COMPOSITIONS AND METHODS FOR ZIKA VIRUS CHARACTERIZATION AND VACCINE DEVELOPMENT

• US 20190323034A1
• Filed: 04/18/2019
• Published: 10/24/2019
• Est. Priority Date: 04/18/2018
• Status: Active Grant

• Current Assignee: Utah State University
• Original Assignee: Utah State University
• Inventors: Lee, Young-Min
• Granted Patent: US 11,254,953 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2039/5254 avirulent or attenuated
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2770/24121 Viruses as such, e.g. new isolates, mutants or their genomic sequences
• CPC Class Codes: C12N 2770/24134 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2770/24143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2770/24151 Methods of production or purification of viral material
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present disclosure relates to compositions and methods for investigating Zika virus (ZIKV) biology and pathogenicity. The present disclosure provides genetically stable viral vectors to produce functional RNA transcripts of ZIKV cDNAs. In particular, the present disclosure provides full-length infectious cDNAs as bacterial artificial chromosomes for spatiotemporally distinct and genetically divergent ZIKVs. The present disclosure also provides methods of generating a genetically engineered attenuated ZIKV using the genetically stable viral vectors described herein.

Claims

1. A genetically stable viral vector comprising:

a Zika virus cDNA;

a RNA polymerase promoter upstream of the 5′

end of the Zika virus cDNA; and

a restriction endonuclease site downstream of the 3′

end of the Zika virus cDNA;

wherein the Zika virus cDNA, the RNA polymerase promoter, and the restriction endonuclease site are cloned into a bacterial artificial chromosome vector, and

wherein the Zika virus cDNA is capable of being transcribed into an RNA transcript that is functional.

2. The vector of claim 1, wherein the Zika virus cDNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:

4, SEQ ID NO;

5, and SEQ ID NO;

6.

3. The vector of claim 1, wherein the Zika virus cDNA comprises a nucleotide sequence with at least 85% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:

4, SEQ ID NO;

5, or SEQ ID NO;

6.

4. The vector of claim 1, wherein the Zika virus cDNA comprises a nucleotide sequence with at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:

4, SEQ ID NO;

5, or SEQ ID NO;

6.

5. The vector of claim 1, wherein the Zika virus cDNA comprises a nucleotide sequence with at least 95% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:

4, SEQ ID NO;

5, or SEQ ID NO;

6.

6. The vector of claim 1, wherein the Zika virus cDNA comprises a nucleotide sequence with at least 99% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:

4, SEQ ID NO;

5, or SEQ ID NO;

6.

7. The vector of claim 1, wherein the RNA polymerase promoter is an SP6 promoter.

8. The vector of claim 1, wherein the RNA polymerase promoter comprises a nucleotide sequence of SEQ ID NO:

7.

9. The vector of claim 1, wherein the restriction endonuclease site does not interfere with the 3′

end of the Zika virus cDNA.

10. The vector of claim 1, wherein the restriction endonuclease site is capable of being cleaved by PsrI or BarI endonucleases.

11. The vector of claim 1, wherein the restriction endonuclease site comprises a nucleotide sequence of SEQ ID NO:

8 or SEQ ID NO;

9.

12. The vector of claim 1, wherein the bacterial artificial chromosome vector is a single- or low-copy number bacterial artificial chromosome vector.

13. The vector of claim 1, wherein the bacterial artificial chromosome is pBeloBac11.

14. The vector of claim 1, wherein the RNA transcript has a N⁷-methyl-guanosine-5′

-triphosphate-5′

-adenosine (m⁷GpppA) cap.

15. The vector of claim 1, comprising

a Zika virus cDNA of strain MR-766 cloned into a bacterial artificial chromosome vector, wherein the sequence of the vector is represented by SEQ ID NO:

1.

16. The vector of claim 1, comprising

a Zika virus cDNA of strain P6-740 cloned into a bacterial artificial chromosome vector, wherein the sequence of the vector is represented by SEQ ID NO:

2.

17. The vector of claim 1, comprising

a Zika virus cDNA of strain PRVABC-59 cloned into a bacterial artificial chromosome vector, wherein the sequence of the vector is represented by SEQ ID NO:

3.

18. A method of generating a genetically engineered attenuated Zika virus comprising:

obtaining a genetically stable viral vector of claim 1; and

altering one or more nucleotides in the Zika virus cDNA to produce a synonymous or non-synonymous codon alteration;

wherein the synonymous or non-synonymous codon alteration produces a Zika virus with compromised virulence.

19. The method of claim 18, wherein the one or more nucleotides are located in a nucleotide sequence of the Zika virus cDNA corresponding to the RNA-dependent RNA polymerase domain of Zika virus NS5 protein.

20. The method of claim 18, wherein the Zika virus cDNA is SEQ ID NO:

2 and wherein the one or more nucleotides comprises cytidine at nucleotide position 9804 altered to uridine.

21. The method of claim 18, wherein altering one or more nucleotides in the Zika virus cDNA replaces a His with Tyr at amino acid position 713 of the Zika virus NS5 protein.

22. The method of claim 18, wherein the Zika virus with compromised virulence has decreased virulence compared to a Zika virus without any altered nucleotide sequences.

23. The method of claim 18, wherein the virulence of the Zika virus with compromised virulence is decreased by at least 1 log.

24. A vaccine comprising a genetically engineered attenuated Zika virus made by the method of claim 18.