Recombinant VZV-TB double-target Fc vaccine

Abstract

The invention relates to the technical field of vaccines, and provides a recombinant VZV-TB double-target Fc vaccine, which comprises three functional elements, namely a mycobacterium tuberculosis (TB) antigen 85B (Ag85B), a varicella-zoster virus (VZV) antigen glycoprotein E (gE) and an immunoglobulin Fc, and the functional elements are directly or indirectly connected through a linker; a mouse is immunized by the recombinant VZV-TB double-target Fc vaccine once a week and four times in total, the immunized mouse is detected to generate antibodies simultaneously aiming at gE and Ag85B in vivo, splenocytes of the mouse are detected by adopting an elispot method, and after the mouse is respectively stimulated by gE and Ag85B antigens, different degrees of cellular immune effects can be simultaneously generated, so that the immune effect of the gE and the Ag85B can be realized, and the immune effect of the gE and the Ag85B can be realized. It is preliminarily proved that the double-target vaccine has the capacity of causing the specific immune response of the organism, and a theoretical and experimental basis is provided for entering human vaccines in the later period.

Description

Technical Field

[0001] The invention relates to the technical field of vaccines, in particular to a recombinant VZV-TB dual-target Fc vaccine. Background Art

[0002] As the average life expectancy in my country continues to grow, the number of people over 65 years old is also increasing. In some developed areas, the elderly population has reached 1 / 3 of the total population, and the health problems of the elderly have increasingly become a focus of attention. Among the elderly population, infections and recurrences of various infectious diseases caused by decreased immune function account for a large proportion, and some of them can currently be prevented through immune strengthening.

[0003] The incidence of tuberculosis in the elderly in China has shown a gradual upward trend in recent years. This trend is related to the decline and decline of the immune function of the elderly. With the increase of age, the immune function gradually weakens, leading to an increase in endogenous recurrence and exogenous re-infection. Another high-incidence disease among middle-aged and elderly people is herpes zoster, and the onset of herpes zoster is also due to immune failure during childhood and the decline of immune function in the elderly. In my country, there are about 1.56 million new cases of herpes zoster in people aged 50 and above each year. People aged 50 and above are a high-risk group for herpes zoster. The older they are, the more likely they are to suffer from herpes zoster and the more serious the condition is. Even if they are healthy, the risk of herpes zoster in middle-aged and elderly people will increase sharply after the age of 50.

[0004] Varicella-zoster virus (VZV) encodes nine membrane glycoproteins, three of which exist in the form of heterodimers. Antigenic glycoprotein E (gE) is the most abundant glycoprotein expressed on the surface of VZV, and gE is the main target of VZV-specific antibodies and T cell responses. gE can promote viral transmission and skin damage, and can therefore be used as a VZV vaccine target antigen. Although there are both tuberculosis vaccines and shingles vaccines on the market, considering the low compliance of the elderly with immunization and vaccination, as well as their general concerns about the cost of vaccination, the development of a one-time multi-antigen vaccine is of great practical significance. Summary of the invention

[0005] The present invention provides a recombinant VZV-TB dual-target Fc vaccine. By immunizing mice with the recombinant VZV-TB dual-target Fc vaccine, it is preliminarily proved that the dual-target vaccine has the ability to induce a specific immune response in the body, which provides a theoretical and experimental basis for the later development of human vaccines to solve the problems raised in the background technology.

[0006] The technical solution of the present invention is as follows:

[0007] A recombinant VZV-TB dual-target Fc vaccine comprises three functional components: Mycobacterium tuberculosis (TB) antigen 85B (Ag85B), varicella-zoster virus (VZV) antigen glycoprotein E (gE) and immunoglobulin (Fc), and these functional components are directly or indirectly connected through a linker.

[0008] As a technical solution of the present invention, the Fc region to which the immunoglobulin belongs can be a constant region amino acid sequence from IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2, wherein IgG4 is preferred;

[0009] IgG2 and IgG4 have weaker ability to induce ADCC and CDC effects, but longer serum half-life, and are very suitable as preventive vaccines; the Fc region can be the normal constant region sequence of IgG4 or a constant region sequence containing mutation points, and these mutations include but are not limited to S36T, V90P, N216A, S10P, E15P, F16V, L17A, D47A and R191K, which can further reduce the ADCC caused by Fc, and the normal cell damage caused by nonspecific cytotoxicity caused by CDC; Fc (S10P) is preferred in the present invention.

[0010] As a technical solution of the present invention, the VZV-TB dual-target Fc vaccine containing three functional elements is a heterodimer Ag85B-gE-Fc, which includes two separate fusion proteins, namely fusion protein 1 and fusion protein 2. Fusion protein 1 includes Ag85B protein and immunoglobulin Fc region (Ag85B-Fc) from N-terminus to C-terminus, and fusion protein 2 includes gE protein and immunoglobulin Fc region (gE-Fc) from N-terminus to C-terminus. The immunoglobulin Fc region contained in fusion protein 1 and the immunoglobulin Fc region contained in fusion protein 2 are derived from immunoglobulins of the same subtype.

[0011] As a technical solution of the present invention, the heterodimer Ag85B-gE-Fc is mixed with an adjuvant and used for immunization;

[0012] The adjuvants used include, but are not limited to, mineral salts, such as aluminum hydroxide (also known as aluminum adjuvants) and aluminum phosphates; oil / water emulsions, such as MF59; particulate adjuvants, such as viroids composed of viral membrane proteins embedded in a double membrane, immune stimulating complexes (ISCOMS), etc.; derivatives from bacterial components, such as bifidobacterium; derivatives from plant components, such as purified saponins (such as QS21); cytokines, such as GM-CSF, IL-12 and IL-2, and novel adjuvants containing nucleic acid components, such as CpGODN, PolyI:C, etc., which can be used to enhance the immune effect of adjuvant components; since the immune response of the elderly is weaker, strong adjuvants such as QS21 can be preferred.

[0013] As a technical solution of the present invention, it includes a nucleic acid molecule, which encodes the fusion protein 1Ag85B-Fc.

[0014] As a technical solution of the present invention, it includes a nucleic acid molecule, which encodes the fusion protein 2gE-Fc.

[0015] As a technical solution of the present invention, it includes a vector, wherein the vector contains a nucleic acid molecule encoding the fusion protein 1Ag85B-Fc.

[0016] As a technical solution of the present invention, it includes a vector, wherein the vector contains a nucleic acid molecule encoding the fusion protein 2gE-Fc.

[0017] As a technical solution of the present invention, it includes a cell, which contains the dimeric fusion protein Ag85B-gE-Fc of the present invention, a nucleic acid molecule encoding the fusion protein or a vector expressing the fusion protein, and is used to produce the fusion protein; the cell is selected from eukaryotic animal cells, preferably CHO and HEK293 cells.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The present invention observes and demonstrates that mice immunized with the recombinant VZV-TB dual-target Fc vaccine produce antibodies against both gE and Ag85B in the mice after immunization. The elispot method is used to detect the spleen cells of the mice. After stimulation with gE and Ag85B antigens respectively, different degrees of cellular immune effects can be simultaneously produced. This preliminarily proves that the dual-target vaccine has the ability to induce specific immune responses in the body, providing a theoretical and experimental basis for the later entry into human vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the structure of the recombinant dual antigen Ag85B-gE-Fc (S10P) of the present invention;

[0021] Figure 2 It is the production of the Ag85B antibody of the present invention;

[0022] Figure 3 is the production of gE antibody of the present invention;

[0023] Figure 4 The secretion of INF-γ from mouse spleen cells stimulated by Ag85B in vitro;

[0024] Figure 5 This is the situation of INF-γ secretion of mouse spleen cells stimulated by gE of the present invention in vitro. DETAILED DESCRIPTION

[0025] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0026] The present invention provides a recombinant VZV-TB dual-target Fc vaccine. In order to prove that the vaccine has an immune effect, the following experiments were conducted.

[0027] Experimental Animals:

[0028] Wild-type BALB / c mice, 6-8 weeks old, were selected and all mice were raised in a specific pathogen-free (SPF) barrier environment.

[0029] Step 1: Construction of Ag85B-Fc (S10P) fusion protein sequence and gene synthesis:

[0030] The Ag85B antigen sequence was connected to the N-terminus of IgG4Fc (S10P) via GGSGGGGSGGGGSGG, and the codons were optimized and the corresponding sequence was synthesized, such as SEQ4.

[0031] Step 2: gE-Fc (S10P) fusion protein sequence construction and gene synthesis:

[0032] The gE extracellular soluble sequence was connected to the N-terminus of IgG4Fc (S10P) via GGSGGGGSGGGGSGG, and the codons were optimized and the corresponding sequence was synthesized, such as: SEQ5.

[0033] The specific sequences involved are as follows:

[0034] The amino terminal sequence encoding Mycobacterium tuberculosis (TB) antigen 85B (Ag85B) is denoted as SEQ1.

[0035] The amino acid sequence of the extracellular soluble region of varicella-zoster virus (VZV) glycoprotein E (gE) is denoted as SEQ2.

[0036] The nucleic acid sequence encoding the Fc segment of human immunoglobulin IgG4 and carrying the S10P mutation is recorded as the amino acid sequence of Fc, recorded as SEQ3.

[0037] Step 3: Construction and expression of heterodimer fusion protein:

[0038] The sequences of Ag85B-Fc (S10P) and gE-Fc (S10P) were cloned into PSec (Invitrogen) plasmid, respectively. The heterodimerization of Ag85B-Fc (S10P) and gE-Fc (S10P) was obtained by the previously reported knob-to-holes (Ridgway, JB, Presta, LG & Carter, P. 'Knobs into holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Engineering, Design and Selection 9, 617-621 (1996)) technology; the plasmid was added to 293F cells at a ratio of 1:1, the supernatant was collected 6 days after transfection, and the fusion protein (Ag85B-gE-Fc (S10P)) was purified using a protein A-agarose gel column.

[0039] Step 4: Vaccine preparation

[0040] The Ag85B-gE-Fc (S10P) fusion protein was added at 50 ug / mouse and 20 ug / mouse of adjuvant QS-21 to mix them to prepare a vaccine for later use.

[0041] Step 5: Animal Immunization

[0042] Nine mice were divided into three groups, each group included three female BALB / c mice aged 6-8 weeks. Group 1: negative control group (normal saline); Group 2: adjuvant control group (QS-21); Group 3: immune group (Ag85B-gE-Fc(S10P)+QS-21); immunization was performed once a week for a total of four times. After 48 hours of the last administration, the animals were euthanized, blood was collected, and spleens were removed for further analysis.

[0043] Step 6: Analysis of specific immune response

[0044] 6.1. Analysis of Ag85B-specific antibodies in serum

[0045] Collect whole blood from mice routinely and place it in an EP tube without anticoagulant. Place it at 4°C overnight and centrifuge it at 1500rpm for 8-10min, then carefully draw out the separated serum. Use 5ug / mL, 50ul / well of Ag85B protein to coat the antigen overnight at 4°C. Shake off the antigen coating solution, tap with absorbent paper to absorb the residual liquid, and wash three times with 250ul / well PBS buffer. Add 200ul 1% BSA-PBS buffer to each well and place it at room temperature for 1h. Discard the blocking solution, and 250ul / well ul / well PBS buffer was used for washing three times, and the serum of mice in each group was taken for gradient dilution at 1:100, 1:200, 1:500, 1:1000, and 1:2000, respectively. 100ul of diluted serum was added to each well, and incubated at 37°C for 1h; the liquid was discarded, and 250ul / well PBS buffer was used for washing three times, and HRP-labeled goat anti-mouse secondary antibody was added, 100ul / well, and incubated at 37°C for 1h; 250ul / well PBS buffer was used for washing three times, and 100ul TMB colorimetric solution was added to each well. After reacting at room temperature in the dark for 15min, 50ul stop solution was added to each well, and the absorbance at 450nm was measured with an enzyme reader. We defined the titer as the maximum dilution corresponding to twice the OD value of the BLANK well, and the production of Ag85B-specific antibodies in the three groups of mice is shown in the figure. Figure 2 .

[0046] 6.2 Analysis of gE-specific antibodies in serum

[0047] Collect whole blood from mice routinely and place it in an EP tube without anticoagulant. Place it at 4°C overnight and centrifuge it at 1500rpm for 8-10 minutes, then carefully draw out the separated serum. Use 5ug / mL, 50ul / well gE protein to coat the antigen overnight at 4°C. Shake off the antigen coating solution, tap with absorbent paper to absorb the residual liquid, and wash three times with 250ul / well PBS buffer. Add 200ul to each well. 1% BSA-PBS buffer, block at room temperature for 1h; discard the blocking solution, wash three times with 250ul / well PBS buffer, aspirate mouse serum and dilute them in a gradient of 1:100, 1:200, 1:500, 1:1000, and 1:2000, add 100ul of diluted serum to each well, and incubate at 37℃ for 1h; discard the liquid, wash three times with 250ul / well PBS buffer, add HRP-labeled goat anti-mouse secondary antibody, 100ul / well, and incubate at 37℃ for 1h; wash three times with 250ul / well PBS buffer, add 100ul TMB colorimetric solution to each well, react at room temperature in the dark for 15min, add 50ul stop solution to each well, and measure the absorbance at 450nm with an enzyme reader.

[0048] 6.3. Ag85B stimulates INF-Y secretion in mouse spleen cells in vitro

[0049] DAY1: (Aseptic operation, spleen cells were collected for one day) Prepare INF-Y antibody diluent A, add antibody solution A to the PVDF ELISPOT plate, 80 μl / well; incubate overnight at 4°C;

[0050] DAY2: Gently pour out the antibody solution A incubated overnight, wash three times with 200ul / well PBS, and block with blocking solution. Inoculate mouse spleen cells into the wells at an appropriate concentration, and add appropriate amounts of positive control stimuli or Ag85B antigen according to the purpose of the experiment, and design a negative control group; culture at 37°C and 5% CO2 for 48-72h;

[0051] DAY3: Remove the cells in the wells, then wash with PBS for 4 times to completely remove the cells, 200μl each time, add detection antibody to each empty space at 100ul / well, incubate for 2h, wash with PBS for 5 times, add precipitating colorimetric solution, incubate at room temperature for 15min, and gently rinse the PVDF membrane three times with tap water to terminate the reaction; turn the PVDF96-well plate upside down on a ventilated operating table for 2 hours or on an ordinary workbench for 24 hours to dry, and scan and count with an instrument.

[0052] 6.4gE stimulates INF-Y secretion of mouse spleen cells in vitro

[0053] DAY1: (Aseptic operation, spleen cells were collected for one day) Prepare INF-γ antibody diluent A, add antibody solution A to the PVDF ELISPOT plate, 80 μl / well, and incubate overnight at 4°C;

[0054] DAY2: Gently pour out the antibody solution A incubated overnight, wash three times with 200ul / well PBS, and block with blocking solution. Inoculate mouse spleen cells into the wells at an appropriate concentration, and add appropriate amounts of positive control stimulators or gE antigens according to the purpose of the experiment, and design a negative control group; culture at 37°C and 5% CO2 for 48-72h;

[0055] DAY3: Remove the cells in the wells, then wash with PBS for 4 times to completely remove the cells, 200μl each time, add detection antibody at 100ul / well to each hole, incubate for 2h, wash with PBS for 5 times, add precipitating colorimetric solution, incubate at room temperature for 15min, gently rinse the PVDF membrane with tap water three times to terminate the reaction, turn the PVDF96-well plate upside down on a ventilated operating table for 2 hours or on an ordinary workbench for 24 hours to dry, and scan and count with an instrument.

[0056] In summary, the present invention provides a recombinant VIZ-TB dual-target Fc vaccine. Mice are immunized with the recombinant VZV-TB dual-target Fc vaccine once a week for a total of 4 times. After the immunization, it is found that the mice produce antibodies against both gE and Ag85B in vivo. The elispot method is used to detect the spleen cells of the mice. After being stimulated with gE and Ag85B antigens respectively, different degrees of cellular immune effects can be simultaneously produced. This preliminarily proves that the dual-target vaccine has the ability to induce specific immune responses in the body, and provides a theoretical and experimental basis for the later entry into human vaccines. Ag85B-Fc fusion protein and gE-Fc fusion protein are constructed separately, and the spontaneous binding ability of the Fc end is utilized to form heterologous dimers of Ag85B-Fc fusion protein and gE-Fc fusion protein, which provides a technical basis for the design and development of multi-target vaccines of "one vaccine for two treatments" or "one vaccine for multiple treatments", and can save economic costs and improve social benefits.

[0057] The embodiments of the present invention are provided for the purpose of illustration and description. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations of the present invention. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A recombinant VIZ-TB dual-target Fc vaccine, comprising: The three functional elements are Mycobacterium tuberculosis (TB) antigen 85B (Ag85B), varicella-zoster virus (VZV) antigen glycoprotein E (gE) and immunoglobulin (Fc), and the three functional elements are directly or indirectly connected through a linker.

2. A recombinant VIZ-TB dual-target Fc vaccine as claimed in claim 1, characterized in that: The Fc region of the immunoglobulin comes from the constant region amino acid sequence of IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2; among them, IgG2 and IgG4 have weaker ability to induce ADCC and CDC effects, but longer serum half-life, and are very suitable as preventive vaccines; the Fc region can be the normal constant region sequence of IgG4 or the constant region sequence containing mutation points, and these mutations include but are not limited to S36T, V90P, N216A, S10P, E15P, F16V, L17A, D47A and R191 K.

3. A recombinant VIZ-TB dual-target Fc vaccine as claimed in claim 1, characterized in that: The VZV-TB dual-target Fc vaccine containing three functional elements is a heterodimer Ag85B-gE-Fc, which includes two separate fusion proteins, namely fusion protein 1 and fusion protein 2. Fusion protein 1 includes Ag85B protein and immunoglobulin Fc region from N-terminus to C-terminus, and fusion protein 2 includes gE protein and immunoglobulin Fc region from N-terminus to C-terminus. The immunoglobulin Fc region contained in fusion protein 1 and the immunoglobulin Fc region contained in fusion protein 2 are derived from immunoglobulins of the same subtype.

4. A recombinant VIZ-TB dual-target Fc vaccine as claimed in claim 3, characterized in that: The heterodimer Ag85B-gE-Fc was mixed with an adjuvant and used for immunization.

5. A recombinant VIZ-TB dual-target Fc vaccine as claimed in claim 1, characterized in that: The adjuvant used for the combination with the heterologous Fc fusion dimer protein includes but is not limited to mineral salts.

6. A recombinant VIZ-TB dual-target Fc vaccine as claimed in claim 3, characterized in that: The invention comprises a nucleic acid molecule encoding a fusion protein 1Ag85B-Fc.

7. A recombinant VIZ-TB dual-target Fc vaccine as claimed in claim 6, characterized in that: The invention comprises a nucleic acid molecule encoding the fusion protein 2gE-Fc.

8. A recombinant VIZ-TB dual-target Fc vaccine as claimed in claim 7, characterized in that: The invention comprises a vector, wherein the vector comprises a nucleic acid molecule encoding fusion protein 1Ag85B-Fc.

9. A recombinant VIZ-TB dual-target Fc vaccine as claimed in claim 8, characterized in that: The invention comprises a vector comprising a nucleic acid molecule encoding a fusion protein 2gE-Fc.

10. A recombinant VIZ-TB dual-target Fc vaccine as claimed in claim 9, characterized in that: The invention comprises a cell, which contains a dimeric fusion protein Ag85B-gE-Fc, a nucleic acid molecule encoding the fusion protein or a vector expressing the fusion protein, and is used for producing the fusion protein; the cell is a eukaryotic animal cell.

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