CN112107683A

CN112107683A - Photochemical inactivation method of coronavirus and vaccine

Info

Publication number: CN112107683A
Application number: CN202011003927.2A
Authority: CN
Inventors: 于斌; 周锡鹏; 张�杰; 姜淼; 吴南屏; 靳昌忠
Original assignee: Boxin Beijing Biotechnology Development Co ltd
Current assignee: Boxin Beijing Biotechnology Development Co ltd
Priority date: 2020-03-04
Filing date: 2020-09-22
Publication date: 2020-12-22

Abstract

The application relates to the field of medicine, in particular to a photochemical inactivation method of coronavirus and a vaccine. The method comprises the following steps: separating coronavirus from clinical sample or culture medium, adding photosensitizer to make photochemical inactivation, concentrating inactivated coronavirus sample to obtain coronavirus inactivated vaccine. The method can prepare the autologous plasma inactivated vaccine, and the prepared vaccine has good immunocompetence.

Description

Photochemical inactivation method of coronavirus and vaccine

Technical Field

The application relates to the field of medicine, in particular to a photochemical inactivation method of coronavirus and a vaccine.

Background

2019 coronavirus epidemic situation (COVID-19), which is caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), is at present seriously threatening public health of human beings.

SARS-CoV-2 was first isolated from human airway epithelial cells and was identified as a new member of the β -CoVs coronavirus. Coronaviruses are enveloped, positive-sense-stranded, single-stranded RNA viruses. Also of the genus β -CoVs is the SARS virus.

Although most diseases caused by coronaviruses are respiratory tract infections, several studies have shown that viral RNA can be detected in the plasma of SARS-CoV, MERS-CoV or SARS-CoV-2 coronaviruses at different times after the onset of symptoms.

The world health organization noted as early as 2003 that blood products are potentially contaminated with transfusion SARS-CoV virus and thus there is a theoretical risk of SARS-CoV or other coronaviruses being transmitted by transfusion. As more and more asymptomatic infections are found in COVID-19 cases, blood safety is of concern.

In addition, due to the current lack of specific drugs and vaccines against SARS-CoV-2. In this case, convalescent serum/plasma therapy of the patient is a rapid and effective treatment. The core idea behind this approach is to ensure complete inactivation of SARS-CoV-2 in the plasma of a plasma donor. Therefore, the reliable and practical plasma SARS-CoV-2 inactivation scheme is urgently needed in clinic at present.

The photochemical method is a common method for killing virus in blood products, and the method is usually adopted for killing virus by adding Methylene Blue (MB) into the blood products and then irradiating the blood products with light. Methylene blue, also known as methylene blue, is a photosensitizer with a maximum absorption peak of 670nm, and is commonly used as an antidote clinically for treating methemoglobinemia and cyanide poisoning caused by nitrite poisoning. Methylene blue carries a positive charge on its surface and can intercalate into DNA/RNA, particularly in association with the negatively charged G-C base pairs of viral nucleic acids. Under the irradiation of light, methylene blue absorbs light energy, is activated from a ground state to a singlet state, and generates singlet oxygen through electron transfer to damage and break nucleic acid, thereby achieving the purpose of killing viruses. Viral envelope and nucleic acid components can be targets for attack, and methylene blue can cause nucleic acid strand breakage under light.

Since 2000, photochemistry has been widely used in European countries such as Germany, Switzerland and, and domestically. The adopted light sources are halogen lamps, metal halide lamps, fluorescent lamps and the like, and the light rays are all full-waveband white light. However, because the excitation efficiency of the white light of the whole wave band to the methylene blue is low, and heat can be generated to destroy blood plasma components, the prior art develops a method for killing various lipid envelope viruses such as HIV-1, SARS-Cov and the like by using single-wavelength light matched with the methylene blue, and the virus killing effect is better.

As a novel coronavirus, the resistance of SARS-CoV-2 to physicochemical conditions is unknown.

Disclosure of Invention

The present invention has been made to solve the above problems.

The invention aims to provide a method for photochemical inactivation of coronaviruses in blood plasma.

The invention also aims to provide a photochemical preparation method of the coronavirus sample inactivated vaccine.

The embodiment of the invention provides a photochemical preparation method of a coronavirus sample inactivated vaccine, which comprises the following steps:

separating coronavirus from a clinical sample or from a culture medium to form a coronavirus sample to be treated;

adding a photosensitizer to the coronavirus sample;

carrying out photochemical inactivation on the coronavirus sample added with the photosensitizer;

and concentrating the inactivated coronavirus sample to obtain the coronavirus inactivated vaccine.

Preferably, the photochemical preparation method of inactivated vaccine for coronavirus sample according to the present invention, wherein the sample of coronavirus is from a clinically isolated sample.

Preferably, the photochemical preparation method of the inactivated vaccine for coronavirus sample according to the present invention is used for preparing coronavirus sample, wherein the coronavirus sample is plasma, respiratory tract secretion, digestive tract excretion, urine or derived from cell culture, tissue culture and chick embryo culture.

Preferably, the photochemical preparation method of the inactivated vaccine for coronavirus sample according to the present invention, wherein the photosensitizer is methylene blue and/or riboflavin.

Preferably, the photochemical preparation method of inactivated vaccine for coronavirus sample according to the present invention, wherein the photosensitizer is at a concentration of 1uM to 4 uM.

Preferably, the photochemical preparation method of inactivated vaccine for coronavirus sample according to the present invention, wherein the isolated coronavirus titer is 3.5-4.5LgTCID₅₀。

Preferably, the photochemical preparation method of the inactivated vaccine for coronavirus sample according to the present invention comprises irradiating light with wavelength of 610-650nm, unit irradiation intensity of 32000-48000lx, and irradiation time of 2-40 min.

Preferably, the photochemical preparation method of the inactivated vaccine for coronavirus sample according to the present invention is to perform a concentration process on the inactivated coronavirus sample by ultracentrifugation, ultrafiltration, molecular sieve column method, ion exchange method, or dialysis method.

The embodiment of the invention provides a photochemical inactivation method of coronavirus in a biological sample, which comprises the steps of adding photosensitizer into plasma containing SARS-CoV-2 virus, and irradiating for 0-40min under 610-650nm light.

Preferably, the method for photochemical inactivation of a coronavirus in a biological sample according to the present invention, wherein the irradiation is performed under 630nm single wavelength light.

Preferably, the method for photochemical inactivation of a coronavirus in a biological sample according to the present invention, wherein the irradiation intensity per unit area is 32000-.

Preferably, the method for photochemical inactivation of a coronavirus in a biological sample according to the present invention, wherein the coronavirus is SARS-CoV-2 virus, MERS-CoV virus or SARS-CoV virus.

Preferably, the method for photochemical inactivation of a coronavirus in a biological sample according to the present invention, wherein the coronavirus-containing plasma is plasma produced from blood of a patient with a coronavirus.

Preferably, the method for photochemical inactivation of a coronavirus in a biological sample according to the present invention, wherein the photosensitizer is methylene blue or riboflavin.

Preferably, the method for photochemical inactivation of a coronavirus in a biological sample according to the present invention, wherein the photosensitizer is added to the biological sample to be inactivated at a final concentration of 1. mu.M-4. mu.M.

The embodiment of the application adopts at least one technical scheme which can achieve the following beneficial effects:

the photochemical preparation method of the coronavirus sample inactivated vaccine disclosed by the embodiment of the invention can be used for preparing the autologous plasma inactivated vaccine, and the prepared vaccine has good immunocompetence.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

FIG. 1 shows the results of SARS-CoV-2 virus infection of host cells (100X) in different treatment groups, wherein the virus control group, MB alone no-light control group, and MB alone no-light control group are shown, respectively.

FIG. 2 shows the effect of inactivating SARS-CoV-2 virus in vitro according to an embodiment of the present invention.

FIG. 3 shows ELISA detection results of photochemical inactivation of methylene blue of the novel coronavirus.

FIG. 4 shows the result of the measurement of the antibody titer of the alpaca immune serum against the new coronavirus spike protein.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail and completely with reference to the following specific embodiments of the present application and the accompanying drawings. It should be apparent that the described embodiments are only some of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The technical solutions provided by the embodiments of the present application are described in detail below with reference to the accompanying drawings.

The embodiment of the invention discloses a photochemical preparation method of a coronavirus sample inactivated vaccine, which comprises the following steps:

s10, separating the coronavirus from the clinical sample or from the culture medium to form a coronavirus sample to be treated;

s20, adding a photosensitizer into the coronavirus sample;

s30, carrying out photochemical inactivation on the coronavirus sample added with the photosensitizer;

and S40, concentrating the inactivated coronavirus sample to obtain the coronavirus inactivated vaccine.

Coronaviruses belong phylogenetically to the order of the nested viruses (Nidovirales) the family of Coronaviridae (Coronaviridae) the genus coronaviruses (Coronavirus). Viruses of the genus coronaviruses are enveloped (envelope) RNA viruses with a linear single-stranded positive strand genome, and are a large group of viruses that are widely found in nature.

The coronavirus has the diameter of about 80-120 nm, the 5 'end of the genome has a methylated cap structure, the 3' end has a poly (A) tail, and the total length of the genome is about 27-32kb, so that the coronavirus is the virus with the largest genome in the currently known RNA viruses. Coronaviruses only infect vertebrates, such as humans, mice, pigs, cats, dogs, wolves, chickens, cows, birds. The virus particles have a diameter of 60 to 200nm, an average diameter of 100nm, are spherical or elliptical, and have polymorphism. The virus has an envelope, spinous processes exist on the envelope, the whole virus is like coronas, and the spinous processes of different coronaviruses have obvious difference. Tubular inclusions are sometimes visible in coronavirus infected cells. 2019 the novel coronavirus is one of the 7 th coronavirus which are known to infect human, and the other 6 are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV, respectively.

Preferably, the sample of coronavirus is any sample from a clinical setting.

Preferably, according to the photochemical preparation method of the inactivated vaccine for coronavirus sample of the present invention, for example, the sample containing coronavirus is plasma, respiratory tract secretion, digestive tract excretion, urine, etc., and the isolated coronavirus-containing cells are obtained by cell culture, tissue culture, chick embryo culture, etc.

A frequently used clinical biological sample is plasma made from the blood of a patient. Blood is a red opaque viscous liquid that flows in human blood vessels and the heart. Blood consists of plasma and blood cells. One liter of plasma contains 900-910 g of water, 65-85 g of protein and about 20 g of low molecular substances, such as various electrolytes and organic compounds, and coronaviruses are also present in the plasma. Therefore, the plasma can be separated from the blood cells by centrifugation or the like to obtain individual plasma to be treated.

Blood cells, including red blood cells, white blood cells and platelets, are of great importance for the metabolism of the human body and have no substantial effect on the preparation of vaccines, so that it is desirable to have a rapid and synchronized return of these blood cells to the patient after isolation. Since the blood cells have an extremely high platelet content and are easily coagulated, it is preferable to add an amount of anticoagulant during the return transfusion in order to maintain the fluidity of the blood cells. In addition, physiological saline can be synchronously input into the patient to replenish substances such as water, electrolyte and the like which are missing in the blood.

The separated plasma to be processed is guided to a collection bag through a catheter, the collection is stopped after a sufficient amount of plasma is collected in the collection bag, the bag is sealed and transferred, and the next operation is ready to be carried out. In order to facilitate the rapid stopping of the collecting operation, the guide pipe can be thermally sealed by a heat sealing machine, so that the bag sealing operation can be simplified.

Next, the plasma to be treated obtained by separation is subjected to inactivation treatment, so that the pathogenicity of the plasma is lost, and an autologous plasma inactivated vaccine is formed. Viral inactivation refers to methods of killing viruses by physical or chemical means, but without damaging their useful antigens in vivo. The physical inactivation method and the chemical inactivation method can be subdivided into various ways, for example, the physical inactivation method can include ultrasonic inactivation, ultraviolet inactivation, ray inactivation, high-pressure inactivation, etc. Chemical inactivation methods may include photochemical inactivation, enzymatic chemical inactivation, and the like. Each inactivation method has its own application range, and cannot achieve the same effect on all viruses and bacteria. Therefore, it is very important to select the inactivation mode appropriately. Through a large number of experiments, the photochemical inactivation has been found to have better effect on the inactivation of coronavirus than other inactivation methods.

Preferably, the coronavirus titer isolated from a clinically isolated sample or a sample obtained by tissue culture is 3.5-4.5LgTCID₅₀。

Before photochemical inactivation, pretreatment is firstly carried out on the plasma to be treated obtained by separation, and mainly comprises adding a photosensitizer into the plasma to be treated to form the plasma to be inactivated. Photosensitizers are also known as sensitizers, photocrosslinkers. In photochemical reactions, the photosensitizer absorbs only photons and transfers energy to molecules that cannot absorb photons, thus promoting chemical reactions, but does not participate in chemical reactions per se, and the original state is restored. The photochemical reaction initiated by the photosensitizer is called a photoreaction. Generally, a photoreaction involving oxygen molecules and accompanied by biological effects is called a photodynamic reaction, and a drug which induces the photodynamic reaction to destroy a cell structure is called a photodynamic drug, i.e., a photosensitizing drug.

The purpose of adding a photosensitizer in this application is to use it to cause photochemical reactions to inactivate coronaviruses. In order to obtain better fluidity and dispersibility of the photosensitizer, the photosensitizer is preferably added to the plasma to be treated in the form of a mixed solution. The photosensitizer may be added to physiological saline to form a mixed solution. The normal saline is a common injection for daily injection, has no side effect on human body basically, and is very suitable to be used as a carrier of the photosensitizer. The mixed solution can be injected into the collection bag through a syringe after the preparation is finished.

There are many types of photosensitizers, and different types of photosensitizers have a large difference in their photochemical inactivation effects on coronaviruses. The photosensitizer is selected from the group consisting of methylene blue, riboflavin and fructus Psoraleae, and can be used alone or in combination to improve inactivation.

The concentration of the photosensitizer is also an important index, and if the addition amount of the photosensitizer is too low, the inactivation effect is possibly poor, and the treatment effect of the final vaccine is also poor. If the concentration of the photosensitizer is too high, waste can be caused on one hand, and on the other hand, the photosensitizer with high concentration can also cause negative effects on human bodies. The concentration of the photosensitizer in the mixed solution after mixing with the sample is preferably 1 micromole/liter to 4 micromole/liter, as determined experimentally. The photosensitizer can be added into the blood plasma to be treated and then shaken for a certain time, so that the photosensitizer can be dispersed in an accelerated way.

And finally, placing the collection bag containing the inactivated plasma into a plasma virus inactivation instrument for photochemical inactivation, thereby finally obtaining the autologous plasma inactivated vaccine. The wavelength, intensity and duration of the irradiating light during the inactivation have important influence on the inactivation effect, and should be carefully selected. According to the experiment, the optimal wavelength range of the irradiation light is 610-650nm, wherein 630nm is the optimal wavelength value. The irradiation intensity per unit area should be maintained at 32000-.

The obtained autologous plasma inactivated vaccine can be stored for several hours at normal temperature. And (4) returning the autologous plasma inactivated vaccine to the patient according to a treatment scheme by selecting a proper time. The feedback process can be completed by an infusion mode, and because the autologous plasma inactivated vaccine obtained by the mode has no rejection or other adverse reactions with a patient, the feedback speed is very high, and the feedback operation of 600ml of the autologous plasma inactivated vaccine can be completed within ten minutes usually. Through experimental detection, the treatment effect of the autologous plasma inactivated vaccine is very ideal.

Example 1

Materials and methods

1. Cells, instruments, reagents, viruses, etc

VERO cells are stored in laboratories of Zhejiang university, and DMEM medium, fetal calf serum and P/S, PBS buffer solution are GIBCO products. Methylene blue is commercially available and human plasma is donated to volunteers. All experimental procedures involving live viruses were performed in the biosafety third-level laboratory (BSL-3). Isolation of SARS-CoV-2 virus

Collecting 1-2mL of COVID-2019 patient sputum, diluting with PBS buffer solution 2 times, and centrifuging at 2000rpm for 20min at 4 deg.C in a biological safety centrifuge to remove most impurities. The supernatant was collected and filtered through a 0.45um filter (Millipore). VERO-E6 cell culture medium from a 25cm2 cell culture flask in logarithmic growth phase was discarded and washed 2 times with PBS buffer. 0.5mL of the filtrate was aspirated and inoculated into a VERO-E6 cell culture flask, supplemented with 2ML DMEM culture solution, placed in a 37 ℃ 5% CO2 incubator and adsorbed for 3 hours while shaking once every 15min to allow the sample to be fully adsorbed on the cell surface. After 3h, 4.5mL of DMEM medium containing 2% FBS was added and placed in a 37 ℃ 5% CO2 incubator with VERO-E6 cell control. Cell morphology Change (CPE) was observed under an inverted microscope every 24 h. Changes from 0-25% cell CPE to "+", 26-50% cell CPE to "+", 51-75% cell CPE to "+ + +", 76-100% cell CPE to "+ + + +", and normal cell morphology to "-". And 200uL of the supernatant was aspirated for viral nucleic acid detection.

Methylene blue light inactivated virus

Taking 180mL of healthy human plasma, adding 20mL of indicator virus according to the ratio of 9:1(V: V), and mixing uniformly. Taking 1 (20mg/2 mL/piece) of clinical Methylene Blue (MB) injection, diluting 13.4 times with normal saline to prepare 2mM/L methylene blue solution, diluting according to 2000, 1000 and 500 times, adding into 200mL plasma virus mixed solution, mixing uniformly to make the final concentration of the mixed methylene blue solution be 1uM, 2uM and 4uM, using BX-1 type AIDS therapeutic apparatus at room temperature to regulate the illumination to be 5.5 ten thousand +/-0.5 ten thousand Lux, and irradiating for 0, 2, 5, 10, 20 and 40min under 630nm single-wavelength light. Approximately 1mL of plasma was taken at each time point, 10-fold diluted in DMEM medium containing 2% FBS, and filtered through a 0.45 μm filter for virus titer detection. And simultaneously setting virus control, simple Methylene Blue (MB) treatment control) and simple illumination control, wherein the virus control group is that only virus is added into plasma, the plasma is placed at room temperature for 40 minutes as untreated control, the simple Methylene Blue (MB) treatment control is that virus and methylene blue 4 mu M are added into the plasma, the plasma is not subjected to illumination treatment, the plasma is placed at room temperature for 40 minutes in a dark place and is used as control of the influence of the methylene blue on the virus, and the simple illumination control is that only virus is added into the plasma, and the illumination is carried out for 40 minutes and is used as control of the influence of the illumination on the virus.

Titration of viral Activity

VERO cells after trypsinization were expressed at 1X10⁴One well, 100ul of medium per well was seeded in 96 well cell culture plates. After the cells grew into monolayers in the 96-well plates, the culture medium was discarded, and the treated plasma was inoculated. Viruses were logarithmically diluted from 10 in 2% FBS DMEM medium^-2-10^-7. Repeat 4 wells for each dilution, 200 ul/well. Normal cell control wells (with cells, no virus) were set. Place 96-well plates in 5% CO₂After 3 hours of incubation in a 37 ℃ incubator, the supernatant was washed off and 200 ul/well of 2% FBS DMEM medium was added. Cytopathic effects were observed every 24 hours until 6 days. TCID calculation by Reed-Muench method₅₀. And (4) sucking cell culture supernatant cultured on the 6 th day, and detecting the viral nucleic acid load.

Blind transmission third generation test

1mL of test group plasma, to which virus and 1, 2, 4uM methylene blue were added and which was irradiated for 40min, was diluted 10-fold with 2% FBS DMEM medium, filtered with 0.45 μm filter (Millipore) and added to VERO cells, 1mL of supernatant was taken every 48 hours, diluted 10-fold with 2% FBS DMEM medium and added to VERO cells, and 3-passage blind cells were performed. And observing whether cytopathic effect appears. In the whole process of blind 3 generations of cells, the cytopathic effect is positive (+) at any time, which indicates that the virus is not completely inactivated; the absence of cytopathic effect is negative (-), indicating that the virus has been completely inactivated.

qRT-PCR assay of viral RNA load in culture supernatants

Nucleic acid extraction: 200uL of virus culture supernatant was collected, and viral nucleic acid was extracted with an EX2400 nucleic acid automatic extractor (of Shanghai, Inc.) using a nucleic acid extraction kit (Cat. Z-M-0044, of Shanghai, Inc.) by MVR01 magnetic bead method in an elution volume of about 50. mu.L.

qRT-PCR: the viral load was examined by qRT-PCR using a novel coronavirus nucleic acid assay kit (Cat. No. Z-RR-0479-02-50, J.Biotech Co., Ltd., Shanghai) with CT values below 35 being considered as effective amplification and above 35 being considered as undetected.

Results

Effect of SARS-CoV-2 infecting VERO cells

100 μ L of 4LgTCID₅₀SARS-CoV-2, m L, was inoculated into 96-well plates full of a monolayer of VERO cells, cultured under conventional conditions, and cytopathic effect (CPE) was observed. The results are shown in FIG. 1D, where SARS-CoV-2 has a strong cytopathic effect, and the virus-infected cells show marked pathological necrosis and extensive wall detachment.

2. Determination of SARS-CoV-2 virus inactivation by methylene blue photochemical method by observing CPE

As shown in FIG. 1, 100. mu.L of 4LgTCID was added₅₀SARS-CoV-2, per m L, was inoculated into a 96-well plate of full monolayer VERO cells, irradiated for an additional 40 minutes with the addition of 1uM, 2uM, 4uM methylene blue, and the control group was incubated under normal conditions with no irradiation and only with no addition of methylene blue, and cytopathic effect (CPE) was observed. The result shows that the curves of the pure illumination group and the virus control group are coincided, the curves of the 1 mu M methylene blue group and the 2 mu M methylene blue group are coincided, the viruses can be completely inactivated by adding 1uM, 2uM and 4uM methylene blue light, the inactivated viruses have no influence on the cell growth, the viruses cannot be inactivated by only the illumination group without the illumination group or the methylene blue, and the cells infected with the viruses have obvious lesion necrosis and large-area wall detachment.

3. Virus titer detection of SARS-CoV-2 virus inactivation by methylene blue photochemical method

According to the result of CPE method, the methylene blue photochemical method is found to inactivate SARS-CoV-2 virus within 40min, and in order to further judge the inactivation efficiency, time gradients of 0min, 2min, 5min, 10min, 20min and 40min are respectively made. As shown in Table 1, the virus titer test found that the virus reduction was 2min, 5min, 10min, 20min and 40min in the virus plasma containing 1. mu.M and 2. mu.M methylene blue>4LgTCID₅₀(ii) a The group containing 4. mu.M methylene blue had an initial titer of only 3.5LgTCID due to the inter-assay lot variation problem₅₀The virus reduction amounts of 2min, 5min, 10min, 20min and 40min were all 3.5LgTCID₅₀。

4. Verification of virus inactivation by blind passage 3

During the whole process of 3 blind generations of cells, no cytopathic effect appears in any generation of cells, which indicates that the virus is completely inactivated.

qRT-PCR assay of viral RNA load in culture supernatants

Fluorescent quantitative PCR has been used as a gold standard for virus detection due to its high sensitivity, which is far higher than cytology detection. The results indicate that viral plasma containing 1. mu.M, 2. mu.M, and 4. mu.M methylene blue can completely inactivate the virus within 2 minutes.

TABLE 1 plasma SARS-CoV-2 Virus titration test for various dilutions of supernatant Virus nucleic acid qRT-PCR detection CT values

Note: the dilution was not detected. ND means that the dilution was not detected

6. ELISA detection for methylene blue photochemical inactivation of new coronavirus antigenicity

(1) A96-well plate was coated with the murine monoclonal antibody against the protein S1 of the New coronavirus in an amount of 5ng per well, 100uL per well volume, and left at 4 ℃ overnight.

(2) The coating solution was aspirated and the plate was washed 3 times with PBST.

(3) Add 200uL of 5% BSA per well and block for 2h at room temperature.

(4) The blocking solution was aspirated and the plate washed 3 times with PBST.

(5) Diluting the inactivated new coronavirus by 10 times with a confining liquid, and sequentially carrying out gradient dilution to 10 times according to the dilution of 10 times^-8. Meanwhile, the HIV-1 virus photochemically inactivated by methylene blue is used as a negative control group, and the same gradient dilution is carried out.

(6) The diluted virus was added to 96-well plates at 100 uL/well and incubated at room temperature for 2 h.

(7) The virus dilutions were aspirated and the plates were washed 3 times with PBST.

(8) Diluting the mouse anti-new coronavirus S2 monoclonal antibody by 1000 times, adding the diluted antibody into a 96-well plate, performing incubation for 1h at room temperature in a dark place at 100 uL/well.

(9) The primary antibody was discarded and the plate washed 3 times with PBST.

(10) The goat anti-mouse secondary antibody marked by HRP is diluted 20000 times, added into a 96-well plate at 100 uL/well, and incubated for 1h at room temperature in the dark.

(11) The secondary antibody was aspirated and the plate washed 3 times with PBST.

(12) 100uL of substrate was added to each well and incubated at room temperature in the dark for 20 min.

(13) 100uL of stop solution was added to each well.

(14) The absorbance at 450nm was read on a plate reader.

As shown in FIG. 3, the new coronavirus chemically inactivated by the sub-blue light can be recognized by anti-new coronavirus S1 and S2 proteins, which indicates that the antigenicity of the new coronavirus is retained by the inactivation method.

7. Methylene blue photochemical inactivation new corona virus immunogenicity detection

(1) Concentrating the inactivated virus solution, determining protein concentration, injecting each alpaca according to the dose of 6-7 mug/kg, mixing the concentrated virus solution with Freund's adjuvant 1:1, injecting subcutaneously at multiple points of neck, immunizing for 2 times, and each time spacing 2 weeks.

(2) 2ml of peripheral blood before immunization and 2 weeks after 1 st and 2 nd immunization were respectively extracted, and the plasma was separated by centrifugation at 200g for 5min at room temperature and stored at-20 ℃.

(3) The inactivated virus was coated onto 96-well plates at 20ng protein per well, 100uL per well volume, and left at 4 ℃ overnight.

(4) The coating solution was aspirated and the plate was washed 3 times with PBST.

(5) Add 200uL of 5% BSA per well and block for 2h at room temperature.

(6) The blocking solution was aspirated and the plate washed 3 times with PBST.

(7) Diluting immune serum by 10 times with blocking solution, and sequentially performing gradient dilution to 10 times according to 10 times dilution^-8。

(8) The diluted immune serum was added to a 96-well plate at 100 uL/well and incubated at room temperature for 2 h.

(9) The primary antibody was discarded and the plate washed 3 times with PBST.

(10) 20000 times dilution of goat anti-alpaca secondary antibody marked by HRP is carried out, 96-well plates are added, 100 uL/well is carried out, and incubation is carried out for 1h at room temperature in a dark place.

(13) 100uL of stop solution was added to each well.

(14) The absorbance at 450nm was read on a plate reader.

As shown in fig. 4, the new coronavirus, photochemically inactivated by methylene blue, retained good immunogenicity.

The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A photochemical preparation method of inactivated vaccine of coronavirus sample, which comprises the following steps:

adding a photosensitizer to the coronavirus sample;

2. The photochemical process for the preparation of inactivated vaccine of coronavirus sample as claimed in claim 1, wherein the sample of coronavirus is from a sample isolated clinically.

3. The photochemical process for preparing inactivated vaccine of coronavirus sample as claimed in claim 1, wherein the coronavirus sample is plasma, airway secretion, digestive tract excretion, urine, or from cell culture, tissue culture, chick embryo culture.

4. The photochemical process for preparing inactivated vaccine of coronavirus sample as claimed in claim 1, wherein the photosensitizer is methylene blue.

5. The photochemical process for preparing inactivated vaccine of coronavirus sample as claimed in claim 1, wherein the photosensitizer is at a concentration of 1uM-4 uM.

6. The photochemical process for the preparation of inactivated vaccine of coronavirus sample as claimed in claim 1, wherein the isolated coronavirus titer is 3.5-4.5LgTCID50/m L.

7. The photochemical preparation method of inactivated vaccine for coronavirus sample as claimed in claim 1, wherein the wavelength of the irradiation light is 610-650nm, the unit irradiation intensity is 32000-48000lx, and the irradiation time is 2-40 min.

8. The photochemical preparation method of inactivated vaccine for coronavirus sample according to claim 1, wherein the concentration method of inactivated coronavirus sample is ultracentrifugation, ultrafiltration, molecular sieve column method, ion exchange method or dialysis method.

9. The photochemical preparation method for inactivated vaccine of coronavirus sample according to claim 1, wherein the coronavirus is SARS-CoV-2 virus, MERS-CoV virus or SARS-CoV virus.

10. A method for photochemical inactivation of coronaviruses, comprising the steps of adding a photosensitizer to plasma containing coronaviruses and irradiating for 0min to 40min under 610nm to 650nm light.