WO2011079817A1

WO2011079817A1 - Dendrimer of tuftsin and influenza matrix protein，and use thereof

Info

Publication number: WO2011079817A1
Application number: PCT/CN2010/080595
Authority: WO
Inventors: 张智清; 刘晓宇; 郭建强; 徐一; 韩苏; 姚立红; 陈爱珺
Original assignee: 中国疾病预防控制中心病毒病预防控制所; 元森泰生物科技（天津）有限公司
Priority date: 2009-12-31
Filing date: 2010-12-31
Publication date: 2011-07-07
Also published as: CN102153654B; CN102153654A

Abstract

The present invention provides a dendrimer or derivative thereof，which comprises extracellular domain of influenza matrix protein 2, linker and TUFTSIN. The formula of the dendrimer is (AA_N)₈-(Lys)₄-(Lys)₂-Lys-Thr-Lys-Pro-Arg or (AA_N)₄-(Lys)₂-Lys-Thr-Lys-Pro-Arg, in which preferably the amino acid sequence of AA_N is Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-Ile-Arg-Asn-Glu-Trp-Gly-Cys-Arg-Cys-Asn-Asp-Ser-Ser-Asp. The dendrimer can be used to detect polyclonal antibodies of influenza matrix protein 2, and to stimulate macrophages so as to activate antigen presenting cells. The present invention further provides a vaccine comprising the dendrimer or derivative thereof for inhibiting influenza virus.

Description

Dendrimer of TUFTSIN and influenza matrix protein and application thereof The present application claims to be filed on December 31, 2009 in China, the application number is 200910312897. 0, the name is the immunologically active peptide-based branched polypeptide and its derivatives and applications Priority of the invention patent application. Technical field

The invention relates to a branched polypeptide with an immunologically active peptide as a carrier and a derivative thereof and application thereof, in particular to a branched polypeptide having immunogenicity of influenza virus. Background technique

The high variability of RNA viruses is a great challenge for the production of vaccines against such viral infections, including influenza viruses, HIV and HCV. The influenza pandemic caused by different types of influenza viruses threatens the world, and the protective effect of existing influenza vaccines is limited by the degree of matching between vaccine strains and actual strains. Therefore, in order to develop a universal vaccine that is effective against various subtypes or mutant strains of influenza A virus, people have turned their attention to the more conserved nuclear proteins (NP) and matrix proteins 2 (M2) of influenza viruses (GS J imenez, R Planchon). , Q Wei, Hum Vacc in, 3 (5) : 157-64 , 2007 ; M Schot saer t, M De Fi let te, W Fier s, Exper t Rev Vacc ines, 8 (4) : 499-508, 2009 ).

The protective effect of the antiviral vaccine is related to the type of vaccine, in addition to the viral protein targeted. Synthetic peptide vaccines, especially epitope peptide vaccines prepared by screening antigenic epitopes of viruses and using synthetic methods have many advantages: avoiding epitopes that are detrimental to immune responses and selecting epitopes that are beneficial for immune responses, while maintaining antigens The immune response can be induced on the basis of specificity; it can be prepared by chemical synthesis, the development time is short, the process cartridge is simple, and the preparation process is safe. After obtaining the viral antigen sequence information, the preparation process can be completed quickly, so it is suitable for coping with new infections and sudden infections. disease. However, the anti-pro-epitope is mostly a short peptide with a small molecular weight, weak antigenicity and immunogenicity, and must be processed and modified to function as a vaccine.

Summary of the invention

The invention provides a branched polypeptide and a derivative thereof and an application thereof, and one of the objects is to provide a A highly immunogenic branched polypeptide.

The structural formula of the branched polypeptide provided by the invention from the amino terminus to the carboxy terminus is

(AA _N ) ₄ - ( Lys ) , -Lys-Thr-Lys-Pro-Arg, the carboxy terminal sequence Thr-Lys-Pro_Arg of the above branched polypeptide is the immunologically active peptide Tuf ts in. The human "is the extracellular domain of matrix protein 2 of influenza A virus. The above branched polypeptide is named as: branched (M2e) 4-Tuf ts in.

Wherein, the sequence of the extracellular region of the matrix protein 2 of the influenza A virus from the end to the carboxy terminus is:

Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-I le-Arg-Asn-G lu-Trp-Gly- Cys- Arg- Cys_Asn_Asp- Ser_Ser-Asp.

Hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation may be carried out on the amino acid side chain group of the branched polypeptide, at the amino terminus or the carboxy terminus, Branched polypeptide derivatives.

The branched polypeptide can be reacted with an acid or a base to form a pharmaceutically acceptable salt compound. An equivalent change can be made to the branched polypeptide of the present invention to obtain the following branched polypeptide:

( Ser-Leu-Leu-Thr-G lu-Val-Glu-Thr-Pro-I le-Arg-Asn-Glu-Trp-Gl y-Cys-Arg-Cys-Asn-Asp-Ser-Ser-Asp ) ₈ - ( Lys ) ₄ _ ( Lys ) ₂ _ Lys -Thr-Lys-Pro-Arg;

( Ser-Leu-Leu-Thr-G lu-Val-Glu-Thr-Pro-I le-Arg-Asn-Glu-Trp-Gl y-Cys-Arg-Cys-Asn-Asp-Ser-Ser-Asp ) ₂ - Lys_Thr- Lys- Pro- Arg.

Another object of the invention is to provide the use of said branched polypeptides and derivatives thereof.

The branched polypeptides and derivatives thereof can be prepared as influenza vaccines.

In the influenza vaccine, the active substance is the branched polypeptide or the derivative of the branched polypeptide.

The influenza vaccine also contains an adjuvant which enhances the antigen-specific immune response to the vaccine component or alters the immune response. A variety of adjuvants can be used, such as AL (0H) ₃ adjuvant.

The above influenza vaccine is a sputum influenza virus vaccine.

The sputum influenza virus includes H1N1 influenza virus, H2N2 influenza virus, H3N2 influenza virus, H5N1 influenza virus, H7N7 influenza virus, and H9N2 influenza virus.

The branched polypeptide of the present invention can specifically bind to the M2 polyclonal antibody, and can specifically bind to macrophages, thereby improving the working efficiency of antigen presenting cells. The vaccine prepared by the branched polypeptide of the present invention induces an animal's immune response with high efficiency and protects the animal from viral infection.

Specific embodiments of the present invention are given in detail by the following examples and the accompanying drawings. DRAWINGS

Figure 1 is a schematic diagram of the branched (M2e) 4- Tuft sin structure.

Figure 2 shows the results of the branched (M2e) 4-Tuftsin HPLC analysis.

Figure 3 shows the results of the branched (M2e) 4-Tuftsin mass spectrometry.

Figure 4 shows the results of the branched (M2e) 4-Tuft sin SDS-PAGE analysis, where 1 represents branching (M2e) 4-Tuftsin; 2 represents branched (M2e) 4-G4.

The figure shows the results of 4-Tuftsin Western-Blot analysis of branched (M2e). In the figure, 1 is branched (M2e) 4- Tuftsin; 2 is branched (M2e) 4- G4.

Figure 6 shows the titer of M2e antibody in serum of immunized mice by ELISA.

Figure 7 shows the results of ELISpot detection of spleen cells from immunized mice.

Figure 8 shows the survival rate of mice in different immunized groups after influenza virus challenge.

Figure 9 is a schematic diagram of the branched (M2e) 8- Tuftsin structure.

Figure 10 shows the results of ELISA for detection of branched (M2e) 8- Tuftsin antigenicity.

Figure 11 shows the binding of FITC-labeled branched (M2e) 8-Tuftsin to macrophages (400

X).

Figure 12 is a graph showing the titer of serum M2e antibody in mice by Example 2 ELISA.

Figure 13 shows the results of the ELISpot experiment of Example 2.

Figure 14 is a graph showing the results of detecting the lung viral load of the mouse of Example 1. The best way to achieve this embodiment 1

1) Synthesis of branched peptides

The four-branched polypeptide (M2e) 4- Tuftsin shown in Figure 1 was synthesized by solid phase synthesis using a peptide synthesizer. The structural formula of the branched peptide from N-terminus to C-terminus was (Ser- Leu-Leu- Thr-Glu_Va 1- Glu-Thr-Pro-Ile-Arg-Asn-Glu-Trp-Gly-Cys-Arg-Cys-Asn-As-Ser-Se r-Asp ) 4 - ( Lys ) ₂ - Lys- Thr- Lys-Pro_Arg.

High performance liquid chromatography and mass spectrometry analysis of the above branched polypeptides, and SDS-polypropylene Amide gel electrophoresis and Western-blot analysis. The knots shown in Figures 2 to 5 show that the peptide synthesized by the peptide synthesizer is a branched polypeptide designed by us, which is named as branched (M2e) 4- Tuftsin, and its molecular weight is 11312 Da.

2) Antigenic identification of branched polypeptides

The ability of the branched (M2e)4-Tuftsin and M2 polyclonal antibodies to specifically recognize and bind was determined by ELISA. The specific methods are as follows:

The concentrations of 0.0625 μ _§ , 0.125 μ 0.25 μ 0.5 μ g and 1 μg of branched (M2e) 4-Tuf ts in were coated into the wells of the ELISA plate, and the recombinant avian influenza virus Μ2 protein expressed in Escherichia coli was used as the Positive control. The mouse anti-Μ2 polyclonal antibody was used as a primary antibody, and the secondary antibody was an alkaline phosphatase-labeled goat anti-mouse immunoglobulin IgG antibody.

The ELISA results showed that the branched (M2e)4-Tuftsin specifically recognizes and binds to the anti-M2 polyclonal antibody when the branched (M2e)4-Tuftsin coating is 0.0625 μg/well to 0.25 μ^/well. a range, proportional to the absorbance value; and branched (M2e) 4-Tuftsin volume at 0.25 μ§ / hole to 1μ _§ / hole, absorbance values reached a plateau. This indicates that the branched (M2e)4-Tuftsin has the sputum influenza virus M2 antigen activity.

3) Binding polypeptide and macrophage binding assay

The control branch peptide (M2e) 4-G4 was synthesized by solid phase synthesis using a peptide synthesizer, and Tufts in was replaced with four glycines.

The structural formula of the branched peptide (M2e) 4-G4 from the N-terminus to the C-terminus is ( Ser- Leu_Leu_Thr-Glu-Val-Glu-Thr-Pro-I le-Arg-Asn-Glu-Trp-Gly-Cys-Arg-Cys- Asn-Asp-Ser-Ser -Asp) ₄ - (Lys) -Lys-Gly-Gly-Gly-Gly ₀

The ability of fluorescein FITC-labeled branched (M2e) 4- Tuftsin to bind to macrophages was determined. Fluorescein FITC-labeled branched peptide (M2e)4-G4 was used as a control. The specific method was as follows: BALB/c mouse peritoneal cavity was taken. Macrophages, cultured in 96-well plate 1640 medium at 37 ° C 5% C0 _{2 for} 7 h, 2 X 107 ml peritoneal macrophages per well, discarded the upper suspension cells, and then placed the adherent macrophages 4 Incubate at °C for 40 minutes.

The above 96-well plates were divided into two groups of 48 wells each.

One group was added with FITC-labeled branched (M2e) 4-Tuftsin, and the final concentration of branched (M2e) 4-Tuftsin was 0.5 mol/L; the other group was added with FITC-labeled branched peptide (M2e)4 per well. - G4, branched (M2e) 4- G4 with a final concentration of 0.5 μmol/L, mixed, cultured at 4 °C 20 minutes.

Then, the cells were washed twice with 1640 medium, and the washed and washed cells were observed under an Olympus 1X-51FL fluorescence microscope.

Under the fluorescence microscopy, the FITC-labeled branched (M2e) 4-Tuf tsin and mouse peritoneal macrophages were incubated with UV light, and green fluorescence was observed on the cell surface, indicating FITC labeling. Branched (M2e) 4-Tuftsin binds to Tuftsin receptor on macrophage surface; FITC-labeled branched peptide (M2e)4-G4 is imaged with mouse peritoneal macrophages in UV light, cell surface is not See green fluorescence, indicating that the control branch peptide (M2e) 4-G4 does not bind to macrophages.

The above experiments demonstrate that the branched (M2e)4-Tuftsin binds to macrophages and confirms the activity of Tuftsin.

4) Immunization of mice with branched peptides

Six-week-old female BALB/c mice were randomly divided into four groups: PBS group, M2e monomer group, (M2e)4-11^3111 group and 2. 4^4 groups, 16 BALB/c mice in each group.

PBS group was injected with PBS and the inside muscle of each Al 10 μ 1 in the two hind legs of mice (0H) ₃ mixture of adjuvant, PBS with Al (0H) ₃ adjuvant and PBS A1 (0¾ ₃ adjuvant The volume ratio is 1:1.

The M2e monomer group was prepared by injecting 10 μl of M2e monomer and Al(0H) ₃ adjuvant into the inner muscles of the hind legs of the mice, and the concentration of the M2e monomer in the influenza vaccine was 5 ug/10 ul. The total amount of M2e monomer injected per mouse was 10 μg/head.

(M2e) The 4_Tuftsin group was prepared by injecting 10 μl of branched (M2e)4-Tuftsin and Al(OH) ₃ adjuvant into the inner muscles of the hind legs of the mice. The influenza vaccine was branched (M2e). 4- Tufts in concentration of 5ug/10uL per mouse (M2e) 4- Tufts in total injection volume is 10μ _§ / only.

(M2e) The 4-G4 group was injected with 10 μM of branched peptide (M2e) in the inner muscles of the hind legs of the mice. 4-G4 and Al (OH) ₃ adjuvant, branched peptide (M2e) 4-G4 and Al (OH) ₃ adjuvant in a mixture of branched peptide (the M2e) 4_G4 concentration of 5ug / 10ul _o branched peptide (the M2e) of each mouse was injected 4-G4 total 10μ _§ / only.

Each group of mice was immunized twice, with each immunization interval of two weeks.

A) Blood was collected from the posterior orbital vein of the mouse on the 14th day after each immunization, and the blood was collected by centrifugation. Clear, - 20 ° C preservation, for serum anti-M2e antibody titer detection.

Serum anti-M2e antibody titers were measured by ELISA on the 14th day after immunization of mice in different immunization groups.

The ELISA results are shown in Figure 6, on the 14th day after the first immunization (indicated by the first immunization in Figure 6) M2e monomer group, branched (M2e) 4-Tuftsin group and branched peptide (M2e) 4- The serum antibody titers in the G4 group were 3, 860, and 180, respectively; on the 14th day after the second immunization (indicated by the second immunization in Figure 6) M2e monomer group, branched (M2e) 4-Tuftsin group The antibody titers in the serum of the 4-G4 group and the branched peptide [M2e] were 4, 54800 and 11940, respectively; the antibody titers after the two immunizations in the PBS group were zero.

The above results indicated that the M2e monomer group, the branched (M2e) 4-Tuftsin group and the branched peptide (M2e) 4-G4 group can induce anti-M2e antibody production in mice, branched (M2e) 4-Tuftsin group and branch. There was a significant difference in the antibody titer between the peptide (M2e)4-G4 group and the M2e monomer group (P < 0.05). In the branched (M2e) 4-Tuftsin group, the level of anti-M2e antibody induced by the branched (M2e)4-G4 group was higher, and there was also a significant difference in antibody titer between them (P < 0.05).

B) Twelve days after the second immunization, 4 mice in each group were sacrificed to obtain spleens, and spleen lymphocytes were isolated for ELISP0T test.

The ELISP0T method was used to detect the number of cells secreting IFN-γ after stimulation of splenic lymphocytes with M2e polypeptide, which indirectly reflected the specific response of mouse Thl cells after immunization.

ELISP0T uses the BD ELISP0T kit.

The results of ELISP0T test are shown in Fig. 7. In the PBS group, the average spleen lymphocytes produced 6 spots per 1 χ 10 ^δ cells, and the M2e monomer group spleen lymphocytes produced 46 spots per 1 χ 10 ^δ cells. , (M2e) 4- Tuftsin group spleen lymphocytes produced an average of 254 spots per 1 χ 10 ⁶ cells, (M2e) 4-G4 group spleen lymphocytes produced an average of 156 spots per 1 χ 10 ⁶ cells .

The above results indicated that both branched (M2e)4_Tuftsin, (M2e) 4-G4 and monomeric M2e could induce specific cellular immunity in mice, and the number of spots produced by the branched M2e group was significantly higher than that of the monomeric M2e group. The difference in sex (P<0.05), while the number of spots induced by the control branch peptide (M2e)4-G4 was significantly lower than that of the branched peptide-containing group containing Tnf tsin.

C) 14 days after the second immunization, each group of remaining mice was challenged with the influenza virus PR8 strain, and the influenza virus attack method was as follows: The mice were anesthetized by the CO ₂ inhalation method, and then 10 drops were directly dropped into the nostrils on both sides thereof. LD ₅ . The influenza virus PR8 strain of virus fluid makes it fully inhaled. On the 5th day after the challenge, 2 mice in each group were sacrificed to take the lungs, weighed, and the lung tissue suspension was prepared. After serial dilution, the MDCK cells were inoculated and the influenza virus titer (TCID ₅ ) was determined. Pulmonary viral load assays showed lg TCID _{5 in the} PBS group. For the 7.21, M2e monomer group lg TCID ₅ . For the 6.32, (26)4-but 11 3111 group (10 ₅ . 5.63, (M2e) 4 - G4 group lg TCID ₅ . 5.9 , this result indicates that the virus is replicated in three M2e immunized mice Efficient inhibition, while the tumor-immunized group of Tuft sin-containing peptides had the lowest lung viral load and was significantly different from the PBS group (Ρ<0.05).

After the challenge, the survival of each group of mice was observed daily, and the survival rate was measured 14 days after the challenge. The mice that died within 24 hours of the challenge were considered as accidental death.

The results of mouse survival were shown in Figure 8. Nine mice died after challenge with PBS, and 7 mice died from M2e monomer. The mice inoculated with branched [M2e]4-Tuftsin only 2 deaths, vaccination (M2e) 4-G4 mice except one accidental death, and 5 deaths, indicating that the two branched peptide immunizations have protective effects on mice, while Tuftsin-containing branched peptide immunization group The protection is best. Example 1

1) Synthesis of branched peptides

The octahedral polypeptide (M2e) 8- Tuftsin shown in Figure 9 was synthesized by solid phase synthesis using a peptide synthesizer. The structural formula of the branched peptide from N to C was (Ser- Leu-Leu- Thr-Glu_Va 1- Glu-Thr-Pro-Ile-Arg-Asn-Glu-Trp-Gly-Cys-Arg-Cys-Asn-As-Ser-Se r-Asp ) s - ( Lys ) - ( Lys ) ₂ - ys_Thr- ys- Pro- Arg.

The branched polypeptides synthesized above were subjected to high performance liquid chromatography and mass spectrometry.

The analysis showed that the peptide synthesized by the peptide synthesizer was a branched polypeptide designed by us, which was named as branched (M2e) 8- Tuftsin with a molecular weight of 22251 Da.

2) Antigenic identification of branched polypeptides

The ability of the branched (M2e) 8-Tuftsin and M2 polyclonal antibodies to specifically recognize and bind was determined by ELISA. The specific methods are as follows:

Branched (M2e) 8-Tuf ts in concentrations of 0.0625 μ _§ , 0.125 μg, 0.25 μg, 0.5 μg, and 1 μg were individually coated into wells of an ELISA plate, and recombinant avian influenza expressed in E. coli The virus Μ2 protein was used as a positive control. Mouse anti-Μ2 polyclonal antibody was used as primary antibody and secondary antibody was alkaline Phosphatase labeled goat anti-mouse immunoglobulin IgG antibody.

The ELISA results are shown in Figure 10. The branched (M2e) 8-Tuf tsin specifically recognizes and binds to the anti-M2 polyclonal antibody when the branched (M2e) 8-Tuftsin coating is 0.0625 μg/well to 0.25. In the g/pore range, the absorbance value is proportional to it, and the branch (M2e) 8- Tuftsin amount is between 0.25 μg/well and 1 μg/well, and the absorbance value reaches the plateau. Description Branched (M2e) 8-Tuftsin has influenza A virus M2 antigen activity.

3) Binding polypeptide and macrophage binding assay

The control branch peptide (M2e) 8-G4 was synthesized by solid phase synthesis using a peptide synthesizer, and Tufts in was replaced with four glycines.

The structural formula of the branched peptide (M2e) 8-G4 from the N-terminus to the C-terminus is ( Ser- Leu-Leu-Thr-Glu-Val - Glu - Thr - Pro - I le-Arg-Asn-Glu-Trp-Gly-Cys- Arg-Cys-Asn-Asp-Ser-Ser -Asp ) ₈ - ( Lys ) 4- ( Lys ) Plant! ^ys - Gly - Gly - Gly - Gly.

The ability of fluorescein FITC-labeled branched (M2e) 8-Tuftsin macrophage to bind was determined using fluorescein FITC-labeled branched peptide (M2e) 8-G4 as a control. The specific method is as follows:

BALB/c mouse peritoneal macrophages were cultured in 96-well plate 1640 medium at 37 ° C 5% C0 _{2 for} 7 h, 2 X 107 ml peritoneal macrophages per well, and the upper suspension cells were discarded. Wall macrophages were cultured at 4 ° C for 40 minutes.

The above 96-well plates were divided into two groups of 48 wells each.

One group was added with FITC-labeled branched (M2e) 8- Tuftsin, and the final concentration of branched (M2e) 8-Tuftsin was 0.5 m·l/L; the other group was added with FITC-labeled branched peptide (M2e) per well. ) 8-G4, branched (M2e) 8- G4 with a final concentration of 0.5 μmol/L, mixed and incubated at 4 ° C for 20 minutes.

The cells were then washed twice with 1640 medium, and the washed cells were observed under an Olympus 1X-51FL fluorescence microscope.

The results under fluorescence microscopy are shown in Figure 11. Figure 11A is an image of the FITC-labeled branched (M2e) 8-Tuftsin and mouse peritoneal macrophages after incubation; Figure 11B is the FI TC-labeled branch ( M2 e) Image of 8 -Tuf tsin and mouse peritoneal macrophages after incubation with ultraviolet light, green fluorescence was observed on the cell surface, and the results of Fig. 11A and Fig. 11B indicate FITC-labeled branch (M2e) 8 - Tuftsin binds to Tuftsin receptor on macrophage surface; Figure 11C shows FITC-labeled branched peptide (M2e) 8-G4 incubated with mouse peritoneal macrophages Image under visible light; Figure 11D is an image of FITC-labeled branched peptide (M2e) 8-G4 co-incubated with mouse peritoneal macrophages under ultraviolet light, no green fluorescence on the cell surface, and scar on Figure 11C and Figure 11D This indicates that the branched peptide (M2e) 8-G4 cannot bind to macrophages.

The above experiments demonstrate that the branched (M2e)8_Tuftsin binds to macrophages and confirms the activity of Tuftsin.

4) Immunization of mice with branched peptides

Six-week-old female BALB/c mice were randomly divided into three groups: PBS group, (M2e) 8-Tuftsin group, and (M2e) 8-G4 group, 20 BALB/c mice in each group.

The PBS group was injected subcutaneously with 30 μl of PBS and incomplete Freund's adjuvant on the inner side of the hind leg of the mouse, and the volume of PBS and incomplete Freund's adjuvant in a mixture of PBS and incomplete Freund's adjuvant. The ratio is 1:1.

(M2e) 8-Tuftsin group is a flu vaccine prepared by injecting 30 μl of branched (M2e) 8-Tuftsin and incomplete Freund's adjuvant into the skin of the two hind legs of the mice. M2e) 8- Tuftsin concentration was 5ug/30uL. The total amount of branching (M2e) 8- Tuftsin injected per mouse was 10Og/mo.

(M2e) The 8-G4 group was injected subcutaneously with 30 μl of branched peptide (M2e) 8-G4 and incomplete Freund's adjuvant in the inner side of the hind leg of the mouse, and the branched peptide (M2e) 8-G4 and The concentration of the branched peptide (M2e) 8_G4 in the mixture of incomplete Freund's adjuvant was 5 ug / 30 ul. The total amount of branched peptide (M2e) 8-G4 injected per mouse was 10 _μg /head.

Each group of mice was immunized three times, with each immunization interval of three weeks.

A) Blood was collected from the posterior orbital vein of the mouse on the 14th day after each immunization, and the blood was collected by centrifugation and stored at - 20 ° C for the detection of anti-M2e antibody titer in serum.

The ELISA results are shown in Figure 12. In the serum of the branched (M2e) 8_Tuftsin group and the branched peptide (M2e) 8-G4 group on the 14th day after the first immunization (indicated by the first immunization in Figure 12) The valences were 1056 and 39 respectively; on the 14th day after the second immunization (indicated by the second immunization in Figure 12), the antibodies in the serum of the branched (M2e) 8-Tuftsin group and the branched peptide (M2e) 8-G4 group The titers were 9370 and 252 respectively; the 14th day after the third immunization (represented after the third immunization in Figure 12) in the serum of the branched (M2e) 8-Tuftsin group and the branched peptide (M2e) 8-G4 group Antibody titers were 14703 and 696; antibody titers after three immunizations of PBS were zero.

The above results indicate that both branched (M2e) S-Tuftsin and branched peptide (M2e) 8-G4 can induce anti-M2e antibody production in mice, branched [M2e] 8-Tuftsin group and branched (M2e) 8-G4. There was a significant difference in antibody titers between the groups and the PBS group (P < 0.05). In the branched (M2e) 8-Tuftsin group, the level of anti-M2e antibody induced by the branched (M2e) 8-G4 group was higher, and there was also a significant difference in antibody titer between them (P < 0.05).

B) 12 days after the third immunization, 5 mice in each group were sacrificed to obtain spleens, and spleen lymphocytes were isolated for ELISP0T test.

The number of cells that specifically stimulate M2e-stimulated IFN-γ by ELISP0T method indirectly reflects the specific response function of 'Th” and murine Th 1 cells.

The ELISP0T method uses the Quick Spot Human IFN-γ ELISP0T pre-coated kit.

The ELISP0T method detects scars as shown in Fig. 13. In the PBS group, the spleen lymphocytes produced an average of 12 spots per 5 10 ⁵ cells, and the (M2e) 8-Tuftsin group produced an average of 5 10 ⁵ cells in the spleen lymphocytes. Of the 124 spots, (M2e) 8-G4 mice produced an average of 30 spots per 5 χ 10 ⁵ cells in the spleen lymphocytes.

It is indicated that the branched (M2e) 8-Tuftsin can induce cellular immunity in mice, and the number of spots produced is significantly different from the number of spots induced by PBS (P<0.05), while the control branch peptide (M2e) 8-G4 is induced. The number of spots is significantly lower than that of the branch peptide containing Tuftsin.

C) 14 days after the third immunization, the remaining mice were challenged with the influenza virus PR8 strain. The influenza virus PR8 challenge method was as follows: The mice were anesthetized by CO 2 inhalation, and then rapidly instilled into the nostrils on both sides. μ 1 5 times TCID ₅ . Influenza virus PR8 virus solution, allowing it to be inhaled adequately. On the 5th day after challenge, 5 mice in each group were sacrificed to take lungs, weighed, and lung tissue suspensions were prepared. After serial dilution, MDCK cells were inoculated and TCID _{5 was} determined. .

Pulmonary viral load assay results are shown in Figure 14, PBS group lg TCID ₅ . Is 6.53, (M2e) 8-Tuftsin&lg TCID ₅ . It is 5.53, (M2e)8 - G4 group lg TCID ₅ . The result was 5.89. This result showed that the lung virus load of the mice with branching peptide containing Tuftsin was the lowest, which was significantly different from that of the PBS group (P<0.05), indicating that the replication of the virus in this group of mice was effectively inhibited. .

After the challenge, the survival rate of 10 mice in each group was observed daily, and the survival rate was 14 days after the challenge. The mice that died within 24 hours of the challenge were regarded as accidental death. The statistical results of mouse survival showed that one mouse died on the 7th day and the 8th day after challenge, and the mice inoculated with the control branch peptide (M2e) 8-G4 had 1 on the 8th day. Death, while mice inoculated with branched (M2e) S-Tuftsin did not die, indicating that the two branched peptide immunizations have protective effects on mice.

Further embodiments of the present invention also provide a branched polypeptide derivative which is on the amino acid side chain group of branched (M2e)4-11!^3111 and branched (^126) 8-Tuftsin, and an amino terminal. Or a derivative obtained by hydroxylation, carboxylation, coagulation, methylation, acetylation, phosphorylation, esterification or glycosylation at the carboxy terminus.

Other embodiments of the present invention also teach the branched (M2e) 4-Tuftsin and branched (M2e) 8-Tuftsin pharmaceutically acceptable salts which are branched (M2e) 4-Tuf ts in and branched ( M2e) 8-Tuftsin is available as a raw material in combination with prior art preparation methods.

The branched (M2 e) 4-Tuf tsin and the branched (M2 e) 8-Tuf tsin proposed by the present invention can be used for preparing a vaccine, in particular, preparing an influenza A virus vaccine, and further, the influenza A virus It is an H1N1 influenza virus, an H2N2 influenza virus, an H3N2 influenza virus, an H5N1 influenza virus, an H7N7 influenza virus or an H9N2 influenza virus.

The present invention also proposes a vaccine comprising branched (M2e) 4-Tuf tsin or branched (M2e) 8-Tuftsin as an active component. The vaccine is a sputum influenza virus vaccine. Further, the influenza A virus vaccine is an H1N1 influenza virus vaccine, an H2N2 influenza virus vaccine, an H3N2 influenza virus vaccine, an H5N1 influenza virus vaccine, an H7N7 influenza virus vaccine or an H9N2 influenza virus. vaccine. Industrial applicability

The branched polypeptide of the present invention can be prepared into an influenza vaccine. The branched polypeptide of the present invention can specifically bind to an M2 polyclonal antibody, and can specifically bind to macrophages to promote treatment of antigen presenting cells. The vaccine prepared from the branched polypeptide of the present invention induces an animal immune response with high efficiency and protects the animal from viral infection.

Claims

The present invention claims a branched polypeptide characterized in that the structure of the branched polypeptide from the amino terminus to the carboxy terminus is as follows;

( AA _N ) ₈ - ( Lys ) - ( Lys ) ₂ -Lys-Thr-Lys-Pro-Arg; or

( AA _N ) 4 - ( Lys ) 2 ^_ Lys-Thr-Lys-Pro-Arg;

The AA _N is the extracellular domain of the matrix protein 2 of the influenza A virus, and the carboxy terminal sequence Thr- Lys_Pro_Arg of the above branched polypeptide is the immunologically active peptide Tuf ts in.

The branched polypeptide according to claim 1, wherein the sequence of the extracellular region of the matrix protein 2 of the influenza A virus from the amino terminus to the carboxy terminus is as follows:

3. A branched polypeptide derivative which is hydroxylated, carboxylated, carbonylated, methylated, acetylated at the amino terminus or the carboxy terminus of the branched amino acid side chain group of claim 1 or 2. , a derivative obtained by phosphorylation, esterification or glycosylation.

A branched polypeptide derivative which is a pharmaceutically acceptable salt of the branched polypeptide according to claim 1 or 2.

5. Use of a branched polypeptide according to any one of claims 1 to 4 for the preparation of a vaccine.

6. Use according to claim 5, characterized in that the vaccine is an influenza A virus vaccine.

The use according to any one of claims 1 to 6, wherein the influenza virus is H1N1 influenza virus, H2N2 influenza virus, H3N2 influenza virus, H5N1 influenza virus, H7N7 influenza virus or H9N2 influenza virus. .

A vaccine, wherein the active substance is the branched polypeptide of any one of claims 1 to 4.

The vaccine according to claim 8, wherein the vaccine is an influenza A virus vaccine.

The vaccine according to claim 9, wherein the influenza A virus is an H1N1 influenza virus, an H2N2 influenza virus, an H3N2 influenza virus, an H5N1 influenza virus, an H7N7 influenza virus or an H9N2 influenza virus.