CN118059227A - Application of oil-in-water emulsion in preparation of rabies immune suit - Google Patents

Abstract

The invention belongs to the field of biomedical engineering, and particularly provides an application of an oil-in-water emulsion in preparation of rabies immune suit, which combines rabies virus antigen with a specific oil-in-water emulsion adjuvant, can quickly excite immune response, effectively improve immune activity, and can obtain good immune effect under the condition of reducing the dosage of antigen or adjuvant. The combined mode can effectively utilize the existing rabies vaccine mode, combines the rabies vaccine of the finished product with the adjuvant product subjected to safety verification, does not need clinical test of a system, only needs to carry out bridging experiment verification effect, and the adopted adjuvant does not contain antibiotics or aluminum, has low risk, can reduce the inoculation times and the inoculation dosage on the premise of ensuring the immune effect, and is hopeful to simplify the immune process, control the production cost and the inoculation cost and reduce the risk of immune side reaction, thereby ensuring the benefits of both producers and patients.

Description

Application of oil-in-water emulsion in preparation of rabies immune suit

Technical Field

The application belongs to the field of biomedical engineering, and particularly relates to an application of an oil-in-water emulsion in preparing rabies immune suit.

Background

Rabies (rabies) is also called water-terrorism (hydrophobia), is an acute infectious disease commonly suffered by people and animals, is mainly transmitted to human beings through the modes of scratch or bite of infected animals (crazy animals) such as dogs, cats, bats, foxes and the like, invades the central nervous system after infection, causes dysfunction, and has typical clinical characteristics including water terrorism, wind fear, hypersalivation, muscle convulsions, spasm, progressive paralysis and the like, and the death rate is almost 100%. At present, no effective treatment means for rabies is available, only prevention can be carried out, and timely vaccination is considered to be the most effective prevention measure at the present stage.

According to the current study, rabies vaccine requires multiple injections to achieve the specific neutralizing antibody level required against the virus, whereas rabies vaccine is peculiar in that most people are immunized after exposure (bitten or scratched), so that multiple vaccination procedures are required to continuously suppress the progress of the course, such as five-needle methods commonly used in China (i.e. one needle for each of days 0,3, 7, 14, 28 of exposure) and four-needle methods approved by the state of the European and American parts (two needles for each of day 0 of exposure and one needle for each of days 7 and 21 of exposure). If the vaccine is not inoculated in time before the incubation period is finished or enough neutralizing antibodies are not produced in time after the incubation period is finished, the vaccine is difficult to play an effective immunization role, the progress of the disease course is likely to be impossible to be blocked, and timely inoculation and rapid and efficient immune mobilization are important conditions for preventing rabies. For severe bites and severe exposure, the dose is usually increased in the early stage, the period of the inoculation is prolonged after the whole injection, and the injection times are increased (serious cases are continuously vaccinated for 10 days and 4 booster immunizations are performed within 90 days). Even for those who need to be immunized prior to exposure for special reasons, at least three injections (0, 7, 21 days each with one needle) are required to meet the requirements. Thus, current rabies immunization strategies require considerable time and economic costs, a not insignificant burden for economically unobtrusive patients.

At present, the existing rabies vaccine is widely considered to have higher immunogenicity, and can cause stronger immune response after inoculation, so that no adjuvant is added into the rabies vaccine on the market. However, there has been a continuing effort in the industry to develop rabies vaccine adjuvants that are expected to accelerate the onset of immune responses after vaccination of patients and achieve protective effects comparable to or even higher than normal doses at lower vaccination doses. The faster progress in the related studies is currently being made by Picard adjuvant (PIKA), and three-phase clinical trials have been conducted in Singapore. In addition, aluminum adjuvants are often used as positive controls in related experiments, and can achieve certain effects. However, the use of Pi Kazuo doses of kanamycin may cause allergies or affect immune responses in the body, and the potential risk of using large amounts of aluminum adjuvants in the short term (e.g., 10 consecutive days of vaccination) has been controversial and therefore is not an ideal choice for the user.

Disclosure of Invention

In order to solve the technical problems, the invention provides an application of an oil-in-water emulsion in preparing rabies immune suit.

The technical scheme of the invention is as follows:

The invention provides an application of an oil-in-water emulsion in preparing a rabies immune suit, wherein the immune suit is used for providing a complete immune program applicable to human bodies; the oil-in-water emulsion comprises squalene, alpha-tocopherol and tween, the immune suit comprises 2-3 groups of rabies immune preparations, each group of rabies immune preparations comprises a first component and a second component which are used in combination, the first component comprises the oil-in-water emulsion, the second component comprises a rabies virus antigen preparation, and the dosage of any one of the first component and the second component is lower than the standard single human dosage.

The invention provides a mode of combining rabies virus antigen and a specific oil-in-water emulsion adjuvant, which can quickly excite immune response, effectively improve immune activity and has a remarkably better effect than other emulsion adjuvants; the group number of rabies immune preparations in each set of immune suit can meet the requirement of one conventional post-exposure immune program (which can be reduced to 3 times) or pre-exposure immune program (which can be reduced to 2 times), and the inoculation times and the inoculation dosage can be reduced on the premise of ensuring the immune effect, so that the immune program is simplified; the content of the antigen can be reduced by a certain extent in actual use, the burden of the antigen on an immune system can be reduced to a certain extent while the immune effect is maintained, and the safety is improved. On the one hand, the production cost of the producer can be controlled, on the other hand, the time and the economic cost of the user can be reduced, the risk of the occurrence of immune side reaction can be reduced, and the use safety can be improved.

Further, the oil-in-water emulsion is obtained by mixing and emulsifying an oil phase and an aqueous phase containing an emulsifier; the oil phase comprises squalene and tocopherol, the aqueous phase is an aqueous solution comprising tween, and the aqueous solution is selected from any one of phosphate buffer, citrate buffer, tris-HCl buffer, acetate buffer or citric acid-phosphate buffer.

In some embodiments, the oil-in-water emulsion comprises 6-15 mg squalene, 6-15 mg alpha-tocopherol, and 3-10 mg tween 80.

In a preferred embodiment, the oil-in-water emulsion comprises 10.69mg squalene, 11.86mg alpha tocopherol and 4.86mg tween 80.

Another preferred embodiment is that the oil-in-water emulsion comprises 5.35mg squalene, 5.93mg alpha tocopherol and 2.43mg tween 80.

The applicant found in the study that the immune effect of the rabies vaccine can be significantly improved when the oil-in-water emulsion adjuvant containing specific components is adopted; when the amount of this particular adjuvant was halved, the immune effect did not appear to be significantly reduced, and remained at a higher level. It can be seen that the idea of reducing the cost by reducing the adjuvant usage is viable.

Further, the volume of the first component of the standard single human dose is 0.2-1 ml.

Further, the volume of the first component of the standard single-person dose is 0.5ml.

In some embodiments, in each set of the rabies virus preparation, the first component and the second component are packaged separately, and the two components are mixed temporarily before use, and the mixed solution is used to vaccinate a subject. The method can save the mixing procedure in the production process, save the cost and improve the stability of the product.

Further, the first component is a finished preparation of attenuated live vaccine, inactivated vaccine, toxoid vaccine, subunit vaccine, vector vaccine, genetic engineering vaccine or nucleic acid vaccine.

Further, the first component is in the form of a lyophilized powder, solution or suspension.

Further, the rabies virus is at least one strain or derivative strain selected from PAS strain, flury strain, SAD strain, aG strain and CTN strain.

At present, all vaccine strains used at home and abroad can be classified into Pasteur strain (PAS), flury strain, SAD strain, aG strain, CTN strain and derivatives thereof, and the five series of vaccine strains can be completely suitable for in-vivo and in-vitro culture.

PAS strain: the strain was isolated in 1882 and was the oldest rabies virus vaccine strain. This strain was passaged only in rabbit cells. There are many strains derived from this strain: the famous PV strain appears in the disease center of the beast of Pan-America and is derived from PAS strain. The CVS strain is derived from a PV strain, and derived strains thereof are CVS-11 strain and CVS-24 strain. In addition, it is widely believed that the PM strain is also derived from the PAS strain. PAS strains and derivatives thereof have been used in the preparation of a large number of human or veterinary rabies vaccines.

Flury strain: the strain was isolated in 1939 in georgia, usa and passaged on chicken embryo cells for attenuation. LEP strains and HEP strains are derived strains thereof.

SAD strain: this strain was isolated in 1935 in alabama, usa. The derivatives CL-60 and Vnukovo-32, ERA, SAD-B19, SAG-1 and SAG-2 are used for the injection immunization of dogs and other animals.

AG strain: the strain was isolated from Beijing in 1931. The aG strain was then obtained by adaptive passage of the kidney cells of the mice and subsequent alternate passage in guinea pig brain and monolayer cell culture.

CTN strain: the strain was isolated from Shandong in 1957. The strain is obtained by continuous culture for a plurality of times in the brain of the mouse and the diploid cell strain of the human embryo lung. The cells were then adapted by successive passaging on Vero cells.

At present, PAS strain and aG strain are mainly used in China, but the vaccine produced by different strains has no obvious difference in immune effect in China and around the world.

In some embodiments, the standard single human dose of the rabies virus antigen preparation is an inactivated rabies vaccine finished product preparation, and the titer is 3-10 IU.

In a preferred embodiment, the second component has a titer of 5IU.

In another preferred embodiment, the second component has a titer of 2.5IU.

The applicant found that when the single dose of the rabies vaccine inactivated preparation was halved, the ideal immune effect could still be obtained with reduced number of vaccinations, and it was found that the idea of reducing the production cost and the risk of use of vaccinated subjects by reducing the antigen dose was equally viable.

In some embodiments, the first component and the second component are each packaged independently in each set of the rabies immune formulation.

In some embodiments, specific methods of using the immune kit to provide a complete immunization program are as follows:

S1: mixing the first component and the second component in a group of rabies immune preparations to prepare a rabies vaccine in a solution or suspension state;

s2: immediately administering the rabies vaccine to the vaccinated subject;

S3: within 28 days, the remaining groups of the rabies immune preparation were formulated into rabies vaccine according to the method described in S1 and administered to the vaccinated subjects.

The antigen component and the adjuvant component are independently packaged and form a packaged form according to single use dosage, and the antigen component and the adjuvant component are directly mixed or directly and respectively injected when in use, so that the effect of instant use can be obtained, no additional preparation process or operation training is needed, the use is convenient and quick, and the cost is further saved.

In some specific embodiments, when the immunogenic component is a lyophilized powder, the rabies immunization kit may further add a separately packaged diluent in the same parts as the immunogenic component, and the lyophilized powder is dissolved by the diluent and then combined with an oil-in-water solvent in order to prevent the occurrence of a situation that the oil-in-water emulsion does not dissolve the lyophilized powder well; preferably, the diluent is water for injection or PBS buffer.

The invention further provides a rabies immune suit, which comprises 2-3 groups of rabies vaccine preparations, wherein each group of preparations consists of an antigen preparation and an adjuvant which are independently packaged, the titer of the antigen preparation is 2-5 IU, and the adjuvant consists of 1-5.35mg squalene, 1-5.93mg alpha-tocopherol and 1-2.43mg Tween 80.

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

The invention provides an application of an oil-in-water emulsion in preparing a rabies immune suit, which combines rabies virus antigen with a specific oil-in-water emulsion adjuvant, can quickly excite immune response, effectively improve immune activity, and can obtain good immune effect under the condition of reducing the dosage of the antigen or the adjuvant. The combined mode can effectively utilize the existing rabies vaccine mode, combines the rabies vaccine of the finished product with the adjuvant product subjected to safety verification, does not need clinical test of a system, only needs to carry out bridging experiment verification effect, and the adopted adjuvant does not contain antibiotics or aluminum, has low risk, can reduce the inoculation times and the inoculation dosage on the premise of ensuring the immune effect, and is hopeful to simplify the immune process, control the production cost and the inoculation cost and reduce the risk of immune side reaction, thereby ensuring the benefits of both producers and patients.

Drawings

FIG. 1 (a) is a graph showing the titre of pseudovirus neutralizing antibodies after 14 days of immunization with the first dose of the different immunization program in example 3;

FIG. 1 (b) shows the neutralizing antibody titres of pseudoviruses after 14 days of full vaccination using different immunization programs in example 3;

FIG. 2 shows the variation of the titre of pseudovirus neutralizing antibodies within 42 days after the first dose of immunization using different immunization programs in example 3;

FIG. 3 (a) is the pseudovirus neutralizing antibody titer profile of example 4 after 14 days of initial immunization with different immunization doses;

FIG. 3 (b) is the neutralizing antibody profile of pseudoviruses after 14 days of full-course immunization with different immunization doses in example 4;

FIG. 4 (a) is a pseudo-virus neutralizing antibody titer profile 14 days after immunization with the different adjuvant first dose in example 5;

FIG. 4 (b) pseudo-virus neutralizing antibody titres 14 days after full immunization with different adjuvants in example 5;

FIG. 5 (a) shows the pseudovirus neutralizing antibody titres 7 days and 14 days after full immunization using the muscle immunization route in example 6;

FIG. 5 (b) shows the neutralizing antibody titres of pseudoviruses after 14 days of full-range immunization using the intraperitoneal route in example 6.

Detailed Description

The invention will be further illustrated by the following non-limiting examples, which are well known to those skilled in the art, that many modifications can be made to the invention without departing from the spirit thereof, and such modifications also fall within the scope of the invention. The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention as embodiments are necessarily varied. The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting, the scope of the present invention being defined in the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Preferred methods and materials of the invention are described below, but any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. The following experimental methods are all methods described in conventional methods or product specifications unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified.

Definition of terms

In the application, the 'finished product preparation' refers to vaccine preparations which are clear in all formulas, can be prepared by a determined method and are packaged, stored and transported, and comprises a commercial rabies vaccine and a rabies vaccine preparation which is still in a clinical test and approval stage and is not yet approved for marketing; including existing inactivated vaccines, and also including inactivated vaccines, recombinant protein vaccines and any other form of rabies vaccine that have been produced into finished products but have not yet been formally marketed. The "commercially available rabies vaccine" refers to a rabies vaccine which is legally sold in any country or region other than china or China and can be purchased through a normal route, and is not limited to the strain, the preparation method or the product dosage form (lyophilized powder or injection) adopted, including but not limited to the division of the traditional Chinese medicine (national medicine standard S20000004), the division of the biological medicine in Henan (national medicine standard S20000044), the division of the Liaoning into large organism (national medicine standard S20043089, national medicine standard S20043090), the division of the Jilin Huikang organism medicine (national medicine standard S20060076), the division of the biological medicine in large Lian Yali peak (national medicine standard S20160003), the division of the Ningyi organism medicine (national medicine standard S20030033), the division of the Ningbo biological medicine industry (national medicine standard S20073014), the division of the Chengdu biological product standard (national medicine standard S20120007), the division of the Dakka biological medicine in Chengkunkan, the division of the Liu medicine standard (national medicine standard S20160006), the division of the biological medicine in Shangkunkan (national medicine standard S20210026) and the like. The rabies vaccines which are marketed at present are all inactivated vaccines, and the immune effects in actual use are not obviously different, so that the rabies vaccines can be mutually replaced in the application.

In the present application, the term "standard single human dose" for an antigen refers to the dose of rabies virus antigen in each dose of vaccine in the "finished formulation", which may be the dose of each dose in the product marketed in the obtained lot, or may be a suitable dose determined during the test. For example, in the embodiment of the present application, an inactivated vaccine is used, and the antigen dose of each dose in the marketed product is 5 IU, and the "standard single human dose" is 5 IU. For adjuvants, since no adjuvant is present in the rabies vaccine currently marketed in batches, the "standard single human dose" should be a suitable dose determined experimentally by the skilled person, for example, the dose of each vaccine described in the documents of patents, journal articles, clinical test data, etc., or the dose that the skilled person can determine or infer from experimental data, and in the present application, it is specifically referred to the dose described in the specification.

The "commercial rabies vaccine" used in the following examples may be in the form of a lyophilized powder or in the form of an injection; because the freeze-dried powder is often matched with a diluent (water for injection) to be prepared and dissolved before use, the use effect of the freeze-dried powder vaccine dissolved by a single dose is considered to be equivalent to that of the injection by the single dose under the condition that the antigen component and the auxiliary material component are the same. For the purpose of controlling a single variable in the experimental design, the same lyophilized powder vaccine and the injection prepared by the same method and the same strain from the same manufacturer were used in the following examples. When the freeze-dried powder vaccine is used, if the set adjuvant cannot or possibly cannot fully dissolve the freeze-dried powder (for example, the volume of the adjuvant is too small), the freeze-dried powder vaccine can be re-dissolved by using a matched diluent which does not exceed the dosage of one person, and then the diluent is mixed with the adjuvant, and the diluent added by default can not influence the immune effect.

The final vaccines used in the examples below are all commercial rabies vaccines, and since the rabies vaccines which have been marketed at present are all inactivated vaccines and the immune effects in actual use do not show significant differences, any "commercial rabies vaccine" may default to the same dose in the present invention (i.e. to different vaccines in a single dose), i.e. the method in the examples below may default to be applicable to any commercial rabies vaccine.

The various adjuvants used in the examples below (including aluminum hydroxide adjuvant, AS02 adjuvant, AS03 adjuvant, MF59 adjuvant, and PIKA adjuvant) are currently existing immunoadjuvants, can be purchased from the market through normal channels, or can be prepared by themselves according to the technical information disclosed in the published materials, and in the examples of the present invention, only one possible solution is provided for the purpose of adequate disclosure, but not the only solution is represented, and any proven or proven solution can be used AS a candidate solution.

In the present application, the term "comprising" generally means containing, summarizing, containing or comprising. In some cases, the meaning of "as", "consisting of … …" is also indicated.

EXAMPLE 1 preparation of the first component

Culturing Vero cells to 1-1.5X10 ⁷ cells/ml in a bioreactor, inoculating rabies virus to the cultured Vero cells according to 1/500-1/100MOI, and performing continuous perfusion culture under the culture conditions: the temperature is 32-36 ℃, the pH is 7.2-7.6, the dissolved oxygen is 40-80%, and the stirring speed is 80-120 r/min. Harvesting virus liquid 2-3 days after virus inoculation, continuously harvesting for 5-20 days, coarse filtering the virus liquid by a 0.65 μm filter element, and concentrating by 15-30 times by a 300-500kD membrane package. Adding beta-propiolactone into the concentrated virus harvest liquid according to the volume ratio of 1:4000, and inactivating rabies virus at the temperature of 4 ℃ for at least 24 hours; then left at 37℃for 2-3 hours to hydrolyze the beta-propiolactone. Purifying the inactivated virus liquid by using a Sepharose 4FF chromatographic column, eluting by using 0.01mol/L phosphate buffer solution, and loading the sample with the volume of 5-10% of the volume of the chromatographic column; the detector is an ultraviolet detector, the detection wavelength is 280nm, the rabies vaccine stock solution is obtained by collecting the first chromatographic peak, and the rabies vaccine for human is obtained after dilution, wherein the standard single human dose is 0.5mL, and the potency is 5 IU.

EXAMPLE 2 preparation of the second component

2.14G squalene and 2.37g alpha-tocopherol were mixed, then 45ml phosphate buffer containing 5wt.% tween 80 was added, mixed, stirred at 12000rpm for 20min at 20 ℃ and then homogenized. And (3) carrying out 2-4 cycles at the homogenizing temperature of 20 ℃ and the homogenizing pressure of 80-120 MPa to prepare the oil-in-water emulsion adjuvant with the particle size of less than 160 nm. A standard single human dose is 0.5ml, and the ingredients include 10.69mg squalene, 11.86mg alpha-tocopherol, 4.86mg Tween 80,3.53mg NaCl,0.09mg KCl,0.51mg Na ₂HPO₄, 0.09mg KH ₂PO₄ and sterilized water for injection.

EXAMPLE 3 comparison of the immune Effect of different immunization programs

BALB/c mice were immunized in groups of 6 by intramuscular injection using the first and second components prepared in examples 1 and 2, each in an amount of 1/10 of the human dose (i.e., 0.5 IU for rabies vaccine, 50 μl for adjuvant prepared in example 2), the immunization schedule for each group being as shown in table 1. Serum was collected on days 3, 7, 14, 21, 28, 35 and 42, respectively, after primary immunization, and serum neutralization antibody detection was performed.

TABLE 1 immunization experiment grouping design

The experimental results are shown in fig. 1 and 2. In fig. 1 (a), after 14 days of initial immunization, the pseudovirus neutralizing antibody titers of each group Gr1 to Gr5 (rabies vaccine+adjuvant prepared in example 2) were significantly higher than Gr6 (conventional unadjuvanted vaccine), indicating that the immunized form of the rabies vaccine+the above adjuvant was able to rapidly elicit a higher level of immune response than the conventional unadjuvanted vaccine, with five-needle immunization procedure (Gr 5) being highest, significantly higher than the remaining groups, gr1 and Gr 4. In fig. 1 (b), after the whole vaccination is completed for 14 days (day 42), the titers of neutralizing antibodies of three groups Gr3 to Gr5 are all significantly higher than those of the other groups, and no significant difference is shown between the three groups, which indicates that the immune effects of the two three-needle immunization programs of 0/3/21 and 0/21 (2+1) are not inferior to the conventional five-needle immunization program; in the other 3 groups, the neutralizing antibody titer of Gr2 was low but still significantly higher than Gr1 and Gr6, probably because the last immunization of Gr2 (day 14) was up to 30 days from the last blood sampling, when the neutralizing antibody level had decreased, not the highest level after immunization.

FIG. 2 shows the change in neutralizing antibodies for each group over a 42 day monitoring period, from which it can be seen that there is no significant difference in neutralizing antibody titers for each group on day 3 after primary immunization, all at a lower level, but starting on day 7, neutralizing antibody titers for Gr 1-Gr 5 started to rise rapidly, pulled apart from Gr6 by a significant difference, after which Gr1 started to grow slowly, still quite different from Gr 2-Gr 5, until day 42 reached the highest level; the highest level of neutralizing antibody titer of four groups Gr 2-Gr 5 appeared on day 21, day 35 and day 42, respectively, and all except Gr2 was day 14 after the last immunization, consistent with the general cognition; the trend of variation of Gr3 and Gr4 was substantially the same, no significant difference was generated, and at day 42, the neutralizing antibody titers of Gr3 and Gr4 were somewhat decreased, but were not significantly different from the highest level of Gr 5.

Therefore, the immune form of the rabies vaccine provided by the application and the adjuvant provided by the application definitely shows the feasibility of reducing the inoculation times and simplifying the immune process on the basis of the traditional five-needle immune process, and particularly, the two forms of 0/3/21 and 0/21 (2+1) can completely replace the five-needle form; whereas for this form 0/21 (2+1), the fact that the immunization dose is doubled at the time of primary immunization provides even a concept of reducing the number of immunization to two by increasing the priming dose, and has great application potential for both pre-exposure immunization and post-exposure immunization.

EXAMPLE 4 comparison of the immune Effect of different immune doses

BALB/c mice were immunized in groups of 6 by intramuscular injection with the first and second components prepared in examples 1 and 2, each in an amount of 1/10 (i.e., 0.5 IU for rabies vaccine, 50 μl for adjuvant prepared in example 2) or 1/20 (i.e., 0.25 IU for rabies vaccine, 25 μl for adjuvant) of the individual groups as designed in table 2. Serum was collected on days 3, 7, 14, 21, and 28 after primary immunization, respectively, and serum-neutralizing antibody detection was performed.

TABLE 2 design of immunization experiment groups

The experimental results are shown in fig. 3, and the pseudovirus neutralizing antibody titer of each group of Gr2 to Gr4 (rabies vaccine+adjuvant prepared in example 2) is always significantly higher than Gr1 (conventional adjuvant-free vaccine). In fig. 3 (a), no significant difference was shown between three groups Gr2 to Gr4 after 14 days of the first immunization; in fig. 3 (b), after 14 days of full inoculation (day 42) each group was highest with Gr2, with Gr3 and Gr4 being slightly lower, but still not showing significant differences from Gr 2. It can be seen that even if one dose of the antigen or adjuvant is halved in the immunized form of the rabies vaccine + the adjuvant prepared in example 2, an excellent immune effect can be obtained without delivering the full dose, which also shows the possibility of reducing the production cost and the immunological side effects by reducing the content of the vaccine active ingredient on the premise of ensuring the immune effect.

Example 5 comparison of the immunization Effect of different adjuvants in combination with rabies vaccine

BALB/c mice were immunized in groups of 6 by intramuscular injection with the first and second components prepared in examples 1 and 2, each in an amount of 1/10 of the human dose (i.e., 0.5 IU for rabies vaccine, 50 μl for adjuvant prepared in example 2, or 50 μl for MF 59), the immunization schedule for each group being designed as shown in table 3. Serum was collected on days 14 and 21, respectively, after primary immunization, and serum neutralization antibody detection was performed.

TABLE 3 design of immunization experiment groups

Experimental results as shown in figure 4, the pseudovirus neutralising antibody titres for Gr1 (rabies vaccine + adjuvant prepared in example 2) were consistently significantly higher than for Gr2 (rabies vaccine + MF 59) and Gr3 (conventional unadjuvanted rabies vaccine) during the monitoring period. In fig. 4 (a), gr2 was not shown to be prominent after 14 days of initial immunization, with neutralizing antibody titers even lower than that of Gr3 without adjuvant; in fig. 4 (b), after 14 days of full vaccination (day 21), there was a large increase in the neutralizing antibody titer of Gr2, but there was still a large gap from Gr 1. Therefore, when the rabies vaccine is matched, the immunostimulation effect of the adjuvant provided by the application is obviously better than that of an MF59 adjuvant.

EXAMPLE 6 comparison of the immune Effect of different immune pathways

BALB/c mice were immunized in groups of 6 by intramuscular injection with the first and second components prepared in examples 1 and 2, each in an amount of 1/10 of the human dose (i.e., 0.5 IU for rabies vaccine, 50 μl for adjuvant prepared in example 2, or 50 μl for MF 59), the immunization schedule for each group being designed as shown in table 4. Serum was collected on days 7 and 14, respectively, after the last immunization, and serum neutralization antibody detection was performed.

TABLE 4 immunization experiment grouping design

The experimental results are shown in FIG. 5. In fig. 5 (a), after 7 days and 14 days of last immunization, gr1 neutralizing antibody titers were consistently higher than Gr2; in fig. 5 (b), the neutralizing antibody titer of Gr3 was also significantly higher than Gr4 and Gr5 after 14 days of full vaccination completion. However, gr1 and Gr3 cannot be directly compared due to the difference of the immunization program, and there is no significant difference only from the detection result. Thus, the immunostimulating effect of the adjuvant prepared in example 2 was significantly higher than MF59, regardless of the immunization route or immunization program, and the adjuvant was applicable to either immunization route when used with the rabies vaccine.

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims (14)

1. Use of an oil-in-water emulsion for the preparation of a rabies immune set, wherein said immune set is used to provide a complete immunization program suitable for use in humans; the oil-in-water emulsion comprises squalene, alpha-tocopherol and tween, the immune suit comprises 2-3 groups of rabies immune preparations, each group of rabies immune preparations comprises a first component and a second component which are used in combination, the first component comprises the oil-in-water emulsion, the second component comprises a rabies virus antigen preparation, and the dosage of any one of the first component and the second component is lower than the standard single human dosage.

2. The use according to claim 1, wherein the oil-in-water emulsion comprises 6-15 mg squalene, 6-15 mg alpha-tocopherol and 3-10 mg tween 80.

3. The use according to claim 2, wherein the oil-in-water emulsion comprises 10.69mg squalene, 11.86mg α -tocopherol and 4.86mg tween 80.

4. The use according to claim 2, wherein the oil-in-water emulsion comprises 5.35mg squalene, 5.93mg α -tocopherol and 2.43mg tween 80.

5. The use according to claim 2, wherein the volume of the first component in each set of rabies virus preparation is 0.2-1 ml.

6. The use according to claim 5, wherein the volume of the first component in each set of the rabies immune formulation is 0.5ml.

7. The use according to claim 1, wherein the rabies virus antigen preparation is a finished preparation of attenuated live vaccine, inactivated vaccine, toxoid vaccine, subunit vaccine, vector vaccine, genetically engineered vaccine or nucleic acid vaccine.

8. The use according to claim 7, wherein the rabies virus antigen preparation is in the form of a lyophilized powder, solution or suspension.

9. The use according to claim 7, wherein the standard single human dose of the rabies virus antigen preparation is a finished inactivated rabies vaccine preparation having a potency of 3-10 IU.

10. The use of claim 9, wherein the second component has a potency of 5IU.

11. The use of claim 9, wherein the second component has a potency of 2.5IU.

12. The use of any one of claims 1 to 11, wherein the first component and the second component are packaged separately in each set of the rabies immune formulation.

13. The use according to claim 12, wherein the specific method of providing a complete immunization program using the immunization package is as follows:

S1: mixing the first component and the second component in a group of rabies immune preparations to prepare a rabies vaccine in a solution or suspension state;

s2: immediately administering the rabies vaccine to the vaccinated subject;

S3: within 28 days, the remaining groups of the rabies immune preparation were formulated into rabies vaccine according to the method described in S1 and administered to the vaccinated subjects.

14. The rabies immune suit is characterized by comprising 2-3 groups of rabies vaccine preparations, wherein each group of preparations consists of an antigen preparation and an adjuvant which are independently packaged, the titer of the antigen preparation is 2-5 IU, and the adjuvant consists of 1-5.35mg squalene, 1-5.93mg alpha-tocopherol and 1-2.43mg Tween 80.