WO2008088846A2

WO2008088846A2 - Substance p and analogs thereof as an immunogenic composition adjuvant

Info

Publication number: WO2008088846A2
Application number: PCT/US2008/000621
Authority: WO
Inventors: Hal Siegel
Original assignee: Immuneregen Biosciences, Inc.
Priority date: 2007-01-18
Filing date: 2008-01-17
Publication date: 2008-07-24
Also published as: EP2125869A2; CA2675753A1; WO2008088846A3

Abstract

Substance P and bioactive analogs thereof can be used as an adjuvant with immunogenic compositions to enhance the immunizing properties of the immunogenic compositions. In some embodiments, the amount of antigen needed in each dose of immunogenic composition is decreased due to the adjuvant activity of the substance P analogs.

Description

SUBSTANCE P AND ANALOGS THEREOF AS AN IMMUNOGENIC COMPOSITION ADJUVANT

1. PRIORITY

[0001] This application claims the benefit of priority to U.S. Provisional Application number 60/885,562 filed January 18, 2007, U.S. Provisional Application number 60/915,358 filed May 1, 2007, and U.S. Provisional Application number 60/955,712 filed August 14, 2007.

2. BACKGROUND OF THE INVENTION

[0002] The development of vaccines to prevent infection in animals has been effective against some diseases, however some animals, including humans, fail to have the required immunological response for protection. Agents that enhance immune response to vaccines, called adjuvants, may be used, but currently employed vaccine adjuvants are outdated and have an unsatisfactory safety profile. There is therefore a need for safe, immuno-stimulatory molecules to improve vaccine technology.

3. SUMMARY OF THE INVENTION

[0003] Provided herein are methods and compositions for use of substance P analogs as adjuvants in immunological compositions for humans. In one embodiment, a method for preventing disease or infection in humans comprises administering an immunogenic composition comprising an antigen and an adjuvant effective amount of a substance P analog. [0004] In one embodiment, a method for preventing a viral disease or infection in a human comprises administering to said human, an immunogenic composition comprising an antigen and an adjuvant effective amount of a substance P analog. In one embodiment, a method for immunizing a human against a viral disease or infection comprises administering to said human, an immunogenic composition comprising an antigen and an adjuvant effective amount of a substance P analog. In one embodiment, a method for inducing an immune response in a human against a viral disease or infection comprises administering to said human, an immunogenic composition comprising an antigen and an adjuvant effective amount of a substance P analog.

[0005] In one embodiment the viral disease or infection is influenza. In a preferred embodiment, the viral disease or infection is influenza A. In a more preferred embodiment, the viral disease or infection is avian influenza. In an even more preferred embodiment, the viral disease or infection is H5N1 or H7N3. In one embodiment, the methods comprise preexposure prevention of avian influenza in a human comprising administering to said human an immunogenic composition comprising an antigen and an adjuvant effective amount of a substance P analog. In one embodiment, the methods comprise post-exposure prophylaxis of avian influenza in a human comprising administering to said human an immunogenic composition comprising an antigen and an adjuvant effective amount of a substance P analog. [0006] In one embodiment, the methods for preventing disease or infection in a human, immunizing, or inducing an immune response in a human, comprise administering to said human an immunogenic composition comprising an antigen and an adjuvant effective amount of a substance P analog. [0007] In certain embodiments, the substance P analog is of Formula (I):

Zi-Xaa'-Xaa^Xaa^Xaa^Xaa^Xaa^Xaa'-Xaa^Xaa'-Xaa^-Xaa^{1 !}-Z₂ (I) or a pharmaceutically acceptable salt thereof, wherein:

Xaa¹ is Arg, Lys, 6-N methyllysine, or (6-N, 6-N) dimethyllysine;

Xaa² is Pro or Ala;

Xaa³ is Lys, Arg, 6-N-methyllysine, or (6-N, 6-N) dimethyllysine;

Xaa⁴ is Pro or Ala;

Xaa⁵ is GIn or Asn; Xaa⁶ is GIn or Asn;

Xaa⁷ is Phe or Phe substituted with chlorine at position 2, 3 or 4; Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 2, 3 or 4; Xaa⁹ is GIy, Pro, Ala, or N-methylglycine;

Xaa¹⁰ is Leu, VaI, He, Norleucine, Met, Met sulfoxide, Met sulfone, N- methylleucine, or N-methylvaline;

Xaa¹ ' is Met, Met sulfoxide, Met sulfone, or Norleucine; Zi is R₂N- or RC(O)NR-; Z₂ is -C(O)NR₂ or -C(O)OR or a salt thereof; each R is independently R is — H, (Ci-C₆) alkyl, (Ci -C₆) alkenyl, (Ci-C₆) alkynyl, (C₅-C₂₀) aryl, (C₆-C₂₆) alkaryl, 5-20 membered heteroaryl or 6-26 membered alkheteroaryl; and each "— " between residues Xaa¹ through Xaa¹ ' independently designates an amide linkage, a substitute amide linkage or an isostere of an amide.

[0008] In one embodiment, the substance P analog can be of Formula (I) as described herein, wherein Xaa¹ is Arg; Xaa² is Pro; Xaa³ is Lys; Xaa⁴ is Pro; Xaa⁵ is GIn; Xaa⁶ is GIn; Xaa⁷ is Phe or Phe substituted with chlorine at position 4; Xaa is Tyr, Phe, or Phe substituted with chlorine at position 4; Xaa⁹ is GIy, Pro or N-methylglycine; Xaa¹⁰ is Leu; and Xaa¹¹ is Met, Met sulfoxide, Met sulfone, or Norleucine.

[0009] In a preferred embodiment, the substance P analog can be of Formula (I) as described herein, wherein the "— " between residues Xaa¹ through Xaa¹¹ designates -C(O)NH-; Zj is H₂N-; and Z₂ is -C(O)NH₂.

[0010] In another preferred embodiment, the substance P analog can be: RPKPQQFFGLM (SEQ ID NO.: 1);

RPKPQQFFGLNIe (SEQ ID NO.: 2); RPKPQQFFPLM (SEQ ID NO. : 3);

RPKPQQFFMeGIyLM (SEQ ID NO. : 4);

RPKPQQFTGLM (SEQ ID NO.: 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO.: 6);

RPKPQQFFGLM(O) (SEQ ID NO.: 7);

RPKPQQFFMeGIyLM(O) (SEQ ID NO.: 8);

RPKPQQFFGLM(O₂) (SEQ ID NO. : 9); or

RPKPQQFFMeGIyLM(O₂) (SEQ ID NO. : 10).

[0011] In an even more preferred embodiment, the substance P analog can be Zi-RPKPQQFFMeGlyM(O₂)-Z₂; wherein Z_x is NH₂ and Z₂ is C(O)NH₂.

4. BRIEF DESCRIPTION OF THE FIGURES

[0012] Fig. 1 provides a plot of antibody titer in F2 immunized mice and F2 + VIPRO VEX® immunized mice against Avian Influenza proteins.

[0013] Fig. 2 provides a plot of antibody titer in F3 immunized mice and F3 + VIPROVEX® immunized mice against Avian Influenza proteins.

[0014] Fig. 3 provides a plot of antibody titer in F2/F3 immunized mice and F2/F3 +

VIPROVEX® immunized mice against Avian Influenza proteins.

[0015] Fig. 4 provides a plot of antibody titer in Control immunized mice against Avian

Influenza proteins.

[0016] Fig. 5 provides a plot of antibody titer in F2 immunized mice and F2 + VIPROVEX^® immunized mice against Spanish Flu proteins.

[0017] Fig. 6 provides a plot of F3 immunized mice and F3 + VIPROVEX® immunized mice against Spanish Flu proteins.

[0018] Fig. 7 provides a plot of antibody titer in F2/F3 immunized mice and F2/F3 + VIPRO VEX® immunized mice against Spanish Flu proteins.

[0019] Fig. 8 provides a plot of antibody titer in Control (D3 and PBS) immunized mice against Spanish Flu proteins.

[0020] Fig. 9 provides a plot of percent body weight in F2 immunized mice and F2 +

VIPRO VEX^® immunized mice after A/VN/1203/04 challenge.

[0021] Fig. 10 provides a plot of percent body weight in F3 immunized mice and F3 +

VIPRO VEX® immunized mice after A/VN/1203/04 challenge.

[0022] Fig. 11 provides a plot of percent body weight in F2/F3 immunized mice and F2/F3 +

VIPRO VEX® immunized mice after A/VN/1203/04 challenge.

[0023] Fig. 12 provides a plot of percent body weight in D3 (control) immunized mice after

A/VN/1203/04 challenge.

[0024] Fig. 13 provides a plot of percent body weight in PBS (control) immunized mice after

A/VN/1203/04 challenge.

[0025] Fig. 14 provides a plot of percent body weight in F2 immunized mice and F2 +

VIPRO VEX^® immunized mice after A/Indo/5/05 challenge.

[0026] Fig. 15 provides a plot of percent body weight in F3 immunized mice and F3 +

VIPRO VEX® immunized mice after A/Indo/5/05 challenge.

[0027] Fig. 16 provides a plot of percent body weight in F2/F3 immunized mice and F2/F3 +

VIPRO VEX® immunized mice after A/Indo/5/05 challenge.

[0028] Fig. 17 provides a plot of percent body weight in D3 (control) immunized mice after

A/Indo/5/05 challenge.

[0029] Fig. 18 provides a plot of percent body weight in PBS (control) immunized mice after

A/Indo/5/05 challenge.

[0030] Fig. 19 provides a plot of the effects of VIPROVEX® + immunogenic composition on animal survival following A/VN/1203/04 challenge. [0031] Fig. 20 provides a plot of the effects of VIPRO VEX® + immunogenic composition on animal survival following A/Indo/5/05 challenge.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 DEFINITIONS

[0032] The term "antigen" refers to peptides, polypeptides, proteins, nucleic acids, or polysaccharides that stimulates an immune response. Such antigens include but are not limited to peptides, polypeptides, proteins, nucleic acids, or polysaccharides of viruses, bacteria, or other microorganisms.

[0033] The term "adjuvant" refers to compounds that can increase, stimulate, induce, enhance, augment or increase the immunogenicity of an antigen. In some embodiments, the adjuvant can assist or potentiate the antigenicity of an antigen.

[0034] The term "adjuvant effective amount" refers to an amount effective to potentiate, augment, enhance, or increase the intrinsic immunogenicity of an antigen.

[0035] The term "alkyl" refers to a saturated branched, straight chain or cyclic hydrocarbon radical. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, and the like. In preferred embodiments, the alkyl groups are (Cj-C₆) alkyl.

[0036] The term "alkenyl" refers to an unsaturated branched, straight chain, or cyclic hydrocarbon radical having at least one carbon-carbon double bond. The radical may be in either the cis or trans conformation about the double bond(s). Typical alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, tert- butenyl, pentenyl, hexenyl, and the like. In preferred embodiments, the alkenyl group is

(C₁-C₆) alkenyl.

[0037] The term "alkynyl" refers to an unsaturated branched, straight chain, or cyclic hydrocarbon radical having at least one carbon-carbon triple bond. Typical alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl, and the like. In preferred embodiments, the alkynyl group is (Cj-C₆) alkynyl. [0038] The term "aryl" refers to an unsaturated cyclic hydrocarbon radical having a conjugated π electron system. Typical aryl groups include, but are not limited to, penta-2,4-diene, phenyl, naphthyl, anthracyl, azulenyl, chrysenyl, coronenyl, fluoranthenyl, indacenyl, idenyl, ovalenyl, perylenyl, phenalenyl, phenanthrenyl, picenyl, pleiadenyl, pyrenyl, pyranthrenyl, rubicenyl, and the like. In preferred embodiments, the aryl group is (C₅-C₂₀) aryl.

[0039] The term "alkaryl" refers to a straight-chain alkyl, alkenyl, or alkynyl group wherein one of the hydrogen atoms bonded to a terminal carbon is replaced with an aryl moiety. Typical alkaryl groups include, but are not limited to, benzyl, benzylidene, benzylidyne, benzenobenzyl, naphthenobenzyl and the like. In preferred embodiments, the alkaryl group is (C₆-C₂₆) alkaryl, i.e., the alkyl, alkenyl, or alkynyl moiety of the alkaryl group is (Ci-C₆) and the aryl moiety is (C₅-C₂₀). In particularly preferred embodiments, the alkaryl group is (C₆-Ci₃) alkaryl, i.e., the alkyl, alkenyl or alkynyl moiety of the alkaryl group is (Ci-C₃) and the aryl moiety is (C₅-Ci₀).

[0040] The term "heteroaryl" refers to an aryl moiety wherein one or more carbon atoms is replaced with another atom, such as N, P, O, S, As, Se, Si, Te, etc. Typical heteroaryl groups include, but are not limited to, acridarsine, acridine, arsanthridine, arsindole, arsindoline, carbazole, .beta.-carboline, chromene, cinnoline, furan, imidazole, indazole, indole, indolizine, isoarsindole, isoarsinoline, isobenzofuran, isochromene, isoindole, isophosphoindole, isophosphinoline, isoquinoline, isothiazole, isoxazole, naphthyridine, perimidine, phenanthridine, phenanthroline, phenazine, phosphoindole, phosphinoline, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, selenophene, tellurophene, thiophene, and xanthene. In preferred embodiments, the heteroaryl group is a 5-20 membered heteroaryl, with 5-10 membered aryl being particularly preferred. [0041] The term "alkheteroaryl" refers to a straight-chain alkyl, alkenyl, or alkynyl group where one of the hydrogen atoms bonded to a terminal carbon atom is replaced with a heteroaryl moiety. In preferred embodiments, the alkheteroaryl group is 6-26 membered alkheteroaryl, i.e., the alkyl, alkenyl, or alkynyl moiety of the alkheteroaryl is (Cj-C₆) and the heteroaryl is a 5-20-membered heteroaryl. In particularly preferred embodiments the alkheteroaryl is 6-13 membered alkheteroaryl, i.e., the alkyl, alkenyl, or alkynyl moiety is a 5-10 membered heteroaryl.

[0042] The term "immunize" refers to the administration of antigenic material to produce an immune response, preferably an immune response that can protect a subject, e.g., a human, from a disease or infection.

[0043] The term "substituted alkyl, alkenyl, alkynyl, aryl alkaryl, heteroaryl, or alkheteroaryl" refers to an alkyl, alkenyl, alkynyl, aryl, alkaryl, heteroaryl, or alkheteroaryl group in which one or more hydrogen atoms is replaced with another substituent. Preferred substituents include -OR, -SR, -NRR, -NO₂, -CN, halogen, -C(O)R, -C(O)OR, and -C(O)NR, wherein each R is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, alkaryl, heteroaryl, or alkheteroaryl.

[0044] The terms "pre-exposure prophylaxis" and "pre-exposure prevention" refer to preventing or protecting a human from developing or acquiring a viral disease or infection prior to the human's exposure to virus. "Pre-exposure prophylaxis" and "pre-exposure prevention" also refer to prevention, protection, prophylaxis of a disease or infection in a human at risk of becoming infected with virus. "Pre-exposure prophylaxis" and "preexposure prevention" also refer to inducing an immune response or immunization of a human against viral infection prior to the human's exposure to virus.

[0045] The terms "post-exposure prophylaxis" and "post-exposure prevention" refer to preventing or protecting a human from developing a viral disease or infection following the human's exposure or suspected exposure to virus but prior to the human's development of symptoms. "Post-exposure prophylaxis" and "post-exposure prevention" also refer to inducing an immune response or immunization of a human against viral infection after the human's exposure or suspected exposure to virus but prior to the human's development of symptoms.

[0046] The term "preventing" refers to reducing the severity, ameliorating a symptom, or reducing the duration of a subsequent infection, disease, or disorder.

5.2 THE METHODS

[0047] Provided herein are methods and compositions for use of substance P analogs in an immunogenic composition. In one embodiment, substance P analogs is used as an adjuvant in an immunogenic composition. In one embodiment, a method for preventing viral disease or infection in humans comprises administering an immunogenic composition comprising an antigen and an adjuvant effective amount of a substance P analog. In a preferred embodiment, the substance P analog adjuvant can decrease the amount of antigen needed in each dose of an immunogenic composition to stimulate an effective immune response in a human. In another preferred embodiment, the substance P analog adjuvant can stimulate an effective immune response to a dose of antigen that is unable to stimulate an immune response in the absence of an adjuvant. In another preferred embodiment, the substance P analog adjuvant can decrease the amount of antigen needed in each dose of an immunogenic composition to stimulate an effective immune response in a human against a viral antigen. In yet another preferred embodiment, the substance P analog adjuvant can decrease the amount of influenza antigen needed in each dose of an immunogenic composition to stimulate an effective immune response in a human against influenza. [0048] The potential efficacy of [Sar⁹, Met (O₂)¹ "]-NH₂-Substance P

(RPKPQQFFMeGIyLM(O)₂) (SEQ ID NO.: 10) (VIPROVEX®, ImmuneRegen BioSciences, Scottsdale, AZ) as an adjuvant results from the unique mechanism through which [Sar⁹, Met (O₂)¹¹]- NH₂-Substance P is believed to modulate the immune system, which is predominately through interactions with a tachykinin-specific receptor, the neurokinin- 1 (NKl) receptor (NKl-R). The NKl receptor is a member of the super-family of 7 transmembrane domain, G-protein coupled receptors located on the plasma membrane of a multiplicity of cells. Three neurokinin receptors have been identified: NKl-R, NK2-R, and NK3-R. [Sar⁹, Met (O₂)¹¹]- NH₂-Substance P has been reported to agonize only NKl receptors, and has been shown to be more than 3 times as potent as the endogenous neuropeptide, substance P, while exhibiting near-identical binding activity (Tousignant C. et al, 1990, Brain Research 524(2): 263-70).

[0049] The mechanism of action through which [Sar⁹, Met (O₂)¹¹]- NH₂-Substance P elicits an immuno-stimulatory effect is likely mediated via the binding with the NKl receptor. While not wishing to be bound by any theory, it is hypothesized that following ligand- receptor interaction, conformational changes within the receptor cause the G-protein to exchange GDP for GTP, which then activates the G-protein causing the hydrolysis of GTP to GDP (Khawaja A.M and Rogers D.F., 1996, Int. J. Biochem Cell Biol 28(7): 721-738). The NKl receptor is coupled to the pertussis-toxin-insensitive Gq/Gl 1 family, which is associated with phosphoinositide metabolism. The intracellular effector is phospholipase C (PLC) enzyme. Activation of the NKl receptor thus initiates phospho inositol breakdown, leading to the creation of inositol 1,4,5-triphosphate (IP3) and diaglycerol (DAG). IP3 induces the release of calcium from intracellular stores by effecting receptors on the sarcoplasmic reticulum. DAG is thought to increase intracellular calcium by increasing the permeability of transmembrane voltage-gated Ca²⁺ channels via protein kinase C (PKC) (Khawaja A.M and Rogers D.F., 1996, Int. J. Biochem Cell Biol 28(7): 721-738). These intracellular signaling events have been shown to induce the activation of the NF-κB pathway. Other theories of substance P analog action include association of agonist occupied receptor with beta-arrestin with resultant activation of a mitogen-activated protein kinase (MAPK) and extracellular regulated kinase (ERX). Studies report that this MAPK/ERK activation, perhaps independently of PKC (and/or through the c-src product), may result in cellular responses such as IL-6 release but not NF-κB elevation. There are, thus, molecular events associated with the pleiotropic actions of substance P and analogs thereof that might underlie different effects based on local environment, co-factors or cellular control state. [0050] Adjuvants can be pharmacologically active compounds intended to have no inherent activity or toxicity on their own, but have the capability to stimulate, induce, enhance, augment, or increase the immune stimulating effects of other compounds. Adjuvants can be immune stimulating molecules, often derived from pathogens to which the immune system has been pre-exposed (Jiang Z.H. and Koganty R.R., 2003, Curr. Med. Chem. 10(15):1423- 1439). An adjuvant can be a counterpart to a novel antigen, exaggerating the immune response initiated by the pathogen and provoking the immunopotentiation required to achieve sufficient humoral or cell-mediated response {See, Kenney R.T. and Edelman R., 2003, Expert Rev. Vaccines 2(2): 167-88; Schijns V.E., 2000, Curr. Opin. Immunol. 12(4): 456- 463, Gupta R.K. et ai, 1993, Vaccine 11(3):293-306; Aprile M.A. and Wardlaw A.C., 1966, Can. J. Public Health 57(8): 343-60; Degen W.G. et al, 2003, Expert Rev. Vaccines 2(2):327-35). In one embodiment, the substance P analog adjuvants can decrease the amount of antigen needed in each dose of an immunogenic composition to stimulate an effective immune response in a human. In one embodiment, the substance P analog adjuvants can decrease the amount of antigen needed in each dose of an immunogenic composition to stimulate an effective immune response in a human by about 10%, about 20%, about 30%, or about 40%. Greater reductions are preferred.

[0051] In another preferred embodiment, the substance P analog adjuvants can increase immunoglobulin levels about 2 fold more than if the human is given an immunogenic composition alone. The substance P analog adjuvants can increase immunoglobulin levels by about 2 fold, about 3 fold, or about 4 fold. Greater increases are preferred. [0052] In one embodiment, a method for preventing a viral disease or infection in a human comprises administering to said human, an immunogenic composition comprising an antigen and an adjuvant effective amount of a substance P analog. In one embodiment, a method for immunizing a human against a viral disease or infection comprises administering to said human, an immunogenic composition comprising an antigen and an adjuvant effective amount of a substance P analog. In one embodiment, a method for inducing an immune response in a human against a viral disease or infection comprises administering to said human, an immunogenic composition comprising an antigen and an adjuvant effective amount of a substance P analog.

[0053] In one embodiment, the viral disease or infection is influenza. In a preferred embodiment, the viral disease or infection is influenza A. In a more preferred embodiment, the viral disease or infection is avian influenza. In an even more preferred embodiment, the viral disease or infection is H5N1 or H7N3. In one embodiment, the methods comprise preexposure prevention of avian influenza in a human comprising administering to said human an immunogenic composition comprising an antigen and an adjuvant effective amount of a substance P analog. In one embodiment, the methods comprise post-exposure prophylaxis of avian influenza in a human comprising administering to said human an immunogenic composition comprising an antigen and an adjuvant effective amount of a substance P analog. [0054] In one embodiment, the methods for preventing disease or infection in a human, immunizing, or inducing an immune response in a human, comprise administering to said human an immunogenic composition comprising an antigen and an adjuvant effective amount of a substance P analog. [0055] In one embodiment, the substance P analog is of Formula (I):

ZrXaa'-Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^-Xaa¹¹ -Z₂ (I) or a pharmaceutically acceptable salt thereof, wherein:

Xaa¹ is Arg, Lys, 6-N methyllysine, or (6-N, 6-N) dimethyllysine;

Xaa² is Pro or Ala;

Xaa³ is Lys, Arg, 6-N-methyllysine, or (6-N, 6-N) dimethyllysine;

Xaa⁴ is Pro or Ala;

Xaa⁵ is GIn or Asn;

Xaa⁶ is GIn or Asn;

Xaa⁷ is Phe or Phe substituted with chlorine at position 2, 3 or 4;

Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 2, 3 or 4;

Xaa⁹ is GIy, Pro, Ala, or N-methylglycine;

Xaa¹¹ is Met, Met sulfoxide, Met sulfone, or Norleucine;

Z₁ is R₂N- or RC(O)NR-;

Z₂ is -C(O)NR₂ or -C(O)OR or a salt thereof; each R is independently R is H, (C₁-C₆) alkyl, (Ci-C₆) alkenyl, (Cj-C₆) alkynyl,

(C₅-C₂₀) aryl, (C₆-C₂₆) alkaryl, 5-20 membered heteroaryl, or 6-26 membered alkheteroaryl; and each "— " between residues Xaa¹ through Xaa¹¹ independently designates an amide linkage, a substitute amide linkage or an isostere of an amide.

[0056] In one embodiment, the substance P analogs can be of Formula (I) wherein Xaa¹ is Arg; Xaa² is Pro; Xaa³ is Lys; Xaa⁴ is Pro; Xaa⁵ is GIn; Xaa⁶ is GIn; Xaa⁷ is Phe or Phe substituted with chlorine at position 4; Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 4; Xaa⁹ is GIy, Pro or N-methylglycine; and Xaa¹⁰ is Leu; and Xaa¹¹ is Met, Met sulfoxide, Met sulfone, or Norleucine. In a preferred embodiment the "— " between residues Xaa¹ through Xaa¹¹ of the substance P analogs designates -C(O)NH-; Zj is H₂N-; and Z₂ is -C(O)NH₂.

[0057] In another preferred embodiment, the substance P analog is selected from the group consisting of:

RPKPQQFFGLM (SEQ ID NO.: 1);

RPKPQQFFGLNIe (SEQ ID NO.: 2);

RPKPQQFFPLM (SEQ ID NO. : 3);

RPKPQQFFMeGIyLM (SEQ ID NO. : 4);

RPKPQQFTGLM (SEQ ID NO.: 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO.: 6);

RPKPQQFFGLM(O) (SEQ ID NO. : 7);

RPKPQQFFMeGIyLM(O) (SEQ ID NO. : 8);

RPKPQQFFGLM(O₂) (SEQ ID NO. : 9); and

RPKPQQFFMeGIyLM(O₂) (SEQ ID NO. : 10).

[0058] In an even more preferred embodiment, the substance P analog is Zi-RPKPQQFFMeGlyM(O₂)-Z₂; wherein Z₁ is NH₂ and Z₂ is C(O)NH₂. [0059] In one embodiment, the immunogenic composition can induce an immune response against a virus. The viral antigen of the immunogenic composition can be inactivated (killed), or attenuated (weakened), or comprised of viral subunits, usually proteins or protein subunits. In one embodiment, the methods and compositions can be an adjuvant in an immunogenic composition against a virus, viral particle, or components or products of a virus. In one embodiment the immunogenic composition can be comprised of synthetic viral particles and components. In a preferred embodiment, the immunogenic composition can be an immunogenic composition that protects or immunizes a human against Influenza A. In a more preferred embodiment, the immunogenic composition can be an immunogenic composition that protects or immunizes a human against Avian Influenza ("bird flu"). In a more preferred embodiment, the immunogenic composition can be an immunogenic composition that protects or immunizes a human against H5N1 or H7N3 Influenza. [0060] In one embodiment, the methods and immunogenic compositions can induce an immune response against other viruses. In a preferred embodiment, the immunogenic composition can induce an immune response against a virus that is a potential bioterrorist threat including, small-pox or hemorrhagic fevers. In one embodiment, the immunogenic composition can induce an immune response against a virus including, but not limited to, Arenaviruses, Bunyaviruses, Flaviruses, Filoviruseses, Caliciviruses, Hepatitis A, Viral Encephalitides, influenza, Dengue virus, encephalitis, West Nile virus, LaCrosse, California encephalitis, Japanese encephalitis, Kyasanur Forus virus, Nipah virus, Crimean-Congo hemorrhagic fever virus, Chikungunya virus, severe acute respiratory syndrome associated coronavirus (SARS-CoV), or yellow fever virus.

[0061] In one embodiment, the immunogenic composition can induce an immune response against a bacterium. The bacterial antigen of the immunogenic composition can be inactivated (killed), or attenuated (weakened), or bacterial antigenic derivatives, usually proteins or protein subunits. In one embodiment, the methods and compositions can be an adjuvant in an immunogenic composition against a bacterium, bacterial particle, or components or products of a bacterium. In one embodiment the immunogenic composition can be comprised of synthetic bacterial particles and components. In one embodiment, the immunogenic composition can be an immunogenic composition that protects or immunizes a human against Escherichia, Enterococcus, Chlamydia, Campylobacter, Bacillus, Salmonella, Shigella, tuberculosis, Staphylococcus, Streptococcus, Vibrio, Pseudomonas aeruginosa, Rickettsia, Neisseria, Mycoplasma, Mycobacterium, or Haemophilus. In another embodiment, the immunogenic composition can be an immunogenic composition that protects or immunizes a human against Bacillus anthracis, Bacillus subtilis, Chlamydia trachomatis, Chlamydia psittaci, Escherichia coli, Haemophilus influenza, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitidis, Salmonella enteritidis, Salmonella sp, Yersinia pestis, Yersinia enterocolitica, Francisella tularensis, Vibrio cholera, Clostridium botulinium, Burkholderia pseudomallei, Coxiella burnetti, Brucella sp., Rickettsia prowazekii, Shigella sp., Listeria monocytogenes, Campylobacter jejuni, or Staphylococcus aureus. In one embodiment, the immunogenic composition can protect a human from anthrax, plague, tularemia, cholera, Q fever, brucellosis, glanders, psittacosis, typhus fever or botulism. Bacterial infection can result directly from bacterial infection or from pathological events caused by a component or activity of the bacterial. For example, Vibrio cholera, the causative agent for cholera exerts a pathological effect in humans by the production of enterotoxin that acts on the mucosal epithelium of the small intestine causing severe dehydration, hypotension and often death. Another example is Bacillus anthracis, the causative agent for anthrax. Once in the blood stream of an animal, B. anthracis releases a tripartite toxin comprised of lethal factor, edema factor and protective antigen which accelerate the tissue destruction, bleeding and eventual death of the animal. The methods and compositions can be used as an adjuvant in an immunogenic composition against such enterotoxins and toxins as described herein. [0062] In one embodiment, the immunogenic composition can induce an immune response against other microorganisms including, but not limited to, protists such as protozoa, fungi, archaea, prions, and parasites. Such antigens of the immunogenic composition can be inactivated (killed), or attenuated (weakened), or antigenic derivatives thereof, usually proteins or protein subunits. In one embodiment, the methods and compositions can be an adjuvant in an immunogenic composition against a microorganism, particles, or components or products thereof. In one embodiment the immunogenic composition can be comprised of synthetic microorganism particles and/or components. In another embodiment, the immunogenic composition can be an immunogenic composition that protects or immunizes a human against Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida dubliniensis, Candida glabrata, Tinea pedis, Tinea corporis, Tinea cruris, Cryptosporidium parvum, Cyclospora caatanensis, Giardia lamblia, Entamoeba histolytica, Toxoplasma, Microsporidia, or Pneumocystis jirovecii.

[0063] In one embodiment, the methods and immunogenic compositions can protect a human from the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Category A, B and C Priority Pathogens.

[0064] Immunogenic composition that can be used against biological toxins, for example, bacterial toxins. Such toxins can be used as biological warfare agents, for example ricin. The immunogenic composition can induce an immune response against nerve poisons, such as those from certain plants and animals. In one embodiment, the immunogenic composition can be a substance P analog and RTA 1-33/44-198, an immunogenic composition developed by the U.S. Army Medical Research Institute of Infectious Diseases against ricin. [0065] Immune response in an animal can be through cellular or humoral pathways. Innate immunity is a non-specific, inherited, basic immune response that does not require cellular activation. Adaptive immunity can be established through antigen-presenting cells (APCs) and T-cell activation (cellular or cell-mediated immunity) (Kenney R.T. and Edelman R., 2003, Expert Rev. Vaccines 2(2):167-88; Schijns V.E., 2001, Vet. Sciences Tomorrow; Grakoui A. et al., 1999, Science 285(5425): 221-7).

[0066] The cellular immune response involves upregulation of inflammatory cytokines via Toll-like receptors (TLRs). TLR signaling induces the production of proinflammatory cytokines, including interleukin-1 (IL-I), interleukin-6 (IL-6), interleukin-12 (IL- 12), and tumor necrosis factor-alpha (TNF-α). These inflammatory cytokines activate the surrounding cells, resulting in the increased production of chemokines or intercellular adhesion molecules (ICAMs). As a consequence of this, inflammatory cells are directed to the sites of TLR activation. The recruited inflammatory cells, typically macrophages or neutrophils, are subsequently activated and envelope pathogens via phagocytosis. These immune cells eliminate infected cells by producing nitric oxide, reactive oxygen species, and defensins (Pazgier M. et al., 2006, Cell. MoI. Life Sci. 63(11):1294-313; Kim C. et al., 2005 Proc. Natl. Acad. ScL USA 102(13): 4830-5. See generally, Villinger F., 2003, Expert Rev. Vaccines 2(2):317-326; Janeway C. et al., 2005, ImmunoBiology 6^th ed. Garland Science Publishing; Schroder K. et al, 2001, Immunobiology 211(6-8):511-24).

[0067] CD4⁺ T-cells are known to differentiate into T_H1 or T_H2 cells (Janeway C. et al. 2005, ImmunoBiology 6^th ed. Garland Science Publishing). The T_H1 response is characterized by the production of interferon-gamma (IFN-γ), a proinflammatory cytokine that is important in both the innate and adaptive immune responses (Schroder K. et al., 2001, Immunobiology 211(6-8):511-524) and the mediation of antiviral or antibacterial immunity. The T_HI response is typical for most TLR ligands, however, TLR2, TLR4 and TLR5 ligands have been noted to induce a T_H2 response under certain conditions. As an example, MyD88- deficient mice failed to generate THI responses when challenged with LPS, a TLR4 ligand, instead mounting T_H2 responses (Kaisho T. et ah, 2002, Int. Immunol. 14(7):695-700). The

T_H2 response is characterized by the production of interleukin-4 (IL-4), which results in the activation of B-cells that produce neutralizing antibodies that result in 'humoral immunity'.

In one embodiment, the immune response can stimulate, enhance or augment cellular or humoral immunity in a human.

[0068] The immune response can be mediated by activation of various kinases (Agrawal S. et al., 2003, J. Immunol., 4984-9), such that the nature of intracellular mediators stimulated via a number of pathways act in a concordant manner to impact the type (T_HI or T_H2; humoral or cellular) and magnitude of immune response.

[0069] In one embodiment, the immunogenic composition comprises adjuvant that stimulates

T_HI or T_H2 responses in a human. In one embodiment, the stimulated T_HI response can result in increased IFN-γ. In one embodiment, the stimulated T_H2 response can result in increased IL-4. In another embodiment, the stimulated T_H2 response can result in increased

B-lymphocytes.

[0070] In one embodiment, the methods and compositions can stimulate immunoglobulin production in a human greater than the immunoglobulin production in a human after challenge with antigen alone. In a preferred embodiment IgG, IgA and IgM is stimulated. In an even more preferred embodiment one or more IgG subclass (e.g., IgGl, IgG2, IgG3 or

IgG4) is stimulated.

[0071] In one embodiment, increases of immunoglobulin of about 10%, about 40%, about

80%, about 100%, about 120%, about 140%, about 160%, about 180%, or about 200% or more are contemplated. Greater increases are preferred.

[0072] NF-κB has been shown to induce the expression of numerous pro-inflammatory genes, subsequently leading to the production of pro-inflammatory cytokines, chemokines, and intercellular adhesion molecules. Thus [Sar⁹, Met (O₂)¹¹J-NH₂ substance P can elicit comparable effects to TLR ligand-based adjuvants, while avoiding the toxicity issues accompanied with TLR ligand-based adjuvants (Gavin A. L. et al., 2006, Science 314(5807): 1936-8).

[0073] In one embodiment, the methods and compositions comprise adjuvants that can be administered serially or in combination with one or more immunogenic compositions. [0074] In one embodiment, the methods and compositions comprise adjuvants that can be administered before, concurrently, or after administration of antigen. In one embodiment, the adjuvants and antigen can be administered in the same or different compositions.

5.3 SUBSTANCE P- ANALOGS

[0075] As will be understood by those of skill in the art, substance P (SEQ ID NO 1) refers to peptide having the sequence: Arg Pro Ly s Pro GIn GIn Phe Phe GIy Leu Met, or the single letter representation RPKPQQFFGLM (SEQ ID NO 1). As such, a substance P analog as described herein refers to a substance P analog that comprises one or more amino acids substitutions relative to SEQ ID NO 1 and can compete with substance P for binding to its receptor (NK-I). In one embodiment, the substance P analog comprises one or more amino acids substitutions relative to SEQ ID NO 1 and can agonize the NK-I (neurokinin) receptor according to an assay conventional to the art, e.g., as described in Shue H. J., et al.,Bioorg. Med. Chem. Lett. 2006, 16(4): 1065-9. The amino acid substitutions can be conservative or non-conservative substitutions. Further, the amino acid substitutions can include substitutions of non-standard amino acids {e.g., amino acids other than the 20 amino acids normally encoded by the genetic code). In one example, the substance P analog can comprise norleucine (NIe). In yet another example, the substance P analog can comprise sarcosine (Sar) or N-methylglycine (MeGIy). In yet another example, the substance P analog can comprise phenylalanine that is substituted with between 1 and 4 chlorines, more preferably 1 chlorine.

[0076] In one embodiment the methionine residue side chain S can be oxidized. In one embodiment the methionine is methionine sulfoxide (-NH-CH(CO)-CH₂-CH₂-S(O)CH₃). In one embodiment the methionine is methionine sulfone or methionine S, S, dioxide, (-NH-

CH(CO)-CH₂-CH₂-S(O₂)CH₃) , also referred to herein as Met(O)₂.

[0077] In one embodiment, the substance P analog can be of Formula (I):

Zi-Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Z₂ (I) or a pharmaceutically acceptable salt thereof, wherein:

Xaa¹ is Arg, Lys, 6-N methyllysine, or (6-N, 6-N) dimethyllysine;

Xaa² is Pro or Ala;

Xaa³ is Lys, Arg, 6-N-methyllysine, or (6-N, 6-N) dimethyllysine;

Xaa⁴ is Pro or Ala;

Xaa⁵ is GIn or Asn;

Xaa⁶ is GIn or Asn;

Xaa⁷ is Phe or Phe substituted with chlorine at position 2, 3 or 4;

Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 2, 3 or 4;

Xaa⁹ is GIy, Pro, Ala, or N-methylglycine;

Xaa¹ ' is Met, Met sulfoxide, Met sulfone, or Norleucine;

Z₁ is R₂N- or RC(O)NR-;

Z₂ is -C(O)NR₂ or -C(O)OR or a salt thereof; each R is independently R is H, (Ci-C₆) alkyl, (C₁-C₆) alkenyl, (Ci-C₆) alkynyl,

(C₅-C₂₀) aryl, (C₆-C₂₆) alkaryl, 5-20 membered heteroaryl or 6-26 membered alkheteroaryl; and each "— " between residues Xaa¹ through Xaa" independently designates an amide linkage, a substitute amide linkage or an isostere of an amide.

[0078] In a preferred embodiment the substance P analogs can be of Formula (I) wherein Xaa¹ is Arg; Xaa² is Pro; Xaa³ is Lys; Xaa⁴ is Pro; Xaa⁵ is GIn; Xaa⁶ is GIn; Xaa⁷ is Phe or Phe substituted with chlorine at position 4; Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 4; Xaa⁹ is GIy, Pro, or N-methylglycine; Xaa¹⁰ is Leu; and Xaa¹¹ is Met, Met sulfoxide, Met sulfone, or Norleucine. In a more preferred embodiment, the "— " between residues Xaa¹ through Xaa¹¹ of the substance P analogs can be -C(O)NH-; and Z₁ is H₂N-; and Z₂ is -C(O)NH₂.

[0079] In yet another preferred embodiment, the substance P analogs can be selected from the group consisting of:

RPKPQQFFGLM (SEQ ID NO. : 1 );

RPKPQQFFGLNIe (SEQ ID NO. : 2);

RPKPQQFFPLM (SEQ ID NO. : 3);

RPKPQQFFMeGIyLM (SEQ ID NO. : 4);

RPKPQQFTGLM (SEQ ID NO. : 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO. : 6);

RPKPQQFFGLM(O) (SEQ ID NO. : 7);

RPKPQQFFMeGIyLM(O) (SEQ ID NO.: 8);

RPKPQQFFGLM(O₂) (SEQ ID NO.: 9); and

RPKPQQFFMeGIyLM(O₂) (SEQ ID NO.: 10).

[0080] In another preferred embodiment, the substance P analog can be Zi-RPKPQQFFMeGlyM(O₂)-Z₂; wherein Z, is NH₂ and Z₂ is C(O)NH₂. [0081] It will be apparent to one skilled in the art that the amino (designated herein as Zi) or carboxy terminus (designated herein as Z₂) of the substance P analogs can be modified. In one embodiment "blocked" forms of the substance P analogs, i.e., forms of the substance P analogs in which the N- and/or C-terminus is blocked with a moiety capable of reacting with the N-terminal -NH₂ or C-terminal -C(O)OH. In some embodiments the N- and/or C- terminal charges of the substance P analogs can be an N-acylated peptide amide, ester, hydrazide, alcohol, and substitutions thereof. In a preferred embodiment, either the N- and/or C-terminus (preferably both termini) of the substance P analogs are blocked. Typical N- terminal blocking groups include RC(O)-, where R is — H, (Ci-C₆) alkyl, (Ci-C₆) alkenyl, (Ci-C₆) alkynyl, (C₅-C₂₀) aryl, (C₆-C₂₆) alkaryl, 5-20 membered heteroaryl or 6-26 membered alkheteroaryl. Preferred N-terminal blocking groups include acetyl, formyl and dansyl. Typical C-terminal blocking groups include -C(O)NRR and -C(O)OR, wherein each R is independently defined as above. Preferred C-terminal blocking groups include those where each R is independently methyl. In another preferred embodiment the C-terminal group is amidated.

[0082] Substituted amides generally include, but are not limited to, groups of the formula -C(O)NR-, wherein R is (C₁-C₆) alkyl, substituted (Ci-C₆) alkyl, (Ci-C₆) alkenyl, substituted (Ci-C₆) alkenyl, (C_rC₆) alkynyl, substituted (Ci-C₆) alkynyl, (C₅-C₂₀) aryl, substituted (C₅-C₂₀) aryl, (C₆-C₂₆) alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 membered alkheteroaryl and substituted 6-26 membered alkheteroaryl.

[0083] Amide isosteres generally include, but are not limited to, -CH₂ NH-, -CH₂ S-, -CH₂CH₂ -, -CH=CH- (cis and trans), -C(O)CH₂ -, -CH(OH)CH₂ - and -CH₂ SO-. Compounds having such non-amide linkages and methods for preparing such compounds are well-known in the art (see, e.g., Spatola, March 1983, Vega Data Vol. 1, Issue 3; Spatola, 1983, "Peptide Backbone Modifications" in: Chemistry and Biochemistry of Amino Acids Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267 (general review); Morley J., 1980, Trends Pharm. Sci. 1 :463-8; Hudson et al, 1979, Int. J. Prot. Res. 14:177- 185 (-CH₂ NH-, -CH₂ CH₂ -); Spatola A.F. et al, 1986, Life Sci. 38(14):1243-9 (-CH₂-S); Harm, 1982, J. Chem. Soc. Perkin Trans. I. 1 :307-314 (-CH=CH-, cis and trans); Almquist et al, 1980, J. Med. Chem. 23:1392-1398 (-COCH₂ -); Jennings- White et al, Tetrahedron. Lett. 23:2533 (-COCH₂-); European Patent Application EP 45665 (1982) CA 97:39405 (-CH(OH)CH₂ -); Holladay et al, 1983, Tetrahedron Lett. 24:4401-4404 (-C(OH)CH₂ -); and Hruby V. J., 1982, Life Sci. 31 :189-99 (-CH₂ -S-)). [0084] Additionally, one or more amide linkages can be replaced with peptidomimetic or amide mimetic moieties which do not significantly interfere with the structure or activity of the peptides. Suitable amide mimetic moieties are described, for example, in Olson G. L. et al, 1993, J. Med. Chem. 36(21):3039-49.

[0085] Substance P (RPKPQQFFGLM; SEQ ID NO: 1) or a bioactive analog thereof such as [Sar⁹, Met (O₂)¹ ^-Substance P can be administered as an adjuvant in an immunogenic composition to prevent viral infection. Other compounds which function in the same way can be identified by their ability to compete with substance P for binding to its receptor (NKl) and for their ability to agonize the NKl receptor. Routine assays for such activities are known in the art and can be used. See, e.g., Shue HJ. , et al.Bioorg. Med. Chem. Lett. 2006, 16(4):1065-9.

[0086] In some embodiments, bioactive analogs are those which act as competitive inhibitors of substance P by binding to the substance P receptor (NKl receptor). The analogs can be agonists of the NK-I receptor. Other derivatives as are known in the art and commercially available (e.g., from Sigma, Inc.) can be used. In addition, substance P fragments and derivatized substance P fragments that substantially retain the overall characteristics and properties of substance P can be used in the methods and compositions for preventing disease or infection in humans. In other embodiments, the substance P analog comprise substitution, deletion, or insertion of one to eight amino acid residues, and preferably from one to three amino acid residues. In addition, functional groups may be modified on the substance P analogs while retaining the same amino acid backbone. Again, routine testing can determine which modifications do not adversely affect biological activity.

5.3 COMPOSITIONS AND KITS

[0087] In one embodiment, compositions for administration of a substance P analog as an adjuvant in conjunction with an immunogenic composition. In one embodiment, an immunogenic composition comprises an adjuvant effective amount of a substance P analog according to Formula (I) as described herein and a pharmaceutically acceptable carrier. [0088] The substance P analogs may be administered by any suitable route that ensures bioavailability in the circulation. This can best be achieved by parenteral routes of administration, including intravenous (IV), intramuscular (IM), intradermal (ID), subcutaneous (SC), and intraperitoneal (IP) injections. However, other routes of administration can be used. For example, absorption through the gastrointestinal tract can be accomplished by oral routes of administration (including but not limited to ingestion, buccal, and sublingual routes) provided appropriate formulations {e.g., enteric coatings) are used to avoid or minimize degradation of the active ingredient, e.g., in the harsh environments of the oral mucosa, stomach, and/or small intestine. Alternatively, transmucosal administration via mucosal tissue such as nasal, vaginal, and rectal modes of administration may be utilized to avoid or minimize degradation in the gastrointestinal tract. In yet another alternative, the formulations can be administered transcutaneously {e.g., transdermally), or by inhalation. It will be appreciated that the preferred route may vary with the condition, age, and compliance of the recipient.

[0089] In a preferred embodiment, the compositions can be pharmaceutical compositions. In a more preferred embodiment, the pharmaceutical compositions can be administered by injection or inhalation. In an even more preferred embodiment, the composition can be administered intramuscularly, intradermally, subcutaneously, transmucosally, or by inhalation.

[0090] The actual dose of the substance P analogs used will vary with the route of administration, and should be adjusted to achieve circulating plasma concentrations of 100 mg/1 to 2 g/1. Data obtained in animal model systems described herein show that the substance P analogs can be administered by injection at a dose between 0.01 mg to 200 mg per immunogenic composition dose. In a more preferred embodiment, the substance P analog can be administered at a dose of about 0.1 mg to about 100 mg per immunogenic composition dose. In a more preferred embodiment, the dose can be about 0.5 mg to about 50 mg. In an even more preferred embodiment, the dose of the substance P analog can be about 0.5 mg to about 15 mg per immunogenic composition dose. See, FDA Guidance Document, Estimating the Safe Starting Dose in Clinical Trials for Therapeutics in Adult Healthy Volunteers, Center for Biologies Evaluation and Research, United States Department of Health and Human Services.

[0091] In one embodiment, the composition can be given via the respiratory system (i.e. by inhalation) via the nose or mouth. In one embodiment the inhalation composition is an aerosol. Aerosols comprised of solubilized agent in a liquid medium or dry powder compositions are known in the art. Aerosol compositions can be administered via nebulizer, metered dose inhalers, (MDIs) or dry powder inhalers (DPIs). MDIs and DPIs can also be referred to as puffers. [0092] In one embodiment, the methods and compositions can be administered in a frequency and duration for prevention of viral infection or immunization against a virus in a human. In one embodiment, the compositions can be administered one time (e.g. single dose). In one embodiment, the compositions can be administered multiple times, for example a booster dose can be given days, weeks, or even years after an initial immunogenic composition plus adjuvant dose is given. In one embodiment, the compositions can be administered intermittently, for example, about every 30 days, about every 60 days, about every 90 days, about every 180 days, about every 360 days, about every 5 years, about every 10 years, and the like.

[0093] The substance P analogs can be used in combination with an immunogenic composition to increase the humoral and/or cellular immune response to the immunogenic composition as an adjuvant. In one embodiment, the immunogenic composition and the substance P analogs can be administered contemporaneously, for example, on the same day. In one embodiment, the an immunogenic composition and the substance P analogs can be administered at intervals, for example, the immunogenic composition given on Day 1 and the substance P analogs can be administered on Day -3, Day -1, Day 2, Day 3, or Day 7, and the like. In one embodiment, the substance P analogs can be given on Day 1 and the an immunogenic composition can be administered later on Day 1, or Day 3, or Day 7 and the like. Such combinations can be administered either before or after exposure to virus either before or after development of symptoms (pre-exposure prevention or post-exposure prevention).

[0094] In one embodiment, a kit comprises administering a pre-exposure preventative or post-exposure preventative agent. Such a kit can have both an antiviral immunogenic composition and at least one substance P analog.

[0095] The immunogenic composition and the substance P analog adjuvant can be in separate, or divided or undivided containers. The two agents can be in liquid, dried, lyophilized, or frozen form, as convenient for the end user and good for shelf life. The treatments can be administered at one time or sequentially, over a period of, for example, one day, one week, one month, six months, or twelve months.

[0096] Suitable devices for administering the aerosol are known in the art and include atomizers and droppers as well as nebulizers and hand-held aerosol metered dose and dry powder inhaler ("puffer") devices. Suitable regimens for administration include daily or multiple daily administrations by aerosol. Other modes of administration include continual intradermal infusion, transdermal infusion, intravenous injection, intramuscular, sublingual, subcutaneous injection, and oral. Suitable compositions of substance P or substance P analogs for administration are any which are pharmaceutically acceptable and in which substance P or substance P analogs retain biological activity. Generally, such compositions are substance P or substance P analogs dissolved in sterile normal saline, buffered normal saline, or sterile water. Other compositions for changing stability, absorption, and half-life characteristics can be used, including liposomal compositions, carrier molecules that physically or chemically retard the release of the adjuvant into physiological spaces, and slow-release (depot) compositions.

[0097] Immunogenic compositions can include "pharmaceutically acceptable carriers," which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Typically these pharmaceutically acceptable carriers are sterile and pyrogen free. Exemplary carriers are large, slowly metabolized macromolecules such as polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplet emulsions or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. In some aspects, these carriers can function as immune-stimulating agents. [0098] Immunogenic compositions can further comprise inactivated or attenuated whole virus, whole inactivated bacteria, or subunits thereof. Subunit immunogenic compositions can comprise, for example, the two major surface antigens of the virus, the hemagglutinin (HA) and the neuraminidase (NA), in more or less purified forms. In one embodiment, the immunogenic composition can comprise an antigen that is conserved across multiple viral species, isolates or clades, such as influenza M2 protein, in more or less purified forms. [0099] According to yet another aspect, the methods comprise a kit for providing preexposure prophylaxis or post-exposure prophylaxis against a viral infection to a human comprising at least substance P analog and an immunogenic composition. In another embodiment, the kit further comprises an antiviral agent. In one embodiment the antiviral agent can be FLUD ASE® (NEX-DAS 181), INVIRIDIN™ (NEX-PIN) or VIRACIDIN™ (NEX-VAC). (NexBio, San Diego, CA). In another preferred embodiment, the antiviral agent can be a neuramidase inhibitor. In a more preferred embodiment, the kit further comprises oseltamivir (TAMIFLU®) or zanamivir (RELENZA®).

6. EXAMPLES Example 1. Adjuvant Activity

[00100] The following experiment was a collaborative effort between GenPhar, Inc.

(Mount Pleasant, South Carolina) and ImmuneRegen Biosciences, Inc. (Scottsdale, Arizona). Gen Phar supplied the CAdVax vectors while ImuneRegen supplied the VIPRO VEX® (ImmuneRegen BioSciences, Scottsdale, AZ) compound. Materials and Methods

[00101] Reagents. VIPROVEX® was obtained from ImmuneRegen in 2 vials, each containing 2 mg of compound and stored at -20°C until use. Prior to administration, 2mg of VIPROVEX® was dissolved in 20μl of 10% acetic acid. This solution was then diluted with sterile PBS to 2ml total volume (lmg/ml working concentration). CAdVax, vector platform based on replication incompetent complex Ad vector, was used to make CAdVax-F2 (avian flu immunogenic composition), F3 (Spanish flu immunogenic composition), and D3 (control vector) immunogenic compositions vectors. (GenPhar) Each vector was diluted in sterile PBS to a working concentration of 2.5 x 10⁹ TCID₅₀/ml just prior to injection. [00102] Animals. Male BALB/c mice (4-6 weeks old) were purchased from Charles

River Laboratories and housed 4 per cage while in the GenPhar animal suite. All mice were maintained according to NIH Guidelines and Gen Phar's IACUC regulations. For immunizations, mice were injected intraperitoneally (IP) with 5 x 10⁸ TCID₅₀ of indicated immunogenic composition vector or PBS placebo in a volume of 200 μl. Combination immunization groups (F2 + F3) received 2.5 x 10 TCID₅₀ of each component for a total of 5 x 10⁸ TCID_5O. Some groups of mice also received VIPRO VEX® inoculations intranasally at 1 μg per gram body weight. VIPRO VEX® was administered immediately after injections of CAdVax vectors.

[00103] Serological Analysis. Blood was collected from each mouse one week prior to immunization and bi-weekly thereafter. Route of collection was retro-orbital sinus under light anesthesia. Blood samples were allowed to clot overnight at 4° C. The next day, samples were centrifuged at 7,000 x g for 10 minutes to remove clots. Serum samples were supplemented with NaN₃ at a final concentration of 0.05% as a preservative, and all samples were stored at 4° C during the study.

[00104] Because H5N1 and the Spanish flu virus can only be handled in biosafety level 3 (BSL3) laboratories, ELISA plates were prepared using lysates derived from CAdVax-infected cells, which requires only BSL2 containment. For coating antigens, BS-C- 1 cells were infected with either C AdVax-F2 or C AdVaxF3 at an MOI of 20 (multiplicity of infection) for 48 hours. (MOI refers to the ratio of infectious virus particles to the number of cells being infected). The CAdVax-F2 lysates contain high levels of the H5N1 flu virus H5, Nl, and Ml proteins, while the CAdVax-F3 lysates contained high levels of the Spanish flu virus Hl, Nl, and Ml proteins. Protein expression levels were confirmed by Western blot analysis (not shown). Cell lysates were collected and protein concentrations determined using the Advanced Protein Assay (Cytoskeleton, Inc.) according to instructions. Maxisorp microtiter plates (Nunc; Rochester, NY) were coated with 5 μg/ml of each lysate and assays were allowed to dry uncovered overnight at 37°C. The next day, plates were washed three times with PBS supplemented with 0.1% TWEEN-20™ (ICI Americas) (PBS-T) and blocked in a solutions of 5% non-fat dry milk (w/v) dissolved in PBS-T for one hour at room temperature. After blocking, plates were washed three times more and incubated with serial dilutions of animal sera or positive control antibodies. Antisera were diluted in PBS-T/5% milk, and incubations were kept for 2 hr at 30°C. Plates were washed three times again and incubated with secondary antibody (HRP-conjugated goat anti-mouse IgG) diluted in 5% milk PBS-T at 1 :5,000 for one hour at 3O⁰C. After secondary antibody, plates were washed a final three times and one-step HRP substrate (Fitzgerald Laboratories) was added for 20 minutes. Reactions were terminated by adding 0.5M HCL and OD readings were measured at 450 nm on a microplate reader. Antibody titers for each mouse were determined by calculating the dilution of serum that corresponded to a signal of two times the background for that particular test.

[00105] Animal ID for shipment. During the study, all mice were identified by systematic ear hole punches. However, since the mice would be mixed in large crates during shipment to Southern Research Institute (SRI, Birmingham, AL), each animal was identified by a numbered steel ear band three days prior to shipping. Mice were grouped and labeled according to Table 1. Due to accidental deaths and humane euthanasia (necessitated by Staphylococcus infections, shown as x** in the Tables) during the study, 70 mice were used for the H5N1 challenge portion of the study. Mice were selected based on highest avian influenza antibody titers. Animals were shipped to SRI in approved shipping containers (Charles River Labs) equipped with bedding, food, and gel packs at approximately 24 mice per crate. RESULTS

[00106] Humoral immune response against avian influenza. When testing immunized mice sera against avian influenza protein lysates, VIPROXEX® administration appeared to have an adjuvant effect on only the group of mice immunized with CAdVax-F3 (Spanish influenza). There was relatively no difference in anti-avian influenza antibody titers between F2-immunized animals and F2-immunized animals that also received VIPRO VEX® (Fig. 1). Similar results were noted for the groups of animals receiving the F2 + F3 immunogenic composition formulation +/- VIPRO VEX® (Fig. 3). However, animals immunized with F3 showed a noticeable increase in antibody titer when given the VIPROVEX® compound (Fig. 2). Control- immunized animals or PBS injected animals showed no reactivity, regardless of VIPROVEX® administration (Fig. 4). [00107] Humoral immune response against Spanish influenza. When testing the immunized mouse sera against Spanish influenza proteins, it appeared that VIPROVEX® enhanced the humoral immune response generated by both F2 and F3 immunogenic compositions (Figs 5 and 6). This adjuvant effect was less noticeable in the F2+F3 group (Fig. 7). As before, VIPROVEX® administration had no effect in control-immunized or PBS-injected mice in regard to anti-Spanish influenza humoral immune response (Fig. 8). [00108] The results presented in Figs. 1-8 are the averages for each group of mice. Individual results for each animal in regard to avian and Spanish influenza antibody titers are presented below in Tables 2-11.

CONCLUSIONS FOR HUMORAL RESPONSE DATA [00109] VIPROVEX® administration appears to have an adjuvant effect on CAdVax-based pandemic influenza immunogenic compositions in mice. In particular, in groups showing lower antibody titers, VIPROVEX® administration does appear to enhance the antibody response by 2-3 fold compared to immunogenic composition only. H5N1 VIRUS CHALLENGE

[00110] On week 9 after primary immunization, all mice were shipped to Southern Research Institute (SRI) in Birmingham, AL. There, the animals were quarantined for a period of 3-5 days prior to H5N1 influenza virus challenge. Animal distribution for the virus challenge experiment is described in Table 1. Groups of animals were challenged intranasally with 1 x 10⁶ EID₅₀ (egg infectious dose 50%) of either influenza A/Vietnam/ 1203/04 or influenza A/Indonesia/5/05. After challenge mice were monitored for clinical signs if infection (such as weight loss) as well as survival.

[00111] In mice challenged with A/VN/1203/04, there was slight display of weight loss in immunized mice, followed by a recovery period where body weight returned to baseline. This was noted for mice immunized with F2, F3, and F2+F3, regardless of VIPROVEX® administration. Figs. 9-11. This indicates that all mice may have developed minor disease symptoms from H5N1 infection. Control or PBS-injected mice, however, showed rapid and severe weight loss after A/VN/1203/04 challenge. Figs. 12-13. It should be noted that D3 and PBS-injected animals did not survive A/VN/1203/04 challenge, and therefore weights were not measured past day 11.

[00112] Additional groups of immunized mice were also challenged with the same dose and route of influenza A/Indonesia/5/05. Similar to the clade 1 Vietnam virus challenge described above, all immunized mice displayed minor weight loss immediately after challenge, followed by recovery of body weight. Figs. 14-16. Also as above, VIPROVEX® administration did not appear to have a beneficial effect on weight loss following influenza challenge. Comparatively, control and PBS-injected mice showed more severe weight loss. Figs. 17-18. The clade 2 Indonesia virus is characterized as less pathogenic than the clade 1 Vietnam strain, thus the control groups' overall weight loss was not as severe as seen in the Vietnam strain challenge.

[00113] When comparing survival among the different groups, 100% of mice immunized with F2 survived lethal challenge from both strains of H5N1 virus (Fig. 19 and Table 12). The effects of VIPRO VEX® on survival of immunized mice after lethal HPAI (highly pathogenic avian influenza) can be seen in Figs. 19 and 20. Kaplan-Mier survival curves for mice immunized with the indicated immunogenic composition with or without VIPRO VEX® (V) administration. Mice were challenged intranasally with 1 x 10⁶ EID₅₀ of HPAI strain A/VN/1203/04 (Fig. 19) or A/Indo/5/05 (Fig. 20). After challenge mice were monitored for survival. Any mouse losing > 25% body weight was humanely euthanized and counted as an infection-mediated fatality. Cohort numbers are listed in graph legends. [00114] One hundred percent of F3 -immunized animals survived lethal challenge, but only from the clade 1 Vietnam strain of H5N1. Only 33% of F3 -immunized mice (n=3) survived challenge from the clade 2 Indonesia strain, while 100% of the F-3 immunized mice that also received VIPRO VEX® (n=2) survived the same challenge (Fig. 20 and Table 12). It should be noted that in the F3+V group, one animal died accidentally, reducing the number in the group from three to two. Animals immunized with F2 + F3 had survival rates of 75% and 100% against AATN/ 1203/04 and A/Indo/5/05, respectively. In comparison, 100% of mice that were immunized with F2+F3 in addition to VIPROVEX® administration survived challenges from both viruses. Animals immunized with the D3 control vector or PBS- injected had very poor survival rates (Table 12). CONCLUSIONS FOR H5N1 CHALLENGE [00115] Animals immunized with the avian influenza immunogenic composition, F2, all survived challenge from either strain of H5N1 virus, regardless of VIPROVEX® administration. Animals immunized with the bivalent avian/Spanish flu (F2+F3) immunogenic composition also showed high levels of protection against both strains of virus. However, animals immunized with the 1918 Spanish flu immunogenic composition (F3) had survival rates of 100% and 33.3% against A/VN/1203/04 and A/Indo/5/05, respectively. The group of animals receiving the same F3 immunogenic composition in addition to VIPROVEX® administration survived 100% from both challenge strains of virus. This may indicate that while the F3 immunogenic composition only offered partial protection (33.3%) from lethal A/Indo/5/05 challenge, VIPROVEX® administration was able to enhance the immune response and provide 100% protection. This is consistent with the antibody response profiles from these same two groups of mice, where VIPROVEX® on average, increased the antibody titers 2 to 3 fold against both avian and Spanish influenza protein (Figs 1-8). Meanwhile, the groups showing little difference in antibody titers between immunogenic composition only and immunogenic composition plus VIPROVEX® (F2, F2+F3) also showed little difference in survival rates.

[00116] Various embodiments have been described. The descriptions and examples are intended to be illustrative and not limiting. Indeed, it will be apparent to those of skill in the art that modifications may be made to the various embodiments described without departing from the spirit or scope of the appended claims set forth below.

[00117] All references cited herein are incorporated herein by reference in their entireties for all purposes. Table 1. Animal ID for shipment. (V= VIPRO VEX®)

Table 2. Individual F2-immunized mice antibody titers against avian influenza proteins. Mouse # Week 0 Week 2 Week 4 Week 6 Week 8

1 0.0 319.5 425.0 3323.3 5415.8

2 0.0 262.5 205.5 1542.2 4438.0

3 0.0 466.5 858.1 2345.0 3065.3

4 0.0 420.7 228.9 1802.1 2794.4

F2 only 5 0.0 87.1 1197.9 3653.0 5782.7

6 0.0 154.6 413.4 1685.8 3253.9

7 0.0 - 909.1 2758.6 3353.3

8 0.0 1951.8 3991.1 5303.5

9 0.0 1507.0 2868.0 3264.2

10 0.0 - 0.0 0.0 0.0

1 0.0 572.5 386.1 2058.3 3415.7

2 0.0 1029.4 951.7 3392.3 2868.0

3 0.0 1126.8 816.8 3179.1 3824.0

F2 + 4 0.0 189.2 1455.1 1993.7 3155.5

5 0.0 0.0 0.0 0.0 0.0

VIPROVEX® 6 0.0 1560.6 0.0 0.0 0.0

7 0.0 - 2581.4 5020.4 5008.8

8 0.0 - 965.9 3498.7 3853.5

9 0.0 X* X X X

10 0.0 - 621.0 2859.6 4272.6

*animal died from injection complications; x = dead Table 3. Individual F3-immunized mice antibody titers against avian influenza proteins.

Mouse # Week O Week 2 Week 4 Week 6 Week 8

1 0.0 91.2 251.8 X* * X

2 0.0 3.5 2.8 177.7 3957.7

3 0.0 14.7 3.8 31.7 520.0

4 0.0 17.9 274.4 531.5 256.2

F3 only 5 0.0 72.7 5.3 19.4 247.3

6 0.0 0.5 1.0 218.7

7 0.0 X* X X X

8 0.0 - 2.5 29.2 834.8

9 0.0 - 8.7 654.2 147.7

10 0.0 - 319.2 1396.5 1718.5

1 0.0 6.1 316.5 539.1 775.2

2 0.0 132.4 576.0 1970.1 3195.6

3 0.0 2.2 1.5 8.6 86.1

F3 + 4 0.0 0.1 283.8 842.8 2760.3

5 0.0 47.0 409.9 913.3 1928.7

VIPROVEX® 6 0.0 51.4 248.7 719.3 848.5

7 0.0 X* X X X

8 0.0 X* X X X

9 0.0 _ 221.8 1036.9 593.7

10 0.0 - 457.2 778.3 885.4

Table 4. Individual F2+F3-immunized mice antibody titers against avian influenza proteins.

Mouse # Week O Week 2 Week 4 Week 6 Week 8

1 0.0 22.9 201.3 985.4 1994.1

2 0.0 171.6 334.6 2667.9 5359.7

3 0.0 45.0 239.2 1531.9 4926.8

4 0.0 615.1 1217.1 3348.1 6216.5

F2+F3 5 0.0 190.2 893.8 4002.1 7453.4

6 0.0 78.4 255.0 1 127.1 5032.5

Only 7 0.0 X* X X X

8 0.0 X* X X X

9 0.0 X* X X X

10 0.0 - 378.9 1748.8 2875.7

1 0.0 61.8 559.2 2498.8 5634.7

2 0.0 243.8 788.4 1992.8 3717.3

3 0.0 285.2 501.7 2049.1 2432.9

F2+F3 + 4 0.0 278.8 817.1 1424.6 2379.3

5 0.0 945.5 1588.5 3601.7 10008.4

VIPROVEX® 6 0.0 65.0 393.8 1409.0 3504.6

7 0.0 X* X X X

8 0.0 X* X X X

9 0.0 - 405.5 2475.8 1645.6

10 0.0 - 48.6 423.9 304.1

Table 5. Individual D3-immunized mice antibody titers against avian influenza proteins.

Mouse # Week O Week 2 Week 4 Week 6 Week 8

1 0.0 0.1 1.4 1.7 6.8

2 0.0 0.0 1.0 2.1 5.9

3 0.0 0.2 7.5 7.2 89.2

4 0.0 0.0 3.5 0.9 17.0

D3 only 5 0.0 0.0 0.0 0.0 52.9

6 0.0 0.0 0.0 0.0 0.1

7 0.0 - 0.0 0.3 10.9

8 0.0 - 0.2 1.2 5.9

9 0.0 - 0.2 0.4 2.3

10 0.0 - 0.2 0.0 0.1

1 0.0 0.1 0.0 0.0 0.0

2 0.0 0.0 2.6 2.5 9.3

3 0.0 0.0 5.1 17.9 0.0

D3 + 4 0.0 0.0 0.2 1.1 0.0

5 0.0 0.0 1.7 1.0 5.9

VIPROVEX® 6 0.0 0.0 1.7 1.6 10.3

7 0.0 - 0.2 0.2 3.4

8 0.0 - 0.5 0.0 6.6

9 0.0 - 0.1 0.3 0.9

10 0.0 - 0.0 0.0 0.2

Table 6. Individual PBS-injected mice antibody titers against avian influenza proteins.

Mouse # Week O Week 2 Week 4 Week 6 Week 8

1 0.0 0.0 0.0 0.0 0.0

2 0.0 0.0 0.0 0.0 0.0

3 0.0 0.0 0.0 0.0 0.0

4 0.0 0.0 0.0 0.0 0.0

PBS only 5 0.0 1.0 1.1 0.0 0.0

6 0.0 0.0 0.0 0.0 0.0

7 0.0 0.0 0.0 0.0

8 0.0 - 0.0 0.0 0.0

9 0.0 0.0 0.0 0.0

10 0.0 - 0.0 0.0 0.0

1 0.0 0.0 0.0 0.0 0.0

2 0.0 0.0 0.0 0.0 0.0

3 0.0 0.0 0.0 0.0 0.0

PBS + 4 0.0 0.0 0.0 0.0 0.0

5 0.0 0.0 0.0 0.0 0.0

VIPROVEX® 6 0.0 0.0 0.0 0.0 0.0

7 0.0 - 0.0 0.0 0.0

8 0.0 - 0.0 0.0 0.0

9 0.0 - 0.0 0.0 0.0

10 0.0 - 0.0 0.0 0.0

Table 7. Individual F2-immunized mice antibody titers against Spanish influenza

Mouse # Week O Week 2 Week 4 Week 6 Week 8

1 0.0 134.0 1090.6 1665.5 2057.5

2 0.0 138.1 356.7 993.9 950.9

3 0.0 531.5 508.8 788.3 820.5

4 0.0 330.8 387.0 1533.1 813.7

F2 only 5 0.0 90.5 84.9 277.9 492.7

6 0.0 418.9 558.5 1435.5 1786.1

7 0.0 - 540.2 2725.0 728.6

8 0.0 - 1031.7 803.8 1096.6

9 0.0 - 263.5 295.6 436.0

10 0.0 - 0.0 0.0 0.0

1 0.0 91.2 681.5 2094.8 1438.3

2 0.0 102.8 1078.8 1421.9 1499.1

3 0.0 1048.3 692.7 2076.3 1742.1

F2 + 4 0.0 40.8 2110.7 563.8 1583.1

5 0.0 0.0 0.0 0.0 0.0

VIPROVEX® 6 0.0 152.3 0.0 0.0 0.0

7 0.0 - 777.7 5338.3 900.4

8 0.0 - 207.5 826.5 433.3

9 0.0 X* X X X

10 0.0 - 522.8 2827.6 1294.0 proteins.

Table 8. Individual F3-immunized mice antibody titers against Spanish influenza proteins.

Mouse # WeekO Week2 Week4 Weekό Week 8

1 0.0 4813.9 X** X X

2 0.0 4590.7 13060.3 477'11.4 294384.8

3 0.0 3356.5 3900.8 9755.2 19578.0

4 0.0 21153.0 3815.6 9621.0 35695.8

F3 only 5 0.0 2199.5 22058.1 210715.4 X**

6 0.0 1543.1 5606.4 19096.5 37110.3

7 0.0 5745.6 10754.0 18991.9

8 0.0 - 9905.9 22161.8 24271.4

9 0.0 - 9158.9 82031.9 X**

10 0.0 - 43078.7 12.9 75994.6

1 0.0 8859.0 23379.3 209111.6 336103.2

2 0.0 12245.3 15472.4 118131.6 268763.4

3 0.0 768.4 5208.9 16017.1 100867.2

F3 + 4 0.0 6318.5 10804.0 66958.3 27440.9

VIPROVEX® 5 0.0 5351.9 38401.2 283840.5

6 0.0 13147.0 30499.0 111332.9 19867545.7

7 0.0 X* X X X

8 0.0 X* X X X

9 0.0 - 31986.9 334373.0 19282.4

10 0.0 - 131534.9 117816.5 17357.3

Table 9. Individual F2+F3-immunized mice antibody titers against Spanish influenza proteins.

Mouse # Week O Week 2 Week4 Weekό Week 8

1 0.0 15996.5 6307.6 6613.1 17915.0

2 0.0 7765.7 9760.8 82755.6 290189.4

3 0.0 2972.3 6379.4 43620.5 163643.9

4 0.0 8755.9 21406.8 415263.5 491049.0

F2+F3 5 0.0 8395.2 14312.1 118927.6 78704.4

6 0.0 9984.1 6385.7 9554.9 24058.4

Only 7 0.0 X* X X X

8 0.0 X* X X X

9 0.0 X* X X X

10 0.0 - 59486.5 324237.7 277483.8

1 0.0 8146.3 8780.1 52322.8 168360.6

2 0.0 8308.6 10610.8 44687.2 73164.4

3 0.0 9904.8 9635.4 40824.2 39127.2

F2+F3 + 4 0.0 35202.3 19300.2 26750.0 156039.7

5 0.0 19528.9 22095.0 273966.8 17607.4

VIPROVEX® 6 0.0 3161.1 10654.2 8029.8 3504.6

7 0.0 X* X X X

8 0.0 X* X X X

9 0.0 28915.5 165414.3 12077.1

10 0.0 - 5191.9 15532.4 3183.4

Table 10. Individual D3- immunized mice antibody titers against Spanish influenza proteins.

Mouse # Week O Week 2 Week 4 Week 6 Week 8

1 0.0 0.9 14.7 12.6 5.1

2 0.0 0.0 3.7 4.9 11.1

3 0.0 0.3 42.8 17.7 9.7

4 0.0 0.1 28.2 13.9 0.0

D3 only 5 0.0 0.0 0.0 0.0 20.9

6 0.0 0.0 0.0 0.0 0.0

7 0.0 - 1.9 10.8 3.2

8 0.0 - 11.9 12.3 5.5

9 0.0 - 7.5 13.3 3.4

10 0.0 - 2.6 0.0 0.0

1 0.0 0.8 0.0 0.0 0.0

2 0.0 0.1 31.3 8.3 3.8

3 0.0 0.1 28.6 43.6 10.0

D3 + 4 0.0 0.1 0.8 1.2 1.7

5 0.0 0.0 3.2 7.2 2.2

VIPROVEX® 6 0.0 0.0 2.5 2.9 1.2

7 0.0 - 2.2 1.3 1.2

8 0.0 - 0.4 0.0 0.2

9 0.0 - 0.0 0.0 0.0

10 0.0 - 0.1 0.2 0.3

Table 11. Individual PBS- immunized mice antibody titers against Spanish influenza proteins.

Mouse # Week O Week 2 Week 4 Week 6 Week 8

1 0.0 0.0 0.0 0.0 0.0

2 0.0 0.0 0.0 0.0 0.0

3 0.0 0.0 0.0 0.0 0.0

4 0.0 0.0 0.0 0.0 0.0

PBS only 5 0.0 1.4 0.2 0.0 0.0

6 0.0 0.0 0.0 0.0 0.0

7 0.0 0.0 0.0 0.0

8 0.0 - 0.0 0.0 0.0

9 0.0 - 0.0 0.0 0.0

10 0.0 - 0.0 0.0 0.0

1 0.0 0.0 0.0 0.0 0.0

2 0.0 0.0 0.0 0.0 0.0

3 0.0 0.0 0.0 0.0 0.0

PBS + 4 0.0 0.0 0.2 0.0 0.0

5 0.0 0.0 0.0 0.0 0.0

VIPROVEX® 6 0.0 0.0 0.0 0.0 0.0

7 0.0 - 0.0 0.0 0.0

8 0.0 0.0 0.0 0.0

9 0.0 - 0.0 0.0 0.0

10 0.0 - 0.0 0.2 0.0

Table 12. Percent survival* after H5N1 virus challenge

* survival determined 14 days after intranasal challenge with 1 x 10 EID₅₀ of H5N1 virus.

Claims

What is claimed is:

1. A method of preventing viral infection of a human, comprising administering to said human, an immunogenic composition comprising an antigen and an adjuvant effective amount of a substance P analog wherein the substance P analog is of Formula (I): Zi-Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^-Xaa¹ '-Z₂ (I) or a pharmaceutically acceptable salt thereof, wherein:

Xaa¹ is Arg, Lys, 6-N methyllysine, or (6-N, 6-N) dimethyllysine;

Xaa² is Pro or Ala;

Xaa³ is Lys, Arg, 6-N-methyllysine, or (6-N, 6-N) dimethyllysine;

Xaa⁴ is Pro or Ala;

Xaa⁵ is GIn or Asn;

Xaa⁶ is GIn or Asn;

Xaa⁷ is Phe or Phe substituted with chlorine at position 2, 3 or 4;

Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 2, 3 or 4;

Xaa⁹ is GIy, Pro, Ala, or N-methylglycine;

Xaa¹¹ is Met, Met sulfoxide, Met sulfone, or Norleucine;

Zi is R₂N- or RC(O)NR-;

Z₂ is -C(O)NR₂ or -C(O)OR or a salt thereof; each R is independently R is H, (Cj-C₆) alkyl, (Ci-C₆) alkenyl, (Ci-C₆) alkynyl,

(C₅-C₂₀) aryl, (C₆-C₂₆) alkaryl, 5-20 membered heteroaryl or 6-26 membered alkheteroaryl; and each "— " between residues Xaa¹ through Xaa¹ ' independently designates an amide linkage, a substitute amide linkage or an isostere of an amide.

2. The method of claim 1 wherein

Xaa¹ is Arg;

Xaa² is Pro;

Xaa³ is Ly s;

Xaa⁴ is Pro;

Xaa⁵ is GIn;

Xaa⁶ is GIn;

Xaa⁷ is Phe or Phe substituted with chlorine at position 4;

Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 4;

Xaa⁹ is GIy, Pro, or N-methylglycine;

Xaa¹⁰ is Leu; and

Xaa¹¹ is Met, Met sulfoxide, Met sulfone, or Norleucine.

3. The method of claim 1 wherein the "— " between residues Xaa¹ through Xaa¹¹ designates

-C(O)NH-;

Zi is H₂N-; and Z₂ is -C(O)NH₂

4. The method of claim 1 wherein the substance P analog is selected from the group consisting of:

RPKPQQFFGLM (SEQ ID NO.: 1);

RPKPQQFFGLNIe (SEQ ID NO. : 2);

RPKPQQFFPLM (SEQ ID NO. : 3);

RPKPQQFFMeGIyLM (SEQ ID NO. : 4);

RPKPQQFTGLM (SEQ ID NO. : 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO.: 6);

RPKPQQFFGLM(O) (SEQ ID NO. : 7); RPKPQQFFMeGIyLM(O) (SEQ ID NO. : 8);

RPKPQQFFGLM(O₂) (SEQ ID NO. : 9); and

RPKPQQFFMeGIyLM(O₂) (SEQ ID NO. : 10).

5. The method of claim 1 wherein the substance P analog is Z₁-RPKPQQFFMeGlyM(O₂)-Z₂; wherein

Z, is NH₂ and Z₂ is C(O)NH₂.

6. The method of claim 1 wherein the viral infection is Influenza A.

7. The method of claim 6 wherein the Influenza A is avian influenza.

8. The method of claim 7 wherein the avian influenza is H5N1.

9. The method of claim 7 wherein the avian influenza is H7N3.

10. The method of claim 1 wherein the method of preventing viral infection is preexposure prevention.

11. The method of claim 1 wherein the method of preventing viral infection is postexposure prevention.

12. The method of claim 1 wherein the immunogenic composition is administered intranasally.

13. The method of claim 1 wherein the immunogenic composition is administered by injection.

14. The method of claim 1 wherein the immunogenic composition is administered intramuscularly.

15. The method of claim 1 wherein the immunogenic composition is administered subcutaneously.

16. The method of claim 1 wherein the immunogenic composition is administered intradermally.

17. A method of immunizing a human against a viral infection comprising administering to said human, an immunogenic composition comprising an antigen and an effective amount of a substance P analog wherein said substance P analog is of Formula (I):

Zi-Xaa'-Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^-Xaa¹ '-Z₂ (I) or a pharmaceutically acceptable salt thereof, wherein:

Xaa¹ is Arg, Lys, 6-N methyllysine, or (6-N, 6-N) dimethyllysine;

Xaa² is Pro or Ala;

Xaa³ is Lys, Arg, 6-N-methyllysine, or (6-N, 6-N) dimethyllysine;

Xaa⁴ is Pro or Ala;

Xaa⁵ is GIn or Asn;

Xaa⁶ is GIn or Asn;

Xaa⁷ is Phe or Phe substituted with chlorine at position 2, 3 or 4;

Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 2, 3 or 4;

Xaa⁹ is GIy, Pro, Ala, or N-methylglycine;

Xaa¹⁰ is Leu, VaI, He, Norleucine, Met, Met sulfoxide, Met sulfone, N- methylleucine, or N-methyl valine;

Xaa¹¹ is Met, Met sulfoxide, Met sulfone, or Norleucine;

Zi is R₂N- or RC(O)NR-;

Z₂ is -C(O)NR₂ or -C(O)OR or a salt thereof; each R is independently R is H, (Ci-C₆) alkyl, (Ci-C₆) alkenyl, (Ci-C₆) alkynyl,

(C₅-C₂₀) aryl, (C₆-C₂₆) alkaryl, 5-20 membered heteroaryl or 6-26 membered alkheteroaryl; and each "— " between residues Xaa through Xaa¹ ' independently designates an amide linkage, a substitute amide linkage or an isostere of an amide.

18. The method of claim 17 wherein: Xaa¹ is Arg;

Xaa² is Pro;

Xaa³ is Ly s;

Xaa⁴ is Pro;

Xaa⁵ is GIn;

Xaa⁶ is GIn;

Xaa⁷ is Phe or Phe substituted with chlorine at position 4;

Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 4;

Xaa⁹ is GIy, Pro, or N-methylglycine;

Xaa¹⁰ is Leu; and

Xaa¹ ' is Met, Met sulfoxide, Met sulfone, or Norleucine.

19. The method of claim 17 wherein the "— " between residues Xaa¹ through Xaa¹¹ designates

-C(O)NH-;

Z₁ is H₂N-; and Z₂ is -C(O)NH₂

20. The method of claim 17 wherein the substance P analog is selected from the group consisting of:

RPKPQQFFGLM (SEQ ID NO. : 1 );

RPKPQQFFGLNIe (SEQ ID NO.: 2);

RPKPQQFFPLM (SEQ ID NO.: 3);

RPKPQQFFMeGIyLM (SEQ ID NO.: 4);

RPKPQQFTGLM (SEQ ID NO.: 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO.: 6);

RPKPQQFFGLM(O) (SEQ ID NO. : 7);

RPKPQQFFMeGIyLM(O) (SEQ ID NO. : 8); RPKPQQFFGLM(O₂) (SEQ ID NO. : 9); and

RPKPQQFFMeGIyLM(O₂) (SEQ ID NO. : 10).

21. The method of claim 17 wherein the substance P analog is Z,-RPKPQQFFMeGlyM(O₂)-Z2; wherein;

Zi is NH₂ and Z₂ is C(O)NH₂.

22. The method of claim 17 wherein the viral infection is Influenza A.

23. The method of claim 22 wherein the Influenza A is avian influenza.

24. The method of claim 23 wherein the avian influenza is H5N1.

25. The method of claim 23 wherein the avian influenza is H7N3.

26. The method of claim 17 wherein the method of immunizing is pre-exposure immunization.

27. The method of claim 17 wherein the method of immunizing is post-exposure immunization.

28. The method of claim 17 wherein the immunogenic composition is administered intranasally.

29. The method of claim 17 wherein the immunogenic composition is administered by injection.

30. The method of claim 17 wherein the immunogenic composition is administered intramuscularly.

31. The method of claim 17 wherein the immunogenic composition is administered subcutaneously.

32. The method of claim 17 wherein the immunogenic composition is administered intradermally.

33. A method of inducing an immune response in a human against a viral infection comprising administering to said human, an immunogenic composition comprising an antigen and an adjuvant effective amount of a substance P analog wherein the substance P analog is of Formula (I):

Zi -Xaa¹ -Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa' ^O-Xaa^! ' -Z₂ (I) or a pharmaceutically acceptable salt thereof, wherein: Xaa¹ is Arg, Lys, 6-N methyllysine, or (6-N, 6-N) dimethyllysine; Xaa² is Pro or Ala;

Xaa³ is Lys, Arg, 6-N-methyllysine, or (6-N, 6-N) dimethyllysine; Xaa⁴ is Pro or Ala; Xaa⁵ is GIn or Asn; Xaa⁶ is GIn or Asn;

Xaa¹ ' is Met, Met sulfoxide, Met sulfone or Norleucine;

Zi is R₂N- or RC(O)NR-;

34. The method of claim 33 wherein:

Xaa¹ is Arg;

Xaa² is Pro;

Xaa is Lys;

Xaa⁴ is Pro;

Xaa⁵ is GIn;

Xaa⁶ is GIn;

Xaa⁷ is Phe or Phe substituted with chlorine at position 4;

Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 4;

Xaa⁹ is GIy, Pro, or N-methylglycine;

Xaa¹⁰ is Leu; and

Xaa¹ ' is Met, Met sulfoxide, Met sulfone, or Norleucine.

35. The method of claim 33 wherein the "— " between residues Xaa¹ through Xaa¹¹ designates

-C(O)NH-;

Z, is H₂N-; and Z₂ is -C(O)NH₂

36. The method of claim 23 wherein the substance P analog is selected from the group consisting of:

RPKPQQFFGLM (SEQ ID NO.: 1);

RPKPQQFFGLNIe (SEQ ID NO.: 2);

RPKPQQFFPLM (SEQ ID NO. : 3);

RPKPQQFFMeGIyLM (SEQ ID NO. : 4);

RPKPQQFTGLM (SEQ ID NO. : 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO. : 6);

RPKPQQFFGLM(O) (SEQ ID NO. : 7); RPKPQQFFMeGIyLM(O) (SEQ ID NO. : 8);

RPKPQQFFGLM(O₂) (SEQ ID NO. : 9); and

RPKPQQFFMeGIyLM(O₂) (SEQ ID NO. : 10).

37. The method of claim 33 wherein the substance P analog is Z₁-RPKPQQFFMeGlyM(O₂)-Z2; wherein

Z₁ is NH₂ and Z₂ is C(O)NH₂.

38. The method of claim 33 wherein the viral infection is Influenza A.

39. The method of claim 38 wherein the Influenza A is avian influenza.

40. The method of claim 39 wherein the avian influenza is H5N1.

41. The method of claim 39 wherein the avian influenza is H7N3.

42. The method of claim 33 wherein the method of inducing an immune response is preexposure prevention.

43. The method of claim 33 wherein the method of inducing an immune response is postexposure prevention.

44. The method of claim 33 wherein the immunogenic composition is administered intranasally.

45. The method of claim 33 wherein the immunogenic composition is administered by injection.

46. The method of claim 33 wherein the immunogenic composition is administered intramuscularly.

47. The method of claim 33 wherein the immunogenic composition is administered subcutaneously.

48. The method of claim 33 wherein the immunogenic composition is administered intradermally.

49. A pharmaceutical composition for preventing viral infection of a human, comprising an antigen and an adjuvant effective amount of a substance P analog wherein the substance P analog is of Formula (I): Zi-Xaa'-Xaa^Xaa^Xaa^Xaa^Xaa^Xaa'-Xaa^Xaa'-Xaa^-Xaa¹ '-Z₂ (I) or a pharmaceutically acceptable salt thereof, wherein:

Xaa¹ is Arg, Lys, 6-N methyllysine, or (6-N, 6-N) dimethyllysine;

Xaa² is Pro or Ala;

Xaa³ is Lys, Arg, 6-N-methyllysine, or (6-N, 6-N) dimethyllysine;

Xaa⁴ is Pro or Ala;

Xaa⁵ is GIn or Asn;

Xaa⁶ is GIn or Asn;

Xaa⁷ is Phe or Phe substituted with chlorine at position 2, 3 or 4;

Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 2, 3 or 4;

Xaa⁹ is GIy, Pro, Ala, or N-methylglycine;

Xaa¹ ' is Met, Met sulfoxide, Met sulfone, or Norleucine;

Zi is R₂N- or RC(O)NR-;

50. The composition of claim 49 wherein Xaa¹ is Arg;

Xaa² is Pro;

Xaa³ is Lys;

Xaa⁴ is Pro;

Xaa⁵ is GIn;

Xaa⁶ is GIn;

Xaa⁷ is Phe or Phe substituted with chlorine at position 4;

Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 4;

Xaa⁹ is GIy, Pro, or N-methylglycine;

Xaa¹⁰ is Leu; and

Xaa¹ ' is Met, Met sulfoxide, Met sulfone, or Norleucine.

51. The composition of claim 49 wherein the "— " between residues Xaa¹ through Xaa¹¹ designates

-C(O)NH-;

Z₁ is H₂N-; and Z₂ is -C(O)NH₂

52. The composition of claim 49 wherein the substance P analog is selected from the group consisting of:

RPKPQQFFGLM (SEQ ID NO. : 1 );

RPKPQQFFGLNIe (SEQ ID NO.: 2);

RPKPQQFFPLM (SEQ ID NO. : 3);

RPKPQQFFMeGIyLM (SEQ ID NO. : 4);

RPKPQQFTGLM (SEQ ID NO. : 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO. : 6);

RPKPQQFFGLM(O) (SEQ ID NO.: 7);

RPKPQQFFMeGIyLM(O) (SEQ ID NO. : 8); RPKPQQFFGLM(O₂) (SEQ ID NO. : 9); and

RPKPQQFFMeGIyLM(O₂) (SEQ ID NO. : 10).

53. The composition of claim 49 wherein the substance P analog is Zi-RPKPQQFFMeGlyM(O₂)-Z₂; wherein

Z, is NH₂ and Z₂ is C(O)NH₂.

54. The composition of claim 49 wherein the viral infection is Influenza A.

55. The composition of claim 54 wherein the Influenza A is avian influenza.

56. The composition of claim 55 wherein the avian influenza is H5N1.

57. The composition of claim 55 wherein the avian influenza is H7N3.

58. The composition of claim 49 wherein the prevention of viral infection of a human is pre-exposure prevention.

59. The composition of claim 49 wherein the prevention of viral infection of a human is post-exposure prevention.

60. The composition of claim 49 wherein the immunogenic composition is administered intranasally.

61. The composition of claim 49 wherein the immunogenic composition is administered by injection.

62. The composition of claim 49 wherein the immunogenic composition is administered intramuscularly.

63. The composition of claim 49 wherein the immunogenic composition is administered subcutaneously.

64. The composition of claim 49 wherein the immunogenic composition is administered intradermally.