Mouse-adapted SARS-CoV-2 viruses and methods of use thereof

Patent metadata

Field	Value
Publication number	US-11225508-B1
Application number	US-202117173617-A
Country	US
Kind code	B1
Filing date	Feb 11, 2021
Priority date	Sep 23, 2020
Publication date	Jan 18, 2022
Grant date	Jan 18, 2022

Abstract

This invention relates to SARS-CoV-2 viruses adapted with nanoluciferase reporter molecules and mouse-adapted SARS-CoV-2 viruses, compositions including the same and methods of use thereof.

First claim

That which is claimed:

1. A recombinant SARS-CoV-2 virus particle comprising
a spike protein, wherein the spike protein comprises the amino acid sequence: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMD LEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSET KCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIA DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGST PCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKN KCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVS VITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEH VNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPT NFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDK NTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQY GDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIP FAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQN AQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAA EIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKN FTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVN NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLN ESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC KFDEDDSEPVLKGVKLHYT (SEQ ID NO:2) or an amino acid sequence at least 90% identical thereto, wherein either said amino acid sequence comprises a substitution of Q498Y and P499T.

2. An isolated nucleic acid molecule encoding the SARS-CoV-2 virus particle of claim 1 .

3. An isolated nucleic acid molecule encoding the SARS-CoV-2 virus particle of claim 1 , wherein the nucleic acid molecule comprises one or more of the following nonsynonymous nucleotide substitutions, wherein the numbering is based on the reference nucleotide sequence of SEQ ID NO:1:
G9479T, C9491T, A12678G, A12884G, C23039A, C9438T, A11847G, A12159G, T27221C, C23054T, A23056C, C23057A, C23059G.

4. The isolated nucleic acid molecule of claim 3 , further comprising one or more synonymous nucleotide substitution(s).

5. The isolated nucleic acid molecule of claim 4 , wherein the synonymous nucleotide substitution is T15102A.

6. The isolated nucleic acid molecule of claim 3 , wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:8.

7. The isolated nucleic acid molecule of claim 3 , wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:9.

8. The isolated nucleic acid molecule of claim 3 , wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:10.

9. The isolated nucleic acid molecule of claim 3 , wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:11.

10. The isolated nucleic acid molecule of claim 3 , wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:12.

11. The isolated nucleic acid molecule of claim 3 , wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:15.

12. The isolated nucleic acid molecule of claim 3 , wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40.

13. A population of SARS-CoV-2 particles comprising the SARS-CoV-2 virus particle of claim 1 .

14. A composition comprising the SARS-CoV-2 virus particle of claim 1 in a pharmaceutically acceptable carrier.

15. A composition comprising the isolated nucleic acid molecule of claim 2 in a pharmaceutically acceptable carrier.

16. A method of producing an immune response to a coronavirus in a subject, comprising administering to the subject an effective amount of the SARS-CoV-2 virus particle of claim 1 .

17. The recombinant SARS-CoV-2 virus particle of claim 1 , wherein the spike protein amino acid sequence further comprises the substitution Q493K.

18. The isolated nucleic acid molecule of claim 2 , wherein the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO:15.

19. The isolated nucleic acid molecule of claim 2 , wherein the nucleic acid molecule encodes
a substitution of Q493K in the spike protein amino acid sequence, and further encodes
a SARS-CoV-2 nsp4 protein comprising a T285I, G309C and/or H313Y substitution(s);
a SARS-CoV-2 nsp7 protein comprising a K2R substitution;
a SARS-CoV-2 nsp8 protein comprising a E23G and/or K196R substitution;
a SARS-CoV-2 nsp9 protein comprising a T67A substitution; and/or
a SARS-CoV-2 ORF6 protein comprising a F7S substitution,

wherein the numbering corresponds to the amino acid sequences of SEQ ID NO:3 (nsp4 protein), SEQ ID NO:4 (nsp7 protein), SEQ ID NO:5 (nsp8 protein), SEQ ID NO:6 (nsp9 protein) or SEQ ID NO:7 (ORF6 protein), respectively.

20. The isolated nucleic acid molecule of claim 19 , wherein the nucleic acid molecule further encodes
a SARS-CoV-2 nsp4 protein comprising the amino acid sequence of SEQ ID NO:3, wherein said amino acid sequence comprises a T285I substitution;
a SARS-CoV-2 nsp7 protein comprising the amino acid sequence of SEQ ID NO:4, wherein said amino acid sequence comprises a K2R substitution;
a SARS-CoV-2 nsp8 protein comprising the amino acid sequence of SEQ ID NO:5, wherein said amino acid sequence comprises a E23G substitution; and
a SARS-CoV-2 ORF6 protein comprising the amino acid sequence of SEQ ID NO:7, wherein said amino acid sequence comprises a F7S substitution.

21. The isolated nucleic acid molecule of claim 20 , wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:8, 9, 10, 11, 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40.

22. The isolated nucleic acid molecule of claim 20 , wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:30.

Assignees

• Univ North Carolina Chapel Hill

Inventors

• Baric Ralph
• Dinnon Iii Kenneth Harold
• Leist Sarah Rebecca

Classifications

• C12N2770/20023

  Virus like particles [VLP] · CPC title

• C12N2770/20022

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

• C12N2770/20021

  Viruses as such, e.g. new isolates, mutants or their genomic sequences · CPC title

• C07K14/005 Primary

  from viruses · CPC title

• A01K2207/15

  Humanized animals · CPC title