Recombinant rotavirus expression system and recombinant rotaviruses

Patent metadata

Field	Value
Publication number	US-2021269488-A1
Application number	US-201917259844-A
Country	US
Kind code	A1
Filing date	Jul 12, 2019
Priority date	Jul 13, 2018
Publication date	Sep 2, 2021
Grant date	—

Abstract

Embodiments herein report compositions, methods, uses and manufacturing procedures for rotavirus constructs and immunogenic compositions thereof. Some embodiments concern compositions that include, but are not limited to, chimeric rotaviruses of use in immunogenic compositions against rotavirus infection as well as against other pathogenic virus infection in a subject. In certain embodiments, constructs of use herein can be generated and used where a rotavirus expression system further includes one or more nucleic acid molecules encoding one or more polypeptides of another pathogen (e.g. another enteric or mucosal pathogen).

First claim

1 . A recombinant rotavirus comprising a gene segment including a nucleotide sequence encoding rotavirus nonstructural protein 3 (NSP3) and a nucleotide sequence encoding a non-rotavirus polypeptide, wherein the NSP3 and the non-rotavirus polypeptide are encoded by a single open reading frame, separated by a self-cleavage protease domain.

2 . The recombinant rotavirus of claim 1 , wherein the self-cleavage protease domain is a 2A cleavage element.

3 . The recombinant rotavirus of claim 1 , wherein the self-cleavage protease domain is a tesco porcine virus 2A (P2A) element.

4 . The recombinant rotavirus of claim 3 , wherein the P2A element has a sequence having at least 80% sequence identity to SEQ ID NO: 1 (SKFQIDKILISGDIELNPGP).

5 . The recombinant rotavirus of claim 1 , wherein the non-rotavirus polypeptide is derived from a virus that causes gastroenteritis.

6 . The recombinant rotavirus of claim 1 , wherein the non-rotavirus polypeptide is derived from a virus selected from the group consisting of: norovirus, enterovirus, poliovirus, coxsackie virus, enterovirus, hepatitis A virus, hepatitis C virus, hepatitis E virus, calicivirus, astrovirus, parechovirus, reovirus, adenovirus, torovirus, picornavirus, and coronavirus.

7 . The recombinant rotavirus of claim 1 , wherein the non-rotavirus polypeptide induces an immunological response against a second virus that is not rotavirus.

8 . The recombinant rotavirus of claim 1 , wherein the non-rotavirus polypeptide induces an immunological response against a virus selected from the group consisting of: norovirus, enterovirus, poliovirus, coxsackie virus, enterovirus, hepatitis A virus, hepatitis C virus, hepatitis E virus, calicivirus, astrovirus, parechovirus, reovirus, adenovirus, torovirus, picornavirus, and coronavirus.

9 . The recombinant rotavirus of claim 1 , wherein the non-rotavirus polypeptide is selected from the group consisting of norovirus VP1 protein, norovirus P domain, and norovirus P2 domain.

10 . The recombinant rotavirus of claim 1 , wherein the recombinant rotavirus is based upon strain G1P[8].

11 . The recombinant rotavirus of claim 1 , wherein the recombinant rotavirus is attenuated.

12 . An immunogenic composition comprising the recombinant rotavirus of claim 1 .

13 . The immunogenic composition of claim 12 , further comprising a pharmaceutically acceptable excipient.

14 . The immunogenic composition of claim 13 , wherein the immunogenic composition is formulated for oral, subcutaneous, or intramuscular administration.

15 . A method for inducing a protective immune response against rotavirus and a second virus in a subject, the method comprising administering an effective amount of the immunogenic composition of claim 12 to the subject.

16 . The method of claim 15 , wherein the immunogenic composition comprises a pharmaceutically acceptable excipient and is formulated for oral, subcutaneous, or intramuscular administration.

17 . The method of claim 15 , wherein the second virus is selected from: norovirus, enterovirus, poliovirus, coxsackie virus, enterovirus, hepatitis A virus, hepatitis C virus, hepatitis E virus, calicivirus, astrovirus, parechovirus, reovirus, adenovirus, torovirus, picornavirus, and coronavirus.

18 . A recombinant rotavirus expression system comprising:
a nonstructural protein 3 (NSP3) expression vector including a nucleotide sequence encoding rotavirus (NSP3) and a nucleotide sequence encoding a non-rotavirus polypeptide, wherein the NSP3 and the non-rotavirus polypeptide are encoded by a single open reading frame, separated by a self-cleavage protease domain; a VP1 expression vector; a VP2 expression vector; a VP3 expression vector; a VP4 expression vector; a VP5 expression vector; a VP6 expression vector; a VP7 expression vector; a NSP1 expression vector; a NSP2 expression vector; a NSP4 expression vector; a NSP5/6 expression vector; and an African Swine Fever Virus NP868R RNA capping enzyme expression vector.

19 . The recombinant rotavirus expression system of claim 18 , wherein each of the NSP3 expression vector, VP1 expression vector, VP2 expression vector, VP3 expression vector, VP4 expression vector, VP5 expression vector, VP6 expression vector, VP7 expression vector, NSP1 expression vector, NSP2 expression vector, NSP4 expression vector, and NSP5/6 expression vector are T7 expression vectors.

20 . The recombinant rotavirus expression system of claim 18 , wherein the self-cleavage protease domain is a 2A cleavage element.

21 . The recombinant rotavirus expression system of claim 18 , wherein the self-cleavage protease domain is a tesco porcine virus 2A (P2A) element.

22 . The recombinant rotavirus expression system of claim 18 , wherein the 2A element has a sequence having at least 80% sequence identity to SEQ ID NO: 1 (SKFQIDKILISGDIELNPGP).

23 . The recombinant rotavirus expression system of claim 18 , wherein the non-rotavirus polypeptide is derived from a virus that causes gastroenteritis.

24 . The recombinant rotavirus expression system of claim 18 , wherein the non-rotavirus polypeptide is derived from a virus selected from the group consisting of: norovirus, enterovirus, poliovirus, coxsackie virus, enterovirus, hepatitis A virus, hepatitis C virus, hepatitis E virus, calicivirus, astrovirus, parechovirus, reovirus, adenovirus, torovirus, picornavirus, and coronavirus.

25 . The recombinant rotavirus expression system of claim 18 , wherein the non-rotavirus polypeptide induces an immunological response against a second virus that is not rotavirus.

26 . The recombinant rotavirus expression system of claim 18 , wherein the non-rotavirus polypeptide induces an immunological response against a virus selected from the group consisting of: norovirus, enterovirus, poliovirus, coxsackie virus, enterovirus, hepatitis A virus, hepatitis C virus, hepatitis E virus, calicivirus, astrovirus, parechovirus, reovirus, adenovirus, torovirus, picornavirus, and coronavirus.

27 . The recombinant rotavirus expression system of claim 18 , wherein the non-rotavirus polypeptide is selected from the group consisting of norovirus VP1 protein, norovirus P domain, and norovirus P2 domain.

28 . The recombinant rotavirus expression system of claim 18 , wherein the rotavirus reverse genetics system is based upon strain G1P[8].

29 . A method for producing a recombinant rotavirus, the method comprising:
transfecting BHK-T7 cells with the recombinant rotavirus expression system of claim 18 ; overseeding the transfected BHK-T7 cells with MA104 cells; preparing a clarified cell lysate; and isolating recombinant rotavirus.

30 . The method of claim 29 , wherein recombinant rotavirus is isolated by plaque purification.

31 . The method of claim 29 , wherein the recombinant rotavirus is attenuated.

Assignees

• Univ Indiana Trustees

Inventors

• Patton John Thomas

Classifications

• A61K39/12 Primary

  Viral antigens · CPC title

• C12N2720/12351

  Methods of production or purification of viral material · CPC title

• A61K39/15

  Reoviridae, e.g. calf diarrhea virus · CPC title

• C07K14/005 Primary

  from viruses · CPC title

• C12N2720/12322

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title