Vaccines comprising novel nanoparticle scaffolds

Abstract

The present invention provides a new modified nanoparticle scaffold sequence derived from an I3-01 protein. The novel scaffold sequences of I3-01 origin of the present invention comprise an extended N-terminal helix relative to known I3-01 proteins or variants thereof. Also provided herein are vaccine constructs comprising a plurality of immunogenic proteins that are displayed on the novel nanoparticle scaffold sequences described herein. The vaccine constructs of the invention include, for example, nanoparticles that display tandem repeats of influenza M2e protein or HCV E2 core protein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] 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 this priority application is incorporated herein by reference in its entirety for all purposes.

[0003] Sequence Listing

[0004] This application contains a sequence listing 2189_1PC_Sequence Listing in XML format incorporated by reference, which was created on November 9, 2023 and contains 37KB of content. Background Art

[0005] Influenza virus (IAV) belongs to the Orthomyxoviridae family and can be divided into four types: A, B, C and D. All influenza viruses are negative-sense single-stranded RNA viruses with an envelope and a segmented genome, of which influenza A and influenza B viruses (IAV and IBV) contain 8 gene segments that encode at least 17 proteins. The most abundant surface glycoprotein, hemagglutinin (HA), allows the virus to bind to host cell receptors and mediate cell entry. Neuraminidase (NA) helps release viral particles by cleaving residues on the surface of the host cell. Matrix-1 protein (M1) helps the virus bud from the plasma membrane of infected cells, and matrix-2 protein (M2) helps maintain pH during viral entry and viral replication in host cells. IAV can be further classified into subtypes based on the antigenic properties of two surface glycoproteins, HA (HA1-18) and NA (NA1-11). IAV can infect many hosts, while IBV is restricted to humans and has differentiated into two lineages (Victoria and Yamagata) through intra-host evolution. Influenza viruses of avian origin recognize α-2,3 sialic acid receptors, while human influenza viruses prefer to bind to α-2,6 sialic acid receptors in the upper respiratory tract. Influenza viruses use two mechanisms to evade the immune system. Antigenic drift consists of small changes introduced into HA and NA under immune pressure and is responsible for the annual epidemics of human influenza. Antigenic shift occurs when IAV undergoes a complete change in the HA and / or NA genes due to its large animal reservoir. Antigenic shift leads to increased transmission of new IAV strains in humans and is the main cause of pandemics.

[0006] Since the 1940s, 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 attenuated live strains. The most common inactivated virus vaccine type is called a split vaccine, in which detergents or chemicals are used to destroy viral particles. Attenuated live vaccines use cold-adapted live viruses that do not replicate at human body temperature and are usually administered intranasally to induce strong local immunity. Subunit vaccines utilize partially purified viral HA or NA proteins after chemical or detergent cracking. The virus strains selected for the quadrivalent vaccine (comprising H1N1, H3N2 and two Flu B strains) are produced in chicken eggs. However, due to the mutation of influenza viruses and the tendency of immune escape, current influenza vaccines must be updated annually to include predicted strains. Strain mismatch usually results in low efficacy, which emphasizes the demand for better vaccines.

[0007] There is a strong unmet need in the medical field for more reliable and effective influenza vaccines. The present invention is directed to this and other unmet needs in the art. Summary of the Invention

[0008] In one aspect, the present invention provides N-terminally extended I3-01 nanoparticle scaffold sequences. These scaffold sequences comprise an N-terminal helix that is extended compared to the N-terminal helix in the original or wild-type I3-01 scaffold sequence. In some embodiments, the novel I3-01-derived NP scaffold sequence of the present invention comprises a heterologous helical motif of about 6 to about 12 amino acid residues, which is fused to the N-terminus of the I3-01 scaffold sequence shown in SEQ ID NO: 27 (I3-01v9). In some of these embodiments, the inserted heterologous helical motif comprises AKLAEELQK (SEQ ID NO: 25), a conservatively modified variant thereof, or a substantially identical sequence. 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. In a related aspect, the present invention provides self-assembling nanoparticles formed by the novel N-terminally extended I3-01 nanoparticle scaffold sequence of the present invention.

[0009] In another aspect, the present invention provides nanoparticle vaccine constructs comprising 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 N-terminal helix that is extended compared to the N-terminal helix in 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 helical motif of about 6 to about 12 amino acid residues, which is fused to the N-terminus of the I3-01 scaffold sequence shown in SEQ ID NO: 27 (I3-01v9). In some embodiments, the extended heterologous helical motif comprises AKLAEELQK (SEQ ID NO: 25), a conservatively modified variant thereof, or a substantially identical sequence. 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. Generally speaking, the immunogenic protein 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).

[0010] 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 extracellular domain (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 displayed M2e tandem repeat comprises a missense mutation at the conserved Cys17 and Cys19 residues. In some of these embodiments, the missense mutation comprises replacing each of the two Cys residues with an amino acid residue having an uncharged polar side chain. In multiple embodiments, each of the Cys residues is independently replaced by an amino acid residue selected from the following: serine, glycine, asparagine, glutamine, threonine, and tyrosine. In some preferred embodiments, both Cys residues in the same M2e sequence are replaced by Ser.

[0011] In some influenza vaccines of the present invention, the displayed immunogenic protein comprises three tandem M2e sequences. In some of these embodiments, the three tandem M2e sequences are independently human M2e sequences, avian / porcine consensus M2e sequences, or human / porcine 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 / porcine consensus M2e sequence in which Cys17 and Cys19 are each replaced by a Ser residue (SEQ ID NO: 30), and a human / porcine consensus M2e sequence in which Cys17 and Cys19 are each replaced by a Ser residue (SEQ ID NO: 31). In some embodiments, the displayed influenza M2e tandem repeat fusion polypeptide comprises, in any order, human M2e in which Cys17 and Cys19 are each replaced by a Ser residue (SEQ ID NO: 29), an avian / porcine consensus M2e sequence in which Cys17 and Cys19 are each replaced by a Ser residue (SEQ ID NO: 30), and a human / porcine consensus M2e sequence in which Cys17 and Cys19 are each replaced by a Ser residue (SEQ ID NO: 31). In some embodiments, the displayed influenza M2e tandem fusion polypeptide comprises SEQ ID NO: 23, SEQ ID NO: 24, conservatively modified variants thereof, or substantially identical sequences. Some of these influenza vaccine constructs comprise the subunit or scaffold sequences set forth in SEQ ID NO: 11, SEQ ID NO: 12, conservatively modified variants thereof, or substantially identical sequences.

[0012] In addition to the immunogenic protein sequence, the vaccine constructs based on the new I3-01 scaffold of the present invention may additionally include a T cell epitope and a locking domain at the C-terminus. For example, they may include the locking domain shown in SEQ ID NO:5 and the T cell epitope shown in SEQ ID NO:6 at the C-terminus. Some of these vaccine constructs include the subunit or scaffold sequences shown in SEQ ID NO:14, SEQ ID NO:15, their conservatively modified variants or substantially the same sequences. Some of the vaccine constructs of the present invention may additionally include an N-terminal leader sequence. Some of these vaccine constructs include the subunit or scaffold sequences shown in SEQ ID NO:17, SEQ ID NO:18, their conservatively modified variants or substantially the same sequences. Some of the vaccine constructs of the present invention may additionally include an N-terminal leader sequence and a T cell epitope and a locking domain at the C-terminus. Some of these vaccine constructs include the subunit or scaffold sequences shown in SEQ ID NO:20, SEQ ID NO:21, their conservatively modified variants or substantially the same sequences.

[0013] In some vaccine constructs of the present invention, the displayed immunogenic protein is an HCV immunogen, for example, E2 core or E1E2 dimer protein. In some of these embodiments, the displayed protein comprises the HCV E2 core as shown in any one of SEQ ID NOs: 32 to 35. In some embodiments, the displayed HCV immunogenic protein comprises tandem copies of two E2 core sequences. In some of these embodiments, the two E2 core sequences are from different HCV isolates. For example, 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 additionally comprise a T cell epitope and a locking domain at the C-terminus. For example, the vaccine construct may comprise the locking domain shown in SEQ ID NO: 5 and / or the T cell epitope shown in SEQ ID NO: 6. Some of these HCV vaccines comprise subunit or scaffold sequences having, from N-terminus to C-terminus, different structural motifs set forth, respectively, in: (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 additional constructs comprise subunit or scaffold sequences that are conservatively modified variants of one of these exemplified scaffold sequences or substantially identical sequences.

[0014] In another aspect, the present invention provides polynucleotide sequences encoding subunits or scaffold sequences of one of the nanoparticle vaccine constructs described herein. The present invention also encompasses vectors or expression constructs containing one or more of these polynucleotide sequences. Also provided are pharmaceutical compositions or kits comprising the nanoparticle vaccine constructs or encoding polynucleotide sequences described herein.

[0015] 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 DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Rational design of I3-01v9a to achieve optimal display of monomeric antigens on the surface of nanoparticles (NPs). The complete amino acid sequence of the I3-01v9a scaffold is shown (SEQ ID NO: 4).

[0017] Figure 2(A) 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). (B) EM images of three HCV E2 core I3-01v9a nanoparticles. (C) Construct design of HCV tandem E2 core I3-01v9a nanoparticles. The linker sequence between the two E2 cores (SEQ ID NO:3) and the linker sequence between the second E2 core and the nanoparticle scaffold (SEQ ID NO:36) are shown. (D) SEC and EM analysis of two tandem E2 core I3-01v9a nanoparticles. SEC profiles of two expression volumes in ExpiCHO cells: 50 ml vs. 200 ml.

[0018] Figure 3 (A) Design of M2e-based vaccine constructs. Left: hM2e structure and sequence (SEQ ID NO: 2). Center: Model of the hM2e-5GS-1TD0 trimer; Right: Models of hM2e-5GS-FR, hM2e-5GS-E2p-LD4-PADRE, and hM2e-5GS-I3-01v9a-LD7-PADRE 1c-SApNPs. (B) Schematic diagram of 1c-SApNP expression and purification. (C) SEC profiles of hM2e trimer and 1c-SApNP. (D) Negative-stain EM micrograph of hM2e 1c-SApNPs purified with Fab148.

[0019] Figure 4 (A) Mouse immunization / challenge schedule. (B) Survival and weight loss after A / Puerto Rico / 8 / 1934 (PR8) H1N1 virus challenge. (C) Survival and weight loss after A / Hong Kong / 1 / 1968 (HK68) H3N2 virus challenge. (D) ELISA of hM2e-specific antibody responses in mouse sera against the hM2e probe.

[0020] Figure 5 (A) Negative-stained EM image of tandem M2e 1c-SApNPs. The M2ex3-5GS-FR sample maintained at 70°C for 10 minutes showed no visible structural changes and bound to anti-M2e antibodies with nearly identical affinity. (B) Survival and weight loss of the alum-adjuvanted group after A / Puerto Rico / 8 / 1934 (PR8) H1N1 virus challenge. (C) Survival and weight loss of the AddaVax-adjuvanted group after A / Puerto Rico / 8 / 1934 (PR8) H1N1 virus challenge. DETAILED DESCRIPTION

[0021] I Overview

[0022] A variety of antigens and vaccine strategies have been explored to develop universal influenza vaccines. The most common antigen is HA, and recent vaccine design efforts have focused on conserved epitopes within the conserved stem region or head domain. These approaches include the use of headless HA, chimeric HA, and mosaic HA. NA is also an attractive target for broadly neutralizing antibodies (bNAbs) against seasonal and pandemic strains. The extracellular domain (M2e) of the M2 protein is a highly conserved target for universal IAV vaccines. Although M2e is small (approximately 23 aa) and non-immunogenic, it can be linked to large vectors to elicit cross-protection and reduce viral replication. Internal proteins (such as nucleoprotein and M1) have been explored for T cell targeting. The use of adjuvants (such as MF59 and AS03) has been shown to significantly improve the efficacy of influenza vaccines. Therefore, when developing universal influenza vaccines targeting subdominant HA stems and M2e, the adjuvant effect must be carefully examined.

[0023] M2e-based vaccines provide protection through mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), which eliminate virus-infected cells. M2e has been linked to a variety of vectors for vaccine development. One of the early M2e vaccines used hepatitis B core protein (HBc) as a vector. Tobacco mosaic virus (TMV) coat protein, keyhole limpet hemocyanin (KLH), rotavirus NSP4, GCN4, bacterial flagellin, and liposomes have been tested in M2e vaccine development. As the search for better vectors continues, many vaccine candidates have advanced to human trials, providing important feedback for future M2e vaccine development. In a Phase I trial (clinicaltrials.gov: NCT00819013), an adjuvanted M2e-HBc fusion protein induced anti-M2e antibodies in 90% of cases and was well tolerated. However, the vaccine-induced anti-M2e antibody response declined rapidly. In a Phase I trial (clinicaltrials.gov: NCT00921206), an M2e-flagellin fusion vaccine was highly immunogenic but caused undesirable side effects at higher doses, such as fever, diarrhea, fatigue, headache, and myalgia. Vaccines combining M2e with multiple cytotoxic T lymphocyte (CTL) epitopes stimulated strong cellular immunity in humans (clinicaltrials.gov: NCT01181336), but the T cell response was narrow and slow, making this vaccine unsuitable for emerging pandemic situations. Therefore, the correct balance between vector, adjuvant, and antibody and T cell responses is a major challenge in M2e vaccine development.

[0024] The inventors designed multilayer single-component self-assembling protein nanoparticles (1c-SApNPs) based on E2p and I3-01 (two bacterial proteins that self-assemble into 60-mers of 22 to 25 nm) as carriers of foreign antigens for vaccine development. These 1c-SApNPs have been successfully applied to HIV-1, HCV, Ebola virus (EBOV) and SARS-CoV-2 to produce nanoparticle vaccines. In the embodiments described herein, the present invention encompasses a new I3-01-derived NP platform that has been shown to have the activity of presenting immunogenic proteins (e.g., influenza M2e protein and HCV immunogen). The present invention also encompasses a broad range of protective vaccines comprising immunogenic proteins (e.g., HCV E2 core protein and influenza M2e protein) displayed on new NP scaffolds. Details of the compositions and methods encompassed by the present invention are described below.

[0025] The nanoparticle scaffolds and vaccine constructs described herein can have a variety of applications in clinical settings. For example, the novel I3-01-derived NP scaffolds described herein (e.g., I3-01v9a (SEQ ID NO: 4)) can be used to present a variety of other monomeric antigens in addition to the HCV and influenza immunogenic proteins exemplified herein. Vaccines constructed therefrom (e.g., HCV vaccines or tandem hM2e vaccines as exemplified herein) can be used as broadly protective vaccines. Using influenza vaccines as an example, they can be added as "performance enhancers" to seasonal vaccines (e.g., HCV or influenza vaccines) to improve protection against endemic (human strains) and pandemic (swine and avian strains) influenza viruses. In other applications, vaccines based on the novel I3-01 scaffold (e.g., vaccines displaying M2e) can be combined with other vaccine formats (e.g., hemagglutinin (HA) stem-based vaccines) to create a truly universal influenza vaccine.

[0026] Relative to related vaccines known in the art, the vaccine of the present invention also has many advantageous properties. Using influenza vaccine to illustrate, the uniform distribution of antigen anchoring sites on the surface and the exposure slightly above the reference level (just-above-sea-level) make the new I3-01 scaffold described herein become the ideal nanoparticle platform for presenting monomeric antigens. Icosahedral symmetry and dense surface display make 1c-SApNP become the ideal carrier for the multivalent display of suitable antigens (e.g., influenza M2e). Designed as a single segment or tandem construct, it can be best displayed on the nanoparticle surface and can produce high-quality antibody responses. In addition, gene fusion and self-assembly combination will result in the robust production of 1c-SApNP in laboratory and industrial environments. As shown herein, after immunoaffinity (Fab148) purification, the vaccine can be produced in ExpiCHO cells with reasonable yield and extremely high purity. Since CHO is one of the main mammalian cell lines for industrial production of protein therapeutics and vaccines, and ExpiCHO is a transient form of this CHO cell line, vaccines obtained from ExpiCHO cells (e.g., influenza M2e vaccine) are expected to have the same properties as those from industrial CHO cell lines. This will enable GMP manufacturing of 1c-SApNP vaccines for human use. In addition, the multilayer structure will ensure the thermal stability of 1c-SApNP (e.g., influenza M2e 1c-SApNP) and allow for multiple delivery routes and combined use with other related vaccines. As exemplified herein, maintaining a high temperature of 70°C for 10 minutes did not cause any structural changes, and ELISA showed almost identical binding to M2e-specific antibodies (e.g., Fab65 and Fab148). The excellent thermal stability of the 1c-SApNP (e.g., influenza M2e 1c-SApNP) of the present invention will allow them to be used in harsh conditions, such as being embedded in self-dissolving microneedles for transdermal immunization.

[0027] Unless otherwise indicated herein, the various compositions and methods of the present invention can be made or performed according to the procedures exemplified herein or conventional practices known in the art. See, for example,

[0028] Methods in Enzymology, Vol. 289: Solid-Phase Peptide Synthesis, JN Abelson, M.I. Simon, G.B. Fields (eds.), Academic Press; 1st ed. (1997) (ISBN-13:978-0121821906); U.S. Pat. Nos. 4,965,343 and 5,849,954; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, (3rd ed. 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, ed. S. L. Berger and A. R. Kimmerl, Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (ed. John E. Coligan, et. al., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (ed. Juan S. Bonifacino et. al., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, ed. Jennie P. Mather and David Barnes, Academic Press, 1st edition, 1998)

[0029] The following section provides additional guidance for practicing the compositions and methods of the present invention.

[0030] II. definition

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The following references provide general definitions of many of the terms used in the present invention for those skilled in the art:

[0032] Academic Press Dictionary of Science and Technology, Morris (ed.), Academic Press (1st ed., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (ed.), 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. (ed.), John Wiley & Sons (3rd ed., 2002); Dictionary of Chemistry, Hunt (ed.), Routledge (1st ed., 1999); Dictionary of Pharmaceutical Medicine, Nahler (ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (eds.), Anmol Publications Pvt. Ltd. Pvt. Ltd. (2002); and A Dictionary of Biologgy (Oxford Paperback Reference), Martin and Hine (eds.), Oxford University Press (4th edition, 2000)

[0033] Further explanation of some of these terms as they apply specifically to the present invention is provided herein.

[0034] Unless the context clearly indicates otherwise, nouns used herein without quantifiers refer to one or more. For example, "Env-derived trimer" can refer to both a single or multiple Env-derived trimer molecules and can be considered equivalent to the phrase "at least one Env-derived trimer."

[0035] As used herein, the terms "antigen" or "immunogen" are used interchangeably to refer to a substance, typically a protein, that is capable of inducing an immune response in a subject. The term also refers to a protein that is immunologically active in the sense that it is capable of eliciting a humoral and / or cellular immune response against the protein upon administration to a subject (either directly or by administering to a subject a nucleotide sequence or vector encoding the protein). Unless otherwise indicated, the term "vaccine immunogen" is used interchangeably with "protein antigen" or "immunogenic polypeptide."

[0036] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to specific nucleic acid sequences, conservatively modified variants refer to those nucleic acids that encode the same or substantially the same amino acid sequence, or, where the nucleic acid does not encode an amino acid sequence, to substantially the same sequence. 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 with conservative amino acid substitutions (amino acid residues are replaced by other amino acid residues with similarly charged side chains). Families of amino acid residues with similarly charged side chains 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).

[0037] Epitope refers to an antigenic determinant. These are specific chemical groups or peptide sequences on a molecule that are antigenic so that they elicit a specific immune response, for example, an epitope is a region of an antigen that responds to B and / or T cells. An epitope can be formed by both contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of a protein.

[0038] The effective amount of a vaccine or other agent is enough to produce a desired response, such as to alleviate or eliminate the signs or symptoms of a disease or disease (such as seasonal influenza). For example, this can be the amount required for suppressing viral replication or measurably changing the outward symptoms of a viral infection (such as an increase in T cell count in the case of an influenza infection). Generally speaking, this amount is enough to measurably suppress viral replication or infectivity. When applied to a subject, a dosage that reaches a target tissue concentration (for example, in lymphocytes) that has been shown to achieve in vitro inhibition of viral replication is generally used. In some embodiments, an "effective amount" is an amount for treating (including preventing) one or more symptoms and / or potential causes (for example, for treating influenza infection) of any disease or disease. In some embodiments, an effective amount is a therapeutically effective amount. In some embodiments, an effective amount is an amount for preventing one or more signs or symptoms of a particular disease or disease from occurring (for example, one or more signs or symptoms associated with a disease).

[0039] The fusion protein used herein is a recombinant protein comprising amino acid sequences from at least two unrelated proteins, which have been linked together by peptide bonds to form a single protein. Unrelated amino acid sequences can be directly linked to each other, or they can be linked using a linker sequence. If the amino acid sequence of the protein used herein is not typically found to be linked together by peptide bonds in its natural environment (e.g., within a cell), the protein used herein is unrelated. For example, the amino acid sequence of the enzyme of the anaerobic bacterium Thermotoga maritima (from which the I3-01 NP scaffold is derived) is not naturally linked to the amino acid sequence of influenza M2e or HCV E2 core by a peptide bond.

[0040] An immunogen is a protein or portion thereof that is capable of inducing an immune response in a mammal (e.g., a mammal infected with or at risk of infection by a pathogen). Administration of an immunogen can result in protective immunity and / or active immunity against the pathogen of interest.

[0041] An immune response refers to the response of cells of the immune system (e.g., B cells, T cells, or monocytes) to stimulation. In some embodiments, the response is specific for a particular antigen ("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, and results in the production of specific antibodies.

[0042] 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 induces a measurable B cell response (eg, production of antibodies) against the immunogenic polypeptide.

[0043] As used herein, amino acid numbering or an 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. Standardized sequence alignment allows the sequences of different orthologs of the same immunogenic protein (e.g., M2e) from other strains or species, or modified forms of the same protein described herein, to be compared with the sequence of the prototype sequence. Utilizing such a standard or standardized amino acid numbering, conserved amino acid residues in immunogenic proteins from various viral strains or modified proteins can be easily identified and named. For example, unless otherwise specified herein, the amino acid numbering of 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 for all influenza strains are referred to as residues Cys17 and Cys19.

[0044] The sequence identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is represented by the identity or similarity between the sequences.Sequence identity can be measured with percentage identity; The higher the percentage, the more identical the sequence.When two sequences are compared and aligned in a comparison window or a specified region to obtain maximum correspondence, as measured by one of the following sequence comparison algorithms or by manual comparison and visual inspection, if the two sequences have the same amino acid residues or nucleotides of a specified percentage (that is, 60% identity in a specified region or over the entire sequence when not specified, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity), then the two sequences are "substantially identical". Optionally, identity is present in a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably in a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0045] When compared using standard methods, homologs or orthologs of nucleic acid or amino acid sequences have relatively high degrees of sequence identity / similarity. Sequence alignment methods for comparison are well known in the art. Various programs and alignment algorithms are described below:

[0046] 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., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al. al., J. Mol. Biol. 215:403-10, 1990

[0047] , showing detailed considerations of sequence alignment methods and homology calculations.

[0048] The term "subject" refers to any animal classified as a mammal, such as humans and non-human mammals. Some examples of non-human animals include dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein. Preferably, the subject is a human.

[0049] The terms "treat" or "alleviate" include administering a compound or agent to a subject to prevent or delay the onset of symptoms, complications, or biochemical markers of a disease (e.g., influenza infection), alleviate symptoms, or prevent or inhibit further development of a disease, condition, or disorder. Subjects in need of treatment include those already suffering from the disease or condition and those at risk of developing the condition. Treatment can be prophylactic (preventing or delaying the onset of the disease, or preventing the manifestation of its clinical or subclinical symptoms), or therapeutic suppression or alleviation of symptoms after disease manifestation.

[0050] Vaccine refers to a pharmaceutical composition that causes a preventive or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Generally speaking, vaccines cause antigen-specific immune responses against pathogens (e.g., viral pathogens) antigens or against cellular components associated with a pathological condition. Vaccines may include polynucleotides (e.g., nucleic acids encoding disclosed antigens), peptides or polypeptides (e.g., disclosed antigens), viruses, cells, or one or more cellular components. In some embodiments of the invention, vaccines or vaccine immunogens or vaccine compositions are expressed by fusion constructs and self-assembled into nanoparticles that display immunogenic polypeptides or proteins on the surface.

[0051] Vaccine (for example, influenza or HCV vaccine) refers to an immunogenic composition capable of stimulating an immune response, which is administered to prevent, improve or treat a disease or infection (for example, influenza virus infection). Vaccines may include, for example, attenuated or killed (for example, lysed) pathogens (for example, viruses), virus-like particles (VLPs) and / or antigenic polypeptides or DNA derived therefrom, or any recombinant form of such immunogenic substances.

[0052] Virus-like particles (VLPs) refer to non-replicative viral capsids derived from any one of several viruses. VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid proteins, coat proteins, shell proteins, surface proteins and / or envelope proteins, or particle-forming polypeptides derived from these proteins. After recombinant expression of proteins in a suitable expression system, VLPs can form spontaneously. Methods for producing specific VLPs are known in the art. After recombinant expression of viral proteins, the presence of VLPs can be detected using conventional techniques known in the art (e.g., by electron microscopy, biophysical characterization, etc.). See, for example, Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) J. Virol. 68: 4503-4505. For example, VLPs can be separated by density gradient centrifugation and / or identified by characteristic density bands. Alternatively, cryo-electron microscopy can be performed on the vitrified aqueous sample of the VLP preparation in question, and images can be recorded under suitable exposure conditions.

[0053] Self-assembling nanoparticles refer to spherical protein shells with a diameter of tens of nanometers and well-defined surface geometry, which are formed by identical copies of non-viral proteins that can automatically assemble into nanoparticles with an outward appearance similar to VLPs. Some known examples include ferritin (FR), which is conserved between species and forms 24 aggressiveness, as well as Bacillus stearothermophilus (B.Stearothermophilus) dihydrolipoyl acyltransferase (E2p), Aquifexaeolicus (Aquifexaeolicus) lumazine synthase (LS), variants in I3-01 sources, and Thermotoga maritima encapsulin, which all form 60 aggressiveness. After protein recombinant expression in an appropriate expression system, self-assembling nanoparticles can form spontaneously. The generation, detection, and characterization methods of nanoparticles can be carried out using the same techniques developed for VLPs.

[0054] III. New NP scaffolds with improved activity

[0055] The present invention provides new nanoparticle scaffold sequences, which are suitable for presenting multiple viral immunogenic proteins to elicit potent neutralizing antibody responses. These scaffold sequences, which have been rationally designed and functionally tested, are based on I3-01 proteins. I3-01 is a modified protein (SEQ ID NO: 22) that can self-assemble into ultrastable nanoparticles. The original ("unextended" or "wild type") I3-01 protein is described in Hsia et al., Nature 535, 136-139, 2016. Several ultrastable nanoparticle scaffolds derived from I3-01 have been previously developed, and used to present viral proteins, such as viral proteins from HIV-1 and HCV. See, for example, WO21 / 021603, WO22 / 035739, U.S. Patent No. 10,906,944 and WO19 / 089817. In order to identify new NP scaffolds with improved activity, the inventors rationally designed known I3-01 variant scaffolds and functionally tested them. The original I3-01 protein and variants known in the art (i.e., the unextended I3-01 scaffold sequence) contain the N-terminal helical motif KMEELFKKHK (SEQ ID NO: 26). The novel scaffold sequences of the present invention are obtained by extending the N-terminal helix of an existing I3-01 variant scaffold (e.g., I3-01v9 (SEQ ID NO: 27)) by grafting heterologous helical motifs, followed by rational design using an ensemble-based protein design program. An 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) is capable of providing optimal surface display of monomeric protein antigens.

[0056] I3-01 sequence without the first Met residue (SEQ ID NO: 22) (N-terminal helix is underlined):

[0057] KMEELFKKHK IVAVLRANSVEEAKKKALAVFLGGVHLIEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKAFVEKIRGCTE

[0058] 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) with 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.

[0059] I3-01v9 (SEQ ID NO: 27) (N-terminal helix is underlined):

[0060] KMEELFKKHK IVAVLRANSVEEAKMKALAVFVGGVHLIEITFTVPDADTVIKELSFLKELGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTIAEVAAKAAAFVEKIRGCTE

[0061] I3-01v9a (SEQ ID NO: 4) (the extension of the N-terminal helix is underlined):

[0062] AKLAEELQKKMEELFKKHKIVAVLRANSVEEAKMKALAVFVGGVHLIEITFTVPDADTVIKELSFLKELGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEK GVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTIAEVAAKAAAFVEKIRGCTE

[0063] In various embodiments, the novel I3-01 derived NP scaffolds of the present invention comprise SEQ ID NO: 4, a conservatively modified variant thereof, or a substantially identical sequence (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 or a substantially identical sequence of SEQ ID NO: 27. In some further embodiments, the entire extended N-terminal helix of the novel I3-01 variant scaffold is identical to the N-terminal helix in I3-01v9a (i.e., AKLAEELQKKMEELFKKHK (SEQ ID NO: 28)), while the remainder of the sequence is a conservatively modified variant or a substantially identical sequence of the corresponding sequence of SEQ ID NO: 27 (i.e., SEQ ID NO: 27 minus the N-terminal helix).

[0064] IV. Immunogenic polypeptides or proteins for producing vaccine compositions

[0065] The novel I3-01-derived nanoparticle scaffolds described herein can be used to construct vaccines that present a variety of different immunogenic proteins, including monomeric and multimeric proteins. These include any protein or polypeptide from a pathogen against which an immune response can be expected. Thus, the vaccine compositions of the present invention can utilize immunogenic polypeptides derived from any virus, bacterium, or other pathogenic organism. Suitable immunogenic polypeptides for use in the present invention can also be derived from non-pathogenic species, including human proteins, against which the immune response elicited can have a therapeutic effect, alleviating disease symptoms or improving overall health. Generally speaking, an immunogenic polypeptide can be any structural or functional polypeptide or peptide comprising at least about 10 amino acid residues. In some embodiments, the immunogenic polypeptide is about 10 to about 10,000 amino acid residues in length. In some embodiments, the immunogenic polypeptide is about 25 to about 2,000 amino acid residues in length. In some embodiments, the immunogenic polypeptide is about 50 to about 500 amino acid residues in length. Thus, the molecular weight of immunogenic polypeptides or proteins suitable for the present invention may be from about 1 kDa to about 1,000 kDa, and preferably from about 2.5 kDa to about 250 kDa. In some more preferred embodiments, the molecular weight of the immunogenic polypeptide employed is from about 5 kDa to about 25 kDa or 50 kDa.

[0066] In some embodiments, the immunogenic polypeptide or protein used in the vaccine composition 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, but are not limited to, glycoproteins (or surface antigens, such as GP120 and GP41) and capsid proteins (or structural proteins, such as P24 protein) of HIV; surface antigens or core proteins of hepatitis A, B, C, D or E virus (e.g., small hepatitis B virus surface antigen (S-HBsAg) and core proteins NS3, NS4 and NS5 antigens of hepatitis C virus); glycoprotein gp350 / 220 of Epstein-Barr virus (EBV), glycoprotein (G protein) or fusion protein (F protein) of respiratory syncytial virus (RSV); surface and core proteins of herpes simplex virus HSV-1 and HSV-2 (e.g., glycoprotein D from HSV-2), surface proteins of poliovirus (e.g., gB, gC, gD, gH and gL), measles virus (e.g., rubella virus ... virus (MV), the glycoprotein G of lymphocytic choriomeningitis virus (LCMV), the fiber and penton base proteins of adenovirus, the S spike of coronavirus, the envelope (E) protein of flaviviruses (e.g., dengue virus, yellow fever virus, and Zika virus), and the non-enveloped capsid proteins of picornaviruses.

[0067] In some preferred embodiments, the immunogen or immunogenic protein displayed on the new I3-01 NP scaffold is a monomeric protein. Some examples of such proteins include, for example, the influenza M2 extracellular domain (M2e) protein exemplified herein. As described in detail below, some embodiments of the influenza vaccine of the present invention encompass NP vaccines comprising a new 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 tandem M2e copies comprise substitutions at the conserved Cys17 and Cys19 residues to prevent the formation of random disulfide bonds.

[0068] Some other embodiments of the present invention relate to HCV vaccines comprising a new I3-01 NP scaffold (e.g., SEQ ID NO: 4) that displays HCV immunogenic proteins. Generally speaking, the HCV immunogenic proteins to be displayed on the NP scaffold are derived from HCV glycoproteins E1 and E2, which form heterodimers on the HCV envelope that mediate viral entry into host hepatocytes. In some embodiments, the displayed HCV protein comprises the E2 core. As known in the art, the E2 core refers to a portion of E2 that forms a 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 variant scaffold of the present invention can be used to display a single copy of the E2 core protein or a tandem E2 core fusion protein. A specific HCV E2 core protein that can be used in the HCV vaccine construct of the present invention is the redesigned E2mc3 protein as described in U.S. Patent No. 11,008,368. E2mc3 from a variety of HCV subtypes or isolates can be used, including the E2mc3 sequences of HCV H77, J6, ED43, and UKN3A1.28c isolates exemplified herein (SEQ ID NOs: 32 to 35, respectively). Conservatively modified variants of these exemplified E2 core sequences or substantially identical sequences can also be used in the HCV vaccine constructs of the present invention.

[0069] 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 further 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 further embodiments, the HCV immunogenic protein displayed by the NP scaffold comprises an E1E2 heterodimer, for example, a rationally redesigned HCV E1E2 dimer. In addition to the HCV-derived immunogenic protein, the HCV vaccine displayed by the novel I3-01 scaffold may further comprise a locking domain and / or a T cell epitope. For example, the vaccine construct may have an LD7 motif (SEQ ID NO: 5) and a PADRE epitope (SEQ ID NO: 6) at the C-terminus, as exemplified herein.

[0070] In the construction of the HCV vaccine of the present invention, any E2 core protein sequence, tandem E2 core fusion molecule, and E1E2 dimer known in the art or that can be easily modified are suitable. Detailed guidance for obtaining such HCV immunogenic proteins and constructing NP vaccines containing such HCV immunogenic proteins is provided below, for example

[0071] WO21 / 021603; McGregor et al., J.Virol.96:e01675-21; Lin et al., FrontImmunol.2022:13:831285; Wang et al., Proc.Natl.Acad.Sci.USA 119:e2112008119, 2022: Clarke et al. al., Plant Biotechnol. J. 15:1611-21, 2017; and Sepulveda-Crespo et al., J. Biomed. Sci 27, 78, 2020

[0072] The various immunogenic proteins or polypeptides displayed on the novel I3-01NP scaffolds of the present invention (e.g., tandem influenza M2e fusion proteins or tandem HCV E2 core fusion proteins) can be obtained or produced according to the protocols exemplified herein or methods known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, (3rd ed., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ed., 2003).

[0073] V NP vaccines containing the novel I3-01NP scaffold

[0074] The present invention provides nanoparticle vaccines with the new I3-01NP scaffold disclosed herein. As described above, some vaccine constructs display HCV immunogenic proteins such as tandem E2 core proteins. Some other vaccine constructs of the present invention display a single copy of the influenza M2 protein extracellular domain (M2e). Several examples of such influenza NP vaccines are exemplified herein. In other embodiments, a fusion polypeptide comprising tandem repeats of the influenza M2e protein is displayed on a novel I3-01-derived nanoparticle scaffold. In these embodiments, the displayed immunogenic protein displayed on the novel I3-01 scaffold sequence is a fusion polypeptide comprising tandem repeats of two or more influenza M2e sequences. In various embodiments, at least one M2e tandem repeat contains missense mutations at the conserved Cys17 and Cys19 residues to prevent the formation of random disulfide bonds. In some of these influenza NP vaccine constructs, the engineered missense mutations replace each of the Cys residues with an amino acid residue containing an uncharged polar side chain. For example, each of the two CY residues in one or more tandem M2e repeats can be independently replaced by serine, glycine, asparagine, glutamine, threonine or tyrosine. In some embodiments, one or both of the Cys residues are replaced by Ser.

[0075] Typically, the tandem M2e fusion polypeptide sequence is fused to the N-terminus of the novel I3-01 scaffold sequence, for example, 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, the GGGG (SEQ ID NO: 9) spacer herein can be used to separate different M2e sequences, as exemplified herein. Some influenza NP vaccines of the present invention comprise three tandem M2e repeats. In various embodiments, the tandem M2e repeats displayed on the novel I3-01 scaffold of the present invention can be the same or different. Orthologous M2e sequences from many species and their modified forms are known in the art. See, for example, Mezhenskaya et al., J. Biomed. Sci. 26, 76, 2019. Thus, for example, each M2e tandem repeat can independently be a human M2e sequence (SEQ ID NO: 2), an avian / porcine consensus M2e sequence (SEQ ID NO: 7), or a human / porcine 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, the tandem M2e repeat sequence in the influenza vaccine of the present invention can include a human M2e sequence, an avian / porcine consensus M2e sequence, and a human / porcine consensus M2e sequence. In these embodiments, the three different Me2 sequences can be linked to the scaffold sequence in any of the six possible sequence orders.

[0076] 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 may retain unmutated residues at Cys17 and Cys19, while the avian / porcine consensus M2e sequence and the human / porcine consensus M2e sequence contain substituted residues at Cys17 and Cys19 (e.g., all replaced with Ser). In some of these embodiments, the mutated Cys residues are all replaced with Ser residues. Thus, the displayed M2e fusion polypeptide can comprise, in any order, unmutated human M2e (SEQ ID NO: 2), the mutated avian / porcine consensus M2e sequence SLLTEVETPTRNGWE S K S SDSSD (SEQ ID NO: 30) and mutated human / pig consensus M2e sequence SLLTEVETPTRSEWE S R SSGSSD (SEQ ID NO: 31). In some embodiments, all three tandem M2e repeats contain substitutions at residues Cys17 and Cys19. Thus, the displayed M2e fusion polypeptides can contain the mutated human M2eSLLTEVETPIRNEWG in any order. S R S NDSSD (SEQ ID NO: 29), mutated avian / porcine consensus M2e sequence SLLTEVETPTRNGWE S K S SDSSD (SEQ ID NO: 30) and mutated human / pig consensus M2e sequence SLLTEVETPTRSEWE S R S SGSSD (SEQ ID NO: 31). As a specific example, the fusion M2e polypeptide may comprise the sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24, conservatively modified variants thereof, or substantially identical sequences.

[0077] In some embodiments, NP vaccines (e.g., tandem influenza M2e vaccines or HCV E2 vaccines) comprising the novel I3-01 variant scaffolds of the present invention may optionally comprise a trimerization motif, e.g., SHP or foldon. Some nanoparticle vaccine compositions may additionally comprise other structural components whose function is to further enhance the stability and antigenicity of the displayed immunogen. In some embodiments, a locking protein domain (LD) may be inserted into the nanoparticle construct, e.g., by covalent fusion with the C-terminus of the nanoparticle subunit. The locking domain may be any dimeric protein capable of forming an interface through specific interactions, e.g., hydrophobic (van der Waals) contacts, hydrogen bonds, and / or salt bridges. An example of a locking domain that can be used in the vaccine of the present invention is LD7 (SEQ ID NO: 5) as exemplified herein. General guidance and various other examples for selecting lock domains (eg, LD4) are described in the art, for example, in WO 19 / 241483, US Pat. No. 10,906,944, and US Pat. No. 11,305,004.

[0078] In some embodiments, the scaffolded influenza vaccine of the present invention may also include T cell epitopes to promote robust T cell responses and guide B cells to develop into bNAbs. The T cell epitope can be located at any position relative to other structural components, as long as it does not affect the presentation of the modified HA protein on the surface of the nanoparticle. Any T cell epitope sequence or peptide known in the art can be used in the practice of the present invention. It includes any polypeptide sequence that contains an MHC class II epitope and can effectively activate CD4+ and CD8+ T cells after immunization, for example, 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; DeGroot 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 (pan D Repitope 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 is described in, for example, 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 are also described in the art, for example, D and TpD epitopes (Fraser et al., Vaccine 32, 2896-2903, 2014).

[0079] The nanoparticle vaccines based on the novel I3-01 scaffold of the present invention can be constructed according to standard recombinant techniques and other methods described in the art, for example, 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 PCT publications WO2017 / 192434, WO2019 / 089817, and WO19 / 241483. In various embodiments, nanoparticle vaccines based on the novel I3-01 scaffold can be constructed by fusing an immunogenic protein of interest (e.g., a tandem HCV E2 core or a tandem influenza M2e polypeptide) to an 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.

[0080] Following recombinant expression (e.g., in ExpiCHO cells as described in detail herein), the nanoparticle vaccines of the invention can be substantially purified by any conventionally practiced procedure. See, e.g., 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. Substantially purified means purified 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 easily tested using standard methods, such as antigenic profiling using known bNAbs and non-Nabs, differential scanning calorimetry (DSC), electron microscopy, binding analysis by ELISA, biolayer interferometry (BLI), surface plasmon resonance (SPR), and co-crystallographic analysis. Some of these assays are exemplified herein for the analysis of new I3-01 scaffolded HCV or influenza vaccines.

[0081] V. Polynucleotides and expression constructs

[0082] The new I3-01 scaffold of the present invention and the vaccine based thereon are generally produced by first producing an expression construct (i.e., an expression vector) comprising the coding sequences of the various structural components described herein that are operably linked. In some embodiments, the vaccine composition of the present invention is based on polynucleotides (e.g., mRNA-based vaccines). Therefore, in some related aspects, the present invention provides polynucleotides (e.g., DNA or RNA) encoding the subunit sequences of the new I3-01 scaffold or the nanoparticle vaccine based on the scaffold, expression vectors comprising such polynucleotides, and host cells (e.g., ExpiCHO cells as exemplified herein) for producing new NP scaffolds and vaccines. The present invention also encompasses fusion polypeptides encoded by the polynucleotides or expressed by the vector.

[0083] Polynucleotides and related vectors can be readily produced using standard molecular biology techniques or the protocols exemplified herein. For example, general protocols for cloning, transfection, transient gene expression, and obtaining stably transfected cell lines are described in the art, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, (3rd ed., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou edition, 2003). Introduction of mutations into a polynucleotide sequence by PCR can be performed as described in, for example, PCR Technology: Principles and Applications for DNA Amplification, HA 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.

[0084] The selection of a specific vector depends on the intended use of the fusion polypeptide. For example, the vector selected must be able to drive the expression of the fusion polypeptide in the desired cell type, whether the cell type is prokaryotic or eukaryotic. Many vectors contain sequences that allow both eukaryotic expression of operably linked gene sequences and prokaryotic vector replication. The vectors that can be used in the present invention can replicate autonomously, that is, the vector exists outside the chromosome, and its replication is not necessarily directly linked to the replication of the host cell's genome. Alternatively, the replication of the vector 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 in stably transfected cell lines. Both viral-based expression vectors and non-viral expression vectors can be used to produce immunogens in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors (usually with expression cassettes for expressing proteins or RNA) and human artificial chromosomes (see, for example, Harrington et al., Nat. Genet. 15: 345, 1997). Useful viral vectors include those based on lentivirus or other retroviruses, adenovirus, adeno-associated virus, cytomegalovirus, herpes virus, SV40, papillomavirus, HBP, Epstein-Barr virus, vaccinia virus, and Semliki Forest virus (SFV). See Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

[0085] Depend on the specific carrier for expressing fusion polypeptide, a variety of known cells or cell lines can be used for practice of the present invention.Host cell can be any cell that can introduce the recombinant vector carrying fusion body of the present invention, and wherein allows the carrier that drives fusion polypeptide expression to be used for the present invention.It can be prokaryotic, for example any one in many bacterial strains, or can be eukaryotic, for example yeast or other fungal cells, insect or amphibian cells or mammalian cells, including for example rodent, monkey or human cells.The cell expressing fusion polypeptide of the present invention can be primary culture cell or can be the cell line of having been established.Therefore, except the cell line (for example, CHO cell) illustrated herein, many other host cell lines known in the art can also be used for practice of the present invention.These include for example multiple Cos cell line, HeLa cell, HEK293, AtT20, BV2 and N18 cell, myeloma cell line, transformed B cell and hybridoma.

[0086] Using mammalian tissue cell culture to express polypeptide has been generally discussed in for example Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987.The carrier expressing fusion polypeptide can be introduced into the host cell of selection by any of many suitable methods known to those skilled in the art.In order to introduce the carrier encoding fusion polypeptide into mammalian cells, the method used will depend on the form of the carrier.For plasmid vectors, the DNA encoding fusion polypeptide sequence can be introduced by any of many transfection methods, and the method comprises for example lipid-mediated transfection (" lipid transfection (lipofection)"), DEAE-dextran-mediated transfection, electroporation or calcium phosphate precipitation. These methods are for example described in detail in Brent et al. above. The lipid transfection reagent and method that are suitable for transient transfection of a wide variety of transformed and untransformed or primary cells are widely available, making lipid transfection become the attractive method for constructs to be introduced into eukaryotic and particularly cultivated mammalian cells. For example, LipofectAMINE TM (Life Technologies) or LipoTaxi TM (Stratagene) kits are available. Other companies that provide reagents and methods for lipid transfection include Bio-Rad Laboratories, Clontech, Glen Research, Life Technologies, JBL Scientific, MBI Fermentas, PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.

[0087] In order to produce recombinant fusion polypeptides in high yield over a long period of time, stable expression is preferred. Instead of using an expression vector containing a viral origin of replication, the host cell can be transformed with a fusion polypeptide coding sequence and a selectable marker controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). The selectable marker in the recombinant vector confers resistance to selection and allows the cell to stably integrate the vector into its chromosome. Commonly used selectable markers include neo (Colberre-Garapin, et al., J. Mol. Biol., 150: 1, 1981), which confers resistance to the aminoglycoside G-418; and hygro (Santerre et al., Gene, 30: 147, 1984), which confers resistance to hygromycin. By appropriate selection, the transfected cells can contain an integrated copy of the fusion polypeptide coding sequence.

[0088] VI. Pharmaceutical compositions and therapeutic applications

[0089] The present invention provides pharmaceutical compositions or immunogenic compositions and related treatment methods using vaccines (e.g., influenza vaccines or HCV vaccines) based on the new I3-01 scaffold. In some embodiments, the vaccine composition can be used to prevent and treat diseases or infections (e.g., HCV infection or influenza). In some embodiments, nanoparticles displaying immunogenic proteins (e.g., tandem M2e or HCV E2 core protein) are included in the pharmaceutical composition. The pharmaceutical composition can be a therapeutic formulation or a prophylactic formulation. Generally speaking, the composition additionally comprises one or more pharmaceutically acceptable carriers, and optionally other therapeutic ingredients (e.g., antibiotics or antiviral drugs). A variety of pharmaceutically acceptable additives can also be used in the composition.

[0090] Some pharmaceutical compositions of the present invention are vaccines. For vaccine compositions, a suitable adjuvant may be additionally included. Some examples of suitable adjuvants include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, MPL TM and IL-12. In some embodiments, the vaccines based on the new I3-01 scaffold of the present invention can be formulated as controlled release or time-release formulations. This can be achieved in a composition comprising a slow-release polymer or by a microencapsulated delivery system or a bioadhesive gel. Various pharmaceutical compositions can be prepared according to standard procedures known in the art. See, for example, Remington's Pharmaceutical Sciences, 19.sup.th Ed., Mack Publishing Company, Easton, Pa., 1995; Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978); U.S. Patent Nos. 4,652,441 and 4,917,893; U.S. Patent Nos. 4,677,191 and 4,728,721; and U.S. Patent No. 4,675,189.

[0091] The therapeutic methods of the present invention relate to applying a suitable vaccine of the present invention (e.g., influenza vaccine or HCV vaccine) to a subject suffering from a disease or infection (e.g., influenza or HCV infection) or at risk of developing a disease or infection (e.g., influenza or HCV infection). Using influenza vaccine as an example, the immunogenic composition of the present invention is typically administered in an amount sufficient to induce an immune response against influenza virus or a group of viruses. For prophylactic applications, the immunogenic composition is provided before any symptoms (e.g., before infection). The prophylactic administration of the immunogenic composition is used to prevent or improve any subsequent infection. Therefore, in some embodiments, the subject to be treated is a subject suffering from influenza virus infection or at risk of developing influenza virus infection, e.g., due to exposure or possible exposure to a virus. After administering a therapeutically effective amount of the disclosed therapeutic composition, the viral infection of the subject, the symptoms associated with the viral infection, or both thereof can be monitored. For therapeutic applications, the immunogenic composition is provided during or after the onset of symptoms of disease or infection (e.g., after influenza symptoms occur, or after diagnosing a viral infection). Thus, the immunogenic composition can be provided before expected exposure to the virus to reduce the severity, duration or extent of the expected infection and / or related disease symptoms, provided after exposure or suspected exposure to the virus, or provided after the actual infection begins. The appropriate amount of the vaccine can be determined based on the specific disease or condition to be treated or prevented, the severity, the age of the subject, and other personal attributes of the specific subject (e.g., the overall state of the subject's health and the robustness of the subject's immune system). The determination of the effective dose is also guided by animal model studies followed by human clinical trials and by an administration regimen that significantly reduces the occurrence or severity of the targeted disease symptoms or conditions in the subject.

[0092] The pharmaceutical composition of the present invention can be combined with other pharmaceutical agents known in the art for treating or preventing diseases or infections (e.g., influenza virus infection). The administration of the pharmaceutical composition and known antiviral agents can be carried out simultaneously or sequentially. The pharmaceutical composition comprising a suitable vaccine of the present invention can be provided as a component of a medicine box. Optionally, such a medicine box comprises additional components, including packaging, instructions, and a variety of other reagents, such as buffer, substrate, antibody or ligand (e.g., control antibody or ligand) and detection reagent. Optional instructions can be provided in the medicine box in addition.

[0093] Example

[0094] The following examples are offered to illustrate but not to limit the invention.

[0095] Example 1 Rational design of the new I3-01v9a nanoparticle scaffold

[0096] The I3-01v9 nanoparticle scaffold was rationally optimized to achieve optimal surface display of monomeric protein antigens ( Figure 1The N-terminus of I3-01v9 forms a wide triangle, which is expected to be used for displaying monomeric antigens ( Figure 1 A). However, the first residue (antigen anchoring site) is located below the surface of the nanoparticle, and therefore a long linker must be used to connect the antigen to the N-terminus of I3-01v9, which will increase structural instability. The goal is to extend the I3-01v9 N-terminal helix so that its first residue is at the same level as the nanoparticle surface. To achieve this goal, the helical backbone of residues 953 to 982 from the c-MYC transcription factor protein (PDB ID: 6G6L) was selected, and the residues E2 and E3 of I3-01v9 were used for structural fitting and grafted onto the I3-01v9 subunit (SEQ ID NO: 27) ( Figure 1 B). The extended N-terminal helix was then truncated to 11 residues so that its first residue was just above the nanoparticle surface ( Figure 1 C). Next, the protein structure sampling program CONCOORD was used to generate 1000 slightly perturbed conformations for the modified I3-01v9 subunit ( Figure 1 D). Thereafter, the amino acids of the first 9 residues in the 11-residue segment were predicted using an ensemble-based protein design program previously used to optimize HIV gp140 and HCV E2 antigens using the RAPDF scoring function based on Cα and Cβ ( Figure 1 E). By combining the data from the predictions using the two energy functions, the final design I3-01v9a (SEQ ID NO: 4) was selected ( Figure 1 F).

[0097] Example 2 Display of HCV antigen on I3-01v9a nanoparticle scaffold

[0098] This example describes the multivalent display of HCV E2 core and tandem E2 core on I3-01v9a nanoparticle scaffolds. The newly designed I3-01v9a nanoparticle scaffold (SEQ ID NO: 4) has been used to present monomeric HCV E2 cores of different genotypes ( Figure 2 A). In negative staining EM, E2mc3I3-01v9a-LD7-PADRE nanoparticles designed for H77 (genotype 1a), HCV1 (genotype 1b), and ED43 (genotype 4) showed good nanoparticle formation ( Figure 2 B). The sequences of different E2mc3 proteins are shown in SEQ ID NOs: 32 to 35, respectively. Based on this result, a tandem E2 core antigen was designed, in which two HCV E2 cores of different genotypes were linked in tandem with a 5GS linker, and the tandem E2 core antigen was linked to I3-01v9a using an enzymatic restriction site "AS" (to facilitate molecular cloning) and another 5GS linker. Figure 2C). Based on this design strategy, H77 (genotype 1) and J6 (genotype 2) E2 cores were co-displayed on I3-01v9a-LD7-PADRE nanoparticles, and ED43 (genotype 4) and UKN3A1.28c (genotype 3) E2 cores were co-displayed on I3-01v9a-LD7-PADRE nanoparticles. Both nanoparticles were expressed in 50 ml or 200 ml of ExpiCHO cells and subsequently characterized by size-exclusion chromatography (SEC) on a Superose 6 column and by negative staining EM ( Figure 2 D). In conclusion, I3-01v9a has been successfully used to display monomeric antigens for vaccine development.

[0099] E2mc3 of H77 isolate (SEQ ID NO: 32):

[0100] QLINTNGSWHINSTALNCNESLNTGWLAGLFYQHKFDSSGCPERASGHYPRPCGIVPAKSVCGPVYCFTPSPVVVGTTDRSGAPTYSWGANDTDVFVLNNTGNWFGCTWMNSTGFTKVCGAPPGGPTDGGSGPWITPRCMVDYPYRLWHYPCTINYTIFKVRMYVGGVEHRLEAACN

[0101] E2mc3 of J6 isolate (SEQ ID NO: 33):

[0102] QLVNTNGSWHINRTALNCNDSLHTGFIASLFYTHSFNS SGCPERASGHYPRQCGVVSAKTVCGPVYCFTPSPVVVGTTDRLGAPTYTWGENETDVFLLNSTGSWFGCTWMNSSGYTKTCGAPPGGPTDGGSGPWLTPRCLIDYPYRLWHYPCTVNYTIFKIRMYVGGVEHRLTAACN

[0103] E2mc3 of ED43 isolate (SEQ ID NO: 34):

[0104] QLINSNGSWHINRTALNCNDSLNTGFLASLFYTHKFNSSGCSERASGHYARPCGIVPASSVCGPVYCFTPSPVVVGTTDHVGVPTYTWGENETDVFLLNSTGAWFGCVWMNSTGFTKTCGAPPGGPTDGGSGPWITPRCLIDYPYRLWHFPCTANFSVFNIRTFVGGIEHRMQAACN

[0105] E2mc3 of UKN3A1.28c isolate (SEQ ID NO: 35):

[0106] QLVNTNGSWHINRTALNCNESINTGFIAGLFYYHKFNSTGCPQRASGHYARPCESVPASKVCGPVYCFTPSPVVVGTTDAKGVPTYTWGANETDVFLLNSLGRWFGCTWMNSTGFTKTCGAPPGGPTDGGAGPWLTPRCMVDYPYRLWHYPCTVNFTLFQVRMFVGGFEHRFTAACN

[0107] Example 3 Design and characterization of a single hM2e (1c-SApNP) vaccine

[0108] The crystal structure of human M2e (hM2e) in complex with monoclonal antibodies Fab65 and Fab148 is available. Fab65-bound hM2e folds into a β-turn (T5-E8) and a 3 10 The hM2e bound by Fab148 adopts a hook-like conformation with an N-terminal β-turn (S2-T5). A trimeric scaffold (PDB ID: 1TD0) was used to present hM2e with a 5GS spacer (S2-D24) ( Figure 3 A), because 1TD0 was used as a C-terminal motif to stabilize EBOV GP trimers in our previous study. Structural modeling showed that the two hM2e peptides on the 1TD0 scaffold spanned 9.1 nm as measured at P10. The hM2e peptide was fused to 24-mer ferritin (FR) and two “multi-layered” 1c-SApNPs: E2p-LD4-PADRE (also known as E2p-L4P) and I3-01v9a-LD7-PADRE (also known as I3-01v9a-L7P), producing 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 ExpiCHO cells and purified by immunoaffinity chromatography (IAC) using antibodies Fab65 and Fab148 ( Figure 3 B). Size exclusion chromatography (SEC) spectra of the hM2e scaffold and three 1c-SApNPs obtained on Superdex 75 and Superose 6 columns, respectively ( Figure 3 C). Although multiple peaks were observed in SEC, IAC-purified 1c-SApNPs showed high purity in negative stain EM images collected at Scripps EM Core, indicating well-formed NPs ( Figure 3 D).

[0109] Example 4 Immunization with a single hM2e vaccine and influenza virus challenge

[0110] The immunogenicity and protective efficacy of M2e-based vaccines were evaluated in a comprehensive mouse study. Briefly, 10 mice / group were immunized by intradermal injection into the footpad with a total of 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. The mice were immunized twice, 3 weeks apart, and blood was collected 2 weeks after each injection. In this study, a group of naive mice was included as a negative control and a second group of mice was immunized with PR8 H1N1 virus (also known as inactivated H1N1 vaccine) inactivated by beta-propiolactone (BPL) to serve as a positive control. Three weeks after the second immunization, the mice were treated with 10×LD 50 The mice were challenged intranasally (in) with a 50% lethal dose (50%) of vaccine-matched A / Puerto Rico / 8 / 1934 (PR8) H1N1 virus (determined in a previous study). The mice were weighed daily and monitored for visible symptoms of infection (including ruffled fur, hunched posture, and / or decreased activity) 14 days post-infection (dpi). Mice that were visibly distressed or had lost 75% of their original body weight were euthanized. After surviving mice recovered to their post-immunization, pre-challenge baseline, they were treated with 10×LD 50 The A / Hong Kong / 1 / 1968 (HK68) H3N2 virus was used for heterologous IAV challenge and monitored 14 dpi. The vaccination / challenge study protocol was Figure 4 Shown in A.

[0111] After the first attack ( Figure 4 B), 8 of the 10 mice in all original mice and 1TD0 trimer group died within 8dpi. In contrast, the survival rate of all three 1c-SApNP groups and the group receiving inactivated PR8 H1N1 vaccine was 100%. The mice receiving the PR8H1N1 vaccine of strain matching lost the least weight, began to regain weight at 6dpi, and returned to their starting weight at 14dpi. The mice receiving hM2e-5GS-FR and hM2e-5GS-I3-01v9a-L7P lost more weight and began to recover at 8dpi, but also returned to their starting weight at 14dpi. The mice receiving hM2e-5GS-1TD0 trimer lost significantly more weight than those in the 1c-SApNP group. The two surviving mice began to regain weight at 9dpi and had not yet returned to their starting weight at 14dpi. After the second attack ( Figure 4 C), 4 of 9 mice in the group receiving the inactivated PR8 H1N1 vaccine died, while all mice vaccinated with hM2e survived the H3N2 challenge. Consistently, mice that received the inactivated H1N1 virus vaccine lost significantly more weight after the H3N2 challenge and began to regain their weight a day later (at dpi 6) at a slower rate compared to the hM2e trimer and 1c-SApNP vaccine groups. Therefore, the challenge data emphasize the effectiveness and broad protection of the M2e1c-SApNP vaccine. To determine whether the protection of mice vaccinated with hM2e was associated with hM2e-specific antibody responses, an enzyme-linked immunosorbent assay (ELISA) was performed on mouse sera at w5 (one week before the first challenge) using an hM2e-5GS-foldon antigen probe ( Figure 4 D). Foldon (PDBID: 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 exhibited no hM2e-specific response. Notably, mice vaccinated with the inactivated H1N1 virus also showed no signal, consistent with the low abundance of M2 in virions 32. While all 1c-SApNP groups exhibited high hM2e-specific antibody titers, two surviving mice in the 1TD0 trimer group developed a detectable hM2e antibody response. Overall, these results demonstrate that 1c-SApNPs displaying M2e effectively protect mice against lethal challenge with two different IAVs, and that this protection is closely associated with M2e-specific antibodies.

[0112] Example 5 Sequences of some single hM2e vaccine constructs

[0113] The sequences of some of the hM2e vaccines described herein have been determined. The complete sequences of the I3-01v9a-based constructs are shown below. In this 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 were mutated in some of the tandem M2e constructs discussed below are also underlined. It should be noted that the first Met residue was removed from the hM2e sequence inserted into the NP constructs described herein. Therefore, although referred to herein (and in the literature) as Cys17 and Cys19, respectively, based on the original complete hM2e sequence, they are actually residues 16 and 18 in the hM2e sequence present in the vaccine constructs. The two bolded and underlined residues represent PCR restriction sites. The double-underlined sequence represents the I3-01v9aNP scaffold subunit sequence (SEQ ID NO: 4). The construct may also optionally include a locking domain and / or a T cell epitope. As illustrated in the construct herein, the locking domain used may be LD7 (SEQ ID NO:5) (shown in double underline and bold), and the T cell epitope may be a PADRE epitope (SEQ ID NO:6) (shown in double underline and italics). Joints or spacers separating the different structural motifs of the nanoparticle construct are shown in the construct sequence herein with italics and underlined residues, for example, GS, GGGG spacers (SEQ ID NO:9) and 5GS joints (SEQ ID NO:3).

[0114] hM2e-5GS-I3-01v9a-LD7-PADRE construct without N-terminal leader and LD / PADRE motif (SEQ ID NO: 10)

[0115]

[0116] hM2e-5GS-I3-01v9a-LD7-PADRE construct containing a C-terminal LD / PADRE motif (SEQ ID NO: 13)

[0117]

[0118] hM2e-5GS-I3-01v9a-LD7-PADRE construct containing an N-terminal leader (SEQ ID NO: 16)

[0119]

[0120] hM2e-5GS-I3-01v9a-LD7-PADRE construct containing an N-terminal leader and a C-terminal LD / PADRE motif (SEQ ID NO: 19)

[0121]

[0122] Example 6 Design, characterization, and challenge studies of tandem M2e vaccines

[0123] Phylogenetic analysis has divided IAVs into several lineages based on their original host species: avian, porcine, or human. Although M2e is highly conserved, small but important sequence differences exist between IAVs from different species, which have been shown to limit cross-protection. Therefore, in addition to seasonal endemic strains, a universal influenza vaccine based on M2e must provide protection against pandemic strains that are usually derived from avian or porcine IAVs. The combined use of M2e sequences from multiple species has been reported previously. Here, a tandem M2e × 3 construct containing human, avian / porcine, and human / porcine M2e sequences was designed, with a GGGG (SEQ ID NO: 9) spacer between consecutive M2e segments. Notably, Cys17 and Cys19 in the second (avian / porcine) and third (human / porcine) repeats were mutated to serine to avoid random disulfide bonds. Optionally, as exemplified herein, Cys1 7 and Cys1 9 in all three repeats can be mutated to serine. The M2e × 3 antigen was fused to lTD0 and three 1c-SApNPs via a 5GS spacer to produce a product called

[0124] Four constructs were constructed: M2e×3-5GS-1TD0, M2e×3-5GS-FR, M2e×3-5GS-E2p-LD4-PADRE (or M2e×3-5GS-E2p-L4P), and M2e×3-5GS-I3-01v9a-LD7-PADRE (or M2e×3-5GS-I3-01v9a-L7P). These four tandem M2e immunogens were transiently expressed in ExpiCHO cells and purified by IAC using a Fab148 antibody column. The Fab148-purified 1c-SApNP samples were analyzed using negative staining EM at Scripps EM Core. Consistent with hM2e 1c-SApNP, all tandem M2e 1c-SApNPs showed good NP formation ( Figure 5 A).

[0125] The immunogenicity and protective efficacy of the tandem M2e vaccine were evaluated in mice following a similar protocol to that used for the hM2e immunogen studies ( Figure 4A). Two adjuvants were tested in this study: aluminum hydroxide (AH) and the oil-in-water emulsion AddaVax. Figure 5 B), after PR8 H1N1 challenge, all naive mice and 4 of 8 mice in the 1TD0 trimer group died within 9 dpi. In contrast, the survival rate of FR 1c-SApNP was 88%, and the survival rate of the two large multilayer 1c-SApNP groups and the group receiving inactivated PR8 H1N1 vaccine was 100%. In terms of weight loss, AH-formulated E2p 1c-SApNP showed the closest effect to the inactivated vaccine and was more effective than other tandem M2e immunogens. For the AddaVax adjuvant group ( Figure 5 C), after PR8 H1N1 challenge, all naive mice and 3 of 8 mice in the 1TD0 trimer group died within 9 dpi. In contrast, the survival rate of all 1c-SApNP groups and the group receiving the inactivated PR8 H1N1 vaccine was 100%. In terms of weight loss, the I3-01v9a 1c-SApNP formulated by AddaVax showed the closest effect to the inactivated vaccine and was more effective than other tandem M2e immunogens. This formulation also outperformed the E2p / AH formulation in preventing weight loss ( Figure 5 B). Overall, the tandem M2e immunogens exhibited broad protection, with the tandem M2e-5GS-1TD0 trimer significantly outperforming its hM2e counterpart, and I3-01v9a 1c-SApNP being the best performing of all immunogens when paired with AddaVax.

[0126] Example 7 Sequences of some tandem M2e×3 immunogen constructs

[0127] 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 these two constructs contains a rationally designed I3-01v9a variant 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 human M2e sequences SLLTEVETPIRNEWG, SLLTEVETPIRNEWG, SLLTEVETPIRNEWG, SLLTEVETPIRNEWG, SLLTEVETPIRNEWG, SLLTEVETPIRNEWG, SLLTEVETPIRNEWG, SLLTEVETPIRNEWG, SLLTEVETPIRNEWG, SLLTEVETPIRNEWG, SLLTEVETPIRNEWG, SLLTEVETPIRNEWG, SLLTEVETPIRNEWG, SLLTEVETPIRNEWG C R C NDSSD (SEQ ID NO: 2; Cys17 and Cys19 are underlined), avian / porcine consensus M2e sequence SLLTEVETPTRNGWE C K CSDSSD (SEQ ID NO: 7; Cys17 and Cys19 are underlined) and human / pig consensus M2e sequence SLLTEVETPTRSEWE C R C SGSSD (SEQ ID NO: 8; Cys17 and Cys19 are underlined). In addition, the two constructs have the conserved Cys17 and Cys19 residues replaced with Ser residues in two or all three of the M2e tandem repeats, respectively. 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 of the constructs may additionally have a leader sequence at the N-terminus. The leader sequence may comprise MGILPSPGMPALLSLVSLLSVLLMGCVAE (SEQ ID NO: 1), as exemplified herein.

[0128] Tandem M2e polypeptide with conserved CYs residues mutated in repeats 2 and 3 (SEQ ID NO: 23):

[0129]

[0130] Tandem M2e polypeptide with conserved CYs residue mutated in all three repeats (SEQ ID NO: 24):

[0131]

[0132] 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 or the substituted Ser residues in the M2e sequences are also underlined. The two bold and underlined residues represent restriction sites for PCR. Linkers or spacers connecting the different motifs of the M2e sequences and / or constructs are italicized and underlined. These include the 5aa GS linker GGGGS (SEQ ID NO: 3) and the GGGG (SEQ ID NO: 9) spacer that separates the tandem M2e sequences. The double-underlined sequences represent the display of the I3-01v9a scaffold sequence. The sequence of the lock domain (LD7) is double-underlined and bolded. Finally, the PATRE T cell epitope is double-underlined and italicized.

[0133] Tandem M2eNP construct with Cys17 / CYs19 mutated to serine in M2e repeats #2 and #3, without the N-terminal leader and C-terminal LD / PADRE motif (SEQ ID NO: 11):

[0134]

[0135] Tandem M2eNP construct with Cys17 / CYs19 mutated to serine in M2e repeats #2 and #3, containing a C-terminal LD / PADRE motif (SEQ ID NO: 14):

[0136]

[0137] Tandem M2e NP construct with Cys17 / CYs19 mutated in both M2e repeats, comprising an N-terminal leader sequence (SEQ ID NO: 17):

[0138]

[0139] Tandem M2e NP construct with Cys17 / CYs19 mutated in two M2e repeats, comprising an N-terminal leader sequence and a C-terminal LD / PADRE motif (SEQ ID NO: 20):

[0140]

[0141] Tandem M2eNP construct with Cys17 / CYs19 mutated to serine in all three M2e repeats, without the N-terminal leader and C-terminal LD / PADRE motif (SEQ ID NO: 12):

[0142]

[0143] Tandem M2eNP construct with Cys17 / CYs19 mutated to serine in all three M2e repeats, containing a C-terminal LD / PADRE motif (SEQ ID NO: 15):

[0144]

[0145] Tandem M2eNP construct with Cys17 / CYs19 mutated to serine in all three M2e repeats, comprising an N-terminal leader sequence (SEQ ID NO: 18):

[0146]

[0147] Tandem M2eNP construct with Cys17 / CYs19 mutated to serine in all three M2e repeats, comprising an N-terminal leader sequence and a C-terminal LD / PADRE motif (SEQ ID NO: 21):

[0148]

[0149]

[0150] ***

[0151] Thus, the invention has been broadly disclosed and illustrated with reference to the representative embodiments described above. It will be appreciated that various modifications can be made thereto without departing from the spirit and scope of the invention.

[0152] It should also be noted that all publications, sequence accession numbers, patents, and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for all purposes to the same extent as if each were individually so indicated. Definitions contained in the text incorporated by reference are excluded to the extent they conflict with definitions in this disclosure.

Claims

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

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

27.

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

4. 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.

5. Self-assembling nanoparticles, which are formed by the N-terminally extended I3-01 nanoparticle scaffold sequence according to 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. The nanoparticle vaccine construct of claim 6, wherein the N-terminally extended I3-01 nanoparticle scaffold sequence comprises a heterologous helical motif of about 6 to about 12 amino acid residues, which is fused to the N-terminus of the I3-01 scaffold sequence shown in SEQ ID NO:

27.

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

9. 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.

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

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

14. The nanoparticle-displayed 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 M2e tandem repeat comprises a missense mutation at the conserved Cys17 and Cys19 residues.

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

17. The nanoparticle vaccine construct of claim 15, wherein each of the Cys residues is independently replaced 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 both of the Cys residues are replaced by Ser.

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

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

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 in which Cys17 and Cys19 are each replaced by a Ser residue (SEQ ID NO: 30), and a human / porcine consensus M2e sequence in which Cys17 and Cys19 are each replaced by a Ser residue (SEQ ID NO: 31).

22. The nanoparticle vaccine construct of claim 20, wherein the influenza fusion polypeptide comprises, in any order, human M2e in which Cys17 and Cys19 are each replaced with a Ser residue (SEQ ID NO: 29), an avian / porcine consensus M2e sequence in which Cys17 and Cys19 are each replaced with a Ser residue (SEQ ID NO: 30), and a human / porcine consensus M2e sequence in which Cys17 and Cys19 are each replaced with a Ser residue (SEQ ID NO: 31).

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

24. The nanoparticle vaccine construct of claim 20, comprising the sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, conservatively modified variants thereof, or substantially identical sequences.

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

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. The nanoparticle vaccine construct of claim 25, comprising SEQ ID NO: 14, SEQ ID NO: 15, conservatively modified variants thereof, or substantially identical sequences.

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

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

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

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

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

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

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

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

36. 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 T cell epitope and a locking domain at the C-terminus.

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. The nanoparticle vaccine construct of claim 37, comprising (1) a subunit sequence comprising, from N-terminus to 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 of the subunit sequence or a substantially identical sequence.

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.

Images