US20020164353A1

US20020164353A1 - Replication-defective adenovirus human type 5 recombinant as a vaccine carrier

Info

Publication number: US20020164353A1
Application number: US09/897,170
Authority: US
Inventors: Hildegund Ertl; James Wilson
Original assignee: University of Pennsylvania Penn
Current assignee: University of Pennsylvania Penn; Wistar Institute of Anatomy and Biology
Priority date: 1995-06-05
Filing date: 2001-07-02
Publication date: 2002-11-07
Also published as: AU6261696A; US6019978A; US6287571B1

Abstract

A replication defective recombinant adenovirus is provided which contains a complete deletion of its E1 gene and at least a partial deletion of its E3 gene, said virus containing in the site of the E1 deletion a sequence comprising a non-adenovirus promoter directing the replication and expression of DNA encoding a heterologous protein from a disease-causing agent, which, when administered to a mammal in said recombinant virus, elicits a substantially complete protective immune response against the agent. Pharmaceutical and veterinary products containing the recombinant adenovirus are provided.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 09/347,060, filed Jul. 2, 1999, which is a continuation of U.S. patent application Ser. No. 08/973,233, filed Dec. 3, 1997, now U.S. Pat. No. 6,019,978, which is a 35 USC §371 application based on International Patent Application No. PCT/US96/09495, filed Jun. 5, 1996, which claims the benefit of priority from U.S. patent application Ser. No. 08/461,837, filed Jun. 5, 1995, now U.S. Pat. No. 5,698,202, and which claims the benefit of priority of U.S. Provisional Patent Application No. 60/000,078, filed Jun. 8, 1995, now abandoned.[0001]
[0002] This invention was supported by the National Institutes of Health Grant Nos. NIH AI 33683-02 and NIH AI 27435-05. The United States government has certain rights in this invention.

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

A replication competent, recombinant adenovirus (Ad) is an adenovirus with intact or functional essential genes, (i.e., E1a, E1b, 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. Pat. 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)). However, doses above 10⁶plaque-forming units (pfu) of this replication-competent virus were required to induce complete protection to viral challenge. Further, the use of these viruses in a live form capable of replicating in vivo is an undesirable attribute of a vaccine component.

In contrast, adenoviruses which have been made replication deficient by deletion of the Ad E1a and E1b genes have been used primarily for gene therapy protocols (See, e.g., Kozarsky and Wilson, Curr. Opin. Genet. Dev., 3:499-503 91993); Kozarsky et al, Som. Cell Mol. Genet., 19:449-458 (1993); see also, International Patent Application No. WO95/00655, published Jan. 5, 1995). Such recombinant, replication deficient adenoviruses have been found to induce cell-mediated immune responses (Y. Yang et al, Proc. Natl. Acad. Sci. USA, 91:4407 (1994) and Y. Yang et al, Immunity, 1:433-442 (August 1994)) and neutralizing antibodies (T. Smith et al, Gene Therapy, 5:397 (1993); K. Kozarsky et al, J. Biol. Chem., 269:13695 (1994)). None of these articles relating to the use of recombinant replication deficient Ad in gene therapy have measured the induction of a protective immune response.

Others have described the insertion of a foreign gene into a replication-defective adenovirus for putative use as a vaccine (See, e.g. T. Ragot et al, J. Gen. Virol., 74:501-507 (1993); M. Eliot et al, J. Gen. Virol., 71:2425-2431 (1990); and S. C. Jacobs et al, J. Virol., 66:2086-2095 (1992)). Jacobs et al, cited above, describes a recombinant E1-deleted, E3 intact, Ad containing encephalitis virus protein NS1 under the control of a heterologous cytomegalovirus (CMV) promoter. When mice were immunized with the recombinant Ad vaccines and challenged with virus, Jacobs et al obtained only partial protection (at most a 75% protection) for an average survival of 15 days. Eliot et al, cited above, describe a recombinant E1-deleted, partially E3-deleted Ad with pseudorabies glycoprotein 50 inserted into the E1 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). Ragot et al, cited above, describe a recombinant E1-deleted, partially E3-deleted Ad with Epstein Barr virus glycoprotein gp340/220 inserted into the E1 deletion site under the control of a homologous Ad promoter. In marmosets (tamarins) after three high dose (5×10⁹pfu, 1×10¹⁰pfu and 2×10¹⁰pfu), intramuscular immunizations and viral challenge, full protection was obtained.

For certain highly infectious diseases, such as rabies, there is a demand for an effective vaccine. Desirably, 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. Currently, a vaccinia rabies glycoprotein (VRG) vaccine is being used for oral wildlife immunization (B. Brochier et al, Vaccine, 12:1368-1371 (1994)). However, doses above 10⁶pfu are required to induce complete protection.

There thus remains a need in the art for a method of vaccinating against various disease states, and particularly rabies, which is safe and highly effective.

SUMMARY OF THE INVENTION

The inventors have surprisingly found 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. For example, 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 ⁴pfu of rabies vaccine vector to induce complete protection. This effect is also accomplished by administration routes other than the oral route.

Thus, in one aspect, 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. Further in the site of the E1a/E1b deletion which renders the Ad replication-defective, the recombinant virus contains a sequence comprising a non-adenovirus promoter directing the replication and expression of the DNA encoding the heterologous protein. For example, an exemplary rAd is Adrab.gp, which contains a rabies gp gene and is useful in a method for treating or preventing rabies.

In another aspect, the invention provides pharmaceutical and veterinary compositions which contain the rAd of the invention.

In still another aspect, the invention provides for the use of the rAd in the manufacture of the compositions described above.

In yet a further aspect, 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. Further in the site of the E1a/E1b deletion which renders the Ad replication-defective, the recombinant virus contains a sequence comprising a non-adenovirus promoter directing the replication and expression of the DNA encoding the heterologous protein.

In another aspect, 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.

Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the 1650 bp rabies glycoprotein gene from Evelyn Rockitniki Abelseth strain excised from the pSG5.ragp plasmid by cleavage with BglII. The 1650 bp sequence spans nucleotide 1178 to 2827 of SEQ ID NO: 1. [0017]
FIG. 1B is a schematic map of the pAd.CMVlacZ (also known as H5.020CMVlacZ) plasmid, which contains adenovirus map units (m.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. 9-16 represented by the long black bar at the lower lefthand portion of the circular plasmid 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. Restriction endonuclease enzymes are represented by conventional designations in this plasmid. NotI digestion removes the LacZ gene from this plasmid. [0018]
FIG. 1C is a schematic map of the plasmid pAdCMV.rabgp which results from blunt end cloning of the BglII fragment of pSG5.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 ([0019] 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. 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. 1D 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. 1 and 9, with the remaining Ad5 m.u. 9-100 having the above-mentioned E3 gene deletion. Restriction endonuclease enzymes are represented by conventional designations. [0020]
FIG. 2 is a bar graph plotting [0021] ³H-thymidine ((3H)TdR) incorporation, measured at counts per minute±standard deviation (cpm±SD), for irradiated splenocytes plated at 5×10⁵cells per well of a round bottom microtiter plate and incubated with 5 (diagonally striped), 1 (cross-hatched) or 0.2 (solid) μg/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. As described in Example 2B, after cloned T cells were added, cells were pulsed two days later for 6 hours with ³H-thymidine, harvested and counted in a β-counter.
FIG. 3A is a graph plotting % specific lysis (means of triplicates±SD) vs. effector:target cell ratio for groups of C3H/He mice inoculated with 2×10[0022] ⁶pfu of Adrab.gp (solid box) or H5.020CMVlacZ (open box), as described in Example 4B. Splenocytes were harvested 14 days later and co-cultured for 5 days with 1 pfu of Adrab.gp virus per cells. Activated lymphocytes were then tested at different E:T ratios on H-2 compatible L929 cells stably transfected with a rabies virus G protein-expressing vector (t.L929rab.gp) in a 4 hour ⁵¹Cr-release assay.
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 [0023] ⁵¹Cr-release assay, as a control.
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. 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. [0024]
FIG. 4B is a graph similar to FIG. 4A above, except the cells were harvested 36 hours after infection. [0025]
FIG. 4C is a graph similar to FIG. 4A above, except the cells were harvested 60 hours after infection. [0026]
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. [0027]
FIG. 4E is a graph similar to FIG. 4D above, except the cells were harvested 36 hours after infection. [0028]
FIG. 4F is a graph similar to FIG. 4D above, except the cells were harvested 60 hours after infection. [0029]
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. [0030]
FIG. 4H is a graph similar to FIG. 4G, except the cells were harvested 36 hours after infection. [0031]
FIG. 4I is a graph similar to FIG. 4G, except the cells were harvested 60 hours after infection. [0032]
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. [0033]
FIG. 4K is a graph similar to FIG. 4J above, except the cells were harvested 36 hours after infection. [0034]
FIG. 4L is a graph similar to FIG. 4J above, except the cells were harvested 60 hours after infection. [0035]
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 μg/mL of purified H5.020CMVlacZ virus. [0036]
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. [0037]
FIG. 6A is a graph plotting mean percentage (%) specific lysis of triplicates±SD vs. E:T cell ratio for C3H/He mice inoculated with 106 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). Mice were sacrificed 4 weeks later and upon restimulation with 1 pfu of Adrab.gp per cell tested on a 4 hour [0038] ⁵¹Cr-release assay on L929 cells stably transfected with pSG5rab.gp. See Example 6.
FIG. 6B is a graph similar to FIG. 6A, except the L929 cells were transfected with pSV2neo. [0039]
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.[0040]

DETAILED DESCRIPTION OF THE INVENTION

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. As discussed herein, the inventors have surprisingly found that use of such a recombinant Ad, described in detail below, provides substantially complete immune protection in vaccinates. [0041]
By “substantially complete” protection is meant when administered in an effective amount, 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. By “substantially all” is meant greater than 90% of the vaccinates. Unexpectedly, 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. For example, immunization of mice with a single dose of as little as 10[0042] ⁴pfu of the recombinant, replication defective Ad containing a rabies glycoprotein has been observed to induce complete protection against rabies infection. Partial protection was seen seven days after immunization.
While not wishing to be bound by theory, the inventors currently believe that 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. For example, although 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. [0043]
With respect to safety, 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, E1-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 E1 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. [0044]
With respect to efficacy, 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. [0045]
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. In contrast to the other replication defective adenovirus vaccines, 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. [0046]
For these reasons, 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. [0047]
I. The Recombinant Adenovirus [0048]
As used herein, the term “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. By “vector or plasmid” is meant the construct comprised of 5′ sequences of the Ad virus (usually Ad m.u. 0-1) deleted of the E1 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. 16 to 100), but rather conventional plasmid sequences. This vector does not contain all of the genes essential to a replicative virus. By “recombinant, replication defective Ad” is meant the infectious recombinant virus, deleted of its E1 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. [0049]
The recombinant virus of the method of the invention is a replication-defective recombinant adenovirus containing a deletion of its E1 gene and at least a partial, functional deletion of its E3 gene. In the site of the E1 deletion 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. [0050]
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, [0051] 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, Md., 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 (Acc. 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 E1a and E1b. See, K. F. Kozarsky and J. M. Wilson, [0052] Curr. Opin. Genet. Dev., 3:499-503 (1993). Similarly, a replication defective adenovirus may be designed by deleting less than the entire E1a and E1b locus, but enough to functionally disable the E1 genes.
An additional characteristic of the 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. [0053]
A preferred recombinant Ad virus may be produced by using a plasmid vector pAd.CMVlacZ as described in FIG. 1B. This plasmid contains adenovirus sequences Ad m.u. 0-1 (i.e., it is fully deleted of E1a and E1b 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. When this vector is manipulated to place a minicassette into the E1 deletion site, and supplied with the remaining 3′ Ad sequences with a full deletion of E3 and cultured in a helper cell line, 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 1D. [0054]
The preferred recombinant Ad of this invention contains a minicassette which uses the cytomegalovirus (CMV) promoter (see, e.g., Boshart et al, [0055] Cell, 41:521-530 (1985)) to control the expression of the inserted heterologous gene. The promoter is inserted in the site of the E1 deletion and directs the replication and expression of the protein encoded by the selected heterologous gene. However, 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 E1 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 β-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.
As discussed above, in the site of the E1 deletion, and under control of a promoter heterologous to Ad, 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. [0056]
The heterologous nucleic acid encodes a protein which is desirably capable of inducing an immune response to a pathogen. Such 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. Particularly, where the condition is human immunodeficiency virus (HIV) infection, the protein is preferably HIV glycoprotein 120 for which sequences are available from GenBank. Where the condition is human papilloma virus infection, the protein is selected from the group consisting of E6, E7 and/or L1 (Seedorf, K. et al, [0057] Virol., 145:181-185 (1985)). Where 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. In addition to these proteins, other virus-associated proteins are readily available to those of skill in the art. Selection of the heterologous proteins is not a limiting factor in this invention.
In a particularly preferred embodiment, the condition is rabies and the protein is the rabies glycoprotein (see, U.S. Pat. No. 4,393,201). A variety of rabies strains are well known and available from academic and commercial sources, including depositories 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. However, this invention is not limited by the selection of the rabies strain. [0058]
In a preferred embodiment, 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 replication defective virus conferred complete protection at a relatively low dose following challenge with rabies virus. [0059]
II. Formulation of Vaccine [0060]
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. [0061]
Optionally, 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. [0062]
III. Administration of Vaccine [0063]
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. [0064]
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. For example, in prophylaxis of rabies, the subcutaneous, intratracheal and intranasal routes are preferred. The route of administration primarily will depend on the nature of the disease being treated. [0065]
Doses or effective amounts of the recombinant replication defective Ad virus will depend primarily on factors such as the condition, the selected gene, the age, weight and health of the animal, and may thus vary among animals. For example, a prophylactically effective amount or dose of the Ad vaccine is generally in the range of from about 100 μl to about 10 ml of saline solution containing concentrations of from about 1×10[0066] ⁴to 1×10⁷plaque forming units (pfu) virus/ml. A preferred dose is from about 1 to about 10 ml saline solution at the above concentrations. The levels of immunity of the selected gene can be monitored to determine the need, if any, for boosters.
Currently, when vaccinating against rabies, the preferred dose is about 10[0067] ⁴pfu of the recombinant virus per mouse, preferably suspended in about 0.1 mL saline. Thus, when vaccinating against rabies infection, a larger animal would preferably be administered about a 1 mL dose containing about 1×10⁵Adrab.gp pfu suspended in saline. Following an assessment of antibody titers in the serum, 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. [0068]

EXAMPLE 1

Production and Purification of Vectors and Viruses [0069]

A. 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. Pat. No. 3,423,505) (ERA) was constructed as follows. See the flowchart of FIGS. 1A to [0070] 1D.
The 1650 bp rabies virus G cDNA (nucleotides 1178 to 2827 of SEQ ID NO: 1) was purified from the pSG5rab.gp plasmid (S. R. Burger et al, [0071] J. Gen. Virol., 72:359-367 (1991)) upon digestion with BglII, and blunt-ended with Klenow to supply the G gene. See also U.S. Pat. No. 4,393,201, issued Jul. 12, 1983.
The pAd.CMVlacZ vector (J. Wilson et al, [0072] 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.
The cDNA encoding the rabies G protein, described above, was inserted into this 5.6 kb fragment via blunt-end cloning to generate pAdCMV.rabgp, which is similar to pAd.CMVlacZ but contains the rabies sequence in place of the lacZ gene. The appropriate orientation of the insert was confirmed by restriction enzyme mapping. pAdCMV.rabgp (SEQ ID NO: 1) contains adenovirus m.u. 0-1 ([0073] 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); 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.
To provide a recombinant virus capable of infecting a cell, 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 NheI. 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, [0074] J. Gen. Virol., 36:59-72 (1977)). The transfected packaging cells were grown in DMEM with 10% FBS without HEPES buffer in a 5% CO₂incubator with an E3 deleted Ad5 DNA (Ad5dl7001l, 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 ClaI to remove the left end, i.e., 917 bp from the 5′ end of the adenovirus sequence, rendering the DNA non-infectious.
Following the co-transfection, only products of homologous recombination which occurred between Ad m.u. 9-16 of the pAdCMV.rabgp and the 5′ deleted-Ad5dl7001 could produce replicative Ad virus in 293 cells. That is, when homologous recombination occurred, the 3′ end of pAd.rabgp from about Ad m.u. 9 to about m.u. 16 and all of the plasmid sequence was swapped with the 3′ end of the 5′ truncated Ad5dl7001 virus, from about Ad m.u. 9 through m.u. 100. [0075]
Several recombinant viral plaques were harvested and tested for expression of the rabies virus G protein as described below. One recombinant, replication defective clone termed Adrab.gp was purified by two rounds of plaque purification and used for further studies and is illustrated schematically in FIG. 1D above. [0076]
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. [0077]

B. H5.010CMVlacZ

The recombinant replication defective Ad, H5.010CMVlacZ, is substantially identical to Adrab.gp, except that this virus contains [0078] 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 NheI and co-transfected into 293 cells with a partially E3 deleted Ad5 DNA (sub 360 DNA, H5sub360), which had been digested with ClaI to eliminate the sequence of m.u. 83.5 to 85. As above, homologous recombination, followed by plaquing 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 [0079] Escherichia coli lacZ gene, a pA site, and Ad5 m.u. 9-83.5 and 85-100.

C. Viral Propagation and Purification

The 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 supernatants were either used directly or they were further purified by CsCl density centrifugation. Viral stocks were titrated on 293 cells using a plaque assay. [0080]

EXAMPLE 2

Immunofluorescence and T Cell Studies [0081]
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. [0082]

A. Indirect Immunofluorescence

To assess the conformation of the G protein as expressed by the Adrab.gp virus, HeLa cells (which had been maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10% FBS, HEPES buffer and antibiotics in a 10% CO[0083] ₂incubator) 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 1112-1, and prepared using a 1:100 to 1:1000 dilution of ascitic fluid) to different conformation-dependent binding sites of the rabies virus G protein. 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, 75:3938-3945 (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 5×10[0084] ⁵cells 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). Alternatively cells adherent to glass cover slips were stained with the same antibody preparations for subsequent analysis with confocal microscopy.
For all of the antibodies, Adrab.gp virus-infected cells exhibited surface staining with the antibody, while cells infected with the control recombinant virus expressing lacZ were negative. [0085]

B. T Cell Proliferation Assay

Further in vitro studies showed that the recombinant virus Adrab.gp induced proliferation of a rabies virus G protein specific T helper cell clone in the presence of syngeneic, γ-irradiated splenocytes (FIG. 2). In a separate experiment, this T cell clone did not proliferate in the presence of H5.010CMVlacZ (data not shown). [0086]
A rabies virus-specific helper T cell clone, obtained from splenocytes of VRG immune C3H/He mice in the inventors' laboratory, was cultured (2×10[0087] ⁴cells/well) in 96-well round-bottom microtiter plate with 5×10⁵irradiated 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 10⁻⁶M 2-mercaptoethanol and 10% rat Concanavalin A supernatant as a lymphokine source as described previously (L. Otvos, Jr., Biochim. Biophys. Acta, 1224:68-76 (1994)). Proliferation of the cloned T cells was assessed 48 hours later by a 6 hour pulse with 0.5 μCi of ³H-thymidine as described in H. C. J. Ertl et al, Eur. J. Immunol., 21:1-10 (1991). Furthermore, mouse fibroblasts infected with the Adrab.gp recombinant virus were rendered susceptible to lysis by rabies virus G protein induced H-2 compatible cytolytic T cells.
Together these in vitro experiments demonstrated that 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. Specifically, 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. [0088]

EXAMPLE 3

Immunization Studies [0089]
In the next set of experiments, 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, Ind.)) or C3H/He mice (The Jackson Laboratories (Bar Harbor, Me.)) were injected subcutaneously (s.c.), orally (per os), intranasally (i.n.), or upon anesthesia and surgical exposure of the trachea intratracheally (i.t.), with the recombinant adenoviruses of the previous examples diluted in 100 to 150 μl of saline. VRG (which had been propagated on HeLa cells as described in T. J. Wiktor et al, [0090] Proc. Natl. Acad. Sci. USA, 81:7194-7198 (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, [0091] J. Virol., 21:626-633 (1977)). Mice were challenged with 10 mean lethal doses (LD₅₀) 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.

A. Virus Neutralizing Antibodies

Groups of ICR mice were immunized in three separate experiments with the different recombinant viruses given at the doses in Table 1 below either i.m., i.n., i.t., or per os. Mice inoculated into the trachea or i.n. were anesthetized prior to vaccination. Mice were bled 10 to 14 days later after a single immunization and serum antibody titers to rabies virus were tested by a neutralization assay. Virus neutralizing antibody (VNA) titers were determined on BHK-21 cells using infectious ERA virus at 1 pfu per cell (B. D. Dietzschold et al, [0092] Virology, 161:29-36 (1987)).
Table 1 below 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. [0093]

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 ⁶pfu. Oral immunization with Adrab.gp or systemic immunization with H5.020CMVlacZ failed to induce a measurable antibody response to rabies virus. The antibody responses to different doses of the recombinant replication-defective Adrab.gp were clearly superior to the response induced by the VRG recombinant. For example, the antibody titers of mice inoculated with as little as 2×10⁴pfu of Adrab.gp were more than 10 times higher than those of mice infected with 2×10⁶pfu of VRG (Table 1).

TABLE 1


Adrab.gp Recombinant Induces Neutralizing Antibodies
to Rabies Virus

		Route of	Time	VNA titer
Vaccine	Dose	Immunizat'n	After	Immunizat'n

Adrab.gp	2 × 10⁶	s.c.	day 10	3,645
Adrab.gp	2 × 10⁵	s.c.	day 10	405
Adrab.gp	2 × 10⁴	s.c.	day 10	405
VRG	2 × 10⁶	s.c.	day 10	45
VRG	2 × 10⁵	s.c.	day 10	15
VRG	2 × 10⁴	s.c.	day 10	5
None	—	—	day 10	<5
Adrab.gp	10⁴	s.c.	day 14	1,215
Adrab.gp	10³	s.c.	day 14	405
Adrab.gp	10²	s.c.	day 14	<5
Adrab.gp	10⁶	i.n.	day 14	1,215
Adrab.gp	10⁶	i.t.	day 14	3,645
Adrab.gp	10⁶	per os	day 14	<5
None	—	—		<5

To ensure that the antibody response was caused by infection recombinant virus rather than by G protein fragments contaminating the virus-containing tissue culture supernatant used for immunization, 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. [0095]

B. Cell-mediated Cytolysis

In addition to neutralizing antibodies, 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, [0096] 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% CO[0097] ₂incubator. L929 cells stably transfected with pSG5rab.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. These cell lines used as target cells for cell-mediated cytolysis assays have been described in detail previously (Z. Q. Xiang et al, J. Virol. Meth., 47:103-116 (1994)).
Briefly, splenocytes were harvested from immunized C3H/He mice. Single cells were prepared and incubated at 6×10[0098] ⁶cells 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% CO₂incubator. The effector cells were then co-cultured with ⁵¹Cr-labeled L929 cells expressing the rabies virus G protein upon stable transfection with the pSG5rab.gp vector at varied effector-to-target cells ratios. To assess spontaneous release, ⁵¹Cr-labeled target cells were incubated with medium; to determine maximal release target cells were co-cultured with 10% sodium dodecyl sulfate. Cell-free supernatants were harvested 4 hours later and radioactivity was measured. Percentage of specific lysis was calculated by using the formula (Y. Yang et al, Immunity, 1:433-442 (1994)):
100×{(Release in presence of effectors−spontaneous release)/(Maximal release−spontaneous release)}
The results are illustrated graphically in 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. [0099]

EXAMPLE 4

Challenge Studies [0100]
Four different experiments were conducted in which mice, immunized as described in Example 3A above, were challenged with 10 LD[0101] ₅₀of rabies virus. Briefly, mice immunized with the Adrab.gp or the VRG recombinant virus were challenged 2 to 5 weeks after immunization with 10 LD₅₀of 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.

The results are illustrated in Table 2 below. Mice immunized with Adrab.gp i.m., i.t., or i.n. using doses ranging from 10 ⁴to 2×10⁶pfu were fully protected against infection; 87% of mice inoculated with 10³pfu were protected. All mice immunized with only 10²pfu of the recombinant adenovirus or inoculated with the H5.020CMVlacZ control virus (2×10⁶pfu) or with Adrab.gp per os developed a fatal rabies virus encephalitis within 10 days after infection. Mice vaccinated with VRG showed partial protection; the group receiving the highest dose, i.e., 2×10⁶pfu, had a mortality rate above 50% raising to ˜90% in mice inoculated with 2×10⁴pfu of VRG.

TABLE 2


Adrab.gp Recombinant Virus Induces Protective Immunity to
Challenge with Rabies Virus

		Route of	%
Vaccine	Dose	immunization	mortality

Adrab.gp	2 × 10⁶	s.c.	0
H5.010CMVlacZ	2 × 10⁶	s.c.	90
Adrab.gp	2 × 10⁶	s.c.	0
Adrab.gp	2 × 10⁵	s.c.	0
Adrab.gp	2 × 10⁴	s.c.	0
VRG	2 × 10⁶	s.c.	56
VRG	2 × 10⁵	s.c.	71
VRG	2 × 10⁴	s.c.	86
None	—	—	100
Adrab.gp	10⁴	s.c.	0
Adrab.gp	10³	s.c.	13
Adrab.gp	10²	s.c.	100
None	—	—	100
Adrab.gp	10⁶	i.n.	0
Adrab.gp	10⁶	i.t.	0
Adrab.gp	10⁶	per os	100
None	—	—	100

EXAMPLE 5

Comparison Studies [0103]
The relationship between the magnitude of an immune response and the amount of antigen available to induce naive T and B cells was studied. As determined by immunofluorescence and subsequent analysis by FACS (FIGS. [0104] 4A-4L), both the VRG and the Adrab.gp recombinants express comparable levels of the rabies virus G protein but the kinetics of expression are different. Cells infected with the VRG virus express high levels of G protein within 12 hours after infection; these levels increased over the next day. By 60 hours the VRG virus has completely lysed a cell population infected with ˜1 pfu of virus per cell.
The same cell line infected with 1 pfu of Adrab.gp per cell shows low expression of the rabies virus G protein on [0105] 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. [0106]
To substantiate this hypothesis, the inventors compared the immune response to rabies proteins upon immunization of mice with a replication-defective E1 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). The ELISAs were conducted in 96-well microtiter plates coated with 0.1 to 0.2 μg per well of ERA-BPL virus or 1-2 μg 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, [0107] Virus Res., 24:297-314 (1992). As shown in FIGS. 5A and 5B, the antibody response to the E1 deleted Adrab.gp virus (solid box) was superior to that of a replication competent Ad virus (open box). This supports the position that long-term expression of viral antigens by a non-lytic virus can induce stronger immune response compared to short-term expression by a replication-competent agent. FIGS. 5A and 5B illustrate that expression of E1 causes a reduction in the antibody response to adenovirus.
These studies demonstrate that the recombinant replication-defective adenovirus used in the present invention shows higher immunogenicity compared to a replication-competent adenovirus. Without wishing to be bound by theory, it is believed that the length of expression of the antigen plays a role in induction of the immune response. In similar studies comparing the replication defective adenovirus vaccine to the VRG vaccine, the Ad vaccine expresses the rabies antigen longer than the VRG recombinant virus vaccine. [0108]

EXAMPLE 6

Further Comparative Studies [0109]
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[0110] ⁵or 10⁶pfu of a replication-competent adenovirus human serotype 5 that had been deleted of the E3 gene. Mice were injected 4 weeks later with 10⁶pfu of Adrab.gp. Control mice were only injected with Adrab.gp (10⁶pfu). Mice were bled 12 days later and neutralizing antibody titers were determined (Table 3).

TABLE 3

The Effect of Pre-Existing Immunity to Adenovirus on the

Rabies VNA Response to the Adrab.gp Vaccine

Pre-immunization

Titer Immunization VNA

None 106 pfu Adrab.gp 3.645

10⁵pfu Ad5d17001 10⁶pfu Adrab.gp 3.645

10⁶pfu Ad517001 10⁶pfu Adrab.gp 1.215

None None <5
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. When tested in comparison to a reference serum provided by the World Health Organization, 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. [0111]
Similar data were obtained for the stimulation of cytolytic T cells to rabies virus-infected cells, pre-immune animals showed somewhat lower lysis compared to the control group (see FIGS. 6A and 6B). FIGS. 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. [0112]

EXAMPLE 7

Additional Challenge Studies [0113]
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[0114] ⁶PFU of Adrab.gp s.c. Immunized mice were challenged 3 (⋄), 7 (□), and 10 (▪) days after vaccination with 10 LD₅₀of 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.
Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto. [0115]
1 2 1 8236 DNA rabies glycoprotein gene CDS (1185)..(2756) 1 gaattcgcta gcatcatcaa taatatacct tattttggat tgaagccaat atgataatga 60 gggggtggag tttgtgacgt ggcgcggggc gtgggaacgg ggcgggtgac gtagtagtgt 120 ggcggaagtg tgatgttgca agtgtggcgg aacacatgta agcgacggat gtggcaaaag 180 tgacgttttt ggtgtgcgcc ggtgtacaca ggaagtgaca attttcgcgc ggttttaggc 240 ggatgttgta gtaaatttgg gcgtaaccga gtaagatttg gccattttcg cgggaaaact 300 gaataagagg aagtgaaatc tgaataattt tgtgttactc atagcgcgta atatttgtct 360 agggagatca gcctgcaggt cgttacataa cttacggtaa atggcccgcc tggctgaccg 420 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata 480 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta 540 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc 600 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac 660 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga 720 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg 780 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg 840 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac 900 cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac 960 cgatccagcc tccggactct agaggatccg gtactcgagg aactgaaaaa ccagaaagtt 1020 aactggtaag tttagtcttt ttgtctttta tttcaggtcc cggatccggt ggtggtgcaa 1080 atcaaagaac tgctcctcag tggatgttgc ctttacttct aggcctgtac ggaagtgtta 1140 cttctgctct aaaagctgcg gaattgtacc cgcggccagg aaag atg gtt cct cag 1196 Met Val Pro Gln 1 gct ctc ctg ttt gta ccc ctt ctg gtt ttt cca ttg tgt ttt ggg aaa 1244 Ala Leu Leu Phe Val Pro Leu Leu Val Phe Pro Leu Cys Phe Gly Lys 5 10 15 20 ttc cct att tac acg ata cta gac aag ctt ggt ccc tgg agc ccg att 1292 Phe Pro Ile Tyr Thr Ile Leu Asp Lys Leu Gly Pro Trp Ser Pro Ile 25 30 35 gac ata cat cac ctc agc tgc cca aac aat ttg gta gtg gag gac gaa 1340 Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp Glu 40 45 50 gga tgc acc aac ctg tca ggg ttc tcc tac atg gaa ctt aaa gtt gga 1388 Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val Gly 55 60 65 tac atc tta gcc ata aaa atg aac ggg ttc act tgc aca ggc gtt gtg 1436 Tyr Ile Leu Ala Ile Lys Met Asn Gly Phe Thr Cys Thr Gly Val Val 70 75 80 acg gag gct gaa acc tac act aac ttc gtt ggt tat gtc aca acc acg 1484 Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr Thr 85 90 95 100 ttc aaa aga aag cat ttc cgc cca aca cca gat gca tgt aga gcc gcg 1532 Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala Ala 105 110 115 tac aac tgg aag atg gcc ggt gac ccc aga tat gaa gag tct cta cac 1580 Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu His 120 125 130 aat ccg tac cct gac tac cgc tgg ctt cga act gta aaa acc acc aag 1628 Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr Lys 135 140 145 gag tct ctc gtt atc ata tct cca agt gta gca gat ttg gac cca tat 1676 Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro Tyr 150 155 160 gac aga tcc ctt cac tcg agg gtc ttc cct agc ggg aag tgc tca gga 1724 Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser Gly 165 170 175 180 gta gcg gtg tct tct acc tac tgc tcc act aac cac gat tac acc att 1772 Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr Ile 185 190 195 tgg atg ccc gag aat ccg aga cta ggg atg tct tgt gac att ttt acc 1820 Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe Thr 200 205 210 aat agt aga ggg aag aga gca tcc aaa ggg agt gag act tgc ggc ttt 1868 Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly Phe 215 220 225 gta gat gaa aga ggc cta tat aag tct tta aaa gga gca tgc aaa ctc 1916 Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys Leu 230 235 240 aag tta tgt gga gtt cta gga ctt aga ctt atg gat gga aca tgg gtc 1964 Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp Val 245 250 255 260 gcg atg caa aca tca aat gaa acc aaa tgg tgc cct ccc gat cag ttg 2012 Ala Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Gln Leu 265 270 275 gtg aac ctg cac gac ttt cgc tca gac gaa att gag cac ctt gtt gta 2060 Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu His Leu Val Val 280 285 290 gag gag ttg gtc agg aag aga gag gag tgt ctg gat gca cta gag tcc 2108 Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu Ser 295 300 305 atc atg aca acc aag tca gtg agt ttc aga cgt ctc agt cat tta aga 2156 Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu Arg 310 315 320 aaa ctt gtc cct ggg ttt gga aaa gca tat acc ata ttc aac aag acc 2204 Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys Thr 325 330 335 340 ttg atg gaa gcc gat gct cac tac aag tca gtc aga act tgg aat gag 2252 Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn Glu 345 350 355 atc ctc cct tca aaa ggg tgt tta aga gtt ggg ggg agg tgt cat cct 2300 Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His Pro 360 365 370 cat gtg aac ggg gtg ttt ttc aat ggt ata ata tta gga cct gac ggc 2348 His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp Gly 375 380 385 aat gtc tta atc cca gag atg caa tca tcc ctc ctc cag caa cat atg 2396 Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His Met 390 395 400 gag ttg ttg gaa tcc tcg gtt atc ccc ctt gtg cac ccc ctg gca gac 2444 Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala Asp 405 410 415 420 ccg tct acc gtt ttc aag gac ggt gac gag gct gag gat ttt gtt gaa 2492 Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe Val Glu 425 430 435 gtt cac ctt ccc gat gtg cac aat cag gtc tca gga gtt gac ttg ggt 2540 Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp Leu Gly 440 445 450 ctc ccg aac tgg ggg aag tat gta tta ctg agt gca ggg gcc ctg act 2588 Leu Pro Asn Trp Gly Lys Tyr Val Leu Leu Ser Ala Gly Ala Leu Thr 455 460 465 gcc ttg atg ttg ata att ttc ctg atg aca tgt tgt aga aga gtc aat 2636 Ala Leu Met Leu Ile Ile Phe Leu Met Thr Cys Cys Arg Arg Val Asn 470 475 480 cga tca gaa cct acg caa cac aat ctc aga ggg aca ggg agg gag gtg 2684 Arg Ser Glu Pro Thr Gln His Asn Leu Arg Gly Thr Gly Arg Glu Val 485 490 495 500 tca gtc act ccc caa agc ggg aag atc ata tct tca tgg gaa tca cac 2732 Ser Val Thr Pro Gln Ser Gly Lys Ile Ile Ser Ser Trp Glu Ser His 505 510 515 aag agt ggg ggt gag acc aga ctg tgaggactgg ccgtcctttc aacgatccaa 2786 Lys Ser Gly Gly Glu Thr Arg Leu 520 gtcctgaaga tcacctcccc ttggggggtt ctttttaaaa aggccgcggg gatccagaca 2846 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct 2906 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac 2966 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg 3026 ttttttcgga tcctctagag tcgacctgca ggctgatctg gaaggtgctg aggtacgatg 3086 agacccgcac caggtgcaga ccctgcgagt gtggcggtaa acatattagg aaccagcctg 3146 tgatgctgga tgtgaccgag gagctgaggc ccgatcactt ggtgctggcc tgcacccgcg 3206 ctgagtttgg ctctagcgat gaagatacag attgaggtac tgaaatgtgt gggcgtggct 3266 taagggtggg aaagaatata taaggtgggg gtcttatgta gttttgtatc tgttttgcag 3326 cagccgccgc cgccatgagc accaactcgt ttgatggaag cattgtgagc tcatatttga 3386 caacgcgcat gcccccatgg gccggggtgc gtcagaatgt gatgggctcc agcattgatg 3446 gtcgccccgt cctgcccgca aactctacta ccttgaccta cgagaccgtg tctggaacgc 3506 cgttggagac tgcagcctcc gccgccgctt cagccgctgc agccaccgcc cgcgggattg 3566 tgactgactt tgctttcctg agcccgcttg caagcagtgc agcttcccgt tcatccgccc 3626 gcgatgacaa gttgacggct cttttggcac aattggattc tttgacccgg gaacttaatg 3686 tcgtttctca gcagctgttg gatctgcgcc agcaggtttc tgccctgaag gcttcctccc 3746 ctcccaatgc ggtttaaaac ataaataaaa aaccagactc tgtttggatt tggatcaagc 3806 aagtgtcttg ctgtctttat ttaggggttt tgcgcgcgcg gtaggcccgg gaccagcggt 3866 ctcggtcgtt gagggtcctg tgtatttttt ccaggacgtg gtaaaggtga ctctggatgt 3926 tcagatacat gggcataagc ccgtctctgg ggtggaggta gcaccactgc agagcttcat 3986 gctgcggggt ggtgttgtag atgatccagt cgtagcagga gcgctgggcg tggtgcctaa 4046 aaatgtcttt cagtagcaag ctgattgcca ggggcaggcc cttggtgtaa gtgtttacaa 4106 agcggttaag ctgggatggg tgcatacgtg gggatatgag atgcatcttg gactgtattt 4166 ttaggttggc tatgttccca gccatatccc tccggggatt catgttgtgc agaaccacca 4226 gcacagtgta tccggtgcac ttgggaaatt tgtcatgtag cttagaagga aatgcgtgga 4286 agaacttgga gacgcccttg tgacctccaa gattttccat gcattcgtcc ataatgatgg 4346 caatgggccc acgggcggcg gcctgggcga agatatttct gggatcacta acgtcatagt 4406 tgtgttccag gatgagatcg tcataggcca tttttacaaa gcgcgggcgg agggtgccag 4466 actgcggtat aatggttcca tccggcccag gggcgtagtt accctcacag atttgcattt 4526 cccacgcttt gagttcagat ggggggatca tgtctacctg cggggcgatg aagaaaacgg 4586 tttccggggt aggggagatc agctgggaag aaagcaggtt cctgagcagc tgcgacttac 4646 cgcagccggt gggcccgtaa atcacaccta ttaccgggtg caactggtag ttaagagagc 4706 tgcagctgcc gtcatccctg agcagggggg ccacttcgtt aagcatgtcc ctgactcgca 4766 tgttttccct gaccaaatcc gccagaaggc gctcgccgcc cagcgatagc agttcttgca 4826 aggaagcaaa gtttttcaac ggtttgagac cgtccgccgt aggcatgctt ttgagcgttt 4886 gaccaagcag ttccaggcgg tcccacagct cggtcacctg ctctacggca tctcgatcca 4946 gcatatctcc tcgtttcgcg ggttggggcg gctttcgctg tacggcagta gtcggtgctc 5006 gtccagacgg gccagggtca tgtctttcca cgggcgcagg gtcctcgtca gcgtagtctg 5066 ggtcacggtg aaggggtgcg ctccgggctg cgcgctggcc agggtgcgct tgaggctggt 5126 cctgctggtg ctgaagcgct gccggtcttc gccctgcgcg tcggccaggt agcatttgac 5186 catggtgtca tagtccagcc cctccgcggc gtggcccttg gcgcgcagct tgcccttgga 5246 ggaggcgccg cacgaggggc agtgcagact tttgagggcg tagagcttgg gcgcgagaaa 5306 taccgattcc ggggagtagg catccgcgcc gcaggccccg cagacggtct cgcattccac 5366 gagccaggtg agctctggcc gttcggggtc aaaaaccagg tttcccccat gctttttgat 5426 gcgtttctta cctctggttt ccatgagccg gtgtccacgc tcggtgacga aaaggctgtc 5486 cgtgtccccg tatacagact tgagaggcct gtcctcgacc gatgcccttg agagccttca 5546 acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg 5606 tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg 5666 aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct 5726 tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc 5786 aggccattat cgccggcatg gcggccgacg cgctgggcta cgtcttgctg gcgttcgcga 5846 cgcgaggctg gatggccttc cccattatga ttcttctcgc ttccggcggc atcgggatgc 5906 ccgcgttgca ggccatgctg tccaggcagg tagatgacga ccatcaggga cagcttcaag 5966 gatcgctcgc ggctcttacc agcctaactt cgatcactgg accgctgatc gtcacggcga 6026 tttatgccgc ctcggcgagc acatggaacg ggttggcatg gattgtaggc gccgccctat 6086 accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa 6146 tggaagccgg cggcacctcg ctaacggatt caccactcca agaattggag ccaatcaatt 6206 cttgcggaga actgtgaatg cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc 6266 catctccagc agccgcacgc ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg 6326 catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta 6386 gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc 6446 gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg 6506 gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc 6566 ctgtggaaca cctacatctg tattaacgaa gcctttctca atgctcacgc tgtaggtatc 6626 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 6686 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 6746 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 6806 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 6866 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 6926 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 6986 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 7046 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 7106 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 7166 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 7226 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 7286 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 7346 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 7406 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 7466 gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 7526 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 7586 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 7646 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 7706 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 7766 gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag 7826 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 7886 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 7946 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 8006 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 8066 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 8126 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca 8186 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa 8236 2 524 PRT rabies glycoprotein gene 2 Met Val Pro Gln Ala Leu Leu Phe Val Pro Leu Leu Val Phe Pro Leu 1 5 10 15 Cys Phe Gly Lys Phe Pro Ile Tyr Thr Ile Leu Asp Lys Leu Gly Pro 20 25 30 Trp Ser Pro Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val 35 40 45 Val Glu Asp Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu 50 55 60 Leu Lys Val Gly Tyr Ile Leu Ala Ile Lys Met Asn Gly Phe Thr Cys 65 70 75 80 Thr Gly Val Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr 85 90 95 Val Thr Thr Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala 100 105 110 Cys Arg Ala Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu 115 120 125 Glu Ser Leu His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val 130 135 140 Lys Thr Thr Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp 145 150 155 160 Leu Asp Pro Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly 165 170 175 Lys Cys Ser Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His 180 185 190 Asp Tyr Thr Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys 195 200 205 Asp Ile Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu 210 215 220 Thr Cys Gly Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly 225 230 235 240 Ala Cys Lys Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp 245 250 255 Gly Thr Trp Val Ala Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro 260 265 270 Pro Asp Gln Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu 275 280 285 His Leu Val Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp 290 295 300 Ala Leu Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu 305 310 315 320 Ser His Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile 325 330 335 Phe Asn Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg 340 345 350 Thr Trp Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly 355 360 365 Arg Cys His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu 370 375 380 Gly Pro Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu 385 390 395 400 Gln Gln His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His 405 410 415 Pro Leu Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu 420 425 430 Asp Phe Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly 435 440 445 Val Asp Leu Gly Leu Pro Asn Trp Gly Lys Tyr Val Leu Leu Ser Ala 450 455 460 Gly Ala Leu Thr Ala Leu Met Leu Ile Ile Phe Leu Met Thr Cys Cys 465 470 475 480 Arg Arg Val Asn Arg Ser Glu Pro Thr Gln His Asn Leu Arg Gly Thr 485 490 495 Gly Arg Glu Val Ser Val Thr Pro Gln Ser Gly Lys Ile Ile Ser Ser 500 505 510 Trp Glu Ser His Lys Ser Gly Gly Glu Thr Arg Leu 515 520

Claims

What is claimed is:

1. A method of inducing an immune response in a mammal against a selected pathogen comprising: administering to a mammalian subject a sufficient amount of a recombinant adenovirus comprising a complete deletion of its E1 gene and at least a functional deletion of its E3 gene and, in the site of the E1 gene deletion, a sequence comprising a cytomegalovirus promoter directing the expression of DNA encoding a protein of said pathogen, which when administered to the subject in said recombinant virus, elicits an immune response against said pathogen.

2. The method according to claim 1, wherein said adenovirus is administered subcutaneously, intranasally, intratracheally, or intramuscularly.

3. A method of inducing an immune response in a mammal against human immunodeficiency virus HIV comprising: administering to a mammalian subject a sufficient amount of a recombinant adenovirus comprising a complete deletion of its E1 gene and at least a functional deletion of its E3 gene, and in the site of the E1 gene deletion, a sequence comprising a cytomegalovirus promoter directing the replication and expression of DNA encoding an HIV protein, which when administered to the subject in said recombinant virus, elicits an immune response against HIV.

4. The method according to claim 3, wherein said adenovirus is administered subcutaneously, intranasally, intratracheally, or intramuscularly.

5. A method of inducing an immune response in a mammal against human papilloma virus comprising: administering to a mammalian subject a sufficient amount of a recombinant adenovirus comprising a complete deletion of its E1 gene and at least a functional deletion of its E3 gene, and, in the site of the E1 gene deletion, a sequence comprising a cytomegalovirus promoter directing the replication and expression of DNA encoding a human papilloma virus protein, which when administered to the subject in said recombinant virus, elicits a protective immune response against human papilloma virus.

6. The method according to claim 5, wherein said adenovirus is administered subcutaneously, intranasally, intratracheally, or intramuscularly.