Methods for inducing extended immune responses

Abstract

The present disclosure relates generally to the field of molecular virology, and particularly relates to methods of inducing extended immune responses by administration of nucleic acid molecules encoding a modified alphavirus virus viral genome or self-replicating RNA (srRNA) construct.

Description

Attorney Docket No.: 058462-520001WOMETHODS FOR INDUCING EXTENDED IMMUNE RESPONSESCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S. Provisional Patent Application Serial No. 63 / 790,436, filed on April 17, 2025. The contents of the above-referenced application is herein expressly incorporated by reference in its entirety, including any drawings.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The material in the accompanying Sequence Listing is hereby incorporated by reference into this application. The accompanying Sequence Listing XML file, named 058462-52000 lWO_Sequence_Listing_ST26. xml, was created on April 15, 2026, and is approximately 73,728 bytes in size.FIELD

[0003] The present disclosure relates to the field of molecular virology and immunology, and particularly relates to methods of inducing an extended immune response in human after administration of nucleic acid molecules encoding modified alphavirus viral genomes or selfreplicating RNA (srRNA). Also provided are methods for expanding T follicular helper cells in humans.BACKGROUND

[0004] Advantages of mRNA vaccines were evident during the SARS-CoV-2 pandemic, due to their ability to elicit protective immunity while accelerating production timelines.However, longitudinal analysis of immune responses over the following four years has shown that antibody titers to the encoded SARS-CoV-2 Spike antigen in mRNA vaccines wane, leaving immunized populations vulnerable to infection. Moreover, comparison of approved mRNA vaccines, Spikevax (mRNA-1273) and (BNT162b2) to Ad26.COV2.S, a viral vector approach, showed differential kinetics, where mRNA vaccines showed a more rapid decline in antibody titers. This limitation has now become apparent for additional targets, such as Respiratory syncytial virus (RSV). RSV vaccines, Arexvy and Abrysvo are approved adjuvanted Fusion (F) protein subunit vaccines and have shown to elicit superior durability of immune responses compared to mRESVIA, the approved mRNA vaccine encoding F. Thus, for two distinct viral antigens, SARS-CoV-2 Spike and RSV F, mRNA as a class may not beAttorney Docket No.: 058462-520001WOadvantaged as a vaccine platform compared to traditional technologies for generation of long-lasting immunity.

[0005] In contrast, vaccines using self-replicating RNA (srRNA) technology, which share beneficial attributes with conventional mRNA such as simplified manufacturing and rapid deployability, have shown to be superior in generation of sustained immunity. Recent studies have shown that antibody titers from SARS-CoV-2 srRNA vaccine Kostaive (ARCT-154), approved in Japan and EU, have better durability compared to mRNA (BNT162.b2) in a head-to-head Phase 3 clinical trial. In addition, srRNA SARS-CoV-2 vaccine candidate GRT-R910, showed 6 month durability of neutralizing antibody titers when administered as a boost to adenoviral primary vaccination series (palmer 2023), whereas antibody titers from mRNA vaccines (BNT162b2 and mRNA1273) declined after just 3 months in a similar boost study (Liu et al J infect 2022). Thus, self-replicating RNA vaccines, as a class, are differentiated from conventional mRNA in their ability to elicit more durable immune responses.

[0006] However, a direct head-to-head comparison of srRNA to a traditional ( / .< ., protein or whole virus) vaccine technology is needed to show the full promise of this platform as a transformative vaccine technology.

[0007] The disclosure provided here provides solutions to the problems existing with previous attempts to compare srRNA to traditional vaccines by comparing a next generation srRNA vaccine for rabies prophylaxis to a commercial inactivated viral vaccine RabAvert in a Phase 1 study (NCT06048770).SUMMARY

[0008] The present disclosure relates generally to the induction of extended immune responses in humans receiving srRNA vaccines that are equal or superior to that induced by a traditional commercial vaccine. Also disclosed are methods for expanding T follicular helper cells in humans receiving a vaccination. The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.

[0009] In one aspect of the disclosure, provided herein is a method of inducing an extended immune response in a human subject. The method includes administering to the human subject a vaccine composition comprising a nucleic acid construct comprising a nucleic acidAttorney Docket No.: 058462-520001WOsequence 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 for a polypeptide construct comprising an envelope glycoprotein G of a rabies virus (RABV-G), a variant thereof, or an antigenic determinant of either thereof, wherein the induced immune response extends beyond 85 days after the administering.

[0010] In some embodiments, the induced immune response extends at least 120 days after administering.

[0011] In some embodiments, the induced immune response extends at least 180 days after administering.

[0012] In some embodiments, the induced immune response extends at least 240 days after administering.

[0013] In some embodiments, the vaccine composition comprises a pharmaceutically acceptable excipient.

[0014] In some embodiments, the vaccine composition is formulated with a delivery vehicle into a delivery system, wherein the delivery system comprises a liposome, a viral replicon particle (VRP), a lipid-based nanoparticle (LNP), a polymer nanoparticle, a physiologic buffer, a microsphere, an immune stimulating complex (ISCOM), a conjugate of bioactive ligand, or a combination of any thereof. In some embodiments, the delivery system comprises a lipid-based nanoparticle (LNP). In some embodiments, the LNP delivery system comprises a cationic lipid, an ionizable cationic lipid, an anionic lipid, or a neutral lipid. In some embodiments, the LNP delivery system comprises an ionizable lipid.

[0015] In some embodiments, the vaccine composition is administered intramuscularly.

[0016] In some embodiments, the administering comprises administering a single dose.

[0017] In some embodiments, the administering comprises administering a first dose and a second dose. In some embodiments, the second dose is administered at least 8 weeks after the first dose.

[0018] In some embodiments, the administering comprises administering about 0.1 mcg of the vaccine composition.

[0019] In some embodiments, the administering comprises administering about 1 mcg of the vaccine composition.

[0020] In some embodiments, the administering comprises administering about 10 mcg of theAttorney Docket No.: 058462-520001WOvaccine composition.

[0021] In some embodiments, the extended immune response comprises a neutralizing antibody response. In some embodiments, the neutralizing antibody response comprises a neutralizing antibody titer of equal or greater than 0.5 lU / mL.

[0022] In some embodiments, the extended immune response comprises T follicular helper cells.

[0023] The method of any preceding claim, wherein the nucleic acid construct of the vaccine composition comprises a coding sequence for a polypeptide construct comprising an antigenic determinant of RABV-G that resides within the N-terminal half of the RABV-G.

[0024] In some embodiments, the nucleic acid construct of the vaccine composition comprises a coding sequence for a polypeptide construct comprising an antigenic determinant of RABV-G that resides within the C-terminal half of the RABV-G.

[0025] In some embodiments, the nucleic acid construct of the vaccine composition comprises a coding sequence for a polypeptide construct comprising an antigenic determinant of RABV-G comprising antigenic site I, antigenic site II, antigenic site III, antigenic site IV, antigenic site minor A of the RABV-G, or a combination of any thereof.

[0026] In some embodiments, the envelope glycoprotein G is of a virulent rabies virus strain or an avirulent rabies virus strain.

[0027] In some embodiments, the envelope glycoprotein G is of a rabies virus strain selected from a Flury LEP strain, a Flury LEP-C strain, a Flury HEP strain, a 1088 strain, a AT6 strain, a CQ92 strain, a CVS-11 strain, a CVS-26 strain, a CVS-26(G-N204S) strain, a CYN1009D strain, a CYN1026D strain, a CYN1029D strain, a CYN1138D strain, a CYN1140D strain, a CYN1141D strain, a CYN1242H strain, a CYN1243D strain, a CYN1244D strain, a CYN1245D strain, a CYN1247D strain, a CYN1249D strain, a CYN1250D strain, a CYN1251D strain, a CYN1252D strain, a CYN1253D strain, a CYN1255D strain, a CYN1256D strain, a CYN1257D strain, a CYN1259D strain, a CYN1260D strain, a CYN1261D strain, a GX4 strain, aH-08-1320 strain, aH-1413-09 strain, a IP 1586 / 10 strain, a IP 2990 / 13 strain, a IP 2991 / 13 strain, a IP 2992 / 13 strain, a IP 3176 / 09 strain, a IP 4005 / 12 strain, a IP 412 / 10 strain, a IP 542 / 10 strain, a IP 7941 / 09 strain, a J strain, a JX-08-47 strain, a JX08-48 strain, a Kyoto strain, a Kyoto(G-S204N) strain, a N.HL strain, a RC.HL strain, a rHEP5.0-CVSG strain, a RRV ON-99-2 strain, a SAD-B19 strain, a SH06 strain, a SHRBV-18 strain, a SNK-CTN strain, a SRV9 strain, a StreetAttorney Docket No.: 058462-520001WOAlabama Dufferin (HCP-SAD) strain, a VRC-RZ2 strain, a ZJ-LA strain, and a ZJ-QZ strain.

[0028] In some embodiments, the envelope glycoprotein G is of a Flury LEP strain.

[0029] In some embodiments, the nucleic acid sequence encoding the modified alphavirus genome or srRNA of the vaccine composition comprises no nucleic acid sequence encoding viral structural proteins.

[0030] In some embodiments, the nucleic acid sequence encoding the polypeptide construct of the vaccine composition is operably linked to a promoter sequence. In some embodiments, the promoter sequence is a 26S subgenomic (.g) promoter.

[0031] In some embodiments, the modified alphavirus genome or srRNA encoded by the nucleic acid construct of the vaccine composition is 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).

[0032] In some embodiments, the nucleic acid construct of the vaccine composition 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 the group consisting of SEQ ID NOS: 4-9.

[0033] In some embodiments, the human subject is having or suspected of having a rabies infection.

[0034] In some embodiments, the composition is administered to the human subject individually as a single agent prophylaxis or therapy (monotherapy) or as a first therapy in combination with at least one additional therapy.

[0035] Also provided herein is a method of expansion of T follicular helper cells in a human subject. The method includes administering to the human subject a vaccine composition comprising 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 for a polypeptide construct comprising an envelope glycoprotein G of a rabies virus (RABV-G), a variant thereof, or an antigenic determinant of either thereof.

[0036] In some embodiments, the T follicular helper cells are CD69+ and CD40L+.Attorney Docket No.: 058462-520001WO

[0037] In some embodiments, the vaccine composition comprises a pharmaceutically acceptable excipient.

[0038] In some embodiments, the vaccine composition is formulated with a delivery vehicle into a delivery system, wherein the delivery system comprises a liposome, a viral replicon particle (VRP), a lipid-based nanoparticle (LNP), a polymer nanoparticle, a physiologic buffer, a microsphere, an immune stimulating complex (ISCOM), a conjugate of bioactive ligand, or a combination of any thereof. In some embodiments, the delivery system comprises a lipid-based nanoparticle (LNP). In some embodiments, the LNP delivery system comprises a cationic lipid, an ionizable cationic lipid, an anionic lipid, or a neutral lipid. In some embodiments, the LNP delivery system comprises an ionizable lipid.

[0039] In some embodiments, the vaccine composition is administered intramuscularly.

[0040] In some embodiments, the administering comprises administering a single dose.

[0041] In some embodiments, the administering comprises administering a first dose and a second dose. In some embodiments, the second dose is administered at least 8 weeks after the first dose.

[0042] In some embodiments, the administering comprises administering about 0.1 mcg of the vaccine composition.

[0043] In some embodiments, the administering comprises administering about 1 mcg of the vaccine composition.

[0044] In some embodiments, the administering comprises administering about 10 mcg of the vaccine composition.

[0045] In some embodiments, the nucleic acid construct of the vaccine composition comprises a coding sequence for a polypeptide construct comprising an antigenic determinant of RABV-G that resides within the N-terminal half of the RABV-G.

[0046] In some embodiments, the nucleic acid construct of the vaccine composition comprises a coding sequence for a polypeptide construct comprising an antigenic determinant of RABV-G that resides within the C-terminal half of the RABV-G.

[0047] In some embodiments, the nucleic acid construct of the vaccine composition comprises a coding sequence for a polypeptide construct comprising an antigenic determinant of RABV-G comprising antigenic site I, antigenic site II, antigenic site III, antigenic site IV, antigenic site minor A of the RABV-G, or a combination of any thereof.

[0048] In some embodiments, the envelope glycoprotein G is of a virulent rabies virus strainAttorney Docket No.: 058462-520001WOor an avirulent rabies virus strain.

[0049] In some embodiments, the envelope glycoprotein G is of a rabies virus strain selected from a Flury LEP strain, a Flury LEP-C strain, a Flury HEP strain, a 1088 strain, a AT6 strain, a CQ92 strain, a CVS-11 strain, a CVS-26 strain, a CVS-26(G-N204S) strain, a CYN1009D strain, a CYN1026D strain, a CYN1029D strain, a CYN1138D strain, a CYN1140D strain, a CYN1141D strain, a CYN1242H strain, a CYN1243D strain, a CYN1244D strain, a CYN1245D strain, a CYN1247D strain, a CYN1249D strain, a CYN1250D strain, a CYN1251D strain, a CYN1252D strain, a CYN1253D strain, a CYN1255D strain, a CYN1256D strain, a CYN1257D strain, a CYN1259D strain, a CYN1260D strain, a CYN1261D strain, a GX4 strain, aH-08-1320 strain, aH-1413-09 strain, a IP 1586 / 10 strain, a IP 2990 / 13 strain, a IP 2991 / 13 strain, a IP 2992 / 13 strain, a IP 3176 / 09 strain, a IP 4005 / 12 strain, a IP 412 / 10 strain, a IP 542 / 10 strain, a IP 7941 / 09 strain, a J strain, a JX-08-47 strain, a JX08-48 strain, a Kyoto strain, a Kyoto(G-S204N) strain, a N.HL strain, a RC.HL strain, a rHEP5.0-CVSG strain, a RRV ON-99-2 strain, a SAD-B19 strain, a SH06 strain, a SHRBV-18 strain, a SNK-CTN strain, a SRV9 strain, a Street Alabama Dufferin (HCP-SAD) strain, a VRC-RZ2 strain, a ZJ-LA strain, and a ZJ-QZ strain.

[0050] In some embodiments, the envelope glycoprotein G is of a Flury LEP strain.

[0051] In some embodiments, the nucleic acid sequence encoding the modified alphavirus genome or srRNA of the vaccine composition comprises no nucleic acid sequence encoding viral structural proteins.

[0052] In some embodiments, the nucleic acid sequence encoding the polypeptide construct of the vaccine composition is operably linked to a promoter sequence. In some embodiments, the promoter sequence is a 26S subgenomic (.g) promoter.

[0053] In some embodiments, the modified alphavirus genome or srRNA encoded by the nucleic acid construct of the vaccine composition is of an alphavirus belonging to the VEEV / EEEV / WEEV group, or the SFV group, or the SINV group.

[0054] 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).

[0055] In some embodiments, the nucleic acid construct of the vaccine composition 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 the groupAttomey Docket No.: 058462-520001WOconsisting of SEQ ID NOS: 4-9.

[0056] In some embodiments, the human subject is having or suspected of having a rabies infection.

[0057] In some embodiments, the composition is administered to the human subject individually as a single agent prophylaxis or therapy (monotherapy) or as a first therapy in combination with at least one additional therapy.BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The features of the present disclosure are set forth with particularity 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 that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0059] FIGs. 1A-1B show long term rabies virus neutralizing antibody (RVNA) responses following immunization with RBI-4000 or Rab Avert. FIG. 1A shows longitudinal measurements of serum geometric mean titers of RVNA as measured by RFFIT assay.Dashed grey line indicates 0.5 lU / mL threshold. FIG. IB shows RVNA responses at six months per cohort. Frequency of subjects within each cohort that maintain detectable RVNA titers but below (light blue) or above (dark blue) the WHO indirect immune measure of protection. Frequency of subjects with no detectable RVNA shown in grey.

[0060] FIG. 2 shows statistical modeling of RVNA decay. Model 1 measures group peak to the start of stable decay for the group. Model 2 uses individual peaks, and the group GMT start of stable decay. Model 3 uses the group mean GMT as the peak and the final study day 240. Model 4 uses the individual RVNA peak and the final study day 240. Model 5 uses the group mean GMT peak and the point at which the first subject decays to the LLOQ. Model 6 measures each individual RVNA peak and the point at which the first subject of the group decays to the LLOQ. Model 7 measures from group peak to the group half-life from the peak RVNA. Model 8 measures from individual peak to individual half-life of RVNA titers.

[0061] FIG.3 shows slope and peak by model and cohort of rabies virus neutralizing antibody response.

[0062] FIGs. 4A-4H show decay analysis of RBI-4000 RVNA titers compared to Rab Avert. Decay rates were calculated based on eight different statistical models. The slopes of RBLAttorney Docket No.: 058462-520001WO4000 treated cohorts were compared to the slope of the RabAvert cohort and the ratio is plotted. Symbols show the group ratio and error bars show 95% CI.

[0063] FIGs. 5A-5C show decay rate modeling of RVNA responses elicited by RBI-4000 compared to RabAvert. RVNA decay rate modeling based on either (FIG. 5A) Model 6 that measured individual subject peak RVNA titers until the first individual reaches lower limit of quantification (LLOQ) of the assay; or (FIG. 5B) Model 8 where individual RVNA titer peak to individual corresponding RVNA titer half-life. Slopes calculated based on these models are shown as a ratio compared to RabAvert for all RBI-4000 groups. Symbols show ratio and error bars show 95% CI for each group. FIG. 5C is a Table that shows half-life values for each cohort from the peak of the RVNA response.

[0064] FIGs. 6A-6B show RBI-4000 primes activation-induced marker (AIM+) positive CD4 T cells. A. Representative flow cytometry plots and gating strategy to identify AIM+ cells in PBMCs of RBI-4000 (10 mcg) and RabAvert 15 days post immunization, followed by ex vivo stimulation with a RABV-G peptide library and immunostaining. B. Frequency of AIM+ CD4+ T cells in all available subjects plotted for RBI-4000 (n= 7) and RabAvert (n= 4), symbols show individual subjects and bars represent mean + / - SD.DETAILED DESCRIPTION OF THE DISCLOSURE

[0065] Rabies virus is endemic in over 150 countries causing 59,000 deaths annually due to its 99% mortality rate if left untreated. As such it has been designated an NIAID priority antigen. Current rabies vaccines are effective for both pre- and post-exposure prophylaxis, however these suffer supply shortages due to complex manufacturing processes which increases the difficulties of administration in resource limited settings. Importantly, in contrast to influenza or SARS-CoV-2 where pre-existing immunity may confound vaccine responses, no previous exposure to rabies is expected, allowing for clear interpretation of vaccine-elicited immune responses in naive individuals. Lastly, a standardized WHO immune metric exists to benchmark efficacy of rabies vaccines, where titers of rabies virus neutralizing antibodies (RVNA) at or above 0.5 lU / mL are considered to be protective and provide a clear immunogenicity readout.

[0066] The results described herein relate to a Phase I study, where RBI-4000 was able to elicit a durable RVNA responses with equal or superior durability to RabAvert, a traditional whole virus vaccine, using decay model analysis. The Phase 1 randomized open label clinicalAttorney Docket No.: 058462-520001WOtrial study enrolled 89 healthy volunteers, age 18-45 years with no prior exposure or vaccination to rabies. The study included four cohorts of ascending doses of RBI-4000 and an active comparator of the commercially available rabies vaccine, Rab Avert. Participants in Cohorts 1 (0.1 mcg), 2 (1 mcg), and 4 (10 mcg) received a prime-boost dosing schedule of RBI-4000, 8 weeks apart, while Cohort 3 participants received a single dose of 10 mcg. The 8-week interval for RBI-4000 dosing was selected as it is optimal for generation of immune responses, specifically for srRNA-based vaccines. RabAvert was administered as per the CDC guidelines for pre-exposure prophylaxis, i.e., two doses 1 week apart, on Days 1 and 8. Safety analysis showed a well-tolerated vaccine with no serious adverse events. RVNA titers were assessed throughout the study at enrollment, and Days 15, 29, 43, 57, 64, 71, 85, 120, 180 for all participants, and partially for Day 240 upon study termination.

[0067] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0068] Although various features of the disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.DEFINITIONS

[0069] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0070] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, comprising 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”.

[0071] The term “portion” as used herein refers to a fraction. With respect to a particularAttorney Docket No.: 058462-520001WOstructure such as a polynucleotide sequence or an amino acid sequence or protein the term “portion” thereof may designate a continuous or a discontinuous fraction of said 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 said amino acid sequence. In addition, or alternatively, if the portion is a discontinuous fraction, said discontinuous fraction is composed of 2, 3, 4, 5, 6, 7, 8, or more parts of a structure (e.g., domains of a protein), each part being a continuous element of the structure. For example, a discontinuous fraction of an amino acid sequence may be composed of 2, 3, 4, 5, 6, 7, 8, or more, for example not more than 4 parts of said amino acid sequence, wherein each part comprises at least 1, at least 2, at least 3, at least 4, at least 5 continuous amino acids, at least 10 continuous amino acids, at least 20 continuous amino acids, or at least 30 continuous amino acids of the amino acid sequence.

[0072] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0073] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%.

[0074] The term “percent identity,” as used herein in the context of two or more nucleic acidsAttorney Docket No.: 058462-520001WOor proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See e.g., the NCBI website at ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the complement of a sequence. This definition also includes sequences that have deletions and / or additions, as well as those that have 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 eta!., J Mol Biol 215:403, 1990). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof.

[0075] It is understood that aspects and embodiments of the disclosure described herein include "comprising", "consisting", 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 elements, steps, or ingredients 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. Any recitation herein of the term "comprising", particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps.

[0076] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular speciesAttorney Docket No.: 058462-520001WOis disclosed, this disclosure is intended to be exemplary only and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes or gene products disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences, are intended to encompass 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, the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides and proteins are human. The term “gene” is also intended to include variants thereof.

[0077] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.Alphaviruses

[0078] Alphaviruses are small, enveloped RNA viruses with a single-stranded, positive-sense RNA genome. The alphavirus genus includes, inter alia, the Sindbis virus (SINV), the Semliki Forest virus (SFV), the 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 are able to infect various vertebrates such as mammalians, rodents, fish, avian species, and larger mammals such as humans and horses as well as invertebrates such as insects. In particular, the Sindbis and the Semliki Forest viruses have been widely studied and the life cycle, mode of replication, etc., of these viruses are well characterized.

[0079] The alphavirus genome is approximately 12 Kb long, and it consists of two open reading frames (ORFs): a 7 Kb frame encoding the nonstructural proteins (nsPs) and a 4 Kb frame encoding the structural polyprotein. The non-structural polyprotein (nsP) is cleaved into four different proteins (nsPl, nsP2, nsP3, and nsP4) which are necessary for theAttorney Docket No.: 058462-520001WOtranscription and translation of viral mRNA inside the cytoplasm of host cells.

[0080] The nsPl protein is an mRNA capping enzyme that possesses both guanine-7-methyltransferase (MTase) and guanylyltransferase (GTase) activities, where they direct the methylation and capping of newly synthesized viral genomic and subgenomic RNAs. The MTase motif in the N-terminal domain of nsPl catalyzes the transfer of the methyl group from S -adenosylmethionine (AdoMet) to the N7 position of a GTP molecule (m7Gppp). GTase then binds the m7Gppp, forming a covalent link with a catalytic histidine (m7Gp-GTase) and releasing PPi. The GTase then transfers the m7Gp molecule to the 5 ’-diphosphate RNA to create m7GpppNp-RNA. The resulting cap structure is essential for viral mRNA translation and prevents the mRNA from being degraded by cellular 5’ exonucleases.Following the N-terminal domain are features that allow the association of the nsPl protein to cellular membranes. The presence of a-helical amphipathic loop and palmitoylation sites allow the nsPl protein and nsPl -containing replication complex to anchor onto the plasma membrane, possibly through nsPl interaction with the membrane’s anionic phospholipids.

[0081] The nsP2 protein possesses numerous enzymatic activities and functional roles. The N-terminal region contains a helicase domain that has seven signature motif of Superfamily 1 (SF1) helicases. It functions as an RNA triphosphatase that performs the first of the viral RNA capping reactions. It also functions as a nucleotide triphosphatase (NTPase), fueling the RNA helicase activity. The C-terminal region of nsP2 contains a papain-like cysteine protease, which is responsible for processing the viral non- structural polyprotein. The protease recognizes conserved motifs within the polyprotein. This proteolytic function is highly regulated and is modulated by other domains of nsP2. The alphavirus nsP2 protein has also been described as a virulence factor responsible for the transcriptional and translational shutoff in infected host cells and the inhibition of interferon (IFN) mediated antiviral responses contributing to the controlling of translational machinery by viral factors.

[0082] The precise role(s) of alphavirus nsP3 protein in the replication complex is less clear. The nsP3 protein has three recognized domains: the N-terminal macrodomain with phosphatase activity and nucleic acid binding ability, the alphavirus unique domain (AUD) and the C-terminal hypervariable domain. It has been demonstrated that the deletion of this domain in SFV nsP3 resulted in low viral pathogenicity, suggesting its importance in viral RNA transcription regulation.

[0083] The nsP4 polymerase is the most highly conserved protein in alphaviruses, with theAttorney Docket No.: 058462-520001WOmost divergent being >50% identical in amino acid sequence when compared with other alphaviral nsP4s. The nsP4 contains the core RNA-dependent RNA polymerase (RdRp) domain at the C-terminal end, determined to be solely responsible for the RNA synthetic properties of the viral replication complex. The RdRp participates in replicating the genomic RNA via a negative strand RNA and transcribing the 26S subgenomic RNA. The N-terminal domain is alphavirus-specific and can be partially disordered structurally.

[0084] The 5' two-thirds of the alphavirus genome encodes a number of non- structural proteins (nsPs) necessary for transcription and replication of viral RNA. These proteins are translated directly from the RNA and together with cellular proteins form the RNA-dependent RNA polymerase essential for viral genome replication and transcription of sgRNA. Four nsPs (nsPl-4) are produced as a single polyprotein constitute the virus' replication machinery. The processing of the polyprotein occurs in a highly regulated manner, with cleavage at the P2 / 3 junction influencing RNA template use during genome replication. This site is located at the base of a narrow cleft and is not readily accessible. Once cleaved, nsP3 creates a ring structure that encircles nsP2. These two proteins have an extensive interface. Mutations in nsP2 that produce noncytopathic viruses or a temperature sensitive phenotypes cluster at the P2 / P3 interface region. P3 mutations opposite the location of the nsP2 noncytopathic mutations prevent efficient cleavage of P2 / 3. This in turn can affect RNA infectivity altering viral RNA production levels.

[0085] The 3’ one-third of the genome comprises sgRNA which serves as a template for translation of all the structural proteins required for forming viral particles: the core nucleocapsid protein C, and the envelope proteins P62 and El that associate as a heterodimer. The viral membrane-anchored surface glycoproteins are responsible for receptor recognition and entry into target cells through membrane fusion. The 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 into E2 and E3 causes a change in the viral surface. Together the El, E2, and sometimes E3, glycoprotein "spikes" form an E1ZE2 dimer or an E1ZE2 / E3 trimer, where E2 extends from the center to the vertices, El fills the space between the vertices, and E3, if present, is at the distal end of the spike. Upon exposure of the virus to the acidity of the endosome, El dissociates from E2 to form an El homotrimer, which is necessary for the fusion step to drive the cellular and viral membranes together. The alphaviral glycoprotein El is a class II viral fusion protein, which is structurally differentAttorney Docket No.: 058462-520001WOfrom the class I fusion proteins found in influenza virus and HIV. The E2 glycoprotein functions to interact with the nucleocapsid through its cytoplasmic domain, while its ectodomain is responsible for binding a cellular receptor. Most alphaviruses lose the peripheral protein E3, while in Semliki viruses it remains associated with the viral surface.

[0086] Alphavirus replication has been reported to take place on membranous surfaces within the host cell. In the first step of the infectious cycle, the 5' end of the genomic RNA is translated into a polyprotein (nsPl-4) with RNA polymerase activity that produces a negative strand complementary to the genomic RNA. The sequence at the 3’ end of the genomic RNA plays an important role in the initiation negative-strand synthesis, where a minimum number of adenylate residues has been identified to be essential for replication to occur. In particular, it has been previously reported that for alphavirus genomes to replicate, there must be at least 11 residues in the poly(A) tail following the 3’ UTR to efficiently initiate minus-strand synthesis, and therefore replication to occur. It has also been previously reported that lengthening the poly(A) tail to 25 residues results in enhanced replication, but no further enhancement of replication was observed when the poly(A) was lengthened further to 34 residues. In addition, internal non-A residues in the poly(A) are most often deleterious to replication, which suggests that enzymatic poly(A) tailing would not benefit srRNA that did not exclusively contain 3’ adenylate residues following the 3’ UTR. It has been previously reported that there is no enhancement of minus-strand synthesis on RNA templates with greater than 25 adenylate residues in the poly(A) tail. In a second step of replication, the negative strand is used as a template for the production of two RNAs, respectively: (1) a positive genomic RNA corresponding to the genome of the secondary viruses producing, by translation, other nsPs and acting as a genome for the virus; and (2) sgRNA encoding the structural proteins of the virus forming the infectious particles. The positive genomic RNA / sgRNA ratio is regulated by proteolytic autocleavage of the polyprotein to nsPl, nsP2, nsP3 and nsP4. In practice, the viral gene expression takes place in two phases. In a first phase, there is main synthesis of positive genomic strands and of negative strands. During the second phase, the synthesis of sgRNA is virtually exclusive, thus resulting in the production of large amount of structural protein.Self-repli eating RNA

[0087] As will be appreciated by the skilled artisan, the term “self-replicating RNA” refers to RNA molecule that contains all of the genetic information required for directing its own selfAttorney Docket No.: 058462-520001WOamplification or self-replication within a permissive cell. To direct its own replication, the srRNA generally (1) encodes polymerase, replicase, or other proteins which may interact with viral or host cell -derived proteins, nucleic acids or ribonucleoproteins to catalyze the RNA amplification process; and (2) contain c / .s-acting RNA sequences required for replication and transcription of the subgenomic RNA (sgRNA). These sequences may be bound during the process of replication to its self-encoded proteins, or non-self-encoded cell-derived proteins, nucleic acids or ribonucleoproteins, or complexes between any of these components. In some embodiments of the disclosure, an alphavirus srRNA construct generally contains the following elements: 5' viral or defective-interfering RNA sequence(s) required in cis for replication, sequences coding for biologically active alphavirus non-structural proteins (e.g., nsPl, nsP2, nsP3, and nsP4), a subgenomic promoter (sg) for the sgRNA, 3' viral sequences required in cis for replication, and optionally a polyadenylate tract (poly(A)). In some instances, a subgenomic promoter (sg) that directs expression of a heterologous sequence can be included in the srRNA construct of the disclosure.

[0088] Further, the term srRNA generally refers to a molecule of positive polarity, or “message” sense, and the srRNA may be of length different from that of any known, naturally-occurring alphavirus. In some embodiments of the present disclosure, the srRNA does not contain at least a portion of the coding sequence for one or more of the alphavirus structural proteins; and / or sequences encoding structural genes can be substituted with heterologous sequences. In those instances, where the srRNA is to be packaged into a recombinant alphavirus particle, it can contain one or more sequences, so-called packaging signals, which serve to initiate interactions with alphavirus structural proteins that lead to particle formation.

[0089] The srRNA constructs of the 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 can 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 5 kb to about 6Attorney Docket No.: 058462-520001WOkb, about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 Kb to about 10 kb, about 6 Kb to about 9 Kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb to about 11 kb, about 7 kb to about 10 kb, about 7 kb to about 9 kb, about 7 kb to about 8 kb, about 8 kb to about 11 kb, about 8 kb to about 10 kb, about 8 kb to about 9 kb, about 9 kb to about 11 kb, about 9 kb to about 10 kb, or about 10 kb to about 11 kb. In some embodiments, the srRNA can have a length of about 6 kb to about 14 kb. In some embodiments, the srRNA can have a length of about 6 kb to about 16 kb.Rabies Virus envelope glycoprotein G (RABV-G)

[0090] Rabies virus is bullet shaped with a length of about 180nm and a diameter of 75 nm. The genome is non-segmented with negative sense single stranded RNA. The genome encodes five structural proteins 3’ N-P-M-G-L 5’: N (nucleoprotein), P (phosphoprotein), M (matrix protein), G (glycoprotein) and L (RNA dependent RNA polymerase). The leader and trailer untranslated regions at the 3’ and 5’ end of the genome flank the structural genes and between the protein encoding regions there are non-coding intergenic sequences; N-P, P-M, M-G, G-L.

[0091] Among the five structural proteins, G is present on the envelope and is the only protein exposed out. Glycoproteins are proteins which are covalently bonded with sugar units. Rabies virus glycoprotein (RABV-G) is a trimeric type I trans-membrane protein which are single pass type with extracellular N-terminus and cytoplasmic C-terminus for cell membrane. The precursor RABV-G is 524 amino acids (aa) in length (522 aa in Mokola virus), consisting of a signal peptide of 19 aa in the N terminal. The mature protein has N-terminal ectodomain (439 aa), a transmembrane segment (22 aa) and a cytoplasmic tail / endodomain / ENDO (44 aa). As the G protein covers the outer surface of the virion envelope and is the only target antigen, which is able to induce virus-neutralizing antibodies.METHOD OF INDUCING AN EXTENDED IMMUNE RESPONSE

[0092] In one aspect, the present disclosure relates to a method of inducing an extended immune response in a human subject. The method involves administering to the human subject a vaccine composition comprising 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 for a polypeptide construct comprising an envelope glycoprotein G of a rabies virus (RABV-G), aAttorney Docket No.: 058462-520001WOvariant thereof, or an antigenic determinant of either thereof. In some embodiments, the extended immune response lasts beyond 85 days after administration of the vaccine composition.

[0093] As used herein, an immune response in a subject can refer to the development of a humoral immune response, a cellular immune response, or a humoral and acellular immune response to an antigen (e.g., RABV-G). Immune responses can be determined using standard immunoassays and neutralization assays, which are known in the art.

[0094] As used herein, an “extended immune response” is used interchangeably with a “durable immune response”. The immune response to a vaccine composition is said to be “durable” if the serum of the human subject, when sampled at future time points following inoculation, maintains the presence serum neutralizing antibodies against the pathogen (e.g., rabies virus) from which the inoculated vaccine composition is derived.

[0095] In the context of the present disclosure, a durable immune response is demonstrated where a subject to which a vaccine composition as described herein has been administered displays detectable rabies virus neutralizing antibody (RVNA) titers for a period of greater than 85 days, 120 days, 150 days, 180 days, 210 days, 240 days, 270 days, 300 days, 330 days, one year, two years, 5 years or longer. In some embodiments, the RVNA titer is equal or greater than 0.5 lU / mL for a period of greater than 85 days, 120 days, 150 days, or 180 days.

[0096] In some embodiments, the extended immune response is a neutralizing antibody response. A neutralizing antibody response is an immune response wherein specialized cells of the immune system recognize the presentation of antigen(s), and launch a specific immune response, which prevents infection of target cells from an agent, for example a virus (e.g., rabies virus). Methods of detecting neutralizing antibody responses are known in the art and may include, for example, assays as described in the Examples herein. For example, neutralizing antibody responses can be measured by Rapid Fluorescent Foci Inhibition Test (RFFIT) after a single dose of the vaccine composition disclosed herein or after multiple doses of the vaccine composition disclosed herein. In some embodiments, multiple RFFIT assays can be performed over a period of about 1 week, 2 weeks, 1 month, 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, to about 12 months. An exemplary RFFIT analysisAttorney Docket No.: 058462-520001WOschedule is shown in the Examples provided herein and includes assessment of RVNA titers prior to vaccine administration, as well as at days 15, 29, 43, 57, 64, 71, 85, 120, 180, and 240. Additional methods for measuring rabies neutralizing antibody titers can also be used in the methods described herein and include, without limitation, a Fluorescent Antibody Virus Neutralization (FAVN) test, a Direct Fluorescent Antibody test (DFAT), Mouse Neutralization Test (MNT), Simplified Fluorescent Inhibition MicroTest (SFIMT), modified counterimmunoelectrophoresis (CIE / SN) technique, Virus Neutralization Test-Indirect Immunoperoxidase (VNT-IIP), Plaque Reduction Neutralization Test (PRNT), High Throughput Neutralization Test (HTNT), immunohistochemistry (IHQ), Latex Agglutination Test (LAT), Enzyme Linked Immunosorbent Assay (ELISA), Rapid Neutralizing Antibody detection test (RAPINA), Rapid Immunochromatographic Test Strip (ICTS), Electrochemiluminescence Test (ECL), and Time-resolving fluorimmunoassay (TRFIA) (Ciconello et al., A comparative review of serological assays for the detection of rabies virusspecific antibodies,” Acta Tropica 2022226:106254). In some embodiments, the neutralizing antibody response is measured using an RFFIT assay.

[0097] In some embodiments, the neutralizing antibody response comprises a neutralizing antibody titer of equal to or greater than 0.5 lU / mL after 85 days of administration of the vaccine composition. In some embodiments, the neutralizing antibody response comprises a neutralizing antibody titer of about 0.6 lU / mL, about 0.7 lU / mL, about 0.8 lU / mL, about 0.9 lU / mL, about 1 lU / mL, about 1.5 lU / mL, about 2 lU / mL, about 2.5 lU / mL, about 3 lU / mL, about 3.5 lU / mL, about 4 lU / mL, about 4.5 lU / mL, about 5 lU / mL, about 5.5 lU / mL, about 6 lU / mL, about 6.5 lU / mL, about 7 lU / mL, about 7.5 lU / mL, about 8 lU / mL, about 8.5 lU / mL, about 9 lU / mL, about 9.5 lU / mL, about 10 lU / mL, about 15 lU / mL, about 20 lU / mL, about 25 lU / mL, about 30 lU / mL, about 35 lU / mL, about 40 lU / mL, about 45 lU / mL, or about 50 lU / mL after 85 days of administration of the vaccine composition.

[0098] In some embodiments, the extended immune response comprises a cellular immune response. In some embodiments, the cellular immune response comprises a CD4+ T helper cell response. T helper (Th) cells are a class of CD4+ cells that function to regulate the proliferation of B cells and B cell responses. Th cells play an importance role in humoral immunity and immunopathology. Follicular helper T cells (TFH) are a defined subset of CD4+ T cells that are essential for helping cognate B cells form and maintain the germinal center (GC) reaction, and for development of humoral immune responses. Therefore, in someAttorney Docket No.: 058462-520001WOembodiments, the CD4+ T helper cell response comprises T follicular helper cells.

[0099] The T cell phenotype can be measured by any standard methods, including detecting cellular phenotypic markers by flow cytometry. T follicular helper cells can be identified by the expression of cellular markers including, but not limited to, CXCR5, CD25, CD69, CD95, CD57, 0X40, and CD40L (Laurent et al., “A novel subset of T-helper cells: follicular T-helper cells and their markers,” Haematologica 95(3):356-358 (2010)). Circulating T follicular helper cells can be detected in the peripheral blood (Locci et al, “Human Circulating PD- I CXCR.3 CXCR.5 Memory Tfh Cells Are Highly Functional and Correlate with Broadly Neutralizing HIV Antibody Responses,” Immunity 39(4):758-769 (2013)). In some embodiments, the circulating T follicular helper cells are CD69+CD40L+ (Reiss, S. et al. “Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells,” PLoS ONE 12, e0186998 (2017)).Vaccine composition

[0100] As described above, in the method of the present disclosure a vaccine composition is administered that includes 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 for a polypeptide construct comprising an envelope glycoprotein G of a rabies virus (RABV-G), a variant thereof, or an antigenic determinant of either thereof.

[0101] The term “construct” can refer to a recombinant molecule, e.g., recombinant nucleic acid or polypeptide, including one or more nucleic acid sequences or amino acid sequences from heterologous sources. For example, polypeptide constructs can be chimeric polypeptide molecules in which two or more amino acid sequences of different origin are operably linked to one another in a single polypeptide construct. Similarly, nucleic acid constructs can be chimeric nucleic acid molecules in which two or more nucleic acid sequences of different origin are assembled into a single nucleic acid molecule. Representative nucleic acid constructs can include any recombinant nucleic acid molecules, linear or circular, single stranded or double stranded DNA or RNA nucleic acid molecules, derived from any source, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid sequences have been operably linked. Two or moreAttorney Docket No.: 058462-520001WOnucleic acid constructs can be contained within a single nucleic acid molecule, such as a single vector, or can be containing within two or more separate nucleic acid molecules, such as two or more separate vectors.

[0102] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein, and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. A nucleic acid molecule can be double-stranded or single-stranded (e.g., a sense strand or an antisense strand). A nucleic acid molecule may contain unconventional or modified nucleotides. The terms “polynucleotide sequence” and “nucleic acid sequence” as used herein interchangeably refer to the sequence of a polynucleotide molecule. The nomenclature for nucleotide bases as set forth in 37 CFR §1.822 is used herein.

[0103] Nucleic acid molecules of the present disclosure can be of 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, for example between about 15 Kb to 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb.

[0104] Non-limiting exemplary embodiments of the methods of the disclosure can include one or more of the following features. In some embodiments, the alphavirus srRNA vector is devoid of at least a portion of the nucleic acid sequence encoding one or more of the viral structural proteins CP, El, E2, E3, and 6K of the alphavirus srRNA vector. In some embodiments, the alphavirus srRNA vector is devoid of a portion of or the entire sequence encoding CP. In some embodiments, the alphavirus srRNA vector is devoid of a portion of or the entire sequence encoding El. In some embodiments, the alphavirus srRNA vector is devoid of a portion of or the entire sequence encoding E2. In some embodiments, the alphavirus srRNA vector is devoid of a portion of or the entire sequence encoding E3. In some embodiments, the alphavirus srRNA vector is devoid of a portion of or the entire sequence encoding 6K. In some embodiments, the alphavirus srRNA vector is devoid of a portion of or the entire sequence encoding a combination of CP, El, E2, E3, and 6K. In some embodiments of the disclosure, the coding sequence for nonstructural proteins nsPl, nsP2, nsP3, and nsP4 of the alphavirus srRNA vector is present, however at least a portion of or the entire sequence encoding one or more structural proteins (e.g., CP, El, E2, E3, and 6K) of theAttorney Docket No.: 058462-520001WOalphavirus srRNA vector is absent.

[0105] In some embodiments, the alphavirus srRNA vector is devoid of a substantial portion of the nucleic acid sequence encoding one or more viral structural proteins. The skilled artisan will understand that a substantial portion of a nucleic acid sequence encoding a viral structural polypeptide can include enough of the nucleic acid sequence encoding the viral structural polypeptide to afford putative identification of that polypeptide, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (see, for example, in “Basic Local Alignment Search Tool”; Altschul SF et al., J. Mol. Biol. 215:403-410, 1993).Accordingly, a substantial portion of a nucleotide sequence comprises enough of the sequence to afford specific identification and / or isolation of a nucleic acid fragment comprising the sequence. For example, a substantial portion of a nucleic acid sequence can include at least about 20%, for example, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% of the full-length nucleic acid sequence.

[0106] In some embodiments, the alphavirus srRNA vector is devoid of the entire sequence encoding viral structural proteins, e.g., the alphavirus srRNA vector includes no nucleic acid sequence encoding the viral structural proteins.

[0107] 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).

[0108] In some embodiments of the methods described herein, the recombinant alphavirus srRNA is of a virus belonging to the Alphavirus genus of the Togaviridae family. In some embodiments, the recombinant alphavirus srRNA is of an alphavirus belonging to the VEEV / EEEV group, or the SFV group, or the SINV group. In some embodiments, the alphavirus is Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O’Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Tonate virusAttorney Docket No.: 058462-520001WO(TONV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu virus (NDUV), Madariaga virus (MADV), or Buggy Creek virus. In some embodiments, the alphavirus is VEEV, EEEV, CHIKV, MADV, WEEV, or TONV. In some embodiments, the alphavirus is VEEV. In some embodiments, the alphavirus is EEEV. In some embodiments, the alphavirus is CHIKV. In some embodiments, the alphavirus is MADV. In some embodiments, the alphavirus is TONV. In some embodiments, the alphavirus is WEEV.

[0109] In some embodiments, the srRNA constructs of the disclosure can include a 5’ cap. The term “5’ cap” as used herein refers to a structure found on the 5’ end of some eukaryotic RNAs, e.g., RNA transcripts, and generally includes a dinucleotide or a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5'- to 5'-triphosphate linkage (also referred to as Gppp or G(5')ppp(5')). In some embodiments, a guanosine nucleoside included in a 5' cap may be modified, for example, by methylation at one or more positions (e.g., at the 7-position) on a base (guanine), and / or by methylation at one or more positions of a ribose. In some embodiments, a guanosine nucleoside included in a 5' cap includes a 2'0 methylation at a ribose (2'0MeG). In some embodiments, a guanosine nucleoside included in a 5' cap includes methylation at the 7-position of guanine (m7G). In some embodiments, a guanosine nucleoside included in a 5' cap includes methylation at the 7-position of guanine and a 2' O methylation at a ribose (m7(2'OMeG)).

[0110] Multiple distinct cap structures can be used to generate the 5' cap of in vitro transcribed srRNA. In some embodiments, providing an srRNA with a 5' cap disclosed herein or a 5' cap analog may be achieved by in vitro transcription, in which a 5' cap is co-transcriptionally expressed into an RNA strand, or may be attached to an RNApost-transcriptionally using capping enzymes. Accordingly, in some embodiments, 5' capping of srRNA can be performed co-transcriptionally with chemical cap analogs (i.e., capping during in vitro transcription). For example, CleanCap® technology provides high efficiency capping (90%+) in a co-transcriptional reaction using commercially available reagents with an AG initiator to provide a natural Cap 1 structure with a 2'-O-methyl group and N7 methyl on separate guanine components. As another example, the Anti-Reverse Cap Analog (ARC A) cap contains a 5 '-5 '-triphosphate guanine-guanine linkage where one guanine contains an N7 methyl group as well as a 2'-O-methyl group. Alternatively, in some embodiments, the srRNA of the disclosure may also be enzymatically capped post-transcriptionally. These may generate a more authentic 5' cap structure that more closelyAttorney Docket No.: 058462-520001WOmimics, either structurally or functionally, the endogenous 5' cap which have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and / or reduced 5' decapping. Numerous synthetic 5' cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see, e.g., Grudzien-Nogalska E. et al., Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P. H. Ed), 2013). Accordingly, in some embodiments of the disclosure, the srRNA is a capped srRNA comprising a 5’-cap. In some embodiments, the 5’-cap is a cap-0, cap-1, cap-2, or cap analogue. In some embodiments, the capped srRNA is a co-transcriptionally capped srRNA. In some embodiments, the capped srRNA is an enzymatically capped srRNA.[OHl] Thus, in some embodiments, the srRNA of the disclosure is co-transcriptionally capped srRNA. Exemplary co-transcriptionally capped srRNAs include, without limitation, anti-reverse cap analogs (ARCAs) and CleanCap® srRNA.

[0112] In other embodiments, the srRNA of the disclosure is enzymatically capped srRNA. Exemplary capping enzymes include, without limitation, Vaccinia virus capping enzyme (VCE) and Faustovirus capping enzyme (FCE).

[0113] In some embodiments, the modified alphavirus genome or srRNA includes a poly(A) tail, and wherein: (a) the poly(A) tail does not comprise a 3’ non-A residue; and / or (b) the lengthened sequence encoding the poly(A) tail is longer than 34 residues. In some embodiments, the lengthened poly(A) tail has a length ranging from about 30 to about 120 adenylate residues.

[0114] In some embodiments, the sequence encoding the modified alphavirus genome or srRNA further includes an additional restriction site incorporated at the end of the sequence encoding the poly(A) tail of the alphavirus genome or srRNA. In instances in which the nucleic acid constructs (e.g. plasmids) are in circular form, the additional restriction site incorporated immediately downstream of the sequence encoding the poly(A) tail may facilitate the linearization of the circular nucleic acid constructs, thereby generating “clean” poly(A) template ends and / or generating nucleic acid products with the same end identity. In some embodiments, such restriction site may allow for generation of de-concatemerized rolling circle amplification (RCA) products or processing of polymerase chain reaction (PCR) products that leave the same end identity. One skilled in the art will appreciate that aAttorney Docket No.: 058462-520001WO“clean” poly(A) template end generally denotes a DNA sequence end with a homopolymeric sequence that templates for an RNA IVT product that terminates by run-off transcription, resulting in an RNA product containing a poly(A) sequence without 3’ non-A residues.

[0115] In one aspect, some embodiments of the methods of the disclosure involve nucleic acid constructs including a modified alphavirus genome or replicon RNA including a poly(A) tail, wherein an additional restriction site is engineered immediately downstream of the sequence encoding the poly(A) tail of the alphavirus genome or replicon RNA. In some embodiments, the additional restriction site is cleavable by a Type IIS restriction enzyme. Examples of Type IIS restriction enzymes suitable for the compositions and methods of the present disclosure include Acul, Alwl, Alw26I, Bael, Bbil, BbsI, BbsI-HF, Bbvl, BccI, BceAI, Bcgl, BciVI, BcoDI, BfuAI, BmrI, Bpml, BpuEI, Bsal, Bsal-HF, BsaI-HFv2, BsaXI, BseGI, BseRI, Bsgl, BsmAI, BsmBI-v2, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, Btsl-v2, and BtsIMutl. Additional suitable Type IIS restriction enzymes include, but are not limited to, CspCI, Earl, Ecil, Eco31I, Esp3I, Faul, FokI, Hgal, HphI, HpyAV, Lpul, MboII, Mlyl, Mmel, Mnll, NmeAIII, PaqCI, Piel, SapI, and SfaNI. In some embodiments, the additional restriction site is cleavable by SapI, Bpil, Bmsl, Mval269I or an isoschizomer of any thereof. In some embodiments, the additional restriction site is cleavable by SapI or an isoschizomer thereof.

[0116] The nucleic acid constructs administered in the methods of the disclosure further include a coding sequence for a polypeptide construct that replaces at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or srRNA. In principle, the nucleic acid constructs disclosed herein can generally include any number of coding sequences for a polypeptide construct. In some embodiments, the nucleic acid constructs 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 for a polypeptide constructs. A coding sequence for a polypeptide construct can be a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and / or translation of the coding sequences in a cell, in vivo and / or ex vivo. The coding sequence for a polypeptide construct can be inserted into a vector for targeting to a desired host cell and / or into a subject. Accordingly, in some embodiments, the term “coding sequence for a polypeptide construct” can be used interchangeably with the term “expression construct.” In some embodiments, a coding sequence for a polypeptide construct can be aAttorney Docket No.: 058462-520001WOnucleic acid construct that includes a gene encoding a protein or functional RNA operably linked to regulatory elements such as, for example, a promoter and / or a termination signal, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the gene. In some embodiments, the coding sequence is operably linked to a promoter sequence. In some embodiments, at least one of the promoter sequences is a subgenomic (sg) promoter. In some embodiments, the sg promoter is a 26S genomic promoter.

[0117] The nucleic acid constructs administered in the methods described herein include a coding sequence for an envelope glycoprotein G of a rabies virus (RABV-G), a variant thereof, or an antigenic determinant of either thereof, which encode polypeptides containing epitopes that are able to elicit an immune response. The variants of RABV-G can encompass coding sequences for polypeptides having an amino acid sequence that is the same or essentially the same as that of the reference protein (e.g., RABV-G) except having at least one amino acid modified, for example, deleted, inserted, or replaced, respectively. The amino acid replacement may be a conservative amino acid substitution, preferably at a non-essential amino acid residue in the protein. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), betabranched side chains (e.g, threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). A variant of a protein may have an amino acid sequence 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 a protein that retains the same function as the protein. The terms “variant”, when used in reference to a nucleic acid sequence, refer to a nucleic acid sequence that differs by one or more nucleotides from another, usually related nucleotide acid sequence. As such, the term “variant” can refer to a change of one or more nucleotides of a reference nucleic acid which includes the insertion of one or more new nucleotides, deletion of one or more nucleotides, and substitution of one or more existing nucleotides. A variantAttorney Docket No.: 058462-520001WOcan also include a point mutation, multiple mutation, single nucleotide polymorphism (SNP), deletion, insertion, and translocation. Thus, variants of the coding sequences described herein include nucleic acids that encode polypeptides that can be, for example, full length, mutated, truncated, inactivated, peptide / epitopes or combinations thereof of RABV-G.

[0118] The nucleic acid constructs administered in the methods described herein can also include a coding sequence for an envelope glycoprotein G of a rabies virus (RABV-G), or an antigenic determinant thereof. In some embodiments, the antigenic determinant resides within the N-terminal half of rabies virus G glycoprotein. Other particular antigenic determinants reside within the C-terminal half of rabies virus. Such antigenic determinants can reside, for example, within 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 rabies virus G glycoprotein, or any interval, portion or range thereof. In one embodiment, the antigenic determinant resides within the N-terminal half of rabies virus G glycoprotein, i.e., between about amino acid residues 19-422. In another embodiment, the antigenic determinant resides 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 as well as alterations thereof, such as substitutions or deletions.

[0119] In some embodiments, the rabies G glycoprotein antigenic determinant comprises one or more epitopes. Non-limiting examples of epitopes include linear epitope, conformational epitope, discontinuous epitope, or combinations of such epitopes. One skilled in the art will understand that the term “epitope” refers to an antigenic determinant that interacts with a specific antigen-binding site of an antigen-binding polypeptide, e.g., a variable region of an antibody molecule, known as a paratope. A single antigen can have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term “epitope” also refers to a site on an antigen to which B cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can be linear or conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes can include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, orAttorney Docket No.: 058462-520001WOsulfonyl groups, and, in certain embodiments, can have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0120] 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, antigenic site minor A, or combinations of such antigenic sites, for example, antigenic site III and minor site A. Antigenic sites of rabies virus have been identified using panels of monoclonal antibodies and their respective monoclonal antibody-resistant virus variants. The majority of 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 at aa 330 to 338 and harbors 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). The conformational antigenic site I was defined by only one monoclonal antibody, 509-6, 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 rabies virus glycoprotein: analysis with monoclonal antibodies,” J. Gen. Virol. 64:843-8451). Antigenic site IV is known to harbor 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 minor site located at position 342 to 343, which is distinct from antigenic site III despite its close proximity.Attorney Docket No.: 058462-520001WO

[0121] The RABV-G of the nucleic acid constructs administered in the methods described herein can be from a virulent rabies virus strain or an avirulent rabies virus strain.

[0122] The anti-rabies vaccines currently in use are either vaccines made from inactivated viruses or vaccines consisting of viral strains with attenuated virulence or recombinant viruses.

[0123] Viruses can be inactivated by a variety of methods, in particular chemical methods such as treatment with formaldehyde or 3 -propiolactone.

[0124] The attenuation of the virulence of viral strains is well known; for example, it may be carried out by successive passages of viral strains on a different host, vector type (e.g. rabbit or mouse) or in cell cultures. Thus, strains are poorly adapted to the host source and therefore less pathogenic to the host, while maintaining their vaccination capacity.

[0125] Avirulent rabies strains often 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 Virus is Unable to Infect Motorneurons In Vivo and In Vitro,” J. Virol. 72(1): 273-278 (1998)). By way of example, antigenic mutants of RABV-G isolated from either the challenge virus standard strain (CVS), CVS derivatives, or the Street- Alabama-Dufferin (SAD) Bern strain, which have the arginine (Arg) at positon 333 in G replaced by either a glutamine (Gin), a glycine (Gly), a leucine, an isoleucine, a methionine, a cysteine, or a serine, are avirulent (Tuffereau et al., “Arginine or lysine in 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, recombinant viruses that have the Arg at position 333 in G replaced by a glutamic acid are 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). Thus, in general, RABV becomes avirulent when the Arg or Lys at position 333 in G is changed to other amino acids.

[0126] In principle, there are no particular limitations to the rabies virus strains from which the envelope glycoprotein G can be derived from. Non-limiting examples of rabies virus strains suitable for the compositions and methods disclosed herein include a Flury LEP strain,Attorney Docket No.: 058462-520001WOa Flury LEP-C strain, a Flury HEP strain, a 1088 strain, a AT6 strain, a CQ92 strain, a CVS-11 strain, a CVS-26 strain, a CVS-26(G-N204S) strain, a CYN1009D strain, a CYN1026D strain, a CYN1029D strain, a CYN1138D strain, a CYN1140D strain, a CYN1141D strain, a CYN1242H strain, a CYN1243D strain, a CYN1244D strain, a CYN1245D strain, a CYN1247D strain, a CYN1249D strain, a CYN1250D strain, a CYN1251D strain, a CYN1252D strain, a CYN1253D strain, a CYN1255D strain, a CYN1256D strain, a CYN1257D strain, a CYN1259D strain, a CYN1260D strain, a CYN1261D strain, a GX4 strain, a H-08-1320 strain, a H-1413-09 strain, a IP 1586 / 10 strain, a IP 2990 / 13 strain, a IP 2991 / 13 strain, a IP 2992 / 13 strain, a IP 3176 / 09 strain, a IP 4005 / 12 strain, a IP 412 / 10 strain, a IP 542 / 10 strain, a IP 7941 / 09 strain, a J strain, a JX-08-47 strain, a JX08-48 strain, a Kyoto strain, a Kyoto(G-S204N) strain, aN.HL strain, a RC.HL strain, a rHEP5.0-CVSG strain, a RRV ON-99-2 strain, a SAD-B19 strain, a SH06 strain, a SHRBV-18 strain, a SNK-CTN strain, a SRV9 strain, a Street Alabama Dufferin (HCP-SAD) strain, a VRC-RZ2 strain, a ZJ-LA strain, and a ZJ-QZ strain. In some embodiments, the envelope glycoprotein G is of a Flury LEP strain.

[0127] The full-length amino acid sequence of a non-limiting RABV-G protein is set forth in SEQ ID NO: 1 as follows:

[0128] MVPQVLLFVPLLGFSLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGC TNLSEFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPD ACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVTDLDPYDKS LHSRVFPGGNCSGITVSSTYCSTNHDYTIWMPENLRLGTSCDIFTNSRGKRASKGGKT CGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSDETKWCPPGQLVNL HDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIF NKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGSDGHVLIPE MQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEVEDFVEVHLPDVHEQVSGVEL GLPNWGKYVLMIAGALIALMLIIFLMTCCRRVNRPESTQSSLGETGRNVSVTSQSGK VISSWESYKSGGETRL

[0129] In some embodiments, the coding sequence for RABV-G in the nucleic acid constructs described herein encodes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid constructs of the disclosure include a nucleic acid sequence encoding a 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 to an amino acidAttorney Docket No.: 058462-520001WOsequence of SEQ ID NO: 1. In some embodiments, the nucleic acid constructs of the disclosure include a nucleic acid sequence encoding a RABV-G having the amino acid sequence of SEQ ID NO: 1, wherein one, two, three, four, or five of the amino acid residues in a SEQ ID NO: 1 is / are substituted by a different amino acid residue. In some embodiments, the coding sequence for RABV-G encodes smaller portions of the amino acid sequence of SEQ ID NO: 1. For example, these smaller portions can include at least 8, 10, 12, 14, 16, 18, 20, 30 or more amino acids of SEQ ID NO: 1.

[0130] In some embodiments, the nucleic acid construct as described herein comprises a 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 of SEQ ID NOs: 4-9. In some embodiments, the nucleic acid construct as described herein comprises a 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 of SEQ ID NO: 4. In some embodiments, the nucleic acid construct as described herein comprises a 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 of SEQ ID NO: 5. In some embodiments, the nucleic acid construct as described herein comprises a 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 of SEQ ID NO: 6. In some embodiments, the nucleic acid construct as described herein comprises a 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 of SEQ ID NO: 7. In some embodiments, the nucleic acid construct as described herein comprises a 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 of SEQ ID NO: 8. In some embodiments, the nucleic acid construct as described herein comprises a 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 of SEQ ID NO: 9.

[0131] In some embodiments, the coding sequence of RABV-G is redesigned and / or optimized for a desired property, such as increased stability, potency, and expression (e.g.,Attorney Docket No.: 058462-520001WOtranslation efficiency), which in turns can maximize the impact of producing, delivering, and administering biotherapeutic. For example, in some embodiments, the coding sequence is optimized for expression at a level higher than the expression level of a reference coding sequence. With respect to sequence-optimization of nucleotide sequences, degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. Hence, the nucleic acid constructs of the present disclosure may also have any base sequence that has been changed from any polynucleotide sequence disclosed herein by substitution in accordance with degeneracy of the genetic code. References describing codon usage are readily publicly available. In some embodiments, polynucleotide sequence variants can be produced for a variety of reasons, e.g., to optimize expression for a particular host (e.g., changing codon usage in the alphavirus mRNA to those preferred by other organisms such as human, non-human primates, hamster, mice, or monkey). Accordingly, in some embodiments, the coding sequence is optimized for expression in a target host cell through the use of codons optimized for expression. The techniques for the construction of synthetic nucleic acid sequences encoding genes using preferred codons optimal for host cell expression may be determined by computational methods analyzing the commonality of codon usage for encoding native proteins of the host cell genome and their relative abundance by techniques well known in the art. The codon usage database (http: / / www.kazusa.or.jp / codon) may be used for generation of codon optimized sequences in mammalian cell environments. Furthermore, a variety of software tools are available to convert sequences from one organism to the optimal codon usage for a different host organism such as the JCat Codon Optimization Tool (www.jcat.de), Integrated DNA Technologies (IDT) Codon Optimization Tool (https: / / www.idtdna.com / CodonOpt) or the Optimizer online codon optimization tool (http: / / genomes.urv.es / OPTIMIZER). Such synthetic sequences may be constructed by techniques known in the art for the construction of synthetic nucleic acid molecules and may be obtained from a variety of commercial vendors.

[0132] In some embodiments, the coding sequence is optimized for enhanced RNA stability and / or expression. The stability of RNA generally relates to the “half-life” of RNA. “Halflife” relates to the period of time which is needed to eliminate half of the activity, amount, or number of molecules. In the context of the present disclosure, the half-life of an RNA isAttorney Docket No.: 058462-520001WOindicative for the stability of said RNA. The half-life of RNA may influence the “duration of expression” of the RNA. Additional information regarding principles, strategies, and methods for use in enhancing RNA stability can be found at, for example, Leppek K. et al., Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics. bioRxiv. (Preprint). Mar 30, 2021. doi: 10.1101 / 2021.03.29.437587.

[0133] As described above, the nucleic acid construct is administered to a human subject in a vaccine composition. Such compositions generally include one or more of the nucleic acid constructs described and provided herein, and a pharmaceutically acceptable excipient, e.g., carrier.

[0134] The term “pharmaceutically acceptable excipient” can refer to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administration of a compound(s) of interest to a subject. As such, “pharmaceutically acceptable excipient” can 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 and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics and additional therapeutic agents) can also be incorporated into the compositions.

[0135] The nucleic acid constructs of the disclosure can be used in a naked form or formulated with a delivery vehicle. Exemplary routes, either using in a free form, e.g., inserted into a nucleic acid, e.g., a vector.

[0136] For use in a vaccine composition of the disclosure, a nucleic acid as described herein can be formulated into or with delivery vehicles. Exemplary delivery vehicles suitable for the compositions and methods of the disclosure include, but are not limited to liposomes (e.g., neutral or anionic liposomes), microspheres, immune stimulating complexes (ISCOMS), lipid-based nanoparticles (LNP), polymer nanoparticles, viral replicon particles (VRPs), or conjugated with bioactive ligands, which can facilitate delivery and / or enhance the immune response. These compounds are readily available to one skilled in the art; for example, see Liposomes: A Practical Approach, RCP New Ed, IRL press (1990). Adjuvants other than liposomes and the like are also used and are known in the art. Adjuvants may protect the antigen (e.g., srRNA construct) from rapid dispersal by sequestering it in a local deposit, orAttorney Docket No.: 058462-520001WOthey may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. An appropriate selection can be made by those skilled in the art, for example, from those described below.

[0137] In some embodiments, the vaccine composition of the disclosure can include one or more of the following: physiologic buffer, a liposome, a lipid-based nanoparticle (LNP), a polymer nanoparticle, a viral replicon particle (VRP), a microsphere, an immune stimulating complex (ISCOM), a conjugate of bioactive ligand, or a combination of any thereof.

[0138] In some embodiments, the vaccine composition is administered to the human subject by a lipid-based nanoparticle (LNP). LNP are generally less immunogenic than viral particles. While many humans have preexisting immunity to viral particles there is no preexisting immunity to LNP. In addition, adaptive immune response against LNP is unlikely to occur which enables repeat dosing of LNP.

[0139] The lipids suitable for the compositions and methods described herein can be cationic lipids, ionizable cationic lipids, anionic lipids, or neutral lipids.

[0140] In some embodiments, the LNP of the 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) as the pH is lowered below the pKa of the ionizable group of the lipid but is more neutral at higher pH values. At pH values below the pKa, the lipid is then able to associate with negatively charged nucleic acids (e.g., oligonucleotides). As used herein, the term "ionizable lipid" includes lipids that assume a positive charge on pH decrease from physiological pH, and any of a number of lipid species that carry a net positive charge at a selective pH, such as physiological pH. Permanently cationic lipids such as DOTMA have proven too toxic for clinical use. The ionizable lipid can be present in lipid formulations according to other embodiments, preferably in 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 other embodiments, about 50 Mol%, in still other embodiments, and about 60 Mol% in yet others (“Mol%” means the percentage of the total moles that is of a particular component). The term “about” in this paragraph signifies a plus or minus range of 5 Mol%. DODMA, or 1 ,2-dioleyloxy-3-dimethylaminopropane, is an ionizable lipid, as is DLin-MC3-DMA or 0-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N- dimethylamino) (“MC3”).

[0141] Exemplary ionizable lipids suitable for the compositions and methods of theAttorney Docket No.: 058462-520001WOdisclosure includes those described in PCT publications WO2020252589A1 and W02021000041A1, U.S. Patent Nos. 8,450,298 and 10,844,028, and LoveK.T. 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. Accordingly, in some embodiments, the LNP of the disclosure includes one or more lipid compounds described in Love K.T. et al., 2010 supra, such as C16-96, C14-110, and C12-200. In some embodiments, the LNP includes an ionizable cationic lipid. Suitable ionizable cationic lipids include the group consisting of ALC-0315, Cl 2-200, LN16, MC3, MD1, SM-102, and a combination of any thereof. In some embodiments, the LNP of the disclosure includes C12-200. The structure of C12-200 lipid is known in the art and described in, e.g., U.S. Patent Nos. 8,450,298 and 10,844,028, which are hereby incorporated by reference in their entirety. In some embodiments the C12-200 is combined with cholesterol, C14-PEG2000, and DOPE. In some embodiments, the C 12-200 is combined with DSPC and DMG-PEG2000.

[0142] In some embodiments, the LNP of the disclosure includes one or more cationic lipids. Suitable cationic lipids include, but are not limited to, 98N12-5, C 12-200, C14-PEG2000, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. In some embodiments, the LNP of the disclosure includes one or more neutral lipids. Non-limiting neutral lipids suitable for the compositions and methods of the disclosure include DPSC, DPPC, POPC, DOPE, and SM. In some embodiments, the LNP of the disclosure includes one or more ionizable lipid compounds described in PCT publications WO2020252589A1 and WO2021000041 Al, which are hereby incorporated by reference in their entirety.

[0143] A number of other lipids or combination of lipids that are known in the art can be used to produce an LNP. Non-limiting examples of lipids suitable for use to produce LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC -cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Non-limiting examples of cationic lipids include 98N12-5, C12-200, C14-PEG2000, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, 7C1, and a combination of any thereof. Nonlimiting examples of neutral lipids include DPSC, DPPC, POPC, DOPE, and SM. Nonlimiting examples of PEG-modified lipids include PEG-DMG, PEG-CerC14, and PEG-CerC20.

[0144] In some embodiments, the LNP of the disclosure includes at least one lipid. Suitable lipids include C12-200, C14-PEG2000, DOPE, DMG-PEG2000, DSPC, DOTMA, DOSPA,Attorney Docket No.: 058462-520001WODOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). In some embodiments the C 12-200 is combined with cholesterol, C14-PEG2000, and DOPE. In some embodiments, the C12-200 is combined with DSPC and DMG-PEG2000.

[0145] 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, the lipid-based nanoparticles have an average diameter of less than about 1000 nm, about 500 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, about 75 nm, about 50 nm, or about 25 nm. In some embodiments, the LNPs have an average diameter ranging from about 70 nm to 100 nm. In some embodiments, the LNPs have an average diameter ranging from about 88 nm to about 92 nm, from 82 nm to about 86 nm, or from about 80 nm to about 95 nm.

[0146] In some embodiments, the vaccine compositions of the disclosure are formulated in a liposome. In some embodiments, the vaccine compositions of the disclosure are formulated in a lipid-based nanoparticle (LNP). In some embodiments, the vaccine compositions of the disclosure are formulated in a polymer nanoparticle.

[0147] As described above, neutral lipids, also known as “structural lipids” or “helper lipids” can also be incorporated into lipid formulations and lipid particles in some embodiments. The lipid formulations and lipid particles can include one or more structural lipids at about 10 to 40 Mol% of the composition. Suitable structural lipids support the formation of particles during manufacture. Structural lipids refer to any one of a number of lipid species that exist in either in an anionic, uncharged or neutral zwitterionic form at physiological pH.Representative structural lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, diacylphosphatidylglycerols, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides.

[0148] Exemplary structural lipids include zwitterionic lipids, for example,di stearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamineAttorney Docket No.: 058462-520001WO(POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl -phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1 -stearoyl-2-oleoyl-phosphatidyethanol amine (SOPE), and 1 ,2-dielaidoyl-sn-glycero-3- phophoethanolamine (trans DOPE).

[0149] In another embodiment, the structural lipid can be any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerols such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleyolphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modifying groups joined to neutral lipids. Other suitable structural lipids include glycolipids (e.g., monosialoganglioside GM1).

[0150] Stabilizing agents can be included in lipid formulations embodiments to ensure integrity of the mixtures. Stabilizing agents are a class of molecules which disrupt or help form the hydrophobic-hydrophilic interactions among molecules. Suitable Stabilizing agents include, but are not limited to, polysorbate 80 (also known as Tween 80, 1UPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2- (2 -hydroxy ethoxy)ethoxy] ethyl octadec-9-enoate), Myij52 (Polyoxyethylene (40) stearate), and Brij™ S10 (Polyoxyethylene (10) stearyl ether). Polyethylene glycol conjugated lipids may also be used. The stabilizing agents may be used alone or in combinations with each other.

[0151] In some embodiments, the stabilizing agents comprises about .1 to 3 Mol% of the overall lipid mixture. In some embodiments, the stabilizing agents comprise about 0.5 to 2.5 Mol% of the overall lipid mixture. In some embodiments, the stabilizing agent is present at greater than 2.5 Mol%. In some embodiments the stabilizing agent is present at 5 Mol%. In some embodiments the stabilizing agent is present at 10 Mol%. In some embodiments, the stabilizing agent 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, and so forth. In other embodiments, the stabilizing agent is 2.6-10 Mol% of the lipid mixture. In other embodiments, the stabilizing agents is present at greater than 10 Mol% of the lipid mixture.

[0152] Steroids can also be included in the lipid compositions for certain applications, and lipid particles made therefrom include sterols, such as cholesterol and phytosterol.Attorney Docket No.: 058462-520001WO

[0153] In some embodiments, the vaccine compositions are formulated for one or more of intranasal administration, transdermal administration, intradermal administration, intraperitoneal administration, intramuscular administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, intravaginal administration, and oral administration.

[0154] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™. (BASF, Parsippany, N. J.), 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 easy syringability exists. It can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be generally to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sucrose, trehalose, and / or sodium chloride 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.

[0155] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0156] In some embodiments, the composition is formulated for one or more of intranasal administration, transdermal administration, intramuscular administration, intranodalAttorney Docket No.: 058462-520001WOadministration, intravenous administration, intraperitoneal administration, oral administration, or intra-cranial administration.Administration

[0157] As described above, the method of the present disclosure involves administration of the vaccine composition to a human subject. In some embodiments, the human subject is one who has, who is suspected of having, or who may be at high risk for developing rabies infection. In some embodiments, the human subject is a patient under the care of a physician.

[0158] The vaccine composition is administered to a subject in a composition having a pharmaceutically acceptable carrier and in an amount effective to stimulate an extended immune response. Generally, a subject can be immunized through an initial series of injections (or administration through one of the other routes described below) and subsequently given boosters to increase the protection afforded by the original series of administrations. The initial series of injections and the subsequent boosters are administered in such doses and over such a period of time as is necessary to stimulate an extended immune response in a subject.

[0159] In some embodiments, the vaccine composition is administered orally, by inhalation, or through a parenteral route. Examples of parenteral routes of administration include, for example, intramuscular, intraocular, intravenous, intranodal, intradermal, subcutaneous, transdermal (topical), transmucosal, intravaginal, and rectal administration. In some embodiments, the composition is administered intramuscularly. Solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, 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 the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as mono- and / or di-basic sodium phosphate, hydrochloric acid or sodium hydroxide (e.g., to a pH of about 7.2-7.8, e.g., 7.5). The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0160] The compositions of this invention can be administered in a single dose or multiple dose schedule. A multiple dose schedule is one in which a primary course of administrationAttorney Docket No.: 058462-520001WOmay include 1 to 10 or more separate doses, followed by other doses administered at subsequent time intervals as necessary to maintain and reinforce the desired effect (e.g., an extended immune response), for example, weekly or in 1 to 4 months for a second dose, and if necessary, a subsequent dose after several months (for example, 4 or 6 months) / year. In some embodiments, the vaccine compositions are administered at weekly intervals, e.g., 1 to 2, 2 to 3, or 3 to 4 doses given at 1 to 2, 2 to 3, or 3-to-4-week intervals. This may be followed with an additional administration every 1, 2, 3, or 4 months. In some embodiments, 2 doses can be administered at an 8-week interval. In some embodiments, this can be followed by administration every 3, 6, 9, or 12 months. This may be followed with an additional administration every 1, 2, 3, or 4 years. In some embodiments, 3 doses can be administered at a 3 to 8 week interval, followed by administration every 3, 6, 9, or 12 months. This may be followed with an additional administration every 1, 2, 3, or 4 years.

[0161] The dosage of the vaccine composition can be determined readily by the skilled artisan, for example, by first identifying doses effective to elicit a prophylactic or therapeutic immune response, e.g., by measuring the serum titer of virus specific immunoglobulins or by measuring the inhibitory ratio of antibodies in serum samples, or urine samples, or mucosal secretions. Said dosages can be determined from animal studies. A non-limiting list of animals used to study the efficacy of vaccines include the guinea pig, hamster, ferrets, chinchilla, mouse and cotton rat, and non-human primates.

[0162] In addition, as described in the Examples herein, human clinical studies can be performed to determine the preferred effective dose for humans by a skilled artisan. Such clinical studies are routine and well known in the art. The precise dose to be employed will also depend on the route of administration. Effective doses may be extrapolated from doseresponse curves derived from in vitro or animal test systems.

[0163] In some embodiments, the vaccine composition is administered at a dose of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, or about 10.0 mcg. In some embodiments, the vaccine composition is administered at 0.1 mcg. In some embodiments, the vaccine composition is administered at 1 mcg. In some embodiments, the vaccine composition is administered at 10 mcg.

[0164] In some embodiments, a single dose of 0.1 mcg is administered to the human subject.Attorney Docket No.: 058462-520001WOIn some embodiments, two doses of 0.1 mcg are administered to the human subject. In some embodiments, the first dose and the second dose are administered about 8 weeks apart. In some embodiments, a single dose of 1.0 mcg is administered to the human subject. In some embodiments, two doses of 1.0 mcg are administered to the human subject. In some embodiments, the first dose and the second dose are administered about 8 weeks apart. In some embodiments, a single dose of 10 mcg is administered to the human subject. In some embodiments, two doses of 10 mcg are administered to the human subject. In some embodiments, the first dose and the second dose are administered about 8 weeks apart.Additional therapies

[0165] In some embodiments, a vaccine composition according to the present disclosure is administered to the subject individually as a single prophylaxis or therapy (monotherapy) or as a first therapy in combination with at least one additional therapy (e.g., second therapy). In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, targeted therapy, and surgery. In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy or surgery. In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy is administered at the same time as the second therapy. 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 rotation. In some embodiments, the first therapy and the second therapy are administered together in a single formulation.METHOD OF EXPANSION OF T FOLLICULAR HELPER CELLS

[0166] In another aspect, the present disclosure relates to a method of expansion of T follicular helper cells in a human subject. The method involves administering to the human subject a vaccine composition comprising 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 for aAttorney Docket No.: 058462-520001WOpolypeptide construct comprising an envelope glycoprotein G of a rabies virus (RABV-G), a variant thereof, or an antigenic determinant of either thereof.T follicular Helper Cells

[0167] As described above, Follicular helper T cells (TFH) are a defined subset of CD4+ T cells that are essential for helping cognate B cells form and maintain the germinal center (GC) reaction, and for development of humoral immune responses. In addition to CXCR5, TFH cells express markers such as CD25, CD69, CD95, CD57 (in humans only), 0X40 (CD 134) and CD40L (CD 154) and induce over-expression of activation-induced cytidine deaminase in B cells. TFH also produce cytokines such as IL-10 and IL-21 that promote B-cell survival and antibody production (King et al., “T follicular helper (TFH) cells in normal and dysregulated immune responses,” Annu Rev. Immunol. 2008;26:741-66). Circulating T follicular helper cells can be detected in the peripheral blood (Locci et al, Human Circulating PD- I CXCR.3 CXCR.5 Memory Tfh Cells Are Highly Functional and Correlate with Broadly Neutralizing HIV Antibody Responses,” Immunity 39(4):758-769 (2013)).

[0168] In some embodiments, the circulating T follicular helper cells are CD69+. In some embodiments, the circulating T follicular helper cells are CD40L+. In some embodiments, the circulating T follicular helper cells are CD69+CD40L+ (Reiss, S. et al. “Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigenspecific CD4 T cells,” PLoSONE 12, e0186998 (2017)).Expansion

[0169] In the method described herein, the T follicular helper cells are expanded by administering to the human subject a vaccine composition comprising a nucleic acid construct comprising a nucleic acid sequence encoding a modified alphavirus genome or selfreplicating 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 for a polypeptide construct comprising an envelope glycoprotein G of a rabies virus (RABV-G), a variant thereof, or an antigenic determinant of either thereof.

[0170] Vaccine compositions and administration regimens for use in the method of expanding T follicular helper cells are described above for the method of inducing extended immune responses.

[0171] In some embodiments, the vaccine composition comprises a nucleic acid comprising aAttorney Docket No.: 058462-520001WOnucleic 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 of SEQ IDNOs: 4-9.

[0172] In some embodiments, the vaccine composition is formulated in an LNP.

[0173] In some embodiments, the vaccine composition is administered intramuscularly.

[0174] In some embodiments, a single dose of 0.1 mcg is administered to the human subject. In some embodiments, two doses of 0.1 mcg are administered to the human subject. In some embodiments, the first dose and the second dose are administered about 8 weeks apart. In some embodiments, a single dose of 1.0 mcg is administered to the human subject. In some embodiments, two doses of 1.0 mcg are administered to the human subject. In some embodiments, the first dose and the second dose are administered about 8 weeks apart. In some embodiments, a single dose of 10 mcg is administered to the human subject. In some embodiments, two doses of 10 mcg are administered to the human subject. In some embodiments, the first dose and the second dose are administered about 8 weeks apart.

[0175] The expansion in T cell subpopulations following methods of the disclosure may result in an increase in number that be relatively small, but is many instances the result is a significant increase, such as for example an increase in cell number of at least about 2 fold, at least about 5 fold, about 20 fold, about 50 fold, at least about 100 fold, about 500 fold, about 1,000 fold, about 2,000 fold, about 5,000 fold, about 10,000 fold, about 20,000 fold, about 30,000 fold, about 40,000 fold, about 50,000 fold, about 75,000 fold, about 100,000 fold, or greater as compared to subjects not receiving the vaccine composition of the disclosure. In some embodiments, administration of the vaccine composition as disclosed herein results in at least about 2 fold, at least about 5 fold, about 20 fold, about 50 fold, at least about 100 fold, about 500 fold, about 1,000 fold, about 2,000 fold, about 5,000 fold, about 10,000 fold, about 20,000 fold, about 30,000 fold, about 40,000 fold, about 50,000 fold, about 75,000 fold, about 100,000 fold, or greater expansion of the T follicular helper cells as compared to a commercial rabies vaccine, such as Rabavert. Such increases in number may be measured at any appropriate time point after administration of the vaccine composition.

[0176] Methods of detecting the expansion of T cells are known in the art and include, without limitation, cell counting, flow cytometry, and molecular techniques like quantitative PCR.

[0177] All publications and patent applications mentioned in this disclosure are hereinAttorney Docket No.: 058462-520001WOincorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0178] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the Applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0179] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure and are to be included within the spirit and purview of this application.

[0180] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.EXAMPLES

[0181] 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 explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. 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, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, C A: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K.Attorney Docket No.: 058462-520001WOB., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.

[0182] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.EXAMPLE 1Materials and Methods

[0183] Preparation of srRNA. Self-replicating RNA (srRNA) was transcribed in vitro using linearized plasmids encoding the transgene(s) under the control of a Venezuelan Equine Encephalitis Virus-based srRNA vector as template (SEQ ID NO: 4). The coding sequences for Flury-LEP-C RABV-G (Genbank: ACL98057.1). For preclinical material, in vitro transcription (IVT) was performed using the Hi Scribe™ T7 Quick High Yield RNA Synthesis Kit (NEB, Cat#E2050S) and purified by LiCl precipitation. The RNA product was capped using Vaccinia Capping System (NEB, Cat# M2080S) and mRNA Cap 2 -O-Methyltransferase (NEB, Cat# M0366S) and purified by LiCl precipitation. For the clinical study, the GMP srRNA drug substance was produced by Curia (Hopkinton, MA, USA).

[0184] LNP formulation. For the clinical study, the GMP srRNA drug product was produced at Precision NanoSystems Inc. (Vancouver, Canada). The RBI-4000 drug product is supplied as a frozen suspension (-80 °C) at 0.1 mg / mL. Prior to patient dosing, it is freshly thawed and diluted with sterile 0.9% sodium chloride USP for injection to the required concentration so that administration of 0.5 mL via intramuscular injection achieves the required dose.

[0185] Clinical trial. RBI-4000-101 was a multicenter, open label, randomized, active control Phase I clinical trial (NCT06048770) to assess the safety and immunogenicity of RBI-4000. The study enrolled 89 healthy volunteers, age 18-45 years with no prior exposure or vaccination to rabies. Participants also had to be seronegative to hepatitis B, hepatitis C and HIV and have no history of immune dysregulation, myocarditis or pericarditis orAttorney Docket No.: 058462-520001WOmalignancy within the past 5 years. All participants had to verify willingness not to initiate a pregnancy for at least 60 days following the completion of vaccination. Enrollment was completed at 2 sites in the United States.

[0186] Upon arrival at the clinical facility, subjects were randomly assigned subject numbers that corresponded to a previously generated randomization scheme. The randomization schedule was generated by Syneos Health using a validated proprietary computer software program (Statistical Analysis System [SAS®]) Version 9.4 and was reviewed by a biostatistician.

[0187] Details of the trial, including demographics, safety, and an interim analysis of immunogenicity, have been previously published Maine et al., “Safety and immunogenicity of an optimized self-replicating RNA platform for low dose or single dose vaccine applications: a randomized, open label Phase I study in healthy volunteers,” Nature Comm.16:4562025). The study was conducted in accordance with the protocol, applicable laws, and regulatory requirements, as well as International Council for Harmonization Good Clinical Practice guidelines, and the consensus ethical principles derived from international guidelines, including the Declaration of Helsinki and Council for International Organizations of Medical Sciences International Ethical Guidelines. The protocol was approved by the central institutional review board (Advarra, Inc., Columbia, MD) prior to study initiation, and written informed consent was obtained from all participants before enrollment.

[0188] Immunogenicity was assessed at predefined time points prior and post immunization to measure serum RVNA using a RFFIT validated assay (Kansas State University Rabies Laboratory) or rabies-specific T cells in total PBMCs using an AIM qualified assay (CellCarta, Montreal, Canada).

[0189] Activation-induced marker assay. The AIM assay was performed by stimulating PBMCs, collected at the indicated timepoints, with an overlapping peptide library spanning the length of RABV-G protein (CellCarta, Montreal, Canada). DMSO was used as a negative control for mock stimulation. Samples with viability below 70% prior to stimulation (after thawing and rest) were excluded from the analysis. All evaluable samples based on this criterion were included in the analysis. Testing was conducted in accordance with the applicable regulatory agencies as stated in the clinical protocol RBI-4000-101. Applicable portions of the Good Clinical Practices (GCP) and Good Clinical Laboratory Practices (GCLP) guidelines were followed.Attorney Docket No.: 058462-520001WO

[0190] Data were acquired on an LSR Fortessa flow cytometer (BD biosciences) and analyzed using FlowJo (BD Biosciences).

[0191] Rabies virus neutralization assay. Sera were used to perform rapid fluorescent foci inhibition tests (RFFIT) to quantify RVNA titers using a validated assay 46 by Kansas State University Rabies Laboratory. The lower limit of detection for this assay is <0.1 lU / ml and any value below this limit is plotted as 0.09.

[0192] Data Collection and statistical analysis. RFFIT and AIM data were collected, analyzed and presented using GraphPad Prism, version 10.1.1. Statistical modeling for RVNA was performed by Stat One LLC (Morrisville, NC, USA). Clinical demographic data were retrieved and processed by Syneos Health using RAVE EDC version 2022.3.1.(Medidata Solutions, New York City, NY, USA). Any statistical comparisons are noted and called out in the figure legend and main text.

[0193] Decay rate modeling. Mixed model regression was used to evaluate the change in the geometric mean of the rabies titer response value (antibodies) in lU / ml (Stat One LLC, Morrisville, NC, USA). Each regression model evaluated log(antibody) values as a function of months from the starting date in the period, the dose cohort, the interaction term of month x cohort and with subject included as a random effect. For all models the LLOQ were included as the log of U the LLOQ (0.05). In total 8 models were created to evaluate change in slope of the antibody level. Four of the models evaluated the change from the group peak to some endpoint while the other four models evaluated the change from the individual subject peaks to some endpoint. The endpoints analyzed across the different models were start of stable decay, study day 240, individual subject values below 0.2 lU / mL, and the halflife of the peak antibody level. The reported statistics for each model included the slope and intercept estimates by cohort, F-statistics for the main effects and day-cohort interaction, exponential values of the slopes and slope 95% Cis, and exponential values of the differences in RBI-4000 slopes relative to control. For the models evaluating half-life, the day of half-life occurrence was also approximated based on the slope of the model.EXAMPLE 2RBI-4000 and RabAvert elicit distinct antibody kinetics with similar titers over 6 months

[0194] This Example describes the experiments performed to analyze antibody kinetics long term in subjects vaccinated with RBI-4000.Attorney Docket No.: 058462-520001WO

[0195] The Phase 1 randomized open label clinical trial study enrolled 89 healthy volunteers, age 18-45 years with no prior exposure or vaccination to rabies. The study included four cohorts of ascending doses of RBI-4000 and an active comparator of the commercially available rabies vaccine, RabAvert. Participants in Cohorts 1 (0.1 mcg), 2 (1 mcg), and 4 (10 mcg) received a prime-boost dosing schedule of RBI-4000, 8 weeks apart, while Cohort 3 participants received a single dose of 10 mcg. The 8-week interval for RBI-4000 dosing was selected as it is optimal for generation of immune responses, specifically for srRNA-based vaccines 9. RabAvert was administered as per the CDC guidelines for pre-exposure prophylaxis, i.e. two doses 1 week apart, on Days 1 and 849. Safety analysis showed a well-tolerated vaccine with no serious adverse events. RVNA titers were assessed throughout the study at enrollment, and Days 15, 29, 43, 57, 64, 71, 85, 120, 180 for all participants, and partially for Day 240 upon study termination.

[0196] Longitudinal analysis of RVNA responses shows distinct kinetics over time between RabAvert, single dose, and two dose RBI-4000 cohorts, due to the differences in the immunization regimen and platform technologies. RVNA responses peak at three distinct time points, with maximal RabAvert-induced RVNA titers observed at Day 15 (7 days postboost), followed by a sharp decline prior to entering stable decay, defined by a flattening of the RVNA titers, at Day 70 (FIG. 1A). During stable decay, gradual decline in RVNA titers is observed until the end of study (Day 240). For RBI-4000 two-dose cohorts, a clear dose response is observed with a peak at Day 71 (14 days post-boost), while the single dose cohort peaked at Day 29 (FIG. 1A). Cohorts 3 and 4 that received the highest dose of RBI-4000 as a single or two-dose schedule (10 mcg prime only or 10 mcg prime-boost), respectively, had a more gradual decline in RVNA titers and entered stable decay at Day 180 compared to lower dose cohorts. Importantly, across all cohorts, individuals with RVNA titers above the WHO protective threshold were still observed at the end of study.

[0197] Evaluation of RVNA titers at Day 180 of the study showed a clear dose response with the majority of individuals with sustained RVNA titers in all cohorts (FIG. IB). A two-dose regimen of RBI-4000 at 1 or 10 mcg results in 100% of individuals with detectable RVNA titers and 61% and 88% being at or above the 0.5 lU / mL protective threshold, respectively for Cohort 2 and 4 (1 mcg x2 and 1-mcg x2). Similarly, 100% of individuals in Cohort 5 (RabAvert) that received RabAvert also had detectable RVNA titers with 58% achieving this threshold.Attorney Docket No.: 058462-520001WOEXAMPLE SDurability of RBI-4000 elicited rabies virus neutralizing antibody responses is equal or exceeds the durability of the commercial comparator vaccine Rab Avert

[0198] This Example describes the comparison of the durability of the immune response to RBI-4000 to that of Rab Avert.

[0199] To assess durability of RVNA responses, eight separate decay analysis models were used (FIG. 2). Models 1 and 2 consider decay from the peak RVNA response until the start of stable decay, Models 3 and 4 analyze decay from the peak until Day 240 and Models 5 and 6 analyze decay from the peak until the first individual reaches the LLOQ of the RVNA detection assay. Alternatively, Models 7 and 8 determined the decay rate from the peak of the RVNA titer to its half-life point. Within each pair of models, decay analysis was performed either as a group (Model 1, 3, 5, and 7) or on an individual participant basis (Model 2, 4, 6, and 8). Additional analysis, such as slope and intercept values for each decay curves are provided in Supplementary FIG. 3.

[0200] In brief, durability analysis showed that vaccine-elicited RVNA responses from RBI-4000 were either equivalent to those from RabAvert (Model 3, 4, and 6) or superior ( / .< ., longer lasting) using Model 1, 2, 5, 7, and 8 (FIGs. 4A-4H). These results held true for all dose levels of RBI-4000 (Cohorts 1, 2, 4) and a prime only administration (Cohort 3). Of these models, we selected to focus more in depth on Models 6 and 8, as the analysis included mathematically quantified endpoints of individual responses in the initial acute phase of decay. Using Model 6, which analyzes the decay rate based on each individual participant in the cohort until the first reaches the LLOQ (defined as 0.1 lU / ml), all doses of RBI-4000 show equal durability to RabAvert (FIG. 5A). Using Model 8, which determines the half-life of the RVNA response post-peak, all RBI-4000 cohorts show significantly lower RVNA titer decay rates, i.e. better durability, compared to RabAvert (FIG. 5B). Specifically, RVNA titer half-lives of RBI-4000 administered cohorts showed at least 6-fold longer duration compared to those that received RabAvert (FIG. 5C).EXAMPLE 4RBI-4000 primes helper T cells with a T follicular helper phenotype

[0201] This Example describes the analysis of T cell phenotypes potentially involved in theAttorney Docket No.: 058462-520001WOdurability of the immune response elicited by RBI-4000.

[0202] Quality and durability of vaccine-elicited antibody responses relies on engagement of the cellular arm of the immune system to potentiate B cells function. It has previously been shown that RBI-4000 can elicit rabies-specific T cells in a dose-specific manner (Maine et al., “Safety and immunogenicity of an optimized self-replicating RNA platform for low dose or single dose vaccine applications: a randomized, open label Phase I study in healthy volunteers,” Nature Comm. 16:4562025).

[0203] The T cell response was further phenotyped to assess the induction of CD4 T helper cells, specifically assessing activation-induced markers (AIM) CD69 and CD40L that are descriptive of antigen-primed T cells associated with potentiating of antibody responses by cognate B cells (Reiss, S. et al. Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells. PLoS ONE 12, e0186998 (2017) (FIG. 6A). PBMCs stimulated with rabies glycoprotein led to significant expansion of antigen-specific AIM+ CD4 T cells in individuals that received RBI-4000. Although some AIM+ CD4 T cells were also observed with RabAvert immunization, the levels were not different from resting (mock-treated) conditions and thus not deemed to be antigen-specific (FIG. 6B). These data show that RBI-4000 is able to induce antigen-specific CD4 T helper cells that can in turn bolster B cell function and antibody production.Discussion of Examples 1-4

[0204] Self-repli eating RNA primes protective and durable immune responses to numerous infectious disease antigens resulting in approved vaccines in Japan and Europe. The combination of high replication of the antigen transgene with the viral nature of the platform leads to strong immune priming. This built in adjuvant effect, absent from non-replicating RNA platforms, triggers innate pathogen sensing pathways which prime effective B and T cell responses (Maine et al., “Safety and immunogenicity of an optimized self-replicating RNA platform for low dose or single dose vaccine applications: a randomized, open label Phase I study in healthy volunteers,” Nature Comm. 16:4562025, Palmer, C. D. et al. GRT-R910: a self-amplifying mRNA SARS-CoV-2 vaccine boosts immunity for >6 months in previously-vaccinated older adults. Nat. Commun. 14, 3274 (2023); Palmer, C. D. et al.Individualized, heterologous chimpanzee adenovirus and self-amplifying mRNA neoantigen vaccine for advanced metastatic solid tumors: phase 1 trial interim results. Nat. Med. 28,Attorney Docket No.: 058462-520001WO1619-1629 (2022). Superior T cell responses to mRNA have been reported clinically and these likely drive improved B cell help, antibody production and longer-lived plasma cell response resulting in improved durability of antibody titers (Palmer, C. D. et al. GRT-R910: a self-amplifying mRNA SARS-CoV-2 vaccine boosts immunity for >6 months in previously-vaccinated older adults. Nat. Commun. 14, 3274 (2023); Palmer, C. D. et al. Individualized, heterologous chimpanzee adenovirus and self-amplifying mRNA neoantigen vaccine for advanced metastatic solid tumors: phase 1 trial interim results. Nat. Med. 28, 1619-1629 (2022); Melo, M. et al. Immunogenicity of RNA Replicons Encoding HIV Env Immunogens Designed for Self-Assembly into Nanoparticles. Mol. Ther. 27, 2080-2090 (2019)).Prolonged antigen availability has been shown to drive improved germinal center responses (Baumjohann et al., “Persistent antigen and germinal center B cells sustain T follicular helper cell responses and phenotype,” Immunity 38(3): 596-605 (2013)) which contribute to the generation of improved and durable B cell responses. The results herein show that RBI-4000 primes high levels of antigen specific Tfh cells which mechanistically could drive the observed durability of RVNA.

[0205] The results herein demonstrate for the first time that srRNA can elicit protective immunity with comparable durability to an approved traditional vaccine platform. In contrast to conventional mRNA technology where in head-to-head clinical studies in terms of durable humoral immunity suggesting that mRNA may be of limited use outside of the SARS-CoV-2 pandemic. In more complex infectious diseases in which the virus uses periods of latency to evade immune detection, such as Epstein-Barr virus or cytomegalovirus, vaccine platforms will need sufficient antibody durability to combat viral reactivation. Similarly in the case of travel vaccination, durability needs to match existing approved vaccines before the benefits of replacing these existing vaccines with newer RNA technology become apparent. RBI-4000 shows equal durability in RVNA titers to RabAvert with levels remaining above the WHO indirect measure of protection for over 6 months. The advantages in manufacturing cost, speed and dose sparing of srRNA compared to cell-based virus production positions RBI-4000 to continue clinical development with the aim to replace existing rabies vaccines.

[0206] Furthermore, it is shown that boosting of seropositive individuals with a single dose of RBI-4000, even as low as 0.1 mcg of RBI-4000, preexisting RVNA titers are boosted and maintained for eight months demonstrating srRNA is a powerful platform for boosting. This is important in the context of seasonal vaccines especially if a single dose can provideAttorney Docket No.: 058462-520001WOprotection for an entire infection season.

[0207] While particular alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. There is no intention, therefore, of limitations to the exact abstract and disclosure herein presented.

Claims

Attorney Docket No.: 058462-520001WOCLAIMS WHAT IS CLAIMED IS:

1. A method of inducing an extended immune response in a human subject, the method comprising:administering to the human subject a vaccine composition comprising a nucleic acid construct comprising a nucleic acid sequence encoding a modified alphavirus genome or selfreplicating 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 for a polypeptide construct comprising an envelope glycoprotein G of a rabies virus (RABV-G), a variant thereof, or an antigenic determinant of either thereof, wherein the induced immune response extends beyond 85 days after the administering.

2. The method of Claim 1, wherein the induced immune response extends at least 120 days after administering.

3. The method of Claim 1, wherein the induced immune response extends at least 180 days after administering.

4. The method of Claim 1, wherein the induced immune response extends at least 240 days after administering.

5. The method of any preceding claim, wherein the vaccine composition comprises a pharmaceutically acceptable excipient.

6. The method of any preceding claim, wherein the vaccine composition is formulated with a delivery vehicle into a delivery system, wherein the delivery system comprises a liposome, a viral replicon particle (VRP), a lipid-based nanoparticle (LNP), a polymer nanoparticle, a physiologic buffer, a microsphere, an immune stimulating complex (ISCOM), a conjugate of bioactive ligand, or a combination of any thereof.

7. The method of Claim 6, wherein the delivery system comprises a lipid-based nanoparticle (LNP).Attorney Docket No.: 058462-520001WO8. The method of Claim 7, wherein the LNP delivery system comprises a cationic lipid, an ionizable cationic lipid, an anionic lipid, or a neutral lipid.

9. The method of Claim 8, wherein the LNP delivery system comprises an ionizable lipid.

10. The method of any preceding claim, wherein the vaccine composition is administered intramuscularly.

11. The method of any preceding claim, wherein the administering comprises administering a single dose.

12. The method of any one of claims 1 to 10, wherein the administering comprises administering a first dose and a second dose.

13. The method of claim 12, wherein the second dose is administered at least 8 weeks after the first dose.

14. The method of any preceding claim, wherein the administering comprises administering about 0.1 mcg of the vaccine composition.

15. The method of any preceding claim, wherein the administering comprises administering about 1 mcg of the vaccine composition.

16. The method of any preceding claim, wherein the administering comprises administering about 10 mcg of the vaccine composition.

17. The method of any preceding claim, wherein the extended immune response comprises a neutralizing antibody response.

18. The method of claim 17, wherein the neutralizing antibody response comprises a neutralizing antibody titer of equal or greater than 0.5 lU / mL.

19. The method of any preceding claim, wherein the extended immune response comprises T follicular helper cells.Attorney Docket No.: 058462-520001WO20. The method of any preceding claim, wherein the nucleic acid construct of the vaccine composition comprises a coding sequence for a polypeptide construct comprising an antigenic determinant of RABV-G that resides within the N-terminal half of the RABV-G.

21. The method of any preceding claim, wherein the nucleic acid construct of the vaccine composition comprises a coding sequence for a polypeptide construct comprising an antigenic determinant of RABV-G that resides within the C-terminal half of the RABV-G.

22. The method of any preceding claim, wherein the nucleic acid construct of the vaccine composition comprises a coding sequence for a polypeptide construct comprising an antigenic determinant of RABV-G comprising antigenic site I, antigenic site II, antigenic site III, antigenic site IV, antigenic site minor A of the RABV-G, or a combination of any thereof.

23. The method of any preceding claim, wherein the envelope glycoprotein G is of a virulent rabies virus strain or an avirulent rabies virus strain.

24. The method of any preceding claim, wherein the envelope glycoprotein G is of a rabies virus strain selected from a Flury LEP strain, a Flury LEP-C strain, a Flury HEP strain, a 1088 strain, a AT6 strain, a CQ92 strain, a CVS-11 strain, a CVS-26 strain, a CVS-26(G-N204S) strain, a CYN1009D strain, a CYN1026D strain, a CYN1029D strain, a CYN1138D strain, a CYN1140D strain, a CYN1141D strain, a CYN1242H strain, a CYN1243D strain, a CYN1244D strain, a CYN1245D strain, a CYN1247D strain, a CYN1249D strain, a CYN1250D strain, a CYN1251D strain, a CYN1252D strain, a CYN1253D strain, a CYN1255D strain, a CYN1256D strain, a CYN1257D strain, a CYN1259D strain, a CYN1260D strain, a CYN1261D strain, a GX4 strain, aH-08-1320 strain, aH-1413-09 strain, a IP 1586 / 10 strain, a IP 2990 / 13 strain, a IP 2991 / 13 strain, a IP 2992 / 13 strain, a IP 3176 / 09 strain, a IP 4005 / 12 strain, a IP 412 / 10 strain, a IP 542 / 10 strain, a IP 7941 / 09 strain, a J strain, a JX-08-47 strain, a JX08-48 strain, a Kyoto strain, a Kyoto(G-S204N) strain, a N.HL strain, a RC.HL strain, a rHEP5.0-CVSG strain, a RRV ON-99-2 strain, a SAD-B19 strain, a SH06 strain, a SHRBV-18 strain, a SNK-CTN strain, a SRV9 strain, a Street Alabama Dufferin (HCP-SAD) strain, a VRC-RZ2 strain, a ZJ-LA strain, and a ZJ-QZ strain.

25. The method of claim 24, wherein the envelope glycoprotein G is of a Flury LEP strain.Attorney Docket No.: 058462-520001WO26. The method of any preceding claim, wherein the nucleic acid sequence encoding the modified alphavirus genome or srRNA of the vaccine composition comprises no nucleic acid sequence encoding viral structural proteins.

27. The method of any preceding claim, wherein the nucleic acid sequence encoding the polypeptide construct of the vaccine composition is operably linked to a promoter sequence.

28. The method of claim 27, wherein the promoter sequence is a 26S subgenomic (sg) promoter.

29. The method of any preceding claim, wherein the modified alphavirus genome or srRNA encoded by the nucleic acid construct of the vaccine composition is of an alphavirus belonging to the VEEV / EEEV / WEEV group, or the SFV group, or the SINV group.

30. The method of claim 29, 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).

31. The method of any preceding claim, wherein the nucleic acid construct of the vaccine composition 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 the group consisting of SEQ ID NOS:4-9.

32. The method of any preceding claim, wherein the human subject is having or suspected of having a rabies infection.

33. The method of any preceding claim, wherein the composition is administered to the human subject individually as a single agent prophylaxis or therapy (monotherapy) or as a first therapy in combination with at least one additional therapy.

34. A method of expansion of T follicular helper cells in a human subject, the method comprising:administering to the human subject a vaccine composition comprising a nucleic acid construct comprising a nucleic acid sequence encoding a modified alphavirus genome or selfreplicating 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 replacedAttorney Docket No.: 058462-520001WOby a coding sequence for a polypeptide construct comprising an envelope glycoprotein G of a rabies virus (RABV-G), a variant thereof, or an antigenic determinant of either thereof.

35. The method of claim 34, wherein the T follicular helper cells are CD69+ and CD40L+.

36. The method of claim 34 or 35, wherein the vaccine composition comprises a pharmaceutically acceptable excipient.

37. The method of any one of claims 34 to 36, wherein the vaccine composition is formulated with a delivery vehicle into a delivery system, wherein the delivery system comprises a liposome, a viral replicon particle (VRP), a lipid-based nanoparticle (LNP), a polymer nanoparticle, a physiologic buffer, a microsphere, an immune stimulating complex (ISCOM), a conjugate of bioactive ligand, or a combination of any thereof.

38. The method of Claim 37, wherein the delivery system comprises a lipid-based nanoparticle (LNP).

39. The method of Claim 38, wherein the LNP delivery system comprises a cationic lipid, an ionizable cationic lipid, an anionic lipid, or a neutral lipid.

40. The method of Claim 39, wherein the LNP delivery system comprises an ionizable lipid.

41. The method of any one of claims 34 to 40, wherein the vaccine composition is administered intramuscularly.

42. The method of any one of claims 34 to 41, wherein the administering comprises administering a single dose.

43. The method of any one of claims 34 to 41, wherein the administering comprises administering a first dose and a second dose.

44. The method of claim 43, wherein the second dose is administered at least 8 weeks after the first dose.Attorney Docket No.: 058462-520001WO45. The method of any one of claims 34 to 44, wherein the administering comprises administering about 0.1 mcg of the vaccine composition.

46. The method of any one of claims 34 to 44, wherein the administering comprises administering about 1 mcg of the vaccine composition.

47. The method of any one of claims 34 to 46, wherein the administering comprises administering about 10 mcg of the vaccine composition.

48. The method of any one of claims 34 to 47, wherein the nucleic acid construct of the vaccine composition comprises a coding sequence for a polypeptide construct comprising an antigenic determinant of RABV-G that resides within the N-terminal half of the RABV-G.

49. The method of any one of claims 34 to 48, wherein the nucleic acid construct of the vaccine composition comprises a coding sequence for a polypeptide construct comprising an antigenic determinant of RABV-G that resides within the C-terminal half of the RABV-G.

50. The method of any one of claims 34 to 49, wherein the nucleic acid construct of the vaccine composition comprises a coding sequence for a polypeptide construct comprising an antigenic determinant of RABV-G comprising antigenic site I, antigenic site II, antigenic site III, antigenic site IV, antigenic site minor A of the RABV-G, or a combination of any thereof.

51. The method of any one of claims 34 to 50, wherein the envelope glycoprotein G is of a virulent rabies virus strain or an avirulent rabies virus strain.

52. The method of any one of claims 34 to 51, wherein the envelope glycoprotein G is of a rabies virus strain selected from a Flury LEP strain, a Flury LEP-C strain, a Flury HEP strain, a 1088 strain, a AT6 strain, a CQ92 strain, a CVS-11 strain, a CVS-26 strain, a CVS-26(G-N204S) strain, a CYN1009D strain, a CYN1026D strain, a CYN1029D strain, a CYN1138D strain, a CYN1140D strain, a CYN1141D strain, a CYN1242H strain, a CYN1243D strain, a CYN1244D strain, a CYN1245D strain, a CYN1247D strain, a CYN1249D strain, a CYN1250D strain, a CYN1251D strain, a CYN1252D strain, a CYN1253D strain, a CYN1255D strain, a CYN1256D strain, a CYN1257D strain, a CYN1259D strain, a CYN1260D strain, a CYN1261D strain, a GX4 strain, aH-08-1320 strain, aH-1413-09 strain, a IP 1586 / 10 strain, a IP 2990 / 13 strain, a IP 2991 / 13 strain, a IP 2992 / 13 strain, a IPAttorney Docket No.: 058462-520001WO3176 / 09 strain, a IP 4005 / 12 strain, a IP 412 / 10 strain, a IP 542 / 10 strain, a IP 7941 / 09 strain, a J strain, a JX-08-47 strain, a JX08-48 strain, a Kyoto strain, a Kyoto(G-S204N) strain, a N.HL strain, a RC.HL strain, a rHEP5.0-CVSG strain, a RRV ON-99-2 strain, a SAD-B19 strain, a SH06 strain, a SHRBV-18 strain, a SNK-CTN strain, a SRV9 strain, a Street Alabama Dufferin (HCP-SAD) strain, a VRC-RZ2 strain, a ZJ-LA strain, and a ZJ-QZ strain.

53. The method of claim 52, wherein the envelope glycoprotein G is of a Flury LEP strain.

54. The method of any one of claims 34 to 53, wherein the nucleic acid sequence encoding the modified alphavirus genome or srRNA of the vaccine composition comprises no nucleic acid sequence encoding viral structural proteins.

55. The method of any one of claims 34 to 54, wherein the nucleic acid sequence encoding the polypeptide construct of the vaccine composition is operably linked to a promoter sequence.

56. The method of claim 55, wherein the promoter sequence is a 26S subgenomic (sg) promoter.

57. The method of any one of claims 34 to 56, wherein the modified alphavirus genome or srRNA encoded by the nucleic acid construct of the vaccine composition is of an alphavirus belonging to the VEEV / EEEV / WEEV group, or the SFV group, or the SINV group.

58. The method of any one of claims 34 to 57, 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).

59. The method of any one of claims 34 to 58, wherein the nucleic acid construct of the vaccine composition 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 the group consisting of SEQ ID NOS:4-9.

60. The method of any one of claims 34 to 59, wherein the human subject is having or suspected of having a rabies infection.Attorney Docket No.: 058462-520001WO61. The method of any one of claims 34 to 60, wherein the composition is administered to the human subject individually as a single agent prophylaxis or therapy (monotherapy) or as a first therapy in combination with at least one additional therapy.

Images