CN102764430A - Nasal spraying mucosa immunizing vaccine composition and preparation method thereof - Google Patents
Nasal spraying mucosa immunizing vaccine composition and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a nasal spraying mucosa immunizing vaccine composition and a preparation method thereof, and belongs to the technical field of biological pharmacy. The vaccine composition consists of the following components based on the total weight of the vaccine composition: 0.01 to 20 percent of immune cytokine, 45 to 80 percent of chitosan quaternary amine salt hydrogel, 0.01 to 20 percent of vaccine antigen, 0.01 to 15 percent of thimerosal, and 0.5 to 2 percent of polyethylene glycol. The components of the nasal spraying mucosa immunizing vaccine composition play an effective superposed synergistic role; and the immune cytokine is used as a mucosa vaccine adjuvant and an immune regulator, the chitosan quaternary amine salt hydrogel is used as a biological adhesive substance and an antigen transfer system, and the polyethylene glycol is used as a stabilizer and used with the vaccine antigen, so that the retention time of the vaccine antigen in the nasal cavity and immune response of the antigen are improved, comprehensive effectiveness of nasal spraying mucosa vaccines is remarkably improved, and irritation of vaccine spray on the nasal mucosa and consumption of the antigen are reduced.
Description
Technical field
The invention belongs to biological pharmacy technical field, be specifically related to a kind of spray nasal mucosa immune vaccine composition and method of making the same.
Background technology
In recent years, the harm of cause of disease mucosa infection property diseases such as SARS, highly pathogenic H5N1 bird flu, influenza A H1N1 is severe day by day.As everyone knows, mostly the whole world nearly 20 years New Developments, burst serious infectious diseases are due to the mucosa infection, and general susceptible, are difficult to control.In addition, reasons such as variation of ecology and environment, the sick entomophila evil life of biography, antibiotic abuse, pathogen variation, superbacteria and supervirus appearance, some controlled infectious disease are revivable again, very easily cause the serious infectious diseases outbreak of epidemic.Therefore WHO authoritative sources foretells, the mankind will face the serious threat of infectious disease.
As everyone knows, the pathogenic infection more than 95% occurs in mucosa or is caused a disease by the mucosa invasion.The mucosa system extensively is distributed in respiratory tract, digestive tract and urogenital tract, and exocrine gland, and respiratory tract accounts for the first place.This system area coverage in vivo is big; Form the first line of defence of body opposing exotic disease substance invasion; Particularly infection induced generation secretory IgA, sIgA of mucosa-immune simulating nature and CTL effect and comprehensive immunne response play a significant role in anti-infectious immunity, and have interlinking between each mucosa.Yet existing injecting immune vaccine approach usually can only the activating system immunne response, and does not have mucosal immune response and cross immunity protective effect, make the vaccine kind, type is single and compliance is poor, and uses limited.Decades in the past; People attempt to design the vaccine dosage form and can produce systemic immunity and can produce mucosa-immune and cell immunoreceptor simultaneously again; But in mucosa vaccine adjuvant and carrier transfer system achieving no breakthrough property progress, particularly in the technical bottleneck that exists aspect dose-effect control, the slow release targeting property.Therefore, spray nasal mucosa vaccine adjuvant and mucosa vaccine delivery system compositions have attracted people's sight, and become the major fields of this field development.
Research in recent years shows; Become the mucosa-immune vaccine adjuvant of potentialization with endotoxins such as antibacterial cholera enterotoxin CT, escherichia coli heat-labile toxin LT; It can not only produce specific antibody and reply, and also can induce stronger mucosal immune response, and the ability of effective enhancing body opposing pathogen infection; But making, himself high immunogenicity and toxin safety in clinical research, go no further; Particularly himself protein macromolecule immunogenicity is strong, and covers purpose antigen, is difficult to become the candidate thus with mucosa vaccine adjuvant composition.Research subsequently; Concentrating on immune cell factor has enhance immunity and replys; And certain adjuvant effect is arranged, but immune cell factor is huge and kind is complicated, and which also definitely is not so far; Do not have adhesiveness yet, and be difficult to the problem that single immune cell factor becomes stabilizing effective mucosa vaccine adjuvant.Report is arranged simultaneously; Chitosan is as a kind of natural cation biopolymer; Because of having bioadhesive, the compatibility and degradability; And its catabolite is nontoxic, non-immunogenicity and non-carcinogenesis, and chitosan has certain mucosa vaccine and transmit carrier effect, becomes very promising vaccine carrier.But chitosan is insoluble in water, and is soluble in dilute acid soln, this to most of albumen, peptide class antigen effect is not good enough, and use and to be restricted.Thus, attempting to develop a kind of mucosa vaccine adjuvant and delivery system composition safe, effective, stable and quality controllable system is the urgent great demand of development mucosa-immune vaccine.
In recent years research shows; Can obtain Quaterisation chitosan and derivant thereof with chemical compound such as (+)-2,3-Epoxy-1-propanol trimethyl ammonia chloride or iodomethane and chitosan reaction, it compare with chitosan have better water-solubility, hydrophilic and cationic, be fit to biomacromolecule class antigens such as albumen, peptide class, nucleic acid greatly; And utilize the cationic property of n-trimethyl chitosan chloride itself; Or, has the responsive responsiveness aquagel of different pH through regulating the interaction between chitosan and the hydrone, can preparing to the hydroxyl on its macromolecular chain, amido modified; Make chitosan solution in host relaxing the bowels with purgatives of warm nature generation gelling, to reach the long-acting release effects of vaccine antigen.And the quaternary ammonium salt aquagel can with nasal epithelial cells and rete malpighii generation strong effect thereof; To guarantee before being removed, making vaccine antigen transhipment passing through nasal mucosa by the mucosa cilium; Can increase the ability of polarity albumen, peptide class cell antigen bypass carrying in addition through the temporary transient opening of tight junction between the epithelial cell; The effect that realization is transmitted carrier effect and targeting property to vaccine antigen at mucosa, thus realize mucosa vaccine carrier transmission effects.
Up to the present, the chitosan of preparation or quaternary ammonium salt aquagel transmit carrier system as the mucosa vaccine and also have fatal shortcoming, have limited development and have used.Mainly show: the first, although chitosan has slow release and immune-enhancing effect, from just playing a role in acid solution, this has limited use for protide, peptide class antigen; It two is, although the n-trimethyl chitosan chloride hydrogel has solved and acid mostly albumen, the antigenic influence of peptide class has been in external shaping implantation or injecting body, has limited quaternary ammonium salt aquagel mucosa range of application; It three is, although the n-trimethyl chitosan chloride hydrogel has certain immunopotentiation, can not reach the effect of vaccine adjuvant performance far away, also needs the assistance of other adjuvants; It four is that the pH response of the responsive n-trimethyl chitosan chloride hydrogel of pH of existing preparation is responsive inadequately, is difficult to respond pH minor variations in the body rapidly and realizes that the discontinuity of vaccine antigen discharges; It five is that vaccine antigen is prone to microbiological contamination and makes its inefficacy or human body is caused other harm in the deposit process.Thus, a kind of compositions of spraying nasal mucosa vaccine transmission carrier and adjuvant of necessary development to realize, comprehensively and effectively immunoprophylaxis safe and simple to the mucosa infection disease, realizes the purpose of immune prevention and control infectious disease.
Summary of the invention
The object of the present invention is to provide a kind of spray nasal mucosa immune vaccine compositions.
The present invention also aims to provide a kind of spray nasal mucosa immune vaccine preparation method of composition.
The object of the invention also is to provide a kind of application in preparation immunoprophylaxis medicine of spraying nasal mucosa immune vaccine compositions.
A kind of spray nasal mucosa immune vaccine compositions, said vaccine combination is made up of the following component that accounts for the vaccine combination gross weight: immune cell factor 0.01-20%, chitosan quaternary amine hydrogel 45-80%, vaccine antigen 0.01-20%, thimerosal 0.01-15%, Polyethylene Glycol 0.5-2%.
Said immune cell factor is one or more among IL-1 α natural or reorganization, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-27, IL-28A, IL-28B, IL-31, the IL-33.
In the proteantigen of the virus antigen that said immune vaccine antigen is deactivation or attenuation, bacterial antigens, natural or reorganization, polysaccharide antigen, antigen nucleic acid, the protein binding polysaccharide antigen one or more.
Said vaccine combination is through spray nose or collunarium, but also the Sublingual splashes into the immunization route inoculation.
Above-mentioned a kind of spray nasal mucosa immune vaccine preparation of compositions method, carry out according to following steps:
(1) preparation of chitosan quaternary ammonium salt
6.0 gram chitosans are scattered in the 90ml distilled water, form the chitosan dispersion liquid, with 75.0 grams 2; 3 epoxypropyl trimethylammonium chloride ammoniums are dissolved in the distilled water and are settled to 30ml, 2,3 epoxypropyl trimethylammonium chloride ammonium salt solutions are added in the chitosan dispersion liquid while stirring form mixed solution; In 80 ℃ of following stirring reactions 6 hours; Mixed solution is poured in the acetone of 4 ℃ of pre-coolings, be placed on 2-8 ℃ and spend the night, wherein the volume ratio of mixed solution and acetone is 1:3～1:4.Deposition after will spending the night is placed under 105 ℃ of conditions and is dried, and obtains white and evacuates solids, is chitosan quaternary ammonium salt;
(2) preparation of chitin quarternary ammonium salt aquagel
Chitosan quaternary ammonium salt 6.0 grams and Polyethylene Glycol 3 grams are dissolved in the acetum of 96ml 0.1mol/L; Be cooled to 4 ℃; Dropwise add the alpha-beta that concentration is 0.1-0.5g/ml-phosphoglycerol sodium solution 24ml under stirring, stir regulator solution pH value to 7.0; This solution is heated to 37 ℃ of insulations 60 minutes, forms chitin quarternary ammonium salt aquagel; Place 2-8 ℃ of preservation then;
(3) in the above-mentioned chitin quarternary ammonium salt aquagel of preparation, add immune cell factor 2-8mg/ml while stirring in proportion; Vaccine antigen 10-20mg/ml; Thimerosal 0.1-3mg/ml; The pH value of regulator solution is 6.8～7.0, is spray nasal mucosa immune vaccine composition solution, puts 2-8 ℃ of preservation.
The above-mentioned a kind of application of nasal mucosa immune vaccine compositions in preparation immunoprophylaxis medicine of spraying.
Beneficial effect of the present invention: the effective stack of performance synergism between the component of spray nasal mucosa immune vaccine compositions of the present invention; Immune cell factor is as mucosa vaccine adjuvant and immunomodulator; Chitosan quaternary amine hydrogel is as biological slime addendum and antigen delivery system; Unite use with vaccine antigen, both increased the time of staying of vaccine antigen in nasal cavity, increased antigenic effect of immune response again; Thereby increased the effectiveness of spray nasal mucosa vaccine significantly, also reduced zest and the antigen consumption of vaccine spray nasal mucosa.
The specific embodiment
Below in conjunction with specific embodiment the present invention is further specified.
Experimental technique among the following embodiment if no special instructions, is conventional method.
Laboratory animal in following examples derives from Military Medical Science Institute's Experimental Animal Center.
Embodiment 1 spray nasal mucosa immune vaccine preparation of compositions
The source of chitosan: chitosan is bought in U.S. sigma company or domestic pharmaceutical grade production company, and molecular weight is 150KD, and is deacetylated more than 85%.
Method for preparing is following:
(1) preparation of chitosan quaternary ammonium salt
6.0 gram chitosans are scattered in the 90ml distilled water, form the chitosan dispersion liquid, with 75.0 grams 2; 3 epoxypropyl trimethylammonium chloride ammoniums are dissolved in the distilled water and are settled to 30ml, 2,3 epoxypropyl trimethylammonium chloride ammonium salt solutions are added in the chitosan dispersion liquid while stirring form mixed solution; In 80 ℃ of following stirring reactions 6 hours; Mixed solution is poured in the acetone of 4 ℃ of pre-coolings, be placed on 2-8 ℃ and spend the night, wherein the volume ratio of mixed solution and acetone is 1:3.Deposition after will spending the night is placed under 105 ℃ of conditions and is dried, and obtains white and evacuates solids, is chitosan quaternary ammonium salt;
(2) preparation of chitin quarternary ammonium salt aquagel
Chitosan quaternary ammonium salt 6.0 grams and Polyethylene Glycol 3 grams are dissolved in the acetum of 96ml 0.1mol/L; Be cooled to 4 ℃; Dropwise add the alpha-beta that concentration is 0.1-0.5g/ml-phosphoglycerol sodium solution 24ml under stirring, stir regulator solution pH value to 7.0; This solution is heated to 37 ℃ of insulations 60 minutes, forms chitin quarternary ammonium salt aquagel; Place 2-8 ℃ of preservation then;
(3) in the above-mentioned chitin quarternary ammonium salt aquagel of preparation, add immune cell factor 4-8mg/ml while stirring in proportion; Vaccine antigen 10-20mg/ml; Thimerosal 0.1-3mg/ml; The pH value of regulator solution is 6.8～7.0, is spray nasal mucosa immune vaccine composition solution, puts 2-8 ℃ of preservation.
The NAT that embodiment 2 different immune cell factors produce as the immune vaccine composition adjuvant
1. the source of immune cell factor: the people IL-1 α of natural or reorganization, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-27, IL-28A, IL-28B, IL-31, IL-33 be totally 26 kinds of cytokines; Purchase (Minneapolis, MN) company in R&D Systems.
2. different immune cell factors are as the preparation of immune vaccine composition adjuvant:
Prepare the vaccine combination that contains above-mentioned immune cell factor respectively in step among the embodiment 1 and ratio, immunizing antigen wherein is respectively influenza trivalent cracking antigen, respiratory syncytial virus VLP antigen, the conjugated protein antigen of whooping cough polysaccharide, the conjugated protein antigen of pneumonia 23 valency polysaccharide, papillary tumor 6 valency VLP antigens, the conjugated protein antigen of hemophilus influenza polysaccharide.With vaccine combination difference collunarium immunity inoculation Balb/c mice, 2 all booster immunizations are 1 time at interval, observe humoral immunization neutralizing antibody effect, and are as shown in table 1.
The NAT that the different immune cell factors of table 1. produce as the immune vaccine composition adjuvant
Visible by table 1, IL-1 β, IL-1 α, IL-2, IL-18 and IL-33 immune cell factor improve the antigen NAT in various degree, and wherein IL-1 β effect is best.
Embodiment 3 immune cell factor IL-1 β produce the effect of mucosa-immune and cellullar immunologic response as the immune vaccine composition adjuvant
Prepare vaccine combination that contains immune cell factor IL-1 β and the vaccine combination that does not contain immune cell factor IL-1 β respectively in step among the embodiment 1 and ratio, immunizing antigen wherein is respectively influenza trivalent cracking antigen, respiratory syncytial virus VLP antigen, the conjugated protein antigen of whooping cough polysaccharide, the conjugated protein antigen of pneumonia 23 valency polysaccharide, papillary tumor 6 valency VLP antigens, the conjugated protein antigen of hemophilus influenza polysaccharide.With vaccine combination difference collunarium immunity inoculation Balb/c mice, 2 all booster immunizations are 1 time at interval, observe mucosa-immune, cellullar immunologic response effect, and are as shown in table 2.
Table 2.IL-1 β produces mucosa-immune and cellullar immunologic response effect as spray nasal mucosa immune vaccine composition adjuvant
Visible by table 2; The mucosa antibody sIgA that IL-1 β can significantly improve vaccine antigen tires and IFN-gamma cells immunne response effect; And sIgA and IFN-γ that the vaccine antigen group that does not add IL-1 produces are lower, thereby show that IL-1 β has the comprehensive response effect of obvious raising antigen as the mucosa vaccine adjuvant.
The fungistatic effect of embodiment 4 spray nasal mucosa immune vaccine compositionss
Method according to embodiment 1 prepares two parts of spray nasal mucosa immune vaccine compositionss respectively, a thimerosal 1mg/ml that adds, and portion does not add thimerosal, places 2-8 ℃ of preservation.
The spray nasal mucosa immune vaccine compositions of adding thimerosal after 4 months has no to be polluted and corruption, and the spray nasal mucosa immune vaccine compositions of not adding thimerosal begins that living contaminants is arranged after preserving 40 days, preserves to begin long bacterium after 4 months.
PEG's increases steady effect in the embodiment 5 spray nasal mucosa immune vaccine compositionss
Method according to embodiment 1 prepares two parts of spray nasal mucosa immune vaccine compositionss respectively, a 2%PEG that adds, and portion does not add PEG, places 2-8 ℃ of preservation.The result shows that the spray nasal mucosa immune vaccine compositions of adding PEG can significantly improve vaccine stability, preserves more than 18 months stable; Do not add the spray nasal mucosa immune vaccine composition stable property of PEG, only preserved less than 10 months.
Embodiment 6 spray nose influenza trivalent split vaccine preparation of compositions and immune effect evaluations
H1N1, H3N2 and Type B influenza virus source: the biological article collecting center (ATCC) of Unite States Standard.
H1N1, H3N2 and Type B influenza virus are inoculated 9-11 age in days chick embryo allantois respectively, behind 37 ℃ of cultivation 48h, collect allantoic fluid virus; Use formalin-inactivated, film bag ultrafiltration and concentration, sucrose gradient centrifugation purification; Trion-100 lytic virus granule; Gradient centrifugation removes sugared purification again, preparation influenza trivalent (H1N1, H3N2 and Type B) lytic virus antigen, and carry out type calibrating, antigenic content, thermal source matter, exogenous factor calibrating.
Press embodiment 1 method for preparing, the preparation chitin quarternary ammonium salt aquagel adds immune cell factor IL-1 β 8mg/ml in proportion while stirring in chitin quarternary ammonium salt aquagel; Influenza trivalent (H1N1, H3N2 and Type B) lytic virus antigen 1 5mg/ml; Thimerosal 1mg/ml, the pH value of regulator solution is 6.8～7.0, is sub-packed in the nasal-spraying immune device; Preparation spray nose influenza trivalent split vaccine is put 2-8 ℃ of preservation.
The preparation chitosan does not have quaternised hydrogel spray nose influenza trivalent split vaccine compositions: press step among the embodiment 1 (2) preparation hydrogel; Wherein chitosan quaternary ammonium salt is replaced with chitosan, the preparation chitosan does not have quaternised hydrogel, in hydrogel, adds immune cell factor IL-1 β 8mg/ml in proportion while stirring; Influenza trivalent (H1N1, H3N2 and Type B) lytic virus antigen 1 5mg/ml; Thimerosal 1mg/ml, the pH value of regulator solution is 6.8～7.0, is sub-packed in the nasal-spraying immune device; The preparation chitosan does not have quaternised hydrogel spray nose influenza trivalent split vaccine compositions, puts 2-8 ℃ of preservation.
Spray nose influenza trivalent split vaccine compositions, injection influenza trivalent split vaccine, the chitosan of the present invention's preparation are not had quaternised hydrogel spray nose influenza trivalent split vaccine collunarium immunity inoculation Balb/c mice, ferret respectively, and 2 all booster immunizations are 1 time at interval.Gather venous blood and separation of serum and lymphocyte then respectively, gather the respiratory tract juice simultaneously, measure serum neutralizing antibody, sIgA, INF-gamma cells immunne response effect, use 100LD subsequently respectively
50Corresponding influenza virus virulent strain aerosol counteracting toxic substances is observed immune protective effect.
Result by table 3 is visible; The chitin quarternary ammonium salt aquagel of the present invention's preparation and the spray nose influenza trivalent split vaccine of immune cell factor IL-1 β all can produce high NAT of tiring in two kinds of animal bodies; And can produce sIgA mucosa-immune and cellullar immunologic response, animal all survives; And injection influenza trivalent split vaccine does not produce sIgA mucosa-immune and INF-gamma cells immunne response, and the antigen consumption is 1/3 of a vaccinate consumption; And chitosan does not have the spray nose influenza trivalent split vaccine immunity of quaternized and hydrogel to fail effectively to produce immunne response and immune protective effect.This shows; Chitin quarternary ammonium salt aquagel has good immunne response and immune protective effect as mucosa vaccine carrier transmission system, IL-1 β as the spray nose influenza trivalent split vaccine of mucosa vaccine adjuvant preparation, and has good cross immunity protection effect.
Table 3. chitin quarternary ammonium salt aquagel transmits carrier, the IL-1 β immune effect as the spray nose influenza trivalent split vaccine of vaccine combination adjuvant preparation as vaccine combination
The preparation and the immune effect evaluation of embodiment 7 nasal-spraying immune influenza trivalent subunit vaccines
H1N1, H3N2 and Type B influenza virus are inoculated 9-11 age in days chick embryo allantois respectively, behind 37 ℃ of cultivation 48h, collect allantoic fluid virus; Use formalin-inactivated, film bag ultrafiltration and concentration, sucrose gradient centrifugation purification; Trion-100 lytic virus granule; Gradient centrifugation removes sugared purification again, preparation influenza trivalent (H1N1, H3N2 and Type B) hemagglutinin (HA), neuraminidase (NA) antigen, and carry out type calibrating, antigenic content, thermal source matter, exogenous factor calibrating.
Press embodiment 1 method for preparing, the preparation chitin quarternary ammonium salt aquagel adds immune cell factor IL-1 β 8mg/ml in proportion while stirring in the chitin quarternary ammonium salt aquagel of preparation; The influenza trivalent hemagglutinin (HA) of above-mentioned preparation and neuraminidase (NA) antigen 1 5mg/ml; Thimerosal 1mg/ml, the pH value of regulator solution is 6.8～7.0, is sub-packed in the nasal-spraying immune device; Preparation nasal-spraying immune influenza trivalent subunit vaccine is put 2-8 ℃ of preservation.
Spray nose influenza trivalent subunit vaccine with preparation; Difference collunarium immunity inoculation Balb/c mice, ferret, 2 all booster immunizations are 1 time at interval, and do contrast with the spray nose influenza trivalent split vaccine among the embodiment 6, injection influenza trivalent split vaccine; Gather venous blood and separation of serum and lymphocyte respectively; Gather respiratory tract different parts juice simultaneously, measure serum neutralizing antibody, respiratory tract sIgA mucosa-immune, INF-gamma cells immunne response effect, use 100LD subsequently respectively
50Corresponding influenza virus virulent strain aerosol counteracting toxic substances is observed immune protective effect.
Result by table 4 is visible; The chitin quarternary ammonium salt aquagel of the present invention's preparation and the spray nose influenza trivalent subunit vaccine of IL-1 β mucosa adjuvant all can produce high-caliber NAT in two kinds of animal bodies; And can produce sIgA mucosa-immune and cellullar immunologic response, animal all survives, and is suitable with spray nose influenza trivalent split vaccine effect; And injection influenza trivalent split vaccine does not produce sIgA mucosa-immune and cellullar immunologic response, and the antigen consumption is 1/4 of a vaccinate consumption; This shows; Chitin quarternary ammonium salt aquagel has good immunne response and immune protective effect as mucosa vaccine carrier transmission system, IL-1 β as the spray nose influenza trivalent subunit vaccine of mucosa vaccine adjuvant preparation, and has good cross immunity protection effect.
Table 4. chitin quarternary ammonium salt aquagel transmits carrier, the IL-1 β immune effect as the spray nose influenza trivalent subunit vaccine of vaccine adjuvant preparation as vaccine
The preparation of embodiment 8 nasal-spraying immune influenza virus sample particle vaccines and immune effect evaluation
With H1N1, H3N2 and Type B influenza virus; Adopt that gene recombination technology, protokaryon or eukaryotic expression system are cloned respectively, recombinated, expression, purification prepare HA-NA-M1 virus-like particle antigen, and carry out type calibrating, antigenic content, thermal source matter, exogenous factor calibrating.
Press embodiment 1 method for preparing, the preparation chitin quarternary ammonium salt aquagel adds immune cell factor IL-1 β 8mg/ml in proportion while stirring in the chitin quarternary ammonium salt aquagel of preparation; HA-NA-M1 virus-like particle antigen 1 5mg/ml; Thimerosal 1mg/ml, the pH value of regulator solution is 6.8～7.0, is sub-packed in the nasal-spraying immune device; Preparation spray nose influenza trivalent VlP vaccine is put 2-8 ℃ of preservation.
The spray nose influenza trivalent VlP vaccine that preparation is had chitin quarternary ammonium salt aquagel and IL-1 β mucosa adjuvant; Difference collunarium immunity inoculation Balb/c mice, ferret; 2 all booster immunizations are 1 time at interval; And do not have the spray nose influenza trivalent VLP vaccine of quaternized and hydrogel to do contrast with injection influenza trivalent split vaccine, chitosan, and gather venous blood and separation of serum and lymphocyte respectively, gather respiratory tract different parts juice simultaneously; Measure serum neutralizing antibody, respiratory tract juice IgA, INF-gamma cells immunne response effect, use 100LD subsequently respectively
50Corresponding influenza virus virulent strain aerosol counteracting toxic substances is observed immune protective effect.
Result by table 5 is visible; The chitin quarternary ammonium salt aquagel of preparation and the spray nose influenza trivalent VLP vaccine of IL-1 β all can produce high-caliber NAT in two kinds of animal bodies; And can produce sIgA and INF-gamma cells immunne response, and animal all survives, and effect is better than spray nose influenza trivalent subunit vaccine effect; And injection influenza trivalent split vaccine does not produce sIgA mucosa-immune and cellullar immunologic response, and the antigen consumption is 1/5 of a vaccinate consumption; And chitosan does not have the spray nose influenza trivalent VLP vaccine immunity of quaternized and hydrogel to fail effectively to produce immunne response and immune protective effect.This shows; Chitin quarternary ammonium salt aquagel has good immunne response and immune protective effect as mucosa vaccine carrier transmission system, IL-1 β as the spray nose influenza trivalent VLP vaccine of mucosa vaccine adjuvant preparation, and has good cross immunity protection effect.
Table 5. chitin quarternary ammonium salt aquagel transmits carrier, the IL-1 β immune effect as the spray nose influenza trivalent VLP vaccine of mucosa vaccine adjuvant preparation as the mucosa vaccine
The preparation and the immune effect evaluation of embodiment 9 spray nasal respiration road syncytial virus sample particle vaccines
A, Type B respiratory syncytial virus: derive from the biological article collecting center (ATCC) of Unite States Standard.
With A, Type B respiratory syncytial virus; Adopt that gene recombination technology, protokaryon, eukaryotic cell expression system clone respectively, recombinate, expression, purification prepare purification respiratory syncytial virus virus-like particle antigen, and carry out type calibrating, antigenic content, thermal source matter, exogenous factor calibrating.
Press embodiment 1 method for preparing, the preparation chitin quarternary ammonium salt aquagel adds immune cell factor IL-1 β 8mg/ml in proportion while stirring in the chitin quarternary ammonium salt aquagel of preparation; Respiratory syncytial virus virus-like particle antigen 1 5mg/ml; Thimerosal 1mg/ml, the pH value of regulator solution is 6.8～7.0, is sub-packed in the nasal-spraying immune device; Preparation spray nasal respiration road syncytial virus sample particle vaccines is put 2-8 ℃ of preservation.
The spray nasal respiration road syncytial virus VlP vaccine difference collunarium immunity inoculation Balb/c mice, the cotton mouse that spray nasal respiration road syncytial virus VLP vaccine, injection respiratory syncytial virus inactivated vaccine, the chitosan of preparation are not had quaternised hydrogel; 2 all booster immunizations are 1 time at interval; Gather venous blood and separation of serum and lymphocyte respectively; Gather respiratory tract different parts juice simultaneously, measure serum neutralizing antibody, respiratory tract juice IgA, cellullar immunologic response effect, use 50LD subsequently respectively
50A, Type B respiratory syncytial virus virulent strain aerosol counteracting toxic substances are observed immune protective effect.
Visible by table 6 result; The spray nasal respiration road syncytial virus VLP vaccine of preparation all can produce high-caliber NAT in two kinds of animal bodies; And can produce sIgA and cellullar immunologic response; Animal all survives, and injection respiratory syncytial virus inactivated vaccine does not produce sIgA mucosa-immune and serum I NF-gamma cells immunne response, and can bring out immunologic injury; And chitosan does not have the spray nasal respiration road syncytial virus VlP vaccine immunity of quaternised hydrogel to fail effectively to produce immunne response and immune protective effect.This shows; Chitin quarternary ammonium salt aquagel has good immunne response and immune protective effect as mucosa vaccine carrier transmission system, IL-1 β as the spray nasal respiration road syncytial virus VLP vaccine of mucosa vaccine adjuvant preparation, and has good cross immunity protection effect.
Table 6. chitin quarternary ammonium salt aquagel transmits carrier, the IL-1 β immune effect as the spray nose RSV VLP vaccine of mucosa vaccine adjuvant preparation as the mucosa vaccine
The preparation and the immune effect evaluation of the conjugated protein vaccine of embodiment 10 spray nose 23 valency pneumonia polysaccharide
23 valency streptococcus pneumoniae strains: derive from the biological article collecting center (ATCC) of Unite States Standard.
With 23 valency streptococcus pneumoniae strains; Adopt bacterial fermentation, polysaccharide extraction, protein-crosslinking technology; Or the conjugated protein technology of biosynthesis polysaccharide, the conjugated protein antigen of preparation purification 23 valency pneumococal polysaccharides, and carry out type calibrating, antigenic content, thermal source matter and exogenous factor calibrating.
Press embodiment 1 method for preparing, the preparation chitin quarternary ammonium salt aquagel adds immune cell factor IL-1 β 8mg/ml in proportion while stirring in the chitin quarternary ammonium salt aquagel of preparation; The conjugated protein antigen 1 5mg/ml of 23 valency pneumococal polysaccharides; Thimerosal 1mg/ml, the pH value of regulator solution is 6.8～7.0, is sub-packed in the nasal-spraying immune device; Prepare the conjugated protein vaccine of 23 valency pneumococal polysaccharides, put 2-8 ℃ of preservation.
The conjugated protein vaccine of spray nose 23 valency pneumococal polysaccharides with preparation; Difference collunarium immunity inoculation Balb/c mice, Rhesus Macacus; 2 all booster immunizations are 1 time at interval; And do not have the conjugated protein vaccine of spray nose 23 valency pneumococal polysaccharides of quaternised hydrogel to do contrast with injection 23 valency pneumococal polysaccharides conjugated protein vaccine, chitosan, and gather venous blood and separation of serum and lymphocyte respectively, gather respiratory tract different parts juice simultaneously; Measure serum neutralizing antibody, respiratory tract sIgA and serum I NF-gamma cells immunne response effect, use 200LD subsequently respectively
50Zones of different different serotypes pneumonia virulent strain aerosol counteracting toxic substances is observed immune protective effect.
Visible by table 7 result; The chitin quarternary ammonium salt aquagel of preparation and the conjugated protein vaccine of spray nose 23 valency pneumococal polysaccharides of IL-1 β all can produce high-caliber NAT in two kinds of animal bodies; And can produce sIgA mucosa-immune and serum I NF-gamma cells immunne response; Animal all survives, and does not produce sIgA mucosa-immune and cellullar immunologic response and inject the conjugated protein vaccine of 23 valency pneumococal polysaccharides, and the protection effect obviously improves; And chitosan does not have the conjugated protein vaccine immunity of spray nose 23 valency pneumococal polysaccharides of quaternized and hydrogel to fail effectively to produce immunne response and immune protective effect.This shows; Chitin quarternary ammonium salt aquagel has good immunne response and immune protective effect as mucosa vaccine carrier transmission system, IL-1 β as the conjugated protein vaccine of spray nose 23 valency pneumococal polysaccharides of mucosa vaccine adjuvant preparation, and has good cross immunity protection effect.
Table 7. chitin quarternary ammonium salt aquagel transmits carrier, the IL-1 β immune effect as the conjugated protein vaccine of spray rhinopneumonitis polysaccharide of mucosa vaccine adjuvant preparation as the mucosa vaccine
The preparation and the immune effect evaluation of embodiment 11 spray nose human papillomavirus VLP vaccines
With 6,8,11,18,56,58 serotype papillomatosis strains (coming from ATCC); Adopt that gene recombination technology, protokaryon, eukaryotic cell expression system clone respectively, recombinate, expression, purification prepare sexavalence human papillomavirus VLP antigen, and carry out calibrating comprehensively such as type calibrating, antigenic content, thermal source matter, exogenous factor.
Press embodiment 1 method for preparing; The preparation chitin quarternary ammonium salt aquagel; In the chitin quarternary ammonium salt aquagel of preparation, add immune cell factor IL-1 β 8mg/ml in proportion while stirring, sexavalence human papillomavirus VLP antigen 1 5mg/ml, thimerosal 1mg/ml; The pH value of regulator solution is 6.8-7.0, places 2-8 ℃ of preservation.
Spray nose sexavalence human papillomavirus VLP vaccine with preparation; Difference collunarium immunity inoculation Balb/c mice, Rhesus Macacus; 2 all booster immunizations are 1 time at interval; And do not have the spray nose sexavalence human papillomavirus VLP vaccine of quaternised hydrogel to do contrast with injection sexavalence human papillomavirus VLP vaccine, chitosan, and gather venous blood and separation of serum and lymphocyte respectively, gather the respiratory tract juice simultaneously; Measure serum neutralizing antibody, respiratory tract juice IgA, cellullar immunologic response effect, use 200LD subsequently respectively
50Zones of different different serotypes human papillomavirus virulent strain aerosol counteracting toxic substances is observed immune protective effect.
The result is as shown in table 8; The spray nose sexavalence human papillomavirus VLP vaccine of preparation all can produce high-caliber NAT in two kinds of animal bodies; And can produce sIgA and cellullar immunologic response; Animal all survives, and injection sexavalence human papillomavirus VLP vaccine does not produce sIgA mucosa-immune and cellullar immunologic response, and the protection effect is obviously low; Chitosan does not have quaternised hydrogel spray nose sexavalence human papillomavirus VLP vaccine immunity to fail effectively to produce immunne response and immune protective effect.This shows; Chitin quarternary ammonium salt aquagel has good immunne response and immune protective effect as mucosa vaccine carrier transmission system, IL-1 β as the spray nose sexavalence human papillomavirus VLP vaccine of mucosa vaccine adjuvant preparation, and has good cross immunity protection effect.
Table 8. chitin quarternary ammonium salt aquagel transmits carrier, the IL-1 β immune effect as the spray nose papillary tumor VLP vaccine of mucosa vaccine adjuvant preparation as the mucosa vaccine
Embodiment 12 spray nasal mucosa immune vaccine immune immune effect evaluations in human body
The experimenter: derive from 302 medical examiners of hospital of PLA, child, adult, old people inoculate and contrast six groups, every group of each 30 people.
The preparation immune cell factor is spray nose influenza trivalent split-virus vaccine, the conjugated protein vaccine of spray nose whooping cough polysaccharide, the conjugated protein vaccine of spray rhinopneumonitis 23 valency polysaccharide, the conjugated protein vaccine of spray nose hemophilus influenza (Hib) polysaccharide of IL-1 β; Nasal-spraying immune is inoculated child, adult, old people respectively; 2 all booster immunizations are 1 time at interval; And do contrast with vaccinate respectively; Gather venous blood and separation of serum and lymphocyte respectively, gather the upper respiratory tract juice simultaneously, measure serum neutralizing antibody, upper respiratory tract sIgA and serum I NF-gamma cells immunne response effect.
As shown in table 9; Spray nose influenza trivalent split-virus vaccine, the conjugated protein vaccine of spray nose whooping cough polysaccharide, the conjugated protein vaccine of spray rhinopneumonitis 23 valency polysaccharide, the conjugated protein vaccine of spray nose Hib polysaccharide all can produce high-caliber NAT in child, adult and old people's all ages and classes section crowd; And can produce mucosa-immune and cellullar immunologic response effect, and injection influenza trivalent split-virus vaccine, the conjugated protein vaccine of whooping cough polysaccharide, the conjugated protein vaccine of pneumonia 24 valency polysaccharide, the conjugated protein vaccine of Hib polysaccharide do not produce effective mucosa-immune and cellullar immunologic response.This shows that chitin quarternary ammonium salt aquagel can produce comprehensive immunne response as the spray nasal mucosa immune vaccine of mucosa vaccine adjuvant preparation as mucosa vaccine carrier transmission system, IL-1 β in human body, and have good cross immunity protection effect.
The immune effect that table 9. chitin quarternary ammonium salt aquagel is planted the crowd as the spray nasal mucosa vaccine of mucosa vaccine adjuvant preparation as mucosa vaccine transmission carrier, IL-1 β
Claims (5)
1. one kind sprays nasal mucosa immune vaccine compositions; It is characterized in that said vaccine combination is made up of the following component that accounts for the vaccine combination gross weight: immune cell factor 0.01-20%, chitosan quaternary amine hydrogel 45-80%, vaccine antigen 0.01-20%, thimerosal 0.01-15%, Polyethylene Glycol 0.5-2%.
2. according to the said a kind of spray nasal mucosa immune vaccine compositions of claim 1; It is characterized in that said immune cell factor is one or more among IL-1 α natural or reorganization, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-27, IL-28A, IL-28B, IL-31, the IL-33.
3. according to the said a kind of spray nasal mucosa immune vaccine compositions of claim 1; It is characterized in that one or more in the proteantigen of the virus antigen that said immune vaccine antigen is deactivation or attenuation, bacterial antigens, natural or reorganization, polysaccharide antigen, antigen nucleic acid, the protein binding polysaccharide antigen.
4. the described a kind of spray nasal mucosa immune vaccine preparation of compositions method of claim 1 is characterized in that, carries out according to following steps:
(1) preparation of chitosan quaternary ammonium salt
6.0 gram chitosans are scattered in the 90ml distilled water, form the chitosan dispersion liquid, with 75.0 grams 2; 3 epoxypropyl trimethylammonium chloride ammoniums are dissolved in the distilled water and are settled to 30ml, 2,3 epoxypropyl trimethylammonium chloride ammonium salt solutions are added in the chitosan dispersion liquid while stirring form mixed solution; In 80 ℃ of following stirring reactions 6 hours; Mixed solution is poured in the acetone of 4 ℃ of pre-coolings, be placed on 2-8 ℃ and spend the night, wherein the volume ratio of mixed solution and acetone is 1:3～1:4.Deposition after will spending the night is placed under 105 ℃ of conditions and is dried, and obtains white and evacuates solids, is chitosan quaternary ammonium salt;
(2) preparation of chitin quarternary ammonium salt aquagel
Chitosan quaternary ammonium salt 6.0 grams and Polyethylene Glycol 3 grams are dissolved in the acetum of 96ml 0.1mol/L; Be cooled to 4 ℃; Dropwise add the alpha-beta that concentration is 0.1-0.5g/ml-phosphoglycerol sodium solution 24ml under stirring, stir regulator solution pH value to 7.0; This solution is heated to 37 ℃ of insulations 60 minutes, forms chitin quarternary ammonium salt aquagel; Place 2-8 ℃ of preservation then;
(3) in the above-mentioned chitin quarternary ammonium salt aquagel of preparation, add immune cell factor 2-8mg/ml while stirring in proportion; Vaccine antigen 10-20mg/ml; Thimerosal 0.1-3mg/ml; The pH value of regulator solution is 6.8～7.0, is spray nasal mucosa immune vaccine composition solution, puts 2-8 ℃ of preservation.
5. according to the said a kind of application of nasal mucosa immune vaccine compositions in preparation immunoprophylaxis medicine of spraying of claim 1.
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