ZOONOTIC DISEASE RNA VACCINES

1 . A zoonotic disease vaccine, comprising a ribonucleic acid (RNA) comprising an open reading frame (ORF) encoding an antigen selected from Lassa virus antigens, Nipah virus antigens, and betacoronavirus antigens, wherein intramuscular (IM) administration of a therapeutically effective amount of the vaccine to a subject induces an immune response in the subject.

2 . The zoonotic disease vaccine of claim 1 , wherein the ORF encodes a Lassa virus antigen.

3 . The zoonotic disease vaccine of claim 2 , wherein the Lassa virus antigen comprises a glycoprotein.

4 . The zoonotic disease vaccine of claim 3 , wherein the Lassa virus antigen comprises a Lassa virus glycoprotein precursor (GPC), a structurally stabilized Lassa virus GPC, an ectodomain of Lassa virus glycoprotein 1 (GP1), or a Lassa virus glycoprotein 2 (GP2).

5 . The zoonotic disease vaccine of claim 4 , wherein the Lassa virus antigen comprises amino acid residues 59-259 of a Lassa virus GPC.

6 . The zoonotic disease vaccine of claim 2 , wherein the Lassa virus antigen comprises a nucleocapsid protein (NP).

7 . The zoonotic disease vaccine of claim 2 , wherein the Lassa virus antigen has an amino acid sequence that has at least 90%, at least 95%, or at least 99% identity to an amino acid sequence identified by any one of SEQ ID NO: 1-3, but does not include wild-type protein sequence.

8 . The zoonotic disease vaccine of claim 2 , wherein the Lassa virus antigen has an amino acid sequence of any one of SEQ ID NO: 1-3.

9 . The zoonotic disease vaccine of claim 2 , wherein the RNA comprising an ORF sequence has at least 90%, at least 95%, or at least 99% identity to a nucleic acid sequence identified by any one of SEQ ID NO: 6, 7 or 9, but does not include wild-type protein sequence.

10 . The zoonotic disease vaccine of claim 2 , wherein the RNA comprising an ORF sequence comprises a nucleic acid sequence of any one of SEQ ID NO: 6, 7 or 9.

11 . The zoonotic disease vaccine of claim 1 , wherein the ORF encodes a Nipah virus antigen and/or a Hendra virus antigen.

12 . The zoonotic disease vaccine of claim 11 , wherein the Nipah virus antigen and/or a Hendra virus antigen comprises a hemagglutinin-neuraminidase protein (HN), a hemagglutinin protein (H), or a glycoprotein (G).

13 . The zoonotic disease vaccine of claim 12 , wherein the Nipah virus antigen and/or a Hendra virus antigen comprises an attachment glycoprotein, optionally a type II membrane protein.

14 . The zoonotic disease vaccine of claim 12 , wherein the Nipah virus antigen and/or a Hendra virus antigen comprises a fusion (F) glycoprotein.

15 . The zoonotic disease vaccine of claim 14 , wherein the F glycoprotein comprises a trimeric class I fusogenic envelope glycoprotein containing two heptad repeat (HR) regions and a hydrophobic fusion peptide.

16 . The zoonotic disease vaccine of any one of claims 11 - 15 , wherein the Nipah virus antigen and/or a Hendra virus antigen is a Nipah virus antigen.

17 . The zoonotic disease vaccine of any one of claims 11 - 15 , wherein the Nipah virus antigen and/or a Hendra virus antigen is a Hendra virus antigen.

18 . The zoonotic disease vaccine of claim 11 , wherein the Nipah virus antigen and/or a Hendra virus antigen has an amino acid sequence that has at least 90%, at least 95%, or at least 99% identity to an amino acid sequence identified by any one of SEQ ID NO: 10-13 but does not include wild-type protein sequence.

19 . The zoonotic disease vaccine of claim 11 , wherein the Nipah virus antigen and/or a Hendra virus antigen has an amino acid sequence of any one of SEQ ID NO: 10-13.

20 . The zoonotic disease vaccine of claim 11 , wherein the RNA comprising an ORF sequence has at least 90%, at least 95%, or at least 99% identity to a nucleic acid sequence identified by SEQ ID NO: 16 or 17, but does not include wild-type protein sequence.

21 . The zoonotic disease vaccine of claim 11 , wherein the RNA comprising an ORF sequence comprises a nucleic acid sequence of SEQ ID NO: 16 or 17.

22 . The zoonotic disease vaccine of claim 1 , wherein the ORF encodes a middle east respiratory syndrome coronavirus (MERS-CoV) antigen and/or a severe acute respiratory syndrome-like coronavirus WIV1 (SL-CoV-WIV1) antigen.

23 . The zoonotic disease vaccine of claim 22 , wherein the MERS-CoV antigen and/or a SL-CoV-WIV1 antigen comprises a betacoronavirus structural protein.

24 . The zoonotic disease vaccine of claim 23 , wherein the betacoronavirus structural protein is spike protein, envelope protein, nucleocapsid protein, or membrane protein.

25 . The zoonotic disease vaccine of claim 24 , wherein the betacoronavirus structural protein is spike protein.

26 . The zoonotic disease vaccine of claim 25 , wherein the betacoronavirus structural protein a S1 subunit of the spike protein or a S2 subunit of the spike protein.

27 . The zoonotic disease vaccine of any one of claims 22 - 26 , wherein the MERS-CoV antigen and/or a SL-CoV-WIV1 antigen is a MERS-CoV antigen.

28 . The zoonotic disease vaccine of any one of claims 22 - 26 , wherein the MERS-CoV antigen and/or a SL-CoV-WIV1 antigen is a SL-CoV-WIV1 antigen.

29 . The zoonotic disease vaccine of claim 22 , wherein the MERS-CoV antigen and/or a SL-CoV-WIV1 antigen has an amino acid sequence that has at least 90%, at least 95%, or at least 99% identity to an amino acid sequence identified SEQ ID NO: 18 but does not include wild-type protein sequence.

30 . The zoonotic disease vaccine of claim 22 , wherein the MERS-CoV antigen and/or a SL-CoV-WIV1 antigen has an amino acid sequence of SEQ ID NO: 18.

31 . The zoonotic disease vaccine of claim 22 , wherein the RNA comprising an ORF sequence has at least 90%, at least 95%, or at least 99% identity to a nucleic acid sequence identified by SEQ ID NO: 18, but does not include wild-type protein sequence.

32 . The zoonotic disease vaccine of claim 22 , wherein the RNA comprising an ORF sequence comprises a nucleic acid sequence of SEQ ID NO: 18.

33 . The zoonotic disease vaccine of any one of claims 1 - 32 , wherein IM administration of a therapeutically effective amount of the vaccine to a subject induces a neutralizing antibody titer in the subject.

34 . The zoonotic disease vaccine of claim 33 , wherein the neutralizing antibody titer is at least 100 neutralizing units per milliliter (NU/mL), at least 500 NU/mL, or at least 1000 NU/mL.

35 . The zoonotic disease vaccine of claim 33 or 34 , wherein the neutralizing antibody titer is sufficient to reduce viral infection of B cells by at least 50% relative to a neutralizing antibody titer of an unvaccinated control subject or relative to a neutralizing antibody titer of a subject vaccinated with a live attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit viral vaccine.

36 . The zoonotic disease vaccine of any one of claims 33 - 35 , wherein the neutralizing antibody titer is induced in the subject following fewer than three doses of the vaccine.

37 . The zoonotic disease vaccine of any one of claims 1 - 36 , wherein a single dose is of 10 μg-100 μg.

38 . The zoonotic disease vaccine of any one of claims 33 - 37 , wherein the neutralizing antibody titer and/or a T cell immune response is sufficient to reduce the rate of asymptomatic viral infection relative to the neutralizing antibody titer of unvaccinated control subjects.

39 . The zoonotic disease vaccine of any one of claims 33 - 38 , wherein the neutralizing antibody titer and/or a T cell immune response is sufficient to prevent viral latency the subject.

40 . The zoonotic disease vaccine of any one of claims 33 - 39 , wherein the neutralizing antibody titer is sufficient to block fusion of virus with epithelial cells and/or B cells of the subject.

41 . The zoonotic disease vaccine of any one of claims 33 - 40 , wherein the neutralizing antibody titer is induced within 20 days following a single 10-100 μg of the vaccine, or within 40 days following a second 10-100 μg dose of the vaccine.

42 . The zoonotic disease vaccine of any one of claims 33 - 40 , wherein IM administration of a therapeutically effective amount of the vaccine to a subject induces a T cell immune response in the subject.

43 . The zoonotic disease vaccine of claim 42 , wherein the T cell immune response comprises a CD4 + T cell immune response and/or a CD8 + T cell immune response.

44 . The zoonotic disease vaccine of any one of claims 1 - 43 , wherein the antigen is expressed on the surface of cells of the subject.

45 . The zoonotic disease vaccine of any one of claims 1 - 44 , wherein the vaccine comprises (a) a ribonucleic acid (RNA) having an open reading frame (ORF) encoding two antigens, or (b) two RNAs, each having an ORF encoding an antigen.

46 . The zoonotic disease vaccine of any one of claims 1 - 45 , wherein the vaccine comprises a RNA having an ORF encoding two antigens formulated in a lipid nanoparticle.

47 . The zoonotic disease vaccine of any one of claims 1 - 46 , wherein the vaccine comprises two RNAs, each having an ORF encoding an antigen, wherein the two RNAs are formulated in a single lipid nanoparticle or wherein the each RNAs is formulated in a single lipid nanoparticle.

48 . The zoonotic disease vaccine of any one of claims 1 - 47 , further comprising at least one additional RNA having an ORF encoding at least one additional antigen.

49 . The zoonotic disease vaccine of any one of claims 46 - 48 , wherein the lipid nanoparticle comprises a molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid

50 . The zoonotic disease vaccine of any one of claims 1 - 49 , wherein the antigen is fused to a signal peptide.

51 . The zoonotic disease vaccine of any one of claims 1 - 50 , wherein the antigen is fused to a scaffold moiety.

52 . The zoonotic disease vaccine of claim 51 , wherein the scaffold moiety is selected from the group consisting of: ferritin, encapsulin, lumazine synthase, hepatitis B surface antigen, and hepatitis B core antigen.

53 . The zoonotic disease vaccine of any one of claims 1 - 52 , wherein the RNA comprises messenger RNA (mRNA).

54 . The zoonotic disease vaccine of any one of claims 1 - 53 , wherein the RNA further comprises a 5′UTR and/or a 3′UTR.

55 . The zoonotic disease vaccine of any one of claims 1 - 54 , wherein the RNA is unmodified.

56 . The zoonotic disease vaccine of any one of claims 1 - 54 , wherein the RNA comprise a modified nucleotide.

57 . The zoonotic disease vaccine of claim 56 , wherein at least 80% of the uracil in the ORF comprise 1-methyl-pseudouridine modification.

58 . A method comprising administering to a subject the zoonotic disease vaccine of any one of claims 1 - 57 in a therapeutically effective amount to induce an immune response in the subject.

59 . The method of claim 58 , wherein the therapeutically effective amount induces a neutralizing antibody titer and/or a T cell immune response in the subject.

60 . The method of claim 59 , wherein efficacy of the vaccine is at least 80% relative to unvaccinated control subjects.

61 . The method of any one of claims 58 - 60 , wherein detectable levels of the antigen are produced in the serum of the subject at 1-72 hours post administration of the vaccine.

62 . The method of any one of claims 59 - 61 , wherein a neutralizing antibody titer of at least 100 NU/ml, at least 500 NU/ml, or at least 1000 NU/ml is produced in the serum of the subject at 1-72 hours post administration of the vaccine.

63 . The method of any one of claims 58 - 62 , wherein the therapeutically effective amount is a total dose of 20 μg-200 μg or a total dose of 50 μg-100 μg.