CN117164678A - Respiratory syncytial virus immune composition and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, and particularly provides a respiratory syncytial virus immune composition, and a preparation method and application thereof. The invention provides a respiratory syncytial virus preF protein CD4T cell epitope peptide FP4. The invention also provides an immune composition of the respiratory syncytial virus, and the active ingredients of the immune composition comprise the respiratory syncytial virus G protein, the preF protein CD4T cell epitope peptide FP4 and/or cyclosporin A. The test proves that: the respiratory syncytial virus immune composition of the invention not only can obviously enhance the antiviral effect of immunized animals, but also can effectively inhibit the excessive cellular immune response.

Description

Respiratory syncytial virus immune composition and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a respiratory syncytial virus immune composition, a preparation method and application thereof.

Background

A negative strand RNA virus of the genus Pneumovirus of the family Paramyxoviridae, genus Pneumovirus, is described as respiratory syncytial virus (Respiratory Syncytial Virus, RSV). The entire genome of RSV may encode up to 10 viral proteins, including three transmembrane glycoproteins: an adhesion-acting G protein, a fusion-acting F protein and a small hydrophobin SH protein, wherein the F protein usually has two forms, namely, a pre-fusion conformation F protein (preF) and a post-fusion conformation F protein (postF).

RSV is a major cause of acute lower respiratory tract infection in infants and elderly, almost all infants under two years old experience RSV infection, and infants and elderly often experience recurrent infections, a disease that severely jeopardizes infant health. The existing drug palivizumab against RSV is only used for congenital heart disease or lung disease in premature infants less than 35 weeks, and is not currently marketed in China. The earliest developed RSV vaccine was formalin inactivated RSV (FI-RSV), which was clinically tested in the 60 Th 20 Th century, and as a result of Treg deletion and unbalanced Th 2-type inflammation, FI-RSV vaccinated infants again experience natural infection with RSV, resulting in more severe vaccine-enhanced disease (vaccine enhanced disease, VED), exacerbating illness leading to death in several infants, and no specific therapeutic and prophylactic vaccine for RSV virus is currently available. Experience of RSV vaccine research failure revealed to us the following two major challenges to be overcome: firstly, eliminating the over-strong immune response of an organism induced by immunogen, and avoiding the exacerbation (VED) of the organism caused by immune vaccine due to inflammatory cell infiltration; second, the vaccine can stimulate the body to generate enough virus neutralizing antibodies, so that the body can be protected when the RSV infection is faced again.

Thus, based on the above findings, it is internationally believed that a safe and effective RSV vaccine should possess both of the following properties: (1) activating high levels of humoral immune responses (neutralizing antibodies), (2) suppressing pathological T cell immune responses to eliminate VED. The method can effectively prevent and control RSV infection and simultaneously avoid serious adverse reaction caused by vaccine. For decades, internationally well known research institutions and businesses have performed much work in this area, but no effective, safe RSV vaccine has been marketed so far.

In view of this, the present invention has been made.

Disclosure of Invention

The first object of the present invention is to provide an epitope peptide which is respiratory syncytial virus preF protein CD 4T cell epitope peptide FP4.

A second object of the present invention is to provide an respiratory syncytial virus immune composition.

The third object of the present invention is to provide a method for preparing an respiratory syncytial virus immune composition.

A fourth object of the present invention is to provide the use of an respiratory syncytial virus immune composition.

In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:

an epitope peptide is respiratory syncytial virus preF protein CD 4T cell epitope peptide FP4, and the amino acid sequence of the epitope peptide is shown as SEQ ID NO. 8.

An immune composition of respiratory syncytial virus comprises active ingredients of respiratory syncytial virus G protein, respiratory syncytial virus preF protein CD 4T cell epitope peptide FP4 and cyclosporin A;

further, the amino acid sequence of the G protein of the respiratory syncytial virus is shown as SEQ ID NO.2, and the amino acid sequence of the CD 4T cell epitope peptide FP4 of the preF protein of the respiratory syncytial virus is shown as SEQ ID NO. 8.

Further, the mass ratio of the respiratory syncytial virus G protein, the respiratory syncytial virus preF protein CD 4T cell epitope peptide FP4 and cyclosporin A is 1: (0.1-10): (0.1-10), preferably 1: (0.5-1.5): (0.5-1.5).

Further, an adjuvant is included;

preferably, the adjuvant comprises at least one of metal ion adjuvants including aluminium hydroxide, aluminium phosphate, zinc aluminium adjuvants, manganese adjuvants and the like, TLRs ligands and cytokine adjuvants including chemokine adjuvants, preferably aluminium adjuvants;

preferably, the mass ratio of the respiratory syncytial virus G protein, the respiratory syncytial virus preF protein CD 4T cell epitope peptide FP4, the cyclosporin A and the adjuvant is 1: (0.1-10): (0.1-10): (5-20), more preferably 1: (0.5-1.5): (0.5-1.5): (9-11).

Further, the respiratory syncytial virus immune composition is in the form of injection, oral preparation, nose drops, spray or transdermal preparation.

The respiratory syncytial virus immune composition is prepared by mixing the respiratory syncytial virus G protein, the respiratory syncytial virus preF protein CD 4T cell epitope peptide FP4 and cyclosporin A.

Further, the respiratory syncytial virus immune composition is prepared by combining respiratory syncytial virus G protein, respiratory syncytial virus preF protein CD 4T cell epitope peptide FP4, cyclosporin A and an adjuvant;

preferably, the adjuvant comprises at least one of metal ion adjuvants including aluminium hydroxide, aluminium phosphate, zinc aluminium adjuvants, manganese adjuvants and the like, TLRs ligands and cytokine adjuvants including chemokine adjuvants, preferably aluminium adjuvants;

preferably, the mass ratio of the respiratory syncytial virus G protein, the respiratory syncytial virus preF protein CD 4T cell epitope peptide FP4, the cyclosporin A and the adjuvant is 1: (0.1-10): (0.1-10): (5-20), more preferably 1: (0.5-1.5): (0.5-1.5): (9-11).

Further, the respiratory syncytial virus immune composition is in the form of injection, oral preparation, nose drops, spray or transdermal preparation.

The application of the epitope peptide FP4 or the respiratory syncytial virus immune composition in preparing products for preventing and/or treating respiratory syncytial virus infection or diseases caused by respiratory syncytial virus infection.

Use of the epitope peptide FP4 or the respiratory syncytial virus immune composition as described above in (1) - (4) below:

(1) Preparing a product that increases the level of respiratory syncytial virus specific IgG antibodies in a mammal;

(2) Preparing a product that reduces the viral load of a mammalian respiratory syncytial virus;

(3) Preparing a product that inhibits proliferation and infiltration of inflammatory cells in the lung of a mammal;

(4) A product is prepared that reduces damage to mammalian lung tissue from respiratory syncytial virus.

An antibody obtained by immunizing an individual with the respiratory syncytial virus immunization composition.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a respiratory syncytial virus preF protein CD 4T cell epitope peptide FP4.

The invention also provides a respiratory syncytial virus immune composition, which uses the major surface protein G of RSV and the preF protein CD 4T cell epitope peptide FP4 as vaccine antigens to activate high-level antibodies, and simultaneously uses an immunomodulator cyclosporin A to induce and inhibit the specific pathological VED reaction of RSV, thereby achieving the purpose of preparing effective and safe RSV preventive vaccine. The vaccine composition of the G combined with the FP4 and the CsA can well inhibit proliferation and invasion of inflammatory cells in the lung of a mammal, reduce damage of respiratory syncytial virus to lung tissues of the mammal, inhibit VED phenomenon common to RSV vaccine, and more importantly, the immune effect of the FP4 has consistency with that of preF protein, and even more unexpected, the FP4 can play a better effect than preF. The FP4 improves the IgG antibody level of the specific G antigen of the mammal respiratory syncytial virus, improves the IgG antibody aiming at the G antigen CCD area, has biological activity and function aiming at the G antigen CCD area, and can improve the neutralizing antibody level, well reduce the pulmonary virus load and inhibit the replication of the virus; the combination of the two achieves a substantially unexpected synergistic effect. And the results of HE staining and PAS staining and slicing of lung tissues show that the G combined CsA and FP4 vaccine group can well inhibit proliferation and infiltration of inflammatory cells in the lung of mammals, reduce damage of respiratory syncytial virus to the lung tissues of the mammals, inhibit the common VED phenomenon of RSV vaccine, and more importantly, the FP4 inhibition VED phenomenon is obviously better than preF protein.

Meanwhile, unexpectedly, the polypeptide FP4 can promote the immune response of the G protein and also promote the immune response of the OVA protein, namely, the FP4 has the function of improving the immune response of various antigens in a broad spectrum.

In addition, in the practical application in industry, the epitope peptide FP4 has simpler structure compared with the full length of the preF protein, so that the scheme is simpler and more economical.

Overall, the vaccine composition combines the advantages of the G protein and the preF protein CD 4T cell epitope peptide FP4 as antigens, even achieves synergistic effect, improves the immunity protection of organisms, has no obvious side effect, reduces the common VED phenomenon of RSV vaccine, and solves the bottleneck problem in the RSV prevention/treatment field. Furthermore, FP4 has a function of broad-spectrum enhancing immune responses to various antigens.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the titers of anti-F specific antibodies IgG on day 28 after immunization of the mice of example 3;

FIG. 2 shows the titers of anti-G specific antibodies IgG on day 28 after immunization of the mice of example 3;

FIG. 3 is a schematic diagram of amino acid sequence and position of CCD region of the protein of example 4G;

FIG. 4 is an anti-G of example 4 day 26 after immunization of mice _153-197 Concentration of IgG;

FIG. 5 is an anti-G of example 4 day 26 after immunization of mice _159-173 Concentration of IgG;

FIG. 6 is an anti-G of example 4 day 26 after immunization of mice _165-179 Concentration of IgG;

FIG. 7 is an anti-G of example 4 day 26 after immunization of mice _171-185 Concentration of IgG;

FIG. 8 is an anti-G of example 4 day 26 after immunization of mice _177-191 Concentration of IgG;

FIG. 9 is an anti-G of example 4 day 26 after immunization of mice _183-197 Concentration of IgG;

FIG. 10 is an antibody activity assay of serum antibodies against the CCD region at day 26 after immunization of the mice of example 5;

FIG. 11 is a sample of the detection of neutralizing antibodies in the serum of the 26 th day after immunization of the mice of example 6;

FIG. 12 is a transfer of CD4 from example 7 mice ⁺ Fold change of anti-G specific antibody IgG before and after T cells;

FIG. 13 is a sample of example 7 mice transferred CD4 ⁺ Plasma cell ratio after T cells;

FIG. 14 is a CD4 transfer from example 7 mice ⁺ post-T cell B220 ⁺ GL7 ⁺ B cell ratio;

FIG. 15 is a transfer of CD4 from example 7 mice ⁺ IgD after T cells ^- IgM ^- B cell ratio;

FIG. 16 is the ability of spleen cells to secrete cytokine IFN-gamma following in vitro peptide stimulation of example 8;

FIG. 17 shows the titers of anti-G specific antibodies IgG on day 28 after immunization of the mice of example 8;

FIG. 18 is a graph of CD19 in lymph node cells at different days after immunization of example 8 mice ⁺ CD69 ⁺ B cell ratio;

FIG. 19 is a graph showing CD19 in spleen cells at different days after immunization of example 8 mice ⁺ CD69 ⁺ B cell ratio;

FIG. 20 shows IgD in spleen cells at day 7 after immunization of mice of example 8 ^- IgM-B cell ratio;

FIG. 21 is a graph of the mean fluorescence intensity of the proportion of CD 80B cells in spleen cells at day 7 after immunization of the mice of example 8;

FIG. 22 is a statistical plot of the results of IgG secreting plasma cells ELISPots in bone marrow cells at day 35 after immunization of the mice of example 8;

FIG. 23 is a visual image of the results of IgG secreting plasma cells ELISPot in bone marrow cells at day 35 after immunization of the mice of example 8;

FIG. 24 is an anti-G of example 9 day 28 after immunization of mice _159-173 Concentration of IgG;

FIG. 25 is an anti-G of example 9 day 28 after immunization of mice _153-197 Concentration of IgG;

FIG. 26 is a graph showing the detection of serum antibody activity against class 131-2G antibody at day 28 after immunization of the mice of example 9;

FIG. 27 is a plot of neutralizing antibody detection in the serum of day 28 after immunization of the mice of example 9;

FIG. 28 is the neutralizing antibody titer at day 33 post immunization of the mice of example 10;

FIG. 29 shows the amount of viral expression in the lung of mice after viral infection in example 10;

FIG. 30 is a HE stained pathological section of the lung of a mouse after virus infection in example 10;

FIG. 31 is a HE staining pathology score of the lungs of mice post-viral infection of example 10;

FIG. 32 is a PAS staining pathological section of the lung of the mice after virus infection in example 10;

FIG. 33 is PAS staining pathological mucus scores of the lungs of mice post-viral infection of example 10;

FIG. 34 is the specific antibody levels against OVA at day 28 after immunization of the mice of example 11.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any method or material similar or equivalent to those described may be used in the present invention.

The invention provides an epitope peptide, which is an epitope peptide FP4 of a respiratory syncytial virus preF protein CD 4T cell, and the amino acid sequence of the epitope peptide is shown as SEQ ID NO. 8.

The invention also provides a respiratory syncytial virus immune composition, the active ingredients of which are respiratory syncytial virus G protein, respiratory syncytial virus preF protein CD 4T cell epitope peptide FP4 and cyclosporin A. The immune composition uses RSV main surface protein G and F protein CD 4T cell epitope peptide FP4 before fusion as vaccine antigen to induce high level antibody, and uses immunomodulator cyclosporin A to inhibit RSV specific pathological VED reaction, so as to prepare effective and safe RSV prophylactic vaccine.

In a preferred embodiment, the amino acid sequence of the G protein of the respiratory syncytial virus is shown as SEQ ID NO.2, and the amino acid sequence of the CD 4T cell epitope peptide FP4 of the preF protein of the respiratory syncytial virus is shown as SEQ ID NO. 8. The inventor surprisingly finds that the G protein of SEQ ID NO.2 and the preF protein CD 4T cell epitope peptide FP4 of SEQ ID NO.8 can activate high-level antibodies as antigens in the research and development process, and simultaneously utilizes an immunomodulator cyclosporin A to induce and inhibit the specific pathological VED reaction of RSV, thereby achieving the purpose of preparing effective and safe RSV preventive vaccine. Wherein, the preF protein CD 4T cell epitope peptide FP4 of SEQ ID NO.8 can obviously promote the antibody of anti-G, has the same technical effect as the preF protein of SEQ ID NO.4, and has better technical effect than the preF protein in the aspect of inhibiting pathological VED reaction, thereby showing that the respiratory syncytial virus immune composition can obtain better immune effect.

Amino acid sequence of G protein (SEQ ID No. 2):

MHKVTPTTAIIQDATSQIKNTTPTYLTQNPQLGISPSNPSEITSQITTILASTTPGVKSTLQSTTVKTKNTTTTQTQPSKPTTKQRQNKPPSKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKKPTLKTTKKDPKPQTTKSKEVPTTKPTEEPTINTTKTNIITTLLTSNTTGNPELTSQMETFHSTSSEGNPSPSQVSTTSEY PSQPSSPPNT PRQ。

amino acid sequence of preF protein (SEQ ID No. 4):

MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGQSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGHHHHHHSAWSHPQFEK。

amino acid sequence of preF protein CD 4T cell epitope peptide FP4 (SEQ ID No. 8):

RTGWYTSVITIELSN

in a preferred embodiment, the mass ratio of respiratory syncytial virus G protein, respiratory syncytial virus preF protein CD 4T cell epitope peptide FP4 and cyclosporin a is 1: (0.1-10): (0.1-10), preferably 1: (0.5-1.5): (0.5-1.5). Wherein the mass ratio may be, but is not limited to, 1:0.1:0.1, 1:0.1: 10. 1:10:0.1, 1:0.5:1.5, 1:1.5:0.5, 1:0.5:0.5, 1:1.5:1.5 or 1:1.1:0.9, preferably 1:1:1.

in a preferred embodiment, the respiratory syncytial virus immune composition further comprises an adjuvant, specifically a metal ion adjuvant such as aluminum hydroxide, aluminum phosphate, zinc aluminum adjuvant, manganese adjuvant, TLRs ligand and/or cytokine, chemokine adjuvant. Further, the mass ratio of the respiratory syncytial virus G protein, the respiratory syncytial virus preF protein CD 4T cell epitope peptide FP4, the cyclosporin A and the adjuvant is 1: (0.1-10): (0.1-10): (5-20), more preferably 1: (0.5-1.5): (0.5-1.5): (9-11). The mass ratio may be, but is not limited to, 1:0.1:0.1: 5. 1:0.1:10: 20. 1:10:0.1: 10. 1:0.5:1.5: 9. 1:1.5:0.5: 11. 1:0.5:0.5: 9. 1:1.5:1.5:11 or 1:1:1:10.

In preferred embodiments, the respiratory syncytial virus immune composition is in the form of an injection, an oral, a nasal drop, a spray, or a transdermal agent.

The invention also provides a preparation method of the respiratory syncytial virus immune composition, which comprises the step of mixing respiratory syncytial virus G protein, respiratory syncytial virus preF protein CD 4T cell epitope peptide FP4 and cyclosporin A. If the raw material of the immune composition further comprises an adjuvant, the immune composition can be obtained by compatibility according to a conventional preparation method. Wherein the adjuvant comprises aluminum hydroxide, aluminum phosphate, zinc aluminum adjuvant, manganese adjuvant, TLRs ligand and/or cytokine and chemokine adjuvant. The respiratory syncytial virus G protein, the respiratory syncytial virus preF protein CD 4T cell epitope peptide FP4, the cyclosporin A and optional adjuvants can be prepared into injection, oral preparation, nasal drops, spray, transdermal preparation and the like according to different requirements.

The invention also provides application of the respiratory syncytial virus immune composition in preparing products for preventing and/or treating respiratory syncytial virus infection.

The invention provides application of the respiratory syncytial virus immune composition in preparing products for preventing and/or treating diseases caused by respiratory syncytial virus infection.

The invention provides application of the respiratory syncytial virus immune composition in preparing a product for improving the respiratory syncytial virus specific IgG antibody level of mammals.

The invention provides application of the epitope peptide FP4 and the respiratory syncytial virus immune composition in preparing products for reducing viral load of mammal respiratory syncytial virus.

The invention provides application of the epitope peptide FP4 and the respiratory syncytial virus immune composition in preparing products for inhibiting proliferation and infiltration of inflammatory cells in the lung of mammals.

The invention provides application of the respiratory syncytial virus immune composition in preparing products for reducing damage of respiratory syncytial virus to lung tissues of mammals.

The invention provides a respiratory syncytial virus immune composition, and a product for inhibiting inflammatory response of mammals.

Each of the above products may be in the form of an vaccination kit, the respiratory syncytial virus immunization composition being administered by a vaccine spray, a vaccine gun, a vaccine needle or an electroporation device.

The invention finally provides an antibody which is obtained after the individual is immunized by the respiratory syncytial virus immune composition provided by the invention.

The invention is further illustrated by the following specific examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and are not to be construed as limiting the invention in any way.

Example 1 preparation of vaccine components

Acquisition of G protein

The coding gene sequence of the respiratory syncytial virus G protein is shown as SEQ ID NO.1, and the amino acid sequence of the G protein is shown as SEQ ID NO. 2. The coding gene sequence of the respiratory syncytial virus G protein shown in SEQ ID NO.1 is synthesized by a total gene synthesis method, the SEQ ID NO.1 is inserted between NcoI and XhoI sites of a pET28a (+) vector to obtain a recombinant expression plasmid, and the recombinant plasmid is transformed into escherichia coli BL21 (DE 3) for expression and purification to obtain the required G protein. The protein lyophilized product was provided by Ai Diwei Xin (su state) biopharmaceutical limited.

Obtaining of pref protein

The coding gene sequence of the respiratory syncytial virus preF protein is shown as SEQ ID NO.3, and the amino acid sequence of the preF protein is shown as SEQ ID NO. 4. The coding gene sequence of respiratory syncytial virus preF protein shown in SEQ ID NO.3 is synthesized by a total gene synthesis method, SEQ ID NO.3 is inserted between EcoR I and Hind III sites of a pcDNA3 vector to obtain a recombinant expression plasmid, and the recombinant plasmid is transfected into CHO cells to be expressed and purified to obtain the required preF protein.

Obtaining of pref protein CD 4T cell epitope peptide

The preF protein sequence (SEQ ID NO. 4) is put into IEDB for prediction, BALB/c mice H2-IAd and H2-IEd alleles are used, the algorithm applies the consensus algorithm in peptide binding to MHC class II molecules, the amino acid length is 15AA, 5 preF protein CD 4T cell epitopes are screened out according to the readiness of the core sequence, and are named as FP1, FP2, FP3, FP4 and FP5 respectively, and 5 polypeptide sequences are subjected to polypeptide synthesis to obtain the preF protein CD 4T cell epitope polypeptide as shown in Table 3.

4. Cyclosporin A (CsA) in the present invention is a product of Taishan chemical pharmaceutical Co., ltd, product catalog number national drug standard H10940111. Vector pET28a (+) is a product of Novagen, cat# 69864-3. The vector pcDNA3 is a product of Invitrogen corporation, catalog number V79020. Coli BL21 (DE 3) is a product of Tiangen Biochemical technology (Beijing) Co., ltd., catalog number CB105-02.

EXAMPLE 2 animal immunization

Female BALB/c mice of 6 week age, free of Specific Pathogen (SPF), were purchased from shanghai jess laboratory animals limited and kept at the basic medical college of complex denier university SPF laboratory animal center. The vaccine composition obtained in example 1 was used for vaccine preparation, the immunization groups and the amounts of the reagents were as shown in Table 1, the preparation procedure was as shown in Table 2, the PBS injection group was the group 1, the group 2 was the group 10. Mu. g G +10. Mu.g CsA, the group 3 was the group 10. Mu.g preF, the group 4 was the group 10. Mu.g G+10. Mu.g CsA+10. Mu.g preF, the day of the first immunization of the mice was defined as day 0, the day of the first immunization was-1, the day of the last immunization was 1, and so on. Day 0, 14 mice were immunized by intramuscular injection, 520 μl each time, two injections, 260 μl each, and one injection per leg. And collecting blood samples of mice on days 26 and 28, and separating serum for detecting specific antibody experiments.

TABLE 1 immunopacketization and reagent usage

Table 2 vaccine configuration procedure

Example 3 antigen-specific antibody detection

To evaluate the effect of the different groups of vaccines prepared in example 2 on the humoral immune response of mice, the titers of antigen-specific antibodies IgG (anti-G, anti-F) in the serum of day 28 after immunization of mice were measured by ELISA.

The specific experimental method comprises the steps of: diluting antigen with antigen coating solution, coating 2 μg/ml G protein or 1 μg/ml preF protein with 96-well plate, adding 100 μl per well to 96-well ELISA plate, and incubating overnight at 2-8deg.C. Closing: the coating was discarded, washed 3 times with PBST, blocked by adding 3% BSA for 1h. Incubation of primary antibody (test serum): the blocking solution was discarded, the plates were washed 3 times with PBST, and the serum to be tested was serially diluted 2-fold with 1% BSA, 100. Mu.L per well and incubated for 1h at 37 ℃. Secondary antibody incubation: the assay solution was discarded, the plate was washed 5 times with PBST, and the secondary antibody (gold anti-mouse IgG-HRP) was diluted with 1% BSA, 100. Mu.L was added to each well, and incubated at 37℃for 1 hour. Color development: the secondary antibody was discarded, washed 5 times with PBST, and developed at room temperature for 10-30 minutes (blue after development) with 100. Mu.L of TMB developing solution. And (3) terminating: 50 mu L of 2M H are added to each well ₂ SO ₄ The color development was terminated (the color turned bright yellow after the termination of the color development). The reading was detected with a microplate reader (A450/A620) within 30 minutes.

Conclusion: the results in FIG. 1 show that immunization of the preF protein group alone, and the combined immunization of the preF protein and G+CsA (G+CsA+preF group) induced high levels of anti-F antibody titres, comparable to each other.

The results in FIG. 2 show that the G+CsA vaccine group can induce high levels of anti-G antibody titer, and that the anti-G antibody titer in the G+CsA+pref group is significantly higher than that in the G+CsA group after combined immunization of preF protein and G+CsA (G+CsA+pref group), indicating that preF protein can promote antibody response of G protein.

Example 4G protein CCD region antigen-specific antibody detection

CCD area (G) _153-197 ) Antigen-specific antibody detection of (2)

Example 3 the results show that the preF protein and g+csa combined immunization promotes the antibody response of the G protein, whereas the Conserved Central Domain (CCD) of the G protein does not have a complex spatial structure, and that antibodies directed against this region recognize a linear conformation. To further explore F protein promotionWhether the response of the G-in protein antibody is also directed against the CCD region (G _153-197 ) With consistent effect, we detected serum antibodies from mice at day 26 post immunization by a relative quantitative ELISA method. Mice were immunized as follows: group 1 was PBS injection group, group 2 was 10. Mu.gG+10. Mu.g CsA group, group 3 was 10. Mu. g G +10. Mu.g CsA+10. Mu.g preF group, and the immunization was the same as in example 2. The ELISA method for detecting serum antibodies comprises the following steps: a96-well plate was incubated at 4℃with 5. Mu.g/mL of CCD polypeptide (G _153-197 Specific sequences are shown in FIG. 3) were coated on 96-well plates, blocked with 3% BSA, incubated at 37℃for 1h with 1:50 dilution of serum, and antibody-binding polypeptides were detected with anti-mouse IgG HRP (1:400). In the experiment, monoclonal antibody 131-2G is used as a standard substance, and the concentration of the antibody of 131-2G in serum within the range of 131-2G is calculated according to standard curve.

Conclusion: as shown in fig. 4, the experimental result shows that a certain amount of specific antibodies can be generated against the G protein CCD region in the g+csa immune group, the concentration of the specific antibodies at the g+csa+pref group CCD site after combined immunization with preF protein is significantly increased, which is consistent with the detection result (fig. 2) of the anti-G protein antibody in example 3, so that the experimental result further shows that preF protein can also increase the antibody level at the G protein CCD site.

2. Antigen-specific antibody detection of multiple CCD polypeptides

The CCD polypeptide used in the above experiments is the 153-197 amino acid sequence of the G protein sequence, which includes a stringent CCD region, a known monoclonal antibody binding region and CX3C motif site; to better determine the binding site of vaccine-increasing antibodies, we synthesized 5 CCD polypeptides by means of overlap (G _159-173 、G ₁₆₅ - ₁₇₉ 、G _171-185 、G _177-191 、G _183-197 Specific sequences are shown in FIG. 3), the binding of the serum antibodies of the mice on day 26 after immunization of the above experiment to the 5 CCD polypeptides was detected by a relative quantitative ELISA method, and the specific method is the same as the above experiment, and the amount of the 5 CCD polypeptides used is 5. Mu.g/ml.

Conclusion: the experimental results are shown in FIGS. 5-9, and found to be similar to the CCD (G) _153-197 ) Antibody results are evidentIn agreement with G _159-173 Polypeptides (FIG. 5) and G _177-191 The binding concentration of serum antibodies to this site was significantly increased after the combined immunization of the polypeptide (FIG. 8), preF protein and G+CsA (G+CsA+pref group) compared to the G+CsA immunized group. And G _177-191 The sequence comprises CX3C motif, the CX3C motif is used as ligand to bind with a cell surface receptor CX3CR1, CX3C in RSV G protein can lead to virus infection of cells through binding with CX3CR1, and CCD antibody generated after G protein immunization can prevent the RSV virus from binding with CX3CR 1. This experiment predicts that combined immunization with preF protein and g+csa promotes antibodies against the CX3C motif in the G protein, thereby blocking binding of the G protein to the host surface CX3CR1 and reducing infection.

Example 5 competitive ELISA detection of the G protein CCD region (G _153-197 ) Antibody biological Activity

To verify the neutralizing site G of immunization _153-197 The biological activity of the antibody is that 131-2G with known neutralizing capacity is used as a standard substance to detect the capacity of immune serum antibody and monoclonal antibody to compete with the same polypeptide epitope. In experiments we made standard curves by gradient dilution of 131-2G, serum at different dilutions competed with 0.25. Mu.g/ml 131-2G, while immune serum was derived from example 4 using 3D3 monoclonal antibody as positive control, serum antibodies after immunization were detected with CCD region by competitive ELISA method (G _153-197 ) Is used for the binding capacity of the polymer. The experimental method comprises the following steps: biotin-labeled CCD (G) _153-197 ) Coated in 96-well plates at 0.125 μg/mL overnight at 4 ℃; after blocking with 3% BSA, diluted serum (1:25, 1:50, 1:100, 1:200, 1:400, 1:800) was added, respectively, incubated at 37℃for 1 hour, 3D3 monoclonal antibody was used as positive control, 131-2G-rabbit Fc monoclonal antibody (0.25. Mu.g/ml) was added to the well plate, incubated at 37℃for 1 hour, and different dilutions of 131-2G-rabit Fc monoclonal antibody were used as standard curve; after 1 hour, the 131-2G concentration-conjugated biotin-labeled CCD was detected with a coat anti-rabit IgG-HRP. Inhibition rate was calculated according to the formula = (1-experimental group/blank group) ×100% of blocking rate.

Conclusion: the experimental results are shown in FIG. 10, which shows that antibodies raised by the immune group compete well with 131-2GIdentical site epitopes. G+csa group serum at 1: the competition rate can reach 50% at the dilution of 25. As serum dilution increased, its competition rate decreased, but was significantly higher than PBS group serum. Meanwhile, the competition rate of immune group (G+CsA+pref group) serum and 131-2G after the preF protein is combined with the immunity G+CsA is 1: near 80% at 25 dilutions, at 1: the competition rate at 100 dilutions was close to 50%, and the competition rate of serum from the G+CsA+pref immune group with 131-2G was significantly higher than that from the G+CsA group. Indicating that in the G+CsA+pref immune group serum there is G _153-197 The epitope-enhanced antibody competes with 131-2G in a similar epitope, and the combined immunization of the preF protein and the G+CsA promotes the antibody against CX3C motif in the G protein, indicating the neutralizing site G generated by the immunization of the G+CsA+preF _153-197 The antibodies have the same biological activity as 131-2G.

EXAMPLE 6 detection of RSV N Gene in THP-1 cells after RSV virus infection

The antibodies generated by the vaccine mainly play a role in neutralizing viruses, and in order to further verify the effect of blocking the combination of RSV and CX3CR1 by immune serum antibodies to prevent RSV infection, whether the immune serum antibodies have immunological functions or not is detected, and the inhibition effect of the immune serum antibodies on the RSV infection THP-1 is detected, wherein immune serum is derived from the example 4. The experimental method comprises the following steps: RSV (5X 10) ⁵ pfu) was mixed with 100 μl of serum diluted 1:50 and incubated at 4deg.C for 2h. THP-1 cells were suspended in 3X 10 ⁶ Per ml of serum-free medium, 100. Mu.L/well was added to the 24-well plate. The virus and serum mixture was added to THP-1 cells, cultured at 37℃for 2 hours, and then cultured in 1640 (containing 2% FBS) medium for 3 days. RNA was extracted from THP-1 cells according to the protocol using a sparkaeasy tissue cell RNA rapid extraction kit; cDNA synthesis was performed on the extracted RNA using a cDNA transcription kit HifairII 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus), according to the methods described herein and using conditions of 25℃for 5min,42℃for 30min, and 85℃for 5 min; real-time quantitative PCR was then performed using qPCR detection kit Hieff qPCR SYBR Green Master Mix (No Rox), PCR reaction conditions: at 95 ℃ and 5min,95 ℃, 10s,60 ℃, 20s,72 ℃ and 20s for 40 cycles, and simultaneously, the plasmid containing the RSV N gene is used as a standard substance to calculate the content of the RSV N gene in the cDNA of the sample to be detected Amount of the components. The primer used is a forward primer TACGGTGGGGAGTCTTAGCA; reverse primer CACCACCCAATTTTTGGGCA.

Conclusion: as shown in FIG. 11, the copy number of RSV N gene in THP-1 cells in PBS group is very high, and after the serum of G+CsA immune group is neutralized, the content of intracellular RSV N gene is relatively reduced, and the content of intracellular RSV N gene after the serum antibody of G+CsA+pref immune group is neutralized is obviously reduced compared with that of G+CsA group, so that preF protein can obviously improve the immune response effect of G+CsA, promote the generation of antibody, improve the neutralizing antibody level of conjugated antibody and inhibit the RSV from infecting THP-1 cells.

EXAMPLE 7 preF protein-specific T cells promote G+CsA vaccine antibody response

The experimental results in examples 1-6 above demonstrate that preF protein promotes the humoral immune response of G protein and that antibody production is mainly due to the conversion of antigen-specific B cells into plasma cells under Tfh and thus secretion of antibodies. Given that preF proteins have no homology to the G protein sequence, we exclude the possibility that preF protein-induced specific B cells secrete G protein antibodies directly, and thus speculate that preF protein-induced T cells promote G protein-specific B cell secretion of antibodies. We therefore hypothesize that specific CD4 helper cells are promoted by the combined immunization of preF protein, and to verify this hypothesis we have passed the mouse spleen CD4 after immunization with preF protein ⁺ T(2×10 ⁷ ) After cell sorting, the cells were transferred to mice immunized with the g+csa vaccine by tail vein injection, and after 5 days, the antibody level and the change in B cell activation and differentiation were detected. Donor immune group: 10 μg G+10 μg CsA (positive control group), 10 μg preF (experimental group), 10 μg OVA (negative control group); immunization mode: mice were immunized 3 times by intramuscular injection on days 0, 14, 28, 4 days after 3 rd immunization, with mouse CD4 ⁺ T cell separation kit (Mouse CD 4) ⁺ T Cell Isolation Kit) CD4 was isolated from spleens of donor mice immunized with G+CsA (positive control group), preF (experimental group), OVA (negative control), respectively ⁺ T cells. Recipient immune group: 10 mu g G +10 mu g CsA; immunization mode: day 0, 14 mice were immunized by intramuscular injection 2 times, 2 nd immunizationOn day 8 post-epidemic, donor immune groups were individually isolated for CD4 ⁺ T cells 2X 10 ⁷ Cells were diluted in 200 μlpbs and transferred intravenously to day 8 recipient immunized mice following day 2 immunization. anti-G IgG in peripheral blood of recipient mice was quantitatively detected by ELISA 1 day before intravenous transfer and 5 days after transfer, and the antibody was increased several times by calculating the difference in the levels of the antibodies before and after transfer. Recipient mouse spleen plasma cells were assayed 5 days after transfer (B220-CD 138) ⁺ ) Germinal center B cells (B220) ⁺ GL7 ⁺ ) And Ig-converted B cells (IgD) ^- IgM ^- )。

Conclusion: the experimental results are shown in FIGS. 12-15, transferring CD4 in OVA immunized donor mice ⁺ After T cells had been in the G+CsA immunized recipient mice, the antibody levels in the recipient mice (FIG. 12) and the levels of 3 types of B cells (FIGS. 13-15) were lower; while transferring CD4 in preF-immunized or G+CsA-immunized donor mice ⁺ After T cells had been immunized into g+csa recipient mice, the levels of antibodies (fig. 12) and 3 types of B cells (fig. 13-15) were significantly increased in the recipient mice, which were significantly higher than in the OVA donor group; wherein the fold change in anti-G IgG antibody levels was even higher than in the G+CsA donor group, we considered that it was possible to immunize against preF to generate CD4 ⁺ T cells promote early activation and class transformation of G protein-specific B cells, promote germinal center formation, and thus promote antibody production by plasma cells.

EXAMPLE 8 CD4 of preF ⁺ Screening and obtaining of T cell epitope peptides

From example 7 we verify that preF protein can be immunologically generated from CD4 ⁺ T cells to promote humoral immune response to G protein, we next speculate that there should be a T cell epitope in the preF protein that can produce this function, we first screened the reported CD4 epitope peptide of preF protein in the literature for this functional epitope peptide, according to the literature and in comparison with our structure of F protein, we screened 1 reported epitope peptide (FP 6) in mice, in addition to the preF protein sequence into IEDB to predict polypeptides with stronger binding to MHC II, BALB/c mice H2-IAd and H2-IEd alleles were used in the prediction process The method uses the consensus algorithm in peptide binding to MHC class II molecules, the amino acid length is 15AA, 10 sequences are selected from the core sequences with the strongest binding capacity, epitopes which are proved to be ineffective or are not based on protein structures in the previous literature are removed, the adjustment is carried out according to the recombination hydrophilicity of the core sequences, and the finally determined sequences are unreported and unreported 5 amino acid polypeptides, namely FP1, FP2, FP3, FP4 and FP5. We performed the next verification with the 1 polypeptide identified as reported and with the 5 polypeptides predicted in IEDB, the polypeptide amino acid sequences are shown in Table 3.

TABLE 3 IEDB predicted F protein CD4 ⁺ T cell epitopes

1. To determine the ability of preF protein immunized mouse spleen cells to secrete the cytokine IFN-gamma after stimulation with 5 polypeptides predicted in vitro, the mice were taken from their spleen 28 days after immunization with preF protein and intracellular cytokines were detected by flow cytometry. The experimental method comprises the following steps: spleen cells from mice immunized with preF protein were stimulated with 40 μg/mL of each polypeptide and PMA (50 ng/mL)/ionomycin (IONO, 1 μg/mL) as positive control for 6 hours, during which time blocking was performed with 1 μg/mL brefeldin a (BD). After further surface staining with anti-CD 4-PECy7 (eBioscience, 25-0041-82) and fixation with formaldehyde at room temperature for 8 min, anti-IFN- γ -BV421 (Biolegend, 505830) was incubated at room temperature (diluted 1:100 in 1 XMembrane-disrupting buffer) for 1 hour, and the final samples were detected by LSRFortessa flow cytometer (BD) and analyzed by FlowJo software (Tree Star, ashland, OR, USA).

Conclusion: the results are shown in FIG. 16, which shows the results with negative control (DMSO) and independent group (OVA _323-339 ) In contrast, FP4 and FP5 stimulated spleen CD4 ⁺ T cells secrete high levels of IFN-gamma, whereas the FP1, FP2 and FP3 stimulated groups did not produce IFN-gamma. Indicating that FP4 and FP5 are CD4 on preF protein ⁺ T cell epitopes.

2. To screen whether a protein which can exert a similar effect to preF protein can promote humoral immune response of G protein, we performed in vivo immunization of the polypeptide with a G protein vaccine (g+csa). Mice were immunized as follows: the immunization procedure and reagent preparation method were the same as in example 2, except that group 1 was G+CsA, group 2 was G+CsA+pref, group 3 was G+CsA+FP1, group 4 was G+CsA+FP2, group 5 was G+CsA+FP3, group 6 was G+CsA+FP4, group 7 was G+CsA+FP5, group 8 was G+CsA+FP6, and the amounts of each reagent were 10. Mu.g. A blood sample of the mice at day 28 was collected and the anti-G IgG titer in the peripheral blood was measured by ELISA (same as in example 3).

Conclusion: as shown in the results of FIG. 17, among the 6 groups of FP1-FP6 polypeptides, the antibody level of the G+CsA+Fp4 group after immunization was highest and was comparable to that of the G+CsA+pref group, indicating that the promotion of FP4 was consistent with preF protein and could promote the humoral immune response of the G+CsA vaccine. Thus we believe that the CD 4T cell epitope peptide of FP4 as a preF protein promotes the humoral immune response of the G protein vaccine. In view of the above, we screened for the CD 4T cell epitope peptide that gave FP4 as a preF protein to promote the immune response of the G protein.

3. To further evaluate the role of FP4 in promoting g+csa immune antibody responses and to investigate possible mechanisms initially, we analyzed early activation of CD69 in the lymph nodes and spleen of mice on days 3, 5, 7 and 10 after primary immunization by flow cytometry ⁺ Proportion of B cells and Ig-converted B cells on day 7 post-immunization (B220 ⁺ IgD ^- IgM-) and B cell surface co-stimulatory molecule CD80. Single cell suspensions were prepared from the spleen or lymph nodes of the immunized mice described above, cells were counted and used Fixable Viability Dye eFluor ^TM 780 (eBioscience, OR, USA) after 15 min staining, cells were incubated in PBS (with 2% FBS) for 15 min with the following anti-mouse antibodies (all diluted 1:200): anti-B220-Pacific Blue (bioleged, 103227), anti-IgM-APC-Cy 7 (bioleged, B194508), anti-IgD-FITC (bioleged, B147607), anti-CD 69-PECy7 (bioleged, 25-0691-81) and anti-CD 80-BV605 (eBioscience, 63-0801-82). The final samples were examined by LSRFortessa flow cytometer (BD) and analyzed by FlowJo software (Tree Star, ashland, OR, USA).

Conclusion: in lymph node cells (FIG. 18), we observed CD19 between groups ⁺ CD69 ⁺ Cell levels were similar at early day 3, but the FP4+G+CsA group increased at day 5 and decreased to a level similar to that of the control groups G+CsA and G+CsA+Fp2 at day 7, with a further increase at day 10. CD19 of group G+CsA+FP4 in spleen cells (FIG. 19) ⁺ CD69 ⁺ Cell levels were significantly higher on day 7 than in the g+csa and g+csa+fp2 groups; the differences between the three groups were more pronounced by day 10. In addition, ig-converted B cells were detected on day 7 post-immunization (B220 ⁺ IgD-IgM-) and B cells expressing co-stimulatory molecule CD80, the results show (as shown in fig. 20-21) that the proportion of Ig-converted B cells in the g+csa+fp4 group and CD 80B cells are significantly higher than in the two control groups g+csa and g+csa+fp2. It was shown that FP4 can promote early B cell activation after g+csa immunization, thereby promoting antibody production.

4. Since B cells differentiate into antibody secreting plasma cells (ASCs), a portion of the plasma cells then circulate back to the bone marrow as persistent memory B cells. We next determined plasma cell levels in the bone marrow of mice at day 35 post immunization by ELISpot. The experimental method comprises the following steps: 96-well plates (Millipore, boston, USA) were prewetted with 35% ethanol, then washed 3 times and coated overnight with 20. Mu.g/mL G-protein at 4 ℃. Plates were washed with RPMI1640 containing 10% heat-inactivated FBS and blocked for 1 hour. After removal of the blocking solution, single cell suspensions from bone marrow preparations were removed at 2.5X10 per well ⁵ Individual cells were added to 96-well plates and incubated in an incubator for 20 hours. After washing the plates, and incubation with 1. Mu.g/mL of anti-mouse IgG detection antibody was performed for 2 hours at room temperature according to the mouse IgG ELISPOT kit (Daidae Biotechnology Co., ltd.). Spots were detected by AID ELIspot reader (AID, germany) and Spot Formation Units (SFU) were calculated per million cells.

Conclusion: as shown in fig. 22-23, G protein-specific antibody secreting plasma cells were significantly increased in the g+csa+pref and g+csa+fp4 immunized groups compared to the g+csa and g+csa+fp2 groups. These data suggest that FP4 may promote the production of persistent B cells following g+csa immunization.

EXAMPLE 9 CD4 of preF ⁺ Immune effect verification of T cell epitope peptide FP4

To further verify the CD4 of the preF protein screened in our example 8 ⁺ Whether the immune response effect of the T cell epitope peptide FP4 on the G protein is consistent with that of the preF protein or not is detected, and whether the immune response effect of the FP4 on the G protein CCD antibody, the biological activity of the antibody on the CCD area and the antibody have an immunological function or not are detected. Experimental grouping: group 1 is PBS group, group 2 is G+CsA group, group 3 is G+CsA+pref group, group 4 is G+CsA+FP4 group, the dosage of each reagent is 10 μg, the immunization process and reagent configuration method are the same as in example 2, the 28 th day mouse blood sample is collected, and antibodies in serum are detected.

Antibody specific to G protein CCD region antigen of epitope peptide FP4 of pref and antibody biological activity detection

In example 8 we have verified the acceleration effect of FP4 on G protein antibodies, thus further detecting the immune response effect of FP4 on G protein CCD antibodies, experimental procedure was consistent with the relative quantitative ELISA method of example 4; meanwhile, the method for detecting the biological activity of the serum antibody after immunization and the antibody of the CCD region is consistent with the competitive ELISA method of example 5.

Conclusion: the results are shown in FIGS. 24-25, and the antibody level of the G+CsA+FP4 group against the CCD region is significantly higher than that of the G+CsA group, even higher than that of the G+CsA+pref group, indicating that the promotion effect of FP4 is even better than that of preF protein, and the immune response of G+CsA can be promoted.

The results of the detection of the biological activity of the antibodies in the CCD region are shown in FIG. 26, and the competition rate curves of the G+CsA+FP4 group and the G+CsA+pref group are basically consistent, which shows that the biological activity of FP4 is similar to preF protein, and the generated serum can better compete with 131-2G, and further shows that the promotion effect of FP4 on G protein is consistent with preF protein.

Detection of RSV N Gene in THP-1 cells after RSV viral infection

In order to examine the immunological function of the antibodies after FP4 immunization, whether or not it could inhibit RSV infection of host cells, we examined the inhibitory effect of immune serum antibodies on RSV infection of THP-1, and the experimental procedure was the same as in example 6.

Conclusion: as shown in FIG. 27, the content of RSV N gene in the cell after neutralization of the serum antibodies of the two immunized groups of G+CsA+FP4 and G+CsA+pref is obviously reduced compared with that of the G+CsA, and the content of RSV N gene in the two immunized groups is basically equivalent, which indicates that the promoting effect of FP4 on the G protein vaccine is equivalent to or even better than that of preF protein, thus obviously improving the immune response effect of G+CsA, promoting the generation of neutralizing antibodies and inhibiting the infection of RSV on THP-1 cells.

EXAMPLE 10 CD4 of preF ⁺ Verification of T cell epitope peptide FP4 for preventing RSV infection

To further examine the practical effect of FP4 combined immunization with g+csa on the prevention of RSV infection in mice, we examined the peripheral blood neutralizing antibodies, pulmonary viral load and pulmonary pathology in mice after 2 muscle immunizations and RSV challenge on day 28, 5 days post challenge. Experimental grouping: the 1 st group is PBS injection group, the 2 nd group is G+CsA group, the 3 rd group is preF group, the 4 th group is G+CsA+pref group, the 5 th group is G+CsA+F4 group, the 6 th group is FI-RSV group, the dosage of each reagent is 10 mug, wherein the preparation method of the 6 th group inactivated vaccine FI-RSV is 10 ⁷ TCID ₅₀ After the RSV virus (American ATCC, category No. VR-26 TM) was reacted with formaldehyde for 72h at 37 ℃, the concentrated inactivated RSV stock solution was purified by high-speed centrifugation for 1h with 50000g, 50. Mu.L of the inactivated stock solution was mixed with an equivalent amount of aluminum adjuvant, and the mixture was placed on a shaking table and subjected to mixing shaking for 30 minutes, and 100. Mu.L of each mouse was injected. Immunization procedure and reagent preparation method were the same as in example 2, and on day 28, mice of each group were nasally infected with RSV virus in an amount of 2X 10 ⁶ pfu/each mouse, euthanized on day 33, blood samples were collected to detect serum neutralizing antibodies, lung tissue was collected for viral load determination and pathology examination.

1. Neutralizing antibody detection

Inactivating the serum to be tested in water bath at 56 ℃ for 30min, diluting with serum-free DMEM medium for 2 times, diluting with 5 gradients, and mixing the serum to be tested with RSV A2 (1×10) ⁴ pfu) was mixed at 4℃for 2 hours, and then Vero cells (2X 10) previously cultured in 96-well plates were added ⁴ /well). After 2 hours DMEM medium containing 4% fetal bovine serum was added to the petri dishes. After 5 days, 80% of the pre-cooled polypropylene is usedThe cells were fixed with ketone and then blocked with 5% bsa. After washing, the goat anti-RSV IgG was added to the well plate and incubated at 37℃for 1h. After washing, the dorkey anti-goat IgG-HRP was added and the reading was detected by an enzyme-labeled instrument (A450/A620).

Conclusion: the detection results of the neutralizing antibodies are shown in fig. 28, and the neutralizing antibody titer of the G+CsA+FP4 group and the neutralizing antibody titer of the G+CsA+pref group after immunization are not obviously different, compared with the neutralizing antibody titer of the G+CsA group, the neutralizing antibody titer of the G protein after virus challenge of mice can be obviously increased by FP4, and RSV infection can be prevented.

2. Pulmonary RSV viral load detection

After 5 days of challenge, mice were anesthetized with isoflurane, lungs were removed and weighed, then tissue homogenized, and lung tissue homogenized RNA was extracted according to the protocol described using RNA extraction Kit e.z.n.a Total RNA Kit I. cDNA synthesis was performed on the extracted RNA using a cDNA transcription kit HifairII 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus), according to the methods described herein and using conditions of 25℃for 5min,42℃for 30min, and 85℃for 5 min; real-time quantitative PCR was then performed using qPCR detection kit Hieff qPCR SYBR Green Master Mix (No Rox), PCR reaction conditions: the total of 40 cycles of 95 ℃, 5min,95 ℃, 10s,60 ℃, 20s,72 ℃ and 20s are carried out, and meanwhile, the content of the RSV N gene in the cDNA of the sample to be detected is calculated by taking the plasmid containing the RSV N gene as a standard substance. The primer used is a forward primer TACGGTGGGGAGTCTTAGCA; reverse primer CACCACCCAATTTTTGGGCA.

Conclusion: the results are shown in fig. 29, in which the virus content of the preF alone immunized group, the g+csa+fp4 group and the g+csa+pref group is significantly reduced, and the virus content of the g+csa+fp4 group is the lowest, further showing that FP4 can significantly enhance the immune response of g+csa after challenge in mice, prevent RSV virus infection, and inhibit pulmonary virus replication.

3. Pathological section, staining and reading of lung

After the lung specimens of mice were fixed with 4% paraformaldehyde at room temperature for 24 hours, paraffin embedding was performed. Staining with Hematoxylin and Eosin (HE) or iodate-Schiff (PAS) after slicing, and evaluating lung inflammation and injury degree by inflammatory cell infiltration and mucous secretion score after photographing and observation, wherein the lung inflammation and injury degree is 0-normal; 1-minimum (min); 2-slight (slight); 3-modem (medium); 4-sever (severe).

Conclusion: results as shown in fig. 30-33, lung section HE staining results (fig. 30) showed that there was a large amount of lymphocyte infiltration around the bronchi of mice after PBS immune group challenge compared to mice without challenge (Non-infusion), and the tracheal wall was thickened; peribronchial cell infiltration is very serious in the FI-RSV immune group, a large amount of lymphocyte infiltration is also present in alveoli, and the tracheal wall is thickened; the lung bronchus of the group G+CsA mice are infiltrated by a certain amount of lymphocytes, but the tracheal wall is not obviously thickened; the mice in the independent immune preF protein group have small amount of lymphocyte infiltration around bronchi, and the bronchus wall is not thickened; few lymphocyte infiltrates around the bronchi of the G+CsA+pref immunized group mice, no thickening of the bronchi wall, and intact alveoli; the lungs of the G+CsA+Fp4 immunized mice have no obvious inflammation and lymphocyte infiltration; the pathology scores of HE staining results are shown in fig. 31, corresponding to the description above, and the lung inflammation and injury levels were found to be low for the g+csa+pref immune group and the g+csa+fp4 immune group compared to the other challenge groups, with scores below 1. The lung slice PAS staining results (FIG. 32) showed that there was a large amount of mucus production around the bronchi in mice in the FI-RSV group; no mucus was produced in the g+csa group; more mucus is present in the panel of immunopraf proteins; and the amount of mucus in the g+csa+pref immunized group is less relative to the immunized preF group alone; there was no mucus produced around the bronchi of the g+csa+fp4 immunized group of mice; the pathological mucus scores of PAS staining results are shown in FIG. 33, corresponding to the descriptions above, and the degree of lung inflammation and injury was found to be better in the G+CsA+Fp4 immunized group than in the G+CsA+pref immunized group, with no mucus production and a score of 0. The total results of HE staining and PAS staining show that the G+CsA+Fp4 immune group has excellent effect, optimal effect of inhibiting the VED reaction of the lung, normal lung tissue, no obvious inflammation and lymphocyte infiltration, and no mucus production around bronchus. Therefore, we believe that the epitope peptide FP4 in the preF protein can well promote g+csa to prevent infection of RSV virus while not eliciting vaccine-induced VED responses.

EXAMPLE 11 CD4 of preF ⁺ T cell epitope peptideImmune response of FP4 to other antigenic proteins

To investigate whether FP4 has the same immunopotentiating effect on other antigen proteins, mice were immunized with 10 μg OVA, 10 μg ova+10 μg FP4, 10 μg ova+100 μg alum, respectively, by means of muscle immunization on day 0 and day 14, blood samples were collected on day 28, and serum was checked for anti-OVA antibody levels by ELISA. The experimental process comprises the following steps: the 96-well plate was incubated with 5. Mu.g/mL OVA overnight at 4 ℃, after PBST washing, blocked with 3% BSA for 1h, serum samples diluted 1:1000 (diluted with 1% BSA) were added, and incubated at 37℃for 1 hour. After washing with PBST, secondary antibody (gold anti-mouse IgG-HRP) was added for 1 hour, and after development with TMB for 10-30 minutes, 50. Mu.L of 2M H was added to each well ₂ SO ₄ The color development was terminated and the absorbance at 450/620nm (OD 450/620) was measured with a microplate reader.

Conclusion: as shown in fig. 34, the antibody level of the FP4+ OVA immune group against OVA was significantly higher than that of OVA alone, indicating that FP4 can significantly promote the immune response of OVA protein, and it can be seen that FP4 can promote not only the immune response of G protein but also the immune response of OVA protein, and thus, unexpectedly, FP4 may have the ability to promote various antigen immune responses, i.e., FP4 has the function of widely enhancing various antigen immune responses.

The results show that the combination of the respiratory syncytial virus G protein and the CD 4T cell epitope peptide FP4 of the preF protein is taken as an antigen, and combined with an immunosuppressant CsA, namely, a G+CsA+F4 vaccine group has a good protection effect on mice, can induce organisms to generate high humoral immune response, and more importantly, the immune effect of the FP4 has consistency with the preF protein, so that the FP4 can even play a better effect than the preF. FP4 not only increases IgG antibody levels against mammalian respiratory syncytial virus-specific G antigen, but also increases IgG antibodies against the G antigen CCD region, which in combination substantially achieve unexpected synergy. The IgG antibody has biological activity and function aiming at the G antigen CCD region, and meanwhile, the FP4 can also improve the level of a neutralizing antibody, can well reduce the pulmonary viral load and inhibit the replication of viruses; and the results of HE staining and PAS staining and slicing of lung tissues show that the G combined CsA and FP4 vaccine group can well inhibit proliferation and infiltration of inflammatory cells in the lung of mammals, reduce damage of respiratory syncytial virus to the lung tissues of the mammals, inhibit the common VED phenomenon of RSV vaccine, and more importantly, the FP4 inhibition VED phenomenon is obviously better than preF protein. Meanwhile, unexpectedly, the polypeptide FP4 can promote the immune response of the G protein and the immune response of the OVA protein, namely, the FP4 has the function of improving the immune response of various antigens in a broad spectrum.

In addition, in the practical application in industry, the epitope peptide FP4 has simpler structure compared with the full length of the preF protein, so that the scheme is simpler and more economical.

In general, the vaccine group combines the advantages of the G protein and the preF protein CD 4T cell epitope peptide FP4 as antigens, improves the immune protection of organisms, has no obvious side effect, and reduces the common VED phenomenon of RSV vaccines. In addition, the polypeptide FP4 has the function of improving the immune response of various antigens in a broad spectrum.

Accordingly, the present invention has developed a respiratory syncytial virus vaccine which can be used for the prevention and/or treatment of diseases caused by respiratory syncytial virus infection. From the known superior effects of this vaccine, it is expected that this vaccine can be fully advanced to the clinic, with the opportunity to fill the gap of RSV vaccines.

While particular embodiments of the present invention have been illustrated and described, it will be appreciated that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Sequence listing

<110> Ai Diwei Xin (Suzhou) biopharmaceutical Co., ltd

Fudan University

<120> respiratory syncytial virus immune composition, preparation method and application thereof

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Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Leu Lys

130 135 140

Thr Thr Lys Lys Asp Pro Lys Pro Gln Thr Thr Lys Ser Lys Glu Val

145 150 155 160

Pro Thr Thr Lys Pro Thr Glu Glu Pro Thr Ile Asn Thr Thr Lys Thr

165 170 175

Asn Ile Ile Thr Thr Leu Leu Thr Ser Asn Thr Thr Gly Asn Pro Glu

180 185 190

Leu Thr Ser Gln Met Glu Thr Phe His Ser Thr Ser Ser Glu Gly Asn

195 200 205

Pro Ser Pro Ser Gln Val Ser Thr Thr Ser Glu Tyr Pro Ser Gln Pro

210 215 220

Ser Ser Pro Pro Asn Thr Pro Arg Gln

225 230

<210> 3

<211> 1572

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atggaactgc tgatcctgaa ggccaacgcc atcaccacca tcctgaccgc cgtgaccttc 60

tgttttgcct ccggccagaa catcaccgaa gagttctacc agtctacctg ctccgccgtg 120

tccaagggat atctgggcgc tctgagaacc ggctggtaca cctctgtgat caccatcgag 180

ctgtccaaca tcaaagaaaa caagtgcaac ggcaccgacg ccaaagtgaa gctgatcaag 240

caagagctgg acaagtacaa gaatgccgtg accgacctgc agctgctgat gcagtctacc 300

cctgctaccg gctctggctc tgctatctgt agcggagtgg ctgtgtgcaa ggtgctgcac 360

ctggaaggcg aagtgaacaa gatcaagagc gccctgctgt ccaccaacaa ggccgtggtg 420

tctctgtcta acggcgtgtc cgtgctgacc tttaaggtgc tggatctgaa gaactacatc 480

gacaaacagc tgctgcccat cctgaacaag cagtcctgca gcatccccaa catcgagaca 540

gtgatcgagt tccagcagaa gaacaaccgg ctgctggaaa tcacccgcga gttctctgtg 600

aatgccggcg tgaccacacc tgtgtccacc tacatgctga ccaactccga gctgctgtcc 660

ctgatcaacg acatgcccat caccaacgac cagaaaaagc tgatgtccaa caacgtgcag 720

atcgtgcggc agcagtccta ctccatcatg tgcattatca aagaagaggt gctggcctac 780

gtggtgcagc tgcctctgta tggcgtgatc gacacccctt gctggaagct gcatacctct 840

ccactgtgca ccaccaacac caaagagggc tccaacatct gcctgaccag aaccgacaga 900

ggctggtact gtgacaacgc cggctccgtc tcattcttcc cacaagccga gacatgcaaa 960

gtgcagtcca accgggtgtt ctgcgacacc atgaactctc ggaccctgcc ttctgaagtg 1020

aacctgtgca acgtggacat cttcaaccct aagtacgact gcaagatcat gacctccaag 1080

accgacgtgt cctccagcgt gatcacctct ctgggagcca tcgtgtcctg ctacggcaag 1140

accaagtgca ccgcctccaa caagaaccgg ggcatcatca agaccttctc caacggctgc 1200

gactacgtca gcaacaaagg cgtggacacc gtgtctgtgg gcaacaccct gtactgcgtg 1260

aacaaacaag agggccagag cctgtacgtg aagggcgagc ccatcatcaa cttctacgac 1320

cctctggtgt tccccagcga cgagttcgat gcctccatca gccaagtgaa cgagaagatc 1380

aaccagtctc tggccttcat ccggaagtcc gatgagctgc tgagcgctat cggcggctat 1440

atccctgagg ctcctagaga tggccaggct tacgtgcgga aggatggcga atgggtgctg 1500

ctgtctacct ttctcggcgg ccaccatcat catcaccact ccgcatggtc ccatcctcag 1560

ttcgagaagt ga 1572

<210> 4

<211> 523

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Gly Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Asp Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Gly Ser Gly Ser Ala Ile Cys Ser Gly

100 105 110

Val Ala Val Cys Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile

115 120 125

Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn

130 135 140

Gly Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn Tyr Ile

145 150 155 160

Asp Lys Gln Leu Leu Pro Ile Leu Asn Lys Gln Ser Cys Ser Ile Pro

165 170 175

Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu

180 185 190

Glu Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val

195 200 205

Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp

210 215 220

Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln

225 230 235 240

Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Cys Ile Ile Lys Glu Glu

245 250 255

Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr

260 265 270

Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys

275 280 285

Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys

290 295 300

Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys

305 310 315 320

Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Arg Thr Leu

325 330 335

Pro Ser Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr

340 345 350

Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile

355 360 365

Thr Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr

370 375 380

Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys

385 390 395 400

Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr

405 410 415

Leu Tyr Cys Val Asn Lys Gln Glu Gly Gln Ser Leu Tyr Val Lys Gly

420 425 430

Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu

435 440 445

Phe Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu

450 455 460

Ala Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser Ala Ile Gly Gly Tyr

465 470 475 480

Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly

485 490 495

Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly His His His His His

500 505 510

His Ser Ala Trp Ser His Pro Gln Phe Glu Lys

515 520

<210> 5

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr

1 5 10

<210> 6

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu Leu Ser

1 5 10 15

<210> 7

<211> 13

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

1 5 10

<210> 8

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn

1 5 10 15

<210> 9

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

1 5 10

<210> 10

<211> 13

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

Val Ser Val Leu Thr Phe Lys Val Leu Asp Leu Lys Asn

1 5 10

Claims (10)

1. An epitope peptide, which is characterized in that the amino acid sequence of the epitope peptide is shown as SEQ ID NO. 8.

2. An immunological composition of respiratory syncytial virus, characterized in that the active ingredient comprises the epitope peptide of claim 1, respiratory syncytial virus G protein and/or cyclosporin a.

3. The respiratory syncytial virus immune composition according to claim 2, wherein the amino acid sequence of the respiratory syncytial virus G protein is shown in SEQ ID No. 2.

4. A respiratory syncytial virus immune composition according to any one of claims 2-3, wherein the mass ratio of respiratory syncytial virus G protein, epitope peptide and cyclosporin a is 1: (0.1-10): (0.1-10), preferably 1: (0.5-1.5): (0.5-1.5).

5. The respiratory syncytial virus immune composition according to any one of claims 2-4, further comprising an adjuvant;

preferably, the adjuvant comprises at least one of a metal ion adjuvant, a TLRs ligand and a cytokine adjuvant;

preferably, the metal ion adjuvant comprises aluminium hydroxide, aluminium phosphate, zinc aluminium adjuvant or manganese adjuvant, more preferably aluminium adjuvant;

preferably, the cytokine adjuvant comprises a chemokine adjuvant;

preferably, the mass ratio of the respiratory syncytial virus G protein, the epitope peptide, the cyclosporin A and the adjuvant is 1: (0.1-10): (0.1-10): (5-20), more preferably 1: (0.5-1.5): (0.5-1.5): (9-11).

6. The respiratory syncytial virus immune composition according to any one of claims 2-5, wherein the respiratory syncytial virus immune composition is in the form of an injection, an oral, a nasal drop, a spray or a transdermal agent.

7. The method for preparing the respiratory syncytial virus immune composition according to any one of claims 2 to 6, wherein the respiratory syncytial virus immune composition is prepared by mixing the respiratory syncytial virus G protein, epitope peptide and cyclosporin a.

8. The preparation method of claim 7, wherein the respiratory syncytial virus immune composition is prepared by combining respiratory syncytial virus G protein, epitope peptide, cyclosporin a and an adjuvant;

Preferably, the adjuvant comprises at least one of a metal ion adjuvant, a TLRs ligand and a cytokine adjuvant;

preferably, the metal ion adjuvant comprises aluminium hydroxide, aluminium phosphate, zinc aluminium adjuvant or manganese adjuvant, more preferably aluminium adjuvant;

preferably, the cytokine adjuvant comprises a chemokine adjuvant;

preferably, the mass ratio of the respiratory syncytial virus G protein, the epitope peptide, the cyclosporin A and the adjuvant is 1: (0.1-10): (0.1-10): (5-20), more preferably 1: (0.5-1.5): (0.5-1.5): (9-11).

9. Use of the epitope peptide of claim 1 or the respiratory syncytial virus immune composition of any one of claims 2-6 for the preparation of a product for the prevention and/or treatment of respiratory syncytial virus infection or a disease caused by respiratory syncytial virus infection.

10. Use of the epitope peptide of claim 1 or the respiratory syncytial virus immune composition of any one of claims 2-6 in (1) - (4) as follows:

(1) Preparing a product that increases the level of antibodies specific for respiratory syncytial virus in a mammal;

(2) Preparing a product that reduces the viral load of a mammalian respiratory syncytial virus;

(3) Preparing a product that inhibits proliferation and infiltration of inflammatory cells in the lung of a mammal;

(4) A product is prepared that reduces damage to mammalian lung tissue from respiratory syncytial virus.