WO2002011761A2 - Vaccine against rsv - Google Patents

Abstract

The present invention relates to a vaccine comprising adjuvanting oligodideoxynucleotides (ODNs), containing at least one CpG dinucleotide and an antigen comprising a peptide sequence bearing at least one epitope of a Paramyxoviridae F protein. In one embodiment, the ODN is admixed or conjugated to an F protein from a respiratory syncytial virus (RSV). The vaccine of the invention may be administered directly to mucosal tissues of the respiratory tract by inhalation or intranasal administration.

Description

VACCINE AGAINST RSV

DESCRIPTION OF THE INVENTION

Field of the Invention

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. In one embodiment, the viral protein is the surface glycoprotein F of a respiratory syncytial virus (RSV), or other member of the family Paramyxoviridae. In one embodiment, the composition is topically administered to nasal and/or pulmonary mucosa by inhalation or intranasal application.

Background of the Invention

The use of 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. For example, 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. Indeed, antibodies directed against cellular DNA have long been implicated in the autoimmune condition, systemic lupus erythematosus.

It is also known that DNA sequences containing certain unmethylated CpG sequences, sometimes called "CpG ODNs" (CpG oligodeoxynucleotides), are highly immunogenic, and can induce the vigorous proliferation and immunoglobulin (Ig) production by B cells. See generally Klinman et al., Vaccine 1999;17:19; and McCluskie and Davis, J. Immun. 1998; 161 :4463 (incorporated by reference). Interestingly, these unmethylated CpG dinucleotides are far more frequent in the genomes of bacteria and viruses than vertebrates and may contribute to the innate responsiveness of the vertebrate's immune responses to bacteria and viruses. Klinman et al., Proc. Natl. Acad. Sci. USA 1996;93:2879; Yi et al. J. Immun. 1996; 157: 5394; Hua Liang et al., J. Clin. Invest. 1996; 98 :1119; Krieg et al., Nature 1995; 374: 546 , each of which is incorporated herein by reference.

Since the interest in CpG DNA began, studies have focused on the possible mechanism of action. In mice, 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-α, CSF, and interferons. These cytokines stimulate natural killer (NK) cells to secrete γ-interferon (IFN-γ) and also increases the lytic activity of NK cells. 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.

In light of the above observations, oligonucleotides, particularly those containing various formulations of CpG motifs, have frequently been suggested as adjuvants in a wide variety of vaccine formulations. See the collected reviews in Immunobiology of Bacterial CpG-DNA (Springer, 2000, H. Wagner ed.) (incorporated by reference in its entirety.) For example, 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. See, H.L. Davis, "Use of CpG DNA for Enhancing Specific Immune Responses," in Immunobiology of Bacterial CpG- DNA (Springer, 2000, H. Wagner ed.).

The use of CpG adjuvants has not, however, been suggested to combat infections by Paramyxoviridae. One clinically important member of this family, respiratory syncytial virus (RSV), is the primary cause of viral bronchiolitis and pneumonia in infants and children throughout the world, and the principal agent of infectious pulmonary disease in infants. 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. Collins et al., "Respiratory Syncytial Virus," in: Fields Virology (Lippincott-Raven 1996, B.N Fields et al., eds.) (incorporated by reference). RSV is increasingly also recognized as the causative agent of serious disease in adults, particularly the elderly (Han et al. J. Infect. Dis. 1999;197:25-30), and patients whose immune systems have been compromised. McCarthy et al. Bone Marrow Transp. 1999; 24-1315-22 .

Immunity to RSV infection is not durable, and infections recur throughout life, often manifesting as a "severe cold." Efforts to generate vaccines effective against RSV have spanned nearly four decades without success. See, Dudas et al., Clin Microbiol Rev 1998; 11 :430-9 . Indeed, the first clinical trials of a candidate vaccine, based on a formalin-inactivated preparation, culminated in a dramatic failure as exposure to the vaccine resulted in markedly enhanced disease upon subsequent natural RSV infection. Kapikian et al., Am. J. Epidemiol. 1969; 89:422-434. Consequently, issues of vaccine safety are paramount, and no candidate non-replicating vaccine has yet received approval for testing in immunologically naive infants, one of the principal target populations for widespread immunization.

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. Virology 1997; 230:155-66); a prototype recombinant vaccine combining RSF protein F epitopes with epitopes of parainfluenza virus type 3 adjuvanted with alum (Run-Pan et al., Bio/Technology 1994;12:813-8 ); an RSV F-G recombinant subunit vaccine adjuvanted with monophosphoryl lipid A, QS-21 , or alum, (Neuzil et al. Vaccine 1997; 15-525-32 ; and a fusion protein containing epitopes of RSV F protein administered to patients with cystic fibrosis. Piedra et al. Ped Infect Dis.1996; 15:23-31. Nevertheless, there are serious concerns that these vaccines may not provide long lasting protection from RSV infection. The Piedra vaccine, for example, generated a significant humoral immune response, but failed to show any significant reduction in RSV infection. Moreover, the remaining studies required standard chemical adjuvants, which are either ineffective or inappropriate for intranasal or interpulmonary administration.

As an alternative to vaccine therapy, Medimune, Inc, (Gaithersburg, MD) promotes 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.

SUMMARY OF THE INVENTION

The present invention relates to 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.

BRIEF DESCRIPTION OF THE DRAWINGS

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. DESCRIPTION OF THE EMBODIMENTS

CPG-ODN COMPOSITION

The CpG-ODNs of the invention, which may be referred to herein as ODNs, 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.

A "CpG dinucleotide" refers to a nucleic acid sequence having a cytosine followed by a guanine linked by a phosphate bond. In one embodiment, the pyrimidine ring of the cytosine is unmethylated. Nevertheless, 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.

For the purpose of this invention, 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.

For example, the base sequence of an ODN may comprise one or more CpG sequences represented by the formula 5' N₁N₂MT-CpG-AKN N 3", wherein M is A or C; K is G or T; and N-i, N₂, N₃, and N₄ are any nucleotides, with the proviso that K is G when M is C, and K is T when M is A. Thus, an HDR may include a sequence represented by the formula 5' n-ιN₂CT-CpG- AGN₃N₄ 3' or the formula 5' N-,N₂AT-CpG-ATN₃N₄ 3'.

In other embodiments the ODN comprises one or more sets of nucleotides of the formula: 5' NιX CpG-X₂N₂ 3', described in WO 98/37939, (incorporated by reference). In these embodiments, at least one nucleotide separates consecutive CpGs; where Xi is adenine, guanine, or thymidine; X₂ is cytosine or thymine; N is any nucleotide and Ni + N₂ is from 0-26 bases. In this embodiment, it is preferred that Ni and N₂ do not contain a CCGG quadramer or more than one CGG trimer, and the entire DNA portion is preferably between 8-30 bases. Similarly, the DNA portion may be described as 5' N₁X₁X2CPGX3X₄N2 3' wherein X1X2 is selected from the group consisting of GpT, GpG, GpA, ApT, and ApA, and X₃X₄ is TpT or CpT.

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. Thus, one or more CpG-ODNs may be selected for their tendency to stimulate the desired response profile. For example, a humoral immune response may be preferentially induced by CpG- ODNs having a minimum of 12 nucleotides containing either 5' N-iN₂N₃AG- CpG-TTN₄N₅N₆ 3' or 5' N₁N2N₃CT-CpG-AGN₄N₅N6 3, wherein Nt* can be any nucleotide and the central CpG is not methylated. Conversely, a cell- mediated immune response may be preferentially promoted by CpG-ODNs having a minimum of 16 nucleotides containing 5' N₁N₂N₃N₄N₅AT-CpG- ATN₆N N₈N₉Nιo 3', wherein N₁-₁₀ 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:

5' NιXι-CpG-X₂N₂ 3' wherein at least one nucleotide separates consecutive CpGs; X₁ is adenine, guanine, or thymidine; X₂ is cytosine or thymidine; N is any nucleotide and Ni + N₂ is from about 0-26 bases with the proviso that Ni and N₂ does not contain a CCGG quadmer or more than one CCG or CGG trimer. In some embodiments X-i, X₂ or both are thymidine; or

5' NXj X₂-CpG-X3 X₄N 3' wherein at least one nucleotide separates consecutive CpGs; X₁ and X₂ is selected from GpT, GpG, GpA, ApT, and ApA; X₃ t is selected from TpT or CpT; N is any nucleotide and Nj+ N₂ is from about 0-26 bases with the proviso that Ni and N₂ does not contain a CCGG quadmer or more than one CCG or CGG trimer. Exemplary nucleotides conforming to these formulae are:

TGACGTT SEQIDNO:1;

GTCGTT SEQ ID NO:2;

GTCGCT SEQ ID NO:3;

TGTCGTT SEQ ID NO:4;

TGTCGCT SEQ ID NO:5;

TCCATGTCGTTCCTGTCGTT SEQ ID NO:6;

TCCTGACGTTCCTGACGTT SEQ ID NO:7;

TCGTCGTTTTGTCGTTTTGTCGTT SEQ ID NO:8;

TCCATGACGTTCCTGACGTT SEQ ID NO:9;

TCCATGTCGCTCCTGATGCT SEQIDNO:10;

TCCATAACGTTCCTGATGCT SEQ ID NO:11 ;

TCCATGACGATCCTGATGCT SEQ ID NO:12;

TCCATGGCGGTCCTGATGCT SEQ ID NO:13;

TCCATGTCGGTCCTGATGCT SEQ ID NO:14;

TCCATAACGTCCCTGATGCT SEQIDNO:15;

TCCATGTCGTTCCTGATGCT SEQ ID NO:16;

GCTAGACGTTAGCGT (ODN1555) SEQ ID No: 17 and;

TCAACGTTGA (ODN 1466) SEQ ID No: 18.

Additional CpG-ODN sequences useful in the practice of this invention described in pending U.S. Application No: 60/128,898, to Klinman et al., filed April 12, 1999, (now PCT US 00/09839) (incorporated by reference) include:

TCGAGCGTTCTC SEQ ID NO: 19

ATCGACTCTCGAGCGTTCT SEQ ID NO: 20

TCGTCGTTTTGTCGTTTTGCTGTT SEQ ID NO: 21

TCTCGAGCGTTCTC SEQ ID NO: 22

TCGACTCTCGAGCGTTCTC SEQ ID NO: 23

ATCGACTAGCGTTCGTTCTC SEQ ID NO: 24

ACTCTCGAGCGTTCTC SEQ ID NO: 25

CTCTCGAGCGTTCTC SEQ ID NO: 26

GTCGACGTTGAC SEQ ID NO: 27

GTCGGCGTTGAC SEQ ID NO: 28

CGACTCTCGAGCGTTCTC SEQ ID NO: 29

GTCGACGCTGAC SEQ ID NO: 30

GTCAGCGTTGAC SEQ ID NO: 31

GACTCTCGAGCGTTCTC SEQ ID NO: 32

GTCGTCGATGAC SEQ ID NO: 33

ATGCACTCTCGAGCGTTCTC SEQ ID NO: 34

CTCGAGCGTTCTC SEQ ID NO: 35

TGCAGCGTTCTC SEQ ID NO: 36

TTTGGCGTTTTT SEQ ID NO: 37

ATCGACTCTCGAGCGTTCTC (ODN K3) SEQ ID NO: 38

AGCGTTTCTCGATCGACCTC SEQ ID NO: 39

GGTGCACCGATGCAGGGGG SEQ ID NO: 40 GTCGTCGACGACGG SEQ ID NO: 41 ;

GGGGGCGTTG SEQ ID NO: 42

ATGCACTCTGCAGCGTTCTC SEQ ID NO: 43

ATCGACTCTCGAGGCTTCTC SEQ ID NO: 44

GGTGCATCGATGCAGGG SEQ ID NO: 45

GGGTCGTCG l l l l GTCGTTTCGTTG SEQ ID NO: 46

AAAGGCGTTAAA SEQ ID NO: 47

CCCGGCGTTCCC SEQ ID NO: 48

GTCATCGATGCA SEQ ID NO: 49

GGTGCATCGATGCAGGGGGG SEQ ID NO: 50

GGGGTCATCGATGAAAAAAA SEQ ID NO: 51

GGTGCATCGATGCAGGGGGG SEQ ID NO: 52

AAGGTCAACGTTGAAAAAAA SEQ ID NO: 53

AAGGTCATCGATGGGGGGGG SEQ ID NO: 54

GGTGCATCGATGCAGGGGGG SEQ ID NO: 55

GGTGCATCGATGCAGGGGGG SEQ ID NO: 56

GGTGCGTCGACGCAGGGGGG SEQ ID NO: 57

GGTGCGTCGATGCAGGGGGG SEQ ID NO: 58

GGTGCGTCGACGCAGGGGGG SEQ ID NO: 59

GGTGCACCGGTGCAGGGGGG SEQ ID NO: 60

GGTGCATCGATGCAGGGGGG SEQ ID NO: 61

GTCAACGTCGAC SEQ ID NO: 62

GTCGGCGTCGAC SEQ ID NO: 63

GGGGTCAACGTTGAGGGGG SEQ ID NO: 64

GTCGGCGCTGAC SEQ ID NO: 65

ATGCACTCTCGAGGCTTCTC SEQ ID NO: 66

AATGCATCGATGCAAAA SEQ ID NO: 67

GTCAGCGTCGAC SEQ ID NO: 68

GTCAACGTTGAC SEQ ID NO: 69

TGCATCGATGCA SEQ ID NO: 70

GGTGCATCGATGCAGGGGG SEQ ID NO: 71

GTCGACGTCGAC SEQ ID NO: 72

GTCGACGCCGAC SEQ ID NO: 73

CCCAACGTTCCC SEQ ID NO: 74

GTCAACGCTGAC SEQ ID NO: 75

GAGCGTTCTC SEQ ID NO: 76

GGGAACGTTGGG SEQ ID NO: 77

GTCAGCGCTGAC SEQ ID NO: 78

GGGGGAACGTTCGGGG SEQ ID NO: 79

GTCGGCGCCGAC SEQ ID NO: 80

GGGGTAACGTTAGGGG SEQ ID NO: 81

GTCAACGCCGAC SEQ ID NO: 82

TGCCTCGAGGCA SEQ ID NO: 83

TTTAACGTTTTT SEQ ID NO: 84

AAAAACGTTAAA SEQ ID NO: 85

GGGGGAAGCTTCGGGG SEQ ID NO: 86

GTCAGCGCCGAC SEQ ID NO: 87

CGAGCGTTCTC SEQ ID NO: 88

GGTGCATCGATGCAGG SEQ ID NO: 89 GGTGCATCGATGCAGGGGGG SEQ ID NO 90

GGTGCATCGATGCAGGGGG SEQ ID NO 91

GCGTCGACGGGG SEQ ID NO 92

GGTGCGTCGTTGCAGGGGG SEQ ID NO 93

GGTGCGCCGATGCAGGGGG SEQ ID NO 94

GGGGGATCGATCGGGG SEQ ID NO 95

GGGGTCGACAGGG SEQ ID NO 96

GGTGCGTCGGTGCAGGGGG SEQ ID NO 97

GGGGGATGCATCGGGG SEQ ID NO 98

GGTGCGTCGATGCAGGGGGG SEQ ID NO 99

GGTGCGTCGATGCAGGGGGG SEQ ID NO 100

GGTGCGTCGATGCAGGGGG SEQ ID NO 101

GGTGCCTCGAGGCAGGGGG SEQ ID NO 102

GGGGGCTCGAGAGGGG SEQ ID NO 103

GGGGTATCGATAGGGG SEQ ID NO 104

GGTGCATCGATGCGAGAGA SEQ ID NO 105

GGTGCATCGACGCAGGGGG SEQ ID NO 106

GGGGTCAACGTTGAGGGGGG SEQ ID NO 107

GGTGCATGCATGCAGGGGGG SEQ ID NO 108

GGGGTCAAGCTTGAGGGGGG SEQ ID NO 109

GGGGTAAGCTTAGGGG SEQ ID NO 110

GGTGCATGCATGCAGGG SEQ ID NO 111

GGTGCATAAATGCAGGGGGG SEQ ID NO 1 12

AATGCATGCATGCAAAA SEQ ID NO 113

GGTGCATGCATGCAGGGGGG SEQ ID NO 1 14

ATCGACTCTGCAGGCTTCTC SEQ ID NO 115

TCGAGGCTTCTC SEQ ID NO 116 and;

ATGCACTCTGCAGGCTTCTC SEQ ID NO 1 17.

Modifications and Analogs of ODNs

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. For example, 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. 1988; 16(8):3209-21 ; Stein, et al., Anal. Biochem. 1990; 188:11 ; Lyer et aI., J. Am. Chem. Soc. 1990; 112:1253-54; and Metelev and Agrawal, Anal. Biochem. 1992; 200:342-346 , each of which is incorporated by reference. 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.

Another modification that renders the ODNs less susceptible to degradation is the inclusion of nontraditional bases such as inosine, as well as acetyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine. Other 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. 5,023,243 and European Patent No. WO 092 574, both of which are incorporated herein by reference). Methods for making other DNA backbone modifications and substitutions are described in Uhlmann and Peyman, Chem. Rev. 1990; 90:544 ; and Goodchild, Bioconjugate Chem. 1990; 1 :165, both of which are incorporated by reference. ODNs containing a diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini, may also be more resistant to degradation.

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). Paramyxoviridae Protein F

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).

Although each member of the Paramyxoviridae is linked by varying degrees of relatedness, 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. In vitro, 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. Moreover, 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. Alternatively, the antigen may comprise one or more synthetic peptides or genetic fusion proteins with viral or non-viral proteins.

In one embodiment, the antigen is a genetic fusion of portions of the F protein and another viral protein, for example, structural proteins G or SH. For example, 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. In one embodiment, 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. Alternatively, 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).

Co-administration and Conjugation

In one embodiment, 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. Alternatively, 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. Hermanson in Bioconjugate Techniques, Academic Press (1996). 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. In these, embodiments, the multiple copies of the ODN and/or F protein may be presented to B cells as a multivalent array.

Additional Immunomodulators and Cell Targeting Elements

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. 09/328,599 entitled: Enhancement of B Cell Activation and Immunoglobulin Secretion by Co-stimulation Of Receptors for Antigen and EBV Gp350/220, filed June 10, 1999, in the names of Drs. James Mond and Andrew Lees (incorporated by reference); (5) peptides encoding lipidation signals, for example, signals for farnesylation, geranylgeranylation, myristolation, or palmitoylation as described in U.S. Patent No. 5,776,675; (6) a universal TCE or Pan DR epitope, as described, for example in U.S. Patent No. 5,114,713 to Sinigaglia; Alexander et al., Immunity 1994; 1 :751-761 ; Ahlborg et al., Infect Immun 2000; 68:2102-9 ; Kaumaya et. al., J Mol Recognit. 1993; 6:81-94 ; Greenstein et al., J. Immunol. 148:3970-7 (1992) (each of which is incorporated by reference); (7) 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-γ, TNF-α, TNF-β, and GM-CSF, especially combinations of GM-CSF with IL-2, and other immunostimulatory combinations described in copending U.S. Application No. 08/568,343, to Mond and Snapper, filed May 10, 2000, entitled: Compositions For Stimulating The Release of Antibody By B Lymphocytes; moreover, cytokines GM-CSF and IL-2 may be particularly advantageous in the context of intranasal vaccination, as detailed in Wortham et al., Infect. Immun. 1998; 66(4): 1513-20; (7) T cell-dependent antigens, and (8) high molecular weight polyvalent carriers, including bacterial polysaccharides, glucans, dextrans polyacrylamide, and poly-amino acids.

In one embodiment, 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. In preferred embodiments, 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).

In another embodiment, the immungenicity 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. Pharmaceutical Compositions

The 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. In addition, a vaccine may comprise an immunostimulatory composition of the invention suspended, dissolved, admixed, adhered, or embedded in a pharmaceutically acceptable carrier. In addition, as used herein, a vaccine may refer to an immunostimulatory composition for administration to an organism for any prophylactic, ameliorative, palliative, or therapeutic purpose.

As used herein, 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.

Proper formulation of the pharmaceutical composition or vaccine is dependent on the route of administration chosen. For example, with intravenous administration by bolus injection or continuous infusion, the compositions are preferably water soluble, and saline is a preferred carrier. For transcutaneous, intranasal, oral, gastric, intravaginal, intrarectal, or other transmucosal administration, penetrants appropriate to the barrier to be permeated may be included in the formulation and are known in the art.

For oral administration, 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).

Alternative delivery systems appropriate for the administration of the disclosed immunostimulatory compounds of the invention include those disclosed in U.S. Patents No. 5,239,660, 4,748,043, 4,667,014, 4,452,775, 3,854,480, and 3,832,252 (each of which is incorporated by reference).

Aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable, intranasal, or aerosol solutions. For administration by aerosol, as by pressurized spray or nebulizer, 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.

Treatment and Administration

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. As used herein, treatment encompasses ameliorative and preventive methods relating to infection with any virus of the family Paramyxoviridae.

Thus, 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. In one embodiment, mucosal application of the composition (e.g., by oral, inhalation, or intranasal administration) or injection into mucosal lymph nodes or Peyer's patches may be employed to promote a predominantly humoral immune response with substantial IgA class switching. In addition, the 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.

Immunostimulatory amount

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. Alternatively, 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. Thus, 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.

Immunization schedule

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 μg 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 μg, preferably from 0.1 to 100 μg, more preferably, from 1 to 50 μg, 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.

Patient

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. Such 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.

Model System

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. 1993; 329:1524-1530; and PREVENT Study Group, Pediatrics 1997; 99:93-99) and the humanized human monoclonal antibody treatments, RESPIGAM® and SYNAGIS® (Medlmune, Inc, Gaithersburg, MD) relied on preclinical data generated solely in cotton rats.

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. In fact, 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. Other human pathogens which have been studied successfully in cotton rats include parainfluenza virus (types 1 , 2 and 3), influenza virus (types A and B), Venezuelan equine encephalitis, epidemic typhus, Mycoplasma pneumoniae, enteroviruses, tuberculosis, rickettsial spotted fever, Rift Valley Fever virus, vesicular stomatits virus and herpes simplex virus. It has also been reported that cotton rats can be infected with HIV-1. Langley et al., Proc. Natl. Acad. Sci. USA, 1998; 95:14355-60.

Inbred cotton rats (Sigmodon hispidus) 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

Example I

F Protein

Human epidermoid carcinoma cells (HEp-2, ATCC No. CCL-23, Manassas, VA) were infected with RSV (Long strain, ATCC No. VR-26) using standard methodology. 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% C0₂, 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. 59, Protein Purification Protocols, (Humana Press, 2000, S. Doonan ed.) (incorporated by reference). F glycoprotein may also be purified as described by Roder et al. in J. Chromatogr. B. Biomed. Sci. Appl. 2000; 737(1 -2):97-106 (incorporated by reference). ODNs

Three representative CpG-ODNs were synthesized on a commercially- available PE/ABI 394 RNA/DNA Synthesizer using standard methods, essentially as recommended by the manufacturer. The representative ODNs are:

ATCGACTCTCGAGCGTTCTC (K3) SEQ ID NO: 38 GCTAGACGTTAGCGT (1555) SEQ ID NO: 17

TCAACGTTGA (1466) SEQ ID NO: 18

Although each ODN, individually, provides adjuvanting activity when admixed with purified F protein, for Examples ll-Vlll below, the term "admixed ODNs" refers to a mixture of equal parts of oligonucleotides K3, 1555, and 1466. In each example below, control groups and untreated groups did not receive any F protein/ODN treatment. Example II

High Dose Challenge

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 μg/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. In combination with CpG-containing ODNs, however, 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.

Example III

Low Dose Challenge

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 μg/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.

Example IV

CpG with F Protein - Intranasal and Intramuscular Routes - Experiment 1

Groups of five S. hispidus cotton rats were immunized intranasally (i.n.) or intramuscularly (i.m.) with an aqueous formulation containing F (50 ng/dose), with or without an admixed ODN formulation (300 μg/dose). Immunizations were carried out on day 0 and again on day 14. The ODN formulation contained a mix of three difference CpG molecules, as indicated above. Immunized and untreated control animals were then challenged i.n. with low dose of RSV (10^4,5 pfu per animal) on day 28 and sacrificed 4 days later. Viral titers in lungs were determined and are shown in Table 1. Prince et al., Am. J. Pathol. 1978; 93:771-92.

TABLE 1

Note: Each data point represents the average of 5 animals per group.

Although CpG formulations enhanced the protective effect of the F and FG vaccines for every combination tested, both this Example and Example V below show that this effect was greatest when the route of immunization was intranasal.

Example V

CpG with F Protein - Intranasal and Intramuscular Routes - Experiment 2

Groups of four S. hispidus cotton rats were immunized intranasally (i.n.) or intramuscularly (i.m.) with an aqueous formulation containing F (50 ng/dose), with or without an admixed ODN formulation (100 μg/dose). Immunizations were carried out on day 0 and again on day 16. Immunized and untreated control animals were challenged i.n. with a high dose of RSV (10⁶ pfu per animal) on day 31 and sacrificed 4 days later. Viral titers in the lungs and the nose along with anti-RSV antibody titers in the serum were determined and are shown in Table 2. TABLE 2

Note: Each data point represents the average of 4 animals per group.

The reduction in viral titers demonstrated in Examples l-V indicates that administration of F protein in combination with an ODN is prophylactic for viral challenge in the cotton rat model system. Consequently, the vaccine of the invention will also be effective in preventing, ameliorating, or palliating viral symptoms in human patients.

Example VI

Vaccine Component Titration - Experiment 1

Groups of five cotton rats were intranasally immunized on day 0 and again on day 10 with an aqueous nose drop solution containing:

Group 2, F (10 ng/dose);

Group 3, F (10 ng/dose) and ODNs (100 μg/dose);

Group 4, F (10 ng/dose) and ODNs (300 μg/dose);

Group 5, F (50 ng/dose);

Group 6, F (50 ng/dose) and ODNs (100 μg/dose); and

Group 7, F (50 ng/dose) and ODNs (300 μg/dose).

Again, the same ODN formulation as used in the previous examples was used here.

Immunized and untreated control animals (Group 1 ) were challenged with a low dose of RSV (10^4'5 pfu/animal) on day 28 and sacrificed 4 days later. Viral titers in lungs were determined and compiled in Figure 3. Once again, inoculation with F alone provided no protective effect against RSV challenge unless combined with an ODN. Comparison of Groups 3 and 6 with Groups 4 and 7 shows that the higher dose of F (50 ng) was more effective in reducing viral load. Example VII

Vaccine Component Titration - Experiment 2

Groups of five cotton rats were intranasally immunized on day 0 and again on day 10 with 100 μl of an aqueous nose drop solution containing:

Group 2, F (10 ng/dose);

Group 3, F (10 ng/dose) and ODNs (100 μg/dose);

Group 4, F (10 ng/dose) and ODNs (300 μg/dose);

Group 5, F (50 ng/dose);

Group 6, F (50 ng/dose) and ODNs (20 μg/dose)

Group 7, F (50 ng/dose) and ODNs (100 μg/dose);

Group 8, F (50 ng/dose) and ODNs (300 μg/dose);

Group 9, F (250 ng/dose);

Group 10, F (250 ng/dose) and ODNs (20 μg/dose); and

Group 11 , F (250 ng/dose) and ODNs (100 μg/dose).

The same ODN formulation as used in the previous examples was used here.

Immunized and untreated control animals (Group 1) were challenged with a low dose of RSV (10⁴⁵ pfu/animal) on day 28 and sacrificed 4 days later. Viral titers in lungs were determined as shown in Table 3.

TABLE 3

Note: Each data point represents the average of 5 animals per group.

As shown in Example VI above, the F protein alone provided no protection from infection. A significant protective effect was apparent when greater than 20 μg of ODNs were combined with the antigen. 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 μg/dose). Immunized and untreated control animals are challenged with a high dose of RSV (10⁶⁵ 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.

The specification is most thoroughly understood in light of the teachings of the references cited within the specification, all of which are hereby incorporated by reference in their entirety. The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan recognizes that many other embodiments are encompassed by the claimed invention and that it is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A vaccine comprising,

1) one or more epitopes of a Paramyxoviridae F protein; and

2) one or more CpG-ODNs.

2. The vaccine of claim 1 , wherein the Paramyxoviridae is of the genus Pneumovirus.

3. The vaccine of claim 2, wherein the Pneumovirus is RSV.

4. The vaccine of claim 1 , wherein the CpG-ODN comprises at least one sequence selected from SEQ ID NO: 1 through SEQ ID NO: 117.

5. A vaccine comprising,

1 ) protein F from RSV; and 2) at least one CpG-ODN comprising at least one sequence selected from SEQ ID NO: 1 through SEQ ID NO: 117.

6. A method for vaccinating a patient comprising administering the vaccine of any of claims 1-6 to a patient.

7. The method of claim 6, wherein the vaccine is administered intranasally.