Bivalent nanoparticle antigen against respiratory syncytial virus and methods of making and using same

Abstract

The application belongs to the field of biological medicine, and particularly relates to a bivalent nanoparticle antigen for preventing respiratory syncytial virus infection and application thereof. The F protein sequences of two subtypes of RSVA and B and the human Ferritin protein sequence are connected in series, the post-F specific I and IV epitope sequences and the pre-F and post-F shared III epitope partial sequences are removed, and the bivalent antigen nanoparticle containing the new F protein of the two subtypes A and B is obtained through the self-assembly of the Ferritin protein. The bivalent nanoparticle antigen has a larger molecular weight, can effectively improve the immunogenicity of the antigen, stimulate the body to produce more neutralizing antibodies, and produce neutralizing antibodies against the two subtypes A and B of viruses, and is more broad-spectrum.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to proteins for preventing respiratory syncytial virus infection and their applications. 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 three, leading to pneumonia or bronchitis, and even death. In immunocompromised and weakened elderly populations, RSV infection can cause severe lower respiratory tract clinical symptoms, thus increasing the risk of death. Therefore, developing a safe, effective, and inexpensive vaccine to prevent RSV 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 RNA, 15.2 kb in length, encoding 11 viral proteins. Based on the RSV genome sequence, it is divided into two subtypes, A and B. Due to the high degree of conservation of the RSV F protein, the differences between different RSV subtypes are only about 10%. Therefore, 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 protein changes from a pre-F conformation with higher potential energy and lower stability to a post-F conformation with lower potential energy and relative stability. During this conformational change, pre-F specific epitopes... The V epitope is blocked, preventing the body from producing antibodies with high neutralizing activity; therefore, the development of pre-F-based RSV vaccines has become a hot research topic in the prevention of RSV infection. Currently, clinically available RSV vaccines exist, all of which are modified based on the monomeric structure of the F protein [Krarup A., et al. A highly stable prefusion RSV F vaccine 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 suffer from problems such as the inability to consistently maintain the pre-F conformation [Crank MC., et al. Science, 2019, 365, 505-509.]. Furthermore, due to the small size and poor immunogenicity of RSV F monomers, we selected a protein backbone as a support to allow the RSV F protein to be displayed on the backbone surface, thereby improving immunogenicity. We chose Ferritin protein. Ferritin protein is present in almost all organisms and has good biocompatibility. It consists of 24 monomers, each of which can be covalently linked to an antigen. Due to its good stability and high efficacy, it is often used in vaccine development. Currently, vaccines made using Ferritin have entered the clinical stage, such as influenza vaccines [Na Kyeong Lee., et al. Ferritin–a multifaceted proteinscaffold for biotherapeutics. Experimental & Molecular Medicine, 2022, 54, 1652–1657]. Summary of the Invention

[0005] To address the problems existing in the prior art, the inventors of this invention modified the RSV-F protein sequence and tandemly linked the modified F protein dimer gene with the human Ferritin protein gene. Specifically, the key design of this invention is as follows: 1. Based on the RSVA and B subtypes of the F protein sequence, new F antigen sequences are 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. The two new F antigen sequences are tandemly linked and then tandemly linked with the human Ferritin sequence to form a new F bivalent antigen nanoparticle. A linker sequence (e.g., two amino acids of GS) may or may not be inserted between the two; preferably, a 12-amino acid linker sequence of 6 GS is inserted. These two design features, on the one hand, enable the novel F bivalent antigen nanoparticles to maintain the pre-F conformation in vivo and in vitro, thereby stimulating the body to produce antibodies specific to the pre-F epitopes of the A and B subtypes and possessing high neutralizing activity; on the other hand, the bivalent nanoparticle antigen monomers of this invention have a larger molecular weight, which can effectively improve the immunogenicity of the antigen and has the advantage of stimulating the body to produce more neutralizing antibodies.

[0006] This invention provides a method for preparing bivalent nanoparticle antigens targeting respiratory syncytial virus (RSV) subtypes A and B, wherein the RSV antigens are obtained by expressing the following antigen-encoding genes:

[0007] The I and IV epitopes specific to post-F and the III epitope common to pre-F and post-F were removed from the A and B subtypes, respectively. The two new F protein genes were tandemly linked and then linked with the human Ferritin gene to obtain the respiratory syncytial virus bivalent nanoparticle antigen encoding gene.

[0008] Preferably, based on the F protein sequence of the RSVA2 strain (preferably its amino acid sequence as shown in SEQ ID NO: 1), the N104-L142 segment (specifically as shown in SEQ ID NO: 2) and the V308-G544 segment (specifically as shown in SEQ ID NO: 3) are removed; a linker sequence (e.g., the two amino acids GS) is inserted between T103 and G143 to link them; S35 is mutated to T, C37 to A, E92 to D, V144 to S, and S215 to P, to obtain the gene of the RSVA2 strain F protein monomer that does not contain the post-F-specific I and IV epitopes and the III epitope portion shared by pre-F and post-F; preferably, the amino acid sequences of the post-F-specific I and IV epitopes and the amino acid sequence of the III epitope portion shared by pre-F and post-F are removed;

[0009] Based on the F protein sequence of RSVB9320 strain (preferably its amino acid sequence as shown in SEQ ID NO: 4), the N104-L142 segment (specifically as shown in SEQ ID NO: 5) and the V308-S552 segment (specifically as shown in SEQ ID NO: 6) were removed; A103 and G143 were directly linked, and C37 was mutated to A, T97 to M, S197 to N, Q202 to R, and K226 to M, to obtain the gene of the F protein monomer of RSV B9320 strain that does not contain the I and IV epitopes specific to post-F and the III epitope shared by pre-F and post-F;

[0010] The M1-S25 segment was removed from the F protein monomer gene of the newly obtained RSVA2 strain, and the M1-Q26 segment was removed from the F protein monomer gene of the newly obtained RSVB9320 strain. The two were then tandemly linked with the human Ferritin (H-chain) gene (specifically as shown in SEQ ID NO: 7) to obtain two new respiratory syncytial virus bivalent nanoparticle antigen encoding genes.

[0011] A linker sequence (e.g., two amino acids of GS) may or may not be inserted between the new F protein dimer gene and the Ferritin protein gene. Preferably, a 12-amino acid linker sequence of 6 GS is inserted.

[0012] Preferably, the amino acid sequence of the novel respiratory syncytial virus bivalent nanoparticle antigen is shown in SEQ ID NO: 8.

[0013] Furthermore, the encoding nucleotide sequence of the stop codon is a single stop codon.

[0014] In a specific embodiment, the respiratory syncytial virus antigen encoding gene is expressed in a host cell using a recombinant expression vector to obtain the respiratory syncytial virus antigen; specifically, the starting vector of the recombinant vector is a mammalian cell expression vector such as pCAGGS vector, and the host cell is a mammalian cell such as 293F cell; optionally, the method further includes the steps of collecting cell supernatant and purifying the respiratory syncytial virus antigen after expression.

[0015] The present invention provides a respiratory syncytial virus antigen obtained by the preparation method described above, preferably having the amino acid sequence shown in SEQ ID NO: 8.

[0016] Furthermore, the encoding gene of the respiratory syncytial virus antigen is provided.

[0017] The present invention also provides a recombinant expression vector containing the aforementioned coding gene and a recombinant host cell. Specifically, the starting vector of the recombinant vector is a mammalian cell expression vector such as the pCAGGS vector, and the host cell is a mammalian cell such as 293F cell.

[0018] The present invention also provides an antigen composition consisting of the respiratory syncytial virus antigen as the active ingredient and an adjuvant, preferably the adjuvant being AddaVax adjuvant.

[0019] The present invention also provides the use of the respiratory syncytial virus antigen in the preparation of reagents for immunizing animals to obtain antibodies.

[0020] The bivalent nanoparticle antigen provided by this invention obtains bivalent antigen nanoparticles containing novel F proteins of both A and B subtypes through the self-assembly of Ferritin protein. These nanoparticles can maintain a pre-F conformation in vivo and in vitro, thereby stimulating the body to produce antibodies specific to the pre-F epitope and possessing high neutralizing activity. The bivalent nanoparticle antigen of this invention has a larger molecular weight, which can effectively improve the immunogenicity of the antigen, stimulate the body to produce more neutralizing antibodies, and because the nanoparticles of this invention contain both A and B subtypes of F protein, they can stimulate the body to produce neutralizing antibodies against both A and B subtypes of viruses, thus exhibiting broader spectrum of activity. Attached Figure Description

[0021] Figure 1 Superdex 200Increase 10 / 300 (GE) molecular sieve chromatography and electrophoresis images of RSV novel F bivalent antigen nanoparticles.

[0022] Figure 2 Schematic diagram of the immunization strategy.

[0023] Figure 3 Results of RSVF-specific IgG antibody titers induced in cotton rats after immunization with RSVF new bivalent antigen nanoparticles.

[0024] Figure 4 Results of RSVA2 virus neutralizing antibody titers induced by immunization of rats with RSV novel F bivalent antigen nanoparticles.

[0025] Figure 5 Results of RSVB9320 virus neutralizing antibody titers induced by immunization of rats with RSV novel F bivalent antigen nanoparticles.

[0026] Figure 6 Results of RSV A2 viral RNA load in lung tissue of rats after re-challenge with RSV novel F bivalent antigen nanoparticles.

[0027] Figure 7Results of RSV B9320 viral RNA load in lung tissue of rats after re-challenge with RSV novel F bivalent antigen nanoparticles. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but this should not be construed as limiting the invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods well known to those skilled in the art. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0029] Example 1: Expression and purification of RSV novel F bivalent nanoparticle antigen

[0030] Based on the F protein sequence of RSVA2 strain (as shown in SEQ ID NO: 1), the F sequence of RSVB9320 strain (as shown in SEQ ID NO: 4), and the amino acid sequence of the RSV F protein monomer that does not contain the I and IV epitopes specific to post-F and the III epitope shared by pre-F and post-F, the amino acid sequence of the new RSVF protein dimer obtained by tandem with the human Ferritin amino acid sequence (as shown in SEQ ID NO: 8), the respiratory syncytial virus antigen encoding gene was obtained after codon optimization.

[0031] The obtained respiratory syncytial virus antigen-encoding gene sequence was then cloned into the pCAGGS vector to obtain the pCAGGS-new F bivalent antigen nanoparticle plasmid. The plasmid was then transfected into a 293F cell expression system for expression, and the cell supernatant was collected and purified after expression.

[0032] Chromatography was performed using Superdex 200 Increase 10 / 300 (GE) molecular sieves, and the elution peak around 40 mL was collected (e.g., Figure 1 SDS-PAGE analysis was performed on the protein sample (shown in the image). The protein size around 40 mL of elution volume was approximately 78 kDa, confirming that the protein obtained from this elution peak was a novel F bivalent antigen nanoparticle.

[0033] Example 2: Antigen-antibody affinity analysis of RSV novel F bivalent nanoparticles

[0034] Using the Biacore 8K biomolecular interaction analysis system, the novel RSV F bivalent antigen nanoparticles obtained in Example 1 were immobilized on an SA chip. Then, various antibody Fab fragments targeting different epitopes of the F protein were flowed onto the chip surface, and the affinity between the antigen and various antibodies was measured.In this embodiment, epitope I 4D7 [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 [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], epitope III MPE8 [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.], epitope IV 101F [McLellan JS. et al. Structure of a major antigenic site on the respiratory syncytial virus fusion glycoprotein in complex with neutralizing antibody 101F. Journal of Virology, 2010, 84:12236-12244.], epitope V CR9501 [Gilman MS. et al. Transient opening of trimeric prefusion RSV F proteins. Nature Communications, 2019, 10:2105.] are selected. Epitope Am22 [McLellan JS. et al. Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody.Scinece,2013,340(6136):1113-1117.]. Biacore 8K analysis revealed the binding and affinity between the novel RSV F bivalent antigen nanoparticles and the antibody (as shown in Table 1). Blank cells indicate that the antigen and antibody do not bind.

[0035] Table 1. Binding and affinity of RSV novel F bivalent antigen nanoparticles to antibodies

[0036]

[0037] Example 3: Immunization of cotton rats with RSV novel F bivalent antigen nanoparticles

[0038] This invention selects AddaVax adjuvant (squalene oil-in-water nanoemulsion, similar to MF59 adjuvant) as a vaccine adjuvant for animal experiments, which will have direct guiding significance for subsequent clinical trials. Therefore, this invention dilutes the antigen obtained in Example 1 with PBS solution to the required concentration, mixes it with AddaVax adjuvant, emulsifies it, and then immunizes 6-8 week old female mice in groups. The immunization grouping, immunogen dosage, and adjuvant usage for each group are shown in Table 2, with blank cells indicating "none". The immunization strategy is as follows: Figure 2 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, 100 μL in each thigh. Blood samples were collected from the orbital fossa 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 experiments.

[0039] Table 2. Immunization grouping, immunogen dosage, adjuvant, and challenge strain for each group.

[0040] Serial Number Number dose adjuvant Challenge strain subtype PBS 6 Addavax A AB-Ferritin 6 3μg Addavax A PBS 6 Addavax B AB-Ferritin 6 3μg Addavax B

[0041] Example 4: ELISA assay for detecting antigen-induced specific antibody titers

[0042] The RSV novel F bivalent antigen nanoparticle protein obtained in Example 1 was diluted to 3 μg / ml with ELISA coating buffer and added to 100 μL of each well in a 96-well ELISA plate (Coring, 3590). After incubation at 4°C for 12 hours, the coating buffer was discarded, and the plate was washed twice with PBS. Then, 100 μL of 5% skim milk powder prepared with PBS was added to each well as blocking buffer, and the plate was blocked at 37°C for 1 hour. After blocking, the blocking buffer was discarded, and 100 μL of serially diluted serum samples obtained in Example 3 were added. Serum samples were serially diluted 4-fold starting from 40-fold with blocking buffer, for a total of 12 dilutions. 100 μL of blocking buffer was added directly to the negative control wells. After incubating at 37°C for 2 hours, the supernatant was discarded, and the plate was washed 3 times with PBST. Then, 50 L of blocking buffer containing chicken anti-mouse IgG secondary antibody diluted 1:5000 was added. After incubating at 37°C for 0.5 hours, the plate was washed 3 times with PBST. Then, 100 L of blocking buffer containing goat anti-chicken tertiary antibody diluted 1:5000 was added. After incubating at 37°C for 1 hour, the plate was washed 4 times with PBST. TMB chromogenic buffer was added for color development. The reaction was terminated by adding 2M hydrochloric acid after an appropriate reaction time. The OD was measured using a microplate reader. 450 Reading value. The antibody titer value is defined as the highest serum dilution where the reaction value is greater than 2.5 times the negative control value. When the reaction value at the lowest dilution (limit of detection) is still less than 2.5 times the negative control value, the sample titer is defined as half of the lowest dilution, i.e., 1:20.

[0043] The results are as follows Figure 3 As shown, throughout the immunization process, the level of specific antibodies in the serum of mice in each immunization group increased with the number of immunizations, reaching its highest level after three immunizations. This result indicates that RSV novel F bivalent antigen nanoparticles can effectively activate the antibody response in mice.

[0044] Example 5: Virus neutralization experiment with immune serum

[0045] The serum obtained in Example 3 was initially diluted 1:5, and then serially diluted 4-fold eight times, each diluted with an equal volume of 200 TCID50. 50RSVA2 and RSVB9320 viruses were mixed, with a final initial serum concentration of 1:10. After co-incubation at 37°C for 1 hour, 100 μL of the mixture was added to 96-well plates with approximately 70% HEp-2 cell coverage. After incubation at 37°C for 4 days, the culture medium was discarded, cells were washed twice with PBS, and fixed with ice-cold 80% acetone solution for 20 minutes. The supernatant was discarded, and cells were washed twice with PBS. 100 μL of 5% skim milk powder prepared with PBS was added to each well, and the plates were blocked at 37°C for 1 hour. After blocking, the blocking solution was discarded, and 100 μL of 3 g / mL Palivizumab primary antibody solution prepared with the blocking solution was added to each well. After incubation at 37°C for 2 hours, the plates were washed three times with PBST. 50 μL of HRP-conjugated goat anti-human IgG secondary antibody diluted 1:5000 with the blocking solution was added to each well, and the plates were incubated at 37°C for 1.5 hours. After washing four times with PBST. Add TMB chromogenic solution for color development, and after an appropriate reaction time, add 2M hydrochloric acid to terminate the reaction. Detect OD using a microplate reader. 450 Reading value. The antibody titer value is defined as the highest dilution of serum where the reaction value is less than 2.5 times the negative control value. When the reaction value at the lowest dilution (limit of detection) is still greater than 2.5 times the negative control value, the sample titer is defined as half of the lowest dilution, i.e., 1:5.

[0046] The results are as follows Figure 4 , Figure 5 As shown, during the entire immunization process, at a dose of 3 μg of AddaVax adjuvant, the neutralizing antibody levels reached 2 after 3 immunizations. 8 The above antibody titer levels indicate that the new RSV F bivalent antigen nanoparticles can effectively stimulate mice to produce neutralizing antibodies against RSV A2 and RSV B9320 viruses, thus achieving immune protection.

[0047] Example 6: Cotton Mouse Virus Challenge Protection Experiment

[0048] The cotton mice from Example 3 were infected with 1×10 mice via nasal drops on day 63. 5 TCID 50 RSVA2 virus and 1×10 5 TCID 50 RSVB9320 virus (such as) Figure 2 As shown in Table 2, the groups were divided into groups. Five days later, the animals were sacrificed, their lungs were removed, and the tissue was homogenized. 200 μL of the homogenate was used to extract RNA. 2 μL of the extracted RNA was used for real-time quantitative PCR amplification. The results are shown below. Figure 6 , Figure 7 As shown, the RNA loads of RSVA2 and RSVB9320 viruses in lung tissue were lower than those in the control group. This result fully demonstrates that the new RSV F bivalent antigen nanoparticles have a good protective effect on the body.

Claims

1. A method for preparing bivalent nanoparticle antigen of respiratory syncytial virus, characterized in that, The respiratory syncytial virus antigen is obtained by expressing the following antigen-encoding gene: The genes of the F protein dimers of RSV A and RSV B subtypes, which have had their post-F-specific I and IV epitopes removed and the III epitope shared by pre-F and post-F removed, are linked together with the human Ferritin gene. Through the self-assembly of the Ferritin protein, a new respiratory syncytial virus bivalent nanoparticle antigen encoding gene is obtained. Preferably, based on the F protein sequence of the RSVA2 strain (preferably its amino acid sequence as shown in SEQ ID NO: 1), the N104-L142 segment (specifically as shown in SEQ ID NO: 2) and the V308-G544 segment (specifically as shown in SEQ ID NO: 3) are removed; a linker sequence (e.g., the two amino acids GS) is inserted between T103 and G143 to connect them; S35 is mutated to T, C37 to A, E92 to D, V144 to S, and S215 to P, to obtain the gene of the RSVA2 F protein monomer that does not contain the I and IV epitopes specific to post-F and the III epitope shared by pre-F and post-F; Based on the F protein sequence of the RSVB9320 strain (preferably its amino acid sequence as shown in SEQ ID NO: 4), the N104-L142 segment (specifically as shown in SEQ ID NO: 5) and the V308-S552 segment (specifically as shown in SEQ ID NO: 6) were removed; A103 and G143 were directly linked, and C37 was mutated to A, T97 to M, S197 to N, Q202 to R, and K226 to M, resulting in the gene of the RSV B9320 F protein monomer that does not contain the I and IV epitopes specific to post-F and the III epitope shared by pre-F and post-F; The M1-S25 segment of the F protein monomer gene of the newly obtained RSVA2 strain was removed (specifically as shown in SEQ ID NO: 9), and the M1-Q26 segment of the F protein monomer gene of the newly obtained RSVB9320 strain was removed. The two were then tandemly synthesized to obtain a dimeric gene. This dimeric gene was then tandemly synthesized with the human Ferritin (H-chain) gene (specifically as shown in SEQ ID NO: 7) to obtain a novel respiratory syncytial virus bivalent nanoparticle antigen encoding gene.

2. The preparation method according to claim 1, characterized in that, The amino acid sequence of the post-F-specific I epitope was removed as F387-D392 (FNPKYD), and the amino acid sequence of the post-F-specific IV epitope was removed as K427-S436 (KNRGIIKTFS); and the portion of the III epitope common to pre-F and post-F, except for the amino acid sequence V40-L45 (VSKGYL), was removed.

3. The preparation method according to claim 1, characterized in that, A linker sequence (e.g., two amino acids of GS) may or may not be inserted between the new F protein dimer gene and the Ferritin protein gene. Preferably, a 12-amino acid linker sequence of 6 GS is inserted.

4. The preparation method according to claim 1, characterized in that, Also, add the nucleotide sequence that encodes a stop codon at the 3' end; Preferably, the nucleotide sequence encoding the stop codon is a single stop codon.

5. The preparation method according to any one of claims 1 to 4, characterized in that, The respiratory syncytial virus antigen encoding gene is expressed in host cells using a recombinant expression vector to obtain the respiratory syncytial virus antigen; specifically, the starting vector of the recombinant vector is a mammalian cell expression vector such as pCAGGS vector, and the host cell is a mammalian cell such as 293F cell; optionally, the method further includes the steps of collecting cell supernatant and purifying the respiratory syncytial virus antigen after expression.

6. The bivalent nanoparticle antigen against respiratory syncytial virus obtained by the preparation method according to any one of claims 1 to 5; Preferably, its amino acid sequence is shown in SEQ ID NO:

8.

7. The encoding gene for the respiratory syncytial virus bivalent nanoparticle antigen as described in claim 6.

8. A recombinant expression vector or recombinant host cell containing the encoding gene as described in claim 7, wherein the originating vector of the recombinant vector is a mammalian cell expression vector such as pCAGGS vector, and the originating host cell of the recombinant host cell is a mammalian cell such as 293F cell.

9. An antigen composition comprising the bivalent nanoparticle antigen against respiratory syncytial virus as described in claim 8 as an active ingredient and an adjuvant, preferably the adjuvant being AddaVax adjuvant.

10. The use of the respiratory syncytial virus bivalent nanoparticle antigen as described in claim 6 in the preparation of a reagent for immunizing animals to obtain antibodies.

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