CN112843092A

CN112843092A - Application of immune cells in treating diseases and preparation method thereof

Info

Publication number: CN112843092A
Application number: CN202110071865.7A
Authority: CN
Inventors: 张海涛; 陈帅
Original assignee: Beijing Xinsong Biotechnology Co ltd
Current assignee: Xinjiang Xinnuo Ark Health Management Co Ltd; Zhu Youjian
Priority date: 2021-01-20
Filing date: 2021-01-20
Publication date: 2021-05-28
Anticipated expiration: 2041-01-20
Also published as: CN112843092B

Abstract

The invention relates to an application of immune cells in treating diseases and a preparation method thereof, wherein the immune cells comprise a monoclonal antibody specifically aiming at varicella-zoster virus envelope glycoprotein, the monoclonal antibody has better binding property and virus inhibiting effect, and meanwhile, the monoclonal antibody and the immune cells are found in a mouse model through experiments, so that the treatment effect on the virus infection of the skin of a mouse can be effectively improved, and the monoclonal antibody has better application value and application prospect.

Description

Application of immune cells in treating diseases and preparation method thereof

Technical Field

The invention relates to the field of biology, and in particular relates to application of immune cells in treating diseases and a preparation method thereof.

Background

Herpes Zoster (HZ) is a varicella-zoster virus (VZV) that remains dormant for a long time in the dorsal root ganglion of the spinal nerve or the cranial nerve sensory ganglion, and after reactivation and growth, migrates along nerve fibers to the skin to cause acute infectious skin inflammation, often manifested as herpes caused by the corresponding segments of the sensory nerve, with severe neuralgia. Generally, there are 4 areas of good hair: in the intercostal nerve, cervical nerve, trigeminal nerve and lumbosacral nerve innervating area, the affected part is usually initially provided with erythema, then becomes papule to soybean-sized papule which is distributed in clusters without fusion, finally becomes clustered blister, the blister wall is tensed and shines, the blister liquid is clear, the periphery is wound with areola, the skin among the clusters of blister is normal, and the skin lesion is mostly arranged in a belt shape along the peripheral nerve on one side. The course of the disease is usually 2-3 weeks, and the blisters dry, scab and fall off, leaving temporary reddish spots or pigmentation.

It was found that one of the important factors for maintaining the cellular immune function is the balance of Th1/Th2, while the Th1/Th2 cytokine levels in HZ patients are just out of balance, which is mainly expressed as: compared with normal human, the level of interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-alpha) representing Th1 type is obviously reduced, the corresponding cellular immune function is weakened, and the level of IL-4 representing Th2 type is obviously increased. The weakened immune function of Th1 cells means that the immune function of specific cells with inhibition effect on herpes virus in organisms is weakened, while interleukin-4 (IL-4) in HZ patients is increased, the function of Th2 cells is enhanced, and the inhibition effect on IL-2 and TNF-alpha biological activity is also enhanced; high levels of IL-4 immune status may result in a reduction in the body's ability to activate lymphocytes and NK cells, reducing their ability to lyse virus-infected cells. That is, after the balance of Th1/Th2 of cells in a VZV infected patient is disrupted, the body-specific immune function is reduced, and the VZV is more easily survived and proliferated in a large amount in the patient, thereby destroying more immune mechanisms of the body and triggering neuropathy of the affected part of the body.

The medicine for treating herpes zoster in acute stage comprises western medicines such as antiviral medicine and non-antiviral medicine, Chinese medicinal preparation and Chinese patent medicine. Common antiviral drugs such as famciclovir and penciclovir are nucleoside anti-herpes virus drugs and can be used for acute-stage treatment of herpes zoster. The antiviral drug acyclovir is also called acyclovir, is a synthesized purine nucleoside analogue, plays an antiviral role mainly by interfering virus deoxyribonucleic acid (DNA) synthesis, is a preferred drug for treating herpes zoster, has two common dosage forms of oral administration and external administration, but generally has low oral bioavailability and can achieve the treatment effect by large dose and multiple oral administrations. Valacyclovir is a prodrug of acyclovir, is good in absorption and fast in conversion, and has the bioavailability 3-4 times higher than that of acyclovir, but the results of the clinical curative effects of valacyclovir and acyclovir on treating herpes zoster compared with those of valacyclovir and acyclovir in the Yangtze river and the like show that the clinical total effective rate of valacyclovir on treating herpes zoster has no statistical significance with the difference of acyclovir. Interferons (IFNs) are broad-spectrum antiviral drugs, classified as alpha- (leukocyte) type, beta- (fibroblast) type, and gamma- (lymphocyte) type. IFN can inhibit the transcription, replication and release of viruses and can increase the activity of natural killer lymphotoxin. The results of combined application of IFN and valacyclovir to herpes zoster of Uylor and the like show that the effective rate of the combined drug group is 97.78 percent, the combined drug group is obviously higher than that of a valacyclovir group used alone, and the occurrence rates of pain relieving, blister stopping and crusting time, PHN and the like are lower than that of the valacyclovir group used alone.

The Chinese patent medicine for clinically treating herpes zoster mainly has the functions of cooling blood, removing toxicity, removing dampness-heat, promoting blood circulation, removing blood stasis, promoting qi circulation, relieving pain and the like. The clinical application of the chamomile to the treatment of herpes zoster by combining the compound isatis root granules, the salvia miltiorrhiza injection and the astragalus injection with the acyclovir eye drops shows that the Chinese patent medicine and the acyclovir eye drops can rapidly relieve pain and relieve blisters, are favorable for nerve recovery and reduce the generation of PHN. The gentian liver-fire-purging pill and the same-kernel bezoar heart-fire-clearing pill are taken together to treat the herpes zoster, and the total effective rate reaches 93.3 percent after 30 days of treatment. The Suwengui is used for treating the herpes zoster by combining the QINGKAILING injection with traditional Chinese medicine external washing, and the result shows that the components of the QINGKAILING injection, such as the bezoar, the buffalo horn, the baical skullcap root, the honeysuckle, the gardenia jasminoides ellis and the like, have the effects of clearing heat, purging pathogenic fire, clearing heat, detoxifying and the like, can improve the cure rate of the herpes zoster, shorten the cure time and control the incidence rate of sequelae. In addition, the Chinese patent medicines such as Shuanghuanglian, Chuanhuning and Xiyanping injection are reported to have better treatment effect on the herpes zoster.

In recent years, antibody drugs for the treatment of viruses have become extremely hot. However, monoclonal antibodies against herpes zoster virus have not been much studied. The research of Wuhan virus research of Chinese academy of sciences in 2018 makes an important progress in the research of screening neutralizing antibodies against herpes simplex virus, and the team successfully screens a new monoclonal antibody m27f, finds that the monoclonal antibody recognizes a new continuous epitope in envelope glycoprotein and has higher activity of neutralizing antibodies. But no further studies of antibodies related to herpes zoster virus were performed.

The CIK cells are a cell group generated by inducing and culturing peripheral blood mononuclear cells in vitro through various cytokines, are easy to culture and rapid to expand, are killer cells with high cytotoxicity and have obvious antiviral activity. CIK cell therapy is one of adoptive immunotherapy, which is an active immune cell with NK cell function obtained by culturing lymphocyte populations separated from peripheral blood, bone marrow or umbilical blood under the induction of various cytokines IL-2, IL-1, IFN-r, anti-CD 3 monoclonal antibodies and the like for a certain time, can identify cells infected by various viruses, inhibits and kills the replication of tumor cells and viruses by directly contacting with target cells and killing or secreting various antiviral cytokines such as IFN-r, TNF-a, IL-6 and the like, effectively regulates the immune function of an organism, and improves the anti-tumor and anti-virus capability of a patient organism. However, the number of immune cells adopted in the prior art for treating herpes viruses is not large, and further development is needed.

Disclosure of Invention

On one hand, the invention overcomes the defects of the prior art and provides a monoclonal antibody which adopts immune cells and is specifically directed at varicella-zoster virus envelope glycoprotein, and the monoclonal antibody has better binding property and virus inhibiting effect.

In one aspect of the present invention, a prepared CIK cell is provided, wherein the CIK cell is prepared by a method conventional in the art. The method for producing the obtained CIK cells can also be used in the method of example 1.

In another aspect, CIK cells are prepared as follows: adding normal peripheral blood into normal saline 1: 1, after dilution, slowly adding the blood mixture onto a Ficoll lymphocyte separation solution along the tube wall by using a suction tube, and centrifuging for 20min at 800g and 4 ℃; carefully sucking the second white cloudy mononuclear cell layer with a pipette into a centrifugal tube, adding normal saline, centrifuging at 4 deg.C for 10min for 3 times at 400g, and adjusting cell concentration to 1.0 × 10⁶Per mL; inoculating the cells to a T175 culture bottle coated with a CD3 monoclonal antibody in advance, adding a BPMI-1640 culture medium containing 10% fetal bovine serum, adding 50 mu g/L of IL-2, 50 mu g/L of IFN-gamma and 25 mu g/L of IL-22, placing the mixture in a 5% CO2 cell culture box at 37 ℃ for induction culture, replacing the culture medium by half every day, and culturing for 10d to obtain the cells, namely the CIK cells.

In another aspect of the invention, a monoclonal antibody specific for varicella-zoster virus envelope glycoprotein is provided, wherein the monoclonal antibody has good binding property and virus inhibition effect. Specifically, the light chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO: 1, the heavy chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO: 2, respectively.

In another aspect of the invention, wherein the monoclonal antibody is an antibody fragment.

Preferably, wherein the antibody fragment is selected from the group consisting of an scFv fragment, an Fab fragment, an F (ab ') 2 fragment and an Fab' fragment.

Further, the monoclonal antibodies of the invention may be humanized.

Furthermore, the invention also provides a coding gene of the anti-varicella-zoster virus monoclonal antibody.

Furthermore, the expression vector of the encoding gene of the anti-varicella-zoster virus monoclonal antibody.

Further, the present invention provides the use of the antibody against varicella-zoster virus for the manufacture of a medicament against varicella-zoster virus.

In a further aspect of the present invention, there is provided a pharmaceutical composition prepared by combining CIK cells with antibodies against varicella-zoster virus.

The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

The dosage form of the medicine is tablets, capsules, granules, pills, injections, soft extracts, suspending agents, dispersing agents, syrups, suppositories, gels, aerosols or patches.

In a final aspect of the invention, there is provided the use of a CIK cell and a varicella-zoster virus antibody in the preparation of a medicament against varicella-zoster virus.

Advantageous effects

The monoclonal antibody specifically aiming at the varicella-zoster virus envelope glycoprotein is developed, has better binding property and virus inhibiting effect, and can effectively improve the treatment effect on the virus infection of the skin of a mouse through experimental discovery in a mouse model together with CIK cells, thereby having better application value and application prospect.

Drawings

FIG. 1 is a Western-blot result chart, in which lanes 1 to 6 show the results of the binding reactions of the monoclonal antibody with VZV-pOKA virus strain, BTV-8, CHUV, EHDV-1, and influenza B virus Malaysia2506/04, respectively.

FIG. 2 is a graph showing the results of ELISA detection of monoclonal antibodies

Detailed Description

The invention discloses application of monoclonal antibody and immune cell in preparing medicament for preventing and/or treating human herpesvirus infection, and the technical personnel can use the content to reference and properly improve process parameters to realize the purpose. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

EXAMPLE 1 preparation of CIK cells

Adding normal peripheral blood 100mL into normal saline 1: 1, diluting, slowly adding the blood mixture onto the Ficoll lymphocyte separation solution along the tube wall with a pipette, and centrifuging at 800g and 4 deg.C for 20 min. Carefully sucking the second white cloudy mononuclear cell layer with a pipette into a centrifugal tube, adding normal saline, centrifuging at 4 deg.C for 10min for 3 times at 400g, and adjusting cell concentration to 1.0 × 10⁶and/mL. Inoculating to T175 culture bottle coated with CD3 monoclonal antibody in advance, adding BPMI-1640 medium containing 10% fetal bovine serum, adding 50 μ g/L IL-2, 50 μ g/L IFN-gamma and 25 μ g/L IL-22, and culturing at 37 deg.C in 5% CO2 cell culture box. The medium was replaced half way through the day. Respectively taking mononuclear cells before induction and induction culturing CIK cells to 10 days by 1 × 10⁶The supernatant was centrifuged off, resuspended in 600. mu.L of PBS and counted. The results showed that the suspended mononuclear cells were uniformly distributedIn the culture flask, the edge is regularly rounded. Under the induction of cytokines, the cells grew into colonies, and the number of cells is shown in table 1.

TABLE 1 CIK cell proliferation in vitro

Number of days	0	2	4	6	8	10
							Number of cells	1.0×10⁶	8.0×10⁶	3.2×10⁷	1.8×10⁸	1.5×10⁹	7.0×10⁹

As can be seen from table 1, CIK cells induced by three factors were able to proliferate faster.

And adding 100 mu L of cell suspension which is proliferated for 10d and originally separated into each flow tube and respectively labeled with a fluorescent monoclonal antibody (CD3-FITC) + (IgG1-PE), (CD8-PE) + (IgG1-FITC), (CD56-PE) + (IgG1-FITC), (CD3-FITC) + (CD56-PE), (CD3-FITC) + (CD8-PE), (IgG1-FITC) + (IgG1-PE) as a negative control, centrifuging and washing by PBS after keeping away from light for 20min at room temperature, adding 200 mu LPBS into each tube, and resuspending on a machine for detection. The results of the phenotypic analysis of CIK cells were obtained as shown in table 2.

TABLE 2 phenotypic analysis of CIK cells (x. + -. s)

Group of	CD3⁺	CD8⁺	CD56⁺	CD3⁺ +CD8⁺	CD3⁺ +CD56⁺
						PBMC cells	67.01±3.17	43.52±4.53	21.86±2.87	32.11±0.97	9.01±1.27
CIK cell	98.09±2.97	76.53±3.12	25.37±3.01	90.31±1.94	25.30±1.83*

As can be seen from Table 2, the proportion of CIK major effector cells CD3+ + CD56+ double positive cells after 10 days of induction culture was greatly increased compared to the non-induced PBMC cells (P < 0.05).

EXAMPLE 2 preparation of monoclonal antibody targeting varicella-zoster Virus envelope glycoprotein

Immunization of varicella-zoster virus envelope glycoprotein high antigen epitope peptide

According to the sequence of the envelope glycoprotein of the varicella-zoster virus, a high antigen epitope peptide is obtained by searching a conservation interval through a hydrophobic index, a structural domain and a sequence alignment of a plurality of strains of B virus, and the sequence is vaatlghfatsvifttyfcgrgldetn. The polypeptide is synthesized by Shanghai's work. 2mg of the dried polypeptide powder was dissolved in 500. mu.l of 2- (N-morpholine) ethanesulfonic acid Monohydrate (MES) buffer, 2mg of BSA was dissolved in 200. mu.l MES buffer, and 10mg of EDC was dissolved in 1ml of ddH₂O, mixing 500. mu.l of polypeptide solution with 200. mu.l of BSA solution, adding 100. mu.l of EDC solution, mixing well for 2h at room temperature, dialyzing with PBS, and storing at-20 ℃ for later use. Fusing the components

The polypeptide is mixed with equal volume of Freund's complete adjuvant, 6 BALB/c female mice (antigen amount 100 ug/mouse) with 7 weeks of age are immunized by multi-point injection on the neck and back, and then 2 nd and 3 rd immunization are carried out respectively on 14 th and 28 th days by the same method and dosage (Freund's complete adjuvant is replaced by Freund's incomplete adjuvant). On day 10 after 3 rd immunization, the mice with the highest antibody level were selected, and 1 booster immunization (100 ug/mouse antigen) was performed by intraperitoneal injection, and splenocytes were taken for cell fusion after 4 days.

Take 1X 10⁸Each splenocyte is matched with (1-2) × 10⁷Myeloma cells in several logarithmic growth phases are mixed at a ratio of 7: 1 in a 50mL centrifuge tube, cell fusion is carried out by using a PEG1500 fusion agent according to a conventional method, a positive hybridoma cell strain is screened by indirect ELISA by using immune polypeptide as a coating antigen, and subcloning is continuously carried out for 3 times by using a limiting dilution method until the hybridoma cell strain capable of stably secreting the antibody is obtained. Using antibody subclass identificationAnd (4) determining the kit, and identifying the monoclonal antibody subclasses according to the instruction.

After spleen lymphocytes of an immunized mouse are fused with SP2/0 cells, 2 hybridoma cell strains which have the best positive effect and stably secrete monoclonal antibodies of anti-varicella-zoster virus envelope glycoprotein high-antigen epitope peptides are finally obtained through 3 times of subcloning and screening. Both the hybridoma cells and their monoclonal antibodies were designated "HHV-5F 3" and "HHV-6D 6".

The obtained monoclonal hybridoma cells are subjected to expanded culture, injected into the abdominal cavity of an F1 mouse (the center of medical laboratory animals in Guangdong province), ascites is collected after the abdominal cavity of the mouse is swollen, and the monoclonal antibody is obtained by purifying through a Protein-A affinity chromatography column. And (3) identifying the monoclonal antibody secreted by the hybridoma cell strain by using an antibody subclass identification kit, and determining that the two monoclonal antibodies consist of an IgG1 heavy chain and a kappa light chain.

Example 3 specific identification of HHV-5F3 monoclonal antibody

Varicella-zoster virus VZV-pOKA virus strain was purchased from the Wuhan virus institute of Chinese academy of sciences; the VZV-pOKA virus strain and other different virus strains BTV-8, CHUV, EHDV-1 and influenza B virus Malaysia2506/04 are respectively infected with BHK cells cultured in a 6-well plate, and cell proteins are extracted about 72 hours when cytopathic effect is obvious. PAGE electrophoresis is carried out on samples with the same amount of protein, then the samples are electrically transferred to a PVDF membrane, 5% skimmed milk is sealed for 1h at room temperature, a primary antibody (HHV-5F3) is incubated overnight at 4 ℃, a secondary antibody is incubated for 1h at room temperature after membrane washing, and chemiluminescence self-development is carried out after membrane washing. The Western-blot results show that the monoclonal antibody HHV-5F3 can specifically recognize VZV-protein and does not react with other viral proteins (FIG. 1).

ELISA: coating was carried out at a protein amount of 200 ng/well (3 wells per sample), and indirect ELISA was carried out according to a conventional method using HHV-5F3 monoclonal antibody as a primary antibody. The specificity of the antibody was also confirmed by the ELISA results of the detection of the monoclonal antibody HHV-5F3, the OD of which reached 0.34 (FIG. 2).

Example 4HHV-5F3 monoclonal antibody affinity and variable region identification

The experiment uses a Fortebio Octet molecular interaction instrument to detect the affinity constant of the single HHV-5F3 monoclonal antibody and the immune polypeptide.

The experimental method for detecting the affinity constant of the HHV-5F3 monoclonal antibody and the immune polypeptide by the Fortebio Octet molecular interaction instrument is as follows: the sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. mu.g/mL of the polypeptide was immobilized on the HIS1K sensor for 40s, the sensor was equilibrated in buffer for 55s, the polypeptide immobilized on the sensor was bound to the antibody at a concentration of 0.1-50nM for 100s, and the antibody was dissociated in buffer for 100 s. The sensor was regenerated using 10mM glycine, pH1.5, for a period of 5s, and repeated 4 times. The detection temperature was 30 ℃ and the frequency was 0.3 Hz. Data were expressed as 1: 1, carrying out model fitting analysis to obtain an affinity constant.

The result shows that the HHV-5F3 monoclonal antibody can be combined with the polypeptide, has the affinity constant of 6.83E-09M, and has better affinity effect.

Total RNA was extracted from the cultured HHV-5F3 monoclonal cell line using Trizol reagent. The total RNA was converted to cDNA using the reverse transcription cDNA kit from Taraka. The cDNA was further supplemented with polyG at the 3' end. The gene amplification of the antibody variable region was performed using the tailed cDNA as a template. The variable region sequences of the light chain and the variable region sequences of the heavy chain of the monoclonal antibody were obtained as follows:

light chain variable region (SEQ ID NO: 1)

Heavy chain variable region (SEQ ID NO: 2):

example 5 experiment of suppression of viruses by HHV-5F3 monoclonal antibody

Vero cells were cultured in DMEM medium containing 2% fetal bovine serum at 5X 10⁴Inoculating 96-well culture plate at concentration of 100 μ L per well, standing at 37 deg.C and 5% CO₂The cell monolayer was formed overnight in the incubator. After discarding the supernatant, the mixture was washed with PBSWashing the cells for 1 time to remove residual fetal calf serum, adding DMEM medium without fetal calf serum to dilute to 100TCID₅₀VZV-pOKA virus (100. mu.L/well) was incubated at 35 ℃ for 2 hours, and then the virus solution was discarded. Preparing HHV-5F3 monoclonal antibody and positive control drug acyclovir into DMEM culture medium containing 2% fetal calf serum at different concentrations, adding into single-layer Vero cell, setting 3 multiple holes, and culturing at 37 deg.C and 5% CO in Vero cell₂Culturing in an incubator for 48h, and observing cytopathic phenomenon every day. After 48h, the supernatant was discarded, 100. mu.L of 10% formaldehyde was added for fixation for 1h, 0.1% (w/v) crystal violet was stained for 15min, and the absorbance was measured by an microplate reader at 570 nm. The rate of virus inhibition was calculated, and EC50 (half effective concentration) of the HHV-5F3 monoclonal antibody against the virus was calculated. The test results are shown in table 3:

TABLE 3 antiviral Activity of HHV-5F3 mAb on VZV-pOKA

Medicine	EC50
		HHV-5F3 monoclonal antibody	0.83μM
Acyclovir	1.26μM

Table 1 the results show that: the HHV-5F3 monoclonal antibody has obvious inhibition effect on cytopathic effect caused by human herpesvirus VZV-pOKA in vitro, has the EC50 of 0.83 mu M, and has the in vitro pharmacodynamic basis of being applied to treatment of herpesvirus infection.

Example 6 mouse experiment of virus inhibition by HHV-5F3 monoclonal antibody

Selecting 30 SPF grade 8 week old male C57 mice with weight of 18-22g, prepared from Shanghai university of medicineA central supply. After the sodium pentobarbital is anesthetized, the backs of the guinea pigs are unhaired, cleaned and disinfected according to a conventional method, and the unhaired parts of the backs of the mice are divided into a plurality of skin areas by a marking pen. Central intradermal injection 10 at each dermatome⁷PFU/mLVZV-pOKA virus strain virus, 0.1mL per injection. During anaesthesia, animals were kept in an incubator at 30 ℃ for 2 h. Respectively relating to a positive control group, an antibody + immune cell group, an immune cell group and a blank control group, wherein the positive control group is an acyclovir group; the antibody group was the monoclonal antibody group of the present invention, the antibody + immune cell group was the monoclonal antibody of the present invention + CIK cells prepared in example 1, the immune cell group was the CIK cells prepared in example 1, and the blank control group was smeared with PBS solution. Applying 30 μ L of the medicinal liquid each time, and continuously administering for 5 days after applying the medicinal liquid 2 times daily until the medicinal liquid is dried. The cure conditions of the pathological changes of the mice are observed day by day. The results are shown in Table 4.

TABLE 4 therapeutic Effect of topical administration on herpes Virus infection of the skin of mice

Administration group	Dosage form	Healing days (Tian)
			Positive control group	10mg/kg/d	10.56±1.38
Antibody group	10mg/kg/d	8.65±1.12
			Antibody + immune cell group	10mg/kg/d+5×10⁹Per kg/d	6.04±1.40
Immune cell group	5×10⁹Per kg/d	12.68±1.34
			Blank control group	-(PBS)	19.32±1.02

The results in table 4 show that, after the skin of the mouse is inoculated with the infectious virus, the cure days of the groups coated with the antibody and the combination of the antibody and the immune cells are both significantly reduced compared with the positive control, particularly the combination of the antibody and the immune cells can be cured within 6.04 days of the minimum pharmaceutical preparation. The antibody and the combination of the antibody and the immune cells have treatment effect on the virus infection of the skin of the mouse, can shorten the recovery time, and have better effect. And by detecting the visceral organs of the mice, no substantive lesion exists, which shows that the safety of the medicine can be guaranteed.

While the invention has been described and illustrated in detail as being sufficient to enable those skilled in the art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein represent preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications thereof and other uses will occur to those skilled in the art. Such modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

Sequence listing

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115

Claims

The application of NIK immune cells in preparing medicaments for preventing and/or treating varicella-zoster virus infection of human beings; the NIK immune cells are prepared by the following method: adding normal peripheral blood into normal saline 1: 1, after dilution, slowly adding the blood mixture onto a Ficoll lymphocyte separation solution along the tube wall by using a suction tube, and centrifuging for 20min at 800g and 4 ℃; carefully sucking the second white cloudy mononuclear cell layer with a pipette into a centrifugal tube, adding normal saline, centrifuging at 4 deg.C for 10min for 3 times at 400g, and adjusting cell concentration to 1.0 × 10⁶Per mL; inoculating to T175 culture bottle coated with CD3 monoclonal antibody in advance, adding BPMI-1640 culture medium containing 10% fetal calf serum, adding 50 μ g/L IL-2, 50 μ g/L IFN-gamma and 25 μ g/L IL-22, culturing at 37 deg.C in 5% CO2 cell culture box, and replacing half the culture every other dayCulturing the cells for 10 days to obtain the NIK immune cells.
2. The use according to claim 1, wherein the disease caused by infection is herpes labialis, pharyngitis, keratitis or sporadic encephalitis.
The application of NIK immune cells and HHV-5F3 monoclonal antibodies in preparing pharmaceutical compositions for treating varicella-zoster virus infection of human beings; wherein the light chain variable region sequence of the antibody is shown in SEQ ID NO: 1, the heavy chain variable region sequence of the antibody is shown as SEQ ID NO: 2 is shown in the specification; the NIK immune cells are prepared by the following method: adding normal peripheral blood into normal saline 1: 1, after dilution, slowly adding the blood mixture onto a Ficoll lymphocyte separation solution along the tube wall by using a suction tube, and centrifuging for 20min at 800g and 4 ℃; carefully sucking the second white cloudy mononuclear cell layer with a pipette into a centrifugal tube, adding normal saline, centrifuging at 4 deg.C for 10min for 3 times at 400g, and adjusting cell concentration to 1.0 × 10⁶Per mL; inoculating the cells in a T175 culture bottle coated with a CD3 monoclonal antibody in advance, adding a BPMI-1640 culture medium containing 10% fetal bovine serum, adding 50 mu g/L of IL-2, 50 mu g/L of IFN-gamma and 25 mu g/LIL-22, placing the mixture in a 5% CO2 cell culture box at 37 ℃ for induction culture, replacing the culture medium by half every day, and culturing for 10d to obtain the NIK immune cells.
4. The use according to any one of claims 1 to 3, wherein the medicament further comprises a pharmaceutically acceptable carrier.
5. The use of claim 4, wherein the pharmaceutical formulation is any one of clinically acceptable oral administration, injection administration or external administration.
6. The use according to claim 5, wherein the medicament is in the form of tablets, capsules, granules, pills, injections, creams, suspensions, dispersions, syrups, suppositories, gels, aerosols or patches.
7. The use according to any one of claims 1 to 3, wherein the active dose of said antibody is 10mg/kg body weight/d of the human body and the dose of said immune cells is 5 x 10⁹One/kg body weight/d.
The HHV-5F3 monoclonal antibody, characterized in that the light chain variable region sequence of said antibody is as set forth in SEQ ID NO: 1, the heavy chain variable region sequence of the antibody is shown as SEQ ID NO: 2, respectively.