Group B Coxsackievirus Compositions and Methods of Use

Abstract

The present disclosure provides group B Coxsackievirus (CVB) compositions that induce an immune response against CVB in an individual, and methods of using such compositions.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 271,546, filed October 25, 2021, U.S. Provisional Patent Application No. 63 / 273,723, filed October 29, 2021, and U.S. Provisional Patent Application No. 63 / 321,911, filed March 21, 2022, which applications are incorporated by reference herein in their entireties.

[0002] Incorporation by Reference of Electronically Submitted Material The sequence listing is provided herewith as "PRVN-V003WO_SEQ_LIST", an XML sequence listing created on October 21, 2022 and measuring 5KB in size. The contents of the XML sequence listing are incorporated herein by reference in their entirety. [Background technology]

[0003] Introduction Group B Coxsackieviruses (CVB) are members of the Picornaviridae family, Enterovirus genus. Six serotypes of CVB (1-6) are recognized: CVB1, CVB2, CVB3, CVB4, CVB5, and CVB6. CVB can cause a variety of diseases, including gastrointestinal disease, myocarditis, pneumonia, aseptic meningitis, encephalitis, and hepatitis. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides group B Coxsackievirus (CVB) compositions that induce an immune response against CVB in an individual, and methods of using such compositions. [Brief description of the drawings]

[0005] [Figure 1] FIG. 1 presents the amino acid sequence of the polyprotein encoded by CVB4 (SEQ ID NO:1). [Figure 2-1] FIG. 1 depicts virus neutralizing titers (VNT) against CVB1-CVB5 in serum of vaccinated human subjects one month after the final dose. [Figure 2-2] Continued from Figure 2-1. [Diagram 3] FIG. 1 depicts peak VNT for each CVB serotype (CVB1-CVB5). [Figure 4] Figures 12A and 12B depict VNT against CVB1 (Figures 4 and 5), CVB2 (Figures 6 and 7), CVB3 (Figures 8 and 9), CVB4 (Figures 10 and 11), and CVV5 (Figures 12 and 13) in the sera of vaccinated human subjects. Subjects were either seropositive for VNT against the indicated CVB serotypes prior to vaccination (Figures 4, 6, 8, 10, and 12) or seronegative prior to vaccination (Figures 5, 7, 9, 11, and 13). [Diagram 5] Figures 12A and 12B depict VNT against CVB1 (Figures 4 and 5), CVB2 (Figures 6 and 7), CVB3 (Figures 8 and 9), CVB4 (Figures 10 and 11), and CVV5 (Figures 12 and 13) in the sera of vaccinated human subjects. Subjects were either seropositive for VNT against the indicated CVB serotypes prior to vaccination (Figures 4, 6, 8, 10, and 12) or seronegative prior to vaccination (Figures 5, 7, 9, 11, and 13). [Figure 6] Figures 12A and 12B depict VNT against CVB1 (Figures 4 and 5), CVB2 (Figures 6 and 7), CVB3 (Figures 8 and 9), CVB4 (Figures 10 and 11), and CVV5 (Figures 12 and 13) in the sera of vaccinated human subjects. Subjects were either seropositive for VNT against the indicated CVB serotypes prior to vaccination (Figures 4, 6, 8, 10, and 12) or seronegative prior to vaccination (Figures 5, 7, 9, 11, and 13). [Figure 7]Figures 12A and 12B depict VNT against CVB1 (Figures 4 and 5), CVB2 (Figures 6 and 7), CVB3 (Figures 8 and 9), CVB4 (Figures 10 and 11), and CVV5 (Figures 12 and 13) in the sera of vaccinated human subjects. Subjects were either seropositive for VNT against the indicated CVB serotypes prior to vaccination (Figures 4, 6, 8, 10, and 12) or seronegative prior to vaccination (Figures 5, 7, 9, 11, and 13). [Figure 8] Figures 12A and 12B depict VNT against CVB1 (Figures 4 and 5), CVB2 (Figures 6 and 7), CVB3 (Figures 8 and 9), CVB4 (Figures 10 and 11), and CVV5 (Figures 12 and 13) in the sera of vaccinated human subjects. Subjects were either seropositive for VNT against the indicated CVB serotypes prior to vaccination (Figures 4, 6, 8, 10, and 12) or seronegative prior to vaccination (Figures 5, 7, 9, 11, and 13). [Figure 9] Figures 12A and 12B depict VNT against CVB1 (Figures 4 and 5), CVB2 (Figures 6 and 7), CVB3 (Figures 8 and 9), CVB4 (Figures 10 and 11), and CVV5 (Figures 12 and 13) in the sera of vaccinated human subjects. Subjects were either seropositive for VNT against the indicated CVB serotypes prior to vaccination (Figures 4, 6, 8, 10, and 12) or seronegative prior to vaccination (Figures 5, 7, 9, 11, and 13). [Figure 10] Figures 12A and 12B depict VNT against CVB1 (Figures 4 and 5), CVB2 (Figures 6 and 7), CVB3 (Figures 8 and 9), CVB4 (Figures 10 and 11), and CVV5 (Figures 12 and 13) in the sera of vaccinated human subjects. Subjects were either seropositive for VNT against the indicated CVB serotypes prior to vaccination (Figures 4, 6, 8, 10, and 12) or seronegative prior to vaccination (Figures 5, 7, 9, 11, and 13). [Figure 11]Figures 12A and 12B depict VNT against CVB1 (Figures 4 and 5), CVB2 (Figures 6 and 7), CVB3 (Figures 8 and 9), CVB4 (Figures 10 and 11), and CVV5 (Figures 12 and 13) in the sera of vaccinated human subjects. Subjects were either seropositive for VNT against the indicated CVB serotypes prior to vaccination (Figures 4, 6, 8, 10, and 12) or seronegative prior to vaccination (Figures 5, 7, 9, 11, and 13). [Figure 12] Figures 12A and 12B depict VNT against CVB1 (Figures 4 and 5), CVB2 (Figures 6 and 7), CVB3 (Figures 8 and 9), CVB4 (Figures 10 and 11), and CVV5 (Figures 12 and 13) in the sera of vaccinated human subjects. Subjects were either seropositive for VNT against the indicated CVB serotypes prior to vaccination (Figures 4, 6, 8, 10, and 12) or seronegative prior to vaccination (Figures 5, 7, 9, 11, and 13). [Figure 13] Figures 12A and 12B depict VNT against CVB1 (Figures 4 and 5), CVB2 (Figures 6 and 7), CVB3 (Figures 8 and 9), CVB4 (Figures 10 and 11), and CVV5 (Figures 12 and 13) in the sera of vaccinated human subjects. Subjects were either seropositive for VNT against the indicated CVB serotypes prior to vaccination (Figures 4, 6, 8, 10, and 12) or seronegative prior to vaccination (Figures 5, 7, 9, 11, and 13). [Figure 14] FIG. 1 depicts CVB neutralizing antibody levels after vaccination with a CVB composition in subjects who were seronegative at baseline. [Figure 15] 1 depicts absolute enterovirus-specific IgG levels in serum of subjects after vaccination with CVB compositions. Data includes serum levels during the vaccination period and up to 6 months after the last dose. Data is expressed as enzyme immunoassay units (EIU). [Figure 16]FIG. 1 depicts enterovirus-specific IgG levels in the serum of subjects after vaccination with a CVB composition, expressed as the fold increase in enterovirus-specific IgG levels relative to the levels measured at baseline for each individual. [Figure 17] Figure 1 depicts CVB neutralizing antibody titers in serum of all subjects (baseline seropositive subjects and baseline seronegative subjects) after vaccination with CVB compositions. Data includes titers during the vaccination period and up to 6 months after the last dose. Titers are expressed on a logarithmic (log2) scale. [Figure 18] FIG. 1 depicts VNT in serum of individuals who were seronegative at baseline and after vaccination with a CVB composition.

[0006] definition The terms "individual" and "patient" are used interchangeably herein to refer to an individual (eg, a human) treated using the methods of the present disclosure.

[0007] As used herein, "treating" and "treatment" refer to treating a disease or condition of interest in a mammal, e.g., a human, having the disease or condition of interest, and include, for example, (i) inhibiting or lessening the severity of the disease or condition, or one or more symptoms thereof, e.g., arresting or slowing the onset or progression of the disease or condition, and / or ameliorating one or more symptoms; (ii) relieving the disease or condition, i.e., causing regression of the disease or condition, or one or more symptoms thereof; and / or (iii) stabilizing the disease or condition.

[0008] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which may include genetically and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The terms "polypeptide", "peptide" and "protein" include fusion proteins, including but not limited to, with heterologous amino acid sequences, fusion proteins with heterologous and homologous leader sequences, with or without an N-terminal methionine residue; immunotagging proteins, and the like.

[0009] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to the polymeric form of nucleotides of any length, be they ribonucleotides or deoxyribonucleotides.Thus, the terms "polynucleotide" and "nucleic acid" include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers that contain purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0010] "Neutralizing antibody" or "virus neutralizing antibody" (VNT) refers to an antibody purified from or present in serum that recognizes a specific antigen (e.g., a polypeptide encoded by a CVB) and inhibits the infectivity of the CVB and the effect(s) of the CVB in a host (e.g., a human).

[0011] Before the invention is further described, it is to be understood that the invention is not limited to the particular embodiments described, as such embodiments may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0012] Where a range of values ​​is presented, it is understood that each intermediate value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit unless the context clearly dictates otherwise, and any other stated or intermediate value within that stated range, is encompassed within the invention. The upper and lower limits of these subranges are independently encompassed within the smaller ranges, which are also encompassed within the scope of the invention, subject to any specifically excluded limits within the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0013] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In carrying out or testing the present invention, any methods and materials similar or equivalent to those described herein can be used, but the preferred methods and materials are described herein. All publications mentioned herein disclose the relevant methods and / or materials for which the publications are cited and are incorporated herein by reference in their entirety as if described.

[0014] Please note that, unless the context clearly dictates otherwise, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents. Thus, for example, a reference to "Group B Coxsackievirus (CVB)" includes a plurality of such CVBs, a reference to "inactivated virus particles (IVPs)" includes a reference to one or more IVPs and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims are drafted to exclude appropriate elements. As such, this statement is intended to serve as a predicate for the use of exclusionary terminology, such as "only," "only," and the like, in the recitation of claim elements or in connection with the use of a "negative" limitation.

[0015] It is understood that certain features of the invention that are described for clarity in the context of a separate embodiment are also presented in combination in a single embodiment. Conversely, various features of the invention that are also described for brevity in the context of a single embodiment may also be presented separately or in any suitable subcombination. All combinations of the embodiments of the invention are specifically embraced by the present invention, and each and every combination is disclosed herein exactly as if it were individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention, and each and every such subcombination is disclosed herein exactly as if it were individually and explicitly disclosed herein.

[0016] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which must be independently confirmed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present disclosure provides group B Coxsackievirus (CVB) compositions that induce an immune response against CVB in an individual, and methods of using such compositions.

[0018] composition The present disclosure provides a CVB composition, where such a composition is also referred to herein as an "immunogenic composition." The CVB composition of the present disclosure may induce an immune response in an individual against one or more CVB serotypes. The CVB composition of the present disclosure may comprise a whole CVB or a portion of a CVB. Thus, the term "CVB composition" includes a) an inactivated virus particle (IVP); b) a CVB virus-like particle (VLP); c) a CVB subunit (e.g., one or more CVB polypeptides); and d) one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. Polypeptides encoded by CVB include VP1, VP2, VP3, VP4, 2A, 2B, 2C, 3A, 3B, 3C, and 3D. A "CVB composition" may include one or more CVB-encoded polypeptides or a nucleic acid comprising a nucleotide sequence encoding one or more CVB-encoded polypeptides. Unless expressly indicated otherwise, a composition comprising a "CVB" refers to a composition comprising a CVB IVP, a CVB VLP, one or more CVB polypeptides, or a nucleic acid(s) comprising a nucleotide sequence encoding one or more CVB polypeptides.

[0019] Group B Coxsackievirus (CVB; also referred to herein and in the literature as "Group B Coxsackievirus" or "CBV") is a group of six serotypes of Coxsackievirus (CVB1-CVB6). Optionally, the compositions of the present disclosure include CVBs of all six serotypes (in the form of CVB IVPs, CVB VLPs, CVB polypeptides, or nucleic acid(s) comprising a nucleotide sequence encoding one or more CVB polypeptides); i.e., optionally, the compositions of the present disclosure include CVB1, CVB2, CVB3, CVB4, CVB5, and CVB6. Optionally, the compositions of the present disclosure include only five serotypes. For example, optionally, the compositions of the present disclosure include CVB1, CVB2, CVB3, CVB4, and CVB5, but not CVB6. Optionally, the compositions of the present disclosure include only a single CVB serotype. For example, optionally, the compositions of the present disclosure include only CVB1. As another example, optionally, the composition of the disclosure includes only CVB2. As another example, optionally, the composition of the disclosure includes only CVB3. As another example, optionally, the composition of the disclosure includes only CVB4. As another example, optionally, the composition of the disclosure includes only CVB5. Optionally, the composition of the disclosure includes CVBs of only two different serotypes (e.g., CVB1 and CVB2; CVB1 and CVB3; CVB1 and CVB4; CVB1 and CVB5; CVB2 and CVB3; CVB2 and CVB4; CVB2 and CVB5; CVB3 and CVB4; CVB3 and CVB5; or CVB4 and CVB5) and does not include CVBs of other serotypes. In some cases, the compositions of the present disclosure contain CVBs of only three different serotypes (e.g., CVB1, CVB2, and CVB3; CVB1, CVB2, and CVB4; CVB1, CVB2, and CVB5; CVB1, CVB3, and CVB4; CVB1, CVB3, and CVB5; CVB2, CVB3, and CVB4; CVB2, CVB3, and CVB5; CVB3, CVB4, and CVB5) and do not contain CVBs of other serotypes.In some cases, the compositions of the disclosure include CVBs of only four different serotypes (e.g., the compositions may exclude CVB3 and CVB6; CVB4 and CVB6; CVB5 and CVB6; any other combination of two CVB serotypes).

[0020] In some cases, the compositions of the disclosure do not include non-CVB enteroviruses. For example, in some cases, the compositions of the disclosure do not include whole virus, inactivated virus, viral proteins (e.g., viral subunits), or nucleic acids encoding viral proteins of non-CVB enteroviruses.

[0021] In some cases, the composition of the present disclosure includes one or more serotypes of CVB, in which the CVB is in an "inactivated" form, meaning that the infectivity of the virus is reduced or eliminated. As used herein, the term "inactivated" also includes viruses that are replication-defective. Replication-defective viruses are viruses that are defective for their constituent proteins. Such viruses can invade cells, deliver some genomes, translate the encoded proteins, and replicate the genome, but cannot encapsidate new particles. Thus, replication-defective viruses cannot spread to neighboring cells or tissues.

[0022] Inactivated CVB (CVB IVP) is produced by propagating the virus in cell culture and purifying the virus from infected cells and culture medium by high-speed centrifugation in a density gradient formed by sucrose or other high-density medium. Alternatively, the virus is purified by chromatography. The infectivity of the purified virus is destroyed by inactivating the virus through chemical treatment (e.g., formalin inactivation, such as formalin inactivation used to make inactivated poliovirus vaccines), irradiation, or heat treatment. Replication-defective viruses are prepared by physical or genetic inactivation, for example, by deleting structural genes in the viral genome and creating a complementing cell line that constitutively expresses the protein encoded by the deleted gene to replicate the defective virus. In some cases, the composition of the present disclosure includes a formalin-inactivated CVB IVP.

[0023] In some cases, the compositions of the disclosure include CVBs of two to five serotypes, where the CVBs are in the form of an IVP, a CVB VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence(s) encoding one or more CVB polypeptides, and the composition comprises: a) IVP 2.0×10 7 pieces~IVP 2.5×10 8 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 1.5×10 8 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 1.5×10 9 Amount of CVB3; d) IVP 2.0×10 7 pieces~IVP 2.5×10 8 amount of CVB4; and e) IVP 4.0×10 7 pieces~IVP 4.0×10 8 Two or more of the quantities CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides, wherein the amount of CVB is present in a volume of about 75 μL to about 600 μL (e.g., about 75 μL to about 100 μL, about 100 μL to about 150 μL, about 150 μL to about 200 μL, about 200 μL to about 250 μL, about 250 μL to about 300 μL, about 300 μL to about 350 μL, about 350 μL to about 400 μL, about 400 μL to about 450 μL, about 450 μL to about 500 μL, about 500 μL to about 550 μL, or about 550 μL to about 600 μL). In some cases, the composition comprises an amount of CVB in a volume of 100 μL. Optionally, the composition comprises an amount of CVB in a volume of 500 μL. Optionally, the composition does not comprise CBV6.

[0024] In some cases, the compositions of the disclosure include CVBs of two to five serotypes, where the CVBs are in the form of an IVP, a CVB VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence(s) encoding one or more CVB polypeptides, and the composition comprises: a) IVP 2.0×10 7 pieces~IVP 5.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 3.0×10 7 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 3.0×10 8 Amount of CVB3; d) IVP 2.0×10 7 pieces~IVP 5.0×10 7 amount of CVB4; and e) IVP 4.0×10 7 pieces~IVP 8.0×10 7 Two or more of the quantities CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. The amount of CVB can be in a volume of about 75 μL to about 150 μL (e.g., about 75 μL to about 80 μL, about 80 μL to about 85 μL, about 85 μL to about 90 μL, about 90 μL to about 110 μL, about 95 μL to about 105 μL, about 95 μL to about 100 μL, about 100 μL to about 110 μL, about 110 μL to about 120 μL, about 120 μL to about 130 μL, about 130 μL to about 140 μL, or about 140 μL to about 150 μL). In some cases, the composition comprises an amount of CVB in a volume of 100 μL. In some cases, the composition does not comprise CBV6.

[0025] In some cases, the compositions of the disclosure include CVBs of two to five serotypes, where the CVBs are in the form of an IVP, a CVB VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence(s) encoding one or more CVB polypeptides, and the composition comprises: a) IVP 1.0×10 8 pieces~IVP 2.5×10 8 Amount of CVB1; b) IVP 5.0×10 7 pieces~IVP 1.5×10 8 Quantity of pieces CVB2; c) IVP 5.0×10 8 pieces~IVP 1.5×10 9 Amount of CVB3; d) IVP 1.0×10 8 pieces~IVP 2.5×10 8 amount of CVB4; and e) IVP 2.0×10 8 pieces~IVP 4.0×10 8 Two or more of the quantities CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. The amount of CVB can be in a volume of about 400 μL to about 450 μL, about 450 μL to about 500 μL, about 500 μL to about 550 μL, or about 550 μL to about 600 μL. Optionally, the composition comprises an amount of CVB in a volume of 500 μL. Optionally, the composition does not comprise CBV6.

[0026] In some cases, the composition of the present disclosure comprises: a) IVP 2.0×10 7 pieces~IVP 5.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 3.0×10 7 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 3.0×10 8 Amount of CVB3; d) IVP 2.0×10 7 pieces~IVP 5.0×10 7 amount of CVB4; and e) IVP 4.0×10 7 pieces~IVP 8.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. The amount of CVB can be in a volume of about 75 μL to about 150 μL (e.g., about 75 μL to about 80 μL, about 80 μL to about 85 μL, about 85 μL to about 90 μL, about 90 μL to about 110 μL, about 95 μL to about 105 μL, about 95 μL to about 100 μL, about 100 μL to about 110 μL, about 110 μL to about 120 μL, about 120 μL to about 130 μL, about 130 μL to about 140 μL, or about 140 μL to about 150 μL). In some cases, the composition comprises an amount of CVB in a volume of 100 μL. In some cases, the composition does not comprise CBV6.

[0027] In some cases, the composition of the present disclosure comprises: a) IVP 3.0×10 7 pieces~IVP 4.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 2.0×10 7 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 2.0×10 8 Amount of CVB3; d) IVP 3.0×10 7 pieces~IVP 4.0×10 7 amount of CVB4; and e) IVP 6.0×10 7 pieces~IVP 6.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. The amount of CVB can be in a volume of about 75 μL to about 150 μL (e.g., about 75 μL to about 80 μL, about 80 μL to about 85 μL, about 85 μL to about 90 μL, about 90 μL to about 110 μL, about 95 μL to about 105 μL, about 95 μL to about 100 μL, about 100 μL to about 110 μL, about 110 μL to about 120 μL, about 120 μL to about 130 μL, about 130 μL to about 140 μL, or about 140 μL to about 150 μL). In some cases, the composition comprises an amount of CVB in a volume of 100 μL. In some cases, the composition does not comprise CBV6.

[0028] In some cases, the composition of the present disclosure comprises: a) CVB1:IVP 3.3×10 7 pieces; b) CVB2: IVP 1.1 x 10 7 pieces; c) CVB3: IVP 1.5 x 10 8 pieces; d) CVB4: IVP 3.2 x 10 7 pcs; and e) CVB5: IVP 6.3 x 107 piece, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. The amount of CVB can be in a volume of about 75 μL to about 150 μL (e.g., about 75 μL to about 80 μL, about 80 μL to about 85 μL, about 85 μL to about 90 μL, about 90 μL to about 110 μL, about 95 μL to about 105 μL, about 95 μL to about 100 μL, about 100 μL to about 110 μL, about 110 μL to about 120 μL, about 120 μL to about 130 μL, about 130 μL to about 140 μL, or about 140 μL to about 150 μL). In some cases, the composition comprises an amount of CVB in a volume of 100 μL. In some cases, the composition does not comprise CBV6.

[0029] In some cases, the composition of the present disclosure comprises: a) CVB1: IVP 1.6×10 8 pieces; b) CVB2: IVP 5.5 x 10 7 pieces; c) CVB3: IVP 4.5 x 10 9 pieces; d) CVB4: IVP 1.6×10 8 pcs; and e) CVB5:IVP 4.8×10 8 piece, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. The amount of CVB can be in a volume of about 400 μL to about 450 μL, about 450 μL to about 500 μL, about 500 μL to about 550 μL, or about 550 μL to about 600 μL. Optionally, the composition comprises an amount of CVB in a volume of 500 μL. Optionally, the composition does not comprise CBV6.

[0030] In some cases, the composition of the present disclosure comprises: a) CVB1: IVP 1.6×10 8 pieces; b) CVB2: IVP 5.5 x 107 pieces; c) CVB3: IVP 7.5 x 10 8 pieces; d) CVB4: IVP 1.6×10 8 pcs; and e) CVB5: IVP 3.1 x 10 8 piece, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. The amount of CVB can be in a volume of about 400 μL to about 450 μL, about 450 μL to about 500 μL, about 500 μL to about 550 μL, or about 550 μL to about 600 μL. Optionally, the composition comprises an amount of CVB in a volume of 500 μL. Optionally, the composition does not comprise CBV6.

[0031] In some cases, the composition of the present disclosure comprises: a) IVP 2.0×10 7 pieces~IVP 2.5×10 8 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 1.5×10 8 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 1.5×10 9 Amount of CVB3; d) IVP 2.0×10 7 pieces~IVP 2.5×10 8 amount of CVB4; and e) IVP 4.0×10 7 pieces~IVP 4.0×10 8 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. The amount of CVB may be from about 75 μL to about 600 μL in volume (e.g., from about 75 μL to about 80 μL, from about 80 μL to about 85 μL, from about 85 μL to about 90 μL, from about 90 μL to about 110 μL, from about 95 μL to about 105 μL, from about 95 μL to about 100 μL, from about 100 μL to about 110 μL, from about 110 μL to about 120 μL, from about 120 μL to about 130 μL, from about 130 μL to about 140 μL). The amount may be in the range of about 140 μL to about 150 μL, about 150 μL to about 200 μL, about 200 μL to about 250 μL, about 250 μL to about 300 μL, about 300 μL to about 350 μL, about 350 μL to about 400 μL, about 400 μL to about 450 μL, about 450 μL to about 500 μL, about 500 μL to about 550 μL, or about 550 μL to about 600 μL. In some cases, the composition does not include CBV6.

[0032] In some cases, the composition of the present disclosure has a total protein amount of about 5 μg to about 250 μg, a) CVB1 in an amount of about 1 μg protein to about 50 μg protein (e.g., about 1 μg to 2 μg, 2 μg to 3 μg, 3 μg to 5 μg, 5 μg to 10 μg, 10 μg to 15 μg, 15 μg to 20 μg, 20 μg to 25 μg, 25 μg to 30 μg, 30 μg to 35 μg, 35 μg to 40 μg, 40 μg to 45 μg, or 45 μg to 50 μg); b) CVB2 in an amount of about 1 μg protein to about 50 μg protein (e.g., about 1 μg to 2 μg, 2 μg to 3 μg, 3 μg to 5 μg, 5 μg to 10 μg, 10 μg to 15 μg, 15 μg to 20 μg, 20 μg to 25 μg, 25 μg to 30 μg, 30 μg to 35 μg, 35 μg to 40 μg, 40 μg to 45 μg, or 45 μg to 50 μg); c) CVB3 in an amount of about 1 μg protein to about 50 μg protein (e.g., about 1 μg to 2 μg, 2 μg to 3 μg, 3 μg to 5 μg, 5 μg to 10 μg, 10 μg to 15 μg, 15 μg to 20 μg, 20 μg to 25 μg, 25 μg to 30 μg, 30 μg to 35 μg, 35 μg to 40 μg, 40 μg to 45 μg, or 45 μg to 50 μg); d) CVB4 in an amount of about 1 μg protein to about 50 μg protein (e.g., about 1 μg to 2 μg, 2 μg to 3 μg, 3 μg to 5 μg, 5 μg to 10 μg, 10 μg to 15 μg, 15 μg to 20 μg, 20 μg to 25 μg, 25 μg to 30 μg, 30 μg to 35 μg, 35 μg to 40 μg, 40 μg to 45 μg, or 45 μg to 50 μg); and e) CVB5 in an amount of about 1 μg protein to about 50 μg protein (e.g., about 1 μg to 2 μg, 2 μg to 3 μg, 3 μg to 5 μg, 5 μg to 10 μg, 10 μg to 15 μg, 15 μg to 20 μg, 20 μg to 25 μg, 25 μg to 30 μg, 30 μg to 35 μg, 35 μg to 40 μg, 40 μg to 45 μg, or 45 μg to 50 μg) The amount of protein may be in the range of about 75 μL to about 600 μL (e.g., about 75 μL to about 80 μL, about 80 μL to about 85 μL, about 85 μL to about 90 μL, about 90 μL to about 110 μL, about 95 μL to about 105 μL, about 95 μL to about 100 μL, about 100 μL to about 110 μL, about 110 μL to about 120 μL, about 120 μL to about 130 μL, about 130 μL to about 140 μL). The amount of CBV6 in the composition may be about 0 μL, about 140 μL to about 150 μL, about 150 μL to about 200 μL, about 200 μL to about 250 μL, about 250 μL to about 300 μL, about 300 μL to about 350 μL, about 350 μL to about 400 μL, about 400 μL to about 450 μL, about 450 μL to about 500 μL, about 500 μL to about 550 μL, or about 550 μL to about 600 μL. In some cases, the composition does not include CBV6.

[0033] In some cases, the compositions of the present disclosure have a total protein amount of about 8 μg protein to about 15 μg protein, a) CVB1 in an amount of about 2 μg protein to about 4 μg protein; b) CVB2 in an amount between about 1 μg protein and about 2.5 μg protein; c) CVB3 in an amount between about 2 μg protein and about 4 μg protein; d) CVB4 in an amount of about 1 μg protein to about 2 μg protein; and e) CVB5 in an amount of about 1 μg protein to about 2.5 μg protein The amount of protein may be in a volume of about 75 μL to about 150 μL (e.g., about 75 μL to about 80 μL, about 80 μL to about 85 μL, about 85 μL to about 90 μL, about 90 μL to about 110 μL, about 95 μL to about 105 μL, about 95 μL to about 100 μL, about 100 μL to about 110 μL, about 110 μL to about 120 μL, about 120 μL to about 130 μL, about 130 μL to about 140 μL, or about 140 μL to about 150 μL). In some cases, the composition comprises an amount of CVB in a volume of 100 μL. In some cases, the composition does not comprise CBV6.

[0034] In some cases, the composition of the present disclosure comprises about 10.7 μg total protein, a) CVB1 in an amount of approximately 2.8 μg protein; b) CVB2 in an amount of about 1.7 μg protein; c) CVB3 in an amount of about 2.7 μg protein; d) CVB4 in an amount of about 1.2 μg protein; and e) CVB5 in an amount of about 2.3 μg protein The amount of protein can be in a volume of about 75 μL to about 150 μL (e.g., about 75 μL to about 80 μL, about 80 μL to about 85 μL, about 85 μL to about 90 μL, about 90 μL to about 110 μL, about 95 μL to about 105 μL, about 95 μL to about 100 μL, about 100 μL to about 110 μL, about 110 μL to about 120 μL, about 120 μL to about 130 μL, about 130 μL to about 140 μL, or about 140 μL to about 150 μL). In some cases, the composition comprises an amount of CVB in a volume of 100 μL. In some cases, the composition does not comprise CBV6.

[0035] In some cases, the compositions of the present disclosure have a total protein amount of about 35 μg protein to about 75 μg protein, a) CVB1 in an amount of about 10 μg protein to about 20 μg protein; b) CVB2 in an amount between about 5 μg protein and about 12.5 μg protein; c) CVB3 in an amount of about 10 μg protein to about 20 μg protein; d) CVB4 in an amount of about 5 μg protein to about 10 μg protein; and e) CVB5 in an amount of about 5 μg protein to about 12.5 μg protein The amount of CVB may be in a volume of about 400 μL to about 450 μL, about 450 μL to about 500 μL, about 500 μL to about 550 μL, or about 550 μL to about 600 μL. In some cases, the composition comprises an amount of CVB in a volume of 500 μL. In some cases, the composition does not comprise CBV6.

[0036] In some cases, the composition of the present disclosure comprises a total protein amount of 53.5 μg, a) CVB1 in an amount of approximately 14 μg protein; b) CVB2 in an amount of about 8.5 μg protein; c) CVB3 in an amount of about 13.5 μg protein; d) CVB4 in an amount of about 6 μg protein; and e) CVB5 in an amount of about 11.5 μg protein The amount of CVB may be in a volume of about 400 μL to about 450 μL, about 450 μL to about 500 μL, about 500 μL to about 550 μL, or about 550 μL to about 600 μL. In some cases, the composition comprises an amount of CVB in a volume of 500 μL. In some cases, the composition does not comprise CBV6.

[0037] A composition of the present disclosure, when administered to an individual, induces a virus neutralizing antibody titer (VNT) of 1 / 8 to 1 / 64,000 as determined by a VNT assay. In some cases, such a VNT is a peak VNT (e.g., as depicted in FIG. 3). In some cases, a composition of the present disclosure, when administered to an individual, induces a VNT of about 1 / 2,000 to about 1 / 4,000 as determined by a VNT assay. In some cases, a composition of the present disclosure, when administered to an individual, induces a VNT of about 1 / 10 to about 1 / 500 as determined by a VNT assay. In some cases, a composition of the present disclosure, when administered to an individual, induces a VNT of about 1 / 500 to about 1 / 1,000 as determined by a VNT assay. In some cases, a composition of the present disclosure, when administered to an individual, induces a VNT of about 1 / 1,000 to about 1 / 2,000 as determined by a VNT assay. In some cases, the compositions of the present disclosure, when administered to an individual, induce about 1 / 2,000 to about 1 / 4,000 of the VNT as determined by a VNT assay. In some cases, the compositions of the present disclosure, when administered to an individual, induce about 1 / 4,000 to about 1 / 8,000 of the VNT as determined by a VNT assay. In some cases, the compositions of the present disclosure, when administered to an individual, induce about 1 / 8,000 to about 1 / 10,000 of the VNT as determined by a VNT assay. In some cases, the compositions of the present disclosure, when administered to an individual, induce about 1 / 10,000 to about 1 / 20,000 of the VNT as determined by a VNT assay.

[0038] As used herein, "virus neutralizing antibody titer" refers to the highest dilution of a sample that still neutralizes the infectivity of the virus in cell culture.Virus neutralizing titer (VNT) is calculated as the last serum dilution that reduces the number of plaques by at least 80% using plaque reduction assay.See, for example, Boone and Albrecht (1983), J.Virol.Methods, 6:193.Thus, for example, a VNT of 1 / 2,000 refers to the amount of antibody in a serum sample that no longer neutralizes the CVB in the sample when the sample is diluted more than 1 / 2,000 ("VNT of 2,000"). For example, in Figures 2 and 3, "2,000" means that a 2000-fold dilution (referred to as "1 / 2,000" or "2,000 VNT") is the highest dilution that reduces infection of cells in culture by at least 80%; "4,000" means that a 4000-fold dilution (referred to as "1 / 4,000" or "4,000 VNT") is the highest dilution that reduces infection of cells in culture by at least 80%.

[0039] Optionally, a single dose of the subject composition is administered to the individual. Optionally, 14 days after the single dose of the composition, the VNT to CVB in the individual is increased 8-10 fold compared to a control. Optionally, 14 days after the single dose of the composition, the VNT to CVB in the individual is increased 3-10 fold compared to a control. Control refers to i) the VNT to CVB in the individual prior to the single dose of the disclosed composition; or ii) a reference control level of VNT to CVB.

[0040] Optionally, a first administration of the subject composition is administered to the individual; a second administration of the subject composition is administered to the individual at a time point after the first administration (e.g., the second administration is administered 7 days to 3 weeks, 7 days to 2 weeks, 2 weeks to 1 month, 1 month to 2 months, 2 months to 3 months, or 3 months to 6 months after the first administration). Optionally, a first administration of the subject composition is administered to the individual; a second administration of the subject composition is administered to the individual one month after the first administration. Optionally, a first administration of the subject composition is administered to the individual; a second administration of the subject composition is administered to the individual two months after the first administration. Optionally, a first administration of the subject composition is administered to the individual; a second administration of the subject composition is administered to the individual three months after the first administration. Optionally, the VNT to CVB in the individual 14 days after the second administration of the composition is increased 8-10 fold compared to a control. In some cases, VNT to CVB in the individual 14 days after the second administration of the composition is increased 3-10 fold compared to a control, which refers to i) the VNT to CVB in the individual prior to the first administration of a composition of the present disclosure; or ii) a reference control level of VNT to CVB.

[0041] The neutralizing titer is provided by neutralizing antibodies against CVB as measured in the serum of the subject. In some cases, an effective dose of the composition of the present disclosure is sufficient to provide a viral neutralizing titer (VNT) of at least 100. In some cases, an effective dose of the composition of the present disclosure is sufficient to provide a VNT of 100 to 500. In some cases, an effective dose of the composition of the present disclosure is sufficient to provide a VNT of more than 500. For example, in some cases, an effective dose of the composition of the present disclosure is sufficient to provide a VNT of 500 to 1,000. As another example, in some cases, an effective dose of the composition of the present disclosure is sufficient to provide a VNT of 1000 to 10,000. In some cases, the effective dose of the composition of the present disclosure is between 1000-2000, 1000-3000, 1000-4000, 1000-5000, 1000-6000, 1000-7000, 1000-8000, 1000-9000, 1000-10,000, 2000-3000, 2000-4000, 2000-5000, 2000-6000, 2000-7000, 2000-8000, 2000-9000, 2000-10,000, 3000-4000, 3000-5000, 3000-6000, 3000-7000, 3000-8000, 3000-9000 , 3,000–10,000, 4,000–5,000, 4,000–6,000, 4,000–7,000, 4,000–8,000, 4,000–9,000, 4,000–10,000, 5,000–6,000, 5,000–7,000, 5,000–8,000, 5,000–9,000; 5,000–10,000, 6,000–7,000, 6,000–8,000, 6,000–9,000, 6,000–10,000, 7,000–8,000, 7,000–9,000, 7,000–10,000, 8,000–9,000, 8,000–10,000, or 9,000–10,000 VNTs.In some cases, an effective dose of a composition of the present disclosure is sufficient to provide a VNT of 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 11000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19000, 20,000, or more. In some cases, the VNT is provided 1-72 hours after administration. For example, VNT is provided 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-72, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-72, 20-30, 20-40, 20-50, 20-60, 20-70, 20-72, 30-40, 30-50, 30-60, 30-70, 30-72, 40-50, 40-60, 40-70, 40-72, 50-60, 50-70, 50-72, 60-70, 60-72, or 70-72 hours after administration. In some cases, the VNT is provided 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 56, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours after administration. In some cases, the VNT is provided within 14 days of administration of a composition of the present disclosure. In some cases, the VNT is effected within 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days of administration of the composition of the present disclosure.

[0042] In some cases, 1 / 8 to 1 / 64,000 of the VNT is maintained for at least 24 weeks, at least 32 weeks, at least 40 weeks, at least 52 weeks, or more than 52 weeks after one or more administrations of a CVB composition of the present disclosure. In some cases, the VNT comprises IgG isotype antibodies (e.g., the VNT comprises at least 50%, at least 60%, at least 75%, at least 85%, or more than 85% IgG isotype antibodies). In some cases, the VNT comprises IgG isotype antibodies, IgM isotype antibodies, and IgA isotype antibodies.

[0043] VNT can be determined using a VNT assay (also referred to as a "plaque reduction assay"). The presence of neutralizing antibodies blocks the infectivity of the virus to cells. To perform a VNT assay, serum obtained from an individual is diluted to various degrees. The diluted serum is mixed with a certain amount of infectious virus optimized for each virus serotype (e.g., 30-100 plaque forming units (PFU) of virus, where PFU represents one infectious virus particle), and the mixture is incubated at room temperature for a certain time (e.g., an initial 60 minutes at 37°C followed by overnight (about 18 hours) incubation). After the incubation period, the mixture is added to a monolayer of cells (e.g., Green Monkey Kidney cells (GMK-AH-1 cells; RRID:CVCL_L878)), and the cell monolayer is incubated with the mixture for a certain time (e.g., 40-48 minutes). After this incubation, the number of virus-induced plaques on the monolayer is counted, for example after staining the cells or by microscopic examination of the cells. A mixture of virus and hyperimmune serum raised against the enterovirus serotype in question (for example serum raised in monkeys or horses) is used as a positive control for specific neutralization of each serotype. Virus without serum is used as a negative control. The neutralization titer (VNT) is calculated as the last serum dilution that reduces the number of plaques by at least 80%. See, for example, Boone and Albrecht (1983), J. Virol. Methods, 6:193.

[0044] The compositions of the disclosure may include the whole CVB virus, whose infectivity has been inactivated, or a subunit vaccine containing viral RNA or cDNA fragments encoding certain antigenic structures, proteins, or peptides, or combinations thereof (such as virus-like particles), of the virus, or the whole virus or individual viral proteins or inactivated forms of the virus.

[0045] In some cases, the compositions of the present disclosure include components of the CVB. A "component" can be an immunogenic polypeptide of the virus, such as a subunit thereof, including a chimeric subunit, or a nucleic acid fragment, such as a portion of the genome of the virus. A component can also be produced or modified recombinantly or synthetically.

[0046] Subunit vaccines can consist of purified or recombinant viral proteins, synthetic peptides corresponding to viral antigenic epitopes, VLPs, or empty viral capsids produced during infection but lacking the viral genome. These subunit vaccines can be administered per se or conjugated to haptens or carriers (e.g., ISCOM particles, chitosan, TLR agonists, biodegradable microparticles).

[0047] As mentioned above, the compositions of the present disclosure may include CVB subunits, such as one or more CVB polypeptides. Polypeptides encoded by CVB include VP1, VP2, VP3, VP4, 2A, 2B, 2C, 3A, 3B, 3C, and 3D. For example, a composition may include one or more CVB polypeptides, where the one or more CVB polypeptides include an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of any polypeptide derived from the entire viral polyprotein (e.g., the CVB4 polyprotein shown in FIG. 1), or a precursor polyprotein. For example, the first 73 amino acids of the CVB4 polyprotein in FIG. 1 form the viral CBV4 VP4 component upon maturation of the polyprotein. The composition can include any CBV4 VP4 polypeptide that includes an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the entire amino acid sequence of the mature form of CBV4 VP4 or a polypeptide fragment. The VLPs are formed using full-length viral proteins, truncated forms of full-length viral proteins, or fusion proteins containing portions of viral proteins.

[0048] As mentioned above, a composition suitable for administration to an individual in need thereof may include one or more nucleic acids comprising a nucleotide sequence(s) encoding one or more CVB polypeptides. Suitable nucleic acids include recombinant expression vectors. Optionally, the one or more nucleic acids are recombinant expression vectors comprising one or more nucleotide sequences encoding one or more CVB polypeptides. Optionally, the recombinant expression vector is a DNA or RNA molecule comprising a nucleotide sequence encoding a gene product (e.g., RNA or polypeptide) of interest. Recombinant expression vectors include viral expression vectors (e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus, adeno-associated virus (AAV), lentivirus, SV40, herpes simplex virus, human immunodeficiency virus (HIV), retrovirus, etc.). Optionally, the recombinant expression vector is a recombinant lentivirus vector. Optionally, the recombinant expression vector is a recombinant HIV vector. Optionally, the recombinant expression vector is a recombinant adenovirus vector. Optionally, the recombinant expression vector is a recombinant AAV vector. In some cases, the nucleotide sequence(s) encoding one or more CVB polypeptides are operably linked to one or more transcriptional control elements, such as a promoter. In some cases, the nucleotide sequence encoding a gene product of interest is operably linked to a promoter functional in a mammalian cell, e.g., a muscle cell, an epithelial cell, a dendritic cell, an antigen-presenting cell, etc.

[0049] Suitable nucleic acids include mRNA. Thus, in some cases, a composition suitable for administration to an individual in need thereof includes one or more RNA molecules (e.g., mRNA) that include one or more nucleotide sequences encoding one or more CVB polypeptides. In some cases, the one or more RNA molecules include at least one 5' cap structure and / or a 5' untranslated region (5'UTR). In some cases, the one or more RNA molecules also include a 3'UTR and / or a 3' tailing sequence (e.g., a poly(adenosine) (polyA) sequence). Examples of 5' cap structures include inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some cases, the one or more RNA molecules include one or more of a nucleoside base modification, a sugar modification, and a backbone modification. In some cases, the compositions include, in addition to the one or more RNA molecules, one or both of: a) a polymer (e.g., polyethylene glycol, polyglycolide, polyvinyl alcohol, polyvinylpyrrolidone, polylactide, poly(lactide-co-glycolide) (PLGA), polycaprolactone, polysorbate, polyethylene oxide, polypropylene oxide, poly(ethylene oxide-co-propylene oxide), poloxamer, poloxamine, poly(oxyethylated)glycerol, poly(oxyethylated)sorbitol, poly(oxyethylated)glucose, polyethyleneimine, dendrimers based on polyamidoamine (PAMAM), and the block copolymer, poly(ethylene glycol)-block-poly(lactic acid-co-glycolic acid) (PEG-b-PLGA)); and b) a lipid.

[0050] In some cases, the composition suitable for administration to an individual in need thereof comprises one or more RNA molecules (e.g., mRNA) comprising one or more nucleotide sequences encoding one or more CVB polypeptides. In some cases, the one or more RNA molecules are administered to an individual in need thereof in an amount such that the level of the encoded CVB polypeptide(s) in the serum of the individual is at least 50 pg / mL at least 2 hours after administration. In some cases, the one or more RNA molecules are administered to an individual in need thereof in an amount such that the level of the encoded CVB polypeptide(s) in the serum of the individual remains above 50 pg / mL for at least 72 hours after administration.

[0051] In some cases, each of the one or more mRNAs is administered in an amount of about 1 μg to about 200 μg, e.g., about 1 μg to about 5 μg, about 5 μg to about 10 μg, about 10 μg to about 15 μg, about 15 μg to about 20 μg, about 20 μg to about 25 μg, about 25 μg to about 30 μg, about 30 μg to about 40 μg, about 40 μg to about 50 μg, about 50 μg to about 60 μg, about 60 μg to about 70 μg, about 70 μg to about 80 μg, about 80 μg to about 90 μg, about 90 μg to about 100 μg, about 100 μg to about 125 μg, about 125 μg to about 150 μg, about 150 μg to about 175 μg, or about 175 μg to about 200 μg.

[0052] For example, in some cases, compositions of the disclosure include nucleic acids (e.g., mRNAs) encoding CVB1, CVB2, CVB3, CVB4, and CVB5, but not CVB6, in which case each of the mRNAs is present in the composition at a concentration of from about 1 μg to about 200 μg, e.g., from about 1 μg to about 5 μg, from about 5 μg to about 10 μg, from about 10 μg to about 15 μg, from about 15 μg to about 20 μg, from about 20 μg to about 30 μg, or from about 30 μg to about 40 μg. It is present in an amount of about 25 μg, about 25 μg to about 30 μg, about 30 μg to about 40 μg, about 40 μg to about 50 μg, about 50 μg to about 60 μg, about 60 μg to about 70 μg, about 70 μg to about 80 μg, about 80 μg to about 90 μg, about 90 μg to about 100 μg, about 100 μg to about 125 μg, about 125 μg to about 150 μg, about 150 μg to about 175 μg, or about 175 μg to about 200 μg.

[0053] In some cases, a composition of the disclosure includes a nucleic acid (e.g., an mRNA) encoding a polypeptide of only a single CVB serotype. For example, in some cases, a composition of the disclosure includes a nucleic acid encoding only a CVB1 polypeptide. As another example, in some cases, a composition of the disclosure includes a nucleic acid encoding only a CVB2 polypeptide. As another example, in some cases, a composition of the disclosure includes a nucleic acid encoding only a CVB3 polypeptide. As another example, in some cases, a composition of the disclosure includes a nucleic acid encoding only a CVB4 polypeptide. As another example, in some cases, a composition of the disclosure includes a nucleic acid encoding only a CVB5 polypeptide. In some cases, the nucleic acid encoding only a polypeptide of a single CVB serotype is mRNA, and the mRNA is present in the composition at about 1 μg to about 200 μg, for example, about 1 μg to about 5 μg, about 5 μg to about 10 μg, about 10 μg to about 15 μg, about 15 μg to about 20 μg, about 20 μg to about 25 μg, about 25 μg to about 30 μg, about 30 μg about 40 μg, about 40 μg to about 50 μg, about 50 μg to about 60 μg, about 60 μg to about 70 μg, about 70 μg to about 80 μg, about 80 μg to about 90 μg, about 90 μg to about 100 μg, about 100 μg to about 125 μg, about 125 μg to about 150 μg, about 150 μg to about 175 μg, or about 175 μg to about 200 μg.

[0054] In some cases, the compositions of the disclosure include nucleic acids (e.g., mRNAs) encoding only two different serotypes (e.g., CVB1 and CVB2; CVB1 and CVB3; CVB1 and CVB4; CVB1 and CVB5; CVB2 and CVB3; CVB2 and CVB4; CVB2 and CVB5; CVB3 and CVB4; CVB3 and CVB5; or CVB4 and CVB5) and do not include nucleic acids encoding CVB polypeptides of any other serotypes. In some cases, the nucleic acids encoding polypeptides of only two CVB serotypes are mRNAs, and each of the mRNAs is present in the composition at about 1 μg to about 200 μg, e.g., about 1 μg to about 5 μg, about 5 μg to about 10 μg, about 10 μg to about 15 μg, about 15 μg to about 20 μg, about 20 μg to about 25 μg, about 25 μg to about 30 μg, about 30 μg to about 40 μg, about 40 μg to about 50 μg, about 50 μg to about 60 μg, about 60 μg to about 70 μg, about 70 μg to about 80 μg, about 80 μg to about 90 μg, about 90 μg to about 100 μg, about 100 μg to about 200 μg, about 100 μg to about 300 μg, about 100 μg to about 400 μg, about 100 μg to about 5 ... The compound is present in an amount of about 100 μg to about 125 μg, about 125 μg to about 150 μg, about 150 μg to about 175 μg, or about 175 μg to about 200 μg.

[0055] In some cases, the compositions of the disclosure include nucleic acids (e.g., mRNA) encoding only three different serotypes (e.g., CVB1, CVB2, and CVB3; CVB1, CVB2, and CVB4; CVB1, CVB2, and CVB5; CVB1, CVB3, and CVB4; CVB1, CVB3, and CVB5; CVB2, CVB3, and CVB4; CVB2, CVB3, and CVB5; CVB3, CVB4, and CVB5) and do not include nucleic acids encoding CVB polypeptides of any other serotypes. In some cases, the nucleic acid encoding the polypeptides of only the three CVB serotypes is an mRNA, and each of the mRNAs is present in the composition at a concentration of about 1 μg to about 200 μg, e.g., about 1 μg to about 5 μg, about 5 μg to about 10 μg, about 10 μg to about 15 μg, about 15 μg to about 20 μg, about 20 μg to about 25 μg, about 25 μg to about 30 μg, about 30 μg, or about 30 μg. The compound is present in an amount of about 100 μg to about 125 μg, about 125 μg to about 150 μg, about 150 μg to about 175 μg, or about 175 μg to about 200 μg.

[0056] In some cases, the compositions of the disclosure include nucleic acids encoding CVB polypeptides of only four different serotypes (e.g., the compositions may exclude CVB3 and CVB6; CVB4 and CVB6; CVB5 and CVB6; or nucleic acids encoding polypeptides of any other combination of two CVB serotypes).

[0057] The above-described CVBs, subunits, VLPs, or nucleic acids are formulated into pharmaceutical compositions that may contain, in addition to the active ingredient that induces immune stimulation, pharma- ceutically acceptable excipients, carriers, haptens, and / or adjuvants. Excipients, carriers, haptens, and adjuvants may include, for example, phenoxyethanol, magnesium chloride, sucrose, thiomersal, formaldehyde, phenol, antibiotics (preservatives), or aluminum salts, polymeric microparticles, ISCOM particles, carrier proteins (e.g., cholera toxin), liposomes, protein micelles (hapten / adjuvant), or TLR agonists.

[0058] The CVB composition may include an adjuvant (e.g., an immunostimulatory amount of an adjuvant). The CVB composition may include an immunostimulatory amount of an adjuvant. Examples of known suitable adjuvants used in humans include, but are not necessarily limited to, alum, aluminum phosphate, aluminum hydroxide, MF59 (4.3% w / v squalene, 0.5% w / v Tween 80™, 0.5% w / v Span 85), CpG-containing nucleic acids (wherein cytosine is unmethylated), QS21, monophosphoryl lipid A (MPL), 3-Q-desacyl-4'-monophosphoryl lipid A (3DMPL), Aquila extract, immune stimulating complexes (ISCOMS; complexes of cholesterol, phospholipids, and Quillaja saponin), LT / CT mutants, poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A, interleukins, and the like. For laboratory animals, Freund's incomplete adjuvant or Freund's complete adjuvant can be used.N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (referred to as nor-MDP, CGP 11637), N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxylphosphoryloxy)-ethylamine (referred to as MTP-PE, CGP 19835A), and RIBI, which contains three components extracted from bacteria: monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene / Tween 80 emulsion, are also suitable for use. As another example, the adjuvant may include another virus (other than CVB) in combination with a composition of the present disclosure, or may include CVB proteins presented by a carrier other than the CVB virus.

[0059] Further exemplary adjuvants that enhance the efficacy of the composition include (1) for example, (a) MF59™ (see, e.g., WO 90 / 14837) containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing MTP-PE) formulated into submicron particles using a Microfluidizer; (b) SAF containing 10% squalene, 0.4% Tween 80, 5% L121, a pluronic block polymer, and thr-MDP that has been Microfluidized or vortexed into a submicron emulsion to create a large particle size emulsion; and (c) 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components, such as monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), e.g., oil-in-water emulsion formulations, such as the RIBI™ Adjuvant System (RAS), (Ribi Immunochem, Hamilton, Mont.), containing MPL+CWS (Detox™), with or without other specific immune stimulants, such as muramyl peptides (see below) or bacterial cell wall components; (2) QS21 or Stimulon™ (Cambridge (2) saponin adjuvants, such as purified extract of Saponaria officinalis (Bioscience, Worcester, Mass.), or particles made therefrom, such as ISCOMs (immunostimulating complexes), lacking additional detergents (e.g., WO00 / 07621); (3) Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA); (4) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO99 / 44636), interferons (e.g., gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF);(5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL), optionally in the substantial absence of alum when used together with pneumococcal saccharide (e.g. WO00 / 56358); (6) combinations of 3dMPL, for example, with QS21 and / or oil-in-water emulsions (e.g. EP-A-0835318, EP-A-0735898, EP-A (7) oligonucleotides containing a CpG motif, containing at least one CG dinucleotide in which the cytosines are unmethylated (see, e.g., WO96 / 02555, WO98 / 16247, WO98 / 18810, WO98 / 40100, WO98 / 55495, WO98 / 37919, and WO98 / 52581); (8) polyoxyethylene ethers or polyoxyethylene ethers. (9) polyoxyethylene sorbitan ester surfactants in combination with octoxynol (WO01 / 21207), or polyoxyethylene alkyl ether surfactants or polyoxyethylene alkyl ester surfactants in combination with at least one additional non-ionic surfactant, such as octoxynol (WO01 / 21152); (10) saponin and an immunostimulatory oligonucleotide (e.g., CpG oligonucleotide) (WO00 / 62800); (11) an immunostimulatory agent and a particle of a metal salt (see, e.g., WO00 / 23105); (12) saponin and an oil-in-water emulsion (see, e.g., WO99 / 11241); (13) saponin (e.g., QS21) + 3dMPL + IM2 (optionally with a sterol) (see, e.g., WO98 / 57659);(14) Other substances that act as immune stimulating agents to enhance the efficacy of the composition, including but not limited to. Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxylphosphoryloxy)-ethylamine (MTP-PE), and the like. Matrix-M™ is also suitable for use; Matrix-M™ is an adjuvant that includes 40 nm nanoparticles that include soapberry, cholesterol, and phospholipids. Adjuvants suitable for administration to humans are particularly adjuvants of interest. In some cases, the adjuvant may be an adjuvant that stimulates CD4 against the immunogen; + It is an adjuvant that enhances helper T cell response. Polyinosine:cytosine (polyI:C) nucleic acid is also suitable for use. PolyI:C is a synthetic double-stranded RNA. Cyclic dinucleotide activators of the STING pathway are also suitable for use. Examples of suitable cyclic dinucleotide adjuvants include, but are not limited to: 1) bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP); 2) bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP); and bis-(3',5')-cyclic dimeric inosine monophosphate (c-di-IMP). Poly(I:C) is also suitable for use.

[0060] The efficacy of an adjuvant may be determined by one or more of: i) measuring the amount of antibodies directed against the immunogenic antigen or its antigenic epitopes (e.g., using the VNT assay described above); ii) measuring the cytotoxic T lymphocyte response to the antigen; and iii) measuring the helper T cell response to the antigen.

[0061] The composition of the present disclosure may contain pharmaceutically acceptable excipients, the variety of which is known in the art and need not be discussed in detail here.Pharmaceutically acceptable excipients are fully described in various publications, including, for example, "Remington: Science and Practice of Pharmacy", 19th edition (1995), or latest edition, Mack Publishing Co; A.Gennaro (2000); "Remington: Science and Practice of Pharmacy", 20th edition, Lippincott, Williams & Wilkins; "Pharmaceutical Dosage Forms and Drug Delivery Systems" (1999), HC Ansel et al., ed., 7th edition, Lippincott, Williams & Wilkins; and "Handbook of Pharmaceutical Excipients" (2000), AH Kibbe et al., ed., 3rd edition, Amer.Pharmaceutical Assoc.

[0062] The pharmaceutical composition may include a pharmaceutically acceptable excipient.In some cases, the subject pharmaceutical composition is a pharmaceutical composition suitable for administration to a subject, for example, a sterile pharmaceutical composition.For example, in some cases, the subject pharmaceutical composition is a pharmaceutical composition suitable for administration to a human subject, for example, in this case, the composition is a sterile composition and does not contain detectable pyrogens and / or other toxins.

[0063] Protein compositions may include other ingredients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium, carbonates, etc. The compositions may contain pharma- ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, hydrochloride salts, sulfates, solvates (e.g., mixed ionic salts, water, organics), hydrates (e.g., water), etc.

[0064] In some cases, the composition of the present disclosure is a liquid composition. Thus, the present disclosure provides a composition (e.g., a liquid composition including a pharmaceutical composition) that includes the CVB described above. In some cases, the composition of the present disclosure includes a) the CVB described above; and b) saline (e.g., 0.9% NaCl). In some cases, the composition is a sterile composition. In some cases, the composition is suitable for administration to a human subject, for example, in this case, the composition is a sterile composition and does not contain detectable pyrogens and / or other toxins. Thus, the present disclosure provides a composition that includes a) the CVB described above; and b) saline (e.g., 0.9% NaCl), in which case the composition is a sterile composition and does not contain detectable pyrogens and / or other toxins.

[0065] Methods for inducing an immune response The present disclosure provides a method of inducing an immune response against CVB in an individual. The method comprises administering to an individual in need thereof an effective amount of a composition of the present disclosure. The composition of the present disclosure is hereinafter referred to as an "immunogenic composition". The present disclosure provides a method of reducing the likelihood that an individual will develop an acute CVB infection. The method comprises administering to an individual in need thereof an effective amount of a composition of the present disclosure. The present disclosure provides a method of reducing the likelihood that an individual will develop a CVB infection-associated disease and / or symptoms of a CVB infection. The method comprises administering to an individual in need thereof an effective amount of a composition of the present disclosure.

[0066] The immunogenic compositions described above are administered parenterally, bucally, orally, intradermally, transdermally, sublingually, intranasally, nasopharyngeally, via inhalation, or rectally, by injection (e.g., intramuscular or subcutaneous injection).

[0067] In some cases, a prime-boost vaccine protocol is used. In some cases, a first (priming) immunogenic composition is administered, and after a period of time, a second (booster) immunogenic composition is administered. The second immunogenic composition is administered one day to one year after administration of the first immunogenic composition. For example, the second immunogenic composition is administered one day to one week, one week to two weeks, two weeks to one month, one month to two months, two months to six months, or six months to one year after administration of the first immunogenic composition.

[0068] The method of the present disclosure can induce an immune response against CVB in an individual. For example, the method of the present disclosure can induce an immune response against one or more of CVB1, CVB2, CVB3, CVB4, and CVB5 in an individual. In some cases, the method of the present disclosure can induce an immune response against CVB1, CVB2, CVB3, CVB4, and CVB5 in an individual.

[0069] In some cases, the disclosed method may reduce the likelihood of acute infection by CVB. In some cases, the disclosed method may reduce the likelihood that an individual will develop a CVB infection-related disease (e.g., CVB-induced gastrointestinal disease, myocarditis, pneumonia, aseptic meningitis, encephalitis, or hepatitis). In some cases, the disclosed method may ameliorate one or more adverse symptoms of CVB infection. In some cases, the disclosed method treats one or more of CVB-induced gastrointestinal disease, myocarditis, pneumonia, aseptic meningitis, encephalitis, and hepatitis.

[0070] In some cases, when an individual to whom an immunogenic composition of the disclosure is administered is at greater risk than the general population of developing type 1 diabetes (T1D), the methods of the disclosure may reduce the likelihood that the individual will develop T1D.

[0071] In some cases, when an individual to whom an immunogenic composition of the disclosure is administered is at greater risk than the general population of developing celiac disease, the methods of the disclosure may reduce the likelihood that the individual will develop celiac disease.

[0072] Suitable subjects for treatment In some cases, individuals suitable for treatment according to the disclosure are between 1 month and 3 months of age (e.g., 1 month, 2 months, or 3 months of age). In some cases, individuals suitable for treatment according to the disclosure are between 3 months and 5 years of age. In some cases, individuals suitable for treatment according to the disclosure are between 3 months and 6 months of age, between 6 months and 1 year of age, between 1 year and 2 years of age, or between 2 years and 5 years of age. In some cases, individuals suitable for treatment according to the disclosure are between 5 years and 11 years of age. In some cases, individuals suitable for treatment according to the disclosure are between 12 years and 17 years of age. In some cases, individuals suitable for treatment according to the disclosure are between 18 years and 25 years of age. In some cases, individuals suitable for treatment according to the disclosure are over 25 years of age.

[0073] Optionally, the individual is over 6 years of age, 2 years or older, 6 years or older, 12 years or older, 18 years or older, 30 years or older, between about 6 and 12 years of age, between about 6 and about 30 years of age, between about 12 and about 30 years of age, or between about 18 and about 30 years of age. Optionally, the individual is a pregnant woman.

[0074] In some cases, individuals suitable for treatment according to the present disclosure are immunologically naive with respect to CVB; i.e., the individual has low or undetectable neutralizing antibodies to CVB. In some cases, individuals suitable for treatment according to the present disclosure have previously been exposed to CVB (e.g., via natural infection) and have detectable antibody titers to CVB.

[0075] In some cases, the individual is at risk (e.g., at a greater risk than the general population) of developing type 1 diabetes (T1D). Individuals at risk (e.g., at a greater risk than the general population) of developing T1D include individuals who are genetically determined to be at a higher risk of developing T1D, such as individuals who are HLA-endowed susceptible to T1D, particularly carriers of HLA DR3 and / or DR4 alleles. Individuals at risk (e.g., at a greater risk than the general population) of developing T1D include mothers or children who are HLA-endowed susceptible to T1D, particularly carriers of HLA DR3 and / or DR4 alleles; individuals with T1D in first- or second-degree relatives; and individuals (e.g., children) who test positive for two or more diabetes-related autoantibodies. In some cases, the subject method reduces the likelihood that an individual will develop T1D.

[0076] In some cases, the individual is at risk of developing celiac disease (e.g., at a greater risk than the general population). Such individuals include individuals who have a genetic predisposition to developing celiac disease; and individuals who have tested positive for celiac disease-associated autoantibodies. In some cases, the individual is a carrier of the HLA-DR3 allele.

[0077] Examples of Non-Limiting Aspects of the Disclosure Mode Set A The aspects including the embodiments of the subject matter described above may be useful alone or in combination with one or more other aspects or embodiments. Below, without limiting the above description, certain non-limiting aspects of the present disclosure are presented. As will be apparent to those skilled in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or subsequent individually numbered aspects. This is intended to endorse all such combinations of aspects, and is not limited to the combinations of aspects explicitly presented below: A composition comprising two to five serotypes of Group B Coxsackievirus (CVB), the CVB being in the form of an inactivated virus particle (IVP), a virus-like particle (VLP), one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides, the composition comprising: A) In a volume of 100 μL, a) IVP 2.0×10 7 pieces~IVP 5.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 3.0×10 7 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 3.0×10 8 Amount of CVB3; d) IVP 2.0×10 7 pieces~IVP 5.0×10 7 amount of CVB4; and e) IVP 4.0×10 7 pieces~IVP 8.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. two or more of; or B) In a volume of 500 μL, a) IVP 1.0×10 8 pieces~IVP 2.5×10 8 Amount of CVB1; b) IVP 5.0×10 7 pieces~IVP 1.5×10 8 Quantity of pieces CVB2; c) IVP 5.0×10 8 pieces~IVP 1.5×10 9 Amount of CVB3; d) IVP 1.0×10 8 pieces~IVP 2.5×10 8 amount of CVB4; and e) IVP 2.0×10 8 pieces~IVP 4.0×10 8 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. Two or more of Including, Does not contain CVB6, composition. Aspect 2. In a volume of 100 μl, a) IVP 2.0×10 7 pieces~IVP 5.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 3.0×10 7 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 3.0×10 8 Amount of CVB3; d) IVP 2.0×10 7 pieces~IVP 5.0×10 7 amount of CVB4; and e) IVP 4.0×10 7 pieces~IVP 8.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. 2. The composition of embodiment 1, comprising: Aspect 3.a) IVP 3.0 x 10 7 pieces~IVP 4.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 2.0×10 7 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 2.0×10 8 Amount of CVB3; d) IVP 3.0×10 7 pieces~IVP 4.0×10 7 amount of CVB4; and e) IVP 6.0×10 7 pieces~IVP 6.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. 3. The composition of embodiment 2, comprising: Embodiment 4. A composition comprising two to five serotypes of a Group B Coxsackievirus (CVB), wherein the CVB is in the form of an inactivated virus particle (IVP), a CVB virus-like particle (VLP), one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides, wherein the composition, when administered to an individual, induces a virus neutralizing antibody titer (VNT) of 1 / 8 to 1 / 64,000 as determined by a VNT assay, and the composition does not include CVB6. Embodiment 5. The composition comprises 2 to 5 serotypes of CVB, the CVB being in the form of an inactivated virus particle (IVP), and the composition comprises: A) In a volume of 100 μL, a) IVP 2.0×10 7 pieces~IVP 5.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 3.0×10 7 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 3.0×10 8 Amount of CVB3; d) IVP 2.0×10 7 pieces~IVP 5.0×10 7 amount of CVB4; and e) IVP 4.0×10 7 pieces~IVP 8.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. two or more of; or B) In a volume of 500 μL, a) IVP 1.0×10 8 pieces~IVP 2.5×10 8 Amount of CVB1; b) IVP 5.0×10 7 pieces~IVP 1.5×10 8 Quantity of pieces CVB2; c) IVP 5.0×10 8 pieces~IVP 1.5×10 9 Amount of CVB3; d) IVP 1.0×10 8 pieces~IVP 2.5×10 8 amount of CVB4; and e) IVP 2.0×10 8 pieces~IVP 4.0×10 8 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. Two or more of 5. The composition of embodiment 4, comprising: Aspect 6.a) IVP 2.0 x 10 7 pieces~IVP 5.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 3.0×10 7 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 3.0×10 8 Amount of CVB3; d) IVP 2.0×10 7 pieces~IVP 5.0×10 7 amount of CVB4; and e) IVP 4.0×10 7pieces~IVP 8.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. 6. The composition of embodiment 5, comprising: Aspect 7.a) IVP 3.0 x 10 7 pieces~IVP 4.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 2.0×10 7 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 2.0×10 8 Amount of CVB3; d) IVP 3.0×10 7 pieces~IVP 4.0×10 7 amount of CVB4; and e) IVP 6.0×10 7 pieces~IVP 6.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. 7. The composition of embodiment 6, comprising: Embodiment 8. The composition according to any one of embodiments 1 to 7, wherein the IVP is inactivated with formalin. Embodiment 9. A composition according to any one of embodiments 1 to 8, comprising an adjuvant. Embodiment 10. The composition according to any one of embodiments 1 to 8, comprising a saline solution. Embodiment 11. A method of inducing an immune response against group B Coxsackievirus (CVB) in an individual, comprising administering to the individual a composition according to any one of embodiments 1 to 10. Embodiment 12 The method of embodiment 11, wherein the composition is administered intramuscularly. Embodiment 13 The method of embodiment 11, wherein the composition is administered subcutaneously. Embodiment 14. A method of reducing the likelihood that an individual will develop an acute Coxsackievirus B (CVB) infection, comprising administering to the individual a composition according to any one of embodiments 1 to 10. Embodiment 15 The method of any one of embodiments 11 to 14, wherein the individual is a newborn. Embodiment 16 The method of any one of embodiments 11 to 14, wherein the individual is a pregnant woman. Embodiment 17 The method of any one of embodiments 11 to 14, wherein the individual is at increased risk of developing type 1 diabetes. Embodiment 18 The method of any one of embodiments 11 to 14, wherein the individual is at increased risk of developing celiac disease. Embodiment 19. The method according to any one of embodiments 11 to 14, wherein the individual is a carrier of the HLA DR3 allele and / or the HLA DR4 allele. Embodiment 20. The method of any one of embodiments 11 to 14, wherein the individual was seropositive for virus neutralizing titers against one or more of CVB1, CVB2, CVB3, CVB4, and CVB5 prior to said administering step. Embodiment 21. The method of any one of embodiments 11 to 14, wherein the individual was seronegative for virus neutralization titers against one or more of CVB1, CVB2, CVB3, CVB4, and CVB5 prior to said administering step.

[0078] Mode Set B The aspects including the embodiments of the subject matter described above may be useful alone or in combination with one or more other aspects or embodiments. Below, without limiting the above description, certain non-limiting aspects of the present disclosure are presented. As will be apparent to those skilled in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or subsequent individually numbered aspects. This is intended to endorse all such combinations of aspects, and is not limited to the combinations of aspects explicitly presented below: A composition comprising two to five serotypes of Group B Coxsackievirus (CVB), the CVB being in the form of an inactivated virus particle (IVP), a virus-like particle (VLP), one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides, the composition comprising: A) In a volume of 100 μL, a) IVP 2.0×10 7 pieces~IVP 5.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 3.0×10 7 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 3.0×10 8 Amount of CVB3; d) IVP 2.0×10 7 pieces~IVP 5.0×10 7 amount of CVB4; and e) IVP 4.0×10 7 pieces~IVP 8.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. two or more of; or B) In a volume of 500 μL, a) IVP 1.0×10 8 pieces~IVP 2.5×10 8 Amount of CVB1; b) IVP 5.0×10 7 pieces~IVP 1.5×10 8 Quantity of pieces CVB2; c) IVP 5.0×10 8 pieces~IVP 1.5×10 9 Amount of CVB3; d) IVP 1.0×10 8 pieces~IVP 2.5×10 8 amount of CVB4; and e) IVP 2.0×10 8 pieces~IVP 4.0×10 8 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. two or more of; or C) a) CVB1 protein in an amount between 1 μg and 50 μg; b) CVB2 protein in an amount between 1 μg and 50 μg; c) CVB3 protein in an amount between 1 μg and 50 μg; d) CVB4 protein in an amount between 1 μg and 50 μg; and e) CVB5 protein in an amount between 1 μg and 50 μg Two or more of Including, Does not contain CVB6, composition. Aspect 2. In a volume of 100 μl, a) IVP 2.0×10 7 pieces~IVP 5.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 3.0×10 7 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 3.0×10 8 Amount of CVB3; d) IVP 2.0×10 7 pieces~IVP 5.0×10 7 amount of CVB4; and e) IVP 4.0×10 7 pieces~IVP 8.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. 2. The composition of embodiment 1, comprising: Aspect 3.a) IVP 3.0 x 10 7 pieces~IVP 4.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 2.0×10 7 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 2.0×10 8 Amount of CVB3; d) IVP 3.0×10 7 pieces~IVP 4.0×10 7 amount of CVB4; and e) IVP 6.0×10 7 pieces~IVP 6.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. 3. The composition of embodiment 2, comprising: Aspect 4. In a volume of 100 μl, a) IVP 3.3×10 7 Amount of CVB1; b) IVP 1.1×10 7 Quantity of pieces CVB2; c) IVP 1.5×10 8 Amount of CVB3; d) IVP 3.2 x 10 7 amount of CVB4; and e) IVP 6.3×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. 2. The composition of embodiment 1, comprising: 5. In a volume of 500 μL, a) IVP 1.6×10 8 Amount of CVB1; b) IVP 5.5×10 7 Quantity of pieces CVB2; c) IVP 7.5×10 8 Amount of CVB3; d) IVP 1.6×10 8 amount of CVB4; and e) IVP 3.1 x 10 8 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. 2. The composition of embodiment 1, comprising: 6. In a volume of 100 μl, a) CVB1 protein in an amount of 2.8 μg; b) CVB2 protein in an amount of 1.7 μg; c) CVB3 protein in an amount of 2.7 μg; d) CVB4 protein in an amount of 1.2 μg; and e) CVB5 protein in an amount of 2.3 μg 2. The composition of embodiment 1, comprising: 7. In a volume of 500 μl, a) CVB1 protein in an amount of 14 μg; b) CVB2 protein in an amount of 8.5 μg; c) CVB3 protein in an amount of 13.5 μg; d) CVB4 protein in an amount of 6.0 μg; and e) CVB5 protein in an amount of 11.5 μg 2. The composition of embodiment 1, comprising: Embodiment 8. A composition comprising two to five serotypes of a Group B Coxsackievirus (CVB), wherein the CVB is in the form of an inactivated virus particle (IVP), a CVB virus-like particle (VLP), one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides, wherein the composition, when administered to an individual, induces a virus neutralizing antibody titer (VNT) of 1 / 8 to 1 / 64,000 as determined by a VNT assay, and the composition does not include CVB6. Embodiment 9. The composition comprises 2 to 5 serotypes of CVB, the CVB being in the form of an inactivated virus particle (IVP), the composition comprising: A) In a volume of 100 μL, a) IVP 2.0×10 7 pieces~IVP 5.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 3.0×10 7 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 3.0×10 8 Amount of CVB3; d) IVP 2.0×10 7 pieces~IVP 5.0×10 7 amount of CVB4; and e) IVP 4.0×10 7 pieces~IVP 8.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. two or more of; or B) a) IVP 1.0×10 8 pieces~IVP 2.5×10 8 Amount of CVB1; b) IVP 5.0×10 7 pieces~IVP 1.5×10 8 Quantity of pieces CVB2; c) IVP 5.0×10 8 pieces~IVP 1.5×10 9 Amount of CVB3; d) IVP 1.0×10 8 pieces~IVP 2.5×10 8 amount of CVB4; and e) IVP 2.0×10 8 pieces~IVP 4.0×10 8 Quantity of CVB5 two or more of; or C) In a volume of 500 μL, a) CVB1 protein in an amount between 1 μg and 50 μg; b) CVB2 protein in an amount between 1 μg and 50 μg; c) CVB3 protein in an amount between 1 μg and 50 μg; d) CVB4 protein in an amount between 1 μg and 50 μg; and e) CVB5 protein in an amount between 1 μg and 50 μg; or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. Two or more of 9. The composition of embodiment 8, comprising: Aspect 10.a) IVP 2.0 x 10 7 pieces~IVP 5.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 3.0×10 7 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 3.0×10 8 Amount of CVB3; d) IVP 2.0×10 7 pieces~IVP 5.0×10 7 amount of CVB4; and e) IVP 4.0×10 7 pieces~IVP 8.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. 10. The composition of embodiment 9, comprising: Aspect 11.a) IVP 3.0 x 10 7 pieces~IVP 4.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces~IVP 2.0×10 7 Quantity of pieces CVB2; c) IVP 1.0×10 8 pieces~IVP 2.0×10 8 Amount of CVB3; d) IVP 3.0×10 7 pieces~IVP 4.0×10 7 amount of CVB4; and e) IVP 6.0×10 7 pieces~IVP 6.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. 11. The composition of embodiment 10, comprising: 12. In a volume of 100 μl, a) IVP 3.3×10 7 Amount of CVB1; b) IVP 1.1 x 10 7 Quantity of pieces CVB2; c) IVP 1.5×10 8 Amount of CVB3; d) IVP 3.2 x 10 7 amount of CVB4; and e) IVP 6.3×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. 10. The composition of embodiment 9, comprising: 13. In a volume of 500 μL, a) IVP 1.6×10 8 Amount of CVB1; b) IVP 5.5×10 7 Quantity of pieces CVB2; c) IVP 7.5×10 8 Amount of CVB3; d) IVP 1.6×10 8 amount of CVB4; and e) IVP 3.1 x 10 8 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. 10. The composition of embodiment 9, comprising: 14. In a volume of 100 μl, a) CVB1 protein in an amount of 2.8 μg; b) CVB2 protein in an amount of 1.7 μg; c) CVB3 protein in an amount of 2.7 μg; d) CVB4 protein in an amount of 1.2 μg; and e) CVB5 protein in an amount of 2.3 μg 10. The composition of embodiment 9, comprising: 15. In a volume of 500 μl, a) CVB1 protein in an amount of 14 μg; b) CVB2 protein in an amount of 8.5 μg; c) CVB3 protein in an amount of 13.5 μg; d) CVB4 protein in an amount of 6.0 μg; and e) CVB5 protein in an amount of 11.5 μg 10. The composition of embodiment 9, comprising: Embodiment 16 The composition according to any one of embodiments 1 to 5 and 8 to 13, wherein the IVP is inactivated with formalin. 17. Adjuvants 17. The composition of any one of the preceding aspects, comprising: Aspect 18. Saline solution 18. The composition of any one of the preceding aspects, comprising: Embodiment 19. A method of inducing an immune response against group B Coxsackievirus (CVB) in an individual, comprising administering to the individual a composition according to any one of embodiments 1 to 18. Embodiment 20 The method of embodiment 19, wherein the composition is administered intramuscularly. Embodiment 21 The method of embodiment 19, wherein the composition is administered subcutaneously. Embodiment 22. A method for reducing the likelihood that an individual will develop an acute Coxsackievirus B (CVB) infection or a disease associated with a CVB infection, comprising administering to the individual a composition according to any one of embodiments 1 to 18. Embodiment 23 The method of any one of embodiments 19 to 22, wherein the individual is a newborn. Embodiment 24 The method of any one of embodiments 19 to 22, wherein the individual is a pregnant woman. Embodiment 25 The method of any one of embodiments 19 to 22, wherein the individual is at increased risk of developing type 1 diabetes. Embodiment 26 The method of any one of embodiments 19 to 22, wherein the individual is at high risk of developing celiac disease. Embodiment 27. The method according to any one of embodiments 19 to 22, wherein the individual is a carrier of the HLA DR3 allele and / or the HLA DR4 allele. Embodiment 28. The method of any one of embodiments 19 to 22, wherein the individual was seropositive for virus neutralizing titers against one or more of CVB1, CVB2, CVB3, CVB4, and CVB5 prior to said administering step. Embodiment 29. The method of any one of embodiments 19 to 22, wherein the individual was seronegative for virus neutralization titers against one or more of CVB1, CVB2, CVB3, CVB4, and CVB5 prior to said administering step.

[0079] Working Example The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight by weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations are used, such as, for example, bp: base pair(s); kb: kilobase(s); pl: picoliter(s); s or sec: second(s); min: minute(s); h or hr: hour(s); aa: amino acid(s); kb: kilobase(s); bp: base pair(s); nt: nucleotide(s); im: intramuscular; ip: intraperitoneal; sc: subcutaneous, etc. EXAMPLES

[0080] Induction of VNT in human subjects A composition containing CVB serotypes 1-5 was administered to normal healthy human volunteers. Two cohorts were randomized 3:1 to receive 100 μL or 500 μL of the composition or a matching placebo. Subjects were administered three doses at monthly intervals. All subjects were analyzed for VNT after completing three doses and followed for one month after the final dose. The composition is a formalin-inactivated vaccine containing CVB serotypes 1-5 in the form of inactivated virus particles (IVP). The IVP is equivalent to the RNA copy number.

[0081] The 100 μL ("low dose") composition contains CVB1-CVB5 in the following amounts: a) CVB1:IVP 3.3×10 7 pieces; b) CVB2: IVP 1.1 x 10 7 pieces; c) CVB3: IVP 1.5 x 10 8 pieces; d) CVB4: IVP 3.2 x 10 7 pcs; and e) CVB5: IVP 6.3 x 10 7 pieces Included.

[0082] The 500 μL ("high dose") composition contains CVB1-CVB5 in the following amounts: a) CVB1: IVP 1.6×10 8 pieces; b) CVB2: IVP 5.5 x 10 7 pieces; c) CVB3: IVP 7.5 x 10 8 pieces; d) CVB4: IVP 1.6×10 8 pcs; and e) CVB5:IVP 3.1×10 8 pieces Included.

[0083] No treatment-emergent serious adverse events (TESAES) were observed in any of the subjects. No treatment-emergent adverse events of special interest (TEAESIS) were observed in any of the subjects.

[0084] A dose-dependent generation of high virus neutralizing titers (VNT) was observed. Blue: low dose; red: high dose. Data are shown in Figure 2. As shown in Figure 2, titers ranging from 1 / 2,000 to 1 / 4,000 were observed. Anti-CVB virus titers present in approximately 40% of subjects account for baseline VNT in all groups. Peak VNT titers of antibodies for each serotype are shown in Figure 3.

[0085] VNT in seropositive and seronegative individuals VNT for each of CVB1 to CVB5 was examined both pre- and post-vaccination. The data are depicted in Figures 4 to 13. Figures 4 to 13 present mean (± standard deviation) curves for anti-CVB virus neutralizing antibody titers, baseline seroconversion, and serotype by visit.

[0086] Figures 4-13 depict VNT against CVB1 (Figures 4 and 5), CVB2 (Figures 6 and 7), CVB3 (Figures 8 and 9), CVB4 (Figures 10 and 11), and CVB5 (Figures 12 and 13) in the serum of vaccinated human subjects. Subjects were either seropositive for VNT against the indicated CVB serotypes prior to vaccination (Figures 4, 6, 8, 10, and 12) or seronegative prior to vaccination (Figures 5, 7, 9, 11, and 13).

[0087] Response rate Response rates were evaluated in vaccinated human subjects. Responders were defined as: a) subjects who were seronegative at baseline and seroconverted (i.e., VNT is ≧1 / 4) at any time point after vaccination; or b) subjects who were positive for antibodies (i.e., subjects with pre-existing antibodies) at baseline and experienced a 4-fold or greater increase in VNT at any time point after vaccination.

[0088] The data is shown in Table 1 below.

[0089] [Table 1]

[0090] Results at 6 months (week 32) Levels of VNT were assayed at various time points (weeks 4, 8, 12, and 32) after the first dose of the CVB vaccine composition. The results are shown in Figures 14, 15, and 17.

[0091] As shown in Figure 14, for subjects who were seronegative at baseline, at week 32 (i.e., 24 weeks after the last dose at week 12), 90% in the high-dose group had titers (VNT) of 1 / 8 or greater against all five serotypes. As shown in Figure 14, at week 32, 75% of subjects in the low-dose group had titers of 1 / 8 or greater against all five serotypes.

[0092] As shown in FIG. 15, a commercial ELISA assay (DRG International) demonstrated a sustained enterovirus-specific IgG response in a dose / time-dependent manner.

[0093] Figure 17 depicts CVB neutralizing antibody titers in all subjects (seropositive subjects at baseline and seronegative subjects at baseline). Titers are presented on a logarithmic (log2) scale. In addition to a dose / time response, durability of response at 32 weeks is evident for all serotypes in the vaccine.

[0094] The data show the persistence of VNT responses against all strains of CVB in the vaccine (CVB1-5). At this final time point (week 32), 6 months after the last dose, the percentage of subjects in the high-dose PRV-01 arm who maintained high titers of VNT was 100% for the four serotypes included in the vaccine and 90% for all five serotypes. These data confirmed the persistence of responses. High titers are defined as ≥ 1 / 8, a level seen to be clinically protective in the only other enterovirus vaccine, the commercial inactivated poliovirus vaccine. Evidence of VNT cross-reactivity against CVB6, a less common CVB serotype not included in the CVB vaccine composition, was observed. In addition to VNT, a commercial ELISA assay showed a dose / time-dependent sustained IgG response after switching from an initial robust IgM response as well as some IgA response (indicative of a degree of mucosal immunity). EXAMPLES

[0095] A composition containing CVB serotypes 1-5 was administered to normal healthy human volunteers. Two cohorts were randomized 3:1 to receive 100 μL or 500 μL of the composition or a matching placebo. Subjects were administered three doses at monthly intervals. Analysis of VNT was performed after all subjects had completed three doses. Final analysis was performed over a 6-month period (up to week 32 of the study) after the last dose of vaccine. The composition is a formalin-inactivated vaccine containing CVB serotypes 1-5 in the form of an IVP.

[0096] The results indicate that high titers of VNT were induced in a dose-dependent manner against all five serotypes. Responses were sustained for up to six months after the last dose of vaccine, with high levels of protective antibodies achieved in 100% of subjects for most serotypes included in the vaccine and in over 90% of subjects for all serotypes. The data are shown in Figures 17 and 18.

[0097] As shown in Figure 16, anti-CVB antibodies, IgG, were produced in a dose-dependent manner. The data support the persistence of IgG responses at week 32. Similar data were obtained for IgM.

[0098] Figure 18 shows dose-dependent production of high titers of VNT for all serotypes (CVB1, CVB2, CVB3, CVB4, and CVB5). For subjects who were seronegative at baseline, at week 32, 100% of subjects had neutralizing antibody titers of 1 / 8 or greater against at least four serotypes included in the vaccine, and over 90% of subjects had neutralizing antibody titers of 1 / 8 or greater against all five serotypes. EXAMPLES

[0099] Neutralizing antibodies were measured against CVB6 and Coxsackievirus type A9 (CAV9) using a plaque neutralizing antibody assay. Neutralizing antibodies were analyzed from baseline serum samples taken at the time of the first vaccination as well as samples taken one month after the third vaccination.

[0100] In total, 64 serum samples, including 32 V2 samples and 32 V11 samples, were analyzed for neutralizing CVB antibodies. The antibody levels at these two time points indicate whether the PRV-101 vaccine induces cross-reactive antibodies in subjects who were initially CVB6 / CAV9 seronegative or increases antibody titers in subjects who were initially seropositive. The results showed that the PRV-101 vaccine induced neutralizing antibodies with some cross-reactivity against CVB6. No cross-reactivity against CAV9 was observed.

[0101] In total, eight study participants developed seroconversion or a four-fold or greater increase in neutralizing antibodies against CVB6. All eight responders were in the vaccination study arm and none in the placebo arm. No study participants developed a response to CVA9. The data indicate that, given the similarities between CVB viruses, a vaccine containing some, but not all six, of the known CVB serotypes may elicit protection against all six CVB serotypes. Thus, an immunogenic composition could be a vaccine based on one to five CVB strains, but also elicit protection against one or more of the CVB strains not present on the vaccine.

[0102] Although the present invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

1. A composition comprising two to five serotypes of group B Coxsackievirus (CVB), wherein the CVB is in the form of an inactivated virus particle (IVP), a virus-like particle (VLP), one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides, the composition comprising: A) In a volume of 100 μL, a) IVP 2.0×10 7 pieces ~ IVP 5.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces ~ IVP 3.0×10 7 Quantity of CVB2; c) IVP 1.0×10 8 pieces ~ IVP 3.0×10 8 Quantity of CVB3; d) IVP 2.0×10 7 pieces ~ IVP 5.0×10 7 an amount of CVB4; and e) IVP 4.0×10 7 Piece ~ IVP 8.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. two or more of: B) In a volume of 500 μL, a) IVP 1.0×10 8 pieces ~ IVP 2.5×10 8 Amount of CVB1; b) IVP 5.0×10 7 pieces ~ IVP 1.5×10 8 Quantity of CVB2; c) IVP 5.0×10 8 pieces ~ IVP 1.5×10 9 Quantity of CVB3; d) IVP 1.0×10 8 pieces ~ IVP 2.5×10 8 an amount of CVB4; and e) IVP 2.0×10 8 Pieces ~ IVP 4.0×10 8 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. two or more of: C) a) CVB1 protein in an amount of 1 μg to 50 μg; b) CVB2 protein in an amount of 1 μg to 50 μg; c) CVB3 protein in an amount of 1 μg to 50 μg; d) CVB4 protein in an amount of 1 μg to 50 μg; and e) CVB5 protein in an amount between 1 μg and 50 μg Two or more of Including, Does not contain CVB6, composition.

2. The composition contains, in a volume of 100 μl: a) IVP 2.0×10 7 pieces ~ IVP 5.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces ~ IVP 3.0×10 7 Quantity of CVB2; c) IVP 1.0×10 8 pieces ~ IVP 3.0×10 8 Quantity of CVB3; d) IVP 2.0×10 7 pieces ~ IVP 5.0×10 7 an amount of CVB4; and e) IVP 4.0×10 7 Piece ~ IVP 8.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. Includes; or The composition contains, in a volume of 100 μl: a) IVP 3.0×10 7 Pieces ~ IVP 4.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 Pieces ~ IVP 2.0×10 7 Quantity of CVB2; c) IVP 1.0×10 8 Pieces ~ IVP 2.0×10 8 Quantity of CVB3; d) IVP 3.0×10 7 Pieces ~ IVP 4.0×10 7 Quantity of CVB4; and e) IVP 6.0×10 7 pieces ~ IVP 6.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. Includes; or The composition contains, in a volume of 100 μl: a) IVP 3.3×10 7 Amount of CVB1; b) IVP 1.1×10 7 Quantity of CVB2; c) IVP 1.5×10 8 Quantity of CVB3; d) IVP 3.2×10 7 an amount of CVB4; and e) IVP 6.3×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. The composition of claim 1 comprising:

3. The composition contains, in a volume of 500 μL: a) IVP 1.6×10 8 Amount of CVB1; b) IVP 5.5×10 7 Quantity of CVB2; c) IVP 7.5×10 8 Quantity of CVB3; d) IVP 1.6×10 8 an amount of CVB4; and e) IVP 3.1×10 8 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. The composition of claim 1 comprising:

4. The composition contains, in a volume of 100 μl: a) CVB1 protein in an amount of 2.8 μg; b) CVB2 protein in an amount of 1.7 μg; c) CVB3 protein in an amount of 2.7 μg; d) CVB4 protein in an amount of 1.2 μg; and e) CVB5 protein in an amount of 2.3 μg Contains; or The composition contains, in a volume of 500 μl: a) CVB1 protein in an amount of 14 μg; b) CVB2 protein in an amount of 8.5 μg; c) CVB3 protein in an amount of 13.5 μg; d) CVB4 protein in an amount of 6.0 μg; and e) CVB5 protein in an amount of 11.5 μg The composition of claim 1 comprising:

5. A composition comprising two to five serotypes of group B coxsackievirus (CVB), wherein the CVB is in the form of an inactivated virus particle (IVP), a CVB virus-like particle (VLP), one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides, wherein the composition, when administered to an individual, induces a virus neutralizing antibody titer (VNT) of 1 / 8 to 1 / 64,000 as determined by a VNT assay, and the composition does not contain CVB6.

6. The composition comprises 2 to 5 serotypes of CVB, the CVB being in the form of inactivated virus particles (IVP), and the composition A) In a volume of 100 μL, a) IVP 2.0×10 7 pieces ~ IVP 5.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces ~ IVP 3.0×10 7 Quantity of CVB2; c) IVP 1.0×10 8 pieces ~ IVP 3.0×10 8 Quantity of CVB3; d) IVP 2.0×10 7 pieces ~ IVP 5.0×10 7 an amount of CVB4; and e) IVP 4.0×10 7 Piece ~ IVP 8.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. two or more of: B) a) IVP 1.0×10 8 pieces ~ IVP 2.5×10 8 Amount of CVB1; b) IVP 5.0×10 7 pieces ~ IVP 1.5×10 8 Quantity of CVB2; c) IVP 5.0×10 8 pieces ~ IVP 1.5×10 9 Quantity of CVB3; d) IVP 1.0×10 8 pieces ~ IVP 2.5×10 8 an amount of CVB4; and e) IVP 2.0×10 8 Pieces ~ IVP 4.0×10 8 Amount of CVB5 two or more of: C) In a volume of 500 μL, a) CVB1 protein in an amount of 1 μg to 50 μg; b) CVB2 protein in an amount of 1 μg to 50 μg; c) CVB3 protein in an amount of 1 μg to 50 μg; d) CVB4 protein in an amount of 1 μg to 50 μg; and e) CVB5 protein in an amount of 1 μg to 50 μg; or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. Two or more of The composition of claim 5 comprising:

7. The composition comprises: a) IVP 2.0×10 7 pieces ~ IVP 5.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 pieces ~ IVP 3.0×10 7 Quantity of CVB2; c) IVP 1.0×10 8 pieces ~ IVP 3.0×10 8 Quantity of CVB3; d) IVP 2.0×10 7 pieces ~ IVP 5.0×10 7 an amount of CVB4; and e) IVP 4.0×10 7 Piece ~ IVP 8.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. Includes; or The composition comprises: a) IVP 3.0×10 7 Pieces ~ IVP 4.0×10 7 Amount of CVB1; b) IVP 1.0×10 7 Pieces ~ IVP 2.0×10 7 Quantity of CVB2; c) IVP 1.0×10 8 Pieces ~ IVP 2.0×10 8 Quantity of CVB3; d) IVP 3.0×10 7 Pieces ~ IVP 4.0×10 7 an amount of CVB4; and e) IVP 6.0×10 7 pieces ~ IVP 6.0×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. Includes; or The composition contains, in a volume of 100 μl: a) IVP 3.3×10 7 Amount of CVB1; b) IVP 1.1×10 7 Quantity of CVB2; c) IVP 1.5×10 8 Quantity of CVB3; d) IVP 3.2×10 7 an amount of CVB4; and e) IVP 6.3×10 7 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. The composition of claim 6 comprising:

8. The composition contains, in a volume of 500 μL: a) IVP 1.6×10 8 Amount of CVB1; b) IVP 5.5×10 7 Quantity of CVB2; c) IVP 7.5×10 8 Quantity of CVB3; d) IVP 1.6×10 8 an amount of CVB4; and e) IVP 3.1×10 8 Quantity of CVB5, or an equivalent amount of a VLP, one or more CVB polypeptides, or one or more nucleic acids comprising a nucleotide sequence encoding one or more CVB polypeptides. The composition of claim 6 comprising:

9. The composition contains, in a volume of 100 μl: a) CVB1 protein in an amount of 2.8 μg; b) CVB2 protein in an amount of 1.7 μg; c) CVB3 protein in an amount of 2.7 μg; d) CVB4 protein in an amount of 1.2 μg; and e) CVB5 protein in an amount of 2.3 μg Contains; or The composition contains, in a volume of 500 μl: a) CVB1 protein in an amount of 14 μg; b) CVB2 protein in an amount of 8.5 μg; c) CVB3 protein in an amount of 13.5 μg; d) CVB4 protein in an amount of 6.0 μg; and e) CVB5 protein in an amount of 11.5 μg The composition of claim 6 comprising:

10. 10. The composition of claim 1 or 5, wherein the IVP is formalin inactivated.

11. The composition of claim 1 or 5, comprising an adjuvant and / or saline.

12. 10. The composition of claim 1 or 5 for use in inducing an immune response against group B Coxsackievirus (CVB) in an individual.

13. The composition of claim 12, wherein the composition is administered intramuscularly or subcutaneously.

14. 10. The composition of claim 1 or 5 for use in reducing the likelihood that an individual will develop an acute group B Coxsackievirus (CVB) infection or a disease associated with CVB infection.

15. 13. The composition of claim 12, wherein the individual is a newborn, the individual is a pregnant woman, the individual is at increased risk of developing type 1 diabetes, the individual is at increased risk of developing celiac disease, the individual is a carrier of the HLA DR3 allele and / or the HLA DR4 allele, the individual was seropositive for virus neutralization titers against one or more of CVB1, CVB2, CVB3, CVB4, and CVB5 prior to said use, or the individual was seronegative for virus neutralization titers against one or more of CVB1, CVB2, CVB3, CVB4, and CVB5 prior to said use.

16. 15. The composition of claim 14, wherein the individual is a newborn, the individual is a pregnant woman, the individual is at increased risk of developing type 1 diabetes, the individual is at increased risk of developing celiac disease, the individual is a carrier of the HLA DR3 allele and / or the HLA DR4 allele, the individual was seropositive for virus neutralization titers against one or more of CVB1, CVB2, CVB3, CVB4, and CVB5 prior to said use, or the individual was seronegative for virus neutralization titers against one or more of CVB1, CVB2, CVB3, CVB4, and CVB5 prior to said use.

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