U.S. patent application number 11/501904 was filed with the patent office on 2010-09-16 for adjuvant combination formulations.
This patent application is currently assigned to Wyeth Holdings Corporation. Invention is credited to Michael Hagen.
Application Number | 20100233117 11/501904 |
Document ID | / |
Family ID | 22461127 |
Filed Date | 2010-09-16 |
United States Patent
Application |
20100233117 |
Kind Code |
A1 |
Hagen; Michael |
September 16, 2010 |
Adjuvant combination formulations
Abstract
The use of 3-O-deacylated monophosphoryl lipid A or
monophosphoryl lipid A and derivatives and analogs thereof, in
combination with a cytokine or lymphokine such as granulocyte
macrophage colony stimulating factor or interleukin-12 is useful as
an adjuvant combination in an antigenic composition to enhance the
immune response in a vertebrate host to a selected antigen.
Inventors: |
Hagen; Michael; (Pittsford,
NY) |
Correspondence
Address: |
WYETH LLC;PATENT LAW GROUP
5 GIRALDA FARMS
MADISON
NJ
07940
US
|
Assignee: |
Wyeth Holdings Corporation
Madison
NJ
|
Family ID: |
22461127 |
Appl. No.: |
11/501904 |
Filed: |
August 8, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10009473 |
Nov 8, 2001 |
7611721 |
|
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PCT/US00/13156 |
May 12, 2000 |
|
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11501904 |
|
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60133963 |
May 13, 1999 |
|
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Current U.S.
Class: |
424/85.2 |
Current CPC
Class: |
A61K 39/39 20130101;
A61K 39/12 20130101; A61P 31/12 20180101; A61P 37/00 20180101; A61K
2039/55522 20130101; A61K 2039/57 20130101; A61K 38/00 20130101;
A61K 39/095 20130101; C12N 2740/16034 20130101; C12N 2760/18534
20130101; A61P 37/04 20180101; A61P 25/28 20180101; A61K 39/21
20130101; A61P 35/00 20180101; A61P 31/04 20180101; C12N 2740/15034
20130101; A61K 39/155 20130101; A61K 2039/55538 20130101; A61K
2039/55572 20130101; A61P 31/16 20180101; A61K 2039/55566 20130101;
A61P 31/18 20180101; A61P 31/20 20180101; A61P 37/08 20180101; C12N
2760/16134 20130101 |
Class at
Publication: |
424/85.2 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61P 31/18 20060101 A61P031/18 |
Claims
1. An antigenic composition consisting of an antigen and an
effective adjuvanting amount of the combination of: (1) a stable
oil-in-water emulsion of 3-0-deacylated monophosphoryl lipid A or
monophosphoryl lipid A (MPL-SE) and (2) interleukin-12 (IL-12),
together with a diluent or carrier, wherein the combination of
adjuvants enhances the immune response in a vertebrate host to said
antigen.
2. The antigenic composition of claim 1, where the antigen is a
polypeptide, peptide or fragment derived from a protein.
3. (canceled)
4. The antigenic composition of claim 1, where the antigen is
derived from a pathogenic virus.
5. A method for increasing the ability of an antigenic composition
containing an antigen from a pathogenic virus to elicit an immune
response in a vertebrate host against said pathogenic virus, which
comprises administering to said host an antigenic composition of
claim 4.
6. A method for increasing the ability of an antigenic composition
containing an antigen from a pathogenic virus to elicit cytotoxic T
lymphocyte responses in a vertebrate host, which comprises
administering to said host an antigenic composition of claim 4.
7. The antigenic composition of claim 4, where the antigen is from
human immunodeficiency virus (HIV).
8. The antigenic composition of claim 7, where the HIV antigen is
an HIV protein, polypeptide, peptide or fragment derived from said
protein.
9. The antigenic composition of claim 8 where the antigen is the
HIV peptide having the amino acid sequence: Lys Gln Ile Ile Asn Met
Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Cys Thr Arg Pro Asn Tyr Asn
Lys Arg Lys Arg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Lys
(SEQ ID NO:1), or Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys
Ala Met Tyr Ala Thr Arg Pro Asn Tyr Asn Lys Arg Lys Arg Ile His Ile
Gly Pro Gly Arg Ala Phe Tyr Thr Thr Lys (SEQ ID NO:2).
10. (canceled)
11. A method for increasing the ability of an antigenic composition
containing an HIV antigen to elicit an immune response to said
antigen in a vertebrate host, which comprises administering to said
host an antigenic composition of claim 7.
12. The method of claim 11, where the HIV antigen is the HIV
peptide having the amino acid sequence: Lys Gln Ile Ile Asn Met Trp
Gln Glu Val Gly Lys Ala Met Tyr Ala Thr Arg Pro Asn Tyr Asn Lys Arg
Lys Arg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Lys (SEQ ID
NO:2).
13. A method for increasing the ability of an antigenic composition
containing an HIV antigen to elicit cytotoxic T lymphocyte
responses in a vertebrate host, which comprises administering to
said host an antigenic composition of claim 7.
14. The method of claim 13, where the HIV antigen is the HIV
peptide having the amino acid sequence: Lys Gln Ile Ile Asn Met Trp
Gln Glu Val Gly Lys Ala Met Tyr Ala Thr Arg Pro Asn Tyr Asn Lys Arg
Lys Arg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Lys (SEQ ID
NO:2).
Description
[0001] This application is a divisional application of U.S. patent
application Ser. No. 10/009,473, filed Nov. 8, 2001, which is a 371
national application of PCT/US00/13156 filed May 12, 2000, which
claims the benefit of U.S. Provisional Application No. 60/133,963,
filed May 13, 1999, the contents of which are incorporated in their
entirety.
FIELD OF THE INVENTION
[0002] This invention relates to the use of 3-O-deacylated
monophosphoryl lipid A or monophosphoryl lipid A and derivatives
and analogs thereof, in combination with a cytokine or lymphokine,
in particular granulocyte macrophage colony stimulating factor or
interleukin-12, as an adjuvant formulation in an antigenic
composition to enhance the immune response in a vertebrate host to
a selected antigen.
BACKGROUND OF THE INVENTION
[0003] The immune system uses a variety of mechanisms for attacking
pathogens. However, not all of these mechanisms are necessarily
activated after immunization. Protective immunity induced by
immunization is dependent on the capacity of the vaccine to elicit
the appropriate immune response to resist or eliminate the
pathogen. Depending on the pathogen, this may require a
cell-mediated and/or humoral immune response.
[0004] The current paradigm for the role of helper T cells in the
immune response is that T cells can be separated into subsets on
the basis of the cytokines they produce, and that the distinct
cytokine profile observed in these cells determines their function.
This T cell model includes two major subsets: TH-1 cells that
produce interleukin-2 (IL-2) and interferon gamma, which augment
both cellular and humoral (antibody) immune responses; and TH-2
cells that produce interleukin-4, interleukin-5 and interleukin-10
(IL-4, IL-5 and IL-10, respectively), which augment humoral immune
responses (Bibliography entry 1).
[0005] It is often desirable to enhance the immunogenic potency of
an antigen in order to obtain a stronger immune response in the
organism being immunized and to strengthen host resistance to the
antigen-bearing agent. In some situations, it is desirable to shift
the immune response from a predominantly humoral (TH-2) response to
a more balanced cellular (TH-1) and humoral (TH-2) response.
[0006] A cellular response involves the generation of a CD8+ CTL
(cytotoxic T-lymphocyte) response. Such a response is desirable for
the development of vaccines against intracellular pathogens.
Protection against a variety of pathogens requires strong mucosal
responses, high serum titers, induction of CTL and vigorous
cellular responses. These responses have not been provided by most
antigen preparations, including conventional subunit vaccines.
Among such pathogens is the human immunodeficiency virus (HIV).
[0007] Thus, there is a need to develop antigenic composition
formulations that are able to generate both humoral and cellular
immune responses in a vertebrate host.
SUMMARY OF THE INVENTION
[0008] Accordingly, it is an object of this invention to utilize
adjuvant combination formulations in antigenic compositions
containing 3-O-deacylated monophosphoryl lipid A (MPL.TM.) or
monophosphoryl lipid A and derivatives and analogs thereof,
combined with a cytokine or lymphokine, in particular
granulocyte-macrophage colony stimulating factor (GM-CSF) or
interleukin-12 (IL-12), or an agonist or antagonist to said
cytokine or lymphokine. An adjuvant is a substance that enhances
the immune response when administered together with an immunogen or
antigen. The adjuvant formulation of this invention is administered
together with a selected antigen in an antigenic composition. The
antigenic compositions of this invention enhance the immune
response in a vertebrate host to that selected antigen. The
selected antigen may be a polypeptide, peptide or fragment derived
(1) from a pathogenic virus, bacterium, fungus or parasite, or (2)
from a cancer cell or tumor cell, or (3) from an allergen so as to
interfere with the production of IgE so as to moderate allergic
responses to the allergen, or (4) from amyloid peptide protein so
as to prevent or treat disease characterized by amyloid deposition
in a vertebrate host. In one embodiment of the invention, the
selected antigen is from HIV. The selected HIV antigen may be an
HIV protein, polypeptide, peptide or fragment derived from said
protein. In a particular embodiment of the invention, the HIV
antigen is a specific peptide. In other embodiments of the
invention, the selected antigen is from Neisseria gonorrhoeae or
Respiratory syncytial virus.
[0009] The MPL.TM. can be present as an aqueous solution, or as a
stabilized oil-in-water emulsion (stable emulsion or SE). In a
preferred embodiment of the invention, the oil-in-water emulsion
contains squalene, glycerol and phosphatidyl choline. In the SE
formulation, the MPL.TM. is mixed with the cytokine or lymphokine
to form the antigenic composition prior to administration. The
cytokine or lymphokine is not required to enter the emulsion. In a
preferred embodiment of the invention, the MPL.TM. is in the SE
form. The antigenic composition may further comprise a diluent or
carrier.
[0010] The invention is also directed to methods for increasing the
ability of an antigenic composition containing a selected antigen
(1) from a pathogenic virus, bacterium, fungus or parasite to
elicit the immune response of a vertebrate host, or (2) from a
cancer antigen or tumor-associated antigen from a cancer cell or
tumor cell to elicit a therapeutic or prophylactic anti-cancer
effect in a vertebrate host, or (3) from an allergen so as to
interfere with the production of IgE so as to moderate allergic
responses to the allergen, or (4) from amyloid peptide protein so
as to prevent or treat disease characterized by amyloid deposition
in a vertebrate host, by including an effective adjuvanting amount
of a combination of a cytokine or lymphokine, in particular MPL.TM.
with GM-CSF or IL-12, or an agonist or antagonist to said cytokine
or lymphokine.
[0011] The invention is further directed to methods for increasing
the ability of an antigenic composition containing a selected
antigen from a pathogenic virus, bacterium, fungus or parasite to
elicit cytotoxic T lymphocytes in a vertebrate host by including an
effective adjuvanting amount of a combination of a cytokine or
lymphokine, in particular MPL.TM. with GM-CSF or IL-12, or an
agonist or antagonist to said cytokine or lymphokine.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 depicts the reciprocal endpoint titers determined
from groups of five Balb/c female mice immunized with 25 .mu.g
T1SP10MN(A)(-Cys), a multiepitope 39 amino acid peptide, formulated
with CFA or IFA (triangles), or with 50 .mu.g MPL.TM. 2% stable
emulsion (SE) (squares). Mice were immunized on day 0 and boosted
on day 28. Peptide-specific IgG, IgG1, and IgG2a titers were
determined in sera collected on day 42 by ELISA.
[0013] FIG. 2 depicts the effect of SE alone, MPL.TM. alone or
MPL.TM. SE on the adjuvanting properties of GM-CSF on
anti-T1SP10MN(A)(-Cys) IgG titers. Groups of five female Balb/c
mice were immunized with 25 .mu.g T1SP10MN(A)(-Cys) on day 0, and
boosted on day 28 with the indicated adjuvant formulations. CFA and
IFA were emulsified with aqueous peptide at a ratio of 1:1. GM-CSF
was used at 10 .mu.g/dose. MPL.TM. was delivered to mice at a final
concentration of 50 .mu.g as an aqueous formulation, or at 25 .mu.g
as part of a stable emulsion with 1% SE. Titers were determined two
weeks after the second immunization. The data represent individual
titers determined from five mice.
[0014] FIG. 3 depicts the results of a viral neutralization assay.
Pooled sera taken at day 42 from mice immunized at days 0 and 28
with the indicated formulations were diluted (1/1600) and added to
dilutions of T cell-adapted HIV.sub.MN prior to addition to AA5
cells in vitro. After seven days culture, the cell culture
supernatants were assayed for viral reverse transcriptase as an
indicator of viral replication.
[0015] FIG. 4 depicts the proliferation of spleen cells from mice
immunized with T1SP10MN(A)(-Cys) and various adjuvant formulations.
Spleen cells were stimulated in vitro for four days with 3.3
.mu.g/ml T1SP10MN(A)(-Cys). The results are shown as the change in
the incorporation of labeled thymidine as a result of in vitro
stimulation with T1SP10MN(A)(-Cys) over the incorporation in the
absence of stimulation (delta cpm).
[0016] FIG. 5 depicts the CTL activity of spleen cells isolated
from mice seven days after secondary immunization. Spleen cells
were harvested from groups of three Balb/c mice immunized on days 0
and 21 with 50 .mu.g T1SP10MN(A)(-Cys) formulated with 50 .mu.g
MPL.TM. in 1% SE with or without 10 .mu.g GM-CSF. Cells were
cultured with the HIV.sub.MN CTL epitope peptide for seven days.
IL-2 was added to the cultures for the last five days. Effector
spleen cells were added to chromium-labelled P815 cells pulsed with
HIV.sub.MN, another strain designated HIV.sub.IIIB or no peptide at
the indicated ratios. Percent CTL activity was calculated as:
specifically released cpm - spontaneously released cpm total
maximum cpm - spontaneously released cpm .times. 100
##EQU00001##
"E:T" means effector to target cell ratio.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Adjuvants, cytokines and lymphokines are immune modulating
compounds which have the ability to enhance and steer the
development and profile of immune responses against various
antigens that are themselves poorly immunogenic. The appropriate
selection of adjuvants, cytokines and lymphokines can induce good
humoral and cellular immune responses that would not develop in the
absence of adjuvant, cytokine or lymphokine. In particular,
adjuvants, cytokines and lymphokines have significant effects in
enhancing the immune response to subunit and peptide antigens in
vaccines. Their stimulatory activity is also beneficial to the
development of antigen-specific immune responses directed against
protein antigens. For a variety of antigens that require strong
mucosal responses, high serum titers, induction of CTL and vigorous
cellular responses, adjuvant and cytokine/lymphokine combinations
provide stimuli that are not provided by most antigen
preparations.
[0018] Numerous studies have evaluated different adjuvant
formulations in animal models, but alum (aluminum hydroxide or
aluminum phosphate) is currently the only adjuvant licensed for
widespread use in humans. One group of adjuvants, stable emulsions,
consisting of various water-in-oil or oil-in-water combinations,
has received considerable attention for their immunopotentiating
ability. These formulations generally consist of various
combinations of metabolizable or inert oils, that act to stabilize
and depot antigen at the site of injection. One such adjuvant is
incomplete Freund's adjuvant (IFA), which includes mineral oil,
water and an emulsifying agent. Complete Freund's adjuvant (CFA) is
IFA plus heat-killed Mycobacteria. A particular concern in using
these types of adjuvants has been injection site-associated
irritation, often the result of mononuclear cell infiltrations
causing granulomatous lesions. Therefore, other compounds and
formulations are being investigated as potential adjuvants.
[0019] One such compound is 3-O-deacylated monophosphoryl lipid A
(MPL.TM.), which is available from Ribi ImmunoChem Research Inc.
(Hamilton, Mont.). MPL.TM. is described in U.S. Pat. No. 4,912,094
(2), which is hereby incorporated by reference.
[0020] Recently, Ribi ImmunoChem Research Inc. has formulated a
metabolizable oil-in-water formulation which, when combined with
MPL.TM., results in the formation of a stabilized emulsion
designated MPL.TM. SE.
[0021] The stabilized emulsion is generated through
microfluidization of MPL.TM. with squalene oil, glycerol and
phosphatidyl choline. The current formulation is a GMP-quality
microfluidized emulsion. Emulsions containing 1 or 2% oil are
described in the experiments below.
[0022] MPL.TM. SE resulted in no discernable injection-site
associated tissue pathology when administered subcutaneously, or
intramuscularly into Balb/c mice. A stabilized emulsion containing
the same components, but without MPL.TM., was also generated for
comparative purposes. Specifically, subcutaneous or intramuscular
immunization with a 40 amino acid HIV peptide T1SP10MN(A)(+Cys), or
with a cysteine-deleted 39 amino acid peptide T1SP10MN(A)(-Cys)
peptide (which lacks the cysteine residue at amino acid number 17
of the 40 amino acid peptide (+Cys)), formulated with the
combination of adjuvants MPL.TM. SE and GM-CSF resulted in no
discernable cellular infiltration or tissue abnormalities two weeks
after immunization.
[0023] Also within the scope of this invention is the use of
monophosphoryl lipid A, a precursor form of MPL.TM., which is also
described in U.S. Pat. No. 4,912,094 (2). Further within the scope
of this invention are derivatives and analogs of MPL.TM. and
monophosphoryl lipid A.
[0024] The incorporation of cytokines and lymphokines into vaccine
formulations has shown promise for the expansion and enhancement of
vaccine potential (3). The cytokine interleukin-12 (IL-12) has been
demonstrated to evoke and enhance cell mediated immunity, through a
shift in T helper cell subset expansion towards a Th1 cytokine
profile (i.e., to IgG2 subclass in the mouse model) (4-6). In mice,
recombinant murine IL-12 has been shown to enhance a Th1 dominated
immune response profile (3).
[0025] IL-12 is produced by a variety of antigen-presenting cells,
principally macrophages and monocytes. It is a critical element in
the induction of TH1 cells from naive T-cells. Production of IL-12
or the ability to respond to it has been shown to be critical in
the development of protective TH1-like responses, for example,
during parasitic infections, most notably Leishmaniasis (7). The
effects of IL-12 are mediated in large part by interferon-gamma
produced by NK cells and T helper cells. Interferon-gamma is
critical for the induction of IgG2a antibodies to T-dependent
protein antigens (8) and IgG3 responses to T-independent antigens
(9). IL-12, originally called natural killer cell stimulatory
factor, is a heterodimeric cytokine (10). The expression and
isolation of IL-12 protein in recombinant host cells is described
in published International Patent Application WO 90/05147 (11).
[0026] Another cytokine that holds potential promise as an adjuvant
is GM-CSF. GM-CSF is a particular type of colony stimulating factor
(CSF). The CSFs are a family of lymphokines that induce progenitor
cells found in the bone marrow to differentiate into specific types
of mature blood cells. As described in U.S. Pat. No. 5,078,996
(12), which is hereby incorporated by reference, GM-CSF activates
macrophages or precursor monoctyes to mediate non-specific
tumoricidal activity. The nucleotide sequence encoding the human
GM-CSF gene has been described (12). A plasmid containing GM-CSF
cDNA has been transformed into E. coli and has been deposited with
the American Type Culture Collection (ATCC), 10801 University
Boulevard, Manassas, Va. 20110-2209, under Accession Number 39900.
As described in U.S. Pat. No. 5,229,496 (13), which is hereby
incorporated by reference, the GM-CSF gene has also been inserted
into a yeast expression plasmid and deposited with the ATCC under
Accession Number 53157. Furthermore, as described in U.S. Pat. No.
5,073,627 (14), which is hereby incorporated by reference, a DNA
sequence encoding GM-CSF having glycosylation sites removed was
deposited with the ATCC under Accession Number 67231.
[0027] GM-CSF has been shown to upregulate protein molecules on
antigen presenting cells known to enhance immune responses (15),
and to affect Ig secretion in sort-purified murine B cells
(16).
[0028] Other cytokines or lymphokines have been shown to have
immune modulating activity, including, but not limited to, the
interleukins 1-alpha, 1-beta, 2, 4, 5, 6, 7, 8, 10, 13, 14, 15, 16,
17 and 18, the interferons-alpha, beta and gamma, granulocyte
colony stimulating factor, and the tumor necrosis factors alpha and
beta.
[0029] Of concern related to the systemic administration of any
cytokine or lymphokine are the biological consequences associated
with cytokine or lymphokine activity. Additionally, cytokine or
lymphokine effects related to the development of antigen-specific
immune responses should be enhanced if local concentrations of
cytokine or lymphokine are maintained.
[0030] In previous studies, GM-CSF and IL-12 have been evaluated
separately; enhancement of various immune response parameters was
observed.
[0031] The invention described herein demonstrates that, through
the combination of an antigen, selected cytokine or lymphokine
adjuvant, and the second adjuvant, MPL.TM. (preferably in a stable
metabolizable emulsion), the immune responses specific for the
antigen are enhanced.
[0032] The antigens selected for inclusion in the antigenic
compositions of this invention comprise peptides or polypeptides
derived from proteins, as well as fragments of any of the
following: saccharides, proteins, poly- or oligonucleotides, or
other macromolecular components. As used herein, a "peptide"
comprises a series of at least six amino acids and contains at
least one antigenic determinant, while a "polypeptide" is a longer
molecule than a peptide, but does not constitute a full-length
protein. As used herein, a "fragment" comprises a portion, but less
than all of a saccharide, protein, poly- or oligonucleotide, or
other macromolecular components. In the case of HIV, the antigenic
compositions of this invention further comprise full-length HIV
proteins.
[0033] The invention is exemplified in a model system using peptide
antigens derived from HIV. These peptides are described in or
derived from U.S. Pat. Nos. 5,013,548 (17) and 5,019,387 (18),
which are hereby incorporated by reference and are now summarized.
These peptides comprise amino acid sequences which correspond to a
region of the HIV envelope protein against which neutralizing
antibodies and T cell responses are produced.
[0034] HIV is a human retrovirus which is the causative agent of
acquired immunodeficiency syndrome (AIDS). HIV infects T
lymphocytes of the immune system by attaching its external envelope
glycoprotein to the CD4 (T4) molecule on the surface of T
lymphocytes, thus using the CD4 (T4) molecule as a receptor to
enter and infect T cells. Attempts to induce a protective immune
response specific for HIV-infection through immunization have been
met with very limited success. A number of approaches are currently
being pursued in an attempt to determine an effective and
protective vaccine strategy. These include using attenuated and
recombinant bacterial vectors that express antigenic epitopes from
HIV (19), recombinant adenovirus (20) or vaccinia virus vectors
(21), DNA vaccines (22), and synthetic peptides that contain
various T and B cell epitopes of HIV (23).
[0035] The HIV external envelope glycoprotein gp120 has been shown
to be capable of inducing neutralizing antibodies in man. The
recombinant protein PB1, which encodes approximately one-third of
the entire gp120 molecule, has been shown to include the part of
the envelope protein that induces the formation of neutralizing
antibodies. However, studies in chimpanzees demonstrated that
neither intact gp120 or PB1 is able to induce the production of
high titers of neutralizing antibodies.
[0036] Short peptides were synthesized by conventional methods
which correspond to antigenic determinants of gp120 and generate an
antibody response against gp120 that neutralize the virus and
induce T-helper and CTL responses against the virus.
[0037] One such peptide is the C4/V3 multiepitope-containing
HIV-1.sub.MN peptide designated T1SP10MN(A)(+Cys), and a
cysteine-deleted variant T1SP10MN(A)(-Cys). These peptides include
Th, T.sub.CTL and B epitopes, but do not induce antibodies which
interfere with CD4 binding. Previously, it has been demonstrated
that these C4/V3 HIV peptides are promising candidates for the
induction of immune responses when administered with CFA, or
CFA-like adjuvants (24-29). These peptides contain epitopes that
have previously been shown to evoke CD4+ Th cell responses in both
mice and humans, and it contains both a principal neutralizing
determinant and a site which is recognized by CD8+ CTL in both
Balb/c mice and humans that are HLA B7+. The 39 amino acid peptide
has recently demonstrated both immunogenicity and safety in
HIV-infected patients (28).
[0038] T1SP10MN(A)(+Cys) has the following sequence of 40 amino
acids:
TABLE-US-00001 (SEQ ID NO: 1) Lys Gln Ile Ile Asn Met Trp Gln Glu
Val Gly Lys Ala Met Tyr Ala Cys Thr Arg Pro Asn Tyr Asn Lys Arg Lys
Arg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Lys (26).
[0039] T1SP10MN(A)(-Cys) has been synthesized without the cysteine
at position 17 and has the following sequence of 39 amino
acids:
TABLE-US-00002 (SEQ ID NO: 2) Lys Gln Ile Ile Asn Met Trp Gln Glu
Val Gly Lys Ala Met Tyr Ala Thr Arg Pro Asn Tyr Asn Lys Arg Lys Arg
Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Lys.
[0040] This cysteine residue is located outside of the functional
epitopes recognized by Th cells, CTL or B cells. Other HIV peptides
from various regions of the viral genome are described in U.S. Pat.
No. 5,861,243 (30), U.S. Pat. No. 5,932,218 (31), U.S. Pat. No.
5,939,074 (32), U.S. Pat. No. 5,993,819 (33), U.S. Pat. No.
6,037,135 (34) and Published European Patent Application Number
671,947 (35), which are also incorporated by reference.
[0041] The HIV antigen may be a protein, polypeptide, peptide or
fragment derived from said protein. The protein may be a
glycoprotein such as gp41, gp120 or gp160. Alternatively, the
protein may be a protein encoded by such genes as gag, pol, vif,
rev, vpr, tat, nef or env. Peptides derived from such proteins will
contain at least one antigenic determinant (epitope) at least six
amino acids in length.
[0042] The immune response to an HIV peptide may be enhanced by
covalently linking (conjugating) the peptide to a pharmaceutically
acceptable carrier molecule. Examples of suitable carrier molecules
include tetanus toxoid, diphtheria toxoid, keyhole limpet
haemocyanin and other peptides corresponding to T cell epitopes of
the HIV gp120 glycoprotein.
[0043] It is currently felt that a successful vaccine strategy
against HIV will need to elicit mucosal immunity to HIV, as well as
a good CTL response. In a recent murine study using the T1SP10MN(A)
multi-epitope peptide, and a mucosal adjuvant, cholera toxin, it
was shown that intranasal immunization induced neutralizing serum
IgG1 antibodies (36). A subsequent study also using HIV-V3 loop
peptides demonstrated the induction of mucosally synthesized IgA
antibody and strong cell mediated responses, including
peptide-specific CTL (37). The functional role of high titers of
systemic and neutralizing antibodies in the prevention of, or
stabilization of HIV-infected individuals is unknown, although high
titers of virus-specific antibody are believed to be important in
preventing viral spread.
[0044] In a preferred embodiment of the invention, a stable
oil-in-water emulsion is formulated which contains MPL.TM., which
is then mixed with the cytokines IL-12 or GM-CSF. The data
presented below demonstrate that these combinations result in high
titers of HIV-neutralizing serum antibodies. The combination of
MPL.TM. SE and GM-CSF induces high titers of antigen-specific IgG
and IgA antibody in the vaginal vault of immunized female mice.
Immunization of mice with either of the T1SP10MN(A) peptides
formulated with MPL.TM. SE and GM-CSF induced a strong cellular
immune response as determined by enhanced antigen specific cellular
proliferation and secretion into culture of cytokines, as well as
the induction of peptide-specific CTL responses.
[0045] Generally, the antigen/adjuvant formulation of MPL.TM. or
MPL.TM. SE combined with GM-CSF or IL-12 and a protein or peptide
of choice induces high titers of antigen-specific and virus
neutralizing antibody, a significant shift in the IgG subclass
ratio to a greater proportion of complement-fixing IgG antibodies
(in favor of IgG2a in mice), elevated production of cytokines and
cellular proliferation from mononuclear cells in response to
antigen stimulation in vitro. These properties were not observed
with formulations of antigen and SE in the absence of MPL.TM.,
either with or without GM-CSF or IL-12. The formulations of this
invention also induce good cellular responses as determined through
induction of CTL.
[0046] A benefit of MPL.TM. SE is that the formulation does not
induce granulomatous accumulation and inflammation at the site of
injection; such injection site reactions are typically induced by
water-in-oil or oil-in-water adjuvant formulations.
[0047] The ability to induce an enhanced immune response through
the stimulatory effects of MPL.TM. in combination with GM-CSF or
IL-12 in the absence of local granulomatous inflammation has not
been reported with other adjuvant formulations currently proposed
for treatment of HIV.
[0048] A series of studies was conducted to compare MPL.TM. (either
with or without SE) plus GM-CSF or IL-12 to each of MPL.TM., SE,
GM-CSF, IL-12 or CFA/IFA individually, or together with an HIV
peptide. A summary of the results will now be presented, followed
by a more detailed discussion.
[0049] In a first experiment, Balb/c mice immunized subcutaneously
with the C4/V3 HIV peptide T1SP10MN(A) (-Cys), formulated with
MPL.TM. SE and GM-CSF, produced serum IgG titers in excess of
10.sup.7 after only two injections. The antibody response was
HIV-neutralizing, and demonstrated significant increases in IgG1,
IgG2a, and IgG2b peptide-specific antibody titers. Spleen cells
stimulated in culture with peptide released elevated levels of
IL-4, IL-5, and interferon-gamma. Collectively, those findings are
indicative of the induction of a balanced Th1/Th2-type response.
IgG and IgA antibodies were generated that were specific for
T1SP10MN(A)(-Cys) in the vaginal lavage fluids of mice immunized
with MPL.TM. SE, and GM-CSF. These findings indicate that the
combination of MPL.TM. SE and GM-CSF with an HIV-peptide antigen
results in the induction of a favorable immune response
profile.
[0050] In this first experiment, Balb/c mice immunized with the HIV
peptide T1SP10MN(A)(-Cys) and an SE-containing adjuvant
formulation, or GM-CSF, generated peptide specific IgG antibody
titers (Table 1). An oil-in-water stable emulsion (SE) consisting
of squalene, glycerol, and an emulsifying agent (phosphatidyl
choline), demonstrated an ability to enhance peptide-specific IgG
titers when mixed with T1SP10MN(A)(-Cys). IgG titers induced
through immunization with 25:g T1SP10MN(A)(-Cys) formulated with SE
induced secondary response titers that were approximately one-fifth
of those induced in mice immunized with peptide and CFA, and
boosted with peptide in IFA. Recipients of CFA/IFA formulated
vaccines routinely developed T1SP10MN(A)(-Cys)-specific IgG titers
in response to a primary immunization. For comparative purposes,
mice were immunized with 25:g of T1SP10MN(A)(-Cys) peptide
alone.
[0051] Aqueous and SE formulations of MPL.TM. were compared with
responses induced through immunization of mice with Freund's
adjuvants or the cytokines IL-12 and GM-CSF. Recipients of
T1SP10MN(A)(-Cys) mixed with IL-12 generally did not generate
peptide-specific antibody titers in several repeat studies. In
contrast, recipients of GM-CSF or MPL.TM. SE plus T1SP10MN(A)(-Cys)
did develop low, but readily detectable IgG antibody titers. The
addition of IL-12 or GM-CSF to formulations containing MPL.TM. SE
plus T1SP10MN(A)(-Cys) peptide induced significantly higher titers
of IgG in response to immunization. Indeed, immunization of mice
with MPL.TM. SE and GM-CSF in combination resulted in secondary
response titers that were consistently greater than those
determined from mice immunized with any other formulation tested.
Peptide-specific IgG titers were higher than those of mice
immunized with even 125 .mu.g of T1SP10MN(A)(-Cys) formulated with
Freund's adjuvants.
[0052] A desirable feature of an HIV-specific immune response is
one that is balanced between cellular and humoral components.
Particular immunoglobulin isotype subclasses have been correlated
with the skewing of T helper cell subset types toward either Th1 or
Th2 predominance. The cytokines that each of these T helper cell
subsets secrete have demonstrated activity in directing IgG
subclass switching. IgG subclass endpoint titers were determined
from pooled sera collected two weeks after the second immunization
(Table 2). Immunization of mice with T1SP10MN(A)(-Cys) alone, or
formulated with either GM-CSF or IL-12, resulted in no or low IgG
subclass titers in several repeat studies. IgG3 antibodies could
not be detected by ELISA. Groups of mice immunized with
T1SP10MN(A)
[0053] (-Cys) emulsified with CFA and boosted with IFA developed
predominantly an IgG1 antibody response specific for
T1SP10MN(A)(-Cys). Formulations of peptide with SE, MPL.TM. SE,
MPL.TM. SE plus IL-12, or MPL.TM. SE plus GM-CSF, also developed
high titers of IgG1 antibody. Recipients of SE-formulated vaccines
repeatedly demonstrated significant IgG1, but not significant IgG2a
or IgG2b titers. The inclusion of MPL.TM. into the SE formulation
resulted in enhanced IgG2a and IgG2b T1SP10MN(A)(-Cys)-specific
antibody titers. The inclusion of either IL-12 or GM-CSF with
MPL.TM. SE and T1SP10MN(A)(-Cys) resulted in a shift in the
IgG1:IgG2a antibody titer ratio. Without cytokine, MPL.TM. SE
formulated vaccines induced similar titers of IgG1 and IgG2a. Both
IL-12 and GM-CSF increased the relative serum concentrations of
peptide-specific IgG2a. Moreover, the combination of MPL.TM. SE and
GM-CSF also induced a significant increase in IgG2b antibody titers
specific for T1SP10MN(A)(-Cys) (47-fold compared to MPL.TM. SE and
74-fold compared to SE). Titers developed in mice immunized with
MPL.TM. SE and GM-CSF together with T1SP10MN(A)(-Cys) peptide were
consistently the highest of any vaccine recipient group.
[0054] To determine if the high titers measured from sera pooled
from mice immunized with T1SP10MN(A)(-Cys), MPL.TM. SE, and GM-CSF
were representative of the individual mice within the group, titers
of individual mice within that group were compared with those of
mice immunized with Freund's adjuvant formulated peptide (FIG. 1).
It was determined that the mean of the individual serum titers for
IgG, IgG1, and IgG2a were similar to the titers measured from serum
pools (data not shown). The co-formulation of T1SP10MN(A)(-Cys)
with MPL.TM. SE and GM-CSF resulted in titers of IgG, IgG1, and
IgG2a that were significantly higher than those induced in mice
immunized with CFA/IFA. All mice immunized with MPL.TM. SE and
GM-CSF formulated peptide developed higher titers of IgG antibody
than those measured from mice immunized with the CFA/IFA
formulation. These results indicated that the combination of
MPL.TM. SE with GM-CSF generated a favorable antibody response
profile as determined by high titers of peptide-specific antibody,
and a favorable IgG subclass distribution. This formulation
routinely induced the highest T1SP10MN(A)(-Cys)-specific titers of
any vaccine formulation used.
[0055] A comparison was made of the anti-T1SP10MN(A) (-Cys) IgG
titers of mice immunized with GM-CSF formulated together with
aqueous MPL.TM., SE or MPL.TM. SE, to determine the effects of
GM-CSF as an adjuvant supplement (FIG. 2). The results suggest that
the combination of MPL.TM. SE with GM-CSF and peptide are unique in
this particular embodiment in the induction of high titer antibody.
MPL.TM. plus GM-CSF elicited titers comparable to CFA/IFA. Thus,
the adjuvanting properties of MPL.TM. and GM-CSF appear to be
synergistic when formulated together where the MPL.TM. is either in
aqueous form or is present as a stable emulsion.
[0056] Next, T1SP10MN(A)(-Cys)-specific antibody titers were
measured in pooled vaginal lavage fluid obtained from mice four
weeks after a second immunization (Table 3). Mice immunized with
MPL.TM. SE plus GM-CSF developed high titers of both IgA and IgG
antibody. Antibody titers in vaginal lavage obtained from mice
immunized with other formulations have not been routinely detected.
Since the ratio of IgG to IgA in the vaginal lavage favors IgG, and
since sIgA was not measured, it cannot be concluded that the IgA
antibody detected in vaginal lavage fluid is locally synthesized by
mucosal tissues. Indeed, it is likely that the IgA and IgG titers
detected are the result of transudated immunoglobulin secreted from
plasma cells located distal to the vaginal mucosa.
[0057] These results demonstrated that mice immunized with an
HIV-peptide T1SP10MN(A)(-Cys), formulated with MPL.TM. SE in
combination with either IL-12 or GM-CSF had high titers of
peptide-specific serum antibody.
[0058] To assess whether those antibody titers were functionally
effective, sera were analyzed for their ability to inhibit the
infection of cells in vitro by a laboratory adapted strain of HIV.
The assay measured the reverse transcriptase activity of virus that
was shed into culture supernatants from cells infected with the
appropriate HIV strain. Serum from mice immunized with MPL.TM. SE
and GM-CSF, or MPL.TM. SE and IL-12, both significantly reduced
viral infectivity (FIG. 3).
[0059] Maximum viral reverse transcriptase units ranged from 9,481
to 10,411. Sera from mice immunized with that formulation inhibited
viral replication. Even at viral dilutions of only 1/20, sera from
mice immunized with MPL.TM. SE and GM-CSF together with
T1SP10MN(A)(-Cys) inhibited viral replication by approximately
fifty percent. The serum neutralization titer for this formulation
was determined to be greater than 1600 as compared to 71 for serum
obtained from mice immunized with the MPL.TM. SE, IL-12 and HIV
peptide formulation.
[0060] Serum anti-T1SP10MN(A)(-Cys) titers from those groups of
mice were similar (albeit somewhat higher) to those elicited
through immunization of mice with CFA and IFA. The sera from mice
immunized with T1SP10MN(A)(-Cys) emulsified with CFA/IFA did not
demonstrate HIV-neutralization in this assay. Sera from mice
immunized with peptide and the combination of MPL.TM. SE plus
GM-CSF as adjuvant, demonstrated greater neutralizing activity than
any other sera. At equivalent dilutions, sera from mice immunized
with MPL.TM. SE plus GM-CSF and the HIV peptide neutralized higher
concentrations of virus than sera from recipients of other vaccine
formulations.
[0061] HIV peptide-specific spleen cell proliferation in culture
was then measured. As a measurement of cellular responsiveness to
T1SP10MN(A) (-Cys), spleen cells were cultured in vitro with
peptide or control proteins. The assay measured .sup.3H-thymidine
incorporated into the DNA of dividing cells (Table 4). Unlike
spleen cells from mice immunized without adjuvant, spleen cells
from mice immunized with T1SP10MN(A)(-Cys) formulated with SE,
proliferated vigorously in response to the peptide. Spleen cells
from vaccine recipients did not respond in culture to an irrelevant
antigen (lysozyme), or with no antigen stimulation. All groups
responded similarly to stimulation with the mitogen ConA. Within
most groups, there was an indication of an antigen-specific dose
dependent proliferative response. At all three doses of peptide,
the highest degree of proliferation was determined for groups of
mice immunized with GM-CSF co-formulated with MPL.TM. SE. The
lowest proliferation responses were measured from spleen cells of
groups of mice immunized with CFA/IFA, or IL-12 plus HIV-peptide
formulated vaccines. Spleen cells from mice immunized with peptide
and either SE or GM-CSF, incorporated similar levels of thymidine
in culture. These results show that the co-formulation of HIV
peptide with MPL.TM. SE together with GM-CSF, provided murine
spleen cells with the highest potential for proliferation in
response to in vitro presentation of antigen.
[0062] Cultured spleen cells were examined for their potential to
secrete into culture supernatants the cytokines IL-4 (Table 5) and
interferon-gamma (Table 6). These cytokines were measured in
culture supernatants harvested after three and six days of in vitro
stimulation with antigen or mitogen. Measurement of IL-4, a T
helper type 2 associated cytokine, showed that whereas all groups
produced detectable levels in response to stimulation with the
mitogen ConA by day 3, only mice immunized with MPL.TM. SE, or
MPL.TM. SE plus GM-CSF, produced IL-4 in response to peptide
stimulation. Only mice immunized with MPL.TM. SE plus GM-CSF and
T1SP10MN(A)(-Cys) secreted into culture detectable levels of IL-4
at all doses of peptide used to stimulate spleen cells. By day six
of culture, spleen cells from mice immunized with peptide together
with MPL.TM. SE plus GM-CSF had secreted higher levels of IL-4 than
those detected from mice immunized with peptide together with
MPL.TM. SE, MPL.TM. SE plus IL-12, or SE. The levels of IL-4 were
even higher than those induced through the stimulation of those
cells in culture with ConA. Spleen cells cultured from mice
immunized with MPL.TM. SE plus GM-CSF also secreted into culture
detectable levels of IL-5 (another T helper type 2 cytokine (not
shown)) in response to six days of stimulation with
T1SP10MN(A)(-Cys). Spleen cells from no other groups produced
detectable IL-5 in these cultures.
[0063] In response to three days of stimulation in culture with
ConA, spleen cells from all groups of mice secreted detectable
levels of interferon-gamma into culture (Table 6). Only cells from
mice immunized with MPL.TM. SE or MPL.TM. SE plus GM-CSF produced
detectable levels of interferon-gamma in response to three days of
stimulation with the HIV peptide. At the end of six days of culture
stimulation, higher concentrations of interferon-gamma were
observed in response to both ConA and peptide. Of interest were the
interferon-gamma levels measured from the culture supernatants of
spleens from mice immunized with MPL.TM. SE, or MPL.TM. SE plus
GM-CSF. Spleen cells from those two recipient groups secreted
markedly higher concentrations of interferon-gamma into culture
supernatants than did spleen cells from mice immunized with peptide
together with MPL.TM. SE plus IL-12, or SE.
[0064] The results of the first experiment demonstrate that the
inclusion of MPL.TM. into a stable oil-in-water emulsion, and then
combining the emulsion with the HIV peptide T1SP10MN(A)(-Cys) and
GM-CSF, results in the induction of neutralizing antibodies.
Moreover, the co-formulation of GM-CSF with MPL.TM. SE and a
vaccine antigen resulted in increased levels of IL-4, IL-5, and
interferon-gamma secreted into culture supernatants and enhanced
the proliferative response of spleen cells stimulated in culture
with the immunizing antigen. That formulation also induced the
highest IgG, IgG2a, and IgG2b titers of any of the vaccine
formulations looked at. Only groups of mice immunized with the
combination of MPL.TM. SE and GM-CSF together with peptide had
detectable IgG and IgA titers in vaginal lavage fluid consistently
over a number of repeat studies. The combination of MPL.TM. SE,
IL-12 and peptide also resulted in increased levels of IgG1 and
IgG2a titers, increased viral neutralization, increased spleen cell
proliferation, and secretion of IL-4 and interferon-gamma.
[0065] The immunization of mice with any single adjuvant formulated
with the HIV peptide did not produce an immune response with the
properties of eliciting a neutralizing antibody.
[0066] It was often observed that immunization of mice with
T1SP10MN(A)(-Cys), together with MPL.TM. SE or MPL.TM. as an
aqueous formulation, induced good titers of antibody. Those
formulations did not, however, consistently induce an immune
response having vaginal antibody titers, neutralizing antibody
titers (or strong CTL responses as described below in Experiment
8). Occasionally, MPL.TM. or MPL.TM. SE combined with peptide
induced measurable titers of IgA and IgG in vaginal lavage fluid.
In some studies, the addition of either IL-12, or GM-CSF to MPL.TM.
in the vaccine formulation resulted in titers that were similar to
those produced in mice immunized with CFA/IFA, or any of the
MPL.TM. SE formulations with or without cytokine. This observation
suggests that the SE form of MPL.TM. is not required for high titer
antiserum specific for the HIV-peptide. The addition of GM-CSF to
the SE vehicle conferred an increase in peptide-specific titers as
compared to mice immunized with SE alone, or with SE and IL-12. In
general, however, the induction of good IgG2a and IgG2b antibody
titers was dependent upon the formulation of the peptide with
MPL.TM. SE and GM-CSF. It is interesting to note that this
formulation induced IgG titers that were similar to those induced
through immunization with other formulations like CFA/IFA and
peptide. The combination of MPL.TM. SE with GM-CSF and peptide was
the only formulation to demonstrate the induction of both high
titer neutralizing antibody and CTL (see Experiment 8). The
inclusion of IL-12 with MPL.TM. SE and peptide also induced a
favorable immune response profile. The results indicated that
MPL.TM. SE co-formulated with cytokines GM-CSF or IL-12 imparted a
qualitative difference in the antibody response as compared to
immunization with CFA and IFA. That difference is believed to be
attributable to elevated levels of IgG2a and IgG2b.
[0067] In a second experiment, the protocols of the first
Balb/c-HIV peptide experiment were followed with minor
modifications. MPL.TM. was also administered in aqueous form, with
or without a cytokine.
[0068] Immunization of Balb/c mice with the HIV peptide
T1SP10MN(A)(-Cys) without adjuvant did not induce significant
titers of antibody. In contrast, formulation of peptide antigen
with various adjuvant/cytokine combinations did result in the
induction of high antibody titers after two immunizations.
[0069] Immunization with peptide and IL-12, or with SE only,
resulted in titers that were indistinguishable from those induced
without adjuvant (Table 7). Recipients of peptide co-formulated
with GM-CSF had modest increases in titer. Compared with recipients
of vaccine containing CFA/IFA, microfluidized MPL.TM. SE
demonstrated similar peptide-specific titer development. Compared
to recipients of CFA/IFA vaccine, MPL.TM. SE induced higher levels
of peptide-specific IgG2a. The immunization schedules used may
affect the antibody titers observed. However, immunization of mice
with MPL.TM. (aqueous) formulated peptide induced high titers of
peptide-specific antibody. Addition of GM-CSF or IL-12 to this
formulation resulted in increased titers of greater than 10.sup.6.
Thus, the combination of an HIV peptide with MPL.TM. and the
cytokines IL-12 or GM-CSF induced high titer antibody specific for
the peptide.
[0070] Only groups of mice immunized with peptide and either
MPL.TM. and IL-12, or MPL.TM. SE and GM-CSF, developed relatively
high titers of antibody detected from the fluids obtained from
vaginal lavage (Table 8). Indeed, only that group of mice immunized
with MPL.TM. and IL-12 with peptide produced peptide-specific
IgA.
[0071] The proliferative capacity of spleen cells in culture was
determined through the incorporation of thymidine in response to in
vitro stimulation with peptide. The data in Table 9 are presented
in such a way as to normalize the proliferation, standardizing to
maximal proliferation as determined through stimulation with ConA.
Spleen cells from mice immunized with MPL.TM. SE together with
either GM-CSF or IL-12, as well as those from mice immunized with
GM-CSF only, demonstrated low levels of peptide-associated
proliferation. In contrast, spleen cells from mice immunized with
peptide combined with MPL.TM. and GM-CSF demonstrated significant
proliferation.
[0072] Cytokine production from spleen cells cultured in vitro was
also measured. For IL-4, only mice immunized with MPL.TM. SE
combined with GM-CSF and T1SP10MN(A)(-Cys) secreted good levels of
IL-4 in response to stimulation with peptide (Table 10). For
interferon-gamma, mice immunized with MPL.TM. SE and either GM-CSF
or IL-12, or aqueous MPL.TM. with GM-CSF or IL-12, produced readily
detectable levels of this cytokine into culture supernatants (Table
11).
[0073] Thus, the combination of cytokines GM-CSF or IL-12 with
MPL.TM. or MPL.TM. SE induced high titers of antibody specific for
the peptide antigen. Titers were similar to those induced through
immunization of mice with peptide and CFA/IFA. The data show that
these combinations also induced the highest proliferative responses
of spleen cells set up in culture, and established populations of
spleen cells that secreted the highest levels of interferon-gamma
in response to peptide stimulation.
[0074] The results of this second experiment indicate that the
coformulation of T1SP10MN(A)(-Cys) with MPL.TM. and the cytokines
GM-CSF or IL-12 induces an immune response profile that is similar
to, or better than, that induced in mice immunized with peptide and
CFA, and boosted with peptide and IFA.
[0075] A histological evaluation (not shown) of the injection site
two weeks after the second immunization showed that mice immunized
with microfluidized MPL.TM. SE did not develop or maintain a
mononuclear cell infiltration into the dermis. Hematoxylin/eosin
stained tissues looked like those prepared from recipients of no
adjuvant. In contrast, Balb/c mice immunized with CFA and IFA as
adjuvants (a water-in-oil emulsion) had a large accumulation of
mononuclear cells in this region. The recipients of MPL.TM. SE
together with GM-CSF and peptide showed a noticeable, but marginal
increase in mononuclear cells as compared to MPL.TM. SE recipients
without GM-CSF. Tissues from mice immunized with GM-CSF and peptide
only were not examined.
[0076] The protocols of the second experiment were followed in a
third experiment, with Swiss-Webster mice used instead of Balb/c
mice. Swiss-Webster mice were used to determine adjuvant effects
with the HIV peptide antigen wherein the MHC-restricted helper T
cell epitope would not influence the immune response. Swiss-Webster
mice are an outbred strain of mice; as such, no cellular studies
were performed. Only reciprocal anti-HIV peptide IgG endpoint
titers and vaginal lavage endpoint reciprocal endpoint IgG and IgA
antibody titers were measured in this experiment. As seen in Tables
12 and 13, the response profile was comparable to that obtained
from the Balb/c mice measured in the first and second
experiments.
[0077] In a fourth experiment, the protocols of the second
experiment were followed with minor variations using Balb/c mice.
As shown in Table 14, the adjuvant formulations of MPL.TM. together
with either GM-CSF or IL-12 elicited a noticeably higher IgG GMT
response than MPL.TM. alone. As shown in Table 15, the adjuvant
formulation of MPL.TM. SE together with GM-CSF elicited a
significant IgG2b subclass response. The adjuvant formulations of
MPL.TM. together with either GM-CSF or IL-12 elicited a noticeably
higher IgG2a subclass response than MPL.TM. alone, while the
formulations of MPL.TM. SE together with either GM-CSF or IL-12
elicited a higher IgG2a subclass response than MPL.TM. alone. As
shown in Table 16, the adjuvant formulations of MPL.TM. together
with either GM-CSF or IL-12 elicited a noticeably higher IgG titers
in vaginal lavage fluids than MPL.TM. alone. Finally, as shown in
FIG. 4, the adjuvant formulations of MPL.TM. together with either
GM-CSF or IL-12, as well as the formulations of MPL.TM. SE together
with either GM-CSF or IL-12, demonstrated a greater proliferation
of spleen cells than MPL.TM. alone or MPL.TM. SE alone,
respectively.
[0078] In a fifth experiment, the protocols of the second
experiment were followed with minor variations using Balb/c mice;
IL-12 was not included in the adjuvant formulations. As shown in
Table 17, adjuvant formulations containing both MPL.TM. and GM-CSF
elicited noticeably higher IgG2a and IgG2b responses than MPL.TM.
alone. Furthermore, adjuvant formulations containing both MPL.TM.
and GM-CSF elicited noticeably higher responses for all IgG
subclasses than MPL.TM. alone.
[0079] In a sixth experiment, the protocols of the second
experiment were followed with minor variations using Balb/c mice;
IL-12 was not included in the adjuvant formulations. The HIV
peptide was 40 amino acids in length, because of the presence of a
cysteine at amino acid position 17. As shown in Table 18, adjuvant
formulations containing both MPL.TM. SE and GM-CSF elicited
significantly higher responses for all IgG subclasses than MPL.TM.
alone and noticeably higher responses for all subclasses than
MPL.TM. SE alone.
[0080] In a seventh experiment, the protocols of the sixth
experiment were followed with minor variations using Balb/c mice;
IL-12 was not included in the adjuvant formulations. As shown in
Table 19, adjuvant formulations containing both MPL.TM. SE and
GM-CSF elicited noticeably higher responses for all IgG subclasses
than MPL.TM. SE, and adjuvant formulations containing both MPL.TM.
and GM-CSF elicited noticeably higher responses for all IgG
subclasses than MPL.TM. alone.
[0081] In an eighth experiment, as a measure of functional cell
mediated immunity, the ability of spleen cells from mice immunized
with MPL.TM. SE, or MPL.TM. SE plus GM-CSF formulated together with
the multi-epitope peptide T1SP10MN(A)(+Cys) to generate
HIV.sub.MN-specific CTL responses was assessed.
[0082] As shown in FIG. 5, spleen cells from mice immunized with
MPL.TM. SE, or MPL.TM. SE plus GM-CSF demonstrated low activity
toward target cells that were either unlabelled or pulse-labeled
with the IIIB CTL epitope. Spleen cells from mice immunized with
T1SP10MN(A)(+Cys) formulated with MPL.TM. SE and GM-CSF together
induced good HIV.sub.MN-specific CTL activity after a single
immunization. HIV.sub.MN-specific CTL-mediated target cell lysis
was markedly enhanced when measured seven days after secondary
immunization (FIG. 5). In separate experiments, mice immunized
without adjuvant did not induce a CTL response. Mice immunized with
aqueous MPL.TM. and peptide generated low (<30%)
peptide-specific CTL responses.
[0083] One difficulty is assessing the potential efficacy of
immunogenic compositions against HIV is that non-human primates
infected with HIV do not develop AIDS-like symptoms. Thus, a
potential animal model does not mimic the human symptomology caused
by HIV. Fortunately, non-human primates infected with Simian
immunodeficiency virus (SIV), which is closely related to HIV, do
develop AIDS-like symptoms.
[0084] This enables SIV antigens to be assessed in non-human
primates. The SIV antigen may be a protein, polypeptide, peptide or
fragment derived from said protein. The protein may be a
glycoprotein such as gp41, gp120 or gp160. Alternatively, the
protein may be a protein encoded by such genes as gag, pol, vif,
rev, vpr, tat, nef or env. Peptides derived from such proteins will
contain at least one antigenic determinant (epitope) at least six
amino acids in length.
[0085] Analogously to HIV, multiepitope SIV peptides are used in
non-human primates. A study was conducted to assess whether various
peptides in combination with MPL.TM. SE and GM-CSF could elicit a
CTL response. Rhesus macaques were immunized subcutaneously at
weeks 0, 4, 8 and 18 with MPL.TM. SE and GM-CSF together with
either of the following sets of three peptides (see Table 20):
[0086] (1) Each peptide contained a Mamu A*01 restricted CTL
epitope as follows:
TABLE-US-00003 Cys Thr Pro Tyr Asp Ile Asn Gln Met (SEQ ID NO: 3)
(gag)(38,39) Ser Thr Pro Pro Leu Val Arg Leu Val (SEQ ID NO: 4)
(pol)(40) Tyr Ala Pro Pro Ile Ser Gly Gln Ile (SEQ ID NO: 5)
(env)(40)
[0087] (2) Alternatively, each of these three peptides was linked
to a promiscuous T-helper epitope having the following
sequence:
TABLE-US-00004 (SEQ ID NO: 6) Glu Leu Tyr Lys Tyr Lys Val Val Lys
Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala (adapted from 41).
[0088] Thus, the three peptides had the following sequences:
TABLE-US-00005 (SEQ ID NO: 7) Glu Leu Tyr Lys Tyr Lys Val Val Lys
Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Cys Thr Pro Tyr Asp Ile
Asn Gln Met (SEQ ID NO: 8) Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile
Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Ser Thr Pro Pro Leu Val Arg
Leu Val (SEQ ID NO: 9) Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu
Pro Leu Gly Val Ala Pro Thr Lys Ala Tyr Ala Pro Pro Ile Ser Gly Gln
Ile.
[0089] Heparinized blood was collected every two weeks and
peripheral blood mononuclear cells analyzed for CTL by the
.sup.51Cr Release assay, tetramer staining of fresh peripheral
blood mononuclear cells (PBMC) and tetramer staining of cultured
PBMC. Tetramer staining of fresh PBMC and cytolytic killing by
.sup.51Cr release did not reveal any activity. However,
immunization of Rhesus macaques with Mamu A*01 restricted Th/CTL
peptide cocktails formulated with MPL.TM. SE and GM-CSF resulted in
the detection of tetramer positive CD8 positive T cells. The
results presented in Tables 21-24 are shown as the percent
positive, tetramer positive CD8+ (Tables 21-23) or CD4+ (Table 24)
T cells detected in PBMC cultured with the respective peptide for
11 days.
[0090] Overall, all four Mamu A*01 positive animals immunized with
the CTL epitopes either with (Rh 55, Rh 142) or without (Rh 73, Rh
80) the Th epitopes demonstrated CD8 positive tetramer positive
cells specific for either gag, pol or env. As expected, no tetramer
positive CD8 cells were detected in the Mamu A*01 negative animals
(Rh 41, Rh 47). SIV gag and env specific immune responses were seen
after priming while pol specific Tetramer positivity was observed
after boosting. Because the final booster dose at 18 weeks did not
further elevate the response, data are not presented after 14 weeks
in Tables 21-24.
[0091] In summary, immunization of Rhesus macaques with Mamu*A01
restricted Th/SIV gag, pol and env CTL epitope peptide cocktails
adjuvanted with MPL.TM. SE and human GM-CSF elicited cellular
responses as evidenced by the sensitive and specific tetramer
assay.
[0092] The Porin B protein of Neisseria gonorrhoeae, also known as
the PIB protein, has been expressed recombinantly (42, which is
hereby incorporated by reference), and is a candidate antigen for
prevention or treatment of infections caused by Neisseria
gonorrhoeae.
[0093] A series of studies was conducted to compare MPL.TM. (either
with or without SE) plus GM-CSF or IL-12, to MPL.TM. (either with
or without SE) alone, together with a modified version of the Porin
B protein of Neisseria gonorrhoeae, in which 16 amino acids at the
amino-terminus are from a phage, followed by the mature form of the
Porin B protein. A summary of the results will now be
presented.
[0094] In a first experiment, Swiss-Webster mice immunized
subcutaneously in the rump with recombinant Porin B protein
generated antigen-specific antibody titers, demonstrating that
Porin B protein is a viable candidate antigen. Addition of GM-CSF
to MPL.TM. and the Porin B protein resulted in elevated serum
antibody IgG and IgG2a titers compared to recipients of MPL.TM. and
the Porin B protein (see Tables 25 and 26).
[0095] In a second experiment, Swiss-Webster mice immunized
subcutaneously in the rump with the Porin B protein plus IL-12 and
MPL.TM. or MPL.TM. SE induced higher antigen-specific antibody
(particularly IgG) compared to recipients of the Porin B protein
plus MPL.TM. or MPL.TM. SE. Higher titers were observed after both
the primary and secondary immunizations. The inclusion of IL-12 in
the formulations resulted in an approximate ten-fold increase in
IgG titers measured in vaginal lavage fluid (see Table 27).
[0096] The purified native fusion (F) protein of Human Respiratory
syncytial virus (RSV) in the native dimeric form is a candidate
antigen for prevention of infections caused by RSV (43, which is
hereby incorporated by reference.
[0097] A series of studies was conducted to compare MPL.TM. (either
with or without SE) plus GM-CSF or IL-12 to each of MPL.TM. (either
with or without SE), aluminum phosphate or Stimulon.TM. QS-21
individually, together with the purified native F protein of RSV. A
summary of the results will now be presented.
[0098] In a first experiment, Balb/c mice immunized intramuscularly
with the native RSV F protein generated antigen-specific antibody
titers, demonstrating that the F protein is a viable candidate
antigen. Addition of GM-CSF to MPL.TM. induced higher endpoint
titers than MPL.TM. alone against F protein after both the primary
and secondary immunizations (see Table 28), and also induced an
enhanced cellular response to in vitro stimulation of spleen cells
than MPL.TM. alone (see Table 29). Addition of GM-CSF to MPL.TM. SE
resulted in an elevated primary IgG response to F protein than
MPL.TM. SE alone (see Table 28).
[0099] A second experiment repeated the protocol of the first
experiment. Addition of GM-CSF to MPL.TM. induced higher endpoint
titers than MPL.TM. alone against F protein after both the primary
and secondary immunizations (see Table 30). Addition of GM-CSF to
MPL.TM. SE also induced higher endpoint titers than MPL.TM. SE
alone against F protein after the primary immunization (see Table
30). The addition of GM-CSF to formulations of F protein plus
MPL.TM. or MPL.TM. SE induced a higher percentage of RSV-specific
splenic CTL activity than that induced by formulations lacking
GM-CSF, as measured from the spleen cells of immunized mice (see
Table 31).
[0100] A third experiment substituted IL-12 for GM-CSF. The
co-formulation of IL-12 with MPL.TM. induced higher titers of IgG
after priming immunization, as compared to the delivery of F
protein with MPL.TM. alone (see Table 32). However, the addition of
IL-12 to MPL.TM. or MPL.TM. SE had no effect on the RSV-specific
CTL activity measured after in vitro stimulation of effector cells
(see Table 33).
[0101] One study was conducted to compare MPL.TM. (either with or
without SE) plus GM-CSF to MPL.TM. (either with or without SE)
individually, together with the Influenza virus NP (nucleocapsid)
protein. There were insufficient quantities of the NP to conduct
experiments to measure antibody titers. Mice immunized with the NP
peptide with or without adjuvants were analyzed for spleen cell
responses to antigen stimulation 14 days after the final
immunization. The inclusion of GM-CSF in the formulations
containing MPL.TM. or MPL.TM. SE resulted in a marked reduction of
CTL activity (data not shown).
[0102] It is unclear why this anomalous result was obtained. There
may have been technical problems in the conduct of the assay.
[0103] The antigenic compositions of the present invention modulate
the immune response by improving the vertebrate host's antibody
response and cell-mediated immunity after administration of an
antigenic composition comprising a selected antigen from a
pathogenic virus, bacterium fungus or parasite, and an effective
adjuvanting amount of MPL.TM. (in an aqueous or stable emulsion
form) combined with a cytokine or lymphokine, in particular GM-CSF
or IL-12. Other cytokines or lymphokines have been shown to have
immune modulating activity, including, but not limited to, the
interleukins 1-alpha, 1-beta, 2, 4, 5, 6, 7, 8, 10, 13, 14, 15, 16,
17 and 18, the interferons-alpha, beta and gamma, granulocyte
colony stimulating factor, and the tumor necrosis factors alpha and
beta.
[0104] Agonists of or antagonists to said cytokines or lymphokines
are also within the scope of this invention. As used herein, the
term "agonist" means a molecule that enhances the activity of, or
functions in the same way as, said cytokines or lymphokines. An
example of such an agonist is a mimic of said cytokines or
lymphokines. As used herein, the term "antagonist" means a molecule
that inhibits or prevents the activity of said cytokines or
lymphokines. Examples of such antagonists are the soluble IL-4
receptor and the soluble TNF receptor.
[0105] As used herein, the term "effective adjuvanting amount"
means a dose of the combination of adjuvants described herein,
which is suitable to elicit an increased immune response in a
vertebrate host. The particular dosage will depend in part upon the
age, weight and medical condition of the host, as well as on the
method of administration and the antigen. In a preferred
embodiment, the combination of adjuvants will utilize MPL.TM. in
the range of 1-100 .mu.g/dose. Suitable doses are readily
determined by persons skilled in the art. The antigenic
compositions of this invention may also be mixed with
immunologically acceptable diluents or carriers in a conventional
manner to prepare injectable liquid solutions or suspensions.
[0106] The antigenic compositions of this invention are
administered to a human or non-human vertebrate by a variety of
routes, including, but not limited to, intranasal, oral, vaginal,
rectal, parenteral, intradermal, transdermal (see, e.g.,
International application WO 98/20734 (44), which is hereby
incorporated by reference), intramuscular, intraperitoneal,
subcutaneous, intravenous and intraarterial. The amount of the
antigen component or components of the antigenic composition will
vary depending in part upon the identity of the antigen, as well as
upon the age, weight and medical condition of the host, as well as
on the method of administration. Again, suitable doses are readily
determined by persons skilled in the art. It is preferable,
although not required, that the antigen and the combination of
adjuvants be administered at the same time. The number of doses and
the dosage regimen for the antigenic composition are also readily
determined by persons skilled in the art. In some instances, the
adjuvant properties of the combination of adjuvants may reduce the
number of doses needed or the time course of the dosage
regimen.
[0107] The combinations of adjuvants of this invention are suitable
for use in antigenic compositions containing a wide variety of
antigens from a wide variety of pathogenic microorganisms,
including but not limited to those from viruses, bacteria, fungi or
parasitic microorganisms which infect humans and non-human
vertebrates, or from a cancer cell or tumor cell. The antigen may
comprise peptides or polypeptides derived from proteins, as well as
fragments of any of the following: saccharides, proteins, poly- or
oligonucleotides, cancer or tumor cells, allergens, amyloid peptide
protein, or other macromolecular components. In some instances,
more than one antigen is included in the antigenic composition.
[0108] Desirable viral vaccines containing the adjuvant
combinations of this invention include those directed to the
prevention and/or treatment of disease caused by, without
limitation, Human immunodeficiency virus, Simian immunodeficiency
virus, Respiratory syncytial virus, Parainfluenza virus types 1-3,
Influenza virus, Herpes simplex virus, Human cytomegalovirus,
Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Human
papillomavirus, poliovirus, rotavirus, caliciviruses, Measles
virus, Mumps virus, Rubella virus, adenovirus, rabies virus, canine
distemper virus, rinderpest virus, coronavirus, parvovirus,
infectious rhinotracheitis viruses, feline leukemia virus, feline
infectious peritonitis virus, avian infectious bursal disease
virus, Newcastle disease virus, Marek's disease virus, porcine
respiratory and reproductive syndrome virus, equine arteritis virus
and various Encephalitis viruses.
[0109] Desirable bacterial vaccines containing the adjuvant
combinations of this invention include those directed to the
prevention and/or treatment of disease caused by, without
limitation, Haemophilus influenzae (both typable and nontypable),
Haemophilus somnus, Moraxella catarrhalis, Streptococcus
pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae,
Streptococcus faecalis, Helicobacter pylori, Neisseria
meningitidis, Neisseria gonorrhoeae, Chlamydia trachomatis,
Chlamydia pneumoniae, Chlamydia psittaci, Bordetella pertussis,
Salmonella typhi, Salmonella typhimurium, Salmonella choleraesuis,
Escherichia coli, Shigella, Vibrio cholerae, Corynebacterium
diphtheriae, Mycobacterium tuberculosis, Mycobacterium
avium-Mycobacterium intracellulare complex, Proteus mirabilis,
Proteus vulgaris, Staphylococcus aureus, Clostridium tetani,
Leptospira interrogans, Borrelia burgdorferi, Pasteurella
haemolytica, Pasteurella multocida, Actinobacillus pleuropneumoniae
and Mycoplasma gallisepticum.
[0110] Desirable vaccines against fungal pathogens containing the
adjuvant combinations of this invention include those directed to
the prevention and/or treatment of disease caused by, without
limitation, Aspergillis, Blastomyces, Candida, Coccidiodes,
Cryptococcus and Histoplasma.
[0111] Desirable vaccines against parasites containing the adjuvant
combinations of this invention include those directed to the
prevention and/or treatment of disease caused by, without
limitation, Leishmania major, Ascaris, Trichuris, Giardia,
Schistosoma, Cryptosporidium, Trichomonas, Toxoplasma gondii and
Pneumocystis carinii.
[0112] Desirable vaccines for eliciting a therapeutic or
prophylactic anti-cancer effect in a vertebrate host, which contain
the adjuvant combinations of this invention, include those
utilizing a cancer antigen or tumor-associated antigen including,
without limitation, prostate specific antigen, carcino-embryonic
antigen, MUC-1, Her2, CA-125 and MAGE-3.
[0113] Desirable vaccines for moderating responses to allergens in
a vertebrate host, which contain the adjuvant combinations of this
invention, include those containing an allergen or fragment
thereof. Examples of such allergens are described in U.S. Pat. No.
5,830,877 (45) and published International Patent Application
Number WO 99/51259 (46), which are hereby incorporated by
reference, and include pollen, insect venoms, animal dander, fungal
spores and drugs (such as penicillin). The vaccines interfere with
the production of IgE antibodies, a known cause of allergic
reactions.
[0114] Desirable vaccines for preventing or treating disease
characterized by amyloid deposition in a vertebrate host, which
contain the adjuvant combinations of this invention, include those
containing portions of amyloid peptide protein (APP). This disease
is referred to variously as Alzheimer's disease, amyloidosis or
amyloidogenic disease. The .beta.-amyloid peptide (also referred to
as A.beta. peptide) is a 42 amino acid fragment of APP, which is
generated by processing of APP by the .beta. and .gamma. secretase
enzymes, and has the following sequence:
TABLE-US-00006 (SEQ ID NO: 10) Asp Ala Glu Phe Arg His Asp Ser Gly
Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser
Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile
Ala.
[0115] In some patients, the amyloid deposit takes the form of an
aggregated A.beta. peptide. Surprisingly, it has now been found
that administration of isolated A.beta. peptide induces an immune
response against the A.beta. peptide component of an amyloid
deposit in a vertebrate host (47). Thus, the vaccines of this
invention include the adjuvant combinations of this invention plus
A.beta. peptide, as well as fragments of A.beta. peptide and
antibodies to A.beta. peptide or fragments thereof. One such
fragment of A.beta. peptide is the 28 amino acid peptide having the
following sequence (48):
TABLE-US-00007 (SEQ ID NO: 11) Asp Ala Glu Phe Arg His Asp Ser Gly
Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser
Asn Lys.
[0116] In the case of HIV and SIV, the antigenic compositions
comprise at least one protein, polypeptide, peptide or fragment
derived from said protein. In some instances, multiple HIV or SIV
proteins, polypeptides, peptides and/or fragments are included in
the antigenic composition.
[0117] The adjuvant combination formulations of this invention are
also suitable for inclusion as an adjuvant in polynucleotide
vaccines (also known as DNA vaccines). Such vaccines may further
include facilitating agents such as bupivicaine (see U.S. Pat. No.
5,593,972 (49), which is hereby incorporated by reference).
[0118] In order that this invention may be better understood, the
following examples are set forth. The examples are for the purpose
of illustration only and are not to be construed as limiting the
scope of the invention.
EXAMPLES
Experiment 1
Immunization of Balb/C Mice with an HIV Peptide and Various
Adjuvants
Example 1
Materials and Methods
Animals
[0119] Female Balb/c mice, aged 7-9 weeks, were purchased from
Taconic Farms, Inc. (Germantown, N.Y.). All mice were housed in a
facility approved by the American Association for Accreditation of
Laboratory Animal Care. Mice were acclimatized to the housing
facility for one week prior to initiation of studies.
Peptides
[0120] The sequence of the multiepitope HIV-1-.sub.MN peptide
T1SP10MN(A)(-Cys) is as follows:
[0121] Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr
Ala Thr Arg Pro Asn Tyr Asn Lys Arg Lys Arg Ile His Ile Gly Pro Gly
Arg Ala Phe Tyr Thr Thr Lys (SEQ ID NO:2). This peptide has been
previously described (28,29), and contains sequences from HIV-1
gp120.sub.MN that evoke CD4+Th cell responses in both mice and
humans, a principal neutralizing determinant, and a site recognized
by CD8+CTL in Balb/c mice. The peptide was provided by Dr. R.
Scearce (Duke University, Durham, N.C.). For CTL analysis, peptides
corresponding to the CTL epitope within the V3 loop of
HIV-1-.sub.IIIB (Arg Gly Pro Gly Arg Ala Phe Val Thr Ile (SEQ ID
NO:12), H-2D.sup.d restricted) or HIV-1-.sub.MN (Ile Gly Pro Gly
Arg Ala Phe Tyr Thr Thr (SEQ ID NO:13), H-2D.sup.d restricted),
were purchased from Genosys Biotechnologies Inc. (The Woodlands,
Tex.). Peptides were solubilized in sterile water, and diluted in
appropriate buffers, or cell culture medium, prior to use.
Adjuvants
[0122] All MPL.TM.-containing adjuvant preparations were obtained
from Ribi ImmunoChem Research, Inc. (Hamilton, Mont.). MPL.TM. was
prepared as an aqueous formulation using triethanolamine (Sigma,
St. Louis, Mo.). After solubilization, MPL.TM. was sonicated as per
manufacturer's instructions to generate an opalescent/clear
solution which was sterile filtered. MPL.TM. SE was provided as a
preformulated squalene based oil-in-water (1-2% oil) emulsion,
having MPL.TM. concentrations ranging from 0-2500 .mu.g/ml.
Aluminum phosphate was prepared in-house. Freund's complete
adjuvant (CFA) and incomplete adjuvant (IFA) were purchased from
Difco Laboratories, Detroit, Mich. T1SP10MN(A) peptides and
Freund's adjuvants were emulsified in a 1:1 ratio using two linked
syringes. Recombinantly expressed murine IL-12 was provided by
Genetics Institute (Cambridge, Mass.). Recombinant murine GM-CSF
was supplied by Immunex (Seattle, Wash.), provided by R&D
Systems (Minneapolis, Minn.), or purchased from Biosource
International (Camarillo, Calif.) as a carrier-free lyophilized
powder.
Immunizations
[0123] Mice were immunized subcutaneously in the rump, in a total
volume of 0.2 ml equally divided on each side of the tail.
Immunizations were administered at varying time intervals, as
indicated below. Antigen and cytokines were diluted in phosphate
buffered saline to the appropriate concentrations and formulated
with adjuvants less than 16 hours prior to immunization, under
sterile conditions. Vaccines were mixed by gentle agitation, and
stored at 4.degree. C. Formulations were mixed by vortex
immediately prior to immunization.
Sample Collections
[0124] Animals were bled prior to initial immunization, and at
indicated time points. Serum was analyzed from individual mice, or
as pools from mice within groups. Vaginal lavage was performed on
euthanized mice to assess antibody levels. This was accomplished by
instillation of 75:l RPMI-10 into the vaginal vault of female mice
using a 200:l pipette. The vault was washed by repeated delivery
and removal of fluid, which was then added to 10:l of FBS. Vaginal
lavage were analyzed as pools.
Cell Preparations
[0125] For proliferation assays and in vitro cytokine analysis,
spleen cells were obtained from mice at the indicated time points.
Single cell suspensions were prepared from pools of 3-5 mice as
indicated in Results. For proliferation and cytokine analysis,
cells were suspended in round bottom 96 well culture plates
precoated overnight with HIV peptide antigens, control proteins, or
RPMI-10 only. Spleen cells were added at 5.times.10.sup.5
cells/well using culture medium having 2.times. supplements. Cell
culture supernatants were harvested from triplicate wells for
cytokine analysis three or six days after culture initiation.
Immediately after supernatant harvest, cultures were pulsed with
.sup.3H-thymidine for 18-24 hours, and harvested to quantify cell
proliferation.
Enzyme-Linked Immunosorbent Assays
[0126] For analysis of HIV peptide-specific antibody and subclass
distribution, peptide was suspended in either carbonate buffer (15
mM Na.sub.2CO.sub.3, 35 mM NaHCO.sub.3, pH 9.6), or PBS, at a
concentration of 1 .mu.g/ml and plated to 96 well microtiter plates
(Nunc) in a volume of 100:l. After overnight incubation at
37.degree. C., plates were washed, and blocked (0.1% gelatin/PBS)
at room temperature for 2-4 hours. ELISA plates were washed with
wash buffer (PBS, 0.1% Tween.TM. 20) before addition of serially
diluted serum (PBS, 0.1% gelatin, 0.05% Tween.TM. 20, 0.02% sodium
azide). After a four hour incubation, wells were washed and
appropriate dilutions of biotinylated anti-isotype/subclass
antibodies were added for incubation at 4.degree. C. overnight.
Wells were washed and incubated with strepavidin--conjugated
horseradish peroxidase. After incubation, wells were washed, and
developed with ABTS. Wells were read at 405 nm. Titers were
standardized using control sera.
[0127] For cytokine analysis, cell culture supernatants were added
to wells coated with BVD6-11B11 (for anti-IL4), or R4-6A2 (for
interferon-gamma). After incubation and washing, wells were
developed using biotin-labeled BVD6-24G2 (for IL4) or XMG 1.2
[0128] (for interferon-gamma). The concentration of cytokines was
determined using a standard curve prepared from recombinant murine
interferon-gamma or interleukin-4. All cytokine reagents were
purchased from Pharmingen (San Diego, Calif.).
HIV-1.sub.MN Neutralization Assays
[0129] Neutralization assays were performed in the laboratory of
Dr. Thomas Matthews at Duke University. Briefly, coded serum was
provided for neutralization of the laboratory virus isolate
HIV-1.sub.MN (NIH). The assay was performed essentially as
previously described (25). Briefly, dilutions of test sera were
aliquoted in wells of a 96-well microtiter plate (25:l/well). An
equal volume of serially diluted virus stock was added to each
well. After incubation, the virus/antibody mixture was added to AA5
target cells. Cells were cultured in 96-well microtiter plates by
addition of fresh medium every other day. Seven days
post-infection, supernatants were assessed for the presence of
viral reverse transcriptase as a measurement of viral replication,
and successful infection or inhibition thereof.
Example 2
Reciprocal Anti-T1SP10MN(A) IgG Endpoint Titers
[0130] Reciprocal anti-HIV peptide-specific IgG endpoint titers
were measured from pooled serum (n=5 Balb/c) at the indicated time
points after initial immunization. Mice were immunized
subcutaneously in the rump with 25 .mu.g of T1SP10MN(A)(-Cys),
unless otherwise indicated, on day 0, and on day 27. For recipients
of Freund's adjuvants, mice were primed with peptide emulsified in
CFA, and boosted with IFA. MPL.TM. SE was provided as an emulsion
containing 2% squalene oil and 50 .mu.g MPL.TM. per dose. SE is an
oil-in-water emulsion vehicle consisting of squalene, glycerol, and
an emulsifying agent. Recombinant murine IL-12 was delivered at 50
ng/mouse. Recombinant murine GM-CSF was delivered at 10
.mu.g/mouse. The results are given in Table 1.
TABLE-US-00008 TABLE 1 Reciprocal anti-T1SP10MN(A)(-Cys) IgG
endpoint titers .mu.g HIV Endpoint Titers Adjuvant peptide WEEK 4
WEEK 6 WEEK 8 None 25 <100 <100 <100 CFA/IFA 25 23,998
137,683 313,200 rIL-12 25 <100 <100 <100 GM-CSF 25 <100
17,579 12,537 SE 25 <100 171,923 76,479 MPL .TM. SE 25 <100
104,331 79,021 MPL .TM. SE + 25 <100 1,313,330 631,688 rIL-12
MPL .TM. SE + 25 14,824 >10,000,000 3,752,870 GM-CSF
Example 3
Reciprocal Anti-T1SP10MN(A)(-Cys) IgG Endpoint Subclass Titers
[0131] Reciprocal endpoint IgG subclass titers were measured from
pooled serum (n=5 Balb/c) six weeks after initial immunization, two
weeks after secondary immunization. Mice were immunized
subcutaneously in the rump with 25 .mu.g of peptide, unless
otherwise indicated. For recipients of Freund's adjuvants, mice
were primed with peptide emulsified in complete Freund's adjuvant,
and boosted with incomplete Freund's adjuvant on weeks four and
six. MPL.TM. SE was provided as an emulsion containing 2% squalene
oil and 50 .mu.g MPL.TM. per dose. SE is an oil-in-water emulsion
vehicle consisting of squalene, glycerol, and an emulsifying agent.
Recombinant murine IL-12 was delivered at 50 ng/mouse. Recombinant
murine GM-CSF was delivered at 10 .mu.g/mouse. The results are
given in Table 2.
TABLE-US-00009 TABLE 2 Reciprocal anti-T1SP10MN(A)(-Cys) IgG
endpoint subclass titers .mu.g HIV Endpoint Titers Adjuvants
peptide IgG1 IgG2a IgG2b None 25 <100 <100 <100 CFA/IFA 25
29,907 143 798 rIL-12 (.05) 25 <100 <100 <100 GM-CSF (10)
25 1,783 <100 <100 SE (2%) 25 74,293 <100 3,331 MPL .TM.
(50) SE (2%) 25 11,441 10,176 5,280 MPL .TM. (50) SE (2%) + 25
169,278 27,161 2,303 rIL-12 (.05) MPL .TM. (50) SE (2%) + 25
3,494,862 954,707 245,828 GM-CSF (10)
Example 4
[0132] Vaginal Lavage IgG and IgA anti-T1SP10MN(A)(-Cys) Antibody
Titers
[0133] Vaginal IgG and IgA anti-peptide antibody titers were
measured from lavage obtained 2 weeks after final immunization.
Groups of 5 Balb/c female mice were immunized with 25 .mu.g
T1SP10MN(A)(-Cys) and the indicated adjuvant formulations on days
0, 28, and 42. Vaginal antibody titers were determined from pooled
lavage fluid. The results are given in Table 3.
TABLE-US-00010 TABLE 3 Vaginal Lavage IgG and IgA
anti-T1SP10MN(A)(-Cys) Antibody Titers Adjuvant IgG IgA None <20
<20 CFA/IFA 20 <20 rIL-12 (.05) <20 <20 GM-CSF (10)
<20 <20 MPL .TM. (50) SE (2%) <20 <20 MPL .TM. (50) SE
(2%) + rIL-12 24 <20 (.05) MPL .TM. (50) SE (2%) + GM-CSF 1,125
113 (10) SE (2%) <20 <20
Example 5
Spleen Cell Proliferation
[0134] Proliferation of spleen cells from mice immunized with
T1SP10MN(A)(-Cys) and various adjuvant formulations was measured.
Groups of five Balb/c female mice were immunized with 25 .mu.g
T1SP10MN(A)(-Cys) and the indicated adjuvants on days 0 and 28.
Spleen cells were established in culture on day 56, and harvested
for measurement of .sup.3H-thymidine incorporation 96 hours later.
Mice were immunized with 50 ng IL-12, 10 .mu.g GM-CSF, 50 .mu.g
MPL.TM. in an aqueous formulation, or as a stable emulsion with 2%
SE. Data are presented as the delta cpm values as compared to
proliferation values measured from cells grown in culture without
stimulation. Background stimulation counts were <800 cpm. The
results are given in Table 4.
TABLE-US-00011 TABLE 4 Spleen cell proliferation Antigen HIV
Peptide HIV Peptide HIV Peptide Con A Lysozyme Medium Adjuvant 10
.mu.g/ml 3.3 .mu.g/ml 1.1 .mu.g/ml 1 .mu.g/ml 30 .mu.g/ml 0
.mu.g/ml None 2,065 2,373 801 50,019 628 -- CFA/IFA 1,236 878 641
53,781 -- -- IL-12 809 692 308 42,612 -- -- GM-CSF 25,821 19,784
14,249 55,578 -- -- MPL .TM. SE 46,275 41,675 40,998 45,443 413 --
MPL .TM. SE + IL- 26,560 19,907 9,600 38,989 934 -- 12 MPL .TM. SE
+ 74,909 66,257 62,798 37,775 366 -- GM-CSF SE 31,327 23,396 20,480
66,949 -- --
Example 6
Secretion of IL-4 by Spleen Cells
[0135] Interleukin-4 secreted into culture by spleen cells
stimulated with 25 .mu.g T1SP10MN(A)(-Cys) was measured. Spleen
cells were harvested from pools of five Balb/c female mice and
cultured with the indicated antigenic stimuli (50 ng IL-12, 10
.mu.g GM-CSF, 50 .mu.g MPL.TM. as indicated) for either three or
six days. Interleukin-4 levels were determined by ELISA, and
compared to a standard having a known concentration. Blank wells
indicate that the assay could not detect Interleukin-4 from those
culture conditions. The lower limit of detection sensitivity was 22
Units/ml. The results are given in Table 5.
TABLE-US-00012 TABLE 5 Secretion of IL-4 by Spleen Cells Antigen
HIV Peptide HIV Peptide HIV Peptide Con A Lysozyme Medium Adjuvant
10 .mu.g/ml 3.3 .mu.g/ml 1.1 .mu.g/ml 1 .mu.g/ml 30 .mu.g/ml 0
.mu.g/ml Three Day Cultures: None 149 CFA/IFA 156 IL-12 156 GM-CSF
159 MPL .TM. SE 297 79 90 134 MPL .TM. SE + IL- 77 12 MPL .TM. SE +
142 151 102 146 GM-CSF SE 197 Six Day Cultures: None 124 CFA/IFA
204 IL-12 146 GM-CSF 143 MPL .TM. SE 163 96 57 63 MPL .TM. SE + IL-
55 76 12 MPL .TM. SE + 511 457 204 76 GM-CSF SE 41 36 88
Example 7
Secretion of Interferon-Gamma by Spleen Cells
[0136] Interferon-gamma secreted into culture by spleen cells
stimulated with 25 .mu.g T1SP10MN(A)(-Cys). Spleen cells were
harvested from pools of five Balb/c female mice and cultured with
the indicated antigenic stimuli (the same as in Example 6) for
either three or six days. Interferon-gamma levels were determined
by ELISA, and compared to a standard having a known concentration.
Blank wells indicate that the assay could not detect
Interferon-gamma from those culture conditions. The lower limit of
detection sensitivity was 4 picograms/ml. The results are given in
Table 6.
TABLE-US-00013 TABLE 6 Secretion of Interferon-gamma by Spleen
Cells Antigen HIV Peptide HIV Peptide HIV Peptide Con A Lysozyme
Medium Adjuvant 10 .mu.g/ml 3.3 .mu.g/ml 1.1 .mu.g/ml 1 .mu.g/ml 30
.mu.g/ml 0 .mu.g/ml Three Day Cultures: None 23.0 CFA/IFA 5.0 IL-12
5.9 GM-CSF 17.6 MPL .TM. SE 6.6 4.2 4.3 18.2 MPL .TM. SE + IL- 9.2
12 MPL .TM. SE + 11.5 5.1 5.3 14.6 GM-CSF SE 8 Six Day Cultures:
None 71.8 CFA/IFA 15.9 IL-12 8.4 GM-CSF 7.3 MPL .TM. SE 190.1 51.5
319.0 187.3 MPL .TM. SE + IL- 18.0 15.6 42.0 83.4 12.7 12 MPL .TM.
SE + 62.2 20.2 39.2 134.7 GM-CSF SE 13.3 54.6
Experiment 2
Immunization of Balb/c Mice with an HIV Peptide and Various
Adjuvants
Example 8
Materials and Methods
Animals
[0137] Female Balb/c mice, aged 7-9 weeks, were used according to
Example 1 above.
Peptides
[0138] The HIV-1-MN peptide T1SP10MN(A) described in Example 1 was
used. The peptide was rehydrated in saline to a concentration of 1
mg/ml.
Adjuvants
[0139] The adjuvants used were as described in Example 1, except
that in some instances the MPL.TM. was retained as an aqueous
formulation instead of using the stable emulsion form.
Immunizations
[0140] Mice were immunized subcutaneously in the rump, in a total
volume of 0.2 ml equally divided on each side of the tail.
Immunizations were administered on days 0 and 21 with 25/4 of the
HIV peptide, together with the indicated amount of adjuvant(s).
Mice receiving CFA/IFA received CFA on day 0 and IFA on day 21.
Dilutions and mixing were as described in Example 1.
Sample Collections
[0141] Sample collections from animals were carried out in
accordance with the protocol of Example 1 one day prior to each
immunization and 14 days after the second immunization.
Cell Preparations
[0142] Cell preparations were generated and handled in accordance
with the protocol of Example 1.
Enzyme-Linked Immunosorbent Assays
[0143] ELISAs were carried out in accordance with the protocol of
Example 1.
HIV-1 Neutralization Assays
[0144] Neutralization assays were again performed at Duke
University in accordance with the protocol of Example 1.
Example 9
Reciprocal Anti-T1SP10MN(A)(-Cys) IgG Endpoint Titers
[0145] Reciprocal anti-peptide IgG endpoint titers were measured
from either geometric means from individual mice (GMT) or from
pooled serum (n=5 Balb/c), obtained 14 days after the second
immunization. IgG1 and IgG2a subclass endpoint titers were also
measured from pooled serum. For recipients of Freund's adjuvants,
mice were primed with 25 .mu.g peptide emulsified in CFA, and
boosted with IFA. MPL.TM. SE was provided as an emulsion containing
1% squalene oil and 50 .mu.g MPL.TM. per dose. Aqueous MPL.TM. was
delivered at 50 .mu.g per dose. Recombinant murine IL-12 was
delivered at 40 ng/mouse. Recombinant murine GM-CSF was delivered
at 10 .mu.g/mouse. The results are given in Table 7.
TABLE-US-00014 TABLE 7 Reciprocal anti-T1SP10MN(A)(-Cys) IgG
endpoint titers Endpoint Titers Adjuvant IgG IgG IgG1 IgG2a
.mu.g/dose (pool) (GMT) (pool) (pool) None <1000 720 <1000
<1000 CFA/IFA 72,387 135,740 126,433 9,023 MPL .TM. SE 183,802
197,808 162,480 98,342 SE 2,426 6,029 1,859 <1000 MPL .TM. SE +
GM- 148,139 133,171 103,298 50,415 CSF MPL .TM. SE + rIL- 182,852
611,076 6,610 111,662 12 GM-CSF 27,333 1,756 14,538 4,864 rIL-12
<1000 500 <1000 <1000 MPL .TM. 219,705 241,918 134,428
7,127 MPL .TM. + 946,695 1,101,449 545,444 12,291 GM-CSF MPL .TM. +
377,972 2,378,702 204,334 12,795 rIL-12
Example 10
Reciprocal Anti-T1SP10MN(A)(-Cys) IgG Endpoint Subclass Titers
[0146] HIV peptide-specific vaginal lavage endpoint reciprocal
endpoint IgG and IgA antibody titers were measured from pooled
serum (n=5 Balb/c) 15 days after secondary immunization. Mice were
immunized as in Example 9. MPL.TM. SE was provided as an emulsion
containing 1% squalene oil and 50 .mu.g MPL.TM. per dose. Aqueous
MPL.TM. was delivered at 50 .mu.g per dose. Recombinant murine
IL-12 was delivered at 40 ng/mouse. Recombinant murine GM-CSF was
delivered at 10 .mu.g/mouse. The results are given in Table 8.
TABLE-US-00015 TABLE 8 Reciprocal anti-T1SP10MN(A)(-Cys) IgG and
IgA endpoint titers Endpoint Titers Adjuvants IgG IgA None <10
<10 CFA/IFA <10 <10 MPL .TM. SE (1%) 32 <10 SE <10
<10 MPL .TM. SE (1%) + GM- 129 <10 CSF MPL .TM. SE (1%) +
IL-12 40 <10 GM-CSF 26 <10 rIL-12 <10 <10 MPL .TM.
<10 <10 MPL .TM. + GM-CSF <10 <10 MPL .TM. + IL-12 260
197
Example 11
Spleen Cell Proliferation
[0147] Spleen cell proliferation in response to in vitro
stimulation with T1SP10MN(A)(-Cys) and various adjuvant
formulations (the same as in Example 10) was measured. Cells were
cultured for a total of 96 hours. .sup.3H-thymidine was added to
cultures for the last 18 hours. Data are presented as a
proliferation index normalized to cells stimulated in culture with
ConA ([mean cpm Antigen/mean cpm ConA]-[mean cpm medium/mean cpm
ConA]).times.100. As a result, cells cultured in medium have a
background proliferation of 0. The results are given in Table 9.
Proliferation values less than cells grown in culture unstimulated
are in parentheses.
TABLE-US-00016 TABLE 9 Spleen cell proliferation Antigen HIV HIV
HIV Peptide Peptide Peptide Con A Lysozyme Adjuvant 10 .mu.g/ml 3.3
.mu.g/ml 1.1 .mu.g/ml 5 .mu.g/ml 10 .mu.g/ml None 0.1 0.2 0.1 98.3
0.4 CFA/IFA 2.0 0.8 0.4 98.0 0.9 MPL .TM. SE 0.7 0.4 (0.2) 99.2 0.4
SE 0.5 0.3 0.1 99.0 0.4 MPL .TM. SE + 4.3 2.7 2.3 99.1 0.8 GM-CSF
MPL .TM. SE + 6.6 4.9 (1.3) 83.8 17.4 IL-12 GM-CSF 6.8 2.7 1.6 99.0
0.1 IL-12 0.2 0.2 0.5 99.1 0.2 MPL .TM. 1.0 1.4 0.5 99.2 0.4 MPL
.TM. + 27.5 19.2 13.3 97.5 0.5 GM-CSF MPL .TM. + 2.3 1.5 1.3 99.4
(0.0) IL-12
Example 12
Secretion of IL-4 by Spleen Cells
[0148] Interleukin-4 secreted into culture by spleen cells
stimulated with T1SP10MN(A)(-Cys) was measured. Cells were cultured
for a total of 96 hours. Cell culture supernatants were analyzed
for IL-4 by ELISA. All values were after subtraction from those
determined from supernatants of cells stimulated with 10 .mu.g of
an irrelevant protein (lysozyme). The results are given in Table 10
in pg/ml. Results which were below the limit of detection, after
subtraction from stimulation induced with lysozyme are indicated as
"bd". The adjuvants were 40 ng IL-12, 10 .mu.g GM-CSF and 50 .mu.g
MPL.TM..
TABLE-US-00017 TABLE 10 Secretion of IL-4 by Spleen Cells Antigen
HIV Peptide HIV Peptide HIV Peptide Con A Adjuvant 10 .mu.g/ml 3.3
.mu.g/ml 1.1 .mu.g/ml 5 .mu.g/ml None bd bd bd 336 CFA/IFA bd bd bd
117 MPL .TM. SE bd bd bd 187 SE bd bd bd 450 MPL .TM. SE + 40 42 24
214 GM-CSF MPL .TM. SE + 5 bd bd 266 IL-12 GM-CSF bd 9 36 226 IL-12
bd bd 15 411 MPL .TM. 5 bd 17 286 MPL .TM. + GM-CSF bd bd bd 241
MPL .TM. + IL-12 bd bd bd 665
Example 13
Secretion of Interferon-Gamma by Spleen Cells
[0149] Interferon-gamma secreted into culture by spleen cells
stimulated with T1SP10MN(A)(-Cys) was measured. Cells were cultured
for a total of 96 hours. Cell culture supernatants were analyzed
for interferon-gamma by ELISA. All values were after subtraction
from those determined from supernatants of cells stimulated with 10
.mu.g of lysozyme. The results are given in Table 11 in units/ml.
Results which were below the limit of detection, after subtraction
from stimulation induced with lysozyme are indicated as "bd". The
adjuvants were the same as those in Example 12.
TABLE-US-00018 TABLE 11 Secretion of Interferon-gamma by Spleen
Cells Antigen HIV Peptide HIV Peptide HIV Peptide Con A Adjuvant 10
.mu.g/ml 3.3 .mu.g/ml 1.1 .mu.g/ml 5 .mu.g/ml None bd bd 1 189
CFA/IFA bd bd 3 193 MPL .TM. SE 2 bd bd 170 SE bd bd bd 130 MPL
.TM. SE + 12 3 5 138 GM-CSF MPL .TM. SE + 23 8 9 168 IL-12 GM-CSF 2
3 4 167 IL-12 4 2 41 179 MPL .TM. 5 bd bd 203 MPL .TM. + GM-CSF bd
20 19 31 MPL .TM. + IL-12 10 4 3 51
Experiment 3
Immunization of Swiss-Webster Mice with an HIV Peptide and Various
Adjuvants
[0150] The protocols of Experiment 2 were followed, with
Swiss-Webster mice used instead of Balb/c mice. Only reciprocal
anti-peptide IgG endpoint titers and vaginal lavage endpoint
reciprocal endpoint IgG and IgA antibody titers were measured in
this experiment.
Example 14
Reciprocal Anti-HIV Peptide IgG Endpoint Titers
[0151] Reciprocal anti-T1SP10MN(A)(-Cys) IgG endpoint titers were
measured from geometric means from individual Swiss-Webster mice
(GMT), obtained 14 days after the second immunization. IgG1 and
IgG2a subclass endpoint titers were also measured from pooled
serum. For recipients of Freund's adjuvants, mice were primed with
peptide emulsified in CFA, and boosted with IFA. MPL.TM. SE was
provided as an emulsion containing 1% squalene oil and 50 .mu.g
MPL.TM. per dose. Aqueous MPL.TM. was delivered at 50 .mu.g per
dose. Recombinant murine IL-12 was delivered at 40 ng/mouse.
Recombinant murine GM-CSF was delivered at 10 .mu.g/mouse. The
results are given in Table 12.
TABLE-US-00019 TABLE 12 Reciprocal anti-HIV peptide IgG endpoint
titers Endpoint Titers IgG IgG1 IgG2a Adjuvant (GMT) (pool) (pool)
None 500 <1000 <1000 CFA/IFA 9,038 62,358 54,053 MPL .TM. SE
15,831 3,835 8,872 SE 625 <1000 <1000 MPL .TM. SE + GM- 1,374
<1000 1,328 CSF MPL .TM. SE + rIL-12 6,142 <1000 2,170 GM-CSF
500 <1000 <1000 rIL-12 500 <1000 <1000 MPL .TM. 1,960
<1000 <1000 MPL .TM. + GM-CSF 58,211 35,724 37,959 MPL .TM. +
rIL-12 5,489 8,535 17,769
Example 15
Reciprocal Anti-HIV Peptide IgG Endpoint Subclass Titers
[0152] HIV peptide-specific vaginal lavage endpoint reciprocal
endpoint IgG and IgA antibody titers were measured from pooled
serum (n=5 Swiss-Webster) 15 days after secondary immunization.
Mice were immunized as in Example 14. MPL.TM. SE was provided as an
emulsion containing 1% squalene oil and 50 .mu.g MPL.TM. per dose.
Aqueous MPL.TM. was delivered at 50 .mu.g per dose. Recombinant
murine IL-12 was delivered at 40 ng/mouse. Recombinant murine
GM-CSF was delivered at 10 .mu.g/mouse. The results are given in
Table 13.
TABLE-US-00020 TABLE 13 Reciprocal anti-HIV peptide IgG and IgA
endpoint titers Endpoint Titers Adjuvants IgG IgA None <10
<10 CFA/IFA 118 <10 MPL .TM. SE (1%) <10 <10 SE <10
<10 MPL .TM. SE (1%) + GM- <10 <10 CSF MPL .TM. SE (1%) +
IL-12 <10 <10 GM-CSF <10 <10 rIL-12 <10 <10 MPL
.TM. <10 <10 MPL .TM. + GM-CSF 25 <10 MPL .TM. + IL-12
<10 <10
Experiment 4
Immunization of Balb/c Mice with an HIV Peptide and Various
Adjuvants
[0153] The protocols of Experiment 2 were followed, except that
mice were immunized on days 0 and 28, and bled for serological
evaluation on days 0, 27 and 41. CFA/IFA was formulated with CFA at
day 0, IFA at day 28. MPL.TM. SE was formulated with 50 .mu.g
MPL.TM. and 2% SE, while 50 ng IL-12 and 10 .mu.g GM-CSF were
used.
Example 16
Reciprocal Anti-HIV Peptide IgG Endpoint Titers
[0154] Reciprocal anti-T1SP10MN(A)(-Cys) IgG endpoint titers were
measured from individual mice and their geometric means (n=5
Balb/c) 41 days after initial immunization, 13 days after secondary
immunization. The results are given in Table 14. The day zero
individual titers were all less than 50. The notation "[no data]"
means the animal died prior to the completion of the protocol. "SD"
means standard deviation.
TABLE-US-00021 TABLE 14 Individual and Geometric Mean IgG Serum
Titers Specific for T1SP10MN(A)(-Cys) Adjuvant Mouse #1 Mouse #2
Mouse #3 Mouse #4 Mouse #5 GMT SD CFA/IFA 2,806,160 4,856,380
148,038 172,947 972,484 805,599 2,025,740 IL-12 594 50 50 50 50 82
243 GM-CSF 50 50 50 50 50 50 0 SE 11,469 [no data] 5,519 12,620 50
2,514 5,813 SE + IL-12 38,042 8,030 35,081 50 37,932 7,271 18,320
SE + GM-CSF 485,985 223,518 63,377 38,050 1,857,860 217,494 761,374
MPL .TM. 50 151,846 249,054 436,378 1,246,470 63,452 490,091 MPL
.TM. + IL-12 1,855,170 1,117,800 1,255,290 692,219 7,001,540
1,660,297 2,614,417 MPL .TM. + GM-CSF 115,527 1,049,310 301,636
316,223 736,959 385,568 380,380 MPL .TM. SE 904,947 5,805,010
291,382 346,835 354,449 716,000 2,396,968 MPL .TM. SE + IL-12
244,171 8,545.550 455,380 377,697 1,095,650 829,707 3,593,727 MPL
.TM. SE + GM- 3,016,000 724,940 1,718,590 1,483,990 28,259 691,033
1,124,414 CSF
Example 17
Reciprocal Anti-HIV Peptide IgG Endpoint Subclass Titers
[0155] Reciprocal endpoint anti-peptide IgG subclass titers were
measured from pooled serum (n=5 Balb/c) 41 days after initial
immunization, 13 days after secondary immunization. The results are
given in Table 15.
TABLE-US-00022 TABLE 15 Reciprocal anti-T1SP10MN(A)(-Cys) IgG
endpoint subclass titers Endpoint Titers Adjuvants IgG1 IgG2a IgG2b
CFA/IFA 359,238 122,107 155,877 IL-12 <100 <100 <100
GM-CSF 5,514 <100 <100 SE 5,011 <100 <100 SE + IL-12
7,331 <100 <100 SE + GM-CSF 67,111 <100 <100 MPL .TM.
33,544 212 <100 MPL .TM. + IL-12 608,163 6,019 <100 MPL .TM.
+ GM-CSF 114,959 8,000 <100 MPL .TM. SE 142,404 29,141 1,564 MPL
.TM. SE + IL-12 164,866 34,439 558 MPL .TM. SE + GM-CSF 274,241
33,843 29,965
Example 18
Vaginal Lavage IgG and IgA Anti-HIV Peptide Antibody Titers
[0156] Vaginal IgG and IgA anti-peptide antibody titers were
measured from lavage 41 days after initial immunization, 13 days
after secondary immunization. The results are given in Table
16.
TABLE-US-00023 TABLE 16 Vaginal Lavage IgG and IgA
anti-T1SP10MN(A)(-Cys) Antibody Titers Adjuvant IgG IgA CFA/IFA 464
13 IL-12 <10 <10 GM-CSF <10 <10 SE <10 <10 SE +
IL-12 <10 <10 SE + GM-CSF 32 14 MPL .TM. 12 <10 MPL .TM. +
IL-12 643 44 MPL .TM. + GM-CSF 211 65 MPL .TM. SE 153 16 MPL .TM.
SE + IL-12 88 30 MPL .TM. SE + GM-CSF 190 53
Example 19
Spleen Cell Proliferation
[0157] Proliferation of spleen cells from mice immunized with
T1SP10MN(A)(-Cys) and various adjuvant formulations was measured.
Spleen cells were stimulated in vitro for four days with 3.3
.mu.g/ml T1SP10MN(A)(-Cys). The results are shown in FIG. 4 as the
change in the incorporation of labeled thymidine as a result of in
vitro stimulation with 3.3 .mu.g/ml T1SP10MN(A)(-Cys) over the
incorporation in the absence of stimulation (delta cpm).
Experiment 5
Immunization of Balb/c Mice with an HIV Peptide and Various
Adjuvants
[0158] The protocols of Experiment 2 were followed, except that
mice were immunized subcutaneously on days 0 and 22, and bled for
serological evaluation on day 42. MPL.TM. SE was formulated with 50
.mu.g MPL.TM. and 1% SE, while 10 .mu.g GM-CSF were used.
Example 20
Reciprocal Anti-HIV Peptide IgG Endpoint Titers
[0159] Reciprocal anti-peptide endpoint IgG subclass titers were
measured from pooled serum (n=5 Balb/c) 42 days after initial
immunization, 13 days after secondary immunization. Geometric means
with standard deviation for IgG were also measured. The results are
given in Table 17.
TABLE-US-00024 TABLE 17 Reciprocal anti-T1SP10MN(A)(-Cys) IgG
Endpoint Titers Adjuvant IgG IgG1 IgG2a IgG2b IgG GMT SD None
<1000 <1000 <1000 <1000 <1000 -- CFA/IFA 390,931
144,564 33,137 13,134 741,966 834,567 GM-CSF 11,639 3,815 <1000
<1000 5,133 32,762 SE <1000 <1000 <1000 <1000
<1000 -- SE + GM-CSF 84,965 55,998 <1000 <1000 28,247
165,628 MPL .TM. 2,635,118 1,314,771 9,688 13,716 2,032,441
5,638,450 MPL .TM. + GM-CSF 835,218 322,441 26,976 35,697 1,133,423
881,331 MPL .TM. SE 1,577,357 642,436 113,917 45,025 1,450,821
5,876,690 MPL .TM. SE + GM- 6,598,573 1,212,160 238,440 214,570
6,418,920 2,925,687 CSF
Experiment 6
Immunization of Balb/c Mice with an HIV Peptide and Various
Adjuvants
[0160] The protocols of Experiment 2 were followed, except that the
HIV peptide contained a cysteine at amino acid position 17, and the
mice (n=3 Balb/c) were immunized subcutaneously on days 0 and 21,
and bled for serological evaluation on days -1 (the day before
first immunization), 13, 20 and 28. MPL.TM. SE was formulated with
50 .mu.g MPL.TM. and 1% SE, while 10 .mu.g GM-CSF were used. The
HIV peptide T1SP10MN(A)(+Cys) (26) contains a cysteine at amino
acid position 17 and is 40 residues long. T1SP10MN(A)(+Cys) was
purchased from Genosys Biotechnologies (The Woodlands, Tex.).
Example 21
Reciprocal Anti-T1SP10MN(A) IgG Endpoint Titers
[0161] Reciprocal anti-T1SP10MN(A) IgG endpoint titers were
measured from individual mice and their geometric means (n=3
Balb/c) 28 days after initial immunization. The results are given
in Table 18.
TABLE-US-00025 TABLE 18 Effect of MPL .TM. SE + GM-CSF on the IgG
Response to HIV Peptide (+Cys) Adjuvant IgG GMT IgG1 GMT IgG2a GMT
IgG2b GMT MPL .TM. SE + 14,275,585 8,942,480 2,097,104 1,437,319
251,235 239,615 210,746 210,178 GM-CSF 13,618,523 2,484,849 225,253
296,429 3,678,324 569,823 243,103 148,621 MPL .TM. SE 1,913,110
5,206,349 644,377 990,194 22,557 114,913 23,141 70,632 11,405,649
1,937,492 152,430 127,600 6,467,553 777,643 441,326 119,338 MPL
.TM. 91,728 350,486 23,249 84,978 500 2,859 500 2,906 529,663
155,199 1,628 1,102 886,156 170,071 28,722 44,531 None <500 --
<500 -- <500 -- <500 -- <500 <500 <500 <500
<500 <500 <500 <500
Experiment 7
Immunization of Balb/c Mice with an HIV Peptide and Various
Adjuvants
[0162] The protocols of Experiment 6 were followed, except that
mice (n=3 Balb/c) were immunized subcutaneously on days 0 and 32,
and bled for serological evaluation on day 38. MPL.TM. SE was
formulated with 50 .mu.g MPL.TM. and 1% SE, while 10 .mu.g GM-CSF
were used.
Example 22
Reciprocal Anti-HIV Peptide IgG Endpoint Titers
[0163] Reciprocal anti-T1SP10MN(A)(+Cys) IgG endpoint titers were
measured from individual mice and their geometric means (n=3
Balb/c) 38 days after initial immunization. The results are given
in Table 19.
TABLE-US-00026 TABLE 19 Effect of MPL .TM. SE + GM-CSF on the IgG
Response to HIV Peptide (+Cys) Adjuvant IgG GMT IgG1 GMT IgG2a GMT
IgG2b GMT MPL .TM. 4,144,648 4,782,117 +/- 472,745 922,507
1,090,760 +/- 224,940 115,290 328,097 +/- 216,753 74,130 64,521 +/-
SE + 5,055,375 985,180 629,615 76,257 13,077 GM-CSF 5,219,387
1,427,916 486,563 47,515 MPL .TM. SE 736,325 559,033 +/- 177,831
288,659 293,160 +/- 297,378 58,506 103,260 +/- 83,698 33,047 27,180
+/- 696,393 809,341 244,559 46,703 13,837 340,712 107,844 76,951
13,010 MPL .TM. + 444,774 1,367,343 +/- 1,494,876 146,146 611,040
+/- 1,549,005 3,342 8,546 +/- 5,769 2,155 7,067 +/- GM-CSF
3,993,897 3,568,062 17,469 14,130 5,152 1,439,116 437,511 10,691
11,590 MPL .TM. 404,755 442,259 +/- 30,080 148,818 115,544 +/-
23,969 11,874 3,429 +/- 4,727 9,685 4,980 +/- 446,952 90,380 1,674
3,605 2,882 478,163 114,686 2,028 3,538 SE <1,000 <1,000
<1,000 <1,000 <1,000 <1,000 <1,000 <1,000
<1,000 <1,000 <1,000 <1,000 1,214 <1,000 <1,000
<1,000 None <1,000 <1,000 <1,000 <1,000 <1,000
<1,000 <1,000 <1,000 <1,000 <1,000 <1,000
<1,000 <1,000 <1,000 <1,000 <1,000
Experiment 8
CTL Analysis in Balb/c Mice
[0164] The protocols of Experiment 6 were followed regarding
immunization of mice. The CTL activity of spleen cells isolated
from mice seven days after secondary immunization was assessed.
MPL.TM. SE was formulated with 50 .mu.g MPL.TM. in 1% SE, with or
without 10 .mu.g GM-CSF, plus 50 .mu.g T1SP10MN(A)(+Cys).
Example 23
CTL Analysis in Balb/c Mice
[0165] For CTL analysis, spleen cells were removed from immunized
mice 14 days after primary, and seven days after secondary
immunization. A protocol previously described (34) was essentially
followed. Briefly, erythrocyte-depleted spleen cells from three
mice per group were pooled. Spleen effector cells
(4.times.10.sup.6/ml) were restimulated in 24 well culture plates
in a volume of 1.5-2 ml for seven days with 1 .mu.g/ml of either
the "MN", or the "IIIB" 10mer CTL epitope peptides. Both CTL
epitopes were restricted to H-2D.sup.d. Cultures were supplemented
with 10 U/ml recombinant murine IL-2 (Biosource) for the last five
days of culture. For analysis of cytotoxic activity, P815 cells
were labeled with Cr.sup.51 and pulsed with 5 .mu.g/ml peptide
(IIIB or MN) for four hours, and added to cultured splenic effector
cells. Three-fold dilutions of effector to target cell ratios were
used, from 100:1 through 3.7:1. Percent CTL activity was calculated
as the percentage of chromium release using ((specific chromium
release-spontaneous chromium release)/(maximal chromium
release-spontaneous chromium release)).times.100. Chromium release
was assessed after a six hour incubation period. The average
spontaneous release of chromium was always less than 15% of maximal
release. The results of data from day 28 are shown in FIG. 5.
Experiment 9
Immunization of Rhesus Macaques with Various SIV Peptides and
Adjuvants
[0166] The MPL.TM. SE and GM-CSF adjuvant formulation was tested in
Rhesus macaques (Macaca mulatta) for its ability to induce
antigen-specific CTL. In this experiment, the adjuvant formulation
was tested with a trivalent peptide immunogen consisting of three
separate Mamu A*01 restricted CTL epitopes (one each from gag, pol,
and env), each synthesized chemically with or without a promiscuous
T-helper epitope from SIV env at the laboratory of Dr. Barton
Haynes, Duke University.
[0167] The peptides containing a Mamu A*01 restricted CTL epitope
were as follows:
TABLE-US-00027 Cys Thr Pro Tyr Asp Ile Asn Gln Met (SEQ ID NO: 3)
(gag) Ser Thr Pro Pro Leu Val Arg Leu Val (SEQ ID NO: 4) (pol) Tyr
Ala Pro Pro Ile Ser Gly Gln Ile (SEQ ID NO: 5) (env)
[0168] Each of these CTL-containing epitopes was also linked to the
T-helper epitope having the following sequence:
TABLE-US-00028 (SEQ ID NO: 6) Glu Leu Tyr Lys Tyr Lys Val Val Lys
Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala
[0169] Thus, the three multiepitope peptides had the following
sequences:
TABLE-US-00029 (SEQ ID NO: 7) Glu Leu Tyr Lys Tyr Lys Val Val Lys
Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Cys Thr Pro Tyr Asp Ile
Asn Gln Met (SEQ ID NO: 8) Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile
Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Ser Thr Pro Pro Leu Val Arg
Leu Val (SEQ ID NO: 9) Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu
Pro Leu Gly Val Ala Pro Thr Lys Ala Tyr Ala Pro Pro Ile Ser Gly Gln
Ile
[0170] CTL analysis was conducted by Mamu A*01 restricted tetramer
staining analysis at the laboratory of Dr. Norman Letvin, Harvard
Medical School.
Animals, Doses and Immunogens:
[0171] Rhesus macaques expressing the HLA-A homologue molecule Mamu
A*01 and the subtype DR.beta.0201 were identified by PCR and housed
at the colony at New Iberia, La.
[0172] The study included three groups of two juvenile Rhesus
macaques (Macaca mulatta) each described in Table 20. Group 1
consisted of two Mamu A*01 positive, DR.beta.0201 negative animals
Rh 73 and Rh 80. These animals were administered the trivalent Mamu
A*01 restricted SIV gag, env and pol CTL epitope peptides (short
peptide cocktail), together with MPL.TM. SE and GM-CSF. Group 2
consisted of two Mamu A*01 positive, DR.beta.0201 positive macaques
which received the Th/SIVCTL gag, pol and env epitope peptides
(long peptide cocktail), together with MPL.TM. SE and GM-CSF. Group
3 comprised of two Mamu A*01 negative, DR.beta.0201 positive
animals inoculated with the Th/SIVCTL gag, pol and env peptides
(long peptide cocktail). Table 20 sets forth the groups by HLA type
and peptide immunogens used.
TABLE-US-00030 TABLE 20 Animals, Doses and Immunogens Group Animal
# HLA type Peptide Immunogens 1 Rh 73, Mamu A*01 + CTL/SIV gag p11C
Rh 80 DR*.beta.0201 - (SEQ ID NO: 3) CTL/SIV pol p68A (SEQ ID NO:
4) CTL/SIV env p41A (SEQ ID NO: 5) 0.75 mg of each peptide 2 Rh 55,
Mamu A*01 + Th1/CTL/SIV gag p11C Rh 142 DR*.beta.0201 + (SEQ ID NO:
7) Th1/CTL/SIV pol p68A (SEQ ID NO: 8) Th1/CTL/SIV env p41A (SEQ ID
NO: 9) 2.4 mg of each peptide 3 Rh 41, Mamu A*01 - Th1/CTL/SIV gag
p11C Rh 47 DR*.beta.0201 + (SEQ ID NO: 7) Th1/CTL/SIV pol p68A (SEQ
ID NO: 8) Th1/CTL/SIV env p41A (SEQ ID NO: 9) 2.4 mg of each
peptide
[0173] All groups were immunized subcutaneously with 1 ml of the
respective peptide cocktail formulated in 50 .mu.g MPL.TM. SE in 1%
oil and 250 .mu.g human GM-CSF at 0, 4, and 8 weeks. The dose of
MPL.TM. SE was increased to 125 .mu.g in 1% oil for the 18 week
immunization. For all groups, 2.4 mg of each of the long peptides
and 0.75 mg of each of the short peptides were dissolved in 900
.mu.l distilled, deionized water. The peptide solution was then
used for reconstitution of the human GM-CSF, and 100 .mu.l of the
MPL.TM. SE formulation was added.
Example 24
CTL Analysis in Rhesus Macaques
[0174] Animals were bled every two weeks and heparinized blood
analyzed for Mamu A*01 restricted CTL by tetramer staining on fresh
and cultured peripheral blood mononuclear cells (PBMC) (50). PBMC
were stimulated with either p11c, p68A, p41A or p46 on day 0 and
then cultured in the presence of IL-2 and analyzed on day 11.
Standard .sup.51Cr release assay was also carried out on cultured
PBMC (50).
[0175] A tetramer assay was carried out as follows: Epitope
peptides p11c from gag, p68A from pol or p68A from pol or p41A from
env were incubated with purified biotinylated Mamu A*01 in the
presence of .beta.2 microglobulin, then attached to avidin and
conjugated to PE (phycoerythrin). This tetramer was then used for
staining macaque CD8+ cells with T cell receptors that recognize
the p11C, p68A or p41A epitopes. A different DR.beta.0201 tetramer
folded around the dominant env p46 epitope allowed for staining of
CD4+ cells that specifically recognized the p46 Th epitope. The
results are shown in Tables 21-24.
TABLE-US-00031 TABLE 21 Percent P11c/SIVgag tetramer positive CD8+
cells Weeks 0 2 4 6 8 9 10 14 Group 1 Rh 73 0.1 3.9 5.1 4.2 2.7 2.6
0.1 2.7 Rh 80 0.1 0.4 0.1 0.6 0.2 0.2 1.4 0.2 Group 2 Rh 55 0.1 3.1
4.5 5.9 4.0 4.0 4.1 2.7 Rh 142 0.2 4.7 2.5 5.4 3.9 3.9 2.5 4.1
Group 3 Rh 41 0 0.2 0.2 0.3 0.2 0.2 0.1 0.1 Rh 47 0.1 0.3 0.1 0.2
0.1 0.1 0.0 0.1
TABLE-US-00032 TABLE 22 Percent p68A/SIV CTL pol tetramer positive
CD8+ cells Weeks 0 2 4 6 8 9 10 14 Group 1 Rh 73 0.1 0.4 0.1 10.1
2.5 0.5 1.8 1.5 Rh 80 0.2 0.2 0.6 2.3 0.5 0.1 0.1 0.1 Group 2 Rh 55
0.1 1.1 1.1 5.5 5.6 1.5 11.7 6.4 Rh 142 0.2 0.6 0.2 1.0 1.8 0.3 2.3
1.2 Group 3 Rh 41 0.1 0.2 0.1 0.3 0.1 0.1 0.1 0.3 Rh 47 0.1 0.1 0.1
0.3 0.3 0.1 0.1
TABLE-US-00033 TABLE 23 Percent P41A/SIV env tetramer positive CD8+
cells Weeks 0 2 4 6 8 9 10 14 Group 1 Rh 73 0.8 3.5 2.5 2.0 3.5 1.7
1.5 1.6 Rh 80 0.2 0.2 3.4 0.5 0.1 0.0 0.2 0.2 Group 2 Rh 55 0.2 1.1
0.4 0.6 0.4 0.2 0.1 0.6 Rh 142 0.3 0.5 0.4 0.6 0.3 0.3 0.2 0.4
Group 3 Rh 41 0 0.2 0.1 0.3 0.0 0.0 0.2 Rh 47 0.1 0.2 0.1 0.2 0.2
0.1 0.0 0.2
TABLE-US-00034 TABLE 24 Percent p46/SIV T helper DR.beta.0201
tetramer positive CD4+ cells Weeks 0 2 4 6 8 9 10 14 Group 1 Rh 73
0.2 0.0 0.0 0.0 0.0 0.0 Rh 80 0.2 0.0 0.0 0.1 0.0 0.0 Group 2 Rh 55
0.2 0.5 0.7 0.4 0.3 0.3 Rh 142 0.2 0.9 0.6 1.0 0.6 0.5 Group 3 Rh
41 0.6 0.4 0.3 0.3 Rh 47 1.2 0.8 1.6 1.2
Experiment 10
Immunization of Swiss-Webster Mice with a Neisseria gonorrhoeae
Porin B Protein and Various Adjuvants
[0176] Outbred Swiss-Webster mice were divided into five groups of
ten mice each. Each group received 1 .mu.g of recombinant Porin B
protein (from strain FA1090 with 16 amino acids at the
amino-terminus from a phage, followed by the mature form of the
Porin B protein). The first group did not receive an adjuvant; the
second group received 50 .mu.g of MPL.TM.; the third group received
MPL.TM. plus 5 .mu.g GM-CSF; the fourth group received 25 .mu.g
MPL.TM. SE; the fifth group received MPL.TM. SE plus 5 .mu.g
GM-CSF. Mice were immunized subcutaneously in the rump with a total
volume of 0.2 ml, divided equally into each of two sites at the
base of the tail/rump. Immunizations were administered at week 0
and week 4.
Example 25
Reciprocal Anti-Porin B Protein IgG Endpoint Subclass Titers
[0177] Mice were bled the day prior to each immunization, and at 13
days after the final immunization. Serum was analyzed from pools
from mice within groups. Reciprocal endpoint anti-Porin B protein
IgG subclass titers were measured from pooled serum (n=10
Swiss-Webster) by vaginal lavage at week 3 and at week 6. The
results are given in Table 25. All day 0 pre-immunization titers
were less than 50.
TABLE-US-00035 TABLE 25 Reciprocal anti-Porin B Protein IgG
Endpoint Sublass Titers Week 3 Week 6 Adjuvant IgG IgG1 IgG2a IgG
IgG1 IgG2a None 4,146 531 293 157,203 4,467 9,782 MPL .TM. 3,381
171 318 431,529 23,465 20,422 MPL .TM. + 7,895 50 980 790,193 2,478
82,690 GM-CSF MPL .TM. 135,016 297 13,339 3,945,614 10,805 342,322
SE MPL .TM. 106,008 725 8,772 3,304,231 31,920 201,787 SE +
GM-CSF
[0178] Individual week 6 IgG titer geometric means against the
recombinant Porin B protein were also determined. The results are
given in Table 26.
TABLE-US-00036 TABLE 26 Individual IgG Titers Adjuvant Geometric
Mean Standard Error None 100,089 63,467 MPL .TM. 217,114 451,611
MPL .TM. + GM-CSF 649,801 353,863 MPL .TM. SE 1,917,908 1,478,357
MPL .TM. SE + GM-CSF 2,144,567 858,184
Experiment 11
Immunization of Swiss-Webster Mice with a Neisseria gonorrhoeae
Porin B Protein and Various Adjuvants
[0179] Outbred Swiss-Webster mice were divided into six groups of
five mice each. Each group received 1 .mu.g of recombinant Porin B
protein (from strain FA1090 with 16 amino acids at the
amino-terminus from a phage, followed by the mature form of the
Porin B protein). The first group did not receive an adjuvant
(protein was formulated in PBS); the second group received 40 ng of
IL-12; the third group received 50 .mu.g of MPL.TM.; the fourth
group received MPL.TM. plus 40 ng IL-12; the fifth group received
25 .mu.g MPL.TM. SE; the sixth group MPL.TM. SE plus 40 ng IL-12.
Mice were immunized subcutaneously in the rump with a total volume
of 0.2 ml. Immunizations were administered at week 0 and week
4.
Example 26
[0180] Reciprocal Anti-Porin B Protein IgG Endpoint Subclass Titers
and Vaginal Lavage IgG and IgA Titers
[0181] Mice were bled the day prior to each immunization, and at 13
days after the final immunization. Serum was analyzed from pools
from mice within groups. Reciprocal endpoint anti-Porin B protein
IgG subclass titers were measured from pooled serum (n=5
Swiss-Webster) by vaginal lavage, and IgG and IgA vaginal wash
titers were measured, each at week 3 and at week 6. The results are
given in Table 27. All day 0 pre-immunization titers were less than
50. The starting dilution for vaginal lavage analysis was 1/5.
TABLE-US-00037 TABLE 27 Reciprocal anti-Porin B Protein IgG
Endpoint Sublass Titers and Vaginal Lavage IgG and IgA Titers Week
3 Week 6 Vaginal Wash Adjuvant IgG IgG1 IgG2a IgG IgG1 IgG2a IgG
IgA None 9,084 1,872 2,872 408,944 8,314 64,500 80 5 IL-12 7,266
2,578 2,071 571,325 6,278 58,552 93 5 MPL .TM. 5,656 500 1,925
265,127 76,640 60,910 54 5 MPL .TM. + 28,274 1,442 11,348 3,747,987
120,112 44,997 88 5 IL-12 MPL .TM. SE 53,056 8,543 17,550 5,133,154
513,236 622,514 338 5 MPL .TM. SE + 757,133 5,622 33,259 10,935,000
210,478 471,552 3,036 5 IL-12
Experiment 12
Immunization of Balb/c Mice with Respiratory Syncytial Virus F
Protein and Various Adjuvants
[0182] Balb/c mice were divided into seven groups of five mice
each. Each group received 3 .mu.g of purified native Human
Respiratory syncytial virus (RSV) F protein (in the dimeric form).
The first group did not receive an adjuvant (protein was formulated
in PBS); the second group received 100 .mu.g of aluminum phosphate
(alum); the third group received 20 .mu.g of Stimulon.TM. QS-21
(Aquila Biopharmaceuticals, Inc., Framingham, Mass.); the fourth
group received 50 .mu.g MPL.TM.; the fifth group received MPL.TM.
plus 5 .mu.g GM-CSF; the sixth group received 25 .mu.g MPL.TM. SE;
the seventh group received MPL.TM. SE plus 5 .mu.g GM-CSF. Mice
were immunized intramuscularly with a total volume of 0.2 ml in the
upper thigh. Immunizations were administered at week 0 and week
4.
Example 27
Reciprocal Anti-RSV F Protein IgG Endpoint Subclass Titers
[0183] Mice were bled the day prior to each immunization, and at 13
days after the final immunization. Serum was analyzed from pools
from mice within groups. Reciprocal endpoint anti-RSV F protein IgG
subclass titers were measured from pooled serum (n=5 Balb/c). The
results are given in Table 28. All day 0 pre-immunization titers
were less than 50.
TABLE-US-00038 TABLE 28 Reciprocal anti-RSV F Protein IgG Endpoint
Sublass Titers Week 3 Week 6 Adjuvant IgG IgG1 IgG2a IgG IgG1 IgG2a
None 18,452 3,698 319 539,156 3,119,905 80,855 Alum 66,710 35,839
4,321 5,417,001 3,226,833 291,474 Stimulon .TM. QS- 313,665 150,988
176,080 12,113,156 2,902,521 4,324,004 21 MPL .TM. 124,197 28,134
11,882 3,310,838 900,863 1,057,108 MPL .TM. + 419,873 91,649 65,453
10,343,803 753,890 688,554 GM-CSF MPL .TM. SE 374,992 44,115
147,366 19,333,189 1,493,284 6,314,264 MPL .TM. SE + 1,748,272
51,966 267,265 30,816,193 1,716,850 2,641,258 GM-CSF
Example 28
Spleen Cell Proliferation
[0184] Spleen cell proliferation in response to in vitro
stimulation with 2.5 .mu.g/ml RSV F protein and various adjuvant
formulations (the same as in Example 27) was measured. Spleen cells
were harvested at 14 days post-secondary immunization and were
established in culture at a density of 5.times.10.sup.5 cells.
Cells were cultured for a total of 96 hours. .sup.3H-thymidine was
added to cultures for the last 18 hours. Data are presented as a
proliferation index normalized to cells stimulated in culture with
ConA ([mean cpm Antigen/mean cpm ConA]-[mean cpm medium/mean cpm
ConA]).times.100. As a result, cells cultured in medium have a
background proliferation of 0. The results are given in Table
29.
TABLE-US-00039 TABLE 29 Spleen cell proliferation Adjuvant
Normalized Proliferation Index None 18.1 Alum 13.1 Stimulon .TM.
QS-21 0.8 MPL .TM. 0.4 MPL .TM. + GM-CSF 20.0 MPL .TM. SE 17.8 MPL
.TM. + SE GM-CSF 16.3
Experiment 13
Immunization of Balb/c Mice with Respiratory Syncytial Virus F
Protein and Various Adjuvants
[0185] The protocol of Experiment 12 was repeated (immunizations at
week 0 and week 4 with RSV F protein with or without various
adjuvants).
Example 29
Reciprocal Anti-RSV F Protein IgG Endpoint Subclass Titers
[0186] Mice were bled the day prior to each immunization, and at 13
days after the final immunization. Serum was analyzed from pools
from mice within groups. Reciprocal endpoint anti-RSV F protein IgG
subclass titers were measured from pooled serum (n=5 Balb/c). The
results are given in Table 30. All day 0 pre-immunization titers
were less than 50.
TABLE-US-00040 TABLE 30 Reciprocal anti-RSV F Protein IgG Endpoint
Sublass Titers Week 3 Week 6 Adjuvant IgG IgG1 IgG2a IgG IgG1 IgG2a
None 6,442 2,808 713 5,195,059 963,203 38,791 Alum 128,695 36,841
1,975 4,285,993 567,972 27,668 Stimulon .TM. QS- 528,036 296,292
176,703 37,221,721 1,823,402 1,724,319 21 MPL .TM. 104,702 21,930
61,253 6,153,833 1,384,927 955,685 MPL .TM. + 262,128 79,888 55,249
21,054,796 3,412,710 2,070,305 GM-CSF MPL .TM. SE 184,246 47,194
180,932 31,731,335 4,376,601 6,406,591 MPL .TM. SE + 375,575 70,422
289,542 27,079,086 2,124,043 6,341,497 GM-CSF
Example 30
Spleen Cell CTL Activity
[0187] Spleen cell CTL (cytotoxic T-lymphocyte) activity as a
consequence of immunization with RSV F protein and the indicated
adjuvants was assessed two weeks after the final immunization. Data
represent the percent specific CTL activity of spleen cells
cultured with RSV-infected target cells at an effector to target
cell ratio of 33:1. Percent specific CTL activity was determined as
in Example 24, subtracting the CTL activity against non-infected
targets from that of activity specific for RSV-infected target
cells. Naive spleen cells were infected with RSV at an MOI
(multiplicity of infection) of 1.5 for two hours as a source of in
vitro stimulator cells. Responder cells from the spleens of
immunized mice were added to simulator cells at a ratio of 5:1, and
cultured for six days. On day 5, target cells (Balb/C MHC-H-2d cell
line) were infected with RSV at an MOI of 10 for two hours and
incubated overnight. On day 6, infected and non-infected target
cells were harvested and pulsed with .sup.51Cr. In vitro effector
cells were then added to target cells at an E:T ratio ranging from
100:1 to 3:1. Chromium release was measured after four hours of
incubation. The results are given in Table 31.
TABLE-US-00041 TABLE 31 Spleen cell CTL activity Adjuvant Percent
CTL Activity None 1 Alum 4 Stimulon .TM. QS-21 53 MPL .TM. 6 MPL
.TM. + GM-CSF 15 MPL .TM. SE 30 MPL .TM. + SE GM-CSF 36
Experiment 14
Immunization of Balb/c Mice with Respiratory Syncytial Virus F
Protein and Various Adjuvants
[0188] Balb/c mice were divided into six groups of five mice each.
Each group received 3 .mu.g of purified native RSV F protein (in
the dimeric form). The first group did not receive an adjuvant
(protein was formulated in PBS); the second group received 40 ng
IL-12; the third group received 50 .mu.g MPL.TM.; the fourth group
received MPL.TM. plus 40 ng IL-12; the fifth group received 25
.mu.g MPL.TM. SE; the sixth group received MPL.TM. SE plus 40 ng
IL-12. Mice were immunized subcutaneously with a total volume of
0.2 ml in the rump, divided equally between two does given on
either side of the tail. Immunizations were administered at week 0
and week 4.
Example 31
Reciprocal Anti-RSV F Protein IgG Endpoint Subclass Titers
[0189] Mice were bled the day prior to each immunization, and at 13
days after the final immunization. Serum was analyzed from pools
from mice within groups. Reciprocal endpoint anti-RSV F protein IgG
subclass titers were measured from pooled serum (n=5 Balb/c). The
results are given in Table 32. All day 0 pre-immunization titers
were less than 50.
TABLE-US-00042 TABLE 32 Reciprocal anti-RSV F Protein IgG Endpoint
Sublass Titers Week 3 Week 6 Adjuvant IgG IgG1 IgG2a IgG IgG1 IgG2a
None 5,332 12,925 500 2,381,899 977,782 76,226 IL-12 13,557 3,442
500 4,459,059 1,345,099 65,951 MPL .TM. 26,179 55,767 8,397
3,467,097 402,128 170,252 MPL .TM. + 186,516 22,321 10,800
1,546,443 420,322 253,465 IL-12 MPL .TM. SE 1,708,358 53,608
144,876 9,075,480 565,403 1,000,459 MPL .TM. SE + IL- 329,050
15,788 69,794 10,935,000 386,639 1,284,274 12
Example 32
Spleen Cell CTL Activity
[0190] Spleen cell CTL activity as a consequence of immunization
with RSV F protein and the indicated adjuvants was assessed two
weeks after the final immunization. Data represent the percent
specific CTL activity of spleen cells cultured with RSV-infected
target cells at an effector to target cell ratio of 33:1. Percent
specific CTL activity was determined as in Example 24, subtracting
the CTL activity against non-infected targets from that of activity
specific for RSV-infected target cells. Naive spleen cells were
infected with RSV at an MOI of 1.5 for two hours as a source of in
vitro stimulator cells. Responder cells from the spleens of
immunized mice were added to simulator cells at a ratio of 5:1, and
cultured for six days. On day 5, target cells (Balb/C MHC-H-2d cell
line) were infected with RSV at an MOI of 10 for two hours and
incubated overnight. On day 6, infected and non-infected target
cells were harvested and pulsed with .sup.51Cr. In vitro effector
cells were then added to target cells at an E:T ratio ranging from
100:1 to 3:1. Chromium release was measured after four hours of
incubation. The results are given in Table 33.
TABLE-US-00043 TABLE 33 Spleen cell CTL activity Adjuvant Percent
CTL Activity None 6 IL-12 22 MPL .TM. 15 MPL .TM. + IL-12 13 MPL
.TM. SE 33 MPL .TM. + SE IL-12 28
Experiment 15
Immunization of Balb/c Mice with the Influenza Virus Nucleocapsid
Protein and Various Adjuvants
[0191] Balb/c mice were divided into six groups of five mice each.
Each group received 1 .mu.g of the Influenza virus NP
(nucleocapsid) protein from the A/dorn/307/72 strain. [check
groups] The first group did not receive an adjuvant (peptide was
formulated in PBS); the second group received 100 .mu.g of aluminum
phosphate (alum); the third group received 50 .mu.g of MPL.TM.; the
fourth group received MPL.TM. plus 5 .mu.g GM-CSF; the fifth group
received 25 .mu.g MPL.TM. SE; the sixth group received MPL.TM. SE
plus 5 .mu.g GM-CSF. Mice were immunized subcutaneously in the rump
with a total volume of 0.2 ml. Immunizations were administered at
week 0 and week 4.
Example 33
Spleen Cell CTL Activity
[0192] Spleen cell CTL activity as a consequence of immunization
with the influenza NP peptide and the indicated adjuvants was
assessed two weeks after the final immunization. The assessment was
carried out following the procedure of Example 32 using
peptide-pulsed target p815 cells (the peptide corresponded to amino
acids 147-155 of NP and had the sequence: Thr Tyr Gln Arg Thr Arg
Ala Leu Val (SEQ ID NO:14). The inclusion of GM-CSF in the
formulations containing MPL.TM. or MPL.TM. SE resulted in a marked
reduction of CTL activity (data not shown).
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Sequence CWU 1
1
14140PRTHuman immunodeficiency virus 1Lys Gln Ile Ile Asn Met Trp
Gln Glu Val Gly Lys Ala Met Tyr Ala 1 5 10 15Cys Thr Arg Pro Asn
Tyr Asn Lys Arg Lys Arg Ile His Ile Gly Pro 20 25 30Gly Arg Ala Phe
Tyr Thr Thr Lys 35 40239PRTHuman immunodeficiency virus 2Lys Gln
Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala 1 5 10
15Thr Arg Pro Asn Tyr Asn Lys Arg Lys Arg Ile His Ile Gly Pro Gly
20 25 30Arg Ala Phe Tyr Thr Thr Lys 3539PRTSimian immunodeficiency
virus 3Cys Thr Pro Tyr Asp Ile Asn Gln Met 1 549PRTSimian
immunodeficiency virus 4Ser Thr Pro Pro Leu Val Arg Leu Val 1
559PRTSimian immunodeficiency virus 5Tyr Ala Pro Pro Ile Ser Gly
Gln Ile 1 5620PRTSimian immunodeficiency virus 6Glu Leu Tyr Lys Tyr
Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala 1 5 10 15Pro Thr Lys
Ala 20729PRTSimian immunodeficiency virus 7Glu Leu Tyr Lys Tyr Lys
Val Val Lys Ile Glu Pro Leu Gly Val Ala 1 5 10 15Pro Thr Lys Ala
Cys Thr Pro Tyr Asp Ile Asn Gln Met 20 25829PRTSimian
immunodeficiency virus 8Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu
Pro Leu Gly Val Ala 1 5 10 15Pro Thr Lys Ala Ser Thr Pro Pro Leu
Val Arg Leu Val 20 25929PRTSimian immunodeficiency virus 9Glu Leu
Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala 1 5 10
15Pro Thr Lys Ala Tyr Ala Pro Pro Ile Ser Gly Gln Ile 20
251042PRTUnknown OrganismDescription of Unknown Organism Human
amyloid peptide protein 10Asp Ala Glu Phe Arg His Asp Ser Gly Tyr
Glu Val His His Gln Lys 1 5 10 15Leu Val Phe Phe Ala Glu Asp Val
Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val
Val Ile Ala 35 401128PRTUnknown OrganismDescription of Unknown
Organism Human amyloid peptide protein 11Asp Ala Glu Phe Arg His
Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15Leu Val Phe Phe
Ala Glu Asp Val Gly Ser Asn Lys 20 251210PRTHuman immunodeficiency
virus 12Arg Gly Pro Gly Arg Ala Phe Val Thr Ile 1 5 101310PRTHuman
immunodeficiency virus 13Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr 1
5 10149PRTInfluenza virus 14Thr Tyr Gln Arg Thr Arg Ala Leu Val 1
5
* * * * *