Self-attenuated prophylactic and therapeutic vaccines against pathogens

1. A live, self-attenuated virus comprising:

a viral genome that expresses one or more viral antigens; and

an artificial microRNA 30 (amiR-30 expression cassette) inserted into a viral neuraminidase (NA) or a viral non-structural (NS) gene segment of the viral genome, as applicable, of the live self-attenuated virus to express an amiR-30 that specifically inhibits the expression of a host gene essential for virus replication in host cells.

2. The virus of claim 1 , wherein the isolated virus further comprises a mammalian-specific artificial microRNA 93 (amiR-93) expression cassette inserted into the viral neuraminidase (NA) or the viral non-structural (NS) gene segment that inhibits viral nucleoprotein (NP) gene expression.

3. The virus of claim 1 , wherein the amiR-30 comprises a sequence that targets expression of the host gene in a miR-30 backbone.

4. The virus of claim 1 , wherein the host gene is at least one of CDC-Like Kinase 1 (CLK1), SON DNA binding protein (SON), cyclin-dependent kinase inhibitor 1B (Cdkn1b), calcium/calmodulin-dependent protein kinase (CaM kinase) II beta (CAMK2B), or vacuolar ATPase (vATPase).

5. The virus of claim 1 , wherein the microRNA is not expressed in avian cells; or the microRNA comprises a miR-93 backbone-based cassette for species-specific microRNA expression.

6. The virus of claim 1 , wherein the influenza virus at least one of: expresses one or more viral antigens that confer protection against H1N1, pandemic H1N1, and H3N2; the virus is packaged into a vaccine; the virus is adapted for pulmonary, oral, nasal, transcutaneous, or mucosal administration; or the virus is adapted for use as a vaccine for prevention of infectious diseases or as therapeutic for post infection treatment.

7. The virus of claim 1 , wherein the virus is packaged into a vaccine and less than 1,000, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 1 viral particle(s) trigger a humoral and a cellular immune response to the one or more viral antigens, or confers protective immunity to the virus.

8. The virus of claim 2 , wherein the mature miR-93 loop is replaced with sequence within the mature miR-93 loop was replaced with one of the following sequences:

(SEQ ID NO: 2)

amiR-93-NP

GTTAACTCCCGTCTTGGACCTCAGTCCTGGGGGCTC ACTCCTCTGCA

TTGTCTCCGAAG TGTGATTACCCAACCTT GGAGCAATGGCAGAGGAA

AAG AGCCCCCGGGACACGTTCTCTCTGCCAATTCTCGAG

(SEQ ID NO: 3)

amiR-93-SON

TCCCGTCTTGGACCTCAGTCCTGGGGGCTC GTGAAATAGTACAAGGT

GCACAG TGTGATTACCCAACCT GGCACTTGTAGCTATTTCTGTG AGC

CCCCGGGACACGTTCTCTCTGCCAATT

(SEQ ID NO: 5)

amiR-30-CLK1

GAAGGTATATTGCTGTTGACAGTGAGCGAC TCCCAATGAGGTCAAAG

AGAAG CTGTGAAGCCACAGATGGG CTTCTCTTTCCTCATTGGGAGC T

GCCTACTGCCTCGGACTTCAAGGGGCTAC

(SEQ ID NO: 6)

amiR-30-Cdkn1b

GAAGGTATATTGCTGTTGACAGTGAGCGAC TAGAAGAATCGTCGGTT

GCAGG CTGTGAAGCCACAGATGGG CCTGCAACCCGATTCTTCTAGC T

GCCTACTGCCTCGGACTTCAAGGGGCTAC

(SEQ ID NO: 7)

amiR-30-CAMK2B

GAAGGTATATTGCTGTTGACAGTGAGCGAC CACATCTGGTCTTGTTT

TTCTG CTGTGAAGCCACAGATGGG CAGAAAAACGACCAGATGTGGC T

GCCTACTGCCTCGGACTTCAAGGGGCTAC

(SEQ ID NO: 8)

amiR-30-vATPase (ATP6V0C)

GAAGGTATATTGCTGTTGACAGTGAGCGAC TAAGGTTATAGATAGCT

GGGAG CTGTGAAGCCACAGATGGG CTCCCAGCTCTATAACCTTAGC T

GCCTACTGCCTCGGACTTCAAGGGGCTAC.

9. The virus of claim 1 , wherein the virus has an EID 50 of 10 or less.

10. The virus of claim 1 , wherein the virus comprises multiple artificial miRNA expression cassettes.

11. The virus of claim 1 , wherein the virus is selected from an influenza, human immunodeficiency virus (HIV), hepatitis B virus (HBV), West Nile virus, Dengue Fever, or Zika virus.

12. A method of making a live, self-attenuated virus comprising:

obtaining an isolated virus comprising a viral genome that expresses one or more viral antigens; and

inserting into the viral genome an artificial microRNA 30 (amiR-30) expression cassette inserted into a viral neuraminidase (NA) or a viral non-structural (NS) gene segment that expresses an amiR-30 that specifically inhibits the expression of a host gene essential for influenza virus replication in host cells.

13. The method of claim 12 , wherein the isolated virus further comprises a mammalian-specific artificial microRNA 93 (amiR-93) expression cassette inserted into the viral neuraminidase (NA) or the viral non-structural (NS) gene segment that inhibits viral nucleoprotein (NP) gene expression.

14. The method of claim 12 , wherein the amiR-30 comprises a sequence that targets expression of the host gene in a miR-30 backbone.

15. The method of claim 12 , wherein the host gene is at least one of CDC-Like Kinase 1 (CLK1), SON DNA binding protein (SON), cyclin-dependent kinase inhibitor 1B (Cdkn1b), calcium/calmodulin-dependent protein kinase (CaM kinase) IIbeta (CAMK2B), or vacuolar ATPase (vATPase).

16. The method of claim 12 , wherein the microRNA is not expressed in avian cells or the microRNA comprises a miR-93 backbone-based cassette for species-specific microRNA expression.

17. The method of claim 12 , wherein the virus at least one of: expresses one or more additional viral antigens that confer protection against H1N1, pandemic H1N1, and H3N2; the virus is packaged into a vaccine; the virus is adapted for pulmonary, oral, nasal, transcutaneous, or mucosal administration; or the virus is adapted to prevent an infectious diseases or as therapeutic for post infection treatment.

18. The method of claim 12 , wherein the virus is packaged into a vaccine and less than 1,000, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 1 viral particle(s) trigger a humoral and a cellular immune response to the one or more viral antigens, or confers protective immunity to the virus.

19. The method of claim 13 , wherein the mature miR-93 loop is replaced with sequence within the mature miR-93 loop was replaced with one of the following sequences:

(SEQ ID NO: 2)

amiR-93-NP

GTTAACTCCCGTCTTGGACCTCAGTCCTGGGGGCTC ACTCCTCTGCA

TTGTCTCCGAAG TGTGATTACCCAACCTT GGAGCAATGGCAGAGGAA

AAG AGCCCCCGGGACACGTTCTCTCTGCCAATTCTCGAG

(SEQ ID NO: 3)

amiR-93-SON

TCCCGTCTTGGACCTCAGTCCTGGGGGCTC GTGAAATAGTACAAGGT

GCACAG TGTGATTACCCAACCT GGCACTTGTAGCTATTTCTGTG AGC

CCCCGGGACACGTTCTCTCTGCCAATT

(SEQ ID NO: 5)

amiR-30-CLK1

GAAGGTATATTGCTGTTGACAGTGAGCGAC TCCCAATGAGGTCAAAG

AGAAG CTGTGAAGCCACAGATGGG CTTCTCTTTCCTCATTGGGAGC T

GCCTACTGCCTCGGACTTCAAGGGGCTAC

(SEQ ID NO: 6)

amiR-30-Cdkn1b

GAAGGTATATTGCTGTTGACAGTGAGCGAC TAGAAGAATCGTCGGTT

GCAGG CTGTGAAGCCACAGATGGG CCTGCAACCCGATTCTTCTAGC T

GCCTACTGCCTCGGACTTCAAGGGGCTAC

(SEQ ID NO: 7)

amiR-30-CAMK2B

GAAGGTATATTGCTGTTGACAGTGAGCGAC CACATCTGGTCTTGTTT

TTCTG CTGTGAAGCCACAGATGGG CAGAAAAACGACCAGATGTGGC T

GCCTACTGCCTCGGACTTCAAGGGGCTAC

(SEQ ID NO: 8)

amiR-30-vATPase (ATP6V0C)

GAAGGTATATTGCTGTTGACAGTGAGCGAC TAAGGTTATAGATAGCT

GGGAG CTGTGAAGCCACAGATGGG CTCCCAGCTCTATAACCTTAGC T

GCCTACTGCCTCGGACTTCAAGGGGCTAC.

20. The method of claim 12 , wherein the virus has an EID 50 of 10 or less.

21. The method of claim 12 , wherein the virus comprises multiple artificial miRNA expression cassettes.

22. The method of claim 12 , wherein the virus is selected from an influenza, human immunodeficiency virus (HIV), hepatitis B virus (HBV), West Nile virus, Dengue Fever, or Zika virus.

23. The method of claim 12 , further comprising testing the live, self-attenuated virus by:

determining if the virus propagates in the viral propagation cell but is attenuated in the viral target species cell.

24. The virus of claim 1 , further comprising:

one or more adjuvants, excipients, or buffers, wherein the therapeutic vaccine is adapted for at least one of: pulmonary, intraalveolar, nasal, transcutaneous, or mucosal administration.

25. A method of treating a patient with a therapeutic vaccine comprising:

identifying that the patient is in need of prophylaxis or treatment of an active viral infection; and

providing the patient with a vaccine comprising a live, attenuated virus that comprises an artificial microRNA 30 (amiR-30) expression cassette inserted into a viral neuraminidase (NA) or a viral non-structural (NS) gene segment that expresses an amiR-30 that specifically inhibits the expression of a host gene essential for influenza virus replication in host cells, in an amount effective to provide prophylaxis against, or treatment of, an active viral infection.