Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02697993 2010-02-26
METHODS OF ENHANCING ADJUVANTICITY OF VACCINE
COMPOSITIONS
CROSS-REFERECE TO RELATED APPLICATIONS
This application claims the priority benefit of U.S. Provisional Patent
Application No. 60/968,731, filed August 29, 2007, which is incorporated
herein by
reference in its entirety.
INTRODUCTION
The fundamental purpose of a vaccine is to provide lasting immunity against
a pathological condition. Ideally vaccines provide functionally active
antibodies,.
elicit cell-mediated immunity, and activate T- and B-lymphocytes with highly
specific reactivity as well as "memory" to provide protection against further
encounters with a pathogen.
Adjuvants are vaccine additives which nonspecifically augment the immune
response. The mechanism by which adjuvants enhance the immune system varies
widely. Adjuvants may be classified as "immunomodulatory" or "antigen delivery
systems." Immunomodulatory adjuvants prime the immune system by regulating
the action of immune cells by altering lymphokine production. Antigen delivery
systems, on the other hand, function to deliver the antigen to the appropriate
immune cells. In addition, adjuvants may enhance the speed or duration of an
immune response, modulate antibody avidity, specificity, isotype or subclass
distribution, stimulate cell mediated immunity, promote mucosal immunity, or
enhance the immune responses in immunologically immature or senescent
individuals. Adjuvants can affect either the humoral or cell-mediated immune
response, or a combination of both.
Immunomodulation refers to the ability of an adjuvant to alter the
lymphokine response by activating differential subsets of inunune cells. Two
major
subsets of CD4+ T lymphocytes, Thl and Th2, play a major role in determining
the
immune response. Thl responses typically induce complement fixing antibody and
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strong delayed-type hypersensitivity (DTH) reactions and are associated with -
)-IFN,
IL-2, and IL-12, while Th2 responses result in high circulating and secretory
antibody levels and are associated with cytokines IL-4, IL-5, IL-6 and IL-10.
Activation of Thl cells also regulates the cellular immune response by
activating
the proliferation of cytotoxic CD8+ T cells and enhanced cytolysis of target
cells.
Lipid species have been investigated for adjuvant properties. Unlike peptide
antigens, lipids are processed and presented to the immune system by the CD1
family of (32 microglobulin-associated molecules. CD1 molecules have evolved
to
capture and process both foreign and self lipid antigens for display to
particular
subsets of T cells. The CD1 presentation pathway triggers.both innate and
adaptive
immune response by activating two complementary CD1 restricted T cell subsets;
natural killer T (NKT) cells that perform adjuvant functions; and non-NKT T-
cells
capable of helper or cytolytic functions. NKT cells express both natural
killer (NK)
cell surface markers and a conserved semi-invariant T-cell receptor (TCR),
Va24-
Ja18N38 in mice and Va24-Ja18Na11 in humans. Accordingly, NKT cells play
an important role in a number of immune functions, including antimicrobial
responses, antitumor immunity, and regulation of the balance between tolerance
and
autoimmunity.
A number of natural and synthetic lipid molecules are processed by antigen-
presenting cells and presented by CD 1 molecules to NKT cells. The
prototypical
compound used to study NKT cell activation in vitro and in vivo is KRN7000,
and
u-galactosyceramide ("aGalCer") derived from marine sponge Agelas mauritianus.
Additional compounds recently identified iinclude isoglobotrihexosylceramide
("iGB3") which is an endogenous glycolipid and PBS-57, a modified 6"amino 6"
deoxygalactosyceramide, as described in PCT Application PCT/US07/66250, the
disclosure of which is incorporated herein by reference. These compounds
activate
NKT cells and upregulate cytokine responses in vitro. However, in the context
of in
vivo vaccinations, little is known regarding the effectiveness of lipid
adjuvanticity
for these compounds.
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Few adjuvants have been approved for use in human vaccines due to toxic
side effects. The most common adjuvants are aluminum and oil adjuvants, but
they
are known to induce side effects such as fever, headache, muscle aches and
pains or
rash. Other disadvantages include a strong local stimulation of the immune
system
leading to pain, redness, swelling or a small lump at the site of injection.
Adjuvants
also have complicated methods of vaccine preparation and may fail to increase
immunogenicity of weak antigens. At present, the choice of adjuvants for human
vaccination reflects a compromise between the requirement for adjuvanticity
and an
acceptable level of side effects. There is a need for new adjuvants with fewer
side
effects that still elicit long-lasting protective immunity.
SUMMARY OF INVENTION
The inventors have discovered that the addition of a synthetic glycolipid,
designated PBS-57, to a vaccine preparation administered to a subject
activates both
a humoral and cellular immune response when administered to the subject.
Accordingly, the invention provides methods of enhancing the immunogenicity of
a
vaccine in a subject, stimulating a humoral inunune response, stimulating a
CD4+ T
cell response and stimulating a CD8+ cytotoxic T cell response in a subject by
co-
administering PBS-57 and a vaccine to a subject.
BRIEF DESCRIPTION OF FIGURES
FIG. 1 is a graph showing the percentage of NKT cells on day 7 in a CD3+
T cell population of cultured peripheral blood lymphocytes (PBL) treated with
various amounts of adjuvant glycolipids aGalCer, PBS20, PBS25 and PBS57.
FIG. 2 is a graph showing the percentage of NKT cells on day 7 in a CD3+
T cell population of cultured PBLs treated with various amounts of aGalCer,
PBS25, PBS57 and PBS83.
FIG. 3 is a graph showing an increase in NKT cells in in vitro cultures of
PBLs isolated from volunteers treated with aGalCer, PBS20, PBS25, PBS57 and
PBS83. The median value is depicted above the graphs.
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FIG. 4A is a graph depicting the percentage of TCRce cells within the
spleen of mice 24 hours after administration of aGalCer, PBS57, and PBS83.
FIG.
4B is a graph of the percentage of NKT cells within the TCRoqS population in
the
spleen of mice 24 hours after administration of the adjuvant glycolipids. The
two-
tailed p value was calculated for each adjuvant compared to the control.
FIG. 5A is a graph of the percentage of CD40+ cells in the CDl lc+ CD8a-
cell population from the spleen of mice 24 hours after intravenous
administration of
adjuvant glycolipids. FIG. 5B is a graph of the percentage of CD40+ cells in
the
CD11 c+ CD8a+ cell population from the spleen of mice 24 hours after
intravenous
administration of adjuvant glycolipids. FIG. 5C is a graph of the percentage
of
CD80+ cells in the CDllc+ CDBcK cell population from the spleen of mice 24
hours after intravenous administration of adjuvant glycolipids. FIG. 5D is a
graph
of the percentage of CD80+ cells in the CDl1c+ CD8ca+- cell population from
the
spleen of mice 24 hours after intravenous administration of adjuvant
glycolipids.
FIG. 5E is a graph of the percentage of CD86+ cells in the CDllc+ CD8cK- cell
population from the spleen of mice 24 hours after intravenous administration
of
adjuvant glycolipids. FIG. 5F is a graph of the percentage of CD86+ cells in
the
CD11 c+ CD8ca+ cell population from the spleen of mice 24 hours after
intravenous
administration of adjuvant glycolipids.
FIG. 6 is a dot plot and a histogram of results of flow cytometric CFSE
staining from an assay to determine the specific lysis of antigen presenting
cells for
mice immunized with ovalbumin (Ova) alone or Ova and aGalCer.
FIG. 7 is a graph depicting the percentage of specific lysis of Ova-specific
target cells in the spleen of mice inununized with adjuvant, with or without
Ova.
FIG. 8 is a graph depicting the percentage of specific lysis of Ova-specific
target cells in the blood of mice injected intravenously ("IV") with Ova in
combination with different concentrations of aGalCer.
FIG. 9 is a graph depicting the percentage of specific lysis of Ova-specific
target cells in the blood of mice injected IV with Ova in combination with
different
concentrations of PBS57.
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FIG. 10 is a graph depicting the percentage of specific lysis of Ova-specific
target cells in the blood of mice injected IV with PBS57 in combination with
different concentrations of Ova.
FIG. 11 is a graph depicting the percentage of specific lysis of Ova-specific
target cells in the blood of mice injected intramuscularly (IM) with Ova in
combination with different concentrations of aGalCer.
FIG. 12 is a graph depicting of the percentage of specific lysis of Ova-
specific target cells in the blood of mice injected IM with Ova in combination
with
different concentrations of PBS57.
FIG. 13 is a graph comparing the percentage of specific lysis of Ova-specific
target cells in the blood of mice injected either IV or IM with different Ova
and
adjuvant combinations.
FIG. 14 is a bar graph depicting enhanced antibody production by mice
inununized with a tetanus toxoid (TT) vaccine composition containing PBS-57.
FIG. 15 is a bar graph depicting enhanced CD8+ T cell responsiveness in
mice immunized intramuscularly with ovalbumin (OVA) in combination with
PBS57.
FIG. 16 is a bar graph depicting enhanced CD8+ T cell responsiveness in
mice immunized subcutaneously with OVA in combination with PBS57.
DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
Adjuvants enhance the immunogenicity of antigens in vaccine preparations
in a variety of ways. In the case of toxins, a good humoral immune response is
required. In the case of intracellular bacteria, a cell-mediated response,
mediated
mainly by cytotoxic T cells and Thl cells, is important. In the case of viral
infections, both humoral and cellular responses are fundamental to control the
infection. The ability of an adjuvant to enhance not only the humoral but also
the
cell-mediated immune response increases the likelihood of developing long-
lasting
immunity. An effective adjuvant also would be useful for combination with a
wide
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variety of antigens. The inventors have found that a glycosphingolipid, PBS-
57, has
the ability to stimulate both a cell-mediated and humoral immune response in
vivo.
In addition, PBS-57 is able to stimulate an immune response against a weak
nominal antigen to produce antibodies and simultaneously provide for cell-
mediated
lysis of cells expressing specific surface antigens.
In one embodiment, the invention provides a method of enhancing the
immunogenicity of a vaccine in a subject by co-administering PBS57 and the
vaccine. As used herein, a"subject" is a mammal, e.g., a mouse, more suitably
a
human. "Enhancing the immunogenicity of a vaccine" refers to the ability of
PBS-
57 to enhance the humoral and/or cell mediated immune response of a subject to
a
vaccine in relation to a suitable control. For purposes of determining whether
immunogenicity is enhanced relative to a control, a quantitative comparison of
the
signal in a sample from a subject vaccinated with antigen and PBS57 can be
compared to the signal in a sample from a subject vaccinated with antigen
alone.
As used herein, immunogenicity may be measured by any assay used by those of
skill in the art to measure the humoral or cell-mediated immune response. For
example, the imrnunogenicity may be measured using an ELISA assay for various
cytokine levels, the performance of which is routine to those skilled in the
art.
In particular embodiments, the immune response is enhanced at least 25%,
at least 30%, at least 50%, at least 60%, at least 65%, at least 70%, at least
75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least
150%, at
least 200%, or at least 400%, relative to a suitable control. A suitable
control may
be a subject treated with a vaccine composition not including PBS57. Percent
enhancement may be calculated using the following formula:
[(value representing subject's immune response after treatment with
composition containing PBS57)-(value representing immune response of
control)/(value representing subject's immune response after treatment with
composition containing PBS57)]x100.
As used herein the term "co-administration" or "co-administering" refers to
administration of at least the adjuvant and the vaccine concurrently, i.e.,
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simultaneously in time, or sequentially, i.e., administration of an adjuvant,
followed
by administration of the vaccine. That is, after administration of the
adjuvant, the
vaccine can be administered substantially immediately after the adjuvant or
the
vaccine can be administered after an effective time period after the adjuvant;
the
effective time period is the amount of time given for realization of maximum
benefit from the administration of the adjuvant. Alternatively, the adjuvant
and
vaccine may be co-formulated.
Vaccine compositions are suitably formulated to include PBS-57.
"Vaccine" refers to a composition which, when administered to a subject,
induces
cellular or humoral immune responses as described herein. Vaccine compositions
may include an antigen or combinations of antigens; the antigen may be a
polypeptide or carbohydrate moiety, or combinations thereof, for example, a
glycoprotein. The antigen is suitably derived from an infectious agent (e.g.,
a
pathogenic microorganism), a tumor, an endogenous molecule (e.g., a "self'
molecule), or, for purposes of study, a nominal antigen, such as ovalbumin
(referred
to herein as "Ova"). Vaccine compositions may also include killed or
attenuated
infectious agents. The vaccine compositions used in conjunction with the
invention
suitably include PBS57 and an antigen. The structure of PBS-57 is shown below:
0
0
OH NH
( CH Z)UC H=C H( CH 2)7C H 3
O Hbi
OH
HO
OH C1aH2s
OH H
PBS57 activates NKT cells in vitro and in vivo. PBS57 contains an amide group
at
the C6 position of the galactose and a cis-double bond in the acyl chain of
the
ceramide portion. Not to be bound by theory, the double bond in the ceramide
side-
chain is thought to facilitate binding to the groove of the CDld molecule and
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increase the solubility of glycosphingolipid. PBS57 has also been shown to
induce
the release of INF-y and IL-4 in vitro.
Vaccine compositions including PBS57 may be formulated using a variety
of preparative methods and inactive ingredients known to those of skill in the
art.
See Remington's Pharmaceutical Sciences, Mack Publishing Co., (2000), which is
incorporated herein by reference. The vaccine may also contain a suitable
antigen
delivery system to target the antigen to immune cells. Suitable antigen
delivery
systems are known in the art, and include, but are not limited to, MVA
(Modified
virus ankara), adenovirus, lentivirus, translocated subunit of pertussis or
shiga toxin,
or antigen encapuslated liposomes. Suitable effective dosage amounts of PBS57
in a
vaccine compositions may be determined by those of skill in the art, but
typically
range from about 1 microgram to about 10,000 micrograms per kilogram of body
weight, although they are typically about 1,000 micrograms or less per
kilogram of
body weight. In some embodiments, the effective dosage amount ranges from
about
10 to about 5,000 micrograms per kilogram of body weight. In another
embodiment, the effective dosage amount ranges from about 50 to about 1,000
micrograms per kilogram of body weight. In another embodiment, the effective
dosage amount ranges from about 75 to about 500 micrograms per kilogram of
body
weight. For purposes of study, a suitable dosage for a mouse is I g PBS57 per
100 1 dose. The PBS57 composition can be administered in a single dose, or may
be administered in multiple doses over a period of weeks or months.
One or more antigens may be included in the compositions with PBS57, or
may be formulated independently. As used herein, an antigen refers to a
molecule
that stimulates an immune response. It will be appreciated that the dosage of
antigen will depend on the specific antigen, and on the age and immune status
of the
subject, as well as other relevant factors that may be determined by those
skilled in
the art.
Suitably, antigens are derived from attenuated or killed infectious agents.
Whole microorganisms or portions thereof (e.g., membrane ghosts; crude
membrane
preparations, lysates and other preparations of microorganisms) may be
utilized.
Suitable infectious agents from which an antigen may be derived include, but
are
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not limited to, pathogens and microorganisms such as bacteria, parasites and
viruses. In some contexts, suitable antigens are obtained or derived from a
viral
pathogen that is associated with human disease including, but not limited to,
HIV/AIDS (Retroviridae, e.g., gp120 molecules for HIV-1 and HIV-2 isolates,
HTLV-I, HTLV-11), influenza viruses (Orthomyxoviridae, e.g., types A, B and
C),
herpes (e.g., herpes simplex viruses, HSV-1 and HSV-2 glycoproteins gB, gD and
gH), rotavirus infections (Reoviridae), respiratory infections (parainfluenza
and
respiratory syncytial viruses), Poliomyelitis (Picornaviridae, e.g.,
polioviruses,
rhinoviruses), measles and mumps (Paramyxoviridae), Rubella (Togaviridae,
e.g.,
rubella virus), hepatitis (e.g., hepatitis viruses types A, B, C, D, E and/or
G),
cytomegalovirus (e.g., gB and gH), gastroenteritis (Caliciviridae), Yellow and
West
Nile fever (Flaviviridae), Rabies (Rhabdoviridae), Korean hemorrhagic fever
(Bunyaviridae), Venezuelan fever (Arenaviridae), warts (Papillomavirus),
simian
immunodeficiency virus, encephalitis virus, varicella zoster virus, Epstein-
Barr
virus, and other virus families, including Coronaviridae, Birnaviridae and
Filoviridae.
Suitable bacterial and parasitic antigens can also be obtained or derived from
known agents responsible for diseases including, but not limited to,
diphtheria,
pertussis, tetanus, tuberculosis, bacterial or fungal pneumonia, otitis media,
gonorrhea, cholera, typhoid, meningitis, mononucleosis, plague, shigellosis or
salmonellosis, Legionnaires' disease, Lyme disease, leprosy, malaria,
hookworm,
Onchocerciasis, Schistosomiasis, Trypanosomiasis, Leishmaniasis, giardiases,
amoebiasis, filariasis, Borrelia, and trichinosis. Still further antigens can
be
obtained or derived from unconventional pathogens such as the causative agents
of
kuru, Creutzfeldt-Jakob disease (CJD), scrapie, transmissible mink
encephalopathy,
and chronic wasting diseases, or from proteinaceous infectious particles such
as
prions that are associated with mad cow disease.
Additional specific pathogens from which antigens can be derived include
M. tuberculosis, Chlamydia, N. gonorrhoeae, Shigella, Salmonella, Vibrio
cholerae, Treponema pallidum, Pseudomonas, Bordetella pertussis, Brucella,
Francisella tularensis, Helicobacter pylori, Leptospira interrogans,
Legionella
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pneumophila, Yersinia pestis, Streptococcus (types A and B), pneumococcus,
meningococcus, Haemophilus influenza (type b), Toxoplasma gondii, Moraxella
catarrhalis, donovanosis , and actinomycosis; fungal pathogens include
candidiasis
and aspergillosis; parasitic pathogens include Taenia, flukes, roundworms,
amebiasis, giardiasis, Cryptosporidium, Schistosoma, Pneumocystis carinii,
trichomoniasis and trichinosis. The present invention can also be used to
provide a
suitable immune response against numerous veterinary diseases, such as foot-
and-
mouth diseases, coronavirus, Pasteurella multocida, Helicobacter, Strongylus
vulgaris, Actinobacillus pleuropneumonia, Bovine Viral Diarrhea Virus (BVDV),
Klebsiella pneumoniae, E. coli, and Bordetella pertussis, parapertussis and
brochiseptica.
In other embodiments, antigens for inclusion in vaccine compositions that
may be used in the present methods are tumor-derived antigens or autologous or
allogeneic whole tumor cells. Suitably, the tumor antigen is a tumor specific
antigen (TSA) or a tumor associated antigen (TAA). Several tumor antigens and
their expression patterns are known in the art and can be selected based on
the
tumor type to be treated. Non-limiting examples of tumor antigens include cdk4
(melanoma), 6-catenin (melanoma), caspase-8 (squamous cell carcinoma), MAGE-1
and MAGE-3 (melanoma, breast, glioma), tyrosinase (melanoma), surface Ig
idiotype (e.g., BCR) (lymphoma), Her-2/neu (breast, ovarian), MUC-1 (breast,
pancreatic) and HPV E6 and E7 (cervical carcinoma). Additional suitable tumor
an tigens include prostate specific antigen (PSA), sialyl Tn (STn), heat shock
proteins and associated tumor peptides (e.g., gp96), ganglioside molecules
(e.g.,
GM2, GD2, and GD3), Carcinoembryonic antigen (CEA) and MART-1.
As appreciated by skilled artisans, vaccines are suitably formulated to be
compatible with the intended route of administration. Examples of suitable
routes
of administration include parenteral, e.g., intravenous, intradermal,
subcutaneous,
intramuscular, oral (e.g., inhalation), transdermal (topical), intranasal,
interperitoneal, transmucosal, and rectal administration. The vaccine may also
include a physiologically acceptable vehicle. A "physiologically acceptable"
vehicle is any vehicle that is suitable for in vivo administration (e.g.,
oral,
CA 02697993 2010-02-26
transdermal or parenteral administration) or in vitro use, i.e., cell culture.
Suitable
physiologically acceptable vehicles for in vivo administration include water,
buffered solutions and glucose solutions, among others. Additional components
of
the compositions may suitably include excipients such as stabilizers,
preservatives,
diluents, emulsifiers or lubricants, in addition to the physiologically
acceptable
vehicle and the antigen. In particular, suitable excipients include, but are
not
limited to, Tween 20, DMSO, sucrose, L-histadine, polysorbate 20 and serum.
Another embodiment of the invention is a method of stimulating a humoral
immune response to an antigen. The method includes co-administering PBS-57 and
the antigen to a subject, as described above. As used herein, a "humoral
immune
response" is the production of antibodies by B cells, and the accessory
process that
accompanies it, including, but not limited to, e.g., Th2 activation and
cytokine
production, germinal center formation and isotype switching, affinity
maturation
production and memory cell generation. For purposes of determining whether a
humoral immune response is activated, a quantitative comparison of the singal
in a
sample from a subject vaccinated with antigen and PBS57 can be compared to a
sample from a subject vaccinated with antigen alone. The humoral immune
response may be evaluated by measuring the effector functions of antibodies,
including pathogen or toxin neutralization, classical compliment activation,
and
opsonin promotion of phagocytosis and pathogen elimination. The antibodies
produced in response to co-administering PBS-57 and an antigen may be of any
type, e.g., IgM, IgA, or IgG. The humoral immune response may be assayed by
any
quantitative method known in the art, e.g., ELISA, single radial
immunodiffusion
assay (SRID), enzyme immunoassay (EIA), or hemagglutination inhibition assay
(HAI).
A further embodiment of the invention is a method of activating CD4+ T
lymphocytes in a subject. As understood in the art, CD4+ T cells, or "T helper
cells," are cells that recognize antigens presented by class II major
histocompatability marker (MHC) on the surface of antigen presenting cells,
and
secrete lymphokines to stimulate both cell-mediated and antibody-mediated
branches of the immune system. CD4+ T cell activation promotes lymphokine
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secretion, immunoglobulin isotype switching, affinity maturation of the
antibody
response, macrophage activation and enhanced activity of natural killer (NK)
and
cytotoxic T cells (CTL). Lymphokines are proteins secreted by lymphocytes that
affect their own activity and/or the activity of other cells. Lymphokines
include, but
are not limited to, interleukins and cytokines, e.g., IL-2, IL-4, IL-5, IL-6,
IL-10, IL-
12, or INF~: For purposes of determining whether a CD4+ T lymphocytes are
activated, a quantitative comparison of the signal in a sample from a subject
vaccinated with antigen and PBS57 can be compared to a sample from a subject
vaccinated with antigen alone. Methods to assay activation CD4+ T cells are
known in the art.
Another embodiment of the invention is a method of activating CD8+ T
lymphocytes in a subject. CD8+ T lymphocytes recognize antigens presented by
Class I MHC molecules (present on all nucleated cells). Engagement of the MHC
class I-peptide complex results in delivery of lytic granules to the target
cell causing
lysis of the target cell. Methods used to assay the activation of CD8+ T cells
are
known in the art, including but not limited to ELISPOT, ELISA, and
cytotoxicity
assays. As used herein, a mouse model is used to monitor the activation of
CD8+ T
cells using a fluorescent assay to measure cell-mediated cytotoxicity, as
described in
Hermans et al, 2004, Journal of Immunologic Methods, 285:25-40, incorporated
by
reference in its entirety. In this assay, mice are immunized on day 0 with the
vaccine with or without PBS57. Syngeneic target cells are created by isolating
splenocytes from a second set of mice and labeling the cells with two separate
cell-
labeling fluorescent dyes or high and low concentrations of a single
fluorescent dye,
e.g., CFSE or CMTMR. One set of target cells is loaded with an antigen-
specific
peptide while a second set of target cells is loaded with an irrelevant
peptide. The
two target cell populations are mixed in equal amounts and injected into
immunized
mice. 24 hours later, mice are sacrificed, and splenocytes and blood samples
are
obtained. The level of each set of target cells is analyzed by flow cytometry.
Activation of CD8+ lymphocytes is determined by comparing the number of target
cells in a sample vaccinated with antigen and PBS57 to the number of target
cells in
a sample from a subject vaccinated with antigen alone.
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Other aspects of the invention will become apparent by consideration of the
following non-limiting examples and accompanying drawings.
EXAMPLES
Example 1: PBS57 activation of NKT cells in vitro
To determine whether PBS-57 is able to stimulate NKT cells in culture,
expansion of NKT cells in culture of peripheral blood lymphocytes (PBL) was
measured. PBLs were derived from two healthy donors and grown in RPMI cell
culture media with 5% AB serum. Cells were plated on 12-well plates at 2 x106
PBL/welU2m1. Wells were treated with negative controls (0.05% Tween20 and 1%
DMSO in PBS or 0.05% Tween20 and 10% DMSO in PBS) or the test adjuvants
(aGalCer, PBS-20, PBS-25, or PBS-57) at a final concentration of 10 ng/ml, 100
ng/ml or I g/m1. The chemical structures for control compounds aGalCer, PBS-
20
and PBS25 are shown below:
HD H
O
HO O
HN OH
Ho o EXatMaW~7o3.09 PBS20
MOL WL:704A3
OH
HO H
O o~/'~~~ E%A~t Aass 80S I0
no HN OH MaL Wt:908.8d
HOO i
Oli
PBS-25
OH
HO 0
HO 0
HN OH
HO aGalCer
aGi OH
All cultures were supplemented with recombinant human IL2 (rhIL-2) to
obtain final concentration of 100UI/ml by day 1. All cells were cultured for
10 days
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at 37 C under 5% COZ. On day 7, half the culture medium (1 ml) was removed for
NKT analysis. Percentage of NKT cells in the CD3+ population was detected by
flow cytometry using fluorescently labeled CD1 tetramers or fluorescently
labeled
antibodies (anti-CD3, anti-V/311, and anti NKT). The NKT population was
identified by flow cytometry as the V(3l1+, Tetramer+ and TCRa 3+ population.
NKT cell percentage was calculated in each sample by subtracting the Vfll 1+
PBS-
57-tetramer+ TCRc43+ from the V(311+ empty-tetramer+ TCRc43+. FIG. 1 shows
the increase of NKT cells as after treatment with PBS-57, aGalCer, PBS-25 and
PBS-20. As shown, treatment of PBLs with PBS57 or aGalCer leads to an
expansion of NKT cells.
In a separate experiment, the ability of PBS-57 compared to other candidate
glycolipids to induce in vitro expansion of NKT cells was measured. Peripheral
blood lymphocytes were derived from two healthy donors and cultured in RPMI
with 5% AB serum. Cells are plated in a 12-well plate with 2 x 106 cells per
well
per 2 ml media. One well received carrier (0.05% Tween2O and 1% DMSO in
PBS) as a negative control, and test wells received aGalCer, PBS-25, PBS-57,
or
PBS-83 at final concentrations of 1 ng/ml, 10 ng/ml and 100 ng/ml. The
structure
of PBS-83 is shown below:
HOHN~
,( O O Chwrooal Formup- C~N~Op
HO_a~~rcQai HN Ettact Mab5:868.45
~ FAoUwllai W amght: 86a.C
P63-8S om
All cultures were supplemented with rhIL-2 to obtain a final concentration of
100
UI/ml by day 1, and cultured for 7 days at 37 C under 5% COZ conditions. At
day
7, the percentage NKT cells was detected by flow cytometry. FIG. 2
demonstrates
the percentage of NKT cells on day 7 for the different glycolipid treatment
conditions. Treatment with PBS57 resulted in at least a two fold increase in
NKT
cells over cells treated with aGalCer.
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Example 2: Expansion of NKT cells in cultures treated with PBS57
NKT staining was performed on a panel of fourteen healthy donor peripheral
blood lymphocytes (PBL) or peripheral blood monocyte (PBMC) samples. PBLs
were isolated from 12 subjects, and PBMCs were isolated from 2 subjects. Each
isolated cell sample was split into two samples. The first sample from each
donor
was stained with 10 l anti-VQ11 FITC (Beckman Coulter), 10 l anti-TCRC43-PC5
(Beckman Coulter), and 2 l PE-labeled PBS-57-CD 1 d-tetramer. The second
sample from each donor was stained with 10 l V/311, 10 l anti-TCRc43 -FITC,
and
2 l PE-labeled empty-CDld-tetramer.
To determine a percentage increase in NKT cells, the number of NKT cells
on day 7 was divided by the number of NKT cells on day 0. As shown in FIG. 3,
incubation with PBS57 results in a substantial increase in NKT cells in PBL
cultures from human volunteers.
Example 3: Analysis of TCR ft, NKT cell populations and dendritic cell
maturation in mice administered PBS-57
To test for adjuvant activity of PBS57 in vivo, mice were injected with test
compounds and assayed for cell activation and expansion at 24 hours. Four
groups
of five C57B1/6J mice were each intravenously administered phosphate buffered
saline (PBS) with carrier (DMSO alone), 0.97 Ag of aGalCer, 1.3 g of PBS57,
or
0.97 itg of PBS83 in a total of 100 l PBS. 24 hours after administration of
the test
compound, the mice were sacrificed and blood samples and splenocytes were
isolated by standard methods. Percentage of NKT cells in the spleen was
evaluated
by flow cytometry with staining using anti-TCRag-FITC and PE-labeled CDId-
tetramer loaded with PBS-57 (PBS57-tetramer) or without lipid (empty-
tetramer).
FIG. 4A shows the increase in percentage of TCRoq'i cells in mice treated with
caGalCer or with PBS57 compared with control mice treated with PBS and carrier
alone. The percentage of NKT cells was determined by subtracting the
percentage
of PBS57-tetramer+ cells from the percentage of empty-tetramer+ cells in the
TCRaO population. FIG. 4B shows the decrease in percentage of NKT cells in the
spleen of mice treated with PBS57 or aGalCer relative to the control. Taken
together, the result shown in FIG. 4A and FIG. 4B demonstrate that PBS57
CA 02697993 2010-02-26
increased the number of TCRoo cells in the spleen of mice as effectively as
aGalCer. The loss of NKT cells in the mice injected with aGalCer or PBS57
relative to control demonstrates a down-expression of TCR on the NKT cell
surface.
Dendritic cells (DCs) were detected in the splenocytes by flow cytometry by
staining with anti-CDl lc R-PE (BD Pharmingen) and anti-CD8cx FITC (Beckman
Coulter). Two subsets of dendritic cells, CD11c+CD8c~- and CD11c+CD8a+
populations, were analyzed further. A decrease of 50% of both subsets of DCs
was
seen in mice treated with PBS57 or aGalCer adjuvants compared to the control.
To
determine if administration of glycolipid induced maturation of the two
subsets of
dendritic cells, the cells were stained with anti-CD40 Biotin/Stepta APC (BD
Bioscience), anti-CD80 Biotin (BD Pharmingen) /Stepta APC (BD Bioscience) and
anti-CD86 Biotin (BD Pharmingen)/Stepta APC (BD Bioscience) antibodies and
analyzed by flow cytometry. FIG. 5A and 5B show the percentage of CD40
positive cells in both the CD11c+CD8cK and CD11c+CD8a+ cells, respectively.
FIG. 5C and 5D shows the percentage of CD80 positive cells in both the
CD11c+CD8cx- and CDl lc+CD8a+ cell populations, respectively. FIG. 5E and 5F
shows the percentage of CD86-positive cells in both CD11c+CD8u- and
CD11c+CD8a+ cells, respectively. Horizontal lines represent the averages.
Taken
collectively, the data show that PBS57 and aGalCer both substantially
increased the
percentage of CD40+, CD80+ and CD86+ expressing cells in both the
CD 11 c+CD8a- and CD 11 c+CD8cx+ populations.
Example 4: Protocol for testing adjuvanticity of PBS-57 in mouse model
A mouse model was used to test the in vivo specific cytotoxic T cell
response (CD8+) elicited by PBS57 in combination with antigen. C57B1/6J
CD45.2 female mice were immunized on day 0 with antigen (Ovalbumin, Ova,
grade VII, Sigma, St. Louis, MO) with or without adjuvant, adjuvant alone, or
carrier alone (control) in a total of 100 l PBS. Test compounds were 1 g
PBS57,
1 g aGalCer, 1 g PBS83 with or without 50 g Ova. Syngeneic target cells
were
prepared by isolating splenocytes from a second set of C57B1/6J CD45.2 female
mice and labeling the cells with either low concentration (0.6 M over 10 min
at
37 C) or high concentration (6 itM over 10 minutes at 37 C) of CFSE
(fluorescent
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dye). The population labeled with high concentration CFSE was pre-loaded with
5
M SIINFEKL peptide (Ova-specific peptide, NeoMPS, Inc, San Diego, CA) over
60 minutes at 37 C. The population labeled with low concentration CFSE was pre-
loaded with 5 l LCMV gp33-41 peptide (non-Ova peptide, NeoMPS, Inc, San
Diego, CA) over 60 minutes at 37'C. Equal numbers of both populations of
target
cells were mixed (1x107 cells of low or high concentration CFSE, 2x107 total
per
100 l) and injected intravenously into each of the immunized mice on day 10.
Mice were sacrificed at day 11, and spleen cells and blood samples from the
orbital
sinus were collected. The mean percentage survival of the peptide-pulsed
target
cells (CFSE labeled) were calculated relative to the control population by
flow
cytometric analysis. FIG. 6B depicts typical flow cytometric data showing the
loss
of Ova-specific peptide loaded target cells. The cytotoxic activity was
expressed as
a percent specific lysis, calculated by subtracting the mean percent survival
of the
Ova-specific target cells from 100. FIG. 7 depicts the percentage of specific
lysis of
target cells in the spleen of immunized mice. Only the combination of Ova and
PBS57 produced cytotoxic lysis of Ova-specific target cells in the spleen.
Example 5: Cytotoxic response after immunization with Ova with varying
concentrations of txGalCer
The in vivo cytotoxicity induced by the CD8+ T cell response to Ova-
peptide loaded target cells was evaluated as described in Example 4 for
varying
aGalCer concentrations. Eleven groups of three mice were intravenously
immunized with 100 l total of PBS alone, 50 g Ova alone, I g of aGalCer
alone,
50 g of Ova with 1 g, 100 ng, 10 ng, 1000 pg, 100 pg, 10 pg, or 0.1 pg
aGalCer.
On day 10, mice were injected intravenously with CFSE-labeled target cells. On
day
11, blood samples were collected from the orbital sinus of cach mouse. The
mean
percent survival and cytotoxic activity was determined as described above.
FIG. 8
depicts the percent specific lysis for each group of mice for the specified
dosages of
aGalCer. The results show that aGalCer with antigen is able to generate a dose
dependent specific cytotoxic effect when injected intraveneously.
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Example 6: Cytotoxic response after immunization with Ova with varying
concentrations of PBS57 intraveneously
The in vivo cytotoxicity induced by CD8+ T cell response stimulated with
different concentrations of PBS-57 of Ova-peptide loaded CFSE-labeled target
cells
was evaluated as described in Example 4. Eleven groups of three mice were
intravenously immunized with 100 l total of PBS alone, 50 g Ova alone, 50 g
Ova with 10 ug PBS57, I g PBS57, 100 ng PBS57, 10 ng PBS57, 1 ng PBS57,
100 pg PBS57, 10 pg PBS57, 1 pg PBS57, or 0.1 pg PBS57. On day 10, mice were
injected intravenously with target cells, and on day 11, blood samples were
collected from the orbital sinus. The mean percent survival and cytotoxic
activity
was determined as described above. FIG. 9 shows the percentage specific lysis
in
the blood for each group of mice immunized under each dosage of PBS57. The
results show that PBS-57 in combination with Ova was able to induce a dose-
dependent specific cytotoxic effect when injected intravenously.
Example 7: Cytotoxic response after immunization with PBS57 with varying
concentrations of Ova intravenously
The cytotoxic response of CD8+ cells against different concentrations of
ovalbumin with constant concentrations of PBS57 was evaluated as described in
Example 4. Ten groups of three mice were injected intravenously on day 0 with
100 l total in PBS of 50 g Ova alone,l. g PBS57 alone, 1 g PBS57 with 50
g
Ova, 10 g Ova, 1 g Ova, 100 ng Ova, 10 ng Ova, 1 ng Ova, 100 pg Ova, or 10
pg
Ova. On day 10, mice were injected with target cells intravenously and on day
11,
mice were sacrificed and blood samples were collected from the orbital sinus
and
spleen cells were isolated. The mean percentage of survival and cytotoxic
activity
was calculated as described above. FIG. 10 shows the percentage of specific
lysis
for each group of mice immunized with each dosage of Ova. The results show the
cytotoxic response to vaccination with both antigen and PBS-57 is dependent on
antigen concentration.
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Example 8: Cytotoxic response after immunization with ceGalCer with varying
concentrations of Ova intramuscularly
The cytotoxic response of CD8+ cells against different concentrations of
ovalbumin with constant concentrations of ckGalCer injected intramuscularly
was
evaluated as described in Example 4. Fourteen groups of three mice were
injected
intramuscularly in the hind left leg on day 0 with 100 iCl total in PBS of 400
g Ova
alone, 1 g aGalCer alone, or 400 g Ova with I g aGalCer, 0.5 g aGalCer,
0.1
g aGalCer, 50 ng aGalCer, 10 ng aGalCer, 5 ng a.GalCer, 1 ng aGalCer, 500 pg
aGalCer, 100 pg ctiGalCer, 50 pg aGalCer or 10 pg aGalCer. On day 10, mice
were
injected intraveneously with target CFSE stained cells, and on day 11 mice
were
sacrificed and blood samples were collected from the orbital sinus were
obtained.
Specific lysis of the SIINFEKL-loaded target cells was monitored by flow
cytometry. The mean percent survival and cytotoxic activity was calculated as
described in Example 4. FIG. 11 shows the percentage of specific lysis in the
blood for each group of mice immunized under each dosage of aGalCer. The
results show that caGalCer is not able to generate a specific cytotoxic T cell
response
when injected with the Ova antigen intramuscularly, as seen by very low
specific
lysis over a broad range of caGalCer concentrations from l g-100pg. These
concentrations of aGalCer elicited a cytotoxic response when injected
intravenously
at I g-10 ng, but did not elicit a cytotoxic response upon intramuscular
administration.
Example 9: Cytotoxic response after immunization with PBS57 with varying
concentrations of Ova intramuscularly
The cytotoxic response of CD8+ cells against intramuscular injection of
PBS57 was analyzed as described in Example 4. Fourteen groups of three mice
were injected intramuscularly in the hind left leg on day 0 with 100 1cl total
in PBS
of PBS alone, 400 g Ova alone, 1itg PBS57 alone, or 400 g Ova with 1 g
PBS57, 0.5 g PBS57, 0.1 Ag PBS57, 50 ng PBS57, 10 ng PBS57, 5 ng PBS57, I
ng PBS57, 500 pg PBS57, 100 pg PBS57, 50 pg PBS57, or 10 pg PBS57. On day
10, mice were injected intravenously with target CFSE labeled cells, and blood
samples from the orbital sinus on day 11. The mean percentage of survival and
cytotoxic activity was determined as described in Example 4. FIG. 12 shows the
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CA 02697993 2010-02-26
specific lysis of the dosage curves of mice immunized intramuscularly with
PBS57
and Ova. The results show that PBS-57 can elicit a specific cytotoxic response
when injected both intravenously and intramuscularly, unlike aGalCer which can
only elicit a cytotoxic response when injected intravenously.
Example 10: Comparison of the cytotoxic response after intramuscular or
intravenous immunization with Ova and PBS57 or cxGalCer
The cytotoxic response to Ova with PBS57 or Ova with aGalCer upon
intravenous and intramuscular administration was evaluated as described in
Example 4. Eight groups of mice were injected on day 0 as follows:
1) 3 mice injected IM with 400 g Ova in 50 l PBS;
2) 3 mice injected by N with 400 g Ova into 50 l PBS;
3) 6 mice injected IM with 400 g Ova and I jig aGalCer in 50 jil PBS;
4) 3 mice injected IV with 400 g Ova and 1 g aGalCer in 50 l PBS;
5) 6 mice injected IM with 400 g Ova and 1 g aGalCer in 50 l PBS;
6) 3 mice injected N with 400 /.tg Ova and 1 g aGalCer in 50 l PBS;
7) 6 mice injected IM with 400 jig Ova and llCg PBS57 in 50 l PBS;
and
8) 3 mice injected N with 400 g Ova and 1 g PBS57 into 50 1 PBS.
Mice were injected with CFSE labeled target cells on day 10. Mice were
sacrificed on day 11 and blood samples were collected. The mean percentage of
survival and cytotoxic activity was determined as described in Example 4. FIG.
13
compares the percentage of specific lysis using different routes of
administration.
The results confirm that aGalCer did not induce a cytotoxic response when
injected
intramuscularly, while PBS57 caused a cytotoxic response regardless of the
route of
administration.
Example 11: PBS57 boosts antibody response to tetanus toxoid in vivo
To determine if the addition of PBS57 to tetanus toxoid immunization
resulted in an enhanced immune response, a group of six mice were immunized
CA 02697993 2010-02-26
intramuscularly at day 0 and day 15 with 10 g tetanus toxoid (TT) or 10 g
tetanus
toxoid in combination with I g PBS57. IgG titers were determined by standard
methods from blood samples drawn on day 0, 15 and 30. The antibody titer was
determined by tetanus toxoid speicific ELISA. As seen in FIG. 14, PBS57
enhanced the antibody response to TT.
Example 12: Comparison of the CD8+ T cell response after intramuscular
immunization with Ova alone or in combination with PBS57
A mouse model was used to test the in vivo specific T cell response (CD8+)
elicited by PBS57 in combination with antigen. C57B1/6J CD45.2 female mice
were immunized intramuscularly on day 0 and 14 with antigen (Ovalbumin, Ova,
grade VII, Sigma, St. Louis, MO) with or without adjuvant, adjuvant alone, or
carrier alone (control) in a total of 100 .l PBS carrier. Test adjuvant was 1
g
PBS57 with or without 50 g Ova. Each group included at least three mice.
Blood
was collected from each mouse at day 21 and isolated cells were subjected to
pentamer staining with H2Kb-SIINFEKL pentamers after blocking the Fc receptor.
FACS analysis was performed including only CD8+ cells in the analysis (CD19+
cells were excluded from the analysis to exclude any B cells). FIG. 15 depicts
typical flow cytometric data showing an increase in Ova-specific CD8+ T cells
after
intramuscular injection with PBS57 and Ova as compared to Ova alone. The
results
are reported as mean +/- standard deviation of H2Kb-SIINFEKL specific CD8+ T
cells in the blood.
Example 13: Comparison of the CD8+ T cell response after subcutaneous
immunization with Ova alone or in combination with PBS57 or aGalCer
A mouse model was used to test the in vivo specific T cell response (CD8+)
elicited by PBS57 in combination with antigen as compared to that of aGalCer.
C57B1/6J CD45.2 female mice were immunized subcutaneously on day 0 and 14
with antigen (Ovalbumin, Ova, grade VII, Sigma, St. Louis, MO) with or without
adjuvant, adjuvant alone, or carrier alone (control) in a total of 100 l PBS
carrier.
Test adjuvant was 1 g PBS57 or aGalCer with or without 50 jug Ova. Each group
included at least three mice. Blood was collected from each mouse at day 21
and
isolated cells were subjected to pentamer staining with H2Kb-SIINFEKL
pentamers
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CA 02697993 2010-02-26
after blocking the Fc receptor. FACS analysis was performed including only
CD8+
cells in the analysis (CD 19+ cells were excluded from the analysis to exclude
any B
cells). FIG. 16 depicts typical flow cytometric data showing an increase in
Ova-
specific CD8+ T cells after intramuscular injection with PBS57 and Ova as
compared to Ova alone or as compared to immunization with aGalCer and Ova.
The results are reported as mean +/- standard deviation of H2K6-SIINFEKL
specific
CD8+ T cells in the blood.
While the compositions and methods of this invention have been described
in terms of exemplary embodiments, it will be apparent to those skilled in the
art
that variations may be applied to the compositions and methods and in the
steps or
in the sequence of steps of the methods described herein without departing
from the
concept, spirit and scope of the invention. More specifically, it will be
apparent that
certain agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or similar results
would
be achieved. All such similar substitutes and modifications apparent to those
skilled
in the art are deemed to be within the spirit, scope and concept of the
invention. In
addition, all patents and publications listed or described herein are
incorporated in
their entirety by reference.
As used in this specification and the appended claims, the singular forms
"a," "an," and "the" include plural referents unless the content clearly
dictates
otherwise. Thus, for example, reference to a composition containing "a
polynucleotide" includes a mixture of two or more polynucleotides. It should
also
be noted that the term "or" is generally employed in its sense including
"and/or"
unless the content clearly dictates otherwise. All publications, patents and
patent
applications referenced in this specification are indicative of the level of
ordinary
skill in the art to which this invention pertains. All publications, patents
and patent
applications are herein expressly incorporated by reference to the same extent
as if
each individual publication or patent application was specifically and
individually
indicated by reference. In case of conflict between the present disclosure and
the
incorporated patents, publications and references, the present disclosure
should
control.
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CA 02697993 2010-02-26
It also is specifically understood that any numerical value recited herein
includes all values from the lower value to the upper value, i.e., all
possible
combinations of numerical values between the lowest value and the highest
value
enumerated are to be considered to be expressly stated in this application.
23
