A fusion protein of respiratory syncytial virus, a subunit vaccine containing the same, and a construction method and application thereof

Abstract

The present invention discloses a respiratory syncytial virus fusion protein, a subunit vaccine containing the same, and methods for constructing and using the same, belonging to the field of protein vaccines. The fusion protein comprises an amino acid sequence as shown in either SEQ ID NO. 2 or SEQ ID NO. 4. The fusion protein vaccine provided by the present invention can generate high levels of neutralizing antibodies and cellular immune responses against respiratory syncytial virus, can recognize viruses of subtypes different from the sequence it contains, and can produce broad-spectrum immune protection against multiple respiratory syncytial viruses.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein vaccines, and in particular relates to a fusion protein of respiratory syncytial virus, a subunit vaccine containing the fusion protein, and a construction method and application thereof. Background Art

[0002] Respiratory syncytial virus (RSV) is a major viral pathogen that can cause respiratory tract infections in young children, the elderly and people with weakened immune systems. RSV is an enveloped, non-segmented, single-stranded, negative-sense RNA virus belonging to the family Paramyxoviridae and the genus Pneumovirus. It mainly consists of two subtypes, A and B, which can infect people alternately. The most effective measure to prevent respiratory syncytial virus is vaccination. However, as of now, there is no RSV vaccine available in China. The use of traditional vaccine research and development strategies and technical routes to produce RSV vaccines has problems such as long cycles, high costs and lags, making it difficult to adapt to the needs of preventing and controlling RSV pandemics. If a broad-spectrum RSV vaccine with cross-protection can be developed, it will change the current passive situation in the world's prevention and control of RSV pandemics.

[0003] In recent years, the discovery of broad-spectrum neutralizing antibodies against respiratory viruses, in-depth research on conserved regions of RSV virus antigens and cellular immune mechanisms, and the optimization of vaccine immunization strategies have all provided new ideas for the development of RSV vaccines. Studies have shown that RSV surface proteins attachment glycoprotein (G) and fusion protein (F) play an important role in mediating RSV infection of the host and are the main targets for vaccine development. Among them, the G protein is poorly conserved, with amino acid similarity of about 53% between RSV A and B subtypes. It mediates RSV adsorption to host cells. It has a central conserved region containing multiple B cell and T cell antigen epitopes, and the immune response induced by it has a cross-protective effect. The amino acid similarity of the F protein is over 90%, mediating the fusion of RSV with host cells. Due to its conserved nature, it has cross-reactivity between different subtypes of RSV virus, providing an experimental basis for a broad-spectrum RSV vaccine against RSV subtypes A and B.

[0004] Interferon (IFN) is a cytokine produced by the body's cells, among which IFN-α is a commonly used vaccine adjuvant with antiviral, antitumor and immunomodulatory properties.

[0005] Currently, there is no vaccine specifically targeting RSV in China. Furthermore, the development and production of RSV vaccines based on traditional vaccine development strategies and technologies is characterized by long development cycles, high costs, and lags in production, making it difficult to adapt to the needs of RSV pandemic prevention and control. Current subunit RSV vaccines under development target only the conserved region of the F protein of the virus, resulting in limited protection and weak immunogenicity. Therefore, a highly effective RSV vaccine is urgently needed. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects of RSV vaccines produced in the prior art, such as insufficient protection, weak immunogens and high production costs, and to provide a fusion protein, a vaccine containing the fusion protein and its application.

[0007] For vaccine proteins with multiple viral epitopes in series, the inventors conducted extensive research on the conserved regions of the RSV virus and found that only epitopes of specific viral subtypes or specific truncated epitope fragments, as well as a specific combination of epitope quantities, can produce the optimal effect.

[0008] In a first aspect, the embodiments of the present application provide a fusion protein, the amino acid sequence of which is shown in SEQ ID NO.2 or SEQ ID NO.4.

[0009] In a second aspect, the embodiments of the present application provide a vaccine comprising the fusion protein of the first aspect.

[0010] In some embodiments, the content of the fusion protein in the vaccine is 80-250 μg / mL; preferably, the content of the fusion protein is 80 μg / mL.

[0011] In a third aspect, the embodiments of the present application provide the use of a fusion protein in the preparation of a drug for preventing or treating respiratory syncytial virus.

[0012] In a fourth aspect, an embodiment of the present application provides a nucleic acid encoding the fusion protein of the first aspect.

[0013] In some embodiments, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 1 or 3.

[0014] In a fifth aspect, the embodiments of the present application provide the use of nucleic acids in the preparation of drugs for preventing or treating respiratory syncytial virus.

[0015] In a sixth aspect, an embodiment of the present application provides a recombinant vector comprising the nucleic acid of the fourth aspect.

[0016] In a seventh aspect, the embodiments of the present application provide use of a recombinant vector in the preparation of a drug for preventing or treating respiratory syncytial virus.

[0017] In an eighth aspect, an embodiment of the present application provides a transformant comprising the recombinant vector of the sixth aspect.

[0018] Beneficial effects:

[0019] The fusion protein provided by the present invention has a wide recognition rate for respiratory syncytial virus and can recognize viruses of subtypes different from the sequence it contains. It can obtain a broad-spectrum immune protection effect against multiple subtypes of RSV viruses, bringing hope for the use of vaccines to actively prevent pandemics caused by unknown RSV viruses and has good clinical application value. In addition, the antibody titer, virus neutralization ability and antigen-antibody affinity produced by it are strong.

[0020] In a preferred embodiment of the present invention, the fusion protein is fused to an interferon, significantly enhancing the immune response and being non-toxic to cells. Furthermore, the fusion protein provided by the present invention can be expressed in large quantities using prokaryotic expression systems, such as Escherichia coli, significantly reducing costs and shortening production cycles, making it highly suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Features, advantages, and technical effects of exemplary embodiments of the present invention will be described below with reference to the accompanying drawings.

[0022] Figure 1 The results of 1.2% agarose gel electrophoresis analysis of the PCR products of the FG and FGI fusion protein encoding gene fragments of the embodiment of the present application are shown, wherein: lane 1 is a DNA marker; lane 2 is the PCR product of the FGI fusion protein gene fragment; lane 3 is the PCR product of the FG fusion protein gene fragment.

[0023] Figure 2 The 12% SDS-PAGE electrophoresis results of the FGI fusion protein after renaturation are shown. Lane 1 is a protein marker; lanes 2-6 are the renatured FGI fusion protein.

[0024] Figure 3 The graph shows the results of detecting the affinity of antibodies induced by FGI fusion protein and RSV-A2 G antigen protein using surface plasmon resonance (SPR) technology in the examples of the present application.

[0025] Figure 4 The graph shows the results of detecting the affinity of antibodies induced by FGI fusion protein and RSV-B (strain 18537) antigen protein using the SPR method in the examples of the present application.

[0026] Figure 5The graph shows the results of detecting the affinity of antibodies induced by FGI fusion protein and RSV-B1 G antigen protein using the SPR method in the examples of the present application.

[0027] Figure 6 The graph shows the results of detecting the affinity of antibodies induced by FGI fusion protein and RSV-A2 F antigen protein using the SPR method in the examples of the present application.

[0028] Figure 7 The figure shows the result of detecting the affinity of the antibody induced by FGI fusion protein and RSV-A (1734) G antigen protein using the SPR method in the examples of the present application. DETAILED DESCRIPTION

[0029] The following detailed description of the embodiments of the present invention is provided in conjunction with the accompanying drawings and examples. The following detailed description of the embodiments and the accompanying drawings are used to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention. That is, the present invention is not limited to the described embodiments.

[0030] The following examples further describe the present disclosure. These examples are intended to be illustrative only, and various modifications and variations within the scope of the present disclosure will be apparent to those skilled in the art. Unless otherwise indicated, the percentages and ratios described in the following examples are by weight, and all reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further processing. The instruments used in the examples are also commercially available.

[0031] Example 1. FG Fusion antigen protein, FGI Design and preparation of fusion antigen protein

[0032] Based on the conserved segments of the F and G proteins of respiratory syncytial virus and interferon (IFN-α), FG fusion antigen proteins and FGI antigen fusion proteins were designed, respectively. The fusion genes encoding them are fg and fgi, respectively. The genes have the base sequences in SEQ ID NO:1 and SEQ ID NO:3 in Table 3, respectively. The FG antigen fusion protein comprises, from nitrogen terminus to carbon terminus, a conserved segment of the F protein, a connecting peptide, and a conserved segment of the G protein. The base sequence of the conserved segment of the F protein is positions 1 to 1062 as shown in SEQ ID NO:1, and the base sequence of the conserved segment of the G protein is positions 1084 to 1707 as shown in SEQ ID NO:1. The connecting peptide has the amino acid sequence shown in SEQ ID NO:7, and the gene encoding it has the base sequences in SEQ ID NO:1, respectively.

[0033] FGI antigen fusion protein: From nitrogen-terminus to carbon-terminus, it comprises, in order, an F protein conserved fragment, a connecting peptide, a G protein conserved fragment, a linking peptide, and an IFN-α sequence. The F protein conserved fragment base sequence is as shown in SEQ ID NO: 3, from positions 1 to 1062; the G protein conserved fragment base sequence is as shown in SEQ ID NO: 3, from positions 1084 to 1707; and the IFN-α fragment base sequence is as shown in SEQ ID NO: 3, from positions 1729 to 2127. The connecting peptide has the amino acid sequence shown in SEQ ID NO: 7, and its encoding gene has the base sequences as shown in SEQ ID NO: 3, from positions 1063 to 1083 and from positions 1708 to 1728.

[0034] According to the characteristics of the prokaryotic expression host bacteria Escherichia coli BL21 (DE3) cells, the codons of the coding genes fg and fgi of the FG and FGI fusion antigen proteins were optimized respectively, and the fusion genes were synthesized by Shanghai Bioengineering Co., Ltd.

[0035] Example 2. Expression and purification of FG fusion antigen protein and FGI fusion antigen protein

[0036] The fusion gene fg (base sequence in SEQ ID NO: 1) and the fusion gene fgi (base sequence in SEQ ID NO: 3) prepared in Example 1 were connected to the prokaryotic expression vector pET-28a plasmid (purchased from Novagen) to express the fusion subunit vaccine FG and the fusion subunit vaccine FGI in Escherichia coli, respectively.

[0037] The specific preparation process of fusion subunit vaccine FGI is as follows:

[0038] The expression vector pET-28a plasmid DNA, fg and fgi gene DNA fragments were double-digested with NcoI (purchased from Dalian Takara Biotechnology Co., Ltd.) and Hind III (purchased from Dalian Takara Biotechnology Co., Ltd.), respectively. After 1.0% agarose gel electrophoresis, the fg and fgi DNA fragments and the pET-28a plasmid DNA large fragment were recovered from the gel and ligated overnight at 16°C with T4 DNA ligase (purchased from Dalian Takara Biotechnology Co., Ltd.) to construct the fusion protein expression vectors pET28a-fg and pET28a-fgi. The ligation products were transformed into Escherichia coli DH5α competent cells (purchased from Dalian Takara Biotechnology Co., Ltd.). Transformants were screened for resistance to kanamycin (purchased from Sigma), and recombinant single clones were selected for expansion. Plasmid DNA was extracted and the correct recombinant clones were identified by double digestion with NcoI and Hind III ( Figure 1). The DNA fragment in lane 1 is 2200 bp in size, consistent with the target DNA fragment fgi, confirming the correct construction of the fusion protein expression vector pET28a-fgi. The recombinant cloned plasmid, verified by double enzyme digestion, was sent to Shanghai Bio-Technology Co., Ltd. for sequencing. The sequenced plasmid was transformed into competent E. coli BL21(DE3) cells (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.).

[0039] (1) Expression of fusion protein FGI: A single BL21 (DE3) colony transformed with the recombinant plasmid pET28a-fgi was inoculated into liquid LB medium containing 60 μg / mL kanamycin at 37°C overnight. The next day, the colony was transferred to the next generation at a ratio of 1:100 (volume ratio) and cultured at 37°C until the OD 600 The pH value was 0.6-0.8, and IPTG (purchased from Sigma) was added at a final concentration of 10 mM and induced at 30°C and 37°C for 4 h.

[0040] (2) Collecting the precipitate containing the fusion protein: The cells after induced expression were collected by centrifugation. Each 100 mL of the original culture was resuspended in 10 mL of equilibrium buffer (20 mM Tirs-HCl, 500 mM NaCl, 50 mM imidazole, pH = 7.9). The cells were ultrasonically disrupted at 0°C until the cell solution became clear. The cells were centrifuged at 10,000 rpm for 10 min to collect the inclusion body precipitate.

[0041] (3) Purify the fusion protein using an AKTA purifier (GE, USA): dissolve the inclusion body protein in 8M urea equilibrium buffer and load the sample (loading speed is 0.2-0.8mL / min) onto the protein purification workstation AKTA Ni 2+ An affinity chromatography column (GE, USA) was rinsed with 10 column volumes of equilibration buffer containing 8 M urea (8 M urea, 20 mM Tirs-HCl, 500 mM NaCl, 50 mM imidazole, pH = 7.9) until the UV detection value stabilized. Finally, the target protein was eluted with elution buffer containing 8 M urea (8 M urea, 20 mM Tirs-HCl, 500 mM NaCl, 250 mM imidazole, pH = 7.9). After the UV detection value began to rise, the denatured target protein was collected and collected until the UV value stabilized.

[0042] (4) Place the dialysis bag containing the denatured target protein in a beaker (500 mL) containing 6M urea refolding solution, stir slowly at 4°C for 4 hours on a magnetic stirrer, pour out 250 mL of the refolding solution, and pump new refolding solution (without urea) into the beaker at a rate of 2 mL per minute, 4°C, 4 hours, repeat 5-6 times, then pump in PBS solution (0.1 M, pH 7.2), 4°C, 4 hours, repeat 3 times, finally place the dialysis bag in PBS solution, stir and dialyze at 4°C for 12 hours, repeat 3 times, and obtain a refolded target protein with high purity. The purity was analyzed by SDS-PAGE, and the results are as follows: Figure 2 As shown, it is demonstrated that the purified fusion protein FGI was prepared.

[0043] The specific preparation of the fusion subunit vaccine FG also adopts the above preparation process.

[0044] Comparative Example

[0045] In order to verify the effect of the immunomodulatory protein IFN-α at different fusion positions of the vaccine protein, this experiment constructed three fusion proteins, IFG (IFNα-FG), FIG (F-IFNα-G), and GFI (GF-IFNα), according to the processes of Examples 1 and 2 above. The amino acid sequences are shown in SEQ ID NOs: 10, 12, and 14, and the DNA base sequences are shown in SEQ ID NOs: 11, 13, and 15. The immune effects were studied by antibody titer and neutralization experiments. The results are shown in Tables 1 and 2. The IFG and GFI treatment groups can induce the production of broad-spectrum antibodies against multiple RSV viruses, but their antibody titers and ability to neutralize viruses are far inferior to those of the FGI (FG-IFNα) treatment group.

[0046] The coding base sequence DNA of all protein molecules in this comparative example was synthesized by Shanghai Shenggong Company. The construction of expression vectors, protein expression and purification, and protein quantification methods of all protein molecules were the same as those in Example 2. The activity detection of all protein molecules was the same as that in Examples 3, 4, and 5.

[0047] Example 3 Antibody titer study

[0048] Serotype studies in mice immunized with respiratory syncytial virus subunit vaccines. Six- to eight-week-old female BALB / c mice were selected as research subjects and intraperitoneally immunized with vaccine proteins (FGI, FG, PBS (control)) on days 0, 14, and 28, respectively. Each mouse received 50 μg of protein, e.g., fusion protein FGI or fusion protein FG, in a 500 μL immunization volume. Serum was collected 14 days after the third immunization (day 42) and serum antibody analysis was performed.

[0049] Five respiratory syncytial virus surface antigen proteins (RSV A2 F, RSV A2 G, RSV BG, RSV B1 G, and RSV A1734 G) purchased from Sino Biological Co., Ltd. (due to experimental conditions, only the above six proteins are used as representatives) were coated on ELISA enzyme-labeled plates at a concentration of 2 μg / mL. The plates were washed four times with PBST, blocked with blocking solution, and washed four times with PBST. Then, they were incubated with serially diluted sera from each immune group at 37°C for 1 hour, washed four times with PBST, and incubated with secondary antibody (HRP-labeled goat anti-mouse IgG (H+L)) at 37°C for 1 hour. The plates were washed four times with PBST. TMB was used for display and the absorbance was measured at 450 nm. A ratio of ≥2.1 between the treatment group and the control group was considered positive.

[0050] Table 1

[0051]

[0052]

[0053] The results are shown in Table 1. The immunoglobulin induced by the FG fusion protein can better recognize and bind to the antigenic proteins of the above five viral subtypes (not limited to these five, if there are more subtypes, it will also bind), and the immunoglobulin content induced by the FGI treatment group is higher than that of the FG treatment group.

[0054] Example 4 Micro-neutralization Experiment

[0055] On the first day, the sera from the different immune groups in Example 3 were diluted in a 2-fold gradient to the corresponding multiples, and 50 μL / well was added to a 96-well plate. Then, 50 μL / well of 100 TCID 50 The virus (represented by RSV A2 strain) was co-incubated at 37°C for 2 hours, and 1.5×10 4 HEp-2 cells (human laryngeal epidermoid carcinoma cells, purchased from ATCC), 100 μ L / well, in 37 ℃ of cultivation for 20 hours, control group is set simultaneously, positive control 50 μ L virus dilution+50 μ L virus+100 μ L HEp-2 cell suspension.Negative control 100 μ L virus dilution+100 μ L HEp-2 cell suspension.On the second day, virus-infected cells are detected with ELISA, first use the mouse monoclonal antibody (purchased from Beijing Yiqiao Shenzhou Science and Technology Co., Ltd.) of anti-RSV-F protein to detect the replication of virus in HEp-2 cells, thereby reflecting the neutralization of serum sample to a certain virus, cell positive control average OD value-cell negative control average OD value / 2+ cell negative control average OD value=cell half infection threshold value, when every hole OD value is lower than cell half infection threshold value, be judged as neutralization reaction positive, the highest dilution of neutralization reaction positive serum is serum neutralizing antibody titer, as shown in Table 2.

[0056] Immune sera were collected from mice in the FG, FGI, F, and G groups immunized alone, mixed with FG and IFN-α, and IFN-α and PBS. The mouse immunization process was the same as in Example 3.

[0057] Under the same immunization dosage and method, the FG-treated group was still able to effectively neutralize the RSV A2 strain after a maximum dilution of 320, while F, G, and IFN-α alone were unable to effectively neutralize these two viruses (i.e., they could only exert a neutralizing effect at a lower dilution factor). This indicates that the fusion protein FG of the present invention has the potential to become a broad-spectrum subunit vaccine (i.e., it can induce the body to produce antibodies against multiple respiratory syncytial virus infections). When the FG fusion protein is used in combination with the immunomodulatory protein IFN-α (coupling or mixed), the induced serotype can effectively neutralize RSV at higher dilution factors, preventing it from infecting HEp-2 cells. The serum from the FG+IFN-α-treated group effectively neutralized the virus after a maximum dilution of 640 times. The FG1-immunized serotype had the strongest neutralizing ability, namely, it could still effectively neutralize RSV after the serum was diluted 1280 times. This indicates that in the presence of the immunomodulatory protein IFN-α, FG can better induce the body to produce immune protection, so FGI fusion protein has a better immune protection effect.

[0058] Table 2

[0059]

[0060] Example 4. Antibody-antigen affinity detection

[0061] On the premise that it is known that FGI fusion protein can induce mice to produce a broad spectrum of antibodies, surface plasmon resonance (SPR) was used to detect antibody-antigen affinity. First, the purified serum antibodies from the FGI group of the above example were placed in PBSP buffer (purchased from BD Company, USA) at a concentration of 1.25nM, and amine-coupled and fixed through goat anti-mouse IgG (H+L) on the surface of the biosensor (GM5). The goat anti-mouse IgG (H+L) sensor channel served as a negative control for each binding effect. Then, the sample to be tested was introduced at a flow rate of 30μL / min, that is, multiple concentrations of RSV-A2 G, RSV-B (strain 18537), RSV-A1 G, RSV-B2F, and RSV-A (1734) G antigen proteins were passed through the running buffer of PBSP (purchased from BD Company, USA). After each binding, the sensor surface was repeatedly washed with pH 2.0 glycine solution (purchased from BD Company, USA) for regeneration. The response unit (RU) reflects the binding of the antibody-antigen complex, and the results are shown in Figure 2. Figure 3-7 As shown, mF1G4 is used to represent FGI fusion protein. Figure 3The results show that the affinity of antibodies induced by different concentrations of FGI fusion protein to RSV-A2 G, RSV-B (strain 18537) G, RSV-B1 G, RSV-B2 F, RSV-A (1734) G and other antigenic proteins are 3.48×10 -9 M, 7.41×10 -9 M, 3.96×10 -9 M, 6.27×10-7M, 5.62×10 -9 M, showing high affinity to RSV-A2, RSV-A (1734), RSV-B (strain 18537), etc. This indicates that FGI fusion protein can induce the production of high-affinity neutralizing antibodies and will exert a good protective effect.

[0062] Comparative experiment

[0063] To verify the effect of the immunomodulatory protein IFN-α fused to different positions in the vaccine protein, this experiment constructed two fusion proteins: IFG (IFNα-FG), FIG (F-IFNα-G), and GFI (GF-IFNα). Their immune effects were studied through antibody titer and neutralization experiments. The results are shown in Tables 1 and 2. The IFG and GFI treatment groups induced the production of broad-spectrum antibodies against multiple RSV viruses, but their antibody titers and virus neutralization abilities were far inferior to those of the FGI (FG-IFNα) treatment group.

[0064] The coding base sequence DNA of all protein molecules in this comparative example was synthesized by Shanghai Shenggong Company. The construction of expression vectors, protein expression and purification, and protein quantification methods of all protein molecules were the same as those in Example 2. The activity detection of all protein molecules was the same as that in Examples 3, 4, and 5.

[0065] Although the present invention has been described with reference to preferred embodiments, various modifications may be made thereto and equivalent components may be substituted without departing from the scope of the present invention. In particular, the various technical features described in the various embodiments may be combined in any manner as long as no structural conflicts exist. The present invention is not limited to the specific embodiments disclosed herein, but encompasses all technical solutions within the scope of the claims.

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

Claims

1. A fusion protein, characterized in that Its amino acid sequence is shown in SEQ ID NO.2 or SEQ ID NO.

4.

2. A vaccine, characterized in that The vaccine comprises the fusion protein according to claim 1.

3. The vaccine according to claim 2, wherein the content of the fusion protein in the vaccine is 80 to 250 μg / ml.

4. Use of the fusion protein according to claim 1 in the preparation of a medicament for preventing or treating respiratory syncytial virus infection. A nucleic acid encoding the fusion protein according to claim 1 . The nucleic acid according to claim 5 , wherein the nucleotide sequence is shown in SEQ ID NO: 1 or 3.

7. Use of the nucleic acid according to claim 5 or 6 in the preparation of a medicament for preventing or treating respiratory syncytial virus infection. A recombinant vector comprising the nucleic acid according to claim 5 or 6.

9. Use of the recombinant vector according to claim 8 in the preparation of a medicament for preventing or treating respiratory syncytial virus infection.

10. A transformant comprising the recombinant vector according to claim 8.

Images