CN113117065A

CN113117065A - Influenza vaccine composition for sublingual mucosal delivery

Info

Publication number: CN113117065A
Application number: CN201911419876.9A
Authority: CN
Inventors: 廖辉; 张庶民; 周荔葆; 马凤森; 刘苗苗; 修雪亮; 徐国标; 王一平; 杨文腰; 吴铮
Original assignee: Liaoning Chengda Biological Co ltd
Current assignee: Liaoning Chengda Biological Co ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2021-07-16

Abstract

The invention discloses an influenza vaccine composition for sublingual mucosal delivery, which comprises: a) an effective dose of an influenza vaccine; b) FimH adjuvant. The invention provides a method for adding the FimH adjuvant with specific content into the influenza vaccine to form the composition for sublingual mucosal delivery, which not only effectively improves the immune response of the influenza vaccine, but also avoids the immune tolerance existing in sublingual mucosal delivery.

Description

Influenza vaccine composition for sublingual mucosal delivery

Technical Field

The invention relates to the technical field of influenza vaccine administration, in particular to an influenza vaccine composition for sublingual mucosal delivery.

Background

Influenza (Influenza) is an acute respiratory disease caused by Influenza virus. Influenza viruses are responsible for high mortality and morbidity worldwide due to their susceptibility to variation, high infectivity, and rapid transmission. Human influenza viruses are mainly classified into type A and type B. Type A (A) is further divided into several subtypes. There are 3 subtypes that spread among the population: form A1, represented by A (H1N 1); form a 2, represented by a (H2N 2); form A3, represented by A (H3N 2). The main clinical manifestations of influenza virus infection are fever, headache, general weakness, and many symptoms of respiratory system such as runny nose, dry cough, pharyngalgia, and myocarditis and pericarditis. Vaccination is currently the primary measure for the prevention of influenza.

The marketed influenza vaccine mainly comprises four types of trivalent and quadrivalent split vaccines, subunit vaccines, whole virus vaccines and attenuated vaccines, and all contains inactivated influenza viruses or antigen components of A1 subtype, A3 subtype and B3 types. The subunit vaccine is prepared by extracting special protein structures of bacteria and viruses by chemical decomposition or proper protein hydrolysis method, and screening out the part with immunological activity. The subunit vaccine has purer antigen components compared with other three kinds of vaccines, and because the subunit vaccine only has a few major surface proteins, the subunit vaccine can avoid the body from generating a plurality of antibodies induced by irrelevant antigens, thereby reducing the side effects of the vaccine and relevant diseases caused by the vaccine. Therefore, the subunit vaccine has wide application prospect.

Mucosal immunity becomes the first line of defense for the host as influenza viruses invade the host by invading mucosal surfaces of the upper respiratory tract. The initial site of infection is effectively controlled by mucosal immunity. Furthermore, mucosal immunization, as described in the literature (Brandtzaeg P. indication of surgery immunity and memory at mucosals surfaces [ J ]. Vaccine,2007, 25(30):0-5484.), not only produces a mucosal local specific immune response, but also is effective in inducing a systemic immune response. In addition, the vaccine is delivered by mucosa, so that the defect that the conventional intramuscular injection (i.m.) needs professional technicians and inoculators and has poor compliance and cross infection caused by insufficient sanitation can be avoided, and the vaccine is more convenient and safer to administer and is very suitable for large-scale immunization during epidemic period. Therefore, the development of mucosal vaccines has become an emerging direction and focus in recent years.

For example, chinese patent publication No. CN 105342982a discloses an influenza vaccine immune preparation administered nasally and a preparation method thereof, and proposes that influenza vaccine can be made into a spray for administration via nasal mucosa. But the technical scheme has the following defects: since the nasal cavity olfactory nerve is associated with central nerve, there is a risk that the drug is transferred to the central nervous system through olfactory nerve when administered to nasal mucosa, and there are also disadvantages of retention and influence on the lower respiratory tract. Therefore, it is important to find a safe and effective mucosal delivery route for influenza vaccine.

The sublingual mucosal administration route (SLIT) has obvious advantages in mucosal administration due to the characteristics of rapid absorption, avoidance of first-pass effect, high bioavailability, convenience in administration, good patient compliance and the like. Unlike injection vaccines, which induce antibody production primarily in blood, SLIT induces antibodies both in the lining of the lung mucosa and in the blood. However, SLIT vaccines have the following problems in the administration route: the immunogenicity of sublingual administration is not enough; ② the mucosal immune system can induce immune tolerance. How to effectively solve the problems is a general concern of the industry.

Dosage forms currently primarily suitable for SLIT administration include: drops, films, pills and sublingual tablets. For example, The literature (Murugappan S, Patil H P, Frijlink H W, et al. Simplifyzing Influent nza Vaccine Dual proteins peptides: Sublingual primers and intramular Boosting of Immune Responses with heterologus and inactive Influenza Vaccine [ J ]. The AAPS Journal,2014,16(2):342 349) explores The preparation of Influenza Vaccine in The form of Sublingual fast disintegrating tablets, which enhances The residence time of The drug on The Sublingual mucosa and also releases The drug rapidly. However, this document does not provide any information on whether the influenza vaccine prepared as a sublingual rapidly disintegrating tablet can exert the effect of avoiding immune tolerance; that is, there is uncertainty as to whether a sublingual formulation of an influenza vaccine can effectively enhance the immune effect of the influenza vaccine.

To enhance the sublingual mucosal immune response, an adjuvant is typically added to the vaccine formulation. For example, the chinese invention patent publication No. CN101524537A provides an influenza oral buccal tablet vaccine, an influenza oral sustained release vaccine and preparation methods thereof. The scheme has the following defects: it is mentioned on page 15 only that adjuvants can be added to the vaccine, but no screening is done for the type and amount of adjuvant to determine if the immunogenicity of the vaccine is improved. As another example, the literature (Spiner J L, Oberoi H S, Yorgensen Y M, et al. Methylglycerol cholesterol and a synthetic TLR4 agonist enzyme immunity responses to an influenza Vaccine J. Vaccine 2015,33(43): 5845. 5853.) explored the addition of the adjuvants CRX-601, chitosan or both to improve the immunogenicity of vaccines, but had the disadvantages: the problem of immune tolerance with SLIT administration is not solved.

In summary, the focus of current vaccines for oral mucosal administration is on how to improve the immunogenicity of antigens. However, numerous studies have found that oromucosal administration results in immune tolerance, which once produced fails to produce specific antibodies and thus fails to carry out a normal immune response. Unfortunately, none of the current literature on oral administration of viral vaccines addresses the issue of development of immune tolerance by oromucosal administration.

The applicant is informed by referring to relevant documents that Dendritic Cells (DCs) are immune cells in oral mucosa, the degree and type of immune response induction of the dendritic cells are closely related to the maturation state of the DCs, and it is generally considered that immature dendritic cells (imdcs) are mainly distributed in non-lymphoid tissues such as oral mucosa, are the first line of defense of mucosal immunity and are related to immune tolerance; mature dendritic cells (mdcs) are distributed mainly in secondary lymphoid tissues and organs such as lymph nodes, are powerful antigen presenting cells, and mainly induce immune activation. Therefore, how to effectively induce the imDC to mDC conversion and promote the increase of the content of mDC is the focus of the present application.

In conclusion, how to provide an influenza vaccine composition which can avoid immune tolerance and effectively induce immune response to realize effective delivery of sublingual mucosa is a technical problem which is not solved by the technical personnel in the field.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an influenza vaccine composition for sublingual mucosal delivery, which solves the defects existing in the prior art by selecting specific types and contents of adjuvants.

Therefore, the invention adopts the following technical scheme:

an influenza vaccine composition for sublingual mucosal delivery comprising: a) an effective dose of an influenza vaccine; b) FimH adjuvant, content 15%.

Preferably, the content ratio of the influenza vaccine to the FimH adjuvant in the influenza vaccine composition is 1: 10.

Preferably, in the influenza vaccine composition, the influenza vaccine is against influenza a and influenza b viruses, and the influenza vaccine is selected from one of trivalent and tetravalent split vaccines, subunit vaccines, whole virus vaccines and attenuated vaccines.

Preferably, the influenza vaccine composition is combined with pharmaceutically acceptable auxiliary materials to be prepared into tablets, drops, films or dripping pills for sublingual mucosal delivery administration.

More preferably, the influenza vaccine composition is combined with pharmaceutically acceptable auxiliary materials to prepare a sublingual rapidly disintegrating tablet, and the sublingual rapidly disintegrating tablet consists of the influenza vaccine, FimH adjuvant and tablet matrix materials, wherein the tablet matrix materials include but are not limited to fillers, excipients, disintegrants and lubricants.

More preferably, the sublingual rapidly disintegrating tablet comprises the following components in parts by mass: 1.5 percent of influenza vaccine, 15 percent of FimH adjuvant, 55 percent of cane sugar, 10 percent of croscarmellose sodium, 18 percent of microcrystalline cellulose and 0.5 percent of superfine silica gel powder.

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

the invention provides a method for adding the FimH adjuvant with specific content into the influenza vaccine to form the composition for sublingual mucosal delivery, which not only effectively improves the immune response of the influenza vaccine, but also avoids the immune tolerance existing in sublingual mucosal delivery. Furthermore, the influenza vaccine composition is prepared into a sublingual rapidly disintegrating tablet, so that the residence time of the drug on the sublingual mucosa can be prolonged, and the drug can effectively present antigen through the mucosa; can also release the drug quickly, has quick and convenient use process, simplifies the inoculation procedure, facilitates the large-area inoculation of the influenza vaccine and is beneficial to the large-scale immunity during the influenza virus epidemic period.

Drawings

FIG. 1 is a graph showing the average body weight gain of each experimental group over 7 days.

FIG. 2 is a graph showing hemagglutination inhibition titers measured in each experimental group under different adjuvants.

FIG. 3 is a graph showing hemagglutination inhibition titers measured in each experimental group at different levels of the same adjuvant.

Detailed Description

The following are specific examples of the present invention and further describe the technical solutions of the present invention, but the scope of the present invention is not limited to these examples. All changes, modifications and equivalents that do not depart from the spirit of the invention are intended to be included within the scope thereof.

Based on safety and possibility consideration, after relevant documents are consulted, several adjuvants are screened out: MF59, aluminum hydroxide, CRX-601, chitosan and FimH.

MF59 and aluminum hydroxide are widely added into various vaccines as common adjuvants, and CRX-601 is also reported to be capable of being used for improving the immunogenicity of influenza vaccines in a synergistic effect with chitosan. FimH is a type I pilus-tipped adhesin component that has been shown to be associated with the early stages of dendritic cells, but FimH is currently used as an adjuvant in very few studies.

In addition, no relevant literature reports on whether the adjuvants MF59, aluminum hydroxide, CRX-601, chitosan and FimH can improve the immunogenicity of influenza vaccines and avoid the immune tolerance of oral mucosa when used alone.

Accordingly, the applicant has conducted the following screening test.

EXAMPLE 1 preparation of Sublingual rapidly disintegrating tablets of influenza vaccine

For the convenience of screening, after the influenza vaccine composition is prepared into the sublingual fast disintegrating tablet, the influence factors on the immunogenicity and the immune tolerance of the influenza vaccine are investigated. It should be noted that the focus of the present invention is to explore the influence of the screening of the adjuvant on the immunogenicity and the immune tolerance of the influenza vaccine, and not to investigate the influence of changing the application dosage form of the influenza vaccine composition on the immunogenicity and the immune tolerance of the influenza vaccine. Therefore, the sublingual rapidly disintegrating tablet of influenza vaccine prepared in example 1 is only an example of a SLIT administration, but the dosage form application of the influenza vaccine composition is not limited to the sublingual rapidly disintegrating tablet indicated in example 1, and pharmaceutically feasible SLIT administration dosage forms such as common tablets, drops, membranes or dropping pills are also included.

The invention relates to a sublingual quick-disintegrating tablet of an influenza vaccine, the prescription of which consists of the influenza vaccine, a FimH adjuvant and a pharmaceutically acceptable tablet matrix material, and the preparation method comprises the following steps:

preparing influenza vaccine according to the prescription amount, taking the influenza vaccine and adjuvant, mixing uniformly, and freeze-drying. And (3) gradually mixing the freeze-dried product with tablet matrix materials (55% of sucrose, 10% of croscarmellose sodium, 18% of microcrystalline cellulose and 0.5% of superfine silica gel) in equal amount, and tabletting to obtain the sublingual rapidly disintegrating tablet of the influenza vaccine.

Through inspection, the sublingual rapidly disintegrating tablets of the influenza vaccine prepared in the embodiment all meet the evaluation standard of the rapidly disintegrating tablets specified in pharmacopoeia.

Example 2 Effect of the types of adjuvants on the immunogenicity of influenza vaccines

In the present example, to examine the influence of the adjuvant type on the immunogenicity of influenza vaccine, FimH adjuvant, MF59 adjuvant, CRX-601 adjuvant, chitosan adjuvant, and aluminum hydroxide adjuvant were selected for comparative study.

The examination method is as follows:

1) taking FimH adjuvant, MF59 adjuvant, CRX-601 adjuvant, chitosan adjuvant and aluminum hydroxide adjuvant according to the prescription amount for later use;

2) the component ratio of the adjuvanted influenza vaccine and the tablet matrix material was fixed, and the dose of sublingual tablets for human use was converted into the dose of sublingual tablets for mice by referring to (yellow-serendian, yellow-shoghui, Chengyang, et al. equivalent dose conversion between animals and human bodies in pharmacological tests [ J ]. China clinical pharmacology and therapeutics, 2004, 9(9):1069-1072.) article, and prepared according to the method in example 1, blank influenza vaccine samples and different kinds of adjuvanted influenza vaccine sample groups were prepared, and the total of 6 experimental groups were recorded as: vaccine group, MF59 adjuvant group, Al (OH)3 adjuvant group, CRX-601 adjuvant group, FimH adjuvant group;

3) immunization and detection: standard female BALB/c mice (6-8 weeks old) were immunized with SLIT using six different samples prepared as described above. Each group had 3 mice, and the weight measurements were performed every 7 days after the first day of inoculation, and the average weight gain of each group over 7 days was calculated, and the results are shown in fig. 1; and tail blood was collected at 14 days, 21 days, 28 days, 35 days, 42 days, and 49 days after the first immunization, and serum was separated to determine the hemagglutination inhibition titer, and the results are shown in FIG. 2.

As can be seen from fig. 1 and 2:

1) after inoculation of the experimental mice, there was a corresponding weight gain in all 6 experimental groups. The influenza vaccine group had minimal weight change, and the chitosan and FimH nearly did not differ in weight gain in the CRX-601 group.

2) Compared with the influenza vaccine group without the adjuvant, the hemagglutination inhibition antibody titer of the influenza vaccine is improved in the other 5 groups with different adjuvants.

3) The potency curve boost for the MF59 adjuvant group and the al (oh)3 adjuvant group were very close before 21 days, with the overall level of the al (oh)3 adjuvant group being slightly higher than the blank vaccine group in the assay before 28 days; a rapid decline occurred after 28 days and leveled off after 42 days.

4) Compared to the two types of adjuvants, al (oh)3 and MF59, the FimH group had a weak lead before day 21, but had a jump increase at 28 days, 35 days and 42 days; the highest titer was reached at 42 days. The FimH group was steadily trending downward compared to the CRX-601 and chitosan groups in the 49 and 56 day assays.

The reason for the analysis is as follows: compared with a blank vaccine group, the adjuvant group has no obvious change of body weight after inoculation, and shows that the side effects of five groups of adjuvants are not obvious to a certain extent and the safety is higher; ② compared with other adjuvant groups, the hemagglutination inhibition titer of the FimH group is far higher than that of the other groups at the peak value and the decline trend is mild, which shows that the FimH adjuvant has better immune effect when being combined with influenza vaccine.

Example 3 Effect of adjuvant classes on sublingual mucosal immune tolerance

In the present example, to examine the influence of adjuvant types on sublingual mucosal immune tolerance, FimH adjuvant, MF59 adjuvant, CRX-601 adjuvant, chitosan adjuvant, and aluminum hydroxide adjuvant were selected for comparative study.

The examination method is as follows:

2) the ratio of the components of the influenza vaccine containing adjuvant and the matrix material of the tablet is fixed, and the corresponding sublingual rapidly disintegrating tablet of the influenza vaccine is prepared according to the preparation method of the example 2. Mice were vaccinated with SLIT. Sublingual mucosa was isolated 0 or 2 hours after SLIT immunization, and cells were collected from the mucosa and analyzed using flow cytometry. 2 indexes of mDC increment and the tendency of imDC conversion into mDC are adopted as indexes for evaluating sublingual mucosal immune tolerance. The results are shown in Table 1.

Table 1. effect of different adjuvants on mDC (mean ± SD, n ═ 3)

Experimental group	mDC increment (%)	Amount of conversion of imDC to mDC (%)
			Vaccine group	1.03±0.24	<0.5
MF59 adjuvant group	1.41±0.18	<0.5
			Al(OH)₃Adjuvant group	2.37±0.21	<0.5
CRX-601 adjuvant group	3.14±0.19	<0.5
			Chitosan adjuvant group	2.93±0.19	<0.5
FimH adjuvant group	12.7±0.21	5.37±0.16

From the combination table 1, it can be seen that:

1) from the index of the increase of the mDC, the FimH adjuvant group achieves a 12-fold increase rate compared with the blank vaccine group, which is far higher than that of the rest adjuvant groups.

2) In the statistics of the conversion amount of imDC to mDC, the conversion amount of the other groups except the FimH group is lower than 0.5%, while the FimH adjuvant group reaches the value of about 5.37%, which is far higher than that of the other groups.

The reason for the analysis is as follows: MF59 is used as a common adjuvant in injection, and the effect of the MF59 added in a sublingual preparation is not good, which shows that the use effect of the adjuvant is different for different administration parts; ② the FimH adjuvant group has the best effect in the mDC increment and the conversion of imDC into mDC, which indicates that FimH as a mucosal adjuvant can effectively induce the maturation of dendritic cells and the presentation of antigen.

Example 4 Effect of adjuvant content on immunogenicity of influenza vaccines

This example aims to examine the influence of the adjuvant content on the sublingual mucosal immune tolerance, and to change the content of FimH adjuvant based on examples 2 and 3 for comparative study.

The examination method is as follows:

1) the adjuvant is selected from FimH, the content of the FimH adjuvant is respectively 5%, 10%, 15%, 20% and 25% for standby;

2) the components of the adjuvant-containing influenza vaccine and the matrix material of the tablet are fixed, and the corresponding sublingual fast disintegrating tablet of the influenza vaccine is prepared according to the preparation method of the example 2. Standard female BALB/c mice (6-8 weeks old) were immunized with SLIT. 3 of each group were inoculated once on day 1 and once again on day 21. And tail blood was collected 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, and 49 days after the first immunization, and serum was separated to determine hemagglutination inhibition titer, and the results are shown in FIG. 3.

As can be seen from fig. 3:

1) when the adjuvant content was 5%, a peak was reached at day 35 and the titer level was lowest during the assay period.

2) At 10% adjuvant, the titer level was significantly increased compared to 5% and reached a peak of about 1500 at day 42, but then dropped rapidly.

3) The peak was reached at day 35 when the adjuvant content was 20% and 25%. But then decreases rapidly and the peak value is lower than the 10% content.

4) When the adjuvant content was 15%, the difference compared to the optimal titer group before day 35 was slightly higher rapidly after day 35, reaching an optimal potency value of over 2000 at day 42. And then the downward trend is smooth.

The analytical results were as follows: the optimum potency is obtained when the adjuvant content is 15%.

EXAMPLE 5 Effect of adjuvant content on sublingual mucosal immune tolerance

The examination method is as follows:

2) the components of the adjuvant-containing influenza vaccine and the matrix material of the tablet are fixed, and the corresponding sublingual fast disintegrating tablet of the influenza vaccine is prepared according to the preparation method of the example 2. Mice were vaccinated with SLIT. Sublingual mucosa was isolated 0 or 2 hours after SLIT immunization, and cells were collected from the mucosa and analyzed using flow cytometry. 2 indexes of mDC increment and the tendency of imDC conversion into mDC are adopted as indexes for evaluating sublingual mucosal immune tolerance. The results are shown in Table 2.

Table 2. effect of different adjuvants on mDC (mean ± SD, n ═ 3)

Content (wt.)	mDC increment (%)	Amount of conversion of imDC to mDC (%)
			5％	7.62±0.18	3.34±0.18
10％	12.5±0.21	5.13±0.16
			15％	17.2±0.23	8.21±0.18
20％	17.3±0.19	8.17±0.19
			25％	16.4±0.19	7.26±0.17

From table 2, it can be seen that:

1) when the adjuvant content is 5%, the mDC increment is only about 7.62%, and the effect is the worst compared with the other four groups.

2) When the adjuvant content was 10%, the mDC gain was 1.6 times more than the 5% content group, with a corresponding increase, but the level was still poor compared to the 20% and 25% groups.

3) The optimal incremental values for mDC were achieved with a small difference between 15% and 20% adjuvant content. The 25% content group did not increase as much as the first two groups.

4) When the adjuvant content was 15% and 20%, the conversion of imDC to mDC was around 8.20%, with the best effect compared to the rest of the group.

The analytical results were as follows: when the adjuvant content is 15% or 20%, there is an optimal maturation rate of dendritic cells. However, in combination with the influence of example 4 on immunogenicity, it was found that the immunogenicity of influenza vaccines can be improved and immune tolerance can be avoided only when the adjuvant content is 15%.

In conclusion, the influenza vaccine sublingual rapidly disintegrating tablet comprises the following components in the best mass ratio: 1.5 percent of influenza vaccine, 15 percent of FimH adjuvant, 55 percent of cane sugar, 10 percent of croscarmellose sodium, 18 percent of microcrystalline cellulose and 0.5 percent of superfine silica gel powder.

Claims

1. An influenza vaccine composition for sublingual mucosal delivery comprising:

a) an effective dose of an influenza vaccine;

b) FimH adjuvant, content 15%.

2. The influenza vaccine composition for sublingual mucosal delivery according to claim 1, characterized in that the ratio of the content of influenza vaccine and FimH adjuvant in the composition is 1: 10.

3. The influenza vaccine composition for sublingual mucosal delivery according to claim 1, wherein the influenza vaccine in the composition is directed against influenza a and b viruses, the influenza vaccine being selected from one of trivalent and tetravalent split vaccines, subunit vaccines, whole virus vaccines, attenuated vaccines.

4. The influenza vaccine composition for sublingual mucosal delivery according to any one of claims 1 to 3, wherein the composition is combined with pharmaceutically acceptable excipients to be prepared into tablets, drops, films or dripping pills for sublingual mucosal delivery administration.

5. The influenza vaccine composition for sublingual mucosal delivery according to claim 5, wherein the composition is combined with pharmaceutically acceptable excipients to prepare sublingual rapidly disintegrating tablets, said sublingual rapidly disintegrating tablets consisting of influenza vaccine, FimH adjuvant and tablet matrix materials, wherein the tablet matrix materials include but are not limited to fillers, excipients, disintegrants, lubricants.

6. The influenza vaccine composition for sublingual mucosal delivery according to claim 5, wherein the sublingual rapidly disintegrating tablet comprises the following components in parts by mass: 1.5 percent of influenza vaccine, 15 percent of FimH adjuvant, 55 percent of cane sugar, 10 percent of croscarmellose sodium, 18 percent of microcrystalline cellulose and 0.5 percent of superfine silica gel powder.