阅读说明：本技术 一种水痘-带状疱疹病毒亚单位纳米疫苗的制备方法和应用 (Preparation method and application of varicella-zoster virus subunit nano vaccine ) 是由 刘利新 陈浩林 陈永明 刘鸿 于 2020-05-29 设计创作，主要内容包括：本发明公开了一种水痘-带状疱疹病毒亚单位纳米疫苗的制备方法和应用。具体公开了一种纳米颗粒,包含水痘-带状疱疹病毒(VZV)gE重组蛋白、免疫佐剂、阳离子脂质和辅助脂质。本发明制备得到的纳米颗粒尺寸均匀,形态规则、外形圆整、表面光滑、分散性好,无明显粘连、破损、坍塌等现象；纳米颗粒中VZV gE重组蛋白具有较高的包封率；所述纳米颗粒施用于动物后,能产生较强的体液免疫,使细胞免疫明显增强,具有较大的应用前景。(The invention discloses a preparation method and application of a varicella-zoster virus subunit nano vaccine. Specifically disclosed is a nanoparticle comprising a varicella-zoster virus (VZV) gE recombinant protein, an immunoadjuvant, a cationic lipid, and a helper lipid. The nano particles prepared by the method have the advantages of uniform size, regular shape, round and smooth appearance, smooth surface, good dispersibility, no obvious adhesion, damage, collapse and other phenomena; the VZV gE recombinant protein in the nano-particles has higher encapsulation efficiency; after the nano-particles are applied to animals, stronger humoral immunity can be generated, the cellular immunity is obviously enhanced, and the application prospect is larger.)

1. A nanoparticle comprising a varicella-zoster virus gE recombinant protein, an immunoadjuvant, a cationic lipid and a helper lipid.

2. The nanoparticle according to claim 1, wherein the nanoparticle vaccine is a liposome nucleocapsid structure, the core is varicella-zoster virus gE recombinant protein and immune adjuvant, and the shell is cationic lipid and helper lipid coated on the core.

3. The nanoparticle according to claim 1 or 2, wherein the varicella-zoster virus gE recombinant protein has an amino acid sequence shown in SEQ ID NO 1.

4. The nanoparticle of claim 1, wherein the immunoadjuvant is IMQ and/or MPLA.

5. The nanoparticle according to claim 1 or 2, wherein the cationic lipid is selected from one or more of trimethyl-2, 3-dioleyloxypropylammonium bromide, dimethyl-2, 3-dioleyloxypropyl-2- (2-spermicarbonamido) ethylammonium trifluoroacetate, dimethyldioctadecylammonium bromide, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, 1, 2-dioleyl-3-succinyl-sn-glycerocholine ester, 3 β - [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol, stearylamine.

6. The nanoparticle according to claim 1 or 2, wherein the helper lipid is selected from one or more of phosphatidylethanolamine, phosphatidylcholine, cholesterol, dioleoylphosphatidylethanolamine.

7. The nanoparticle according to claim 1 or 2, wherein the varicella-zoster virus gE recombinant protein: an immunological adjuvant: cationic lipid: the mass ratio of the auxiliary lipid is 6-9: 0.4-4: 30-40: 10 to 14.

8. The nanoparticle of any one of claims 1 to 7, wherein the preparation of the nanoparticle comprises the following steps:

s1, providing a solution containing cationic lipid, a solution containing helper lipid, a solution containing varicella-zoster virus gE recombinant protein and a solution containing immune adjuvant;

s2, enabling the solution containing the cationic lipid, the solution containing the auxiliary lipid, the solution containing the varicella-zoster virus gE recombinant protein, the solution containing the immunologic adjuvant and deionized water to respectively enter a mixing area through different sample introduction channels to reach the mixing area, and mixing to obtain a nanoparticle solution;

and S3, removing the organic solvent to obtain the nano-particle aqueous solution.

9. Use of the nanoparticles according to any one of claims 1 to 7 for the preparation of an immunogenic composition for the treatment of a disease associated with varicella-zoster virus infection.

10. The use of claim 9, wherein the immunogenic composition further comprises a pharmaceutically acceptable excipient.

Technical Field

The invention relates to the technical field of biological medicines. More particularly, relates to a varicella-zoster virus subunit nano vaccine, a preparation method and an application thereof.

Background

Varicella-zoster virus (VZV), a double-stranded DNA virus, human herpes virus type 3 (HHV-3), belongs to the sub-family of the alpha herpes viruses of the herpes virus family, and is the only natural host. The primary infection caused by VZV is manifested as varicella (varicella) and lies within the sensory ganglia of the host, which when reactivated can cause shingles (HZ) to be more common in adults and the elderly. VZV virus has only one serotype, and animal and chicken embryos are not sensitive to VZV, proliferate in human or monkey fibroblasts, and slowly produce cytopathic effects, form multinucleated giant cells, and eosinophilic inclusion bodies are visible in infected nuclei.

Herpes zoster is a recurrent infection of VZV that remains latent in the body. When the body is subjected to certain stimulation, such as heating, cold and mechanical compression, and the cellular immune function is damaged or reduced by using immunosuppressive agent, X-ray irradiation, leukemia, tumor and the like, the latent virus is activated, the virus descends along the axis of a sensory nerve to reach the skin cells dominated by the nerve for proliferation, and then the herpes zoster which is in series connection and is shaped like a belt is generated on the skin along the pathway of the sensory nerve, so the herpes zoster is named. When herpes zoster occurs, patients usually have symptoms such as fever and hypodynamia, the patients begin to red at local skin and are accompanied by burning sensation and nerve pain sensation, and the local pain sensation is very sensitive and has severe pain within 1-4 weeks. After suffering from chickenpox, the organism can produce specific humoral immunity and cellular immunity, and can not be infected any more for the whole life. However, the virus can not be eliminated due to long-term latent in ganglia, so that the virus can not be prevented from activating to generate herpes zoster.

Because no specific therapeutic drug aiming at chicken pox and herpes zoster exists at present, broad-spectrum antiviral drugs such as acyclovir are mostly used for clinical treatment, narcotic or non-narcotic analgesics, anticonvulsants and antidepressant drugs are also used for relieving the strong neuralgia brought by herpes zoster, the work of preventing and controlling VZV infection is very important, and vaccination is the most important and most effective way for preventing and controlling VZV at present. The varicella and herpes zoster vaccines on the market are mostly developed based on the Oka strain VZV live attenuated virus, and only differ from each other in the dose of the virus used for each vaccination and the number of vaccinations. In addition to live attenuated vaccines, subunit vaccines and DNA vaccines of VZV are currently also becoming a more interesting subject of research.

The safety of conventional inactivated or inactivated vaccines and the resulting systemic immune storm are unavoidable in many vaccine systems. However, with the development of modern molecular biology and biochemistry, the advantages and applications of subunit protein vaccines and polypeptide vaccines are widely determined. The subunit vaccine is prepared by extracting special protein structures of bacteria and viruses by chemical decomposition or controlled proteolysis, and screening out fragments with immunological activity. Compared with a whole virus vaccine, the subunit vaccine has higher safety and better stability; meanwhile, the subunit vaccine has durability in immunity, and long-term immunity can be obtained by one-time inoculation without repeated multiple times of immunity enhancement.

VZV plays a very important role in the replication of cell infection. The virus is replicated from the DNA of the nucleus to the nucleocapsid to assemble out the nucleus, then to the cortex assembly modification of endoplasmic reticulum and Golgi apparatus, and finally the virus is infected by cell-out and intercellular fusion, a series of processes need to go through multiple times of envelope and de-envelope, and the glycoprotein existing in the virus envelope plays an important role in the processes. There are 9 VZV glycoproteins currently identified, gB, gC, gE, gH, gI, gK, gL, gM, gN. Among them, gE glycoprotein is the glycoprotein with the highest expression level of VZV, plays a major role in the replication and assembly process of virus, and mediates the spread of virus among cells. It was found that in the serum of patients with chickenpox and shingles in convalescent phase, VZV antibodies are directed primarily against gE, gB and gH, especially gE-induced cellular and humoral immunity, protecting the animals from viral attack. In addition, VZV gE monoclonal antibodies can mediate antibody-dependent cellular cytotoxicity and neutralize viral infectivity in the presence of exogenous complement. Given that VZV gE is highly immunogenic and induces an immune response against VZV, it has become one of the major candidate antigens for VZV subunit vaccines and DNA vaccines. The HZ subunit vaccine Shingrix with gE as the main component is approved by the Food and Drug Administration (FDA) to be marketed at the end of 2017 and is used for preventing HZ and complications thereof in the elderly people over 50 years old.

Studies have shown that gE protein alone is not capable of inducing a strong cellular immune response in animal models and must be adjuvanted to enhance the immune response to gE. Patent CN201780072995.4 discloses a varicella zoster virus vaccine composition, which contains surface protein (gE) of varicella zoster virus and aluminum salt immunopotentiating adjuvant with specific ratio, and has the problem of weak immunogenicity although the immune effect is enhanced.

Disclosure of Invention

The present invention is directed to overcoming the above-mentioned drawbacks and deficiencies of the prior art and to providing a nanoparticle.

Another object of the present invention is to provide a method for preparing the above nanoparticles.

A third object of the present invention is to provide the use of the above nanoparticles.

In order to achieve the purpose, the invention is realized by the following scheme:

a nanoparticle comprising a varicella-zoster virus (VZV) gE recombinant protein, an immune adjuvant, a cationic lipid, and a helper lipid.

According to the invention, varicella-zoster virus (VZV) gE glycoprotein is used as an antigen of a vaccine, a cationic phospholipid and VZV gE recombinant protein are subjected to electrostatic interaction, auxiliary lipid such as cholesterol is used as a stabilizer, and a specific immune adjuvant is encapsulated to form liposome nanoparticles containing VZV gE protein and the specific immune adjuvant.

The "encapsulation" is not limited to placing the VZV gE recombinant protein and immune adjuvant completely inside the nanoparticle. In the nanoparticle of the invention, the VZV gE recombinant protein and the immunoadjuvant can be completely positioned in the nanoparticle or partially positioned on the surface of the nanoparticle.

Preferably, the nanoparticle vaccine is a liposome nucleocapsid structure, the core is varicella-zoster virus (VZV) gE recombinant protein and immune adjuvant, and the shell is cationic lipid and auxiliary lipid coated on the core.

Preferably, the varicella-zoster virus gE recombinant protein has an amino acid sequence shown as SEQ ID NO. 1. The invention obtains the VZV gE protein through deleting the gene sequence of the transmembrane region in the full-length segment of the VZV gE to carry out in-vitro recombination expression, and designs the VZV gE protein new antigen epitope.

Preferably, the immunoadjuvant is IMQ and/or MPLA.

Preferably, the cationic lipid is selected from trimethyl-2, 3-dioleoyloxypropylammonium bromide (DOTAP), trimethyl-2, 3-dioleyloxypropylammonium bromide (DOTMA), dimethyl-2, 3-dioleyloxypropyl-2- (2-spermicarbonamido) ethylammonium trifluoroacetate (DOSPA), dimethyldioctadecylammonium bromide (DDAB), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), 1, 2-dioleoyl-3-succinyl-sn-glycerocholine ester (DOSC), 3 β - [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol (DC-Chol), Stearylamine (SA).

More preferably, the cationic lipid is trimethyl-2, 3-dioleoyloxypropylammonium bromide (DOTAP).

Preferably, the helper lipid is selected from one or more of Phosphatidylethanolamine (PE), Phosphatidylcholine (PC), cholesterol (Chol), Dioleoylphosphatidylethanolamine (DOPE).

More preferably, the helper lipid is cholesterol (Chol).

Preferably, the VZV gE recombinant protein: an immunological adjuvant: cationic lipid: the mass ratio of the auxiliary lipid is 6-9: 0.4-4: 30-40: 10 to 14.

More preferably, when the adjuvant is IMQ, the VZV gE recombinant protein: an immunological adjuvant: cationic lipid: the mass ratio of the auxiliary lipid is 8:3:36: 12; when the adjuvant is MPLA, the VZV gE recombinant protein: an immunological adjuvant: cationic lipid: the mass ratio of the auxiliary lipid is 8:0.4:36: 12. When the adjuvant is IMQ and MPLA, the VZV gE recombinant protein: immunological adjuvant IMQ: immunological adjuvant MPLA: cationic lipid: the mass ratio of the auxiliary lipid is 8:3:0.4:36: 12.

Preferably, the nanoparticles are approximately spherical.

Preferably, the nanoparticles have a particle size of 30 to 200nm, such as 30 to 50nm, 50 to 80nm, 80 to 100nm, 100 to 150nm or 150 to 200 nm.

Preferably, the Zeta potential of the nanoparticles is from +10 to +35mV, for example from +10 to +15mV, +15 to +20mV, +20 to +25mV, +25 to +30mV, +30 to +35 mV.

Preferably, the entrapment rate of the VZV gE protein in the nanoparticle is 80% to 100%, such as 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100%.

The invention also provides a preparation method of the nanoparticle, which comprises the following steps:

s1, providing a solution containing cationic lipid, a solution containing helper lipid, a solution containing VZV gE recombinant protein and a solution containing immune adjuvant;

s2, enabling the solution containing the cationic lipid, the solution containing the auxiliary lipid, the solution containing the VZV gE recombinant protein and the solution containing the immune adjuvant to respectively reach a mixing area through different sample feeding channels, and mixing to obtain a nanoparticle solution;

and S3, removing the organic solvent to obtain the nano-particle aqueous solution.

Preferably, the method is performed in an apparatus comprising a first channel, a second channel, a third channel, a fourth channel, and a mixing zone. In a preferred embodiment, the apparatus is a multi-inlet vortex mixer, for example a four-inlet vortex mixer. The techniques and apparatus are described in the inventor's earlier patent application No. PCT/US 2017/014080.

Preferably, when the adjuvant is IMQ, the solution comprising the cationic lipid, the solution comprising the helper lipid, and the solution comprising the immunoadjuvant IMQ are mixed through the first channel, the solution of VZV gE recombinant protein is passed through the second channel, a separate channel, and pure water is passed through the third and fourth channels, and the flow rates of each channel are the same.

Preferably, when the adjuvant is MPLA, the solution comprising the cationic lipid, the solution comprising the helper lipid, the solution comprising the immunoadjuvant MPLA are mixed through the first channel, the solution of VZV gE recombinant protein is passed through the second channel, a separate channel, pure water is passed through the third and fourth channels, and the flow rates of each channel are the same.

Preferably, when the adjuvant is IMQ and MPLA, the solution comprising the cationic lipid, the solution comprising the helper lipid, the solution comprising the immunoadjuvant IMQ and MPLA are mixed through the first channel, the solution of VZV gE recombinant protein is passed through the second channel, a separate channel, pure water is passed through the third and fourth channels, and the flow rates of each channel are the same.

Preferably, the solution comprising the cationic lipid, the solution comprising the helper lipid, the solution comprising the VZV gE recombinant protein and the solution comprising the immunoadjuvant create a vortex in the mixing zone.

Preferably, the flow rate of each channel is the same and is 1-20 mL/min, such as 1mL/min, 5mL/min, 8mL/min, 10mL/min, 15mL/min or 20 mL/min.

More preferably, the flow rates of the solution comprising the cationic lipid, the solution comprising the helper lipid, the solution comprising the VZV gE recombinant protein, the solution comprising the immunoadjuvant, and pure water in the channel are all 10 mL/min.

Preferably, the solution comprising VZV gE recombinant protein has a pH of 7.2 to 7.4.

Preferably, the method further comprises step 4: the solution comprising the nanoparticles is subjected to lyophilization concentration, for example by addition of a lyoprotectant.

Preferably, the concentration ratio of the solution containing the cationic lipid, the solution containing the helper lipid, the solution containing the VZV gE recombinant protein and the solution containing the immune adjuvant is 3-4 mg/mL: 1.0-1.4 mg/mL: 600-800 μ g/mL: 40-400 μ g/mL.

Preferably, when the adjuvant is IMQ, the concentration ratio of the solution containing the cationic lipid, the solution containing the helper lipid, the solution containing the VZVgE recombinant protein and the solution containing the immunological adjuvant IMQ is 3.4-3.8 mg/mL: 1.2 mg/mL: 600-800 μ g/mL: 300. mu.g/mL.

Preferably, when the adjuvant is MPLA, the concentration ratio of the solution comprising the cationic lipid, the solution comprising the helper lipid, the solution comprising the VZVgE recombinant protein, the solution comprising the immunological adjuvant MPLA is 3.4-3.8 mg/mL: 1.2 mg/mL: 600-800 μ g/mL: 40-50 μ g/mL.

Preferably, when the adjuvant is IMQ and MPLA, the concentration ratio of the solution containing the cationic lipid, the solution containing the helper lipid, the solution containing the VZV gE recombinant protein, the solution containing the immune adjuvant IMQ and the solution containing the immune adjuvant MPLA is 3.4-3.8 mg/mL: 1.2 mg/mL: 600-800 μ g/mL: 280-300 mu g/mL: 300 to 400 μ g/mL.

Preferably, in step 1, the solution containing the cationic lipid is an ethanol solution.

Preferably, in step 1, the solution containing the helper lipid is an ethanol solution.

Preferably, in the step 1, the solution containing the immune adjuvant is an ethanol solution.

Preferably, in the step 1, the solution containing VZV gE recombinant protein is Hepes buffer solution (pH 7.2 ± 0.2).

The nanoparticle co-loaded with VZV gE recombinant protein and immune adjuvant of the invention can cause stronger immune response than free VZVgE recombinant protein and adjuvant. Therefore, the invention also claims the use of said nanoparticles for the preparation of immunogenic compositions for diseases associated with VZV infection.

Preferably, the disease associated with VZV infection is varicella and/or herpes zoster.

The invention also provides an immunogenic composition comprising the nanoparticles of the invention.

Preferably, the immunogenic composition further comprises pharmaceutically acceptable excipients, such as excipients, preservatives, antibacterial agents and/or additional immunological adjuvants.

Preferably, the immunogenic composition is a vaccine.

Preferably, the immunogenic composition is for use in the prevention and/or treatment of a disease associated with VZV infection, such as varicella, herpes zoster, in a subject.

Preferably, the subject is a mammal, e.g., bovine, equine, porcine, canine, feline, rodent, primate; for example, the subject is a human.

Preferably, the immunogenic composition further comprises a second immunogenic agent. For example, the immunogenic composition further comprises a VZV other protein than the VZV gE recombinant protein. For example, the immunogenic composition further comprises inactivated and deactivated VZV. For example, the immunogenic composition may also comprise other pathogenic microorganisms (including live, inactivated or attenuated) than VZV. For example, the immunogenic composition further comprises a portion of other pathogenic microorganisms than VZV.

In one aspect, the invention also provides a method of preventing and/or treating a disease associated with VZV infection in a subject, comprising administering to the subject a nanoparticle or immunogenic composition (e.g., vaccine) of the invention.

Preferably, the disease associated with VZV infection is varicella, herpes zoster.

Preferably, the subject is a mammal, e.g., bovine, equine, porcine, canine, feline, rodent, primate; for example, the subject is a human.

In one aspect, the present application provides a method of eliciting or enhancing an immune response to VZV in a subject, comprising administering to the subject a nanoparticle or immunogenic composition (e.g., a vaccine) of the invention.

Preferably, the subject is a mammal, e.g., bovine, equine, porcine, canine, feline, rodent, primate; the subject is the subject C57BL/6 mouse.

In one aspect, the present application provides the use of a nanoparticle or immunogenic composition (e.g. vaccine) of the invention for eliciting or enhancing an immune response to VZV in a subject.

Preferably, the subject is a mammal, e.g., bovine, equine, porcine, canine, feline, rodent, primate; for example, the subject is the subject C57BL/6 mouse.

The liposome nanoparticle of the invention takes recombinant protein antigen VZV gE protein as a basis, and an adjuvant and an antigen are loaded together. The nanoparticle is similar to the size of pathogenic microorganism, so that the nanoparticle is more easily captured by Antigen Presenting Cells (APC) and presents antigen to T cells, T cells and B cells are promoted to mature and activate, and the immune effect is enhanced.

The immune effect of the liposome nanoparticle vaccine wrapping VZV gE recombinant protein, double immune adjuvants IMQ and MPLA is obviously better than that of a mixture of an aluminum adjuvant and free VZV gE recombinant protein. The nano vaccine can well stimulate Th1 type immunity and activate Th2 type immunity; can increase the expression of IFN-gamma, TNF-alpha and IL-2 of CD4+ and CD8+ lymphocyte T cells, thereby enhancing the cellular immune effect mediated by the T cells.

Compared with the prior art, the invention has the beneficial effects that:

(1) the varicella-zoster virus gE recombinant protein is used as a vaccine antigen to prepare the nano vaccine, and the prepared nano particles have regular shape, round and smooth appearance, good dispersibility, no obvious phenomena of adhesion, breakage, collapse and the like, and the VZVgE recombinant protein has higher encapsulation efficiency.

(2) After the nano particles are applied to animals, stronger humoral immunity can be generated, the cellular immunity is obviously enhanced, and the immune effect is superior to that of free antigen/adjuvant mixed injection and the existing vaccine containing aluminum adjuvant; and has the function of targeting lymph nodes, and improves the enrichment of the vaccine in the lymph nodes and the intake of antigen presenting cells.

(3) The nano-particles can be prepared by a simple method, have stable quality and are easy for industrial production.

Drawings

Fig. 1 is an exemplary illustration of step 2 in the method of preparing nanoparticles according to the present invention.

FIG. 2 shows the morphology results of 4 types of nanoparticles prepared in examples 3 to 5 of the present invention under a transmission electron microscope. A is the form of nanoparticle NPS wrapping VZVgE recombinant protein, cationic lipid and auxiliary lipid Chol under a transmission electron microscope; b is the form of nanoparticle NPS-I wrapping VZV gE recombinant protein, cationic lipid, auxiliary lipid Chol and immunologic adjuvant IMQ under a transmission electron microscope; c is the form of nanoparticle NPS-M wrapping VZV gE recombinant protein, cationic lipid, auxiliary lipid Chol and immunological adjuvant MPLA under a transmission electron microscope; d is the form of the nanoparticle NPS-I-M wrapping VZV gE recombinant protein, cationic lipid, auxiliary lipid Chol, immune adjuvant IMQ and immune adjuvant MPLA under a transmission electron microscope. As shown in the figure, the four nanoparticles have regular shapes, round shapes, smooth surfaces, good dispersibility, no obvious phenomena of adhesion, breakage, collapse and the like.

FIG. 3 shows the results of particle size measurements of 4 types of nanoparticles prepared in examples 3 to 5 of the present invention. As shown in the figure, four kinds of nanoparticles have narrow particle size distribution and are symmetrical in particle size distribution.

FIG. 4 shows the IgG titer (FIG. 4A), IgG1 titer (FIG. 4B), IgG2C titer (FIG. 4C), and IgG2C/IgG1 ratio (FIG. 4D) in the serum of each group of mice on day 7 after the first immunization of the mice in example 8 of the present invention.

FIG. 5 shows the IgG titer (FIG. 5A), IgG1 titer (FIG. 5B), IgG2C titer (FIG. 5C), and IgG2C/IgG1 ratio (FIG. 5D) in the serum of each group of mice on day 14 after the first immunization of the mice in example 8 of the present invention. Experimental results show that after mice are immunized by using the nanoparticles disclosed by the invention, the polarization of T cells can be promoted, and the effect of cellular immunity is enhanced.

FIG. 6 shows the IgG titer (FIG. 6A), IgG1 titer (FIG. 6B), IgG2C titer (FIG. 6C), and IgG2C/IgG1 ratio (FIG. 6D) in the serum of each group of mice on day 14 after the second immunization of the mice in example 8 of the present invention. Experimental results show that after mice are immunized by using the nanoparticles disclosed by the invention, the polarization of T cells can be promoted, and the effect of cellular immunity is enhanced.

FIG. 7 shows the IgG titer (FIG. 7A), IgG1 titer (FIG. 7B), IgG2C titer (FIG. 7C), and IgG2C/IgG1 ratio (FIG. 7D) in the serum of each group of mice on day 14 after the mice were immunized a third time in example 8 of the present invention. Experimental results show that after mice are immunized by using the nanoparticles disclosed by the invention, the polarization of T cells can be promoted, and the effect of cellular immunity is enhanced.

FIG. 8 shows the IgG titer (FIG. 8A), IgG1 titer (FIG. 8B), IgG2C titer (FIG. 8C), and IgG2C/IgG1 ratio (FIG. 8D) in the serum of each group of mice on day 32 after the mice were immunized the third time in example 8 of the present invention.

FIG. 9 shows the expression levels of IFN-. gamma.and TNF-. alpha.in CD4+ and CD8+ lymphoid T cells after antigen stimulation in various groups of mice. The results showed that the expression levels of IFN-. gamma.and TNF-. alpha.were significantly different in group J (NPS-I-M nanoparticle administration) compared to group A (PBS-administration negative control). The experimental result shows that the nanoparticles (NPS-I-M nanoparticles are applied) can increase the expression of IFN-gamma and TNF-alpha of CD4+ and CD8+ lymphocyte T cells after mice are immunized, thereby enhancing the cellular immune effect mediated by the T cells.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.