A fusion protein useful for rotavirus vaccination.

Abstract

To provide recombinantly constructed polypeptides useful for preparing vaccines, particularly for reducing one or more clinical signs caused by rotavirus infection. [Solution] The present invention relates to recombinantly constructed polypeptides useful for preparing vaccines, and in particular for reducing one or more clinical signs caused by rotavirus infection. More specifically, the present invention relates to a fusion protein comprising (i) an immunogenic fragment of the rotavirus VP8 protein and (ii) an immunoglobulin Fc fragment, such as an IgG Fc fragment, in the direction from the N-terminus to the C-terminus, wherein the fusion protein is useful in a method for reducing one or more clinical signs, mortality, or fecal excretion caused by rotavirus infection in animals of the genus Suis.

Description

[Technical Field]

[0001] Background of the Invention Technical field The present invention relates to recombinantly constructed polypeptides useful for preparing vaccines, and in particular for reducing one or more clinical signs caused by rotavirus infection. More specifically, the present invention relates to a fusion protein comprising (i) an immunogenic fragment of the rotavirus VP8 protein and (ii) an immunoglobulin Fc fragment, such as an IgG Fc fragment, in the direction from the N-terminus to the C-terminus, wherein the fusion protein is useful in a method for reducing one or more clinical signs, mortality, or fecal excretion caused by rotavirus infection in swine animals. [Background technology]

[0002] Background information Rotavirus is a double-stranded RNA virus belonging to the Reoviridae family. Rotavirus infection is known to cause gastrointestinal illness and is considered the most common cause of gastroenteritis in young children. Rotavirus is transmitted via the fecal-oral route and infects the cells lining the small intestine. Infected cells produce enterotoxins that induce gastroenteritis, leading to severe diarrhea and sometimes death from dehydration. Rotavirus has a genome consisting of 11 segments of double-stranded RNA (dsRNA) and is currently classified into eight groups (A-H) based on antigenicity and the sequence of 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 subsequent publications referred to herein are incorporated in their entirety by reference). The rotavirus genome encodes six structural proteins (VP1-VP4, VP6, and VP7) and six non-structural proteins (NSP1-NSP6), where 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, different 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 structural proteins VP7 and VP4. VP7 and VP4 are components of the outermost protein layer (outer capsid) and both possess neutralizing epitopes. VP7 is a glycoprotein that forms the outer layer or surface of the virion (hence denoted as "G"). VP7 determines the G-type strain, and the names G serotype and G genotype are identical. VP4 is protease-sensitive (hence denoted as "P") and determines the P-type of the virus. In contrast to the G-type, the number of P serotypes and genotypes assigned is different (Santos N. et Hoshino Y., 2005, Reviews in Medical Virology, 15, 29-56). Therefore, the P serotype is represented as P followed by an assigned number, and the P genotype is represented as P followed by an assigned number in parentheses (e.g., "P[7]" or "P

[13] "). Strains belonging to the same genotype have amino acid sequence identity higher than 89% (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)).

[0004] Rotavirus is a major cause of gastroenteritis, particularly in pigs that possess antibodies against rotavirus groups A and C, which are present in nearly 100% of pigs (Vlasova et al. Viruses. 9(3): 48 (2017)). Currently, only modified live vaccines or dead vaccines are available for rotavirus A. The inability to culture rotavirus C in the laboratory hinders the development of vaccines against this group, which in turn increases the appeal of recombinant vaccines.

[0005] The preparation of recombinant anti-rotavirus vaccines is hampered by the complexity of the rotavirus capsid, which consists 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 intermediate layer, which is formed by 260 trimers of VP6 with T=13 symmetry. The resulting symmetry mismatch between VP2 and VP6 gives rise to five distinct VP6 trimer positions and three distinct pore types. In the absence of VP2, VP6 readily forms regular high molecular weight microtubules and spheres in a salt and pH-dependent manner, which may indicate a byproduct of viral assembly. In the capsid, the VP6 layer is covered by 260 Ca2+-dependent trimers of VP7, which act as clamps holding the VP4 spike in place. VP7 is glycosylated or a G-type antigen and contains a neutralizing epitope. Most neutralizing antibodies are thought to act by recognizing only the VP7 trimer and then preventing the dissociation of the VP7 trimer, thereby blocking spike release. The rotavirus spike exists as a trimer of 60 VP4s, inserted into the VP6 layer only in type II pores. VP4 contains a neutralizing epitope and is a type P antigen, and the spike base VP5 is converted by trypsin. * , and VP5 after cutting * Cell interaction head VP8 remains associated with it. *The spikes are cleaved. Trypsin treatment stimulates the spikes for cell entry, while they undergo a large-scale structural rearrangement, exposing the active site for receptor binding in the host cell. Ignoring the complexity of the above assembly process, achieving stoichiometric expression of rotavirus capsid proteins under environmental conditions that promote proper assembly is difficult.

[0006] Given the difficulties of rotavirus capsid assembly, there was interest in a subunit vaccine approach. VP7 and VP4 are two proteins containing 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. Trypsinization of VP4 would result in the VP8 domain or VP8 * The VP8 protein, also known as VP8, contains a neutralizing epitope, is a monomer, its structure has been determined at high resolution (Dormitzer et al. EMBO J. 21(5): 885-897 (2002)), and it is described as being very stable. Furthermore, within the VP8 protein, the lectin-like domains (aa65~224) are thought to interact with host receptors and be involved in the attachment of the virus to host cells (Rodriguez et al., PloS Pathog. 10(5):e1004157 (2014)).

[0007] An approach for developing a rotavirus subunit vaccine for children is described, in which the N-terminus is tetanus toxoid universal CD4. + T cell epitope (aa830~844)P2 linked to the truncated VP8 protein (VP8 *The amino acid residues 64 (or 65) to 223 of the protein were 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]) induced an insufficient response to atypical 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)).

[0008] In an alternative approach, the N-terminal truncated VP8 protein "VP8-1" (aa26~241) was fused at the N-terminus or C-terminus with the pentamerized non-toxic B subunit (CTB) of cholera toxin. Of the resulting pentamerized fusion proteins (CTB-VP8-1, VP8-1-CTB), only CTB-VP8-1 (i.e., VP8-1 fused to CTB at the N-terminus) was considered a viable candidate for further development compared to VP8-1-CTB, and this was demonstrated in mouse models with GM1 or VP8 * The specific neutralizing monoclonal antibody showed higher binding activity to the conformation sensitive to the target, inducing a higher titer neutralizing antibody and conferring higher protective efficacy (Xue et al. Hum Vaccin Immunother. 12(11) 2959-2968 (2016)). However, given the difficulty of rotavirus capsid assembly, there is interest in alternative subunit vaccine approaches, particularly since subunit vaccines are generally considered very safe. Furthermore, recombinant expression of effective rotavirus subunit antigens, which would allow for the easy production of vaccine antigens for rotaviruses that are difficult to culture, is highly desired. Moreover, since rotavirus is a major cause of gastroenteritis in Suis animals, there is a particularly strong need for subunit vaccines for Suis animals that contain antigens that enable efficacy equivalent to, or even greater than, the MLV rotavirus vaccines currently commercially available for Suis animals. [Modes for carrying out the invention]

[0009] Description of the Invention The solutions to the above technical problems are achieved by the descriptions and embodiments characterized in the claims. Therefore, the present invention will be implemented in a different embodiment according to the claims. This invention is based on the remarkable finding that administration to female pigs of a polypeptide containing an IgG Fc fragment and a rotavirus VP8 protein fragment linked at the C-terminus, i.e., a polypeptide with an elongated N-terminus lectin-like domain, significantly reduced diarrhea and fecal excretion in their offspring after rotavirus exposure, via the passive transfer of neutralizing antibodies.

[0010] In the first embodiment, therefore, the present invention is - Immunogenic fragments of rotavirus VP8 protein, and - Immunoglobulin Fc fragments With respect to polypeptides containing the above, the polypeptide is also referred to as "the polypeptide of the present invention" hereafter in this specification. In the context of the present invention, we have also unexpectedly discovered that, once such polypeptides are produced in cells, they can be released from the cells and subsequently recovered from the supernatant surrounding the cells rather than from the cells themselves. A further advantage of the polypeptide of the present invention is that, if desired, it can be prepared as a single polypeptide containing / presenting two immunogenic fragments of different rotaviruses, thereby eliminating the need to separately prepare two different monovalent polypeptides that then need to be combined for the same purpose. Preferably, the immunoglobulin Fc fragment described herein is - The C-terminus of the immunogenicity fragment of the rotavirus VP8 protein, or - The N-terminus of the immunogenicity fragment of the rotavirus VP8 protein. It is connected to.

[0011] In particular, the immunoglobulin Fc fragment is preferably, - The C-terminus of the immunogenicity fragment of the rotavirus VP8 protein via the linker portion, or - The N-terminus of the immunogenicity fragment of the rotavirus VP8 protein via the linker moiety. It is connected to.

[0012] In another preferred embodiment, the immunoglobulin Fc fragment described herein is - 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 - A peptide bond is established 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, thereby connecting the N-terminal It is connected to. Most preferably, the immunoglobulin Fc fragment described herein is ligated to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein. Therefore, the polypeptide of the present invention is particularly, - Immunogenic fragments of rotavirus VP8 protein, and - A polypeptide comprising an immunoglobulin Fc fragment, wherein the immunoglobulin Fc fragment is linked to the C-terminus of an immunogenic fragment of the rotavirus VP8 protein.

[0013] As used herein, the term "polypeptide" specifically refers to any chain of amino acid residues linked together by peptide bonds and does not refer to a product of a specific length. For example, "polypeptide" can refer to a long chain of amino acid residues, such as 150 to 600 amino acid residues in length or longer. The term "polypeptide" includes polypeptides having one or more post-translational modifications, where post-translational modifications include, for example, glycosylation, phosphorylation, lipidation (e.g., myristoylation, etc.), acetylation, ubiquitination, sulfation, ADP-ribosylation, hydroxylation, Cys / Met oxidation, carboxylation, methylation, etc. The terms "polypeptide" and "protein" are used interchangeably in the context of the present invention.

[0014] It is understood that the term "immunogenic fragment" specifically refers to a fragment of a protein that at least partially retains the immunogenicity of the protein from which it is derived. Thus, it is understood that an "immunogenic fragment of the rotavirus VP8 protein" specifically refers to a fragment of the rotavirus VP8 protein that at least partially retains the immunogenicity of the full-length VP8 protein. As used herein, the term "VP8 protein", when described herein, is understood to be equivalent to "VP8 domain", "VP8 * ", or "VP8 fragment of VP4", which are frequently used in the context of rotavirus. As used herein, the term "immunoglobulin Fc fragment" refers to a protein containing the heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of an immunoglobulin, more specifically, a protein that does not contain the variable regions of the heavy and light chains of an immunoglobulin, as well as the light chain constant region 1 (CL1). This may further include the hinge region of an immunoglobulin or a portion of the hinge region (i.e., the hinge region of the heavy chain constant region). Also, the immunoglobulin Fc fragment may contain a part or all of the heavy chain constant region 1 (CH1). As used herein, the term "immunoglobulin Fc fragment" is understood to be equivalent to the "immunoglobulin Fc domain".

[0015] As used herein, the term "linked to" specifically refers 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 what is meant by linking include (1) indirect linking of an immunoglobulin Fc fragment to the C-terminus of an immunogenic fragment of the rotavirus VP8 protein by means of an intervening moiety that is directly linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein and also binds to the immunoglobulin Fc fragment, and (2) direct linking of an immunoglobulin Fc fragment to the C-terminus of an immunogenic fragment of the rotavirus VP8 protein by means of a covalent bond. The terms "linked to" and "linked with" are used interchangeably in the context of the present invention.

[0016] In particular, " - an immunogenic fragment of a rotavirus VP8 protein, and - an immunoglobulin Fc fragment A polypeptide comprising the immunoglobulin Fc fragment being linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein", as used herein, in particular, "In the direction from the N-terminus to the C-terminus, - Amino acid sequence of immunogenicity fragment of rotavirus VP8 protein, and - Amino acid sequence of immunoglobulin Fc fragments The expression "polypeptide containing", or "- Immunogenic fragments of rotavirus VP8 protein, and - Immunoglobulin Fc fragment linked to the C-terminus of the immunogenic fragment The expression "polypeptides containing" It is understood that this is equivalent to [the other].

[0017] In the most preferred embodiment, the immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein via a linker moiety. The linker portion, as described herein, is preferably a peptide linker in the context of the present invention. 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 linking two variable proteins and / or domains, for example, an immunogenic fragment of the rotavirus VP8 protein and an immunoglobulin Fc fragment.

[0018] In a particular preferred embodiment, the immunoglobulin Fc fragment is ligated to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein via a linker moiety, where, - 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 portion 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 portion. Furthermore, it is preferable 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.

[0019] The polypeptide of the present invention will be understood to be, in particular, a fusion protein. As used herein, the term “fusion protein” means a protein formed by fusing (i.e., conjugating) all or part of two or more non-identical polypeptides. Typically, fusion proteins are prepared using recombinant DNA techniques by conjugating end-to-end polynucleotides encoding two or more polypeptides. More specifically, the term “fusion protein” means a protein translated from a nucleic acid transcript prepared by combining a first nucleic acid sequence encoding a first polypeptide and at least a second nucleic acid encoding a second polypeptide, where the fusion protein is not a naturally occurring protein. A nucleic acid construct may encode two or more polypeptides that are conjugated in the fusion protein. In another preferred embodiment, the present invention relates to polypeptides, in particular the polypeptides mentioned above, wherein the polypeptide is of formula xyz (wherein x consists of or contains an immunogenic fragment of the rotavirus VP8 protein. y is the linker part, z is an immunoglobulin Fc fragment. This provides a polypeptide, which is a fusion protein.

[0020] Formula xyz should be understood to mean, in particular, that the C-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein is linked by the linker moiety, preferably via peptide bonds 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 by the linker moiety, preferably via peptide bonds with the C-terminal amino acid residue of the linker moiety. The expression "x consists of an immunogenic fragment of the rotavirus VP8 protein" is understood to be equivalent, in particular, to "x is an immunogenic fragment of the rotavirus VP8 protein" when used herein. In a preferred embodiment, an immunogenic fragment of the rotavirus VP8 protein, as referred to herein, is preferably capable of inducing an immune response to rotavirus in a subject to whom the immunogenic fragment of the rotavirus VP8 protein is administered. In another preferred embodiment, the immunogenicity fragment of the rotavirus VP8 protein is a polypeptide having a length of 50 to 200, preferably 140 to 190 amino acid residues.

[0021] The rotaviruses referred to herein are preferably selected from the group consisting of rotavirus A and rotavirus C. Therefore, when referred to herein, immunogenic fragments of rotavirus VP8 protein are preferably selected from the group consisting of immunogenic fragments of rotavirus A VP8 protein and immunogenic fragments of rotavirus C VP8 protein. The terms "rotavirus A" and "rotavirus C," when used herein, refer to rotavirus A and rotavirus C, respectively, as defined by the ICTV (summarized by Matthijnssens et al. Arch Virol 157:1177-1182 (2012)).

[0022] In another preferred embodiment, the rotavirus referred to herein is porcine rotavirus. In a particularly preferred embodiment, the rotavirus referred to herein is rotavirus A. Therefore, the immunogenic fragment of the rotavirus VP8 protein, as described herein, is preferably the immunogenic fragment of the rotavirus A VP8 protein. In a more preferred embodiment, the immunogenic fragment of the rotavirus VP8 protein comprises the lectin-like domain of the rotavirus VP8 protein. When referred to herein, “lectin-like domain of the rotavirus VP8 protein” is understood to preferably be the lectin-like domain of the rotavirus A VP8 protein.

[0023] The term "lectin-like domain of rotavirus VP8 protein" specifically refers to residues 65-224 of the rotavirus VP8 protein, which correspond to the amino acid sequence consisting of amino acid residues 65-224 of the rotavirus VP8 protein, where the amino acid residues 65-224 of the rotavirus VP8 protein are preferably amino acid residues 65-224 of the rotavirus A VP8 protein. Therefore, the "lectin-like domain of the rotavirus VP8 protein" preferably consists of the amino acid sequence of amino acid residues 65-224 of the rotavirus VP8 protein, particularly the rotavirus A VP8 protein.

[0024] Preferably, the immunogenic fragment of the rotavirus VP8 protein is a lectin-like domain with an extended N-terminus of the rotavirus VP8 protein, where the N-terminal extension is 1 to 20 amino acid residues long, particularly 5 to 15 amino acid residues. Most preferably, the immunogenic fragment of the rotavirus VP8 protein is a lectin-like domain with an extended N-terminus of the rotavirus VP8 protein, where the N-terminal extension is 8 amino acid residues long. The N-terminal extension amino acid residues are preferably individual amino acid sequences of lengths adjacent to the N-terminal amino acid residue of the lectin-like domain in the amino acid sequence of the rotavirus VP8 protein. Therefore, in certain embodiments, the immunogenic fragment of the rotavirus VP8 protein, as referred herein, preferably consists of the amino acid sequence of amino acid residues 60-224, 59-224, 58-224, 57-224, 56-224, 55-224, 54-224, 53-224, 52-224, 51-224, 50-224, or 49-224 of the rotavirus VP8 protein, particularly the rotavirus A protein.

[0025] Most preferably, the immunogenic fragment of the rotavirus VP8 protein, as referred to herein, consists of the amino acid sequence of amino acid residues 57-224 of the rotavirus VP8 protein, particularly the 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 the wild-type rotavirus VP8 protein, particularly the wild-type rotavirus A VP8 protein. The wild-type rotavirus VP8 protein is preferably the protein shown in Sequence ID No. 1.

[0026] In 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, particularly rotavirus A. Therefore, when referred to herein, the immunogenic fragment of the rotavirus VP8 protein is preferably selected from the group consisting of the immunogenic fragment of the genotype P[6]rotavirus VP8 protein, the immunogenic fragment of the genotype P[7]rotavirus VP8 protein, and the immunogenic fragment of the genotype P

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

[13] rotavirus A VP8 protein.

[0027] The terms “genotype P[6] rotavirus,” “genotype P[7] rotavirus,” “genotype P

[13] rotavirus,” and “genotype P

[23] rotavirus,” when used herein, refer in particular to the established VP4(P) genotyping classifications 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); and Gorziglia et al. Proc Natl Acad Sci US A. 87(18):7155-9 (1990).

[0028] Most preferably, the rotavirus referred to herein is genotype P[7]rotavirus. Therefore, when referred to herein, the immunogenic fragment of the rotavirus VP8 protein is most preferably an immunogenic fragment of genotype P[7]rotavirus VP8 protein, in particular an immunogenic fragment of genotype P[7]rotavirus A VP8 protein. The rotavirus VP8 protein referred to herein most preferably comprises or consists of an amino acid sequence having sequence identity of at least 90%, preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% with the sequence of SEQ ID NO: 1. When referred to herein, the lectin-like domain of the rotavirus VP8 protein preferably comprises or consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO: 2. In one example, the immunogenic fragment of the rotavirus VP8 protein consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO: 3. In another preferred embodiment, the immunogenic fragment of the rotavirus VP8 protein consists of, or is, a portion of the rotavirus VP8 protein, particularly a consensus sequence of a portion of the rotavirus A VP8 protein.

[0029] As used herein, the term “consensus sequence” refers in particular to a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (see, for example, Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family of proteins, each position in the consensus sequence is occupied by the most frequently occurring amino acid at that position in the family. The term “consensus sequence” therefore represents a putative amino acid sequence (or nucleotide sequence). A consensus sequence represents multiple similar sequences. Each position in the consensus sequence corresponds to the most frequently occurring amino acid residue (or nucleotide base) at that position, determined by aligning three or more sequences.

[0030] Preferably, the consensus sequence of a portion of the rotavirus VP8 protein is, when referred to herein, - A step of translating multiple nucleotide sequences that encode a portion of the rotavirus VP8 protein into an amino acid sequence. - Preferably, the amino acid sequence is aligned with a known rotavirus VP8 protein by using MUSCLE sequence alignment software UPGMB clustering and default gap penalty parameters. - The steps of subjecting the aligned sequences to phylogenetic analysis and creating neighbor-jointed phylogenetic reconstructions based on the rotavirus VP8 protein sequence, in particular, the steps of importing the aligned amino acid sequences into MEGA7 software for phylogenetic analysis and creating neighbor-jointed phylogenetic reconstructions based on the rotavirus VP8 protein sequence, - A step to calculate the optimal tree using the Poisson correction method with a phylogenetic 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 association higher than 70% are considered significant. - A step of designating nodes with a distance of approximately 10% and a bootstrap cluster association higher than 70% as a cluster, and - A step of creating 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. It can be obtained by methods including,

[0031] For example, in this context, the immunogenic fragment of the rotavirus VP8 protein preferably consists of an amino acid sequence having sequence identity of at least 90%, preferably at least 95%, more preferably at least 98%, or even more preferably at least 99%, with a sequence selected from the group consisting of SEQ ID NOs: 4 and SEQ ID NOs: 5. In a further preferred embodiment, the rotavirus referred to herein is rotavirus C. In this embodiment, the immunogenic fragment of the rotavirus VP8 protein is preferably the immunogenic fragment of the rotavirus C VP8 protein. In the context of this embodiment, the immunogenic fragment of the rotavirus VP8 protein preferably consists of an amino acid sequence having sequence identity of at least 90%, preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% with the sequence of SEQ ID NO: 6.

[0032] According to the present invention, the immunogenic fragment of the rotavirus VP8 protein is therefore preferably, - Immunogenic fragments of rotavirus A VP8 protein, in particular any of the immunogenic fragments of rotavirus A VP8 protein described herein, or - A portion of the rotavirus VP8 protein, for example, a consensus sequence of a portion of the rotavirus A VP8 protein, preferably, in the context of the consensus sequence, any of the immunogenic fragments of the rotavirus VP8 protein described herein, or - Immunogenic fragments of rotavirus C VP8 protein, in particular any of the immunogenic fragments of rotavirus C VP8 protein described herein. It consists of, or is.

[0033] In a particular preferred embodiment, the immunogenic fragment of the rotavirus VP8 protein is a polypeptide comprising an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 3, SEQ ID NOs: 4, SEQ ID NOs: 5, and SEQ ID NOs: 6.

[0034] The immunoglobulin Fc fragments described herein are preferably at least 220 amino acid residues in length, and most preferably 220 to 250 amino acid residues in length. According to another particular preferred embodiment, the immunoglobulin Fc fragments described herein are not glycosylated. The term “not glycosylated,” as used herein, means, in particular, that the immunoglobulin Fc fragments do not have oligosaccharide molecules attached to them. Preferably, the immunoglobulin Fc fragment, when referred to herein, is an immunoglobulin, - heavy chain constant region 2 (CH2), and - heavy chain constant region 3 (CH3), and - Optionally, the hinge area or a portion of the hinge area It includes or consists of.

[0035] In another preferred embodiment, the immunoglobulins referred to herein are selected from the group consisting of IgG, IgA, IgD, IgE, and IgM. Therefore, the immunoglobulin Fc fragment is preferably selected from the group consisting of IgG Fc fragment, IgA Fc fragment, IgD Fc fragment, IgE Fc fragment, and IgM Fc fragment. In its most preferred embodiment, the immunoglobulin Fc fragment described herein is an IgG Fc fragment. When IgG is referred to herein, it is preferably selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgG5, and IgG6. Accordingly, according to another preferred embodiment, the immunoglobulin Fc fragment referred to herein is selected from the group consisting of IgG1 Fc fragment, IgG2 Fc fragment, IgG3 Fc fragment, IgG4 Fc fragment, IgG5 Fc fragment, and IgG6 Fc fragment.

[0036] Most preferably, the immunoglobulin Fc fragment is a protein encoded by a certain genome in which intestinal cells are susceptible to infection by rotavirus from which the immunogenic fragment of the rotavirus VP8 protein referred to herein originates. For example, if the rotavirus VP8 protein fragment is a porcine rotavirus VP8 protein fragment, the immunoglobulin Fc fragment is preferably an immunoglobulin Fc fragment encoded by the porcine genome. In another example, if the rotavirus VP8 protein fragment is a chicken rotavirus VP8 protein fragment, the immunoglobulin Fc fragment is preferably an immunoglobulin Fc fragment encoded by the chicken genome. More specifically, the immunoglobulin Fc fragment is preferably a IgG Fc fragment from a genus of the genus Suis. In a further preferred embodiment, the immunoglobulin Fc fragment comprises or consists of an amino acid sequence having sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or particularly 100%, of a sequence selected from the group consisting of SEQ ID NOs: 7 and SEQ ID NOs: 8.

[0037] The linker moieties or peptide linkers referred to herein are preferably amino acid sequences with a length of 1 to 50 amino acid residues, and more particularly amino acid sequences with a length of 3 to 20 amino acid residues. For example, the linker moiety may be a peptide linker with a length of 3, 8, or 10 amino acid residues. Depending on the purpose, shorter linkers may be desired to reduce the risk of proteolysis between fusion protein partners. Accordingly, the peptide linkers described in the context of the present invention are preferably, each having a length of 1 to 5 amino acid residues, more preferably 2 to 4 amino acid residues, and most preferably 3 amino acid residues. In a preferred embodiment, the linker portion comprises, or consists of, a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, and 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.

[0038] Preferably, the polypeptide of the present invention has an N-terminal methionine residue adjacent to the N-terminal amino acid residue of the immunogenic fragment of the rotavirus VP8 protein. In another preferred embodiment, the polypeptide of the present invention comprises a further immunogenic fragment of rotavirus VP8 protein ligated to the C-terminus of the immunoglobulin Fc fragment. Further immunogenic fragments of the rotavirus VP8 protein are preferably, - Immunogenic fragments of rotavirus A VP8 protein, in particular any of the immunogenic fragments of rotavirus A VP8 protein described herein, or - A portion of the rotavirus VP8 protein, for example, a consensus sequence of a portion of the rotavirus A VP8 protein, preferably, in the context of the consensus sequence, any of the immunogenic fragments of the rotavirus VP8 protein described herein, or - Immunogenic fragments of rotavirus C VP8 protein, in particular any of the immunogenic fragments of rotavirus C VP8 protein described herein. It consists of, or is.

[0039] In particular, the further immunogenic fragment of the rotavirus VP8 protein preferably comprises, or consists of, an amino acid sequence having sequence identity of at least 90%, preferably at least 95%, more preferably at least 98%, or even more preferably at least 99%, with a sequence selected from the group consisting of SEQ ID NOs: 2 to 6. In a particular preferred embodiment, the further immunogenic fragment of the rotavirus VP8 protein differs from the immunogenic fragment of the rotavirus VP8 protein in which the C-terminus is linked to the immunoglobulin Fc fragment. The further immunogenic fragment of the rotavirus VP8 protein is preferably ligated to the C-terminus of the immunoglobulin Fc fragment via a linker moiety, particularly via any of the linker moieties described herein. Preferably, the further immunogenic fragment of the rotavirus VP8 protein is ligated to the linker moiety 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 linker moiety. Alternatively, it may be preferable that a further immunogenic fragment of the rotavirus VP8 protein is ligated 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.

[0040] In a particular preferred embodiment, the polypeptide of the present invention is a protein comprising, or comprising, an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 12, SEQ ID NOs: 13, SEQ ID NOs: 14, SEQ ID NOs: 15, and SEQ ID NOs: 16. Preferably, the polypeptide of the present invention is a protein comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0041] The expressions "consisting of an amino acid sequence" or "consists of an amino acid sequence," as used herein, are understood to also relate to any co-translational and / or post-translational modifications of an amino acid sequence that are influenced by the cell on which the protein or protein domain is expressed. Therefore, as used herein, unless otherwise expressly stated, the expressions "consisting of an amino acid sequence" or "consists of an amino acid sequence" also refer to amino acid sequences having modifications of amino acid residues, preferably selected from the group consisting of glycosylation, phosphorylation, and acetylation, that are brought about in protein biosynthesis and / or protein processing.

[0042] With respect to the term "at least 90%", when referred to in the context of the present invention, the term is understood to preferably mean "at least 91%", more preferably "at least 92%", even more preferably "at least 93%", or in particular "at least 94%". With respect to the term "at least 95%", when referred to in the context of the present invention, the term is understood to preferably mean "at least 96%", more preferably "at least 97%", even more preferably "at least 98%", or in particular "at least 99%". With respect to the term "at least 99%", when referred to in the context of the present invention, it is understood that the term preferably refers to "at least 99.2%", more preferably to "at least 99.4%", even more preferably to "at least 99.6%", or in particular to "at least 99.8%". The term "having 100% sequence identity" is understood to be equivalent to the term "identical" when used herein.

[0043] Sequence identity percentage has recognized significance in the art, and there are many methods for measuring the identity between two polypeptide or polynucleotide sequences. See, for example, 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 are systematized 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 with affine gap search with a gap-open penalty of -12 and a gap-stretch penalty of -2. For the purposes of the present invention, nucleotide sequences are aligned using the Clustal W method in DNASTAR Inc.'s MegAlign software version 11.1.0(59),419, using a set of default alignment parameters in this program (gap penalty = 15.0, gap length penalty = 6.66, and delayed mismatch sequence (%) = 30%, DNA transfer weight = 0.50, and DNA weight matrix = IUB), and respectively, protein / amino acid sequences are aligned using the Clustal W method in DNASTAR Inc.'s MegAlign software version 11.1.0(59),419, using a set of default alignment parameters in this program (Gonnet series protein weight matrix with gap penalty = 10.0, gap length penalty = 0.2, and delayed mismatch sequence (%) = 30%).

[0044] Where used herein, the term “sequence identity of sequence X with sequence” is understood to be equivalent to the term “sequence identity of sequence X over the length of sequence X” or the term “sequence identity of sequence X over the entire length of sequence X,” respectively. In this context, “X” is any integer selected from 1 to 25 such that “sequence X” represents any of the sequence numbers referred to herein.

[0045] The expression “a group consisting of sequence numbers [...], ... and sequence numbers [...]” is interchangeable with “a group consisting of sequences of sequence numbers [...], ... and sequences of sequence numbers [...]” when used herein. In this context, "[...]" is a placeholder for sequence numbers. For example, the expression “a group consisting of sequence number 3, sequence number 4, sequence number 5 and sequence number 6” is interchangeable with “a group consisting of sequences of sequence number 3, sequence number 4, sequence number 5 and sequence number 6.”

[0046] According to another particular preferred embodiment, the polypeptide of the present invention is - Immunogenic fragments of rotavirus VP8 protein, in particular any of the immunogenic fragments of rotavirus VP8 protein described herein, - The N-terminal methionine residue adjacent to the N-terminal amino acid residue of the immunogenicity fragment of the rotavirus VP8 protein, and - Immunoglobulin Fc fragments, in particular any of the immunoglobulin Fc fragments described herein. And, The immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein, particularly via a linker moiety, and the linker moiety is preferably one of the linker moieties described herein, and - Optionally, in particular, a further immunogenic fragment of the rotavirus VP8 protein linked to the C-terminus of the immunoglobulin Fc fragment via a linker moiety, wherein the further immunogenic fragment of the rotavirus VP8 protein is preferably one of the further immunogenic fragments of the rotavirus VP8 protein described herein, and the linker moiety is preferably one of the linker moieties described herein. It consists of.

[0047] In a further preferred embodiment, the polypeptide of the present invention forms a dimer with a further polypeptide of the present invention. Most preferably, the polypeptide of the present invention forms a homodimer with a second identical polypeptide. Therefore, it is particularly understood that the term “polypeptide of the present invention” further encompasses any dimer composed of two polypeptides of the present invention, and in particular, any homodimer composed of two identical polypeptides of the present invention. In another particular preferred embodiment, the present invention provides a polymer comprising or composed of a plurality of polypeptides of the present invention, wherein the polymer is also referred to hereafter as the “polymer of the present invention”. Preferably, the polymer of the present invention is a homodimer formed by one polypeptide of the present invention and a second identical polypeptide of the present invention.

[0048] The term "multimer of the present invention" can refer to any mixture of different multimers of the present invention, for example, - A homodimer formed by one polypeptide of the present invention and a second identical polypeptide of the present invention, and - One or more polymers formed by three or more polypeptides of the same invention It is particularly understood that the mixture further encompasses this. The present invention further provides immunogenic compositions comprising the polypeptide and / or polymer of the present invention, wherein the immunogenic composition is also referred to as "the immunogenic composition of the present invention" hereafter.

[0049] Therefore, in a preferred example, the immunogenic composition of the present invention is - A monomer comprising one polypeptide of the present invention, and - A homodimer comprising two identical polypeptides of the present invention, and - Optionally, a homotrimer consisting of three identical polypeptides of the present invention This includes, and here preferably, - Each of the two identical polypeptides of the present invention, and - Optionally, each of the three identical polypeptides of the present invention The polypeptide of the present invention contains or consists of the same amino acid sequence as described above.

[0050] The immunogenic composition of the present invention preferably contains the polypeptide of the present invention 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. In another preferred embodiment, the immunogenic composition of the present invention contains the polypeptide of the present invention in 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 possibly 2 mL, of the immunogenic composition of the present invention is administered to the subject. Therefore, the dose of the immunogenic composition of the present invention administered to the 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.

[0051] The immunogenic compositions of the present invention are preferably administered systemically or topically. Conventionally used preferred routes of administration are parenteral or oral administration, such as intramuscular, intradermal, intravenous, intraperitoneal, subcutaneous, intranasal, and inhalation. However, depending on the properties and mechanism of action of the compound, the immunogenic compositions may also be administered by other routes. Intramuscular administration of the immunogenic composition is most preferred. The immunogenic compositions of the present invention preferably further comprise pharmaceutically or veterinarily acceptable carriers or excipients. As used herein, “pharmaceutically or veterinarily acceptable carriers” include all kinds of solvents, dispersions, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption retarders, and the like. In some preferred embodiments, particularly embodiments comprising lyophilized immunogenic compositions, the stabilizers for use in the present invention include stabilizers for lyophilization or freeze-drying.

[0052] In some embodiments, the immunogenic composition of the present invention contains an adjuvant. As used herein, "adjuvants" may include aluminum hydroxide and aluminum phosphate, saponins, e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsions, oil-in-water emulsions, and water-in-oil emulsions. Emulsions may be based in particular on light liquid paraffin oil (European Pharmacopoeia type); isoprenoid oils such as squalane or squalene; alkenes, in particular oils obtained from oligomerization of 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; and esters of branched fatty acids or alcohols, in particular isostearate esters. The oil is used in combination with an emulsifier to form an emulsion. The emulsifier is preferably a nonionic surfactant, particularly sorbitan, mannides (e.g., anhydrous mannitol oleate), glycols, polyglycerols, propylene glycols, and esters of oleic acid, isostearic acid, ricinoleic acid, or hydroxystearic acid, which may be ethoxylated, as well as polyoxypropylene-polyoxyethylene copolymer blocks, particularly Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, DES), John Wiley and Sons, NY, pp51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997).An example of an adjuvant is the SPT emulsion described on page 147 of "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.

[0053] Further examples of adjuvants are polymers of acrylic acid or methacrylic acid, and compounds selected from copolymers of maleic anhydride and alkenyl derivatives. Favorable adjuvant compounds are polymers of acrylic acid 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). Those skilled in the art may also refer to U.S. Patent 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 radicals having at least two carbon atoms. Preferred radicals contain 2 to 4 carbon atoms, e.g., vinyl, allyl, and other ethylenically unsaturated groups. The unsaturated radicals themselves may contain other substituents, such as methyl. Products marketed under the name CARBOPOL® (BF Goodrich, Ohio, USA) are particularly suitable. They are crosslinked with allyl clucrose or allyl pentaerythritol. Among these, Carbopol 974P, 934P, and 971P can be mentioned. The use of CARBOPOL® 971P is most preferred. Among these, the copolymer of maleic anhydride and alkenyl derivatives is copolymer EMA (Monsanto), which is a copolymer of maleic anhydride and ethylene. The solubility of these polymers in water preferably yields an acidic solution neutralized to physiological pH to give an adjuvant solution incorporated into the immunogenic composition, immunological composition, or vaccine composition itself.

[0054] Further preferred 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 CA), monophosphoryl lipid A, abridine lipid-amine adjuvants, heat-labile enterotoxins (recombinant or otherwise) derived from Escherichia coli, cholera toxin, IMS1314 or muramyl dipeptide, or naturally occurring or recombinant cytokines or their analogues, or stimulants of endogenous cytokine release.

[0055] The adjuvant is expected to 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%. Examples of diluents include water, physiological saline, dextrose, ethanol, and glycerol. Examples of isotonic agents include sodium chloride, dextrose, mannitol, sorbitol, and lactose. Examples of stabilizers include albumin and alkali salts of ethylenediaminetetraacetic acid.

[0056] In a particularly preferred embodiment, the present invention also provides an immunogenic composition, in particular the immunogenic composition of the present invention, wherein the immunogenic composition is - The polypeptide and / or polymer of the present invention, and - Pharmaceutically or veterinarily acceptable carriers or excipients, and - Optionally, adjuvant It includes or will include. In the context of the present invention, the adjuvant is 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 elicits an immunogenic response in a host to which the immunogenic composition is administered. Such an immunogenic response may be a cellular immune response and / or an antibody-mediated immune response to the immunogenic composition according to the present invention. The host is also referred to as “subject.” Preferably, any host or subject described or referred to herein is an animal. When used herein, the term "animal" refers in particular to mammals, preferably animals of the genus Suis, more preferably pigs, and most preferably piglets.

[0057] Typically, an "immunological response" may include, 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 against one or more antigens contained in the immunogenic composition of the present invention. Preferably, the host presents either a protective immunological response or a therapeutic response. A "protective immunological response" is demonstrated by a reduction or absence of one or more clinical signs normally presented by the infected host, a faster recovery time and / or a reduced duration of infection, or a lower pathogenic titer in the infected host's tissues, fluids, or excretions. When referred to herein, “pathogen” or “specific pathogen” refers in particular to rotaviruses from which immunogenic fragments of the rotavirus VP8 protein originate. For example, when referred to herein, the pathogen is rotavirus A or rotavirus C.

[0058] An immunogenic composition is described as a "vaccine" when the host exhibits a protective immunological response that enhances resistance to new infections and / or reduces the clinical severity of the disease. Where used herein, “antigen” refers to a component that elicits an immunological response in a host to such an antigen or an immunologically active component thereof in an immunological composition or vaccine of interest. In particular, where used herein, the term “antigen” refers to a protein or protein domain that, when administered to a host, can elicit an immunological response in the host.

[0059] 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 by or associated with a particular pathogen infection. Accordingly, 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 typically associated with or caused by an infection with a pathogen in a group of animals that have received an effective amount of the immunogenic composition provided herein, compared to a group of animals that have not received such an immunogenic composition. "Treatment and / or prevention" generally includes the administration of an effective amount of the polypeptide or immunogenic composition of the present invention to a subject or population of subjects that requires or can benefit from such treatment / prevention. The term "treatment" refers to the administration of an effective amount of the immunogenic composition at a time when a subject in a population or at least some of the animals has already been infected with such pathogen and such animals have already shown several clinical signs caused by or related to such pathogen infection. The term "prevention" refers to the administration to a subject prior to any infection of such subject by the pathogen, or when at least all animals in such animals or population of animals have not shown any clinical signs caused by or related to such pathogen infection.

[0060] The term “effective amount,” as used herein, means, but is not limited to, an amount of antigen, in particular the polypeptide and / or polymer of the present invention, that elicits or can elicit 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%, most preferably at least 95%, compared to any subject that was untreated or treated with an immunological composition that was applicable prior to the present invention but subsequently infected with the particular pathogen.

[0061] As used herein, the term "clinical signs" refers to signs of infection of a subject from a particular pathogen. The clinical signs of infection depend on the pathogen selected. Examples of such clinical signs, but not limited to, include diarrhea, vomiting, fever, abdominal pain, and dehydration. The reduction in the appearance or severity of one or more clinical signs caused by or associated with a specific pathogen infection in a subject can be achieved by administering one or more doses of the immunogenic composition of the present invention to the subject.

[0062] The term "reduces fecal excretion" means, but is not limited to, a reduction in the number of RNA copies of pathogenic viruses such as rotavirus per 1 mL of feces, or a reduction in the number of plaque-forming colonies per deciliter of feces, in the feces of subjects receiving the composition of the present invention by at least 50% compared to subjects not receiving the composition and who could become infected. More preferably, the fecal excretion level is reduced by at least 90%, preferably at least 99.9%, more preferably at least 99.99%, and even more preferably at least 99.999% in subjects receiving the composition of the present invention. The term "fecal excretion," as used herein, is used in accordance with its obvious and ordinary meaning in medicine and virology, and refers to the production and release of viruses from the cells of an infected subject into the environment via the feces of that subject.

[0063] The polypeptide of the present invention is preferably a recombinant protein, particularly a recombinant baculovirus expression protein. The term "recombinant protein," as used herein, refers in particular to a protein produced by recombinant DNA techniques, where generally, the 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 host cells to produce a heterologous protein. Thus, the term "recombinant protein," as used herein, refers in particular to a protein molecule expressed from a recombinant DNA molecule. The term "recombinant DNA molecule," as used herein, refers to a DNA molecule composed of DNA segments joined together by molecular biological techniques. Suitable systems for recombinant protein production include, but are not limited to, insect cells (e.g., baculoviruses), prokaryotic cell lines (e.g., Escherichia coli), fungi (e.g., Myceliophthora thermophile, Aspergillus oryzae, Ustilago maydis), yeasts (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 lysis).

[0064] In another aspect, the present invention provides a polynucleotide comprising a sequence encoding the polypeptide of the present invention, wherein the polynucleotide is also referred to hereafter as "the polynucleotide according to the present invention," and is preferably an isolated polynucleotide. Preferably, the polynucleotide according to the present invention comprises a sequence selected from the group consisting of SEQ ID NOs: 17, SEQ ID NOs: 18, SEQ ID NOs: 19, SEQ ID NOs: 20, and SEQ ID NOs: 21, and a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or particularly 100% sequence identity.

[0065] The production of polynucleotides described herein is within the realm of skill in the art and can be carried out according to the recombinant techniques described in 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 further embodiment, the present invention provides a vector containing a polynucleotide encoding the polypeptide of the present invention.

[0066] "Vector" and "vector containing a polynucleotide encoding the polypeptide of the present invention" refer to a suitable expression vector, preferably a baculovirus expression vector, for the purposes of the present invention, which is then used to transfect a host cell or, in the case of a baculovirus expression vector, to infect a host cell in order to produce a DNA-encoded protein or polypeptide. Vectors, and methods for constructing and / or using vectors (or recombinants) for expression, are described in U.S. Patents 4,603,112, 4,769,330, 5,174,993, 5,505,941, 5,338,683, 5,494,807, 4,722,848, 5,942,235, 5,364,773, 5,762,938, 5,770,212, 5,942,235, 382,425, and PCT Publications 94 / 16716, 96 / 39491, and 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. Patent 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; US Patent Application No. 920,197 filed on 16 October 1986; European Patent Application Publication No. 265785; US Patent No. 4,769,331 (recombinant herpesvirus); 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; International Publication No. 98 / 00166; U.S. applications Nos. 08 / 675,556 and 08 / 675,566 (recombinant adenovirus), both filed and approved on July 3, 1996; 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. Patents No. 5,591,639, 5,589,466 and 5,580,859, as well as International Publications 90 / 11092, 93 / 19183, 94 / 21797, 95 / 11307 and 95 / 20660; in particular, Tang et al., Nature and Furth et al., relating to DNA expression vectors.They can be prepared or made by methods disclosed in Analytical Biochemistry, or by similar methods. See also International Publication No. 98 / 33510; Ju et al., Diabetologia, 41: 736-739, 1998 (Lentiviral Expression System); Sanford et al., U.S. Patent No. 4,945,050; Fischbach et al. (Intracel); International Publication No. 90 / 01543; Robinson et al., Seminars in Immunology vol.9, pp.271-283 (1997) (DNA Vector System); Szokka et al., U.S. Patent No. 4,394,448 (Method of Inserting DNA into Living Cells); McCormick et al., U.S. Patent No. 5,677,178 (Use of Cytopathic Viruses); and U.S. Patent No. 5,928,913 (Vectors for Gene Delivery), as well as other literature cited herein.

[0067] Preferred viral vectors include baculoviruses such as BaculoGold (BD Biosciences Pharmingen, San Diego, CA), especially if the producing cells are insect cells. While baculovirus expression systems are preferred, it will be understood by those skilled in the art that other expression systems, including those described above, can serve the purpose of the present invention, i.e., for the expression of recombinant proteins.

[0068] Accordingly, the present invention also provides a baculovirus containing a polynucleotide comprising a sequence encoding the polypeptide of the present invention. The baculovirus, also referred to hereafter as "baculovirus according to the present invention," is preferably an isolated baculovirus. Furthermore, the present invention also provides plasmids, preferably expression vectors, comprising polynucleotides containing sequences encoding the polypeptide of the present invention. The plasmids, also referred to hereafter as "plasmids according to the present invention," are in particular isolated plasmids. The present invention also provides cells infected with and / or containing a baculovirus comprising a polynucleotide comprising a sequence encoding the polypeptide of the present invention, or a plasmid comprising a polynucleotide comprising a sequence encoding the polypeptide of the present invention, preferably an expression vector. The cells, also referred to hereafter as “cells according to the present invention,” are preferably isolated cells.

[0069] The term "isolated," when used in the context of isolated cells, refers to cells that exist separately from their natural environment by human intervention and are therefore not natural products. In another embodiment, the present invention also relates to the use of polypeptides, polymers, baculoviruses, immunogenic compositions, polynucleotides, virus-like particles, plasmids, and / or cells for pharmaceutical, preferably vaccine preparation.

[0070] In this context, the present invention also provides a method for producing the polypeptide of the present invention, wherein the method comprises the step of infecting cells, preferably insect cells, with the baculovirus according to the present invention. Furthermore, the present invention also provides a method for generating the polypeptide of the present invention, wherein the method comprises the step of transfecting cells with the plasmid according to the present invention. The polypeptide of the present invention is preferably expressed in a sufficiently high amount for stable self-assembly of virus-like particles, which can then be used for vaccination.

[0071] The terms “vaccination” or “vaccinating” as used herein mean, but are not limited to, a process comprising administering an antigen, for example, an antigen contained in an immunogenic composition, to a subject, where the antigen, for example, a polypeptide or polymer of the present invention, when administered to the subject, induces or can induce a protective immunological response in the subject. The present invention also provides polypeptides or immunogenic compositions of the present invention for use as pharmaceuticals, preferably as vaccines.

[0072] In particular, the polypeptide or immunogenic composition of the present invention is provided for use in a method of reducing or preventing one or more clinical signs or diseases caused by rotavirus infection, wherein the rotavirus is preferably a rotavirus from the group having a genome encoding an immunogenic fragment of the rotavirus VP8 protein. The polypeptide or immunogenic composition of the present invention is provided for use in a method of reducing or preventing fecal excretion caused by rotavirus infection, wherein the virus is preferably a rotavirus from the group having a genome encoding an immunogenic fragment of the rotavirus VP8 protein. Therefore, in a particular example, if the immunogenic fragment of the rotavirus VP8 protein referred to herein is encoded by the genome of rotavirus A, the polypeptide or immunogenic composition of the present invention is for use in a method of reducing or preventing one or more clinical signs, mortality, fecal excretion, or diseases caused by infection with rotavirus A.

[0073] More specifically, the polypeptides or immunogenic compositions of the present invention are provided for use in methods to reduce or prevent one or more clinical signs, mortality or fecal excretion caused by rotavirus infection in a subject, or for use in methods to treat or prevent rotavirus infection in a subject. When referred to herein, rotavirus infection refers specifically to infection with rotavirus A or rotavirus C. Furthermore, the polypeptides or immunogenic compositions of the present invention are provided for use in methods for inducing an immune response to rotavirus in a subject. The subjects, as referred to herein, are preferably mammals, such as Suis or Bovidae, or birds, such as chickens. In particular, the subjects are pigs, where the pigs are preferably piglets or sows, such as pregnant sows. Most preferably, the subjects are pregnant sows in the context of inducing an immune response to rotavirus in the subjects. Most preferably, the subjects are piglets in the context of reducing or preventing one or more clinical signs, mortality or fecal excretion caused by rotavirus infection in the subjects, or treating or preventing rotavirus infection in the subjects.

[0074] In one preferred embodiment, the polypeptide or immunogenic composition of the present invention is for use in a method of reducing or preventing one or more clinical signs, mortality or fecal excretion caused by rotavirus infection in piglets, wherein the piglets are lactated by a sow to which the immunogenic composition has been administered. The sow to which the immunogenic composition has been administered is preferably a sow to which the immunogenic composition has been administered while the sow is pregnant, in particular while pregnant with the piglets. Furthermore, the present invention relates to a method for treating or preventing rotavirus infection, reducing, preventing or treating one or more clinical signs, mortality or fecal excretion caused by rotavirus infection, or preventing or treating a disease caused by rotavirus infection, comprising the step of administering the polypeptide or immunogenic composition of the present invention to a subject. Furthermore, preferably, a method is provided for inducing the production of antibodies specific to rotavirus in pregnant sows, wherein the method comprises the step of administering the polypeptide of the present invention or the immunogenic composition of the present invention to the sow.

[0075] 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, wherein the method is - The steps of administering the polypeptide of the present invention or the immunogenic composition of the present invention to a sow, and - The piglet is nursed by the sow. The present invention provides a method comprising, wherein the sow is preferably, in particular, a sow that is pregnant with the pig.

[0076] Preferably, the two aforementioned methods are - A step of administering the polypeptide of the present invention or the immunogenic composition of the present invention to a sow that is pregnant with the piglets, - The step of causing the sow to give birth to the piglets, - The piglet is nursed by the sow. Includes. The present invention also provides a method for reducing one or more clinical signs, mortality, or fecal excretion caused by rotavirus infection in piglets, wherein the piglets are lactated by a sow administered with the polypeptide or immunogenic composition of the present invention.

[0077] When one or more clinical signs are referred to herein, preferably, - diarrhea, - Rotavirus colonization, especially intestinal rotavirus colonization. - Lesions, especially macroscopic lesions, and - Reduction in average daily weight gain It is selected from the group consisting of the following. For example, one or more clinical signs referred to herein are rotavirus colonization of the intestines, particularly the small intestine. For example, one or more clinical signs referred to herein are intestinal lesions, particularly macroscopic intestinal lesions.

[0078] According to another particular preferred embodiment, the polypeptide or immunogenic composition of the present invention is for use in any of the methods described above, where, - The rotavirus infection is caused by genotype P

[23] rotavirus and / or genotype P[7]rotavirus, - The rotavirus infection is caused by genotype P

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

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

[23] rotavirus and / or genotype P[7]rotavirus, Preferably, each of the polypeptides of the present invention is any of the polypeptides of the present invention described herein, comprising an immunogenic fragment of the genotype P[7]rotavirus VP8 protein, comprising 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, or the immunogenic composition of the present invention comprises any of the polypeptides of the present invention described herein, comprising an immunogenic fragment of the genotype P[7]rotavirus VP8 protein, comprising 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.

[0079] In a particular aspect, “infection with genotype P

[23] rotavirus and / or genotype P[7]rotavirus” means, as used herein, infection with genotype P

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

[23] rotavirus and / or genotype P[7]rotavirus” means, as referred to herein, infection with genotype P

[23] rotavirus and genotype P[7]rotavirus. In a particular aspect, “immune response to genotype P

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

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

[0080] In a particular embodiment, “antibodies specific to genotype P

[23] rotavirus and / or genotype P[7]rotavirus” is, as referred to herein, antibodies specific to genotype P

[23] rotavirus. In another preferred embodiment, “antibodies specific to genotype P

[23] rotavirus and / or genotype P[7]rotavirus” means, when referred to herein, an antibody specific to genotype P

[23] rotavirus and an antibody specific to genotype P[7]rotavirus.

[0081] In a further embodiment, the polypeptides of the present invention or the immunogenic compositions of the present invention are administered to animals, preferably pregnant sows, to induce the production of antibodies specific to rotavirus C. Preferably, in this further embodiment, each of the polypeptides of the present invention is any of the polypeptides of the present invention described herein, comprising an immunogenic fragment of the rotavirus C VP8 protein, in particular having 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, or the immunogenic compositions of the present invention are any of the polypeptides of the present invention described herein, comprising an immunogenic fragment of the rotavirus C VP8 protein, comprising 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.

[0082] The present invention provides a method for producing polypeptides and / or polymers of the present invention, wherein the method further comprises the step of transfecting cells with plasmids of the present invention. Furthermore, a method for producing the polypeptide and / or polymer of the present invention is also provided, wherein the method comprises the step of infecting cells, preferably insect cells, with the baculovirus of the present invention.

[0083] Furthermore, the present invention relates to a method for producing the immunogenic composition of the present invention, wherein the method is (a) A step of enabling infection of susceptible cells in a culture with a vector comprising a nucleic acid sequence encoding the polypeptide of the present invention, wherein the polypeptide is expressed by the vector, (b) Subsequently, a step of recovering the polypeptide, particularly from the supernatant of the cultured cells, wherein the cell debris is separated from the polypeptide via a separation step, preferably a separation step including microfiltration through at least one filter, preferably two filters, wherein at least one filter preferably has a pore size of about 1 to about 20 μm and / or about 0.1 μm to about 4 μm. (c) Inactivating the vector by adding binary ethyleneimine (BEI) to the mixture from step (b), (d) A step of neutralizing the BEI by adding sodium thiosulfate to the mixture obtained from step (c), and (e) A step of concentrating the polypeptide in the mixture obtained from step (d) by removing the liquid portion from the mixture by a filtration step using a filter having a filter membrane having a molecular weight cutoff of about 5 kDa to about 100 kDa, preferably about 10 kDa to about 50 kDa, and (f) Optionally, the mixture remaining after step (e) is mixed with further components selected from the group consisting of pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and combinations thereof. This includes methods.

[0084] In step (a) of the above method, the cells are preferably insect cells, and the vector is preferably the baculovirus of the present invention. In step (b) of the method described above, the polypeptide is most preferably recovered in the supernatant of the cultured cells rather than from inside the cells. Furthermore, the present invention provides an immunogenic composition and a use of the immunogenic composition in any of the methods described herein, wherein the immunogenic composition is obtained by a method described later for generating the immunogenic composition of the present invention.

[0085] Furthermore, the present invention is - Immunogenic fragments of rotavirus VP8 protein, and - Heterodimerization domain The present invention provides a polypeptide comprising the above, wherein the heterodimerization domain is ligated to the C-terminus of the immunogenicity fragment of the rotavirus VP8 protein. The term “dimerizing domain,” as used herein, particularly relates to an amino acid sequence that can specifically bind to or associate with one further dimerizing domain to form, for example, a dimer. In one example, a dimerizing domain is an amino acid sequence that can bind to or homoassociate with one other dimerizing domain having the same amino acid sequence to form a homodimer. A dimerizing domain may contain one or more cysteine ​​residues, and as a result, disulfide bonds may or may be formed between the associated dimerizing domains.

[0086] In this context, "heterodimerization domain" particularly refers to a dimerization domain derived from an entity other than rotavirus from which the immunogenic fragment of the rotavirus VP8 protein referred to herein originates. For example, a heterodimerization domain is a dimerization domain encoded by the genome of a virus other than rotavirus, or preferably by the genome of a eukaryotic or prokaryotic cell, particularly a mammalian or avian cell. Preferably, the heterodimer domain is a dimer domain encoded by a genome of a certain type that is susceptible to infection by rotavirus from which the immunogenic fragment of the rotavirus VP8 protein referred to herein originates. For example, if the rotavirus VP8 protein fragment is a porcine rotavirus VP8 protein fragment, the heterodimer domain is preferably a dimer domain encoded by the porcine genome. In another example, if the rotavirus VP8 protein fragment is a chicken rotavirus VP8 protein fragment, the heterodimer domain is preferably a dimer domain encoded by the chicken genome.

[0087] In another preferred embodiment, the heterodimerizing domains can each form a homodimer, or can form one. In a preferred example, the heterodimerizing domain referred to herein is a coiled-coil domain, in particular a leucine zipper domain. The leucine zipper domain is preferably a c-Jun leucine zipper domain, for example, a porcine c-Jun leucine zipper domain. [Examples]

[0088] The following embodiments are intended solely to illustrate the present disclosure and are not intended to limit the scope of the claims. (Example 1) Design, production, and testing of fusion proteins: Building design: The rotavirus A VP4 sequence was originally obtained from a fecal sample of a genus Suis, and it most closely matches the GenBank sequence JX971567.1, classifying it as the P[7] genotype. We used amino acids 57-224 of VP4 (SEQ ID NO: 3), also named "AVP8" below, which has an elongated N-terminus of 8 amino acids but corresponds to the lectin-like domain of the VP8 protein. The linker is Gly-Gly-Ser (SEQ ID NO: 9). The genus Suis IgG Fc sequence (SEQ ID NO: 7) matches amino acids 242-470 of the IgG heavy chain constant precursor (GenBank sequence BAM75568.1). We received (SEQ ID NO: 17) the IDT Gblock encoding AVP8, the Gly-Gly-Ser linker, and the genus Suis IgG Fc sequence, all codon-optimized for insect cells, and named AVP8-IgG Fc as described herein. The protein encoded by AVP8-IgG Fc (SEQ ID NO: 12) is also referred to herein as the "AVP8-IgG Fc protein".

[0089] Cloning, expression, and purification: AVP8-IgG Fc was cloned using TOPO, then inserted into the baculovirus transfer plasmid pVL1393 using the BamHI and NotI restriction sites, and subsequently co-transfected Sf9 cells with BaculoGold to produce recombinant baculovirus. The AVP8-IgG Fc protein was produced as follows: 1 L of Sf+ cells in a 3 L spinner flask were infected with used medium recovered at 4 DPI at a 0.2 MOI, centrifuged at 15,000 g for 20 minutes, and filtered through a 0.2 μm filter. 1 mL of MabSelect SuRE LX resin slurry (GE Healthcare, catalog no. 17-5474-01) was added, and the mixture was incubated overnight at 4°C with gentle agitation. The resin was recovered by filtration, washed with 4 × 10 mL of Gentle binding buffer (Pierce, catalog number 21012), and eluted with 7 × 5 mL of Gentle elution buffer (Pierce, catalog number 21027). The fractions were combined and dialyzed at 4°C against 3.5 L of TBS with a single buffer exchange. The concentration (80 μg / mL) was determined by a BCA assay (Thermo Scientific, catalog number 23227).

[0090] Serological studies: AVP8-IgG Fc protein purified with Protein A was formulated with Emulsigen D containing 87.5% antigen and 12.5% ​​adjuvant. Pigs approximately 7 weeks old received a 2 mL dose via IM on the lateral side of the neck at a boost at day 21. Serum samples were collected weekly for 7 weeks. Serum from pigs vaccinated with AVP8-IgG Fc protein was evaluated by ELISA (Figure 1) as described below ("Protocol for ELISA") and by viral neutralization assay (Figure 2) as described below ("Protocol for Viral Neutralization Assay"). Compared to an unrelated vaccine control, IgG ELISA results from pigs vaccinated with AVP8-IgG Fc protein showed an increase in the SP ratio, peaking at day 14 and rising again after the boost at day 21. Viral neutralizing titers similarly increased at days 7 and 14, followed by a second peak at day 28 after the boost at day 21.

[0091] Protocol for ELISA For IgA ELISA, a 96-well ELISA plate conjugated with culture medium proteins was coated with whole rotavirus antigen diluted 1:16 in 1×PBS. The plate was incubated overnight at 4°C. After incubation, the plate was washed with 1×PBST and then blocked with casein blocking solution at 37°C for 1 hour. After washing, 100 μL of primary antibody, diluted to a final dilution of 1:40 in blocking buffer, was added to the plate and incubated at 37°C for 1 hour. After washing, the wells were coated with 100 μL of horseradish peroxidase (HRP) conjugate goat anti-bovine IgA diluted 1:3200 and incubated at 37°C for 1 hour. After washing, the plate was developed with 3,5,3',5'-tetramethylbenzidine at room temperature for 15 minutes, and the reaction was stopped with 1N HCl before optical density (CD) measurement at 450 nm. Samples containing positive and negative controls are run in two replicates in wells, and the results are reported as the average of the ratio (SN) / (PN) of (sample-negative control) to (positive control-negative control).

[0092] For IgG ELISA, a 96-well ELISA plate coated with medium protein was coated with whole rotavirus antigen diluted 1:8 with 1×PBS. The plate was incubated overnight at a temperature of 4°C. After incubation, the plate was washed using 1×PBST and then blocked with a blotting grade blocking solution at 37°C for 1 hour. After washing, 100 μL of the primary antibody diluted to a final dilution of 1:625 in blocking buffer was added to the plate and incubated at 37°C for 1 hour. After washing, the wells were coated with 100 μl of 1:8000 diluted horseradish peroxidase (HRP)-conjugated goat anti-rat IgG and incubated at 37°C for 1 hour. After washing, the plate was developed with 3,5,3’,5’-tetramethylbenzidine at room temperature for 10 minutes, and the reaction was stopped with 1N HCl before measuring the optical density (OD) at 450 nm. Samples including positive and negative controls were run in duplicate in the wells, and the results were reported as the mean of the ratio (sample - negative control) to (positive control - negative control), (S - N) / (P - N).

[0093] Protocol for virus neutralization assay All serum and milk samples were heat-inactivated at 56°C for 30 minutes. The 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 isolate (titer 7.0 log TCID 50The stock ( / mL) was diluted 1:25,000 in rotavirus growth medium. A total of 200 μl of diluted serum was added to 200 μl of diluted virus, and the mixture was incubated at 37°C ± 5% CO2 for 1 hour. The growth medium was aseptically removed from a 96-well plate on which MA104 cells had been seeded for 3-4 days. After incubation, 200 μl of the virus-serum mixture was transferred to a cell culture plate. The cells were incubated at 37°C ± 5% CO2 for 72 hours. The stock and diluted viruses were titrated on the day of use to confirm the dilutions to be used in the assay. After incubation, the supernatant was discarded, and the plate was washed once with 200 μL / well of 1×PBS. After fixation, 100 μL / well of 50% / 50% acetone / methanol was added. The plates were incubated at room temperature for 15 minutes, air-dried, and then rehydrated with 100 μL / well of 1×PBS. The primary antibody (rabbit anti-rotavirus A polyclonal serum, prepared internally) was diluted 1:1000 in 1×PBS. 100 μL / well of the diluted primary antibody was added, and the plates were incubated at 37°C ± 5% CO2 for 1 hour. After incubation, the plates were washed twice with 100 μL / well of 1×PBS. The secondary antibody (Jackson ImmunoResearch FITC-labeled goat anti-rabbit IgG, catalog number 111-095-003) was diluted 1:100 in 1×PBS. 100 μL / well of the diluted secondary antibody was added, and the plates were incubated at 37°C ± 5% CO2 for 1 hour. After incubation, the plates were washed twice with 100 μL / well of 1×PBS. The plates were read for the presence of fluorescence using an ultraviolet microscope. The assay was performed on diluted virus titers (prepared using the Reed-Muench method) of 2.8 ± 0.5 log TCID. 50 A value of / mL was considered effective. In addition, known positive and negative samples were included as controls in each assay. Serum titers were reported as the highest dilution for which no staining was observed.

[0094] (Example 2) Load study: The primary objective of this study was to evaluate whether conventional 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 herein as "placebo," to conventional sows conferred passive protection to the pigs against pathogenic rotavirus A load. Furthermore, for comparison, a commercially available MLV rotavirus vaccine (ProSystem® Rota, Merck Animal Health), also referred herein as "commercial product" or "commercial vaccine," was used in the study. The prototype vaccine was produced in the same manner as the production described above in Example 1, but using different volumes and longer incubation periods used for infection, as described in the section "Generation of IgG:AVP8" below. The commercial product was used according to the label instructions provided by the manufacturer for the vaccine ProSystem® TGE / Rota (dosage and instructions, and recommended methods for oral vaccination of Suis animals).

[0095] 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. Sows T02 and T04 were mixed among three rooms. Sows T06 and T07 were housed in two separate rooms. All sows were vaccinated with appropriate materials via the appropriate routes listed in Table 1. Sows T07 remained unvaccinated (strict control). Serum was collected periodically from sows throughout the vaccination period and assayed for evidence of seroconversion. Fecal samples were collected pre-farming and screened by RT-qPCR to confirm that the mothers were not actively shedding rotavirus pre-farming. Overall health observations were recorded daily for each sow. Farming was allowed to occur spontaneously until the sows reached 114 days of gestation. After this point, farming 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. When the pigs reached 0-5 days of age, they were induced to bleed, fecal swabs were collected, and the pigs were loaded (except T07). At loading, the pigs were administered 5 mL of sodium bicarbonate intragastricly, followed by 5 mL of loading material intragastricly. Throughout the loading period, all animals were monitored daily for the presence of intestinal disease (diarrhea and behavioral changes). Fecal samples were collected regularly throughout the loading period. Two days after loading (DPC 2), approximately one-third of the pigs from each litter were euthanized. After euthanasia, necropsies were performed, and the pigs were evaluated for macroscopic lesions. Intestinal sections were collected for microscopic and immunohistochemical evaluation. Intestinal swabs were collected for RT-qPCR evaluation. At DPC 21, all remaining pigs were weighed, induced to bleed, and fecal swabs were collected. After sample collection, the pigs were euthanized. The pigs were evaluated for macroscopic lesions, and intestinal swabs were collected.

[0096] [Table 1]

[0097] Throughout the study, serum VN titers in sows from T07 (strict control) remained constant or decreased, demonstrating the absence of exposure and the effectiveness of the study (virus neutralization was assessed as described above in Example 1 ("Protocol for Virus Neutralization Assay"), and the results are shown in Figure 3). During the vaccination phase, the highest median serum VN titer was observed in sows vaccinated with the IgG:AVP8 (T04) prototype vaccine. In this group, a single dose administered 6 weeks prior to farrowing resulted in a more than fourfold increase in titer in 3 / 5 animals with T04 (IgG:AVP8) by D14. Prior to the time of pig loading, 5 / 5 animals with T04 (IgG:AVP8) had a more than fourfold increase in titer. Sows in the placebo group (T02) did not show a significant increase (<2x) in serum VN titer during the vaccination phase. Sows in the T06 (commercial vaccine) group did not show a significant increase (<2x) in serum VN titer until D35. Before the pig loading, both T06 (commercial vaccine) sows showed a 4x increase in titer. After lateral exposure to the loading material, serum VN titer increased in sows in the T02 (placebo) and T06 (commercial vaccine) groups. Conversely, serum VN titer in sows in the T04 (IgG:AVP8) group remained constant or decreased in 4 / 5 of the sows. Regarding colostrum and milk VN titer, in the T04 group (IgG:AVP8), VN titer was highest at farrowing, decreased in pre-load samples, and further decreased in post-load samples. In the placebo group (T02), VN titer was low at farrowing and before loading, but increased after lateral exposure to the loading material.

[0098] VN titers in pre-feeding pig serum were high (>1280) in most T04 (IgG:AVP8) pigs, indicating passive transfer of immunity from sow to pig. Conversely, titers were low (<1280) in most T02 (placebo) and T06 (commercial vaccine) pigs.

[0099] Throughout the entire loading phase, the highest mortality rate was observed in T02 (placebo), with 8 / 57 (14.0%) of the pigs that died. Conversely, only 1 / 46 (2.2%) of pigs died in T04 (IgG:AVP8), 1 / 22 (4.5%) of pigs died in T06 (commercial vaccine), and 1 / 27 (3.7%) of pigs died in T07 (strict control). Clinical signs of diarrhea were not observed in T07 (strict control) pigs throughout the study. Clinical signs of diarrhea in T02 (placebo) pigs began on day 1 or 2 after loading and resolved in most animals by DPC10. Overall, clinical signs of diarrhea were observed at least once during the study in 44 / 57 (77.2%) of animals in T02 (placebo). Of these 44 animals, diarrhea was considered severe in 29 (65.9%). In contrast, clinical signs of diarrhea were reduced in T04 (IgG:AVP8) pigs. See Table 2 below for a summary of the clinical diarrhea outcomes by group.

[0100] [Table 2]

[0101] Prior to loading, no rotavirus A RNA was detected by RT-qPCR, demonstrating the validity of the study. In addition, throughout the study, no rotavirus A RNA was detected by RT-qPCR in female or pig sows from T07 (strict control). In pigs after loading, shedding was most prevalent in T02 (placebo). In most pigs, shedding began in 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 detected RNA were reduced (see Figure 4 for median log rotavirus A RNA genome copies (gc) / mL in feces per study day), and the tests were conducted as described below ("Protocol for Rota A qRT-PCR").

[0102] A subset of pigs randomly selected from each group was euthanized and necropped using DPC2. The pigs were evaluated for macroscopic intestinal lesions (thin wall, gas-distended small intestine, pure liquid content, etc.), microscopic lesions (atrophic intestine), and the presence of rotavirus A-specific staining by immunohistochemical testing (IHC). Table 3 below shows the number of pigs with intestinal lesions at necropation by group. The load was considered successful because 84.2% (16 / 19) of the placebo group (T02) had macroscopic lesions, and 63.2% (12 / 19) of those were stained. Most interesting was the absence of rotavirus A staining in animals in only 1 / 15 of the pigs in T04 (IgG:AVP8). In addition, T04 (IgG:AVP8) showed a reduced percentage of pigs with macroscopic lesions compared to T02 (placebo) and the commercial product (T06).

[0103] [Table 3]

[0104] The average daily weight gain (kg) 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).

[0105] [Table 4] In conclusion, conventional vaccination of sows with the IgG:AVP8 prototype vaccine (containing the polypeptide of SEQ ID NO: 12) at 6 weeks and 2 weeks prior to farrowing results in high neutralizing antibody titers in sow serum and colostrum. These neutralizing antibodies were passively transmitted to the pigs postnatally, 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. Specifically, pigs born from vaccinated sows showed reduced fecal shedding of rotavirus A RNA, reduced mortality, reduced clinical signs of diarrhea, reduced rotavirus A colonization in DPC2, reduced macroscopic lesions in DPC2, and increased ADWG compared to pigs born from placebo controls and commercially available vaccines.

[0106] Protocol for rota A 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, catalog number 1725140) was used for the assay. See Table 5 below for primer and probe information.

[0107] [Table 5]

[0108] Real-time RT-PCR was performed in a 20 μl reaction mixture containing 5 μl extracted total nucleic acid, 1 μl of each probe (5 μM), 1 μl of each primer (10 μM), 10 μl 2×RT-PCR mix, 0.5 μl iScript reverse transcriptase, and 0.5 μl DEPC-treated water. The reaction was performed using a CFX96 real-time PCR detection system (BioRad) under the following conditions: initial reverse transcription at 50°C for 10 minutes, followed by initial denaturation at 95°C for 3 minutes, denaturation for 40 cycles at 95°C for 15 seconds, and annealing and extension at 60°C for 45 seconds. To prepare relative quantitative data, serial dilutions of two rotavirus A g-blocks were included in each run. Each of the equal volumes of g-blocks was used as a starting concentration of 5.0 × 10⁶. 7 The genome copy / μL was used and included in the run. Optical data was analyzed using CFX Manager software. For each decision, a threshold line was automatically calculated using the regression setting for the cycle threshold (Ct) determination mode. Baseline subtraction was performed automatically using the baseline subtraction mode. Curves with baseline final values ​​less than 10 were manually corrected.

[0109] IgG:AVP8 production 1 x 10 in a 5L shaker flask 6Two liters of Sf+ (Spodoptera frugiperda) cells at an appropriate concentration of cells / mL were infected with 1.7 mL of recombinant baculovirus stock containing rotavirus A VP8 core-bovine IgG Fc fusion protein (BaculoGold(BG) / pVL1393-AVP8-IgG; 1.18 × 10⁸ TCID50 / mL). The shaker flask was incubated at 28°C ± 2°C for 5 days with constant agitation at 90 rpm. The cells and medium were transferred sterile to 3 × 1 L centrifuge bottles, and the cells were pelleted at 10,000 g at 4°C for 20 minutes. The obtained supernatant was passed through a 0.2 μm filter (Thermo Scientific, catalog number 567-0020) and then incubated overnight at 4°C with 2.5 mL of MabSelect SuRe LX Protein A resin (GE Healthcare, catalog number 17-5474-01) with gentle agitation. The resin was collected by 0.2 μm filtration (Thermo Scientific, catalog number 567-0020) and then washed with 12 × 10 mL of Gentle Ag / Ab binding buffer (Thermo Scientific, catalog number 21012). AVP8-IgG was eluted from the resin using 7 × 10 mL of Gentle Ag / Ab elution buffer (Thermo Scientific, catalog number 21027). AVP8-IgG was dialyzed in 3.5 L of 20 mM Tris pH 7.5 and 150 mM NaCl with a single buffer change. The remaining baculovirus was inactivated with 5 mM BEI at 37°C for 24 hours. The resulting material was diluted to a target concentration of 70 μg / mL in 1×PBS (Gibco catalog number 10010-023). The diluted material was formulated using 12.5% ​​Emulsigen D.

[0110] (Example 3) Serological studies: The primary objective of this study was to evaluate whether conventional administration of a prototype vaccine containing the AVP8-IgG Fc protein (SEQ ID NO: 12) and a control vaccine referred to herein as "placebo" to conventional sows induced 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 prepared in the same manner as the preparations described above in Examples 1 and 2, but using different volumes and longer incubation periods used for infection, as described below in the section "Vaccine Production: IgG-AVP8".

[0111] A total of 20 sows were included in the study. The sows were randomized to one of four treatment groups, as described in Table 6 below. The sows were mixed throughout the study. All sows were intramuscularly vaccinated with appropriate material on D0 and D21, as listed in Table 4. Serum was collected periodically from the sows throughout the study and assayed for evidence of seroconversion by viral neutralization assay. Overall health observations were recorded daily for each sow. The study was concluded on D42.

[0112] [Table 6]

[0113] Throughout the study, serum VN titers in sows from T06 and T07 (placebo group) remained constant or decreased, demonstrating the lack of exposure and the effectiveness of the study (virus neutralization was assessed as described above in Example 1 ("Protocol for Virus Neutralization Assay"), with modifications that evaluated increased dilution from 1:40 to 1:40,960). During the vaccination phase, sows vaccinated with IgG-AVP8 / Emulsigen D (T02) and IgG-AVP8 / Carbopol (T03) prototype vaccines showed a significant increase in titer (>4-fold). For both groups (T02 and T03), the group-average titer was above 640 after one vaccination and remained above 640 throughout the study period. In contrast, sows in the placebo group (T06 and T07) did not show a significant increase (<2-fold) in serum VN titer throughout the study. In conclusion, conventional vaccination of sows with the IgG-AVP8 prototype vaccine (containing the polypeptide of SEQ ID NO: 12) at 6 weeks and 2 weeks before farrowing results in high neutralizing antibody titers in the sow serum.

[0114] Vaccine generation: IgG-AVP8 1.00 x 10 in a 10L Sartorius Biostat B glass jacket container 6 8 L of Sf+ cells at individual cells / mL, mixed with 15 mL of BG / pVL1393-AVP8-IgG, 1.19 x 10⁴ cells. 8The cells were infected with TCID 50 / mL at an MOI of 0.22. The bioreactor was run at 27°C with agitation at 100 rpm and oxygen spurged at 0.3 slpm. The containers were collected at 6 DPI, centrifuged at 10,000 g and 4°C for 20 minutes, and the supernatant was filtered through a 0.8 / 0.2 μm filter (GE Healthcare, catalog no. 6715-7582). 2750 mL of the clarified supernatant was inactivated with 5 mM BEI at 27°C for 5 days. After neutralization of the remaining BEI with sodium thiosulfate, 2750 mL was concentrated to 225 mL approximately 12 times using a 10 kDa hollow fiber filter (GE, catalog no. UFP-10-C-4MA). The concentration was determined to be 255 μg / mL.

[0115] [Table 7]

[0116] [Table 8]

[0117] (Example 4) The primary objective of this study was to evaluate whether animals vaccinated with IgG-AVP8 (including 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 demonstrate the protective ability of the AVP8-IgG Fc protein (SEQ ID NO: 12) against other isolates. In short, fever-inactivated serum from IgG-AVP8-vaccinated pigs was diluted 2-fold in MEM starting at 1:200 in dilution blocks A through G. Row H did not contain serum. Various G and P types of rotavirus A were added to separate dilution blocks, columns 1 through 11, at 6.0 Log. 10 TCID 50Starting at 1 / mL, the solution was diluted 1.5-fold across the dilution plate. Column 12 did not contain the virus. 250 μL of virus and 250 μL of serum from the corresponding wells were mixed and incubated at 37°C for 1 hour. After 1 hour of incubation, a monolayer of MA104 cells was covered with 100 μL of the virus-serum mixture and incubated at 37°C for 72 hours. The cells were stained with IFA and read for the presence of the virus. The presence of the virus was recorded as "+" on the plate, and the absence of the virus was recorded as "0". These results were then transferred to Table 8. The following six rotavirus A isolates were compared in 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] , also neutralized the virus, indicating that the G-type was not important in neutralizing the virus in this assay.

[0118] [Table 9-1] [Table 9-2]

[0119] In conclusion, animals vaccinated with IgG-AVP8 (containing the AVP8-IgG Fc protein (SEQ ID NO: 12)) would cross-neutralize rotavirus genotypes P[7] and P

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

[0120] (Example 5) Proof of a conceptual experiment in animals of the genus *Suis*: A total of 40 animals will be used in this study. Pigs will be randomized into four treatment groups of 10 pigs each. The pigs will be mixed throughout the study. Overall health monitoring, pre-screening of serum samples, and pre-screening of fecal samples will be performed before treatment to confirm the health of the animals, determine the baseline serological response to rotavirus A, and confirm the absence of active rotavirus A infection before or at the time of vaccination. At day 0 (D0) of the study, animals will be intramuscularly vaccinated with the following materials: T01: IgG-P[7]AVP8 vaccine (containing polypeptide of SEQ ID NO: 12), T02: IgG-P

[13] AVP8 vaccine (containing polypeptide of SEQ ID NO: 14), T03: P[7]AVP8-IgG-P

[13] AVP8 vaccine (containing polypeptide of SEQ ID NO: 16), T04: placebo. Serum samples will be collected on days 0, 7, 14, 21, 28, 36, 42, and 49 of the study. All animals will be humanely euthanized by autopsy on D49 of the study. Serum samples will be tested by a viral neutralization assay to determine the serological response to the vaccine prototype over time. Animals vaccinated in T01 will have antibodies neutralizing rotavirus genotypes P[7] and P

[23] , animals vaccinated in T02 will have antibodies neutralizing rotavirus genotype P

[13] , and animals vaccinated in T03 will have antibodies neutralizing rotavirus genotypes P[7], P

[13] , and P

[23] .

[0121] (Example 6) SDS PAGE: SDS-PAGE of Protein A-purified AVP8-IgG Fc protein (SEQ ID NO: 12) product with and without DTT (Figure 5A): The method for preparing samples for SDS-PAGE imaging was, in short, as follows: The baculovirus recovered supernatant was inactivated with 10 mM BEI at 37°C for 36 hours and then neutralized. The samples were then purified using Protein A resin. All samples were then denatured using NuPAGE 4×LDS sample buffer (Invitrogen catalog number NP0007) with either 25 mM DTT (final) or an equal volume of water, and heated at 95°C for 10 minutes. The samples were stained by running them on a 4-12% SDS-PAGE gel (Invitrogen catalog number NP0335BOX) at 180V for 45 minutes (eStain L1, GenScript catalog number M00548-1; destaining catalog number M00549-1). As a result, it was found that in the lane run with the reduced (+DTT (dithiothreitol)) sample, mainly one band (monomer AVP8-IgG Fc protein, considered in conjunction with the Western blot results described below) was observed. Additional bands were observed in the lane run with the non-reduced sample (-DTT). Each of the additional bands was in a molecular weight range that was a multiple of the monomer.

[0122] Western blot: Western blot of anti-sinus animal IgG Fc fragments (Figure 5B): The AVP8-IgG Fc protein (SEQ ID NO: 12) product generated in a bioreactor was collected in 1 mL samples before BEI addition. The samples were centrifuged at 20,000 g and 4°C for 5 minutes, the supernatant was decanted into a new tube, and both the 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 volumes of pellet and supernatant were passed over an SDS-PAGE under reducing conditions (+DTT) and transferred to a PVDF membrane. Western blots were examined with 1:1000 dilution HRP-conjugated goat anti-sinus animal to detect the sinus animal IgG Fc fragments. As a result, unexpectedly, the AVP8-IgG Fc protein was not found in the cell pellet samples. Instead, all AVP8-IgG Fc proteins (SEQ ID NO: 12) were favorably found in the cell culture supernatant samples.

[0123] (Example 7) Consensus sequence generation: The consensus sequences for Sequence ID No. 4 (based on genotype P[6] rotavirus VP8 protein) and Sequence ID No. 5 (based on genotype P

[13] rotavirus VP8 protein) were constructed as described below.

[0124] Sequences were compiled from publicly available rotavirus VP4 nucleotide sequences from the NCBI Virus Variation database and from internally induced rotavirus isolate sequences. Additional metadata about the sequences, including isolate name, isolate type P, geographical origin, and isolation date (where available), was also compiled. The 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. Unaligned VP5 amino acids were trimmed and discarded. The aligned VP8 protein sequences were imported into MEGA7 software for phylogenetic analysis, and neighbor-joined phylogenetic reconstructions were constructed based on the VP8 protein sequences. Optimal trees were computer-generated (n=100) using a Poisson correction method with a phylogenetic bootstrap test, plotted at a constant scale with branch lengths equal to evolutionary distance, at a unit of site-level amino acid substitutions, across all 170 positions. Nodes with a bootstrap cluster association higher than 70% were considered significant. Nodes with a distance of approximately 10% and a bootstrap cluster association higher than 70% were designated as clusters. Abnormal sequences that did not fit into larger clusters were individually evaluated for sequence quality and P-type origin. Suspiciously low-quality sequences were removed from the analysis, while sequences from P-type, which are rarely observed in rotaviruses of the genus *Sinosus*, were retained. Clusters used to construct the consensus sequence were selected based on the desired product protection profile and in vitro serum cross-neutralization studies. The consensus sequence was constructed by the maximum frequency at each aligned position, and amino acid residues were selected based on reported epidemiological data, along with the product protection profile, when equivalent proportions of amino acids were observed at the aligned positions.

[0125] (Example 8) Load study: The primary objective of this study was to evaluate whether conventional administration to maternal animals 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 herein as "placebo," conferred passive protection to pigs against pathogenic rotavirus A loading. The prototype vaccine was produced in the same manner as the production described above in Example 1, but using different volumes and different purification methods used for infection, as described in the section "Generation of IgG#AVP8" below.

[0126] A total of 20 sows were included in the study. The sows were randomized to two treatment groups and one strict control group, as described in Table 9 below. Sows T01 and T03 were mixed among three rooms. Sows T07 were housed in separate rooms. All sows were vaccinated with appropriate materials via the appropriate routes listed in Table 9. Sows T07 remained unvaccinated (strict control). Serum was collected periodically from the sows throughout the vaccination period and assayed for evidence of seroconversion. Fecal samples were collected pre-farrowing and screened by RT-qPCR to confirm that the sows were not actively shedding rotavirus pre-farrowing. Overall health observations were recorded daily for each sow. Farring was allowed to occur spontaneously until the sows reached 114 days of gestation. After this point, 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. When the pigs reached 1–5 days of age, they were induced to bleed, fecal swabs were collected, and the pigs were loaded (except for T07). At loading, the pigs were administered 5 mL of sodium bicarbonate intragastricly, followed by 1 mL of loading material intragastricly. Throughout the loading period, all animals were monitored daily for the presence of intestinal disorders (diarrhea and behavioral changes). Fecal samples were collected 1 day after loading (DPC1). At DPC2, all pigs from T01 and T03 were euthanized. Intestinal sections were collected for microscopic and immunohistochemical evaluation.

[0127] [Table 10]

[0128] Throughout the study, the serum VN titer of mothers from T07 (strict control) increased less than fourfold, demonstrating the absence of exposure and the effectiveness of the study. (Virus neutralization was assessed as described above in Example 1 ("Protocol for Virus Neutralization Assay"), and the results are shown in Table 10 and Figure 6.) During the vaccination phase, the highest mean serum VN titer was observed in mothers vaccinated with the prototype vaccine IgG#AVP8 (T03 group). In this group, a single dose administered 6 weeks prior to calving resulted in a more than fourfold increase in titer in 6 / 8 animals in T03 (IgG#AVP8) by D14. Mothers in the T01 group (placebo) did not have a significant increase (<2x) in serum VN titer during the vaccination phase. Mother colostrum VN titer: Mothers in the T03 group (IgG#AVP8) had a higher mean VN titer compared to mothers in the T01 group (placebo).

[0129] [Table 11]

[0130] VN titers in pre-loaded pig serum were high (>1280) in most pigs in the T03 group (IgG number AVP8), indicating passive transfer of immunity from mother to pig. Conversely, titers were low (<1280) in most pigs in the T02 group (placebo). In the T01 (placebo) and T03 (IgG#AVP8) groups, pigs were defined as affected if rotavirus antigen was detected by immunohistochemical testing (IHC) in at least one intestinal section and the animal had an abnormal fecal score at least one day after loading. The frequency distribution is listed in Table 1 below. Based on the use of this case definition, maternal vaccination with the prototype vaccine IgG#AVP8 (T03 group) at 6 weeks and 2 weeks prior to farrowing prevented rotavirus-related disease in pigs after loading with heterologous rotavirus AP[7] loading material; prevention rate, 0.926, 95% confidence interval 0.734, 0.979.

[0131] [Table 12]

[0132] In conclusion, conventional maternal vaccination with the prototype vaccine IgG#AVP8 (containing the polypeptide of SEQ ID NO: 12) at 6 weeks and 2 weeks prior to farrowing results in high neutralizing antibody titers in sow serum and colostrum. These neutralizing antibodies were passively transmitted to pigs postnatally, as evidenced by the detection of high titers (>1280) in the serum of pigs from vaccinated mothers. The presence of high neutralizing antibody titers in pigs provides clinical protection. Specifically, a small number of pigs born from vaccinated mothers were considered affected compared to pigs born from placebo controls.

[0133] Generation of IgG#AVP8 Two 10L Sartorius Biostat B glass-jacketed containers contain 1.00 x 10 6Sf+ cells were seeded in 3 L of cells / mL. Three days after seeding, each container was infected with a MOI of 0.1, and the volume of each container was adjusted to 8 L using Ex-cell 420 serum-free medium (SAFC catalog no. 14420C-1000 mL). The bioreactor was run at 27°C with agitation at 100 rpm, using a CCA covered with dissolved oxygen set to 40% or above 40% and 1.3 slpm. The containers were collected seven days after inoculation, the liquid was centrifuged at 10,000 g and 4°C for 20 minutes, and the supernatant was filtered through a 0.8 / 0.2 μm filter (GE Healthcare, catalog no. 6715-7582). The clarified supernatant (8 L / container) was inactivated in Sartorius Biostat B glass-jacketed containers with 5 mM BEI 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, catalog number UFP-10-C-5A). The concentrated material was dialyzed with 5 volumes (3500 mL) of 1×PBS. The vaccine was formulated using 12.5% ​​Emulsigen D, 28% concentrated material, and 59.5% 1×PBS (volume:volume).

[0134] (Example 9) Proof of a conceptual experiment in animals of the genus *Suis*: A total of 20 animals will be used in this study. The pigs will be randomized into two treatment groups of 10 pigs each. The pigs will be mixed throughout the study. Overall health monitoring, pre-screening of serum samples, and pre-screening of fecal samples will be performed before treatment to confirm the health of the animals, determine the baseline serological response to rotavirus C, and confirm the absence of active rotavirus C infection before or at the time of vaccination. On day 0 (D0) and D28 of the study, the animals will be intramuscularly vaccinated with the following materials: T01: IgG-CVP8 vaccine (containing polypeptide of SEQ ID NO: 15), T02: placebo. Serum samples will be collected on days 0, 7, 14, 21, 28, 36, and 42 of the study. All animals will be humanely euthanized by necropsy on D42 of the study. Serum samples will be tested by ELISA to determine the serological response to the vaccine prototype over time. Animals vaccinated with T01 had higher average levels of antibodies against rotavirus C than animals vaccinated with T02, although this did not involve an increase in titer. [Brief explanation of the drawing]

[0135] [Figure 1] This figure shows the serum IgG response of pigs vaccinated against porcine rotavirus A with either AVP8-IgG Fc protein formulated with Emulsigen D (labeled "AVP8-IgG") or placebo ("unrelated control"). [Figure 2] This figure shows the results of a VN (viral neutralization) assay performed to detect and quantify antibodies capable of neutralizing porcine rotavirus A virus in samples from pigs vaccinated with either AVP8-IgG Fc protein formulated with Emulsigen D (labeled "AVP8-IgG") or placebo ("unrelated control"). [Figure 3]This figure shows the average VN titer for rotavirus in sow serum by group and study day. In the figure, study days D0 and D28 represent the time points "6 weeks and 2 weeks before farrowing" (i.e., when the investigational drug was administered to study groups T02 and T04, respectively), while study days D7, D28, and D35 represent the time points "5 weeks, 2 weeks, and 1 week before farrowing" (i.e., when the commercially available vaccine was administered to T06). [Figure 4] This figure shows the median log rotavirus A RNA genome copy (gc) / mL in the feces of each group on the day of the study. [Figure 5] A) This figure shows the SDS-PAGE of a sample of protein A-purified AVP8-IgG Fc protein (SEQ ID NO: 12) product, either reduced with dithiothreitol ("+DTT") or not reduced ("-DTT"); B) This figure shows a Western blot of the AVP8-IgG Fc protein (SEQ ID NO: 12) bioreactor product, in which the sample was centrifuged and separated into a cell pellet fraction ("pellet") and a supernatant fraction ("supernatant"). After a freeze-thaw process, these were flowed onto an SDS-PAGE under reducing conditions ("+DTT"), transferred to a PVDF membrane, and examined with HRP-conjugated goat anti-sinus animals to detect the sinus IgG Fc fragment. [Figure 6] This figure shows the average VN titer for rotavirus in sow serum by group and study date. In the figure, study dates D0 and D28 represent the time points "6 weeks and 2 weeks before farrowing" (i.e., when the investigational drug was administered to study groups T01 and T03, respectively).

[0136] In the sequence listing / origin and geographical origin (where applicable): Sequence ID 1 corresponds to the sequence of the (genotype P[7]) rotavirus VP8 protein originating from a farm in North Carolina, USA. Sequence ID 2 corresponds to the lectin-like domain sequence of the (genotype P[7]) rotavirus VP8 protein originating from a farm in North Carolina, USA. Sequence ID 3 corresponds to the sequence of an immunogenic fragment of the (genotype P[7]) rotavirus VP8 protein originating from a farm in North Carolina, USA. Sequence ID 4 corresponds to the sequence of the immunogenicity fragment of the rotavirus VP8 protein, i.e., the consensus sequence of a portion of the rotavirus VP8 protein (based on genotype P[6]).

[0137] Sequence ID 5 corresponds to the sequence of the immunogenic fragment of the rotavirus VP8 protein, that is, the consensus sequence of a portion of the consensus sequence of the immunogenic fragment of the rotavirus VP8 protein (based on genotype P

[13] ). Sequence ID 6 corresponds to the sequence of the immunogenic fragment of the rotavirus C VP8 protein. Sequence ID 7 corresponds to the sequence of an IgG Fc fragment from a wild boar (Philadelphia genus). Sequence ID 8 corresponds to the sequence of a guinea pig IgG Fc fragment. Sequence ID 9 corresponds to the linker sequence.

[0138] Sequence ID 10 corresponds to the linker sequence. Sequence ID 11 corresponds to the linker sequence. Sequence ID 12 corresponds to the sequence of a polypeptide (fusion protein) that contains the sequences of Sequence ID 3, Sequence ID 9, and Sequence ID 7. Sequence ID 13 corresponds to the sequence of a polypeptide (fusion protein) that contains the sequences of Sequence ID 4, Sequence ID 9, and Sequence ID 7. Sequence ID 14 corresponds to the sequence of a polypeptide (fusion protein) that contains the sequences of Sequence ID 5, Sequence ID 9, and Sequence ID 7.

[0139] Sequence ID 15 corresponds to the sequence of a polypeptide (fusion protein) that contains the sequences of Sequence ID 6, Sequence ID 9, and Sequence ID 7. Sequence ID 16 corresponds to the sequence of a polypeptide (fusion protein) that contains the sequences of Sequence ID 3, Sequence ID 9, Sequence ID 7, Sequence ID 10, and Sequence ID 5. Sequence ID 17 corresponds to the polynucleotide sequence encoding the polypeptide (fusion protein) of Sequence ID 12. Sequence ID 18 corresponds to the polynucleotide sequence encoding the polypeptide (fusion protein) of Sequence ID 13. Sequence ID 19 corresponds to the polynucleotide sequence encoding the polypeptide (fusion protein) of Sequence ID 14. Sequence ID 20 corresponds to the polynucleotide sequence encoding the polypeptide (fusion protein) of Sequence ID 15. Sequence ID 21 corresponds to the polynucleotide sequence encoding the polypeptide (fusion protein) of Sequence ID 16. Sequence IDs 22-25: Primer and probe sequences (Table 5).

[0140] The following sections are also disclosed herein. Accordingly, this disclosure further includes embodiments characterized by the following sections. 1. Immunogenic fragments of rotavirus VP8 protein, and - Immunoglobulin Fc fragments Polypeptides containing this material. 2. The immunoglobulin Fc fragment is ligated to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein, or The immunoglobulin Fc fragment is ligated to the N-terminus of the immunogenicity fragment of the rotavirus VP8 protein. Polypeptides as described in item 1. 3. 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 N-terminus of the immunogenicity fragment of the rotavirus VP8 protein via a linker moiety. A polypeptide as described in 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. A polypeptide as described in any one of items 1 to 3. 5. The polypeptide according to any one of claims 1 to 4, wherein the immunoglobulin Fc fragment is ligated to the C-terminus of the immunogenicity fragment of the rotavirus VP8 protein.

[0141] 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. A polypeptide as described in any one of items 1 to 5. 7. The polypeptide described in any one of items 1 to 6, wherein the polypeptide is a fusion protein. 8. The polypeptide is of formula xyz (wherein, x consists of an immunogenic fragment of the rotavirus VP8 protein. y is the linker part, z is an immunoglobulin Fc fragment. A polypeptide that is a fusion protein, in particular a polypeptide as described in any one of items 1 to 7. 9. The polypeptide according to any one of claims 1 to 8, wherein the immunogenic fragment of the rotavirus VP8 protein can induce an immune response to rotavirus in a subject to whom the immunogenic fragment of the rotavirus VP8 protein is administered. 10. The polypeptide according to any one of items 1 to 9, wherein the immunogenicity fragment of the rotavirus VP8 protein has a length of 50 to 200, preferably 140 to 190 amino acid residues.

[0142] 11. The polypeptide according to any one of items 1 to 10, wherein the rotavirus is porcine rotavirus. 12. The polypeptide 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 polypeptide according to any one of items 1 to 12, wherein the rotavirus is rotavirus A. 14. The polypeptide according to any one of items 1 to 13, wherein the immunogenicity fragment of the rotavirus VP8 protein contains the lectin-like domain of the rotavirus VP8 protein.

[0143] 15. The polypeptide according to any one of items 1 to 14, wherein the immunogenicity fragment of the rotavirus VP8 protein is a lectin-like domain with an extended N-terminus of the rotavirus VP8 protein, and the N-terminal extension is 1 to 20 amino acid residues long, preferably 5 to 15 amino acid residues long. 16. The polypeptide according to item 14 or 15, wherein the lectin-like domain of the rotavirus VP8 protein consists of the amino acid sequence of amino acid residues 65-224 of the rotavirus VP8 protein. 17. The polypeptide according to item 15 or 16, wherein the N-terminal extension amino acid sequence is an individual amino acid sequence of length adjacent to the N-terminal amino acid residue of the lectin-like domain in the amino acid sequence of the rotavirus VP8 protein. 18. The immunogenic fragment of the rotavirus VP8 protein is Rotavirus VP8 protein, amino acid residues 60-224, 59-224, 58-224, 57-224, 56-224, 55-224, 54-224, 53-224, 52-224, 51-224, 50-224, or 49-224 A polypeptide consisting of the amino acid sequence described in any one of items 1 to 17. 19. The polypeptide according to any one of items 1 to 18, wherein the immunogenicity fragment of the rotavirus VP8 protein consists of the amino acid sequence of amino acid residues 57 to 224 of the rotavirus VP8 protein.

[0144] 20. The polypeptide according to any one of claims 16 to 19, wherein the numbering of the amino acid residues refers to the amino acid sequence of the wild-type rotavirus VP8 protein, particularly the wild-type rotavirus A VP8 protein, and the wild-type rotavirus VP8 is preferably the protein shown in SEQ ID NO: 1. 21. The polypeptide 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. 22. The polypeptide according to any one of claims 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 with the sequence of SEQ ID NO: 1.

[0145] 23. The polypeptide according to any one of claims 14 to 22, wherein the lectin-like domain of the rotavirus VP8 protein consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO: 2. 24. The polypeptide according to any one of claims 1 to 23, wherein the immunogenic fragment of the rotavirus VP8 protein comprises 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.

[0146] 25. The immunogenic fragment of the rotavirus VP8 protein consists of, or is a portion of, the consensus sequence of the rotavirus VP8 protein, particularly a portion of the rotavirus A VP8 protein. The consensus sequence of a portion of the rotavirus VP8 protein is preferably, - A step of translating multiple nucleotide sequences that encode a portion of the rotavirus VP8 protein into an amino acid sequence. - Preferably, the amino acid sequence is aligned with a known rotavirus VP8 protein by using MUSCLE sequence alignment software UPGMB clustering and default gap penalty parameters. - The steps of subjecting the aligned sequences to phylogenetic analysis and creating neighbor-jointed phylogenetic reconstructions based on the rotavirus VP8 protein sequence, in particular, the steps of importing the aligned amino acid sequences into MEGA7 software for phylogenetic analysis and creating neighbor-jointed phylogenetic reconstructions based on the rotavirus VP8 protein sequence, - A step to calculate the optimal tree using the Poisson correction method with a phylogenetic 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 association higher than 70% are considered significant. - A step of designating nodes with a distance of approximately 10% and a bootstrap cluster association higher than 70% as a cluster, and - A step of creating 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. Methods that may be obtained, including A polypeptide as described in any one of items 1 through 24.

[0147] 26. The polypeptide according to any one of claims 1 to 25, wherein the immunogenicity fragment of the rotavirus VP8 protein comprises an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 4 and SEQ ID NOs: 5. 27. The polypeptide according to any one of items 1 to 26, wherein the rotavirus is rotavirus C. 28. The polypeptide according to claims 1 to 27, wherein the immunogenic fragment of the rotavirus VP8 protein comprises 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. 29. The immunogenic fragment of the rotavirus VP8 protein is - Immunogenic fragments of rotavirus A VP8 protein as defined in any one or more of paragraphs 9 to 24, or - A portion of the rotavirus VP8 protein, in particular a consensus sequence of a portion of the rotavirus A VP8 protein, as specified in any one of paragraphs 9-13, 25, and 26, or - Immunogenic fragments of the rotavirus C VP8 protein as specified in any one of paragraphs 9-12, 27, and 28. A polypeptide consisting of, or being, one of the following items 1 to 28.

[0148] 30. The polypeptide according to any one of claims 1 to 29, wherein the immunogenicity fragment of the rotavirus VP8 protein comprises an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 3, SEQ ID NOs: 4, SEQ ID NOs: 5, and SEQ ID NOs: 6. 31. The immunoglobulin Fc fragment is at least 220 amino acid residues long, preferably 220 to 250 amino acid residues long, and / or The immunoglobulin Fc fragment is not glycosylated. A polypeptide as described in any one of items 1 to 30. 32. The polypeptide according to any one of claims 1 to 31, wherein the immunoglobulin Fc fragment comprises or consists of a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3), and optionally, a hinge region or a portion of a hinge region of the immunoglobulin. 33. The polypeptide 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 polypeptide according to any one of items 1 to 33, wherein the immunoglobulin Fc fragment is an immunoglobulin Fc fragment encoded by a certain genome that makes intestinal cells susceptible to infection by rotavirus derived from an immunogenic fragment of the rotavirus VP8 protein. 35. The polypeptide according to any one of items 1 to 34, wherein the immunoglobulin Fc fragment is a IgG Fc fragment of a genus Suis.

[0149] 36. The polypeptide according to any one of claims 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 particularly 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 7 and SEQ ID NOs: 8. 37. The polypeptide 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 polypeptide according to any one of claims 3 to 37, wherein the linker portion comprises, or consists of, an amino acid sequence having at least 66%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or particularly 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. 39. The polypeptide according to any one of claims 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.

[0150] 40. The polypeptide according to any one of claims 5 to 39, wherein the polypeptide comprises a further immunogenic fragment of the rotavirus VP8 protein ligated to the C-terminus of the immunoglobulin Fc fragment. 41. - Immunogenic fragment of rotavirus VP8 protein (1), - Immunoglobulin Fc fragments, and - Further immunogenic fragments of the rotavirus VP8 protein (2) Polypeptides containing, in particular, polypeptides described in any one of claims 1 to 40, The immunoglobulin Fc fragment is ligated to the C-terminus of the immunogenicity fragment (1), A further immunogenic fragment (2) of the rotavirus VP8 protein is ligated to the C-terminus of the immunoglobulin Fc fragment. Polypeptide.

[0151] 42. Further immunogenic fragments of the rotavirus VP8 protein are - Immunogenic fragments of rotavirus A VP8 protein as defined in any one or more of paragraphs 9 to 24, or - A consensus sequence of a part of the rotavirus VP8 protein, particularly a part of the rotavirus A VP8 protein, as defined in any one or more of items 9 to 13, 25, and 26, or - An immunogenic fragment of the rotavirus C VP8 protein as defined in any one or more of items 9 to 12, 27, and 28 The polypeptide according to item 40 or 41, which consists of or is such. 43. A 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 with a sequence selected from the group consisting of SEQ ID NOs: 2 to 6, and / or The polypeptide according to any one of items 40 to 42, wherein a further immunogenic fragment of the rotavirus VP8 protein is different from an immunogenic fragment of the rotavirus VP8 protein whose C-terminus is linked to the immunoglobulin Fc fragment.

[0152] 44. A further immunogenic fragment of the rotavirus VP8 protein is linked to the C-terminus of the immunoglobulin Fc fragment via a linker portion, and the linker portion is preferably a linker portion as defined in item 37 or 38, or A 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. The polypeptide according to any one of items 40 to 43. 45. The polypeptide is - An immunogenic fragment of the rotavirus VP8 protein, particularly an immunogenic fragment of the rotavirus VP8 protein as defined in any one or more of items 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 - Immunoglobulin Fc fragments, in particular, immunoglobulin Fc fragments as defined in any one or more of items 31-36. And, The immunoglobulin Fc fragment is linked to the C-terminus of the immunogenic fragment of the rotavirus VP8 protein, particularly via a linker moiety, and the linker moiety is preferably a linker moiety as defined in item 37 or 38, and - Optionally, in particular, a further immunogenic fragment of the rotavirus VP8 protein linked to the C-terminus of the immunoglobulin Fc fragment via a linker moiety, wherein the further immunogenic fragment of the rotavirus VP8 protein is preferably a further immunogenic fragment as defined in any one or more of items 41 to 44, and the linker moiety is preferably a linker moiety as defined in item 37 or 38. A polypeptide comprising any one of items 1 to 44.

[0153] 46. ​​The polypeptide according to any one of claims 1 to 45, wherein the polypeptide comprises, or is a protein comprising, an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or particularly 100%, sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 12, SEQ ID NOs: 13, SEQ ID NOs: 14, SEQ ID NOs: 15, and SEQ ID NOs: 16. 47. The polypeptide according to any one of items 1 to 46, wherein the polypeptide is a recombinant protein, in particular a recombinant baculovirus expression protein. 48. The polypeptide according to any one of claims 1 to 47, wherein the polypeptide forms a homodimer with a second identical polypeptide. 49. A polymer comprising or composed of a plurality of polypeptides as described in any one of items 1 to 48, wherein the polymer is preferably a homodimer formed by a polypeptide described in any one of items 1 to 48 and a second identical polypeptide.

[0154] 50. An immunogenic composition comprising a polypeptide as described in any one of items 1 to 48 and / or a polymer as described in item 49. 51. The immunogenic composition according to claim 50, further comprising a pharmaceutically or veterinarily acceptable carrier or excipient. 52. The immunogenic composition according to claim 50 or 51, further comprising an adjuvant. 53. - Polypeptides described in any one of items 1 to 48 and / or polymers described in item 49, and - Pharmaceutically or veterinarily acceptable carriers or excipients, - Optionally, adjuvant An immunogenic composition containing or comprising the same. 54. The immunogenic composition according to item 52 or 53, wherein the adjuvant is an emulsified oil-in-water adjuvant. 55. The immunogenic composition according to item 52 or 53, wherein the adjuvant is a carbomer.

[0155] 56. A polynucleotide comprising a nucleotide sequence encoding a polypeptide as described in any one of items 1 to 48. 57. The polynucleotide according to claim 56, wherein the polynucleotide comprises 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, and a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or particularly 100% sequence identity. Plasmids comprising polynucleotides containing a sequence encoding a polypeptide as described in any one of sections 58.1 to 48, preferably an expression vector. 59. A plasmid comprising a polynucleotide containing a sequence encoding a polypeptide as described in any one of sections 1 to 48, preferably a cell comprising an expression vector.

[0156] 60. A baculovirus containing a polynucleotide comprising a sequence encoding a polypeptide as described in any one of paragraphs 1 to 48. 61. Cells containing a baculovirus, preferably insect cells, that contain a polynucleotide comprising a polypeptide sequence described in any one of items 1 to 48. 62. For the preparation of pharmaceuticals, preferably vaccines, - Polypeptides described in any one of items 1 to 48, - The polymer described in item 49, - Immunogenic compositions as described in any one of items 50 to 55, - Polynucleotides as described in item 56 or 57, - Plasmids described in item 58, - Baculoviruses as described in item 60, and / or - Cells as described in item 59 or 61 Use. 63. A polypeptide according to any one of items 1 to 48 or an immunogenic composition according to any one of items 50 to 55, for use as a pharmaceutical. 64. A polypeptide according to any one of items 1 to 48 or an immunogenic composition according to any one of items 50 to 55, for use as a vaccine.

[0157] 65. A polypeptide according to any one of claims 1 to 48 or an immunogenic composition according to any one of claims 50 to 55, for use in a method for inducing an immune response to rotavirus in a subject. 66. For use in reducing or preventing one or more clinical signs, mortality or fecal shedding caused by rotavirus infection in subjects, or for use in treating or preventing rotavirus infection in subjects, A polypeptide as described in any one of items 1 to 48, or an immunogenic composition as described in any one of items 50 to 55. 67. The polypeptide or immunogenic composition according to item 65 or 66, wherein the subject is a mammal or a bird, and the bird is preferably a chicken. 68. The polypeptide or immunogenic composition according to any one of items 65 to 67, wherein the subject is a mammal, and the mammal is preferably an animal of the genus Sus scrofa or an animal of the subfamily Bovinae. 69. The polypeptide or immunogenic composition according to any one of items 65 to 68, wherein the subject is a pig, and the pig is preferably a piglet or a sow.

[0158] 70. The polypeptide or immunogenic composition according to item 65, wherein the subject is a pregnant sow. 71. The polypeptide or immunogenic composition according to item 66, wherein the subject is a piglet. 72. A polypeptide according to any one of items 1 to 48 or an immunogenic composition according to any one of items 50 to 55 for use in a method of reducing or preventing one or more clinical signs, mortality or fecal excretion caused by rotavirus infection in piglets, wherein the piglets are suckled by a sow to which the immunogenic composition has been administered. 73. The polypeptide or immunogenic composition according to item 72, wherein the sow to which the immunogenic composition has been administered is a sow that was pregnant, particularly a sow to which the immunogenic composition was administered while pregnant with the piglets. 74. A method for the treatment or prevention of rotavirus infection, reduction, prevention or treatment of one or more clinical signs, mortality or fecal excretion caused by rotavirus infection, or prevention or treatment of a disease caused by rotavirus infection, the method comprising administering a polypeptide according to any one of items 1 to 48 or an immunogenic composition according to any one of items 50 to 55 to a subject. 75. A method for inducing the production of antibodies specific to rotavirus in sows, the method comprising administering a polypeptide according to any one of items 1 to 48 or an immunogenic composition according to any one of items 50 to 55 to the sow.

[0159] 76. A method for reducing or preventing one or more clinical signs, mortality, or fecal shedding caused by rotavirus infection in piglets, The method described above is - A step of administering to a sow a polypeptide described in any one of items 1 to 48 or an immunogenic composition described in any one of items 50 to 55, and - The piglet is nursed by the sow. Methods that include... 77. The method according to paragraph 76, wherein the sow is pregnant, in particular a sow pregnant with a piglet. 78. - A step of administering a polypeptide according to any one of items 1 to 48 or an immunogenic composition according to any one of items 50 to 55 to a sow that is pregnant with the piglets. - The step of causing the sow to give birth to the piglets, - The piglet is nursed by the sow. The method described in paragraph 76 or 77, including the method described in paragraph 76 or 77. 79. A method for reducing one or more clinical signs, mortality or fecal excretion caused by rotavirus infection in piglets, wherein the piglets are lactated by a sow administered with a polypeptide according to any one of items 1 to 48 or an immunogenic composition according to any one of items 50 to 55.

[0160] 80. One or more of the above clinical signs are present. - diarrhea, - Rotavirus colony formation, - Lesions, especially macroscopic lesions, - A decrease in the average daily weight gain, and - Gastroenteritis A polypeptide or immunogenic composition according to any one of claims 66 to 73, selected from the group consisting of the above, or the method according to any one of claims 74 to 79. 81. The polypeptide or immunogenic composition according to item 80, or the method according to item 80, wherein the rotavirus colonization is intestinal rotavirus colonization and / or the lesion is an intestinal lesion.

[0161] 82. - The rotavirus infection is caused by genotype P

[23] rotavirus and / or genotype P[7]rotavirus. - The rotavirus infection is caused by genotype P

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

[23] rotavirus and / or genotype P[7]rotavirus. A polypeptide or immunogenic composition as described in any one of items 65-73, 80, and 81, or the method described in any one of items 74-81.

[0162] 83. The polypeptide according to claim 82, wherein the polypeptide comprises an immunogenic fragment of the genotype P[7]rotavirus VP8 protein, and the polypeptide is preferably a polypeptide as defined in any one of claims 21-26 and 29-48. 84. The immunogenic composition or method according to claim 82, wherein the immunogenic composition comprises a polypeptide as defined in any one of claims 21-26 and 29-48, and the immunogenic fragment of the rotavirus VP8 protein is an immunogenic fragment of genotype P[7]rotavirus VP8 protein.

[0163] 85. The polypeptide according to item 83 or the immunogenic composition or method according to item 84, wherein the immunogenic fragment of the genotype P[7]rotavirus VP8 protein comprises 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. 86. A method for producing a polypeptide and / or a polymer as described in any one of sections 1 to 48, comprising the step of transfecting a cell with a plasmid as described in section 58. 87. A method for producing a polypeptide and / or a polymer according to any one of items 1 to 48, comprising the step of infecting cells, preferably insect cells, with a baculovirus according to item 60.

[0164] 88. A method for producing an immunogenic composition as described in any one of paragraphs 50 to 55, wherein the method is (a) A step that enables infection of a susceptible cell in a culture with a vector comprising a nucleic acid sequence encoding a polypeptide described in any one of items 1 to 48, wherein the polypeptide is expressed by the vector, (b) Subsequently, a step of recovering the polypeptide, particularly in the cell culture supernatant, wherein the cell debris is separated from the polypeptide via a separation step, preferably a separation step comprising microfiltration through at least one filter, preferably two filters, wherein at least one filter preferably has a pore size of about 1 to about 20 μm and / or about 0.1 μm to about 4 μm. (c) Inactivating the vector by adding binary ethyleneimine (BEI) to the mixture from step (b), (d) A step of neutralizing the BEI by adding sodium thiosulfate to the mixture obtained from step (c), and (e) A step of concentrating the polypeptide in the mixture obtained from step (d) by removing the liquid portion from the mixture by a filtration step using a filter having a filter membrane having a molecular weight cutoff of about 5 kDa to about 100 kDa, preferably about 10 kDa to about 50 kDa, and (f) Optionally, the mixture remaining after step (e) is mixed with further components selected from the group consisting of pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and combinations thereof. Methods that include... 89. Immunogenic compositions as defined in any one of the following sections, 50-55, 63-73 and 80-85, the use described in section 62, or the method described in any one of the following sections, 74-82, 84 and 85, which may be obtained by the method described in section 88.

[0165] 90. - Immunogenic fragments of rotavirus VP8 protein, and - Heterodimerization domain A polypeptide comprising the above, wherein the heterodimerizing domain is linked to the C-terminus of the immunogenicity fragment of the rotavirus VP8 protein. 91. The polypeptide according to item 90, wherein the heterodimerizing domain is a coiled-coil domain, in particular a leucine zipper.

Claims

1. - Immunogenic fragments of rotavirus VP8 protein, and - Immunoglobulin Fc fragment Polypeptides containing this material.

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 polypeptide according to claim 1.

3. Equation x - y - z (In the formula, x consists of an immunogenic fragment of the rotavirus VP8 protein. y is the linker part, z is an immunoglobulin Fc fragment. A polypeptide that is a fusion protein, particularly the polypeptide according to claim 1 or 2.

4. The rotavirus is porcine rotavirus, and / or The rotavirus is selected from the group consisting of rotavirus A and rotavirus C. The polypeptide according to any one of claims 1 to 3.

5. The polypeptide according to any one of claims 1 to 4, wherein the immunogenic fragment of the rotavirus VP8 protein is a lectin-like domain with an extended N-terminus of the rotavirus VP8 protein, and the N-terminal extension is 1 to 20 amino acid residues long, preferably 5 to 15 amino acid residues long.

6. The polypeptide according to any one of claims 1 to 5, wherein the rotavirus is selected from the group consisting of genotype P[7] rotavirus, genotype P[6] rotavirus, and genotype P[13] rotavirus.

7. The immunogenic fragment of the rotavirus VP8 protein consists of, or is a portion of, the consensus sequence of the rotavirus VP8 protein, particularly a portion of the rotavirus A VP8 protein. The consensus sequence of a portion of the rotavirus VP8 protein is preferably, - A step of translating multiple nucleotide sequences that encode a portion of the rotavirus VP8 protein into an amino acid sequence. - Preferably, the amino acid sequence is aligned with a known rotavirus VP8 protein by using the MUSCLE sequence alignment software UPGMB clustering and default gap penalty parameters. - The steps of subjecting the aligned sequences to phylogenetic analysis and creating neighbor-jointed phylogenetic reconstructions based on the rotavirus VP8 protein sequence, in particular the steps of importing the aligned amino acid sequences into MEGA7 software for phylogenetic analysis and creating neighbor-jointed phylogenetic reconstructions based on the rotavirus VP8 protein sequence, - A step of calculating the optimal tree using the Poisson correction method with a phylogenetic 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 association of more than 70% are considered significant. - A step of designating nodes having a distance of approximately 10% and a bootstrap cluster association higher than 70% as a cluster, and - A step of creating 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. It can be obtained by a method including, The polypeptide according to any one of claims 1 to 6.

8. The polypeptide according to any one of claims 1 to 7, wherein the immunogenicity fragment of the rotavirus VP8 protein comprises an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 3, SEQ ID NOs: 4, SEQ ID NOs: 5, and SEQ ID NOs:

6.

9. The immunoglobulin Fc fragment is an immunoglobulin Fc fragment encoded by a certain genome that makes intestinal cells susceptible to infection by rotavirus derived from an immunogenic fragment of the rotavirus VP8 protein, and / or The immunoglobulin Fc fragment is preferably a IgG Fc fragment from a genus Suis, and / or 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 particularly 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 7 and SEQ ID NOs:

8. The polypeptide according to any one of claims 1 to 8.

10. The linker portion is an amino acid sequence having a length of 1 to 50 amino acid residues, and / or The linker portion contains, 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 particularly 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 9, SEQ ID NOs: 10, and SEQ ID NOs:

11. The polypeptide according to any one of claims 1 to 9.

11. The further immunogenic fragment of rotavirus VP8 protein is linked to the C-terminus of the immunoglobulin Fc fragment, wherein the further immunogenic fragment of rotavirus VP8 protein is preferably linked to the C-terminus of the immunoglobulin Fc fragment via a linker moiety, the linker moiety being, in particular, the linker moiety defined in claim 10, or Further immunogenic fragments of the rotavirus VP8 protein are 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, and The further immunogenic fragment of the rotavirus VP8 protein preferably contains, or consists of, an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 2 to 6, and / or The further immunogenic fragment of the rotavirus VP8 protein is preferably different from the immunogenic fragment of the rotavirus VP8 protein in which the C-terminus is linked to the immunoglobulin Fc fragment. The polypeptide according to any one of claims 2 to 10.

12. A polypeptide according to any one of claims 1 to 11, comprising, or comprising, a protein, an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or particularly 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 12, SEQ ID NOs: 13, SEQ ID NOs: 14, SEQ ID NOs: 15, and SEQ ID NOs:

16.

13. A polymer comprising or composed of a plurality of polypeptides according to any one of claims 1 to 12, wherein the polymer is preferably a homodimer formed by the polypeptide according to any one of claims 1 to 12 and a second identical polypeptide.

14. An immunogenic composition comprising the polypeptide described in any one of claims 1 to 12 and / or the polymer described in claim 13.

15. A polynucleotide comprising a nucleotide sequence encoding a polypeptide according to any one of claims 1 to 12, wherein the polynucleotide preferably comprises 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, and a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or particularly 100% sequence identity.

16. A polypeptide according to any one of claims 1 to 12 or an immunogenic composition according to claim 14, for use as a pharmaceutical, preferably for use as a vaccine.

17. For use in reducing or preventing one or more clinical signs, mortality or fecal shedding caused by rotavirus infection in subjects, or for use in treating or preventing rotavirus infection in subjects, and / or For use in methods for inducing an immune response to rotavirus in a subject, A polypeptide according to any one of claims 1 to 12, or an immunogenic composition according to claim 14.

18. A method for reducing or preventing one or more clinical signs, mortality, or fecal shedding caused by rotavirus infection in piglets, wherein the method is - The step of administering to a sow the polypeptide according to any one of claims 1 to 12 or the immunogenic composition according to claim 14, and - The piglet is nursed by the sow. Methods that include...

19. The aforementioned 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 A polypeptide or immunogenic composition according to claim 17, selected from the group consisting of the above, or the method according to claim 18.

20. - The rotavirus infection is caused by genotype P[23] rotavirus and / or genotype P[7] rotavirus. - The rotavirus infection is caused by genotype P[23] rotavirus and / or genotype P[7] rotavirus, or - The immune response to the rotavirus is an immune response to genotype P[23] rotavirus and / or genotype P[7] rotavirus. The polypeptide or immunogenic composition according to claim 17 or 19, or the method according to claim 18 or 19.

21. The polypeptide comprises an immunogenic fragment of the genotype P[7]rotavirus VP8 protein, or the immunogenic composition comprises a polypeptide comprising an immunogenic fragment of the genotype P[7]rotavirus VP8 protein. Preferably, the immunogenicity fragment of the genotype P[7]rotavirus VP8 protein consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% sequence identity with the sequence of SEQ ID NO:

3. The polypeptide or immunogenic composition according to claim 20, or the method according to claim 20.

22. A method for producing the immunogenic composition described in claim 14, wherein the method is (a) A step of enabling infection of a susceptible cell in a culture with a vector comprising a nucleic acid sequence encoding a polypeptide according to any one of claims 1 to 12, wherein the polypeptide is expressed by the vector, (b) A step thereafter to recover the polypeptide, particularly in the cell culture supernatant, wherein the cell debris is separated from the polypeptide via a separation step, preferably a separation step including microfiltration through at least one filter, preferably two filters, wherein at least one filter preferably has a pore size of about 1 to about 20 μm and / or about 0.1 μm to about 4 μm. (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 of concentrating the polypeptide in the mixture obtained from step (d) by removing the liquid portion from the mixture by a filtration step using a filter having a filter membrane having a molecular weight cutoff of about 5 kDa to about 100 kDa, preferably about 10 kDa to about 50 kDa, and (f) Optionally, the mixture remaining after step (e) is mixed with further components selected from the group consisting of pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and combinations thereof. Methods that include...

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