RESPIRATORY SYNCYTIAL VIRUS RECOMBINANT F PROTEIN AND VACCINE COMPOSITION CONTAINING SAME

Abstract

The present invention provides a respiratory syncytial virus (RSV) recombinant fusion protein (F protein) in which a polymerization domain derived from a foreign protein is bound to the C terminal of a fusion protein (F protein) lacking a transmembrane domain of a wild-type respiratory syncytial virus (RSV) fusion protein (F protein). The recombinant fusion protein of the present invention is soluble and can retain an F protein trimer. Excellent immune-inducing effects can be expected from the recombinant fusion protein of the present invention, and vaccine composition containing same.

Claims

1. A recombinant fusion protein (F protein) of respiratory syncytial virus (RSV), wherein a polymerization domain derived from a heterologous protein is linked at the C-terminal of a fusion protein (F protein) with the transmembrane domain of the wild-type respiratory syncytial virus (RSV) fusion protein (F protein) deleted.

2. The recombinant fusion protein of respiratory syncytial virus according to claim 1, wherein the fusion protein (F protein) with the transmembrane domain of the wild-type respiratory syncytial virus (RSV) fusion protein (F protein) deleted has SEQ ID NO 2, and further has the FLGFLLGVG sequence at positions 137-145 modified of the amino acid sequence of SEQ ID NO 2.

3. The recombinant fusion protein of respiratory syncytial virus according to claim 2, wherein the FLGFLLGVG sequence at positions 137-145 is substituted with i) AAGAAAGAG; ii) QNGQNNGSG; iii) NSGNSSGGG; or iv) TLSKKRKRR.

4. The recombinant fusion protein of respiratory syncytial virus according to claim 1, wherein the fusion protein with the transmembrane domain of the wild-type respiratory syncytial virus (RSV) fusion protein (F protein) deleted is an amino acid sequence selected from SEQ ID NOS 7-22.

5. The recombinant fusion protein of respiratory syncytial virus according to claim 1, wherein the polymerization domain derived from a heterologous protein is a polypeptide, and the polypeptide is a bacteriophage T4 fibritin foldon domain polypeptide having SEQ ID NO 23.

6. The recombinant fusion protein of respiratory syncytial virus according to claim 5, wherein the recombinant fusion protein of respiratory syncytial virus further has a recombinant D1 domain polypeptide of the flagellin protein having SEQ ID NO 24 linked to the C-terminal of the bacteriophage T4 fibritin foldon domain polypeptide having SEQ ID NO 23.

7. The recombinant fusion protein of respiratory syncytial virus according to claim 1, wherein the recombinant fusion protein of respiratory syncytial virus has the N-terminal of the polymerization domain derived from a heterologous protein linked to the C-terminal of the fusion protein (F protein), and the linkage between the fusion protein and the polymerization domain is covalent bonding by a linker.

8. The recombinant fusion protein of respiratory syncytial virus according to claim 7, wherein the linker is a KLSG linker having an amino acid sequence of KLSG.

9. The recombinant fusion protein of respiratory syncytial virus according to claim 8, wherein the C-terminal of the KLSG linker is covalently bonded to the N-terminal of the bacteriophage T4 fibritin foldon domain polypeptide having SEQ ID NO 23.

10. The recombinant fusion protein of respiratory syncytial virus according to claim 9, wherein the recombinant fusion protein of respiratory syncytial virus has an amino acid sequence selected from SEQ ID NOS 28-48.

11. The recombinant fusion protein of respiratory syncytial virus according to claim 6, wherein the C-terminal of the bacteriophage T4 fibritin foldon domain polypeptide having SEQ ID NO 23 is linked to the recombinant D1 domain polypeptide of the flagellin protein having SEQ ID NO 24 by a GGGS linker having an amino acid sequence of GGGS.

12. The recombinant fusion protein of respiratory syncytial virus according to claim 7, wherein the recombinant fusion protein of respiratory syncytial virus has the N-terminal of the bacteriophage T4 fibritin foldon domain polypeptide having SEQ ID NO 23 linked to the C-terminal of the fusion protein (F protein) by a KLSG linker, and has the N-terminal of the recombinant D1 domain polypeptide of the flagellin protein having SEQ ID NO 24 linked to the C-terminal of the bacteriophage T4 fibritin foldon domain polypeptide having SEQ ID NO 23 by a GGGS linker.

13. The recombinant fusion protein of respiratory syncytial virus according to claim 12, wherein the recombinant fusion protein of respiratory syncytial virus has an amino acid sequence selected from SEQ ID NOS 49-69.

14. A recombinant fusion protein of respiratory syncytial virus, wherein the recombinant D1 domain polypeptide of the flagellin protein having SEQ ID NO 24 is linked at the C-terminal of the fusion protein (F protein) with the transmembrane domain of the wild-type respiratory syncytial virus (RSV) fusion protein (F protein) deleted.

15. The recombinant fusion protein of respiratory syncytial virus according to claim 14, wherein the fusion protein (F protein) with the transmembrane domain of the wild-type respiratory syncytial virus (RSV) fusion protein (F protein) deleted has an amino acid sequence selected from SEQ ID NOS 3-22.

16. The recombinant fusion protein of respiratory syncytial virus according to claim 14, wherein the N-terminal of the recombinant D1 domain polypeptide of the flagellin protein having SEQ ID NO 24 is linked to the C-terminal of the fusion protein (F protein), and the linkage between the fusion protein and the recombinant D1 domain polypeptide of the flagellin protein is covalent bonding by a KLGGGS linker.

17. The recombinant fusion protein of respiratory syncytial virus according to claim 16, wherein the recombinant fusion protein of respiratory syncytial virus has an amino acid sequence selected from SEQ IDS NO 70-90.

18. A nucleic acid encoding the recombinant fusion protein of respiratory syncytial virus according to claim 1.

19. A cell or a virus comprising the nucleic acid according to claim 18.

20. A vaccine composition capable of inducing immune response in a host, comprising the recombinant fusion protein of respiratory syncytial virus according to claim 1 and a pharmaceutically acceptable carrier, wherein the recombinant fusion protein of respiratory syncytial virus comprised in the vaccine composition has a post-fusion F protein linked to a polymerization domain or a recombinant D1 domain of the flagellin protein.

21. (canceled)

22. A medicine for treating or preventing respiratory syncytial viral infection, comprising the recombinant fusion protein according to claim 1 as an antigen.

Description

DESCRIPTION OF DRAWINGS

[0167] FIG. 1 schematically shows the concept of the present disclosure.

[0168] FIG. 2 A) schematically shows foldon linked to the C-terminal of SK-FP4 by a KLSG linker, and B) schematically shows SK_fla linked to the C-terminal of SK-FP4 by a KLGGGS linker.

[0169] FIG. 3 schematically shows foldon and SK_fla linked to the C-terminal of SK-FP4. The sequence of linkage is F4-KLSG-foldon-GGGS-flagellin.

[0170] FIG. 4 shows the expression of the recombinant F protein of the present disclosure.

[0171] FIG. 5 shows a result of purifying the recombinant F protein of the present disclosure.

[0172] FIG. 6 shows a result of analyzing FP4 and FP4-foldon and FP4-flagellin fusion proteins by TEM.

[0173] FIG. 7 shows a schedule of administration of the recombinant F protein of the present disclosure and blood sampling.

[0174] FIG. 8 shows a result of analyzing the soluble post-fusion F protein-specific IgG total antibody titer of mouse antiserum.

[0175] FIG. 9 shows a result of analyzing the RSV A2 neutralizing antibody titer of mouse antiserum.

MODE FOR DISCLOSURE

[0176] Hereinafter, the present disclosure will be described in detail through examples, etc. in order to help understanding. However, the examples according to the present disclosure may be changed into various other forms and it should not be interpreted that the scope of the present disclosure is limited by the examples. The examples of the present disclosure are provided such that the description of the present disclosure is more complete to those having ordinary knowledge in the art.

[0177] 1. Preparation of RSV Recombinant F Protein

[0178] Preparation of sF Protein

[0179] (1) In order to obtain an RSV recombinant F protein which is stable and soluble, wild-type RSV was prepared as follows. It is well known to those skilled in the art that the following mutations are applied to all RSV subtypes including wild-type RSV-A2, RSVB and RSV-A long. The wild-type RSV F protein of RSV-B GenBank Accession No. AEQ63641.1 was mutated.

[0180] (2) A transmembrane domain of the wild-type F protein was removed. The wild-type F protein with the transmembrane domain removed was named SK-Seq 1_dTM and was represented by SEQ ID NO 2. Then, the amino acid sequence of the fusion peptide, which corresponds to the positions 137-145 of SEQ ID NO 2, was changed. SK-FP4 of SEQ ID NO 5 (an example of sF protein) was obtained by substituting the amino acid sequence FLGFLLGVG at the positions with NSGNSSGGG.

[0181] Preparation of Polymerization Domain Derived from Heterologous Protein

[0182] A domain which is derived from a heterologous protein and can help maintenance of the trimer form of the F protein was introduced to the C-terminal of SK-PF4 of SEQ ID NO 1, which is one of sF proteins.

[0183] (1) Preparation of Foldon Domain

[0184] A foldon domain of the fibritin protein of bacteriophage T4 was prepared.

[0185] The domain was represented by SEQ ID NO 23.

[0186] (2) Preparation of Recombinant D1 Domain of Flagellin

[0187] The flagellin protein is a building block of the flagellar filament and is encoded by the fliC gene. The flagellin protein of Salmonella enterica was used. The flagellin protein which is encoded by the FliC gene possessed by the serotype group D (Dublin) of Salmonella enterica ssp. enterica was used for experiment.

[0188] The recombinant D1 domain of flagellin to be fused with FP4 was designed by linking the amino acid sequences 54-176 and 413-454 of the D1 domain of the FliC gene possessed by the serotype group D (Dublin) of Salmonella enterica ssp. enterica with a linker G.

[0189] The domain was named SK_fla and was represented by SEQ ID NO 24.

[0190] (3) Preparation of Linker

[0191] A linker that can be link the domain to the C-terminal of SK-FP4 was prepared.

[0192] Foldon was linked to the C-terminal of SK-FP4 with a KLSG linker (see FIG. 2A).

[0193] SK_fla was linked to the C-terminal of foldon with a KLGGGS linker (see FIG. 2B). Then, foldon and SK_fla were linked with a GGGS linker (see FIG. 3).

[0194] Designing of Recombinant Polymerization Domain-Bound Fusion Protein

[0195] A recombinant fusion protein of RSV (RFP-RSV) was designed as follows.

[0196] Foldon and/or SK_fla was linked to the sF protein by a linker. As a result, RFP-RSVs having SEQ ID NOS 28-90 were obtained.

[0197] Among them, FP4 having SEQ ID NO 5 was designed as follows.

[0198] (1) Designing of FP4-Foldon Fusion Protein

[0199] Foldon was linked to the C-terminal of FP4 by a KLSG linker (see SEQ ID NO 31).

[0200] (2) Designing of FP4-Flagellin Fusion Protein

[0201] SK_fla was linked to the C-terminal of FP4 by a KLGGGS linker (see SEQ ID NO 73).

[0202] (3) Designing of FP4-Foldon-Flagellin Fusion Protein

[0203] After fusing foldon to the C-terminal of FP4 with a KLSG linker, flagellin was fused with a GGGS linker (see SEQ ID NO 52).

[0204] 2. Production of Recombinant Baculovirus

[0205] (1) Preparation of Recombinant Baculovirus

[0206] For easy differentiation of the designed recombinant fusion protein, the DNA sequence of a gene encoding the recombinant fusion protein was optimized. A DNA sequence having SEQ ID NO 91, a DNA sequence having SEQ ID NO 92 and a DNA sequence having SEQ ID NO 93 were cloned respectively into the pFastBac™1 transfer vector. Then, recombinant baculovirus (rBV_FP4-foldon, rBV_FP4-flagellin, and rBV_FP4-foldon-flagellin) was prepared using the recombinant pFastBac™1 and the bac-to-bac® baculovirus expression system (Invitrogen).

[0207] Table 1 shows the plaque titer of the recombinant baculovirus stocks.

TABLE-US-00001 TABLE 1 Recombinant baculovirus P0 titration P1 titration P2 titration rBV_FP4-foldon 8.8 × 10.sup.5 pfu/ml 5.8 × 10.sup.7 pfu/ml 1.2 × 10.sup.7 pfu/ml rBV_FP4-flagellin 3.0 × 10.sup.6 pfu/ml 3.4 × 10.sup.7 pfu/ml 1.0 × 10.sup.7 pfu/ml rBV_FP4-foldon-flagellin 1.1 × 10.sup.6 pfu/ml 1.1 × 10.sup.8 pfu/ml 1.6 × 10.sup.8 pfu/ml

[0208] (2) Expression of FP4-Foldon, FP4-Flagellin and FP4-Foldon-Flagellin Fusion Proteins

[0209] Sf9 insect cells were cultured at 27° C. using the insect-XPRESS™ medium (Lonza).

[0210] When the cell concentration reached 1.5×10.sup.6 cells/mL, the cells were infected with the recombinant baculovirus expressing the FP4-foldon, FP4-flagellin or FP4-foldon-flagellin fusion protein at MOI of 0.5. On days 3 and 5 after the infection, the expression of the fusion protein in the medium and the cells was analyzed by western blot under reducing condition (detection was made using the mouse monoclonal antibody 2F7). Protein expression was confirmed in both the cells and the medium, and protein degradation was observed with time. The result is shown in FIG. 4.

[0211] 3. Purification of Protein

[0212] (1) Purification of FP4-Foldon Protein

[0213] Sf9 cells were cultured in a 1-L spinner flask. When the cell concentration reached 1.5×10.sup.6 cells/mL, the Sf9 cells were infected with FP4-foldon rBV P1 at MOI of 0.5, and the medium was recovered 3 days later through centrifugation 6000 g for 15 minutes. TMAE anion exchange chromatography was conducted through column equilibration with 50 mM Tris (pH 8). After loading the prepared culture medium into the column, the flow-through sample was recovered. The flow-through sample was buffer-exchanged and concentrated by UF/DF using a buffer (20 mM Tris, 150 mM NaCl, pH 7.4). A Lentil Lectins column was equilibrated by applying a binding buffer (20 mM Tris, 0.5 M NaCl, 1 mM CaCl.sub.2), 1 mM MnCl.sub.2, pH 7.4). After adding 1 mM MnCl.sub.2 and 1 mM CaCl.sub.2), the sample was loaded into the column.

[0214] The protein was eluted by flowing an elution buffer (50 mM sodium phosphate, 100 mM NaCl, 0.5 M methyl-D-mannopyranoside, pH 6.8). The recovered sample was buffer-exchanged and concentrated by UF/DF using PBS. 0.1% Tween-80 was added to the finally recovered protein sample.

[0215] (2) The FP4-flagellin and FP4-foldon-flagellin proteins were also purified by the same method. But, unlike in (1), TMAE anion exchange chromatography was conducted through column equilibration with 50 mM sodium phosphate (pH 6.7).

[0216] The result of protein purification is shown in FIG. 5.

[0217] 4. Analysis of Protein Structure by Electron Microscopy

[0218] The purified protein sample was mounted on a disc and negative staining was performed. The protein was observed with an electronic microscope.

[0219] The result is shown in FIG. 6. The soluble FP4 not fused with foldon looked smaller and thinner than the FP4-foldon fusion protein as a whole. The FP4-foldon had a lollipop-like shape.

[0220] 5. Test of Immunogenicity

[0221] (1) Inoculation of FP4-Foldon, FP4-Flagellin and FP4-Foldon-Flagellin Fusion Proteins to Mouse and Gathering of Serum

[0222] The purified FP4-foldon, FP4-flagellin or FP4-foldon-flagellin fusion protein and FP4 were formulated with aluminum hydroxide. The prepared formulation was administered 2 times to 6-week-old female Balb/c mouse via IM route at 30 μg/mouse with a 2-week interval. Details are given in Table 2.

TABLE-US-00002 TABLE 2 Antigen Aluminum Groups Description content hydroxide content Mouse n Group 1 FP4-foldon 30 μg 200 μg 5 Group 2 FP4-flagellin 30 μg 200 μg 5 Group 3 FP4-foldon-flagellin 30 μg 200 μg 5 Group 4 FP4 30 μg 200 μg 5 Group 5 PBS  0 μg 200 μg 5

[0223] (2) Analysis of F Protein-Specific Antibody Titer of Mouse Antiserum

[0224] The soluble F-specific IgG antibody titer of mouse antiserum was measured by indirect ELISA. After coating the soluble F protein on the bottom of a 96-well plate, with 200 ng per well, and binding 4-fold serially diluted antiserum, anti-mouse IgG-HRP antibody was bound thereto and OD value was measured after adding TMB substrate. After analyzing the reaction curve for each mouse with a 4-parameter logistic model, EC.sub.50 antibody titer and GMT antibody titer for 5 mice were calculated. The EC.sub.50 antibody titer and GMT for soluble post-fusion F protein (post-F)-specific IgG are shown in Table 3 and FIG. 8.

TABLE-US-00003 TABLE 3 Groups Antigens #1 #2 #3 #4 #5 GMT Group 1 FP4-foldon 3400 1345 2399 3099 3644 2622 Group 2 FP4-flagellin 2264 1752 2413 1922 773 1701 Group 3 FP4-foldon + flagellin 5812 6200 1780 4636 1826 3523 Group 4 FP4 3306 2946 1823 3398 985 2264 Group 5 PBS/alum 72 50 11 130 79 53

[0225] As can be seen from Table 3 and FIG. 8, Groups 1 and 3 showed increased total antibody titer as compared to FP4. Group 2 having only flagellin linked showed very low GMT as compared to Group 4 treated with FP4 alone. That is to say, it can be seen from the above result that total antibody titer was increased as the foldon trimerization domain was bound. Through this, it was confirmed that the recombinant fusion protein of the present disclosure can play an important role in all immune responses induced by antigens.

[0226] (3) Analysis of RSV Neutralizing Antibody Titer of Fusion Protein Mouse Antiserum

[0227] A monolayer of Vero cells was cultured on a 24-well plate and infected with RSV A2 virus that had been pre-incubated with 2-fold serially diluted antiserum. Three days later, the infected cells were fixed and immunostained with palivizumab, and then immunofocus was counted. The number of immunofocus of each group depending on serum dilution factor is shown in Table 4. The average immunofocus of Group 5, which was inoculated with PBS-alum, was 67.6. The serum dilution factor (ND.sub.50) at which the immunofocus was half, i.e., 33.8, was defined as neutralizing antibody titer. The focus forming unit and ND.sub.50 depending on serum dilution factor are shown in Table 4 and FIG. 9.

TABLE-US-00004 TABLE 4 Serum dilution factor Groups Antigens 10 20 40 80 160 320 ND.sub.50 Group 1 FP4-foldon 28 37 45 46.5 64.5 68 18 Group 2 FP4-flagellin 20 35.5 45 57 50 60 23 Group 3 FP4-foldon + flagellin 11.5 23 32 46.5 63.5 56.5 47 Group 4 FP4 27.5 35 50.5 55.5 67.5 56.5 18.5 Group 5 PBS/alum 60.5 68 59.5 65 78.5 74 ND

[0228] As can be seen from Group 3, the recombinant F protein wherein both foldon and flagellin proteins were linked resulted in remarkably increased neutralizing antibody production. The neutralizing antibody titer ratio of FP4-foldon-flagellin and FP4-foldon was 2.6, which means that the RSV neutralizing antibody titer was increased by 2.6 times through fusion with flagellin.

[0229] The neutralizing antibody titer ratio of FP4-foldon-flagellin and FP4-flagellin was 2.04, which means that the RSV neutralizing antibody titer was increased by 2.04 times through fusion with foldon.

INDUSTRIAL APPLICABILITY

[0230] The present disclosure may provide a vaccine against respiratory syncytial virus. The present disclosure may also provide a composition for preventing or treating respiratory syncytial virus infection.