Compositions and methods for preventing and treating rabies virus infection

Abstract

The present disclosure generally relates to the field of molecular virology, the present invention relates to nucleic acid molecules, and in particular to nucleic acid molecules encoding modified alphavirus viral genomes or self-replicating RNA (srRNA) constructs, recombinant cells and pharmaceutical compositions containing said nucleic acid molecules and the use of such nucleic acid molecules, recombinant cells and compositions for producing desired products in cell cultures or in vivo. Also provided are methods for eliciting an immune response in a subject in need thereof, as well as methods for preventing and / or treating a rabies virus infection.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Patent Application No. 17 / 720,002, filed on April 13, 2022, the disclosure of which is incorporated herein by reference in its entirety (including any drawings). Incorporation by Reference into the Sequence Listing

[0002] The material in the attached sequence listing is hereby incorporated into this application by reference. The attached sequence listing file is named 2023-04-06Sequence_Listing_ST26 058462-510001WO.xml, created on April 6, 2023, and is 72,426 bytes in size. Technical Field

[0003] The present disclosure relates to the fields of molecular virology and immunology, and in particular to nucleic acid molecules encoding modified alphavirus genomes or self-replicating RNA (srRNA), recombinant cells and pharmaceutical compositions containing the nucleic acid molecules, and the use of such nucleic acid molecules, recombinant cells and compositions for producing desired products in cell culture or in vivo. Also provided are methods for eliciting an immune response in a subject in need thereof, and methods for preventing and / or treating rabies virus infection. Background Art

[0004] Rabies is a viral zoonosis that is endemic in more than 100 countries and regions and poses a threat to more than 3 billion people. After the onset of clinical symptoms without post-exposure prophylaxis, the disease is always fatal (WHO epidemiological record 2010. 32 (85): 309-320. Rabies vaccine: WHO position paper). The WHO estimates that there are 55,000 rabies-related deaths each year and more than 10 million people receive post-exposure treatment (PET).

[0005] Currently available rabies vaccines include the most widely used but high-risk neural tissue vaccines, or the safer but more expensive cell culture and embryonated egg vaccines (CCEEV). Risks associated with neural tissue vaccines include: induction of autoimmune central nervous system disease due to their inherent myelin content; requirement for multiple injections; and unreliable efficacy. WHO does not recommend the use of neural tissue vaccines and strongly encourages increased supply of modern, high-quality vaccines to poor populations. Avian embryo vaccines and cell culture vaccines contain inactivated, purified virus and do not contain neural proteins. Although cell culture production methods are safer and more immunogenic than neural tissue vaccines, they are time-consuming and resource-intensive, and the associated cost burden largely limits their use to developed countries despite current WHO recommendations.

[0006] Preexposure prophylaxis (PrEP) using cell culture vaccines is safe and recommended for individuals at increased risk (e.g., laboratory personnel, veterinarians, animal trainers, wildlife workers, and travelers to rabies-endemic areas) but is largely limited to developed countries because of cost. In addition, antirabies vaccines are recommended for travelers to African and Asian countries where rabies is endemic.

[0007] The current problem is that these vaccines are in short supply and at some point these vaccines can only be used for post-exposure prophylaxis and not for preventive vaccination. However, preventive vaccination is important for travelers to developing countries where rabies Ig for post-exposure prophylaxis may not be available.

[0008] Therefore, there is a need for safe and effective rabies vaccines that are readily available.

[0009] The disclosure provided herein provides solutions to problems present in previous attempts to produce rabies vaccines and potentially provides improved methods for the treatment and prevention of rabies infection. Summary of the invention

[0010] The present disclosure generally relates to the development of immunotherapeutics (such as recombinant nucleic acid constructs and pharmaceutical compositions comprising recombinant nucleic acid constructs) for the prevention and management of rabies virus infection. In particular, as described in more detail below, some embodiments of the present disclosure provide nucleic acid constructs containing sequences encoding modified alphavirus genomes or self-replicating RNA (srRNA), wherein at least a portion of the nucleic acid sequences encoding viral structural proteins of the modified alphavirus genome or srRNA has been replaced by the coding sequence of a polypeptide construct comprising an antigenic determinant of the envelope glycoprotein G (RABV-G) of rabies virus, its variants, or either. Also disclosed are recombinant cells that have been engineered to contain one or more nucleic acid constructs disclosed herein; methods for producing target molecules; and pharmaceutical compositions comprising one or more of the following: (a) nucleic acid constructs of the present disclosure, (b) recombinant cells of the present disclosure, or (c) pharmaceutical compositions of the present disclosure. Particular aspects of the present disclosure further provide compositions and methods for eliciting an immune response in a subject in need thereof, and / or for preventing and / or treating rabies virus infection. The foregoing summary is illustrative only and is not intended to be limiting in any way. In addition to the illustrative embodiments and features described herein, other aspects, embodiments, objects, and features of the present disclosure will become fully apparent from the drawings, detailed description, and claims.

[0011] In one aspect of the present disclosure, provided herein is a nucleic acid construct comprising a nucleic acid sequence encoding a modified alphavirus genome or self-replicating RNA (srRNA), wherein at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or srRNA has been replaced by a coding sequence of a polypeptide construct comprising an antigenic determinant of the envelope glycoprotein G of rabies virus (RABV-G), a variant thereof, or either of the two.

[0012] In some embodiments, the antigenic determinant is located within the N-terminal half of RABV-G.

[0013] In some embodiments, the antigenic determinant is located within the C-terminal half of RABV-G.

[0014] In some embodiments, the antigenic determinant comprises antigenic site I, antigenic site II, antigenic site III, antigenic site IV, small antigenic site A, or a combination of any thereof, of the RABV-G.

[0015] In some embodiments, the envelope glycoprotein G is the envelope glycoprotein G of a virulent rabies virus strain or an avirulent rabies virus strain.

[0016] In some embodiments, the envelope glycoprotein G is an envelope glycoprotein G of a rabies virus strain selected from the group consisting of Flury LEP, Flury LEP-C, Flury HEP, 1088, AT6, CQ92, CVS-11, CVS-26, CVS-26 (G-N204S), CYN1009D, CYN1026D, CYN1029D, CYN1138D, CYN1140D, CYN1141D, CYN1242H, CYN1243D, CYN1244D, CYN1245 D strain, CYN1247D strain, CYN1249D strain, CYN1250D strain, CYN1251D strain, CYN1252D strain, CYN1253D strain, CYN1255D strain, CYN 1256D strain, CYN1257D strain, CYN1259D strain, CYN1260D strain, CYN1261D strain, GX4 strain, H-08-1320 strain, H-1413-09 strain, IP 1586 / 10 strain, IP 2990 / 13 strain, IP 2991 / 13 strain, IP 2992 / 13 strain, IP 3176 / 09 strain, IP 4005 / 12 strain, IP 412 / 10 strain, IP 542 / 10 strain, IP 7941 / 09 strain, J strain, JX-08-47 strain, JX08-48 strain, Kyoto strain, Kyoto (G-S204N) strain, N.HL strain, RC.HL strain, rHEP5.0-CVSG strain, RRV ON-99-2 strain, SAD-B19 strain, SH06 strain, SHRBV-18 strain, SNK-CTN strain, SRV9 strain, Street Alabama Dufferin (HCP-SAD) strain, VRC-RZ2 strain, ZJ-LA strain and ZJ-QZ strain. In some embodiments, the envelope glycoprotein G is the envelope glycoprotein G of the Flury LEP strain.

[0017] In some embodiments, the polypeptide construct comprises a molecular alteration that stabilizes an antigenic determinant of the RABV-G, a variant thereof, or either.

[0018] In some embodiments, the modified alphavirus genome or srRNA does not include a nucleic acid sequence encoding a viral structural protein.

[0019] In some embodiments, the nucleic acid sequence encoding the polypeptide construct is operably linked to a promoter sequence. In some embodiments, the promoter sequence is a 26S subgenomic (sg) promoter.

[0020] In some embodiments, the modified alphavirus genome or srRNA is a modified alphavirus genome or srRNA of an alphavirus belonging to the VEEV / EEEV / WEEV group, or the SFV group or the SINV group. In some embodiments, the alphavirus is Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Chikungunya virus (CHIKV), Western equine encephalitis virus (WEEV) or Sindbis virus (SINV).

[0021] In some embodiments, the nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 6-11.

[0022] In one aspect, provided herein is a recombinant cell comprising a nucleic acid construct as disclosed herein. In some embodiments, the recombinant cell is a mammalian cell or an insect cell.

[0023] In yet another aspect, provided herein is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a nucleic acid construct of the present disclosure.

[0024] In some embodiments, the composition is formulated into a delivery system with a delivery vehicle, wherein the delivery system includes a liposome, a viral replicon particle (VRP), a lipid-based nanoparticle (LNP), a polymer nanoparticle, a physiological buffer, a microsphere, an immunostimulatory complex (ISCOM), a conjugate of a bioactive ligand, or a combination of any thereof. In some embodiments, lipid is present in a mass ratio of about 100:1 to about 4:1 of lipid and RNA. In some embodiments, the lipid-based nanoparticle has an average diameter of about 25nm to about 1000nm. In some embodiments, the composition is formulated as a vaccine or an adjuvant. In some embodiments, the composition is formulated for intramuscular administration.

[0025] In another aspect, provided herein is a method for inducing an immune response or treating rabies infection in a subject in need thereof. The method comprises administering to the subject a composition comprising a nucleic acid construct of the present disclosure. In some embodiments, the method is a method for inducing an immune response. In some embodiments, the immune response is a neutralizing antibody response. In some embodiments, the neutralizing antibody response comprises a neutralizing antibody titer equal to or greater than 0.5 IU / mL. In some embodiments, the composition is administered to the subject as a single prevention or therapy (monotherapy) alone or as a first therapy in combination with at least one additional therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description and accompanying drawings which set forth illustrative embodiments utilizing the principles of the present disclosure, in which:

[0027] Figure 1 It is a bar graph showing RNA replication from different srRNA vectors encoding rabies virus glycoprotein (RABV-G). srRNA was transfected into 7.7E5 BHK-21 cells with 50 or 500 ng by nuclear transfection, and cells were collected 15 hours later and stained with anti-dsRNA antibodies conjugated with AF488. The frequency of dsRNA positive cells was determined by flow cytometry (FC).

[0028] Figure 2 It is a bar graph showing the relative expression of rabies virus glycoprotein (RABV-G) from different srRNA vectors. The srRNA encoding the RABV-G gene was transfected into 7.7E5 BHK-21 cells by nuclear transfection at 50 or 500 ng, and cells were collected 15 hours later and stained with anti-RabG antibody (1c5) conjugated with AF647. The mean fluorescence intensity (MFI) of the transfected cells was determined by flow cytometry (FC).

[0029] Figure 3 It is shown that srRNA based rabies vaccines can generate T cell responses in vivo (ELISpot).T cell responses measured by IFNγ ELISpot 14 days after a single dose of srRNA-RABV-G (0.15ug) are shown. Figure 3 Results are shown along with corresponding statistics for wild-type RABV-G (**<0.01).

[0030] Figure 4 It is shown that rabies vaccines based on srRNA can produce protective antibody responses in vivo. Neutralizing antibody responses measured by rapid fluorescent focus inhibition test (RFFIT) 14 days after a single dose of srRNA-RABV-G (0.15ug) are shown. Neutralizing antibody titers of 0.5IU / ml that are considered to be protective are depicted with blue lines. Statistical data (**<0.01; *<0.05) are shown. DETAILED DESCRIPTION

[0031] The present disclosure generally relates to a nucleic acid construct expressing a variant or antigenic determinant of RABV-G for the purpose of preventive and therapeutic treatment of rabies virus infection. These constructs solve the problem that current rabies vaccines need to use a 3-dose regimen to induce immune protection in a short period of time and need to be regularly inoculated with booster shots due to the poor persistence of immune response. This article particularly provides a gene expression system with excellent expression potential, which is suitable for expressing in recombinant cells the coding sequence of a polypeptide construct comprising an antigenic determinant of the envelope glycoprotein G (RABV-G) of rabies virus, its variants, or either of the two. For example, some embodiments of the present disclosure relate to a nucleic acid construct (e.g., expression construct and vector) containing a modified alphavirus genome or srRNA, wherein at least a portion of the nucleic acid sequence encoding the viral structural protein of the modified alphavirus genome or srRNA has been replaced by a coding sequence of a polypeptide construct comprising an antigenic determinant of the envelope glycoprotein G (RABV-G) of rabies virus, its variants, or either of the two. A recombinant cell is further provided, which is genetically engineered to contain one or more of the nucleic acid molecules disclosed herein. Biological materials and recombinant products derived from such recombinant cells are also within the scope of the present application. Also provided are compositions and methods useful for eliciting an immune response or treating rabies infection in a subject in need thereof.

[0032] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0033] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although for clarity, the present disclosure may be described herein in the context of multiple separate embodiments, the present disclosure may also be implemented in a single embodiment. definition

[0034] Unless otherwise defined, all technical terms, symbols and other scientific terms or expressions used herein are intended to have the meanings commonly understood by those skilled in the art to which this application belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and these definitions contained herein need not be interpreted as representing substantial differences from the meanings commonly understood in the art. Many of the techniques and procedures described or mentioned herein are well understood by those skilled in the art and are generally adopted by those skilled in the art using conventional methods.

[0035] Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. For example, the term "cell" includes one or more cells, including mixtures thereof. "A and / or B" is used herein to include all of the following alternatives: "A", "B", "A or B", and "A and B".

[0036] As used herein, the terms "administration" and "administering" refer to the delivery of a biologically active composition or formulation by an administration route including, but not limited to, intranasal, transdermal, intravenous, intraarterial, intramuscular, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration or a combination thereof. The terms include, but are not limited to, administration by a medical professional and self-administration.

[0037] The terms "cell", "cell culture" and "cell line" refer not only to a specific subject cell, cell culture or cell line, but also to the offspring or potential offspring of such a cell, cell culture or cell line, without regard to the number of transfers or passages in culture. It should be understood that not all offspring are exactly the same as the parent cell. This is because certain modifications may occur in the offspring due to mutations (e.g., intentional or unintentional mutations) or environmental influences (e.g., methylation or other epigenetic modifications), so that the offspring may actually be different from the parent cell, but still be included in the scope of the term as used herein, as long as the offspring retains the same function as the original cell, cell culture or cell line.

[0038] The term "construct" refers to a recombinant molecule, for example, a recombinant nucleic acid or polypeptide, including one or more nucleic acid sequences or amino acid sequences from a heterologous source. For example, a polypeptide construct can be a chimeric polypeptide molecule, in which two or more amino acid sequences from different sources are operably connected to each other in a single polypeptide construct. Similarly, a nucleic acid construct can be a chimeric nucleic acid molecule, in which the nucleic acid sequences from two or more different sources are assembled into a single nucleic acid molecule. A representative nucleic acid construct can include any recombinant nucleic acid molecule, which is a linear or circular, single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source (such as a plasmid, a clay, a virus, a self-replicating polynucleotide molecule, a bacteriophage), capable of genome integration or self-replication, including nucleic acid molecules in which one or more nucleic acid sequences are operably connected. Two or more nucleic acid constructs can be included in a single nucleic acid molecule (such as a single vector), or can be included in two or more independent nucleic acid molecules (such as two or more independent vectors).

[0039] The terms "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" of a composition (e.g., a nucleic acid construct, srRNA, recombinant cell, and / or pharmaceutical composition) of the present disclosure generally refer to an amount sufficient to enable the composition to achieve the stated purpose (e.g., to achieve the effect of its administration, stimulate an immune response, prevent or treat a disease, or alleviate one or more symptoms of a disease, disorder, infection, or health condition) relative to the absence of the composition. An example of an "effective amount" is an amount sufficient to facilitate the treatment, prevention, or alleviation of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount." "Allowance" of a symptom means a reduction in the severity or frequency of one or more symptoms or the elimination of one or more symptoms. The exact amount of the composition (including a "therapeutically effective amount") will depend on the purpose of the treatment, and will be determined by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (Vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th edition, 2003, Gennaro, ed., Lippincott, Williams & Wilkins).

[0040] As used herein, the term "naked" is substantially free of other macromolecules, such as lipids, polymers and proteins, when referring to nucleic acids. "Naked" nucleic acids (such as self-replicating RNA) are not formulated with other macromolecules to improve cellular uptake. Therefore, naked nucleic acids are not encapsulated, absorbed or combined by liposomes, microparticles, nanoparticles, cationic emulsions, etc.

[0041] As used herein, the term "operably connected" represents the physical or functional connection between two or more elements (e.g., polypeptide sequence or polynucleotide sequence), which allows them to operate in their expected manner. For example, when used in the context of the coding sequence and promoter sequence in nucleic acid molecules as described herein or nucleic acid molecules, the term "operably connected" means that the coding sequence and promoter sequence are in the frame and in the appropriate space and distance away, so as to allow the corresponding combination of transcription factors or RNA polymerase to exert an influence on transcription. It should be understood that the elements operably connected can be continuous or discontinuous (e.g., connected to each other by a joint). In the context of polypeptide constructs, "operably connected" refers to the physical connection (e.g., directly or indirectly connected) between amino acid sequences (e.g., different sections, parts, regions or domains) to provide the desired activity of constructs. The operably connected sections, parts, regions and domains of polypeptides disclosed herein or nucleic acid molecules can be continuous or discontinuous (e.g., connected to each other by a joint).

[0042] As used herein, the term "portion" refers to a fraction. The term "portion" with respect to a specific structure thereof (such as a polynucleotide sequence or an amino acid sequence or a protein) can represent a continuous or discontinuous portion of the structure. For example, a portion of an amino acid sequence comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% and at least 90% of the amino acids of the amino acid sequence. Additionally or alternatively, if the portion is a discontinuous portion, the discontinuous portion is composed of 2, 3, 4, 5, 6, 7, 8 or more portions of a structure (such as a domain of a protein), each of which is a continuous element of the structure. For example, a discontinuous portion of an amino acid sequence may consist of 2, 3, 4, 5, 6, 7, 8 or more, such as no more than 4, portions of the amino acid sequence, wherein each portion comprises at least 1, at least 2, at least 3, at least 4, at least 5 consecutive amino acids, at least 10 consecutive amino acids, at least 20 consecutive amino acids, or at least 30 consecutive amino acids of the amino acid sequence.

[0043] In the case of providing a range of values, it should be understood that unless the context clearly specifies otherwise, each intermediate value (to one tenth of the unit of the lower limit) between the upper and lower limits of the range and any other stated or intermediate values ​​in the stated range are all included in the present disclosure. These upper and lower limits of smaller ranges can be independently included in smaller ranges and are also included in the present disclosure, subject to any definite exclusion of limits in the stated range. In the case where the stated range includes one or two limits, the scope excluding any one or two of those included limits is also included in the present disclosure.

[0044] Certain ranges are presented herein by numerical values ​​preceded by the term "about". The term "about" is used herein to provide textual support for the precise number thereafter and the number of the number close to or approximating the number after the term. When determining whether a number is close to or approximate to a specifically listed number, the close or approximate unlisted number can be a number that provides the basic equivalent form of the specifically listed number in the context in which it is presented. If the approximate degree is not clear according to the context, "about" means within plus or minus 10% of the value provided, or rounded to the nearest significant figure, including the value provided in all cases. In some embodiments, the term "about" indicates a specified value ± at most 10%, at most ± 5%, or at most ± 1%.

[0045] In the context of two or more nucleic acids or proteins, the term "percent identity" as used herein refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acids (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity when compared and aligned over a comparison window or specified region to obtain maximum correspondence), as measured using BLAST or BLAST 2.0 sequence comparison algorithms using default parameters as described below or by manual alignment and visual inspection. See, for example, the NCBI website at ncbi.nlm.nih.gov / BLAST. Such sequences are then referred to as "substantially identical". This definition also relates to or can be applied to the complement of a sequence. This definition also includes sequences with deletions and / or additions and those with substitutions. Sequence identity can be calculated using published techniques and widely available computer programs such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol 215:403, 1990). Sequence identity can be measured using sequence analysis software using its default parameters, such as the sequence analysis software package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wisconsin 53705).

[0046] As used herein, the term "pharmaceutically acceptable excipient" refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administering one or more compounds of interest to a subject. Thus, a "pharmaceutically acceptable excipient" may encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc., that are compatible with drug administration. Supplementary active compounds (e.g., antibiotics and additional therapeutic agents) may also be incorporated into the composition.

[0047] As used herein, "subject" or "individual" includes animals, such as humans (e.g., human individuals) and non-human animals. In some embodiments, a "subject" or "individual" is a patient under the care of a doctor. Therefore, the subject can be a human patient or individual who suffers from, is at risk of suffering from, or is suspected of suffering from one or more symptoms of a health condition (e.g., rabies infection) and / or a health condition. The subject can also be an individual who is diagnosed as having a risk of a health condition at the time of diagnosis or thereafter. The term "non-human animal" includes all vertebrates, such as mammals (e.g., rodents, such as mice, non-human primates) and other mammals (e.g., sheep, dogs, cattle, chickens) and non-mammals (e.g., amphibians, reptiles, etc.).

[0048] It should be understood that aspects and embodiments of the present disclosure described herein include "comprising aspects and embodiments," "consisting of aspects and embodiments," and "consisting essentially of aspects and embodiments." As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any element, step, or ingredient not specified in the claimed composition or method. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. The term "comprising" in any recitation herein, particularly in the description of components of a composition or in the description of steps of a method, is understood to cover those compositions and methods consisting essentially of and consisting of the recited components or steps.

[0049] All genes, gene names and gene products disclosed herein are intended to correspond to homologs of any species to which the compositions and methods disclosed herein are applicable. Therefore, the term includes, but is not limited to, genes and gene products from humans and mice. It should be understood that when disclosing genes or gene products from specific species, the disclosure is only intended to be exemplary and should not be interpreted as restrictive unless the context in which it appears clearly indicates. Therefore, for example, for genes or gene products disclosed herein, the term relates to mammalian nucleic acids and amino acid sequences in some embodiments, and is intended to cover homologous and / or orthologous genes and gene products from other animals (including but not limited to other mammals, fish, amphibians, reptiles and birds). In some embodiments, genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides and proteins are human. The term "gene" is also intended to include variants thereof.

[0050] It should be understood that certain features of the present disclosure described in the context of a separate embodiment for clarity may also be provided in combination in a single embodiment. On the contrary, various features of the present disclosure described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination. All combinations of embodiments belonging to the present disclosure are specifically encompassed in the present disclosure and disclosed herein as if each and every combination were individually and clearly disclosed. In addition, all sub-combinations of various embodiments and elements thereof are also specifically encompassed in the present disclosure and disclosed herein as if each and every such sub-combination were individually and clearly disclosed herein. Alphavirus

[0051] Alphaviruses are small enveloped RNA viruses with a single-stranded positive-sense RNA genome. The genus Alphaviruses includes, in particular, Sindbis virus (SINV), Semliki Forest virus (SFV), Ross River virus (RRV), Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV), which are all closely related and can infect various vertebrates (such as mammals, rodents, fish, birds) and large mammals (such as humans and horses) as well as invertebrates (such as insects). In particular, Sindbis virus and Semliki Forest virus have been extensively studied, and the life cycle, replication pattern, etc. of these viruses are all well characterized.

[0052] The alphavirus genome is approximately 12Kb long and consists of two open reading frames (ORFs): a 7Kb frame encoding nonstructural proteins (nsP) and a 4Kb frame encoding structural polyproteins. The nonstructural polyprotein (nsP) is cleaved into four different proteins (nsP1, nsP2, nsP3 and nsP4), which are essential for the transcription and translation of viral mRNA in the host cell cytoplasm.

[0053] The nsP1 protein is an mRNA capping enzyme with guanine-7-methyltransferase (MTase) and guanylate transferase (GTase) activities that direct the methylation and capping of newly synthesized viral genomic and subgenomic RNAs. The MTase motif in the N-terminal domain of nsP1 catalyzes the transfer of a methyl group from S-adenosylmethionine (AdoMet) to the N7 position of a GTP molecule (m7Gppp). GTase then binds m7Gppp, forming a covalent linkage with the catalytic histidine (m7Gp-GTase) and releasing PPi. GTase then transfers the m7Gp molecule to the 5'-diphosphate RNA, generating m7GpppNp-RNA. The resulting cap structure is essential for viral mRNA translation and protects the mRNA from degradation by cellular 5' exonucleases. Following the N-terminal domain are features that allow the nsP1 protein to associate with the cell membrane. The presence of the α-helical amphipathic loop and the palmitoylation site allows the nsP1 protein and the replication complex containing nsP1 to be anchored to the plasma membrane, probably through the interaction of nsP1 with membrane anionic phospholipids.

[0054] The nsP2 protein has multiple enzymatic activities and functional roles. The N-terminal region contains a helicase domain with seven characteristic motifs of superfamily 1 (SF1) helicases. It functions as an RNA triphosphatase that performs the first viral RNA capping reaction. The nsP2 protein also functions as a nucleotide triphosphatase (NTPase), enhancing RNA helicase activity. The C-terminal region of nsP2 contains a papain-like cysteine ​​protease that is responsible for processing the viral nonstructural polyprotein. The protease recognizes conserved motifs within the polyprotein. This protease is highly regulated and is modulated by other domains of nsP2. Alphavirus nsP2 proteins have also been described as virulence factors, responsible for transcriptional and translational shutdown in infected host cells, and for suppressing interferon (IFN)-mediated antiviral responses, thereby contributing to viral factors controlling the translation machinery.

[0055] The exact role or roles of the alphavirus nsP3 protein in the replication complex are less clear. The nsP3 protein has three recognized domains: an N-terminal macrodomain with phosphatase activity and nucleic acid binding ability, an alphavirus unique domain (AUD), and a C-terminal hypervariable domain. It has been demonstrated that deletion of this domain in SFV nsP3 results in low viral pathogenicity, indicating its importance in the regulation of viral RNA transcription.

[0056] Compared to other alphavirus nsP4s, the nsP4 polymerase is the most highly conserved protein among alphaviruses, with the most divergent amino acid sequences being >50% identical. nsP4 contains a core RNA-dependent RNA polymerase (RdRp) domain at the C-terminus that has been identified as solely responsible for the RNA synthesis properties of the viral replication complex. The RdRp participates in the replication of genomic RNA as well as the transcription of the 26S subgenomic RNA via negative strand RNA. The N-terminal domain is alphavirus specific and may be partially disordered in structure.

[0057] The 5'-end two-thirds of the alphavirus genome encodes a variety of nonstructural proteins (nsPs) necessary for viral RNA transcription and replication. These proteins are translated directly from RNA and, together with cellular proteins, form an RNA-dependent RNA polymerase that is essential for viral genome replication and sgRNA transcription. Four nsPs (nsP1-4) are produced as a single polyprotein, constituting the viral replication machinery. The processing of the polyprotein occurs in a highly regulated manner, with cleavage at the P2 / 3 junction affecting the use of RNA templates during genome replication. This site is located at the bottom of a narrow cleft and is not easily accessible. Once cleaved, nsP3 creates a ring structure that surrounds nsP2. The two proteins have an extensive interface. Mutations in nsP2 that produce non-cytopathic viruses or temperature-sensitive phenotypes cluster in the P2 / P3 interface region. P3 mutations relative to the position of nsP2 non-cytopathic mutations prevent efficient cleavage of P2 / 3. This in turn affects RNA infectivity, thereby altering viral RNA production levels.

[0058] The 3' end of the genome contains sgRNA, which is used as a translation template for all structural proteins required to form viral particles (core nucleocapsid protein C and envelope proteins P62 and E1 associated as heterodimers). The surface glycoproteins anchored by the viral membrane are responsible for receptor recognition and enter the target cell through membrane fusion. sgRNA is transcribed from the p26S subgenomic promoter present at the 3' end of the RNA sequence encoding the nsp4 protein. The proteolytic maturation of P62 to E2 and E3 can cause changes in the surface of the virus. E1, E2, and sometimes E3, glycoprotein "spikes" together form E1 / E2 dimers or E1 / E2 / E3 trimers, in which E2 extends from the center to the apex, E1 fills the space between the apex, and E3 (if present) is located at the distal end of the spike. When the virus is exposed to the acidity of the endosome, E1 dissociates from E2 to form an E1 homotrimer, which is necessary to drive the fusion step of the cell membrane with the viral membrane. The alphavirus glycoprotein E1 is a class II viral fusion protein that is structurally distinct from the class I fusion proteins found in influenza virus and HIV. The E2 glycoprotein functions through its cytoplasmic domain to interact with the nucleocapsid, while its extracellular domain is responsible for binding to cellular receptors. Most alphaviruses have lost the peripheral protein E3, while in Semliki virus it remains associated with the viral surface.

[0059] Alphavirus replication is reported to occur on the membrane surface within the host cell. In the first step of the infection cycle, the 5' end of the genomic RNA is translated into a polyprotein (nsP1-4) with RNA polymerase activity, which produces a negative strand complementary to the genomic RNA. The sequence at the 3' end of the genomic RNA plays an important role in initiating negative strand synthesis, where a minimum number of adenylate residues have been identified as necessary for replication to occur. In particular, it has been previously reported that for the alphavirus genome to be replicated, there must be at least 11 residues in the poly(A) tail following the 3'UTR to effectively initiate negative strand synthesis, and thus replication to occur. It has been previously reported that extending the poly(A) tail to 25 residues resulted in enhanced replication, but no further enhancement of replication was observed when the poly(A) was further extended to 34 residues. In addition, internal non-A residues in the poly(A) are generally detrimental to replication, suggesting that enzymatic poly(A) tailing has no benefit for srRNAs that do not contain only 3' adenylate residues following the 3'UTR. It has been previously reported that there is no enhancement of negative strand synthesis on RNA templates with more than 25 adenylic acid residues in the poly (A) tail. In the second step of replication, the negative strand is used as a template to produce the following two RNAs: (1) a positive genomic RNA corresponding to the genome of the secondary virus that produces other nsPs by translation and serves as the viral genome; and (2) sgRNA encoding the structural proteins of the virus that forms the infectious particles. The positive genomic RNA / sgRNA ratio is regulated by the proteolytic self-cleavage of the polyprotein and nsP1, nsP2, nsP3 and nsP4. In fact, viral gene expression is carried out in two stages. In the first stage, the positive genomic strand and the negative strand are mainly synthesized. During the second stage, the synthesis of sgRNA is actually exclusive, thus resulting in the production of a large amount of structural proteins. Self-replicating RNA

[0060] As will be understood by the skilled artisan, the term "self-replicating RNA" refers to an RNA molecule that contains all the genetic information required to direct its own self-amplification or self-replication within a permissive cell. To direct its own replication, the srRNA typically (1) encodes a polymerase, replicase, or other protein that may interact with a viral or host cell-derived protein, nucleic acid, or ribonucleoprotein to catalyze the RNA amplification process; and (2) contains cis-acting RNA sequences required for the replication and transcription of subgenomic RNA (sgRNA). These sequences may bind to their own encoded proteins or non-self-encoded cell-derived proteins, nucleic acids, or ribonucleoproteins, or complexes between any of these components during the replication process. In some embodiments of the present disclosure, the alphavirus srRNA construct typically contains the following elements: one or more 5' viral or defective interfering RNA sequences required for cis replication, sequences encoding biologically active alphavirus nonstructural proteins (e.g., nsP1, nsP2, nsP3, and nsP4), the subgenomic promoter (sg) of the sgRNA, 3' viral sequences required for cis replication, and an optional polyadenylic acid stretch (poly(A)). In some cases, a subgenomic promoter (sg) that directs expression of a heterologous sequence may be included in the srRNA constructs of the disclosure.

[0061] In addition, the term srRNA generally refers to a molecule of positive polarity or "information" sense, and the length of the srRNA may be different from the length of any known, naturally occurring alphavirus srRNA. In some embodiments of the present disclosure, the srRNA does not contain at least a portion of the coding sequence for one or more alphavirus structural proteins; and / or the sequence encoding the structural gene may be replaced by a heterologous sequence. In these cases, when the srRNA is packaged into a recombinant alphavirus particle, it may contain one or more sequences, so-called packaging signals, for initiating interaction with the alphavirus structural proteins, thereby leading to particle formation.

[0062] The srRNA constructs of the present disclosure generally have a length of at least about 2 kb. For example, the srRNA can have a length of at least about 2 kb, at least about 3 kb, at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb, or more than 12 kb. In some embodiments, the srRNA may have a length of about 4 kb to about 20 kb, about 4 kb to about 18 kb, about 5 kb to about 16 kb, about 6 kb to about 14 kb, about 7 kb to about 12 kb, about 8 kb to about 16 kb, about 9 kb to about 14 kb, about 10 kb to about 18 kb, about 11 kb to about 16 kb, about 5 kb to about 18 kb, about 6 kb to about 20 kb, about 5 kb to about 10 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about In some embodiments, the srRNA may have a length of about 6 kb to about 14 kb. In some embodiments, the srRNA may have a length of about 6 kb to about 14 kb. In some embodiments, the srRNA may have a length of about 6 kb to about 16 kb. Rabies virus envelope glycoprotein G (RABV-G)

[0063] The rabies virus is bullet-shaped, about 180 nm in length and 75 nm in diameter. The genome is a non-segmented, negative-sense, single-stranded RNA. The genome encodes five structural proteins: 3'NPMGL 5'; N (nucleoprotein), P (phosphoprotein), M (matrix protein), G (glycoprotein) and L (RNA-dependent RNA polymerase). The leading and trailing untranslated regions at the 3' and 5' ends of the genome flank the structural genes, and there are non-coding intergenic sequences between the protein coding regions; NP, PM, MG, GL.

[0064] Of the five structural proteins, G is present on the envelope and is the only protein that is exposed. Glycoproteins are proteins covalently bound to sugar units. Rabies virus glycoprotein (RABV-G) is a type I trimeric transmembrane protein, a single transmembrane type with an extracellular N-terminus and a cytoplasmic C-terminus relative to the cell membrane. The length of the precursor RABV-G is 524 amino acids (aa) (522 aa in Mokola virus) and consists of a 19 aa signal peptide at the N-terminus. The mature protein has an N-terminal extracellular domain (439 aa), a transmembrane segment (22 aa), and a cytoplasmic tail / intracellular domain / ENDO (44 aa). Since the G protein covers the outer surface of the viral particle envelope and is the only target antigen, it is able to induce virus neutralizing antibodies. Compositions of the Disclosure

[0065] As described in more detail below, one aspect of the present disclosure relates to a nucleic acid construct containing a sequence encoding a modified alphavirus genome or srRNA, wherein at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or srRNA has been replaced by a coding sequence of a polypeptide construct comprising an antigenic determinant of the envelope glycoprotein G of rabies virus (RABV-G), a variant thereof, or either thereof. Recombinant cells and cell cultures are also provided, which have been engineered to include a nucleic acid construct as disclosed herein. Nucleic acid construct

[0066] As described in more detail below, one aspect of the present disclosure relates to a nucleic acid construct comprising a nucleic acid sequence encoding a modified alphavirus genome or srRNA, wherein at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or srRNA has been replaced by a coding sequence of a polypeptide construct comprising an antigenic determinant of the envelope glycoprotein G of rabies virus (RABV-G), a variant thereof, or either thereof. In some embodiments, the sequence encoding the nucleic acid construct can be operably linked, for example, placed under the control of an element required for expression (e.g., a promoter sequence), which allows the srRNA construct to be expressed in a host cell, a subject, or an in vitro cell-free expression system.

[0067] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to both RNA molecules and DNA molecules, including nucleic acid molecules comprising: cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules comprising nucleic acid analogs. Nucleic acid molecules can be double-stranded or single-stranded (e.g., sense strand or antisense strand). Nucleic acid molecules can contain unconventional or modified nucleotides. As used herein, the terms "polynucleotide sequence" and "nucleic acid sequence" refer interchangeably to the sequence of a polynucleotide molecule. The nucleotide base nomenclature shown in 37 CFR § 1.822 is used herein.

[0068] The nucleic acid molecules of the present disclosure can have any length, including, for example, between about 1.5 Kb and about 50 Kb, between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, such as between about 15 Kb and 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, between about 5 Kb and about 25 Kb, or between about 30 Kb and about 50 Kb.

[0069] Non-limiting exemplary embodiments of the methods of the present disclosure may include one or more of the following features. In some embodiments, the alphavirus srRNA vector lacks at least a portion of the nucleic acid sequence encoding one or more of the viral structural proteins CP, E1, E2, E3, and 6K of the alphavirus srRNA vector. In some embodiments, the alphavirus srRNA vector lacks part or all of the sequence encoding CP. In some embodiments, the alphavirus srRNA vector lacks part or all of the sequence encoding E1. In some embodiments, the alphavirus srRNA vector lacks part or all of the sequence encoding E2. In some embodiments, the alphavirus srRNA vector lacks part or all of the sequence encoding E3. In some embodiments, the alphavirus srRNA vector lacks part or all of the sequence encoding 6K. In some embodiments, the alphavirus srRNA vector lacks part or all of the sequence encoding a combination of CP, E1, E2, E3, and 6K. In some embodiments of the present disclosure, coding sequences for the nonstructural proteins nsP1, nsP2, nsP3, and nsP4 of the alphavirus srRNA vector are present, however, at least a portion or the entire sequence encoding one or more structural proteins (e.g., CP, E1, E2, E3, and 6K) of the alphavirus srRNA vector is absent.

[0070] In some embodiments, the alphavirus srRNA vector lacks the main part of the nucleic acid sequence encoding one or more viral structural proteins. It will be appreciated by those skilled in the art that the main part of the nucleic acid sequence encoding the viral structural polypeptide may include enough nucleic acid sequence encoding the viral structural polypeptide to provide a putative identification of the polypeptide, or by manual evaluation of the sequence by those skilled in the art, or by computer automatic sequence comparison and identification using algorithms such as BLAST (see, for example, "Basic Local Alignment Search Tool"; Altschul SF et al., J. Mol. Biol. 215: 403-410, 1993). Therefore, the main part of the nucleotide sequence includes enough sequence to provide specific identification and / or separation of nucleic acid fragments comprising the sequence. For example, the main part of the nucleic acid sequence may include at least about 20% of the full-length nucleic acid sequence, such as about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%.

[0071] In some embodiments, the alphavirus srRNA vector lacks the entire sequence encoding the viral structural protein, for example, the alphavirus srRNA vector does not contain a nucleic acid sequence encoding the viral structural protein.

[0072] In some embodiments, the alphavirus srRNA vector comprises a sequence comprising CTGGAGACGTGGAGGAGAACCCTGGACCT (SEQ ID NO: 2). In some embodiments, the alphavirus vector comprises a sequence comprising GACCGCTACGCCCCAATGACCCGACCAGC (SEQ ID NO: 3). In some embodiments, the alphavirus srRNA vector comprises a sequence comprising CTGGAGACGTGGAGGAGAACCCTGGACCT (SEQ ID NO: 2) and a sequence comprising GACCGCTACGCCCCAATGACCCGACCAGC (SEQ ID NO: 3).

[0073] The nucleic acid construct of the present disclosure further includes the following coding sequence of the polypeptide construct, and the coding sequence replaces at least a part of the nucleic acid sequence of the viral structural protein of the modified alphavirus genome or srRNA encoding. In principle, the nucleic acid construct disclosed herein can generally include any number of coding sequences of the polypeptide construct. In some embodiments, the nucleic acid construct disclosed herein can include at least one, at least two, at least three, at least four, at least five or at least six coding sequences of the polypeptide construct. The coding sequence of the polypeptide construct can be a construct of genetic material, which includes the coding sequence and enough regulatory information to guide the appropriate in vivo and / or in vitro transcription and / or translation of the coding sequence in the cell. The coding sequence of the polypeptide construct can be inserted into the vector for targeting the desired host cell and / or in the subject. Therefore, in some embodiments, the term "coding sequence of the polypeptide construct" can be used interchangeably with the term "expression construct". In some embodiments, the coding sequence of the polypeptide construct can be such a nucleic acid construct, which includes a gene or functional RNA of a protein encoding operably connected to a regulatory element (e.g., a promoter and / or a termination signal, and optionally, any other nucleic acid sequence or a combination of other nucleic acid sequences that affect gene transcription or translation).

[0074] The nucleic acid constructs described herein include the coding sequence of the antigenic determinant of the envelope glycoprotein G (RABV-G) of rabies virus, its variants or either, and the coding sequence encodes a polypeptide containing an epitope capable of eliciting an immune response. The variant of RABV-G may encompass the coding sequence of a polypeptide having an amino acid sequence identical or substantially identical to the amino acid sequence of a reference protein (e.g., RABV-G), except having at least one modified (e.g., deleted, inserted or substituted, respectively) amino acid. Amino acid substitutions may be conservative amino acid substitutions, preferably at non-essential amino acid residues in a protein. "Conservative amino acid substitutions" are substitutions in which an amino acid residue is substituted by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains are known in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Variants of a protein may have an amino acid sequence that is at least about 80%, 90%, 95% or 99%, preferably at least about 90%, more preferably at least about 95% identical to the amino acid sequence of the protein. Preferably, a variant is a functional variant of the protein that retains the same function as the protein. When used in reference to a nucleic acid sequence, the term "variant" refers to a nucleic acid sequence that has one or more nucleotide differences from another, generally related, nucleotide sequence. Therefore, the term "variant" can refer to a change in one or more nucleotides of a reference nucleic acid, including the insertion of one or more new nucleotides, the deletion of one or more nucleotides, and the replacement of one or more existing nucleotides. Variants can also include point mutations, multiple mutations, single nucleotide polymorphisms (SNPs), deletions, insertions, and translocations. Therefore, variants of coding sequences as described herein include nucleic acids encoding polypeptides, which can be, for example, full-length, mutant, truncated, inactivated peptides / epitopes of RABV-G or combinations thereof.

[0075] Nucleic acid constructs as described herein can also include the coding sequence of the envelope glycoprotein G (RABV-G) of rabies virus or its antigenic determinant. In some embodiments, the antigenic determinant is located in the N-terminal half of the rabies virus G glycoprotein. Other specific antigenic determinants are located in the C-terminal half of rabies virus. Such antigenic determinants can be located in, for example, amino acids 1-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-524 of the rabies virus G glycoprotein, or in any interval, part or range thereof. In one embodiment, the antigenic determinant is located in the N-terminal half of the rabies virus G glycoprotein, that is, between about amino acid residues 19-422. In another embodiment, the antigenic determinant is located within the C-terminal half of the rabies virus G glycoprotein, i.e., between about amino acid residues 1-19. In another embodiment, the antigenic determinant of the rabies G glycoprotein comprises amino acid residues 336-442. In one embodiment, the rabies G glycoprotein comprises amino acid residue 336 and changes thereof, such as substitutions or deletions.

[0076] In some embodiments, the rabies G glycoprotein antigenic determinant comprises one or more epitopes. Non-limiting examples of epitopes include linear epitopes, conformational epitopes, discontinuous epitopes, or combinations of such epitopes. It will be understood by those skilled in the art that the term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site (e.g., a variable region of an antibody molecule, referred to as a paratope) of an antigen-binding polypeptide. A single antigen may have more than one epitope. Therefore, different antibodies may bind to different regions on an antigen and may have different biological effects. The term "epitope" also refers to the site on an antigen to which B cells respond. It also refers to the region in an antigen that is bound by an antibody. An epitope may be defined as structural or functional. Functional epitopes are typically subsets of structural epitopes and have those residues that directly promote affinity for interaction. An epitope may be linear or conformational, that is, composed of nonlinear amino acids. In certain embodiments, an epitope may include a determinant, i.e., a chemically active surface group (such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group) of a molecule, and in certain embodiments, may have specific three-dimensional structural features, and / or specific charge features.

[0077] In another related embodiment, the rabies G glycoprotein antigenic determinant comprises or consists of antigenic site I, antigenic site II, antigenic site III, antigenic site IV, small antigenic site A, or a combination of such antigenic sites (e.g., antigenic site III and small site A). Antigenic sites of rabies virus have been identified using a panel of monoclonal antibodies and their corresponding monoclonal antibody-resistant virus variants. Most rabies virus neutralizing monoclonal antibodies are directed against antigenic site II (Benmansour et al., "Antigenicity of Rabies Virus Glycoprotein," J. Virol. 65:4198-4203 (1991), which is hereby incorporated by reference), which is a discontinuous conformational epitope comprising aa 34 to 42 and aa 198 to 200 (Prosniak et al. 2003. Development of a cocktail of recombinant-expressed human rabies virus-neutralizing monoclonal antibodies for postexposure prophylaxis of rabies. J. Infect. Dis. 188:53-56). Antigenic site III is a continuous conformational epitope located at aa 330 to 338 and contains two charged residues, K330 and R333, that affect viral pathogenicity (Coulon et al. 1998. An avirulent mutant of rabies virus is unable to infect motoneurons in vivo and in vitro. J. Virol. 72: 273-278; Dietzschold et al. 1983. Characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus. Proc. Natl. Acad. Sci. USA 80: 70-74; Seif et al. 1985. Rabies virulence: effect on pathogenicity and sequence characterization of rabies virus mutations affecting antigenic site III of the glycoprotein. J. Virol. 53: 926-934).Conformational antigenic site I is defined by only one monoclonal antibody, 509-6, and is located at aa 231 (Benmansour et al., "Antigenicity of Rabies Virus Glycoprotein," J. Virol. 65:4198-4203 (1991); Lafon et al. 1983. Antigenic sites on the CVS rabiesvirus glycoprotein: analysis with monoclonal antibodies. J. Gen. Virol. 64:843-8451). Antigenic site IV is known to contain overlapping linear epitopes (Bunschoten et al., 1989. Characterization of a new virus-neutralizing epitope that denotes a sequential determinant on the rabies virus glycoprotein. J. Gen. Virol. 70: 291-298; Luo et al. 1997. A virus-neutralizing epitope on the glycoprotein of rabies virus that contains Trp251 is a linear epitope. Virus Res. 51: 35-41; Ni et al. 1995. Mapping and characterization of a sequential epitope on the rabies virus glycoprotein which is recognized by a neutralizing monoclonal antibody, RG719. Microbiol. Immunol. 39: 693-702). Benmansour et al. also described the presence of a small site located at positions 342 to 343, which is different from antigenic site III despite being very close.

[0078] The RABV-G of the nucleic acid construct described herein may be derived from a virulent rabies virus strain or an avirulent rabies virus strain.

[0079] Currently used anti-rabies vaccines are either made from inactivated viruses or vaccines composed of weakened virus strains or recombinant viruses.

[0080] Viruses can be inactivated by a variety of methods, particularly chemical methods such as treatment with formaldehyde or 3-propiolactone.

[0081] The weakening of the virulence of a virus strain is well known; for example, it can be carried out by serial passage of the virus strain in different hosts, carrier types (e.g., rabbits or mice) or cell cultures. Thus, while maintaining its vaccination capacity, the strain has poor adaptability to the host source and is therefore less pathogenic to the host.

[0082] Avirulent rabies virus strains typically include one or more mutations in the envelope glycoprotein G (see, e.g., Flamand et al., “Avirulent Mutants of Rabies Virus and Their Use as Live Vaccine,” Trends Microbiol. 1(8):317-20 (1993); Coulon et al., “An Avirulent Mutant of Rabies Virusis Unable to Infect Motorneurons In Vivo and In Vitro,” J. Virol. 72(1):273-278 (1998)). For example, antigenic mutants of RABV-G isolated from the challenge virus standard strain (CVS), CVS derivatives or Street-Alabama-Dufferin (SAD) Bern strain are avirulent, wherein the arginine (Arg) at position 333 in the antigenic mutant G is replaced by glutamine (Gln), glycine (Gly), leucine, isoleucine, methionine, cysteine ​​or serine (Tuffereau et al., Arginine or lysine at position 333 of ERA and CVS glycoprotein is necessary for rabies virulence in adult mice. Virology. 1989; 172: 206-212; Seif et al., Rabies virulence: effect on pathogenicity and sequence characterization of rabies virus mutations affecting antigenic site III of the glycoprotein. J Virol. 1985; 53: 926-934). In addition, the recombinant virus in which the Arg at position 333 in G was replaced by glutamic acid was also avirulent (McGettigan et al., Second-generation rabies virus-based vaccine vectors expressing human immunodeficiency virus type 1 gag have greatly reduced pathogenicity but are highly immunogenic. J Virol. 2003; 77: 237-244).Therefore, in general, when Arg or Lys at position 333 in G is changed to other amino acids, RABV becomes avirulent.

[0083] In principle, there is no particular limitation on the rabies virus strain from which the envelope glycoprotein G can be derived. Non-limiting examples of rabies virus strains suitable for use in the compositions and methods disclosed herein include Flury LEP, Flury LEP-C, Flury HEP, 1088, AT6, CQ92, CVS-11, CVS-26, CVS-26 (G-N204S), CYN1009D, CYN1026D, CYN1029D, CYN1138D, CYN1140D, CYN1141D, CYN1242H, CYN1243D, CYN1244D, CYN1245 D strain, CYN1247D strain, CYN1249D strain, CYN1250D strain, CYN1251D strain, CYN1252D strain, CYN1253D strain, CYN1255D strain, CYN 1256D strain, CYN1257D strain, CYN1259D strain, CYN1260D strain, CYN1261D strain, GX4 strain, H-08-1320 strain, H-1413-09 strain, IP 1586 / 10 strain, IP 2990 / 13 strain, IP 2991 / 13 strain, IP 2992 / 13 strain, IP 3176 / 09 strain, IP 4005 / 12 strain, IP 412 / 10 strain, IP 542 / 10 strain, IP 7941 / 09 strain, J strain, JX-08-47 strain, JX08-48 strain, Kyoto strain, Kyoto (G-S204N) strain, N.HL strain, RC.HL strain, rHEP5.0-CVSG strain, RRVON-99-2 strain, SAD-B19 strain, SH06 strain, SHRBV-18 strain, SNK-CTN strain, SRV9 strain, Street Alabama Dufferin (HCP-SAD) strain, VRC-RZ2 strain, ZJ-LA strain and ZJ-QZ strain. In some embodiments, the envelope glycoprotein G is the envelope glycoprotein G of the Flury LEP strain.

[0084] The full-length amino acid sequence of a non-limiting RABV-G protein is shown below in SEQ ID NO: 1: MVPQVLLFVPLLGFSLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLS EFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTTFKRKHFRPTPDACR AAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVTDLDPYDKSLHS RVFPGGNCSGITVSSTYCSTNHDYTIWMPENLRLGTSCDIFTNSRGKRASKGGKTCG FVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSDETKWCPPGQLVNLH DFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFN KTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGSDGHVLIPE MQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEVEDFVEVHLPDVHEQVSGVE LGLPNWGKYVLMIAGALIALMLIIFLMTCCRRVNRPESTQSSLGETGRNVSVTSQSG KVISSWESYKSGGETRL

[0085] In some embodiments, the coding sequence of RABV-G in the nucleic acid construct described herein encodes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid construct of the present disclosure comprises a nucleic acid sequence encoding RABV-G having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid construct of the present disclosure comprises a nucleic acid sequence encoding RABV-G having an amino acid sequence of SEQ ID NO: 1, wherein one, two, three, four or five amino acid residues in SEQ ID NO: 1 are substituted by different amino acid residues. In some embodiments, the coding sequence of RABV-G encodes a smaller portion of the amino acid sequence of SEQ ID NO: 1. For example, these smaller portions may include at least 8, 10, 12, 14, 16, 18, 20, 30 or more amino acids of SEQ ID NO: 1.

[0086] In some embodiments, the nucleic acid construct as described herein comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NOs: 6-11.

[0087] In some embodiments, the coding sequence of RABV-G is redesigned and / or optimized to obtain desired properties, such as increased stability, efficacy and expression (e.g., translation efficiency), which in turn can maximize the impact of producing, delivering and administering biotherapeutics. For example, in some embodiments, the coding sequence is optimized to be expressed at a level higher than the expression level of the reference coding sequence. With regard to sequence optimization of nucleotide sequences, the degeneracy of the genetic code provides the possibility of replacing at least one base of a gene sequence encoding a protein with a different base without causing the amino acid sequence of the polypeptide produced by the gene to be changed. Therefore, the nucleic acid construct of the present disclosure can also have any base sequence changed from any polynucleotide sequence disclosed herein by substitution according to the degeneracy of the genetic code. References describing codon usage are readily publicly available. In some embodiments, polynucleotide sequence variants can be generated for a variety of reasons, such as to optimize expression of a specific host (e.g., changing the codon usage in alphavirus mRNA to those preferred by other organisms (e.g., humans, non-human primates, hamsters, mice or monkeys)). Therefore, in some embodiments, the coding sequence is optimized for expression in a target host cell by using codons optimized for expression. The technology for constructing the synthetic nucleic acid sequence of the coding gene using the preferred codons that are best used for host cell expression can be determined by the degeneracy of the codon usage of the natural protein encoding the host cell genome and the calculation method of its relative abundance by the technology well known in the art. The codon usage database (http: / / www.kazusa.or.jp / codon) can be used to generate the codon optimized sequence in the mammalian cell environment. In addition, various software tools can be used to convert the sequence of an organism to the best codon for different host organisms, such as JCat codon optimization tool (www.jcat.de), integrated DNA technology (IDT) codon optimization tool (https: / / www.idtdna.com / CodonOpt) or optimizer online codon optimization tool (http: / / genomes.urv.es / OPTIMIZER). This synthetic sequence can be constructed by the technology known in the art for constructing synthetic nucleic acid molecules, and can be obtained from various commercial suppliers.

[0088] In some embodiments, the coding sequence is optimized for enhancing RNA stability and / or expression. The stability of RNA is generally related to the "half-life" of RNA. "Half-life" refers to the time required to eliminate half of the activity, quantity or quantity of a molecule. In the context of the present disclosure, the half-life of RNA indicates the stability of the RNA. The half-life of RNA may affect the "duration of expression" of RNA. Additional information about rules, strategies and methods for enhancing RNA stability can be found in, for example, Leppek K. et al., Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics. bioRxiv. (Preprint). March 30, 2021. doi: 10.1101 / 2021.03.29.437587. Recombinant cells

[0089] The nucleic acid construct of the present disclosure can be introduced into a host cell to produce a recombinant cell containing the nucleic acid molecule. Therefore, a prokaryotic cell or eukaryotic cell containing a nucleic acid construct encoding a modified alphavirus genome as described herein is also a feature of the present disclosure. In related aspects, some embodiments disclosed herein relate to a method for transforming cells, comprising introducing a nucleic acid construct as provided herein into a host cell (such as an animal cell), and then selecting or screening the transformed cell. The nucleic acid construct of the present disclosure can be introduced into a cell by methods known to those skilled in the art, such as viral infection, transfection, conjugation, protoplast fusion, liposome transfection, electroporation, nuclear transfection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, direct microinjection, nanoparticle-mediated nucleic acid delivery, etc.

[0090] In one aspect, some embodiments of the present disclosure relate to recombinant cells, for example, recombinant animal cells comprising nucleic acid constructs as described herein. For example, nucleic acid constructs can be stably integrated into the host genome, or can be replicated as episomes, or present in recombinant host cells as mini-ring expression vectors for stable or transient expression. Therefore, in some embodiments disclosed herein, the nucleic acid constructs are maintained and replicated as episomal units in recombinant host cells. In some embodiments, the nucleic acid constructs are stably integrated into the genome of the recombinant cells. Stable integration can be accomplished using classical random genome recombination techniques or more accurate genome editing techniques (such as CRISPR / Cas9 or TALEN genome editing guided by guide RNA). In some embodiments, nucleic acid molecules are present in recombinant host cells as mini-ring expression vectors for stable or transient expression.

[0091] In some embodiments, the recombinant cell is a prokaryotic cell (such as the bacterium Escherichia coli (E. coli)) or a eukaryotic cell (such as an insect cell (e.g., a mosquito cell or an Sf21 cell) or a mammalian cell (e.g., a COS cell, a NIH 3T3 cell, or a HeLa cell)). In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vitro. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is an animal cell of a vertebrate or an animal cell of an invertebrate. In some embodiments, the recombinant cell is a mammalian cell. Suitable recombinant cells include monkey kidney CV1 cells (COS-7) transformed by SV40, human embryonic kidney cells (e.g., HEK 293 or HEK 293 cells), baby hamster kidney cells (BHK), mouse supporting cells (e.g., TM4 cells), monkey kidney cells (CV1), human cervical cancer cells (HeLa), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse breast tumor (MMT 060562), TRI cells, FS4 cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NS0 mouse myeloma cells, human epidermoid laryngeal cells, human fibroblasts, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human macrophages, human RAW 264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells.

[0092] In some embodiments, the recombinant cell is an insect cell, for example, a cell of an insect cell line. In some embodiments, the recombinant cell is an Sf21 cell. Other suitable insect cell lines include, but are not limited to, cell lines established from the insect orders Diptera, Lepidoptera, and Hemiptera, and may be derived from different tissue sources. In some embodiments, the recombinant cell is a cell of a Lepidopteran cell line. In the past few decades, the availability of the Lepidopteran insect cell line has increased with about 50 lines per decade. More information about available Lepidopteran insect cell lines can be found in, for example, Lynn DE, Available lepidopteran insect celllines. Methods Mol Biol. 2007; 388: 117-38, and the document is incorporated herein by reference. In some embodiments, the recombinant cell is a mosquito cell, e.g., a cell of a mosquito species within the genera Anopheles (An.), Culex (Cx.), and Aedes (subgenus Stegomyia) (Ae.). Exemplary mosquito cell lines suitable for use in the compositions and methods described herein include cell lines from the following mosquito species: Aedes aegypti, Aedes albopictus, Aedes pseudoscutellaris, Aedes triseriatus, Aedes vexans, Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Culex quinquefasciatus, Culex theileri, Culextritae niorhynchus, Culex bitae niorhynchus, and Toxorhynchites amboinensis. Suitable mosquito cell lines include, but are not limited to, CCL-125, Aag-2, RML-12, C6 / 26, C6 / 36, C7-10, AP-61, AtGRIP-1, AtGRIP-2, UM-AVE1, Mos.55, Sua1B, 4a-3B, Mos.43, MSQ43, and LSB-AA695BB. In some embodiments, the mosquito cell is a cell of the C6 / 26 cell line.

[0093] In another aspect, provided herein is a cell culture comprising at least one recombinant cell as disclosed herein and a culture medium. Typically, the culture medium can be any suitable culture medium for culturing cells as described herein. Techniques for transforming various host cells and species mentioned above are known in the art and are described in the technical and scientific literature. Therefore, a cell culture comprising at least one recombinant cell as disclosed herein is also within the scope of the present application. Methods and systems suitable for producing and maintaining cell cultures are known in the art.

[0094] Recombinant polypeptides produced by the methods disclosed herein are also within the scope of the present disclosure.

[0095] Non-limiting exemplary embodiments of the disclosed methods for producing recombinant polypeptides may include one or more of the following features. In some embodiments, the method for producing a recombinant polypeptide of the present disclosure further comprises isolating and / or purifying the produced polypeptide. In some embodiments, the method for producing a polypeptide of the present disclosure further comprises structurally modifying the produced polypeptide to extend the half-life. Pharmaceutical composition

[0096] The nucleic acid constructs, recombinant cells, and recombinant polypeptides of the present disclosure can be incorporated into compositions (including pharmaceutical compositions). Such compositions generally include one or more of nucleic acid constructs, recombinant cells, recombinant polypeptides described and provided herein, and pharmaceutically acceptable excipients such as carriers. In some embodiments, the compositions of the present disclosure are formulated for preventing, treating, or managing rabies infection. For example, the compositions of the present disclosure can be formulated as preventive compositions, therapeutic compositions, or pharmaceutical compositions or mixtures thereof comprising pharmaceutically acceptable excipients. In some embodiments, the compositions of the present disclosure are formulated for use as vaccines. In some embodiments, the compositions of the present application are formulated for use as adjuvants.

[0097] Thus, in one aspect, provided herein is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and: a) a nucleic acid construct of the present disclosure; b) a recombinant cell of the present disclosure; and / or c) a recombinant polypeptide of the present disclosure.

[0098] The non-limiting exemplary embodiments of the pharmaceutical composition of the present disclosure may include one or more of the following features. The nucleic acid construct of the present disclosure can be used in naked form or formulated with a delivery vehicle. Exemplary approaches, or use in free form (e.g., inserting nucleic acid (e.g., carrier)). For example, as described in more detail below, nucleic acid constructs as described herein can be used as vaccines.

[0099] For use in the pharmaceutical composition of the present disclosure, nucleic acid or recombinant cells as described herein can be formulated into or formulated with a delivery vehicle. Exemplary delivery vehicles suitable for the compositions and methods of the present disclosure include, but are not limited to, liposomes (e.g., neutral or anionic liposomes), microspheres, immunostimulatory complexes (ISCOMS), lipid-based nanoparticles (LNP), polymer nanoparticles, viral replicon particles (VRP) or conjugated with bioactive ligands, which can promote delivery and / or enhance immune response. These compounds are readily available to those skilled in the art; for example, see Liposomes: A Practical Approach, RCP New Ed, IRL press (1990). Adjuvants other than liposomes are also used, and are known in the art. Adjuvants can protect antigens from rapid diffusion by isolating antigens (e.g., srRNA constructs) in local deposits, or they can contain materials that stimulate host secretion of factors that are chemotactic to macrophages and other components of the immune system. Those skilled in the art can appropriately select, for example, from those described below.

[0100] Thus, in some embodiments, the compositions of the present disclosure may include one or more of the following: physiological buffers, liposomes, lipid-based nanoparticles (LNPs), polymer nanoparticles, viral replicon particles (VRPs), microspheres, immunostimulatory complexes (ISCOMs), conjugates of biologically active ligands, or any combination thereof.

[0101] In some embodiments, the nucleic acid constructs of the present disclosure can be delivered to cells or subjects via lipid-based nanoparticles (LNPs). The immunogenicity of LNPs is generally lower than that of viral particles. Although many people have pre-existing immunity to viral particles, they do not have pre-existing immunity to LNPs. In addition, adaptive immune responses to LNPs are unlikely to occur, so LNPs can be administered repeatedly.

[0102] Lipids suitable for use in the compositions and methods described herein can be cationic lipids, ionizable cationic lipids, anionic lipids, or neutral lipids.

[0103] In some embodiments, the LNP of the present disclosure can include one or more ionizable lipids. As used herein, the term "ionizable lipid" refers to a lipid that is cationic or becomes ionizable (protonated) but more neutral at a higher pH value when pH is reduced to the pKa of the ionizable group of lipid. At a pH value lower than pKa, lipid can associate with negatively charged nucleic acid (e.g., oligonucleotide). As used herein, the term "ionizable lipid" is included in a lipid that presents a positive charge when pH is reduced from physiological pH, and any of a variety of lipids that carry a net positive charge at a selective pH (e.g., physiological pH). Permanent cationic lipids such as DOTMA have been shown to be too toxic for clinical use. Ionizable lipids may be present in lipid formulations according to other embodiments, preferably at a ratio of about 30 to about 70 Mol%, in some embodiments about 30 Mol%, in other embodiments, about 40 Mol%, in other embodiments, about 45 Mol%, in other embodiments, about 47.5 Mol%, in still other embodiments, about 50 Mol%, and in yet other embodiments about 60 Mol% ("Mol%" means the percentage of the moles of a particular component to the total moles). The term "about" in this paragraph represents a positive or negative range of 5 Mol%. DODMA, or 1,2-dioleoyloxy-3-dimethylaminopropane, is an ionizable lipid, as is DLin-MC3-DMA, or O-(Z,Z,Z,Z-triacontane-6,9,26,29-tetraene-19-yl)-4-(N,N-dimethylamino) ("MC3").

[0104] Exemplary ionizable lipids suitable for the compositions and methods of the present disclosure include ionizable lipids described in PCT Publications WO 2020252589 A1 and WO 2021000041 A1, U.S. Patent Nos. 8,450,298 and 10,844,028, and Love KT et al., Proc Natl Acad Sci USA, Feb. 2, 2010 107 (5) 1864-1869, all of which are hereby incorporated by reference in their entirety. Therefore, in some embodiments, the LNP of the present disclosure includes Love KT et al., one or more lipid compounds described in 2010 supra, such as C16-96, C14-110 and C12-200. In some embodiments, LNP includes ionizable cationic lipids. Suitable ionizable cationic lipids include the group consisting of: ALC-0315, C12-200, LN16, MC3, MD1, SM-102 and any combination thereof. In some embodiments, the LNP of the present disclosure includes C12-200. The structure of C12-200 lipids is known in the art and is described in, for example, U.S. Patent Nos. 8,450,298 and 10,844,028, which are hereby incorporated by reference in their entirety. In some embodiments, C12-200 is combined with cholesterol, C14-PEG2000 and DOPE. In some embodiments, C12-200 is combined with DSPC and DMG-PEG2000.

[0105] In some embodiments, the LNP of the present disclosure includes one or more cationic lipids. Suitable cationic lipids include, but are not limited to, 98N12-5, C12-200, C14-PEG2000, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. In some embodiments, the LNP of the present disclosure includes one or more neutral lipids. Non-limiting neutral lipids suitable for the compositions and methods of the present disclosure include DPSC, DPPC, POPC, DOPE, and SM. In some embodiments, the LNP of the present disclosure includes one or more ionizable lipid compounds described in PCT Publications WO 2020252589 A1 and WO 2021000041 A1, and the document is hereby incorporated by reference in its entirety.

[0106] Many other lipids known in the art or the combination of lipids can be used for producing LNP.The non-limiting example of the lipid suitable for producing LNP comprises DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE and GL67A-DOPE-DMPE-polyethylene glycol (PEG).The non-limiting example of cationic lipid comprises 98N12-5, C12-200, C14-PEG2000, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, 7C1 and the combination of any thereof.The non-limiting example of neutral lipid comprises DPSC, DPPC, POPC, DOPE and SM.The non-limiting example of the lipid modified by PEG comprises PEG-DMG, PEG-CerC14 and PEG-CerC20.

[0107] In some embodiments, the LNP of the present disclosure includes at least one lipid. Suitable lipids include C12-200, C14-PEG2000, DOPE, DMG-PEG2000, DSPC, DOTMA, DOSPA, DOTAP, DMRIE, DC cholesterol, DOTAP cholesterol, GAP-DMORIE-DPyPE and GL67A-DOPE-DMPE polyethylene glycol (PEG). In some embodiments, C12-200 is combined with cholesterol, C14-PEG2000 and DOPE. In some embodiments, C12-200 is combined with DSPC and DMG-PEG2000.

[0108] In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 100: 1 to about 3: 1, about 70: 1 to 10: 1 or 16: 1 to 4: 1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 16: 1 to 4: 1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 20: 1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 8: 1. In some embodiments, lipid-based nanoparticles have an average diameter of less than about 1000nm, about 500nm, about 250nm, about 200nm, about 150nm, about 100nm, about 75nm, about 50nm or about 25nm. In some embodiments, the average diameter range of LNP is about 70nm to 100nm. In some embodiments, the average diameter range of LNP is about 88nm to about 92nm, 82nm to about 86nm or about 80nm to about 95nm.

[0109] In some embodiments, the compositions of the present disclosure are formulated in liposomes. In some embodiments, the compositions of the present disclosure are formulated in lipid-based nanoparticles (LNPs). In some embodiments, the compositions of the present disclosure are formulated in polymer nanoparticles.

[0110] As described above, in some embodiments, neutral lipids, also referred to as "structural lipids" or "helper lipids," can also be incorporated into lipid formulations and lipid particles. Lipid formulations and lipid particles can include one or more structural lipids that account for about 10 to 40 Mol% of the composition. Suitable structural lipids support the formation of particles during the manufacturing process. Structural lipids refer to any of the many lipids that exist in anionic, uncharged or neutral zwitterionic forms at physiological pH. Representative structural lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylglycerol, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0111] Exemplary structured lipids include zwitterionic lipids such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-ditransoleoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE).

[0112] In another embodiment, the structural lipid can be any lipid with negative charge at physiological pH. These lipids include phosphatidylglycerol, such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modifying groups connected to neutral lipids. Other suitable structural lipids include glycolipids (e.g., monosialoganglioside GM1).

[0113] Stabilizers can be included in lipid formulation embodiments to ensure the integrity of the mixture. Stabilizers are molecules that destroy or help form hydrophobic-hydrophilic interactions between molecules. Suitable stabilizers include, but are not limited to, polysorbate 80 (also known as Tween 80, lUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolane-2-yl]-2-(2-hydroxyethoxy)ethoxy]octadec-9-enoic acid ethyl ester), Myrj52 (polyoxyethylene (40) stearate), and Brij TM S10 (polyoxyethylene (10) stearyl ether). Polyethylene glycol-conjugated lipids may also be used. Stabilizers may be used alone or in combination with one another.

[0114] In some embodiments, the stabilizer constitutes about 1 to 3Mol% of the entire lipid mixture. In some embodiments, the stabilizer constitutes about 0.5 to 2.5Mol% of the entire lipid mixture. In some embodiments, the stabilizer is present at more than 2.5Mol%. In some embodiments, the stabilizer is present at 5Mol%. In some embodiments, the stabilizer is present at 10Mol%. In some embodiments, the stabilizer is about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, etc. In other embodiments, the stabilizer is 2.6-10Mol% of the lipid mixture. In other embodiments, the stabilizer is present at more than 10Mol% of the lipid mixture.

[0115] Steroids may also be included in lipid compositions for certain applications, and lipid particles made from steroids include sterols, such as cholesterol and phytosterols.

[0116] In some embodiments, the therapeutic compositions described herein (e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions) are incorporated into a therapeutic composition for use in a method of preventing or treating a subject suffering from, or suspected of suffering from, rabies infection. A subject suspected of suffering from rabies infection may be a subject bitten by an animal suffering from, or suspected of suffering from, rabies infection, such as a raccoon, skunk, fox, coyote, bat, dog, cat, etc.

[0117] In some embodiments, the composition is an immunogenic composition, for example, a composition that can stimulate an immune response in a subject. In some embodiments, the immunogenic composition is formulated as a vaccine. In some embodiments, the pharmaceutical composition is formulated as an adjuvant. In some embodiments, the immunogenic composition is formulated as a biotherapeutic, for example, a vehicle for gene delivery of different molecules with biological activity. Non-limiting examples of biotherapeutics include cytokines, chemokines and other soluble immunomodulators, enzymes, peptide and protein agonists, peptide and protein antagonists, hormones, receptors, antibodies and antibody derivatives, growth factors, transcription factors and gene silencing / editing molecules. In some embodiments, the pharmaceutical composition is formulated as an adjuvant.

[0118] In some embodiments, the immunogenic composition is substantially non-immunogenic or minimally immunogenic, e.g., a composition that minimally stimulates an immune response in a subject. In some embodiments, the non-immunogenic or minimally immunogenic composition is formulated as a biotherapeutic. In some embodiments, the pharmaceutical composition is formulated for one or more of intranasal administration, transdermal administration, intraperitoneal administration, intramuscular administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, intravaginal administration, and oral administration.

[0119] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, New Jersey) or phosphate buffered saline (PBS), tris (tromethamine) and HEPES. In these cases, the composition should be sterile and should be fluid to the extent that it is easy to inject. It can be stable under the conditions of manufacture and storage, and can be preserved against the contaminating effects of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, appropriate fluidity can be maintained by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant (e.g., sodium lauryl sulfate). Preventing the effects of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, isotonic agents such as sugars, polyols (such as mannitol, sorbitol), sucrose, trehalose and / or sodium chloride will generally be included in the composition. In some embodiments, the composition comprises tris and sucrose.Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0120] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount into an appropriate solvent optionally with one or a combination of the ingredients listed above, followed by filtered sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above.

[0121] In some embodiments, the composition is formulated for one or more of intranasal administration, transdermal administration, intramuscular administration, intranodal administration, intravenous administration, intraperitoneal administration, oral administration, or intracranial administration. Methods of the present disclosure

[0122] The administration of any one of the therapeutic compositions described herein (e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions) can be used to treat related health conditions, such as rabies virus infection. In some embodiments, nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions as described herein can be used to induce an immune response in a subject in need. In some embodiments, nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions as described herein can be incorporated into a therapeutic agent for use in treating a subject who suffers from, is suspected of suffering from, or may have a high risk of suffering from rabies infection. In some embodiments, the subject is a patient under the care of a doctor.

[0123] Thus, in one aspect, provided herein is a method for inducing an immune response in a subject in need thereof, the method comprising administering to the subject a composition comprising: a) a nucleic acid construct of the present disclosure; b) a recombinant cell of the present disclosure; c) a recombinant polypeptide of the present disclosure; and / or d) a pharmaceutical composition of the present disclosure.

[0124] In some embodiments, the immune response comprises a T cell response. The T cell response may comprise the production of interferon gamma (IFNγ). The production of IFNγ may be measured using several methods known in the art, including but not limited to ELISpot assays.

[0125] In some embodiments, the immune response is a neutralizing antibody response. Neutralizing antibody response is an immune response in which the specialized cells of the immune system recognize the presentation of one or more antigens and initiate a specific immune response, which prevents target cells from being infected with antigens, such as viruses (e.g., rabies virus). The method for detecting neutralizing antibody responses is known in the art, and may include, for example, the assay described in the embodiments herein. For example, neutralizing antibody responses can be measured by a rapid fluorescent focus inhibition test (RFFIT) 14 days after a single dose of nucleic acid constructs disclosed herein. Neutralizing antibody responses can also be demonstrated by injecting srRNA constructs described herein to animals (e.g., mice or non-human primates) and testing the ability of serum from animals to neutralize virus-infected cells. In some embodiments, the neutralizing antibody response comprises a neutralizing antibody titer equal to or greater than 0.5IU / mL.

[0126] In another aspect, provided herein is a method for preventing and / or treating rabies infection in a subject in need thereof, the method comprising prophylactically or therapeutically administering to the subject a composition comprising: a) a nucleic acid construct of the present disclosure; b) a recombinant cell of the present disclosure; c) a recombinant polypeptide of the present disclosure; and / or d) a pharmaceutical composition of any one of the present disclosure.

[0127] In some embodiments, the disclosed compositions are formulated to be compatible with their intended routes of administration. For example, the nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions of the present disclosure may be administered orally, by inhalation or by parenteral routes. Examples of parenteral routes of administration include, for example, intramuscular, intraocular, intravenous, intranodular, intradermal, subcutaneous, transdermal (topical), transmucosal, intravaginal and rectal administration. In some embodiments, the composition is administered intramuscularly. Solutions or suspensions for parenteral applications may include the following components: sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, phosphates, tris, sucrose and agents for adjusting tension (such as sodium chloride or dextrose). The pH can be adjusted with an acid or base (such as sodium dihydrogen phosphate and / or disodium phosphate, hydrochloric acid or sodium hydroxide) (e.g., to a pH of about 7.2-7.8, such as 7.5). The parenteral preparation can be enclosed in an ampoule, a disposable syringe or a multiple dose vial made of glass or plastic.

[0128] It can be administered once or more per day to once or more per week; including administration of therapeutic compositions described herein, such as nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions, every other day. Treatment of a subject with a therapeutically effective amount of a subject nucleic acid construct, recombinant cell, recombinant polypeptide and / or pharmaceutical composition of the present disclosure may include a single treatment, or may include a series of treatments. In some embodiments, the composition is administered at weekly intervals, such as 1 to 2, 2 to 3 or 3 to 4 doses at intervals of 1 to 2, 2 to 3 or 3 to 4 weeks. Additionally, it may be administered every 1, 2, 3 or 4 months. In some embodiments, 3 doses may be administered intramuscularly at intervals of 3 to 8 weeks, followed by intramuscular administration every 3, 6, 9 or 12 months. Additionally, it may be administered every 1, 2, 3 or 4 years. With regard to nucleic acid constructs and recombinant polypeptides, the therapeutically effective amount (e.g., effective dose) of a nucleic acid construct or recombinant polypeptide of the present disclosure depends on the selected nucleic acid construct or recombinant polypeptide.

[0129] As discussed above, a therapeutically effective amount includes an amount of a therapeutic composition sufficient to promote a specific effect when administered to a subject, such as a subject suffering from, suspected of suffering from, or at risk of suffering from a health condition (e.g., rabies infection). In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of symptoms of a disease, to alter the course of symptoms of a disease (e.g., but not limited to, to slow the progression of symptoms of a disease), or to reverse symptoms of a disease.

[0130] Treatment is considered to be effective treatment if at least any one or all of the signs or symptoms of the disease are improved or ameliorated. Efficacy can also be measured by failure of individual deterioration (e.g., cessation or at least slowing of disease progression) as assessed by hospitalization or the need for medical intervention. Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of a disease in a subject or animal (some non-limiting examples include humans or mammals) and includes: (1) inhibiting the disease, e.g., stopping or slowing the progression of symptoms; or (2) alleviating the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of symptom development.

[0131] In some embodiments, the ability of the compositions described herein (e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions) to play a role in treating rabies infection can be tested in a suitable animal model system before use in humans. Such animal model systems include, but are not limited to, mice, rats, hamsters, monkeys, etc. This can be measured by the ability of post-exposure prophylactic vaccination to prevent disease and death from rabies virus infection.

[0132] In some embodiments, the rabies virus strains described herein are used to challenge an animal that is subsequently administered a composition described herein (e.g., a nucleic acid construct, a recombinant cell, a recombinant polypeptide, and / or a pharmaceutical composition). Exemplary rabies virus strains include, but are not limited to, Flury LEP strain, Flury LEP-C strain, Flury HEP strain, 1088 strain, AT6 strain, CQ92 strain, CVS-11 strain, CVS-26 strain, CVS-26 (G-N204S) strain, CYN1009D strain, CYN1026D strain, CYN1029D strain, CYN1138D strain, CYN1140D strain, CYN1141D strain, CYN1242H strain, CYN1243D strain, CYN1244D strain, CYN1245D strain, CYN1246D strain, CYN1247D strain, CYN1248D strain, CYN1249D strain, CYN1250D strain, CYN1251D strain, CYN1252D strain, CYN1253D strain, CYN1254D strain, CYN1255D strain, CYN1256D strain, CYN1257D strain, CYN1258D strain, CYN1259D strain, CYN1260D strain, CYN1261D strain, CYN1262D strain, CYN1263D strain, CYN1264D strain, CYN1265D strain, CYN1266D strain, CYN12664D strain, CYN1267D strain, CYN1268D strain, CYN1269D strain, CYN1269D strain, CYN N1247D strain, CYN1249D strain, CYN1250D strain, CYN1251D strain, CYN1252D strain, CYN1253D strain, CYN1255D strain, CYN1256D strain, C YN1257D strain, CYN1259D strain, CYN1260D strain, CYN1261D strain, GX4 strain, H-08-1320 strain, H-1413-09 strain, IP1586 / 10 strain, IP 2990 / 13 strain, IP 2991 / 13 strain, IP 2992 / 13 strain, IP 3176 / 09 strain, IP 4005 / 12 strain, IP412 / 10 strain, IP 542 / 10 strain, IP 7941 / 09 strain, J strain, JX-08-47 strain, JX08-48 strain, Kyoto strain, Kyoto (G-S204N) strain, N.HL strain, RC.HL strain, rHEP5.0-CVSG strain, RRV ON-99-2 strain, SAD-B19 strain, SH06 strain, SHRBV-18 strain, SNK-CTN strain, SRV9 strain, Street Alabama Dufferin (HCP-SAD) strain, VRC-RZ2 strain, ZJ-LA strain and ZJ-QZ strain. In some embodiments, the rabies virus used is the challenge virus standard strain (CVS)-11.

[0133] For example, the animal is infected with a rabies virus strain, and the composition described herein, such as a nucleic acid construct, a recombinant cell, a recombinant polypeptide and / or a pharmaceutical composition, is administered on the 0th, 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th and / or 14th day after infection. In some embodiments, the composition is in an LNP formulation. In some embodiments, the addition of human rabies immunoglobulin (HRIG) or similar rabies neutralizing immunoglobulin (e.g., horse rabies immunoglobulin, ERIG) can be combined with srRNA treatment. In some embodiments, the recommended dose (HRIG of 20IU / kg body weight or ERIG of 40IU / kg body weight) is administered. The disease signs of the animal are monitored every day until about 35 days after the virus is inoculated (DPI) or the control non-treatment group is lost. Rabies virus infection can be monitored by real-time reverse transcriptase polymerase chain reaction (RT-qPCR). Alternatively, according to standard methods, direct fluorescent antibody (DFA) test can be used to determine the rabies virus staining in fixed tissues.

[0134] In some embodiments, the nucleic acid construct, recombinant cell, recombinant polypeptide and / or pharmaceutical composition of the present disclosure can be applied to a subject in a composition with a pharmaceutically acceptable carrier and in an amount that effectively stimulates an immune response. In general, the subject can be immunized by an initial series of injections (or administered by one of the other routes described below), and then a reinforcing agent is given to increase the protection provided by the initial series of administrations. The initial series of injections and subsequent reinforcing agents are administered at a dose necessary for stimulating the subject's immune response and over a time period necessary for stimulating the subject's immune response. In some embodiments of the disclosed method, the subject is a mammal. In some embodiments, the mammal is a human subject. As described above, pharmaceutically acceptable carriers suitable for injection use include sterile aqueous solutions (water soluble) or dispersions, and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. In these cases, the composition must be sterile and should be a fluid of a degree that is easy to inject. The composition must also be stable under manufacturing and storage conditions and must be preserved against the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), a suitable mixture thereof, and a vegetable oil. Suitable fluidity can be maintained, for example, by using a coating (e.g., lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. Preventing the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.).

[0135] Sterile injectable solutions can be prepared by incorporating the nucleic acid construct, recombinant cells and / or recombinant polypeptide in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

[0136] When nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions as described above are suitably protected, they can be administered orally, for example, with inert diluents or assimilable edible carriers. Nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions and other ingredients can also be encapsulated in hard or soft shell gelatin capsules, compressed into tablets, or directly incorporated into the diet of an individual. For oral therapeutic administration, the active compound can be combined with an excipient and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. Other treatments

[0137] In some embodiments, the composition according to the present disclosure is applied to the subject as a single prevention or therapy (monotherapy) alone or as a first therapy in combination with at least one other therapy (e.g., a second therapy). In some embodiments, the second therapy is selected from chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery. In some embodiments, the second therapy is selected from chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, or surgery. In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy and the second therapy are administered simultaneously. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapy and the second therapy are administered in turn. In some embodiments, the first therapy and the second therapy are administered together with a single formulation. Reagent test kit

[0138] Also provided herein are various kits for practicing the methods described herein and written instructions for making and using the kits. Specifically, some embodiments of the present disclosure provide kits for eliciting an immune response in a subject. Some other embodiments relate to kits for treating methods of cancer in a subject in need thereof. For example, in some embodiments, provided herein are kits comprising one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions as provided and described herein, and written instructions for preparing and using them.

[0139] In some embodiments, the kit of the present disclosure further comprises one or more means for administering any of the provided nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions to a subject. For example, in some embodiments, the kit of the present disclosure further comprises one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) for administering any of the provided nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions to a subject. In some embodiments, the kit may have one or more additional therapeutic agents, which may be administered simultaneously or sequentially with other kit components for a desired purpose, such as for diagnosing, preventing or treating a condition in a subject in need thereof.

[0140] Any of the above kits may further comprise one or more additional reagents, wherein such additional reagents may be selected from: dilution buffer; reconstitution solution, wash buffer, control reagent, control expression vector, negative control, positive control, reagents suitable for in vitro production of nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions provided by the present disclosure.

[0141] In some embodiments, the components of the kit can be in separate containers. In some other embodiments, the components of the kit can be combined in a single container. Therefore, in some embodiments of the present disclosure, the kit includes one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions as provided and described herein in one container (e.g., in a sterile glass or plastic vial) and an additional therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0142] In another embodiment, the kit includes a combination of the compositions described herein, including one or more nucleic acid constructs, recombinant cells and / or recombinant polypeptides of the present disclosure in combination with one or more additional therapeutic agents, which are formulated together in a single common container, optionally in a pharmaceutical composition.

[0143] If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit may include a device (e.g., an injection device or catheter) for performing such administration. For example, the kit may include one or more hypodermic needles or other injection devices as discussed above containing one or more nucleic acid constructs, recombinant cells and / or recombinant polypeptides of the present disclosure.

[0144] In some embodiments, the kit may also include instructions for practicing the methods disclosed herein using the components of the kit. For example, the kit may include a drug instruction sheet containing information about the pharmaceutical composition and dosage form in the kit. Typically, such information helps patients and physicians to effectively and safely use the encapsulated pharmaceutical composition and dosage form. For example, the following information about the combination of the present disclosure may be provided in the drug instruction sheet: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdose, appropriate dosage and administration, how to supply, appropriate storage conditions, references, manufacturer / distributor information, and intellectual property information.

[0145] The instructions for practicing the method are usually recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastics, etc. The instructions can be present in a test kit as a package insert, in a label of a container of a test kit or its component (for example, associated with packaging or sub-packaging), etc. The instructions can exist as an electronic storage data file present in a suitable computer-readable storage medium (for example, CD-ROM, floppy disk, flash drive, etc.). In some cases, the actual instructions are not present in the test kit, but can be provided for obtaining the instructions from a remote source (for example, via the Internet). The example of this embodiment is a test kit including a website, in which the instructions can be viewed and / or can be downloaded from the website. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

[0146] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0147] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states the views asserted by their authors, and the applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It should be clearly understood that although a number of information sources are referred to herein, including scientific journal articles, patent documents and textbooks; this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0148] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and substitutions will be apparent to those skilled in the art after reviewing this disclosure and are to be included within the spirit and scope of this application.

[0149] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration only and are not intended to limit the scope of the disclosure or the claims in any way. Example

[0150] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are fully explained in the literature, such as Sambrook, J., & Russell, DW (2012). Molecular Cloning: A Laboratory Manual (4th Edition). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J. and Russel, DW (2001). Molecular Cloning: A Laboratory Manual (3rd Edition). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to herein as "Sambrook"); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, DM et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, MG et al. (1995). Viral Vectors: Gene Therapy and NeuroscienceApplications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The ImmunologyMethods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue: Laboratory Procedures in Biotechnology. New Culture York, NY: Wiley; Mullis, KB, Ferré, F. and Gibbs, R. (1994). PCR: The Polymerase Chain Reaction.Boston: Birkhauser Publisher; Greenfield, EA (2014). Antibodies: A Laboratory Manual (2nd Edition). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, SL et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements up to 2014); and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV, the disclosures of which are incorporated herein by reference. .

[0151] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration only and are not intended to limit the scope of the disclosure or the claims in any way. Example 1 Construction of alphavirus vector

[0152] This example describes experiments performed to construct a basic alphavirus vector (eg, without a heterologous gene of interest) that was subsequently used to construct a vector expressing a gene of interest (eg, RABV-G). EEEV basic carrier

[0153] The basic EEEV vector (i.e., without the heterologous gene of interest) was constructed as follows: The basic EEEV vector was synthesized de novo from a reference sequence (Genbank EF151502) with several modifications in four approximately 4 kb sections (Twist Bioscience). Silent mutations G301A, A3550C, G4516A, G5725A, G7399A mutations were incorporated to eliminate restriction enzyme sites. Unique restriction enzyme sites (SpeI, 5'-A'CTAG, T-3') were incorporated to replace the coding sequence of the native EEEV structural gene (wherein the 5'A matches the position of the structural polyprotein ATG start codon and the 3'T matches the position of the structural polyprotein stop codon TAA). The 5' adapter sequence (5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3'; SEQ ID NO: 2) was inserted upstream of the SpeI site, and the 3' adapter sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC-3'; SEQ ID NO: 3) was inserted downstream of the SpeI site for subsequent Gibson Procedure (Gibson et al., Nat. Methods 6, 343-345, 2009). The bacteriophage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO: 4) was included upstream of the EEEV genomic sequence, and the downstream contained a poly(A) sequence, followed by a SapI site upstream of the cleavage recognition site. Immediately downstream of the SapI site was the T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 5), followed by a unique restriction enzyme site (NotI, 5'-GC'GGCC, GC-3'). These parts were combined in a five-piece Gibson The reactions (linearized pYL backbone and the four synthetic fragments) were combined to generate the EEEV base vector. CHIKV base vector

[0154] The basic CHIKV S27 vector was synthesized de novo in four approximately 4 kb sections (Twist Bioscience, ThermoFisher GeneArt) from a reference sequence (Genbank AF369024) with a silent A5366G mutation and a unique restriction site (SpeI, 5'-A'CTAG, T-3') that replaces the coding sequence of the CHIKV structural gene (wherein the 5'A matches the position of the ATG start codon of the structural polyprotein and the 3'T matches the position of the stop codon TAA of the structural polyprotein). A 5' adapter sequence (5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3'; SEQ ID NO: 2) was inserted upstream of the SpeI site, and a 3' adapter sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC-3'; SEQ ID NO: 3) was inserted downstream of the SpeI site for subsequent Gibson Procedure. The bacteriophage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO: 4) was included upstream of the CHIKV genomic sequence, and the downstream contained a poly(A) sequence, followed by a SapI site upstream of the cleavage recognition site. Immediately downstream of the SapI site was the T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 5), followed by a unique restriction enzyme site (NotI, 5'-GC'GGCC, GC-3'). The sections were assembled in a five-piece Gibson The reactions (linearized pYL backbone and the four synthetic fragments) were combined to generate the CHIKV S27 base vector.

[0155] The CHIKV DRDE base vector was similarly constructed from the reference sequence (Genbank EF210157), except that the S27 3'UTR was used in place of the DRDE 3'UTR. SINV base vector

[0156] The basic SINV Girdwood vector was synthesized de novo in four approximately 4 kb sections (Twist Bioscience, Thermo Fisher GeneArt) from the Girdwood strain reference sequence (Genbank MF459683) with unique restriction enzyme sites (SpeI, 5'-A'CTAG, T-3') replacing the coding sequence of the SINV structural gene (wherein 5'A is the next nucleotide after the P2A sequence after nucleotide 93 of the structural polyprotein gene, and 3'T matches the position of the stop codon TGA of the structural polyprotein). The bacteriophage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO: 4) was included upstream of the SINV genomic sequence, and the downstream contained a poly (A) sequence, followed by a SapI site upstream of the cleavage recognition site. Immediately downstream of the SapI site is a T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 5), followed by a unique restriction enzyme site (NotI, 5'-GC'GGCC, GC-3'). The pYL backbone and four synthetic fragments were combined in a reaction (eg, linearized pYL backbone and four synthetic fragments) to generate the SINV Girdwood base vector.

[0157] The base SINV AR86 vector was similarly constructed from the reference sequence (Genbank U38305), except that the nsP2 coding sequence was derived from the Girdwood reference sequence. VEE basic carrier

[0158] The basic VEE vector was synthesized de novo in four approximately 4 kb sections (Twist Bioscience, Thermo Fisher GeneArt) from the TC-83 strain reference sequence (Genbank L01443) with a silent A2087G mutation and a unique restriction site (SpeI, 5'-A'CTAG, T-3') that replaces the coding sequence of the VEE structural gene (wherein the 5'A is the next nucleotide after the P2A sequence after nucleotide 93 of the structural polyprotein gene, and the 3'T matches the position of the stop codon TGA of the structural polyprotein). The 5' adapter sequence (5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3'; SEQ ID NO: 2) was inserted upstream of the SpeI site, and the 3' adapter sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC-3'; SEQ ID NO: 3) was inserted downstream of the SpeI site for subsequent Gibson Procedure. The bacteriophage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO:4) was included upstream of the VEE genomic sequence, and the downstream contained a poly(A) sequence, followed by a SapI site upstream of the cleavage recognition site. Immediately downstream of the SapI site was the T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO:5), followed by a unique restriction enzyme site (NotI, 5'-GC'GGCC, GC-3'). The sections were assembled in a five-piece Gibson The pYL backbone and four synthetic fragments were combined in a reaction (e.g., linearized pYL backbone and four synthetic fragments) to generate the VEE base vector.

[0159] The RABV-G transgene was synthesized as (IDT) with flanks homologous to the 5' and 3' adapter sequences and cloned by Gibson Insertion into the SpeI linearized base vector to generate the final vector. Example 2 In vitro evaluation of modified alphavirus vectors

[0160] This example describes the results of in vitro experiments performed to evaluate RNA replication and RABV-G expression levels of the synthetic alphavirus srRNA constructs described in Example 1 above and to investigate any differential behavior (eg, RNA replication and protein expression).

[0161] In vitro transcription : Using 5'ARCA cap (HiScribeTM T7 ARCA mRNA kit, NEB) or by capless transcription (HiScribe TM RNA was prepared from the SapI linearized plasmid template by in vitro transcription using bacteriophage T7 polymerase (T7 High Yield RNA Synthesis Kit, NEB) followed by addition of 5' cap 1 (Vaccinia Capping System, mRNA cap 2'-O-methyltransferase, NEB). RNA was then extracted using phenol / chloroform or purified by column purification ( RNA was purified using the RNA Cleanup Kit (NEB). RNA concentration was determined by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0162] RNA replication: Transform RNA into BHK-21 or Vero cells by electroporation (e.g., 4D-Nucleofector TM , Lonza). 15-22 hours after transformation, cells were fixed and permeabilized (eBioscience TM Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and stained with PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Scicons) to quantify the frequency of dsRNA+ cells and the mean fluorescence intensity (MFI) of dsRNA in individual cells by fluorescent flow cytometry. Figure 1 Shown in.

[0163] Protein expression : Transform RNA into BHK-21 or Vero cells by electroporation (e.g., 4D-Nucleofector TM , Lonza). 15-22 hours after transformation, cells were fixed and permeabilized (eBioscience TM Foxp3 / transcription factor staining buffer set, Invitrogen) and stained with AF647-conjugated anti-RABV-G antibody (1c5). The mean fluorescence intensity (MFI) of AF647 was used as a readout of RABV-G expression. The results are shown in Figure 2 middle. Example 3 In vivo evaluation of modified alphavirus vectors

[0164] This example describes the results of in vivo experiments performed to evaluate the srRNA constructs described herein.

[0165] In these experiments, synthetic srRNA constructs derived from various alphavirus strains were designed and subsequently evaluated.

[0166] Mice and injections BALB / c mice were purchased from Charles River Labs, Envigo or Jackson Laboratories. On the day of dosing, 0.15 or 1.5 μg of the material was injected intramuscularly into one quadriceps muscle or divided into two quadriceps muscles. The carrier was formulated with LNP. The body weight and other general observations of the animals were monitored throughout the study. For immunogenicity studies, animals were dosed only on day 0 or on day 0 and day 21.

[0167] LNP Formulations srRNA was formulated in lipid nanoparticles using a microfluidic mixer, and particle size, polydispersity were analyzed using dynamic light scattering and encapsulation efficiency. Lipids were suspended in ethanol. RNA was suspended in 250 mM NaOAc (pH 4.0) at a concentration of 82 ug / ml and mixed at a flow rate of 3:1 (aqueous: organic).

[0168] ELISpot. To measure the magnitude of RABV-G-specific T cell responses, IFNγ ELISpot analysis was performed using the Mouse IFNγ ELISpot PLUS Kit (HRP) (MabTech) according to the manufacturer's instructions. Results of the mouse IFNγ detection ELISpot assay, measured by spot-forming units corresponding to responding splenic T cells, were compared 14 days after intramuscular injection of srRNA encoding RABV-G. Figure 3 Total T cell responses (plotted as spot forming units per million cell counts) are shown on the y-axis.

[0169] Neutralizing antibody response obtained by rapid fluorescent focus inhibition test: In order to measure the neutralizing antibody titer produced by the vaccine, serum was obtained from mice 14 days after intramuscular administration, diluted, mixed, and incubated with a standard amount of rabies virus in a multi-chamber slide. After a short incubation, cells susceptible to rabies virus infection and replication were added to the serum-virus mixture and incubated overnight (about 16-24h) to allow unneutralized virus to infect cells and replicate. The cells were then fixed and stained for the production of rabies virus using a fluorescent microscope. Up to 20 microscope fields were read for each serum sample in total and compared with a slide with a reference control serum-virus mixture. The infected cells for each serum dilution were counted and used to determine the neutralization titer. The final RFFIT result is the calculation result of the count multiplied by the serum dilution factor. The result of RFFIT can be expressed as a serum titer or in international units (IU) of antibodies per milliliter of serum, which is achieved by comparing the neutralizing activity of the test serum with the neutralizing activity of a reference serum with a known IU concentration ( Figure 4 ). Example 4 Modified alphavirus vectors as rabies virus post-exposure prophylaxis for the treatment of rabies infection Internal evaluation

[0170] This example describes in vivo experiments performed to evaluate the srRNA constructs described herein.

[0171] In these experiments, synthetic srRNA constructs derived from various alphavirus strains were designed and subsequently evaluated for their ability to prevent disease and mortality from rabies virus infection via post-exposure prophylactic vaccination.

[0172] Induce rabies virus. The challenge virus standard strain (CVS)-11 was purchased from the American Type Culture Collection (ATCC) and grown in baby hamster kidney (BHK)-21 cells.

[0173] LNP Formulations srRNA was formulated in lipid nanoparticles using a microfluidic mixer, and particle size, polydispersity were analyzed using dynamic light scattering and encapsulation efficiency. Lipids were suspended in ethanol. RNA was suspended in 250 mM NaOAc (pH 4.0) at a concentration of 82 μg / ml and mixed at a flow rate of 3:1 (aqueous: organic).

[0174] Mice and injections BALB / c mice were purchased from Charles River Labs, Envigo or Jackson Laboratories. On the day before srRNA administration (day -1), 10 2.5Mice were inoculated intranasally with a 50% cell culture infectious dose of CVS-11. On the day of srRNA administration (day 0, or day 0 and day 14), 0.1, 1, or 10 μg of material was injected intramuscularly into one quadriceps muscle or divided into two quadriceps muscles. srRNA was formulated with LNP. Animals were monitored for weight and clinical observation throughout the study. For some groups, the addition of human rabies immune globulin (HRIG) or a similar rabies neutralizing immunoglobulin can be combined with srRNA treatment.

[0175] Disease progression. Mice were observed for signs of disease every day until about 35 days after virus inoculation (DPI) or until the control non-treatment group was lost. Typical disease patterns that progressed were scored as follows: separation from the group (score 1), slow movements / not very active (score 2), paw paresis (score 3), uncoordinated movements (score 4), no spontaneous movements (score 5), no response to stimulation (score 6), and a terminal stage characterized by mice burying their heads in the cage pads and slow breathing (score 7). Disease progression was represented by drawing daily scores of the DPI function. The incubation period was defined as the time period from virus inoculation to the first appearance of disease signs. When mice reached 6 points, they were euthanized by cervical dislocation. The results were represented by Kaplan-Meier survival curves.

[0176] Viral load. Use real-time reverse transcriptase polymerase chain reaction (RT-qPCR) to confirm the rabies virus infection in the brain. According to the manufacturer's instructions (RNeasy test kit, Qiagen), the brain is homogenized and RNA is extracted. Alternatively, according to standard methods, direct fluorescent antibody (DFA) test can be used to determine the rabies virus staining in the fixed tissue.

[0177] Although specific alternatives to the present disclosure have been disclosed, it should be understood that various modifications and combinations are possible and are considered within the true spirit and scope of the appended claims.Therefore, there is no intention to be limited to the exact abstract and disclosure presented herein.

Claims

1. A nucleic acid construct comprising a nucleic acid sequence encoding a modified alphavirus genome or a self-replicating RNA (srRNA), wherein at least a portion of the nucleic acid sequence encoding a viral structural protein of the modified alphavirus genome or srRNA has been replaced by a coding sequence of a polypeptide construct comprising an antigenic determinant of the envelope glycoprotein G of rabies virus (RABV-G), a variant thereof, or either of the two.

2. The nucleic acid construct of claim 1, wherein the antigenic determinant is located within the N-terminal half of the RABV-G.

3. The nucleic acid construct of claim 1, wherein the antigenic determinant is located within the C-terminal half of the RABV-G.

4. The nucleic acid construct of claim 1, wherein the antigenic determinant comprises antigenic site I, antigenic site II, antigenic site III, antigenic site IV, small antigenic site A, or a combination of any of the RABV-G.

5. The nucleic acid construct according to any preceding claim, wherein the envelope glycoprotein G is the envelope glycoprotein G of a virulent rabies virus strain or an avirulent rabies virus strain.

6. The nucleic acid construct according to any preceding claim, wherein the envelope glycoprotein G is an envelope glycoprotein G of a rabies virus strain selected from the group consisting of Flury LEP, Flury LEP-C, Flury HEP, 1088, AT6, CQ92, CVS-11, CVS-26, CVS-26 (G-N204S), CYN1009D, CYN1026D, CYN1029D, CYN1138D, CYN1140D, CYN1141D, CYN1242H, CYN1243D, CYN1244D, CYN1245 D strain, CYN1247D strain, CYN1249D strain, CYN1250D strain, CYN1251D strain, CYN1252D strain, CYN1253D strain, CYN1255D strain, CYN 1256D strain, CYN1257D strain, CYN1259D strain, CYN1260D strain, CYN1261D strain, GX4 strain, H-08-1320 strain, H-1413-09 strain, IP 1586 / 10 strain, IP 2990 / 13 strain, IP 2991 / 13 strain, IP 2992 / 13 strain, IP 3176 / 09 strain, IP4005 / 12 strain, IP 412 / 10 strain, IP 542 / 10 strain, IP 7941 / 09 strain, J strain, JX-08-47 strain, JX08-48 strain, Kyoto strain, Kyoto (G-S204N) strain, N.HL strain, RC.HL strain, rHEP5.0-CVSG strain, RRV ON-99-2 strain, SAD-B19 strain, SH06 strain, SHRBV-18 strain, SNK-CTN strain, SRV9 strain, Street Alabama Dufferin (HCP-SAD) strain, VRC-RZ2 strain, ZJ-LA strain and ZJ-QZ strain. The nucleic acid construct according to claim 6 , wherein the envelope glycoprotein G is the envelope glycoprotein G of the Flury LEP strain.

8. The nucleic acid construct of any preceding claim, wherein the polypeptide construct comprises a molecular alteration that stabilizes an antigenic determinant of the RABV-G, a variant thereof, or either.

9. The nucleic acid construct according to any preceding claim, wherein the modified alphavirus genome or srRNA does not comprise a nucleic acid sequence encoding a viral structural protein.

10. A nucleic acid construct according to any preceding claim, wherein the nucleic acid sequence encoding the polypeptide construct is operably linked to a promoter sequence.

11. The nucleic acid construct of claim 10, wherein the promoter sequence is a 26S subgenomic (sg) promoter.

12. The nucleic acid construct according to any preceding claim, wherein the modified alphavirus genome or srRNA is a modified alphavirus genome or srRNA of an alphavirus belonging to the VEEV / EEEV / WEEV group, or the SFV group or the SINV group.

13. The nucleic acid of claim 12, wherein the alphavirus is Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Chikungunya virus (CHIKV), Western equine encephalitis virus (WEEV), or Sindbis virus (SINV).

14. A nucleic acid construct according to any preceding claim, wherein the nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 6-11.

15. A recombinant cell comprising the nucleic acid construct according to any one of claims 1-14. The recombinant cell according to claim 15 , wherein the recombinant cell is a mammalian cell or an insect cell.

17. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the nucleic acid construct according to any one of claims 1 to 14.

18. The pharmaceutical composition of claim 17, wherein the composition is formulated into a delivery system with a delivery vehicle, wherein the delivery system comprises liposomes, viral replicon particles (VRPs), lipid-based nanoparticles (LNPs), polymer nanoparticles, physiological buffers, microspheres, immunostimulatory complexes (ISCOMs), conjugates of biologically active ligands, or a combination of any thereof.

19. The pharmaceutical composition of claim 18, wherein the LNP delivery system comprises a cationic lipid, an ionizable cationic lipid, an anionic lipid, or a neutral lipid.

20. The pharmaceutical composition of any one of claims 18-19, wherein the lipid is present in a mass ratio of lipid to RNA of about 100:1 to about 4:

1.

21. The pharmaceutical composition of any one of claims 18-20, wherein the lipid-based nanoparticles have an average diameter of about 25 nm to about 1000 nm.

22. The pharmaceutical composition according to any one of claims 18-21, wherein the composition is formulated as a vaccine or an adjuvant.

23. The pharmaceutical composition of claim 17, wherein the composition is formulated for intramuscular administration.

24. A method of inducing an immune response or treating rabies infection in a subject in need thereof, the method comprising administering to the subject a composition comprising a nucleic acid construct according to any one of claims 1-14.

25. The method according to claim 24, wherein the method is a method for inducing an immune response.

26. The method of claim 25, wherein the immune response is a neutralizing antibody response.

27. The method of claim 26, wherein the neutralizing antibody response comprises a neutralizing antibody titer greater than 0.5 IU / mL.

28. The method of claim 24, wherein the subject has or is suspected of having a rabies infection.

29. The method of claim 24, wherein the composition is administered to the subject as a single agent prophylactic or therapeutic (monotherapy) alone or as a first therapy in combination with at least one additional therapy.

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