Recombinant rotavirus and its production method and production system

Abstract

Disclosed are novel recombinant rotaviruses and methods for making same, systems for producing same, and methods for using same to induce an immune response in a subject against rotavirus infection and infection or to vaccinate a subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 414,283, filed October 7, 2022, and U.S. Provisional Patent Application No. 63 / 510,958, filed June 29, 2023, the entire contents of each of which are incorporated by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under AI144881 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing A Sequence Listing is submitted herewith as a Sequence Listing xml file entitled "144578_00383.xml", 55,776 bytes in size, created on October 9, 2023. The Sequence Listing was submitted electronically via the Patent Center and is incorporated by reference in its entirety. [Background technology]

[0004] Reverse genetics systems have been developed for several rotavirus strains, including the monkey strain SA11, the rhesus strain RRV, the murine strain rD6 / 2-2g, the bovine RF, the avian strain PO-13, the human G1P[8] strain KU, the human G1P[8] strain CDC-9, and the human G4P[8] strain Odelia. The most widely used rotavirus vaccine (Rotarix) is manufactured by GSK and formulated from the human G1P[8] strain RIX4414. In fact, approximately 75% of children vaccinated against rotavirus receive Rotarix. The demand for rotavirus vaccine doses is expected to reach nearly 300 million by 2030. Therefore, there is a need in the art for novel recombinant rotaviruses. Summary of the Invention

[0005] In one aspect of the present disclosure, compositions are provided. In some embodiments, the composition comprises a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript, and the recombinant rotavirus protein is a RIX4414 strain rotavirus protein.

[0006] Aspects of the present disclosure provide a collection of polynucleotides. In some embodiments, each of the polynucleotides of the collection comprises a sequence encoding at least one RIX4414 strain rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5, wherein the polynucleotides of the collection encode each of the VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 proteins, and each of the sequences encoding one rotavirus protein is operably linked to a promoter.

[0007] In some embodiments, the infectious particle comprises a sequence encoding a recombinant rotavirus protein, the polynucleotide encodes a positive-strand viral transcript, and the recombinant rotavirus protein is a RIX4414 strain rotavirus protein.

[0008] In some embodiments, the infectious particles are produced by transfecting cells with a collection of polynucleotides, each of the polynucleotides in the collection comprising a sequence encoding at least one RIX4414 strain rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5, and the polynucleotides of the collection encode each of the VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 proteins, and each of the sequences encoding one rotavirus protein is operably linked to a promoter.

[0009] In some embodiments, the pharmaceutical composition comprises an infectious particle comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript and the recombinant rotavirus protein is a RIX4414 strain rotavirus protein.

[0010] In some embodiments, the method comprises administering to a subject a pharmaceutical composition comprising an infectious particle comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript and the recombinant rotavirus protein is a RIX4414 strain rotavirus protein.

[0011] Aspects of the present disclosure provide methods for inducing an immune response against one or more microorganisms in a subject. In some embodiments, the method comprises administering a pharmaceutical composition comprising an infectious particle comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript, and the recombinant rotavirus protein is a RIX4414 strain rotavirus protein to the subject to induce an immune response against one or more microorganisms.

[0012] In some embodiments, the method comprises administering a pharmaceutical composition comprising an infectious particle comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript, and the recombinant rotavirus protein is a RIX4414 strain rotavirus protein to a subject to elicit an immune response in the subject against a pathogen or to vaccinate the subject against one or more pathogens.

[0013] Aspects of the present disclosure provide methods for vaccinating a subject against one or more pathogens. In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising an infectious particle comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript, and the recombinant rotavirus protein is a RIX4414 strain rotavirus protein, vaccinating the subject against one or more pathogens.

[0014] In some embodiments, the cell comprises a composition comprising a sequence encoding at least one RIX4414 strain rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5, wherein the polynucleotides of the collection encode each of the VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 proteins, and each of the sequences encoding one rotavirus protein is operably linked to a promoter or infectious particle comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript, and the recombinant rotavirus protein is a RIX4414 strain rotavirus protein.

[0015] Aspects of the present disclosure provide methods for producing rotavirus in vitro. In some embodiments, the method includes introducing a composition comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript and the recombinant rotavirus protein is a RIX4414 strain rotavirus protein, allowing cells to express one or more rotavirus proteins selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5, incubating the cells for a sufficient time to produce rotavirus, and recovering the virus produced by the cells to produce rotavirus in vitro.

[0016] In some embodiments, a method for producing rotavirus in vitro comprises introducing a collection of polynucleotides, made by transfecting a cell, wherein each of the polynucleotides in the collection comprises a sequence encoding at least one RIX4414 strain rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5, wherein the polynucleotides of the collection encode each of the VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 proteins, and each of the sequences encoding one rotavirus protein is operably linked to a promoter; and recovering virus produced by the cell to produce rotavirus in vitro.

[0017] Aspects of the present disclosure provide a system for producing a recombinant rotavirus. In some embodiments, the system comprises: (a) a composition comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript, and the recombinant rotavirus protein is a RIX4414 strain rotavirus protein; and (b) a cell capable of expressing the composition of (a).

[0018] In some embodiments, the system for producing a recombinant rotavirus comprises: (a) a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript, and the recombinant rotavirus protein is a RIX4414 strain rotavirus protein; and (b) a cell capable of expressing the assembly of (a). [Brief explanation of the drawings]

[0019] [Figure 1]Polyacrylamide gel demonstrating the size of genome segments of rotavirus isolates. Lane 1: Recombinant RIX4414 (human G1P[8]). Lane 2: Recombinant reassortants of RIX4414 and Odelia. Lane 3: Recombinant reassortants of RIX4414 and Odelia. Lane 4: Recombinant Odelia (human G4P[8]). [Figure 2A] Figures 2A and 2B show the recovery of recombinant RIX4414-like viruses by reverse genetics. Genome segments purified by Trizol extraction from (A) recombinant SA11 virus (rSA11wt), RIX4414-like virus (rRIX4414L), a reassortant of RIX4414-like virus and Odelia (rOdelia), and rOdelia, and (B) rRIX4414L and RIX4414 derived from the RV1 vaccine (vRIX4414), were separated by electrophoresis on a 10% polyacrylamide gel and stained with ethidium bromide. The positions of genome segments 1 to 11 in (A) rSA11wt and (B) rRIX4414L are indicated. The recombinant viruses rSA11wt and rOdelia were generated as previously described (8, 10). For the reassortant strain rRIX4414L / rOdelia, segments 1, 2, 3, 5, and 6 are derived from Odelia, and the remainder is derived from RIX4414 (8). [Figure 2B]Figures 2A and 2B show the recovery of recombinant RIX4414-like viruses by reverse genetics. Genome segments purified by Trizol extraction from (A) recombinant SA11 virus (rSA11wt), RIX4414-like virus (rRIX4414L), a reassortant of RIX4414-like virus and Odelia (rOdelia), and rOdelia, and (B) rRIX4414L and RIX4414 derived from the RV1 vaccine (vRIX4414), were separated by electrophoresis on a 10% polyacrylamide gel and stained with ethidium bromide. The positions of genome segments 1 to 11 in (A) rSA11wt and (B) rRIX4414L are indicated. The recombinant viruses rSA11wt and rOdelia were generated as previously described (8, 10). For the reassortant strain rRIX4414L / rOdelia, segments 1, 2, 3, 5, and 6 are derived from Odelia, and the remainder is derived from RIX4414 (8). DETAILED DESCRIPTION OF THE INVENTION

[0020] The introduction of rotavirus (RV) vaccines has reduced the incidence of severe gastroenteritis among young children in many countries. As an approach to generating rotavirus-based vaccines that confer protection against multiple pathogens, we investigated the feasibility of using RV as an expression vector for foreign proteins. This approach incorporates a 2A stop-restart translation element and a foreign ORF into RV segment 7 (NSP3) RNA, such that the segment acquires the ability to encode two separate proteins, NSP3 and a heterologous protein. To apply this technology, we developed a reverse genetics system for the human G1P[8] rotavirus strain (RIX4414) (formulated into the widely used ROTARIX vaccine). Through studies of RIX4414 and RIX4414 / SA11 reassortant viruses, we determined that RIX4414 segment 7 RNA could be reengineered to express the NSP3 and capsid proteins of other RNA viruses, including norovirus (NoV) and SARS-CoV-2. For example, inserting 0.9 and 1.6 kb of NoV sequence into RIX4414 segment 7 allows expression of the NoV P and VP1 capsid proteins, respectively. Furthermore, inserting 0.8 and 2.1 kb of SARS-CoV-2 sequence into RIX4414 segment 7 allows expression of the RBD and S1 domains of the SARS-CoV-2 spike protein, respectively. Further analysis of these and other recombinant RVs indicates that insertions of up to 1.1 kb of segment 7 sequence are genetically stable. These results indicate that RIX4414 can be used as a potential pediatric vaccine vector, enabling the development of combination vaccines against NoV and other pathogens.

[0021] The inventors have created a novel reverse genetics system for producing recombinant attenuated rotavirus, such as rotavirus strain RIX4414 (more commonly known as the strain found in the ROTARIX live-attenuated rotavirus vaccine). Furthermore, the inventors disclose herein that the novel reverse genetics system can be used to generate recombinant rotaviruses containing heterologous polynucleotide sequences, such as polynucleotide sequences encoding additional antigens, e.g., norovirus antigens. The inventors believe that the disclosed compositions, pharmaceutical compositions, infectious particles, methods, and systems may enable combination vaccination against the two most common causes of severe gastroenteritis in children.

[0022] definition The disclosed subject matter may be further described using the following definitions and terminology: The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0023] As used in this specification and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. For example, the term "substituent" should be construed to mean "one or more substituents" unless the context clearly dictates otherwise.

[0024] As used herein, "about," "approximately," "substantially," and "substantially / significantly" are understood by those of ordinary skill in the art and will vary to some extent with the context in which they are used. If there are uses of the terms that are not clear to persons of ordinary skill in the art given the context in which they are used, "about" and "approximately" will mean up to plus or minus 10% of the particular term, and "substantially and significantly" will mean plus or minus more than 10% of the particular term.

[0025] As used herein, the terms "include" and "including" have the same meaning as the terms "comprise" and "comprising." The terms "comprise" and "comprising" should be interpreted as "open" transitional terms that allow for the inclusion of additional components beyond those recited in the claims. The terms "consist" and "consisting of" should be interpreted as "closed" transitional terms that do not permit the inclusion of additional components beyond those recited in the claims. The term "consisting essentially of" should be interpreted as partially closed, allowing for the inclusion of only additional components that do not fundamentally alter the nature of the claimed subject matter.

[0026] The phrase "such as" should be interpreted as "for example, including." Furthermore, the use of any and all exemplary phrases, including but not limited to "such as," is intended merely to better clarify the invention and does not purport to limit the scope of the invention unless otherwise claimed.

[0027] Additionally, when a convention similar to "at least one of A, B, and C, etc." is used, generally, such a configuration is intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together). It will be further understood by those skilled in the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B."

[0028] All terms such as "up to," "at least," "greater than," "less than," and the like, are inclusive of the recited numbers and refer to ranges that can be subsequently divided into ranges and subranges. Ranges include individual members. Thus, for example, a group having 1 to 3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, etc.

[0029] The modal verb "may" refers to the preferred use or selection of one or more options or choices among several described embodiments or features contained therein. When options or choices regarding the specific embodiments or features contained are not disclosed, the modal verb "may" refers to a positive action regarding how to make or use aspects of the described embodiments or features contained therein, or to a final decision to use a particular skill regarding the described embodiments or features contained therein. In this latter context, the modal verb "may" has the same meaning and significance as the auxiliary verb "can."

[0030] As used herein, a "subject in need thereof" may refer to a subject at risk of rotavirus infection. In some embodiments, the disclosed compositions, methods, and infectious particles comprise a heterologous polynucleotide encoding an additional, non-rotavirus protein or peptide. Thus, in some embodiments, a subject in need thereof may refer to a subject at risk of rotavirus infection and / or infection by another pathogen, and the heterologous polynucleotide encodes an antigen, e.g., a protein or peptide, from a pathogen that is not rotavirus.

[0031] The term "subject" may be used interchangeably with the terms "individual" and "patient" and includes human and non-human mammalian subjects.

[0032] The phrases "% sequence identity," "percent identity," or "% identity" refer to the percentage of amino acid residues that match between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, described in more detail below, generally preserve charge and hydrophobicity at the substitution site, thus preserving the structure (and therefore function) of the polypeptide. Percent amino acid sequence identity can be determined as understood in the art. See, for example, U.S. Pat. No. 7,396,664, incorporated herein by reference in its entirety. A commonly used, freely available suite of sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the website of NCBI, Bethesda, Maryland. The BLAST software suite includes various sequence analysis programs, including "blastp," which is used to align a known amino acid sequence with other amino acid sequences from various databases.

[0033] The nucleic acids, proteins, and / or other compositions described herein can be purified. As used herein, "purified" means separate from a majority of other compounds or entities, and includes partially purified or substantially purified. Purity can be expressed on a weight basis and can be determined using various analytical techniques, including, but not limited to, gravimetric mass spectrometry, HPLC, and the like.

[0034] Polypeptide sequence identity can be measured over the length of the entire defined polypeptide sequence, for example, as defined by a particular SEQ ID NO, or over a shorter length, for example, over the length of a fragment obtained from the larger defined polypeptide sequence, for example, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 contiguous residues. It will be understood that such lengths are exemplary only, and that any fragment length supported by the sequences set forth herein in the tables, figures, or sequence listing can be used to describe the length over which percent identity can be measured.

[0035] As used herein, the terms "polypeptide," "protein," and "peptide" are used interchangeably and refer to a polymer of three or more amino acids. Thus, for example, a protein can include two proteins joined (fused) together. Additionally, a protein can refer to a portion or fragment of a protein, for example, the SARS-CoV-2 surface glycoprotein or SARS-CoV-2 S1 protein, which is a fragment of the "S" protein.

[0036] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Nucleic acid generally refers to a polymer comprising nucleotides or nucleotide analogs linked together via backbone bonds, e.g., but not limited to, phosphodiester bonds. Nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acids (RNA), such as messenger RNA (mRNA) and transfer RNA (tRNA). Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides, are linear molecules in which adjacent nucleotides are linked to each other via phosphodiester bonds. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA and single-stranded and / or double-stranded DNA. Nucleic acids can be naturally occurring, e.g., present in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. Alternatively, a nucleic acid molecule can be a non-naturally occurring molecule, e.g., recombinant DNA or RNA, artificial chromosome, engineered genome or fragment thereof, or synthetic DNA, RNA, DNA / RNA hybrid, or can contain non-naturally occurring nucleotides or nucleosides. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems, and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs, such as analogs with chemically modified bases or sugars, and backbone modifications. Nucleic acid sequences are presented in a 5' to 3' direction unless otherwise indicated.In some embodiments, nucleic acids are selected from the group consisting of naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methyluridine, C5-methyl- ... cytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine and 2-thiocytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0037] As used herein, the term "hybridization" refers to the formation of a duplex structure by two single-stranded nucleic acids through complementary base pairing. Hybridization can occur between completely complementary nucleic acid strands or between "substantially complementary" nucleic acid strands containing small regions of mismatch. Conditions under which hybridization of completely complementary nucleic acid strands is particularly favorable are called "stringent hybridization conditions" or "sequence-specific hybridization conditions." Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions. The degree of mismatch allowed can be controlled by appropriate adjustment of the hybridization conditions. Those skilled in the art of nucleic acid technology can determine duplex stability empirically, taking into account several variables, including, for example, the length and base pair composition of the oligonucleotide, ionic strength, and the incidence of mismatched base pairs, following guidance provided by those skilled in the art (see, e.g., Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Wetmur, 1991, Critical Review in Biochem. and Mol. Biol. 26(3 / 4):227-259; and Owczarzy et al., 2008, Biochemistry, 47:5336-5353, which are incorporated herein by reference).

[0038] Recombinant rotavirus compositions Thus, in a first aspect, disclosed herein is a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript.

[0039] As used herein, "RIX4414" refers to a strain of live attenuated rotavirus. In contrast, the present disclosure provides a "recombinant RIX4414" rotavirus produced by the disclosed reverse genetics system, which is distinct from the RIX4414 strain.

[0040] As used herein, "RIX4414 strain rotavirus protein" refers to a protein derived from the RIX4414 strain of rotavirus.

[0041] In some embodiments, the polynucleotide is operably linked to a promoter to allow expression of said polynucleotide in a cell, eg, a mammalian cell.

[0042] As used herein, "operably linked" refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship between a transcriptional control element (promoter) and a transcribed sequence. For example, a promoter is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate cell. Generally, promoter transcriptional regulatory elements operably linked to a sequence are physically adjacent to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory elements, such as enhancers, need not be physically adjacent to or located in close proximity to the coding sequence whose transcription they enhance. Exemplary promoters include the T7 bacteriophage promoter (SEQ ID NO: 14) and the T3 bacteriophage promoter (SEQ ID NO: 15). Suitable promoters can be selected from promoters known in the art. In some embodiments, the cell is a mammalian cell selected from MA-104 cells, Vero cells, and BHK-1 cells.

[0043] In some embodiments, the polynucleotide comprises a sequence encoding a rotavirus protein, i.e., selected from sequences encoding rotavirus VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5. The sequences encoding RIX4414 strain VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 proteins are presented herein as SEQ ID NOs: 1-11, respectively. Accordingly, the inventors contemplate that the disclosed compositions may comprise a polynucleotide comprising any one of SEQ ID NOs: 1-11, or a functional variant thereof (e.g., nucleic acid sequence variants that encode the same amino acids due to redundancy in the genetic code, e.g., or variants that result in different amino acid sequences but encode proteins or polypeptides having the same function), or a variant having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to any one of the polypeptides encoded by SEQ ID NOs: 1-11.

[0044] The inventors have discovered that a reverse genetics system can be used to generate a RIX4414 strain rotavirus that expresses a heterologous polynucleotide sequence by fusing the heterologous polynucleotide sequence to a sequence encoding the rotavirus NSP3 protein. Accordingly, the inventors herein disclose that in some embodiments, the disclosed compositions comprise a polynucleotide comprising a sequence encoding NSP3, e.g., (SEQ ID NO:9), and further comprise a heterologous polynucleotide sequence.

[0045] The heterologous polynucleotide may encode a Norovirus protein or peptide or a SARS-CoV-2 protein or peptide. The heterologous polynucleotide may be encoded by SEQ ID NOs: 16-26, or a sequence that is 85% similar, 86% similar, 87% similar, 88% similar, 89% similar, 90% similar, 91% similar, 92% similar, 93% similar, 94% similar, 95% similar, 96% similar, 97% similar, 98% similar, or 99% similar to one of SEQ ID NOs: 16-26, respectively (see Table 1 for complete sequences), NoV GII.4 MDA-145 VP1, NoV GII.4 MDA-145 P, NoV GII.4 MDA-145 P2, NoV GII.4 Cincinnati (Cin) VP1, NoV GII.4 Cincinnati (Cin) P, NoV GII.4 Sydney (Syd) VP1, NoV GII.4 Sydney (Syd)P may encode the SARS-CoV-2 S1 portion of the spike (S) protein, the SARS-CoV-2 RBD region of the S1 protein, the SARS-CoV-2 extended RBD region of the S1 protein, and the SARS-CoV-2 glycosylated S1 portion of the spike (S) protein including the C-terminal CTMI domain of the S2 portion of the spike (S) protein.

[0046] Furthermore, Philip AA, Patton JT. 2022. Generation of Recombinant Rotaviruses Expressing Human Norovirus Capsid Proteins. Journal of Virology 96:No 22, published October 31, 2022, which is incorporated herein by reference in its entirety, demonstrates that the inventors have successfully expressed norovirus (NoV) capsid proteins as heterologous polynucleotides in the RIX4414 reverse genetics system disclosed herein (see, e.g., Figure 5 of Phillip and Patton, 2022).

[0047] [Table 1] TIFF2025534877000003.tif209162TIFF2025534877000004.tif210161TIFF2025534877000005.tif210161TIFF2025534877000006.tif210161TIFF2025534877000007.tif209161TIFF2025534877000008.tif211161TIFF2025534877000009.tif211161TIFF2025534877000010.tif80161

[0048] In some embodiments, the heterologous polynucleotide encodes a protein or peptide. In some embodiments, a sequence encoding NSP3, e.g., SEQ ID NO: 9, further comprises a heterologous polynucleotide fused to its 3' end, such that the heterologous polynucleotide encodes the protein or peptide in-frame with the NSP3 sequence, thereby enabling transcription of a single mRNA encoding both NSP3 and the heterologous polynucleotide. In some embodiments, the polynucleotide comprising the sequence encoding NSP3 and the heterologous polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a self-cleaving peptide, e.g., the porcine Teschovirus P2A element (SEQ ID NO: 13). Thus, in some embodiments, the disclosed compositions comprise a polynucleotide encoding NSP3 fused 5' to 3' in-frame to a sequence encoding a self-cleaving peptide, which is fused in-frame to a heterologous polynucleotide sequence encoding a peptide or protein. Thus, transcription and translation of such a composition results in the production in a cell of a fusion protein comprising, from its N-terminus to its C-terminus, a self-cleaving peptide fused to the peptide or protein encoded by the heterologous polynucleotide, e.g., a rotavirus NSP3 protein fused to SEQ ID NO: 13. After translation, the fusion protein self-cleaves to yield two distinct proteins: (1) a functional rotavirus NSP3 protein and (2) a protein or peptide encoded by the heterologous polynucleotide. In some embodiments, the composition includes a sequence encoding a linker, e.g., a flexible linker, positioned in-frame 3' to the sequence encoding the NSP3 protein and 5' to the cleavage site. Without being limited by any theory or mechanism, the inventors believe that the addition of a flexible linker between the NSP3 protein and the cleavage site improves cleavage. In some embodiments, the linker is (GAG), where n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. n Linker (also called GAG linker), or n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (GSG) nlinker (also called GSG linker).

[0049] In some embodiments, the sequence encoding the cleavage site encodes a protease cleavage site, eg, a thrombin cleavage site, eg, SEQ ID NO:12.

[0050] The inventors further contemplate that the heterologous polynucleotide sequences described above include sequences encoding proteins or peptides derived from infectious organisms, such as norovirus or SARS-CoV-2. Thus, in some embodiments, the disclosed compositions include sequences encoding rotavirus NSP3 fused in-frame to a heterologous polynucleotide encoding a norovirus protein or peptide, such as norovirus VP1, or a SARS-CoV-2 protein or peptide, such as a SARS-CoV-2 surface glycoprotein.

[0051] In some embodiments, the composition comprises a polynucleotide comprising a sequence encoding a recombinant rotavirus NSP3, e.g., SEQ ID NO: 9, wherein the polynucleotide encodes a positive-strand viral transcript, and the polynucleotide further comprises a heterologous polynucleotide in-frame with the sequence encoding the recombinant rotavirus NSP3, wherein the heterologous polynucleotide encodes a peptide or protein comprising a norovirus or SARS-CoV-2 peptide or protein, or a fragment thereof. In some embodiments, the composition further comprises a sequence encoding a self-cleaving peptide, e.g., a sequence encoding SEQ ID NO: 13, fused in-frame between the polynucleotide and the heterologous polynucleotide. Thus, transcription and translation of the composition yields, from N-terminus to C-terminus, a functional rotavirus NSP3 protein, a self-cleaving linker, e.g., SEQ ID NO: 13, and a norovirus protein or peptide, e.g., norovirus VP1, or a SARS-CoV-2 protein or peptide, or a fragment thereof.

[0052] infectious particles In another aspect of the present disclosure, an infectious particle is provided. In some embodiments, the infectious particle comprises a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript.

[0053] As used herein, an "infectious particle" refers to any particle capable of causing infection of an organism or cell. Exemplary infectious particles include, but are not limited to, virus particles or virions. The terms "virus," "virus particle," and "virion" are used interchangeably herein.

[0054] Without wishing to be limited, the present disclosure provides a composition comprising a polynucleotide encoding a rotavirus protein operably linked to a promoter, for example, a T7 promoter (SEQ ID NO: 13). In some embodiments, the composition may be used in a reverse genetics approach to generate a recombinant rotavirus, for example, the recombinant rotavirus strain RIX4414. Thus, in some embodiments, the recombinant rotavirus may comprise the disclosed composition.

[0055] The disclosed infectious particles, e.g., recombinant rotaviruses, can contain one or more heterologous proteins or peptides, such as norovirus or SARS-CoV-2 proteins or peptides, as described above. The heterologous proteins or peptides can be encoded in the infectious particle, e.g., virus, genome and subsequently produced during viral replication. Such infectious particles containing heterologous proteins or peptides can be advantageous for eliciting an immune response in a subject or as a vaccine composition.

[0056] Pharmaceutical Composition The inventors herein disclose compositions, methods, and systems useful for producing recombinant rotaviruses that may be suitable for administration to a subject. Accordingly, in another aspect of the present disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical composition comprises an infectious particle comprising a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript. In some embodiments, the pharmaceutical composition comprises an infectious particle produced by transfecting a cell with a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript.

[0057] The disclosed compositions and methods may be administered as pharmaceutical compositions, and therefore, pharmaceutical compositions incorporating the compounds are considered embodiments of the disclosed compositions. Such compositions can take any pharmaceutically acceptable physical form. Illustratively, they may be orally administered pharmaceutical compositions. Such pharmaceutical compositions contain an effective amount of the disclosed compositions, which amount is related to the daily dose of the administered composition. Each dosage unit may contain the daily dose of a given composition, or each dosage unit may contain a fraction of the daily dose, such as half or one-third of the dose. The amount of each composition contained in each dosage unit may depend, in part, on other factors, such as the identity of the specific composition selected for treatment and the indication for which it is intended. The disclosed pharmaceutical compositions may be formulated to provide rapid, sustained, or delayed release of the active ingredient after administration to a patient using well-known procedures.

[0058] The pharmaceutical compositions can be utilized in methods of inducing an immune response or vaccinating against a pathogen, e.g., rotavirus, norovirus, SARS-CoV-2. As used herein, the terms "treat" or "treating" mean, respectively, alleviating symptoms, eliminating the cause of resulting symptoms, either temporarily or permanently, and / or preventing or delaying the onset of symptoms resulting from the specified disease or disorder, or reversing the progression or severity of symptoms. Thus, the methods disclosed herein encompass both therapeutic and prophylactic administration. By way of example, a subject can be at risk for infection by a pathogen, e.g., rotavirus, norovirus, SARS-CoV-2, and administration of the disclosed pharmaceutical compositions induces a protective immune response or vaccinates against the pathogen.

[0059] As used herein, the term "effective amount" refers to an amount or dose of a compound, upon single or multiple administration to a subject, that provides the desired effect in the subject being diagnosed or treated. The disclosed methods can include administering an effective amount of the disclosed compounds (e.g., when present in a pharmaceutical composition) to induce an immune response against a pathogen, e.g., rotavirus, norovirus, SARS-CoV-2, or vaccinating against the pathogen.

[0060] The effective amount can be easily determined by the attending physician, who is skilled in the art, by using known techniques and by observing results obtained under similar circumstances. In determining the effective amount or dose of the composition to be administered, several factors can be taken into account by the attending diagnostician, such as the species of the subject; its size, age, and general health; the degree or severity of the involvement of the disease or disorder involved; the response of the individual subject; the specific composition to be administered; the mode of administration; the bioavailability characteristics of the administered preparation; the selected dosage regimen; the use of concomitant medications; and other relevant circumstances.

[0061] Oral administration is an exemplary route of administering the compositions and methods disclosed herein. Other exemplary routes of administration include transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or rectal routes. The route of administration can vary in any way, limited by the physical properties of the compound used and the convenience of the subject and caregiver.

[0062] As one skilled in the art will appreciate, suitable formulations include those suitable for more than one route of administration. For example, a formulation may be suitable for both intrathecal and intracerebral administration. Alternatively, suitable formulations include those suitable for only one route of administration, as well as those suitable for one or more routes of administration but not one or more others. For example, a formulation may be suitable for oral, transdermal (percutaneous), intravenous, intramuscular, intranasal, buccal, and / or intrathecal administration, but not for intracerebral administration.

[0063] The inactive ingredients and formulation of the pharmaceutical compositions are conventional. Conventional methods of formulation used in pharmacy can be used herein. All conventional types of compositions can be used, including tablets, chewable tablets, capsules, liquids, parenteral solutions, nasal sprays or powders, troches, suppositories, transdermal patches, and suspensions. Generally, the compositions contain from about 0.5% to about 50% total compound, depending on the desired dose and the type of composition used. However, the amount of compound is best defined as an "effective amount," i.e., the amount of compound that provides the desired dose to a patient in need of such treatment. The activity of the compounds used in the compositions and methods disclosed herein is not expected to depend significantly on the nature of the composition; therefore, the compositions can be selected and formulated primarily or solely for convenience and economy.

[0064] Capsules are prepared by mixing the compound with a suitable diluent and filling the appropriate amount of the mixture into capsules. Typical diluents include inert powdered substances (such as starch), powdered cellulose (especially crystalline and microcrystalline cellulose), sugars (such as fructose, mannitol, and sucrose), flours, and similar edible powders.

[0065] Tablets are prepared by direct compression, wet granulation, or dry granulation. These formulations usually incorporate diluents, binders, lubricants, and disintegrants (in addition to the compound). Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts (such as sodium chloride), and powdered sugar. Powdered cellulose derivatives can also be used. Typical tablet binders include substances such as starch, gelatin, and sugars (such as lactose, fructose, glucose, etc.). Natural and synthetic gums, including acacia, alginate, methylcellulose, polyvinylpyrrolidine, etc., can also be used. Polyethylene glycol, ethylcellulose, and waxes can also function as binders.

[0066] Tablet can be coated with sugar, for example, as flavor enhancer and sealant.Compound can also be formulated as chewable tablet by using a large amount of pleasant-tasting substance such as mannitol in the formulation.For example, to ensure that patients use dosage form, and also avoid the difficulty that some patients experience when swallowing solid objects, can also use instantaneous dissolving tablet-like formulation.

[0067] Lubricants can be used in tablet formulations to prevent the tablet and punches from sticking to the die. Lubricants can be selected from slippery solids such as talc, magnesium and calcium stearate, stearic acid, and hydrogenated vegetable oils.

[0068] Tablets can also contain disintegrants. Disintegrants are substances that swell when wet, causing the tablet to disintegrate and release the compound. These include starch, clay, cellulose, algin and gum. Further examples include corn and potato starch, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation exchange resin, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate and carboxymethylcellulose.

[0069] The composition can be formulated as an enteric preparation, for example, to protect the active ingredient from the strong acid content of the stomach.Such a preparation can be made by coating a solid dosage form with a polymer film that is insoluble in acidic environments and soluble in basic environments.Exemplary films include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate and hydroxypropylmethylcellulose acetate succinate.

[0070] The compounds can also be delivered using transdermal patches. The transdermal patch can include a resin composition in which the compound dissolves or partially dissolves, and a film that protects the composition and holds the resin composition in contact with the skin. Other, more complex patch compositions can also be used, such as those with a membrane perforated with multiple pores through which the drug is pumped by the action of osmotic pressure.

[0071] As will also be understood by those skilled in the art, formulations can be prepared using materials (e.g., active excipients, carriers (e.g., cyclodextrins), diluents, etc.) that have properties (e.g., purity) that make the formulation suitable for administration to humans, or the formulations can be prepared with materials that have purity and / or other properties that make the formulation suitable for administration to non-human subjects but not for administration to humans.

[0072] Methods for generating recombinant rotavirus Another aspect of the present disclosure provides methods for producing rotavirus in vitro. In some embodiments, the methods include introducing a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, where the polynucleotide encodes a positive-strand viral transcript, allowing cells to express one or more rotavirus proteins selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5, incubating the cells for a sufficient time to produce rotavirus, and recovering the virus produced by the cells to produce rotavirus in vitro.

[0073] cell The inventors herein disclose cells comprising the disclosed compositions, which can also be used in the disclosed methods and systems. Accordingly, in another aspect of the present disclosure, cells are provided. In some embodiments, the cells comprise a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript. In some embodiments, the cells are selected from MA-104 cells, Vero cells, and BHK-1 cells.

[0074] Rotavirus vaccine strains have traditionally been grown using Vero cells. This method of producing rotavirus has been found to be suitable for producing rotavirus for administration to subjects. Thus, in some embodiments, the cells are Vero cells.

[0075] In some embodiments, the cells disclosed herein further comprise a heterologous RNA polymerase, which binds to a promoter of the disclosed compositions and catalyzes sequence-dependent RNA polymerization based on the compositions when the compositions are introduced into the cells. As used herein, "heterologous RNA polymerase" refers to an RNA polymerase that is not present in the cell without introduction by molecular biological techniques, such as transduction, transfection, lipofection, etc. In some embodiments, the heterologous RNA polymerase comprises T7 bacteriophage RNA polymerase or T3 bacteriophage RNA polymerase, more commonly known as simply T7 polymerase and T3 polymerase, respectively.

[0076] Thus, in some embodiments, the cells further comprise T7 RNA polymerase or T3 RNA polymerase. In some embodiments, such cells are derived from BHK-1 cells but express a heterologous RNA polymerase, T7 bacteriophage RNA polymerase, and are therefore referred to as, for example, BHK-T7 cells. Thus, as used herein, a "BHK-T7 cell" is a BHK-1 cell that expresses a heterologous RNA polymerase, T7 bacteriophage RNA polymerase.

[0077] Methods for eliciting an immune response The present disclosure provides compositions, methods for their manufacture, and infectious particles. Accordingly, in another aspect of the present disclosure, a method for eliciting an immune response is provided. In some embodiments, the method comprises administering a pharmaceutical composition comprising an infectious particle comprising a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript.

[0078] In some embodiments, the method comprises administering a pharmaceutical composition comprising infectious particles produced by transfecting cells with a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript.

[0079] In some embodiments, methods of eliciting an immune response are provided. In some embodiments, the methods of eliciting an immune response comprise administering a pharmaceutical composition comprising infectious particles comprising a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript. In some embodiments, the methods of eliciting an immune response comprise administering a pharmaceutical composition comprising infectious particles produced by transfecting a cell with a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript.

[0080] How to vaccinate a subject Another aspect of the present disclosure provides methods for vaccinating a subject against one or more pathogens. In some embodiments, the method comprises administering a pharmaceutical composition comprising infectious particles comprising a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript. In some embodiments, the method comprises administering a pharmaceutical composition comprising infectious particles produced by transfecting a cell with a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript.

[0081] System for generating recombinant rotavirus In another aspect of the present disclosure, a system for producing a recombinant rotavirus is provided. In some embodiments, the system includes: (a) a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript; and (b) a cell capable of expressing the composition of (a). [Example]

[0082] The following examples are illustrative and should not be construed as limiting the scope of the claimed subject matter.

[0083] Example 1 - Generation of novel recombinant rotaviruses based on RIX4414 using reverse genetics We report the development of a reverse genetics system for the human rotavirus G1P[8]RIX4414 strain. This invention allows for the modification of the rotavirus strain used to formulate the widely used Rotarix vaccine and provides a route to generating modified RIX4414 strains that express capsid proteins of other infectious viruses, including norovirus. Rotavirus vaccines formulated with RIX4414-expressing norovirus capsid proteins, used in the leu of RIX4414, can provide immunological protection against rotavirus and norovirus, the two most common causes of severe viral gastroenteritis in children.

[0084] The RIX4414 reverse genetics system was developed as follows. Based on data available in NCBI GenBank (SEQ ID NOs: 1-11), the sequences of 11 genome segments of the RIX4414 strain were predicted. These sequences were used to construct 11 pUC19-based pT7 transcription vectors, each expressing one of the RIX4414 positive-strand RNAs. Recombinant RIX4414 was produced using the RIX4414 pT7 plasmid, generally following the reverse genetics protocol previously described in AAPhillips et al. (2020). The RIX4414 pT7 transcription plasmid and pCMV-NP868R capping enzyme plasmid were transfected into BHK-T7 cells as described previously. After 2 days, MA104 cells were overseeded onto the transfected BHK-T7 cells. After 3 days, the BHK-T7 / MA104 cell culture was overseeded with Vero cells (which is unique to the RIX4414 reverse genetics system). After 8 days, the BHK-T7 / MA104 cell mixture was freeze-thawed three times and used to amplify the recombinant virus. When the cell culture reached full infection, lysates were prepared from the cells, and rRIX4414 was recovered by plaque isolation.

[0085] Example 2 - T7 expression plasmid for producing recombinant human G1P[8] rotavirus composed of the RIX44143 sequence of the RV1 (Rotarix®, GSK) vaccine strain The most widely used rotavirus vaccine, RV1 (Rotarix®, GSK), is formulated from the human G1P[8] virus RIX4414 (1, 2). Over 24 million children received the RV1 vaccine in 2021 (2) to protect against rotavirus gastroenteritis. A challenge to rotavirus immunization efforts is that RV1 and other rotavirus vaccines achieve efficacy in low-income countries (50–64%), which can be substantially lower than in high- and middle-income countries (85–98%) (3, 4). Here, we report the development of a T7 expression plasmid that allows the recovery of recombinant RIX4414-like viruses by reverse genetics. This method may enable the generation of modified forms of the RV1 vaccine with improved performance in low-income countries.

[0086] The rotavirus genome consists of 11 double-stranded RNA segments (5). The rotavirus strain RIX4414 (originally designated 89-12) was isolated from a child with acute gastroenteritis in 1989 and serially passaged in cell culture to facilitate the introduction of attenuating mutations (6). In this study, we used the sequence information for RIX4414 available in GenBank to design 11 pT7 expression plasmids, each containing a cDNA sequence corresponding to one of the RIX4414 genome segments (Table 2). Where sequence information for RIX4414 was missing, the sequence information for the prototype human G1P[8]Wa virus was used instead for portions of the 5' and 3' untranslated regions (Table 2). Because the original pT7 / RIX4414 VP2 plasmid was not functional in the reverse genetics system, a modified plasmid was created by slightly modifying the RIX4414 VP2 coding region to include residues common to other human G1 / 4P[8] virus strains (e.g., Wa, KU (7), Odelia (8)) (Table 2). The RIX4414 cDNA sequence was placed downstream of the T7 promoter and upstream of the hepatitis D virus ribozyme sequence in the pT7 plasmid (9, 10). The pT7 plasmid was generated by Azenta Life Sciences using a pUC19 backbone.

[0087] The RIX4414 pT7 plasmid supported the recovery of recombinant (r)RIX4414-like viruses using a slightly modified reverse genetics procedure (11, 12). Briefly, individual wells of a 12-well plate containing BHK-T7 cells were transfected with a plasmid mixture containing 0.8 μg of each RIX4414 T7 plasmid (except for the NSP2 and NSP5 plasmids, which were 4.8 μg each), 1.6 μg of the pCMV-NP868R RNA capping plasmid, and 1.6 μg of the pcDNA-T7 RNA polymerase plasmid. Two days later, the transfected BHK-T7 cells were overseeded with 10 MA104 cells / well. Three days later, the BHK-T7 / MA104 cell culture was overseeded with 10 Vero cells / well. After 8 days, rRIX4414-like viruses contained in the cell lysates were amplified using Vero cells and plaque-isolated using MA104 cells (13). The RNA genomic profile of the rRIX4414-like viruses is shown in Figure 1. The genome sequence of the rRIX4414-like isolate was confirmed by Nanopore sequencing (14). Based on sequence analysis, the rRIX4414-like viruses shared >99% nucleotide and amino acid sequence identity with the RIX4414 vaccine virus, making the rRIX4414 reverse genetics system an ideal tool for investigating genetic alterations that may improve RV1 performance in low-income countries.

[0088] [Table 2] TIFF2025534877000012.tif229161

[0089] Each of the above references is incorporated herein by reference.

[0090] In the foregoing description, it will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein may suitably be practiced in the absence of any element or elements, or limitation or limitations, not specifically disclosed herein. The terms and expressions used are used as terms of description and not of limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, while the invention has been illustrated by specific embodiments and optional features, it should be understood that modifications and / or variations of the concepts disclosed herein may be made by those skilled in the art, and that such modifications and variations are deemed to be within the scope of the invention.

[0091] Numerous patent and non-patent literature may be cited herein. The cited references are incorporated herein by reference in their entirety. If there is a discrepancy in the definition of a term herein compared to the definition of a term in a cited reference, the term should be construed in accordance with the definition herein.

[0092] [Table 3] TIFF2025534877000014.tif248161

Claims

1. A composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-strand viral transcript, and the recombinant rotavirus protein is a RIX4414 strain rotavirus protein.

2. The composition of claim 1 , wherein the polynucleotide is operably linked to a promoter.

3. 3. The composition of claim 2, wherein the promoter is a T7 promoter, and optionally the T7 promoter comprises SEQ ID NO:

14.

4. 3. The composition of claim 2, wherein the promoter is a T3 promoter, and optionally the T3 promoter comprises SEQ ID NO:

15.

5. The composition of claim 1, wherein the polynucleotide comprises any one of SEQ ID NOs: 1 to 11.

6. The composition of claim 1 , wherein the polynucleotide comprises a sequence encoding rotavirus NSP3.

7. The composition of claim 5, wherein the sequence encoding rotavirus NSP3 comprises SEQ ID NO:

9.

8. The composition of claim 5 or 6, wherein the polynucleotide further comprises a heterologous polynucleotide.

9. 8. The composition of claim 7, wherein the heterologous polynucleotide encodes a protein in frame with the NSP3 ORF.

10. The composition of any one of claims 7 to 8, wherein the heterologous polynucleotide encodes a peptide or protein.

11. The composition of claim 10 , wherein the heterologous polynucleotide encodes a reporter.

12. The composition of claim 11 , wherein the peptide or protein comprises a microbial peptide or protein.

13. The composition of claim 12 , wherein the peptide or protein comprises a bacterial peptide or protein.

14. The composition of claim 12 , wherein the peptide or protein comprises a viral peptide or protein.

15. 14. The composition of claim 13, wherein the peptide or protein comprises a Norovirus (NoV) or SARS-CoV-2 peptide or protein.

16. 15. The composition of claim 14, wherein the Norovirus peptide or protein comprises a Norovirus VP1 protein or a fragment thereof.

17. The composition of any one of claims 1 to 16, wherein the polynucleotide comprises a sequence encoding a cleavage site.

18. 18. The composition of claim 17, wherein the cleavage site is a protease cleavage site.

19. 19. The composition of claim 18, wherein the cleavage site is a thrombin cleavage site (SEQ ID NO: 12).

20. 18. The composition of claim 17, wherein the cleavage site is a self-cleaving peptide sequence.

21. 21. The composition of claim 20, wherein the self-cleaving peptide sequence is the porcine Teschovirus 2A element (SEQ ID NO: 13).

22. The composition of claim 7 , wherein the polynucleotide comprises a sequence encoding a linker.

23. 23. The composition of claim 22, wherein the linker is a flexible linker selected from a GAG linker or a GSG linker.

24. 1. A collection of polynucleotides, each of the polynucleotides comprising a sequence encoding at least one RIX4414 strain rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5, wherein a polynucleotide of the collection encodes each of the VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 proteins, and each of the sequences encoding one rotavirus protein is operably linked to a promoter.

25. 25. The collection of claim 24, wherein the sequence encoding at least one rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 and NSP5 comprises SEQ ID NOs: 1 to 11, respectively.

26. 26. The collection of claim 25, wherein the sequence encoding at least one rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 and NSP5 consists of SEQ ID NOs: 1 to 11, respectively.

27. An infectious particle comprising the composition of claim 1.

28. An infectious particle comprising the composition of any one of claims 3 to 5.

29. 15. An infectious particle comprising the composition of claim 14.

30. An infectious particle produced by transfecting a cell with a composition according to any one of claims 1 to 23.

31. A pharmaceutical composition comprising an infectious particle according to any one of claims 27 to 30.

32. 1. A method comprising:

26. A method comprising administering to a subject the pharmaceutical composition of claim 25.

33. 1. A method of eliciting an immune response to one or more microorganisms in a subject, comprising:

32. A method comprising administering the pharmaceutical composition of claim 31 to a subject to elicit an immune response against the one or more microorganisms.

34. 34. The method of claim 33, wherein the one or more microorganisms include a norovirus.

35. 34. The method of claim 33, wherein the one or more microorganisms include rotavirus and norovirus.

36. 1. A method comprising:

32. A method comprising administering to a subject the pharmaceutical composition of claim 31.

37. 1. A method comprising:

32. A method comprising administering to a subject the pharmaceutical composition of claim 31 to induce an immune response in the subject against a pathogen or to vaccinate the subject against one or more pathogens.

38. 1. A method of vaccinating a subject against one or more pathogens, comprising:

32. A method comprising administering to a subject the pharmaceutical composition of claim 31 to vaccinate the subject against said one or more pathogens.

39. 39. The method of claim 37 or 38, wherein the one or more pathogens include norovirus.

40. 39. The method of claim 37 or 38, wherein the one or more pathogens include rotavirus and norovirus.

41. A cell comprising a composition according to any one of claims 1 to 23 or an infectious particle according to any one of claims 27 to 30.

42. A cell comprising the aggregate according to any one of claims 24 to 26.

43. The cell according to claim 41 or 42, wherein the cell is an MA-104 cell, a Vero cell, or a BHK-1 cell.

44. 44. The cell of claim 43, which expresses a heterologous RNA polymerase.

45. 45. The cell of claim 44, wherein the heterologous RNA polymerase is selected from T7 RNA polymerase and T3 RNA polymerase.

46. 1. A method for producing rotavirus in vitro, comprising:

24. A method comprising: introducing a composition according to any one of claims 1 to 23 into a cell; allowing the cell to express one or more rotavirus proteins selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 and NSP5; incubating the cell for a time sufficient to produce rotavirus; and recovering the virus produced by the cell to produce the rotavirus in vitro.

47. 1. A method for producing rotavirus in vitro, comprising:

31. A method comprising: introducing the assembly of any one of claims 27 to 30 into cells; incubating the cells for a time sufficient to produce rotavirus; and recovering the virus produced by the cells to produce the rotavirus in vitro.

48. 48. The method of claim 4 or 47, wherein the cells contain T7 RNA polymerase and optionally African swine fever virus capping enzyme.

49. 49. The method of claim 48, wherein the cell is selected from MA-104 cells, Vero cells, and BHK-1 cells.

50. 50. The method of claim 49, wherein the cell expresses a heterologous RNA polymerase.

51. 51. The method of claim 50, wherein the heterologous RNA polymerase is selected from T7 RNA polymerase and T3 RNA polymerase.

52. 41. The method of claim 40, wherein the cells are BHK-1 cells containing T7 RNA polymerase and optionally containing African swine fever virus capping enzyme.

53. A system for producing a recombinant rotavirus, comprising: (a) the composition of any one of claims 1 to 23; and (b) a system comprising a cell capable of expressing the composition of (a).

54. A system for producing a recombinant rotavirus, comprising: (a) an assembly according to any one of claims 27 to 30; and (b) A system comprising a cell capable of expressing the assembly of (a).

55. 55. The system of claim 53 or 54, wherein the cell comprises a heterologous RNA polymerase and optionally comprises African swine fever virus capping enzyme.

56. 56. The system of claim 55, wherein the cells comprise cells derived from a cell line selected from MA-104 cells, Vero cells, and BHK-1 cells.

57. 57. The system of claim 56, wherein the cells comprise BHK-1 cells containing T7 RNA polymerase.

58. 58. The system of claim 57, wherein the cells include BHK-1 cells, Vero cells, and MA-104 cells containing T7 RNA polymerase.

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