U.S. patent application number 09/897170 was filed with the patent office on 2002-11-07 for replication-defective adenovirus human type 5 recombinant as a vaccine carrier.
This patent application is currently assigned to The Trustees of the University of Pennsylvania. Invention is credited to Ertl, Hildegund C. J., Wilson, James M..
Application Number | 20020164353 09/897170 |
Document ID | / |
Family ID | 26667200 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020164353 |
Kind Code |
A1 |
Ertl, Hildegund C. J. ; et
al. |
November 7, 2002 |
Replication-defective adenovirus human type 5 recombinant as a
vaccine carrier
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.
Inventors: |
Ertl, Hildegund C. J.;
(Villanova, PA) ; Wilson, James M.; (Gladwyne,
PA) |
Correspondence
Address: |
HOWSON AND HOWSON
ONE SPRING HOUSE CORPORATION CENTER
BOX 457
321 NORRISTOWN ROAD
SPRING HOUSE
PA
19477
US
|
Assignee: |
The Trustees of the University of
Pennsylvania
Philadelphia
PA
|
Family ID: |
26667200 |
Appl. No.: |
09/897170 |
Filed: |
July 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09897170 |
Jul 2, 2001 |
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09347060 |
Jul 2, 1999 |
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6287571 |
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09347060 |
Jul 2, 1999 |
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08973233 |
Dec 3, 1997 |
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6086501 |
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08973233 |
Dec 3, 1997 |
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PCT/US96/09495 |
Jun 5, 1996 |
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PCT/US96/09495 |
Jun 5, 1996 |
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08461837 |
Jun 5, 1995 |
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5698202 |
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60000078 |
Jun 8, 1995 |
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Current U.S.
Class: |
424/199.1 ;
424/204.1; 424/233.1; 435/235.1; 435/320.1; 536/23.72 |
Current CPC
Class: |
A61P 31/18 20180101;
C12N 2760/20134 20130101; C12N 2710/10343 20130101; C07K 14/005
20130101; A61P 37/04 20180101; A61K 2039/53 20130101 |
Class at
Publication: |
424/199.1 ;
424/204.1; 424/233.1; 435/235.1; 435/320.1; 536/23.72 |
International
Class: |
C07H 021/04; A61K
039/12; A61K 039/23; A61K 039/235; C12N 007/00; C12N 007/01; C12N
015/00; C12N 015/09; C12N 015/63; C12N 015/70; C12N 015/74 |
Goverment Interests
[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.
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.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] 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 .sctn.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.
FIELD OF THE INVENTION
[0003] 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
[0004] 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).
[0005] 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 10.sup.6 plaque-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.
[0006] 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.
[0007] 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.times.10.sup.9 pfu,
1.times.10.sup.10 pfu and 2.times.10.sup.10 pfu), intramuscular
immunizations and viral challenge, full protection was
obtained.
[0008] 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 10.sup.6 pfu are required to induce complete
protection.
[0009] 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
[0010] 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.sup.4 pfu of rabies vaccine vector
to induce complete protection. This effect is also accomplished by
administration routes other than the oral route.
[0011] 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.
[0012] In another aspect, the invention provides pharmaceutical and
veterinary compositions which contain the rAd of the invention.
[0013] In still another aspect, the invention provides for the use
of the rAd in the manufacture of the compositions described
above.
[0014] 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.
[0015] 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.
[0016] 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
[0017] 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.
[0018] 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.
[0019] 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
(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.
[0020] 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.
[0021] FIG. 2 is a bar graph plotting .sup.3H-thymidine ((3H)TdR)
incorporation, measured at counts per minute.+-.standard deviation
(cpm.+-.SD), for irradiated splenocytes plated at 5.times.10.sup.5
cells per well of a round bottom microtiter plate and incubated
with 5 (diagonally striped), 1 (cross-hatched) or 0.2 (solid)
.mu.g/ml of betapropionolactone-inactivated Evelyn Rockitniki
Abelseth rabies strain (ERA-BPL) or approximately 1 (diagonally
striped), 0.1 (cross-hatched), and 0.01 (solid) pfu of Adrab.gp per
cell or medium only as a negative control for 60 minutes at
37.degree. C. As described in Example 2B, after cloned T cells were
added, cells were pulsed two days later for 6 hours with
.sup.3H-thymidine, harvested and counted in a .beta.-counter.
[0022] 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.times.10.sup.6 pfu 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 .sup.51Cr-release assay.
[0023] 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 .sup.51Cr-release
assay, as a control.
[0024] 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.
[0025] FIG. 4B is a graph similar to FIG. 4A above, except the
cells were harvested 36 hours after infection.
[0026] FIG. 4C is a graph similar to FIG. 4A above, except the
cells were harvested 60 hours after infection.
[0027] 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.
[0028] FIG. 4E is a graph similar to FIG. 4D above, except the
cells were harvested 36 hours after infection.
[0029] FIG. 4F is a graph similar to FIG. 4D above, except the
cells were harvested 60 hours after infection.
[0030] 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.
[0031] FIG. 4H is a graph similar to FIG. 4G, except the cells were
harvested 36 hours after infection.
[0032] FIG. 4I is a graph similar to FIG. 4G, except the cells were
harvested 60 hours after infection.
[0033] 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.
[0034] FIG. 4K is a graph similar to FIG. 4J above, except the
cells were harvested 36 hours after infection.
[0035] FIG. 4L is a graph similar to FIG. 4J above, except the
cells were harvested 60 hours after infection.
[0036] 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 .mu.g/mL of
purified H5.020CMVlacZ virus.
[0037] 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.
[0038] 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 .sup.51Cr-release assay on
L929 cells stably transfected with pSG5rab.gp. See Example 6.
[0039] FIG. 6B is a graph similar to FIG. 6A, except the L929 cells
were transfected with pSV2neo.
[0040] 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.
DETAILED DESCRIPTION OF THE INVENTION
[0041] 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.
[0042] 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.sup.4 pfu 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] I. The Recombinant Adenovirus
[0049] 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.
[0050] 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.
[0051] Any Ad that infects the target cells is appropriate for use
in this invention. Desirable adenoviruses are human type C
adenoviruses, including serotypes Ad2 and Ad5. The DNA sequences of
a number of adenovirus types, including type Ad5, are available
from GenBank (Accession No. M73260). The adenovirus sequences may
be obtained from any known adenovirus type, including the presently
identified 41 human types (Horwitz et al, Virology, 2d ed., B. N.
Fields, Raven Press, Ltd., New York (1990)). Similarly,
adenoviruses known to infect other animals may also be employed in
this invention. The selection of the adenovirus type and strain is
not anticipated to limit the following invention. A variety of
adenovirus strains are available from the American Type Culture
Collection, Rockville, 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.
[0052] 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, 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.
[0053] 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.
[0054] 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.
[0055] The preferred recombinant Ad of this invention contains a
minicassette which uses the cytomegalovirus (CMV) promoter (see,
e.g., Boshart et al, 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 .beta.-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.
[0056] 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.
[0057] 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, 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.
[0058] 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.
[0059] 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.
[0060] II. Formulation of Vaccine
[0061] 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.
[0062] 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.
[0063] III. Administration of Vaccine
[0064] 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.
[0065] 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.
[0066] 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 .mu.l to about 10 ml of
saline solution containing concentrations of from about
1.times.10.sup.4 to 1.times.10.sup.7 plaque forming units (pfu)
virus/ml. A preferred dose is from about 1 to about 10 ml saline
solution at the above concentrations. The levels of immunity of the
selected gene can be monitored to determine the need, if any, for
boosters.
[0067] Currently, when vaccinating against rabies, the preferred
dose is about 10.sup.4 pfu 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.times.10.sup.5
Adrab.gp pfu suspended in saline. Following an assessment of
antibody titers in the serum, optional booster immunizations may be
desired.
[0068] 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.
EXAMPLE 1
[0069] Production and Purification of Vectors and Viruses
A. Adrab.gp
[0070] 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
1D.
[0071] 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, 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.
[0072] The pAd.CMVlacZ vector (J. Wilson et al, Hum. Gene Ther.,
5:501-519 (1994); K. Kozarsky et al, J. Biol. Chem.,
269:13695-13702 (1994)), which contains Ad5 m.u. 0-1, followed by
the cytomegalovirus (CMV) enhancer/promoter, the beta galactosidase
(lacZ) gene, a polyadenylation signal (pA), adenovirus m.u. 9-16
and plasmid sequences from plasmid pAT153 including an origin of
replication and ampicillin resistance gene, was completely digested
with NotI to remove the lacZ gene and provide an .about.5.6 kb
backbone.
[0073] 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
(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.
[0074] 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, J. Gen. Virol.,
36:59-72 (1977)). The transfected packaging cells were grown in
DMEM with 10% FBS without HEPES buffer in a 5% CO.sub.2 incubator
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.
[0075] 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.
[0076] 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.
[0077] 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.
B. H5.010CMVlacZ
[0078] The recombinant replication defective Ad, H5.010CMVlacZ, is
substantially identical to Adrab.gp, except that this virus
contains E. coli lacZ in place of the rabies G protein and only a
partial deletion of E3.
[0079] 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 Escherichia coli lacZ gene, a pA site, and
Ad5 m.u. 9-83.5 and 85-100.
C. Viral Propagation and Purification
[0080] 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.
EXAMPLE 2
[0081] Immunofluorescence and T Cell Studies
[0082] 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.
A. Indirect Immunofluorescence
[0083] 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.sub.2 incubator) were
infected for 48 hours with 1 pfu of Adrab.gp virus per cell or as a
control with H5.020CMVlacZ. Cells were stained 24 hours later by an
indirect immunofluorescence assay using three MAbs (designated
523-11, 509-6, and 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.
[0084] 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.times.10.sup.5 cells per well. Cells were harvested at varied
times after infection by treatment with trypsin and incubated for
60 minutes on ice with the MAbs identified above. Cells were washed
once with phosphate-buffered saline (PBS) and then incubated with a
FITC-labeled goat anti-mouse immunoglobulin (Ig) antibody. Cells
were washed and analyzed by a fluorescence activated cell sorter
(FACS). Alternatively cells adherent to glass cover slips were
stained with the same antibody preparations for subsequent analysis
with confocal microscopy.
[0085] 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.
B. T Cell Proliferation Assay
[0086] 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,
.gamma.-irradiated splenocytes (FIG. 2). In a separate experiment,
this T cell clone did not proliferate in the presence of
H5.010CMVlacZ (data not shown).
[0087] A rabies virus-specific helper T cell clone, obtained from
splenocytes of VRG immune C3H/He mice in the inventors' laboratory,
was cultured (2.times.10.sup.4 cells/well) in 96-well round-bottom
microtiter plate with 5.times.10.sup.5 irradiated syngeneic C3H/He
splenocytes pretreated with different antigen preparations (1, 0.1
and 0.01 pfu Adrab.gp per cell) in DMEM supplemented with 2% FBS
and 10.sup.-6 M 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 .mu.Ci of .sup.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.
[0088] 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.
EXAMPLE 3
[0089] Immunization Studies
[0090] 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 .mu.l of saline. VRG (which
had been propagated on HeLa cells as described in T. J. Wiktor et
al, 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.
[0091] The challenge virus standard (CVS)-24 strain of rabies
virus, that is antigenically closely related to the ERA strain but
shows higher virulence in mice, was derived from brain suspensions
of infected newborn ICR mice (T. J. Wiktor et al, J. Virol.,
21:626-633 (1977)). Mice were challenged with 10 mean lethal doses
(LD.sub.50) of CVS-24 virus given intramuscularly (i.m.) into the
masseter muscle; they were observed for the following 3 weeks for
symptoms indicative of a rabies virus infection. Mice that
developed complete bilateral hind leg paralysis (proceeding death
by 24 to 48 hours) were euthanized for humanitarian reasons.
A. Virus Neutralizing Antibodies
[0092] 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, Virology, 161:29-36 (1987)).
[0093] 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.
[0094] 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.sup.6 pfu. 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.times.10.sup.4 pfu of Adrab.gp were more than
10 times higher than those of mice infected with 2.times.10.sup.6
pfu of VRG (Table 1).
1TABLE 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 .times. 10.sup.6 s.c. day 10 3,645 Adrab.gp
2 .times. 10.sup.5 s.c. day 10 405 Adrab.gp 2 .times. 10.sup.4 s.c.
day 10 405 VRG 2 .times. 10.sup.6 s.c. day 10 45 VRG 2 .times.
10.sup.5 s.c. day 10 15 VRG 2 .times. 10.sup.4 s.c. day 10 5 None
-- -- day 10 <5 Adrab.gp 10.sup.4 s.c. day 14 1,215 Adrab.gp
10.sup.3 s.c. day 14 405 Adrab.gp 10.sup.2 s.c. day 14 <5
Adrab.gp 10.sup.6 i.n. day 14 1,215 Adrab.gp 10.sup.6 i.t. day 14
3,645 Adrab.gp 10.sup.6 per os day 14 <5 None -- -- <5
[0095] 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.
B. Cell-mediated Cytolysis
[0096] 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, J. Virol. Meth., 47:103-116 (1994)).
[0097] 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.sub.2 incubator.
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)).
[0098] Briefly, splenocytes were harvested from immunized C3H/He
mice. Single cells were prepared and incubated at 6.times.10.sup.6
cells per well with 1 pfu per cell of the Adrab.gp recombinant
virus in 1.6 ml of DMEM supplemented with 10-6 M 2-mercaptoethanol
and 2% FBS for 5 days in a humidified 10% CO.sub.2 incubator. The
effector cells were then co-cultured with .sup.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,
.sup.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.times.{(Release in presence of effectors-spontaneous
release)/(Maximal release-spontaneous release)}
[0099] 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.
EXAMPLE 4
[0100] Challenge Studies
[0101] Four different experiments were conducted in which mice,
immunized as described in Example 3A above, were challenged with 10
LD.sub.50 of rabies virus. Briefly, mice immunized with the
Adrab.gp or the VRG recombinant virus were challenged 2 to 5 weeks
after immunization with 10 LD.sub.50 of the virulent CVS-24 strain
of rabies virus given i.m. into the masseter muscle. Mice that
subsequently developed complete bilateral hind leg paralysis
indicative of a terminal rabies virus infection were euthanized for
humanitarian reasons. Survivors were observed for a total of 21
days.
[0102] 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.sup.4
to 2.times.10.sup.6 pfu were fully protected against infection; 87%
of mice inoculated with 10.sup.3 pfu were protected. All mice
immunized with only 10.sup.2 pfu of the recombinant adenovirus or
inoculated with the H5.020CMVlacZ control virus (2.times.10.sup.6
pfu) 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.times.10.sup.6 pfu, had a mortality rate above 50%
raising to .about.90% in mice inoculated with 2.times.10.sup.4 pfu
of VRG.
2TABLE 2 Adrab.gp Recombinant Virus Induces Protective Immunity to
Challenge with Rabies Virus Route of % Vaccine Dose immunization
mortality Adrab.gp 2 .times. 10.sup.6 s.c. 0 H5.010CMVlacZ 2
.times. 10.sup.6 s.c. 90 Adrab.gp 2 .times. 10.sup.6 s.c. 0
Adrab.gp 2 .times. 10.sup.5 s.c. 0 Adrab.gp 2 .times. 10.sup.4 s.c.
0 VRG 2 .times. 10.sup.6 s.c. 56 VRG 2 .times. 10.sup.5 s.c. 71 VRG
2 .times. 10.sup.4 s.c. 86 None -- -- 100 Adrab.gp 10.sup.4 s.c. 0
Adrab.gp 10.sup.3 s.c. 13 Adrab.gp 10.sup.2 s.c. 100 None -- -- 100
Adrab.gp 10.sup.6 i.n. 0 Adrab.gp 10.sup.6 i.t. 0 Adrab.gp 10.sup.6
per os 100 None -- -- 100
EXAMPLE 5
[0103] Comparison Studies
[0104] 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. 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 .about.1 pfu of virus per cell.
[0105] The same cell line infected with 1 pfu of Adrab.gp per cell
shows low expression of the rabies virus G protein on day 1. The
level of expression increases until days 3 to 4 after infection and
then reaches plateau levels (data shown for days 1 to 3 in FIG. 4A
through FIG. 4L). The replication-defective recombinant
adenoviruses are non-lytic and maintain stable infection and
expression of virus-encoded proteins for extended periods of time.
In tissue culture, expression has been shown for 7 days in vivo;
using the H5.010CMVlacZ recombinant virus, stable levels of
expression were demonstrated in immunocompromised mice for 10
months.
[0106] 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.
[0107] 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 .mu.g per well of ERA-BPL virus or
1-2 .mu.g per well of purified H5.010CMVlacZ virus, using an
alkaline phosphatase conjugated goat anti-mouse Ig as second
antibody as described in detail in Xiang and Ertl, Virus Res.,
24:297-314 (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.
[0108] 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.
EXAMPLE 6
[0109] Further Comparative Studies
[0110] 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.sup.5 or
10.sup.6 pfu of a replication-competent adenovirus human serotype 5
that had been deleted of the E3 gene. Mice were injected 4 weeks
later with 10.sup.6 pfu of Adrab.gp. Control mice were only
injected with Adrab.gp (10.sup.6 pfu). Mice were bled 12 days later
and neutralizing antibody titers were determined (Table 3).
3TABLE 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 10.sup.5 pfu Ad5d17001
10.sup.6 pfu Adrab.gp 3.645 10.sup.6 pfu Ad517001 10.sup.6 pfu
Adrab.gp 1.215 None None <5
[0111] 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.
[0112] 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.
EXAMPLE 7
[0113] Additional Challenge Studies
[0114] 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.sup.6 PFU of
Adrab.gp s.c. Immunized mice were challenged 3 (.diamond.), 7
(.quadrature.), and 10 (.box-solid.) days after vaccination with 10
LD.sub.50 of rabies virus given i.m. Naive mice (X) served as
controls. Mice were observed for four weeks to record mortality. As
shown in FIG. 7, mice vaccinated with Adrab.gp virus 10 days
previously were completely protected; while more than half of the
animals were protected as early as seven days after a single
injection. Mice vaccinated three days before challenge succumbed to
the infection.
[0115] 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.
Sequence CWU 1
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
* * * * *