Novel RNA and DNA immunovisuation techniques for against alvavirus and other new and

Abstract

Various vaccine systems or platforms have been proposed. Since these vaccine systems or platforms are not optimal, the art requires improved systems or platforms, including efficient, safe and economical systems or platforms.

Description

[0001] By Peter Pushko (Frederick, MD, USA) and Irina Terejakova (Frederick, MD, USA), Madigan Corporation, 8420 Gas House Pike, Suite S, Frederick, Maryland, USA

[0002] Assigned to

[0003] Madigan Corporation

[0004] 8420 Gas House Pike, Suite S

[0005] Frederick,MD 21701

[0006] +1-301-378-8321 (telephone)

[0007] +1-301-378-8322 (Fax)

[0008] ppushko@medigen-usa.com

[0009] SHEPPARD MULLIN RICHTER&HAMPTON LLP

[0010] 2099 Pennsylvania Avenue, NW

[0011] Suite 100

[0012] Washington, DC 20006-6801

[0013] Agent File Number: 79NF-353168-WO

[0014] Cross-reference to prior applications

[0015] This application claims priority to U.S. Provisional Application No. 63 / 321,652, filed on March 18, 2022, which is hereby incorporated by reference in its entirety and assigned to the assignee of this application.

[0016] Government interests

[0017] This protected subject matter was made in part with U.S. government support under Grant No. R43AI152717, provided by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health. The U.S. government may have certain rights in this protected subject matter. Technical Field

[0018] iRNA and / or iDNA vaccine systems and / or platforms for stimulating an immune response against one or more pathogens, and methods of making and using the novel systems and / or platforms. Background Art

[0019] Various vaccine systems or platforms have been proposed. Because these vaccine systems or platforms are not optimal, there is a need in the art for improved systems or platforms, including effective, safe, and economical systems or platforms.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Countries with current or past localized CHIKV transmission reported by CDC are shown. Countries with only documented imported cases are not included.

[0022] Figure 2 An overview of iRNA immunization is shown. The rearranged CHIKV iRNA is placed downstream of the optimized hybrid hCMV promoter sequence. In tissues injected with iDNA, transcription from the promoter produces full-length rearranged iRNA, which is able to initiate limited replication of attenuated live virus particles, thereby inducing a specific immune response.

[0023] Figure 3 The genetic structure of the prototype 181 / 25 virus (top), the rearranged CHIKV iRNA (middle), and the iDNA plasmid encoding the rearranged iRNA are shown. The attenuating mutations are shown as asterisks and the two 26S promoters are shown as empty arrows.

[0024] Figure 4A and 4B A genetic map depicting the pMG5040 plasmid encoding the full-length rearranged RNA is shown. Figure 4A The approximate positions of the CMV promoter (solid arrow), 26S promoter (empty arrow), and attenuation mutations (asterisks) are shown. The top panel shows the wild-type CHIKV genome. The middle panel shows the CHIKV pMG5040 plasmid encoding the rearranged full-length infectious CHIKV RNA under the transcriptional control of the CMV promoter. The bottom panel shows the plasmid pMG5040 containing two attenuation mutations from the IND vaccine 181 / 25. Figure 4B A 1% agarose / TAE gel of the pMG5040 plasmid compared to a control VEEV vaccine is shown. Lane M is a 1 kB Plus DNA ladder purchased from Thermo; lane 1 is the VEE control pMG4020; and lane 2 is CHIKVpMG5040.

[0025] Figure 5The use of iRNA to induce immunity is shown. The vaccine induces innate immunity and triggers an adaptive immune response against attenuated, rearranged live CHIK ViRNA.

[0026] Figure 6 Growth curves of CHIKV vaccines in Vero cells infected with virus (dashed line) or Vero cells transfected with iDNA (solid line) are shown. Cells were infected with 181 / 25 virus or transfected by electroporation with preparations of plasmids #7 and #39 encoding 181 / 25.

[0027] Fig. 7A and 7B The preparation of V5040 CHIKV in Vero cells is shown. Fig. 7A Plaque morphology of attenuated V5040 and 181 / 25 vaccine viruses is shown. Figure 7B Shown is a comparison of the growth kinetics of V5040 in Vero cells infected with V5040 or transfected with pMG5040, with CHIKV 181 / 25 vaccine virus used as a control.

[0028] Fig. 8A and 8B The immunogenicity of V5040 virus in mice was shown. Fig. 8A Western blots using antiserum from BALB / c mice inoculated with V5040 virus are shown. Mouse serum was probed at a dilution of 1:100 and then probed with AP-conjugated goat anti-mouse IgG (H+L) secondary antibody (1:1000 dilution). Lane 1 shows SeeBlue Plus2 standard; Lane 2 shows control Vero cell lysate; Lane 3 shows blank; and Lane 4 shows lysate of Vero cells infected with CHIKV 181 / 25 virus. The predicted band of E2 is indicated. Figure 8B Vero cells and IFA using antiserum from BALB / c mice inoculated with V5040 virus are shown. Vero cells are inoculated in chamber slides and infected with V5040 with MOI 0.01. After infection for 24 hours, the infected monolayer cells are fixed with acetone and detected with mouse serum diluted in 1:25, and then detected with goat anti-mouse IgG (H+L) secondary antibody (1:25 dilution) conjugated with FITC. The infected Vero monolayer cells form the cell focus (indicated by arrows) of infection V5040 dyed green. The nucleus is dyed red using VectaShield mounting medium containing propidium iodide.

[0029] Fig. 9Neutralizing antibodies from C57BL / 6 mice inoculated with V5040 or CHIKV 181 / 25 are shown. Antiserum collected from terminal blood sampling of mice inoculated with V5040 or CHIKV 181 / 25 was tested for PRNT50 determination of its corresponding homologous virus. Circles and squares indicate antiserum of CHIKV 181 / 25 and V5040, respectively. Each circle or square represents serum from a single mouse (n=8 / group / time point). Bars and whiskers represent mean values ​​and ± their standard deviations. Ns represents not significant. ** represents P<0.0021, as determined by two-way ANOVA and Sidak multiple comparison test (Prism).

[0030] Fig.10 Table 1 describing the CHIKV 181 / 25 iDNA vaccine in BALB / c mice is shown.

[0031] Fig.11 Table 2 is shown, describing the immunogenicity of V5040 CHIKV vaccine in BALB / c and C57BL / 6 mice. PFU indicates plaque forming unit; sc indicates subcutaneous; WB indicates Western blot; IFA indicates indirect immunofluorescence assay; PRNT50 indicates plaque reduction neutralization assay; nt indicates not tested.

[0032] Fig.12 The genetic structure of the pMG4020 plasmid encoding the V4020 VEEV vaccine virus with a rearranged genome is shown, including the 5' cap, major open reading frames (nsP1-4, GP, C), 26S subgenomic promoter (empty arrow), 3'-Poly (A), and the positions of attenuating mutations indicated by asterisks. The figure is not drawn to scale.

[0033] Fig.13 The immunogenicity of pMG4020 and V4020 vaccines in rabbits is shown. Pre-bleeding serum (day 0) was used to compare the PRNT titers of serum on days 7 and 21 after vaccination with the PRNT titers before bleeding. Statistical analysis was performed using the mean logarithm of the titer (day 21) and a one-sided Student's t-test. V4020 indicates live attenuated virus; pMG4020 indicates iDNA plasmid; SC indicates subcutaneous administration using a syringe; TD indicates transdermal administration using a 3M (Kindeva) hMTS device; PRNT indicates plaque reduction neutralization test. Summary of the invention

[0034] Described herein are several iRNA and / or iDNA vaccine systems and / or platforms for eliciting an immune response against one or more pathogens, as well as methods of making and using the novel systems or platforms.

[0035] Disclosed herein is an infectious RNA (iRNA) that is capable of directly establishing a live attenuated alphavirus in vivo and directly inducing an immune response in vivo after administration of the iRNA. Exemplary embodiments of alphaviruses include CHIKV, VEEV, YFV, JEV, SARS-CoV-2, and combinations thereof. Exemplary embodiments also include newly emerged variants thereof.

[0036] The iRNA is characterized in that, when applied to a subject in need, it is attenuated and non-virulent (notvirulent). Attenuation is achieved by one or more mutations, including at least one point mutation (e.g., attenuated point mutation) and at least one rearrangement. In a preferred embodiment, the rearrangement is achieved in a laboratory, preferably by a skilled genetic engineer. Preferably, the rearrangement occurs between the capsid protein and the glycoprotein. Because the design-in rearrangement in the iRNA will not be restored during the drug production process and / or after being administered to a subject in need, the iRNA is characterized by safe and stable attenuation, and the risk of returning to a more virulent virus strain is negligible. In an exemplary embodiment, the iRNA encodes a full-length live attenuated alphavirus. In an exemplary embodiment, the iRNA achieves safe and stable attenuation without the need to delete most of the genome of the alphavirus. Most of the genome deletions are, for example, deletions of about 60bp.

[0037] In an exemplary embodiment, the further feature of iRNA is to have an ultra-low effective vaccine dose that causes a strong immune response in a subject in need. Since iRNA is infectious and can self-replicate in host cells, a strong immune response can be caused by applying nanogram-level iRNA. In an exemplary embodiment, a single iRNA can cause a strong immune response. In other words, iRNA can cause a strong immune response without the need for iDNA, a vehicle (e.g., liposome), VLP, and / or a viral vector carrying iRNA. In an exemplary embodiment, iRNA can be combined with a non-toxic pharmacological excipient and / or a non-toxic adjuvant suitable for use in a subject in need. In an exemplary embodiment, iRNA can be encapsulated in nanoparticles, such as lipid nanoparticles, including liposomes. In other exemplary embodiments, iRNA can be directly applied to a subject by the corresponding iDNA encoding the iRNA. In certain embodiments, iRNA is applied by injection in a suitable position of the subject. In other embodiments, iRNA is applied by microneedles or using a needle-free method. In certain embodiments, different iRNAs and iDNAs can be combined to create a multivalent composition capable of eliciting an immune response against one or more (if not all) different encoding viruses in a subject. In another embodiment, different iRNAs and iDNAs can be encapsulated in nanoparticles (e.g., lipid nanoparticles) to achieve the same effect.

[0038] In exemplary embodiments, the iRNA is produced using GMP practices, and in certain embodiments, the iRNA is in a pharmaceutically acceptable formulation for administration to a subject in need thereof.

[0039] Disclosed herein is a novel iRNA and methods for preparing and using the same. An infectious RNA (iRNA) molecule may include RNA encoding an attenuated virus. The attenuated virus may be a newly emerged virus. The attenuated virus may be a positive-strand RNA virus, preferably a positive-strand RNA virus selected from one or more Ribovirus, Orthornavirus, Kitrinovirus, Lenarvirus, Pisuvirus, Pisonivirus, Stelpavirus, Togavirus, and combinations thereof; more preferably, selected from the following:

[0040] one or more alphaviruses, such as Western Equine Encephalitis Virus (WEEV), Venezuelan Equine Encephalitis Virus (VEEV), Eastern Equine Encephalitis Virus (EEEV), Sindbis Virus (SINV), Semliki Forest Virus (SFV), and Chikungunya Virus (CHIKV);

[0041] one or more flaviviruses, such as hepatitis C virus, yellow fever virus (YFV), West Nile virus, dengue virus, and Japanese encephalitis virus (JEV);

[0042] One or more arenaviruses, such as Lassa virus, Guanaritovirus, Junin virus, Lujo virus, Machupo virus, Sabia virus, and Whitewater Arroyo virus;

[0043] One or more coronaviruses, such as Middle East Respiratory Syndrome (MERS) virus, Severe Acute Respiratory Syndrome (SARS) virus, and SARS-coronavirus 2 (SARS-CoV-2);

[0044] One or more Picomaviruses, such as rhinoviruses and common cold viruses;

[0045] one or more retroviruses, such as simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), simian retrovirus type D (SRV), and simian T-lymphotropic virus (STLV),

[0046] and combinations thereof.

[0047] In certain embodiments, the positive-strand RNA virus is selected from one or more CHIKV, VEEV, YFV, JEV and combinations thereof. In certain other embodiments, the positive-strand RNA virus is selected from one or more CHIKV, VEEV and combinations thereof. In certain preferred embodiments, the positive-strand RNA virus is CHIKV. In certain other preferred embodiments, the positive-strand RNA virus is VEEV. In certain preferred embodiments, the positive-strand RNA virus is selected from 181 / 25 CHIKV, V5040 CHIKV, TC85 VEEV, V4020 VEEV and combinations thereof. The attenuated virus is encoded by the sequence of any one of SEQ.1 to 4.

[0048] iRNA molecules disclosed herein can encode all gene products of attenuated viruses, preferably encode full-length RNA from attenuated viruses.iRNA molecules are preferably recombinant RNA molecules, including iRNA molecules encoding one or more attenuated mutations.One or more attenuated mutations can be point mutations, rearrangements, and / or combinations thereof.Specifically, one or more attenuated mutations do not include non-rearranged deletions exceeding 180bp, and the non-rearranged deletions are preferably no more than about 175bp, more preferably no more than about 170bp, more preferably no more than about 170bp, more preferably no more than about 165bp, more preferably no more than about 160bp, more preferably no more than about 155bp, more preferably no more than about 150bp, more preferably no more than about 125bp, more preferably no more than about 100bp, more preferably no more than about 75bp, more preferably no more than about 55bp, more preferably no more than about 50bp, more preferably no more than about 25bp, more preferably no more than about 20bp, more preferably no more than about 15bp, more preferably no more than about 10bp, more preferably no more than about 5bp, more preferably no more than about 0bp. In certain preferred embodiments, the one or more attenuating mutations do not include non-rearrangement deletions of nucleotides encoding no more than 58 amino acids, preferably no more than about 55 amino acids, more preferably no more than about 50 amino acids, more preferably no more than about 45 amino acids, more preferably no more than about 40 amino acids, more preferably no more than about 35 amino acids, more preferably no more than about 30 amino acids, more preferably no more than about 25 amino acids, more preferably no more than about 20 amino acids, more preferably no more than about 15 amino acids, more preferably no more than about 10 amino acids, more preferably no more than about 5 amino acids, and more preferably no more than about 0 amino acids.

[0049] In certain preferred embodiments, when the attenuated virus is CHIKV, the iRNA molecule encodes a full-length nsP3 gene, and / or when the attenuated virus is CHIKV, the iRNA molecule encodes a full-length 6K gene. In certain preferred embodiments, the iRNA molecule encodes two attenuating point mutations and / or rearrangements. Rearrangement may occur between the capsid gene and the glycoprotein gene. One or more point mutations may be located at the glycoprotein gene and / or at the envelope gene. One or more point mutations may be located at the E2 gene of CHIKV. Exemplary attenuating point mutations include one or both of the Thr12Ile and Gly82Arg mutations of the E2 gene of CHIKV. In other exemplary embodiments, one or more mutations are selected from SEQ.1 and / or SEQ.3.

[0050] The designed-in mutation in the iRNA is substantially stable. It has a significantly reduced reversion rate, significant resistance to reversion, and / or a combination of the two. Preferably, the mutation is highly stable, has a highly reduced reversion rate, a high resistance to reversion, and / or a combination thereof. Preferably, the mutation does not revert to a more virulent virus. Preferably, the mutation has no detectable reversion.

[0051] In certain preferred embodiments, the designed introduced mutation is substantially stable, or highly stable, or there is no detectable reversion after the iRNA is directly or indirectly administered to a subject in need. Preferably, the mutation is substantially stable, or highly stable, or there is no detectable reversion over the entire treatment process after the iRNA is directly or indirectly administered to a subject in need.

[0052] In certain preferred embodiments, the mutation does not impair replication of the attenuated virus in mammalian cells, preferably primate cells, more preferably human cells. In certain preferred embodiments, the mutation is stable.

[0053] In certain preferred embodiments, the iRNA molecule comprises one or more subgenomic promoters. One or more, preferably two or more subgenomic promoters are operably connected to the iRNA molecule. Preferably, the one or more subgenomic promoters are RNA polymerase promoters, preferably eukaryotic promoters or phage promoters. Preferably, the one or more subgenomic promoters are one or more 26S promoters. Preferably, the one or more subgenomic promoters are two 26S promoters. Preferably, each subgenomic promoter is operably connected to a gene encoded by the iRNA molecule. Preferably, the first subgenomic promoter is operably connected to a capsid gene, and the second subgenomic promoter is operably connected to a glycoprotein gene. Preferably, the glycoprotein gene is E3-E2-6K-E1. In certain preferred embodiments, the iRNA molecule has the sequence of SEQ.1 and / or SEQ.3.

[0054] Disclosed herein are infectious DNA (iDNA) molecules encoding any of the iRNA molecules disclosed herein. The iDNA is operably linked to a DNA-dependent RNA polymerase production promoter. In certain preferred embodiments, the production promoter is suitable for manufacturing the encoded iRNA molecule. Preferably, the manufactured iRNA molecule is pharmaceutically acceptable. In certain preferred embodiments, the production promoter is a bacteriophage promoter or a prokaryotic promoter. In certain embodiments, the promoter is selected from T3, T7 and 26S. In certain preferred embodiments, the production promoter is different from a subgenomic promoter. In certain preferred embodiments, the iDNA molecule is carried by a DNA plasmid. In certain preferred embodiments, the iDNA molecule is carried by a pT7 plasmid. In certain preferred embodiments, the iDNA molecule is carried by a pMG plasmid. In preferred embodiments, the DNA plasmid is a pMG4020 plasmid and / or a pMG5040 plasmid. In certain preferred embodiments, the iDNA has the sequence of SEQ.2 and / or SEQ.4.

[0055] The iDNA may also comprise a eukaryotic promoter, preferably the promoter is optimized, and preferably the promoter is a mammalian promoter, more preferably a CMV promoter, more preferably an optimized hybrid human CMV (hCMV) promoter.

[0056] In certain preferred embodiments, the iDNA molecule encodes a full-length RNA from an attenuated virus. In certain preferred embodiments, a eukaryotic promoter and / or a production promoter is located upstream of the iRNA encoded by the iDNA molecule. In certain preferred embodiments, the iDNA molecule is stable, preferably thermostable and / or genetically stable.

[0057] In certain preferred embodiments, the iDNA molecules are characterized as being sterile, and / or having about 95% of the iDNA molecules being supercoiled, and / or having an A selected from the group consisting of 260 / A 280 Ratios: about 1.6 to about 2.2, about 1.7 to about 2.1, about 1.8 to about 2.0, and about 1.9. In certain preferred embodiments, the iDNA molecules are capable of inducing an immune response, preferably, the immune response is an innate immune response, an adaptive immune response, and / or a combination thereof; more preferably, the immune response is a persistent immunity. The acquired immunity is preferably a broad cross-neutralizing immune response. In a preferred embodiment, the iDNA molecules are capable of inducing an immune response with a single dose.

[0058] In certain preferred embodiments, the iDNA molecule is pharmaceutically acceptable. The iDNA molecule can be manufactured in vitro or in vivo, or manufactured by direct synthesis. Preferably, the iDNA molecule is manufactured in vitro under GMP conditions and manufactured for vaccine purposes.

[0059] In certain preferred embodiments, the iDNA is formulated into a composition that also includes a pharmaceutically acceptable non-toxic component. The component can be saline and / or a buffer; preferably, the saline is a phosphate buffered solution. In certain embodiments, the component is a carrier, preferably a pharmaceutically acceptable carrier. Preferred compositions containing the iDNA are preferably pharmaceutically acceptable.

[0060] In certain preferred embodiments, the composition and / or iDNA molecule is suitable for direct administration to a subject in need. In certain preferred embodiments, the composition and / or iDNA molecule is capable of inducing an immune response in a subject in need through iRNA, preferably, the immune response is an innate immune response, or an adaptive immune response, and / or a combination thereof; more preferably, the immune response is a persistent immune response; more preferably, the immune response is a broad cross-neutralization immune response. In a preferred embodiment, the composition and / or iDNA molecule is capable of inducing an immune response in one or more doses, preferably in a single dose.

[0061] In certain preferred embodiments, the compositions and / or iDNA molecules are suitable for use as vaccines. In certain preferred embodiments, the iDNA molecules are suitable for use as vaccines. In certain preferred embodiments, the viruses are suitable for preventing and / or reducing and / or significantly reducing one or more symptoms of a subject in need thereof. The subject is preferably a mammal, more preferably a human being.

[0062] In certain preferred embodiments, the iDNA molecules are used to prevent, reduce or significantly reduce viral infection and / or prevent or alleviate the symptoms of viral infection; and preferably, the symptoms are selected from fever, headache, rash, nausea, myalgia, arthralgia, respiratory failure, cardiovascular disease, hepatitis, skin effects, central nervous system abnormalities and combinations thereof.

[0063] Disclosed herein is a composition comprising an iRNA molecule disclosed herein, and further comprising a pharmaceutically acceptable non-toxic component. The component may be saline and / or a buffer; preferably, the saline is a phosphate buffered solution. In certain preferred embodiments, the iRNA molecule is pharmaceutically acceptable. In certain preferred embodiments, the iRNA molecule is manufactured in vitro or in vivo or by direct synthesis; preferably, the iRNA molecule is manufactured in vitro under GMP conditions and manufactured for vaccine use.

[0064] In certain preferred embodiments, the iRNA molecules are made using the iDNA disclosed herein, and preferably, the composition is pharmaceutically acceptable. In certain preferred embodiments, the composition and / or the iRNA molecules are suitable for direct administration to a subject in need. In certain preferred embodiments, the composition and / or the iRNA molecules are capable of inducing an immune response after direct administration to a subject in need, preferably, the immune response is an innate immune response, or an adaptive immune response, and / or a combination thereof; preferably, the immune response is a persistent immunity; preferably, the immune response is a broad cross-neutralization immune response. In preferred embodiments, the composition and / or the iRNA molecules are capable of inducing an immune response with one or more doses, preferably a single dose.

[0065] In certain preferred embodiments, the composition and / or iRNA molecule is suitable for use as a vaccine. In certain preferred embodiments, the composition and / or iRNA molecule is suitable for preventing and / or reducing and / or significantly reducing one or more symptoms of a subject in need. The subject is preferably a mammal, more preferably a human. In certain preferred embodiments, the iRNA molecule is used to prevent, reduce or significantly reduce viral infection and / or prevent or alleviate the symptoms of viral infection. Preferably, the symptom is selected from fever, headache, rash, nausea, myalgia, arthralgia, respiratory failure, cardiovascular disease, hepatitis, skin effects, central nervous system abnormalities and combinations thereof.

[0066] Disclosed herein is a vehicle comprising infectious genetic material. In certain preferred embodiments, the vehicle encapsulates infectious genetic material. In certain preferred embodiments, the infectious genetic material comprises an iRNA molecule disclosed herein, an iDNA molecule disclosed herein, a composition disclosed herein and / or a combination thereof. In certain preferred embodiments, the vehicle is a nanoparticle, which comprises liposomes and lipid nanoparticles, and comprises lipid nanoparticles based on cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) lipids. In certain preferred embodiments, the lipid nanoparticle comprising infectious genetic material is formed by mixing an iRNA molecule with a PEGylated and cationic lipid.

[0067] In certain preferred embodiments, the amount of initial infectious genetic material encapsulated by the lipid nanoparticle is more than about 60%, preferably more than about 70%, preferably more than about 80%, preferably more than about 90%, preferably more than about 91%, preferably more than about 92%, preferably more than about 93%, preferably more than about 94%, preferably more than about 95%, preferably more than about 96%, preferably more than about 97%, preferably more than about 98%, preferably more than about 99%, preferably more than about 99.9%. In certain preferred embodiments, the average diameter of the lipid nanoparticle is from about 5nm to about 200nm; preferably from about 10nm to about 150nm; preferably from about 20nm to about 100nm; preferably from about 30nm to about 90nm; preferably from about 40nm to about 85nm; preferably from about 50nm to about 80nm; preferably from about 60nm to about 75nm; preferably about 75nm. In certain preferred embodiments, the lipid nanoparticle is a unilamellar vesicle. In certain preferred embodiments, the lipid nanoparticles contain an infectious genetic material in a range selected from about 1 to about 100, about 1 to about 90, about 1 to about 80, about 1 to about 70, about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 1 infectious genetic material / lipid nanoparticle.

[0068] In certain preferred embodiments, the vector is capable of delivering infectious genetic material to a subject in need thereof. In certain preferred embodiments, the vector is capable of delivering infectious genetic material to the cytoplasm of cells of a subject in need thereof. In certain preferred embodiments, the vector is capable of inducing an immune response, preferably an innate immune response, an adaptive immune response, and / or a combination thereof; preferably persistent immunity; and preferably a broad cross-neutralizing immune response.

[0069] In certain preferred embodiments, the vehicle is capable of inducing an immune response with a single dose. In certain preferred embodiments, the vehicle is suitable for vaccine use. In certain preferred embodiments, the vehicle also comprises a non-toxic excipient and / or a non-toxic adjuvant.

[0070] In certain preferred embodiments, the vector containing the infectious genetic material can be stably stored at ambient temperature and / or ultra-low temperature; more preferably, the vector containing the infectious genetic material can be stably stored without ultra-low temperature. In certain preferred embodiments, the vector containing the infectious genetic material can be stably stored for more than 12 weeks after lyophilization, such as about 12 weeks to about 52 weeks, and about 12 weeks to about 24 weeks.

[0071] Disclosed herein is a virus comprising infectious genetic material. The infectious genetic material is an iRNA molecule disclosed herein, an iDNA molecule disclosed herein, a composition disclosed herein, and / or a combination thereof. In certain embodiments, the virus is a recombinant virus, is homogenously pure, is a live attenuated virus, and / or contains stable and attenuated mutations.

[0072] In certain preferred embodiments, the virus is capable of inducing an immune response, preferably, the immune response is an innate immune response, an adaptive immune response and / or a combination thereof; more preferably, the immune response is a persistent immunity; and more preferably, the immune response is a broad cross-neutralization immune response. In certain preferred embodiments, the virus is capable of inducing an immune response with a single dose.

[0073] In certain preferred embodiments, the virus is suitable for use in a vaccine. In certain preferred embodiments, the virus is suitable for preventing and / or reducing and / or significantly reducing one or more symptoms of a subject in need thereof. The subject is preferably a mammal, more preferably a human; preferably, the virus is suitable for administration by injection, including intramuscular injection, and / or subcutaneous injection, and / or a combination thereof. In certain preferred embodiments, the vehicle is used to prevent, reduce or significantly reduce viral infection and / or prevent or alleviate the symptoms of viral infection. In certain preferred embodiments, the symptoms are selected from fever, headache, rash, nausea, myalgia, arthralgia, respiratory failure, cardiovascular disease, hepatitis, skin effects, central nervous system abnormalities, and combinations thereof.

[0074] Disclosed herein is a vaccine comprising (i) infectious genetic material and / or a vehicle and (ii) a non-toxic pharmacological excipient thereof. In certain embodiments, the pharmacological excipient may be a pharmacologically acceptable carrier. The infectious genetic material is an iRNA molecule disclosed herein, an iDNA molecule disclosed herein, a composition disclosed herein, a vehicle disclosed herein, and / or a combination thereof. In certain preferred embodiments, the infectious genetic material is homogeneously pure. In certain preferred embodiments, the infectious genetic material has a low percentage of single nucleotide polymorphisms. In certain preferred embodiments, the pharmacological excipient comprises a preservative and / or saline; preferably, the saline is a phosphate buffered solution. In certain preferred embodiments, the vaccine further comprises an adjuvant. In certain preferred embodiments, the vaccine is a multivalent vaccine.

[0075] In certain preferred embodiments, the vaccine comprises an effective amount of attenuated virus. In certain preferred embodiments, the effective dose of the vaccine is selected from the following: about 1 ng to about 500,000 ng, about 5 ng to about 250,000 ng, about 6 ng to about 100,000 ng, about 7 ng to about 50,000 ng, about 8 ng to about 10,000 ng, about 9 ng to about 5,000 ng, about 10 ng to about 1,000 ng, about 10 ng to about 500 ng, about 10 ng to about 250 ng, about 10 ng to about 100 ng, about 100 ng, about 10 ng to about 50 ng, About 50 ng to about 500 ng, about 50 ng to about 1,000 ng, about 100 ng to about 1,000 ng, about 100 ng to about 5,000 ng, about 100 ng to about 10,000 ng, about 500 ng to about 1,000 ng, about 500 ng to about 5,000 ng, about 500 ng to about 10,000 ng, about 500 ng to about 50,000 ng, about 500 ng to about 75,000 ng, and about 500 ng to about 100,000 ng of infectious genetic material.

[0076] In certain preferred embodiments, the effective dose of the vaccine is selected from the group consisting of about 1 ng to about 500,000 ng, about 5 ng to about 250,000 ng, about 6 ng to about 100,000 ng, about 7 ng to about 50,000 ng, about 8 ng to about 10,000 ng, about 9 ng to about 5,000 ng, about 10 ng to about 100,000 ng, about 10 ng to about 75,000 ng, about 10 ng to about 50,000 ng, about 10 ng to about 25,000 ng, about 10 ng to about 10,000 ng, about 10 ng to about 7,500 ng, about 10 ng to about 50,000 ng, about 10 ng to about 25,000 ng, about 10 ng to about 10,000 ng, about 10 ng to about 7,500 ng, about 10 ng to about 50,000 ng, about 10 ng to about The iRNA molecules can be from about 0 ng to about 2,500 ng, from about 10 ng to about 1,000 ng, from about 10 ng to about 500 ng, from about 10 ng to about 250 ng, from about 10 ng to about 120 ng, from about 10 ng to about 100 ng, from about 10 ng to about 50 ng, from about 10 ng to about 75 ng, from about 50 ng to about 500 ng, from about 50 ng to about 1,000 ng, from about 100 ng to about 1,000 ng, from about 100 ng to about 5,000 ng, from about 100 ng to about 10,000 ng, from about 500 ng to about 1,000 ng, from about 500 ng to about 5,000 ng, from about 500 ng to about 10,000 ng, from about 500 ng to about 50,000 ng, from about 500 ng to about 75,000 ng, and from about 500 ng to about 100,000 ng.

[0077] In certain preferred embodiments, the effective dose of the vaccine is selected from the following: about 1 ng to about 500,000 ng, about 5 ng to about 250,000 ng, about 6 ng to about 100,000 ng, about 7 ng to about 50,000 ng, about 8 ng to about 10,000 ng, about 9 ng to about 5,000 ng, about 10 ng to about 1,200 ng, about 10 ng to about 1,100 ng, about 10 ng to about 1,000 ng, about 10 ng to about 1,200 ng, about 10 ng to about 500 ng, about 10 ng to about 250 ng, about 10 ng to about 120 ng, about 10 ng to about 100 ng, about 1 The iDNA molecules include but are not limited to, for example, iDNA molecules of about 0 ng to about 50 ng, about 50 ng to about 500 ng, about 50 ng to about 1,000 ng, about 100 ng to about 1,000 ng, about 100 ng to about 1,100 ng, about 100 ng to about 1,200 ng, about 100 ng to about 5,000 ng, about 100 ng to about 10,000 ng, about 500 ng to about 1,000 ng, about 500 ng to about 5,000 ng, about 500 ng to about 10,000 ng, about 500 ng to about 50,000 ng, about 500 ng to about 75,000 ng, and about 500 ng to about 100,000 ng.

[0078] In certain preferred embodiments, the effective dose of the vaccine is selected from the group consisting of about 0.5 ng to about 100,000 ng, about 0.5 ng to about 75,000 ng, about 0.5 ng to about 50,000 ng, about 0.5 ng to about 25,000 ng, about 0.5 ng to about 10,000 ng, about 0.5 ng to about 7.500 ng, about 0.5 ng to about 5,000 ng, about 0.5 ng to about 2,500 ng, about 0.5 ng to about 1,000 ng, about 0.5 ng to about 500 ng, about 0.5 ng to about 250 ng, about 0.5 ng to about 500 ng. ng to about 120 ng, about 1 ng to about 100,000 ng, about 1 ng to about 75,000 ng, about 1 ng to about 50,000 ng, about 1 ng to about 25,000 ng, about 1 ng to about 10,000 ng, about 1 ng to about 7,500 ng, about 1 ng to about 5,000 ng, about 1 ng to about 2,500 ng, about 1 ng to about 1,000 ng, about 1 ng to about 500 ng, about 1 ng to about 250 ng, about 1 ng to about 120 ng, about 10 ng to about 120 ng, about 10 ng to about 119 ng, about 10 ng to about 199 ng. g to about 118ng, about 10 ng to about 117ng, about 10 ng to about 116ng, about 10 ng to about 115ng, about 10 ng to about 114ng, about 10 ng to about 113ng, about 10 ng to about 112ng, about 10 ng to about 111ng, about 10 ng to about 110ng, about 10 ng to about 109ng, about 10 ng to about 108ng, about 10 ng to about 107ng, about 10 ng to about 106ng, about 10 ng to about 105ng, about 10 ng to about 104ng, about 10 ng to about 103ng, about 10 The iDNA molecule comprises: 1. an attenuated live CHIKV, 2. an attenuated live CHIKV, 3. an attenuated live CHIKV, 4. an attenuated live CHIKV, 5. an attenuated live CHIKV, 6. an attenuated live CHIKV, 7. an attenuated live CHIKV, 8. an attenuated live CHIKV, 9. an attenuated live CHIKV, 10. an attenuated live CHIKV, 11. an attenuated live CHIKV, 12. an attenuated live CHIKV, 13. an attenuated live CHIKV, 14. an attenuated live CHIKV, 15. an attenuated live CHIKV, 16. an attenuated live CHIKV, 17. an attenuated live CHIKV, 18. an attenuated live CHIKV, 19. an attenuated live CHIKV, 20.

[0079] In certain preferred embodiments, the effective dose of the vaccine is selected from the group consisting of about 0.5 ng to about 100,000 ng, about 0.5 ng to about 75,000 ng, about 0.5 ng to about 50,000 ng, about 0.5 ng to about 25,000 ng, about 0.5 ng to about 10,000 ng, about 0.5 ng to about 7.500 ng, about 0.5 ng to about 5,000 ng, about 0.5 ng to about 2,500 ng, about 0.5 ng to about 1,000 ng, about 0.5 ng to about 500 ng, about 0.5 ng to about 250 ng, about 0.5 ng to about 500 ng. ng to about 120 ng, about 1 ng to about 100,000 ng, about 1 ng to about 75,000 ng, about 1 ng to about 50,000 ng, about 1 ng to about 25,000 ng, about 1 ng to about 10,000 ng, about 1 ng to about 7,500 ng, about 1 ng to about 5,000 ng, about 1 ng to about 2,500 ng, about 1 ng to about 1,000 ng, about 1 ng to about 500 ng, about 1 ng to about 250 ng, about 1 ng to about 120 ng, about 10 ng to about 120 ng, about 10 ng to about 119 ng, about 10 n g to about 118ng, about 10 ng to about 117ng, about 10 ng to about 116ng, about 10 ng to about 115ng, about 10 ng to about 114ng, about 10 ng to about 113ng, about 10 ng to about 112ng, about 10 ng to about 111ng, about 10 ng to about 110ng, about 10 ng to about 109ng, about 10 ng to about 108ng, about 10 ng to about 107ng, about 10 ng to about 106ng, about 10 ng to about 105ng, about 10 ng to about 104ng, about 10 ng to about 103ng, about 10 ng to about 102ng, about 10ng to about 101ng, about 10ng to about 100ng, about 15ng to about 120ng, about 15ng to about 119ng, about 20ng to about 118ng, about 25ng to about 117ng, about 30ng to about 116ng, about 40ng to about 115ng, about 50ng to about 114ng, about 60ng to about 113ng, about 70ng to about 112ng, about 80ng to about 111ng, about 90ng to about 110ng of an iRNA molecule, wherein the iRNA molecule encodes an attenuated live CHIKV.

[0080] In certain preferred embodiments, the effective dose of the vaccine is selected from the group consisting of about 1 ng to about 200,000 ng, about 2 ng to about 150,000 ng, about 3 ng to about 100,000 ng, about 4 ng to about 90,000 ng, about 5 ng to about 80,000 ng, about 6 ng to about 70,000 ng, about 7 ng to about 60,000 ng, about 8 ng to about 50,000 ng, about 9 ng to about 40,000 ng, about 10 ng to about 30,000 ng, about 10 ng to about 20,000 ng, about 10 ng to about 19,000 ng, about 10 ng to about 18,000 ng, about 10 ng to about 17,000 ng, about 10 ng to about 18,000 ng. to about 16,000 ng, about 10 ng to about 15,000 ng, about 10 ng to about 10,000 ng, about 10 ng to about 5,000 ng, about 10 ng to about 1,000 ng, about 10 ng to about 500 ng, about 100 ng to about 500 ng, about 100 ng to about 1,000 ng, about 500 ng to about 5,000 ng, about 500 ng to about 10,000 ng, about 500 ng to about 25,000 ng, about 500 ng to about 50,000 ng, about 500 ng to about 100,000 ng, and about 500 ng to about 150,000 ng of an iDNA molecule, wherein the iDNA molecule encodes a live, attenuated VEEV.

[0081] In certain preferred embodiments, the effective dose of the vaccine is selected from the group consisting of about 1 ng to about 200,000 ng, about 2 ng to about 150,000 ng, about 3 ng to about 100,000 ng, about 4 ng to about 90,000 ng, about 5 ng to about 80,000 ng, about 6 ng to about 70,000 ng, about 7 ng to about 60,000 ng, about 8 ng to about 50,000 ng, about 9 ng to about 40,000 ng, about 10 ng to about 30,000 ng, about 10 ng to about 20,000 ng, about 10 ng to about 19,000 ng, about 10 ng to about 18,000 ng, about 10 ng to about 17,000 ng, about 10 ng to about 18,000 ng, about 10 ng to about 19 ...8,000 ng, about 10 ng to about 18,000 ng, about 10 ng to about 18,000 ng, about 10 ng to about 18,000 ng, about 10 ng to about 18,0 About 16,000 ng, about 10 ng to about 15,000 ng, about 10 ng to about 10,000 ng, about 10 ng to about 5,000 ng, about 10 ng to about 1,000 ng, about 10 ng to about 500 ng, about 100 ng to about 500 ng, about 100 ng to about 1,000 ng, about 500 ng to about 5,000 ng, about 500 ng to about 10,000 ng, about 500 ng to about 25,000 ng, about 500 ng to about 50,000 ng, about 500 ng to about 100,000 ng, and about 500 ng to about 150,000 ng of an iRNA molecule, wherein the iRNA molecule encodes an attenuated live VEEV.

[0082] In certain preferred embodiments, the effective dose has a concentration selected from the following: about 10 3 PFU to 10 7 PFU, about 5x10 in 20ul 3 PFU to 5x10 6 PFU, about 10 in 20ul 4 PFU to 10 6 PFU, about 5x10 in 20ul 4 PFU to 5x10 5 PFU, about 6x10 in 20ul 4 PFU to 4x10 5 PFU, about 7x10 in 20ul 4 PFU to 3x10 5 PFU, about 8x10 in 20ul 4 PFU to 2x10 5 PFU, about 9x10 in 20ul 4 PFU is about 1x10 5 PFU and about 1x10 in 20ul 5 PFU of infectious genetic material.

[0083] In certain preferred embodiments, the effective dose is selected from the following: about 10 PFU to about 10,000 PFU, about 10 PFU to about 5,000 PFU, about 10 PFU to about 3,100 PFU, about 10 PFU to about 3,000 PFU, about 10 PFU to about 2,500 PFU, about 50 PFU to about 5,000 PFU, about 50 PFU to about 3,100 PFU, about 75 PFU to about 2,500 PFU, about 100 PFU to about 1,000 PFU, about 200 PFU to about 800 PFU, about 300 PFU to about 700 PFU, about 400 PFU to about 600 PFU, about 500 PFU of infectious genetic material.

[0084] In certain preferred embodiments, the effective amount of a single dose of the vaccine can provide a therapeutic benefit to a subject in need. In certain preferred embodiments, the vaccine has lower adverse reactions to subjects in need of the vaccine. In certain preferred embodiments, the infectious genetic material is used to prevent, reduce or significantly reduce viral infection and / or prevent or alleviate the symptoms of viral infection. In certain preferred embodiments, the symptoms are selected from fever, headache, rash, nausea, myalgia, arthralgia, respiratory failure, cardiovascular disease, hepatitis, skin effects, central nervous system abnormalities and combinations thereof. In certain preferred embodiments, the vaccine has lower adverse reactions to subjects in need of the vaccine. In certain preferred embodiments, the vaccine is administered by injection, including intramuscular injection and / or subcutaneous injection; by microinjection, including transdermal microinjection; and / or a combination thereof. The vaccine can also be administered by microneedle injection or needle-free administration.

[0085] Disclosed herein is a vaccine comprising an attenuated virus disclosed herein and a non-toxic pharmacological excipient thereof. In certain preferred embodiments, the pharmacological excipient comprises a preservative and / or saline; preferably, the saline is a phosphate buffered solution. In certain preferred embodiments, the vaccine further comprises an adjuvant. In certain preferred embodiments, the vaccine is a multivalent vaccine. In certain preferred embodiments, the vaccine comprises an effective amount of the attenuated virus.

[0086] In certain preferred embodiments, the effective dose has a concentration selected from the following: about 10 3 PFU to 10 7 PFU, about 5x10 in 20ul 3 PFU to 5x10 6 PFU, about 10 in 20ul 4 PFU to 10 6 PFU, about 5x10 in 20ul 4 PFU to 5x10 5 PFU, about 6x10 in 20ul 4PFU to 4x10 5 PFU, about 7x10 in 20ul 4 PFU to 3x10 5 PFU, about 8x10 in 20ul 4 PFU to 2x10 5 PFU, about 9x10 in 20ul 4 PFU to 1x10 5 PFU, about 1x10 in 20ul 5 PFU of attenuated virus.

[0087] In certain preferred embodiments, the effective dose is selected from the group consisting of about 1 PFU to about 100,000 PFU, about 2 PFU to about 100,000 PFU, about 5 PFU to about 50,000 PFU, about 10 PFU to about 10,000 PFU, about 10 PFU to about 5,000 PFU, about 10 PFU to about 3,100 PFU, about 10 PFU to about 3,000 PFU, about 10 PFU to about 2,500 PFU, about 50 PFU to about 5,000 PFU, about 60 PFU to about 2,500 PFU, about 70 PFU to about 1,500 PFU, about 75 PFU to about 1 , 300 PFU, about 80 PFU to about 1,200 PFU, about 90 PFU to about 1,100 PFU, about 100 PFU to about 1,000 PFU, about 200 PFU to about 800 PFU, about 300 PFU to about 700 PFU, about 400 PFU to about 600 PFU, about 500 PFU, about 50,000 PFU to about 500,000 PFU, about 10,000 PFU to about 100,000 PFU, about 5,000 PFU to about 50,000 PFU, about 2,500 PFU to about 25,000 PFU and about 10,000 PFU of attenuated virus.

[0088] In certain preferred embodiments, the effective dose is selected from the group consisting of about 500,000 to about 5,000,000, about 100,000 to about 1,000,000, about 50,000 to about 500,000, about 25,000 to about 250,000, and about 100,000 of attenuated virus.

[0089] In certain preferred embodiments, the effective amount of a single dose of the vaccine can provide a therapeutic benefit to a subject in need. In certain preferred embodiments, the vaccine has lower adverse reactions to subjects in need of the vaccine. In certain preferred embodiments, the infectious genetic material is used to prevent, reduce or significantly reduce viral infection and / or prevent or alleviate the symptoms of viral infection. In certain preferred embodiments, the symptoms are selected from fever, headache, rash, nausea, myalgia, arthralgia, respiratory failure, cardiovascular disease, hepatitis, skin effects, central nervous system abnormalities and / or combinations thereof. In certain preferred embodiments, the vaccine has lower adverse reactions to subjects in need of the vaccine. In certain preferred embodiments, the vaccine is administered by injection, including intramuscular injection and / or subcutaneous injection; administered by microinjection, including transdermal microinjection; and / or a combination thereof. In certain embodiments, the vaccine is administered by microneedle injection and / or needle-free administration.

[0090] Disclosed herein is a method for preventing and / or reducing and / or significantly reducing viral infection, comprising directly administering to a subject in need thereof an effective amount of an iRNA molecule disclosed herein, an iDNA molecule disclosed herein, a composition disclosed herein, a vector disclosed herein, an attenuated virus disclosed herein, a vaccine disclosed herein, and / or a combination thereof.

[0091] Disclosed herein is a method for preventing and / or reducing and / or significantly reducing viral infection, comprising directly administering to a subject in need thereof an effective amount of an iRNA molecule disclosed herein, an iDNA molecule disclosed herein, a composition disclosed herein, a vector disclosed herein, an attenuated virus disclosed herein, a vaccine disclosed herein, and / or a combination thereof.

[0092] Disclosed herein is a method for making the iDNA molecules disclosed herein, comprising cloning a stably attenuated RNA virus into a plasmid.

[0093] Disclosed herein is a method of making a composition disclosed herein, comprising mixing an iRNA disclosed herein with a pharmaceutically acceptable non-toxic component.

[0094] Disclosed herein is a method of making a vector disclosed herein, comprising mixing an iRNA disclosed herein, an iDNA molecule disclosed herein, a composition disclosed herein, and / or a combination thereof with a lipid and an emulsifier.

[0095] Disclosed herein is a method of producing the virus disclosed herein, comprising transfecting a eukaryotic cell with an iDNA molecule disclosed herein.

[0096] Disclosed herein is a method for making a vaccine disclosed herein, comprising mixing any of the iRNA molecules disclosed herein, the iDNA molecules disclosed herein, the compositions disclosed herein, the vector disclosed herein, the attenuated viruses disclosed herein, and / or a combination thereof with a non-toxic pharmacological excipient.

[0097] Disclosed herein is a method for making a multivalent vaccine, comprising mixing one or more iRNA molecules disclosed herein, iDNA molecules disclosed herein, compositions disclosed herein, vectors disclosed herein, attenuated viruses disclosed herein, and / or combinations thereof with a non-toxic pharmacological excipient; wherein the one or more attenuated live viruses encoded in the iRNA and / or iDNA are different.

[0098] The disclosed vaccine, including the V5040 vaccine with the rearranged V5040 viral genome, is a safe and effective new attenuated live vaccine for CHIKV. In certain embodiments, the use of CHIKV iDNA plasmids can be used to prepare a new attenuated live CHIKV vaccine with a rearranged RNA genome with improved safety. In certain embodiments of the CHIKV vaccine, the genomic RNA is rearranged to encode a capsid gene located downstream of the glycoprotein gene. In certain embodiments, the attenuated mutations derived from the experimental CHIKV 181 / 25 vaccine are engineered into the E2 gene of the CHIKV vaccine to improve safety. In certain embodiments, a DNA copy of the rearranged CHIKV genomic RNA with an attenuated mutation is cloned into the iDNA plasmid pMG5040 downstream of the CMV promoter. After in vitro transfection, an iDNA plasmid (such as pMG5040) can effectively create a replication-competent CHIKV virus (such as a V5040 virus) having a rearranged genome and an attenuated E2 mutation. In certain embodiments, CHIKV vaccines (eg, vaccines containing V5040 virus) demonstrate safety and immunogenicity in a murine model. Vaccination with the CHIKV vaccines disclosed herein, including subcutaneous administration of the V5040 vaccine, elicits a CHIKV-specific immune response.

[0099] Disclosed herein is an infectious DNA (iDNA) plasmid capable of creating an attenuated live CHIKV vaccine in vivo. The disclosed vaccine, including a V5040 vaccine with a rearranged V5040 viral genome, is a safe and effective new attenuated live vaccine for CHIKV. In certain embodiments, the use of the CHIKV iDNA plasmid can be used to prepare a new attenuated live CHIKV vaccine with a rearranged RNA genome with improved safety. In certain embodiments of the CHIKV vaccine, the genomic RNA is rearranged to encode a capsid gene located downstream of the glycoprotein gene. In certain embodiments, the attenuated mutation derived from the experimental CHIKV 181 / 25 vaccine is engineered into the E2 gene of the CHIKV vaccine to improve safety. In certain embodiments, a DNA copy of the rearranged CHIKV genomic RNA with an attenuated mutation is cloned into the iDNA plasmid pMG5040 downstream of the CMV promoter. After in vitro transfection, iDNA plasmids (such as pMG5040) effectively create replication-competent CHIKV viruses (such as V5040 viruses) having rearranged genomes and attenuated E2 mutations. In certain embodiments, CHIKV vaccines (such as vaccines containing V5040 viruses) have been shown to be safe and immunogenic in mouse models. Vaccination using CHIKV vaccines disclosed herein, including subcutaneous administration of V5040 vaccines, has triggered a CHIKV-specific immune response. DETAILED DESCRIPTION

[0100] Explanation and Definitions

[0101] Unless otherwise indicated, the description employs conventional methods of chemistry, biochemistry, molecular biology, immunology, and pharmacology and terms having ordinary meanings to those skilled in the art (unless otherwise defined / described herein). All publications, references, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

[0102] As used in this specification and the appended claims, the following general rules apply. The singular forms "a", "an", and "the" include plural references unless the content clearly dictates otherwise. General nomenclature rules for classifying organisms also apply. That is, the names of orders, families, genera, and species are in italics.

[0103] The following terms used herein should have specific meanings. As understood by those skilled in the art, the term "about" has its straightforward and common meaning, i.e., "approximately". The term "about" used herein means approximately, within the range of, roughly, or around. When the term "about" is used with a numerical range, it modifies the range by extending the upper and lower boundaries of the listed numerical values. In general, the terms "about" or "approximately" used herein are used to modify numerical values ​​that are 20% higher or lower than the stated value, with a variation range of. The terms "include", "comprise", "contain", "includes, but is not limited to", or "characterized by" are inclusive or open-ended, and do not exclude additional, unlisted elements.

[0104] As used herein, the following terms shall have specific meanings.

[0105] "Atenuated" means reduced virulence.

[0106] "Infectious" means capable of self-replication.

[0107] "Infectious genetic material" refers to iDNA, iRNA, and combinations thereof.

[0108] “iDNA” refers to infectious DNA. Infectious DNA encodes viral genetic material that is able to replicate itself in a host cell.

[0109] "iRNA" refers to infectious RNA. Infectious RNA encodes viral genetic material that is able to replicate itself in a host cell.

[0110] "V5040" refers to the live attenuated vaccine virus V5040 prepared by using the pMG5040 plasmid.

[0111] "pMG5040" refers to a plasmid derived from the iDNA plasmid p181 / 25. pMG5040 is based on the pUC backbone vector and encodes the full-length reassortant RNA genome of the V5040 vaccine virus.

[0112] "Rearranged" means that the order of all or part of a gene has been rearranged. Examples of rearrangements can be found in Figure 3 , Figure 4A and Fig.12 .

[0113] A "subject" is a vertebrate, such as a mammal, including but not limited to humans, primates, rodents, farm animals, sports animals (e.g., horses), and pets. In certain embodiments, the subject is a human. In other embodiments, the method can be used for experimental animals (e.g., monkeys of all species), veterinary applications, and / or the development of animal models of disease. In certain embodiments, the vaccine is a VEE (e.g., TC-83) vaccine and the subject is a horse.

[0114] "Subject in need thereof" refers to any subject, patient or individual who can benefit from the compounds, compositions or methods described herein. The subject in need of the present invention is preferably an animal, such as a mouse, ferret, chicken, pig, etc.; preferably a mammal or bird; most preferably a human.

[0115] "Therapeutically effective dose" or "pharmaceutically effective dose" or "therapeutically effective amount" or "pharmaceutically effective amount" refers to the dose or amount of the administered drug that produces the desired effect. The exact dose will depend on the purpose of the treatment and can be determined by those skilled in the art using known techniques.

[0116] The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0117] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle that is administered with the iDNA. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal origin, plant origin or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, combinations thereof, and the like. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, combinations thereof, and the like. If desired, the composition may also contain a small amount of a wetting agent or emulsifier, or a pH buffer. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations, combinations thereof, and the like. The composition may be formulated as a suppository using conventional adhesives and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and combinations thereof. Examples of suitable pharmaceutical carriers are described by EW Martin in "Remington's Pharmaceutical Sciences".

[0118] "Pharmaceutically acceptable carrier" refers to, but is not limited to, a vehicle containing infectious genetic material that can be injected into a mammalian host without producing adverse reactions. Suitable pharmaceutically acceptable carriers known in the art include, but are not limited to, gold particles, sterile water, saline, glucose, dextrose or buffered solutions, combinations thereof, and the like. The carrier may include adjuvants, including, but not limited to, diluents, stabilizers (i.e., sugars and amino acids), preservatives, wetting agents, emulsifiers, pH buffers, viscosity enhancing additives, colorants, combinations thereof, and the like.

[0119] "Long-lasting immunity" means that the immunity elicited by the molecule, composition, vector, virus and / or vaccine protects the subject from the same or substantially similar viruses during the subject's lifetime for at least about 3 months, more preferably at least about 6 months, more preferably at least about 9 months, more preferably at least about 12 months, more preferably at least about 15 months, more preferably at least about 18 months, more preferably at least about 21 months, more preferably at least about 24 months, more preferably at least about 1 year, more preferably at least about 3 years, more preferably at least about 5 years, more preferably at least about 10 years, more preferably at least about 15 years, more preferably at least about 20 years, more preferably at least about 25 years, more preferably at least about 30 years, more preferably at least about 35 years, more preferably at least about 40 years, more preferably at least about 40 years, more preferably at least about 50 years, more preferably at least about 60 years, more preferably at least about 70 years, more preferably at least about 80 years, more preferably at least about 90 years, more preferably at least about 100 years.

[0120] “CDC” means the Centers for Disease Control and Prevention.

[0121] “WHO” means the World Health Organization.

[0122] "IM" when referring to administration of a vaccine means intramuscular injection.

[0123] "pc" refers to post-challenge when referring to the period of time after a subject is challenged with the pathogen of interest.

[0124] “NHP” refers to non-human primates.

[0125] "CMV" refers to cytomegalovirus.

[0126] “CHIKV” refers to Chikungunya fever virus, a mosquito-borne alphavirus that causes Chikungunya fever.

[0127] "VEEV" stands for Venezuelan equine encephalitis virus.

[0128] "EEEV" stands for Eastern equine encephalitis virus.

[0129] "SINV" refers to Sindbis virus.

[0130] "SFV" refers to Semliki Forest Virus.

[0131] "YF" stands for yellow fever.

[0132] "YFV" refers to yellow fever virus.

[0133] "SIV" refers to simian immunodeficiency virus.

[0134] “HIV” stands for human immunodeficiency virus.

[0135] "SRV" refers to simian type D retrovirus.

[0136] "STLV" refers to simian T-lymphotropic virus.

[0137] “WEEV” stands for Western equine encephalitis virus.

[0138] “JEV” stands for Japanese encephalitis virus.

[0139] "ORF" refers to open reading frame.

[0140] "LNP" refers to lipid nanoparticle.

[0141] "IVT" refers to in vitro transcription.

[0142] "IFA" refers to immunofluorescence assay.

[0143] "PBS" refers to phosphate buffered saline.

[0144] "PRR" refers to pattern recognition receptor.

[0145] "TLR" refers to Toll-like receptor.

[0146] "RIG-I" refers to retinoic acid inducible gene I.

[0147] "RLR" refers to RIG-1-like receptor.

[0148] "NOD" refers to nucleotide-binding oligomerization domain.

[0149] "NLR" refers to the NOD-like receptor family of proteins.

[0150] "DOTAP" refers to the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane.

[0151] "IFN" refers to interferon.

[0152] "CMI" refers to cell-mediated immunity.

[0153] "sc" means subcutaneous.

[0154] "FITC" refers to fluorescein isothiocyanate.

[0155] "PRNT" refers to plaque reduction neutralization assay.

[0156] “PRNT50 ” refers to the 50% plaque reduction neutralization assay. This is the serum concentration required to reduce the number of plaques by 50% compared to the virus in the absence of serum.

[0157] "hCMV" refers to the human CMV promoter.

[0158] "Live attenuated" means that the virus is able to reproduce in host cells and the virulence of the virus is reduced.

[0159] "Vaccine" refers to a composition that reduces, prevents, significantly reduces and / or significantly inhibits viral infection, and / or reduces and / or prevents symptoms caused by viruses. Vaccines achieve this goal by enhancing the resistance of the subject's immune system to the virus. Examples of vaccines include DNA, RNA, liposomes, viruses (replicating or non-replicating), replicons, virus-like particles, proteins, and portions of proteins. A multivalent vaccine is a vaccine that can prevent, reduce, significantly reduce and / or significantly inhibit infection by more than one different type of virus, and / or can reduce and / or prevent symptoms caused by more than one different type of virus.

[0160] "GP" refers to glycoprotein.

[0161] "nsp1-4" refers to nonstructural proteins 1-4.

[0162] "181 / 25" refers to the virus or genetic material encoding the attenuated vaccine candidate CHIK 181 / 25 (TSI-GSD-218), which is produced by the CHIKV 15561 strain and is at risk of reverting to a virulent form. The attenuation is caused by two point mutations.

[0163] "C" refers to the capsid protein.

[0164] "mcg" means micrograms.

[0165] "PFU" refers to plaque forming units.

[0166] iRNA vaccines against positive-strand RNA viruses

[0167] Disclosed herein is a novel iRNA platform / system for vaccine use against positive-strand RNA viruses. In certain embodiments, an iRNA vaccine may be a self-replicating RNA encoding all proteins of a virus. In certain embodiments, an iRNA vaccine may encode an attenuated virus. In certain embodiments, an iRNA vaccine comprises a rearranged gene, which may include an attenuated mutation resistant to a reverse mutation. In some embodiments, an iRNA vaccine may be rapidly synthesized from a known pathogen sequence without obtaining or manipulating a pathogenic virus to attenuate it using a classical method. In some embodiments, the vaccine may provide protective immunity against one or more positive-strand RNA viruses in a single dose. In some embodiments, an iRNA vaccine is delivered by a DNA plasmid. In some embodiments, an iRNA vaccine is delivered by nanoparticles (e.g., lipid nanoparticles). In some embodiments, an iRNA vaccine is suitable for viral outbreaks and / or epidemics.

[0168] In some embodiments, iRNA vaccines can be used for positive-strand RNA viruses in the realm Riboviria, including emerging viruses. In some embodiments, iRNA vaccines can be used for positive-strand RNA viruses in the kingdom Orthornavirae. In some embodiments, iRNA vaccines can be used for positive-strand RNA viruses in the phylum Kitrinoviricota, the phylum Lenarviricota, and / or the phylum Pisuviricota. In some embodiments, iRNA vaccines can be used for positive-strand RNA viruses in the class Pisoniviricetes and / or the class Stelpavirictes.

[0169] In some embodiments, iRNA vaccines can be used for positive-strand RNA viruses in the Togaviridae family.

[0170] In some embodiments, iRNA vaccines can be used for positive-strand RNA viruses in the genus Alphavirus, such as Western Equine Encephalitis Virus (WEEV), Venezuelan Equine Encephalitis Virus (VEEV), Eastern Equine Encephalitis Virus (EEEV), Sindbis Virus (SINV), Semliki Forest Virus (SFV), and Chikungunya Virus (CHIKV).

[0171] In some embodiments, iRNA vaccines can be used for positive-strand RNA viruses in the Flaviviridae family, such as hepatitis C virus.

[0172] In some embodiments, iRNA vaccines can be used for positive-strand RNA viruses in the Flavivirus genus, such as Yellow Fever (YF) virus, West Nile virus, Dengue virus, and Japanese encephalitis virus (JEV).

[0173] In some embodiments, iRNA vaccines can be used for positive-strand RNA viruses in the Arenaviridae family, such as Lassa virus, Guanarito virus, Junin virus, Lujo virus, Machupo virus, Sabia virus, and Whitewater Arroyo virus.

[0174] In some embodiments, iRNA vaccines can be used for positive-strand RNA viruses in the Coronaviridae family, such as MERS, SARS, and SARS-CoV-2.

[0175] In some embodiments, iRNA vaccines can be used for positive-strand RNA viruses in the Picornaviridae family, such as rhinoviruses (ie, common cold viruses).

[0176] In some embodiments, iRNA vaccines can be used for positive-strand RNA viruses in the Retroviridae family, such as simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), simian type D retrovirus (SRV), and / or simian T-lymphotropic virus (STLV).

[0177] In certain embodiments, the platform combines the advantages of the mRNA platform / system with the advantages of attenuated live virus vaccines. For example, the platform can enable in vitro production of vaccines, achieve high efficiency, single-dose immunization, and reduce vaccine costs.

[0178] Effective vaccination using gene (DNA and mRNA) vaccines remains one of the most important goals of current vaccinology. Although they have been clinically proven to be safe and effective, typical DNA and mRNA vaccines sometimes still require large doses, multiple booster vaccinations, advanced adjuvants, complex delivery formulations and / or specialized equipment, such as in vivo electroporation.

[0179] From the perspective of vaccine antigens, both DNA and mRNA vaccines express subunit antigens, which are weak immunogens in most cases, with low immunogenicity and require adjuvants to improve immunogenicity and efficacy. In epidemic / pandemic scenarios, multiple booster doses of DNA or mRNA vaccines will increase response time and lead to higher morbidity and mortality. In addition, for mRNA vaccines, ultra-low cold chain complicates their use, especially in countries with limited ultra-low cold chain ultrastructure.

[0180] The present application describes a self-replicating "infectious" RNA (iRNA) platform / system based on iRNA encoding a complete attenuated live virus. The vaccine is suitable for positive-sense RNA viruses, including but not limited to: alphaviruses, such as Western Equine Encephalitis Virus (WEEV), Venezuelan Equine Encephalitis Virus (VEEV), Eastern Equine Encephalitis Virus (EEEV), Sindbis Virus (SINV), Semliki Forest Virus (SFV) and Chikungunya Virus (CHIKV); Flaviviruses, such as Yellow Fever Virus, West Nile Virus, Dengue Virus, Japanese Encephalitis Virus and Hepatitis C Virus; Arenaviruses, such as Lassa Virus, Guanarito Virus, Junin Virus, Lujo Virus, Machupo Virus, Sabia Virus, Whitewater Arroyo Virus and combinations thereof, etc.

[0181] In some embodiments, attenuated mutations derived from known attenuated live viruses and attenuation strategies that prevent, reduce, significantly reduce and / or significantly inhibit the production of pathogenic viruses are introduced to improve vaccine safety. In certain embodiments, the system / platform includes genetic rearrangements of viral genes within iRNA, which prevent, reduce, significantly reduce or significantly inhibit the return to the wild-type genome of pathogenic viruses. In certain embodiments, by placing the full-length iRNA under the transcriptional control of a mammalian promoter, the iRNA molecule is encoded in a DNA plasmid called iDNA to improve the thermal stability and genetic stability of the iRNA. When such iDNA enters the cell, it synthesizes iRNA, thereby initiating the replication of the attenuated live vaccine virus. Attenuated live vaccines account for about half of all approved vaccines, and the FDA has recently approved several live vaccines, including vaccines for influenza, herpes zoster and rotavirus, confirming the safety of the platform.

[0182] Live CHIKV vaccines can effectively control viral outbreaks and save human lives. Live attenuated vaccines account for approximately half of all vaccines approved in the United States. Live vaccines Zostavax, RotaTeq (Merck), FluMist (AstraZeneca), and Rotarix (GSK) have all been recently approved, demonstrating that live attenuated vaccine platforms can be configured to meet stringent FDA safety standards.

[0183] In some embodiments, the novel iRNA system / platform is based on the full-length, engineered genomic iRNA of one or more positive-strand RNA viruses. The iRNA encodes all proteins required for one or more viral replications. In order to improve safety, the iRNA of one or more positive-strand RNA viruses can be engineered with rearranged genes, which represent attenuated mutations resistant to reverse mutations. In certain embodiments, the iRNA is expressed in cells, initiates the replication of attenuated live viruses, and represents an effective emergency vaccine for the outbreak and prevalence of positive-strand RNA viruses. In some embodiments, the iRNA vaccine can provide protective immunity by a single dose inoculation. In some embodiments, the iRNA vaccine has the potential advantages of synthetic manufacturing, high purity, genetic stability, simple production, single dose inoculation, and / or lasting immunity.

[0184] iRNA Chikungunya Vaccine

[0185] In some embodiments, the iRNA vaccine comprises a self-replicating RNA encoding all proteins of a live attenuated CHIKV virus.

[0186] Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes chikungunya fever. CHIKV is a priority pathogen for CEPI and WHO. CHIKV causes widespread human infections and epidemics in Asia, Africa, and more recently in the Americas. CHIKV outbreaks are also spreading and continue to spread around the world ( Figure 1 ). A vaccine is needed to control outbreaks and prevent epidemics of CHIKV fever, but to date there is no licensed CHIKV vaccine.

[0187] CHIKV is primarily transmitted to humans by Aedes aegypti. In addition, a new mosquito vector, Aedes albopictus, was identified during an epidemic in 2005–2006 in the Indian Ocean Reunion Islands that affected more than one-third of the island's population and caused 284 deaths. CHIKV outbreaks include an outbreak in India from 2005 to 2006, with an estimated 1.3 million infections, and outbreaks in the Americas since 2014, with more than 150,000 confirmed cases of CHIKV infection in the Caribbean alone. CHIKV is also widely spread in Africa and Southeast Asia. With increased global travel, the risk of CHIKV spreading rapidly to non-endemic areas also increases. Some travelers are viremic, and Aedes albopictus is common in urban areas in the United States, raising concerns about an immunologically naive population. Local transmission cases have been reported in both Europe and the United States. Climate change also favors the geographic expansion of CHIKV. Given the current large outbreaks and global distribution of Aedes aegypti and Aedes albopictus, CHIKV has become a global public health threat.

[0188] CHIKV can cause fever, headache, rash, nausea, myalgia, and arthralgia. Complications include respiratory failure, cardiovascular disease, hepatitis, skin effects, and central nervous system problems. More than 50% of patients with severe infection are over 65 years old, and more than 33% of these patients die. Current treatment for CHIKV is only for symptoms of the disease and may include anti-inflammatory drugs, fluids, and bed rest. Antiviral drugs and drugs that limit the spread of the virus between cells may be useful but are not yet approved by the FDA. Joint pain associated with fever may persist for months or years and, in some patients, develops into arthritis.

[0189] A CHIKV vaccine is needed to prevent CHIKV transmission. However, there is currently no CHIKV vaccine or specific antiviral therapy licensed in the United States.

[0190] For this situation, CHIKV iRNA-based vaccines have many advantages. To meet the need for safe and effective vaccines, we describe an iDNA and / or iRNA vaccine system to prevent CHIKV and related diseases.

[0191] GMP batches of novel iRNA CHIKV, Yellow Fever, and Japanese Encephalitis vaccines have shown favorable toxicity and immunogenicity profiles in preclinical studies in relevant animal models, including non-human primates (NHPs). The iRNA platform is applicable to different types of potentially epidemic or pandemic RNA viruses, including emerging viruses.

[0192] iRNA vaccines delivered via DNA plasmids can be safe, immunogenic, and effective against positive-strand RNA viral infections of at least alphaviruses, flaviviruses, and arenaviruses. This DNA plasmid is sometimes referred to as iDNA because it is designed to express iRNA. In the iDNA plasmid, the iRNA is encoded downstream of the RNA polymerase promoter, which drives the synthesis of the iRNA in the cell.

[0193] This article describes a new type of plasmid DNA that expresses the full-length genomic RNA of attenuated CHIKV from a eukaryotic promoter, a strategy called iDNA. The iDNA plasmid can be transfected in cell culture to make an attenuated live virus vaccine, or can be directly administered to patient tissues to produce attenuated live viruses in vivo. In the latter case, the iDNA plasmid is absorbed by a limited number of cells, and the CHIKV genomic RNA is transcribed in these cells. Then, CHIKV proteins are synthesized, the genomic RNA is packaged into viral particles, and the attenuated live CHIKV is assembled and secreted from the cells. In immunocompetent BALB / c mice, the prototype 181 / 25iDNA plasmid vaccine showed safety and protective efficacy.

[0194] We also describe the use of CHIKV iDNA plasmids as a reverse genetics system to engineer novel CHIKV vaccines. To improve the safety of live attenuated CHIKV vaccines, V5040 CHIKV encoding a rearranged RNA genome was prepared. Gene rearrangements in many viruses have an attenuating effect and are highly resistant to reversion. However, it is impossible to predict whether rearrangements will have a negative impact on viral replication. Therefore, until now, it was unclear whether attenuated live CHIKV iDNA, iRNA, or viral vaccines could be genetically rearranged without compromising the infectivity and replication of CHIKV. Here, we show for the first time that infectious genetic material (e.g., iRNA and iDNA) encoding live attenuated CHIKV can be made and can replicate efficiently in mammalian cells.

[0195] We placed the CHIKV capsid gene downstream of the glycoprotein gene, which is controlled by a duplicate subgenomic 26S promoter. In addition, the E2 glycoprotein region also contains two attenuated mutations derived from the prototype 181 / 25 vaccine. We found that after the pMG5040 plasmid was transfected into Vero cells, the live virus with the rearranged genome could replicate. Therefore, the rearranged CHIKV iDNA pMG5040 can establish attenuated live CHIKV V5040 in Vero cell culture. In addition, inoculation of mice with V5040 confirmed its safety and triggered CH1KV-specific serum antibodies. Therefore, we have developed a novel V5040 vaccine suitable for use as a CHIKV vaccine.

[0196] Gene rearrangements can potentially be used to engineer safer live attenuated vaccines. Gene rearrangements are expected to be highly resistant to reversion because reversion requires multiple independent mutations. Here, we exploited gene rearrangements to develop a novel live attenuated CHIKV vaccine based on the investigational 181 / 25 live attenuated CHIKV vaccine. In earlier studies, the experimental CHIKV vaccine clone 181 / 25 was reported to be highly immunogenic in a phase II clinical trial; however, mild transient arthralgia was observed in some patients. The vaccine was evaluated in 59 healthy volunteers with a seroconversion rate of 98%. However, mild transient arthralgia occurred in up to 8% of subjects. Reversion mutations were detected in viremic patients vaccinated with the 181 / 25 IND vaccine, suggesting a need for improved safety. Rearrangements may also represent additional attenuating mutations.

[0197] We also describe an iRNA vaccine against a positive-strand RNA virus, wherein the iRNA vaccine is delivered to a subject via lipid nanoparticles (LNP), iDNA plasmid in vivo delivery systems, and combinations thereof.

[0198] Vaccines based on Chikungunya VLPs and live attenuated measles vectored vaccines have been qualified for Phase II clinical trials. Live attenuated vaccines prepared by using reverse genetics have been described, and the 181 / 25 Chikungunya vaccine has been tested in Phase I / II clinical trials. Recently, Valneva tested a live attenuated Chikungunya vaccine in Phase III clinical trials. A promising treatment approach using DNA to create monoclonal antibodies capable of neutralizing the virus has also been reported. However, no CHIKV vaccine has been approved to date. Although an investigational live attenuated CHIKV vaccine (clone 181 / 25) developed in the United States was found to be highly immunogenic in Phase II clinical trials, mild transient arthralgia was observed in some patients, indicating a need for improved safety. To meet the need for safe and effective vaccines, a novel iRNA platform / system based on full-length engineered genomic iRNAs of live attenuated CHIKV is described herein. This iRNA encodes all proteins required for viral replication. In order to improve safety, CHIKV iRNA has been engineered with rearranged genes, which represent attenuated mutations that can resist reversion mutations. In certain embodiments, iRNA is expressed in cells, starts the replication of attenuated live CHIKV, and represents an effective emergency vaccine for CHIKV outbreaks and epidemics due to providing immunity by single-dose inoculation. Previous candidate vaccines also have high manufacturing costs, safety and immunogenicity problems. CHIKV iRNA vaccine described herein has the potential advantages of synthetic production and high purity, genetic stability, production simplicity, single-dose inoculation and lasting immunity.

[0199] Early attempts to use attenuated CHIKV produced by several point mutations as a vaccine were stopped due to severe side effects (most likely due to attenuation reversion). Since then, others have used reverse genetics to delete most of the CHIKV genome to reduce the possibility of attenuated viruses reverting to more pathogenic viruses. To achieve this goal, 183 nucleotides in the CHIKV genome located in the nsP3 gene and the 6K gene were deleted. However, deleting most of the nucleotides encoding 60 amino acids is not ideal. For example, the nsP3 protein plays an important role in the alphavirus replicase. For another example, most of the deletions in the C-terminal hypervariable region of nsP3 affect the interaction with several host proteins. Therefore, it is necessary to carry out stable attenuated mutations in CHIKV that are resistant to reversion and can confer the full range of CHIKV immunogens to maximize the immunogenic characteristics and protect against pathogenic CHIKV.

[0200] Several other experimental CHIKV vaccines have been described. These include live chimeric alphaviruses carrying CHIKV structural proteins, and formalin-inactivated vaccines adjuvanted with aluminum hydroxide. In addition, a virus-like particle (VLP) CHIKV vaccine protects non-human primates against CHIKV challenge and is currently in clinical trials. DNA-based vaccines are a promising vaccination strategy. However, many experimental vaccines require two or more vaccinations to elicit a protective immune response, which may be a disadvantage when rapid control of viral outbreaks is required. A single-dose CHIKV vaccine that provides long-term protection could provide enormous benefits to global health. A measles virus-vectored CHIKV vaccine is currently in clinical trials. In addition, the successful completion of a batch-to-batch Phase 3 clinical trial of a single-dose Chikungunya vaccine candidate, CHIKVΔ5nsP3 (VLA1553), was announced.

[0201] The applicant has discovered a rearranged iDNA-derived V5040 vaccine designed to improve safety and induce effective immunity after a single injection. The live attenuated vaccine has a good cost-effectiveness ratio and innovative safety characteristics. It can be used to vaccinate individuals at risk of CHIKV infection and can also be quickly deployed during a virus outbreak to establish immunity against CHIKV in endemic and non-endemic areas.

[0202] Applicants have discovered iRNA and iDNA CHIKV vaccines based on V5040 and pMG5040. In proof-of-concept experiments, we showed that vaccination of BALB / C mice with 181 / 25 protected them from challenge with CHIKV. However, pMG5040 has not been tested for vaccination in iDNA form. Advanced safety, immunogenicity, and efficacy studies in non-human primates can be used for preclinical evaluation of CHIKV vaccines. Non-human primates have been successfully used to evaluate the safety and immunogenicity of live CHIKV and other vaccines. Another future project is to confirm that no reverse mutations occur in vivo. To address this issue, in vivo genetic stability studies (multiple passages in tissues and adult mouse brains) can be performed to provide additional safety information as described previously.

[0203] The iRNA platform is highly innovative. iRNA can be delivered via lipid nanoparticles (LNP) or DNA plasmids ( Figure 2). The proposed iRNA method represents a hybrid of live attenuated vaccines, mRNA and DNA vaccines, whereby vaccine plasmids encoding iRNA are administered in vivo to initiate the synthesis of live attenuated viruses. We have previously demonstrated the feasibility of preparing iRNA live virus vaccines created by iDNA for positive RNA viruses and delivering them in vivo for safe and effective vaccination. Here, we describe a vaccine system / platform for emerging viruses with epidemic and pandemic potential. In some embodiments, the vaccine system / platform is an advanced synthetic iRNA technology. In certain embodiments, iRNA or plasmids encoding iRNA viral vaccines can be synthesized rapidly and synthetically from known pathogen sequences without obtaining or manipulating pathogenic viruses to attenuate them using classical methods. Therefore, iRNA vaccines as described herein can have a transformative impact on the field of vaccinology. Our platform also provides a powerful tool to elucidate the contribution of specific mutations or genetic features to safety and immunogenicity. In particular, plasmids can be mixed to defend against multiple pathogens. The iRNA platform / system described herein can help improve the effective and rapid response to future epidemics by providing immune protection through a single dose of immunization.

[0204] The applicant has demonstrated the feasibility of iDNA method to prepare CHIKV attenuated live vaccine with rearranged genome. The applicant believes that iDNA vaccine can be directly used for vaccination by injection into the muscle of vaccine recipients. This iDNA vaccine can combine the advantages of traditional DNA immunization, but it uses a small amount of DNA to establish an effective attenuated live vaccine. iDNA converts a small amount of cells in muscle into a cell-scale vaccine "factory". iDNA uses mature plasmid bacterial production and manufacturing technology, and plasmids are easier to store, control and operate than live virus stocks. Since iDNA plasmids represent genetically defined homogeneous clones, the viruses produced after vaccination will undergo the least replication cycle, so the possibility of reverse mutation is reduced compared with traditional manufacturing. Due to the immunostimulatory effect of DNA vaccines, iDNA vaccination can also bring additional advantages in immunogenicity. In addition to improved safety and immunogenicity, CHIKV iDNA vaccines also have potential advantages such as high purity, genetic stability, simple production, no need for cold chain, single-dose vaccination and lasting immunity. Therefore, iDNA can combine the advantages of DNA immunization and the high efficiency of attenuated live vaccines.

[0205] iRNA VEEV vaccine

[0206] VEEV is a mosquito-borne alphavirus belonging to the family Togaviridae. VEEV causes human disease outbreaks and equine epidemics, primarily in South, Central, and North America. Climate and ecological changes and international travel increase the risk of VEEV re-emergence. In addition, VEEV is a potential bioterrorism threat. Currently, there are no approved vaccines for human use against VEEV, and the potential risk of VEEV outbreaks necessitates the development of safe and effective vaccines.

[0207] VEEV vaccines have also been improved. As with CHIKV and other alphaviruses, due to the high mutation rates of RNA viruses, there is concern that live vaccines are composed of heterogeneous populations and may be at risk for acquiring reversion mutations during multiple passages during vaccine production, resulting in the regeneration of more virulent viruses. For example, the U.S. Food and Drug Administration (FDA) did not approve TC83, a live attenuated VEEV vaccine developed in the 1960s, due to safety concerns. The TC83 vaccine contains two attenuating mutations, 5'A3 and E2-Arg120. Genetic reversion is associated with the risk of adverse reactions. Intrinsically stable iDNA plasmids represent genetically defined vaccines that can offer many safety advantages compared to standard viral vaccines that represent viral populations.

[0208] The novel V4020 VEEV iDNA vaccine was engineered with improved safety, including additional attenuation strategies to prevent reversion. The V4020 vaccine was prepared using an iDNA infectious clone encoding a full-length rearranged genomic RNA downstream of an optimized CMV promoter. The recombinant plasmid pMG4020 encoding the V4020 vaccine virus genomic RNA has been demonstrated to establish replication of the live attenuated V4020 vaccine virus in vitro and in vivo. The safety, immunogenicity, and protection of the V4020 virus against VEEV challenge were demonstrated in BALB / c mice and non-human primates. Small doses of iDNA can be used to create live attenuated virus. However, to create V4020 virus in vivo, iDNA was previously delivered using transfection reagents or by electroporation, similar to traditional DNA vaccines.

[0209] In certain embodiments, iDNA vaccines encoding attenuated live VEEV can be effectively delivered in vivo using a single dose inoculation of naked iDNA plasmids using a microneedle device. For example, the pMG4020 plasmid can encode a live attenuated vaccine V4020 for VEEV. The V4020 virus may contain structural gene rearrangements, as well as attenuated mutations that are genetically engineered to prevent reversion mutations. pMG4020 can be administered in vivo to, for example, rabbits, etc. by injection. The injection can include the use of a hollow microstructured transdermal system (hMTS) microneedle device. In certain other embodiments, the MicronJet600 or hMTS platform can be used to deliver iDNA. In other embodiments, iDNA can be delivered using a needle-free delivery system. No adverse events of vaccination were observed after administration. In addition, animals receiving the pMG4020 plasmid have successfully achieved seroconversion and have higher plaque reduction neutralization test (PRNT) antibody titers, which are similar to the antibody titers observed in animals receiving the V4020 virus instead of the pMG4020 iDNA plasmid. This supports that naked iDNA vaccines can be successfully delivered in vivo using a microneedle device and a single-dose vaccination.

[0210] In certain embodiments, vaccines based on iRNA can be used as a single-dose attenuated live vaccine for VEEV virus. The advantage is a single-dose vaccine, which can produce effective protection after a single dose, a fully synthetic vaccine, and low manufacturing, storage and transportation costs. In certain embodiments, vaccines based on iRNA can be used to fight VEEV virus and another positive-strand RNA virus with a single dose. In certain embodiments, vaccines based on iRNA can be delivered by DNA plasmids and / or lipid carriers (e.g., lipid nanoparticles).

[0211] iRNA YFV vaccine

[0212] YFV vaccine has also been improved. In certain embodiments, vaccines based on iRNA can be used as single-dose attenuated live vaccines for YF virus. The advantage is a single-dose vaccine, which can produce effective protection after a single dose, a fully synthetic vaccine, and low manufacturing, storage and transportation costs. In certain embodiments, vaccines based on iRNA can be used to combat YF virus and another positive-strand RNA virus with a single dose. In certain embodiments, vaccines based on iRNA can be delivered by DNA plasmids and / or lipid carriers (e.g., lipid nanoparticles).

[0213] iRNA JEV vaccine

[0214] JEV vaccine has also been improved. In certain embodiments, vaccines based on iRNA can be used as single-dose attenuated live vaccines for JEV. Its advantages are single-dose vaccines, effective protection can be produced after a single dose, fully synthetic vaccines, and low manufacturing, storage and transportation costs. In certain embodiments, vaccines based on iRNA can be used to combat JEV virus and another positive-strand RNA virus with a single dose. In certain embodiments, vaccines based on iRNA can be delivered by DNA plasmids and / or lipid carriers (e.g., lipid nanoparticles).

[0215] iRNA SARS-CoV-2 Vaccine

[0216] SARS-CoV-2 vaccines have also been improved. In certain embodiments, iRNA-based vaccines can be used as a single-dose live attenuated vaccine against the SARS-CoV-2 virus that causes COVID-19 infection. The advantages are a single-dose vaccine that produces effective protection after a single dose, a fully synthetic vaccine, and low manufacturing, storage, and transportation costs. In certain embodiments, iRNA-based vaccines can be used to fight the SARS-CoV-2 virus and another positive-strand RNA virus with a single dose. In certain embodiments, iRNA-based vaccines can be delivered via DNA plasmids and / or lipid carriers (e.g., lipid nanoparticles).

[0217] iRNA multivalent vaccine

[0218] Multivalent vaccines have also been improved. In certain embodiments, vaccines based on iRNA can be used as a mixture of single-dose attenuated live vaccines against multiple viruses. The advantages are single-dose vaccines, effective protection can be produced after a single dose, fully synthetic vaccines, and low manufacturing, storage and transportation costs. In certain embodiments, vaccines based on iRNA can be used to fight VEEV virus and another positive-strand RNA virus with a single dose. In certain embodiments, multivalent vaccines based on iRNA can be delivered via DNA plasmids and / or lipid carriers (e.g., lipid nanoparticles).

[0219] Example

[0220] Example 1

[0221] Production and immunogenicity of CHIKV iRNA plasmid vaccine

[0222] We prepared a 181 / 25 CHIKV iRNA vaccine prototype ( Figure 3 ). Due to its clinical history, the 181 / 25 vaccine is a good starting point for CHIKV vaccine development. This plasmid established CHIKV iRNA and vaccine virus in vitro ( Figure 3 and Fig. 8A). We developed an iDNA approach as a new infectious cloning plasmid in which the full-length viral RNA genome is transcribed from the CMV promoter. By using the iDNA approach, we prepared iDNA encoding the prototype 181 / 25 CHIKV vaccine. The latter encodes the 181 / 25 genomic RNA downstream of the CMV promoter in the plasmid. The plasmid established a CHIKV 181 / 25-like vaccine virus in cell culture and in vivo. Inoculation of BALB / C mice with the 181 / 25iDNA plasmid protected the mice from CHIKV challenge. The attenuated mutations in the iDNA-generated virus were confirmed by DNA sequencing, and no reversion was detected. In addition, the iDNA-derived virus is expected to have lower heterogeneity (%SNP) at the attenuated mutation sites E2-12 and E2-82 compared to the prototype 181 / 25 virus. In previous studies, iDNA-derived RNA had fewer SNPs compared to the 181 / 25 virus vaccine, suggesting that it has potential safety advantages over traditional attenuated live viruses.

[0223] Furthermore, vaccination of BALB / C mice with this prototype vaccine protected them from CHIKV challenge ( Fig.10 , Table 1). The attenuating mutations in the iRNA-generated viruses were confirmed by genotyping and DNA sequencing, and no reversion was detected.

[0224] Studies involving mice were performed according to approved institutional animal protocols. BALB / c mice (4-8 weeks old, Noble Life Sciences, Woodbine, MD) were anesthetized with isoflurane before vaccination. Mice (n = 5 to 8 animals per group, female, two independent replicates) were vaccinated subcutaneously (sc) in the dorsal region with vaccines and controls at a dose of 10 4 and 10 5 PFU. An exemplary vaccine is the V5040 vaccine virus and an exemplary control is the 181 / 25 vaccine. After vaccination, animals were observed daily for signs of infection, morbidity, and discomfort. Blood samples were collected from the retro-orbital sinus on days 0 (before blood collection), 2-4 days, and 28 days. Viremia was assessed on days 2-4 after vaccination by direct plaque assay or by virus amplification in Vero cells followed by plaque assay. For virus amplification using co-culture, 20 μL of serum in 2 mL of complete medium was used to infect 75 cm 2 Vero cells in the flask were shaken for 1 hour, then 20 mL of culture medium was added and incubation continued for 48 hours. The supernatant was harvested and tested for virus by plaque assay.

[0225] Example 2

[0226] Production and immunogenicity of VEEV iRNA plasmid vaccine

[0227] We also prepared an iDNA vaccine from the alphavirus TC83 VEEV (Venezuelan equine encephalitis virus). 24 and 48 hours after cultured cells were transfected with the iDNA plasmid, immunofluorescence assays revealed that at 24 hours, some cells expressed the TC83 antigen, while at 48 hours, the entire cell population expressed the antigen ( Figure 3 After TC83 iDNA was injected into BALB / c mice, the animals produced virus-neutralizing antibodies ( Figure 5 ) and were protected from challenge. As expected, the unrelated control provided no protection. These results demonstrate the feasibility of iRNA vaccines delivered by plasmids.

[0228] In one example, we prepared a rearranged V4020 vaccine from TC83, an alphavirus vaccine against VEEV. We found that the V4020 vaccine was safe, immunogenic, and effective in mice and non-human primates.

[0229] Example 3

[0230] Generation and immunogenicity of a next-generation CHIKV iRNA vaccine further attenuated by genome rearrangement

[0231] An exemplary next generation iRNA CHIKV vaccine is based on the 181 / 25 investigational vaccine sequence. In immunocompetent BALB / c mice, the prototype vaccine showed safety and protective efficacy. In addition, after transfection in Vero cells, the exemplary next generation CHIKV vaccine virus had fewer mutations than the 181 / 25 virus vaccine, indicating that this method is safer than vaccination with live attenuated virus. Since the IND 181 / 25 virus vaccine caused adverse reactions in early clinical trials (see Example 1), strategies to improve safety are needed.

[0232] Our exemplary next generation CHIKV iRNA vaccine comprises genome rearrangement as additional attenuation mutation.Gene rearrangement in many viruses has attenuation, and has high resistance to reversion.The genome rearrangement of the iRNA-based vaccine (V5040) delivered by LNP and / or iDNA can improve safety by (i) additional attenuation and / or (ii) prevention, reduction and / or significant reduction and / or significant suppression of potential reversion.

[0233] To produce the exemplary next generation V5040 attenuated live CHIKV, we rearranged the structural genes and the 181 / 25GP containing the E2 mutation. The gene structure of CHIKV pMG5040 and V5040 RNA is shown in Figure 4A As shown, the structural genes of the V5040 RNA genome are rearranged compared with the wild-type CHIKV genome.

[0234] We designed the pMG5040 plasmid to create the replication of live attenuated V5040 virus in mammalian cells. In the pMG5040 iDNA plasmid, a DNA copy of the CHIKV vaccine virus genome was rearranged, in which the capsid gene was placed downstream of the glycoprotein gene using a dual subgenomic promoter. The rearrangement preserved the protein sequence and immunogenic epitopes. Until now, it was unclear whether the CHIKV 181 / 25 live attenuated vaccine could be rearranged without impairing its replication. Therefore, in this study, we demonstrated that V5040 virus replicated efficiently in Vero cells transfected with pMG5040. The rearranged V5040 virus was derived from pMG5040, and the viral growth of V5040 in cell culture was evaluated. Finally, the safety and immunogenicity of V5040 were demonstrated by vaccinating mice.

[0235] To prepare the live attenuated virus vaccine V5040, in one example, we transfected Vero cells with the pMG5040 plasmid. The Vero cell line (American Type Culture Collection, Manassas, VA) was cultured in a 37°C, 5% CO2 humidified incubator in αMEM supplemented with 10% fetal bovine serum (FBS) and gentamicin sulfate (10 μg / ml) (Life Technologies, Carlsbad, CA). Transfection was accomplished by electroporation or other means. After transfection of Vero cells with pMG5040, the CMV promoter directed the transcription of functional viral genomic RNA from the 5' end to the 3' end poly-A sequence.

[0236] In an exemplary embodiment, Vero cells are transfected with 100 ng pMG5040 and incubated at 37°C. Transfection medium is harvested 48 hours after transfection, concentrated and partially purified by ultracentrifugation, and then resuspended in phosphate buffered saline (PBS) at pH 7.4. V5040 titers are determined by standard plaque assays using serial dilutions of the virus in Vero cell monolayers in 6-well tissue culture plates and stained with neutral red. In order to detect low-titer viruses in samples (below the detection limit of direct plaque assay, 25 PFU / ml), the test samples are co-cultured with Vero cells to amplify low viral loads. Vero cells are infected with transfection medium at a multiplicity of infection of 0.01 to produce V5040 passage 1 (P1) virus. P1 virus is harvested 48 hours after infection (hpi) and the titer of V5040 P1 virus is determined. The virus is aliquoted and stored at -80°C until use in vitro or in vivo.

[0237] To determine viral growth kinetics, 75 cm 2 cells were infected with 100 PFU or 1000 PFU of V5040 virus or CHIKV 181 / 25 control virus. 2 Vero cells in shake flasks. CHIKV 181 / 25 vaccine virus (control) was obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) at the University of Texas Medical Branch (UTMB) in Galveston, Texas. In the same virus growth kinetics experiment, Vero cells were also transfected with 100 ng pMG5040 by electroporation. Growth medium samples were collected every 12 hours.

[0238] Example 4

[0239] Administration of CHIKV iRNA vaccine

[0240] In one example, the iRNA of the engineered live attenuated CHIKV vaccine V5040 can be administered intramuscularly (IM) as a single-dose formulation. The advantage of the single-dose form is that the outbreak and the rapidly spreading epidemic can be controlled without booster vaccination, and it is intended to be used as an emergency vaccine to prevent the spread of CHIKV epidemics worldwide. Initially, the vaccine can be targeted at initial responders, medical staff, healthy adults and travelers. When safety is confirmed, the vaccine can be considered for other populations, including vaccination in epidemic areas such as India, Asia and Central America.

[0241] Example 5

[0242] Genome-rearranged attenuated non-CHIKV iRNA vaccine

[0243] The principles disclosed herein (including plasmids, vectors, cells, production methods and administration methods) can also be used to manufacture and use iRNA-based YFV and JEV vaccines of genome rearrangement. For example, the capsid gene and envelope gene of the YFV gene can be rearranged to introduce stable attenuation, as disclosed herein.

[0244] Example 6

[0245] Genome-rearranged attenuated multivalent vaccine

[0246] The full-length attenuated RNA disclosed herein can also be used to prepare iRNA-based vaccines against genome rearrangements of a variety of positive-strand RNA viruses.

[0247] Prepare an iRNA-based vaccine V5040 for CHIKV with attenuated genomic rearrangement, and compare CHIKV iRNA delivered in vitro and in vivo by LNP and iDNA. Gene rearrangement places the capsid gene downstream of the glycoprotein, which is an attenuated mutation in alphaviruses and is highly resistant to reversion because multiple independent mutations are required to revert to wild-type CHIKV. The plaque phenotype, growth curve, and genetic stability (NGS) of iRNA-derived V5040 can be first examined in vitro in Vero cells 3, and then the safety, immunogenicity, and efficacy in mice are compared side by side using LNP and iDNA delivery.

[0248] CHIKV iRNAs were able to create a live attenuated V5040 CHIKV vaccine in vitro and in vivo. We demonstrated that vaccination of BALB / C mice with a prototype 181 / 25-based CHIKV vaccine protected the mice from challenge with CHIKV. Adverse reactions previously detected with the prototype 181 / 25 vaccine could be reduced when additional attenuating mutations were used, as well as strategies to prevent potential reversion mutations. The 181 / 25 vaccine was provided by the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) through the University of Texas Medical Branch (UTMB) in Galveston, Texas. Gene rearrangements in many viruses, including alphaviruses, are highly resistant to reversion. Therefore, the attenuating rearrangements in the V5040 iRNA were placed in the capsid gene downstream of the glycoprotein gene ( Figure 3 ). The rearranged CHIKV iRNA may have improved safety, immunogenicity, and efficacy, requiring only a single IM vaccination using iRNA delivered by LNP and / or iDNA. As a control, we can use the 181 / 25IND vaccine virus. The rearranged CHIKV iRNA sequence can be confirmed by sequencing. The rearranged CHIKV iRNA can be prepared in a plasmid downstream of T7 RNA polymerase. CHIKV cDNA plasmids containing iRNAs located downstream of the CMV promoter are available from previous studies. Full-length DNA clones from CHIKV and VEEV (a related alphavirus) can also be prepared.

[0249] Example 7

[0250] Plasmid encoding rearranged CHIKV iRNA V5040

[0251] Molecular biology. The capsid (C) gene is cloned downstream of the glycoprotein using a dual subgenomic promoter. For example, the structural gene region can be divided into two open reading frames (ORFs), one expressing the GP gene E3-E2-6K-E1 and the other expressing only the C gene (see Figure 3). Each structural ORF is expressed from its own 26S subgenomic promoter and includes translation initiation and termination codons. The genes are rearranged, wherein the GP gene is placed before the C gene in the CHIKV plasmid. A full-length DNA copy of the engineered and rearranged genomic RNA can be cloned into a DNA vector plasmid containing a T7 phage promoter to produce iRNA in vitro. In addition, the CHIKV iRNA is cloned downstream of the CMV promoter to transcribe the rearranged genomic RNA in mammalian cells.

[0252] Rearrangement has retained the protein sequence, and thereby retained the immunogenic epitope. The vaccine V5040iRNA of rearrangement can produce clone-purified progeny virus and homogeneous virus, and these viruses are well-characterized, attenuated and have unique genetic characteristics. This homogeneous population virus has a safety advantage than the traditional attenuated live vaccine of the mixed population composed of heterogeneous subgroups (quasispecies). In addition, similar to other viruses, rearrangement can be attenuated and resist reversion, because multiple independent mutations are needed to revert to the wild-type sequence.

[0253] As another example, the CHIKV structural gene region is divided into two open reading frames (ORFs), one expressing the GP gene E3-E2-6K-E1 and the other expressing only the C gene. Each structural ORF is expressed from its own 26S subgenomic promoter and includes translation initiation and termination codons. The C and GP genes are rearranged in V5040, with the GP gene located before the C gene ( Figure 4A ). Artificially rearranged genomes result in attenuation of many viruses and are resistant to reversion because multiple independent mutations are required to restore the wild-type viral sequence.

[0254] The full-length functional RNA genome of the V5040 vaccine virus is encoded in the pMG5040 plasmid downstream of an optimized CMV promoter. pMG5040 was prepared by redesigning a prototype CHIKV iDNA clone. For example, the pMG5040 plasmid was derived from the iDNA plasmid p181 / 25. pMG5040 is based on the pUC backbone vector and encodes the full-length rearranged RNA genome of the V5040 vaccine virus ( Figure 4A). The capsid (C) gene was cloned downstream of the glycoprotein gene by overlapping PCR and expressed using a dual subgenomic promoter. An ATG codon was introduced at the 5' end of the E3 gene within the glycoprotein (GP) gene, and a TGA stop codon was introduced at the 3' end of the C gene. The full-length genomic cDNA of the V5040 virus was placed in pMG5040 under the transcriptional control of an optimized CMV promoter. The hepatitis D ribozyme was introduced downstream of the V5040 cDNA to ensure that the genomic RNA transcript was cleaved after the synthetic poly (A) at the 3' end of the virus. The cDNA of V5040 also retains the attenuated mutations Thr12Ile and Gly82Arg (both in the E2 gene) derived from the 181 / 25 prototype vaccine sequence.

[0255] The iDNA plasmid pMG5040 containing the full-length CHIKV iDNA of rearrangement was isolated from Escherichia coli. The CHIKV sequence was confirmed by DNA sequencing. Bioinformatics analysis was carried out to ensure that the CHIKV sequence of rearrangement does not interfere with the plasmid production in Escherichia coli or the translation of the viral genome in eukaryotic cells. We confirmed the existence of true (authentic) ORF, and there is no strong internal bacterial promoter and transcription site, which may cause the expression of potential toxic proteins and inhibit the plasmid growth in Escherichia coli. ORF was predicted using NCBIORF Finder software. Potential bacterial promoters were predicted using BPROM software, which is a bacterial promoter recognition program with an accuracy rate of about 80% and specificity. In addition, by using the software developed in the Berkeley Drosophila Genome Project (Berkeley Drosophila Genome Project), potential splicing sites that may cause RNA degradation in the nucleus were screened in the rearranged sequence. The amino acid sequence of the CHIKV protein is consistent with GenBank 181 / 25TSI-GSD-218CHIKV 181 / 25 vaccine #L37661, but with an additional Met at the beginning of the GP gene and a stop codon at the end of the C gene ( Figure 4A ).

[0256] The resulting plasmid, pMG5040, was propagated in E. coli Stbl3 cells (Thermo, Carlsbad, CA) using standard Luria Broth (LB) medium in the presence of kanamycin. The plasmid was isolated using an endotoxin-free DNA isolation method (Qiagen, Valencia, CA) or a similar DNA isolation method according to the manufacturer's instructions. Finally, pMG5040 was formulated in phosphate buffered saline (PBS) to a concentration of approximately 1 mg / ml. This process produced transfection-grade, sterile DNA with a supercoiled DNA content of 95%, an A260 / A280 ratio of approximately 1.9, and minimal residual endotoxin, RNA, genomic DNA, and protein impurities.

[0257] Example 8

[0258] In vitro assessment of iRNA production by viruses

[0259] iRNA can be generated in vitro using pT7-V5040 as a template, followed by runoff transcription in vitro using T7 polymerase. DOTAP lipid-based LNPs can be used to encapsulate iRNA for vaccination. iDNApCMV-V5040 can be formulated in PBS for vaccination. 181 / 25 vaccine virus can be used as a control. Vero (ATCC CCL-81.5) cells can be (1) transfected with iRNA-LNP, (2) transfected with iDNA using Fugene transfection reagent, or (3) infected with control 181 / 25 CHIKV. Vero cells in serum-free medium are often used for vaccine manufacturing and testing. Live attenuated virus samples can be collected from the culture medium every 6 hours for 96 hours. Reassortant viruses harvested from iRNA-transfected or iDNA-transfected Vero cells can be characterized by plaque phenotype, growth curve, and genetic stability (by Illumina NGS). The control can be a standard IND 181 / 25 virus vaccine. Genetic and phenotypic characterization of the three viruses may include: kinetic parameters of replication in tissue culture, antigenic properties, genetic stability, and molecular heterogeneity determined by the following in vitro assays: (1) plaque assay and phenotype; (2) viral growth curves; (3) Western blot; (4) immunofluorescence; and (5) NGS sequencing of each virus, including a 181 / 25 control to assess population heterogeneity and genetic stability in mammalian cells.

[0260] For example, serum collected on the 28th day after inoculation was used to determine CHIKV-specific antibody responses by Western blot and indirect immunofluorescence assay (IFA). For example, mouse serum was probed by Western blot with lysate of Vero cells infected with CHIKV 181 / 25 at a dilution of 1:100, and then probed with goat anti-mouse IgG (H+L) secondary antibody (1:1000 dilution) conjugated with alkaline phosphatase (AP). As another example, IFA, Vero cells inoculated in chamber slides were infected with V5040 at MOI 0.01. Cell foci infected with V5040 appeared in Vero monolayer cells 24 hours after infection. The monolayer was fixed with acetone and probed with mouse serum diluted 1:25, and then probed with goat anti-mouse IgG (H+L) secondary antibody (1.25 dilution) conjugated with fluorescein isothiocyanate (FITC). Cell nuclei were stained using VectaShied mounting medium containing propidium iodide (Vector Laboratories, Inc., Newark, CA).

[0261] Example 9

[0262] In vivo vaccination of CHIKV V5040 iRNA vaccine.

[0263] Mice can be inoculated with (1) iRNA formulated in LNP, (2) iRNA formulated in iDNA, or (3) 181 / 25 virus. Based on the clinical and pathological similarities to human CHIKV infection, C57BL / 6 and CD-1 mice provide a useful and realistic model for studying CHIKV infection and inoculation.

[0264] We can optimize and evaluate iRNA vaccination in 14-day-old male and female C57BL / 6 mice (Harlan Sprague-Dawley, Indianapolis, IN). iRNA is an innovative technology for creating attenuated live viruses. The optimal method for delivering iRNA in vivo can be determined. iRNA delivery can be optimized in C57BL / 6 mice (8 per group, male and female) using a single dose vaccination with 50 μg of each vaccine (1) LNP-formulated iRNA, (2) iRNA formulated with iDNA using an in vivo transfection reagent (In vivo-jetPEI, PolyPlus, Illkirch, France), and (3) control 181 / 25 virus. The recommended dose of 50 μg is similar to that of mRNA vaccines. iRNA vaccine efficacy can be measured in vivo by evaluating viremia after vaccination, and can also be measured by the magnitude of the immune response to CHIKV antigens as assessed by analysis of CHIKV neutralizing antibodies (plaque reduction neutralization test, PRNT) and cell-mediated responses (ELISPOT). Animals can be challenged with neurovirulent CHIKV to demonstrate the protective effects of the proposed V5040 CHIKV iRNA vaccine as described elsewhere. Groups of 8 animals can provide sufficient statistical power to generate valid results.

[0265] For example, as described above, in the plaque reduction neutralization assay (PRNT50), C57BL / 6J mice (5-6 weeks old, Jackson Laboratories) were vaccinated with test vaccines (105PFU / mouse) or PBS. Animals (n=8 / group, same sex) were sacrificed at different time points (3, 7, 14, 21 and 41 days after vaccination) and whole blood was collected by terminal bleeding. For PRNT, diluted homologous virus was mixed with an equal volume of serially diluted serum (1:64 to 1:2048, two-fold dilution) and incubated at 37°C for one hour. The virus-serum mixture was added to Vero 76 cells grown at confluence in 24-well plates and incubated at 37°C for 1h to adsorb the virus. The cells were washed once with PBS and then covered with covering medium (1XEMEM containing 2% FBS, 0.75% methylcellulose). After incubation in a CO2 incubator at 37°C for four days, cells were fixed and visualized with crystal violet staining solution (2% paraformaldehyde, 10% ethanol and 1% crystal violet). The number of viral focus forming units was standardized according to the mean value of the mock group, and the PRNT50 was calculated using a dose-response analysis of a four-parameter logistic model (XLfit, IBDS, UK). Statistical analysis was performed using GraphPad Prism (9.4.1).

[0266] Preparation and evaluation of vaccines based on iRNA and comparison of LNP and iDNA methods of iRNA delivery in vitro and in vivo.Similar to other rearranged alphaviruses, the replication rate of V5040 virus is slower than that of classic 181 / 25 virus.In addition, compared with its 181 / 25 vaccine, the iRNA-derived V5040 virus can have high homogeneity (% single nucleotide polymorphism, SNP).The rearranged sequence is not detected to return to the wild-type sequence.The method (LNP or iDNA) of iRNA delivery in vivo can be compared.The iRNA prepared by LNP can directly establish the vaccine in the cytoplasm, while the iRNA prepared by iDNA requires the nuclear mechanism to synthesize iRNA and be transported to the cytoplasm by nuclear pores.However, LNP-iRNA may require ultra-low temperature storage / transportation, and iDNA can be formulated to store at ambient temperature.When delivered by LNP or iDNA, iRNA can activate innate immunity through the TLR mechanism, and effectively induce the specific immune response to CHIKV vaccine.

[0267] CHIKV is a cytoplasmic virus and the genomic RNA replicates only in the cytoplasm. Similar to the 181 / 25 control virus, LNP-formulated iRNA can also establish replication of V5040 in the cytoplasm. In contrast, iDNA-delivered iRNA must be introduced into the nucleus to synthesize the iRNA. iRNA V5040 can be created from LNP-formulated or iDNA-formulated iRNA because only a few intact iRNA copies in the cytoplasm are required to initiate replication of live attenuated CHIKV. When LNP or iDNA vaccination results in low immunogenicity, adjuvants and / or microneedles can be added to enhance the immunogenicity of iRNA vaccines. The iDNA strategy for (+)-stranded RNA viruses can be a feasible approach ( Figure 6 ). Figure 6 It was shown that only 10 ng of iDNA-formulated iRNA was sufficient to establish CHIKV replication.

[0268] Example 10

[0269] The platform applicability of iRNA technology against JEV and YFV and multivalency was evaluated in small animals.

[0270] The iRNA platform and in vivo delivery method developed in the above examples are suitable for other emerging viral pathogens with epidemic potential, such as YFV and JEV. A YFV iRNA vaccine based on the live attenuated vaccine 17D can be prepared. Similarly, a JEV iRNA vaccine can be prepared using the JEV vaccine SA-14-14-2. Three iRNA vaccines can be mixed to form a multivalent iRNA vaccine.

[0271] 48 animals (C57BL / 6, equal numbers of males and females) can be divided into 4 groups (12 mice per group) and vaccinated by the optimal method (see Aim 1) with 50 μg of each iRNA vaccine: (1) V5040 CHIKV iRNA; (2) JEV iRNA; (3) YFV iRNA, and (4) mixed CHIKV, YFV, JEV iRNA. Each iRNA formulation can be delivered using the optimal iRNA delivery method described above. The doses selected are close to current mRNA vaccines. Control animals (Group 5) can be injected with 10 5 PFU of 181 / 25 virus vaccine. Blood samples are collected every 2-7 days for viremia, NGS, and serology. NGS of viruses from viremic mice can compare genetic variations and determine the potential recovery rate of the vaccine. Immunogenicity can be determined by analyzing the neutralizing immune response (PRNT) and CMI (ELISPOT) to the corresponding virus. Statistical analysis can be performed using Student's t-test.

[0272] Embodiment 11

[0273] Safety, immunogenicity and efficacy of iRNA-based CHIKV vaccines

[0274] We can also evaluate the safety, immunogenicity, and efficacy studies of iRNA-based CHIKV vaccines in relevant animal models, including CD-1 mice and cynomolgus macaque non-human primates. In previous Phase II clinical trials, the CHIKV 181 / 25 vaccine was highly immunogenic (98% seroconversion rate) and well tolerated. However, up to 8% of vaccine recipients experienced mild transient adverse reactions, indicating the need for safer CHIKV vaccines. The CHIKV V5040 vaccine based on iRNA containing gene rearrangements to attenuate and ensure vaccine safety can meet this need. Importantly, the rearrangements not only attenuate but also resist reversion. Reversion mutations were detected in viremic patients vaccinated with the traditional 181 / 25 IND vaccine. Therefore, the proposed rearrangement and iRNA approach would improve two potential safety issues: (i) improve the safety of CHIKV vaccines through additional attenuation, as described for other viruses with rearranged genomes9, (ii) prevent accidental reversion to virulent genotypes, and (iii) improve immunogenicity by stimulating the innate immune response through the adjuvant properties of iRNA itself.

[0275] Even with highly attenuated rearrangements, the immunogenicity and protective immune responses of CHIKV vaccines can be improved by using iRNA technology. Unlike live virus vaccines, iRNA vaccines can self-adjuvant and stimulate innate immune responses before vaccine particles are established ( Figure 5 ). Therefore, similar to mRNA vaccines, after administration of iRNA vaccines, iRNA can be recognized by multiple pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-1)-like receptors (RLRs), and nucleotide-binding oligomerization domain (NOD)-like receptor family proteins (NLRs), leading to the production of IFN and proinflammatory cytokines, thus providing enhanced antigen presentation capacity when producing CHIKV vaccine antigens17.

[0276] Example 12

[0277] CHIKV V5040 iRNA vaccine was administered and challenged in outbred CD-1 mice.

[0278] The CD-1 mouse model can be used to test the safety, immunogenicity and efficacy of the iRNA CHIKV V5040 vaccine in a dose escalation study to determine the optimal dose of the iRNA vaccine. Briefly, forty-eight outbred CD-1 mice (12 mice per group, male and female) can be inoculated with the iRNA described herein.

[0279] The groups were as follows: (i) v5040 iRNA 0.5 μg intramuscular dose, (ii) v5040 iDNA 5 μg dose; (iii) v5040 iDNA 50 μg dose; (iv) control group sham-vaccinated. Due to the current mRNA vaccines, the upper limit dose of 50 μg was selected.

[0280] Blood samples are collected every 2-7 days for viremia, NGS, and serology. Immunogenicity can be determined by analyzing innate immunity, neutralizing immune responses (PRNT), and CMI (ELISPO). 5 The mice were challenged with CHIKV subcutaneously (d28) with a dose of PFU. CD-1 mice challenged subcutaneously with CHIKV developed lethargy, difficulty walking, hind limb dragging, and decreased weight gain within 7-10 days after infection. Within the first 6-7 days, animals showed high levels of viremia and leg muscle (10 6 -10 8 PFU / mL) CHIKV. After the challenge, the clinical symptoms of infection and body weight of the animals can be observed every day. The morbidity and viremia can be recorded and statistically analyzed.

[0281] Example 13

[0282] Safety, immunogenicity, and efficacy of CHIKV V5040 iRNA vaccine in NHPs.

[0283] The cynomolgus macaque (Macaca fascicularis) may represent an ideal model for evaluating the safety of CHIKV vaccines. In previous studies, multiple parameters during CHIKV infection in NHPs have been measured to assess the effectiveness of potential vaccines in impeding the development of CHIKV infection.

[0284] Cynomolgus monkeys of similar age (>3 years), weighing 3-6 kg, free of infection with simian immunodeficiency virus (SIV), simian retrovirus type D (SRV), simian T-lymphotropic virus (STLV), and alphavirus antibodies (by hemagglutination inhibition assay) against western equine encephalitis virus (WEEV), Venezuelan virus (VEEV), eastern equine encephalitis virus (EEEV), Sindbis virus (SINV), Semliki Forest virus (SFV), and CHIKV were available. This study was approved by the Institutional Animal Care and Use Committee, and all animals were handled in accordance with the guidelines of the American Association for Accreditation of Laboratory Animal Care.

[0285] The CHIKV V5040 iRNA vaccine can be randomized to cynomolgus monkey cohorts; one cohort (n=4) can be vaccinated intramuscularly with the iRNA with the optimal formulation and dose (see objectives 1 and 2), and the second cohort (n=4) can be vaccinated with the control vaccine 181 / 25. The third cohort (n=7) can be sham vaccinated intramuscularly with saline. Anesthetized animals can be vaccinated intramuscularly in the upper deltoid muscle with a single vaccination of iRNA in saline or 5.0 log10 PFU of control vaccine in a volume of 100 μL. Animals can be observed for any signs of clinically recognizable adverse reactions and bled on days 1-3, 15, and 50 after vaccination. Serum samples can be collected. Immunogenicity can be determined by analyzing neutralizing immune responses (PRNT) and CMI (ELISPOT). Statistical analysis can be performed using pre-bleeding serum as a control. The cross-reactivity of antisera can be examined by evaluating the neutralization effect of CHIKV strains provided by UTMB and / or the CEPI network.

[0286] On day 52 after inoculation, as described elsewhere, a single subcutaneous inoculation of WT CHIKV-LR (5.0 log10 PFU in a volume of 100 μL) in the upper deltoid muscle of anesthetized cynomolgus monkeys can be used for challenge. Blood can be collected on days 1-3, 6, 9, 13, and 35 after challenge, at the termination of the experiment, and at autopsy. Morbidity and mortality can be determined and statistically analyzed using the Student's t-test. Tissues can be placed in 10% zinc-formalin for histopathological analysis. Some tissues can also be placed in RNAlater solution and frozen for viral titration by plaque assay: axillary, bronchial, and inguinal lymph nodes.

[0287] The use of two mouse models (C57BL / 6 and outbred CD-1) and one NHP model can increase rigor and produce statistically valid results. The iRNA vaccine is safe and can induce significant, broad cross-neutralizing immune responses due to the immunostimulatory effects of iRNA activating innate immunity. In addition to improved safety and immunogenicity, the rearranged V5040 CHIKV iRNA also has advantages in manufacturing, storage, genetic stability and transportation, without the need to use a cold chain for iDNA preparations.

[0288] Embodiment 14

[0289] Chemistry, Manufacturing and Controls (CMC) Development

[0290] Manufacturing Process and Analytical Methods: iRNA production is performed in vitro using an in vitro transcription (IVT) reaction. The formulation containing LNP is described below. The formulation of iRNA in iDNA is achieved by cloning the iRNA downstream of the CMV promoter to transcribe the iRNA in vivo. The feasibility of iRNA-based vaccines in small animals has been demonstrated. Advanced development including testing NHPs, developing manufacturing processes, and GMP manufacturing, release testing, and IND-supportive preclinical studies can all be performed in accordance with industry procedures.

[0291] iRNA vaccines can be prepared using T7 RNA polymerase transcription in a GMP facility to prepare GMP-grade vaccines for Phase 1-2 clinical trials in accordance with FDA and ICH regulations. After quality control testing, iRNA can be formulated with LNPs. To promote effective delivery of iRNA in vivo, we will complex iRNA with PEGylated and cationic lipids to form LNPs. For this purpose, an ethanol dialysis method can be used. The LNP will include the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). Lipids in ethanol can bind and emulsify RNA in citrate buffer, and then dialysis is used to remove ethanol and promote LNP-iRNA self-assembly. Under these conditions, LNP encapsulates approximately 95% of RNA. As determined by light scattering and cryo-electron microscopy, LNPs are mainly unilamellar vesicles with an average diameter of 75nm. Assuming that the LNP is a uniform population of this size, each particle can contain approximately 1 iRNA molecule.

[0292] iDNA preparations represent plasmids encoding iRNAs located downstream of the CMV promoter. Established methods can be used to manufacture plasmid iDNAs from E. coli for production of bacterial cell banks, fermentation, harvesting / lysis of biomass, and downstream DNA purification. The process produces a sterile DNA product, 95% of which is supercoiled DNA, with an A260 / A280 ratio of about 1.9, and minimal residual endotoxin, RNA, genomic DNA, and protein impurities. Quality control can include (i) endotoxin testing, (ii) agarose gel, (iii) SDS-PAGE; (iv) restriction enzyme analysis, and (v) DNA sequencing.

[0293] Storage requirements for the final product: LNP-formulated iRNA vaccines can be formulated by lyophilization for long-term storage at refrigerated temperatures. mRNA-LNP can be lyophilized, and the physicochemical properties of the lyophilized material do not change significantly after 12 weeks at room temperature and at least 24 weeks at 4°C.

[0294] As with any DNA vaccine, iDNA-formulated iRNA vaccines can be formulated for storage at ambient temperature.

[0295] Programs to transfer RNA vaccine candidate technology to vaccine manufacturers in developing countries: The advantages of the proposed iRNA-based technology are low vaccine cost, rapid production, affordable transportation and storage costs, and user-friendly manufacturing.

[0296] Due to the immunostimulatory effects of iRNA and live attenuated virus, iRNA-based vaccines can induce a broader immune response than standard mRNA or DNA vaccines. In addition to improved safety and immunogenicity, rearranged iRNA also has advantages in manufacturing, storage, genetic stability and transportation, without the need for a cold chain.

[0297] Embodiment 15

[0298] In vitro rescue of V5040 attenuated live CHIKV vaccine from pMG5040 iDNA plasmid

[0299] The iDNA plasmid containing the full-length iDNA was isolated from Escherichia coli ( Figure 4B ) and evaluated in cultured cells. It is expected that genomic RNA transcription from iDNA will occur in the nucleus and that genomic RNA will be transported to the cytoplasm where translation and viral synthesis will occur. To evaluate whether V5040 CHIKV with rearranged structural genes can be established in vitro from plasmids, pMG5040 was transfected into Vero cells (ATCC CCL-81.5). Vero cells are frequently used in vaccine manufacturing. Expression of replication-competent (infectious) virus from transfected cells in culture was confirmed by plaque assay. Fig. 7A and 7B As shown, cells incubated with culture medium transfected with pMG5040 (see Materials and Methods) developed plaques on agarose overlays 12-24 hours after infection ( Fig. 7A and 7B ). Similar to the plaques produced by CHIKV 181 / 25, V5040 plaques were heterogeneous and appeared to be smaller in diameter. V5040 virus harvested from the supernatant 48 hours after transfection had an infectious titer of 108 PFU / mL, indicating that replication-competent virus was successfully rescued from the pMG5040 iDNA plasmid and that V5040 replicated efficiently in Vero cells. The expression of CH1KV-specific antigens in V5040-infected cells was confirmed by IFA using CH1KV-specific antiserum VR-1241AF, as described elsewhere.

[0300] In the next experiment, we compared the activity of V5040 virus in mice infected with infectious V5040 (100 PFU or 1000 PFU, respectively, representing an MOI of 10 -5 and 10 -4 ) or Vero cells transfected with pMG5040 plasmid. As a control, we used an experimental CHIKV 181 / 25 vaccine.

[0301] Culture medium samples from transfected and infected cells were collected every 12 h and viral titers were quantified by plaque assay. Figure 7B As shown, 10 -5 Infection with an MOI of 10 resulted in efficient viral replication, which was comparable to CHIKV 181 / 25 kinetics and reached 108 PFU / ml at 48 h post infection. -4 The infection under the same conditions has similar dynamics, reaching a peak at 60 hours after infection. In cells transfected with pMG5040, most cells expressed CHIKV antigens 48 hours after transfection, and the infectious virus in the culture medium was close to 108PFU / ml at 72 hours. Without being bound by a particular theory, we believe that the delay in peak titers in cells transfected with pMG5040 compared to cells infected with V5040 can be explained by the time associated with cell recovery after pMG5040 transfection, DNA penetration of the nucleus of the transfected cells, RNA transcription and transport to the cytoplasm to start V5040 replication. Therefore, compared with the control CHIKV 181 / 25 virus, the replication of the V5040 virus from the plasmid is expected to be delayed, resulting in peak titers appearing at a later time point. Similar observations have been made for related reassortant alpha viruses.

[0302] According to our experiments, we observed that infection with 100-1000 PFU of V5040 virus or transfection with 100 ng of pMG5040 iDNA effectively initiated the in vitro replication of attenuated live CHIKV.

[0303] Example 16

[0304] Immunogenicity of V5040 vaccine in mice

[0305] To further ensure the attenuation and safety of the experimental V5040 vaccine, the attenuating mutations E2 Thr12Ile and Gly82Arg derived from the CHIKV 181 / 25 vaccine currently under clinical evaluation were introduced into the GP region of pMG5040 and V5040.

[0306] To test the safety and immunogenicity of the reassortant V5040 CHIKV vaccine, we used BALB / c and C57BL / 6 mice. In the first experiment, BALB / c mice were vaccinated subcutaneously with a single dose of 4 PFU or 10 5V5040 virus was prepared from the growth medium of Vero cells transfected with pMG5040. Similarly, 181 / 25 virus was administered as a control. After injection, all mice remained healthy with no detectable adverse effects, such as changes in body weight or behavior. Serum samples were collected as described in Materials and Methods. No viremia was detected in mice inoculated with V5040 by direct plaque assay (detection limit 25 PFU / ml). However, low viremia was detected by co-culture assay in 40% of the inoculated mice ( Fig.11 , Table 2). On day 28, mice seroconverted ( Fig. 8A and 8B ;as well as Fig.11 , Table 2). Similarly, BALB / c mice were subcutaneously inoculated with a single dose of 10 5 PFU of V5040 virus did not present any safety issues and produced an antibody response ( Fig.11 , Table 2).

[0307] Next, we evaluated the neutralizing activity of the V5040 vaccine using the PRNT50 assay with antisera from vaccinated C57BL / 6 mice. As a control, we used the 181 / 25 vaccine. Both vaccines showed measurable neutralizing activity starting on day 7 after a single dose of the vaccine ( Fig. 9 ). The iDNA-based V5040 vaccine induced stronger and more durable PRNT50 neutralization titers compared to the 181 / 25 CHIKV vaccine. While approximately 50% (4-5 / 8 mice per group) of 181 / 25-vaccinated mice showed measurable neutralizing activity (>1:32), almost all V5040-vaccinated mice exhibited PRNT50>1:128 at 14 days post-vaccination. Interestingly enough, V5040 showed better persistence of neutralizing activity, showing significantly higher PRNT50 titers at 42 days post-vaccination compared to CHIKV 181 / 25 (p<0.0007, two-way ANOVA, mixed effects model).

[0308] Embodiment 17

[0309] iDNA VEEV vaccine administered via microneedle injection

[0310] Nucleic acid vaccines can be delivered using the hMTS platform, which is designed for delivering non-viscous liquid formulations. According to the manufacturer's instructions, the cartridges of the hMTS device are aseptically filled with 0.5 ml of vaccine (pMG4020 iDNA or V4020 virus). Three hMTS devices are equipped with pMG4020, and one hMTS device is equipped with a control V4020 live virus vaccine. The filled cartridges are crimped to properly seal the cartridges. Before administering the vaccine to the animal, the cartridge containing the vaccine is inserted into the syringe to align with the spring-driven vaccine delivery plunger structure. The hMTS syringe contains twelve pre-installed hollow microneedle arrays to administer the vaccine to the injection site.

[0311] Animal studies involving experimental rabbits were performed according to approved institutional protocols (Noble Life Sciences No. 493). New Zealand white rabbits were purchased from Charles River. The animals were divided into two groups of three. Rabbits (2.5-3 kg, adult, female) were anesthetized before vaccination. One group of three animals was vaccinated with the pMG4020 plasmid encoding the V4020 virus. The other group was vaccinated with the V4020 virus. The vaccines (pMG4020iDNA plasmid or V4020 virus) were administered using the hMTS microneedle device according to the manufacturer's instructions. To minimize the number of animals for ethical and animal welfare reasons, one control rabbit was injected with the V4020 vaccine virus using the hMTS device, and one control rabbit was injected with the V4020 vaccine virus using a standard syringe needle through the subcutaneous (SC) administration route.

[0312] Vaccines were administered on day 0. Animals were inoculated with 0.5 ml of pMG4020iDNA (20 mg) or V4020 virus vaccine (10 4 PFU) or 0.5 ml (10 4 PFU) V4020 vaccine, titer is 2x10 4 PFU / ml. The human dose of TC83 vaccine is 10 5 PFU, so considering the weight difference, 10 was selected for rabbits. 4 The dose of PFU.

[0313] Animals were generally anesthetized using a combination of ketamine (50 mg / kg) and xylazine (10 mg / kg), and the injection site was shaved clean with scissors and then a razor before applying the device and injection. The skin at the injection site was stretched and fixed to a sturdy support rod. The device is attached to the skin via a self-adhesive surface, and the pre-loaded vaccine cargo is delivered from the cartridge through the microneedles by pressing the injection button on the device. As a control, one animal was subcutaneously administered 0.5 ml of the attenuated VEE vaccine strain V4020, the same amount and concentration as in the hMTS microneedle array device.

[0314] After vaccination, animals were observed daily. Blood samples were collected on days 0, 7, and 21. Rabbits were bled via the ear artery on days 0 (vaccination), 7, and 21 to assess viremia (day 7) and humoral responses to the vaccine (neutralizing antibodies and IFA). In previous studies, viremia was detectable for up to 8 days after subcutaneous inoculation of cotton rats with 3 log10 PFU VEEV.

[0315] The results showed that iDNA encoding a live attenuated VEEV vaccine delivered by iDNA using this approach was immunogenic in vivo.

[0316] Embodiment 18

[0317] iDNA CHIKV vaccine administered by microneedle injection

[0318] iDNA of full-length attenuated CHIKV can be delivered in vivo in a manner similar to that described in Example 17. The results show that iDNA encoding live attenuated CHIKV vaccine delivered by iDNA using this method is immunogenic in vivo.

[0319] Embodiment 19

[0320] Immunogenicity after direct administration of iRNA in vivo

[0321] BALB / c mice (4-8 weeks old, 5 mice per group, Noble Life Sciences, Woodbine, MD) were anesthetized with isoflurane before inoculation. Each experiment was performed at least twice to ensure repeatability. 0.1 or 10ug of full-length attenuated live CHIKV iRNA prepared using (i) saline, (ii) liposome formulations was inoculated subcutaneously (sc) to mice, or CHIKV iRNA (same dose) was inoculated transdermally (id) using microneedles. As a control, CHIKV 181 / 25 vaccine virus was administered subcutaneously with a standard needle. As another control, iDNA encoding iRNA (same dose) was used. Inoculation was completed in the dorsal region. Alternatively, iRNA inoculation was performed by intramuscular injection (im) at the same dose, followed by in vivo electroporation at the injection site. After inoculation, the clinical symptoms and body weight of the infection of the animals were observed every day. Blood samples were collected from the retroorbital sinus 2-5 days after vaccination to detect CHIKV (viremia) in the blood, and immune responses were detected on the 28th day. The mice were then transferred to a BSL3 facility and treated subcutaneously with 1x 10 4 PFU dose of virulent CHIKV was used to challenge the virus to detect whether the inoculation of iRNA or control virus can cause protection. After the challenge, the clinical symptoms and body weight of the infection of the animals were observed every day. The incidence rate (weight, behavioral changes) was determined. Serum was collected on the 3rd day after the challenge, and representative mice of each challenge group were killed on the 3rd day to check the viral titer in the tissue. The results show that iRNA can induce an immune response when given by various routes of administration and dosage.

[0322] References

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Claims

1. An infectious RNA (iRNA) molecule comprising RNA encoding an attenuated virus; Optionally, the attenuated virus is a newly emerging virus; Preferably, the attenuated virus is a positive-strand RNA virus; More preferably, the positive-strand RNA virus is selected from one or more of a ribovirus, a positive RNA virus, a Kitrinovirus, a Lenarvirus, a Pisuvirus, a Pisonivirus, a Stelpavirus, a Togavirus, and combinations thereof; More preferably, the positive-strand RNA virus is selected from the following: one or more alphaviruses, such as Western Equine Encephalitis Virus (WEEV), Venezuelan Equine Encephalitis Virus (VEEV), Eastern Equine Encephalitis Virus (EEEV), Sindbis Virus (SINV), Semliki Forest Virus (SFV), and Chikungunya Virus (CHIKV), one or more flaviviruses, such as hepatitis C virus, yellow fever virus (YFV), West Nile virus, dengue virus, and Japanese encephalitis virus (JEV), One or more arenaviruses, such as Lassa, Guanarito, Junin, Lujo, Machupo, Sabia, and Whitewater Arroyo, one or more coronaviruses, such as Middle East Respiratory Syndrome (MERS) virus, Severe Acute Respiratory Syndrome (SARS) virus, and SARS-coronavirus 2 (SARS-CoV-2), one or more microRNA viruses, such as rhinoviruses and common cold viruses, one or more retroviruses, such as simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), simian retrovirus type D (SRV), and simian T-lymphotropic virus (STLV), and combinations thereof; More preferably, the positive-strand RNA virus is selected from one or more of CHIKV, VEEV, YFV, JEV and combinations thereof; More preferably, the positive-strand RNA virus is selected from one or more of CHIKV, VEEV, and combinations thereof; More preferably, the positive-strand RNA virus is CHIKV; More preferably, the positive-strand RNA virus is VEEV; More preferably, the positive-strand RNA virus is selected from 181 / 25 CHIKV, V5040 CHIKV, TC85 VEEV, V4020 VEEV and combinations thereof; and More preferably, the attenuated virus is encoded by the sequence of any one of SEQ.1 to 4.

2. The iRNA molecule according to claim 1, wherein the iRNA molecule encodes all gene products of the attenuated virus; Preferably, the iRNA molecule encodes a full-length RNA from an attenuated virus; Preferably, the iRNA molecule is a recombinant RNA molecule; Preferably, the iRNA molecule encodes one or more attenuating mutations; More preferably, the one or more attenuating mutations are point mutations, rearrangements and / or combinations thereof; More preferably, the one or more attenuating mutations do not include a non-rearranged deletion of more than 180 bp, and the non-rearranged deletion is preferably no more than about 175 bp, more preferably no more than about 170 bp, more preferably no more than about 170 bp, more preferably no more than about 165 bp, more preferably no more than about 160 bp, more preferably no more than about 155 bp, more preferably no more than about 150 bp, more preferably no more than about 125 bp, more preferably no more than about 100 bp, more preferably no more than about 75 bp, more preferably no more than about 55 bp, more preferably no more than about 50 bp, more preferably no more than about 25 bp, more preferably no more than about 20 bp, more preferably no more than about 15 bp, more preferably no more than about 10 bp, more preferably no more than about 5 bp, more preferably no more than about 0 bp; More preferably, the one or more attenuating mutations do not include non-rearranged deletions of nucleotides encoding no more than 58 amino acids, preferably no more than about 55 amino acids, more preferably no more than about 50 amino acids, more preferably no more than about 45 amino acids, more preferably no more than about 40 amino acids, more preferably no more than about 35 amino acids, more preferably no more than about 30 amino acids, more preferably no more than about 25 amino acids, more preferably no more than about 20 amino acids, more preferably no more than about 15 amino acids, more preferably no more than about 10 amino acids, more preferably no more than about 5 amino acids, more preferably no more than about 0 amino acids; More preferably, when the attenuated virus is CHIKV, the iRNA molecule encodes a full-length nsP3 gene; More preferably, when the attenuated virus is CHIKV, the iRNA molecule encodes the full-length 6K gene; More preferably, the iRNA molecule encodes two attenuating point mutations and / or rearrangements, Preferably, the rearrangement is between the capsid gene and the glycoprotein gene; Preferably, the one or more point mutations are located in the glycoprotein gene; More preferably, the one or more point mutations are located in the envelope gene; and More preferably, the one or more point mutations are located in the E2 gene of CHIKV, and are preferably one or two of Thr12Ile and Gly82Arg mutations of the E2 gene of CHIKV; More preferably, the one or more point mutations are one or more mutations selected from SEQ.1 and / or SEQ.3; More preferably, the mutation is substantially stable, has a significantly reduced reversion rate, significant resistance to reversion, and / or a combination thereof; Preferably, the mutation is highly stable, has a highly reduced reversion rate, is highly resistant to reversion, and / or a combination thereof; More preferably, the mutation does not revert to a more virulent virus; and More preferably, the mutation has no detectable reversion; Preferably, the mutation is substantially stable, or highly stable, or has no detectable reversion after direct or indirect administration of the iRNA to a subject in need thereof; Preferably, the mutation is substantially stable, or highly stable, or has no detectable reversion over the entire treatment course after direct or indirect administration of the iRNA to a subject in need thereof; More preferably, the mutation does not impair replication of the attenuated virus in mammalian cells, preferably primate cells, more preferably human cells; More preferably, the mutation is stable; Preferably, the iRNA molecule comprises one or more subgenomic promoters, wherein one or more, preferably two or more, subgenomic promoters are operably linked to the iRNA molecule, Preferably, the one or more subgenomic promoters are RNA polymerase promoters, preferably eukaryotic promoters or bacteriophage promoters; Preferably, the one or more subgenomic promoters are one or more 26S promoters; More preferably, the one or more subgenomic promoters are two 26S promoters; Preferably, each subgenomic promoter is operably linked to a gene encoded by an iRNA molecule; More preferably, the first subgenomic promoter is operably linked to the capsid gene and the second subgenomic promoter is operably linked to the glycoprotein gene; and More preferably, the glycoprotein gene is E3-E2-6K-E1; and More preferably, the iRNA molecule has the sequence of SEQ.1 and / or SEQ.

3.

3. An infectious DNA (iDNA) molecule encoding any one of the iRNA molecules of claims 1 to 2, wherein the iDNA is operably linked to a DNA-dependent RNA polymerase-producing promoter, Preferably, the production promoter is suitable for producing the encoded iRNA molecule; preferably, the iRNA molecule produced is pharmaceutically acceptable; Preferably, the production promoter is a bacteriophage promoter; Preferably, the production promoter is a prokaryotic promoter; Preferably, the production promoter is selected from T3, T7 and 26S; Preferably, the production promoter is different from the subgenomic promoter; Preferably, the iDNA molecule is carried in a DNA plasmid; Preferably, the iDNA molecule is carried in a pT7 plasmid; Preferably, the iDNA molecule is carried in a pMG plasmid; More preferably, the DNA plasmid is pMG4020 plasmid and / or pMG5040 plasmid; More preferably, the iDNA has the sequence of SEQ.2 and / or SEQ.4; in, The iDNA further comprises a eukaryotic promoter, preferably, the promoter is optimized; More preferably, it is a mammalian promoter, More preferably, it is a CMV promoter, and More preferably, it is an optimized hybrid human CMV (hCMV) promoter; Preferably, the iDNA molecule encodes a full-length RNA from an attenuated virus; Preferably, the eukaryotic promoter and / or the production promoter is located upstream of the iRNA encoded by the iDNA molecule; Preferably, the iDNA molecule is stable, preferably thermostable and / or genetically stable; Preferably, the iDNA molecules are characterized as being sterile, and / or about 95% of the iDNA molecules are supercoiled, and / or have an A selected from the group consisting of about 1.6 to about 2.2, about 1.7 to about 2.1, about 1.8 to about 2.0, and about 1.

9. 260 / A 280 ratio; Preferably, the iDNA molecule is capable of inducing an immune response; Preferably, the immune response is an innate immune response, an adaptive immune response and / or a combination thereof; More preferably, the immune response is a persistent immunity; More preferably, a broadly cross-neutralizing immune response; and More preferably, the iDNA molecule is capable of inducing an immune response with a single dose; Preferably, the iDNA molecule is pharmaceutically acceptable; Preferably, the iDNA molecule is produced in vitro or in vivo or by direct synthesis; more preferably, the iDNA molecule is produced in vitro; More preferably, the iDNA is manufactured under GMP conditions; More preferably, the iDNA is made for vaccine use; Preferably, the iDNA is formulated in a composition that further comprises a pharmaceutically acceptable non-toxic component; Optionally, the component is saline and / or a buffer; and preferably, the saline is a phosphate buffer solution; More preferably, the composition is pharmaceutically acceptable; Preferably, the composition and / or iDNA molecule is suitable for direct administration to a subject in need thereof; Preferably, the composition and / or iDNA molecule is capable of inducing an immune response in a subject in need thereof via iRNA. Preferably, the immune response is an innate immune response, or an adaptive immune response, and / or a combination thereof; More preferably, the immune response is a persistent immunity; More preferably, the immune response is a broadly cross-neutralizing immune response; and More preferably, the composition and / or iDNA molecule is capable of inducing an immune response in one or more doses, preferably a single dose; Preferably, the composition and / or iDNA molecule is suitable for use as a vaccine; Preferably, the iDNA molecule is suitable for use as a vaccine; Preferably, the virus is suitable for preventing and / or reducing and / or significantly reducing one or more symptoms in a subject in need thereof, The subject is preferably a mammal, more preferably a human; and preferably, the iDNA molecule is used to prevent, reduce or significantly reduce viral infection and / or prevent or alleviate the symptoms of viral infection; and Preferably, the symptom is selected from the group consisting of fever, headache, rash, nausea, myalgia, arthralgia, respiratory failure, cardiovascular disease, hepatitis, skin effects, central nervous system abnormalities, and combinations thereof.

4. A composition comprising the iRNA molecule of any one of claims 1 to 2, and further comprising a pharmaceutically acceptable non-toxic component; Optionally, the component is saline and / or a buffer; and preferably, the saline is a phosphate buffer solution; Preferably, the iRNA molecule is pharmaceutically acceptable; Preferably, the iRNA molecule is produced in vitro or in vivo or by direct synthesis; more preferably, the iRNA molecule is produced in vitro; More preferably, the iRNA is manufactured under GMP conditions; More preferably, the iRNA is manufactured for vaccine use; Preferably, the iRNA molecule is produced using the iDNA described in any one of claims 3; More preferably, the composition is pharmaceutically acceptable; Preferably, the composition and / or iRNA molecule is suitable for direct administration to a subject in need thereof; Preferably, the composition and / or iRNA molecule is capable of inducing an immune response upon direct administration to a subject in need thereof. Preferably, the immune response is an innate immune response, or an adaptive immune response, and / or a combination thereof; More preferably, the immune response is a persistent immunity; More preferably, the immune response is a broadly cross-neutralizing immune response; and More preferably, the composition and / or iRNA molecule is capable of inducing an immune response in one or more doses, preferably a single dose; Preferably, the composition and / or iRNA molecule is suitable for use as a vaccine; Preferably, the composition and / or iRNA molecule is suitable for preventing and / or reducing and / or significantly reducing one or more symptoms in a subject in need thereof. The subject is preferably a mammal, more preferably a human; and preferably, the iRNA molecule is used to prevent, reduce or significantly reduce viral infection and / or prevent or alleviate the symptoms of viral infection; and Preferably, the symptom is selected from the group consisting of fever, headache, rash, nausea, myalgia, arthralgia, respiratory failure, cardiovascular disease, hepatitis, skin effects, central nervous system abnormalities, and combinations thereof.

5. A vector containing infectious genetic material; Preferably, the vector encapsulates the infectious genetic material; Preferably, the infectious genetic material is the iRNA molecule of any one of claims 1 to 2, the iDNA molecule of claim 3, the composition of claim 4 and / or a combination thereof; Preferably, the vehicle is a nanoparticle, which includes liposomes and lipid nanoparticles, and includes lipid nanoparticles based on the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) lipid; Preferably, the lipid nanoparticles comprising the infectious genetic material are formed by mixing the iRNA molecules with PEGylated and cationic lipids; Preferably, the amount of initial infectious genetic material encapsulated by the lipid nanoparticles is more than about 60%, preferably more than about 70%, preferably more than about 80%, preferably more than about 90%, preferably more than about 91%, preferably more than about 92%, preferably more than about 93%, preferably more than about 94%, preferably more than about 95%, preferably more than about 96%, preferably more than about 97%, preferably more than about 98%, preferably more than about 99%, preferably more than about 99.9%; Preferably, the average diameter of the lipid nanoparticles is from about 5 nm to about 200 nm; preferably from about 10 nm to about 150 nm; preferably from about 20 nm to about 100 nm; preferably from about 30 nm to about 90 nm; preferably from about 40 nm to about 85 nm; preferably from about 50 nm to about 80 nm; preferably from about 60 nm to about 75 nm; preferably about 75 nm; Preferably, the lipid nanoparticles are unilamellar vesicles; Preferably, the lipid nanoparticles contain an infectious genetic material in each lipid nanoparticle in a range selected from the group consisting of about 1 to about 100, about 1 to about 90, about 1 to about 80, about 1 to about 70, about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 1 infectious genetic material / lipid nanoparticle; Preferably, the vector is capable of delivering infectious genetic material to a subject in need thereof; Preferably, the vector is capable of delivering infectious genetic material into the cytoplasm of cells of a subject in need thereof; Preferably, the vehicle is capable of inducing an immune response, Preferably, the innate immune response, the adaptive immune response and / or a combination thereof; More preferably, the immunity is long-lasting; and More preferably, it is a broadly cross-neutralizing immune response; Preferably, the vector is capable of inducing an immune response in a single dose; Preferably, the vehicle is suitable for vaccine use; Preferably, the vehicle further comprises a non-toxic excipient and / or a non-toxic adjuvant; Preferably, the vector containing the infectious genetic material is storage stable at ambient temperature and / or ultra-low temperature; More preferably, the vector containing the infectious genetic material is storage-stable without ultra-low temperatures; and Preferably, the vector comprising the infectious genetic material is stable for more than 12 weeks after lyophilization, optionally from about 12 weeks to about 52 weeks, optionally from about 12 weeks to about 24 weeks.

6. A virus containing infectious genetic material; wherein the infectious genetic material is the iRNA molecule of any one of claims 1 to 2, the iDNA molecule of claim 3, the composition of claim 4, and / or a combination thereof; wherein the virus is a recombinant virus; Preferably, the virus is homogenously pure, and / or attenuated, and / or contains stabilizing and attenuating mutations; Preferably, the virus is capable of inducing an immune response; Preferably, the immune response is an innate immune response, an adaptive immune response and / or a combination thereof; More preferably, the immune response is a persistent immunity; and More preferably, the immune response is a broadly cross-neutralizing immune response; More preferably, the virus is capable of inducing an immune response in a single dose; Preferably, the virus is suitable for use in a vaccine; Preferably, the virus is suitable for preventing and / or reducing and / or significantly reducing one or more symptoms in a subject in need thereof, The subject is preferably a mammal, more preferably a human; Preferably, the virus is suitable for administration by injection, including intramuscular injection, and / or subcutaneous injection, and / or a combination thereof; Preferably, the vector is used to prevent, reduce or significantly reduce viral infection and / or prevent or alleviate the symptoms of viral infection; and Preferably, the symptom is selected from the group consisting of fever, headache, rash, nausea, myalgia, arthralgia, respiratory failure, cardiovascular disease, hepatitis, skin effects, central nervous system abnormalities, and combinations thereof.

7. A vaccine comprising (i) infectious genetic material and / or vector and (ii) a non-toxic pharmacological excipient thereof, wherein the infectious genetic material is the iRNA molecule of any one of claims 1 to 2, the iDNA molecule of claim 3, the composition of claim 4, the vector of claim 5, and / or a combination thereof; Preferably, the infectious genetic material is homogeneously pure; Preferably, the infectious genetic material has a low percentage of single nucleotide polymorphisms; Preferably, the pharmacological excipient comprises a preservative and / or saline; and preferably, the saline is a phosphate buffered solution; Preferably, the vaccine further comprises an adjuvant; Preferably, the vaccine is a multivalent vaccine; Preferably, the vaccine comprises an effective amount of attenuated virus; Preferably, the effective dose of the vaccine is selected from about 1 ng to about 500,000 ng, about 5 ng to about 250,000 ng, about 6 ng to about 100,000 ng, about 7 ng to about 50,000 ng, about 8 ng to about 10,000 ng, about 9 ng to about 5,000 ng, about 10 ng to about 1,000 ng, about 10 ng to about 500 ng, about 10 ng to about 250 ng, about 10 ng to about 100 ng, about 10 ng to about 500 ng, about 10 ng to about 500 ng, about 10 ng to about 250 ng, about 10 ng to about 100 ng, about 10 ng to about 50 ng, about 10 ng to about 50 ng. ng to about 500 ng, about 50 ng to about 1,000 ng, about 100 ng to about 1,000 ng, about 100 ng to about 5,000 ng, about 100 ng to about 10,000 ng, about 500 ng to about 1,000 ng, about 500 ng to about 5,000 ng, about 500 ng to about 10,000 ng, about 500 ng to about 50,000 ng, about 500 ng to about 75,000 ng, and about 500 ng to about 100,000 ng of infectious genetic material; Preferably, the effective dose of the vaccine is selected from about 1 ng to about 500,000 ng, about 5 ng to about 250,000 ng, about 6 ng to about 100,000 ng, about 7 ng to about 50,000 ng, about 8 ng to about 10,000 ng, about 9 ng to about 5,000 ng, about 10 ng to about 100,000 ng, about 10 ng to about 75,000 ng, about 10 ng to about 50,000 ng, about 10 ng to about 25,000 ng, about 10 ng to about 10,000 ng, about 10 ng to about 7,500 ng, about 10 ng to about 50,000 ng, about 10 ng to about 25,000 ng, about 10 ng to about 10,000 ng, about 10 ng to about 7,500 ng, about 10 ng to about 5,000 ng, about 10 ng to about about 2,500 ng, about 10 ng to about 1,000 ng, about 10 ng to about 500 ng, about 10 ng to about 250 ng, about 10 ng to about 120 ng, about 10 ng to about 100 ng, about 10 ng to about 50 ng, about 10 ng to about 75 ng, about 50 ng to about 500 ng, about 50 ng to about 1,000 ng, about 100 ng to about 1,000 ng, about 100 ng to about 5,000 ng, about 100 ng to about 10,000 ng, about 500 ng to about 1,000 ng, about 500 ng to about 5,000 ng, about 500 ng to about 10,000 ng, about 500 ng to about 5,000 ng, about 500 ng to about 10,000 ng, about 500 ng to about 50,000 ng, about 500 ng to about 75,000 ng, and about 500 ng to about 100,000 ng of an iRNA molecule; Preferably, the effective dose of the vaccine is selected from about 1 ng to about 500,000 ng, about 5 ng to about 250,000 ng, about 6 ng to about 100,000 ng, about 7 ng to about 50,000 ng, about 8 ng to about 10,000 ng, about 9 ng to about 5,000 ng, about 10 ng to about 1,200 ng, about 10 ng to about 1,100 ng, about 10 ng to about 1,000 ng, about 10 ng to about 1,200 ng, about 10 ng to about 500 ng, about 10 ng to about 250 ng, about 10 ng to about 120 ng, about 10 ng to about 100 ng, about 10 ng to about 1,200 ng about 50 ng, about 50 ng to about 500 ng, about 50 ng to about 1,000 ng, about 100 ng to about 1,000 ng, about 100 ng to about 1,100 ng, about 100 ng to about 1,200 ng, about 100 ng to about 5,000 ng, about 100 ng to about 10,000 ng, about 500 ng to about 1,000 ng, about 500 ng to about 5,000 ng, about 500 ng to about 10,000 ng, about 500 ng to about 50,000 ng, about 500 ng to about 10,000 ng, about 500 ng to about 50,000 ng, about 500 ng to about 75,000 ng, and about 500 ng to about 100,000 ng of iDNA molecules; Preferably, the effective dose of the vaccine is selected from about 0.5 ng to about 100,000 ng, about 0.5 ng to about 75,000 ng, about 0.5 ng to about 50,000 ng, about 0.5 ng to about 25,000 ng, about 0.5 ng to about 10,000 ng, about 0.5 ng to about 7.500 ng, about 0.5 ng to about 5,000 ng, about 0.5 ng to about 2,500 ng, about 0.5 ng to about 1,000 ng, about 0.5 ng to about 500 ng, about 0.5 ng to about 250 ng, about 0.5 ng to about 120 ng, about 1 ng to about 100,000 ng, about 1 ng to about 75,000 ng, about 1 ng to about 50,000 ng, about 1 ng to about 25,000 ng, about 1 ng to about 10,000 ng, about 1 ng to about 7,500 ng, about 1 ng to about 5,000 ng, about 1 ng to about 2,500 ng, about 1 ng to about 1,000 ng, about 1 ng to about 500 ng, about 1 ng to about 250 ng, about 1 ng to about 120 ng, about 10 ng to about 120 ng, about 10 ng to about 119 ng, about 10 ng to about 10 ng, about 10 ng to about 109 ng, about 10 ng to about 101 ng, about 10 ng to about 102 ng, about 10 ng to about 103 ng, about 10 ng to about 104 ng, about 10 ng to about 105 ng, about 10 ng to about 106 ng, about 10 ng to about 107 ng, about 10 ng to about 108 ng, about 10 ng to about 109 ng, about 10 ng to about 101 ng, about 10 ng to about 102 ng, about 10 ng to about 103 ng, about 104 ng, about 10 ng to about 105 ng, about 10 ng to about 106 ng ng to about 102 ng, about 10 ng to about 101 ng, about 10 ng to about 100 ng, about 15 ng to about 120 ng, about 15 ng to about 119 ng, about 20 ng to about 118 ng, about 25 ng to about 117 ng, about 30 ng to about 116 ng, about 40 ng to about 115 ng, about 50 ng to about 114 ng, about 60 ng to about 113 ng, about 70 ng to about 112 ng, about 80 ng to about 111 ng, about 90 ng to about 110 ng of an iDNA molecule, wherein the iDNA molecule encodes a live attenuated CHIKV; Preferably, the effective dose of the vaccine is selected from about 0.5 ng to about 100,000 ng, about 0.5 ng to about 75,000 ng, about 0.5 ng to about 50,000 ng, about 0.5 ng to about 25,000 ng, about 0.5 ng to about 10,000 ng, about 0.5 ng to about 7.500 ng, about 0.5 ng to about 5,000 ng, about 0.5 ng to about 2,500 ng, about 0.5 ng to about 1,000 ng, about 0.5 ng to about 500 ng, about 0.5 ng to about 250 ng, about 0.5 ng to about 120 ng, about 1 ng to about 100,000 ng, about 1 ng to about 75,000 ng, about 1 ng to about 50,000 ng, about 1 ng to about 25,000 ng, about 1 ng to about 10,000 ng, about 1 ng to about 7,500 ng, about 1 ng to about 5,000 ng, about 1 ng to about 2,500 ng, about 1 ng to about 1,000 ng, about 1 ng to about 500 ng, about 1 ng to about 250 ng, about 1 ng to about 120 ng, about 10 ng to about 120 ng, about 10 ng to about 119 ng, about 10 ng to about 10 ng, about 10 ng to about 109 ng, about 10 ng to about 101 ng, about 10 ng to about 102 ng, about 10 ng to about 103 ng, about 10 ng to about 104 ng, about 10 ng to about 105 ng, about 10 ng to about 106 ng, about 10 ng to about 107 ng, about 10 ng to about 108 ng, about 10 ng to about 109 ng, about 10 ng to about 101 ng, about 10 ng to about 102 ng, about 10 ng to about 103 ng, about 104 ng, about 10 ng to about 105 ng, about 10 ng to about 106 ng ng to about 102 ng, about 10 ng to about 101 ng, about 10 ng to about 100 ng, about 15 ng to about 120 ng, about 15 ng to about 119 ng, about 20 ng to about 118 ng, about 25 ng to about 117 ng, about 30 ng to about 116 ng, about 40 ng to about 115 ng, about 50 ng to about 114 ng, about 60 ng to about 113 ng, about 70 ng to about 112 ng, about 80 ng to about 111 ng, about 90 ng to about 110 ng of an iRNA molecule, wherein the iRNA molecule encodes an attenuated live CHIKV; Preferably, the effective dose of the vaccine is selected from about 1 ng to about 200,000 ng, about 2 ng to about 150,000 ng, about 3 ng to about 100,000 ng, about 4 ng to about 90,000 ng, about 5 ng to about 80,000 ng, about 6 ng to about 70,000 ng, about 7 ng to about 60,000 ng, about 8 ng to about 50,000 ng, about 9 ng to about 40,000 ng, about 10 ng to about 30,000 ng, about 10 ng to about 20,000 ng, about 10 ng to about 19,000 ng, about 10 ng to about 18,000 ng, about 10 ng to about 17,000 ng, about 10 ng to about 19,000 ng, about 10 ng to about 18 ...8,000 ng, about 10 ng to about 18,000 ng, about 10 ng to about 18,000 ng, about 10 ng to about 18 about 10 ng to about 15,000 ng, about 10 ng to about 10,000 ng, about 10 ng to about 5,000 ng, about 10 ng to about 1,000 ng, about 10 ng to about 500 ng, about 100 ng to about 500 ng, about 100 ng to about 1,000 ng, about 500 ng to about 5,000 ng, about 500 ng to about 10,000 ng, about 500 ng to about 25,000 ng, about 500 ng to about 50,000 ng, about 500 ng to about 100,000 ng, and about 500 ng to about 150,000 ng of an iDNA molecule, wherein the iDNA molecule encodes a live, attenuated VEEV; Preferably, the effective dose of the vaccine is selected from about 1 ng to about 200,000 ng, about 2 ng to about 150,000 ng, about 3 ng to about 100,000 ng, about 4 ng to about 90,000 ng, about 5 ng to about 80,000 ng, about 6 ng to about 70,000 ng, about 7 ng to about 60,000 ng, about 8 ng to about 50,000 ng, about 9 ng to about 40,000 ng, about 10 ng to about 30,000 ng, about 10 ng to about 20,000 ng, about 10 ng to about 19,000 ng, about 10 ng to about 18,000 ng, about 10 ng to about 17,000 ng, about 10 ng to about 16 ,000 ng, about 10 ng to about 15,000 ng, about 10 ng to about 10,000 ng, about 10 ng to about 5,000 ng, about 10 ng to about 1,000 ng, about 10 ng to about 500 ng, about 100 ng to about 500 ng, about 100 ng to about 1,000 ng, about 500 ng to about 5,000 ng, about 500 ng to about 10,000 ng, about 500 ng to about 25,000 ng, about 500 ng to about 50,000 ng, about 500 ng to about 100,000 ng, and about 500 ng to about 150,000 ng of an iRNA molecule, wherein the iRNA molecule encodes an attenuated live VEEV; Preferably, the effective dose has a concentration selected from the following: about 10 3 PFU to 10 7 PFU, about 5x10 in 20ul 3 PFU to 5x10 6 PFU, about 10 in 20ul 4 PFU to 10 6 PFU, about 5x10 in 20ul 4 PFU to 5x10 5 PFU, about 6x10 in 20ul 4 PFU to 4x10 5 PFU, about 7x10 in 20ul 4 PFU to 3x10 5 PFU, about 8x10 in 20ul 4 PFU to 2x10 5 PFU, about 9x10 in 20ul 4 PFU to 1x10 5 PFU and about 1x10 in 20ul 5 PFU of infectious genetic material; Preferably, the effective dose is selected from about 10 PFU to about 10,000 PFU, about 10 PFU to about 5,000 PFU, about 10 PFU to about 3,100 PFU, about 10 PFU to about 3,000 PFU, about 10 PFU to about 2,500 PFU, about 50 PFU to about 5,000 PFU, about 50 PFU to about 3,100 PFU, about 75 PFU to about 2,500 PFU, about 100 PFU to about 1,000 PFU, about 200 PFU to about 800 PFU, about 300 PFU to about 700 PFU, about 400 PFU to about 600 PFU, about 500 PFU of infectious genetic material; Preferably, a single dose of the vaccine is in an effective amount that provides a therapeutic benefit to a subject in need thereof; Preferably, the vaccine has low adverse effects on subjects in need thereof; Preferably, the infectious genetic material is used to prevent, reduce or significantly reduce viral infection and / or prevent or alleviate the symptoms of viral infection; Preferably, the symptom is selected from the group consisting of fever, headache, rash, nausea, myalgia, arthralgia, respiratory failure, cardiovascular disease, hepatitis, skin effects, central nervous system abnormalities, and combinations thereof; Preferably, the vaccine has low adverse effects on subjects in need thereof; Preferably, the vaccine is administered by injection, including intramuscular injection and / or subcutaneous injection; by microinjection, including transdermal microinjection; and / or a combination thereof; and Optionally, the vaccine is administered by microneedle injection.

8. A vaccine comprising the attenuated virus according to claim 6 and a non-toxic pharmacological excipient thereof; Preferably, the pharmacological excipient comprises a preservative and / or saline; and preferably, the saline is a phosphate buffered solution; Preferably, the vaccine further comprises an adjuvant; Preferably, the vaccine is a multivalent vaccine; Preferably, the vaccine comprises an effective amount of the attenuated virus; Preferably, the effective dose has a concentration selected from the following: about 10 3 PFU to 10 7 PFU, about 5x10 in 20ul 3 PFU to 5x10 6 PFU, about 10 in 20ul 4 PFU to 10 6 PFU, about 5x10 in 20ul 4 PFU to 5x10 5 PFU, about 6x10 in 20ul 4 PFU to 4x10 5 PFU, about 7x10 in 20ul 4 PFU to 3x10 5 PFU, about 8x10 in 20ul 4 PFU to 2x10 5 PFU, about 9x10 in 20ul 4 PFU to 1x10 5 PFU, about 1x10 in 20ul 5 PFU of attenuated virus; Preferably, the effective dose is selected from about 1 PFU to about 100,000 PFU, about 2 PFU to about 100,000 PFU, about 5 PFU to about 50,000 PFU, about 10 PFU to about 10,000 PFU, about 10 PFU to about 5,000 PFU, about 10 PFU to about 3,100 PFU, about 10 PFU to about 3,000 PFU, about 10 PFU to about 2,500 PFU, about 50 PFU to about 5,000 PFU, about 60 PFU to about 2,500 PFU, about 70 PFU to about 1,500 PFU, about 75 PFU to about 1,300 PFU. about 80 PFU to about 1,200 PFU, about 90 PFU to about 1,100 PFU, about 100 PFU to about 1,000 PFU, about 200 PFU to about 800 PFU, about 300 PFU to about 700 PFU, about 400 PFU to about 600 PFU, about 500 PFU, about 50,000 PFU to about 500,000 PFU, about 10,000 PFU to about 100,000 PFU, about 5,000 PFU to about 50,000 PFU, about 2,500 PFU to about 25,000 PFU, and about 10,000 PFU of attenuated virus; Preferably, the effective dose is selected from about 500,000 to about 5,000,000, about 100,000 to about 1,000,000, about 50,000 to about 500,000, about 25,000 to about 250,000, and about 100,000 of an attenuated virus; Preferably, a single dose of the vaccine is in an effective amount that provides a therapeutic benefit to a subject in need thereof; Preferably, the vaccine has low adverse effects on subjects in need thereof; Preferably, the infectious genetic material is used to prevent, reduce or significantly reduce viral infection and / or prevent or alleviate the symptoms of viral infection; Preferably, the symptom is selected from fever, headache, rash, nausea, myalgia, arthralgia, respiratory failure, cardiovascular disease, hepatitis, skin effects, central nervous system abnormalities and / or combinations thereof; Preferably, the vaccine has low adverse effects on subjects in need thereof; Preferably, the vaccine is administered by injection, including intramuscular injection and / or subcutaneous injection; by microinjection, including transdermal microinjection; and / or a combination thereof; and Optionally, the vaccine is administered by microneedle injection.

9. A method for preventing and / or reducing and / or significantly reducing viral infection, comprising directly administering to a subject in need thereof an effective amount of the iRNA molecule of any one of claims 1 to 2, the iDNA molecule of claim 3, the composition of claim 4, the vector of claim 5, the attenuated virus of claim 6, the vaccine of claim 7 or 8, and / or a combination thereof.

10. A method for preventing and / or reducing and / or significantly reducing viral infection, comprising directly administering to a subject in need thereof an effective amount of the iRNA molecule of any one of claims 1 to 2, the iDNA molecule of claim 3, the composition of claim 4, the vector of claim 5, the attenuated virus of claim 6, the vaccine of claim 7 or 8, and / or a combination thereof.

11. A method for producing the iDNA molecule of claim 3, comprising cloning a stably attenuated RNA virus into a plasmid.

12. A method for producing the composition of claim 4, comprising mixing the iRNA of any one of claims 1 to 2 with a pharmaceutically acceptable non-toxic component.

13. A method for making the vector of claim 5, comprising mixing the iRNA of any one of claims 1 to 2, the iDNA molecule of claim 3, the composition of claim 4, and / or a combination thereof with a lipid and an emulsifier.

14. A method for producing the virus of claim 6, comprising transfecting eukaryotic cells with the iDNA molecule of claim 12.

15. A method for producing the vaccine of claim 7 or 8, comprising mixing the iRNA molecule of any one of claims 1 to 2, the iDNA molecule of claim 3, the composition of claim 4, the vector of claim 5, the attenuated virus of claim 6 and / or a combination thereof with a non-toxic pharmacological excipient.

16. A method for making a multivalent vaccine, comprising mixing one or more iRNA molecules according to any one of claims 1 to 2, the iDNA molecules according to claim 3, the composition according to claim 4, the vector according to claim 5, the attenuated virus according to claim 6 and / or a combination thereof with a non-toxic pharmacological excipient; wherein the one or more attenuated live viruses encoded in the iRNA and / or iDNA are different.

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