U.S. patent application number 10/794876 was filed with the patent office on 2004-09-02 for recombinant adenovirus vaccines.
This patent application is currently assigned to Wyeth. Invention is credited to Chanda, Pranab K., Davis, Alan R., Hung, Paul P., Lee, Shaw-Guang L., Lubeck, Michael D., Murthy, Shridhara C. S., Natuk, Robert J..
Application Number | 20040170647 10/794876 |
Document ID | / |
Family ID | 27493445 |
Filed Date | 2004-09-02 |
United States Patent
Application |
20040170647 |
Kind Code |
A1 |
Davis, Alan R. ; et
al. |
September 2, 2004 |
Recombinant adenovirus vaccines
Abstract
This invention provides a method of protecting a primate from an
infectious organism by stimulating the production of antibodies or
cell mediated immunity to the infectious organism which comprises
administering to said primate intranasally, intramuscularly, or
subcutaneously, live recombinant adenoviruses in which the virion
structural protein is unchanged from that in the native adenovirus
from which the recombinant adenovirus is produced, and which
contain the gene coding for the antigen corresponding to said
antibodies or inducing said cell mediated immunity. Preferably, the
infectious organism is HIV and the primate is a human.
Inventors: |
Davis, Alan R.; (Missouri
City, TX) ; Lubeck, Michael D.; (Highland Mills,
NY) ; Natuk, Robert J.; (Raritan, NJ) ;
Chanda, Pranab K.; (West Windsor, NJ) ; Murthy,
Shridhara C. S.; (Ann Arbor, MI) ; Lee, Shaw-Guang
L.; (Villanova, PA) ; Hung, Paul P.; (Bryn
Mawr, PA) |
Correspondence
Address: |
WYETH
PATENT LAW GROUP
FIVE GIRALDA FARMS
MADISON
NJ
07940
US
|
Assignee: |
Wyeth
Madison
NJ
|
Family ID: |
27493445 |
Appl. No.: |
10/794876 |
Filed: |
March 5, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10794876 |
Mar 5, 2004 |
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09457421 |
Dec 7, 1999 |
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09457421 |
Dec 7, 1999 |
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08276289 |
Jul 20, 1994 |
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08276289 |
Jul 20, 1994 |
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08105232 |
Aug 11, 1993 |
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08105232 |
Aug 11, 1993 |
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07926491 |
Aug 7, 1992 |
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Current U.S.
Class: |
424/199.1 ;
424/93.2 |
Current CPC
Class: |
Y02A 50/30 20180101;
C12N 2740/16122 20130101; C12N 2740/16134 20130101; C12N 2710/10343
20130101; C07K 14/005 20130101; C12N 15/86 20130101; C12N
2740/16222 20130101; A61K 39/00 20130101; Y02A 50/464 20180101;
C12N 2740/16234 20130101 |
Class at
Publication: |
424/199.1 ;
424/093.2 |
International
Class: |
A61K 048/00; A61K
039/12 |
Claims
What is claimed is:
1. A method of protecting a primate against HIV-1 infection
comprising intranasal or intramuscular administration to said
primate of an intranasal or intramuscular dosage of a recombinant
adenovirus having a deletion in the E3 gene and an expression
cassette containing a major late promoter, a tripartite leader
sequence, part or all of the HIV-1 gp160 sequence and a
polyadenylation signal sequence, said cassette being inserted into
said recombinant adenovirus between the E4 promoter and the
inverted terminal repeat of said recombinant adenovirus.
2. The method of claim 1 wherein said intranasal or intramuscular
administration of recombinant adenovirus is followed by one or more
intranasal or intramuscluar booster administrations of said
recombinant adenovirus.
3. The method of claim 2 wherein said adenovirus is a serotype 4, 5
or 7 serotype adenovirus.
4. The method of claim 3 wherein said expression cassette
additionally comprises part of all of the coding sequence for the
HIV-1 rev gene inserted in frame after the HIV-1 gp160 sequence and
before the polyadenylation signal sequence.
5. The method of claim 4 wherein said HIV-1 gp160 sequence is the
MN strain gp160 sequence or the LAV strain gp160 sequence.
6. The method of claim 4 wherein said HIV-1 gp160 sequence is
replaced by a sequence encoding the gag-pro region of HIV-1.
7. The method of claim 2 wherein said one or more intranasal or
intramuscular booster administrations of said adenovirus are
followed by an intramuscular injection of at least one booster
immunization with an HIV-1 subunit antigen preparation.
8. The method of claim 7 wherein said HIV-1 subunit antigen
preparation contains an HIV-1 gag and/or env polypeptide
sequence.
9. The method of claim 1 wherein said intranasal dosage
administered is in the range of 1.times.10.sup.7 pfu of virus.
10. The method of claim 1 wherein said intramuscular dosage
administered is in the range of 1.times.10.sup.7 to
2.times.10.sup.9 pfu of virus.
11. The method of claim 9 wherein said intranasal booster is
administered in a dosage in the range of 1.times.10.sup.7 to
1.times.10.sup.8 pfu of virus.
12. The method of claim 10 wherein said intramuscular booster is
administered in a dosage in the range of 1.times.10.sup.10 to
8.times.10.sup.10 pfu of virus.
13. The method of claim 8 wherein said subunit antigen preparation
contains between 200 .mu.g and 0.5 mg of HIV-1 polypeptide.
14. A method of protecting a primate against HIV-1 infection
comprising the steps of (i) intranasal or intramuscular
administration to said primate of an intranasal or intramuscular
dosage of a recombinant adenovirus serotype 4, 5 or 7 having a
deletion in the E3 gene and an expression cassette containing a
major late promoter, a tripartite leader sequence, part or all of
the HIV-1 gp160 sequence, part of all of the coding sequence for
the HIV-1 rev gene inserted in frame after the HIV-1 gp160 sequence
and a polyadenylation signal sequence, said cassette being inserted
into said recombinant adenovirus between the E4 promoter and the
inverted terminal repeat of said recombinant adenovirus; and (ii),
followed by one or more intranasal or intramuscular booster
administrations of said recombinant adenovirus.
15. The method of claim 14 wherein said primate is a human.
16. The method of claim 15 wherein said HIV-1 gp 160 sequence is
replaced by a sequence encoding the gag-pro region of HIV-1.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation in part of Ser. No.
08/276,289, filed Jul. 20, 1994, which is a continuation in part of
Ser. No. 08/105,232, filed Aug. 11, 1993, which is a
continuation-in-part of Ser. No. 07/926,491, filed Aug. 7,
1992.
BACKGROUND OF THE INVENTION
[0002] A major goal of biomedical research is to provide protection
against viral disease through immunization. One approach has been
to use killed vaccines. However, large quantities of material are
required for killed vaccine in order to retain sufficient antigenic
mass. In addition, killed vaccines are often contaminated with
undesirable products during their preparation. Heterologous live
vaccines, using appropriately engineered adenovirus, which is
itself a vaccine, seems like an excellent immunogen [Chanock R.,
JAMA, 195, 151 (1967)]. Our invention concerns vaccines using
adenovirus as a vector.
[0003] Presently marketed adenovaccine comprises live, infectious
adenoviruses in an enteric-coated dosage form. Upon administration
to the patient to be vaccinated, the virus is carried past the
upper-respiratory system (where disease-producing infection is
thought to occur), and is released in the intestine. In the
intestine, the virus reproduces in the gut wall, where, although it
is not capable of causing adenoviral disease, nevertheless induces
the formation of adenovirus antibodies, thus conferring immunity to
adenoviral disease. In our invention, live, infectious adenovirus
which has been engineered to contain genes coding for antigens
produced by other disease-causing organisms. Upon release the virus
will reproduce and separately express both the adenoviral antigen
and the pathogen antigen, thereby inducing the formation of
antibodies or induce cell mediated immunity to both adenovirus and
the other disease-causing organism. By "live virus" is meant, in
contradistinction to "killed" virus, a virus which is, either by
itself or in conjunction with additional genetic material, capable
of producing identical progeny. By "infectious" is meant having the
capability to deliver the viral genome into cells.
[0004] Roy, in European Patent Publication 80,806 (1983), proposed
a method for producing immunity to microbial diseases by the
administration of a microbe containing a foreign gene which will
express an antigen of a second microbe to which immunity is
conferred. He states that preferred oral preparations are enteric
coated. Dubelcco proposed recombinant adenovirus vaccines in which
the surface protein of adenovirus is modified to contain in its
structure a segment of foreign protein which will produce a desired
biological response on administration to animals. [PCT
International Publication WO 83/02393 (1983)]. Davis discloses oral
vaccines derived from recombinant adenoviruses. [UK Patent GB
2166349 B].
[0005] Human immunodeficiency virus type 1 (HIV-1) has been
etiologically associated with acquired immunodeficiency syndrome
(AIDS) and related disorders. [Barre-Sinoussi, F., Science 220: 868
(1983); Gallo, R., Science 224: 500 (1984); Popovic, M., Science
224: 497 (1984); Sarngadharan, M., Science 224: 506 (1984)]. AIDS
is now a worldwide epidemic for which, currently, there is no
vaccine or cure. Most of the effort for vaccine development has
focused on the envelope (env) glycoprotein as an antigen which
might provide protective immunity. Antisera prepared against
purified gp 120 can neutralize HIV-1 in vitro. [Crowl, R., Cell 41:
979 (1985); Putney, S., Science 234: 1392 (1986); Ho, D., J. Virol.
61: 2024 (1987); Nara, P., Proc. Natl. Acad. Sci. USA 84: 3797
(1987)]. HIV-1 envelope antigen has been produced in different
expression systems including Escherichia coli [Crowl, R., Cell 41:
979 (1985); Chang, T., Bio/Technology 3: 905 (1985); Dawson, G., J.
Infect. Dis. 157: 149 (1988)] as well as mammalian [Chakrabarti,
S., Nature 320: 535 (1986); Dewar, R., J. Virol. 63: 129 (1989);
Rekosh, D., Proc. Natl. Acad. Sci. USA 85: 334 (1988); Whealy, M.,
J. Virol. 62: 4185 (1988)] yeast [Barr, P., Vaccine 5: 90 (1987)]
and insect cells [Hu, S., Nature 328: 721 (1978); Rusche, J., Proc.
Natl. Acad. Sci. USA 84: 6294 (1987)].
[0006] Live recombinant vaccinia virus expressing the entire HIV-1
env glycoprotein [Hu, S., J. Virol. 61: 3617 (1987)] or purified
recombinant gp 120 env glycoprotein [Berman, P., Proc. Natl. Acad.
Sci. USA 85: 5200 (1988)] were evaluated in chimpanzees as vaccine
candidates. Active immunization with these vaccines induced a good
cell-mediated immune response as well as cytotoxic T-cell activity
to the env antigen [Zarling, J., J. Immunol. 139: 988 (1987)]. All
experimental animals seroconverted as assayed by ELISA and Western
blotting. However, immunized chimpanzees developed no or only low
titers of neutralizing antibody to HIV-1. Challenge with live virus
failed to protect chimpanzees against these vaccines. Type-specific
HIV-1 neutralizing antibodies were found in chimpanzees early in
infection against a variable domain (V3) within the C-terminus half
of gp 120 [Goudsmit, J., Proc. Natl. Acad. Sci. USA 85: 4478
(1988)]. The recombinant gp 120 made in insect cells has also been
shown to induce humoral immune response in goat (Rusche J., Proc.
Natl. Acad. Sci. USA 84: 6294 (1987)]. Zagury [Nature 332: 728
(1988)] have demonstrated both anamnestic humoral and cellular
immune reaction in humans using a vaccine virus recombinant
expressing gp 160. [Chakrabarti, S., Nature 320: 535 (1986); Hahn,
B., Proc. Natl. Acad. Sci. USA 82: 4813 (1985)]. Both
group-specific cell-mediated immunity and cell-mediated
cytotoxicity against infected T4 cells were also found. These
results indicate that an immune state against HIV-1 can be obtained
in humans using recombinant env-based vaccine. Recently, Desrosiers
has shown that vaccination with inactivated whole simian
immunodeficiency virus (SIV) can protect macaques against challenge
with live SIV. [Proc. Natl. Acad. Sci. USA 86: 6353 (1989)]. These
data provide hope that vaccine protection against human AIDS virus,
HIV-1, infection may be possible.
[0007] Chanda discloses high level expression of the envelope
glycoproteins of HIV-1 in the presence of rev gene using
helper-independent adenovirus type 7 recombinants. [Virology 175:
535 (1990)]. Vernon discloses the ultrastructural characterization
of HIV-1 gag subunit in a recombinant adenovirus vector system. [J.
Gen. Virology 72: 1243 (1991)]. Vernon also discloses the
preparation of the HIV-1 recombinant adenoviruses Ad7-rev-gag and
Ad4-rev-gag.
SUMMARY OF THE INVENTION
[0008] This invention provides a method of producing antibodies or
cell mediated immunity to an infectious organism in a warm blooded
mammal which comprises administering to said warm blooded mammal
intranasally, intramuscularly, or subcutaneously, live recombinant
adenoviruses in which the virion structural protein is unchanged
from that in the native adenovirus from which the recombinant
adenovirus is produced, and which contain the gene coding for the
antigen corresponding to said antibodies or inducing said cell
mediated immunity. The warm blooded mammal is preferably a primate,
most preferably a human.
[0009] In its preferred embodiments, this invention provides a
method of producing antibodies to human immunodeficiency virus
(HIV-1), hepatitis B, hepatitis C, human papilloma virus,
respiratory syncytial virus, rotavirus, or parainfluenza virus in a
warm blooded mammal which comprises administering to said warm
blooded mammal intranasally, intramuscularly, or subcutaneously,
live recombinant adenoviruses in which the virion structural
protein is unchanged from that in the native adenovirus from which
the recombinant adenovirus is produced and which contain the gene
coding for, respectively, human immunodeficiency virus, hepatitis
B, hepatitis C, human papilloma virus, respiratory syncytial virus,
rotavirus, or parainfluenza virus.
[0010] This invention also provides composition for producing
antibodies or cell mediated immunity to an infectious organism in a
warm blooded mammal, comprising live recombinant adenoviruses in
which the virion structural protein is unchanged from that in the
native adenovirus from which the recombinant adenovirus is
produced, and which contain the gene coding for the antigen
corresponding to said antibodies or inducing said cell mediated
immunity, said composition being formulated in an intranasal,
intramuscular, or subcutaneous dosage form.
[0011] Although this specification specifically refers to
adenovirus of types 4, 5, or 7, live, infectious adenovirus of any
type may be employed in this invention. Additionally, while the
specification specifically refers to adenoviruses having an early
region 3 (E3) deletion, adenoviruses which are attenuated, contain
a temperature sensitive lesion, or a E1 deletion may also be used
as a vector. Similarly, although specific reference has been made
to vaccines producing antibodies to HIV, hepatitis B, hepatitis C,
human papilloma virus, respiratory syncytial virus, rotavirus, or
parainfluenza virus, our invention provides vaccines against any
infectious agent containing an antigen to which a warm-blooded
animal will produce antibodies or cell mediated immunity, and which
antigen is coded for by a gene composed of up to about 3000 base
pairs. Thus, for example, included within the scope of the
invention are immunization against such diseases as influenza,
hepatitis A, cholera, E. coli, pertussis, diphtheria, tetanus,
shigellosis, gonorrhea, mycoplasma pneumonia, and the like.
[0012] In one embodiment, the method of treatment includes
administering the recombinant adenovirus both prophylactically to
an HIV-1 susceptible mammal and as immunotherapy following
detection of HIV in said mammal. Regimens containing the following
recombinant adenoviruses were used to produce the anti-HIV
responses.
[0013] In a preferred embodiment, the method is a method of
protecting a primate against HIV-1 infection comprising intranasal
or intramuscular administration to said primate of an intranasal or
intramuscular dosage of a recombinant adenovirus having a deletion
in the E3 gene and an expression cassette containing a major late
promoter, a tripartite leader sequence, part or all of the HIV-1
gp160 sequence and a polyadenylation signal sequence. Preferably
the primate is a human. The expression cassette is inserted into
the recombinant adenovirus between the E4 promoter and the inverted
terminal repeat. Optionally the intranasal or intramuscular
administration of recombinant adenovirus is followed by one or more
intranasal or intramuscular booster administrations of the
recombinant adenovirus. The recombinant adenovirus is a serotype 4,
5 or 7 serotype adenovirus and optionally the expression cassette
additionally comprises part of all of the coding sequence for the
HIV-1 rev gene inserted in frame after the HIV-1 gp160 sequence and
before the polyadenylation signal sequence. The HIV-1 gp160
sequence can be from the MN strain gp160 sequence or the LAV strain
gp160 sequence. In an alternative embodiment, the HIV-1 gp160
sequence is replaced by a sequence encoding the gag-pro region of
HIV-1 In either embodiment, when the initial administration is
followed by one or more intranasal or intramuscular booster
administrations of the recombinant adenovirus, the last booster
administration may be followed by an intramuscular injection of at
least one booster immunization with an HIV-1 subunit antigen
preparation, preferably containing an HIV-1 gag and/or env
polypeptide sequence. For intranasal administration, the intranasal
dosage administered is in the range of 1.times.10.sup.7 pfu of
virus and for intramuscular administration, the intramuscular
dosage administered is in the range of 1.times.10.sup.7 to
2.times.10.sup.9 pfu of virus. The intranasal booster is
administered in a dosage in the range of 1.times.10.sup.7 to
1.times.10.sup.8 pfu of virus and the intramuscular booster is
administered in a dosage in the range of 1.times.10.sup.10 to
8.times.10.sup.10 pfu of virus. When a subunit antigen booster is
employed, the subunit antigen preparation contains between 200
.mu.g and 0.5 mg of HIV-1 polypeptide.
1 Virus Name Descriptive Name ATCC Name Ad7-env Ad7-tplenv-tplHrev
VR-2299 Ad7-gag Ad7-tplgag-tplHrev VR-2393 Ad7-gag-1 Ad7-rev-gag
VR-2392 Ad4-env Ad4-tplenv-tplHrev VR-2293 Ad4-gag
Ad4-tplgag-tplHrev VR-2391 Ad4-gag-1 Ad4-rev-gag VR-2390 Ad5-env
Ad5-tplenv-tplHrev VR-2297 Ad5-gag Ad5-tplgag-tplHrev VR-2298
Ad7-env.sub.MN Ad7-tplenv.sub.MN-tplHrev VR- Ad4-env.sub.MN
Ad4-tplenv.sub.MN-tplHrev VR- Ad5-env.sub.MN
Ad5-tplenv.sub.MN-tplHrev VR-
[0014] Referring to the above table Ad4, Ad5, and Ad7 refer to
human adenoviruses types 4, 5, and 7 respectively in which the E3
region has been deleted. Env refers to the HIV envelope
glycoprotein (gp 160) gene. Gag refers to the HIV gag/pro gene. Rev
refers to the HIV regulatory gene. Hrev refers to an altered
version of the rev gene where the nucleotide sequences were changed
without changing the amino acid sequence employing codons that were
frequently used in human genes. The sequence of Hrev is set forth
in FIG. 2. Tpl refers to the upstream adenovirus tripartite leader
sequence with an intervening sequence between the first and second
leaders that are positioned in front of the recombinant genes. The
constructs designated Ad7-env, Ad7-gag, Ad7-gag-1, Ad4-env,
Ad4-gag, Ad4-gag-1, Ad5-env, and Ad5-gag contain either the gag or
the env gene from the LAV strain of HIV and the constructs
Ad7-env.sub.MN, Ad4-env.sub.MN, and Ad5-env.sub.MN contain the env
gene from the MN strain of HIV. The recombinant adenoviruses made
from the LAV and MN strains of HIV-1 are illustrative of
recombinant adenoviruses covered by this invention. This invention
also covers recombinant adenoviruses which include the env and/or
gag genes from other strains of HIV-1.
[0015] Both the Ad-env and Ad-env.sub.MN adenoviruses were shown to
replicate in human A549 cells and expressed recombinant env antigen
in vitro demonstrating their capability of generating cell
mediated, humoral, and secretory immunity in a mammal.
[0016] As described in detail below, intranasal administration of
Ad-HIV recombinant viruses to naive chimpanzees resulted in both
priming and boosting of both humoral and cell-mediated immune
responses directed at HIV recombinant antigens. The recombinant
adenoviruses administered to chimpanzees were shown to produce
antibodies to the env and gag proteins of HIV. IgG antibodies
specific for HIV were observed in nasal, saliva, and vaginal
secretions following administration of the recombinant adenoviruses
and IgA antibodies specific for HIV were observed in nasal and
saliva secretions. The first set of recombinant viruses (Ad7)
appeared to be shed the longest period of time and induce the best
anti-Ad antibody response. The results also showed that
administration of Ad-HIV vaccines by the intranasal route was
superior to administration of enteric-coated recombinant viruses by
the oral route.
[0017] Optimum immune responses directed at HIV antigens required
primary infection one booster immunization with a heterotypic
recombinant Ad-HIV to elicit strong anti-HIV binding antibodies.
Intranasal administration of the Ad-HIV viruses effectively primed
chimpanzees to respond with high titered neutralizing antibodies to
HIV-1 following subsequent HIV-1 subunit protein booster
immunization.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows the inhibition of gp120 binding to CD4 by sera
induced in dogs by recombinant Ad-HIV vaccines.
[0019] FIG. 2 illustrates the DNA sequence of the expression
cassette containing the HIV gp160 coding sequence and the Hrev
coding sequence inserted into the E3 deleted region of Adenovirus
serotype 7 as described in Example 6.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Preparation of Representative Recombinant Adenoviruses
[0021] The following Examples show the construction of
representative recombinant adenoviruses of this invention. The
recombinant viruses were propagated on A549 cells and subsequently
titered on A549 cells.
EXAMPLE 1
Ad7-gag-1
[0022] The construction of recombinant adenoviruses containing the
gene for the HIV envelope protein has been described [Chanda, P.,
Virology 175: 535 (1990)]; a similar procedure was used to
incorporate gag and pro [see Vernon, S., J. Gen. Virology 72: 1243
(1991)]. Briefly, a DNA fragment containing the entire gag and pro
coding regions (bp 335 to 2165) of HIV-1 strain LAV [Wain-Hobson,
S., Cell 40: 9, (1985)] was constructed with a unique Sa/I site in
front of the AUG codon of the gag gene and an XbaI site at bp 2165,
for the insertion of the viral rev-responsive element (rre; bp 7178
to 7698). A 2.37 kb SaI fragment containing the three HIV-1
sequences was inserted at a SaI site in an expression cassette
containing the adenovirus type 7 (Ad7) major late promoter (MLP),
the tripartite leader (TPL) with an intervening sequence between
the first and second leaders, and the hexon polyadenylation site
(poly A) as described in Chanda [Virology 175: 535 (1990)]. The
cassette was inserted 159 bp from the right end of an Ad7 genome
[Sussenbach, The Adenoviruses, Ginsberg, ed., Plenum Press, pp.
34-124 (1984)] containing the HIV-1 rev gene [Feinberg, M., Cell
46: 807 (1986); Sodroski, J., Nature 321: 412 (1986)] in a deleted
[79.5 to 88.4 map units (m.u.)] E3 region [Chanda, P., Virology
175: 535 (1990)].
EXAMPLE 2
Ad4-gag-1
[0023] Following the procedure for the construction of the
Ad7-gag-1 recombinant adenovirus in Example 1, a similar expression
cassette containing analogous Ad4 sequences and the three HIV
coding regions were inserted at a site 139 bp from the right end of
an Ad4 genome which contained HIV-1 rev in an E3 deletion between
76 and 86 m.u.
EXAMPLE 3
Ad5-env
[0024] Ad5-tplenv-tplHrev contains the entire coding sequence of
HIV-1 (LAV strain) gp160 and a modified version of the rev gene,
called Hrev. Both the env as well as rev gene are preceded by a
synthetic copy of the Ad5 tripartite leader (Ad5-tpl). Ad5-tpl was
chemically synthesized and was cloned in pTZ vector. Then the gp
160 DNA sequence was inserted behind the Ad5-tpl to create
Ad5-tplenv/PTZ18R clone. The Hrev (.about.360 bp) was also
chemically synthesized where the nucleotide sequences were changed
without changing the amino acid sequence with the help of the codon
usage. This was done to avoid homologous recombination as some
identical sequences exist between env and rev. In an analogous way
like Ad5-tplenv construct, Hrev gene was also inserted behind tpl
in pTZ18R vector to create the plasmid, Ad5-tplHrev. The entire
sequence containing Ad5-tplHrev was excised out and then inserted
behind Ad5-tplenv to create the plasmid, Ad5-tplenv-tplHrev. This
plasmid was then inserted in the deleted E3 region of Ad5 Marietta
strain (78.8-85.7 mu deletion) at 78.8 mu. This plasmid was
linearized with BglI enzyme and then mixed with 0-87 mu SnaB1
fragment that was derived from the wild-type purified Ad5 virus.
After A549 cells were transfected with the DNA mixtures,
recombinant virus plaques were picked, plaque purified three times,
and their genomic structures were confirmed by restriction
endonuclease site analysis of DNA extracted from infected cells by
the method of Hirt. [J. Mol Bio. 26: 365 (1967)].
EXAMPLE 4
Ad5-gag
[0025] Ad5-tplgag-tplHrev contains the entire gag and pro region as
well as the modified rev gene, Hrev. A copy of the Ad5 synthetic
tripartite leader was placed in front of the gag and Hrev genes. A
DNA fragment containing the entire gag and pro regions (bp 335 to
2165 of LAV strain of HIV-1) was constructed with a unique SalI
site in front of AUG codon of the gag gene and an xba site at bp
2165, for the insertion of the viral rev-responsive element (rre;
bp 7178-7698). Two separate plasmids Ad5-tplgag as well as
Ad5-tplHrev were constructed in a similar way as described for
Ad5-tplenv-tplHrev. Then the Ad5-tplHrev fragment was inserted
behind Ad5-tplgag to create the plasmid Ad5-tplgag-tplHrev. Then
the fragment Ad5-tplgag-tplHrev was inserted at the unique XbaI
site at map position 78.8 of the Ad5 Marietta strain with an E3
deletion (78.8-85.7 mu E3 deletion). Then the final plasmid
containing the Ad5 sequence was linearized and then mixed with the
0-87 mu SnaB1 viral fragment for transfection. Recombinant plaques
were picked up, plaque purified three times, and were checked by
Hirt analysis of DNA extracted from the infected cells.
EXAMPLE 5
Enteric Coated Capsules
[0026] Recombinant adenoviruses were grown in A549 cells and
harvested following 3 cycles of freeze-thawing. Clarified infected
cell lysates were lypholized and 60 to 100 mg were packed into #2
gelatin capsules using a 1 ml syringe plunger under dehumidified
conditions. The capsules were coated with a 10% cellulose acetate
phthalate in acetone/100% Ethanol (1:1) by manually dipping each
end 6 times with air drying between dips. A coating between 69 to
77 mg of cellulose acetate phthalate was formed under these
conditions. Sample capsules were tested for resistance to simulated
gastric fluid (0.32% pepsin, 0.2% NaCl pH 1.2) at 37.degree. C.
using a VanKel Disintegration Testor apparatus for 1 hr. The
capsules were inspected for holes or cracks and transferred to a 15
ml tube containing 10 ml of simulated intestinal fluid (1.0%
pancreatin, 0.05 M monobasic potassium phosphate pH 7.5) and
rotated at 37.degree. C. All capsules tested were resistant to
simulated gastric fluid for 1 hr at 37.degree. C. with agitation
and began to dissolve within 15 min. in simulated intestinal fluid.
The amount of virus was titrated on confluent A549 cell monolayers
by a plaque assay and the viral DNA stability confirmed by Hirt
analysis.
EXAMPLE 6
Ad7-env.sub.MN
[0027] The construction of recombinant adenoviruses containing the
coding sequence of the env (gp 160) gene of MN strain of HIV-1 is
described briefly as follows: The 125 bp (6243 to 6367) fragment of
the amino (NH 2) terminus of the env (gp160) gene including the
initiation codon (ATG) as well as consensus Kozak sequence was
amplified by polymerase chain reaction (PCR) from the clone pMNST
1-8-9. This fragment was then cloned in pGEM vector and the
resultant clone was designated as pGEMMNenv. The following
fragments of DNA were isolated by digesting with the restriction
enzymes KpnI and XbaI from the clone PAd5tpl.sub.MNenv 223 (6367 bp
to 8816 bp), XhoI+KpnI fragment from PGEMenv and salI+XbaI fragment
from pAd7tpl 18RD. All of these fragments were ligated together and
the resultant clone was designated as pAd7tpl.sub.MNenv. This
plasmid was then digested with XbaI and treated with calf intestine
alkaline phosphatase (CIAP). The NheI+XbaI fragment of Hrev gene
was then isolated from the plasmid, pAd7tplHrev 18RD. The clone
that was obtained after ligating these two fragments together was
designated as pAD7tpl.sub.MNenvtplHrev. This plasmid was then
digested with NheI+XbaI and then ligated to the E3 deletion plasmid
of Ad7, pAd7.DELTA.E3 (68 m.u. to 100 m.u. deletion) that was also
digested with XbaI and then treated with CIAP. The resultant
plasmid was designated as pAD7.DELTA.E3tpl.sub.MNenvtpl.sub.MNHrev.
This plasmid was digested with EcoRI and mixed with the EcoRI (0-87
m.u.) fragment of the Ad7 genomic DNA. A549 cells were then
transfected with these DNAs. Recombinant plaques obtained from in
vivo recombination were identified by the appropriate restriction
digestion analyses of the Hirt DNA. The plaques were also
identified by the production of gp160, gp120, and gp41 using
appropriate antibodies on Western blots.
[0028] FIG. 2 illustrates the complete DNA sequence of the
expression cassette containing the HIV gp160 coding sequence and
the Hrev coding sequence inserted into the E3 deleted region of
Adenovirus serotype 7 as described above. The first 200 bp
tripartite leader sequence begins at bp 88, the HIV gp 160 sequence
extends from bp 306 through bp 2879, the second tripartite leader
sequence extends from bp 2886 through bp 3085 and the Hrev sequence
extends from bp 3099 through 3449 in the Ad7 deleted E3 region.
EXAMPLE 7
Ad4-env.sub.MN and Ad5-env.sub.MN
[0029] The construction of Ad4 and Ad5 recombinants are the same as
that of Ad7-env.sub.MN except that for Ad4, EcoRI digested DNA from
pAd4.DELTA.E3tpl.sub.MNenvtplHrev was combined with the BclI (0-87
m.u.) fragment from the Ad4 genomic DNA to produce the recombinant
Ad4 virus. Similarly for Ad5, MluI-digested DNA from
pAd5.DELTA.E3tpl.sub.MNenvtplHr- ev was combined with the SpeI
(0-75 m.u.) fragment of Ad5 genomic DNA to produce the recombinant
Ad5 adenovirus. Like Ad7, both Ad4 and Ad5 recombinants were
obtained from A549 cells.
EXAMPLE 8
Subunit Antigen Preparation
[0030] gp-120.sub.MN was prepared according to Kaufman, R. J.,
Nucleic Acid Res. 19: 4485 (1991) and was used in SAF-m adjuvant
(Allison, A. C., J. Imm. Meth. 95: 157 (1986). gp-120.sub.SF2 was
prepared according to Scandella, C. J., AIDS Res. Human
Retroviruses 9: 1233 (1993) and was used in MF-59 adjuvant (Keitel,
W., Vaccine 11: 909 (1993)). HA-env.sub.Kl17K was prepared
according to Kalayan, N., Vaccine 12: 753 (1994) and was used in
SAF-m adjuvant.
[0031] Measurement of Repilcation and Antigen Expression
[0032] Human A549 cells were infected (MOI 10:1) with recombinant
adenovirus types 4, 5, and 7 that contained either the LAV or MN
env genes. At 34 hours post-infection, virus titer and env antigen
expression was determined in duplicate samples. One dish of
infected cells was subjected to 3 cycles of freeze thawing and the
cell lysate was tested for the presence of infectious virus by
plaque assay. The second culture dish was washed, detergent
solubilized, and an aliquot of the cell lysate was loaded on to a
10% polyacrylamide gel. Following electrophoresis, the separated
proteins were transferred to nitrocellulose by a Western blot
apparatus. The transferred proteins were immunostained with
anti-env reagents. A known standard, recombinant gp160, was added
prior to electrophoresis. The resulting immunoblot was scanned by a
densitometer and the amount of recombinant env determined. There
were no significant differences seen between wild type adenoviruses
and the recombinant adenoviruses expressing either the LAV or MN
env gene. Both types of recombinant adenoviruses, LAV or MN,
produced similar amounts of env antigen. Therefore, both types of
Ad-env recombinants, LAV and MN, were able to grow in human A549
cells as well as their corresponding wild type adenovirus, and were
able to express recombinant env antigen. These results therefore
demonstrate that both the LAV and MN adenovirus recombinants are
capable of generating cell mediated, humoral, and secretory
immunity in a mammal. The data obtained are summarized in the table
below.
2 ADENOVIRUS REPLICATION AND ANTIGEN EXPRESSION Adenovirus pfu/cell
.times. 10.sup.2 .mu.g env/10.sup.6 cells Ad4 wild type 5.4 0
Ad4-env 9.1 2.1 Ad4-env.sub.MN 6.8 2.7 Ad5 wild type 22 0 Ad5-env
86 5.4 Ad5-env.sub.MN 18 5.7 Ad7 wild type 18 0 Ad7-env 11 3.1
Ad7-env.sub.MN 7.8 3.6
[0033] Treatment Regimens
[0034] Immunogenicity of the recombinant adenoviruses for HIV was
evaluated in chimpanzees under four treatment regimens (1, 2, 3,
and 6), and in dogs two treatment regimens (4 and 5). Protection
against HIV-1 infection was evaluated in chimpanzees in the sixth
treatment regimen. The first regimen consisted of administering the
recombinant adenovirus orally via an enterically coated capsule
(Example 5) at 0, 7, and 26 weeks followed by an env+gag subunit
protein booster using alum as an adjuvant. The second regimen
consisted of further treating the chimpanzees that received regimen
1 at 46 and 58 weeks with additional boosters of recombinant
adenovirus administered intranasally. The third treatment regimen
consisted of administering recombinant adenovirus intranasally to
naive chimpanzees at weeks 0, 24, and 52 followed by an env subunit
booster at week 75. The fourth treatment regimen consisted of
administering recombinant adenoviruses derived from both the LAV
and MN strains of HIV-1 to dogs.
[0035] The fifth treatment regiment consisted of administering env
subunit boosters to either previously immunized or control dogs.
Each treatment group consisted of 6 previously immunized dogs and 2
control dogs. Of the previously immunized dogs, six had received
Treatment Regimen 4 (Group A); six had received Treatment Regimen 4
(Group D); six had received Ad-env.sub.HXB2 (expressing a portion
of the HIV env V3 loop, derived from the LAV strain of HIV); and
twelve had previously received Ad-env.sub.HXB2 (expressing a
portion of the HIV env V3 loop, derived from the MN strain of HIV)
(prepared according to Robert-Guroff, M., J. Virol 68: 3459 (1994)
and Veronese, F. D., J. Biol. Chem. 268: 25894 (1993)).
[0036] The sixth treatment regimen consisted of administering
Ad-env.sub.MN recombinants to chimpanzees, followed by 0, 1 or 2
Ad-env.sub.MN booster immunizations using heterologous Ad vectors.
The chimpanzees were then given one or two booster immunizations
with env (gp120.sub.SF2) subunit antigen preparations, followed by
a challenge with the SF2 strain of HIV.
[0037] The following table summarizes treatment regimens 1 and
2.
3 TREATMENT REGIMENS 1 AND 2 Immunization Time Chimpanzees 1 and 2
Chimpanzee 3 Regimen 1 Primary* 0 weeks 1.5 .times. 10.sup.7 pfu
Ad7-env 1.5 .times. 10.sup.7 pfu 2.0 .times. 10.sup.9 pfu Ad7-gag-1
Ad7-env 1st Booster* 7 weeks 1.1 .times. 10.sup.10 pfu Ad4-env 1.1
.times. 10.sup.10 pfu 1.0 .times. 10.sup.10 pfu Ad4-gag-1 Ad4-env
2nd Booster* 26 weeks 7.9 .times. 10.sup.10 pfu Ad5-env 7.9 .times.
10.sup.10 pfu Ad5-env 3rd Booster.sup.+ 34 weeks 200 ug env in 0.2%
alum 200 .mu.g env in alum 500 ug env in 0.2% alum 0.2% Regimen 2
1st Intranasal 46 weeks 1.0 .times. 10.sup.8 pfu Ad7-env 1.0
.times. 10.sup.8 pfu Boost 1.0 .times. 10.sup.8 pfu Ad7-gag Ad7-env
2nd Intranasal 58 weeks 1.0 .times. 10.sup.8 pfu Ad4-env 1.0
.times. 10.sup.8 pfu Boost 1.0 .times. 10.sup.8 pfu Ad4-gag Ad4-env
*Each dose was administered in enteric-coated gelatin capsules on 3
consecutive days. .sup.+Administered intramuscularly.
[0038] The following table summarizes treatment regimen 3.
4 TREATMENT REGIMEN 3 Immunization Time Chimpanzees 4 and 5
Chimpanzee 6 Primary* 0 weeks 1.0 .times. 10.sup.7 pfu Ad7-env 1.5
.times. 10.sup.7 pfu Ad7- 1.0 .times. 10.sup.7 pfu Ad7-gag env 1st
Booster* 24 weeks 1.0 .times. 10.sup.7 pfu Ad4-env 1.5 .times.
10.sup.7 pfu Ad4- 1.0 .times. 10.sup.7 pfu Ad4-gag env 2nd Booster*
52 weeks 1.0 .times. 10.sup.7 pfu Ad5-env 1.5 .times. 10.sup.7 pfu
Ad5- 1.0 .times. 10.sup.7 pfu Ad5-gag env 3rd Booster.sup.+ 75
weeks 0.5 mg env 0.5 mg env *Administered intranasally.
.sup.+Administered intramuscularly.
[0039] The following Table summarizes treatment regimen 4.
5 TREATMENT REGIMEN 4 Group A Group B Group C Group D Immunization
Time (n = 6) (n = 3) (n = 3) (n = 6) Primary* 0 weeks
Ad7-env.sub.MN Ad7-env Ad7-env.sub.MN + Ad7-env Ad5-env.sub.MN 1st
Booster* 12 weeks Ad5-env.sub.MN Ad5-env Ad5-env.sub.MN + Ad5-env
Ad4-env.sub.MN *Each recombinant adenovirus was administered
intratracheally at a dose of 1 .times. 10.sup.9 per dog.
[0040] The following summarizes treatment regimen 5. Each group
consisted of 6 dogs that were previously immunized, as described
above, and 2 control dogs. Each group received 50 .mu.g of the
subunit in adjuvant at 0 weeks (20-28 weeks from the last Ad-env
administration). Group A received gp120.sub.SF2 in MF59 adjuvant;
Group B received CHO-derived gp120.sub.MN (antibody purified) in
SAF-m; Group C received Ad5-gp160.sub.MN-derived gp160.sub.MN
(lentil lectin purified) in SAF-m; Group D received
Ad5-gp160.sub.MN-derived gp160.sub.MN (lentil lectin purified) in
MF59; and Group E received HA-env.sub.K17K (expressing a portion of
the HIV env V3 loop). Twelve weeks later dogs were identically
boosted with the same subunit, with the exception of Group D dogs
which were reboosted with the HA-env.sub.Kl7K.
[0041] The following table summarizes treatment regimen 6.
6 TREATMENT REGIMEN 6 Time Chimpanzee Number Immunization (weeks) 7
8 & 9 10 11 12 Primary 0 Ad5-env.sub.MN.sup.+ Ad5-env.sub.MN
Ad5-env.sub.MN, Ad5-env.sub.MN Ad5 wild type Ad7-env.sub.MN,
Ad4-env.sub.MN 1st Booster 12 -- Ad7-env.sub.MN Ad5-env.sub.MN,
Ad7-env.sub.MN Ad7 wild type Ad7-env.sub.MN, Ad4-env.sub.MN 2nd
Booster 24 -- -- -- Ad4-env.sub.MN Ad4 wild type Subunit Boost 26
gp120.sub.SF2.sup.* -- -- -- -- Subunit Boost 38 gp120.sub.SF2
gp120.sub.SF2 gp120.sub.SF2 -- Subunit Boost 48 -- -- --
gp120.sub.SF2 MF59 Challenge # HIV.sub.SF2 HIV.sub.SF2 --
HIV.sub.SF2 HIV.sub.SF2 .sup.+All Ad-env and Ad viruses were
administered at a dose of 1.0 .times. 10.sup.7 pfu/virus
intranasally. *50 .mu.g HIV gp120.sub.SF2 formulated in MF59
adjuvant was administered intramuscularly. .sup.#Chimpanzees 7, 8,
and 9 were challenged at 40 weeks; 11 and 12 were challenged at 52
weeks, and 10 was not challenged.
[0042] Measurement of Immunogenicity: Treatment Regimen 1
[0043] Chimpanzee Inoculations
[0044] Three chimpanzees (2 males and 1 female) that were screened
negative for the presence of neutralizing antibodies to human
adenoviruses type 4, and 7 were evaluated using treatment regimen
1. Enteric-coated capsules containing recombinant adenoviruses were
given using a stomach tube under anesthesia on three consecutive
days. Two chimpanzees (1 and 2) received both env and gag
recombinant viruses while the third chimp (3) received only env
recombinant viruses.
[0045] Adenovirus-derived subunit preparations containing env or
gag gene products were purified from infected A549 cell cultures
[see Vernon, S., J. Gen. Virology 72: 1243 (1991) and Natuk, R.,
Proc. Natl. Acad. Sci. USA 89: 7777 (1992)]. Recombinant antigens
were formulated with alum adjuvant and administered
intramuscularly, 200 ug/dose env and 500 ug/dose gag particles.
[0046] Whole blood, serum, and stool samples were collected at
different times during the course of the experiment. Whole blood
was processed to obtain white blood cell populations for FACS, HIV
CTL (using recombinant vaccinia viruses expressing HIV-env,
HIV-gag, or the lac gene products), and for lymphoproliferative
assays to purified HIV recombinant gp160, gp120, and p24. Serum and
stool specimens were stored at -70.degree. C. until use.
[0047] Detection of Recombinant Adenoviruses in Stool Specimens
[0048] Chimpanzee stool specimens were thawed and 10% (V/V)
suspensions were made into antibiotic containing DMEM. Clarified
stool suspensions were used to infect confluent A549 cell
monolayers in 60 mm tissue culture dishes. After a 1 hr adsorption
period the unbound material was washed away and the monolayers were
overlaid with an 0.5% agar overlay medium. Plaques were allowed to
develop for 7-10 days and plaques were visualized by neutral red
staining, counted and the agar overlay was gently removed taking
care not to disturb the cell monolayer. The cell sheet was
transferred to nitrocellulose filter membranes (Millipore Type HA,
0.45 um), presoaked in 20.times.SSC and placed on the cell layer
and left in contact with the cell monolayer for 2 to 4 minutes. The
filters were peeled off, air-dried, and baked for 2 hr in a vacuum
oven at 80.degree. C. Nitrocellulose filters were washed twice in
3.times.SSc/0.1% SDS at room temperature and prehybridized and
hybridized according to standard procedures [Poncet, D., J. Virol.
Methods 26: 27 (1989)]. .sup.32P-labeled oligo-probes were added to
the hybridization buffer (1.times.10.sup.6 CPM) and incubated
overnight at 42.degree. C. DNA probes were prepared by which could
detect either Ad4 fiber, Ad5 fiber, Ad7 fiber, HIV-env or HIV-gag
specific sequences. [Wain-Hobson, Cell 40: 9 (1985)]. The filters
were washed, autoradiographed, and hybridization signals were
counted.
[0049] Adenovirus Neutralization Test Procedures
[0050] Serial 2-fold dilutions (starting with 1:4) of
heat-inactivated (56.degree. C. fdor 30 min.) dog serum were made
in 96-well microtiter plates (0.05 ml/well) and were mixed with
0.05 ml media containing 30-100 TCID.sub.50 virus for 1 hr at
37.degree. C. To each well 0.05 ml of media containing
2.times.10.sup.4 A549 cells were added and the plates were
incubated at 37.degree. C. 5% CO.sub.2 for 7-10 days. All samples
were done in duplicate. Virus and uninfected cell controls were
included in each assay for determining the end point in test sera.
Titers were expressed as the reciprocal of the lowest dilution at
which 50% cytopathic effect was observed.
[0051] Detection of Anti-HIV Antibodies by ELISA and Western
Blotting
[0052] Detection of anti-HIV antibodies Chimpanzee antibody
responses to HIV-1 antigens were measured by testing various
dilutions by commercial ELISA and Western blot kits as instructed
by the manufacturers (DuPont, Wilmington, Del.).
[0053] Results
[0054] Feces were collected from each chimpanzee prior to and after
virus inoculation and stored at -70.degree. C. Ten percent
suspensions were prepared from each sample and were used to infect
confluent A549 cell monolayers. After 7-10 days viral plaques were
identified by neutral red staining and the cell monolayers were
transferred to nitrocellulose membranes. Representative samples
were hybridized with various labeled oligo-probes to detect
sequences specific for Ad4, Ad5, Ad7, HIV-env, or HIV-gag genes.
Identification of specific recombinant Ad-HIV viruses could be
determined by this plaque hybridization technique. None of the
recombinant viruses were shed into the feces for longer than 7 days
p.i. Peak titers were always associated with 1-3 day samples and
most likely represented the non-adsorbed virus inoculum. Previous
chimp studies using Ad-HBsAg recombinants had indicated that
Ad-HBsAg recombinants could be detected for 30-40 days p.i. With
the enteric capsule route of administration, it appeared that these
recombinant viruses did not replicate well in vivo.
[0055] Seroconversion to the serotype of the adenovirus vectors
employed was determined by neutralization test procedures. Very low
to modest anti-adenovirus serum titers were measured to all 3
serotypes used in each of the chimpanzees.
[0056] Seroconversion to recombinant HIV gene products were
determined by either ELISA or Western blotting techniques. No ELISA
response was detected in any of the chimpanzees prior to the second
booster inoculation with the Ad5-env recombinant. Two weeks
following Ad5-env inoculation anti-env responses could be measured
in 2 of the 3 animals. Intramuscular injection of gag and/or env
preparations had a slight boosting effect in 1 of the 3 animals.
Western blot analysis appeared to be much more sensitive than the
ELISA and had the further advantage of identification of which env
and/or gag gene products were being recognized as being
immunogenic. Low serum antibody titers were measured following both
the primary Ad7 recombinant and first booster with Ad4 recombinants
viruses. A significant increase in serum titer to env gene products
was observed following the second booster immunization with the
Ad5-env recombinant. Significant increases in the 2 animals which
received gag gene products were seen following injection with
subunit preparations. Despite relatively good Western blot titers
to HIV antigens, only 1 of the 3 animals responded with serum
neutralizing antibodies. This response in chimpanzee 2 was very low
(titer of 10 to 20). These results are summarized in the following
table.
7 RESULTS OBTAINED USING TREATMENT REGIMEN 1 Recombinant Western
Blot Peak Peak Chimp Recombinant Virus Shedding Peak Anti-Adeno
anti-HIV Titers Anti-HIV Number Virus Stools (Days) Neutralizing
Titer env gag Neutralizing Titer 1 Ad7-env, Ad7-gag-1 2, 2 128 --
20 <10 Ad4-env, Ad4-gag-1 2, 2 8 -- 20 <10 Ad5-env 7+ 128 100
-- <10 subunit: env + gag 100 1000 <10 2 Ad7-env, Ad7-gag-1
3, 2 64 -- 20 <10 Ad4-env, Ad4-gag-1 1, 7 128 20 100 <10
Ad5-env 7+ 64 10000 -- 20 subunit: env + gag 1000 10000 10 3
Ad7-env 2 6 20 N/A* <10 Ad4-env 1 128 20 N/A <10 Ad5-env 7+
512 1000 N/A <10 subunit: env 100 N/A <10 *N/A = not
applicable.
[0057] Cell-mediated immunity was measured in peripheral blood
mononuclear cell population obtained from chimpanzees. HIV specific
CTL activity was measured by determining lysis of syngenic target
cells that were infected with vaccinia virus recombinants that
express either the HIV-env gene products, the HIV-gag gene
products, or the lac gene product (control for nonspecific
cytotoxicity). A hint of HIV specific CTL-like activity was
measured in this way.
[0058] Lymphoproliferative assays were performed to determine
whether purified recombinant env (gp160, gp120) or gag (p24)
preparations were capable of stimulating blastogenesis. No
proliferation was measured after the primary inoculum and only 1 of
the 3 animals show a lymphoproliferative response following
administration of the first boost with Ad4 recombinant viruses. All
3 animals responded with proliferative responses after the second
booster (Ad5-env) and the third boost (subunit preparations).
[0059] Measurement of Immunogenicity: Treatment Regimen 2
[0060] Chimpanzee Inoculations and Collection of Data
[0061] Three chimpanzees (2 males and 1 female) that were
previously inoculated with Ad-HIV recombinant viruses in
enteric-coated capsules and boosted with adenovirus-derived gag
and/or env subunits (treatment regimen 1) were infected
intranasally with Ad7-HIV viruses (week 46) and 12 weeks later
(week 58) with Ad4-HIV viruses. Recombinant adenoviruses were given
in tissue culture media diluted with phosphate saline buffer
dropwise into the nostrils of chimpanzees under anesthesia. Two
chimpanzees (numbers 1 and 2) received both env and gag recombinant
viruses while the third chimp (number 3) received only env
recombinant viruses.
[0062] Whole blood, serum, and stool samples were collected at
different times during the course of the experiment, and processed
as described in Regimen 1. Adenovirus detection in stool samples or
nasal swabs, adenovirus neutralization test procedures, and
detection of anti-HIV antibodies were performed according to the
procedures described in Regimen 1.
[0063] Results
[0064] The first intranasal booster with Ad7 recombinants was given
in one dose of 1.times.10.sup.8 pfu's/chimpanzee. At the time of
virus administration chimpanzees 3, 1, and 2 had serum anti-Ad7
neutralization titers of <4, 8, and 64 respectively from
previous oral immunizations. Nasal swabs and stool samples were
examined for the presence of shed recombinant viruses by a plaque
hybridization technique. Recombinant Ad7-env was detected in nasal
swabs up to 7 days p.i. in two of the animals. Recombinant Ad7-env
and Ad7-gag were found to be present in stool samples from 5 to 12
days p.i. There was a correlation between the serum titer to Ad7
and the ability to detect recombinant viruses in nasal swabs and
stool specimens. The two animals which displayed marginal anti-HIV
antibody response were greatly augmented by the intranasal boost.
The third animal was boosted to a lesser extent. Low titered
neutralizing antibodies directed at HIV could now be detected in
all three animals. Secretory antibodies were detected in nasal swab
specimens which contained anti-gag and/or env binding antibodies.
No signs or symptoms of respiratory disease were observed in these
animals as a result of intranasal administration of the Ad7
recombinant viruses.
[0065] Three months later these chimpanzees were immunized with Ad4
recombinants at a single dose of 1.times.10.sup.8
pfu's/chimpanzee/virus. These animals had serum anti-Ad4
neutralization titers between 128 to 256 from previous oral
immunization at the time of intranasal challenge. At 3 days
post-infection 2 of the animals (2 and 3) had a slight cough. The
third animal (number 1) died on day 5 from a bacterial pneumonia
(Streptococcus pneumoniae was isolated). The other two animals
presented harsh sounds by auscultation and S. pneumoniae was
isolated from both chimpanzees. Antibiotic treatments were
initiated and both chimpanzees recovered.
[0066] Upon retrospective examination of this situation several
observations could be made. At the time of intranasal
administration chimpanzee number 1 was already experiencing a fever
and an abnormal Complete Blood Count. There was a disproportionate
number of polymorphonuclear cells present and a 5% level of band
cells (immature polymorphonuclear cells) taken together, this
information indicated that there was a significant bacterial
infection taking place prior to virus inoculation. Autopsy
specimens taken from the lung, liver, spleen, and serum all tested
negative for the presence of infectious adenovirus by tissue
culture using 3 blind passages on susceptible A549 cell monolayers.
Similar findings were obtained by plaque hybridization techniques.
Lung and liver paraffin embedded samples tested negative for the
presence of adenovirus antigens using a commercial
immunofluorescent kit for adenovirus antigens. Inclusion bodies
were observed in H&E stained lung sections. There was a
disagreement by experts as to whether these inclusions were caused
by adenovirus or not. Several weeks later another chimpanzee
experienced a similar fate at the same primate center and died.
While it was likely that administration of recombinant adenoviruses
had a only a minor role, if any, in causing the death of chimpanzee
number 1 it was considered prudent to administer antibiotics
prophylactically prior to and after any further intranasal
administration of adenovirus recombinants to chimpanzees.
[0067] The following table shows the results obtained using
treatment Regimen 1 and the Ad7-recombinants in Regimen 2.
8 RESULTS OBTAINED USING TREATMENT REGIMENS 1 AND 2 Recombinant
Western Blot Peak Peak Chimp Recombinant Virus Shedding Peak
Anti-Adeno anti-HIV Titers Anti-HIV Number Virus Stools (Days)
Neutralizing Titer env gag Neutralizing Titer 1 Regimen 1 Ad7-env,
Ad7-gag-1 2, 2 128 -- 20 <10 Ad4-env, Ad4-gag-1 2, 2 8 -- 20
<10 Ad5-env 7+ 128 100 -- <10 subunit: env + gag 100 1000
<10 Regimen 2 Ad7-env, Ad7-gag 12 512 10000 10000 10 2 Regimen 1
Ad7-env, Ad7-gag-1 3, 2 64 -- 20 <10 Ad4-env, Ad4-gag-1 1, 7 128
20 100 <10 Ad5-env 7+ 64 10000 -- 20 subunit: env + gag 1000
10000 10 Regimen 2 Ad7-env, Ad7-gag 9 8192 1000 10000 20 3 Regimen
1 Ad7-env 2 6 20 N/A* <10 Ad4-env 1 128 20 N/A <10 Ad5-env 7+
512 1000 N/A <10 subunit: env 100 N/A <10 Regimen 2 Ad7-env 7
256 10000 N/A 10 *N/A = not applicable.
[0068] Measurement of Immunogenicity: Treatment Regimen 3
[0069] Chimpanzee Inoculations and Collection of Data
[0070] Three chimpanzees (2 males and 1 female) that were screened
negative for the presence of neutralizing antibodies to human
adenoviruses type 4, 5, and 7 were evaluated using treatment
regimen 3. Two chimpanzees (numbers 4 and 5) received both env and
gag recombinant viruses while the third chimp (number 6) received
only env recombinant viruses. Antibiotics were administered
prophylactically to the chimpanzees and no respiratory disorders
were observed.
[0071] Whole blood, serum, and stool samples were collected at
different times during the course of the experiment, and processed
as described in Regimen 1. Adenovirus detection in stool samples or
nasal swabs, adenovirus neutralization assays, and detection of
anti-V antibodies were performed according to the procedures
described in Regimen 1.
[0072] Detection of Inhibition of Gp120 Binding to CD4 Binding
Sites
[0073] This assay is designed to measure the ability of chimpanzee
anti-env antibodies to block the interaction of the HIV gp120
antigen with in natural ligand CD4. Various dilutions of chimpanzee
sera were incubated with purified recombinant gp120 (1 ug/ml)
37.degree. C. for 1 hour. HeLa CD4 positive cells
(5.times.10.sup.5) were added to this mixture and incubated at
4.degree. C. for 1 hour. The cells were washed 3 times with PBS-5%
BSA and mixed with a FITC-labeled monoclonal antibody directed at
the CD4 antigen (same site the gp120 binds to) and incubated at
4.degree. C. for 1 hour. The cells were washed three times with the
PBS-5% BSA and analyzed by flow cytometry.
[0074] Results
[0075] 1st Immunization with Ad7-recombinants: Recombinant viruses
were shed into feces for 22 to 34 days post-infection. No
recombinant viruses were detected in nasal secretions taken at 2
weeks post-infection. Seroconversion to the serotype of the
adenovirus vectors employed was determined by neutralization
assays. Excellent anti-adenovirus serum titers were measured in all
3 chimpanzees to Ad7 serotypes used in each of the chimpanzees.
Seroconversion to recombinant HIV gene products were determined by
Western blotting. Four weeks following the primary immunization
with Ad7-recombinants anti-env and anti-gag responses could be
measured in 2 of the 3 chimpanzees. By 20 weeks post-infection all
3 animals had measurable antibodies to HIV antigens. Secretory
antibodies were not found in nasal swabs taken within the first 4
weeks following primary immunization. All 3 chimpanzees failed to
mount detectable anti-HIV neutralizing antibody responses.
[0076] 1st Booster Immunization with Ad4-recombinants: Recombinant
Ad4 viruses were shed into feces for 14-28 days post-infection.
Examination of nasal swabs indicated that recombinant Ad4 viruses
could be detected in all 3 chimpanzees for at least 7 days
post-infection. Significant anti-Ad4 responses were mounted against
the Ad4 serotype following intranasal administration. The magnitude
was slightly lower then that measured against the Ad7-recombinant
viruses. Excellent booster responses to gag and/or env antigens
were measured in all three animals. Low titered (1:2) anti-gag
and/or anti-env responses were measured in nasal swabs from
Chimpanzees 4 and 5. Still no anti-HIV neutralizing antibodies were
measured in any of the animals.
[0077] 2nd Booster Immunization with Ad5-recombinants: Recombinant
Ad5 viruses were shed into feces for 8 days post-infection. No
recombinant viruses could be detected in nasal swabs at 0, 1, or 2
weeks post-inoculation. Anti-HIV IgG and IgA antibody response
against env and gag could be measured in nasal swabs taken from 2
of 3 chimpanzees following Ad5-recombinant booster immunization by
Western blot analysis. IgG and IgA anti-env and/or anti-gag
antibodies were detected in saliva samples collected from 2 of 3
chimpanzees. Anti-env and -gag antibodies of the IgG class were
detected in vaginal swabs taken from the single female
chimpanzee.
[0078] Several samples which contained the greatest amount of
anti-HIV antibodies of the IgA class were examined for the presence
of secretory component. This was accomplished by substitution of
polyclonal anti-secretory component (human) for polyclonal anti-IgA
(human) in the HIV Western blot assay. Secretory anti-HIV IgA,
containing secretory component, was detected in both nasal swabs
and saliva samples in 1 of 3 chimpanzees.
[0079] 3rd Booster Immunization with env Subunit: The strongest
anti-env antibody responses were measured following subunit
administration of these chimpanzees that had been primed with live
recombinant adenoviruses. Anti-env antibody responses were detected
in both serum and in various secretory samples collected from the
nasal-oral cavity, vagina, and rectum. Peak antibody titers were
detected at 4 weeks post administration with env subunit.
[0080] Serum anti-HIV neutralizing antibody titers of 320-640 were
observed in all 3 chimpanzees. Antibodies directed against the
gp120 V3 loop were detected by ELISA and against the gp120 CD4
binding site were detected by a FACS blocking assay. All three
chimpanzees produced high ELISA titers (1000-9000) directed at the
V3 loop (a region which contains the major neutralization
determinant for HIV).
[0081] Chimpanzee sera collected at the height of the neutralizing
response was evaluated for the presence of anti-CD4 binding site
antibodies. All three animals had acquired antibodies that were
capable of blocking the interaction between gp120 with CD4. The CD4
binding site is a conformational epitope and antibodies directed at
this site are believed to be important in blocking uptake up
cell-free HIV and perhaps capable of inhibiting gp 120-CD4
syncytium induction. The results are shown in FIG. 1.
[0082] Nasal swab anti-env antibody titers of the IgG and IgA
classes of immunoglobulins were boosted in 3 of 3 and 2 of 3
chimpanzees, respectively, following booster immunization with the
env subunit. Similar results were observed in the saliva samples
taken from these chimpanzees. Two of three chimpanzees had IgG
anti-env antibodies present in rectal swabs and the single female
chimpanzee had a strong IgG anti-env booster response measured in
vaginal swabs. The presence of anti-HIV antibodies in mucosal
secretions is critical because certain mucosal surfaces represent
major sites for HIV infection.
[0083] Summary Tables: The following table shows the results
obtained using treatment regimen 3.
9 RESULTS OBTAINED USING TREATMENT REGIMEN 3 Recombinant Western
Blot Peak Peak Chimp Recombinant Virus Shedding Peak Anti-Adeno
anti-HIV Titers Anti-HIV Number Virus Stools (Days) Neutralizing
Titer env gag Neutralizing Titer 4 Ad7-env, Ad7-gag 22, 22 1024 100
1000 <10 Ad4-env, Ad4-gag 14, 14 128 10000 10000 <10 Ad5-env,
Ad5-gag 8, 8 32 10000 10000 20 subunit: env N/A* N/A >10000
10000 640 5 Ad7-env, Ad7-gag 34, 27 1024 100 10000 <10 Ad4-env,
Ad4-gag 14, 14 512 10000 10000 <10 Ad5-env, Ad5-gag 8, 8 32
10000 10000 20 subunit: env N/A N/A 1000 10000 320 6 Ad7-env 34
1024 100 N/A <10 Ad4-env 28 512 10000 N/A <10 Ad5-env, gag 8,
8 32 10000 N/A 40 subunit: env N/A N/A >10000 N/A 320 *N/A = not
applicable.
[0084] The following table summarizes anti-HIV responses detected
in chimpanzee secretions following intranasal booster immunization
with the Ad5-HIV recombinants and after the intramuscular subunit
boost (week 23 post boost).
10 ANTI-HIV RESPONSES DETECTED IN SECRETIONS Antigen Weeks
Secretion Analyzed Chimp Rec- Post Nasal Saliva Vaginal Number
ognized Boost** IgA IgG IgA IgG IgA IgG 4 env 0 --* 360 -- -- -- 1
180 360 -- -- -90 2 180 2880 20 20 -90 4 720 1440 -- 20 -360 23 --
-- -- 80 -- 24 90 720 -- -- -- 25 90 2880 20 160 -180 27 90 1440 --
160 -720 gag 0 -- 180 -- -- -- 1 360 360 -- 20 -90 2 720 2880 -- 20
-90 4 720 720 -- 20 -90 23 90 -- -- -- -- 24 90 90 -- -- -- 25 --
90 -- -- -- 27 90 360 -- -- -- 5 env 0 -- -- -- -- N/A.sup.+ N/A 1
-- 90 -- -- N/A N/A 2 -- 2880 -- -- N/A N/A 4 -- 360 -- -- N/A N/A
23 -- -- -- -- N/A N/A 24 -- 360 -- 20 N/A N/A 25 90 2880 20 80 N/A
N/A 27 -- 720 -- 320 N/A N/A gag 0 -- -- -- -- N/A N/A 1 -- 90 --
-- N/A N/A 2 90 1440 -- -- N/A N/A 4 -- 360 -- -- N/A N/A 23 90 --
-- -- N/A N/A 24 90 -- -- -- N/A N/A 25 90 90 -- -- N/A N/A 27 --
-- -- -- N/A N/A 6 env 0 -- -- -- -- N/A N/A 1 -- -- -- -- N/A N/A
2 -- 1440 20 20 N/A N/A 4 -- 360 -- -- N/A N/A 23 -- -- -- -- N/A
N/A 24 -- 180 -- -- N/A N/A 25 -- 720 -- 20 N/A N/A 27 -- 720 -- --
N/A N/A *--equals less than 90 for nasal and vaginal swabs and less
than 20 for saliva samples. .sup.+N/A = not applicable. **Post
Ad5-boost. Subunit boost was administered 23 weeks after Ad5
boost.
[0085] Measurement of Immunogenicity: Treatment Regimen 4
[0086] Dog Inoculations and Collection of Data
[0087] Recombinant adenovirus was administered according to the
table shown above for Treatment Regimen 4. Serum was collected at
different times during the course of the experiment, and processed
as described in Regimen 1. Adenovirus neutralization test
procedures were performed according to the procedures described in
Regimen 1. Detection of anti-HIV antibodies was performed according
to the procedure described in Regimen 1 except that biotinolylated
goat anti-dog IgG.sub.(H+L) was substituted for biotinylated goat
anti-human IgG.sub.(H+L).
[0088] Serum samples were taken from immunized dogs at regular
intervals after primary immunization and booster immunizations.
Seroconversion to the serotype of the adenovirus vector employed
was determined by neutralization test procedures. All of the dogs
responded with strong anti-adenovirus titer to Ad7 vectors. Weaker
anti-Ad5 responses were seen following Ad5 primary or booster
inoculation. Seroconversion to env antigens was measured by Western
blot and by HIV neutralization assays. Some dogs were able to
produce low titer anti-env antibodies following primary
immunization with recombinant Ad-env (LAV or MN). Significant
booster responses to env antigen were observed in almost all of the
dogs following heterotypic boosting with another recombinant Ad-env
(LAV or MN) virus expressing the same type of env antigen.
[0089] Dogs that were primed with Ad7-env.sub.MN and boosted with
Ad5-env.sub.MN had an average anti-HIV.sub.MN serum titer of
>180 (range 45->270) at 4 weeks post-boost. Dogs receiving
the Ad5-env.sub.MN and Ad4-env.sub.MN combination had an average
anti-HIV.sub.MN serum titer of >170 (range 45->270) at this
same time. There were no cross protective antibodies directed at
the HIV.sub.LAV strain in any of these dogs. Dogs receiving the
Ad7-env and Ad5-env combination had an average anti-HIV.sub.LAV
serum titer of 55 (range 20-85) at 4 weeks post-boost and none of
these dogs had anti-HIV.sub.MN titers. In at least one of the three
dogs receiving the "recombinant cocktail" that contained both MN
and LAV recombinant viruses had a anti-HIV.sub.MN serum titer of 90
and an anti-HIV.sub.LAV titer of 50. The other two dogs had
anti-HIV.sub.LAV titers of 45 and 15.
[0090] These results demonstrate that the recombinant Ad-HIV.sub.MN
viruses all elicit neutralizing antibodies directed at the MN
strain of HIV. Low neutralizing titers were seen in 2 of 6 dogs in
Groups 1 and 1 of 6 in Group 4 following the first immunization
with Ad-env.sub.MN recombinants. Low to high neutralization titers
were measured in all of the dogs in these two groups following
booster immunization with heterotypic recombinant viruses. The
neutralization titers produced were type specific and did not cross
react with the LAV strain of HIV. When compared directly to other
dogs treated with LAV recombinant Ad-env viruses, Ad-env.sub.MN
recombinant viruses appeared to elicit higher type-specific
neutralization titers in the dog standard pharmacological test
procedure. Finally, the use of a "recombinant cocktail" which
contains both MN and LAV recombinants appears to be capable of
eliciting neutralizing antibodies to both strains of HIV.
[0091] Measurement of Immunogenicity: Treatment Regimen 5
[0092] HIV Subunit Administration in Dogs
[0093] Thirty laboratory dogs that were either previously immunized
twice with Ad-env recombinants (12-18 week intervals, with the 2nd
immunization 20-28 weeks prior to the 1st subunit immunization) and
ten (10) control dogs that have never been exposed to Ad-env
recombinants were injected with one of five different HIV-env
subunit preparations according to the description shown above for
Treatment Regimen 5. All immunizations were administered by the
subcutaneous route. Serum was collected at different times during
the course of the experiment, and processed as described in Regimen
1. Adenovirus neutralization test procedures were performed
according to the procedures described in Regimen 1.
[0094] Results
[0095] The results that were obtained are described below and
provided in a summary table that follows.
[0096] 1st Subunit Administration. All subunit vaccines
administered to Ad-env "primed" dogs boosted anti-HIV.sub.MN
neutralizing antibody responses. Two subunit preparations, A and C,
were both examined for their ability to induce cross neutralizing
responses to HIV.sub.SF2. Heterologous boosting (i.e.,
Ad-env.sub.MN primed and gp-120.sub.SF2 boost) as well as
homologous boosting (Ad-env.sub.MN primed and gp160.sub.MN boost)
both stimulated anti-HIV.sub.SF2 neutralizing antibody responses.
Control dogs from groups B and C produced anti-env binding
antibodies to HIV-env. Neutralizing antibody responses were not
observed in control dogs following the first subunit
administration.
[0097] 2nd Subunit Administration. Administration of the second
subunit did not appear to be as effective as a boosting agent
compared to the first subunit administration. Group B dogs
exhibited the greatest serum neutralizing antibody response (3-4
fold increase) of the five groups following the second booster
immunization. Groups A and C showed two-fold increases following
their second subunit administrations, while the HA-env antigen
failed to significantly alter the geometric mean neutralizing titer
of either Group D or E. Controls from all five groups produced
anti-env binding antibodies. Functional neutralizing anti-HIV
antibodies were observed only in the groups B, C, and D controls.
Group A and E controls still failed to produce neutralizing
antibody responses after the second subunit administration.
[0098] In summary, these results demonstrate that strong
neutralizing antibody responses were elicited in all groups that
were previously "primed" with Ad-HIV recombinants. After priming,
high neutralizing antibody titers were observed in groups that were
boosted heterologously (with gp120.sub.SF2) and homologously (with
gp120.sub.MN). In the primed dogs, neutralizing antibodies were
generated to both the MN and SF2 strains of HIV. Neutralizing
antibody titers were still observed at twelve weeks, prior to the
second boost. After the second boost, significant increases in
neutralizing antibodies were observed in both gp120-boosted groups
(Groups A and B).
[0099] Summary Table
[0100] The following table shows the results obtained using
Treatment Regimen 5.
11 HIV SUBUNIT IMMUNIZATION IN Ad-HIV PRIMED DOGS Peak Second Titer
After Anti-HIV.sub.MN Responses* Group.sup.+ n First Subunit
Subunit 2nd Ad-HIV 0 wk 2 wk 12 wk 14 wk A 6 gp120.sub.SF2
gp120.sub.SF2 122 16 357 84 270 2 gp120.sub.SF2 gp120.sub.SF2 -- --
-- -- -- B 6 gp120.sub.MN gp120.sub.MN 141 17 883 229 472 2
gp120.sub.MN gp120.sub.MN -- -- -- -- 156 C 6 gp160.sub.MN
gp160.sub.MN 88 29 369 55 68 2 gp160.sub.MN gp160.sub.MN -- -- --
-- 100 D 6 gp160.sub.MN HA-env 88 25 391 87 83 2 gp160.sub.MN
HA-env -- -- -- -- 93 E 6 HA-env HA-env 189 41 431 62 110 2 HA-env
HA-env -- -- -- -- -- .sup.+Each group consisted of 6 dogs that
were previously immunized twice with Ad-Env.sub.MN and 2 control
dogs that were not immunized. *Reciprocal geometric mean
neutralization titer to HIV.sub.MN. Reciprocal geometric mean
neutralization titers to of 98 and 42 to HIV.sub.SF2 were observed
for the previously immunized dogs of groups A and C respectively,
at 2 weeks.
[0101] Measurement of Immunogenicity: Treatment Regimen 6
[0102] Chimpanzee Inoculations and Collection of Data
[0103] Six female chimpanzees were selected on the basis of their
serological profiles to human adenoviruses types 4, 5, and 7, and
were treated according to the table shown above for Treatment
Regimen 6. Their selection was based on a "best fit" for having the
lowest possible serum neutralization titers directed at the various
Ad-env vaccine combinations that were designated to be
administered. Four chimpanzees that were either seronegative or
weakly seropositive received either 1, 2, or 3 consecutive
intranasal immunizations with recombinant Ad-env (12 week
intervals) (Chimpanzees 7, 8, 9, and 11). One chimpanzee that was
strongly seropositive (titers of 128 to all 3 Ad serotypes;
Chimpanzee 10) was given a mixture of all 3 recombinants (each at a
dose of 1.times.10 pfu) as a primary immunization and boosted 12
weeks later with the same mixture. All of the Ad-env immunized
chimpanzees received an intramuscular immunization boost with 50
.mu.g of gp120.sub.SF2 HIV-env subunit formulated in MF59 adjuvant
(MF59 adjuvant is described in Vaccine 11: 909 (1993)). One control
chimpanzee (number 12) received 3 consecutive intranasal
immunizations with wild-type human adenoviruses (12 week intervals)
and an intramuscular immunization with the MF59 adjuvant alone at
week 48. Antibiotics were administered prophylactically to all of
the chimpanzees and no respiratory disorders were observed.
[0104] Whole blood, serum, and stool samples were collected at
different times during the course of the experiment, and processed
as described in Regimen 1. Adenovirus detection in stool samples,
nasal or pharyngeal swab samples were done either by a plaque
hybridization assay (described in Regimen 1) or by PCR technology
(see below). Adenovirus neutralization assays and detection of
anti-HIV antibodies were performed according to the procedures
described in Regimen 1. Chinese hamster ovary cell (CHO)-derived
gp120 or commercially purchased (American Biotechnologies,
Cambridge, Mass.) HIV V3.sub.MN peptides were used as substitute
antigen reagents in antibody binding assays.
[0105] PCR detection of recombinant Ad-env in chimpanzee stool
samples was carried out with a commercially purchased PCR kit
according to the supplier's instructions (Perkin Elmer Cetus,
Norwalk Conn.). Briefly, about 250 .mu.l of the stool samples was
heated to 95.degree. C. for 5 minutes and centrifuged in a
microfuge at top speed for 2-3 minutes. The supernatant was saved.
1-10 .mu.l per PCR reaction was used. Several tubes of master mix
were prepared from the PCR kit and kept frozen at -20.degree. C.
For a 10 reaction tube, sterile water (615 .mu.l), 10 X buffer (100
.mu.l), dATP (20 .mu.l), dCTP (20 .mu.l), dGTP (20_.mu.l), and dTTP
(20 .mu.l) were mixed to make up the master mix. For each reaction,
79.5 .mu.l of the master mix were used. On the day of the first
PCR, a tube of master mix (10 rx) was thawed. To the master mix
were added 10 .mu.l of each of the oligomers, 5 .mu.l of native Taq
DNA polymerase, 50 .mu.l water. The solution was mixed and about 90
.mu.l was distributed to each reaction tube. The PCR was carried
out in a 0.5 ml eppendorf tube. To each tube was added 10 .mu.l of
the stool supernatant. Thirty (30) cycles of PCR amplification were
run at 95.degree. C. for 1 hour, 45.degree. C. for 1.5 hours, and
72.degree. C for 2 hours. A second PCR was performed with a 2.5
.mu.l aliquot of the first PCR product as a DNA template and a
corresponding oligo pair as primers. After 30 cycles of
amplification, 10 .mu.l of the reaction product was run on a 1.2%
argose gel. A 800 bp DNA band was observed as a positive control
for Ad7-env. The following primer pairs were used for nested
PCR.
12 Template Gene 1st PCR 2nd PCR DNA Size HIV-1 gp 120.sub.MN
5166/5209 5164/5208 800 bp Ad4 fiber 5467/5468 5469/5470 782 bp Ad5
fiber 5625/5523 5624/5522 423 bp Ad7 fiber 5505/5504 5503/5502 978
bp
[0106] HIV specific CTL activity was measured by determining lysis
of syngenic target cells that were infected with vaccinia virus
recombinants that express either the HIV-env gene products, the
HIV-gag gene products, or the lac gene product (control for
nonspecific cytotoxicity).
[0107] Results
[0108] 1st Immunization with Ad5-recombinants: Recombinant Ad5
virus was shed into fecal, pharyngeal, and/or nasal specimens for
0-7 days collected from chimpanzees that were seronegative or
weakly seropositive to Ad5. Only the Ad5 recombinant was detected
in the strongly seropositive chimpanzee immunized with the mixture
of three recombinants. Wild-type adenovirus was shed for 56 days in
the control chimpanzee that was weakly seropositive to Ad5.
Significant anti-Ad5 responses were produced in most of the
chimpanzees, with the strongest response produced in the control
animal immunized with the wild-type Ad5. Three of the four
chimpanzees (numbers 7, 9 and 11) immunized with the single Ad5
recombinant produced weak anti-env antibody responses. Functional
serum neutralizing anti-HIV antibodies were detected only in
chimpanzee 5, which was originally seronegative to Ad5. Secretory
anti-IgG anti-env antibodies were detected in vaginal, nasal, and
saliva specimens collected from chimpanzee 11. Sporadic detection
of env-specific CTL activity (specific lysis=10%) was observed in
in vitro stimulated peripheral blood lymphocyte (PBL) populations
obtained from chimpanzees 7, 8, 9, and 10 following the primary
immunization with Ad5-env. Significant CTL responses were not
observed in PBL obtained from chimpanzee 11.
[0109] 2nd Immunization with Ad7-recombinants. Recombinant Ad7
viruses were shed into fecal, pharyngeal, and/or nasal specimens
for 7-10 days in the three chimpanzees (numbers 8, 9, and 11) that
were immunized with the Ad7-env alone and for 7 days in the
chimpanzee (number 10) that was strongly seropositive to all 3
recombinant adenoviruses. Wild-type Ad7 was shed for 14 days in the
control chimpanzee (number 12). Significant anti-Ad7 responses were
developed in all Ad7 immunized animals with the best response
observed in the control chimpanzee immunized with wild-type virus.
Significant anti-env responses were boosted in 2 (numbers 9 and 11)
of the 3 chimpanzees boosted with Ad7-env alone, while
insignificant changes were observed in the animal given the mixed
adenovirus preparation. Importantly, the two chimpanzees both
contained functional neutralizing antibodies to HIV.sub.MN.
Chimpanzee 11 also had a very low cross-negative neutralizing
antibody response directed at HIV.sub.SF2. Nasal and saliva
specimens collected from this chimpanzee also became positive for
anti-env IgG antibodies. Vaginal anti-env IgG antibody responses
were also boosted in chimpanzee 11. Still, anti-env antibody
responses were not observed in any of the secretory fluids
collected from the other chimpanzees. Again, only sporadic
detection of env-specific CTL responses were detected in in vitro
stimulated PBL populations prepared from chimpanzees 8, 9, and 10.
As before, significant CTL responses were not observed in PBL
populations obtained from chimpanzee 11.
[0110] 3rd Immunization with Ad4-env recombinants. Recombinant
Ad4-env was shed in stools for up to 3 days in the single animal
(number 11) that was immunized with the Ad4-env alone. Ad4-env
shedding was not detected in the strongly anti-Ad seropositive
chimpanzee (number 10) after either immunization with the mixed
Ad-env preparation. Wild-type Ad4 was shed for 7 days in chimpanzee
12. Both chimpanzees 11 and 12 made excellent anti-Ad4 antibody
responses. The second booster immunization in chimpanzee 11
resulted with a significant boost in the anti-env antibody
responses, including anti-HIV.sub.MN neutralizing antibody
response. Nasal, vaginal, and saliva anti-env IgG antibody
responses were boosted in samples collected from chimpanzee 11.
Despite the generation of an excellent humoral anti-env immune
response in chimpanzee 11, significant CTL responses were not
observed.
[0111] 1st Subunit boost. The heterologous gp120.sub.SF2 subunit
antigen preparation was administered to chimpanzee 7, 26 weeks
after the primary Ad5-env immunization. The subunit immunization
was very successful in boosting the anti-env antibody response. A
high titered neutralizing anti-HIV.sub.MN response (>400) was
observed along with a lower anti-HIV.sub.SF2 response (100). The
subunit administration also elicited strong anti-env IgG antibody
responses in nasal and vaginal secretions, as well as weaker
anti-env responses in rectal secretions. One (chimpanzee 9) of the
two animals given the (Ad5-env)/(Ad7-env) combination also showed
excellent anti-env booster antibody responses following subunit
administration. This animal had similar anti-HIV.sub.MN and
anti-HIV.sub.SF2 neutralizing titers as seen in chimpanzee 1. Weak
anti-env IgG responses were observed in nasal, rectal, saliva, and
vaginal secretions collected from this animal. The other chimpanzee
(number 8) had a much weaker, but still significant anti-env
response induced following subunit administration, but this
response did not include functional neutralizing antibodies to HIV.
Nor were anti-env antibodies detected in any of the secretions
collected from this chimpanzee. Chimpanzee 11 which received the
(Ad5-env)/(Ad7-env)/(Ad4-env- ) combination also showed an
excellent anti-env antibody booster response. This included a very
high neutralization titer (>1400) to HIV.sub.MN and high
(>400) titers to HIV.sub.SF2. Excellent anti-env IgG antibody
responses were observed in vaginal, nasal, and saliva specimens. A
weak anti-env response was observed in pharyngeal secretions. The
subunit did not have a significant effect on the anti-env antibody
response (serum or secretory) of chimpanzee 10 (the strongly
anti-Ad seropositive animal). Sporadic anti-env CTL activity was
detected in in vitro stimulated PBL populations collected only from
chimpanzees 7 and 8 following subunit administration. Similar
analysis of in vitro stimulated lymph node cells (obtained from a
lymph node biopsy located in close proximity to the subunit
inoculation site) revealed that cells obtained only from chimpanzee
8 (basically a non-humoral responder) contained significant CTL
activity directed at both env.sub.SF2 and env.sub.MN.
[0112] 2nd Subunit boost. Only chimpanzee 7 received a second
subunit immunization. This second immunization resulted with a
significant boost in the anti-env antibody response, including high
titered anti-HIV.sub.MN (>200) and low (<100)
anti-HIV.sub.SF2 neutralizing antibody responses. Excellent
anti-env IgG responses were observed in vaginal, nasal, and rectal
specimens. Sporadic anti-HIV CTL activity was also seen in PBL
populations.
[0113] HIV.sub.SF2 Challenge of Immunized and Control Chimpanzees.
A cell-free HIV.sub.SF2 challenge was administered intravenously to
five of the six chimpanzees (7, 8, 9, 11, 12). The challenge stock
dilution of 1/40 was shown to productively infect control
chimpanzees within 3 to 4 weeks. The chimpanzees were monitored for
signs of HIV infection for a period of 10 weeks. HIV could be
co-cultured from PBLs obtained from control chimpanzee 12 collected
at 4 and 6 weeks post-challenge. Anti-gag antibody responses were
readily measurable (another indication of HIV infection since the
recombinant vaccines lacked gag determinants) in serum samples
collected at 6, 8, and 10 weeks post-challenge. All other
chimpanzees were protected from the HIV challenge at 10 weeks.
[0114] These results demonstrate that the intranasal administration
of the Ad-env recombinants (particularly Ad7-env.sub.MN,
Ad5-env.sub.MN, Ad4-env.sub.MN or a combination thereof) elicited
the production of neutralizing antibodies against HIV-1.
Neutralizing antibodies were produced following the first
administration of the Ad-env recombinants, and the titer was
increased through the use of one or more booster intranasal
immunizations with the Ad-env recombinants. Antibody response to
both the MN and SF2 strains of HIV was further boosted through the
administration of one or more inoculations with an env (gp120)
subunit antigen preparation (particularly gp120.sub.SF2). Most
importantly, protection against HIV-1 infection was demonstrated
following the administration of the Ad-env/subunit booster
treatment regimen.
Sequence CWU 1
1
1 1 3655 DNA Adenovirus 1 agacccttcc tcctctgatc caggactcta
actctacctt accagcacca tccactacta 60 accttcccga aactaacaag
cttctagcac tgtcttccgg atcgctctcc aggagcgcca 120 gctgttgggc
tcgcggttga gaaggtattc ttcgtgatcc ttccagtact cttcgagggg 180
aaacccgtct ttttctgcac ggtactccgc gcaaggacct gattgtctca agatccacgg
240 gatctgaaaa cctttcgacg aaagcgtcta accagtcgca atcgcaagaa
gcttgtcgag 300 ccaccatgag agtgaagggg atcaggagga attatcagca
ctggtgggga tggggcacga 360 tgctccttgg gttattaatg atctgtagtg
ctacagaaaa attgtgggtc acagtctatt 420 atggggtacc tgtgtggaaa
gaagcaacca ccactctatt ttgtgcatca gatgctaaag 480 catatgatac
agaggtacat aatgtttggg ccacacaagc ctgtgtaccc acagacccca 540
acccacaaga agtagaattg gtaaatgtga cagaaaattt taacatgtgg aaaaataaca
600 tggtagaaca gatgcatgag gatataatca gtttatggga tcaaagccta
aagccataac 660 cccactctgt gttactttaa attgcactga tttgaggaat
actactaata ccaataatag 720 tactgctaat aacaatagta atagcgaggg
aacaataaag ggaggagaaa tgaaaaactg 780 ctctttcaat atcaccacaa
gcataagaga taagatgcag aaagaatatg cacttcttta 840 taaacttgat
atagtatcaa tagataatga tagtaccagc tataggttga taagttgtaa 900
tacctcagtc attacacaag cttgtccaaa gatatccttt gagccaattc ccatacacta
960 ttgtgccccg gctggttttg cgattctaaa atgtaacgat aaaaagttca
gtggaaaagg 1020 atcatgtaaa aatgtcagca cagtacaatg tacacatgga
attaggcaac tcaactgctg 1080 ttaaatggca gtctagcaga agaagaggta
gtaattagat ctgagaattt cactgataat 1140 gctaaaacca tcatagtaca
tctgaatgaa tctgtacaaa ttaattgtac aagacccaac 1200 tacaataaaa
gaaaaaggat acatatagga ccagggagag cattttatac aacaaaaaat 1260
ataataggaa ctataagaca agcacattgt aacattagta gagcaaaatg gaatgacact
1320 ttaagacaga tagttagcaa attaaaagaa caatttaaga ataaaacaat
agtctttaat 1380 caatcctcag gaggggaccc agaaattgta atgcacagtt
ttaattgtgg aggggaattt 1440 ttctactgta atacatcacc actgtttaat
agtacttgga atggtaataa tacttggaat 1500 aatactacag ggtcaaataa
caatatcaca cttcaatgca aaataaaaca aattataaac 1560 atgtggcagg
aagtaggaaa agcaatgtat gcccctccca ttgaaggaca aattagatgt 1620
tcatcaaata ttacagggct actattaaca agagatggtg gtaaggacac ggacacgaac
1680 gacaccgaga tcttcagacc tggaggagga gatatgaggg acaattggag
aagtgaatta 1740 tataaatata aagtagtaac aattgaacca ttaggagtag
cacccaccaa ggcaaagaga 1800 agagtggtgc agagagaaaa aagagcagcg
ataggagctc tgttccttgg gttcttagga 1860 gcagcaggaa gcactatggg
cgcagcgtca gtgacgctga cggtacaggc cagactatta 1920 ttgtctggta
tagtgcaaca gcagaacaat ttgctgaggg ccattgaggc gcaacagcat 1980
atgttgcaac tcacagtctg gggcatcaag cagctccagg caagagtcct ggctgtggaa
2040 agatacctaa aggatcaaca gctcctgggg ttttggggtt gctctggaaa
actcatttgc 2100 accactactg tgccttggaa tgctagttgg agtaataaat
ctctggatga tatttggaat 2160 aacatgacct ggatgcagtg ggaaagagaa
attgacaatt acacaagctt aatatactca 2220 ttactagaaa aatcgcaaac
ccaacaagaa aagaatgaac aagaattatt ggaattggat 2280 aaatgggcaa
gtttgtggaa ttggtttgac ataacaaatt ggctgtggta tataaaaata 2340
ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata
2400 gtgaatagag ttaggcaggg atactcacca ttgtcgttgc agacccgccc
cccagttccg 2460 aggggacccg acaggcccga aggaatcgaa gaagaaggtg
gagagagaga cagagacaca 2520 tccggtcgat tagtgcatgg attcttagca
attatctggg tcgacctgcg gagcctgttc 2580 ctcttcagct accaccacag
agacttactc ttgattgcag cgaggattgt ggaacttctg 2640 ggacgcaggg
ggtgggaagt cctcaaatat tggtggaatc tcctacagta ttggagtcag 2700
gaactaaaga gtagtgctgt tagcttgctt aatgccacag ctatagcagt agctgagggg
2760 acagataggg ttatagaagt actgcaaaga gctggtagag ctattctcca
catacctaca 2820 agaataagac agggcttgga aagggctttg ctataatcta
gcactgtctt ccggatcgct 2880 gtccaggagc gccagctgtt gggctcgcgg
ttgagaaggt attcttcgct gtccaggagc 2940 gccagctgtt gggctcgcgg
ttgagaaggt attcttcgtg atccttccag tactcttcga 3000 ggggaaaccc
gtctttttct gcacggtgtg atccttccag tactcttcga ggggaaaccc 3060
gtctttttct gcacggtact ccgcgcaagg acctgattgt ctcaagatcc acgggatctg
3120 aaaacctttc gacgaaagcg tctaaccagt cgcaatcgca agaagcttgt
cgactatggc 3180 aggaagaagc ggagacagcg acgaagacct cctcaaggca
gtcagactca tcaagtttct 3240 ctatcaaagc aaccccccac ctaaccctga
aggcacaagg caagctaggc ggaacaggag 3300 gaggcggtgg agggaaaggc
aaaggcaaat tcactccatc tccgagagga ttctgtccac 3360 ctacctcggc
aggtccgcgg aacccgtccc cctgcaactg ccccccctgg aaagactgac 3420
cctggactgc aatgaagact gcggcacctc cggaacccaa ggagtcggct ccccccagat
3480 cctggtcgag tcccccaccg tgctggaatc cggcaccaag gagtagtcga
ctctagaagg 3540 tgcacctaca ccctgctaaa gaccctatgc ggcctaagag
acctgctacc catgaattaa 3600 aaattaataa aaaatcactt acttgaaatc
agcaataagg tctctgtttg gaaat 3655
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