Rabies G protein and its uses

Abstract

Polypeptides are provided that include a rabies G protein ectodomain. The rabies G protein ectodomain can have a deletion of the fusion loop domain. Also provided are nanoparticle vaccines against rabies virus. Also provided are pharmaceutical compositions, methods of manufacture, and methods of use, for example, to immunize a subject to generate a protective immune response against rabies virus. The present disclosure generally relates to vaccines against rabies virus.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 371,148, filed August 11, 2022, which is incorporated herein by reference in its entirety.

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application contains a Sequence Listing that has been submitted via EFS-WEB in .XML format and is incorporated herein by reference in its entirety. The .XML copy, created on August 7, 2023, is named 061291-507001WO_SeqList_ST26.xml and is 236 kilobytes in size.

[0003] The present disclosure relates generally to vaccines against rabies virus. [Background technology]

[0004] Rabies virus (RV) is a deadly viral pathogen transmitted to humans primarily through the bite of infected dogs. Estimates suggest that rabies causes over 55,000 deaths annually, primarily among children in Asia and Africa. Global annual direct expenditures on rabies treatment, including antibodies for post-exposure prophylaxis, exceed US$1 billion, and the total economic impact of rabies is estimated to exceed US$8 billion. Because rabies is endemic to wild animals (e.g., foxes, skunks, ferrets, and bats), eradication of the pathogen is unlikely.

[0005] RV is a lyssavirus of the Rhabdoviridae family. Lyssaviruses have a 12-kb nonsegmented RNA genome encoding five viral proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (or large protein, L). RVs consist of two structural and functional units: an internal helical nucleocapsid and an external envelope. The nucleocapsid consists of a ribonucleoprotein complex containing genomic RNA and tightly associated N, L, and P proteins. The lipid envelope is derived from the host cytoplasmic membrane during budding. G protein spikes, composed of trimers of glycosylated ectodomains, bind the virus to host cell receptors and facilitate fusion of the viral membrane with the host membrane. The M protein forms oligomers that bind to the exterior of the nucleocapsid, providing rigidity to the virion structure and providing a binding platform for the viral G protein, a known target of protective immunity. The G protein is a class III viral fusion protein.

[0006] Vaccines against rabies have been available for over 100 years, but their cost and complex immunization schedules have reduced access. Despite the international momentum to eliminate rabies deaths using available tools, current vaccines are suboptimal for controlling RV infection and disease. Drawbacks of current products include low product yield and purity, poor heat stability, sensitivity to freeze-thaw cycles, and challenging regimens (including multiple doses delivered via intradermal immunization).

[0007] Therefore, there is a need for improved vaccines against rabies. Summary of the Invention [Means for solving the problem]

[0008] In one embodiment, the disclosure provides a polypeptide comprising a rabies G protein ectodomain, wherein the rabies G protein ectodomain comprises a deletion of a fusion loop domain of the rabies G protein ectodomain, the deleted fusion loop domain being from about residue 70 to about residue 200 of the rabies G protein ectodomain, as numbered according to SEQ ID NO:53.

[0009] In a variation, the disclosure provides a polypeptide comprising a rabies G protein ectodomain, wherein the rabies G protein ectodomain comprises a deletion of a fusion loop domain of the rabies G protein ectodomain, wherein the deleted fusion loop domain is located between about residue 50 to about residue 180, about residue 70 to about residue 180, about residue 80 to about residue 180, about residue 90 to about residue 180, or about residue 100 to about residue 180 of the rabies G protein ectodomain, as numbered according to SEQ ID NO:53; about residue 50 to about residue 190, about residue 70 to about residue 190, about residue 80 to about residue 190, about residue 90 to about residue 190, or about residue 100 to about residue 180 0 to about residue 190; about residue 50 to about residue 200, about residue 70 to about residue 200, about residue 80 to about residue 200, about residue 90 to about residue 200, or about residue 100 to about residue 200; about residue 50 to about residue 210, about residue 70 to about residue 210, about residue 80 to about residue 210, about residue 90 to about residue 210, or about residue 100 to about residue 210; about residue 50 to about residue 220, about residue 70 to about residue 220, about residue 80 to about residue 220, about residue 90 to about residue 220, or about residue 100 to about residue 220.

[0010] In some embodiments of the polypeptide, the deleted fusion loop domain is residue 66 to residue 207 of the rabies G protein ectodomain, numbered according to SEQ ID NO:53.

[0011] In some embodiments of the polypeptide, the rabies G protein ectodomain comprises a first polypeptide segment linked to a second polypeptide segment, wherein the first polypeptide segment shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with residues 208-417 of SEQ ID NO:53, and the second polypeptide segment shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with residues 208-417 of SEQ ID NO:53.

[0012] In some embodiments of the polypeptide, the polypeptide is a fusion protein comprising, in N-terminus to C-terminus order, a first polypeptide segment, a polypeptide linker, and a second polypeptide segment.

[0013] In some embodiments of the polypeptide, the rabies G protein ectodomain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs:77-81.

[0014] In some embodiments of the polypeptide, the polypeptide comprises a multimerization domain.

[0015] In some embodiments of the polypeptide, the multimerization domain is a trimerization domain.

[0016] In some embodiments of the polypeptide, the multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with FoldOn (SEQ ID NO: 58).

[0017] In some embodiments of the polypeptide, the multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a polypeptide sequence selected from SEQ ID NOs: 1, 4, 5, 7, 9, 18, 19, 21, 24, 25, 26, 29, 30, 31, 34, 36, 37, 39, 42, 43, 44, 45, 46, 47, 48, 49, 50, and 51, and the interface residues identified in Table 3 are conserved.

[0018] In some embodiments of the polypeptide, the multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 7).

[0019] In some embodiments of the polypeptide, the multimerization domain is I53-50A-Δcys (SEQ ID NO: 67).

[0020] In some embodiments of the polypeptide, the polypeptide shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 93-96.

[0021] In some embodiments of the polypeptide, the multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-dn5B (SEQ ID NO: 75).

[0022] In some embodiments of the polypeptide, the polypeptide shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:101 or SEQ ID NO:102.

[0023] In some embodiments of the polypeptide, the multimerization domain is a ferritin polypeptide capable of forming ferritin particles.

[0024] In some embodiments of the polypeptide, the polypeptide shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with construct F-ferritin (SEQ ID NO: 103).

[0025] In some embodiments of the polypeptide, the multimerization domain is a particle-forming domain.

[0026] In another aspect, the present disclosure provides a nanoparticle comprising a polypeptide according to any of the embodiments. In some embodiments, the polypeptide comprises a rabies G protein ectodomain according to any of the embodiments.

[0027] In some embodiments of the nanoparticle, the nanoparticle is a protein-based virus-like particle (pbVLP) or nanostructure.

[0028] In some embodiments of the nanoparticles, the nanoparticles are free of lipid components.

[0029] In some embodiments of the nanoparticles, the nanoparticles comprise a lipid component.

[0030] In some embodiments of the nanoparticle, the nanoparticle comprises a second polypeptide component.

[0031] In some embodiments of the nanoparticles, the nanoparticles comprise a second polypeptide component and a first polypeptide component, and the nanoparticles are self-assembling nanoparticles comprising the first and second polypeptide components symmetrically arranged with point group symmetry.

[0032] In some embodiments of the nanoparticle, (a) a first polypeptide component comprises any of the polypeptides of the present disclosure, and the first polypeptide component forms a first homomeric complex via a multimerization domain of the polypeptide; (b) a second polypeptide component comprises a second multimerization domain, and the second polypeptide component forms a second homomeric complex via the second multimerization domain of the polypeptide; (c) the first homomeric complex and the second homomeric complex assemble to form a nanoparticle with point group symmetry; (d) the nanoparticle is devoid of other polypeptide components; and / or (e) the nanoparticle is devoid of a lipid component.

[0033] In some embodiments of the nanoparticles, the nanoparticles have icosahedral symmetry.

[0034] In some embodiments of the nanoparticles, the first multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 7) or I53-50AΔCys (SEQ ID NO: 67), and the second multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50B (SEQ ID NO: 8) or I53-50B.4PosT1 (SEQ ID NO: 34).

[0035] In some embodiments of the nanoparticle, the first multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-dn5B (SEQ ID NO: 75) and the second multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-dn5A (SEQ ID NO: 74).

[0036] In some embodiments of the nanoparticles, the first polypeptide component shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a polypeptide sequence selected from SEQ ID NOs: 93-96, and the second polypeptide component shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50B (SEQ ID NO: 8) or I53-50B.4PosT1 (SEQ ID NO: 34).

[0037] In some embodiments of the nanoparticles, the first polypeptide component shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a polypeptide sequence selected from SEQ ID NOs: 101-102, and the second polypeptide component shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-dn5A (SEQ ID NO: 74).

[0038] In another aspect, the present disclosure provides a pharmaceutical composition for use as a vaccine comprising any of the polypeptides or nanoparticles of the present disclosure, and optionally one or more pharmaceutically acceptable excipients.

[0039] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises at least one adjuvant.

[0040] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises an oil-in-water emulsion.

[0041] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises a squalene-based oil-in-water emulsion.

[0042] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises a toll-like receptor (TLR) immunostimulatory agent.

[0043] In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises squalene, SLA, GLA, R848, IMQ, 3M-052, CpG, saponin (QS21), or a combination thereof.

[0044] In another aspect, the present disclosure provides a polynucleotide comprising a polynucleotide sequence encoding any of the polypeptides or nanoparticles of the present disclosure.

[0045] In another aspect, the present disclosure provides a vector comprising a polynucleotide comprising a polynucleotide sequence encoding any of the polypeptides or nanoparticles of the present disclosure.

[0046] In another aspect, the present disclosure provides a method of generating an immune response in a subject infected with rabies virus, the method comprising administering any of the polypeptides, nanoparticles, pharmaceutical compositions, polynucleotides, or vectors of the present disclosure in an amount effective to generate an immune response.

[0047] In another aspect, the present disclosure provides a method of immunizing a subject infected with rabies virus against infection by rabies, comprising administering any of the polypeptides, nanoparticles, pharmaceutical compositions, polynucleotides, or vectors of the present disclosure in an amount effective to generate an immune response.

[0048] In another aspect, the present disclosure provides a method of administering post-exposure prophylaxis to a subject infected with rabies virus, the method comprising administering any of the polypeptides, nanoparticles, pharmaceutical compositions, polynucleotides, or vectors of the present disclosure in an amount effective to generate an immune response.

[0049] In some embodiments of the method, the immune response comprises a humoral immune response.

[0050] In some embodiments of the method, the immune response comprises a polyclonal antibody response against rabies G protein.

[0051] In some embodiments of the method, the immune response comprises a neutralizing antibody response to the rabies virus.

[0052] In some embodiments of the method, the method generates a protective immune response against rabies virus.

[0053] In some embodiments of the method, the method generates neutralizing antibodies against rabies virus.

[0054] In some embodiments of the method, the administering step comprises intramuscular or subcutaneous injection.

[0055] In some embodiments of the method, the method results in the production of rabies-specific neutralizing antibodies in a subject in need thereof.

[0056] In some embodiments of the method, the method results in an increase in rabies-specific neutralizing antibodies in a subject in need thereof that is at least about a 2-fold, at least about a 3-fold, at least about a 4-fold, at least about a 5-fold, at least about a 10-fold, at least about a 15-fold, at least about a 20-fold, or at least about a 25-fold increase compared to rabies-specific neutralizing antibodies in the same subject prior to the administering step.

[0057] In some embodiments of the method, the method generates a neutralization titer of at least 0.5 IU / mL in a rapid fluorescent focus inhibition test (RFFIT) and / or a fluorescent antibody virus neutralization (FAVN) test.

[0058] In some embodiments of the methods, the subject is a non-human animal.

[0059] In some embodiments of the methods, the subject is a companion animal.

[0060] In some embodiments of the methods, the subject is a human.

[0061] In another aspect, the present disclosure provides a host cell comprising a polynucleotide comprising a polynucleotide sequence encoding any of the polypeptides or nanoparticles of the present disclosure.

[0062] In another aspect, the present disclosure provides a method of producing a vaccine, the method comprising culturing a host cell of the present disclosure in a culture medium such that the host cell secretes a first polypeptide component into the culture medium, purifying the first polypeptide component from the culture medium, mixing the first polypeptide component with a second polypeptide component, wherein the first and second polypeptide components self-assemble to form nanoparticles, mixing, and / or purifying the nanoparticles.

[0063] In another aspect, the present disclosure provides a kit comprising a polypeptide, nanoparticle, polynucleotide, vector, or pharmaceutical composition of the present disclosure. [Brief explanation of the drawings]

[0064] [Figure 1] FIG. 1 is a diagram of the sequence of the rabies G protein with secondary structure elements indicated. [Figure 2] FIG. 1 shows one-component nanoparticles and two-component nanoparticles (without rabies G protein). [Figure 3]

[0023] Figure 1 shows a diagram of a two-component protein-based virus-like particle (pbVLP). The multimerization domains I53-50A + I53-50B or I53-dn5B + I53-dn5A can be used to form VLPs. As shown, the VLPs have icosahedral (I53) symmetry. [Figure 4] 1 is an image of an SDS-PAGE analysis of pbVLP (construct F-CompA+CompB, I53-50 version). [Figure 5] 1 is a cryo-electron microgram of VLPs showing that the VLPs form the expected icosahedral particles. [Figure 6] FIG. 1 is a graph of biolayer interferometry (BLI) using two neutralizing antibodies, D1-25 and 1112-1 (RD-Biotech), which recognize antigenic sites III and II of the rabies G protein, respectively, both without freezing ("pre-freeze") or after freeze-thawing ("post-freeze"). [Figure 7] Graph showing potency ELISA assay using two neutralizing antibodies (D1-25 and 1112-1 (RD-Biotech)). Construct F CompA transiently expressed in Expi293 (HEK) or CHO-K1 cells, along with VLPs assembled with CompA, was evaluated in a potency ELISA. [Figure 8] Graph of neutralization titers in serum on day 35 for Construct C VLP (AddaVax group) and ImRab3 groups, with geometric mean titers of 37.87 IU / ml and 17.9 IU / ml, respectively. Construct C VLP formulated with Alhydrogel or Construct C components induced low but detectable titers, with geometric mean titers of 0.25 IU / ml and 0.78 IU / ml, respectively. [Figure 9]This graph shows that comparable neutralizing titers were induced by constructs C (I53-50 and dn5), B, and F, with all animals showing titers above the correlate of protection (0.5 IU / ml) as indicated by sera on day 35. Geometric mean titers induced by VLPs ranged from 21.3 to 34.3 IU / ml, with no statistically significant differences between groups (Mann-Whitney test). Titers observed with VLPs were comparable to those with ImRab3. [Figure 10] Figure 1 shows that serum on day 35 showed neutralization geometric mean titers ranging from 74.8 to 211 IU / ml for Construct C and Construct F. All Construct C and Construct F titers were not statistically significant between groups (Mann-Whitney test). All animals (ICR mice) administered Construct C and Construct F had titers above the correlate of protection, except for one animal at the 1 μg dose level of Construct F, which was at background levels (<0.2 IU / ml). Titers observed with Construct C and Construct F were comparable to ImRab3 at day 35 (geometric mean 71.1). [Figure 11] This graph shows that serum on day 35 showed that neutralizing titers of Construct C exceeded the protective correlate in 17 / 18 animals (Syrian golden hamsters) at doses of 3 μg, 1 μg, and 0.3 μg, with geometric mean titers ranging from 2.2 to 6.7 IU / mL. At the lowest dose of Construct C (0.1 μg), titers exceeded the protective correlate in 2 / 6 animals. The three highest doses of Construct F (3 μg, 1 μg, and 0.3 μg) produced comparable titers in all 18 animals (that exceeded the protective correlate), with geometric mean titers ranging from 4.8 to 5.7 IU / mL, and at the lowest dose, 3 / 6 animals exceeded the protective correlate. [Figure 12] 1 is a graph showing neutralization titers of day 35 sera for construct F I53-50 VLP, construct F dn5 VLP, or construct F-CompA formulated with aqueous buffer, Alhydrogel, or AddaVax. Construct F I53-50 VLP formulated with AddaVax induced significantly higher titers. [Figure 13] 1 is a graph showing survival rates of animals administered saline and either challenged with rabies virus, immunized with Construct F VLP, or immunized with RabAvert. [Figure 14] 10 is a graph showing neutralizing antibody titers from serum on day 34 of animals administered either saline, Construct F VLP, or RabVert. [Table A] DETAILED DESCRIPTION OF THE INVENTION

[0065] The present disclosure provides polypeptides, nanoparticles, and related compositions useful for generating an immune response against rabies G protein, as well as methods for making and using them. Without being bound by theory, the rabies G protein is believed to contain a fusion loop domain that includes two fusion loops and flanking sequences that together form a complex tertiary structure. Deletion of this fusion loop domain may, at least in some cases, enhance the expression, stability, and / or assembly ability of the rabies G protein. Furthermore, at least in some cases, the antigenic sites of the rabies G protein are preserved.

[0066] Several neutralizing antibody epitopes for the rabies G protein have been described. Antigenic site I includes residues 226-231, site II is a discontinuous epitope consisting of residues 34-42 and 198-200, site III consists of residues 226-231 (discontinuous epitopes 198-200 and 330-338) (Kuzmina et al. J Antivir Antiretrovir 5:22013 (2013)), and site IV contains the crucial residue 251 (Luo et al. Microbiol. Immunol. 39:693-702 (1995)). Furthermore, the neutralizing antibody RG719 binds to residues 249-268 (Ni et al. Microbiol. Immunol. 39(9):693-702 (1995)). These are examples and not a complete list of epitopes reported for neutralizing antibodies against rabies G protein.

[0067] Because neutralizing epitopes are generally conformational epitopes, peptides corresponding to the epitope do not bind to antibodies and therefore do not induce neutralizing titers; however, Dietzschold et al., J. Virol., 595-602 (1982) described the generation of neutralizing titers following immunization with three different peptides of rabies G protein cleaved by CNBr.

[0068] Unlike other glycoproteins (e.g., influenza HA), which lack a proteolytic site, the rabies G protein contains two small loops (residues 91–97 and 139–145) that form a bipartite peptide involved in fusion.

[0069] Polypeptides The native sequence of the rabies G protein (GenBank P15199.2) is shown below, with the signal sequence underlined and italicized, and the transmembrane and intracellular portions underlined (SEQ ID NO: 53). [ka]

[0070] The native signal sequence is post-translationally cleaved when the protein is expressed. An alternative signal sequence may replace the native signal sequence for expression of the ectodomain, or in some embodiments, no signal sequence is used. Thus, the native rabies G protein ectodomain has the following sequence: [ka]

[0071] However, without being bound by theory, the C-terminus of the rabies G protein may be further C-terminally truncated to around residue 400, around residue 410, or around residue 420.

[0072] Without being bound by theory, Figure 1 shows the predicted secondary structure and disulfide bonding pattern of the rabies G protein, numbered starting from the N-terminus of the protein after removal of the signal peptide. The fusion loop domain is believed to comprise approximately residue 70 to approximately residue 200 of the rabies G protein ectodomain, as numbered according to SEQ ID NO:53, or approximately residue 50 to approximately residue 180 of the rabies G protein ectodomain, as numbered according to Figure 1.

[0073] The present disclosure demonstrates the expression of rabies G protein antigens as soluble trimers and as components of protein-based VLPs. Without being bound by theory, the antigens described herein may be superior to known rabies G protein antigens in that they may be more stable, may be expressed in higher yields, may be less toxic to host cells, and / or may be more suitable for display in virus-like particles, including, but not limited to, protein-based virus-like particles.

[0074] In some embodiments, the rabies G protein ectodomain comprises one or more internal insertions and / or deletions. In particular, the rabies G protein ectodomain can comprise a deletion of part, all, or substantially all of the fusion loop domain of the rabies G protein ectodomain. In some embodiments, the rabies G protein ectodomain comprises one or more, two or more, or three or more amino acid substitutions.

[0075] In one embodiment, the disclosure provides a polypeptide comprising a rabies G protein ectodomain, wherein the rabies G protein ectodomain comprises a deletion of a fusion loop domain of the rabies G protein ectodomain, the deleted fusion loop domain being from about residue 70 to about residue 200 of the rabies G protein ectodomain, as numbered according to SEQ ID NO:53.

[0076] The deleted fusion loop domains are from about residue 50 to about residue 180, from about residue 70 to about residue 180, from about residue 80 to about residue 180, from about residue 90 to about residue 180, or from about residue 100 to about residue 180 of the rabies G protein ectodomain, as numbered according to SEQ ID NO: 53; from about residue 50 to about residue 190, from about residue 70 to about residue 190, from about residue 80 to about residue 190, from about residue 90 to about residue 190, or from about residue 100 to about residue 190; from about residue 50 to about residue 200, from about residue 70 to about residue 200, It can be around residue 200, around residue 80 to around residue 200, around residue 90 to around residue 200, or around residue 100 to around residue 200; around residue 50 to around residue 210, around residue 70 to around residue 210, around residue 80 to around residue 210, around residue 90 to around residue 210, or around residue 100 to around residue 210; around residue 50 to around residue 220, around residue 70 to around residue 220, around residue 80 to around residue 220, around residue 90 to around residue 220, or around residue 100 to around residue 220.

[0077] In some embodiments of the polypeptide, the deleted fusion loop domain is residue 66 to residue 207 of the rabies G protein ectodomain, numbered according to SEQ ID NO:53.

[0078] In some embodiments, the rabies G protein ectodomain is SEQ ID NO: 55, or a variant thereof. [ka] wherein X represents any amino acid or is absent (i.e., one or more of the X residues may be deleted to remove all or part of this loop in the predicted structure). Exemplary variants may share at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 55 over the entire length of SEQ ID NO: 55, excluding the segment represented by X. The polypeptide linker may be any suitable linker. A Gly-Ser linker, such as the polypeptide sequence GSGSGSG, may be used. Other suitable linkers include Ser-Ser-Ile-Ser-Asn and Gly-Ser-Gly-Ser-Gly-Ser-Gly.

[0079] Deletion of the fusion loop domain may leave a first polypeptide and a second polypeptide segment, which may be linked by a polypeptide linker. In a variation, the two polypeptide segments are co-expressed without a polypeptide linker. In a further variation, the two polypeptide segments are expressed separately and combined together. Chemical linkage may be used to hold the two polypeptide segments together.

[0080] For example, the rabies G protein ectodomain is SEQ ID NO: 56: [ka] a first polypeptide segment sharing at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with and / or SEQ ID NO: 57: [ka] The polypeptide may comprise a second polypeptide segment that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide. The linker can be any suitable linker. A Gly-Ser linker, such as the polypeptide sequence GSGSGSG, can be used.

[0081] In some embodiments of the polypeptide, the rabies G protein ectodomain comprises a first polypeptide segment linked to a second polypeptide segment, wherein the first polypeptide segment shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with residues 208-417 of SEQ ID NO:53, and the second polypeptide segment shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with residues 208-417 of SEQ ID NO:53.

[0082] In some embodiments of the polypeptide, the polypeptide is a fusion protein comprising, in N-terminus to C-terminus order, a first polypeptide segment, a polypeptide linker, and a second polypeptide segment.

[0083] In some embodiments, the rabies G protein ectodomain comprises a deletion, truncation, or substitution (e.g., with a linker) of amino acid residues G68 to F211, I70 to T212, S71 to T211, S71 to F211, A72 to P207, I73 to P207, K74 to P207, D137 to V144, L141 to V144, or P136 to K148 relative to the reference polypeptide sequence SEQ ID NO: 53. The polypeptide linker sequence, when present, may replace 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more amino acid residues. The linker sequence may be shorter than the sequence it replaces. For example, residues G68 through P207 can be replaced with a linker of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues, optionally a series of glycine, serine, threonine, or alanine residues, or a series of glycine and serine residues (i.e., a glycine-serine linker).

[0084] The C-terminus of the polypeptide can be at a variety of suitable residues within the ectodomain of the rabies G protein. In variants, the polypeptide can retain the transmembrane and / or intracellular segments of the rabies G protein. In some embodiments, the C-terminal residue is any residue within the predicted non-helical region (residues 355-445). In some embodiments, the rabies G protein ectodomain is at residues 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 48 Ending in 26, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, or 461.

[0085] In some embodiments, the C-terminal truncation is a deletion from residue 418 to the C-terminus of the rabies G protein relative to the reference sequence SEQ ID NO: 53, also referred to as a C-terminal truncation at residue 417.

[0086] In some embodiments, the rabies G protein ectodomain comprises a sequence that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with TVFKEGDEAEDFVEVHLPD (SEQ ID NO: 64).

[0087] In some embodiments, the rabies G protein ectodomain comprises a sequence that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with TVFKEGDEAEDFVEVHLPDVYKQISGVDLGLP (SEQ ID NO: 65).

[0088] In some embodiments, the rabies G protein ectodomain comprises a sequence that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with TVFKEGDEAEDFVEVHLPDVYKQISGVDLGLPNWGKY (SEQ ID NO: 66).

[0089] Various additional modifications of the rabies G protein ectodomain may be included. Using the methods described in the Examples, modifications of the amino acid sequence can be made and tested to identify those that increase (or decrease) expression, ability to generate stable cell lines, protein stability, nanoparticle assembly, and / or antigenicity. For example, but not by way of limitation, cysteine ​​(Cys) residues in the sequence can be removed by substitution with alternative residues such as serine (Ser), glycine (Gly), or alanine (Ala).

[0090] A variety of rabies G proteins may be used, including but not limited to the ectodomains in Table 1. [Table 1-1] [Table 1-2] [Table 1-3]

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

[0091] In some embodiments, the rabies G protein ectodomain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 104-170.

[0092] Exemplary rabies G protein ectodomain sequences with deletions of the fusion loop domain are shown in Table 2. [Table 2-1] [Table 2-2]

[0093] In some embodiments of the polypeptide, the rabies G protein ectodomain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs:77-81.

[0094] In some embodiments of the polypeptide, the polypeptide comprises a multimerization domain. In some embodiments of the polypeptide, the multimerization domain is a trimerization domain. In some embodiments of the polypeptide, the multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with FoldOn (SEQ ID NO: 58). In some embodiments of the polypeptide, the multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a polypeptide sequence selected from SEQ ID NOs: 1, 4, 5, 7, 9, 18, 19, 21, 24, 25, 26, 29, 30, 31, 34, 36, 37, 39, 42, 43, 44, 45, 46, 47, 48, 49, 50, and 51, and the interface residues identified in Table 3 are conserved. In some embodiments of the polypeptide, the multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 7). In some embodiments of the polypeptide, the multimerization domain is I53-50A-Δcys (SEQ ID NO: 67). In some embodiments of the polypeptide, the polypeptide shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 93-96.In some embodiments of the polypeptide, the multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-dn5B (SEQ ID NO: 75). In some embodiments of the polypeptide, the polypeptide shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 101 or SEQ ID NO: 102. In some embodiments of the polypeptide, the multimerization domain is a ferritin polypeptide capable of forming ferritin particles. In some embodiments of the polypeptide, the polypeptide shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with construct F-ferritin (SEQ ID NO: 103). In some embodiments of the polypeptide, the multimerization domain is a particle-forming domain.

[0095] Subunit vaccines The polypeptides of the present disclosure can be used as subunit vaccines. For example, the polypeptides can include a trimerization domain, such as a FoldOn or GCN4 trimerization domain, or an I53-50A or I53-dn5B trimerization domain. The I53-50A or I53-dn5B trimerization domain can be used to generate soluble trimers of the rabies G protein ectodomain in the absence of their respective partners (I53-50B and I53-dn5A). Such soluble trimers can be used as assay reagents (e.g., validation standards) or as subunit vaccines. For example, the disclosure provides a pharmaceutical composition comprising a polypeptide, wherein the polypeptide comprises, in order from N-terminus to C-terminus, a rabies G protein ectodomain, the rabies G protein ectodomain comprising a deletion of the fusion loop domain of the rabies G protein ectodomain; a polypeptide linker; and optionally a trimerization domain selected from a FoldOn, GCN4, I53-50A, or I53-dn5B trimerization domain.

[0096] nanoparticles The present disclosure relates, in part, to polypeptides for use as components of nanoparticles. The nanoparticles can be nanoparticles containing lipid components, or preferably nanoparticles that are substantially free of lipid components or nanoparticles that are devoid of lipid components. For example, the nanoparticles can be protein-based virus-like particle (pbVLP) nanostructures. An exemplary nanostructure is shown in Figure 2, without the rabies G protein. Further exemplary nanostructures or pbVLPs are shown in Figure 3.

[0097] The nanoparticles of the present disclosure may present an antigen capable of eliciting an immune response against the rabies virus. In some embodiments, the nanoparticles of the present disclosure are useful for preventing or reducing the severity of rabies infection. In particular, the nanoparticles of the present disclosure present the ectodomain of the rabies G protein. The ectodomain may be non-covalently or covalently bound to the core of the nanoparticle, including as a fusion protein or by other means disclosed herein. In some embodiments, a linker connects the ectodomain to a first polypeptide comprising a multimerization domain. The linker may be any chemical linkage, including, but not limited to, a polypeptide used to form a fusion at the N- or C-terminus of the ectodomain with the first polypeptide. The rabies G protein may optionally be presented along the axis of symmetry of the VLP. In some embodiments, the G protein is C-terminally linked to a first polypeptide comprising a trimerization domain. An intervening trimerization tag (e.g., a FoldOn tag) may be present.

[0098] Protein-based virus-like particles The nanoparticles of the present disclosure can be protein-based virus-like particles (pbVLPs). The pbVLPs of the present invention can comprise a multimeric protein assembly adapted for displaying the ectodomain of a rabies G protein. The pbVLPs of the present invention comprise at least one polypeptide component present in multiple copies in the pbVLP. The multimerization domain of a first polypeptide component can be derived from a naturally occurring protein sequence by substitution of at least one amino acid residue. This first component can form the entire core of the pbVLP. Alternatively, the core of the pbVLP can comprise a second polypeptide component, or a third, fourth, fifth, etc. polypeptide component, such that the VLP comprises two, three, four, five, six, seven, or more components, each of which is present in multiple copies. In some cases, the first polypeptide component comprising a polypeptide comprising a rabies G protein ectodomain forms a trimer related by three-fold rotational symmetry, and the second polypeptide component forms a pentamer related by five-fold rotational symmetry. In such cases, the VLP forms an "icosahedral particle" with I53 symmetry. The two polypeptide components are arranged together so that the members of each homomeric complex are related to each other by symmetry operators. A general computational method for designing self-assembling protein materials, including symmetric docking of protein components in a target symmetric structure, is disclosed in U.S. Patent Publication No. US2015 / 0356240 A1.

[0099] The term "core" of a pbVLP is used herein to describe the central portion of the pbVLP that links together several copies of the rabies G protein ectodomain displayed by the pbVLP. In some embodiments, the first component comprises a polypeptide comprising the rabies G protein ectodomain, a polypeptide linker, and a first multimerization domain. In other embodiments, the polypeptide comprising the rabies G protein ectodomain is non-covalently or covalently linked to the first multimerization domain. For example, an antibody or antigenic fragment thereof can be fused to the first multimerization domain and configured to bind to the rabies G protein ectodomain or a chemical tag on the ectodomain. The streptavidin-biotin (or neuravidin-biotin) system can be used. Alternatively, various chemical linkers can be used. The advantage of designing the core to be a universal platform is that one or more multimerization domains comprising the core can be designed and optimized in advance and then tested with various ectodomains. Of course, in some cases the same polypeptide may form part of the "core" and then extend outward as an adapter for attachment of the rabies G protein ectodomain or to include the ectodomain (i.e., a fusion protein). In embodiments of the present disclosure, the polypeptide comprises additional polypeptide sequences in addition to the rabies G protein ectodomain. In certain embodiments, the ectodomain is glycosylated, either naturally or with alternative oligosaccharides (e.g., oligosaccharides specific to the host cell used to express the antigen).

[0100] The multimerization domain(s) drive the self-assembly of pbVLPs. In some cases, even though the core can independently self-assemble in the absence of the ectodomain, self-assembly can be further facilitated by ectodomain multimerization. Without being bound by theory, rabies G protein forms a trimer in its native state. The presentation of the ectodomain on the VLP, at least in some embodiments, reduces the thermodynamic barrier to assembly and / or the equilibrium ratio of assembled to disassembled VLP components. In some cases, the trimeric ectodomain positioned along the three-fold axis of the VLP properly trimerizes in part because the ectodomain is presented on the three-fold axis of the VLP core, promoting proper folding and conformational stability of the ectodomain and making VLP self-assembly a cooperative process, in that the VLP is stabilized in the assembled form, at least in part, by non-covalent or covalent interactions between the trimeric units. In some cases, particularly when cysteine ​​residues are positioned to form intramolecular disulfide bonds, the assembly can optionally be further stabilized by introducing mutations into the antigen or VLP components. In some examples, dimeric, trimeric, tetrameric, pentameric, or hexamer antigens are displayed on cores designed with matching 2-, 3-, 4-, 5-, or 6-fold symmetry axes, such that the core accommodates the arrangement of multimeric antigens with the antigen's natural symmetry.

[0101] In some embodiments, protein-based VLPs can comprise a symmetric core. These include, but are not limited to, designed VLPs. For example, the protein ferritin has been used to generate symmetric protein-based VLPs using naturally occurring ferritin sequences. Ferritin-based VLPs are distinguished from designed VLPs in that no protein engineering is required beyond fusing a viral protein (here, the rabies G protein ectodomain) to the ferritin molecule to form a symmetric VLP from ferritin. Protein design methods can be used to generate similar one-component and two-component nanostructures based on template structures (e.g., structures deposited in protein data banks) or de novo (i.e., by computationally designing a new protein with a desired structure but with little or no homology to naturally occurring proteins). Such one-component and two-component nanostructures can then be used as the core of a designed VLP. The terms "protein nanoparticle" or "nanoparticle" and "nanostructure" may be used to refer to the protein-based VLPs described herein.

[0102] The VLP may be a ferritin-based VLP. In some embodiments, the protein complex is a protein-based VLP (including ferritin, E2p, I3-01, and I3-01 variants) such as those described in U.S. Patent Publication No. US2020 / 0009244 A1 and International Patent Publication Nos. WO2022 / 046583 A1 and WO2021 / 210984 A1, the disclosures of which are incorporated herein by reference. Protein-based VLPs may use various coupling techniques to attach antigens to the VLP core, including, but not limited to, the SpyCatcher system, as described in, for example, Escolano et al. Nature 570:468-473 (2019), He et al. Sci Adv. 7(12):eabf1591 (2021), and Tan et al. Nat. Commun. 12(1):542 (2021). The protein-based VLP can be, for example, a lumazine synthase nanoparticle, as described in Geng et al. PLoS Pathog. 17(9):e1009897 (2021). The protein-based VLP can be, for example, a ferritin nanoparticle, as described in Joyce et al. bioRxiv 2021.05.09.443331 and U.S. Patent Publication No. US2019 / 0330279 A1.

[0103] In the present disclosure, protein-based VLPs are distinguished from nanoparticle vaccines more generally, as the term "nanoparticle" vaccine is used in the art to refer to protein- or glycoprotein-based vaccines (see, e.g., U.S. Pat. No. 9,441,019), polymerized liposomes (see, e.g., U.S. Pat. No. 7,285,289), surfactant micelles (see, e.g., U.S. Patent Publication No. US 2004 / 0038406 A1), and synthetic biodegradable particles (see, e.g., U.S. Pat. No. 8,323,696). Use of the disclosed rabies G protein ectodomain is contemplated in any of these formats.

[0104] The protein-based VLPs of the present disclosure are distinguishable from VLPs that display rabies G protein on the surface of micellar particles containing detergent (e.g., NP-9), or alternatively, those made by extracting antigenic proteins from live virus while preserving the lipid components of the viral envelope. In contrast, the protein-based VLPs described herein are free or substantially free of lipids and detergents. Furthermore, the symmetrical display of rabies G protein in some embodiments of the protein-based VLPs of the present disclosure may generate a superior immune response to the G protein compared to other VLPs.

[0105] The term "virus-like particle" or "VLP" refers to a molecular assembly that resembles a virus but is non-infectious and displays antigenic proteins or antigenic fragments of viral proteins or glycoproteins. A "protein-based VLP" refers to a VLP formed from proteins or glycoproteins and substantially free of other components (e.g., lipids). Protein-based VLPs may contain post-translational and chemical modifications, but should be distinguished from micellar VLPs and VLPs formed by extraction of viral proteins from live or inactivated live virus preparations. The term "designed VLP" refers to a VLP containing one or more polypeptides generated by computational protein design. An exemplary designed VLP is a VLP containing the nanostructure shown in Figure 2. The term "symmetric VLP" refers to a protein-based VLP with a symmetric core, such as those shown in Figures 2 and 3. These include, but are not limited to, designed VLPs.

[0106] The term "icosahedral particle" refers to designed VLPs having a core with icosahedral symmetry (e.g., particles designated I53 and I52 in Table 3). I53 refers to an icosahedral particle constructed from pentamers and trimers. I52 refers to an icosahedral particle constructed from pentamers and dimers. T33 refers to a tetrahedral particle constructed from two sets of trimers. T32 refers to a tetrahedral particle constructed from trimers and dimers.

[0107] Antigens may be non-covalently or covalently attached to the core of the protein-based VLP, including as a fusion protein or by other means disclosed herein. Multimeric antigens may optionally be presented along the axis of symmetry of the VLP. Also provided are proteins and nucleic acid molecules encoding such proteins, formulations, and methods of use.

[0108] A non-limiting example of one embodiment is shown in Figure 3, which depicts a G protein genetically fused to the multimerization domain of a VLP, optionally recombinantly expressed in a host cell (e.g., 293F cells), along with a pentameric homomeric complex of a second component, optionally recombinantly expressed in the same or a different host cell (e.g., E. coli cells). These two components self-assemble into a VLP that displays 20 G protein trimers around an icosahedral core. In this embodiment, the core has a universal design. In some embodiments, the VLP is further linked to a polypeptide or other agent that can act as an adjuvant. In some embodiments, the antigen (first component) and / or the second component comprise one or more T cell epitopes, optionally including heterologous T cell epitopes.

[0109] Other possible arrangements of polypeptides of the present disclosure are shown in Figure 2. In some embodiments, the VLP is adapted for display of up to 8 trimers; 8 trimers and 12 dimers; 6 tetramers and 12 dimers; 6 tetramers and 8 trimers; 20 trimers and 30 dimers; 4 trimers and 6 dimers; 4 first trimers and 4 second trimers, or 8 trimers; 12 pentamers and 20 trimers; 12 pentamers and 30 dimers; or 4 trimers. In some cases, one of the axes of symmetry is not used for antigen presentation; thus, in some embodiments, the VLP is adapted for display of up to 8 trimers, 12 dimers, 6 tetramers, 20 trimers, 30 dimers, 4 trimers, 6 dimers, 8 trimers, or 12 pentamers. In some cases, monomeric antigens are displayed, and thus the VLP is adapted to display up to 12, 24, 60, or 70 monomeric antigens. In some cases, the VLP contains multiple intermixed polypeptides, such that otherwise identical polypeptides in the core of the VLP display different antigens or no antigens. Thus, depending on the ratio of polypeptides, the VLP is adapted to display 1 to 130 antigens (e.g., on an I52 particle), and each of the displayed antigens can be the same or a different member of the intermixed population in any proportion selected. These antigens can be coexpressed in a recombinant expression system and allowed to self-assemble before purification. Alternatively, the antigens can be expressed separately and then mixed together either before or after purification from the expression host and associated contaminants. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more antigens are presented.Non-limiting representative VLPs are provided in Bale et al. Science 353:389-94 (2016), Heinze et al. J. Phys. Chem B. 120:5945-5952 (2016), King et al. Nature 510:103-108 (2014), and King et al. Science 336:1171-71 (2012).

[0110] In some cases, VLPs are adapted to display the same antigen from two or more different rabies strains. In a non-limiting example, the same VLP displays a mixed population of homotrimeric protein antigens or mixed heterotrimers of protein antigens from different rabies strains. In further embodiments, particles can be engineered to display proteins from diverse sources, such as the rabies G protein ectodomain and one or more trimeric glycoproteins of HIV-1, HIV-2, EBV, CMV, RSV, influenza, Ebola, Marburg, Dengue, SARS, MERS, Hanta, or Zika viruses. In some embodiments, VLPs comprise a trimeric glycoprotein of a virus related evolutionarily or by sequence identity to any of these representative viruses, including, but not limited to, herpesvirus, orthomyxovirus, paramyxovirus, pneumovirus, filovirus, flavivirus, reovirus, or retrovirus. In one embodiment, the VLP comprises the extracellular domain(s) of a transmembrane protein or glycoprotein, or an antigenic fragment thereof.

[0111] When mixed VLPs are generated, it may be advantageous to ensure homomerization in a strain-specific manner rather than allowing heterodimerization, such as displaying all strain 1 G proteins on one triad of the T33 particle while displaying all strain 2 G proteins on the other triad of the T33 particle. This can be achieved by using a VLP containing two or more types of multiple polypeptides as the core of the VLP, allowing each type of multiple polypeptide to bind different antigens. Alternatively, VLPs may be engineered with one or more symmetry-breaking mutations, such as knob-in-hole mutations or intermolecular disulfide mutations, which have the effect of preventing trimerization between different antigens. In this case, the VLP will display multimeric antigens from different strains at symmetrically equal positions on the VLP, but each position on the VLP will be occupied by homomers from the same strain, with only a small proportion of inter-strain heteromeric antigens. In some cases, the antigens themselves can be genetically engineered to prevent inter-strain heterodimerization. In one embodiment, VLPs are engineered to prevent heteromerization of two structurally conserved but antigenically distinct antigenic proteins, such as, for example, strain 1 G protein and strain 2 G protein, or rabies G protein and a non-rabies antigenic protein. Furthermore, when mixed VLPs are made and antigens are presented as fusion proteins, the VLP will contain three or more different proteins because the fusion proteins share the same (or equivalent) domains used to form the core of the VLP, with one different antigenic domain for each antigen presented on the VLP.

[0112] Linker The VLPs of the present disclosure display antigenic proteins in a variety of ways, including genetic fusion or other means disclosed herein. As used herein, "linked to" or "coupled to" refers to any means known in the art for associating two polypeptides. This association can be direct or indirect, reversible or irreversible, weak or strong, covalent or non-covalent, and selective or non-selective. In some embodiments, a "polypeptide linker" is described, which can refer to a linker between two portions of a G protein.

[0113] In some embodiments, the linkage is achieved by genetic engineering to create an N- or C-terminal fusion of the rabies G protein ectodomain to a multimerization domain.

[0114] In some embodiments, the linkage is achieved by post-translational covalent attachment of the rabies G protein ectodomain to the multimerization domain of the first component of the VLP. In some cases, chemical cross-linking is used to non-specifically bind an antigen to a VLP polypeptide. In some cases, chemical cross-linking is used to specifically bind an antigenic protein to a VLP polypeptide (e.g., to a first polypeptide or a second polypeptide). A variety of specific and non-specific cross-linking chemistries are known in the art, such as click chemistry and other methods. Generally, any cross-linking chemistry used to link two proteins can be adapted for use in the VLPs disclosed herein. In particular, chemistries used to create immunoconjugates or antibody-drug conjugates can be used. In some cases, VLPs are created using cleavable or non-cleavable linkers. Processes and methods for conjugating antigens to carriers are provided, for example, in U.S. Patent Publication No. US2008 / 0145373 A1.

[0115] In one embodiment, binding to the VLP is achieved by non-covalent attachment. In some cases, the rabies G protein ectodomain is engineered to be negatively charged on at least one surface, and the core polypeptide is engineered to be positively charged on at least one surface, or to be positively and negatively charged, respectively. This facilitates intermolecular association between the antigenic protein and the core polypeptide through electrostatic forces. In some cases, shape complementarity is used to induce linkage of the antigenic protein to the core. This shape complementarity can be pre-existing or rationally designed. In one embodiment, the antigen is biotinylated and the polypeptide comprises streptavidin, or vice versa. In one embodiment, streptavidin is presented as a tetramer on the four-fold axis of the core by genetic fusion or otherwise, and the biotinylated antigen is a monomer, dimer, or tetramer, allowing association to the core in a configuration appropriate for natural antigen multimerization. In some cases, a protein-based adapter is used to capture the antigenic protein. Optionally, the polypeptide is fused to a protein that can bind to a complementary protein that is fused to the antigenic protein.

[0116] The immune response to rabies G can be controlled by altering the positioning of the ectodomain relative to the core. Depending on how the antigenic protein is attached to the core of the VLP, the antigenic protein can be presented in various positions. In some embodiments, the antigenic protein is presented such that one or more known epitopes are positioned at or toward the distal end of the antigenic protein, making these epitopes preferentially accessible to the immune system. In some cases, the positioning replicates the positioning of viral proteins relative to the virus. Thus, in the case of rabies G protein, the antigenic protein (rabies G protein ectodomain) can be positioned so that the epitope identified in Marissen et al. (2005) J Virol. 79(8):4672-4678 is at the distal end of the protein. The positioning selection can direct the immune system to one or other epitope.

[0117] In some embodiments, epitope preference is controlled by other means, such as positioning of glycans on the VLP by addition or removal of the N-linked glycan sequence motif NX-[T / S] at predetermined positions within the amino acid sequence of any of the polypeptides of the VLP, including within the amino acid sequence of the antigenic protein.

[0118] In some cases, epitopes found at intermediate distances from the proximal to the distal end are preferred over more distally located epitopes, depending on various considerations, including, but not limited to, the overall shape of the VLP, surface hydrophobicity, surface charge, and competitive binding of proteins endogenously present in the subject or exogenously provided in the vaccine composition. The present disclosure encompasses all known methods for the rational design of protein structures, and the foregoing is not intended to be limiting.

[0119] VLP polypeptide sequence The polypeptides of the present disclosure can comprise a variety of amino acid sequences. U.S. Patent Publication No. US2015 / 0356240 A1 describes various methods for designing protein assemblers. As described in U.S. Patent Publication No. US2016 / 0122392 A1 and International Patent Publication No. WO2014 / 124301 A1, isolated polypeptides of SEQ ID NOs: 1-51 have been designed to self-assemble in pairs to form VLPs, such as icosahedral particles. Additional suitable VLPs are described in U.S. Patent Publication No. US2022 / 0072120 A1. This design method entailed designing suitable interface residues for each member of a polypeptide pair that can assemble to form a VLP. The VLPs thus formed contain symmetrically repeated, non-natural, non-covalent polypeptide-polypeptide interfaces that position the first and second assemblers into VLPs, such as those with icosahedral symmetry. Thus, in one embodiment, the first and second polypeptides (i.e., the two core polypeptides of the VLP) are selected from the group consisting of SEQ ID NOs: 1-51. In each case, the sequences do not include the N-terminal methionine residue that is present in the full-length protein but is typically removed to create fusions. In various embodiments, one or more additional residues are deleted from the N-terminus and / or additional residues are added to the N-terminus (e.g., to form a helical extension). [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6]

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

[0120] Table 3 shows the amino acid sequences of the first and second multimerization domains. In each case, the pair of sequences together form I53 multimers with icosahedral symmetry. The right-most column of Table 3 identifies the residue numbers in each representative polypeptide identified as being present at the interface of the resulting assembled virus-like particle (i.e., "Identified Interface Residues"). As can be seen from the table, the number of interface residues in the representative polypeptides of SEQ ID NOS: 1-34 ranges from 4 to 13. In various embodiments, the first polypeptide and the second polypeptide comprise an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 identified interface positions (depending on the number of interface residues in a given polypeptide) to the amino acid sequence of a polypeptide selected from the group consisting of SEQ ID NOs: 1-34. SEQ ID NOs: 35-51 represent other amino acid sequences of the multimerization domain. In other embodiments, the first polypeptide and / or second polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to the amino acid sequence of a polypeptide selected from the group consisting of SEQ ID NOs: 1-51, and at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an identified interface position.

[0121] As is true for proteins in general, polypeptides are expected to tolerate some variations in the designed sequence that do not interfere with subsequent assembly into virus-like particles, particularly when such variations involve conservative amino acid substitutions. As used herein, "conservative amino acid substitutions" means that hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) may only be substituted with other hydrophobic amino acids, hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) may only be substituted with other hydrophobic amino acids with bulky side chains, amino acids with positively charged side chains (Arg, His, Lys) may only be substituted with other amino acids with positively charged side chains, amino acids with negatively charged side chains (Asp, Glu) may only be substituted with other amino acids with negatively charged side chains, and amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) may only be substituted with other amino acids with polar, uncharged side chains.

[0122] In various embodiments of the VLPs of the invention, the first polypeptide and the second polypeptide (or vice versa) comprise polypeptides having an amino acid sequence selected from the following pairs or modified versions thereof (i.e., permissible modifications disclosed for the polypeptides of the invention: an isolated polypeptide comprising an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to the amino acid sequence set forth by a SEQ ID NO: and / or identical at at least one of the identified interface positions): SEQ ID NO: 1 and SEQ ID NO: 2 (I53-34A and I53-34B); SEQ ID NO:3 and SEQ ID NO:4 (I53-40A and I53-40B); SEQ ID NO:3 and SEQ ID NO:24 (I53-40A and I53-40B.1); SEQ ID NO:23 and SEQ ID NO:4 (I53-40A.1 and I53-40B); SEQ ID NO: 35 and SEQ ID NO: 36 (genus I53-40A and I53-40B); SEQ ID NO:5 and SEQ ID NO:6 (I53-47A and I53-47B); SEQ ID NO:5 and SEQ ID NO:27 (I53-47A and I53-47B.1); SEQ ID NO:5 and SEQ ID NO:28 (I53-47A and I53-47B.1NegT2); SEQ ID NO:25 and SEQ ID NO:6 (I53-47A.1 and I53-47B); SEQ ID NO:25 and SEQ ID NO:27 (I53-47A.1 and I53-47B.1); SEQ ID NO:25 and SEQ ID NO:28 (I53-47A.1 and I53-47B.1NegT2); SEQ ID NO:26 and SEQ ID NO:6 (I53-47A.1NegT2 and I53-47B); SEQ ID NO:26 and SEQ ID NO:27 (I53-47A.1NegT2 and I53-47B.1); SEQ ID NO: 26 and SEQ ID NO: 28 (I53-47A.1NegT2 and I53-47B.1NegT2); SEQ ID NO: 37 and SEQ ID NO: 38 (genus I53-47A and I53-47B); SEQ ID NO:7 and SEQ ID NO:8 (I53-50A and I53-50B); SEQ ID NO:7 and SEQ ID NO:32 (I53-50A and I53-50B.1); SEQ ID NO:7 and SEQ ID NO:33 (I53-50A and I53-50B.1NegT2); SEQ ID NO: 7 and SEQ ID NO: 34 (I53-50A and I53-50B.4PosT1); SEQ ID NO:29 and SEQ ID NO:8 (I53-50A.1 and I53-50B); SEQ ID NO:29 and SEQ ID NO:32 (I53-50A.1 and I53-50B.1); SEQ ID NO:29 and SEQ ID NO:33 (I53-50A.1 and I53-50B.1NegT2); SEQ ID NO:29 and SEQ ID NO:34 (I53-50A.1 and I53-50B.4PosT1); SEQ ID NO: 30 and SEQ ID NO: 8 (I53-50A.1NegT2 and I53-50B); SEQ ID NO: 30 and SEQ ID NO: 32 (I53-50A.1NegT2 and I53-50B.1); SEQ ID NO: 30 and SEQ ID NO: 33 (I53-50A.1NegT2 and I53-50B.1NegT2); SEQ ID NO: 30 and SEQ ID NO: 34 (I53-50A.1NegT2 and I53-50B.4PosT1); SEQ ID NO: 31 and SEQ ID NO: 8 (I53-50A.1PosT1 and I53-50B); SEQ ID NO: 31 and SEQ ID NO: 32 (I53-50A.1PosT1 and I53-50B.1); SEQ ID NO: 31 and SEQ ID NO: 33 (I53-50A.1PosT1 and I53-50B.1NegT2); SEQ ID NO: 31 and SEQ ID NO: 34 (I53-50A.1PosT1 and I53-50B.4PosT1); SEQ ID NO: 39 and SEQ ID NO: 40 (genus I53-50A and I53-50B); SEQ ID NO: 9 and SEQ ID NO: 10 (I53-51A and I53-51B); SEQ ID NO:11 and SEQ ID NO:12 (I52-03A and I52-03B); SEQ ID NO: 13 and SEQ ID NO: 14 (I52-32A and I52-32B); SEQ ID NO: 15 and SEQ ID NO: 16 (I52-33A and I52-33B) SEQ ID NO: 17 and SEQ ID NO: 18 (I32-06A and I32-06B); SEQ ID NO: 19 and SEQ ID NO: 20 (I32-19A and I32-19B); SEQ ID NO:21 and SEQ ID NO:22 (I32-28A and I32-28B); SEQ ID NO:23 and SEQ ID NO:24 (I53-40A.1 and I53-40B.1); SEQ ID NO: 41 and SEQ ID NO: 42 (T32-28A and T32-28B); SEQ ID NO: 43 and SEQ ID NO: 44 (T33-09A and T33-09B); SEQ ID NO: 45 and SEQ ID NO: 46 (T33-15A and T33-15B); SEQ ID NO: 47 and SEQ ID NO: 48 (T33-21A and T33-21B); SEQ ID NO: 49 and SEQ ID NO: 50 (T33-28A and T32-28B); and SEQ ID NO: 51 and SEQ ID NO: 44 (T33-31A and T33-09B (also referred to as T33-31B)).

[0123] In some embodiments, one or more rabies G protein ectodomains are expressed as a fusion protein with a first multimerization domain.

[0124] Non-limiting examples of designed protein complexes useful in the protein-based VLPs of the present disclosure include those disclosed in U.S. Patent No. US9,630,994, International Patent Publication No. WO2018187325A1, U.S. Patent Publication No. US2018 / 0137234A1, and U.S. Patent Publication No. US2019 / 0155988A2, each of which is incorporated herein by reference in its entirety.

[0125] In various embodiments of the VLPs of the present disclosure, the first multimerization domain and the second multimerization domain (or vice versa) comprise polypeptides having amino acid sequences selected from the following pairs or modified versions thereof (i.e., permissible modifications as disclosed for the polypeptides of the present invention: an isolated polypeptide comprising an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to the amino acid sequence set forth by a SEQ ID NO: and / or identical at at least one of the identified interface positions): SEQ ID NO: 68 and SEQ ID NO: 69 (T33-dn2A and T33-dn2B); SEQ ID NO: 70 and SEQ ID NO: 71 (T33-dn5A and T33-dn5B); SEQ ID NO: 72 and SEQ ID NO: 73 (T33-dn10A and T33-dn10B); or SEQ ID NO: 74 and SEQ ID NO: 75 (I53-dn5A and I53-dn5B).

[0126] In some embodiments, the polypeptide comprises a multimerization domain that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-dn5A (SEQ ID NO: 74).

[0127] For example, the second component may comprise I53-dn5A or a functional variant thereof.

[0128] In some embodiments, the antigen comprises a polypeptide that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-dn5B (SEQ ID NO: 75).

[0129] For example, the first component may comprise I53-dn5B or a functional variant thereof, optionally fused N-terminally to the rabies G protein ectodomain.

[0130] Various protein nanostructures are known in the art, for example, as described in U.S. Patent Publication Nos. US2015 / 0356240 A1, US2016 / 0122392 A1, US20180030429 A1, US20190341124 A1, and US2022 / 0072120 A1, the contents of which are incorporated herein by reference. In some embodiments, the protein nanostructure comprises, as an organizing domain, a variant of KDPG aldolase (Protein Data Bank code 1WA3) engineered to self-assemble into protein nanostructures. In its native form, 1WA3 non-covalently assembles to form trimers via a first interface (trimer interface). When 20 copies of the trimer (60 monomers) are computationally docked to form a one-component icosahedral protein nanostructure, five 1WA3 monomers contact each other through a second interface (the pentamer interface). By introducing amino acid substitutions, the pentamer interface can be stabilized, allowing the protein nanostructure to spontaneously self-assemble, for example, in expressing cells or when isolated trimers (or monomers) are mixed under suitable conditions.

[0131] In some embodiments, the organizing domain comprises an amino acid substitution that removes a cysteine ​​residue. In some embodiments, the organizing domain comprises a substitution of C76A and / or C100A with SEQ ID NOs: 175-181. In some embodiments, the organizing domain comprises a substitution of C76A, C100A, C165A, and / or C203A with SEQ ID NOs: 175-181. Exemplary organizer domain sequences are shown in Table 3. In each case, the N-terminal MK is optional and is not included in calculating sequence identity, but is shown for numbering purposes only. That is, the MK is included in the reference sequence but is not necessarily included in the organizing domain of the nanostructure.

[0132] In some embodiments, organizing domains trimerize with other organizing domains to form trimeric "components" of protein nanostructures.

[0133] In some embodiments, the organizing domain is a ferritin polypeptide. In some embodiments, the organizing domain of a ferritin protein nanostructure comprises a polypeptide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the following sequences:

[0134] MLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKVELIGNENHGLYLADQYVKGIAKSRKS (SEQ ID NO: 182).

[0135] MLKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYPTFNFLQWYVSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDAQN (SEQ ID NO: 183).

[0136] NFHQDCEAGLNRTVNLKFHSSYVYLSMASYFNRDDVALSNFAKFFRERSEEEKEHAEKLIEYQNQRGGRVFLQSVEKPERDDWANGLEALQTALKLQKSVNQALLDLHAVAADKSDPHMTDFLESPYLSESVETIKKLGDHITSLKKLWSSHPGMAEYLFNKHTLG (SEQ ID NO: 184).

[0137] QFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 185).

[0138] SGESQVRQNFKPEMEEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYPTFNFLQWYVSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDGS (SEQ ID NO: 186).

[0139] In some embodiments, the first and second components optionally comprise an additional trimerization tag (e.g., a FoldOn tag or a GCN4 trimer). In some embodiments, the linker sequence comprises a foldon, and the foldon sequence is GYIPEAPRDG QAYVRKDGEWVLLSTFL (SEQ ID NO: 58). In some embodiments, the linker can comprise a Gly-Ser linker (i.e., a linker consisting of glycine and serine residues) of any suitable length. In some embodiments, the Gly-Ser linker can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length.

[0140] In some embodiments, the VLP comprises a trimeric assembly of antigens comprising a first polypeptide comprising a first multimerization domain and a second polypeptide comprising a second multimerization domain. The first multimerization domain comprises a protein-protein interface that induces three copies of the first polypeptide to self-associate to form a trimeric component. In a VLP having two or more components, each copy of the first multimerization domain further comprises a surface-exposed interface that interacts with a complementary surface-exposed interface on the second multimerization domain. Similarly, the second multimerization domain is adapted to multimerize with the first multimerization domain (of the first polypeptide of the first component). As described by King et al. (Nature 510, 103-108, 2014), Bale et al. (Science 353, 389-394, 2016), and patent publications WO2014124301A1 and US20160122392A1, the complementary protein-protein interface between the first and second multimerization domains drives the assembly of multiple copies of the trimer-assembling domain and the second assembly domain into a target VLP. In some embodiments, each copy of the trimer-assembling domain of a VLP has an antigenic protein or antigenic fragment thereof linked thereto (e.g., as a gene fusion), and these VLPs display the full valency of the protein. In other embodiments, the VLPs of the present disclosure contain one or more copies of a first multimerization domain with an antigenic protein or antigenic fragment thereof (e.g., as a genetic fusion), as well as one or more first multimerization domains without the antigenic protein, and these VLPs display partial valency of G proteins. The first multimerization domain can be any polypeptide sequence that forms trimers and interacts with the second multimerization domain to drive assembly into a target VLP. In some embodiments, the VLPs comprise a first polypeptide and a second polypeptide selected from those disclosed in US20130274441A1, US2015 / 0356240A1, US2016 / 0122392A1, and WO2018 / 187325A1 (each of which is incorporated by reference in its entirety).

[0141] In some embodiments of the VLPs of the present disclosure, the antigenic protein and the core of the VLP may be genetically fused together so that they are both present in a single polypeptide. The bond between the protein and the core allows the antigenic protein or its antigenic fragment to be displayed on the outside of the VLP. Therefore, the attachment point to the core must be outside the core of the virus-like particle that is formed. A wide variety of polypeptide sequences can be used to link the protein or its antigenic fragment to the core of the virus-like particle. In some cases, the linker comprises a polypeptide sequence. Any suitable linker polypeptide may be used. In some embodiments, the linker imposes a strict relative positioning of the antigenic protein (e.g., ectodomain) or its antigenic fragment relative to the core. In some embodiments, the linker allows for flexible linking of the antigenic protein (e.g., ectodomain) or its antigenic fragment relative to the core. In some embodiments, the linker comprises an additional trimerization domain (e.g., the foldon domain of T4 fibritin) to help stabilize the trimeric form of the G protein, e.g., GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 58) or a functional variant thereof.

[0142] In some embodiments, the linker can comprise a Gly-Ser linker of any suitable length (i.e., a linker composed of glycine and serine residues). In some embodiments, the Gly-Ser linker can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length. In some embodiments, the Gly-Ser linker can comprise or consist of the amino acid sequence GSGGSGSGSGGSGSG (SEQ ID NO:59), GGSGGSGS (SEQ ID NO:60), or GSGGSGSG (SEQ ID NO:61). In some embodiments, the linker comprises the sequence GSGSGSG (SEQ ID NO:62). In some embodiments, the linker comprises the sequence GSGSGSGSGSGSGSSG (SEQ ID NO:63).

[0143] Exemplary fusion proteins containing I53-50A are shown in Table 4. In each case, the N-terminal signal peptide or C-terminal purification tag can be replaced with known alternatives. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9]

[0144] In some embodiments, the fusion protein shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a polypeptide sequence selected from SEQ ID NOs: 82, 83, 85, 87, 89, 91, 93-96.

[0145] Exemplary fusion proteins containing I53-dn5B are shown in Table 5. In each case, the N-terminal signal peptide or C-terminal purification tag can be replaced with known alternatives. [Table 5-1] [Table 5-2] [Table 5-3]

[0146] In some embodiments, the fusion protein shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a polypeptide sequence selected from SEQ ID NOs: 97, 99, 101, 102.

[0147] Exemplary fusion proteins containing ferritin are shown in Table 6. The N-terminal signal peptide or C-terminal purification tag can be replaced with known alternatives. [Table 6]

[0148] In some embodiments, the fusion protein shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a polypeptide sequence selected from SEQ ID NO: 103.

[0149] Assembly of VLPs In some embodiments, a single component self-assembles into a VLP. In some embodiments, one or more purified samples of a first component and a second component for use in forming a VLP are mixed in an approximately equimolar molar ratio under aqueous conditions (e.g., I53-50A / B icosahedral VLP). The first and second components interact with each other (via the multimerization domain, and optionally via the ectodomain) to drive assembly of the target VLP. Successful assembly of the target VLP can be confirmed by analyzing the in vitro assembly reaction using common biochemical or biophysical methods used to assess the physical size of proteins or protein aggregates, including, but not limited to, size-exclusion chromatography, native (non-denaturing) gel electrophoresis, dynamic light scattering, multi-angle light scattering, analytical ultracentrifugation, negative stain electron microscopy, cryo-electron microscopy, or X-ray crystallography. If necessary, assembled VLPs can be purified from other species or molecules present during in vitro assembly reactions using preparative techniques commonly used to isolate proteins by their physical size, including, but not limited to, size-exclusion chromatography, preparative ultracentrifugation, tangential flow filtration, or preparative gel electrophoresis. The presence of antigenic proteins in VLPs can be assessed by commonly used techniques for determining the identity of protein molecules in aqueous solution, including, but not limited to, SDS-PAGE, mass spectrometry, protein sequencing, ELISA, surface plasmon resonance, biolayer interferometry, or amino acid analysis. The accessibility of proteins on the outside of the particles, and their conformation or antigenicity, can be assessed by commonly used techniques for detecting the presence and conformation of antigens, including, but not limited to, monoclonal antibody binding, conformation-specific monoclonal antibodies, surface plasmon resonance, biolayer interferometry, or antigen-specific antisera.

[0150] In various embodiments, the VLPs of the present disclosure comprise two or more distinct first polypeptides carrying different antigenic proteins as genetic fusions. These VLPs co-present multiple different proteins on the same VLP. These multi-antigen VLPs are produced by in vitro assembly using a mixture of two or more antigens, each containing a multimerization domain. The proportion of each antigen in the mixture determines the average valency of each antigenic protein in the resulting VLP. The presence and average valency of each antigen in a given sample can be assessed by quantitative analysis using the techniques described above to assess the presence of antigenic proteins in full-valency VLPs.

[0151] In various embodiments, the VLPs are about 20 nanometers (nm) to about 40 nm in diameter, with a lumen about 15 nm to about 32 nm in diameter, and pores within the protein shell measuring about 1 nm to about 14 nm in their longest dimension.

[0152] In some embodiments, the VLP has icosahedral symmetry. In such embodiments, the VLP may comprise 60 copies of the first component and 60 copies of the second component. In one such embodiment, the number of identical first polypeptides in each first assembly is different from the number of identical first polypeptides in each second assembly. For example, in some embodiments, the VLP comprises 12 first assembly members and 20 second assembly members. In such embodiments, each first assembly member may comprise, for example, 5 copies of the same first component and each second assembly member may comprise, for example, 3 copies of the same second component. In some embodiments, the VLP comprises 12 first assembly members and 30 second assembly members. In such embodiments, each first assembly member may comprise, for example, 5 copies of the same first component and each second assembly member may comprise, for example, 2 copies of the same second component. In a further embodiment, the VLP comprises 20 first assemblers and 30 second assemblers, where each first assembler may comprise, for example, three copies of the same first component, and each second assembler may comprise, for example, two copies of the same second component, all of which are capable of forming protein-based VLPs with icosahedral symmetry.

[0153] In various further embodiments, the oligomeric state of the first and second multimerization domains is as follows: I53-34A: trimer + I53-34B: pentamer, I53-40A: pentamer + I53-40B: trimer, I53-47A: trimer + I53-47B: pentamer, I53-50A: trimer + I53-50B: pentamer, I53-51A: trimer + I53-51B: pentamer, I32-06A: dimer + I32-06B: trimer, I32-19A: trimer + I32-19B: dimer, I32-28A: trimer + I32-28B: dimer, I52-03A: pentamer + I52-03B: dimer, I52-32A: dimer + I52-32B: pentamer, and I52-33A: Pentamer + I52-33B: Dimer.

[0154] In some embodiments, the first multimerization domain (of the first polypeptide) comprises a sequence that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A or a variant thereof: [ka]

[0155] In some embodiments, the second multimerization domain (of the second polypeptide) shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 7 or an antigenic fragment thereof.

[0156] The I53-50A protein sequence has two intramonomer disulfide bonds. In some embodiments, the cysteine ​​residues are mutated to residues that do not contain thiol groups (e.g., alanine or serine). Removal of the thiol groups can promote correct protein folding without impairing multimerization. In some embodiments, the multimerization domain of the first polypeptide comprises amino acid substitutions at one or more of positions 74, 97, 163, and 201 relative to SEQ ID NO: 7, as shown herein. [ka]

[0157] In some embodiments, the multimerization domain of the first polypeptide comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 86: C74A, C97A, C163A, and C201A. In some embodiments, the multimerization domain of the first polypeptide comprises SEQ ID NO: 86 or a variant thereof. [ka]

[0158] In some embodiments, the multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:7 or SEQ ID NO:86, or an antigenic fragment thereof, and comprises one, two, three, or four amino acid substitutions selected from C74A, C97A, C163A, and C201A. Alternatively, the substitutions can be of C to any amino acid other than A, T, S, L, I, or C.

[0159] The polypeptides provided herein may contain one or more conservative amino acid substitutions. The term "conservative amino acid substitution" is well known in the art and refers to replacing a particular amino acid with one that has similar properties (e.g., similar charge or hydrophobicity). Conservative mutations may include, but are not limited to, substitutions of amino acid residues that have similar charge or hydrophobicity but different size or bulk (e.g., providing cavity-filling function). A non-limiting list of representative conservative amino acid substitutions is shown in the table below. [Table B]

[0160] Alternatively, non-conservative amino acid substitutions may be preferred. For example, eliminating flexible portions in the secondary structure of native rabies G protein can be achieved by adding cysteine ​​residues (or vice versa). A "non-conservative substitution" refers to the substitution of one class of amino acid for another class of amino acid, e.g., the substitution of Ala with Asp, Asn, Glu, or Gln. Further non-limiting examples of non-conservative substitutions include the substitution of non-polar (hydrophobic) amino acid residues such as isoleucine, valine, leucine, alanine, and methionine with polar (hydrophilic) residues such as cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of polar residues with non-polar residues. Substitution of D-Cys with D-Ala, D-Ser, or D-Tyr (or another residue) can be used to eliminate intramolecular disulfide bonds, which may improve protein stability or expression in some cases. Substitution with D-Cys can be used to create disulfide bonds to stabilize proteins or to lock proteins into desired conformations.

[0161] In some embodiments, the nanoparticles comprising the polypeptide comprise a multimerization domain. In some embodiments, the nanoparticles comprising a multimerization domain are trimerization domains. In some embodiments, the nanoparticles comprising a multimerization domain share at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with FoldOn (SEQ ID NO: 58). In some embodiments, a nanoparticle comprising a multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a polypeptide sequence selected from SEQ ID NOs: 1, 4, 5, 7, 9, 18, 19, 21, 24, 25, 26, 29, 30, 31, 34, 36, 37, 39, 42, 43, 44, 45, 46, 47, 48, 49, 50, and 51, and conserved interface residues identified in Table 3. In some embodiments, the nanoparticles comprising a multimerization domain share at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 7). In some embodiments, the nanoparticles comprising a multimerization domain are I53-50A-Δcys (SEQ ID NO: 67). In some embodiments, the nanoparticles comprising a polypeptide share at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 93-96.In some embodiments, the nanoparticles comprising the multimerization domain share at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-dn5B (SEQ ID NO: 75). In some embodiments, the nanoparticles comprising the polypeptide share at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 101 or SEQ ID NO: 102. In some embodiments, the nanoparticles comprising the multimerization domain are ferritin polypeptides capable of forming ferritin particles. In some embodiments, the nanoparticles comprising the polypeptide share at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the construct F-ferritin (SEQ ID NO: 103). In some embodiments, the nanoparticles comprising the multimerization domain are particle-forming domains.

[0162] In another aspect, the present disclosure provides a nanoparticle comprising a polypeptide according to any embodiment, or a nanoparticle comprising a rabies G protein ectodomain, hi some embodiments, the polypeptide comprises a rabies G protein ectodomain according to any embodiment.

[0163] In some embodiments of the nanoparticle, the nanoparticle is a protein-based virus-like particle (pbVLP) or nanostructure.

[0164] In some embodiments of the nanoparticles, the nanoparticles are free of a lipid component. In some embodiments of the nanoparticles, the nanoparticles comprise a lipid component. In some embodiments of the nanoparticles, the nanoparticles comprise a second polypeptide component.

[0165] In some embodiments of the nanoparticles, the nanoparticles comprise a second polypeptide component and a first polypeptide component, and the nanoparticles are self-assembling nanoparticles comprising the first and second polypeptide components symmetrically arranged with point group symmetry.

[0166] In some embodiments of the nanoparticle, (a) a first polypeptide component comprises any of the polypeptides of the present disclosure, and the first polypeptide component forms a first homomeric complex via a multimerization domain of the polypeptide; (b) a second polypeptide component comprises a second multimerization domain, and the second polypeptide component forms a second homomeric complex via the second multimerization domain of the polypeptide; (c) the first homomeric complex and the second homomeric complex assemble to form a nanoparticle with point group symmetry; (d) the nanoparticle is devoid of other polypeptide components; and / or (e) the nanoparticle is devoid of a lipid component.

[0167] In some embodiments of the nanoparticles, the nanoparticles have icosahedral symmetry.

[0168] In some embodiments of the nanoparticles, the first multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 7) or I53-50AΔCys (SEQ ID NO: 67), and the second multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50B (SEQ ID NO: 8) or I53-50B.4PosT1 (SEQ ID NO: 34).

[0169] In some embodiments of the nanoparticle, the first multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-dn5B (SEQ ID NO: 75) and the second multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-dn5A (SEQ ID NO: 74).

[0170] In some embodiments of the nanoparticles, the first polypeptide component shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a polypeptide sequence selected from SEQ ID NOs: 93-96, and the second polypeptide component shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50B (SEQ ID NO: 8) or I53-50B.4PosT1 (SEQ ID NO: 34).

[0171] In some embodiments of the nanoparticles, the first polypeptide component shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a polypeptide sequence selected from SEQ ID NOs: 101-102, and the second polypeptide component shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-dn5A (SEQ ID NO: 74).

[0172] Delivery Vehicle In some embodiments, a polynucleotide (e.g., mRNA) encoding a protein nanostructure comprising components including viral protein monomers of a trimeric viral antigen is formulated in a delivery vehicle. In some embodiments, the delivery vehicle is a non-viral vector. In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP). In some embodiments, the delivery vehicle is a liposome. In some embodiments, the delivery vehicle is a polymeric non-viral vector such as spermine, polyethyleneimine, chitosan, or polyurethane. In some embodiments, the delivery vehicle is a polymeric delivery system such as polyamidoamine (PAA), polybeta-aminoester (PBAE), or polyethyleneimine (PEI). In some embodiments, the delivery vehicle is a ferritin nanoparticle. In some embodiments, the delivery vehicle is an encapsulin.

[0173] In some embodiments, a polynucleotide (e.g., mRNA) encoding a protein nanostructure comprising components including viral protein monomers of a trimeric viral antigen is formulated in a nanoparticle. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the polynucleotide is formulated in a lipid-polycation complex called a cationic LNP. By way of non-limiting example, the polycation can include a cationic peptide or polypeptide, such as, but not limited to, polylysine, polyornithine, and / or polyarginine. In some embodiments, the polynucleotide is formulated in an LNP containing a non-cationic lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0174] In various embodiments, the lipid nanoparticles have an average diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In various embodiments, the LNPs are substantially non-toxic. In certain embodiments, the polynucleotides, when present in the LNPs, are resistant in aqueous solution to degradation by nucleases.Polynucleotide-containing lipids and LNPs and methods for their preparation are described in, for example, U.S. Patent Nos. 8,569,256, 5,965,542, and U.S. Patent Publication Nos. 2016 / 0199485, 2016 / 0009637, 2015 / 0273068, 2015 / 0265708, 2015 / 0203446, 2015 / 0005363, 2014 / 0308304, 2014 / 0200257, 2013 / 086373, 2013 / 0338210, 2013 / 0338211, 2013 / 0338221, 2013 / 0338231, 2013 / 0338241, 2013 / 0338252, 2013 / 0338261, 2013 / 0338272, 2013 / 0338281, 2013 / 0338291, 2013 / 0338292, 2013 / 0338293, 2013 / 0338294, 2013 / 0338295, 2013 / 0338296, 2013 / 0338297, 2013 / 0338298, 2013 / 033829 ... / 0323269, 2013 / 0245107, 2013 / 0195920, 2013 / 0123338, No. 2013 / 0022649, No. 2013 / 0017223, No. 2012 / 0295832, No. 2012 / 018358 No. 1, No. 2012 / 0172411, No. 2012 / 0027803, No. 2012 / 0058188, No. 2011 / 03 No. 11583, No. 2011 / 0311582, No. 2011 / 0262527, No. 2011 / 0216622, No. 201 1 / 0117125, 2011 / 0091525, 2011 / 0076335, 2011 / 0060032, Same No. 2010 / 0130588, No. 2007 / 0042031, No. 2006 / 0240093, No. 2006 / 00837 No. 80, No. 2006 / 0008910, No. 2005 / 0175682, No. 2005 / 017054, No. 2005 / 01 No. 18253, No. 2005 / 0064595, No. 2004 / 0142025, No. 2007 / 0042031, No. 199 and PCT Publication Nos. WO99 / 39741, WO2017 / 004143, WO2017 / 075531, WO2015 / 199952, WO2014 / 008334, WO2013 / 086373, WO2013 / 086322, WO2013 / 016058, WO2013 / 086373, WO2011 / 141705, and WO2001 / 07548, the contents of which are incorporated herein by reference.

[0175] Additional exemplary lipids and LNPs and their production are described in the art, e.g., in U.S. Patent Application No. US2012 / 0276209; Semple et al., 2010, Nat Biotechnol., 28(2):172-176; Akinc et al., 2010, Mol Ther., 18(7):1357-1364; Basha et al., 2011, Mol Ther, 19(12):2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34):18440-18450; Lee et al., 2012, Int J Cancer., 131(5):E781-90; Belliveau et al., 2012, Mol Ther Nucleic Acids, 1:e37; Jayaraman et al. al., 2012, Angew Chem Int Ed Engl., 51(34):8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids.2, e139; Maier et al., 2013, Mol Ther., 21(8):1570-1578; and Tam et al., 2013, Nanomedicine, 9(5):665-74, each of which is incorporated herein by reference. Lipids and their production can be found, for example, in U.S. Publication Nos. 2015 / 0376115 and 2016 / 0376224, the contents of which are incorporated herein by reference.

[0176] Nucleic acids, vectors, and cells In another aspect, the present disclosure provides isolated nucleic acids encoding the antigens, first components, and / or second components of the present disclosure. Isolated nucleic acid sequences can comprise RNA or DNA. As used herein, an "isolated nucleic acid" is a nucleic acid that is removed from its normal surrounding nucleic acid sequences in a genome or cDNA sequence. Such isolated nucleic acid sequences can contain additional sequences useful for facilitating expression and / or purification of the encoded protein, including, but not limited to, polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, transport signals, secretion signals, nuclear localization signals, and plasma membrane localization signals. It will be clear to one of skill in the art based on the teachings herein which nucleic acid sequences encode the proteins of the present disclosure.

[0177] In a further aspect, the present disclosure provides a recombinant expression vector comprising an isolated nucleic acid of any embodiment or combination of embodiments of the present disclosure operably linked to a suitable control sequence. An "expression vector" includes a vector in which a nucleic acid coding region or gene is operably linked to any control sequence capable of effecting expression of the gene product. A "control sequence" operably linked to a nucleic acid sequence of the present disclosure is a nucleic acid sequence capable of effecting expression of a nucleic acid molecule. Control sequences need not be contiguous with the nucleic acid sequence, so long as they function to direct expression of the nucleic acid sequence. Thus, for example, non-translated but transcribed intervening sequences may be present between the promoter sequence and the nucleic acid sequence, and the promoter sequence would still be considered "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type known in the art, including, but not limited to, plasmid and viral-based expression vectors. The control sequences used to drive expression of the nucleic acid sequences of the present disclosure in mammalian systems can be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of several inducible promoters, including but not limited to, tetracycline, ecdysone, steroid responsive). The construction of expression vectors for use in transfecting cells is also well known in the art and can thus be accomplished through standard techniques.(See, e.g., Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, NJ; and Ambion 1998 Catalog (Ambion, Austin, TX).) Expression vectors must be replicable in the host organisms either as episomes or by integration into the host chromosomal DNA.

[0178] In another aspect, the present disclosure provides a host cell transfected or transduced with a recombinant expression vector disclosed herein, which may be either a prokaryotic or eukaryotic cell. The cell may be transiently or stably transfected or transduced. Transfection or transduction of the expression vector into such prokaryotic and eukaryotic cells may be accomplished by any technique known in the art, including, but not limited to, standard bacterial transformation, calcium phosphate co-precipitation, electroporation, or liposome-, DEAE-dextran-, polycation-, or viral-mediated transfection. (See, e.g., Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press); Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (RI Freshney, 1987, Liss, Inc., New York, NY)).

[0179] In another aspect, the present disclosure provides methods for producing an antigen, component, or VLP according to the present disclosure. In some embodiments, the method comprises (a) culturing a host according to this aspect of the disclosure under conditions conducive to expression of the polypeptide, and (b) optionally recovering the expressed polypeptide.

[0180] In some embodiments, the present disclosure provides methods of producing a vaccine, comprising culturing host cells comprising a polynucleotide comprising a sequence encoding an antigen of the present disclosure in a culture medium such that the host cells secrete the antigen into the culture medium; optionally purifying the antigen from the culture medium; mixing the antigen with a second component, where the second component multimerizes with the antigen to form a VLP; and optionally purifying the VLP.

[0181] In some embodiments, the present disclosure provides a method of producing a vaccine, comprising culturing a host cell comprising one or more polynucleotides comprising sequences encoding both components of any one of the VLPs of the present disclosure, such that the host cell secretes the first component and the second component into the culture medium; and optionally purifying the VLP from the culture medium.

[0182] Exemplary host cells include E. coli cells, 293 and 293F cells, HEK293 cells, Sf9 cells, Chinese hamster ovary (CHO) cells, and any other cell line used in the production of recombinant proteins.

[0183] In various embodiments, the first component is expressed in a manufacturing method according to the present disclosure (e.g., in 293F or CHO cells grown in suspension) at about 0.5 mg / L, about 1.0 mg / L, about 2.5 mg / L, about 5 mg / L, about 10 mg / L, about 25 mg / L, about 50 mg / L, about 250 mg / L, about 500 mg / L, about 1000 mg / L, or more. In various embodiments, the first component expresses an expression level of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the rabies G protein (optionally the same ectodomain as that of the VLP) in the same or a similar expression system. In various embodiments, the first component expresses an expression level of rabies G protein (optionally the same ectodomain as that of the VLP) of at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 150%, at least 175%, or at least 200% in the same or similar expression system.

[0184] In some embodiments, the rabies G protein ectodomain of the first component is in the pre-fusion conformation, or a substantial proportion of the rabies G protein ectodomain is in the pre-fusion conformation. In various embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the rabies G protein ectodomain of the first component is in the pre-fusion conformation. In some embodiments, the rabies G protein ectodomain of the VLP is in the pre-fusion conformation, or a substantial proportion of the rabies G protein ectodomain is in the pre-fusion conformation. In various embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the rabies G protein ectodomain of the VLP is in the pre-fusion conformation.

[0185] In some embodiments, the proportion of rabies G protein ectodomain in the pre-fusion state is determined by binding to conformation-specific antibodies (e.g., D1-25 and 1112-1). In some embodiments, the proportion of rabies G protein ectodomain in the first component in the pre-fusion conformation is at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 150%, at least 175%, or at least 200% higher than the proportion in a reference component or the proportion in a reference protein that is not linked to a multimerization domain. In some embodiments, the proportion of rabies G protein ectodomain in the first component in the pre-fusion conformation is at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 150%, at least 175%, or at least 200% higher than the proportion in a VLP (e.g., a micellar VLP).

[0186] In another aspect, the present disclosure provides a polynucleotide comprising a polynucleotide sequence encoding any of the polypeptides or nanoparticles of the present disclosure.

[0187] In some embodiments, the polynucleotide encoding the fusion protein shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NOs: 84, 86, 88, 90, 92, 98, 100.

[0188] In some embodiments, the polynucleotide is from about 500 nucleotides to about 10,000 nucleotides. In some embodiments, the polynucleotide is from about 1,000 nucleotides to about 10,000 nucleotides. In some embodiments, the polynucleotide is from about 1,000 nucleotides to about 9,000 nucleotides. In some embodiments, the polynucleotide is from about 1,000 nucleotides to about 8,000 nucleotides. In some embodiments, the polynucleotide is from about 1,000 nucleotides to about 7,000 nucleotides. In some embodiments, the polynucleotide is from about 1,000 nucleotides to about 6,000 nucleotides. In some embodiments, the polynucleotide is from about 1,000 nucleotides to about 5,000 nucleotides. In some embodiments, the polynucleotide is from about 1,000 nucleotides to about 4,000 nucleotides. In some embodiments, the polynucleotide is from about 1,000 nucleotides to about 3,000 nucleotides. In some embodiments, the polynucleotide is from about 1,000 nucleotides to about 2,000 nucleotides. In some embodiments, the polynucleotide is from about 1,000 nucleotides to about 1,500 nucleotides. In some embodiments, the polynucleotide is from about 1000 nucleotides to about 1400 nucleotides. In some embodiments, the polynucleotide is from about 1000 nucleotides to about 1300 nucleotides. In some embodiments, the polynucleotide is from about 1000 nucleotides to about 1200 nucleotides. In some embodiments, the polynucleotide is from about 1000 nucleotides to about 1100 nucleotides. In some embodiments, the polynucleotide is from about 1100 nucleotides to about 1900 nucleotides.

[0189] In another aspect, the present disclosure provides a vector comprising a polynucleotide comprising a polynucleotide sequence encoding any of the polypeptides or nanoparticles of the present disclosure.

[0190] In another aspect, the present disclosure provides a kit comprising a polypeptide, nanoparticle, polynucleotide, vector, or pharmaceutical composition of the present disclosure.

[0191] Vaccines and Administration The present disclosure also provides vaccines comprising the VLPs described herein. Such compositions can be used to raise antibodies in mammals (e.g., humans). Vaccine compositions of the present disclosure typically include a pharmaceutically acceptable carrier, a detailed description of such carriers is available in Remington: The Science and Practice of Pharmacy.

[0192] The pH of the composition is usually about 4.5 to about 11, about 5 to about 11, about 5.5 to about 11, about 6 to about 11, about 5 to about 10.5, about 5.5 to about 10.5, about 6 to about 10.5, about 5 to about 10, about 5.5 to about 10, about 6 to about 10, about 5 to about 9.5, about 5.5 to about 9.5, about 6 to about 9.5, about 5 to about 9, about 5.5 to about 9, about 6 to about 9, about 5 to about 8.5, about 5.5 to about 8.5, about 6 to about 8.5, about 5 to about 8, about 5.5 to about 8, about 6 to about 8, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, etc. A stable pH can be maintained through the use of a buffer such as a Tris buffer, a citrate buffer, a phosphate buffer, or a histidine buffer. Thus, the composition generally includes a buffer.

[0193] The composition may be sterile and / or pyrogen-free. The composition may be isotonic with respect to a human standard.

[0194] The pharmaceutical composition may contain an effective amount of the antigen polypeptide. An "effective amount" is an amount effective to induce an antibody response to the antigen when administered to a subject. This amount may vary depending on the health and physical condition of the individual being treated, their age, the capacity of their immune system to synthesize antibodies, the degree of protection desired, the vaccine formulation, the attending physician's evaluation of the medical situation, and other relevant factors. It is expected that the amount will fall within a relatively broad range that can be determined through routine testing. The antigen content of the compositions of the present disclosure is generally expressed in terms of mass of protein per dose. Doses of 10 to 500 μg (e.g., 50 μg) per antigen may be useful.

[0195] In another aspect, the present disclosure provides a pharmaceutical composition for use as a vaccine, comprising any of the polypeptides or nanoparticles of the present disclosure, and optionally one or more pharmaceutically acceptable excipients. In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises at least one adjuvant. In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises an oil-in-water emulsion. In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises a squalene-based oil-in-water emulsion. In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises a toll-like receptor (TLR) immunostimulator. In some embodiments of the pharmaceutical composition, the pharmaceutical composition comprises squalene, SLA, GLA, R848, IMQ, 3M-052, CpG, saponin (QS21), or a combination thereof.

[0196] The antigenic polypeptide or nanoparticle may be the only active agent in the composition (e.g., formulated as an aqueous vaccine), or the composition may further include one or more other agents suitable for the intended use, including, but not limited to, adjuvants to generally stimulate the immune system and improve the overall immune response.

[0197] The vaccine composition may contain an immunological adjuvant. Representative adjuvants include, but are not limited to, the following: 1. mineral-containing compositions, 2. oil emulsions, 3. saponin formulations, 4. virosomes and virus-like particles, 5. bacterial or microbial derivatives, 6. bioadhesives and mucoadhesives, 7. liposomes, 8. polyoxyethylene ether and polyoxyethylene ester formulations, 9. polyphosphazenes (PCPPs), 10. muramyl peptides, 11. imidazoquinolone compounds, 12. thiosemicarbazone compounds, 13. tryptanthrin compounds, 14. human immunomodulators, 15. lipopeptides, 16. benzonaphthyridines, 17. microparticles, and 18. immunostimulatory polynucleotides (such as RNA or DNA; e.g., CPG-containing oligonucleotides).

[0198] In some embodiments, the adjuvant is an oil-in-water emulsion, such as a squalene-based oil-in-water emulsion, or an aluminum hydroxide adjuvant. In some embodiments, the adjuvant comprises Allhydrogel. For example, the composition can include an aluminum salt adjuvant or an oil-in-water emulsion.

[0199] For example, the composition may include an aluminum salt adjuvant, an oil-in-water emulsion (e.g., an oil-in-water emulsion containing squalene, such as MF59, SWE, or AS03), a TLR9 agonist (e.g., a CpG oligodeoxynucleotide), a TLR7 agonist (e.g., an imidazoquinoline or imiquimod), or a combination thereof. In some embodiments, the adjuvant is a combination of an aluminum salt and CPG1018. Suitable aluminum salts include hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates), aluminum (see, e.g., Vaccine Design. (1995) eds. Powell & Newman, Chapters 8 and 9, ISBN: 030644867X. Plenum), or mixtures thereof. The salt can be in any suitable form (e.g., gel, crystalline, amorphous, etc.), one example being adsorption of an antigen to the salt. The Al salt in the composition for administration to a patient may be used in combination with an aluminum salt. +++ The concentration of Al may be less than 5 mg / ml, e.g., <4 mg / ml, <3 mg / ml, <2 mg / ml, <1 mg / ml, etc. In some embodiments, Al +++ The ranges are 0.3-1 mg / ml, 0.3-2 mg / ml, 0.3-3 mg / ml, or 0.3-4 mg / ml. In some embodiments, a maximum of 0.85 mg / dose is used. Aluminum hydroxide and aluminum phosphate adjuvants are suitable for use in the disclosed invention. In a preferred embodiment, the pharmaceutical compositions provided herein comprise aluminum hydroxide as an adjuvant. In some embodiments, the pharmaceutical compositions provided herein comprise 500 μg of aluminum hydroxide.

[0200] Representative adjuvants include 3M-052, Adju-Phos™, Alhydrogel™, Adjumer™, albumin-heparin microparticles, algal glucan, algammulin, alum, antigen preparations, AS-2 adjuvant, ASO1, ASO3, autologous dendritic cells, autologous PBMCs, Avridine™, B7-2, BAK, BAY R1005, and BECC. TLR-4 agonist, bupivacaine, bupivacaine-HCl, BWZL, calcitriol, calcium phosphate gel, CCR5 peptide, CFA, cholera holotoxin (CT) and cholera toxin B subunit (CTB), cholera toxin A1-subunit-protein AD fragment fusion protein, CpG, CPG-1018, CRL1005, cytokine-containing liposomes, D-murapalmitin, DDA, DHEA, diphtheria toxoid, DL-PGL, DMPC, DMPG, DOC / alum complex, fowlpox, Freund's complete adjuvant, gamma inulin, Gerbu adjuvant, GM-CSF, GMDP, hGM-CSF, hIL-12 (N222L), hTNF-alpha, IFA, IFN-gamma in pcDNA3, IL-12 DNA, IL-12 plasmid, IL-12 / GMCSF plasmid (Sykes), IL-2 in pcDNA3, IL-2 / Ig plasmid, IL-2 / Ig protein, IL-4, IL-4 in pcDNA3, Imiquimod™, ImmTher™, immunoliposomes containing antibodies to costimulatory molecules, interferon-gamma, interleukin-1 beta, interleukin-12, interleukin-2, interleukin-7, ISCOM(s)™, Iscoprep 7.0.3™, keyhole limpet hemocyanin, lipid-based adjuvant, liposome, loxoribine, LT(R192G), LT-OA or LT oral adjuvant, LT-R192G, LTK63, LTK72, Matrix-M™ adjuvant, MF59, MONTANIDE ISA 51, MONTANIDE ISA 720, MPL™, MPL-SE, MTP-PE, MTP-PE liposomes, muramethide, murapalmitin, NAGO, nCT native cholera toxin, non-ionic surfactant vesicles, non-toxic mutant E112K of cholera toxin mCT-E112K, p-hydroxybenzoic acid methyl ester, pCIL-10, pCIL12, pCMVmCAT1, pCMVN, Peptomer-NP, Pleuran, PLG, PLGA, PGA, and PLA, Pluronic® L121, PMMA, PODDS™, poly rA:poly rU, polysorbate 80, protein cochleate, QS-21, Quadri A saponin, Quil-A, Rehydragel HPA, Rehydragel Adjuvants include, but are not limited to, LV, RIBI, Ribi-like adjuvant systems (MPL, TMD, CWS), S-28463, SAF-1, Sclavo peptides, Sendai proteoliposomes, Sendai-containing lipid matrices, Span® 85, Specol, squalane 1, squalene 2, stearyl tyrosine, SWE, tetanus toxoid (TT), Theramide™, threonyl muramyl dipeptide (TMDP), Ty particles, and Walter Reed liposomes. The choice of adjuvant depends on the subject being treated. Preferably, a pharmaceutically acceptable adjuvant is used. In a preferred embodiment, the adjuvant is aluminum hydroxide gel (e.g., Alhydrogel™). In a preferred embodiment, the adjuvant is SWE. In a preferred embodiment, the adjuvant is MF59.

[0201] In some embodiments, the adjuvant is a squalene emulsion.

[0202] In some embodiments, the adjuvant is a TLR4 immunostimulator (e.g., SLA, GLA), for example, as described in Van Hoeven at al. PLoS One. 11(2): e0149610 (2016).

[0203] In some embodiments, the adjuvant is a TLR7 / 8 immunostimulant (e.g., R848, IMQ, 3M-052), e.g., as described in Dowling D. ImmunoHorizons(6):185-197(2018).

[0204] In some embodiments, the adjuvant is a TLR9 immunostimulator (CpG), for example, as described in Bode et al. Expert Rev Vaccines. 10(4):499-511 (2011).

[0205] In some embodiments, the adjuvant is a saponin (QS21), e.g., as described in Zhu et al. Nat Prod Chem Res. 3(4):e113 (2016).

[0206] In some embodiments, the vaccine comprises a combination of two or more adjuvants (eg, a squalene emulsion and alum or a TLR4 immunostimulatory agent).

[0207] One suitable immunological adjuvant comprises a compound of formula (I), as defined in WO 2011 / 027222, or a pharmaceutically acceptable salt thereof, adsorbed to an aluminium salt. Many additional adjuvants can be used, including those disclosed in Powell & Newman (1995).

[0208] The compositions may contain antibacterial agents, particularly when packaged in multi-dose formats. Antibacterial agents such as thiomersal and 2-phenoxyethanol are commonly found in vaccines, although it may sometimes be desirable to use mercury-free preservatives, or no preservatives at all.

[0209] The composition may include a surfactant, such as a polysorbate, for example, polysorbate 80. The surfactant is generally present at low levels, for example, less than 0.01%.

[0210] The composition may include a sodium salt (e.g., sodium chloride) to provide tonicity. A concentration of 10±2 mg / ml, e.g., about 9 mg / ml NaCl is typical.

[0211] In some embodiments, the buffer in the vaccine composition is Tris buffer, histidine buffer, phosphate buffer, citrate buffer, or acetate buffer. The composition may also include a lyoprotectant, such as sucrose, sorbitol, or trehalose. In certain embodiments, the composition includes a preservative, such as benzalkonium chloride, benzethonium, chlorhexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the composition includes a bulking agent, such as glycine. In still other embodiments, the composition comprises a surfactant, such as, for example, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, poloxamer 188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, or a combination thereof. The composition may also comprise a tonicity adjuster, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood. Exemplary tonicity adjusters include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the composition further comprises a stabilizer, e.g., a molecule that substantially prevents or reduces chemical and / or physical instability of the VLP in lyophilized or liquid form. Representative stabilizers include, but are not limited to, sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0212] In some embodiments, the present disclosure provides pharmaceutical compositions comprising one or more pharmaceutically acceptable excipients.

[0213] In some embodiments, the pharmaceutical composition is a stable emulsion.

[0214] In some embodiments, the present disclosure provides pharmaceutical compositions comprising one or more adjuvants, in some embodiments, the one or more adjuvants comprise a TLR4 immunostimulatory agent, such as monophosphoryl lipid A (MPL), glucopyranosyl lipid A (GLA), and / or soluble leishmanial antigen (SLA).

[0215] Also provided herein is a unit dose of the pharmaceutical composition described herein. In some embodiments, the unit dose is about 1 μg to about 5 μg, about 5 μg to about 10 μg, about 10 μg to about 15 μg, about 15 μg to about 20 μg, about 20 μg to about 30 μg, about 30 μg to about 40 μg, about 40 μg to about 50 μg, about 50 μg to about 60 μg, about 60 μg to about 70 μg, about 70 μg to about 80 μg, about 80 μg to about 90 μg, about 90 μg to about 100 μg, or about 100 μg to about 110 μg. The protein complex may be 0 μg, about 110 μg to about 120 μg, about 120 μg to about 130 μg, about 130 μg to about 140 μg, about 140 μg to about 150 μg, about 150 μg to about 200 μg, about 200 μg to about 250 μg, about 250 μg to about 300 μg, about 300 μg to about 350 μg, about 350 μg to about 400 μg, about 400 μg to about 450 μg, or about 450 μg to about 500 μg. In some embodiments, a unit dose comprises about 1 μg, about 2 μg, about 5 μg, about 10 μg, about 15 μg, about 25 μg, about 50 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 200 μg, about 250 μg, or about 300 μg of protein complex. In some embodiments, a unit dose comprises 25 μg, 75 μg, 150 μg, 250 μg, or about 300 μg of protein complex. The abbreviation "μg" may be used interchangeably with the abbreviation "mcg," which refers to micrograms of material.

[0216] The pH of the formulation may also vary. Generally, the pH is about 6.2 to about 8.0. In some embodiments, the pH is about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, or about 8.0. The pH may be within a range of values. Thus, in some embodiments, the pH is about 6.2 to about 8.0, about 6.2 to about 7.8, about 6.2 to about 7.6, about 6.2 to about 7.4, about 6.2 to about 7.2, about 6.2 to about 7.0, about 6.2 to about 6.8, about 6.2 to about 6.6, or about 6.2 to about 6.4. In other embodiments, the pH is about 6.4 to about 8.0, about 6.4 to about 7.8, about 6.4 to about 7.6, about 6.4 to about 7.4, about 6.4 to about 7.2, about 6.4 to about 7.0, about 6.4 to about 6.8, or about 6.4 to about 6.6. In still other embodiments, the pH is about 6.6 to about 8.0, about 6.6 to about 7.8, about 6.6 to about 7.6, about 6.6 to about 7.4, about 6.6 to about 7.2, about 6.6 to about 7.0, or about 6.6 to about 6.8. In still other embodiments, the pH is about 6.8 to about 8.0, about 6.8 to about 7.8, about 6.8 to about 7.6, about 6.8 to about 7.4, about 6.8 to about 7.2, or about 6.8 to about 7.0. In still other embodiments, it is about 7.0 to about 8.0, about 7.0 to about 7.8, about 7.0 to about 7.6, about 7.0 to about 7.4, about 7.0 to about 7.2, about 7.2 to about 8.0, about 7.2 to about 7.8, about 7.2 to about 7.6, about 7.2 to about 7.4, about 7.4 to about 8.0, about 7.4 to about 7.8, about 7.4 to about 7.6, about 7.6 to about 8.0, or about 7.6 to about 7.8.

[0217] In some embodiments, the pharmaceutical composition can include one or more salts, such as sodium chloride, sodium phosphate, or a combination thereof. Generally, each salt is present in the formulation at about 10 mM to about 200 mM. Thus, in some embodiments, any salt present is present at about 10 mM to about 200 mM, about 20 mM to about 200 mM, about 25 mM to about 200 mM, about 30 mM to about 200 mM, about 40 mM to about 200 mM, about 50 mM to about 200 mM, about 75 mM to about 200 mM, about 100 mM to about 200 mM, about 125 mM to about 200 mM, about 150 mM to about 200 mM, or about 175 mM to about 200 mM. In other embodiments, any salt present is present at about 10 mM to about 175 mM, about 20 mM to about 175 mM, about 25 mM to about 175 mM, about 30 mM to about 175 mM, about 40 mM to about 175 mM, about 50 mM to about 175 mM, about 75 mM to about 175 mM, about 100 mM to about 175 mM, about 125 mM to about 175 mM, or about 150 mM to about 175 mM. In still other embodiments, any salt present is present at about 10 mM to about 150 mM, about 20 mM to about 150 mM, about 25 mM to about 150 mM, about 30 mM to about 150 mM, about 40 mM to about 150 mM, about 50 mM to about 150 mM, about 75 mM to about 150 mM, about 100 mM to about 150 mM, or about 125 mM to about 150 mM. In still other embodiments, any salts present are present at about 10 mM to about 125 mM, about 20 mM to about 125 mM, about 25 mM to about 125 mM, about 30 mM to about 125 mM, about 40 mM to about 125 mM, about 50 mM to about 125 mM, about 75 mM to about 125 mM, or about 100 mM to about 125 mM. In some embodiments, any salts present are present at about 10 mM to about 100 mM, about 20 mM to about 100 mM, about 25 mM to about 100 mM, about 30 mM to about 100 mM, about 40 mM to about 100 mM, about 50 mM to about 100 mM, or about 75 mM to about 100 mM. In still other embodiments, any salt present is present at about 10 mM to about 75 mM, about 20 mM to about 75 mM, about 25 mM to about 75 mM, about 30 mM to about 75 mM, about 40 mM to about 75 mM, or about 50 mM to about 75 mM.In still other embodiments, any salts present are present at about 10 mM to about 50 mM, about 20 mM to about 50 mM, about 25 mM to about 50 mM, about 30 mM to about 50 mM, or about 40 mM to about 50 mM. In other embodiments, any salts present are present at about 10 mM to about 40 mM, about 20 mM to about 40 mM, about 25 mM to about 40 mM, about 30 mM to about 40 mM, about 10 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 10 mM to about 25 mM, about 20 mM to about 25 mM, or about 10 mM to about 20 mM. In some embodiments, sodium chloride is present in the formulation at about 100 mM. In some embodiments, sodium phosphate is present in the formulation at about 25 mM.

[0218] The formulation may further comprise a solubilizing agent, such as a non-ionic surfactant, including, but not limited to, polysorbate 80 (Tween® 80), Triton® X-100, and polysorbate 20.

[0219] In some embodiments, the vaccine is a pediatric vaccine, which may be formulated with a polypeptide, virus-like particle, nucleic acid, expression vector, and / or adjuvant, including, but not limited to, vaccines against hepatitis B, rotavirus, diphtheria-tetanus-pertussis ("DTaP"), polio, influenza, and measles-mumps-rubella ("MMR").

[0220] In another aspect, the present disclosure provides a method of inducing an immune response against rabies, comprising administering to a subject in need thereof an immunologically effective amount of an immunogenic composition described herein comprising a VLP described herein.

[0221] In certain embodiments, the immune response comprises the production of neutralizing antibodies against the infectious agent, hi certain embodiments, the neutralizing antibodies are complement-independent.

[0222] The immune response may include a humoral immune response, a cellular immune response, or both. In some embodiments, an immune response is induced against each antigenic protein delivered. The cellular immune response may include a helper T cell (Th) response, a CD8+ cytotoxic T cell (CTL) response, or both. In some embodiments, the immune response includes a humoral immune response, and the antibodies are neutralizing antibodies. Neutralizing antibodies block viral infection of cells. The virus also infects epithelial cells and fibroblasts. In some embodiments, the immune response reduces or prevents infection of both cell types. The neutralizing antibody response may be complement-dependent or complement-independent. In some embodiments, the neutralizing antibody response is complement-independent. In some embodiments, the neutralizing antibody response is cross-neutralizing, i.e., antibodies generated against the administered composition neutralize virus strains other than the strain used in the composition.

[0223] A useful measure of antibody potency in the art is the "50% neutralization titer." To determine the 50% neutralization titer, serum from an immunized animal is diluted to determine the extent to which the diluted serum retains its ability to block 50% of the virus from entering cells. For example, a titer of 700 means that the serum retains its ability to neutralize 50% of the virus after being diluted 700 times. Therefore, a higher titer indicates a stronger neutralizing antibody response. In some embodiments, the 50% neutralization titer is within a range having a lower limit of about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, or about 7000. The 50% neutralization titer is about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, about 7000, about 8000, about 9000, about 10000, about 11000, about 12000, about 130 The range may have an upper limit of about 1000, about 14,000, about 15,000, about 16,000, about 17,000, about 18,000, about 19,000, about 20,000, about 21,000, about 22,000, about 23,000, about 24,000, about 25,000, about 26,000, about 27,000, about 28,000, about 29,000, or about 30,000. For example, a 50% neutralization titer may be about 3,000 to about 25,000. With respect to the disclosed titers, "about" means the recited value ±10%.

[0224] The World Health Organization (WHO) has defined protective correlates for rabies virus using the rapid fluorescent focus inhibition test (RFFIT) or fluorescent antibody virus neutralization test (FAVN) assay. RFFIT is a serum neutralization (inhibition) test. FAVN refers to the RFFIT method adapted to a microtiter plate (e.g., 96-well) format. This assay uses a fixed amount of rabies virus incubated with serial dilutions of serum before infecting BHK-21 cells. The serum titer is the dilution at which 100% of the virus is neutralized in 50% of the wells. Titers are expressed in IU / ml by comparison with the neutralization dilution of a reference standard. In an exemplary method, serum from a vaccinated subject is diluted 5-fold (1 part serum to 4 parts diluent) and then serially diluted 5-fold. The serially diluted samples are mixed and incubated with a standardized amount of live rabies virus. Each diluted sample is then used to infect a culture of target cells. The results of this test can be expressed as an endpoint titer of rabies virus neutralizing antibody (RVNA) (e.g., 1:50) or as a value of RVNA potency (e.g., 0.5 IU). IU stands for International Units and is calculated by comparing the titer to that of a standard reference serum; i.e., the sample titer divided by the reference serum titer multiplied by the IU / mL value of the reference serum. The 1978 joint WHO / IABS symposium (Dev Biol Stand. 1978;40:1-288) defined a protective correlate of rabies neutralizing antibody to the G protein of ≥0.5 IU / mL, based on data demonstrating 100% protection from virus challenge in dogs and cats with neutralizing titers of 0.2 IU / mL and 0.1 IU / mL, respectively. A conservative value of 0.5 IU / mL was selected as the minimum value for an adequate immunizing titer.

[0225] In some embodiments, the virus-like particles of the present disclosure generate an immune response of about 0.5 IU / mL, about 1.0 IU / mL, about 1.5 IU / mL, about 2.0 IU / mL, about 10 IU / mL, about 25 IU / mL, about 50 IU / mL, about 100 IU / mL, or more. In some embodiments, the virus-like particles of the present disclosure generate an immune response of about 0.5 IU / mL or more.

[0226] The composition of the present disclosure is generally administered directly to the subject.Direct delivery can be achieved by parenteral injection (for example, subcutaneously, intraperitoneally, intravenously, intramuscularly, or into the interstitial space of tissue), orally, intranasally, or any other suitable route.For example, intramuscular administration can be used, for example, in the thigh or upper arm.Injection can be performed by needle (for example, hypodermic needle), or alternatively, needleless injection can be used.The typical intramuscular dose is about 0.5ml.

[0227] Administration can be by a single-dose schedule or a multiple-dose schedule. Multiple doses may be used in a primary immunization schedule and / or a booster immunization schedule. In a multiple-dose schedule, various doses may be given by the same or different routes, e.g., a parenteral prime and a transmucosal boost, a transmucosal prime and a parenteral boost, etc. Multiple doses are typically administered at intervals of at least one week (e.g., about two weeks, about three weeks, about four weeks, about six weeks, about eight weeks, about ten weeks, about twelve weeks, about sixteen weeks, etc.). Multiple doses may also be administered at intervals of at least one month (e.g., about two months, about three months, about four months, about six months, about eight months, about ten months, about twelve months, about sixteen months, etc.). A second or subsequent dose may be administered at longer intervals, e.g., about one year or about two years after the preceding dose.

[0228] Immunization can involve 1 to 10 or more administrations (e.g., injections) of the composition, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more administrations. Generally, the initial administration may not elicit a detectable immune response, as each subsequent administration will enhance the immune response generated by the previous administration.

[0229] If the vaccine is used prophylactically, the human is preferably a child (e.g., a toddler or infant), teenager, or adult, and if the vaccine is used therapeutically, the human is preferably a teenager or adult. Pediatric vaccines may be administered to adults, e.g., to assess safety, dosage, immunogenicity, etc.

[0230] The vaccines of the present disclosure may be prophylactic (i.e., prevent disease) or therapeutic (i.e., reduce or eliminate symptoms of disease). The term prophylactic may be considered as reducing the severity of a particular condition or preventing its onset. For the avoidance of doubt, the term prophylactic vaccine may also refer to a vaccine that ameliorates the effects of future infections, for example, by reducing the severity or duration of such infections.

[0231] The isolated and / or purified VLPs described herein can be administered alone or as either the prime or boost in a mixed-modality regimen in which an RNA or DNA primer is followed by a protein boost. Advantages of an RNA prime / protein boost strategy compared to a protein prime / protein boost strategy include, for example, increased antibody titers, a more balanced IgG1:IgG2a subtype profile, induction of a TH1-type CD4+ T cell-mediated immune response similar to that of viral particles, and reduced production of non-neutralizing antibodies. RNA prime can increase the immunogenicity of a composition, regardless of whether the composition contains an adjuvant.

[0232] VLPs of the present disclosure can be administered, for example, before, concurrently with, or after vaccination with rabies immune globulin (RIG) for post-exposure prophylaxis (PEP).

[0233] In the RNA prime / protein boost strategy, RNA and protein are directed to the same target antigen.Suitable examples of RNA delivery methods include viral-like replicon particles (VRP), alphavirus RNA, replicon encapsulated in lipid nanoparticles (LNP), or formulated RNA, such as replicon formulated with cationic nanoemulsion (CNE).Suitable cationic oil-in-water nanoemulsions are disclosed in WO2012 / 006380, which include, for example, an oil core (e.g., containing squalene) and cationic lipids (e.g., DOTAP, DMTAP, DSTAP, DC-cholesterol, etc.).

[0234] Alternatively, to achieve a prime-boost effect, two doses of VLPs can be administered at a predetermined interval. The predetermined interval can be 1, 2, 3, 4, 6, 7, 10, or 14 days, or 3-5 days, 7-10 days, or 10-14 days, etc. The predetermined interval can also be 1, 2, 3, 4, or 6 weeks, or 2-3 weeks, 3-4 weeks, or 5-6 weeks, etc. The predetermined interval can also be 1, 2, 3, or 4 months.

[0235] In some embodiments, the RNA molecule is encapsulated within, bound to, or adsorbed onto a cationic lipid, a liposome, a cochleate, a virosome, an immune stimulating complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil-in-water emulsion, a water-in-oil emulsion, an emulsome, a polycationic peptide, a cationic nanoemulsion, or a combination thereof.

[0236] The present disclosure further provides combination vaccines, including vaccines that contain both rabies VLPs and vaccines against one or more of typhoid, hepatitis A, polio, influenza, hepatitis B, yellow fever, Japanese encephalitis, parvovirus, distemper, adenovirus, parainfluenza, influenza, measles, Lyme disease, coronavirus, vesicular stomatitis virus, herpes simplex virus, baculovirus, thogotovirus, and bornaviridae.

[0237] Also provided herein are kits and instructions for administering the nucleic acids (e.g., RNA), purified proteins, and purified VLPs described herein. The present disclosure also provides delivery devices pre-filled with the compositions or vaccines disclosed herein.

[0238] The pharmaceutical compositions described herein can be administered in combination with one or more additional therapeutic agents.Additional therapeutic agents can include, but are not limited to, antibiotics or antibacterial agents, antiemetics, antifungals, anti-inflammatory agents, antivirals, immunomodulators, cytokines, antidepressants, hormones, alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, topoisomerase inhibitors, cytostatics, anti-invasive agents, antiangiogenic agents, growth factor function inhibitors, viral replication inhibitors, viral enzyme inhibitors, anticancer drugs, α-interferon, β-interferon, ribavirin, hormones, and other toll-like receptor modulators, immunoglobulins (Ig) and antibodies that modulate Ig function (for example, anti-IgE (omalizumab)).

[0239] In certain embodiments, the compositions disclosed herein may be used as a pharmaceutical, e.g., for use in inducing or enhancing an immune response in a subject, e.g., a mammal, in need thereof.

[0240] In certain embodiments, the compositions disclosed herein can be used in the manufacture of a medicament for inducing or enhancing an immune response in a subject, e.g., a mammal, in need thereof.

[0241] One way to confirm the effectiveness of therapeutic treatment involves monitoring infection with an infectious agent after administration of a composition or vaccine disclosed herein. One way to confirm the effectiveness of prophylactic treatment involves monitoring the immune response to the antigen systemically (e.g., monitoring the level of IgG1 and IgG2a production) and / or mucosally (e.g., monitoring the level of IgA production). Typically, antigen-specific serum antibody responses are determined after immunization but before challenge, whereas antigen-specific mucosal antibody responses are determined after immunization and after challenge.

[0242] How to use In another aspect, the present disclosure provides a method of generating an immune response in a subject infected with rabies virus, the method comprising administering any of the polypeptides, nanoparticles, pharmaceutical compositions, polynucleotides, or vectors of the present disclosure in an amount effective to generate an immune response.

[0243] In another aspect, the present disclosure provides a method of immunizing a subject infected with rabies virus against infection by rabies, comprising administering any of the polypeptides, nanoparticles, pharmaceutical compositions, polynucleotides, or vectors of the present disclosure in an amount effective to generate an immune response.

[0244] In another aspect, the present disclosure provides a method of administering post-exposure prophylaxis to a subject infected with rabies virus, the method comprising administering any of the polypeptides, nanoparticles, pharmaceutical compositions, polynucleotides, or vectors of the present disclosure in an amount effective to generate an immune response.

[0245] In some embodiments of the method, the immune response comprises a humoral immune response.

[0246] In some embodiments of the method, the immune response comprises a polyclonal antibody response against rabies G protein.

[0247] In some embodiments of the method, the immune response comprises a neutralizing antibody response to the rabies virus.

[0248] In some embodiments of the method, the method generates a protective immune response against rabies virus.

[0249] In some embodiments of the method, the method generates neutralizing antibodies against rabies virus.

[0250] In some embodiments of the method, the administering step comprises intramuscular or subcutaneous injection.

[0251] In some embodiments of the method, the method results in the production of rabies-specific neutralizing antibodies in a subject in need thereof.

[0252] In some embodiments of the method, the method results in an increase in rabies-specific neutralizing antibodies in a subject in need thereof that is at least about a 2-fold, at least about a 3-fold, at least about a 4-fold, at least about a 5-fold, at least about a 10-fold, at least about a 15-fold, at least about a 20-fold, or at least about a 25-fold increase compared to rabies-specific neutralizing antibodies in the same subject prior to the administering step.

[0253] In some embodiments of the method, the method generates a neutralization titer of at least 0.5 IU / mL in a rapid fluorescent focus inhibition test (RFFIT) and / or a fluorescent antibody virus neutralization (FAVN) test.

[0254] In some embodiments of the methods, the subject is a non-human animal.

[0255] In some embodiments of the methods, the subject is a companion animal.

[0256] In some embodiments of the methods, the subject is a human.

[0257] In another aspect, the present disclosure provides a host cell comprising a polynucleotide comprising a polynucleotide sequence encoding any of the polypeptides or nanoparticles of the present disclosure.

[0258] In another aspect, the present disclosure provides a method of producing a vaccine, the method comprising culturing a host cell of the present disclosure in a culture medium such that the host cell secretes a first polypeptide component into the culture medium, purifying the first polypeptide component from the culture medium, mixing the first polypeptide component with a second polypeptide component, wherein the first and second polypeptide components self-assemble to form nanoparticles, mixing, and / or purifying the nanoparticles.

[0259] definition All publications, patents, and patent applications, including any drawings and appendices therein, are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application, drawing, or appendix was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0260] The terms "a" or "an" refer to one or more of the entity in question, i.e., may refer to multiple referents. Thus, the terms "a," "an," "one or more," and "at least one" are used interchangeably herein. Furthermore, reference to "an element" by the indefinite article "a" or "an" does not exclude the presence of a plurality of elements, unless the context clearly requires that there be only one of that element.

[0261] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range, for example, within ±20%, ±10%, or ±5%. When used in conjunction with a range or series of values, the term "about" applies to the endpoints of the range or each of the recited values ​​in the series, unless otherwise specified. As used herein, the terms "about" and "approximately" are used equivalently. When used herein to refer to the deletion of the fusion loop domain of rabies G protein, the term "about" can mean ±1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 residues.

[0262] The term "antigen" refers to a polypeptide or polypeptide complex that contains at least one component designed to elicit an immune response.

[0263] The term "polypeptide" refers to a series of amino acid residues linked by peptide bonds, optionally with one or more post-translational modifications (e.g., glycosylation) and / or other modifications (including, but not limited to, polypeptide moieties used as markers, such as fluorescent tags, or conjugation of covalently attached adjuvants).

[0264] The term "infection" refers to both symptomatic and asymptomatic infection.

[0265] The term "ectodomain" refers to the part of a transmembrane protein or glycoprotein that, in the protein's native state, is on the outside of the cellular or viral membrane.

[0266] The term "variant" refers to a polypeptide that has one or more insertions, deletions, or amino acid substitutions compared to a reference polypeptide, but retains one or more properties of the reference protein. When the term "variant" is used, the variant shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference protein.

[0267] The term "antigenic variant" refers to a variant that shares one or more epitopes with a reference polypeptide and / or that generates the same or a similar immune response as the reference polypeptide when administered to a subject.

[0268] The term "functional variant" refers to a variant that exhibits the same or similar functional effect(s) as a reference polypeptide. For example, a functional variant of a multimerization domain can promote multimerization to the same extent as, or to a similar extent to, the reference multimerization domain and / or can multimerize with the same cognate multimerization domain as the reference multimerization domain.

[0269] The term "linker" refers to a chemical linkage (i.e., a covalent bond or series of covalent bonds with intervening chemical moieties). A "polypeptide linker" refers to a linker consisting of a polypeptide inserted between two other polypeptide chains.

[0270] The term "domain" refers to any part of a polypeptide that has tertiary structure.

[0271] The terms "multimerization domain" and "multimerization" refer to the ability of a polypeptide, or a domain of a polypeptide, to form dimers, trimers, tetramers, pentamers, or hexamers, and / or to form heteromers with other multimerization domains.

[0272] The term "trimerization domain" refers to a multimerization domain that forms trimers.

[0273] The term "VLP-forming domain" refers to a multimerization domain that, alone or together with other multimerization domains, forms a symmetric protein complex.

[0274] The term "fragment" refers to a polypeptide having one or more N-terminal or C-terminal truncations compared to a reference polypeptide.

[0275] The term "functional fragment" refers to a functional variant of a fragment.

[0276] The term "amino acid substitution" refers to the replacement of a single amino acid residue in a sequence with another amino acid residue. Standard abbreviations for amino acid substitutions are used. For example, V94R refers to the substitution of valine (V) with arginine (R) in the reference sequence. The abbreviation Arg94 refers to any sequence in which the 94th residue compared to the reference sequence is arginine (Arg).

[0277] The term "helix" or "helical" refers to an α-helical secondary structure known to occur or predicted to occur in polypeptides. For example, a sequence may be described as helical if computational modeling suggests that the sequence is likely to adopt a helical conformation.

[0278] The term "polypeptide component" refers to a polypeptide that can assemble into nanoparticles under appropriate conditions.

[0279] The term "vaccine" refers to a composition that can be used in generating an immune response in a subject.

[0280] The term "pharmaceutically acceptable excipient" means an excipient that is biologically or pharmacologically compatible for in vivo use in animals or humans, and can mean an excipient approved by a regulatory agency of the U.S. federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, or more specifically, in humans.

[0281] The term "excipient," as used herein, refers to an inert substance commonly used as a diluent, vehicle, preservative, binder, or stabilizer for a drug that confers beneficial physical properties to a formulation, such as increased protein stability, increased protein solubility, and reduced viscosity. Examples of excipients include, but are not limited to, proteins (e.g., but not limited to, serum albumin), amino acids (e.g., but not limited to, aspartic acid, glutamic acid, lysine, arginine, and glycine), surfactants (e.g., but not limited to, SDS, Tween® 20, Tween® 80, polysorbates, and nonionic surfactants), sugars (e.g., but not limited to, glucose, sucrose, maltose, and trehalose), polyols (e.g., but not limited to, mannitol and sorbitol), fatty acids, and phospholipids (e.g., but not limited to, alkylsulfonates and caprylates).

[0282] The term "adjuvant" refers to a pharmaceutically acceptable substance that enhances the immune response to an antigen when administered in combination with an antigen, or when administered before, during, or after administration of the antigen to a subject.

[0283] The term "TLR4 immunostimulatory agent" refers to an adjuvant, e.g., monophosphoryl lipid A (MPL), glucopyranosyl lipid A (GLA), and / or soluble leishmanial antigen (SLA), that stimulates Toll-like receptor 4 (TLR4) in a subject's immune cells to modulate the immune response.

[0284] The term "effective amount" refers to the amount of a composition that, when administered to a subject for treating a condition, disorder, or condition, is sufficient to effect such treatment, or that, when administered to a subject for generating an immune response, is sufficient to generate such an immune response. An "effective amount" will vary depending on the active ingredient, the condition, disorder, or condition being treated and its severity, and the age, weight, physical condition, and responsiveness of the subject being treated.

[0285] The term "immune response" refers to the elicitation of activity of one or more immune cell types in a subject. Immune responses include, for example, T cell responses and B cell responses.

[0286] The term "humoral immune response" refers to an immune response that results in the generation of plasma or serum antibodies (eg, IgG).

[0287] The term "protective immune response" refers to an immune response that prevents infection and / or reduces the severity of a pathogen upon subsequent challenge of the subject, or generates a level of immune response that correlates with protection. For example, vaccination can generate a protective immune response if it results in the production of neutralizing antibodies in the plasma or serum of a subject (e.g., a human, pet, or agricultural animal) that are present in an amount that protects the subject from subsequent infection and / or that is observed to protect a test subject (e.g., a Syrian golden hamster (SGH)).

[0288] The term "polyclonal antibody response" refers to an antibody response that includes antibodies with multiple specificities and / or variations in antibody sequence.

[0289] The term "neutralization" (e.g., a "neutralizing antibody response") refers to an antibody that prevents infection by a pathogen and / or reduces the level of infection. A neutralizing antibody response can be measured either in an in vitro assay (e.g., infection of cells in culture by a pathogen in the presence of the antibody) or in an in vivo assay (e.g., by determining a protective dose of antibody by administering the antibody to a subject prior to challenge with an infectious dose of the pathogen).

[0290] The term "predetermined time" refers to a time interval appropriately selected to observe a particular effect. The predetermined time can be selected before or during an experiment or procedure.

[0291] The term "post-exposure prophylaxis" refers to the administration of an antigenic composition (e.g., a vaccine) to a subject previously exposed to and / or infected with a pathogen to induce an immune response that protects against infection by the pathogen and / or to reduce the severity of one or more symptoms of infection by the pathogen.

[0292] The term "administering" refers to providing a composition to a subject in a manner that allows the composition to have its intended effect. Administration for vaccination or post-exposure prophylaxis can be by intramuscular injection, intravenous injection, intraperitoneal injection, subcutaneous injection, or any other suitable route.

[0293] The terms "immunization" and "immunizing" refer to administering a composition to a subject in an amount sufficient to elicit a desired immune response (e.g., a humoral immune response) after one or more administration steps. As used herein, the term "immunizing" includes post-exposure prophylaxis.

[0294] The term "subject" refers to a human or non-human animal to which a composition may be administered for vaccination, treatment, or other purposes. In some embodiments, the non-human animal is a non-human primate, including, but not limited to, a rabbit, hamster, gerbil, pig, cow, sheep, goat, guinea pig, rat, mouse, squirrel, wolf, fox, horse, zebra, giraffe, elephant, cat, dog, llama, or ferret.

[0295] The term "manufacturing" refers to producing a recombinant polypeptide or virus-like particle at any scale, including but not limited to at least 25 mL, 50 mL, 1 L, 2 L, 1,000 L, 50,000 L, or larger.

[0296] The terms "culturing" and "culture medium" refer to standard cell culture and recombinant protein expression techniques.

[0297] The term "host cell" refers to any cell that can be used for the expression of a recombinant polypeptide.

[0298] The term "secrete" refers to the ability of a host cell to secrete a polypeptide into the medium in which the host cell is cultured.

[0299] The term "signal sequence" refers to a polypeptide sequence that is typically located at the N-terminus of a polypeptide expressed in a host cell and that directs the polypeptide to a specific cellular compartment. A signal sequence can be a secretion signal that causes the host cell to secrete the polypeptide into the medium in which the host cell is cultured. A variety of signal sequences are known, and selecting an appropriate signal sequence is within the skill of one in the art. Signal sequences are short peptides (typically 16-30 amino acids in length) that are cleaved either during translocation or after translocation is complete, generating a free signal peptide and mature protein.

[0300] The term "mixing" refers to bringing two solutions into contact and allowing the solutions to mix.

[0301] The term "purify" refers to separating a molecule from other substances present in a composition. Polypeptides can be purified by affinity (e.g., using an antibody or tag, e.g., His-tag capture resin), by charge (e.g., ion exchange chromatography), by size (e.g., preparative ultracentrifugation, size exclusion chromatography), or by other methods.

[0302] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to a polymeric form of nucleotides of more than about 100 nucleotides, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0303] The terms "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a certain percentage of identical amino acid residues or nucleotides when compared and aligned to maximize correspondence. Methods for aligning sequences for comparison are well known in the art. After alignment, the number of matches is determined by counting the number of positions where identical nucleotides or amino acid residues occur in both sequences. The percent sequence identity is determined by dividing the number of matches in the alignment by the length of the reference sequence and then multiplying the result by 100. For example, a peptide sequence that has 1166 matches when aligned with a reference sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166 ÷ 1554 × 100 = 75.0). As these terms are used herein, gaps in the alignment do not reduce the percent sequence identity. Unless otherwise specified, optimal alignment of sequences for comparison is performed by the global alignment algorithm of Needleman and Wunsch, Mol. Biol. 48:443 (1970), as implemented by EMBOSS Needle (World Wide Web ebi.ac.uk / Tools / psa / emboss_needle / ) (Madeira et al. Nucleic Acids Res. 50(W1):W276-W279(2022)).Other alignment methods may also be used, including, but not limited to, those described in Devereux, et al., Nucleic Acids Res. 12:387-95 (1984), Atschul et al. J. Mo. Biol. 215:403-10 (1990) (BLAST), Carrillo and Lipman Siam J. Appl. Math. 48(5) (1988), Computational Molecular Biology (Lesk, AM, ed., 1989), Biocomputing Informatics and Genome Projects, (Smith, DW, ed., 1993), Computer Analysis of Sequence Data, Part I, (Griffin and Griffin, eds., 1994), Sequence Analysis in Molecular Biology (von Heinje, 2012), and Sequence Analysis Primer (Gribskov and Devereux, J., eds. 1993). Sequence identity is calculated using an implementation of the Needleman-Wunsch algorithm provided by the National Library of Medicine (World Wide Web blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=GlobalAln).

[0304] The term "treating" refers to one or more of alleviating, ameliorating, delaying, reducing, reversing, ameliorating, or managing at least one symptom of a condition in a subject. The term "treating" can also refer to one or more of arresting a condition, delaying its onset (i.e., the period before clinical manifestation of the condition), or reducing the risk of its occurrence or worsening.

[0305] As used herein, "substantially" or "substantially" refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is "substantially" enclosed means that the object is completely enclosed or nearly completely enclosed. The exact degree of acceptable deviation from absolute completeness may, in some cases, depend on the specific context. Generally speaking, however, the proximity of completion is such that the overall result is the same as if absolute and total completion had been achieved. The use of "substantially" is equally applicable when used in the negative sense to refer to the complete or nearly complete absence of an action, characteristic, property, state, structure, item, or result. For example, a composition that is "substantially free" of other active agents is completely devoid of other active agents, or nearly completely devoid of other active agents, so that its effect is the same as if it were completely devoid of other active agents. In other words, a composition that is "substantially free" of a component or element or another active agent may still contain such item as long as there is no measurable effect of that item.

[0306] As used herein, the term "companion animal" refers to any animal whose diet is controlled by an owner, breeder, or caretaker. In some embodiments, the companion animal is an animal selected from the group consisting of a dog, a cat, a rabbit, a hamster, a gerbil, a ferret, and a guinea pig. In some embodiments, the companion animal is a dog or a cat.

[0307] This description includes information that may be useful in understanding the present invention. Nothing made herein should be construed as an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any specifically or implicitly referenced publication is available as prior art. [Example]

[0308] Example 1 Various modified forms of rabies G protein were designed and tested as fusion proteins with the multimerization domain I53-50A. Many had poor expression and / or the protein aggregated. Some of the designs tested are shown in Table 7.

[0309] Human codon-optimized polynucleotide sequences were generated by gene synthesis and cloned into expression vectors. Each expression vector was individually expressed in Expi293 cells by transient transfection. Supernatants were collected 4 days after transfection.

[0310] Western blots were performed using an anti-His6 monoclonal antibody. Conditioned medium (12 μl) from transient transfections was mixed with 4 μl of reducing sample loading buffer and heated to 95°C for 5 minutes. Samples were loaded onto a NuPAGE 4-12% Bis-Tris protein gel and run at 120V for 2 hours. Proteins were transferred to an Immuno-Blot PDVF membrane (Bio-Rad). After transfer, the blot was blocked with 1X casein buffer (Bio-Rad) at room temperature for 1 hour with shaking. The membrane was then incubated with an HRP-conjugated anti-His6 monoclonal antibody at room temperature for 2 hours with shaking. The membrane was washed three times (15 minutes each) with PBS containing 0.1% Tween® 20. The membrane was then incubated with HRP substrate (Pierce ECL plus kit) for 2 minutes and imaged.

[0311] Expression levels were assessed by visual inspection of Western blots and designated as no expression (X) or low expression (*) to high expression (****), no expression to >10 mg / L. [Table 7]

[0312] Construct A was the wild-type ectodomain sequence of rabies G fused to I53-50A and was expressed at low levels (<2 mg / L). The resulting protein was highly aggregated.

[0313] Construct B, which has two deletions within the fusion loop domain, showed greatly improved expression and did not aggregate like the wild-type sequence.

[0314] Deletion of the complete fusion loop domain was surprisingly well tolerated, as constructs with C-terminal truncations to residue 417 (constructs C and F) or to residue 454 (constructs D and E) were all well expressed.

[0315] The rabies G protein has been reported to be toxic in mammalian cells. To test the ability to stably express selected rabies G proteins, polynucleotides encoding construct B or construct F were transfected into CHO-K1 cells, and stable pools were selected using glutamine synthetase as a selectable marker. Cells expressing construct B did not survive 14 days under glutamine selection. The other three test constructs also did not yield stable cell lines. Using a fed-batch system at a 25 mL scale, stable cell lines of construct F were successfully selected, with expression of CompA-construct F exceeding 400 mg / L.

[0316] Example 2 The I53-50A multimerization tag can form trimers by itself. I53-50A trimers are known to self-assemble into icosahedral particles when mixed with pentameric I53-50B. Constructs B, C, and F (produced by transient transfection) were assembled with I53-50B to generate VLPs. All assembled into monodisperse VLPs of the expected size. Representative characterization of construct F VLPs is shown in Figure 4 (SDS-PAGE), Figure 5 (negative stain electron microscopy), Figure 6 (BLI), and Figure 7 (ELISA).

[0317] To characterize the rabies G protein on the resulting particles, we tested the binding of neutralizing antibodies D1-25 and 1112-1 (RD-Biotech), which recognize antigenic sites III and II, respectively, by biolayer interferometry (BLI). Both neutralizing antibodies bind to construct F particles regardless of whether they are stored at room temperature or subjected to freeze / thawing (Figure 6).

[0318] Binding of the soluble trimeric construct F ("VLP") or soluble trimeric construct F ("CompA") was also tested with samples prepared from transiently transfected Expi293 cells ("HEK") or a stable CHO-K1 cell line ("CHO-K1"). Wells of a 96-well plate were coated overnight at 4°C with 2 μg / mL of 1112-1 monoclonal antibody (100 μl) in carbonate-bicarbonate buffer (pH 9.6). The plate was washed three times with 300 μl of PBS containing 0.05% Tween® 20. After blocking with 100 μl of PBS containing 2% BSA at 37°C for 1 hour, the plate was washed three times as described above. Trimeric construct F (construct F compA) or construct F particles (construct F VLP) were added in a volume of 100 μl, and the plate was gently shaken at 37°C for 1 hour. The plate was washed three times with 200 μl of PBS containing 0.05% Tween® 20. Biotinylated D1-25 (100 μl) in PBS containing 0.05% Tween®-20 and 0.1% BSA was added, and the plate was gently shaken at 37°C for 1 hour. After washing the plate three times with 300 μl of PBS containing 0.05% Tween® 20, 100 μl of a 1:5000 dilution of HRP-conjugated streptavidin was added to each well and shaken at room temperature for 1 hour. The plate was washed six times with 200 μl of PBS containing 0.05% Tween® 20, followed by the addition of 100 μl of TMB substrate and development for 10 minutes in the dark. The reaction was stopped by adding 100 μl of 0.1 M HCl, and the absorbance at 450 nm was measured on a plate reader. The sandwich ELISA system gives a positive signal only if the sample binds to both the 1112-1 capture antibody and the D1-25 detection antibody.

[0319] All CompA and VLP samples showed positive signals in the sandwich ELISA (Figure 7). This indicates that the fusion loop domain-deleted rabies G protein is stably folded in an antigenic conformation, whether as a trimer or assembled into particles. The sample source (293F cells or CHO cells) did not affect binding. A higher signal was observed for particles than for trimers (Figure 7).

[0320] Example 3 A study was conducted in naive BALB / c mice to investigate the immunogenicity of a rabies G protein containing a deletion of residues 66-207 displayed on a bicomponent virus-like particle (VLP). The experiment in Example 2 was performed using the I53-50A / I53-50B multimerization domain pair. In this example, a different particle was used in which the rabies G protein ectodomain was fused to the I53-dn5B multimerization domain, which forms a trimer, and then assembled with the pentameric I53-dn5A multimerization domain to form a particle.

[0321] The rabies G protein ectodomain fused to the I53-dn5B trimerization domain (SEQ ID NO:97) ("Construct C-Component") was expressed and purified, then mixed with I53-dn5A to assemble into particles. The trimeric I53-dn5B protein complex was formulated with an oil-in-water adjuvant (Addavax™). The assembled I53-dn5B / dn5A particles were formulated with an oil-in-water adjuvant (Addavax™) or an aluminum hydroxide (alum) adjuvant (Alhydrogel™). The commercially available adjuvanted rabies vaccine IMRAB®3 ("ImRab3") was used as a control.

[0322] A mouse immunogenicity study was conducted, involving four groups of eight female Balb / c mice immunized on days 0 and 21. Mice were immunized twice with 2.5 μg of construct C VLP, 4 μg of construct C-dn5B (twice the antigen content of the VLP), or ImRab3 at one-tenth the commercial dose. Blood samples were collected on days 0, 21, and 35 and processed to serum for analysis in the Rapid Fluorescent Focus Inhibition Test (RFFIT) for neutralizing antibodies. Day 0 serum was pooled by group. Neutralizing antibody titer data were plotted, showing the geometric mean with the geometric standard deviation (SD). Group statistical analysis was performed using the nonparametric Mann-Whitney test (GraphPad Prism V.9.3.1).

[0323] Sera on day 35 showed robust neutralization titers for Construct C VLPs with oil-in-water adjuvants and the ImRab3 control, with geometric mean titers of 37.87 and 17.9 IU / ml, respectively (Figure 8). Construct C VLPs formulated with alum (Construct C-dn5) or Construct C VLPs formulated with the trimeric Construct C-dn5B component (Construct C-CompA) induced low but detectable titers, with geometric mean titers of 0.25 IU / ml and 0.78 IU / ml, respectively (Figure 8). The recognized correlate of protection for rabies is 0.5 IU / ml.

[0324] Example 4 A study was conducted to investigate the immunogenicity of different rabies G antigens in naive BALB / c mice. Two VLP platforms were compared, including Construct C displayed in either I53-50 or dn5. Construct F and Construct B (I53-50 VLPs) were also included. The purpose of this study was to evaluate the induction of neutralizing titers by Constructs B, C, and F. Mice were immunized twice with 0.1 μg of each VLP formulated with Addavax, an oil-in-water adjuvant. One group was immunized with the commercially available pet rabies vaccine MRAB®3 ("ImRab3") administered at 1 / 10 the canine dose. Serum samples were subjected to the RFFIT (rapid fluorescent focus inhibition) test to determine neutralizing antibody titers against rabies. Neutralizing antibody titer data were plotted, showing the geometric mean with the geometric SD. Group statistical analysis was performed using the nonparametric Mann-Whitney test (GraphPad Prism V.9.3.1).

[0325] Five groups of eight female BALB / c mice were immunized on days 0 and 21. Blood samples were collected on days 0, 21, and 35 and processed to serum for analysis in the RFFIT test for neutralizing antibodies. Day 0 serum was pooled by group.

[0326] Neutralizing titers were induced at comparable levels by constructs C (I53-50 and dn5), B, and F (Figure 9), with titers exceeding the protective correlate (0.5 IU / ml) in all animals, as shown by serum on day 35. Geometric mean titers induced by VLPs ranged from 21.3 to 34.3 IU / ml, with no statistically significant differences between groups (Mann-Whitney test). Titers observed with VLPs were comparable to those with ImRab3.

[0327] Example 5 A study was conducted to investigate the immunogenicity of two rabies G antigens presented on bicomponent virus-like particles (VLPs) in naive ICR mice. Construct C I53-50 particles or Construct F I53-50 particles formulated with an oil-in-water adjuvant (Addavax™) were evaluated at doses of 3 μg, 1 μg, 0.3 μg, and 0.1 μg compared to an ImRab3 control administered at 1 / 10 the dog dose. A rapid fluorescent focus inhibition test (RFFIT) test was performed to determine neutralizing antibody titers against rabies.

[0328] Nine groups of eight female outbred ICR mice were immunized on days 0 and 21. Blood samples were collected on days 0, 21, and 35 and processed to serum for analysis. Day 0 serum was pooled by group. Neutralizing antibody titer data were plotted to show the geometric mean with geometric SD. Group statistical analysis was performed using the nonparametric Mann-Whitney test (GraphPad Prism V.9.3.1).

[0329] Day 0 serum samples yielded titers below 0.2 IU / ml in all groups. Day 35 serum showed neutralization geometric mean titers ranging from 74.8 to 211 IU / ml for Construct C and Construct F (Figure 10). Because dose titration resulted in comparable titers, the data indicate that 100 ng of rabies VLP formulated with Addavax induces the maximal humoral response in naive ICR mice. All animals receiving Construct C and Construct F had titers above the correlate of protection, except for one animal at the 1 μg dose level of Construct F, which was at background levels (<0.2 IU / ml). Titers observed with Construct C and Construct F were comparable to ImRab3 at day 35 (geometric mean 71.1).

[0330] Example 6 A study investigating the immunogenicity of two rabies G antigens presented on bicomponent virus-like particles (VLPs) in naive Syrian golden hamsters (SGH) was conducted essentially as described in Example 5, except for the control animals. Nine groups of six female SGH were immunized on days 0 and 21. Blood samples were collected on days 0, 21, and 35 and processed to serum for analysis in the RFFIT test for neutralizing antibodies. Day 0 serum was pooled by group. Neutralizing antibody titer data were plotted, showing geometric means with geometric SD. Group statistical analysis was performed using the nonparametric Mann-Whitney test (GraphPad Prism V.9.3.1).

[0331] Pooled serum samples on day 0 yielded titers less than 0.2 IU / ml in all groups. Sera on day 35 showed that neutralizing titers of Construct C exceeded the protective correlate in 17 / 18 animals at doses of 3 μg, 1 μg, and 0.3 μg, with geometric mean titers ranging from 2.2 to 6.7 IU / ml (Figure 11). At the lowest dose of Construct C (0.1 μg), titers exceeded the protective correlate in 2 / 6 animals. The three highest doses of Construct F (3 μg, 1 μg, and 0.3 μg) yielded comparable titers in all 18 animals (all above the protective correlate), with geometric mean titers ranging from 4.8 to 5.7 IU / ml; at the lowest dose, 3 / 6 animals exceeded the protective correlate.

[0332] Example 7 To investigate the immunogenicity of construct F presented to either I53-50 or dn5 as a trimeric soluble protein or VLP, studies were conducted in naive BALB / c mice. Construct F was fused to CompA (I53-50, SEQ ID NO: 95) or dn5B (dn5, SEQ ID NO: 97) and expressed as a soluble trimeric protein. The trimeric protein was also combined with the appropriate pentameric subunit to form VLPs. Mice were immunized twice on days 0 and 21 with each trimeric protein or VLP at the doses and formulations shown in Table 8. Trimeric soluble proteins were administered at equivalent antigen content as 0.5 μg and 0.1 μg VLPs. One group was immunized with the commercially available companion animal rabies vaccine MRAB®3 ("ImRab3") at 1 / 10 the canine dose. Serum samples were subjected to the RFFIT test (rapid fluorescent focus inhibition test) to determine neutralizing antibody titers against rabies. Neutralizing antibody titer data were plotted, and the geometric mean was shown with the geometric SD. Group statistical analysis was performed using the nonparametric Mann-Whitney test (GraphPad Prism V.10.0.0).

[0333] Eleven groups of eight female BALB / c mice were immunized on days 0 and 21. Blood samples were collected on days 0, 21, and 35 and processed to serum for analysis in the RFFIT test for neutralizing antibodies. Day 0 serum was pooled by group.

[0334] Sera on day 35 showed that neutralizing titers were induced to various degrees above the baseline titer on day 0 (<0.2 IU / ml). Construct F I53-50 VLP formulated with aqueous buffer or Alhydrogel induced low titers, with only some titers above the protective correlate (0.5 IU / ml), with geometric mean titers of 0.56 and 0.52 IU / ml, respectively (Figure 12). Construct F I53-50 VLP formulated with AddaVax induced significantly higher titers (p=0.0002, Mann-Whitney test), with geometric mean titers of 63.11 and 47.44 IU / ml at the 0.1 μg and 0.2 μg doses, respectively, with all animals exceeding the protective correlate (Figure 12). The groups receiving CompA-Construct F (soluble trimeric protein) had geometric mean titers of 1 IU / ml and 0.65 IU / ml, which were significantly lower than those receiving I53-50 VLPs containing Construct F (p=0.0002, Mann-Whitney test) (Figure 12). Construct F dn5 VLPs formulated with AddaVax induced a broad distribution of titers, suggesting that the quality of the antigen in the VLP preparation was suboptimal. Geometric mean titers were 2.65 and 1.03 IU / ml at the 0.1 μg and 0.2 μg doses, respectively, with the majority of animals exceeding the protective correlate (Figure 12). The groups receiving dn5B-Construct F (soluble trimeric protein) had geometric mean titers of 0.91 IU / ml and 1.56 IU / ml, similar to those receiving dn5 VLPs containing Construct F (Figure 12). The titers observed with I53-50 VLP were comparable to those with ImRab3 (Figure 12). This data demonstrates that rabies G antigen presentation is affected by adjuvant and formatting in the VLP format better than soluble protein. [Table 8]

[0335] Example 8 A study was conducted in Syrian golden hamsters to evaluate the ability of construct F VLPs to protect against subsequent challenge with rabies virus. Five groups of 10 female Syrian golden hamsters were included in the study. Construct F VLPs (I53-50) were administered at doses of 2 μg or 0.2 μg formulated with AddaVax on days 0 and 21. RabAvert, a commercially available human rabies vaccine, was administered at 1 / 20 the human dose on days 0, 7, and 21. Two groups received saline; one of these groups was not challenged with rabies virus. All other groups were challenged with rabies virus intramuscularly on day 35. Blood samples were collected on days 0, 21, 34, and on the day of sacrifice and processed for serum. Brains from each animal were collected on the day of sacrifice. Serum samples were subjected to the RFFIT (rapid fluorescent focus inhibition test) test to determine neutralizing antibody titers against rabies. Neutralizing antibody titer data were plotted, and geometric means with geometric SDs were shown. Group statistical analysis was performed using the nonparametric Mann-Whitney test (GraphPad Prism V.10.0.0). Direct fluorescent antibody (DFA) testing was performed on brain specimens to detect the presence of rabies virus. All animals after rabies virus challenge were monitored for clinical signs of infection for 30 days and euthanized upon reaching a humane endpoint (i.e., paralysis).

[0336] Of the animals injected with saline and challenged with rabies virus, 7 / 10 animals were euthanized due to clinical signs of infection 30 days after challenge (Figure 13). Of the 20 animals immunized with Construct F VLP, none were euthanized due to reaching a humane endpoint (Figure 13). None of the animals immunized with RabAvert similarly reached a humane endpoint (Figure 13). DFA test results indicated the presence of rabies virus only in the brains of the seven control animals that were euthanized due to reaching a humane endpoint. All other animals were negative for rabies virus. Neutralizing antibody titers from serum samples on day 34 indicated that the titers in saline-injected animals were at the lower limit of detection of the assay (Figure 14). All animals immunized with Construct F VLP (day 0 and day 21 doses) had titers at or above the protective correlate (Figure 14). The RabAvert-treated groups (treated on days 0, 7, and 21) also demonstrated titers equal to or greater than the correlates of protection (Figure 14). This study demonstrates that construct F VLPs can fully protect animals from rabies virus challenge.

[0337] While the invention has been described in relation to specific suggested embodiments thereof, it is to be understood that further modifications are possible, and this application is intended to cover any variations, uses, or applications of the invention which generally follow from the principles of the invention, including such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains and which may apply to the essential features described above and fall within the scope of the appended claims.

Claims

1. a polypeptide comprising a rabies G protein ectodomain, said rabies G protein ectodomain comprising a deletion of the fusion loop domain of said rabies G protein ectodomain; the deleted fusion loop domain is from about residue 70 to about residue 200 of the rabies G protein ectodomain, as numbered according to SEQ ID NO:53; or The deleted fusion loop domain is selected from the group consisting of: from about residue 50 to about residue 180, from about residue 70 to about residue 180, from about residue 80 to about residue 180, from about residue 90 to about residue 180, or from about residue 100 to about residue 180 of the rabies G protein ectodomain, as numbered according to SEQ ID NO:53; from about residue 50 to about residue 190, from about residue 70 to about residue 190, from about residue 80 to about residue 190, from about residue 90 to about residue 190, or from about residue 100 to about residue 190; from about residue 50 to about residue 200, from about residue 70 to about residue 200, the polypeptide is from about residue 50 to about residue 210, from about residue 70 to about residue 210, from about residue 80 to about residue 210, from about residue 90 to about residue 210, or from about residue 100 to about residue 210; from about residue 50 to about residue 220, from about residue 70 to about residue 220, from about residue 80 to about residue 220, from about residue 90 to about residue 220, or from about residue 100 to about residue 220.

2. 2. The polypeptide of claim 1, wherein the deleted fusion loop domain is residue 66 to residue 207 of the rabies G protein ectodomain, as numbered according to SEQ ID NO:

53.

3. 3. The polypeptide of claim 1 or claim 2, wherein the rabies G protein ectodomain comprises a first polypeptide segment linked to a second polypeptide segment; the first polypeptide segment shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with residues 20-65 of SEQ ID NO:53; The polypeptide wherein the second polypeptide segment shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with residues 208-417 of SEQ ID NO:

53.

4. The polypeptide of claim 3, wherein the polypeptide is a fusion protein comprising, in order from N-terminus to C-terminus, the first polypeptide segment, a polypeptide linker, and the second polypeptide segment.

5. 5. The polypeptide of claim 4, wherein the rabies G protein ectodomain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs:77-81.

6. The polypeptide of any one of claims 1 to 5, wherein the polypeptide comprises a multimerization domain.

7. The polypeptide of claim 6 , wherein the multimerization domain is a trimerization domain.

8. 8. The polypeptide of claim 7, wherein the multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with FoldOn (SEQ ID NO: 58).

9. 8. The polypeptide of claim 6 or claim 7, wherein the multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a polypeptide sequence selected from SEQ ID NOs: 1, 4, 5, 7, 9, 18, 19, 21, 24, 25, 26, 29, 30, 31, 34, 36, 37, 39, 42, 43, 44, 45, 46, 47, 48, 49, 50, and 51, and wherein the interface residues identified in Table 3 are conserved.

10. 10. The polypeptide of claim 9, wherein the multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 7).

11. The polypeptide of claim 10, wherein the multimerization domain is I53-50A-Δcys (SEQ ID NO: 67).

12. 12. The polypeptide of claim 10 or claim 11, wherein the polypeptide shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 93-96.

13. 8. The polypeptide of claim 6 or claim 7, wherein the multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-dn5B (SEQ ID NO:75).

14. 12. The polypeptide of claim 10 or claim 11, wherein the polypeptide shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:101 or SEQ ID NO:

102.

15. 8. The polypeptide of claim 6 or claim 7, wherein the multimerization domain is a ferritin polypeptide capable of forming ferritin particles.

16. 12. The polypeptide of claim 10 or claim 11, wherein the polypeptide shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with construct F-ferritin (SEQ ID NO: 103).

17. The polypeptide according to any one of claims 6 to 16, wherein the multimerization domain is a particle-forming domain.

18. A nanoparticle comprising the polypeptide according to any one of claims 1 to 17.

19. The nanoparticle of claim 18, wherein the polypeptide comprises a rabies G protein ectodomain according to any one of claims 1 to 17.

20. 20. The nanoparticle of claim 18 or 19, wherein the nanoparticle is a protein-based virus-like particle (pbVLP) or nanostructure.

21. The nanoparticle of any one of claims 18 to 20, wherein the nanoparticle is free of lipid components.

22. The nanoparticle of claim 18 , wherein the nanoparticle comprises a lipid component.

23. The nanoparticle of any one of claims 18 to 22, wherein the nanoparticle comprises a second polypeptide component.

24. The nanoparticle according to any one of claims 18 to 21, wherein the nanoparticle comprises a second polypeptide component and the first polypeptide component, and the nanoparticle is a self-assembling nanoparticle comprising the first and second polypeptide components symmetrically arranged with point group symmetry.

25. 24. The nanoparticle of claim 23, (a) the first polypeptide component comprises the polypeptide of any one of claims 1 to 17, and the first polypeptide component forms a first homomeric complex via the multimerization domain of the polypeptide; (b) the second polypeptide component comprises a second multimerization domain, and the second polypeptide component forms a second homomeric complex via the second multimerization domain of the polypeptide; (c) the first homomeric complex and the second homomeric complex assemble to form the nanoparticle having point group symmetry; (d) the nanoparticles are free of other polypeptide components; and / or (e) The nanoparticles, wherein the nanoparticles are free of lipid components.

26. 26. The nanoparticle of claim 25, wherein the nanoparticle has icosahedral symmetry.

27. 27. The nanoparticle of claim 26, the first multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO:7) or I53-50AΔCys (SEQ ID NO:67); The nanoparticle, wherein the second multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50B (SEQ ID NO: 8) or I53-50B.4PosT1 (SEQ ID NO: 34).

28. 27. The nanoparticle of claim 26, the first multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-dn5B (SEQ ID NO:75); The nanoparticle, wherein the second multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-dn5A (SEQ ID NO: 74).

29. The nanoparticle according to any one of claims 18 to 26, the first polypeptide component shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a polypeptide sequence selected from SEQ ID NOs:93-96; The nanoparticle, wherein the second polypeptide component shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50B (SEQ ID NO:8) or I53-50B.4PosT1 (SEQ ID NO:34).

30. The nanoparticle according to any one of claims 18 to 26, the first polypeptide component shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a polypeptide sequence selected from SEQ ID NOs: 101-102; The nanoparticle, wherein the second polypeptide component shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-dn5A (SEQ ID NO: 74).

31. A pharmaceutical composition for use as a vaccine comprising a polypeptide according to any one of claims 1 to 17 or a nanoparticle according to any one of claims 18 to 30, optionally comprising one or more pharmaceutically acceptable excipients.

32. 32. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition comprises at least one adjuvant.

33. 33. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition comprises an oil-in-water emulsion.

34. 34. The pharmaceutical composition of claim 33, wherein the pharmaceutical composition comprises a squalene-based oil-in-water emulsion.

35. 33. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition comprises a toll-like receptor (TLR) immunostimulant.

36. 33. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition comprises squalene, SLA, GLA, R848, IMQ, 3M-052, CpG, saponin (QS21), or a combination thereof.

37. A polynucleotide comprising a polynucleotide sequence encoding the polypeptide of any one of claims 1 to 17 or the nanoparticle of any one of claims 18 to 30.

38. A vector comprising a polynucleotide comprising a polynucleotide sequence encoding any of the polypeptides of any of claims 1 to 17 or the nanoparticles of any of claims 18 to 30.

39. 1. A method of generating an immune response in a subject infected with rabies virus, comprising: The method comprises administering a polypeptide according to any one of claims 1 to 17, a nanoparticle according to any one of claims 18 to 29, a pharmaceutical composition according to any one of claims 30 to 35, a polynucleotide according to claim 36, or a vector according to claim 37 in an amount effective to generate an immune response.

40. 1. A method for immunizing a subject against infection by rabies for a subject infected with rabies virus, comprising: The method comprises administering a polypeptide according to any one of claims 1 to 17, a nanoparticle according to any one of claims 18 to 29, a pharmaceutical composition according to any one of claims 30 to 35, a polynucleotide according to claim 36, or a vector according to claim 37 in an amount effective to generate an immune response.

41. 1. A method of administering post-exposure prophylaxis to a subject infected with rabies virus, comprising: The method comprises administering a polypeptide according to any one of claims 1 to 17, a nanoparticle according to any one of claims 18 to 29, a pharmaceutical composition according to any one of claims 30 to 35, a polynucleotide according to claim 36, or a vector according to claim 37 in an amount effective to generate an immune response.

42. The method of any one of claims 39 to 41, wherein the immune response comprises a humoral immune response.

43. 43. The method of any one of claims 39 to 42, wherein the immune response comprises a polyclonal antibody response against rabies G protein.

44. 44. The method of any one of claims 39 to 43, wherein the immune response comprises a neutralizing antibody response against rabies virus.

45. 45. The method of any one of claims 39 to 44, wherein the method generates a protective immune response against rabies virus.

46. 46. ​​The method of any one of claims 39 to 45, wherein the method generates neutralizing antibodies against rabies virus.

47. 47. The method of any one of claims 39 to 46, wherein the administering step comprises intramuscular or subcutaneous injection.

48. 48. The method of any one of claims 39 to 47, wherein said method results in the production of rabies-specific neutralizing antibodies in said subject in need thereof.

49. 49. The method of any one of claims 39-48, wherein said method results in an increase in rabies-specific neutralizing antibodies in said subject in need thereof that is at least about a 2-fold, at least about a 3-fold, at least about a 4-fold, at least about a 5-fold, at least about a 10-fold, at least about a 15-fold, at least about a 20-fold, or at least about a 25-fold increase compared to rabies-specific neutralizing antibodies in the same subject prior to said administering step.

50. 50. The method of any one of claims 39 to 49, wherein the method generates a neutralization titer of at least 0.5 IU / mL in a Rapid Fluorescent Focus Inhibition Test (RFFIT) and / or a Fluorescent Virus Antibody Neutralization (FAVN) test.

51. 51. The method of any one of claims 39 to 50, wherein the subject is a non-human animal.

52. 52. The method of any one of claims 39 to 51, wherein the subject is a companion animal.

53. The method of any one of claims 39 to 52, wherein the subject is a human.

54. A host cell comprising a polynucleotide comprising a polynucleotide sequence encoding the nanoparticle of any one of claims 1 to 17 or any one of claims 18 to 30.

55. 1. A method of producing a vaccine, comprising: Culturing the host cell of claim 51 in a culture medium such that the host cell secretes the first polypeptide component into the culture medium; purifying said first polypeptide component from said culture medium; mixing the first polypeptide component with a second polypeptide component, wherein the first and second polypeptide components self-assemble to form nanoparticles; and / or purifying the nanoparticles. The method comprising:

56. A kit comprising the polypeptide of any one of claims 1 to 17, the nanoparticle of any one of claims 18 to 30, the pharmaceutical composition of any one of claims 31 to 36, the polynucleotide of claim 37, or the vector of claim 38.

Images