A recombinant antigen combining respiratory syncytial virus and influenza A virus and its application

Abstract

This invention belongs to the field of biomedicine, specifically relating to a recombinant antigen combining respiratory syncytial virus (RSV) and influenza A virus, and its application. Based on the F protein of RSVA subtype strains and the HA protein of influenza virus H1N1, this invention obtains a recombinant F protein sequence by removing the post-F-specific I and IV epitope sequences and the shared III epitope sequence between pre-F and post-F. This recombinant F protein then replaces the head region of the influenza virus HA protein, with the two sides connected by linkers to construct an F-HA chimeric protein. It can maintain the pre-F trimer conformation in vivo and in vitro, thereby stimulating the body to produce antibodies specific to the pre-F epitope and possessing high neutralizing activity. Simultaneously, the presence of the stem region of the influenza HA protein provides protection against influenza virus infection. The recombinant antigen of this invention has the advantages of stimulating the body to produce antibodies against both viruses and has the advantage of high yield.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a recombinant antigen of respiratory syncytial virus and influenza A virus and its application. Background Technology

[0002] Respiratory syncytial virus (RSV) is one of the most common pathogens causing lower respiratory tract infections in infants and young children worldwide. Almost all children will be infected with RSV at least once before the age of two. RSV can induce diseases such as pneumonia or bronchitis, and in severe cases, even death. Furthermore, in immunocompromised and weakened elderly populations, RSV infection can cause severe lower respiratory tract clinical symptoms and significantly increase the risk of death. Influenza virus (IV) can also induce severe respiratory illnesses. Based on the differences between its nucleoprotein and matrix protein, influenza viruses can be divided into four types: A, B, C, and D. Influenza A virus (IAV) is the dominant type circulating in the population, causing the infectious respiratory disease influenza. Major influenza outbreaks and recurring seasonal influenza pose enormous health threats and economic losses, representing one of the greatest public health security challenges globally. Therefore, developing safe, effective, and inexpensive vaccines to prevent RSV and IAV infection has become one of the most urgent problems to be solved worldwide.

[0003] RSV belongs to the Paramyxoviridae family and the Pneumovirus genus. Its genome is a single-stranded negative-sense virus, 15.2 kb in length, encoding 11 viral proteins. Based on the RSV genome sequence, it is divided into two subtypes, A and B. Because the RSV F protein is highly conserved, with only about 10% difference between different RSV subtypes, the F protein is the main antigen for the design and development of broad-spectrum RSV vaccines.

[0004] During the fusion of the RSV F protein with the host cell membrane, the pre-F conformation, characterized by higher potential energy and lower stability, transforms into a post-F conformation, characterized by lower potential energy and relative stability. During this conformational change, the pre-F specific epitopes Ø and V are masked, preventing the body from producing antibodies with high neutralizing activity. Therefore, developing pre-F-based RSV vaccines has become a hot research topic in RSV infection prevention. Currently, clinically available RSV vaccines exist, all of which are modified from the monomeric structure of the F protein (Krarup A., et al. A highly stable prefusion RSV Fvaccine derived from structural analysis of the fusion mechanism. Nature Communications, 2015, 6:8143; Crank MC., et al. A proof of concept for structure-based vaccine design targeting RSV in humans. Science, 2019, 365,505-509.) to maintain the pre-F conformation. However, according to literature reports, these protein vaccines all have problems such as low yield (below 6 mg / L) (Joyce MG., et al. Iterative structure-based improvement of afusion-glycoprotein vaccine against RSV. Nature Structural & Molecular biology, 2016, 23(9):811-820.) and inability to maintain the pre-F conformation (Crank MC., et al. Science, 2019, 365, 505-509.).

[0005] Influenza viruses are enveloped viruses with single-stranded, negative-sense RNA, belonging to the Orthomyxoviridae family. Influenza virus particles are pleomorphic, appearing as spherical or filamentous particles of varying lengths. Influenza virus particles consist of three parts: an envelope, a matrix layer, and a ribonucleoprotein complex. The envelope surface is embedded with two glycoproteins, HA and NA. HA proteins play crucial roles in receptor binding, inducing membrane fusion, packaging, pathogenicity, and immune recognition, making them important targets for vaccine development. However, because influenza virus HA proteins are highly susceptible to antigenic drift and mutation, the circulating strains each year are difficult to predict, leading to antigenic mismatches between candidate vaccines and circulating strains, resulting in reduced protective efficacy. Therefore, developing a universal influenza vaccine with broad cross-protection is of great significance.

[0006] The influenza virus HA protein has always been a major vaccine immunogen. The HA protein consists of a highly variable head domain and a highly conserved stem domain (Wiley DC, Wilson IA, Skehel JJ. Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature. 1981;289(5796):373-378.). Therefore, the stem domain is an important target for developing universal influenza vaccines. In 2015, Impaglia et al. generated a stable and correctly folded headless HA by introducing a stable conformational mutation at the proximal membrane of the HA (Impagliazzo, A., Milder, F., Kuipers, H., Wagner, MV, Zhu, XY, Hoffman,RMB, van Meersbergen, R., Huizingh, J., Wanningen, P., Verspuij, J., et al. (2015). A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen. Science 349, 1301-1306.), overcoming the difficulty of maintaining a stable structure after the HA protein loses its head domain. Furthermore, the headless HA can induce stem-specific antibodies through an Fc-dependent mechanism and provide immune protection to mice, laying the foundation for the development of a universal influenza virus vaccine.

[0007] Therefore, addressing the bottlenecks in vaccine development for RSV and influenza (IV) viruses, such as low yield and insufficient conformational stability of RSV pre-F protein vaccines, and the tendency of influenza virus HA protein to undergo antigenic drift leading to short-lasting vaccine protection, the development of a safe, effective, and cost-efficient combination vaccine that can simultaneously cover both pathogens has become a research hotspot and core direction in the field of respiratory virus vaccines. Summary of the Invention

[0008] To address the problems existing in the prior art, the inventors of this invention achieved this invention through research on the modification and recombination fusion of the RSVF protein sequence and the influenza A virus HA protein sequence. Specifically, the key design of this invention is as follows: 1. Based on the RSV A subtype F protein sequence, a new F antigen sequence is obtained by removing the post-F-specific I and IV epitope sequences and the III epitope portion shared by pre-F and post-F; 2. Based on the influenza A virus HA protein sequence, a stable HA protein stem region sequence is obtained through partial truncation and mutation; 3. The above two proteins are recombinated together to obtain a stable combined recombinant protein sequence. These three design points enable the new F protein to maintain its pre-F conformation in vivo and in vitro, forming a new and stable recombinant chimeric immunogen. This stimulates the body to produce antibodies specific to the RSV A virus pre-F epitope and possessing high neutralizing activity, while simultaneously providing protection against influenza virus infection.

[0009] On the one hand, the present invention provides a combined recombinant antigen for preparing a combined specific antibody against respiratory syncytial virus and influenza A virus, the amino acid sequence of which is shown in SEQ ID NO: 8.

[0010] On the other hand, the present invention provides a method for preparing the above-mentioned combined recombinant antigen, characterized in that the preparation method includes removing the I and IV epitope sequences of the post-F protein of respiratory syncytial virus (RSV) F protein and the III epitope sequence common to pre-F and post-F to obtain a recombinant F protein sequence; and linking the recombinant F protein sequence with the HA protein sequence of influenza A virus to obtain a combined recombinant antigen of RSV and influenza A virus.

[0011] Preferably, the respiratory syncytial virus F protein is the F protein of RSV A2 strain; the influenza A virus is the A / Victoria / 2570 / 2019 strain; Preferably, the recombinant F protein sequence is shown in SEQ ID NO: 11.

[0012] Furthermore, the I and IV epitope sequences of the post-F protein with the respiratory syncytial virus F protein removed are shown in SEQ ID NO: 2; the III epitope sequence shared by the pre-F and post-F protein with the respiratory syncytial virus F protein removed is shown in SEQ ID NO: 3.

[0013] Furthermore, the HA protein sequence is obtained by selecting two HA1 sequences and two HA2 sequences and mutating them; the two HA1 sequences are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively; the two HA2 sequences are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively. Preferably, the mutation is to mutate the 4th position K to C, the 17th position V to K, and the 20th position I to K in the sequence shown in SEQ ID NO: 5, to obtain the mutated HA1 sequence; Preferably, the mutation is to mutate the 10th position I to T, the 63rd position F to Y, the 66th position V to I, the 68th position K to C, the 70th position F to Y, and the 73rd position L to S in the sequence shown in SEQ ID NO: 6, to obtain the mutated HA2 sequence.

[0014] Furthermore, the recombinant F protein sequence is linked to the influenza A virus HA protein sequence by sequentially linking the sequence shown in SEQ ID NO: 4, the sequence shown in SEQ ID NO: 11, the mutated HA1 sequence, the mutated HA2 sequence, and the sequence shown in SEQ ID NO: 7. Preferably, linker sequences are inserted on both sides of the recombinant F protein sequence during the ligation process; Preferably, during the ligation process, a GCN4 sequence is inserted between the mutated HA1 sequence and the mutated HA2 sequence; Preferably, the combined recombinant antigen further includes a tag encoding a T4 trimer and / or six histidine tags.

[0015] On the other hand, the present invention provides a nucleic acid molecule, characterized in that the nucleic acid molecule encodes the aforementioned recombinant antigen, and the sequence of the nucleic acid molecule is shown in SEQ ID NO: 10.

[0016] On the other hand, the present invention provides the application of the above-mentioned combined recombinant antigen in reagents for preparing combined specific antibodies against respiratory syncytial virus and influenza A virus.

[0017] On the other hand, the present invention provides a combined specific antibody against respiratory syncytial virus and influenza A virus, wherein the combined specific antibody is capable of specifically binding to the aforementioned combined recombinant antigen.

[0018] Furthermore, the combined specific antibody is obtained by immunizing animals with the antigen described above or the antigen encoded by the nucleic acid molecule described above.

[0019] Furthermore, the combined specific antibody is a polyclonal antibody.

[0020] The technical solution of this invention has the following advantages: The combined recombinant antigen provided by this invention can maintain the pre-F trimer conformation in vivo and in vitro, thereby stimulating the body to produce antibodies specific to the pre-F epitope and with high neutralizing activity. Simultaneously, the presence of the stem region of the influenza HA protein can provide protection against influenza virus infection. The combined recombinant antigen of this invention has the advantages of stimulating the body to produce antibodies against both viruses and has the advantage of high yield. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 Hiload 16 / 60superdex 200 PG (GE) molecular sieve chromatography and electrophoresis of the recombinant antigen protein of respiratory syncytial virus and influenza A virus.

[0023] Figure 2 : Results of stability testing of recombinant antigen protein.

[0024] Figure 3 : Schematic diagram of immunization and virus attack strategies.

[0025] Figure 4 Results of specific IgG antibody titers induced in mice after immunization with recombinant antigen.

[0026] Figure 5 Results of RSV A2 virus neutralizing antibody titers induced in mice after immunization with recombinant antigen.

[0027] Figure 6 Results of RSV B9320 virus neutralizing antibody titers induced in mice after immunization with recombinant antigen.

[0028] Figure 7 Changes in body weight and survival rate of mice after challenge with A / PR / 8 / 34 (H1N1).

[0029] Figure 8Virus content in various tissues of mice after challenge with A / PR / 8 / 34 (H1N1).

[0030] Figure 9 Results of live virus titers in various tissues after rat immunization with recombinant antigen dimer RSV. Detailed Implementation

[0031] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0032] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0033] Example 1: Expression and purification of recombinant antigen protein of respiratory syncytial virus and influenza A virus Based on the full-length F protein sequence of the RSVA2 strain (as shown in SEQ ID NO: 1), the amino acid sequence N104-V147 of the post-F-specific I and IV epitopes (as shown in SEQ ID NO: 2) and the amino acid sequence V308-S552 of the RSV F protein monomer (as shown in SEQ ID NO: 3) containing the III epitope shared by pre-F and post-F were removed to obtain the amino acid sequence of the new RSVF protein monomer (specifically shown in SEQ ID NO: 11). Based on the HA protein sequence of the influenza A virus A / Victoria / 2570 / 2019 strain, two HA1 sequences (specifically shown in SEQ ID NO: 4 and 5) and two HA2 sequences (specifically shown in SEQ ID NO: 6 and 7) were selected. Three amino acid mutations were introduced in the SEQ ID NO: 5 sequence to maintain construction stability; the mutations were to change K at position 4 to C, V at position 17 to K, and I at position 20 to K in the SEQ ID NO: 5 sequence. Six amino acid mutations were introduced in the SEQ ID NO: 6 sequence to maintain construction stability; the mutations were to change I at position 10 to T, F at position 63 to Y, V at position 66 to I, K at position 68 to C, F at position 70 to Y, and L at position 73 to S in the SEQ ID NO: 6 sequence. The four HA protein sequences, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, are all stem region sequences of the influenza A virus HA protein. The amino acid sequence of the newly obtained RSVF protein monomer was linked to the four HA protein sequences mentioned above in the following order: SEQ ID NO: 4, SEQ ID NO: 11, mutated SEQ ID NO: 5, mutated SEQ ID NO: 6, SEQ ID NO: 7. Furthermore, linker sequences (e.g., the two amino acids GS) were inserted at both ends of the amino acid sequence of the new RSVF protein monomer (specifically, as shown in SEQ ID NO: 11), and a GCN4 sequence was inserted between the mutated SEQ ID NO: 6 and SEQ ID NO: 7 to obtain the coding sequence for the combined recombinant antigen of respiratory syncytial virus and influenza A virus (as shown in SEQ ID NO: 8). A T4 trimer tag and six histidine tags were added to the 3' end to obtain the amino acid sequence of the combined recombinant antigen (as shown in SEQ ID NO: 9). The amino acid sequence of the combined recombinant antigen was codon-optimized to obtain the coding gene for the combined recombinant antigen of respiratory syncytial virus and influenza A virus (as shown in SEQ ID NO: 10). The above sequences are shown in Table 1.

[0034] Table 1 Sequence

[0035] The gene encoding the recombinant antigen of respiratory syncytial virus and influenza A virus was then cloned into the pCAGGS vector to obtain the pCAGGS-recombinant antigen plasmid. The pCAGGS-recombinant antigen plasmid was then transfected into a 293F cell expression system for expression. After expression, the cell supernatant was collected and purified to obtain the purified product.

[0036] The purified product was subjected to chromatography using a Hiload 16 / 60 Superdex 200 PG molecular sieve. The elution peak sample was collected when the elution volume reached 60 ml, and then analyzed by SDS-PAGE (e.g., ...). Figure 1 (As shown). The protein size at an elution volume of 60 ml exceeded 170 kDa under non-reducing conditions (without DTT) and was approximately 75 kDa under reducing conditions (with DTT), proving that the sample obtained from the elution peak at an elution volume of 60 ml was the combined recombinant antigen trimer (hereinafter referred to as the combined recombinant antigen protein). Under laboratory conditions, approximately 35 mg of the combined recombinant antigen protein can be purified per liter of cell culture.

[0037] Experimental Example 1: Stability Analysis of Recombinant Antigen Combined with Respiratory Syncytial Virus and Influenza A Virus The fresh recombinant antigen protein obtained in Example 1 was placed in an environment at 4°C for 10, 40, 60, and 80 days, respectively, and the protein degradation was then detected by SDS-PAGE (e.g., Figure 2 (As shown). After standing at 4°C for 80 days, the protein still maintained a stable trimer state; after standing at 4°C for 40 days, most of the recombinant antigen monomers under reducing conditions did not undergo breakage or degradation.

[0038] Protein thermostability was detected using a protein stability analyzer and nanoDSF technology (see Real-Hohn, Antonio et al. "nanoDSF: In vitro Label-Free Method to Monitor Picornavirus Uncoating and Test Compounds Affecting Particle Stability." Frontiers in microbiology vol.11 1442. 26 Jun. 2020). The results (e.g.) were obtained. Figure 2As shown in the figure, the measured Tm value was 73.4℃, and the temperature at which the protein began to change was 53.5℃. These two results indicate that the combined recombinant antigen has a high Tm value and can remain stable without degradation for a relatively long time even under non-freezing conditions.

[0039] Experiment Example 2: Affinity Analysis of Recombinant Antigen-Antibody Combined with Respiratory Syncytial Virus and Influenza A Virus Using the Biacore 8K biomolecular interaction analysis system, the combined recombinant antigen protein obtained in Example 1 was immobilized on an SA sensor chip. Then, various antibody Fab fragments targeting different epitopes of the F protein and the stem region of the HA protein were flowed onto the chip surface to determine the affinity between the combined recombinant antigen protein and various antibodies. This embodiment uses the following antibodies: Epitope I 4D7 (see Flynn JA., et al. Stability Characterization of a Vaccine Antigen Based on the Respiratory Syncytial Virus Fusion Glycoprotein. PLoS ONE, 2016, 11(10):e0164789), Epitope II Palivazumab (see The IMPACT-RSV Study Group. Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. Pediatrics, 1998, 102, 531-537), and Epitope III MPE8 (see Gilman MS. et al. Characterization of a prefusion-specific antibody that recognizes a quaternary, cleavage-dependent epitope on the RSV fusion glycoprotein. PLoS Pathogen, 2015). 11:e1005035), IV epitope 101F antibody (see McLellan JS. et al. Structure of a major antigenic site on the respiratory syncytialvirus fusion glycoprotein in complex with neutralizing antibody 101F. Journal of Virology, 2010, 84: 12236-12244), V epitope CR9501 antibody (see Gilman MS. et al.).Transient opening of trimeric prefusion RSV F proteins. Nature Communications, 2019, 10:2105), Ø epitope Am22 antibody (see McLellan JS. et al. Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Scinece, 2013, 340(6136):1113-1117), HA stem region binding antibody FI6 antibody (see Limberis MP, Racine T, Kobasa D, et al. Vectored expression of the broadly neutralizing antibody FI6 in mouse airway provides partial protection against a new avian influenza A virus, H7N9. Clin Vaccine Immunol.2013;20(12):1836-1837.). Following Biacore 8K analysis (see Li J, Ma X, Xu Z, et al. Rational design of respiratory syncytial virus dimeric Fsubunit vaccines in protein and mRNA forms. EBioMedicine 2025, 119: 105902.), the binding and affinity of the combined recombinant antigen and antibody against respiratory syncytial virus and influenza A virus were obtained (as shown in Table 2). Blank cells indicate no antigen-antibody binding. According to the results in Table 2, the combined recombinant antigen protein exhibits affinity for the antibodies binding to epitope II (Palivazumab), epitope III (MPE8), epitope V (CR9501), epitope Ø (Am22), and HA stem region (FI6), demonstrating that the combined recombinant antigen protein can be effectively recognized by specific functional antibodies and possesses the potential to elicit a protective immune response.

[0040] Table 2. Binding and affinity of recombinant antigen and antibody to respiratory syncytial virus (RSV) and influenza A virus.

[0041] Experiment Example 3: Immunization of mice with recombinant antigen of respiratory syncytial virus and influenza A virus Preparation of the immunogen: MF59 (AddaVax used below is an MF59-like adjuvant) is a commonly used adjuvant approved by the SFDA. This invention selects this adjuvant as the vaccine adjuvant for animal experiments, which will have direct guiding significance for subsequent clinical trials. In this invention, the combined recombinant antigen protein obtained in Example 1 is diluted with PBS solution to the required concentration, then mixed with AddaVax adjuvant and emulsified to obtain the immunogen.

[0042] Immunization of mice: Mice used in this experiment were purchased from Vitaliva, weighing 16-20g. Six- to eight-week-old female BALB / c mice were immunized in groups using an immunogen. The immunization groupings, immunogen dosages, and adjuvants used in each group are shown in Table 3 (where 3μg-AddaVax and 10μg-AddaVax indicate that the content of the recombinant antigen protein in the immunogen obtained by mixing the diluted recombinant antigen protein with AddaVax adjuvant is 3μg or 10μg). The immunization strategy is as follows: Figure 3 As shown, each mouse received an immunogen three times via intramuscular injection in the thigh on days 0, 21, and 42, with each injection consisting of 100 μl, 50 μl in each thigh. Blood samples were collected from the orbital rim on days 19, 40, and 56. After standing, the blood samples were centrifuged at 3000 rpm for 10 minutes to obtain serum, which was then inactivated in a 56 °C water bath for 30 minutes and stored at -80 °C for specific antibody titer detection and RSV neutralization assays.

[0043] Table 3. Immunization grouping, immunogen dosage, adjuvant, and challenge strain for each group.

[0044] 1. RSV virus neutralization experiment with immune serum In the RSV virus neutralization experiment using immune serum, experimental and control groups were set up, and the specific method is as follows: Positive control wells: Serum samples obtained from experimental groups 1, 2, 5, 6, 9, and 10 were initially diluted 1:5, and then further diluted 4-fold to create 7 different concentrations. Each concentration was then mixed with an equal volume of 200 TCID50 solution. 50RSVA2 and RSVB9320 were mixed and incubated at 37 °C for 1 hour to obtain a mixture. 100 μl of the mixture was added to the positive control wells of a 96-well plate containing approximately 70% HEp-2 cells. After incubation at 37 °C for 2 hours, the mixture was discarded. Then, 100 μl of DMEM medium containing 2% FBS was added to the positive control wells. After incubation at 37 °C for 4 days, the DMEM medium was discarded, and HEp-2 cells were washed twice with PBS. After washing, the HEp-2 cells were fixed with ice-cold 80% acetone solution for 20 minutes. The supernatant was discarded, and the HEp-2 cells were washed a second time with PBS. 100 μl of blocking solution prepared with 5% skim milk powder in PBS was added to each positive control well, and the cells were blocked at 37 °C for 1 hour. After blocking, discard the blocking solution, and add 100 μl / well of 3 μg / ml Palivazumab primary antibody solution (prepared with blocking solution) to the positive control wells. Incubate at 37 °C for 1 hour, then wash the positive control wells three times with PBST to obtain cells incubated with the primary antibody. Add diluted HRP-conjugated goat anti-human IgG secondary antibody (prepared by diluting blocking solution at a volume ratio of 1:5000) to the cells incubated with the primary antibody. Incubate at 37 °C for 1 hour, then wash the positive control wells four times with PBST to obtain cells incubated with the secondary antibody. Add TMB chromogenic solution to the cells incubated with the secondary antibody for color development. After an appropriate reaction time, add 2M hydrochloric acid to terminate the reaction. Detect OD on a microplate reader. 450 Read value.

[0045] Negative control wells: Serum from control groups 1 and 2 were used in accordance with the experimental procedure for the positive control wells to obtain OD values ​​for detection on the microplate reader. 450 Read value.

[0046] Antibody titer is defined as the highest serum dilution where the reaction value of the positive control well is greater than 2.1 times the reaction value of the negative control well. The lowest detection dilution is 1:10. If the reaction value of the positive control well is still less than 2.1 times the reaction value of the negative control well at this dilution, the sample titer is defined as half of the lowest dilution, i.e., 1:5.

[0047] The results are as follows Figure 5 , Figure 6 As shown, during the entire immunization process, at a dose of 3 μg-AddaVax, after three immunizations, the level of neutralizing antibodies against both RSV subtypes reached 2. 8 The antibody titer levels were approximately [value missing]. This result indicates that the combined recombinant antigen of respiratory syncytial virus and influenza A virus can effectively stimulate mice to produce neutralizing antibodies, thereby achieving immune protection.

[0048] II. Mouse A / PR / 8 / 34(H1N1) Challenge Protection Experiment Mice were infected with 10LDA / PR / 8 / 34(H1N1) virus via intranasal instillation on day 63 (immunization and challenge strategies as follows). Figure 3 As shown in Table 3, the grouping was as follows: one group was weighed for 14 consecutive days (i.e., day 77 of the immune challenge strategy), and the other group was sacrificed after 6 days (i.e., day 69 of the immune challenge strategy). Lung and nasal turbinate tissues were removed, homogenized, and centrifuged at 3000 rpm for 15 minutes. The supernatant was collected. Viral RNA was extracted using a viral RNA purification kit (MagaBio plus Viral RNA Purification Kit BSC86) and detected by qPCR.

[0049] The results are as follows Figure 7 , Figure 8 As shown, after challenge with the virus, mice in control groups 3 and 4 (treated only with adjuvant PBS-AddaVax) experienced significant weight loss and some died. In contrast, mice in experimental groups 3, 4, 7, and 8 (treated with the immunogen) only experienced slight fluctuations in weight loss, which quickly returned to normal levels, and no deaths occurred. Similarly, qPCR results also showed that the viral load in the experimental group tissues was lower than that in the control group. This result fully demonstrates that the combined recombinant antigen of respiratory syncytial virus and influenza A virus has a good protective effect against influenza A virus infection.

[0050] Experiment Example 4: ELISA assay for detecting antigen-induced specific antibody titers Positive control wells: The combined recombinant antigen protein obtained in Example 1 was diluted to 3 μg / ml with ELISA coating buffer. The diluted combined recombinant antigen protein was added to the positive control wells of a 96-well ELISA plate (Coring, 3590), 100 μl per well. The plate was incubated at 4 °C for 12 hours. The ELISA coating buffer was then discarded, and the positive control wells were washed twice with PBS. Next, 100 μl of blocking buffer (5% skim milk powder prepared with PBS) was added to each positive control well, and the plate was blocked at 37 °C for 1 hour. After blocking, the blocking solution was discarded. 100 μl of serially diluted mouse serum samples obtained in Example 3 were added to the positive control wells. All serum samples were serially diluted 4-fold starting from 50-fold with the blocking solution, for a total of 12 dilutions. After incubation at 37 °C for 1 hour, the supernatant was discarded, and the positive control wells were washed three times with PBST. Then, diluted goat anti-mouse IgG secondary antibody was added to the positive control wells. This diluted goat anti-mouse IgG secondary antibody was obtained by diluting the blocking solution at a volume ratio of 1:5000. After incubation at 37 °C for 1 hour, the positive control wells were washed four times with PBST. TMB chromogenic solution was then added to the positive control wells for color development. After an appropriate reaction time, 2M hydrochloric acid was added to terminate the reaction. The OD was detected using a microplate reader. 450 Read value.

[0051] Negative control wells: Add 100 μl of blocking buffer to the negative control wells of a 96-well ELISA plate (Coring, 3590), incubate at 37 °C for 1 hour, discard the supernatant, wash the negative control wells three times with PBST, then add diluted goat anti-mouse IgG secondary antibody (obtained by diluting blocking buffer at a volume ratio of 1:5000) to the negative control wells, incubate at 37 °C for 1 hour, and wash the negative control wells four times with PBST. Then add TMB chromogenic solution to the negative control wells for color development, and stop the reaction by adding 2M hydrochloric acid after an appropriate reaction time. Detect OD using a microplate reader. 450 Read value.

[0052] Antibody titer is defined as the highest serum dilution where the reaction value of the positive control well is greater than 2.1 times the reaction value of the negative control well. The lowest detection dilution is 1:5. If the reaction value at this dilution is still less than 2.1 times the reaction value of the negative control well, the sample titer is defined as half of the lowest dilution, i.e., 1:25.

[0053] The results are as follows Figure 4 As shown, throughout the immunization process, the level of specific antibodies in the serum of mice in each immunization group increased significantly with the increase of the number of immunizations, and the antibody level was the highest after 3 immunizations, reaching 10. 6The results indicate that the combined recombinant antigen of respiratory syncytial virus and influenza A virus can effectively activate the antibody response in mice.

[0054] Experiment Example 5: Immunization of cotton rats with recombinant antigen of respiratory syncytial virus and influenza A virus MF59 (AddaVax, used below, is an MF59-like adjuvant) is a commonly used adjuvant approved by the SFDA. This invention selects this adjuvant as a vaccine adjuvant for animal experiments, which will have direct guiding significance for subsequent clinical trials. In this invention, the combined recombinant antigen protein obtained in Example 1 was diluted with PBS solution to the required concentration, mixed with AddaVax adjuvant, emulsified, and then used to immunize 6-8 week old female cotton rats in groups. The immunization grouping, immunogen dosage, and adjuvant usage for each group are shown in Table 4. The immunization strategy is as follows: Figure 3 As shown, each cotton mouse received three immunizations via intramuscular injection in the thigh on days 0, 21, and 42, with each injection consisting of 200 μl of vaccine and 100 μl injected into each thigh.

[0055] Table 4. Immunization grouping, immunogen dosage, adjuvant, and challenge strain for each group.

[0056] RSV challenge protection experiment in cotton rats: On day 63, the cotton rats obtained in this example were infected with RSV by intranasal drops, and 10 rats were infected with RSV. 5 TCID 50 RSVA2 virus and 10 5 TCID 50 RSVB9320 virus (immunity and attack strategies such as...) Figure 3 As shown in Table 3, the cells were sacrificed after 5 days, and lung, trachea, and nasal turbinate tissues were extracted. The lung, trachea, and nasal turbinate tissues were homogenized and then serially diluted 3-fold to obtain diluted tissue homogenates. 100 μl of the diluted tissue homogenate was added to a 96-well plate containing approximately 70% HEp-2 cell coverage. The 96-well plate was incubated at 37°C with 5% CO2 for 1 hour. The supernatant tissue homogenate was then discarded, and 100 μl of DMEM medium containing 2% fetal bovine serum was added to each well. The 96-well plate was incubated at 37°C with 5% CO2 for 4 days, and the viral titer (TCID) was measured. 50 The result is as follows: Figure 9 As shown, the results of virus cultures from the lungs, nasal turbinates, and trachea all indicated that, regardless of whether RSVA2 or RSVB9320 virus was used, the viral load in all tissues of the vaccine-treated mice was near the detection line (dashed line in the figure), approximately 2. 2.3The viral titer was 2, while the average viral titer of the protein-free adjuvant-only group (PBS) was 2. 7-10 This result fully demonstrates that the combined recombinant antigen of respiratory syncytial virus and influenza A virus has a good protective effect against RSV infection.

[0057] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A combined recombinant antigen for preparing a combined specific antibody against respiratory syncytial virus and influenza A virus, characterized in that, The amino acid sequence of the combined recombinant antigen is shown in SEQ ID NO:

8.

2. The method for preparing the combined recombinant antigen according to claim 1, characterized in that, The preparation method includes removing the I and IV epitope sequences of post-F and the III epitope sequence common to pre-F and post-F to obtain a recombinant F protein sequence; and linking the recombinant F protein sequence with the HA protein sequence of influenza A virus to obtain a combined recombinant antigen of respiratory syncytial virus and influenza A virus. Preferably, the respiratory syncytial virus F protein is the F protein of RSV A2 strain; the influenza A virus is the A / Victoria / 2570 / 2019 strain; Preferably, the recombinant F protein sequence is shown in SEQ ID NO:

11.

3. The preparation method according to claim 2, characterized in that, The I and IV epitope sequences of the post-F of the respiratory syncytial virus F protein are shown in SEQ ID NO: 2; the III epitope sequence shared by the pre-F and post-F of the respiratory syncytial virus F protein is shown in SEQ ID NO:

3.

4. The preparation method according to claim 2, characterized in that, The HA protein sequence was obtained by selecting two HA1 sequences and two HA2 sequences and mutating them; the two HA1 sequences are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively; the two HA2 sequences are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively. Preferably, the mutation is to mutate the 4th position K to C, the 17th position V to K, and the 20th position I to K in the sequence shown in SEQ ID NO: 5, to obtain the mutated HA1 sequence; Preferably, the mutation is to mutate the 10th position I to T, the 63rd position F to Y, the 66th position V to I, the 68th position K to C, the 70th position F to Y, and the 73rd position L to S in the sequence shown in SEQ ID NO: 6, to obtain the mutated HA2 sequence.

5. The preparation method according to any one of claims 2-4, characterized in that, The recombinant F protein sequence is linked to the influenza A virus HA protein sequence by sequentially linking the sequence shown in SEQ ID NO: 4, the sequence shown in SEQ ID NO: 11, the mutated HA1 sequence, the mutated HA2 sequence, and the sequence shown in SEQ ID NO:

7. Preferably, linker sequences are inserted on both sides of the recombinant F protein sequence during the ligation process; Preferably, during the ligation process, a GCN4 sequence is inserted between the mutated HA1 sequence and the mutated HA2 sequence; Preferably, the combined recombinant antigen further includes a tag encoding a T4 trimer and / or six histidine tags.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the combined recombinant antigen according to any one of claims 1-5, and the sequence of the nucleic acid molecule is shown in SEQ ID NO:

10.

7. The use of the combined recombinant antigen according to any one of claims 1-5 in the reagent for preparing a combined specific antibody against respiratory syncytial virus and influenza A virus.

8. A combined specific antibody against respiratory syncytial virus and influenza A virus, characterized in that, The combined specific antibody can specifically bind to the combined recombinant antigen described in claim 1.

9. The combined specific antibody according to claim 8, characterized in that, The combined specific antibody is obtained by immunizing an animal with the antigen described in claim 1 or the antigen encoded by the nucleic acid molecule described in claim 6.

10. The combined specific antibody according to claim 8, characterized in that, The combined specific antibody is a polyclonal antibody.

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