FUSION PROTEIN COMPRISING BP26 AND ANTIGENIC POLYPEPTIDE

Abstract

The present disclosure relates to a fusion protein comprising BP26 and an antigenic polypeptide, and to a nanoarchitecture comprising same. A vaccine composition comprising the fusion protein, nanoarchitecture, or combination thereof of the present disclosure can be used to effectively prevent or treat pathogens or cancer, and thus can be used as a multi-purpose vaccine platform.

Claims

1. A fusion protein comprising BP26 and an antigenic polypeptide.

2. The fusion protein of claim 1, wherein the BP26 comprises the amino acid sequence of SEQ ID NO: 1 or 2.

3. The fusion protein of claim 1, wherein the antigen is a pathogen-derived antigen or a tumor-derived antigen.

4. The fusion protein of claim 3, wherein the pathogen is selected from the group consisting of a virus, a bacterium, a rickettsia, a fungus, and a protozoa.

5. The fusion protein of claim 3, wherein the pathogen-derived antigenic polypeptide is M2e.

6. The fusion protein of claim 5, wherein the M2e comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, or 6.

7. The fusion protein of claim 1, wherein the fusion protein comprises at least one copy of the antigenic polypeptide.

8. The fusion protein of claim 1, wherein the antigenic polypeptides are continuously or discontinuously linked in the fusion protein.

9. A nucleic acid molecule comprising a nucleotide sequence encoding for the fusion protein of claim 1.

10. A recombinant vector comprising the nucleic acid of claim 9.

11. A host cell comprising the recombinant vector of claim 10.

12. A nanoarchitecture comprising the fusion protein of claim 1.

13. The nanoarchitecture of claim 12, wherein the nanoarchitecture comprises two or more copies of a fusion protein comprising BP26 and an antigenic polypeptide.

14. The nanoarchitecture of claim 12, wherein the nanoarchitecture comprises 8 or 16 copies of a fusion protein comprising BP26 and an antigenic polypeptide.

15. A vaccine composition comprising the fusion protein of claim 1, a nanoarchitecture comprising a fusion protein comprising BP26 and an antigenic polypeptide, or a combination thereof.

16. The vaccine composition of claim 15, wherein the vaccine composition is for preventing infection from an infectious pathogen.

17. The vaccine composition of claim 15, wherein the vaccine composition is for a cancer vaccine.

18. The vaccine composition of claim 15, wherein the vaccine composition further comprises an adjuvant.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0165] FIG. 1 shows a BP26-based nanoarchitecture displaying an influenza M2e epitope.

[0166] FIG. 2A shows a fusion protein including BP26 and M2e.

[0167] FIG. 2B shows the expression of BP26-M2e (×4) and BP26-M2e (×8) as analyzed by size exclusion chromatography.

[0168] FIG. 2C shows the expression of BP26-WT, BP26-M2e (×4) and BP26-M2e (×8) as analyzed by SDS-PAGE.

[0169] FIG. 2D shows transmission electron microscopy (TEM) images of BP26-M2e (×4) and BP26-M2e (×8) (scale bar=100 nm).

[0170] FIG. 2E shows the hydrodynamic diameter of the nanoarchitecture as measured by dynamic light scattering (DLS).

[0171] FIG. 2F shows the results of ELISA analysis to confirm the accessibility and reactivity of the anti-M2e antibody to the multi-tandem (tandem) repeat structure of the M2e epitope displayed on the surface of the BP26 nanoarchitecture.

[0172] FIG. 3A shows an experimental method for confirming the antibody production in mice by the nanoarchitecture.

[0173] FIG. 3B shows titers of anti-M2e antibodies present in the sera after immunization with nanoarchitectures as measured by ELISA.

[0174] FIG. 3C shows titers of anti-M2e IgG in sera of immunized mice (days 14, 35, and 56) as measured by ELISA.

[0175] FIG. 3D shows titers of M2e-specific IgG1 and IgG2a in sera of immunized mice (day 56) as measured by ELISA.

[0176] FIG. 3E shows titers of anti-BP26 IgG in sera of immunized mice (day 56) as measured by ELISA.

[0177] FIG. 3F shows Effect of pre-existing anti-BP26 antibodies on M2e-specific humoral responses elicited by BP26-M2e nanovaccine. (A) Immunization schedule. (B) Anti-M2e IgG titer in the sera of BP26-M2e(×8) immunized or non-immunized control mice. Titers of M2e-specific antibody were measured by ELISA. (*P<0.05, **P<0.01, ***P<0.001, n.s.=not significant, one-way ANOVA with post hoc Tukey's test).

[0178] FIG. 4A shows anti-M2e antibody binding to M2e displayed on influenza virus-infected cells.

[0179] FIG. 4B shows confocal laser-scanning microscopic images accounting for the binding capability of anti-M2e antibody for influenza virus-infected MDCK cells (scale bar=100 μm).

[0180] FIG. 4C shows the binding capability of anti-M2e antibody to influenza virus-infected MDCK cells as measured by whole-cell ELISA.

[0181] FIG. 4D shows the binding capability of anti-M2e antibody to influenza virus-infected MDCK cells as measured by Western blot analysis.

[0182] FIG. 5A shows the immunization schedule of Balb/c mice to evaluate the protective effect of the BP26-M2e nano vaccine against influenza virus.

[0183] FIG. 5B shows clinical scores for influenza virus-infected mice.

[0184] FIG. 5C shows measurements of survival rate to evaluate the protective effect of BP26-M2e nano vaccine against influenza virus.

[0185] FIG. 5D shows body weight changes to evaluate the protective effect of the BP26-M2e nano vaccine against influenza virus.

[0186] FIG. 5E shows clinical scores to evaluate the protective effect of BP26-M2e nano vaccine against influenza virus.

[0187] FIG. 5F shows rectal temperature measurements to evaluate the protective effect of BP26-M2e nano vaccine against influenza virus.

[0188] FIG. 5G shows residual lung viral titers in immunized mice measured 3 days after virus challenge.

[0189] FIG. 5H shows Protective immunity of BP26-M2e nanovaccine after A/California/04/2009 (H1N1 pdm09) challenge. (A) An immunization schedule for determining protective effects against influenza virus (n=12 mice per group). (B) Survival curves of A/CA/04/09-infected mice. (C) Body weight changes of influenza A virus-infected mice. (D) Clinical score of influenza A virus-infected mice. (E) Rectal temperature changes of influenza A virus-infected mice.

[0190] FIG. 5I shows Histology of lung tissues isolated from influenza A virus-challenged mice. Lung tissues were isolated from immunized mice with each modality at day 5 post infection with A/CA/04/09 and assessed by H&E staining. Images are representatives of each immunization group (n=3 mice per group). Scale bar=50 μm.

[0191] FIG. 6 shows analysis of binding modes of anti-M2e antibodies in the sera of BP26-M2e(×8) immunized mice. (A) Titer of anti-influenza A virus IgG in the sera of PBS-treated or BP26-M2e(×8) immunized mice was measured by ELISA. Sera from A/CA/04/09-infected mice were used as a positive control. Optical density of pre-immune sera on day −1 is ˜0.13 (data not shown). (B) Whole-cell ELISA was conducted to examine binding of anti-M2e antibodies in the sera of PBS or BP26-M2e(×8) immunized mice to influenza A virus-infected MDCK cells.

[0192] FIG. 7 shows that Anti-M2e antibodies in the sera of BP26-M2e nanovaccine-immunized mice can induce NK cell-mediated ADCC. (A) A schematic depiction of anti-M2e antibody-dependent NK cell-mediated cytotoxicity assay. Effector cells (NK cells) and target cells (influenza virus-infected MDCK cells) were co-incubated in the presence of sera from either PBS-treated mice or BP26-M2e(×8) immunized mice. (B) Representative flow cytometry results of ADCC assay. (C) Percentages of target cell-specific lysis at an E:T ratio of 2:1 or 1:2.

[0193] FIG. 8 is a schematic diagram of a protein complex constructed to confirm the use of BP26 of the present disclosure as a cancer vaccine.

[0194] FIG. 9 a diagram of an immunization schedule for evaluating the efficacy of the BP26-containing cancer vaccine of the present disclosure.

[0195] FIG. 10 is a plot of volumes of B16F10 tumor cells and shows degrees of growth of tumor cells.

[0196] FIG. 11 shows a flow cytometry gating strategy of the present disclosure.

[0197] FIG. 12 is a plot of the % of CD4+ T cells secreting IFN-γ after immunization with the cancer vaccine containing BP26 of the present disclosure.

[0198] FIG. 13 shows a fusion protein displaying 10 tandem repeats of MHC class II neoantigen peptide M30 (15 amino acids, VDWENVSPELNSTDQ) from mouse melanoma cell line B16-F10 at the C-term of BP26 protein.

[0199] FIG. 14 is data on the purification and expression of the BP26-LQ-M30 (×10) fusion protein.

[0200] FIG. 15 shows the Vaccine immunization schedule.

[0201] FIG. 16 shows the B16F10 tumor growth curve after inoculation with the BP26-LQ-M30 (×10) fusion protein.

[0202] FIG. 17 shows the B16F10 tumor volume on the 18th day after tumor inoculation.

[0203] FIG. 18 shows the antigen-specific T cell immune response by inoculation with the BP26-LQ-M30 (×10) fusion protein.

[0204] FIG. 19 shows that the adjuvant (e.g. CpG, poly(I:C)) improves the antitumor efficacy of the BP26-LQ-M30 (×10) fusion protein.

[0205] FIG. 20 shows the efficacy of administration of the BP26-LQ-M30 (×10) fusion protein compared to the positive control group.

[0206] FIG. 21 shows the B16F10 individual tumor growth curve of each group shown in FIG. 20.

DETAILED DESCRIPTION

[0207] Hereinafter, the present disclosure will be described in more detail through examples. These examples are only for illustrating the present disclosure in more detail, and it will be apparent to those skilled in the art that the scope of the present disclosure is not limited by these examples according to the gist of the present disclosure.

ExampleS

[0208] Unless otherwise stated, “%” used to indicate the concentration of a specific substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and (volume/volume) % for liquid/liquid throughout the specification.

[0209] Animals, Cells, and Viruses

[0210] Female Balb/c mice were purchased from Orient Bio (Korea) and housed under pathogen-free conditions. Animal care and experimental procedures were approved by the Animal Experimental Ethics Committees of the Korea Advanced Institute of Science and Technology (KAIST) (Accreditation No.: KA2020-56). MDCK cells were cultured 37° C. in MEM medium (Welgene, Gyeongsan, Korea) supplemented with 1% penicillin/streptomycin and 10% heat-inactivated fetal bovine serum (FBS; Welgene) under a 5% CO2 condition. The influenza A viruses A/PR/8, A/CA/04/09 and A/Aquatic bird/Korea were used.

Example 1: Cloning, Expression, and Purification of BP26-M2e Recombinant Proteins

[0211] The BP26 sequence is as follows (Table 1).

TABLE-US-00001 TABLE 1 SEQ ID category Sequence NO. BP26 QENQMTTQPARIAVTGEGMMTASPDMAILNLSVLRQA 1 KTAREAMTANNEAMTKVLDAMKKAGIEDRDLQTGGIDI QPIYVYPDDKNNLKEPTITGYSVSTSLTVRVRELANVGK ILDESVTLGVNQGGDLNLVNDNPSAVINEARKRAVANAI AKAKTLADAAGVGLGRVVEISELSRPPMPMPIARGQFR TMLAAAPDNSVPIAAGENSYNVSVNVVFEIK BP26 having His MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSE 2 tag and TEV NLYFQGSQENQMTTQPARIAVTGEGMMTASPDMAILN cleavage site LSVLRQAKTAREAMTANNEAMTKVLDAMKKAGIEDRD inserted thereto LQTGGIDIQPIYVYPDDKNNLKEPTITGYSVSTSLTVRV (sequence used RELANVGKILDESVTLGVNQGGDLNLVNDNPSAVINEA for cloning) RKRAVANAIAKAKTLADAAGVGLGRVVEISELSRPPMP MPIARGQFRTMLAAAPDNSVPIAAGENSYNVSVNVVFE IK

[0212] Tandem repeats of the M2e(×4) sequence were synthesized using a gene synthesizer (Bioneer, Korea) and incorporated into the pUC vector. The four M2e repeats (SLLTEVETPIRNEWGSRSNDSSD, SEQ ID NO: 3) in the sequence were separated by an amino acid linker consisting of GGGSG. The nucleotide sequence of M2e (×4) is as follows, and was codon-optimized to maximize expression in an E. coli system. BamHI and Xho I restriction sites were incorporated to the N- and C-termini, respectively.

TABLE-US-00002 Nucleic acid sequence of M2e (SEQ ID NO: 7): AGCCTGCTGACCGAAGTCGAGACTCCGATCCGTAATGAATGGGGCT CTCGTTCTAACGACTCGTCGGAT

[0213] The expression vector for BP26-M2e (×4) was constructed by subcloning into a modified pET28a vector containing an N-terminal His-tag and a tobacco etch virus (TEV) protease cleavage site after the His tag, and the expression vector for BP26-M2e (×8) was constructed using an in-fusion cloning kit (Takara, Japan) according to the manufacturer's protocols. Recombinant proteins were expressed in BL21 (DE3) RIPL E. coli for 16 hours at 18° C. after induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and were purified by Ni-NTA affinity chromatography (Qiagen, Hilden, Germany). After cleavage of the N-terminal His-tag with TEV protease, proteins were further purified using Ni-NTA resin, a HiTrap Q-SP cation exchange column, and Superdex 200 26/60 size-exclusion chromatography (GE Healthcare, Illinois, USA).

[0214] Expression and purification of recombinant proteins were confirmed by SDS-PAGE.

[0215] The main epitope of M2e was fused to monomeric BP26, which undergoes self-assembly into an octamer and then further assembles into a hexadecameric hollow barrel-like architecture, termed a nanobarrel, in which M2e antigens are displayed around the rim of the nanobarrel (FIG. 1).

[0216] Four and eight tandem repeats of M2e were genetically fused to the C-terminus of monomeric BP26 using a short flexible linker (GGGSG) to yield two fusion proteins, designated BP26-M2e (×4) and BP26-M2e (×8), respectively (FIG. 2A). Both fusion proteins were expressed in E. coli in high yield and subsequently purified by size-exclusion chromatography (FIG. 2B). SDS-PAGE analysis clearly distinguished the two proteins based on their molecular size (FIG. 2C).

Example 2: Characterization of BP26-M2e Nanoarchitecture

2-1. Morphology of BP26-M2e (×4) and BP26-M2e (×8) Evaluated by Transmission Electron Microscopy

[0217] The morphology of BP26-M2e (×4) and BP26-M2e (×8) was evaluated by negative staining transmission electron microscopy (TEM).

[0218] The transmission electron microscopy (TEM) analysis of the self-assembled structures of these fusion proteins revealed that both BP26-M2e (×4) and BP26-M2e (×8) formed discrete nanoparticles with mean diameters of 16.4 nm and 19.2 nm, respectively, and contained a hollow cavity (hole) at its center (FIG. 2D); the hole in the latter was smaller than that in the former, presumably because the larger M2e tandem repeats in the latter protruded around the hole. These structural observations indicate that the presence of M2e antigen does not interfere with the BP26 self-assembly process, despite the fact that the size of the M2e(×8) repeat (˜23 kDa) is similar to that of a BP26 monomer (˜28 kDa), suggesting that there might be no need for complicated vaccine module design and expression screening.

2-2. Hydrodynamic Size of Nanoarchitectures

[0219] The hydrodynamic size of nanobarrels was determined by dynamic light scattering (DLS) at ambient temperature using a Zetasizer Nano range system (Malvern, Worcestershire, UK).

[0220] Hydrodynamic size is defined as “the size of an imaginary solid sphere that diffuses in the same way as the particle being measured” as measured by DLS.

[0221] The hydrodynamic size of BP26-based nanobarrels, measured by dynamic light scattering (DLS), was ˜10.97 nm for wild-type (WT) BP26 and increased to ˜14.07 nm for BP26-M2e (×4) and ˜22.42 nm for BP26-M2e (×8) (FIG. 2E).

2-3. Accessibility of Anti-M2e Antibody to M2e Epitope on BP26 Nanoarchitecture Surface

[0222] The accessibility of the anti-M2e antibody to M2e epitopes on the surface of BP26 nanobarrel was confirmed using ELISA. BP26-WT, BP26-M2e (×4), and BP26-M2e (×8) were coated onto 96-well plates at different concentrations (M2e concentration, 0.01-1000 pmol) and incubated overnight at 4° C. The plates were then blocked with PBS with Tween-20 (PBST) containing 2% bovine serum albumin (BSA) for 1 hour at room temperature and then incubated with mouse anti-M2e IgG antibody (14C2 clone, 1:1000 dilution; Santa Cruz Biotechnology, Texas, USA) for 2 hours at room temperature. Plates were subsequently washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (1:5000 dilution; Santa Cruz Biotechnology) for 1 hour at room temperature, after which TMB substrate solution was added to each well and changes were detected colorimetrically by measuring absorbance at 450 nm using a microplate reader (VersaMax™; Molecular Devices, California, USA).

[0223] The ELISA results revealed much higher reactivity of the anti-M2e antibody against BP26-M2e (×8) than BP26-M2e (×4) (FIG. 2F), implying that increasing M2e tandem repeats may result in more efficient recognition of displayed antigen by BCR on B cells, leading to greater B cell responses.

Example 3: Confirmation of Antibody Production in Mice by Nanoarchitectures

3-1. Immunization of Mice Using Nanoarchitectures

[0224] Six-week-old female BALB/c mice were immunized using a homologous prime-boost regimen. Mice were divided into five groups: PBS buffer vehicle, (BP26-WT+M2e)/Alum (BP26-WT, 20 μg; M2e, 7.3 μg), BP26-M2e (×4) (25 μg), BP26-M2e (×8) (18 μg), and BP26-M2e (×8)/Alum (BP26-M2e: 18 μg). Mice in each group were immunized thrice at intervals of three weeks by subcutaneous injection into both footpads.

[0225] The doses were adjusted so that immunizing M2e was administered in equimolar amounts. For the groups containing alum, the antigen solution and 30% AlOH solution were mixed in equal amounts. A 30% AlOH solution was prepared by diluting an Al.sub.2O.sub.3 solution (Rehydragel HPA, Reheis, Berkeley Heights, N.J.) with dH2O to adjust the pH to 7.

3-2. Determination of M2e Antibody Production by ELISA

[0226] Mice were divided into 5 treatment groups as described above and immunized three times at 3-week intervals. To determine the humoral immune response, sera were isolated from blood collected retro-orbitally into Serum Separator Tubes (BD) at four time points: day −1 (pre-immunization) and days 14, 35, and 56 (i.e., 2 weeks after each immunization). M2e-specific antibody titers were determined by ELISA.

[0227] 96-well plates were coated with M2e antigen and incubated overnight at 4° C. Plates were washed and blocked with PBST containing 2% BSA for 1 hour at room temperature, and then incubated with diluted serum samples for 2 hours at room temperature. Plates were subsequently washed and incubated with HRP-conjugated goat anti-mouse IgG, IgG1 or IgG2a (1:5000 dilution; Santa Cruz Biotechnology) as secondary antibodies for 1 hour at room temperature.

[0228] The immunogenicity of the BP26-M2e nanoarchitectures was evaluated in Balb/c mice immunized thrice at 3-week intervals via subcutaneous injection. Titers of anti-M2e antibody were measured in sera collected on days 14, 35 and 56 (FIG. 3A).

[0229] A physical mixture of soluble M2e and BP26-WT did not induce any detectable antibody production regardless of the number of immunization, even with the use of an alum adjuvant.

[0230] In contrast, immunization with BP26-M2e nanoarchitectures induced the production of anti-M2e IgG antibodies; notably, antibody titers increased more than 100-fold after multiple boosting immunizations. In addition, the BP26-M2e (×8)-immunized group had higher anti-M2e IgG titers than the BP26-M2e (×4)-immunized group. While nanoarchitecture-based immunization per se induced a high level of antibody production, efficiency was further enhanced by additional use of alum adjuvant (FIGS. 3B and 3C).

[0231] In addition, an analysis of M2e-specific antibody responses according to IgG1 and IgG2a isotypes showed that BP26-M2e (×8) induced significantly greater IgG1 and IgG2a responses than did BP26-M2e (×4) (FIG. 3D).

3-3. Determination of BP26 Antibody Production by ELISA

[0232] To evaluate the immunogenicity of the BP26 carrier, 96-well plates were coated with BP26 protein and incubated overnight at 4° C. Plates were washed, blocked, and incubated with serum samples as described supra. Plates were then washed and incubated with HRP-conjugated goat anti-mouse IgG (1:5000 dilution) as secondary antibody for 1 hour at room temperature.

[0233] Since BP26 is the major immunodominant antigen of Brucella, antibodies specific for the BP26 carrier were generated in the immune group (FIG. 3E); However, this anti-BP26 antibody did neither interfere with the M2e-specific antibody response, nor induced complications such as autoimmune responses in immunized animals.

[0234] These results clearly indicate that genetic fusion of M2e antigen to BP26 and its self-assembled nanoarchitecture is critical for inducing robust antibody production (humoral responses) against the low-immunogenic M2e of influenza virus and rather, is most likely to protect against the zoonotic Brucella infection.

[0235] To verify this hypothesis we examined whether pre-existing anti-BP26 antibodies could compromise the humoral responses induced by BP26-M2e(×8) nanobarrel immunization. One group of mice was immunized with BP26-WT/Alum three weeks in advance to generate anti-BP26 antibodies, followed by BP26-M2e(×8) immunization, and the other group of mice was immunized with BP26-M2e(×8) only.

[0236] Two weeks after BP26-M2e(×8) immunization, the titer of anti-M2e antibodies in the sera of each group was measured by ELISA (FIG. 3F, A).

[0237] Although a considerable level of antibodies against the immunogenic BP26 carrier was generated by BP26-WT/Alum immunization, there was little difference in the titers of anti-M2e antibodies between the two groups (FIG. 3F, B), indicating that pre-existing anti-BP26 antibody is unlikely to interfere with the M2e-specific antibody response.

Example 4: Binding Capacity of Anti-M2e Antibody to Influenza Virus-Infected Cells

[0238] Immunocytochemistry (ICC), ELISA, and Western blot were performed to determine whether the anti-M2e antibody of the immunized mouse serum could recognize influenza-infected cells expressing M2e on the plasma membrane (FIG. 4a).

4-1. Confirmation of Anti-M2e Antibody Binding to Influenza Virus-Infected Cells Through Immunocytochemistry

[0239] In order to confirm the binding of the anti-M2e antibody to the influenza virus-infected cells, immunocytochemical analysis using confocal microscopy was performed. To evaluate the binding capacity of anti-M2e antibodies, MDCK cells were seeded and grown on coverslips IN 24-well plates, grown at a density of 2×105 cells per well in 0.5 mL medium and allowed to adhere overnight. MDCK cells were then infected with influenza A virus strains (A/PR/8, A/CA/04/09 or A/Aquatic bird/Korea) in influenza infection medium (DMEM medium supplemented with MEM-vitamin, gentamicin and 4% BSA) and cultured for 20 hours. Cells were washed with PBS and fixed with 10% formalin solution for 10 min. Thereafter, cells were incubated for 2 hours at room temperature with sera containing anti-M2e IgG antibody (1:100 dilution) obtained from immunized mice. Cells were washed with PBS and fixed with 10% formalin solution for 10 min. Cells were incubated with sera containing immunized mice anti-M2e IgG antibody (1:100 dilution) at room temperature for 2 hours. The serum containing the M2e IgG antibody was obtained on day 56 from M2e (×8) immunized mice. Incubation was made with anti-M2e IgG antibody for 2 hours and then with Alexa Fluor 594-conjugated donkey anti-mouse secondary antibody (1:200 dilution; Abcam) for 1 hour at room temperature. Nuclei were stained with Hoechst 33342 (1:5000 dilution). All samples were imaged using a confocal laser scanning microscope (LSM 780; Carl Zeiss).

[0240] Because M2e, unlike the large glycoproteins hemagglutinin (HA) and neuraminidase (NA), is a small, low-abundance protein buried in the membrane of virions, the anti-M2e antibody is known to exert its antiviral effect by inducing antibody-dependent cellular cytotoxicity (ADCC) upon binding to infected cell membranes, rather than by directly neutralizing the viral infection. That is, protection from viral infection is expected to be directly related to the ability of anti-M2e antibody to bind the plasma membrane of virus-infected cells.

[0241] The virus strain and M2e amino acid sequence used in the experiment are as follows (Table 2).

TABLE-US-00003 TABLE 2 Virus strain Subtype M2e amino acid sequence SEQ ID NO: A/PR/8 H1N1 SLLTEVETPIRNEWGCRCNDGSD 4 A/CA/04/09 H1N1 SLLTEVETPTRNGWECKCSDSSD 5 A/Aquatic bird/Korea H5N2 SLLTEVETPTRSEWECRCSDSSD 6

[0242] Confocal laser-scanning microscopic imaging revealed that the anti-M2e antibody in immunized mouse sera exhibited marked binding to influenza virus-infected MDCK cells, regardless of virus subtype, whereas little binding was observed for non-infected MDCK cells (FIG. 4B).

4-2. Confirmation of Anti-M2e Antibody Binding to Influenza Virus-Infected Cells Through Whole-Cell ELISA

[0243] Specific binding of the anti-M2e antibody was confirmed by whole-cell ELISA (FIG. 4c).

[0244] For whole-cell ELISA, MDCK cells were seeded at a density of 1×104 cells per well into 96-well plates containing 0.2 mL medium each well and allowed to adhere overnight. MDCK cells were infected with influenza virus strains (multiplicity of infection=0.1) and incubated with sera containing anti-M2e IgG antibody (1:100 dilution) from mice immunized. The sera containing the anti-M2e IgG antibody was obtained from M2e (×8) immunized mice on day 56. Cells were then incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (1:5000 dilution) for 1 hour at room temperature, and absorbance was read at 450 nm using a microplate reader.

[0245] The ELISA data revealed that the anti-M2e antibody in immunized mouse sera exhibited binding to influenza virus-infected MDCK cells, regardless of virus subtype, whereas little binding was observed for non-infected MDCK cells (FIG. 4C).

4-3. Confirmation of Anti-M2e Antibody Binding to Influenza Virus-Infected Cells Through Western Blotting

[0246] Specific binding of the anti-M2e antibody was confirmed by western blotting.

[0247] For Western blot analysis, MDCK cells were seeded at a density of 5×105 cells per well into 6-well plates containing 2 mL medium each well, incubated overnight, then infected with influenza virus strains (multiplicity of infection=1). Virus-infected cells were harvested and lysed using protein extraction solution (PRO-PREP™, iNtRON Biotechnology) according to the manufacturer's instructions, after which the protein concentration was determined by Bradford assay. Proteins in whole-cell extracts were separated by SDS-PAGE on 20% gels and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked by incubating with 5% skim milk in TBS-T for 2 hours at room temperature and then incubated for 2 hours at room temperature with sera containing anti-M2e IgG antibody (1:200 dilution), obtained from immunized mice. The membrane was incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (1:5000 dilution) for 1 hour at room temperature, after which immunoreactive proteins were imaged using a ChemiDoc XRS Imaging System (BIO-RAD, Hercules, Calif.).

[0248] It was revealed from the data of western blot analysis that the anti-M2e antibody in immunized mouse sera exhibited binding to influenza virus-infected MDCK cells, regardless of virus subtype, whereas little binding was observed for non-infected MDCK cells (FIG. 4D).

[0249] Collectively, these findings indicate that immunization of mice with BP26-M2e nanovaccine generates an anti-M2e antibody that can specifically bind to mammalian cells infected with various influenza viruses, thereby suggesting the possibility that BP26-M2e nanoarchitectures could be developed as a universal vaccine against influenza.

Example 5: Evaluation of Vaccine Effect Through Virus Inoculation Experiment

[0250] For the virus inoculation experiment, mice were immunized in the same manner as in Example 3-1 as follows:

[0251] Mice were divided into five groups: PBS buffer vehicle, (BP26-WT+M2e)/Alum (BP26-WT, 20 μg; M2e, 7.3 μg), BP26-M2e (×4) (25 μg), BP26-M2e (×8) (18 μg), and BP26-M2e (×8)/Alum (BP26-M2e: 18 μg). Mice in each group were immunized thrice at intervals of three weeks by subcutaneous injection into both footpads.

[0252] Mice were immunized thrice at 3-week intervals by intranasal administration of 30 μL of PBS containing a lethal dose (4×LD50) of A/PR/8 influenza virus two weeks after the last immunization (FIG. 5A). Mice were monitored every day for survival rates, body weight changes, clinical scores, and rectal temperatures for 16 days following the challenge infection. Mice that lost 25% or more of their initial body weight were euthanized humanely and included as experimental endpoints. Clinical scores were determined using the criteria described in FIG. 5B. Because influenza virus-infected mice unlike humans are reported to become hypothermic, rectal temperature was also measured.

[0253] Control influenza virus-infected mice treated with phosphate buffered saline (PBS) showed a loss in body weight by 30% or more, a sharp increase in clinical score, and drop in rectal temperature within 10 days after virus challenge.

[0254] Immunization with a physical mixture of BP26-WT and M2e together with alum adjuvant produced a trend similar to that observed in the PBS-treated group.

[0255] However, immunization with BP26-M2e (×4) increased survival rate to 60%, despite a significant loss in body weight; other symptoms gradually subsided and had abated by 8 days after the challenge infection.

[0256] Immunization with BP26-M2e (×8) further significantly increased the survival of mice to 80% while causing only mild symptoms (slightly ruffled fur) and a much lower clinical score compared with immunization with BP26-M2e (×4) which contained shorter antigen repeats.

[0257] Addition of alum adjuvant to BP26-M2e(×8) nanoarchitectures protected all mice from lethal influenza virus challenge (100% survival), together with slight body weight loss and faster recovery (FIGS. 5C to 5F).

Example 6: Measurement of Lung Viral Titer

[0258] To further confirm the protective immunity conferred by the BP26-M2e nanovaccine, lung viral titers were measured.

[0259] Mice were immunized in the same manner as in Example 3-1 as follows:

[0260] Mice were divided into five groups: PBS buffer vehicle, (BP26-WT+M2e)/Alum (BP26-WT, 20 μg; M2e, 7.3 μg), BP26-M2e (×4) (25 μg), BP26-M2e (×8) (18 μg), and BP26-M2e (×8)/Alum (BP26-M2e: 18 μg). Mice in each group were immunized thrice at intervals of three weeks by subcutaneous injection into both footpads.

[0261] Mice were immunized thrice at 3-week intervals by intranasal administration of 30 μL of PBS containing a lethal dose (4×LD50) of A/PR/8 influenza virus two weeks after the last immunization. Mice were euthanized three days after virus challenge and lung homogenate suspensions were obtained. Lung viral titers were determined in MDCK cells using a 50% tissue culture infectious dose (TCID50) assay.

[0262] Immunization with a physical mixture of BP26-WT+M2e and alum adjuvant failed to reduce lung viral titers, whereas immunization with either BP26-M2e (×8) alone or together with alum led to a significant reduction in lung viral titers (FIG. 5G).

[0263] Taken together, these results indicate that the BP26-M2e nanovaccine can generate strong cross-protective immunity against influenza virus infection, even without the use of a conventional adjuvant. Moreover, the protective efficacy can be tuned by controlling the length of the displayed antigen.

[0264] To examine the possibility of BP26-M2e nanobarrel as a universal vaccine against influenza virus infection, we evaluated cross-protection efficacy of the nanovaccine against another influenza A virus strain. Mice were immunized three times at 3-week intervals with PBS vehicle, (BP26-WT+M2e)/Alum, BP26-M2e(×8), or BP26-M2e(×8)/Alum. Two weeks after the final immunization, mice were challenged with a lethal dose (4×LD.sub.50) of the 2009 pandemic strain, A/California/04/2009 (H1N1pdm09) (FIGS. 5H, A).

[0265] Protective efficacy was evaluated by measuring survival rates, body weight, clinical score and rectal temperature for 14 days post viral infection. PBS- and (BP26-WT+M2e)/Alum-immunized mice showed drastic body weight loss, a sharp increase in clinical score, and decrease of rectal temperature, thus all mice died or were euthanized within 9 days after viral infection (FIGS. 5H, B-E).

[0266] In contrast, immunization with BP26-M2e(×8) and BP26-M2e(×8)/Alum showed significantly increased survival rate to ˜75% and 100%, respectively, and led to restoration of body weight, clinical score and rectal temperature of mice to the normal state. Histological analysis on lung tissues of the virus challenged mice revealed that both PBS- and (BP26-WT+M2e)/Alum-immunized groups developed severe pulmonary edema and peribronchiolar and perivascular inflammation, whereas such signs of inflammation were considerably reduced in the lungs of both BP26-M2e(×8) and BP26-M2e(×8)/Alum immunized mice (FIG. 5I).

[0267] From the result, the present inventors have developed a cross-protective universal vaccine platform against influenza A virus infection based on a protein nanoarchitecture formed by self-assembly of the Brucella outer membrane protein BP26. Genetic engineering of BP26 enabled generation of a barrel-shaped nanovaccine displaying the viral antigen M2e (BP26-M2e). Immunization of mice with BP26-M2e nanobarrel vaccines induced high-level production of anti-M2e antibodies that could specifically bind to influenza virus-infected cells and effectively protect mice from influenza infection even without the use of a conventional adjuvant.

[0268] The immune response to BP26-M2e nanobarrel vaccines can be tuned by controlling the length of tandem repeats of the M2e epitope. The BP26-based nanobarrel vaccines can be designed to display relatively large antigens as well as multiple epitopes to optimize or maximize humoral and cellular responses against various viruses. Furthermore, BP26-based vaccines can be produced in high yield through simple expression in E. coli, and thus have high commercialization potential for human or animal use.

[0269] The strong immune response induced by the present disclosure is due to the nanoarchitecture formed by BP26. Since there is no limitation on the polypeptide fused to BP26 by the linker, the nanoarchitecture of the present disclosure can induce immunity to various pathogens.

[0270] Therefore, it is expected that the unique features of the BP26-based nanobarrel system as a versatile vaccine platform may enable rapid development of antiviral vaccines against various bacteria and viruses, including SARS-CoV-2, influenza viruses, and the like.

Example 7: BP26-M2e Nanovaccines Induce Both Antibody-Dependent Cellular Cytotoxicity and T Cell Responses Against Influenza Virus-Infected Cells

[0271] The present inventors investigated mechanisms by which BP26-M2e nanovaccine exerts protection effects against influenza infection. We first examined whether anti-M2e antibodies in the sera of BP26-M2e(×8) immunized mice could be bound to influenza A virus directly and neutralize them. We found that the anti-M2e antibodies did not recognize the virus at all (FIG. 6, A and B). This finding suggests that anti-M2e antibodies are not virus-neutralizing antibodies, which is in good agreement with findings reported previously. Because anti-M2e antibodies can be bound to influenza virus-infected cells as shown in FIG. 4 and FIG. 6 (B), it is expected that the antibody-dependent cellular cytotoxicity (ADCC) may be involved in immune protection of the nanobarrel vaccine. Natural killer (NK) cells are known to play a crucial role in ADCC for influenza virus-infected cells and thus, we evaluated NK cell-mediated ADCC by flow cytometry. A/PR/8 influenza virus-infected MDCK cells were stained with carboxyfluorescein succinimidyl ester (CFSE) and incubated with the sera of either PBS-treated or BP26-M2e(×8) immunized mice. NK cells (effector) isolated from mouse splenocytes were added to CFSE-labeled, virus-infected MDCK cells (target) at an effector to target (E:T) ratio of 2:1 or 1:2. Anti-M2e antibodies-dependent NK cells-mediated cytotoxicity was assessed using a live and dead assay (FIG. 7). Sera of BP26-M2e(×8) immunized mice resulted in much greater ADCC than that of PBS-treated control mice at the two-tested E:T ratios (viability of MDCK cells in the absence of NK cells was ˜3.75%; data not shown) (FIG. 6B-C). These findings suggest that BP26-M2e nanovaccine induces generation of non-neutralizing M2e-specific antibodies that engage in NK cell-mediated ADCC capable of destroying M2e-exposed influenza virus-infected cells.

[0272] On the other hand, we further examined whether BP26-M2e nanovaccine can also induce T cell responses..sup.36-38 For evaluation of M2e antigen-specific T cell responses, mice were immunized three times at 3-week interval with PBS, a physical mixture of (BP26-WT+M2e)/Alum, BP26-M2e(×8), or BP26-M2e(×8)/Alum and sacrificed three weeks after the last immunization (Figure S8A). Splenocytes were isolated from the immunized mice and restimulated with M2e antigen peptide. Intracellular cytokine staining (ICS) was performed to measure IFN-γ producing CD8.sup.+ and CD4.sup.+ T cells using flow cytometry (Figure S8B). While both PBS control and the physical mixture of (BP26-WT+M2e)/Alum failed to induce M2e-specific T cell responses, BP26-M2e(×8) nanovaccine led to appreciable increase in the population of IFN-γ secreting CD8.sup.+ and CD4.sup.+ T cells; as expected, addition of alum adjuvant to BP26-M2e(×8) nanovaccine further enhanced the antigen-specific T cell responses (Figure S8C-D). Taken together, these results of mechanism studies suggest that BP26-M2e nanovaccine may exert its immune protection efficacy against influenza virus by engaging in anti-M2e antibodies-mediated ADCC as well as by inducing M2e-specific T cell responses.

Example 8: Preparation of BP26-Containing Cancer Vaccine

[0273] The present inventors tried to prepare a cancer vaccine composition using a nanoarchitecture containing BP26. Specifically, the present inventors prepared a protein complex having a structure as shown in FIG. 6.

[0274] As shown in FIG. 8, a fusion protein was designed that displays M30 (27 amino acids), an MHC class II neoantigen peptide of mouse melanoma cell line B16-F10, in 6 tandem repeats on the C-term side of the BP26 protein.

[0275] The amino acid sequence of M30 peptide and the nucleotide sequence encoding therefor are shown in Table 3.

TABLE-US-00004 TABLE 3 SEQ ID Category Sequence NO: Amino acid PSKPSFQEFVDWENVSPELNSTDQPFL  8 sequence of M30 Nucleotide CCGAGCAAACCGAGCTTCCAAGAGTTTGTGGACTGGG  9 sequence of M30 AAAACGTTAGCCCGGAGCTGAACAGCACCGATCAACC (1) GTTCCTG Nucleotide CCGAGCAAGCCGAGCTTCCAAGAATTTGTGGACTGGG 10 sequence of M30 AGAACGTTAGCCCGGAACTGAACAGCACCGACCAACC (2) GTTTCTG Nucleotide CCTTCTAAGCCGAGCTTCCAGGAGTTTGTGGACTGGG 11 sequence of M30 AGAATGTCTCTCCTGAGCTGAACAGCACTGACCAACC (3) GTTCCTG Nucleotide CCTTCTAAACCGAGCTTCCAGGAATTTGTGGACTGGGA 12 sequence of M30 AAATGTGTCTCCTGAACTGAACAGCACTGATCAACCGT (4) TTCTG Nucleotide CCTTCCAAACCGAGCTTCCAGGAGTTTGTGGACTGGG 13 sequence of M30 AAAACGTATCTCCCGAGCTGAACAGCACAGACCAACC (5) GTTCCTG Nucleotide CCTTCAAAGCCGAGCTTCCAAGAGTTTGTGGACTGGG 14 sequence of M30 AGAATGTGAGCCCGGAGCTGAATAGCACCGACCAACC (6) GTTCCTG

[0276] There was a connection via the flexible linker between BP26 and M30 and between M30 and M30. In particular, since the M30 peptide can act as an MHC class II epitope to induce M30 antigen-specific CD4+ T cell response, development trend has been shifted from conventional anticancer vaccines focusing on the induction of CD8+ T cell-based antitumor immune responses toward anticancer vaccines capable of inducing CD4+ T cell responses.

Example 9: Efficacy Evaluation of BP26-Bearing Cancer Vaccine 9-1. Inoculation of Cancer Vaccine Inoculation and Efficacy on Tumor Growth Inhibition

[0277] FIG. 9 is a diagram showing an immunization schedule for evaluating the efficacy of the BP26-bearing cancer vaccine of the present disclosure.

[0278] As shown in FIG. 9, the present inventors inoculated the cells through subcutaneous injection of B16-F10 cancer cells (day 0). Then, immunization was conducted by administering the cancer vaccine composition (BP26 and M30 tandem repeat fusion protein, BP26-M30) prepared in Example 8 of the present disclosure at a dose of 46.8 μg/head a total of 2 times (day 4 and day 8) at intervals of four days from day 4 after inoculation of tumor cells. Five days after the last immunization, the mice were sacrificed, the spleen was excised, and splenocytes were isolated. For other experimental groups, an aqueous mixture (BP26-WT+M30) of BP26-WT (25.3 μg/head) and M30 peptide (20 μg/head) and a mixture of the cancer vaccine of the present disclosure and a vaccine adjuvant, CpG ODN (BP26-M30, 46.8 μg/head+CpG, 10 μg/head) were administered, respectively. The CpG ODN is an oligonucleotide composed of the nucleotide sequence of SEQ ID NO: 15 (5′-TCC ATG ACG TTC CTG ACG TT-3′) and having a phosphorothioate backbone and was purchased from Genotech (Daejeon, Korea). As a control, PBS was administered (Control). In addition, the tumor volume was measured every 2 days after tumor cell inoculation until sacrifice.

[0279] The results are shown in FIG. 10.

[0280] FIG. 10 shows volumes of B16F10 tumor cells, accounting for degrees of growth of the tumor cells. As shown in FIG. 10, immunization of mice with the cancer vaccine composition (BP26-M30) of the present disclosure was observed to suppress tumor growth, compared to the other experimental group, i.e., the aqueous mixture of BP26-WT and M30 peptide (BP26-WT+M30). In addition, it was found that the cancer vaccine composition of the present disclosure suppressed tumor growth similarly to the mouse group (BP26-M30+CpG) to which the vaccine adjuvant CpG ODN was additionally administered.

9-2. Inoculation of Cancer Vaccine and Activation of Cellular Immune Response

[0281] The present inventors checked the percent of CD4+ T cells secreting IFN-γ to examine whether the M30 antigen-specific T cell immune response was induced, that is, to confirm the immunogenicity of the cancer vaccine.

[0282] Antigen-specific T cell responses were determined by ex vivo restimulation of splenocytes with M30 peptide (10 μg/ml), and INF-γ produced by CD4+ T cells was quantified as determined by intracellular cytokine staining (ICS).

[0283] In brief, GolgiStop™ or GolgiPlug™ (BD Biosciences) was added to each tube to inhibit intracellular transport of cytokines. The cells were then incubated for 5 hours. The cells were immunostained with Fixable Viability Dye eFluor450™ (eBioscience, San Diego, Calif., USA) for 20 min at 4° C. to distinguish dead cells and then with anti-CD3 PE/Cy7 and anti-CD4 FITC antibodies for 20 min at 4° C. For intracellular cytokine staining, cells were permeabilized using a Cytofix/Cytoperm™ solution (BD Biosciences) and incubated with PE-conjugated anti-IFN-γ antibody. The samples were then washed and analyzed using flow cytometry.

[0284] FIG. 11 shows a flow cytometry gating strategy for confirming proportions of IFN-γ-producing CD4+ T cells through flow cytometry for splenocytes. FSC×SSC gating was used to obtain single cells based on size and granularity, and dead cells were excluded to analyze only live cells. CD3, CD4 and CD8 were used as T cell markers. Finally, INF-γ secretion from CD3+CD4+ T cells was confirmed.

[0285] FIG. 12 is a plot of the % of CD4+ T cells secreting IFN-γ after immunization with the cancer vaccine containing BP26 of the present disclosure.

[0286] As shown in FIG. 12, the BP26-M30 cancer vaccine of the present disclosure was found to induce M30 antigen-specific CD4+ T cell response, compared to the control group and the BP26-WT+M30 aqueous mixture-administered group. In addition, when BP26-M30 and CpG ODN adjuvant were co-administered, the % of CD4+ T cells secreting IFN-γ was further improved, indicating that antigen-specific cellular immune response could be more effectively induced.

[0287] Taken together, the data obtained above exhibited that the BP26 platform of the present disclosure can effectively induce antigen-specific cellular immune responses when displaying cancer neoantigen peptide as well as M2e peptide of influenza virus, implying the possibility that the BP26 platform can be used not only as an influenza vaccine but also as a cancer vaccine.

Example 10. Optimization of Cancer Vaccine

[0288] The present inventors tried to optimize the design of the cancer vaccine prepared in Example 8.

[0289] The present inventors designed a fusion protein displaying 10 tandem repeats of MHC class II neoantigen peptide M30 (15 amino acids, VDWENVSPELNSTDQ) from mouse melanoma cell line B16-F10 at the C-term of BP26 protein (FIG. 13). Between BP26 and M30 and between M30 and M30 were linked through LQ, a cathepsin cleavage linker. M30 peptide can induce M30 antigen-specific CD4+ T cell response by acting as an MHC class II epitope. Conventional anticancer vaccines have focused on CD8+ T cell-based antitumor immune responses. Therefore, the present inventors tried to develop an anticancer vaccine capable of inducing CD4+ T cell response as well as CD8+ T cell-based antitumor immune response.

[0290] FIG. 14 is data on the purification and expression of the BP26-LQ-M30 (×10) fusion protein. As shown in FIG. 14, the BP26-LQ-M30 (×10) fusion protein was successfully expressed and purified. Purification was performed via FPLC, and SDS-PAGE showed that only the fusion protein was isolated.

[0291] FIG. 15 shows the Vaccine immunization schedule. As shown in FIG. 15, immunization was performed 3 times at intervals of 4 days from the 4th day after cell inoculation through subcutaneous injection of B16-F10 cancer cells.

[0292] FIG. 16 shows the B16F10 tumor growth curve after inoculation with the BP26-LQ-M30 (×10) fusion protein.

[0293] As shown in FIG. 16, when the fusion protein of BP26 and M30 tandem repeat (i.e., BP26-LQ-M30 (×10)) was immunized to mice, tumor growth was inhibited compared to a soluble mixture of BP26-WT and M30 peptides. In addition, tumor growth was further suppressed when the vaccine adjuvant CpG ODN group was added.

[0294] FIG. 17 shows the B16F10 tumor volume on the 18th day after tumor inoculation.

[0295] FIG. 18 shows the antigen-specific T cell immune response by inoculation with the BP26-LQ-M30 (×10) fusion protein.

[0296] Seven days after the final immunization on D12, the mice were sacrificed, and immune cells, splenocytes, were isolated from the spleen. After giving M30 peptide ex vivo restimulation to the isolated splenocytes, the CD4+ T cell population secreting interferon-gamma (IFN-γ) was analyzed by intracellular cytokine staining (ICS). As a positive control, PMA/lonomycin ex vivo stimulation was additionally performed.

[0297] When M30 stimulation was performed, it was confirmed that the M30-antigen-specific CD4 T cell immune response was induced only in the BP26-LQ-M30 (×10)+CpG group (data on the left). When PMA/lonomycin stimulation was given, no difference could be confirmed in all groups. from the above result

[0298] From the results, it was confirmed that an antigen-specific T cell immune response was induced by inoculation with the BP26-LQ-M30 (×10) fusion protein.

Example 11. Optimization of Cancer Vaccine Immunization

[0299] In Example 10, the antitumor efficacy of the BP26-LQ-M30 (×10) fusion protein was verified. In this example, tests for optimizing vaccine administration were performed. Specifically, an experiment was conducted to find an optimal adjuvant capable of maximizing the antitumor efficacy of the BP26-LQ-M30 (×10) fusion protein.

[0300] The antitumor efficacy was confirmed when CpG ODN, a conventional adjuvant, and poly(I:C), an adjuvant mainly used in neoantigen vaccines, were used together. Results are shown in FIG. 19.

[0301] As shown in FIG. 19, when the adjuvant was administered together with the BP26-LQ-M30 (×10) fusion protein, the antitumor efficacy was improved, and the efficacy was particularly excellent when used together with CpG ODN.

[0302] FIG. 20 shows the efficacy of administration of the BP26-LQ-M30 (×10) fusion protein compared to the positive control group.

[0303] As a positive control group, the tumor volumes of the groups (M30pep+CpG and M30pep+poly(I:C)) administered with 5 times the dose of antigen and immunostimulant were compared. In the case of the positive control group, the administered dose was 5 times, but it showed antitumor efficacy similar to that of the BP26-LQ-M30 (×10) fusion protein+immune enhancer administration group.

[0304] FIG. 21 shows the B16F10 individual tumor growth curve of each group shown in FIG. 20.

INDUSTRIAL APPLICABILITY

[0305] The present disclosure relates to a fusion protein comprising BP26 and an antigenic polypeptide.

[0306] This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted concurrently herewith as the sequence listing text file entitled “000338uscoa_SequenceListing.NRL”, file size 182 kilobytes (KB), created on 25 Apr. 2023. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).