Immunogenic composition for vaccination against rotavirus

Abstract

The present invention relates to immunogenic compositions comprising recombinantly constructed polypeptides suitable for the preparation of vaccines, in particular vaccines for reducing one or more clinical signs caused by rotavirus infection. More specifically, the present invention is directed to an immunogenic composition containing: (i) a fusion protein comprising in the N-terminal to C-terminal direction (A) an immunogenic fragment of a rotavirus VP8 protein and (B) an immunoglobulin Fc fragment such as, for example, an IgG Fc fragment; and (ii) an immunogenic substance different from the fusion protein, wherein the immunogenic composition is suitable for use in a method of reducing one or more clinical signs, death or faecal excretion caused by rotavirus infection in a porcine animal.

Description

Background of the Invention Technical Field

[0002] The present invention relates to an immunogenic composition comprising a recombinantly constructed polypeptide, which is suitable for reducing one or more clinical signs caused by rotavirus infection. More specifically, the present invention relates to an immunogenic composition, which contains: (i) a fusion protein comprising (A) an immunogenic fragment of a rotavirus VP8 protein and (B) an immunoglobulin Fc fragment such as an IgG Fc fragment in the N-terminal to C-terminal direction; and (ii) an immunogenic substance different from the fusion protein, wherein the immunogenic composition is suitable for use in a method of reducing one or more clinical signs, mortality or fecal excretion caused by rotavirus infection in pigs. Background Art

[0003] Rotavirus is a double-stranded RNA virus of the genus Reoviridae. Rotavirus infection is known to cause gastrointestinal disease and is considered the most common cause of gastroenteritis in infants. Rotavirus is transmitted by the fecal-oral route and infects cells that pass through the small intestine. Infected cells produce enterotoxins that induce gastroenteritis, leading to severe diarrhea and sometimes death from dehydration.

[0004] Rotaviruses have a genome composed of 11 double-stranded RNA (dsRNA) segments and are currently divided into eight groups (AH) based on the antigenic properties of the internal viral capsid protein 6 (VP6) and sequence-based classification as defined by the International Committee on Taxonomy of Viruses (ICTV) and outlined by Matthijnssens et al. (Arch Virol 157: 1177-1182 (2012)), wherein this disclosure and the following disclosures mentioned herein are incorporated by reference in their entirety.

[0005] The rotavirus genome encodes six structural proteins (VP1 to VP4, VP6 and VP7) and six non-structural proteins (NSP1 to NSP6), of which genome segments 1 to 10 each encode one rotavirus protein, and genome segment 11 encodes two proteins (NSP5 and NSP6).

[0006] In the case of rotavirus A, different strains can be classified into genotypes (defined by comparative sequence determination and / or nucleic acid hybridization data) or serotypes (defined by serological determination) based on the structural proteins VP7 and VP4. VP7 and VP4 are components of the outermost protein layer (external capsid), and both carry neutralizing epitopes. VP7 is a glycoprotein (hence "G") that forms the outer layer or surface of the virion. VP7 determines the G type of the virus strain and the names of the G serotype and the G genotype are the same. VP4 is protease-sensitive (hence "P") and determines the P type of the virus. Compared with the G type, the numbers specified for the P serotype and genotype are different (Santos N.et Hoshino Y., 2005, Reviews in Medical Virology, 15, 29-56). Therefore, the P serotype is called P, followed by the specified number, and the P genotype is called P, followed by the number specified in square brackets (e.g., "P[7]" or "P

[13] "). Virus strains belonging to the same genotype have greater than 89% amino acid sequence identity (Estes and Kapikian. Rotaviruses. In Knipe, DM; Howley, PM Fields Virology, 5th ed.; Wolters Kluwer / Lippincott Williams & Wilkins Health: Philadelphia, PA, USA (2007); Gorziglia et al. Proc Natl Acad Sci US A. 87(18):7155-9 (1990)).

[0007] Rotavirus in particular is also a major cause of gastroenteritis in pigs with antibodies against nearly 100% of group A and C rotaviruses present in pigs (Vlasova et al. Viruses. 9(3):48 (2017)). Currently, modified live or killed vaccines are only available against rotavirus A. The inability to grow rotavirus C in the laboratory has hampered the development of vaccines against this group, increasing the attractiveness of recombinant vaccines.

[0008] The production of recombinant anti-rotavirus vaccines is hampered by the complexity of the rotavirus capsid, which is composed of four proteins arranged in three layers. The innermost layer is composed of 60 VP2 dimers with T=1 symmetry. For the proper ordering of the middle layer, a VP2 layer is required, which is formed by 260 VP6 trimers with T=13 symmetry. The resulting symmetry mismatch between VP2 and VP6 produces five different VP6 trimer positions and three different pore types. In the absence of VP2, VP6 readily forms ordered high-molecular-weight microtubules and spheres in a salt- and pH-dependent manner that may represent a byproduct of viral assembly. In the capsid, the VP6 layer is covered by 260 Ca2+-dependent trimers of VP7, which act as a clamp to hold the VP4 spike protein in place. VP7 is a glycosylated or G-type antigen and contains neutralizing epitopes. Most neutralizing antibodies recognize only trimeric VP7 and are thought to work by preventing the dissociation of VP7 trimers, thereby blocking the release of the spike protein. The rotavirus spike protein exists as a trimer of 60 VP4s, which are inserted into the VP6 layer only at the type II pore. VP4 contains a neutralizing epitope and is a P-type antigen, which is cleaved by trypsin into the spike protein base VP5* and the cell interaction head VP8*, which remains associated with VP5* after cleavage. Trypsin digestion allows the spike protein to enter the cell, during which the spike protein undergoes a deep structural rearrangement to expose the active site for receptor binding on the host cell. Ignoring the complexity of the above assembly method, it is difficult to achieve the stoichiometric expression of the rotavirus capsid protein under environmental conditions that promote appropriate assembly.

[0009] In view of the difficulty of rotavirus capsid assembly, attention has been paid to subunit vaccine approaches. VP7 and VP4 are two proteins containing neutralizing epitopes, but the use of VP7 will be complicated by its glycosylation and calcium-dependent trimerization. The use of VP4 is complicated by its trimerization, trypsin digestion and range of potential conformational states. The VP8 protein, also referred to as the VP8 domain or VP8*, is produced by trypsin digestion of VP4, contains a neutralizing epitope, is a monomer, and its structure has been determined to be high resolution (Dormitzer et al. EMBO J. 21 (5): 885-897 (2002)) and has been described as highly stable.

[0010] In addition, the VP8 protein has a lectin-like domain (aa65-224) that is thought to interact with host receptors and be involved in viral attachment to host cells (Rodriguez et al., PloS Pathog. 10(5):e1004157 (2014)).

[0011] Methods for developing a rotavirus subunit vaccine for children have been described in which the N-terminus is linked to the universal CD4 +A truncated VP8 protein (amino acid residues 64 (or 65)-223 of VP8*) with the T cell epitope (aa830-844) P2 was produced in Escherichia coli (Wen et al. Vaccine. 32(35):4420-7 (2014)) and tested in infants and young children (Groome et al. Lancet Infect Dis. 17(8):843-853 (2017)). However, since the use of a monovalent subunit vaccine (based on a truncated VP8 protein of rotavirus genotype P[8]) elicited adverse reactions against heterotypic rotavirus strains, a trivalent vaccine formulation (comprising three proteins for combining genotypes P[4], P[6], and P[8] antigens) has also recently been tested (Groome et al. Lancet Infect Dis. S1473-3099(20)30001 (2020)).

[0012] In another method, the N-terminus or C-terminus of the N-terminally truncated VP8 protein "VP8-1" (aa26-241) was fused to the pentameric non-toxic B subunit of cholera toxin (CTB). Among the resulting pentameric fusion proteins (CTBA-VP8-1, VP8-1-CTB), only CTB-VP8-1 (i.e., VP8-1 fused to CTB at the N-terminus) was considered a viable candidate for further development in a mouse model, as compared to VP8-1-CTB, which exhibited higher binding activity with GM1 or with conformationally sensitive neutralizing monoclonal antibodies specific for VP8*, and induced higher titers of neutralizing antibodies and conferred higher protective efficacy (Xue et al. Hum Vaccin Immunother. 12 (11) 2959-2968 (2016)).

[0013] However, in view of the difficulties in rotavirus capsid assembly, attention has been paid to alternative subunit vaccine approaches, especially because subunit vaccines are generally considered to be very safe. In addition, there is a strong need for recombinant expression of effective rotavirus subunit antigens, which allows for simple production of vaccine antigens for such rotaviruses that are difficult to culture. Moreover, because rotavirus is a major cause of swine gastroenteritis, there is a particular need for subunit vaccines for pigs that include antigens that are comparable in efficacy to or even more effective than the currently commercially available MLV rotavirus vaccines for pigs. Summary of the invention

[0014] The solution to the above technical problem is achieved through the implementation schemes characterized in the specification and claims.

[0015] Thus, the invention in its different aspects is implemented according to the claims.

[0016] The present invention is based on the surprising discovery that administration of a polypeptide comprising a fragment of the rotavirus VP8 protein, i.e., an N-terminally extended lectin-like domain and an IgG Fc fragment linked to the C-terminus, to sows after challenge with rotavirus can significantly reduce diarrhea and fecal excretion in their offspring via passive transfer of neutralizing antibodies, even when the polypeptide is mixed with additional immunogenic substances prior to administration.

[0017] Therefore, in a first aspect, the present invention relates to an immunogenic composition comprising

[0018] (i) a polypeptide comprising:

[0019] - an immunogenic fragment of the rotavirus VP8 protein, and

[0020] - immunoglobulin Fc fragment,

[0021] and

[0022] (ii) at least one immunogenic substance different from said polypeptide,

[0023] The immunogenic composition is also referred to as "the immunogenic composition of the present invention" hereinafter.

[0024] Therefore, the immunogenic composition of the present invention is specifically a composition comprising two components (i; ii), namely

[0025] (i) a polypeptide comprising:

[0026] - an immunogenic fragment of the rotavirus VP8 protein, and

[0027] - immunoglobulin Fc fragment,

[0028] wherein said polypeptide is also referred to hereinafter as "component (i) of the immunogenic composition of the invention" or "component (i)", respectively, and

[0029] (ii) at least one immunogenic substance different from said polypeptide,

[0030] The at least one immunogenic substance is hereinafter also referred to as "component (ii) of the immunogenic composition of the present invention" or "component (ii)", respectively.

[0031] The polypeptide comprises:

[0032] - an immunogenic fragment of the rotavirus VP8 protein, and

[0033] - immunoglobulin Fc fragment,

[0034] It is described herein and in the context of the present invention is also hereinafter referred to as "polypeptide of the invention".

[0035] Therefore, component (i) of the immunogenic composition of the invention is a polypeptide of the invention.

[0036] Preferably, component (ii) consists of two, three or more immunogenic substances, wherein all of said immunogenic substances

[0037] is different from component (i), and

[0038] Different from each other.

[0039] In the context of the present invention, it was also unexpectedly found that, since the polypeptides of the present invention are released from the cells when produced in the cells and can subsequently be recovered from the supernatant surrounding the cells rather than from the cells themselves, the immunogenic composition can be simply produced. Subsequently, the recovered polypeptides can be easily mixed with other components to produce the immunogenic composition of the present invention, the other components including at least one immunogenic substance different from the polypeptide.

[0040] Another advantage of the polypeptide of the invention is that it can, if desired, be prepared as one polypeptide comprising / presenting two immunogenic fragments of different rotaviruses, thereby obviating the need to separately prepare two different monovalent polypeptides which then need to be combined for the same purpose.

[0041] Preferably, the immunoglobulin Fc fragment as described herein is linked to

[0042] - the C-terminus of the immunogenic fragment of the rotavirus VP8 protein, or

[0043] - The N-terminus of the immunogenic fragment of the rotavirus VP8 protein.

[0044] Specifically, the immunoglobulin Fc fragment is preferably

[0045] - linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein via a linker moiety, or

[0046] - linked to the N-terminus of the immunogenic fragment of the rotavirus VP8 protein via a linker moiety.

[0047] In another preferred aspect, the immunoglobulin Fc fragment as described herein

[0048] - linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein via a peptide bond between the N-terminal amino acid residue of the immunoglobulin Fc fragment and the C-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein, or

[0049] - linked to the N-terminus of the immunogenic fragment of the rotavirus VP8 protein via a peptide bond between the C-terminal amino acid residue of the immunoglobulin Fc fragment and the N-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein.

[0050] Most preferably, the immunoglobulin Fc fragment as described herein is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein.

[0051] Therefore, the polypeptide of the present invention is particularly a polypeptide comprising:

[0052] - an immunogenic fragment of the rotavirus VP8 protein, and

[0053] - immunoglobulin Fc fragment,

[0054] The immunoglobulin Fc fragment is connected to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein.

[0055] The term "polypeptide" as used herein refers in particular to any chain of amino acid residues linked together by peptide bonds, and does not refer to a specific length of the product. For example, a "polypeptide" may refer to a long chain of amino acid residues, for example, 150 to 600 amino acid residues or longer in length. The term "polypeptide" includes polypeptides having one or more post-translational modifications, wherein the post-translational modifications include, for example, glycosylation, phosphorylation, lipidation (e.g., myristoylation, etc.), acetylation, ubiquitination, sulfation, ADP ribosylation, hydroxylation, Cys / Met oxidation, carboxylation, methylation, etc. The terms "polypeptide" and "protein" are used interchangeably in the context of the present invention.

[0056] The term "immunogenic fragment" is particularly understood to refer to a fragment of a protein that at least partially retains the immunogenicity of the protein from which it is derived. Thus, an "immunogenic fragment of a rotavirus VP8 protein" is particularly understood to refer to a fragment of a rotavirus VP8 protein that at least partially retains the immunogenicity of the full-length VP8 protein.

[0057] "Immunogenic substance" refers to any substance that can elicit a humoral and / or cellular immune response, and particularly refers to a molecule such as a peptide or polypeptide that can elicit, generate or produce an immune response in an animal.

[0058] As described herein, the term "VP8 protein" is to be understood as being equivalent to, inter alia, a "VP8 domain", "VP8*" or a "VP8 fragment of VP4", as is frequently used in the context of rotaviruses.

[0059] As used herein, the term "immunoglobulin Fc fragment" refers to a protein containing the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of an immunoglobulin, and more specifically, does not contain the heavy chain and light chain variable regions and the light chain constant region 1 (CL1) of an immunoglobulin. It may further include the hinge region of an immunoglobulin or a portion of the hinge region (i.e., the hinge region at the heavy chain constant region). In addition, the immunoglobulin Fc fragment may contain a portion or all of the heavy chain constant region 1 (CH1).

[0060] It will be understood that the term "immunoglobulin Fc fragment" as used herein is equivalent to "immunoglobulin Fc domain".

[0061] The term "linked to" as used herein refers in particular to any means for linking the immunoglobulin Fc fragment to the C-terminus or N-terminus of the immunogenic fragment of the rotavirus VP protein within a polypeptide. Examples of linking means include (1.) the immunoglobulin Fc fragment is indirectly linked to the C-terminus of the immunogenic fragment of the rotavirus VP 8 protein through an insertion portion, the insertion portion is directly linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein, and also binds to the immunoglobulin Fc fragment; and (2.) the immunoglobulin Fc fragment is directly linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein by a covalent bond. The terms "linked to" and "linked to..." are used interchangeably in the context of the present invention.

[0062] In particular, it is to be understood that the phrase "a polypeptide comprising:

[0063] - an immunogenic fragment of the rotavirus VP8 protein, and

[0064] - immunoglobulin Fc fragment,

[0065] wherein the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein,"

[0066] As used herein, the following expressions are particularly equivalent:

[0067] "A polypeptide comprising, in the N-terminal to C-terminal direction:

[0068] - the amino acid sequence of an immunogenic fragment of the rotavirus VP8 protein, and

[0069] -Amino acid sequence of immunoglobulin Fc fragment",

[0070] or equivalent to the following

[0071] "Contains the following peptides

[0072] - an immunogenic fragment of the rotavirus VP8 protein, and

[0073] - an immunoglobulin Fc fragment linked to the C-terminus of said immunogenic fragment".

[0074] According to a most preferred aspect, the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein via a linker moiety.

[0075] A linker moiety as described herein in the context of the present invention is preferably a peptide linker.

[0076] The term "peptide linker" as used herein refers to a peptide comprising one or more amino acid residues. More specifically, the term "peptide linker" as used herein refers to a peptide capable of connecting two variable proteins and / or domains, such as an immunogenic fragment of a rotavirus VP8 protein and an immunoglobulin Fc fragment.

[0077] In a particularly preferred aspect, the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein via a linker moiety, wherein

[0078] - the immunogenic fragment of the rotavirus VP8 protein is linked to the linker moiety via a peptide bond between the N-terminal amino acid residue of the linker moiety and the C-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein, and

[0079] - The linker moiety is linked to the immunoglobulin Fc fragment via a peptide bond between the N-terminal amino acid residue of the immunoglobulin Fc fragment and the C-terminal amino acid residue of the linker moiety.

[0080] Furthermore, the immunoglobulin Fc fragment may be linked to the immunogenic fragment of the rotavirus VP8 protein, preferably via a peptide bond between the N-terminal amino acid residue of the immunoglobulin Fc fragment and the C-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein.

[0081] It will be appreciated that the polypeptides of the invention are in particular fusion proteins.

[0082] As used herein, the term "fusion protein" represents a protein formed by fusing (i.e., joining) all or part of two or more different polypeptides. Typically, fusion proteins are made by joining polynucleotides encoding two or more polypeptides end-to-end using recombinant DNA technology. More specifically, the term "fusion protein" therefore refers to a protein translated from a nucleic acid transcript produced by combining a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid encoding at least a second polypeptide, wherein the fusion protein is not a naturally occurring protein. A nucleic acid construct may encode two or more polypeptides joined in a fusion protein.

[0083] In another preferred aspect, the polypeptide of the present invention is a fusion protein of formula xyz, wherein

[0084] x consists of or contains an immunogenic fragment of rotavirus VP8 protein;

[0085] y is a linker moiety; and

[0086] z is the immunoglobulin Fc fragment.

[0087] Formula xyz should be understood in particular as meaning that the C-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein is connected to the linker portion, preferably via a peptide bond, to the N-terminal amino acid residue of the linker portion, and the N-terminal amino acid residue of the immunoglobulin Fc fragment is connected to the linker portion, preferably via a peptide bond, to the C-terminal amino acid residue of the linker portion.

[0088] As described herein, the expression "x consists of an immunogenic fragment of a rotavirus VP8 protein" is particularly understood to be equivalent to "x is an immunogenic fragment of a rotavirus VP8 protein".

[0089] In a preferred aspect, the immunogenic fragment of rotavirus VP8 protein as mentioned herein is preferably capable of inducing an immune response against rotavirus in a subject to which the immunogenic fragment of rotavirus VP8 protein is administered.

[0090] In another preferred aspect, the immunogenic fragment of the rotavirus VP8 protein is a polypeptide having a length of 50 to 200, preferably 140 to 190 amino acid residues.

[0091] The rotavirus mentioned herein is preferably selected from the group consisting of rotavirus A and rotavirus C. Therefore, as mentioned herein, the immunogenic fragment of rotavirus VP8 protein is preferably selected from the group consisting of: an immunogenic fragment of rotavirus A VP8 protein and an immunogenic fragment of rotavirus C VP8 protein.

[0092] The terms "rotavirus A" and "rotavirus C" as referred to herein, respectively, refer to rotavirus A and rotavirus C, respectively, as defined by ICTV (summarized by Matthijnssens et al. Arch Virol 157: 1177-1182 (2012)).

[0093] According to another preferred aspect, the rotavirus mentioned herein is porcine rotavirus.

[0094] In a particularly preferred aspect, the rotavirus referred to herein is rotavirus A. Therefore, the immunogenic fragment of the rotavirus VP8 protein as described herein is preferably an immunogenic fragment of the rotavirus A VP8 protein.

[0095] In another preferred aspect, the immunogenic fragment of rotavirus VP8 protein comprises the lectin-like domain of rotavirus VP8 protein.As mentioned herein, "the lectin-like domain of rotavirus VP8 protein" should preferably be understood as the lectin-like domain of rotavirus A VP8 protein.

[0096] The term "lectin-like domain of rotavirus VP8 protein" particularly refers to residues 65-224 of rotavirus VP8 protein or corresponds to an amino acid sequence consisting of amino acid residues 65-224 of rotavirus VP8 protein, respectively, and wherein the amino acid residues 65-224 of rotavirus VP8 protein are preferably amino acid residues 65-224 of rotavirus A VP8 protein.

[0097] Therefore, the "lectin-like domain of rotavirus VP8 protein" preferably consists of the amino acid sequence of amino acid residues 65-224 of rotavirus VP8 protein, especially rotavirus A VP8 protein.

[0098] Preferably, the immunogenic fragment of the rotavirus VP8 protein is an N-terminal extended lectin-like domain of the rotavirus VP8 protein, wherein the N-terminal extension length is 1 to 20 amino acid residues, especially 5 to 15 amino acid residues. Most preferably, the immunogenic fragment of the rotavirus VP8 protein is an N-terminal extended lectin-like domain of the rotavirus VP8 protein, wherein the N-terminal extension length is eight amino acid residues.

[0099] The N-terminal extended amino acid sequence is preferably an amino acid sequence of the respective lengths of the N-terminal amino acid residues flanking the lectin-like domain in the amino acid sequence of the rotavirus VP8 protein.

[0100] Therefore, in a specific aspect, the immunogenic fragment of the rotavirus VP8 protein as mentioned herein preferably consists of the following: the amino acid sequence of amino acid residues 60-224, amino acid residues 59-224, amino acid residues 58-224, amino acid residues 57-224, amino acid residues 56-224, amino acid residues 55-224, amino acid residues 54-224, amino acid residues 53-224, amino acid residues 52-224, amino acid residues 51-224, amino acid residues 50-224 or amino acid residues 49-224 of the rotavirus VP8 protein, in particular the rotavirus A protein.

[0101] Most preferably, as mentioned herein, the immunogenic fragment of the rotavirus VP8 protein consists of the amino acid sequence of amino acid residues 57-224 of the rotavirus VP8 protein, in particular the rotavirus A protein.

[0102] The above amino acid residue numbers (eg, "65-224" or "57-224") preferably refer to the amino acid sequence of wild-type rotavirus VP8 protein, especially wild-type rotavirus A VP8 protein. The wild-type rotavirus VP8 protein is preferably the protein shown in SEQ ID NO:1.

[0103] According to another preferred aspect, the rotavirus mentioned herein is a rotavirus selected from the group consisting of: genotype P[6] rotavirus, genotype P[7] rotavirus and genotype P

[13] rotavirus, in particular rotavirus A. Thus, as mentioned herein, the immunogenic fragment of the rotavirus VP8 protein is preferably selected from the group consisting of: an immunogenic fragment of a rotavirus VP8 protein of genotype P[6], an immunogenic fragment of a rotavirus VP8 protein of genotype P[7] and an immunogenic fragment of a rotavirus VP8 protein of genotype P

[13] , and in particular selected from the group consisting of: an immunogenic fragment of a rotavirus A VP8 protein of genotype P[6], an immunogenic fragment of a rotavirus A VP8 protein of genotype P[7] and an immunogenic fragment of a rotavirus A VP8 protein of genotype P

[13] .

[0104] As used herein, the terms "genotype P[6] rotavirus", "genotype P[7] rotavirus", "genotype P

[13] rotavirus" and "genotype P

[23] rotavirus" refer specifically to the established VP4 (P) genotype classification of rotaviruses (e.g., P[6], P[7], P

[13] or P

[23] ), which are described in Estes and Kapikian. Rotaviruses. In Knipe, DM; Howley, PM Fields Virology, 5th ed.; Wolters Kluwer / Lippincott Williams & Wilkins Health: Philadelphia, PA, USA (2007); Gorziglia et al. Proc Natl Acad Sci USA. 87(18):7155-9 (1990).

[0105] Most preferably, the rotavirus referred to herein is a genotype P[7] rotavirus. Therefore, as referred to herein, the immunogenic fragment of the rotavirus VP8 protein is most preferably an immunogenic fragment of the genotype P[7] rotavirus VP8 protein, especially an immunogenic fragment of the genotype P[7] rotavirus A VP8 protein.

[0106] The rotavirus VP8 protein mentioned herein most preferably comprises or consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:1.

[0107] As mentioned herein, the lectin-like domain of the rotavirus VP8 protein preferably comprises or consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO: 2.

[0108] In one example, the immunogenic fragment of the rotavirus VP8 protein consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:3.

[0109] In another preferred aspect, the immunogenic fragment of the rotavirus VP8 protein consists of or is the consensus sequence of: a portion of the rotavirus VP8 protein, in particular a portion of the rotavirus A VP8 protein.

[0110] As used herein, the term "consensus sequence" refers to a sequence formed by the most frequently occurring amino acids (or nucleotides) in a family of related sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family of proteins, each position in the consensus sequence is occupied by the amino acid that most frequently occurs at that position in the family. The term "consensus sequence" therefore refers to an inferred amino acid sequence (or nucleotide sequence). A consensus sequence represents a plurality of similar sequences. Each position in the consensus sequence corresponds to the most frequently occurring amino acid residue (or nucleotide base) at that position as determined by aligning three or more sequences.

[0111] Preferably, the consensus sequence of a portion of the rotavirus VP8 protein as mentioned herein can be obtained by a method comprising the following steps:

[0112] - translating a plurality of nucleotide sequences encoding a portion of the rotavirus VP8 protein into amino acid sequences,

[0113] - aligning the amino acid sequence with known rotavirus VP8 proteins, preferably by using the UPGMB clustering method of the MUSCLE sequence alignment software and default gap penalty parameters,

[0114] - performing phylogenetic analysis on the aligned sequences and generating a neighbor joining phylogenetic reconstruction based on the rotavirus VP8 protein sequence, in particular importing the aligned amino acid sequences into MEGA7 software for phylogenetic analysis and generating a neighbor joining phylogenetic reconstruction based on the rotavirus VP8 protein sequence,

[0115] - Optimal trees were calculated using Poisson correction and bootstrap retest of phylogeny (n=100),

[0116] - The optimal tree was scaled with branch lengths equal to the evolutionary distance in units of amino acid substitutions per site over a total of 170 positions,

[0117] - nodes with a bootstrapped cluster association greater than 70% are considered significant,

[0118] - nodes with a distance of about 10% and a bootstrapped re-clustering association greater than 70% are designated as clusters, and

[0119] - selecting a cluster and generating a consensus sequence by identifying the maximum frequency of each aligned position within said cluster,

[0120] - and optionally, in cases where an equal proportion of amino acids is observed in the aligned positions, selecting amino acid residues based on reported epidemiological data as well as a predetermined product protection profile.

[0121] For example, in this case, the immunogenic fragment of the rotavirus VP8 protein preferably consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:5.

[0122] In another preferred aspect, the rotavirus referred to herein is rotavirus C. According to this aspect, the immunogenic fragment of the rotavirus VP8 protein is preferably an immunogenic fragment of the rotavirus C VP8 protein.

[0123] In the context of this aspect, the immunogenic fragment of the rotavirus VP8 protein preferably consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:6.

[0124] According to the present invention, the immunogenic fragment of the rotavirus VP8 protein thus preferably consists of or is:

[0125] - an immunogenic fragment of a rotavirus A VP8 protein, in particular any of the immunogenic fragments of a rotavirus A VP8 protein described herein, or

[0126] a consensus sequence of a portion of a rotavirus VP8 protein, such as a consensus sequence of a portion of a rotavirus A VP8 protein, preferably in the context of a consensus sequence, any of the immunogenic fragments of a rotavirus VP8 protein described herein, or

[0127] - an immunogenic fragment of a rotavirus C VP8 protein, in particular any of the immunogenic fragments of a rotavirus C VP8 protein described herein.

[0128] In a particularly preferred aspect, the immunogenic fragment of the rotavirus VP8 protein is a polypeptide consisting of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0129] The immunoglobulin Fc fragment described herein is preferably at least 220 amino acid residues in length, and most preferably 220 to 250 amino acid residues in length.

[0130] According to another particularly preferred aspect, the immunoglobulin Fc fragment described herein is not glycosylated. As used herein, the term "not glycosylated" especially means that the immunoglobulin Fc fragment has no oligosaccharide molecules attached thereto.

[0131] Preferably, the immunoglobulin Fc fragment as referred to herein comprises or consists of:

[0132] - the heavy chain constant region 2 (CH2) of an immunoglobulin, and

[0133] - heavy chain constant region 3 (CH3) of an immunoglobulin,

[0134] - and optionally the hinge region or a part of the hinge region of an immunoglobulin.

[0135] According to another preferred aspect, the immunoglobulin mentioned herein is selected from the group consisting of IgG, IgA, IgD, IgE and IgM. Therefore, the immunoglobulin Fc fragment is preferably selected from the group consisting of: IgG Fc fragment, IgA Fc fragment, IgDFc fragment, IgE Fc fragment and IgM Fc fragment.

[0136] According to a most preferred aspect, the immunoglobulin Fc fragment described herein is an IgG Fc fragment.

[0137] As mentioned herein, IgG is preferably selected from the group consisting of: IgG1, IgG2, IgG3, IgG4, IgG5 and IgG6. Therefore, according to another preferred aspect, the immunoglobulin Fc fragment mentioned herein is selected from the group consisting of: IgG1 Fc fragment, IgG2 Fc fragment, IgG3 Fc fragment, IgG4 Fc fragment, IgG5 Fc fragment and IgG6 Fc fragment.

[0138] Most preferably, the immunoglobulin Fc fragment is a protein encoded by the genome of a species whose intestinal cells are susceptible to rotavirus infection from which an immunogenic fragment of the rotavirus VP8 protein as mentioned herein is derived. For example, if the fragment of the rotavirus VP8 protein is a fragment of the porcine rotavirus VP8 protein, the immunoglobulin Fc fragment is preferably an immunoglobulin Fc fragment encoded by the porcine genome. According to another example, if the fragment of the rotavirus VP8 protein is a fragment of the chicken rotavirus VP8 protein, the immunoglobulin Fc fragment is preferably an immunoglobulin Fc fragment encoded by the chicken genome.

[0139] More specifically, the immunoglobulin Fc fragment is preferably a porcine IgG Fc fragment.

[0140] In another preferred aspect, the immunoglobulin Fc fragment comprises or consists of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8.

[0141] The linker moiety or peptide linker mentioned herein, respectively, is an amino acid sequence preferably having a length of 1 to 50 amino acid residues, in particular a length of 3 to 20 amino acid residues. For example, the linker moiety can be a peptide linker having a length of 3, 8 or 10 amino acid residues.

[0142] Depending on the purpose, short linkers may be needed to reduce the risk of proteolysis between fusion protein partners. Therefore, the peptide linkers described in the context of the present invention preferably have a length of 1 to 5 amino acid residues, more preferably 2 to 4 amino acid residues and most preferably three amino acid residues, or are respectively composed of them.

[0143] According to a preferred aspect, the linker portion comprises or consists of an amino acid sequence having at least 66%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11.

[0144] As referred to herein, "SEQ ID NO: 9" refers to the amino acid sequence (single letter code for amino acid residues): GGS, whose conventional three letter code is Gly-Gly-Ser.

[0145] Therefore, in the context of the present invention, "SEQ ID NO: 9" refers to the amino acid sequence Gly-Gly-Ser or correspondingly "Gly Gly Ser" as shown for the sequence SEQ ID NO: 9 in the following sequence listing.

[0146] Preferably, the polypeptide of the invention has an N-terminal methionine residue flanked by the N-terminal amino acid residue of an immunogenic fragment of a rotavirus VP8 protein.

[0147] According to another preferred aspect, the polypeptide of the present invention comprises another immunogenic fragment of the rotavirus VP8 protein linked to the C-terminus of the immunoglobulin Fc fragment.

[0148] Another immunogenic fragment of the rotavirus VP8 protein preferably consists of or is:

[0149] - an immunogenic fragment of a rotavirus A VP8 protein, in particular any of the immunogenic fragments of a rotavirus A VP8 protein described herein, or

[0150] a portion of a rotavirus VP8 protein, such as a consensus sequence of a portion of a rotavirus A VP8 protein, preferably in the context of a consensus sequence, any of the immunogenic fragments of a rotavirus VP8 protein described herein, or

[0151] - an immunogenic fragment of a rotavirus C VP8 protein, in particular any of the immunogenic fragments of a rotavirus C VP8 protein described herein.

[0152] Specifically, the other immunogenic fragment of the rotavirus VP8 protein preferably comprises or consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 2 to 6.

[0153] In a particularly preferred aspect, the other immunogenic fragment of the rotavirus VP8 protein is different from the immunogenic fragment of the rotavirus VP8 protein that is C-terminally linked to the immunoglobulin Fc fragment.

[0154] The other immunogenic fragment of the rotavirus VP8 protein is preferably connected to the C-terminus of the immunoglobulin Fc fragment via a linker portion, in particular via any of the linker portions described herein. Preferably, the other immunogenic fragment of the rotavirus VP8 protein is connected to the linker portion via a peptide bond between the N-terminal amino acid residue of the other immunogenic fragment of the rotavirus VP8 protein and the C-terminal amino acid residue of the linker portion.

[0155] Optionally, the other immunogenic fragment of the rotavirus VP8 protein is linked to the C-terminus of the immunoglobulin Fc fragment via a peptide bond between the N-terminal amino acid residue of the other immunogenic fragment of the rotavirus VP8 protein and the C-terminal amino acid residue of the immunoglobulin Fc fragment.

[0156] In a particularly preferred aspect, the polypeptide of the present invention is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0157] Preferably, the polypeptide of the present invention is a protein comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.

[0158] It is to be understood that the expression "consisting of an amino acid sequence" or "consists of an amino acid sequence", as used herein, also specifically relates to any one or more co-translational and / or post-translational modifications of an amino acid sequence, respectively, which are affected by the cell expressing the protein or protein domain. Thus, unless explicitly mentioned otherwise, the expression "consisting of an aminoacid sequence" or "consists of an amino acid sequence", as described herein, also relates to an amino acid sequence having one or more modifications, affected by the cell expressing the protein or protein domain, respectively, in particular modifications of amino acid residues produced in protein biosynthesis and / or protein processing, preferably selected from the group consisting of glycosylation, phosphorylation and acetylation.

[0159] Concerning the term "at least 90%" as mentioned in the context of the present invention, it should be understood that the term preferably means "at least 91%", more preferably "at least 92%", even more preferably "at least 93%" or especially "at least 94%".

[0160] Concerning the term "at least 95%" as mentioned in the context of the present invention, it should be understood that the term preferably means "at least 96%", more preferably "at least 97%", even more preferably "at least 98%" or especially "at least 99%".

[0161] Concerning the term "at least 99%" as mentioned in the context of the present invention, it should be understood that the term preferably means "at least 99.2%", more preferably "at least 99.4%", even more preferably "at least 99.6%" or especially "at least 99.8%".

[0162] More specifically, the term "at least 99% sequence identity" refers to 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity.

[0163] As used herein, the term "having 100% sequence identity" should be understood to be equivalent to the term "being identical".

[0164] The percentage of sequence identity has a meaning recognized in the art and there are a variety of methods to measure the identity between two polypeptide or polynucleotide sequences. See, for example, Lesk, Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, Biocomputing: Informatics And Genome Projects, Academic Press, New York, (1993); Griffin and Griffin, Computer Analysis Of Sequence Data, Part I, Humana Press, New Jersey, (1994); von Heinje, Sequence Analysis In Molecular Biology, Academic Press, (1987); and Gribskov and Devereux, Sequence Analysis Primer, M Stockton Press, New York, (1991). Methods for aligning polynucleotides or polypeptides are encoded in computer programs including the GCG program package (Devereux et al., Nuc. Acids Res. 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al., J. Molec. Biol. 215:403 (1990)), and the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711), which uses the local homology algorithm of Smith and Waterman (Adv. App. Math., 2:482-489 (1981)). For example, the computer program ALIGN employing the FASTA algorithm may be used, with an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2.For the purposes of the present invention, the nucleotide sequences were aligned using the Clustal W method in the MegAlign software version 11.1.0 (59), 419 of DNASTAR, using the default multiple sequence alignment parameter settings in the program (gap penalty = 15.0, gap length penalty = 6.66, delayed divergent sequence (%) = 30%, DNA conversion weight = 0.50 and DNA weight matrix = IUB), and the protein / amino acid sequences were aligned using the Clustal W method in the MegAlign software version 11.1.0 (59), 419 of DNASTAR, using the default multiple sequence alignment parameter settings in the program (Gonnet continuous protein weight matrix, where gap penalty = 10.0, gap length penalty = 0.2 and delayed divergent sequence (%) = 30%).

[0165] As used herein, it is particularly understood that the term "sequence identity to the sequence of SEQ ID NO: X" is equivalent to the term "sequence identity to the sequence of SEQ ID NO: X over the length of SEQ ID NO: X" or equivalently to the term "sequence identity to the sequence of SEQ ID NO: X over the entire length of SEQ ID NO: X", respectively. In this context, "X" is any integer selected from 1 to 25, such that "SEQ ID NO: X" represents any one of the SEQ ID NOs mentioned herein.

[0166] As used herein, the phrase "a group consisting of SEQ ID NO: [...], ... and SEQ ID NO: [...]" is interchangeable with "a group consisting of a sequence of SEQ ID NO: [...], ... and a sequence of SEQ ID NO: [...]". In this context, "[...]" is a placeholder for the number of the sequence. For example, the phrase "a group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6" is interchangeable with "a group consisting of a sequence of SEQ ID NO:3, a sequence of SEQ ID NO:4, a sequence of SEQ ID NO:5 and a sequence of SEQ ID NO:6".

[0167] According to another particularly preferred aspect, the polypeptide of the present invention consists of:

[0168] - an immunogenic fragment of a rotavirus VP8 protein, in particular any of the immunogenic fragments of a rotavirus VP8 protein described herein,

[0169] - an N-terminal methionine residue flanking the N-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein, and

[0170] - an immunoglobulin Fc fragment, in particular any one of the immunoglobulin Fc fragments described herein,

[0171] - wherein the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein, in particular via a linker moiety, wherein the linker moiety is preferably any one of the linker moieties described herein,

[0172] - and optionally another immunogenic fragment of the rotavirus VP8 protein linked to the C-terminus of the immunoglobulin Fc fragment, in particular via a linker portion, wherein the other immunogenic fragment of the rotavirus VP8 protein is preferably any one of the other immunogenic fragments of the rotavirus VP8 protein described herein, and wherein the linker portion is preferably any one of the linker portions described herein.

[0173] In yet another preferred aspect, the polypeptide of the present invention forms a dimer with another polypeptide of the present invention. Most preferably, the polypeptide of the present invention forms a homodimer with another identical polypeptide.

[0174] Therefore, it is especially to be understood that the term "polypeptide of the invention" further encompasses any dimer composed of two polypeptides of the invention, and especially encompasses any homodimer composed of two identical polypeptides of the invention.

[0175] According to another particularly preferred aspect, the present invention provides a multimer, which comprises or consists of a plurality of polypeptides of the present invention, and wherein the multimer is also referred to hereinafter as "the multimer of the present invention".

[0176] Preferably, the multimer of the present invention is a homodimer formed by one polypeptide of the present invention and another identical polypeptide of the present invention.

[0177] In particular, it is to be understood that the term "multimer of the invention" further encompasses any mixture of different multimers of the invention, such as a mixture of:

[0178] - a homodimer formed by a polypeptide of the present invention and another identical polypeptide of the present invention, and

[0179] - One or more multimers formed by more than two of the same polypeptides of the invention.

[0180] Therefore, in a particularly preferred embodiment, component (i) of the immunogenic composition of the present invention is present in

[0181] - a monomer consisting of a polypeptide of the present invention, and / or

[0182] - a homodimer consisting of two identical polypeptides of the invention,

[0183] - and / or optionally in a homotrimer consisting of three identical polypeptides of the invention,

[0184] Among them, preferably

[0185] - each of said two identical polypeptides of the invention,

[0186] - and optionally each of the three identical polypeptides of the invention,

[0187] It comprises or consists of the same amino acid sequence as the one polypeptide of the present invention.

[0188] Or more generally, the present invention also encompasses an immunogenic composition comprising:

[0189] (i) a polypeptide comprising:

[0190] - an immunogenic fragment of the rotavirus VP8 protein, and

[0191] - immunoglobulin Fc fragment,

[0192] Optionally, the polypeptide is included in a multimer comprising or consisting of a plurality of the polypeptides,

[0193] and

[0194] (ii) at least one immunogenic substance different from the polypeptide.

[0195] At least one immunogenic substance different from the polypeptide is described herein, the polypeptide preferably consists of two or more immunogenic substances, wherein all of the immunogenic substances

[0196] - is different from said polypeptide, and

[0197] - are different from each other.

[0198] Therefore, component (ii) of the immunogenic composition of the present invention preferably consists of two or more immunogenic substances, wherein each of said immunogenic substances is different from component (i) of the immunogenic composition of the present invention, and wherein all said immunogenic substances are different from each other.

[0199] Preferably, at least one immunogenic substance different from said polypeptide is at least one immunogenic substance comprising or consisting of a reovirus antigen different from said immunogenic fragment. Thus, component (ii) is preferably at least one immunogenic substance comprising or consisting of a reovirus antigen different from said immunogenic fragment of component (i).

[0200] As used herein, the term "reovirus antigen" refers in particular to a peptide or protein comprising an epitope recognized by an antibody specific for a virus belonging to the family Reoviridae, or a portion thereof.

[0201] In a preferred aspect, at least one immunogenic substance different from said polypeptide consists of two or more immunogenic substances, each of said substances comprising or consisting of a reovirus antigen, and wherein all said antigens are different from said immunogenic fragment and are different from each other. Thus, component (ii) preferably consists of two or more immunogenic substances, each of said substances comprising or consisting of a reovirus antigen, and wherein all said antigens are different from said immunogenic fragment of component (i) and are different from each other.

[0202] In another preferred aspect, at least one immunogenic substance different from the polypeptide is at least one protein comprising or consisting of a reovirus antigen different from the immunogenic fragment. Therefore, component (ii) is preferably at least one protein comprising or consisting of a reovirus antigen different from the immunogenic fragment of component (i).

[0203] In a further preferred aspect, at least one immunogenic substance different from said polypeptide consists of two or more proteins, each of said proteins comprising or consisting of a reovirus antigen, and wherein all said reovirus antigens are different from said immunogenic fragment and are different from each other. Thus, component (ii) preferably consists of two or more proteins, each of said proteins comprising or consisting of a reovirus antigen, and wherein all said antigens are different from said immunogenic fragment of component (i) and are different from each other.

[0204] Preferably, at least one immunogenic substance different from said polypeptide is at least one immunogenic substance comprising or consisting of a rotavirus antigen different from said immunogenic fragment. Thus, component (ii) is preferably at least one immunogenic substance comprising or consisting of a rotavirus antigen different from said immunogenic fragment of component (i).

[0205] As used herein, the term "rotavirus antigen" refers in particular to a peptide or protein comprising an epitope recognized by an antibody specific for a virus belonging to the genus Rotavirus, or a portion thereof.

[0206] In a preferred aspect, at least one immunogenic substance different from said polypeptide consists of two or more immunogenic substances, each of said substances comprising or consisting of a rotavirus antigen, and wherein all said antigens are different from said immunogenic fragment and are different from each other. Thus, component (ii) preferably consists of two or more immunogenic substances, each of said substances comprising or consisting of a rotavirus antigen, and wherein all said antigens are different from said immunogenic fragment of component (i) and are different from each other.

[0207] In another preferred aspect, at least one immunogenic substance different from said polypeptide is at least one protein comprising or consisting of a rotavirus antigen different from said immunogenic fragment. Thus, component (ii) is preferably at least one protein comprising or consisting of a rotavirus antigen different from said immunogenic fragment of component (i).

[0208] In a further preferred aspect, at least one immunogenic substance different from said polypeptide consists of two or more proteins, each of said proteins comprising or consisting of a rotavirus antigen, and wherein all said rotavirus antigens are different from said immunogenic fragment and are different from each other. Thus, component (ii) preferably consists of two or more proteins, each of said proteins comprising or consisting of a rotavirus antigen, and wherein all said antigens are different from said immunogenic fragment of component (i) and are different from each other.

[0209] In a particularly preferred aspect, the immunogenic composition of the present invention is preferably an immunogenic composition wherein

[0210] (i) the polypeptide is a first polypeptide;

[0211] and

[0212] (ii) the at least one immunogenic substance different from the polypeptide is at least one other polypeptide different from the first polypeptide.

[0213] Preferably, the at least one immunogenic substance comprises or is

[0214] o a second polypeptide comprising or consisting of a Reovirus antigen different from the immunogenic fragment,

[0215] and optionally comprises a third polypeptide different from said first and second polypeptides,

[0216] And optionally comprises a fourth polypeptide that is different from all of the first to third polypeptides.

[0217] Preferably, the third polypeptide comprises a Reovirus antigen that is different from both the immunogenic fragment and the Reovirus antigen of the second polypeptide.

[0218] The fourth polypeptide preferably comprises a reovirus antigen other than all of:

[0219] - said immunogenic fragment,

[0220] - a reovirus antigen of a second polypeptide, and

[0221] - A third polypeptide of a reovirus antigen.

[0222] In another particularly preferred aspect,

[0223] Component (i) of the immunogenic composition of the present invention is a first polypeptide

[0224] and component (ii) of the immunogenic composition of the present invention is at least one other polypeptide different from the first polypeptide,

[0225] And preferably, component (ii)

[0226] - comprises or is a second polypeptide comprising a rotavirus antigen different from the immunogenic fragment of component (i), and

[0227] - preferably comprises a third polypeptide different from said first and second polypeptides, and

[0228] - optionally comprising a fourth polypeptide which is different from all of said first to third polypeptides.

[0229] Preferably, the third polypeptide comprises a rotavirus antigen different from both the immunogenic fragment of component (i) and the rotavirus antigen of the second polypeptide.

[0230] According to a preferred aspect, the second polypeptide is any one of the polypeptides of the invention as described herein, with the proviso that the second polypeptide is different from the first polypeptide,

[0231] And preferably, the third polypeptide is any one of the polypeptides of the present invention as described herein, with the proviso that the third polypeptide is different from both the first and second polypeptides,

[0232] And optionally, the fourth polypeptide is any one of the polypeptides of the present invention as described herein, with the proviso that the fourth polypeptide is different from all of the first to third polypeptides.

[0233] In another preferred aspect, the immunogenic composition of the present invention is preferably the following immunogenic composition, wherein

[0234] (i) the polypeptide is a first polypeptide, which comprises:

[0235] - a first immunogenic fragment of the rotavirus VP8 protein, and

[0236] - Immunoglobulin Fc fragment;

[0237] and

[0238] (ii) the at least one immunogenic substance different from the polypeptide comprises or consists of:

[0239] - a second polypeptide comprising a second immunogenic fragment of a rotavirus VP8 protein, wherein said second immunogenic fragment is different from said first immunogenic fragment,

[0240] - and preferably a third polypeptide comprising a third immunogenic fragment of a rotavirus VP8 protein, wherein said third immunogenic fragment is different from both said first and second immunogenic fragments,

[0241] - and optionally a fourth polypeptide comprising a fourth immunogenic fragment of a rotavirus VP8 protein, wherein said fourth immunogenic fragment is different from all of said first to third immunogenic fragments.

[0242] Preferably, each of the second to fourth immunogenic fragments is independently selected from the group consisting of:

[0243] - an immunogenic fragment of a rotavirus A VP8 protein, in particular any of the immunogenic fragments of a rotavirus A VP8 protein described herein,

[0244] a portion of a rotavirus VP8 protein, such as a consensus sequence of a portion of a rotavirus A VP8 protein, preferably in the context of a consensus sequence, any of the immunogenic fragments of a rotavirus VP8 protein described herein, and

[0245] - an immunogenic fragment of a rotavirus C VP8 protein, in particular any of the immunogenic fragments of a rotavirus C VP8 protein described herein,

[0246] In particular, with the proviso that all of said second to fourth immunogenic fragments are different from said first immunogenic fragment and from each other.

[0247] More specifically,

[0248] The first immunogenic fragment is selected from the group consisting of: X7, X13, X6 and XC, and / or

[0249] The second immunogenic fragment is selected from the group consisting of: X7, X13, X6 and XC, and / or

[0250] The third immunogenic fragment is selected from the group consisting of: X7, X13, X6 and XC, and / or

[0251] The fourth immunogenic fragment is selected from the group consisting of: X7, X13, X6 and XC;

[0252] in

[0253] X7 represents an immunogenic fragment of genotype P[7] rotavirus VP8 protein,

[0254] X13 represents an immunogenic fragment of genotype P

[13] rotavirus VP8 protein,

[0255] X6 represents an immunogenic fragment of genotype P[6] rotavirus VP8 protein, and

[0256] XC represents the immunogenic fragment of rotavirus C VP8 protein,

[0257] In particular, with the proviso that all of said second to fourth immunogenic fragments are different from said first immunogenic fragment and from each other.

[0258] Therefore, as mentioned in this article,

[0259] "X7" represents the immunogenic fragment of genotype P[7] rotavirus VP8 protein,

[0260] "X13" represents the immunogenic fragment of genotype P

[13] rotavirus VP8 protein,

[0261] "X6" represents an immunogenic fragment of the genotype P[6] rotavirus VP8 protein, and

[0262] "XC" represents the immunogenic fragment of rotavirus C VP8 protein.

[0263] As mentioned herein, X7 preferably consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:3.

[0264] As mentioned herein, X13 preferably consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:5.

[0265] As mentioned herein, X6 preferably consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:4.

[0266] As mentioned herein, XC preferably consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:6.

[0267] In particular, at least one immunogenic substance different from said polypeptide comprises or consists of a second polypeptide comprising a second immunogenic fragment of the rotavirus VP8 protein, and

[0268] Each of the first and second immunogenic fragments is independently selected from the group consisting of: X7, X13, X6 and XC,

[0269] Provided that when the first immunogenic fragment is X7, the second immunogenic fragment is selected from the group consisting of X13, X6 and XC,

[0270] Provided that when the first immunogenic fragment is X6, the second immunogenic fragment is selected from the group consisting of X7, X13 and XC,

[0271] Provided that when the first immunogenic fragment is X13, the second immunogenic fragment is selected from the group consisting of X7, X6 and XC,

[0272] And provided that when the first immunogenic fragment is XC, then the second immunogenic fragment is selected from the group consisting of X7, X13 and X6.

[0273] More specifically, at least one immunogenic substance different from said polypeptide comprises or consists of:

[0274] a second polypeptide comprising a second immunogenic fragment of a rotavirus VP8 protein, and

[0275] a third polypeptide comprising a third immunogenic fragment of a rotavirus VP8 protein,

[0276] and each of the first to third immunogenic fragments is independently selected from the group consisting of X7, X13, X6 and XC,

[0277] Provided that when the first immunogenic fragment is X7, then the second immunogenic fragment is X13 and the third immunogenic fragment is selected from the group consisting of X6 and XC,

[0278] Provided that when the first immunogenic fragment is X6, then the second immunogenic fragment is X7 and the third immunogenic fragment is selected from the group consisting of X13 and XC,

[0279] Provided that when the first immunogenic fragment is X13, the second immunogenic fragment is X7 and the third immunogenic fragment is selected from the group consisting of X6 and XC,

[0280] And provided that when the first immunogenic fragment is XC, then the second immunogenic fragment is X7 and the third immunogenic fragment is selected from the group consisting of X13 and X6.

[0281] More specifically, at least one immunogenic substance different from said polypeptide comprises or consists of:

[0282] a second polypeptide comprising a second immunogenic fragment of a rotavirus VP8 protein, and

[0283] a third polypeptide comprising a third immunogenic fragment of a rotavirus VP8 protein, and

[0284] a fourth polypeptide comprising a fourth immunogenic fragment of a rotavirus VP8 protein,

[0285] wherein each of the first to fourth immunogenic fragments is independently selected from the group consisting of X7, X6, X13 and XC,

[0286] Provided that when the first immunogenic fragment is X7, then the second immunogenic fragment is X13, the third immunogenic fragment is X6, and the fourth immunogenic fragment is XC,

[0287] Provided that when the first immunogenic fragment is X6, then the second immunogenic fragment is X7, the third immunogenic fragment is X13, and the fourth immunogenic fragment is XC,

[0288] Provided that when the first immunogenic fragment is X13, then the second immunogenic fragment is X7, the third immunogenic fragment is X6, and the fourth immunogenic fragment is XC,

[0289] And provided that when the first immunogenic fragment is XC, then the second immunogenic fragment is X7, the third immunogenic fragment is X13, and the fourth immunogenic fragment is X6.

[0290] Preferably,

[0291] The first immunogenic fragment mentioned herein is an immunogenic fragment (X7) of genotype P[7] rotavirus VP8 protein, and / or

[0292] The second immunogenic fragment mentioned herein is an immunogenic fragment of genotype P

[13] rotavirus VP8 protein (X13), and / or

[0293] The third immunogenic fragment mentioned herein is an immunogenic fragment (X6) of genotype P[6] rotavirus VP8 protein, and / or

[0294] The fourth immunogenic fragment mentioned herein is the immunogenic fragment (XC) of rotavirus C VP8 protein.

[0295] Most preferably, the first immunogenic fragment is an immunogenic fragment (X7) of genotype P[7] rotavirus VP8 protein, and

[0296] The second immunogenic fragment is an immunogenic fragment (X13) of genotype P

[13] rotavirus VP8 protein, and

[0297] The third immunogenic fragment is an immunogenic fragment (X6) of genotype P[6] rotavirus VP8 protein,

[0298] And optionally, the fourth immunogenic fragment is an immunogenic fragment (XC) of rotavirus C VP8 protein.

[0299] Preferably,

[0300] The first immunogenic fragment consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO: 3, and

[0301] The second immunogenic fragment consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO: 5, and

[0302] The third immunogenic fragment consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO: 4, and

[0303] Optionally, the fourth immunogenic fragment consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:6.

[0304] In another preferred aspect,

[0305] As mentioned herein, the first polypeptide is selected from the group consisting of: R7, R13, R6 and RC, and / or

[0306] As mentioned herein, the second polypeptide is selected from the group consisting of: R7, R13, R6 and RC, and / or

[0307] The third polypeptide is selected from the group consisting of: R7, R13, R6 and RC, and / or

[0308] The fourth polypeptide is selected from the group consisting of: R7, R13, R6 and RC;

[0309] in

[0310] R7 is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 12,

[0311] R13 is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 14,

[0312] R6 is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 13, and

[0313] RC is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 15,

[0314] Specifically, the proviso is that all of the second to fourth polypeptides are different from the first polypeptide and from each other.

[0315] Preferably, at least one immunogenic substance different from the first polypeptide comprises or consists of a second polypeptide of the rotavirus VP8 protein, and

[0316] Each of the first and second polypeptides is independently selected from the group consisting of: R7, R13, R6 and RC,

[0317] Provided that when the first polypeptide is R7, the second polypeptide is selected from the group consisting of R13, R6 and RC,

[0318] Provided that when the first polypeptide is R6, the second polypeptide is selected from the group consisting of R7, R13 and RC,

[0319] Provided that when the first polypeptide is R13, the second polypeptide is selected from the group consisting of R7, R6 and RC,

[0320] And provided that when the first polypeptide is RC, the second polypeptide is selected from the group consisting of R7, R13 and R6.

[0321] Specifically, at least one immunogenic substance different from the first polypeptide comprises or consists of:

[0322] a second polypeptide, and

[0323] The third polypeptide,

[0324] and each of the first to third polypeptides is independently selected from the group consisting of R7, R13, R6 and RC,

[0325] Provided that when the first polypeptide is R7, the second polypeptide is R13 and the third polypeptide is selected from the group consisting of R6 and RC,

[0326] Provided that when the first polypeptide is R6, the second polypeptide is R7 and the third polypeptide is selected from the group consisting of R13 and RC,

[0327] Provided that when the first polypeptide is R13, the second polypeptide is R7 and the third polypeptide is selected from the group consisting of R6 and RC,

[0328] And provided that when the first polypeptide is RC, then the second polypeptide is R7 and the third polypeptide is selected from the group consisting of R13 and R6.

[0329] More specifically, at least one immunogenic substance different from said polypeptide comprises or consists of:

[0330] a second polypeptide, and

[0331] A third polypeptide, and

[0332] The fourth polypeptide,

[0333] wherein each of the first to fourth polypeptides is independently selected from the group consisting of: R7, R6, R13 and RC,

[0334] Provided that when the first polypeptide is R7, the second polypeptide is R13, the third polypeptide is R6, and the fourth polypeptide is RC,

[0335] Provided that when the first polypeptide is R6, the second polypeptide is R7, the third polypeptide is R13, and the fourth polypeptide is RC,

[0336] Provided that when the first polypeptide is R13, the second polypeptide is R7, the third polypeptide is R6, and the fourth polypeptide is RC,

[0337] And provided that when the first polypeptide is RC, the second polypeptide is R7, the third polypeptide is R13, and the fourth polypeptide is R6.

[0338] According to a preferred aspect,

[0339] The first polypeptide mentioned herein is a protein comprising an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with the sequence of SEQ ID NO: 12, or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with the sequence of SEQ ID NO: 12, and

[0340] The second polypeptide mentioned herein is a protein comprising an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 13 and SEQ ID NO: 15, or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 13 and SEQ ID NO: 15, and

[0341] Preferably, the third polypeptide mentioned herein is a protein comprising an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 13 and SEQ ID NO: 15, or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 13 and SEQ ID NO: 15,

[0342] And optionally, the fourth polypeptide mentioned herein is a protein comprising an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 13 and SEQ ID NO: 15, or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 13 and SEQ ID NO: 15.

[0343] Specifically, the immunogenic composition of the present invention preferably comprises:

[0344] (i) a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with the sequence of SEQ ID NO: 12, and

[0345] (ii) a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 13 and SEQ ID NO: 15.

[0346] Most particularly, the immunogenic composition of the present invention preferably comprises:

[0347] (i) a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 12, and

[0348] (ii) a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 14, and

[0349] A protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 13,

[0350] And optionally a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 15.

[0351] The immunogenic composition of the invention preferably comprises each of the substances of component (i) and component (ii) at a concentration of at least 100 nM, preferably at least 250 nM, more preferably at least 500 nM and most preferably at least 1 μM.

[0352] According to another preferred aspect, the immunogenic composition of the present invention contains each of the substances of component (i) and component (ii) at a concentration of 100 nM to 50 μM, preferably 250 nM to 25 μM and most preferably 1 to 10 μM.

[0353] Specifically, 1 mL or, as the case may be, 2 mL of the immunogenic composition of the present invention is administered to the subject. Thus, the dose of the immunogenic composition of the present invention to be administered to the subject preferably has a volume of 1 mL or 2 mL.

[0354] Preferably, one or two doses of the immunogenic composition are administered to the subject.

[0355] The immunogenic compositions of the present invention are preferably administered systemically or topically. Suitable routes of administration conventionally used are parenteral or oral administration, such as intramuscular, intradermal, intravenous, intraperitoneal, subcutaneous, intranasal, and inhalation. However, depending on the nature and mode of action of the compound, the immunogenic composition may also be administered by other routes. Most preferably, the immunogenic composition is administered intramuscularly. The immunogenic composition of the present invention preferably further comprises a pharmaceutically or veterinarily acceptable carrier or excipient.

[0356] As used herein, "pharmaceutically or veterinarily acceptable carriers" include any and all solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delay agents, and the like. In some preferred embodiments and particularly embodiments including lyophilized immunogenic compositions, stabilizers for use in the present invention include stabilizers for lyophilization or freeze drying.

[0357] In some embodiments, the immunogenic compositions of the invention contain an adjuvant.

[0358] As used herein, "adjuvants" may include aluminum hydroxide and aluminum phosphate, saponins such as Quil A, QS-21 (Cambridge Biotech, Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Birmingham, AL), water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions. Emulsions may be based, inter alia, on light liquid paraffin oil (European Pharmacopeia type); isoprenoid oils such as squalane or squalene; oils resulting from oligomerization of olefins, especially isobutylene or decene; esters of acids or alcohols containing linear alkyl groups, especially vegetable oils, ethyl oleate, propylene glycol di(caprylate / caprate), glyceryl tri(caprylate / caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, especially isostearates. Oils are used in combination with emulsifiers to form emulsions. Emulsifiers are preferably nonionic surfactants, especially (optionally ethoxylated) sorbitan, mannide (e.g., anhydrous mannitol oleate), ethylene glycol, polyglycerol, propylene glycol and esters of oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, and polyoxypropylene-polyoxyethylene copolymer blocks, especially Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, DES), John Wiley and Sons, NY, pp. 51-94 (1995), and Todd et al., Vaccine 15: 564-570 (1997). Exemplary adjuvants are the SPT emulsion described on page 147 of "Vaccine Design, The Subunit and Adjuvant Approach", eds. M. Powell and M. Newman, Plenum Press, 1995, or the emulsion MF59 described on page 183 of the same book.

[0359] Another example of an adjuvant is a compound selected from polymers of acrylic acid or methacrylic acid and copolymers of maleic anhydride and alkenyl derivatives. Advantageous adjuvant compounds are polymers of acrylic acid or methacrylic acid crosslinked, in particular, with polyalkenyl ethers of sugars or polyols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Those skilled in the art may also refer to U.S. Pat. No. 2,909,462, which describes such acrylic acid polymers crosslinked with polyhydroxy compounds having at least 3 hydroxyl groups, preferably no more than 8 hydroxyl groups, at least three of which have hydrogen atoms replaced by unsaturated aliphatic groups having at least 2 carbon atoms. Preferred groups are groups containing 2 to 4 carbon atoms, such as vinyl, allyl and other olefinically unsaturated groups. The unsaturated group itself may contain other substituents, such as methyl. The name The products sold by Carbopol (BF Goodrich, Ohio, USA) are particularly suitable. They are crosslinked with allyl sucrose or allyl pentaerythritol. In particular, Carbopol 974P, 934P and 971P may be mentioned. Most preferably, the use of 971P. Copolymers of maleic anhydride and alkenyl derivatives are in particular copolymers EMA (Monsanto), which are copolymers of maleic anhydride and ethylene. These polymers are dissolved in water to produce an acid solution, which is preferably neutralized to physiological pH in order to obtain an adjuvant solution incorporated into the immunogenic, immune or vaccine composition itself.

[0360] Other suitable adjuvants from which the adjuvant can be selected include, but are not limited to, the RIBI adjuvant system (Ribi Corporation), Block copolymers (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from Escherichia coli (E. coli) (recombinant or otherwise), cholera toxin, IMS1314 or muramyl dipeptide, or naturally occurring or recombinant cytokines or analogs thereof, or endogenous cytokine release stimulators, etc.

[0361] It is contemplated that the adjuvant may be added in an amount of about 100 μg to about 10 mg per dose, preferably in an amount of about 100 μg to about 10 mg per dose, more preferably in an amount of about 500 μg to about 5 mg per dose, even more preferably in an amount of about 750 μg to about 2.5 mg per dose, and most preferably in an amount of about 1 mg per dose. Alternatively, the adjuvant may be added in a concentration of about 0.01% to 50%, preferably in a concentration of about 2% to 30%, more preferably in a concentration of about 5% to 25%, still more preferably in a concentration of about 7% to 22%, and most preferably in a concentration of 10% to 20%, based on the volume of the final product.

[0362] "Diluents" may include water, physiological saline, dextrose, ethanol, glycerol and the like. Isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol and lactose, etc. Stabilizers include albumin and alkali metal salts of ethylenediaminetetraacetic acid, etc.

[0363] According to a particularly preferred aspect, the present invention also provides an immunogenic composition, in particular the immunogenic composition of the present invention, wherein the immunogenic composition comprises or consists of:

[0364] - component (i),

[0365] - component (ii),

[0366] - a pharmaceutically or veterinarily acceptable carrier or excipient,

[0367] - and optionally an adjuvant.

[0368] In the context of the present invention, the adjuvant is preferably selected from the group consisting of emulsified oil-in-water adjuvants and carbomers.

[0369] The term "immunogenic composition" refers to a composition comprising at least one antigen, which elicits an immune response in a host to which the immunogenic composition is administered. Such an immune response may be a cellular and / or antibody-mediated immune response to the immunogenic composition according to the invention. The host is also described as a "subject". Preferably, any of the hosts or subjects described or referred to herein is an animal.

[0370] As used herein, the term "animal" refers in particular to a mammal, preferably a swine, more preferably a pig, most preferably a piglet.

[0371] Typically, an "immune response" includes, but is not limited to, one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells and / or cytotoxic T cells and / or γ-δ T cells specific for one or more antigens included in the immunogenic composition of the invention. Preferably, the host will present a protective immune response or a therapeutic response.

[0372] A "protective immune response" will be evidenced by a reduction or absence of one or more clinical signs normally presented by an infected host, an accelerated recovery time and / or a reduced duration of infection, or a reduction in pathogen titers in the tissues or body fluids or excreta of an infected host.

[0373] As referred to herein, "pathogen" or "specific pathogen" particularly relates to the rotavirus from which the immunogenic fragment of the rotavirus VP8 protein is derived. For example, the pathogen as referred to herein is rotavirus A or rotavirus C.

[0374] An immunogenic composition is described as a "vaccine" where the host mounts a protective immune response such that resistance to new infection will be enhanced and / or the clinical severity of disease will be reduced.

[0375] As used herein, "antigen" refers to, but is not limited to, a component that elicits an immune response in a host to an immunogenic composition or vaccine of interest comprising such antigen or an immunologically active component thereof. Specifically, the term "antigen" as used herein refers to a protein or protein domain that, if administered to a host, can elicit an immune response in the host.

[0376] The term "treatment and / or prevention" refers to a reduction in the incidence of a specific pathogen infection in a herd, or a reduction in the severity of one or more clinical signs caused by or associated with a specific pathogen infection. Therefore, the term "treatment and / or prevention" also refers to a reduction in the number of animals infected with a specific pathogen in a herd (= a reduction in the incidence of a specific pathogen infection) or a reduction in the severity of one or more clinical signs typically associated with or caused by a pathogen infection in a group of animals in which the animals have received an effective amount of an immunogenic composition as provided herein, compared to a group of animals in which the animals have not received such an immunogenic composition.

[0377] "Treatment and / or prevention" generally involves administering an effective amount of the immunogenic composition of the present invention to a subject or group of subjects that is in need of such treatment / prevention or that may benefit from such treatment / prevention. The term "treatment" refers to administering an effective amount of the immunogenic composition once at least some of the animals in a subject or herd have been infected with such pathogens and wherein such animals have exhibited some clinical signs caused by or associated with infection with such pathogens. The term "prevention" refers to administering to a subject before such subject is infected with any pathogen or at least when all animals in such animal or group of animals do not exhibit one or more clinical signs caused by or associated with infection with such pathogens.

[0378] As used herein, the term "effective amount" represents but is not limited to the amount of an antigen, particularly a polypeptide of the present invention, that causes or is capable of causing an immune response in a subject. Such an effective amount is capable of reducing the incidence of infection with a specific pathogen in a herd or reducing the severity of one or more clinical signs of infection with a specific pathogen. Preferably, the incidence or severity of one or more clinical signs is reduced by at least 10%, more preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95%, compared to an untreated or untreated immunogenic composition available prior to the present invention, but subsequently infected with a specific pathogen.

[0379] As used herein, the term "clinical signs" refers to symptoms of a subject being infected with a particular pathogen. The clinical signs of infection depend on the selected pathogen. Examples of such clinical signs include, but are not limited to, diarrhea, vomiting, fever, abdominal pain, and dehydration.

[0380] Reducing the incidence or severity of one or more clinical symptoms caused by or associated with infection with a specific pathogen in a subject can be achieved by administering to the subject one or more doses of an immunogenic composition of the invention.

[0381] The term "reducing fecal excretion" represents, but is not limited to, reducing the number of RNA copies of pathogenic viruses (such as rotavirus) per mL of feces or the number of plaque-forming colonies per deciliter of feces, and the feces of the subject receiving the composition of the present invention is reduced by at least 50% compared to the subject who does not receive the composition and can be infected. More preferably, the fecal excretion level in the subject receiving the composition of the present invention is reduced by at least 90%, preferably at least 99.9%, more preferably at least 99.99% and even more preferably at least 99.999%.

[0382] As used herein, the term "fecal shedding" is used according to its ordinary meaning in medicine and virology and refers to the production of virus from the subject's cells and the release of virus from an infected subject to the environment via the subject's stool.

[0383] The polypeptide of the present invention is preferably a recombinant protein, especially a protein expressed by a recombinant baculovirus.

[0384] As used herein, the term "recombinant protein" refers in particular to a protein produced by recombinant DNA techniques, wherein the DNA encoding the expressed protein is usually inserted into a suitable expression vector, which is then used to transform or, in the case of a viral vector, infect a host cell to produce the heterologous protein. Thus, as used herein, the term "recombinant protein" refers in particular to a protein molecule expressed from a recombinant DNA molecule. As used herein, a "recombinant DNA molecule" refers to a DNA molecule composed of DNA segments joined together by means of molecular biological techniques. Suitable systems for producing recombinant proteins include, but are not limited to, insect cells (e.g., baculovirus), prokaryotic systems (e.g., Escherichia coli), fungi (e.g., Myceliophthora thermophile, Aspergillus oryzae, Ustilagomaydis), yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris), mammalian cells (e.g., Chinese hamster ovary, HEK293), plants (e.g., safflower), algae, avian cells, amphibian cells, fish cells, and cell-free systems (e.g., rabbit reticulocyte lysate).

[0385] According to another aspect, the present invention provides a polynucleotide comprising a sequence encoding a polypeptide of the present invention, wherein the polynucleotide, hereinafter also referred to as "a polynucleotide according to the present invention" is preferably an isolated polynucleotide.

[0386] Preferably, the polynucleotide according to the present invention comprises a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or in particular 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.

[0387] The production of the polynucleotides described herein is within the skill in the art and can be performed, among other occasions, according to the recombinant techniques described in Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Amusable et al., 2003, Current Protocols In Molecular Biology, Greene Publishing Associates & Wiley Interscience, NY; Innis et al. (eds.), 1995, PCR Strategies, Academic Press, Inc., San Diego; and Erlich (ed.), 1994, PCR Technology, Oxford University Press, New York, all of which are incorporated herein by reference.

[0388] In another aspect, the present invention provides a vector comprising a polynucleotide encoding a polypeptide of the present invention.

[0389] For the purposes of the present invention, "vector" and "vector containing a polynucleotide encoding a polypeptide of the present invention" refer to a suitable expression vector, preferably a baculovirus expression vector, which is then used to transfect or, in the case of a baculovirus expression vector, to infect a host cell to produce the protein or polypeptide encoded by the DNA. Vectors and methods for preparing and / or using vectors (or recombinants) for expression can be made or performed, in particular, by the following or similar methods disclosed in: U.S. Pat. Nos. 4,603,112, 4,769,330, 5,174,993, 5,505,941, 5,338,683, 5,494,807, 4,722,848, 5,942,235, 5,364,773, 5,762,938, 5,770,212, 5,942,235, 382,425, PCT Publications WO 94 / 16716, WO 96 / 39491, WO 95 / 30018; Paoletti, “Applications of pox virus vectors to vaccination: An update”, PNAS USA 93:11349-11353, October 1996; Moss, “Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety”, PNAS USA 93:11341-11348, October 1996; Smith et al., U.S. Pat. No. 4,745,051 (recombinant baculovirus); Richardson, CD (ed.), Methods in Molecular Biology 39, “Baculovirus Expression Protocols” (1995 Humana Press); Smith et al., “Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector”, Molecular and Cellular Biology, December 1983, Vol. 3, No. 12, pp. 2156-2165;Pennock et al., "Strong and Regulated Expression of Escherichia coli B-Galactosidase in Infect Cells with a Baculovirus vector", Molecular and Cellular Biology, March 1984, Vol. 4, No. 3, p. 406; EPA 0 370 573; U.S. Application No. 920,197, filed October 16, 1986; European Patent Application No. 265 785; U.S. Patent No. 4,769,331 (recombinant herpes virus); Roizman, "The function of herpes simplex virus genes: A primer for genetic engineering of novel vectors", PNAS USA 93:11307-11312, October 1996; Andreansky et al., "The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors", PNAS USA 93:11307-11312, October 1996 93:11313-11318, October 1996; Robertson et al., "Epstein-Barr virus vectors for gene delivery to B lymphocytes", PNAS USA 93:11334-11340, October 1996; Frolov et al., "Alphavirus-based expression vectors: Strategies and applications", PNAS USA 93:11371-11377, October 1996; Kitson et al., J. Virol. 65, 3068-3075, 1991; U.S. Patent Nos. 5,591,439 and 5,552,143; WO 98 / 00166; U.S. Application Serial Nos. 08 / 675,556 and 08 / 675,566, both filed on July 3, 1996 (recombinant adenovirus); Grunhaus et al., 1992, “Adenovirus as cloning vectors”, Seminars in Virology (Vol. 3) pp. 237-52, 1993;Ballay et al., EMBO Journal, Vol. 4, pp. 3861-65, Graham, Tibtech 8, 85-87, April 1990; Prevec et al., J. Gen Virol. 70, 42434; PCT WO 91 / 11525; Felgner et al. (1994), J. Biol. Chem. 269, 2550-2561, Science, 259: 1745-49, 1993; and McClements et al., "Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease", PNAS USA 93:11414-11420, October 1996; and U.S. Patent Nos. 5,591,639, 5,589,466, and 5,580,859, and WO 90 / 11092, WO 93 / 19183, WO 94 / 21797, WO 95 / 11307, WO 95 / 20660; Tang et al., Nature, and Furth et al., Analytical Biochemistry, relating to DNA expression vectors. See also WO 98 / 33510; Ju et al., Diabetologia, 41:736-739, 1998 (lentiviral expression system); Sanford et al., U.S. Pat. No. 4,945,050; Fischbach et al. (Intracel); WO 90 / 01543; Robinson et al., Seminars in Immunology, Vol. 9, pp. 271-283 (1997), (DNA vector system); Szoka et al., U.S. Pat. No. 4,394,448 (methods for inserting DNA into living cells); McCormick et al., U.S. Pat. No. 5,677,178 (use of cytopathic viruses); and U.S. Pat. No. 5,928,913 (vectors for gene delivery); and other references cited herein. ;

[0390] Preferred viral vectors include baculovirus, such as BaculoGold (BD Biosciences Pharmingen, San Diego, CA), specifically, provided that the production cells are insect cells. Although the baculovirus expression system is preferred, it will be appreciated by those skilled in the art that other expression systems including those described above will effectively achieve the purpose of the present invention, i.e., the expression of recombinant proteins.

[0391] Therefore, the present invention also provides a baculovirus containing a polynucleotide, wherein the polynucleotide comprises a sequence encoding a polypeptide of the present invention. The baculovirus, hereinafter also referred to as "the baculovirus according to the present invention", is preferably an isolated baculovirus.

[0392] In addition, the present invention therefore also provides a plasmid, preferably an expression vector, which comprises a polynucleotide comprising a sequence encoding a polypeptide of the present invention. The plasmid, hereinafter also referred to as "plasmid according to the present invention", is especially an isolated plasmid.

[0393] The present invention also provides a cell infected by and / or containing a baculovirus, which comprises a polynucleotide comprising a sequence encoding a polypeptide of the present invention; or a plasmid, preferably an expression vector, which comprises a polynucleotide comprising a sequence encoding a polypeptide of the present invention. The cell, hereinafter also referred to as a "cell according to the present invention", is preferably an isolated cell.

[0394] The term "isolated" when used in the context of an isolated cell is a cell that has been removed from its natural environment by the hand of man and therefore is not a product of nature.

[0395] In a further aspect, the present invention also relates to the use of the immunogenic composition of the present invention for the preparation of a medicament, preferably a vaccine.

[0396] In this context, the present invention also provides a method for producing an immunogenic composition of the present invention, wherein the method comprises the step of infecting cells, preferably insect cells, with a baculovirus according to the present invention.

[0397] Furthermore, the present invention also provides a method for producing the polypeptide of the present invention, wherein the method comprises the step of transfecting cells with the plasmid according to the present invention.

[0398] The polypeptides of the present invention are preferably expressed in high amounts that are sufficiently stable to self-assemble into virus-like particles that can subsequently be used for vaccination.

[0399] As used herein, the terms "vaccination" and "vaccinating" represent, but are not limited to, a method comprising administering an antigen (such as an antigen included in an immunogenic composition) to a subject, wherein the antigen (e.g., a polypeptide of the present invention) elicits or is capable of eliciting a protective immune response in the subject when administered to the subject.

[0400] The present invention also provides an immunogenic composition of the present invention for use as a medicament, preferably as a vaccine.

[0401] Specifically, the immunogenic composition of the present invention is provided for use in a method for reducing or preventing one or more clinical signs or diseases caused by rotavirus infection, wherein the rotavirus is preferably a rotavirus in the group of genomes having an immunogenic fragment encoding the rotavirus VP8 protein. In particular, the immunogenic composition of the present invention is provided for use in a method for reducing or preventing fecal discharge caused by rotavirus infection, wherein the virus is preferably a rotavirus in the group of genomes having an immunogenic fragment encoding the rotavirus VP8 protein. Therefore, in a specific example, if the immunogenic fragment of the rotavirus VP8 protein as mentioned herein is encoded by the genome of rotavirus A, the immunogenic composition of the present invention is used to reduce or prevent one or more clinical signs, death, fecal discharge or disease caused by rotavirus A infection.

[0402] More specifically, the immunogenic compositions of the invention are provided for use in methods of reducing or preventing one or more clinical signs, mortality, or fecal excretion caused by rotavirus infection in a subject or for use in methods of treating or preventing rotavirus infection in a subject.

[0403] Rotavirus infection as referred to herein refers in particular to infection with rotavirus A or rotavirus C.

[0404] Furthermore, the immunogenic compositions of the invention are provided for use in inducing an immune response against rotavirus in a subject.

[0405] As mentioned herein, the subject is preferably a mammal, such as a pig or a cow; or a bird, such as a chicken. Specifically, the subject is a pig, and wherein the pig is preferably a piglet or a sow, such as a pregnant sow. Most preferably, in the case of inducing an immune response against rotavirus in a subject, the subject is a pregnant sow. In the case of reducing or preventing one or more clinical signs, mortality or fecal discharge caused by rotavirus infection in a subject or treating or preventing rotavirus infection in a subject, the subject is most preferably a piglet.

[0406] According to a preferred aspect, the immunogenic composition of the present invention is used in a method for reducing or preventing one or more clinical signs, mortality or fecal excretion caused by rotavirus infection in piglets, wherein the piglets are suckled by sows to which the immunogenic composition has been administered. The sows to which the immunogenic composition has been administered are preferably sows to which the immunogenic composition has been administered when the sows are already pregnant, especially pregnant with the piglets.

[0407] In addition, the present invention relates to a method for treating or preventing rotavirus infection, reducing, preventing or treating one or more clinical signs, death or fecal excretion caused by rotavirus infection, or preventing or treating a disease caused by rotavirus infection, comprising administering the immunogenic composition of the present invention to a subject.

[0408] Furthermore, a method for inducing the production of antibodies specific for rotavirus in, preferably a pregnant sow, is provided, wherein the method comprises administering to the sow an immunogenic composition of the invention.

[0409] In addition, the present invention provides a method for reducing or preventing one or more clinical signs, mortality or fecal excretion caused by rotavirus infection in piglets, wherein the method comprises:

[0410] - administering the immunogenic composition of the invention to a sow, and

[0411] - allowing said sow to suckle said piglets,

[0412] And wherein the sow is preferably a pregnant sow, especially pregnant with the piglets.

[0413] Preferably, the two aforementioned methods comprise the following steps:

[0414] - administering the immunogenic composition of the present invention to a sow pregnant with said piglet,

[0415] - allowing said sow to give birth to said piglets, and

[0416] - allowing the sow to suckle the piglets.

[0417] Additionally, provided is a method of reducing one or more clinical signs, mortality, or fecal shedding caused by rotavirus infection in piglets, wherein the piglets are suckled by sows to which the immunogenic composition of the invention has been administered.

[0418] The one or more clinical signs as mentioned herein are preferably selected from the group consisting of:

[0419] -diarrhea,

[0420] - rotavirus colonization, especially enteric rotavirus colonization,

[0421] - Lesions, especially macroscopic ones, and

[0422] - Reduced average daily weight gain.

[0423] According to one example, the one or more clinical signs mentioned herein are rotavirus colonization of the intestine, especially the small intestine. According to another example, the one or more clinical signs mentioned herein are intestinal lesions, especially macroscopic intestinal lesions.

[0424] According to another particularly preferred aspect, the immunogenic composition of the present invention is used in any of the methods described above, wherein

[0425] - the rotavirus infection is infection with genotype P

[23] rotavirus and / or genotype P[7] rotavirus,

[0426] - the rotavirus infection is infection with genotype P

[23] rotavirus and / or genotype P[7] rotavirus,

[0427] - said immune response against rotavirus is an immune response against genotype P

[23] rotavirus and / or genotype P[7] rotavirus, or

[0428] - the antibody specific for rotavirus is an antibody specific for rotavirus genotype P

[23] and / or rotavirus genotype P[7],

[0429] And preferably, the immunogenic composition of the present invention comprises any one of the polypeptides of the present invention described herein comprising an immunogenic fragment of the genotype P[7] rotavirus VP8 protein, wherein the immunogenic fragment is especially composed of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:3.

[0430] In a specific aspect, the "infectious genotype P

[23] rotavirus and / or genotype P[7] rotavirus" as referred to herein is an infectious genotype P

[23] rotavirus.

[0431] In another preferred aspect, the "infectious rotavirus of genotype P

[23] and / or rotavirus of genotype P[7]" as referred to herein refers to an infectious rotavirus of genotype P

[23] and rotavirus of genotype P[7].

[0432] In a specific aspect, the "immune response against genotype P

[23] rotavirus and / or genotype P[7] rotavirus" as referred to herein is an immune response against genotype P

[23] rotavirus.

[0433] In another preferred aspect, the "immune response against genotype P

[23] rotavirus and / or genotype P[7] rotavirus" as referred to herein is an immune response against genotype P

[23] rotavirus and genotype P[7] rotavirus.

[0434] In a specific aspect, an "antibody specific for rotavirus genotype P

[23] and / or rotavirus genotype P[7]" as referred to herein is an antibody specific for rotavirus genotype P

[23] .

[0435] In another preferred aspect, the "antibodies specific for rotavirus genotype P

[23] and / or rotavirus genotype P[7]" as referred to herein comprise or are antibodies specific for rotavirus genotype P

[23] and antibodies specific for rotavirus genotype P[7].

[0436] In another aspect, the immunogenic composition of the invention is administered to induce the production of antibodies specific for rotavirus C in animals, preferably in pregnant sows. Preferably, in this other aspect, the immunogenic composition of the invention comprises any of the polypeptides of the invention described herein comprising an immunogenic fragment of a rotavirus C VP8 protein, in particular consisting of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO: 15.

[0437] The invention further provides a method of producing an immunogenic composition of the invention, wherein the method comprises transfecting a cell with a plasmid of the invention.

[0438] Furthermore, a method of producing an immunogenic composition of the invention is provided, wherein the method comprises infecting a cell, preferably an insect cell, with a baculovirus of the invention.

[0439] Furthermore, the present invention relates to a method for producing the immunogenic composition of the present invention, wherein the method comprises the following steps:

[0440] (a) allowing susceptible cells in culture to be infected with a vector comprising a nucleic acid sequence encoding a polypeptide of the present invention, wherein the polypeptide is expressed by the vector;

[0441] (b) thereafter recovering the polypeptide, in particular in the supernatant of the cultured cells, wherein cell debris is preferably separated from the polypeptide via a separation step, the separation step preferably comprising microfiltration through at least one filter, preferably two filters, wherein the pore size of the at least one filter is preferably from about 1 to about 20 μm and / or from about 0.1 μm to about 4 μm;

[0442] (c) deactivating the carrier by adding diethyleneimine (BEI) to the mixture of step (b);

[0443] (d) neutralizing the BEI by adding sodium thiosulfate to the mixture produced in step (c); and

[0444] (e) concentrating the polypeptides in the mixture produced in step (d) by removing a portion of the liquid from the mixture through a filtering step using a filter having a membrane having a molecular weight cutoff of between about 5 kDa and about 100 kDa, preferably between about 10 kDa and about 50 kDa;

[0445] And wherein the method comprises the following steps:

[0446] (f) admixing the mixture remaining after step (e) with at least one immunogenic substance different from the polypeptide;

[0447] (g) and optionally blending the mixture remaining after step (f) with another component selected from the group consisting of a pharmaceutically acceptable carrier, an adjuvant, a diluent, an excipient and a combination thereof,

[0448] Or wherein the method comprises the following steps:

[0449] (f) blending the mixture remaining after step (e) with another component selected from the group consisting of a pharmaceutically acceptable carrier, adjuvant, diluent, excipient, and combinations thereof, and

[0450] (g) admixing the mixture remaining after step (f) with at least one immunogenic substance different from the polypeptide.

[0451] For said admixture with at least one immunogenic substance different from said polypeptide, at least one immunogenic substance different from said polypeptide as described herein is used, respectively,

[0452] or any one of component (ii).

[0453] In step (a) of the method, the cell is preferably an insect cell and the vector is preferably a baculovirus of the present invention.

[0454] In step (b) of the method, the polypeptide is most preferably recovered in the supernatant of the cultured cells, rather than from the interior of the cells.

[0455] Furthermore, the present invention provides an immunogenic composition of the present invention and use of said immunogenic composition in any of the methods described herein, wherein said immunogenic composition is obtainable by the aforementioned method for producing an immunogenic composition of the present invention. Example

[0456] The following examples are intended only to illustrate the present invention and should not limit the scope of the claims in any way.

[0457] Example 1

[0458] Design, production and testing of fusion proteins:

[0459] Construct design:

[0460] The rotavirus A VP4 sequence was originally obtained from a pig fecal sample, which most closely matched the GenBank sequence JX971567.1 and was classified as the P[7] genotype. VP4 amino acids 57-224 (SEQ ID NO:3) were used, also designated "AVP8" hereinafter, and correspond to the lectin-like domain of the VP8 protein, but with the N-terminus extended by eight amino acid residues. The linker portion was Gly-Gly-Ser (SEQ ID NO:9). The porcine IgG Fc sequence (SEQ ID NO:7) matched amino acids 242-470 of the IgG heavy chain constant precursor (Genbank sequence BAM75568.1). The IDT Gblock, Gly-Gly-Ser linker, and porcine IgG Fc sequence encoding AVP8 (SEQ ID NO:17) were received, all of which were optimized for insect cells and designated AVP8-IgG Fc herein. The protein encoded by AVP8-IgG Fc (SEQ ID NO: 12) is also referred to herein as "AVP8-IgG Fc protein."

[0461] Cloning, expression and purification:

[0462] AVP8-IgG Fc was TOPO cloned and then inserted into the baculovirus transfer plasmid pVL1393 using BamHI and NotI restriction sites, followed by co-transfection with BaculoGold into Sf9 cells to produce recombinant baculovirus. Production of AVP8-IgG Fc protein was performed as follows: 1 L of Sf+ cells in a 3L spinner flask was infected with spent medium harvested 4 DPI at 0.2 MOI, centrifuged at 15,000 g for 20 minutes and filtered through 0.2 μm. 1 mL of MabSelect SuRE LX resin slurry (GE Healthcare, catalog number 17-5474-01) was added and incubated overnight at 4°C with moderate agitation. The resin was re-harvested by filtration, washed with 4×10 mL of mild binding buffer (Pierce, catalog number 21012) and eluted in 7×5 mL volumes of mild elution buffer (Pierce, catalog number 21027). Fractions were pooled and dialyzed against 3.5 L TBS with one buffer change at 4° C. A BCA assay (Thermo Scientific, cat. no. 23227) was performed to determine the concentration (80 μg / mL).

[0463] Serological studies:

[0464] Protein A purified AVP8-IgG Fc protein was formulated with Emulsigen D with 87.5% antigen and 12.5% ​​adjuvant. Pigs at approximately seven weeks of age received a 2 mL dose via IM in the flank of the neck with a boost 21 days later. Serum samples were collected weekly for seven weeks. The serum samples were analyzed by ELISA ( Figure 1 ) and the virus neutralization assay as described below ("Protocol for virus neutralization assay") Figure 2 ) evaluated sera from pigs vaccinated with AVP8-IgG Fc protein. Compared with the unrelated vaccine control, the IgG ELISA results from pigs vaccinated with AVP8-IgG Fc protein showed a peak increase in SP ratio on day 14 and increased again after a boost on day 21. Virus neutralization titers similarly showed an increase on days 7 and 14, followed by a second peak on day 28 after a boost on day 21.

[0465] Protocol for ELISA

[0466] For IgA ELISA, media protein-bound 96-well ELISA plates were coated with complete rotavirus antigen diluted in 1×PBS 1:16. The plates were incubated overnight at 4°C. After incubation, the plates were washed with 1×PBST and then blocked with casein blocking solution for 1 hour at 37°C. After washing, 100 μL of primary antibody diluted to a final dilution of 1:40 in blocking buffer was added to the plates and incubated for 1 hour at 37°C. After washing, the wells were coated with 100 μL of a 1:3200 dilution of horseradish peroxidase (HRP)-conjugated goat anti-swine IgA and incubated for one hour at 37°C. After washing, the plates were developed with 3,5,3',5'-tetramethylbenzidine at room temperature for 15 minutes and the reaction was terminated with 1N HCl before optical density (OD) measurement at 450 nm. Samples including positive and negative controls were run in duplicate wells and results are reported as the mean of the (sample - negative control) to (positive - negative control) ratios (SN) / (PN).

[0467] For IgG ELISA, a media protein-bound 96-well ELISA plate was coated with complete rotavirus antigen diluted in 1×PBS 1:8. The plates were incubated overnight at 4°C. After incubation, the plates were washed with 1×PBST and then blocked with ink-dot blocking solution for 1 hour at 37°C. After washing, 100 μL of primary antibody diluted to a final dilution of 1:625 in blocking buffer was added to the plate and incubated for 1 hour at 37°C. After washing, the wells were coated with 100 μL of a 1:8000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-swine IgG and incubated for one hour at 37°C. After washing, the plates were developed with 3,5,3',5'-tetramethylbenzidine at room temperature for 10 minutes and the reaction was terminated with 1N HCl before optical density (OD) measurement at 450 nm. Samples including positive and negative controls were run in duplicate wells and results are reported as the mean of the (sample - negative control) to (positive - negative control) ratio (SN) / (PN).

[0468] Protocol for virus neutralization assay

[0469] All serum and milk samples were heat inactivated at 56°C for 30 minutes. Samples were serially diluted from 1:40 to 1:2,560 in rotavirus growth medium (MEM + 2.5% HEPES + 0.3% trypsin phosphate broth + 0.02% yeast + 10 μg / mL trypsin). Rotavirus A isolate (titer 7.0 log TCID 50 / mL) was diluted 1:25,000 into rotavirus growth medium. A total of 200 μl of diluted serum was added to 200 μl of diluted virus; the mixture was incubated for one hour at 37°C ± 5% CO2. The growth medium was aseptically removed from a three- to four-day-old 96-well plate seeded with MA104 cells. After incubation, 200 μl of the virus-serum mixture was transferred to the cell plate. The cells were incubated at 37°C ± 5% CO2 for 72 hours. The stock solution and diluted virus were titrated on the day of use to determine the dilution used in the assay. After incubation, the supernatant was discarded and the plate was washed once with 200 microliters / well 1× PBS. For fixation, 100 microliters / well of 50% / 50% acetone / methanol was added. The plate was incubated at room temperature for 15 minutes, air-dried, and then rehydrated with 100 microliters / well 1× PBS. The primary antibody (rabbit anti-rotavirus A polyclonal serum, produced in-house) was diluted 1:1000 in 1×PBS. 100 μL / well of the diluted primary antibody was added and the plate was incubated for one hour at 37°C ± 5% CO2. After incubation, the plate was washed twice with 100 μL / well 1×PBS. The secondary antibody (Jackson ImmunoResearch FITC-labeled goat anti-rabbit IgG, catalog number 111-095-003) was diluted 1:100 in 1×PBS. 100 μL / well of the diluted secondary antibody was added and the plate was incubated for one hour at 37°C ± 5% CO2. After incubation, the plate was washed twice with 100 μL / well 1×PBS. The plate was read for the presence of fluorescence using a UV microscope. If the titer of the diluted virus (generated using the Reed-Muench method) is found to be 2.8 ± 0.5 log TCID 50 / mL, the assay was considered valid. In addition, known positive and negative samples were included in each assay as controls. Serum titers are reported as the highest dilution where no staining was observed.

[0470] Example 2

[0471] Anti-virus research:

[0472] The primary objective of this study was to evaluate the effectiveness of a conventional sow-administered prototype vaccine, also referred to herein as "IgG:AVP8," comprising the AVP8-IgG Fc protein (SEQ ID NO: 12), and an unrelated control vaccine, referred to herein as "placebo," in providing passive protection to pigs against a virulent rotavirus A challenge. In addition, for comparison, a commercially available MLV rotavirus vaccine ( Rota, Merck Animal Health), also referred to herein as "commercial product" or "commercial vaccine". The prototype vaccine was produced in a manner similar to that described in Example 1 above, but with different volumes used for infection and longer incubation periods, as described in the section "Production of IgG:AVP8" below. The commercial product was used for the vaccine TGE / Rota was used according to the label instructions provided by the manufacturer (dosage and directions, as well as the recommended method for oral vaccination of pigs).

[0473] A total of 16 sows were included in the study. The sows were randomly divided into three treatment groups and a strict control group, as described in Table 1 below. The sows in T02 and T04 were mixed between three rooms. The sows in T06 and T07 were housed in two separate rooms. All sows were inoculated with appropriate substances by appropriate routes as listed in Table 1. The sows in T07 remained unvaccinated (strict control). Serum was collected regularly from sows throughout the vaccination period and evidence of seroconversion was determined. Fecal samples were collected before delivery and screened by RT-qPCR to confirm that the female livestock did not actively excrete rotavirus before delivery. The overall health status observations of each sow were recorded daily. Delivery was allowed to proceed naturally until the sows reached the 114th day of pregnancy. Thereafter, delivery was induced. At delivery, piglets were selected for the trial. Only piglets that were healthy at birth were marked, handled according to the facility standard operating procedures and included in the trial. When the pigs were zero to five days old, blood was drawn, fecal swabs were collected, and the pigs were challenged (excluding T07). During the challenge, a 5 mL dose of sodium bicarbonate was administered intragastricly to the pigs, followed by a 5 mL dose of the challenge material. During the entire challenge period, all animals were monitored daily for intestinal disease (diarrhea and behavioral changes). Fecal samples were collected regularly during the entire challenge period. Two days after the challenge (DPC 2), about one-third of the pigs from each litter were euthanized. After euthanasia, autopsies were performed and the macroscopic lesions of the pigs were evaluated. Intestinal sections were collected for microscopic and immunohistochemical evaluations. Intestinal swabs were collected for RT-qPCR evaluation. At DPC 21, all remaining pigs were weighed, blood was drawn, and fecal swabs were collected. After sample collection, the pigs were euthanized. The macroscopic lesions of the pigs were evaluated and intestinal swabs were collected.

[0474] Table 1: Study design

[0475]

[0476]

[0477] *IM = intramuscular, IN = intranasal

[0478] Throughout the study, serum VN titers from sows in T07 (strict control) remained constant or decreased, indicating a lack of exposure and a valid study (as described above in Example 1 to assess virus neutralization ("Protocol for Virus Neutralization Assay"), the results are shown in Figure 3 ). During the vaccination phase, the highest mean VN titers were observed in the sera of sows vaccinated with the IgG:AVP8 (T04) prototype vaccine. In this group, one dose administered six weeks before farrowing resulted in a four-fold or greater increase in titers by D14 in 3 / 5 animals in T04 (IgG:AVP8). Prior to pig challenge, 5 / 5 animals in T04 (IgG:AVP8) had a four-fold or greater increase in titers. During the vaccination phase, there was no significant increase (<2-fold) in serum VN titers of sows in the placebo group (T02). Until D35, there was no significant increase (<2-fold) in serum VN titers of sows in T06 (commercial vaccine). Prior to pig challenge, the titers of two sows in T06 (commercial vaccine) increased fourfold. After exocarpal exposure to the challenge substance, VN serum titers of sows in T02 (placebo) and T06 (commercial vaccine) increased. Conversely, VN serum titers remained constant or decreased in sows in T04 (IgG: AVP8) in 4 / 5 sows. Regarding colostrum and milk VN titers, in group T04 (IgG: AVP8), VN titers were highest at farrowing, decreased in pre-challenge samples and further decreased in post-challenge samples. In the placebo group (T02), VN titers were low before farrowing and challenge, but increased after exoskeleton exposure to the challenge agent.

[0479] VN titers in pig sera before challenge were high (>1280) in most pigs in T04 (IgG: AVP8), indicating passive transfer of immunity from sows to pigs. In contrast, most of the pigs in T02 (placebo) and T06 (commercial vaccine) had low titers (<1280).

[0480] Throughout the challenge phase, the highest number of deaths was observed in T02 (placebo), with 8 / 57 (14.0%) pigs dying. In contrast, only 1 / 46 (2.2%) pigs died in T04 (IgG: AVP8), 1 / 22 (4.5%) pigs died in T06 (commercial vaccine), and 1 / 27 (3.7%) pigs died in T07 (strict control). No clinical signs of diarrhea were observed in pigs in T07 (strict control) throughout the study. Clinical signs of diarrhea began in pigs in T02 (placebo) on day 1 or 2 after challenge (DPC) and resolved in most animals by DPC10. Overall, clinical signs of diarrhea were observed in 44 / 57 (77.2%) animals in T02 (placebo) at least once during the study. Of these 44 animals, diarrhea was considered severe in 29 (65.9%) animals. In contrast, clinical signs of diarrhea were reduced in pigs in T04 (IgG:AVP8). See Table 2 below for a summary of clinical diarrhea results for each group.

[0481] Table 2: Percentage of animals with abnormal diarrhea (ever) in each group

[0482]

[0483]

[0484] *Includes pigs with a score of 1 or 2 at least once during the study divided by the total number of pigs in each group

[0485] ** Includes pigs with a score of 2 at least once during the study divided by the total number of pigs ever abnormal

[0486] Prior to challenge, no rotavirus A RNA was detected by RT-qPCR, indicating a valid study. In addition, no rotavirus A RNA was detected by RT-qPCR in sows or pigs from T07 (strict control) throughout the study. In pigs following challenge, shedding was most common in T02 (placebo). In most pigs, shedding began on DPC1-3 and continued until DPC14. Of greatest concern was the reduction in shedding observed in T04 (IgG: AVP8) compared to T02 (placebo) and T06 (commercial vaccine). Both the percentage of shedding and the median amount of RNA detected were reduced (see Figure 4 , group median log rotavirus A RNA genome copies (gc) / mL stool) on study day; testing was performed as described below ("Protocol for Rotavirus A qRT-PCR").

[0487] A randomly selected subset of pigs from each group was euthanized and necropsied at DPC2. Pigs were evaluated for the presence of macroscopic intestinal lesions (thin-walled, gas-dilated small intestines, pure fluid content, etc.), microscopic lesions (atrophic enteritis), and rotavirus A specific staining by immunohistochemistry (IHC). Table 3 below presents the number of pigs with intestinal lesions at necropsy by group. The challenge was considered successful because 84.2% (16 / 19) of the pigs in the placebo group (T02) had macroscopic lesions and 63.2% (12 / 19) of those had staining. Of greatest concern was the lack of rotavirus A staining in only 1 / 15 pigs in T04 (IgG: AVP8). In addition, the percentage of pigs with macroscopic lesions in T04 (IgG: AVP8) was reduced compared to T02 (placebo) and the commercial product (T06).

[0488] Table 3. Percentage of animals with intestinal lesions and IHC staining at necropsy by group.

[0489]

[0490] *Represents the number of pigs with intestinal lesions at DPC2 divided by the total number of pig necropsies at DPC2

[0491] ** Score 1 = <10% of villi contain antigen, Score 2 = 10% to 50% of villi contain antigen, Score 3 = >50% of villi contain antigen

[0492] §Not applicable, as pigs from T07 were not necropsy-examined

[0493] The average daily weight gain (in kg) of the surviving pigs was calculated and is presented in Table 4 below. The highest numerical benefit in ADWG was observed in pigs from T04 (IgG: AVP8). The increase in ADWG after vaccination was significantly different compared to T02 (placebo).

[0494] Table 4. Mean daily weight gain in kg (standard deviation) by group.

[0495]

[0496] In summary, vaccination of conventional sows with the IgG:AVP8 prototype vaccine (comprising the polypeptide of SEQ ID NO: 12) six weeks and two weeks prior to farrowing produced higher neutralizing antibody titers in sow serum and colostrum. These neutralizing antibodies were passively transmitted to pigs after birth, as demonstrated by the detection of high titers (>1280) in pig serum from vaccinated sows. The presence of high neutralizing antibody titers in pigs resulted in clinical protection. Specifically, pigs born from vaccinated sows had reduced fecal excretion of rotavirus A RNA, reduced mortality, reduced clinical signs of diarrhea, reduced colonization of rotavirus A at DPC2, reduced macroscopic lesions at DPC2, and increased ADWG compared to pigs born from placebo controls and commercial vaccines.

[0497] Protocol for rotavirus A qRT-PCR

[0498] To measure rotavirus A RNA in stool samples, a quantitative one-step RT-PCR kit (iTaq Universal One-Step RT-PCR Kit; BioRad, catalog number 1725140) was used. For information on primers and probes, see Table 5 below.

[0499] Table 5: Primer (F / R) and probe (Pr1 / Pr2) information

[0500]

[0501] Real-time RT-PCR was performed in a 20 μl reaction containing 5 μl of extracted total nucleic acid, 1 μl of each probe (5 μM), 1 μl of each primer (10 μM), 10 μl of 2×RT-PCR mixture, 0.5 μl of iScript reverse transcriptase, and 0.5 μl of DEPC-treated water. The reaction was performed using a CFX96 real-time PCR detection system (BioRad) under the following conditions: initial reverse transcription at 50°C for 10 min, followed by initial denaturation at 95°C for 3 min, 40 cycles of denaturation at 95°C for 15 s, and annealing and extension at 60°C for 45 s. To generate relative quantitative data, serial dilutions of two rotavirus Ag blocking solutions were included in each run. 5.0×10 7 Each of the g blocking solutions was included in the run with 100 genome copies / microliter as the starting concentration. Optical data were analyzed using CFX Manager software. For each assay, the critical value line was automatically calculated using the regression setting of the cycle critical value (Ct) assay mode. Baseline subtraction was automatically performed using the baseline subtracted mode. Curves with baseline end values ​​less than 10 were manually corrected.

[0502] Production of IgG:AVP8

[0503] 1.7 mL of rotavirus A VP8 core-swine IgG Fc fusion protein (BaculoGold (BG) / pVL1393-AVP8-IgG; 1.18×10 8 TCID50 / mL) of recombinant baculovirus stock was used to infect approximately 1×10 6 2L Sf+ (Spodoptera frugiperda) cells at a concentration of cells / mL. The shake flask was incubated for five days at 28°C ± 2°C under continuous stirring at 90rpm. The cells and culture medium were aseptically transferred to 3×1L centrifuge bottles, and the cells were precipitated at 10,000g for 20 minutes at 4°C. The resulting supernatant was passed through a 0.2 μm filter (Thermo Scientific, catalog number 567-0020), and then incubated overnight with 2.5mL of MabSelect SuRe LX protein A resin (GE Healthcare, catalog number 17-5474-01) at 4°C under moderate stirring. The resin was recovered by 0.2 μm filtration (Thermo Scientific, catalog number 567-0020), and then washed with a gentle Ag / Ab binding buffer (Thermo Scientific, catalog number 21012) with a volume of 12×10mL. AVP8-IgG was eluted from the resin using 7×10 mL volumes of Mild Ag / Ab Elution Buffer (Thermo Scientific, Cat. No. 21027). AVP8-IgG was dialyzed against 3.5 L of 20 mM Tris pH 7.5, 150 mM NaCl with one buffer change. Residual baculovirus was inactivated with 5 mM BEI at 37°C for 24 hours. The resulting material was diluted to a target concentration of 70 μg / mL in 1×PBS (Gibco, Cat. No. 10010-023). The diluted material was formulated with 12.5% ​​Emulsigen D.

[0504] Example 3

[0505] Serological studies:

[0506] The primary objective of this study was to evaluate whether administration of a prototype vaccine (including AVP8-IgG Fc protein (SEQ ID NO: 12)) and a control vaccine (referred to herein as "placebo") to conventional sows generates a serological response against rotavirus A. The prototype vaccine (comprising Emulsigen D or Carbopol as adjuvant, see Tables 7A and 7B below), also referred to herein as "IgG-AVP8" was produced in a manner similar to that described above in Examples 1 and 2, but with different volumes used for infection and longer incubation periods, as described below in the section "Vaccine Production: IgG-AVP8".

[0507] A total of 20 sows were included in the study. The sows were randomly divided into four treatment groups, as described in Table 6 below. Throughout the study, the sows were mixed. All sows were intramuscularly vaccinated with appropriate materials at D0 and D21, as listed in Table 4. Serum was collected from sows regularly throughout the study and evidence of seroconversion was determined by virus neutralization assays. Observations of the overall health status of each sow were recorded daily. The study was terminated at D42.

[0508] Table 6: Study design

[0509]

[0510] *IM = Intramuscular

[0511] Throughout the study, serum VN titers from sows in T06 and T07 (placebo groups) remained constant or decreased, indicating a lack of exposure and efficacy studies (virus neutralization was assessed as described above in Example 1 ("Protocol for Virus Neutralization Assay"), with modifications of increasing dilutions evaluated - 1:40 to 1:40,960). During the vaccination phase, titers increased significantly (>4-fold) in sows vaccinated with the IgG-AVP8 / Emulsigen D (T02) and IgG-AVP8 / Carbopol (T03) prototype vaccines. For both groups (T02 and T03), the mean titers in each group were above 640 after one vaccination and remained above 640 throughout the study period. In contrast, sows in the placebo groups (T06 and T07) had no significant increase (<2-fold) in serum VN titers throughout the study.

[0512] In conclusion, vaccination of conventional sows with the IgG-AVP8 prototype vaccine (comprising the polypeptide of SEQ ID NO: 12) six and two weeks before farrowing produced high neutralizing antibody titers in sow sera.

[0513] Vaccine production: IgG-AVP8

[0514] For an MOI of 0.22, 1.19 × 108 15 mL BG / pVL1393-AVP8-IgG infected 8 L of a jacketed 10 L Sartorius Biostat B glass vessel with TCID50 / mL of 1.00 × 10 6 The Sf+ cells of individual cells / mL. The bioreactor was operated at 27°C under 100rpm stirring and bubbling oxygen at 0.3slpm. The container was harvested at 6DPI, centrifuged at 10,000g and 4°C for 20 minutes, and the supernatant was filtered with 0.8 / 0.2μm (GE Healthcare, catalog number 6715-7582). 2750mL of the clear supernatant was inactivated for five days with 5mM BEI at 27°C. After the residual BEI was neutralized with sodium thiosulfate, 2750mL was concentrated to about 12× to 225mL using a 10kDa hollow fiber filter (GE, catalog number UFP-10-C-4MA). The concentration was determined to be 255μg / mL.

[0515] Table 7A. Vaccine formulations

[0516] Components Target volume concentration AVP8-IgG protein antigen 16.5mL 27.5％ PBS Dilution 31.5mL 52.5％ Carbopol Adjuvant 12mL 20％

[0517] Table 7B. Vaccine formulations

[0518] Components Target volume concentration AVP8-IgG protein antigen 16.5mL 27.5％ PBS Dilution 36mL 60％ Emulsigen D Adjuvant 7.5mL 12.5％

[0519] Example 4

[0520] The primary objective of this study was to evaluate whether animals vaccinated with IgG-AVP8 (including AVP8-IgG Fc protein (SEQ ID NO: 12)) could cross-neutralize various rotavirus A serotypes / genotypes of various G and P types except P[7], from which the AVP8-IgG Fc protein was designed. This would indicate the ability of the AVP8-IgG Fc protein (SEQ ID NO: 12) to be protective against other isolates.

[0521] Briefly, heat-inactivated sera from pigs vaccinated with IgG-AVP8 were diluted 2-fold in MEM starting at 1:200 in dilution blocking buffer in columns A to G. Column H contained no sera. In another dilution blocking buffer, various rotavirus A types G and P were present at 6.0 Log from rows 1 to 11. 10 TCID 50 / mL dilution 1.5 times. The 12th row does not contain virus. 250 μL of virus and 250 μL of serum from the corresponding wells are combined and incubated at 37°C for 1 hour. After incubation for 1 hour, 100 μL of virus-serum mixture is overlaid on a monolayer of MA104 cells and incubated at 37°C for 72 hours and stained by IFA and read for the presence of virus. The presence of virus is recorded as '+' on the plate and the lack of virus is recorded as '0". These results are then transferred to Table 8.

[0522] The following six rotavirus A isolates were compared to this assay: G9P[7], G9P

[23] , G4P

[23] , G3P[7], G5P[7], and G4P[7]. The results in Table 1 indicate that the P type P

[23] cross-neutralizes P[7]. All G types including P[7] or P

[23] also neutralize, indicating that the G types are not significant in the neutralization of the virus in this assay.

[0523] Table 8: Study design and results

[0524]

[0525]

[0526]

[0527] In conclusion, animals vaccinated with IgG-AVP8, including AVP8-IgG Fc protein (SEQ ID NO: 12), cross-neutralized rotavirus genotypes P[7] and P

[23] . The G type played a significant role in virus neutralization.

[0528] Example 5

[0529] Proof of concept experiment in pigs:

[0530] A total of 40 animals were used in this study. Pigs were randomized into four treatment groups of 10 pigs per group. Pigs were pooled throughout the study. General health observations, pre-screening serum samples, and pre-screening fecal samples were obtained prior to treatment to confirm animal health, determine baseline serological responses to rotavirus A, and confirm the absence of active rotavirus A infection prior to or at the time of vaccination. On study day 0 (D0), animals were vaccinated intramuscularly with the following: T01: IgG-P[7]AVP8 vaccine (comprising the polypeptide of SEQ ID NO: 12); T02: IgG-P

[13] AVP8 vaccine (comprising the polypeptide of SEQ ID NO: 14); T03: P[7]AVP8-IgG-P

[13] AVP8 vaccine (comprising the polypeptide of SEQ ID NO: 16); T04: placebo. Serum samples were collected on study days 0, 7, 14, 21, 28, 36, 42, and 49. All animals were humanely euthanized at necropsy on study D49. Serum samples were tested by virus neutralization assay to determine serological responses to the vaccine prototypes over time. Animals vaccinated with T01 had antibodies neutralizing rotavirus genotypes P[7] and P

[23] , animals vaccinated with T02 had antibodies neutralizing rotavirus genotype P

[13] , and animals vaccinated with T03 had antibodies neutralizing rotavirus genotypes P[7], P

[13] , and P

[23] .

[0531] Example 6

[0532] SDS PAGE:

[0533] In the presence and absence of DTT ( Figure 5 A)) SDS-PAGE of Protein A Purified AVP8-IgG Fc Protein (SEQ ID NO: 12) Product: The method for generating samples for SDS-PAGE imaging is briefly described as follows: The supernatant was harvested for 36 hours at 37°C by inactivating baculovirus with 10 mM BEI and then neutralized. The samples were then purified using Protein A resin. All samples were then denatured using NuPAGE 4×LDS sample buffer (Invitrogen, catalog number NP0007) with 25 mM DTT (final) or an equal volume of water and heated at 95°C for 10 minutes. The samples were run on a 4%-12% SDS-PAGE gel (Invitrogen, catalog number NP0335BOX) at 180V for 45 minutes and stained (eStain L1, GenScript catalog number M00548-1; destain catalog number M00549-1).

[0534] Thus, it was found that in the band with reduced (+DTT (dithiothreitol)) samples, mainly one band was seen (monomeric AVP8-IgG Fc protein, which is considered in conjunction with the results of the Western Blot described below). Other bands were visible in the band run with the non-reduced sample (-DTT). The molecular weight range of the additional bands was each a multiple of the monomer.

[0535] Western Blot:

[0536] Anti-swine IgG Fc fragment Western blot ( Figure 5 B)): AVP8-IgG Fc protein (SEQ ID NO: 12) product produced in the bioreactor was harvested with 1 mL sample before BEI addition. The sample was centrifuged at 20,000g and 4°C for 5 minutes, the supernatant was decanted into a new tube, and the pellet and supernatant were stored at -70°C. The pellet and supernatant were thawed, the pellet was resuspended in 1 mL 8M urea, and then equal amounts of the pellet and supernatant were run on SDS-PAGE under reducing conditions (+DTT) and transferred to PVDF membrane. Western Blot was probed with a 1:1000 dilution of HRP-conjugated goat anti-swine to detect porcine IgG Fc fragment.

[0537] Thus, unexpectedly, no AVP8-IgG Fc protein was found in the cell pellet samples. In fact, advantageously all of the AVP8-IgG Fc protein (SEQ ID NO: 12) was found in the cell culture supernatant samples.

[0538] Example 7

[0539] Generation of consensus sequences:

[0540] The consensus sequences of SEQ ID NO:4 (based on genotype P[6] rotavirus VP8 protein) and SEQ ID NO:5 (based on genotype P

[13] rotavirus VP8 protein) were generated as described below:

[0541] Sequences were compiled from publicly available porcine rotavirus VP4 nucleotide sequences from the NCBI Virus Variation Database and internally derived rotavirus isolate sequences. Additional metadata for the sequences were also compiled, including metadata for the following: isolate name, isolate P type, geographic origin, and isolation date when available. The nucleotide sequences were translated into protein sequences and aligned to known VP8 proteins using the MUSCLE sequence alignment software UPGMB clustering method and default gap penalty parameters. Unaligned VP5 amino acids were trimmed and discarded. The VP8 aligned protein sequences were imported into the MEGA7 software for phylogenetic analysis, and neighbor-joining phylogenetic reconstructions were generated based on the VP8 protein sequences. The optimal tree (n=100) was calculated and scaled using the Poisson correction method and the self-help re-drawing test of phylogeny, where the branch length was equal to the evolutionary distance in units of amino acid substitutions per site at a total of 170 positions. Nodes with a self-help re-drawing clustering association greater than 70% were considered significant. Nodes with about 10% distance and greater than 70% self-help re-extraction cluster association are designated as clusters. Outlier sequences that are not fitted into large clusters are evaluated independently for sequence quality and P-type sources. Suspected low-quality sequences are removed from the analysis, and sequences from P-types that are rarely observed in porcine rotavirus are retained. Clusters that are selected for the generation of consensus sequences are selected based on the required product protection profile and in vitro serum cross neutralization studies. Consensus sequences are generated according to the maximum frequency of each comparison position, where equal proportions of amino acids are observed in the comparison position, amino acid residues are selected based on reported epidemiological data and product protection profiles.

[0542] Example 8

[0543] Anti-virus research:

[0544] The primary objective of this study was to evaluate whether administration of a prototype vaccine, also referred to herein as "IgG#AVP8", comprising an AVP8-IgG Fc protein (SEQ ID NO: 12), and an unrelated control vaccine, referred to herein as "placebo", to conventional dams, provided passive protection to pigs against a virulent rotavirus A challenge. The prototype vaccine was produced in a manner similar to that described in Example 1 above, but with different volumes used for infection and different purification methods, as described in the section "Production of IgG#AVP8" below.

[0545] A total of 20 females were included in the study. The females were randomly divided into two treatment groups and a strict control group, as described in Table 9 below. The females in T01 and T03 were mixed between three rooms. The females in T07 were housed in a separate room. All females were inoculated with appropriate substances by appropriate routes as listed in Table 9. The females in T07 remained unvaccinated (strict control). Serum was regularly collected from females during the entire vaccination period and evidence of seroconversion was determined. Fecal samples were collected before delivery and screened by RT-qPCR to confirm that the females did not actively excrete rotavirus before delivery. The overall health status observations of each sow were recorded daily. Delivery was allowed to proceed naturally until the sows reached the 114th day of pregnancy. Thereafter, delivery was induced. At delivery, piglets were selected for the trial. Only piglets that were healthy at birth were marked, handled according to the facility standard operating procedures and included in the trial. When the pigs were one to five days old, blood was drawn, fecal swabs were collected, and the poison pigs (excluding T07) were challenged. At the time of the challenge, a 5 mL dose of sodium bicarbonate was administered intragastricly to the pigs, followed by a 1 mL dose of the challenge substance. During the entire challenge period, all animals were monitored daily for intestinal disease (diarrhea and behavioral changes). Fecal samples were collected one day after the challenge (DPC1). At DPC2, all pigs in T01 and T03 were euthanized. Intestinal sections were collected for microscopic and immunohistochemical evaluations.

[0546] Table 9: Study Design

[0547]

[0548] *IM = Intramuscular

[0549] Throughout the study, serum VN titers from dams T07 (strict control) increased less than 4-fold, indicating a lack of exposure and a valid study (as described above in Example 1 ("Protocol for Virus Neutralization Assay") to assess virus neutralization, the results are shown in Tables 10 and Figure 6 During the vaccination phase, the highest mean VN titers were observed in the serum of dams vaccinated with the prototype vaccine IgG#AVP8 (group T03). In this group, one dose administered six weeks before farrowing resulted in a four-fold or greater increase in titers by D14 in 6 / 8 animals in T03 (IgG#AVP8). None of the dams in group T01 (placebo) had a significant increase in serum VN titers (<2-fold) during the vaccination phase. Maternal colostrum VN titers: Dams in group T03 (IgG#AVP8) had higher mean VN titers than dams in group T01 (placebo).

[0550] Table 10: VN results

[0551]

[0552]

[0553] *DOF = Day of Delivery

[0554] VN titers in pig sera before challenge were high (>1280) in most pigs in group T03 (IgG#AVP8), indicating passive transfer of immunity from the dam to the pigs. In contrast, most of the titers were low (<1280) in pigs in T02 (placebo).

[0555] In groups T01 (placebo) and T03 (IgG#AVP8), pigs were defined as infected if rotavirus antigen was detected by immunohistochemistry (IHC) in at least one intestinal section and the animal had an abnormal fecal score for at least one day after challenge. The frequency distribution is listed in Table 11 below. Based on the use of this diagnostic criteria, vaccination of dams with the prototype vaccine IgG#AVP8 (group T03) 6 weeks and 2 weeks before farrowing protected against rotavirus-associated disease in pigs following challenge with heterologous rotavirus AP[7] challenge material; prevention fraction 0.926, 95% confidence interval 0.734, 0.979.

[0556] Table 11. Frequency distribution of diagnostic criteria

[0557]

[0558] *Diagnostic criteria: Pigs were considered infected if one or more of the ileal or jejunal tissue samples were IHC positive for rotavirus A (score > 0) and had at least one abnormal fecal score on any day after challenge. Score 0 = uninfected; 1 = infected

[0559] In summary, vaccination of conventional dams with the prototype vaccine IgG#AVP8 (comprising a polypeptide of SEQ ID NO: 12) six weeks and two weeks prior to farrowing resulted in high neutralizing antibody titers in sow serum and colostrum. These neutralizing antibodies were passively transmitted to pigs after birth, as demonstrated by the detection of high titers (>1280) in pig serum from vaccinated dams. The presence of high neutralizing antibody titers in pigs resulted in clinical protection. Specifically, fewer pigs born from vaccinated dams were considered infected compared to pigs born from placebo controls.

[0560] Production of IgG#AVP8

[0561] Two 10 L jacketed Sartorius Biostat B glass vessels were placed at 1.00 × 10 63L Sf+ cells were inoculated with 10 cells / mL. Three days after inoculation, each container was infected with an MOI of 0.1 and the volume of each container was adjusted to 8L using Ex-cell 420 serum-free medium (SAFC catalog number 14420C-1000mL). The bioreactor was operated at 27°C under 100rpm stirring, with dissolved oxygen set at 40% or higher, and CCA coating at 1.3slpm. The container was collected 7 days after inoculation; the fluid was centrifuged for 20 minutes at 10,000g at 4°C, and the supernatant was filtered at 0.8 / 0.2 μm (GE Healthcare, catalog number 6715-7582). Clarified supernatant (8 liters / container) was inactivated for three days with 5mM BEI in a jacketed Sartorius Biostat B glass container at 37°C. After inactivation, residual BEI was neutralized with sodium thiosulfate. After neutralization, 7000 mL was concentrated approximately 10× to 700 mL using a 10 kDa hollow fiber filter (GE, catalog number UFP-10-C-5A). The concentrated material was diafiltered with 5 volumes (3500 mL) of 1× PBS. The vaccine was formulated with 12.5% ​​Emulsigen D, 28% concentrated material, and 59.5% 1× PBS (vol:vol).

[0562] Example 9

[0563] Proof of concept experiment in pigs:

[0564] A total of 20 animals were used for this study. Pigs were randomized into two treatment groups of 10 pigs each. Pigs were pooled throughout the study. General health observations, pre-screening serum samples, and pre-screening fecal samples were obtained before treatment to confirm animal health, determine baseline serological responses to rotavirus C, and confirm no active rotavirus C infection before or at the time of vaccination. On study day 0 (D0) and D28, animals were vaccinated intramuscularly with the following: T01: IgG-P CVP8 vaccine (polypeptide comprising SEQ ID NO: 15); T02: placebo. Serum samples were collected on study days 0, 7, 14, 21, 28, 36, and 42. All animals were humanely euthanized at necropsy on study D42. Serum samples were tested by ELISA to determine serological responses to the vaccine prototype over time. The average level of anti-rotavirus C antibodies in animals vaccinated with T01 was higher than that in animals vaccinated with T02, and their titers did not increase.

[0565] Example 10

[0566] Proof of concept experiment in pigs:

[0567] The primary objective of this study was to evaluate whether administration of a prototype vaccine, also referred to herein as "IgG:AVP8P[6,7,13]", comprising three AVP8-IgG Fc proteins (SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14), and an unrelated control vaccine, referred to herein as "placebo", to conventional dams provided passive protection to pigs against a virulent rotavirus A challenge. The components of the prototype vaccine were produced in a manner similar to that described in Example 1 above and subsequently mixed as described in the section "Production of IgG:AVP8P[6,7,13]" below.

[0568] A total of 32 females were included in the study. The females were randomly divided into two treatment groups and a strict control group, as described in Table 12 below. The females in T01 and T03 were mixed between seven rooms. The females in T04 were housed in separate rooms. All females were vaccinated with appropriate substances by appropriate routes as listed in Table 12. The females in T04 remained unvaccinated (strict control). Serum was collected regularly from the females during the entire vaccination period and evidence of seroconversion was determined by virus neutralization (VN) and enzyme-linked immunosorbent assay (ELISA). Fecal samples were collected before delivery and screened by RT-qPCR to confirm that the females did not actively excrete rotavirus before delivery. During this study, RT-qPCR was performed as described in Example 2 ("Protocol of Rotavirus A qRT-PCR") above. The overall health observations of each sow were recorded daily. Parturition was allowed to proceed naturally until the sows reached the 115th day of pregnancy. Thereafter, parturition was induced. At parturition, piglets were selected for the trial. Only piglets that are healthy at birth are marked, processed according to facility standard operating procedures and included in the experiment. When pigs are two to five days old, blood is drawn, fecal swabs are collected, and pigs are challenged (excluding T04). During the challenge, 5 mL of sodium bicarbonate is administered intragastricly to the pigs, followed by 1 mL of the challenge material intragastricly. Throughout the challenge period, all animals are monitored daily for intestinal disease (diarrhea and behavioral changes). Fecal samples are collected one day after the challenge (DPC1) and DPC3. At DPC3, all pigs in T01 and T03 are euthanized. Intestinal sections are collected for microscopic and immunohistochemical evaluation.

[0569] Table 12: Study Design

[0570]

[0571] *IM = Intramuscular

[0572] Throughout the study, serum VN titers from dams T04 (strict control) increased 4-fold or less, indicating a lack of exposure and a valid study (as described below ("Protocol for Virus Neutralization Assay") to assess virus neutralization, the results are shown in Tables 13 and Figure 7 During the vaccination period, none of the dams in group T01 (placebo) had a significant increase in serum VN titers (≤2-fold). On D14, D21, D27, and D47, VN titers in the serum of dams vaccinated with the prototype vaccine IgG: AVP8 P[6,7,13] (group T03) were significantly higher (p<0.01) compared to dams vaccinated with placebo (group T01). The mean colostrum VN titers of dams in group T03 (IgG: AVP8 P[6,7,13]) were also significantly higher compared to dams in group T01 (placebo).

[0573] Table 13: VN results

[0574]

[0575]

[0576] VN titers in pig sera before challenge were high (>1280) in most pigs in group T03 (IgG: AVP8 P[6,7,13]), indicating passive transfer of immunity from the dam to the pigs. In contrast, none of the pigs in T01 (placebo) had high VN titers (>1280).

[0577] Similar to the VN data, increased amounts of anti-rotavirus AVP8 P[6] and anti-rotavirus AVP8 P

[13] antibodies were detected at D47 in dams vaccinated with the IgG:AVP8 P[6,7,13] prototype (group T03), but not in dams vaccinated with the placebo (group T01). These assays were performed as described below ("Protocol for IgG:AVP8 P

[13] ELISA" and "Protocol for IgG:AVP8 P[6] ELISA", respectively). The results are shown in Figure 8 and Fig. 9 middle.

[0578] In groups T01 (placebo) and T03 (IgG: AVP8 P[6,7,13]), pigs were defined as infected if rotavirus antigen was detected by immunohistochemistry (IHC) in at least one intestinal section and the animal had an abnormal fecal score for at least one day after challenge. The frequency distribution is listed in Table 14 below. Based on the use of this diagnostic criteria, vaccination of dams with the prototype vaccine IgG: AVP8 P[6,7,13] (group T03) 6 weeks and 2 weeks before farrowing protected against rotavirus-associated disease in pigs following challenge with heterologous rotavirus AP[7] challenge material; fraction of protection 0.567, 95% confidence interval -0.086, 0.827.

[0579] Table 14. Frequency distribution of diagnostic criteria

[0580]

[0581] *Diagnostic criteria: If one or more of the ileal or jejunal tissue samples are positive for rotifer disease

[0582] Virus A IHC positive (score>0) and at least one abnormal stool on any day after challenge

[0583] If the stool score is lower than 0, the pig is considered infected. Score 0 = not infected; 1 = infected

[0584] In summary, vaccination of conventional dams six and two weeks prior to farrowing with the prototype vaccine IgG: AVP8 P[6,7,13] (polypeptides comprising SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14) resulted in high neutralizing antibody titers in the serum and colostrum of sows. These neutralizing antibodies were passively transmitted to pigs after birth, as demonstrated by the detection of high titers (>1280) in pig sera from vaccinated dams. The presence of high neutralizing antibody titers in pigs resulted in clinical protection. Specifically, fewer pigs born to vaccinated dams were considered infected compared to pigs born to placebo controls.

[0585] Production of IgG:AVP8 P[6,7,13]

[0586] The prototype vaccine contained three fractions. The production of each fraction was performed separately and described below. The vaccine was formulated with 12.0% Emulsigen D, 7% IgG:AVP8 P[7] fraction, 4.4% IgG:AVP8 P[6] fraction, 1.8% IgG:AVP8 P

[13] fraction and 47.8% 1×PBS (vol:vol).

[0587] IgG:AVP8 P[7] fraction: 0.75×10 63L Sf+ cells of 10L Sartorius Biostat B glass container with 10 cells / mL were inoculated. Two days after inoculation, the container was infected with an MOI of 0.1 and the volume of the container was adjusted to 8L using Ex-cell 420 serum-free medium (SAFC catalog number 14420C-1000mL). The bioreactor was operated at 27°C under 100rpm stirring, with dissolved oxygen set to 40% or higher, and CCA coating at 1.3slpm. The container was collected 7 days after inoculation; the fluid was centrifuged at 10,000g for 20 minutes at 4°C, and the supernatant was filtered at 0.8 / 0.2μm (GE Healthcare, catalog number 6715-7582). The clarified supernatant (8 liters / container) was inactivated with 5mM BEI for three days in a Sartorius Biostat B glass container with a jacket at 37°C. After inactivation, the residual BEI was neutralized with sodium thiosulfate. After neutralization, approximately 7000 mL was concentrated approximately 10× to 700 mL using a 10 kDa hollow fiber filter (GE, Cat. No. UFP-10-C-5A). The concentrated material was diafiltered with 5 volumes (3500 mL) of 1× PBS.

[0588] IgG:AVP8 P[6] fraction: 0.75×10 6 3L Sf+ cells of 10L Sartorius Biostat B glass container with 10 cells / mL were inoculated. Two days after inoculation, the container was infected with an MOI of 0.1 and the volume of the container was adjusted to 8L using Ex-cell 420 serum-free medium (SAFC catalog number 14420C-1000mL). The bioreactor was operated at 27°C with 100rpm stirring, wherein the dissolved oxygen was set to 40% or above, and the CCA coating was at 1.3slpm. The container was collected 8 days after inoculation; the fluid was centrifuged at 10,000g for 20 minutes at 4°C, and the supernatant was filtered at 0.8 / 0.2μm (GE Healthcare, catalog number 6715-7582). The clarified supernatant (8 liters / container) was inactivated for 24 hours with 10mM BEI in a Sartorius Biostat B glass container with a jacket at 37°C. After inactivation, the residual BEI was neutralized with sodium thiosulfate. After neutralization, approximately 7000 mL was concentrated approximately 10× to 600 mL using a 10 kDa hollow fiber filter (GE, Cat. No. UFP-10-C-5A). The concentrated material was diafiltered with 5 volumes (3500 mL) of 1× PBS.

[0589] IgG:AVP8 P

[13] fraction: 0.75×10 63L Sf+ cells of 10 cells / mL were inoculated into a jacketed 10L Sartorius Biostat B glass container. Two days after inoculation, the container was infected with an MOI of 0.1 and the volume of the container was adjusted to 8L using Ex-cell 420 serum-free medium (SAFC catalog number 14420C-1000mL). The bioreactor was run at 27°C with 100rpm stirring, with dissolved oxygen set at 40% or above, and CCA coating at 1.3slpm. The container was collected 7 days after inoculation; the fluid was centrifuged at 10,000g for 20 minutes at 4°C, and the supernatant was filtered at 0.8 / 0.2μm (GE Healthcare, catalog number 6715-7582). The clarified supernatant (8 liters / container) was inactivated for 24 hours in a jacketed Sartorius Biostat B glass container at 37°C with 10mM BEI. After inactivation, the residual BEI was neutralized with sodium thiosulfate. After neutralization, approximately 7000 mL was concentrated approximately 10× to 750 mL using a 10 kDa hollow fiber filter (GE, Cat. No. UFP-10-C-5A). The concentrated material was diafiltered with 5 volumes (3500 mL) of 1× PBS.

[0590] IgG:AVP8 P

[13] ELISA protocol:

[0591] Nuc MaxiSorb 96-well ELISA plates (Thermo) were coated with rotavirus AVP8 P

[13] purified protein diluted 1:10 in 1×PBS. The plates were incubated at 37°C for 1 hour. After incubation, the plates were washed with 1×PBST and then blocked with 3% BSA in PBST for 1 hour at 37°C. After washing, 100 μL of test serum diluted to a final dilution of 1:512 in 1×PBS was added to the plates and incubated at 37°C for 1 hour. After washing, the wells were coated with 100 μL of a 1:25,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-swine IgG antibody (Jackson ImmunoResearch) and incubated at 37°C for one hour. After washing, the plates were developed with 3,5,3',5'-tetramethylbenzidine (1-Step UltraTMB-ELISA, Thermo) for 10 minutes at room temperature and the reaction was stopped with stop solution (Sigma) before optical density (OD) measurement at 450 nm. Samples including positive and negative controls were run in duplicate wells and the results were reported as the average of the (sample-negative control) to (positive-negative control) ratio (SN) / (PN).

[0592] IgG:AVP8 P[6]ELISA protocol:

[0593] Nuc MaxiSorb 96-well ELISA plates (Thermo) were coated with rotavirus AVP8 P[6] purified protein diluted 1:10 in 1×PBS. The plates were incubated at 37°C for 1 hour. After incubation, the plates were washed with 1×PBST and then blocked with 3% BSA in PBST for 1 hour at 37°C. After washing, 100 μL of test serum diluted to a final dilution of 1:512 in 1×PBS was added to the plates and incubated at 37°C for 1 hour. After washing, the wells were coated with 100 μL of a 1:25,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-swine IgG antibody (Jackson ImmunoResearch) and incubated at 37°C for one hour. After washing, the plates were developed with 3,5,3',5'-tetramethylbenzidine (1-Step UltraTMB-ELISA, Thermo) for 10 minutes at room temperature and the reaction was stopped with stop solution (Sigma) before optical density (OD) measurement at 450 nm. Samples including positive and negative controls were run in duplicate wells and the results were reported as the average of the (sample-negative control) to (positive-negative control) ratio (SN) / (PN).

[0594] Protocol for virus neutralization assay

[0595] All serum and milk samples were heat inactivated at 56°C for 30 minutes. Samples were serially diluted from 1:40 to 1:40,960 in rotavirus growth medium (MEM + 2.5% HEPES + 0.3% trypsin phosphate broth + 0.02% yeast + 10 μg / mL trypsin). Antibiotics were added to the medium for milk sample testing. Rotavirus A isolates (titer approximately 6.5 log TCID 50 / mL) was diluted 1:78,000 into rotavirus growth medium. A total of 250 μl of diluted serum was added to 250 μl of diluted virus; the mixture was incubated for one hour at 37°C ± 5% CO2. Growth medium was aseptically removed from a three- to four-day-old 96-well plate seeded with MA104 cells. After incubation, 200 μl of the virus-serum mixture was transferred to the cell plate. Cells were incubated at 37°C ± 5% CO2 for 3-4 days. The stock solution and diluted virus were titrated on the day of use to determine the dilution used in the assay. After incubation, the supernatant was discarded. For fixation, 100 microliters / well of 50% / 50% acetone / methanol was added. The plate was incubated at room temperature for 15 minutes, air-dried, and then rehydrated with 100 microliters / well 1× PBS. The primary antibody (rabbit anti-rotavirus A polyclonal serum, produced in-house) was diluted 1:1000 in 1× PBS. Add 100 μl / well of diluted primary antibody and incubate the plate at 37°C ± 5% CO2 for one hour. After incubation, wash the plate twice with 100 μl / well 1× PBS. Dilute the secondary antibody (Jackson ImmunoResearch FITC-labeled goat anti-rabbit IgG, catalog number 111-095-003) at 1:100 in 1× PBS. Add 100 μl / well of diluted secondary antibody and incubate the plate at 37°C ± 5% CO2 for one hour. After incubation, wash the plate twice with 100 μl / well 1× PBS. Read the plate for the presence of fluorescence using a UV microscope. If the titer of the diluted virus (generated using the Reed-Muench method) is found to be 2.8±0.5log TCID 50 / mL, the assay was considered valid. In addition, known positive and negative samples were included in each assay as controls. Serum titers are reported as the highest dilution where no staining was observed.

[0596] Embodiment 11

[0597] Proof of concept experiment in pigs:

[0598] The primary objective of this study was to evaluate whether administration of a prototype vaccine, also referred to herein as "IgG:AVP8P[6,7,13]+IgG:CVP8," including three AVP8-IgG Fc proteins (SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14) and one CVP8-IgG Fc protein (SEQ ID NO: 15), and an unrelated control vaccine, referred to herein as "placebo," to conventional dams provided passive protection to pigs against virulent rotavirus A challenge. A total of 24 dams were included in the study. The dams were randomized into two treatment groups and a strict control group, as described in Table 15 below. Dams in T01 and T02 were intermixed between the three rooms. Dams in T03 were housed in separate rooms. All dams were vaccinated with the appropriate substances by the appropriate route as listed in Table 15. Dams in T03 remained unvaccinated (strict control). Serum was collected from the dams regularly throughout the vaccination period and evidence of seroconversion was determined. Fecal samples were collected before delivery and screened by RT-qPCR to confirm that the sows did not actively excrete rotavirus before delivery. The overall health observations of each sow were recorded daily. Delivery was allowed to proceed naturally until the sows reached the 114th day of gestation. Thereafter, delivery was induced. At delivery, piglets were selected for the trial. Only piglets that were healthy at birth were marked, processed according to the facility standard operating procedures and included in the trial. When the pigs were one to five days old, blood was drawn, fecal swabs were collected, and the pigs were challenged (excluding T03). At the time of challenge, 5 mL of sodium bicarbonate was administered to the pigs' stomachs, followed by 1 mL of the challenge substance administered intragastricly. Throughout the challenge period, all animals were monitored daily for intestinal disease (diarrhea and behavioral changes). Fecal samples were collected one day after the challenge (DPC1). At DPC3, all pigs in T01 and T02 were euthanized. Intestinal sections were collected for microscopic and immunohistochemical evaluation. Dams in group T01 (IgG:AVP8 P[6,7,13]+IgG:CVP8) had higher mean antibody levels against rotavirus A genotypes PP[6], P[7] and P

[13] , and rotavirus C compared to vaccinated T02 (placebo) animals that did not have titer increases. Dams in group T01 (IgG:AVP8 P[6,7,13]+IgG:CVP8) had fewer infected piglets (determined by clinical signs and immunohistochemical evaluation) compared to dams in group T02 (placebo).

[0599] Table 15: Study Design

[0600]

[0601] *IM = Intramuscular

[0602] List of Figures

[0603] Figure 1 : Serum IgG responses of pigs vaccinated against porcine rotavirus A vaccination with AVP8-IgG Fc protein formulated with Emulsigen D (referred to as "AVP8-IgG" in the label) or placebo ("irrelevant control").

[0604] Figure 2 : Results of a VN (Virus Neutralization) assay performed to detect and quantify antibodies capable of neutralizing porcine rotavirus A virus in samples from pigs vaccinated with AVP8-IgG Fc protein formulated with Emulsigen D (referred to as "AVP8-IgG" in the label) or placebo ("non-relevant control").

[0605] Figure 3 : Mean VN titers against rotavirus in sow sera according to group and study day, where study days D0 and D28 represent the time points "six weeks and two weeks before farrowing" (i.e., when the investigational product was administered to study groups T02 and T04, respectively), and study days D7, D28 and D35 represent the time points "five weeks, two weeks and one week before farrowing" (i.e., when the commercial vaccine was administered T06).

[0606] Figure 4 : Group median log rotavirus A RNA genome copies (gc) / mL in feces according to study day.

[0607] Figure 5 : A) SDS-PAGE of protein A purified AVP8-IgGFc protein (SEQ ID NO: 12) product samples with dithiothreitol reduction ("+DTT") or non-reduction ("-DTT"); B) Western Blot of AVP8-IgG Fc protein (SEQ ID NO: 12) bioreactor product, wherein the sample was centrifuged to separate the cell pellet fraction ("pellet") and the supernatant fraction ("supernatant"), which was run on SDS-PAGE under reducing conditions (+DTT) after the freeze-thaw process, transferred to PVDF membrane and probed with HRP-conjugated goat anti-swine antibody to detect porcine IgG Fc fragment.

[0608] Figure 6 : Mean VN titers against rotavirus in sow sera according to group and study day, where study days D0 and D28 represent the time points "six and two weeks before farrowing" (i.e. when the test product was administered to study groups T01 and T03, respectively).

[0609] Figure 7: Mean VN titers against rotavirus in sow serum according to group and study day, where study day D0 represents the time point of the first administration of the test product to study groups T01 and T03, respectively.

[0610] Figure 8 Results of an “IgG:AVP8 P[6] ELISA” in which ELISA plates coated with rotavirus A VP8 P[6] protein were incubated with diluted test sera and in which anti-rotavirus AVP8 P[6] antibodies were subsequently detected by using HRP-conjugated goat anti-swine IgG antibodies.

[0611] Fig. 9 Results of an “IgG:AVP8 P

[13] ELISA” in which ELISA plates coated with rotavirus A VP8 P

[13] protein were incubated with diluted test sera and in which anti-rotavirus AVP8 P

[13] antibodies were subsequently detected by using HRP-conjugated goat anti-swine IgG antibodies.

[0612] In the sequence listing / Source and geographical origin (where applicable):

[0613] SEQ ID NO: 1 corresponds to the sequence of rotavirus VP8 protein (genotype P[7]) originating from a farm in North Carolina, USA,

[0614] SEQ ID NO:2 corresponds to the sequence of the lectin-like domain of the rotavirus VP8 protein (genotype P[7]) originating from a farm in North Carolina, USA,

[0615] SEQ ID NO:3 corresponds to the sequence of an immunogenic fragment of the rotavirus VP8 protein (genotype P[7]) originating from a farm in North Carolina, USA,

[0616] SEQ ID NO:4 corresponds to the sequence of an immunogenic fragment of the rotavirus VP8 protein, i.e., the consensus sequence of a portion of the rotavirus VP8 protein (based on genotype P[6]),

[0617] SEQ ID NO:5 corresponds to the sequence of an immunogenic fragment of the rotavirus VP8 protein, i.e., the consensus sequence of a portion of an immunogenic fragment of the rotavirus VP8 protein (based on genotype P

[13] ),

[0618] SEQ ID NO:6 corresponds to the sequence of an immunogenic fragment of rotavirus C VP8 protein,

[0619] SEQ ID NO:7 corresponds to the sequence of a porcine IgG Fc fragment,

[0620] SEQ ID NO:8 corresponds to the sequence of guinea pig IgG Fc fragment,

[0621] SEQ ID NO:9 (Gly-Gly-Ser) corresponds to the sequence of the linker portion,

[0622] SEQ ID NO: 10 corresponds to the sequence of the linker portion,

[0623] SEQ ID NO: 11 corresponds to the sequence of the linker portion,

[0624] SEQ ID NO: 12 corresponds to the sequence of a polypeptide (fusion protein) comprising the sequences of SEQ ID NO: 3, SEQ ID NO: 9 (Gly-Gly-Ser) and SEQ ID NO: 7,

[0625] SEQ ID NO: 13 corresponds to the sequence of a polypeptide (fusion protein) comprising the sequences of SEQ ID NO: 4, SEQ ID NO: 9 (Gly-Gly-Ser) and SEQ ID NO: 7,

[0626] SEQ ID NO: 14 corresponds to the sequence of a polypeptide (fusion protein) comprising the sequences of SEQ ID NO: 5, SEQ ID NO: 9 (Gly-Gly-Ser) and SEQ ID NO: 7,

[0627] SEQ ID NO: 15 corresponds to the sequence of a polypeptide (fusion protein) comprising the sequences of SEQ ID NO: 6, SEQ ID NO: 9 (Gly-Gly-Ser) and SEQ ID NO: 7,

[0628] SEQ ID NO: 16 corresponds to the sequence of a polypeptide (fusion protein) comprising the sequences of SEQ ID NO: 3, SEQ ID NO: 9 (Gly-Gly-Ser), SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 5,

[0629] SEQ ID NO: 17 corresponds to the sequence of a polynucleotide encoding the polypeptide (fusion protein) of SEQ ID NO: 12,

[0630] SEQ ID NO: 18 corresponds to the sequence of a polynucleotide encoding the polypeptide (fusion protein) of SEQ ID NO: 13,

[0631] SEQ ID NO: 19 corresponds to the sequence of a polynucleotide encoding the polypeptide (fusion protein) of SEQ ID NO: 14,

[0632] SEQ ID NO:20 corresponds to the sequence of a polynucleotide encoding the polypeptide (fusion protein) of SEQ ID NO:15,

[0633] SEQ ID NO:21 corresponds to the sequence of a polynucleotide encoding the polypeptide (fusion protein) of SEQ ID NO:16,

[0634] SEQ ID NO: 22-25: Primer and probe sequences (Table 5).

[0635] The following clauses are also disclosed herein. Therefore, the present invention further includes aspects characterized by the following clauses:

[0636] 1. An immunogenic composition comprising:

[0637] (i) a polypeptide comprising:

[0638] - an immunogenic fragment of the rotavirus VP8 protein, and

[0639] - Immunoglobulin Fc fragment;

[0640] and

[0641] (ii) at least one immunogenic substance different from the polypeptide.

[0642] 2. The immunogenic composition of clause 1, wherein the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein,

[0643] Or wherein the immunoglobulin Fc fragment is linked to the N-terminus of the immunogenic fragment of the rotavirus VP8 protein.

[0644] 3. The immunogenic composition of clause 1 or 2, wherein

[0645] The immunoglobulin Fc fragment is connected to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein via a linker portion,

[0646] Or wherein the immunoglobulin Fc fragment is linked to the N-terminus of the immunogenic fragment of the rotavirus VP8 protein via a linker moiety.

[0647] 4. The immunogenic composition of any one of clauses 1 to 3, wherein the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein via a peptide bond between the N-terminal amino acid residue of the immunoglobulin Fc fragment and the C-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein,

[0648] Or wherein the immunoglobulin Fc fragment is linked to the N-terminus of the immunogenic fragment of the rotavirus VP8 protein through a peptide bond between the C-terminal amino acid residue of the immunoglobulin Fc fragment and the N-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein.

[0649] 5. The immunogenic composition of any one of clauses 1 to 4, wherein the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein.

[0650] 6. The immunogenic composition of any one of clauses 1 to 5, wherein the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein via a linker moiety,

[0651] Or wherein the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein through a peptide bond between the N-terminal amino acid residue of the immunoglobulin Fc fragment and the C-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein.

[0652] 7. The immunogenic composition of any one of clauses 1 to 6, wherein the polypeptide is a fusion protein.

[0653] 8. The immunogenic composition of any one of clauses 1 to 7, wherein the polypeptide is a fusion protein of formula xyz, wherein

[0654] x consists of an immunogenic fragment of the rotavirus VP8 protein;

[0655] y is a linker moiety; and

[0656] z is the immunoglobulin Fc fragment.

[0657] 9. The immunogenic composition of any one of clauses 1 to 8, wherein the immunogenic fragment of the rotavirus VP8 protein is capable of inducing an immune response against rotavirus in a subject to which the immunogenic fragment of the rotavirus VP8 protein is administered.

[0658] 10. The immunogenic composition of any one of clauses 1 to 9, wherein the immunogenic fragment of the rotavirus VP8 protein is 50 to 200, preferably 140 to 190 amino acid residues in length.

[0659] 11. The immunogenic composition of any one of clauses 1 to 10, wherein the rotavirus is porcine rotavirus.

[0660] 12. The immunogenic composition of any one of clauses 1 to 11, wherein the rotavirus is selected from the group consisting of rotavirus A and rotavirus C.

[0661] 13. The immunogenic composition of any one of clauses 1 to 12, wherein the rotavirus is rotavirus A.

[0662] 14. The immunogenic composition of any one of clauses 1 to 13, wherein the immunogenic fragment of the rotavirus VP8 protein comprises the lectin-like domain of the rotavirus VP8 protein.

[0663] 15. An immunogenic composition as described in any one of clauses 1 to 14, wherein the immunogenic fragment of the rotavirus VP8 protein is an N-terminal extended lectin-like domain of the rotavirus VP8 protein, wherein the length of the N-terminal extension is 1 to 20 amino acid residues, preferably 5 to 15 amino acid residues.

[0664] 16. The immunogenic composition of clause 14 or 15, wherein the lectin-like domain of the rotavirus VP8 protein consists of the amino acid sequence of amino acid residues 65-224 of the rotavirus VP8 protein.

[0665] 17. The immunogenic composition of clause 15 or 16, wherein the N-terminally extended amino acid sequence is an amino acid sequence of the respective length of the N-terminal amino acid residues flanking the lectin-like domain in the amino acid sequence of the rotavirus VP8 protein.

[0666] 18. The immunogenic composition of any one of clauses 1 to 17, wherein the immunogenic fragment of the rotavirus VP8 protein consists of the following amino acid sequence:

[0667] Amino acid residues 60-224, amino acid residues 59-224, amino acid residues 58-224, amino acid residues 57-224, amino acid residues 56-224, amino acid residues 55-224, amino acid residues 54-224, amino acid residues 53-224, amino acid residues 52-224, amino acid residues 51-224, amino acid residues 50-224 or amino acid residues 49-224 of rotavirus VP8 protein.

[0668] 19. The immunogenic composition of any one of clauses 1 to 18, wherein the immunogenic fragment of the rotavirus VP8 protein consists of the amino acid sequence of amino acid residues 57-224 of the rotavirus VP8 protein.

[0669] 20. An immunogenic composition as described in any one of items 16 to 19, wherein the numbering of the amino acid residues refers to the amino acid sequence of wild-type rotavirus VP8 protein, especially wild-type rotavirus A VP8 protein, and wherein the wild-type rotavirus VP8 is preferably the protein shown in SEQ ID NO: 1.

[0670] 21. The immunogenic composition of any one of clauses 1 to 20, wherein the rotavirus is selected from the group consisting of genotype P[7] rotavirus, genotype P[6] rotavirus and genotype P

[13] rotavirus.

[0671] 22. An immunogenic composition as described in any one of clauses 1 to 21, wherein the rotavirus VP8 protein comprises or consists of an amino acid sequence that has at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:1.

[0672] 23. An immunogenic composition as described in any one of clauses 14 to 22, wherein the lectin-like domain of the rotavirus VP8 protein consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:2.

[0673] 24. The immunogenic composition of any one of clauses 1 to 23, wherein the immunogenic fragment of the rotavirus VP8 protein consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:3.

[0674] 25. The immunogenic composition of any one of clauses 1 to 24, wherein the immunogenic fragment of the rotavirus VP8 protein consists of or is the consensus sequence of: a portion of a rotavirus VP8 protein, in particular a portion of a rotavirus AVP8 protein,

[0675] And wherein the consensus sequence of a part of the rotavirus VP8 protein is preferably obtained by a method comprising the following steps:

[0676] - translating a plurality of nucleotide sequences encoding a portion of the rotavirus VP8 protein into amino acid sequences,

[0677] - aligning the amino acid sequence with known rotavirus VP8 proteins, preferably by using the UPGMB clustering method of the MUSCLE sequence alignment software and default gap penalty parameters,

[0678] - performing a phylogenetic analysis on the aligned sequences and generating a neighbor joining phylogenetic reconstruction based on the rotavirus VP8 protein sequence, in particular importing the aligned amino acid sequences into MEGA7 software for phylogenetic analysis and generating a neighbor joining phylogenetic reconstruction based on the rotavirus VP8 protein sequence,

[0679] - Optimal trees were calculated using Poisson correction and bootstrap retests of phylogeny (n=100),

[0680] - The optimal tree was scaled with branch lengths equal to the evolutionary distance in units of amino acid substitutions per site over a total of 170 positions,

[0681] - nodes with a bootstrapped cluster association greater than 70% are considered significant,

[0682] - nodes with a distance of about 10% and a bootstrapped re-clustering association greater than 70% are designated as clusters, and

[0683] - selecting a cluster and generating a consensus sequence by identifying the maximum frequency of each aligned position within said cluster,

[0684] - and optionally, in cases where an equal proportion of amino acids is observed in the aligned positions, selecting amino acid residues based on reported epidemiological data as well as a predetermined product protection profile.

[0685] 26. An immunogenic composition as described in any one of items 1 to 25, wherein the immunogenic fragment of the rotavirus VP8 protein consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:5.

[0686] 27. The immunogenic composition of any one of clauses 1 to 26, wherein the rotavirus is rotavirus C.

[0687] 28. The immunogenic composition of clauses 1 to 27, wherein the immunogenic fragment of the rotavirus VP8 protein consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:6.

[0688] 29. The immunogenic composition of any one of clauses 1 to 28, wherein the immunogenic fragment of the rotavirus VP8 protein consists of or is:

[0689] - an immunogenic fragment of a rotavirus A VP8 protein as defined in any one or more of clauses 9 to 24, or

[0690] - a consensus sequence of a portion of a rotavirus VP8 protein, in particular a portion of a rotavirus A VP8 protein, as defined in any of clauses 9 to 13, 25 and 26, or

[0691] - an immunogenic fragment of a rotavirus C VP8 protein as defined in any of clauses 9 to 12, 27 and 28.

[0692] 30. The immunogenic composition of any one of clauses 1 to 29, wherein the immunogenic fragment of the rotavirus VP8 protein consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0693] 31. The immunogenic composition of any one of clauses 1 to 30,

[0694] The immunoglobulin Fc fragment has a length of at least 220 amino acid residues, preferably a length of 220 to 250 amino acid residues,

[0695] And / or wherein the immunoglobulin Fc fragment is not glycosylated.

[0696] 32. The immunogenic composition of any one of clauses 1 to 31, wherein the immunoglobulin Fc fragment comprises or consists of the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of an immunoglobulin and optionally the hinge region or a portion of the hinge region.

[0697] 33. The immunogenic composition of any one of clauses 1 to 32, wherein the immunoglobulin is selected from the group consisting of IgG, IgA, IgD, IgE and IgM.

[0698] 34. The immunogenic composition of any one of clauses 1 to 33, wherein the immunoglobulin Fc fragment is an immunoglobulin Fc fragment encoded by the genome of a species whose intestinal cells are susceptible to rotavirus infection, and the immunogenic fragment of the rotavirus VP8 protein is derived from the rotavirus.

[0699] 35. The immunogenic composition of any one of clauses 1 to 34, wherein the immunoglobulin Fc fragment is a porcine IgG Fc fragment.

[0700] 36. The immunogenic composition of any one of clauses 1 to 35, wherein the immunoglobulin Fc fragment comprises or consists of an amino acid sequence that has at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8.

[0701] 37. The immunogenic composition of any one of clauses 3 to 36, wherein the linker moiety is an amino acid sequence of 1 to 50 amino acid residues in length.

[0702] 38. The immunogenic composition of any one of clauses 3 to 37, wherein the linker portion comprises or consists of an amino acid sequence that has at least 66%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 9 (Gly-Gly-Ser), SEQ ID NO: 10 and SEQ ID NO: 11.

[0703] 39. The immunogenic composition of any one of clauses 5 to 38, wherein the polypeptide has an N-terminal methionine residue flanked by the N-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein.

[0704] 40. The immunogenic composition of any one of clauses 5 to 39, wherein the polypeptide comprises a further immunogenic fragment of the rotavirus VP8 protein linked to the C-terminus of the immunoglobulin Fc fragment.

[0705] 41. The immunogenic composition of any one of clauses 1 to 40, wherein the polypeptide comprises:

[0706] - an immunogenic fragment of the rotavirus VP8 protein (1),

[0707] - immunoglobulin Fc fragment, and

[0708] - another immunogenic fragment of the rotavirus VP8 protein (2),

[0709] wherein the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment (1),

[0710] And wherein another immunogenic fragment (2) of the rotavirus VP8 protein is linked to the C-terminus of the immunoglobulin Fc fragment.

[0711] 42. The immunogenic composition of clause 40 or 41, wherein the other immunogenic fragment of the rotavirus VP8 protein consists of or is:

[0712] - an immunogenic fragment of a rotavirus A VP8 protein as defined in any one or more of clauses 9 to 24; or

[0713] - a consensus sequence of a portion of a rotavirus VP8 protein, in particular a portion of a rotavirus A VP8 protein, as defined in any of clauses 9 to 13, 25 and 26; or

[0714] - an immunogenic fragment of a rotavirus C VP8 protein as defined in any one or more of clauses 9 to 12, 27 and 28.

[0715] 43. The immunogenic composition of any one of clauses 40 to 42, wherein the other immunogenic fragment of the rotavirus VP8 protein comprises or consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 2 to 6,

[0716] And / or wherein the another immunogenic fragment of the rotavirus VP8 protein is different from the immunogenic fragment of the rotavirus VP8 protein that is C-terminally linked to the immunoglobulin Fc fragment.

[0717] 44. The immunogenic composition of any one of clauses 40 to 43,

[0718] wherein the other immunogenic fragment of the rotavirus VP8 protein is linked to the C-terminus of the immunoglobulin Fc fragment via a linker moiety, wherein the linker moiety is preferably a linker moiety as defined in clause 37 or 38,

[0719] Or wherein the other immunogenic fragment of the rotavirus VP8 protein is linked to the C-terminus of the immunoglobulin Fc fragment via a peptide bond between the N-terminal amino acid residue of the other immunogenic fragment of the rotavirus VP8 protein and the C-terminal amino acid residue of the immunoglobulin Fc fragment.

[0720] 45. The immunogenic composition of any one of clauses 1 to 44, wherein the polypeptide consists of:

[0721] - an immunogenic fragment of a rotavirus VP8 protein, in particular an immunogenic fragment of a rotavirus VP8 protein as defined in any one or more of clauses 9 to 30,

[0722] - an N-terminal methionine residue flanking the N-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein, and

[0723] - an immunoglobulin Fc fragment, in particular an immunoglobulin Fc fragment as defined in any one or more of clauses 31 to 36,

[0724] wherein the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein, in particular via a linker moiety, wherein the linker moiety is preferably a linker moiety as defined in clause 37 or 38,

[0725] - and optionally another immunogenic fragment of a rotavirus VP8 protein linked to the C-terminus of said immunoglobulin Fc fragment, in particular via a linker portion, wherein said another immunogenic fragment of a rotavirus VP8 protein is preferably another immunogenic fragment as defined in any one or more of clauses 41 to 44, and wherein said linker portion is preferably a linker portion as defined in clause 37 or 38.

[0726] 46. ​​An immunogenic composition as described in any one of items 1 to 45, wherein the polypeptide is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0727] 47. The immunogenic composition of any one of clauses 1 to 46, wherein the polypeptide is a recombinant protein, in particular a recombinant baculovirus-expressed protein.

[0728] 48. The immunogenic composition of any one of clauses 1 to 47, wherein the polypeptide forms a homodimer with another identical polypeptide.

[0729] 49. The immunogenic composition of any one of clauses 1 to 48, wherein component (i) is present in a multimer comprising or consisting of a plurality of said polypeptides, and wherein said multimer is preferably a homodimer formed by said polypeptide and another identical polypeptide.

[0730] 50. The immunogenic composition of any one of clauses 1 to 49, wherein at least one immunogenic substance other than said polypeptide consists of two or more immunogenic substances, wherein all said immunogenic substances

[0731] is different from the polypeptide, and

[0732] Different from each other.

[0733] 51. The immunogenic composition of any one of clauses 1 to 50, wherein at least one immunogenic agent different from said polypeptide is at least one immunogenic agent comprising a Reovirus antigen different from said immunogenic fragment.

[0734] 52. An immunogenic composition as described in any one of clauses 1 to 51, wherein at least one immunogenic substance different from said polypeptide consists of two or more immunogenic substances, each of which comprises a reovirus antigen, and wherein all of said reovirus antigens are different from said immunogenic fragment and different from each other.

[0735] 53. The immunogenic composition of any one of clauses 1 to 52, wherein at least one immunogenic substance different from said polypeptide is at least a protein comprising a reovirus antigen different from said immunogenic fragment.

[0736] 54. An immunogenic composition as described in any one of items 1 to 53, wherein at least one immunogenic substance different from the polypeptide consists of two or more proteins, each of which contains a reovirus antigen, and wherein all of the reovirus antigens are different from the immunogenic fragment and different from each other.

[0737] 55. The immunogenic composition of any one of clauses 1 to 54, wherein component (ii) consists of at least one immunogenic substance comprising a rotavirus antigen different from said immunogenic fragment of component (i).

[0738] 56. The immunogenic composition of any one of clauses 1 to 55, wherein component (ii) consists of two or more immunogenic substances, wherein each of said substances comprises a rotavirus antigen, and wherein all said rotavirus antigens are different from said immunogenic fragment of component (i) and are different from each other.

[0739] 57. The immunogenic composition of any one of clauses 1 to 56, wherein component (ii) consists of at least one protein comprising a rotavirus antigen different from said immunogenic fragment of component (i).

[0740] 58. The immunogenic composition of any one of clauses 1 to 57, wherein component (ii) consists of two or more proteins, wherein each of said proteins comprises a rotavirus antigen, and wherein all said rotavirus antigens are different from said immunogenic fragment of component (i) and are different from each other.

[0741] 59. The immunogenic composition of one or more of clauses 1 to 58, wherein

[0742] The polypeptide is a first polypeptide;

[0743] and

[0744] The at least one immunogenic substance different from the polypeptide is at least one other polypeptide different from the first polypeptide.

[0745] 60. The immunogenic composition of clause 59, wherein at least one immunogenic substance different from said polypeptide comprises or is:

[0746] - a second polypeptide different from said first polypeptide,

[0747] and preferably comprises a third polypeptide different from the first and second polypeptides, and

[0748] Optionally comprises a fourth polypeptide which is different from all of said first to third polypeptides.

[0749] 61. The immunogenic composition of clause 59 or 60, wherein at least one immunogenic substance different from said polypeptide comprises or is:

[0750] - a second polypeptide comprising a Reovirus antigen different from said immunogenic fragment,

[0751] and preferably comprises a third polypeptide different from the first and second polypeptides, and

[0752] Optionally comprises a fourth polypeptide which is different from all of said first to third polypeptides.

[0753] 62. The immunogenic composition of clause 61, wherein the third polypeptide comprises a reovirus antigen that is different from both the immunogenic fragment and the reovirus antigen of the second polypeptide,

[0754] And wherein optionally, the fourth polypeptide comprises a reovirus antigen different from all of the following:

[0755] - said immunogenic fragment,

[0756] - a reovirus antigen of a second polypeptide, and

[0757] - A third polypeptide of a reovirus antigen.

[0758] 63. The immunogenic composition of one or more of clauses 51 to 54 and 61 to 62, wherein the reovirus antigen is a rotavirus antigen and the reovirus antigens are respectively rotavirus antigens.

[0759] 64. The immunogenic composition according to one or more of clauses 60 to 63,

[0760] wherein the second polypeptide is a polypeptide as defined in any one of clauses 1 to 49, and wherein the second peptide is different from the first polypeptide,

[0761] and wherein preferably the third polypeptide is a polypeptide as defined in any one of clauses 1 to 49, and wherein the third polypeptide is different from both the first and second polypeptide,

[0762] And wherein optionally the fourth polypeptide is a polypeptide as defined in any one of clauses 1 to 49, and wherein the fourth polypeptide is different from all of the first to third polypeptides.

[0763] 65. The immunogenic composition of one or more of clauses 1 to 64, wherein

[0764] The polypeptide is a first polypeptide, which comprises:

[0765] - a first immunogenic fragment of the rotavirus VP8 protein, and

[0766] - Immunoglobulin Fc fragment;

[0767] and

[0768] At least one immunogenic substance different from the polypeptide comprises or consists of:

[0769] a second polypeptide comprising a second immunogenic fragment of a rotavirus VP8 protein, wherein the second immunogenic fragment is different from the first immunogenic fragment,

[0770] and preferably a third polypeptide comprising a third immunogenic fragment of a rotavirus VP8 protein, wherein said third immunogenic fragment is different from both said first and second immunogenic fragments,

[0771] and optionally a fourth polypeptide comprising a fourth immunogenic fragment of a rotavirus VP8 protein, wherein the fourth immunogenic fragment is different from all of the first to third immunogenic fragments.

[0772] 66. The immunogenic composition of clause 65, wherein each of the second to fourth immunogenic fragments is independently selected from the group consisting of:

[0773] - an immunogenic fragment as defined in any one or more of clauses 9 to 24,

[0774] - a consensus sequence of a portion of a rotavirus VP8 protein, in particular a portion of a rotavirus A VP8 protein, as defined in any one or more of clauses 9 to 13, 25 and 26,

[0775] and

[0776] - an immunogenic fragment of a rotavirus C VP8 protein as defined in any one or more of clauses 9 to 12, 27 and 28,

[0777] In particular, with the proviso that all of said second to fourth immunogenic fragments are different from said first immunogenic fragment and from each other.

[0778] 67. The immunogenic composition of clause 65 or 66, wherein

[0779] The first immunogenic fragment is selected from the group consisting of: X7, X13, X6 and XC, and / or

[0780] The second immunogenic fragment is selected from the group consisting of: X7, X13, X6 and XC, and / or

[0781] The third immunogenic fragment is selected from the group consisting of: X7, X13, X6 and XC, and / or

[0782] The fourth immunogenic fragment is selected from the group consisting of: X7, X13, X6 and XC;

[0783] in

[0784] X7 represents an immunogenic fragment of genotype P[7] rotavirus VP8 protein,

[0785] X13 represents an immunogenic fragment of genotype P

[13] rotavirus VP8 protein,

[0786] X6 represents an immunogenic fragment of genotype P[6] rotavirus VP8 protein,

[0787] and

[0788] XC represents an immunogenic fragment of rotavirus C VP8 protein.

[0789] 68. The immunogenic composition of clause 67, wherein at least one immunogenic substance different from said polypeptide comprises or is:

[0790] a second polypeptide comprising a second immunogenic fragment of a rotavirus VP8 protein,

[0791] and wherein each of the first and second immunogenic fragments is independently selected from the group consisting of: X7, X13, X6 and XC,

[0792] Provided that when the first immunogenic fragment is X7, the second immunogenic fragment is selected from the group consisting of X13, X6 and XC,

[0793] Provided that when the first immunogenic fragment is X6, the second immunogenic fragment is selected from the group consisting of X7, X13 and XC,

[0794] Provided that when the first immunogenic fragment is X13, the second immunogenic fragment is selected from the group consisting of X7, X6 and XC,

[0795] And provided that when the first immunogenic fragment is XC, then the second immunogenic fragment is selected from the group consisting of X7, X13 and X6.

[0796] 69. The immunogenic composition of clause 67 or 68, wherein at least one immunogenic substance different from said polypeptide comprises or consists of:

[0797] a second polypeptide comprising a second immunogenic fragment of a rotavirus VP8 protein, and

[0798] a third polypeptide comprising a third immunogenic fragment of a rotavirus VP8 protein,

[0799] wherein each of the first to third immunogenic fragments is independently selected from the group consisting of X7, X13, X6 and XC,

[0800] Provided that when the first immunogenic fragment is X7, then the second immunogenic fragment is X13 and the third immunogenic fragment is selected from the group consisting of X6 and XC,

[0801] Provided that when the first immunogenic fragment is X6, then the second immunogenic fragment is X7 and the third immunogenic fragment is selected from the group consisting of X13 and XC,

[0802] Provided that when the first immunogenic fragment is X13, the second immunogenic fragment is X7 and the third immunogenic fragment is selected from the group consisting of X6 and XC,

[0803] And provided that when the first immunogenic fragment is XC, then the second immunogenic fragment is X7 and the third immunogenic fragment is selected from the group consisting of X13 and X6.

[0804] 70. The immunogenic composition of one or more of clauses 67 to 69, wherein at least one immunogenic substance different from said polypeptide comprises or consists of:

[0805] a second polypeptide comprising a second immunogenic fragment of a rotavirus VP8 protein, and

[0806] a third polypeptide comprising a third immunogenic fragment of a rotavirus VP8 protein, and

[0807] a fourth polypeptide comprising a fourth immunogenic fragment of a rotavirus VP8 protein,

[0808] and wherein each of the first to fourth immunogenic fragments is independently selected from the group consisting of X7, X13, X6 and XC,

[0809] Provided that when the first immunogenic fragment is X7, then the second immunogenic fragment is X13, the third immunogenic fragment is X6, and the fourth immunogenic fragment is XC,

[0810] Provided that when the first immunogenic fragment is X6, then the second immunogenic fragment is X7, the third immunogenic fragment is X13, and the fourth immunogenic fragment is XC,

[0811] Provided that when the first immunogenic fragment is X13, then the second immunogenic fragment is X7, the third immunogenic fragment is X6, and the fourth immunogenic fragment is XC,

[0812] And provided that when the first immunogenic fragment is XC, then the second immunogenic fragment is X7, the third immunogenic fragment is X13, and the fourth immunogenic fragment is X6.

[0813] 71. The immunogenic composition of one or more of clauses 67 to 70, wherein

[0814] X7 consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO: 3, and / or

[0815] X13 consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:5, and / or

[0816] X6 consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO: 4, and / or

[0817] XC consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:6.

[0818] 72. The immunogenic composition of one or more of clauses 65 to 71, wherein the first immunogenic fragment is an immunogenic fragment of a genotype P[7] rotavirus VP8 protein, and

[0819] The second immunogenic fragment is an immunogenic fragment of genotype P

[13] rotavirus VP8 protein,

[0820] And preferably, the third immunogenic fragment is an immunogenic fragment of genotype P[6] rotavirus VP8 protein, and

[0821] And optionally, the fourth immunogenic fragment is an immunogenic fragment of rotavirus C VP8 protein.

[0822] 73. The immunogenic composition of one or more of clauses 65 to 72, wherein

[0823] The first immunogenic fragment consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO: 3, and

[0824] The second immunogenic fragment consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO: 5,

[0825] And preferably, the third immunogenic fragment consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO: 4, and

[0826] And optionally, the fourth immunogenic fragment consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:6.

[0827] 74. The immunogenic composition of one or more of clauses 59 to 73, wherein

[0828] The first polypeptide is selected from the group consisting of: R7, R13, R6 and RC, and / or

[0829] The second polypeptide is selected from the group consisting of: R7, R13, R6 and RC, and / or

[0830] The third polypeptide is selected from the group consisting of: R7, R13, R6 and RC, and / or

[0831] The fourth polypeptide is selected from the group consisting of: R7, R13, R6 and RC;

[0832] in

[0833] R7 is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 12,

[0834] R13 is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 14,

[0835] R6 is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 13, and

[0836] RC is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 15.

[0837] 75. The immunogenic composition of one or more of clauses 59 to 74, wherein

[0838] At least one immunogenic substance different from the polypeptide comprises or is a second polypeptide of the rotavirus VP8 protein, and wherein

[0839] Each of the first and second polypeptides is independently selected from the group consisting of: R7, R13, R6 and RC,

[0840] Provided that when the first polypeptide is R7, the second polypeptide is selected from the group consisting of R13, R6 and RC,

[0841] Provided that when the first polypeptide is R6, the second polypeptide is selected from the group consisting of R7, R13 and RC,

[0842] Provided that when the first polypeptide is R13, the second polypeptide is selected from the group consisting of R7, R6 and RC,

[0843] And provided that when the first polypeptide is RC, the second polypeptide is selected from the group consisting of R7, R13 and R6.

[0844] 76. The immunogenic composition of clause 74 or 75, wherein

[0845] At least one immunogenic substance different from said polypeptide comprises or consists of a second polypeptide of rotavirus VP8 protein, and wherein

[0846] Each of the first and second polypeptides is independently selected from the group consisting of: R7, R13 and R6,

[0847] Provided that when the first polypeptide is R7, the second polypeptide is selected from the group consisting of R13 and R6,

[0848] Provided that when the first polypeptide is R6, the second polypeptide is selected from the group consisting of R7 and R13,

[0849] And provided that when the first polypeptide is R13, the second polypeptide is selected from the group consisting of R7 and R6.

[0850] 77. The immunogenic composition of one or more of clauses 74 to 76, wherein

[0851] The first polypeptide is R7 and the second polypeptide is selected from the group consisting of R13 and R6.

[0852] 78. The immunogenic composition of one or more of clauses 74 to 77, wherein

[0853] At least one immunogenic substance different from the polypeptide comprises or consists of:

[0854] a second polypeptide, and

[0855] The third polypeptide,

[0856] and wherein each of the first to third polypeptides is independently selected from the group consisting of R7, R13, R6 and RC,

[0857] Provided that when the first polypeptide is R7, the second polypeptide is R13 and the third polypeptide is selected from the group consisting of R6 and RC,

[0858] Provided that when the first polypeptide is R6, the second polypeptide is R7 and the third polypeptide is selected from the group consisting of R13 and RC,

[0859] Provided that when the first polypeptide is R13, the second polypeptide is R7 and the third polypeptide is selected from the group consisting of R6 and RC,

[0860] And provided that when the first polypeptide is RC, then the second polypeptide is R7 and the third polypeptide is selected from the group consisting of R13 and R6.

[0861] 79. The immunogenic composition of one or more of clauses 74 to 78, wherein

[0862] The first polypeptide is R7, and

[0863] The second polypeptide is R13, and

[0864] The third polypeptide is selected from the group consisting of R6 and RC.

[0865] 80. The immunogenic composition of one or more of clauses 74 to 79, wherein

[0866] At least one immunogenic substance different from the polypeptide comprises or consists of:

[0867] a second polypeptide, and

[0868] The third polypeptide,

[0869] and wherein each of the first to third polypeptides is independently selected from the group consisting of: R7, R13 and R6,

[0870] Provided that when the first polypeptide is R7, the second polypeptide is R13 and the third polypeptide is R6,

[0871] Provided that when the first polypeptide is R6, the second polypeptide is R7 and the third polypeptide is R13,

[0872] Provided that when the first polypeptide is R13, then the second polypeptide is R7 and the third polypeptide is R6.

[0873] 81. The immunogenic composition of one or more of clauses 74 to 80, wherein

[0874] At least one immunogenic substance different from the polypeptide comprises or consists of:

[0875] a second polypeptide, and

[0876] A third polypeptide, and

[0877] The fourth polypeptide,

[0878] and wherein each of the first to fourth polypeptides is independently selected from the group consisting of: R7, R6, R13 and RC,

[0879] Provided that when the first polypeptide is R7, the second polypeptide is R13, the third polypeptide is R6, and the fourth polypeptide is RC,

[0880] Provided that when the first polypeptide is R6, the second polypeptide is R7, the third polypeptide is R13, and the fourth polypeptide is RC,

[0881] Provided that when the first polypeptide is R13, the second polypeptide is R7, the third polypeptide is R6, and the fourth polypeptide is RC,

[0882] And provided that when the first polypeptide is RC, the second polypeptide is R7, the third polypeptide is R13, and the fourth polypeptide is R6.

[0883] 82. The immunogenic composition of one or more of clauses 74 to 81, wherein

[0884] The first polypeptide is R7, and

[0885] The second polypeptide is R13, and

[0886] The third polypeptide is R6, and

[0887] The fourth polypeptide is RC.

[0888] 83. An immunogenic composition, in particular the immunogenic composition of any one of clauses 1 to 82, comprising:

[0889] - a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with the sequence of SEQ ID NO: 12, and

[0890] - A protein comprising or consisting of an amino acid sequence that has at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 13 and SEQ ID NO: 15.

[0891] 84. An immunogenic composition, in particular the immunogenic composition of any one of clauses 1 to 83, comprising:

[0892] - a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with the sequence of SEQ ID NO: 12, and

[0893] - A protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity to the sequence of SEQ ID NO: 14.

[0894] 85. The immunogenic composition of clause 84, comprising:

[0895] - A protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 13.

[0896] 86. The immunogenic composition of clause 84 or 85, comprising:

[0897] - A protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 15.

[0898] 87. The immunogenic composition of any one of clauses 1 to 86, wherein the immunogenic composition further comprises a pharmaceutically or veterinarily acceptable carrier or excipient.

[0899] 88. The immunogenic composition of any one of clauses 1 to 87, wherein the immunogenic composition further comprises an adjuvant.

[0900] 89. The immunogenic composition of any one of clauses 1 to 88, comprising:

[0901] - a pharmaceutically or veterinarily acceptable carrier or excipient,

[0902] - and optionally an adjuvant.

[0903] 90. An immunogenic composition according to clause 88 or 89, wherein the adjuvant is an emulsified oil-in-water adjuvant.

[0904] 91. An immunogenic composition as described in item 88 or 89, wherein the adjuvant is carbomer.

[0905] 92. Use of the immunogenic composition of any one of clauses 1 to 91 for the preparation of a medicament, preferably a vaccine.

[0906] 93. The immunogenic composition of any one of clauses 1 to 91 for use as a medicament.

[0907] 94. The immunogenic composition of any one of clauses 1 to 91 for use as a vaccine.

[0908] 95. The immunogenic composition of any one of clauses 1 to 91 for use in a method of inducing an immune response against rotavirus in a subject.

[0909] 96. The immunogenic composition of any one of clauses 1 to 91 for use in a method of reducing or preventing one or more clinical signs, mortality or fecal excretion caused by rotavirus infection in a subject or for use in a method of treating or preventing infection by rotavirus in a subject.

[0910] 97. The immunogenic composition of clause 95 or 96, wherein the subject is a mammal or a bird, and wherein the bird is preferably a chicken.

[0911] 98. The immunogenic composition of any one of clauses 95 to 97, wherein the subject is a mammal, and wherein the mammal is preferably a pig or a cow.

[0912] 99. The immunogenic composition of any one of clauses 95 to 98, wherein the subject is a pig, and wherein the pig is preferably a piglet or a sow.

[0913] 100. The immunogenic composition of clause 95, wherein the subject is a pregnant sow.

[0914] 101. The immunogenic composition of clause 96, wherein the subject is a piglet.

[0915] 102. The immunogenic composition of any one of clauses 1 to 91 for use in a method of reducing or preventing one or more clinical signs, mortality or faecal shedding caused by rotavirus infection in piglets, wherein the piglets are suckled by sows to which the immunogenic composition has been administered.

[0916] 103. The immunogenic composition of clause 102, wherein the sow to which the immunogenic composition has been administered is a sow to which the immunogenic composition has been administered while the sow was pregnant, especially while carrying the piglet.

[0917] 104. A method for treating or preventing rotavirus infection, reducing, preventing or treating one or more clinical signs, mortality or fecal excretion caused by rotavirus infection, or preventing or treating a disease caused by rotavirus infection, comprising administering to a subject an immunogenic composition of any one of clauses 1 to 91.

[0918] 105. A method for inducing the production of antibodies specific for rotavirus in a sow, wherein the method comprises administering to the sow the immunogenic composition of any one of clauses 1 to 91.

[0919] 106. A method for reducing or preventing one or more clinical signs, mortality or fecal shedding caused by rotavirus infection in piglets, wherein the method comprises:

[0920] - administering to a sow an immunogenic composition according to any one of clauses 1 to 91, and

[0921] - allowing the sow to suckle the piglets.

[0922] 107. The method of clause 106, wherein the sow is a pregnant sow, in particular pregnant with the piglet.

[0923] 108. The method of clause 106 or 107, comprising the steps of:

[0924] - administering the immunogenic composition of any one of clauses 1 to 91 to a sow pregnant with said piglet,

[0925] - allowing said sow to give birth to said piglets, and

[0926] - allowing the sow to suckle the piglets.

[0927] 109. A method of reducing one or more clinical signs, mortality or fecal excretion caused by rotavirus infection in piglets, wherein the piglets are suckled by sows to which the immunogenic composition of any one of clauses 1 to 91 has been administered.

[0928] 110. The immunogenic composition of any one of clauses 96 to 103 or the method of any one of clauses 104 to 109, wherein the one or more clinical signs are selected from the group consisting of:

[0929] -diarrhea,

[0930] - rotavirus colonization,

[0931] - Lesions, especially macroscopic lesions,

[0932] - Reduced average daily weight gain, and

[0933] - Gastroenteritis.

[0934] 111. The immunogenic composition of clause 110 or the method of clause 110, wherein the rotavirus colonization is rotavirus colonization of the intestine, and / or wherein the lesions are intestinal lesions.

[0935] 112. The immunogenic composition of any one of clauses 95 to 103, 110 and 111 or the method of any one of clauses 104 to 111, wherein

[0936] - the rotavirus infection is infection with genotype P

[23] rotavirus and / or genotype P[7] rotavirus,

[0937] - the rotavirus infection is infection with genotype P

[23] rotavirus and / or genotype P[7] rotavirus,

[0938] - said immune response against rotavirus is an immune response against genotype P

[23] rotavirus and / or genotype P[7] rotavirus, or

[0939] - The antibody specific for rotavirus is an antibody specific for rotavirus of genotype P

[23] and / or rotavirus of genotype P[7].

[0940] 113. An immunogenic composition or method as described in item 112, wherein the immunogenic composition comprises a polypeptide as defined in any one of items 21 to 26 and 29 to 48, wherein the immunogenic fragment of the rotavirus VP8 protein is an immunogenic fragment of the genotype P[7] rotavirus VP8 protein.

[0941] 114. An immunogenic composition as described in item 113, wherein the immunogenic fragment of the genotype P[7] rotavirus VP8 protein consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:3.

[0942] 115. A method of producing an immunogenic composition according to any one of clauses 1 to 91, wherein the method comprises the following steps:

[0943] (a) allowing infection of susceptible cells in culture with a vector comprising a nucleic acid sequence encoding a polypeptide as defined in any one of clauses 1 to 48, wherein the polypeptide is expressed by the vector;

[0944] (b) thereafter recovering the polypeptide, in particular in a cell culture supernatant, wherein cell debris is preferably separated from the polypeptide by a separation step, said separation step preferably comprising microfiltration through at least one filter, preferably two filters, wherein at least one filter preferably has a pore size of about 1 to about 20 μm and / or about 0.1 μm to about 4 μm;

[0945] (c) deactivating the carrier by adding diethyleneimine (BEI) to the mixture of step (b);

[0946] (d) neutralizing the BEI by adding sodium thiosulfate to the mixture produced in step (c); and

[0947] (e) concentrating the polypeptides in the mixture produced in step (d) by removing a portion of the liquid from the mixture through a filtering step using a filter having a membrane having a molecular weight cutoff of between about 5 kDa and about 100 kDa, preferably between about 10 kDa and about 50 kDa;

[0948] And wherein the method comprises the following steps:

[0949] (f) admixing the mixture remaining after step (e) with at least one immunogenic substance different from the polypeptide;

[0950] (g) and optionally blending the mixture remaining after step (f) with another component selected from the group consisting of a pharmaceutically acceptable carrier, an adjuvant, a diluent, an excipient and a combination thereof,

[0951] Or wherein the method comprises the following steps:

[0952] (f) blending the mixture remaining after step (e) with another component selected from the group consisting of a pharmaceutically acceptable carrier, adjuvant, diluent, excipient, and combinations thereof, and

[0953] (g) admixing the mixture remaining after step (f) with at least one immunogenic substance different from the polypeptide.

[0954] 116. The method of clause 115, wherein the at least one immunogenic substance is at least one immunogenic substance as defined in any one of clauses 50 to 76.

[0955] 117. A method as described in item 115 or 116, wherein in step (a), the polypeptide as defined in any one of items 1 to 48 is a protein comprising an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 12, or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 12.

[0956] 118. The method of any one of clauses 115 to 117, wherein the at least one immunogenic substance different from the polypeptide is at least one protein selected from the group consisting of:

[0957] A protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 14,

[0958] A protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 13, and

[0959] A protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 15.

[0960] 119. The immunogenic composition of any one of clauses 1 to 91, 93 to 103 and 110 to 114, the use of clause 92 or the method of any one of clauses 104 to 112, 113 and 114, wherein the immunogenic composition is obtainable by the method of any one of clauses 115 to 118.

[0961]

[0962]

[0963]

[0964]

[0965]

[0966]

[0967]

[0968]

[0969]

[0970]

[0971]

[0972]

[0973]

[0974]

[0975]

[0976]

[0977]

[0978]

[0979]

[0980]

[0981]

[0982]

[0983]

[0984]

[0985]

[0986]

[0987]

[0988]

[0989]

[0990]

[0991]

[0992]

[0993]

Claims

1. An immunogenic composition comprising: (i) a polypeptide comprising: - an immunogenic fragment of the rotavirus VP8 protein, and - immunoglobulin Fc fragment, as well as (ii) at least one immunogenic substance different from the polypeptide.

2. The immunogenic composition of claim 1, wherein the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein via a linker moiety, Or wherein the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein through a peptide bond between the N-terminal amino acid residue of the immunoglobulin Fc fragment and the C-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein.

3. The immunogenic composition of claim 1 or 2, wherein the polypeptide is a fusion protein of formula xyz, wherein x consists of an immunogenic fragment of the rotavirus VP8 protein; y is a linker moiety; and z is the immunoglobulin Fc fragment.

4. The immunogenic composition of any one of claims 1 to 3, wherein the rotavirus is porcine rotavirus, And / or wherein the rotavirus is selected from the group consisting of: rotavirus A and rotavirus C.

5. The immunogenic composition of any one of claims 1 to 4, wherein the immunogenic fragment of the rotavirus VP8 protein is an N-terminal extended lectin-like domain of the rotavirus VP8 protein, wherein the length of the N-terminal extension is 1 to 20 amino acid residues, preferably 5 to 15 amino acid residues.

6. The immunogenic composition of any one of claims 1 to 5, wherein the rotavirus is selected from the group consisting of genotype P[7] rotavirus, genotype P[6] rotavirus, and genotype P[13] rotavirus.

7. The immunogenic composition according to any one of claims 1 to 6, wherein the immunogenic fragment of the rotavirus VP8 protein consists of or is the following: a consensus sequence of a part of the rotavirus VP8 protein, in particular a consensus sequence of a part of the rotavirus AVP8 protein, And wherein the consensus sequence of a part of the rotavirus VP8 protein is preferably obtained by a method comprising the following steps: - translating a plurality of nucleotide sequences encoding a portion of the rotavirus VP8 protein into amino acid sequences, - aligning the amino acid sequence with known rotavirus VP8 proteins, preferably by using the UPGMB clustering method of the MUSCLE sequence alignment software and default gap penalty parameters, - performing phylogenetic analysis on the aligned sequences and generating a neighbor joining phylogenetic reconstruction based on the rotavirus VP8 protein sequence, in particular importing the aligned amino acid sequences into MEGA7 software for phylogenetic analysis and generating a neighbor joining phylogenetic reconstruction based on the rotavirus VP8 protein sequence, - Optimal trees were calculated using Poisson correction and bootstrap retest of phylogeny (n=100), - The optimal tree was scaled with branch lengths equal to the evolutionary distance in units of amino acid substitutions per site over a total of 170 positions, - nodes with a bootstrapped cluster association greater than 70% are considered significant, - nodes with a distance of about 10% and a bootstrapped re-clustering association greater than 70% are designated as clusters, and - selecting a cluster and generating a consensus sequence by identifying the maximum frequency of each aligned position within said cluster, - and optionally, in cases where an equal ratio of amino acids is observed in the aligned positions, selecting amino acid residues based on reported epidemiological data in combination with a predetermined product protection profile.

8. The immunogenic composition of any one of claims 1 to 7, wherein the immunogenic fragment of the rotavirus VP8 protein consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98% or even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO:

6.

9. The immunogenic composition of any one of claims 1 to 8, wherein the immunoglobulin Fc fragment is an immunoglobulin Fc fragment encoded by the genome of a species whose intestinal cells are susceptible to infection by the rotavirus, and the immunogenic fragment of the rotavirus VP8 protein is derived from the rotavirus, and / or wherein the immunoglobulin Fc fragment is preferably a porcine IgG Fc fragment, And / or wherein the immunoglobulin Fc fragment comprises or consists of: An amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO:

8.

10. The immunogenic composition of any one of claims 1 to 9, wherein the linker moiety is an amino acid sequence of 1 to 50 amino acid residues in length, And / or wherein the linker moiety comprises or consists of: An amino acid sequence having at least 66%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 9 (Gly-Gly-Ser), SEQ ID NO: 10 and SEQ ID NO:

11.

11. The immunogenic composition of any one of claims 1 to 10, wherein the polypeptide is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO:

16.

12. The immunogenic composition of any one of claims 1 to 11, wherein the at least one immunogenic substance different from the polypeptide consists of two or more immunogenic substances, wherein all of the immunogenic substances is different from the polypeptide, and Different from each other.

13. The immunogenic composition of any one of claims 1 to 12, wherein component (ii) consists of two or more immunogenic substances, wherein each of said substances comprises a rotavirus antigen, and wherein all of said rotavirus antigens are different from said immunogenic fragment of component (i) and from each other.

14. The immunogenic composition of any one of claims 1 to 13, wherein The polypeptide is a first polypeptide, which comprises: - a first immunogenic fragment of the rotavirus VP8 protein, and - Immunoglobulin Fc fragment; and The at least one immunogenic substance different from the polypeptide comprises or consists of: a second polypeptide comprising a second immunogenic fragment of a rotavirus VP8 protein, wherein the second immunogenic fragment is different from the first immunogenic fragment, and preferably a third polypeptide comprising a third immunogenic fragment of a rotavirus VP8 protein, wherein said third immunogenic fragment is different from both said first and second immunogenic fragments, and optionally a fourth polypeptide comprising a fourth immunogenic fragment of a rotavirus VP8 protein, wherein the fourth immunogenic fragment is different from all of the first to third immunogenic fragments.

15. The immunogenic composition of claim 14, wherein the at least one immunogenic substance different from the polypeptide comprises or consists of: a second polypeptide comprising a second immunogenic fragment of a rotavirus VP8 protein, and a third polypeptide comprising a third immunogenic fragment of a rotavirus VP8 protein, and wherein each of the first to third immunogenic fragments is independently selected from the group consisting of X7, X6, X13 and XC, in X7 represents an immunogenic fragment of genotype P[7] rotavirus VP8 protein, X6 represents an immunogenic fragment of genotype P[6] rotavirus VP8 protein, X13 represents an immunogenic fragment of genotype P[13] rotavirus VP8 protein, and XC indicates the immunogenic fragment of rotavirus C VP8 protein; Provided that when the first immunogenic fragment is X7, then the second immunogenic fragment is X6 and the third immunogenic fragment is selected from the group consisting of X13 and XC, Provided that when the first immunogenic fragment is X6, then the second immunogenic fragment is X7 and the third immunogenic fragment is selected from the group consisting of X13 and XC, Provided that when the first immunogenic fragment is X13, then the second immunogenic fragment is X7 and the third immunogenic fragment is selected from the group consisting of X6 and XC, And provided that when the first immunogenic fragment is XC, then the second immunogenic fragment is X7 and the third immunogenic fragment is selected from the group consisting of X6 and X13.

16. The immunogenic composition of claim 14 or 15, wherein the at least one immunogenic substance different from the polypeptide comprises or consists of: a second polypeptide, and The third polypeptide, and wherein each of the first to third polypeptides is independently selected from the group consisting of R7, R13, R6 and RC, in R7 is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 12, R13 is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 14, R6 is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 13, and RC is a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 15; Provided that when the first polypeptide is R7, the second polypeptide is R13 and the third polypeptide is selected from the group consisting of R6 and RC, Provided that when the first polypeptide is R6, the second polypeptide is R7 and the third polypeptide is selected from the group consisting of R13 and RC, Provided that when the first polypeptide is R13, the second polypeptide is R7 and the third polypeptide is selected from the group consisting of R6 and RC, And provided that when the first polypeptide is RC, then the second polypeptide is R7 and the third polypeptide is selected from the group consisting of R13 and R6.

17. The immunogenic composition of any one of claims 1 to 16, comprising: (i) a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 12, and (ii) a protein comprising or consisting of an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or especially 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 13 and SEQ ID NO:

15.

18. The immunogenic composition of any one of claims 1 to 17 for use as a medicament, preferably as a vaccine.

19. The immunogenic composition of any one of claims 1 to 17 for use in a method of reducing or preventing one or more clinical signs, mortality or fecal excretion caused by rotavirus infection in a subject or for use in a method of treating or preventing infection by rotavirus in a subject, and / or for use in a method of inducing an immune response against rotavirus in a subject.

20. A method for reducing or preventing one or more clinical signs, mortality or fecal shedding caused by rotavirus infection in piglets, wherein the method comprises: - administering to a sow the immunogenic composition of any one of claims 1 to 17, and - allowing the sow to suckle the piglets.

21. The immunogenic composition of claim 19 or the method of claim 20, wherein the one or more clinical signs are selected from the group consisting of: -diarrhea, - Rotavirus colonization, especially in the intestine, - Lesions, especially macroscopic lesions, - Reduced average daily weight gain, and - Gastroenteritis.

22. The immunogenic composition of claim 19 or 21 or the method of claim 20 or 21, wherein - the rotavirus infection is infection with genotype P[23] rotavirus and / or genotype P[7] rotavirus, - the rotavirus infection is infection with genotype P[23] rotavirus and / or genotype P[7] rotavirus, or - The immune response against rotavirus is an immune response against genotype P[23] rotavirus and / or genotype P[7] rotavirus.

23. A method for producing an immunogenic composition as described in any one of claims 1 to 17, wherein the method comprises the following steps: (a) allowing infection of susceptible cells in culture with a vector comprising a nucleic acid sequence encoding a polypeptide as defined in any one of claims 1 to 11, wherein the polypeptide is expressed by the vector; (b) thereafter recovering the polypeptide, in particular in a cell culture supernatant, wherein preferably cell debris is separated from the polypeptide by a separation step, preferably comprising microfiltration through at least one filter, preferably two filters, wherein the pore size of the at least one filter is preferably from about 1 to about 20 μm and / or from about 0.1 μm to about 4 μm; (c) deactivating the carrier by adding diethyleneimine (BEI) to the mixture of step (b); (d) neutralizing the BEI by adding sodium thiosulfate to the mixture produced in step (c); and (e) concentrating the polypeptides in the mixture produced in step (d) by removing a portion of the liquid from the mixture through a filtration step using a filter having a membrane having a molecular weight cutoff of between about 5 kDa and about 100 kDa, preferably between about 10 kDa and about 50 kDa; And wherein the method comprises the following steps: (f) admixing the mixture remaining after step (e) with at least one immunogenic substance different from the polypeptide; (g) and optionally blending the mixture remaining after step (f) with another component selected from the group consisting of a pharmaceutically acceptable carrier, adjuvant, diluent, excipient and combinations thereof, Or wherein the method comprises the following steps: (f) blending the mixture remaining after step (e) with another component selected from the group consisting of a pharmaceutically acceptable carrier, adjuvant, diluent, excipient, and combinations thereof, and (g) admixing the mixture remaining after step (f) with at least one immunogenic substance different from the polypeptide.

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