Vaccine adjuvant and application

Abstract

The invention provides a novel vaccine adjuvant and application of the novel vaccine adjuvant in preparation of the vaccine adjuvant. The BCG-PSN and vaccine induced RBD specific IgG antibody and neutralizing antibody level are equivalent to those of an aluminum adjuvant, the BCG-PSN and vaccine induce Th1 biased T cell reaction, the induced T cell response level is obviously higher than that of the aluminum adjuvant, and K18-hACE2 mice are protected from being affected by Delta variant induced lung lesions. As a vaccine adjuvant, BCG-PSN has wide compatibility and safety, and has no toxic or side effect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of BCG polysaccharide nucleic acid in the preparation of adjuvants or vaccine compositions for novel coronavirus (COVID-19) vaccines. Background Technology

[0002] Immunization is directly related to the prevention of viral infection, reduction of viral transmission, and decrease in the incidence of severe disease. Inactivated COVID-19 vaccines have been widely used and proven effective against SARS-CoV-2. However, inactivated vaccines alone induce low levels of immunity, making the addition of appropriate vaccine adjuvants crucial for vaccine efficacy. Adjuvants are substances that enhance specific immune responses, activating the innate immune system and thus enhancing adaptive immune responses to antigens. Despite extensive research on vaccine adjuvants, the number of commercially available human vaccine adjuvants is currently limited, such as aluminum hydroxide, MF59, AS01, AS03, AS04, and CpG 1018. Aluminum adjuvants are the most commonly used, acting as antigen storage agents while improving antigen uptake and promoting antigen presentation. However, aluminum adjuvants (aluminum hydroxide) only enhance immune responses to a limited extent, primarily promoting humoral immunity (Th2) with minimal impact on cellular immunity (Th1), limiting their application in vaccines targeting cellular immunity. Aluminum adjuvants (aluminum hydroxide) can also induce IL-1 production, causing adverse reactions such as headache, joint pain, muscle pain, and injection site reactions. To overcome the drawbacks of routine use of aluminum adjuvants alone, it is necessary to explore a novel COVID-19 vaccine adjuvant and its immunization strategy. Summary of the Invention

[0003] This invention addresses the shortcomings of existing aluminum adjuvant applications by providing a novel vaccine adjuvant and its uses.

[0004] A vaccine adjuvant comprising BCG polysaccharide nucleic acid extracted from BCG.

[0005] Preferably, the ratio of polysaccharide to nucleic acid in the BCG polysaccharide nucleic acid is 5-7:1-3.

[0006] Preferably, the ratio of polysaccharide to nucleic acid in the BCG polysaccharide nucleic acid is 6:1.

[0007] Preferably, the ratio of polysaccharide to nucleic acid in the BCG polysaccharide nucleic acid is 7:1.

[0008] Preferably, the ratio of polysaccharide to nucleic acid in the BCG polysaccharide nucleic acid is 7:3.

[0009] Preferably, the ratio of polysaccharide to nucleic acid in the BCG polysaccharide nucleic acid is 5:2.

[0010] Preferably, the ratio of polysaccharide to nucleic acid in the BCG polysaccharide nucleic acid is 6:3.

[0011] The present invention provides a vaccine composition comprising a vaccine and the aforementioned vaccine adjuvant; the vaccine is preferably a novel coronavirus vaccine.

[0012] This invention proposes a vaccine composition comprising a vaccine and the aforementioned vaccine adjuvant (BCG polysaccharide nucleic acid) and aluminum adjuvant; the vaccine is preferably a novel coronavirus vaccine.

[0013] This invention also proposes the use of BCG polysaccharide nucleic acid in the preparation of the aforementioned vaccine adjuvants or vaccine compositions. BCG polysaccharide nucleic acid induces a Th1-biased T cell response, with the induced T cell response level being significantly stronger than that induced by aluminum adjuvant.

[0014] BCG-PSN is an immunomodulatory substance extracted from BCG using the hot phenol method. It contains polysaccharides and nucleic acids (such as CpG motifs, including GACGTT, AACGTT, GTCGTT, and TTCGT). The main components of the polysaccharide are glucan, mannan, and arabinomannan. Polysaccharides are major components of the BCG cell wall cytoskeleton and can effectively induce dendritic cells and act as adjuvants. The BCG genomic DNA contains a rich CpG DNA matrix, which can function as an adjuvant for Th1 responses. BCG-PSN can induce Th1-mediated immune responses by promoting the differentiation of T cells into Th1 subsets and activating the Toll-like receptor (TLR) signaling pathway, thereby inducing the secretion of Th1 cytokines (such as IL-2 and IFN-γ).

[0015] The beneficial effects of this invention are as follows: This invention uses the original SARS-CoV-2 strain antigen combined with aluminum adjuvant and / or BCG-PSN for inoculation, and measures are taken to detect antibody levels, germinal center responses (GC), and the number and function of memory T cells in the spleen. The BCG-PSN vaccine adjuvant group significantly increased IgG antibody levels, induced neutralizing antibody levels comparable to those induced by aluminum adjuvant, and induced a Th1-biased T cell response, with significantly stronger T cell responses than aluminum adjuvant. It also significantly enhanced spleen GC structure and protected K18-hACE2 mice from lung lesions induced by the Delta variant. As a vaccine adjuvant, compared to metal salt adjuvants such as aluminum adjuvant, which are not easily metabolized in vivo and have the risk of accumulation, BCG-PSN has broad compatibility and safety, and no toxic side effects. The immune composition obtained in this invention (BCG polysaccharide nucleic acid adjuvant + SARS-CoV-2 original strain antigen (COVID-19 inactivated vaccine)) has a better immune effect than the effect of using COVID-19 inactivated vaccine alone, and also better than the immune effect of vaccines using aluminum adjuvant. Attached Figure Description

[0016] Figure 1 Immune procedure diagram

[0017] Figure 2 Antibody response in immunized mice. Serum samples were collected from BALB / c mice (A to F) and K18-hACE2 mice (G to L) on day 28, and serum samples were collected at different time points (F and L). (A and G) Anti-S protein IgG antibody level, (B and H) Anti-RBD IgG antibody level, (C) Anti-RBD IgG2a:IgG1, (D and J) Anti-RBD IgG1, (E) IgG2a antibody level, (F) Anti-RBD IgM antibody level, (I) Anti-RBD IgG2c:IgG1, (L) to assess the dynamic changes in anti-RBD IgG antibody levels over 6 consecutive months. n = 5 or 10 mice. Bar graphs represent mean ± SD. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, no significance (nes): P>0.05.

[0018] Figure 3 Antibody response of immune serum to SARS-CoV-2 (Delta) and neutralizing antibody levels against SARS-CoV-2 (original strain) and its variants (Delta). (A) Level of anti-RBD IgG (Delta) in BALB / c mouse serum at D28. (B) Neutralizing antibody (NAb) response in BALB / c mouse serum at D28. (C) Level of anti-RBD IgG (Delta) in K18hACE2 mouse serum at D28. (D) NAb response in K18-hACE2 mouse serum at D28. (E) Level of anti-RBD IgG (Delta) in K18-hACE2 mouse serum 32 weeks after the first immunization. (F) NAb response in K18-hACE2 mouse serum 32 weeks after the first immunization. Bars represent mean ± SD. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, no significance (nes): P>0.05.

[0019] Figure 4 Hematoxylin-eosin (HE) staining and immunofluorescence of spleen from K18-hACE2 mice immunized on day 28. (A) HE staining of spleen. WP: white pulp; MZ: marginal zone; RP: red pulp of spleen; (B) Immunofluorescence showing the reproductive center (GC) reaction of K18-hACE2 mice.

[0020] Figure 5 T cell response in K18-hACE2 mice. (A, B) Detection of IFN-γ ELISpot and IL-2 ELISpot in mice from different groups. (C) Cytokine levels on day 28 of different immunization regimens. Bar graphs represent mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, no significant difference (nes): P>0.05.

[0021] Figure 6 Flow cytometry analysis revealed CD4+ levels in K18-hACE2 mice on day 28 and week 25. 4+ and CD 8+ Memory T cells. (A) Ratio of CD4+ and CD8+ memory T cells at day 28 and week 25. (B) CD4+ and CD8+ memory T cells at day 28. 4+ and CD 8+ Memory T cells. Bar graphs represent mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, not significant (nes): P>0.05.

[0022] Figure 7 The BCG-PSN adjuvant vaccine protected K18-hACE2 mice from SARS-CoV-2 infection. Eight weeks after the third booster immunization, K18-hACE2 mice were intranasally vaccinated with 10 6 TCID 50 Delta variant. (A) Body weight changes were assessed daily until day 5 post-infection. (B) Lung tissue was collected on day 5 post-Delta virus infection, and ORF1ab RNA levels were detected by qRT-PCR. (C) Cytokine levels in the supernatant of mouse lung tissue on day 5 post-infection. (D) Hematoxylin-eosin staining and imaging of lung tissue sections on day 5 post-infection. Data are results from three independent experiments. Bars represent mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, no significant difference (nes): P>0.05.

[0023] Figure 8 Changes in body weight of K18-hACE2 mice, bars represent mean ± SD. Detailed Implementation

[0024] Example 1: BCG polysaccharide-nucleic acid vaccine adjuvant and its enhancing effect on the immunogenicity of SARS-CoV-2 antigen. 1. Materials and methods

[0025] 1.1 Adjuvants and antigens

[0026] The COVID-19 inactivated vaccine was provided by Zhejiang Tianyuan Biopharmaceutical Co., Ltd. The vaccine adjuvant (BCG polysaccharide-nucleic acid) was provided by Hunan Siqi Biopharmaceutical Co., Ltd. Vaccine adjuvant 1 (BCG-PSN-1) contained 99% polysaccharide purity, with a sample concentration of 400 ug / ml. Vaccine adjuvant 2 (BCG-PSN-2) was a polysaccharide-nucleic acid mixture (polysaccharide 280 ug / ml, nucleic acid 120 ug / ml, polysaccharide-nucleic acid ratio = 7:3). Vaccine adjuvant 3 (BCG-PSN-3) was a polysaccharide-nucleic acid mixture (polysaccharide 340 ug / ml, nucleic acid 60 ug / ml, polysaccharide-nucleic acid ratio = 5.67:1 ≈ 6:1). Alum adjuvant was purchased from Thermo Fisher Scientific (77161).

[0027] 1.2 Vaccines and Immunization Programs

[0028] Before the experiment, the mice were allowed to acclimatize to the environment for one week. Each group (n=10) of 6-8 week old female BALB / c mice were intraperitoneally injected with 500 μl of vaccine (some groups received aluminum adjuvant Alum, some did not) or PBS, immunized twice, with an interval of 2 weeks (days 0 and 14). Figure 1 (Table 1). K18-hACE2 mice: Each group (n=10) of 6-week-old K18-hACE2 mice were intraperitoneally injected with 500 μl of vaccine (some groups with aluminum adjuvant, some without aluminum adjuvant Alum) or PBS, immunized three times (on days 0, 14, and 168). Figure 1 (Table 1). The vaccine adjuvant (BCG-PSN adjuvant) and the inactivated vaccine were administered separately. Two doses of BCG-PSN adjuvant were administered on the first and third days after each dose of inactivated vaccine. Blood and spleen samples were collected at different time points after immunization. Figure 1 Blood samples were collected on days 0, 7, 13, 21, 28, 42, 56, 84, 112, 140, 167, and 175. Spleen samples were collected on days 28 and 175, and lung samples were collected on day 229. Samples were prepared according to Table 1.

[0029] Table 1 Immunization Schedule

[0030]

[0031] 1.3 Enzyme-linked immunosorbent assay

[0032] Serum IgG and IgG antibody levels were detected using enzyme-linked immunosorbent assay (ELISA). SARS-CoV-2 RBD or S protein was coated onto an ELISA plate and incubated overnight at 4°C. Blocking buffer was used for 2 hours at room temperature. Serum samples were diluted, added to wells, and incubated for 2 hours. After three washes, biotin-bound anti-mouse IgG, IgG1, IgG2a, IgG2c, or IgM was added to the wells and incubated for 1 hour. After three washes, streptavidin (HRP) (abcam) was added and incubated for 40 minutes. After five washes, tetramethylbenzidine (TMB, 100 μL) substrate solution was added for development. After 5 minutes, sulfuric acid was added to terminate the reaction. The absorbance (OD) was measured at 450 nm, and the antibody content in the sample was calculated. The result was interpreted (OD ratio ≥ 2.1 was considered positive).

[0033] 1.4 Micro-neutralization detection

[0034] The serum was initially diluted 1:10, and then serially diluted 2-fold (columns 1-10). 50 μL of diluted serum was mixed with 50 μL of SARS-CoV-2 virus (100 TCID50). 50 Mix and incubate at 35°C and 5% CO2 for 2 hours. Then, add 100 μl of Vero cell suspension (per well). 4 Cells were cultured at 35°C and 5% CO2 for 5 days. After the culture was completed, the results were confirmed by evaluating the cytopathic effect (CPE).

[0035] 1.5 ELISPOT Detection

[0036] Add freshly isolated spleen cells to the wells of the ELISPOT plate, 4 × 10⁶ cells per well. 5 Individual cells were processed according to the ELISPOTplus kit, following the manufacturer's instructions. T cell responses were induced with 5 μg / mL SARS-CoV-2 mixed antigen. Negative control wells were treated with culture medium only, while positive control wells were treated with PMA / ionomycin. Spot Imaging Analyzer ( Analyzers were used to perform spot counting analysis.

[0037] 1.6 Histopathology and Immunofluorescence

[0038] Mouse spleen and lung tissues were fixed in 4% paraformaldehyde for 2 days, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The sections were first deparaffined with xylene, then rehydrated with a series of concentrations of alcohol. The sections were then placed in citrate buffer and subjected to antigen retrieval in a microwave oven for 15 minutes. Subsequently, the slides were washed three times with distilled water. The slides were incubated with 3% bovine serum albumin at room temperature for 20 minutes, then sequentially incubated with peanut lectin (PNA, 1:100), streptavidin-Cy3 (Streptavidin-Cy3, 1:300), and B220 (1:200). Horseradish peroxidase (HRP) labeling was then performed using Perkin Elmer multicolor fluorescent staining reagent, followed by nuclear staining, mounting, and finally image acquisition using an EVOS M7000 microscope scanning system.

[0039] 1.7 Cytokine Analysis

[0040] Immunized mouse spleen cells were cultured in 96-well plates, with 4 × 10⁶ cells per well. 5 Freshly isolated spleen cells were stimulated with a 5 μg / mL SARS-CoV-2 peptide library for 20 hours, and the supernatant was collected. Lung tissue obtained on day 5 post-infection was homogenized with 10 mg of lung tissue in 80 μl PBS, and the resulting supernatant was collected for analysis. Cytokines were detected using the V-PLEX Proinflammatory Panel 1 kit.

[0041] 1.8 Flow cytometry

[0042] Splenic cells were collected, passed through a 70 μm filter, and 1 × 10⁻⁶ cells were collected per well. 6 Cells were cultured in 96-well plates. Dead cells were removed with FVS700 dye. Spleen samples were then incubated with anti-CD3-FITC (1:50), anti-CD4-PE-cy7 (1:50), anti-CD8-BV605 (1:50), anti-CD44-BUV395 (1:50), and anti-CD62L-BV510 (1:50) at 4°C for 30 minutes. Samples were analyzed using a CytoFLEX flow cytometer.

[0043] 1.9 K18-hACE2 mouse infection test

[0044] K18-hACE2 mice were administered Figure 1 Immunization was performed according to Table 1. On day 224, the Delta variant virus (1×10⁻⁶) was administered. 6 TCID 50 (Nose-immunized mice) Five days after infection, all animals were euthanized and lung tissue was collected for subsequent viral RNA detection and histopathological examination.

[0045] 1.10 Quantitative analysis of SARS-CoV-2 viral RNA

[0046] Take lung tissue samples (20 mg) from infected mice, add 200 μl of sterile PBS to the lung tissue for homogenization, extract viral RNA from the lung tissue with trizol, and detect it using a novel coronavirus (2019-nCoV) qRT-PCR kit.

[0047] 1.12 Statistical Analysis

[0048] Data were analyzed using GraphPad Prism. Analysis of variance, Mann-Whitney test, or t-test were used to determine statistical significance between different groups.

[0049] 2. Experimental Results

[0050] 2.1 BCG-PSN enhances humoral immune response

[0051] We first evaluated whether different adjuvants could promote the immunogenicity of the inactivated vaccine. Fourteen days after the second dose (D28), we assessed the humoral immune response. Compared with the antigen alone, the BCG-PSN adjuvant increased IgG and IgM antibody titers in BALB / c and K18-hACE2 mice. Figure 2 Aluminum adjuvant and BCG-PSN-1 induced antibody responses favored IgG1 (). Figure 2 The antibody responses induced by BCG-PSN-2 and BCG-PSN-3 are biased towards IgG2a ( Figure 2 The combination of Alum / BCG-PSN-3 induced the highest levels of IgM, IgG, IG1, and IgG2a antibodies. Figure 2 Furthermore, the level of RBD-specific antibodies remained high even after 6 months. Figure 2 ).

[0052] 2.2 The inactivated COVID-19 vaccine with BCG-PSN as adjuvant has broad-spectrum neutralizing activity.

[0053] The neutralization experiment results were consistent with those of the enzyme-linked immunosorbent assay (ELISA). The level of neutralizing antibodies in the BCG-PSN group was higher than that in the antigen-only group after the second immunization (D28). Figure 3(A to D). Compared with the antigen group, the Alum / BCG-PSN-3 group induced higher levels of neutralizing antibodies (NAb) against the original SARS-CoV-2 strain in serum, with a geometric mean titer (GMT) of 1114 in BALB / c mice and 1940 in K18-hACE2 mice, both higher than the Alum or BCG-PSN groups. Neutralizing antibody levels against the Delta variant decreased in all groups compared to the original strain; however, the decrease observed in the Alum / BCG-PSN-3 group was smaller (the GMT decreased 3.4-fold in the Alum / BCG-PSN-3 group at day 28, compared to 4.0-fold to 6.0-fold decreases in the other groups). Figure 3 All groups showed low neutralizing efficacy against the Omeprone variant. Following the third immunization, the Alum / BCG-PSN-3 group had higher levels of anti-RBD IgG (Delta) and neutralizing antibodies than the Alum group. Figure 3 In K18-hACE2 mice, the GMT value of the original SARS-CoV-2 strain reached 5113.

[0054] 2.3 H&E staining and immunofluorescence of spleen

[0055] The BCG-PSN-2 and Alum / BCG-PSN-3 groups showed higher lymphocyte counts in the spleen's white pulp (WP) and marginal zone (MZ). A large number of erythrocytes were observed in the spleen's red pulp (RP). The erythrocyte volume in the spleen of the BCG-PSN group was larger than that of the Alum or antigen groups. Figure 4 A), indicating that BCG-PSN enhances the immune response. Consistent with previous experimental results regarding humoral immune responses, immunofluorescence examination of spleen sections showed strong activation of GC structures within the spleen, particularly in the BCG-PSN-2 and Alum / BCG-PSN-3 groups. Figure 4 B).

[0056] 2.4 Inactivated COVID-19 vaccines with BCG-PSN as adjuvant can enhance antigen-specific immune responses.

[0057] Vaccine-induced T-cell responses were assessed 14 days after the second immunization (D28) and 7 days after the third immunization (W25). Spleen cells were collected for ELISApot analysis of IFN-γ and IL-2. Figure 5(A and B). Compared with Alum adjuvant, both BCG-PSN / Alum and BCG-PSN alone induced a significant increase in IFN-γ and IL-2 responses at D28 (p<0.05), and this response further increased after the third immunization (W25). Multiple cytokine analyses showed that the vaccine composition containing BCG-PSN adjuvant induced a superior T-cell response compared to the Alum adjuvant vaccine, with higher IFN-γ secretion levels in the BCG-PSN-2 group than in the BCG-PSN-3 group. Consistent with previous results regarding humoral immune responses, the BCG-PSN-2 and BCG-PSN-3 groups induced increased production of Th1 cytokines IFN-γ / IL-2 / TNF-α, while Alum / BCG-PSN-3 induced balanced production of Th1 and Th2 cytokines. Figure 5 C).

[0058] 2.5 COVID-19 inactivated vaccines adjuvanted with BCG-PSN can induce high levels of memory T cells.

[0059] Compared with the Alum adjuvant group, the percentage of effector memory T cells (CD44+CD62L-T cells) in spleen cells of the BCG-PSN group was significantly increased after the second injection (D28) and the third injection (W25). Figure 6 The levels of CD4+ memory T cells induced by BCG-PSN adjuvant plus vaccine were comparable to those promoted by aluminum adjuvant, suggesting that BCG-PSN adjuvant plus vaccine can provide long-term protection for the body.

[0060] 2.6 Evaluation of the protective efficacy of vaccination

[0061] Eight weeks after the third immunization, K18-hACE2 mice were intranasally inoculated with the Delta variant, and their tissues were collected for virological evaluation. Mice were sacrificed on day 5 post-infection to analyze viral concentration and histopathological condition. K18-hACE2 mice in the BCG-PSN-2 and BCG-PSN-3 groups did not experience weight loss within 5 days post-infection, while mice in the antigen and aluminum adjuvant groups experienced rapid weight loss exceeding 5% within 5 days post-infection. Figure 7 A). Further evidence from lung virus titer assays, histopathological analysis, and lung and cytokine assays confirmed that K18-hACE2 mice vaccinated with BCG-PSN-2, BCG-PSN-3, and Alum / BCG-PSN-3 showed reduced SARS-CoV-2 infection rates, demonstrating the immunoprotective effect of BCG-PSN adjuvant + vaccine. Figure 7 BCG-PSN adjuvant plus vaccine protects hACE2 mice from lung lesions caused by Delta variant.

[0062] 2.7 Safety of Adjuvants

[0063] The body weight of K18-hACE2 mice was measured for up to 6 months, and the results showed that the BCG-PSN adjuvant had no significant toxic effects on the mice. Figure 8 ).

Claims

1. A vaccine adjuvant, characterized in that, A vaccine comprising a BCG polysaccharide nucleic acid extracted from BCG.

2. The vaccine adjuvant of claim 1, characterized in that, The polysaccharide to nucleic acid ratio in the BCG polysaccharide nucleic acid is 5-7:1-3.

3. The vaccine adjuvant of claim 2, characterized in that, The polysaccharide to nucleic acid ratio in the BCG polysaccharide nucleic acid is 6:

1.

4. The vaccine adjuvant of claim 2, characterized in that, The polysaccharide to nucleic acid ratio in the BCG polysaccharide nucleic acid is 7:

1.

5. The vaccine adjuvant of claim 2, wherein, The polysaccharide to nucleic acid ratio in the BCG polysaccharide nucleic acid is 7:

3.

6. The vaccine adjuvant of claim 2, characterized in that, The polysaccharide to nucleic acid ratio in the BCG polysaccharide nucleic acid is 5:

2.

7. Use according to claim 2, characterized in that, The polysaccharide to nucleic acid ratio in the BCG polysaccharide nucleic acid is 6:

3.

8. A vaccine composition, characterized in that, A vaccine and vaccine adjuvant of any one of claims 1-7.

9. The vaccine composition of claim 8, wherein, The vaccine is a vaccine for COVID-19.

10. Use of a BCG polysaccharide nucleic acid in the manufacture of a vaccine adjuvant or vaccine composition of any one of claims 1-9.

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