WO2023082213A1

WO2023082213A1 - Novel coronavirus protein antigen nanovaccine, preparation method therefor and application thereof.

Info

Publication number: WO2023082213A1
Application number: PCT/CN2021/130486
Authority: WO
Inventors: 陈填烽; 许利耕; 赖浩强
Original assignee: 暨南大学
Priority date: 2021-11-13
Filing date: 2021-11-13
Publication date: 2023-05-19

Abstract

Disclosed are a novel coronavirus protein antigen nanovaccine, a preparation method therefor and an application thereof. The preparation method comprises: respectively dissolving lentinan, sodium selenite and vitamin C in high pressure sterilized water to respectively prepare a lentinan stock solution, a sodium selenite stock solution and a vitamin C stock solution; adding RBD into the sodium selenite stock solution and stirring, adding the mixture into the lentinan stock solution and continuing to stir, then adding the resulting mixture into high-pressure sterilized water and stirring to enable the polymer to be fully and uniformly mixed with the RBD and the sodium selenite, so as to form a mixed solution; and dropwise adding the vitamin C stock solution into the mixed solution, stirring at low temperature after dropwise adding, and finally putting the solution obtained after reaction into a dialysis bag to obtain a novel coronavirus protein antigen nanovaccine. According to the present invention, by utilizing the advantages of a nano-selenium carrier to load RBD for effective presentation, the immunogenicity of the RBD vaccine is improved, and the body is induced to produce a potent immune response.

Description

New coronavirus protein antigen nano vaccine and its preparation method and application

technical field

The invention belongs to the field of nano-selenium vaccines, in particular to novel coronavirus protein antigen nano-vaccine and its use for preventing novel coronavirus epidemics.

Background technique

Novel coronavirus pneumonia (COVID-19) has become one of the most serious infectious diseases in human history. It is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has killed more than 180 million people. people infected. Targeted research and development of SARS-CoV-2 vaccines is the most effective way to block the spread of COVID-19. Although human adenovirus vector vaccines, inactivated vaccines, mRNA vaccines, and peptide vaccines have been approved for use, due to the Therefore, the development of new safe and effective vaccines is of great significance to contain the epidemic.

The new coronavirus mainly mediates the combination of the virus and the host cell angiotensin-converting enzyme 2 (ACE2) through the spike protein, in which the receptor binding domain (RBD) of the spike protein is the core region of the new coronavirus and the receptor binding, targeting RBD antibody can effectively block virus infection, and it is also an important vaccine antigen. However, due to the small molecular weight of RBD, the immunogenicity is not high, and there are few reactive epitopes containing T cells, which limits the RBD vaccine to a certain extent. role in containing the epidemic.

The important role of selenium compounds in the prevention and treatment of cancer and anti-virus has been verified by the results of a large number of epidemiological, preclinical and clinical intervention studies. Compared with other nanomaterial carriers, nano-selenium has superior biological safety and immune regulation functions. If the advantages of nanotechnology or nanocarriers are used to load RBD for effective presentation, the immunogenicity of RBD vaccines can be improved, and the body can be induced to produce a strong immune response, which is a new field worthy of research.

Contents of the invention

In view of the deficiencies in the prior art described above, the primary purpose of the present invention is to provide the preparation of a novel coronavirus protein antigen nano-vaccine using nano-selenium as a carrier.

Another object of the present invention is to provide a novel coronavirus protein antigen nano-vaccine using nano-selenium as a carrier.

Another object of the present invention is to provide a novel coronavirus protein antigen nano-vaccine for preventing novel coronavirus outbreaks.

The preparation method of the new coronavirus protein antigen nano vaccine provided by the invention comprises the following steps:

(1) Prepare stock solution, take lentinan and sodium selenite respectively and dissolve in autoclaved water to make lentinan stock solution and sodium selenite stock solution respectively;

(2) Prepare a mixed solution, add the receptor binding region (RBD) of the spike protein of the new coronavirus into the sodium selenite stock solution and stir, then add the lentinan stock solution and autoclaved water and continue stirring Mix well to form the mixed solution;

(3) Add vitamin C solution dropwise to the mixed solution, and stir at low temperature to make a solution containing RBD@SeNPs nano-vaccine, and then perform dialysis and filtration treatment to obtain the new coronavirus protein antigen with nano-selenium as the carrier nano vaccine.

Further, step (2) includes: adding 1-1.5 mg of the receptor binding region (RBD) of the spike protein of the new coronavirus to 0.5-4 mL of the sodium selenite stock solution, and stirring the container on ice for 10-60 minutes , and then add 1-3mL of the lentinan stock solution and autoclaved water, and then stir and mix for 5-30 minutes to form the mixed solution.

Further, the mass ratio of the RBD to the selenium contained in the sodium selenite is 1:2-16.

Further, after preparing the lentinan stock solution and the sodium selenite stock solution described in step (1), they are all put into a refrigerator and stored at low temperature for future use.

Further, the concentration of the lentinan stock solution is 18-22mg/mL, and the concentrations of the sodium selenite stock solution and the vitamin C solution are both 95-105mM.

Further, the dialysis process in step (3) is to use a dialysis bag under the condition of 10,000-20,000 kDa to dialysis in autoclaved water for 12-24 hours.

The present invention also provides a novel coronavirus protein antigen nano-vaccine, which is prepared by the above-mentioned preparation method, and is a novel coronavirus protein antigen nano-vaccine with nano-selenium as a carrier.

The present invention also provides the application of the novel coronavirus protein antigen nano-vaccine prepared by the above-mentioned preparation method in the field of biomedicine, specifically using the synergy and synergistic effect of nano-selenium as a drug carrier to improve the immunogenicity of the RBD vaccine , induce the body to produce a strong immune response, which can prevent the new coronavirus epidemic.

The above scheme of the present application brings the following beneficial effects:

(1) The preparation method of the novel coronavirus protein antigen nano-vaccine of this application adopts the modern vaccine technology combined with RBD@SeNPs, and utilizes the carrier advantage of nano-selenium to load RBD for effective presentation, which improves the immunogenicity of the RBD vaccine, and can then induce The body mounts a powerful immune response. The preparation method provided by this application is simple, fast, safe and reliable, and easy for large-scale production. In addition, the nano-selenium of the present invention is a food-grade substance with good safety and stability, which makes the new coronavirus protein antigen nano-vaccine more potential clinical application. Transform application prospects.

(2) Compared with other nanomaterial carriers, nano-selenium has superior biological safety and immune regulation functions. Selenium mainly participates in selenoproteins in the form of selenium-containing amino acids. Existing studies have shown that compared with other forms of selenium, such as sodium selenite, sodium selenate, organic selenium and selenomethionine, nano-selenium can More efficient conversion into selenium-containing amino acids, thereby more efficient regulation of selenoprotein expression. Therefore, the novel coronavirus protein antigen nano-vaccine with nano-selenium as the carrier provided in the examples of the present application is of great significance and application value for improving the immunogenicity of RBD, preventing the new coronavirus, improving the body's immune activity and antagonizing the new coronavirus infection.

(3) At present, there is no literature report on the application of nano-selenium in vaccines, and according to the research of the inventors, the effect of using nano-selenium as a carrier to load RBD or combining RBD for the prevention and treatment of new coronavirus is better than that of RBD alone combined with sub- Preventive and curative effects of sodium selenate, sodium selenate, organic selenium, and selenomethionine. At the same time, the inventors have proved through experiments that the vaccine combined with RBD@SeNPs, on the one hand, RBD protein is an antigenic substance, can act as a vaccine to activate the body to produce antibodies against RBD, and at the same time, selenium can maintain the redox level of immune cells; On the one hand, selenium can also regulate the immune activity of immune cells, thereby inducing the body to produce a strong and lasting immune response. The inventors found that the antibody titers of IgG1 and IgG2a produced in mice induced by RBD@SeNPs vaccine were comparable to those induced by RBD combined with sodium selenite, RBD combined with sodium selenate, RBD combined with organic selenium and RBD combined with selenomethionine to induce IgG1 Compared with IgG2a antibody titers, they are 60-90 times, 180-200 times, 60-180 times and 50-90 times respectively.

Description of drawings

Fig. 1 is the hydrated particle size distribution of the independent nano-selenium in the embodiment of the application;

Fig. 2 is the hydrated particle size distribution of the nanoparticles of the RBD@SeNPs nanovaccine in the embodiment of the present application;

Fig. 3 is the Zeta potential of the single nano-selenium in the embodiment of the present application, and the Zeta potential of the RBD@SeNPs nano-vaccine; in the figure, SeNPs is a single nano-selenium, and RBD@SeNPs is the RBD@SeNPs nano-vaccine;

Fig. 4 is the antibody titer of IgG1 in the mouse serum after the 7th day after the RBD@SeNPs nano-vaccine in the embodiment of the application stopped immunization;

Figure 5 is the antibody titer of IgG2a in mouse serum after the 7th day after the RBD@SeNPs nano-vaccine in the embodiment of the present application stopped immunization;

Figure 6 is the antibody titer of IgM in the mouse serum after the 7th day after the RBD@SeNPs nano-vaccine in the embodiment of the application stopped immunization;

Fig. 7 is the antibody titer of IgG1 in the serum of the mice after the 21st day after the RBD@SeNPs nano-vaccine in the embodiment of the present application stopped immunization;

Fig. 8 is the antibody titer of IgG2a in the serum of mice on the 21st day after immunization with RBD@SeNPs nano-vaccine in the example of the present application.

Detailed ways

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.

Embodiment 1, the preparation method of the novel coronavirus protein antigen nano-vaccine provided by the present invention, comprises the following steps:

(1) Preparation of lentinan stock solution: Weigh 200 mg of lentinan and dissolve it in 10 mL of autoclaved water to prepare a lentinan stock solution with a concentration of 20 mg/mL and store it in a refrigerator at 4°C for later use.

(2) Preparation of sodium selenite stock solution and vitamin C stock solution: 172.94 mg of sodium selenite (Na ₂ SeO ₃ ) and 176.13 mg of vitamin C (Vc) were dissolved in 10 mL of autoclaved water, each A sodium selenite stock solution and a vitamin C stock solution with a concentration of 100 mM were prepared and stored in a refrigerator at 4°C for future use.

(3) Preparation of mixed solution: Take 1 mg RBD and add it to 0.5 mL sodium selenite stock solution, stir the container on ice for 15 minutes, add 1 mL of lentinan stock solution and continue stirring for 15 minutes, add autoclaved water to 8 mL, and Stir on a magnetic stirrer for 5 minutes to fully mix the lentinan, RBD and sodium selenite to form a mixed solution.

(4) Preparation of RBD@SeNPs nano-vaccine: Then, 2 mL of vitamin C stock solution was added dropwise to the mixed solution, and after the vitamin C stock solution was added dropwise, it was stirred at 4°C for 8 hours to prepare a nano-vaccine containing RBD@SeNPs The solution.

(5) Dialysis: place the reacted solution in a dialysis bag of 20,000 kDa and dialyze for 12 hours to remove unreacted lentinan, sodium selenite and vitamin C.

(6) Storage: Finally, collect the RBD@SeNPs nano-vaccine in a centrifuge tube and store it at 4°C.

(7) Detection: Digest the RBD@SeNPs nano-vaccine according to the national standard method (GB5009.93-2017), then measure the selenium content by atomic fluorescence spectrometer, and measure the RBD content by BCA protein concentration determination kit (Beiyuntian Bio, P0010S) .

(8) Experimental comparison effect

(i) Grouping of experimental animals

BALB/C mice (40, female) were divided into normal saline autoclaved water group, RBD antigen immunization group, nano-selenium group, RBD@SeNPs nano-vaccine immunization group, and aluminum adjuvant (aluminum adjuvant + RBD) group. 5 groups, 8 in each group.

(ii) Drug dispersion method

Drugs were dispersed with autoclaved water; 25 microliters of drugs were administered per mouse by subcutaneous injection.

(iii) Experimental content

Each mouse was given 20 micrograms/mouse of RBD and 40 micrograms/mouse of nano-selenium. The dosing frequency is once every two weeks, for a total of 3 doses. On the 7th and 21st days after the third dose of vaccine injection, blood was collected by taking blood from the eyeball. The blood was centrifuged at 3000 rpm and 4°C for 10 minutes after standing at room temperature for 1.5 hours. Transfer the serum to another EP tube, and store the collected serum in a -80°C refrigerator.

Spread RBD in a 96-well plate at 5 μg/ml, 100 μl/well, overnight at 4°C. The next day, the 96-well plate was taken out, the supernatant was discarded, and the plate was washed with PBST (PBS containing 0.5% Tween 20) after being patted dry, 5 minutes/time, and after washing 3 times, 5% bovine serum albumin (dissolved in phosphate buffer), 100 μl/well. Place the plate in a 37°C incubator and block for 2 hours. After 2 hours, take out the plate, and wash the plate with PBST (PBS containing 0.5% Tween 20), 5 minutes/time, after washing 3 times, add different serial dilutions of serum (starting at 1:400, sequentially dilute to 1:1638400 times) 100 μl/well. Place the plate in a 37°C incubator and incubate for 2 hours. After 2 hours, take out the plate, and wash the plate with PBST (PBS containing 0.5% Tween 20), 5 minutes/time, after washing 4 times, dilute the enzyme-labeled secondary antibody 1:10000 times with enzyme diluent, and then wash each well Add 100 μL, cover the enzyme-linked plate with self-adhesive, and incubate in a 37°C incubator for 30 minutes. Discard the secondary antibody, wash the plate 5 times with washing solution (PBST), 20-30 seconds each time, and pat dry. Then add 100 μL of TMB color development solution to each well, shake gently, and develop color in the dark at 37°C for 10-15 minutes, then add 50 μL of stop solution to each well, and detect at 450 nm with a microplate reader (630 nm is the reference wavelength). OD values less than 0.12 were considered negative.

(iv) Experimental results

The results are shown in Figure 1-3. The hydrated particle size of the single nano-selenium is 72.25 nanometers, and the Zeta potential is -22.5mV. After adding RBD, the hydrated particle size of the nanoparticles in the system is 293.8 nanometers, and the Zeta potential is 0.057 mV, these results indicate that RBD interacts with nano-selenium, and nano-selenium is loaded with RBD. The present invention successfully prepared a new coronavirus protein antigen nano-vaccine with nano-selenium as a carrier.

In order to further verify the activity of the RBD@SeNPs nanovaccine, we intervened mice by intradermal injection. As shown in Figure 4-6, we found that the IgG1 antibody titer in the serum of mice immunized with RBD@SeNPs nano-vaccine (i.e. the novel coronavirus protein antigen nano-vaccine with nano-selenium as the carrier) was 60% higher than that of the RBD immunized group alone. times, 16 times higher than that of aluminum adjuvant group, and the content of IgG2a and IgM in serum was also consistent with the antibody titer trend of IgG1.

In addition, as shown in Figures 7-8, the antibody induced by RBD@SeNPs nanovaccine could maintain a higher titer for a longer period of time after 21 days of cessation of vaccine immunization.

These results indicated that the introduction of nanoselenium as a nanocarrier can significantly enhance the immunogenicity of RBD and improve the immune response of the body.

Embodiment 2, the preparation method of the novel coronavirus protein antigen nano-vaccine provided by the present invention, comprises the following steps:

(1) Preparation of lentinan stock solution: Weigh 215 mg of lentinan and dissolve it in 11 mL of autoclaved water to prepare a lentinan stock solution with a concentration of 19.55 mg/mL and store it in a refrigerator at 4°C for later use.

(2) Preparation of sodium selenite stock solution and vitamin C stock solution: take 181 mg of sodium selenite (Na ₂ SeO ₃ ) and 187 mg of vitamin C (Vc) and dissolve them in 11 mL of autoclaved water, respectively, and make The sodium selenite stock solution with a concentration of 95.11mM and the vitamin C stock solution with a concentration of 96.59mM were stored in a refrigerator at 4°C for use.

(3) Preparation of mixed solution: Take 1.1 mg RBD and add it to 0.9 mL sodium selenite stock solution, stir on ice for 16 minutes, add 1 mL of lentinan stock solution and continue stirring for 16 minutes, add autoclaved water to 9 mL, and Stirring on a magnetic stirrer for 5 minutes makes the lentinan, RBD and sodium selenite evenly mixed to form a mixed solution.

(4) Preparation of RBD@SeNPs nano-vaccine: Then add 2.5mL vitamin C stock solution dropwise to the mixed solution, and after the vitamin C stock solution is added dropwise, stir at 4°C for 9 hours to prepare RBD@SeNPs nano-vaccine Vaccine solution.

(5) Dialysis: place the reacted solution in a dialysis bag of 10000 kDa, and dialyze for 24 hours to remove unreacted lentinan, sodium selenite and vitamin C.

(6) Storage: The final collected RBD@SeNPs nano-vaccine was placed in a centrifuge tube and stored at 4°C.

(7) experiment

Experiment method and experiment content are the same as embodiment 1.

The hydrated particle size of the single nano-selenium is 72.56 nanometers, and the Zeta potential is -22.3mV. After the RBD is added, the hydrated particle size of the nanoparticles in the system is 294.1 nanometers, and the Zeta potential is 0.058mV.

The IgG1 antibody titer in the serum of mice immunized with RBD@SeNPs nanovaccine was 60 times higher than that of the RBD immunized group alone, and 16 times higher than that of the aluminum adjuvant group. same trend. After 21 days of cessation of vaccine immunization, the antibodies induced by RBD@SeNPs nanovaccine could maintain a higher titer for a longer period of time.

Example 3, sodium selenite, sodium selenate, selenomethionine and organic selenium were physically mixed at a ratio of RBD to selenium of 1:2-1:16 to prepare a vaccine. Animal immunization methods and related index detection methods are the same as in Example 1.

The experimental results found that the antibody titers of IgG1 and IgG2a produced in mice induced by vaccines combined with RBD@SeNPs were significantly higher than those induced by RBD combined with sodium selenite, RBD combined with sodium selenate, RBD combined with organic selenium, and RBD combined with selenomethionine to induce IgG1 Compared with IgG2a antibody titers, they are 60-90 times, 90-180 times, 180-200 times and 50-90 times respectively.

The above experiments prove that the vaccine combined with RBD@SeNPs exerts the synergistic and synergistic effect of nano-selenium as a drug carrier, improves the immunogenicity of RBD vaccine, and induces a strong immune response in the body.

The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present invention can easily think of changes or Replacement should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims

The preparation method of the new coronavirus protein antigen nano vaccine is characterized in that it comprises the following steps:

(1) Prepare stock solution, take lentinan and sodium selenite and dissolve them in autoclaved water respectively, and make lentinan stock solution and sodium selenite stock solution respectively;

(2) Prepare a mixed solution, add the receptor binding region (RBD) of the spike protein of the new coronavirus into the sodium selenite stock solution and stir, then add the lentinan stock solution and autoclaved water and continue stirring Mix well to form the mixed solution;

(3) Add vitamin C solution dropwise to the mixed solution, and stir at low temperature to make a solution containing RBD@SeNPs nano-vaccine, and then perform dialysis and filtration treatment to obtain the new coronavirus protein antigen with nano-selenium as the carrier nano vaccine.
The preparation method according to claim 1, wherein step (2) comprises: adding 1-1.5 mg of the receptor binding domain (RBD) of the spike protein of the new coronavirus to 0.5-4 mL of the sodium selenite stock solution , the container was stirred on ice for 10-60 minutes, and then 1-3 mL of the lentinan stock solution and autoclaved water were added and stirred for 5-30 minutes to form the mixed solution.
according to the described preparation method of claim 1, it is characterized in that, the mass ratio of the selenium element contained in described RBD and sodium selenite is 1:2-16.
According to the described preparation method of claim 1, it is characterized in that, after making the lentinan stock solution and the sodium selenite stock solution described in the step (1), they are all put into a refrigerator and stored at low temperature for subsequent use.
The preparation method according to claim 1, wherein the concentration of the lentinan stock solution is 18-22mg/mL, and the concentrations of the sodium selenite stock solution and the vitamin C solution are both 95-105mM.
The preparation method according to claim 1, characterized in that, the dialysis process in step (3) is to use a dialysis bag under the condition of 10000-20000kDa to dialysis in autoclaved water for 12-24 hours.
The novel coronavirus protein antigen nano vaccine is characterized in that it is prepared by the preparation method described in any one of claims 1-6, and is a novel coronavirus protein antigen nano vaccine with nano-selenium as a carrier.
Adopt the preparation method described in any one of claim 1-6 to make the new coronavirus protein antigen nano vaccine, the application in the field of biomedicine.
According to the application according to claim 8, it is characterized in that it is specifically preventing the novel coronavirus epidemic.