Recombinant rotavirus and method and system for producing same

Abstract

Disclosed are novel recombinant rotaviruses and methods of making the same, systems for generating the viruses, and methods of using the viruses to elicit an immune response in a subject or vaccinate a subject against rotavirus infection and infection.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 414,283 filed on October 7, 2022 and U.S. Provisional Patent Application No. 63 / 510,958 filed on June 29, 2023, the entire contents of each of which are incorporated herein by reference.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED DEVELOPMENT RESEARCH

[0004] This invention was made with government support from the National Institutes of Health under Grant No. AI 144881. The government has certain rights in this invention.

[0005] Sequence Listing

[0006] The present application is accompanied by a sequence listing, which is submitted as an xml file named "144578_00383.xml", with a size of 55,776 bytes and created on October 9, 2023. The sequence listing is submitted electronically through Patent Center, and its entire contents are incorporated herein by reference. Background Art

[0007] Reverse genetics systems have been developed for multiple rotavirus strains, including simian strain SA11, rhesus strain RRV, murine strain rD6 / 2-2g, bovine RF, avian strain PO-13, human G1P[8] strain KU, human G1P[8] strain CDC-9, and human G4P[8] strain Odelia. The most widely used rotavirus vaccine (Rotarix) is produced by GSK and is formulated with 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 new recombinant rotaviruses. Summary of the invention

[0008] In one aspect of the present disclosure, a composition is provided. In some embodiments, the composition comprises a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript, wherein the recombinant rotavirus protein is a rotavirus protein of the RIX4414 strain.

[0009] In one aspect of the present disclosure, a polynucleotide collection is provided. In some embodiments, each polynucleotide 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 in the collection encode each of VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 and NSP5 proteins, wherein each sequence encoding a rotavirus protein is operably linked to a promoter.

[0010] In one aspect of the present disclosure, an infectious particle is provided. In some embodiments, the infectious particle comprises a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript, wherein the recombinant rotavirus protein is a rotavirus protein of the RIX4414 strain.

[0011] In some embodiments, the infectious particles are prepared by transfecting cells with a collection of polynucleotides, each polynucleotide 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, wherein the polynucleotides in the collection encode each of the VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 and NSP5 proteins, wherein each sequence encoding a rotavirus protein is operably linked to a promoter.

[0012] In one aspect of the present disclosure, a pharmaceutical composition is provided. In some embodiments, the pharmaceutical composition comprises infectious particles containing a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript, wherein the recombinant rotavirus protein is a rotavirus protein of the RIX4414 strain.

[0013] In one aspect of the present disclosure, a method is provided. In some embodiments, the method comprises: administering to a subject a pharmaceutical composition comprising infectious particles comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript, wherein the recombinant rotavirus protein is a rotavirus protein of the RIX4414 strain.

[0014] In one aspect of the present disclosure, a method of eliciting an immune response against one or more microorganisms in a subject is provided. In some embodiments, the method comprises: administering to the subject a pharmaceutical composition comprising infectious particles comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript, wherein the recombinant rotavirus protein is a rotavirus protein of the RIX4414 strain, to induce an immune response against the one or more microorganisms.

[0015] In some embodiments, the method comprises administering to a subject a pharmaceutical composition comprising infectious particles comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript, wherein the recombinant rotavirus protein is a RIX4414 strain rotavirus protein, to elicit an immune response in the subject against a pathogen or to vaccinate the subject against one or more pathogens.

[0016] In one aspect of the present disclosure, a method for vaccinating a subject against one or more pathogens is provided. In some embodiments, the method comprises: administering to the subject a pharmaceutical composition comprising infectious particles containing a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript, wherein the recombinant rotavirus protein is a rotavirus protein of the RIX4414 strain, to vaccinate the subject against one or more pathogens.

[0017] In one aspect of the present disclosure, a cell is provided. 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, wherein each sequence encoding a rotavirus protein is operably linked to a promoter or an infectious particle, wherein the promoter or infectious particle comprises a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript, wherein the recombinant rotavirus protein is a RIX4414 strain rotavirus protein. -

[0018] In one aspect of the present disclosure, a method for generating rotavirus in vitro is provided. In some embodiments, the method comprises: introducing a composition comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript, wherein the recombinant rotavirus protein is a RIX4414 strain rotavirus protein; causing 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 sufficient time to generate rotavirus; and harvesting the virus produced by the cell to generate rotavirus in vitro.

[0019] In some embodiments, the method for generating rotavirus in vitro comprises: introducing a polynucleotide collection into cells, wherein each polynucleotide 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 in the collection encode each of VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 and NSP5 proteins, wherein each sequence encoding a rotavirus protein is operably linked to a promoter; incubating the cells for a sufficient time to produce rotavirus; and harvesting the virus produced by the cells to generate rotavirus in vitro.

[0020] In one aspect of the present disclosure, a system for generating a recombinant rotavirus is provided. In some embodiments, the system comprises: (a) a composition comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript, wherein the recombinant rotavirus protein is a RIX4414 strain rotavirus protein; and (b) a cell capable of expressing the composition of (a).

[0021] In some embodiments, a system for producing a recombinant rotavirus comprises: (a) a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript, wherein the recombinant rotavirus protein is a RIX4414 strain rotavirus protein; and (b) cells capable of expressing the collection of (a). BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1A polyacrylamide gel illustrating the size of rotavirus isolate genome segments is shown. Lane 1: recombinant RIX4414 (human G1P[8]) Lane 2: recombinant reassortant of RIX4414 and Odelia. Lane 3: recombinant reassortant of RIX4414 and Odelia. Lane 4: recombinant Odelia (human G4P[8]).

[0023] Figure 2A and 2B The recovery of recombinant RIX4414-like viruses by reverse genetics is shown. Genome segments purified by Trizol extraction from (A) recombinant SA11 virus (rSA11wt), RIX4414-like virus (rRIX4414L), genetic reassortment strains of RIX4414-like virus and Odelia (rOdelia) and rOdelia and (B) RIX4414 and rRIX4414L derived from RV1 vaccine (vRIX4414) were resolved by electrophoresis on 10% polyacrylamide gels and stained with ethidium bromide. The positions of genomic segments 1-11 of (A) rSA11wt and (B) rRIX4414L are indicated. The recombinant viruses rSA11wt and rOdelia were generated as described previously (8, 10). For the reassortant strain rRIX4414L / rOdelia, segments 1, 2, 3, 5, and 6 were derived from Odelia, and the rest were from RIX4414 (8). DETAILED DESCRIPTION

[0024] The introduction of rotavirus (RV) vaccines has reduced the incidence of severe gastroenteritis in young children in many countries. As an approach to generate rotavirus-based vaccines that can protect against a variety of pathogens, the inventors investigated the possibility of using RV as a foreign protein expression vector. The method incorporates the 2A stop-restart translation element and the foreign ORF into the RV segment 7 (NSP3) RNA, allowing the segment to acquire the ability to encode two different proteins: NSP3 and a heterologous protein. To apply this technology, the inventors 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 genetic reassortment viruses, the inventors determined that RIX4414 segment 7 RNA could be reengineered to express capsid proteins and NSP3 from other RNA viruses, including norovirus (NoV) and SARS-CoV-2. For example, 0.9 and 1.6 kb of NoV sequences were inserted into RIX4414 segment 7 to express NoV P and VP1 capsid proteins, respectively. In addition, 0.8 and 2.1 kb of SARS-CoV-2 sequences were inserted into RIX4414 segment 7 to express the RBD and S1 domains of the SARS-CoV-2 spike protein, respectively. Further analysis of these and other recombinant RVs showed that insertions of segment 7 sequences up to 1.1 kb were genetically stable. These results suggest that RIX4414 can be used as a potential pediatric vaccine vector, enabling the development of combination vaccines against NoV and other pathogens.

[0025] The inventors have generated a novel reverse genetics system for producing recombinant attenuated rotaviruses, such as rotavirus strain RIX4414 (commonly referred to as the strain present in the ROTARIX live-attenuated rotavirus vaccine). In addition, the inventors disclose herein that the novel reverse genetics system can be used to generate recombinant rotaviruses comprising 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 can enable combined vaccination against the two most common causes of severe gastroenteritis in children.

[0026] definition

[0027] The subject matter of the present disclosure can be further described using the following definitions and terms. The definitions and terms used herein are for describing particular embodiments only and are not intended to be limiting.

[0028] As used in this specification and claims, the singular forms "a", "an" and "the" include plural forms unless the context clearly indicates otherwise. For example, the term "a substituent" should be interpreted as "one or more substituents" unless the context clearly indicates otherwise.

[0029] As used herein, "about," "approximately," "substantially," and "significantly" will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which the term is used. If the term is unclear to one of ordinary skill in the art in the context in which it is used, "about" and "approximately" will mean plus or minus 10% of the particular term, and "substantially" and "significantly" will mean plus or minus more than 10% of the particular term.

[0030] As used herein, the terms "include" and "comprising" have the same meaning as the terms "containing" and "comprising." The terms "include" and "comprising" should be interpreted as "open" transition terms, which allow the inclusion of additional components other than those listed in the claims. The terms "consisting of" and "consisting of..." should be interpreted as "closed" transition terms, which do not allow the inclusion of additional components other than those described in the claims. The term "consisting essentially of..." should be interpreted as partially closed, allowing only the inclusion of additional components that do not fundamentally change the claimed subject matter.

[0031] The phrase "such as" should be interpreted as "for example, including". In addition, the use of any and all exemplary language used herein (including but not limited to "for example / such as / such as") is merely to better illustrate the present invention, and is not intended to limit the scope of protection claimed by the present invention, unless otherwise indicated.

[0032] Furthermore, in those instances where a convention similar to "at least one of A, B, and C, etc." is used, generally speaking, such construction is intended to be in the sense that one of ordinary skill in the art would understand the convention to be (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together). Those skilled in the art would further understand that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification or drawings, should be understood to contemplate the possibility of including one, any one, or both of the terms. For example, the phrase "A or B" would be understood to include the possibility of "A" or "B" or "A and B."

[0033] All language, such as "up to," "at least," "greater than," "less than," etc., is inclusive of the recited numbers and refers to ranges that can then be subdivided into ranges and subranges. Ranges include each individual member. Thus, for example, a group having 1-3 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, and so on.

[0034] The modal verb "may" refers to the preferred use or selection of one or more options or choices among the multiple embodiments or included features described. If the options or choices regarding a specific embodiment or feature included are not disclosed, the modal verb "may" refers to an affirmative action regarding how to make or use the embodiment or feature included, or a definite decision regarding the use of a specific skill with respect to the embodiment or feature included. In the latter case, the modal verb "may" has the same meaning and connotation as the auxiliary verb "may / could".

[0035] 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 at risk of infection by another pathogen, wherein the heterologous polynucleotide encodes an antigen, such as a protein or peptide, from a non-rotavirus pathogen.

[0036] The term "subject" is used interchangeably with the terms "individual" and "patient" and includes both human and non-human mammalian subjects.

[0037] The phrases "% sequence identity", "percent identity" or "% identity" refer to the percentage of amino acid residue matches 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 (explained in more detail below) generally retain the charge and hydrophobicity of the substitution site, thereby retaining the structure (and function) of the polypeptide. The percentage identity of an amino acid sequence can be determined as understood in the art. (See, e.g., U.S. Patent No. 7,396,664, the entire contents of which are incorporated herein by reference.) The Base Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) of the United States provides a set of commonly used and freely available sequence comparison algorithms, which can be obtained from multiple sources, including the NCBI website in Bethesda, Maryland. The BLAST software suite includes various sequence analysis programs, including "blastp", which is used to compare known amino acid sequences with other amino acid sequences from various databases.

[0038] The nucleic acids, proteins and / or other compositions described herein may be purified. As used herein, "purified" means separated from most other compounds or entities, and encompasses partial purification or substantial purification. Purity can be expressed by weight as measured by weight, and can be determined using various analytical techniques, such as, but not limited to, mass spectrometry, HPLC, etc.

[0039] Polypeptide sequence identity can be measured over the length of a completely defined polypeptide sequence, e.g., a polypeptide sequence defined by a specific SEQ ID number, or can be measured over a shorter length, e.g., a fragment extracted from a larger, defined polypeptide sequence, e.g., 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 consecutive residues. Such lengths are exemplary only, and it should be understood that any fragment length supported by the sequences shown in this article, the tables, the figures, or the sequence listing can be used to describe the length over which percent identity can be measured.

[0040] As used herein, the terms "polypeptide," "protein / protein," and "peptide" are used interchangeably herein to refer to a polymer of 3 or more amino acids. Thus, for example, a protein may include two proteins linked (fused) together. In addition, a protein may refer to a portion or fragment of a protein, such as the SARS-CoV-2 S1 protein, which is a fragment of the SARS-CoV-2 surface glycoprotein or "S" protein.

[0041] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to compounds comprising a core base and an acidic portion (e.g., a nucleoside, a nucleotide, or a nucleotide polymer). Nucleic acid generally refers to a polymer comprising nucleotides or nucleotide analogs connected together by a backbone connection (such as, but not limited to, a phosphodiester bond). Nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), such as messenger RNA (mRNA), transfer RNA (tRNA), etc. Typically, polymeric nucleic acids, such as nucleic acid molecules comprising three or more nucleotides, are linear molecules in which adjacent nucleotides are interconnected by 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" are used interchangeably to refer to nucleotide polymers (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 acid can be naturally occurring, for example, in the case of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid or other naturally occurring nucleic acid molecule. On the other hand, nucleic acid molecule can be a non-naturally occurring molecule, for example, a recombinant DNA or RNA, an artificial chromosome, an engineered genome or its fragment, or a synthetic DNA, RNA, DNA / RNA hybrid, or comprises a non-naturally occurring nucleotide or nucleoside. In addition, the terms "nucleic acid", "DNA", "RNA" and / or similar terms include nucleic acid analogs, i.e., analogs with the exception of the phosphodiester backbone. Nucleic acid can be purified from natural sources, produced using a recombinant expression system and optionally purified, chemically synthesized, etc. Where appropriate, for example, in the case of a chemically synthesized molecule, nucleic acid can include nucleoside analogs, such as analogs with chemically modified bases or sugar and backbone modifications. Unless otherwise indicated, nucleotide sequences are presented in 5' to 3' directions.In some embodiments, the nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propargyluridine, C5-propargylcytidine, 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); inserted 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 bonds).

[0042] As used herein, the term "hybridization" refers to the formation of a double-stranded structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between completely complementary nucleic acid chains, or between "substantially complementary" nucleic acid chains containing a small amount of mismatched regions. The conditions for hybridization of completely complementary nucleic acid chains are called "stringent hybridization conditions" or "sequence-specific hybridization conditions." Stable double strands of substantially complementary sequences can be obtained under less stringent hybridization conditions; the degree of mismatch that can be tolerated can be controlled by appropriately adjusting the hybridization conditions. Those skilled in the art of nucleic acid technology can determine duplex stability empirically taking into account a variety of variables, including, for example, the length and base pair composition of the oligonucleotide, ionic strength, and the incidence of mismatched base pairs, following the guidance provided 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, the entire contents of which are incorporated herein by reference).

[0043] Recombinant rotavirus composition

[0044] 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-sense viral transcript.

[0045] As used herein, "RIX4414" refers to a live attenuated strain of rotavirus. In contrast, the present disclosure provides a "recombinant RIX4414" rotavirus produced by a disclosed reverse genetics system, which is distinguished from the RIX4414 strain.

[0046] As used herein, "RIX4414 strain rotavirus protein" refers to a protein derived from the RIX4414 strain of rotavirus.

[0047] In some embodiments, a polynucleotide is operably linked to a promoter to allow expression of the polynucleotide in a cell (eg, a mammalian cell).

[0048] As used herein, "operably connected" refers to the functional relationship between two or more nucleic acid (e.g., DNA) segments. Generally, it refers to the functional relationship between a transcriptional regulatory element (promoter) and a transcribed sequence. For example, if a promoter stimulates or regulates the transcription of a coding sequence in an appropriate cell, the promoter is operably connected to a coding sequence. In general, promoter transcriptional regulatory elements operably connected to a sequence are physically continuous with the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory elements (such as enhancers) do not need to be physically continuous or adjacent to the coding sequence that they enhance transcription. Exemplary promoters include T7 phage promoter (SEQ ID NO: 14) and T3 phage 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 and is selected from MA-104 cells, Vero cells, and BHK-1 cells.

[0049] In some embodiments, the polynucleotide comprises a sequence encoding a rotavirus protein, i.e., a sequence selected from the group consisting of rotavirus VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5. The sequences encoding the RIX4414 strain VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 proteins are provided herein as SEQ ID NOs: 1-11, respectively. Thus, the inventors contemplate that the disclosed compositions may include a polynucleotide comprising any one of SEQ ID NOs: 1-11, or a functional variant thereof (e.g., a nucleic acid sequence variant that encodes the same amino acid due to redundancy in the genetic code, or a variant that results in a different amino acid sequence but encodes a protein or polypeptide with 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 with any one of the polypeptides encoded by SEQ ID NOs: 1-11.

[0050] The inventors have found that by fusing a heterologous polynucleotide sequence with a sequence encoding the rotavirus NSP3 protein, a reverse genetics system can be used to generate a RIX4414 strain rotavirus expressing a heterologous polynucleotide sequence. Therefore, the inventors disclose herein that, in some embodiments, the disclosed composition comprises a polynucleotide comprising a sequence encoding NSP3 (e.g., (SEQ ID NO: 9)), and further comprises a heterologous polynucleotide sequence.

[0051] The heterologous polynucleotide can encode a Norovirus protein or peptide or a SARS-CoV-2 protein or peptide. The heterologous polynucleotide can encode NoV GII.4MDA-145VP1, NoV GII.4MDA-145P, NoV GII.4MDA-145P2, NoV GII.4 Cincinnati (Cin) VP1, NoV GII.4 Cincinnati (Cin) P, NoV GII.4 Sydney (Syd) VP1, NoV GII.4 Sydney (Syd) P, the S1 portion of the SARS-CoV-2 spike (S) protein, the RBD region of the SARS-CoV-2 S1 protein, the extended RBD region of the SARS-CoV-2 S1 protein, the glycosylated S1 portion of the SARS-CoV-2 spike (S) protein (including the C-terminal CTMI domain of the S2 portion of the spike (S) protein), which can be encoded by SEQ ID NO: 16-26, respectively, or with SEQ ID NO: 27. One of NO:16-26 has a sequence with 85% similarity, 86% similarity, 87% similarity, 88% similarity, 89% similarity, 90% similarity, 91% similarity, 92% similarity, 93% similarity, 94% similarity, 95% similarity, 96% similarity, 97% similarity, 98% similarity or 99% similarity (see Table 1 for complete sequence).

[0052] In addition, Philip AA, Patton JT. 2022. Generation of Recombinant Rotaviruses Expressing Human Norovirus Capsid Proteins. Journal of Virology 96: Issue 22, published on October 31, 2022, the entire contents of which are incorporated herein by reference, which describes that the inventors have successfully expressed Norovirus (NoV) capsid protein as a heterologous polynucleotide in the RIX4414 reverse genetics system disclosed herein, see, for example, Figure 5 of Phillip and Patton, 2022.

[0053] Table 1. Exemplary heterologous polynucleotides.

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] In some embodiments, the heterologous polynucleotide encodes a protein or peptide. In some embodiments, the sequence encoding NSP3, such as SEQ ID NO:9, further comprises a heterologous polynucleotide fused to the 3' end of the sequence, such that the heterologous polynucleotide encodes a protein or peptide in frame with the NSP3 sequence, thereby allowing transcription of a single mRNA encoding both NSP3 and the heterologous polynucleotide. In some embodiments, a polynucleotide comprising a sequence encoding NSP3 and a heterologous polynucleotide comprising a sequence encoding a cleavage site. In some embodiments, the cleavage site is a self-cleaving peptide, such as the porcine Teschovirus 2A element (SEQ ID NO:13). Therefore, in some embodiments, the disclosed composition comprises, from 5' to 3', a polynucleotide encoding NSP3, which is fused in frame with a sequence encoding a self-cleaving peptide, which is fused in frame with a heterologous polynucleotide sequence encoding a peptide or protein. Thus, transcription and translation of such compositions in cells results in the production of a fusion protein comprising, from N-terminus to C-terminus, a rotavirus NSP3 protein fused to a self-cleaving peptide (e.g., SEQ ID NO: 13), which is fused to a peptide or protein encoded by a heterologous polynucleotide; after translation, the fusion protein self-cleaves to produce two separate proteins (1) a functional rotavirus NSP3 protein and (2) a protein or peptide encoded by a heterologous polynucleotide. In some embodiments, the composition further comprises a sequence encoding a linker, e.g., a flexible linker located 3' and in frame with the sequence encoding the NSP3 protein and located 5' of the cleavage site. Without being bound by any theory or mechanism, the inventors believe that adding a flexible linker between the NSP3 protein and the cleavage site can improve the cleavage effect. In some embodiments, the linker is (GAG) n linker (also known as GAG linker), where n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or (GSG) n Linkers (also known as GSG linkers) where n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0069] In some embodiments, the cleavage site encoding sequence encodes a protease cleavage site, such as a thrombin cleavage site (eg, SEQ ID NO: 12).

[0070] The inventors further contemplate that the above-mentioned heterologous polynucleotide sequence comprises a sequence encoding a protein or peptide derived from an infectious organism (e.g., norovirus or SARS-CoV-2). Thus, in some embodiments, the disclosed composition comprises a sequence encoding rotavirus NSP3, which is fused in frame with a heterologous polynucleotide encoding a norovirus protein or peptide (e.g., norovirus VP1) or a SARS-CoV-2 protein or peptide (e.g., SARS-CoV-2 surface glycoprotein).

[0071] 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-sense viral transcript, wherein 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, such as a sequence encoding SEQ ID NO: 13, which is fused in frame between the polynucleotide and the heterologous polynucleotide. Thus, transcription and translation of the composition produces a functional rotavirus NSP3 protein from N-terminus to C-terminus, 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. -

[0072] Infectious particles

[0073] 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-sense viral transcript.

[0074] As used herein, "infectious particle" refers to any particle that can cause infection in an organism or cell. Exemplary infectious particles include, but are not limited to, viral particles, virions, etc. The terms "virus," "viral particle," and "virion" are used interchangeably herein.

[0075] Without wishing to be limited, the present disclosure provides compositions comprising a polynucleotide encoding a rotavirus protein operably linked to a promoter, such as a T7 promoter (SEQ ID NO: 13). In some embodiments, the composition can be used in a reverse genetics approach to generate a recombinant rotavirus, such as a recombinant rotavirus strain RIX4414. Thus, in some embodiments, a recombinant rotavirus can comprise a disclosed composition.

[0076] The disclosed infectious particles (e.g., recombinant rotaviruses) may comprise 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 may be encoded in the genome of the infectious particle (e.g., virus) and subsequently produced during viral replication. Such infectious particles comprising heterologous proteins or peptides may advantageously elicit an immune response in a subject or as a vaccine composition.

[0077] Pharmaceutical composition

[0078] The inventors disclose herein compositions, methods and systems that can be used to prepare recombinant rotaviruses suitable for administration to a subject. Therefore, in another aspect of the present disclosure, a pharmaceutical composition is provided. In some embodiments, the pharmaceutical composition comprises infectious particles, the infectious particles comprising a composition comprising a polynucleotide containing a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript. In some embodiments, the pharmaceutical composition comprises infectious particles prepared by transfecting cells with a composition comprising a polynucleotide containing a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript.

[0079] The disclosed compositions and methods can be administered as pharmaceutical compositions, and thus pharmaceutical compositions incorporating compounds are considered embodiments of the disclosed compositions. Such compositions can take any physical form that is pharmaceutically acceptable; for example, they can be pharmaceutical compositions for oral administration. Such pharmaceutical compositions contain an effective amount of the disclosed composition, which is related to the daily dose of the composition to be administered. Each dosage unit may contain a daily dose of a given composition, or each dosage unit may contain a portion of the daily dose (such as one-half or one-third of the dose). The amount of each composition contained in each dosage unit depends in part on the characteristics of the specific composition selected for treatment and other factors (such as the indication for which it is intended). The disclosed pharmaceutical compositions can be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to a patient by known methods.

[0080] The pharmaceutical composition can be used in a method for vaccinating or eliciting an immune response against a pathogen (e.g., rotavirus, norovirus, SARS-CoV-2). As used herein, the terms "treat" or "treat" each mean temporarily or permanently alleviating symptoms, eliminating the cause of the resulting symptoms, and / or preventing or slowing the appearance of symptoms caused by a specified disease or condition or reversing its progression or severity. Therefore, the methods disclosed herein encompass therapeutic and prophylactic administration. For example, a subject may be at risk of being infected by a pathogen (e.g., rotavirus, norovirus, SARS-CoV-2), and the administration of the disclosed pharmaceutical composition induces a protective immune response or vaccination against the pathogen.

[0081] As used herein, the term "effective amount" refers to the amount or dosage of a compound that provides a desired effect in a subject under diagnosis or treatment when a single dose or multiple doses are administered to the subject. The disclosed methods may include administering an effective amount of a disclosed compound (e.g., those present in a pharmaceutical composition) to elicit an immune response against a pathogen (e.g., rotavirus, norovirus, SARS-CoV-2), or vaccinating against a pathogen.

[0082] As a person skilled in the art, the attending diagnostician can easily determine the effective amount by using known techniques and by observing the results obtained under similar circumstances. In determining the effective amount or dosage of the composition to be administered, the attending diagnostician can consider many factors, such as: the species of the subject; its size, age and general health; the extent or severity of the disease or condition involved; the response of the individual subject; the specific composition to be administered; the mode of administration; the bioavailability characteristics of the administered formulation; the dosage regimen selected; the use of concomitant drugs; and other relevant circumstances.

[0083] Oral administration is an exemplary route for administering the compositions and methods disclosed herein. Other exemplary routes of administration include transdermal, percutaneous, intravenous, intramuscular, intranasal, oral, intrathecal, intracerebral or rectal routes. The route of administration can be arbitrarily changed according to the physical properties of the compound used and the convenience of the subject and the caregiver.

[0084] Those skilled in the art will appreciate that suitable formulations include those suitable for more than one route of administration. For example, the formulation may be a formulation suitable for both intrathecal and intracerebral administration. Alternatively, suitable formulations include those suitable for only one route of administration, and those suitable for one or more routes of administration but not for one or more other routes of administration. For example, the formulation may be a formulation suitable for oral, transdermal, percutaneous, intravenous, intramuscular, intranasal, oral and / or intrathecal administration but not for intracerebral administration.

[0085] The inert ingredients and formulation of the pharmaceutical composition are conventional. The formulation methods commonly used in pharmaceutical science are used herein. All common types of compositions can be used, including tablets, chewable tablets, capsules, solutions, parenteral solutions, intranasal sprays or powders, lozenges, suppositories, transdermal patches and suspensions. In general, the composition contains a total of about 0.5% to about 50% of the compound, depending on the desired dose and the type of composition to be used. However, the amount of the compound is best defined as an "effective amount", that is, the amount of the compound that provides the desired dose to the patient in need of such treatment. It is believed that the activity of the compounds used in the compositions and methods disclosed herein does not depend to a large extent on the properties of the composition, and therefore, the composition can be selected and formulated mainly or only for the purpose of convenience and economy.

[0086] Capsules are prepared by mixing the compound with a suitable diluent and filling an appropriate amount of the mixture into capsules. Commonly used diluents include inert powdered substances (such as starch), powdered cellulose (especially crystalline and microcrystalline cellulose), sugars (such as fructose, mannitol and sucrose), cereal flours and similar edible powders.

[0087] Tablets are prepared by direct compression, wet granulation or dry granulation. Their formula usually comprises diluent, binder, lubricant and disintegrant (except compound). Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or calcium sulfate, inorganic salts (such as sodium chloride) and powdered sugar. Powdered cellulose derivatives can also be used. Typical tablet binders include various materials (such as starch, gelatin and sugar (such as lactose, fructose, glucose etc.)). Natural and synthetic gums can also be used, including gum arabic, alginate, methylcellulose, polyvinylpyrrolidine etc. Polyethylene glycol, ethyl cellulose and wax can also be used as binders.

[0088] Tablets may be coated with sugar, for example, as a flavor enhancer and sealant. The compound may be formulated as a chewable tablet by using a large amount of a pleasant tasting substance (such as mannitol) in the formulation. A fast dissolving tablet-like formulation may also be employed, for example, to ensure that the patient can consume the dosage form and avoid the difficulty some patients encounter when swallowing solid objects.

[0089] Lubricants may be used in tablet formulations to prevent tablets and pellets from sticking to the mold. Lubricants may be selected from such slippery solids as talc, magnesium and calcium stearate, stearic acid, and hydrogenated vegetable oils.

[0090] Tablets may also contain disintegrants. Disintegrants are substances that swell when exposed to moisture, causing the tablet to break apart and release the compound. They include starches, clays, celluloses, algins, and gums. By way of further illustration, corn and potato starch, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponges, cation exchange resins, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate, and carboxymethylcellulose may be used.

[0091] The composition can be formulated as an enteric formulation, for example to protect the active ingredient from the strong acid content of the stomach. Such a formulation can be prepared by coating the solid dosage form with a polymer film that is insoluble in an acidic environment but soluble in an alkaline environment. Exemplary films include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate.

[0092] Transdermal patches can also be used to deliver compounds. Transdermal patches can include a resin composition in which the compound will dissolve or partially dissolve; and a protective composition, a thin film that keeps the resin composition in contact with the skin. Other more complex patch compositions can also be used, such as those having a membrane pierced with a plurality of holes, through which the drug is pumped by osmosis.

[0093] It will also be understood by those skilled in the art that formulations can be prepared using materials (e.g., active excipients, carriers (such as cyclodextrins), diluents, etc.) that have properties (e.g., purity) that make the formulations suitable for human administration. In addition, formulations can also be prepared using materials that have purity and / or other properties that make the formulations suitable for administration to non-human subjects, but not to humans.

[0094] Methods for generating recombinant rotavirus

[0095] In another aspect of the present disclosure, a method for generating rotavirus in vitro is provided. In some embodiments, the method comprises introducing a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript; causing 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 sufficient time to generate rotavirus; and harvesting the virus produced by the cell to generate rotavirus in vitro.

[0096] cell

[0097] The inventors disclose herein cells comprising the disclosed compositions, which can also be used in the disclosed methods and systems. Therefore, 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 sense viral transcript. In some embodiments, the cells are selected from MA-104 cells, Vero cells, and BHK-1 cells.

[0098] Traditionally, Vero cells are used to culture rotavirus vaccine strains. This method of producing rotavirus has been found to be suitable for generating rotavirus for administration to a subject. Thus, in some embodiments, the cell is a Vero cell.

[0099] In some embodiments, the cells disclosed herein further comprise a heterologous RNA polymerase, wherein the heterologous RNA polymerase binds to a promoter in the disclosed composition and catalyzes sequence-dependent RNA polymerization based on the composition when the composition is introduced into the cell. As used herein, "heterologous RNA polymerase" refers to an RNA polymerase that is not introduced by molecular biology techniques (e.g., transduction, transfection, lipofection, etc.) and does not exist in the cell. In some embodiments, the heterologous RNA polymerase includes T7 bacteriophage RNA polymerase or T3 bacteriophage RNA polymerase, more commonly referred to as T7 polymerase and T3 polymerase, respectively.

[0100] Therefore, in some embodiments, the cell further comprises T7 RNA polymerase or T3 RNA polymerase. In some embodiments, such cells are referred to as, for example, BHK-T7 cells, because they are derived from BHK-1 cells, but express heterologous RNA polymerase T7 bacteriophage RNA polymerase. Therefore, as used herein, "BHK-T7 cells" are BHK-1 cells expressing heterologous RNA polymerase T7 bacteriophage RNA polymerase.

[0101] Methods of triggering an immune response

[0102] The present disclosure provides compositions, methods for preparing compositions, and infectious particles. Therefore, 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 infectious particles, the infectious particles comprising a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive sense viral transcript.

[0103] In some embodiments, the method comprises administering a pharmaceutical composition comprising infectious particles prepared by transfecting cells with a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript.

[0104] In some embodiments, a method of eliciting an immune response is provided. In some embodiments, the method of eliciting an immune response comprises administering a pharmaceutical composition comprising infectious particles, the infectious particles comprising a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript. In some embodiments, the method of eliciting an immune response comprises administering a pharmaceutical composition comprising infectious particles, the infectious particles being prepared by transfecting cells with a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript.

[0105] Methods of vaccinating a subject

[0106] In another aspect of the present disclosure, a method for vaccinating a subject against one or more pathogens is provided. In some embodiments, the method comprises administering a pharmaceutical composition comprising infectious particles, the infectious particles comprising a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript. In some embodiments, the method comprises administering a pharmaceutical composition comprising infectious particles, the infectious particles being prepared by transfecting cells with a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript.

[0107] System for generating recombinant rotavirus

[0108] In another aspect of the present disclosure, a system for generating a recombinant rotavirus is provided. In some embodiments, the system comprises: (a) a composition comprising a polynucleotide comprising a sequence encoding a recombinant rotavirus protein, wherein the polynucleotide encodes a positive-sense viral transcript; and (b) a cell capable of expressing the composition of (a).

[0109] Example

[0110] The following examples are illustrative and should not be construed as limiting the scope of the claimed subject matter.

[0111] Example 1 - Generation of novel recombinant rotavirus based on RIX4414 using reverse genetics

[0112] The inventors report the development of a reverse genetics system for the human rotavirus G1P[8] strain RIX4414. The invention enables us to modify the rotavirus strain used to formulate the widely used Rotarix vaccine and provides a means to generate modified RIX4414 strains expressing capsid proteins of other infectious viruses, including norovirus. As an alternative to RIX4414, a rotavirus vaccine formulated with norovirus capsid proteins expressing RIX4414 could provide immune protection against rotavirus and norovirus, the two most common causes of severe viral gastroenteritis in children.

[0113] The RIX4414 reverse genetics system was developed as follows. The sequences of 11 genomic segments of the RIX4414 strain were predicted based on data available in NCBI GenBank (SEQ ID NOs: 1-11). These sequences were used to construct 11 pUC19-based pT7 transcription vectors, each expressing one of the RIX4414 positive RNAs. The RIX4414 pT7 plasmid was used to produce recombinant RIX4414, generally following the reverse genetics protocol previously described in AA Phillips et al. 2020. As before, the RIX4414 pT7 transcription plasmid and the pCMV-NP868R capping enzyme plasmid were transfected into BHK-T7 cells. Two days later, the transfected BHK-T7 cells were overseeded with MA104 cells. Three days later, the BHK-T7 / MA104 cell culture was overseeded with Vero cells (which is unique to the RIX4414 reverse genetics system). Eight days later, the BHK-T7 / MA104 cell mixture was frozen and 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.

[0114] Example 2 - For producing a mixture comprising RV1 ( T7 expression plasmid of recombinant human G1P[8] rotavirus containing the RIX44143 sequence of the GSK vaccine strain

[0115] RV1( The most widely used rotavirus vaccine, RV1 (GSK), is formulated with the human G1P[8] virus RIX4414 (1,2). To prevent rotavirus gastroenteritis, more than 24 million children were vaccinated with RV1 in 2021 (2). One challenge facing rotavirus immunization efforts is that the effectiveness of RV1, as well as other rotavirus vaccines, in low-income countries (50-64%) can be significantly lower than in high- and middle-income countries (85-98%) (3,4). The inventors report here the development of a T7 expression plasmid that allows the recovery of recombinant RIX4414-like viruses by reverse genetics. With this approach, it may be possible to generate a modified form of the RV1 vaccine with improved performance in low-income countries.

[0116] The rotavirus genome consists of 11 segments of double-stranded RNA (5). Rotavirus strain RIX4414 (originally named 89-12) was isolated from a child with acute gastroenteritis in 1989 and serially propagated in cell culture to facilitate the introduction of attenuating mutations (6). In this study, the inventors used the RIX4414 sequencing information in GenBank to design 11 pT7 expression plasmids, each containing a cDNA sequence corresponding to one of the RIX4414 genomic segments (Table 2). In the absence of RIX4414 sequence information (relative to portions of the 5' and 3' untranslated regions), sequence information from the prototype human G1P[8]Wa virus was used instead (Table 2). Since the original pT7 / RIX4414 VP2 plasmid was nonfunctional in the reverse genetics system, a modified plasmid was prepared with slight modifications in 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). In the pT7 plasmid, the RIX4414 cDNA sequence is located downstream of the T7 promoter and upstream of the hepatitis D virus ribozyme sequence (9,10). The pT7 plasmid was generated by Azenta Life Sciences using the pUC19 backbone.

[0117] The RIX4414 pT7 plasmid supports the recovery of recombinant (r)RIX4414-like viruses using a slightly modified reverse genetics procedure (11, 12). Briefly, each well of a 12-well plate containing BHK-T7 cells was transfected with a plasmid mixture containing 0.8 mg of each RIX4414 T7 plasmid (except for the NSP2 and NSP5 plasmids, which were 4.8 mg each), 1.6 mg of the pCMV-NP868R RNA capping plasmid, and 1.6 mg of the pcDNA-T7 RNA polymerase plasmid. Two days later, the transfected BHK-T7 cells were incubated with 10 5 MA104 cells / well were overseeded. Two days later, the transfected BHK-T7 / MA104 cell cultures were incubated with 10 5 Vero cells / well were overseeded. Eight days later, rRIX4414-like viruses contained in the cell lysate were amplified using Vero cells and plaque isolated using MA104 cells (13). The RNA genome profile of the rRIX4414-like virus 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 virus shares >99% nucleotide and amino acid sequence identity with the RIX4414 vaccine virus, making the rRIX4414 reverse genetics system an ideal tool for studying genetic changes that may improve RV1 performance in low-income countries.

[0118] References

[0119] 1. Ward RL, Bernstein DI. Rotarix: arotavirus vaccine for the world. Clin Infect Dis. 2009 Jan 15;48(2):222-8.

[0120] 2. UNICEF, 2022. “Rotavirus Vaccine: Supply and Demand Update.” UNICEF Supply Division, January 2022.

[0121] 3. Jonesteller CL, Burnett E, Yen C, Tate JE, Parashar UD. Effectiveness of Rotavirus Vaccination: A Systematic Review of the First Decade of Global Postlicensure Data, 2006-2016. Clin Infect Dis. 2017; 65(5): 840-850.

[0122] 4. Bergman H, Henschke N, Hungerford D, Pitan F, Ndwandwe D, Cunliffe N, Soares-Weiser K. Vaccines for preventingrotavirus diarrhoea: vaccines in use. Cochrane Database Syst Rev. 2021 Nov 17;11(11):CD008521.

[0123] 5. Trask SD, McDonald SM, Patton JT. Structural insights into the coupling of virion assembly and rotavirus replication. Nat Rev Microbiol. 2012 Jan 23;10(3):165-77. doi:10.1038 / nrmicro2673. Erratum: Nat Rev Microbiol. 2014 Jan;12(1):70.

[0124] 6. Bernstein DI, Smith VE, Sherwood JR, Schiff GM, Sander DS, DeFeudis D, Spriggs DR, Ward RL. Safety and immunogenicity of live, attenuated human rotavirus vaccine 89-12. Vaccine. 1998 Feb;16(4):381-7.

[0125] 7. Komoto S, Fukuda S, Kugita M, Hatazawa R, Koyama C, Katayama K, Murata T, Taniguchi K. Generation of Infectious Recombinant Human Rotaviruses from Just 11 Cloned cDNAs Encoding the Rotavirus Genome. J Virol. 2019 Apr 3;93(8):e02207-18.

[0126] 8. Kawagishi T, Nurdin JA, Onishi M, Nouda R, Kanai Y, Tajima T, Ushijima H, Kobayashi T. Reverse Genetics System for a Human Group A Rotavirus. J Virol. 2020 Jan 6;94(2):e00963-19.

[0127] 9. Kanai Y, Komoto S, Kawagishi T, Nouda R, Nagasawa N, Onishi M, Matsuura Y, Taniguchi K, Kobayashi T. Entirely plasmid-based reverse genetics system for rotaviruses. Proc Natl Acad Sci U SA. 2017 Feb 28;114(9):2349-2354.

[0128] 10. Philip AA, Perry JL, Eaton HE, Shmulevitz M, Hyser JM, Patton JT. Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System. J Virol. 2019 Nov 26;93(24):e01616-19.

[0129] 11. Philip AA, Dai J, Katen SP, Patton JT. Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins. J Vis Exp. 2020Apr 17; (158).

[0130] 12. Philip AA, Patton JT. (Generation of Recombinant RotavirusesExpressing Human Norovirus Capsid Proteins). J Virol. 2022 Nov 23;96(22):e0126222.

[0131] 13. Arnold M, Patton JT, McDonald SM. Culturing, storage, and quantification of rotaviruses. Curr Protoc Microbiol. 2009 November; Chapter 15: Unit 15C.3.

[0132] 14. Faizuloev E, Mintaev R, Petrusha O, Marova A, Smirnova D, Ammour Y, Meskina E, Sergeev O, Zhavoronok S, Karaulov A, Svitich O, Zverev V. New approach of genetic characterization of group A rotaviruses by the nanopore sequencing method. J Virol Methods. 2021 Jun;292:114114.

[0133] Each of the above references is incorporated herein by reference.

[0134] In the foregoing description, it is obvious to those of ordinary skill 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 exemplarily described herein can be appropriately implemented in the absence of any one or more elements, one or more limitations not specifically disclosed herein. The terms and expressions used herein are used as illustrative and non-restrictive terms, and the use of these terms and expressions does not exclude any equivalent features or parts of the features or parts thereof shown and described, and it should be recognized that various modifications are likely to fall within the scope of the present invention. Therefore, it should be understood that although the present invention is described by specific embodiments and optional features, those skilled in the art may modify and / or change the concepts disclosed herein, and therefore it should be considered that such modifications and changes fall within the scope of the present invention.

[0135] Many patents and non-patent references are cited herein. The cited references are incorporated herein by reference in their entirety. If the definition of a term in this specification is inconsistent with the definition of the term in the cited reference, the term should be interpreted based on the definition in this specification.

[0136]

[0137]

[0138]

Claims

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

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

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

14.

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

15.

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

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. 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 7-8, wherein the heterologous polynucleotide encodes a peptide or a 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. The composition of claim 13, wherein the peptide or protein comprises a Norovirus (NoV) or SARS-CoV-2 peptide or protein.

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

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

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

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

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

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

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

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

24. A collection of polynucleotides, wherein each polynucleotide 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 in the collection encode each of the VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 and NSP5 proteins, wherein each sequence encoding a rotavirus protein is operably linked to a promoter.

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

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

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

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

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

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

31. A pharmaceutical composition comprising the infectious particle of any one of claims 27-30.

32. A method comprising: Administering the pharmaceutical composition of claim 25 to a subject.

33. A method of eliciting an immune response in a subject to one or more microorganisms, the method comprising: The pharmaceutical composition of claim 31 is administered to a subject to elicit an immune response to one or more microorganisms.

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

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

36. A method comprising: Administering the pharmaceutical composition of claim 31 to a subject.

37. A method comprising: The pharmaceutical composition of claim 31 is administered to a subject to elicit an immune response in the subject to a pathogen or to vaccinate the subject against one or more pathogens.

38. A method of vaccinating a subject against one or more pathogens, the method comprising: The pharmaceutical composition of claim 31 is administered to a subject to vaccinate the subject against one or more pathogens.

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

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

41. A cell comprising the composition of any one of claims 1-23 or the infectious particle of any one of claims 27-30.

42. A cell comprising the collection of any one of claims 24-26.

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

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

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

46. ​​A method for producing rotavirus in vitro, comprising: introducing the composition of any one of claims 1 to 23 into a cell; The cells are caused to express one or more rotavirus proteins selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 and NSP5; the cells are incubated for a sufficient time to produce rotavirus; and the virus produced by the cells is harvested to generate rotavirus in vitro.

47. A method for producing rotavirus in vitro, comprising: introducing the collection of any one of claims 27-30 into a cell; incubating the cells for a time sufficient to produce rotavirus; The virus produced by the cells is then harvested to generate rotavirus in vitro.

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

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

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

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

52. The method of claim 40, wherein the cell is a BHK-1 cell comprising T7 RNA polymerase and optionally comprising an African swine fever virus capping enzyme.

53. A system for generating a recombinant rotavirus, comprising: (a) a composition as claimed in any one of claims 1 to 23; as well as (b) a cell capable of expressing the composition of (a).

54. A system for generating a recombinant rotavirus, comprising: (a) a collection as claimed in any one of claims 27 to 30; as well as (b) A cell capable of expressing the collection of (a).

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

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

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

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

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