LIPOSOME COMPOSITIONS UNDERSTANDING AN ADJUVANT AND USES OF THESE COMPOSITIONS AND USES OF THOSE COM

专利摘要:
liposome compositions comprising an adjuvant that activates or increases the activity of tlr2 and uses thereof. the invention relates to compositions comprising liposomes, an antigen capable of inducing a humoral immune response, a vehicle comprising a continuous phase of a hydrophobic substance, and an adjuvant that activates or increases the activity of tlr2. the invention also relates to the use of such compositions in inducing a humoral response and methods for their use in the treatment of a disease, disorder or malaise ameliorated by a humoral immune response.
公开号:BR112014007927B1
申请号:R112014007927-7
申请日:2012-10-05
公开日:2021-04-13
发明作者:Marc Mansour；Lisa Diana Macdonald；Genevieve Mary Weir；Leeladhar Sammatur；Kendall Sharp
申请人:Immunovaccine Technologies Inc；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED REQUESTS
[001] This application claims priority benefit to United States Provisional Patent Application No. 61 / 544,020, filed on October 6, 2011, which is hereby incorporated by reference in its entirety. FIELD OF THE INVENTION
[002] The present application relates to vaccine compositions that enhance the production of antigen-specific antibodies in immunized individuals. BACKGROUND OF THE INVENTION
[003] Immune responses induced by vaccination can be broadly categorized into humoral or cellular types. A humoral response is typically desired to protect against viral or bacterial invaders, so immunity against virally infected cells and cancer cells typically involves a cell-mediated response. Humoral immunity is characterized by high levels of antibody production by B cells, so cellular immunity is characterized by increased activation of cytotoxic CD8 T lymphocytes.
[004] The type of immunity induced by a vaccine depends largely on the type of adjuvant included in the vaccine. Adjuvants based on palmitic acid, such as dipalmitoyl-S-glyceryl-cysteine (PAM2Cys) and tripalmitoyl-S-glyceryl-cysteine (PAMaCys), and variants of these, have been reported to enhance humoral and cellular responses against a variety of antigens. For practical reasons, the solubility of such adjuvants has typically been improved with the addition of hydrophilic non-immunogenic amino acid residues (Lysines, for example). Such adjuvants have been mixed with antigen, but in many instances, adjuvants based on palmitic acid have been covalently linked to antigens prior to administration to an individual. Palmitic acid adjuvants were also codistributed with antigen using liposomes as vehicles. Protein-based and carbohydrate-based antigens have been combined with palmitic acid adjuvants to produce antibody and T cell responses. The use of palmitic acid adjuvants for cancer applications is well documented, with activity mediated primarily by cellular responses.
[005] Although derivatives of palmitic acid are known to proliferate B cells, induce isotypic switching, induce differentiation of human B lymphocytes into plasma cells of IgG secretion and increase the expression of various costimulatory molecules (MHC I, II, CD80 , etc), reports of palmitic acid adjuvants inducing antibody responses have varied in the literature from being able to enhance antibody responses as they are not particularly useful for generating such responses.
[006] Thus, there remains a need to develop vaccine compositions for generating strong humoral responses against a variety of antigens. The present invention provides vaccine compositions that contain a lipid-based adjuvant, and are particularly useful for inducing a high level of antibodies in humanized individuals. SUMMARY OF THE INVENTION
[007] In one aspect, a composition is provided comprising: liposomes; an antigen capable of inducing a humoral immune response; a vehicle comprising a continuous phase of a hydrophobic substance; and an adjuvant that activates or increases the activity of toll-like receptor 2 (TLR2), for example, by interaction with a TLR2 dimer, such as TLR1 / 2 or TLR2 / 6.
[008] In an embodiment of the composition as described herein, the adjuvant is a lipid-based adjuvant.
[009] In an embodiment of the composition as described herein, the lipid-based adjuvant is a palmitic acid adjuvant.
[0010] In one embodiment of the composition as described herein, the lipid-based adjuvant comprises dipalmitoyl-S-glyceryl-cysteine (PAM2Cys) or tripalmitoyl-S-glyceryl-cysteine (PAM3Cys); or the lipid-based adjuvant is Pam-2-Cys-Ser- (Lys) 4 or Pam-3-Cys- Ser- (Lys) 4.
[0011] In another embodiment of the composition as described herein, the lipid-based adjuvant is, or comprises: the diacylated synthetic lipoprotein FSL-1 (Pam2CGDPKHPKSF), a synthetic lipoprotein derived from Mycoplasma salivarium, or the macrophage-activating lipopeptide (MALP-2) of Mycoplasma fermentans.
[0012] In one embodiment, the composition of the invention may comprise an adjuvant, as described herein, in combination with at least one other suitable adjuvant.
[0013] In another embodiment of the composition as described herein, the antigen is a polypeptide or a carbohydrate.
[0014] In one embodiment of the composition as described herein, the antigen comprises a B cell epitope, or a plurality of B cell epitopes.
[0015] In another embodiment of the composition as described herein, the antigen is a cancer antigen bound to the membrane surface; a toxin; an allergen, such as pollen; or an amyloid protein.
[0016] In another embodiment of the composition as described herein, the liposome comprises a phospholipid or unesterified cholesterol.
[0017] In another embodiment, the composition as described herein is capable of inducing a humoral immune response in an individual with a single dose.
[0018] The present invention in an additional aspect provides a method for treating or preventing a disease or disorder ameliorated by a humoral immune response, said method comprising administering the composition as described herein to an individual.
[0019] In another aspect, the present invention provides a method for treating or preventing an infectious disease; a cancer involving a cancer antigen attached to the membrane surface; or a disease or disorder where it is desirable to sequester circulating antigen, such as an amyloid protein for treating, for example, Alzheimer's disease, said method comprising administering the composition as described herein to an individual.
[0020] In another aspect, the present invention provides a method for neutralizing a toxin, virus, bacteria or allergen, with an antibody, said method comprising administering the composition as described herein to an individual.
[0021] In one embodiment of the invention, the subject referred to here is a mammal.
[0022] In another embodiment of the invention, the individual referred to here is a human.
[0023] According to another aspect, the present invention relates to a kit useful for treating or preventing a disease or disorder, as described herein, or neutralizing a toxin, virus, bacteria or allergen, with an antibody, in the which kit comprises a composition as described herein, and instructions for its use.
[0024] According to another aspect, the present invention relates to a method of producing a composition of the present invention as described herein.
[0025] Other aspects and characteristics of the present invention will become apparent to those skilled in the art after reviewing the following description of the specific embodiments of the invention in conjunction with the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES
[0026] In the figures, which illustrate embodiments of the invention by way of example only:
[0027] Figure 1 illustrates the humoral immune response generated by a vaccine produced according to the invention ("Vaccine A"). Two groups of mice (n = 8 or 9) were vaccinated as follows: Group 1, the mice were vaccinated with a single dose of 1 microgram of rHA and 1 microgram of Pam-3-Cys-Ser- (Lys) 4 in one 50 microliter dose formulated as a water / liposome / P3C-free hydrophobic carrier vaccine (Vaccine A). In Group 2, the mice were treated with 1 microgram of rHA and 50 micrograms of alum per control dose of 50 microliters of alum vaccine; the mice were challenged 28 days post-vaccination. Humoral immune responses were measured by ELISA as described above. For each treatment group, log 10 values of endpoint antibody titers were averaged, and standard deviations calculated for each time point.
[0028] Figure 2 illustrates the effect of a single administration of a vaccine produced according to the invention ("Vaccine B"). Three groups of mice (n = 9 or 11) were vaccinated as follows: In Group 1, the mice were vaccinated with a single dose of 1 microgram of PT and 1 microgram of Pam-3-Cys-Ser- (Lys) 4 in a 50 microliter dose formulated as a water / liposome / P3C free hydrophobic carrier vaccine (Vaccine B). In Group 2 and Group 3, the mice were treated with 1 microgram of PT and 100 micrograms of alum per 100 microliters of control dose alum vaccine; in group 2, the mice received a single dose, in group 3, the mice were populated on days 21 and 31. In group 4, the mice remained unvaccinated. Mice were challenged 56 days post-vaccination with aerosol inoculation with Bordetella pertussis and bacterial lung counts established 8 and 15 days post-challenge. For each treatment group, the log 10 values of colony-forming units per lung were averaged, and standard deviations calculated for each time point.
[0029] Figure 3 illustrates the effect of a single administration of a vaccine produced according to the invention ("Vaccine C"). Two groups of rabbits (N = 8) were vaccinated as follows: In Group 1, rabbits were vaccinated with a single dose of 8 micrograms of rPA and 2 micrograms of Pam-3-Cys in a dose of 100 microliths formulated as liposome / P3C / hydrophobic carrier vaccine (Vaccine C). In Group 2, rabbits were treated with 8 micrograms of rPA and 350 micrograms of aluminum hydroxide per 100 microliters of control dose alum vaccine; rabbits were challenged at 28 and 84 days post-vabination. Humoral immune responses were measured by ELISA as described above. For each treatment group, the Log10 values of endpoint antibody titers were averaged, and standard deviations calculated for each time point.
[0030] Figure 4 illustrates that Vaccine A of the invention, specifically comprising an antigen, liposomes, a palmitic acid adjuvant and a hydrophobic vehicle, is capable of simulating maximum immunogenicity. Four groups of mice (N = 10) were vaccinated as follows: In Group 1, the mice were vaccinated with a single dose of 1 microgram of rHA and 1 microgram of Pam-3-Cys in a 50 microliter dose formulated as a liposome / P3C / hydrophobic carrier vaccine (Vaccine A, the invention). In Group 2, mice were treated with 1 microgram of rHA and 1 microgram of P3C per 50 microliter dose. In Group 3, mice were treated with 1 microgram of rHA and 1 microgram of P3C per 50 microliter dose formulated as an aqueous / liposome / P3C vaccine. In Group 4, mice were treated with 1 microgram of rHA formulated as a liposome / hydrophobic carrier vaccine. Humoral responses were measured by ELISA as described above. For each treatment group, log 10 values for endpoint antibody titers were averaged, and standard deviations calculated for each time point.
[0031] Figure 5 illustrates that Vaccine D of the invention, comprising a lipid-based adjuvant (i.e., Pam-3-Cys), is able to enhance the immune response to inactivated viral vaccine formulations. Panel (A) shows the clinical classification, and panel (B) shows the total survival of mice challenged with influenza A / PR / 8/34 (H1N1) 28 days after a single vaccination. In Group 1, the mice were treated with 50 microliters of saline. In Group 2, the mice were treated with 50 microliters of 2.56 x 10A3 TCID50 A / PR / 8/34 with alum. In Group 3, mice were treated with 50 microliters of 2.56 x 10A3 TCID50 A / PR / 8/34 and 1 microgram of Pam-3-Cys formulated as a liposome / P3C / hydrophobic carrier vaccine (Vaccine D). Following the viral challenge, the clinical signs of each rat were followed every day for 10 days, and classified on the basis of physical appearance, posture, level of activity / behavior, body temperature, body weight, and hydration. Any rat with a rating of> 12 (out of a possible total 18) was euthanized.
[0032] Figure 6 illustrates that Vaccine A of the invention is capable of stimulating a specific immune response that is significantly stronger than a comparable vaccine prepared with a different adjuvant (liposomes / IMQ / hydrophobic vehicle). Two groups of mice (N = 9) were vaccinated as follows: In Group 1, mice were vaccinated with a single dose of 1 microgram of rHA and 1 microgram of Pam-3-Cys in a dose of 50 microliters formulated as a liposome / P3C / hydrophobic carrier vaccine (Vaccine A). In Group 2, the mice were treated with 1 microgram of rHA and 1 microgram of Imiquimod per 50 microliter dose formulated as a liposome / IMQ / hydrophobic carrier vaccine (Control Vaccine). Humoral immune responses were measured by ELISA as described above. For each treatment group, log 10 values for endpoint antibody titers were calculated. Statistical analysis performed by unpaired t-test, P <0.005.
[0033] Figure 7 illustrates that both Pam3Cys and Pam2Cys are capable of inducing potent B cell proliferation. Purified B cells from C57BL6 mice were stimulated for three days in vitro with Pam2Cys (A), Pam3Cys (B), Poly l: C (C) or LPS (D) in three different concentrations in the presence of anti-Ig & anti-CD40. Proliferation was measured by incorporating [3 H] -thymidine, quantified as counts per minute (CPM). N = 2-10, statistical analysis performed by ANOVA. DETAILED DESCRIPTION
[0034] The type of immunity induced by a vaccine depends largely on the type of adjuvant included in the vaccine. The magnitude and duration of such a response depends on the type of adjuvant used, as well as the composition or method by which the antigen and adjuvant are presented to the immune system. For example, live attenuated viruses can be used to deliver genes to antigens of interest, which are then produced in vivo to be promptly presented by antigen presenting cells; Liposomes can be used to co-distribute antigen and adjuvant directly to antigen presenting cells to trigger a better immune response than bare antigen and adjuvant are capable of.
[0035] The present invention provides vaccine compositions that use an adjuvant that activates or increases the activity of TLR2 to generate unexpectedly strong antibody responses. In some embodiments, the vaccine compositions of the invention were able to protect an individual from a disease agent with only a single dose, so, as shown in the examples here, control vaccines were unable to produce such levels of antibody, and are unable to protect individuals vaccinated to the same degree.
[0036] The compositions of the invention, combining an antigen capable of inducing a humoral immune response, an adjuvant that activates or increases the activity of TLR2, liposomes and a vehicle comprising the continuous phase of a hydrophobic substance, provide surprisingly more antibody titrations higher than aqueous aluminum based control compositions. In addition, a single dose of a composition of the invention was able to effectively protect mice from bacterial challenge, and allows them to completely get rid of lung infection, which was not seen for aqueous aluminum based control compositions.
[0037] The ability to have robust and long-lasting humoral immune responses with at least one immunization using the components of the described composition of the invention (for example, Examples 1 to 4) illustrates the particular utility of these compositions in a wide range of applications medical conditions, such as, for example, those described here. As shown in the examples here, the compositions of the invention can produce a strong and enhanced immune response at least before three weeks post-immunization, and the immune response is long-lived with antibody titers remaining high for at least twenty-four weeks post-immunization (for example, Figure 3).
[0038] The compositions of the invention, comprising antigen, liposomes, an adjuvant that activates or increases the activity of TLR2, and a vehicle comprising the continuous phase of a hydrophobic substance, robust and long-lasting humoral immune responses can occur. For example, the data described in Example 4 here show that antibody titers generated by the mice in Group 1 (a vaccine of the invention) were significantly higher than the antibody titers generated by the mice in the control groups without liposomes (Group 2) , without hydrophobic vehicle (Group 3), or without lipid-based adjuvant (Group 4).
[0039] Thus, the vaccine compositions of the invention containing a lipid-based adjuvant are capable of generating strong humoral immune responses, with high levels of antibody production, in immunized individuals.
[0040] The compositions as described herein may be useful for treating or preventing diseases and / or disorders ameliorated by humoral immune responses (for example, involving B cells and antibody production). The compositions find application in any example where it is desired to administer an antigen to an individual to induce a humoral immune response or antibody production.
[0041] As used herein, "inducing" an immune response is to induce and / or potentiate an immune response. The reduction of an immune response involves examples where the immune response is enhanced, elevated, enhanced or strengthened to the benefit of the host relative to the prior state of the immune response, for example, prior to administration of a composition of the invention.
[0042] A humoral immune response, as opposed to cell-mediated immunide, is mediated by secreted antibodies that are produced in cells of the B lymphocyte lineage (B cells). Such secreted antibodies bind to antigens, such as, for example, those on the surfaces of foreign substances and / or pathogens (for example, viruses, bacteria, etc.) and mark them for destruction.
[0043] An "antibody" is a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes, or fragments of immunoglobulin genes. Recognized immunoglobulin genes include K, A, α, Y, δ, ε, and μ constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either K or À. Heavy chains are classified as Y, μ, α, δ, or ε, which, in turn, define the classes of immunoglobulin, IgG, IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin structural unit (antibody) comprises a protein containing four polypeptides. Each structural unit of the antibody is composed of two identical pairs of polypeptide chains, each having a "light" chain and a "heavy" chain. The N-terminus of each chain defines a variable region mainly responsible for the recognition of the antigen. The structural units of the antibody (for example, of the IgA and IgM classes) can also be combined in oligomeric forms with each other and additional polypeptide chains, for example, as IgM pentamers in association with the J chain polypeptide.
[0044] Antibodies are the antigen-specific glycoprotein products of a subset of white blood cells called B lymphocytes (B cells). Antigen engagement with antibody expressed on the surface of B cells can induce an antibody response comprising stimulating B cells to become activated, to support mitosis, and to differentiate terminally into plasma cells, which are specialized for antibody synthesis and secretion antigen-specific.
[0045] B cells are the only producers of antibodies during an immune response, and are thus a key element for effective humoral immunity. In addition to the production of large amounts of antibodies, B cells also act as antigen presenting cells, and can present antigen to T cells, such as CD4 helper T or CD8 cytotoxic, thereby propagating the immune response.
[0046] B cells, as well as T cells, are part of the adaptive immune response that is essential for the effectiveness of the vaccine. During an active, induced immune response, either by vaccination, or by natural infection, antigen-specific B cells are activated and clonally expanded. During expansion, B cells evolve to have a higher affinity for the epitope. The proliferation of B cells can be induced indirectly by activated T-helper cells, and also directly through stimulation of receptors, such as toll-like receptors (TLRs).
[0047] Antigen presenting cells, such as dendritic cells and B cells, are removed to vaccination sites, and can interact with antigens and adjuvants contained in the vaccine. The adjuvant stimulates cells to become activated, and the antigen provides a model for the target. Different types of adjuvants provide different stimulation signals to cells. For example, Poly 1: C (a TLR3 agonist) can activate dendritic cells, but not B cells. Adjuvants, such as Pam3Cys, Pam2Cys and FSL-1, are especially adept at activating and initiating B cell proliferation, which is expected to facilitate the production of an antibody response (Moyle et a /., CurrMed Chem, 2008; So., J Immunol, 2012).
[0048] As used herein, the term "antibody response" refers to an increase in the amount of antigen-specific antibodies in an individual's body in response to the introduction of the antigen into the individual's body.
[0049] One method of evaluating an antibody response is to measure the antibody titers reactive with a particular antigen. This can be accomplished using a variety of methods known in the art, such as enzyme-linked immunosorbent assay (ELISA) of antibody-containing substances obtained from animals. For example, titers of serum antibodies that bind to a particular antigen can be determined in an individual, both before and after exposure to the antigen. A statistically significant increase in the titer of antigen-specific antibodies following exposure to the antigen would indicate that the individual mounted an antibody response to the antigen.
[0050] Other assays that can be used to detect the presence of an antigen specific antibody include, without limitation, immunological assays (for example, radioimmunoassay (RIA)), immunoprecipitation assays, and protein staining assays (for example , Western blot); and neutralization assays (for example, neutralization of viral infectivity in an in vitro or in vivo assay).
[0051] The compositions of the present invention, by stimulating strong antibody responses, may be able to protect an individual from a disease, disorder or malaise associated with an antigen capable of inducing a humoral immune response.
[0052] Without limitation, these include, for example, infectious diseases, cancers involving a cancer antigen attached to the membrane surface that are recognized by an antibody, diseases where it is desirable to sequester circulating antigen, similar to amyloid protein (for example , Alzheimer's disease); neutralization of toxins with an antibody; neutralization of viruses or bacteria with an antibody; or neutralization of allergens (eg pollen) for the treatment of allergies.
[0053] "Humoral immune response", as referred to here, refers to antibody production and the accompanying accessory processes, such as, for example, T-helper 2 (Th2) cell activation and cytokine production, switching of isotype, affinity maturation, and memory cell activation. It also refers to an antibody's effective functions, such as, for example, toxin neutralization, classic complement activation, and promotion of phagocytosis and elimination of pathogenesis. The humoral immune response is aided by CD4 + Th2 cells and, therefore, the activation or generation of this type of cell is also indicative of a humoral immune response, as mentioned here.
[0054] A "humoral immune response", as mentioned here, may also involve the generation and / or activation of T-helper cells 17 (Th17). Th17 cells are a subset of effector-auxiliary T-lymphocytes characterized by the secretion of host defense cytokines, such as IL-17, IL-17F and IL-22. Th17 cells are considered developmentally distinct from Th1 and Th2 cells, and have been postulated to facilitate the humoral immune response, such as, for example, providing an important function in antimicrobial immunity and protection against infections. Its production of IL-22 is thought to stimulate epithelial cells to produce antimicrobial proteins, and production of IL-17 may be involved in the recruitment, activation and migration of neutrophils to protect against host infection by various bacterial and fungal species.
[0055] A humoral immune response is the primary mechanism for active infectious disease vaccines. However, a humoral immune response can also be useful to fight cancer. Unlike a cancer vaccine designed to produce a cytotoxic CD8 T cell response that can recognize and destroy cancer cells, B cell-mediated responses can target cancer cells through other mechanisms that can and, for example, cooperate with a CD8 T cell cytotoxin for maximum benefit. Examples of B cell-mediated anti-tumor response mechanisms (for example, humoral-mediated immune response) include, but are not limited to: 1) Antibodies produced by B cells that bind to surface antigens found on tumor cells, or other cells that influence tumorigenesis. Such antibodies can, for example, induce target cell death through antibody dependent cell mediated cytotoxicity (ADCC), or complement fixation, potentially resulting in the release of additional antigens that can be recognized by the immune system; 2) Antibodies that bind to receptors on tumor cells to block their stimulation and, in effect, neutralize their effects; 3) Antibodies that bind to factors released by, or associated with, tumor cells, or cells associated with tumor to modulate a signaling pathway, or cell that supports cancer; and 4) Antibodies that bind to intracellular targets and mediate anti-tumor activity through a currently unknown mechanism.
[0056] Various methods can be used to demonstrate the induction of humoral immunity following vaccination. These can be broadly classified in the detection of: i) specific antigen presenting cells; ii) specific effector cells and their functions; and iii) release of soluble mediators, such as cotokines. i) Antigen presenting cells: Dendritic cells and B cells (and, to a lesser extent, macrophages) are equipped with special immunostimulatory receptors that allow enhanced T cell activation, and are called professional antigen presenting cells (APC). These immunostimulatory molecules (also called co-stimulatory molecules) are regulated superiorly in these cells following infection or vaccination, during the antigen presentation process for effector cells, such as CD4 and CD8 cytotoxic T cells. Such co-stimulatory molecules (such as, CD80, CD86, MHC class I or MHC class II) can be detected by using flow cytometry with fluorochrome-conjugated antibodies directed against these molecules, along with antibodies that specifically identify APC (such as CD11c for dendritic cells). ii) CD4 + "helper" T cells: CD4 + lymphocytes, or helper T cells, are immune-mediating responses, and play an important role in establishing and maximizing the capacities of the adaptive immune response. These cells have no cytotoxic activity or phagocytic activity; and they cannot kill infected cells or treat pathogens, but, in essence, they "control" the immune response by directing other cells to perform these tasks. Two types of effector CD4 + T helper cell responses can be induced by a professional APC, designated Th1 and Th2, each designed to eliminate different types of pathogens.
[0057] T cell receptors that express T Helper cells (TCR) that recognize antigen bound to Class II MHC molecules. The activation of a naive helper T cell causes it to release cytokines, which influences the activity of many cell types, including the APC that activates them. The two populations of Th cell, Th1 and Th2, differ in the pattern of effector proteins (cytokines) produced. In general, Th1 cells assist the cellular immune response by activating macrophages and cytotoxic T cells; whereby Th2 cells promote the humoral immune response by stimulation of B cells for conversion to plasma cells, and by the formation of antibodies. A response regulated by Th2 cells can predominantly enhance the production of lgG1 in mice (lgG2 in humans). Measuring the cytokines associated with Th1 or Th2 responses will give a measure of successful vaccination. This can be achieved by specific ELISA designed for Th1 cytokines, such as IFN-Y, IL-2, IL-12, TNF-α and others, or Th2 cytokines, such as IL-4, IL-5, IL10, among others.
[0058] Another Th cell population is the Th17 cell. The measurement of cytokines associated with Th17 cells can also give a measurement of a successful vaccination. This can be achieved, for example, by a specific ELISA designated for Th17 cytokines, such as IL-17, IL-17F and IL-22. iii) Measurement of cytokines: released from regional lymph nodes give a good indication of successful immunization. As a result of the presentation and maturation of APC antigen and immune effector cells, such as CD4 and CD8 T cells, several cytokines are released by lymph node cells. By culturing these LNCs in vitro in the presence of antigen, an antigen-specific immune response can be detected by measuring the release of certain important cytokines, such as IL-4, IL-5, and IL10, to detect a humoral immune response. This can be done by ELISA using culture supernatants and recombinant cytokines as standards.
[0059] Successful immunization can additionally be terminated in a number of additional ways known to the skilled artisan, including, but not limited to, hemagglutination inhibition (HAIJ and serum neutralization inhibition assays to detect functional antibodies ; challenge studies, in which vaccinated individuals are challenged with the associated pathogenesis to determine the effectiveness of vaccination; and the use of fluorescence-activated cell classification (FACS) to determine the cell population that expresses a specific cell surface marker , for example, in the identification of activated or memory lymphocytes. Also, the vaccine's effectiveness in stimulating a humoral immune response can be assessed by ELISA detection of antigen-specific antibody levels in the serum of immunized individuals. can also determine whether immunization with a composition of the invention induced a humoral (or mediated antibody) response using o other known methods. See, for example, Current Protocols in Immunology Coligan et al., Ed. (Wiley Interscience, 2007). The term "infectious disease", as used herein, can refer, for example, to any communicable disease, contagious disease, or communicable disease, resulting from the infection, presence and / or growth of pathogenic biological agents.
[0060] Without limitation, an infectious pathogen can include, for example, viruses, bacteria, fungi, protozoa, and parasites. Non-limiting examples of infectious diseases include influenza (for example, influenza virus infection), respiratory tract infections, such as, for example, bronchiolitis and pneumonia (for example, respiratory syncytial virus infection), tetanus or whooping cough (for example, example, infection by Bordetella pertussis), anthrax (eg infection by Bacillus anthracis) and malaria (eg infection by Plasmodium malariae, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale or Plasmodium knowlesi).
[0061] As used herein, the terms "cancer", "cancer cells", "tumor" and "tumor cells", (used interchangeably) refer to cells that exhibit abnormal growth, characterized by a significant loss to control cell proliferation or cells that have been immortalized. The term "cancer" or "tumor" includes metastatic cancer, as well as non-metastatic cancer. A cancer can be diagnosed using criteria generally accepted in the art, including the presence of a malignant tumor.
[0062] A "toxin", as used herein, refers to any substance produced by living cells or organisms (for example, plants, animals, microorganisms, etc.) that is capable of causing disease or malaise, or an infectious substance, or a recombinant or synthesized molecule capable of adverse effect. Toxins can be, for example, small molecules, peptides, or proteins. Toxins include drug substances, such as, for example, cocaine.
[0063] An "allergen", as used herein, refers to any substance that may cause an allergy. The allergen can be derived from, without limitation, cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptides and non-peptide polysaccharide analogues, and other molecules, small molecules, lipids, glycolipids, and carbohydrates from plants, animals, fungi, insects, food, drugs, dust, and mites. Allergens include, but are not limited to, environmentally airborne allergens; plant pollens (for example, tasna / rhinitis); herb pollen allergens; grass pollen allergens; Johnson grass; tree pollen allergens; ryegrass; arachnid allergens (for example, allergens from domestic dust mites); storage mite allergens; Japanese cedar pollen / hay fever; fungal spore boil / allergens; animal allergens (eg, dog, guinea pig, hamster, gerbil, rat, mouse, etc., allergens); food allergens (for example, crustaceans; nuts; citrus fruits; flour; coffee); insect allergens (for example, fleas, cockroach); poisons: (Hymenoptera, yellow jacket, bee honey, wasp, hornet, ant); bacterial allergens (for example, streptococcus antigens; parasite allergens, such as Ascaris antigen); viral antigens; drug allergens (for example, penicillin); hormones (for example, insulin); enzymes (for example, streptokinase); and drugs or chemicals capable of acting as incomplete antigens or haptens (for example, acid anhydrides and isociates).
[0064] Where a hapten is used in a composition of the invention, it can be attached to a vehicle, such as, for example, a protein, to form a hapten carrier addition product. The hapten carrier addition product is able to initiate a humoral immune response, so the hapten itself would not induce antibody production. Non-limiting examples of haptens are aniline, urushiol (a toxin in ivy poison), hydralazine, fluorescein, biotin, digoxigenin and dinitrophenol.
[0065] "Treating" or "treating", or "preventing" or "preventing", as referred to herein, refers to an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired results may include, but are not limited to, alleviating or improving one or more symptoms or conditions, decreasing the extent of the disease, stabilizing the disease state, preventing disease development, preventing disease spread, delaying or slowing down the progression of the disease, delaying or slowing down the onset of the disease, conferring protective immunity against an agent and improving or palliating the state of the disease. "Treatment" or "prevention" may also mean prolonging a patient's survival beyond that expected in the absence of treatment, and may also mean temporarily inhibiting disease progression, although more preferably, it involves preventing the occurrence of disease, such as pre - prevention of infection in an individual.
[0066] The individual to be treated can be any vertebrate, preferably a mammal, more preferably, a human. Adjuvants
[0067] Suitable adjuvants in the composition of the invention are adjuvants that activate or increase a TLR2 activity. In some embodiments, the adjuvant is a lipid-based adjuvant, which involves any adjuvant comprising at least a portion of the lipid or lipid component.
[0068] As used herein, the term "lipid moiety" or "lipid component" refers to any fatty acid (for example, fatty acyls) or derivatives thereof, including, for example, triglycerides, diglycerides, and monoglycerides. Exemplary fatty acids include, without limitation, palmitoyl, myristoyl, stearoyl and decanoyl groups, or any saturated or unsaturated fatty acyl group C2 to C30, preferably any saturated or unsaturated acyl fatty group C14 to C22, and, more preferably, a fatty acyl group saturated or unsaturated C16. Thus, as referred to herein, the term "lipid-based adjuvant" encompasses any adjuvant comprising a fatty acyl group or derivative thereof.
[0069] The lipid-based adjuvants of the present invention contain at least at least one lipid moiety, or a synthetic / semi-synthetic lipid moiety analog, which can be coupled to an amino acid, an oligopeptide, or in other molecules (for example, a carbohydrate, a glycan, a polysaccharide, biotin, rhodamine, etc.). Thus, without limitation, the lipid-based adjuvant can be, for example, a lipoamino acid, a lipopeptide, a lipoglycan, a lipopolysaccharide, or a lipoteic acid. In addition, a lipid moiety, or a structure containing a lipid moiety, can be coupled covalently or non-covalently to an antigen to create antigenic compounds with adjuvant composition properties. For example, and without limitation, the lipid-based portion may comprise a cation (e.g., nickel) to provide a positive charge for non-covalent coupling.
[0070] In some embodiments, the lipid portion or lipid component may occur naturally, such as, for example, a cell wall component (e.g., lipoprotein) of a Gram-positive or Gram-negative bacterium, Rhodopseudomonas viridis, or mycoplasma. In other embodiments, the lipid portion, or lipid component, can be synthetic or semi-synthetic.
[0071] The lipid-based adjuvant may comprise palmitic acid (PAM) as at least one of the lipid portions or components of the adjuvant. Such lipid-based adjuvants are referred to herein as a "palmitic acid adjuvant". Palmitic acid is a low molecular weight lipid found in the immunologically reactive Braun lipoprotein of Escherichia coli. Other common chemical names for palmitic acid include, for example, hexadecanoic acid in the IUPAC nomenclature, and 1-Pentadecanecarboxylic acid. The molecular formula of palmitic acid is CH3 (CH2) i4CO2H. As will be understood by those skilled in the art, it is possible that the lipid chain of palmitic acid may be altered. Exemplary compounds that can be used here as adjuvants to palmitic acid, and methods for their synthesis, are described, for example, in United States Patent Publications US 2008/0233143; US 2010/0129385; and US 2011/0200632, the disclosures of which are incorporated herein.
[0072] As described above for lipid portions generally, a palmitic acid adjuvant contains, at a minimum, at least a portion of palmitic acid, which can be coupled to an amino acid, an oligopeptide, or other molecules. A portion of palmitic acid, or a structure containing palmitic acid, can be coupled covalently or non-covalently to an antigen to create antigenic compounds with adjuvant composition properties. The palmitic acid portion, or a chemical structure containing palmitic acid, can be conjugated to a cysteine peptide (Cys) to allow for various structural configurations of the adjuvant, including linear and branched structures. The cysteine residue was commonly extended by polar residues, such as serine (Ser) and / or lysine (Lys) at the C-terminus to create adjuvant compounds with improved solubility. Palmitic acid containing adjuvant compounds can be mixed with an antigen, associated with antigen through non-covalent interactions, or alternatively, covalently attached to an antigen, or directly, or with the use of a linker / spacer, to generate enhanced immune responses . Most commonly, two portions of palmitic acid are attached to a glyceryl support and cysteine residue to create dipalmitoyl-S-glyceryl-cysteine (PAM2Cys), or tripalmitoyl-S-glyceryl-cysteine (PAM3Cys), which can also be used in multiple configurations, as described above.
[0073] Palmitic acid adjuvants are known to activate B cells causing rapid antibody proliferation and production. B cells recognize the antigen co-distributed with the adjuvant in the vaccine formulation, and through maturity of affinity it will proliferate with increased specificity for the antigen. Activated B cells are known to secrete large amounts of soluble immunoglobulin antibodies that can bind to soluble targets, such as bacteria, present in the blood. The effector functions of the antibody are i) opsonization; ii) antibody dependent cell mediated cytotoxicity (ADCC); iii) complement activation; iv) neutralization.
[0074] While most B cells will mature in plasma cells that secrete antibody, a portion must differentiate into memory B cells that persist after the immune response has controlled infection. This provides long-term immunity against subsequent exposure to the pathogen. Ideally, a prophylactic vaccine should induce a strong memory B-cell population.
[0075] Therefore, in one embodiment, the adjuvant in the composition of the invention is any type of adjuvant comprising a portion of palmitic acid or component. The portion of palmitic acid can be modified or manipulated to improve its stability in vitro or in vivo, intensify its binding to receptors (such as, for example, toll-like receptors, as described below), or intensify its biological activity.
[0076] In a particular embodiment, the palmitic acid adjuvant can comprise PAM2Cys.
[0077] In another particular embodiment, the palmitic acid adjuvant may comprise PAM3Cys.
[0078] In another particular embodiment, the palmitic acid adjuvant can be Pam-2-15 Cys-Ser- (Lys) 4, or Pam-3-Cys-Ser- (Lys) 4. Such palmitic acid adjuvants are available, for example, as resource reagents, from EMC Microcollections GmbH (Germany) and InvivoGen (San Diego, California, USA).
[0079] Also available from EMC Microcollections are several Pam-2-Cys-Ser- (Lys) 4 and Pam-3-Cys-Ser- (Lys) 4 analogs, including labeled analogs. These analogs are involved here and include, without limitation, PAM3Cys-SKKKK (p-irradiated), R -PAM3Cys-SKKKK, S- PAM3Cys-SKKKK, PAM3Cys-SKKKK (Biotin-Aca-Aca), PAM3Cys-SKKKK (Fluorescein-Aca -Aca), PAM3Cys-SKKKK (Rhodamine-Aca-Aca), PAM3Cys-SKKKK-FLAG-tag, PHC-SKKKK, PHC-SKKKK (Biotin- Aca-Aca), PAM3Cys-SSNAKIDQLSSDVQT, PAM3Cys- SSTKST PAM3Cys- SSGSKPSGG PLPDAK, Pam3Cys-SSGNKSAPSSSASSS, PAM3Cys- GSHQMKSEGHANMQL, Pam3Cys-SSSNNDAAGNGAAQT, Pam3Cys-KQNVSSLDEKNSVSV, Pam3Cys-NNSGKDGNTSA NSAD, Pam3Cys-NNGGPELKSDEVAKS, Pam3Cys-SQEPAAPAAEATPAG, Pam3Cys-SSSKSSDSSAPKAYG, Pam3Cys-AQEKEAKSELDYDQT, Pam2Cys-SKKKK (mixture of RR and stereoisomers of RS), S- Pam2Cys-SKKKK (RR stereoisomers), S-Pam2Cys-SKKKK (RS stereoisomers), PamCys (Pam) -SKKKK, Pam2Cys-SKKKK (Biotin-Aca-Aca) -NH2, Pam2Cys -SKKKK (Fluorescein-Aca-Aca) -NH2, PAM2Cys-SKKKK (Rhodamine-Aca-Aca) -NH2, and PAM2Cys-SKKKK- FLAG-tag. Where appropriate, the palmitic acid adjuvant, or analogue thereof, can be used as stereochemically defined compounds, or as a mixture of stereoisomers.
[0080] The adjuvant is one that activates or increases the activity of toll like receptors (TLRs), and, preferably, activates or increases an activity of TLR2. As used herein, an adjuvant that "activates" or "increases the activity" of a TLR includes any adjuvant; in some embodiments, a lipid-based adjuvant acts as a TLR agonist. In addition, the activation or enhancement of a TLR2 activity involves its activation in any monomeric, homodimeric or heterodimeric form, and, particularly, includes the activation of TLR2 as a heterodimer with TLR1 or TLR6 (ie, TLR1 / 2 or TLR2 / 6), as described in further detail below.
[0081] TLRs are a conserved family of transmembrane enveloping receptors found mainly in leukocytes, such as dendritic cells (DCs) and macrophages, professional antigen presenting cells. TLRs have evolved specifically to recognize and induce an immune response to pathogenesis associated with molecular patterns, such as, for example, bacterial lipoproteins and lipopeptides and viral double-stranded RNA. More than 10 distinct TLRs have been identified in mice and humans, although the ligand and signaling trajectories are not yet known to some (see Table 1 below). There are 13 TLRs identified in humans, numbered 1 to 13.

[0082] TLRs typically form homodimers, with the exception of TLR2 which forms a heterodimer with TLR1 or TLR6 resulting in different ligand specificity. TLR2 acts as an intermediary in downstream signaling, so that these heterodimers are often referred to collectively as TLR2 (Takeuchi, O. and S. Akira, Cell, 2010, 140 (6): p. 805-20). The stimulation of TLRs in DCs results in superior regulation of MHC and co-stimulatory molecules, which intensify the function that these cells' antigen presents, as well as the production of Th1-type cytokines, and promotion of cross presentation (Lahiri et al., Vaccine, 2008, 26 (52): p. 6777-83; Welters et al, Vaccine, 2007, 25 (8): p. 1379-89; Matsumoto et al., Adv Drug Deliv Rev, 2008, 60 (7): p. 805 -12; Blander, JM, Ann Rheum Dis, 2008, 67 Suppl 3: p. Iii44-9). Because stimulation through TLRs has a direct effect on eliciting the immune response, TLR antagonists have been studied as potential adjuvants (Barchet et al., CurrOpin Immunol, 2008, 20 (4): p. 389-95).
[0083] TLRs have a conserved cytosolic domain called the Toll-interleukin 1 receptor (TIR) that is associated with an adapter molecule that facilitates signaling trajectories downstream that lead to cell activation. TLRs can be broadly categorized by the adptor protein that they are associated with MyD88 or TRIF. TLR4 can only signal through both paths. Both signaling trajectories converge on the activation of the transcription factor NF-KB (Ouyang et al., Biochem Biophys Res Commun, 2007, 354 (4): p. 1045-51). Several studies have shown that although the different TLRs share some downstream simalization molecules, each receptor produces a unique profile of pro-inflammatory mediators (Welters et al., Vaccine, 2007, 25 (8): p. 137989; Seya et al ., Evid Based Complement Alternat Med, 2006, 3 (1): p. 31-8 and discussion 133-7; Ghosh et al., Cell Immunol, 2006, 243 (1): p. 48-57; Re, F and Strominger, JL, J Immunol, 2004, 173 (12): p. 754855; Avril et al., J Immun other, 2009, 32 (4): p. 353-62). The total downstream path for TLR receptors is not fully elucidated, but differences in activation may be the result of ligand resistance, subcellular location of the receptor, cell type, and the presence of interferon regulatory factors (IRF).
[0084] Palmitic acid adjuvants have been reported to signal via toll-like receptor 2 (TLR2). For example, PAM2Cys is recognized by the heterodimer of TLR2 and TLR6. Also as an example, PAM3Cys, which is recognized by the heterodimer of TLR1 and TLR2, triggers an anti-bacterial response typified by human-moral activity. In contrast, double stranded virus RNA is recognized by TLR3, and induces an antiviral response that is usually characterized by interferon release and T cell activity. The mediation of cellular responses has been associated with TLR2.
[0085] Pam3Cys has been tested in a variety of animal models, and in a Phase I clinical trial in humans with no reported side effects (Moyle, PM and Toth. I., Curr Med Chem, 2008, 15 (5): p 506-16; Wiedemann et al., J Pathol, 1991, 164 (3): p. 265-71). In a classification of TLR agonists in murine DCs, stimulation with Pam3Cys in vitro produces high levels of the pro-inflammatory cytokines IL-12p40, IL-6 and TNFa that were achieved with only small amounts of the adjuvant relative to other TLR agonists tested (Welters et al., Vacina, 2007, 25 (8): p. 1379-89).
[0086] As will be appreciated by those skilled in the art, the present invention involves adjuvants that activate or increase the activity of a TLR, or act as an agonist for a TLR, particularly a lipid-based adjuvant. In a particular embodiment, the lipid-based adjuvant activates or increases the activity of TLR2. Without limitation, such lipid-based adjuvants may be a palmitic acid adjuvant that activates or increases the activity of a TLR, such as a palmitic acid adjuvant comprising PAM2Cys or PAM3Cys.
[0087] Other exemplary TLR2 agonists that can be used as a lipid-based adjuvant in the composition of the invention include, without limitation, cell wall components, such as lipoteic acid and Gram-positive bacterial lipoprotein, and lipoarabi- mycobacterium nomannan. A number of these cell wall components are available from InvivoGen (San Diego, California, USA), such as M. smegmatis lipoarabinomannan (LAM-MS), M. smegmatis lipomannan (LM-MS), P. gingivalis lipopolysaccharide (LPS-PG Ultrapure), and lipoteicoic acid from B. subtilis (LTA-BS) and S. aureus (LTA-SA). In some embodiments, the lipid-based adjuvant that activates or increases the activity of TLR2 may involve a heat-dead bacterium that comprises any one or more of the cell wall components described above. Such heat-killed bacteria is available, for example, from InvivoGen (San Diego, California, USA).
[0088] Synthetic lipoproteins that act as TLR agonists are also involved by the invention, and include, without limitation, the palmitic acid adjuvants and analogs described above, and synthetic diacetylated FSL-1 lipoprotein available from InvivoGen (San Diego, California, USA ) and EMC Microcollections GmbH (Germany). FSL-1 (Pam2CGDPKHPKSF) is a synthetic lipoprotein that represents the N-terminal part of the 44-kDa lipoprotein LP44 of Mycoplasma salivium. FSL-1 comprises PAM2Cys, and has a similar structure as macrophage-activating lipopeptide-2 (MALP-2), a lipopeptide derived from Mycoplasma fermentans. It is postulated that FSL-1 and MALP-2, containing a lipolyzed N-terminal diacetylated cysteine residue, are recognized by TLR2 and TLR6 dimer 9TLR2 / 6). Synthetic MALP-2 is available from Enzo Life Sciences (Farmingdale, New York, USA).
[0089] In one embodiment, the lipid-based adjuvant of the invention comprises FSL-1 or MALP-2, or the lipid-based adjuvant is FSL-1 or MALP-2. Where appropriate, FSL-1 or MALP-2 can be used as stereochemically defined compounds, or as a mixture of stereoisomers. FSL-1 or MALP-2 can be labeled (for example, biotin, Fluorescein, Rhodamine, etc.). FSL-1 is also available as a collection of FSL-1 Ala scan (EMC Microcollections) comprising nine different FSL-1-Ala compounds. Each of these FSL-1-Ala molecules is involved here individually, or in combination.
[0090] Additional embodiments of the lipid-based adjuvants of the invention may include substructures of TLR2 ligands, such as monoacylated lipopeptides. Without limitation, these may include, for example, Pam-Dhc-SKKKK, Pam-CSKKKK, Pam-Dhc-GDPKHPKSF or Pam-CGDPKHPKSF (EMC Microcollections).
[0091] Other lipid-based adjuvants that activate or increase TLR2 activity can be identified, for example, by using the InvivoGen activation reporting system (San Diego, California, USA) HEK-Blue® TLR2. This system allows assessment of the ability of potential TLR2 ligands to stimulate TLR2, either in human cells (hTLR2), or in murine cells (mTLR2).
[0092] In some embodiments, the lipid-based adjuvant of the compositions of the invention is one that activates or increases the activity of only TLR2, TLR1 and TLR2 heterodimer (TLR1 / 2), and / or TLR2 and TLR6 heterodimer (TLR2 / 6), while other TLRs are not activated. In a further embodiment, the lipid-based adjuvant activates or increases only the heterodimer activity of TLR1 / 2 and / or TLR2 / 6, but does not activate other TLRs.
[0093] The composition of the invention can comprise an adjuvant as described above, in combination with at least one other suitable adjuvant. Exemplary embodiments of at least one other adjuvant involve, but are by no means limited to, organic and inorganic compounds, polymers, proteins, peptides, sugars from synthetic biological or non-biological sources (including, but not limited to, virosomes , particles similar to viruses, viruses and bacteria from their components).
[0094] Additional examples of compatible adjuvants may include, without limitation, chemokines, Toll like receptor agonists, colony stimulating factors, cytokines, 1018 ISS, aluminum salts, Amplivax, AS04, AS15, ABM2, Adjumer, Algammulin, AS01B, AS02 (SBASA), AS02A, BCG, Calcitriol, Chitosan, Cholera toxin, CP- 870,893, CpG, polylC, CyaA, Dimethyldioctadecylammonium bromide (DDA), Dibutyl phthalate (DBP), dSLIM, Inulin gamma, GM-CSF, GM-CSF , Glycerol, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISCOM, ISCOMA-TRIX, Juvlmmune, LipoVac, LPS, lipid core protein, MF59, monophosphoryl lipid A, Montanide® IMS1312, Montanide®-based adjuvants, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system, other palmitoil-based molecules, PLG microparticles, resiquimod, squalene, SLR172, YF-17 DBCG, QS21, QuilA, P1005 , Poloxamer, Saponin, synthetic polynucleotides, Zymosan, toxin pertussis.
Consequently, the compositions may comprise one or more pharmaceutically acceptable adjuvants, where at least one of the adjuvants in the composition is an adjuvant that activates or increases the activity of TLR2.
[0096] In another embodiment, the antigen can be coupled to a lipid moiety, such as, for example, a palmitic acid moiety, to provide the adjuvant property. The composition can also comprise pharmaceutically acceptable excipients, diluents, etc., as known in the art. See, for example, Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985) and the United States Pharmacopoeia: The National Formulary (USP 24 NF19), published in 1999.
[0097] In one embodiment, such suitable additional adjuvants may comprise a CpG-containing oligodeoxynucleotide (CpG ODN), such as 5'-TCCATGACGTTCC TGACGTT-3 '. The person skilled in the art can select an appropriate CpG based on the target species and effectiveness.
[0098] The amount of adjuvant used depends on the amount of antigen and the type of adjuvant. A person skilled in the art can readily determine the amount of adjuvant needed in a particular application by empirical testing. Antigens
[0099] The compositions of the invention comprise one or more antigens. As used herein, the term "antigen" refers to a substance that can specifically bind to an antibody. Suitable antigens in the composition are those that are capable of inducing a humoral immune response in an individual.
[00100] Antigens useful in the compositions of the invention include, without limitation, polypeptides, carbohydrates, a microorganism or a part thereof, such as inactivated or killed, attenuated, living, virus or protozoan bacteria, or part thereof. The antigen can be, for example, a biological pathogen, a toxin, an allergen, a peptide, a suitable native, non-native, recombinant or denatured protein, or polypeptide, or a fragment thereof, or an epitope that it is capable of producing a humoral immune response in an individual.
[00101] As used herein and in the claims, the term "antigen" also includes a polynucleotide that encodes the polypeptide that functions as an antigen. Nucleic acid-based vaccination strategies are known, in which a vaccine composition containing a polynucleotide is administered to an individual. The antigenic polypeptide encoded by the polynucleotide is expressed in the individual, such that the antigenic polypeptide is ultimately present in the individual, only as if the vaccine composition itself contains the polypeptide. For the purpose of the present invention, the term "antigen", where the context determines, involves such polynucleotides that encode the polypeptide that functions as the antigen.
[00102] Polypeptides or fragments thereof which may be useful as antigens in the invention include, without limitation, those derived from Cholera toxoid, tetanus toxoid, diphtheria toxoid, hepatitis B surface antigen, hemagglutinin (e.g., recombinant hemaglutinin protein H5N1), recombinant anthrax protective antigen (List Biologies; Campbell, CA), neuraminidase, influenza M protein, PfHRP2, pLDH, aldolase, MSP1, MSP2, AMA1, Der-p-1, Der-f-1, Adipophyllin, AFP, AIM-2, ART-4, BAGE, a-fetus protein, BCL-2, Bcr-Abl, BING-4, CEA, CPSF, CT, cyclin D1Ep-CAM, EphA2, EphA3, ELF- 2, FGF- 5, G250, Gonadotropin Release Hormone, HER-2, intestinal carboxyl esterase (iCE), IL13Ra2, MAGE-1, MAGE-2, MAGE-3, MART-1, MART-2, M-CSF, MDM-2 , MMP-2, MUC-1, NY-EOS-1, MUM-1, MUM-2, MUM-3, pertussis toxoid protein, p53, PBF, PRA-ME, PSA, PSMA, RAGE-1, RNF43, RU1 , RU2AS, SART-1, SART-2, SART-3, SAGE-1, SCRN 1, SOX2, SOX10, STEAP1, survivin, Telomerase, TGF (3RII, TRAG-3, TRP -1, TRP-2, TERT and WT1.
[00103] Viruses, or parts thereof, useful as antigens in the invention, include, without limitation, Cowpoxvirus, Vaccinia virus, Pseudocowpox virus, human herpes virus 1, human herpes virus 2, Cytomegalovirus, human AF adenovirus, Polyomavirus, human papillomavirus, Parvovirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, human immunodeficiency virus, Ortoreovirus, Rotavirus, Ebolavirus, influenza paravirus, influenza virus (for example, influenza virus H5N1, influenza virus A, influenza B virus, influenza virus C), measles virus, mumps virus, rubella virus, Pneumovirus, human respiratory disease virus, rabies virus, California encephalitis virus, Japanese encephalitis virus, Hantaan virus, lymphocytic choriomingitis virus, Coronavirus, Enterovirus, Rhinovirus, Poliovirus, Norovirus, Flavivirus, Dengue virus, Nile Valley fever virus, yellow fever virus, and chickenpox.
[00104] In one embodiment, a composition of the invention can be used to treat and / or prevent an infection of the influenza virus in an individual in need thereof. Influenza is a double-stranded RNA virus in the Orthomyxoviridae family, and is often characterized based on two large glycoproteins outside the viral particle, hemagglutinin (HA) and neuraminidase (NA). Numerous HA subtypes of influenza A have been identified (Kawaoka et al., Virology (1990) 179: 759-767; Webster et al., "Antigenic variation among type A influenza viruses," p. 127-168. In: P. Palese and DW Kingsbury (ed.), Genetics of influenza viruses, Springer-Verlag, New York).
[00105] Bacteria or parts thereof useful as antigens in the invention include, without limitation, Anthrax (Bacillus anthracis), Brucella, Boretella pertussis, Candida, Chlamydia pneumoniae, Chlamydia psittaci, Cholera, Clostridium botulinum, Coccidioides immitis, Cryptococcus, Cryptococcus, Escherichia coli 0157: H7, Enterohemorrhagic Escherichia coli, Enterotoxigenic Escherichia coli, Haemophilus influenzae, Helico-bacter pylori, Legionella, Leptospira, Listeria, Meningococcus, Myco-plasma pneumoniae, Mycobacterium, Pertussis, Pneumonia, Pneumonia, Pneumonia, Pneumonia, Yersinia enterocolitica.
[00106] The antigen may alternatively be of protozoan origin, for example, from the genus Plasmodium (Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale or Plasmodium knowlesi), which cause malaria.
[00107] The antigen may alternatively be a naturally occurring toxin or synthesized toxin, such as a drug substance (for example, cocaine).
[00108] The term "polypeptide" involves any chain of amino acids, regardless of length (for example, at least 6, 8, 10, 12, 14, 16, 18, or 20 amino acids), or post-translational modification ( for example, glycosylation or phosphorylation) and include, for example, natural proteins, synthetic and recombinant polypeptides and peptides, epitopes, hybrid molecules, variants, homologues, analogs, peptides, peptidomimetics, etc. A variant or derivative, therefore, includes cancellations, including truncations and fragments; insertions and additions, for example, conservative substitutions, site-directed mutants and allelic variants; and modifications, including pepttoids having one or more non-amino acyl groups (e.g., sugar, lipid, etc.) covalently linked to the peptide and post-translational modifications. As used herein, the term "conserved amino acid substitutions" or "conservative substitutions" refer to the substitution of amino acid for another at a given location on the peptide, where the substitution can be made without substantial loss of the relevant function. In making such changes, substitutions of similar amino acid residues can be made on the basis of relative similarity of side chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions can be evaluated for their effect in peptide function by routine testing. Specific non-limiting examples of a conservative substitution include the following examples:

[00109] Polypeptides or peptides that have substantial identity to a preferred antigen sequence can be used. Two strings are considered to have substantial identity if, when optimally aligned (with allowances allowed), they share at least approximately 50% sequence identity, or if the strings share defined functional motifs. In alternative embodiments, optimally aligned strings can be considered to be substantially identical (that is, to have substantial identity) if they share at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity over a specified region. The term "identity" refers to the similarity of sequence between two polypeptide molecules. Identity can be determined by comparing each position in the aligned sequences. A degree of identity between amino acid sequences is a function of the number of identical amino acids or of matching in the positions shared by the sequences, for example, over a specified region. Optimal sequence alignment for identity comparisons can be conducted using a variety of algorithms, as they are known in the art, including the ClustalW program, available at http: //clustalw.qenome.ad.ip, Smith's local homology algorithm and Waterman, 1981, Adv. Appl. Math 2: 482, Needleman and Wunsch's homology alignment algorithm, 1970, J. Mol. Biol. 48: 443, the search for similarity method by Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wl, U.S.A.). The sequence identity can also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215: 403-10 (using published fault settings). For example, the "BLAST 2 Sequences" tool, available through the National Center for Biotechnology Information (via the internet at http://www.ncbi.nlm.nih.gov/ BLAST / bl2seq / wblast2.cgi) can be used, selecting a "blastp" program in the following fault settings: expected limit 10; word size 3; BLOSUM 62 matrix; existence of clearance costs 11, extension 1. In another embodiment, the technician in the subject can readily and correctly align any given sequence and deduce sequence identity and / or homology by mere visual inspection.
[00110] Polypeptides and peptides used to practice the invention can be isolated from natural sources, be synthetic, or be recombinantly generated polypeptides. Peptides and proteins can be recombinantly expressed in vitro or in vivo. The peptides and polypeptides used to practice the invention can be produced and isolated using any method known in the art. Polypeptides and peptides used to practice the invention can also be synthesized, in whole or in part, using chemical methods well known in the art. See, for example, Caruthers (1980) Nucleic Acids Res. Symp. 215-223; Horn (1980) Nucleic Acids Res. Symp. 225-232; Banga, A. K, Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, Pa. For example, the peptide synthesis can be performed using various solid phase techniques (see, for example , Roberge (1995) Science 269: 202; Merrifield (1997) Methods Enzymol. 289: 313), and automatic synthesis can be achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer .
[00111] In some embodiments, the antigen can be a purified antigen, for example, from about 25% to 50% pure, from about 50% to about 75% pure, from about 75% to about 85% pure, from about 85% to about 90% pure, from about 90% to about 95% pure, from about 95% to about 98% pure, from about 98% to about 99% pure, or greater than 99% pure.
[00112] As noted above, the term "antigen" also includes a polynucleotide that encodes the polypeptide that functions as an antigen. As used herein and in the claims, the term "polynucleotide" involves a chain of nucleotides of any length (for example, 9, 12, 18, 24, 30, 60, 150, 300, 600, 1500 or more nucleotides), or number of strands (for example, single strand or double strand). Polynucleotides can be DNA (for example, genomic DNA or cDNA) or RNA (for example, mRNA) or combinations of these. They can occur naturally or be synthetic (for example, chemically synthesized). It is contemplated that the polynucleotide may contain modifications of one or more nitrogenous bases, pentose sugars, or phosphate groups, in the nucleotide chain. Such modifications are well known in the art, and may be for the purpose of, for example, improving the po-linucleotide stability.
[00113] The polynucleotide can be distributed in several forms. In some embodiments, a naked polynucleotide can be used, either in linear form, or inserted into a plasmid, such as an expression plasmid. In other embodiments, a live vector, such as a viral or bacterial vector, can be used.
[00114] One or more regulatory sequences that assist in the transcription of DNA into RNA and / or translation of RNA into a polypeptide may be present. In some examples, such as in the case of a polynucleotide that is a messenger RNA (mRNA) molecule, regulatory sequences related to the transcription process (for example, a promoter) are not required, and protein expression can be carried out in the absence of a prosecutor. The person skilled in the art may include appropriate regulatory sequences as circumstances require.
[00115] In some embodiments, the polynucleotide is present in an expression cassette, in which it is operably linked to the regulatory sequences that will allow the polynucleotide to be expressed in the individual to whom the composition of the invention is administered. The choice of the expression cassette depends on the individual to whom the composition is administered, as well as the desired characteristics for the expressed polypeptide.
[00116] Typically, an expression cassette includes a promoter that is functional in the individual, and can be either constitutive or inducible; a ribosome binding site; a start codon (ATG) if necessary; the polynucleotide that encodes the polypeptide of interest; a stop codon; and, optionally, a 3 'terminal region (translation and / or transcription terminator). Additional sequences, such as a region that encodes a signal peptide, can be included. The polynucleotide encoding the polypeptide of interest can be homologous or heterologous to any of the regulatory sequences in the expression cassette. The sequences to be expressed along with the polypeptide of interest, such as a signal peptide coding region, are typically located adjacent to the polynucleotide that encodes the protein to be expressed and placed in the correct reading frame. The open reading frame constituted by the polynucleotide that encodes the protein to be expressed alone or together with any other sequence to be expressed (for example, the signal peptide), is placed under the control of the promoter so that transcription and translation occur in the individual to whom the composition is administered.
[00117] The amount of antigen used in a single treatment with a composition as described here can vary, depending on the type of antigen and the size of the individual. A person skilled in the art will be able to determine, if undue experimentation, the effective amount of antigen to use in a particular application. The term "effective amount", as used herein, means an effective amount, in dosages and for periods of time necessary, to achieve the desired result.
[00118] In another embodiment, the antigen may be or comprise a B cell epitope capable of inducing a humoral immune response. For example, the antigen can be or comprise a B cell epitope derived from a virus, such as, for example, influenza virus, or respiratory syncytial virus.
[00119] In another embodiment, the B cell epitope may be an epitope derived from the H5N1 influenza virus hemagglutinin glycoprotein.
[00120] In another embodiment, the antigen may be or comprise a B cell epitope derived from a bacterium, such as, for example, Bordetella pertussis or Bacillus anthracis.
[00121] In another embodiment, the B cell epitope may be an epitope of the toxoid pertussis protein produced by Bordetella pertussis.
[00122] In another embodiment, the B cell epitope may be an epitope of the recombinant protective anthrax antigen.
[00123] In another embodiment, the antigen may be or comprise a B cell epitope associated with an infectious disease.
[00124] In another embodiment, the antigen may be or comprise a B cell epitope derived from a protozoan, such as from the genus Plasmodium.
[00125] In another embodiment, the antigen can be a cancer protein or tumor-associated protein, such as, for example, a cancer antigen bound to the membrane surface that is capable of being recognized by an antibody.
[00126] Cancers that can be treated and / or prevented by the use or administration of a composition of the invention, include, without limitation, carcinoma, adenocarcinoma, lymphoma, leukemia, sarcoma, blastoma, myeloma, and germ cell tumors. In one embodiment, the cancer can be caused by a pathogen, such as a virus. Viruses linked to the development of cancer are known to the person skilled in the art and include, but are not limited to, human papillomavirus (HPV), John Cunningham virus (JCV), human herpes virus 8, Epstein Barr Virus (EBV), polyomavirus from Merkel cell, Hepatitis C virus and human T cell leukemia virus -1. A composition of the invention can be used for either cancer treatment or prophylaxis, for example, in reducing the severity of cancer, or in preventing recurrences of cancer. Cancers that may benefit from the compositions of the invention include any malignant cell that expresses one or more tumor specific antigens.
[00127] In another embodiment, the antigen can be a toxin or an allergen that is capable of being neutralized by an antibody. In one embodiment, the toxin is a drug substance, such as, for example, cocaine.
[00128] In another embodiment, the antigen may be an antigen associated with a disease where it is desirable to sequester the circulating antigen, such as, for example, an amyloid protein (for example, Alzheimer's disease). Thus, a composition of the invention may be suitable for use in the treatment and / or prevention of a neurodegenerative disease in an individual in need thereof, in which the neurodegenerative disease is associated with the expression of an antigen. The individual may have a neurodegenerative disease, or he may be at risk of developing a neurodegenerative disease. Neurodegenerative diseases that can be treated and / or prevented by the use or administration of a composition of the invention include, without limitation, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS). For example, Alzheimer's disease is characterized by the association of R.- amyloid plaques and / or tau proteins in the brains of patients with Alzheimer's disease (see, for example, Goedert and Spillantini, Science, 314: 777-781, 2006). Herpes simplex virus type 1 has also been proposed to play a causative role in people carrying susceptible versions of the apoE gene (Itzhaki and Wozniak, J Alzheimers Dis 13: 393-405, 2008).
[00129] In a further embodiment, the composition may comprise a mixture of B cell epitopes as antigens for inducing a humoral immune response. B cell epitopes can be ligated to form a single polypeptide.
[00130] In another embodiment, the antigen can be any peptide or polypeptide that is capable of inducing a specific humoral immune response to a specific conformation in target tumor cells. Auxiliary epitopes T
[00131] T helper epitopes are a sequence of amino acids (natural or unnatural amino acids) that have T helper activity. T helper epitopes are recognized by T helper lymphocytes, which play an important role in establishing and maximizing the capacities of the immune system, and are involved in the activation and targeting of other immune cells, such as, for example, switching B-cell antibody class.
[00132] An auxiliary epitope T can consist of a continuous or discontinuous epitope.
[00133] Consequently, not all amino acids in a T helper are necessary parts of the epitope. Consequently, T helper epitopes, including analogs and T helper epitope segments, are capable of enhancing or stimulating an immune response. Immunodominant T helper epitopes are largely reactive in animal and human populations with widely divergent MHC types (Celis et al. (1988) J. Immunol. 140: 1808-1815; Demotz et al. (1989) J. Immunol. 142: 394 -402; Chong et al. (1992) Infect. Immun. 60: 46404647). The T helper domain of the individual peptides is from about 10 to about 50 amino acids and, preferably, from about 10 to about 30 amino acids. When multiple T helper epitopes are present, then each T helper epitope acts independently.
[00134] In one embodiment, the composition described herein may also comprise at least one auxiliary epitope T. In some examples, auxiliary epitope T may form part of the antigen. In particular, if the antigen is of sufficient size, it may contain an epitope that functions as an auxiliary epitope T. In other embodiments, the auxiliary epitope T is a separate molecule from the antigen.
[00135] In another embodiment, auxiliary T epitope analogs can include substitutions, deletions and insertions of one to about 10 amino acid residues in auxiliary T epitope. Auxiliary T segments are contiguous portions of an auxiliary T epitope that are sufficient to enhance or stimulate an immune response. An example of T helper segments is a series of overlapping peptides that are derived from a single longer peptide.
[00136] Sources of T helper epitopes for use in the present invention include, for example, hepatitis B surface antigen helper T cell epitopes, toxin pertussis helper T cell epitopes, viral protein F helper T cell epitopes of measles, auxiliary T cell epitope of Chlamydia trachomitis major external protein, diphtheria toxin auxiliary T cell epitopes, Plasmodium falciparum circumsporozoite auxiliary T cell epitopes, Schistosoma mansoni auxiliary T cell epitopes, isomerase epitopes, epitopes helper T cell cells Escherichia coli TraT, and immune enhancement analog segments and segments of any of these T helper epitopes.
[00137] In one embodiment, the T helper epitope is a universal T helper epitope. A universal T helper epitope, as used herein, refers to a peptide or other immunogenic molecule, or a fragment thereof, that binds to a multiplicity of MHC class II molecules in a way that activates T cell function in a way class II (CD4 + T cells).
[00138] In another embodiment, the T helper epitope can be a universal T helper epitope, such as PADRE (pan-DR epitope) comprising the AKXVAAWTLKAAA peptide sequence, in which X can be cyclohexylalanyl. PADRE specifically has a CD4 + T helper epitope, that is, it stimulates induction of a PADRE-specific CD4 + T helper response.
[00139] Tetanus toxoide has T helper epitopes that operate in a similar manner as PRIEST. Tetanus and diphtheria toxins have universal epitopes for human CD4 + cells. (Diethelm-Okita, B.M. et al., Universal epitopes for human CD4 + cells on tetanus and diphtheria toxins. J. Infect. Diseases, 181: 1001-1009, 2000). In another embodiment, the T helper epitope may be a toxoid tetanus peptide, such as F21E comprising the FNNFTVS-FWLRVPKVSASHLE peptide sequence (amino acids 947-967).
[00140] In another embodiment, the auxiliary epitope T is fused to at least one antigen (i.e., a peptide), or a mixture of antigens, to produce a fusion peptide. Vehicles
The composition vehicle comprises a continuous phase of a hydrophobic substance, preferably a liquid hydrophobic substance. The continuous phase can be an essentially pure hydrophobic substance, or a mixture of hydrophobic substances. In addition, the vehicle may be a water emulsion in a hydrophobic substance, or a water emulsion in a mixture of hydrophobic substances, provided that the hydrophobic substance constitutes the continuous phase. In yet another embodiment, the vehicle can function as an adjuvant.
[00142] The hydrophobic substances that are useful in the compositions, as described herein, are those that are pharmaceutically and / or immunologically acceptable. The vehicle is preferably a liquid, but certain hydrophobic substances which are not liquid at atmospheric temperature, can be liquefied, for example, by heating, and are also useful in this invention. In one embodiment, the hydrophobic vehicle may be a phosphate-buffered saline emulsion / Freund's Incomplete Adjuvant (PBS / FIA).
[00143] Oil or water-in-oil emulsions are particularly suitable vehicles for use in the present invention. The oils must be pharmaceutically and / or immunologically acceptable. Suitable oils include, for example, mineral oils (especially light or low-viscosity mineral oil, such as Drakeol® 6VR), vegetable oils (for example, soybean oil), almond oils (for example, peanut oil), or mixtures of these. In one embodiment, the oil is a manganese oleate in a mineral oil solution, commercially available as Montanide® ISA 51. Animal fats and artificial hydrophobic polymeric materials, particularly those that are liquid at atmospheric temperature, or that can be liquefied relatively easily, can also be used.
[00144] In the embodiments here where the composition is described as a water-free ("water-free”) liposome suspension, it is possible that the hydrophobic carrier of these "water-free" compositions may still contain small amounts of water, provided that water is present in the non-continuous phase of the vehicle. For example, individual components of the composition may have water bound which may not be completely removed by processes, such as freeze-drying or evaporation, and certain hydrophobic vehicles may contain small amounts of water dissolved therein. Generally, compositions of the invention that are described as "free of water" contain, for example, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %, 1%, 0.5%, 0.1%, 0.05% or 0.01% water on a weight / weight basis of the total weight of the vehicle component of the composition. Liposomes
[00145] Liposomes are completely closed lipid bilayer membranes containing a entrained aqueous volume. Liposomes can be unilamellar vesicles (having a single bilayer membrane), or multilamellar vesicles characterized by multi-membrane bilayers, each bilayer may or may not be separated from the next by an aqueous layer. A general discussion of liposomes can be found in Gregoriadis G. Immunol. Todia, 11: 89-97, 1990; and Frezard, F., Braz. J. Med. Bio. Res., 32: 181-189, 1999. As used herein and in the claims, the term "liposomes" is intended to encompass all such vesicular structures, as described above, including, without limitation, those described in the art as "niosomes" , "transferssomas" and "virosomes".
[00146] Although any liposomes can be used in this invention, including liposomes produced from archaebacterial lipids, particularly useful liposomes use phospholipids and unesterified cholesterol in the liposome formulation. Cholesterol is used to stabilize liposomes, and any other compound that stabilizes liposomes can replace cholesterol. Other liposome stabilizing compounds are known to those skilled in the art. For example, saturated phospholipids produce liposomes with higher transition temperatures indicating increased stability.
[00147] The phospholipids that are preferably used in the preparation of liposomes are those with at least one major group selected from the group consisting of phosphoglycerol, phosphoethanolamine, phosphoserine, phosphocholine and phosphoinositol. More preferred are liposomes that comprise lipids that are 94-100% phosphoditylcholine. Such lipids are commercially available in Phospholipon® 90 G lecithin. When non-esterified cholesterol is also used in the liposome formulation, cholesterol is used in an amount equivalent to about 10% by weight of phospholipid. If a compound other than cholesterol is used to stabilize liposomes, a person skilled in the art can readily determine the amount needed in the composition.
[00148] Liposome compositions can be obtained, for example, by using natural lipids, synthetic lipids, sphingolipids, ether lipids, sterols, cardiolipin, cationic lipids, and poly (ethylene glycol) modified lipids, and others polymers. Synthetic lipids can include the following fatty acid constituents; lauroil, miristoil, palmitoil, estearoil, araquidoil, oleoil, linoleoil, erucolil, or combinations of these fatty acids. Compositions
[00149] Additional embodiments of the present invention include methods of producing a composition of the invention comprising liposomes; an antigen capable of inducing a humoral immune response; a vehicle comprising a continuous phase of a hydrophobic substance; and an adjuvant that activates or increases the activity of TLR2.
[00150] Methods for producing liposomes are well known in the art. See, for example, Gregoriadis (1990) and Frezard (1999), both cited earlier. Any suitable method for producing liposomes can be used in the practice of the invention, or liposomes can be obtained from a commercial source. Liposomes are typically prepared by hydrating the liposome components that will form the lipid bilayer (for example, phospholipids and cholesterol) with an aqueous solution, which can be pure water, or a solution of one or more components dissolved in water, for example , phosphate buffered saline (PBS), phosphate-free saline, or any other physiologically compatible aqueous solution.
[00151] In one embodiment, a liposome component, or mixture of liposome components, such as a phospholipid (eg, Phospholipon® 90G) and cholesterol, can be solubilized in an organic solvent, such as a mixture of chloroform and methanol , followed by filtration (for example, a 0.2 μm PTFE filter) and drying, for example, by rotary evaporation, to remove solvents.
[00152] Hydration of the resulting lipid mixture can be effected by, for example, injecting the lipid mixture into an aqueous solution or sonicating the lipid mixture, and an aqueous solution. During liposome formation, the liposome components form single (unilamellar) bilayers, or multiple (multi-lamellar) bilayers, which surround a volume of the aqueous solution with which the liposome components are hydrated.
[00153] In some embodiments, the liposomes are then dehydrated, such as by freeze-drying or lyophilization.
[00154] The liposomes are combined with the vehicle comprising a continuous hydrophobic phase. This can be done in a variety of ways.
[00155] If the vehicle is composed only of a hydrophobic substance, or a mixture of hydrophobic substances (for example, use of 100% mineral oil vehicle), the liposomes can simply be mixed with the hydrophobic substance, or if there are substances multiple hydrophobic, mixed with any one or a combination thereof.
[00156] If instead the vehicle comprising a continuous phase of a hydrophobic substance contains a discontinuous aqueous phase, the vehicle will typically take the form of an emulsion of the aqueous phase in the hydrophobic phase, such as a water-in-oil emulsion. Such compositions can contain an emulsifier to stabilize the emulsion, and to promote a constant distribution of the liposomes. In this particular, emulsifiers can be useful even if a water-free vehicle is used, for the purpose of promoting a constant distribution of the liposomes in the vehicle. Typical emulsifiers include mannide oleate (Arlacel ™ A), lecithin (for example, S100 lecithin), a phospholipid, Tween ™ 80, and Spans ™ 20, 80, 83 and 85. Typically, the volume ratio (v / v) from hydrophobic substance to emulsifier is in the range of about 5: 1 to about 15: 1 with a ratio of about 10: 1 being preferred.
[00157] Liposomes can be added to the finished emulsion, or they can be present in, or in the aqueous phase, or in the hydrophobic phase before emulsification.
[00158] The antigen can be introduced at several different stages of the formulation process. More than one type of antigen can be incorporated into the composition (for example, an inactivated virus, live attenuated virus, protein or polypeptide).
[00159] In some embodiments, the antigen is present in the aqueous solution used to hydrate the components that are used to form the lipid bilayers of the liposomes (e.g., phospholipid (s) and cholesterol). In this case, the antigen will be encapsulated in the liposome, present in its aqueous interior. If the resulting liposomes are not washed or dried, such that there is a residual aqueous solution present that is ultimately mixed with the vehicle comprising a continuous phase of a hydrophobic substance, it is possible that additional antigen may be present outside the liposomes in the final product. . In a related technique, the antigen can be mixed with the components used to form the lipid bilayers of the liposomes, before hydration with the aqueous solution. The antigen can also be added to the preformed liposomes, in which case the antigen can be actively loaded into the liposomes, or attached to the surface of the liposomes, or the antigen can remain external to the liposomes. In such embodiments, prior to the addition of antigen, the preformed liposomes can be empty liposomes (for example, containing no encapsulated antigen or lipid-based adjuvant), or the preformed liposomes can contain lipid-based adjuvant incorporated in the or associated with liposomes. These steps can preferably take place before mixing with the vehicle comprising a continuous phase of a hydrophobic substance.
[00160] In an alternative approach, the antigen can instead be mixed with the vehicle comprising a continuous phase of a hydrophobic substance, before, during or after the vehicle is combined with the liposomes. If the vehicle is an emulsion, the antigen can be mixed with either or both of the aqueous phase or the hydrophobic phase before emulsification. Alternatively, the antigen can be mixed with the vehicle after emulsification.
[00161] The technique of combining the antigen with the vehicle can be used together with encapsulation of the antigen in the liposomes, as described above, such that the antigen is present both within the liposomes and in the vehicle comprising a continuous phase of a hydrophobic substance.
[00162] The procedures described above for introducing the antigen into the composition also apply to the adjuvant of the compositions of the present invention. That is, the adjuvant can be introduced into, for example, any one or more of: (1) the aqueous solution used to hydrate the components that are used to form the lipid bilayers of the liposomes; (2) the aqueous solution after formation of the lipid bilayers of the liposomes; (3) the components used to form the lipid bilayers of the liposomes; or (4) the vehicle comprising a continuous phase of a hydrophobic substance, before, during or after the vehicle is combined with the liposomes. If the vehicle is an emulsion, the adjuvant can be mixed with either or both of the aqueous phase, or the hydrophobic phase before, during or after emulsification.
[00163] The technique of combining the adjuvant with the vehicle can be used together with encapsulation of the adjuvant in the liposomes, or with addition of the adjuvant to the liposomes, such that the adjuvant is present inside and / or outside the liposomes, and in the vehicle comprising a continuous phase of a hydrophobic substance.
[00164] The adjuvant can be incorporated into the composition together with the antigen in the same processing step, or separately, in a different processing step. For example, the antigen and the adjuvant can both be present in the aqueous solution used to hydrate the liposome components of lipid bilayer formation, such that both the antigen and adjuvant become encapsulated in the liposomes. Alternatively, the antigen can be encapsulated in the liposomes, and the adjuvant mixed with the vehicle comprising a continuous phase of a hydrophobic substance. In a further embodiment, the adjuvant can be incorporated into the composition after the antigen encapsulation step by passing the liposome-antigen preparation through a manual mini-extruder and then mixing the liposome-antigen preparation obtained with the base adjuvant. of lipid in, for example, phosphate buffer. The adjuvant can also be incorporated into the composition, either alone or together with antigen, after the liposomes have been formed, such that the adjuvant can be combined, or remains external to the liposomes. The adjuvant can also be incorporated into, or associated with, liposomes prior to the addition of antigen, with the antigen remaining outside the reformed liposomes or loaded into / associated with the liposomes by further processing. In such embodiments, the resulting liposome-antigen-adjuvant preparation can be lyophilized and then reconstituted in the vehicle comprising a continuous phase of a hydrophobic substance. It will be appreciated that many such combinations are possible.
[00165] If the composition contains one or more additional adjuvants, such additional adjuvants may be incorporated into the composition in a similar manner, as described above for the adjuvant, or by a combination of several such methods, as may be suitable for the adjuvant (s) additional (s).
[00166] Stabilizers, such as sugars, antioxidants, or preservatives, which maintain biological activity or enhance chemical stability to prolong the useful life of antigen, adjuvant, liposomes or continuous hydrophobic vehicle, can be added to such compositions.
[00167] In some embodiments, a mixture of antigen / adjuvant can be used, in which case the antigen and adjuvant are incorporated into the composition at the same time. An "antigen / adjuvant mixture" refers to an embodiment in which the antigen and adjuvant are in the same diluent at least before incorporation into the composition. The antigen and adjuvant in an antigen / adjuvant mixture can, but need not be, chemically bound, such as by covalent bonding.
[00168] In some embodiments, the vehicle comprising a continuous phase of a hydrophobic substance can have adjuvant activity. Freund's Incomplete Adjuvant is an example of a hydrophobic vehicle with an adjuvant effect. As used herein and in the claims, when the term "adjuvant" is used, it is intended to indicate the presence of an adjuvant in addition to any adjuvant activity provided by the vehicle comprising a continuous phase of a hydrophobic substance.
[00169] In one embodiment, to formulate a composition of the invention, a homogeneous mixture of S100 lecithin and cholesterol (eg 10: 1 w: w) is hydrated in the presence of an antigen, optionally in phosphate buffer, to form liposomes with encapsulated antigen. The liposome preparation can then be extruded, optionally via a manual mini-extruder, and mixed with the adjuvant, optionally in phosphate buffer, to incorporate the adjuvant. This suspension can then be lyophilized and reconstituted in a vehicle comprising a continuous phase of a hydrophobic substance to form a water-free liposome suspension.
[00170] In some embodiments, the composition can be formulated by hydrating a homogeneous mixture of S100 lecithin and cholesterol (eg 10: 1 p: p) in the presence of an antigen and a suitable adjuvant (eg Pam-3 -Cys), optionally, in phosphate buffer, to form liposomes with encapsulated and adjuvant antigen. The liposome / antigen / adjuvant preparation can then be diluted sufficiently, and optionally using water, and lyophilized. Lyophilized liposomes can and then be reconstituted in a vehicle comprising a continuous phase of a hydrophobic substance (for example, mineral oil or Montanide® ISA 51) to form a water-free liposome suspension.
[00171] In some embodiments, the composition can be formulated by hydrating a homogeneous mixture of dioleoyl-phosphoditylcholine (DOPC) and cholesterol (eg 10: 1 p: p) in the presence of an antigen and a suitable adjuvant (eg , Pam-3-Cys-Ser- (Lys) 4), optionally in phosphate buffer, to form encapsulated liposomes with antigen and adjuvant. The liposome / antigen / adjuvant preparation can then be lyophilized, and the resulting product reconstituted in a vehicle comprising a continuous phase of a hydrophobic substance (for example, mineral oil or Montanide® ISA 51) to form a liposome suspension. free of water.
[00172] Alternatively, the antigen or antigen / adjuvant complex, can be associated with, in contact with, or separated from liposomes, and not encapsulated in liposomes. The liposome encapsulation efficacy of some hydrophilic antigens or complexes of hydrophilic / adjuvant antigen may be poor, so that after being placed in a hydrophobic environment, or more freeze-drying of the antigen, it becomes associated with the outer surface of the liposomes. This represents another embodiment of the invention.
[00173] In an additional embodiment, an antigen (peptide or polypeptide) having a B cell and PADRE epitope (fused to the antigen or separated), can be encapsulated together in the liposomes. In another embodiment, more than one antigen can be placed together on the same liposomes. In a further embodiment, other substances can be used instead of PADRE, which have a T helper epitope, for example, toxoid tetanus peptide (s). In another embodiment, an adjuvant, preferably a palmitic acid-based adjuvant comprising PAM2Cys or PAMaCys, can be encapsulated in the liposomes as well. The liposomes are preferably suspended in PBS. This suspension is then emulsified in a hydrophobic vehicle, such as, for example, ISA51, or mineral oil. The result is that liposomes containing the antigen (s) and adjuvant (s) are suspended in PBS which, in turn, are emulsified in a hydrophobic vehicle, for example, ISA51 or mineral oil.
[00174] In one embodiment, antibody titers obtained from mice injected intramuscularly with a single dose of a composition of the invention comprising liposomes / recombinant H5N1 he-maglutinin protein (antigen) / Pam-3-Cys-Ser- (Lys) 4 (adjuvant) / hydrophobic vehicle (Vaccine A) were significantly strained at three, four and eight weeks post-immunization compared to mice treated (and challenged) with an aqueous aluminum-based control vaccine (Figure 1). For example, Vaccine A of the invention was able to generate endpoint antibody titers in the three and four weeks post-vaccination of up to 1 / 2.48,000 and up to 1 / 8,192,000 to eight weeks post-vaccination, so titrations endpoint antibody to control vaccine were only up to 1 / 512,000, 1 / 256,000 and 1 / 4,096,000 at three, four and eight weeks post-vaccination, respectively. These results indicate that the compositions of the invention are capable of generating, after a single dose, an enhanced humoral immune response in vivo compared to the single control vaccine or aqueous aluminum-based control vaccine.
[00175] In one embodiment, immunizing mice by single treatment with a composition of the invention comprising liposomes / pertussis toxoid protein (antigen) / Pam-3-Cys-Ser- (Lys) 4 (adjuvant) / hydrophobic vehicle ( Vaccine B) was able to reduce bacterial lung counts from as high as 6.2 x 10 M cfu per lung on day 8 post-challenge with Bordetella pertussis to 0 cfu per lung on day 15 post-challenge (Figure 2). These results indicate that a single dose of a composition of the invention effectively protects mice from bacterial challenge, and allows them to completely treat the infection of the lungs.
[00176] In one embodiment, antibody titers obtained from rabbits injected intramuscularly with a single dose of a composition of the invention comprising liposomes / recombinant antigen anthrax protector (antigen) / Pam-3-Cys (adjuvant) / hydrophobic vehicle (Vaccine C) were significantly intensified compared to rabbits treated with a control vaccine based on aqueous aluminum at the previous tenpo points (pre-challenge) (Figure 3). For example, Vaccine C of the invention was able to generate endpoint antibody titers in the three and four weeks post-vaccination of up to 1 / 2,048,000 and up to 1 / 8,192,000 in eight weeks post-vaccination, so titrations endpoint antibody to control vaccine were only up to 1 / 64,000, 1 / 256,000 and 1 / 2,048,000 at three, four and eight weeks post-vaccination, respectively. These results indicate that the compositions of the invention are capable of generating a surprisingly strong humoral immune response in vivo earlier than three weeks following a single vaccination.
[00177] In one embodiment, antibody titers obtained from mice injected intramuscularly with a single dose of Vaccine A of the invention (Group 1) were significantly enhanced compared to single-dose administration of liposome-free control compositions (Group 2) without hydrophobic vehicle (Group 3), or without lipid-based adjuvant (Group 4) (Figure 4). For example, Vaccine C of the invention was able to generate endpoint antibody titers in the eight weeks post-vaccination of up to 1 / 2,048,000, while Groups 2, 3 and 4 were only able to generate antibody titers of up to 1 / 2,048,000. end point at the same time point of 1 / 64,000. 1 / 128,000 and 1 / 128,000, respectively. These results show that the compositions of the invention each comprising: an antigen, liposomes, a lipid-based adjuvant and a vehicle comprising a continuous phase of a hydrophobic substance, are capable of providing robust and long-lasting humoral immune responses in vivo.
[00178] The compositions as described herein can be formulated in a form that is suitable for oral, nasal, rectal or parenteral administration. Parenteral administration includes intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, transepithelial, intrapulmonary, intrathecal, and topical modes of administration. The preferred routes are intramuscular, subcutaneous and intradermal, to achieve a deposit effect.
[00179] The person skilled in the art can determine suitable treatment regimens, routes of administration, dosages, etc., for any particular application in order to achieve the desired result. Factors that can be taken into account include, for example, the nature of the antigen; the state of the disease to be prevented or treated; the age, physical condition, body weight, sex and diet of the individual; and other clinical factors. See, for example, "Vaccine Handbook", edited by Researcher's Associates (Gaku-yuu-kai) of The National Institute of Health (1994); "Manual of Prophylactic Inoculation, 8th edition", edited by Mikio Kimura, Munehiro Hirayama, and Harumi Sakai, Kindai Shuppan (2000); "Minimum Requirements for Biological Products", edited by Association of Biologicals Manufacturers of Japan (1993).
[00180] The optimum amount of adjuvant and antigen to induce an optimal immune response may depend on a number of factors including, without limitation, the composition, the disease, the individual, and can be readily determined by the person skilled in the art using standard studies including , for example, observations of antibody titers, and other immunogenic responses in the host.
[00181] The compositions, as described here, can be effective when administered in a single application.
[00182] In another embodiment, the compositions, as described herein, can be used in combination, before or after, with other therapies. Kits and Reagents
[00183] The present invention is optionally provided to a user as a kit. For example, a kit of the invention contains one or more of the compositions of the invention. The kit may further comprise one or more additional reagents, packaging material, containers for retaining kit components, and a preferred instruction set or methods for detailing the user manual for using the kit components. Embodiments of the Invention
[00184] Particular embodiments of the invention include, without limitation, the following:
[00185] 1. A composition comprising, consisting of, or consisting essentially of: liposomes; an antigen capable of inducing a humoral immune response; a vehicle comprising a continuous phase of a hydrophobic substance; and an adjuvant that activates or increases a TLR2 activity, preferably a lipid-based adjuvant.
[00186] 2. The composition of paragraph 1, in which the adjuvant activates or increases the activity of toll-like receptor 2 (TLR2), or a dimer of TLR2, such as TLR1 / 2 or TLR2 / 6.
[00187] 3. The composition of paragraph 1, in which the adjuvant only activates or increases the activity of a toll-like receptor (TLR) selected from TLR2, TLR1 / 2 heterodimeric and TLR2 / 6 heterodimeric, but not active or increases the activity of other LRs.
[00188] 4. The composition according to any of paragraphs 1 to 3, in which the adjuvant is a compound comprising, consisting of, or consisting essentially of, at least a portion of natural, synthetic or semi-synthetic lipid, lipid component , or analog or derivative thereof, including, for example, a lipoamino acid, a lipoglycan, a lipopolysaccharide, a lipoteic acid, or a cell wall component of a Gram-positive or Gram-negative bacterium, Rhodopseudomonas viridis, or mycoplasma.
[00189] 5. The composition according to any of paragraphs 1 to 4, in which the adjuvant comprises, consists of, or essentially consists of PAM2Cys-Ser- (Lys) 4, PAM3Cys-Ser- (Lys) 4 , PAM3Cys- SKKKK (post-irradiated), S -PAM3Cys-SKKKK, S-PAM3Cys-SKKKK, PAM3Cys-SKKKK (Biotin-Aca-Aca), PAM3Cys-SKKKK (Fluorescein-Aca-Aca), PAMKC-SKK-Rod Aca-Aca), PAM3Cys-SKKKK- FLAG-tag, PHC-SKKKK, PHC-SKK KK (Biotin-Aca-Aca), PAM3Cys- SSNAKIDQLSSDVQT, PAM3Cys-SSNKSTTGSGETTTA, PAM3Cys- PST3SYS Pam3Cys-GSHQMKSEGHANMQL, PAM3Cys- SSSNNDAAGNGAAQT, Pam3Cys-KQNVSSLDEKNSVSV, PAM3Cys- NNSGKDGNTSANSAD, Pam3Cys-NNGGPE LKSDEVAKS, PAM3Cys- SQEPAAPAAEATPAG, Pam3Cys-SSSKSSDSSAPKAYG, PAM3Cys- AQEKEAKSELDYDQT, PAM2Cys-SKKKK (mixture of RR and RS sômeros estereoi-), S PAM2Cys-SKKKK (RR Stereoisomer), S- PAM2Cys-SKKKK (RS Stereoisomer), PAMCys (PAM) -SKKKK, PAM2Cys-SKKKK (Biotin-Aca-Aca) -NH2, PAM2Cys-SKKK-Flu- Aca) -NH2, PAM2Cys-SKK KK (Rhodamine-Aca-Aca) -NH2, PAM2Cys-SKK KK-FLAG-tag, PAM-Dhc-SKKKK, PAM-CSKKKK, PAM-Dhc-GDPKHPKSF, PAM-CGDPKH PKSF, FSL-1 (Pam2CGDPKHPKSF), FSL 1-Ala, macrophage-activating lipopeptide-2 (MALP-2), M. smegmatis lipoarabinomannan (LAM-MS), M. smegmatis lipomannan (LM-MS), P. gingivalis lipopolysaccharide (LPS -PG Ultrapure), B. subtilis lipoteic acid (LTA-BS) or S. aureus (LTA-SA), or derivatives and analogues thereof, or is a heat-dead bacterium that comprises the cell wall component of a bacterium Gram-positive or Gram-negative.
[00190] 6. The composition according to any of paragraphs 1 to 5, in which the adjuvant is an adjuvant for palmitic acid.
[00191] 7. The composition according to any of paragraphs 1 to 6, in which the adjuvant comprises, consists of, or essentially consists of dipalmitoyl-S-glyceryl-cysteine (PAM2Cys) or tripalmitoyl-S-glyceryl-cysteine (PAM3Cys).
[00192] The composition, according to any of paragraphs 1 to 6, in which the adjuvant is PAM2Cys-Ser- (Lys) 4, PAM3Cys-Ser- (Lys) 4, FSL-1 or MALP-2.
[00193] The composition of paragraph 8, in which the adjuvant is PAM2Cys-Ser- (Lys) 4.
[00194] The composition of paragraph 8, in which the adjuvant is PAM3Cys-Ser- (Lys) 4.
[00195] 11. The composition according to any of paragraphs 1 to 10, which further comprises at least one other suitable adjuvant in addition to the adjuvant that activates or increases the activity of TLR2.
[00196] 12. The composition, according to any of paragraphs 1 to 11, in which the antigen is a polypeptide or a carbohydrate.
[00197] 13. The composition according to any of paragraphs 1 to 12, in which the antigen comprises, consists of, or essentially consists of, a B cell epitope, or a plurality of B cell epitopes.
[00198] 14. The composition of paragraph 13, in which the B cell epitope is derived from a virus or bacteria.
[00199] 15. The composition of paragraph 14, in which the B cell epitope is derived from influenza viruses, Bordetella pertussis or Bacillus anthracis.
[00200] 16. The composition of paragraph 15, in which the B cell epitope is an epitope of an H5N1 influenza virus hemagglutinin protein, an epitope of pertussis toxoid protein, or an epitope of a recombinant anthrax protective antigen.
[00201] 17. The composition, according to any of paragraphs 1 to 13, in which the antigen is: an antigen associated with an infectious disease; a cancer antigen attached to the membrane surface; a toxin; an allergen, such as pollen; or an antigen associated with a disease where it is desirable to sequester the circulating antigen, such as, for example, an amyloid protein.
[00202] 18. The composition of paragraph 17, in which the infectious disease is influenza, a respiratory tract infection caused by human respiratory syncytial viruses, pertussis, anthrax or malaria.
[00203] 19. The composition of paragraph 17, in which the toxin is a drug substance, such as cocaine.
[00204] 20. The composition, according to any of paragraphs 1 to 13, in which the antigen is a hapten carrier addition product.
[00205] 21. The composition, according to any of paragraphs 1 to 20, in which the liposome comprises, consists of, or essentially consists of a phospholipid or non-esterified cholesterol.
[00206] 22. The composition, according to any of paragraphs 1 to 21, in which the antigen is encapsulated in the liposomes, or both the antigen and the adjuvant are encapsulated in the liposomes.
[00207] 23. The composition, according to any of paragraphs 1 to 22, which is a water-free liposome suspension.
[00208] 24. The composition, according to any of paragraphs 1 to 23, in which the composition is capable of inducing a humoral immune response with a single dose.
[00209] The composition of paragraph 24, in which the humoral immune response is characterized by production of antigen-specific antibody.
[00210] The composition of paragraph 25 which is capable of generating the antigen-specific antibody at an antibody titer of up to about 1 / 2,048,000 for about three weeks post-vaccination of an individual.
[00211] The composition of paragraph 25 that is capable of generating the antigen-specific antibody at an antibody titer of up to about 1 / 8,192,000 for about eight weeks after an individual's vaccination.
[00212] 28. The composition, according to any of paragraphs 24 to 27, in which the humoral immune response is associated with the activation or generation of T-helper 2 cells (Th2) or T-helper cells 17 (Th17).
[00213] 29. The composition, according to any of paragraphs 1 to 28, for the treatment or prevention of a disease or disorder ameliorated by a humoral immune response.
[00214] 30. The composition, according to any of paragraphs 1 to 28, for the treatment or prevention of: an infectious disease; a cancer involving a cancer antigen attached to the membrane surface; or a disease or disorder where it is desirable to hijack circulating antigen, such as Alzheimer's disease.
[00215] 31. The composition, according to any of paragraphs 1 to 28, for neutralizing a toxin, virus, bacteria or allergen, with an antibody.
[00216] 32. A method for treating or preventing an illness or disorder ameliorated by a humoral immune response, said method comprising, consisting of, or consisting essentially of administering the composition according to any of paragraphs 1 to 28 to an individual .
[00217] 33. A method for treating or preventing an infectious disease; a cancer involving a cancer antigen attached to the membrane surface; or a disease or disorder where it is desirable to sequester circulating antigen, such as Alzheimer's disease, said method comprising, consisting of, or essentially consisting of administering the composition according to any of paragraphs 1 to 28 to an individual.
[00218] 34. The method of paragraph 33, in which the infectious disease is influenza, an infection of the respiratory tract caused by human respiratory syncytial viruses, pertussis, anthrax or malaria.
[00219] 35. A method for neutralizing a toxin, virus, bacteria or allergen, with an antibody, said method comprising, consisting of, or consisting essentially of administering the composition according to any of paragraphs 1 to 28 to a individual.
[00220] The method of paragraph 35, in which the toxin is a drug substance, such as cocaine.
[00221] The method, according to any of paragraphs 32 to 36, in which the individual is a mammal, preferably a human.
[00222] A useful kit for treatment or prevention of a disease or disorder improved by a humoral immune response; or useful for treating or preventing an infectious disease; a cancer involving a cancer antigen attached to the membrane surface; or a disease or disorder where it is desirable to hijack circulating antigen, such as Alzheimer's disease; or useful for neutralizing a toxin, virus, bacteria or allergen, with an antibody, in which the kit comprises, consists of, or essentially consists of, the composition according to any of paragraphs 1 to 28, and instructions for its use thereof.
[00223] 39. A method of preparing the composition of paragraph 1, comprising, consisting of, or consisting essentially of: hydrating a homogeneous mixture of S100 lecithin and cholesterol in the presence of the antigen to form a liposome preparation with encapsulated antigen; extruding and then mixing the liposome preparation with the adjuvant; lyophilization and reconstitution of the resulting product in a vehicle comprising a continuous phase of a hydrophobic substance. In an alternate embodiment, the hydration step can be performed in the presence of a homogeneous mixture of dioleoyl-phosphoditylcholine (DOPC) and cholesterol.
[00224] 40. A method of preparing the composition of paragraph 1, comprising, consisting of, or consisting essentially of: hydration of a homogeneous mixture of dioleoyl-phosphoditylcholine (DOPC) and cholesterol in the presence of the antigen and the adjuvant to form a preparation liposome with encapsulated antigen and adjuvant; lyophilization and reconstitution of the liposome preparation in a vehicle comprising a continuous phase of a hydrophobic substance. In an alternate embodiment, the hydration step can be performed in the presence of a homogeneous mixture of S100 lecithin and cholesterol.
[00225] The invention is further illustrated by the following non-limiting examples. EXAMPLES Example 1
[00226] Female pathogen-free CD1 mice, 6-8 weeks old, were obtained from Charles River Laboratories (St Constant, QC, Canada), and were housed according to institutional guidelines with water and food ad libitum, under air circulation controlled with filter.
[00227] H5N1 recombinant hemagglutinin protein was purchased from Protein Sciences (Meridien, CT, USA). This recombinant protein has an approximate molecular weight of 72,000 daltons, and corresponds to hemagglutinin glycoprotein, an antigenic protein present on the surface of the H5N1 flu virus. This recombinant protein, hereinafter referred to as rHA, was used as a model antigen to test the effectiveness of vaccine formulations. The rHA was used at 1 microgram per 50 microliter dose.
[00228] The effectiveness of the vaccine was evaluated by an enzyme-linked immunosorbent assay (ELISA), a method that allows the detection of levels of antigen-specific antibody in the serum of immunized animals. Performing the ELISA on serum collected from immunized mice at a regular interval (every four weeks, for example), is useful for monitoring antibody responses to a given vaccine formulation. Briefly, a 96-well microtiter plate is coated with antigen (rHA, 1 microgram / milliliter) overnight at 4 degrees Celsius, blocked with 3% gelatin for 30 minutes, then incubated overnight at 4 degrees Celsius with Serial dilutions of serum, typically starting at a dilution of 1/2000. A secondary reagent (protein G conjugated to alkaline phosphatase, EMD chemicals, Gibbstown, NJ, USA) is then added to each well at a 1/500 dilution for one hour at 37 degrees Celsius. After a 60-minute incubation with a solution containing 1 milligram / milliliter of 4-nitrophenyl phosphate disodium salt hexahydrate (Sigma-Aldrich Chemie GmbH, Switzerland), the absorbance of 405 nanometers from each well is measured using a plate reader. microtiter (ASYS Hitech GmbH, Austria). Endpoint titrations are calculated as described in Frey A. etal. (Journal of Immunological Methods, 1998, 221: 35-41). The calculated titrations represent the highest dilution at which a statistically significant increase in absorbance is observed in serum samples from immunized mice, versus serum samples from naive non-immunized control mice. The titrations are presented as Log10 values of the end point dilution.
[00229] To formulate vaccine corresponding to the invention, a homogeneous 10: 1 p: p mixture of S100 lecithin and cholesterol (Lipoid GmbH, Germany) was hydrated in the presence of rHA in phosphate buffer to form liposomes with encapsulated rHA. Soon, 20 micrograms of rHA in 775 microliters of 50 millimolar phosphate buffer (pH 7.4) was added to 132 milligrams of the S100 lecithin / cholesterol mixture to form approximately 900 microliters of a liposome suspension that encapsulates the antigen of rHA. The liposome preparation was then extruded by passing the material through a manual mini-extruder (Avanti, Alabaster, AL, USA) fitted with a 200 nanometer polycarbonate membrane. To incorporate the adjuvant, the sized liposome mixture was thoroughly mixed with 20 micrograms of Pam-3-Cys-Ser- (Lys) 4 adjuvant (referred to as P3C) (EMC Microcollections GmbH, Germany) in 100 microliters of phosphate buffer. After diluting the final mixture in half using water, the liposome suspension was lyophilized using the Virtis Advantage freeze dryer (SP Industries, Warminister, PA, USA). For every 1 milliliter of original liposome suspension containing rHA and P3C, 800 microliters of a mineral oil vehicle equivalent to Freund's incomplete adjuvant (known as Montanide® ISA 51, supplied by Seppic, France) were used to reconstitute the lyophilized liposomes for form a water-free liposome suspension. Each vaccine dose consists of 50 microliters of the formulation described above containing liposomes, rHA antigen, P3C adjuvant, and the mineral oil carrier. This vaccine formulation will be referred to as free of water / liposome / P3C / hydrophobic vehicle.
[00230] The effectiveness of the water-free liposome formulation described above was compared to the effectiveness of a control vaccine consisting of 1 microgram of rHA and 50 micrograms of aluminum hydroxide (alhydrogel, Sigma, Mississauga, ON, Canada, hereafter called alum) in 50 microliters of 50 millimolar phosphate buffer (pH 7.4). A group of mice (N = 9) were injected once (no challenge) with 1 microgram of rHA antigen and 1 microgram of P3C adjuvant formulated in 50 microliters of free water / liposome / P3C / hydrophobic vehicle, as described above. In Group 2, mice (N = 8) were vaccinated twice (day 0 and day 28) with 1 microgram of rHA and 50 micrograms of an alum adjuvant suspended in 50 millimolar phosphate buffer. All mice were vaccinated intramuscularly in the flank region and serum samples were collected at 3, 4, and 8 weeks post-immunization. The antibody titers of rHA in these sera were examined by ELISA, as described above.
[00231] The results of this experiment are shown in Figure 1. In Group 2, the mice generate a detectable antigen-specific antibody response following administration of an aluminum adjuvant control vaccine. The mice in group 1, vaccinated with the free formulation of water / liposome / P3C / hydrophobic vehicle, produced significantly enhanced endpoint titrations compared to those in group 2. The mice in Group 2 generated titrations up to 1 / 512,000 (Log10 value of 5 , 71) and up to 1 / 256,000 (Log10 value of 5.41) in three and four weeks respectively (before stimulation) and up to 1 / 4,096,000 (Log10 equal to 6.61) at eight weeks post-vaccination (after stimulus). The presence of such antibody responses confirms a genuine immunogenerated response as a result of vaccination. The Group 1 mice, vaccinated with the vaccine corresponding to the invention, were able to generate endpoint titrations reaching up to 1 / 2,048,000 (Log10 value of 6.31) in three and four weeks post-vaccination and 1 / 8,192. 000 (a Log10 value of 6.91) at eight weeks post-immunization. These results indicate that single-dose formulations free of water / liposome / hydrophobic vehicle containing a palmitic acid adjuvant are capable of generating an enhanced immune response in vivo compared to a single aqueous aluminum-based control vaccine (week data points 3 and week 4) or stimulated (week 8 point data). Example 2
[00232] Young adult female Balb / C mice, free of pathogenesis, were obtained from Charles River Laboratories (St Constant, QC, Canada), and were housed according to institutional guidelines with water and food ad libitum, under air circulation controlled by filter.
[00233] The toxoid pertussis protein was provided with Biocine (Connaught Biosciences, Toronto, ON, Canada). This multisubunit protein has a molecular weight of approximately 106 kilo-daltons, and corresponds to an antigenic toxin produced by Bordetella pertussis, the bacterium that causes coughing. This protein, hereinafter referred to as PT, was used as a model antigen to test the effectiveness of vaccine formulations. PT was used at 1 microgram per 50 microliter dose.
[00234] The vaccine's effectiveness was assessed by live bacterial challenge with Bordetella pertussis. The mice were challenged by aerosol inoculation with 9.1 x 10A8 Bordetella pertussis, 56 days post-vaccination. Several mice were sacrificed immediately to establish baseline bacterial lung counts. The remaining mice were monitored and sacrificed at eight and fifteen days post-challenge, and bacterial lung counts were established.
[00235] To formulate the vaccine corresponding to the invention, a homogeneous 10: 1 p: p mixture of DOPC and cholesterol (Lipoid GmbH, Germany) was hydrated in the presence of PT and Pam-3-Cys-Ser- (Lys) 4 ( designated P3C) in phosphate buffer to form liposomes with encapsulated PT and P3C. Briefly, 20 micrograms each of PT and P3C in 850 microliters of 50 millimolar phosphate buffer were added to 132 milligrams of the S100 lecithin / cholesterol mixture to form approximately one milliliter of a liposome suspension that encapsulates PT antigen and adjuvant of P3C. The liposome preparation was then lyophilized using the Virtis Advantage freeze dryer (SP Industries, Warminister, PA, USA). For an entire milliliter of original liposome suspension containing rHAand P3C, 800 microliters of a mineral oil vehicle equivalent to Freund's incomplete adjuvant (known as Montanide® ISA 51, supplied by Seppic, France) was used to reconstitute the lyophilized liposomes to form a water-free liposome suspension. Each vaccine dose consists of 50 microliters of the formulation described above containing liposomes, PT antigen, P3C adjuvant, and the mineral oil carrier. This vaccine formulation will be referred to as free of water / liposome / P3C / hydrophobic vehicle.
[00236] The effectiveness of the water-free liposome formulation described above was compared to the effectiveness of a control vaccine consisting of 1 microgram of PT and 100 micrograms of aluminum hydroxide adjuvant (Alhydrogel, Sigma, Mississauga, ON, Canada, from here) hereinafter called alum) in 100 microliters of 50 milliliters of phosphate buffer (pH 7.0). A group of mice (N = 11) were injected once (no challenge) with 1 microgram of PT antigen and 1 microgram of P3C adjuvant formulated in 50 microliters of free water / liposome / P3C / hydrophobic vehicle, as described above . Group 2 mice (N = 9) and group 3 mice (N = 9) were vaccinated once or three times (day 0, day 21, and day 31) with 1 microgram of PT and 100 micrograms of adjuvant from alum suspended in 100 microliters of phosphate buffer. The mice were vaccinated intramuscularly in the flank region. Group 4 mice (N = 10) remained unvaccinated for the duration of the study. All mice were challenged on day 56 post-immunization and bacterial lung counts established 8 and 15 days post-challenge, as described above.
[00237] The results of this experiment are shown in Figure 2. The mice in Group 4 (naive) were not able to treat the infection, bacterial counts were as high as 2.5 x 10A5 cfu per lung at 8 days post-challenge, and 4.7 x IOA3 cfu per lung at 15 days post-challenge. Group 2 mice, vaccinated with a dose of the control vaccine with alum adjuvant, have bacterial lung counts as high as 8.9 x 10 3 O3 and 3.1 x 10A 2 cfu per lung at 8 and 15 days post-challenge respectively . Group 3 mice vaccinated with three doses of the control have lung counts as high as 3.5 x IOA3 and 1.8 x 10A3 cfu per lung at the same respective time points. Group 1 mice, vaccinated with a single dose of the vaccine corresponding to the invention, have a bacterial lung count as high as 6.2 x 10 M cfu per lung at 8 days post-challenge and 0 cfu per lung in all animals in the day 15 post-challenge. A single dose of the vaccine corresponding to the invention effectively protects mice from bacterial challenge, and allows them to completely treat lung infection. Example 3
[00238] Female pathogen free rabbits, New Zealand White, 23 kg in weight, were obtained from Charles River Laboratories (St Constant, QC, Canada), and were housed according to institutional guidelines with water and food ad libitum, under filter-controlled air circulation.
[00239] The recombinant protective anthrax antigen was purchased from List Biologies (Campbell, CA). This recombinant protein has an approximate molecular weight of 83,000 daltons, and corresponds to the protective antigen protein, a cell-binding component of the three-protein exotoxin produced by a Bacillus anthracis. This recombinant protein, hereinafter referred to as rPA, was used as a model antigen to test the efficacy of vaccine formulations. rPA was used at 8 micrograms per 100 microliter dose.
[00240] The effectiveness of the vaccine was evaluated by an enzyme-linked immunosorbent assay (ELISA), a method that allows the detection of levels of antigen-specific antibody in the serum of immunized animals. Performing an ELISA on serum collected from immunized mice at a regular interval (every four weeks, for example), is useful for monitoring antibody responses to a given vaccine formulation. ELISA was performed as outlined in Example 1, using 1 microgram / milliliter rPA as the coating antigen.
[00241] To formulate the vaccine corresponding to the invention, a homogeneous 10: 1 p: p mixture of DOPC lecithin and cholesterol (Lipoid GmbH, Germany) was hydrated in the presence of rPA and Pam-3-Cys (P3C) to form liposomes with encapsulated rHA and P3C. Briefly, 80 micrograms of rPA and 20 micrograms of P3C in 850 microliters of sterile water were added to 132 milligrams of the DOPC lecithin / cholesterol mixture to form approximately one milliliter of a liposome suspension that encapsulates the rHA antigen and P3C adjuvant. After dilution to a sufficient amount using sterile water, the liposome suspension was lyophilized using the Virtis Advantage freeze dryer (SP Industries, Warminister, PA, USA). For every 1 milliliter of original liposome suspension containing rPA and P3C, 800 microliters of a mineral oil vehicle equivalent to Freund's incomplete adjuvant (known as Montanide® ISA 51, supplied by Seppic, France) were used to reconstitute the lyophilized liposomes for form a water-free liposome suspension. Each dose of vaccine consists of 100 microliters of the formulation described above containing liposomes, rPA antigen, P3C adjuvant, and the mineral oil carrier. This vaccine is called Vaccine C (invention).
[00242] The effectiveness of the vaccine formulation described above was compared to the effectiveness of a control vaccine consisting of 8 micrograms of rPA and 350 micrograms of aluminum hydroxide adjuvant (Alhydrogel) (Sigma, Mississauga, ON, Canada) in 100 microliters of sterile water. A group of rabbits (N = 8) was injected once (no challenge) with 8 micrograms of rPA antigen and 2 micrograms of P3C adjuvant formulated in 100 microliters of vaccine formulation, as described above (Group 1). Rabbits in Group 2 (N = 8) were vaccinated three times (day 0, 28 and 84) with 8 micrograms of rPA and 350 micrograms of alum adjuvant suspended in sterile water. All rabbits were vaccinated intramuscularly in the right gastrocnemius muscle, and serum samples were collected at 3, 4, 8, 12, 16, 20 and 24 weeks post-immunization. The antibody titers of rPA in these sera were examined by ELISA, as described above.
[00243] The results of this experiment are shown in Figure 3. Rabbits in Group 2 generated a detectable antigen-specific antibody response following administration of a control vaccine with alum adjuvant. Rabbits in Group 1, vaccinated with the Vaccine C formulation, produced significantly enhanced endpoint titrations compared to those in Group 2, at previous time points (provocation). Group 2 rabbits generated titrations up to 1 / 64,000 (average Log10 value of 4.66) and up to 1 / 256,000 (average log 10 value of 4.73) in three and four weeks respectively (before challenge) and up to 1 / 2,048,000 (average log 10 equal to 5.86) at eight weeks post-vaccination (after challenge). The presence of such antibody responses confirms a genuine immunogenerated response as a result of vaccination. Group 1 rabbits, vaccinated with the vaccine corresponding to the invention, were able to generate endpoint titrations reaching up to 1 / 2,048,000 (mean log value 0 of 6.20 and 6.09) in three and four weeks post -vaccination and 1 / 8,192,000 (mean log 10 value of 6.53) in eight weeks post-immunization. These results showing that a single dose of liposome / hydrophobic vehicle formulations containing a Pam-3-Cys adjuvant are capable of generating an average of 34.6 times and 22.9 times (in three and four weeks respectively) more antibodies in vivo than can be achieved with an aqueous alum control vaccine, demonstrate an ability to produce a surprisingly strong immune response earlier than three weeks following a single vanication. Example 4
[00244] Female pathogenic CD1 mice, 6-8 weeks old, were obtained from Charles River Laboratories (St Constant, QC, Canada), and were housed according to institutional guidelines with water and food ad libitum, under filter-controlled air circulation.
[00245] The recombinant hemagglutinin H5N1 protein was purchased from Protein Sciences (Meridien, CT, USA). This recombinant protein has a molecular weight of approximately 72,000 daltons, and corresponds to the glycoprotein of hemagglutinin, an antigenic protein present on the surface of the H5N1 flu virus. This recombinant protein, hereinafter referred to as rHA, was used as a model antigen to test the effectiveness of vaccine formulations. rHA was used at 1 microgram per 50 microliter dose.
[00246] The efficacy of the vaccine was evaluated by an enzyme-linked immunosorbent assay (ELISA), a method that allows the detection of levels of antigen-specific antibody in the serum of immunized animals. Performing an ELISA on sera collected from immunized mice at a regular interval (every four weeks, for example), is useful for monitoring antibody responses to a given vaccine formulation. ELISA was performed as described in Example 1.
[00247] To formulate the vaccine corresponding to the invention, a 10: 1 p: p homogeneous mixture of S100 lecithin and cholesterol (Lipoid GmbH, Germany) was hydrated in the presence of rHA and Pam-3-Cys (P3C) in phosphate buffer for form liposomes with encapsulated rHA and P3C. Briefly, 20 micrograms each of rHA and P3C in 850 microliters of 50 millimolar phosphate buffer were added to 132 milligrams of the S100 lecithin / cholesterol mixture to form approximately one milliliter of a liposome suspension that encapsulates the rHA antigen and adjuvant of P3C. The liposome preparation was diluted in a sufficient amount with sterile water and then lyophilized using the Virtis Advantage freeze dryer (SP Industries, Warminister, PA, USA). For an entire milliliter of original liposome suspension containing rHA and P3C, 800 microliters of the mineral oil vehicle (Montanide® ISA 51, supplied by Seppic, France), was used to reconstitute the lyophilized liposomes to form a water-free liposome suspension . Each dose of vaccine consists of 50 microliters of the formulation described above containing liposomes, rHA antigen, P3C adjuvant, and the mineral oil carrier. This vaccine formulation will be referred to as a liposome / P3C / hydrophobic vehicle. This formulation was used to vaccinate Group 1 mice (n = 10).
[00248] Group 2 mice (n = 10) were treated with 1 microgram of rHA and 1 microgram of P3C per 50 microliter dose, in the absence of liposomes / hydrophobic vehicle. Group 3 mice (n = 10) were treated with 1 microgram of rHA and 1 microgram of P3C per 50 microliter dose formulated as an aqueous vaccine / liposome / P3C, in the absence of a hydrophobic vehicle. Group 4 mice (n = 10) were treated with 1 microgram of rHA formulated as a liposome / hydrophobic carrier vaccine in the absence of P3C. All mice were vaccinated intramuscularly in the flank region, and serum samples were collected at 3, 4, 8, 12, 16 and 20 weeks post-immunization. The antibody titers of rHA in these sera were examined by ELISA as described.
[00249] The efficacies of these vaccine formulations have been tested to assess the relative contribution of the components of these vaccine formulations (see Figure 4). The titers of the mice in all control groups (groups 2, 3 and 4) were consistently lower than the titers of group 1, vaccinated with Vaccine A, indicating that all components of this formulation may be important for enhanced immunogenicity. For example, at week 8 post-vaccination, mice in group 1 (vaccinated with Vaccine A) were able to generate end point titers reaching up to 1 / 2,048,000 (mean Log 10 value of 5.65), so the mice in group 2, 3 and 4 were able to generate endpoint titrations of 1 / 64,000 (mean value of Log 10 of 4.41), 1 / 128,000 (mean value of Log 10 of 4.44) and 1 / 128,000 (average Log 10 value of 4.69), respectively. The titers generated by the mice in group one were significantly higher (p <0.0001, by a variance analysis) than the titers generated in any of the three control vaccine groups. This indicates an involvement of all components of vaccine A formulation, specifically antigen, liposomes, an adjuvant of palmitic acid and a hydrophobic vehicle, in simulation of maximum immunogenicity of this formulation. Example 5
[00250] Female, pathogen-free Balb / C mice, 6-12 weeks of age, were obtained from Charles River Laboratories (St Constant, QC, Canada), and were housed according to institutional guidelines with water and food ad libitum, under filter-controlled air circulation.
[00251] The antigen used in the vaccine formulations was an inactivated influenza A / PR / 8/34 (H1N1) strain. A viral stock was prepared by spreading on chicken eggs. A viral stock aliquot of A / PR / 8/34 was quickly thawed and placed in a 56-degree Celcius water bath for 30 minutes to allow heat inactivation of the virus.
[00252] The vaccines were administered on day zero under isoflurane anesthetic, intramuscularly in the thigh muscle (one dose of vaccine was divided into two injections, one per leg). The mice were weighed during the week after vaccination to ensure that the vaccine itself does not cause disease.
On day 28, the mice were anesthetized using isoflurane and inoculated intranasally with 10 x MLD50 virus (two separate administrations of 25 microliters each divided equally in each nostril). The mice were then monitored for 10 days by measuring weight, temperature, and hydration, and by observing appearance, posture, and behavior. Mice that reached predetermined points of morbidity were euthanized.
[00254] The mice in group 1 (n = 10) were vaccinated with saline only, and served as a negative control vaccine.
[00255] Mice in group 2 (n = 10) were vaccinated with 2.56 X 10A3 of TCID50 of the A / PR / 8/34 (H1N1) heat inactivated Flu strain formulated in Alhydrogel.
[00256] Mice in group 3 (n = 10) were vaccinated with a liposome / P3C / hydrophobic carrier vaccine. Briefly, a homogeneous 10: 1 (w: w) mixture of S100 lecithin and cholesterol (Lipoid GmbH, Germany) was hydrated in the presence of A / PR / 8/34 heat inactivated influenza strain and sterile water to form approximately 850 microliters of liposomes with encapsulated antigen. Pam-3-Cys (P3C) adjuvant was then added, liposomes well mixed, and the mixture diluted to a sufficient amount with sterile water before being lyophilized using the Virtis Advantage freeze dryer (SP Industries, Warminister, PA, USA). For every 1 milliliter of original liposome suspension containing A / PR / 8/34 and P3C, 800 microliters of a mineral oil vehicle (Montanide® ISA 51, Seppic, France) were used to reconstitute the lyophilized liposomes to form a suspension of water-free liposome. Each dose volume was 50 microliters and contained liposomes, strain of influenza A / PR / 8/34 (2.56 X 10A3 TCID50), P3C (1 microgram), and the mineral oil vehicle.
[00257] The results of this experiment are shown in Figure 5. The mice in Group 1 vaccinated with saline only quickly developed clinical signs of influenza infection, and all succumbed to infection by day four. The mice in group 2, vaccinated with antigen formulated in Alhydrogel, showed moderately severe clinical symptoms under flu infection, with 30% of animals succumbing to the infection. However, the mice in group 3, vaccinated with the liposome / P3C / hydrophobic carrier vaccine, have relatively moderate clinical symptoms, and 100% survived influenza infection.
[00258] These observations demonstrated that Pam-3-Cys formulated in the vaccine of the invention can enhance the immune response to inactivated viral vaccine formulations, as demonstrated by the enhanced control of the virus after infection. Example 6
[00259] Female pathogenic CD1 mice, 6-8 weeks old, were obtained from Charles River Laboratories (St Constant, QC, Canada), and were housed in accordance with institutional guidelines with water and food ad libitum, under filter-controlled air circulation.
[00260] As in Examples 1 and 4, hemagglutinin recombinant H5N1 protein, corresponding to hemagglutinin glycoprotein on the surface of the H5N1 influenza virus, was purchased from Protein Sciences (Meridien, CT, USA). This recombinant protein, hereinafter referred to as rHA, was used as a model antigen to test the effectiveness of vaccine formulations. rHA was used at 1 microgram per 50 microliter dose.
[00261] The efficacy of the vaccine was evaluated by an enzyme-linked immunosorbent assay (ELISA), a method that allows the detection of levels of antigen-specific antibody in the serum of immunized animals. Performing the ELISA on serum collected from immunized mice at a regular interval (every four weeks, for example), is useful for monitoring antibody responses to a given vaccine formulation. ELISA was performed as described in Example 1.
[00262] Both vaccines in this example were formulated as described in Example 4. In summary, for the vaccine corresponding to the invention, rHA antigen and Pam-3-Cys adjuvant (P3C) in 50 millimolar phosphate buffer were used to moisturize S100 lecithin and cholesterol. The final liposome preparation was lyophilized and then reconstituted with ISA51. The final vaccine consists of 50 microliters of a formulation containing liposomes, 1 microgram of rHA antigen, 1 microgram of P3C adjuvant, and the mineral oil carrier. This vaccine formulation will be referred to as liposome / P3C / hydrophobic vehicle. This formulation was used for vaccinated Group 1 mice (n = 9).
[00263] The Group 2 mice (n = 9) were treated with a formulation containing liposomes, 1 microgram of rHA, 1 microgram of Imiquimod adjuvant (IMQ), and the mineral oil vehicle. This vaccine will be referred to as liposome / IMQ / hydrophobic vehicle. In summary, the rHA antigen and IMQ adjuvant (InvivoGen, San Diego, CA, USA) in 50 millimolar phosphate buffer were used to hydrate S100 lecithin and cholesterol. The final liposome preparation was lyophilized and then reconstituted with ISA51.
[00264] All mice were vaccinated intramuscularly in the flank region, and serum samples were collected at 4 weeks post-immunization. The titers of rHA antibody in these sera were examined by ELISA as described in Example 1.
[00265] The efficacies of these vaccine formulations were tested to assess the relative contribution of P3C adjuvant to the liposome / hydrophobic carrier formulation (see Figure 6). Group 2 mice generate a detectable antigen-specific antibody response following administration of a control vaccine with Imiquimod adjuvant. The mice in Group 1, vaccinated with liposome / P3C / hydrophobic vehicle photmulation, produced significantly enhanced endpoint titrations, compared to those in group 2 (P <0.005). Group 2 mice generated titrations up to 1 / 128,000 (Log10 value of 5.11) in 28 days (4 weeks) post-vaccination. As noted in example 1, the presence of such antibody responses confirms a genuine immunogenerated response as a result of vaccination. The Group 1 mice, vaccinated with the vaccine corresponding to the invention, were able to generate endpoint titrations reaching up to 1 / 1,024,000 (Log1 value of 6.01) in four weeks post-immunization. This data indicates that the vaccine corresponding to the invention (liposomes / P3C / hydrophobic vehicle) is capable of stimulating a specific humoral immune response that is significantly stronger than a vaccine comparable with a different adjuvant (lymphomas / IMQ / hydrophobic vehicle) . Example 7
[00266] Female C57BL6 mice, free of pathogenesis, 6-8 s and older, were obtained from Charles River Laboratories (St Constant, QC, Canada), and were housed according to institutional guidelines with water and food ad libitum , under filter-controlled air circulation.
[00267] Untreated "naive" mice were terminated and spleens collected. A single cell suspension was prepared from splenocytes and lysed red sabule cells using ACK lysis buffer. B cells were isolated using a Miltenyi negative selection magnetic isolation kit (Auburn, CA, USA). The cells were resuspended in complete RPMI medium containing 10% FBS, 1% penicillin-streptomycin, 1% L-glutamine and 0.1% b-mercaptoethanol (c-RPMI) at a final concentration of 2x106 cells / ml. B cells were added to the wells of a 96-well plate (2x105 cells / well) with anti-Ig (2.5 µg / ml; BD Biosciences, Mississauga, Canada) and anti-CD40 (1 µg / ml; BD Biosciences). B cells were stimulated with the following triplicate adjuvants: Pam2Cys (EMC Microcollections, Tuebingen, Germany), Pam3Cys (EMC Microcollections), Poly 1: C (Thermo Fisher, Ml, USA) and LPS (Sigma-Aldrich, Oakville , Canada), or no adjuvant. Each adjuvant was dosed in 10 µg / ml, 1 µg / ml and 0.1 µg / ml, except LPS which was dosed in 100 ng / ml, 10 ng / ml and 1 ng / ml. The cells were incubated at 37 ° C / 5% CO2 for 3 days. Eighteen animals before the end of the experiment, [3H] -thymidine was added to each well at a final concentration of 0.2 uCi / well. The plates were collected using Titertek Cell Harvester (Skatron Instruments, Sterling, VA, USA) on the filter membranes which were then counted with a Beckman LS6000IC liquid scintillation counter (Beckman Coulter Inc., Mississauga, ON, Canada). Proliferation was quantified by calculating the average of triplicate counts per minute (CPM) representing the incorporation of [3H] -thymidine.
[00268] The results of B cell adjuvant stimulation show that while Pam3Cys and Pam2Cys can induce potent B cell proliferation, Poly 1: C does not (see Figure 7). LPS, included as a positive control, is known to induce low concentration B cell proliferation. Based on these results, it is reasonable to expect that vaccines containing Pam2Cys or Pam3Cys would have similar effects on B cells in vivo, and would be able to facilitate the production of similar levels of antigen-specific antibodies.
[00269] All publications and patent applications cited in this specification are incorporated herein by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date, and should not be construed as an admission that the present invention is not entitled to anticipate such publication by virtue of the previous invention.
[00270] Although the foregoing invention has been described in some detail by way of illustration and example for the sake of clarity of understanding, it is readily apparent to that person skilled in the art in the light of the teachings of this invention that certain changes and modifications can be made to it without escape the spirit or scope of the attached claims.
[00271] It should be noted that as used in this specification and in the appended claims, the singular forms "one", "one", and "o" include plural references, unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.
[00272] The phrase "and / or", as used herein in the specification and in the claims, must be understood to mean "either or both" of the elements thus conjugated, that is, elements that are present together in some cases and present disjunctively present in other cases. The multiple elements listed with "and / or" must be constructed in the same way, that is, "one or more" of the elements thus conjugated. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open language, such as "comprising", may refer, in one embodiment, to A only (optionally including other elements than B); in another embodiment, for B only (optionally including elements other than A); in yet another embodiment, for both A and B (optionally including other elements); etc.
[00273] As used herein in the specification and claims, "or" must be understood to involve the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as being inclusive, that is, the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional items not listed.
[00274] As used herein, whether in the specification or in the appended king-vindications, the transitional terms "comprising", "including", "leading", "having", "containing", "involving", and the like , are to be understood as being inclusive or open (that is, to mean including, but not limited to), and they do not exclude undetermined elements, materials or method steps. Only the transitional phrases "consisting of" and "essentially consisting of", respectively, are closed or semi-closed transitional phrases with respect to the claims. The transitional phrase "consisting of" excludes any element, step, or ingredient that is not specifically determined. The transitional phrase "consisting essentially of" limits the scope to the specified elements, materials or steps, and to those that do not materially affect the basic feature (s) of the invention disclosed and / or claimed herein.

权利要求:
Claims (20)
[0001]
1. Water-free composition, characterized by the fact that it comprises an adjuvant that activates or increases the activity of Toll-type 2 receptor (TLR2) and lyophilized liposomes with an encapsulated antigen, reconstituted in a vehicle comprising a continuous phase of a hydrophobic substance , in which the antigen comprises a B cell epitope and is capable of inducing a humoral immune response; and wherein the adjuvant comprises dipalmitoyl-S-glyceryl-cysteine (PAM2Cys) or tripalmitoyl-S-glyceryl-cysteine (PAM3Cys).
[0002]
2. Composition according to claim 1, characterized by the fact that the adjuvant activates or increases the activity of TLR2 by a TLR2 dimer of TLR1 / 2 or TLR2 / 6.
[0003]
Composition according to claim 1 or 2, characterized by the fact that the adjuvant is dipalmitoyl-S-glyceryl-cysteine (PAM2-Cys) -SKKKK.
[0004]
Composition according to claim 1 or 2, characterized by the fact that the adjuvant is tripalmitoyl-S-glyceryl-cysteine (PAM3-Cys) -SKKKK.
[0005]
Composition according to any one of claims 1 to 4, characterized in that the antigen is a polypeptide or a carbohydrate.
[0006]
Composition according to any one of claims 1 to 5, characterized in that the antigen comprises a plurality of B cell epitopes.
[0007]
Composition according to any one of claims 1 to 6, characterized in that the B cell epitope is derived from a virus or bacteria.
[0008]
8. Composition according to claim 7, characterized by the fact that the virus or bacterium is the influenza virus (Influenza), Bordetella pertussis, or Bacillus anthracis.
[0009]
Composition according to claim 7 or 8, characterized in that the B cell epitope is an epitope of an influenza virus hemagglutinin protein (Influenza H5N1), an epitope of pertussis toxoid protein, or an epitope of a recombinant protective anthrax antigen.
[0010]
10. Composition according to any one of claims 1 to 6, characterized by the fact that the antigen is: an antigen associated with an infectious disease; a cancer antigen attached to the membrane surface; a toxin; an allergen; or an antigen associated with a disease where it is desirable to hijack the circulating antigen.
[0011]
11. Composition according to claim 10, characterized by the fact that the allergen is pollen.
[0012]
Composition according to claim 10, characterized in that the antigen associated with a disease where it is desirable to sequester the circulating antigen, is an antigen of an amyloid protein.
[0013]
Composition according to any one of claims 1 to 12, characterized in that both the antigen and the adjuvant are encapsulated in the lyophilized liposomes reconstituted in a vehicle comprising a continuous phase of a hydrophobic substance.
[0014]
Composition according to any one of Claims 1 to 13, characterized in that the antigen is not coupled covalently or not covalently to the adjuvant.
[0015]
Composition according to any one of claims 1 to 14, characterized in that the hydrophobic substance is mineral oil.
[0016]
16. Composition according to any one of claims 1 to 15, characterized in that the composition is capable of inducing a humoral immune response with a single dose.
[0017]
17. Use of a composition as defined in any one of claims 1 to 16, characterized in that it is for preparing a medicament for the treatment or prevention of a disease or disorder in ameliorated by a humoral immune response.
[0018]
18. Use of a composition as defined in any of claims 1 to 16, characterized in that it is for preparing a medicament for the treatment or prevention of an infectious disease, a cancer involving an antigen bound to the membrane on the surface of the cancer, or a disease or disorder where sequestration of circulating antigen is desirable.
[0019]
19. Use according to claim 18, characterized by the fact that the disease or disorder where it is desirable to sequester the circulating antigen, is Alzheimer's disease.
[0020]
Use of a composition according to any one of claims 1 to 16, characterized in that it is for preparing a medicament for neutralizing a toxin, virus, bacteria or allergen, with an antibody.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-07-16| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-09-17| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-02-02| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-04-13| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 05/10/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161544020P| true| 2011-10-06|2011-10-06|

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US61/544,020|2011-10-06|

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PCT/CA2012/050705|WO2013049941A1|2011-10-06|2012-10-05|Liposome compositions comprising an adjuvant that activates or increases the activity of tlr2 and uses thereof|

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