U.S. patent application number 14/429123 was filed with the patent office on 2015-09-10 for vaccine compositions and methods of use.
The applicant listed for this patent is Frank BEDU-ADDO, Eric JACOBSON, Kenya JOHNSON. Invention is credited to Frank Bedu-Addo, Eric Jacobson, Kenya Johnson.
Application Number | 20150250872 14/429123 |
Document ID | / |
Family ID | 50341992 |
Filed Date | 2015-09-10 |
United States Patent
Application |
20150250872 |
Kind Code |
A1 |
Bedu-Addo; Frank ; et
al. |
September 10, 2015 |
VACCINE COMPOSITIONS AND METHODS OF USE
Abstract
The present disclosure provides vaccine compositions comprising
at least one adjuvant and at least one antigen, wherein the
adjuvant is a cationic lipid. The disclosure also provides methods
of treating a disease in a mammal, methods of preventing a disease
in a mammal, and methods of effecting antigen cross presentation to
induce a humoral immune response and a cellular immune response in
a mammal utilizing the vaccine compositions. Cross presentation of
various antigens can be achieved by formulating the specific
antigens with cationic lipids possessing adjuvant properties.
Inventors: |
Bedu-Addo; Frank; (Bethel,
CT) ; Jacobson; Eric; (Cincinnati, OH) ;
Johnson; Kenya; (Mason, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BEDU-ADDO; Frank
JACOBSON; Eric
JOHNSON; Kenya |
Bethel,
Cincinnati,
Mason, |
CT
OH
OH |
US
US
US |
|
|
Family ID: |
50341992 |
Appl. No.: |
14/429123 |
Filed: |
September 23, 2013 |
PCT Filed: |
September 23, 2013 |
PCT NO: |
PCT/US2013/061132 |
371 Date: |
March 18, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61703814 |
Sep 21, 2012 |
|
|
|
Current U.S.
Class: |
424/210.1 ;
424/209.1; 424/234.1; 424/274.1 |
Current CPC
Class: |
A61K 39/02 20130101;
Y02A 50/412 20180101; A61P 37/00 20180101; A61P 31/12 20180101;
A61K 39/39 20130101; A61K 2039/70 20130101; A61P 31/10 20180101;
A61K 2039/55555 20130101; A61K 39/145 20130101; C12N 2760/16034
20130101; A61P 31/16 20180101; A61K 39/0005 20130101; C12N
2760/16134 20130101; A61K 2039/55511 20130101; A61K 39/0002
20130101; C12N 2760/16234 20130101; A61K 39/12 20130101; Y02A 50/30
20180101; A61P 31/04 20180101 |
International
Class: |
A61K 39/39 20060101
A61K039/39; A61K 39/145 20060101 A61K039/145; A61K 39/00 20060101
A61K039/00; A61K 39/12 20060101 A61K039/12; A61K 39/02 20060101
A61K039/02 |
Claims
1. A vaccine composition comprising at least one adjuvant and at
least one pathogenic antigen, wherein the adjuvant is a cationic
lipid.
2. The vaccine composition of claim 1 wherein the cationic lipid is
a non-steroidal cationic lipid.
3. The vaccine composition of claim 1 wherein the cationic lipid is
selected from the group consisting of DOTAP, DOTMA, DOEPC, and
combinations thereof.
4. The vaccine composition of claim 1 wherein the adjuvant is an
enantiomer of the cationic lipid.
5. The vaccine composition of claim 4 wherein the enantiomer is
R-DOTAP or S-DOTAP.
6. The vaccine composition of claim 1 wherein one or more antigens
is a viral antigen.
7. The vaccine composition of claim 1 wherein one or more antigens
is a bacterial or fungal antigen.
8. The vaccine composition of claim 1 wherein at least one antigen
is an antigen from a conserved region of a pathogen.
9. The vaccine composition of claim 8 wherein the vaccine
composition is a universal vaccine.
10. The vaccine composition of claim 7 wherein the vaccine
composition is an influenza vaccine, and wherein the influenza
vaccine comprises a glycoprotein antigen found on the surface of an
influenza virus.
11. The vaccine composition of claim 10 wherein the antigen is a
hemagglutinin antigen.
12. The vaccine composition of claim 11 wherein the hemagglutinin
antigen comprises an epitope region HA.sub.818-526.
13. The vaccine composition of claim 10 wherein the influenza
vaccine is a neuraminidase subunit vaccine.
14. A method of effecting antigen cross presentation to induce a
humoral immune response and a cellular immune response in a mammal,
said method comprising the step of administering an effective
amount of a vaccine composition to the mammal, wherein the vaccine
composition comprises at least one adjuvant and at least one
antigen, and wherein the adjuvant is a cationic lipid.
15. The method of claim 14 wherein the humoral immune response is
an antibody response.
16. The method of claim 14 wherein the cellular immune response is
a T cell response.
17. The method of claim 16 wherein the T cell response is a CD 8+ T
cell response.
18. The method of claim 14 wherein the cationic lipid is a
non-steroidal cationic lipid.
19. The method of claim 14 wherein the cationic lipid is selected
from the group consisting of DOTAP, DOTMA, DOEPC, and combinations
thereof.
20. The method of claim 14 wherein the adjuvant is an enantiomer of
a cationic lipid.
21-30. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 USC
.sctn.119(e) of U.S. Provisional Application Ser. No. 61/703,814,
filed on Sep. 21, 2012, the entire disclosure of which is
incorporated herein by reference.
TECHNICAL FIELD
[0002] Despite an increasing amount of research and interest in the
field of immunology, there is currently a lack of vaccines that are
adequately effective against various infectious pathogens or
diseases such as malaria, HIV, hepatitis C, influenza, and
tuberculosis. For example, current influenza vaccines induce
antibodies against two main surface proteins from the virus,
hemagglutinin and neuraminidase. Thus, current influenza vaccines
only effectively protect against infection by strains of the virus
that express versions of these proteins present in the vaccine.
However, these two surface proteins frequently change as a
consequence of mutations and re-assortment. Accordingly, influenza
vaccines must be reformulated each year to contain the
hemagglutinin and neuraminidase surface proteins of the newly
formed virus strains.
[0003] Moreover, influenza virus infections, especially pandemic
strains such as H1N1 and H5N1, represent an ever increasing global
health risk. The risk is significantly greater in the elderly and
in persons with chronic diseases, often leading to higher mortality
rates in these patient populations. Vaccination has been a
successful means of controlling disease. However, due to the
potentially limited availability of vaccines in a pandemic due to
current methods of production, and also the limited efficacy in the
elderly, more efficient production methods as well as more
effective influenza vaccines are being sought. Influenza and other
vaccines against infectious pathogens that will be effective
against multiple strains of the pathogens, referred to as
"universal vaccines" are actively being sought. Furthermore,
efficacy of the current influenza vaccines varies significantly.
Due to the health risks associated with pandemic strains of
influenza in particular, safe and effective adjuvants that are
compatible with influenza antigens and which can enable effective
dose sparing of current antigen stocks are also being actively
sought.
[0004] Pathogens such as malaria, HIV, hepatitis C, and
tuberculosis are intracellular, requiring the induction of strong
cellular immunity (including cytotoxic responses (CTL)) to remove
the infected cells. It is well established that the development of
antibody responses can be stimulated by traditional adjuvants such
as alum and Freund's adjuvant. It is also well established that
some adjuvants can elicit T-cell responses when formulated with
T-cell epitope peptides. However, most current adjuvants lack the
ability, when formulated with whole proteins or with viral or
bacterial subunit vaccines (as well as live and attenuated virus
vaccines), to internalize and process the antigens for presentation
via both MHC Class I and Class II to induce both cellular and
antibody mixed immune responses. It is now understood that many
vaccines will need to stimulate both humoral and cellular immune
responses to be adequately effective. Co-generation of MHC class I
restricted CD8+Tcells is now known to be essential for vaccines
aimed at viral and other intracellular infections. Accordingly, an
obstacle exists for developing vaccines that are based on
attenuated pathogens and non-living vectors containing recombinant
antigens, as it is necessary for such agents to access both MHC
class I- and class II-restricted pathways of antigen
processing.
[0005] In particular, methods to improve the protective efficacy of
subunit and live pathogen vaccines against various bacterial and
viral pathogens by enabling "cross-presentation" involving the
processing of an exogenous protein through the class I and class II
processing pathways are highly desirable. Enabling of
"cross-presentation" through the class I and class II processing
pathways yields both antibody and T-cell responses.
BACKGROUND AND SUMMARY OF THE INVENTION
[0006] As described above, immunity has been difficult to induce
against the proteins found in emerging strains of influenza, such
as those in H5N1 viruses that cause avian flu. It is commonly
believed that difficulties occur partly because of the existence of
memory cells that can recognize annual, but not new, viral strains.
A primary response is required, however, to protect against newly
emerging virus strains as they are more antigenically distinct from
the annual influenza strains. Such a primary response usually
requires the addition of an adjuvant.
[0007] Addition of adjuvants (e.g., MF59, AS03, or aluminum salts)
to influenza vaccines increases antibody titers and persistence.
However, these approaches do not provide cross-reactivity to
distinct subtypes of the virus. CD8+ T cells recognize less
variable parts of the virus and could provide a more cross-reactive
response that could be induced by new vaccines.
[0008] There has been a recent shift in the focus of influenza
vaccine development, as well as vaccines for other pathogens,
towards the generation of memory CD8+ T cells that may be able to
provide more cross-reactive protection. As mentioned above, the
antigens that CD8+ T cells recognize are found in less variable
portions of the virus. Several approaches have been attempted. For
example, peptides recognized by CD8+ T cells have been combined
with a lipid moiety, Pam-2-Cys, that activates a TLR on DCs to
prime protective CD8+ T cells. This vaccine generates protective
CD8+ T cells that migrate to the lung when administered via
intranasal delivery.
[0009] CD8+ T cells are specific to detect agents, such as viruses,
that invade the cytoplasm, and the requirements for presentation of
antigen to CD8+ T cells differ from those for the CD4 helper
T-cells. Antigens are transported to the cell surface by molecules
encoded in the MHC. Internalized antigen is carried to the cell
surface by MHC class II, which promotes activation of CD4+ T-cells.
In contrast, endogenous antigen reaches the cell surface by MHC
class I, which activates CD8+ T-cells. To activate cytotoxic
T-cells (CD8+), antigen internalized by DCs must cross to the MHC
class I pathway before reappearing on the cell surface, a process
known as cross-presentation, for which specific subsets of DCs are
specialized. Adjuvant systems that are able to activate antigen
cross-presentation are actively being sought and are essential in
the development of new generation vaccines.
[0010] Several other infections, such as hepatitis, HIV, and
malaria, for example, exist for which antibodies provide
insufficient protection. In these cases, both humoral immunity,
mediated by antibodies, and cell-mediated immunity, which depends
on cytotoxic T cells or T cells that activate immune cells by means
of cytokines, may be required for effective protection.
[0011] Dendritic cells (DCs) are the primary antigen-presenting
cells in the initiation of T cell responses, and are therefore a
major target for adjuvant use. In the presence of an infection,
signals are sent to DCs directly by pattern-recognition receptors
(PRRs) for microbial constituents, and indirectly by inflammatory
cytokines released by other innate immune cells that recognize
microbial constituents. These signals induce maturation of the DCs
and their migration to secondary lymphoid organs where they are
able to interact with and activate naive T cells. DC maturation
involves increased processing of microbial proteins and their
presentation to T cells on major histocompatibility complex (MHC)
molecules.
[0012] Some adjuvants have been demonstrated to activate signals
that induce T helper cell (T.sub.H1) responses, characterized by
IFN.gamma.-producing T helper cells that activate antimicrobial
effects at the effector site. Adjuvants such as the saponins drive
T.sub.H1 responses and are believed to work by inducing IL-12 in
DCs. Aluminum salts, however, do not directly induce signaling
through TLRs and do not stimulate IL-12 production by DCs. Instead,
aluminum-based adjuvants have been found to drive T.sub.H2
responses.
[0013] Adjuvants work by various mechanisms and the ability to
effect cross-presentation is ultimately dependent on the adjuvant's
mechanism. Some mechanisms by which an adjuvant effect is achieved
include retention of the antigen locally at the site of injection
to produce a slow-release depot effect, thus enabling sustained
release of the antigen to the antigen presenting cells. Adjuvants
can also at as chemo-attractants to attract cells of the immune
system to the antigen depot and subsequently stimulate such cells
to elicit immune responses. The most commonly used adjuvant to date
has been Alum (Aluminum hydroxide and aluminum phosphate). Most
adjuvants including Alum are effective in only enhancing the
antibody responses to antigens. Adjuvants such as MPL can activate
antibody responses, and when formulated with T-cell epitope
peptides, have also been demonstrated to elicit CTL responses.
[0014] As described above, although some adjuvants such as the
cationic lipids and MPL can elicit T-cell responses when formulated
with peptides, the use of peptide fragments rather than whole
antigens is a severe limitation because different peptide fragments
are recognized by the T cells of different individuals. As a
result, a very large number of different fragments would have to be
identified and included in such a vaccine. In addition, the ability
of peptides to elicit protective antibody responses is known to be
weak and non-existent with several peptides.
[0015] A promising approach is to induce CTL to internal proteins
such as NP which are highly conserved among different viruses.
Hemagglutinin (HA) T cell epitopes also show less variation than
antibody epitopes. However, existing inactivated vaccines like
Fluzone consist of mostly HA protein and yet do not generate
significant CD8 T cell responses.
[0016] The killing of infected cells by both CTLs and T.sub.H1
cells is reported to be effective in clearing an infection due to
an intracellular pathogen. However, in certain cases, (e.g.
infection of the liver by the hepatitis B virus),
IFN.gamma.-producing CD8+ T cells offer more effective protection
because the virus can be cleared with minimal host cell death.
Similarly, IFN.gamma.-producing CD8+ T cells are shown to be
associated with protection in individuals vaccinated with the RTS,
S malaria vaccine. This vaccine contains a protein from the
parasite fused to a surface protein from the hepatitis B virus. It
is reported that both humoral and cell-mediated immunity targeting
multiple antigens expressed at different stages of the parasite's
lifecycle are required for protection against malaria infection.
The adjuvant system used in the most successful malarial vaccine is
AS02, a combination adjuvant preparation that contains both a
saponin adjuvant component and the TLR agonist MPL formulated in a
particulate system. Notably, both the saponin and MPL adjuvants
together were required to induce cross presentation and hence a
modest level of protection in immunized individuals. In contrast,
however, vaccines using the same antigen with aluminum hydroxide
and MPL (AS04) or in an oil-in-water emulsion (AS03) induced high
levels of antibody but failed to protect against infection.
[0017] Although live attenuated viral and bacterial vaccines can
activate all arms of the immune system, adjuvants have so far not
reached this goal. By combining adjuvants, such as aluminum salts
with MPL, or by using prime-boost strategies with DNA and then
viral or bacterial vectors, both humoral and cell-mediated
responses can potentially be activated. However, such multiple
adjuvant systems are complex and have the potential for formulation
and safety difficulties.
[0018] Therefore, there exists a need for new vaccine compositions
that effectively induce broadly cross-protective immunity to
different subtypes of a pathogen, for example an influenza virus.
Moreover, new and effective methods of treating and preventing
disease, such as those caused by bacteria, viruses, and fungi are
also very desirable. Accordingly, the present disclosure provides
vaccine compositions and method of using the compositions that
exhibit desirable properties and provide related advantages for
cross-presentation of one or more antigens and wherein a humoral
and/or a cellular immune response is achieved.
[0019] The present disclosure provides vaccine compositions
comprising at least one adjuvant and at least one antigen, wherein
the adjuvant is a cationic lipid. The disclosure also provides
methods of treating a disease in a mammal, methods of preventing a
disease in a mammal, and methods of A method of effecting antigen
cross presentation to induce a humoral immune response and a
cellular immune response in a mammal utilizing the vaccine
compositions. Cross presentation of various antigens can be
achieved by formulating the specific antigens with cationic lipids
possessing adjuvant properties.
[0020] The vaccine compositions and methods according to the
present disclosure provide several advantages compared to other
compositions and methods in the art. First, the vaccine
compositions can induce broadly cross-protective immunity to
different subtypes of influenza, as well as development of a
universal influenza vaccine that can provide protection against
multiple influenza strains.
[0021] Second, the vaccine compositions demonstrate strong
increases in both humoral and cell-mediated responses and can
provide a simple adjuvant platform for developing a new generation
of simple vaccines that do not require adjuvant combinations or
viral vectors. This approach to eliciting "cross-presentation" in
the development of anti-viral and anti-bacterial vaccines could
provide a novel and cost effective approach to the development of
vaccines that provide improved protection and cure of various
diseases.
[0022] Third, the influenza vaccine compositions can provide a new
approach to developing a universal influenza vaccine without the
need for the use of multiple T-cell epitope peptides due to the
enhanced cellular CD8+ T-cell response to the HA protein and
resulting "cross-reactivity" among strains in which the CD8 T-cell
epitopes are known to be conserved.
[0023] The following numbered embodiments are contemplated and are
non-limiting:
[0024] 1. A vaccine composition comprising at least one adjuvant
and at least one antigen, wherein the adjuvant is a cationic
lipid.
[0025] 2. The vaccine composition of clause 1 wherein the cationic
lipid is a non-steroidal cationic lipid.
[0026] 3. The vaccine composition of clause 1 or clause 2 wherein
the cationic lipid is selected from the group consisting of DOTAP,
DOTMA, DOEPC, and combinations thereof.
[0027] 4. The vaccine composition of any one of clauses 1 to 3
wherein the cationic lipid is DOTAP.
[0028] 5. The vaccine composition of any one of clauses 1 to 3
wherein the cationic lipid is DOTMA.
[0029] 6. The vaccine composition of any one of clauses 1 to 3
wherein the cationic lipid is DOEPC.
[0030] 7. The vaccine composition of any one of clauses 1 to 6
wherein the adjuvant is an enantiomer of the cationic lipid.
[0031] 8. The vaccine composition of clause 7 wherein the
enantiomer is purified.
[0032] 9. The vaccine composition of clause 7 or clause 8 wherein
the enantiomer is R-DOTAP or S-DOTAP.
[0033] 10. The vaccine composition of clause 7 or clause 8 wherein
the enantiomer is R-DOTAP.
[0034] 11. The vaccine composition of clause 7 or clause 8 wherein
the enantiomer is S-DOTAP.
[0035] 12. The vaccine composition of clause 7 or clause 8 wherein
the enantiomer is R-DOTMA or S-DOTMA.
[0036] 13. The vaccine composition of clause 7 or clause 8 wherein
the enantiomer is R-DOTMA.
[0037] 14. The vaccine composition of clause 7 or clause 8 wherein
the enantiomer is S-DOTMA.
[0038] 15. The vaccine composition of clause 7 or clause 8 wherein
the enantiomer is R-DOEPC or S-DOEPC.
[0039] 16. The vaccine composition of clause 7 or clause 8 wherein
the enantiomer is R-DOEPC.
[0040] 17. The vaccine composition of clause 7 or clause 8 wherein
the enantiomer is S-DOEPC.
[0041] 18. The vaccine composition of any one of clauses 1 to 17
wherein one or more antigens is a protein-based antigen.
[0042] 19. The vaccine composition of any one of clauses 1 to 17
wherein one or more antigens is a peptide-based antigen.
[0043] 20. The vaccine composition of any one of clauses 1 to 19
wherein one or more antigens is selected from the group consisting
of a viral antigen, a fungal antigen, a bacterial antigen, and a
pathogenic antigen.
[0044] 21. The vaccine composition of any one of clauses 1 to 19
wherein one or more antigens is a viral antigen.
[0045] 22. The vaccine composition of any one of clauses 1 to 19
wherein one or more antigens is a fungal antigen.
[0046] 23. The vaccine composition of any one of clauses 1 to 19
wherein one or more antigens is a bacterial antigen.
[0047] 24. The vaccine composition of any one of clauses 1 to 19
wherein one or more antigens is a pathogenic antigen.
[0048] 25. The vaccine composition of any one of clauses 1 to 24
wherein at least one antigen is an antigen from a conserved region
of the pathogen.
[0049] 26. The vaccine composition of clause 24 wherein the
pathogenic antigen is a synthetic or recombinant antigen.
[0050] 27. The vaccine composition of any one of clauses 1 to 20
wherein at least one antigen is selected from the group consisting
of RAHYNIVTF (SEQ. ID. NO: 1), GQAEPDRAHYNIVTF (SEQ. ID. NO: 2),
KSSGQAEPDRAHYNIVTF (SEQ. ID. NO: 3), YMLDLQPETT (SEQ. ID. NO: 4),
KSSYMLDLQPETT (SEQ. ID. NO: 5), KSSMHGDTPTLHEYMLDLQPETT (SEQ. ID.
NO: 6), KSSLLMGTLGIVCPICSQKP (SEQ. ID. NO: 7), KVPRNQDWL (SEQ. ID.
NO: 8), SYVDFFVWL (SEQ. ID. NO: 9), KYICNSSCM (SEQ. ID. NO: 10),
and KSSKVPRNQDWL (SEQ. ID. NO: 11).
[0051] 28. The vaccine composition of any one of clauses 1 to 20
wherein at least one antigen is selected from the group comprising
of gp100 (KVPRNQDWL [SEQ. ID. No. 8]), TRP2 (SYVDFFVWL [SEQ. ID.
No. 9]), and p53 (KYICNSSCM [SEQ. ID. No. 10]), and combinations
thereof.
[0052] 29. The vaccine composition of any one of clauses 1 to 20
wherein the antigens are gp100 (KVPRNQDWL [SEQ. ID. No. 8]) and
TRP2 (SYVDFFVWL [SEQ. ID. No. 9]).
[0053] 30. The vaccine composition of any one of clauses 1 to 20
wherein the antigen is RAHYNIVTF (SEQ. ID. NO: 1).
[0054] 31. The vaccine composition of any one of clauses 1 to 20
wherein the antigen is GQAEPDRAHYNIVTF (SEQ. ID. NO: 2).
[0055] 32. The vaccine composition of any one of clauses 1 to 20
wherein the antigen is KSSGQAEPDRAHYNIVTF (SEQ. ID. NO: 3).
[0056] 33. The vaccine composition of any one of clauses 1 to 20
wherein the antigen is YMLDLQPETT (SEQ. ID. NO: 4).
[0057] 34. The vaccine composition of any one of clauses 1 to 20
wherein the antigen is KSSYMLDLQPETT (SEQ. ID. NO: 5).
[0058] 35. The vaccine composition of any one of clauses 1 to 20
wherein the antigen is KSSMHGDTPTLHEYMLDLQPETT (SEQ. ID. NO:
6).
[0059] 36. The vaccine composition of any one of clauses 1 to 20
wherein the antigen is KSSLLMGTLGIVCPICSQKP (SEQ. ID. NO: 7).
[0060] 37. The vaccine composition of any one of clauses 1 to 20
wherein the antigen is KVPRNQDWL (SEQ. ID. NO: 8).
[0061] 38. The vaccine composition of any one of clauses 1 to 20
wherein the antigen is SYVDFFVWL (SEQ. ID. NO: 9).
[0062] 39. The vaccine composition of any one of clauses 1 to 20
wherein the antigen is KYICNSSCM (SEQ. ID. NO: 10).
[0063] 40. The vaccine composition of any one of clauses 1 to 20
wherein the antigen is KSSKVPRNQDWL (SEQ. ID. NO: 11).
[0064] 41. The vaccine composition of any one of clauses 1 to 40
wherein at least one antigen is selected from the group consisting
of a lipoprotein, a lipopeptide, and a protein or peptide modified
with an amino acid sequence having an increased hydrophobicity or a
decreased hydrophobicity.
[0065] 42. The vaccine composition of any one of clauses 1 to 41
wherein one or more antigens is a lipidated antigen or an antigen
modified to increase hydrophobicity of the antigen.
[0066] 43. The vaccine composition of any one of clauses 1 to 42
wherein at least one antigen is a modified protein or peptide.
[0067] 44. The vaccine composition of any one of clauses 1 to 43
wherein the modified protein or peptide is bonded to a hydrophobic
group.
[0068] 45. The vaccine composition of any one of clauses 1 to 44
wherein the modified protein or peptide bonded to a hydrophobic
group further comprises a linker sequence between the antigen and
the hydrophobic group.
[0069] 46. The vaccine composition of clause 45 wherein the
hydrophobic group is a palmitoyl group.
[0070] 47. The vaccine composition of any one of clauses 1 to 46
wherein at least one antigen is an unmodified protein or
peptide.
[0071] 48. The vaccine composition of any one of clauses 1 to 47
wherein the vaccine composition is a universal vaccine.
[0072] 49. The vaccine composition of any one of clauses 1 to 48
wherein the vaccine composition is an anti-viral vaccine.
[0073] 50. The vaccine composition of any one of clauses 1 to 48
wherein the vaccine composition is an anti-fungal vaccine.
[0074] 51. The vaccine composition of any one of clauses 1 to 48
wherein the vaccine composition is an anti-bacterial vaccine.
[0075] 52. The vaccine composition of any one of clauses 1 to 48
wherein the vaccine composition is an influenza vaccine.
[0076] 53. The vaccine composition of clause 52 wherein the
influenza vaccine is a universal influenza vaccine.
[0077] 54. The vaccine composition of clause 52 or clause 53
wherein the influenza vaccine comprises a glycoprotein antigen
found on the surface of an influenza virus.
[0078] 55. The vaccine composition of clause 54 wherein the antigen
is a hemagglutinin antigen.
[0079] 56. The vaccine composition of clause 55 wherein the
hemagglutinin antigen comprises an epitope region
HA.sub.518-526.
[0080] 57. The vaccine composition of clause 55 wherein the
influenza vaccine is a neuraminidase subunit vaccine.
[0081] 58. The vaccine composition of any one of clauses 52 to 57
wherein the influenza vaccine is an H3N2 vaccine.
[0082] 59. The vaccine composition of any one of clauses 52 to 57
wherein the influenza vaccine is an N1N1 vaccine.
[0083] 60. The vaccine composition of any one of clauses 52 to 57
wherein the influenza vaccine is a Brisbane vaccine.
[0084] 61. The vaccine composition of any one of clauses 52 to 57
wherein the influenza vaccine is an H1N1 vaccine.
[0085] 62. The vaccine composition of any one of clauses 52 to 61
wherein the influenza vaccine comprises one or more protein
antigens from one or more influenza viruses.
[0086] 63. The vaccine composition of any one of clauses 52 to 62
wherein the influenza vaccine comprises an inactivated virus (e.g.
an inactivated whole virus).
[0087] 64. The vaccine composition of any one of clauses 52 to 61
wherein the influenza vaccine comprises an attenuated virus.
[0088] 65. The vaccine composition of any one of clauses 52 to 61
wherein the influenza vaccine comprises a disrupted virus.
[0089] 66. The vaccine composition of any one of clauses 52 to 61
wherein the influenza vaccine comprises a recombinant virus.
[0090] 67. The vaccine composition of any one of clauses 1 to 67
wherein the vaccine composition is capable of inducing a humoral
immune response.
[0091] 68. The vaccine composition of clause 67 wherein the humoral
immune response is an antibody response.
[0092] 69. The vaccine composition of any one of clauses 1 to 68
wherein the vaccine composition is capable of inducing a humoral
immune response against a conserved region of a pathogen.
[0093] 70. The vaccine composition of any one of clauses 1 to 69
wherein the vaccine composition is capable of inducing a cellular
immune response.
[0094] 71. The vaccine composition of clause 70 wherein the
cellular immune response is a T cell response.
[0095] 72. The vaccine composition of clause 71 wherein the T cell
response is a CD 8+ T cell response.
[0096] 73. The vaccine composition of any one of clauses 1 to 72
wherein the vaccine composition is capable of inducing a cellular
immune response against a conserved region of a pathogen.
[0097] 74. The vaccine composition of any one of clauses 1 to 73
wherein the vaccine composition is capable of inducing a humoral
immune response and a cellular immune response in the patient.
[0098] 75. The vaccine composition of any one of clauses 1 to 74
wherein the vaccine composition is capable of cross-presentation of
one or more antigens.
[0099] 76. The vaccine composition of any one of clauses 1 to 75
wherein the vaccine composition generates a humoral immune response
and a cellular immune response.
[0100] 77. The vaccine composition of any one of clauses 1 to 76
wherein the vaccine composition induces an immune response in a
mammal by activating the mitogen-activated protein (MAP) kinase
signaling pathway.
[0101] 78. The vaccine composition of clause 77 wherein the MAP
kinase signaling pathway is activated by stimulating at least one
of extracellular signal-regulated kinase ("ERK")-1, ERK-2, and
p38.
[0102] 79. The vaccine composition of any one of clauses 1 to 78
wherein the vaccine composition enhances functional
antigen-specific CD8+ T lymphocyte response in a mammal.
[0103] 80. The vaccine composition of clause 79 wherein the mammal
is a human.
[0104] 81. A method of treating a disease in a mammal, said method
comprising the step of administering an effective amount of a
vaccine composition to the mammal, wherein the vaccine composition
comprises at least one adjuvant and at least one antigen, and
wherein the adjuvant is a cationic lipid.
[0105] 82. The method of clause 81 wherein the disease is a
pathogenic disease.
[0106] 83. The method of clause 81 or clause 82 wherein the disease
is caused by multiple strains of a pathogen.
[0107] 84. The method of any one of clauses 81 to 83 wherein the
disease is influenza.
[0108] 85. The method of any one of clauses 81 to 84 wherein the
method induces a humoral immune response in the mammal.
[0109] 86. The method of clause 85 wherein the humoral immune
response is an antibody response.
[0110] 87. The method of clause 85 or clause 86 wherein the humoral
immune response is against a conserved region of a pathogen.
[0111] 88. The method of any one of clauses 81 to 87 wherein the
method induces a cellular immune response in the mammal.
[0112] 89. The method of clause 88 wherein the cellular immune
response is a T cell response.
[0113] 90. The method of clause 89 wherein the T cell response is a
CD 8+ T cell response.
[0114] 91. The method of any one of clauses 88 to 90 wherein the
cellular immune response is against a conserved region of a
pathogen.
[0115] 92. The method of any one of clauses 88 to 91 wherein the
method induces a humoral immune response and a cellular immune
response in the mammal.
[0116] 93. The method of any one of clauses 88 to 92 wherein the
cationic lipid is a non-steroidal cationic lipid.
[0117] 94. The method of any one of clauses 88 to 93 wherein the
cationic lipid is selected from the group consisting of DOTAP,
DOTMA, DOEPC, and combinations thereof.
[0118] 95. The method of any one of clauses 88 to 94 wherein the
cationic lipid is DOTAP.
[0119] 96. The method of any one of clauses 88 to 94 wherein the
cationic lipid is DOTMA.
[0120] 97. The method of any one of clauses 88 to 94 wherein the
cationic lipid is DOEPC.
[0121] 98. The method of any one of clauses 88 to 97 wherein the
adjuvant is an enantiomer of the cationic lipid.
[0122] 99. The method of clause 98 wherein the enantiomer is
purified.
[0123] 100. The method of clause 98 or clause 99 wherein the
enantiomer is R-DOTAP or S-DOTAP.
[0124] 101. The method of clause 98 or clause 99 wherein the
enantiomer is R-DOTAP.
[0125] 102. The method of clause 98 or clause 99 wherein the
enantiomer is S-DOTAP.
[0126] 103. The method of clause 98 or clause 99 wherein the
enantiomer is R-DOTMA or S-DOTMA.
[0127] 104. The method of clause 98 or clause 99 wherein the
enantiomer is R-DOTMA.
[0128] 105. The method of clause 98 or clause 99 wherein the
enantiomer is S-DOTMA.
[0129] 106. The method of clause 98 or clause 99 wherein the
enantiomer is R-DOEPC or S-DOEPC.
[0130] 107. The method of clause 98 or clause 99 wherein the
enantiomer is R-DOEPC.
[0131] 108. The method of clause 98 or clause 99 wherein the
enantiomer is S-DOEPC.
[0132] 109. The method of any one of clauses 81 to 109 wherein one
or more antigens is a protein-based antigen.
[0133] 110. The method of any one of clauses 81 to 109 wherein one
or more antigens is a peptide-based antigen.
[0134] 111. The method of any one of clauses 81 to 110 wherein one
or more antigens is selected from the group consisting of a viral
antigen, a fungal antigen, a bacterial antigen, and a pathogenic
antigen.
[0135] 112. The method of any one of clauses 81 to 110 wherein one
or more antigens is a viral antigen.
[0136] 113. The method of any one of clauses 81 to 110 wherein one
or more antigens is a fungal antigen.
[0137] 114. The method of any one of clauses 81 to 110 wherein one
or more antigens is a bacterial antigen.
[0138] 115. The method of any one of clauses 81 to 110 wherein one
or more antigens is a pathogenic antigen.
[0139] 116. The method of any one of clauses 81 to 115 wherein at
least one antigen is an antigen from a conserved region of the
pathogen.
[0140] 117. The method of clause 115 or clause 116 wherein the
pathogenic antigen is a synthetic or recombinant antigen.
[0141] 118. The method of any one of clauses 81 to 117 wherein at
least one antigen is selected from the group consisting of
RAHYNIVTF (SEQ. ID. NO: 1), GQAEPDRAHYNIVTF (SEQ. ID. NO: 2),
KSSGQAEPDRAHYNIVTF (SEQ. ID. NO: 3), YMLDLQPETT (SEQ. ID. NO: 4),
KSSYMLDLQPETT (SEQ. ID. NO: 5), KSSMHGDTPTLHEYMLDLQPETT (SEQ. ID.
NO: 6), KSSLLMGTLGIVCPICSQKP (SEQ. ID. NO: 7), KVPRNQDWL (SEQ. ID.
NO: 8), SYVDFFVWL (SEQ. ID. NO: 9), KYICNSSCM (SEQ. ID. NO: 10),
and KSSKVPRNQDWL (SEQ. ID. NO: 11).
[0142] 119. The method of any one of clauses 81 to 117 wherein at
least one antigen is selected from the group comprising of gp100
(KVPRNQDWL [SEQ. ID. No. 8]), TRP2 (SYVDFFVWL [SEQ. ID. No. 9]),
and p53 (KYICNSSCM [SEQ. ID. No. 10]), and combinations
thereof.
[0143] 120. The method of any one of clauses 81 to 117 wherein the
antigens are gp100 (KVPRNQDWL [SEQ. ID. No. 8]) and TRP2 (SYVDFFVWL
[SEQ. ID. No. 9]).
[0144] 121. The method of any one of clauses 81 to 117 wherein the
antigen is RAHYNIVTF (SEQ. ID. NO: 1).
[0145] 122. The method of any one of clauses 81 to 117 wherein the
antigen is GQAEPDRAHYNIVTF (SEQ. ID. NO: 2).
[0146] 123. The method of any one of clauses 81 to 117 wherein the
antigen is KSSGQAEPDRAHYNIVTF (SEQ. ID. NO: 3).
[0147] 124. The method of any one of clauses 81 to 117 wherein the
antigen is YMLDLQPETT (SEQ. ID. NO: 4).
[0148] 125. The method of any one of clauses 81 to 117 wherein the
antigen is KSSYMLDLQPETT (SEQ. ID. NO: 5).
[0149] 126. The method of any one of clauses 81 to 117 wherein the
antigen is KSSMHGDTPTLHEYMLDLQPETT (SEQ. ID. NO: 6).
[0150] 127. The method of any one of clauses 81 to 117 wherein the
antigen is KSSLLMGTLGIVCPICSQKP (SEQ. ID. NO: 7).
[0151] 128. The method of any one of clauses 81 to 117 wherein the
antigen is KVPRNQDWL (SEQ. ID. NO: 8).
[0152] 129. The method of any one of clauses 81 to 117 wherein the
antigen is SYVDFFVWL (SEQ. ID. NO: 9).
[0153] 130. The method of any one of clauses 81 to 117 wherein the
antigen is KYICNSSCM (SEQ. ID. NO: 10).
[0154] 131. The method of any one of clauses 81 to 117 wherein the
antigen is KSSKVPRNQDWL (SEQ. ID. NO: 11).
[0155] 132. The method of any one of clauses 81 to 131 wherein at
least one antigen is selected from the group consisting of a
lipoprotein, a lipopeptide, and a protein or peptide modified with
an amino acid sequence having an increased hydrophobicity or a
decreased hydrophobicity.
[0156] 133. The method of any one of clauses 81 to 132 wherein one
or more antigens is a lipidated antigen or an antigen modified to
increase hydrophobicity of the antigen.
[0157] 134. The method of any one of clauses 81 to 133 wherein at
least one antigen is a modified protein or peptide.
[0158] 135. The method of clause 134 wherein the modified protein
or peptide is bonded to a hydrophobic group.
[0159] 136. The method of clause 134 or clause 135 wherein the
modified protein or peptide bonded to a hydrophobic group further
comprises a linker sequence between the antigen and the hydrophobic
group.
[0160] 137. The method of clause 136 wherein the hydrophobic group
is a palmitoyl group.
[0161] 138. The method of any one of clauses 81 to 137 wherein at
least one antigen is an unmodified protein or peptide.
[0162] 139. The method of any one of clauses 81 to 138 wherein the
vaccine composition is a universal vaccine.
[0163] 140. The method of any one of clauses 81 to 138 wherein the
vaccine composition is an anti-viral vaccine.
[0164] 141. The method of any one of clauses 81 to 138 wherein the
vaccine composition is an anti-fungal vaccine.
[0165] 142. The method of any one of clauses 81 to 138 wherein the
vaccine composition is an anti-bacterial vaccine.
[0166] 143. The method of any one of clauses 81 to 138 wherein the
vaccine composition is an influenza vaccine.
[0167] 144. The method of any one of clauses 81 to 138 wherein the
influenza vaccine is a universal influenza vaccine.
[0168] 145. The method of clause 143 or clause 144 wherein the
influenza vaccine comprises a glycoprotein antigen found on the
surface of an influenza viruses.
[0169] 146. The method of clause 145 wherein the antigen is a
hemagglutinin antigen.
[0170] 147. The method of clause 146 wherein the hemagglutinin
antigen comprises an epitope region HA.sub.518-526.
[0171] 148. The method of clause 143 or clause 144 wherein the
influenza vaccine is a neuraminidase subunit vaccine.
[0172] 149. The method of any one of clauses 143 to 148 wherein the
influenza vaccine is an H3N2 vaccine.
[0173] 150. The method of any one of clauses 143 to 148 wherein the
influenza vaccine is an N1N1 vaccine.
[0174] 151. The method of any one of clauses 143 to 148 wherein the
influenza vaccine is a Brisbane vaccine.
[0175] 152. The method of any one of clauses 143 to 148 wherein the
influenza vaccine is an H1N1 vaccine.
[0176] 153. The method of any one of clauses 143 to 152 wherein the
influenza vaccine comprises one or more protein antigens from one
or more influenza viruses.
[0177] 154. The method of any one of clauses 143 to 153 wherein the
influenza vaccine comprises an inactivated virus (e.g. an
inactivated whole virus).
[0178] 155. The method of any one of clauses 143 to 152 wherein the
influenza vaccine comprises an attenuated virus.
[0179] 156. The method of any one of clauses 143 to 152 wherein the
influenza vaccine comprises a disrupted virus.
[0180] 157. The method of any one of clauses 143 to 152 wherein the
influenza vaccine comprises a recombinant virus.
[0181] 158. The method of any one of clauses 81 to 157 wherein the
vaccine composition induces an immune response in a mammal by
activating the mitogen-activated protein (MAP) kinase signaling
pathway.
[0182] 159. The method of clause 158 wherein the MAP kinase
signaling pathway is activated by stimulating at least one of
extracellular signal-regulated kinase ("ERK")-1, ERK-2, and
p38.
[0183] 160. The method of any one of clauses 143 to 159 wherein the
vaccine composition enhances functional antigen-specific CD8+ T
lymphocyte response in a mammal.
[0184] 161. The method of any one of clauses 81 to 138 wherein the
mammal is a human.
[0185] 162. A method of preventing a disease in a mammal, said
method comprising the step of administering an effective amount of
a vaccine composition to the mammal, wherein the vaccine
composition comprises at least one adjuvant and at least one
antigen, and wherein the adjuvant is a cationic lipid.
[0186] 163. The method of clause 162 wherein the disease is a
pathogenic disease.
[0187] 164. The method of clause 162 or clause 163 wherein the
disease is caused by multiple strains of a pathogen.
[0188] 165. The method of any one of clauses 162 to 164 wherein the
disease is influenza.
[0189] 166. The method of any one of clauses 162 to 165 wherein the
method induces a humoral immune response in the mammal.
[0190] 167. The method of clause 166 wherein the humoral immune
response is an antibody response.
[0191] 168. The method of clause 166 or clause 167 wherein the
humoral immune response is against a conserved region of a
pathogen.
[0192] 169. The method of any one of clauses 162 to 168 wherein the
method induces a cellular immune response in the mammal.
[0193] 170. The method of clause 169 wherein the cellular immune
response is a T cell response.
[0194] 171. The method of clause 170 wherein the T cell response is
a CD 8+ T cell response.
[0195] 172. The method of any one of clauses 169 to 171 wherein the
cellular immune response is against a conserved region of a
pathogen.
[0196] 173. The method of any one of clauses 162 to 172 wherein the
method induces a humoral immune response and a cellular immune
response in the mammal.
[0197] 174. The method of any one of clauses 162 to 173 wherein the
cationic lipid is a non-steroidal cationic lipid.
[0198] 175. The method of any one of clauses 162 to 174 wherein the
cationic lipid is selected from the group consisting of DOTAP,
DOTMA, DOEPC, and combinations thereof.
[0199] 176. The method of any one of clauses 162 to 175 wherein the
cationic lipid is DOTAP.
[0200] 177. The method of any one of clauses 162 to 175 wherein the
cationic lipid is DOTMA.
[0201] 178. The method of any one of clauses 162 to 175 wherein the
cationic lipid is DOEPC.
[0202] 179. The method of any one of clauses 162 to 178 wherein the
adjuvant is an enantiomer of a cationic lipid.
[0203] 180. The method of clause 179 wherein the enantiomer is
purified.
[0204] 181. The method of clause 179 or clause 180 wherein the
enantiomer is R-DOTAP or S-DOTAP.
[0205] 182. The method of clause 179 or clause 180 wherein the
enantiomer is R-DOTAP.
[0206] 183. The method of clause 179 or clause 180 wherein the
enantiomer is S-DOTAP.
[0207] 184. The method of clause 179 or clause 180 wherein the
enantiomer is R-DOTMA or S-DOTMA.
[0208] 185. The method of clause 179 or clause 180 wherein the
enantiomer is R-DOTMA.
[0209] 186. The method of clause 179 or clause 180 wherein the
enantiomer is S-DOTMA.
[0210] 187. The method of clause 179 or clause 180 wherein the
enantiomer is R-DOEPC or S-DOEPC.
[0211] 188. The method of clause 179 or clause 180 wherein the
enantiomer is R-DOEPC.
[0212] 189. The method of clause 179 or clause 180 wherein the
enantiomer is S-DOEPC.
[0213] 190. The method of any one of clauses 162 to 189 wherein one
or more antigens is a protein-based antigen.
[0214] 191. The method of any one of clauses 162 to 190 wherein one
or more antigens is a peptide-based antigen.
[0215] 192. The method of any one of clauses 162 to 191 wherein one
or more antigens is selected from the group consisting of a viral
antigen, a fungal antigen, a bacterial antigen, and a pathogenic
antigen.
[0216] 193. The method of any one of clauses 162 to 191 wherein one
or more antigens is a viral antigen.
[0217] 194. The method of any one of clauses 162 to 191 wherein one
or more antigens is a fungal antigen.
[0218] 195. The method of any one of clauses 162 to 191 wherein one
or more antigens is a bacterial antigen.
[0219] 196. The method of any one of clauses 162 to 191 wherein one
or more antigens is a pathogenic antigen.
[0220] 197. The method of any one of clauses 162 to 196 wherein at
least one antigen is an antigen from a conserved region of the
pathogen.
[0221] 198. The method of any one of clauses 162 to 197 wherein the
pathogenic antigen is a synthetic or recombinant antigen.
[0222] 199. The method of any one of clauses 162 to 198 wherein at
least one antigen is selected from the group consisting of
RAHYNIVTF (SEQ. ID. NO: 1), GQAEPDRAHYNIVTF (SEQ. ID. NO: 2),
KSSGQAEPDRAHYNIVTF (SEQ. ID. NO: 3), YMLDLQPETT (SEQ. ID. NO: 4),
KSSYMLDLQPETT (SEQ. ID. NO: 5), KSSMHGDTPTLHEYMLDLQPETT (SEQ. ID.
NO: 6), KSSLLMGTLGIVCPICSQKP (SEQ. ID. NO: 7), KVPRNQDWL (SEQ. ID.
NO: 8), SYVDFFVWL (SEQ. ID. NO: 9), KYICNSSCM (SEQ. ID. NO: 10),
and KSSKVPRNQDWL (SEQ. ID. NO: 11).
[0223] 200. The method of any one of clauses 162 to 198 wherein at
least one antigen is selected from the group comprising of gp100
(KVPRNQDWL [SEQ. ID. No. 8]), TRP2 (SYVDFFVWL [SEQ. ID. No. 9]),
and p53 (KYICNSSCM [SEQ. ID. No. 10]), and combinations
thereof.
[0224] 201. The method of any one of clauses 162 to 198 wherein the
antigens are gp100 (KVPRNQDWL [SEQ. ID. No. 8]) and TRP2 (SYVDFFVWL
[SEQ. ID. No. 9]).
[0225] 202. The method of any one of clauses 162 to 198 wherein the
antigen is RAHYNIVTF (SEQ. ID. NO: 1).
[0226] 203. The method of any one of clauses 162 to 198 wherein the
antigen is GQAEPDRAHYNIVTF (SEQ. ID. NO: 2).
[0227] 204. The method of any one of clauses 162 to 198 wherein the
antigen is KSSGQAEPDRAHYNIVTF (SEQ. ID. NO: 3).
[0228] 205. The method of any one of clauses 162 to 198 wherein the
antigen is YMLDLQPETT (SEQ. ID. NO: 4).
[0229] 206. The method of any one of clauses 162 to 198 wherein the
antigen is KSSYMLDLQPETT (SEQ. ID. NO: 5).
[0230] 207. The method of any one of clauses 162 to 198 wherein the
antigen is KSSMHGDTPTLHEYMLDLQPETT (SEQ. ID. NO: 6).
[0231] 208. The method of any one of clauses 162 to 198 wherein the
antigen is KSSLLMGTLGIVCPICSQKP (SEQ. ID. NO: 7).
[0232] 209. The method of any one of clauses 162 to 198 wherein the
antigen is KVPRNQDWL (SEQ. ID. NO: 8).
[0233] 210. The method of any one of clauses 162 to 198 wherein the
antigen is SYVDFFVWL (SEQ. ID. NO: 9).
[0234] 211. The method of any one of clauses 162 to 198 wherein the
antigen is KYICNSSCM (SEQ. ID. NO: 10).
[0235] 212. The method of any one of clauses 162 to 198 wherein the
antigen is KSSKVPRNQDWL (SEQ. ID. NO: 11).
[0236] 213. The method of any one of clauses 162 to 212 wherein at
least one antigen is selected from the group consisting of a
lipoprotein, a lipopeptide, and a protein or peptide modified with
an amino acid sequence having an increased hydrophobicity or a
decreased hydrophobicity.
[0237] 214. The method of any one of clauses 162 to 213 wherein one
or more antigens is a lipidated antigen or an antigen modified to
increase hydrophobicity of the antigen.
[0238] 215. The method of any one of clauses 162 to 213 wherein at
least one antigen is a modified protein or peptide.
[0239] 216. The method of clause 215 wherein the modified protein
or peptide is bonded to a hydrophobic group.
[0240] 217. The method of clause 215 or clause 216 wherein the
modified protein or peptide bonded to a hydrophobic group further
comprises a linker sequence between the antigen and the hydrophobic
group.
[0241] 218. The method of clause 217 wherein the hydrophobic group
is a palmitoyl group.
[0242] 219. The method of any one of clauses 162 to 218 wherein at
least one antigen is an unmodified protein or peptide.
[0243] 220. The method of any one of clauses 162 to 219 wherein the
vaccine composition is a universal vaccine.
[0244] 221. The method of any one of clauses 162 to 220 wherein the
vaccine composition is an anti-viral vaccine.
[0245] 222. The method of any one of clauses 162 to 219 wherein the
vaccine composition is an anti-fungal vaccine.
[0246] 223. The method of any one of clauses 162 to 219 wherein the
vaccine composition is an anti-bacterial vaccine.
[0247] 224. The method of any one of clauses 162 to 219 wherein the
vaccine composition is an influenza vaccine.
[0248] 225. The method of clause 224 wherein the influenza vaccine
is a universal influenza vaccine.
[0249] 226. The method of clause 224 or clause 225 wherein the
influenza vaccine comprises a glycoprotein antigen found on the
surface of an influenza viruses.
[0250] 227. The method of clause 226 wherein the antigen is a
hemagglutinin antigen.
[0251] 228. The method of clause 227 wherein the hemagglutinin
antigen comprises an epitope region HA.sub.518-526.
[0252] 229. The method of any one of clauses 224 to 228 wherein the
influenza vaccine is a neuraminidase subunit vaccine.
[0253] 230. The method of any one of clauses 224 to 229 wherein the
influenza vaccine is an H3N2 vaccine.
[0254] 231. The method of any one of clauses 224 to 229 wherein the
influenza vaccine is an N1N1 vaccine.
[0255] 232. The method of any one of clauses 224 to 229 wherein the
influenza vaccine is a Brisbane vaccine.
[0256] 233. The method of any one of clauses 224 to 229 wherein the
influenza vaccine is an H1N1 vaccine.
[0257] 234. The method of any one of clauses 224 to 233 wherein the
influenza vaccine comprises one or more protein antigens from one
or more influenza viruses.
[0258] 235. The method of any one of clauses 224 to 234 wherein the
influenza vaccine comprises an inactivated virus (e.g. an
inactivated whole virus).
[0259] 236. The method of any one of clauses 224 to 233 wherein the
influenza vaccine comprises an attenuated virus.
[0260] 237. The method of any one of clauses 224 to 233 wherein the
influenza vaccine comprises a disrupted virus.
[0261] 238. The method of any one of clauses 224 to 233 wherein the
influenza vaccine comprises a recombinant virus.
[0262] 239. The method of any one of clauses 162 to 238 wherein the
vaccine composition induces an immune response in a mammal by
activating the mitogen-activated protein (MAP) kinase signaling
pathway.
[0263] 240. The method of clause 240 wherein the MAP kinase
signaling pathway is activated by stimulating at least one of
extracellular signal-regulated kinase ("ERK")-1, ERK-2, and
p38.
[0264] 241. The method of any one of clauses 162 to 240 wherein the
vaccine composition enhances functional antigen-specific CD8+ T
lymphocyte response in a mammal.
[0265] 242. The method of any one of clauses 162 to 241 wherein the
mammal is a human.
[0266] 243. A method of effecting antigen cross presentation to
induce a humoral immune response and a cellular immune response in
a mammal, said method comprising the step of administering an
effective amount of a vaccine composition to the mammal, wherein
the vaccine composition comprises at least one adjuvant and at
least one antigen, and wherein the adjuvant is a cationic
lipid.
[0267] 244. The method of clause 243 wherein the humoral immune
response is an antibody response.
[0268] 245. The method of clause 243 or clause 244 wherein the
humoral immune response is against a conserved region of a
pathogen.
[0269] 246. The method of any one of clauses 243 to 245 wherein the
cellular immune response is a T cell response.
[0270] 247. The method of clause 246 wherein the T cell response is
a CD 8+ T cell response.
[0271] 248. The method of any one of clauses 243 to 247 wherein the
cellular immune response is against a conserved region of a
pathogen.
[0272] 249. The method of any one of clauses 243 to 248 wherein the
cationic lipid is a non-steroidal cationic lipid.
[0273] 250. The method of any one of clauses 243 to 249 wherein the
cationic lipid is selected from the group consisting of DOTAP,
DOTMA, DOEPC, and combinations thereof.
[0274] 251. The method of any one of clauses 243 to 250 wherein the
cationic lipid is DOTAP.
[0275] 252. The method of any one of clauses 243 to 250 wherein the
cationic lipid is DOTMA.
[0276] 253. The method of any one of clauses 243 to 250 wherein the
cationic lipid is DOEPC.
[0277] 254. The method of any one of clauses 243 to 249 wherein the
adjuvant is an enantiomer of a cationic lipid.
[0278] 255. The method of clause 254 wherein the enantiomer is
purified.
[0279] 256. The method of clause 254 or clause 255 wherein the
enantiomer is R-DOTAP or S-DOTAP.
[0280] 257. The method of clause 254 or clause 255 wherein the
enantiomer is R-DOTAP.
[0281] 258. The method of clause 254 or clause 255 wherein the
enantiomer is S-DOTAP.
[0282] 259. The method of clause 254 or clause 255 wherein the
enantiomer is R-DOTMA or S-DOTMA.
[0283] 260. The method of clause 254 or clause 255 wherein the
enantiomer is R-DOTMA.
[0284] 261. The method of clause 254 or clause 255 wherein the
enantiomer is S-DOTMA.
[0285] 262. The method of clause 254 or clause 255 wherein the
enantiomer is R-DOEPC or S-DOEPC.
[0286] 263. The method of clause 254 or clause 255 wherein the
enantiomer is R-DOEPC.
[0287] 264. The method of clause 254 or clause 255 wherein the
enantiomer is S-DOEPC.
[0288] 265. The method of any one of clauses 243 to 264 wherein one
or more antigens is a protein-based antigen.
[0289] 266. The method of any one of clauses 243 to 264 wherein one
or more antigens is a peptide-based antigen.
[0290] 267. The method of any one of clauses 243 to 266 wherein one
or more antigens is selected from the group consisting of a viral
antigen, a fungal antigen, a bacterial antigen, and a pathogenic
antigen.
[0291] 268. The method of any one of clauses 243 to 266 wherein one
or more antigens is a viral antigen.
[0292] 269. The method of any one of clauses 243 to 266 wherein one
or more antigens is a fungal antigen.
[0293] 270. The method of any one of clauses 243 to 266 wherein one
or more antigens is a bacterial antigen.
[0294] 271. The method of any one of clauses 243 to 266 wherein one
or more antigens is a pathogenic antigen.
[0295] 272. The method of any one of clauses 243 to 271 wherein at
least one antigen is an antigen from a conserved region of the
pathogen.
[0296] 273. The method of clause 271 or clause 272 wherein the
pathogenic antigen is a synthetic or recombinant antigen.
[0297] 274. The method of any one of clauses 243 to 273 wherein at
least one antigen is selected from the group consisting of
RAHYNIVTF (SEQ. ID. NO: 1), GQAEPDRAHYNIVTF (SEQ. ID. NO: 2),
KSSGQAEPDRAHYNIVTF (SEQ. ID. NO: 3), YMLDLQPETT (SEQ. ID. NO: 4),
KSSYMLDLQPETT (SEQ. ID. NO: 5), KSSMHGDTPTLHEYMLDLQPETT (SEQ. ID.
NO: 6), KSSLLMGTLGIVCPICSQKP (SEQ. ID. NO: 7), KVPRNQDWL (SEQ. ID.
NO: 8), SYVDFFVWL (SEQ. ID. NO: 9), KYICNSSCM (SEQ. ID. NO: 10),
and KSSKVPRNQDWL (SEQ. ID. NO: 11).
[0298] 275. The method of any one of clauses 243 to 273 wherein at
least one antigen is selected from the group comprising of gp100
(KVPRNQDWL [SEQ. ID. No. 8]), TRP2 (SYVDFFVWL [SEQ. ID. No. 9]),
and p53 (KYICNSSCM [SEQ. ID. No. 10]), and combinations
thereof.
[0299] 276. The method of any one of clauses 243 to 273 wherein the
antigens are gp100 (KVPRNQDWL [SEQ. ID. No. 8]) and TRP2 (SYVDFFVWL
[SEQ. ID. No. 9]).
[0300] 277. The method of any one of clauses 243 to 273 wherein the
antigen is RAHYNIVTF (SEQ. ID. NO: 1).
[0301] 278. The method of any one of clauses 243 to 273 wherein the
antigen is GQAEPDRAHYNIVTF (SEQ. ID. NO: 2).
[0302] 279. The method of any one of clauses 243 to 273 wherein the
antigen is KSSGQAEPDRAHYNIVTF (SEQ. ID. NO: 3).
[0303] 280. The method of any one of clauses 243 to 273 wherein the
antigen is YMLDLQPETT (SEQ. ID. NO: 4).
[0304] 281. The method of any one of clauses 243 to 273 wherein the
antigen is KSSYMLDLQPETT (SEQ. ID. NO: 5).
[0305] 282. The method of any one of clauses 243 to 273 wherein the
antigen is KSSMHGDTPTLHEYMLDLQPETT (SEQ. ID. NO: 6).
[0306] 283. The method of any one of clauses 243 to 273 wherein the
antigen is KSSLLMGTLGIVCPICSQKP (SEQ. ID. NO: 7).
[0307] 284. The method of any one of clauses 243 to 273 wherein the
antigen is KVPRNQDWL (SEQ. ID. NO: 8).
[0308] 285. The method of any one of clauses 243 to 273 wherein the
antigen is SYVDFFVWL (SEQ. ID. NO: 9).
[0309] 286. The method of any one of clauses 243 to 273 wherein the
antigen is KYICNSSCM (SEQ. ID. NO: 10).
[0310] 287. The method of any one of clauses 243 to 273 wherein the
antigen is KSSKVPRNQDWL (SEQ. ID. NO: 11).
[0311] 288. The method of any one of clauses 243 to 287 wherein at
least one antigen is selected from the group consisting of a
lipoprotein, a lipopeptide, and a protein or peptide modified with
an amino acid sequence having an increased hydrophobicity or a
decreased hydrophobicity.
[0312] 289. The method of any one of clauses 243 to 288 wherein one
or more antigens is a lipidated antigen or an antigen modified to
increase hydrophobicity of the antigen.
[0313] 290. The method of any one of clauses 243 to 289 wherein at
least one antigen is a modified protein or peptide.
[0314] 291. The method of clause 290 wherein the modified protein
or peptide is bonded to a hydrophobic group.
[0315] 292. The method of clause 290 or clause 291 wherein the
modified protein or peptide bonded to a hydrophobic group further
comprises a linker sequence between the antigen and the hydrophobic
group.
[0316] 293. The method of clause 292 wherein the hydrophobic group
is a palmitoyl group.
[0317] 294. The method of any one of clauses 243 to 293 wherein at
least one antigen is an unmodified protein or peptide.
[0318] 295. The method of any one of clauses 243 to 294 wherein the
vaccine composition is a universal vaccine.
[0319] 296. The method of any one of clauses 243 to 295 wherein the
vaccine composition is an anti-viral vaccine.
[0320] 297. The method of any one of clauses 243 to 295 wherein the
vaccine composition is an anti-fungal vaccine.
[0321] 298. The method of any one of clauses 243 to 295 wherein the
vaccine composition is an anti-bacterial vaccine.
[0322] 299. The method of any one of clauses 243 to 295 wherein the
vaccine composition is an influenza vaccine.
[0323] 300. The method of clause 299 wherein the influenza vaccine
is a universal influenza vaccine.
[0324] 301. The method of clause 299 or clause 300 wherein the
influenza vaccine comprises a glycoprotein antigen found on the
surface of an influenza viruses.
[0325] 302. The method of clause 301 wherein the antigen is a
hemagglutinin antigen.
[0326] 303. The method of clause 302 wherein the hemagglutinin
antigen comprises an epitope region HA.sub.518-526.
[0327] 304. The method of any one of clauses 299 to 303 wherein the
influenza vaccine is a neuraminidase subunit vaccine.
[0328] 305. The method of any one of clauses 299 to 304 wherein the
influenza vaccine is an H3N2 vaccine.
[0329] 306. The method of any one of clauses 299 to 304 wherein the
influenza vaccine is an N1N1 vaccine.
[0330] 307. The method of any one of clauses 299 to 304 wherein the
influenza vaccine is a Brisbane vaccine.
[0331] 308. The method of any one of clauses 299 to 304 wherein the
influenza vaccine is an H1N1 vaccine.
[0332] 309. The method of any one of clauses 299 to 308 wherein the
influenza vaccine comprises one or more protein antigens from one
or more influenza viruses.
[0333] 310. The method of any one of clauses 299 to 309 wherein the
influenza vaccine comprises an inactivated virus (e.g. an
inactivated whole virus).
[0334] 311. The method of any one of clauses 299 to 308 wherein the
influenza vaccine comprises an attenuated virus.
[0335] 312. The method of any one of clauses 299 to 308 wherein the
influenza vaccine comprises a disrupted virus.
[0336] 313. The method of any one of clauses 299 to 308 wherein the
influenza vaccine comprises a recombinant virus.
[0337] 314. The method of any one of clauses 243 to 313 wherein the
vaccine composition induces an immune response in a mammal by
activating the mitogen-activated protein (MAP) kinase signaling
pathway.
[0338] 315. The method of clause 314 wherein the MAP kinase
signaling pathway is activated by stimulating at least one of
extracellular signal-regulated kinase ("ERK")-1, ERK-2, and
p38.
[0339] 316. The method of any one of clauses 243 to 315 wherein the
vaccine composition enhances functional antigen-specific CD8+ T
lymphocyte response in a mammal.
[0340] 317. The method of any one of clauses 243 to 316 wherein the
mammal is a human.
BRIEF DESCRIPTION OF THE DRAWINGS
[0341] FIG. 1 shows results of a hemagglutination inhibition assay
against H3N2 with a commercial influenza vaccine and the cationic
lipid-based influenza vaccines.
[0342] FIG. 2 shows results of a hemagglutination inhibition assay
against H1N1 with a commercial influenza vaccine and the cationic
lipid-based influenza vaccines.
[0343] FIG. 3 shows results of a hemagglutination inhibition assay
against B Brisbane with a commercial influenza vaccine and the
cationic lipid-based influenza vaccines.
[0344] FIG. 4 shows that R-DOTAP enhances the T cell response to an
internal class I restricted epitope of hemagglutinin. BALB/c mice
were vaccinated with the H5N1 vaccine (inactivated A/Vietnam 2004)
alone, or adjuvanted with either CFA (emulsion) or cationic
lipid.
[0345] FIG. 5 shows that DOTMA and DOEPC enhance the T cell
response to a class I restricted epitope of the human
papillomavirus Strain 16. C57BL/6 mice were vaccinated with the
various formulations consisting of the cationic lipid adjuvants or
Montanide.TM. and the peptide HPV-16 E743-57. Superior T-cell
enhancement results with the use of the cationic lipids compared to
Montanide.TM..
[0346] Various embodiments of the invention are described herein as
follows. In one embodiment described herein, a vaccine composition
is provided. The vaccine composition comprises at least one
adjuvant and at least one antigen, wherein the adjuvant is a
cationic lipid.
[0347] In another embodiment, a method of treating a disease in a
mammal is provided. The method comprises the step of administering
an effective amount of a vaccine composition to the mammal, wherein
the vaccine composition comprises at least one adjuvant and at
least one antigen, and wherein the adjuvant is a cationic
lipid.
[0348] In yet another embodiment, a method of preventing a disease
in a mammal is provided. The method comprises the step of
administering an effective amount of a vaccine composition to the
mammal, wherein the vaccine composition comprises at least one
adjuvant and at least one antigen, and wherein the adjuvant is a
cationic lipid.
[0349] In yet another embodiment, a method of effecting antigen
cross presentation to induce a humoral immune response and a
cellular immune response in a mammal is provided. The method
comprises the step of administering an effective amount of a
vaccine composition to the mammal, wherein the vaccine composition
comprises at least one adjuvant and at least one antigen, and
wherein the adjuvant is a cationic lipid.
[0350] In the various embodiments, the vaccine composition
comprises at least one adjuvant and at least one antigen, wherein
the adjuvant is a cationic lipid. As used herein, the term
"adjuvant" refers to a substance that enhances, augments and/or
potentiates a mammal's immune response to an antigen. Doses of the
adjuvant are known to those of ordinary skill in the art, as well
as those exemplified in PCT/US2008/057678 (Stimulation of an Immune
Response by Cationic Lipids), PCT/US2009/040500 (Stimulation of an
Immune Response by Enantiomers of Cationic Lipids), both herein
incorporated by reference in their entirety.
[0351] In some embodiments described herein, the adjuvant is an
immunomodulator. As used herein, the term "immunomodulator" refers
to an immunologic modifier that enhances, directs, and/or promotes
an immune response in a mammal.
[0352] In some embodiments described herein, the adjuvant is a
nanoparticle. As used herein, the term "nanoparticle" refers to a
particle having a size measured on the nanometer scale. As used
herein, the "nanoparticle" refers to a particle having a structure
with a size of less than about 1,000 nanometers. In some
embodiments, the nanoparticle is a liposome.
[0353] In some embodiments described herein, the adjuvant is a
cationic lipid. As used herein, the term "cationic lipid" refers to
any of a number of lipid species which carry a net positive charge
at physiological pH or have a protonatable group and are positively
charged at pH lower than the pKa.
[0354] Cationic lipid-based nanoparticles have been shown to be
potent immuno-modifying adjuvants in addition to their ability to
act as effective delivery systems, as demonstrated in
PCT/US2008/057678 (Stimulation of an Immune Response by Cationic
Lipids), PCT/US2009/040500 (Stimulation of an Immune Response by
Enantiomers of Cationic Lipids), both herein incorporated by
reference in their entirety. The cationic lipid adjuvants in
vaccine formulations containing short and long T-cell epitope
peptides as expected were demonstrated to elicit superior T-cell
immune responses without antibody immune responses.
[0355] Suitable cationic lipid according to the present disclosure
include, but are not limited to:
3-.beta.[.sup.4N-(.sup.1N,.sup.8-diguanidino
spermidine)-carbamoyl]cholesterol (BGSC);
3-.beta.[N,N-diguanidinoethyl-aminoethane)-carbamoyl]cholesterol
(BGTC); N,N.sup.1N.sup.2N.sup.3Tetra-methyltetrapalmitylspermine
(cellfectin);
N-t-butyl-N'-tetradecyl-3-tetradecyl-aminopropion-amidine
(CLONfectin); dimethyldioctadecyl ammonium bromide (DDAB);
1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide
(DMRIE);
2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-p-
-ropanaminium trifluorocetate) (DOSPA);
1,3-dioleoyloxy-2-(6-carboxyspermyl)-propyl amide (DOSPER);
4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole (DPIM)
N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3-dioleoyloxy-1,4-butane-
-diammonium iodide) (Tfx-50); N-1-(2,3-dioleoyloxy)
propyl-N,N,N-trimethyl ammonium chloride (DOTMA) or other
N--(N,N-1-dialkoxy)-alkyl-N,N,N-trisubstituted ammonium
surfactants; 1,2 dioleoyl-3-(4'-trimethylammonio)
butanol-sn-glycerol (DOBT) or
cholesteryl(4'trimethylammonia)butanoate (ChOTB) where the
trimethylammonium group is connected via a butanol spacer arm to
either the double chain (for DOTB) or cholesteryl group (for
ChOTB); DORI
(DL-1,2-dioleoyl-3-dimethylaminopropyl-.beta.-hydroxyethylammonium)
or DORIE
(DL-1,2-O-dioleoyl-3-dimethylaminopropyl-.beta.-hydroxyethylammoniu-
-m) (DORIE) or analogs thereof as disclosed in WO 93/03709;
1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC);
cholesteryl hemisuccinate ester (ChOSC); lipopolyamines such as
dioctadecylamidoglycylspermine (DOGS) and dipalmitoyl
phosphatidylethanolamylspermine (DPPES),
cholesteryl-3.beta.-carboxyl-amido-ethylenetrimethylammonium
iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl
carboxylate iodide, cholesteryl-3-O-carboxyamidoethyleneamine,
cholesteryl-3-.beta.-oxysuccinamido-ethylenetrimethylammonium
iodide,
1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3-.beta.-oxysu-
ccinate iodide, 2-(2-trimethylammonio)-ethylmethylamino
ethyl-cholesteryl-3-.beta.-oxysuccinate iodide,
3-.beta.-N--(N',N'-dimethylaminoethane) carbamoyl cholesterol
(DC-chol), and 3-.beta.-N-(polyethyleneimine)-carbamoylcholesterol;
O,O'-dimyristyl-N-lysyl aspartate (DMKE);
O,O'-dimyristyl-N-lysyl-glutamate (DMKD);
1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide
(DMRIE); 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC);
1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC);
1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC);
1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPEPC);
1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC);
1,2-dioleoyl-3-trimethylammonium propane (DOTAP); dioleoyl
dimethylaminopropane (DODAP); 1,2-palmitoyl-3-trimethylammonium
propane (DPTAP); 1,2-distearoyl-3-trimethylammonium propane
(DSTAP), 1,2-myristoyl-3-trimethylammonium propane (DMTAP); and
sodium dodecyl sulfate (SDS). Furthermore, structural variants and
derivatives of the any of the described cationic lipids are also
contemplated.
[0356] In some embodiment, the cationic lipid is selected from the
group consisting of DOTAP, DOTMA, DOEPC, and combinations thereof.
In other embodiments, the cationic lipid is DOTAP. In yet other
embodiments, the cationic lipid is DOTMA. In other embodiments, the
cationic lipid is DOEPC. In some embodiments, the cationic lipid is
purified. In other embodiments, the cationic lipid is a
non-steroidal cationic lipid.
[0357] In some embodiments, the cationic lipid is an enantiomer of
a cationic lipid. The term "enantiomer" refers to a stereoisomer of
a cationic lipid which is a non-superimposable mirror image of its
counterpart stereoisomer, for example R and S enantiomers. In
various examples, the enantiomer is R-DOTAP or S-DOTAP. In one
example, the enantiomer is R-DOTAP. In another example, the
enantiomer is S-DOTAP. In some embodiments, the enantiomer is
purified. In various examples, the enantiomer is R-DOTMA or
S-DOTMA. In one example, the enantiomer is R-DOTMA. In another
example, the enantiomer is S-DOTMA. In some embodiments, the
enantiomer is purified. In various examples, the enantiomer is
R-DOPEC or S-DOPEC. In one example, the enantiomer is R-DOPEC. In
another example, the enantiomer is S-DOPEC. In some embodiments,
the enantiomer is purified.
[0358] In various embodiments described herein, the composition
further comprises one or more antigens. As used herein, the term
"antigen" refers to any agent (e.g., protein, peptide,
polysaccharide, glycoprotein, glycolipid, nucleic acid, or
combination thereof) that, when introduced into a mammal having an
immune system (directly or upon expression as in, e.g., DNA
vaccines), is recognized by the immune system of the mammal and is
capable of eliciting an immune response. As defined herein, the
antigen-induced immune response can be humoral or cell-mediated, or
both. An agent is termed "antigenic" when it is capable of
specifically interacting with an antigen recognition molecule of
the immune system, such as an immunoglobulin (antibody) or T cell
antigen receptor (TCR).
[0359] In some embodiments, one or more antigens is a protein-based
antigen. In other embodiments, one or more antigens is a
peptide-based antigen. In various embodiments, one or more antigens
is selected from the group consisting of a viral antigen, a
bacterial antigen, and a pathogenic antigen. A "microbial antigen,"
as used herein, is an antigen of a microorganism and includes, but
is not limited to, infectious virus, infectious bacteria,
infectious parasites and infectious fungi. Microbial antigens may
be intact microorganisms, and natural isolates, fragments, or
derivatives thereof, synthetic compounds which are identical to or
similar to naturally-occurring microbial antigens and, preferably,
induce an immune response specific for the corresponding
microorganism (from which the naturally-occurring microbial antigen
originated). In one embodiment, the antigen is a cancer antigen. In
one embodiment, the antigen is a viral antigen. In another
embodiment, the antigen is a fungal antigen. In another embodiment,
the antigen is a bacterial antigen. In various embodiments, the
antigen is a pathogenic antigen. In some embodiments, the
pathogenic antigen is a synthetic or recombinant antigen.
[0360] In some embodiments of the present disclosure, at least one
antigen comprises a sequence selected from the group consisting of
RAHYNIVTF (SEQ. ID. NO: 1), GQAEPDRAHYNIVTF (SEQ. ID. NO: 2),
KSSGQAEPDRAHYNIVTF (SEQ. ID. NO: 3), YMLDLQPETT (SEQ. ID. NO: 4),
KSSYMLDLQPETT (SEQ. ID. NO: 5), MHGDTPTLHEYMLDLQPETT (SEQ. ID. NO:
6), LLMGTLGIVCPICSQKP (SEQ. ID. NO: 7), KVPRNQDWL (SEQ. ID. NO: 8),
SYVDFFVWL (SEQ. ID. NO: 9), KYICNSSCM (SEQ. ID. NO: 10), and
KSSKVPRNQDWL (SEQ. ID. NO: 11). In one embodiment, at least one
antigen comprises the sequence RAHYNIVTF (SEQ. ID. NO: 1). In
another embodiment, at least one antigen comprises the sequence
GQAEPDRAHYNIVTF (SEQ. ID. NO: 2). In yet another embodiment, at
least one antigen comprises the sequence KSSGQAEPDRAHYNIVTF (SEQ.
ID. NO: 3). In some embodiments, KSSGQAEPDRAHYNIVTF (SEQ. ID. NO:
3) is modified to further comprise a hydrophobic group. In one
embodiment, the hydrophobic group is a palmitoyl group.
[0361] In other embodiments, at least one antigen comprises the
sequence YMLDLQPETT (SEQ. ID. NO: 4). In another embodiment, at
least one antigen comprises the sequence KSSYMLDLQPETT (SEQ. ID.
NO: 5). In yet another embodiment, KSSYMLDLQPETT (SEQ. ID. NO: 5)
is modified to further comprise a hydrophobic group. In one
embodiment, the hydrophobic group is a palmitoyl group.
[0362] In other embodiments, at least one antigen comprises the
sequence KSSMHGDTPTLHEYMLDLQPETT (SEQ. ID. NO: 6). In another
embodiment, KSSMHGDTPTLHEYMLDLQPETT (SEQ. ID. NO: 6) is modified to
further comprise a hydrophobic group. In one embodiment, the
hydrophobic group is a palmitoyl group.
[0363] In other embodiments, at least one antigen comprises the
sequence KSSLLMGTLGIVCPICSQKP (SEQ. ID. NO: 7). In some
embodiments, KSSLLMGTLGIVCPICSQKP (SEQ. ID. NO: 7) is modified to
further comprise a hydrophobic group. In one embodiment, the
hydrophobic group is a palmitoyl group.
[0364] In some embodiments, at least one antigen comprises the
sequence KVPRNQDWL (SEQ. ID. NO: 8). In other embodiments, at least
one antigen comprises the sequence SYVDFFVWL (SEQ. ID. NO: 9). In
yet other embodiments, at least one antigen comprises the sequence
KYICNSSCM (SEQ. ID. NO: 10). In another embodiment, at least one
antigen comprises the sequence KSSKVPRNQDWL (SEQ. ID. NO: 11). In
some embodiments, KSSKVPRNQDWL (SEQ. ID. NO: 11) is modified to
further comprise a hydrophobic group. In one embodiment, the
hydrophobic group is a palmitoyl group.
[0365] In one embodiment, the antigen comprises the sequence
selected from the group comprising of gp100 (KVPRNQDWL [SEQ. ID.
No. 8]), TRP2 (SYVDFFVWL [SEQ. ID. No. 9]), and p53 (KYICNSSCM
[SEQ. ID. No. 10]), and combinations thereof.
[0366] In one embodiment, the antigens comprise the gp100 sequence
0 (KVPRNQDWL [SEQ. ID. No. 8]) or the TRP2 sequence (SYVDFFVWL
[SEQ. ID. No. 9]).
[0367] In various embodiments, at least one antigen is selected
from the group consisting of a lipoprotein, a lipopeptide, and a
protein or peptide modified with an amino acid sequence having an
increased hydrophobicity or a decreased hydrophobicity. In some
embodiments, one or more antigens is an antigen modified to
increase hydrophobicity of the antigen. In one embodiment, at least
one antigen is a modified protein or peptide. In some embodiments,
the modified protein or peptide is bonded to a hydrophobic group.
In other embodiments, the modified protein or peptide bonded to a
hydrophobic group further comprises a linker sequence between the
antigen and the hydrophobic group. In some embodiments, the
hydrophobic group is a palmitoyl group. In yet other embodiments,
at least one antigen is an unmodified protein or peptide.
[0368] In some embodiments described herein, the vaccine
composition is a universal vaccine. As used herein, a "universal"
vaccine can protect mammals against a broad range of pathogens, for
example a broad range of influenza viruses, and may be effective
across multiple strains of a pathogen. Successful development of a
universal influenza vaccine could protect mammals against a broad
variety of related pathogens rather than just a few. A universal
vaccine could potentially be used "off-the-shelf" and could provide
some protection against newly emerging pathogens. For example, a
universal influenza vaccine influenza virus could provide some
protection against newly emerging viruses experts had not
identified during worldwide surveillance of these viruses. A
universal vaccine could decrease the severity of disease, speed up
the ability of the body to clear itself of the pathogen, and reduce
the fatality rate of infections until a specific vaccine against
that pathogen is available.
[0369] In some embodiments described herein, the vaccine
composition is an anti-viral vaccine. In some embodiments described
herein, the vaccine composition is an anti-fungal vaccine. In some
embodiments described herein, the vaccine composition is an
anti-bacterial vaccine.
[0370] In some embodiments described herein, the vaccine
composition is an influenza vaccine. In other embodiments described
herein, the influenza vaccine is a universal influenza vaccine. It
is demonstrated in the present disclosure that the cationic lipids
induce significantly enhanced antibody protection when formulated
with the inactivated H3N2, N1N1, and Brisbane strains of the
influenza virus. There is a well-established CD8 T cell epitope
within hemagglutinin (HA) from the mouse-adapted PR8 strain of
virus (H1N1): HA.sub.518-526, IYSTVASSL, K.sup.d restricted.
Vaccination with this epitope has been shown to protect mice from
lethal infection. This epitope is also shared in the H5N1 virus
A/Vietnam/2004 containing full-length hemagglutinin. Immunization
with H5 can induce cross-protective CD8 immunity to H1N1 in mice,
and thus is considered a good model for cross protective immunity.
Effective cross-presentation of the inactivated H5N1 vaccine when
formulated with a cationic lipid is shown to lead to significantly
enhanced CTL against the CD8 epitope IYSTVASSL. The ability of the
cationic lipids to cause the exogenous HA proteins from the
inactivated virus to be internalized, processed and presented as a
peptide via the MHC-class I pathway in addition to presenting the
proteins via the MHC class II pathway provides a novel approach to
the development of an effective universal influenza vaccine based
on recombinant HA proteins or live attenuated and inactivated
viruses.
[0371] In various embodiments described herein, the influenza
vaccine comprises a glycoprotein antigen found on the surface of an
influenza virus. In one embodiment, the antigen is a hemagglutinin
antigen. In other embodiments, the hemagglutinin antigen comprises
an epitope region HA.sub.518-526.
[0372] In various embodiments described herein, the influenza
vaccine is a neuraminidase subunit vaccine. In other embodiments
described herein, influenza vaccine is an H3N2 vaccine. In yet
other embodiments described herein, influenza vaccine is an N1N1
vaccine. In other embodiments described herein, influenza vaccine
is a Brisbane vaccine. In yet other embodiments described herein,
influenza vaccine is an H1N1 vaccine.
[0373] In various embodiments described herein, the influenza
vaccine comprises one or more protein antigens from one or more
influenza viruses. In other embodiments described herein, the
influenza vaccine comprises an inactivated virus (e.g. an
inactivated whole virus). In yet other embodiments described
herein, the influenza vaccine comprises an attenuated virus. In
some embodiments described herein, the influenza vaccine comprises
a disrupted virus. In other embodiments described herein, the
influenza vaccine comprises a recombinant virus.
[0374] In various embodiments described herein, the vaccine
composition is capable of inducing a humoral immune response. As
used herein, the term "humoral immune response" is related to the
aspect of immunity that is mediated by macromolecules found in
extracellular fluids such as secreted antibodies, complement
proteins and certain antimicrobial peptides. In some embodiments,
the humoral immune response is an antibody response. In various
embodiments, the vaccine composition is capable of inducing a
humoral immune response against a conserved region of a
pathogen.
[0375] In various embodiments described herein, the vaccine
composition is capable of inducing a cellular immune response. As
used herein, the term "cellular immune response" is related to the
activation of phagocytes, antigen-specific cytotoxic T-lymphocytes,
the release of various cytokines in response to an antigen, and the
like. In some embodiments, the cellular immune response is a T cell
response. In certain embodiments, the T cell response is a CD 8+ T
cell response. In various embodiments, the vaccine composition is
capable of inducing a cellular immune response against a conserved
region of a pathogen.
[0376] In various embodiments described herein, the vaccine
composition is capable of effecting antigen cross presentation to
induce a humoral immune response and a cellular immune response in
the patient. In certain embodiments, the vaccine composition is
capable of cross-presentation of one or more antigens. In other
embodiments, the vaccine composition generates a humoral immune
response and a cellular immune response.
[0377] In various embodiments described herein, the vaccine
composition induces an immune response in a mammal by activating
the mitogen-activated protein (MAP) kinase signaling pathway.
Induction of an immune response by adjuvants such as cationic
lipids are described, for example, in PCT/US2008/057678
(WO/2008/116078; "Stimulation of an Immune Response by Cationic
Lipids") and PCT/US2009/040500 (WO/2009/129227; "Stimulation of an
Immune Response by Enantiomers of Cationic Lipids"), the entire
disclosures of both incorporated herein by reference. In some
embodiments, the MAP kinase signaling pathway is activated by
stimulating at least one of extracellular signal-regulated kinase
("ERK")-1, ERK-2, and p38. In other embodiments, the composition
enhances functional antigen-specific CD8+ T lymphocyte response.
The term "mammal" is well known to those of skill in the art. In
one embodiment, the mammal is a human.
[0378] In one embodiment described herein, a method of treating a
disease in a mammal is provided. The method comprises comprising
the step of administering an effective amount of a vaccine
composition to the mammal, wherein the vaccine composition
comprises at least one adjuvant and at least one antigen, and
wherein the adjuvant is a cationic lipid. The previously described
embodiments of the vaccine composition are applicable to the method
of treating a disease in a mammal described herein.
[0379] In some embodiments, "treatment," "treat," and "treating,"
as used herein with reference to infectious pathogens, refer to a
prophylactic treatment which increases the resistance of a subject
to infection with a pathogen or decreases the likelihood that the
subject will become infected with the pathogen; and/or treatment
after the subject has become infected in order to fight the
infection, e.g., reduce or eliminate the infection or prevent it
from becoming worse. In one embodiment, the method is a
prophylactic treatment.
[0380] In some embodiments, the disease is a pathogenic disease. In
other embodiments, the disease is caused by multiple strains of a
pathogen. In certain embodiments, the disease is influenza.
[0381] In various embodiments, the method induces a humoral immune
response in the mammal. In some embodiments, the humoral immune
response is an antibody response. In other embodiments, the humoral
immune response is against a conserved region of a pathogen.
[0382] In various embodiments, the method induces a cellular immune
response in the mammal. In some embodiments, the cellular immune
response is a T cell response. Ion other embodiments, the T cell
response is a CD 8+ T cell response. In certain embodiments, the
cellular immune response is against a conserved region of a
pathogen. In other embodiments, the method induces a humoral immune
response and a cellular immune response in the mammal.
[0383] In various embodiments, the mammal is a human. In some
embodiments, the administration activates an immune response via
the MAP kinase signaling pathway in cells of the immune system of
the mammal. In various embodiments, the MAP kinase signaling
pathway is activated by stimulating at least one of ERK-1, ERK-2,
and p38.
[0384] In other embodiments, the immune response activates
cytotoxic T lymphocytes in the mammal. In one embodiment, the
cytotoxic T lymphocytes are CD8+ T cells. In another embodiment,
the administration enhances functional antigen-specific CD8+ T
lymphocyte response. In yet another embodiment, the immune response
activates an antibody response in the mammal. In other embodiments,
the immune response activates interferon-gamma (IFN-.alpha.) in the
mammal.
[0385] In one embodiment described herein, a method of preventing a
disease in a mammal is provided. The method comprises comprising
the step of administering an effective amount of a vaccine
composition to the mammal, wherein the vaccine composition
comprises at least one adjuvant and at least one antigen, and
wherein the adjuvant is a cationic lipid. The previously described
embodiments of the vaccine composition and the method of treating a
disease in a mammal are applicable to the method of preventing a
disease in a mammal described herein.
[0386] In one embodiment described herein, a method of effecting
antigen cross presentation to induce a humoral immune response and
a cellular immune response in a mammal is provided. The method
comprises the step of administering an effective amount of a
vaccine composition to the mammal, wherein the vaccine composition
comprises at least one adjuvant and at least one antigen, and
wherein the adjuvant is a cationic lipid. The previously described
embodiments of the vaccine composition, the method of treating a
disease in a mammal, and the method of preventing a disease in a
mammal are applicable to the method of effecting antigen cross
presentation to induce a humoral immune response and a cellular
immune response in a mammal described herein.
EXAMPLE 1
Formulation of Influenza Vaccine
[0387] Sterile water for injection (WFI) or a buffer was used in
all liposome preparation procedures. In the present example,
R-DOTAP was used as an exemplary cationic lipid. Liposomes used
these studies were made using lipid films. Lipid films were made in
glass vials by (1) dissolving the lipids in an organic solvent such
as chloroform, and (2) evaporating the chloroform solution under a
steady stream of dry nitrogen gas. Traces of organic solvent were
removed by keeping the films under vacuum overnight. The lipid
films were then hydrated by adding the required amount of WFI or
buffer to make a final concentration of 4 mM or 8 mM R-DOTAP
cationic lipid. The suspensions were then extruded to a size of 200
nm and stored at 4.degree. C. Other cationic lipids and methods
used in general liposome preparation that are well known to those
skilled in the art may also be used.
[0388] A commercial influenza vaccine formulation containing three
influenza antigens B Brisbane, A/California/07/2009 (H1N1)
A/Perth/16/2009 (H3N2) was diluted to 60 .mu.g/ml or 12 .mu.g/ml in
PBS and then mixed 1:1 v/v with 8 mM or 4 mM R-DOTAP or PBS to
produce 30 and 6 .mu.g/ml in PBS, with 4 mM DOTAP, or 2 mM DOTAP,
or PBS. Mixing was performed by pipetting up and down. no emulsion
was created. Solution was slightly turbid, but transparent, typical
of DOTAP formulations. No precipitate was visible
EXAMPLE 2
Evaluation of the Protective Potency of a Cationic Lipid-Based
Influenza Vaccine: Protective Hemagglutination Inhibition Assay
Against a/Perth/16/2009 (H3N2)
[0389] C57BL/6J mice were injected subcutaneously in the shaved
flank with 100 .mu.l to deliver a final dose of 3 .mu.g or 0.6
.mu.g of the antigen in either PBS, 4 mM R-DOTAP or 2 mM R-DOTAP.
The mice were injected on day 0, then again with the identical
formulation on day 21. Tail vein bleeds were performed on days 14
and 35.
[0390] Serum was stored frozen at -80.degree. C. prior to testing.
Samples were coded with respect to the treatment groups. A
Hemagglutination inhibition assay was performed against the viruses
A/Perth/16/2009 (H3N2) to quantify the anti-influenza antibody
induction and resulting protective efficacy of the vaccines.
[0391] Four mice were tested per group:
1. Naive 2. 3 ug+PBS 3. 3 ug+4 mM R-DOTAP 4. 3 ug+2 mM R-DOTAP 5.
0.6 ug+PBS 6. 0.6 ug+4 mM R-DOTAP 7. 0.6 ug+2 mM R-DOTAP
[0392] The results are shown in FIG. 1. After the first injection
(day 14 bleed), the commercial vaccine demonstrated no protective
antibody production against the H3N2 virus. In contrast, the
cationic lipid-based vaccine however demonstrated a significant
increase in HAI titers. After injection 2 (day 35 bleed), the high
antigen dose vaccine shows about an 8-10 fold increase in antibody
induction potency with high or low amounts of R-DOTAP. After
injection 2 (day 35 bleed), the low antigen dose vaccine
demonstrated about a 40-fold increase in antibody induction potency
with either of the vaccine formulations containing the high or low
amounts of R-DOTAP. The low dose antigen vaccine with R-DOTAP
increased potency about 8-fold compared to the high antigen dose
commercial vaccine.
EXAMPLE 3
Evaluation of the Protective Potency of a Cationic Lipid-Based
Influenza Vaccine: Protective Hemagglutination Inhibition Assay
Against Pandemic Influenza Strain A/California/07/2009 (H1N1)
[0393] C57BL/6J mice were injected subcutaneously in the shaved
flank with 100 .mu.l to deliver a final dose of 3 .mu.g or 0.6
.mu.g of the antigen in either PBS, 4 mM R-DOTAP or 2 mM R-DOTAP.
The mice were injected on day 0, then again with the identical
formulation on day 21. Tail vein bleeds were performed on days 14
and 35.
[0394] Serum was stored frozen at -80.degree. C. prior to testing.
Samples were coded with respect to the treatment groups. A
Hemagglutination inhibition assay was performed against the virus
A/California/07/2009 (H1N1) to quantify the antibody induction and
protective efficacy of the vaccines.
[0395] Four mice were tested per group:
1. Naive 2. 3 ug+PBS 3. 3 ug+4 mM R-DOTAP 4. 3 ug+2 mM R-DOTAP 5.
0.6 ug+PBS 6. 0.6 ug+4 mM R-DOTAP 7. 0.6 ug+2 mM R-DOTAP
[0396] The results are shown in FIG. 2. After the first injection
(day 14 bleed), the cationic lipid-based vaccine demonstrated a
superior increase in HAI titers. After injection 2 (day 35 bleed)
the R-DOTAP based vaccine demonstrated a 2-8 fold increase in
antibody induction potency depending on antigen and cationic lipids
dose. After injection 2 (day 35 bleed), the low antigen dose
vaccine with R-DOTAP is at least as potent as the high antigen dose
commercial vaccine containing a 5-fold higher antigen dose.
EXAMPLE 4
Evaluation of the Protective Potency of a Cationic Lipid-Based
Influenza Vaccine: Protective Hemagglutination Inhibition Assay
Against Influenza Strain B Brisbane
[0397] C57BL/6J mice were injected subcutaneously in the shaved
flank with 100 .mu.l to deliver a final dose of 3 .mu.g or 0.6
.mu.g of the antigen in either PBS, 4 mM R-DOTAP or 2 mM R-DOTAP.
The mice were injected on day 0, then again with the identical
formulation on day 21. Tail vein bleeds were performed on days 14
and 35.
[0398] Serum was stored frozen at -80.degree. C. prior to testing.
Samples were coded with respect to the treatment groups. A
Hemagglutination inhibition assay was performed against the virus B
Brisbane to quantify the antibody induction and protective efficacy
of the vaccines.
[0399] Four mice were tested per group:
1. Naive 2. 3 ug+PBS 3. 3 ug+4 mM R-DOTAP 4. 3 ug+2 mM R-DOTAP 5.
0.6 ug+PBS 6. 0.6 ug+4 mM R-DOTAP 7. 0.6 ug+2 mM R-DOTAP
[0400] The results are shown in FIG. 3. After the first injection
(day 14 bleed), little difference between vaccines is observed with
no vaccine providing significant titers. After injection 2 (day 35
bleed), the R-DOTAP based vaccine demonstrated a 4-35 fold increase
in potency depending on antigen and cationic lipids dose. After
injection 2 (day 35 bleed), the low antigen dose vaccine depending
on R-DOTAP concentration is 4-8 times more potent than the high
antigen dose commercial vaccine containing a 5-fold higher antigen
dose.
EXAMPLE 5
Induction of CD8 T Cell Responses Following Vaccination with
R-DOTAP H5N1 Influenza Vaccine
[0401] There is considerable interest in developing an influenza
vaccine to induce broadly cross-protective immunity to different
subtypes of influenza. Existing TIV vaccines like Fluzone consist
of mostly HA protein and do not generate significant CD8 T cell
responses. Examples 2-4 show that R-DOTAP can greatly enhance the
antibody response to HA after Fluzone vaccination.
[0402] There is a well-established CD8 T cell epitope within
hemagglutinin from the mouse-adapted PR8 strain of virus (H1N1):
HA.sub.518-526, IYSTVASSL, K.sup.d restricted. The peptide
IYSTVASSL is used in an IFN.gamma. ELISPOT assay, along with an
irrelevant peptide to assess CD8 responses.
Approach:
[0403] Complete Freund's Adjuvant (CFA) was used as a positive
control since CFA is known to offer cross-presentation of antigens
will also stimulate CD8 T cell responses to whole ovalbumin. CFA
cannot be used in vaccines due its induction of severe and
potentially lethal inflammatory responses.
BALB/c mice, 5 mice/group Vaccinate on Day 0, boost on Day 7,
perform ELISPOT on day 14.
[0404] A. Naive
[0405] B. CFA only
[0406] C. H5N1 vaccine, 3 ug/mouse
[0407] D. H5N1 vaccine, 3 ug/mouse+CFA
[0408] E. H5N1 vaccine, 3 ug/mouse+R-DOTAP 4 mM
[0409] F. R-DOTAP only (4 mM)
Day 14:
[0410] Sacrifice, remove spleens and perform ELISPOT with the
HA.sub.518-526 peptide and an unrelated peptide.
ELISPOT Assay
[0411] IFN-gamma ELISPOT plates; 2.5.times.105 splenocytes/well,
stimulatory peptides: HA.sub.518-526 and HPV E6.sub.29-38
(irrelevant peptide), both at 10 mM. The ELISPOT plates were
developed and the plates scanned and IFN-gamma spots counted.
Conclusions:
[0412] Specific ELISPOTS were obtained to the HA.sub.518-526
epitope after vaccination with H5N1 alone, and greater number of
spots were obtained after adjuvanting with CFA or R-DOTAP (FIG. 4).
CFA enhanced the H5N1 spots only modestly, whereas R-DOTAP
stimulated a 2-fold enhancement of the response. The response was
specific: very low numbers of spots in the no-peptide wells or in
response to the irrelevant peptide. However, there were significant
"background" spots in the wells from CFA vaccinated mice (up to 25
spots). This is in keeping with the high level of non-specific
immune activation following CFA immunization.
[0413] Since vaccination was performed with the inactivated H5N1
vaccine containing full-length hemagglutinin and assayed for the T
cell response to an internal, class I-restricted peptide epitope,
this is an indicator of "cross-presentation" involving the
processing of an exogenous protein through the class I processing
pathway. Therefore, R-DOTAP is demonstrated to significantly
enhance cross presentation of an internal HA epitope that is known
to be cross-protective in mouse experiments.
EXAMPLE 6
Evaluation of Antibody Responses to a Multi-Epitope Peptide
Formulated with R-DOTAP
[0414] HLA-A2 mice were injected subcutaneously with R-DOTAP
formulated with HPV-16 E7 peptide (aa43-57). The mice were
vaccinated on days 1, 21, and 42 and blood was drawn on day 57 and
evaluated by ELISA for the induction of IgG and IgM antibodies to
the peptide vaccine.
Results:
TABLE-US-00001 [0415] TABLE 1 Individual Antibody Immune Response
Results (E7.sub.43-57) - Log Titers Pretest Day 57 Dose Group
Animal # Pretest IgG Day 57 IgG IgM IgM Group 1 104 <2 <2
<2 <2 0.086 mg R- 110 <2 <2 <2 <2 DOTAP 854 <2
<2 <2 <2 0.00 mg 941 <2 <2 <2 <2 Peptide 969
<2 <2 <2 <2 981 <2 <2 <2 2 982 <2 <2
<2 <2 987 <2 <2 <2 2 Group 2 105 <2 <2 <2
<2 0.086 mg R- 106 <2 <2 <2 <2 DOTAP 720 n/a <2
n/a <2 0.02 mg 851 <2 <2 <2 2 Peptide 984 <2 2 <2
2 988 <2 <2 <2 <2 992 <2 <2 <2 2 996 <2
<2 <2 <2
Conclusions:
[0416] When cationic lipid adjuvants are formulated with a T-cell
epitope peptide antibody responses are negligible. However, strong
CTL responses are observed.
EXAMPLE 7
Comparison of Immune Response in Cationic Lipid and Adjuvanted
Vaccine Formulations
[0417] The T-cell immune responses using vaccine formulations
comprising varying cationic lipid nanoparticles and varying antigen
assemblies were evaluated by ELISPOT. In this example, the vaccine
formulations were be formulated using various cationic lipid
nanoparticles DOEPC and DOTMA, and compared with the emulsion
adjuvant Montanide.TM..
[0418] Various different vaccine formulations were evaluated in the
present example. In one formulation, the antigen comprised the
peptide antigen palmitoy-KSSGQAEPDRAHYNIVTF [SEQ. ID. No. 3] (0.11
mM), and the cationic lipid DOEPC (1 mM). In a second formulation,
the antigen comprised the peptide antigen
palmitoy-KSSGQAEPDRAHYNIVTF [SEQ. ID. No. 3] (0.11 mM), and the
cationic lipid DOTMA (1 mM). In a third formulation, the antigen
assembly comprised the modified peptide antigen [SEQ. ID. No. 3]
(0.11 mM) and the emulsion adjuvant Montanide.TM..
[0419] T-cell potency of the various vaccine formulations was
evaluated by determining the antigen-specific immune response via
ELISPOT to the T-cell epitope peptide HPV-16 E7.sub.49-57 RAHYNIVTF
[SEQ. ID. No. 2].
Conclusions:
[0420] Specific ELISPOTS were obtained to the E7.sub.49-57 epitope
after vaccination of DOTMA, DOEPC and Montanide.TM., each
formulated with SEQ1. A greater number of spots was obtained after
formulating with the cationic lipids DOTMA or DOEPC compared to the
Montanide.TM. adjuvant (see FIG. 5). This example demonstrates show
that the cationic lipids act as potent immunomodulatory adjuvants
and induce superior CD8+ T-cell immune responses compared to the
emulsion adjuvant Montanide.TM..
EXAMPLE 8
Induction of CD8 T Cell Responses Following Vaccination with DOTMA
or DOEPC H5N1 Influenza Vaccine
[0421] There is a well-established CD8 T cell epitope within
hemagglutinin from the mouse-adapted PR8 strain of virus (H1N1):
HA.sub.518-526, IYSTVASSL, K.sup.d restricted. The peptide
IYSTVASSL is used in an IFN.gamma. ELISPOT assay, along with an
irrelevant peptide to assess CD8 responses. In the present example,
DOTMA or DOEPC (including enantiomers of each) may be used as the
exemplary cationic lipids.
Approach:
[0422] Complete Freund's Adjuvant (CFA) can be used as a positive
control since CFA is known to offer cross-presentation of antigens
will also stimulate CD8 T cell responses to whole ovalbumin. CFA
cannot be used in vaccines due its induction of severe and
potentially lethal inflammatory responses.
BALB/c mice, 5 mice/group can be evaluated Vaccinate on Day 0,
boost on Day 7, perform ELISPOT on day 14.
[0423] A. Naive
[0424] B. CFA only
[0425] C. H5N1 vaccine, 3 ug/mouse
[0426] D. H5N1 vaccine, 3 ug/mouse+CFA
[0427] E. H5N1 vaccine, 3 ug/mouse+R-DOTAP 4 mM
[0428] F. R-DOTAP only (4 mM)
Day 14:
[0429] Sacrifice, remove spleens and perform ELISPOT with the
HA.sub.518-526 peptide and an unrelated peptide.
ELISPOT Assay
[0430] IFN-gamma ELISPOT plates; 2.5.times.105 splenocytes/well,
stimulatory peptides: HA.sub.518-526 and HPV E6.sub.29-38
(irrelevant peptide), both at 10 mM. The ELISPOT plates can be
developed and the plates can be scanned and IFN-gamma spots can be
counted.
Sequence CWU 1
1
1119PRTHomo sapiens 1Arg Ala His Tyr Asn Ile Val Thr Phe 1 5
215PRTHomo sapiens 2Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile
Val Thr Phe 1 5 10 15 318PRTArtificial SequenceElongated peptide
3Lys Ser Ser Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val 1
5 10 15 Thr Phe 410PRTHomo sapiens 4Tyr Met Leu Asp Leu Gln Pro Glu
Thr Thr 1 5 10 513PRTArtificial SequenceElongated peptide 5Lys Ser
Ser Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr 1 5 10 623PRTArtificial
SequenceElongated peptide 6Lys Ser Ser Met His Gly Asp Thr Pro Thr
Leu His Glu Tyr Met Leu 1 5 10 15 Asp Leu Gln Pro Glu Thr Thr 20
720PRTArtificial SequenceElongated peptide 7Lys Ser Ser Leu Leu Met
Gly Thr Leu Gly Ile Val Cys Pro Ile Cys 1 5 10 15 Ser Gln Lys Pro
20 89PRTArtificial SequenceElongated peptide 8Lys Val Pro Arg Asn
Gln Asp Trp Leu 1 5 99PRTHomo sapiens 9Ser Tyr Val Asp Phe Phe Val
Trp Leu 1 5 109PRTArtificial SequenceElongated peptide 10Lys Tyr
Ile Cys Asn Ser Ser Cys Met 1 5 1112PRTArtificial SequenceElongated
peptide 11Lys Ser Ser Lys Val Pro Arg Asn Gln Asp Trp Leu 1 5
10
* * * * *