Patent Application
20190358318
The material in the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing text file, name PDS_16_1045WO_US_CON_Sequence_Listing, was created on Aug. 6, 2019, and is 3 kb. The file can be accessed using Microsoft Word on a computer that uses Windows OS.
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.
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 influence antigens and which can enable effective dose sparing of current antigen stocks are also being actively sought.
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.
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.
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.
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.
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.
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 IL 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.
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.
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 naïve T cells. DC maturation involves increased processing of microbial proteins and their presentation to T cells on major histocompatibility complex (MHC) molecules.
Some adjuvants have been demonstrated to activate signals that induce T helper cell (TH1) responses, characterized by IFNγ-producing T helper cells that activate antimicrobial effects at the effector site. Adjuvants such as the saponins drive TH1 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 TH2 responses.
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.
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.
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.
The killing of infected cells by both CTLs and TH1 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), IFNγ-producing CD8+ T cells offer more effective protection because the virus can be cleared with minimal host cell death. Similarly, IFNγ-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.
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.
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.
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.
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.
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.
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.
The following numbered embodiments are contemplated and are non-limiting:
1. A vaccine composition comprising at least one adjuvant and at least one antigen, wherein the adjuvant is a cationic lipid.
2. The vaccine composition of clause 1 wherein the cationic lipid is a non-steroidal cationic lipid.
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.
4. The vaccine composition of any one of clauses 1 to 3 wherein the cationic lipid is DOTAP.
5. The vaccine composition of any one of clauses 1 to 3 wherein the cationic lipid is DOTMA.
6. The vaccine composition of any one of clauses 1 to 3 wherein the cationic lipid is DOEPC.
7. The vaccine composition of any one of clauses 1 to 6 wherein the adjuvant is an enantiomer of the cationic lipid.
8. The vaccine composition of clause 7 wherein the enantiomer is purified.
9. The vaccine composition of clause 7 or clause 8 wherein the enantiomer is R-DOTAP or S-DOTAP.
10. The vaccine composition of clause 7 or clause 8 wherein the enantiomer is R-DOTAP.
11. The vaccine composition of clause 7 or clause 8 wherein the enantiomer is S-DOTAP.
12. The vaccine composition of clause 7 or clause 8 wherein the enantiomer is R-DOTMA or S-DOTMA.
13. The vaccine composition of clause 7 or clause 8 wherein the enantiomer is R-DOTMA.
14. The vaccine composition of clause 7 or clause 8 wherein the enantiomer is S-DOTMA.
15. The vaccine composition of clause 7 or clause 8 wherein the enantiomer is R-DOEPC or S-DOEPC.
16. The vaccine composition of clause 7 or clause 8 wherein the enantiomer is R-DOEPC.
17. The vaccine composition of clause 7 or clause 8 wherein the enantiomer is S-DOEPC.
18. The vaccine composition of any one of clauses 1 to 17 wherein one or more antigens is a protein-based antigen.
19. The vaccine composition of any one of clauses 1 to 17 wherein one or more antigens is a peptide-based antigen.
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.
21. The vaccine composition of any one of clauses 1 to 19 wherein one or more antigens is a viral antigen.
22. The vaccine composition of any one of clauses 1 to 19 wherein one or more antigens is a fungal antigen.
23. The vaccine composition of any one of clauses 1 to 19 wherein one or more antigens is a bacterial antigen.
24. The vaccine composition of any one of clauses 1 to 19 wherein one or more antigens is a pathogenic antigen.
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.
26. The vaccine composition of clause 24 wherein the pathogenic antigen is a synthetic or recombinant antigen.
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), KSSMHGDTPTLHEYMLDLQPET (SEQ. ID. NO: 6), KSSLLMGTIGIVCPICSQKP (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).
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.
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]).
30. The vaccine composition of any one of clauses 1 to 20 wherein the antigen is RAHYNIVTF (SEQ. ID. NO: 1).
31. The vaccine composition of any one of clauses 1 to 20 wherein the antigen is GQAEPDRAHYNIVTF (SEQ. ID. NO: 2).
32. The vaccine composition of any one of clauses 1 to 20 wherein the antigen is KSSGQAEPDRAHYNIVTF (SEQ. ID. NO: 3).
33. The vaccine composition of any one of clauses 1 to 20 wherein the antigen is YMLDLQPETT (SEQ. ID. NO: 4).
34. The vaccine composition of any one of clauses 1 to 20 wherein the antigen is KSSYMLDLQPETT (SEQ. ID. NO: 5).
35. The vaccine composition of any one of clauses 1 to 20 wherein the antigen is KSSMHGDTPTLHEYMLDLQPETT (SEQ. ID. NO: 6).
36. The vaccine composition of any one of clauses 1 to 20 wherein the antigen is KSSLLMGTLGIVCPICSQKP (SEQ. ID. NO: 7).
37. The vaccine composition of any one of clauses 1 to 20 wherein the antigen is KVPRNQDWL (SEQ. ID. NO: 8).
38. The vaccine composition of any one of clauses 1 to 20 wherein the antigen is SYVDFFVWL (SEQ. ID. NO: 9).
39. The vaccine composition of any one of clauses 1 to 20 wherein the antigen is KYICNSSCM (SEQ. ID. NO: 10).
40. The vaccine composition of any one of clauses 1 to 20 wherein the antigen is KSSKVPRNQDWL (SEQ. ID. NO: 11).
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.
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.
43. The vaccine composition of any one of clauses 1 to 42 wherein at least one antigen is a modified protein or peptide.
44. The vaccine composition of any one of clauses 1 to 43 wherein the modified protein or peptide is bonded to a hydrophobic group.
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.
46. The vaccine composition of clause 45 wherein the hydrophobic group is a palmitoyl group.
47. The vaccine composition of any one of clauses 1 to 46 wherein at least one antigen is an unmodified protein or peptide.
48. The vaccine composition of any one of clauses 1 to 47 wherein the vaccine composition is a universal vaccine.
49. The vaccine composition of any one of clauses 1 to 48 wherein the vaccine composition is an anti-viral vaccine.
50. The vaccine composition of any one of clauses 1 to 48 wherein the vaccine composition is an anti-fungal vaccine.
51. The vaccine composition of any one of clauses 1 to 48 wherein the vaccine composition is an anti-bacterial vaccine.
52. The vaccine composition of any one of clauses 1 to 48 wherein the vaccine composition is an influenza vaccine.
53. The vaccine composition of clause 52 wherein the influenza vaccine is a universal influenza vaccine.
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.
55. The vaccine composition of clause 54 wherein the antigen is a hemagglutinin antigen.
56. The vaccine composition of clause 55 wherein the hemagglutinin antigen comprises an epitope region HA518-526.
57. The vaccine composition of clause 55 wherein the influenza vaccine is a neuraminidase subunit vaccine.
58. The vaccine composition of any one of clauses 52 to 57 wherein the influenza vaccine is an H3N2 vaccine.
59. The vaccine composition of any one of clauses 52 to 57 wherein the influenza vaccine is an N1N1 vaccine.
60. The vaccine composition of any one of clauses 52 to 57 wherein the influenza vaccine is a Brisbane vaccine.
61. The vaccine composition of any one of clauses 52 to 57 wherein the influenza vaccine is an H1N1 vaccine.
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.
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).
64. The vaccine composition of any one of clauses 52 to 61 wherein the influenza vaccine comprises an attenuated virus.
65. The vaccine composition of any one of clauses 52 to 61 wherein the influenza vaccine comprises a disrupted virus.
66. The vaccine composition of any one of clauses 52 to 61 wherein the influenza vaccine comprises a recombinant virus.
67. The vaccine composition of any one of clauses 1 to 67 wherein the vaccine composition is capable of inducing a humoral immune response.
68. The vaccine composition of clause 67 wherein the humoral immune response is an antibody response.
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.
70. The vaccine composition of any one of clauses 1 to 69 wherein the vaccine composition is capable of inducing a cellular immune response.
71. The vaccine composition of clause 70 wherein the cellular immune response is a T cell response.
72. The vaccine composition of clause 71 wherein the T cell response is a CD 8+ T cell response.
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.
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.
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.
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.
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.
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.
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.
80. The vaccine composition of clause 79 wherein the mammal is a human.
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.
82. The method of clause 81 wherein the disease is a pathogenic disease.
83. The method of clause 81 or clause 82 wherein the disease is caused by multiple strains of a pathogen.
84. The method of any one of clauses 81 to 83 wherein the disease is influenza.
85. The method of any one of clauses 81 to 84 wherein the method induces a humoral immune response in the mammal.
86. The method of clause 85 wherein the humoral immune response is an antibody response.
87. The method of clause 85 or clause 86 wherein the humoral immune response is against a conserved region of a pathogen.
88. The method of any one of clauses 81 to 87 wherein the method induces a cellular immune response in the mammal.
89. The method of clause 88 wherein the cellular immune response is a T cell response.
90. The method of clause 89 wherein the T cell response is a CD 8+ T cell response.
91. The method of any one of clauses 88 to 90 wherein the cellular immune response is against a conserved region of a pathogen.
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.
93. The method of any one of clauses 88 to 92 wherein the cationic lipid is a non-steroidal cationic lipid.
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.
95. The method of any one of clauses 88 to 94 wherein the cationic lipid is DOTAP.
96. The method of any one of clauses 88 to 94 wherein the cationic lipid is DOTMA.
97. The method of any one of clauses 88 to 94 wherein the cationic lipid is DOEPC.
98. The method of any one of clauses 88 to 97 wherein the adjuvant is an enantiomer of the cationic lipid.
99. The method of clause 98 wherein the enantiomer is purified.
100. The method of clause 98 or clause 99 wherein the enantiomer is R-DOTAP or S-DOTAP.
101. The method of clause 98 or clause 99 wherein the enantiomer is R-DOTAP.
102. The method of clause 98 or clause 99 wherein the enantiomer is S-DOTAP.
103. The method of clause 98 or clause 99 wherein the enantiomer is R-DOTMA or S-DOTMA.
104. The method of clause 98 or clause 99 wherein the enantiomer is R-DOTMA.
105. The method of clause 98 or clause 99 wherein the enantiomer is S-DOTMA.
106. The method of clause 98 or clause 99 wherein the enantiomer is R-DOEPC or S-DOEPC.
107. The method of clause 98 or clause 99 wherein the enantiomer is R-DOEPC.
108. The method of clause 98 or clause 99 wherein the enantiomer is S-DOEPC.
109. The method of any one of clauses 81 to 109 wherein one or more antigens is a protein-based antigen.
110. The method of any one of clauses 81 to 109 wherein one or more antigens is a peptide-based antigen.
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.
112. The method of any one of clauses 81 to 110 wherein one or more antigens is a viral antigen.
113. The method of any one of clauses 81 to 110 wherein one or more antigens is a fungal antigen.
114. The method of any one of clauses 81 to 110 wherein one or more antigens is a bacterial antigen.
115. The method of any one of clauses 81 to 110 wherein one or more antigens is a pathogenic antigen.
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.
117. The method of clause 115 or clause 116 wherein the pathogenic antigen is a synthetic or recombinant antigen.
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), KSSMHGDTPTLHEYMLDLQPET (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).
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.
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]).
121. The method of any one of clauses 81 to 117 wherein the antigen is RAHYNIVTF (SEQ. ID. NO: 1).
122. The method of any one of clauses 81 to 117 wherein the antigen is GQAEPDRAHYNIVTF (SEQ. ID. NO: 2).
123. The method of any one of clauses 81 to 117 wherein the antigen is KSSGQAEPDRAHYNIVTF (SEQ. ID. NO: 3).
124. The method of any one of clauses 81 to 117 wherein the antigen is YMLDLQPETT (SEQ. ID. NO: 4).
125. The method of any one of clauses 81 to 117 wherein the antigen is KSSYMLDLQPETF (SEQ. ID. NO: 5).
126. The method of any one of clauses 81 to 117 wherein the antigen is KSSMHGDTPTLHEYMLDLQPET (SEQ. ID. NO: 6).
127. The method of any one of clauses 81 to 117 wherein the antigen is KSSLLMGTLGIVCPICSQKP (SEQ. ID. NO: 7).
128. The method of any one of clauses 81 to 117 wherein the antigen is KVPRNQDWL (SEQ. ID. NO: 8).
129. The method of any one of clauses 81 to 117 wherein the antigen is SYVDFFVWL (SEQ. ID. NO: 9).
130. The method of any one of clauses 81 to 117 wherein the antigen is KYICNSSCM (SEQ. ID. NO: 10).
131. The method of any one of clauses 81 to 117 wherein the antigen is KSSKVPRNQDWL (SEQ. ID. NO: 11).
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.
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.
134. The method of any one of clauses 81 to 133 wherein at least one antigen is a modified protein or peptide.
135. The method of clause 134 wherein the modified protein or peptide is bonded to a hydrophobic group.
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.
137. The method of clause 136 wherein the hydrophobic group is a palmitoyl group.
138. The method of any one of clauses 81 to 137 wherein at least one antigen is an unmodified protein or peptide.
139. The method of any one of clauses 81 to 138 wherein the vaccine composition is a universal vaccine.
140. The method of any one of clauses 81 to 138 wherein the vaccine composition is an anti-viral vaccine.
141. The method of any one of clauses 81 to 138 wherein the vaccine composition is an anti-fungal vaccine.
142. The method of any one of clauses 81 to 138 wherein the vaccine composition is an anti-bacterial vaccine.
143. The method of any one of clauses 81 to 138 wherein the vaccine composition is an influenza vaccine.
144. The method of any one of clauses 81 to 138 wherein the influenza vaccine is a universal influenza vaccine.
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.
146. The method of clause 145 wherein the antigen is a hemagglutinin antigen.
147. The method of clause 146 wherein the hemagglutinin antigen comprises an epitope region HA518-526.
148. The method of clause 143 or clause 144 wherein the influenza vaccine is a neuraminidase subunit vaccine.
149. The method of any one of clauses 143 to 148 wherein the influenza vaccine is an H3N2 vaccine.
150. The method of any one of clauses 143 to 148 wherein the influenza vaccine is an H1N1 vaccine.
151. The method of any one of clauses 143 to 148 wherein the influenza vaccine is a Brisbane vaccine.
152. The method of any one of clauses 143 to 148 wherein the influenza vaccine is an H1N1 vaccine.
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.
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).
155. The method of any one of clauses 143 to 152 wherein the influenza vaccine comprises an attenuated virus.
156. The method of any one of clauses 143 to 152 wherein the influenza vaccine comprises a disrupted virus.
157. The method of any one of clauses 143 to 152 wherein the influenza vaccine comprises a recombinant virus.
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.
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.
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.
161. The method of any one of clauses 81 to 138 wherein the mammal is a human.
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.
163. The method of clause 162 wherein the disease is a pathogenic disease.
164. The method of clause 162 or clause 163 wherein the disease is caused by multiple strains of a pathogen.
165. The method of any one of clauses 162 to 164 wherein the disease is influenza.
166. The method of any one of clauses 162 to 165 wherein the method induces a humoral immune response in the mammal.
167. The method of clause 166 wherein the humoral immune response is an antibody response.
168. The method of clause 166 or clause 167 wherein the humoral immune response is against a conserved region of a pathogen.
169. The method of any one of clauses 162 to 168 wherein the method induces a cellular immune response in the mammal.
170. The method of clause 169 wherein the cellular immune response is a T cell response.
171. The method of clause 170 wherein the T cell response is a CD 8+ T cell response.
172. The method of any one of clauses 169 to 171 wherein the cellular immune response is against a conserved region of a pathogen.
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.
174. The method of any one of clauses 162 to 173 wherein the cationic lipid is a non-steroidal cationic lipid.
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.
176. The method of any one of clauses 162 to 175 wherein the cationic lipid is DOTAP.
177. The method of any one of clauses 162 to 175 wherein the cationic lipid is DOTMA.
178. The method of any one of clauses 162 to 175 wherein the cationic lipid is DOEPC.
179. The method of any one of clauses 162 to 178 wherein the adjuvant is an enantiomer of a cationic lipid.
180. The method of clause 179 wherein the enantiomer is purified.
181. The method of clause 179 or clause 180 wherein the enantiomer is R-DOTAP or S-DOTAP.
182. The method of clause 179 or clause 180 wherein the enantiomer is R-DOTAP.
183. The method of clause 179 or clause 180 wherein the enantiomer is S-DOTAP.
184. The method of clause 179 or clause 180 wherein the enantiomer is R-DOTMA or S-DOTMA.
185. The method of clause 179 or clause 180 wherein the enantiomer is R-DOTMA.
186. The method of clause 179 or clause 180 wherein the enantiomer is S-DOTMA
187. The method of clause 179 or clause 180 wherein the enantiomer is R-DOEPC or S-DOEPC.
188. The method of clause 179 or clause 180 wherein the enantiomer is R-DOEPC.
189. The method of clause 179 or clause 180 wherein the enantiomer is S-DOEPC.
190. The method of any one of clauses 162 to 189 wherein one or more antigens is a protein-based antigen.
191. The method of any one of clauses 162 to 190 wherein one or more antigens is a peptide-based antigen.
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.
193. The method of any one of clauses 162 to 191 wherein one or more antigens is a viral antigen.
194. The method of any one of clauses 162 to 191 wherein one or more antigens is a fungal antigen.
195. The method of any one of clauses 162 to 191 wherein one or more antigens is a bacterial antigen.
196. The method of any one of clauses 162 to 191 wherein one or more antigens is a pathogenic antigen.
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.
198. The method of any one of clauses 162 to 197 wherein the pathogenic antigen is a synthetic or recombinant antigen.
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), KSSMHGDTPTLHEYMLDLQPETI (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).
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.
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]).
202. The method of any one of clauses 162 to 198 wherein the antigen is RAHYNIVTF (SEQ. ID. NO: 1).
203. The method of any one of clauses 162 to 198 wherein the antigen is GQAEPDRAHYNIVTF (SEQ. ID. NO: 2).
204. The method of any one of clauses 162 to 198 wherein the antigen is KSSGQAEPDRAHYNIVTF (SEQ. ID. NO: 3).
205. The method of any one of clauses 162 to 198 wherein the antigen is YMLDLQPETT (SEQ. ID. NO: 4).
206. The method of any one of clauses 162 to 198 wherein the antigen is KSSYMLDLQPETT (SEQ. ID. NO: 5).
207. The method of any one of clauses 162 to 198 wherein the antigen is KSSMHGDTPTLHEYMLDLQPET (SEQ. ID. NO: 6).
208. The method of any one of clauses 162 to 198 wherein the antigen is KSSLLMGTLGTVCPICSQKP (SEQ. ID. NO: 7).
209. The method of any one of clauses 162 to 198 wherein the antigen is KVPRNQDWL (SEQ. ID. NO: 8).
210. The method of any one of clauses 162 to 198 wherein the antigen is SYVDFFVWL (SEQ. ID. NO: 9).
211. The method of any one of clauses 162 to 198 wherein the antigen is KYICNSSCM (SEQ. ID. NO: 10).
212. The method of any one of clauses 162 to 198 wherein the antigen is KSSKVPRNQDWL (SEQ. ID. NO: 11).
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.
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.
215. The method of any one of clauses 162 to 213 wherein at least one antigen is a modified protein or peptide.
216. The method of clause 215 wherein the modified protein or peptide is bonded to a hydrophobic group.
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.
218. The method of clause 217 wherein the hydrophobic group is a palmitoyl group.
219. The method of any one of clauses 162 to 218 wherein at least one antigen is an unmodified protein or peptide.
220. The method of any one of clauses 162 to 219 wherein the vaccine composition is a universal vaccine.
221. The method of any one of clauses 162 to 220 wherein the vaccine composition is an anti-viral vaccine.
222. The method of any one of clauses 162 to 219 wherein the vaccine composition is an anti-fungal vaccine.
223. The method of any one of clauses 162 to 219 wherein the vaccine composition is an anti-bacterial vaccine.
224. The method of any one of clauses 162 to 219 wherein the vaccine composition is an influenza vaccine.
225. The method of clause 224 wherein the influenza vaccine is a universal influenza vaccine.
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.
227. The method of clause 226 wherein the antigen is a hemagglutinin antigen.
228. The method of clause 227 wherein the hemagglutinin antigen comprises an epitope region HA518-526.
229. The method of any one of clauses 224 to 228 wherein the influenza vaccine is a neuraminidase subunit vaccine.
230. The method of any one of clauses 224 to 229 wherein the influenza vaccine is an H3N2 vaccine.
231. The method of any one of clauses 224 to 229 wherein the influenza vaccine is an N1N1 vaccine.
232. The method of any one of clauses 224 to 229 wherein the influenza vaccine is a Brisbane vaccine.
233. The method of any one of clauses 224 to 229 wherein the influenza vaccine is an H1N1 vaccine.
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.
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).
236. The method of any one of clauses 224 to 233 wherein the influenza vaccine comprises an attenuated virus.
237. The method of any one of clauses 224 to 233 wherein the influenza vaccine comprises a disrupted virus.
238. The method of any one of clauses 224 to 233 wherein the influenza vaccine comprises a recombinant virus.
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.
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.
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.
242. The method of any one of clauses 162 to 241 wherein the mammal is a human.
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.
244. The method of clause 243 wherein the humoral immune response is an antibody response.
245. The method of clause 243 or clause 244 wherein the humoral immune response is against a conserved region of a pathogen.
246. The method of any one of clauses 243 to 245 wherein the cellular immune response is a T cell response.
247. The method of clause 246 wherein the T cell response is a CD 8+ T cell response.
248. The method of any one of clauses 243 to 247 wherein the cellular immune response is against a conserved region of a pathogen.
249. The method of any one of clauses 243 to 248 wherein the cationic lipid is a non-steroidal cationic lipid.
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.
251. The method of any one of clauses 243 to 250 wherein the cationic lipid is DOTAP.
252. The method of any one of clauses 243 to 250 wherein the cationic lipid is DOTMA.
253. The method of any one of clauses 243 to 250 wherein the cationic lipid is DOEPC.
254. The method of any one of clauses 243 to 249 wherein the adjuvant is an enantiomer of a cationic lipid.
255. The method of clause 254 wherein the enantiomer is purified.
256. The method of clause 254 or clause 255 wherein the enantiomer is R-DOTAP or S-DOTAP.
257. The method of clause 254 or clause 255 wherein the enantiomer is R-DOTAP.
258. The method of clause 254 or clause 255 wherein the enantiomer is S-DOTAP.
259. The method of clause 254 or clause 255 wherein the enantiomer is R-DOTMA or S-DOTMA.
260. The method of clause 254 or clause 255 wherein the enantiomer is R-DOTMA.
261. The method of clause 254 or clause 255 wherein the enantiomer is S-DOTMA.
262. The method of clause 254 or clause 255 wherein the enantiomer is R-DOEPC or S-DOEPC.
263. The method of clause 254 or clause 255 wherein the enantiomer is R-DOEPC.
264. The method of clause 254 or clause 255 wherein the enantiomer is S-DOEPC.
265. The method of any one of clauses 243 to 264 wherein one or more antigens is a protein-based antigen.
266. The method of any one of clauses 243 to 264 wherein one or more antigens is a peptide-based antigen.
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.
268. The method of any one of clauses 243 to 266 wherein one or more antigens is a viral antigen.
269. The method of any one of clauses 243 to 266 wherein one or more antigens is a fungal antigen.
270. The method of any one of clauses 243 to 266 wherein one or more antigens is a bacterial antigen.
271. The method of any one of clauses 243 to 266 wherein one or more antigens is a pathogenic antigen.
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.
273. The method of clause 271 or clause 272 wherein the pathogenic antigen is a synthetic or recombinant antigen.
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).
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.
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]).
277. The method of any one of clauses 243 to 273 wherein the antigen is RAHYNIVTF (SEQ. ID. NO: 1).
278. The method of any one of clauses 243 to 273 wherein the antigen is GQAEPDRAHYNIVTF (SEQ. ID. NO: 2).
279. The method of any one of clauses 243 to 273 wherein the antigen is KSSGQAEPDRAHYNIVTF (SEQ. ID. NO: 3).
280. The method of any one of clauses 243 to 273 wherein the antigen is YMLDLQPETT (SEQ. ID. NO: 4).
281. The method of any one of clauses 243 to 273 wherein the antigen is KSSYMLDLQPETT (SEQ. ID. NO: 5).
282. The method of any one of clauses 243 to 273 wherein the antigen is KSSMHGDTPTLHEYMLDLQPETT (SEQ. ID. NO: 6).
283. The method of any one of clauses 243 to 273 wherein the antigen is KSSLLMGTLGIVCPICSQKP (SEQ. ID. NO: 7).
284. The method of any one of clauses 243 to 273 wherein the antigen is KVPRNQDWL (SEQ. ID. NO: 8).
285. The method of any one of clauses 243 to 273 wherein the antigen is SYVDFFVWL (SEQ. ID. NO: 9).
286. The method of any one of clauses 243 to 273 wherein the antigen is KYICNSSCM (SEQ. ID. NO: 10).
287. The method of any one of clauses 243 to 273 wherein the antigen is KSSKVPRNQDWL (SEQ. ID. NO: 11).
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.
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.
290. The method of any one of clauses 243 to 289 wherein at least one antigen is a modified protein or peptide.
291. The method of clause 290 wherein the modified protein or peptide is bonded to a hydrophobic group.
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.
293. The method of clause 292 wherein the hydrophobic group is a palmitoyl group.
294. The method of any one of clauses 243 to 293 wherein at least one antigen is an unmodified protein or peptide.
295. The method of any one of clauses 243 to 294 wherein the vaccine composition is a universal vaccine.
296. The method of any one of clauses 243 to 295 wherein the vaccine composition is an anti-viral vaccine.
297. The method of any one of clauses 243 to 295 wherein the vaccine composition is an anti-fungal vaccine.
298. The method of any one of clauses 243 to 295 wherein the vaccine composition is an anti-bacterial vaccine.
299. The method of any one of clauses 243 to 295 wherein the vaccine composition is an influenza vaccine.
300. The method of clause 299 wherein the influenza vaccine is a universal influenza vaccine.
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.
302. The method of clause 301 wherein the antigen is a hemagglutinin antigen.
303. The method of clause 302 wherein the hemagglutinin antigen comprises an epitope region HA518-526.
304. The method of any one of clauses 299 to 303 wherein the influenza vaccine is a neuraminidase subunit vaccine.
305. The method of any one of clauses 299 to 304 wherein the influenza vaccine is an H3N2 vaccine.
306. The method of any one of clauses 299 to 304 wherein the influenza vaccine is an N1N1 vaccine.
307. The method of any one of clauses 299 to 304 wherein the influenza vaccine is a Brisbane vaccine.
308. The method of any one of clauses 299 to 304 wherein the influenza vaccine is an H1N1 vaccine.
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.
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).
311. The method of any one of clauses 299 to 308 wherein the influenza vaccine comprises an attenuated virus.
312. The method of any one of clauses 299 to 308 wherein the influenza vaccine comprises a disrupted virus.
313. The method of any one of clauses 299 to 308 wherein the influenza vaccine comprises a recombinant virus.
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.
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.
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.
317. The method of any one of clauses 243 to 316 wherein the mammal is a human.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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)-carbamoylicholesterol (BGTC); 15 N,N.sup. 1 N.sup.2N.sup.3Tetra-methyltetrapalmitylspermine (cellfectin); N-t-butyl-N′-tetradecyl-3-tetradecyl-aminopropion-ami dine (CLONfectin); dimethyldioctadecyl ammonium bromide (DDAB); 1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DMRIE); 2,3-dio leoylo xy-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′-tetramethyl-N,N′-bis(2-hydroxyethyl)-2,3-diolcoyloxy-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); 0,0′-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); 1,2-10 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.
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.
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.
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).
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.
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.
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, KSSYMLDLQPETI (SEQ. ID. NO: 5) is modified to further comprise a hydrophobic group. In one embodiment, the hydrophobic group is a palmitoyl group.
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.
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.
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.
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.
In one embodiment, the antigens comprise the gp100 sequence 0 (KVPRNQDWL [SEQ. ID. No. 8]) or the TRP2 sequence (SYVDFFVWL [SEQ. ID. No. 9]).
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.
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.
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.
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): HA518-576, IYSTVASSL, Kd 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.
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 HA518-526.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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-a) in the mammal.
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.
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.
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° 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.
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 μg/ml or 12 μg/ml in PBS and then mixed 1:1 v/v with 8 mM or 4 mM R-DOTAP or PBS to produce 30 and 611 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
C57BL/6J mice were injected subcutaneously in the shaved flank with 100 ul to deliver a final dose of 3 μg or 0.6 μg 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.
Serum was stored frozen at −80° 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.
Four mice were tested per group:
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
The results are shown in
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.
C57BL/6J mice were injected subcutaneously in the shaved flank with 100μl to deliver a final dose of 3 μg or 0.6 μg 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.
Serum was stored frozen at −80° 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.
Four mice were tested per group:
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
The results are shown in
C57BL/6J mice were injected subcutaneously in the shaved flank with 100l to deliver a final dose of 3 μg or 0.6 μg 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.
Serum was stored frozen at −80° 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.
Four mice were tested per group:
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
The results are shown in
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.
There is a well-established CD8 T cell epitope within hemagglutinin from the mouse-adapted PR8 strain of virus (H1N1): HA518-526, IYSTVASSL, Kd restricted. The peptide IYSTVASSL is used in an IFNγ ELISPOT assay, along with an irrelevant peptide to assess CD8 responses.
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.
A. Naive
B. CFA only
C. H5N1 vaccine, 3 ug/mouse
D. H5N1 vaccine, 3 ug/mouse+CFA
E. H5N1 vaccine, 3 ug/mouse+R-DOTAP4 mM
F. R-DOTAP only (4 mM)
Sacrifice, remove spleens and perform ELISPOT with the HA518-526 peptide and an unrelated peptide.
IFN-gamma ELISPOT plates; 2.5×105 splenocytes/well, stimulatory peptides: HA518-526 and HPV E629-38 (irrelevant peptide), both at 10 mM. The ELISPOT plates were developed and the plates scanned and IFN-gamma spots counted.
Specific ELISPOTS were obtained to the HA518-526 epitope after vaccination with H5N1 alone, and greater number of spots were obtained after adjuvanting with CFA or R-DOTAP (
Since vaccination was performed with the inactivated H5N1 vaccine containing full-length hemagglutinin and assayed for the T cell response to an internal, class 1-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.
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.
When cationic lipid adjuvants are formulated with a T-cell epitope peptide antibody responses are negligible. However, strong CTL responses are observed.
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™.
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™.
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 E749-57 RAHYNIVTF [SEQ. ID. No. 2].
Specific ELISPOTS were obtained to the E749-57 epitope after vaccination of DOTMA, DOEPC and Montanide™, each formulated with SEQI. A greater number of spots was obtained after formulating with the cationic lipids DOTMA or DOEPC compared to the Montanide™ adjuvant (see
There is a well-established CD8 T cell epitope within hemagglutinin from the mouse-adapted PR8 strain of virus (H1N1): HA518-526, IYSTVASSL, Kd restricted. The peptide IYSTVASSL is used in an IFNγ 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.
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.
A. Naive
B. CFA only
C. H5N1 vaccine, 3 ug/mouse
D. H5N1 vaccine, 3 ug/mouse+CFA
E. H5N1 vaccine, 3 ug/mouse+R-DOTAP4 mM
F. R-DOTAP only (4 mM)
Sacrifice, remove spleens and perform ELISPOT with the HA518-526 peptide and an unrelated peptide.
IFN-gamma ELISPOT plates; 2.5×105 splenocytes/well, stimulatory peptides: HA518-526 and HPV E629-38 (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.
| Number | Date | Country | |
|---|---|---|---|
| 61703814 | Sep 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14429123 | Mar 2015 | US |
| Child | 16388291 | US |
