CN111840538A - Preparation method and application of varicella-zoster virus subunit nano vaccine - Google Patents
Preparation method and application of varicella-zoster virus subunit nano vaccine Download PDFInfo
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- A61K39/12—Viral antigens
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1277—Processes for preparing; Proliposomes
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- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- A61K2039/55511—Organic adjuvants
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6018—Lipids, e.g. in lipopeptides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
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.
Description
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.
EXAMPLE 1 preparation Condition screening (pH screening) of NPS nanoparticles
1. Reagent: the VZV recombinant protein is produced by expression of an escherichia coli expression system and is obtained by dissolution and renaturation, and the amino acid sequence of the VZV recombinant protein is shown as SEQ ID NO. 1.
Other reagents were commercially available.
2. The preparation process comprises the following steps:
(1) cationic lipid trimethyl-2, 3-dioleoyloxypropylammonium bromide (DOTAP) and helper lipid cholesterol (Chol) are co-dissolved in ethanol to obtain an ethanol solution with a DOTAP concentration of 0.75mg/mL and a Chol concentration of 0.25 mg/mL. Dispersing VZV gE recombinant protein in Hepes buffer solutions with different pH values (pH is 6.4, pH is 7.4, and pH is 7.9) to obtain the VZVgE recombinant protein with the concentration of 0.1-0.2 mg/mL.
(2) Respectively filling a solution containing cationic lipid DOTAP and auxiliary lipid Chol, a VZV gE recombinant protein solution, ultrapure water 1 and ultrapure water 2 into four injectors, wherein the ethanol solution containing the cationic lipid DOTAP and the auxiliary lipid Chol is a channel 1, the VZV gE recombinant protein solution is a channel 2, and the ultrapure water 1 and the ultrapure water 2 respectively enter channels 3 and 4; and respectively placing four injectors on a high-pressure pump, wherein the volume of the solution in each channel is 5mL, the solution in each injector passes through 1-4 channels respectively, and the flow rate of each channel is the same and is 10 mL/min. And starting the high-pressure pump to obtain a solution of the liposome nanoparticles wrapping the VZV gE recombinant protein.
(3) And (3) carrying out rotary evaporation or dialysis on the organic solvent to obtain an aqueous solution (named as NPS-1, NPS-2 and NPS-3 nanoparticle solutions) of the liposome nanoparticles wrapping the VZV gE recombinant protein.
(4) The average particle size of NPS-1, NPS-2, NPS-3 nanoparticles was measured using a malvern particle sizer (with a dynamic light scattering detector) and the results are shown in table 1.
TABLE 1
The average particle size of the above 3 types of nanoparticles was measured using a malvern particle sizer (with dynamic light scattering detector) and the results are shown in table 1. By screening different pH to prepare NPS nanoparticles, we finally determined that the optimal pH for preparing nanoparticles was 7.4. The prepared nano particles have uniform particle size and good dispersibility. Subsequent experiments to prepare nanoparticles VZVgE recombinant protein was dispersed in Hepes buffer solution at pH 7.4.
Example 2 preparation Condition screening (protein concentration screening) of NPS nanoparticles
1. Reagent: the VZV recombinant protein is produced by expression of an escherichia coli expression system and is obtained by dissolution and renaturation, and the amino acid sequence of the VZV recombinant protein is shown as SEQ ID NO. 1.
Other reagents were commercially available.
2. The preparation process comprises the following steps:
(1) cationic lipid trimethyl-2, 3-dioleoyloxypropylammonium bromide (DOTAP) and helper lipid cholesterol (Chol) are co-dissolved in ethanol to obtain an ethanol solution with the DOTAP concentration of 3.6mg/mL and the Chol concentration of 1.2 mg/mL. The VZVgE recombinant protein was dispersed in Hepes buffer solution (pH 7.2 ± 0.2) to give VZV gE recombinant protein concentrations of 0.6mg/mL, 0.7mg/mL and 0.8 mg/mL.
(2) Respectively filling a solution containing cationic lipid DOTAP and auxiliary lipid Chol, a VZV gE recombinant protein solution, ultrapure water 1 and ultrapure water 2 into four injectors, wherein the ethanol solution containing the cationic lipid DOTAP and the auxiliary lipid Chol is a channel 1, the VZV gE recombinant protein solution is a channel 2, and the ultrapure water 1 and the ultrapure water 2 respectively enter channels 3 and 4; and respectively placing four injectors on a high-pressure pump, wherein the volume of the solution in each channel is 5mL, the solution in each injector passes through 1-4 channels respectively, and the flow rate of each channel is the same and is 10 mL/min. And starting the high-pressure pump to obtain a solution of the liposome nanoparticles wrapping the VZV gE recombinant protein.
(3) And (3) carrying out rotary evaporation or dialysis on the organic solvent to obtain an aqueous solution (named as NPS-4, NPS-5 and NPS-6 nanoparticle solution) of the liposome nanoparticles wrapping the VZV gE recombinant protein.
(4) The average particle size of NPS-4, NPS-5, NPS-6 nanoparticles was measured using a malvern particle sizer (with a dynamic light scattering detector) and the results are shown in table 2.
TABLE 2
The average particle size of the above 3 types of nanoparticles was measured using a malvern particle sizer (with dynamic light scattering detector) and the results are shown in table 2. By screening different protein concentrations to prepare NPS nanoparticles, we finally determined the optimal for nanoparticle preparation. The prepared nano particles have uniform particle size and good dispersibility. In the subsequent experiment, VZVgE recombinant protein is dispersed in Hepes buffer solution with pH 7.4 to prepare VZV gE recombinant protein solution with initial concentration of 0.8 mg/mL.
EXAMPLE 3 preparation of NPS-I nanoparticles
1. Reagent: the VZV recombinant protein is produced by expression of an escherichia coli expression system and is obtained by dissolution and renaturation, and the amino acid sequence of the VZV recombinant protein is shown as SEQ ID NO. 1.
Other reagents were commercially available.
2. The preparation process comprises the following steps:
(1) cationic lipid trimethyl-2, 3-dioleoyloxypropylammonium bromide (DOTAP), helper lipid cholesterol (Chol) and immunologic adjuvant IMQ are co-dissolved in ethanol to obtain an ethanol solution with the DOTAP concentration of 3.6mg/mL, the Chol concentration of 1.2mg/mL and the IMQ concentration of 0.3 mg/mL. The VZV gE recombinant protein was dispersed in Hepes buffer solution (pH 7.2 ± 0.2) to give a VZV gE recombinant protein concentration of 0.8 mg/mL.
(2) Respectively filling a solution containing cationic lipid DOTAP, auxiliary lipid Chol and immune adjuvant IMQ, a VZV gE recombinant protein solution, ultrapure water 1 and ultrapure water 2 into four injectors, wherein the ethanol solution containing the cationic lipid DOTAP, the auxiliary lipid Chol and the immune adjuvant IMQ is a channel 1, the VZV gE recombinant protein solution is a channel 2, and the ultrapure water 1 and the ultrapure water 2 respectively enter channels 3 and 4; and respectively placing four injectors on a high-pressure pump, wherein the volume of the solution in each channel is 5mL, the solution in each injector passes through 1-4 channels respectively, and the flow rate of each channel is the same and is 10 mL/min. And starting the high-pressure pump to obtain a solution of liposome nanoparticles wrapping VZVgE recombinant protein and an immunologic adjuvant IMQ.
(3) And (3) carrying out rotary evaporation or dialysis on the organic solvent to obtain an aqueous solution (named as NPS-I nanoparticle solution) of the liposome nanoparticles wrapping the VZV gE recombinant protein and the immunologic adjuvant IMQ.
3. Referring to the operations and parameters of steps (1) - (3), an aqueous solution of liposome nanoparticles encapsulated VZV gE recombinant protein without immune adjuvant (named NPS nanoparticle solution) was prepared using an ethanol solution containing cationic lipid DOTAP and helper lipid Chol, VZV gE recombinant protein solution, ultrapure water.
Example 4 preparation of NPS-M nanoparticles
The preparation process comprises the following steps:
(1) co-dissolving cationic lipid DOTAP, auxiliary lipid Chol and immunological adjuvant MPLA in ethanol solution to obtain ethanol solution with DOTAP concentration of 3.6mg/mL, Chol concentration of 1.2mg/mL and MPLA concentration of 0.04 mg/mL. The VZV gE recombinant protein was dispersed in Hepes buffer solution (pH 7.2 ± 0.2) to give a VZV gE recombinant protein concentration of 0.8 mg/mL.
(2) Respectively filling an ethanol solution containing cationic lipid DOTAP, helper lipid Chol and immunological adjuvant MPLA, a VZV gE recombinant protein solution, ultrapure water 1 and ultrapure water 2 into four injectors, wherein the ethanol solution containing the cationic lipid DOTAP, the helper lipid Chol and the immunological adjuvant MPLA is a channel 1, the VZV gE recombinant protein solution is a channel 2, and the ultrapure water 1 and the ultrapure water 2 respectively enter channels 3 and 4; and respectively placing four injectors on a high-pressure pump, and enabling the solution in each injector to pass through 1-4 channels. The volume of the solution in each channel was 5mL, and the flow rate in each channel was the same and was 10 mL/min. And starting the high-pressure pump to obtain a solution of liposome nanoparticles wrapping VZV gE recombinant protein and immune adjuvant MPLA.
(3) And (3) carrying out rotary evaporation or dialysis on the organic solvent to obtain an aqueous solution (named as NPS-M nanoparticle solution) of the liposome nanoparticles wrapping the VZV gE recombinant protein and the immunological adjuvant MPLA.
EXAMPLE 5 preparation of NPS-I-M nanoparticles
The preparation process comprises the following steps:
(1) dissolving cationic lipid DOTAP, auxiliary lipid Chol, immune adjuvants IMQ and MPLA to obtain ethanol solution with DOTAP concentration of 3.6mg/mL, Chol concentration of 1.2mg/mL, IMQ concentration of 0.3mg/mL and MPLA concentration of 0.04 mg/mL. And dispersing the VZV gE recombinant protein into Hepes buffer solution to obtain the VZV gE recombinant protein with the concentration of 0.8 mg/mL.
(2) Respectively filling an ethanol solution containing cationic lipid DOTAP, auxiliary lipid Chol, immune adjuvant IMQ and MPLA, a VZVgE recombinant protein solution, ultrapure water 1 and ultrapure water 2 into four injectors, wherein the ethanol solution containing the cationic lipid DOTAP, the auxiliary lipid Chol, the immune adjuvant IMQ and the MPLA is a channel 1, the VZV gE recombinant protein solution is a channel 2, and the ultrapure water 1 and the ultrapure water 2 respectively enter channels 3 and 4; the four injectors are respectively placed on a high-pressure pump, the solution in each injector respectively passes through 1-4 channels, the volume of the solution in each channel is 5mL, and the flow rate of each channel is the same and is 10 mL/min. And starting the high-pressure pump to obtain a solution of liposome nanoparticles wrapping VZV gE recombinant protein, double immune adjuvants IMQ and MPLA.
(3) And (3) carrying out rotary evaporation or dialysis on the organic solvent to obtain an aqueous solution (named as NPS-I-M nanoparticle solution) of the liposome nanoparticles wrapping the VZV gE recombinant protein and the double immune adjuvants IMQ and MPLA.
Example 6 morphological characterization of nanoparticles, particle size testing and potential testing
1. Morphological characterization
Observing the liposome NPS nanoparticles wrapped with VZV gE recombinant protein and not containing immune adjuvant, the liposome NPS-I nanoparticles wrapped with VZV gE recombinant protein and immune adjuvant IMQ, the liposome NPS-M nanoparticles wrapped with VZV gE recombinant protein and immune adjuvant MPLA and the liposome NPS-I-M nanoparticles wrapped with VZV gE recombinant protein, double immune adjuvants IMQ and MPLA by using a transmission electron microscope, wherein the result is shown in figure 2, the forms of the four nanoparticles are respectively shown in figures 2A, 2B, 2C and 2D, and the four nanoparticles are regular in form, round in shape, smooth in surface, good in dispersity, free of obvious adhesion, breakage, collapse and the like.
2. Particle size test and Zeta potential test:
the average particle size and Zeta potential of the VZV gE recombinant protein-encapsulated, immunoadjuvant-free liposomal NPS nanoparticles, the VZV gE recombinant protein-encapsulated, immunoadjuvant IMQ-encapsulated liposomal NPS-I nanoparticles, the VZV gE recombinant protein-encapsulated, immunoadjuvant MPLA-encapsulated liposomal NPS-M nanoparticles, and the VZV gE recombinant protein-encapsulated, double-immunoadjuvant IMQ-and MPLA-encapsulated liposomal NPS-I-M nanoparticles were tested using a malvern particle sizer (with a dynamic light scattering detector), and the results are shown in table 1.
TABLE 1
FIGS. 3A, 4B, 4C, 4D are particle size distribution diagrams of VZV gE recombinant protein-encapsulated, liposomal NPS nanoparticle without immunoadjuvant, liposomal NPS-I nanoparticle with VZV gE recombinant protein-encapsulated, immunoadjuvant IMQ-encapsulated, liposomal NPS-M nanoparticle with VZV gE recombinant protein-encapsulated, immunoadjuvant MPLA-encapsulated, and liposomal NPS-I-M nanoparticle with VZV gE recombinant protein-encapsulated, double immunoadjuvant IMQ-encapsulated, MPLA, respectively. As can be seen from table 2 and fig. 3, the four kinds of nanoparticles have narrow particle size distributions, and the particle size distributions are symmetrical.
Example 7 calculation of encapsulation efficiency of VZV gE recombinant protein in nanoparticles
1. And (3) putting 5mL of NPS nanoparticle solution into a 300kDa ultrafiltration tube, centrifuging for 30min at 4 ℃ and 3000rpm, taking down the filtrate, detecting the content of free VZV gE recombinant protein in the lower filtrate by using a Bradford protein detection kit, and calculating the encapsulation rate of the VZV gE recombinant protein in the nanoparticles according to the following formula.
Encapsulation efficiency of VZV gE recombinant protein is w0-w1/w0X 100% where w0Is the total amount of VZV gE recombinant protein added; w is a1The total amount of free VZV gE recombinant protein in the following filtrate.
2. The encapsulation efficiency of VZV gE recombinant proteins in NPS-I nanoparticles, NPS-M nanoparticles and NPS-I-M nanoparticles was determined according to the method in 1.
The results of the encapsulation efficiency measurement are shown in Table 2.
TABLE 2
From the results in table 2, it can be seen that, in the four nanoparticles of the present invention, the encapsulation efficiency of VZV gE recombinant protein is higher, which is beneficial for the nanoparticles to induce strong immune effect.
Example 8 evaluation of the immune Effect of nanoparticles in mice
1. Immunization regimen
Female C57BL/6 mice at 5-8 weeks were divided into A, B, C, D, E, F, G, H, I, J ten groups of 10 mice each. Mice were immunized by tail root subcutaneous injection according to the immunization protocol in table 3, and boosted once two weeks, and again once a week apart, for a total of 3 immunizations.
TABLE 3
2. Evaluation of immune Effect (1) IgG detection in mouse serum
Orbital bleeds were performed on days 7, 14, 28, 42, and 60 after the first immunization, and sera were isolated and assayed by Elisa for IgG titers in the sera.
And (3) detection process:
1) 5 u g/mL VZV gE recombinant protein antigen coated in 96-well plates, each hole 100 u L, 4 degrees overnight coating.
2) The overnight coated plate was washed 3 times with PBST, 200. mu.L each, and blocked with 200. mu.L of 3% BSA at 37 ℃ for 2 h.
3) Taking 2 mu L immune serum or negative control serum, diluting to 200 mu L, then sequentially diluting in multiple proportion, adding into the hole coated with the antigen, and incubating for 2h at room temperature.
4) Washed 5 times, processed to IgG-HRP concentration, 100. mu.L per well, and incubated at room temperature for 2 h.
5) Washing 5 times, adding 100 μ L TMB substrate per well, incubating in dark for 20min, and incubating with 200 μ L2 MH2SO4The reaction was terminated and examined at 450nmAnd measuring the OD value.
6) The titer is calculated and if the ratio of the average absorbance (P) of the sample well to the average absorbance (N) of the negative control (group a) (i.e., P/N) is greater than 2.1, the sample well is determined to be positive.
The results of the detection are shown in FIGS. 4A, 5A, 6A, 7A, and 8A.
Figure 4A shows the IgG titers in the sera of the groups of mice at day 7 after the first immunization. The results show that the serum IgG titers of mice in group J (administered NPS-I-M nanoparticles) were higher than those in group D (administered free VZV gE recombinant protein + free MPLA), with significant differences (P < 0.05); and is significantly different (P < 0.01) than group B (administered free VZV gE recombinant protein) and group C (administered free VZV gE recombinant protein + free IMQ).
Figure 5A shows the IgG titers in the sera of the groups of mice at day 14 after the first immunization. The results show that the serum IgG titers of mice in group J (administered NPS-I-M nanoparticles) were higher than those in group B (administered free VZV gE recombinant protein), group C (administered free VZV gE recombinant protein + free IMQ), group D (administered free VZV gE recombinant protein + free MPLA), group F (administered free VZV gE recombinant protein + aluminum adjuvant), group G (administered NPS nanoparticles), with very significant differences (P < 0.001); and also higher than group E (free VZV gE recombinant protein + free IMQ + free MPLA), with significant difference (P < 0.05).
Figure 6A shows the IgG titers in the sera of the groups of mice at day 28 after the first immunization. The results show that the serum IgG titers of mice in group J (administered NPS-I-M nanoparticles) were higher than those in group B (administered free VZV gE recombinant protein), group C (administered free VZV gE recombinant protein + free IMQ), group D (administered free VZV gE recombinant protein + free MPLA), group E (administered free VZV gE recombinant protein + free IMQ + free MPLA), group F (administered free VZV gE recombinant protein + aluminum adjuvant), group G (administered NPS nanoparticles), group H (administered NPS-I nanoparticles), with very significant differences (P < 0.001).
Figure 7A shows the IgG titers in the sera of the groups of mice at day 42 after the first immunization. The results show that the serum IgG titers of the J group (administered NPS-I-M nanoparticles) mice reached 2.5 x 10^6, which is very significant different (P < 0.01) than the E group (free VZV gE recombinant protein + free IMQ + free MPLA).
Figure 8A shows the IgG titers in the sera of the groups of mice at day 60 after the first immunization. The results show that the serum IgG titers of mice in group J (administered NPS-I-M nanoparticles) were higher than in all other groups, with a significant difference (P < 0.001), significantly higher than in groups B (administered free VZV gE recombinant protein) and C (administered free VZV gE recombinant protein + free IMQ).
Experimental results show that after the liposome NPS-I-M nanoparticle wrapping VZV gE recombinant protein, double immune adjuvants IMQ and MPLA is used for immunizing a mouse, humoral immunity can be enhanced, and the titer of an antibody can be improved. The immune effect of the nanoparticle of the invention is obviously superior to that of a mixture of free VZV gE recombinant protein and an aluminum adjuvant.
(2) Detection of IgG subtypes in mouse serum
Orbital bleeds were performed on days 7, 14, 28, 42, and 60 after the first immunization, and sera were separated, and Elisa detected titers of IgG1 and IgG2c in sera, and calculated values of IgG2c/IgG1, as shown in fig. 4-9B-D.
FIG. 4B shows IgG1 titers in the sera of groups of mice at day 7 after the first immunization; figure 4C shows the IgG2C titers in the sera of groups of mice at day 7 after the first immunization; FIG. 4D shows the values of IgG2c/IgG1 in the sera of groups of mice on day 7 after the first immunization. The results show that the serum IgG1 titers of mice in group J (administered NPS-I-M nanoparticles) were higher than those in group B (administered free VZV gE recombinant protein), group C (administered free VZV gE recombinant protein + free IMQ), group D (administered free VZV gE recombinant protein + free MPLA), with significant differences (P < 0.05); group J (NPS-I-M nanoparticle-administered) mice had higher serum IgG2C titers than group B (free VZV gE recombinant protein administered), group C (free VZV gE recombinant protein + free IMQ), with significant differences (P < 0.01); group J (administered NPS-I-M nanoparticles) mice had higher serum IgG2c/IgG1 than group B (administered free VZV gE recombinant protein).
FIG. 5B shows IgG1 titers in the sera of groups of mice at day 14 after the first immunization; FIG. 5C shows IgG2C titers in the sera of groups of mice at day 14 after the first immunization; FIG. 5D shows the values of IgG2c/IgG1 in the sera of groups of mice at day 14 after the first immunization. The results show that the serum IgG1 titers of mice in group J (administered NPS-I-M nanoparticles) were higher than those in group B (administered free VZVgE recombinant protein), group C (administered free VZV gE recombinant protein + free IMQ), with a very significant difference (P < 0.01); mice in group J (administered NPS-I-M nanoparticles) had higher serum IgG2C titers than in group B (administered free VZV gE recombinant protein), group C (administered free VZV gE recombinant protein + free IMQ), group D (administered free VZV gE recombinant protein + free MPLA), with a very significant difference (P < 0.01), than in group E (administered free VZV gE recombinant protein + free IMQ + free MPLA), group F (administered free VZV gE recombinant protein + aluminum adjuvant), group G (administered NPS nanoparticles), with a significant difference (P < 0.05); group J (administered NPS-I-M nanoparticles) mice had significantly different sera IgG2C/IgG1 than group B (administered free VZV gE recombinant protein) and group C (administered free VZV gE recombinant protein + free IMQ) (P < 0.05).
FIG. 6B shows IgG1 titers in the sera of groups of mice at day 28 after the first immunization; FIG. 6C shows IgG2C titers in the sera of groups of mice at day 28 after the first immunization; FIG. 6D shows the values of IgG2c/IgG1 in the sera of groups of mice at day 28 after the first immunization. The results show that the serum IgG1 and IgG2C titers of mice in group J (administered NPS-I-M nanoparticles) were all higher than those in group B (administered free VZV gE recombinant protein), group C (administered free VZV gE recombinant protein + free IMQ), group D (administered free VZV gE recombinant protein + free MPLA), group E (administered free VZV gE recombinant protein + free IMQ + free MPLA), group F (administered free VZV gE recombinant protein + aluminum adjuvant), group G (administered NPS nanoparticles), group H (administered NPS-I nanoparticles), with very significant differences (P < 0.001); the serum IgG2c/IgG1 in mice in group J (administered NPS-I-M nanoparticles) was higher than in group E (free VZV gE recombinant protein + free IMQ + free MPLA), group F (administered free VZV gE recombinant protein + aluminum adjuvant), with a very significant difference (P < 0.01).
FIG. 7B shows IgG1 titers in the sera of groups of mice at day 42 after the first immunization; figure 7C shows the IgG2C titers in the sera of groups of mice at day 42 after the first immunization; FIG. 7D shows the values of IgG2c/IgG1 in the sera of groups of mice at day 42 after the first immunization. The results show that the serum IgG1 titers of mice in group J (administered NPS-I-M nanoparticles) were higher than those in group B (administered free VZVgE recombinant protein), group C (administered free VZV gE recombinant protein + free IMQ), group D (administered free VZV gE recombinant protein + free MPLA), group E (administered free VZV gE recombinant protein + free IMQ + free MPLA), group F (administered free VZV gE recombinant protein + aluminum adjuvant), group G (administered NPS nanoparticles), group H (administered NPS-I nanoparticles), with a very significant difference (P < 0.001), and higher than those in group I (administered NPS-M nanoparticles), with a very significant difference (P < 0.01); the serum IgG2C titers of group J (administered NPS-I-M nanoparticles) mice were higher than those of group B (administered free VZV gE recombinant protein), group C (administered free VZV gE recombinant protein + free IMQ), group D (administered free VZV gE recombinant protein + free MPLA), group E (administered free VZV gE recombinant protein + free IMQ + free MPLA), group F (administered free VZV gE recombinant protein + aluminum adjuvant), with very significant differences (P < 0.001); group J (administered NPS-I-M nanoparticles) mice had higher serum IgG2C/IgG1 than group B (administered free VZV gE recombinant protein), group C (administered free VZV gE recombinant protein + free IMQ), group D (administered free VZV gE recombinant protein + free MPLA), with very significant differences (P < 0.01).
FIG. 8B shows IgG1 titers in the sera of groups of mice at day 60 after the first immunization; figure 8C shows the IgG2C titers in the sera of groups of mice at day 60 after the first immunization; FIG. 8D shows the values of IgG2c/IgG1 in the sera of groups of mice at day 60 after the first immunization. The results show that the serum IgG1 titers of mice in group J (administered NPS-I-M nanoparticles) were higher than those in group C (administered free VZVgE recombinant protein + free IMQ), group D (administered free VZV gE recombinant protein + free MPLA), with a very significant difference (P < 0.01); mice in group J (administered NPS-I-M nanoparticles) had higher serum IgG2C titers than in group C (administered free VZV gE recombinant protein + free IMQ), group D (administered free VZVgE recombinant protein + free MPLA), group E (administered free VZV gE recombinant protein + free IMQ + free MPLA), group F (administered free VZV gE recombinant protein + aluminum adjuvant), group G (administered NPS nanoparticles), group H (administered NPS-I nanoparticles), with very significant differences (P < 0.01).
The experiment results show that after the liposome NPS-I-M nanoparticle wrapping VZV gE recombinant protein, double immune adjuvants IMQ and MPLA disclosed by the invention is used for immunizing a mouse, compared with a free antigen and adjuvant mixture, the liposome NPS-I-M nanoparticle can enhance humoral immunity and improve the titer of an antibody. The liposome NPS-I-M nanoparticle wrapping VZV gE recombinant protein, double immune adjuvants IMQ and MPLA can not only stimulate Th1 type immunity well, but also activate Th2 immunity.
(3) Mouse peripheral blood C84+, CD8+ T cell intracellular factor detection
And (3) detection process: on day 31 after the first immunization, 100 ul/mouse peripheral blood of each group was collected, and erythrocytes were removed by erythrocyte lysate. CD3, CD4, CD8 were labeled by surface antibodies. IFN-. gamma.TNF-. alpha.was then stained intracellularly. Flow cytometer detection and analysis
FIG. 9 shows the expression levels of IFN-. gamma.and TNF-. alpha.in the T lymphocytes of CD4+ and CD8+ after the mice of each group were immunized with antigen, and the specific test results are shown in FIGS. 9A to 9D. The results showed that the expression levels of IFN- γ and TNF- α in group J (NPS-I-M nanoparticle administration) were significantly different from those in group a (PBS-administered negative control), and also significantly different from those in other groups, group B (free VZV gE recombinant protein administration + free IMQ), group C (free VZV gE recombinant protein administration + free IMQ), group D (free VZV gE recombinant protein administration + free MPLA), group E (free VZV gE recombinant protein administration + free IMQ + free MPLA), group F (free VZV gE recombinant protein administration + aluminum adjuvant), group G (NPS nanoparticle administration), group H (NPS-I nanoparticle administration), and group I (NPS-M nanoparticle administration). 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.
Claims (10)
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.
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