus VP7 followed by SV40 splicing signals may be constructed. Each of these is flanked by Xbal sites for insertion into the adenovirus ΔE1, ΔE3, or ΔE1ΔE3 vectors.
Either mouse monoclonal antibodies or rabbit antisera are used to detect expression of recombinant ΔE1 and ΔE3 virus stocks containing HB.sub.S A.sub.g or VP7 DNA sequences. Counterstaining is with goat anti-mouse or anti-rabbit FITC.
EXAMPLE 4 Immunogenic Nature of the Recombinant Adenovirus Live, lyophilized recombinant adenovirus contained in an enteric coated capsule is assessed for immunogenicity by administration (104-105 50% infectious dose/tablet) to hamsters or chimpanzees and testing for antibody levels and protection from challenge.
EXAMPLE 5 Detailed example of a recombinant that expresses authentic HB.sub.S A.sub.g As a detailed example of the construction of one adenovirus recombinant, the HB.sub.S A.sub.g gene of the adw subtype from 26 bp upstream of the HB.sub.S A.sub.g translation initiation codon and 131 bp downstream from the translation termination codon was flanked by upstream sequences from the Ad2 major late promoter (+33 to 400 bp; Solnick, D., Cell, 24, 135-143 (1981) and by downstream sequences from SV40 (2753 to 2516 bp; Tooze, J. (Ed.) Molecular Biology of Tumor Viruses, Cold Spring Harbor Laboratory pp. 801-829 (1980)). This plasmid is termed p6XH (see above). These sequences were inserted into the unique Xbal site plasmid pAC that contains an insert of Ad5 DNA extending from the Eco RI linker at the left end of the adenovirus genome to the Hind III site at Ad5 map coordinate 17.0 (Gluzman, Y., Reichl, H., and Solnick, D., 1982, in (Y. Gluzman, Ed.) Eukaryotic Viral Vectors, Cold Spring Harbor Laboratories, p. 187-192). The new plasmids (pACH-2 and pACH-9) with the cassette containing the Ad2 major late promoter--HB.sub.S A.sub.g gene--SV40 processing signals in either orientation, were cleaved with Hind III. The Hind III cleavage product of each was combined with the large Xbal fragment of the adenovirus mutant ΔEl extending from map coordinates 9.1 to 100 (Gluzman, Y., Reichl, H., and Solnick, D., 1982 in (Y. Gluzman, ed.) Eukaryotic Viral Vectors, Cold Spring Harbor Laboratories, pp. 187-192). This DNA mixture was transfected (Graham, F. L. and ven der Eb, A. J. Virology 52, 456-467 (1973)) onto 293 cells (Graham, F. L., Smiley, J., Russell, W. C., and Nairn, R., J. Gen. Virol., 36, 59-72 (1977)) and cells were overlaid with agar for plaque detection. Approximately 10-14 days later, 54 plaques were picked and virus stocks generated from each. These viruses were screened for the presence of HB.sub.S A.sub.g DNA by hybridization to a 32P-labeled HB.sub.S A.sub.g DNA probe. Positive plaques (49 out of 54) were next infected onto monolayers of 293 cells and the expression of authentic HB.sub.S A.sub.g was detected in cell lysates by both radioimmunoassay (AUSRIA, Abbott Laboratories, Inc. or NML-HB.sub.S A.sub.g RIA, Nuclear-Medical Laboratories) and by immunoprecipitation of 35S-radiolabeled HB.sub.S A.sub.g using a monoclonal antibody to HB.sub.S A.sub.g (anti-a subtype, Boehringer Mannheim Biochemicals).
When these stock viruses (HM1 and HM2) were infected on a human embryonic kidney (293) cell line (Graham, F. L., Smiley, J., Russell, W. C. and Nairn, R. (1977) J. Gen. Virol. 36, 59-72) we found, after 40 h infection, approximately 1 μg HB.sub.S A.sub.g (based upon radioimmunoassay and comparison of cpm to NML-HB.sub.S A.sub.g kit positive control) per 5 infected cells. HM1 virus yielded approximately 60% of this amount. We found that the HB.sub.S A.sub.g polypeptides produced by these viruses were glycosylated (P2) and non-glycosylated (P1) forms (Marion, P. L., Salazar, F. H., Alexander, J. J. and Robinson, W. (1979) J. Virol. 32, 796-802; Peterson D. L. (1981) J. Biol. Chem. 256, 6975-6983). At 40 h after infection most of the HB.sub.S A.sub.g (78%) was secreted from cells into the culture medium as a particle (density=1.20 g/ml) the same or nearly the same as the 22 nm particle (Gerin, J. L., Purcell, R. H., Hoggan, M. D., Holland, P. V. and Chanock, R. M. (1969) J. Virol. 4, 763-768; Gerin, J. L., Holland, P. V., and Purcell, R. H. (1971) J. Virol. 7, 569-576) observed in human serum. HM2 yielded approximately 40% more HB.sub.S A.sub.g than HM1. However, when HM2 was compared to the previously described hybrid adenovirus, ΔE1H, a 70-fold increase in HB.sub.S A.sub.g polypeptide was noted by HM2 virus.
EXAMPLE 7 Recombinant Adenovirus Ad7HZ2-28 Ad7HZ2-28 was isolated after transfection of A549 cells with two overlapping Ad7 DNA fragments that recombined in transfected cells and produced a complete, infectious recombinant adenovirus. The two DNA fragments contributing to the recombination were (1) the EcoRI A fragment derived from genomic DNA map units 0 to 87; and (2) a cloned DNA fragment that extends from map units 68 to 100 and contains the hepatitis B virus surface antigen gene. The construction of the plasmid, pWyAd7E3HSB-C, for the second fragment is described below using standard techniques in molecular biology as summarized in (molecular cloning: A Laboratory Manual, Maniatis, T., et at. (1982) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
EXAMPLE 8 Recombinant Adenovirus Ad4HHXHS Example of a recombinant adenovirus type 4, Ad4HH the hepatitis B virus surface antigen gene with a synthetic splice acceptor sequence, which was inserted into the E3 region of adenovirus 4.
The recombinant adenovirus, Ad4HH surface antigen gene, 12 bp Ad5 hexon splice acceptor and the hepatitis B surface antigen gene inserted into the HpaI site of the E3 region of Ad4. The construction of Ad4HH
The recombinant adenovirus, Ad4E3HH cotransfection of 10 μg pAd4HpSpHS and 5 μg Bc1A fragment of Ad4 (0-87 mu) into A549 cells as described (Berkner and Sharp, [1983], Nucl. Acids Res. 11, 6003). Plaques were picked into 2 ml media, and 1 ml was amplified on A549 cells. The supernatants were screened for the secretion of HBV surface antigen by a radioimmunoassay (Organon Technika Corp., Irving, Tex.). The recombinant adenovirus, Ad4HH significant quantities of Hepatitis B surface antigen. Ad4HH been deposited with the American Type Culture Collection and has been designated ATCC VR 2210.
EXAMPLE 9 Recombinant Adenoviruses WyAd7H 6 and WyAd7H 7
EXAMPLE 10 Recombinant Adenoviruses WyAd7ChH1-16 and WyAd7ChH2-8 A different expression cassette was developed by Chiron and was introduced into the plasmid which- contained the adenovirus type 7 EcoRI B fragment with a unique XbaI site at 159 bp from the right hand end of the genome. Adenovirus DNA (from plasmid WyAdR1A-17) fragment XhoI (nucleotide 5643) to the HindIII site was cloned into pBR327. The tripartite leader was designed based on the published adenovirus type 7 sequence from the PvuII site (nucleotide 5934) within the first leader segment through the end of the first segment (nucleotide 5944), containing the second leader segment (nucleotide 6962-7033), and the third leader segment (nucleotide 9477-9563) and ending with a Hind III site. The poly A site for the hexon gene of adenovirus type 7, the major late promoter and the tripartite leader were cloned into the plasmid which contained viral DNA with a unique XbaI site near the inverted terminal repeat. The DNA constructions contained the promoter oriented in either the left hand orientation or the right hand orientation. The HBSAg gene was obtained from the pHMH5.3C plasmid as described in Example 9 and was cloned into the plasmid listed above.
EXAMPLE 11 Recombinant Adenovirus WyAd7IHH-I The intron between the first and second part of the tripartite leader sequence was isolated from the adenovirus type 7 genome by digesting the adenovirus DNA with XhoI and purifying the fragment from agarose gels (5,643 to 8,168 base pairs on the adenovirus genome). The fragment was inserted by digestion with XbaI and ligation into a cassette which contained the major late promoter, the tripartite leader sequence and the SV40 poly A site. The leader sequence was joined by digestion with ScaI and ligation. The intron was further modified by treatment with BssHII which removed a fragment that corresponded to between 6,178 and 6,517 base pairs on the adenovirus genome DNA. The entire cassette was inserted into the XbaI site that had been constructed at 159 base pairs from the right-hand end of the adenovirus genome. The major HB.sub.S A.sub.g gene was obtained from pHMHS.3C as described in Example 9 and inserted into the SalI site. The hexon polyA site was substituted for the SV40 polyA site by digesting the plasmids with SpeI and exchanging fragments between pAd7ChHI-1 and pAd7ChHA-6 (left-hand orientation) as well as pAd7IChH3-10 and pAd7ChHB-31 (right-hand orientation). The SpeI sites were found near the right-hand end of the adenovirus genome and in the HB.sub.S A.sub.g gene. The plasmids were also treated with StuI and HindIII linkers were added to the blunt ends by ligation. After digestion with HindIII another fragment was removed from the intron which corresponded to between 6,135 and 6,819 base pairs of the adenovirus genome.
EXAMPLE 12 Recombinant Adenovirus Ad4iHR Recombinant virus Ad4iHR has a cassette for production of HB.sub.S A.sub.g positioned at a SalI site found in Ad4 138 bp from the extreme right-hand terminus. The cassette contains (1) the Ad4 major late promoter (MLP), (2) followed by the first leader of the Ad4 tripartite leader CFPL), (3) followed by the first intron of the tripartite leader (TL), followed by (4) the second two exons of the Ad4 TPL, followed by (5) the HB.sub.S A.sub.g gene, followed by (6) a processing and polyadenylation signal from SV40 virus. It was prepared and positioned in the rightward orientation at the unique SalI site as follows: Ad4 is known to have an XhoI site at 15.9 map units (Ginsberg, E. [ed], 1984, The Adenoviruses, Plenum Press, N.Y.), and it was determined by restriction enzyme mapping to have a ScaI site at 19.7 map units. DNA fragment 1 (approx. 1300 bp) from XhoI to ScaI contains the Ad4 major late promoter, the first leader of the TPL and the entire intron between the first and second exon of this TPL and one-half of the second exon.
DNA fragment 2 from this ScaI to a TaqI site 100 bp downstream of this ScaI site in Ad4 was obtained from a cDNA clone of the Ad4 TPL and was determined by DNA sequencing to contain the second-half of the second exon and two-thirds of the third exon for the TPL. This cDNA clone was obtained by standard methods (Maniatis, T. et al [1985], Molecular Cloning: A Laboratory, Cold Spring Harbor, N.Y.) with the use of the oligonucleotide 5'TCTFCAAGGGGGAACCCG3' as probe. Use of the oligonucleotide was based upon the published sequence in this region (exon 2 of the TPL in Ad7 DNA found in (Ginsberg et al. 1984 ibid)). In DNA fragment 2, this TagI site was converted to a SalI site by treatment with Klenow DNA polymerase 1 and ligation of a SalI linker. DNA fragment 3, the HB.sub.S A.sub.g gene, was obtained from pHMHS.3C by SalI digestion inserted into the SalI site of pBR328 (Soberon, X., et at. [1980], Gene 9, 287-305) to create ChpAd7SalBHΔ.
Hepatitis B virus DNA was obtained from a plasmid that contains the hepatitis B virus genome cloned at the Eco RI site. The numbering of the nucleotides in this hepatitis B virus genome is identical to that of Ono, et al [1983] Nucleic Acids Res. �, 1747-1757. The fragment of the hepatitis B virus genome that contains the genetic code for hepatitis B virus major surface antigen, the major envelope protein, lies between an FnuD II site at nucleotide 131 and a Hpa I site at nucleotide 966. This DNA fragment was excised by digestion with FnuD II and Hpa I, Sal I linkers were added, and it was cloned into a pBR322 derivative to create the plasmid, pHMHS.3C. DNA fragment 4 was the SV40 processing and polyadenylation signal, extending from 2753-2516 bp on the SV40 map (Tooze, I. [ed]), 1980 DNA Tumor Viruses, Cold Spring Harbor Laboratory, N.Y.) with a SalI site at 2,753 bp and a XhoI site at 2,516 pb.
EXAMPLE 13 Recombinant Adenoviruses Ad4(di)HR and Ad4(di)HL Recombinant virus Ad4diHL and Ad4diHR were made in the same fashion as Ad4iHR except that the 1,000 bp intron between TPL exon 1 and TPL exon 2 was trimmed by deleting 500 bp from a StyI site approx. 500 bp from the 5' XhoI site to a SacII site approx. 1,000 bp from the 5' XhoI site. In addition, both orientations of this DNA fragment (rightward and leftward) were used. When Ad549 cells were infected with suspensions of Ad4(di)HR and Ad4(di)HL, HB.sub.S A.sub.g activity was demonstrated in both cases.
EXAMPLE 14 Recombinant Adenoviruses Ad4XiHR, Ad4X(di)HR Recombinant viruses Ad4XiHR and Ad4X(di)HR were made in exactly the same fashion as Ad4iHR and Ad4(di)HR except that a synthetic 112 bp Ad4 hexon processing and polyadenylation signal on a SalI to XhoI DNA fragment replaced the SV40 polyadenylation signal. The DNA sequence used for this signal was determined by DNA sequence analysis of the published sequence of the Ad2 hexon polyA site (LeMoullec, J. M. et al [1983], J. Virol. 48: 127) and DNA sequence analysis of the corresponding region in Ad4. By comparing the two sequences, one was able to determine which elements appear to be necessary for function. When A549 cells were infected with a plaque suspension of Ad4XiHR and Ad4X(di)HR, HB.sub.S A.sub.g activity was demonstrated in both instances.
EXAMPLE 15 Recombinant Antivirus WyAd7LAV# 17 This is an example of a recombinant adenovirus type 7, WyAd7LAV#17, that contains lymphadenopathy associated virus DNA within an expression cassette inserted between the E4 region and the right ITR of the adenovirus genome and that replicates in human cells, directing the expression of LAV envelope protein.
EXAMPLE 16 Recombinant Adenoviruses AD5 HBsAg78.5 and Ad5 HBsAG E3 Adenovirus type S (Ad5) (ATCC VR-5) was cloned into the Bam HI site of the bacterial plasmid pBR322 (Pharmacia Inc., catalog 27-4902-01) as described (Berkner, K. L., and Sharp, P. A., Nucleic Acids Res. 11, 6003 [1983]) except that Bam HI linkers (GGGATCCC, Pharmacia lnc.) were used instead of Eco Rl linkers. The resulting plasmid, p60W-43, was modified by excision of the Xba 1 fragment between adenovirus map unit (MU) 78.5 and MU 84.7 from p60W-43 grown in a Dem E. coli strain (NEB208, available from New England Biolabs). The Xba 1 fragment was separated from the modified plasmid, p60W E3 by electrophoresis in a low melting agarose gel and the modified plasmid was recovered, ligated, and propagated in E. coli (general techniques of the art are described in Maniatis, T., Fritsch, E. F., Sambrook, J., [1982J Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). These recombinant adenovirus plasmids, p60W-43 and p60W E3-6, are prepared for insertion of heterologous DNA by digesting plasmid DNA grown in a Dam.sup.+ strain of E. coli (New England Biolabs) with Xba 1 and treating the digested DNA with alkaline phosphatase.
EXAMPLE 17 Recombinant Adenovirus WyAd7IHH-3 Adenovirus vector was further modified by the addition of an EcoRI site at 80 map units. This site was produced by digesting with Stul and the ligation of EcoRI linkers. After digesting with EcoRI and religation, a fragment of adenovirus DNA was deleted which was from 84 to 87 map units.
EXAMPLE 18 Recombinant Adenoviruses WyAd7IHENV-4, 11, and 42 Constructs which contained the envelope gene from the AIDS virus were made by removing the HBsAg gene from other cassettes (pAd7 HEH-3, pAd7HRHL-5 and pAd7IHH-11) by digestion with SalI and ligation with the LAV-ENV gene which had been purified from pUCLenv.8. The envelope gene was the same as was used in the preparation of WyAd7LAV#17.
EXAMPLE 19 Recombinant Adenovirus WyAd7IHART-4 WyAd7IHART-4 was isolated by transfecting A549 cells with two overlapping DNA fragments that recombined in vivo to generate a complete recombinant vital genome capable of replicating and producing infectious recombinant adenovirus (Davis, A. R. et al. [1985] Proc. Natl. Acad. Sci. 82, 7560-7564). The two DNA fragments used for recombination were (1) the EcoRI "A" fragment derived from adenovirus type 7 genomic DNA that extends from map units (m.u.) 0 to 87 and (2) a cloned Ad7 DNA fragment that extends from m.u. 70 to 100 and contains REV(ART/TRS) gene. The construction of the plasmid, WyAd7 BH1-11.ART-4 for the second fragment is described below using standard techniques in molecular biology as summarized in (molecular cloning: A Laboratory Manual, Maniaties, T., et al. (1982) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
EXAMPLE 20 Recombinant Adenovirus WyAd71HART-5,ENV-42 Recombinant adenovirus type 7, WyAd71HART-5.ENV-42 that contains REV(ART/TRS) gene in the deleted (80-87 m.u.) E3 region and ENV gene in the cassette that was inserted between the E4 and ITR region was isolated by transfecting A549 cells with two overlapping DNA fragments that recombined in vivo to generate a complete recombinant viral genome capable of replicating and producing infectious recombinant adenovirus. The two DNA fragments used for recombination were (1) The EcoRI "A" fragment derived from adenovirus type 7 genomic DNA that extends from map units (m.u.) 0 to 87 and (2) a cloned DNA fragment that extends from 70 to 100 m.u. which contains both REV and ENV gene. The construction of the plasmid, WyAd71HART-5.ENV-42 is described below. The plasmid pAd7 HEH-3 which contains HBsAg in the terminal cassette was digested with SalI to remove HBsAg and then ligated to (SalI-Xhol) fragment obtained from pUCLenv.8 which contained the HIV ENV gene to generate the plasmid, WyAd71HENV-42. The plasmid pAd7 HEH-3 contains 80-87 m.u. E3 deletion with a EcoRI site in it. The plasmid, WyAd71HENV.42 was then digested with EcoRI and then ligated to EcoRI fragments of ART gene which was derived from plasmid, pTZ18RART4.R1 which contains the ART gene flanked by EcoRI restriction sites.
EXAMPLE 21 Recombinant Adenovirus WyAd7ΔE3 r80-88)TPL-S-35: (WyAd7delE3H) This is an example of a recombinant adenovirus type 7, that contains synthetic WyAd7 cDNA copy of TPL and HBsAg DNA inserted within endogenous E3 mRNAs having a large deletion and produces high level of HBsAg protein mediated by the internal TPL sequences positioned immediately upstream to HBsAg gene. The E3 region protein coding sequences downstream to the HBsAg insertion site has been completely deleted in this recombinant. The recombinant virus isolation was carried out by recombining in vivo two overlapping WyAd7 DNA fragments transfected into A549 cells (Berkner, K. L. and Sharp, P., Nucleic Acids Res. 11:6003, 1983; and Davis, A. R., et at., Proc. Natl. Acad. Sci. USA, 82:7560, 1985). One of the DNA fragments was purified from WyAd7 vaccine strain (55142) as EcoRI A fragment spanning map units 0-87 portion of the viral genome. The second fragment was ClaI digested recombinant plasmid pAd7 SalIB (ΔE3 80-88) TPL-S-35. This contains a DNA segment of synthetic WyAd7 TPL appended to HBsAg gene and WyAd7 DNA between m.u. 68 to 100 as SalIB fragment, but without most of the E3 region sequences between m.u. 80-88. The construction of this recombinant plasmid involved the standard molecular cloning techniques (Molecular Cloning: A Laboratory Manual, Eds. Maniatis, T., et al. [1982] Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and the details are as follows:
(b) Generation of a plasmid with large deletion (m.u. 80-88) in E3 Region of WyAd7: From a pBR328 vector plasmid that contained WyAd7 SalI B DNA (m.u. 68-m.u. 100) fragment with the 80-84 m.u. deletion in E3 region a derivative was constructed. This was called pAd7SalIB (68-100)ΔE3 (80-84)-12, in which the HindIII, BamHI, NheI sites of pBR328 were deleted. Then the Ad7 E3 region sequence between the StuI site present in a linker at 80 m.u. and the ApaLI site (located upstream to Ad7 E3B polyadenylation signal) at �88 m.u. was deleted to yield pAd7SalIB (68-100)ΔE3 (80-88)-78.
EXAMPLE 22 Recombinant Adenovirus WyAd4(3.11)ΔE3TPLH (WyAd4delE3H) The recombinant virus construction strategy is similar to that in example 21, except that the WyAd4 synthetic TPL was appended to HBsAg gene and placed within a 3.11 kilo base pair deletion in E3 region of Ad4 genome. The steps involved in the construction are as follows:
EXAMPLE 23 Recombinant Adenovirus WyAd7H Ad7-HBsAg recombinant with complete E3 region: The recombinant contains the HBsAg and regulatory sequences inserted at the 3' end non-coding E3B region and retains all the E3 protein coding region as they are in vaccine strain WyAd7. The construction of this recombinant involved the following steps:
(a) Creating a new site of insertion at 3' non-coding part of E3B region: A synthetic SpeI linder/adapter was inserted at the BspMI site located between Ad7E3 region last open-reading frame stop codon and the E3B polyadenylation signal sequence in a subclone of E3b/L5 region and called pGEM3Zf(+) (Ad7E3BSpeI). The sequence of Ad7E3B region it contains is between SacI (�88 m.u.) and HpaI (�90 m.u.) that was subcloned into pGEM3Zf(+) vector from a plasmid containing m.u. 60-m.u. 100 of WyAd7DNA. The SpeI linker/adapter also provides stop condons in all the three reading frames and duplicates 10 bp of E3B sequences on either side of SpeI site to retain possible E3B polyadenylation functions.
(b) Source of HBsAg sequence and the regulatory elements as a cassette: The cassette containing-XbaI site, BamHI site, synthetic Ad7 hexon related splice acceptor, hexon leader, HBsAg coding sequence, SalI site, Ad7 hexon polyadenylation signal sequence and XbaI site was isolated as a DNA fragment with XbaI sites at both of the ends from a parent plasmid, pAd7SHLHPAΔX-7. The internal XbaI site within the HBsAg coding sequence was removed by site-directed mutagenesis of plasmid pAd7SAH The pAd7SAH contain synthetic Ad7 hexon like splice acceptor, hexon leader, HBsAg coding region and Ad7 synthetic hexon polyadenylation signal sequences, that were obtained from pAd7SAH these plasmids the HBsAg (adw) coding sequence was originated from pAd7TP-HS-11, wherein a SalI site was added immediately downstream to stop codon of HBsAg open reading frame. The prior source of adw type HBsAg sequence and synthetic Ad7 hexon polyadenylation signal sequences were from pAd7HEH18R-153-4, while it was a derivative of pAd7NCTPLH 18R-7.
(c) Insertion of HBsAg sequence as a cassette into the new site at 3' non-coding part of E3B region: The cassette containing Ad7 hexon related splice acceptor, hexon leader, HBsAg and hexon polyadenylation signal sequence with XbaI sites at both of the ends was isolated from the plasmid pAd7SHLHPAΔX-7 (described above, Section -b). It was cloned into the synthetic SpeI site of pGEM3Zf(+) (Ad7E3BSpeI) and yielded the plasmid p3Zf(+) (Ad7E3B-xsa-xl-S-xpa)T3. From this plasmid, the SaclHincII DNA fragment containing E3B region (�88 and �90 m.u.) and the HBsAg cassette sequences was cloned into SacI-HpaI sites (�88 and �90 m.u.) of a plasmid[Ad7(84-100)]5a6, that contained Ad7 genome between map units 84 and 100. The resulting recombinant plasmid was called p18R [Ad7(84100)E3B-S]-G14. The pUC18R[Ad7(84-100)]5a6 was a derivative of pAd7SalIB(68-100) clone, that contains WyAd7 genome from 68 m.u. to 100 m.u. in pUC18R vector.
EXAMPLE 24 Recombinant Adenovirus WyAd4H
(b) Construction of Ad4TPL-HBsAg cassette containing plasmid-pAdTPLHx-SHpA-10: The cassette containing NheI site, Ad4TPL, Ad4 hexon leader, SalI site, HBsAg, SalI site, XbaI site, Ad4 hexon polyadenylation signal and NheI site has been put together by several steps of subcloning into pTZ18RD vector. In summary (i) the NheI site, Ad4TPL, Ad4 hexon leader--were obtained by plasmids namely pAd4TPLA HBsAg-64 (derived from pAd4TPLH- 18RD-47, pAd4TPL- 18RD-38, and pAd7NH- 18R-2) and pAd4TPLHxL-5. (ii) The HBsAg sequence was obtained from pAdSTPLS-8 and pAd7TPL-HS-HpA-52 (derived from pAd7TPLHS-11 and pAd7HEH18R-153-4). The HBsAg sequence was edited such that SalI site is created immediately upstream to AUG start codon and another SalI site is brought immediately downstream to TAA stop codon of HBsAg. (iii) The Ad4 hexon polyadenylation signal was obtained from pAd4TPLH 15 (derivative of Ad4TPL-S-HpA-42, and pAd4TPLHpA-38).
(d) Generation of Ad4-TPL-HBsAg recombinant plasmid: The TPL-HBsAg cassette within Ad4E3B region fragment SacI and SphI was purified as BclI (87.1 m.u.)cassette-BclI (�87.4 m.u.)DNA fragment from the above plasmid p18R(Ad4E3B-TPL-xl-S-xpA)32812. This fragment was cloned into the BclI (�87.1 m.u./BclI (�87.4 m.u.) sites of large plasmid pAd4 (71-100) 25.4 that contained HindIII (m.u. �71) - EcoRI (m.u. 100) fragment of WyAd4 vaccine strain. The resulting recombinant large plasmid is called pAd4 (77-100) TPL-S-412G1.
EXAMPLE 25 Ad7-eny.sub.MN The construction of recombinant adenoviruses containing the coding sequence of the env (gp 160) gene of MN strain of HIV-1 is described briefly as follows: The 125 bp (6243 to 6367) fragment of the amino (NH.sub.2) terminus of the env (gp160) gene including the initiation codon (ATG) as well as consensus Kozak sequence was amplified by polymerase chain reaction (PCR) from the clone pMNST 1-8-9. This fragment was then cloned in pGEM vector and the resultant clone was designated as pGEMMNenv. The following fragments of DNA were isolated by digesting with the restriction enzymes KpnI and XbaI from the clone PAd5tpl.sub.MN env 223 (6367 bp to 8816 bp), XhoI+KpnI fragment from PGEMenv and salI+XbaI fragment from pAd7tpl 18RD. All of these fragments were ligated together and the resultant clone was designated as pAd7tpl.sub.MN env. This plasmid was then digested with XbaI and treated with calf intestine alkaline phosphatase (CIAP). The NheI+XbaI fragment of Hrev gene was then isolated from the plasmid, pAd7tplHrev 18RD. The clone that was obtained after ligating these two fragments together was designated as pAD7tpl.sub.MN envtplHrev. This plasmid was then digested with NheI+XbaI and then ligated to the E3 deletion plasmid of Ad7, pAd7ΔE3 (68 m.u. to 100 m.u. deletion) that was also digested with XbaI and then treated with CIAP. The resultant plasmid was designated as pAD7ΔE3tpl.sub.MN envtpl.sub.MN Hrev. This plasmid was digested with EcoRI and mixed with the EcoRI (0-87 m.u.) fragment of the Ad7 genomic DNA. A549 cells were then transfected with these DNAs. Recombinant plaques obtained from in vivo recombination were identified by the appropriate restriction digestion analyses of the Hirt DNA. The plaques were also identified by the production of gp160, gp120, and gp41 using appropriate antibodies on Western blots.
EXAMPLE 26 Ad4-env.sub.MN and Ad5-eny.sub.MN The construction of Ad4 and Ad5 recombinants are the same as that of Ad7-env.sub.MN except that for Ad4, EcoRI digested DNA from pAd4ΔE3tpl.sub.MN envtplHrev was combined with the BclI (0-87 m.u.) fragment from the Ad4 genomic DNA to produce the recombinant Ad4 virus. Similarly for Ad5, MluI-digested DNA from pAd5ΔE3tpl.sub.MN envtplHrev was combined with the SpeI (0-75 m.u.) fragment of Ad5 genomic DNA to produce the recombinant Ad5 adenovirus. Like Ad7, both Ad4 and Ad5 recombinants were obtained from A549 cells.
ADENOVIRUS REPLICATION AND ANTIGEN EXPRESSION ______________________________________Adenovirus   pfu/cell                     &#956;g env/10.sup.6 cells______________________________________Ad4 wild type        5.4         0Ad4-env      9.1         2.1Ad4-env.sub.MN        6.8         2.7Ad5 wild type        22          0Ad5-env      86          5.4Ad5-env.sub.MN        18          5.7Ad7 wild type        18          0Ad7-env      11          3.1Ad7-env.sub.MN        7.8         3.6______________________________________
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