Immunogenic compositions useful for vaccination against rotavirus - Patents.com

Abstract

The present invention relates to an immunogenic composition comprising a recombinantly constructed polypeptide useful for preparing a vaccine, in particular for reducing one or more clinical signs caused by rotavirus infection. More specifically, the present invention is directed to an immunogenic composition comprising (i) a fusion protein comprising, in the N-to-C-terminal direction, (A) an immunogenic fragment of a rotavirus VP8 protein and (B) an immunoglobulin Fc fragment, e.g., an IgG Fc fragment, and (ii) an immunogenic substance different from said fusion protein, said immunogenic composition being usable in a method for reducing one or more clinical signs, mortality or fecal shedding caused by rotavirus infection in a porcine animal.

Description

[Technical field]

[0001] The present invention relates to an immunogenic composition comprising a recombinantly constructed polypeptide useful for reducing one or more clinical signs caused by rotavirus infection. More specifically, the present invention is directed to an immunogenic composition comprising (i) a fusion protein comprising, in the N-to-C-terminal direction, (A) an immunogenic fragment of a rotavirus VP8 protein and (B) an immunoglobulin Fc fragment, such as an IgG Fc fragment, and (ii) an immunogenic substance distinct from said fusion protein, which can be used in a method of reducing one or more clinical signs, mortality or fecal shedding caused by rotavirus infection in a porcine animal. [Background technology]

[0002] Background information Rotaviruses are double-stranded RNA viruses that comprise a genus within the Reoviridae family. Rotavirus infections are known to cause gastrointestinal illness and are considered the most common cause of gastroenteritis in young children. Rotaviruses are transmitted by the fecal-oral route and infect cells lining the small intestine. Infected cells produce enterotoxins that induce gastroenteritis, resulting in severe diarrhea and sometimes death from dehydration. Rotaviruses have a genome composed of 11 segments of double-stranded RNA (dsRNA) and are currently classified into eight groups (A-H) based on antigenicity and a classification based on the sequence of the internal viral capsid protein 6 (VP6), as defined by the International Committee on Taxonomy of Viruses (ICTV) and summarized by Matthijnssens et al. (Arch Virol 157:1177-1182 (2012)) (this publication and the following publications mentioned herein are incorporated by reference in their entirety). The rotavirus genome encodes six structural proteins (VP1-VP4, VP6, and VP7) and six nonstructural proteins (NSP1-NSP6); genome segments 1-10 each encode one rotavirus protein, and genome segment 11 encodes two proteins (NSP5 and NSP6).

[0003] In the context of rotavirus A, the various strains can be classified as genotypes (defined by comparative sequence analysis and / or nucleic acid hybridization data) or serotypes (defined by serological assays) based on the structural proteins VP7 and VP4. VP7 and VP4 are components of the outermost protein layer (outer capsid) and both carry neutralizing epitopes. VP7 is a glycoprotein that forms the outer layer or surface of the virion (hence the designation "G"). VP7 determines the G type of strains, and the assigned numbers for G serotypes and G genotypes are identical. VP4 is sensitive to proteases (hence the designation "P") and determines the P type of the virus. In contrast to G types, the assigned numbers for P serotypes and genotypes are different (Santos N. et Hoshino Y., 2005, Reviews in Medical Virology, 15, 29-56). Thus, P serotypes are represented as P followed by an assigned number, and P genotypes are represented by P followed by an assigned number in parentheses (e.g., "P[7]" or "P

[13] "). 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)). Rotaviruses are also a major cause of gastroenteritis in swine animals, especially with antibodies against rotaviruses of groups A and C present in almost 100% of pigs (Vlasova et al. Viruses. 9(3): 48 (2017)). Currently, only modified live or killed vaccines are available against rotavirus A. The inability to culture rotavirus C in the laboratory hampers the development of a vaccine against this group, which in turn makes recombinant vaccines more attractive.

[0004] The creation of recombinant anti-rotavirus vaccines is hindered by the complexity of the rotavirus capsid, which is composed of four proteins arranged in three layers. The innermost layer is composed of 60 dimers of VP2 with T=1 symmetry. The VP2 layer is necessary for the proper ordering of the middle layer, which is formed by 260 trimers of VP6 with T=13 symmetry. The resulting symmetry mismatch between VP2 and VP6 results in five distinct VP6 trimer positions and three distinct pore types. In the absence of VP2, VP6 readily forms ordered high molecular weight microtubules and globules in a salt- and pH-dependent manner, which may represent a by-product of virus assembly. In the capsid, the VP6 layer binds 260 Ca of VP7, which acts as a clamp to hold the VP4 spike in place. 2+ The VP7 is glycosylated or is a G-type antigen and contains neutralizing epitopes. The majority of neutralizing antibodies recognize only trimeric VP7 and are thought to act by preventing dissociation of the VP7 trimer, which then blocks spike release. The rotavirus spike exists as 60 trimers of VP4 that are inserted into the VP6 layer only in type II pores. VP4 contains neutralizing epitopes, is a P-type antigen, and is synthesized by trypsin into the spike base VP5. * , and after cleavage, VP5 * Cell-interacting head VP8 remains associated with *Trypsin treatment primes the spike for cell entry, during which it undergoes extensive structural rearrangements to expose active sites for receptor binding in the host cell. Ignoring the complexity of the assembly process, it is difficult to achieve stoichiometric expression of rotavirus capsid proteins in environmental conditions that promote proper assembly.

[0005] Given the difficulties of rotavirus capsid assembly, there has been interest in subunit vaccine approaches. VP7 and VP4 are two proteins that contain neutralizing epitopes; however, the use of VP7 would be complicated by its glycosylation and calcium-dependent trimerization. The use of VP4 would be complicated by its trimerization, trypsinization, and range of potential conformational states. The VP8 domain, or VP8, generated by trypsinization of VP4. * The VP8 protein, also named VP8 protein, contains neutralizing epitopes, is monomeric, its structure has been determined at high resolution (Dormitzer et al. EMBO J. 21(5): 885-897 (2002)), and is described as very stable. Furthermore, within the VP8 protein, a lectin-like domain (aa65-224) is thought to interact with host receptors and be involved in virus attachment to host cells (Rodriguez et al., PloS Pathog. 10(5):e1004157 (2014)).

[0006] An approach to develop a rotavirus subunit vaccine for children is described, which contains the tetanus toxoid universal CD4 at the N-terminus. + A truncated VP8 protein (VP8 *A monovalent subunit vaccine (based on the truncated VP8 protein of rotavirus genotype P[8]) was produced in Escherichia coli (Wen et al. Vaccine. 32(35): 4420-7 (2014)) and tested in infants (Groome et al. Lancet Infect Dis.17(8):843-853 (2017)). However, this use of a monovalent subunit vaccine (based on the truncated VP8 protein of rotavirus genotype P[8]) elicited insufficient responses against heterotypic rotavirus strains, and a trivalent vaccine formulation (containing three proteins to combine genotypes P[4], P[6], and P[8] antigens) was recently tested (Groome et al. Lancet Infect Dis. S1473-3099(20)30001 (2020)). In another approach, the N-terminally truncated VP8 protein "VP8-1" (aa 26-241) was fused at the N-terminus or C-terminus to the pentamerized non-toxic B subunit of cholera toxin (CTB). Of the resulting pentameric fusion proteins (CTB-VP8-1, VP8-1-CTB), only CTB-VP8-1 (i.e., VP8-1 fused N-terminally to CTB) was considered a viable candidate for further development compared to VP8-1-CTB, which showed a strong anti-viral effect against GM1 or VP8 in mouse models. * It showed higher avidity for conformations sensitive to specific neutralizing monoclonal antibodies, elicited higher titers of neutralizing antibodies, and conferred higher protective efficacy (Xue et al. Hum Vaccin Immunother. 12(11) 2959-2968 (2016)).

[0007] However, in view of the difficulties of rotavirus capsid assembly, there is interest in alternative subunit vaccine approaches, especially since subunit vaccines are generally considered to be very safe. Also, there is a strong desire for recombinant expression of effective rotavirus subunit antigens, which allows for simple production of vaccine antigens for such rotaviruses, which are difficult to culture. Furthermore, since rotaviruses are the major cause of gastroenteritis in swine animals, there is a high need to have subunit vaccines for swine animals, especially those containing antigens that allow for efficacy comparable to or even better than the MLV rotavirus vaccines currently on the market for swine animals. Summary of the Invention

[0008] Description of the Invention The solution to the above technical problem is achieved by the descriptions and embodiments characterized in the claims. The invention in its different aspects is therefore practiced according to the appended claims. [Brief description of the drawings]

[0009] [Figure 1] Serum IgG responses against porcine rotavirus A in pigs vaccinated with either AVP8-IgG Fc protein formulated with Emulsigen D (labeled "AVP8-IgG") or placebo ("irrelevant control"). [Diagram 2] FIG. 1 shows the results of a VN (virus neutralization) assay performed to detect and quantitate antibodies capable of neutralizing porcine rotavirus A virus in samples from pigs vaccinated with AVP8-IgG Fc protein formulated with Emulsigen D (labeled "AVP8-IgG") or placebo ("irrelevant control"). [Diagram 3]Figure 1 shows the mean VN titers against rotavirus in sow sera by group and study day, where study days D0 and D28 represent the "6 weeks and 2 weeks prior to farrowing" time points (i.e. when the investigational product was administered to the T02 and T04 study groups, respectively) and study days D7, D28 and D35 represent the "5 weeks, 2 weeks and 1 week prior to farrowing" time points (i.e. when the commercial vaccine was administered to T06). [Figure 4] FIG. 1 shows group median log rotavirus A RNA genome copies (gc) / mL in stool by study day. [Diagram 5] A) SDS-PAGE of Protein A purified AVP8-IgG Fc protein (SEQ ID NO: 12) product samples either reduced with dithiothreitol ("+DTT") or non-reduced ("-DTT"); B) Western blot of AVP8-IgG Fc protein (SEQ ID NO: 12) bioreactor product, where samples were centrifuged to separate cell pellet fraction ("pellet") and supernatant fraction ("supernatant"), followed by a freeze-thaw process, and then run on SDS-PAGE under reducing conditions (+DTT), transferred to a PVDF membrane, and probed with HRP-conjugated goat anti-porcine to detect the porcine IgG Fc fragment. [Figure 6] Mean VN titers against rotavirus in sow sera by group and study day, where study days D0 and D28 represent the "6 weeks and 2 weeks prior to farrowing" time points (i.e. when the investigational product was administered to the T01 and T03 study groups, respectively). [Figure 7] Mean VN titers against rotavirus in sow sera by group and study day, where study day D0 represents the time of first administration of investigational product to the T01 and T03 study groups, respectively. [Figure 8] Results of the "IgG:AVP8 P[6] ELISA," in which ELISA plates coated with rotavirus A VP8 P[6] protein were incubated with diluted test sera, and then anti-rotavirus AVP8 P[6] antibodies were detected by using an HRP-conjugated goat anti-porcine IgG antibody. [Figure 9] Results of the "IgG:AVP8 P

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

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

[13] antibodies were detected by using an HRP-conjugated goat anti-porcine IgG antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention is based on the surprising finding that administration to sows of a fragment of the rotavirus VP8 protein, i.e. a polypeptide comprising an N-terminally extended lectin-like domain, linked at its C-terminus to an IgG Fc fragment, significantly reduced diarrhea and fecal shedding in their offspring after challenge with rotavirus, via passive transfer of neutralizing antibodies, even when said polypeptide was mixed with additional immunogenic substances prior to administration. In a first aspect, the present invention therefore provides a method for producing a method for treating a cancer cell comprising the steps of: (i)-An immunogenic fragment of the rotavirus VP8 protein, and - Immunoglobulin Fc fragment A polypeptide comprising and (ii) at least one immunogenic substance different from said polypeptide; The immunogenic composition comprising: Said immunogenic composition is hereinafter also referred to as "immunogenic composition of the invention".

[0011] Thus, the immunogenic composition of the present invention comprises, in particular, two components (i, ii): (i)-An immunogenic fragment of the rotavirus VP8 protein, and - Immunoglobulin Fc fragment A polypeptide comprising: Said polypeptides are hereinafter also referred to as "component (i) of the immunogenic composition of the invention" or "component (i)", respectively; (ii) at least one immunogenic substance different from the polypeptide, Said at least one immunogenic substance is hereinafter referred to as "component (ii) of the immunogenic composition of the invention" or "immunogenic substance (ii)" respectively. A composition comprising:

[0012] As described herein in the context of the present invention, - an immunogenic fragment of the rotavirus VP8 protein, and - an immunogenic fragment of the rotavirus VP8 protein, which is an immunoglobulin Fc fragment A polypeptide comprising the amino acid sequence of Thus, component (i) of the immunogenic composition of the invention is a polypeptide of the present disclosure. Preferably, component (ii) consists of two, three or more immunogenic substances, all of said immunogenic substances being Different from component (i), and Different from each other. In the context of the present invention, it has also been unexpectedly discovered that such immunogenic compositions can be easily produced as the polypeptides of the present disclosure, once produced in cells, are released from the cells and then recovered from the supernatant surrounding the cells, rather than from the cells themselves. The recovered polypeptides can then be easily mixed with further components, including at least one immunogenic substance different from said polypeptides, to produce the immunogenic compositions of the present invention.

[0013] A further advantage of the polypeptides of the present disclosure is that, if desired, they can be prepared as one polypeptide containing / presenting two immunogenic fragments of different rotaviruses, thereby making it unnecessary to separately prepare two different monovalent polypeptides that then have to be combined for the same purpose. Preferably, the immunoglobulin Fc fragment, as described herein, is - the C-terminus of an immunogenic fragment of said rotavirus VP8 protein, or - the N-terminus of an immunogenic fragment of said rotavirus VP8 protein is linked to. In particular, the immunoglobulin Fc fragment preferably comprises - via a linker moiety to the C-terminus of said immunogenic fragment of a rotavirus VP8 protein, or - the N-terminus of said immunogenic fragment of said rotavirus VP8 protein via a linker moiety is linked to.

[0014] In another preferred embodiment, the immunoglobulin Fc fragment, as described herein, is - 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 - 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; is linked to. Most preferably, an immunoglobulin Fc fragment, as described herein, is linked to the C-terminus of said immunogenic fragment of rotavirus VP8 protein.

[0015] Thus, the polypeptides of the present disclosure are, inter alia: - an immunogenic fragment of the rotavirus VP8 protein, and - Immunoglobulin Fc fragment is a polypeptide comprising The immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein. The term "polypeptide" as used herein refers specifically 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, "polypeptide" can refer to a long chain of amino acid residues, e.g., 150-600 amino acid residues long, or longer. The term "polypeptide" includes polypeptides having one or more post-translational modifications, e.g., 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.

[0016] The term "immunogenic fragment" is understood to refer in particular 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 understood to refer in particular to a fragment of a rotavirus VP8 protein that at least partially retains the immunogenicity of the full-length VP8 protein. "Immunogenic substance" refers to any substance capable of eliciting a humoral and / or cellular immune response, particularly a molecule such as a peptide or polypeptide that is capable of eliciting, generating or producing an immune response in an animal. The term "VP8 protein" as used herein includes "VP8 domain", "VP8 protein ... * " or "VP8 fragment of VP4." The term "immunoglobulin Fc fragment" as used herein refers to a protein that contains the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of an immunoglobulin, more specifically, does not contain the variable regions of the heavy and light chains and the light chain constant region 1 (CL1) of the immunoglobulin. It may further contain the hinge region of the immunoglobulin, or a portion of the hinge region (i.e., the hinge region of the heavy chain constant region). An immunoglobulin Fc fragment may also contain part or all of the heavy chain constant region 1 (CH1).

[0017] When the term "immunoglobulin Fc fragment" is used herein, it is understood to be equivalent to "immunoglobulin Fc domain." The term "linked to" as used herein refers in particular to any means for connecting an immunoglobulin Fc fragment to the C-terminus or N-terminus of an immunogenic fragment of a rotavirus VP protein within a polypeptide. Examples of linking means include (1.) indirect linking of an immunoglobulin Fc fragment to the C-terminus of an immunogenic fragment of a rotavirus VP8 protein by an intervening moiety that is directly linked to the C-terminus of said immunoglobulin Fc fragment and that also binds said immunoglobulin Fc fragment, and (2.) direct linking of an immunoglobulin Fc fragment to the C-terminus of an immunogenic fragment of a rotavirus VP8 protein by a covalent bond. The terms "linked to" and "linked with" are used interchangeably in the context of the present invention. In particular, "an immunogenic fragment of the rotavirus VP8 protein, and - Immunoglobulin Fc fragment A polypeptide comprising wherein the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein" is As used herein, in particular, "in the N-to-C-terminal direction, - the amino acid sequence of an immunogenic fragment of the rotavirus VP8 protein, and - Amino acid sequence of immunoglobulin Fc fragment "a polypeptide comprising "- an immunogenic fragment of the rotavirus VP8 protein, and - an immunoglobulin Fc fragment linked to the C-terminus of said immunogenic fragment It is understood that the term "a polypeptide comprising

[0018] In a most preferred embodiment, the immunoglobulin Fc fragment is linked to the C-terminus of said immunogenic fragment of rotavirus VP8 protein via a linker moiety. A linker moiety, as described herein, is in the context of the present invention preferably a peptide linker. 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.

[0019] In a particularly preferred embodiment, the immunoglobulin Fc fragment is linked to the C-terminus of said immunogenic fragment of the rotavirus VP8 protein via a linker moiety, - 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; - 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. It may also be preferred that the immunoglobulin Fc fragment is linked to 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. It will be understood that the polypeptides of the present disclosure are, inter alia, fusion proteins.

[0020] As used herein, the term "fusion protein" refers to a protein formed by fusing (i.e., linking) all or part of two or more polypeptides that are not the same. Typically, fusion proteins are made using recombinant DNA techniques by linking polynucleotides encoding two or more polypeptides end-to-end. More specifically, the term "fusion protein" thus refers to a protein translated from a nucleic acid transcript made by combining a first nucleic acid sequence encoding a first polypeptide and at least a second nucleic acid encoding a second polypeptide; a fusion protein is not a naturally occurring protein. A nucleic acid construct may encode two or more polypeptides that are linked in a fusion protein. In another preferred embodiment, the polypeptide of the present disclosure has the formula xyz, x consists of or comprises an immunogenic fragment of the rotavirus VP8 protein, y is a linker moiety, z is an immunoglobulin Fc fragment It is a fusion protein of It should be understood that the formula xyz in particular means that the C-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein is linked to the linker moiety, preferably via a peptide bond with the N-terminal amino acid residue of the linker moiety, and that the N-terminal amino acid residue of the immunoglobulin Fc fragment is linked to the linker moiety, preferably via a peptide bond with the C-terminal amino acid residue of the linker moiety. The expression "x consists of an immunogenic fragment of a rotavirus VP8 protein" is understood as being equivalent to "x is an immunogenic fragment of a rotavirus VP8 protein" as used herein.

[0021] In a preferred embodiment, an immunogenic fragment of a rotavirus VP8 protein, as referred to herein, is preferably capable of inducing an immune response against rotavirus in a subject to which said immunogenic fragment of a rotavirus VP8 protein is administered. In another preferred embodiment, the immunogenic fragment of a rotavirus VP8 protein is a polypeptide which is 50 to 200, preferably 140 to 190, amino acid residues in length. The rotavirus referred to herein is preferably selected from the group consisting of rotavirus A and rotavirus C. Thus, the immunogenic fragment of a rotavirus VP8 protein as referred to herein is preferably selected from the group consisting of an immunogenic fragment of a rotavirus A VP8 protein and an immunogenic fragment of a rotavirus C VP8 protein. The terms "Rotavirus A" and "Rotavirus C", as referred to herein, relate to Rotavirus A and Rotavirus C, respectively, as defined by ICTV (summarized by Matthijnssens et al. Arch Virol 157:1177-1182 (2012)).

[0022] According to another preferred aspect, the rotavirus referred to herein is a porcine rotavirus. In a particular preferred embodiment, the rotavirus referred to herein is rotavirus A. Thus, an immunogenic fragment of a rotavirus VP8 protein as referred to herein is preferably an immunogenic fragment of a rotavirus A VP8 protein. In a further preferred embodiment, the immunogenic fragment of the rotavirus VP8 protein comprises the lectin-like domain of the rotavirus VP8 protein. It is understood that the "lectin-like domain of the rotavirus VP8 protein" as referred to herein is preferably the lectin-like domain of the rotavirus A VP8 protein. The term "lectin-like domain of rotavirus VP8 protein" refers in particular to residues 65 to 224 of rotavirus VP8 protein, which correspond to the amino acid sequence consisting of amino acid residues 65 to 224 of rotavirus VP8 protein, and said amino acid residues 65 to 224 of rotavirus VP8 protein are preferably amino acid residues 65 to 224 of rotavirus A VP8 protein.

[0023] Therefore, the "lectin-like domain of rotavirus VP8 protein" preferably consists of the amino acid sequence of amino acid residues 65 to 224 of rotavirus VP8 protein, in particular, rotavirus A VP8 protein. Preferably, the immunogenic fragment of a rotavirus VP8 protein is an N-terminally extended lectin-like domain of a rotavirus VP8 protein, said N-terminal extension being 1 to 20 amino acid residues in length, in particular 5 to 15 amino acid residues in length, most preferably, the immunogenic fragment of a rotavirus VP8 protein is an N-terminally extended lectin-like domain of a rotavirus VP8 protein, said N-terminal extension being 8 amino acid residues in length. The amino acid sequence of said N-terminal extension is preferably a respective length of amino acid sequence adjacent to the N-terminal amino acid residue of the lectin-like domain in the amino acid sequence of the rotavirus VP8 protein. Thus, in a particular aspect, an immunogenic fragment of a rotavirus VP8 protein, as referred to herein, preferably consists of the amino acid sequence of amino acid residues 60 to 224, amino acid residues 59 to 224, amino acid residues 58 to 224, amino acid residues 57 to 224, amino acid residues 56 to 224, amino acid residues 55 to 224, amino acid residues 54 to 224, amino acid residues 53 to 224, amino acid residues 52 to 224, amino acid residues 51 to 224, amino acid residues 50 to 224, or amino acid residues 49 to 224 of a rotavirus VP8 protein, in particular a rotavirus A protein.

[0024] Most preferably, an immunogenic fragment of a rotavirus VP8 protein, as referred to herein, consists of the amino acid sequence of rotavirus VP8 protein, in particular, amino acid residues 57 to 224 of rotavirus A protein. The above numbering of amino acid residues (e.g. "65-224" or "57-224") preferably refers to the amino acid sequence of a wild-type rotavirus VP8 protein, in particular a wild-type rotavirus A VP8 protein. Said wild-type rotavirus VP8 protein is preferably the protein shown in SEQ ID NO:1. According to a further preferred embodiment, the rotavirus referred to 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, the immunogenic fragment of a rotavirus VP8 protein as referred to herein is preferably selected from the group consisting of an immunogenic fragment of a genotype P[6] rotavirus VP8 protein, an immunogenic fragment of a genotype P[7] rotavirus VP8 protein and an immunogenic fragment of a genotype P

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

[13] rotavirus A VP8 protein.

[0025] The terms "genotype P[6] rotavirus", "genotype P[7] rotavirus", "genotype P

[13] rotavirus" and "genotype P

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

[13] or P

[23] ) as 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 US A. 87(18):7155-9 (1990). Most preferably, the rotavirus referred to herein is a genotype P[7] rotavirus. Thus, the immunogenic fragment of a rotavirus VP8 protein referred to herein is most preferably an immunogenic fragment of a genotype P[7] rotavirus VP8 protein, in particular an immunogenic fragment of a genotype P[7] rotavirus A VP8 protein.

[0026] The rotavirus VP8 protein referred to in this specification 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 to the sequence of SEQ ID NO:1. The lectin-like domain of rotavirus VP8 protein, as referred to herein, 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. In one example, an immunogenic fragment of a 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 to the sequence of SEQ ID NO:3.

[0027] In another preferred embodiment, the immunogenic fragment of a rotavirus VP8 protein consists of a consensus sequence of a portion of a rotavirus VP8 protein, in particular a portion of a rotavirus A VP8 protein, or is an immunogenic fragment of said sequence. As used herein, the term "consensus sequence" refers specifically to a sequence formed from 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 occurs most frequently at that position in the family. The term "consensus sequence" thus represents a putative amino acid sequence (or nucleotide sequence). A consensus sequence represents a plurality of similar sequences. Each position in a 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.

[0028] Preferably, the consensus sequence of a portion of the rotavirus VP8 protein, as referred to herein, is - translating the plurality of nucleotide sequences encoding portions of the rotavirus VP8 protein into amino acid sequences; - aligning said amino acid sequence with known rotavirus VP8 proteins, preferably by using the MUSCLE sequence alignment software UPGMB clustering and default gap penalty parameters; - subjecting said aligned sequences to phylogenetic tree analysis and generating a neighbor-joining phylogenetic tree reconstruction based on the rotavirus VP8 protein sequences, in particular importing said aligned amino acid sequences into MEGA7 software for phylogenetic tree analysis and generating a neighbor-joining phylogenetic tree reconstruction based on the rotavirus VP8 protein sequences, - calculating the optimal tree using the Poisson correction method with bootstrap testing of the phylogenetic tree (n=100); - drawing a scaled optimal tree across all 170 positions, in units of amino acid substitutions per site, with branch lengths equal to the evolutionary distances; - considering as significant those nodes with bootstrap cluster associations higher than 70%; - designating as clusters those nodes having a distance of approximately 10% and a bootstrap cluster association of greater than 70%; and - generating a consensus sequence by selecting clusters and identifying the maximum frequency per aligned position within the clusters; - optionally selecting amino acid residues based on reported epidemiological data in conjunction with predefined product protection profiles in cases where an equivalent proportion of amino acids is observed at the aligned positions; The method may be obtained by a method comprising:

[0029] For example, in this context, an immunogenic fragment of a 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 to a sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:5. In a further preferred embodiment, the rotavirus referred to herein is rotavirus C. According to this embodiment, the immunogenic fragment of a rotavirus VP8 protein is preferably an immunogenic fragment of a rotavirus C VP8 protein. In the context of this embodiment, an immunogenic fragment of a 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 to the sequence of SEQ ID NO:6.

[0030] According to the invention, an immunogenic fragment of the rotavirus VP8 protein is therefore preferably an immunogenic fragment of the rotavirus A VP8 protein, in particular any of the immunogenic fragments of the rotavirus A VP8 protein described herein, or a portion of a rotavirus VP8 protein, for example a portion of a rotavirus A VP8 protein, preferably a consensus sequence of any of the immunogenic fragments of a rotavirus VP8 protein described herein in the context of the consensus sequence, or - an immunogenic fragment of the rotavirus C VP8 protein, in particular any of the immunogenic fragments of the rotavirus C VP8 protein described herein; It consists of or is the In a particularly preferred embodiment, the immunogenic fragment of a 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 to 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.

[0031] The immunoglobulin Fc fragments described herein are preferably at least 220 amino acid residues in length, most preferably 220-250 amino acid residues in length. According to another particular preferred embodiment, the immunoglobulin Fc fragment described herein is non-glycosylated. The term "non-glycosylated" as used herein means, inter alia, that the immunoglobulin Fc fragment does not have an oligosaccharide molecule attached thereto. Preferably, an immunoglobulin Fc fragment, as referred to herein, is an immunoglobulin Fc fragment of an immunoglobulin. - heavy chain constant region 2 (CH2), and - heavy chain constant region 3 (CH3), and - optionally a hinge region or a portion of a hinge region It comprises or consists of: According to another preferred embodiment, the immunoglobulin referred to herein is selected from the group consisting of IgG, IgA, IgD, IgE and IgM.Accordingly, the immunoglobulin Fc fragment is preferably selected from the group consisting of an IgG Fc fragment, an IgA Fc fragment, an IgD Fc fragment, an IgE Fc fragment and an IgM Fc fragment.

[0032] According to a most preferred embodiment, the immunoglobulin Fc fragment described herein is an IgG Fc fragment. IgG, as referred to herein, is preferably selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgG5 and IgG6. Thus, according to another preferred embodiment, the immunoglobulin Fc fragment referred to herein is selected from the group consisting of an IgG1 Fc fragment, an IgG2 Fc fragment, an IgG3 Fc fragment, an IgG4 Fc fragment, an IgG5 Fc fragment and an IgG6 Fc fragment. Most preferably, the immunoglobulin Fc fragment is a protein encoded by the genome of a species whose intestinal cells are susceptible to infection by the rotavirus from which the immunogenic fragment of the rotavirus VP8 protein referred to 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.

[0033] More particularly, the immunoglobulin Fc fragment is preferably a porcine IgG Fc fragment. In a further preferred embodiment, 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 in particular 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8. A linker moiety or peptide linker, respectively, as referred to herein is preferably an amino acid sequence that is 1 to 50 amino acid residues in length, in particular an amino acid sequence that is 3 to 20 amino acid residues in length. For example, the linker moiety can be a peptide linker that is 3, 8 or 10 amino acid residues in length. Depending on the purpose, a short linker may be desirable to reduce the risk of proteolysis between the fusion protein partners. Thus, the peptide linkers described in the context of the present invention preferably each have a length of 1-5 amino acid residues, more preferably 2-4 amino acid residues, and most preferably 3 amino acid residues, or consist of residues of that length.

[0034] According to a preferred embodiment, 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 in particular 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. "SEQ ID NO:9," as referred to herein, refers to the amino acid sequence (single letter code for amino acid residues): GGS, which in the traditional three letter code is Gly-Gly-Ser. Thus, "SEQ ID NO:9" in the context of the present invention refers to the amino acid sequence Gly-Gly-Ser, or, respectively, "Gly Gly Ser", as shown in the sequence of SEQ ID NO:9 in the sequence listing below. Preferably, a polypeptide of the disclosure has an N-terminal methionine residue adjacent to the N-terminal amino acid residue of an immunogenic fragment of a rotavirus VP8 protein. According to another preferred embodiment, the polypeptide of the present disclosure comprises an additional immunogenic fragment of a rotavirus VP8 protein linked to the C-terminus of said immunoglobulin Fc fragment.

[0035] The immunogenic fragment of said further rotavirus VP8 protein is preferably an immunogenic fragment of the rotavirus A VP8 protein, in particular any of the immunogenic fragments of the rotavirus A VP8 protein described herein, or - a portion of a rotavirus VP8 protein, for example a consensus sequence of a portion of a rotavirus A VP8 protein, preferably any of the immunogenic fragments of a rotavirus VP8 protein described herein in the context of the consensus sequence, or - an immunogenic fragment of a rotavirus C VP8 protein, in particular any of the immunogenic fragments of a rotavirus C VP8 protein described herein; or an immunological fragment thereof. In particular, the immunogenic fragment of the further rotavirus VP8 protein preferably comprises an amino acid sequence having preferably at least 90%, preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2 to 6, or consists of said amino acid sequence.

[0036] In a particularly preferred embodiment, said further immunogenic fragment of a rotavirus VP8 protein is different from the immunogenic fragment of a rotavirus VP8 protein which is C-terminally linked to said immunoglobulin Fc fragment. The immunogenic fragment of the further rotavirus VP8 protein is preferably linked to the C-terminus of the immunoglobulin Fc fragment via a linker moiety, in particular via any of the linker moieties described herein. Preferably, the immunogenic fragment of the further rotavirus VP8 protein is linked to the linker moiety via a peptide bond between the N-terminal amino acid residue of the immunogenic fragment of the further rotavirus VP8 protein and the C-terminal amino acid residue of the linker moiety. Alternatively, it may be preferred that the immunogenic fragment of the further 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 immunogenic fragment of the further rotavirus VP8 protein and the C-terminal amino acid residue of the immunoglobulin Fc fragment. In a particularly preferred embodiment, the polypeptide of the present disclosure 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% sequence identity to 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.

[0037] Preferably, the polypeptide of the present disclosure 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. It is understood that the expressions "consisting of an amino acid sequence" or "consists of an amino acid sequence", respectively, as used herein, also relate to any co-translational and / or post-translational one or more modifications of an amino acid sequence that are in particular influenced by the cell in which the protein or protein domain is expressed. Thus, the expressions "consisting of an amino acid sequence" or "consists of an amino acid sequence", respectively, as used herein, unless expressly stated otherwise, also cover an amino acid sequence having one or more modifications brought about by the cell in which the protein or protein domain is expressed, in particular modifications of amino acid residues brought about in protein biosynthesis and / or protein processing, preferably selected from the group consisting of glycosylation, phosphorylation and acetylation.

[0038] With regard to the term "at least 90%", when referred to in the context of the present invention, it is understood that said term preferably relates to "at least 91%", more preferably "at least 92%", even more preferably "at least 93%" or in particular "at least 94%". With regard to the term "at least 95%", when referred to in the context of the present invention, it is understood that said term preferably relates to "at least 96%", more preferably "at least 97%", even more preferably "at least 98%" or in particular "at least 99%". With regard to the term "at least 99%", when referred to in the context of the present invention, it is understood that said term preferably relates to "at least 99.2%", more preferably "at least 99.4%", even more preferably "at least 99.6%" or in particular "at least 99.8%".

[0039] 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. It is understood that the term "having 100% sequence identity" as used herein is equivalent to the term "identical." Percent sequence identity has an art-recognized meaning, and there are many methods for measuring identity between two polypeptide or polynucleotide sequences. See, e.g., Lesk, Ed., Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, Ed., Biocomputing: Informatics And Genome Projects, Academic Press, New York, (1993); Griffin & Griffin, Eds., Computer Analysis Of Sequence Data, Part I, Humana Press, New Jersey, (1994); von Heinje, Sequence Analysis In Molecular Biology, Academic Press, (1987); and Gribskov & Devereux, Eds., Sequence Analysis Primer, M Stockton Press, New York, (1991). Methods for aligning polynucleotides or polypeptides have been codified 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, which utilizes the FASTA algorithm, can be used, using an affine gap search with a gap opening penalty of -12 and a gap extension penalty of -2. For the purposes of the present invention, nucleotide sequences are aligned using the Clustal W method in MegAlign software, version 11.1.0(59),419 by DNASTAR Inc., using the default multiple alignment parameters set in this program (gap penalty=15.0, gap length penalty=6.66, and delayed mismatched sequences (%)=30%, DNA transfer weight=0.50, and DNA weight matrix=IUB), and protein / amino acid sequences are aligned using the Clustal W method in MegAlign software, version 11.1.0(59),419 by DNASTAR Inc., using the default multiple alignment parameters set in this program (gap penalty=10.0, gap length penalty=0.2, and Gonnet series protein weight matrix with delayed mismatched sequences (%)=30%), respectively.

[0040] As used herein, the term "sequence identity to the sequence of SEQ ID NO:X" is specifically understood to be equivalent to the terms "sequence identity to the sequence of SEQ ID NO:X over the length of SEQ ID NO:X" or "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, and thus "SEQ ID NO:X" represents any of the SEQ ID NOs referred to herein. The expression "the group consisting of SEQ ID NO:[...], ... and SEQ ID NO:[...]" is interchangeable with "the group consisting of sequence SEQ ID NO:[...], ... and sequence SEQ ID NO:[...]" in this context is a placeholder for the number of the sequence. For example, the expression "the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6" is interchangeable with "the group consisting of sequence SEQ ID NO:3, sequence SEQ ID NO:4, sequence SEQ ID NO:5 and sequence SEQ ID NO:6".

[0041] According to another particularly preferred aspect, the polypeptide of the present disclosure comprises: - an immunogenic fragment of a rotavirus VP8 protein, in particular any of the immunogenic fragments of a rotavirus VP8 protein described herein, - an N-terminal methionine residue adjacent to the N-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein, and - an immunoglobulin Fc fragment, in particular any of the immunoglobulin Fc fragments described herein, an immunoglobulin Fc fragment, in particular linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein via a linker moiety, preferably any of the linker moieties described herein; and - optionally, in particular an immunogenic fragment of a further rotavirus VP8 protein linked to the C-terminus of said immunoglobulin Fc fragment via a linker moiety, said immunogenic fragment of said further rotavirus VP8 protein being preferably any of the immunogenic fragments of a further rotavirus VP8 protein described herein, said linker moiety being preferably any of the linker moieties described herein. It consists of:

[0042] In yet further preferred embodiments, the polypeptides of the present disclosure form dimers with additional polypeptides of the present disclosure. Most preferably, the polypeptides of the present disclosure form homodimers with additional identical polypeptides. Thus, it is specifically understood that the term "polypeptide of the disclosure" further encompasses any dimer composed of two polypeptides of the disclosure, and in particular encompasses any homodimer composed of two identical polypeptides of the disclosure. According to another particularly preferred aspect, the present disclosure provides a multimer comprising or consisting of a plurality of polypeptides of the present disclosure, said multimer being hereinafter also referred to as "multimer of the present disclosure". Preferably, the multimers of this disclosure are homodimers formed by one polypeptide of this disclosure with an additional identical polypeptide of this disclosure. In particular, the term "multimer of the present disclosure" refers to any mixture of different multimers of the present disclosure, e.g. - homodimers formed by one polypeptide of the present disclosure with a further identical polypeptide of the present disclosure, and - one or more multimers formed by more than two of the same polypeptides of the present disclosure It is understood that the term further encompasses mixtures of.

[0043] Thus, in a particular preferred embodiment, component (i) of the immunogenic composition of the invention is - a monomer consisting of one of the polypeptides of the present disclosure, and / or - a homodimer consisting of two identical polypeptides of the present disclosure, and / or - optionally a homotrimer consisting of three identical polypeptides of the present disclosure exists in Preferably, - each of said two identical polypeptides of the disclosure, and - optionally, each of said three identical polypeptides of the disclosure comprises the same amino acid sequence as one of the polypeptides of the present disclosure or consists of said amino acid sequence. Or more generally, the present invention relates to a method for producing a composition comprising: (i)-An immunogenic fragment of the rotavirus VP8 protein, and - Immunoglobulin Fc fragment A polypeptide comprising: Optionally, the polypeptide is comprised in a multimer that comprises or is composed of a plurality of the polypeptides, and (ii) at least one immunogenic substance different from said polypeptide; The present invention also includes an immunogenic composition comprising:

[0044] The at least one immunogenic substance different from said polypeptide described herein preferably consists of two or more immunogenic substances, all of said immunogenic substances being different from the polypeptide; and Different from each other. Thus, component (ii) of the immunogenic composition of the invention preferably consists of two or more immunogenic substances, each of said immunogenic substances being different from component (i) of the immunogenic composition of the invention, and all of said immunogenic substances being different from each other. Preferably, said 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 the immunogenic fragment of said component (i).

[0045] As used herein, the term "reovirus antigen" refers specifically to a peptide or protein, or portion thereof, that contains an epitope recognized by an antibody specific for a virus belonging to the Reoviridae family. In a preferred embodiment, said 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, all of said antigens being different from said immunogenic fragment and 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, all of said antigens being different from said immunogenic fragment and different from each other. In another preferred embodiment, the at least one immunogenic substance different from said polypeptide is at least one protein comprising or consisting of a reovirus antigen different from said immunogenic fragment.Thus, component (ii) is preferably at least one protein comprising or consisting of a reovirus antigen different from the immunogenic fragment of said component (i).

[0046] In an even further preferred embodiment, said 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, all of said reovirus antigens being different from said immunogenic fragment and 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, all of said antigens being different from said immunogenic fragment and different from each other. Preferably, said at least one immunogenic substance different from said polypeptide is at least one immunogenic substance comprising a rotavirus antigen different from said immunogenic fragment. Thus, component (ii) is preferably at least one immunogenic substance comprising a rotavirus antigen different from said immunogenic fragment of component (i). The term "rotavirus antigen" as used herein refers in particular to a peptide or protein, or a part thereof, that contains an epitope recognized by an antibody specific for a virus belonging to the genus Rotavirus.

[0047] In a preferred embodiment, said 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, all of said antigens being different from said immunogenic fragment and 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, all of said antigens being different from said immunogenic fragment and different from each other. In another preferred embodiment, said 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 the immunogenic fragment of said component (i). In an even further preferred embodiment, said 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, all of said rotavirus antigens being different from said immunogenic fragment and 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, all of said antigens being different from said immunogenic fragment and different from each other.

[0048] In a particularly preferred embodiment, the immunogenic composition of the present invention is preferably an immunogenic composition comprising: (i) the polypeptide is a first polypeptide, (ii) the at least one immunogenic agent distinct from said polypeptide is at least one additional polypeptide distinct from said first polypeptide. Preferably, the at least one immunogenic agent is a second polypeptide comprising or consisting of a reovirus antigen different from said immunogenic fragment; or include, optionally, a third polypeptide different from both said first and second polypeptides; Optionally, it contains a fourth polypeptide that is different from all of the first to third polypeptides. Preferably, the third polypeptide comprises a reovirus antigen that is distinct from both the immunogenic fragment and the reovirus antigen of the second polypeptide.

[0049] The fourth polypeptide is preferably - said immunogenic fragment, - a reovirus antigen of a second polypeptide, and - a reovirus antigen of a third polypeptide The reovirus antigens are distinct from all of the above. In another particularly preferred embodiment, Component (i) of the immunogenic composition of the invention is a first polypeptide, Component (ii) of the immunogenic composition of the invention is at least one additional polypeptide different from said first polypeptide, Preferably, component (ii) is - comprises or is a second polypeptide comprising a rotavirus antigen different from the immunogenic fragment of component (i), - a third polypeptide, preferably different from both said first and second polypeptides, - optionally comprising a fourth polypeptide different from all of said first to third polypeptides.

[0050] Preferably, said third polypeptide comprises a rotavirus antigen which is different from both the immunogenic fragment of component (i) and the rotavirus antigen of the second polypeptide. According to a preferred embodiment, the second polypeptide is any of the polypeptides of the present disclosure as described herein, with the proviso that the second polypeptide is different from the first polypeptide, Preferably, said third polypeptide is any of the polypeptides of the present disclosure as described herein, with the proviso that said third polypeptide is different from both said first and second polypeptides; Optionally, the fourth polypeptide is any of the polypeptides of the present disclosure as described herein, with the proviso that the fourth polypeptide is different from all of the first through third polypeptides.

[0051] In another preferred embodiment, the immunogenic composition of the present invention is preferably an immunogenic composition (i) the polypeptide is - a first immunogenic fragment of the rotavirus VP8 protein, and - Immunoglobulin Fc fragment a first polypeptide comprising (ii) at least one immunogenic substance different from the polypeptide, - a second polypeptide comprising a second immunogenic fragment of a rotavirus VP8 protein, said second immunogenic fragment being different from said first immunogenic fragment, and - a third polypeptide, preferably comprising a third immunogenic fragment of a rotavirus VP8 protein, said third immunogenic fragment being different from both said first and second immunogenic fragments, and - optionally a fourth polypeptide comprising a fourth immunogenic fragment of a rotavirus VP8 protein, said fourth immunogenic fragment being different from all of said first to third immunogenic fragments; or consisting of said polypeptide.

[0052] Preferably, each of the second to fourth immunogenic fragments is - an immunogenic fragment of a rotavirus A VP8 protein, in particular any of the immunogenic fragments of a rotavirus A VP8 protein described herein, - a portion of a rotavirus VP8 protein, such as a consensus sequence of a portion of a rotavirus A VP8 protein, preferably any of the immunogenic fragments of a rotavirus VP8 protein described herein in the context of the consensus sequence, and - an immunogenic fragment of the rotavirus C VP8 protein, in particular any of the immunogenic fragments of the rotavirus C VP8 protein described herein; are individually selected from the group consisting of In particular, however, all of the second to fourth immunogenic fragments are different from the first immunogenic fragment and different from each other.

[0053] More specifically, The first immunogenic fragment is selected from the group consisting of X7, X13, X6 and XC; and / or the second immunogenic fragment is selected from the group consisting of X7, X13, X6 and XC; and / or the third immunogenic fragment is selected from the group consisting of X7, X13, X6 and XC; and / or the fourth immunogenic fragment is selected from the group consisting of X7, X13, X6 and XC; X7 represents an immunogenic fragment of the genotype P[7] rotavirus VP8 protein; X13 represents an immunogenic fragment of the genotype P

[13] rotavirus VP8 protein; X6 represents an immunogenic fragment of the genotype P[6] rotavirus VP8 protein; XC represents an immunogenic fragment of the rotavirus C VP8 protein, In particular, however, all of the second to fourth immunogenic fragments are different from the first immunogenic fragment and different from each other. Thus, when referred to herein, "X7" means an immunogenic fragment of the genotype P[7] rotavirus VP8 protein; "X13" means an immunogenic fragment of the genotype P

[13] rotavirus VP8 protein; "X6" means an immunogenic fragment of the genotype P[6] rotavirus VP8 protein; "XC" means an immunogenic fragment of rotavirus C VP8 protein.

[0054] X7, as referred to herein, 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 to the sequence of SEQ ID NO:3. X13, as referred to herein, 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 to the sequence of SEQ ID NO:5. X6, as referred to herein, 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. XC, as referred to herein, 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.

[0055] In particular, said at least one immunogenic substance different from said polypeptide comprises or consists of a second polypeptide comprising a second immunogenic fragment of a rotavirus VP8 protein, each of the first and second immunogenic fragments is individually selected from the group consisting of X7, X13, X6 and XC; With the proviso that when the first immunogenic fragment is X7, the second immunogenic fragment is selected from the group consisting of X13, X6 and XC; With the proviso that when the first immunogenic fragment is X6, the second immunogenic fragment is selected from the group consisting of X7, X13 and XC; With the proviso that if the first immunogenic fragment is X13, the second immunogenic fragment is selected from the group consisting of X7, X6 and XC; However, when the first immunogenic fragment is XC, the second immunogenic fragment is selected from the group consisting of X7, X13 and X6.

[0056] More specifically, the at least one immunogenic substance different from the polypeptide is a second polypeptide comprising a second immunogenic fragment of a rotavirus VP8 protein, and a third polypeptide comprising a third immunogenic fragment of the rotavirus VP8 protein. or consisting of the polypeptide Each of the first to third immunogenic fragments is individually selected from the group consisting of X7, X13, X6, and XC; With the proviso that when the first immunogenic fragment is X7, the second immunogenic fragment is X13, and the third immunogenic fragment is selected from the group consisting of X6 and XC; With the proviso that if the first immunogenic fragment is X6, the second immunogenic fragment is X7, and the third immunogenic fragment is selected from the group consisting of X13 and XC; With the proviso that if 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; With the proviso that if the first immunogenic fragment is XC, the second immunogenic fragment is X7, and the third immunogenic fragment is selected from the group consisting of X13 and X6.

[0057] Even more particularly, said at least one immunogenic substance different from said polypeptide is 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 a fourth polypeptide comprising a fourth immunogenic fragment of the rotavirus VP8 protein; or consisting of the polypeptide Each of the first to fourth immunogenic fragments is individually selected from the group consisting of X7, X6, X13, and XC; With the proviso that if the first immunogenic fragment is X7, the second immunogenic fragment is X13, the third immunogenic fragment is X6, and the fourth immunogenic fragment is XC; With the proviso that if the first immunogenic fragment is X6, the second immunogenic fragment is X7, the third immunogenic fragment is X13, and the fourth immunogenic fragment is XC; With the proviso that if the first immunogenic fragment is X13, the second immunogenic fragment is X7, the third immunogenic fragment is X6, and the fourth immunogenic fragment is XC; However, if the first immunogenic fragment is XC, the second immunogenic fragment is X7, the third immunogenic fragment is X13, and the fourth immunogenic fragment is X6.

[0058] Preferably, the first immunogenic fragment referred to herein is an immunogenic fragment of the VP8 protein of genotype P[7] rotavirus (X7); and / or The second immunogenic fragment referred to herein is an immunogenic fragment of the genotype P

[13] rotavirus VP8 protein (X13); and / or The third immunogenic fragment referred to herein is an immunogenic fragment (X6) of the genotype P[6] rotavirus VP8 protein; and / or The fourth immunogenic fragment referred to herein is the immunogenic fragment of the rotavirus C VP8 protein (XC). Most preferably, the first immunogenic fragment is an immunogenic fragment of the genotype P[7] rotavirus VP8 protein (X7); the second immunogenic fragment is an immunogenic fragment of genotype P

[13] rotavirus VP8 protein (X13); the third immunogenic fragment is an immunogenic fragment (X6) of genotype P[6] rotavirus VP8 protein; Optionally, said fourth immunogenic fragment is an immunogenic fragment of rotavirus C VP8 protein (XC).

[0059] Preferably, 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 to the sequence of SEQ ID NO: 3; 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 to the sequence of SEQ ID NO:5; 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 to the sequence of SEQ ID NO: 4; 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 to the sequence of SEQ ID NO:6.

[0060] In another preferred embodiment, The first polypeptide, as referred to herein, is selected from the group consisting of R7, R13, R6 and RC; and / or The second polypeptide, as referred to herein, is selected from the group consisting of R7, R13, R6 and RC; and / or the third polypeptide is selected from the group consisting of R7, R13, R6 and RC; and / or the fourth polypeptide is selected from the group consisting of R7, R13, R6, and RC; 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 in particular 100% sequence identity to 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 in particular 100% sequence identity to 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 in particular 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 13, 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 in particular 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 15, In particular, provided that all of said second to fourth polypeptides are different from said first polypeptide and different from each other.

[0061] Preferably, the at least one immunogenic substance different from the first polypeptide comprises or consists of a second polypeptide of a rotavirus VP8 protein, each of the first and second polypeptides is individually selected from the group consisting of R7, R13, R6, and RC; With the proviso that when said first polypeptide is R7, said second polypeptide is selected from the group consisting of R13, R6, and RC; With the proviso that when the first polypeptide is R6, the second polypeptide is selected from the group consisting of R7, R13, and RC; With the proviso that when said first polypeptide is R13, said second polypeptide is selected from the group consisting of R7, R6, and RC; With the proviso that when said first polypeptide is RC, said second polypeptide is selected from the group consisting of R7, R13 and R6.

[0062] Specifically, the at least one immunogenic substance different from the first polypeptide is A second polypeptide, and Third Polypeptide or consisting of the polypeptide each of the first to third polypeptides is individually selected from the group consisting of R7, R13, R6, and RC; with the proviso that if the first polypeptide is R7, the second polypeptide is R13, and the third polypeptide is selected from the group consisting of R6 and RC; with the proviso that if the first polypeptide is R6, the second polypeptide is R7, and the third polypeptide is selected from the group consisting of R13 and RC; with the proviso 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; With the proviso that if said first polypeptide is RC, said second polypeptide is R7, and said third polypeptide is selected from the group consisting of R13 and R6.

[0063] More specifically, the at least one immunogenic substance different from the polypeptide is A second polypeptide, and a third polypeptide, and The fourth polypeptide or consisting of the polypeptide each of the first to fourth polypeptides is individually selected from the group consisting of R7, R6, R13, and RC; with the proviso that, if the first polypeptide is R7, then the second polypeptide is R13, the third polypeptide is R6, and the fourth polypeptide is RC; With the proviso that, if the first polypeptide is R6, then the second polypeptide is R7, the third polypeptide is R13, and the fourth polypeptide is RC; with the proviso that, if the first polypeptide is R13, then the second polypeptide is R7, the third polypeptide is R6, and the fourth polypeptide is RC; With the proviso that if said first polypeptide is RC, said second polypeptide is R7, said third polypeptide is R13, and said fourth polypeptide is R6.

[0064] According to a preferred embodiment, The first polypeptide referred to herein 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 in particular 100% sequence identity with the sequence of SEQ ID NO: 12, The second polypeptide referred to herein 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 in particular 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, Preferably, the third polypeptide referred to herein 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 in particular 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, Optionally, the fourth polypeptide referred to herein 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 in particular 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.

[0065] in particular, (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 in particular 100% sequence identity with the sequence 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 in particular 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. Preferred is an immunogenic composition of the invention comprising:

[0066] More specifically, (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 in particular 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 in particular 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 14, and 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 in particular 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 13, 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 in particular 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 15. Preferred is an immunogenic composition of the invention comprising:

[0067] The immunogenic compositions of the invention preferably comprise each of components (i) and each of the substances of 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. According to another preferred aspect, the immunogenic composition of the invention contains each of the components (i) and each of the substances of the components (ii) at a concentration of 100 nM to 50 μM, preferably 250 nM to 25 μM, and most preferably 1 to 10 μM. In particular, 1 mL, or in some cases, 2 mL, of the immunogenic composition of the invention is administered to a subject. Thus, the dose of the immunogenic composition of the invention administered to a subject preferably has a volume of 1 mL or 2 mL. Preferably, one or two doses of the immunogenic composition are administered to the subject. The immunogenic composition of the present invention is preferably administered systemically or locally. The preferred routes of administration commonly used are parenteral or oral administration, such as intramuscular, intradermal, intravenous, intraperitoneal, subcutaneous, intranasal, and inhalation. However, depending on the nature and mechanism of action of the compound, the immunogenic composition may also be administered by other routes. It is most preferred that the immunogenic composition is administered intramuscularly. The immunogenic compositions of the invention preferably further comprise a pharma- ceutically or veterinarily acceptable carrier or excipient. As used herein, "pharmaceutically or veterinarily acceptable carriers" includes any and all solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption retarding agents, etc. In some preferred embodiments, particularly those involving lyophilized immunogenic compositions, stabilizers for use in the present invention include stabilizers for lyophilization or freeze-drying.

[0068] In some embodiments, the immunogenic compositions of the invention contain an adjuvant. "Adjuvants" as used herein may include aluminum hydroxide and aluminum phosphate, saponins such as Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions. The emulsions may be based on light liquid paraffin oil (European Pharmacopoeia type), isoprenoid oils such as squalane or squalene, oils obtained from the oligomerization of alkenes, especially isobutene or decene, esters of acids or alcohols containing linear alkyl groups, more specifically 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 isostearate esters. The oil is used in combination with an emulsifier to form an emulsion. The emulsifier is preferably a non-ionic surfactant, in particular sorbitan, mannide (e.g., anhydrous mannitol oleate), glycol, polyglycerol, propylene glycol, and oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid esters, which may be ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular Pluronic products, in particular L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed.Stewart-Tull, DES), JohnWiley and Sons, NY, pp51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997).An exemplary adjuvant is the SPT emulsion described on page 147 in "Vaccine Design, The Subunit and Adjuvant Approach" edited by M. Powell and M. Newman, Plenum Press, 1995, or the emulsion MF59 described on page 183 of the same book.

[0069] Further examples of adjuvants are compounds selected from polymers of acrylic or methacrylic acid and copolymers of maleic anhydride and alkenyl derivatives. Advantageous adjuvant compounds are polymers of acrylic or methacrylic acid that are crosslinked, in particular with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol.8, No.2, June 1996). The skilled artisan may also refer to US Pat. No. 2,909,462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least three hydroxyl groups, preferably not more than eight hydroxyl groups, in which at least three hydroxyl hydrogen atoms are replaced by unsaturated aliphatic groups having at least two carbon atoms. Preferred groups are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. The unsaturated groups may themselves contain other substituents, such as methyl. Particularly suitable are the products sold under the name CARBOPOL® (BF Goodrich, Ohio, USA). They are crosslinked with allyl sucrose or allyl pentaerythritol. Among them, Carbopol 974P, 934P and 971P may be mentioned. The use of CARBOPOL® 971P is most preferred. Among them, the copolymer of maleic anhydride and alkenyl derivative is the copolymer EMA (Monsanto), which is a copolymer of maleic anhydride and ethylene. The dissolution of these polymers in water results in an acid solution that is preferably neutralized to physiological pH to give an adjuvant solution that is incorporated into the immunogenic, immunological or vaccine composition itself.

[0070] Additional suitable adjuvants that may be selected include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), block copolymers (CytRx, Atlanta Ga), SAF-M (Chiron, Emeryville Calif.), monophosphoryl lipid A, avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, IMS1314 or muramyl dipeptide, or naturally occurring or recombinant cytokines or analogs thereof, or stimulators of endogenous cytokine release, among many others. It is expected that the adjuvant may be added in an amount of about 100 μg to about 10 mg per dose, preferably about 100 μg to about 10 mg per dose, more preferably about 500 μg to about 5 mg per dose, even more preferably about 750 μg to about 2.5 mg per dose, and most preferably about 1 mg per dose. Alternatively, the adjuvant may be at a concentration of about 0.01 to 50% of the volume of the final product, preferably about 2% to 30%, more preferably about 5% to 25%, even more preferably about 7% to 22%, and most preferably 10% to 20%.

[0071] "Diluents" may include water, saline, dextrose, ethanol, glycerol, etc. Isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkali salts of ethylenediaminetetraacetic acid, among others. According to a particularly preferred aspect, the present invention also provides an immunogenic composition, in particular an immunogenic composition of the invention, said immunogenic composition comprising: component (i), component (ii), - a pharma- ceutically or veterinarily acceptable carrier or excipient, and - optionally an adjuvant It comprises or consists of: Adjuvants in the context of the present invention are preferably selected from the group consisting of emulsified oil-in-water adjuvants and carbomers. The term "immunogenic composition" refers to a composition comprising at least one antigen that induces an immunological response in a host to which the immunogenic composition is administered. Such an immunological response may be a cellular immune response and / or an antibody-mediated immune response to the immunogenic composition according to the invention. The host is also described as a "subject". Preferably, any host or subject described or referred to herein is an animal. The term "animal", as used herein, particularly relates to mammals, preferably porcine animals, more preferably pigs, most preferably piglets.

[0072] Typically, an "immunological response" includes, but is not limited to, one or more of the following effects: production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells and / or gamma-delta T cells specifically directed against one or more antigens comprised in the immunogenic composition of the invention. Preferably, the host mounts either a protective immunological response or a therapeutic response. A "protective immunological response" is demonstrated by either a reduction or absence of one or more clinical signs normally displayed by an infected host, a more rapid recovery time and / or a reduced duration of infection, or a lower pathogen titer in the tissues or body fluids or excretions of the infected host. "Pathogen" or "particular pathogen" as referred to herein relates in particular to the rotavirus from which the immunogenic fragment of the rotavirus VP8 protein is derived. For example, as referred to herein, the pathogen is rotavirus A or rotavirus C.

[0073] If the host mounts a protective immunological response such that resistance to new infection is enhanced and / or the clinical severity of disease is reduced, the immunogenic composition is described as a "vaccine." "Antigen" as used herein refers to a component that induces an immunological response in a host to a subject immunological composition or vaccine that contains such antigen or an immunologically active component thereof, without limitation. In particular, the term "antigen" as used herein refers to a protein or protein domain that, when administered to a host, can induce an immunological response in the host. The term "treatment and / or prevention" refers to reducing the occurrence of a particular pathogen infection in a population, or reducing the severity of one or more clinical signs caused or associated with a particular pathogen infection. Thus, the term "treatment and / or prevention" refers to reducing the number of animals in a population that become infected with a particular pathogen (=reducing the occurrence of a particular pathogen infection), or reducing the severity of one or more clinical signs normally associated with or caused by an infection by a pathogen in a group of animals in which the animals have received an effective amount of an immunogenic composition provided herein, compared to a group of animals in which the animals have not received such immunogenic composition.

[0074] "Treatment and / or prevention" generally involves administration of an effective amount of the immunogenic composition of the present invention to a subject or a population of subjects in need of or who may benefit from such treatment / prevention. The term "treatment" refers to administration of an effective amount of the immunogenic composition at a time when subjects or at least some animals of a population are already infected with such pathogen and such animals are already showing some clinical signs caused by or associated with such pathogen infection. The term "prevention" refers to administration to a subject prior to any infection of such subjects with a pathogen or at least when all animals in such animal or group of animals are not showing one or more clinical signs caused by or associated with infection by such pathogen. The term "effective amount" as used herein means, without limitation, an amount of an antigen, particularly a polypeptide of the present disclosure, that induces or can induce an immune response in a subject. Such an effective amount can reduce the occurrence of a particular pathogen infection in a population or reduce the severity of one or more clinical signs of a particular pathogen infection. Preferably, one or more clinical signs are reduced in occurrence or severity 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 either untreated or subjects treated with the immunological composition applicable before the present invention but subsequently infected with a particular pathogen.

[0075] The term "clinical signs" as used herein refers to signs of infection in a subject from a particular pathogen. Clinical signs of infection depend on the pathogen selected. Examples of such clinical signs include, but are not limited to, diarrhea, vomiting, fever, abdominal pain, and dehydration. Reduction in the appearance or reduction in the severity of one or more clinical signs caused by or associated with a particular pathogen infection in a subject can be achieved by administration to the subject of one or more doses of an immunogenic composition of the invention. The term "reducing fecal shedding" refers to, but is not limited to, a reduction in the number of RNA copies of a pathogenic virus, such as rotavirus, per mL of feces, or the number of plaque-forming colonies per deciliter of feces, which is reduced in the feces of a subject receiving the composition of the present invention by at least 50% compared to a subject who has not received the composition and may be infected. More preferably, fecal shedding levels are reduced by at least 90%, preferably at least 99.9%, more preferably at least 99.99%, and even more preferably at least 99.999% in subjects receiving the composition of the present invention.

[0076] The term "fecal shedding", as used herein, is used according to its plain and ordinary meaning in medicine and virology and refers to the production and release of virus from a subject's cells into the environment from an infected subject via the subject's feces. The polypeptides of the present disclosure are preferably recombinant proteins, particularly recombinant baculovirus-expressed proteins. The term "recombinant protein" as used herein refers specifically to a protein produced by recombinant DNA techniques; typically, DNA encoding the expressed protein is 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, the term "recombinant protein" as used herein refers specifically to a protein molecule expressed from a recombinant DNA molecule. A "recombinant DNA molecule" as used herein refers to a DNA molecule that is composed of segments of DNA joined together by molecular biological techniques. Suitable systems for the production of recombinant proteins include, but are not limited to, insect cells (e.g., baculovirus), prokaryotic systems (e.g., E. coli), fungi (e.g., Myceliophthora thermophile, Aspergillus oryzae, Ustilago maydis), yeast (e.g., Saccaromyces 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 hemolysate).

[0077] According to another aspect, the present disclosure provides a polynucleotide comprising a sequence encoding a polypeptide of the present disclosure, said polynucleotide hereinafter also referred to as a "polynucleotide according to the present disclosure", preferably an isolated polynucleotide. Preferably, a polynucleotide according to the present disclosure comprises a nucleotide 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:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.

[0078] Production of the polynucleotides described herein is within the skill of the art and can be carried out according to recombinant techniques described in, among others, Sam brook 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. In a still further aspect, the disclosure provides a vector containing a polynucleotide encoding a polypeptide of the disclosure.

[0079] "Vector" and "vector containing a polynucleotide encoding a polypeptide of the disclosure", for purposes 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, infect a host cell so as to produce the protein or polypeptide encoded by the DNA. Vectors and methods for making and / or using vectors (or recombinants) for expression are described in U.S. Patent 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, and 382,425; PCT publications WO 94 / 16716, WO 96 / 39491, and 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 (Editor), Methods in Molecular Biology 39, "Baculovirus Expression Protocols" (1995 Humana Press Inc.); 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, p.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;European Patent Application No. 0370573;U.S. Application No. 920,197, filed October 16, 1986;European Patent Application Publication No. 265785;U.S. Patent No. 4,769,331 (recombinant herpes viruses);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: 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 Nos. 08 / 675,556 and 08 / 675,566, both filed July 3, 1996 and allowed (recombinant adenovirus);Grunhaus et al., 1992, "Adenovirus as cloning vectors," Seminars in Virology (Vol.3) p.237-52, 1993;Ballay et al.EMBO Journal, vol.4, p.3861-65, Graham, Tibtech 8, 85-87, April, 1990;Prevec et al., J.Gen Virol.70, 42434;PCT International Publication No. 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, as well as WO 90 / 11092, WO 93 / 19183, WO 94 / 21797, WO 95 / 11307, WO 95 / 20660; inter alia, Tang et al., Nature and Furth et al., concerning DNA expression vectors., Analytical Biochemistry, or in a manner analogous thereto. See also WO 98 / 33510; Ju et al., Diabetologia, 41: 736-739, 1998 (lentiviral expression systems); 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 systems); Szokka et al., U.S. Pat. No. 4,394,448 (method of 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), as well as other references cited herein.

[0080] Preferred viral vectors include baculoviruses, such as BaculoGold (BD Biosciences Pharmingen, San Diego, Calif.), particularly if the production cells are insect cells. Although the baculovirus expression system is preferred, it will be understood by those of skill in the art that other expression systems, including those described above, will work for the purposes of the present invention, i.e., for expression of recombinant proteins. Thus, the present disclosure also provides a baculovirus containing a polynucleotide comprising a sequence encoding a polypeptide of the present disclosure. Said baculovirus, hereinafter also referred to as "the baculovirus according to the present disclosure", is preferably an isolated baculovirus.

[0081] Furthermore, the present disclosure therefore also provides a plasmid, preferably an expression vector, comprising a polynucleotide comprising a sequence encoding a polypeptide of the present disclosure. Said plasmid, hereinafter also referred to as "plasmid according to the present disclosure", is in particular an isolated plasmid. The present disclosure also provides a cell infected with and / or containing a baculovirus comprising a polynucleotide comprising a sequence encoding a polypeptide of the present disclosure, or a plasmid comprising a polynucleotide comprising a sequence encoding a polypeptide of the present disclosure, preferably an expression vector. Said cell, hereinafter also referred to as "a cell according to the present disclosure", is preferably an isolated cell. The term "isolated" when used in reference to an isolated cell is a cell that exists apart from its natural environment by the hand of man, and thus is not a product of nature. In yet another aspect, the present invention relates to the use of the immunogenic composition of the present invention for preparing a medicament, preferably a vaccine.

[0082] In this context, the present invention also provides a method for producing the immunogenic composition of the invention, said method comprising the step of infecting a cell, preferably an insect cell, with a baculovirus according to the invention. Furthermore, the present disclosure also provides a method of producing a polypeptide of the present disclosure, the method comprising transfecting a cell with a plasmid according to the present disclosure. The polypeptides of the present disclosure are preferably expressed in sufficiently high amounts for stable self-assembly into virus-like particles, which can then be used for vaccination. The term "vaccination" or "vaccinating", as used herein, refers, without limitation, to a process that includes the administration of an antigen, e.g., an antigen comprised in an immunogenic composition, to a subject, where said antigen, e.g., a polypeptide of the present disclosure, when administered to a subject, induces or is capable of eliciting a protective immunological response in said subject. The present invention also provides an immunogenic composition of the invention for use as a medicament, preferably as a vaccine.

[0083] In particular, the immunogenic compositions of the invention are provided for use in a method of reducing or preventing one or more clinical signs or disease caused by rotavirus infection, wherein the rotavirus is preferably of the genus Rotavirus having a genome encoding an immunogenic fragment of the rotavirus VP8 protein. The immunogenic compositions of the invention are provided for use in a method of reducing or preventing fecal shedding caused by rotavirus infection, wherein the virus is preferably of the genus Rotavirus having a genome encoding an immunogenic fragment of the rotavirus VP8 protein. Thus, in one particular example, when the immunogenic fragment of the rotavirus VP8 protein referred to herein is encoded by the genome of Rotavirus A, the immunogenic compositions of the invention are for use in a method of reducing or preventing one or more clinical signs, mortality, fecal shedding or disease caused by infection with Rotavirus A. More specifically, the immunogenic compositions of the invention are provided for use in a method of reducing or preventing one or more clinical signs, mortality or fecal shedding caused by rotavirus infection in a subject, or for use in a method of treating or preventing infection by rotavirus in a subject. Rotavirus infection, as referred to herein, refers in particular to infection with rotavirus A or rotavirus C.

[0084] Furthermore, the immunogenic compositions of the invention are provided for use in methods for inducing an immune response against rotavirus in a subject. The subject as referred to herein is preferably a mammal, for example a porcine or bovine animal, or an avian, for example a chicken. In particular, the subject is a pig, and the pig is preferably a piglet or a sow, for example a pregnant sow. Most preferably, in the context of inducing an immune response against rotavirus in a subject, the subject is a pregnant sow. In the context of reducing or preventing one or more clinical signs, mortality or fecal shedding caused by rotavirus infection in a subject, or treating or preventing infection by rotavirus in a subject, the subject is most preferably a piglet. According to a preferred aspect, the immunogenic composition of the invention is for use in a method for reducing or preventing one or more clinical signs, mortality or fecal shedding caused by rotavirus infection in piglets, wherein the piglets are suckled by a sow to which the immunogenic composition has been administered, preferably a sow to which the immunogenic composition has been administered whilst the sow is pregnant, in particular whilst it is pregnant with the piglets.

[0085] Furthermore, the present invention relates to a method for the treatment or prevention of a rotavirus infection, the reduction, prevention or treatment of one or more clinical signs, mortality or fecal shedding caused by a rotavirus infection, or the prevention or treatment of a disease caused by a rotavirus infection, comprising the step of administering to a subject an immunogenic composition of the present invention. Also preferably provided is a method for inducing the production of rotavirus-specific antibodies in a pregnant sow, said method comprising the step of administering to said sow an immunogenic composition of the invention. Furthermore, the present invention relates to a method for reducing or preventing one or more clinical signs, mortality or fecal shedding caused by rotavirus infection in piglets, said method comprising: - administering an immunogenic composition of the invention to a sow; and - allowing the sow to suckle the piglets wherein the sow is preferably a sow which is particularly pregnant with the piglet.

[0086] Preferably, the two methods include: - administering the immunogenic composition of the invention to a sow gestating said piglet, - allowing the sow to give birth to the piglets; and - allowing the sow to suckle the piglets Includes. Also provided is a method for reducing one or more clinical signs, mortality or fecal shedding caused by rotavirus infection in piglets, wherein the piglets are suckled by a sow to which the immunogenic composition of the invention has been administered. One or more clinical signs as referred to herein are preferably: - diarrhea, - rotavirus colonization, in particular intestinal rotavirus colonization, - Lesions, especially gross lesions, and - Reduced average daily weight gain is selected from the group consisting of:

[0087] According to one example, the one or more clinical signs referred to herein are rotavirus colonization of the intestine, in particular the small intestine. According to another example, the one or more clinical signs referred to herein are intestinal lesions, in particular gross intestinal lesions. According to another particularly preferred embodiment, the immunogenic composition of the invention is a method as described above, comprising the steps of: - the rotavirus infection is an infection with genotype P

[23] rotavirus and / or genotype P[7] rotavirus, - the rotavirus infection is an infection with genotype P

[23] rotavirus and / or genotype P[7] rotavirus, - the immune response to rotavirus is an immune response to genotype P

[23] rotavirus and / or genotype P[7] rotavirus, or - the rotavirus-specific antibody is an antibody specific for genotype P

[23] rotavirus and / or genotype P[7] rotavirus, Preferably, the immunogenic composition of the present invention comprises any of the polypeptides disclosed herein comprising an immunogenic fragment of genotype P[7] rotavirus 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 to the sequence of SEQ ID NO:3.

[0088] In a particular embodiment, "infection with genotype P

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

[23] rotavirus. In another preferred embodiment, "infection with genotype P

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

[23] rotavirus and genotype P[7] rotavirus. In a particular embodiment, an "immune response to genotype P

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

[23] rotavirus. In another preferred embodiment, "immune response to genotype P

[23] rotavirus and / or genotype P[7] rotavirus" as referred to in this specification is an immune response to genotype P

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

[0089] In a particular embodiment, "antibodies specific for genotype P

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

[23] rotavirus. In another preferred embodiment, "antibodies specific for genotype P

[23] rotavirus and / or genotype P[7] rotavirus" as referred to in this specification includes or is an antibody specific for genotype P

[23] and an antibody specific for genotype P[7] rotavirus. In a further embodiment, the immunogenic composition of the invention is administered in animals, preferably in pregnant sows, to induce the production of antibodies specific for Rotavirus C. Preferably, in this further embodiment, said immunogenic composition of the invention comprises any of the polypeptides of the disclosure described herein, comprising an immunogenic fragment of 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, respectively.

[0090] The present invention further provides a method of producing an immunogenic composition of the present invention, said method comprising the step of transfecting a cell with a plasmid of the present invention. Also provided is a method of producing the immunogenic composition of the present invention, comprising the step of infecting a cell, preferably an insect cell, with a baculovirus of the present disclosure.

[0091] The present invention also provides a method of making the immunogenic composition of the present invention, comprising the steps of: (a) infecting susceptible cells in culture with a vector comprising a nucleic acid sequence encoding a polypeptide of the present disclosure, wherein said polypeptide is expressed by said vector; (b) subsequently recovering said polypeptide, in particular in the supernatant of said cultured cells, preferably wherein the cell debris is separated from said polypeptide via a separation step, preferably comprising microfiltration through at least one filter, preferably two filters, at least one filter preferably having a pore size of about 1 to about 20 μm and / or about 0.1 μm to about 4 μm, (c) inactivating the vector by adding binary ethyleneimine (BEI) to the mixture of step (b); (d) neutralizing the BEI by adding sodium thiosulfate to the mixture resulting from step (c); and (e) concentrating the polypeptides in the mixture resulting from step (d) by removing a liquid portion from the mixture by a filtration step utilizing filtration with a filter membrane having a molecular weight cut-off of about 5 kDa to about 100 kDa, preferably about 10 kDa to about 50 kDa. Including, (f) mixing the mixture remaining after step (e) with at least one immunogenic substance different from said polypeptide; and (g) optionally mixing the remaining mixture after step (f) with additional components selected from the group consisting of pharma- ceutically acceptable carriers, adjuvants, diluents, excipients, and combinations thereof. or (f) mixing the remaining mixture after step (e) with additional components selected from the group consisting of pharma- ceutically acceptable carriers, adjuvants, diluents, excipients, and combinations thereof; and (g) mixing the mixture remaining after step (f) with at least one immunogenic substance different from said polypeptide. The present invention relates to the method comprising the steps of:

[0092] With respect to said step of mixing with at least one immunogenic substance different from said polypeptide, in particular any of those described herein, at least one immunogenic substance different from said polypeptide; or Component (ii) are used, respectively. In step (a) of the method, the cell is preferably an insect cell and the vector is preferably a baculovirus of the present disclosure. 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. Furthermore, the present invention provides an immunogenic composition of the invention and the use of said immunogenic composition in any of the methods described herein, said immunogenic composition being obtainable by the method of producing an immunogenic composition of the invention as described above. EXAMPLES

[0093] The following examples are intended only to illustrate the present disclosure. They do not limit the scope of the claims in any way. Example 1 Fusion protein design, production and testing: Building design: The rotavirus A VP4 sequence was originally obtained from a fecal sample of a swine animal, which most closely matches GenBank sequence JX971567.1 and is classified as a P[7] genotype. VP4 amino acids 57-224 (SEQ ID NO:3), hereafter also named "AVP8", were used, which corresponds to the lectin-like domain of the VP8 protein but with an N-terminal extension of 8 amino acid residues. The linker portion is Gly-Gly-Ser (SEQ ID NO:9). The porcine IgG Fc sequence (SEQ ID NO:7) matches 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, all codon-optimized for insect cells, were received (SEQ ID NO:17) and are herein named AVP8-IgG Fc. The protein encoded by AVP8-IgG Fc (SEQ ID NO: 12) is also referred to herein as "AVP8-IgG Fc protein."

[0094] Cloning, expression and purification AVP8-IgG Fc was TOPO cloned and subsequently inserted into the baculovirus transfer plasmid pVL1393 using BamHI and NotI restriction sites, then co-transfected with BaculoGold into Sf9 cells to generate recombinant baculovirus. AVP8-IgG Fc protein was produced as follows: 1 L of Sf+ cells in a 3 L spinner flask was infected at 0.2 MOI with spent media harvested 4 DPI, centrifuged at 15,000g for 20 min, and 0.2 μm filtered. 1 mL of MabSelect SuRE LX resin slurry (GE Healthcare, Cat. No. 17-5474-01) was added and incubated overnight at 4°C with moderate agitation. The resin was recaptured by filtration, washed with 4×10 mL of Gentle Binding Buffer (Pierce, Cat. No. 21012), and eluted in 7×5 mL volumes of Gentle Elution Buffer (Pierce, Cat. No. 21027). Fractions were combined and dialyzed against 3.5 L of TBS at 4° C. with one buffer exchange. A BCA assay (Thermo Scientific, Cat. No. 23227) was performed to determine the concentration (80 μg / mL).

[0095] Serological research: Protein A purified AVP8-IgG Fc protein was formulated in Emulsigen D containing 87.5% antigen and 12.5% ​​adjuvant. Pigs approximately 7 weeks old received a 2 mL dose by IM in the flank of the neck with a boost 21 days later. Serum samples were collected weekly for 7 weeks. Sera from pigs vaccinated with AVP8-IgG Fc protein were evaluated by ELISA (Figure 1) as described below ("Protocol for ELISA") and by virus neutralization assay (Figure 2) as described below ("Protocol for Virus Neutralization Assay"). Compared to irrelevant vaccine control, IgG ELISA results from pigs vaccinated with AVP8-IgG Fc protein showed an increase in SP ratio with a peak on day 14, which rose again after the 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 the boost on day 21.

[0096] Protocol for ELISA For IgA ELISA, 96-well ELISA plates with media protein binding were coated with total rotavirus antigen diluted 1:16 in 1x PBS. The plates were incubated overnight at a temperature of 4°C. After incubation, the plates were washed using 1x PBST and then blocked with casein blocking solution for 1 h 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 h at 37°C. After washing, the wells were coated with 100 μl of horseradish peroxidase (HRP)-conjugated goat anti-pig IgA diluted 1:3200 and incubated for 1 h at 37°C. After washing, the plates were developed with 3,5,3',5'-tetramethylbenzidine for 15 min at room temperature and the reaction was stopped with 1N HCl before optical density (CD) measurement at 450 nm. Samples including positive and negative controls are run in duplicate wells and results are reported as the average of the ratio (SN) / (PN) of (sample-negative control) to (positive control-negative control).

[0097] For IgG ELISA, 96-well ELISA plates with media proteins bound were coated with total rotavirus antigen diluted 1:8 in 1×PBS. The plates were incubated overnight at a temperature of 4° C. After incubation, the plates were washed using 1×PBST and then blocked with blotting grade blocking solution at 37° C. for 1 h. After washing, 100 μL of primary antibody diluted to a final dilution of 1:625 in blocking buffer was added to the plates and incubated at 37° C. for 1 h. After washing, the wells were coated with 100 μl of horseradish peroxidase (HRP)-conjugated goat anti-pig IgG diluted 1:8000 and incubated at 37° C. for 1 h. After washing, the plates were developed with 3,5,3′,5′-tetramethylbenzidine for 10 min at room temperature, the reaction was stopped with 1N HCl, and the optical density (CD) was measured at 450 nm. Samples including positive and negative controls are run in duplicate wells and results are reported as the average of the ratio (SN) / (PN) of (sample-negative control) to (positive control-negative control).

[0098] Protocol for virus neutralization assay All serum and milk samples were heat inactivated at 56°C for 30 min. Samples were serially diluted from 1:40 to 1:2,560 in rotavirus growth medium (MEM + 2.5% HEPES + 0.3% tryptose phosphate broth + 0.02% yeast + 10 μg / mL trypsin). Rotavirus A isolates (titer 7.0 log TCID 50100μl / mL) was diluted 1:25,000 in rotavirus growth medium. A total of 200μl of diluted serum was added to 200μl of diluted virus and the mixture was incubated for 1 hour at 37°C ± 5% CO2. Growth medium was aseptically removed from 3-4 day old 96-well plates seeded with MA104 cells. After incubation, 200μl of virus-serum mixture was transferred to cell culture plates. Cells were incubated for 72 hours at 37°C ± 5% CO2. Stock and diluted viruses were titrated on the day of use to confirm the dilutions used in the assay. After incubation, the supernatant was discarded and the plates were washed once with 200μL / well 1x PBS. After fixation, 100μL / well 50% / 50% acetone / methanol was added. Plates were incubated at room temperature for 15 minutes, air-dried, and then rehydrated with 100 μL / well of 1×PBS. Primary antibody (rabbit anti-rotavirus A polyclonal serum, generated in-house) was diluted 1:1000 in 1×PBS. 100 μL / well of diluted primary antibody was added and plates were incubated for 1 hour at 37° C.±5% CO2. After incubation, plates were washed twice with 100 μL / well of 1×PBS. Secondary antibody (Jackson ImmunoResearch FITC-labeled goat anti-rabbit IgG Cat. No. 111-095-003) was diluted 1:100 in 1×PBS. 100 μL / well of diluted secondary antibody was added and plates were incubated for 1 hour at 37° C.±5% CO2. After incubation, plates were washed twice with 100 μL / well of 1×PBS. Plates were read for the presence of fluorescence using an ultraviolet microscope. The assay showed a titer of diluted virus (generated using the Reed-Muench method) of 2.8±0.5 log TCID 50 Serum titers were considered valid if they were found to be > 0.01 / mL. In addition, known positive and negative samples were included in each assay as controls. Serum titers were reported as the highest dilution at which no staining was observed.

[0099] Example 2 Load study: The primary objective of this study was to evaluate whether administration of a prototype vaccine, also referred to herein as "IgG:AVP8", containing the AVP8-IgG Fc protein (SEQ ID NO: 12), and an unrelated control vaccine, also referred to herein as "placebo", to conventional sows conferred passive protection to the pigs against a pathogenic rotavirus A challenge. Additionally, for comparison, a commercially available MLV rotavirus vaccine (ProSystem® Rota, Merck Animal Health), also referred to herein as the "commercial product" or "commercial vaccine", was used in the study. The prototype vaccine was produced similarly to the production described above in Example 1, but with a different volume used for infection and a longer incubation period, as described in the section "Production of IgG:AVP8" below. The commercially available product was used according to the label instructions (dosage and instructions and recommended method for oral vaccination of porcine animals) provided by the manufacturer for the vaccine ProSystem® TGE / Rota.

[0100] A total of 16 sows were included in the study. The sows were randomized into three treatment groups and one strict control group, as described in Table 1 below. T02 and T04 sows were mixed among the three rooms. T06 and T07 sows were housed in two separate rooms. All sows were vaccinated with the appropriate material by the appropriate route listed in Table 1. T07 sows remained unvaccinated (strict control). Serum was collected from the sows periodically throughout the vaccination period and assayed for evidence of seroconversion. Fecal samples were collected prior to farrowing and screened by RT-qPCR to ensure that the dams were not actively shedding rotavirus prior to farrowing. Overall health observations were recorded daily for each sow. Parturition was allowed to occur naturally until the sows reached 114 days of gestation. After this time, farrowing was induced. Piglets were enrolled in the study at the time of farrowing. Only piglets that were healthy at birth were tagged, processed according to the facility's standard operating procedures, and included in the study. Once the pigs were 0-5 days of age, they were bled, fecal swabs were collected, and pigs were challenged (except T07). At the time of challenge, pigs were administered a 5 mL dose of sodium bicarbonate intragastrically followed by a 5 mL dose of challenge material intragastrically. Throughout the entire challenge period, all animals were monitored daily for the presence of enteric disease (diarrhea, and behavioral changes). Fecal samples were collected periodically throughout the entire challenge period. Two days post-challenge (DPC 2), approximately one-third of the pigs from each litter were euthanized. After euthanasia, necropsies were performed and pigs were evaluated for gross lesions. Intestinal sections were collected for microscopic and immunohistological evaluation. Intestinal swabs were collected for RT-qPCR evaluation. On DPC 21, all remaining pigs were weighed, bled, and fecal swabs were collected. After sample collection, the pigs were euthanized. The pigs were evaluated for gross lesions and intestinal swabs were collected.

[0101] [Table 1]

[0102] Throughout the study, serum VN titers of sows from T07 (strict control) remained constant or decreased, indicating lack of exposure and a valid study (virus neutralization was assessed as described above in Example 1 ("Protocol for virus neutralization assay" and results are shown in Figure 3). During the vaccination phase, the highest median serum VN titers were observed in sows vaccinated with the IgG:AVP8 (T04) prototype vaccine. In this group, one dose administered 6 weeks before farrowing resulted in a 4-fold or greater increase in titers in 3 / 5 animals in T04 (IgG:AVP8) by D14. Prior to the time of pig challenge, 5 / 5 animals in T04 (IgG:AVP8) had a 4-fold or greater increase in titers. Sows in the placebo group (T02) had a 4-fold or greater increase in serum VN titers during the vaccination phase. T06 (commercial vaccine) sows did not have a significant increase (<2-fold) in serum VN titers until D35. Prior to the time of pig challenge, both T06 (commercial vaccine) sows had a 4-fold increase in titers. After lateral exposure to the challenge material, VN serum titers increased in T02 (placebo) and T06 (commercial vaccine) sows. Conversely, VN serum titers in T04 (IgG:AVP8) sows remained constant or decreased in 4 / 5 sows. With regard to colostrum and milk VN titers, in the T04 group (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 at farrowing and pre-challenge, but increased after lateral exposure to the challenge material.

[0103] VN titers in prechallenge pig sera were high (>1280) in the majority of pigs in T04 (IgG:AVP8), indicating passive transfer of immunity from sow to pig. Conversely, most titers in pigs in T02 (placebo) and T06 (commercial vaccine) were low (<1280).

[0104] Throughout the challenge phase, the highest mortality was observed in T02 (placebo) with 8 / 57 (14.0%) of pigs dying. Conversely, in T04 (IgG:AVP8), only 1 / 46 (2.2%) pigs died, in T06 (commercial vaccine), 1 / 22 (4.5%) pigs died, and in T07 (strict control), 1 / 27 (3.7%) pigs died. No clinical signs of diarrhea were observed in T07 (strict control) pigs throughout the study. Clinical signs of diarrhea in T02 (placebo) pigs began on day 1 or 2 post-challenge (DPC1) and resolved in the majority of animals by DPC10. Overall, clinical signs of diarrhea were observed in 44 / 57 (77.2%) of T02 (placebo) animals 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 T04 (IgG:AVP8) pigs. See Table 2 below for a summary of clinical diarrhea results by group.

[0105] [Table 2]

[0106] Prior to challenge, there was no detection of rotavirus A RNA by RT-qPCR, indicating a valid study. In addition, there was no detection of rotavirus A RNA by RT-qPCR in sows or pigs from T07 (strict control) throughout the study. In pigs after challenge, shedding was most prevalent in T02 (placebo). In most pigs, shedding started on DPC1-3 and continued until DPC14. Most interesting 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 for group median log rotavirus A RNA genome copies (gc) / mL in feces by study day), the study was performed as described below ("Protocol for Rota A qRT-PCR"). A randomly selected subset of pigs from each group was euthanized and necropsied on DPC2. Pigs were evaluated for the presence of gross intestinal lesions (thin wall, gas-distended small intestine, pure liquid content, etc.), microscopic lesions (shrunken intestine), and rotavirus A specific staining by immunohistochemistry (IHC). Table 3 below shows the number of pigs per group that had intestinal lesions at the time of necropsy. The challenge was considered successful as 84.2% (16 / 19) of pigs in the placebo group (T02) had gross lesions, of which 63.2% (12 / 19) were stained. Most interesting was the lack of rotavirus A staining of the animals in only 1 / 15 pigs in T04 (IgG:AVP8). In addition, there was a reduction in the percentage of pigs with gross lesions in T04 (IgG:AVP8) compared to T02 (placebo) and the commercial product (T06).

[0107] [Table 3]

[0108] The average weight gain (kg) per day for surviving pigs was calculated and is shown 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).

[0109] [Table 4] In conclusion, vaccination of conventional sows 6 weeks and 2 weeks prior to farrowing with the IgG:AVP8 prototype vaccine (comprising the polypeptide of SEQ ID NO:12) results in high neutralizing antibody titers in the serum and colostrum of the sows. These neutralizing antibodies were passively transferred to the pigs after birth as evidenced by the detection of high titers (>1280) in the serum of pigs from vaccinated sows. The presence of high neutralizing antibody titers in pigs results in clinical protection. In particular, pigs born to vaccinated sows had reduced fecal shedding of Rotavirus A RNA, reduced mortality, reduced clinical signs of diarrhea, reduced Rotavirus A colonization at DPC2, reduced gross lesions at DPC2, and increased ADWG compared to pigs born to placebo controls and commercial vaccines.

[0110] Protocol for RotaA qRT-PCR To determine rotavirus A RNA in fecal samples, a quantitative one-step RT-PCR kit (iTaq Universal One-Step RT-PCR Kit; BioRad, Cat. No. 1725140) was used for the assay. See Table 5 below for primer and probe information. [Table 5]

[0111] Real-time RT-PCR was performed in 20 μl reactions 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 mix, 0.5 μl of iScript reverse transcriptase and 0.5 μl of DEPC-treated water. Reactions were 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, denaturation at 95° C. for 15 s, and annealing and extension at 60° C. for 45 s for 40 cycles. To generate relative quantification data, serial dilutions of the two rotavirus A g-blocks were included in each run. Equal amounts of each of the g-blocks were added at a starting concentration of 5.0×10 7 Genome copies / μL were used to include in the run. Optical data was analyzed using CFX Manager software. For each determination, a threshold line was automatically calculated using the regression settings for cycle threshold (Ct) determination mode using baseline subtraction mode. Baseline subtraction was performed automatically. Curves with a baseline final value of less than 10 were manually corrected.

[0112] IgG:PCV2 production Approximately 1 x 10 in a 2 L shaker flask of 5 L 6Sf+ (Spodoptera frugiperda) cells at a concentration of 10 cells / mL were infected with 1.7 mL of recombinant baculovirus stock containing rotavirus A VP8 core-swine IgG Fc fusion protein (BaculoGold (BG) / pVL1393-AVP8-IgG; 1.18 x 108 TCID50 / mL). Shaker flasks were incubated at 28°C ± 2°C with constant agitation at 90 rpm for 5 days. Cells and media were aseptically transferred to 3 x 1 L centrifuge bottles and cells were pelleted at 10,000 g for 20 min at 4°C. The resulting supernatant was passed through a 0.2 μm filter (Thermo Scientific, Catalog No. 567-0020) and then incubated with 2.5 mL of MabSelect SuRe LX Protein A resin (GE Healthcare Catalog No. 17-5474-01) at 4° C. overnight with moderate agitation. The resin was collected by 0.2 μm filtration (Thermo Scientific, Catalog No. 567-0020) and washed with 12×10 mL volumes of Gentle Ag / Ab Binding Buffer (Thermo Scientific, Catalog No. 21012). AVP8-IgG was eluted from the resin using 7×10 mL volumes of Gentle Ag / Ab Elution Buffer (Thermo Scientific, Catalog No. 21027). AVP8-IgG was dialyzed against 3.5 L of 20 mM Tris pH 7.5, 150 mM NaCl with one buffer exchange. The remaining baculovirus was inactivated with 5 mM BEI for 24 hours at 37° C. 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.

[0113] Example 3 Serological studies: The primary objective of this study was to evaluate whether administration of a prototype vaccine comprising AVP8-IgG Fc protein (SEQ ID NO: 12) and a control vaccine, referred to herein as "placebo", to conventional sows would result in a serological response to Rotavirus A. The prototype vaccine, also referred to herein as "IgG-AVP8" (containing either Emulsigen D or Carbopol as an adjuvant, see Tables 7 and 7B below) was produced similarly to the production 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".

[0114] A total of 20 sows were included in the study. The sows were randomized into four treatment groups as described in Table 6 below. The sows were intermixed throughout the study. All sows were vaccinated intramuscularly on D0 and D21 with the appropriate material as listed in Table 4. Serum was collected periodically from the sows throughout the study and assayed for evidence of seroconversion by virus neutralization assay. Overall health observations were recorded daily for each sow. The study was terminated on D42.

[0115] [Table 6]

[0116] Serum VN titers of sows from T06 and T07 (placebo groups) remained constant or decreased throughout the study, indicating lack of exposure and valid study (virus neutralization was evaluated as described above in Example 1 (Protocol for Virus Neutralization Assay) with the modification that increasing dilutions from 1:40 to 1:40,960 were evaluated). During the vaccination phase, sows vaccinated with IgG-AVP8 / Emulsigen D (T02) and IgG-AVP8 / Carbopol (T03) prototype vaccines had significant increases in titers (>4-fold). For both groups (T02 and T03), group mean titers were above 640 after one vaccination and remained above 640 throughout the study period. In contrast, sows in the placebo groups (T06 and T07) did not have significant increases in serum VN titers throughout the study (<2-fold). In conclusion, vaccination of conventional sows 6 weeks and 2 weeks before farrowing with the IgG-AVP8 prototype vaccine (comprising the polypeptide of SEQ ID NO: 12) results in high neutralizing antibody titers in the serum of the sows.

[0117] Vaccine production: IgG-AVP8 8L of Sf+ cells at 1.00x10^6 cells / mL in a 10L Sartorius Biostat B glass jacketed vessel were infected with 15mL of BG / pVL1393-AVP8-IgG, 1.19x10^8 TCID50 / mL at an MOI of 0.22. The bioreactor was run at 27°C with 100 rpm agitation and oxygen diffused at 0.3 slpm. The vessel was harvested 6 DPI, centrifuged at 10,000g and 4°C for 20 minutes, and the supernatant was 0.8 / 0.2 μm filtered (GE Healthcare, Cat. No. 6715-7582). 2750mL of the clarified supernatant was inactivated with 5mM BEI at 27°C for 5 days. After neutralization of the remaining BEI with sodium thiosulfate, the 2750 mL was concentrated approximately 12 times to 225 mL using a 10 kDa hollow fiber filter (GE, catalog number UFP-10-C-4MA). The concentration was determined to be 255 μg / mL.

[0118] [Table 7A] [Table 7B]

[0119] Example 4 The primary objective of this study was to evaluate whether animals vaccinated with IgG-AVP8 (containing the AVP8-IgG Fc protein (SEQ ID NO: 12)) could cross-neutralize various rotavirus A serotypes / genotypes of different G and P types other than P[7] for 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.

[0120] Briefly, heat-inactivated sera from pigs vaccinated with IgG-AVP8 were diluted 2-fold in MEM starting at 1:200 in dilution blocks from row A to row G. Row H contained no serum. In separate dilution blocks, rotavirus A of various G and P types were diluted 6.0 Log 10 TCID 50 Starting at 100 / mL, the wells were diluted 1.5-fold across the dilution plate. Column 12 contained no virus. 250 μL of virus and 250 μL of serum from the corresponding wells were combined and incubated at 37° C. for 1 hour. After the 1 hour incubation, 100 μL of the virus-serum mixture was overlaid onto a monolayer of MA104 cells, incubated at 37° C. for 72 hours, stained by IFA, and read for the presence of virus. The presence of virus was recorded as a "+" on the plate and the absence of virus was recorded as a "0". These results were then transferred to Table 8. Six rotavirus A isolates were compared using this assay: G9P[7], G9P

[23] , G4P

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

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

[23] , were also neutralized, indicating that the G type was not important in neutralizing the virus in this assay.

[0121] [Table 8] TIFF2025511739000010.tif221153 TIFF2025511739000011.tif78153 In conclusion, animals vaccinated with IgG-AVP8 (containing the AVP8-IgG Fc protein (SEQ ID NO: 12)) cross-neutralize rotavirus genotypes P[7] and P

[23] . G type did not play a significant role in virus neutralization.

[0122] Example 5 Proof-of-concept studies in porcine animals: A total of 40 animals will be used in this study. Pigs will be randomized into 4 treatment groups with 10 pigs per group. Pigs will be intermingled throughout the study. General health observations, pre-screening serum samples, and pre-screening fecal samples will be obtained prior to treatment to ensure animal health, determine baseline serological response to Rotavirus A, and ensure no active Rotavirus A infection prior to and at the time of vaccination. On study day zero (D0), animals will be vaccinated intramuscularly with the following materials: 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 will be obtained on study days 0, 7, 14, 21, 28, 36, 42, and 49. All animals will be humanely euthanized at necropsy on study D49. Serum samples will be tested by virus neutralization assay to determine the serological response to the vaccine prototypes over time. Animals vaccinated with T01 have antibodies that neutralize rotavirus genotypes P[7] and P

[23] , animals vaccinated with T02 have antibodies that neutralize rotavirus genotype P

[13] , and animals vaccinated with T03 have antibodies that neutralize rotavirus genotypes P[7], P

[13] , and P

[23] .

[0123] Example 6 SDS PAGE: SDS-PAGE of Protein A purified AVP8-IgG Fc protein (SEQ ID NO: 12) product with and without DTT (FIG. 5A): The method for preparing samples for SDS-PAGE images was briefly as follows: Baculovirus harvest supernatant was inactivated with 10 mM BEI for 36 hours at 37° C. and then neutralized. Samples were then purified using Protein A resin. All samples were then denatured using NuPAGE 4×LDS sample buffer (Invitrogen Cat. No. NP0007) with either 25 mM DTT (final) or an equal volume of water and heated at 95° C. for 10 minutes. Samples were run on 4-12% SDS-PAGE gels (Invitrogen Cat. No. NP0335BOX) at 180V for 45 minutes and stained (eStain L1, GenScript Cat. No. M00548-1; destain Cat. No. M00549-1).

[0124] As a result, we found that in the lanes run with the reduced (+DTT (dithiothreitol)) sample, we saw primarily one band (monomeric AVP8-IgG Fc protein, which we considered in conjunction with the Western blot results described below). In the lanes run with the non-reduced sample (-DTT), we saw additional bands. The additional bands were in the molecular weight range of multiple monomers, respectively.

[0125] Western Blot: Western blot of anti-porcine IgG Fc fragment (FIG. 5B): AVP8-IgG Fc protein (SEQ ID NO: 12) product produced in the bioreactor was collected in a 1 mL sample prior to the addition of BEI. The sample was centrifuged at 20,000 g and 4° C. for 5 min, the supernatant was decanted into a new tube, and both pellet and supernatant were stored at −70° C. The pellet and supernatant were thawed, the pellet was resuspended in 1 mL of 8 M urea, and then equal amounts of pellet and supernatant were run on SDS-PAGE under reducing conditions (+DTT) and transferred to a PVDF membrane. The Western blot was probed with a 1:1000 dilution of HRP-conjugated goat anti-porcine to detect porcine IgG Fc fragment. As a result, unexpectedly, no AVP8-IgG Fc protein was found in the cell pellet sample, instead, advantageously, all AVP8-IgG Fc protein (SEQ ID NO: 12) was found in the cell culture supernatant sample.

[0126] Example 7 Generation of consensus sequences: The consensus sequences of SEQ ID NO:4 (based on the genotype P[6] rotavirus VP8 protein) and SEQ ID NO:5 (based on the genotype P

[13] rotavirus VP8 protein) were generated as described below. Sequences were collected from publicly available swine rotavirus VP4 nucleotide sequences from the NCBI Virus Variation database and from internally derived rotavirus isolate sequences. Additional metadata for the sequences was also compiled, including metadata for isolate name, isolate P type, geographic origin, and date of isolation, if available. Nucleotide sequences were translated into protein sequences and aligned with known VP8 proteins using MUSCLE sequence alignment software UPGMB clustering and default gap penalty parameters. Non-aligned VP5 amino acids were trimmed and discarded. The aligned protein sequences of VP8 were imported into MEGA7 software for phylogenetic analysis, and a neighbor-joining phylogenetic tree reconstruction was generated based on the VP8 protein sequence. An optimal tree was computed (n=100) using the Poisson correction method with bootstrap testing of the phylogenetic tree and drawn to scale with branch lengths equal to the evolutionary distances in units of amino acid substitutions per site across all 170 positions. Nodes with bootstrap cluster relatedness higher than 70% were considered significant. Nodes with a distance of approximately 10% and bootstrap cluster association higher than 70% were designated as clusters. Outlier sequences that did not fit into the larger clusters were individually evaluated for sequence quality and P-type origin. Suspected low-quality sequences were removed from the analysis, while sequences from P-types rarely observed in swine rotaviruses were retained. Clusters used to generate consensus sequences were selected based on desired product protection profiles and in vitro serum cross-neutralization studies. Consensus sequences were generated by maximum frequency per aligned position, and amino acid residues were selected based on reported epidemiological data in conjunction with product protection profiles, where a comparable proportion of amino acids were observed at aligned positions.

[0127] Example 8 Load study: The primary objective of this study was to evaluate whether administration of a prototype vaccine, also referred to herein as "IgG#AVP8", containing the AVP8-IgG Fc protein (SEQ ID NO: 12) and an unrelated control vaccine, referred to herein as "placebo", to normal dams conferred passive protection to pigs against a pathogenic Rotavirus A challenge. The prototype vaccine was produced similarly to the production described above in Example 1, but with different volumes used for infection and different purification methods, as described in the section "Production of IgG#AVP8" below.

[0128] A total of 20 dams were included in the study. Dams were randomized into two treatment groups and one strict control group, as described in Table 9 below. T01 and T03 dams were mixed among the three rooms. T07 dams were housed in separate rooms. All dams were vaccinated with the appropriate material by the appropriate route listed in Table 9. T07 dams remained unvaccinated (strict control). Serum was collected from dams periodically throughout the vaccination period and assayed for evidence of seroconversion. Fecal samples were collected prior to farrowing and screened by RT-qPCR to ensure that dams were not actively shedding rotavirus prior to farrowing. Overall health observations were recorded daily for each sow. Parturition was allowed to occur naturally until the sows reached 114 days of gestation. After this time, farrowing was induced. Piglets were enrolled in the study at the time of farrowing. Only piglets that were healthy at birth were tagged, processed according to the facility's standard operating procedures, and included in the study. Once the pigs were 1-5 days of age, they were bled, fecal swabs were collected, and the pigs were challenged (except T07). At the time of challenge, the pigs were administered a 5 mL dose of sodium bicarbonate intragastrically followed by a 1 mL dose of the challenge material intragastrically. Throughout the challenge period, all animals were monitored daily for the presence of enteric disease (diarrhea, and behavioral changes). Fecal samples were collected 1 day post-challenge (DPC1). On DPC2, all pigs from T01 and T03 were euthanized. Intestinal sections were collected for microscopic and immunohistological evaluation.

[0129] [Table 9]

[0130] Throughout the study, serum VN titers of dams from T07 (strict control) increased less than 4-fold, indicating lack of exposure and a valid study (virus neutralization was assessed as described above in Example 1 (Protocol for virus neutralization assay) and results are shown in Table 10 and Figure 6). During the vaccination phase, the highest mean serum VN titers were observed in dams vaccinated with the prototype vaccine IgG#AVP8 (group T03). In this group, one dose administered 6 weeks prior to parturition resulted in a 4-fold or greater increase in titers in 6 / 8 animals in T03 (IgG#AVP8) by D14. Dams in group T01 (placebo) did not have a significant increase (<2-fold) in serum VN titers during the vaccination phase. Dams colostrum VN titers: Dams in group T03 (IgG#AVP8) had higher mean VN titers compared to dams in group T01 (placebo).

[0131] [Table 10]

[0132] VN titers in prechallenge pig serum were high (>1280) in the majority of pigs in group T03 (IgG#AVP8), indicating passive transfer of immunity from dam to pig. Conversely, titers in the majority of pigs in T02 (placebo) were low (<1280). In groups T01 (placebo) and T03 (IgG#AVP8), pigs were defined as affected if rotavirus antigen was detected by immunohistochemistry (IHC) in at least one intestinal section and the animals had abnormal fecal scores at least 1 day after challenge. The frequency distribution is listed in Table 11 below. Based on the use of the definition in this case, vaccination of the dams 6 weeks and 2 weeks before farrowing with the prototype vaccine IgG#AVP8 (group T03) prevented rotavirus-associated disease in pigs after challenge with the heterologous rotavirus AP[7] challenge material; prevention proportion 0.926, 95% confidence interval 0.734, 0.979.

[0133] [Table 11]

[0134] In conclusion, vaccination of normal dams 6 weeks and 2 weeks prior to farrowing with prototype vaccine IgG#AVP8 (comprising the polypeptide of SEQ ID NO: 12) results in high neutralizing antibody titers in the serum and colostrum of sows. These neutralizing antibodies were passively transferred to pigs after birth as evidenced by the detection of high titers (>1280) in the serum of pigs from vaccinated dams. The presence of high neutralizing antibody titers in pigs confers clinical protection. Specifically, fewer pigs born to vaccinated dams appeared to be affected compared to pigs born to placebo controls.

[0135] Generation of IgG#AVP8 Two 10L Sartorius Biostat B glass jacketed vessels were inoculated with 3L of Sf+ cells at 1.00x10^6 cells / mL. Three days after inoculation, each vessel was infected at an MOI of 0.1 and the volume of each vessel was adjusted to 8L using Ex-cell 420 serum-free medium (SAFC Cat. No. 14420C-1000mL). The bioreactor was run at 27°C with 100 rpm agitation, dissolved oxygen setting of 40% or higher, and a CCA overlay of 1.3 slpm. The vessels were harvested 7 days after inoculation, the liquid centrifuged at 10,000g and 4°C for 20 minutes, and the supernatant was 0.8 / 0.2μm filtered (GE Healthcare, Cat. No. 6715-7582). The clarified supernatant (8 L / vessel) was inactivated with 5 mM BEI in a Sartorius Biostat B glass jacketed vessel at 37° C. for 3 days. After inactivation, the remaining BEI was neutralized with sodium thiosulfate. After neutralization, 7000 mL was concentrated to 700 mL approximately 10 times using a 10 kDa hollow fiber filter (GE, Cat. No. UFP-10-C-5A). The concentrated material was diafiltered against 5 volumes (3500 mL) of 1×PBS. The vaccine was formulated using 12.5% ​​Emulsigen D, 28% concentrated material, and 59.5% 1×PBS (vol:vol).

[0136] Example 9 Proof-of-concept studies in porcine animals: A total of 20 animals will be used in this study. Pigs will be randomized into two treatment groups with 10 pigs per group. Pigs will be intermingled throughout the study. General health observations, pre-screening serum samples, and pre-screening fecal samples will be obtained prior to treatment to ensure animal health, determine baseline serological response to Rotavirus C, and ensure no active Rotavirus C infection prior to and at the time of vaccination. On study day zero (D0) and D28, animals will be vaccinated intramuscularly with the following materials: T01: IgG-CVP8 vaccine (comprising the polypeptide of SEQ ID NO: 15), T02: placebo. Serum samples will be obtained on study days 0, 7, 14, 21, 28, 36, and 42. All animals will be humanely euthanized at necropsy on study D42. Serum samples will be tested by ELISA to determine serological response to the vaccine prototype over time. Animals vaccinated with T01 have higher mean levels of antibodies against Rotavirus C than animals vaccinated with T02 (which had no increased titers).

[0137] Example 10 Proof-of-concept studies in porcine animals: The primary objective of this study was to evaluate whether administration of a prototype vaccine, also referred to herein as "IgG:AVP8 P[6,7,13]", containing 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 normal dams conferred passive protection in pigs against a pathogenic Rotavirus A challenge. The components of the prototype vaccine were produced similarly to the production described above in Example 1 and then mixed as described in the section below, "Production of IgG:AVP8 P[6,7,13]".

[0138] A total of 32 dams were included in the study. The dams were randomized into two treatment groups and one strict control group, as described in Table 12 below. T01 and T03 dams were mixed among seven rooms. T04 dams were housed in separate rooms. All dams were vaccinated with the appropriate material by the appropriate route listed in Table 12. T04 dams remained unvaccinated (strict control). Serum was collected from dams periodically throughout the vaccination period and assayed for evidence of seroconversion by virus neutralization (VN) and enzyme-linked immunosorbent assay (ELISA). Fecal samples were collected before farrowing and screened by RT-qPCR to ensure that the dams were not actively shedding rotavirus before farrowing. Within this study, RT-qPCR was performed as described above in Example 2 ("Protocol for Rota A qRT-PCR"). Overall health observations were recorded daily in each sow. Parturition was allowed to occur naturally until the sows reached 115 days of gestation. After this time, parturition was induced. Piglets were enrolled in the study at the time of parturition. Only piglets that were healthy at birth were tagged, processed according to the facility's standard operating procedures, and included in the study. When the pigs were 2-5 days old, they were bled, fecal swabs were collected, and the pigs were challenged (except T04). At the time of challenge, the pigs were administered a 5 mL dose of sodium bicarbonate intragastrically followed by a 1 mL dose of the challenge material intragastrically. Throughout the challenge period, all animals were monitored daily for the presence of enteric disease (diarrhea, and behavioral changes). Fecal samples were collected 1 day post-challenge (DPC1) and at DPC3. At DPC3, all pigs from T01 and T03 were euthanized. Intestinal sections were collected for microscopic and immunohistological evaluation.

[0139] [Table 12]

[0140] Throughout the study, serum VN titers of dams from T04 (strict control) increased less than 4-fold, indicating lack of exposure and a valid study (virus neutralization was assessed as described below (Protocol for virus neutralization assay) and results are shown in Table 13 and Figure 7). Dams in group T01 (placebo) did not have a significant increase (≦2-fold) in serum VN titers during the vaccination phase. Serum VN titers from dams vaccinated with the prototype vaccine IgG:AVP8 P[6,7,13] (group T03) were significantly higher (p<0.01) than dams vaccinated with placebo (group T01) at D14, D21, D27, and D47. Mean colostrum VN titers from dams in group T03 (IgG:AVP8 P[6,7,13]) were also significantly higher compared to dams in group T01 (placebo).

[0141] [Table 13]

[0142] VN titers in prechallenge pig serum were high (>1280) in the majority of pigs in group T03 (IgG:AVP8 P[6,7,13]), indicating passive transfer of immunity from dams to pigs. Conversely, VN titers in pigs in T01 (placebo) were not high (>1280). Similar to the VN data, increasing amounts of anti-rotavirus AVP8 P[6] and anti-rotavirus AVP8 P

[13] antibodies were detected in dams vaccinated with the IgG:AVP8 P[6,7,13] prototype (group T03) but not in dams vaccinated with placebo (group T01) by D47. 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 Figures 8 and 9.

[0143] In groups T01 (placebo) and T03 (IgG:AVP8 P[6,7,13]), pigs were defined as affected if rotavirus antigen was detected by immunohistochemistry (IHC) in at least one intestinal section and the animals had abnormal fecal scores at least 1 day after challenge. The frequency distribution is listed in Table 14 below. Based on the use of the definition in this case, vaccination of the dams 6 weeks and 2 weeks before farrowing with the prototype vaccine IgG:AVP8 P[6,7,13] (group T03) prevented rotavirus-associated disease in pigs after challenge with the heterologous rotavirus AP[7] challenge material; prevention proportion 0.567, 95% confidence interval -0.086, 0.827. [Table 14]

[0144] In conclusion, vaccination of normal dams 6 weeks and 2 weeks prior to farrowing with the prototype vaccine IgG:AVP8 P[6,7,13] (comprising polypeptides of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14) results in high neutralizing antibody titers in the serum and colostrum of sows. These neutralizing antibodies were passively transferred to the pigs after birth, as evidenced by the detection of high titers (>1280) in the serum of pigs from vaccinated dams. The presence of high neutralizing antibody titers in pigs confers clinical protection. Specifically, only a minority of pigs born to vaccinated dams appeared to be affected compared to pigs born to placebo controls. IgG:AVP8 P[6,7,13] production The prototype vaccine contained three fractions, the production of each fraction was performed separately and is 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).

[0145] IgG:AVP8 P[7] fraction: One 10L Sartorius Biostat B glass jacketed vessel was inoculated with 3L of Sf+ cells at 0.75x10^6 cells / mL. Two days after inoculation, the vessel was infected at an MOI of 0.1 and the vessel volume was adjusted to 8L using Ex-cell 420 serum-free medium (SAFC Cat. No. 14420C-1000mL). The bioreactor was run at 27°C with 100 rpm agitation, dissolved oxygen setting of 40% or higher, and a CCA overlay of 1.3 slpm. The vessel was harvested 7 days after inoculation, the liquid centrifuged at 10,000g and 4°C for 20 minutes, and the supernatant was 0.8 / 0.2μm filtered (GE Healthcare, Cat. No. 6715-7582). The clarified supernatant (8 L / vessel) was inactivated with 5 mM BEI in a Sartorius Biostat B glass jacketed vessel at 37° C. for 3 days. After inactivation, the remaining BEI was neutralized with sodium thiosulfate. After neutralization, approximately 7000 mL was concentrated to 700 mL approximately 10 times using a 10 kDa hollow fiber filter (GE, Cat. No. UFP-10-C-5A). The concentrated material was diafiltered against 5 volumes (3500 mL) of 1×PBS.

[0146] IgG:AVP8 P[6] fraction: One 10L Sartorius Biostat B glass jacketed vessel was inoculated with 3L of Sf+ cells at 0.75x10^6 cells / mL. Two days after inoculation, the vessel was infected at an MOI of 0.1 and the vessel volume was adjusted to 8L using Ex-cell 420 serum-free medium (SAFC Cat. No. 14420C-1000mL). The bioreactor was run at 27°C with 100 rpm agitation, dissolved oxygen setting of 40% or higher, and a CCA overlay of 1.3 slpm. The vessel was harvested 8 days after inoculation, the liquid centrifuged at 10,000g and 4°C for 20 minutes, and the supernatant was 0.8 / 0.2μm filtered (GE Healthcare, Cat. No. 6715-7582). The clarified supernatant (8 L / vessel) was inactivated with 10 mM BEI in a Sartorius Biostat B glass jacketed vessel at 37° C. for 24 hours. After inactivation, the remaining BEI was neutralized with sodium thiosulfate. After neutralization, approximately 7000 mL was concentrated to 600 mL approximately 10 times using a 10 kDa hollow fiber filter (GE, Cat. No. UFP-10-C-5A). The concentrated material was diafiltered against 5 volumes (3500 mL) of 1×PBS.

[0147] IgG:AVP8 P

[13] fraction: One 10L Sartorius Biostat B glass jacketed vessel was inoculated with 3L of Sf+ cells at 0.75x10^6 cells / mL. Two days after inoculation, the vessel was infected at an MOI of 0.1 and the vessel volume was adjusted to 8L using Ex-cell 420 serum-free medium (SAFC Cat. No. 14420C-1000mL). The bioreactor was run at 27°C with 100 rpm agitation, dissolved oxygen setting of 40% or higher, and a CCA overlay of 1.3 slpm. The vessel was harvested 7 days after inoculation, the liquid was centrifuged at 10,000g and 4°C for 20 minutes, and the supernatant was 0.8 / 0.2μm filtered (GE Healthcare, Cat. No. 6715-7582). The clarified supernatant (8 L / vessel) was inactivated with 10 mM BEI in a Sartorius Biostat B glass jacketed vessel at 37° C. for 24 hours. After inactivation, the remaining BEI was neutralized with sodium thiosulfate. After neutralization, approximately 7000 mL was concentrated to 750 mL approximately 10 times using a 10 kDa hollow fiber filter (GE, Cat. No. UFP-10-C-5A). The concentrated material was diafiltered against 5 volumes (3500 mL) of 1×PBS.

[0148] Protocol for IgG:AVP8 P

[13] ELISA: Nuc MaxiSorb 96-well ELISA plates (Thermo) were coated with rotavirus A VP8 P

[13] purified protein diluted 1:10 in 1x PBS. Plates were incubated at 37°C for 1 hour. After incubation, plates were washed using 1x 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 1x PBS was added to the plate and incubated at 37°C for 1 hour. After washing, wells were coated with 100 μl of 1:25,000 diluted horseradish peroxidase (HRP)-conjugated goat anti-porcine IgG antibody (Jackson ImmunoResearch) and incubated at 37°C for 1 hour. After washing, the plates were developed with 3,5,3',5'-tetramethylbenzidine (1-Step Ultra TMB-ELISA, Thermo) for 10 min at room temperature, and the optical density (CD) at 450 nm was measured after the reaction was stopped with stop solution (Sigma). Samples including positive and negative controls were run in duplicate wells, and the results are reported as the average of the ratio (SN) / (PN) of (sample-negative control) and (positive control-negative control).

[0149] Protocol for IgG:AVP8 P[6] ELISA: Nuc MaxiSorb 96-well ELISA plates (Thermo) were coated with rotavirus A VP8 P[6] purified protein diluted 1:10 in 1x PBS. Plates were incubated at 37°C for 1 hour. After incubation, plates were washed using 1x 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 1x PBS was added to the plate and incubated at 37°C for 1 hour. After washing, wells were coated with 100 μl of 1:25,000 diluted horseradish peroxidase (HRP)-conjugated goat anti-porcine IgG antibody (Jackson ImmunoResearch) and incubated at 37°C for 1 hour. After washing, the plates were developed with 3,5,3',5'-tetramethylbenzidine (1-Step Ultra TMB-ELISA, Thermo) for 10 min at room temperature, and the optical density (CD) at 450 nm was measured after the reaction was stopped with stop solution (Sigma). Samples including positive and negative controls were run in duplicate wells, and the results are reported as the average of the ratio (SN) / (PN) of (sample-negative control) and (positive control-negative control).

[0150] Protocol for virus neutralization assay All serum and milk samples were heat inactivated at 56°C for 30 min. Samples were serially diluted from 1:40 to 1:40,960 in rotavirus growth medium (MEM + 2.5% HEPES + 0.3% tryptose 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 50100 μL / mL) was diluted 1:78,000 in rotavirus growth medium. A total of 250 μl of diluted sample was added to 250 μl of diluted virus and the mixture was incubated for 1 hour at 37°C ± 5% CO2. Growth medium was aseptically removed from 3-4 day old 96-well plates seeded with MA104 cells. After incubation, 200 μl of virus-serum mixture was transferred to cell culture plates. Cells were incubated for 3-4 days at 37°C ± 5% CO2. Stock and diluted virus were titrated on the day of use to confirm the dilution used in the assay. After incubation, the supernatant was discarded. For fixation, 100 μL / well of 50% / 50% acetone / methanol was added. Plates were incubated for 15 minutes at room temperature, air dried, and then rehydrated with 100 μL / well of 1x PBS. Primary antibody (rabbit anti-rotavirus A polyclonal serum, generated in-house) was diluted 1:1000 in 1x PBS. 100 μL / well of diluted primary antibody was added and the plate was incubated for 1 hour at 37°C ± 5% CO2. After incubation, the plate was washed twice with 100 μL / well of 1x PBS. Secondary antibody (Jackson ImmunoResearch FITC-labeled goat anti-rabbit IgG Cat. No. 111-095-003) was diluted 1:100 in 1x PBS. 100 μL / well of diluted secondary antibody was added and the plate was incubated for 1 hour at 37°C ± 5% CO2. After incubation, the plate was washed twice with 100 μL / well of 1x PBS. Plates were read for the presence of fluorescence using a UV microscope. The assay yielded a titer of diluted virus (generated using the Reed-Muench method) of 2.8 ± 0.5 log TCID 50 Serum titers were considered valid if they were found to be > 0.01 / mL. In addition, known positive and negative samples were included in each assay as controls. Serum titers were reported as the highest dilution at which no staining was observed.

[0151] Example 11 Proof-of-concept studies in porcine animals: The primary objective of this study is to evaluate whether administration of a prototype vaccine, also referred to herein as "IgG:AVP8 P[6,7,13]+IgG:CVP8", containing 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 normal dams confers passive protection to pigs against a pathogenic Rotavirus A challenge. A total of 24 dams are included in the study. The dams are randomized into two treatment groups and one strict control group, as described in Table 15 below. T01 and T02 dams are mixed among the three rooms. T03 dams are housed in a separate room. All dams are vaccinated with the appropriate material by the appropriate route listed in Table 15. T03 dams remain unvaccinated (strict control). Serum is collected from dams periodically throughout the vaccination period and assayed for evidence of seroconversion. Fecal samples are collected prior to farrowing and screened by RT-qPCR to ensure that dams are not actively shedding rotavirus prior to farrowing. Overall health observations are recorded daily for each sow. Farrowing is allowed to occur naturally until the sow reaches day 114 of gestation. Farrowing is induced from this time onwards. Piglets are enrolled in the study at the time of farrowing. Only piglets that are healthy at birth are tagged, processed according to the facility's standard operating procedures and included in the study. When pigs are 1-5 days of age, they are bled, fecal swabs collected and the pigs are challenged (except T03). At the time of challenge, pigs are administered a 5 mL dose of sodium bicarbonate intragastrically followed by a 1 mL dose of the challenge material intragastrically. All animals are monitored daily throughout the entire challenge period for the presence of enteric disease (diarrhea and behavioral changes). Fecal samples are collected 1 day post-challenge (DPC1). On DPC3, all pigs from T01 and T02 are euthanized. Intestinal sections are collected for microscopic and immunohistological evaluation.Dams in group T01 (IgG:AVP8 P[6,7,13]+IgG:CVP8) have higher mean levels of antibodies against rotavirus A genotypes P[6], P[7] and P

[13] , and rotavirus C, than animals vaccinated with T02 (placebo), which had no increased titers. Dams in group T01 (IgG:AVP8 P[6,7,13]+IgG:CVP8) have fewer affected piglets (determined by clinical signs and immunohistological evaluation) compared to dams in group T02 (placebo).

[0152] [Table 15]

[0153] In the sequence listing / Derivation and (if applicable) Geographic Origin: SEQ ID NO:1 corresponds to the sequence of a rotavirus VP8 protein (genotype P[7]) originating from a farm in North Carolina, USA. SEQ ID NO:2 corresponds to the sequence of the lectin-like domain of rotavirus VP8 protein (genotype P[7]) originating from a farm in North Carolina, USA. SEQ ID NO: 3 corresponds to the sequence of an immunogenic fragment of the VP8 protein of a rotavirus (genotype P[7]) originating from a farm in North Carolina, USA. 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]). 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 the immunogenic fragment of the rotavirus VP8 protein (based on genotype P

[13] ). SEQ ID NO: 6 corresponds to the sequence of an immunogenic fragment of the rotavirus C VP8 protein. SEQ ID NO: 7 corresponds to the sequence of a porcine IgG Fc fragment. SEQ ID NO: 8 corresponds to the sequence of a guinea pig IgG Fc fragment. SEQ ID NO:9 (Gly-Gly-Ser) corresponds to the sequence of the linker portion.

[0154] SEQ ID NO: 10 corresponds to the sequence of the linker portion. SEQ ID NO: 11 corresponds to the sequence of the linker portion. SEQ ID NO:12 corresponds to the sequence of a polypeptide (fusion protein) comprising the sequences SEQ ID NO:3, SEQ ID NO:9 (Gly-Gly-Ser) and SEQ ID NO:7. SEQ ID NO:13 corresponds to the sequence of a polypeptide (fusion protein) comprising the sequences SEQ ID NO:4, SEQ ID NO:9 (Gly-Gly-Ser) and SEQ ID NO:7. SEQ ID NO:14 corresponds to the sequence of a polypeptide (fusion protein) comprising the sequences SEQ ID NO:5, SEQ ID NO:9 (Gly-Gly-Ser) and SEQ ID NO:7. SEQ ID NO:15 corresponds to the sequence of a polypeptide (fusion protein) comprising the sequences SEQ ID NO:6, SEQ ID NO:9 (Gly-Gly-Ser) and SEQ ID NO:7. 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. SEQ ID NO:17 corresponds to the sequence of a polynucleotide encoding the polypeptide of SEQ ID NO:12 (fusion protein). SEQ ID NO:18 corresponds to the sequence of a polynucleotide encoding the polypeptide of SEQ ID NO:13 (fusion protein). SEQ ID NO:19 corresponds to the sequence of a polynucleotide encoding the polypeptide of SEQ ID NO:14 (fusion protein). SEQ ID NO:20 corresponds to the sequence of a polynucleotide encoding the polypeptide of SEQ ID NO:15 (fusion protein). SEQ ID NO:21 corresponds to the sequence of a polynucleotide encoding the polypeptide of SEQ ID NO:16 (fusion protein). SEQ ID NOs: 22 to 25: primer and probe sequences (Table 5).

[0155] The following clauses are also disclosed herein: Accordingly, the present disclosure further includes aspects characterized by the following clauses: 1.(i)-An immunogenic fragment of the rotavirus VP8 protein, and - Immunoglobulin Fc fragment A polypeptide comprising and (ii) at least one immunogenic substance different from said polypeptide; An immunogenic composition comprising: 2. The immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein; or the immunoglobulin Fc fragment is linked to the N-terminus of the immunogenic fragment of the rotavirus VP8 protein; The immunogenic composition described in item 1.

[0156] 3. The immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein via a linker moiety; or The immunoglobulin Fc fragment is linked to the N-terminus of the immunogenic fragment of the rotavirus VP8 protein via a linker moiety. The immunogenic composition according to item 1 or 2. 4. 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; or the immunoglobulin Fc fragment is 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; The immunogenic composition according to any one of items 1 to 3. 5. The immunogenic composition according to any one of items 1 to 4, wherein the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein.

[0157] 6. The immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein via a linker moiety; or 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; The immunogenic composition according to any one of items 1 to 5. 7. The immunogenic composition according to any one of items 1 to 6, wherein the polypeptide is a fusion protein. 8. The polypeptide has the formula xyz, x consists of an immunogenic fragment of the rotavirus VP8 protein, y is a linker moiety, z is an immunoglobulin Fc fragment The immunogenic composition according to any one of Items 1 to 7, which is a fusion protein of

[0158] 9. The immunogenic composition according to any one of items 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. 10. The immunogenic composition according to any one of items 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. 11. The immunogenic composition according to any one of items 1 to 10, wherein the rotavirus is a porcine rotavirus. 12. The immunogenic composition according to any one of items 1 to 11, wherein the rotavirus is selected from the group consisting of rotavirus A and rotavirus C. 13. The immunogenic composition according to any one of items 1 to 12, wherein the rotavirus is rotavirus A. 14. The immunogenic composition according to any one of items 1 to 13, wherein the immunogenic fragment of the rotavirus VP8 protein comprises a lectin-like domain of the rotavirus VP8 protein.

[0159] 15. The immunogenic composition according to any one of items 1 to 14, wherein the immunogenic fragment of rotavirus VP8 protein is an N-terminally extended lectin-like domain of rotavirus VP8 protein, and the N-terminal extension is 1 to 20 amino acid residues in length, preferably 5 to 15 amino acid residues in length. 16. The immunogenic composition according to item 14 or 15, wherein the lectin-like domain of rotavirus VP8 protein consists of the amino acid sequence of amino acid residues 65 to 224 of rotavirus VP8 protein. 17. The immunogenic composition according to item 15 or 16, wherein the amino acid sequence of the N-terminal extension is an amino acid sequence of a respective length adjacent to the N-terminal amino acid residues of the lectin-like domain in the amino acid sequence of the rotavirus VP8 protein. 18. The immunogenic composition according to any one of items 1 to 17, wherein the immunogenic fragment of the rotavirus VP8 protein consists of the amino acid sequence of amino acid residues 60 to 224, amino acid residues 59 to 224, amino acid residues 58 to 224, amino acid residues 57 to 224, amino acid residues 56 to 224, amino acid residues 55 to 224, amino acid residues 54 to 224, amino acid residues 53 to 224, amino acid residues 52 to 224, amino acid residues 51 to 224, amino acid residues 50 to 224, or amino acid residues 49 to 224 of the rotavirus VP8 protein.

[0160] 19. The immunogenic composition according to any one of items 1 to 18, wherein the immunogenic fragment of the rotavirus VP8 protein consists of the amino acid sequence of amino acid residues 57 to 224 of the rotavirus VP8 protein. 20. The immunogenic composition according to any one of clauses 16 to 19, wherein the numbering of the amino acid residues refers to the amino acid sequence of a wild-type rotavirus VP8 protein, in particular a wild-type rotavirus A VP8 protein, and the wild-type rotavirus VP8 is preferably the protein shown in SEQ ID NO: 1. 21. The immunogenic composition according to any one of items 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.

[0161] 22. The immunogenic composition according to any one of clauses 1 to 21, wherein 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 to the sequence of SEQ ID NO:1. 23. The immunogenic composition according to 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 to the sequence of SEQ ID NO:2. 24. The immunogenic composition according to 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 to the sequence of SEQ ID NO: 3.

[0162] 25. The immunogenic fragment of a rotavirus VP8 protein consists of or is a consensus sequence of a portion of a rotavirus VP8 protein, in particular a portion of a rotavirus A VP8 protein, The consensus sequence of a portion of the rotavirus VP8 protein is preferably - translating the plurality of nucleotide sequences encoding portions of the rotavirus VP8 protein into amino acid sequences; - aligning said amino acid sequence with known rotavirus VP8 proteins, preferably by using the MUSCLE sequence alignment software UPGMB clustering and default gap penalty parameters; - subjecting said aligned sequences to a phylogenetic tree analysis and generating a neighbor-joining phylogenetic tree reconstruction based on the rotavirus VP8 protein sequence, in particular importing said aligned amino acid sequences into MEGA7 software for phylogenetic tree analysis and generating a neighbor-joining phylogenetic tree reconstruction based on the rotavirus VP8 protein sequence, - calculating the optimal tree using the Poisson correction method with bootstrap testing of the phylogenetic tree (n=100); - drawing a scaled optimal tree across all 170 positions, in units of amino acid substitutions per site, with branch lengths equal to the evolutionary distances; - considering as significant those nodes with bootstrap cluster associations higher than 70%; - designating as clusters those nodes having a distance of approximately 10% and a bootstrap cluster association of greater than 70%; and - generating a consensus sequence by selecting clusters and identifying the maximum frequency per aligned position within the clusters; - optionally selecting amino acid residues based on reported epidemiological data in conjunction with predefined product protection profiles when an equivalent proportion of amino acids are observed at the aligned positions. The immunogenic composition according to any one of items 1 to 24, which can be obtained by a method comprising the steps of:

[0163] 26. The immunogenic composition according to any one of clauses 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 to a sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:5. 27. The immunogenic composition according to any one of items 1 to 26, wherein the rotavirus is rotavirus C. 28. The immunogenic composition according to any one of items 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 to the sequence of SEQ ID NO:6. 29. The immunogenic fragment of the rotavirus VP8 protein, - an immunogenic fragment of the rotavirus A VP8 protein as defined in any one or more of paragraphs 9 to 24, or - a consensus sequence of a part of a rotavirus VP8 protein, in particular a part of a rotavirus A VP8 protein, as defined in any one of paragraphs 9 to 13, 25 and 26, or - an immunogenic fragment of the rotavirus C VP8 protein as defined in any one of paragraphs 9 to 12, 27 and 28; Item 29. The immunogenic composition according to any one of Items 1 to 28, which consists of, or is an immunological fragment thereof.

[0164] 30. The immunogenic composition according to 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 to 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. 31. The immunoglobulin Fc fragment is at least 220 amino acid residues in length, preferably 220 to 250 amino acid residues in length, and / or The immunoglobulin Fc fragment is non-glycosylated, The immunogenic composition according to any one of items 1 to 30. 32. The immunogenic composition according to any one of the preceding claims, wherein the immunoglobulin Fc fragment comprises or consists of heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3), and optionally, an immunoglobulin hinge region or a portion of a hinge region.

[0165] 33. The immunogenic composition according to any one of items 1 to 32, wherein the immunoglobulin is selected from the group consisting of IgG, IgA, IgD, IgE and IgM. 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 infection with a rotavirus from which the immunogenic fragment of a rotavirus VP8 protein is derived. 35. The immunogenic composition according to any one of items 1 to 34, wherein the immunoglobulin Fc fragment is a porcine IgG Fc fragment. 36. The immunogenic composition according to any one of clauses 1 to 35, 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 in particular 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8.

[0166] 37. The immunogenic composition according to any one of items 3 to 36, wherein the linker portion is an amino acid sequence having a length of 1 to 50 amino acid residues. 38. The immunogenic composition according to any one of items 3 to 37, wherein the linker portion comprises an amino acid sequence having at least 66%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or particularly 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 9 (Gly-Gly-Ser), SEQ ID NO: 10, and SEQ ID NO: 11, or consists of the amino acid sequence. 39. The immunogenic composition of any one of clauses 5 to 38, wherein the polypeptide has an N-terminal methionine residue adjacent to the N-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein. 40. The immunogenic composition of any one of clauses 5 to 39, wherein the polypeptide comprises an additional immunogenic fragment of a rotavirus VP8 protein linked to the C-terminus of the immunoglobulin Fc fragment.

[0167] 41. The polypeptide, - an immunogenic fragment of the rotavirus VP8 protein (1), - an immunoglobulin Fc fragment, and - Further immunogenic fragments of the rotavirus VP8 protein (2) Including, the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment (1); a further immunogenic fragment (2) of the rotavirus VP8 protein is linked to the C-terminus of the immunoglobulin Fc fragment; The immunogenic composition according to any one of items 1 to 40. 42. The further immunogenic fragment of the rotavirus VP8 protein, - an immunogenic fragment of a rotavirus A VP8 protein as defined in any one or more of paragraphs 9 to 24, or - a consensus sequence of a part of a rotavirus VP8 protein, in particular a part of a rotavirus A VP8 protein, as defined in any one or more of paragraphs 9 to 13, 25 and 26, or - an immunogenic fragment of a rotavirus C VP8 protein as defined in any one or more of paragraphs 9 to 12, 27 and 28; 42. The immunogenic composition according to claim 40 or 41, which consists of, or is an immunogenic fragment thereof.

[0168] 43. The further 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 to a sequence selected from the group consisting of SEQ ID NOs: 2 to 6; and / or the further immunogenic fragment of the rotavirus VP8 protein is different from the immunogenic fragment of the rotavirus VP8 protein C-terminally linked to the immunoglobulin Fc fragment, 43. The immunogenic composition according to any one of items 40 to 42. 44. The further immunogenic fragment of the rotavirus VP8 protein is linked to the C-terminus of the immunoglobulin Fc fragment via a linker moiety, which is preferably a linker moiety as defined in paragraph 37 or 38, or the further 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 further immunogenic fragment of the rotavirus VP8 protein and the C-terminal amino acid residue of the immunoglobulin Fc fragment; 44. The immunogenic composition according to any one of items 40 to 43.

[0169] 45. The polypeptide, - 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 paragraphs 9 to 30, - an N-terminal methionine residue adjacent to the N-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein, and - an immunoglobulin Fc fragment, in particular an immunoglobulin Fc fragment as defined in any one or more of items 31 to 36, Immunoglobulin Fc fragment, in particular linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein via a linker moiety, said linker moiety being preferably a linker moiety as defined in paragraph 37 or 38, and - optionally, in particular via a linker moiety, a further immunogenic fragment of a rotavirus VP8 protein linked to the C-terminus of said immunoglobulin Fc fragment, said further immunogenic fragment of a rotavirus VP8 protein being preferably a further immunogenic fragment as defined in any one or more of clauses 41 to 44, and said linker moiety being preferably a linker moiety as defined in clause 37 or 38. The immunogenic composition according to any one of items 1 to 44, comprising:

[0170] 46. ​​The immunogenic composition according to any one of items 1 to 45, wherein the polypeptide comprises an amino acid 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 to 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, or is a protein consisting of said amino acid sequence. 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. 48. The immunogenic composition of any one of clauses 1-47, wherein the polypeptide forms a homodimer with an additional identical polypeptide. 49. The immunogenic composition according to any one of the preceding claims, wherein component (i) is present in a multimer comprising a plurality of said polypeptides or composed of said polypeptides, said multimer being preferably a homodimer formed by said polypeptide and a further identical polypeptide. 50. At least one immunogenic substance different from the polypeptide consists of two or more immunogenic substances, all of which are different from the polypeptide; and Different from each other, The immunogenic composition according to any one of items 1 to 49.

[0171] 51. The immunogenic composition of any one of clauses 1 to 50, wherein the at least one immunogenic substance different from the polypeptide is at least one immunogenic substance comprising a reovirus antigen different from the immunogenic fragment. 52. The immunogenic composition of any one of clauses 1 to 51, wherein the at least one immunogenic substance different from the polypeptide consists of two or more immunogenic substances, each of the substances comprising a reovirus antigen, and all of the reovirus antigens are different from the immunogenic fragment and different from each other. 53. The immunogenic composition of any one of clauses 1 to 52, wherein the at least one immunogenic substance different from the polypeptide is at least a protein comprising a reovirus antigen different from the immunogenic fragment. 54. The immunogenic composition of any one of clauses 1 to 53, wherein at least one immunogenic substance different from the polypeptide consists of two or more proteins, each of the proteins comprising a reovirus antigen, and all of the reovirus antigens are different from the immunogenic fragment and different from each other.

[0172] 55. The immunogenic composition according to any one of the preceding claims, wherein component (ii) consists of at least one immunogenic substance comprising a rotavirus antigen different from the immunogenic fragment of component (i). 56. The immunogenic composition according to any one of the preceding claims, wherein component (ii) consists of two or more immunogenic substances, each of which comprises a rotavirus antigen, and all of the rotavirus antigens are different from the immunogenic fragment of component (i) and different from each other. 57. The immunogenic composition according to any one of the preceding claims, wherein component (ii) consists of at least one protein comprising a rotavirus antigen different from the immunogenic fragment of component (i). 58. The immunogenic composition according to any one of the preceding claims, wherein component (ii) consists of two or more proteins, each of said proteins comprising a rotavirus antigen, and all of said rotavirus antigens are distinct from the immunogenic fragment of component (i) and distinct from each other.

[0173] 59. The polypeptide is a first polypeptide, At least one immunogenic substance different from the polypeptide is at least one additional polypeptide different from the first polypeptide; Item 59. The immunogenic composition according to any one or more of items 1 to 58. 60. At least one immunogenic substance different from the polypeptide is - a second polypeptide different from said first polypeptide; or said second polypeptide, Preferably, the third polypeptide is different from both the first and second polypeptides, Optionally, a fourth polypeptide different from all of the first to third polypeptides. 60. The immunogenic composition of claim 59. 61. At least one immunogenic substance different from the polypeptide is - a second polypeptide comprising a reovirus antigen different from said immunogenic fragment. or said second polypeptide, Preferably, the third polypeptide is different from both the first and second polypeptides, Optionally, a fourth polypeptide different from all of the first to third polypeptides. Item 61. The immunogenic composition of item 59 or 60.

[0174] 62. The third polypeptide comprises a reovirus antigen that is distinct from both the immunogenic fragment and the reovirus antigen of the second polypeptide; Optionally, the fourth polypeptide comprises: - said immunogenic fragment, - a reovirus antigen of a second polypeptide, and - a reovirus antigen of a third polypeptide containing reovirus antigens distinct from all of Item 62. The immunogenic composition of item 61. 63. The immunogenic composition according to any one or more of clauses 51 to 54 and 61 to 62, wherein the reovirus antigen is a rotavirus antigen, respectively.

[0175] 64. The second polypeptide is a polypeptide as defined in any one of items 1 to 49, wherein the second peptide is different from the first polypeptide; Preferably, the third polypeptide is a polypeptide as defined in any one of items 1 to 49, and the third polypeptide is different from both the first and second polypeptides, Optionally, the fourth polypeptide is a polypeptide as defined in any one of items 1 to 49, and the fourth polypeptide is different from all of the first to third polypeptides. 64. The immunogenic composition according to any one or more of items 60 to 63. 65. The polypeptide, - a first immunogenic fragment of the rotavirus VP8 protein, and - Immunoglobulin Fc fragment a first polypeptide comprising At least one immunogenic substance different from the polypeptide, a second polypeptide comprising a second immunogenic fragment of a rotavirus VP8 protein, said second immunogenic fragment being different from said first immunogenic fragment; and a third polypeptide, preferably comprising a third immunogenic fragment of a rotavirus VP8 protein, said third immunogenic fragment being different from both said first and second immunogenic fragments; and Optionally, a fourth polypeptide comprising a fourth immunogenic fragment of a rotavirus VP8 protein, said fourth immunogenic fragment being different from all of said first to third immunogenic fragments. Item 65. The immunogenic composition according to any one or more of items 1 to 64, comprising or consisting of said polypeptide.

[0176] 66. Each of the second to fourth immunogenic fragments is - an immunogenic fragment as defined in any one or more of paragraphs 9 to 24, - a consensus sequence of a part of a rotavirus VP8 protein, in particular a part of a rotavirus A VP8 protein, as defined in any one or more of paragraphs 9 to 13, 25 and 26, and - an immunogenic fragment of a rotavirus C VP8 protein as defined in any one or more of paragraphs 9 to 12, 27 and 28; are individually selected from the group consisting of In particular, with the proviso that all of the second to fourth immunogenic fragments are different from the first immunogenic fragment and different from each other, Item 66. The immunogenic composition of item 65.

[0177] 67. The first immunogenic fragment is selected from the group consisting of X7, X13, X6 and XC, and / or the second immunogenic fragment is selected from the group consisting of X7, X13, X6 and XC; and / or the third immunogenic fragment is selected from the group consisting of X7, X13, X6 and XC; and / or the fourth immunogenic fragment is selected from the group consisting of X7, X13, X6 and XC; X7 represents an immunogenic fragment of the genotype P[7] rotavirus VP8 protein; X13 represents an immunogenic fragment of the genotype P

[13] rotavirus VP8 protein; X6 represents an immunogenic fragment of the genotype P[6] rotavirus VP8 protein; XC represents an immunogenic fragment of the rotavirus C VP8 protein, Item 67. The immunogenic composition of item 65 or 66.

[0178] 68. At least one immunogenic substance different from the polypeptide is A second polypeptide comprising a second immunogenic fragment of the rotavirus VP8 protein. or said second polypeptide, each of the first and second immunogenic fragments is individually selected from the group consisting of X7, X13, X6 and XC; With the proviso that when the first immunogenic fragment is X7, the second immunogenic fragment is selected from the group consisting of X13, X6 and XC; With the proviso that when the first immunogenic fragment is X6, the second immunogenic fragment is selected from the group consisting of X7, X13 and XC; With the proviso that if the first immunogenic fragment is X13, the second immunogenic fragment is selected from the group consisting of X7, X6 and XC; With the proviso that when the first immunogenic fragment is XC, the second immunogenic fragment is selected from the group consisting of X7, X13 and X6. Item 68. The immunogenic composition of item 67.

[0179] 69. At least one immunogenic substance different from the polypeptide a second polypeptide comprising a second immunogenic fragment of a rotavirus VP8 protein, and a third polypeptide comprising a third immunogenic fragment of the rotavirus VP8 protein. or consisting of the polypeptide each of the first to third immunogenic fragments is individually selected from the group consisting of X7, X13, X6, and XC; With the proviso that if the first immunogenic fragment is X7, the second immunogenic fragment is X13, and the third immunogenic fragment is selected from the group consisting of X6 and XC; With the proviso that if the first immunogenic fragment is X6, the second immunogenic fragment is X7, and the third immunogenic fragment is selected from the group consisting of X13 and XC; With the proviso that if 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; With the proviso that if the first immunogenic fragment is XC, the second immunogenic fragment is X7, and the third immunogenic fragment is selected from the group consisting of X13 and X6. Item 67 or 68. The immunogenic composition of item 67 or 68.

[0180] 70. At least one immunogenic substance different from the polypeptide is 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 a fourth polypeptide comprising a fourth immunogenic fragment of the rotavirus VP8 protein; or consisting of the polypeptide each of the first to fourth immunogenic fragments is individually selected from the group consisting of X7, X13, X6, and XC; With the proviso that if the first immunogenic fragment is X7, the second immunogenic fragment is X13, the third immunogenic fragment is X6, and the fourth immunogenic fragment is XC; With the proviso that if the first immunogenic fragment is X6, the second immunogenic fragment is X7, the third immunogenic fragment is X13, and the fourth immunogenic fragment is XC; With the proviso that if the first immunogenic fragment is X13, the second immunogenic fragment is X7, the third immunogenic fragment is X6, and the fourth immunogenic fragment is XC; With the proviso that if the first immunogenic fragment is XC, the second immunogenic fragment is X7, the third immunogenic fragment is X13, and the fourth immunogenic fragment is X6. 70. The immunogenic composition according to any one or more of items 67 to 69.

[0181] 71. 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 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 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 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; 71. The immunogenic composition according to any one or more of items 67 to 70.

[0182] 72. The first immunogenic fragment is an immunogenic fragment of a genotype P[7] rotavirus VP8 protein; the second immunogenic fragment is an immunogenic fragment of a genotype P

[13] rotavirus VP8 protein; Preferably, the third immunogenic fragment is an immunogenic fragment of a genotype P[6] rotavirus VP8 protein; Optionally, the fourth immunogenic fragment is an immunogenic fragment of a rotavirus C VP8 protein. 72. The immunogenic composition according to any one or more of items 65 to 71. 73. 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; 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; 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; 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. 73. The immunogenic composition according to any one or more of items 65 to 72.

[0183] 74. The first polypeptide is selected from the group consisting of R7, R13, R6 and RC; and / or the second polypeptide is selected from the group consisting of R7, R13, R6 and RC; and / or the third polypeptide is selected from the group consisting of R7, R13, R6 and RC; and / or the fourth polypeptide is selected from the group consisting of R7, R13, R6, and RC; 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 in particular 100% sequence identity to 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 in particular 100% sequence identity to 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 in particular 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 13, 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 in particular 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 15. 74. The immunogenic composition according to any one or more of items 59 to 73.

[0184] 75. At least one immunogenic substance different from the polypeptide comprises or is a second polypeptide of a rotavirus VP8 protein; each of the first and second polypeptides is individually selected from the group consisting of R7, R13, R6, and RC; With the proviso that when said first polypeptide is R7, said second polypeptide is selected from the group consisting of R13, R6, and RC; With the proviso that when the first polypeptide is R6, the second polypeptide is selected from the group consisting of R7, R13, and RC; With the proviso that when said first polypeptide is R13, said second polypeptide is selected from the group consisting of R7, R6, and RC; With the proviso that when the first polypeptide is RC, the second polypeptide is selected from the group consisting of R7, R13, and R6; 75. The immunogenic composition according to any one or more of items 59 to 74.

[0185] 76. At least one immunogenic substance different from the polypeptide comprises or consists of a second polypeptide of a rotavirus VP8 protein; each of the first and second polypeptides is individually selected from the group consisting of R7, R13, and R6; With the proviso that when the first polypeptide is R7, the second polypeptide is selected from the group consisting of R13 and R6; With the proviso that when the first polypeptide is R6, the second polypeptide is selected from the group consisting of R7 and R13; With the proviso that when the first polypeptide is R13, the second polypeptide is selected from the group consisting of R7 and R6. Item 76. The immunogenic composition of item 74 or 75.

[0186] 77. The first polypeptide is R7 and the second polypeptide is selected from the group consisting of R13 and R6. 77. The immunogenic composition according to any one or more of items 74 to 76. 78. At least one immunogenic substance different from the polypeptide A second polypeptide, and Third Polypeptide or consisting of the polypeptide each of the first to third polypeptides is individually selected from the group consisting of R7, R13, R6, and RC; with the proviso that if the first polypeptide is R7, the second polypeptide is R13, and the third polypeptide is selected from the group consisting of R6 and RC; with the proviso that if the first polypeptide is R6, the second polypeptide is R7, and the third polypeptide is selected from the group consisting of R13 and RC; with the proviso 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; With the proviso that when the first polypeptide is RC, the second polypeptide is R7, and the third polypeptide is selected from the group consisting of R13 and R6. 78. The immunogenic composition according to any one or more of items 74 to 77.

[0187] 79. The first polypeptide is R7; the second polypeptide is R13, the third polypeptide is selected from the group consisting of R6 and RC; 79. The immunogenic composition according to any one or more of items 74 to 78. 80. At least one immunogenic substance different from the polypeptide is A second polypeptide, and Third Polypeptide or consisting of the polypeptide each of the first to third polypeptides is individually selected from the group consisting of R7, R13, and R6; With the proviso that, when the first polypeptide is R7, the second polypeptide is R13, and the third polypeptide is R6; With the proviso that, when the first polypeptide is R6, the second polypeptide is R7, and the third polypeptide is R13; With the proviso that, when the first polypeptide is R13, the second polypeptide is R7, and the third polypeptide is R6. 80. The immunogenic composition according to any one or more of paragraphs 74 to 79.

[0188] 81. At least one immunogenic substance different from the polypeptide is A second polypeptide, and a third polypeptide, and The fourth polypeptide or consisting of the polypeptide each of the first to fourth polypeptides is individually selected from the group consisting of R7, R6, R13, and RC; with the proviso that, if the first polypeptide is R7, then the second polypeptide is R13, the third polypeptide is R6, and the fourth polypeptide is RC; With the proviso that, if the first polypeptide is R6, then the second polypeptide is R7, the third polypeptide is R13, and the fourth polypeptide is RC; with the proviso that, if the first polypeptide is R13, then the second polypeptide is R7, the third polypeptide is R6, and the fourth polypeptide is RC; With the proviso that when the first polypeptide is RC, the second polypeptide is R7, the third polypeptide is R13, and the fourth polypeptide is R6. 81. The immunogenic composition according to any one or more of items 74 to 80.

[0189] 82. The first polypeptide is R7; the second polypeptide is R13, the third polypeptide is R6, the fourth polypeptide is RC; 82. The immunogenic composition according to any one or more of items 74 to 81. 83.- Proteins 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 in particular 100% sequence identity with the sequence SEQ ID NO: 12, as well as - 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 in particular 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. An immunogenic composition, particularly the immunogenic composition according to any one of items 1 to 82, comprising:

[0190] 84.- 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 in particular 100% sequence identity with the sequence SEQ ID NO: 12, and 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 in particular 100% sequence identity with the sequence SEQ ID NO: 14; An immunogenic composition, particularly the immunogenic composition according to any one of items 1 to 83, comprising: 85.- 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 in particular 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 13. 85. The immunogenic composition of claim 84, comprising:

[0191] 86.- 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 in particular 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 15. 86. The immunogenic composition of claim 84 or 85, comprising: 87. The immunogenic composition of any one of clauses 1 to 86, further comprising a pharma- ceutically or veterinarily acceptable carrier or excipient. 88. The immunogenic composition of any one of clauses 1 to 87, further comprising an adjuvant. 89.- Pharmaceutically or veterinarily acceptable carriers or excipients, and - optionally an adjuvant The immunogenic composition according to any one of items 1 to 88, comprising: 90. The immunogenic composition of clause 88 or 89, wherein the adjuvant is an emulsified oil-in-water adjuvant. 91. The immunogenic composition of paragraph 88 or 89, wherein the adjuvant is carbomer.

[0192] 92. Use of an immunogenic composition according to any one of clauses 1 to 91 for preparing a medicament, preferably a vaccine. 93. The immunogenic composition according to any one of clauses 1 to 91 for use as a medicament. 94. The immunogenic composition of any one of clauses 1 to 91 for use as a vaccine. 95. The immunogenic composition of any one of clauses 1 to 91, for use in a method for inducing an immune response against rotavirus in a subject. 96. The immunogenic composition of any one of clauses 1 to 91 for use in a method for reducing or preventing one or more clinical signs, mortality or fecal shedding caused by rotavirus infection in a subject, or for use in a method for treating or preventing infection by rotavirus in a subject. 97. The immunogenic composition of paragraph 95 or 96, wherein the subject is a mammal or bird, and the bird is preferably a chicken.

[0193] 98. The immunogenic composition of any one of clauses 95 to 97, wherein the subject is a mammal, and the mammal is preferably a porcine or bovine animal. 99. The immunogenic composition of any one of paragraphs 95 to 98, wherein the subject is a pig, and the pig is preferably a piglet or a sow. 100. The immunogenic composition of paragraph 95, wherein the subject is a pregnant sow. 101. The immunogenic composition of paragraph 96, wherein the subject is a piglet. 102. The immunogenic composition of any one of paragraphs 1 to 91 for use in a method for reducing or preventing one or more clinical signs, mortality or fecal shedding caused by rotavirus infection in piglets, wherein the piglets are suckled by a sow to which the immunogenic composition has been administered. 103. The immunogenic composition of paragraph 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 is pregnant, in particular while the sow is pregnant with the piglet.

[0194] 104. A method for treating or preventing rotavirus infection, reducing, preventing or treating one or more clinical signs, mortality or fecal shedding caused by rotavirus infection, or preventing or treating a disease caused by rotavirus infection, comprising the step of administering to a subject an immunogenic composition described in any one of paragraphs 1 to 91. 105. A method for inducing the production of rotavirus-specific antibodies in a female pig, the method comprising the step of administering to the female pig an immunogenic composition described in any one of paragraphs 1 to 91. 106. A method for reducing or preventing one or more clinical signs, mortality, or fecal shedding caused by rotavirus infection in piglets, comprising: - administering to a sow an immunogenic composition according to any one of claims 1 to 91; and - allowing the sow to suckle the piglets The method comprising: 107. The method according to paragraph 106, wherein the sow is pregnant, in particular a sow pregnant with the piglet.

[0195] 108. - Administering the immunogenic composition according to any one of paragraphs 1 to 91 to a sow pregnant with said piglet, - allowing the sow to give birth to the piglets; and - allowing the sow to suckle the piglets 108. The method of claim 106 or 107, comprising: 109. A method for reducing one or more clinical signs, mortality or fecal shedding caused by rotavirus infection in piglets, wherein the piglets are suckled by a sow to which the immunogenic composition of any one of paragraphs 1 to 91 has been administered. 110. The one or more clinical signs are: - diarrhea, - Rotavirus colonization, - Lesions, especially gross lesions, - A reduction in average daily weight gain, and - Gastroenteritis The immunogenic composition according to any one of items 96 to 103 or the method according to any one of items 104 to 109, wherein the immunogenic composition is selected from the group consisting of:

[0196] 111. The immunogenic composition of clause 110 or the method of clause 110, wherein the rotavirus colonization is intestinal rotavirus colonization and / or the lesion is an intestinal lesion. 112.- The rotavirus infection is an infection with genotype P

[23] rotavirus and / or genotype P[7] rotavirus, - the rotavirus infection is an infection with genotype P

[23] rotavirus and / or genotype P[7] rotavirus, - the immune response to rotavirus is an immune response to genotype P

[23] rotavirus and / or genotype P[7] rotavirus, or - the rotavirus-specific antibody is an antibody specific for genotype P

[23] rotavirus and / or genotype P[7] rotavirus, The immunogenic composition according to any one of items 95 to 103, 110 and 111, or the method according to any one of items 104 to 111.

[0197] 113. The immunogenic composition or method according to paragraph 112, wherein the immunogenic composition comprises a polypeptide as defined in any one of paragraphs 21 to 26 and 29 to 48, and the immunogenic fragment of a rotavirus VP8 protein is an immunogenic fragment of a genotype P[7] rotavirus VP8 protein. 114. The immunogenic composition of clause 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 to the sequence of SEQ ID NO:3.

[0198] 115. A method for producing an immunogenic composition according to any one of claims 1 to 91, comprising: (a) infecting a susceptible cell in culture with a vector comprising a nucleic acid sequence encoding a polypeptide as defined in any one of items 1 to 48, wherein said polypeptide is expressed by said vector; (b) subsequently recovering said polypeptide, in particular in the cell culture supernatant, whereby preferably the cell debris is separated from said polypeptide via a separation step, preferably comprising microfiltration through at least one filter, preferably two filters, whereby 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, (c) inactivating the vector by adding binary ethyleneimine (BEI) to the mixture of step (b); (d) neutralizing the BEI by adding sodium thiosulfate to the mixture resulting from step (c); and (e) concentrating the polypeptides in the mixture resulting from step (d) by removing a liquid portion from the mixture by a filtration step utilizing filtration with a filter membrane having a molecular weight cut-off of about 5 kDa to about 100 kDa, preferably about 10 kDa to about 50 kDa. Including, (f) mixing the mixture remaining after step (e) with at least one immunogenic substance different from said polypeptide; and (g) optionally mixing the remaining mixture after step (f) with additional components selected from the group consisting of pharma- ceutically acceptable carriers, adjuvants, diluents, excipients, and combinations thereof. or (f) mixing the remaining mixture after step (e) with additional components selected from the group consisting of pharma- ceutically acceptable carriers, adjuvants, diluents, excipients, and combinations thereof; and (g) mixing the mixture remaining after step (f) with at least one immunogenic substance different from said polypeptide. The method comprising:

[0199] 116. The method of clause 115, wherein the at least one immunogenic agent is at least one immunogenic agent defined in any one of clauses 50 to 76. 117. The method according to paragraph 115 or 116, wherein in step (a), the polypeptide as defined in any one of paragraphs 1 to 48 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 in particular 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 12.

[0200] 118. The at least one immunogenic substance different from 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 in particular 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 14, 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 in particular 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 13, and 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 in particular 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 15. Item 118. The method according to any one of Items 115 to 117, wherein the protein is at least one protein selected from the group consisting of: 119. The immunogenic composition according to any one of clauses 1 to 91, 93 to 103 and 110 to 114, the use according to clause 92, or the method according to any one of clauses 104 to 112, 113 and 114, wherein the immunogenic composition can be obtained by a method according to any one of clauses 115 to 118.

Claims

1. (i) - An immunogenic fragment of rotavirus VP8 protein, wherein the immunogenic fragment of rotavirus VP8 protein is an N-terminally elongated lectin-like domain of rotavirus VP8 protein, and the N-terminal elongation consists of 1 to 20 amino acid residues, and - Immunoglobulin Fc fragment polypeptides including and (ii) at least one additional immunogenic substance different from the polypeptide, wherein the at least one additional immunogenic substance is a fusion protein comprising a VP8 protein fragment from a different rotavirus genotype, It is effective in inducing an immune response against at least two rotavirus genotypes. Immunogenic composition.

2. The immunoglobulin Fc fragment is linked to the C-terminus of the immunogenicity fragment of the rotavirus VP8 protein via a linker portion, or 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. The immunogenic composition according to claim 1.

3. The polypeptide is given by formula x-y-z (wherein, x consists of an immunogenic fragment of the rotavirus VP8 protein. y is the linker part, z is an immunoglobulin Fc fragment. The immunogenic composition according to claim 1, wherein the fusion protein is...

4. The rotavirus is porcine rotavirus, and The rotavirus is selected from the group consisting of rotavirus A and rotavirus C. The immunogenic composition according to claim 3.

5. The immunogenic composition according to claim 4, wherein the rotavirus is selected from the group consisting of genotype P[7] rotavirus, genotype P[6] rotavirus, and genotype P[13] rotavirus.

6. The immunogenic fragment of the rotavirus VP8 protein consists of a consensus sequence of a portion of the rotavirus VP8 protein, particularly a consensus sequence of a portion of the rotavirus A VP8 protein, or is the aforementioned consensus sequence. The consensus sequence of a portion of the rotavirus VP8 protein is - A step of translating multiple nucleotide sequences that encode a portion of the rotavirus VP8 protein into an amino acid sequence. - A step of aligning the aforementioned amino acid sequence with a known rotavirus VP8 protein, - The steps of subjecting the aligned sequence to phylogenetic analysis and constructing a neighbor-joint phylogenetic tree reconstruction based on the rotavirus VP8 protein sequence, in particular the steps of importing the aligned amino acid sequence into MEGA7 software for phylogenetic analysis and constructing a neighbor-joint phylogenetic tree reconstruction based on the rotavirus VP8 protein sequence, - A step to calculate the optimal tree using the Poisson correction method with a phylogenetic tree bootstrap test (n=100), - A step of drawing an optimal tree at a constant scale, using branch lengths equal to the evolutionary distance, for each amino acid substitution unit at all 170 positions. - A step in which nodes with a bootstrap cluster relevance higher than 70% are considered significant. - A step of designating nodes with a distance of approximately 10% and a bootstrap cluster relevance of more than 70% as a cluster, and - Steps to create a consensus sequence by selecting clusters and identifying the maximum frequency for each aligned position within the clusters, and - Optionally, when equivalent proportions of amino acids are observed at aligned positions, the step of selecting amino acid residues based on reported epidemiological data in conjunction with a predefined product protection profile. The immunogenic composition according to claim 5, which can be obtained by a method comprising the above.

7. The immunogenic composition according to claim 6, wherein the immunogenic fragment of the rotavirus VP8 protein comprises 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, and an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

8. The immunoglobulin Fc fragment is an immunoglobulin Fc fragment encoded by a certain genome, in which intestinal cells are susceptible to infection by rotavirus derived from an immunogenic fragment of the rotavirus VP8 protein, and The immunoglobulin Fc fragment is a porcine animal IgG Fc fragment, and The immunoglobulin Fc fragment contains an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 7 and SEQ ID NOs: 8, or consists of the aforementioned amino acid sequence. The immunogenic composition according to claim 7.

9. The linker portion is an amino acid sequence having a length of 1 to 50 amino acid residues, and The linker portion contains an amino acid sequence having at least 66%, at least 80%, at least 90%, at least 95%, or 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, or consists of the above amino acid sequence. The immunogenic composition according to claim 8.

10. The immunogenic composition according to claim 9, wherein the polypeptide comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 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, or is a protein comprising the amino acid sequence.

11. At least one immunogenic substance different from the polypeptide consists of two or more immunogenic substances, and all of the immunogenic substances are Unlike the aforementioned polypeptide, and Different from each other, The immunogenic composition according to claim 10.

12. The immunogenic composition according to claim 11, wherein component (ii) consists of two or more immunogenic substances, each of which contains a rotavirus antigen, and all of the rotavirus antigens are different from and distinct from the immunogenic fragment of component (i).

13. The polypeptide described above, - The first immunogenic fragment of the rotavirus VP8 protein, and - Immunoglobulin Fc fragment The first polypeptide contains, At least one immunogenic substance different from the polypeptide is A second polypeptide comprising a second immunogenic fragment of rotavirus VP8 protein, wherein the second immunogenic fragment is different from the first immunogenic fragment, and A third polypeptide comprising a third immunogenic fragment of the rotavirus VP8 protein, wherein the third immunogenic fragment is different from both the first and second immunogenic fragments, and Optionally, a fourth polypeptide comprising a fourth immunogenic fragment of the rotavirus VP8 protein, wherein the fourth immunogenic fragment is different from all of the first to third immunogenic fragments. containing or consisting of the polypeptide The immunogenic composition according to claim 12.

14. At least one immunogenic substance different from the polypeptide is A second polypeptide containing a second immunogenic fragment of the rotavirus VP8 protein, and A third polypeptide containing a third immunogenic fragment of the rotavirus VP8 protein. containing or consisting of the polypeptide, Each of the first to third immunogenic fragments is individually selected from the group consisting of X7, X6, X13, and XC. X7 represents the immunogenic fragment of the genotype P[7] rotavirus VP8 protein, X6 represents the immunogenic fragment of the genotype P[6] rotavirus VP8 protein, X13 represents the immunogenicity fragment of the genotype P[13] rotavirus VP8 protein. XC represents the immunogenic fragment of the rotavirus C VP8 protein. However, if the first immunogenic fragment is X7, the second immunogenic fragment is X6, and the third immunogenic fragment is selected from the group consisting of X13 and XC. However, if the first immunogenic fragment is X6, the second immunogenic fragment is X7, and the third immunogenic fragment is selected from the group consisting of X13 and XC. However, if 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. However, if the first immunogenic fragment is XC, the second immunogenic fragment is X7, and the third immunogenic fragment is selected from the group consisting of X6 and X13. The immunogenic composition according to claim 13.

15. At least one immunogenic substance different from the polypeptide is The second polypeptide, and Third polypeptide containing or consisting of the polypeptide, Each of the first to third polypeptides is individually selected from the group consisting of R7, R13, R6, and RC. R7 is a protein comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 12, R13 is a protein comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 14, R6 is a protein comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 13, RC is a protein comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 15, However, if the first polypeptide is R7, the second polypeptide is R13, and the third polypeptide is selected from the group consisting of R6 and RC. However, if the first polypeptide is R6, the second polypeptide is R7, and the third polypeptide is selected from the group consisting of R13 and RC. However, if the first polypeptide is R13, the second polypeptide is R7, and the third polypeptide is selected from the group consisting of R6 and RC. However, if the first polypeptide is RC, the second polypeptide is R7, and the third polypeptide is selected from the group consisting of R13 and R6. The immunogenic composition according to claim 14.

16. (i) A protein comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity with a sequence selected from the group consisting of Sequence ID No. 12, and (ii) A protein comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 14, SEQ ID NOs: 13, and SEQ ID NOs: 15, or the amino acid sequence said amino acid sequence. The immunogenic composition according to claim 15, comprising:

17. The immunogenic composition according to claim 16, for use as a pharmaceutical or as a vaccine.

18. The immunogenic composition according to claim 16, for use in a method to reduce or prevent one or more clinical signs, mortality or fecal excretion caused by rotavirus infection in a subject, or for use in a method to treat or prevent rotavirus infection in a subject, and for use in a method to induce an immune response to rotavirus in a subject.

19. A method for reducing or preventing one or more clinical signs, mortality, or fecal shedding caused by rotavirus infection in piglets, - The step of administering the immunogenic composition described in claim 16 to a sow, and - The step of having the sow nurse the piglets. The method, including the method described above.

20. One or more clinical signs, - diarrhea, - Rotavirus colonization, especially intestinal rotavirus colonization, - Lesions, especially macroscopic lesions, - A decrease in the average daily weight gain, and - Gastroenteritis An immunogenic composition according to claim 18 or the method according to claim 19, selected from the group consisting of the above.

21. - The immune response to rotavirus is an immune response to genotype P[23] rotavirus or genotype P[7] rotavirus. The method according to claim 20.

22. A method for producing the immunogenic composition described in claim 16, (a) A step of infecting susceptible cells in culture with a vector comprising a nucleic acid sequence encoding a polypeptide as defined in any one of claims 1 to 10, wherein the polypeptide is expressed by the vector, (b) Subsequently, a step of recovering the polypeptide in particular the cell culture supernatant, wherein the cell fragments are separated from the polypeptide via a separation step comprising microfiltration through at least one filter, the at least one filter having a pore size of about 1 to about 20 μm. (c) Inactivating the vector by adding binary ethyleneimine (BEI) to the mixture from step (b), (d) A step of neutralizing BEI by adding sodium thiosulfate to the mixture obtained from step (c), and (e) A step to concentrate the polypeptide in the mixture obtained from step (d) by removing the liquid portion from the mixture using a filtration step that utilizes a filter membrane having a molecular weight cutoff of approximately 5 kDa to approximately 100 kDa. Includes, (f) The step of mixing the remaining mixture after step (e) with at least one immunogenic substance different from the polypeptide, and (g) Optionally, the remaining mixture after step (f) is mixed with further components selected from the group consisting of pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and combinations thereof. including, or (f) The step of mixing the remaining mixture after step (e) with further components selected from the group consisting of pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and combinations thereof, (g) A step of mixing the remaining mixture after step (f) with at least one immunogenic substance different from the polypeptide. The method, including the method described above.

23. The immunogenic composition according to claim 4, wherein the N-terminal elongation is 5 to 15 amino acid residues in length.

24. The immunogenic composition according to claim 6, wherein the step of aligning the amino acid sequence with a known rotavirus VP8 protein is performed by using the MUSCLE sequence alignment software UPGMB clustering and default gap penalty parameters.

25. The method according to claim 17, wherein microfiltration is performed through two filters.

26. The method according to claim 17, wherein at least one filter has a pore size of about 0.1 μm to about 4 μm.

27. ​​The method according to claim 17, wherein the filter film has a molecular weight cutoff of about 10 kDa to about 50 kDa.

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