Vaccine for preventing and / or treating respiratory syncytial virus

Abstract

The invention belongs to the technical field of medicine and biology, and particularly relates to a vaccine for preventing and / or treating respiratory syncytial virus. Respiratory syncytial virus Pre F protein is selected as a skeleton, and the C end of the skeleton is connected with a G protein CCD part fragment. According to the invention, double neutralizing antibodies of RSV preF protein and G protein can be generated at the same time, and double effects of preventing viruses from infecting a host and preventing residual viruses from spreading again are achieved. Further, different adjuvants are utilized to improve humoral immune response and reduce vaccine enhanced pathological injury (VED) at the same time. Therefore, the invention has practical value in preventing or treating respiratory syncytial disease virus infection.

Description

Technical Field

[0001] The present invention belongs to the fields of medicine and biotechnology, and particularly relates to a vaccine for preventing and / or treating respiratory syncytial virus. Background Art

[0002] Respiratory syncytial virus (RSV) is a highly contagious virus that spreads through airborne droplets and close contact. Although the virus is easily infective in non-human primates and may cause severe diseases, humans are the only natural host of RSV, which is mostly seen in newborns and infants within 6 months, with the highest incidence rate in infants aged 2 - 3 months. It is the most important pathogen causing viral acute lower respiratory diseases in infants worldwide. RSV belongs to the Paramyxoviridae family and the Pneumovirus genus, and is a negative-strand RNA virus. The entire genome of RSV can encode up to 10 viral proteins, including three transmembrane glycoproteins: the G protein that plays an adhesion role, the F protein that plays a fusion role, and the small hydrophobic protein SH protein.

[0003] The initial research on RSV vaccines started in the 1960s. Researchers used formalin-inactivated RSV (FI-RSV) as a vaccine to inoculate infants. Subsequently, observations found that infants inoculated with FI-RSV were more likely to be reinfected with RSV compared to those not inoculated with FI-RSV. Moreover, after being reinfected with RSV, infants inoculated with FI-RSV not only did not receive immune protection from the vaccine, but instead showed more severe infection conditions, and the aggravated conditions led to the death of the infants. Therefore, the failure of RSV vaccine research has revealed two major problems that need to be overcome: Firstly, eliminating the overly strong immune response of the body induced by the immunogen and avoiding the aggravation of the disease (VED) caused by the immune vaccine due to inflammatory cell infiltration; Secondly, the vaccine can stimulate the body to produce a sufficient amount of virus-neutralizing antibodies, so as to protect the body when facing RSV infection again.

[0004] Currently, the research antigens of RSV vaccines are based on the selection of the F (Fusion) protein. According to different conformations, the F protein is divided into the pre-fusion F protein (Pre F) and the post-fusion F protein (Post F). Research has shown that the level of neutralizing antibodies induced by the Pre F protein is much higher than that of the Post F. During the development of the F protein vaccine, it was found that the structural stability of the Pre F recombinant protein is not high. In addition, there is also the G protein on the surface of the RSV virus envelope, which is also a membrane protein related to virus infection. The main receptor-binding region is located in the central conserved, unglycosylated cysteine-rich domain (CCD), and this cysteine-rich domain is stabilized by a pair of disulfide bonds. The RSV virus mediates the adhesion of cells and the virus by binding the G protein to the cell surface receptor CX3CR1. At the same time, the central conserved region (CCD) has a high homology in the two subtypes, and this region can induce high neutralizing antibodies and can significantly reduce the symptoms of weight loss and pulmonary pathological damage caused by virus infection. Non-clinical studies using monoclonal antibodies targeting the CCD amino acid sequence of the G protein have shown effectiveness as a post-infection treatment. In a mouse model using the RSV strain A2, the monoclonal antibody (131-2G) of the murine anti-G protein CCD antibody administered on the 3rd day after infection can effectively reduce the entry of inflammatory cells into the respiratory tract.

[0005] Based on the above research results and the characteristics of the virus, it is considered internationally that a safe and effective RSV vaccine should simultaneously possess the following two characteristics: ① activating a high level of humoral immune response, ② inhibiting pathological T cell immune response to eliminate VED. Only in this way can it effectively prevent and control RSV infection while avoiding serious adverse reactions caused by the vaccine. For decades, many well-known research institutions and enterprises internationally have successively carried out a lot of work in this field, but there is still a need to develop more effective vaccines, such as vaccines that can improve immunity while reducing pathological damage. Summary of the Invention

[0006] Based on retaining the F protein antigen, the present invention further combines the design of the central conserved domain of the G protein, and uses different adjuvants to improve the humoral immune response while reducing pathological damage. In view of this, the present invention is specifically proposed.

[0007] The present invention provides a vaccine antigen for preventing and / or treating respiratory syncytial virus, which is obtained by selecting the Pre F protein of respiratory syncytial virus as the backbone and connecting a partial fragment of the CCD of the G protein to its C-terminus.

[0008] Preferably, it selects the first 513-574 amino acids at the N-terminus of the Pre F protein of respiratory syncytial virus as the backbone, and more preferably selects the first 513 amino acids at the N-terminus of the Pre F protein of respiratory syncytial virus, that is, the first to 513 amino acids, as the backbone.

[0009] More preferably, the G protein CCD partial fragment is the fragment of amino acids 158 - 191 of the G protein.

[0010] In a preferred embodiment, the amino acid sequence of the N-terminal 1 - 513 amino acids of the respiratory syncytial virus Pre F protein is as shown in SEQ ID NO: 1, and the amino acid sequence of the G protein CCD partial fragment is as shown in SEQ ID NO: 2.

[0011] Further preferably, the C-terminus of the scaffold is connected to the G protein CCD partial fragment through a Foldon trimer and a linker peptide. Preferably, the amino acid sequence of the Foldon trimer is as shown in SEQ ID NO: 3, and the amino acid sequence of the linker peptide is as shown in SEQ ID NO: 4.

[0012] Furthermore, it also includes a purification tag sequence, such as a histidine His tag. Specifically, 6 histidines are added to the C-terminus of the G protein CCD partial fragment through a GS linker peptide. Preferably, the amino acid sequence of the vaccine antigen is as shown in SEQ ID NO: 5.

[0013] The present invention also provides the coding nucleic acid of the above-mentioned vaccine antigen.

[0014] The present invention further provides an expression vector and a recombinant host cell containing the above-mentioned coding nucleic acid.

[0015] The present invention also provides the application of the above-mentioned vaccine antigen in the preparation of a vaccine for preventing and / or treating respiratory syncytial virus. Preferably, the vaccine is obtained by inoculating the vaccine antigen into the muscle or mucosa of an animal.

[0016] The present invention additionally provides an antigen composition composed of the above-mentioned vaccine antigen as an active ingredient and an adjuvant. Preferably, the adjuvant is Alum, CsA, MF59, or a combination of Alum and CsA.

[0017] By further combining the design of the central conserved domain of the G protein on the basis of retaining the F protein antigen, while retaining the pre-fusion trimeric structural conformation of preF and fully inducing neutralizing antibodies against the native RSV F protein, the central conserved domain of the RSV G protein is expressed in a triple form, thereby efficiently inducing the production of neutralizing antibodies against the adhesion site of the RSV G protein and the receptor cell. Based on this, the present invention can simultaneously produce dual neutralizing antibodies against the RSV preF protein and the G protein, with the dual effects of preventing the virus from infecting the host and avoiding the re-spread of the residual virus. Further, different adjuvants are used to enhance the humoral immune response while reducing pathological damage. Therefore, the present invention has practical value in preventing or treating respiratory syncytial virus infection.

[0018] Drawings of the specification

[0019] Figure 1 Serum specific antibody IgG levels against Pre F5 antigen after immunization with the Pre F5 and Pre F5-CCD recombinant protein candidate vaccines provided for Example 3;

[0020] Figure 2 Serum specific antibody IgG levels against G-CCD antigen after immunization with the Pre F5-CCD recombinant protein candidate vaccine provided for Example 3;

[0021] Figure 3 Specific antibody IgA levels against Pre F5 antigen in genital tract lavage fluid after immunization with the Pre F5 and Pre F5-CCD recombinant protein candidate vaccines provided for Example 3;

[0022] Figure 4 Serum neutralizing antibody levels after immunization with the PreF5 and Pre F5-CCD recombinant protein candidate vaccines provided for Example 4;

[0023] Figure 5 Lung viral load in mice after RSV virus infection provided for Example 5;

[0024] Figure 6 HE-stained pathological sections and pathological scores of lung tissues in mice after RSV virus infection provided for Example 5;

[0025] Figure 7 PAS-stained pathological sections and pathological scores of lung tissues in mice after RSV virus infection provided for Example 5. Detailed implementation manners

[0026] The concept, specific structure and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments and drawings, so as to fully understand the purpose, scheme and effects of the present invention. It should be noted that the features in the embodiments of the present application can be combined with each other without conflict.

[0027] The present invention will be further described below in conjunction with the drawings.

[0028] Example 1 Preparation of vaccine antigen components

[0029] 1. Obtaining of RSV Pre F5-CCD protein

[0030] The RSV Pre F5-CCD recombinant protein vaccine in the present invention selects the amino acids 1-513 of the respiratory syncytial virus Pre F protein as the backbone and obtains a polypeptide fragment with the amino acid sequence shown in SEQ ID NO: 1 after deletion mutation.

[0031] Among them, the deletion mutation makes the segment between amino acid positions 104 and 144 of the natural F protein into the mature F protein, and at the same time, 4-site mutations are carried out, namely S155C, S290C, A149C, and Y458C to form stable disulfide bonds, so as to maintain the pre-fusion conformation form of the F protein; and mutations at positions S46G and K465Q are carried out to reduce the charge repulsion at the trimer interface of the F protein trimer and further maintain the stabilized conformation.

[0032] And the partial fragment of the G protein CCD (amino acids 158-191, the amino acid sequence is shown in SEQ ID NO: 2) is connected to the C-terminus of the Pre F protein through Foldon trimer (SEQ ID NO: 3) and Linker with the amino acid residue composition of GGGSGGGSS (SEQ ID NO: 4), and then 6 histidines His are added through the GS linker at the end of SEQ ID NO: 2, thereby constructing the amino acid sequence of the Pre F-CCD protein (SEQ ID NO: 5), denoted as Pre F5-CCD. SEQ ID NO.5 was entrusted to GenScript Biotech Corporation in Suzhou for synthesis, transient expression and purification to obtain the required Pre F5-CCD protein.

[0033] 2. Obtaining of RSV Pre F5 protein

[0034] The amino acid sequence of the respiratory syncytial virus Pre F5 protein described in the present invention is the sequence obtained by connecting SEQ ID NO: 1 and SEQ ID NO: 3 as shown in SEQ ID NO: 6. SEQ ID NO.6 was entrusted to Nanjing Genscript Biotech Co., Ltd. for expression and purification to obtain the required Pre F5 protein.

[0035] The relevant sequence information is as follows:

[0036] Amino acid sequence of PreF protein (SEQ ID NO: 1): MELLILKANAITTILTAVTFCFASGQNITEEF YQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGQSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL。

[0037] Amino acid sequence of the CCD region of G protein (SEQ ID NO: 2): KPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPN。

[0038] Amino acid sequence of Foldon trimer (SEQ ID NO: 3): SAIGGYIPEAPRDGQAYVRKDGEWVLL STFL。

[0039] Amino acid sequence of the linker peptide (SEQ ID NO: 4): GGGSGGGSS。

[0040] Amino acid sequence of PreF-CCD protein (SEQ ID NO:5): MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGQSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGGSGGGSSKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNGSHHHHHH。

[0041] PreF5 protein sequence (SEQ ID NO: 6): MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGQSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL。

[0042] Example 2 Animal Immunization

[0043] 1. The experimental animals were 6 - 8 - week - old female BALB / c mice. The day of the first immunization of the mice was defined as day 0, the day before immunization was - 1 day, and the day after immunization was day 1, and so on. The immunization groups and reagent dosages are shown in Table 1. The mice were immunized on days 0 and 14 respectively, and each mouse was immunized with 100 μL each time.

[0044] 2. Blood was collected from the orbital venous plexus on days 7, 14, 21, and 26, 100 - 200 μL of blood each time. The whole blood was allowed to stand at 37°C for 2 hours or stand overnight at 2 - 8°C. Centrifuge at 4000 rpm for 10 min at 2 - 8°C to separate the serum, and store it at - 20°C for the detection of specific antibodies in the serum. The challenge time was 28 days after the primary immunization, and the viral load and pathological detection were 5 days after the challenge (33 days after the primary immunization).

[0045] Table 1 Immunization groups, administration methods, and administration doses

[0046]

[0047] Note: i.m.: intramuscular injection; i.n.: intranasal instillation; i.na : Nasal delivery of an atomization drug delivery device.

[0048] Example 3 Detection of antigen-specific antibodies

[0049] To evaluate the effects of the vaccines prepared in different groups in Example 2 on the humoral immune response of mice, the titers of antigen (Pre F5, G-CCD polypeptide)-specific antibodies in the sera of mice at different times after immunization were detected by relative quantitative ELISA. The specific experimental method is as follows:

[0050] Coating: Dilute the antigen with antigen coating solution, coat 1 μg / mL PreF protein or 1 μg / mL Biotin-RSV G162-195 polypeptide + 1 μg / mL streptavidin in a 96-well plate, add 100 μL per well to the 96-well ELISA plate, and incubate overnight at 2-8 °C. Blocking: Discard the coating solution, wash 3 times with PBST, add 150 μL per well of 5% (v / v) BSA to block for 1 h. Incubation with primary antibody (test serum): Discard the blocking solution, wash the plate 3 times with PBST, serially dilute the test serum 11 times with 3% (v / v) BSA in 3-fold increments, add 100 μL per well, and incubate at 37 °C for 1 h. Incubation with secondary antibody: Discard the test solution, wash the plate 3 times with PBST, dilute the secondary antibody (Goat anti-mouse IgG-HRP, Abcam, Ab6789, 1:20000) with 3% (v / v) BSA, add 100 μL per well, and incubate at 37 °C for 1 h. Color development: Discard the secondary antibody, wash 6 times with PBST, add 100 μL of TMB color development solution per well, and develop color at room temperature for 10 min (turns blue after color development). Termination: Add 50 μL of 2 M H2SO4 per well to terminate color development (turns bright yellow after termination of color development). Detect and read the absorbance (A450 - A620) with an ELISA reader within 10 min.

[0051] Conclusion: The experimental results showed that in the candidate vaccine intramuscular immunization group of mice, on the 7th and 14th days after the primary immunization, the immunization with PreF5 protein alone could induce the production of PreF protein-specific antibodies (no significant difference compared with the PBS group), the level of PreF5-specific antibodies in the sera of the Pre F5 protein combined with Alum or MF59 adjuvant groups was significantly higher than that in the PreF5 protein alone immunization group, and there was no significant difference in the level of PreF5-specific binding antibodies between the Pre F5 + Alum and Pre F5 + MF59 groups ( Figure 1(A - B). On the 7th and 12th days after the second immunization (Day 21 and 26 after the primary immunization), the levels of PreF5 - specific antibodies in the sera of the PreF5 protein combined with Alum and MF59 adjuvant groups were significantly higher than those in the PreF5 protein alone - immunized group, and on the 7th day after the second immunization, the serum antibody level of the mice in the PreF5+MF59 group was significantly higher than that in the PreF5+Alum group (p < 0.05, Figure 1 in C and D).

[0052] In the muscle - immunized group of candidate vaccines, on the 7th and 14th days after the primary immunization, the PreF5 - CCD protein alone immunization could induce the production of PreF protein - specific and G - CCD polypeptide - specific binding antibodies; on the 7th and 14th days after the primary immunization, the levels of PreF5 - specific binding antibodies and G - CCD polypeptide - specific binding antibodies in the PreF5 - CCD protein combined with Alum, Alum+CsA, and MF59 adjuvant groups were significantly higher than those in the PreF5 - CCD protein alone - immunized group, and there was no significant difference in the binding antibody levels among the PreF5 - CCD protein combined with Alum, Alum+CsA, and MF59 adjuvant groups (p < 0.01, Figure 1 in A and B, Figure 2 in A and B). On the 7th and 14th days after the primary immunization, the level of PreF5 - specific antibodies in the sera of the mice in the PreF5 - CCD+CsA group was significantly lower than that in the PreF5 - CCD+Alum group ( Figure 1 in A and B). On the 14th day after the primary immunization, the level of G - CCD polypeptide - specific antibodies in the sera of the mice in the PreF5 - CCD+CsA group was significantly lower than that in the PreF5 - CCD+Alum group ( Figure 2 in B). On the 7th and 12th days after the second immunization (Day 21, 26), the level of PreF5 - specific antibodies in the sera of the mice in the PreF5 - CCD protein alone - immunized group was significantly higher than that in the PBS control group ( Figure 1 in C and D). On the 7th and 12th days after the second immunization (Day 21, 26), the levels of PreF5 - specific binding antibodies and G - CCD polypeptide - specific binding antibodies in the PreF5 - CCD protein combined with Alum, Alum+CsA, and MF59 adjuvant groups were significantly higher than those in the PreF5 - CCD protein - immunized group. On the 7th day after the second immunization, the level of PreF5 - specific binding antibodies in the sera of the mice in the PreF5 - CCD+MF59 group was significantly higher than that in the PreF5 - CCD+Alum ( Figure 1 in C and D, Figure 2 in A and B). On the 7th and 12th days after the second immunization (Day 21, 26), the level of PreF5 - specific binding antibodies in the sera of the mice in the PreF5 - CCD+CsA group was significantly lower than that in the PreF5 - CCD+Alum group ( Figure 1 in C and D).

[0053] The results of the muscle immunization experiment indicated that the candidate vaccines formulated with PreF5 protein in combination with Alum or MF59 adjuvant could significantly enhance the specific humoral immune level of PreF5 protein in the immunized animals. The candidate vaccines formulated with PreF5-CCD protein in combination with Alum, Alum + CsA or MF59 adjuvant could also significantly enhance the specific humoral immune level of PreF5-CCD protein.

[0054] Meanwhile, the experimental results showed that in the mucosal immunization groups of the candidate vaccines, on the 7th and 14th days after the primary immunization and on the 7th and 12th days after the second immunization, the PreF5 protein alone immunization group failed to induce the production of PreF5 protein-specific antibodies, suggesting that mucosal immunization with PreF5 protein could not induce effective humoral immunity ( Figure 1 ). On the 14th day after the primary immunization and on the 7th and 12th days after the second immunization, the levels of PreF5-specific antibodies in the sera of the mice in the Pre F5 + MF59 intranasal immunization group were significantly higher than those in the PreF5 protein immunization group (p < 0.01, p < 0.05, p < 0.05, see Figure 1 B, C, D in it). On the 7th day after the second immunization, the level of PreF5-specific antibodies in the sera of the mice in the Pre F5 + MF59 nasal spray immunization group was significantly higher than that in the PreF5 protein immunization group (p < 0.01, see Figure 1 C in it). On the 7th and 14th days after the primary immunization and on the 7th and 12th days after the second immunization, there were no significant differences in the levels of PreF5-specific antibodies in the sera of the mice in the Pre F5 + MF59 intranasal immunization group and the PreF5 + MF59 nasal spray immunization group (see Figure 1 A, B, C, D in it).

[0055] In the mucosal immunization groups of the candidate vaccines, on the 7th and 14th days after the primary immunization and on the 7th and 12th days after the second immunization, the PreF5-CCD protein alone immunization group failed to produce antibodies specific to PreF5 protein and G-CCD polypeptide, suggesting that mucosal immunization with PreF5-CCD protein could not effectively induce specific humoral immunity. On the 14th day after the primary immunization and on the 12th day after the second immunization, the levels of PreF5-specific antibodies in the sera of the mice in the Pre F5-CCD + MF59 intranasal immunization group were significantly higher than those in the PreF5-CCD protein immunization group (p < 0.01, p < 0.05, see Figure 1 B and D in it). On the 12th day after the second immunization, the level of PreF5-specific antibodies in the sera of the mice in the Pre F5-CCD + MF59 nasal spray immunization group was significantly higher than that in the PreF5-CCD protein immunization group (p < 0.05, see Figure 1In D). On the 7th and 14th days after the primary immunization, and on the 7th and 12th days after the secondary immunization, there were no significant differences in the levels of PreF5-specific antibodies in the sera of mice in the Pre F5-CCD+MF59 intranasal immunization group and the Pre F5-CCD+MF59 nasal spray nebulization immunization group ( Figure 1 In A, B, C, D).

[0056] Meanwhile, the levels of IgA antibodies in the genital tract lavage fluid of mice were detected. In the candidate vaccine mucosal immunization group of mice, on the 12th day after the secondary immunization, high levels of PreF5-specific IgA antibodies were produced in the genital tract lavage fluid of mice in the PreF5-CCD+MF59 intranasal immunization group and the nasal spray nebulization group. Figure 3 )

[0057] The results of the mucosal immunization experiment suggest that when the candidate vaccine is used to immunize mice mucosally, the MF59 adjuvant can significantly improve the humoral immune levels of PreF5 and PreF5-CCD proteins. Both intranasal immunization and nasal spray nebulization immunization methods are applicable to mucosal immunization.

[0058] Example 4 Detection of neutralizing antibodies in the sera of mice after immunization with the candidate vaccine

[0059] To determine the levels of neutralizing antibodies in the sera of mice immunized with the candidate vaccine prepared in Example 2, this study used a virus micro-neutralization assay to detect the sera on the 12th day (Day 26) after the secondary immunization.

[0060] Specific experimental procedure: Trypsinize HEp-2 cells and dilute them with DMEM medium containing 4% (volume ratio) fetal bovine serum (FBS) to 2×10 4 cells / 100 μL and add them to a 96-well cell culture plate, and incubate overnight in a carbon dioxide incubator. The next day, take the sera on the 26th day after the primary immunization. After inactivating the sera to be tested at 56 °C in a water bath for 30 min, dilute them with serum-free DMEM medium to (volume ratio) 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024. After diluting 6 gradients, mix the sera to be tested with RSV A2 virus (100 TCID 50)Mix and spread them in a 96-well plate at 37 °C, and place them at room temperature for 1 h. At the same time, set up virus control wells and cell control wells. Add the mixed solution of serum and virus solution to the 96-well plate coated with Hep-2 cells, 100 μL per well, with two replicates for each dilution. After 5 days, discard the culture medium in the 96-well plate, fix the cells with pre-cooled 80% acetone, and then block the cells with 5% (volume ratio) BSA. After washing, add rabbit anti-Pre F IgG to the wells and incubate at 37 °C for 1 h. After washing, add Goat anti-rabbit IgG-HRP for color development, and detect the readings (A450 / A620) with an enzyme-linked immunosorbent assay reader. And calculate according to the following formula:

[0061] (OD value of virus control well - OD value of the sample to be tested well) / (OD value of virus control well - OD value of cell control well) * 100 > 50%, indicating that the serum at this dilution has neutralizing activity.

[0062] Conclusion: The experimental results showed that in the candidate vaccine intramuscular immunization groups of PreF5+MF59 and PreF5+Alum, mice produced high levels of neutralizing antibodies, and there was no significant difference in the antibody levels between the two groups. Among them, the serum neutralizing antibody level of the PreF5+MF59 group was significantly higher than that of the PreF5 protein immunization group ( Figure 4 ).

[0063] In the candidate vaccine intramuscular immunization groups of PreF5-CCD+MF59, PreF5-CCD+Alum, and PreF5-CCD+Alum+CsA, the serum neutralizing antibody levels of mice were significantly higher than those of the PreF5-CCD protein immunization group, and there was no significant difference in the antibody levels among these three groups of mice. The serum neutralizing antibody level of the PreF5-CCD+CsA group was significantly lower than that of the PreF5-CCD+Alum+CsA and PreF5-CCD+Alum groups ( Figure 4 ).

[0064] In the candidate vaccine mucosal immunization group of PreF5+MF59, the serum neutralizing antibody level of mice was significantly higher than that of the PreF5 protein immunization group, and there was no significant difference compared with the intramuscular immunization group ( Figure 4 ).

[0065] The experimental results suggest that MF59 and Alum can significantly increase the serum neutralizing antibody level of mice immunized with PreF5 protein. MF59, Alum, and Alum+CsA can all significantly increase the serum neutralizing antibody level of mice immunized with PreF5-CCD protein.

[0066] Example 5 Protective effect of the candidate vaccine on mice against RSV infection

[0067] To confirm the protective effect of the candidate vaccine on mice during RSV challenge after immunization. In this example, on the 14th day (Day28) after the second immunization with the candidate vaccine, the mice were challenged with RSV, and the changes in body weight of the mice 5 days after the challenge, the viral load in the lungs 5 days later, and the pathological changes in the lungs were detected. At 28 days after the primary immunization, the experimental mice were anesthetized by inhalation of isoflurane. 50 μL of the virus solution (5×10 6 PFU) was aspirated with a pipette and slowly dropped into the nasal cavity of the anesthetized experimental mice. After the experimental animals completely inhaled autonomously, the experimental mice were placed horizontally until they woke up and then the remaining experiments were continued.

[0068] 1. Detection of viral load in the lungs of mice after RSV challenge

[0069] Experimental procedure: 5 days after the challenge, the mice were euthanized. The chest cavity was opened with surgical scissors, the lungs were removed and weighed, 300 μL of sterile PBS was added, and then homogenized with a tissue homogenizer at 60 Hz for 90 s and aliquoted at 100 μL / tube. One tube was taken out, centrifuged at 12,000 rpm for 1 min, the supernatant was aspirated, 100 μL of sterile PBS was added, and the SteadyPure Viral DNA / RNA Extraction Kit was used to extract the viral RNA from the homogenate according to the instructions. The cDNA Transcription Kit Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) was used to synthesize cDNA from the extracted RNA. According to the instructions, genomic DNA was removed at 42 °C for 2 min, and then synthesis was carried out under the conditions of 25 °C for 5 min, 42 °C for 30 min, and 85 °C for 5 min; then the qPCR Detection Kit Hieff qPCR SYBR Green Master Mix (No Rox) was used for real-time quantitative PCR. The PCR reaction conditions were: 95 °C for 5 min for 1 cycle, 95 °C for 10 s, 58 °C for 20 s, 72 °C for 20 s, for 40 cycles. At the same time, a plasmid containing the RSV N gene was used as a standard to calculate the content of the RSV N gene in the cDNA of the test sample. The primers used were: forward primer: 5’-CATCCAGCAAATACACCATCCA-3’ (SEQ ID No: 7); reverse primer: 5’-TTCTGCACATCATAATTAGGAGTATCAA-3’ (SEQ ID No: 8).

[0070] Conclusion: The experimental results showed that 5 days after the challenge, the viral load in the lungs of the mice in the PBS challenge group was significantly higher than that in the PBS non-challenged group (p < 0.01, Figure 5 ).

[0071] Compared with the PBS challenge group, the viral loads in the lungs of mice in the PreF5, PreF5+Alum, and PreF5+MF59 groups immunized with the candidate vaccine by intramuscular injection were significantly reduced. The viral loads in the lungs of mice in the PreF5-CCD, PreF5-CCD+Alum, PreF5-CCD+CsA, PreF5-CCD+Alum+CsA, and PreF5-CCD+MF59 groups were significantly reduced, and there were no significant differences in the viral loads among the groups( Figure 5 ).

[0072] Compared with the PBS challenge group, the viral loads in the lungs of mice in the PreF5, PreF5+MF59 intranasal immunization group, and PreF5+MF59 nasal spray atomization group of the candidate vaccine mucosal immunization group were significantly reduced. The viral loads in the lungs of mice in the PreF5-CCD, PreF5-CCD+MF59 intranasal immunization group, and PreF5-CCD+MF59 nasal spray atomization group were significantly reduced, and there were no significant differences in the viral loads among the groups (see Figure 5 ).

[0073] In addition, the viral loads in the lungs of mice in the muscle immunization groups of PreF5 and PreF5-CCD proteins combined with various adjuvants were significantly lower than those in the mucosal immunization groups. The viral load in the lungs of mice in the PreF5-CCD+MF59 muscle immunization group was significantly lower than that in the mucosal immunization group.

[0074] 2. Pathological changes in the lung tissues of mice after RSV challenge

[0075] To confirm whether the candidate vaccine would cause the VED phenomenon in the mouse challenge protection experiment, the lung tissues of mice 5 days after challenge were fixed with 4% paraformaldehyde at room temperature for 24 h and then embedded in paraffin. After sectioning, they were stained with hematoxylin and eosin (HE) or periodic acid-Schiff (PAS), photographed and observed, and the degree of lung inflammation and injury was evaluated by pathological scoring.

[0076] Conclusion: The results of pathological sections showed that severe pathological changes occurred in the lung tissues of mice in the FI-RSV immunization group after challenge, manifested as a large number of inflammatory cell infiltrations around the bronchi and thickening of the bronchial walls. The pathological score of the lung tissues of this group of mice was significantly higher than that of the PBS non-challenged group (p<0.001, Figure 6In mice immunized with the PreF5 protein candidate vaccine, the lung tissues of mice in the PreF5+MF59 intramuscular immunization group were similar to those in the FI-RSV group, with infiltration of inflammatory cells around the bronchi and accompanied by thickening of the bronchial wall. In the PreF5, PreF5+Alum intramuscular immunization groups, the PreF5+MF59 intranasal immunization and nasal spray atomization immunization groups, the inflammatory lesions in the lung tissues of mice were significantly improved compared with the FI-RSV immunization group, the infiltrating inflammatory cells were significantly reduced, the tracheal wall was significantly thinned and smooth, and their pathological scores were significantly lower than those of the FI-RSV immunization group (p<0.01, p<0.05, p<0.05, see Figure 6 in A, B).

[0077] In mice immunized with the PreF5-CCD protein candidate vaccine, there was infiltration of inflammatory cells to varying degrees around the bronchi in the lung tissues of mice in the PreF5-CCD, PreF5-CCD+MF59, PreF5-CCD+Alum, PreF5-CCD+CsA intramuscular immunization groups. In the PreF5-CCD+MF59 intranasal immunization and nasal spray atomization immunization groups, and the PreF5-CCD+Alum+CsA intramuscular immunization group, the inflammatory lesions in the lung tissues of mice were significantly weaker than those in the FI-RSV immunization group, and their pathological scores were significantly lower than those of the FI-RSV immunization group (p<0.01, Figure 6 in A, B).

[0078] The pathological changes in the lung tissues of mice were further observed by PAS staining. The results showed that there was a large amount of positive accumulation of PAS staining on the inner wall of the bronchi in the lung tissues of mice in the FI-RSV immunization group after virus challenge, and the pathological score of the lung tissues of this group of mice was significantly higher than that of the PBS non-challenged group (p<0.01, see Figure 7 in A, B). In mice immunized with the PreF5 protein candidate vaccine, the lung tissues of mice in the PreF5+MF59, PreF5+Alum intramuscular immunization groups were similar to those in the FI-RSV group, with a large amount of positive signals of PAS staining on the inner wall of the bronchi, and there was no significant difference in the pathological scores between these two groups and the FI-RSV group. In the PreF5+MF59 intranasal immunization group, the positive PAS staining on the inner wall of the bronchi in the lung tissues of mice was reduced, approaching that of the PBS challenged group, and its pathological score was significantly lower than that of the FI-RSV immunization group (p<0.001, Figure 7 in A, B). In mice immunized with the PreF5-CCD protein candidate vaccine, the positive signal of PAS staining on the inner wall of the bronchi in the lung tissues of mice in the PreF5-CCD+MF59 intramuscular immunization group was reduced, approaching that of the PBS challenged group, and its pathological score was significantly lower than that of the FI-RSV immunization group (p<0.05, see Figure 7 in A, B).

[0079] Combined with the antibody level, the results of the pulmonary viral load, and the results of HE pathological sections, it was shown that MF59 combined with PreF5 and PreF5-CCD protein for intramuscular immunization could enhance the humoral immune level induced by the protein in mice, reduce the viral load in the lungs of mice during RSV infection, but might not significantly reduce the pulmonary inflammatory response. When MF59 was combined with PreF5 and PreF5-CCD protein for mucosal immunization, it could enhance the humoral immune level induced by the protein in mice, reduce the viral load in the lungs of mice during RSV infection, but would not cause pulmonary inflammatory response. The experimental results suggested that MF59 adjuvant was more suitable for mucosal immunization.

[0080] In addition, both Alum and Alum+CSA combined with PreF5-CCD protein for intramuscular immunization could enhance the humoral immune level induced by the protein in mice and reduce the viral load in the lungs of mice during RSV infection. However, the pulmonary inflammatory response in the Alum+CsA combined with PreF5-CCD group was significantly lower than that in the Alum adjuvant group (p<0.01, Figure 6 in A, B), and the experimental results suggested that the Alum+CsA adjuvant had a better effect than the Alum adjuvant.

Claims

1. A vaccine antigen for preventing and / or treating respiratory syncytial virus, characterized in that, It is obtained by selecting the Respiratory Syncytial Virus (RSV) Pre F protein as the backbone and connecting the CCD partial fragment of the G protein to its C-terminus.

2. The vaccine antigen according to claim 1, characterized in that, It is selected with the first 513 - 574 amino acids at the N-terminus of the RSV Pre F protein as the backbone, preferably with the first 513 amino acids at the N-terminus of the RSV Pre F protein, i.e., the 1st - 513th amino acids, as the backbone. Further preferably, the following mutations are also made to the F protein: a deletion mutation at positions 104 to 144, and mutations at positions S155C, S290C, A149C, Y458C, S46G, and K465Q simultaneously.

3. The vaccine antigen according to claim 2, characterized in that, The CCD partial fragment of the G protein is the fragment of the 158th - 191st amino acids of the G protein.

4. The vaccine antigen according to claim 3, characterized in that, The amino acid sequence of the 1st - 513th amino acids at the N-terminus of the RSV Pre F protein is as shown in SEQ ID NO: 1, and the amino acid sequence of the CCD partial fragment of the G protein is as shown in SEQ ID NO:

2.

5. The vaccine antigen according to claim 3, characterized in that, The C-terminus of the backbone is connected to the CCD partial fragment of the G protein through a Foldon trimer and a linker peptide. Preferably, the amino acid sequence of the Foldon trimer is as shown in SEQ ID NO: 3, and the amino acid sequence of the linker peptide is as shown in SEQ ID NO:

4.

6. The vaccine antigen according to any one of claims 1 to 5, characterized in that, It also includes a purification tag sequence, such as a histidine His tag. Specifically, 6 histidines are added to the C-terminus of the CCD partial fragment of the G protein through a GS linker peptide. Preferably, the amino acid sequence of the vaccine antigen is as shown in SEQ ID NO:

5.

7. A coding nucleic acid of the vaccine antigen according to any one of claims 1 to 6.

8. An expression vector and a recombinant host cell containing the coding nucleic acid according to claim 7.

9. Use of the vaccine antigen according to any one of claims 1 to 6 in the preparation of a vaccine for preventing and / or treating respiratory syncytial virus, preferably, the vaccine is obtained by inoculating the vaccine antigen into the muscle or mucosa of an animal.

10. An antigen composition comprising the vaccine antigen according to any one of claims 1 to 6 as an active ingredient and an adjuvant, preferably, the adjuvant is Alum, CsA, MF59, or a combination of Alum and CsA.

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