Note: Descriptions are shown in the official language in which they were submitted.
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WO 99/44635 PCT/US99/04678
ENHANCEMENT OF IMMUNITY BY INTRANASAL INOCULATION OF IL-
12
RELATED APPLICATIONS)
This application is a Continuation-in-Part of U.S. Application No.
09/035,188 filed March 5, 1998, the entire teachings of which are incorporated
herein by reference.
BACKGROUND OF THE INVENTION
Mucosal surfaces are the major portals of entry for bacteria and viruses, and
therefore constitute the first line of defense for the host. As such,
immunization
strategies that enhance mucosal immunity have practical significance for
preventing
infectious disease. Most parenterally administered vaccines, however, are only
partially effective at inducing optimal mucosal immunity. Thus, adjuvants that
can
enhance mucosal immunity and be delivered in a safe, non-invasive manner are
needed.
SUMMARY OF THE INVENTION
As described herein, intranasal interleukin-12 (IL-12) treatment can
effectively enhance antigen-specific immune responses and enhance immunization
strategies for mucosal vaccines. Thus, the present invention relates to
methods of
enhancing and/or inducing an immune response (e.g., systemic, mucosal) to a
2 0 pathogen in a host (e.g., mammalian, including human), which comprises
administering intranasally (i.n.) to the host an effective amount of IL-12 and
an
antigen (e.g., a protein, carbohydrate, lipid, recombinant DNA, whole
organism,
toxin, organic molecule) of the pathogen. As described herein, the immune
response
can be antigen-specific. In addition, the immune response can result in
enhanced
2 5 expression of a Thl-type cytokine response (e.g., expression of interferon-
'y) and/or
a humoral response (e.g., IgG2a, IgG2b, IgG3).
In one embodiment, the present invention relates to a method of enhancing
an immune response to a pathogen in a host, which comprises administering i.n.
to
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the host an effective amount of IL-12 and an antigen of the pathogen. In
another
embodiment, the present invention relates to a method of inducing an immune
response to a pathogen in a host, which comprises administering i.n. to the
host an
effective amount of IL-12 and an antigen of the pathogen.
In a particular embodiment, the present invention relates to a method of
inducing an immune response to a mucosal pathogen in a host, which comprises
administering i.n. to the host an effective amount of IL-12 and an antigen of
the
pathogen.
Also encompassed by the present invention is a method of inducing a Thl
like immune response to a pathogen in a host, comprising administering i.n. to
the
host an effective amount of IL-12 and an antigen of the pathogen.
The present invention also relates to a method of enhancing a mucosal
immune response to a pathogen in a host, which comprises administering i.n. to
the
host an effective amount of IL-12 and an antigen of the pathogen.
The finding that IL-12 administered i.n. is effective for augmenting antigen
specific-responses in both mucosal and systemic compartments as described
herein,
demonstrates that i.n. administration of IL-12 can be used to obtain a potent
vaccine
adjuvant effect in immunization strategies against pathogens, such as mucosal
pathogens.
2 0 BRIEF DESCRIPTION OF THE FIGURES
Figure lA-1C are bar graphs showing the effects of IL-12 administered
intranasally (i.n.) on respiratory mucosal immune responses; the data are
presented
as average optical density (O.D.) +/- SEM with four mice per group;
bronchoalveolar lavage (BAL) were tested at dilutions corresponding to the
linear
portions of the titration curve (1:64 for IgGl and IgA, 1:8 for IgG2a).
Figure 2A-2E are graphs of reciprocal serum dilution versus O.D. 405 nm
showing the effects of IL-12 administered i.n, on systemic antibody responses;
solid
symbols represent animals injected with DNP-OVA plus II,-12 and open symbols
represent animals injected with DNP-OVA plus phosphate buffered saline (PBS);
3 o each line represents binding of antibody from an individual mouse.
Figures 3A-3B are bar graphs showing the effects of IL-12 administered i.n.
on total Ig levels; the data are presented as average O.D. +/- SEM with four
mice per
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group; sera were tested at dilutions corresponding to the linear portions of
the
titration curve (1:6400 for IgGl, 1:200 for IgG2a).
Figures 4A-4F are bar graphs showing the effects of parenteral (i.p.) and i.n.
administration of IL-12 on fecal mucosal responses; the data shown represent
day 21
antibody responses for IgA and day 28 responses for IgG isotypes, the peak of
each
reactive response; the data are presented as average O.D. +/- SEM with 3-4
mice per
group.
Figures 5A-5B are graphs of reciprocal serum dilution versus O.D. 405 nm
showing the effects of IL-12 administered i.n. on systemic antibody responses;
mice
were immunized i.n. on day 0 with purified hemagglutinin and neuraminidase
derived from influenza virus (HANA) and treated i.n. with either IL-12 (closed
triangles) or phosphate buffered saline (PBS) vehicle (open circles) on days
0, 1, 2
and 3; serum anti-HANA antibody levels on day 14 were determined by isotype-
specific ELISA using HANA-coated microtiter plates; each line represents
binding
of antibody from an individual mouse.
Figures 6A-6B are graphs of reciprocal serum dilution versus O.D. 405 nm
showing the effects of IL-12 administered i.n. on systemic antibody responses;
mice
were immunized i.n. on day 0 with HANA and treated i.n. with either IL-12
(closed
triangles) or PBS vehicle (open circles) on days 0, 1, 2 and 3 and boosted on
day 14;
2 0 serum anti-HANA antibody levels on day 28 were determined by isotype-
specific
ELISA using HANA-coated microtiter plates; each line represents binding of
antibody from an individual mouse.
Figure 7A-7B are graphs of reciprocal serum dilution versus O.D. 405 nm
showing the effects of IL-12 administered i.n. on respiratory mucosal
responses;
2 5 mice were immunized on day 0 with HANA and treated with either IL-12 or
PBS
vehicle on days 0, 1, 2 and 3 and boosted on days 14 and 28; on day 28 the
mice also
received IL-12 or vehicle; mice were sacrificed on day 35, and BAL fluid was
assayed for anti-HANA antibody levels by ELISA using HANA coated microtiter
plates; each line represents binding of antibody from an individual mouse.
3 0 Figures 8A-8B are graphic representations showing the effects of IL-12
administered i.n. on early systemic antibody responses to the subunit
influenza
vaccine. Mice were immunized i.n. on day 0 with H1N1 subunit influenza
vaccine,
and treated i.n. with either IL-12 (closed triangles) or PBS vehicle (open
circles} on
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days 0, 1, 2 and 3. Serum anti-H1N1 antibody levels on day 14 were determined
by
isotype-specific ELISA using HIN1-coated microtiter plates. Each line
represents
binding of antibody from an individual mouse (4 mice per group). The
difference in
binding between mice immunized with vaccine and IL-12 and those immunized with
vaccine and PBS vehicle was significant atp <0.05 for IgG2a.
Figures 9A-9E are graphic representations showing the effects of IL-12
administered i.n. on late systemic antibody responses to the subunit influenza
vaccine. Mice were immunized i.n. on day 0 with H1N1 subunit influenza
vaccine,
treated i.n, with either IL-12 (closed triangles) or PBS vehicle (open
circles) on days
0, 1, 2 and 3, and boosted with vaccine on days 14 and 28. On day 28, the mice
received a second treatment with IL-12 or vehicle. Serum anti-H1N1 antibody
levels on day 35 were determined by isotype-specific ELISA using H1N1-coated
microtiter plates. Each line represents binding of antibody from an individual
mouse (4 mice per group). The differences in binding between mice immunized
with vaccine and IL-12 and those immunized with vaccine and PBS vehicle were
significant atp >0.05 for IgG2a, total Ab and total Ig.
Figures l0A-lOD are graphic representations showing the effects of IL-12
administered i.n. on respiratory mucosal responses. Mice were immunized i.n.
on
day 0 with H1N1 subunit influenza vaccine, treated i.n. with either IL-12 or
PBS
2 o vehicle on days 0, 1, 2 and 3, and boosted with vaccine on days 14 and 28.
On day
28, the mice received a second treatment with IL-12 or vehicle. Mice were
sacrificed on day 35 and BAL fluid was assayed for anti-H1N1 antibody levels
by
ELISA using H1N1-coated microtiter plates. Each line represents binding of
antibody from an individual mouse (4 mice per group). The differences in
binding
2 5 between mice immunized with vaccine and IL-12 and those immunized with
vaccine
and PBS vehicle were significant atp <0.05 for total Ab, IgGI, IgG2a and IgA.
Figures 1 lA-11D are graphic representations showing that co-administration
of influenza subunit vaccine plus IL-12 protects mice from a subsequent
influenza
virus infection. Mice were immunized i.n. with H1N1 subunit vaccine plus IL-12
3 0 (closed triangles), vaccine plus PBS vehicle (open circles), IL-12 only
(open
diamonds) or PBS vehicle only (open squares). All mice (8 per group) were then
challenged i.n. 4-5 weeks later with 103 pfu (A) or 2 x 103 pfu (B) of
A/PRJ8/34
influenza virus. The mice were monitored daily for mortality and weight loss.
The
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differences in survival between mice immunized with vaccine and IL-12 and
those
immunized with vaccine and PBS were significant atp <0.0$.
Figures 12A-12B are graphic representations showing that IL-12 induced
protection against influenza virus infection is mediated by B cells. ,uMT mice
were
immunized i.n. on with H1N1 subunit vaccine plus IL-12 (closed triangles),
vaccine
plus PBS vehicle (open circles) or PBS vehicle only (open diamonds). Wild type
(WT) mice were pre-treated with PBS vehicle (open squares). All mice (8 per
group) were then challenged i.n. 6-7 weeks later with 103 pfu of AlPR/8/34
influenza virus. The mice were monitored daily for mortality and weight loss.
Figure 13 is a graphic representation showing passive transfer of serum from
mice immunized with the subunit influenza vaccine plus IL-12 confers
protection
against influenza virus challenge. Sera were collected from mice immunized
with
the H1N1 subunit influenza vaccine plus IL-12 (closed triangles), vaccine plus
PBS
(open circles) or PBS vehicle only (open squares). Pooled serum was diluted
1:10 in
sterile PBS and injected i.p. at a dose of 0.1 ml/mouse. All mice (7-8 per
group)
were then challenged i.n. $ hours later with 103 pfu of A/PR/8/34 influenza
virus.
The differences in survival between mice immunized with vaccine and IL-12 and
those immunized with vaccine and PBS vehicle were significant atp <0.05.
Figures 14A-14B are graphic representations showing passive transfer of
2 0 BAL fluid i.n. from mice immunized with the subunit influenza vaccine plus
IL-12
confers protection against influenza virus challenge. BAL fluids were
collected
from mice immunized with the H1NI subunit influenza vaccine plus IL-12 (closed
triangles), vaccine plus PBS (open circles) or PBS vehicle only (open
squares). All
mice (8 per group) were then inoculated i.n. with pooled BAL fluid and 2 x 103
pfu
2 5 of A/PR/8/34 influenza virus. The differences in survival between mice
immunized
with vaccine and IL-12 and those immunized with vaccine and PBS were
significant
atp <0.05.
DETAILED DESCRIPTION OF THE INVENTION
As described herein, systemic and mucosal cytokine and antibody production
3 0 in mice immunized with a hapten-carrier antigen has been examined. The
results
show that IL-12 administered i.n. induces Thl-like cytokine and antibody
patterns in
both spleens and lungs of treated mice. The findings demonstrate that i.n.
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inoculation of IL-12 is a powerful means to influence both mucosal and
systemic
immunity.
Thus, the present invention relates to methods of enhancing and/or inducing
immunity to a pathogen (one or more) in a host, which comprises administering
i.n.
to the host an effective amount of IL-12 and an antigen of the pathogen (e.g.,
a
mucosal pathogen). The methods of the present invention can be used to enhance
an
immune response to an antigen in a mammalian host, such as a primate (e.g.,
human), marine, feline, canine, bovine or porcine host.
As used herein, the terms "enhance" and/or "enhancing" refer to the
strengthening (augmenting) of an existing immune response to a pathogen. The
term also refers to the initiation of (initiating, inducing) an immune
response to a
pathogen.
An antigen (one or more) for use in the methods of the present invention
includes (or can be obtained from), but is not limited to, proteins or
fragments
thereof (e.g., proteolytic fragments), peptides (e.g., synthetic peptides,
polypeptides),
glycoproteins, carbohydrates (e.g., polysaccharides), lipids, glycolipids,
hapten
conjugates, recombinant DNA, whole organisms (killed or attenuated) or
portions
thereof, toxins and toxoids (e.g., tetanus, diphtheria, cholera) and/or
organic
molecules. Particular examples of antigens for use in the present invention
include
2 0 hemagglutinin and neuraminidase obtained or derived from the influenza
virus.
The antigen can be obtained or derived from a variety of pathogens or
organisms, such as bacteria (e.g., bacillus, Group B streptococcus,
Bordetella,
Listeria, Bacillus anthracis, S. pneumoniae, N. meningiditis, H. influenza),
viruses
(e.g., hepatitis, measles, poliovirus, human immunodeficiency virus, influenza
virus,
2 5 parainfluenza virus, respiratory syncytial virus), mycobacteria (M.
tuberculosis),
parasites (Leishmania, Schistosomes, Tranpanosomes, toxoplasma, pneumocystis)
and fungi (e.g., Candida, Cryptococcus, Coccidiodes, Aspergillus), against
which an
immune response is desired in a host. The antigen of a pathogen can be
obtained
using skills known in the art. For example, the antigen can be isolated
(purified,
3 0 essentially pure) directly from the pathogen, derived using chemical
synthesis or
obtained using recombinant methodology. In addition, the antigen can be
obtained
from commercial sources. A suitable antigen for use in the present invention
is one
that includes at least one B and/or T cell epitope (e.g., T helper cell or
cytolytic T
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WO 99/44635 PCT/US99/04678
cell epitope). Other suitable antigens useful in the compositions of the
present
invention can be determined by those of skill in the art.
IL-12 is a recently characterized heterodimeric cytokine that has a molecular
weight of 75 kDa and is composed of disulfide-bonded 40 kDa and 35 kDa
subunits.
It is produced by antigen presenting cells such as macrophages and dendritic
cells,
and binds to receptors on activated T, B and NK cells (Desai, B.B., et al., J.
Immunol., 148:3125-3132 (1992); Vogel, L.A., et al., Int. Immunol., 8:1955-
1962
(1996)). It has several effects including 1) enhanced proliferation of T cells
and NK
cells, 2) increased cytolytic activities of T cells, NK cells, and
macrophages,
3) induction of IFN-'y production and to a lesser extent, TNF-a and GM-CSF,
and
4) activation of Thl cells (Trinchieri, G., et al., Blood, 84:4008-4027
(1994). IL-12
has been shown to be an important costimulator of proliferation in Thl clones
(Kennedy et al., Eur. J. Immunol. 24:2271-2278, 1994) and leads to increased
production of IgG2a antibodies in serum when administered i.p. (Morris, S.C.,
et al.,
J. Immunol. 152:1047-1056 (1994); Germann, T.M., et al., Eur. J. Immunol.,
25:823-829 (1995); Sher, A., et al., Ann. N. Y. Acad. Sci., 795:202-207
(1996);
Buchanan, J.M., et al., Int. Imm., 7:1519-1528 (1995); Metzger, D.W., et al.,
Eur. J.
Immunol., 27:1958-1965 (1997)). Administration of IL-12 i.p. can also
temporarily
decrease production of IgGl antibodies (Morns, S.C., et al., J. Immunol.
152:1047-
1056 (1994); McKnight, A.J., J. Immunol. 152:2172-2179 (1994); Buchanan, J.M.,
et al., Int. Imm., 7:1519-1528 (1995)), indicating suppression of the Th2
response.
The purification and cloning of IL-12 are disclosed in PCT publication nos. WO
92/05256 and WO 90/05147, and in European patent publication no. 322,827
(identified as "CLMF").
As used herein, "interleukin-12" and "IL-12" refer to interleukin 12 protein,
its individual subunits, multimers of its individual subunits, functional
fragments of
IL-12, and functional equivalents and/or analogues of "interleukin-12" and "IL-
12".
As defined herein, functional fragments of IL-12 are fragments which, when
administered i.n., modulate an immune response against an antigen in a host.
As
3 0 also defined herein, functional fragments or equivalents of "interleukin-
12" and "IL-
12" include modified IL-12 protein such that the resulting IL-12 product has
activity
similar to the IL-12 described herein (e.g., the ability to enhance an immune
response when administered i.n.). Functional equivalents or fragments of
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WO 99/44635 PCT/US99/04678
"interleukin-12" also include nucleic acid sequences (e.g., DNA, RNA) and
portions
thereof, which encode a protein or peptide having the IL-12 function or
activity
described herein (e.g., the ability to enhance an immune response when
administered
i.n.). In addition, the term includes a nucleotide sequence which through the
degeneracy of the genetic code encodes a similar peptide gene product as IL-12
and
has the IL-12 activity described herein. For example, a functional equivalent
of
"interleukin-12" and "IL-12" includes a nucleotide sequence which contains a
"silent" codon substitution (e.g., substitution of one codon encoding an amino
acid
for another codon encoding the same amino acid) or an amino acid sequence
which
1 o contains a "silent" amino acid substitution (e.g., substitution of one
acidic amino
acid for another acidic amino acid}.
IL-12 suitable for use in the methods of the present invention can be obtained
from a variety of sources or synthesized using known skills. For example, IL-
12 can
be purified (isolated, essentially pure) from natural sources (e.g.,
mammalian, such
as human sources), produced by chemical synthesis or produced by recombinant
DNA techniques. In addition, the IL-12 for use with the present invention can
be
obtained from commercial sources.
An effective amount of IL-12 is administered i.n. in the methods of the
present invention which is an amount that induces and/or enhances an immune
2 o response to an antigen in the host. In particular, "an effective amount of
IL-12" is an
amount such that when administered i.n. with an antigen to a host, enhances an
immune response to the antigen in the host as described herein, relative to
the
immune response to the antigen in a host when an effective amount of IL-12 is
not
administered i.n. to the host. That is, an "effective amount" of IL-12 is an
amount
2 5 that, when administered i.n. with an antigen, it enhances an immune
response to an
antigen in a host as described herein, relative to the immune response to the
antigen
if IL-12 is not administered i.n. to the host.
The IL-12 and/or the antigen can be administered i.n. as a prophylactic
vaccine or a therapeutic vaccine. That is, the IL-12 can be administered
either
3 o before (to prevent) or after (to treat) the effects of a pathogen which
has appeared
and/or manifested in a host. Thus, the IL-12 and/or antigen can be
administered to a
host who either exhibits the disease state caused by a pathogen from which the
antigen is obtained or derived, or does not yet exhibit the disease state
caused by a
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pathogen from which the antigen is obtained or derived. Thus, the IL-12 and/or
antigen can be administered to a host either before or after the disease state
is
manifested in the host and can result in prevention, amelioration, elimination
or a
delay in the onset of the disease state caused by the pathogen from which the
antigen
is obtained or derived.
As described herein the IL-12 and the antigen are administered i.n. to a host.
Any convenient route of i.n. administration can be used. For example,
absorption
through epithelial or mucocutaneous linings (e.g., administering the IL-12
and/or
antigen using a nasal mist; administering the IL-12 and/or antigen to the eye
using
1 o an eye dropper wherein the IL-12 and/or antigen drains into the nasal
cavity) can be
used. In addition, the IL-12 and antigen can be administered together with
other
components or biologically active agents, such as adjuvants (e.g., alum),
pharmaceutically acceptable surfactants (e.g., glycerides), liposomes,
excipients
(e.g., lactose), carriers, diluents and vehicles. If desired, certain
sweetening,
flavoring andlor coloring agents can also be added.
Further, the IL-12 and/or the antigen, in the embodiment wherein the antigen
is a protein (peptide), can be administered i.n. by in vivo expression of
polynucleotides encoding such into a host. For example, the IL-12 or the
antigen
can be administered to a host using a live vector, wherein the live vector
containing
2 0 IL-12 and/or antigen nucleic acid sequences is administered i.n. under
conditions in
which the IL-12 and/or antigen are expressed in vivo. A host can also be
injected
i.n. with a vector which encodes and expresses an antigen in vivo in
combination
with IL-12 protein or peptide, or in combination with a vector which encodes
and
expresses the IL-12 protein in vivo. Alternatively, a host can be injected
i.n. with a
2 5 vector which encodes and expresses IL-12 in vivo in combination with an
antigen in
peptide or protein form, or in combination with a vector which encodes and
expresses an antigen in vivo. A single vector containing the sequences
encoding an
antigen and the IL-12 protein are also useful in the methods of the present
invention.
Several expression vector systems are available commercially or can be
3 0 reproduced according to recombinant DNA and cell culture techniques. For
example, vector systems such as the yeast or vaccinia virus expression
systems, or
virus vectors can be used in the methods and compositions of the present
invention
(Kaufman, R.J., A J. ofMeth. in Cell and Molec. Biol., 2:221-236 (1990)).
Other
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WO 99!44635 PCT/US99/04678
techniques using naked plasmids or DNA, and cloned genes encapsulated in
targeted
liposomes or in erythrocyte ghosts, can be used to introduce IL-12
polynucleotides
into the host (Freidman, T., Science, 244:1275-1281 (1991); Rabinovich, N.R.,
et
al., Science, 265:1401-1404 (1994)). The construction of expression vectors
and the
transfer of vectors and nucleic acids into various host cells can be
accomplished
using genetic engineering techniques, as described in manuals like Molecular
Cloning and Current Protocols in Molecular Biology, which are hereby
incorporated
by reference, or by using commercially available kits (Sambrook, J., et al.,
Molecular Cloning, Cald Spring Harbor Press, 1989; Ausubel, F.M., et al.,
Current
Protocols in Molecular Biology, Greene Publishing Associates and Wiley-
Interscience, 1989).
As described herein, i.n. administration of IL-12 and an antigen to a host
enhances an immune response in the recipient host. For example, the present
invention relates to a method of inducing a Thl-like immune response to a
pathogen
in a host, comprising administering i.n, to the host an effective amount of IL-
12 and
an antigen of the pathogen. The present invention also relates to a method of
enhancing a mucosal immune response to a pathogen in a host, comprising
administering i.n. to the host an effective amount of IL-12 and an antigen of
the
pathogen. The methods described herein can result in enhanced expression of
IFN-
2 0 y. In addition, a humoral response can be induced and/or enhanced in a
host, which
can result in enhanced expression of IgG2a, IgG2b and/or IgG3 antibody. The
immune response can be antigen-specific.
In the methods of enhancing an immune response to an antigen in a host, an
effective amount of IL-12 is administered i.n. to the host, which is an amount
that
2 5 enhances and/or induces an immune response to the antigen in the host and
results in
the improved condition of the host (i.e., the disease or disorder caused by
the
presence of the pathogen from which the antigen is obtained or derived, is
prevented, eliminated or diminished). The amount of IL-12 used to enhance an
immune response to an antigen in a host will vary depending on a variety of
factors,
3 0 including the size, age, body weight, general health, sex and diet of the
host, and the
time of administration, duration or particular qualities of the disease state.
Suitable
dose ranges of IL-12 are generally about O.SUg to about 150 ~g per kg body
weight.
In one embodiment, the dose range is from about 2.75 ~.g to about 100 ~g per
kg
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body weight. In another embodiment, the dose range is from about S ug to about
SOp.g per kg body weight. Effective dosages may be extrapolated from dose-
response curves derived in vitro or in animal model test systems.
In the methods of the present invention, an effective amount of IL-12 is
administered i.n. in combination with an antigen. That is, the IL-12 is
administered
at a time closely related to immunization of the host with an antigen, so that
an
immune response to the antigen is induced or enhanced in the host relative to
the
immunization of a host in which IL-12 is nat administered. Thus, the IL-12 can
be
administered i.n. prior to, preferably just prior to, immunization; at the
time of
immunization (i.e., simultaneously); or after immunization (subsequently). In
addition, the IL-12 can be administered i.n. prior to immunization with the
antigen
followed by subsequent administrations of IL-12 after immunization with the
antigen.
As described herein, IL-12 given i.n. and in a non-invasive manner, redirects
the mucosal compartment of the immune system toward Thl type cytokine and
antibody profiles. As also described herein, i.n. delivery of IL-12 modulates
the
patterns of cytokine and antibody expression in distant systemic compartments
of
the immune system.
Mice immunized i.n. with DNP-OVA plus IL-12 displayed enhanced levels
2 0 of IFN-y mRNA in the lungs after 6 hours with maximal expression noted at
24
hours. There was a similar enhancement of IFN-y mRNA in the spleen after i.n.
administration of IL-12. IFN-y is a potent immunoregulator of Th cell subsets
and
their effector functions (Trinchieri, G., et al., ReS. Immunol., 146:423-431
(1995);
Trinchieri, G., Immunol. Today, 14:335-338 (1993)). Specifically, IFN-'y has
been
2 5 shown to activate macrophages and mediate isotype switching to IgG2a and
IgG3
antibody production which is characteristic of Thl-type immune responses
(Snapper, C.M., et al., Science, 236:944-947 (1987); Snapper, C.M., et al., J.
Immunol., 140:2121-2127 (1988); Finkelman, F.D., et al., J. immunol., 140:1022-
1027 (1988)). Sinularly, an important negative regulator of T-cell responses
is
30 interleukin-10 (IL-10) (Meyaard, L., et al., .l. Immunol., 156:2776-2782
(I996)). IL
l0 is mainly produced by T cells and monocytes and exerts its regulatory
effects
through its actions on antigen presenting cells (Fiorentino, D.F., et al., J.
Immunol.,
146:3444-3451 (1991); Ding, L., et al., J. Immunol., 148:3133-3139 (1992)).
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Recently, several investigators have found that IL-12 is able to induce human
T cells
to secrete IL-10 (Meyaard, L., et al., J. Immunol., 156:2776-2782 (1996);
Daftarian,
P.M., et al., J. Immunol., 157:12-20 (1996); Gerosa, F., et al., J. Exp. Med.,
183:2559-2569 (1996)). In light of these studies, the ability of IL-12 given
i.n. to
induce IL-10 mRNA in both the lungs and spleens was assessed. The results
clearly
show the ability of IL-12 to induce IL-10 mRNA. However, maximal expression
was only noted at 24 hours post inoculation. The delay in the induction of IL-
10
mRNA expression after IL-12 treatment suggests that this cytokine is involved
in a
feedback mechanism designed to modulate the effects of IL-12/IFN-y. IL-5 mRNA
as a specific marker for Th2 differentiation was also analyzed, and a clear
reduction
of IL-5 mRNA in the lungs of mice treated with IL-12 was found. The findings
are
consistent with others (Trinchieri, G., et al., Res. Immunol., 146:423-431
(1995);
Trinchieri, G., Immunol. Today, 14:335-338 (1993); Manetti, R., et al., J.
Exp. Med.,
177:1199-1204 (1993); Hsieh, C.S., et al., Science, 260:547-549 (1993)) who
examined the effects of IL-12 given i.p. on systemic immunity and adds further
support to the immunoregulatory functions of IL-12. The results clearly
demonstrate that i.n. IL-12 administration can induce a Thl-type cytokine
response
in both systemic and mucosal compartments.
Since cytokines that are elaborated in vivo can determine the profile of
2 0 antibodies produced during an immune response (Finkelman, F.D., et al.,
Ann. Rev.
Immunol., 8:303-333 (1990)), antigen-specific antibody levels in BAL, sera and
fecal extracts were examined. Intranasal delivery of antigen and IL-12
resulted in
clear enhancement of BAL IgG2a antibody levels. This is the first evidence
that i.n.
IL-12 administration can modulate respiratory antibody responses in mice. Yang
et
2 5 al. (Yang, Y., et al., Nature Med., 1:890-893 (1995)) previously
demonstrated that
intratracheal inoculation of IL-12 and recombinant adenovirus results in a
reduction
of antigen-specific IgA in BAL without any alteration in IgG levels. However,
the
effects of IL-12 in this system were not thoroughly characterized in terms of
IgG
isotypes and therefore there is little information about the role that IL-12
may play
3 0 in respiratory antibody responses. Furthermore, the intratracheal route
was invasive
and not relevant to vaccination protocols. The findings described herein are
significant in terms of host defense as protection of the lower respiratory
tract
against viral infections has been correlated with IgG antibodies (Palladino,
G., et al.,
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J. Virol., 69:2075-2081 (1995)). Furthermore, marine antibodies of the IgG2a
isotype are known to be very efficient at opsonization and complement
fixation, the
primary mechanisms thought to be involved in clearance of respiratory
pathogens
such as S. pneumoniae and N. meningitides.
Previous work showed that IL-12 given i.p. can alter the isotype-restricted
antibody response of mice to hen eggwhite lysozyme (HEL) (Buchanan, J.M., et
al.,
Int. Immunol., 7:1519-1528 (1995); Metzger, D.W., et al., Ann. N. Y. Acad.
Sci.,
795:100-115 (1996)). Parenteral injections of IL-12 plus HEL greatly elevated
HEL-specific serum IgG2a and temporarily suppressed IgGI antibody production.
l0 In addition, others (McKnight, A.J., et al., J. Immunol., 152:2172-2179
(1994);
Moms, S.C., et al., J. Immunol., 152:1047-1056 (1994); Germann, T., et al.,
Eur. J.
Immunol., 25:823-829 (1995); Wynn, T.A., et al., J. Immunol., 157:4068-4078
(1996); Bliss, J., et al., J. Immunol., 156:887-894 (1996)) have demonstrated
that i.p.
IL-12 administration enhances serum IgG2a, IgG2b and IgG3 antibody responses
to
protein antigens.
Described herein is the fact that IL-12 delivered i.n. by a non-invasive route
is capable of influencing serum antibody responses in a similar manner. Mice
that
were immunized i.n. with antigen and IL-12 had markedly elevated levels of
serum
IgG2a, IgG2b and IgG3 compared to animals receiving antigen only. The observed
2 0 increases in IgG2a and IgG3 levels are consistent with the ability of IL-
12 to induce
IFN-y, which is a potent switch factor for both IgG2a and IgG3 antibody
responses
(Metzger, D.W., et al., Eur. J. Immunol., 27:1958-1965 (1997}; Snapper, C.M.,
et
al., Science, 236:944-947 (1987); Snapper, C.M., et al., J. Exp. Med.,
175:1367-
1371 (1992); Collins, J.T., et al., Int. Immunol., 5:885-891 (1993)). In
addition, the
2 5 initial IgGI suppression seen with IL-12 treatment was lost by day 28, in
agreement
with previous findings (Buchanan, J.M., et al., Int. Immunol., 7:1519-1528
(1995);
Metzger, D.W., et al., Ann. N. Y. Acad. Sci., 795:100-115 (1996)). These
results
demonstrate that IL-12 can be delivered i.n. in a non-invasive fashion to
influence
humoral responses in a manner similar to parenteral administration. Thus, i.n.
IL-12
3 0 administration would be a safer and effective adjuvant for protein vaccine
delivery.
As also described herein, it was found that IL-12 administered i.n. or
parenterally resulted in enhancement of fecal IgG2a antibody levels. In
contrast, i.n.
treatment with IL-12 resulted in reduced IgA expression while parenteral
delivery of
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PCT/US99/04678
IL-12 enhanced IgA levels. These results show an important differential effect
of
IL-12 given via two different routes of administration. Recently, in contrast
to the
data described herein, Okada et al. (Okada, E., et al., ,l. Immunol., 159:3638-
3647
(1997)) reported that i.n. immunization with an HIV DNA vaccine in an IL-12
expressing plasmid did not modify fecal IgA antibody levels. Furthermore,
Marinaro et al. (Marinaro, M., et al., J. Exp. Med., 185:415-427 (1997))
reported
that oral delivery of IL-12 in encapsulated liposomes did not alter IgA
levels,
whereby parenteral administration resulted in reduction of fecal IgA
responses. In
the Marinaro et al. study, mice were immunized orally with antigen for both
routes
of delivery of IL-12, and, as such, it would be difficult to make a direct
comparison
with the findings described herein which utilized different routes of delivery
of
antigen plus IL-12. The results clearly show the ability of IL-12 to
differentially
affect fecal antibody responses depending on the route of immunization.
There is continued interest in developing safer, more potent and better
2 5 targeted vaccine adjuvants against a range of infectious diseases (Van
Regenmortel,
M., ASMNews, 63:136-139 (1997}). This is in part because the adjuvants
currently
approved for human use such as alum lack the ability to elicit cell-mediated
immunity which is crucial for protection against particular diseases (Gupta,
R.K., et
al., "The role of adjuvants and delivery systems in modulation of immune
response
2 0 to vaccines In Novel Strategies in Design and Production of Vaccines, Eds.
Cohen,
S. and Shafferman, A., Plenum Press, New York, 1996, pp. 105-113}. In the
context
of vaccine development, the activation of the appropriate Th cells is integral
in
modulation of the immune response. For example, Thl type immune responses
have been shown to be protective against Leishmania (Muller, L, et al.,
Immunol.
2 5 Rev., 112:95-113 (1989)) and Listeria (Kratz, S.S., et al., J. Immunol.,
141:598-606
(1988)) infections. IL-12 is a key cytokine in immune regulation by its
ability to
direct Th cells towards a Thl phenotype with enhancement of IFN-'y secretion
and
elevation of IgG2a antibody levels. As such, the findings described herein
show that
the i.n. use of IL-12 as an adjuvant enhances vaccine immunity. Moreover,
there are
3 0 no suitable mucosal adjuvants for clinical use at the current time. An
immediate
application for IL-12 given by this route would be for use in conjunction with
nasal
influenza vaccines currently in clinical trials. Since protection against
influenza is
mediated by IgG antibody (Palladino, G., et al., J. Virol., 69:2075-2081
(1995)), co-
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WO 99/44635 15
administration of IL-12 i.n. would be a means to augment both mucosal and
systemic antibody responses towards influenza. In this regard, as shown
herein, i.n.
administration of a subunit influenza vaccine plus IL-12 markedly enhances
systemic and respiratory IgG2a levels.
As described herein a non-invasive i.n. delivery system was used to evaluate
the ability of IL-12 to modulate both mucosal and systemic components of the
immune system. Mice immunized i.n. with DNP conjugated to OVA (DNP-OVA)
in combination with CTB and IL-12 were found to have elevated levels of IFN-y
and IL-10 mRNA transcripts in both lungs and spleens compared to mice not
1 o receiving IL-12. In addition, expression of lung IL-S mRNA was inhibited.
Analysis of BAL after IL-12 treatment revealed a significant increase in IgG2a
and
unaltered IgGl and IgA anti-OVA antibody levels. Serum IgG2a, IgG2b and IgG3
anti-DNP antibody levels were significantly increased by IL-12 given i.n.,
while
serum IgGl antibody levels were suppressed, results that are similar to those
seen
after systemic antigen plus IL-12 administration. Delivery of IL-12 i.n. also
enhanced fecal IgG2a and suppressed IgA levels, in contrast to parenteral
treatment
which increased both fecal IgG2a and IgA antibody expression. These results
show
that i.n. IL-12 treatment can effectively modulate antigen-specific immune
responses
and enhance immunization strategies for mucosal vaccines.
2 0 In summary, the results clearly demonstrate the effectiveness of IL-12
administered i.n. for augmenting antigen specific-responses in both mucosal
and
systemic compartments. The findings show that IL-12 can be used as a potent
vaccine adjuvant for immunization strategies against mucosal pathogens.
Thus, the methods and described herein can be used to treat and/or prevent a
2 5 disease or condition associated with a pathogen having one or more
antigens in a
host. The methods described herein can utilize an effective amount of IL-12 in
combination with a single antigen or multiple antigens which can be derived
from
the same pathogen, from different strains of a pathogen or from different
pathogens.
Thus, IL-12 and one or more antigens can be used to prevent and/or treat one
or
3 0 more disease or condition associated with the pathogens) from which the
antigens)
is derived.
The present invention is illustrated by the following examples, which are not
intended to be limiting in any way.
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EXEMPLIFICATION
EXAMPLE 1: MODULATION OF MUCOSAL AND SYSTEMIC IMMUNITY
BY INTRANASAL INTERLEUKIN 12 DELIVERY
Materials and Methods
Mice
Six to eight week-old female BALB/c mice were obtained from the National
Cancer Institute (Bethesda, MD). Mice were housed in the animal facility at
the
Medical College of Ohio, and provided food and water ad libitum. Animal care
and
experimental procedures were in compliance with the Institutional Animal Care
and
Use Committee (IACUC) of the Medical College of Ohio.
INTRANASAL IMMUNIZATION PROTOCOL
Intranasal treatments were performed on mice that had been anesthetized
intraperitoneally (i.p.) with a combination of ketamine-HCL (Fort Dodge
Laboratories, Fort Dodge, IO) and Xylazine (Bayer Corporation, Shawnee
Mission,
KA) at concentrations of 80 mg and 16 mg per mouse, respectively. On day 0,
mice
were immunized i.n. with 50 ~l of sterile phosphate-buffered saline (PBS)
containing 100 ~g of dinitrophenyl hapten conjugated to ovalbumin (DNP-OVA;
Biosearch Technologies, San Raphael, CA) and 10 ~,g cholera toxin B-subunit
(CTB; Sigma, St. Louis, MO). This was followed on days 0, 1, 2 and 3 with
2 o intranasal i.n. of 1 ~g of recombinant marine IL-12 in PBS containing 1%
normal
BALB/c mouse serum (PBS-NMS) or, in the case of control mice, with PBS-NMS
only. Mice were boosted i.n. with the same amount of DNP-OVA and CTB on days
14 and 28. On day 28, the mice also received 1 ~,g of IL-12 in PBS-NMS or PBS
NMS only. For i.p. inoculations, mice were immunized with 100 ~g of DNP-OVA
in complete Freund's adjuvant (CFA; Life Technologies, Gaithersburg, MD) on
day
0, followed by injection of 1 ~.g of IL-12 in PBS-NMS on days 0, 1, 2 and 3.
Control mice received antigen and PBS-NMS only. Mice were boosted by the same
route on days 14 and 28 with DNP-OVA in incomplete Freund's adjuvant (IFA;
Life
Technologies). On day 28, the mice were also injected i.p. with IL-12 in PBS-
NMS
3 0 or PBS-NMS only. Sera were prepared by bleeding mice from the orbital
plexus.
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RNA ISOLATION
Total RNA isolation from snap frozen spleens and lungs was performed with
Trizol reagent (Gibco-BRL Gaithersburg, MA) according to the manufacturer's
instructions. Briefly, the frozen tissues were homogenized with a mortar and
pestle,
and immediately transferred into polystyrene tubes containing 2.0 ml of Trizol
reagent. The homogenized samples were incubated for 5 minutes at room
temperature to allow dissociation of the nucleoprotein complexes and
centrifuged at
12,OOOg for 10 minutes at 4°C. The supernatant fluids were mixed for 15
seconds
with 0.4 ml of chloroform, incubated for 1 S minutes on ice, and centrifuged
at
12,OOOg for 15 minutes at 4°C. Following centrifugation, the RNA in the
aqueous
phase was precipitated at -20°C for one hour by the addition of 1.0 ml
isopropanol.
The samples were centrifuged for 15 minutes at 12,000g and the RNA pellet was
washed twice with 1.0 ml of 75% ethanol. The pellet was air-dried for 2-S
minutes,
solubilized in DEPC-treated water, and stored at -80°C. The
concentration of total
RNA was calculated using the A260 value for single-stranded RNA (1 A260 unit =
40 ~g of single stranded RNA/ml). The final preparation of total RNA yielded a
260/280 ratio of 1.7-2Ø
FIRST STRAND cDNA SYNTHESIS
First strand cDNA synthesis was performed following the manufacturer's
2 o instructions (Gibco-BRL). Briefly, 1 ~g of oligo(dT), 3 ~g of total RNA,
and sterile
DEPC-treated water were added to a sterile eppendorf tube to a final volume of
11
~,1. The mixture was incubated at 70°C for 10 minutes and then chilled
on ice.
Subsequently, the following components were added in order: 4.Opl of SX first
strand buffer, 2 ~l of 0.1 M DDT, and 1 ul of dNTP mixture (10 mM each of
dATP,
2 5 dGTP, dCTP and dTTP). The contents of the tube were mixed gently and
incubated
at 42°C for 2 minutes, followed by the addition of 1 pl (200 Ln of
Superscript II
reverse transcriptase (RT). The reaction mixture was gently mixed and
incubated at
42°C for one hour, then terminated by incubation at 70°C for 15
minutes.
POLYMERASE CHAIN REACTION (PCR)
3 0 A 50 ~.1 reaction mixture was prepared in a sterile eppendorf tube with
the
following components: 31.30 ~1 DEPC treated water, 10.0 ~1 of S times Tris-HCL
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WO 99/44635 PCT/US99/04678
buffer (optimal magnesium and pH were determined for each primer set), 2 ~.1
of
cDNA from the first strand synthesis, 2 ~.l primer (20 ~m stock
concentration), 5.0
~1 of dNTP mix (2.5 mM dATP, 2.5 mM dCTP, 2.5 mM dGTP, and 2.5 mM dTTP,
pH 8.0) (Invitrogen Corporation), and 0.5 ~.1 (2.5 U) of Taq DNA polymerise
(Gibco-BRL). The tubes were placed into the wells of the Perkin Elmer Thermal
Cycler 480 (Perkin Elmer Cetus, Norwalk, CT), incubated at 95°C for 5
minutes and
then subjected to the following amplification profile: 1 minute at
95°C, 1 minute at
56°C and 1 minute at 72°C for a duration of 35 cycles. 'This
followed by an
incubation at 72°C for 10 minutes followed by a soak cycle at
4°C. The PCR
products were separated on a 2.5% agarose gel and stained with ethidium
bromide.
The bands were visualized and photographed using UV transillumination.
Hypoxanthine phosphoribosyl transferase (HPRT) was used as a housekeeping
control to ensure equal loading of RNA in all lanes and a 100 by DNA ladder
(Gibco-BRL) was used as a molecular weight marker.
Primer Sequences
HPRT
5' GTT GGA TAC AGG CCA GAC TTT GTT G 3' (SEQ ID NO: 1 )
5' GAT TCA ACT TGC GCT CAT CTT AGG C 3' (SEQ ID NO: 2)
IL-5
2 0 5' GAC AAG CAA TGA GAC GAT GAG 3' (SEQ ID NO: 3)
5' GTT ATC CTT GGC TAC ATT ACC 3' (SEQ ID NO: 4)
IL-10
5' ATG CAG GAC TTT AAG GGT TAC TTG GGT T 3' (SEQ ID NO: 5)
S' ATT TCG GAG AGA GGT ACA AAC GAG GTT T 3' (SEQ ID NO: 6)
2 5 IF'N-'y
5' TGA ACG CTA CAC ACT GCA TCT TGG 3' (SEQ ID NO: 7)
5' CGA CTC CTT TTC CGC TTC CTG AG 3' (SEQ ID NO: 8)
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COLLECTION OF BRONCHOALVEOLAR LAVAGE (BAL) AND FECAL
EXTRACTS
_ For collection of BAL, the mice were sacrificed and their tracheas were
exposed and intubated using a 0.58 mm OD polyethylene catheter (Becton
Dickinson, Sparks, MD). The lungs were lavaged two to three times with PBS
containing 5 mM EDTA. Approximately 1.5 ml of lavage fluid was obtained per
mouse and blood contamination was monitored using Hemastix (Bayer Corporation,
Elkhart, Il~. The recovered BAL fluid was centrifuged at 12,OOOg for 5 minutes
at
4°C and the supernatant was stored at -70°C until use. Fecal
extracts were prepared
by the method of deVos and Dick (deVos, T., et al., J. Immunol. Meth., 141:285-
288
(1991). Briefly, 0.1 g of fecal material from each mouse was mixed with 1 ml
PBS
and allowed to incubate for 15 minutes at room temperature. The sample was
subsequently vortexed for 5 minutes and centrifuged at 12,000 x g for 10
minutes.
The supernatant was then stored at -70°C.
DETECTION OF ANTIBODY AND ISOTYPE LEVELS BY ELISA
Anti-DNP and anti-OVA antibody levels were determined by ELISA as
described (Buchanan, J.M., et al., Int. Immunol., 7:1519-1528 (1995); Metzger,
D.W., Eur. J. Immunol., 27:1958-1965 (1997)). Briefly, microtiter plates
(Nalge
Nunc International, Rochester, NY) were coated overnight with 10 ~g/ml DNP-
bovine serum albumin (BSA) or 100 ~g/ml of OVA in PBS. The plates were
washed with PBS containing 0.1% (w/v) gelatin and 0.05% (v/v) Tween 20. Serial
dilutions of serum or BAL fluid were then added and the plates were incubated
for 2
hours at room temperature. The plates were again washed and incubated for 1
hour
with goat anti-mouse IgGI, IgG2a, IgG2b or IgG3 conjugated to alkaline
2 5 phosphatase (Southern Biotechnology Associates, Birmingham, AL). The
plates
were washed and p-nitrophenyl phosphatase substrate was added to obtain
optimal
color development. The plates were read at 405 nm with an ELISA microplate
reader (Bio-Tek Instruments, Winooski, VT). To detect IgA, the wells were
incubated with goat anti-mouse IgA conjugated to biotin (Sigma, St. Louis,
MO),
3 0 washed, and incubated with alkaline phosphatase conjugated to streptavidin
(Biorad,
Richmond, CA) before addition of substrate. Total immunoglobulins were
measured in the sane fashion except that the plates were coated with 10 pg/ml
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WO 99/44635 PCT/US99/04678
affinity purified goat anti-mouse Ig (Southern Biotechnology Associates)
(Buchanan, J.M., et al., Int. Immunol., 7:1519-1528 (1995)). In all cases,
appropriate working dilutions and isotype specificities of the secondary
antibody
conjugates were determined using purified myeloma proteins of known isotypes
(Sigma, St. Loius, MO). Furthermore, antigen specificity of the assays was
established using plates coated with BSA only. Statistical significance was
determined using a two-tailed Student t-test. Data were considered
statistically
significant ifp values were <0.05.
RESULTS
1 o Intranasal IL-12 delivery induces a Thl-like response in the lungs and
spleens
To determine whether intranasal delivery of antigen plus IL-12 would
modulate cytokine mRNA expression in the lungs, mice were immunized with DNP-
OVA and CTB +/- IL-12, and levels of cytokine mRNA in the lungs of individual
animals were analyzed by RT-PCR after 6 and 24 hours. There was found to be a
sharp increase in the expression of IFN-y mRNA in mice 6 hours after treatment
with IL-12 and this expression remained elevated for at least 24 hours
compared to
immunized mice not exposed to IL-12. There were no differences in IL-10 mRNA
expression noted in the lungs of IL-12 treated mice after 6 hours but
increased
expression was observed 24 hours post inoculation. Since IFN-y mRNA has been
2 0 found to downregulate Th2 type cytokines such as IL-5 (Mosmann, T.R., et
al.,
Annu. Rev. Immunol., 7:145-173 (1989); Coffman, R.L., et al., Immunol. Rev.,
123:189-207 (1991)), expression of IL-S mRNA was also examined and a strong
decline by 6 hours, which was still apparent after 24 hours, was observed.
Cytokine expression in the lungs was compared to that in spleens after i.n.
2 5 inoculation of antigen plus IL-12. There was an enhancement of splenic IFN-
y
mRNA expression 6 hours after treatment with IL-12. This increase was still
pronounced at 24 hours whereas untreated mice had nearly undetectable levels
of
IFN-y mRNA at this time point. Increases of IL-10 mRNA levels were also
detected in the spleens of IL-12 treated mice, with maximal expression at 24
hours
3 0 compared to untreated controls. The ability of IL-12 given i.p. to induce
systemic
IL-10 expression was previously shown by others (Meyaard, L., et al., J.
Immunol.,
156:2776-2782 (1996); Daftarian, P.M., et al., J. Immunol., 157:12-20 (1996);
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WO 99/44635 PCT/US99/04678
Gerosa, F., et al., J. Exp. Med., 183:255902569 (1996)). Finally, no IL-5 was
detected in the spleens of either IL-12 treated or control mice in contrast to
the lungs
_ where IL-5 mRNA was detected after i.n. antigen treatment but suppressed by
co
administration of IL-12. Simultaneous amplification of HPRT mRNA confirmed
that equal amounts of RNA were utilized in all of the RT-PCR reactions. These
results clearly demonstrate that i.n. administration of IL-12 can modulate
antigen-
driven cytokine responses in both mucosal and systemic compartments, resulting
in
significant enhancement of IFN-y and IL-10 mRNA expression. These findings
also
provide strong evidence for the ability of i.n. delivery of IL,-12 to
downregulate the
expression of the Th2-associated cytokine, IL-5.
Intranasal IL-12 administration modulates respiratory antibody responses
Previous work (Buchanan, R.L, et al., Int. Immunol., 7:1519-1528 {1995);
Metzger, D.W., et al., Ann. N. Y. Acad. Sci., 795:100-115 (1996); McKnight,
A.J., et
al., J. Immunol., 152:2172-2179 (1994); Morns, S.C., et al., J. Immunol.,
152:1047
1056 (1994); Germann, T., et al., Eur. J. Immunol., 25:823-829 (1995); Wynn,
T.A.,
et al., .l. Immunol., 157:4068-4078 (1996); Bliss, J., et al., J. Immunol.,
156:887-894
(1996)) demonstrated the ability of parenteral delivery of IL-12 to enhance
serum
IgG2a antibody responses to protein and hapten-tamer antigens. IL-12 also
temporarily suppresses IgGI production (Buchanan, J.M., et al., Int. Immunol.,
7:1519-1528 (1995); Metzger, D.W., et al., Ann. N. Y. Acad. Sci., 795:100-115
(1996)). It has now been found, as described herein, that i.n. administration
of IL-12
modulates respiratory antibody responses in a similar fashion. BAL fluids were
collected on day 35 of the immune response and analyzed by ELISA. Mice that
were immunized with DNP-OVA and treated i.n. with IL-12 showed a dramatic
2 5 enhancement (p<0.05) in IgG2a anti-OVA antibody levels compared to
immunized
mice not exposed to IL-12 (Figures lA-1C). There were no differences in IgGl
or
IgA anti-OVA antibody levels between control and experimental groups. Blood
contamination was ruled out by the absence of albumin in respiratory
secretions.
These results provide the first evidence for the ability of i.n. delivery of
IL-12 to
3 o alter a respiratory antibody response.
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Intranasal IL-12 administration modulates serum antibody responses
ELISA analyses of day 14 sera revealed that i.n. inoculation of DNP-OVA
and IL-12 also caused a significant increase (p<0.05) in serum IgG2a anti-DNP
antibody levels compared to control mice that received DNP-OVA and vehicle
(Figures 2A-2E). In addition, there was significant enhancement (p<0.05) of
serum
IgG2b and IgG3 anti-DNP antibody levels after IL-12 treatment. Importantly,
the
serum IgG2a, IgG2b and IgG3 anti-DNP responses were still elevated 28 days
after
i.n. IL-12 treatment. There was also suppression of day 14 serum IgGI anti-DNP
antibody production in IL-12 treated mice but little change in IgA anti-DNP
1 o antibody levels. However, the initial IgGl suppression observed with II,-
12
treatment was lost by day 28 of the immune response showing that the
suppression
of IgGI was only a temporary effect. The effects of i.n. IL-12 treatment on
serum
levels of total (nonspecific) IgGI and IgG2a were also examined. It was found
that
IL-12 treated mice had a corresponding increase in serum IgG2a and a decrease
in
IgGI 14 days after treatment (Figures 3A-3B). This pattern was still observed
four
weeks after IL-12 inoculation.
Influence of IL-12 on fecal antibody
The effects of IL-12 given i.n. or i.p. on fecal antibody responses was
examined. Mice that received antigen and IL-12 by either route had
significantly
2 o higher levels (p<0.05) of fecal IgG2a anti-DNP antibody levels compared to
immunized mice not exposed to IL-12 (Figures 4A-4F). In fact, mice that
received
only antigen parenterally had no detectable IgG2a in fecal extracts. While
parenteral
treatment with antigen and IL-12 also resulted in enhancement of fecal IgA
levels
(p<0.05), i.n. delivery of IL-12 resulted in a decrease (p<0.05) of IgA
antibody
2 5 levels. There were no significant differences in fecal IgGl antibody
levels between
II,-12 treated and control groups after parenteral or i.n. routes of
immunization.
These results show that i.n. delivery of IL-12 and antigen induces shifts in
IgG
production similar to those seen after parenteral injection of IL-12. However,
only
parenteral administration of IL-12 results in enhanced mucosal IgA antibody
levels.
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Effects of IL-12 on systemic antibody responses using purified hemaglutinin
and
neuraminidase derived from influenza virus
The effects of IL-12 administered i.n. on systemic antibody responses were
examined using purified hemaglutinin and neuraminidase derived from influenza
virus (HANA). Mice were immunized i.n. on day 0 with HANA and treated i.n.
with either IL-12 or PBS vehicle on days 0, 1, 2 and 3. Serum anti-HANA
antibody
levels on day 14 were determined by isotype-specific ELISA using HAHA-coated
microtiter plates. See Figures SA-SB. In addition, mice were immunized i.n. on
day
0 with HANA and treated i.n. with either IL-12 or PBS vehicle on days 0, 1, 2
and 3
and boosted on day 14. Serum anti-HANA antibody levels on day 28 were
determined by isotype-specific ELISA using HAHA-coated microtiter plates. See
Figures 6A-6B.
Effects of IL-12 administered i.n. on respiratory mucosal responses
The effects of IL-12 administered i.n. on respiratory mucosal responses were
examined. Mice were immunized on day 0 with HANA and treated with either IL-
12 or PBS vehicle on days 0, 1, 2 and 3 and boosted on days 14 and 28; on day
28
the mice also received IL-12 or vehicle. Mice were sacrificed on day 3S, and
BAL
fluid was assayed for anti-HANA antibody levels by ELISA using HANA coated
microtiter plates. See Figures 7A-7B.
2 o EXAMPLE 2 INTRANASAL INTERLEUKIN-12 IS A POWERFUL ADJUVANT
FOR PROTECTIVE MUCOSAL I1~1MCTNITY
Methods
Mice
Six-to eight-week old female BALB/c mice were obtained from The
2 5 National Cancer Institute (Bethesda, MD). CS7BL/6 IgM deficient (uMT) mice
were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed
in the animal facility at the Medical College of Ohio and provided food and
water ad
libitum. All animal care and experimental procedures were performed in
compliance
with the Institutional Animal Care and Use Committee (IACUC) guidelines.
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Immunizations
Intranasal treatments were performed on mice that had been anesthetized i.p.
with a combination of Ketamine HCL (Fort Dodge Laboratories, Fort Dodge, IO)
and Xylazine (Bayer Corporation, Shawnee Mission, KA). Mice were immunized
i.n. on day 0 with 25 ,ul of sterile PBS containing 1 ~cg of subunit influenza
vaccine
which consisted of soluble hemagglutinin subtype 1 (Hl) and neuraminidase
subtype 1 (Nl) purified from influenza virus A/PR8/34 (provided by Dr. Doris
Bucher, New York Medical College, New York, NY). This was followed on days 0,
1, 2 and 3 with i.n. inoculation of 1 ,ug of recombinant marine IL-12 in PBS
containing 1% normal BALB/c mouse serum (PBS-NMS) or in the case of control
mice, with PBS-NMS only. Mice were boosted i.n. with the same amount of
vaccine on days 14 and 28. On day 28, the mice also received IL-12 in PBS-NMS
or PBS-NMS only. No toxicity was observed with this treatment regimen. Sera
were prepared by bleeding mice from the orbital plexus.
RNA Isolation and RT-PCR
Total RNA isolation from snap frozen spleens and lungs was performed with
the Ambion Total RNA Isolation Kit (Austin, TX) according to the
manufacturer's
instructions. Briefly, the frozen tissues were homogenized with a mortar and
pestle
and immediately transferred into tubes containing 1.0 ml of denaturation
solution.
2 0 Following phenol-chloroform extraction, the homogenized samples were
centrifuged
at 10,000 x g for 10 minutes at 4°C. The supernatants were subjected to
another
round of phenol-chloroform extraction and the resulting RNA was precipitated
with
isopropanol, washed twice with 75% ethanol and solubilized in DEPC-treated
water.
The concentration of total RNA was determined by spectrophotometric analysis
at
2 5 260 nm. Three micrograms of total RNA were reverse transcribed into cDNA
using
a reverse transcription kit (Life Technologies, Gaithersburg, MD) utilizing
oligo
(dT),~,8 primers. The resulting cDNA was amplified using specific primers for
IFN-
y and IL-10 with hypoxanthine phosphoribosyl transferase (HPRT) primers as a
control. The sense and antisense primers utilized had the following sequences:
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~''Y
5'-TGAACGCTACACACTGCATCTTGG-3' (SEQ ID NO: 7) and
5'-CGACTCCTTTTCCGCTTCCTGAG-3' (SEQ ID NO: 8);
IL-10
5'-ATGCAGGACTTTAAGGGTTACTTGGGTT-3' (SEQ ID NO: 5) and
5'-ATTTCGGAGAGAGGTACAAACGAGGTTT-3' (SEQ ID NO: 6);
HPRT
5'-GTTGGATACAGGCCAGACTTTGTTG-3' (SEQ ID NO: 1) and
5'-GATTCAACTTGCGCTCATCTTAGGC-3' (SEQ ID NO: 2).
PCR amplification was performed by mixing 2 ,ul of cDNA, 0.25 mM dNTPs
(Invitrogen Corporation, San Diego, CA), 0.8 ~cM primer and 2.5 U of Taq DNA
Polymerase (Life Technologies) in a final volume of 50 ~cl in 60 mM Tris-HCl
(pH
8.5), 15 mM (NH4)2 504, 0.4 mM MgCl2. The mixtures were incubated at
95°C for
5 minutes and then subjected to the following amplification profile: 1 minute
at
95 ° C, 1 minute at 56 °C and 1 minute at 72 ° C for a
duration of 35 cycles. This was
followed by a final extension for 10 minutes at 72°C. The PCR products
were
separated on a 2.5% agarose gel, stained with ethidium bromide and visualized
by
UV transillumination.
Ribonuclease Protection Assay
2 0 Cytokine mRNA levels were determined utilizing the RiboQuant multi-probe
ribonuclease protection assay system (Pharmingen, San Diego, CA) according to
the
manufacturer's instructions. Briefly, 10 ~g of total RNA was hybridized to a
32P
labeled RNA probe overnight at 56°C. The single-stranded nucleic acid
was
digested with ribonuclease for 45 minutes at 30°C, subjected to phenol-
chloroform
2 5 extraction, and resolved on a 6% denaturing polyacrylamide gel. Transcript
levels
were quantified on a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale;
CA). Total RNA was normalized to the housekeeping gene glyceraldehyde 3-
phosphate dehydrogenase and relative cytokine mRNA levels were expressed as
arbitrary values.
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Collection of Bronchoalveolar Lavage Fluid
For collection of BAL fluid, the mice were sacrificed and their tracheas
intubated using a 0.58 mm OD polyethylene catheter (Becton Dickinson, Sparks,
MD). The lungs were then lavaged two to three times with PBS containing 5 mM
EDTA. The recovered BAL fluid was centrifuged at 12,000 x g for 5 minutes at
4°C and the supernatant was stored at -70°C until use.
Detection of Antibody and Isotype Levels by ELISA
Anti-H1N1 levels in serum and BAL were determined by ELISA essentially
as described (Buchanan, J.M., et al., Int. Immunol., 7:1519-1528 (1995);
Buchanan,
R.M.,et all. ,l. Immunol., 161:5525-5533 (1998)) with minor modifications.
Briefly,
microtiter plates (Nalge Nunc International, Rochester, NY) were coated
overnight
with 1 ~cg/ml of H1N1 in PBS. The plates were washed with PBS containing 0.3%
Brij-35 (Sigma, St. Louis, MO) and blocked for 1 hour at room temperature with
PBS containing 5% fetal calf serum (Hyclone Laboratories, Logan, UT) and 0.1%
Brij-35. Serial dilutions of serum were added and the plates incubated for 2
hours at
room temperature. The plates were washed and incubated with goat anti-mouse
IgGl or IgG2a conjugated to alkaline phosphatase (Southern Biotechnology
Associates, Birmingham, AL). After incubation for 1 hour, the plates were
washed
and p-nitrophenyl phosphatase substrate was added to obtain color development.
2 0 Plates were read at 405 nm with an ELISA microplate reader (Bio-Tek
Instruments,
Winooski, VT). To detect IgA, the plates were incubated with goat anti-mouse
IgA
conjugated to biotin (Sigma), then washed and incubated with alkaline
phosphatase-
conjugated streptavadin (BIO RAD, Richmond, CA). Total immunoglobulins were
measured in the same fashion except that the plates were coated with 10 ,ug/ml
2 5 affinity-purified goat anti-mouse Ig (Southern Biotechnology Associates)
(Buchanan, J.M., et al., Int. Immunol., 7:1519-1528 (1995)). In all cases,
appropriate working dilutions and isotype specificities of the secondary
antibody
conjugates were determined using purified myeloma proteins of known isotypes
(Sigma). Statistical significance was determined using a two-tailed Student t-
test.
3 0 Data were considered statistically significant ifp values were <0.05.
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Virus Challenge
For protection studies, mice were immunized i.n. on day 0 with 25 ,ul of PBS
containing 1 ~cg of HIN1 subunit influenza vaccine. This was followed on days
0,
1, 2 and 3 with i.n. inoculation of 1 ~cg of IL-12 in PBS-NMS or with PBS-NMS
only. Some mice received only IL-12 in PBS-NMS or only PBS-NMS (no H1N1
subunit vaccine). Approximately 4-5 weeks after primary immunization, viral
challenge was performed using infectious A/PR8/34 influenza virus (provided by
Dr. Doris Bucher) administered i.n. to anesthetized mice in 40 ~cl of sterile
PBS.
The mice were weighed daily and monitored for morbidity and mortality.
Passive Transfer of Sera and BAL Fluid
For passive transfer experiments, sera were obtained on day 28 after i.n.
immunization with the H1N1 subunit vaccine. Mice were injected i.p. with 100
,ul
of a 1:10 dilution of pooled serum and challenged 5 hours later with
infectious
influenza virus i.n. BAL fluid collected from mice on day 35 after i.n.
immunization
with H1N1 subunit influenza vaccine was centrifuged to remove cells and the
supernatant was administered i.n. to anesthetized mice together with virus in
a total
volume of 40 ,ul.
Results
Intranasal IL-I2 Administration Induces Expression of Thl Type Cytokine
2 0 Responses in the Lungs and Spleens of Immunized Mice
IL-12 given parenterally has profound regulatory effects on the immune
system through its ability to preferentially activate Thl and NK cells, and
induce
IFN-'y production (Trinchieri, G., et al., Res. Immunol., 146:423-431 (1995);
Gately,
M.K., et al., Annu. Rev. Immunol., 16:495-521 (1998)). As described herein,
the
2 5 effects of i.n. administration of IL-12 on respiratory cytokine gene
expression have
now been examined. Analysis of cytokine mRNA expression in the lungs of
individual mice (3 mice per group) after a single i.n. inoculation of IL-12 or
PBS
vehicle and H1N1 subunit influenza vaccine. Mice were sacrificed 24 hours or
48
hours after treatment, and total lung RNA was assayed for the expression of
the
3 0 indicated cytokines by RT-PCR: IL-10 (455 bp), IFN-y (459 bp) and HPRT
(162
bp). It was found the i.n. treatment of mice with H1N1 subunit influenza
vaccine
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and IL-12 had an enhancing effect on expression of lung IFN-'y mRNA levels
within
24 hours compared to immunization with vaccine only. This increase in IFN-y
mRNA levels was still evident 48 hours after IL-12 inoculation.
It has been previously demonstrated (Meyaard, L., et al., J. Immunol., ,
156:2776-2782 (1996); Daftarian, P.M., et al., J. Immunol., 157:12-20 (1996);
Gerosa, F., et al., J. Exp. Med., 183:2559-2569 (1996)) that treatment with IL-
12
enhances expression of IL-10 mRNA. As described herein, i.n. delivery of H1N1
vaccine plus IL-12 also caused a dramatic increase in lung IL-10 mRNA
expression.
In contrast, there was an absence of IL-10 mRNA in mice that received vaccine
only. IL-10 mRNA expression was still significantly elevated after 48 hours in
the
lungs of IL-12 treated mice compared to animals that received the vaccine
alone.
The expression of IL-5 mRNA was also examined and no differences were found
after IL-12 treatment.
To determine if local mucosal delivery of IL-12 could modulate a distant
systemic compartment, cytokine mRNA patterns in the spleens of immunized mice
were examined. Analysis of cytokine mRNA expression in the spleens of
individual
mice (3 mice per group) after a single i.n. inoculation of IL-12 or PBS
vehicle and
H1N1 subunit influenza vaccine. Mice were sacrificed 24 hours or 48 hours
after
treatment, and total splenic RNA was assayed for the expression of the
indicated
2 o cytokines by RT-PCR: IL-10 (455 bp), IFN-y (459 bp), and HPRT (162 bp).
Intranasal administration of H1N1 subunit vaccine plus IL-12 resulted in a
substantial increase in splenic IFN-'y mRNA expression within 24 hours
compared
to mice that received vaccine alone. Elevated levels of IFN-y were still
evident at
48 hours in IL-12 treated mice. Splenic IL-10 mRNA levels remained elevated at
2 5 both 24 hours and 48 hours after IL-12 treatment. Finally, no IL-5 mRNA
was
detected in the spleens of either IL-12 treated or control animals.
Simultaneous
amplification of HPRT mRNA confirmed that equal amounts of RNA were utilized
in all RT-PCR reactions. To further quantify the levels of cytokine mRNA
transcripts observed after i.n. immunization with influenza vaccine, cytokine
mRNA
3 0 levels in the lungs and spleens were analyzed by ribonuclease protection
assay. It
was found that IFN-y mRNA levels were increased 2-fold in the lungs of animals
24
hours and 48 hours after treatment with H1N1 plus IL-12 compared to mice that
received vaccine alone (the Table). Furthermore, IL-10 mRNA expression was
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PCT/US99/04678
enhanced 5-fold in the lungs after IL-12 treatment. In the spleens of these
animals,
IFN-'y mRNA was elevated 5-fold at 24 hours and 2-fold at 48 hours after IL-12
_ treatment. Similarly, splenic IL-10 mRNA levels were increased 8-fold at 24
hours
and 5-fold at 48 hours after IL-12 treatment.
Table. IFN-y and IL-10 mRNA Levels in the Lungs and Spleens of Mice
Immunized with Influenza Subunit Vaccine*
Lungs
Time Cytokine H1N1 + H1N1 + Fold
PBS IL-12 Increase
IFN-'y 380 t 830 t 61 2.2
6.7
24 hours
IL-10 1.7 ~ 8.7 ~ 1.3 S.1
0.8
IFN-y 340 ~ 630 ~ 152 1.9
110
l0 48 hours
IL-10 1.610.5 8.613.0 5.4
Spleens
Time Cytokine H1N1 + H1N1 + Fold
PBS IL-12 Increase
____________1___________I____________1___________l_______________
IFN-y 410 t 1900 t 4.6
115
24 hours 400
IL-10 2. 9 ~ 2214.3 7.6
1.9
IFN-y 570 t 1000 t 1.8
109
48 hours 170
IL-10 2.7 ~ 1315.0 4.8
1.6
*Mice were sacrificed 24 hours and 48 hours after i.n. treatment with
H1N1 subunit influenza vaccine t Il-12. Total RNA was isolated and
IFN-y and IL-10 transcript levels were analyzed by multiplex
ribonuclease protection assay. Relative RNA levels were quantitated
on a phosphorimager and normalized to glyceraldehyde 3-phosphate
2 0 dehydrogenase. The cytokine mRNA levels are expressed as
arbritary units ~ SE.
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Co-administration of an Intranasal Vaccine Plus IL-12 has Potent Effects on
Systemic Antibody Responses
It was previously demonstrated that parenteral administration of IL-12 alters
isotype-restricted antibody responses to hen eggwhite lysozyme (Buchanan,
J.M., et
al., Int. Immunol., 7:1519-1528 (1995)). In addition, as described in Example
1, IL
12 delivered i.n. modulates both mucosal and systemic immunity to the DNP
hapten.
In this example it has been demonstrated that IL-12 delivered i.n. has similar
effects
on antibody responses to H1N1 influenza vaccine. Fourteen days after
immunization with vaccine by itself or together with IL-12, there was little
if any,
detectable serum IgGI anti-HINT antibody (Figures 8A-8B). In contrast, IgG2a
anti-H1N1 antibody levels were markedly enhanced after IL-12 treatment
compared
to mice that received vaccine alone. Therefore, i.n. IL-12 treatment resulted
in early
activation of serum IgG2a antibody responses.
Similar analysis were performed on day 35 sera to determine the long-term
effects of i.n. IL-12 treatment. At this time point, IL-12-treated mice had 6-
fold
higher levels of total anti-H1N1 serum antibody than mice immunized with the
vaccine alone (Figures 9A-9E). Moreover, there was an increase in total (non-
specific) Ig after i.n. IL-12 treatment. IgG2a antibody levels were still
dramatically
enhanced in mice that received IL-12. Furthermore, IgGl anti-H1N1 antibodies,
2 0 evident in both experimental and control groups, were moderately elevated
in IL-12
treated mice compared to mice receiving only vaccine, an observation which is
consistent with our previous findings (Buchanan, J.M., et al., Int. Immunol.,
7:1519-
1528 (1995)). There was no IgA detected in the sera of any of the mice. The
results
clearly demonstrate the ability of IL-12 co-administered as an adjuvant and
2 5 delivered in a non-invasive form to enhance serum antibody levels.
Intranasal IL-12 Delivery Enhances Respiratory Antibody Levels
The antibody responses in BAL fluid from i.n. immunized mice were also
assessed. Analysis of BAL fluid collected on day 35 of the immune response
revealed that IL-12-treated mice had enhanced mucosal antibody responses to
H1N1
3 0 subunit influenza vaccine. As a group, i.n. IL-12 treatment resulted in 15-
fold
increases in total anti-H1N1 respiratory antibody production compared to mice
immunized with vaccine alone (Figures l0A-lOD). In addition, there was a 13-
fold
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increase in total nonspecific Ig in the BAL fluid of mice that received H1N1
plus IL-
12 i.n. Importantly, animals that were immunized and treated with IL-12
displayed
elevated BAL fluid IgA anti-H1N1 antibody levels compared to animals not
exposed
to II,-12. This result is in stark contrast to the absence of detectable IgA
in the
circulation of these mice. It was also found that levels of both IgGl and
IgG2a anti-
H1N1 antibodies were dramatically enhanced in BAL fluid after IL-12
administration compared to mice that received vaccine alone. These results
firmly
establish the influence of IL-12 delivered i.n. in augmenting respiratory
antibody
expression.
IL-12 Administration Increases the Protective Effects of Influenza Subunit
Vaccination
The effects of co-administrating IL-12 and H1N1 i.n. on survival and clinical
outcome after challenge with influenza virus were also assessed. Mice were
immunized i.n. with H1N1 vaccine on day 0 and treated with 1 ,ug of IL-12 or
PBS
vehicle on days 0, 1, 2 and 3. Some mice received only IL-12 or PBS vehicle.
Four
to five weeks after immunization, the mice were inoculated i.n. with
infectious
A/PR8/34 influenza virus and monitored daily for morbidity and mortality. In
the
first experiment, a dose of virus was used that allowed 50% survival of mice
after
exposure to just vaccine (Figures 11A-11B). It was found that inclusion of IL-
12
2 0 during vaccination resulted in 100% survival and significant reduction in
illness, as
evidenced by reduced weight loss compared to mice that received vaccine alone.
Mice that were pre-treated with IL-12 or PBS-NMS only (no H1N1 subunit
vaccine)
displayed progressive weight loss and all died within 11 days after virus
challenge.
In a second experiment, a larger dose of virus was used for challenge such
2 5 that vaccination with H1N1 alone afforded little if any significant
protection
(Figures 11C-11D). In this case, it was found that vaccination with H1N1 and
IL-12
resulted in SO% survival after challenge. Recovery from infection in the
surviving
mice was evidenced by regaining body weight. As expected, animals that
received
IL-12 or PBS-NMS alone did not survive virus challenge. Hence, co-
administration
3 0 of IL-12 and the H1N1 subunit influenza vaccine i.n. increased the
efficacy of the
vaccine and conferred significant protection against lethal doses of live
influenza
virus.
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WO 99/44635 PCT/US99/04678
Enhanced Protection Against Influenza Infection after Vaccination with H1N1
Plus
IL-12 is Antibody-mediated
To ascertain the role of humoral immunity in protection from influenza virus
infection, the responses of ~cMT mice, which lack B cells to IL-12 treatment
was
examined (Kitamura, D., et al., Nature, 350:423-426 (1991)}. It was found that
all
,uMT mice pre-treated with PBS alone, vaccine alone or vaccine plus IL-12
succumbed to infection by day 10 (Figures 12A-12B). Wild-type mice pre-treated
with PBS alone died twelve days after infection. In addition, all mice
displayed a
steady, progressive loss of body weight. Thus, the enhanced protection
conferred by
IL-12 treatment is a result of augmented B cell function.
To further determine if protection against influenza virus observed in mice
inoculated i.n. with vaccine and IL-12 was mediated by antibody, we
transferred
pooled serum from these mice into naive animals, which were then challenged
with
A/PR8/34 influenza virus 5 hours later. Of the animals that received serum
from
mice inoculated with vaccine or PBS-NMS only, all succumbed to infection
(Figure
13). However, animals that received serum from mice immunized with the vaccine
plus IL-12 exhibited 50% survival after viral challenge.
Whether antibodies generated in the respiratory secretions of immunized
mice played a crucial role in protection against influenza virus infection was
also
2 o determined. BAL fluid recovered from unvaccinated animals or animals
immunized
with H1N1 t IL-12 was administered i.n. to naive mice together with live
virus.
The results showed that virus challenge together with passive transfer of BAL
fluid
from mice that were treated with PBS-NMS alone resulted in 100% death by day 7
(Figures 14A-14B). Virus challenge in the presence of BAL fluid from mice
immunized with H1N1 alone resulted in survival of only one of 8 infected mice.
However, 100% of the animals that received BAL fluid from mice treated with
H1N1 plus IL-12 were protected against virus infection. These mice exhibited
no
transient weight loss over the course of the infection while both of the other
treatment groups displayed progressive weight loss leading to death.
Furthermore,
3 0 mice that received BAL fluid from animals immunized with vaccine alone had
viral
lung titers of 103 pfu on day 4 after infection while mice that received BAL
fluid
from animals treated with vaccine plus IL-12 had viral lung titers of <100
pfu.
Finally, the overall health of virus-challenged animals that received BAL
fluid from
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mice vaccinated with H1N1 plus IL-12 remained noticeably better than mice
which
received BAL fluid from animals vaccinated with H1N1 alone. Thus, passive
_ transfer of BAL fluid i.n. from mice immunized with H1N1 subunit vaccine
plus IL-
12 provided dramatic protection against influenza virus challenge.
Discussion
As described herein, IL-12 delivered i.n. with an influenza subunit vaccine
serves as a potent mucosal adjuvant and confers increased protection against
subsequent viral infection. Use of B cell deficient mice and passive transfer
of
serum or BAL fluid demonstrated that the protection induced by IL-12 is
mediated
1 o by antibody.
Analysis of cytokine mRNA production after i.n. treatment of mice with II,-
12 revealed an enhancement of IFN-y mRNA expression in both lungs and spleen
within 24 hours. IFN-y has a variety of immunoregulatory fimctions, which
include
induction of the Thl cell differentiation and activation of NK cells (Boehm,
U., et
al., Annu. Rev. Immunol., 15:749-795 (1998)). In addition, IFN-y enhances the
production of opsonizing marine antibodies such as IgG2a (Buchanan, J.M., et
al.,
Int. Immunol., 7:1519-1528 (1995); Metzger, D.W., et al., Eur. J. Immunol.,
27:1958-1965 (1997); McKnight, A.J., et al., J. Immunol., 152:2172-2179
(1994);Wynn, T.A., et al., J. Immunol., 157:4068-4078 (1996)). IL-10 mRNA
2 0 expression was also induced in lungs and spleens by i.n. treatment with IL-
12. IL-
10 is mainly produced by T cells and monocytes, and has been shown to inhibit
Thl
cell differentiation (Fiorentino, D.F., et al., J. Immunol., 146:3444-3451
(1991);
Ding, L., et al., J. Immunol., 148:3133-3139 (1992)). Others (Meyaard, L., et
al., J.
Immunol., 156:2776-2782 (1996); Daftarian, P.M., et al., J. Immunol., 157:12-
20
{1996); Gerosa, F., et al., J. Exp. Med., 183:2559-2569 (1996)) have shown
induction of IL-10 after treatment with IL-12, an observation which suggests a
feedback mechanism designed to downregulate the inflammatory effects of IL-12
and IFN-y.
In Example 2, the effects of i.n. IL-12 on responses to a clinically relevant
3 0 influenza subunit vaccine was examined. IL-12 treatment was found to have
a
dramatic effect on the early onset of the humoral response, as reflected by
significant
enhancement of IgG2a anti-H1N1 antibody levels. In comparison, animals that
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received vaccine alone did not develop early IgG2a responses. There was little
detectable IgGl antibody during the early phase of the immune response in
animals
that received vaccine alone or vaccine and IL-12. After 35 days, IgG2a levels
were
still enhanced in IL-12 treated mice and IgGI levels were also somewhat
elevated,
an observation that is in agreement with previous findings in the lysozyme
system
(Buchanan, J.M., et al., Int. Immunol., 7:1519-1528 (I995)). These results
demonstrate the long-lasting effects of IL-12 delivered i.n. and provide
further
evidence for the use of this route of administration for augmenting systemic
humoral
immunity.
IL-12 i.n. administration also resulted in significant increases in
respiratory
antibody levels, including IgG and IgA anti-HIN1 antibody levels. IgA is the
predominant antibody in mucosal secretions, and is thought to play a major
role in
preventing attachment of pathogens to mucosal epithelial surfaces (Lamm, M.E.,
Annu. Rev. Immunol., 51:311-340 (1997)).
As also described herein, passive transfer of serum or BAL fluid collected
from mice immunized with subunit influenza vaccine and IL-12 resulted in
significant protection from morbidity and mortality. The ability of IL-12 to
augment
antibody levels and enhance protection against influenza virus infection is
completely abrogated in ,uMT mice. The augmented protection conferred by
passive
2 0 transfer of BAL fluid observed here is likely to be the result of
significantly
enhanced respiratory antibody levels observed after i.n. IL-12 treatment.
Adjuvants that have been used to enhance mucosal immune responses
include microbial products such as CT and LT, which have been utilized in a
variety
of delivery systems (Staats, H.F., et al., Curr. Opin. Immunol., 6:572-583
(1994);
2 5 Elson, C.O., In Mechanisms in the Pathogenesis of Enteric Disease, Paul,
P.S., et
al., eds., (NY:Plenum Press), pages 373-385 (1997)). CT is a potent inducer of
the
Th2-type responses, whereas LT elicits a mixed Thl and Th2 response (Marinaro,
M., et al., J. Exp. Med., 185:415-427 (1997); Takahashi, L, et al., Infect.
Dis.,
173:627-635 (1996)). However, these adjuvants cause severe diarrhea, and are
not
3 0 suitable for use as mucosal adjuvants in humans. There is also a recent
report
suggesting CT actually suppresses IL-12 production and IL-12 receptor
expression
(Braun, M., et al., J. Exp. Med., in press (1999)). Furthermore, in
respiratory
synctial virus lung infections, Thl responses are protective while Th2
responses
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WO 99144635 -35-
result in lung pathology (Graham, B.S., et al., .I. Clin. Invest., 88:1026-
1033 (1991);
Graham, B.S., et al., J. Immunol., 151:2032-2040 (1991). The ability of IL-12
_ administered i.n. to enhance the protective efficacy of an influenza vaccine
is
therefore of direct relevance for mucosal vaccination protocols.
EQUIVALENTS
While this invention has been particularly shown and described with
references to preferred embodiments thereof, it will be understood by those
skilled
in the art that various changes in form and details may be made therein
without
departing from the spirit and scope of the invention as defined by the
appended
claims. Those skilled in the art will recognize or be able to ascertain using
no more
than routine experimentation, many equivalents to the specific embodiments of
the
invention described specifically herein. Such equivalents are intended to be
encompassed in the scope of the claims.
