Vaccine for treating or preventing hepatitis B virus infection

Abstract

The invention relates to the technical field of biological medicines, in particular to a vaccine for treating or preventing hepatitis B virus infection. The vaccine comprises a hepatitis B surface antigen and a composite adjuvant, wherein the composite adjuvant is prepared from CpG oligodeoxynucleotide and saponin QS-21, and the composite adjuvant is prepared from CpG oligodeoxynucleotide and saponin QS-21; the nucleotide sequence of the CpG oligodeoxynucleotide is as shown in SEQ ID NO. 1. According to the vaccine provided by the invention, an HBsAg antibody can be generated in a normal mouse body, and HBV in an HBV-carrier mouse can be effectively eliminated, so that the vaccine has the effect of treating or preventing hepatitis B virus infection. According to the vaccine provided by the invention, two adjuvants, namely CpG oligodeoxynucleotide and saponin QS-21, are adopted, so that the vaccine can be used for synergistically activating an immune effect, enhancing the activation of B cells and permanently transforming into plasma cells, and meanwhile, the vaccine can be used for resisting immune tolerance of T cells, realizing the removal of hepatitis B viruses and realizing functional cure of clinical hepatitis B treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a vaccine for treating or preventing hepatitis B virus infection. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Hepatitis B virus (HBV) infection is a serious public health problem worldwide, and a major cause of chronic hepatitis B, cirrhosis, and hepatocellular carcinoma. Commonly used drugs for the clinical treatment of chronic HBV infection include nucleoside analogs and interferon. However, nucleoside analogs cannot completely eliminate cccDNA in hepatocytes, and long-term use easily leads to drug-resistant mutant strains and rebound after discontinuation. Interferon is not suitable for asymptomatic HBV carriers, and in chronic HBV patients, the HBeAg seroconversion rate is only 33% after six months of use, indicating limited therapeutic efficacy and significant side effects.

[0004] Currently, the main method for preventing hepatitis B is prevention through hepatitis B vaccination. Preventive vaccines are primarily used on individuals who have never been infected, as the naturally occurring viral proteins can directly serve as vaccine antigens. However, while existing hepatitis B vaccines offer good protection against HBV infection, they are less effective in treating and clearing the virus in already infected individuals. This is mainly because the body has developed specific immune tolerance to the hepatitis B virus, preventing the immune system from effectively eliminating the virus. Currently, there are no effective treatments or methods for hepatitis B virus carriers or infected individuals, especially those with chronic hepatitis B.

[0005] Therapeutic hepatitis B vaccines aim to break immune tolerance in patients with chronic hepatitis B virus (HBV), restore their immune response to achieve functional cure, and complete antigen clearance and seroconversion. Currently, the main therapeutic hepatitis B vaccines can be divided into four categories: protein vaccines, DNA vaccines, viral vector vaccines, and dendritic cell (DC) vaccines. However, all of these vaccines have certain limitations and do not achieve significant therapeutic effects. Summary of the Invention

[0006] To overcome the above problems, the present invention provides a vaccine for treating or preventing hepatitis B virus infection.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a vaccine for treating or preventing hepatitis B virus infection, comprising: hepatitis B surface antigen and a compound adjuvant; The compound adjuvant consists of CpG oligodeoxynucleotides and saponin QS-21. The nucleotide sequence of CpG oligodeoxynucleotide is shown in SEQ ID NO.1.

[0008] In one or more embodiments, the hepatitis B surface antigen is HBsAg produced by recombinant yeast or recombinant CHO cells, or an antigen containing HBsAg preS1 protein and / or preS2 protein.

[0009] In one or more embodiments, the mass ratio of CpG oligodeoxynucleotides to saponin QS-21 is 2:1.

[0010] In one or more embodiments, in a vaccine for treating or preventing hepatitis B virus infection, the concentration of hepatitis B surface antigen is 2 μg / injection, and the amount of compound adjuvant added is 15 μg / injection.

[0011] A second aspect of the present invention provides a vaccine for treating or preventing hepatitis B virus infection as described in the first aspect for use in the preparation of a pharmaceutical composition for treating or preventing hepatitis B virus infection.

[0012] In one or more embodiments, the vaccine for treating or preventing hepatitis B virus infection is administered via intramuscular, subcutaneous, or intradermal injection.

[0013] A third aspect of the invention provides a vaccine for treating or preventing hepatitis B virus infection as described in the first aspect for use in the preparation of a pharmaceutical composition for treating or preventing diseases caused by hepatitis B virus infection.

[0014] In one or more embodiments, the disease caused by the hepatitis B virus infection is selected from hepatitis B, cirrhosis, and liver cancer.

[0015] In one or more embodiments, the vaccine for treating or preventing hepatitis B virus infection is administered via intramuscular, subcutaneous, or intradermal injection.

[0016] A fourth aspect of the present invention provides a pharmaceutical composition comprising the vaccine for treating or preventing hepatitis B virus infection as described in the first aspect.

[0017] The beneficial effects of this invention are as follows: The vaccine provided by this invention not only induces HBsAg antibodies in normal mice but also effectively clears HBV from HBV-carrier mice, thus exhibiting therapeutic or preventative effects against hepatitis B virus infection. The vaccine utilizes two adjuvants: CpG oligodeoxynucleotides and saponin QS-21, which synergistically activate the immune response, enhance B cell activation, and persistently transform into plasma cells. Simultaneously, it resists T cell immune tolerance, achieving hepatitis B virus clearance and functional cure in clinical hepatitis B treatment. Specifically, the vaccine significantly increases the proportion of various dendritic cells (DCs) and CD80 expression levels in HBV-carrier mice, thereby activating innate immunity. It enhances the function of antigen-specific CD4+ T, CD8+ T cells, and NK cells in HBV-carrier mice, reversing their depletion state. Furthermore, it activates antigen-specific CD4+ T and CD8+ T cells in HBV-carrier mice, enhancing their ability to secrete IL-21 and TNF. Attached Figure Description

[0018] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0019] Figure 1 The levels of IgG antibodies and antibody subtypes induced by the vaccine in normal mice; Figure 2 The inhibitory effect of the vaccine on HBsAg in the serum of HBV-carrier mice; Figure 3 This study aimed to analyze the functional effects of the vaccine on dendritic cells (DCs) in HBV-carrier mice. AF represents the proportion of DCs in the spleen as determined by flow cytometry, and GL represents the CD80 MFI level of each DC. Figure 4 This study aimed to analyze the functional response of the vaccine to HBV-carrier mouse antigen-specific CD4+ T cells and CD8+ T cells. Specifically, A represents the proportion of HBV-specific CD4+ T cells in the spleen as detected by flow cytometry; B represents the TIM-3 MFI level in HBV-specific CD4+ T cells in the spleen; C represents the proportion of HBV-specific CD8+ T cells in the spleen as detected by flow cytometry; D represents the TIM-3 MFI level in HBV-specific CD8+ T cells in the spleen; and E represents the TIM-3 MFI level in NK cells in the spleen as detected by flow cytometry. Figure 5The image shows the ICS results of the vaccine on HBV-carrier mice, where A represents the detection of HBV-specific CD4 in the liver by flow cytometry. + B represents the level of IL-21 secreted by T cells; B is the level of HBV-specific CD4 in the liver detected by flow cytometry. + The proportion of T cells secreting TNF; C represents the percentage of HBV-specific CD8+ cells in the liver detected by flow cytometry. + The proportion of T cells that secrete TNF. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0021] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0022] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0023] HBV-carrier mouse model construction: Normal adult C57BL / 6J mice, aged 5-6 weeks, were injected with 0.3 × 10⁻⁶ mg / L via high-pressure tail vein injection over 5-8 seconds. 10 The HBV plasmid (pUF1-HBV-D-AYW1.3 plasmid, purchased from Guangzhou Paizhen Biotechnology Co., Ltd.) was injected in a volume of 100 μL. After 6 weeks, mouse serum was collected. Mice with serum HBsAg concentrations greater than 500 ng / mL were designated as HBV-carrier mice.

[0024] Example 1 Verification of the preventive efficacy of vaccines: Normal adult C57BL / 6J mice aged 5-6 weeks were used. The experimental group was injected intramuscularly with a vaccine for the treatment or prevention of hepatitis B virus infection (HBsAg+AS01B). HBsAg+AS01B consists of the following components: HBsAg 2 μg and compound adjuvant (CpG1018S 10 μg; QS21 5 μg). The blank control group was injected with the same volume of PBS. The mice were immunized three times at two-week intervals. Blood samples were collected at week 4 to detect antibody and genotyping levels.

[0025] The results are as follows Figure 1 As shown, compared with the blank control group, the experimental group was able to induce mice to produce antibodies against HBsAg, and the antibody subtype IgG2b level was higher. This indicates that vaccine immunization can induce mice to produce antibodies, and the mice tend to activate the Th1 immune response.

[0026] Example 2 Verification of the therapeutic efficacy of the vaccine: HBV-carrier mice were used. The experimental group was injected intramuscularly with HBsAg+AS01B, while the blank control group was injected with the same volume of PBS. The mice were immunized three times at two-week intervals. Blood samples were collected on day 7 and day 70 to detect antibody and typing levels.

[0027] The results are as follows Figure 2 As shown, compared with the blank control group, the experimental group was able to significantly reduce the level of HBsAg in the peripheral blood serum of HBV-carrier mice.

[0028] Example 3 HBV-carrier mice were used. The experimental group was injected intramuscularly with HBsAg+AS01B, while the blank control group was injected with the same volume of PBS. The mice were immunized three times at two-week intervals. After the three immunizations, mononuclear cells were isolated from the spleen of the mice. The differences in the proportions of DC, CD8a+CD103+cDC1, CD8a cDC1, CD103+cDC1, cDC2, and CD11b+axl+cDC2 cells were detected by flow cytometry. The expression level of CD80 on each DC cell was also detected by flow cytometry.

[0029] The results are as follows Figure 3 As shown, compared with the blank control group, the experimental group significantly increased the proportion of each DC cell and the expression level of CD80 in HBV-carrier mice.

[0030] Example 4 HBV-carrier mice were used. The experimental group was injected intramuscularly with HBsAg+AS01B, while the blank control group was injected with the same volume of PBS. The mice were immunized three times at one-week intervals. After the three immunizations, mononuclear cells were isolated from the spleen of the mice. The difference in the proportion of antigen-specific CD4+ T and CD8+ T cells was detected by flow cytometry. The expression level of TIM-3 on antigen-specific CD4+ T, CD8+ T cells and NK cells was detected by flow cytometry.

[0031] The results are as follows Figure 4 As shown, compared with the blank control group, the proportion of antigen-specific CD4+ T cells and CD8+ T cells was significantly increased in the spleen of mice after immunotherapy. Furthermore, vaccine immunization could reduce the expression of the immune checkpoint TIM-3, reversing the immune exhaustion state induced by chronic HBV infection. Therefore, HBV recombinant protein vaccine immunization can enhance the function of antigen-specific CD4+ T cells, CD8+ T cells, and NK cells in HBV-carrier mice, reversing their exhaustion state.

[0032] Example 5 HBV-carrier mice were used. The experimental group was injected intramuscularly with HBsAg+AS01B, while the blank control group was injected with the same volume of PBS. The mice were immunized three times at one-week intervals. After the three immunizations, mononuclear cells were isolated from the spleen of the mice. The difference in the proportion of antigen-specific CD4+ T and CD8+ T cells was detected by flow cytometry. The secretion levels of IL-21 and TNF by antigen-specific CD4+ T and CD8+ T cells were also detected by flow cytometry.

[0033] The results are as follows Figure 5 As shown, compared with the blank control group, the vaccine immunized and activated antigen-specific CD4+ T and CD8+ T cells in HBV-carrier mice, and enhanced their secretion of IL-21 and TNF.

[0034] The vaccine provided by this invention uses two adjuvants, CpG oligodeoxynucleotides and saponin QS-21, which can synergistically activate the immune response, enhance B cell activation, and persistently transform into plasma cells. Simultaneously, it can resist T cell immune tolerance, achieving hepatitis B virus clearance and functional cure in clinical hepatitis B treatment. Specifically, the vaccine provided by this invention can significantly increase the proportion of various dendritic cells (DCs) and the expression level of CD80 in HBV-carrier mice, thereby activating innate immunity; it can enhance the function of antigen-specific CD4+ T, CD8+ T cells, and NK cells in HBV-carrier mice, reversing their depletion state; and it can activate antigen-specific CD4+ T and CD8+ T cells in HBV-carrier mice, enhancing their ability to secrete IL-21 and TNF.

[0035] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A vaccine for treating or preventing hepatitis B virus infection, characterized in that, include: Hepatitis B surface antigen and compound adjuvant; The compound adjuvant consists of CpG oligodeoxynucleotides and saponin QS-21. The nucleotide sequence of CpG oligodeoxynucleotide is shown in SEQ ID NO.

1.

2. The vaccine as described in claim 1, characterized in that, The hepatitis B surface antigen is HBsAg produced by recombinant yeast or recombinant CHO cells, or antigen containing HBsAg preS1 protein and / or preS2 protein.

3. The vaccine as described in claim 1, characterized in that, The mass ratio of CpG oligodeoxynucleotides to saponin QS-21 is 2:

1.

4. The vaccine as described in claim 1, characterized in that, The concentration of hepatitis B surface antigen is 2 μg / injection, and the amount of compound adjuvant added is 15 μg / injection.

5. The vaccine according to any one of claims 1 to 4 is used to prepare a pharmaceutical composition for treating or preventing hepatitis B virus infection.

6. The application as described in claim 5, characterized in that, The vaccine is administered via intramuscular, subcutaneous, or intradermal injection.

7. The vaccine according to any one of claims 1 to 4 is used to prepare a pharmaceutical composition for treating or preventing diseases caused by hepatitis B virus infection.

8. The application as described in claim 7, characterized in that, The vaccine is administered via intramuscular, subcutaneous, or intradermal injection.

9. The application as described in claim 7, characterized in that, The diseases caused by hepatitis B virus infection are selected from hepatitis B, cirrhosis, and liver cancer.

10. A pharmaceutical composition, characterized in that, Includes the vaccine as described in any one of claims 1 to 4.

Images