WO2002011761A2 - Vaccin contre le virus respiratoire syncytial (rs) - Google Patents

Vaccin contre le virus respiratoire syncytial (rs) Download PDF

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WO2002011761A2
WO2002011761A2 PCT/US2001/041633 US0141633W WO0211761A2 WO 2002011761 A2 WO2002011761 A2 WO 2002011761A2 US 0141633 W US0141633 W US 0141633W WO 0211761 A2 WO0211761 A2 WO 0211761A2
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seq
vaccine
cpg
protein
rsv
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PCT/US2001/041633
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WO2002011761A3 (fr
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James J. Mond
Gregory Prince
Dennis M. Klinman
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Henry M. Jackson Foundation For The Advancement Of Military Medicine
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Publication of WO2002011761A3 publication Critical patent/WO2002011761A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A61K2039/555—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
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a method for the treatment of viral disease by administering a composition comprising oligonucleotides containing unmethylated CpG dinucleotides and a viral protein.
  • the viral protein is the surface glycoprotein F of a respiratory syncytial virus (RSV), or other member of the family Paramyxoviridae.
  • the composition is topically administered to nasal and/or pulmonary mucosa by inhalation or intranasal application.
  • nucleic acids as immunostimulatory molecules has recently gained acceptance.
  • the immunoreactive properties of nucleic acids are determined by their base composition, modifications, and helical orientation.
  • humoral immune responses to cellular DNAs have been implicated in unusual DNA structures, such as Z-DNA, which can induce significant antibody responses in experimental animals.
  • Double stranded nucleic acids comprising DNA, RNA, and inter-strand DNA:RNA hybrids all have the potential for generating a humoral immune response. Eliat and Anderson, Mol. Immunol. 1994; 31 :1377.
  • antibodies directed against cellular DNA have long been implicated in the autoimmune condition, systemic lupus erythematosus.
  • CpG ODNs CpG oligodeoxynucleotides
  • CpG DNA induces proliferation in almost all (>95%) B cells. These oligonucleotides stimulate immunoglobulin (Ig) secretion and may act by increasing the secretion of IL-6 and IL-12 from B cells. This B cell activation by CpG DNA is T cell independent and antigen non-specific. In addition to its direct effects on B cells, CpG DNA also directly activates monocytes, macrophages, and dendritic cells to secrete a variety of cytokines including IL-6, IL-12, GM-CSF, TNF- ⁇ , CSF, and interferons.
  • Ig immunoglobulin
  • cytokines stimulate natural killer (NK) cells to secrete ⁇ -interferon (IFN- ⁇ ) and also increases the lytic activity of NK cells.
  • IFN- ⁇ ⁇ -interferon
  • Examples of applications covering these aspects can be found in International Patent Applications WO 95/26204, WO 96/02555, WO 98/11211 , WO 98/18810, WO 98/37919, WO 98/40100, WO 98/52581 , and PCT/US98/047703; and U.S. Patent No. 5,663,153, each of which is incorporated by reference.
  • oligonucleotides particularly those containing various formulations of CpG motifs, have frequently been suggested as adjuvants in a wide variety of vaccine formulations.
  • CpG ODNs effectively stimulate mucosal immunity when administered intranasally yet show far less toxicity than the commonly employed cholera toxin (CT) and heat-labile enterotoxin (LT) adjuvants.
  • CT cholera toxin
  • LT heat-labile enterotoxin
  • CpG adjuvants have not, however, been suggested to combat infections by Paramyxoviridae.
  • RSV respiratory syncytial virus
  • Severe episodes of RSV-mediated pulmonary disease has been implicated in deaths of infants from 6 weeks to 2 years of age, most particularly in those who are premature, have bronchopulmonary dysplasia, or congenital heart conditions. See Peter L.
  • Recent attempts at generating efficacious RSV vaccines have focused on G or F protein epitopes administered intramuscularly or intraperitoneally without adjuvants or with standard adjuvants. These include: the use of a prokaryotically expressed recombinant fusion protein containing a respiratory syncytial virus G protein fragment adjuvanted with Alhydrogel, (Power et al.
  • SYNAGIS® a prophylactic humanized monoclonal antibody specific for RSV, that can be given monthly to high-risk infants. Johnson et al. J Infect Dis 1997; 176:1215-24 . While highly effective in reducing hospitalization from RSV, SYNAGIS® cannot economically be given to the normal risk population of infants, children and adults, who nonetheless suffer from repeated RSV infections throughout life. Moreover, the cost of this therapy, which can approach $4,000 per year per patient, places it out of the reach of even high-risk patients in most countries throughout the world. Thus, there is a need for improved treatments for Paramyxoviridae-related illness, including RSV infections and RSV-related diseases, and, in particular, a need for safe, and efficacious vaccines, which provides long term protection from these viral diseases.
  • the present invention relates to an immunostimulatory composition
  • an immunostimulatory composition comprising adjuvanting oligodideoxynucleotides (ODNs), containing at least one CpG dinucleotide and an antigen comprising a viral peptide sequence bearing at least one epitope of a Paramyxoviridae protein F.
  • ODNs adjuvanting oligodideoxynucleotides
  • Figure 1 depicts viral titers in the lungs of untreated and vaccinated cotton rats following infection with a high dose of RSV.
  • Figure 2 depicts viral titers in the lungs of untreated and vaccinated cotton rats following infection with a low dose of RSV.
  • Figure 3 shows the effect of various vaccine formulations on the viral titer of RSV-challenged cotton rats.
  • the CpG-ODNs of the invention may be about 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 50, 75, 100 or more nucleotides in length.
  • the optimal length and sequence of ODNs used in a vaccine for a particular host may be determined empirically; however, for facilitating uptake into cells, less than 40 nucleotides is preferred.
  • Each ODN contains one or more CpG dinucleotides.
  • CpG dinucleotide refers to a nucleic acid sequence having a cytosine followed by a guanine linked by a phosphate bond.
  • the pyrimidine ring of the cytosine is unmethylated.
  • CpG motifs having a methylated cytosine can be effective immunostimulators under certain conditions, (Goeckeritz et al., Internat. Immunol. 1999; 11 :1693 (incorporated by reference)); thus, CpG motifs as used herein, preferably, not necessarily have an unmethylated cytosine.
  • suitable ODNs may comprise DNA sequences, or synthetic hybrid DNA/RNA polynucleotides (HDRs) as described in U.S. Provisional Application 60/209797, filed June 7, 2000, and incorporated herein by reference, in the entirety.
  • the base sequence of ODNs, as used herein, can be determined empirically according to well known techniques in the art, and may be determined or designed according to various canonical formulae, such as those described in U.S. Patents No. 6,008,200 and 5,856,462 each of which is incorporated by reference in their entirety.
  • the base sequence of an ODN may comprise one or more CpG sequences represented by the formula 5' N 1 N 2 MT-CpG-AKN N 3", wherein M is A or C; K is G or T; and N-i, N 2 , N 3 , and N 4 are any nucleotides, with the proviso that K is G when M is C, and K is T when M is A.
  • an HDR may include a sequence represented by the formula 5' n- ⁇ N 2 CT-CpG- AGN 3 N 4 3' or the formula 5' N-,N 2 AT-CpG-ATN 3 N 4 3'.
  • the ODN comprises one or more sets of nucleotides of the formula: 5' N ⁇ X CpG-X 2 N 2 3', described in WO 98/37939, (incorporated by reference).
  • at least one nucleotide separates consecutive CpGs; where Xi is adenine, guanine, or thymidine; X 2 is cytosine or thymine; N is any nucleotide and Ni + N 2 is from 0-26 bases.
  • Ni and N 2 do not contain a CCGG quadramer or more than one CGG trimer, and the entire DNA portion is preferably between 8-30 bases.
  • DNA portion may be described as 5' N 1 X 1 X2CPGX3X 4 N2 3' wherein X1X2 is selected from the group consisting of GpT, GpG, GpA, ApT, and ApA, and X 3 X 4 is TpT or CpT.
  • CpG-ODNs When used in the context of the present invention, it may be preferable to stimulate a predominantly humoral immune response, a predominantly cell- mediated immune response, or an immune response having substantial humoral and cell-mediated components.
  • one or more CpG-ODNs may be selected for their tendency to stimulate the desired response profile.
  • a humoral immune response may be preferentially induced by CpG- ODNs having a minimum of 12 nucleotides containing either 5' N-iN 2 N 3 AG- CpG-TTN 4 N 5 N 6 3' or 5' N 1 N2N 3 CT-CpG-AGN 4 N 5 N6 3, wherein Nt* can be any nucleotide and the central CpG is not methylated.
  • a cell- mediated immune response may be preferentially promoted by CpG-ODNs having a minimum of 16 nucleotides containing 5' N 1 N 2 N 3 N 4 N 5 AT-CpG- ATN 6 N N 8 N 9 N ⁇ o 3', wherein N 1 - 10 can be any nucleotide and the central CpG is not methylated.
  • CpG ODNs applicable to the present invention further include nucleotides described by Kreig and Kline in WO 98/18810. These ODNs are from about 7-30 nucleotides in length having either of the following formulae:
  • X 1 is adenine, guanine, or thymidine
  • X 2 is cytosine or thymidine
  • N is any nucleotide
  • Ni + N 2 is from about 0-26 bases with the proviso that Ni and N 2 does not contain a CCGG quadmer or more than one CCG or CGG trimer.
  • X-i, X 2 or both are thymidine; or
  • N is any nucleotide and Nj+ N 2 is from about 0-26 bases with the proviso that Ni and N 2 does not contain a CCGG quadmer or more than one CCG or CGG trimer.
  • Exemplary nucleotides conforming to these formulae are:
  • GCTAGACGTTAGCGT (ODN1555) SEQ ID No: 17 and;
  • ODNs may be synthesized de novo by any technique known in the art, for example those described in U.S. Patent No. 5,935,527, (incorporated herein by reference), preferably, with any suitable modification which can render the polynucleotide resistant to in vivo degradation resulting from, e.g., exonuclease or endonuclease digestion.
  • the phosphate backbone may be modified by phosphorothioate backbone modification wherein one of the non-bridging oxygens is replaced with sulfur, as set forth in International Patent Application WO 95/26204; U.S. Patent No. 5,003,097; Stein et al., Nuc. Acids Res.
  • Phosphorothioate modifications can occur anywhere in the polynucleotide, for example, throughout the polynucleotide or at either or both termini. In one embodiment, the last two or three 3' and/or 5" nucleotides are liked with phosphorothioate bonds.
  • the ODNs also can be modified to contain a secondary structure (e.g., stem loop structure) such that it is resistant to degradation.
  • modified nucleotides include nonionic analogs, such as alkyl or aryl phosphonates (i.e., the charged phosphonate oxygen is replaced with an alkyl or aryl group, as set forth in U.S. Patent No. 4,469,863, incorporated by reference), phosphodiesters and alkylphosphotriesters (i.e., the charged oxygen atom is alkylated, as set forth in U.S. Patent No.
  • ODNs may be ionically or covalently conjugated to appropriate molecules using techniques which are well known in the art, for example, those described by S.S. Wong in Chemistry of Protein Conjugation and Cross- Linking, CRC Press (1991 ) and Greg T. Hermanson in Bioconjugate Techniques, Academic Press (1996), both of which are incorporated by reference in their entirety.
  • Appropriate molecules include high molecular weight molecules such as polysaccharides, poly-L-lysine, carboxymethylcellulose, polyethylene glycol, or polypropylene glycol, haptenic groups, peptides, and antigens.
  • a variety of coupling or cross-linking agents can be used, e.g., protein A, carbodiimide, and N-succinimidyl-3-(2- pyridyldithio) propionate (SPDP).
  • SPDP N-succinimidyl-3-(2- pyridyldithio) propionate
  • the family Paramyxoviridae contains four genera: genus Paramyxovirus, which includes Sendai viruses, such as human parainfluenza viruses 1 and 3; genus Rubulavirus, which includes mumps virus and simian virus 5, Newcastle disease virus (avian parainfluenza viruses type 1 ) and avian parainfluenza viruses types 2-9, and human parainfluenza viruses 2 and 4; genus Morbillivirus, represented by measles virus; and genus Pneumovirus, encompassing respiratory syncytial virus (RSV), bovine respiratory syncytial virus (BRSV), ovine RSV (ORSV), caprine RSV (CRSV) pneumonia virus of mice (PVM), and turkey rhinotracheitis virus (TRTV).
  • Sendai viruses such as human parainfluenza viruses 1 and 3
  • genus Rubulavirus which includes mumps virus and simian virus 5, Newcastle disease virus (avian parainfluenza viruses type 1 ) and avian
  • protein F exhibits unambiguous sequence and functional relatedness among all family members, to the point of providing a hallmark for classification into the Paramyxoviridae family.
  • Ruigrok et al. J. Gen. Virol. 1991 ; 72:191-194; Ruigrok et al., EMBO J. 1986; 5:41-49; Spriggs et al. Virology 1986; 152:241-251 ; Collins et al., Proc. Natl. Acad. Sci. USA 1984; 81 -.7683-87. Consistent with this high degree of inter-species conservation, protein F also shows little variability within a viral species or strain.
  • An F protein comprises a spike glycoprotein, important for viral penetration at the plasma membrane.
  • Naturally-occurring F proteins are transmembrane proteins of about 529-565 amino acids.
  • the F protein mediates cell fusion, hemolytic activity and syncytium formation. Consequently, F protein is surface-accessible and available for antibody binding or immune cell recognition. This property, coupled with its low sequence variability, makes F an ideal candidate for a prophylactic or ameliorative vaccine.
  • protein F is relatively heat-stable, allowing for the production and distribution of such vaccines in developing countries where general lack of refrigeration limits the shelf-life and utility of heat-labile vaccines.
  • the immunostimulatory composition of the invention thus comprises Paramyxoviridae F protein or other antigen presenting at least one epitope of a Paramyxoviridae F protein (collectively, an F antigen). It is preferred that the epitope correspond to a portion of the F protein which is normally exposed on the surface of an infected cell at some point in the viral life cycle.
  • Naturally-occurring F proteins may be purified by standard methods. See, for example, Piedra et a., Vaccine 1995; 13L1095-1111.
  • the antigen may comprise one or more synthetic peptides or genetic fusion proteins with viral or non-viral proteins.
  • the antigen is a genetic fusion of portions of the F protein and another viral protein, for example, structural proteins G or SH.
  • another viral protein for example, structural proteins G or SH.
  • chimeric FG glycoproteins have been evaluated in rodent models. See, for example, Brideau et al., Vaccine 1991 , 9:863-64; Connors et al., Vaccine 1992, 10:475-84; Wathen et al., J. Infect. Dis. 1991 , 163:477-82.
  • an appropriate epitope is selected by testing a peptide encompassing the epitope for the ability to prevent or reduce syncytial formation, or other evidence neutralizing activity in vitro.
  • an appropriate epitope may be selected by determining whether an antibody generated against the epitope is capable of preventing or reducing syncytial formation, hemolysis, or other evidence neutralizing activity in vitro, essentially as described in Walsh and Hruska, J. Virol 1983; 47:171-177; Beeler and Coelingh, J. Virol. 1989; 63:2941-2950 (both of which are incorporated by reference).
  • the ODN and F antigen are co-administered. In another embodiment, the ODN and F antigen are administered separately, the time between applications ranging from a few minutes, to several hours, or days.
  • one or a multiplicity of ODNs may be directly, or indirectly complexed or covalently bound to or more copies of at least one F antigen prior to administration.
  • the ODN, F antigen or both may also be directly or indirectly complexed, or covalently bound to one or more other antigenic substances.
  • Methods for covalent conjugation are known in the art and include those described in S.S. Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press (1991 ) and Greg T.
  • the ODN and F protein may be incorporated into a conjugate vaccine, as discussed, for example, by Dick and Bueret in Conjugate Vaccines, Contrib. Microbiol. Immunol. 1989; 10:48-114;, Cruse JM and Lewis RE, Jr. eds.) (incorporated by reference) or the "dual conjugate" compositions of Lees et al., Vaccine 1994; 1160-66 ; U.S. Patent Nos. 5,585,100 and 5,955,079 to Mond and Lees, incorporated herein by reference.
  • the multiple copies of the ODN and/or F protein may be presented to B cells as a multivalent array.
  • the immune response elicited by the ODN and F protein-containing compositions of the invention may be further enhanced by the administration of immunomodulators and/or cell targeting moieties, which may be co- administered with, and/or directly or indirectly, chemically or recombinantly conjugated to the F protein or ODN.
  • Preferred additional entities include, for example, (1 ) LPS and detoxified lipopolysaccharides or derivatives thereof, (2) muramyl dipeptides, (3) carbohydrates and lipids (including cationic and anionic lipids, sterols, and the like) that may interact with cell surface determinants to target the construct to immunologically relevant cells; (4) proteins or polypeptides having specific immunological stimulatory activity including, for example, CD40 ligand, and fragments thereof, and polypeptides which bind to the CR2 receptor, including those described in copending U.S. Application No.
  • antibodies that interact with cell surface components including, but not limited to, antibodies directed to CR2, CR2 receptors or other components of the antigen receptor complex, CD40 or CD40 ligand, and MHC components; (6) one or more interleukins or interleukin fusion proteins, including, but not limited to IL-1 , IL-2, IL-3, IL-4, IL- 5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-15, GM-CSF, IFN- ⁇ , TNF- ⁇ , TNF- ⁇ , and GM-CSF, especially combinations of GM-CSF with IL-2, and other immunostimulatory combinations described in copending U.S.
  • the immunogenicity of the F protein may be further enhanced by the co-administration of an adjuvanting lipoprotein, as described in the copending application, incorporated herein by reference: Induction and Enhancement of the Immune Response to Type 2 T Cell-independent Antigens Conjugated to Lipid or Lipid-containing Moieties of Mond and Snapper, Serial No. 09/039,247, filed March 16, 1998.
  • the lipoprotein is covalently conjugated to the target protein, hapten, or composition, using, for example the methods described in U.S. Patent No. 5,693,326 to Lees (incorporated herein by reference).
  • the immacheicity of the F protein is further enhanced by co-administering the immunological composition of the invention with traditional adjuvants (such as alum, Freund's complete and incomplete adjuvants, Alhydrogel, LPS, cholera toxins, heat-labile enterotoxin, BCG, DETOX, Titermax Gold, and the like), as is commonly practiced in the art. Nevertheless, the use of these adjuvants is contraindicated when the administering the vaccine by intranasal or interpulmonary methods, particularly in human subjects.
  • traditional adjuvants such as alum, Freund's complete and incomplete adjuvants, Alhydrogel, LPS, cholera toxins, heat-labile enterotoxin, BCG, DETOX, Titermax Gold, and the like
  • compositions of the invention may be considered immunostimulatory compositions, pharmaceutical compositions, vaccines, or, immunological compositions in that they elicit the production of antibodies directed against at least one epitope of a Paramyxoviridae F protein.
  • a vaccine may comprise an immunostimulatory composition of the invention suspended, dissolved, admixed, adhered, or embedded in a pharmaceutically acceptable carrier.
  • a vaccine may refer to an immunostimulatory composition for administration to an organism for any prophylactic, ameliorative, palliative, or therapeutic purpose.
  • a pharmaceutical composition or vaccine may comprise at least one immunological composition, preferably dissolved or suspended in a pharmaceutically acceptable carrier or vehicle.
  • Any pharmaceutically acceptable carrier can be employed for administration of the composition. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, 18th Edition (A. Gennaro, ed., 1990) Mack Pub., Easton, Pa., incorporated by reference.
  • Carriers can be sterile liquids, such as water, polyethylene glycol, dimethyl sulfoxide (DMSO), oils, including petroleum oil, animal oil, vegetable oil, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Carriers can be in the form of mists, sprays, powders, waxes, cremes, suppositories, implants, salves, ointments, patches, poultices, films, or cosmetic preparations.
  • DMSO dimethyl sulfoxide
  • compositions are preferably water soluble, and saline is a preferred carrier.
  • penetrants appropriate to the barrier to be permeated may be included in the formulation and are known in the art.
  • the active ingredient may be combined with carriers suitable for inclusion into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like.
  • Time-sensitive delivery systems are also applicable for the administration of the compositions of the invention.
  • Representative systems include polymer base systems such as poly(lactide-glycoside), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid and polyanhydrides. These and like polymers may be formulated into microcapsules according to methods known in the art, for example, as taught in U.S. Patent No. 5,075,109 (incorporated by reference).
  • Aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable, intranasal, or aerosol solutions.
  • suitable propellants may be added as understood by those familiar with the art.
  • the immunological composition may also be formulated with solubilizing agents; emulsifiers; stabilizers; dispersants; flavorants; adjuvants; carriers; topical anesthetics such as Iidocaine, xylocaine, and the like; antibiotics; and antiviral, anti-fungal, anti-parasitic, or anti-tumor compounds, whether known or suspected.
  • the present invention encompasses methods of treating a patient in need of immune stimulation by administering to the patient the immunogenic composition of the invention.
  • treatment encompasses ameliorative and preventive methods relating to infection with any virus of the family Paramyxoviridae.
  • treatment comprises administering an immunostimulatory amount of any of the immunostimulatory compositions of the invention by any method familiar to those of ordinary skill in the art, including intravenously, intraperitoneally, intracorporeally, intra-articularly, intraventricularly, intrathecally, intramuscularly, subcutaneously, topically, tonsillarly, mucosally, intranasally, transdermally, intravaginally, orally, by inhalation, and by lavage or gavage.
  • mucosal application of the composition e.g., by oral, inhalation, or intranasal administration
  • injection into mucosal lymph nodes or Peyer's patches may be employed to promote a predominantly humoral immune response with substantial IgA class switching.
  • topical administration to mucosa of the upper and/or lower respiratory tract via inhalation of mists, powders, or sprays, or by intranasal administration of nose drops, swabs, powders, sprays, mists, aerosols, and the like is an economical alternative to traditional administration by injection, and may be advantageous in a developing country setting where disposable or sterilized reusable syringes and needles are not always available, and the potential transfer of HIV, hepatitis, and other blood-borne diseases is a concern.
  • An immunostimulatory (efficacious) amount refers to that amount of the composition that is able to stimulate a humoral and/or cellular immune response in a patient which is sufficient to prevent, ameliorate, palliate, or otherwise treat a pathogenic challenge by virus of the family Paramyxoviridae.
  • an immunostimulatory amount is that amount which measurably lowers the infectious titer of a subsequent viral challenge as compared to the titer obtained if administered in the absence of the ODN, or in the absence of an F antigen epitope.
  • an immunostimulatory amount refers to that amount of an ODN-containing composition of the invention that is able to elicit a detectable protective effect against a pathogenic challenge by a Paramyxoviridae virus.
  • the amount of an immunostimulatory composition to be administered and the frequency of administration can be determined empirically and will take into consideration the age and size of the patient being treated, and the condition or disease to be addressed.
  • ODN adjuvants are known to be safe and well tolerated even at very high doses. Mice given doses of up to 500 ⁇ g of an ODN polynucleotide, for example, showed no adverse effects. H.L. Davis, "Use of CpG DNA for Enhancing Specific Immune Responses," in Immunobiology of Bacterial CpG-DNA (Springer, 2000, H. Wagner ed.).
  • An appropriate dose of the composition in a mouse is within the range of 0.01 to 2000 ⁇ g, preferably from 0.1 to 100 ⁇ g, more preferably, from 1 to 50 ⁇ g, per inoculum.
  • the amount of composition administered may be considerably higher, particularly in human patients. Secondary booster immunizations may be given at intervals ranging from one week to many months later.
  • the invention also relates to the treatment of a patient, hereby defined as any person or non-human animal in need of immune stimulation, or to any subject for whom treatment may be beneficial, including humans and non- human animals.
  • non-human animals to be treated include all domesticated and feral vertebrates which can be infected with a virus of the family Paramyxoviridae, including, but not limited to: cotton rats, mice, rats, rabbits, fish, birds, hamsters, dogs, cats, swine, sheep, horses, cattle, and non-human primates.
  • Examples II through VIII, below, relate to the testing of an exemplary Paramyxoviridae vaccine combination using RSV as a model system. Because the vaccine characterization requires far more than ascertaining whether it elicits high titer antibodies, the efficacy and safety of the vaccine combination described herein is evaluated using the cotton rat (Sigmodon hispidus and Sigmodon fulviventer) animal model, basically as described in Prince et al., Lab. Invest. 1999;79:1385-92; and Prince et al., Am. J. Pathol. 1978; 93:771-91 (both of which are incorporated herein by reference in the entirety).
  • Cotton rats are susceptible to RSV, supporting viral replication in upper and lower respiratory tissues and exhibiting a similar range of nasal and pulmonary infection as seen in human patients. Consequently, these animals are regarded as the model of choice for many types of RSV studies (Prince et al., Am. J. Pathol, 1978; 93:771-791 ; Prince et al., Virus Res.1985; 3:193-206; and Prince et al., J. Virol. 1985; 55:517-520 (each of which is incorporated by reference). Indeed, clinical trials of pooled human RSV immune globin (Groothuls et al., N. Engl. J. Med.
  • the cotton rat In addition to its utility as a model for RSV infection, the cotton rat is unique among small laboratory animals in its susceptibility to a wide variety of other human infectious agents. Its first use is the study of human infection was reported in 1937, when its susceptibility to endemic ("scrub") typhus was described. During World War II the cotton rat was used to prepare a vaccine against endemic typhus, which was given to British troops in Southeast Asia. In 1939 the cotton rat became the first non-primate model of paralytic poliomyelitis and was the model of choice for polio for over a decade.
  • the cotton rat's unique susceptibility to human viruses has led to its use in pathogenesis studies, as well as gene therapy studies that employ human adenovirus as a delivery system for a therapeutic gene.
  • use of the cotton rat in gene therapy has proven so reliable that the Food and Drug Administration now requires studies in cotton rats of certain forms of gene therapy prior to approval to test them in humans.
  • Inbred cotton rats are currently produced by Virion Systems, Inc., Bethesda, Md., for commercial sale. Virion Systems, Inc. is licensed by the United States Department of Agriculture to produce cotton rats for commercial sale. Breeding stock of the same species is also available from the National Center for Research Resources, Bethesda, Md., which is part of the National Institutes of Health. The present invention is illustrated by the following Examples, which are not intended to be limiting in any way. EXAMPLES
  • HEp-2 Human epidermoid carcinoma cells
  • RSV Long strain, ATCC No. VR-26
  • Prince et al. Am. J. Pathol. 1978; 93:771-92 (incorporated by reference).
  • Infected cells were incubated at 37°C in an atmosphere of 5% C0 2 , for 3-4 days, and then subjected to one freeze-thaw cycle.
  • F glycoprotein was purified from these cell lysates by ion-exchange and lectin column chromatography, essentially as described in I. West and O. Goldring, "Lectin Affinity Chromatography," in Methods in Molecular Biology, Vol.
  • each ODN individually, provides adjuvanting activity when admixed with purified F protein
  • the term "admixed ODNs" refers to a mixture of equal parts of oligonucleotides K3, 1555, and 1466.
  • control groups and untreated groups did not receive any F protein/ODN treatment.
  • Cotton rats were intranasally (i.n.) immunized on day 0 and again on day 16 with an aqueous nose drop solution containing F (50 ng/dose), with or without admixed ODNs (300 ⁇ g/dose).
  • Immunized and untreated control animals were challenged with a high dose of RSV (10 6,5 pfu/animal) on day 31 and sacrificed 4 days later.
  • Viral titers in lungs were determined and compiled in Figure 1.
  • Figure 1 shows that inoculation with F alone was ineffective in reducing viral infection.
  • the vaccine reduced viral titers by 20-fold or more, thus, indicating that administration of F protein in combination with an ODN is prophylactic for viral challenge.
  • This data also indicates suggests that the administration of the vaccine to the upper respiratory tract elicits a protective immune response in treated individuals.
  • Cotton rats were intranasally immunized on day 0 and again on day 16 with an aqueous nose drop solution containing F (50 ng/dose), with or without admixed ODNs (300 ⁇ g/dose). Immunized and untreated control animals were challenged with a low dose of RSV (10 4,5 pfu/animal) on day 31 and sacrificed 4 days later. Viral titers in lungs were determined and compiled in Figure 2. Figure 2 shows that inoculation with F alone was ineffective in reducing viral infection. In combination with CpG-containing ODNs, however, the vaccine reduced viral titers by 20-fold or more.
  • Each data point represents the average of 5 animals per group.
  • Each data point represents the average of 4 animals per group.
  • Each data point represents the average of 5 animals per group.
  • Example VIII ODN Vaccine with Chimeric RSV Proteins
  • Cotton rats are immunized i.n. on day 0 and again on day 16 with an aqueous nose drop solution containing chimeric FG protein (50 ng/dose) (Wathen et al., J. Gen. Virol. 1989, 70:2625-35)(incorporated by reference), with or without admixed ODNs (300 ⁇ g/dose).
  • Immunized and untreated control animals are challenged with a high dose of RSV (10 65 pfu/animal) on day 31 and sacrificed 4 days later. Viral titers in lungs are determined as described in Example IV. Viral titers will be reduced by 20-fold or more when chimeric FG protein is administered in combination with ODNs, thus indicating that this vaccine is prophylactic for viral challenge.

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Abstract

L'invention concerne un vaccin comprenant des oligodidésoxynucléotides (ODN) adjuvants, renfermant au moins un dinucléotide CpG et un antigène qui comporte une séquence peptidique porteuse d'au moins un épitope de protéine F de Paramyxoviridae. Selon une variante, l'ODN est mélangé et conjugué à une protéine F de virus RS. Le vaccin peut être administré directement au tissu des muqueuses des voies respiratoires, par inhalation ou voie intranasale.
PCT/US2001/041633 2000-08-10 2001-08-09 Vaccin contre le virus respiratoire syncytial (rs) WO2002011761A2 (fr)

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WO2003103708A1 (fr) * 2002-06-05 2003-12-18 Gotovax Ab Composition bifonctionnelle contenant un cpg ou un oligo-/polynucleotide et une toxine ou une enterotoxine
US20090099122A1 (en) * 2001-12-20 2009-04-16 The Government of the United States of America as represented by the Secretary of the USE OF CpG OLIGODEOXYNUCLEOTIDES TO INDUCE EPITHELIAL CELL GROWTH

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WO1999061056A2 (fr) * 1998-05-22 1999-12-02 Loeb Health Research Institute At The Ottawa Hospital Methodes et produits permettant d'induire une immunite au niveau des muqueuses
WO2000062802A2 (fr) * 1999-04-20 2000-10-26 Smithkline Beecham Biologicals Sa Vaccin

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WO1996002555A1 (fr) * 1994-07-15 1996-02-01 The University Of Iowa Research Foundation Oligonucleotides immunomodulateurs
WO1999061056A2 (fr) * 1998-05-22 1999-12-02 Loeb Health Research Institute At The Ottawa Hospital Methodes et produits permettant d'induire une immunite au niveau des muqueuses
WO2000062802A2 (fr) * 1999-04-20 2000-10-26 Smithkline Beecham Biologicals Sa Vaccin

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TEBBEY P W ET AL: "A NOVEL AND EFFECTIVE INTRANASAL IMMUNIZATION STRATEGY FOR RESPIRATORY SYNCYTIAL VIRUS" VIRAL IMMUNOLOGY, MARY ANN LIEBERT, INC., NEW YORK, US, vol. 12, no. 1, 1999, pages 41-45, XP001055775 ISSN: 0882-8245 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090099122A1 (en) * 2001-12-20 2009-04-16 The Government of the United States of America as represented by the Secretary of the USE OF CpG OLIGODEOXYNUCLEOTIDES TO INDUCE EPITHELIAL CELL GROWTH
US8466116B2 (en) * 2001-12-20 2013-06-18 The Unites States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of CpG oligodeoxynucleotides to induce epithelial cell growth
WO2003103708A1 (fr) * 2002-06-05 2003-12-18 Gotovax Ab Composition bifonctionnelle contenant un cpg ou un oligo-/polynucleotide et une toxine ou une enterotoxine
AU2003241251B2 (en) * 2002-06-05 2008-07-24 Duotol Ab Biofunctional CpG or oligo-/polynucleotide and toxin or enterotoxin containing composition
KR101043341B1 (ko) 2002-06-05 2011-06-21 듀오톨 에이비 생체 관능성 씨피지 또는 올리고-/폴리뉴클레오티드 및독소 또는 장독소 함유 조성물

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