Vaccines containing novel nanoparticle scaffolds

Abstract

The present invention provides novel engineered nanoparticle scaffold sequences derived from the I3-01 protein. Compared to known I3-01 proteins or variants thereof, the novel I3-01-derived scaffold sequences of the present invention contain an extended N-terminal helix. Vaccine constructs containing various immunogenic proteins displayed on the novel nanoparticle scaffold sequences described herein are also provided herein. Vaccine constructs of the present invention include, for example, nanoparticles displaying tandem repeats of influenza M2e protein or HCV E2 core protein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 385,224, filed November 29, 2022, currently pending. The entire disclosure of the priority application is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence Listing This application includes by reference a sequence listing, 2189_1PC_Sequence Listing, in XML format, created on November 9, 2023, which is 37KB in content. [Background technology]

[0003] Influenza viruses (IAV) belong to the Orthomyxoviridae family and can be classified into four types: A, B, C, and D. All influenza viruses are enveloped, negative-sense, single-stranded RNA viruses with segmented genomes. Influenza A and B viruses (IAV and IBV) contain eight gene segments encoding at least 17 proteins. The most abundant surface glycoprotein, hemagglutinin (HA), enables the virus to bind to host cell receptors and mediates cell entry. Neuraminidase (NA) aids in the release of virus particles through cleavage of residues on the surface of host cells. Matrix 1 protein (M1) aids in viral budding from the plasma membrane of infected cells, and matrix 2 protein (M2) promotes viral entry and maintenance of pH during viral replication in host cells. IAV can be further classified by subtype based on the antigenic properties of two surface glycoproteins, HA (HA1-18) and NA (NA1-11). While IAVs can infect many hosts, IBVs are restricted to humans and have diverged into two lineages (Victoria and Yamagata) through intra-host evolution. While influenza viruses of avian origin recognize α-2,3 sialic acid receptors, human influenza viruses preferentially bind to α-2,6 sialic acid receptors in the upper respiratory tract. Influenza viruses utilize two mechanisms to evade the immune system. Antigenic drift, consisting of small changes introduced into the HA and NA under immune pressure, is the cause of annual human influenza epidemics. Antigenic shift occurs when complete changes in the HA and / or NA genes occur in IAVs due to their large animal reservoir. Antigenic shift results in novel IAV strains with increased infectivity in humans and is a major cause of pandemics.

[0004] Since 1940, seasonal influenza vaccines have been used as an efficient and cost-effective tool to minimize influenza epidemics and improve public health. Current vaccines use inactivated or live-attenuated strains. The most common type of inactivated virus vaccine, called a split vaccine, uses detergents or chemicals to disrupt virus particles. Live-attenuated vaccines use cold-adapted live viruses that do not replicate at human body temperature and are generally administered intranasally to induce strong local immunity. Subunit vaccines utilize viral HA or NA proteins that are partially purified after chemical or detergent splitting. The virus strains selected for the quadrivalent vaccine, including H1N1, H3N2, and two Flu B strains, are produced in chicken eggs. However, due to the tendency of influenza viruses to mutate and evade immune responses, current influenza vaccines must be updated annually to include predicted strains. Strain mismatches often result in low efficacy, highlighting the need for better vaccines. Summary of the Invention [Problem to be solved by the invention]

[0005] There is a strong unmet need in the medical field for more reliable and effective influenza vaccines. The present invention addresses this need and other unmet needs in the art. [Means for solving the problem]

[0006] In one aspect, the present invention provides N-terminally extended I3-01 nanoparticle scaffold sequences. These scaffold sequences comprise an extended N-terminal helix relative to the N-terminal helix of the original or wild-type I3-01 scaffold sequence. In some embodiments, the novel I3-01-derived NP scaffold sequences of the present invention comprise a heterologous helix motif of about 6 to about 12 amino acid residues fused to the N-terminus of the I3-01 scaffold sequence set forth in SEQ ID NO: 27 (I3-01v9). In some of these embodiments, the inserted heterologous helix motif comprises AKLAEELQK (SEQ ID NO: 25), a conservatively modified variant thereof, or a substantially identical sequence thereof. Some of the N-terminally extended I3-01 nanoparticle scaffold sequences of the present invention comprise SEQ ID NO: 4, a conservatively modified variant thereof, or a substantially identical sequence thereof. In a related aspect, the present invention provides self-assembled nanoparticles formed with the novel N-terminally extended I3-01 nanoparticle scaffold sequences of the present invention.

[0007] In another aspect, the present invention provides nanoparticle vaccine constructs containing an immunogenic protein or polypeptide immunogen fused to an N-terminally extended I3-01 nanoparticle scaffold sequence. The N-terminally extended I3-01 nanoparticle scaffold sequence in these NP vaccine constructs comprises an extended N-terminal helix compared to the N-terminal helix of the original or wild-type I3-01 scaffold sequence. In some of these vaccine constructs, the N-terminally extended I3-01 nanoparticle scaffold sequence comprises a heterologous helix motif of about 6 to about 12 amino acid residues fused to the N-terminus of the I3-01 scaffold sequence set forth in SEQ ID NO: 27 (I3-01v9). In some embodiments, the extended heterologous helix motif comprises AKLAEELQK (SEQ ID NO: 25), a conservatively modified variant thereof, or a substantially identical sequence thereof. In some vaccine constructs, the N-terminally extended I3-01 nanoparticle scaffold sequence comprises SEQ ID NO: 4, a conservatively modified variant thereof, or a substantially identical sequence thereof. Typically, the immunogenic protein to be displayed in the vaccine construct is fused to the N-terminus of the N-terminally extended I3-01 nanoparticle scaffold sequence via a linker at its C-terminus. In some embodiments, the linker used comprises GGGGS (SEQ ID NO: 3).

[0008] In some vaccine constructs of the present invention, the displayed polypeptide immunogen is an influenza fusion polypeptide containing two or more tandem repeats of the influenza M2 protein ectodomain (M2e). In some other constructs, the displayed polypeptide immunogen is an HCV immunogenic protein. In some embodiments, the displayed tandem influenza M2e repeats are separated by a peptide spacer, e.g., GGGG (SEQ ID NO: 9). In some influenza vaccine constructs, at least one of the displayed M2e tandem repeats contains a missense mutation at the conserved Cys17 and Cys19 residues. In some of these embodiments, the missense mutation comprises a substitution of each of the two Cys residues with an amino acid residue having an uncharged polar side chain. In various embodiments, each of the Cys residues is independently substituted with an amino acid residue selected from the group consisting of serine, glycine, asparagine, glutamine, threonine, and tyrosine. In some preferred embodiments, two Cys residues in the same M2e sequence are both substituted with Ser.

[0009] In some influenza vaccines of the invention, the displayed immunogenic protein contains three tandem M2e sequences. In some of these embodiments, the three tandem M2e sequences are independently human M2e sequences, avian / pig consensus M2e sequences, or human / pig consensus M2e sequences, except for missense mutations at residues Cys17 and Cys19 in at least two of the three tandem M2e sequences. In some of these embodiments, the displayed influenza fusion polypeptide comprises, in any order, human M2e (SEQ ID NO: 2), an avian / pig consensus M2e sequence (SEQ ID NO: 30), each with Cys17 and Cys19 substituted with Ser residues, and a human / pig consensus M2e sequence (SEQ ID NO: 31), each with Cys17 and Cys19 substituted with Ser residues. In some embodiments, the displayed influenza M2e tandem repeat fusion polypeptide comprises, in any order, human M2e (SEQ ID NO:29), each with Cys17 and Cys19 substituted with a Ser residue, an avian / porcine consensus M2e sequence (SEQ ID NO:30), and a human / porcine consensus M2e sequence (SEQ ID NO:31), each with Cys17 and Cys19 substituted with a Ser residue. In some embodiments, the displayed influenza M2e tandem fusion polypeptide comprises SEQ ID NO:23, SEQ ID NO:24, a conservatively modified variant thereof, or a substantially identical sequence thereof. Some of these influenza vaccine constructs comprise the subunit or scaffold sequence set forth in SEQ ID NO:11, SEQ ID NO:12, a conservatively modified variant thereof, or a substantially identical sequence thereof.

[0010] In addition to the immunogenic protein sequence, the novel I3-01 scaffold-based vaccine constructs of the present invention can further comprise a locking domain and a T cell epitope at the C-terminus. For example, they can comprise the locking domain set forth in SEQ ID NO:5 and the T cell epitope set forth in SEQ ID NO:6 at the C-terminus. Some of these vaccine constructs comprise the subunit or scaffold sequence set forth in SEQ ID NO:14 or SEQ ID NO:15, conservatively modified variants thereof, or substantially identical sequences thereof. Some vaccine constructs of the present invention can further comprise an N-terminal leader sequence. Some of these vaccine constructs comprise the subunit or scaffold sequence set forth in SEQ ID NO:17 or SEQ ID NO:18, conservatively modified variants thereof, or substantially identical sequences thereof. Some vaccine constructs of the present invention can further comprise an N-terminal leader sequence and a locking domain and a T cell epitope at the C-terminus. Some of these vaccine constructs comprise the subunit or scaffold sequence set forth in SEQ ID NO:20 or SEQ ID NO:21, conservatively modified variants thereof, or substantially identical sequences thereof.

[0011] In some vaccine constructs of the present invention, the displayed immunogenic protein is an HCV immunogen, such as an E2 core or E1E2 dimer protein. In some of these embodiments, the displayed protein contains an HCV E2 core set forth in any one of SEQ ID NOS: 32-35. In some embodiments, the displayed HCV immunogenic protein comprises two tandem copies of an E2 core sequence. In some of these embodiments, the two E2 core sequences are derived from different HCV isolates. For example, the two tandem E2 core sequences may comprise SEQ ID NOS: 32 and 33, or SEQ ID NOS: 34 and 35, respectively. Some HCV vaccine constructs of the present invention further comprise a locking domain and a T cell epitope at the C-terminus. For example, the vaccine construct may comprise a locking domain set forth in SEQ ID NOS: 5 and / or a T cell epitope set forth in SEQ ID NOS: 6. Some of these HCV vaccines contain subunit or scaffold sequences with different structural motifs set forth, from N- to C-terminus, respectively: (a) SEQ ID NO:4, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:5, and SEQ ID NO:6, or (b) SEQ ID NO:4, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:5, and SEQ ID NO:6. Some other constructs contain subunit or scaffold sequences that are conservatively modified variants or substantially identical sequences of one of these exemplified scaffold sequences.

[0012] In another aspect, the present invention provides a polynucleotide sequence encoding one subunit or scaffold sequence of the nanoparticle vaccine constructs described herein. Vectors or expression constructs having one or more of these polynucleotide sequences are also encompassed by the present invention. Pharmaceutical compositions or kits comprising the nanoparticle vaccine constructs or encoding polynucleotide sequences described herein are further provided in the present invention.

[0013] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims. [Brief explanation of the drawings]

[0014] [Figure 1-1] 1 is a rational design of I3-01v9a to achieve optimal display of monomeric antigens on nanoparticle (NP) surfaces. The complete amino acid sequence of the I3-01v9a scaffold (SEQ ID NO: 4) is shown. [Figure 1-2] 1 is a rational design of I3-01v9a to achieve optimal display of monomeric antigens on nanoparticle (NP) surfaces. The complete amino acid sequence of the I3-01v9a scaffold (SEQ ID NO: 4) is shown. [Figure 2A] Construct design of HCV E2 core I3-01v9a nanoparticles. Restriction sites and linkers between the E2 core and the nanoparticle scaffold are shown (SEQ ID NO: 36). [Figure 2B] EM images of three HCV E2 core I3-01v9a nanoparticles. [Figure 2C] 1 shows the construct design of HCV tandem E2 core I3-01v9a nanoparticles. The connecting sequences between the two E2 cores (SEQ ID NO: 3) and between the second E2 core and the nanoparticle scaffold (SEQ ID NO: 36) are shown. [Figure 2D] SEC and EM analysis of two tandem E2 core I3-01v9a nanoparticles. SEC profile of two expression volumes in ExpiCHO cells: 50 ml vs. 200 ml. [Figure 3A] M2e-based vaccine construct design. Left: hM2e structure and sequence (SEQ ID NO: 2). Center: Model of hM2e-5GS-1TD0 trimer; Right: Models of hM2e-5GS-FR, hM2e-5GS-E2p-LD4-PADRE, and hM2e-5GS-I3-01v9a-LD7-PADRE 1c-SApNP. [Figure 3B] Schematic diagram of the expression and purification of 1c-SApNP. [Figure 3C] SEC profiles of hM2e trimer and 1c-SApNP. [Figure 3D] 10 is a negative staining EM micrograph of Fab148-purified hM2e 1c-SApNP. [Figure 4A] Mouse immunization / challenge schedule. [Figure 4B] Survival and weight loss after A / Puerto Rico / 8 / 1934 (PR8) H1N1 virus challenge. [Figure 4C] Survival rate and weight loss after A / Hong Kong / 1 / 1968 (HK68) H3N2 virus challenge. [Figure 4D] ELISA of hM2e-specific antibody responses in mouse sera to hM2e probes. [Figure 5A] Negative stain EM image of tandem M2e 1c-SApNPs. The M2ex3-5GS-FR sample held at 70 °C for 10 min showed no visible change in structure and bound to anti-M2e antibodies with nearly identical affinity. [Figure 5B] Survival rate and weight loss in the alum adjuvant group after A / Puerto Rico / 8 / 1934 (PR8) H1N1 virus challenge. [Figure 5C] Survival and weight loss in the AddaVax adjuvant group following A / Puerto Rico / 8 / 1934 (PR8) H1N1 virus challenge. DETAILED DESCRIPTION OF THE INVENTION

[0015] I. Overview Various antigens and vaccine strategies have been investigated to develop a universal influenza vaccine. The most common antigen is HA, and recent vaccine design efforts have focused on the conserved stem region or conserved epitopes within the head domain. These approaches include using headless HA, chimeric HA, and mosaic HA. NA is also an attractive target for broadly neutralizing antibodies (bNAbs) against seasonal and pandemic strains. The ectodomain of the M2 protein (M2e) is a highly conserved target for universal IAV vaccines. M2e is small (approximately 23 aa) and non-immunogenic, but can be conjugated to large carriers to induce cross-protection and reduce viral replication. For T cell targeting, internal proteins such as the nucleoprotein and M1 have been investigated. The use of adjuvants such as MF59 and AS03 has been shown to significantly improve influenza vaccine efficacy. Therefore, adjuvant effects must be carefully tested in the development of universal influenza vaccines targeting the subdominant HA stem and M2e.

[0016] M2e-based vaccines confer protection through mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), which eliminate virus-infected cells. M2e has been conjugated to various carriers for vaccine development. One of the early M2e vaccines used hepatitis B core protein (HBc) as a carrier. Tobacco mosaic virus (TMV) coat protein, keyhole limpet hemocyanin (KLH), rotavirus NSP4, GCN4, bacterial flagellin, and liposomes have since been tested in M2e vaccine development. As the search for better carriers continues, numerous vaccine candidates have advanced to human trials, thus providing important feedback for future M2e vaccine development. Adjuvanted M2e-HBc fusion protein induced anti-M2e antibodies in 90% of cases in a phase I trial (clinicaltrials.gov: NCT00819013) and was well tolerated. However, vaccine-induced anti-M2e antibody responses rapidly declined. Although the M2e-flagellin fusion vaccine was highly immunogenic, higher doses caused undesirable side effects, such as fever, diarrhea, fatigue, headache, and myalgia, in a Phase I trial (clinicaltrials.gov: NCT00921206). Vaccines combining M2e with multiple cytotoxic T lymphocyte (CTL) epitopes were able to stimulate strong cell-mediated immunity in humans (clinicaltrials.gov: NCT01181336). However, the T cell response was narrow and slow, making this vaccine inappropriate for emerging pandemics. Therefore, carriers, adjuvants, and the correct balance between antibody and T cell responses are major challenges facing M2e vaccine development.

[0017] We designed multilayered, single-component self-assembling protein nanoparticles (lc-SApNPs) based on two bacterial proteins, E2p and I3-01, which self-assemble into 22-25 nm 60-mers as carriers of foreign antigens for vaccine development. These lc-SApNPs were successfully applied to HIV-1, HCV, Ebola virus (EBOV), and SARS-CoV-2 to create nanoparticle vaccines. In the embodiments described herein, the invention encompasses a novel I3-01-derived NP platform that has demonstrated activity displaying immunogenic proteins, such as influenza M2e protein and HCV immunogens. The invention also encompasses broadly protective vaccines containing immunogenic proteins (e.g., HCV E2 core protein and influenza M2e protein) displayed on the novel NP scaffold. Details for making and using the compositions and methods encompassed by the invention are described below.

[0018] The described nanoparticle scaffolds and vaccine constructs can have a variety of applications in clinical settings. For example, the novel I3-01-derived NP scaffolds described herein, such as I3-01v9a (SEQ ID NO: 4), can be used to display a variety of other monomeric antigens in addition to the HCV and influenza immunogenic proteins exemplified herein. Vaccines constructed in this manner, such as the HCV vaccines or tandem hM2e vaccines exemplified herein, can be used as broadly protective vaccines. Using influenza vaccines as an example, they can be added to seasonal vaccines (e.g., HCV or influenza vaccines) as "performance boosters" to improve protection against epidemic (human strains) and pandemic (swine and avian strains) influenza viruses. In other applications, novel I3-01 scaffold-based vaccines (e.g., M2e-displayed vaccines) can be combined with other vaccine modalities, such as hemagglutinin (HA) stem-based vaccines, to create truly universal influenza vaccines.

[0019] The vaccines of the present invention also have several advantageous properties compared to related vaccines known in the art. Using influenza vaccines as an example, the uniform distribution of antigen-immobilization sites on the surface and the very high level of surface exposure make the novel I3-01 scaffold described herein an ideal nanoparticle platform for presenting monomeric antigens. The icosahedral symmetry and high-density surface display make 1c-SApNPs an ideal carrier for the multivalent display of suitable antigens, such as influenza M2e. The single-segment or tandem construct design allows for optimal display on the nanoparticle surface and can generate high-quality antibody responses. Furthermore, gene fusion combined with self-assembly allows for robust production of 1c-SApNPs in laboratory and industrial settings. As demonstrated herein, vaccines can be produced in ExpiCHO cells with reasonable yields and extremely high purity after immunoaffinity (Fab148) purification. CHO is one of the main mammalian cell lines used in the industrial production of protein therapeutics and vaccines. Because ExpiCHO is a transient version of this CHO cell line, vaccines obtained from ExpiCHO cells (e.g., influenza M2e vaccines) are expected to have the same properties as those obtained from industrial CHO cell lines. This enables GMP production of 1c-SApNP vaccines for human use. Furthermore, the multilayer structure ensures the thermal stability of 1c-SApNPs (e.g., influenza M2e 1c-SApNPs), allowing for various delivery routes and combination with other related vaccines. As exemplified herein, high-temperature incubation at 70°C for 10 minutes did not cause any structural changes, and ELISA showed nearly identical binding to M2e-specific antibodies such as Fab65 and Fab148. The excellent thermal stability of the 1c-SApNPs (e.g., influenza M2e 1c-SApNPs) of the present invention allows them to be used in harsh conditions, such as when embedded in self-dissolving microneedles for transdermal immunization.

[0020] Unless otherwise specified herein, the various compositions and methods of the present invention can all be produced or carried out according to the procedures exemplified herein or routinely practiced methods known in the art. For example, see Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, JN Abelson, M.I. Simon, G.B. Fields (Editors), Academic Press; 1st edition (1997) (ISBN-13:978-0121821906); U.S. Patent Nos. 4,965,343 and 5,849,954; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, (3 rded., 2000); Brent et al., Current Protocols in Molecular Biology, John Wiley&Sons, Inc. (ringbou ed., 2003); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol.152, SLBerger and ARKimmerl Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan, et.al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.) and Culture of Animal Cells: A Manual of Basic Technique by R.Ian See Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998). The following chapters provide further guidance for carrying out the compositions and methods of the present invention.

[0021] II. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1999). st ed.,1992);Oxford Dictionary of Biochemistry and Molecular Biology,Smith et al.(Eds.),Oxford University Press(revised ed.,2000);Encyclopaedic Dictionary of Chemistry,Kumar(Ed.),Anmol Publications Pvt.Ltd.(2002);Dictionary of Microbiology and Molecular Biology,Singleton et al. al.(Eds.),John Wiley&Sons(3 rd ed.,2002);Dictionary of Chemistry,Hunt(Ed.),Routledge(1 st ed., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4 th ed., 2000). Further clarification of some of these terms as they apply specifically to the present invention is provided herein.

[0022] As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly dictates otherwise. For example, "Env-derived trimers" can refer to both single or multiple Env-derived trimer molecules and can be considered equivalent to the phrase "at least one Env-derived trimer."

[0023] As used herein, the terms "antigen" and "immunogen" are used interchangeably to refer to a substance, typically a protein, that can induce an immune response in a subject. The terms also refer to a protein that is immunologically active in the sense that, when administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector encoding the protein), it can induce a humoral and / or cellular immune response against the protein. Unless otherwise specified, the term "vaccine immunogen" is used interchangeably with "protein antigen" or "immunogenic polypeptide."

[0024] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that encode identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. With respect to polypeptide sequences, "conservatively modified variants" refer to variants that have conservative amino acid substitutions, i.e., amino acid residues substituted with other amino acid residues having side chains with similar charges. Families of amino acid residues with side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0025] Epitope refers to an antigenic determinant. It is a specific chemical group or peptide sequence on a molecule that is antigenic and induces a specific immune response. For example, an epitope is the region of an antigen to which B cells and / or T cells respond. Epitopes can be formed from both contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein.

[0026] An effective amount of a vaccine or other agent is an amount sufficient to produce a desired response, such as reducing or eliminating the signs or symptoms of a condition or disease, such as seasonal influenza. For example, this may be the amount necessary to inhibit viral replication or to measurably change an outward symptom of viral infection, such as an increase in T cell count in the case of influenza infection. Generally, this amount is sufficient to measurably inhibit viral replication or infectivity. When administered to a subject, a dosage that achieves a target tissue concentration (e.g., in lymphocytes) shown to achieve in vitro inhibition of viral replication is generally used. In some embodiments, an "effective amount" is an amount that treats (including prevents) one or more symptoms and / or underlying causes of any of the disorders or diseases, e.g., to treat influenza infection. In some embodiments, an effective amount is a therapeutically effective amount. In some embodiments, an effective amount is an amount that prevents the onset of one or more signs or symptoms of a particular disease or condition, e.g., one or more signs or symptoms associated with the disease.

[0027] As used herein, a fusion protein is a recombinant protein containing amino acid sequences from at least two unrelated proteins linked together via peptide bonds to form a single protein. The unrelated amino acid sequences can be linked directly to each other or can be linked using a linker sequence. As used herein, proteins are unrelated if their amino acid sequences are not normally found linked together via peptide bonds in one or more of their natural environments (e.g., the interior of a cell). For example, the amino acid sequence of an enzyme from the anaerobic bacterium Thermotoga maritima, from which the I3-01 NP scaffold is derived, is not naturally linked via peptide bonds to the amino acid sequence of influenza M2e or HCV E2 core.

[0028] An immunogen is a protein or portion thereof that can induce an immune response in a mammal, such as a mammal infected with or at risk of infection by a pathogen. Administration of the immunogen can result in protective and / or prophylactic immunity against the pathogen of interest.

[0029] An immune response refers to the response of cells of the immune system, such as B cells, T cells, or monocytes, to a stimulus. In some embodiments, the response is specific to a particular antigen (an "antigen-specific response"). In some embodiments, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. In some other embodiments, the response is a B cell response, resulting in the production of specific antibodies.

[0030] An immunogenic composition refers to a composition comprising an immunogenic polypeptide that induces a measurable CTL response against a virus expressing the immunogenic polypeptide, or that induces a measurable B cell response (such as the production of antibodies) against the immunogenic polypeptide.

[0031] As used herein, amino acid numbering or amino acid numbering system refers to the numbering or linear position of amino acid residues in an immunogenic protein or polypeptide (e.g., influenza M2e) from a prototype strain or species. Normalized sequence alignment allows for comparison of sequences of different orthologs of the same immunogenic protein (e.g., M2e) from other strains or species, or engineered versions of the same protein described herein, with that of the prototype sequence. Using such standard or normalized amino acid numbering, conserved amino acid residues in immunogenic proteins from various viral strains or engineered proteins can be easily identified and designated. For example, unless otherwise specified herein, the amino acid numbering for the M2e protein can be based on the consensus sequence of the human influenza M2e protein. According to this numbering, the conserved Cys residues to be mutated are designated residues Cys17 and Cys19 for all influenza strains.

[0032] Sequence identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percent identity; the higher the percentage, the more identical the sequences. Two sequences are "substantially identical" if, when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, the two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a designated region, or, if not specified, over the entire sequence). Identity may exist over a region at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0033] Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence identity / similarity when aligned using standard methods. Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al., Computer Appls. in the Biosciences 8,155-65, 1992; and Pearson et al. al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990 presents a detailed discussion of sequence alignment methods and homology calculations.

[0034] The term "subject" refers to any animal classified as a mammal, e.g., a human or non-human mammal. Examples of non-human animals include dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Unless otherwise specified, the terms "patient" or "subject" are used interchangeably herein. Preferably, the subject is a human.

[0035] The terms "treating" or "alleviating" include the administration of a compound or agent to a subject to prevent or delay the onset of symptoms, complications, or biochemical manifestations of a disease (e.g., influenza infection), alleviate symptoms, or halt or inhibit further progression of a disease, condition, or disorder. Subjects in need of treatment include those already suffering from a disease or disorder as well as those at risk of developing the disorder. Treatment can be prophylactic (to prevent or delay the onset of a disease or to prevent the manifestation of its clinical or subclinical symptoms), or can be therapeutic suppression or alleviation of symptoms after the manifestation of a disease.

[0036] A vaccine refers to a pharmaceutical composition that induces a preventative or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine induces an antigen-specific immune response against an antigen of a pathogen, such as a viral pathogen, or a cellular component correlated with a pathological condition. A vaccine may comprise a polynucleotide (such as a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (such as a disclosed antigen), a virus, a cell, or one or more cellular components. In some embodiments of the present invention, the vaccine or vaccine immunogen or vaccine composition is expressed from a fusion construct and self-assembles into nanoparticles that display the immunogenic polypeptide or protein on their surface.

[0037] A vaccine (e.g., an influenza or HCV vaccine) refers to an immunogenic composition capable of stimulating an immune response that is administered to prevent, mitigate, or treat disease or infection (e.g., influenza virus infection). Vaccines can include, for example, attenuated or killed (e.g., split) pathogens (e.g., viruses), virus-like particles (VLPs) and / or antigenic polypeptides or DNA derived therefrom, or any recombinant version of such immunogenic substances.

[0038] Virus-like particles (VLPs) refer to non-replicating viral shells derived from any of several viruses. VLPs are generally composed of one or more viral proteins, such as, but not limited to, proteins called capsid, coat, shell, surface, and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can spontaneously form upon recombinant expression of proteins in an appropriate expression system. Methods for producing specific VLPs are known in the art. The presence of VLPs after recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as electron microscopy, biophysical characterization, etc. See, e.g., Baker et al. (1991) Biophys. J. 60:1445-1456; and Hagensee et al. (1994) J. Virol. 68:4503-4505. For example, VLPs can be isolated by density gradient centrifugation and / or identified by characteristic density banding. Alternatively, cryo-electron microscopy can be performed on vitrified aqueous samples of the VLP preparation in question and images recorded under appropriate exposure conditions.

[0039] Self-assembled nanoparticles refer to ball-shaped protein shells with well-defined surface geometries formed by identical copies of non-viral proteins that can automatically assemble into nanoparticles with diameters of tens of nanometers and similar appearances to VLPs. Known examples include ferritin (FR), which is conserved across species and forms a 24-mer, as well as B. stearothermophilus dihydrolipoyl acyltransferase (E2p), Aquifex aeolicus lumazine synthase (LS), I3-01-derived variants, and Thermotoga maritima encapsulin, all of which form 60-mers. Self-assembled nanoparticles can form spontaneously upon recombinant expression of proteins in appropriate expression systems. Methods for generating, detecting, and characterizing nanoparticles can be performed using the same techniques developed for VLPs.

[0040] III. Novel NP scaffolds with improved activity The present invention provides novel nanoparticle scaffold sequences suitable for displaying various viral immunogenic proteins to elicit potent neutralizing antibody responses. These rationally designed and functionally tested scaffold sequences are based on the I3-01 protein. I3-01 is an engineered protein (SEQ ID NO: 22) that can self-assemble into ultrastable nanoparticles. The original ("unextended" or "wild-type") I3-01 protein was described by Hsia et al., Nature 535, 136-139, 2016. Several ultrastable nanoparticle scaffolds derived from I3-01 have previously been developed and used to display viral proteins, such as those derived from HIV-1 and HCV. See, for example, International Publication Nos. WO 21 / 021603, WO 22 / 035739, U.S. Pat. No. 10,906,944, and WO 19 / 089817. To identify novel NP scaffolds with improved activity, we rationally designed and functionally tested known I3-01 mutant scaffolds. The original I3-01 protein and variants known in the art (i.e., unextended I3-01 scaffold sequences) contain the N-terminal helix motif KMEELFKKHK (SEQ ID NO: 26). The novel scaffold sequences of the present invention were obtained by extending the N-terminal helix of an existing I3-01 variant scaffold, such as I3-01v9 (SEQ ID NO: 27), by grafting a heterologous helix motif, followed by rational design using an ensemble-based protein design program. One example of a novel scaffold is I3-01v9a (SEQ ID NO: 4), as exemplified herein. As described in more detail in the Examples, the resulting novel variant I3-01 scaffold (e.g., SEQ ID NO: 4) can provide optimal surface display of monomeric protein antigens.

[0041] I3-01 sequence (SEQ ID NO: 22) without the first Met residue (N-terminal helix is ​​underlined): [ka]

[0042] In various embodiments, the novel I3-01-derived NP scaffolds of the present invention comprise an I3-01 variant sequence (e.g., SEQ ID NO: 27), except for the addition of a helical motif of about 6 to about 12 amino acid residues at the N-terminus. This inserted helical motif results in an extension of the original N-terminal helix KMEELFKKHK (SEQ ID NO: 26) in the I3-01 protein. In some embodiments, the inserted helical motif comprises AKLAEELQK (SEQ ID NO: 25), a conservatively modified variant thereof, or a substantially identical sequence thereof.

[0043] I3-01v9 (SEQ ID NO: 27) (N-terminal helix underlined): [ka]

[0044] I3-01v9a (SEQ ID NO: 4) (N-terminal helix extension underlined): [ka]

[0045] In various embodiments, the novel I3-01-derived NP scaffolds of the invention comprise SEQ ID NO:4, a conservatively modified variant thereof, or a substantially identical sequence thereof (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identical). In some of these embodiments, the helical motif inserted at the N-terminus is identical to SEQ ID NO:25, while the remainder of the scaffold sequence is a conservatively modified variant of SEQ ID NO:27 or a substantially identical sequence of SEQ ID NO:27. In some other embodiments, the entire extended N-terminal helix of the novel I3-01 variant scaffold is identical to the N-terminal helix of I3-01v9a, i.e., AKLAEELQKKMEELFKKHK (SEQ ID NO:28), while the remainder of the sequence is a conservatively modified variant of or a substantially identical sequence to the corresponding sequence of SEQ ID NO:27 (i.e., SEQ ID NO:27 minus the N-terminal helix).

[0046] IV. Immunogenic Polypeptides or Proteins for Producing Vaccine Compositions The novel I3-01-derived nanoparticle scaffolds described herein can be used to construct vaccines that present many different immunogenic proteins, including monomeric polypeptides and multimeric proteins. These include any protein or polypeptide derived from a pathogen to which an immune response may be desired. Thus, vaccine compositions of the present invention can utilize immunogenic polypeptides derived from any virus, bacterium, or other pathogenic organism. Suitable immunogenic polypeptides for the present invention can also be derived from non-pathogenic species, including human proteins, to which the elicited immune response may have a therapeutic effect, alleviate disease symptoms, or improve general health. Generally, immunogenic polypeptides can be any structural or functional polypeptide or peptide comprising at least about 10 amino acid residues. In some embodiments, immunogenic polypeptides comprise a length of about 10 to about 10,000 amino acid residues. In some embodiments, immunogenic polypeptides comprise a length of about 25 to about 2,000 amino acid residues. In some embodiments, immunogenic polypeptides comprise a length of about 50 to about 500 amino acid residues. Thus, immunogenic polypeptides or proteins suitable for the present invention can have a molecular weight of about 1 kDa to about 1,000 kDa, preferably about 2.5 kDa to about 250 kDa. In some more preferred embodiments, the immunogenic polypeptides used have a molecular weight of about 5 kDa to about 25 kDa or about 50 kDa.

[0047] In some embodiments, the immunogenic polypeptide or protein used in the vaccine compositions of the present invention may be derived from a viral surface or core protein (target polypeptide). There are many known viral proteins that are important for viral infection of host cells. Examples include the glycoproteins (or surface antigens, e.g., GP120 and GP41) and capsid proteins (or structural proteins, e.g., P24 protein) of HIV; the surface antigens or core proteins of hepatitis A, B, C, D, or E viruses (e.g., small hepatitis B virus surface antigen (S-HBsAg) and the core proteins, NS3, NS4, and NS5 antigens of hepatitis C virus); the glycoprotein gp350 / 220 of Epstein-Barr virus (EBV), the glycoprotein (G protein) or fusion protein (F protein) of respiratory syncytial virus (RSV); and the surface proteins of herpes simplex viruses HSV-1 and HSV-2. These include, but are not limited to, proteins and core proteins (e.g., glycoprotein D from HSV-2), surface proteins of poliovirus (e.g., gB, gC, gD, gH, and gL), envelope glycoprotein hemagglutinin (H) and fusion protein (F) of measles virus (MV), glycoprotein G of lymphocytic choriomeningitis virus (LCMV), fiber protein and penton base protein of adenovirus, S spike of coronavirus, envelope (E) proteins of flaviviruses such as dengue virus, yellow fever virus, and Zika virus, and non-enveloped capsid proteins of picornaviruses.

[0048] In some preferred embodiments, the immunogen or immunogenic protein displayed on the novel I3-01 NP scaffold is a monomeric protein. Examples of such proteins include, for example, the influenza M2 ectodomain (M2e) protein exemplified herein. As described in more detail below, some embodiments of the influenza vaccines of the present invention include NP vaccines containing a novel I3-01 scaffold (e.g., SEQ ID NO: 4) displaying tandem repeats (e.g., 2, 3, 4, or more copies) of the M2e protein. In some of these embodiments, one or more of the tandem M2e copies contain substitutions at the conserved Cys17 and Cys19 residues to prevent random disulfide bond formation.

[0049] Some other embodiments of the present invention relate to HCV vaccines containing the novel I3-01 NP scaffold (e.g., SEQ ID NO: 4) that displays an HCV immunogenic protein. Typically, the HCV immunogenic protein to be displayed on the NP scaffold is derived from the HCV glycoproteins E1 and E2, which form a heterodimer on the HCV envelope that mediates viral entry into host hepatocytes. In some embodiments, the displayed HCV protein contains the E2 core. The E2 core, as well understood in the art, refers to the portion of E2 that forms the three-dimensional structure recognized by the broadly neutralizing antibody AR3C Fab (Law et al., Nat. Med. 2008;14:25, 2008). As exemplified herein, the I3-01 mutant scaffold of the present invention can be used to display either a single copy of the E2 core protein or a tandem E2 core fusion protein. One specific HCV E2 core protein that can be used in the HCV vaccine construct of the present invention is the redesigned E2mc3 protein described in U.S. Patent No. 11,008,368. As exemplified herein, E2mc3 from various HCV subtypes or isolates can be used, including the E2mc3 sequences of HCV H77, J6, ED43, and UKN3A1.28c isolates (SEQ ID NOS: 32-35, respectively). Conservatively modified variants or substantially identical sequences of these exemplified E2 core sequences can also be used in the HCV vaccine constructs of the invention.

[0050] In some embodiments, the displayed HCV immunogenic protein is a tandem E2 core fusion protein comprising SEQ ID NO:32 and SEQ ID NO:33, in any order. In some other embodiments, the displayed HCV immunogenic protein is a tandem E2 core fusion protein comprising SEQ ID NO:34 and SEQ ID NO:35, in any order. In some other embodiments, the HCV immunogenic protein displayed by the NP scaffold comprises an E1E2 heterodimer, e.g., a rationally redesigned HCV E1E2 dimer. In addition to the HCV-derived immunogenic protein, the novel I3-01 scaffold-displayed HCV vaccine can further contain a locking domain and / or a T cell epitope. For example, the vaccine construct can have an LD7 motif (SEQ ID NO:5) and a PADRE epitope (SEQ ID NO:6) at the C-terminus, as exemplified herein.

[0051] In the construction of the HCV vaccine of the present invention, any E2 core protein sequence, tandem E2 core fusion molecule and E1E2 dimer that are known in the art or can be easily manipulated are suitable.Detailed guidelines for obtaining such HCV immunogenic proteins and constructing NP vaccines containing them are provided in, for example, WO 21 / 021603; McGregor et al., J.Virol.96:e01675-21; Lin et al., Front Immunol.2022;13:831285; Wang et al., Proc.Natl.Acad.Sci.USA 119:e2112008119,2022; Clarke et al., Plant Biotechnol.J.15:1611-21,2017; and Sepulveda-Crespo et al., J.Biomed.Sci 27,78,2020. Various immunogenic proteins or polypeptides (e.g., tandem influenza M2e fusion proteins or tandem HCV E2 core fusion proteins) for display on the novel I3-01 NP scaffold of the present invention can be obtained or produced according to protocols exemplified herein or methods well known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, (3 rd ed., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou ed., 2003).

[0052] V. NP Vaccine Containing the Novel I3-01 NP Scaffold The present invention provides nanoparticle vaccines having the novel I3-01 NP scaffold disclosed herein. As described above, some of the vaccine constructs display an HCV immunogenic protein, such as a tandem E2 core protein. Some other vaccine constructs of the present invention display a single copy of the influenza M2 protein ectodomain (M2e). Several examples of such influenza NP vaccines are illustrated herein. In yet other embodiments, a fusion polypeptide containing tandem repeats of the influenza M2e protein is displayed on the novel I3-01-derived nanoparticle scaffold. In these embodiments, the immunogenic protein displayed on the novel I3-01 scaffold sequence is a fusion polypeptide containing two or more tandem repeats of the influenza M2e sequence. In various embodiments, at least one of the M2e tandem repeats contains missense mutations at the conserved Cys17 and Cys19 residues to prevent random disulfide bond formation. In some of these influenza NP vaccine constructs, the engineered missense mutations are the replacement of each Cys residue with an amino acid residue containing an uncharged polar side chain. For example, each of the two Cys residues in one or more of the tandem M2e repeats can be independently substituted with serine, glycine, asparagine, glutamine, threonine, or tyrosine. In some embodiments, one or both of the Cys residues are substituted with Ser.

[0053] Typically, the tandem M2e fusion polypeptide sequence is fused to the N-terminus of the novel I3-01 scaffold sequence via a linker motif, such as GGGGS (SEQ ID NO: 3) as exemplified herein. Preferably, the tandem M2e repeats in the displayed fusion polypeptide are separated by a short linker or spacer. For example, as exemplified herein, the GGGG (SEQ ID NO: 9) spacer described herein can be used to separate different M2e sequences. Some influenza NP vaccines of the present invention contain three tandem M2e repeats. In various embodiments, the tandem M2e repeats displayed on the novel I3-01 scaffold of the present invention can be identical or different. Orthologous M2e sequences from many species and modified versions thereof are known in the art. See, for example, Mezhenskaya et al., J. Biomed. Sci. 26, 76, 2019. Thus, for example, each of the M2e tandem repeats can independently be a human M2e sequence (SEQ ID NO: 2), an avian / pig consensus M2e sequence (SEQ ID NO: 7), or a human / pig consensus M2e sequence (SEQ ID NO: 8). When M2e sequences from different sources are used in a tandem M2e molecule, the different M2e motifs can be linked in any order. As an example, a tandem M2e repeat sequence in an influenza vaccine of the present invention can contain a human M2e sequence, an avian / pig consensus M2e sequence, and a human / pig consensus M2e sequence. In these embodiments, the three different M2e sequences can be linked to the scaffold sequence in any of six possible sequence orders.

[0054] In some embodiments, at least two of the three tandem M2e repeats contain substitutions at residues Cys17 and Cys19. For example, the human M2e sequence can retain non-mutated residues at Cys17 and Cys19, while the avian / pig consensus M2e sequence and the human / pig consensus M2e sequence contain substituted residues (e.g., all Ser substitutions) at Cys17 and Cys19. In some of these embodiments, all mutated Cys residues are substituted with Ser residues. Thus, the displayed M2e fusion polypeptide can contain, in any order, non-mutated human M2e (SEQ ID NO: 2), mutated avian / pig consensus M2e sequence SLLTEVETPTRNGWE, and / or SLLTEVETPTRNGWE. S K S SDSSD (SEQ ID NO: 30), and the mutated human / pig consensus M2e sequence SLLTEVETPTRSEWE S R S SGSSD (SEQ ID NO: 31). In some embodiments, all three tandem M2e repeats contain substitutions at residues Cys17 and Cys19. Thus, the displayed M2e fusion polypeptide may contain, in any order, mutant human M2e SLLTEVETPIRNEWG S R S NDSSD (SEQ ID NO: 29), variant avian / pig consensus M2e sequence SLLTEVETPTRNGWE S K S SDSSD (SEQ ID NO: 30), and the mutated human / pig consensus M2e sequence SLLTEVETPTRSEWE S R S SGSSD (SEQ ID NO: 31). As a specific example, the fusion M2e polypeptide may contain the sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24, a conservatively modified variant thereof, or a substantially identical sequence thereof.

[0055] In some embodiments, NP vaccines containing the novel I3-01 mutant scaffold of the present invention (e.g., tandem influenza M2e vaccines or HCV E2 vaccines) may contain a trimerization motif, such as SHP or Foldon. Some nanoparticle vaccine compositions can further contain other structural components that function to further enhance the stability and antigenicity of the displayed immunogen. In some embodiments, a locking protein domain (LD) can be inserted into the nanoparticle construct, for example, by covalent fusion to the C-terminus of the nanoparticle subunit. A locking domain can be any dimeric protein capable of forming an interface through specific interactions, such as hydrophobic (van der Waals) contacts, hydrogen bonds, and / or salt bridges. One example of a locking domain that can be used in the vaccines of the present invention is LD7 (SEQ ID NO: 5), as exemplified herein. General guidelines for selecting locking domains and various other examples (e.g., LD4) are described in the art, for example, in WO 19 / 241483, U.S. Pat. No. 10,906,944, and U.S. Pat. No. 11,305,004.

[0056] In some embodiments, the scaffold-containing influenza vaccines of the present invention can also contain T cell epitopes to promote robust T cell responses and direct B cell development toward bNAbs. The T cell epitopes can be positioned at any position relative to other structural components as long as they do not affect the presentation of the engineered HA protein on the nanoparticle surface. Any T cell epitope sequence or peptide known in the art can be used in the practice of the present invention. These include any polypeptide sequence that contains an MHC class II epitope and can effectively activate CD4+ and CD8+ T cells upon immunization, such as a T helper epitope that activates CD4+ T helper cells. See, e.g., Alexander et al., Immunity 1,751-761, 1994; Ahlers et al., J. Clin. Invest. 108:1677-1685, 2001; Fraser et al., Vaccine 32,2896-2903, 2014; De Groot et al., Immunol. Cell Biol. 8:255-269, 2002; and Gene Ther. 21:225-232, 2014. In some embodiments, the T cell epitope inserted into the nanoparticle vaccine construct is the universal pan DR epitope peptide (PADRE), AKFVAAWTLKAAA (SEQ ID NO: 6), as exemplified herein for influenza and HCV vaccines. More detailed information on T cell epitopes suitable for the present invention can be found, for example, in Hung et al., Mole. Ther. 15:1211-19, 2007; Wu et al., J. Biomed. Sci. 17:88, 2010; and Bissati et al., npj Vaccines 2:24, 2017. Other examples of suitable T cell epitopes, such as the D and TpD epitopes (Fraser et al., Vaccine 32, 2896-2903, 2014), have also been described in the art.

[0057] The novel I3-01 scaffold-based nanoparticle vaccines of the present invention can be constructed according to standard recombinant techniques and other methods described in the art, such as He et al., Nat.Comm. 7, 12041, 2016; Kong et al., Nat.Comm. 7, 12040, 2016; He et al., Sci Adv. 4(11):eaau6769, 2018; and International Publication Nos. WO 2017 / 192434, WO 2019 / 089817, and WO 19 / 241483. In various embodiments, the novel I3-01 scaffold-based nanoparticle vaccines can be constructed by fusing an immunogenic protein of interest (e.g., a tandem HCV E2 core or tandem influenza M2e polypeptide) to the I3-01 scaffold subunit. Preferably, the C-terminus of the immunogenic protein sequence is fused to the N-terminus of the nanoparticle subunit sequence. In some embodiments, a short peptide linker or spacer (eg, SEQ ID NOs: 3 and 9) can be inserted between the immunogenic protein sequence and the nanoparticle subunit sequence or between tandem copies of the immunogenic protein.

[0058] Upon recombinant expression (e.g., in ExpiCHO cells as detailed herein), the nanoparticle vaccines of the present invention can be substantially purified by any of the routine procedures. See, for example, Guide to Protein Purification, Ed. Deutscher, Meth. Enzymol. 185, Academic Press, San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982. Substantial purification refers to purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95%, or 98% pure. Once purified, the antigenicity and other properties of the vaccine can also be readily examined using standard methods, such as antigen profiling using known bNAb and non-NAb antibodies, differential scanning calorimetry (DSC), electron microscopy, binding analysis by ELISA, biolayer interferometry (BLI), surface plasmon resonance (SPR), and co-crystallography. Some of these assays are exemplified herein for analyzing the novel I3-01 scaffold HCV or influenza vaccines.

[0059] V. Polynucleotides and Expression Constructs The novel I3-01 scaffolds of the present invention and vaccines based thereon are typically made by first generating an expression construct (i.e., an expression vector) containing operably linked coding sequences for the various structural components described herein. In some embodiments, the vaccine compositions of the present invention are polynucleotide-based (e.g., mRNA-based vaccines). Accordingly, in some related aspects, the present invention provides polynucleotides (e.g., DNA or RNA) encoding subunit sequences of the novel I3-01 scaffold or scaffold-based nanoparticle vaccines, expression vectors harboring such polynucleotides, and host cells (e.g., ExpiCHO cells, as exemplified herein) for producing the novel NP scaffolds and vaccines. Fusion polypeptides encoded by polynucleotides or expressed from vectors are also encompassed by the present invention.

[0060] Polynucleotides and related vectors can be readily prepared using standard molecular biology techniques or the protocols exemplified herein. For example, general protocols for cloning, transfection, transient gene expression, and obtaining stable transfected cell lines are described in the art, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, (3 rded., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou edition, 2003). Introducing mutations into polynucleotide sequences by PCR can be carried out, for example, as described in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al., (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res, 19:967, 1991; and Eckert et al., PCR Methods and Applications, 1:17, 1991.

[0061] The selection of a particular vector depends on the intended use of the fusion polypeptide. For example, the selected vector must be capable of driving the expression of the fusion polypeptide in the desired cell type, regardless of whether that cell type is prokaryotic or eukaryotic. Many vectors contain sequences that enable both prokaryotic vector replication and eukaryotic expression of operably linked gene sequences. Vectors useful in the present invention can replicate autonomously, i.e., the vector exists extrachromosomally and its replication is not necessarily directly linked to the replication of the host cell's genome. Alternatively, vector replication can be linked to the replication of the host's chromosomal DNA; for example, the vector can be integrated into the host cell's chromosome, as achieved by retroviral vectors and stably transfected cell lines. Both viral and non-viral expression vectors can be used to produce immunogens in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors, and human artificial chromosomes (see, e.g., Harrington et al., Nat. Genet. 15:345, 1997), which typically contain expression cassettes for protein or RNA expression. Useful viral vectors include lentiviruses or other retroviruses, adenoviruses, adeno-associated viruses, cytomegaloviruses, herpes viruses, SV40-based vectors, papillomaviruses, HBP Epstein-Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV)-based vectors. See Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

[0062] Depending on the particular vector used to express the fusion polypeptide, various known cells or cell lines can be used in practicing the present invention. Host cells can be any cell into which a recombinant vector carrying the fusion of the present invention can be introduced and into which the vector can drive expression of the fusion polypeptide useful in the present invention. They can be prokaryotic, such as any of several bacterial strains, or eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells, including rodent, monkey, or human cells. Cells expressing the fusion polypeptide of the present invention can be primary cells or established cell lines. Thus, in addition to the cell lines exemplified herein (e.g., CHO cells), numerous other capable host cell lines known in the art can also be used in practicing the present invention. These include, for example, various Cos cell lines, HeLa cells, HEK293, AtT20, BV2, and N18 cells, myeloma cell lines, transformed B cells, and hybridomas.

[0063] The use of mammalian tissue cell cultures to express polypeptides is generally discussed, for example, in Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. Fusion polypeptide expression vectors can be introduced into selected host cells by any of several suitable methods known to those skilled in the art. The method used to introduce a vector encoding a fusion polypeptide into mammalian cells depends on the form of the vector. In the case of a plasmid vector, DNA encoding the fusion polypeptide sequence can be introduced by any of several transfection methods, including, for example, lipid-mediated transfection ("lipofection"), DEAE-dextran-mediated transfection, electroporation, or calcium phosphate precipitation. These methods are described in detail, for example, in Brent et al., supra. Lipofection reagents and methods suitable for transient transfection of a wide variety of transformed and non-transformed or primary cells are widely available, making lipofection an attractive method for introducing constructs into eukaryotic cells, particularly mammalian cells in culture. For example, LipofectAMINE™ (Life Technologies) or LipoTaxi™ (Stratagene) kits are available. Other companies that provide reagents and methods for lipofection include Bio-Rad Laboratories, Clontech, Glen Research, Life Technologies, JBL Scientific, MBI Fermentas, PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.

[0064] Stable expression is preferred for long-term, high-yield production of recombinant fusion polypeptides. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with a fusion polypeptide coding sequence and a selection marker controlled by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.). The selection marker in the recombinant vector confers resistance to selection and allows cells to stably integrate the vector into their chromosomes. Commonly used selection markers include neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., J. Mol. Biol., 150:1, 1981), and hygro, which confers resistance to hygromycin (Santerre et al., Gene, 30:147, 1984). Through appropriate selection, transfected cells can contain integrated copies of the fusion polypeptide coding sequence.

[0065] VI. Pharmaceutical Compositions and Therapeutic Uses The present invention provides pharmaceutical or immunogenic compositions and related therapeutic methods using vaccines, such as influenza or HCV vaccines, based on the novel I3-01 scaffold. In some embodiments, the vaccine compositions can be used to prevent and treat disease or infection (e.g., HCV infection or influenza). In some embodiments, nanoparticles displaying immunogenic proteins (e.g., tandem M2e or HCV E2 core proteins) are included in the pharmaceutical composition. The pharmaceutical composition can be either a therapeutic or prophylactic formulation. Typically, the composition further comprises one or more pharmaceutically acceptable vehicles and may further comprise other therapeutic components (e.g., antibiotics or antivirals). Various pharmaceutically acceptable additives can also be used in the composition.

[0066] Some of the pharmaceutical compositions of the present invention are vaccines. In the case of vaccine compositions, a suitable adjuvant can be further included. Examples of suitable adjuvants include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, MPL™, and IL-12. In some embodiments, the novel I3-01 scaffold-based vaccine of the present invention can be formulated as a controlled-release or timed-release formulation. This can be achieved in a composition containing a slow-release polymer, or via a microencapsulated delivery system or bioadhesive gel. Various pharmaceutical compositions can be prepared according to standard procedures well known in the art. See, e.g., Remington's Pharmaceutical Sciences, 19th Ed. Mack Publishing Company, Easton, Pa., 1995; Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed. Marcel Dekker, Inc., New York, 1978); U.S. Pat. Nos. 4,652,441 and 4,917,893; U.S. Pat. Nos. 4,677,191 and 4,728,721; and U.S. Pat. No. 4,675,189.

[0067] Therapeutic methods of the present invention involve administering a suitable vaccine of the present invention (e.g., an influenza vaccine or HCV vaccine) to a subject having or at risk of developing a disease or infection (e.g., influenza or HCV infection). Using influenza vaccines as an example, the immunogenic compositions of the present invention are typically administered in an amount sufficient to induce an immune response against an influenza virus or group of viruses. For prophylactic use, the immunogenic composition is provided prior to any symptoms, e.g., prior to infection. Prophylactic administration of the immunogenic composition serves to prevent or mitigate subsequent infection. Thus, in some embodiments, the subject being treated is one who has or is at risk of developing an influenza virus infection, e.g., due to exposure or potential exposure to the virus. After administering a therapeutically effective amount of the disclosed therapeutic composition, the subject can be monitored for viral infection, symptoms associated with viral infection, or both. For therapeutic use, the immunogenic composition is provided at or after the onset of symptoms of the disease or infection, e.g., after the onset of influenza symptoms or after diagnosis of a viral infection. Therefore, the immunogenic composition can be provided before expected exposure to the virus, after exposure or suspected exposure to the virus, or after the actual onset of infection to reduce the expected severity, duration, or extent of infection and / or related disease symptoms.The appropriate amount of vaccine can be determined based on the specific disease or condition to be treated or prevented, its severity, the age of the subject, and other personal attributes of the specific subject (e.g., the general state of health of the subject and the robustness of the subject's immune system).The determination of effective dosage is further guided by animal model tests, followed by human clinical trials, and is guided by an administration protocol that significantly reduces the occurrence or severity of target disease symptoms or conditions in the subject.

[0068] The pharmaceutical compositions of the present invention can be combined with other agents known in the art for treating or preventing disease or infection (e.g., influenza virus infection). The pharmaceutical composition and the known antiviral agent can be administered simultaneously or sequentially. The pharmaceutical composition containing the appropriate vaccine of the present invention can be provided as a component of a kit. Such kits may include additional components, including packaging, instructions, and various other reagents, such as buffers, substrates, antibodies or ligands, e.g., control antibodies or ligands, and detection reagents. Optional instructions can also be provided in the kit.

[0069] [Example] The following examples are offered to illustrate, but not to limit, the present invention.

[0070] [Example 1] Rational design of novel I3-01v9a nanoparticle scaffolds To achieve optimal surface display of monomeric protein antigens, we rationally optimized the I3-01v9 nanoparticle scaffold (Figure 1). The N-terminus of I3-01v9 forms a broad triangle, which is desirable for displaying monomeric antigens (Figure 1A). However, the first residue (the antigen anchoring site) is below the nanoparticle surface. Therefore, a long linker must be used to connect the antigen to the N-terminus of I3-01v9, which increases structural instability. Our goal was to extend the N-terminal helix of I3-01v9 so that its first residue is at the same level as the nanoparticle surface. To achieve this goal, we selected the helical backbone from residues 953–982 of the c-MYC transcription factor protein (PDB ID: 6G6L) and grafted it onto the I3-01v9 subunit (SEQ ID NO: 27) using residues E2 and E3 of I3-01v9 for structural compatibility (Figure 1B). The extended N-terminal helix was then truncated to 11 residues so that its first residue was immediately above the nanoparticle surface (Figure 1C). Next, we used the protein structure sampling program CONCOORD to generate 1,000 slightly perturbed conformations of the modified I3-01v9 subunit (Figure 1D). We then used an ensemble-based protein design program, previously used to optimize HIV gp140 and HCV E2 antigens, to predict the first nine amino acids of the 11-residue segment using Cα- and Cβ-based RAPDF scoring functions (Figure 1E). We selected the final design, I3-01v9a (SEQ ID NO: 4), by combining the data from predictions using both energy functions (Figure 1F).

[0071] [Example 2] Display of HCV antigens on I3-01v9a nanoparticle scaffolds This example describes the multivalent display of HCV E2 core and tandem E2 core on the I3-01v9a nanoparticle scaffold. The newly designed I3-01v9a nanoparticle scaffold (SEQ ID NO: 4) was used to display monomeric HCV E2 core of diverse genotypes (Figure 2A). In negative stain EM, E2mc3 I3-01v9a-LD7-PADRE nanoparticles designed for H77 (genotype 1a), HCV1 (genotype 1b), and ED43 (genotype 4) showed well-formed nanoparticles (Figure 2B). The sequences of the different E2mc3 proteins are shown in SEQ ID NOs: 32–35, respectively. Based on this success, we designed a tandem E2 core antigen in which two HCV E2 cores of different genotypes were linked in tandem with a 5GS linker, and this tandem E2 core antigen was linked to I3-01v9a with an enzyme restriction site (AS) (to facilitate molecular cloning) and another 5GS linker (Figure 2C). Based on this design strategy, we co-displayed H77 (genotype 1) and J6 (genotype 2) E2 cores on I3-01v9a-LD7-PADRE nanoparticles, and co-displayed ED43 (genotype 4) and UKN3A1.28c (genotype 3) E2 cores on I3-01v9a-LD7-PADRE nanoparticles. These two nanoparticles were expressed in 50 ml or 200 ml ExpiCHO cells and then characterized by size-exclusion chromatography (SEC) on a Superose 6 column and negative-stain electron microscopy (NEEM) (Figure 2D). In summary, I3-01v9a was successfully used to display monomeric antigens for vaccine development.

[0072] E2mc3 of isolate H77 (SEQ ID NO: 32): [ka]

[0073] E2mc3 of J6 isolate (SEQ ID NO: 33): [ka]

[0074] E2mc3 of isolate ED43 (SEQ ID NO: 34): [ka]

[0075] E2mc3 of isolate UKN3A1.28c (SEQ ID NO: 35): [ka]

[0076] [Example 3] Design and characterization of a single hM2e (1c-SApNP) vaccine Crystal structures are available for human M2e (hM2e) complexed with monoclonal antibodies Fab65 and Fab148. hM2e bound to Fab65 exhibits a β-turn (T5-E8) and 3-terminal cleavage sites. 10hM2e folds into a compact conformation containing helices (I11-W15). Upon binding to Fab148, hM2e adopts a hook structure with an N-terminal β-turn (S2-T5). We used a trimeric scaffold (PDB ID: 1TD0) to display hM2e (S2-D24) with a 5GS spacer, since 1TD0 has been used in previous studies as a C-terminal motif to stabilize the EBOV GP trimer (Figure 3A). Structural modeling indicates that two hM2e peptides on the 1TD0 scaffold span 9.1 nm, measured at P10. The hM2e peptide was fused to 24-mer ferritin (FR) and two "multilayered" 1c-SApNPs, E2p-LD4-PADRE (also called E2p-L4P) and I3-01v9a-LD7-PADRE (also called I3-01v9a-L7P), resulting in vaccine particles of 20.9 nm, 29.1 nm, and 32.4 nm, respectively (Figure 3A). Four hM2e immunogens, one trimer, and three 1c-SApNPs, were transiently expressed in 25 ml of ExpiCHO cells and purified by immunoaffinity chromatography (IAC) using antibodies Fab65 and Fab148 (Figure 3B). Size-exclusion chromatography (SEC) profiles were obtained for the hM2e scaffold and the three 1c-SApNPs on Superdex 75 and Superose 6 columns, respectively (Figure 3C). Although multiple peaks were observed in SEC, IAC-purified 1c-SApNPs showed high purity in negative-stain EM images collected at the Scripps EM Core, indicating well-formed NPs (Figure 3D).

[0077] [Example 4] Single hM2e vaccine immunization and influenza virus challenge The immunogenicity and protective efficacy of the M2e-based vaccine were evaluated in a comprehensive mouse study. Briefly, 10 mice per group were immunized intradermally in the footpad with 10 μg (2.5 μg / footpad) of hM2e-5GS-1TD0, hM2e-5GS-FR, hM2e-5GS-E2p-LD4-PADRE, or hM2e-5GS-I3-01v9a-LD7-PADRE mixed with aluminum phosphate. Mice were immunized twice, 3 weeks apart, and blood was collected 2 weeks after each injection. In this study, a group of naive mice served as a negative control, and a second group of mice was immunized with β-propiolactone (BPL)-inactivated PR8 H1N1 virus (also known as inactivated H1N1 vaccine) and used as a positive control. Three weeks after the second immunization, mice were immunized with 10×LD , the median lethal dose (determined in previous studies). 50 Mice were challenged intranasally (in) with 10×LD vaccine-adapted A / Puerto Rico / 8 / 1934 (PR8) H1N1 virus. Mice were weighed daily and monitored for visible symptoms of infection (including ruffled hair, hunched posture, and / or decreased activity) for 14 days post-infection (dpi). Mice that were visibly distressed or had lost 75% of their original body weight were euthanized. After surviving mice returned to their pre-challenge baseline after immunization, we administered 10×LD vaccine-adapted A / Puerto Rico / 8 / 1934 (PR8) H1N1 virus intranasally (in). Mice were weighed daily and monitored for visible symptoms of infection (including ruffled hair, hunched posture, and / or decreased activity) for 14 days post-infection (dpi). Mice were euthanized if they were visibly distressed or had lost 75% of their original body weight. After surviving mice returned to their pre-challenge baseline after immunization, we administered 10×LD vaccine-adapted A / Puerto Rico / 8 / 1934 (PR8) H1N1 virus intranasally (in). 50 A heterologous IAV challenge with A / Hong Kong / 1 / 1968 (HK68) H3N2 virus was performed and monitored for 14 dpi. The immunization / challenge study schedule is shown in Figure 4A.

[0078] After the first challenge (Figure 4B), all naive mice and 8 of 10 mice in the 1TD0 trimer group died by 8 dpi. In contrast, survival rates were 100% for all three 1c-SApNP groups, as well as the group receiving the inactivated PR8 H1N1 vaccine. Mice receiving the strain-matched PR8 H1N1 vaccine lost the least weight, began to regain weight by 6 dpi, and returned to their starting weight by 14 dpi. Mice receiving hM2e-5GS-FR and hM2e-5GS-I3-01v9a-L7P lost more weight, began to regain weight by 8 dpi, and returned to their starting weight by 14 dpi. Mice receiving hM2e-5GS-1TD0 trimer lost significantly more weight than the 1c-SApNP group. The two surviving mice began to regain weight by 9 dpi and did not regain their starting weight by 14 dpi. After the second challenge (Figure 4C), four of nine mice in the group administered the inactivated PR8 H1N1 vaccine died, whereas all hM2e-immunized mice survived the H3N2 challenge. Consistently, compared with the hM2e trimer and 1c-SApNP vaccine groups, mice administered the inactivated H1N1 virus vaccine lost significantly more weight upon H3N2 challenge and began to regain weight at a slower pace one day later (dpi 6). Thus, our challenge data highlight the efficacy and broad protection of the M2e 1c-SApNP vaccine. To determine whether protection in hM2e-immunized mice correlated with hM2e-specific antibody responses, we performed an enzyme-linked immunosorbent assay (ELISA) on mouse sera at w5, 1 week before the first challenge, using the hM2e-5GS-Foldon antigen probe (Figure 4D). Foldon (PDB ID: 4NCU) was used in this antigen probe to avoid detection of 1TD0-specific antibodies in the hM2e-5GS-1TD0 trimer group. As expected, naive mice did not show an hM2e-specific response. Notably, mice administered an inactivated H1N1 virus vaccine also showed no signal, consistent with low abundance of M2 in virions.All 1c-SApNP groups showed high hM2e-specific antibody titers, but two surviving mice in the 1TD0 trimer group developed detectable hM2e antibody responses. Overall, our results demonstrate that M2e-displaying 1c-SApNPs can effectively protect mice from lethal challenge with both different IAVs, and this protection is closely correlated with M2e-specific antibodies.

[0079] [Example 5] Some sequences of the single hM2e vaccine construct We have sequenced several of the hM2e vaccines described herein. The full sequence of the I3-01v9a-based construct is shown below. In the sequence, the underlined sequence represents the leader sequence (SEQ ID NO: 1). The italicized sequence represents the 23-residue hM2e (SEQ ID NO: 2). Two conserved Cys residues in the hM2e sequence that are mutated in some of the tandem M2e constructs discussed below are also underlined. Note that the first Met residue was removed from the hM2e sequence inserted into the NP constructs described herein. As a result, although referred to herein (and in the literature) as Cys17 and Cys19, respectively, based on the original complete hM2e sequence, they are actually the 16th and 18th residues in the hM2e sequence present in the vaccine construct. The two bolded and underlined residues indicate restriction sites for PCR. The double-underlined sequence represents the I3-01v9a NP scaffold subunit sequence (SEQ ID NO: 4). The construct may also include a locking domain and / or a T cell epitope. As exemplified in the constructs herein, the locking domain used may be LD7 (SEQ ID NO: 5) (shown double underlined and in bold), and the T cell epitope may be the PADRE epitope (SEQ ID NO: 6) (shown double underlined and in italic font). Linkers or spacers separating different structural motifs of the nanoparticle construct, such as the GS, GGGG spacer (SEQ ID NO: 9) and the 5GS linker (SEQ ID NO: 3), are shown in italic and underlined residues in the construct sequences herein.

[0080] hM2e-5GS-I3-01v9a-LD7-PADRE construct (SEQ ID NO: 10) without the N-terminal leader and LD / PADRE motif [ka]

[0081] hM2e-5GS-I3-01v9a-LD7-PADRE construct containing the C-terminal LD / PADRE motif (SEQ ID NO: 13) [ka]

[0082] hM2e-5GS-I3-01v9a-LD7-PADRE construct containing the N-terminal leader (SEQ ID NO: 16) [ka]

[0083] hM2e-5GS-I3-01v9a-LD7-PADRE construct (SEQ ID NO: 19) containing an N-terminal leader and a C-terminal LD / PADRE motif [ka]

[0084] [Example 6] Design, characterization, and challenge testing of a tandem M2e vaccine Phylogenetic analysis divides IAVs into several lineages based on their original host species: avian, swine, or human. Although M2e is highly conserved, small but significant sequence differences exist between IAVs from different species, which have been shown to limit cross-protection. Therefore, an M2e-based universal influenza vaccine must protect against pandemic strains, which often arise from avian or swine IAVs, in addition to seasonal epidemic strains. The combination of M2e sequences from multiple species has been previously reported. Here, we designed a tandem M2e x3 construct containing human, avian / swine, and human / swine M2e sequences with a GGGG (SEQ ID NO: 9) spacer between consecutive M2e segments. Notably, Cys17 and Cys19 in the second (avian / swine) and third (human / swine) repeats were mutated to serine to avoid random disulfide bonds. As exemplified herein, Cys17 and Cys19 in all three repeats may also be mutated to serine. The M2ex3 antigen was fused to 1TD0 and three 1c-SApNPs using a 5GS spacer, resulting in four constructs designated M2ex3-5GS-1TD0, M2ex3-5GS-FR, M2ex3-5GS-E2p-LD4-PADRE (or M2ex3-5GS-E2p-L4P), and M2ex3-5GS-I3-01v9a-LD7-PADRE (or M2ex3-5GS-I3-01v9a-L7P). These four tandem M2e immunogens were transiently expressed in ExpiCHO cells and purified by IAC using a Fab148 antibody column. Fab148-purified 1c-SApNP samples were analyzed using negative-stain EM at the Scripps EM Core. Consistent with hM2e 1c-SApNP, all tandem M2e 1c-SApNPs exhibited well-formed NPs ( Figure 5A ).

[0085] The immunogenicity and protective efficacy of the tandem M2e vaccine were evaluated in a mouse study following a schedule similar to that of the hM2e immunogen (Figure 4A). Two adjuvants, aluminum hydroxide (AH) and an oil-in-water emulsion, AddaVax, were tested in this study. In the AH adjuvant group (Figure 5B), after PR8 H1N1 challenge, all naive mice and 4 of 8 mice in the 1TD0 trimer group died by 9 dpi. In contrast, survival rates were 88% in the FR 1c-SApNP group and 100% in the two large multilayer 1c-SApNP groups, as well as in the group administered the inactivated PR8 H1N1 vaccine. With regard to weight loss, the AH-formulated E2p 1c-SApNPs most closely resembled the inactivated vaccines and appeared to be more effective than the other tandem M2e immunogens. For the AddaVax adjuvant group (Figure 5C), after PR8 H1N1 challenge, all naive mice and 3 of 8 mice in the 1TD0 trimer group died by 9 dpi. In contrast, survival rates were 100% for all 1c-SApNP groups, as well as for groups receiving the inactivated PR8 H1N1 vaccine. With respect to weight loss, AddaVax-formulated I3-01v9a 1c-SApNP appeared most similar to the inactivated vaccine and more effective than other tandem M2e immunogens. This formulation was also superior to the E2p / AH formulation in preventing weight loss (Figure 5B). Overall, the tandem M2e immunogens demonstrated broad protection, with the tandem M2e-5GS-1TD0 trimer significantly superior to its hM2e counterpart, and I3-01v9a 1c-SApNP performing the best of all immunogens when combined with AddaVax.

[0086] [Example 7] Sequences of several tandem M2ex3 immunogen constructs The amino acid sequences of two exemplary tandem M2e vaccine constructs (M2e×3-5GS-I3-01v9a-LD7-PADRE; also known as M2e×3-5GS-I3-01v9a-L7P) are shown below (SEQ ID NOs: 11 and 12). Each of the two constructs contains a rationally designed I3-01v9a mutant scaffold (SEQ ID NO: 4) and a tandem M2e molecule with three M2e sequences (SEQ ID NO: 23 or SEQ ID NO: 24). The three M2e sequences are the human M2e sequence SLLTEVETPIRNEWG, respectively. C R C NDSSD (SEQ ID NO: 2; Cys17 and Cys19 are underlined), avian / porcine consensus M2e sequence SLLTEVETPTRNGWE C K C SDSSD (SEQ ID NO: 7; Cys17 and Cys19 are underlined) and the human / pig consensus M2e sequence SLLTEVETPTRSEWE C R C SGSSD (SEQ ID NO: 8; Cys17 and Cys19 are underlined). Furthermore, these two constructs each have the conserved Cys17 and Cys19 residues substituted with Ser residues in two or all three of the M2e tandem repeats. In addition to the NP scaffold sequence and the displayed tandem M2e molecules, the constructs also contain the locking domain LD7 (SEQ ID NO: 5) and the universal PADRE T cell epitope (SEQ ID NO: 6). Finally, each construct can further have a leader sequence at the N-terminus. The leader sequence can contain MGILPSPGMPALLSLVSLLSVLLMGCVAE (SEQ ID NO: 1), as exemplified herein.

[0087] Tandem M2e polypeptide with mutated conserved Cys residues in repeats 2 and 3 (SEQ ID NO: 23): [ka]

[0088] Tandem M2e polypeptide with the conserved Cys residue mutated in all three repeats (SEQ ID NO: 24): [ka]

[0089] In each of the two exemplary tandem M2e NP construct sequences shown below, each of the three tandem M2e sequences is italicized. The conserved Cys17 and Cys19 residues in the M2e sequences or the substituted Ser residues are also underlined. Two bolded and underlined residues indicate PCR restriction sites. Linkers or spacers connecting different motifs in the M2e sequences and / or constructs are italicized and underlined. These include the 5aa GS linker GGGGS (SEQ ID NO: 3) and GGGG (SEQ ID NO: 9) spacers that separate the tandem M2e sequences. The double-underlined sequence represents the display I3-01v9a scaffold sequence. The sequence of the locking domain (LD7) is double-underlined and bold. Finally, the PATRE T cell epitope is shown in double-underlined and italicized font.

[0090] Tandem M2e NP construct (SEQ ID NO: 11) in which Cys17 / Cys19 in M2e repeats #2 and #3 were mutated to serine, without the N-terminal leader and C-terminal LD / PADRE motif: [ka]

[0091] Tandem M2e NP construct with Cys17 / Cys19 mutated to serine in M2e repeats #2 and #3 containing a C-terminal LD / PADRE motif (SEQ ID NO: 14): [ka]

[0092] Tandem M2e NP construct with Cys17 / Cys19 mutations in two M2e repeats, including the N-terminal leader sequence (SEQ ID NO: 17): [ka]

[0093] Tandem M2e NP construct with Cys17 / Cys19 mutations in two M2e repeats, containing an N-terminal leader sequence and a C-terminal LD / PADRE motif (SEQ ID NO: 20): [ka]

[0094] Tandem M2e NP construct with Cys17 / Cys19 mutated to serine in all three M2e repeats (SEQ ID NO: 12), without the N-terminal leader and C-terminal LD / PADRE motifs: [ka]

[0095] Tandem M2e NP construct with Cys17 / Cys19 mutated to serine in all three M2e repeats containing a C-terminal LD / PADRE motif (SEQ ID NO: 15): [ka]

[0096] Tandem M2e NP construct with Cys17 / Cys19 mutated to serine in all three M2e repeats, including the N-terminal leader sequence (SEQ ID NO: 18): [ka]

[0097] Tandem M2e NP construct with Cys17 / Cys19 mutated to serine in all three M2e repeats, containing an N-terminal leader sequence and a C-terminal LD / PADRE motif (SEQ ID NO: 21): [ka]

[0098] Thus, the present invention has been broadly disclosed and illustrated with reference to the exemplary embodiments set forth above. It will be understood that various modifications can be made thereto without departing from the spirit and scope of the invention.

[0099] It should be further noted that all publications, sequence accession numbers, patents, and patent applications cited herein are expressly incorporated by reference in their entirety for all purposes, as if each were individually so indicated. Definitions contained in the text incorporated by reference are excluded to the extent that they conflict with definitions in this disclosure.

Claims

1. An N-terminally extended I3-01 nanoparticle scaffold sequence comprising an extended N-terminal helix compared to the N-terminal helix in the original I3-01 scaffold sequence.

2. 2. The N-terminally extended I3-01 nanoparticle scaffold sequence of claim 1, comprising a heterologous helix motif of about 6 to about 12 amino acid residues fused to the N-terminus of the I3-01 scaffold sequence set forth in SEQ ID NO:

27.

3. 3. The N-terminally extended I3-01 nanoparticle scaffold sequence of claim 2, wherein the heterohelix motif comprises AKLAEELQK (SEQ ID NO: 25), a conservatively modified variant thereof, or a substantially identical sequence thereof.

4. 3. The N-terminally extended I3-01 nanoparticle scaffold sequence of claim 2, comprising SEQ ID NO: 4, a conservatively modified variant thereof, or a substantially identical sequence thereof.

5. 10. A self-assembled nanoparticle formed using the N-terminally extended I3-01 nanoparticle scaffold sequence of claim 1.

6. A nanoparticle vaccine construct comprising a polypeptide immunogen fused to an N-terminally extended I3-01 nanoparticle scaffold sequence, wherein the N-terminally extended I3-01 nanoparticle scaffold sequence comprises an extended N-terminal helix compared to the N-terminal helix in the original I3-01 scaffold sequence.

7. 7. The nanoparticle vaccine construct of claim 6, wherein the N-terminally extended I3-01 nanoparticle scaffold sequence comprises a heterologous helix motif of about 6 to about 12 amino acid residues fused to the N-terminus of the I3-01 scaffold sequence set forth in SEQ ID NO:

27.

8. 7. The nanoparticle vaccine construct of claim 6, wherein the heterologous helix motif comprises AKLAEELQK (SEQ ID NO: 25), a conservatively modified variant thereof, or a substantially identical sequence thereof.

9. 7. The nanoparticle vaccine construct of claim 6, wherein the N-terminally extended I3-01 nanoparticle scaffold sequence comprises SEQ ID NO: 4, a conservatively modified variant thereof, or a substantially identical sequence thereof.

10. The nanoparticle vaccine construct of claim 6, wherein the polypeptide immunogen is fused to the N-terminus of the N-terminally extended I3-01 nanoparticle scaffold sequence via a linker at its C-terminus.

11. The nanoparticle vaccine construct of claim 10, wherein the linker comprises GGGGS (SEQ ID NO: 3).

12. 7. The nanoparticle vaccine construct of claim 6, wherein the polypeptide immunogen comprises (a) an influenza fusion polypeptide containing two or more tandem repeats of influenza M2 protein ectodomain (M2e) or (b) an HCV immunogenic protein.

13. The nanoparticle vaccine construct of claim 12, wherein the tandem repeats of M2e are separated by a peptide spacer.

14. The nanoparticle displaying an immunogenic protein of claim 13, wherein the peptide spacer comprises GGGG (SEQ ID NO: 9).

15. The nanoparticle vaccine construct of claim 12, wherein at least one of the tandem repeats of M2e contains a missense mutation at the conserved Cys17 and Cys19 residues.

16. 16. The nanoparticle vaccine construct of claim 15, wherein the missense mutation comprises a substitution of each of the two Cys residues with an amino acid residue having an uncharged polar side chain.

17. 16. The nanoparticle vaccine construct of claim 15, wherein each of the Cys residues is independently substituted with an amino acid residue selected from the group consisting of serine, glycine, asparagine, glutamine, threonine, and tyrosine.

18. The nanoparticle vaccine construct of claim 15, wherein the two Cys residues are both substituted with Ser.

19. 13. The nanoparticle vaccine construct of claim 12, wherein the influenza fusion polypeptide comprises three tandem M2e sequences.

20. 20. The nanoparticle vaccine construct of claim 19, wherein the three tandem M2e sequences are independently human M2e sequences, avian / pig consensus M2e sequences, or human / pig consensus M2e sequences, except for missense mutations at residues Cys17 and Cys19 in at least two of the three tandem M2e sequences.

21. 21. The nanoparticle vaccine construct of claim 20, wherein the influenza fusion polypeptide comprises, in any order, human M2e (SEQ ID NO: 2), an avian / porcine consensus M2e sequence (SEQ ID NO: 30) with Cys 17 and Cys 19 each substituted with a Ser residue, and a human / porcine consensus M2e sequence (SEQ ID NO: 31) with Cys 17 and Cys 19 each substituted with a Ser residue.

22. 21. The nanoparticle vaccine construct of claim 20, wherein the influenza fusion polypeptide comprises, in any order, human M2e (SEQ ID NO: 29), with Cys 17 and Cys 19 each substituted with a Ser residue; an avian / porcine consensus M2e sequence (SEQ ID NO: 30), with Cys 17 and Cys 19 each substituted with a Ser residue; and a human / porcine consensus M2e sequence (SEQ ID NO: 31), with Cys 17 and Cys 19 each substituted with a Ser residue.

23. 21. The nanoparticle vaccine construct of claim 20, wherein the influenza fusion polypeptide comprises SEQ ID NO:23, SEQ ID NO:24, a conservatively modified variant thereof, or a substantially identical sequence thereof.

24. 21. The nanoparticle vaccine construct of claim 20, comprising the sequence shown in SEQ ID NO: 11, SEQ ID NO: 12, a conservatively modified variant thereof, or a substantially identical sequence thereof.

25. The nanoparticle vaccine construct of claim 20, further comprising a locking domain and a T cell epitope at the C-terminus.

26. 26. The nanoparticle vaccine construct of claim 25, wherein the locking domain comprises SEQ ID NO:5 and the T cell epitope comprises SEQ ID NO:

6.

27. 26. The nanoparticle vaccine construct of claim 25, comprising SEQ ID NO: 14, SEQ ID NO: 15, a conservatively modified variant thereof, or a substantially identical sequence thereof.

28. 21. The nanoparticle vaccine construct of claim 20, further comprising an N-terminal leader sequence.

29. 29. The nanoparticle vaccine construct of claim 28, comprising SEQ ID NO: 17, SEQ ID NO: 18, a conservatively modified variant thereof, or a substantially identical sequence thereof.

30. 21. The nanoparticle vaccine construct of claim 20, further comprising an N-terminal leader sequence, and a locking domain and a T-cell epitope at the C-terminus.

31. 31. The nanoparticle vaccine construct of claim 30, comprising SEQ ID NO: 20, SEQ ID NO: 21, a conservatively modified variant thereof, or a substantially identical sequence thereof.

32. The nanoparticle vaccine construct of claim 12, wherein the HCV immunogenic protein comprises an E2 core or an E1E2 dimer.

33. The nanoparticle vaccine construct of claim 32, wherein the HCV E2 core comprises any one of SEQ ID NOs: 32-35.

34. 33. The nanoparticle vaccine construct of claim 32, wherein the HCV immunogenic protein comprises two tandem copies of the E2 core sequence.

35. 35. The nanoparticle vaccine construct of claim 34, wherein the two E2 core sequences are derived from different HCV isolates.

36. 35. The nanoparticle vaccine construct of claim 34, wherein the two E2 core sequences comprise SEQ ID NOs: 32 and 33 or SEQ ID NOs: 34 and 35, respectively.

37. The nanoparticle vaccine construct of claim 34, further comprising a locking domain and a T cell epitope at the C-terminus.

38. 38. The nanoparticle vaccine construct of claim 37, wherein the locking domain comprises SEQ ID NO:5 and the T cell epitope comprises SEQ ID NO:

6.

39. 38. The nanoparticle vaccine construct of claim 37, comprising (1) a subunit sequence comprising, from the N-terminus to the C-terminus: (a) SEQ ID NO:4, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:5, and SEQ ID NO:6, or (b) SEQ ID NO:4, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:5, and SEQ ID NO:6; or (2) a conservatively modified variant thereof or a substantially identical sequence thereof.

40. A polynucleotide sequence encoding a subunit sequence of the nanoparticle vaccine construct of claim 6.

41. A vector comprising the polynucleotide sequence of claim 40.

42. A pharmaceutical composition comprising the nanoparticle vaccine construct of claim 6 or the polynucleotide sequence of claim 40.

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