SELF-REPLICATING RNA AND USES THEREOF SEQUENCE LISTING This application is filed together with a Sequence Listing in electronic form, the entire contents of which are incorporated herein by reference.

Abstract

The present disclosure relates to self-replicating RNAs encoding antigens derived from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and uses thereof. Specifically, the present disclosure provides self-replicating RNAs or monocistronic self-replicating RNAs comprising a nucleotide sequence encoding an antigen operably linked to a subgenomic promoter, wherein the antigen is derived from SARS-CoV-2, and the antigen is the spike (S) protein or the nucleocapsid (N) protein.

Description

[Technical field]

[0001] The present disclosure relates to self-replicating RNA encoding antigens derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and uses thereof. [Background technology]

[0002] In late 2019, a novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) was identified in humans in China, causing the severely infectious coronavirus disease 2019 (COVID-19). Since December 2019, the global spread of the virus has reached all countries, and the World Health Organization (WHO) declared the outbreak a pandemic on March 11, 2020. Human infections with this virus exhibit a wide clinical spectrum with high transmissibility. Global infection and mortality rates continue to rise, with the number of infected exceeding 54 million, 35 million recovered, and over 1.3 million deaths to date.

[0003] Vaccines are a key health intervention to prevent this infectious disease. This pandemic has seen an unprecedented development of multiple vaccines, with over 200 vaccines developed so far, more than 30 in clinical trials, and multiple vaccines in phase 3. The majority of vaccines developed attempt to elicit the immune system to recognize the SARS-COV-2 spike protein (or S protein), since early studies of recombinant SARS-CoV proteins in a hamster challenge model demonstrated that this approach was immunogenic and protective.

[0004] However, there are currently no approved therapeutic and / or prophylactic vaccines against SARS-CoV-2. Thus, there remains a need to develop specific and efficient vaccines against SARS-CoV-2. It is also desirable that such vaccines can be produced in sufficient quantities, especially during a pandemic, and more quickly than the current egg-based techniques used to manufacture influenza vaccines. Summary of the Invention

[0005] The present disclosure is based on the inventors' identification of self-replicating RNA against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens.

[0006] The inventors' discoveries provide the basis for self-replicating RNAs against SARS-CoV-2 antigens. The inventors' discoveries also provide the basis for monocistronic self-replicating RNAs against SARS-CoV-2 antigens. Furthermore, the inventors' discoveries provide the basis for methods of treating, preventing, or delaying the progression of a disease or disorder in a subject, such as SARS-COV-2 infection, COVID-19, and / or acute respiratory distress syndrome (ARDS).

[0007] Thus, the present disclosure provides a self-replicating RNA comprising a nucleotide sequence encoding an antigen operably linked to a subgenomic (SG) promoter, wherein the antigen is derived from SARS-CoV-2.

[0008] The present disclosure also provides a monocistronic self-replicating RNA comprising a nucleotide sequence encoding an antigen operably linked to an SG promoter, wherein the antigen is derived from SARS-CoV-2.

[0009] In one example, the antigen is the spike (S) protein or the nucleocapsid (N) protein. In one example, the antigen is the SARS-CoV-2 N protein or S protein from SARS-CoV-2 strain 2019-nCoV / USA-WA1 / 2020.

[0010] In one example, the antigen is the S protein. For example, the S protein is encoded by the sequence shown in SEQ ID NO:1.

[0011] In another example, the S protein is a mutant S protein.

[0012] In one example, the mutant S protein comprises a mutation in the receptor binding domain. For example, the mutations include S438F, N439K, N440K, L441I, K444R, V445A, V445I, G446V, G446S, N450K, L452R, L452P, L455F, K458N, N460T, D467V, I468F, I468T, I468V, E471O, I472V, A475V, G476S, S477G, S477I, S477N, S477R, T478I, In one example, the mutant S protein comprises a mutation in the receptor binding domain selected from the group consisting of N439K, N439L, L452R, S477N, T478I, V483A, and E484D.

[0013] In one example, the mutant S protein is P337S, F338L, F338C, G339D, E340K, V341I, A344S, T345S, R346K, A348S, A348T, W353R, N354D, N354K, N354S, S359N, D364Y, V367F, S373L, V382L, P384L, P384S, T385A, T393P, V395I, F400C, R403K, R403S, D403Y, V403R, V403K, V403S, D403Y, V403R, V403K, V403S, D403Y, V403R, V403K, V403R ... and D614G.

[0014] In one example, the mutant S protein is L18F, D80A, T95I, Y144S, Y145N, D215G, P337S, F338L, F338C, G339D, E340K, V341I, A344S, T345S, R346K, A348S, A348T, W353R, N354D, N354K, N354S, S359N, D364Y, V367F, S373L, V382L, P384L, P384S, T385A, T393P, V39 5I, F400C, R403K, R403S, D405V, R408I, Q414E, Q414K, Q414P, Q414R, T415S, K417N, K417T, K417R, I418V, Y421S, Y423 C, Y423F, Y423S, D427Y, S438F, N439K, N440K, L441I, K444R, V445A, V445I, G446V, G446S, N450K, L452R, L452P, L455F, K458N, N460T, D467V, I468F, I468T, I468V, E471O, I472V, A475V, G476S, S477G, S477I, S477N, S477R, T478I, T478K, P 479L, P479S, N481D, N481H, V483F, V483A, E484D, E484K, E484K, E484O, G485S, Y489H, Y489D, Y489F, Y489C, Y489N, F49 0L, F490S, P491R, Q493L, S494P, Y495N, T500N, N501S, N501Y, Y505H, Y508H, R509K, V510L, V511E, V512L, L518I, H519O, A520S, A520V, P521R, P521S, A522P, A522S, A570D, D614G, P680H, P681H, A701V, T716I, and D950N.

[0015] In one example, the mutant S protein (i) lacks a furin cleavage site at the S1 / S2 boundary and contains an RRAR to QQAA mutation at residues corresponding to nucleotides 682-685 of SEQ ID NO:18, and / or (ii) lacks a furin cleavage site at the S2' site, and / or (iii) contains a D to G mutation at residue corresponding to nucleotide 614 of SEQ ID NO:18, and (iv) contains an insertion of two proline residues between residues corresponding to nucleotides 986 and 987 of SEQ ID NO:18.

[0016] In one example, the S protein lacks a furin cleavage site at the S1 / S2 boundary and contains an RRAR to QQAA mutation at residues corresponding to nucleotides 682-685 of SEQ ID NO: 18. For example, the mutant S protein is encoded by the sequence shown in SEQ ID NO:2.

[0017] In one example, the S protein lacks a furin cleavage site at the S2' site.

[0018] In one example, the S protein contains a D to G mutation at the residue corresponding to nucleotide 614 of SEQ ID NO: 18. For example, the mutant S protein is encoded by the sequence shown in SEQ ID NO:7.

[0019] In one example, the S protein contains an insertion of two proline residues between residues corresponding to nucleotides 986 and 987 of SEQ ID NO:18.

[0020] In one example, the S protein (i) lacks a furin cleavage site at the S1 / S2 boundary and contains an RRAR to QQAA mutation at residues corresponding to nucleotides 682-685 of SEQ ID NO: 18, and (ii) lacks a furin cleavage site at the S2' site. For example, the mutant S protein is encoded by the sequence shown in SEQ ID NO:5.

[0021] In one example, the S protein (i) lacks a furin cleavage site at the S1 / S2 boundary and contains an RRAR to QQAA mutation at residues corresponding to nucleotides 682-685 of SEQ ID NO: 18, and (ii) contains a D to G mutation at residue corresponding to nucleotide 614 of SEQ ID NO: 18. For example, the mutant S protein is encoded by the sequence set forth in SEQ ID NO:4.

[0022] In one example, the S protein (i) lacks a furin cleavage site at the S1 / S2 boundary and contains an RRAR to QQAA mutation at residues corresponding to nucleotides 682-685 of SEQ ID NO: 18, and (ii) contains an insertion of two proline residues between residues corresponding to nucleotides 986 and 987 of SEQ ID NO: 18. For example, the mutant S protein is encoded by the sequence set forth in SEQ ID NO:3.

[0023] In one example, the S protein (i) lacks a furin cleavage site at the S1 / S2 boundary and contains an RRAR to QQAA mutation at residues corresponding to nucleotides 682-685 of SEQ ID NO: 18, (ii) lacks a furin cleavage site at the S2' site, and (iii) contains a D to G mutation at residue corresponding to nucleotide 614 of SEQ ID NO: 18. For example, the mutant S protein is encoded by the sequence set forth in SEQ ID NO:6.

[0024] In one example, the S protein (i) lacks a furin cleavage site at the S1 / S2 boundary and contains a RRAR to QQAA mutation at residues corresponding to nucleotides 682-685 of SEQ ID NO:18, (ii) lacks a furin cleavage site at the S2' site, and (iii) contains an insertion of two proline residues between residues corresponding to nucleotides 986 and 987 of SEQ ID NO:18.

[0025] In one example, the S protein (i) lacks a furin cleavage site at the S2' site and (ii) contains a D to G mutation at the residue corresponding to nucleotide 614 of SEQ ID NO:18.

[0026] In one example, the S protein (i) lacks a furin cleavage site at the S2' site and (ii) contains an insertion of two proline residues between residues corresponding to nucleotides 986 and 987 of SEQ ID NO:18.

[0027] In one example, the S protein (i) lacks a furin cleavage site at the S2' site, (ii) contains a D to G mutation at a residue corresponding to nucleotide 614 of SEQ ID NO:18, and (iii) contains an insertion of two proline residues between residues corresponding to nucleotides 986 and 987 of SEQ ID NO:18.

[0028] In one example, the S protein contains (i) a D to G mutation at a residue corresponding to nucleotide 614 of SEQ ID NO:18, and (ii) an insertion of two proline residues between residues corresponding to nucleotides 986 and 987 of SEQ ID NO:18.

[0029] In one example, the S protein (i) lacks a furin cleavage site at the S1 / S2 boundary and contains an RRAR to QQAA mutation at residues corresponding to nucleotides 682-685 of SEQ ID NO:18, (ii) lacks a furin cleavage site at the S2' site, (iii) contains a D to G mutation at residue corresponding to nucleotide 614 of SEQ ID NO:18, and (iv) contains an insertion of two proline residues between residues corresponding to nucleotides 986 and 987 of SEQ ID NO:18.

[0030] In one example, the S protein contains an N to Y mutation at a residue corresponding to nucleotide 501 of SEQ ID NO:18.

[0031] In one example, the S protein contains a deletion of two residues corresponding to nucleotides 69 and 70 of SEQ ID NO:18.

[0032] In one example, the S protein contains a deletion of one residue corresponding to nucleotide 144 of SEQ ID NO:18.

[0033] In one example, the S protein contains (i) an RRAR to QQAA mutation at residues corresponding to nucleotides 682-685 of SEQ ID NO:18, (ii) a deletion of two residues corresponding to nucleotides 69 and 70 of SEQ ID NO:18, (iii) a deletion of one residue corresponding to nucleotide 144 of SEQ ID NO:18, (iv) an N to Y mutation at residue corresponding to nucleotide 501 of SEQ ID NO:18, and (v) a D to G mutation at residue corresponding to nucleotide 614 of SEQ ID NO:18. For example, the mutant S protein is encoded by the sequence set forth in SEQ ID NO:19.

[0034] In one example, the S protein contains a deletion of three residues corresponding to nucleotides 242-244 of SEQ ID NO:18.

[0035] In one example, the S protein contains a K to N mutation at the residue corresponding to nucleotide 417 of SEQ ID NO:18.

[0036] In one example, the S protein contains an E to K mutation at the residue corresponding to nucleotide 484 of SEQ ID NO:18.

[0037] In one example, the S protein contains (i) an RRAR to QQAA mutation at a residue corresponding to nucleotides 682-685 of SEQ ID NO:18, (ii) a deletion of three residues corresponding to nucleotides 242-244 of SEQ ID NO:18, (iii) a K to N mutation at a residue corresponding to nucleotide 417 of SEQ ID NO:18, (iv) an E to K mutation at a residue corresponding to nucleotide 484 of SEQ ID NO:18, (v) an N to Y mutation at a residue corresponding to nucleotide 501 of SEQ ID NO:18, and (vi) a D to G mutation at a residue corresponding to nucleotide 614 of SEQ ID NO:18. For example, the mutant S protein is encoded by the sequence set forth in SEQ ID NO:20.

[0038] In one example, the S protein comprises (i) an RRAR to QQAA mutation at a residue corresponding to nucleotides 682-685 of SEQ ID NO:18, (ii) a deletion of two residues corresponding to nucleotides 69 and 70 of SEQ ID NO:18, (iii) a deletion of three residues corresponding to nucleotides 242-244 of SEQ ID NO:18, (iv) a K to N mutation at a residue corresponding to nucleotide 417 of SEQ ID NO:18, (v) an E to K mutation at a residue corresponding to nucleotide 484 of SEQ ID NO:18, (vi) an N to Y mutation at a residue corresponding to nucleotide 501 of SEQ ID NO:18, and (vii) a D to G mutation at a residue corresponding to nucleotide 614 of SEQ ID NO:18. For example, the mutant S protein is encoded by the sequence set forth in SEQ ID NO:21.

[0039] In one example, the S protein contains an A to D mutation at the residue corresponding to nucleotide 570 of SEQ ID NO:18.

[0040] In one example, the S protein contains a P to H mutation at a residue corresponding to nucleotide 680 of SEQ ID NO:18.

[0041] In one example, the S protein contains a T to I mutation at a residue corresponding to nucleotide 716 of SEQ ID NO:18.

[0042] In one example, the S protein comprises (i) an RRAR to QQAA mutation at residues corresponding to nucleotides 682-685 of SEQ ID NO:18, (ii) a deletion of two residues corresponding to nucleotides 69 and 70 of SEQ ID NO:18, (iii) a deletion of one residue corresponding to nucleotide 144 of SEQ ID NO:18, (iv) an N to Y mutation at residue corresponding to nucleotide 501 of SEQ ID NO:18, (v) an A to D mutation at residue corresponding to nucleotide 570 of SEQ ID NO:18, (vi) a D to G mutation at residue corresponding to nucleotide 614 of SEQ ID NO:18, (vii) a P to H mutation at residue corresponding to nucleotide 680 of SEQ ID NO:18, and (viii) a T to I mutation at residue corresponding to nucleotide 716 of SEQ ID NO:18. For example, the mutant S protein is encoded by the sequence set forth in SEQ ID NO:22.

[0043] In one example, the S protein contains an L to F mutation at the residue corresponding to nucleotide 18 of SEQ ID NO:18.

[0044] In one example, the S protein contains a D to A mutation at the residue corresponding to nucleotide 80 of SEQ ID NO:18.

[0045] In one example, the S protein contains a D to G mutation at a residue corresponding to nucleotide 215 of SEQ ID NO:18.

[0046] In one example, the S protein includes an A to V mutation at a residue corresponding to nucleotide 701 of SEQ ID NO:18.

[0047] In one example, the S protein comprises (i) an RRAR to QQAA mutation at a residue corresponding to nucleotides 682-685 of SEQ ID NO:18, (ii) an L to F mutation at a residue corresponding to nucleotide 18 of SEQ ID NO:18, (iii) a D to A mutation at a residue corresponding to nucleotide 80 of SEQ ID NO:18, (iv) a D to G mutation at a residue corresponding to nucleotide 215 of SEQ ID NO:18, (v) a deletion of three residues corresponding to nucleotides 242-244 of SEQ ID NO:18, (vi) a K to N mutation at a residue corresponding to nucleotide 417 of SEQ ID NO:18, (vii) an E to K mutation at a residue corresponding to nucleotide 484 of SEQ ID NO:18, (viii) an N to Y mutation at a residue corresponding to nucleotide 501 of SEQ ID NO:18, (ix) a D to G mutation at a residue corresponding to nucleotide 614 of SEQ ID NO:18, and (x) an A to V mutation at a residue corresponding to nucleotide 701 of SEQ ID NO:18. For example, the mutant S protein is encoded by the sequence set forth in SEQ ID NO:23.

[0048] In one example, the mutant S protein (i) lacks a furin cleavage site at the S1 / S2 boundary and comprises an RRAR to QQAA mutation at residues corresponding to nucleotides 682-685 of SEQ ID NO:18, and / or (ii) lacks a furin cleavage site at the S2' site, and / or (iii) comprises a D to G mutation at residue corresponding to nucleotide 614 of SEQ ID NO:18, and / or (iv) comprises an insertion of two proline residues between residues corresponding to nucleotides 986 and 987 of SEQ ID NO:18, and / or (v) comprises an N to Y mutation at residue corresponding to nucleotide 501 of SEQ ID NO:18, and / or (vi) comprises a deletion of two residues corresponding to nucleotides 69 and 70 of SEQ ID NO:18, and / or (vii) comprises a deletion of one residue corresponding to nucleotide 144 of SEQ ID NO:18, and / or (viii) comprises a deletion of three residues corresponding to nucleotides 242-244 of SEQ ID NO:18, and / or (ix) and / or (x) an E to K mutation at a residue corresponding to nucleotide 484 of SEQ ID NO:18, and / or (xi) an A to D mutation at a residue corresponding to nucleotide 570 of SEQ ID NO:18, and / or (xii) a P to H mutation at a residue corresponding to nucleotide 680 of SEQ ID NO:18, and / or (xiii) a T to I mutation at a residue corresponding to nucleotide 716 of SEQ ID NO:18, and / or (xix) an L to F mutation at a residue corresponding to nucleotide 18 of SEQ ID NO:18, and / or (xx) a D to A mutation at a residue corresponding to nucleotide 80 of SEQ ID NO:18, and / or (xxi) a D to G mutation at a residue corresponding to nucleotide 215 of SEQ ID NO:18, and / or (xxii) an A to V mutation at a residue corresponding to nucleotide 701 of SEQ ID NO:18.

[0049] In one example, the mutant S protein is encoded by a sequence shown in any one of SEQ ID NOs: 2-7.

[0050] In one example, the mutant S protein is encoded by the sequence shown in SEQ ID NO:2.

[0051] In one example, the mutant S protein is encoded by the sequence shown in SEQ ID NO:3.

[0052] In one example, the mutant S protein is encoded by the sequence shown in SEQ ID NO:4.

[0053] In one example, the mutant S protein is encoded by the sequence shown in SEQ ID NO:5.

[0054] In one example, the mutant S protein is encoded by the sequence shown in SEQ ID NO:6.

[0055] In one example, the mutant S protein is encoded by the sequence shown in SEQ ID NO:7.

[0056] In one example, the mutant S protein is encoded by a sequence shown in any one of SEQ ID NOs: 2-7 and / or 19-23.

[0057] In one example, the mutant S protein is encoded by a sequence set forth in any one of SEQ ID NOs: 19-23.

[0058] In one example, the mutant S protein is encoded by the sequence shown in SEQ ID NO:19.

[0059] In one example, the mutant S protein is encoded by the sequence shown in SEQ ID NO:20.

[0060] In one example, the mutant S protein is encoded by the sequence shown in SEQ ID NO:21.

[0061] In one example, the mutant S protein is encoded by the sequence shown in SEQ ID NO:22.

[0062] In one example, the mutant S protein is encoded by the sequence shown in SEQ ID NO:23.

[0063] In one example, the antigen is the N protein. For example, the N protein is encoded by the sequence shown in SEQ ID NO:8.

[0064] In one example, the SG promoter is a native SG promoter. For example, the native SG promoter is a promoter that is native to the RNA virus (e.g., alphavirus) from which it is derived and / or based. In one example, the native SG promoter is a native alphavirus SG promoter.

[0065] In one example, the native SG promoter is a minimal SG promoter. For example, the minimal SG promoter is the minimal sequence required for initiation of transcription. In one example, the minimal native SG promoter is 49 nucleotides in length. In one example, the minimal native SG promoter is encoded by a sequence that comprises or consists of the sequence shown in SEQ ID NO:9.

[0066] In one example, the self-replicating RNA is derived from an alphavirus, for example, the alphavirus is selected from the group consisting of Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis virus (VEE), and combinations thereof.

[0067] In one example, the self-replicating RNA is derived from Semliki Forest Virus (SFV).

[0068] In one example, the self-replicating RNA is from Sindbis virus (SIN).

[0069] In one example, the self-replicating RNA is derived from Venezuelan Equine Encephalitis virus (VEE).

[0070] In one example, the disclosure provides a self-replicating RNA encoded by a sequence set forth in any one of SEQ ID NOs: 10-17.

[0071] In one example, the disclosure provides a self-replicating RNA encoded by the sequence set forth in SEQ ID NO: 10 (Co5).

[0072] In one example, the disclosure provides a self-replicating RNA encoded by the sequence set forth in SEQ ID NO:11 (Co6).

[0073] In one example, the disclosure provides a self-replicating RNA encoded by the sequence set forth in SEQ ID NO: 12 (Co16).

[0074] In one example, the disclosure provides a self-replicating RNA encoded by the sequence set forth in SEQ ID NO: 13 (Co17).

[0075] In one example, the disclosure provides a self-replicating RNA encoded by the sequence set forth in SEQ ID NO: 14 (Co48).

[0076] In one example, the disclosure provides a self-replicating RNA encoded by the sequence set forth in SEQ ID NO: 15 (Co49).

[0077] In one example, the disclosure provides a self-replicating RNA encoded by the sequence set forth in SEQ ID NO: 16 (Co58).

[0078] In one example, the disclosure provides a self-replicating RNA encoded by the sequence set forth in SEQ ID NO: 17 (Co59).

[0079] In one example, the disclosure provides a self-replicating RNA encoded by the sequence set forth in SEQ ID NO:24 (Co77).

[0080] In one example, the disclosure provides a self-replicating RNA encoded by the sequence set forth in SEQ ID NO:25 (Co78).

[0081] In one example, the disclosure provides a self-replicating RNA encoded by the sequence set forth in SEQ ID NO:26 (Co79).

[0082] In one example, the disclosure provides a self-replicating RNA encoded by the sequence set forth in SEQ ID NO:27 (Co80).

[0083] In one example, the disclosure provides a self-replicating RNA encoded by the sequence set forth in SEQ ID NO:28 (Co81).

[0084] The present disclosure also provides an immunogenic composition comprising the self-replicating RNA of the present disclosure. For example, the composition of the present disclosure can induce an immune response in a subject when administered. For example, administration of the composition induces a humoral and / or cell-mediated immune response. In one example, the composition induces a humoral immune response in a subject. For example, the humoral immune response is an antibody-mediated immune response. In another example, the composition induces a cell-mediated immune response. For example, the cell-mediated immune response includes the activation of antigen-specific cytotoxic T cells.

[0085] In one example, the immunogenic composition comprises multiple self-replicating RNAs, each encoding a different polypeptide antigen sequence. For example, the different polypeptide antigen sequences are from strains of the same virus (e.g., encoding antigens from the same SARS-CoV-2 strain). In one example, the different polypeptide antigen sequences are from different strains of the same virus (e.g., encoding antigens from different SARS-CoV-2 strains). In one example, the different polypeptide antigen sequences are from different viruses (e.g., encoding an antigen from SARS-CoV-2 and an antigen from an unrelated virus, e.g., influenza).

[0086] The present disclosure also provides a pharmaceutical composition comprising an immunogenic composition of the present disclosure and a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers suitable for use in the present disclosure will be apparent to those of skill in the art and / or are described herein.

[0087] In one example, the pharmaceutical composition further comprises lipid nanoparticles (LNPs), polymeric microparticles, and oil-in-water emulsions, for example, the self-replicating RNA is encapsulated, bound, or adsorbed to the LNPs, polymeric microparticles, and oil-in-water emulsions.

[0088] In one example, the pharmaceutical composition further comprises a LNP. For example, the self-replicating RNA is encapsulated in the LNP. In another example, the self-replicating RNA is bound to the LNP. In a further example, the self-replicating RNA is adsorbed to the LNP.

[0089] In one example, the pharmaceutical composition further comprises a polymeric microparticle. For example, the self-replicating RNA is encapsulated in the polymeric microparticle. In another example, the self-replicating RNA is bound to the polymeric microparticle. In a further example, the self-replicating RNA is adsorbed to the polymeric microparticle.

[0090] In one example, the pharmaceutical composition further comprises an oil-in-water emulsion. For example, the self-replicating RNA is encapsulated in the oil-in-water emulsion. In another example, the self-replicating RNA is bound to the oil-in-water emulsion. In a further example, the self-replicating RNA is adsorbed to the oil-in-water emulsion. In a further example, the self-replicating RNA is resuspended in the oil-in-water emulsion.

[0091] The present disclosure also provides an immunogenic or pharmaceutical composition of the present disclosure for use as a vaccine.

[0092] The present disclosure further provides a polynucleotide encoding the self-replicating RNA vaccine of the present disclosure. For example, the polynucleotide is DNA. In one example, the present disclosure provides a DNA encoding the self-replicating RNA vaccine of the present disclosure.

[0093] The present disclosure further provides an immunogenic composition or pharmaceutical composition of the present disclosure for use in treating or preventing or delaying the progression of a disease or condition selected from the group consisting of SARS-2-CoV-2 infection, COVID-19, ARDS, and combinations thereof. For example, the present disclosure provides an immunogenic composition or pharmaceutical composition of the present disclosure for use in treating SARS-2-CoV-2 infection, COVID-19, ARDS, and combinations thereof. In one example, the present disclosure provides an immunogenic composition or pharmaceutical composition of the present disclosure for use in preventing SARS-2-CoV-2 infection, COVID-19, ARDS, and combinations thereof. In another example, the present disclosure provides an immunogenic composition or pharmaceutical composition of the present disclosure for use in delaying the progression of SARS-2-CoV-2 infection, COVID-19, ARDS, and combinations thereof.

[0094] In one example, the disclosure provides an immunogenic composition or pharmaceutical composition of the disclosure for use in treating or preventing or delaying the progression of COVID-19. For example, the disclosure provides an immunogenic composition or pharmaceutical composition of the disclosure for use in treating COVID-19. In another example, the disclosure provides an immunogenic composition or pharmaceutical composition of the disclosure for use in preventing COVID-19. In a further example, the disclosure provides an immunogenic composition or pharmaceutical composition of the disclosure for use in delaying the progression of COVID-19.

[0095] In one example, the disclosure provides an immunogenic composition or pharmaceutical composition of the disclosure for use in treating or preventing or delaying the progression of SARS-CoV-2 infection. For example, the disclosure provides an immunogenic composition or pharmaceutical composition of the disclosure for use in treating SARS-CoV-2 infection. In another example, the disclosure provides an immunogenic composition or pharmaceutical composition of the disclosure for use in preventing SARS-CoV-2 infection. In a further example, the disclosure provides an immunogenic composition or pharmaceutical composition of the disclosure for use in delaying the progression of SARS-CoV-2.

[0096] In one example, the present disclosure provides an immunogenic composition or pharmaceutical composition of the present disclosure for use in treating or preventing or delaying the progression of ARDS. For example, the present disclosure provides an immunogenic composition or pharmaceutical composition of the present disclosure for use in treating ARDS. In another example, the present disclosure provides an immunogenic composition or pharmaceutical composition of the present disclosure for use in preventing ARDS. In a further example, the present disclosure provides an immunogenic composition or pharmaceutical composition of the present disclosure for use in delaying the progression of ARDS.

[0097] The present disclosure provides a method of treating, preventing, or delaying the progression of a disease or condition in a subject, comprising administering to a subject in need thereof an immunogenic or pharmaceutical composition of the present disclosure. In one example, the present disclosure provides a method of treating a disease or condition in a subject, comprising administering to a subject in need thereof an immunogenic or pharmaceutical composition of the present disclosure. In another example, the present disclosure provides a method of preventing a disease or condition in a subject, comprising administering to a subject in need thereof an immunogenic or pharmaceutical composition of the present disclosure. In a further example, the present disclosure provides a method of delaying the progression of a disease or condition in a subject, comprising administering to a subject in need thereof an immunogenic or pharmaceutical composition of the present disclosure.

[0098] In one example, there is provided a use of a self-replicating RNA of the present disclosure in the manufacture of a medicament for treating or preventing or slowing the progression of a disease or condition in a subject in need thereof. For example, there is provided a use of a self-replicating RNA of the present disclosure in the manufacture of a medicament for treating a disease or condition in a subject in need thereof. In another example, there is provided a use of a self-replicating RNA of the present disclosure in the manufacture of a medicament for preventing a disease or condition in a subject in need thereof. In a further example, there is provided a use of a self-replicating RNA of the present disclosure in the manufacture of a medicament for slowing the progression of a disease or condition in a subject in need thereof.

[0099] In one example, the subject is suffering from a disease or condition. In one example, the subject has been diagnosed with a disease or condition. In one example, the subject is undergoing treatment for a disease or condition.

[0100] In one example, the disease or condition is selected from the group consisting of SARS-CoV-2 infection, COVID-19, ARDS, and combinations thereof. In one example, the disease or condition is SARS-CoV-2 infection. In another example, the disease or condition is COVID-19. In a further example, the disease or condition is ARDS. In one example, the ARDS is associated with SARS-CoV-2 infection and / or COVID-19. For example, the disease or condition is ARDS. In one example, the ARDS is associated with SARS-CoV-2 infection. In another example, the disease or condition is ARDS. In one example, the ARDS is associated with COVID-19.

[0101] In one example of any of the methods described herein, the self-replicating RNA of the disclosure is administered before or after the onset of SARS-CoV-2 infection, COVID-19, and / or ARDS in the subject. In one example of any of the methods described herein, the self-replicating RNA of the disclosure is administered before the onset of SARS-CoV-2 infection, COVID-19, and / or ARDS in the subject. In one example of any of the methods described herein, the self-replicating RNA of the disclosure is administered after the onset of SARS-CoV-2 infection, COVID-19, and / or ARDS in the subject.

[0102] In one example of any of the methods described herein, the self-replicating RNA of the disclosure is administered after detection of SARS-CoV-2 infection, COVID-19, and / or ARDS in a subject. In one example of any of the methods described herein, the self-replicating RNA of the disclosure is administered after detection of SARS-CoV-2 infection. In another example, the self-replicating RNA of the disclosure is administered after detection of SARS-CoV-2 infection, but before the onset of COVID-19. In a further example of any of the methods described herein, the self-replicating RNA of the disclosure is administered after detection of COVID-19. In one example of any of the methods described herein, the self-replicating RNA of the disclosure is administered after detection of COVID-19, but before the onset of ARDS. In another example of any of the methods described herein, the self-replicating RNA of the disclosure is administered after detection of ARDS.

[0103] In one example, the subject is at risk of developing COVID-19 or ARDS. For example, the subject is at risk of developing COVID-19. In a further example, the subject is at risk of developing ARDS.

[0104] In one example, the compositions of the present disclosure are administered in an amount sufficient to reduce the severity of or prevent the onset of one or more symptoms of SARS-CoV-2 infection, COVID-19, and / or ARDS, which symptoms will be apparent to one of skill in the art and / or described herein.

[0105] The present disclosure provides a method of inducing an immune response in a subject, the method comprising administering to a subject in need thereof a self-replicating RNA, an immunogenic composition, or a pharmaceutical composition of the present disclosure.

[0106] The present disclosure also provides the use of a self-replicating RNA, immunogenic composition, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for inducing an immune response in a subject in need thereof.

[0107] In one example, the self-replicating RNA, immunogenic composition, or pharmaceutical composition of the present disclosure induces a humoral and / or cell-mediated immune response. In one example, the composition induces a humoral immune response in a subject. For example, the humoral immune response is an antibody-mediated immune response. For example, the production of neutralizing antibodies. In another example, the composition induces a cell-mediated immune response. For example, the cell-mediated immune response includes the activation of antigen-specific cytotoxic T cells. For example, the T cells are CD4 T cells and / or CD8 T cells. In one example, the T cells are CD4 T cells. In another example, the T cells are CD8 T cells. In a further example, the T cells are CD4 and CD8 T cells.

[0108] In one example, administration of a self-replicating RNA, immunogenic composition, or pharmaceutical composition of the disclosure induces a CD4 T cell-mediated immune response.

[0109] In one example, administration of a self-replicating RNA, immunogenic composition, or pharmaceutical composition of the disclosure induces a CD8 T cell-mediated immune response.

[0110] In one example, administration of a self-replicating RNA, immunogenic composition, or pharmaceutical composition of the disclosure induces a CD4 and CD8 T cell-mediated immune response.

[0111] In one example, the CD4 T cell mediated immune response is a Th0, Th1, and / or Th2 response. For example, the CD4 T cell mediated immune response is a Th0 response. In another example, the CD4 T cell mediated immune response is a Th1 response. In a further example, the CD4 T cell mediated immune response is a Th2 response. In one example, the CD4 T cell mediated immune response is a Th0 and Th1 response. In another example, the CD4 T cell mediated immune response is a Th0 and Th2 response. In a further example, the CD4 T cell mediated immune response is a Th1 and Th2 response. In another example, the CD4 T cell mediated immune response is a Th0, Th1, and Th2 response.

[0112] In one example, the Th0 responsive cytokine expresses interleukin 2 (IL2+) and / or tumor necrosis factor alpha (TNFa+) and / or is negative for interferon gamma (IFNg-), IL5-, and / or IL13-. For example, the cytokine is IL2+. In another example, the cytokine is TNFa+. In one example, the cytokine is IFNg-. In another example, the cytokine is IL5-. In a further example, the cytokine is IL13-.

[0113] In one example, the Th1 response cytokine expresses interferon gamma (IFNg+) and / or is negative for IL5- and / or IL13-. For example, the cytokine is IFNg+. In another example, the cytokine is IL5-. In a further example, the cytokine is IL13-.

[0114] In one example, the Th2 response cytokine expresses IL5+ and / or IL13+ and / or is negative for IFNg. For example, the cytokine is IL5+. In a further example, the cytokine is IL13+. For example, the cytokine is IFNg-.

[0115] The present disclosure also provides a polynucleotide encoding the self-replicating RNA of the present disclosure. For example, the polynucleotide is a recombinant DNA. In one example, the recombinant DNA is a plasmid. In one example, the plasmid comprises a sequence shown in any one of SEQ ID NOs: 10 to 17.

[0116] The present disclosure also provides kits comprising at least one self-replicating RNA of the present disclosure, optionally in a delivery system and / or a pharma- ceutically acceptable carrier or diluent, packaged with instructions for use in treating or preventing or delaying the progression of a disease or disorder (e.g., SARS-CoV-2 infection, COVID-19, and / or ARDS) in a subject.

[0117] The present disclosure also provides kits comprising at least one self-replicating RNA of the present disclosure, optionally in a delivery system and / or a pharma- ceutically acceptable carrier or diluent, packaged with instructions for administering the RNA to a subject suffering from or at risk for a disease or disorder (e.g., SARS-CoV-2 infection, COVID-19, and / or ARDS).

[0118] In one example, the self-replicating RNA, immunogenic composition, or pharmaceutical composition of the disclosure is supplied in a vial. In another example, the self-replicating RNA, immunogenic composition, or pharmaceutical composition of the disclosure is supplied in a syringe. [Brief description of the drawings]

[0119] [Figure 1] 1 is a series of graphical representations showing antigen-specific T cell induction by Co16, showing the % induced net (antigen-specific) cytokine-producing CD4 and CD8 T cells for (A) S1-specific CD4 T cells, (B) S1-specific CD8 T cells, (C) S2-specific CD4 T cells, (D) S2-specific CD8 T cells, and (E) N-specific CD4 T cells. [Diagram 2] A series of graphical representations showing the neutralization capacity of constructs against (A) the reference Whuan sequence, (B) alpha variants (B.1.1.7, UK strain), (C) beta variants (B.1.351, South African strain), (D) gamma variants (P.1, Brazilian strain), and (E) delta variants (B.1.617.2, Indian strain). [Diagram 3] A series of graphical representations showing that all constructs generated total Ig responses at high and low doses against (A) the reference Whuan sequence, (B) alpha variants (B.1.1.7, UK strain), (C) beta variants (B.1.351, South African strain), (D) gamma variants (P.1, Brazilian strain), and (E) delta variants (B.1.617.2, Indian strain). [Figure 4]1 is a graphical representation showing that all constructs generated S-specific B cells that reacted with all variant B cell receptor-specific probes. Non-specific controls (i.e., no bait and negative control HA H1) showed low levels of background binding. [Diagram 5] FIG. 1 is a series of graphical representations showing that all constructs induced antigen-specific (A) CD4 and (B) CD8 T cells that react with S1 and S2 epitopes. [Table 1-1] [Table 1-2] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0120] general Throughout this specification, unless specifically stated otherwise or the context otherwise requires, references to a single step, composition, group of steps, or group of compositions shall be interpreted as encompassing one and more (i.e., one or more) of that step, composition, group of steps, or group of compositions.

[0121] Those skilled in the art will understand that the present disclosure is susceptible to variations and modifications other than those specifically described. The present disclosure is to be understood to include all such variations and modifications. The present disclosure also includes all of the steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of such steps or features.

[0122] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0123] Any example of the present disclosure herein shall apply mutatis mutandis to any other example of the present disclosure unless otherwise specified. In other words, any specific example of the present disclosure may be combined with any other specific example of the present disclosure (except where mutually exclusive).

[0124] Any instance of this disclosure disclosing a specific feature or group of features, or a method or method step, will be construed as providing explicit support for disclaiming the specific feature or group of features, or method or method step.

[0125] Unless specifically defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0126] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. BDHames(editors),DNA Cloning:A Practical Approach,Volumes 1-4,IRL Press(1995 and 1996),and FMAusubel et al.(editors),Current Protocols in Molecular Biology,Greene Pub.Associates and Wiley-Interscience(1988,including all updates until present),Ed Harlow and David Lane(editors)Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory,(1988),and JEColigan et (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present), and other sources.

[0127] The term "and / or," e.g., "X and / or Y," is understood to mean either "X and Y" or "X or Y," and should be interpreted as providing explicit support for both meanings or either meaning.

[0128] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0129] As used herein, the term "derived from" shall be interpreted to indicate that a particular integer may be obtained from a particular source, but not necessarily directly from that source. Similarly, the term "based on" shall be interpreted to indicate that a particular integer may be developed or used from a particular source, but not necessarily directly from that source.

[0130] Selected Definitions As used herein, the term "self-replicating RNA" refers to an RNA virus-based construct that has been engineered to allow expression of heterologous RNA and proteins. Self-replicating RNA (e.g., in the form of naked RNA) can be amplified in a host cell and result in expression of a desired gene product in the host cell.

[0131] As used herein, the term "monocistronic" with respect to self-replicating RNA refers to an RNA that encodes one polypeptide.

[0132] The term "naked" as used herein refers to a nucleic acid that is substantially free of other macromolecules, such as lipids, polymers, and proteins. A "naked" nucleic acid, such as a self-replicating RNA, is not formulated with other macromolecules to improve cellular uptake. Thus, a naked nucleic acid is not encapsulated, absorbed, or bound to a LNP, a liposome, a polymeric microparticle, or an oil-in-water emulsion.

[0133] As used herein, the term "nucleotide sequence" or "nucleic acid sequence" will be understood to mean a series of contiguous nucleotides (or bases) covalently linked to a phosphodiester backbone. Conventionally, sequences are presented from the 5' to the 3' end unless otherwise specified.

[0134] As used herein, the term "antigen" refers to a molecule or structure that contains one or more epitopes that induce, elicit, enhance, or boost a cellular and / or humoral immune response. Antigens can include, for example, proteins and peptides from pathogens such as viruses, bacteria, fungi, protozoa, plants, or from tumors.

[0135] As used herein, the term "operably linked to" refers to positioning a subgenomic promoter relative to a nucleic acid such that expression of the nucleic acid is controlled or regulated by the element.

[0136] As used herein, the term "subgenomic promoter" (SG, also known as "junction region" promoter) refers to a promoter that directs the expression of a heterologous nucleotide sequence and regulates protein expression.

[0137] The term "polypeptide" or "polypeptide chain" will be understood to mean a series of consecutive amino acids linked by peptide bonds. For example, a protein shall be taken to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains covalently or non-covalently bonded to each other (i.e., a polypeptide complex). A series of polypeptide chains can be covalently linked using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0138] The term "recombinant" is understood to mean the product of artificial genetic recombination.

[0139] As used herein, the terms "disease," "disorder," or "condition" refer to a disruption or interference with normal function and include, but are not limited to, any particular condition, a disease or disorder.

[0140] As used herein, a subject "at risk" of developing a disease or condition may or may not have detectable disease or symptoms of a disease, and may or may not exhibit detectable disease or symptoms of a disease prior to treatment according to the present disclosure. "At risk" indicates that the subject has one or more risk factors, which are measurable parameters that correlate with development of a disease or condition, known in the art and / or described herein.

[0141] As used herein, the terms "treating," "treat," or "treatment" include administering an RNA or composition described herein to thereby reduce or eliminate at least one symptom of a particular disease or condition.

[0142] As used herein, the terms "preventing," "prevent" or "prevention" include providing prophylaxis against the occurrence or recurrence of a particular disease or condition in an individual. An individual may be predisposed or at risk of developing the disease, but may not yet be diagnosed with the disease.

[0143] As used herein, the term "slowing the progression of" includes reducing or slowing the progression of a disease or condition and / or at least one symptom of a disease or condition in an individual.

[0144] An "effective amount" refers to at least an amount effective, at a dosage and for a period of time necessary, to achieve a desired result. For example, the desired result may be a therapeutic or prophylactic result. An effective amount may be provided in one or more administrations. In some examples of the present disclosure, the term "effective amount" refers to an amount necessary to effect a treatment of a disease or condition previously described herein. In some examples of the present disclosure, the term "effective amount" refers to an amount necessary to effect a change associated with a disease or condition previously described herein. An effective amount may vary depending on the disease or condition being treated or the factor being modified, and depending on the weight, age, racial background, sex, health and / or physical condition, and other factors associated with the mammal being treated. Typically, an effective amount will fall within a relatively broad range (e.g., a "dosage" range) that can be determined by a medical practitioner through routine testing and experimentation. Thus, the term is not to be construed to limit the present disclosure to a specific amount, e.g., weight or number, of RNA. An effective amount may be administered in a single dose, or in doses repeated once or several times over the course of a treatment period.

[0145] "Therapeutically effective amount" is the minimum concentration required to bring about at least a measurable improvement in a particular disease or condition. The therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the RNA of the present disclosure to induce a desired response in an individual. The therapeutically effective amount is also one in which any toxic or harmful effects of the RNA are outweighed by the therapeutically beneficial effects.

[0146] As used herein, the term "prophylactically effective amount" shall be taken to mean an amount of RNA of the present disclosure sufficient to prevent or inhibit or delay the onset of one or more detectable symptoms of a disease or disorder described herein.

[0147] As used herein, the term "subject" shall be taken to mean any animal, including humans, e.g., mammals. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0148] self-replicating RNA The present disclosure provides self-replicating RNAs (also known as replicons). For example, the present disclosure provides monocistronic self-replicating RNAs.

[0149] Those skilled in the art will understand that the self-replicating RNA of the present disclosure is based on the genomic RNA of an RNA virus. The RNA should be positive (+) strand, and thus can be directly translated after delivery to a cell without the need for an intervening replication step (e.g., reverse transcription). Translation of the RNA results in the production of nonstructural proteins (NSPs) that combine to form a replicase complex (i.e., RNA-dependent RNA polymerase). The complex then amplifies the original RNA, producing both antisense and sense transcripts, resulting in the production of multiple daughter RNAs that can then be translated and transcribed, enhancing overall protein expression.

[0150] In one example, a self-replicating RNA of the present disclosure includes nonstructural proteins, 5' and 3' untranslated regions (UTRs), and a native subgenomic promoter of an RNA virus.

[0151] In one example, the self-replicating RNA comprises one or more nonstructural proteins of an RNA virus. For example, the RNA comprises at least one or more genes selected from the group consisting of a viral replicase (or viral polymerase), a viral protease, a viral helicase, and other nonstructural viral proteins. For example, the self-replicating RNA comprises a viral replicase (or viral polymerase).

[0152] It will be apparent to those skilled in the art that the RNA suitable for use in the present disclosure may also include a 5' untranslated region (5' UTR), a 3' untranslated region (3' UTR), and / or a coding or translation sequence. In addition, the RNA may include a 5' cap structure, a chain terminating nucleotide, a stem loop (e.g., a histone stem loop), a 3' tailing sequence (e.g., a polyadenylation signal or one or more polyA tails). In another example, the self-replicating RNA includes a 5' terminal UTR and a 3' terminal UTR of an RNA virus. It will be apparent to those skilled in the art that the terms 5' and 3' UTR also encompass the terms 5' and 3' conserved sequence elements (CSEs). In one example, the self-replicating RNA includes a 5' terminal CSE and a 3' terminal CSE.

[0153] The self-replicating RNA of the present disclosure is not capable of inducing the production of infectious viral particles, for example, the self-replicating RNA of the present disclosure does not contain viral genes encoding structural proteins necessary for the production of viral particles.

[0154] In one example, the self-replicating RNA is derived from or based on an alphavirus. Suitable alphaviruses will be apparent to one of skill in the art and / or are described herein.

[0155] In another example, the self-replicating RNA is derived from or based on a virus other than an alphavirus, e.g., a positive stranded RNA virus. Positive stranded RNA viruses suitable for use in the present disclosure will be apparent to one of skill in the art and include, for example, a picornavirus, a flavivirus, a rubivirus, a pestivirus, a hepacivirus, a calicivirus, or a coronavirus.

[0156] Alphaviruses In one example, the self-replicating RNA of the present disclosure is derived from (or based on) an alphavirus.

[0157] Alphaviruses are the only genus in the Togaviridae family and are enveloped viruses with a positive-sense single-stranded RNA genome. One skilled in the art will appreciate that the alphavirus genome contains two open reading frames (ORFs), nonstructural and structural. The first ORF encodes four nonstructural proteins (NSP1, NSP2, NSP3, and NSP4) required for viral RNA transcription and replication. The second encodes three structural proteins that assemble as a heterodimer: the core nucleocapsid protein C, and the envelope proteins P62 and E1. The viral membrane-anchored surface glycoproteins are responsible for receptor recognition and entry into target cells through membrane fusion.

[0158] In one example, the self-replicating RNA of the present disclosure comprises a viral replicase (or viral polymerase). For example, the viral replicase is an alphavirus replicase, such as the alphavirus protein NSP4.

[0159] In one example, a self-replicating RNA of the present disclosure does not encode one or more alphavirus structural proteins (e.g., capsid and / or envelope glycoproteins), e.g., the self-replicating RNA is incapable of producing RNA-containing alphavirus virions (i.e., infectious virus particles).

[0160] In one example, the self-replicating RNA comprises a native alphavirus SG promoter. For example, the native alphavirus SG promoter is a minimal SG promoter (i.e., the minimal sequence required for initiation of transcription) and comprises the sequence set forth in SEQ ID NO:9.

[0161] One of skill in the art would be aware of suitable alphaviruses for use in the present disclosure. Exemplary alphaviruses include, but are not limited to, Venezuelan equine encephalitis virus (VEE, e.g., Trinidad donkey, TC83CR), Semliki Forest virus (SFV), Sindbis virus (SIN), Ross River virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Chikungunya virus, SAAR86 virus, Everglades virus, Mucambo virus, Barmah Forest virus, Middelburg virus, Pixuna virus, O'nyong-nyong virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Wataroa virus, Bambangki virus, Kiziragati virus, Highland J virus, Fort Morgan virus, Ndum virus, and Boggy Creek virus. The term "alphavirus" also includes chimeric alphaviruses (e.g., as described by Perri et al, (2003) J. Virol. 77(19):10394-403), which contain genomic sequences from more than one alphavirus.

[0162] Subgenomic promoters The present disclosure provides a self-replicating RNA comprising a nucleotide sequence encoding an antigen operably linked to an SG promoter.

[0163] SG promoters (also known as "junction region" promoters) suitable for use in the present disclosure will be apparent to those of skill in the art and / or are described herein.

[0164] In one example, the SG promoter is derived from or based on an alphavirus SG promoter. For example, the SG promoter is a native alphavirus SG promoter. In one example, the native SG promoter is a minimal SG promoter. For example, the minimal SG promoter is the minimal sequence required for initiation of transcription.

[0165] 5' untranslated region (5'UTR) In one example, the self-replicating RNA comprises a 5'-UTR of an RNA virus.

[0166] As used herein, the term "5'-untranslated region" or "5'-UTR" refers to the non-coding region of an mRNA located 5' to the translation initiation sequence (AUG).

[0167] In one example, the 5'UTR is the 5'UTR of Venezuelan Equine Encephalitis Virus (VEEV) or a modified form thereof. For example, the 5'UTR comprises the sequence shown in SEQ ID NO:29.

[0168] In one example, the 5'UTR comprises at least one microRNA binding site, an AU-rich element (ARE), a GC-rich element, a stem-loop, and combinations thereof.

[0169] MicroRNA binding sites As used herein, the term "microRNA binding site" refers to a sequence within a polynucleotide (e.g., within a DNA or RNA transcript) that has sufficient complementarity to all or a region of a microRNA (miRNA) to interact with, associate with, or bind to the miRNA.

[0170] As used herein, the term "microRNA" or "miRNA" refers to a 19-25 nucleotide long non-coding RNA that binds to the 5'UTR of a polynucleotide and downregulates gene expression (e.g., by inhibiting translation). The presence of a microRNA binding site in the 5'UTR of the present disclosure can function to inhibit translation of the 5'-UTR.

[0171] Suitable miRNAs for use in the present disclosure will be apparent to one of skill in the art and / or are described herein.

[0172] In one example, the miRNA binding site includes binding sites for tissue-specific microRNAs or those that regulate biological processes, such as the miRNAs of liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), bone marrow cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-id, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126). For example, the microRNA that regulates biological processes such as angiogenesis (miR-132). Further exemplary miRNAs and miRNA binding sites are disclosed in US Patent Application Serial No. 14 / 043,927.

[0173] AU rich elements (ARE) As used herein, the term "AU-rich element (ARE)" or "AU-rich element (ARE)" refers to a region of a nucleotide sequence that contains a stretch of adenosines (A) and uridines (U). Exemplary AREs include, for example, cytoplasmic myc (c-myc), myoblast determining protein 1 (myoD), c-Jun, myogenin, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-α), or combinations thereof.

[0174] In one example, the ARE contains a specific binding site for Human Antigen R or "HuR" (also known as Elavl1). HuR is known to bind to the ARE increasing mRNA stability.

[0175] GC-rich elements As used herein, the term "GC-rich element" refers to a nucleotide sequence that has a high amount of guanine (G) and / or cytosine (C) relative to adenine (A) and thymine (T) / uracil (U). The presence of GC-rich elements in a polynucleotide (e.g., mRNA) can stabilize the mRNA.

[0176] In one example, the GC-rich element comprises a sequence that is 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30 nucleotides in length.

[0177] In one example, a GC-rich element contains 30%-40%, or 40%-50%, or 50%-60%, or 60%-70% cytosine. For example, a GC-rich element contains 30%-40% cytosine. For example, a GC-rich element contains 40%-50% cytosine. For example, a GC-rich element contains 50%-60% cytosine. For example, a GC-rich element contains 60%-70% cytosine.

[0178] In one example, the GC-rich element comprises 30%, or 40%, or 50%, or 60%, or 70% cytosine. For example, the GC-rich element comprises 30% cytosine. For example, the GC-rich element comprises 40% cytosine. For example, the GC-rich element comprises 50% cytosine. For example, the GC-rich element comprises 60% cytosine. For example, the GC-rich element comprises 60% cytosine. For example, the GC-rich element comprises 70% cytosine.

[0179] In one example, the GC-rich element is at least 50% cytosines.

[0180] In one example, the GC-rich element is at least 60% cytosines.

[0181] In one example, the GC-rich element is at least 70% cytosines.

[0182] In one example, the GC rich element comprises the nucleotide sequence CCCCGGCGCC. In another example, the GC rich element comprises the nucleotide sequence CCCCGGC. In a further example, the GC rich element comprises the nucleotide sequence GCGCCCCGCGGCGCCCCGCGCG.

[0183] In one example, the GC-rich element comprises the nucleotide sequence set forth in SEQ ID NO: 31-33. In one example, the GC-rich element comprises the nucleotide sequence set forth in SEQ ID NO: 31. In another example, the GC-rich element comprises the nucleotide sequence set forth in SEQ ID NO: 32. In a further example, the GC-rich element comprises the nucleotide sequence set forth in SEQ ID NO: 33.

[0184] Stem Loop As used herein, the term "stem loop" refers to a nucleotide sequence that comprises intramolecular base pairing of two adjacent complete or partial reverse complementary sequences that form a stem loop. Stem loops can occur in single-stranded DNA or, more commonly, in RNA. Stem loops can also be referred to as hairpins or hairpin loops, which usually consist of a stem and a terminal loop in a continuous sequence, and the stem is formed by two adjacent complete or partial reverse complementary sequences separated by a short sequence that builds the loop into a stem loop structure.

[0185] The stability of a paired stem-loop is determined by its length, the number of mismatches or bulges it contains, and the nucleotide composition of the paired region.

[0186] In one example, the loop of the stem-loop is 3 to 10 nucleotides in length. For example, the loop of the stem-loop is 3 to 8, or 3 to 7, or 3 to 6, or 4 to 5 nucleotides in length.

[0187] In one example, the loop of the stem-loop is 4 nucleotides in length.

[0188] In one example, the stem loop is a histone stem loop, for example, the histone stem loop comprises or consists of the nucleotide sequence set forth in SEQ ID NO:34.

[0189] Kozak consensus sequence As used herein, the term "Kozak consensus sequence" refers to a nucleotide sequence identified in eukaryotic genes that contains an initiation codon (also called a translation initiation codon) that is recognized by the ribosome, thereby facilitating translation of the gene.

[0190] Exemplary Kozak consensus sequences are known in the art and / or described herein. In one example, the Kozak consensus sequence is set forth in SEQ ID NO: 35. In another example, the Kozak consensus sequence is set forth in SEQ ID NO: 36. In one example, the Kozak consensus sequence is ACCATGG. In another example, the Kozak consensus sequence is ACCATG.

[0191] 3' untranslated region (3'-UTR) In one example, the self-replicating RNA comprises the 3'-UTR of an RNA virus.

[0192] As used herein, the term "3'-UTR" refers to the region of an mRNA located 3' to the translation termination codon (ie, the stop codon).

[0193] In one example, the 3'UTR is the 3'UTR of the Sindbis virus (SINV) or a modified form thereof. For example, the 3'UTR comprises the sequence shown in SEQ ID NO:30.

[0194] In one example, the 3'-UTR of the present disclosure further comprises at least one microRNA binding site, an AU-rich element (ARE), a GC-rich element, a triple helix, a stem loop, one or more stop codons, or a combination thereof.

[0195] Stop codon As used herein, the term "stop codon" refers to a trinucleotide sequence within an mRNA that signals the termination of protein synthesis by the ribosome.

[0196] In one example, a polynucleotide of the present disclosure comprises at least one stop codon at the 5' end of the 3'-UTR, e.g., the stop codon is selected from UAG, UAA, and UGA.

[0197] In one example, the polynucleotide contains two consecutive stop codons that contain the sequence UGAUGA.

[0198] In one example, the polynucleotide contains two consecutive stop codons that contain the sequence UAAUAG.

[0199] 3' tailing sequence The RNA of the present disclosure comprises one or more 3' tailing sequences located at the 3' end of the 3'UTR.

[0200] As described herein, the term "3' tailing sequence" or "3' tailing sequence" refers to a nucleotide sequence (e.g., a polyadenylation signal) that directs the addition of non-coding nucleotides to the 3' end of an mRNA or a nucleotide sequence (e.g., a polyA sequence) located at the 3' end of an mRNA. One of skill in the art will understand that a 3' tailing sequence and / or the product of a 3' tailing sequence in an mRNA functions to stabilize the mRNA and / or prevent degradation of the mRNA.

[0201] As used herein, the term "interruption linker" with respect to a polyA or polyC sequence of the present disclosure refers to a single nucleotide or nucleotide sequence that binds to and interrupts a stretch of consecutive adenosine or cytosine nucleotides in a polyA or polyC sequence. For example, an interruption linker in a polyA sequence is a single nucleotide or nucleotide sequence that consists of or includes nucleotides other than adenosine nucleotides. For example, an interruption linker in a polyC sequence is a single nucleotide or nucleotide sequence that consists of or includes nucleotides other than cytosine nucleotides.

[0202] In one example, the one or more 3' tailing sequences are selected from the group consisting of a polyA sequence, a polyadenylation signal, a G-quadruplex, a polyC sequence, a stem loop, and combinations thereof.

[0203] PolyA sequence As used herein, the term "polyA sequence" refers to a nucleotide sequence of adenine (A) located at the 3' end of an mRNA. In the context of the present disclosure, the polyA sequence may be located within an mRNA or a DNA (e.g., a DNA plasmid that serves as a template for generating an mRNA by transcription of a vector).

[0204] Suitable polyA sequences for use in the present disclosure will be apparent to one of skill in the art and / or are described herein. In one example, the polyA sequence comprises any length (e.g., 10-300) of consecutive (i.e., consecutive) adenosine nucleotides. For example, the polyA sequence comprises 36 consecutive adenosine nucleotides. In one example, the polyA sequence comprises the sequence shown in SEQ ID NO:37.

[0205] In one example, the polyA sequence comprises consecutive adenosine nucleotides separated by one or more interruption linkers. In one example, the polyA sequence comprises consecutive adenosine nucleotides without an interruption linker.

[0206] Polyadenylation Signal As used herein, the term "polyadenylation signal" refers to a nucleotide sequence that induces polyadenylation. Polyadenylation is typically understood to be the addition of a polyA sequence to RNA (e.g., to a premature mRNA to generate a mature mRNA). The polyadenylation signal may be located within a nucleotide sequence at the 3' end of the polynucleotide (e.g., mRNA) to be polyadenylated.

[0207] Suitable polyadenylation signals for use in the present disclosure will be apparent to those of skill in the art and / or are described herein.

[0208] In one example, the polyadenylation signal comprises a hexamer consisting of adenine and uracil / thymidine nucleotides. In one example, the hexamer sequence comprises or consists of AAUAAA.

[0209] In one example, the 3' tailing sequence includes a polyadenylation signal but does not include a polyA sequence.

[0210] G-quadruplex As used herein, the term "G-quadruplex" or "G4" refers to a nucleotide sequence rich in guanine residues that forms a four-stranded secondary structure. For example, a G-quadruplex is a cyclic hydrogen-bonded array of four guanine nucleotides formed by a G-rich sequence in both DNA and RNA.

[0211] In one example, the 3' tailing sequence comprises a polyA sequence and a G-quadruplex. For example, the 3' tailing sequence comprises a polyA sequence attached to a G-quadruplex to produce a polyAG quartet.

[0212] Poly C sequence As used herein, the term "polyC sequence" refers to a nucleotide sequence of cytosine (C) located at the 3' end of an mRNA. In the context of the present disclosure, the polyC sequence may be located within an mRNA or DNA (e.g., a DNA plasmid that serves as a template for generating an mRNA by transcription of a vector).

[0213] Suitable polyC sequences for use in the present disclosure will be apparent to those of skill in the art and / or are described herein.

[0214] In one example, the one or more 3' tailing sequences each include one or more poly-C sequences containing 10 to 300 consecutive cytosine nucleotides. For example, the one or more poly-C sequences each include 10 to 20, or 20 to 30, or 30 to 40, or 40 to 50, or 50 to 60, or 60 to 70, or 70 to 80, or 80 to 90, or 90 to 100, or 100 to 125, or 125 to 150, or 150 to 175, or 175 to 200, or 200 to 225, or 225 to 250, or 250 to 275, or 275 to 300 consecutive cytosine nucleotides. For example, the one or more polyC sequences each contain 10, or 20, or 30, or 40, or 50, or 60, or 70, or 80, or 90, or 100, or 125, or 175, or 200, or 225, or 250, or 275, or 300 consecutive cytosine nucleotides.

[0215] In one example, the one or more poly-C sequences are separated by an interrupted linker, e.g., the fourth nucleotide sequence including the one or more 3' tailing sequences includes, in 5' to 3' order, consecutive cytosine nucleotides, an interrupted linker, and further consecutive cytosine nucleotides.

[0216] In one example, the interruption linker is 10 to 50, or 50 to 100, or 100 to 150 nucleotides in length. For example, the interruption linker is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 25, or 30, or 35, or 40, or 45, or 50, or 55, or 60, or 65, or 70, or 75, or 80, or 85, or 90, or 95, or 100, or 110, or 120, or 130, or 140, or 150 nucleotides in length.

[0217] 5' Cap In one example, the self-replicating RNA comprises a 5'-end cap structure.

[0218] As used herein, the term "5' cap structure" refers to a structure at the 5' end of an mRNA that is involved in nuclear export and binds to mRNA cap-binding protein (CBP). The 5' cap structure is known to stabilize the mRNA through the association of CBP with poly(A) binding protein to form a mature mRNA. Thus, the presence of the 5' cap structure in the mRNA of the present disclosure can further increase the stability of the mRNA compared to an mRNA that does not contain a 5' cap.

[0219] Exemplary 5' cap structures include, for example, anti-reverse cap analog (ARCA), N7,2'-0-dimethyl-guanosine (mCAP), inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, N6,2'-O-dimethyladenosine, 7-methylguanosine (m7G), Cap1, and Cap2.

[0220] Typically, endogenous mRNAs are 5'-capped with guanosine through a (5)'-ppp-(5)'-triphosphate linkage attached to the 5'-terminal nucleotide of the mRNA. The guanosine cap can then be methylated to 7-methylguanosine (m7G) generating 7mG(5')ppp(5')N,pN2p (cap 0 structure), where N represents the first and second 5'-terminal nucleotides of the mRNA. The cap 0 structure can be further 2'-O-methylated to produce 7mG(5')ppp(5')NlmpNp (cap 1), and / or 7mG(5')-ppp(5')NlmpN2mp (cap 2).

[0221] In one example, a polynucleotide of the disclosure includes an endogenous cap.

[0222] As used herein, the term "endogenous cap" refers to a 5' cap synthesized in a cell. For example, an endogenous cap is a natural 5' cap or a wild-type 5' cap. For example, an endogenous cap is a cap0, cap1, or cap2 structure.

[0223] In one example, a polynucleotide of the disclosure includes an analog of the endogenous cap (also referred to as a cap analog).

[0224] As used herein, the term "analog thereof" in the context of an endogenous cap or a "cap analog" refers to a synthetic 5' cap. Cap analogs can be used to produce 5'-capped mRNA in an in vitro transcription reaction. Cap analogs can be synthesized and / or attached chemically (i.e., non-enzymatically) or enzymatically to a nucleotide (e.g., the 5'-terminal nucleotide of an mRNA). Exemplary cap analogs are commercially available and include, for example, 3"-O-Me-m7G(5')ppp(5')G, G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G (New England BioLabs). In one example, the cap analog is N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (i.e., anti-reverse cap analog (ARCA)).

[0225] In one example, the 5' cap structure is a non-hydrolyzable cap structure, which can prevent decapping of the mRNA and increase the half-life of the mRNA.

[0226] In one example, the non-hydrolyzable cap structure comprises a modified nucleotide selected from the group consisting of α-thio-guanosine nucleotides, α-methyl-phosphonates, seleno-phosphates, and combinations thereof. In one example, the modified nucleotide is attached to the 5' end of the mRNA through an α-phosphorothioate bond. Methods for attaching modified nucleotides to the 5' end of the mRNA will be apparent to those skilled in the art. For example, using Vaccinia Capping Enzyme (New England Biolabs).

[0227] antigen The self-replicating RNA of the present disclosure comprises a nucleotide sequence that encodes an antigen (e.g., a pathogenic antigen). For example, the antigen can induce an immune response in a subject.

[0228] In one example, the self-replicating RNA of the present disclosure comprises a nucleotide sequence encoding an antigen from SARS-CoV-2.

[0229] Method of production Suitable methods for producing the self-replicating RNA of the present disclosure will be apparent to those of skill in the art and / or are described herein.

[0230] In one example, the self-replicating RNA is produced using plasmid DNA. Those skilled in the art will understand that plasmid DNA is relatively stable. Briefly, competent bacterial cells (e.g., Escherichia coli) cells are transformed with a DNA plasmid that encodes the self-replicating RNA of the present disclosure. Individual bacterial colonies are isolated, and the resulting plasmid DNA is amplified in E. coli cultures.

[0231] In one example, the plasmid DNA is isolated after fermentation. For example, the plasmid DNA is isolated using a commercially available kit (e.g., Maxiprep DNA kit) or other routine methods known to those skilled in the art. After isolation, the plasmid DNA is linearized by restriction digestion (i.e., using a restriction enzyme). The restriction enzyme is removed using methods known in the art, including, for example, phenol / chloroform extraction and ethanol precipitation.

[0232] In one example, mRNA is produced by in vitro transcription from a linearized DNA template using an RNA polymerase (e.g., T7 RNA polymerase). After in vitro transcription, the DNA template is removed by DNase digestion. One skilled in the art will appreciate that synthetic mRNA capping is performed to correct for mRNA processing and contribute to mRNA stabilization. In one example, the mRNA is enzymatically 5'-capped. For example, the 5'-cap is a Cap 0 structure or a Cap 1 structure. In one example, the 5'-cap is a Cap 0 structure, for example, a 5'-cap (i.e., Cap 0) consists of an inverted 7-methylguanosine connected to the remainder of the mRNA via a 5'-5' triphosphate bridge. In one example, the 5'-cap is a Cap 1 structure, for example, a 5'-cap (i.e., Cap 1) consists of a Cap 0 with an additional methylation at the 2'O position of the initiating nucleotide.

[0233] In one example, mRNA is purified. Various methods for purifying mRNA will be clear to those skilled in the art. For example, mRNA is purified using lithium chloride (LiCl) precipitation. In another example, mRNA is purified using tangential flow filtration (TFF). After purification, mRNA is resuspended in, for example, nuclease-free water.

[0234] composition The present disclosure provides an immunogenic composition comprising a self-replicating RNA of the present disclosure.

[0235] The present disclosure also provides a pharmaceutical composition comprising an immunogenic composition of the present disclosure and a pharma- ceutically acceptable carrier.

[0236] It will be apparent to one of skill in the art and / or as described herein that the self-replicating RNA of the present disclosure can be present as naked RNA or in combination with lipids, polymers, or other delivery systems that facilitate entry into cells.

[0237] Delivery System In one example, the pharmaceutical composition of the present disclosure further comprises a LNP, a polymeric microparticle, and an oil-in-water emulsion, for example, the self-replicating RNA is encapsulated, bound, or adsorbed to the LNP, polymeric microparticle, or oil-in-water emulsion.

[0238] Lipid Nanoparticles In one example, the pharmaceutical composition of the present disclosure further comprises an LNP.

[0239] It will be apparent that the term "lipid nanoparticle" refers to any lipid composition, including but not limited to liposomes or vesicles, in which an aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., single, unilamellar, or multiple, multilamellar) micelle-like lipid nanoparticle having a non-aqueous core and a solid lipid nanoparticle, the solid lipid nanoparticle lacking a lipid bilayer.

[0240] Lipid nanoparticles suitable for use in the present disclosure will be apparent to those of skill in the art and / or are described herein. The lipids can have anionic, cationic, or zwitterionic hydrophilic head groups.

[0241] In one example, the lipid nanoparticles include a PEG lipid, a sterol structure lipid, and / or a neutral lipid. In one example, the lipid nanoparticles further include a cationic lipid. In one example, the lipid nanoparticles do not include a cationic lipid.

[0242] In one example, the LNP comprises a PEG-lipid, for example, the PEG-lipid is selected from the group consisting of PEG-c-DMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, and combinations thereof.

[0243] In one example, the LNP comprises a structured lipid, for example, the structured lipid is selected from the group consisting of cholesterol fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and alpha-tocopherol, and combinations thereof.

[0244] In one example, the LNP comprises a neutral lipid. Exemplary phospholipids (anionic or zwitterionic) for use in the present disclosure include, for example, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol. For example, neutral lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3 ... Choline (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin, and combinations thereof.

[0245] In one example, the LNP comprises a cationic lipid. Exemplary cationic lipids include, but are not limited to, dioleoyltrimethylammoniumpropane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 2,5-bis((9z,12z)-octadeca-9,12,dien-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750). In one example, the phospholipid is 2,5-bis((9z,12z)-octadeca-9,12,dien-1-yloxyl)benzyl-4-(dimethylamino)butonoate (LKY750). Exemplary zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids, such as dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and dodecylphosphocholine. The lipids can be saturated or unsaturated.

[0246] Polymer Microparticles In one example, the pharmaceutical composition of the present disclosure further comprises polymeric microparticles.

[0247] Those skilled in the art will recognize that various polymers can form microparticles that encapsulate or adsorb the self-replicating RNA of the present disclosure.It will be clear that the use of substantially non-toxic polymers means that the particles are safe, and the use of biodegradable polymers means that the particles can be metabolized after delivery to avoid long-term persistence.Useful polymers are also sterilizable to aid in the preparation of pharmaceutical grade formulations.

[0248] Exemplary non-toxic and biodegradable polymers include, but are not limited to, poly(α-hydroxy acids), polyhydroxybutyric acid, polylactones (including polycaprolactone), polydioxanones, polyvalerolactones, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinyl-pyrrolidinones or polyester-amides, and combinations thereof.

[0249] Cationic oil-in-water emulsion In one example, the pharmaceutical composition of the present disclosure further comprises a cationic oil-in-water emulsion.

[0250] Suitable oils for use in oil-in-water emulsions will be clear to those skilled in the art and / or described herein. For example, the emulsions include one or more oils, for example, derived from animal (e.g., fish) or vegetable sources (e.g., nuts, seeds, grains). Those skilled in the art will recognize that biocompatible and biodegradable oils are preferentially used. Exemplary animal oils (i.e., fish oils) include cod liver oil, shark liver oil, and whale oil. Exemplary vegetable oils include peanut oil, coconut oil, olive oil, soybean oil, jojoba oil, safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil, corn oil.

[0251] In addition to oil, the oil-in-water emulsion also contains cationic lipids that promote the formation and stabilization of the emulsion. Suitable cationic lipids will be clear to those skilled in the art and / or described herein. Exemplary cationic lipids include, but are not limited to, 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3'-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecyl-ammonium (DDA), 1,2-dimyristoyl-3-trimethyl-ammonium propane (DMTAP), dipalmitoyl [C16:0] trimethylammonium propane (DPTAP), and distearoyl rutyl ammonium propane (DSTAP).

[0252] In some examples, the oil-in-water emulsion also includes a nonionic surfactant and / or a zwitterionic surfactant.Those skilled in the art will recognize suitable surfactants for use in the present disclosure.Exemplary surfactants include, but are not limited to, polyoxyethylene sorbitan ester surfactants (e.g., polysorbate 20 and polysorbate 80), and copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO).

[0253] Pharmaceutically acceptable carriers Preferably, in the composition or method for administering the self-replicating RNA of the present disclosure to a subject, the self-replicating RNA is combined with a pharma- ceutically acceptable carrier, as understood in the art.Thus, one example of the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising the self-replicating RNA of the present disclosure (and any delivery system) combined with a pharma- ceutically acceptable carrier.

[0254] Generally, a "carrier" refers to a solid or liquid filler, binder, diluent, encapsulating material, emulsifier, wetting agent, solvent, suspending agent, coating, or lubricant that can be safely administered to any subject, e.g., a human. Depending on the particular route of administration, a variety of acceptable carriers known in the art can be used, e.g., as described in Remington's Pharmaceutical Sciences (Mack Publishing Co. NJUSA, 1991).

[0255] The self-replicating RNA of the present disclosure is useful for parenteral, topical, oral, or local administration, intramuscular administration, aerosol administration, or transdermal administration for prophylactic or therapeutic treatment. In one example, the self-replicating RNA is administered parenterally, such as intramuscularly, subcutaneously, or intravenously. For example, the self-replicating RNA is administered intramuscularly.

[0256] The formulation of the self-replicating RNA to be administered will vary according to the route of administration and formulation (e.g., solution, emulsion, capsule) selected. A suitable pharmaceutical composition containing the self-replicating RNA to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Those skilled in the art know a variety of suitable aqueous carriers, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution, and glycine. Intravenous vehicles can include additives, preservatives, or fluid, nutrient, or electrolyte replenishers (see generally Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The composition may optionally contain pharma- ceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, and toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The self-replicating RNA may be stored in a liquid phase, or lyophilized for storage and reconstituted in a suitable carrier prior to use according to lyophilization and reconstitution techniques known in the art.

[0257] The optimal concentration of the active ingredient in the selected medium can be determined empirically according to procedures known to those skilled in the art and will depend on the desired final pharmaceutical formulation.

[0258] When formulated, the compositions of the present disclosure will be administered in a manner compatible with the dosage formulation and in a therapeutically / prophylactically effective amount. The dosage range for administration of the molecules of the present disclosure is large enough to produce the desired effect. For example, the composition comprises an effective amount of self-replicating RNA. In one example, the composition comprises a therapeutically effective amount of self-replicating RNA. In another example, the composition comprises a prophylactically effective amount of self-replicating RNA.

[0259] The dosage should not be so large as to cause adverse side effects.Generally, the dosage varies with the age, condition, sex, and degree of disease in the patient, and can be determined by those skilled in the art.The dosage can be adjusted by the individual physician in the event of any complication.

[0260] Dosage can range from about 0.1 mg / kg to about 300 mg / kg, e.g., from about 0.2 mg / kg to about 200 mg / kg, e.g., from about 0.5 mg / kg to about 20 mg / kg, over one or more days, with one or more doses administered per day.

[0261] In some instances, the self-replicating RNA is administered at an initial (or loading) dose that is higher than the subsequent (maintenance) doses. For example, the self-replicating RNA is administered at an initial dose of about 10 mg / kg to about 30 mg / kg. The self-replicating RNA is then administered at a maintenance dose of about 0.0001 mg / kg to about 10 mg / kg. The maintenance dose can be administered every 7 to 35 days, for example, every 7 days or 14 days or 28 days.

[0262] In some examples, a dose escalation regime is used, where the self-replicating RNA is administered initially at a lower dose than that used in subsequent doses. This dosage regime is useful when the subject suffers from an adverse event early on.

[0263] In the case of subjects not responding adequately to treatment, multiple doses may be administered per week. Alternatively, or in addition, increasing doses may be administered.

[0264] The subject may be re-treated with the self-replicating RNA by giving a set of two or more exposures or doses, such as at least about two exposures of the binding protein, e.g., about 2-60 exposures, more particularly about 2-40 exposures, and most particularly about 2-20 exposures.

[0265] In one example, a subject is treated with a first dose of a self-replicating RNA on day 0, followed by a second dose of the self-replicating RNA on day 21. For example, the first and second doses are administered 21 days (or 3 weeks) apart.

[0266] In another example, a subject is treated with a first dose of a self-replicating RNA on day 0, followed by a second dose of the self-replicating RNA on day 28. For example, the first and second doses are administered 28 days (or 4 weeks) apart.

[0267] In one example, optional re-treatment may be given when signs or symptoms of the disease return.

[0268] In another example, any retreatment may be given at defined intervals. For example, subsequent exposures may be administered at various intervals, such as, for example, about 24-28 weeks or 48-56 weeks or more. For example, such exposures are administered at intervals of about 24-26 weeks, or about 38-42 weeks, or about 50-54 weeks, respectively.

[0269] In the case of subjects not responding adequately to treatment, multiple doses may be administered per week. Alternatively, or in addition, increasing doses may be administered.

[0270] In another example, for subjects experiencing adverse reactions, the initial (or loading) dose can be split over several days in a week, or over a number of consecutive days.

[0271] Administration of the self-replicating RNA according to the disclosed methods can be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to those of skill in the art. Administration of the self-replicating RNA can be essentially continuous over a preselected period of time, or can be in a series of spaced doses, for example, either during or after the onset of the condition.

[0272] Screening assays Suitable methods for selecting self-replicating RNAs of the present disclosure are available to those of skill in the art. Assays can be performed to evaluate the efficiency and effectiveness of the RNA, including, for example, serology and immune response.

[0273] antigen expression In one example, the self-replicating RNA is assessed for expression of a gene of interest.

[0274] For example, antigen expression is detected using an antibody against the gene of interest. In one example, the number of cells positive for antigen expression is measured, for example, by fluorescence-activated cell sorting (FACS). In another example, the mean fluorescence intensity (MFI) is determined, for example, using FACS. In a further example, a specific potency value or transfection success probability per unit mass of RNA is calculated.

[0275] Microneutralization assay In one example, the self-replicating RNA (naked and / or formulated) is evaluated for antibody responses. For example, the self-replicating RNA is evaluated using a microneutralization assay. Methods for performing a microneutralization assay will be apparent to one of skill in the art. In one example, the microneutralization assay is a short form assay. For one example, a viral fluorescent focus-based microneutralization assay is performed. In another example, the microneutralization assay is a long form assay.

[0276] Antigen-specific T cell response In one example, the self-replicating RNA is assessed for its ability to induce an antigen-specific T cell response. Methods for assessing the induction of an antigen-specific T cell response will be apparent to one of skill in the art and / or are described herein.

[0277] For example, antigen-specific T cell detection is carried out in spleen culture. Briefly, spleen cell culture is established in T cell medium, and the cell culture is either stimulated with antigen peptide or not stimulated. In one example, antigen-specific T cell response is determined using flow cytometry.

[0278] Neutralization assay The self-replicating RNAs of the present disclosure can be screened in vitro for their ability to bind to the SARS-CoV-2 S protein RBD and neutralize the binding of the S protein RBD to ACE2. Suitable assays will be apparent to those of skill in the art and include, for example, a Vero microneutralization assay, a sVNT assay, or a pseudovirus neutralization assay (e.g., using HEK-293T cells or HeLa-ACE2 cells).

[0279] In one example, the neutralization assay is a Vero microneutralization assay. Briefly, SARS-Cov-2 wild-type virus is passaged in Vero cells (i.e., the Vero line isolated from kidney epithelial cells extracted from African green monkeys). Two-fold serial dilutions of the test protein are incubated at 100 TCID for 1 h. 50 (i.e., median tissue culture infectious dose) of SARS-CoV-2, residual viral infectivity is assessed in Vero cells, and viral cytopathic effects are read, for example, on day 5. Neutralizing antibody titers are calculated using the Reed / Muench method as previously described (Houser et al., 2016; Subbarao et al., 2004).

[0280] In one example, the neutralization assay is a surrogate neutralization test (sVNT). Briefly, the wells of a plate are coated with hACE2 protein in a carbonate-bicarbonate coating buffer (e.g., pH 9.6). HRP-conjugated SARS-CoV-2 and HRP-conjugated SARS-CoV-RBD, pre-incubated with the test protein, are added to the hACE2 at different concentrations and incubated, for example, for 1 hour at room temperature. Unbound HRP-conjugated antigen is removed by washing. A colorimetric signal is developed in an enzymatic reaction of HRP with a chromogenic substrate, for example, 3,3',5,5'-tetramethylbenzidine (TMB). In one example, absorbance readings at 450 nm and 570 nm are obtained.

[0281] In one example, the neutralization is a pseudovirus neutralization assay. Briefly, HIV reporter virus pseudotyped with SARS-2-spike protein is produced by co-transfection of SARS-2-COV-2 spike plasmid with virus backbone plasmid (e.g., pDR-NL Δenv FLUC) into, for example, HEK-293T cells. Pseudovirus is harvested after transfection and clarified by filtration. Viral stock titer, reported as relative luciferase unit infectious dose (RLU), is calculated by limiting dilution infection in Hela-hACE2 cells, measuring luciferase activity as a readout of virus infection.

[0282] Methods of treatment or prevention The present disclosure provides methods of using the immunogenic or pharmaceutical compositions of the present disclosure as vaccines.

[0283] The present disclosure also provides a method of treating, preventing, or delaying the progression of a disease or condition in a subject, comprising administering an immunogenic or pharmaceutical composition of the present disclosure, for example, the disease or condition is selected from the group consisting of SARS-CoV-2 infection, COVID-19, ARDS, and combinations thereof.

[0284] Coronavirus disease 2019 (COVID-19) The disclosure provides, for example, methods for treating, preventing, or slowing the progression of COVID-19.

[0285] The present disclosure also provides, for example, methods of treating, preventing, or delaying the progression of a SARS-CoV-2 infection. In some examples of the present disclosure, the subject has a SARS-CoV-2 infection but does not have clinically diagnosed COVID-19.

[0286] COVID-19 is an infectious disease caused by SARS-CoV-2. Common symptoms include fever, cough, fatigue, shortness of breath, and loss of smell and taste. The majority of cases result in mild symptoms, but some progress to ARDS. The time from exposure to onset of symptoms is typically around 5 days, but can range from 2 to 14 days. Currently, there is no vaccine or specific antiviral treatment for COVID-19, and management includes symptomatic treatment, supportive care, isolation, and experimental measures.

[0287] Thus, in some examples, the subject has a SARS-CoV-2 infection. In one example, the subject has COVID-19, for example, severe COVID-19. In particular, severe COVID-19 often causes ARDS. The methods of the present disclosure can be used to treat, prevent, or delay the progression of ARDS in subjects suffering from severe COVID-19.

[0288] Acute respiratory distress syndrome (ARDS) The disclosure provides, for example, methods of treating, preventing, or slowing the progression of ARDS in a subject.

[0289] ARDS is a life-threatening condition characterized by bilateral pulmonary infiltrates, severe hypoxemia, and disruption of the alveolar-capillary barrier (i.e., pulmonary vascular leakage), leading to noncardiogenic pulmonary edema. Currently, there is no effective pharmacological therapy.

[0290] Infectious etiology is the main cause of ARDS, including influenza and coronavirus infection. Thus, in one embodiment of the present disclosure, ARDS is associated with coronavirus infection, such as SARS-COV infection. In one embodiment, ARDS is associated with SARS-CoV-2 infection.

[0291] ARDS is classified according to the Berlin definition, which includes: (1) Presentation within 1 week of clinical attack or onset of respiratory symptoms; (2) Acute hypoxemic respiratory failure as determined by a PaO2 / FiO2 ratio of 300 mmHg or less with at least 5 cm of continuous positive airway pressure (CPAP) or positive end-expiratory pressure (PEEP), where PaO2 is the partial pressure of oxygen in arterial blood and FiO2 is the fraction of inspired oxygen; (3) Bilateral shadows on lung radiographs not completely explained by exudation, clotting, or atelectasis; and (4) Edema / respiratory failure not entirely explained by cardiac failure or fluid overload.

[0292] In one example, the subject has or is suffering from ARDS (i.e., the subject meets the Berlin definition of ARDS). For example, the subject is in need of (i.e., in need of) treatment.

[0293] In one example, the subject has or suffers from symptoms associated with ARDS. Symptoms associated with ARDS and methods for identifying subjects at risk of developing ARDS will be apparent to those skilled in the art and / or described herein. For example, the subject has one or more or all of the following symptoms: a) Respiratory frequency greater than 30 breaths per minute; b) Oxygen saturation (SpO2) of 93% or less on room air 2 ), c) The ratio of arterial oxygen pressure to the fraction of inspired oxygen below 300 mmHg (PaO 2 / FiO 2 ), d) SpO2 less than 218 2 / FiO2 Ratio, and e) Radiographic pulmonary infiltrates exceeding 50% in volume.

[0294] Currently, ARDS is classified as mild, moderate, or severe, with associated increased mortality. The severity of ARDS can be classified according to the Berlin definition as follows: (i) Mild ARDS: PaO of 200-300 mmHg on at least 5 cm of CPAP or PEEP 2 / FiO 2 , (ii) Moderate ARDS: PaO of 100-200 mmHg with at least 5 cm of PEEP 2 / FiO 2 , and (iii) Severe ARDS: PaO ≤ 100 mmHg with at least 5 cm of PEEP 2 / FiO 2 .

[0295] In one example, the ARDS is mild ARDS, in another example, the ARDS is moderate ARDS, and in a further example, the ARDS is severe ARDS.

[0296] The methods of the present disclosure can be used to prevent the onset of ARDS in addition to treating existing ARDS. Thus, in one example, the subject does not have ARDS.

[0297] kit Another example of the present disclosure provides a kit containing a self-replicating RNA of the present disclosure, useful for treating or preventing or slowing the progression of a disease or disorder such as those mentioned above.

[0298] In one example, the kit includes (a) a container containing the self-replicating RNA, optionally in a delivery system and / or a pharma- ceutically acceptable carrier or diluent, and (b) a package insert with instructions for treating, preventing, or delaying the progression of a disease or disorder (e.g., COVID-19 or ARDS) in a subject.

[0299] According to this example of the disclosure, the package insert is on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The container holds or contains a composition that is effective against a disease or disorder of the disclosure and can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a self-replicating RNA. The label or package insert indicates that the composition is used to treat a suitable subject, for example, a subject suffering from or susceptible to influenza, influenza virus infection, SARS-CoV-2 infection, COVID-19, and / or ARDS, and specific guidance is provided regarding dosage and intervals of treatment, as well as any other medical medications. The kit can further include an additional container containing a pharma- ceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and / or dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0300] The present disclosure includes the following non-limiting examples. EXAMPLES

[0301] Example 1: Generation of self-replicating RNA DNA templates encoding self-replicating RNAs were produced in competent Escherichia coli cells transformed with DNA plasmids. Individual bacterial colonies were isolated and the resulting plasmid DNA was amplified in E. coli cultures. After fermentation, plasmid DNA was isolated using a Maxiprep DNA kit and linearized by restriction digestion. Restriction enzymes were then removed using phenol / chloroform extraction and ethanol precipitation.

[0302] mRNA was generated by in vitro transcription from a linearized DNA template using T7 RNA polymerase. The DNA template was then removed by DNase digestion. Enzymatic capping was performed with Cap 0 to provide functional mRNA. The resulting mRNA was purified and resuspended in nuclease-free water.

[0303] Self-replicating RNA was prepared using spike (S) and nucleocapsid (N) antigens from SARS-CoV-2 strain 2019-nCoV / USA-WA1 / 2020. The following constructs were prepared: NSP1-4.SGP.S(wt)(Co5) NSP1-4.SGP.N(wt)(Co6) ·NSP1-4.SGP.S(RRAR→QQAA)(Co16) NSP1-4.SGP.S (RRAR → QQAA and 986P / 987P) (Co17) ·NSP1-4.SGP.S(D614G)(Co48) NSP1-4.SGP.S (RRAR → QQAA and D614G) (Co49) NSP1-4.SGP.S (RRAR→QQAA and S2') (Co58) NSP1-4.SGP.S (RRAR→QQAA, S2' and D614G) (Co59)

[0304] Example 2: In vitro characterization of self-replicating RNA The self-replicating RNA produced in Example 1 was evaluated for expression of a gene of interest.

[0305] Two-fold serial dilutions of unformulated (naked) or LNP-formulated self-amplifying mRNA constructs were electroporated or transfected into a baby hamster kidney (BHK) cell line. After 17-19 hours, cells were harvested and stained for either S or N antigen expression using anti-S or anti-N antibodies. The number of cells positive for antigen expression and the mean fluorescence intensity (MFI) were measured by FACS. Data were analyzed to calculate specific potency values ​​(probability of successful transfection per mass unit of RNA).

[0306] The in vitro activity and potency of unformulated RNA and LNP was determined by FAC based on S and N expression and is shown in Table 1 below: [Table 2]

[0307] Antibody response To assess antibody responses, sera were collected at the end of the study (i.e., 42 days after the first or 21 days after the last, second vaccine dose) and tested by microneutralization assay (Table 2) and ACE2 binding (Table 3).

[0308] For all serological assays, sera were treated identically with Vibrio cholerae neuraminidase, also known as receptor-destroying enzyme (RDE) (Denka Seiken Co. Ltd., Tokyo, Japan), and diluted in PBS to a starting dilution of 1:10. Sheep serum against H5N1 virus (FDA / CBER Kensington lot nu.H5-Ag-1115) was used as a positive control serum for the 3 assays.

[0309] Microneutralization assay Viral fluorescent focus-based microneutralization (FFA MN) assays were performed using an in-house developed protocol. RDE-treated test mouse samples and positive control sera were heat-inactivated and diluted to a starting dilution of 1:40 in PBS and serially diluted 4-fold in neutralization medium (comprised of Minimum Essential Medium D-MEM (GIBCO) supplemented with 1% BSA (Rockland, BSA-30), 100 U / mL penicillin, and 100 μg / mL streptomycin (GIBCO)) using U-Bottom 96-well plates (BD Falcon). Virus was diluted to approximately 1,000-1,500 fluorescent focus-forming units (FFU) / well (20,000-30,000 FFU / mL) in neutralization medium and added at a 1:1 ratio to the diluted sera.

[0310] 37°C, 5% CO 2 After 2 h of incubation at 4 °C, plates containing MDCK 33016-PF cells (Half Area 96-well plates, Corning) were inoculated with the mixture and incubated at 37 °C, 5% CO 2 The plates were incubated overnight for 16-18 hours at 4°C for 12-24 hours. MDCK 33016-PF cells were seeded 6-8 hours earlier at 3.0E4 / well (3.0E6 / plate) in cell growth medium (composed of D-MEM supplemented with 10% HyClone fetal bovine serum-FBS (Gibco), 100 U / mL penicillin, and 100 μg / mL streptomycin). After overnight incubation and prior to immunostaining, cells were fixed with a cold mixture of acetone and methanol.

[0311] The virus was visualized using separate 1-h incubations at room temperature of a monoclonal antibody specific for the spike (S) protein and an Alexa Fluor 488 goat anti-mouse IgG (H+L) Ab (Invitrogen cat. no. A11001) diluted in PBS buffer containing 0.05% Tween-20 (Sigma) and 2% BSA (fraction V, Calbiochem, 2960, 1194C175). The S protein was quantified by a CTL Immunospot Analyzer (Cellular Technology Limited, Shaker Heights, Cleveland, OH) using a fluorescein isothiocyanate (FITC) fluorescence filter set at excitation and emission wavelengths of 482 and 536 nm. Fluorescence foci were enumerated by using the software Immunospot 7.0.12.1 Professional Analyzer DC using a custom analysis module. Data was continuously recorded by the software into an Excel data analysis spreadsheet, and the 60% focus reduction endpoint was then calculated (for each plate) from the average focus number in virus control wells, and the 60% focus reduction neutralization titer was calculated (for each sample) by linear interpolation between the wells just above and below the 60% endpoint. [Table 3]

[0312] Inhibition of ACE2 binding Inhibition of ACE2 binding was also evaluated, and the results are shown in Table 3. [Table 4]

[0313] Inhibition of ACE2 binding was also evaluated using a surrogate virus neutralization test (sVNT) that detects neutralizing antibodies without the need to use any live virus or cells. Using the viral spike (S) protein and the receptor binding domain (RBD) protein from the host cell receptor ACE2, this test is designed to closely mimic virus-host interactions by direct protein-protein interactions in ELISA plates. The highly specific interaction is then neutralized, i.e., blocked, by specific NAbs in the animal serum in the same way as in conventional VNT.

[0314] The results are shown in Table 3A. [Table 5]

[0315] S and N protein antibodies Antibodies specific for the N protein were also evaluated by ELISA. The results are shown in Table 4. Antibodies specific for the S protein were also evaluated by ELISA. The results are shown in Table 5. [Table 6] [Table 7]

[0316] Pseudovirus Neutralization In addition to the sVNT assay, a pseudovirus neutralization assay was used to evaluate neutralization capacity. The pseudovirus assay is used to demonstrate the ability of the constructs to prevent virus entry into cells. The results are shown in Table 6. [Table 8]

[0317] Cell-Mediated Immune Response The self-replicating RNAs Co5, Co6, Co16 (S(QQAA)), and Co17 (S(QQAA, PP) were evaluated for their ability to induce antigen-specific T cell responses.

[0318] Antigen-specific T cell detection was performed on spleen cultures. Briefly, spleen cells were dissociated in dissociation solution (MACS BSA stock 1:20 with autoMACS rinse solution) and enriched at 4E7 cells / ml. Briefly, spleen cell cultures were established in 96-well plates in T cell medium containing RPMI, NEAA, pen / strep, and βME and incubated at 37°C / 5% CO. 2 Cell cultures were cultured with 100-μl of CoV-2 IgG. Anti-CD28 (clone 37.51, BD Biosciences #553294) and anti-CD107a (clone #1D4B, Biolegend #121618) were added to each well. Cell cultures were either stimulated or unstimulated. To stimulate the cultures N pep mix (spanning amino acid residues 1-419 of CoV-2 full-length N protein), S pep mix 1 (spanning amino acid residues 1-643 of CoV-2 full-length S protein), S pep mix 2 (spanning amino acid residues 633-1273 of CoV-2 full-length S protein) were added with CoV-1 S peptide (CYGVSATKL) or CoV-2 S peptide (CYGVSPTKL). After 2 hours of stimulation, Golgi Plug (containing Brefeldin A, BD Biosciences #555029) was added to each well. The cells were incubated at 37° C. for a total of 6 hours, after which the cells were transferred to 4° C. and stored overnight.

[0319] Antigen-specific T cell responses were determined using flow cytometry. Briefly, Fc block mixture (clone 2.4G2, BD Biosciences #553142) was added to each well, followed by extracellular stains (Brilliant stain buffer plus (BD Biosciences #566385), ICOS BV711 (clone C398.4A, Biolegend #313548), CD44 BUV395 (clone IM7, BD Biosciences #740215), CD3 BV786 (clone 145-2C11, BD Biosciences #564379), CD4 APC-H7 (clone GK1.5, BD Biosciences #560181), CD8 AF700 (clone 53-6.7, BD Biosciences #557959), and staining buffer). Cells were stained with UltraComp eBeads (eBiosciences #01-222-42) according to the manufacturer's protocol and incubated for 30 minutes at 4° C. and protected from light. Cells were washed with staining buffer, centrifuged, resuspended in staining buffer, and data were acquired using a flow cytometer.

[0320] Antigen-specific CD4 and CD8 T cell responses were observed to both N and S constructs. CD4 T cells elicited by sa-mRNA vaccines were mostly Th0 (IL2+ and / or TNFa+, IFNg-, IL5-, IL13-) and Th1 (IFNg+, IL5-, IL13-), with little or no Th2 (IL5+ and / or IL13+, IFNg-) (Figure 1). Similar frequencies of S1 and S2-reactive CD4 T cells were found, but for CD8 T cells, S1-reactive T cells predominated over S2-reactive T cells with a broad range of cytokine phenotypes, triple-, double-, and single-cytokine producing CD8+ T cells.

[0321] IgG subclasses To characterize the type of immune response generated, i.e., Th1 vs. Th2 type responses, S-specific IgG1 and IgG2a IgG subclasses were assessed by ELISA. Little difference was observed between IgG1 and IgG2a responses (Table 7). [Table 9]

[0322] Example 5: Protective effect of immunization with self-replicating RNA To assess the protective effect of immunization, hamsters were immunized with Co16 at a dose of 3 μg RNA / hamster or 0.3 μg RNA / hamster on days 1 and 22. All animals were challenged intranasally with SARS-CoV-2 US virus 28 days after the second immunization and sacrificed 4 days later, with lungs and nasal turbinates collected for infectious virus measured in the lungs and nasal turbinates.

[0323] In hamsters, doses of 3.0 and 0.3 μg elevated neutralization titers with GMTs of 394 and 270, respectively.

[0324] To assess lung protection from viral infection, mean virus recovery from the lungs was compared for hamsters immunized with Co16 and control hamsters immunized with PBS. The virus titer from the control hamsters was 5,011,872 TCID50 / gr., whereas the mean virus recovery from vaccinated hamsters was below the quantification limit of the assay, <20 TCID50 / gr., demonstrating complete protection of the lower respiratory tract with all vaccines included in the study.

[0325] To assess protection of the upper respiratory tract, virus recovery from the nasal turbinates was measured with a mean virus recovery from control hamsters of 120,226,443 TCID50 / gr. Viral titers were 10.0 and 10.2 mg / g for hamsters immunized with Co16 at doses of 3.0 and 0.3 μg, respectively. 4 ~10 55-fold, 1,995 and 9,120 TCID50 / gr. These results demonstrated that sa-mRNA S significantly reduced the viral infection in the upper respiratory tract.

[0326] Example 6: Dual dosing of SARS-CoV-2 with self-replicating RNA SARS-CoV-2 S and N antigens do not immunologically cross-react. To evaluate antibody immune responses in preclinical animal models, female BALB / c mice were immunized with a dose of 1 μg on day 0 and a second dose on day 21. Animals were sacrificed on day 42 and serum was obtained and tested for neutralizing antibodies and antibodies that inhibit the binding of the S protein to the ACE2 receptor.

[0327] The following first and second dose combinations were evaluated: PBS-Co6(N) ·PBS-Co16(S,RRAR→QQAA) ·Co6-Co6 Co6-Co16 Co6-PBS ·Co16-Co16 Co16-Co6 Co16-PBS

[0328] S and N protein antibodies Antibodies specific for the S and N proteins were assessed by ELISA on day 42. The results are shown in Table 8. [Table 10]

[0329] Homologous prime / boost was more effective than heterologous prime / boost for anti-S responses, whereas heterologous prime / boost was more effective than homologous prime / boost for anti-N responses. In addition, boosting with S (i.e., Co16) increases anti-N responses compared to boosting with PBS.

[0330] In the absence of boosting, anti-S antibodies increased from day 21 to day 42 (data not shown).

[0331] Inhibition of ACE2 binding Inhibition of ACE2 binding was also evaluated, and the results are shown in Table 9. [Table 11]

[0332] Microneutralization assay WT virus neutralization was also assessed, and the results are shown in Table 10. [Table 12]

[0333] Cell-Mediated Immune Response Antigen-specific T cell responses were also evaluated: CD4 and CD8 T cell responses were observed after vaccination with both homologous and heterologous antigens.

[0334] Example 6: Generation of self-replicating RNA variants Self-replicating RNA was prepared using spike (S) antigens from SARS-CoV-2 variant strains, namely, UK alpha strain (B.1.1.7), South African beta strain (B.1.351). The following constructs were prepared: ·NSP1-4.SGP.S(RRAR→QQAA)(Co16) NSP1-4.SGP.S (RRAR → QQAA and D614G) (Co49) NSP1-4.SGP.S (RRAR→QQAA, S2' (R815N) and D614G) (Co59) ·NSP1-4.SGP.S(RRAR→QQAA, Δ69-70, ΔY144, N501Y, D614G)(Co77) ·NSP1-4.SGP.S(RRAR→QQAA, Δ242-244, K417N, E484K, N501Y, D614G)(Co78) ·NSP1-4.SGP.S(RRAR→QQAA, Δ69-70, Δ242-244, K417N, E484K, N501Y, D614G)(Co79) ·NSP1-4.SGP.S(RRAR→QQAA, Δ69-70, ΔY144, N501Y, A570D, D614G, P680H, T716I)(Co80) ·NSP1-4.SGP.S(RRAR→QQAA, L18F, D80A, D215G, Δ242-244, K417N, E484K, N501Y, D614G, A701V) (Co81)

[0335] The in vitro activity and potency of the LNP-formulated RNA was determined as described above and is shown in Table 11 below: [Table 13]

[0336] As shown in Table 12, all constructs have in vitro activity and potency. [Table 14]

[0337] Neutralization assay To determine the neutralizing capacity of the constructs, microneutralization assays were performed against the reference Whuan sequence as well as alpha (B.1.1.7, UK strain), beta (B.1.351, South African strain), gamma (P.1, Brazilian strain), and delta (B.1.617.2, Indian strain) variants.

[0338] As shown in FIG. 2, all constructs generated an immune response against all strains.

[0339] Inhibition of ACE2 binding Inhibition of ACE2 binding was also evaluated. The results are shown in Table 13. All constructs inhibited ACE2 binding at day 42. [Table 15]

[0340] IgG ELISA All constructs generated total Ig responses at high and low doses as shown in Figure 3. All constructs generated highly cross-reactive responses.

[0341] Cell-Mediated Immune Response The frequencies of B cells induced by the variant constructs were characterized.

[0342] As shown in Figure 4 and Table 14, all constructs generated S-specific B cells that reacted with all variant B cell receptor-specific probes. Non-specific controls (i.e., no bait and negative control HA H1) showed low levels of background binding.

[0343] Antigen-specific T cell responses were determined using flow cytometry as described above. Peptide pools (as described above) were matched to the original CoV-2 strain, not the variant strain. All constructs induced antigen-specific CD4 and CD8 T cells that reacted with the S1 and S2 epitopes (Figure 5 and Table 14). CD4 T cells were mostly Th0 (IL2+ and / or TNFa+, IFNg-, IL5-, IL13-) and Th1 (IFNg+, IL5-, IL13-), with little or no Th2 (IL5+ and / or IL13+, IFNg-). [Table 16]

Claims

1. 1. A self-replicating RNA comprising a nucleotide sequence encoding an antigen operably linked to a subgenomic promoter, wherein the antigen is derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the antigen is a mutant spike (S) protein lacking a furin cleavage site at the S2' site and comprising a D to G mutation at the residue corresponding to nucleotide 614 of SEQ ID NO:

18.

2. 1. A monocistronic self-replicating RNA comprising a nucleotide sequence encoding an antigen operably linked to a subgenomic promoter, wherein the antigen is derived from SARS-CoV-2, and the antigen is a mutant spike (S) protein lacking a furin cleavage site at the S2' site, and comprising a D to G mutation at the residue corresponding to nucleotide 614 of SEQ ID NO:

18.

3. A self-replicating RNA described in claim 1 or 2, which contains two proline residues corresponding to nucleotides 986 and 987 of sequence number 18.

4. A self-replicating RNA described in claim 1 or 2, further comprising a Kozak consensus sequence shown in sequence number 36.

5. A self-replicating RNA described in claim 1 or 2, further comprising a subgenomic (SG) promoter, the SG promoter comprising the sequence shown in sequence number 9.

6. The self-replicating RNA of claim 1 or 2, wherein the self-replicating RNA is derived from an alphavirus.

7. 7. The self-replicating RNA of claim 6, wherein the alphavirus is selected from the group consisting of Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis virus (VEE), and combinations thereof.

8. The self-replicating RNA described in claim 7, which comprises the 5'-untranslated region of Venezuelan equine encephalitis (VEE) virus.

9. An immunogenic composition comprising the self-replicating RNA of claim 1 or 2.

10. A pharmaceutical composition comprising the immunogenic composition of claim 9 and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 10, wherein the self-replicating RNA is encapsulated, bound to, or adsorbed to an LNP or polymeric microparticle.

12. 11. The pharmaceutical composition of claim 10 for use as a vaccine.

13. 11. The pharmaceutical composition of claim 10 for use in treating or preventing or delaying progression of a disease or condition selected from the group consisting of SARS-CoV-2 infection, coronavirus disease 2019 (COVID-19), acute respiratory disease syndrome (ARDS), and combinations thereof.

14. A polynucleotide encoding the self-replicating RNA of claim 1 or 2.

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