WO2009039125A1 - Use of ocular neuropeptides as immune adjuvants - Google Patents

Abstract

The present invention is directed to methods and compositions relating to immune adjuvants. The present invention is also related to ocular neuropeptides used in compositions and methods of modulating immune responses.

Description

USE OF OCULAR NEUROPEPTIDES AS IMMUNE ADJUVANTS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Application Serial No.: 60/972,834, filed September 17, 2007, incorporated by reference herein in its entirety.

GOVERNMENT INTERESTS:

This invention was made with U.S. Government support (NIH Grants No. ROl EYOl 0752) and the U.S. Government may therefore have certain rights in the invention.

BACKGROUND:

Commonly known vaccinations purposely induce an immune response to a pathogen. Some vaccines are composed of dead or attenuated forms of the pathogen. Other vaccines are mixed with an adjuvant, which modifies the immunogenicity of the vaccines directed against intracellular pathogens. The effect of adjuvants may be enhanced by using bacterial products, which can signal macrophages or dendritic cells to become more effective antigen presenting cells (APCs). This, in turn, increases the production of inflammatory cytokines and provides a potent inflammatory response. However, such bacterial-based adjuvants can cause severe inflammatory reactions that are detrimental to humans. Therefore, it would be valuable to discover a way to take advantage of the positive effects of adjuvants while alleviating any deleterious inflammatory response to a given vaccine.

Induction of the mammalian immune response involves the interaction of innate and adaptive immunity. Innate immunity is an early line of inflammatory defense against infectious and toxic agents. Innate immunity is constantly present and does not increase with repeated exposure to a given pathogen. Innate immunity is mediated by the mechanism of inflammation, e.g., macrophages, dendritic cells and Langerhans cells, in response to various pathogen associated molecules. Initiation of a specific type of inflammatory response depends on which "toll-like receptor" (TLR) is activated on these cells. There are nine TLR in mammals, and each has a range of specific pathogen- associated molecules to which it will bind. Endotoxin of Gram(-) bacteria stimulate macrophages and dendritic cells through TLR4, causing an extreme inflammatory response that can lead to symptoms of septic shock. In contrast, the peptidoglycan of Gram(+) bacteria stimulates macrophages and dendritic cells through TLR2, inducing an inflammatory response that is significantly less than the endotoxin TLR4-mediated inflammation. Nitric oxide generation is an indication of antimicrobial activity by macrophages and dendritic cells, which represents an activation of innate immunity.

Adaptive immunity is the response of antigen-specific lymphocytes to antigen, which also includes the development of immunological memory. Adaptive immune responses are generated by clonal selection of antigen-specific T lymphocytes, the T cells. There are generally considered two types of T cells. ThI cells mediate immune defenses against intracellular pathogens, and also mediate autoimmune disease and hypersensitivity. Th2 cells mediate immune defenses against toxins and parasites by helping B cell production of antibodies and are associated with mediating allergies. The activation of T cells occurs through antigen presenting cells, which are also involved in innate immunity. The T cells bind to a presented antigen through their antigen receptor resulting in the release of cytokines or other binding molecules from the antigen- presenting cells to induce a particular T cell activity. TLRs are also involved in adaptive immunity. Antigen presenting cells stimulated through TLR4 promote the activation of ThI cells. Antigen presenting cells stimulated through TLR2 promote the activation of Th2 cells. The conditions for the activation of ThI or Th2 cells are considered mutually exclusive because the condition for one T cell type is suppressive for the other. Therefore, the activation of ThI cells is associated with an extreme innate inflammatory response and Th2 cells cannot be activated under these conditions. Interferon-gamma ("IFN-γ") production is an indication of ThI cell activation.

There is a need to be able to control the level of activity of innate and/or adaptive immunity in an individual in order to select for a particular response appropriate to that individual's treatment. There is also a need for the development of adjuvants to enhance immunizations as well as prevent side effects of immunizations or prevent side effects from the delivery of antigens to an animal. There is a need for adjuvants that can modulate an immune response in an animal. The present invention fulfills these needs as well as others.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides methods of modulating an immune response to an antigen comprising administering to an animal an immunogenic composition comprising an antigen and an adjuvant, wherein the adjuvant comprises an ocular neuropeptide or fragment or analogue thereof.

In some embodiments, the present invention provides methods of promoting an immunogenic specific ThI response comprising administering to an individual an immunogenic composition comprising calcitonin gene-related peptide ("CGRP").

In some embodiments, the present invention provides methods of suppressing an immunogenic specific ThI response and promoting a Th2 response comprising administering to an individual an immunogenic composition comprising alpha- melanocyte stimulating hormone ("α-MSH").

In some embodiments, the present invention provides methods of inhibiting ThI cell autoimmune disease and graft rejection comprising administering to an individual in need of prevention or inhibition of said autoimmune disease or graft rejection a composition comprising α-MSH and/or somatostatin ("SOM").

In some embodiments, the present invention provides methods of identifying other neuropeptides that can function as an adjuvant comprising: a) contacting a cell with an adjuvant candidate and an inflammatory causing agent; b) determining whether said adjuvant candidate is an adjuvant, wherein said determining comprises: measuring nitric oxide (NO) production; or measuring IL- 12 (interleukin 12) production; or determining whether said contacted cells stimulate IFN-γ production in T cells; or determining whether said contacted cells stimulate IL-4 (interleukin 4) production in T cells; or combinations thereof; c) comparing the results of step (b) with cells in contact with the inflammatory causing agent without the adjuvant candidate, wherein a change indicates that the adjuvant candidate is an adjuvant. In some embodiments, the present invention provides methods of inducing an antibody response without a cellular immune response or an inflammatory response at the site of administration comprising administering an immunogenic composition comprising an antigen and an adjuvant, wherein said adjuvant comprises α-MSH.

In some embodiments, the present invention provides methods of inducing an antibody response or a cellular immune response without an inflammatory response at the site of administration comprising administering an immunogenic composition comprising an antigen and an adjuvant, wherein said adjuvant comprises CGRP.

In some embodiments, the present invention provides methods of inducing an antibody response with an inflammatory response at the site of administration without a cellular immune response comprising administering an immunogenic composition comprising an antigen and an adjuvant wherein said adjuvant comprises SOM.

In some embodiments, the present invention provides methods of generating an immune response in an individual with a live or attenuated virus comprising administering an immunogenic composition comprising an antigen and an adjuvant wherein said adjuvant comprises SOM. In some embodiments, the present invention provides compositions comprising an antigen and an adjuvant, wherein said adjuvant is α-MSH, CGRP, VIP, SOM, or combinations thereof including active fragments and analogues thereof.

In some embodiments, the present invention provides kits for identifying adjuvants. In some embodiments, the present invention provides methods of modulating an immune response in a re-immunization to an antigen comprising administering to an individual an immunogenic composition comprising the antigen and an adjuvant, wherein the adjuvant is α-MSH, CGRP, SOM, or fragments or analogues thereof, or combinations thereof.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1: Use of α-MSH as an adjuvant. α-MSH was used as an adjuvant to measure its effect on vaccine associated swelling as well as its impact on IFN-γ, IL-4, and TGF-(S production. α-MSH suppressed vaccine associated swelling at the site of injection (Figure IA) and suppressed IFN-γ production (Figure IB), but not IL-4 and TGF-/3 production. DETAILED DESCRIPTION

As used herein, the term "adjuvant" refers to a substance that is administered with an antigen (e.g. the target of the vaccination) that modulates an immune response to the antigen.

As used herein, the term "antigen" refers to a target of immunization or a target for which an immune response is desired. An antigen can be any substance that one desires to immunize an individual against, or to generate an immune response against. The immunization can comprise an adaptive immune response and/or an innate immune response. Examples of types of antigens include, but are not limited to, an allergen, a toxin, a pathogen, a viral protein, a bacterial protein, a virus (e.g. live, attenuated, and/or dead), a bacterium (e.g. live, attenuated, and/or dead), and the like. More specifically, examples of antigens include, but are not limited to, smallpox, polio, tetanus, pertussis, animal dander, pollen, HIV, HSV, Hepatitis A, B, or C, west-nile virus, anthrax, botulinum toxin, avian flu virus, SARS virus, non-avian flu virus, and the like.

The methods and compositions described herein can be administered to any vertebrate. Examples of vertebrates include, but are not limited to, mammals and non- mammals (e.g. birds). Examples of mammals include, humans, non-human primates, horses, dogs, cats, and the like. A "human" can also be referred to as an individual. As used herein, the term "adaptive immunity" refers to the response of antigen- specific lymphocytes to an antigen, which can also include the development of immunological memory.

The present invention relates to the use of neuropeptides constitutively found in the eye to modulate innate and adaptive immunity. The effect of neuropeptides on innate and adaptive immunity result in their ability to be used as adjuvants according to the invention, which can be referred to as "immune adjuvants."

For example, immune adjuvants can suppress the side effects of vaccinations, which include but are not limited to the infection of nonvaccinated individuals, the induction of deadly inflammatory responses, allergic responses, and inflammation at the site of administration. With the use of immune adjuvants, or in some embodiments immunosuppressive cytokines, vaccinations or immunizations can be tailored regarding the specific need such as, but not limited to, cellular immunity, antibody immunity, induction of an inflammatory response at the site of immunization to prevent spread of virus in a live virus vaccination, or prevention of allergic or inflammatory responses to the antigen. In some embodiments, the present invention provides methods of modulating an immune response to an antigen comprising administering to an individual an immunogenic composition comprising an antigen and an immune adjuvant, wherein the adjuvant comprises an ocular neuropeptide. An ocular neuropeptide is a neuropeptide that is found in the eye and can modulate an immune response, such as, but not limited to, a ThI cell mediated immunity and/or a Th2 cell mediated immunity. In some embodiments, the adjuvant comprises an ocular neuropeptide composition that comprises α-MSH (alpha-melanocyte stimulating hormone), CGRP (Calcitonin gene-related peptide), VIP (Vasoactive Intestinal Peptide), SOM (Somatostatin), or combinations thereof. In some embodiments 1, 2, 3, or 4 ocular neuropeptides are used. The peptides described herein are known to one of ordinary skill in the art and are also described in, for example, Taylor AW. Neuroimmunomodulation in immune privilege: Role of neuropeptides in ocular immunosuppression, Neuroimmunomodulation. 2002, 10:189- 198, which is hereby incorporated herein by reference in its entirety.

Additionally, in some embodiments, active fragments of or analogues of the peptides can also be used. Active fragments or analogues of the various neuropeptides are known to one of skill in the art. For example, active fragments or analogues of VIP are described in Gonzalez-Rey et al., Vasoactive intestinal peptide family as a therapeutic target for Parkinson's disease. Expert Opin Ther Targets, 9(5):923-9 (2005); and Sergejeva et al., A synthetic VIP peptide analogue inhibits neutrophil recruitment in rat airways in vivo. Regulatory Peptides. 117(2): 149-54. (2004), each of which is hereby incorporated herein by reference in its entirety. Active fragments or analogues of somatostatin have also been used. Examples of analogue somatostatin peptides are described in Helyes et al. Anti-inflammatory effect of synthetic somatostatin analogues in the rat. Br J Pharmacol.;l34(7):l57\-9 (2001), which is hereby incorporated herein by reference in its entirety. Examples of analogue CGRP peptides are described in Malis et al. Modulatory effect of two novel CGRP receptor antagonists on nasal vasodilatator/ responses to exogenous CGRP, capsaicin, bradykinin and histamine in anaesthetised pigs. Regulatory Peptides. 101 (1-3): 101 -8 (2001); and Saha et al., Role of conformational constraints of position 7 of the disulphide bridge of h-alpha-CGRP derivatives in their agonist versus antagonist properties. Journal of Peptide Research. 52(2):112-20 (1998), each of which is hereby incorporated herein by reference in its entirety. Examples of α- MSH analogue peptides are described in Schioth et al. New Melanocortin 1 Receptor Binding Motif Based on the C-Terminal Sequence of alpha-Melanocyte-Stimulating Hormone. Basic Clin Pharmacol Toxicol. 99(4):287-93 (2006); Masman et al. Synthesis and conformational analysis of His-Phe-Arg-Trp-NH(2) and analogues with antifungal properties. BioorgMed C 'hem AA(Tl):! ^'604-1 ^'614 (2006); Fung et al. Design of cyclic and other templates for potent and selective peptide alpha-MSH analogues. Curr Opin Chem Biol. 9(4):352-8 (2005); Bonetto et al. Isolation and characterization of antagonist and agonist peptides to the human melanocortin 1 receptor. Peptides. 26(11):2302-13 (2005); Todorovic et al. N-terminal fatty acylated His-dPhe-Arg-Trp-NH(2) tetrapeptides: influence of fatty acid chain length on potency and selectivity at the mouse melanocortin receptors and human melanocytes. J Med Chem. 48(9):3328-36 (2005); Hoggard et al. Peripherally administered [Nle4,D-Phe7]-alpha-melanocyte stimulating hormone increases resting metabolic rate, while peripheral agouti-related protein has no effect, in wild type C57BL/6 and ob/ob mice. J MoI Endocrinol. 33(3):693-703 (2004); Chen et al. Phenylguanidines as selective nonpeptide melanocortin-5 receptor antagonists. J Med Chem. 29;47(16):4083-8 (2004); and Han et al. De novo design, synthesis, and pharmacology of alpha-melanocyte stimulating hormone analogues derived from somatostatin by a hybrid approach. J Med Chem. 47(6): 1514-26 (2004), each of which is hereby incorporated herein by reference in its entirety. The immune response that is modulated can be any immune response that is triggered upon administering the adjuvant and an antigen. Examples of immune responses include, but are not limited to, production of antibodies (e.g. Th2 cell mediated immunity) and/or cellular immunity (e.g. ThI cell mediated immunity), and/or IFN-γ production. As used herein, the term "modulating an immune response" refers to an immune response being suppressed or enhanced. In some embodiments, the term "enhanced" can also be referred to as "promoted" and the term "suppressed" can be referred to as "inhibited." In some embodiments, the response that is being modulated is modulated by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%. In some embodiments, the percent modulation is determined by comparing the administration of the antigen without the immune adjuvant to the administration of the antigen with the immune adjuvant. For example, if the ThI mediated response is determined to be a certain level "X," and that level is modulated (e.g. suppressed or enhanced) by 50%, then the immune adjuvant is said to have modulated an immune response by 50% or by at least 50%. Methods of measuring immune responses are well known to one of skill in the art.

In some embodiments, the antibody production is enhanced or suppressed. In some embodiments the cellular immunity is enhanced or suppressed. In some embodiments, the method enhances antibody production, while suppressing cellular immunity. In some embodiments, the methods disclosed herein enhance cellular immunity and suppress antibody production. In some embodiments cellular immunity or antibody production is enhanced. In some embodiments both cellular immunity and antibody production are suppressed.

In some embodiments where enhancement of antibody production and suppression of cellular immunity is desired the immune adjuvant comprises α-MSH and/or SOM.

In some embodiments where both enhancement of antibody production (e.g., Th2 cell mediated immunity) or cellular immunity (e.g., ThI cell mediated immunity) is desired the immune adjuvant comprises CGRP. In some embodiments, the methods of modulating an immune response comprise inhibiting an inflammatory immune response at the site of administration. In some embodiments, where suppression of an inflammatory immune response at the site of administration is desired the immune adjuvant comprises α-MSH and/or CGRP. In some embodiments, the immune response that is modulated is IFN-γ production. As noted above, IFN-7 production is an indication of ThI activation. In some embodiments, IFN-γ production is suppressed. In some embodiments, IFN-γ production is enhanced.

As discussed above, in some embodiments the administration of an immune adjuvant and an antigen can either enhance or suppress a Th2 mediated immune response. In some embodiments, where enhancement of a Th2 response is desired the immune adjuvant comprises SOM.

Methods of administration are well known to one of skill in the art and can be done by any method. Methods of administration include, but are not limited to, injection (e.g. intramuscular), oral administration, needleless injections, traditional syringes, and the like.

In some embodiments, the present invention provides methods of enhancing an immunogenic specific ThI response comprising administering to an individual an immunogenic composition comprising CGRP.

As discussed above, it is believed that a ThI response and Th2 response are mutually exclusive such that if there is a ThI response, there cannot be a Th2 response. Accordingly, in some embodiments, to activate or enhance a Th2 response, or promote favorable conditions for a Th2 response, the ThI response can be inhibited. Therefore, in some embodiments, the present invention comprises methods of inhibiting an immunogenic specific ThI, thereby allowing a Th2 response, or converting a ThI immune mediated response into a Th2 response, comprising administering to an individual an immunogenic composition comprising α-MSH.

In some embodiments, the present invention comprises methods of inhibiting ThI cell autoimmune disease and graft rejection comprising administering to an individual in need of prevention or inhibition of the autoimmune disease or graft rejection a composition comprising α-MSH or SOM.

In some embodiments, the present invention provides methods of inducing an antibody response without a cellular immune response or an inflammatory response at the site of administration comprising administering a composition comprising an antigen and an immune adjuvant, wherein the immune adjuvant comprises α-MSH. In some embodiments, the present invention provides methods of inhibiting or not inducing an inflammatory response at the site of administration comprising administering a composition comprising an antigen and an adjuvant, wherein the adjuvant comprises CGRP and/or α-MSH.

In some embodiments, the present invention provides methods of increasing inflammatory response at the site of administration comprising a composition comprising an antigen and an adjuvant, wherein the adjuvant comprises SOM.

As discussed above, when a live vaccine is used the present invention can be used to prevent the transmission of the live vaccine to an uninfected individual. Prevention of the transmission of the live vaccine can be achieved by inducing inflammation at the site of administration which will decrease the likelihood of transmitting a live vaccine to an uninfected individual. Accordingly, in some embodiments, the present invention provides methods of inducing an antibody response, with an inflammatory response at the site of administration, without a cellular immune response, comprising administering a composition comprising SOM.

The present invention also provides compositions comprising an antigen and an adjuvant, wherein the adjuvant is α-MSH, CGRP, VIP, or SOM.

The amounts of the adjuvants that are used in the compositions or the methods described herein can be any amount that is sufficient to modulate the immune response(s) as described herein. One of skill in the art can determine the amount that is sufficient by the routine of injection and with the guide of the examples described herein, hi some embodiments, the amount of the neuropeptide is less than 20 μg, less than 1 ng, less than 200 peg, between about 1 peg to about 1 ng, between about 40 peg to about 1 ng. The amount of the peptide can also be equivalent to what is present physiologically in an individual. The amount present physiologically in an individual can be determined by any method known to one of skill in the art. In some embodiments, the compositions of the present invention can comprise other adjuvants, such as, but not limited to, silica or aluminum salts, Freund's adjuvant, and the like.

The present invention also provides methods of re-immunization. As used herein, the term "re-immunization" refers to a method of administering to an individual an antigen subsequent to a first administration. The re-immunization can be used to boost an immune response to the antigen or alter the type of immune response, which can confer increased immunity to the antigen. In some embodiments, the method comprises a method of re-immunization wherein the initial immunization comprised a ThI cell mediated response, and the re-immunization is used to induce a Th2 cell mediated response. The change in Th cell mediated responses can be accomplished, for example, by administering a composition comprising the antigen and an adjuvant of the invention. Where the initial response is a ThI cell mediated response, and the re-immunization is desired to be a Th2 cell mediated response, the adjuvant can comprise, for example, α- MSH and/or SOM. hi the instance where the initial immunization induced a Th2 cell mediated response and the re-immunization is desired to induce a ThI mediate response, the adjuvant can comprise, for example, CGRP. The re-immunization can also be done under conditions to inhibit inflammation at the site of administration (e.g., at the injection site). In such cases where inflammation is to be inhibited, a composition comprising an antigen and an adjuvant, wherein the adjuvant is, but not limited to, α-MSH and/or CGRP can be administered.

The present invention can also be used to identify other neuropeptides that can be used to modulate an immune response as those described herein. In some embodiments, the method comprises contacting an innate immune cell (e.g. antigen presenting cell) and/or a T cell with an adjuvant candidate and an inflammatory causing agent and measuring nitric oxide (NO) production by the innate immune cell and/or IFN-γ production by the T cell and comparing the NO and/or IFN-γ production to expected TLR4 and/or TLR2 activation events. As noted above, NO production is an indication of activation of innate immunity; IFN-γ production is an indication of ThI cell activation. In some embodiments, the method comprises determining whether the cells contacted with the adjuvant candidate and the inflammatory causing agent stimulate IFN-γ and/or IL-4 production in T cells. IL-4 production in the absence of IFN-γ production indicates activation of Th2 cells. In some embodiments, the method comprises measuring IL- 12 production produced from the innate immune cells contacted with a adjuvant candidate and an inflammatory causing agent. IL- 12 production indicates innate immunity that supports ThI cell activation. In some embodiments, the method comprises comparing the results of where the cell is contacted without the adjuvant candidate, wherein a change indicates that the adjuvant candidate is an adjuvant. Methods of measuring the responses described herein are routine and any such method can be used. An "inflammatory causing agent" refers to an agent that can cause inflammation when exposed to a cell (e.g. a macrophage cell). In some embodiments, the inflammatory causing agent is a bacterial product of Gram(-) bacteria, Gram(+) bacteria; Mycobacterium tuberculosis, or combinations thereof. In some embodiments, the method comprises contacting a macrophage with an adjuvant candidate and an inflammatory causing agent and measuring nitric oxide (NO) production and/or IL- 12 production and/or demonstrating that such treated macrophages stimulate IFN-γ or IL-4 production in T cells, and comparing these effects with macrophages in contact with the inflammatory causing agent without the adjuvant candidate. IL- 12 and/or IL-4 production are indicators of immune responses, which indicate the type of response that will be generated. The following non-limiting embodiments describe how the production or suppression of the various compositions indicate the type of immune response that is either modulated or generated by an adjuvant or adjuvant candidate.

In some embodiments, suppression of NO with stimulated IL- 12 production with T cell production of IFN-γ indicates that the candidate adjuvant will suppress local inflammation while promoting cellular immunity.

In some embodiments, suppression of NO and IL- 12 production with T cell production of IL-4 and suppressed IFN-γ production indicates that the candidate adjuvant will suppress local inflammation while promoting antibody immunity. In some embodiments, suppression of NO and IL- 12 production with T cell production of IFN-γ indicates that the candidate adjuvant will suppress local inflammation while promoting cellular immunity.

The present invention also provides kits for identifying adjuvants. In some embodiments, the kit comprises reagents for measuring nitric oxide and/or IFN- γ production. In some embodiments, the kit comprises an inflammatory causing agent. In some embodiments, the inflammatory causing agent is M. tuberculosis. In some embodiments, the kit comprises an antigen. In some embodiments, the kit comprises a ThI and/or Th2 cell line. The kit can also comprise a monocytic leukemia cell line, wherein the leukemia cell line can serve both as an antigen presenting cell and a macrophage. The kit can also include an instruction manual that details how the assay can be performed. In some embodiments, the kit comprises control neuropeptides. The kit can also comprise instructions on how to understand and interpret the results as presented in Table 1 to determine if a tested substance is an immune adjuvant.

In some embodiments, the present invention provides kits for immunizations. In some embodiments, the kit comprises an immune adjuvant (e.g. positive control) that comprises a 2x solution of one or a combination of neuropeptides in an inert stabilizing buffer, which can be, for example, be supplied as a pre-made solution. A Ix concentration of one or a combination of the neuropeptides can be provided as a lyophilized neuropeptide in a vial into which the antigen solution is added and mixed. The neuropeptide can be chosen based on the condition and type of immunization desired. The neuropeptide concentration can range from its ocular physiological levels to super physiological levels to enhance its immunomodulating activity. A immunogenic composition, including, but not limited to, a vaccine can be made by combining in a syringe, or other equivalent object, an equal volume of the immune adjuvant solution (the neuropeptide) with an equal volume of a 2x solution of the antigen. The composition can then be injected into the appropriate tissue site for vaccination

The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results.

Examples: Example 1: Desiccated M. tuberculosis bacteria (3μg), an antigen with innate immune activating activity (a stimulator of TLR-4 and TLR-2 on innate immune cells); or 160 μg ovalbumin (OVA), an antigen without innate immune activating activity, were added to cultures of 5 x 10⁵ adherent mouse spleen cells treated with aqueous ocular neuropeptides in 200 μl culture media, e.g., alpha-melanocyte stimulating hormone (α-MSH), vasoactive intestinal peptide (VIP), calcitonin gene related peptide (CGRP) or somatostatin (SOM) at their aqueous humor concentration (30 pg/ml, 2 ng/ml, 2 ng/ml, 200 pg/ml respectively). After a 24 hour incubation at 37⁰C, 5% CO₂, the adherent cells were washed and 4 x 10⁵ M tuberculosis specific or OVA specific ThI cells were added to the cultures. The cultures were incubated at 37°C, 5% CO₂ The culture supernatants were assayed 48 hours later for IFN-γ production by ELISA. IFN-γ production is an indication of ThI cell activation.

Cultures of the mouse monocytic leukemia cell line J774, an innate immune cell, were treated with α-MSH, VIP, CGRP or SOM (30 pg/ml, 2 ng/ml, 2 ng/ml, 200 pg/ml respectively) and activated via the TLR4 with the pathogen molecule E.coli endotoxin (0.3 μg), or via the TLR2-activating pathogen molecule S. aureus cell wall (40 μg), or via both TLR-4 and TL-2 with desiccated M. tuberculosis (3 μg) in 200 μl of culture media. The cultures were incubated for 24 hours at 37°C, 5% CO₂. The culture supernatants were assayed for nitric oxide generation by Griess reagent. Nitric oxide generation is an indication of antimicrobial activity by macrophages and dendritic cells, which represents an activation of innate immunity. Antigen presenting cells (APC) that processed and presented antigen with innate immune stimulation were suppressed in activating ThI cells when these APC were treated with α-MSH or SOM, but not suppressed when treated with CGRP or VIP. (See Table I.) Nitric oxide production induced by endotoxin activated J774 cells was suppressed only by α-MSH and CGRP. In regulating innate immunity, α-MSH suppressed all TLR4 associated activity, CGRP suppressed only antimicrobial activity, SOM suppressed only the promoted activation of ThI cells, and VIP had no effect on TLR4 associated innate immunity.

Table I. Antigen Presenting Cell (APC) presentation of antigen and innate inflammatory response.

This study shows that the ocular microenvironment contains a multiplicity of factors, which can be manipulated by a clinician to suppress activation of ThI cells and/or innate immunity. Each factor influences discrete stages of the interface between innate and adaptive immunity.

Example 2:

A series of experiments was conducted to test whether the in vitro findings predict the outcome of using ocular neuropeptides in a cytokine adjuvant in vivo. For this experiment, adult healthy BALB/CJ mice were used. The mice were given a cutaneous injection into one foot with one of three different adjuvant preparations. There were five mice per group. Referring to Figure 1, Group A mice were injected with an emulsification that was 50% metabolic oil (Sequalene) with desiccated Mycobacterium tuberculosis bacteria (200 μg) and 20 μg of α-MSH in sterile physiological saline. Group B mice were injected with an emulsification that was 50% Sequalene with Mycobacterium tuberculosis bacteria (200 μg) in sterile physiological saline and no α- MSH. Group C mice were injected with standard complete Freund's adjuvant, which is mineral oil, and Mycobacterium tuberculosis bacteria (200 μg) and no α-MSH (standard mouse immunization procedure to induce an inflammatory immune response). The injections were 50 μl of the mixtures above. The Mycobacterium tuberculosis bacteria were desiccated and non- viable. We used Mycobacterium tuberculosis bacteria as the antigen because it can act as both a stimulator of innate immune inflammation and as an antigen target for IFN-γ producing (inflammatory) adaptive immunity.

Based on the in vitro experiments, a vaccination scheme containing α-MSH should (1) suppress the inflammation at the site of injection and (2) suppress the activation of immune cells from the draining lymph node that produce interferon-gamma (IFN-γ).

The inflammation of the injected foot of each mouse was examined. Since the use of complete adjuvant causes significant inflammation and swelling that last over the 7 days of the experiment, the feet were examined a couple of times over the 7 days for signs of inflammation. At 48 hours after the injection, it was found that the feet of mice injected with the immunization reagents containing α-MSH (Group A) showed very little signs of inflammation when compared to the two other groups of mice. The inflammation caused by using a Sequalene based immunization (Groups A & B) was less than using the standard Freund's adjuvant(Group C). The inflammation was quantified by measuring the foot swelling on Day 6, a day before the draining lymph nodes were collected to examine the type of immunity induced by the immunization schemes. Using a caliper, the extent of swelling in the injected foot (bottom to top) was measured and compared to the uninfected foot of the same mouse. The difference of the thickness was calculated and the results presented in Figure IA as delta-mm +/- Standard Deviation. The addition of α-MSH significantly suppressed the swelling to a level that was almost insignificant to normal (a value of 0 delta-mm). Therefore, as predicted by the in vitro experiments, α-MSH suppresses the local inflammatory response to an injected immunization that is known to mediate activation of inflammatory innate immunity. To determine whether the inflammatory immune cells are induced by the immunization schemes, draining lymph node cells were collected seven days after the immunization. The cells were used in a standard lymph node cell activation assay to characterize their cytokine profile. This was done by culturing the cells with (+) antigen (3 μg of Mycobacterium tuberculosis) or without (-) antigen and incubating the cultures for 48 hours. This assay mimics the conditions that activate an effector immune response, which defines the type of immunity induced by the immunization. The culture supernatants were assayed for specific lymphokines produced by activated T cells ~ IFN-γ, IL-4 and TGF-β. These three lymphokines are hallmarks of specific immune responses, that can predict the type of immunity that will be mounted by our immune system should we reencounter the antigen or pathogen. The results of these assays are presented in Figure IB. The inclusion of α-MSH (Group A) in the immunization injection significantly suppressed the induction of IFN-γ. α-MSH did not affect the production of other cytokines (IL-4 and TGF-β).

Therefore, a substance (Sequalene based vaccination) containing α-MSH that is injected with an antigen in vivo modifies the immune response to the antigen by suppressing a ThI inflammatory response and enhancing a Th2 response, as predicted by the previous in vitro experimentations.

Example 3: Identification of adjuvants

Adherent cells obtained from mouse spleens (macrophages and dendritic cells), a macrophage cell line, or a specific T cell line are used. To test for the effect on innate immunity, macrophages are treated with a neuropeptide to be tested and any of the following inflammatory causing agents: Endotoxin (of Gram(-) bacteria) to activate only TLR4; Gram(+) Staphylococcus aureus to stimulate only TLR2; or Mycobacterium tuberculosis to stimulate both TLR4 and TLR2 simultaneously, are used as an internal control. Twenty-four hours later, the cultures are assayed for nitric oxide production to determine the level of antimicrobial activity.

To identify adjuvants that have an effect on adaptive immunity, 5 x 10⁵ adherent spleen cells are treated with a candidate adjuvant to be tested and fed antigen with 0.3 μg Endotoxin (Gram(-) bacteria) to activate only TLR4; or 20 μg Gram(+) Staphylococcus aureus to stimulate only TLR2; or 3 μg Mycobacterium tuberculosis to stimulate both TLR4 and TLR2 simultaneously, are used as an internal control. The total culture media volumes are 200 μl and the cultures are incubated for 24 hours at 37°C, 5% CO₂. Twenty- four hours later, the cell cultures are washed and 4 x 10⁵ ThI cells specific for the antigen are added to the cell cultures. The total culture media volumes are 200 μl and the cultures are incubated at 37°C, 5% CO₂. Forty-eight hours later, the culture supernatants are assayed for IFN-γ to see if ThI cells were activated. Additionally, IL-4 production is assayed to see if Th2 cells were activated. Similarly, TGF-β production may be assayed to determine if there is activation of regulatory T cells,i.e., T cells that suppress other T cells, such as ThI and Th2).

By comparing the production of IFN-γ and the nitric oxide generation to TRL4 and TLR2 stimulation, the following results can be determined: whether the agent inhibits the activation of ThI cells through the suppression of TLR4 pathways; whether the agent inhibits the activation of ThI cells independent of innate immunity; whether TM cell activation is promoted without innate immunity; or whether all inflammatory activity is suppressed. This procedure permits one of skill in the art to use the present invention to define and/or identify additional immune adjuvant strategies for vaccination in a manner that elicits the most appropriate response with minimal side effects or the induction of ineffectual immunity.

For example, various scenarios, which are summarized in Table II below, are possible for determining immunization protocols for manipulating a host's immunity in response to a vaccination.

Table II. Neuropeptide effect on immunity.

Other neuropeptides are tested according to the assays described herein and used in an analogous manner, depending on the screening assay results.

The disclosures of each and every patent, patent application, publication, and accession number cited herein are hereby incorporated herein by reference in their entirety.

While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

What is claimed is:

1. A method of modulating an immune response to an antigen comprising administering to an animal an immunogenic composition comprising an antigen and an adjuvant, wherein the adjuvant comprises an ocular neuropeptide, or active fragment or analogue thereof.

2. The method of claim 1 wherein said ocular neuropeptide is α-MSH, CGRP, VIP, SOM, or combinations thereof.

3. The method of claim 1 wherein the immune response comprises a promotion of antibody production and a suppression of the cellular immunity.

4. The method of claim 3 wherein said neuropeptide is α-MSH.

5. The method of claim 3 wherein said neuropeptide is SOM.

6. The method of claim 1 wherein the immune response comprises promoting antibody production or cellular immunity.

7. The method of claim 6 wherein said neuropeptide is GCRP.

8. The method of claim 1 wherein an inflammatory immune response at the site of administration is suppressed or not induced.

9. The method of claim 6 wherein said neuropeptide is α-MSH.

10. The method of claim 6 wherein said neuropeptide is CGRP.

11. The method of claim 1 wherein the modulation of the immune response comprises the suppression of IFN-γ.

12. The method of claim 1 wherein the modulated immune response is a Th2 response, wherein said Th2 response is promoted.

13. The method of claim 12 wherein said neuropeptide is SOM.

14. The method of claim 1 wherein said immunogenic composition is administered by injection.

15. A method of promoting an immunogenic specific ThI response comprising administering to an individual an immunogenic composition comprising CGRP.

16. A method of inhibiting an immunogenic specific ThI response and promoting a Th2 response comprising administering to an individual an immunogenic composition comprising α-MSH.

17. A method of inhibiting or inhibiting ThI cell autoimmune disease and graft rejection comprising administering to an individual in need of prevention or inhibition of said autoimmune disease or graft rejection a composition comprising α-MSH and/or SOM.

18. A method of determining whether a neuropeptide is an adjuvant comprising a) contacting a cell with a neuropeptide and an inflammatory causing agent; b) determining whether said adjuvant candidate is an adjuvant, wherein said determining comprises: measuring nitric oxide (NO) production; or measuring IL- 12 production; or determining whether said contacted cells stimulate IFN-γ production in T cells; or determining whether said contacted cells stimulate IL-4 production in T cells; or combinations thereof; c) comparing the results of step (b) with cells in contact with the inflammatory causing agent without the adjuvant candidate, wherein a change indicates that the adjuvant candidate is an adjuvant.

19. The method of claim 18, wherein suppression of NO, stimulation of IL- 12 production, and T cell production of IFN-γ indicates that the candidate adjuvant will suppress local inflammation while promoting cellular immunity.

20. The method of claim 18, wherein suppression of NO and IL- 12 production, production of IL-4 and suppression of IFN-γ production indicates that the candidate adjuvant will suppress local inflammation while promoting antibody immunity.

21. The method of claim 18, wherein stimulation NO production indicates that the candidate adjuvant will not suppress local inflammation while promoting either cellular or antibody immunity.

24. The method of claim 18 wherein said cell is a macrophage.

25. The method of claim 18 wherein said inflammatory causing agent are bacterial products of Gram(-) bacteria), Gram(+); Mycobacterium tuberculosis, or combinations thereof.

26. A method of inducing an antibody response without a cellular immune response or an inflammatory response at the site of administration comprising administering an immunogenic composition comprising an antigen and an adjuvant, wherein said adjuvant comprises α-MSH.

27. The method of claim 26 wherein said antigen is a toxin or allergen.

28. A method of inducing an antibody response or a cellular immune response without an inflammatory response at the site of administration comprising administering an immunogenic composition comprising an antigen and an adjuvant, wherein said adjuvant comprises CGRP.

29. The method of claim 28 wherein said antigen is a viral protein.

30. A method of inducing an antibody response with an inflammatory response at the site of administration without a cellular immune response comprising administering an immunogenic composition comprising an antigen and an adjuvant wherein said adjuvant comprises SOM.

31. The method of claim 30 wherein said antigen is a live virus.

32. A method of generating an immune response in an individual with a live or attenuated virus comprising administering an immunogenic composition comprising an antigen and an adjuvant wherein said adjuvant comprises SOM.

33. A composition comprising an antigen and an adjuvant, wherein said adjuvant is α-MSH, CGRP, VIP, SOM, or active fragments or analogues thereof, or combinations thereof.

34. The composition of claim 33 wherein said antigen is a live virus, attenuated virus, allergen, toxin, or viral protein.

35. A kit for identifying adjuvants comprising: a reagent for nitric oxide production and/or IFN-γ production; an antigen; a ThI and/or Th2 cell line; and a monocytic leukemia cell line, wherein said leukemia cell line can serve both as an antigen presenting cell and a macrophage. 36 A method of modulating an immune response in a re-immunization to an antigen comprising administering to an individual an immunogenic composition comprising the antigen and an adjuvant, wherein the adjuvant is α-MSH, CGRP, SOM, or fragments or analogues thereof, or combinations thereof.

37. The method of claim 36 wherein the re-immunization changes the immune response from an initial ThI response to a Th2 response.

38. The method of claim 36 wherein the re-immunization changes the immune response of a Th2 response that occurred in the initial administration to a ThI response.

39. The method of claim 36 wherein to prevent inflammation at the site of administration the adjuvant comprises α-MSH and/or CGRP.