Monovalent nanoparticle antigen aiming at respiratory syncytial virus as well as preparation method and application of monovalent nanoparticle antigen

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a monovalent nanoparticle antigen for preventing respiratory syncytial virus infection and application thereof. Based on F protein sequences of RSVA and B subtype strains and a human Ferritin protein sequence, two F proteins are respectively connected in series with a human Ferritin protein, and meanwhile, special I and IV epitope sequences of post-F and common III epitope partial sequences of pre-F and post-F are removed. New F monovalent antigen nanoparticles of A and B subtypes are respectively obtained through the self-assembly effect of Ferritin protein, and are maintained in pre-F conformation in vivo and in vitro, so that an organism is stimulated to generate an antibody which is specific to pre-F epitope and has high and medium neutralization activity. The monovalent nanoparticle antigen monomer has larger molecular weight, can effectively improve the immunogenicity of the antigen, and has the advantage of stimulating an organism to generate more neutralizing antibodies.

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 RSVF 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.].

[0005] Furthermore, due to the small size and poor immunogenicity of RSV F monomers, we selected a protein scaffold as a support to allow the RSV F protein to be displayed on the surface of the scaffold, thereby improving immunogenicity. We chose Ferritin protein. Ferritin 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 protein scaffold for biotherapeutics. Experimental & Molecular Medicine, 2022, 54, 1652–1657]. Summary of the Invention

[0006] To address the problems existing in the prior art, the inventors of this invention modified the RSV-F protein sequence and tandem the modified F protein gene with the human Ferritin protein gene. Specifically, the key design of this invention is as follows: 1. Based on the RSV 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 with the human Ferritin sequence to form nanoparticles of two new F monovalent antigens. A linker sequence (e.g., two amino acids of GS) may or may not be inserted between them; preferably, a 12-amino acid linker sequence of 6 GS is inserted. These two design features, on the one hand, enable the novel F monovalent 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 monovalent 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.

[0007] This invention provides methods for preparing two monovalent nanoparticle antigens targeting respiratory syncytial virus (RSV) subtypes A and B, respectively, wherein the RSV antigens are obtained by expressing the following antigen-encoding genes:

[0008] The genes of RSV F protein monomers were obtained by removing the I and IV epitopes specific to post-F and the III epitope common to pre-F and post-F, respectively. The genes of the two antigen monomers were then linked together with the human Ferritin gene to obtain the respiratory syncytial virus monovalent nanoparticle antigen encoding gene.

[0009] 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, and C37 is mutated to A 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 portion shared by pre-F and post-F; preferably, the amino acid sequences of the I and IV epitopes specific to post-F and the amino acid sequence of the III epitope portion shared by pre-F and post-F are removed;

[0010] 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, resulting in the gene of the RSV B9320F 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.

[0011] The obtained new RSVA2 F protein monomer genes and RSVB9320 F protein monomer genes were tandemly linked with the human Ferritin (H-chain) gene after removing the M1-S25 segment (specifically as shown in SEQ ID NO: 10) to obtain two new respiratory syncytial virus monovalent nanoparticle antigen encoding genes.

[0012] A linker sequence (e.g., two amino acids GS) may or may not be inserted between the new F protein monomer gene and the Ferritin protein gene. Preferably, a 12-amino acid linker sequence of 6 GS is inserted.

[0013] Preferably, the amino acid sequences of the two novel respiratory syncytial virus monovalent nanoparticle antigens are shown in SEQ ID NO: 8 and SEQ ID NO: 9.

[0014] Furthermore, the encoding nucleotide sequence of the stop codon is a single stop codon.

[0015] 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.

[0016] The present invention provides a respiratory syncytial virus antigen obtained by the preparation method described above, preferably having the amino acid sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9.

[0017] Furthermore, the encoding gene of the respiratory syncytial virus antigen is provided.

[0018] 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.

[0019] 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.

[0020] The present invention also provides the use of the respiratory syncytial virus antigen in the preparation of reagents for immunizing animals to obtain antibodies.

[0021] The monovalent nanoparticle antigen provided by this invention can maintain its pre-F conformation in vivo and in vitro, thereby stimulating the body to produce antibodies specific to the pre-F epitope and with high neutralizing activity. It has the advantage of stimulating the body to produce more neutralizing antibodies and improving antigen immunogenicity. Attached Figure Description

[0022] Figure 1A Superdex 200 Increase 10 / 300 (GE) molecular sieve chromatography and electrophoresis images of RSVA-type novel F monovalent antigen nanoparticles.

[0023] Figure 1B Superdex 200 Increase 10 / 300 (GE) molecular sieve chromatography and electrophoresis images of RSVB-type novel F monovalent antigen nanoparticles.

[0024] Figure 2 Schematic diagram of the immunization strategy.

[0025] Figure 3 Results of RSVF-specific IgG antibody titers induced in BALB / c mice after immunization with RSVF monovalent antigen nanoparticles.

[0026] Figure 4 Results of RSVA2 virus neutralizing antibody titers induced in BALB / c mice after immunization with RSV novel F monovalent antigen nanoparticles.

[0027] Figure 5 Results of RSVB9320 virus neutralizing antibody titers induced in BALB / c mice after immunization with RSV novel F monovalent antigen nanoparticles.

[0028] Figure 6 Results of viral RNA load in various tissues of BALB / c mice after re-challenge with RSV novel F monovalent antigen nanoparticles. Detailed Implementation

[0029] 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.

[0030] Example 1: Expression and purification of RSV novel F antigen nanoparticles

[0031] 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 RSV F protein monomers that do 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 sequences of the new RSVF protein monomers were tandemly linked with the human Ferritin amino acid sequence (as shown in SEQ ID NO: 8 and SEQ ID NO: 9). After codon optimization, the respiratory syncytial virus antigen encoding gene was obtained.

[0032] The obtained respiratory syncytial virus antigen-encoding gene sequence was then cloned into the pCAGGS vector to obtain the pCAGGS-new F monovalent 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.

[0033] Chromatography was performed using Superdex 200 Increase 10 / 300 (GE) molecular sieves, and the elution peak around 40 mL was collected (e.g., Figure 1A and Figure 1B SDS-PAGE analysis was performed on the protein sample (shown in the image). The protein size was approximately 50 kDa around 40 mL of elution volume, confirming that the protein obtained from this elution peak was a novel F monovalent antigen nanoparticle.

[0034] Example 2: Affinity analysis of RSV new F divalent antigen dimer antibody

[0035] Using the Biacore 8K biomolecular interaction analysis system, the novel RSV F monovalent 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, I epitope 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.], II epitope 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], III epitope 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.], IV epitope 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.], V epitope 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 monovalent antigen nanoparticles and the antibody (as shown in Table 1). Blank cells indicate that the antigen and antibody do not bind.

[0036] Table 1. Binding and affinity of RSV novel F monovalent antigen nanoparticles to antibodies.

[0037]

[0038] Example 3: Immunization of BALB / c mice with RSV novel F monovalent antigen nanoparticles

[0039] 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. Therefore, this invention diluted the antigen obtained in Example 1 with PBS solution to the required concentration, then mixed it with AddaVax adjuvant and emulsified it. This emulsified the mixture in groups of 6-8 week old female BALB / c mice. The immunization groupings, immunogen dosages used in each group, and adjuvant details are shown in Table 2. Blank cells indicate "none". The immunization strategy is as follows: Figure 2 As shown, each BALB / c mouse received three immunizations 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 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 at 56°C for 30 minutes and stored at -80°C for specific antibody titer detection and RSV neutralization assays.

[0040] Table 2. Immune grouping, immunogen dosage, adjuvant, and challenge strain for each group.

[0041] Serial Number Number dose adjuvant Challenge strain subtype PBS 6 PBS Addavax A Type A antigen 6 2μg Addavax A B antigen 6 2μg Addavax A

[0042] Example 4: ELISA assay for detecting antigen-induced specific antibody titers

[0043] The RSV novel F monovalent antigen nanoparticle protein obtained in Example 1 was diluted to 3 μg / ml with ELISA coating buffer and added to each well of an ELISA 96-well 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 the blocking buffer, and the plate was blocked at 37°C for 1 hour. After blocking, the blocking buffer was discarded, and 100 μL of the serially diluted serum sample obtained in Example 3 was added. The serum samples were serially diluted 4-fold starting from 40-fold with the blocking buffer, for a total of 12 dilutions. 100 μL of blocking buffer was added directly to the negative control wells. After incubation at 37°C for 2 hours, the supernatant was discarded, and the plate was washed 3 times with PBST. Then, goat anti-BALB / c mouse IgG secondary antibody diluted 1:5000 with the blocking buffer was added, and the plate was incubated at 37°C for 1.5 hours, followed by washing 4 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 with a reaction value greater than 2.1 times the negative control value. When the reaction value at the lowest dilution (limit of detection) is still less than 2.1 times the negative control value, the sample titer is defined as half of the lowest dilution, i.e., 1:20.

[0044] The results are as follows Figure 3 As shown, throughout the immunization process, the level of specific antibodies in the serum of BALB / c mice in each immunization group increased with the number of immunizations, reaching its highest level after three immunizations. This result indicates that the RSV novel F monovalent antigen nanoparticles can effectively activate the antibody response in BALB / c mice.

[0045] Example 5: Virus neutralization experiment with immune serum

[0046] The serum obtained in Example 3 was initially diluted 1:10, and then serially diluted 4-fold eight times, each diluted with an equal volume of 200 TCID50. 50RSVA2 and RSVB9320 were mixed. After co-incubating at 37°C for 1 hour, 100 μL of the mixture was added to a 96-well plate with approximately 70% HEp-2 cell coverage. After incubating at 37°C for 4 days, the culture medium was discarded, the cells were washed twice with PBS, and fixed with ice-cold 80% acetone solution for 20 minutes. The supernatant was discarded, and the 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 Palivizumab primary antibody solution at a concentration of 3 μg / mL was added to each well. After incubating at 37°C for 2 hours, the plates were washed three times with PBST. HRP-conjugated goat anti-human IgG secondary antibody diluted 1:5000 with the blocking solution was added, 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 with a reaction value greater than 2.1 times the negative control value. When the reaction value at the lowest dilution (limit of detection) is still less than 2.1 times the negative control value, the sample titer is defined as half of the lowest dilution, i.e., 1:5.

[0047] The results are as follows Figure 4 , Figure 5 As shown, during the entire immunization process, at a dose of 2 μg of AddaVax adjuvant, the neutralizing antibody levels reached 10 after three immunizations. 4 The above antibody titer levels indicate that the RSV novel F monovalent antigen nanoparticles can effectively stimulate BALB / c mice to produce neutralizing antibodies, thereby achieving immune protection for the body.

[0048] Example 6: BALB / c mouse challenge protection experiment

[0049] The BALB / c mice from Example 3 were infected by intranasal instillation on day 63, with 5 × 10⁶ mice. 4 TCID 50 RSVA2 virus (such as) Figure 2 As shown in Table 2, the groups were divided into groups. Five days later, the animals were sacrificed, and their lungs and nasal bones were removed. After tissue homogenization, 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 As shown, the RSVA2 viral RNA load in both the lungs and nasal cheekbones was lower than that in the control group. This result fully demonstrates that the novel RSV F monovalent antigen nanoparticles have a good protective effect on the body.

Claims

1. A method for preparing two monovalent nanoparticle antigens targeting respiratory syncytial virus subtypes A and B, respectively, characterized in that, The respiratory syncytial virus antigen is obtained by expressing the following antigen-encoding gene: The genes of the F protein monomers of RSV A and RSV B subtypes, which have had the I and IV epitopes unique to post-F and the III epitope shared by pre-F and post-F removed, were tandemly linked with the human Ferritin gene. Through the self-assembly of Ferritin protein, two new respiratory syncytial virus monovalent nanoparticle antigen encoding genes were 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, and C37 is mutated to A, resulting in a gene for the RSVA2 F protein monomer that does not contain the post-F specific I and IV epitopes or the III epitope shared by pre-F and post-F; or 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 obtained new RSVA2 F protein monomer genes and RSVB9320 F protein monomer genes were tandemly linked with the human Ferritin (H-chain) gene after removing the M1-S25 segment (specifically as shown in SEQ ID NO: 10) to obtain two new respiratory syncytial virus monovalent nanoparticle antigen encoding genes.

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 GS) may or may not be inserted between the new F protein monomer 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, They also added a stop codon encoding nucleotide sequence to the 3' end.

5. The preparation method according to claim 4, characterized in that, The nucleotide sequence encoding the stop codon is one stop codon; Preferably, 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. Respiratory syncytial virus monovalent nanoparticle antigen 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 and SEQ ID NO:

9.

7. The encoding gene of the respiratory syncytial virus monovalent 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 respiratory syncytial virus monovalent nanoparticle antigen as described in claim 6 as an active ingredient and an adjuvant, preferably the adjuvant being AddaVax adjuvant.

10. The use of the respiratory syncytial virus monovalent nanoparticle antigen as described in claim 9 in the preparation of a reagent for immunizing animals to obtain antibodies.

Images