Use of icos protein in the preparation of vaccine adjuvant potentiators

Abstract

The application discloses application of an agonist of ICOSL in preparation of a vaccine, and finds that under follicular helper T cell (T FH ) deficiency conditions, ICOS protein acts as an agonist of ICOSL protein, a nanoparticle vaccine coupled with the agonist of ICOSL can activate ICOSL molecules, directly acts on ICOSL to promote B cell proliferation and expression of anti-apoptosis related genes, can efficiently promote antibody response and differentiation and proliferation of antibody secreting cells; a vaccine coupled with ICOS protein is prepared, targets ICOSL signaling pathways through the coupled ICOS protein molecules, and shows stronger protection effect in anti-coronavirus and HIV vaccines, and especially has better immune effect on immunodeficient / deficient people.

Description

Technical Field

[0001] The present invention relates to the field of immunology technology, and more specifically, to the use of ICOSL agonists in the preparation of vaccines. Background Art

[0002] During epidemics or outbreaks of infectious diseases, vaccination is an important means of prevention and control. Vaccination can stimulate the immune system to produce protective immunity, prevent pathogen infection, inhibit the occurrence and spread of disease, reduce morbidity and mortality, reduce the number of infections, and mitigate the negative impact on social life.

[0003] After a person is vaccinated, the vaccine is first recognized and taken up by antigen-presenting cells (APCs) in the immune system, such as dendritic cells (DCs) or macrophages. After processing, the antigen peptides are presented to T lymphocytes or B lymphocytes. Among them, B lymphocytes differentiate into germinal center B cells (GCB) to form germinal centers after receiving antigen stimulation, and then undergo antibody affinity maturation with the help of T helper cells, and continue to differentiate into plasma cells and memory B cells. After activation, CD4 T lymphocytes will differentiate into a special group of subpopulations, namely follicular helper CD4 T (T follicular helper, T FH ) cells, which can interact with GCB cells to regulate the maturation of GCB cells and the production of high-affinity neutralizing antibodies.

[0004] T FH B cells express a variety of important molecules that interact with B cells, such as CD40L, IL-21, IL-4, and ICOS. These cytokines also play a role in promoting B cell proliferation, activation, and differentiation during B cell activation. This is particularly true for individuals with immune deficiencies, including those whose immune systems are impaired due to genetic or acquired diseases, as well as individuals whose immune function is deficient or declining due to aging. Therefore, the development of targeted vaccines for these individuals is of great practical significance.

[0005] In recent years, the emergence of nanoparticle vaccines has provided new strategies and new ideas for breaking through the limitations of traditional vaccines. Nanoparticle vaccines have become a hot topic in the field of vaccine research and development in recent years due to their unique design and efficient antigen presentation capabilities. Ferritin is a natural protein that exists in almost all organisms and can self-assemble into a 24-mer nanoparticle structure. The rich surface and highly ordered repetitive symmetrical structure of ferritin nanoparticles make them ideal for antigen delivery, helping to induce efficient immune responses and improve the efficacy of vaccines. Related research has developed influenza virus, new coronavirus and nasopharyngeal carcinoma virus vaccines based on ferritin nanoparticles, all of which have now entered the clinical trial stage.

[0006] Therefore, it is of great practical significance to develop personalized vaccines for people with immune deficiency / insufficiency to improve the efficacy of vaccines. Summary of the Invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of an agonist of ICOSL in the preparation of a vaccine.

[0008] The first object of the present invention is to provide use of an agonist of ICOSL protein in the preparation of a vaccine or a vaccine adjuvant.

[0009] The second object of the present invention is to provide use of an agonist of ICOSL protein in the preparation of a vaccine or a synergist of a vaccine adjuvant.

[0010] The third object of the present invention is to provide a vaccine or adjuvant containing an agonist of ICOSL protein.

[0011] The fourth object of the present invention is to provide use of an agonist and antigen composition of ICOSL protein in the preparation of a vaccine.

[0012] A fifth object of the present invention is to provide an antigen with enhanced immunogenicity.

[0013] The sixth object of the present invention is to provide an antigen of the new coronavirus with enhanced immunogenicity.

[0014] The seventh object of the present invention is to provide an HIV virus antigen with enhanced immunogenicity.

[0015] The eighth object of the present invention is to provide the use of any of the antigens in preparing vaccines

[0016] The ninth object of the present invention is to provide a vaccine with enhanced immunogenicity.

[0017] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0018] The present invention found that in follicular helper T cells (TFH ) deficiency conditions, the COVID-19 nanoparticle vaccine that simultaneously couples ICOS protein and RBD protein can activate ICOSL (Inducible T cell co-stimulator ligand, NCBI Ref: NP_NP_001269979.1) molecules, which can effectively promote antibody responses and the differentiation and proliferation of antibody-secreting cells. It also further clarifies the specific molecular mechanism by which ICOSL protein regulates B cell responses and its role as a new adjuvant target for anti-COVID-19 or HIV vaccines under conditions of immune deficiency or insufficiency. By coupling ICOS protein molecules to target the ICOSL signaling pathway, stronger protective effects are demonstrated in vaccines against COVID-19 and HIV.

[0019] The present invention is intended to protect the following applications:

[0020] Use of an agonist of ICOSL protein in the preparation of a vaccine or a vaccine adjuvant.

[0021] Use of an agonist of ICOSL protein in the preparation of a vaccine or a synergist of a vaccine adjuvant.

[0022] Preferably, the agonist of ICOSL is ICOS protein (NCBI Ref: NP_036224.1).

[0023] Preferably, the vaccine is for immunodeficient people and / or people with insufficient immune function and / or decreased immune function.

[0024] Preferably, the vaccine is against T FH People with cell defects.

[0025] Preferably, the potentiator improves the immune effect of the vaccine.

[0026] Preferably, the potentiator promotes the antibody response and / or the differentiation and / or proliferation of antibody secreting cells.

[0027] Therefore, a vaccine or adjuvant containing an agonist of ICOSL is also desired.

[0028] Preferably, the agonist of ICOSL protein is ICOS protein.

[0029] And, the use of ICOSL protein agonist and antigen composition in the preparation of vaccines.

[0030] Preferably, the agonist of ICOSL protein is ICOS protein.

[0031] Specifically, the present invention claims protection for the following products:

[0032] An antigen with enhanced immunogenicity, wherein the antigen is obtained by spontaneous chemical bonding of component 1 and component 2 via Gv-Sd, wherein component 1 is a mixture of a protein having an amino acid sequence such as SEQ ID NO: 12 and a fusion protein containing an immunogen, wherein the fusion protein containing the immunogen is a protein expressed by fusion of an immunogen and an SD peptide segment, and the amino acid sequence of the SD peptide segment is shown in SEQ ID NO: 8; and component 2 is a protein having an amino acid sequence such as SEQ ID NO: 5.

[0033] An antigen of a new coronavirus with enhanced immunogenicity, wherein the antigen is obtained by spontaneous chemical bonding of component 1 and component 2 through Gv-Sd, wherein component 1 is a mixture of a protein with an amino acid sequence such as SEQ ID NO: 12 and a protein with an amino acid sequence such as SEQ ID NO: 9; and component 2 is a protein with an amino acid sequence such as SEQ ID NO: 5.

[0034] An HIV antigen with enhanced immunogenicity, wherein the antigen is obtained by combining component 1 and component 2 through spontaneous chemical bonds of Gv-Sd, wherein component 1 is a mixture of a protein with an amino acid sequence such as SEQ ID NO: 12 and a protein with an amino acid sequence such as SEQ ID NO: 18; and component 2 is a protein with an amino acid sequence such as SEQ ID NO: 5.

[0035] Further claimed is the use of any of said antigens in the preparation of a vaccine.

[0036] and a vaccine with enhanced immunogenicity, which is prepared using any of the antigens.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention found that in follicular helper T cells (T FH ) deficiency conditions, ICOS acts as an agonist of ICOSL. Nanoparticle vaccines coupled with ICOSL agonists can activate ICOSL molecules, directly act on ICOSL to promote B cell proliferation and the expression of anti-apoptosis related genes, and can efficiently promote antibody responses and the differentiation and proliferation of antibody-secreting cells; ICOS-coupled vaccines are prepared, and the ICOSL signaling pathway is targeted by the coupled ICOS molecules, showing a stronger protective effect in vaccines against the new coronavirus and HIV viruses, especially for people with immune deficiency / insufficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1Figure 1 shows the construction of a SARS-CoV-2 nanoparticle vaccine. A is a schematic diagram of the conjugation of RBD to molecules such as CD40, IL-21, and ICOS on the surface of a single nanoparticle, where the GV / SD can undergo autocatalytic conjugation. B is the expression analysis of purified ferritin, RBD, CD40, IL-21, and ICOS proteins, as well as the confirmation of successful conjugation to ferritin by protein gel. *RBD conjugated with ferritin**CD40L conjugated with ferritin***IL-21 conjugated with ferritin****ICOS conjugated with ferritin; C is a scanning electron microscopy analysis of the assembled nanoparticles' morphology and size uniformity.

[0040] Figure 2 Conjugated ICOS can enhance the efficacy of SARS-CoV-2 RBD nanoparticle vaccine in T FH Antibody immune response in CD4-deficient mice; A is CD4 cre Bcl6 fl / fl Mouse immunization strategy; B is the ELISA test of RBD-specific IgG titer in mouse plasma at the 6th week of immunization; C is the ELISA test of RBD-specific IgA titer in mouse plasma at the 6th week of immunization; D is the ELISA test of RBD-specific IgG titer in mouse plasma at each time point.

[0041] Figure 3 ICOS-coupled SARS-CoV-2 RBD nanoparticles promote the induction of high-quality antibody production in TFH-deficient mice; A is an ELISA combined with 7M urea elution analysis of the high-affinity antibody content in plasma 6 weeks after immunization; B is a pseudovirus neutralization test to test the neutralizing active antibody titer in mouse plasma 6 weeks after immunization.

[0042] Figure 4 Immunizing CD4 with ICOS-RBD nanoparticle vaccine Cre Bcl6 fl / fl The number of B cells and antibody secreting cells in mice increased significantly after 14 days of immunization; A is the number of CD4 Cre Bcl6 fl / fl Mouse CD3 + T quantity and B220 + B is the flow cytometric analysis of CD4 Cre Bcl6 fl / fl Mouse CD3 + T number and ratio analysis, C is CD4 14 days after immunization Cre Bcl6 fl / fl Mouse B220 + B is the number and proportion analysis; D is the CD4Cre Bcl6 fl / fl Analysis of the number and proportion of GL7+GC B cells in mouse spleen; E is the number of CD4 Cre Bcl6 fl / fl Analysis of the number and proportion of CD138+ plasmablasts in mouse spleen; F is the number of CD4+ plasmablasts 14 days after immunization. Cre Bcl6 fl / fl Analysis of the number and proportion of total B cells in mouse spleen; G is CD4 Cre Bcl6 fl / fl Analysis of the number and proportion of memory B cells in mouse spleen; H is the ELIspot detection of CD4 10 weeks after immunization Cre Bcl6 fl / fl The number of RBD-specific antibody-secreting B cells in mouse bone marrow.

[0043] Figure 5 ICOS significantly enhanced B cell responses; A shows the binding of purified ICOS protein to ICOSL on B cells (red); B shows that ICOSL blockade significantly inhibited the production of SARS-CoV-2 RBD-specific IgG antibodies induced by ICOS-RBD nanoparticles; C shows that the percentage of plasmablasts (B220+CD138+) induced by ICOS-RBD nanoparticles decreased after treatment with ICOSL antibody blocker.

[0044] Figure 6 ICOS nanoparticles can promote the survival and proliferation of primary B cells in vitro; A shows that ICOS can bind to 293T cells expressing ICOSL; B to D are detections of ICOS nanoparticles stimulating mouse spleen B cells in vitro, including in vitro cell proliferation and counting analysis (B), BrdU insertion experiment cell cycle detection analysis (C), and Annexin V / PI staining cell apoptosis flow cytometry analysis (D).

[0045] Figure 7 Analysis of the molecular mechanism of ICOS activating B cells; A: After ferritin (HPF) and ICOS nanoparticles were treated in vitro for 2 hours, the phosphorylation modification of PKC, AKT, and NF-κB and the total protein expression levels were detected by WB; B: After ferritin (HPF) and ICOS nanoparticles were treated in vitro for 48 hours, the total RNA was extracted from the primary B cells isolated from mice, and the expression levels of cell cycle and apoptosis-related genes were detected by qPCR.

[0046] Figure 8Schematic diagram of vaccination, serum collection, and intranasal viral infection of Cd4cre Bcl6fl / fl mice. A: Mice were immunized with HPF, RBD-HPF, and ICOS-RBD-HPF vaccines at weeks 0 and 2, and intranasally infected with the Omicron EG.5 strain at week 4. Mice were euthanized on day 5 postinfection and subsequently analyzed. B: RBD-specific IgG titers in sera collected from immunized mice at week 4 were measured by ELISA, serially diluted, and expressed as the reciprocal of the endpoint serum dilution. N = 4-5 per group. C: Lungs were harvested from mice on day 5 postinfection, and the number of viral RNA copies in each mouse lung was determined by qRT-PCR and plotted as log10 copies per ml. D and E: Lungs were harvested from mice on day 5 postinfection, and H&E and immunohistochemical staining show pathological changes and viral replication in the mouse lungs. Scale bar, 100 μm.

[0047] Figure 9 The following table evaluates the immune effect of ICOS-coupled HIV nanoparticle vaccine (MD39) under immunodeficient conditions; A is a schematic diagram of the assembly of ICOS-coupled HIV nanoparticle vaccine; B and C are Coomassie blue staining (B) and WB (C, anti-HIVgp120) confirming the successful assembly of ICOS HIV nanoparticle vaccine *MD39**MD39 after Ferritin coupling***ICOS after Ferritin coupling; D. Schematic diagram of the immunization strategy for CD4CreBcl6fl / fl mice; E and F are ELISA tests for HIV-specific IgG antibody titers in mice at the fourth week of immunization (E), and their changes over time (F); G is ELIA combined with 7M urea elution to detect the proportion of HIV-specific high-affinity IgG content; H is ELISpot detection of the number of HIV env-specific antibody-secreting cells in the bone marrow of mice immunized for 8 weeks. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0049] Tris-NaCl buffer: 20 mM Tris-HCl 50 mM NaCl

[0050] Example 1 Preparation of COVID-19 Nanoparticle Vaccine

[0051] 1. Experimental Methods

[0052] Helicobacter pylori ferritin (HPF) is unlikely to induce autoantibodies, so HPF is used as the core of the nanoparticle vaccine. The GvoTagOpti-SdCatcher (Gvo-SD) system takes advantage of the ability of the ferritin nanoparticle surface to simultaneously couple multiple molecules to the T FH Important cellular functional proteins (CD40L, ICOS, and IL-21) were displayed simultaneously with the codon-optimized SARS-CoV-2 RBD on the same ferritin nanoparticles ( Figure 1 A in the figure is its schematic diagram), CD40L-RBD nanoparticle vaccine, ICOS-RBD nanoparticle vaccine and IL12-RBD nanoparticle vaccine against SARS-CoV-2 were constructed.

[0053] The specific method is:

[0054] 1. Construction of recombinant protein expression plasmid

[0055] The DNA encoding Gvo (the nucleotide sequence of which is shown in SEQ ID NO: 1, and the amino acid sequence encoded by it is shown in SEQ ID NO: 2) was ligated with the DNA sequence of HPF (the nucleotide sequence of which is shown in SEQ ID NO: 3, and the amino acid sequence encoded by it is shown in SEQ ID NO: 4) to obtain Gvo-HPF (the nucleotide sequence of which is shown in SEQ ID NO: 5, and the amino acid sequence encoded by it is shown in SEQ ID NO: 6). A 6×His tag was introduced at the C-terminus to obtain Gvo-HPF-His, which was digested with NcoI and XhoI and ligated into the pET-28a vector using homologous recombination cloning to obtain the plasmid pET28a-Gvo-HPF-His.

[0056] The DNA encoding SD (the nucleotide sequence of which is shown in SEQ ID NO: 7 and the amino acid sequence encoded by it is shown in SEQ ID NO: 8) was ligated to the RBD sequence, ICOS sequence and IL-21 sequence, respectively, to obtain RBD-SD (the nucleotide sequence of which is shown in SEQ ID NO: 9 and the amino acid sequence encoded by it is shown in SEQ ID NO: 10), ICOS-SD (the nucleotide sequence of which is shown in SEQ ID NO: 11 and the amino acid sequence encoded by it is shown in SEQ ID NO: 12) and IL-21-SD (the nucleotide sequence of which is shown in SEQ ID NO: 13 and the amino acid sequence encoded by it is shown in SEQ ID NO: 14). NO: 14), a 6×His tag was further introduced at the C-terminus to obtain RBD-SD-His, ICOS-SD-His, and IL-21-SD-His, which were then double-digested with XhoI and XbaI and ligated to the pCDNA3.1(+) vector using T4 enzyme to obtain plasmids pcDNA3.1-RBD-SD-His and pET28a-ICOS-SD-His; and double-digested with NcoI and XhoI and ligated to the pET28a vector using enzyme to obtain pET28a-IL-21-SD-His.

[0057] Since CD40L is a type II membrane protein, the SD sequence was genetically fused to the N-terminus of CD40L and downstream of the secretory signal peptide (SP) to obtain SD-CD40L (the nucleotide sequence is shown in SEQ ID NO: 15, and the amino acid sequence encoded by it is shown in SEQ ID NO: 16). A 6×His tag was introduced at the C-terminus, and SD-CD40L-His was double-digested with NcoI and XhoI and ligated to the pET28a vector using T4 enzyme to obtain the plasmid pET28a-SD-CD40L-His.

[0058] 2. Expression of Recombinant Protein

[0059] The recombinant eukaryotic expression plasmid pcDNA3.1-RBD-SD-His with correct sequencing was transiently transfected into 293F cells in good growth condition. 500 mL of cell resuspension was transfected and a transfection mixture was prepared (plasmid premix: 20 mL of Opti serum-free medium + 0.625 mg of recombinant eukaryotic expression plasmid pcDNA3.1-RBD-SD-His; PEI MAX premix: 20 mL of Opti serum-free medium + PEI 1.875mL), invert several times, mix thoroughly, and then let each stand for 5 minutes. Then, add the PEI MAX premix to the plasmid premix, mix gently, and let stand for 15 minutes to obtain the transfection system. Slowly add 40mL of the transfection system dropwise to the resuspended cells, shaking the suspension cell culture flask continuously and slowly during the addition process. After the transfection system is completely added, culture the cells under normal conditions. 5 days after transfection, collect the cells into a 50mL centrifuge tube and centrifuge at 10,000 rpm for 10 minutes at room temperature. Transfer the supernatant to a new 50mL centrifuge tube. The target protein (RBD-SD-His) is in the cell supernatant.

[0060] The above-mentioned correctly sequenced recombinant prokaryotic expression plasmids pET28a-SD-CD40L-His, recombinant prokaryotic expression plasmids pET28a-ICOS-SD-His and recombinant prokaryotic expression plasmids pET2a-IL-21-SD-His were transformed into the Rosetta (DE3) competent expression strain, and the activated bacterial solution was inoculated into 1L TB resistance medium and cultured on a shaking platform at 37°C and 220 rpm for about 4 hours until the absorbance OD600 reached 0.5; IPTG inducer with a final concentration of 1mM was added, and the culture was induced on a shaking platform at 16°C and 220 rpm for 16 to 24 hours; the bacterial solution was collected, centrifuged at 4000 rpm for 10 minutes, the supernatant was discarded, and 50 mL of The bacteria were resuspended in Tris-NaCl buffer; the bacterial suspension was disrupted under high pressure; the disrupted bacterial solution was centrifuged at 12000 rpm at 4°C for 30 min, and the insoluble precipitate was removed. The target proteins (SD-CD40L-His, ICOS-SD-His and IL-21-SD-His) were in the supernatant of the lysate.

[0061] The correctly sequenced recombinant prokaryotic expression plasmid pET28a-Gvo-HPF-His was transformed into the Rosetta (DE3) competent expression strain, and the activated bacterial solution was inoculated into 1L TB resistance medium and cultured on a shaking platform at 37°C and 220 rpm for about 4 hours until the absorbance OD600 reached 0.5; IPTG inducer was added at a final concentration of 1mM, and the culture was induced on a shaking platform at 16°C and 220 rpm for 16 to 24 hours; the bacterial solution was collected and centrifuged at 3000 rpm for 10 minutes, the bacteria were collected, ultrasonically disrupted, and the precipitate was discarded by centrifugation. The retained supernatant was diluted to a concentration of 1μg / μL, and then the supernatant was placed in a 70°C water bath. After 15 minutes, it was taken out and centrifuged to remove the E. coli protein precipitate. The target protein (Gvo-HPF-His) was in the supernatant.

[0062] 3. Purification of recombinant protein

[0063] Because the recombinant protein expression plasmids all contained a 6×His tag at the C-terminus, the His tag was bound to a nickel affinity column, followed by competitive elution with imidazole, to purify the target proteins (RBD-SD-His, Gvo-HPF-His, SD-CD40L-His, ICOS-SD-His, and IL-21-SD-His). A 3 mL Ni-NTA column preserving ethanol was loaded onto the column. The plug at the bottom of the column was removed, and the ethanol was washed away with 5 column volumes of deionized water. The column was then equilibrated with 5 column volumes of Tris-NaCl buffer. The treated sample was added to the column, which was capped and incubated on a shaker for 1 hour. The column was then washed with 5 column volumes of Tris-NaCl buffer containing 20 mM imidazole. The target protein was then eluted with Tris-NaCl buffer containing 100, 200, and 500 mM imidazole, respectively. The purity of the target protein was assessed by SDS-PAGE.

[0064] Subsequently, the target proteins (RBD-SD-His, Gvo-HPF-His, SD-CD40L-His, ICOS-SD-His and IL-21-SD-His) were further purified by size exclusion chromatography (SEC) and concentrated by ultrafiltration centrifugation. The supernatant was collected and the expression of the target proteins (RBD-SD-His, Gvo-HPF-His, SD-CD40L-His, ICOS-SD-His and IL-21-SD-His) was identified by Coomassie Brilliant Blue staining.

[0065] 4. Preparation of Nanoparticle Vaccines

[0066] The purified RBD-SD was mixed with SD-CD40L, ICOS-SD, and IL-21-SD monomers at a molar ratio of 1:1; the resulting products were then incubated with Gvo-HPF in Tris-NaCl buffer at room temperature overnight for self-assembly to prepare the respective nanoparticles (RBD NP, ICOS-RBD NP, IL-21-RBD NP, and CD40L-RBD NP).

[0067] The incubated product was concentrated by ultrafiltration, and then the protein concentration was tested using a BCA protein concentration assay kit, and then the nanoparticle coupling was detected by Coomassie brilliant blue staining.

[0068] The purity and uniformity of the nanoparticles were characterized by transmission electron microscopy (TEM).

[0069] 2. Experimental Results

[0070] The identification results of each target protein and each nanoparticle Coomassie Brilliant Blue staining are shown in Figure 1 B in the figure, the results of SEM analysis are shown in Figure 1 C in the figure shows that the self-assembled nanoparticles of uniform size were obtained. The particles were polyhedral spherical, and RBD, CD40L, ICOS and IL-21 were all displayed in the form of spikes on the surface of HPF nanoparticles, successfully preparing the new coronavirus nanoparticle vaccine.

[0071] Example 2 Immune Response of Novel Coronavirus Nanoparticle Vaccine under Immune Deficiency Conditions

[0072] 1. Experimental Methods

[0073] Used CD4 cre Bcl6 fl / fl Mice (Kristin Hollister, et al. Insights into the Role of Bcl6 in Follicular Th Cells Using a New Conditional Mutant Mouse Model, JImmunol (2013) 191 (7): 3705-3711. https: / / doi.org / 10.4049 / jimmunol.1300378), in which T cells cannot differentiate into T cells due to the knockout of the Bcl6 gene. FH The cells are unable to form germinal centers in vivo and their antibody production capacity is impaired, so they are used to simulate immunodeficiency conditions.

[0074] After the target protein was concentrated, mice were immunized with RBD NP, ICOS-RBD NP, IL-21-RBD NP and CD40L-RBD NP prepared in Example 1. Each group was diluted to 100 μl with 20 mM Tri-HCl 50 mM NaCl (pH = 7.5), and emulsified with an equal volume of Alum adjuvant. 10 μg RBD equivalent was injected per mouse. cre Bcl6 fl / flMice were immunized in groups. Each mouse was inoculated with a volume of 200 μl by subcutaneous injection. Boost injections were given on the 14th day. On the 14th and 28th days after the initial immunization, blood was collected from the mice's orbits. After the serum was allowed to stand for a period of time to precipitate, it was centrifuged at 4°C, 2800 rpm for 15 minutes and used for the Anti-RBD IgG ELISA test. See the procedure for details. Figure 2 A in.

[0075] 2. Experimental Results

[0076] The results are as follows Figure 2 The results showed that 2 weeks after Prime / Boost immunization, the RBD-specific IgG levels in the CD40L-RBD NP immunization group, ICOS-RBD NP immunization group, and IL-21-RBD NP immunization group increased by 9.4 times, 63 times, and 13 times, respectively, compared with the RBD NP immunization group. Figure 2 The RBD-specific IgA titer in the ICOS-RBD NP-immunized group was 18-fold or 9-fold higher than that in the CD40L-RBD NP-immunized group and the IL-21-RBD NP-immunized group, respectively. IgA levels are directly related to mucosal immunity that prevents infection ( Figure 2 C). The ICOS-RBD NP immunization group also showed the highest RBD-specific IgG antibody titer in long-term observation ( Figure 2 The above results show that ICOS-coupled nanoparticles can significantly enhance the efficacy of SARS-CoV-2 RBD nanoparticle vaccines in T FH Antibody responses in β-deficient mice.

[0077] Example 3 Affinity and neutralizing activity of antibodies produced by novel coronavirus nanoparticle vaccines

[0078] 1. Experimental Methods

[0079] 1. Detection of the content ratio of high-affinity antibodies

[0080] Six weeks after the initial immunization, the serum of the mice immunized in Example 2 was assayed for antigen-specific antibody titers by ELISA. During the assay, 7M urea concentration was added for elution. Antibodies that were washed away were low-affinity antibodies, while antibodies that remained bound to the plate after 7M urea treatment were high-affinity antibodies. The proportion of high-affinity antibodies was calculated by comparing the ELISA readings of the 7M urea-treated and untreated samples.

[0081] Specifically, the ELISA plate was coated with 5 μg / ml SARS-CoV-2RBD antigen protein in 0.1 M bicarbonate buffer (pH 9.3) at 4 ° C overnight. After washing three times with PBS, it was blocked with 5% skim milk / PBS at room temperature for 1 hour. It was washed again with PBST (containing 1% Tween-20) three times, and the animal serum of each group of mice in Example 2 was serially diluted in PBS, and then the plate was incubated at 37 ° C for 1 hour. After incubation with diluted serum, the plate was washed twice with PBST. The wells were incubated with PBST or PBST (containing 7M urea) for 10 minutes at room temperature. After washing with PBST, a 1:4000 dilution of HRP-labeled goat anti-mouse IgG was added to detect antigen-specific antibodies in mouse serum. After incubation for another 1 hour, the plate was washed with PBST. Subsequently, 50 μL of HRP substrate TMB solution (eBioscience) was added to each well, reacted in the dark, and the reaction was terminated with stop solution (Solarbio) after the reaction was fully developed. Absorbance was measured at 450 nm. GraphPad Prism 9.0 software was used to perform nonlinear regression analysis of the data to calculate endpoint titers. The affinity index for each serum sample was calculated by dividing the readings from the PBST (7 M urea) incubation by the readings from the PBST incubation alone.

[0082] 2. SARS-CoV-2 pseudovirus neutralizing antibody test

[0083] The SARS-CoV-2 Spike protein was inserted into the pcDNA3.1 expression vector using a pseudovirus system based on a lentiviral vector (Ma X, Nanoparticle Vaccines Based on the Receptor Binding Domain (RBD) and Heptad Repeat (HR) of SARS-CoV-2 Elicit Robust Protective Immune Responses. Immunity. 2020 Dec 15; 53(6): 1315-1330.e9.doi:10.1016 / j.immuni.2020.11.015. Epub 2020 Nov 25. PMID: 33275896 Free PMC article.) to obtain the expression vector of the SARS-CoV-2 Spike protein.

[0084] 293T cells were co-transfected with an expression vector for the SARS-CoV-2 Spike protein, along with pHIV-luciferase and psPAX2 plasmids. Five hours after transfection, cells were washed twice with PBS and then cultured in serum-free DMEM medium. After 48 hours, the supernatant was collected, centrifuged to remove cell debris, and lysed in a small volume of serum-free DMEM to obtain the HIV-luc / SARS-CoV-2-S pseudovirus.

[0085] The HIV-luc / SARS-CoV-2-S pseudovirus effectively mimics the invasion of cells by wild-type SARS-CoV-2. Upon infection of producer or target cells, the expression of the luciferase reporter gene accurately reflects the outcome of viral infection, enabling precise and rapid readout of experimental results. This pseudovirus serves as an excellent system for monitoring antibody neutralization potency.

[0086] After the serum from the immunized mice was allowed to stand at room temperature for a period of time to precipitate, the antibody serum was obtained by centrifugation at 2800 rpm for 15 minutes at 4°C. The resulting antibody serum was diluted 2-fold, mixed with HIV-luc / SARS-CoV-2-S pseudovirus, and incubated at 37°C for 1 hour. The mixture was added to a 96-well plate containing hACE2-HEK293T cells at a density of approximately 70%. After 48 hours, the culture medium was discarded, the cells were washed twice with PBS, and cell lysate was added to measure luciferase activity. The pVNT50 result was the dilution concentration at which the antibody serum inhibited luciferase activity by 50% compared to the luciferase activity in the untreated group.

[0087] 2. Experimental Results

[0088] result Figure 3 As shown, the high-affinity antibodies in the ICOS-RBD NP immunization group increased by 46% compared with the RBD NP immunization group ( Figure 3 A in the SARS-CoV-2 pseudovirus neutralizing antibody test showed that ICOS-RBDNP immunization induced nAb titers that were 38-fold and 32-fold higher than those of CD40L-RBD NP and IL-21-RBD NP, respectively ( Figure 3 (B) These results suggest that conjugated ICOS enhances high-quality antibody production induced by SARS-CoV-2 RBD nanoparticle vaccine immunization in the setting of immunodeficiency.

[0089] Example 4 ICOS significantly enhanced B cell responses

[0090] 1. Experimental Methods

[0091] CD4CreBcl6fl / fl mice were immunized with the RBD NP and ICOS-RBD NP prepared in Example 1. Fourteen days later, the percentage and number of total T cells in the lymph nodes of the ICOS-RBD NP-immunized and RBD NP-immunized CD4CreBcl6fl / fl mice were measured. The specific detection method was as follows: After the target protein was concentrated, mice were immunized with 10 μg of RBD protein.

[0092] RBD NP or ICOS-RBD NP was diluted to 100 μl with pH 7.5 Tris-NaCl buffer and emulsified with an equal volume of Alum adjuvant. cre Bcl6 fl / fl Mice were immunized in groups. Each mouse was inoculated with 200 μl of the vaccine by subcutaneous injection. Fourteen days after immunization, spleens, lymph nodes, and bone marrow cells were collected and stained with flow cytometry antibodies to analyze mouse T cells (CD3+), B cells (B220+), B cell memory cells (IgD-CD38+), and plasmablast subsets (B220+CD138+).

[0093] 2. Experimental Results

[0094] CD4 cells were immunized with RBD NP and ICOS-RBD NP respectively. Cre Bcl6 fl / fl Mice, 14 days later, there was no significant difference in the percentage and number of total T cells in the lymph nodes between the ICOS-RBD NP immunization group and the RBD NP immunization group ( Figure 4 However, the percentage of B cells in the lymph nodes of the ICOS-RBD NP immunization group increased by 37% and the number of B cells increased by 48% ( Figure 4 A and

[0095] C), which indicates that ICOS directly affects the proliferation of B cells. Although there was no significant change in the germinal center B (GCB) cells in the spleen of mice immunized with ICOS-RBD NP ( Figure 4 D), the percentage and number of plasmablasts in the ICOS-RBD NP immunization group increased by 53% and 76%, respectively ( Figure 4 E in the figure), plasmablasts secrete antibodies and are associated with long-lived plasma cells in the bone marrow, providing support for long-term protection. In addition, the number of B cells in the spleen of the ICOS-RBD NP immunization group increased by 82% compared with the RBD NP immunization group ( Figure 4 F), the number of memory B cells (MBC) increased by 1.5 times compared with the RBD NP immunization group ( Figure 4 G in ).

[0096] In T FH In the absence of cells, RBD NP alone could hardly induce the production of antibody-secreting B cells (ASCs). However, ICOS-RBD NP significantly increased the number of RBD-specific ASCs. Figure 4 These results indicate that ICOS FH B cell responses, particularly the production of antibody-secreting cells, were significantly enhanced in the deficient mice.

[0097] Example 5 Binding of ICOS Protein to ICOSL Protein

[0098] 1. Experimental Methods

[0099] To further determine the correlation between the binding of ICOS and ICOSL and the activation and proliferation of B cells, anti-ICOSL blocking antibody was used to pre-treat CD4 Cre Bcl6 fl / fl The mice were immunized for 24 hours with the RBD-ICOS NP prepared in Example 1. The dose was equivalent to 10 μg of RBD protein. After 14 days of immunization, the mouse serum was separated for antigen-specific antibody titer ELISA analysis.

[0100] Among them, anti-ICOSL blocking antibody, 500 μg / mouse (Biolegend clone HK5.3 Catlog 107412) (PMID: J Immunol 12960306, PNAS 15014176)

[0101] 2. Experimental Results

[0102] Flow cytometry showed that the purified ICOS protein binds to ICOSL on B cells ( Figure 5 A in Figure 1). 14 days after immunization, the results showed that ICOSL blocking significantly inhibited the production of SARS-CoV-2 RBD-specific IgG antibodies induced by ICOS-RBD nanoparticles compared with the unblocked group ( Figure 5 In addition, ICOSL blockade also reduced ICOS-RBD nanoparticle-induced plasmablasts (B220 + CD138 + )percentage( Figure 5 C), which shows that ICOS-RBD nanoparticles can be FH In the case of cells, immune responses are enhanced through ICOSL on B cells.

[0103] Example 6 ICOS can promote the survival and proliferation of primary B cells in vitro

[0104] 1. Experimental Methods

[0105] Direct treatment of CD4 with ICOS NP or HPF NP in vitro Cre Bcl6 fl / fl Mouse spleen isolation B cells.

[0106] Then, ICOS NP, CD40L NP, IL21 NP and HPF NP were directly used to separate the NPs from the spleen of mice. B cells were co-cultured. After 48 hours, some cells were stained with anti-Annexin V and PI, and cell apoptosis was analyzed by flow cytometry; some cells were incubated with BrdU for 2 hours, stained with anti-BrdU and DAPI, and cell cycle was analyzed by flow cytometry.

[0107] HPF NPs were prepared by incubating the Gvo-HPF prepared in Example 1 in Tris-NaCl buffer at room temperature overnight to allow self-assembly;

[0108] The preparation method of CD40L NP is as follows: SD-CD40L prepared in Example 1 is incubated with Gvo-HPF in Tris-NaCl buffer at room temperature overnight to perform self-assembly;

[0109] The preparation method of IL21 NP is as follows: IL-21-SD prepared in Example 1 is incubated with Gvo-HPF in Tris-NaCl buffer at room temperature overnight to allow self-assembly;

[0110] The preparation method of ICOS NP is as follows: ICOS-SD prepared in Example 1 is incubated with Gvo-HPF in Tris-NaCl buffer at room temperature overnight to perform self-assembly.

[0111] 2. Experimental Results

[0112] Compared with HPF NP treatment, ICOS NP can significantly enhance the proliferation of mouse primary B cells in vitro ( Figure 6 A and B in ).

[0113] Cell cycle analysis showed that ICOS NP promoted the advancement of B cells from G1 phase to S phase, and the proportion of S phase was 6 times higher than that of HPF NP group or IL-21 NP group ( Figure 6 C). In the HPF NP-treated group, the survival rate of B cells was only 35%, while that of B cells in the ICOS group exceeded 50% ( Figure 6 D) in.

[0114] These results indicate that ICOS can promote the survival and proliferation of primary B cells in vitro.

[0115] Example 7: ICOS activates ICOSL to promote B cell proliferation and anti-apoptosis

[0116] 1. Experimental Methods

[0117] 1. Western Blot analysis of key proteins related to B cell proliferation

[0118] Mouse spleen B cells were isolated using anti-CD19 magnetic beads, and then directly co-cultured with ICOSNP, CD40L NP and IL21 NP at a concentration of 50 μg / mL. After 48 hours, some cells were stained with anti-Annexin V and PI, and cell apoptosis was analyzed by flow cytometry; some cells were incubated with BrdU for 2 hours, stained with anti-BrdU and DAPI, and the cell cycle was analyzed by flow cytometry; some cells were lysed with Trizol to extract RNA, and the mRNA was reversed into cDNA for mRNA qPCR quantitative detection of related genes; some cells were lysed with RIPA to extract protein, which was separated by SDS-PAGE and analyzed by Western Blot with related antibodies.

[0119] The preparation methods of ICOS NP, CD40L NP and IL21 NP were the same as those in Example 6.

[0120] 2. Experimental Results

[0121] The results showed that the phosphorylation levels of PKC, AKT, NF-κB and other proteins in B cells were significantly increased after ICOS NP treatment ( Figure 7 At the same time, qPCR was also used to detect the expression of cell cycle-related genes, showing that the expression of Ccna2, Ccnd1, and Cdk4 was increased in B cells after ICOS NP treatment ( Figure 7 In particular, ICOS NP treatment increased the expression of the anti-apoptotic gene Mcl1 in B cells ( Figure 7 B) shows that ICOS NP can promote cell cycle and maintain cell survival phenotype.

[0122] Example 8 Protective Ability of SARS-CoV-2 ICOS-RBD Nanoparticle Vaccine in Immunodeficient Mice

[0123] The Beta, Delta or Omicron subtypes of the new coronavirus can directly infect wild-type mice due to the N501Y mutation on the Spike that increases the affinity for mouse ACE2. It can also directly infect CD4 Cre Bcl6 fl / fl mouse.

[0124] 1. Experimental Methods

[0125] To evaluate the effect of ICOS-carrying nanoparticles on CD4 Cre Bcl6 fl / fl To investigate the protective effect of HPF, RBD and ICOS-RBD nanoparticle vaccines on mice, mice were immunized twice in a prime-boost manner, with subcutaneous vaccination of CD4 Cre Bcl6 fl / fl Mice were immunized for 4 weeks and the serum of mice was tested by ELISA at 4 weeks. The SARS-CoV-2 Omicron EG.5 strain was then infected intranasally and the mice were euthanized on the 5th day after virus infection. The lungs of the mice were lysed with Trizol to extract total RNA, and the viral mRNA copy number was analyzed using the new coronavirus mRNA detection kit. The lungs of the mice were taken for H&E staining and IHC staining with anti-SARS-CoV-2N protein antibody ( Figure 8 A).

[0126] 2. Experimental Results

[0127] The results are as follows Figure 7 The results showed that the ICOS-RBD nanoparticle vaccine group produced higher RBD-specific IgG antibody titers than the HPF and RBD groups ( Figure 8 (B) After Omicron EG.5 infection, the average viral RNA in the lungs of HPF mice was 9.02×10 per μg of total RNA. 6 However, the ICOS-RBD nanoparticle vaccine group reduced viral replication by approximately 1660 or 10-fold compared to the HPF or RBD nanoparticle vaccine groups ( Figure 8 C).

[0128] H&E staining showed that the lungs of mice immunized with ICOS-RBD vaccine had significantly less inflammation than those in other groups ( Figure 8 D) in.

[0129] At the same time, the expression of SARS-CoV-2 nucleocapsid (N) antigen was always the lowest in the lungs of mice immunized with ICOS-RBD ( Figure 8 E).

[0130] In summary, ICOS-RBD NP induced a significant immune response and effectively suppressed CD4Cre Bcl6 fl / fl SARS-CoV-2 infection of mice.

[0131] Example 9 Preparation of HIV Nanoparticle Vaccine

[0132] 1. Experimental Methods

[0133] Because in HIV-1 infected patients, the virus directly infects CD 4+ T cells and can also damage T FH The cells cause severe immune deficiency / insufficiency, so the antibody levels of HIV patients after vaccination are severely reduced compared with healthy people.

[0134] The MD39 immunogen contains the N332 supersite epitope in the V3 loop of the HIV-1 gp120 outer membrane and can be used to induce broad-spectrum neutralizing antibodies against HIV. HIV-1 MD39 was selected as a model antigen to prepare nanoparticle vaccines and conjugated with ICOS ( Figure 9 A in the figure is its schematic diagram), and this is used to evaluate the effect of conjugated ICOS on the immune effect of HIV vaccine.

[0135] The ICOS-MD39 nanoparticle vaccine and the MD39 nanoparticle vaccine were constructed as follows:

[0136] The pcDNA3.1-MD39-SD-His eukaryotic expression plasmid was constructed according to the method of Example 1 (Steichen, J.M. et al. HIV Vaccine Design to Target Germline Precursors of Glycan-Dependent Broadly Neutralizing Antibodies. Immunity 45, 483-496, doi: 10.1016 / j.immuni.2016.08.016(2016).) (wherein the nucleotide sequence of MD39-SD-His is shown in SEQ ID NO: 17, and the amino acid sequence encoded by it is shown in SEQ ID NO: NO: 18) and transiently transfected into HEK293F cells. 5 days after transfection, the cell supernatant was collected. MD39-SD-His and ICOS-SD-His (prepared in Example 1) were then coupled to Gvo-HPF nanoparticles according to the method of Example 1. Coomassie brilliant blue staining and protein immunoblotting were then used to detect the expression of the above-mentioned target proteins and the assembly of the HIV-1 nanoparticle vaccine.

[0137] 2. Experimental Results

[0138] The results are as follows Figure 9B to C in the figure show that the nanoparticle vaccine (ICOS-MD39NP and MD39 NP) against HIV was successfully assembled.

[0139] Example 10 Immune Effects of HIV Nanoparticle Vaccine on Immunodeficient Mice

[0140] 1. Experimental Methods

[0141] ICOS-MD39 NP and MD39 NP prepared in Example 9 were used to immunize CD4 Cre Bcl6 fl / fl Mice, detection of HIV-1 antigen-specific serum IgG titer, urea-eluted high-affinity antibody titer, and Elispot analysis of HIV-1 antigen-specific antibody secreting cell number, Figure 9 D in is a flow chart.

[0142] 2. Experimental Results

[0143] The results showed that the levels of HIV Env-specific IgG and high-affinity antibodies in the ICOS-MD39 immunization group were significantly higher than those in the MD39 immunization group alone, and the levels could be maintained for several weeks ( Figure 9 ELISpot assay showed that the number of HIVEnv antigen-specific antibody-secreting cells increased significantly 8 weeks after immunization.

[0144] The above results indicate that ICOS can also enhance the antibody production and the number of antibody-secreting cells of HIV nanoparticle vaccines under immunodeficient conditions, which is beneficial for the vaccine of HIV-infected patients.

Claims

1. A novel coronavirus antigen with enhanced immunogenicity, characterized in that: The antigen is obtained by spontaneous chemical bonding of component 1 and component 2 through Gv-Sd, wherein component 1 is a mixture of a protein with an amino acid sequence such as SEQ ID NO: 12 and a protein with an amino acid sequence such as SEQ ID NO: 10; and component 2 is a protein with an amino acid sequence such as SEQ ID NO:

6.

2. Use of the novel coronavirus antigen according to claim 1 in the preparation of a novel coronavirus vaccine.

3. An HIV-1 viral antigen with enhanced immunogenicity, characterized in that: The antigen is obtained by spontaneous chemical bonding of component 1 and component 2 through Gv-Sd, wherein component 1 is a mixture of a protein with an amino acid sequence such as SEQ ID NO: 12 and a protein with an amino acid sequence such as SEQ ID NO: 18; and component 2 is a protein with an amino acid sequence such as SEQ ID NO:

6.

4. Use of the HIV-1 virus antigen according to claim 3 in the preparation of HIV-1 virus vaccine.

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