WO2011053789A2 - Pharmaceutical composition and methods to enhance cytotoxic t-cell recognition and maintain t-cell memory against a pathogenic disease - Google Patents

Abstract

The present invention provides methods and pharmaceutical compositions comprising a peptide sequence of endogenous angiotensin I or biologically active fragments thereof in combination with an antigen or immunogenic portion of an antigen for inducing a robust cytotoxic T lymphocyte response and maintaining an ongoing antigen-specific cytotoxic T lymphocyte memory, and their uses for treating or preventing pathogenic diseases.

Description

PHARMACEUTICAL COMPOSITION AND METHODS TO ENHANCE CYTOTOXIC T-CELL RECOGNITION AND MAINTAIN T-CELL MEMORY AGAINST A PATHOGENIC DISEASE

The application claims priority of US provisional application number 61/256,760 filed on October 30, 2009 and is included herein in its entirety by reference.

COPYRIGHT NOTICE

A portion of the disclosure of this patent contains material that is subject to copyright protection. The copyright owner has no objection to the reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent files or records, but otherwise reserves all copyright rights whatsoever.

BACKGROUND OF THE INVENTION

Field of the Invention

[001 ] The present invention relates to compositions and methods for an immunostimulatory effect. In particular, the present invention relates to the combination of an immunostimulatory antigen in combination with a peptide adjuvant that can stimulate a Th1 cytotoxic T lymphocyte (CTL) response characterized by an increased IFN-γ, induction of antigen cross-presentation, immunoglobulin class-switching, and enhanced presentation of the antigen by the MHC class I - TCR complex.

Description of Related Art

[002] Vaccination is the single most valuable tool used to protect against infectious diseases by inducing humoral immunity, or an antigen specific response. Also, vaccination is becoming significant for treating syngeneic tumors. Vaccine formulations contain antigens that induce immunity against pathogenic agents and may be enhanced by using adjuvants that can selectively stimulate immuno-regulatory responses. In many cases, these approaches have been very successful at inducing immune protection, mainly based on antibody responses. Yet, the only adjuvants currently approved by the U.S. FDA are aluminum based mineral salts which are generally safe, but comparative studies show they are weak adjuvants for antibody induction to protein subunits and poor adjuvants for cell mediated immunity.

[003] To develop vaccines against more difficult pathogens, which often establish chronic infections, e.g. HIV, HCV, TB and malaria, the induction of CMI is likely to be necessary. A vaccine for immunizing mammals that stimulates the CMI system which responds to endogenous antigen is presented by the MHC class I processing pathway. Cells can process foreign proteins found in the cell cytosol and display relevant peptide epitopes using this processing pathway. The MHC class I processing pathway involves digestion of the antigen by the proteasome complex and transport of the resulting peptides into the endoplasmic reticulum, where they bind to nascent MHC class I molecules.

[004] Cytotoxic T lymphocytes (CTLs) specifically recognize the foreign antigen displayed by the MHC class I molecules and lyse the antigen-presenting cells. A population of memory T cells is also established that can react to presentation of the specific antigen. The cellular immune system is thus primed to swiftly respond to an intracellular infection by a pathogenic organism such as a virus or tumor associated antigen. Several studies have reported the ability to immunize mammalian hosts with peptides to induce virus-specific or bacteria- specific CTLs.

[005] There is strong evidence from studies, in both humans and animal models, that a robust CD8+ CTL response can control HIV-1 infection. Thus, numerous peptide-based vaccines aimed at eliciting CTL responses for prophylaxis or treatment of cancer and pathogenic infection has been proposed. Currently, the most common therapies include chemotherapy, radiation therapy, or hormonal therapy. These conventional therapies have proven toxic and dangerous to the immune system; treatments kill the majority of cells within a tumor but unaffected cells often reestablish the abnormal pattern of proliferation. Thus, alternative methods of cancer treatment including prevention and prophylactic vaccines are of great research interest.

[006] Despite the foregoing advances in the art of immunotherapy to treat pathogenic infection and cancer, there remains a clear need for additional tools to supplement and enhance existing vaccines and treatment methods. The use of peptide immunogens to treat or prevent viral infections and cancer holds considerable promise. However, additional developments are needed to enhance the efficacy of peptide vaccines and extend their application to a broader range of clinical settings.

[007] The normal measure of T cell mediated-immunity has relied upon demonstration of antigen-specific T cell activation. According to the current practice regarding T cell immunity; positive results are associated with tetramer binding by antigen specific T cells and potent epitope dominate pathogen response. Additionally, the presence of (in vivo) antigen specific antibodies can be detected in plasma. Thus, potent systemic T cell response, systemic antibody response, and antigen specific tetramer binding are considered as all required for scientific demonstration of activity.

SUMMARY OF THE INVENTION

[008] The present invention vaccine compositions comprise a novel immuno- adjuvant ligand in combination with one or more antigens.

[009] More specifically, the present invention involves a novel innate immune response of these compositions, based on the observation that activation of a specific family of regulatory transmembrane G-coupled protein receptors increases innate and adaptive immune surveillance for the co-administered antigen(s). There are at least 4 unique major epitopes of this Ang-GCPR (AT1 , AT2, AT4, and AT7).

[010] As part of the ligand-GCP receptor regulatory mechanism, receptor signaling by MAPK can initiate a Th1 mediated innate immune response propagated by a (PRR) pattern recognition receptor genetically-programmed to elicit a cell mediated immune (CMI) response against the parent antigen. [01 1] Adaptive responses such as stimulated a Th1 cytotoxic T lymphocyte (CTL) response characterized by an increased IFN-γ, induction of antigen cross- presentation, allow for immunoglobulin class-switching, and enhancing presentation of the antigen by the MHC class I - TCR complex are also observed.

[012] The unexpected observation of the present invention is directed to methods and compositions for enhancing a protective or therapeutic mucosal CTL response to a pathogenic virus, bacterium, parasite, protozoan, or tumor associated antigen in a mammal. This response can include induction of CMI against a non-infectious antigen fragment of an otherwise infectious antigen.

[013] The G-coupled protein receptors related to the present invention are naturally occurring regulators of cellular growth, repair, and apoptosis. This regulatory pathway is a highly conserved in nearly every vertebrate. Activation of the receptor up regulates the immunologic activity of immune cells, such as but not limited to: NK Cells, CD4+, CD8+, CD3, M0, dendritic cells, and human progenitor cells.

[014] Ligand activated receptor signaling stimulates the phosphorylation of several tyrosine-containing proteins, such as mitogen-activated protein (MAPK) kinase p38, ERK1/ERK2, JNK and the JAK--STAT pathway; an effect attenuated by Z receptor antagonists.

[015] T cells and NK cells are fully equipped with ligand processing receptors to self-regulate their unregulated participation in the immune response. Chemotaxis is enhanced by ligand binding, indicating that activation of the receptor family mediates an inflammatory amplification system in vertebrates. Recent evidence demonstrates activity of this GCP receptor in dendritic cell (DC) activation, presentation, and mobilization (maturation). Ligand binding and receptor activation is believed to augment mitogen and anti-CD3-stimulated T and NK cell proliferation. INDUCTION OF TH1 CYTOKINES

[016] In the present invention, the composition provides that the peptide adjuvant directs a Th1 dominate CTL and is characterized by increased interferon-γ. Th1 immune responses direct the clonal expansion of activated macrophages, NK cells, and CD8+ cells, promote MHC Class I antigen presentation, dendritic cell mobilization and maturation, phagocytosis, plus the secretion of certain non-specific Ig isotypes. Persons skilled in the art will readily appreciate from a reading of the present disclosure that a Th1 immune response results in other Th1 cytokines including, but not limited to, IL-2, IL-3, IL-12, INF-γ, TNF-a, TNF-β, TGF-β, and GM-CSF.

[017] The present invention discloses surprisingly that co-presentation of an infectious or non-infectious antigen with ligands to these Ang-GCP receptors ( in one embodiment doses from 10 meg to 1000 meg of vaccine (0.14 to 14 mcg/kg)) enhance the cellular immune response to both innate stimulants and adaptive anti-specific T cell antigens. Specifically, the increase cellular activation can be demonstrated by measuring Th1 cytokine release in either peripheral blood mononuclear cells or in whole blood incubations following in vitro endotoxin challenge.

[018] More specifically, the present invention provides a composition capable of enhancing pattern recognition receptor responses to innate stimuli such as endotoxin as demonstrated by changes in phosphorylation, receptor signaling, or TLR mRNA relative to the na^'fve ET response without adjuvant and antigen pretreatment.

Toll-Like Receptors

[019] The activation of toll like receptor 4 (TLR4) is associated with increased release of TNF-a, IL-Ι β, and IL-6 into the circulation, as well as, increased NK cell activation. Blockade of this Ang-GCPR inhibits the TLR4 phosphorylation following experimental endotoxemia in mice. The blockade of the GCPR can protect animals from the lethal cytokine storm following ET shock; indicating the receptor's active participation in the innate response mediated via TLR. This receptor mediates proinflammatory properties by activating NF-κΒ transcription factor nuclear translocation and inducing the expression of chemokines.

PARTICIPATION IN INNATE IMMUNE REACTIONS

[020] More specifically, the present invention provides for compositions and methods for directing antigen-specific CTL responses to the pathologic antigen. The invention is based on an unexpected discovery that certain angiotensin- related peptides, termed immunostimulatory adjuvants, can activate innate immunity, increase dendritic cell activation, enhance cytokinesis and

presentation of antigen by MHC class I TCR complexes, direct autocrine and paracrine Thi cytokine/chemokine synthesis, and up-regulate acquired immunity and potential immunoglobulin class-switching.

MHC I CROSS-PRESENTATION of FRAGMENTS (ANTIGEN)

[021 ] The invention further provides for compositions and methods for induction of a CTL response to an antigen via classic MHC class I presentation by an infectious antigen or by cross-presentation by a non-infectious antigen. In addition, the invention provides compositions and methods for activation and maintenance of an antigen specific cytotoxic T lymphocyte response in an individual; methods for decreasing the number of infectious pathogens in an individual; methods for decreasing tumor load in an individual; and methods of treating an infectious disease in an individual.

ANTIGENS

[022] In one embodiment of the present invention, the antigen is comprised of a peptide pool. Antigen pools for specific pathogens in other embodiments may be composed of: 1 ) Digested fragments of pathogen which, in one embodiment, are at least 10 mer and in another they are between 10 mer and 100 mer; 2) fragments from multiple pathogens are mixed or pooled into the antigen pool; and 3) fragment length can be optimized for each specific pathogen to maintain broad based innate immune balance. Cytokine profiling using a whole blood model is used to adjust antigen and adjuvant dose. [023] The present invention relates to a composition consisting of an

immunostimulatory antigen in combination with a peptide adjuvant that can stimulate a Th1 cytotoxic T lymphocyte (CTL) response characterized by an increased IFN-γ, induction of antigen cross-presentation, allow for

immunoglobulin class-switching, and enhancing presentation of the antigen by the MHC class I - TCR complex. This unexpected observation is directed to methods and compositions for enhancing a protective or therapeutic mucosal CTL response to a pathogenic virus, bacterium, parasite, protozoan, or tumor associated antigen in a mammal. In the present invention, the peptide adjuvant is derived from both natural and non-natural peptide sequences of angiotensin I, which have the ability to bind with an angiotensin type 1 , 2, 4, or 7 receptors as an agonist or a competitive antagonist.

[024] One embodiment of the invention is a pharmaceutical composition containing an antigen or an immunologic portion of the antigen and a peptide adjuvant formulated for mucosal delivery which is capable of inducing a cytotoxic T lymphocyte response in a mammal characterized by a Th1 cytokine response.

[025] In another embodiment, the invention provides methods for

administering to mucosal tissue, a peptide adjuvant and antigen that infects an antigen presenting or dendritic cell and is processed in the cytosol and

presented by MHC Class I - TCR complex. Alternatively, the antigen may be endocytosed and cross-presented by an antigen presenting cell via the MHC Class I complex. In this embodiment, the adjuvant directs the release of Th1 cytokines, mobilization and maturation of dendritic cells, stimulation of na^'fve T cells, and up-regulation of cell mediated immunity. The activation of acquired immunity following an innate immune signal further provides neutralizing antigen- specific immunoglobulins and activation of immunoglobulin class-switching of antibody production.

BRIEF DESCRIPTION OF THE DRAWINGS

[026] Fig. 1 is a diagram of the endogenous Renin-Angiotensin Metabolic Cascade. ACE = angiotensin-converting enzyme; ACE2 = angiotensin- converting enzyme Ang monocarboxylase Ang I = angiotensin decapeptide; Ang 11= antiotensin octapeptide; Ang (1 -9) angiotensin nonopetpide; Ang (1 -7)= angiotensin heptapeptide; Ang (1 -5) = angiotensin decapeptide; Ang III = angiotensin 2-8; Ang IV = angiotensin 3-8; AT1 = angiotensin II type 1 receptor; AT2 = angiotensin II type 2 receptor ;Mas = GCPR receptor by the Mas proto- oncogene, and which mediates angiotensin-(1-7) effects; AT17P7-S =

angiotensin-(1-7) receptor that is inhibited by pro7-angiotensin-(1-7); PCP = procarboxypeptidase; PEP = prolylendopeptidase; NEP = neutral

endopeptidase.

DETAILED DESCRIPTION OF THE INVENTION

[027] While this invention is susceptible to embodiment in many different forms, there is shown in the drawings and will herein be described in detail specific embodiments, with the understanding that the present disclosure of such embodiments is to be considered as an example of the principles and not intended to limit the invention to the specific embodiments shown and described. In the description below, like reference numerals are used to describe the same, similar or corresponding parts in the several views of the drawings. This detailed description defines the meaning of the terms used herein and specifically describes embodiments in order for those skilled in the art to practice the invention.

Definitions

[028] The terms "a" or "an", as used herein, are defined as one or as more than one. The term "plurality", as used herein, is defined as two or as more than two. The term "another", as used herein, is defined as at least a second or more. The terms "including" and/or "having", as used herein, are defined as comprising (i.e., open language). The term "coupled", as used herein, is defined as connected, although not necessarily directly, and not necessarily mechanically.

[029] Reference throughout this document to "one embodiment," "certain embodiments," and "an embodiment" or similar terms means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of such phrases or in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments without limitation.

[030] The term "or" as used herein is to be interpreted as an inclusive or meaning any one or any combination. Therefore, "A, B or C" means any of the following: "A; B; C; A and B; A and C; B and C; A, B and C". An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

[031 ] The drawings featured in the figures are for the purpose of illustrating certain convenient embodiments of the present invention, and are not to be considered as limitation thereto. Term "means" preceding a present participle of an operation indicates a desired function for which there is one or more embodiments, i.e., one or more methods, devices, or apparatuses for achieving the desired function, and that one skilled in the art could select from these or their equivalent in view of the disclosure herein and use of the term "means" is not intended to be limiting.

[032] As used herein, "adaptive immunity" refers to the adaptive immune system which is composed of highly specialized, systemic cells and processes that eliminate or prevent pathogenic challenges. It involves two types of receptors generated by somatic mechanisms during the development of an organism. The adaptive immune system refers to both cellular immunity (CMI or cell-mediated immunity) and humoral immunity. It is adaptive immunity because the body's immune system prepares itself for future challenge.

[033] As used herein, "adaptive immune response" refers to a response involving characteristics of "adaptive immunity" described above and provides the vertebrate immune system with the ability to recognize and remember specific pathogens and to mount stronger attacks each time the pathogen is encountered.

[034] The term "adjuvant" and "peptide adjuvant" is well understood in the art and refers to a molecule which is specifically recognized by a component of the immune system, e.g., an antibody or a T-cell antigen receptor. In the present invention, they are capable of stimulating a Thi cytokine response. As used herein, the term "adjuvant" encompasses peptide chains related to the group comprising a mammalian naturally occurring angiotensin I; a biologically active fragment of angiotensin I excluding angiotensin II; and biologically active peptide sequences and fragments homologous to angiotensin I and, biologically active fragments, of the above with the proviso that positions 3, 4 and 5 in angiotensin I is conserved. In one embodiment there is at least 80% sequence homology and in other embodiments there is 85%, 90%, 95%, 98%, and 99% homology with angiotensin I. By biologically active is meant that the homologous sequence has the activity of angiotensin I. The adjuvant can be considered biologically activated after administered to the mammal, if any endogenous peptidase or amidase cleaves any amino acids and the peptide can bind an angiotensin receptor as defined herein.

[035] The peptide adjuvant can be selected from the group consisting of or comprising a mammalian naturally occurring angiotensin I, a biologically active fragment of angiotensin I excluding angiotensin II and peptide sequences and fragments homologous by at least 80% to angiotensin I and biologically active fragments thereof with the proviso that positions 3, 4 and 5 in angiotensin I is conserved. It can also be selected from the group comprising SEQ ID NO 1 , It can also be not conjugated to a carrier protein or it can be biologically activated after administration by endogenous amidase or peptidase, e.g. the amidase or peptidase can be selected from the group comprising angiotensin converting enzyme 1 , angiotensin converting enzyme 2, chymase, neutral endopeptidase, and aminopeptidase.

[036] The terms "angiotensin" and "RAS" are well understood in the art and refer to peptide molecules which control the renin-angiotensin system (RAS). Angiotensin peptides have a critical role in the cardiovascular system. The most notable sequences of angiotensin peptides are angiotensin I (1 -10 mer), angiotensin II (1 -8 mer), angiotensin 7 (1 -7 mer), angiotensin 5 (1 -5 mer), angiotensin III (2-8 mer), and angiotensin IV (3-8 mer). Angiotensin II is a well- known bioactive substance involved in the regulation of blood pressure and is involved in the exaggeration of cardiovascular disease. Angiotensin 7 and angiotensin 5 are considered to counter regulate actions of angiotensin II; as is angiotensin III and angiotensin IV.

[037] As used herein, the phrase "angiotensin receptors" refers to a series of G-protein coupled cell surface receptors which are expressed on nearly all tissues. The receptors are modulated or expressed in disease and with excess angiotensin II expression. The angiotensin receptors are identified as

angiotensin receptor- type 1 (AT1 ), a type 2 (AT2), a type 4 (AT4), and Mas or type 7 (AT7). Angiotensin peptides are capable of binding all receptors with varying avidity. AT1 acts primarily as an up-regulating cardiovascular receptor as is associated with cardio-renal disease if not regulated. AT2 is a counter regulatory receptor and called a fetal growth receptor. AT4 is a counter regulatory receptor. The Mas receptor (AT7) acts in some tissues as a counter regulatory receptor and in others as a regulatory receptor.

[038] As used herein the terms "antigen" and "epitope" are well understood in the art and refer to the portion of a macromolecule which is specifically recognized by a component of the immune system, e.g., an antibody or a T-cell antigen receptor. As used herein, the term "antigen" encompasses antigenic epitopes, e.g., immunogenically active fragments of an antigen which are antigenic epitopes. Epitopes are recognized by antibodies in solution, e.g., free from other molecules. Epitopes are recognized by T-cell antigen receptors when the epitope is associated with a class I or class II major histocompatibility complex molecule.

[039] The antigen can be a whole virus, an attenuated virus, an inactivated virus, a viral fragment, recombined viral vectors, virus like particles and the like. The antigen can be a non-immunogenically active fragment of an antigen when used without the adjuvant (and active in its presence). Examples of virus include human immunodeficiency virus, simian immunodeficiency virus, human papillomavirus, herpes simplex virus, influenza A virus subtypes H1 N1 , H1 N2, H3N1 , H3N2, and H2N3, and a rotavirus. [040] The antigen component of the composition min one embodiment may consist of peptide pools of fragments (10-100 mer) digested from an infectious pathogen or TAA.

[041 ] Antigen pools for specific pathogens in other embodiments may be composed of: 1 ) Digested fragments of pathogen which are at least 10 mer and in one embodiment between 10 mer and 100 mer; 2) Fragments from multiple pathogens are mixed or pooled into the antigen pool. Fragment length can be optimized for each specific pathogen to maintain broad based innate immune balance. Cytokine profiling using a whole blood model are used to adjust antigen and adjuvant dose.

[042] In one embodiment of the present invention, the antigen or peptide pool and or the adjuvant given can be optimized at a dose and frequency determined and optimized by inducible/measurable cytokine patterns.

[043] In one embodiment the antigen is derived from extracellular turmor associated antigens. Examples include wherein the tumor associated antigen is selected from the group comprising alphafetoprotein, carcinoembryonic antigen, CA-125, epithelial tumor antigen , human epidermal growth factor receptor, vascular endothelial growth factor, and prostate specific antigen. The antigen can also be from a pathogenic organism, such as bacterium, a parasitic organism and a protozoan. Examples are wherein the pathogenic organism is selected from the group comprising a mycobacterium, yersinia, salmonella, rickettsiae, Cryptosporidium, and leishmaniasis

[044] As used herein, the phrase "inducible/measurable cytokine patterns" refers to a quantitative measurement, in vivo or ex vivo, of cytokines induction in a sample, such as human whole blood using standard methods for measuring cytokine induction, such as flow cytometry which demonstrates

immunostimulatory components of the present vaccine composition invention differing from those the test control (e.g. endotoxin). Such inducible measurable cytokine patterns are within the skill in the art from the description herein.

[045] "Cancer," "neoplasm," "tumor," and "carcinoma" are used

interchangeably herein and refer to cells which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. Cancerous cells can be benign or malignant.

[046] As used herein, "cell which is susceptible to infection by a pathogen" is a cell which can be infected by a pathogen that establishes infection or otherwise causes disease or symptoms of disease in a host by interaction with a chemokine receptor.

[047] "Immunoglobulin class switching" or "isotype switching" refers to a biological mechanism that changes a B cell's production of antibody from one class to another, for example, from an isotype called IgA to an isotype called IgG.

[048] "Cross presentation" is a term well understood in the art, and refers to the ability of certain antigen-presenting cells to take up, process and present extracellular antigens with MHC class I molecules to CD8+ T cells (cytotoxic T cells). "Cross-priming" describes the stimulation of naive cytotoxic CD8+ T cells by this process. This process is necessary for immunity against most tumors and against viruses that do not infect antigen-presenting cells. In cross presentation, endocytosed proteins are transported out of this compartment into the cytoplasm by unknown mechanisms. There they are processed by the proteasome into peptides, which are transported by the TAP transporter into the endoplasmic reticulum or back into the same endosomes, where they associate with MHC class I molecules.

[049] As used herein the term "cytokines" refers to small secreted proteins which mediate and regulate immunity, inflammation, and hematopoiesis. They must be produced de novo in response to an immune stimulus. They generally (although not always) act over short distances and short time spans and at very low concentration. They act by binding to specific membrane receptors, which then signal the cell via second messengers, often tyrosine kinases, to alter its behavior (gene expression). Responses to cytokines include increasing or decreasing expression of membrane proteins (including cytokine receptors), proliferation, and secretion of effector molecules. [050] As used herein, the phrase "cytotoxic T lymphocyte response" or "CTL response" refers to the response of cytotoxic T cell (also known as TC, CTL, T- Killer cell, cytolytic T cell, CD8+ T-cells or killer T cell) that are capable of killing cells that are infected with viruses (or other pathogens), or are otherwise damaged or dysfunctional. Most cytotoxic T cell responses require a T-cell receptors (TCRs) that can recognize a specific antigenic peptide bound to Class I MHC molecules. The affinity between CD8+ and the MHC molecule keeps the T cell and the target cell bound closely together during antigen-specific activation.

[051 ] "Dendritic cell" and "antigen presenting cell" or "APC" are terms well understood in the art and refer to a lymphocyte derived from myeloid or lymphoid origin. Immature dendritic cells contain high intracellular MHC II in the form of MHC class II rich compartments (MIICs), express of CD1 a, actively endocytosis for certain particulates and proteins, contain surface FcgR, actively phagocytose, have low/absent adhesive and costimulatory molecules (CD40/54/58/80/86), have low/absent CD25, CD83, p55, DEC-205, 2A1 antigen, are responsive to GM-CSF, but not M-CSF and G-CSF, and will have their maturation inhibited by IL-10. Dendritic cells are located predominately in mucosal tissues.

[052] As used herein, "functional CD4+-T cell" refers to a CD4+-T cell capable of providing T cell help, directly or indirectly, to affect one or more of the following responses: CTL activation; antibody production; macrophage

activation; mast cell growth; and eosinophil growth and differentiation.

[053] As used herein, "functional CD8+-T cell" refers to a CD8+-T cell capable of direct cell mediated cytotoxicity without prior exposure to an antigen. CD8+ T cells are derived from na^'fve T cells after interaction in the thymus with mature dendritic cells presenting an antigenic fragment via the MHC class I - T cell receptor complex on the antigen presenting cell.

[054] The terms "individual," "host," "subject" or "patient" used herein, refer to any mammalian subject, In one embodiment, humans for whom diagnosis, treatment, or therapy is desired. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on. [055] The terms "increasing," "inducing," and "enhancing," used

interchangeably herein with reference to a CTL response, refer to any increase in a CTL response over background, and include inducing a CTL response over an absence of a measurable CTL response, or increasing CTL response over a previously measurable CTL response.

[056] As used herein, "innate immunity" refers to the innate immune system which comprises the cells and mechanisms that defend the host from infection by other organisms, in a non-specific manner. The cells of the innate system recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host. Innate immune systems provide immediate defense against infection, and are found in all classes of plant and animal life.

[057] As used herein, "innate immune response" refers to a response involving the characteristics of "innate immunity" described above.

[058] As used herein, "major histocompatibility complex I" or "MHC I" refers to a cell surface complex, more specifically a membrane spanning molecule composed of two proteins. The spanning protein is approximately 350 amino acids (mer) in length, with approximately 70 amino acids at the carboxylic end, comprising the transmembrane and cytoplasmic portions. MHC class I molecules are found on every nucleated cell of the body (and thus, not on red blood cells). Their function is to display fragments of proteins from within the cell to T cells, so that healthy cells will be left alone and cells with foreign proteins will be attacked by the immune system. Because MHC class I molecules present peptides derived from cytosolic proteins, the pathway of MHC class I presentation is often called the cytosolic or endogenous pathway.

[059] As used herein "major histocompatibility complex II" or "MHC II" refers to a cell surface complex, composed of two membrane spanning proteins. These proteins are approximately 30 kD in size, and made of two globular domains. MHC Class II molecules are found only on a few specialized cell types, including macrophages, dendritic cells and B cells, all of which are professional antigen- presenting cells (APCs). The peptides presented by class II molecules are derived from extracellular proteins (not cytosolic as in class I); hence, the MHC class ll-dependent pathway of antigen presentation is called the endocytic or exogenous pathway. Loading of class II molecules must occur inside the cell; extracellular proteins are endocytosed, digested in lysosomes, and bound by the class II MHC molecule prior to the molecule's migration to the plasma

membrane.

[060] As used herein, "mucosal" and "mucosa" are used interchangeably herein and refer to immune inductive sites collectively termed the mucosa associated lymphoid tissue (MALT). The MALT is further subdivided into the nasal- or nasopharynx-associated lymphoid tissue (NALT), the bronchus- associated lymphoid tissue (BALT), and the gut-associated lymphoid tissue (GALT). These sites are anatomically separated but functionally connected by the common mucosal immune system, so that induction of an immune response at one site leads to an effector response at a different mucosal site, mediated by homing receptors on induced memory T and B cells.

[061 ] As used herein, "pathogen-associated molecular pattern" or "PAMP" refers to a molecular pattern found in microorganisms. A PAMP is capable of triggering an innate immune response when it binds to a PRR. These

immunostimulatory structures, or derivatives thereof, are potential initiators of innate immune response, and therefore, ligands for PRRs, including toll-like receptors (TLR).

[062] As used herein, "pattern recognition receptor" or "PRR" refers to a member of a family of receptors, which, when binding to a PAMP can stimulate an innate immune response.

[063] As used herein, "peptidase" and "amidase" refer to plasma and cellular enzymes that: a) cleave extracellular peptides; b) cleave intracellular peptides; and c) cleave antigens for MHC Class I presentation. Peptidases cleave carboxyl groups from peptides and amidase cleaves amide groups from peptides. In the use described in the present invention, ACE 1 (CD143) is a dipeptidase, ACE2 is a monopeptidase, Neutral endopeptidase is a tripeptidase named neprilysin, and chymase is enzymatically inactive in normal vascular tissue and may produce angiotensin II only in damaged or atherosclerotic arterial walls when released by mast cells, and aminopeptidase A aminopeptidase M cleave angiotensin III and angiotensin IV, respectively.

[064] A "peptide associated with a pathogenic organism," as used herein, is a peptide (or fragment or analog thereof) that is normally a part of a pathogenic organism, or is produced by a pathogenic organism. Generally, a peptide associated with a pathogenic organism is one that is recognized as foreign by a normal individual with a healthy, intact immune system, e.g., the peptide is displayed together with an MHC Class I molecule on the surface of a cell, where it is recognized by a CD8+ lymphocyte.

[065] "Peptide pool" and "antigen peptide pool," used interchangeably herein, refer to mixtures of similar sized fragments digested from pathogens to sizes between 10-100 mer. Peptide pools can be species, specific or non-specific. They can be mixtures of different peptide sources and can be length optimized as desired. Such optimization is within the skill in the art. Peptide pools are generated and optimized using in vitro cytokine release kinetic modeling.

[066] "Polypeptide," "peptide," and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. These terms include polypeptide chains modified or derivatized in any manner, including, but not limited to, glycosylation, formylation, cyclization, acetylation, phosphorylation, and the like. These terms include naturally-occurring peptides, synthetic peptides, and peptides comprising one or more amino acid analogs. The terms also include fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues, immunologically tagged proteins, and the like.

[067] The terms "preventing," "reducing," and "inhibiting," used

interchangeably herein in the context of pathogen infection, refer to reducing the incidence of pathogen infection of a cell which is susceptible to infection by the pathogen. Reducing pathogen infection refers to reducing any parameter or event which leads to pathogen entry into a cell, including, but not limited to, reducing co-receptor mediated fusion; reducing entry of the pathogen into the cell; reducing binding of the pathogen to a cell-surface chemokine receptor; and reducing binding of the pathogen to a cell-surface CD4 molecule. The terms also refer to reducing susceptibility of a cell to infection by a pathogen. The terms also refer to reducing any undesired effect of binding of a pathogen to the cell.

[068] As used herein, "T cell receptor" or "TCR" refer to a molecule found on the surface of T lymphocytes (or T cells) that is, in general, responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. The TCR is a heterodimer consisting of an alpha and beta chain in 95% of T cells, whereas 5% of T cells have TCRs consisting of gamma and delta chains. Engagement of the TCR with antigen and MHC results in activation of its T lymphocyte through a series of biochemical events mediated by associated enzymes, co-receptors, specialized accessory molecules and activated or released transcription factors.

[069] As used herein, "Th1 " and "Th1 cytokines" refer to immune cells classified on the pattern of cytokines that they secrete and the immune function they exhibit. The Th1 cell secretes mainly IL-2, IL-3, IL-12, INF-γ, TNF-a, TNF-β, TGF-β, and GM-CSF. Th1 cells direct the clonal expansion of activate

macrophages, NK cells, and CD8+ cells, MHC Class I antigen presentation, dendritic cell mobilization and maturation, phagocytosis, plus the secretion of certain Ig isotypes.

[070] As used herein, "Th2" and "Th2 cytokines" refer to immune cells classified on the pattern of cytokines that they secrete and the immune function they exhibit. The Th1 cell secretes mainly IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, and G-CSF, TGF-β, and GM-CSF. Th2 cells direct the clonal expansion of B cells, production on antigen-specific antibodies, down-regulate innate immunity, and favor isotype switching in the humoral immune response. In addition, Th2 cytokines depress macrophage activation and cell mediated immunity. [071 ] As used herein, "toll-like receptor" or "TLR" refers to a class of proteins that play a key role in the innate immune system. They are single, membrane- spanning, non-catalytic receptors that recognize structurally conserved

molecules derived from microbes. TLRs recognize microbes that have breached physical barriers such as the skin or intestinal tract mucosa, and activate immune cell responses.

[072] TLRs are a type of pattern recognition receptor (PRR) and recognize molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs). TLRs together with the lnterleukin-1 receptors form a receptor superfamily, known as the "lnterleukin-1 Receptor/Toll-Like Receptor

Superfamily"; all members of this family have in common a so-called TIR (Toll-IL- 1 receptor) domain.

[073] TLRs are present in both vertebrates and invertebrates. Molecular building blocks of the TLRs are represented in bacteria and plants; and in the latter kingdom are well known to be required for host defense against infection. The TLRs thus appear to be one of the most ancient, conserved components of the immune system.

[074] As used herein, the terms "treatment" and "treating", and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly humans, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease, but has not yet been diagnosed as having it; (b) inhibiting the disease (e.g., arresting its development); and (c) relieving the disease (e.g., causing regression of the disease; or completely or partially removing symptoms of the disease).

[075] "Tumor-associated antigen" refers to surface molecules that are differentially expressed in tumor cells relative to noncancerous cells of the same cell type. As used herein, "tumor-associated antigen" includes not only complete tumor-associated antigens, but also epitope-comprising portions (fragments) thereof. A tumor-associated antigen (TAA) may be one found in nature, or may be a synthetic version of a TAA found in nature, or may be a variant of a naturally-occurring TAA, e.g., a variant which has enhanced immunogenic properties.

[076] "Vaccine" refers to a composition comprising an antigen, and optionally other ancillary molecules, the purpose of which is to administer such

compositions to a subject to stimulate an immune response specifically against the antigen and to engender immunological memory that leads to mounting of an immune response should the subject encounter that antigen at some future time. Examples of other ancillary molecules are adjuvants, which are non-specific immunostimulatory, molecules, and other molecules that improve the

pharmacokinetic and/or pharmacodynamic properties of the antigen.

Conventionally, a vaccine usually consists of the organism that causes a disease (suitably attenuated or killed) or some part of the pathogenic organism as the antigen. Attenuated organisms, such as attenuated viruses or attenuated bacteria, are manipulated so that they lose some or all of their ability to grow in their natural host. There is now a range of biotechnological approaches used for producing vaccines (see, e.g., W. Bains (1998) Biotechnology From A to Z, Second Edition, Oxford University Press).

Embodiments

Methods and Compositions for Generating a CTL Response to an Antigen Associated with Disease in a Mammal by a Thi Dominate Immune

Mechanism

[077] T lymphocytes capable of antigen recognition are generally classified as "CD4+" or "CD8+," depending on whether a CD4 or a CD8 molecule is displayed on the cell surface. In general, CD4+ T cells provide signals to activate other cells, e.g., CD4+ cells activate CD8+ cells, to induce B cell to produce

antibodies, or to activate macrophages. In contrast, CD8+ cells are cytotoxic, and recognize antigens displayed with MHC Class I molecules on the cell surface.

[078] Upon activation by IL-2, na^'fve Th cells become Th₀, since they have both Thi and Th₂ characteristics. Th₀ cells undergo clonal expansion towards Thi following the release of INF-γ and TNF-a by Th₀ cells or towards Th₂ following the release of IL-4. Thi cells secrete IL-2, 11-12, INF-γ, and down regulated Th2 cells with additional IL-2. Th₂ cells secrete IL-3, IL-4, IL-5, IL-6, and down regulated Th1 cells with additional IL-4. While the categorization of T cells as Thi , Th₂, or Th₀ is helpful in describing the differences in immune response, it should be understood that it is more accurate to view the T cells and the responses they mediate as forming a continuum, with T n and Th₂ cells at opposite ends of the scale, and Th₀ cells providing the middle of the spectrum.

[079] Therefore, it should be understood that the use of these terms herein is only to describe the predominant nature of the immune response elicited, and is not meant to be limiting to an immune response that is only of the type indicated. Thus, for example, reference to a "type-l" or "Th1 " immune response does not exclude the presence of a "type-2" or "Th2" immune response, and vice versa.

[080] CD8+ T cells represent important mediators in defense against many infectious agents. Priming of CD8+ T cell responses requires that these cells initially recognize pathogen-derived antigenic peptides on the surface of professional antigen-presenting cells (APCs). Since infections can be restricted to non-APCs (for example, certain viruses selectively infect epithelial cells), priming of CD8+ T cell responses is a logistical challenge for the immune system. When a cell is infected with a microbe or has been transformed into a tumor, the peptide-MHC class I repertoire will now include non-self or new peptides derived from the infection or the neoplastic process. In most cell types, peptides presented by MHC-I are derived from endogenously synthesized proteins. In professional antigen presenting cells (APCs), such as dendritic cells (DCs), macrophages, and B lymphocytes, evidence exists for an additional MHC-I restricted pathway presenting peptides from extracellular origin. Antigen- presenting cells capable of cross-presentation are primarily dendritic cells, but macrophages, B lymphocytes and liver sinusoidal endothelial cells have also been shown to be able to do so.

[081 ] Two mechanisms have been described by which this cross-presentation can occur. Firstly, antigens internalized by phagocytosis or pinocytosis have been shown to intersect the ER - MHC class I loading pathway after

phagosomes or pinosomes contact ER-like compartments. Similarly to direct presentation, this process is TAP dependent. While this is thought to be the main pathway for cross-presentation, a second, TAP-independent mechanism has also been reported. In this, cross presentation occurs when peptides from the acquired antigen are loaded into recycling MHC class I molecules in endosomes.

[082] To cope with invaders that do not infect APCs directly, a strategy called cross-priming, in which APCs acquire antigen for presentation by capturing infected cells, is employed. Effective stimulation of T cell responses by cross- priming necessitates that APCs not only internalize exogenous antigens and present these to CD8+ T cells, but also that the APCs receive signals that activate (or license) them to induce a productive immune response.

[083] Significant evidence supports the view that dendritic cells (DCs) are the crucial APCs for the initiation of T cell responses, including the induction of cross-priming. However, it is also clear that DCs are heterogeneous and play multiple roles in the immune system. DC function is integrally linked with the ability of these cells to alter their properties in response to environmental signals. In general, DCs are considered to convert from an immature state, in which they are poor stimulators of immune responses, to a mature state, where they exhibit potent T cell stimulatory ability, upon detection of infection or other signs of potential danger to the host. Depending on the specific maturation signals they receive, DCs can drive different types of immune responses. In addition to such functional plasticity, it is evident that distinct subsets of DCs exist. Accordingly, heterogeneity amongst DCs is associated with variation in their ability to induce cross-priming.

[084] Aside from presentation of antigen and expression of appropriate levels of co-stimulatory molecules, a key requisite for DCs to stimulate a functional T cell response is their proper positioning within tissues. DCs must gain access to the T cell zones of secondary lymphoid organs in order to interact with na^'fve T cells. Although some immature DCs are located in lymph nodes and spleen under steady state conditions, these cells are also found in large numbers in peripheral tissues at potential sites of pathogen entry, where they serve a sentinel function. Movement of DCs to lymphoid organs is dependent on upregulation of the chemokine receptor CCR7, which renders DCs sensitive to CCL19 and CCL21 ; these chemokines control DC exit from peripheral tissues and direct their migration towards the T cell area of lymphoid organs. Induction of CCR7 expression is commonly associated with DC maturation, linking the receipt of infectious or danger signals with an ability to move to locations where it is possible to initiate an immune response.

[085] Dendritic cells are a major source of many cytokines, namely, interferon- alpha (IFN-a), IL-1 , IL-6, IL-7, IL-12 and IL-15 and also produce macrophage inflammatory protein (MIP-1 y), all of which are important in the elicitation of a primary immune response. Also, there is evidence that the cytokine secretion pattern of the plastic-adherent monocyte-derived DCs (grown in GM-CSF and IL- 4) can be induced along the Thi (IL-12) or Th₂ (IL-10) cytokine secretory pathway, lnterleukin-12 production is critical for the promotion of an effective cellular immune response by activating and differentiating T lymphocyte to the Th1 pathway. Its secretion appears to be inhibited by various tumor-derived substances, including nitric oxide (NO), prostaglandin E2 (PGE2), IL-10, IFN-a itself, the p40 homodimer of IL-12, and transforming growth factor b (TGF-β), which is regarded predominantly as an immunosuppressive cytokine.

[086] In humans, the IFN-γ protein is encoded by the IFNG gene. IFN-γ is the hallmark cytokine of Thi cells (whereas Th₂ cells produce IL-4 and Thi₇ cells produce IL-17). NK cells and CD8+ cytotoxic T cells also produce IFN-γ. IFN-y suppresses osteoclast formation by rapidly degrading the RANK adaptor protein TRAF6 in the RANK-RANKL signaling pathway, which otherwise stimulates the production of NFKB.

[087] IFN-γ is a cytokine critical for innate and adaptive immunity against viral and intracellular bacterial infections and for tumor control. Aberrant IFN-y expression is associated with a number of auto inflammatory and autoimmune diseases. The importance of IFN-γ in the immune system stems in part from its ability to inhibit viral replication directly, but most importantly derives from its immunostimulatory and immunomodulatory effects. IFN-γ is produced

predominantly by natural killer (NK) and natural killer T (NKT) cells as part of the innate immune response, and by CD4 and CD8 cytotoxic T lymphocyte (CTL) effector T cells once antigen-specific immunity develops.

[088] IFN-γ has antiviral, immunoregulatory, and anti-tumor properties.

Amongst the effects are: 1 ) increase antigen presentation of macrophages, 2) activate and increase lysosome activity in macrophages, 3) suppress Th₂ cell activity, 4) cause normal cells to increase expression of class I MHC molecules, 5) promotes adhesion and binding required for leukocyte migration, 6) promotes NK cell activity, and 7) activates APCs and promotes Thi differentiation by upregulating the transcription factor TGF-β.

[089] The renin-angiotensin system (RAS) is responsible for the homeostasis of arterial blood pressure and water and electrolyte balance. One of the best- known components of this system is the biologically active peptide angiotensin II (Ang II). Angiotensin II is a major regulator of fluid and sodium balance and hemodynamics, but also of cellular growth and cardiovascular remodeling. Other metabolic actions of Ang II include pro-inflammatory modulation, increased insulin secretion, B-cell apoptosis, reduction of gluconeogenesis and hepatic glucose output and increased plasma triglycerides.

[090] The key biochemical steps involved in the generation of angiotensin peptides involve the biotransformation of angiotensinogen I by renin into the decapeptide angiotensin I (Ang I). This initiates the cascade where the prohormone is cleaved into the octapeptide Ang II primarily by angiotensin converting enzyme (ACE1 ), the nonapeptide Ang-(1-9) by angiotensin- converting enzyme 2 (ACE2), to A (1 -7) by the a number of endopeptidases including neprilysin, prolyl-oligopeptidase, and thimet endopeptidase.

[091 ] Ang II and Ang-(1-7) can further be converted into Ang-(1-7) and Ang- (1-5) by the actions of ACE2 and ACE1 respectively, to counteract the activity of Ang II. Additionally, Ang III and Ang IV may be generated from Ang II by a number of aminopeptidases as an additional counter-regulatory pathway for the effects of Ang II.

[092] It is known that Ang-(1 -7) often counteracts the effects caused by Ang II, making it an excellent target for experimental and pharmacological research. A (1 -7) potentiates the hypotensive effects of bradykinin, and increases the release of prostacyclin and nitric oxide directly or indirectly, thereby elicits vasodilator, antiproliferative, natriuretic, diuretic actions and improves cardiac function. The structural activity associated with the loss in AT1 mediated agonist effect is considered the removal of the terminal carboxyl phenylalanine at position 8 of Ang II. A (1 -7) autoregulates the RAS dynamics and restores the physiological homeostasis systemically and locally.

[093] The activities of the numerous angiotensin peptides are mediated through a number of G-coupled protein receptors. These receptors are

indentified as angiotensin type 1 receptor (AT1 ), the angiotensin type 2 receptor (AT2), the angiotensin type 4 receptor (AT4), and the angiotensin type 7 receptor (AT7) which is identified as a pro-oncogene Mas receptor.

[094] Ang II exerts its primary actions via AT1 and dynamically modulates its function with feedback-control via the AT2 and AT7 receptors and through a shift in production from Ang II to the counter-regulatory peptide A (1 -7); which in principle, but not invariably, mediate opposite functions. In contrast to Ang II, A (1 -7) exerts its primary activities via the AT7 or Mas receptor. Angiotensin peptides are capable of binding to all the specific angiotensin receptors. During this process, the specific binding can either transduce an activating signal or compete with the primary receptor signaling pathway.

[095] This CTL response is further amplified when dendritic cells and other professional antigen presenting cells (APCs) generate ACE1 , ACE2, increase the expressions of AT1 , AT2, and AT7 as well as release Ang II when

encountering "non-self peptides presented in these MHC Class I complexes. The effect of Ang II in this enhanced immune activation results in increase T cell, DC cell, and NK cell migration to the local area, enhancement in endocytosis and allostimulatory activation of na^'fve T cells. A further activity provides for an increase in the synthesis and release of Th1 cytokines such as INF-γ, TNF-a, TGF-β, IL-12, IL-2, and IL-8. Also observed is a shift in the ration of Th1/Th2 cells and enhanced proliferation of dendritic cells and activation in cellular phosphorylation of MAPK such as p38, JNK, and ERK 1/2.

[096] A significant unexpected observation in this cascade is that the administration of angiotensin type 1 receptor antagonists and ACE1 inhibitors results in deactivation or attenuation of the immune events. This effect is demonstrated to be independent of AT2 or AT7 receptor blockade. The maturation of dendritic cells while activated by Ang II, is only slightly affected by AT1 blockade and appears to be regulated predominantly by AT2 activation by Ang II binding to that receptor.

[097] While Ang II has been demonstrated to induce cross presentation, T cell, DC cell, and NK cell function and mobilization, the effect on Ang II has been unexpectedly shown to be independent of binding to AT1 or AT2 and dependant on AT7 binding. However, all of the putative effects of angiotensin on immune cells are only observed if a cell is infected with a microbe or has been

transformed into a tumor and the peptides have no effect on quiescent immune cells.

[098] The findings of the present invention are unexpected to one skilled in the art since Ang (1 -7) effects are counter intuitive with angiotensin blocking agents. That is, it is not obvious to one skilled in the art that the activation of innate immune functions observed with Ang II might be mimicked by the counter- regulatory angiotensin peptides which activate non-AT1 receptors, the primary Ang II receptor.

[099] The present invention makes use of this novel observation allowing for enhanced immune function modulated through a non-AT1 receptor and with a compound devoid of hypertensive properties. This finding allows one practicing the art to administer to a mucosal tissue a peptide adjuvant in combination with an antigen, or immunogenic active portion of an antigen, that infects APCs or DCs and is processed in the cytosol and presented by MHC Class I - TCR complex. The invention teaches that the antigen may be endocytosed and cross- presented by an antigen presenting cell via the MHC Class I complex.

Additionally, this activity directs the release of Th1 cytokines, mobilization and maturation of dendritic cells, stimulation of na^'fve T cells, and up-regulation of cell mediated immunity. The activation of acquired immunity following an innate immune signal further provides neutralizing antigen-specific immunoglobulins and activation of immunoglobulin class-switching of antibody production.

[0100] The present invention provides for exogenous administration as well as endogenous activation of the numerous fragments that are cleaved within the RAS pathway. The vasoactive angiotensin II is not suitable for a peptide adjuvant due to the potent sequelae attributed to its structure.

[0101 ] In one embodiment of the present invention, it is taught that

conservation of amino acid positions 3-4-5 (Val-Tyr-lle) is required to practice the invention. This minimum un-substitutable sequence is necessary for peptide adjuvant binding and immunologic activity.

[0102] In another embodiment, the peptide adjuvants are Asp-Arg-Val-Tyr-lle- His-Pro (Ang-(1 -7)), Asp-Arg-Val-Tyr-lle (Ang-(1 -5)), Arg-Val-Tyr-lle-His-Pro (Ang 2-7), Arg-Val-Tyr-lle (Ang 2-5), Val-Tyr-lle-His-Pro-Phe (Ang 3-8), Val-Tyr- lle-His-Pro (Ang 3-7), Val-Tyr-lle-His (Ang 3-6) ; and, Val-Tyr-lle (Ang 3-5).

[0103] In yet another embodiment, the peptide adjuvants in the composition are Asp-Arg-\ a/-Tyr-//e-His-Pro-Phe-His-Leu (Ang 1 -10), Asp-Arg-Val-Tyr-lle- His-Pro-Phe-His (Ang 1 -9) , Asp-Arg-\ a/-7yr-//e-His (Ang 1 -6), Arg-V al-Tyr-lle- His-Pro-Phe-His-Leu (Ang 2-10), Arg-\ a/-7yr-//e-His-Pro-Phe-His (Ang 2-9), Arg- \ a/-7yr-//e-His-Pro-Phe (Ang 2-8), Arg-Val-Tyr-lle His (Ang 2-6), \/a/-7yr-//e-His- Pro-Phe-His-Leu (Ang 3-10), \/a/-7yr-//e-His-Pro-Phe-His (Ang 3-9).

[0104] In one embodiment, the dosage of peptide adjuvant consisting of Asp- Arg-Val-Tyr-lle-His-Pro (Ang-(1 -7)) should be greater than 1 mcg/kg/dose and less than 100 mcg/kg/dose.

[0105] The dosage and frequency of administration of this present invention can be varied over wide limits. The amount of the peptide adjuvant administered depends upon absorption, distribution, and clearance by the host. The dosage of the peptide adjuvant should be sufficient to produce a Th1 immune response and maintain antigen-specific cell mediated cytotoxicity over extended periods. The frequency of chronic administration can be determined with a minimum of experimentation using conventional dose-response analytical techniques or by scaling up from studies based on animal models of disease. In addition, the use of in vitro cytotoxicity assays can be helpful in optimizing the treatment regimen.

[0106] Due to the basic biochemistry associated with generation suitable peptide adjuvants, one skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the compounds and methods of use thereof described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims herein.

[0107] The present invention provides for compositions and methods for directing antigen-specific CTL responses to the pathologic antigen. This aspect of the invention is based on the unexpected discovery that certain angiotensin- related peptides, termed immunostimulatory adjuvants, can activate innate immunity, increase dendritic cell activation, enhance cytokinesis and

presentation of antigen by MHC class I TCR complexes, direct autocrine and paracrine T n cytokine/chemokine synthesis, and eventually the up-regulation of acquired immunity and potential for immunoglobulin class-switching.

[0108] The invention further provides for compositions and methods for induction of a CTL response to an antigen via classic MHC class I presentation by an infectious antigen or by cross-presentation by a non-infectious antigen. In addition, the invention provides compositions and methods for activation and maintenance of an antigen specific cytotoxic T lymphocyte response in an individual; methods for decreasing the number of infectious pathogens in an individual; methods for decreasing tumor load in an individual; and methods of treating an infectious disease in an individual.

[0109] According to the present invention, an "effective amount" of a vaccine composition is one that is sufficient to achieve a desired biological effect. It is recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of an antigen and peptide adjuvant composition of the invention to achieve the desired biologic effect will be determined based on a number of variables. The dosage may be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue

experimentation.

[01 10] In the present invention, the composition provides that the peptide adjuvant directs a Th1 dominate CTL and is characterized by increased interferon-γ. Th1 immune responses direct the clonal expansion of activated macrophages, NK cells, and CD8+ cells, promote MHC Class I antigen presentation, dendritic cell mobilization and maturation, phagocytosis, plus the secretion of certain non-specific Ig isotypes. Persons skilled in the art will readily appreciate from a reading of the present disclosure that a Th1 immune response results in other Th1 cytokines including, but not limited to, IL-2, IL-3, IL-12, INF-γ, TNF-a, TNF-β, TGF-β, and GM-CSF.

[01 1 1 ] Th1 cytokines maximize the killing efficacy of the macrophages and the proliferation of cytotoxic CD8+ T cells. The Type 1 cytokine IFN-γ increases the production of interleukin-12 by dendritic cells and macrophages, and via positive feedback, IL-12 stimulates the production of IFN-γ in helper T cells, thereby promoting the Th1 profile.

Antigens

[01 12] In one embodiment, the antigen or an immunogenic active portion of an antigen of this present invention can be derived from pathogenic virus, bacterium, parasite, or protozoan comprising a whole, an attenuated, an inactivated, or a fragment of a pathogen. Alternatively, the antigen, or

immunogenic active portion of the antigen may be derived from tumor associated antigen (e.g., alphafetoprotein, carcinoembryonic antigen, CA-125, epithelial tumor antigen, human epidermal growth factor receptor, vascular endothelial growth factor, and prostate specific antigen). One skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the compounds and methods of use thereof described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims herein.

Antigen Response

[01 13] Determination of an effective response varies for specific antigens. In brief, these methods include determining in vitro or in vivo measurement from pre-treatment levels of: a) an effective cell mediated cytotoxic response of infected cells; further defined as a > 20% lysis in less than 24 hours; b) inhibition and continued down-regulation of cellular proliferation as measured by H3- thymidine incorporation in a culture of the infected cells; c) enhanced mixed lymphocyte reaction of > 20% for the specific antigen; d) induction and continued presence of a T cell specific cytokine from baseline > 2 fold; e) enhancement and continued presence of local or systemic anti-directed IgA and IgG; or f) protection from supra-infection.

Antigen Administration

[01 14] The immunostimulatory antigen in combination with a peptide adjuvant may be administered before, simultaneously with, or after the subject is exposed to antigen. Administration of the composition to the mammal may occur via a parenteral or non-parenteral route, e.g., orally, nasally, rectally, intravaginally, intravenously, subcutaneously, dermally, or peritoneally, and by inhalation. The frequency or interval for repeated administration is desirable to maintain CTL responses. The appropriate interval for administration of the composition is dependant on the antigen and route selected.

[01 15] In more detailed aspects, the immunogenic compositions of the invention may include a formulation that is pharmaceutically elegant and delivered as a tablet, capsule, troche, emulsion, suspension, inhaler, non- parenteral solution, parenteral solution or lyophylate, enema, suppository, or gel preparation, wherein the soluble antigen is admixed with a homogenous emulsion or gel carrier, e.g., a polyoxyethylene gel. Alternatively, the soluble antigen may be admixed with compatible foam. [01 16] In other aspects, the immunogenic compositions of the invention are formulated comprising a base or carrier specifically adapted for the delivery of the antigen and adjuvant. The mucosal solid composition should comprise at least two base materials to optimize structural and delivery performance. In other aspects, the solid composition includes a stabilizing agent to minimize

degradation of the soluble antigen.

[01 17] To optimize delivery, the immunogenic compositions of the invention also include an absorption-promoting agent, for example a microcapsule, nanoparticle, glycolic acid, albumin, non-immunogenic protein, surfactant, mixed micelle, enamine, nitric oxide donor, sodium salicylate, glycerol ester of acetoacetic acid, cyclodextrin or beta-cyclodextrin derivative, or medium-chain fatty acid.

Antigen

[01 18] The present invention further provides that the antigen, or immunogenic active portion of an antigen can be added to the composition as a whole live pathogen, an attenuated pathogen, an inactivated pathogen, a protein or peptide fragment from the pathogen, a virus like particle, a tumor associated antigen, a tumor associated cell surface receptor, protein or peptide fragments of the tumor antigen, recombinant peptides or proteins of the "wild-type" antigen or antigen fragment, and unvectored nuclear materials such as DNA, RNA, or antisense. One skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the compounds and methods of use thereof described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

[01 19] Generally, a peptide associated with a pathogenic organism is one that is recognized as foreign by an individual and is displayed together with an MHC Class I molecule - TCR complex on the surface of an APC or atypical cell. In this presented fashion, the antigen fragment is recognized by a cytotoxic T cell such as a CD8+ lymphocyte. 1. Attenuated Pathogen

[0120] In one embodiment, the antigen is an attenuated pathogen. The pathogen can be expressed or grown using contemporary cell culture methods. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, appropriate techniques for growing pathogens in a culture or co-culture system in vitro.

[0121 ] Furthermore, the present invention provides methods for attenuation or inactivation of the pathogen which can be achieved (i) by introducing selected mutations or deletions in the genome, and/or (ii) by chemical or pharmacological treatment of the pathogen. Any modification of the genome that attenuates or inactivates the pathogen is determined by testing infectivity a cell culture, where, using conventional techniques, the level of infectious pathogen present in the culture supernatant is essentially reduced or eliminated compared to a wild type of pathogen.

[0122] Additionally, methods are provided for administering an attenuated pathogen and peptide adjuvant to a mucosal site. The composition can be formulated as an inhalation in a buffered saline containing a bacteriostatic agent. The solution is delivered by a positive pressure assisted inhaler. Further mycobacterium tuberculosis is attenuated by heat in phenol preservative.

[0123] The effective amount is sufficient to induce antigen specific CTL response against the bacteria in a host to which the effective amount of the composition is administered. Thus, both protective and therapeutic immunity may be imparted by the methods of the invention to an individual as evidenced by the absence of clinical indications of disease, or as evidenced by absence of, or reduction in, determinants of pathogenicity, including the absence or reduction in persistence of the infection and/or the absence of pathogenesis and clinical disease, or diminished severity thereof, as compared to individuals not treated by the method of the invention.

[0124] In another embodiment, it is possible to treat an established HIV infection in an individual. In brief, the present invention shows that the anti-HIV-1 specific CTL response can be effectively activated without virus replication. The HIV-1 virus can be attenuated from the complete viral envelope glycoprotein and can contain anyone or more of the gag, pol, and env sequences. In this embodiment, the virus is inactivated by chemical denaturing with gluteraldehyde. The inactive virus and peptide adjuvant are suspended in a rectal suppository covered with a mucoadhesive agent such as methylcellulose.

[0125] Examples of enveloped viruses that can be employed in the invention are retroviruses, such as FeLV, FIV, HIV-I, HIV-2, SIV, MuLV, and GLV; herpes viruses, such as EBV, HSV, CMV, BHV-I, BHV-4, and pseudorabies virus; and paramyxovirus, such as Sendai virus, Newcastle disease virus, human parainfluenza 2 and 3, and mumps viruses. Other viruses can be human papillomavirus, influenza A virus subtypes H1 N1 , H1 N2, H3N1 , H3N2, and H2N3, and a rotavirus.

[0126] The dosage and frequency of administration can be varied over wide limits. The amount of the viral particles administered depends upon absorption, distribution, and clearance by the host. The dosage of the viral particles should be sufficient to produce and maintain antigen-specific cell mediated cytotoxicity over extended periods as well as provide for a reduction in viral plasma load within an acceptable interval. The frequency of chronic administration by rectal suppository can be determined with a minimum of experimentation using conventional dose-response analytical techniques or by scaling up from studies based on animal models of disease. In addition, the use of in vitro cytotoxicity assays can be helpful in optimizing the treatment regimen.

2. Viral Particles

[0127] This invention utilizes a "viral particle" that comprises a viral core and a viral envelope or another surface protein. The core can include the viral structural and immunogenic proteins. For retroviruses, the core can be

composed of gag and pol gene products. The viral envelope glycoprotein, or another surface protein, can be a component of the viral membrane, which allows viral binding and entry into target cells, in the case of this invention, professional antigen presenting cells (APCs). The viral surface protein can be endogenous or exogenous to the wild type virus and is selected according to its ability to bind and to fuse with the membrane of APCs. In the case of

retroviruses, the retroviral env gene product itself is an excellent candidate for mediating viral entry, since it also acts as an immunogenic component of the viral particle.

[0128] The viral particle is employed in the method of the invention in an effective amount sufficient to provide an adequate concentration of the drug to prevent or inhibit infection of the host in vivo or to prevent or at least inhibit the spread of the virus in vivo. Thus, the effective amount can be easily determined from the literature relating to the virus of interest.

[0129] The effective amount is sufficient to induce protective immunity against the virus in a host to which the effective amount of the viral particle is

administered. Thus, the protective immunity imparted by the method of the invention imparts, to an individual, protection from disease, particularly infectious disease associated with viral infection, as evidenced by the absence of clinical indications of disease, or as evidenced by absence of, or reduction in,

determinants of pathogenicity, including the absence or reduction in persistence of the infectious virus in vivo, and/or the absence of pathogenesis and clinical disease, or diminished severity thereof, as compared to individuals not treated by the method of the invention.

[0130] It will be understood that the viral particles can be used in combination with other prophylactic or therapeutic substances. For example, mixtures of different viral particles can be employed in the method of the invention. Similarly, mixtures of viral particles can be employed in the same composition. The viral particles can also be combined with other vaccinating agents for the

corresponding disease, such as microbial immunodominant, immunopathological and immunoprotective epitope-based vaccines or inactivated attenuated, or subunit vaccines.

[0131 ] The viral particle can be used in therapy in the form of pills, tablets, lozenges, troches, capsules, suppositories, injectable in ingestible solutions, and the like in the treatment of cytopathic and pathological conditions in humans and susceptible non-human primates and other animals. Specifically, the host or patient can be an animal susceptible to infection by the virus, and is a mammal. The mammal is selected from the group consisting of a rodent, especially a mouse, a dog, a cat, a bovine, a pig, and a horse. In one embodiment, the mammal is a human.

[0132] Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium carbonate, magnesium stearate, sodium stearate, glycerol monstearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained- release formulations and the like. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. The pharmaceutical compositions contain an effective

therapeutic amount of the viral particle together with a peptide adjuvant suitable amount of carrier so as to provide the form for proper administration to the host.

3. Cancer

[0133] Cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal cells. Cancer is caused by both external factors (tobacco, chemicals, radiation, and infectious organisms) and internal factors (inherited mutations, hormones, immune conditions, and mutations that occur from metabolism). Cancer is treated with surgery, radiation, chemotherapy, hormone therapy, biological therapy, and targeted therapy.

[0134] Mutations of normal cells occur on a daily basis. In most cases, the cell mediate immune process (cytotoxic T cells) recognize and destroy the abnormal cells through and interaction between CTL and non-self expressed peptides presented by the MHC Class I complex on the abnormal cell. While the exact mechanism is not fully known, the development of mutations in normal cellular tissue remains undetected by the CTL. This results in uncontrolled cellular growth of abnormal cells, the release of vascular growth and development factors by the cancer to support the rapid growth, and infiltration of normal tissue by malignant cells. [0135] The present invention provides methods and compositions for the treatment of cancer and defects in cell mediate immune surveillance.

Accordingly, the antigens contemplated in the invention can include cell surface receptors such as human epidermal growth factor receptor, vascular endothelial growth factor, and prostate specific antigen. Further contemplated are shed cell surface antigens such as alphafetoprotein, carcinoembryonic antigen, CA-125, epithelial tumor antigen , human epidermal growth factor receptor, vascular endothelial growth factor , prostate specific antigen, and the like. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the compounds and methods of use thereof described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims herein.

[0136] The present invention provides for methods for inducing CTL activation using exogenous tumor associated antigen (TAA) and a peptide adjuvant which promotes a Th1 immune response, clonal expansion of CTL, cross presentation of the exogenous antigen through MHC Class I processing, activation of na^'fve T cells, activation of tumor specific cell mediated cytotoxicity, induction of helper-T cells and acquired immune responses leading to the generation of anti-idiotypic immunoglobulins, class switching of Ig synthesis, and potentiation of antibody dependant cell mediated cytotoxicity.

[0137] In one embodiment of the present invention, activation of tumor immune surveillance is achieved by a composition containing EGFR peptide fragments from 8 to 20 amino acids in length, digested from the intact recombinant protein. The digestion is performed with a mixture of proteases, amidases, and peptidases. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, appropriate techniques for digesting recombinant proteins into optimal length peptide fragments capable of being processed by APCs and presented with MHC Class I complexes.

[0138] The TAA peptide fragments employed in this method of the present invention are administered in a microencapsulated carrier as taught by D'Souza US Patent 7,105,158 included herein by reference. The microcapsules are suspended in pharmaceutically elegant parenteral formulation in an effective amount sufficient to provide an adequate exposure to the antigen to directly kill and reduce or at least inhibit malignant cell growth in an individual.

[0139] In another embodiment, the composition of the present invention is administered intranasally in a physiologically buffered solution of

carboxymethylcellulose. In yet another embodiment, the composition is administered in an oral liposome.

[0140] The dosage and frequency of administration can be varied over wide limits. The amount of the TAA peptide administered depends upon absorption, distribution, and clearance by the host. The quantity of antigen and adjuvant should be sufficient to produce and maintain antigen-specific cell mediated cytotoxicity over extended periods as well as provide for a reduction in tumor burden or inhibition of tumor growth. The frequency of chronic administration can be determined with a minimum of experimentation using conventional dose- response analytical techniques or by scaling up from studies based on animal models of disease. In addition, the use of in vitro cytotoxicity assays can be helpful in optimizing the treatment regimen.

[0141 ] The methods are effective to reduce a tumor load by at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 75%, at least about 85%, or at least about 90%, up to total eradication of the tumor, when compared to a suitable control. Thus, in these embodiments, an "effective amount" of an immunostimulatory antigen in combination with a peptide adjuvant is an amount sufficient to reduce a tumor load by at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 75%, at least about 85%, or at least about 90%, up to total eradication of the tumor, when compared to a suitable control. In an experimental animal system, a suitable control may be a

genetically identical animal not treated with the immunostimulatory antigen. In non-experimental systems, a suitable control may be the tumor load present before administering the immunostimulatory antigen. Other suitable controls may be a placebo control. [0142] Whether a tumor load has been decreased can be determined using any known method, including, but not limited to, measuring solid tumor mass; counting the number of tumor cells using cytological assays; fluorescence- activated cell sorting (e.g., using antibody specific for a tumor associated antigen); computed tomography scanning, magnetic resonance imaging, and/or x-ray imaging of the tumor to estimate and/or monitor tumor size; measuring the amount of tumor-associated antigen in a biological sample, e.g., blood; and the like.

Methods and Compositions for Administering Vaccines to Mucosal Cells

[0143] The invention provides methods for induction of a mucosal CTL response to any exogenous soluble antigen via a process of cross-presentation. The methods generally involve administering to mucosa in an individual an immunostimulatory antigen in combination with a peptide adjuvant (either alone or in combination with one or more antigens) in an amount effective to increase an antigen-specific CTL response in the individual and/or to decrease a tumor load in an individual and/or to prevent and/or reduce an infectious disease in an individual.

[0144] Mucosal immune inductive sites are collectively termed the mucosa- associated lymphoid tissue (MALT). The MALT is further subdivided into the nasal- or nasopharynx-associated lymphoid tissue (NALT), the bronchus- associated lymphoid tissue (BALT), and the gut-associated lymphoid tissue (GALT). These sites are anatomically separated but functionally connected by the common mucosal immune system, so that induction of an immune response at one site leads to an effector response at a different mucosal site, mediated by homing receptors on induced memory T and B cells. Strangely enough, the MALT is not all inclusive, and the female reproductive tract is not associated with the MALT. However, DCs, which take up antigens from the lumen, can home to an associated draining lymph node, in the case of the female genital tract, the iliac lymph node. For vaccine design, the choice of mucosal inductive site is critical in determining the distal effector site to which induced memory cells will home. The magnitude of the response is also dependent to some extent on the type of immunogen, adjuvant, and delivery method. [0145] Conventional and pharmaceutically acceptable routes of administration include intranasal, , intratracheal, intratumoral, subcutaneous, intradermal, topical application, rectal, nasal, oral and other parenteral routes of

administration. Routes of administration may be combined, if desired, or adjusted depending upon the immunostimulatory nucleic acid and/or the desired effect on the immune response. The immunostimulatory nucleic acid composition can be administered in a single dose or in multiple doses, and may encompass administration of booster doses, to elicit and/or maintain the desired effect on the immune response.

[0146] Immunostimulatory antigens can be administered to a host using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated by the invention include, but are not necessarily limited to, enteral, parenteral, or inhalational routes.

[0147] Inhalational routes may be used in cases of pulmonary involvement, particularly in view of the activity of certain immunostimulatory antigens as a mucosal adjuvant. Inhalational routes of administration (e.g., intranasal, intrapulmonary, and the like) are particularly useful in stimulating an immune response for prevention or treatment of infections of the respiratory tract. Such means include inhalation of aerosol suspensions or insufflation of the

polynucleotide compositions of the invention. Nebulizer devices, metered dose inhalers, and the like suitable for delivery of polynucleotide compositions to the nasal mucosa, trachea and bronchioli are well-known in the art and will therefore not be described in detail here. For general review in regard to intranasal drug delivery, see, e.g., Chien, Novel Drug Delivery Systems, Ch. 5 (Marcel Dekker, 1992).

[0148] In a preferred embodiment, intranasal (IN) vaccination is used due to ease of administration and the lesser amount of antigen needed to induce an immune response. Depending on the mode of delivery and the nature of the vaccine, potential side effects resulting from vaccine reaching the olfactory bulb and brain must be ruled out. Nasal vaccines target the NALT, leading to responses locally and in the lung and female reproductive tract. Nasal immunizations have consistently elicited IgA and IgG responses and antigen- specific cytotoxic T lymphocytes (CTL) in the cervico-vaginal mucosa, but low to no responses in the intestine and rectal mucosa. Therefore, to elicit

comprehensive protection, a combination approach with IN/IM or IN/IR or IN/Oral immunization is embodied by the present invention.

[0149] Novel IN formulations are embodied by compositions of the present invention using an oil-in-water nanoemulsion. A relatively new approach embodied utilizes IN vaccination with recombinant bacteria such as

mycobacterium or salmonella.

[0150] Compositions contemplated herein can be applied either intratracheally (IT) or by aerosol through the nose target mainly the BALT, a diverse inductive site with large follicles similar to Peyer's patches at the bronchial bifurcations. Aerosol administration with a nebulizer or inhaler can elicit potent immune responses, due to the high surface area of the lung. Antibody responses induced in the BALT are mostly of the IgA type, and effector and memory T and B cells induced there can home to distant mucosal sites. That aerosol vaccination via the BALT is embodied in the present invention can be provided with the molecularly attenuated vaccine vector (NYVAC) encoding an HIV clade C envelope.

[0151 ] This embodiment is further provided for IT administration followed by IM boosting to elicit strong cellular immunity when the antigen consists of an envelope protein to provide for durable protection against a mucosal SIV and a reduction in chronic viremia.

[0152] Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, , intraorbital, intraspinal, intrastemal, and routes, i.e., any route of administration other than through the alimentary canal. Parenteral administration can be carried to effect systemic or local delivery of immunostimulatory antigens.

[0153] Systemic administration typically involves intradermal, subcutaneous, systemically absorbed topical or mucosal administration of pharmaceutical preparations. Mucosal administration includes administration to the respiratory tissue, e.g., by inhalation, nasal drops, ocular drop, etc.; anal or vaginal routes of administration, e.g., by suppositories; and the like.

Nanoparticles

[0154] In one embodiment, the present invention is formulated as a

nanopartide using the ligand angiotensin 1 -7 as a targeting ligand. Additionally, methods for formulating nanoparticles with and without ligand -directed targeting by the chemical presentation of the adjuvant on the surface are provided in the present invention.

[0155] In this embodiment, nanoparticles can be formulated from cyclodextrin, PEG, or other pharmaceutically acceptable particulate backbone, wherein; the antigen is complexed to the back-bone to present internally, while; the adjuvant ligand, specifically, to present a carboxyl terminus. Both, protective and therapeutic immunity are imparted by the method of the invention.

[0156] In this specific embodiment, the innate amplification receptor is being targeted the ligand angiotensin, complexed to the specific antigen-nanoparticle package, which is also of size range that provides the desired ratio of the percent of nanopartide that is eliminated by the phagocytic route compared to the total drug given. The ratio range is from 100% phagocytic to 100% non- phagocytic.

[0157] The phagocytosed nanoparticles will be expected to release their antigen content locally following the death of the phagocyte. Transport of the nanopartide by phagocytic cells provides the ability to deliver the contents of the nanopartide to any tissue that can be reached by phagocytic cells. This includes the delivery of nanoparticles across the blood brain barrier

[0158] A variety of parenteral and mucosal combination strategies can be embodied by the present invention. IN or in combined IN/IM regimens can be used to elicit IgG and IgA responses in sera and vaginal secretions as well as cellular immunity. Further, when priming by IN routes is followed by a rest period, IM immunization significantly boosted cellular and humoral immune responses. Combination oral/IM immunization using attenuated Listeria monocytogenes recombinants in combination with a viral pathogenic antigen can be practiced by the teachings herein.

[0159] In further embodiments, intradermal priming with recombinant live attenuated mycobacterium bovis BCG expressing SIV nef, gag, or env, followed by oral or rectal boosting can be used to induce antibody and cellular immune responses to HIV.

[0160] Immunostimulatory antigens can also be delivered to the subject by enteral administration. Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e.g., using a suppository) delivery.

Oral

[0161 ] Oral immunization can be problematic due to inefficient uptake of the vaccine vector due to the presence of food and other microorganisms, low pH, and abundant proteolytic enzymes. In the present invention, recombinant Salmonella enterica serovar Typhi, expressing HIV Gag or Env or both, can be administered intranasally.

[0162] Oral vaccination elicits mucosal immune responses mainly in the gut and mammary and salivary glands. Major obstacles for oral vaccination include the dose and stability of the vaccine during passage through the Gl tract. The vaccine becomes diluted by saliva and gastric fluids, and inactivated by the acid pH of the stomach and digestive enzymes. The current invention provides that enteric-coated capsules, which are resistant to stomach acid but dissolve in the neutral pH of the intestine, can be administered to overcome these problems.

[0163] In one embodiment, oral administration with a recombinant Lactococcus lactis expressing surface bound HIV env is be used to produce systemic cellular immunity and systemic and mucosal humoral immune responses.

Oral Spray

[0164] In an embodiment, oral spray vaccination may target the tonsils rather than the Gl tract. As an example, an HIV gag recombinant envelope combined with a non-pathogenic protozoan parasite vector, Leishmania tarentolae, can be used to provide cross-priming for HIV specific therapy.

Intrarectal

[0165] Whereas earlier studies indicated that intrarectal (IR) or colonic vaccination elicited only local immunity, recent studies have shown more distal responses. Colonic antigen administration can be performed to induce IgA antibodies in the colon, vagina and serum IgG levels. This embodiment can be enabled with HIV envelope proteins in a foam or troche.

[0166] Methods of administration of immunostimulatory antigens through the skin or mucosa include, but are not necessarily limited to, topical application of a suitable pharmaceutical preparation, transdermal transmission, injection and epidermal administration. For transdermal transmission, absorption promoters or iontophoresis are suitable methods. For review regarding such methods, those of ordinary skill in the art may wish to consult Chien, supra at Ch. 7.

Iontophoresis transmission may be accomplished using commercially available "patches" which deliver their product continuously via electric pulses through unbroken skin for periods of several days or more. An exemplary patch product for use in this method is the LECTRO PATCHTM (manufactured by General Medical Company, Los Angeles, Calif.) which electronically maintains reservoir electrodes at neutral pH and can be adapted to provide dosages of differing concentrations, to dose continuously and/or to dose periodically.

[0167] Epidermal administration can be accomplished by mechanically or chemically irritating the outermost layer of the epidermis sufficiently to provoke an immune response to the irritant. An exemplary device for use in epidermal administration employs a multiplicity of very narrow diameter, short tynes which can be used to scratch immunostimulatory antigens coated onto the tynes into the skin. The device included in the MONO-VACCTM tuberculin test

(manufactured by Pasteur Merieux, Lyon, France) is suitable for use in

epidermal administration of immunostimulatory antigens.

[0168] The immunostimulatory antigen in combination with a peptide adjuvant may be administered before, simultaneously with (e.g., in admixture with antigen, or covalently or non-covalently bound, directly or via a linker, to an antigen or antigenic epitope), or after the subject is exposed to antigen. Exposure to antigen may occur by intentionally introducing the antigen into the subject via a systemic or mucosal route, e.g., intranasally, intrarectally, intravenously, subcutaneously, intradermally, or intraperitoneally, and the like, e.g., by a clinician. Alternatively, exposure to antigen may occur accidentally or naturally (e.g., by happenstance), e.g., by the usual routes of exposure of a subject to plant, animal, and other antigens, such as by inhalation, accidental skin exposure, ingestion, and the like.

Methods of Preventing an Infectious Disease in an Individual

[0169] The present invention further provides methods for preventing or treating an infectious disease in an individual, comprising administering a formulation comprising an immunostimulatory antigen in combination with a peptide adjuvant to the individual, in an amount effective to prevent or treat the disease. The methods are particularly useful for preventing or treating infectious diseases caused by intracellular pathogens, such as viruses, intracellular bacteria, fungi and parasites (e.g. protozoans). In particular, opportunistic infections can be treated using the methods of the invention.

[0170] Methods of the invention for reducing pathogen entry into a cell susceptible to pathogen infection are also useful for treating a pathogen infection. Treating a pathogen infection, as used herein, includes, but is not limited to, preventing an infection in an individual who does not yet have a clinically detectable infection; reducing the probability of an infection in an individual who does not yet have a clinically detectable infection; reducing spread of pathogen from an infected cell to a cell not yet infected but susceptible to infection; improving one or more indicia of an infection. For example, treating an HIV infection, includes, but is not limited to, preventing HIV infection, reducing the probability of HIV infection, reducing the spread of HIV from an infected cell to a susceptible cell, reducing viral load in an HIV-infected individual, reducing an amount of virally encoded polypeptide(s) in an HIV-infected individual, and increasing CD4 T cell count in an HIV-infected individual. [0171 ] Methods of determining whether the methods of the invention are effective in reducing pathogen-induced disease in a susceptible cell include any known test for infection by a given pathogen, including, but not limited to, measuring the number of pathogens in a biological sample from a host, e.g., by using a PCR with primers specific for a nucleotide sequence in the pathogen; counting the number of pathogens in the host; detecting or measuring a polypeptide or other product produced by the pathogen; and measuring an indicia of pathogen infection.

[0172] For example, methods of determining whether the methods of the invention are effective in reducing HIV entry into a cell, and/or treating an HIV infection, are any known test for indicia of HIV infection, including, but not limited to, measuring viral load, e.g., by measuring the amount of HIV in a biological sample, e.g., using a polymerase chain reaction (PCR) with primers specific for an HIV polynucleotide sequence; detecting and/or measuring a polypeptide encoded by HIV, e.g., p24, gp120, reverse transcriptase, using, e.g., an immunological assay with an antibody specific for the polypeptide; and

measuring CD4 cell count in the individual. Methods of assaying an HIV infection (or any indicia associated with an HIV infection) are known in the art, and have been described in numerous publications such as HN Protocols (Methods in Molecular Medicine, 17) N. L. Michael and J. H. Kim, eds. (1999) Humana Press.

[0173] Thus, in these embodiments, an "effective amount" of an

immunostimulatory antigen in combination with a peptide adjuvant is an amount sufficient to treat an infectious disease, e.g., to reduce the number of pathogens and/or reduce a parameter associated with the presence of a pathogen, by at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 75%, at least about 85%, or at least about 90%, up to total eradication of the infectious disease, when compared to a suitable control.

[0174] In an experimental animal system, a suitable control may be a genetically identical animal not treated with the immunostimulatory antigen. In non-experimental systems, a suitable control may be the infectious disease present before administering the immunostimulatory antigen. Other suitable controls may be a placebo control.

[0175] Whether an infectious disease has been treated can be determined in any of a number of ways, including but not limited to, measuring the number of infectious agents in the individual being treated, using methods standard in the art; measuring a parameter caused by the presence of the pathogen in the individual, e.g., measuring the levels of a toxin produced by the pathogen;

measuring body temperature; measuring the level of any product produced by the pathogen; measuring or assessing any undesired physiological parameter associated with the presence of an infectious agent in an individual.

[0176] Measuring the number of infectious agents can be accomplished by any conventional assay, such as those typically used in clinical laboratories, for evaluating numbers of pathogens present in a biological sample obtained from an individual. Such methods have been amply described in the literature, including, e.g., Medical Microbiology 3rd Ed., (1998) P. R. Murray et al., eds. Mosby-Year Book, Inc. A level of a product, including a toxin, produced by a pathogen can be measured using conventional immunological assays, using antibody which detects the product, including, but not limited to enzyme-linked immunosorbent assays (ELISA), radioimmunoassays. Other assays include in vivo assays for toxins.

Methods of Preventing or Treating an Infectious Disease in an Individual

[0177] The present invention further provides methods for reducing entry of a pathogen, e.g., an immunodeficiency virus, into a cell. The methods generally involve contacting the cell with an immunostimulatory antigen. The methods are useful for reducing infection with an immunodeficiency virus in an individual.

[0178] In the context of methods of reducing pathogen entry into a susceptible cell, an effective amount of an immunostimulatory antigen in combination with a peptide adjuvant is one that increases chemokine secretion from a cell and reduces infection by the pathogen into the same cell or cells in the vicinity of the chemokine-producing cell. The cell secreting chemokine and the cell susceptible to infection by the pathogen may be the same cell, but need not be. [0179] The present invention provides methods and compositions for ongoing cell mediated immunity (CMI) surveillance. CMI can be elicited to infected cells wherein the antigen is occult, upon renewed antigen budding from the cell.

Methods of Increasing Chemokine Secretion

[0180] The present invention provides methods for increasing chemokine production and secretion by a cell. The methods are useful for treating various disorders which are mediated by cells expressing chemokine receptors. In some embodiments, the methods are carried out in vitro or ex vivo. In these

embodiments, the methods generally involve contacting the cell with an immunostimulatory antigen in combination with a peptide adjuvant in an amount sufficient to increase secretion of a chemokine. In other embodiments, the methods are carried out in vivo. Furthermore, the methods generally involve administering to an individual an immunostimulatory antigen in combination with a peptide adjuvant in an amount sufficient to increase secretion of a chemokine.

[0181 ] In some embodiments, the invention provides methods for increasing chemokine production and secretion in an antigen non-specific manner. In these embodiments, cells are contacted with, or individuals are administered with, immunostimulatory antigen in combination with a peptide adjuvant without antigen.

[0182] The methods of the invention increase secretion of a chemokine from a cell that normally produces a chemokine, particularly those cells that are susceptible to infection by a pathogen. Cells that normally produce chemokines include, but are not limited to, T lymphocytes, macrophages, monocytes, dendritic cells and related antigen-presenting cells (APCs), B lymphocytes, epithelial cells, fibroblasts, endothelial cells, basophils, eosinophils, neutrophils, natural killer cells, and bone marrow stem cells.

[0183] Chemokines whose secretion is increased by contacting a cell that normally produces a chemokine with an immunostimulatory antigen in combination with a peptide adjuvant include, but are not limited to, MIP-1 a, and ΜΙΡ-1 β. Other chemokines which may have increased secretion in response to immunostimulatory nucleic acid include, but are not necessarily limited to, RANTES, SDF-1 , MCP-1 , MCP-2, MCP-3, MCP-4, eotaxin, eotaxin-2,1 - 309/TCA3, ATAC, HCC-1 , HCC-2, HCC-3, LARC/MIP-3a, PARC, TARC, ΟΚβ4, ΟΚβ6, ΟΚβ7, ΟΚβ8, ΟΚβ9, ΟΚβΜ , ΟΚβ12, and ΟΚβ13, CIO, an interleukin-8 (IL- 8) family member; GROa, GRO , GROY, mouse KC, mouse MIP-2, ENA-78, GCP-2, PBP/CTAPIII/ -TG/NAP-2, IP-IO/mouse CRG, Mig, PBSF/SDF-1 , a member of the platelet factor 4 (PF4) family, lymphotactin, or an equivalent in any mammalian species of any of the foregoing.

[0184] In particular embodiments, production and secretion of a chemokine is antigen-specific. The term "antigen-specific" is one well understood in the art, and refers to chemokine production in response to the antigen with which the individual is immunized, or to closely related ("cross-reactive") antigens, e.g., antigens that share one or more epitopes with the immunizing antigen. In in vivo embodiments, the method generally involves administering to an individual an immunostimulatory antigen in combination with a peptide adjuvant and an antigen, wherein the immunostimulatory antigen in combination with a peptide adjuvant is administered in an amount sufficient to increase secretion of a chemokine in an antigen-specific manner.

[0185] Whether chemokine secretion is increased in an antigen specific manner can be readily determined by those skilled in the art using standard methods. As one non-limiting example, splenocytes from an individual immunized with immunostimulatory antigen in combination with a peptide adjuvant plus antigen are cultured in the presence of the immunizing antigen, and secretion of chemokines measured using any known method, as described below.

[0186] In vitro and ex vivo methods of the invention comprise contacting a cell that normally produces a chemokine with an immunostimulatory antigen. In these embodiments, contacting a cell that normally produces a chemokine with an immunostimulatory antigen in combination with a peptide adjuvant increases chemokine secretion from the cell by at least about 10%, at least about 25%, at least about 30%, at least about 50%, at least about 75%, at least about 100% (or two-fold), at least about five fold, at least about 10 fold, at least about 15 fold, at least about 25 fold, at least about 50 fold, at least about 75 fold, at least about 100 fold, at least about 200 fold, at least about 300 fold, at least about 400 fold, at least about 500 fold, at least about 600 fold, at least about 700 fold, at least about 800 fold, at least about 900 fold, at least about 1000 fold, at least about 2000 fold, at least about 3000 fold, at least about 4000 fold, at least about 5000 fold, or at least about 10,000 fold or more, when compared the level of secretion of the chemokine from the cell not contacted with the immunostimulatory antigen.

[0187] In vivo methods of the invention comprise administering to an individual an immunostimulatory antigen in combination with a peptide adjuvant in an amount sufficient to increase secretion of a chemokine from a cell that normally produces a chemokine. A "sufficient amount," used interchangeably in this context with "an effective amount," is an amount of immunostimulatory antigen in combination with a peptide adjuvant sufficient to increase chemokine secretion such that the level of chemokine produced is increased by at least about 10%, at least about 25%, at least about 30%, at least about 50%, at least about 75%, at least about 100% (or two-fold), at least about five fold, at least about 10 fold, at least about 15 fold, at least about 25 fold, at least about 50 fold, at least about 75 fold, at least about 100 fold, at least about 200 fold, at least about 300 fold, at least about 400 fold, at least about 500 fold, at least about 600 fold, at least about 700 fold, at least about 800 fold, at least about 900 fold, at least about 1000 fold, at least about 2000 fold, at least about 3000 fold, at least about 4000 fold, at least about 5000 fold, or at least about 10,000 fold or more, when compared the level of chemokine in the individual before being administered with the immunostimulatory antigen.

[0188] Whether, and to what extent, an immunostimulatory antigen in combination with a peptide adjuvant increases chemokine secretion from a cell that normally produces (e.g., is capable of producing) can be readily determined using any known assay method. The amount of chemokine secreted from a cell can be determined quantitatively (e.g., the amount secreted measured) or semi- quantitatively (e.g., the amount secreted relative to a control determined). Levels of chemokine can be determined using any method known in the art, including a biochemical assay, an immunological assay, or a biological assay. Immunological assays include, but are not limited to, radioimmunoassays, and enzyme-linked immunosorbent assays (ELISA), a number of which are commercially available. Assays can be conducted in vitro, e.g., by adding an immunostimulatory antigen in combination with a peptide adjuvant to the cell culture medium of an in vitro cell culture, and, after a suitable time (e.g., about 10 minutes to about 24 hours), determining the level of chemokine in the cell culture supernatant.

[0189] Biological assays include, but are not limited to, in vitro assays to detect pathogen binding to and/or entry into a cell bearing a chemokine receptor on its surface, which receptor serves as a receptor or co-receptor for infection by the pathogen or as a receptor or co-receptor for a pathogen derived ligand that elicits disease symptoms or causes disease. Any known assay to determine infection of a cell with a pathogen can be used. For example, binding or infection by an immunodeficiency virus can be detected by syncitia formation, cytopathic effects, production of an immunodeficiency virus-encoded polypeptide, e.g. p24, and/ or reverse transcriptase, and/or gp120.

Formulations

[0190] In general, immunostimulatory antigens are prepared in a

pharmaceutically acceptable composition for delivery to a host. In one

embodiment, they are formulated for mucosal delivery. Pharmaceutically acceptable carriers for use with the immunostimulatory antigens of the invention may include sterile aqueous of non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol,

polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, and microparticles, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.

Intravenous vehicles include fluid and nutrient replenishers, electrolyte

replenishers (such as those based on Ringer's dextrose), and the like. A composition comprising an immunostimulatory antigen in combination with a peptide adjuvant may also be lyophilized using means well known in the art, for subsequent reconstitution and use according to the invention.

[0191 ] In general, the pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions comprising the therapeutically active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. In one embodiment, as discussed above, the

immunostimulatory antigen in combination with a peptide adjuvant formulation comprises an additional anti-mycobacterial agent.

[0192] Immunostimulatory antigens can be administered in the absence of agents or compounds that might facilitate uptake by target cells (e.g., as a "naked" polynucleotide, e.g., a polynucleotide that is not encapsulated by a viral particle, a liposome, or any other macromolecule). Immunostimulatory antigens can be administered with compounds that facilitate uptake of immunostimulatory antigens by target cells (e.g., by macrophages) or otherwise enhance transport of an immunostimulatory antigen in combination with a peptide adjuvant to a treatment site for action. Absorption promoters, detergents and chemical irritants (e.g., keratinolytic agents) can enhance transmission of an immunostimulatory antigen in combination with a peptide adjuvant composition into a target tissue (e.g., through the skin). For general principles regarding absorption promoters and detergents which have been used with success in mucosal delivery of organic and peptide-based drugs, see, e.g., Chien, Novel Drug Delivery

Systems, Ch. 4 (Marcel Dekker, 1992).

[0193] Examples of suitable nasal absorption promoters, in particular, are set forth at Chien, supra at Ch. 5, Tables 2 and 3. Suitable agents for use in the method of this invention for mucosal/nasal delivery are also described in Chang, et. al., Nasal Drug Delivery, "Treatise on Controlled Drug Delivery", Ch. 9 and Tables 3-4B thereof, (Marcel Dekker, 1992). Suitable agents which are known to enhance absorption of drugs through skin are described in Sloan, Use of

Solubility Parameters from Regular Solution Theory to Describe Partitioning Driven Processes, Ch. 5, "Prodrugs: Topical and Ocular Drug Delivery" (Marcel Dekker, 1992), and at places elsewhere in the text. All of these references are incorporated herein for the sole purpose of illustrating the level of knowledge and skill in the art concerning drug delivery techniques.

[0194] A colloidal dispersion system may be used for targeted delivery of the immunostimulatory antigens to specific tissue. Colloidal dispersion systems include macro molecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0195] Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 urn can encapsulate a substantial percentage of an aqueous buffer comprising large macromolecules. RNA and DNA can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., (1981 ) Trends Biochem. Sci., 6:77). The composition of the liposome is usually a combination of phospholipids, particularly high -phase-transition temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidyl-glycerol, phosphatidylcholine, phosphatidylserine,

phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.

Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoyl- phosphatidylcholine and distearoyl-phosphatidylcholine. [0196] Where desired, targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand, such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the

composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.

[0197] The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various well known linking groups can be used for joining the lipid chains to the targeting ligand (see, e.g., Yanagawa, et al., (1988) Nuc. Acids Symp. Ser., 19:189;

Grabarek, et al., (1990) Anal. Biochem., 185:131 ; Staros, et al., (1986) Anal. Biochem. 156:220 and Boujrad, et al., (1993) Proc. Natl. Acad. Sci. USA, 90:5728). Targeted delivery of immunostimulatory antigens can also be achieved by conjugation of the immunostimulatory antigens to the surface of viral and non- viral recombinant expression vectors, to an antigen or other ligand, to a monoclonal antibody or to any molecule which has the desired binding

specificity.

[0198] An immunostimulatory antigen in combination with a peptide adjuvant can be administered to an individual in combination (e.g., in the same

formulation or in separate formulations) with another therapeutic agent

("combination therapy") or can be administered in a separate formulation. When administered in separate formulations, an immunostimulatory nucleic acid molecule and another therapeutic agent can be administered substantially simultaneously (e.g., within about 60 minutes, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, or about 1 minute of each other) or separated in time by about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, or about 72 hours, or more.

[0199] Therapeutic agents that can be administered in combination therapy, such as anti-inflammatory, anti-viral, antifungal, antimycobacterial, antibiotic, amoebicidal, trichomonocidal, analgesic, antineoplastic, antihypertensives, antimicrobial and/or steroid drugs, to treat antiviral infections. In some embodiments, patients with a viral or bacterial infection are treated with a combination of one or more immunostimulatory antigens with one or more of the following; betalactam antibiotics, tetracyclines, chloramphenicol, neomycin, gramicidin, bacitracin, sulfonamides, nitrofurazone, nalidixic acid, cortisone, hydrocortisone,

betamethasone, dexamethasone, fluocortolone, prednisolone, triamcinolone, indomethacin, sulindac, acyclovir, amantadine, rimantadine, recombinant soluble CD4 (rsCD4), anti-receptor antibodies (e.g., for rhinoviruses), nevirapine, cidofovir (Vistide TM), trisodium phosphonoformate (Foscamet™), famcyclovir, pencydovir, valacydovir, nucleic acid/replication inhibitors, interferon, zidovudine (AZT, Retrovir™), didanosine (dideoxyinosine, ddl, Videx™), stavudine (d4T, Zerit™), zalcitabine (dideoxycytosine, ddC, HividTM), nevirapine (ViramuneTM), lamivudine (Epivir™, 3TC), protease inhibitors, saquinavir (Invirase TM,

FortovaseTM), ritonavir (Norvir™), nelfinavir (Viracept™), efavirenz (SustivaTM), abacavir (ZiagenTM), amprenavir (Agenerase TM) indinavir (Crixivan TM), ganciclovir, AzDU, delavirdine (Rescriptor™), kaletra, trizivir, rifampin,

clathiromycin, erythropoietin, colony stimulating factors (G-CSF and GM-CSF), non-nucleoside reverse transcriptase inhibitors, nucleoside inhibitors,

adriamycin, fluorouracil, methotrexate, asparaginase and combinations thereof.

Routes of Administration Dosages

[0200] Although the dosage used will vary depending on the clinical goals to be achieved, a suitable dosage range is one which provides up to about 1 fAg to about 1 ,000 fAg or about 10,000 fAg of immunostimulatory antigen in

combination with a peptide adjuvant can be administered in a single dosage. Alternatively, a target dosage of immunostimulatory antigen in combination with a peptide adjuvant can be considered to be about 1 -10 fAM in a sample of host blood drawn within the first 24-48 hours after administration of immunostimulatory antigens. Based on current studies, immunostimulatory antigens are believed to have little or no toxicity at these dosage levels.

[0201 ] It should be noted that the immunotherapeutic activity of

immunostimulatory antigens is generally dose-dependent. Therefore, to increase immunostimulatory antigens potency by a magnitude of two, each single dose is doubled in concentration. Increased dosages may be needed to achieve the desired therapeutic goal. The invention, thus, contemplates administration of "booster" doses to provide and maintain a desired immune response. For example, immunostimulatory antigens may be administered at intervals ranging from at least every two weeks to every four weeks (e.g., monthly intervals) (e.g., every four weeks).

EXAMPLES

[0202] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0203] Vaginal suppositories were prepared using Urovac™, a vaccine containing heat-killed bacteria from 10 human pathogenic strains. Virulence factors present in the strains were determined using polymerase chain reaction primers specific for pathogenicity genes. The vaccine also contained 1 strain each of Proteus mirabilis, Morganella morganii, Klebsiella pneumoniae and Enterococcus faecalis. The 14 bacterial strains were combined in equal numbers (1 x 108 each) in a polyethylene glycol base to form suppositories containing a total of 1 x 10 bacteria each. Placebo suppositories were prepared with polyethylene glycol base only.

[0204] Each suppository consisted of 2 g suppository base ^aWitepsol H-15° (Wiler Fine Chemicals Ltd., London, Ontario, Canada) containing triglycerides (85%), diglycerides (15%), and monoglycerides (1 %), and 500 mg plasmid (167 mg pSLIAgD and 333 mg pSLIAtgD) in 180 ml ddH2O, and Angiotensin 1 -9 100 meg. The suppository base was melted at 56°C, and the plasmids in ddH2O were added and vortexed. The resulting suspension was cooled while being vortexed and poured into molds shortly before hardening. To assess that the plasmids remained supercoiled, a suppository was heated at 39°C and the DNA was collected and run on a 1 % agarose gel, either directly or after ethanol precipitation.

Preparation of vaccine microspheres using spray dryer

[0205] Typhoid Vi capsular polysaccharide antigen-loaded albumin-chitosan matrix (Kramer, 1974; van der Lubben et al., 2001 ) microspheres were prepared with 4.5% (w/v) BSA and 0.5% (w/v) chitosan solution in deionized water using a Buchi 191 mini spray dryer (Brinkman Instruments, Westbury, NY) (Bejugam et al., 2008). Two vaccine formulations with low and high drug loading, TF1 and TF2, were prepared with 0.1 mcg/mg and typhoid Vi antigen loading,

respectively. Spray drying was performed using compressed air from an in- house supply line (700 NL/h). The filtered air was aspirated at 95% and the inlet temperature was set at 1 10°C, giving an outlet temperature of 73°C. Curing of the microsphere matrix was performed through chemical cross-linking using 1 % glutaraldehyde for 6hrs at room temperature (RT).

Enteric coating of capsules

[0206] Prior to usage, HPMC capsules were evaluated for uniformity of weight and material integrity by light microscopy. Disintegration time of uncoated capsules was also evaluated in ddH20. Under constant stirring in a 500-ml sterile beaker, talc (Mg₃Si₄Oio (OH)₂, Sigma, St. Louis, MO) was added slowly and gradually to an aqueous solution of isopropyl alcohol (95.6%, v/v). A 4:1 ratio (8.8%, w/w) of Eudragit® L100 and S100 methacrylic acid co-polymers (Degussa Rohm Pharma Polymers, Darmstadt, Germany) was added very slowly to the talc suspension. The plasticizer triethyl citrate (TEC, Sigma) was added drop-wise to the suspension, covered with Parafilm, and mixed for an additional hour to ensure homogeneity. Both sides of the capsules were coated with three wet coats of the enteric polymer dispersion using a ProCoater (Torpac Inc., Fairfield, NJ).

Preparation of nasal powder formulations

[0207] The following amounts of spray-dried mixtures of Amioca® and

Carbopol® 974P were dispersed in 15ml distilled water and neutralized (except SD 100/0) to pH 7.4 with 1 M or 2M NaOH. Five micrograms heat-inactivated X47 influenza virus and 0.5_g adjuvant LTR192G/mg powder were added to each dispersion. Negative control powders (SD 25/75 and SD 100/0) were prepared without adding antigen. To obtain a powder, the aqueous dispersion was freeze- dried in an Amsco-Finn Aqua GT4 freeze dryer (Amsco, Hurth, Germany). The dispersion was frozen to 228K within 175min at 1000 mbar. Primary drying was performed at 258K and a pressure of 0.8-1 mbar for 13 h, followed by secondary drying at an elevated temperature (283 K) and reduced pressure (0.1-0.2 mbar) for 7 h. After freeze-drying, the powder was manually milled at low relative humidity (20%) and ambient temperature. It was stored in a desiccator at 4-8 °C until use.

[0208] Preparation of liquid nasal PBS vaccines Heat-inactivated X47 influenza virus (H₃N₂) and LTR192G adjuvant were dissolved in LPS-free phosphate buffered saline (pH 7.4) and angiotensin 1 -9 at 75 meg at 700ug/ml and 70ug/ml, respectively, of which 70ul per nostril was administered.

Biological Assay

[0209] Peripheral blood mononuclear cells (PBMC) cultures are infected with a virus stock. Virus is harvested when p24 or reverse transcriptase (RT) is detected in the supernatant. Dilutions of a solution (e.g., a cell culture

supernatant) are mixed with target phytohemagglutinin (PHA-) and Unstimulated PBMCs and incubated at 37° C. for 30 minutes, and are then exposed to an equal volume of virus supernatant containing 1000 times the median tissue culture infectious dose (TCID50), and reincubated at 37° C for 3 hours. Input virus is then washed out before adding growth medium containing appropriate chemokine concentrations. The cultures are incubated at 37 C. for up to 12 days with medium changes twice weekly but without further addition of chemokine. Virus production into the supernatant is assessed by measurement of RT activity using a sensitive nonradioactive method (e.g., a commercially available assay, e.g., the Retrosys RT activity kit from Innovagen AB, Lund, Sweden). Simmons et al. (1997) Science 276:276-279. 40.

Methods of Preventing an Infectious Disease in an Individual

[0210] An embodiment of the present invention further provides methods for preventing a viral infectious disease in an individual, comprising administering a formulation comprising an immunostimulatory antigen fragment, e.g. formulated for mucosal administration, in combination with a peptide adjuvant to the individual, in an amount effective to prevent or treat the disease. The methods are particularly useful for preventing or treating infectious diseases caused by intracellular pathogens, such as viruses, intracellular bacteria, fungi and parasites (e.g. protozoans). In particular, opportunistic infections can be treated using the methods of the invention.

Example 1

Model Vaccine Compositions for Preventing SIV in Adult Rhesus Monkeys

Animals

[021 1 ] (Macaca mulatta) are screened for the presence of the Mamu-A^*01 allele using a PCR-based technique.

Vaccine Preparation.

[0212] Liposomal dispersions are prepared by reverse phase evaporation.

Previously optimized components of liposomes are utilized in this study.

Concerning neutral liposomes (F1 ), phospholipid (lipoid E 80) and cholesterol (2:1 ) are dissolved in chloroform: diethyl ether (1 :1 ). Each peptide pool is comprised of 15-amino-acid peptides overlapping by 1 1 amino acids. The pools covered the entire SIVmac239 Gag protein and HIV-1 89.6P (KB9) Env protein. Each peptide in a pool is present at a l - g/ml concentration. Aqueous phase (buffer solution contains Ang-(1 -5)) is added such that the organic-to aqueous phase ratio is 6:1 . The mixture is sonicatied at 40°C either for 5 or 10 min in an ultrasonic bath. A stable emulsion is produced, from which the organic solvent is slowly removed at reduced pressure at 45°C by a rotary evaporator. Liposomal dispersions formed are maintained for 1 hour at a temperature exceeding the phospholipid transition temperature (40°C). The composition of the negatively charged liposomes (F2) is phospholipid, cholesterol, and dicetylphosphate (2:1 :0.5 molar ratio) and the same procedure is followed.

Liposome preparation.

[0213] Different ratios of Ang-(1 -7) and SIVmac239 Gag protein pools

fragmented by proteolysis , DSPC, and CHOL are mixed and dissolved in 10 mL of mixture of methanol and chloroform (1 :1 , VA/) and dried in a rotary evaporator at 65 °C for 1 hr to form a lipid film. The molar ratio of Ang-(1 -7) to lipid content of liposomes is between 0.1 and 4%. The lipid film is then hydrated with 30 mL of 10 % (mA/) sucrose solution and stirred for one hour. The liposome suspension is extruded through polycarbonate filters of 100 nm pore size for 10 cycles (Liposofast® Extruder, Avestin Inc.). The suspension is then freeze-dried for 48 hrs at -40 °C (Lyotrap Plus, LTE Scientific Limited) to obtain a fine powder of liposomes. For the preparation of pegylated liposomes, appropriate amount of DSPC/DSPE- PEG/CHOL (70/5/25) or (65/10/25) are dissolved in a mixture of methanol and chloroform (1 :1 , VA/) and dried in a rotary evaporator.

[0214] Liposomes are prepared by the thin layer evaporation technique. All lipids of liposomes are dissolved in chloroform/methanol = 2/1 (v/v%), dried under reduced pressure, and stored in vacuo for at least 1 hr. The resulting thin lipid firm is hydrated with PBS (pH 7.4). Liposomes are subjected to freezing and thawing for 3 cycles with liquid nitrogen, and extruded 5-times through

polycarbonate double-membrane filters (pore-size, 200 nm; Nucleopore, Costar). Free lipids of the liposomal suspension are separated by centrifugation.

Liposomal pellets are resuspended in PBS (pH 7.4). Before the characterization of liposomes, the stability is checked, and only stable liposomes are

characterized.

Intranasal vaccine liposome composition.

[0215] Intranasal pulsing of vaccine liposomes is performed prior to intrarectal SIV administration. In brief, animals are delivered an aerosolized dose of vaccine daily for 3 days prior to the viral challenge.

SIV challenge stocks.

[0216] The viruses employed in this study include cell-free uncloned SIVmac251 and SIVsmE660. The stock of SIVmac251 is expanded on human PBMCs, and the stock of SIVsmE660 is expanded on rhesus monkey PBMC. Monkeys are exposed to virus by intrarectal inoculation through a cluster of 6 weekly administrations. Using three monkeys/dose/virus stocks, groups of animals are exposed to 6 X 10⁷, 6 X 10⁶, and 6 X 10⁵ copies of virus with each

administration. The cohorts of nine SIVmac251 -exposed and nine SIVsmE660- exposed monkeys are monitored for evidence of infection by assessing plasma samples obtained on a weekly schedule for SIV gag virus RNA.

Intrarectal exposure to SIV.

[0217] Animals are placed in a sternal position with the pelvis propped up at an approximately 45° angle after anesthetization (10 mg/kg of body weight ketamine intramuscularly (i.m.) and 0.5 mg/kg xylazine i.m.). A lubricated infant feeding catheter is inserted into the rectum of the animal approximately 4 to 6 inches without causing any injury. The animal is returned to its cage and kept tilted at a 45° angle until it fully recovered from anesthesia.

Plasma SIV RNA levels.

[0218] Plasma viral RNA levels are measured by an ultrasensitive branched DNA amplification assay with a detection limit of 125 copies per ml (Bayer

Diagnostics). Antibodies.

[0219] All reagents are validated and titrated using rhesus monkey PBMC. The antibodies(BD Biosciences) used are anti-tumor necrosis factor alpha (TNF-a)- fluorescein isothiocyanate (FITC) (MAb1 1 ), anti-gamma interferon (IFN-γ)- phycoerythrin (PE)-Cy7 (B27), anti-interleukin-2 (IL-2)-allophycocyanin (APC) (MQ1 -17H12), anti-MIP-1 a-PE (D21 -1351 ), anti-CD4-AmCyan (L200), anti- CD3-Pacific Blue (SP34-2), and anti-CD8a-Alexa Fluor 700 (RPA-T8).

IFN-Y ELISPOT assays.

[0220] ELISPOT assays, multiscreen 96-well plates are coated overnight with 100 μΙ per well of 5- g/ml anti-human IFN-γ (B27; BD Pharmingen) in endotoxin- free Dulbecco's PBS (D-PBS). The plates then are washed 3 times with D-PBS containing 0.25% Tween 20, blocked for 2 h with D-PBS containing 5% fetal bovine serum (FBS) to remove the Tween 20, and incubated with peptide pools and 2 X 10⁵ PBMC in triplicate in 100-μΙ reaction volumes. Each peptide pool comprises of 15-amino-acid peptides overlapping by 1 1 amino acids. The pools covered the entire SIVmac239 Gag protein and the HIV-1 89.6P (KB9) Env protein. Each peptide in a pool is present at a l -pg/ml concentration. Following a 18-h incubation at 37°C, the plates are washed and incubated with 2 g/ml biotinylated rabbit anti-human IFN-γ for 2 h at room temperature, then are washed 6 times, and incubated for 2.5 h with a 1 :500 dilution of streptavidin-AP. After 5 washes, the plates are developed with nitroblue tetrazolium-5-bromo-4- chloro-3-indolylphosphate chromogen (Pierce) and the reaction is stopped by washing the plates with tap water. After plates air dry, they are read with an ELISPOT reader. ELISPOT assays are performed on PBMC obtained on weeks 6, 7, 21 , and 27 following the first exposure to virus.

Intracellular cytokine assays.

[0221 ] PBMC are incubated at 37°C in a 5% CO₂ environment for 6 h in the presence of RPMI 1640-10% FCS medium alone (unstimulated), a pool of 15- mer Gag peptides (5 g/ml each peptide), or staphylococcal enterotoxin B (5 g/ml; Sigma-Aldrich) as a positive control. All cultures contain monensin and 1 g/ml anti-CD49d (BD Biosciences). The cultured cells are stained with monoclonal antibodies (MAbs) specific for cell surface molecules (CD3, CD4, CD8, CD28, and CD95) and a hematoxylin-eosin dye to discriminate live from dead cells. Then, cells are permeabilized and stained with antibodies specific for IFN-Y, TNF-a, and IL-2. Labeled cells are fixed in 1 .5% formaldehyde-PBS. Samples are collected and analyzed using FlowJo software. Approximately 200,000 to 1 ,000,000 events are collected per sample. The only samples considered positive are those for which the percentage of cytokine-staining cells is at least twice that of the background or where there is a distinct population of cells brightly positive for cytokine. ICS assays are performed on PBMC obtained on weeks 21 and 27 following the first exposure to virus.

Tetramer staining.

[0222] Soluble tetrameric Mamu-A^*01/p1 1 C complex is prepared as described previously. PE-labeled tetrameric Mamu-A^*01/p1 1 C complex in conjunction with FITC-labeled anti-human CD8a, PerCP-Cy5.5-labeled anti-human CD4, and APC-labeled anti-rhesus CD3 (FN18) MAbs are used to stain p1 1 C-specific CD8a T cells as described previously. Thawed PBMC are washed in RPMI 1640 medium containing 12% FBS (R12). PBMC (5 X 10⁶) are resuspended in 100 μΙ of PBS and directly stained with the reagent mixture, washed in 4 ml of PBS containing 2% FBS, and fixed in 0.5 ml of PBS containing 1 .5% formaldehyde.

[0223] PBMC (4 X 10⁶) in 1 ml of R12 are cultured in the presence of l -pg/ml SIV Gag p1 1 C (CTPYDINQM). On day 3 of culture, 1 ml of 40-U/ml human recombinant IL-2 (Hoffman-La Roche) is added. On day 12 of culture, peptide- stimulated PBMC are centrifuged over a Ficoll gradient and washed. Peptide- stimulated PBMC (5 X 10⁵) are re-suspended in 100 μΙ PBS and stained with 1 g of PE-labeled tetrameric Mamu-A^*01/p1 1 C complex in conjunction with FITC- labeled anti-human CD8a and APC-labeled anti-rhesus CD3 (FN 18) MAb. The samples are washed using procedures set forth above and analyzed by four- color flow cytometry system. Gated CD3+ CD8+ T cells are examined for staining with tetrameric Mamu-A^*01/p1 1 C.

Collection of rectal secretions. [0224] Weck-Cel cellulose sponges, pre-moistened with 50 μΙ D-PBS are used as previously described to absorb secretions from the rectum of each animal. Secretions are eluted from sponges by centrifugation in the presence of 100 μΙ 0.5% Igepal detergent in PBS containing protease inhibitors. After assessing blood contamination through measurement of hemoglobin, 20 μΙ goat serum (GS) is added to the secretion.

Measurement of SIV-specific mucosal IgA antibodies.

[0225] Total IgA, anti-SIV gp130, or anti-SIV gag and pol antibodies are measured by ELISA using high-protein-binding microtiter plates, coated overnight with 50 pg/well affinity-purified goat anti-monkey IgA antibody, 100 ng/well SIVmac251 rgp130, or a 1/400 dilution (250 ng total protein per well) of SIVmac251 viral lysate. Antibodies measured with this ELISA are referred to as being SIV gag/pol-specific. The following day, plates are washed with PBS containing 0.05% Tween 20 (PBST), blocked with 5% GS in PBST, and then loaded with twofold serial dilutions of standards and secretions in 5% GS-PBST. The standard in the total IgA ELISA is a normal monkey serum containing a known amount of IgA. The standards in the SIV ELISA are two preparations of IgG-depleted pooled serum from DNA modified vaccinia virus Ankara-vaccinated macaques that had been found to have high titers of serum IgA antibodies against SIV lysate or gp130 after challenge. The concentration of IgA antibody in each SIV standard had been calibrated relative to the total IgA standard by coating portions of a microtiter plate with each of the relevant coating reagents and developing them as described below.

[0226] The plates are developed by consecutive treatment with biotinylated affinity-purified goat anti-monkey IgA, avidin-labeled peroxidase, and 2,2'- azinobis (3-ethylbenzthiazolinesulfonic acid) as described previously. After absorbance values at 414 nm are recorded, IgA antibody concentrations in specimens are interpolated from standard curves using the SoftMaxPro computer program. The concentration of SIV gag/pol- or gp130-specific IgA subsequently is divided by the concentration of total IgA in the secretion to obtain the specific activity. The secretion is determined to be positive for anti-SIV IgA antibody if the specific activity is greater than or equal to the mean specific activity plus 3 standard deviations measured with 20 negative control secretions obtained from SIVnaive rhesus macaques.

Example 2

Compositions as a Therapeutic Treatment for SIV Infected Animals

[0227] Using the procedures set forth above to induce SIV in a primate, the vaccine is evaluated when administered every day therapeutically in an intranasal vaccine liposome. Activity is assessed using mRNA viral load measurements.

Example 3

Antigen-specific Systemic and Mucosal Responses in a Mouse

Immunization Model

[0228] To evaluate the adjuvant activity of a natural peptide product when delivered using the needle-free method of intranasal mucosal immunization, a normally non-immunogenic antigen fragment (rPA) to anthrax is used in combination with an adjuvant selected from the fragments Ang-(1 -7), Ang-(1 -5), All, or Ang 2-8, which are formulated for an inhalation system as a micro- particulate emulsion. Animals are dosed using a close fitting mask via oral inhalation of aerosols generated from an MDI. Animals are exposure to constant aerosol concentration and duration. Briefly, C3H/HeN female mice (5 per group) are immunized with recombinant anthrax protective antigen (rPA) at 2.0 g per dose on days 0, 7 and 21 . Mice are vaccinated with rPA alone or combined with Ang-(1 -7) (CPC Scientific) at doses of 5, 15, 45 or 135 g at each immunization. CT (1 g) serves a positive control adjuvant that induces Th₂-biased immune responses, and CpG (20 g) are used as a positive control adjuvant that induces Thi-biased immune responses. Serum samples are collected on days 14 and 42 and tested for the presence of anti-rPA IgG, lgG1 , lgG2a, IgE and IgA by ELISA. Mucosal samples (vaginal lavage, fecal extract) are collected on day 42 and tested for the presence of anti-rPA IgG and IgA. Day 42 serum is tested for its ability to neutralize anthrax lethal toxin using a macrophage toxicity assay prior to necropsy. On day 42, mice are euthanized and splenocytes harvested for in vitro restimulation with rPA. To evaluate the antigen-specific Th₁/Th₂/Th₁₇ profile induced by Ang-(1 -7), cell culture supernatants from cells cultured in media alone or media containing rPA are harvested at 60 hrs after restimulation and tested for the presence of IL-4, IL-5, IL-6, IL-10, IL-17 and IFN-γ using a multiplex bead-based immunoassay. ELISA titers are compared for cytokine production in each mouse group are determined by subtracting the cytokine concentrations measured in cells cultured in media alone from the cytokine concentrations measured in cells cultured in media + rPA. The inclusion of CT allows us to determine if Ang-(1 -7) provides adjuvant activity (at any dose tested) comparable to that provided by CT. Studies are repeated to confirm the observation and provide an n=10 for each group. Ang-(1 -7) doses are modified if the proposed dose response range indicated does not identify an Ang-(1 -7) dose that provides maximal adjuvant activity.

Example 4

Persistence of Ang-(1-7)-adjuvanted, Vaccine-induced Antibodies over

Time

[0229] To determine the durability of systemic and mucosal antibody responses induced when using Ang-(1 -7) as the adjuvant, we are using the lowest Ang-(1 - 7) doses that induces maximal lethal toxin neutralizing antibody responses at day 42, determined in the prior experiment. The study is repeated using 10 C3H/HeN female mice per group (antigen alone, antigen + CT, antigen + CpG and antigen + Ang-(1 -7)). Serum and mucosal samples are collected at 1 , 2, 4, and 8 months after the final vaccination to determine if Ang-(1 -7) induces antigen-specific antibody responses that persist over time.

Example 5

Induction of Epitope-specific CD8 Responses Using Ang-(1 -7)

[0230] To evaluate the ability of Ang-(1 -7) to induce epitope-specific CD8 responses as a nasal vaccine adjuvant, BALB/c female mice (15 per group) are nasally immunized on days 0, 7, 14 and 28 with 10 g peptide immunogen (KQIINMWQEVGKAMYA-TRPNNNTRKSIRIQRGPGRAFVTI) to be synthesized alone or combined with Ang-(1 -7) at various doses. CT is the positive control. On days 21 and 35, peripheral blood is collected into anticoagulant tubes for tetramer staining using the H-2Dd tetramers containing the H-2Dd CD8 epitope RGPGRAFVTI, as previously described. On day 42, lymphocytes are isolated from the spleen, cervical lymph node and lung and tested for the presence of epitope-specific T cell responses using tetramer assays and IFN-γ ELISPOT.

Example 6

Viral challenge to by Production by Induction of Epitope-specific Immune

Responses Using Adjuvant, Ang-(1 -7)

[0231 ] If successful at the induction of epitope-specific immune responses using Ang-(1 -7) as an adjuvant, immunization studies are repeated using the control groups and the best dose of Ang-(1 -7) (lowest dose that induces maximal epitope-specific CD8 responses). Viral challenge studies are performed using recombinant vaccinia viruses expressing the RGPGRAFVTI epitope to determine if Ang-(1 -7)-adjuvanted CTL epitope immune responses provide protection against a viral infection. To determine if Ang-(1 -7) is as effective as CT for the induction of epitope-specific CD8 responses, the epitope-specific CD8

responses of the Ang-(1 -7) groups and the CT group are compared using ANOVA and Tukey's multiple comparison tests.

Example 7

Angiotensin Receptors Required for the Adjuvant Activity of Ang-(1-7)

[0232] To determine if the AT1 R angiotensin receptor is required for the adjuvant activity of Ang-(1 -7), the role of ATR1 in the adjuvant activity is evaluated in Ang- (1 -7), wild-type (C57BL/6) and ATR1 -deficient mice by nasal immunization with rPA alone or combined with CT, CpG or Ang-(1 -7). Cellular and humoral responses are monitored as described in Experiment 1 , using 5 mice per group and the experiment is repeated to confirm the results. The rPA-specific humoral and cellular immune responses are evaluated to determine if Ang-(1 -7) demonstrates adjuvant activity in mice deficient in AT1 R. New information is provided on the significance of the angiotensin system in the induction of systemic and mucosal immune responses after nasal immunization. CT and CpG are used as controls to determine if AT1 R influences the adjuvant activity of these classic adjuvants. AT1 R inhibitors, such as losartan may also be used in subsequent studies to confirm the results observed in AT1 R-deficient mice.

Example 8

In Vivo TLR Signaling and the Angiotensin GCPR Complex

[0233] To demonstrate the activity of the angiotensin receptor and the

exogenous angiotensin ligand in innate immune responses and TLR4 receptor signaling in the rat spleen, male Wistar Hanover rats are injected with vehicle or candesartan (1 mg/kg/day sc; Astra-Zeneca) for 3 consecutive days. On day 3, experimental animals from vehicle and candesartan groups are injected with LPS (50 pg/kg ip; Escherichia coli serotype 055:B5, Sigma). The remaining animals from vehicle and candesartan groups are injected with sterile saline. At 3 h after LPS or saline injection, the animals are killed by fast decapitation. Trunk blood is collected into polyethylene tubes with EDTA (1 mg/ml blood). Blood is immediately centrifuged at 1 ,800 g for 15 min at 4°C, and the plasma is stored at -80°C. Spleens are dissected, snap frozen in cold isopentane, and stored at - 80°C.

Measurement of mRNA expression by real-time PCR

[0234] Total RNA is isolated individually from the homogenized rat spleen using TRIzol, purified using an RNeasy Mini kit and treated with DNase I according to the manufacturer's instructions. cDNA is synthesized by reverse transcription using the Superscript III First-Strand Synthesis kit according to the

manufacturer's instructions. Quantitative real-time PCR is performed on DNA Engine Opticon using SYBR Green PCR Master Mix. The amplification conditions consists of one denaturation- activation cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and at 56 or 60°C for 60 s. Serial dilutions of cDNA from the same source are used to obtain a calibration curve. The individual targets for each sample are quantified by determination of the cycle threshold and calibration curves. At the end of amplification, the specificity of the PCR is confirmed by melting temperature determination of the PCR product. The relative amount of the target is normalized with the housekeeping gene 18S rRNA and expressed as arbitrary units.

Western blot analysis

[0235] Spleen samples are homogenized in RIPA buffer (1 :10 wt/vol)

supplemented with 10% glycerol, 1 mM dithiothreitol, 5 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride, and protease inhibitor cocktail. The homogenates are centrifuged for 15 min at 14,000 g at 4°C, and supernatants are saved and stored at -80°C until further processing. Protein concentration is measured by a commercial bicinchoninic acid protein assay kit (Pierce).

Samples are electrophoresed on 10% or 4-12% Nupage Novex Bis-Tris Midi Gel (Invitrogen), and then transferred to polyvinylidene difluoride membranes (Bio- Rad). The membranes are blocked with blocking buffer (Sigma) at room temperature for 60 min and then probed with the primary antibodies overnight at 4°C. After they are washed with Tris-buffered saline [10 mM Tris-CI (pH 7.5) and 100 mM NaCI] containing 0.1 % Tween 20, the membranes are incubated with horseradish peroxidase-conjugated goat anti-mouse (1 : 10,000 -20,000 dilution; (Jackson ImmunoResearch Laboratories) or donkey anti-rabbit IgG (1 :2,000 - 5,000 dilution; GE Healthcare) for 120 min at room temperature. The proteins are visualized by the SuperSignal chemiluminescent substrates (Pierce) and detected by exposure to Kodak XAR film. The intensity of each band is

quantified by densitometry using Scion Image software. β-Actin monoclonal antibody (1 :10,000 dilution; Sigma) is used as a sample loading control. The primary antibodies are as follows: CD14 rabbit polyclonal antibody (1 :200 dilution; Santa Cruz Biotechnologies), TLR4 rabbit polyclonal antibody (1 :200 dilution; Santa Cruz Biotechnologies), COX-2 rabbit polyclonal antibody (1 :1 ,000 dilution; Cayman Chemical, Ann Arbor, Ml), iNOS mouse monoclonal antibody (1 :2, 500 dilution; BD Pharmingen), and gp91 phox (Nox2) rabbit polyclonal antibody (1 :1 ,000 dilution; Millipore). iNOS activity.

[0236] iNOS activity is determined using a commercially available kit (Cayman Chemical) based on the conversion of L-[³H] arginine to L-[³H]citrulline. 25 g of proteins are incubated for 30 min at 37°C in the incubation buffer supplemented with 1 mM NAD(P)H (Sigma), 0.2 uCi of L-[2,3,4,5-3H] arginine per sample (60 nM; GE Healthcare Bio-Sciences), and 10 μΜ L-arginine (Sigma).

Statistical analysis

[0237] Values are means ± SE for groups of seven to nine animals measured individually. Statistical differences are determined by using one-way ANOVA followed by the multiple comparisons Newman-Keuls test.

Example 9

MHC Class I Internalization in Dendritic Cells

[0238] This example determines intracellular compartmentalization and cross- presentation in vivo. Splenic DCs are isolated, cultured overnight at 37 C in complete RPMI and the next day washed in PBS 3 times, aliquoted and labeled with Fc blocker 2.4G2 Fee III/ II for 30 min at 4 C to exclude binding of antibodies to Fc receptor, followed by labeling with AF6-88.5 (H- 2Kb) specific monoclonal antibody conjugated to fluorescein isothiocyanate (FITC) for 30 min at O C in 96-well plates. Next, they are incubated at 37 C and at different time points as indicated; they were chilled to 4 C on ice before fixing in 2%

paraformaldehyde. Recovered DCs are pipetted onto coverslips, mounted on slides and examined with a Nikon multiphoton immuno-fluorescent confocal microscope (ICM). To assess the kinetics of internalization of MHC-I molecules, DCs are isolated, cultured at 37 C and the next day they were washed and stained with Fc blocker followed by AF6-88.5 (H-2Kb) and 36-7-5 (H-2Kk) antibodies (BD Biosciences) conjugated to FITC and phycoerythrin (PE) respectively, at 4 C for 30 min in 96-well plates. Next, sample cells are placed at 37 C, whereas control cells are placed at O C and after different time points DCs are washed 3 times, fixed in ethanol to preserve FITC fluorescence and resuspended in 2% FCS PBS to reach an equal pH of 7.4 and prevent further quenching. DCs with fluorescently labeled H-2Kb molecules are examined using FACSCaliburTM (Becton Dickinson). Data is analyzed using FlowJo software to examine the H-2Kb and H-2Kk molecules following incubation at 37 C as an indication of MHC-I internalization.

Intracellular DC colocalization and image quantification.

[0239] Spleen-derived DCs from transgenic mice are aliquoted in 96-well plates and stained with H-2Kb-FITC antibody after blocking the Fc receptor. Next, DCs are resuspended in 200 ml_ of 37 C pre-warmed completed RPMI, mounted onto Poly-D-lysine precoated coverslips and incubated at 37pC, allowing antibody-bound H-2Kb molecules to internalize. At the end of the last time point, all coverslips containing DCs are treated with 2% BSA in PBS followed by fixation with 2% paraformaldehyde. Spleen-derived DCs are then permeabilized with 0.1 % saponin in 2% BSA PBS followed by incubation with goat anti-mouse EEA1 or LAMP-1 primary antibodies (Santa Cruz Biotechnology). Secondary Alexa-568-conjugated rabbit anti-goat antibody (Molecular Probes) is used as a detection reagent. Isotype control antibodies are used in all confocal microscopy experiments to confirm the specificity of antibody staining. To visualize the acquisition of exogenous OVA peptide by internalized H-2Kb molecules, DCs surface-labeled with H-2Kb- FITC antibody, as described above are incubated at 37 C in complete RPMI containing either 5 mg/mL ovalbumin protein or bovine serum albumin control protein. Next, DCs are mounted onto coverslips and incubated at 37 C for 6 hrs, allowing ovalbumin protein uptake, processing and loading onto H-2Kb molecules. Incubation is halted by chilling the coverslips in ice-cold PBS. DCs are then fixed and permeabilized as described and following Fc blocking for 30 min, they are co-stained with goat anti-mouse EEA1 , LAMP-1 or rat anti-mouse Giantin (Golgi marker) and with anti-H-2Kb/OVA257-264 antibody. Goat anti-mouse or rabbit anti-goat coupled to Alexa-568 (Molecular Probes) are used to detect the endosomes-EEA1 or lysosomes-LAMP-1 and goat anti-Rat coupled to Alexa-568 is used to detect Giantin-Golgi. Goat anti- mouse Alexa-647 labeled is used to visualize the H-2Kb/OVA257-264

complexes. DC surface-derived MHC-I are detected by locating intracellular fluorescent-green punctate dots present. Fluorescence is visualized by ICM using the 488-nm (green) 568-nm (red) and 633-nm (blue) laser lines for excitation of the appropriate fluorochromes. Data is analyzed using lmageJ.1 to select single slices and Adobe Photoshop 9.0 to merge images obtained upon excitation of fluorochromes obtained by red, green and blue channels.

Colocalization of 3 different molecules is evaluated by the presence, intensity and distribution of the white color resulting from the overlapping of green, red and blue. For quantification of co-localization, a total of approximately 50 DCs are examined at 606-magnification. Quantitative confocal image analysis is done by single cell identification using Openlab software and the relative fluorescent intensity of green, red, blue, yellow, purple, light blue and white pixels is assessed. The relative fluorescent intensity of all individual colors is expressed as percent of total fluorescence intensity.

Example 10

Angiotensin Structure Activity Relationship

[0240] The relative potency of the various fragments of angiotensin ligand for augmenting an innate Thi immune response can be demonstrated in an in vitro system. This example demonstrates the relative activity in the production of Thi cytokines, Th₂ cytokines, and endocytic and allostimulatory activity. Briefly, human PBMCs are freshly separated and placed in cultures with the angiotensin peptides (Ang 1 -10, Ang II, Ang-(1 -7), Ang-(1 -5), Ang 2-8, Sham) at

concentrations of 10 mcg/mL to 100 mcg/mL. Dilutions of a solution (e.g., a cell culture supernatant) are mixed with target phytohemagglutinin (PHA-) and Unstimulated PBMCs and incubated at 37° C for 30 min.

Flow cytometry.

[0241 ] Cells are washed twice with PBS supplemented with 2% FCS and resuspended in PBS supplemented with 10% FCS. FITC- and PE-conjugated mAbs are added at saturating concentrations for 30 min at 4°C, and two additional washes are performed. Human cells are stained with the following mAbs: anti-CD1 a FITC, anti-CD14 PE, anti-CD40 PE, anti-CD-86 FITC, and anti- HLA-DR FITC. Mouse cells are stained with the following mAbs: anti-CD1 1 c PE, anti-CD40 FITC, and anti-la FITC. Cell surface antigen expression is evaluated by single or double immunofluorescence staining, and analysis is performed using a FACS flow cytometer and CellQuest software. Analysis of AT1 and AT2 receptor expression is performed by using saturating concentrations of rabbit polyclonal antibodies (Santa Cruz Biotechnology) and FITC-goat anti-rabbit IgG (Sigma).

Allogeneic T cell proliferation.

[0242] Human DCs are extensively washed with complete medium and irradiated (3000 rad from a ¹³⁷Cs source) and 1 10⁴ cells are cocultured with 2 χ 10⁵ responding cells from freshly isolated allogeneic PBMC for 5 days in U-bottom 96-well plates. Thymidine incorporation is measured on day 5 by a 16 h pulse with [3H]-thymidine (1 pCi/well, specific activity, 5 Ci/mM, DuPont). Mixed leukocyte reactions with mouse DC are performed as follows. Mouse DC are irradiated (3000 rad from a 137Cs source), and 5 χ 10⁴ cells are cocultured with 5 x 10⁵ allogeneic splenic cells (C57BL) for 4 days.

Endocytosis of HRP

[0243] Endocytosis of HRP is performed as described previously. Briefly, DCs are incubated at 37°C in complete medium in the presence of 100 g/ml of HRP. After 30 min the cells are collected; washed 4 times in PBS containing 1 % FCS and 4 times in PBS alone with one tube change; lysed with 0.05% Triton X-100 in 10 mM Tris buffer, pH 7.4, for 30 min; and the enzyme activity of the lysate is measured using o-phenylendiamine and H2O2 as substrates with reference to a standard curve, at 492 nm.

Claims

We claim:

1 . An immunogenic composition for inducing cytotoxic T lymphocyte response in a mammal comprising: a. an antigen; and b. a peptide adjuvant capable of stimulation of a Thi cytokine response; wherein the composition is formulated in a pharmaceutically acceptable carrier for administration of the formulation to the mucosal tissue of the mammal.

2. The composition according to claim 1 , wherein the composition is

formulated for administration in a pharmaceutically acceptable carrier selected from the group comprising a tablet, a capsule, a dry-powder inhaler, a metered-dose inhaler, a troche, a sterile solution, a sterile lyophilized powder, an enema, a foam, a suppository, and a gel.

3. The composition according to claim 1 , wherein the antigen is from a virus that is pathogenic in the mammal selected from the group comprising a whole virus, an attenuated virus, an inactivated virus, a viral fragment, recombinant viral vectors, and a virus like particle.

4. The composition according to claim 3, wherein the virus is selected from the group comprising: human immunodeficiency virus, simian

immunodeficiency virus, human papillomavirus, herpes simplex virus, influenza A virus subtypes H1 N1 , H1 N2, H3N1 , H3N2, and H2N3, and a rotavirus.

5. The composition according to claim 1 , wherein the antigen is derived from extracellular tumor associated antigens.

6. The composition according to claim 5, wherein the tumor associated

antigen is selected from the group comprising alphafetoprotein,

carcinoembryonic antigen, CA-125, epithelial tumor antigen , human epidermal growth factor receptor, vascular endothelial growth factor, and prostate specific antigen.

7. The composition according to claim 1 , wherein the antigen is from a

pathogenic organism selected from the group comprising a bacterium, a parasitic organism, and a protozoan.

8. The composition according to claim 7, wherein the pathogenic organism is selected from the group comprising a mycobacterium, yersinia, salmonella, rickettsiae, Cryptosporidium, and leishmaniasis.

9. The composition according to claim 1 , wherein the antigen is an

immunogenically active fragment of an antigen.

10. The composition according to claim 1 , wherein the antigen is a non- immunogenically active fragment of an antigen when used without the peptide adjuvant.

1 1 .The composition according to claim 1 wherein the antigen comprises a peptide pool selected from the group comprising: a) 10 to 100 mer peptide fragments digested from an infectious

pathogen or TAA; b) fragments from multiple pathogens; and c) a pool of fragments of a selected optimized length.

12. The composition according to claim 1 , wherein the peptide adjuvant is

selected from the group comprising a) a mammalian naturally occurring angiotensin I, b) a biologically active fragment of angiotensin I excluding angiotensin II, and c) biologically active peptide sequences and fragments homologous to angiotensin I, and d) biologically active fragments of a) through c) with the proviso that positions 3, 4 and 5 in angiotensin I is conserved.

13. The composition according to claim 1 , wherein the peptide adjuvant is a sequence selected from the group comprising:

Asp-Arg-Va/-7yr-//e-His-Pro-Phe-His-Leu;

Asp-Arg-Va/-7yr-//e-His-Pro-Phe-His;

Asp-Arg-Va/-7yr-//e-H is-Pro;

As p-Arg - Val- Tyr-lle-H is , As p-Arg - Val- Tyr-lle;

Arg-Va/-7yr-//e-His-Pro-Phe-His-l_eu;

Arg-Va/-7yr-//e-His-Pro-Phe-His;

Arg-Va/-7yr-//e-His-Pro-Phe;

Arg - Val-Tyr-lle-H is-Pro, Arg - Val-Tyr-lle;

Va/-7yr-//e-His-Pro-Phe-His-l_eu;

Va/-7yr-//e-His-Pro-Phe-His, V¾/-7yr-//e-His-Pro-Phe, Val-Tyr-lle- is- Pro;

V¾/-7yr-//e-His; and, Val-Tyr-lle.

14. The composition according to claim 1 , wherein the peptide adjuvant is not conjugated to a carrier protein.

15. The composition according to claim 1 , wherein the peptide adjuvant is biologically activated after the composition is administered to the mammal by an endogenous amidase or peptidase.

16. The composition according to claim 15, wherein the endogenous amidase or peptidase is selected from the group comprising angiotensin converting enzyme 1 , angiotensin converting enzyme 2, chymase, neutral

endopeptidase, and aminopeptidase.

17. The composition according to claim 1 , wherein the peptide adjuvant binds a cell surface receptor selected from the group comprising an angiotensin type 1 receptor, an angiotensin type 2 receptor, an angiotensin type 4 receptor, a Mas receptor, and a renin-prorenin receptor.

18. The composition according to claim 1 , wherein the composition is

formulated for administration via a route selected from the group comprising inhalation, intranasal, oral, rectal, vaginal, subcutaneous, and transdermal.

19. The composition according to claim 1 , wherein the composition is

formulated for administration with a carrier selected from the group comprising a binder, a base, a carrier, a diluent, an enteric coating, an excipient, an oncotic agent, a stabilizing agent, a cyclodextrin or beta- cyclodextrin derivatives, a propellant, a medium-chain fatty acid, a PLGA microparticle, a nanoparticle, a liposome, an absorption-promoting agent, a surfactant, and a mixed micelle.

20. A method for inducing a desired cytotoxic T lymphocyte response in a mammal comprising administering to mucosal tissue of the mammal at a desired interval with a composition comprising: a) an antigen; and b) a peptide adjuvant capable of stimulation of a T n cytokine response; wherein the composition is formulated in a pharmaceutically acceptable carrier for administration of the formulation to the mucosal tissue of the mammal.

21 . The method according to claim 20, wherein the composition stimulates an innate immune response, characterized by enhanced innate immune signaling proteins, in a mammal thereby enhancing the adaptive immune response to a foreign or self-antigen.

22. The method according to claim 21 , wherein the composition is coadministered with a PAMP.

23. The method according to claim 20, wherein the cytotoxic T lymphocyte immune response in a mammal is antigen-specific and characterized by adaptive T n cytokines.

24. The method according to claim 20, wherein the peptide adjuvant

administered is selected from the group comprising a) a mammalian naturally occurring angiotensin I, b) a biologically active fragment of angiotensin I excluding angiotensin II, and c) biologically active peptide sequences and fragments homologous to angiotensin I, and d) biologically active fragments of a) through c) with the proviso that positions 3, 4 and 5 in angiotensin I is conserved.

25. The method according to claim 20, wherein the peptide adjuvant administered is selected from the group of amino acid sequences comprising:

Asp-Arg-Va/-7yr-//e-His-Pro-Phe-His-Leu;

Asp-Arg-Va/-7yr-//e-His-Pro-Phe-His;

Asp-Arg-Va/-7yr-//e-H is-Pro;

As p-Arg - Val- Tyr-lle-H is ;

Asp-Arg-Va/-7yr-//e, Arg-Va/-7yr-//e-His-Pro-Phe-His-Leu, Arg-Va/- 7yr-//e-His-Pro-Phe-His;

Arg-Va/-7yr-//e-His-Pro-Phe;

Arg - Val-Tyr-lle-H is-Pro, Arg - Val-Tyr-lle;

Va/-7yr-//e-His-Pro-Phe-His-Leu, Va/-7yr-//e-His-Pro-Phe-His, Val-Tyr- //e-His-Pro-Phe, Val-Tyr-lle- is-Pro; Val-Tyr-lle- \s; and, Val-Tyr-lle.

26. The method according to claim 20, wherein the peptide adjuvant administered to the mammal is biologically activated by an endogenous amidase or peptidase.

27. The method according to claim 26 wherein the endogenous amidase or peptidase is selected from the group comprising angiotensin converting enzyme 1 , angiotensin converting enzyme 2, chymase, neutral endopeptidase, and aminopeptidase.

28. The method according to claim 20, wherein the peptide adjuvant administered binds a cell surface receptor selected from the group comprising an angiotensin type 1 receptor, an angiotensin type 2 receptor, an angiotensin type 4 receptor, a Mas receptor, and a renin-prorenin receptor.

29. The method according to claim 20, wherein the composition is administered to the mammal in a pharmaceutically acceptable carrier selected from the group comprising a tablet, a capsule, a dry-powder inhaler, a metered-dose inhaler, a troche, a sterile solution, a sterile lyophilized powder, an enema, a foam, a suppository, and a gel.

30. The method according to claim 20, wherein the composition is administered via a route selected from the group comprising inhalation, intranasal, oral, rectal, vaginal, and transdermal, .

31 . The method according to claim 20, wherein the composition is administered to the mucosal tissue of the mammal at a selected interval sufficient to maintain memory of the antigen-specific cytotoxic T lymphocyte response.

32. The method according to claim 20, wherein the peptide adjuvant administered induces a Thi dominant response.

33. The method according to claim 20, wherein after administration of the composition induces mucosal dendritic cell uptake, MHC class 1 presentation of the antigen and initiates Thi cytokine signal initiation of dendritic cell activation, mobilization and maturation necessary for cytotoxic T cell activation, and surveillance of cytotoxic T cells in the mammal.

34. The method according to claim 20, wherein the selected antigen comprises a peptide pool selected from the group comprising: a) 10 to 100 mer peptide fragments digested from an infectious pathogen or TAA; b) fragments from multiple pathogens; and c) a pool of fragments of a selected optimized length.

35. A method according to claim 21 , wherein the adaptive immune response is in response to an antigen selected from the group comprising: human immunodeficiency virus, simian immunodeficiency virus, human

papillomavirus, herpes simplex virus, influenza A virus subtypes H1 N1 , H1 N2, H3N1 , H3N2, and H2N3, and a rotavirus.

36. A method of preventing or treating a viral disease comprising administering an immunostimulatory antigen fragment mucosally to an individual in combination with a peptide adjuvant.

37. A method according to claim 36, wherein the virus is an immunodeficiency virus.