[go: up one dir, main page]

WO2011053789A2 - Composition pharmaceutique et procédés pour améliorer la reconnaissance de lymphocytes t cytotoxiques et maintenir la mémoire à lymphocytes t contre une maladie pathogène - Google Patents

Composition pharmaceutique et procédés pour améliorer la reconnaissance de lymphocytes t cytotoxiques et maintenir la mémoire à lymphocytes t contre une maladie pathogène Download PDF

Info

Publication number
WO2011053789A2
WO2011053789A2 PCT/US2010/054726 US2010054726W WO2011053789A2 WO 2011053789 A2 WO2011053789 A2 WO 2011053789A2 US 2010054726 W US2010054726 W US 2010054726W WO 2011053789 A2 WO2011053789 A2 WO 2011053789A2
Authority
WO
WIPO (PCT)
Prior art keywords
antigen
angiotensin
pro
peptide
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2010/054726
Other languages
English (en)
Other versions
WO2011053789A3 (fr
Inventor
James Cameron Oliver
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of WO2011053789A2 publication Critical patent/WO2011053789A2/fr
Publication of WO2011053789A3 publication Critical patent/WO2011053789A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/07—Bacillus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0031—Rectum, anus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to compositions and methods for an immunostimulatory effect.
  • 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- ⁇ , induction of antigen cross-presentation, immunoglobulin class-switching, and enhanced presentation of the antigen by the MHC class I - TCR complex.
  • CTL cytotoxic T lymphocyte
  • 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.
  • MHC class I processing pathway 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.
  • Cytotoxic T lymphocytes 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.
  • a pathogenic organism such as a virus or tumor associated antigen.
  • T cell mediated-immunity has relied upon demonstration of antigen-specific T cell activation.
  • T cell immunity positive results are associated with tetramer binding by antigen specific T cells and potent epitope dominate pathogen response.
  • the presence of (in vivo) antigen specific antibodies can be detected in plasma.
  • potent systemic T cell response, systemic antibody response, and antigen specific tetramer binding are considered as all required for scientific demonstration of activity.
  • the present invention vaccine compositions comprise a novel immuno- adjuvant ligand in combination with one or more antigens.
  • 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).
  • Ang-GCPR AT1 , AT2, AT4, and AT7.
  • 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.
  • PRR a cytotoxic T lymphocyte
  • CTL cytotoxic T lymphocyte
  • 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.
  • 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.
  • 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.
  • MAPK mitogen-activated protein
  • 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
  • the composition provides that the peptide adjuvant directs a Th1 dominate CTL and is characterized by increased interferon- ⁇ .
  • 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.
  • Th1 immune response results in other Th1 cytokines including, but not limited to, IL-2, IL-3, IL-12, INF- ⁇ , TNF-a, TNF- ⁇ , TGF- ⁇ , and GM-CSF.
  • 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.
  • 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.
  • 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.
  • TLR4 toll like receptor 4
  • 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- ⁇ transcription factor nuclear translocation and inducing the expression of chemokines.
  • 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
  • MHC class I TCR complexes direct autocrine and paracrine Thi cytokine/chemokine synthesis, and up-regulate acquired immunity and potential immunoglobulin class-switching.
  • 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.
  • 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.
  • 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.
  • 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- ⁇ , induction of antigen cross-presentation, allow for
  • 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.
  • 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.
  • the invention provides methods for
  • a peptide adjuvant and antigen that infects an antigen presenting or dendritic cell and is processed in the cytosol and
  • the antigen may be endocytosed and cross-presented by an antigen presenting cell via the MHC Class I complex.
  • 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.
  • Fig. 1 is a diagram of the endogenous Renin-Angiotensin Metabolic Cascade.
  • ACE angiotensin-converting enzyme
  • ACE2 angiotensin- converting enzyme
  • 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-converting enzyme
  • Ang 11 an
  • angiotensin-(1-7) receptor that is inhibited by pro7-angiotensin-(1-7);
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • AT1 angiotensin receptor- type 1
  • AT2 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.
  • 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.
  • 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.
  • 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).
  • virus examples 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.
  • 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.
  • 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.
  • 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.
  • the antigen is derived from extracellular turmor associated antigens.
  • 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
  • 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).
  • test control e.g. endotoxin
  • inducible measurable cytokine patterns are within the skill in the art from the description herein.
  • Cancerous cells can be benign or malignant.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • cytotoxic T lymphocyte 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.
  • 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.
  • 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.
  • 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
  • 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.
  • CTL response refers 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.
  • 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.
  • innate immune response refers to a response involving the characteristics of "innate immunity” described above.
  • major histocompatibility complex 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.
  • MHC II major histocompatibility complex II
  • APCs professional antigen- presenting cells
  • 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
  • mucosal and mucosa are used interchangeably herein and refer to immune inductive sites collectively termed the mucosa associated lymphoid tissue (MALT).
  • MALT mucosa associated lymphoid tissue
  • NALT nasal- or nasopharynx-associated lymphoid tissue
  • BALT bronchus- associated lymphoid tissue
  • GALT gut-associated lymphoid tissue
  • pathogen-associated molecular pattern refers to a molecular pattern found in microorganisms.
  • a PAMP is capable of triggering an innate immune response when it binds to a PRR.
  • immunostimulatory structures are potential initiators of innate immune response, and therefore, ligands for PRRs, including toll-like receptors (TLR).
  • TLR toll-like receptors
  • 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.
  • 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.
  • ACE 1 (CD143) is a dipeptidase
  • ACE2 is a monopeptidase
  • Neutral endopeptidase is a tripeptidase named neprilysin
  • 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
  • aminopeptidase A aminopeptidase M cleave angiotensin III and angiotensin IV, respectively.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • pathogen infection refers 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.
  • T cell receptor refers 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.
  • MHC major histocompatibility complex
  • 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.
  • 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- ⁇ , TNF-a, TNF- ⁇ , TGF- ⁇ , and GM-CSF.
  • Th1 cells direct the clonal expansion of activate
  • NK cells NK cells
  • CD8+ cells MHC Class I antigen presentation, dendritic cell mobilization and maturation, phagocytosis, plus the secretion of certain Ig isotypes.
  • 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- ⁇ , 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.
  • Th2 cytokines depress macrophage activation and cell mediated immunity.
  • TLR toll-like receptor
  • TLRs recognize microbes that have breached physical barriers such as the skin or intestinal tract mucosa, and activate immune cell responses.
  • 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
  • TIR Toll-IL- 1 receptor
  • 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.
  • treatment refers 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 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).
  • 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.
  • Vaccine refers to a composition comprising an antigen, and optionally other ancillary molecules, the purpose of which is to administer such
  • ancillary molecules are adjuvants, which are non-specific immunostimulatory, molecules, and other molecules that improve the
  • 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.
  • biotechnological approaches used for producing vaccines (see, e.g., W. Bains (1998) Biotechnology From A to Z, Second Edition, Oxford University Press).
  • 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.
  • CD4+ T cells provide signals to activate other cells, e.g., CD4+ cells activate CD8+ cells, to induce B cell to produce
  • CD8+ cells are cytotoxic, and recognize antigens displayed with MHC Class I molecules on the cell surface.
  • Th 0 cells Upon activation by IL-2, na ' fve Th cells become Th 0 , since they have both Thi and Th 2 characteristics. Th 0 cells undergo clonal expansion towards Thi following the release of INF- ⁇ and TNF-a by Th 0 cells or towards Th 2 following the release of IL-4. Thi cells secrete IL-2, 11-12, INF- ⁇ , and down regulated Th2 cells with additional IL-2. Th 2 cells secrete IL-3, IL-4, IL-5, IL-6, and down regulated Th1 cells with additional IL-4.
  • T cells While the categorization of T cells as Thi , Th 2 , or Th 0 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 Th 2 cells at opposite ends of the scale, and Th 0 cells providing the middle of the spectrum.
  • 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.
  • APCs professional antigen presenting cells
  • DCs dendritic cells
  • macrophages macrophages
  • 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.
  • 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.
  • cross-priming 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.
  • DCs dendritic cells
  • APCs dendritic cells
  • DC function is integrally linked with the ability of these cells to alter their properties in response to environmental signals.
  • 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.
  • DCs can drive different types of immune responses.
  • distinct subsets of DCs exist. Accordingly, heterogeneity amongst DCs is associated with variation in their ability to induce cross-priming.
  • DCs 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.
  • 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.
  • IFN-a interferon- alpha
  • IL-1 interferon- alpha
  • IL-6 interferon- alpha
  • IL-12 interferon- alpha
  • MIP-1 y macrophage inflammatory protein
  • 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 Th 2 (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.
  • nitric oxide NO
  • PGE2 prostaglandin E2
  • IL-10 IFN-a itself
  • TGF- ⁇ transforming growth factor b
  • the IFN- ⁇ protein is encoded by the IFNG gene.
  • IFN- ⁇ is the hallmark cytokine of Thi cells (whereas Th 2 cells produce IL-4 and Thi 7 cells produce IL-17).
  • NK cells and CD8+ cytotoxic T cells also produce IFN- ⁇ .
  • 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.
  • IFN- ⁇ 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- ⁇ 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- ⁇ is produced
  • NK natural killer
  • NKT natural killer T
  • CTL cytotoxic T lymphocyte
  • IFN- ⁇ has antiviral, immunoregulatory, and anti-tumor properties.
  • the effects are: 1 ) increase antigen presentation of macrophages, 2) activate and increase lysosome activity in macrophages, 3) suppress Th 2 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- ⁇ .
  • the renin-angiotensin system 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).
  • Ang 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.
  • angiotensinogen I by renin into the decapeptide angiotensin I (Ang I).
  • Ang I decapeptide angiotensin I
  • ACE1 angiotensin converting enzyme
  • ACE2 angiotensin-converting enzyme 2
  • a (1 -7) by the a number of endopeptidases including neprilysin, prolyl-oligopeptidase, and thimet endopeptidase.
  • 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.
  • 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.
  • 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.
  • 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.
  • AT1 angiotensin type 1 receptor
  • AT2 angiotensin type 2 receptor
  • AT4 angiotensin type 4 receptor
  • AT7 angiotensin type 7 receptor
  • 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.
  • 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.
  • 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- ⁇ , TNF-a, TGF- ⁇ , IL-12, IL-2, and IL-8.
  • Th1/Th2 cells 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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/a/
  • the dosage of peptide adjuvant consisting of Asp- Arg-Val-Tyr-lle-His-Pro should be greater than 1 mcg/kg/dose and less than 100 mcg/kg/dose.
  • 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.
  • the use of in vitro cytotoxicity assays can be helpful in optimizing the treatment regimen.
  • 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
  • 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.
  • 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.
  • 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.
  • 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
  • the composition provides that the peptide adjuvant directs a Th1 dominate CTL and is characterized by increased interferon- ⁇ .
  • 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.
  • Th1 immune response results in other Th1 cytokines including, but not limited to, IL-2, IL-3, IL-12, INF- ⁇ , TNF-a, TNF- ⁇ , TGF- ⁇ , and GM-CSF.
  • Th1 cytokines maximize the killing efficacy of the macrophages and the proliferation of cytotoxic CD8+ T cells.
  • the Type 1 cytokine IFN- ⁇ increases the production of interleukin-12 by dendritic cells and macrophages, and via positive feedback, IL-12 stimulates the production of IFN- ⁇ in helper T cells, thereby promoting the Th1 profile.
  • 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.
  • 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).
  • 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.
  • Determination of an effective response varies for specific antigens.
  • 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.
  • 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.
  • 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.
  • 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.
  • the solid composition includes a stabilizing agent to minimize
  • 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.
  • 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.
  • 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.
  • 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
  • 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
  • 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.
  • 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.
  • compositions 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • enveloped viruses examples include 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.
  • 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
  • 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.
  • 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.
  • the use of in vitro cytotoxicity assays can be helpful in optimizing the treatment regimen.
  • 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.
  • the core can be
  • 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
  • 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.
  • 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.
  • the effective amount can be easily determined from the literature relating to the virus of interest.
  • 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
  • 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.
  • the viral particles can be used in combination with other prophylactic or therapeutic substances.
  • mixtures of different viral particles can be employed in the method of the invention.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • cell surface receptors such as human epidermal growth factor receptor, vascular endothelial growth factor, and prostate specific antigen.
  • 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.
  • 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.
  • TAA tumor associated antigen
  • 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.
  • 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.
  • composition of the present invention is administered intranasally in a physiologically buffered solution of
  • composition is administered in an oral liposome.
  • 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.
  • the use of in vitro cytotoxicity assays can be helpful in optimizing the treatment regimen.
  • 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.
  • a suitable control may be a
  • a suitable control may be the tumor load present before administering the immunostimulatory antigen.
  • Other suitable controls may be a placebo control.
  • 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.
  • 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.
  • Mucosal immune inductive sites are collectively termed the mucosa- associated lymphoid tissue (MALT).
  • MALT mucosa- associated lymphoid tissue
  • NALT nasal- or nasopharynx-associated lymphoid tissue
  • BALT bronchus- associated lymphoid tissue
  • GALT gut-associated lymphoid tissue
  • DCs which take up antigens from the lumen
  • 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.
  • Conventional and pharmaceutically acceptable routes of administration include intranasal, , intratracheal, intratumoral, subcutaneous, intradermal, topical application, rectal, nasal, oral and other parenteral routes of
  • 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.
  • 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.
  • routes of administration contemplated by the invention include, but are not necessarily limited to, enteral, parenteral, or inhalational routes.
  • 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
  • Such means include inhalation of aerosol suspensions or insufflation of the
  • 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.
  • intranasal drug delivery see, e.g., Chien, Novel Drug Delivery Systems, Ch. 5 (Marcel Dekker, 1992).
  • intranasal (IN) vaccination is used due to ease of administration and the lesser amount of antigen needed to induce an immune response.
  • 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
  • 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
  • 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.
  • NYVAC molecularly attenuated vaccine vector
  • 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.
  • 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.
  • 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.
  • the present invention is formulated as a
  • nanopartide using the ligand angiotensin 1 -7 as a targeting ligand is provided in the present invention.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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 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.
  • recombinant Salmonella enterica serovar Typhi expressing HIV Gag or Env or both, can be administered intranasally.
  • 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.
  • 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 vaccination may target the tonsils rather than the Gl tract.
  • 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.
  • 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.
  • a suitable pharmaceutical preparation for transdermal transmission, absorption promoters or iontophoresis are suitable methods.
  • absorption promoters or iontophoresis are suitable 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.
  • 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
  • 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.
  • a systemic or mucosal route e.g., intranasally, intrarectally, intravenously, subcutaneously, intradermally, or intraperitoneally, and the like, e.g., by a clinician.
  • 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.
  • 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).
  • infectious diseases caused by intracellular pathogens, such as viruses, intracellular bacteria, fungi and parasites (e.g. protozoans).
  • opportunistic infections can be treated using the methods of the invention.
  • 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 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.
  • 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.
  • 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.
  • 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 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
  • PCR polymerase chain reaction
  • 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.
  • a suitable control may be a genetically identical animal not treated with the immunostimulatory antigen.
  • a suitable control may be the infectious disease present before administering the immunostimulatory antigen.
  • Other suitable controls may be a placebo control.
  • 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.
  • 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.
  • ELISA enzyme-linked immunosorbent assays
  • Other assays include in vivo assays for toxins.
  • 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.
  • 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.
  • 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.
  • CMI cell mediated immunity
  • 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.
  • the methods are carried out in vitro or ex vivo. In these
  • 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.
  • the invention provides methods for increasing chemokine production and secretion in an antigen non-specific manner.
  • cells are contacted with, or individuals are administered with, immunostimulatory antigen in combination with a peptide adjuvant without antigen.
  • 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.
  • 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 ⁇ -1 ⁇ .
  • 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, ⁇ 4, ⁇ 6, ⁇ 7, ⁇ 8, ⁇ 9, ⁇ , ⁇ 12, and ⁇ 13, 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
  • production and secretion of a chemokine is antigen-specific.
  • 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.
  • 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.
  • chemokine secretion is increased in an antigen specific manner can be readily determined by those skilled in the art using standard methods.
  • 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.
  • In vitro and ex vivo methods of the invention comprise contacting a cell that normally produces a chemokine with an immunostimulatory antigen.
  • 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
  • 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
  • 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.
  • a suitable time e.g., about 10 minutes to about 24 hours
  • Bio 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.
  • 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.
  • immunostimulatory antigens are prepared in a
  • compositions of the invention 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.
  • non-aqueous solvents are propylene glycol,
  • 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
  • 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.
  • 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.
  • the 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
  • immunostimulatory antigen in combination with a peptide adjuvant formulation comprises an additional anti-mycobacterial agent.
  • 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.
  • target cells e.g., by macrophages
  • Absorption promoters, detergents and chemical irritants can enhance transmission of an immunostimulatory antigen in combination with a peptide adjuvant composition into a target tissue (e.g., through the skin).
  • a target tissue e.g., through the skin.
  • Suitable nasal absorption promoters 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
  • 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.
  • 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.
  • phospholipids or other lipids may also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidyl-glycerol, phosphatidylcholine, phosphatidylserine,
  • phosphatidylethanolamine phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
  • 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.
  • 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 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 ligand.
  • composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • the surface of the targeted delivery system may be modified in a variety of ways.
  • 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;
  • 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
  • An immunostimulatory antigen in combination with a peptide adjuvant can be administered to an individual in combination (e.g., in the same
  • 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.
  • 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.
  • 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,
  • clathiromycin erythropoietin
  • colony stimulating factors G-CSF and GM-CSF
  • non-nucleoside reverse transcriptase inhibitors nucleoside inhibitors
  • adriamycin adriamycin, fluorouracil, methotrexate, asparaginase and combinations thereof.
  • 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
  • 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.
  • 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 contemplates administration of "booster" doses to provide and maintain a desired immune response.
  • 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).
  • Vaginal suppositories were prepared using UrovacTM, 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.
  • Each suppository consisted of 2 g suppository base a Witepsol 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.
  • 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,
  • 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).
  • HPMC capsules 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 (Mg 3 Si 4 Oio (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.
  • talc Mg 3 Si 4 Oio (OH) 2 , Sigma, St. Louis, MO
  • 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).
  • 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.
  • PBMC peripheral blood mononuclear cells
  • Virus is harvested when p24 or reverse transcriptase (RT) is detected in the supernatant. Dilutions of a solution (e.g., a cell culture
  • 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).
  • a sensitive nonradioactive method e.g., a commercially available assay, e.g., the Retrosys RT activity kit from Innovagen AB, Lund, Sweden.
  • 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).
  • infectious diseases caused by intracellular pathogens such as viruses, intracellular bacteria, fungi and parasites (e.g. protozoans).
  • opportunistic infections can be treated using the methods of the invention.
  • [021 1 ] (Macaca mulatta) are screened for the presence of the Mamu-A * 01 allele using a PCR-based technique.
  • Liposomal dispersions are prepared by reverse phase evaporation.
  • 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.
  • 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.).
  • 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.
  • 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
  • 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.
  • 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 10 7 , 6 X 10 6 , and 6 X 10 5 copies of virus with each
  • 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.
  • Plasma SIV RNA levels Plasma SIV RNA levels.
  • Plasma viral RNA levels are measured by an ultrasensitive branched DNA amplification assay with a detection limit of 125 copies per ml (Bayer
  • 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- ⁇ )- 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).
  • TNF-a anti-tumor necrosis factor alpha
  • FITC anti-gamma interferon
  • PE phycoerythrin
  • IL-2 anti-interleukin-2
  • API
  • ELISPOT assays multiscreen 96-well plates are coated overnight with 100 ⁇ per well of 5- g/ml anti-human IFN- ⁇ (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 10 5 PBMC in triplicate in 100- ⁇ 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- ⁇ 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.
  • ELISPOT assays are performed on PBMC obtained on weeks 6, 7, 21 , and 27 following the first exposure to virus.
  • PBMC are incubated at 37°C in a 5% CO 2 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.
  • MAbs monoclonal antibodies
  • 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.
  • 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 PBMC (5 X 10 6 ) are resuspended in 100 ⁇ 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.
  • PBMC (4 X 10 6 ) in 1 ml of R12 are cultured in the presence of l -pg/ml SIV Gag p1 1 C (CTPYDINQM).
  • CTPYDINQM l -pg/ml SIV Gag p1 1 C
  • 40-U/ml human recombinant IL-2 Hoffman-La Roche
  • peptide- stimulated PBMC are centrifuged over a Ficoll gradient and washed.
  • Peptide- stimulated PBMC (5 X 10 5 ) are re-suspended in 100 ⁇ 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.
  • 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 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.
  • 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.
  • compositions as a Therapeutic Treatment for SIV Infected Animals
  • the vaccine is evaluated when administered every day therapeutically in an intranasal vaccine liposome. Activity is assessed using mRNA viral load measurements.
  • 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.
  • 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 Th 2 -biased immune responses
  • 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.
  • mice are euthanized and splenocytes harvested for in vitro restimulation with rPA.
  • Th 1 /Th 2 /Th 17 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- ⁇ 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.
  • CT computed tomography
  • 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.
  • mice 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.
  • 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.
  • 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- ⁇ ELISPOT.
  • 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.
  • mice male Wistar Hanover rats are injected with vehicle or candesartan (1 mg/kg/day sc; Astra-Zeneca) for 3 consecutive days.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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
  • ⁇ -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 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
  • iNOS activity is determined using a commercially available kit (Cayman Chemical) based on the conversion of L-[ 3 H] arginine to L-[ 3 H]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 ⁇ L-arginine (Sigma).
  • 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%
  • DCs are pipetted onto coverslips, mounted on slides and examined with a Nikon multiphoton immuno-fluorescent confocal microscope (ICM).
  • ICM Nikon multiphoton immuno-fluorescent confocal microscope
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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
  • 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.
  • 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).
  • Human DCs are extensively washed with complete medium and irradiated (3000 rad from a 137 Cs source) and 1 10 4 cells are cocultured with 2 ⁇ 10 5 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 ⁇ 10 4 cells are cocultured with 5 x 10 5 allogeneic splenic cells (C57BL) for 4 days.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Otolaryngology (AREA)
  • Dispersion Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des procédés et des compositions pharmaceutiques comprenant une séquence peptidique d'angiotensine I endogène ou des fragments biologiquement actifs de celle-ci en combinaison avec un antigène ou une partie immunogène d'un antigène pour induire une réponse à lymphocytes T cytotoxiques robuste et maintenir une mémoire à lymphocytes T cytotoxiques antigène-spécifique persistante, et leurs utilisations pour traiter ou prévenir des affections pathogènes.
PCT/US2010/054726 2009-10-30 2010-10-29 Composition pharmaceutique et procédés pour améliorer la reconnaissance de lymphocytes t cytotoxiques et maintenir la mémoire à lymphocytes t contre une maladie pathogène Ceased WO2011053789A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US25676009P 2009-10-30 2009-10-30
US61/256,760 2009-10-30

Publications (2)

Publication Number Publication Date
WO2011053789A2 true WO2011053789A2 (fr) 2011-05-05
WO2011053789A3 WO2011053789A3 (fr) 2011-09-15

Family

ID=43923001

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2010/054726 Ceased WO2011053789A2 (fr) 2009-10-30 2010-10-29 Composition pharmaceutique et procédés pour améliorer la reconnaissance de lymphocytes t cytotoxiques et maintenir la mémoire à lymphocytes t contre une maladie pathogène

Country Status (1)

Country Link
WO (1) WO2011053789A2 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140205631A1 (en) * 2013-01-23 2014-07-24 University Of Southern California Stimulation of vaccination by angiotensin peptides
WO2014134621A3 (fr) * 2013-03-01 2014-11-06 Colorado State University Research Foundation Procédés et compositions permettant d'améliorer une réponse immunitaire, de bloquer la migration des monocytes, d'amplifier l'immunité d'un vaccin et d'inhiber la croissance d'une tumeur et des métastases
US8975290B2 (en) 2013-03-01 2015-03-10 Colorado State University Research Foundation Methods and compositions for enhancing an immune response, blocking monocyte migration, amplifying vaccine immunity and inhibiting tumor growth and metastasis
US9320735B2 (en) 2008-02-26 2016-04-26 Colorado State University Research Foundation Myeloid derived suppressor cell inhibiting agents
CN108603171A (zh) * 2015-12-23 2018-09-28 基因医疗免疫疗法有限责任公司 新一代抗原-特异性tcr
WO2019238962A1 (fr) * 2018-06-14 2019-12-19 University College Cork - National University Of Ireland, Cork Peptide pour le traitement de maladies
CN114891074A (zh) * 2022-05-10 2022-08-12 中山大学·深圳 一种季节性甲型流感通用病毒样颗粒及其制备方法与应用
US12144898B2 (en) 2020-04-09 2024-11-19 Finncure Oy Virus-like particles for preventing the spreading and lowering the infection rate of viruses
US12194157B2 (en) 2020-04-09 2025-01-14 Finncure Oy Carrier for targeted delivery to a host

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2001271926A1 (en) * 2000-07-13 2002-01-30 University Of Southern California Methods for promoting dendritic cell proliferation or differentiation
US20070053923A1 (en) * 2003-01-30 2007-03-08 Ki-Seok Park Dna vaccine composition with enhanced immunogenicity
GB0427267D0 (en) * 2004-12-13 2005-01-12 Maria Teresa De Magistris Peptide adjuvants
CA2696068A1 (fr) * 2007-08-14 2009-02-19 Thymon, L.L.C. Compositions et procedes pour le traitement et la prophylaxie de l'hypertension

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9320735B2 (en) 2008-02-26 2016-04-26 Colorado State University Research Foundation Myeloid derived suppressor cell inhibiting agents
WO2014116587A1 (fr) * 2013-01-23 2014-07-31 University Of Southern California Stimulation de vaccination par des peptides d'angiotensine
US20140205631A1 (en) * 2013-01-23 2014-07-24 University Of Southern California Stimulation of vaccination by angiotensin peptides
US10206983B2 (en) 2013-03-01 2019-02-19 Colorado State University Research Foundation Methods and compositions for enhancing an immune response, blocking monocyte migration, amplifying vaccine immunity and inhibiting tumor growth and metastasis
WO2014134621A3 (fr) * 2013-03-01 2014-11-06 Colorado State University Research Foundation Procédés et compositions permettant d'améliorer une réponse immunitaire, de bloquer la migration des monocytes, d'amplifier l'immunité d'un vaccin et d'inhiber la croissance d'une tumeur et des métastases
US8975290B2 (en) 2013-03-01 2015-03-10 Colorado State University Research Foundation Methods and compositions for enhancing an immune response, blocking monocyte migration, amplifying vaccine immunity and inhibiting tumor growth and metastasis
US9539314B2 (en) 2013-03-01 2017-01-10 Colorado State University Research Foundation Methods and compositions for enhancing an immune response, blocking monocyte migration, amplifying vaccine immunity and inhibiting tumor growth and metastasis
CN108603171A (zh) * 2015-12-23 2018-09-28 基因医疗免疫疗法有限责任公司 新一代抗原-特异性tcr
WO2019238962A1 (fr) * 2018-06-14 2019-12-19 University College Cork - National University Of Ireland, Cork Peptide pour le traitement de maladies
US12144898B2 (en) 2020-04-09 2024-11-19 Finncure Oy Virus-like particles for preventing the spreading and lowering the infection rate of viruses
US12194157B2 (en) 2020-04-09 2025-01-14 Finncure Oy Carrier for targeted delivery to a host
US12311061B2 (en) 2020-04-09 2025-05-27 Finncure Oy Methods of fabricating carriers for targeted delivery to a host
US12409149B2 (en) 2020-04-09 2025-09-09 Finncure Oy Methods of targeted delivery to a host using a carrier
CN114891074A (zh) * 2022-05-10 2022-08-12 中山大学·深圳 一种季节性甲型流感通用病毒样颗粒及其制备方法与应用

Also Published As

Publication number Publication date
WO2011053789A3 (fr) 2011-09-15

Similar Documents

Publication Publication Date Title
WO2011053789A2 (fr) Composition pharmaceutique et procédés pour améliorer la reconnaissance de lymphocytes t cytotoxiques et maintenir la mémoire à lymphocytes t contre une maladie pathogène
Si et al. Intranasal delivery of adjuvant-free peptide nanofibers elicits resident CD8+ T cell responses
Debin et al. Intranasal immunization with recombinant antigens associated with new cationic particles induces strong mucosal as well as systemic antibody and CTL responses
Mann et al. Lipid vesicle size of an oral influenza vaccine delivery vehicle influences the Th1/Th2 bias in the immune response and protection against infection
JP4359654B2 (ja) 抗原特異的免疫応答を生起させる遺伝子発現ベクターおよびその使用方法
US6534062B2 (en) Methods for increasing a cytotoxic T lymphocyte response in vivo
Jones et al. HIV-1 vaccination by needle-free oral injection induces strong mucosal immunity and protects against SHIV challenge
US7488491B2 (en) Use of glycosylceramides as adjuvants for vaccines against infections and cancer
TW202144383A (zh) 冠狀病毒免疫原性組合物及其等之用途
Luci et al. Dendritic cell-mediated induction of mucosal cytotoxic responses following intravaginal immunization with the nontoxic B subunit of cholera toxin
US9364563B2 (en) Methods and compositions for liposomal formulation of antigens and uses thereof
JP2025061056A (ja) 異なる核酸アジュバントの組み合わせによる、新規Ｔｈ１誘導性アジュバントおよびその用途
EP2701734B1 (fr) Formulations liposomales
Bakkari et al. Toll-like receptor-4 (TLR4) agonist-based intranasal nanovaccine delivery system for inducing systemic and mucosal immunity
US11628208B2 (en) System and method for microneedle delivery of microencapsulated vaccine and bioactive proteins
US10786558B2 (en) Oral dissolving films
Wilmar et al. The cationic lipid, diC14 amidine, extends the adjuvant properties of aluminum salts through a TLR-4-and caspase-1-independent mechanism
Cho et al. Enhanced humoral and cellular immune responses after sublingual immunization against human papillomavirus 16 L1 protein with adjuvants
Kakutani et al. Mucosal vaccination using claudin-4-targeting
US20140093537A1 (en) Immunogenic compositions comprising nanoemulsion and methods of administering the same
US10849962B2 (en) Method and apparatus for microneedle transdermal delivery
JP6152944B2 (ja) 束縛免疫原性組成物およびその用途
Francis et al. Increasing cellular immunogenicity to peptide-based vaccine candidates using a fluorocarbon antigen delivery system
Domm et al. Robust antigen-specific humoral immune responses to sublingually delivered adenoviral vectors encoding HIV-1 Env: association with mucoadhesion and efficient penetration of the sublingual barrier
KR20210079319A (ko) 흡입용 건조 약학적 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10827527

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10827527

Country of ref document: EP

Kind code of ref document: A2