An influenza vaccine composition for sublingual mucosal delivery

Abstract

This invention discloses an influenza vaccine composition for sublingual mucosal delivery, comprising: a) an effective dose of influenza vaccine; and b) FimH adjuvant. This invention provides a method for selecting a specific amount of FimH adjuvant and adding it to an influenza vaccine composition for sublingual mucosal delivery, which not only effectively enhances the immune response to the influenza vaccine but also avoids the immune tolerance present with sublingual mucosal delivery.

Description

Technical Field

[0001] This invention relates to the field of influenza vaccine delivery technology, and more specifically to an influenza vaccine composition for sublingual mucosal delivery. Background Technology

[0002] Influenza is an acute respiratory illness caused by the influenza virus. Due to its high mutation rate, strong infectivity, and rapid spread, the influenza virus causes high mortality and morbidity rates worldwide. Human influenza viruses are mainly classified into two types: type A and type B. Type A (A) is further divided into several subtypes. Three subtypes circulate in the human population: type A1, represented by A(H1N1); type A2, represented by A(H2N2); and type A3, represented by A(H3N2). The main clinical manifestations of influenza virus infection are fever, headache, and general weakness, often accompanied by respiratory symptoms such as runny nose, dry cough, and sore throat, and may also lead to complications such as myocarditis and pericarditis. Currently, vaccination is the primary measure for preventing influenza.

[0003] Currently available influenza vaccines mainly include four types: trivalent and quadrivalent split vaccines, subunit vaccines, whole-virus vaccines, and live attenuated vaccines. All contain inactivated influenza viruses or antigenic components from influenza A subtypes 1, 3, and B. Subunit vaccines are made by extracting specific protein structures from bacteria and viruses through chemical decomposition or appropriate protein hydrolysis methods, selecting the immunologically active portions. Compared to the other three types of vaccines, subunit vaccines have purer antigenic components. Furthermore, because subunit vaccines contain only a few major surface proteins, they can avoid the production of many antibodies induced by irrelevant antigens, thereby reducing vaccine side effects and vaccine-related diseases. Therefore, subunit vaccines have broad application prospects.

[0004] Since influenza viruses invade the host by invading the mucosal surfaces of the upper respiratory tract, mucosal immunity becomes the host's first line of defense. Mucosal immunity effectively controls the initial site of infection. Furthermore, according to the literature (Brandtzaeg P. Induction of secretory immunity and memory at mucosal surfaces[J]. Vaccine,2007, 25(30):0-5484.), mucosal immunity can not only generate a local specific immune response in the mucosa but also effectively induce a systemic immune response. In addition, mucosal delivery of vaccines avoids the disadvantages of conventional intramuscular injection (IM), such as the need for skilled personnel and poor patient compliance, as well as cross-infection caused by inadequate hygiene. This makes vaccine administration more convenient, safer, and highly suitable for large-scale immunization during epidemics. Therefore, the development of mucosal vaccines has become a new and important direction in recent years.

[0005] For example, Chinese invention patent CN 105342982A discloses a nasal-administered influenza vaccine immunizing agent and its preparation method, proposing that the influenza vaccine can be made into a spray for administration via the nasal mucosa. However, this technical solution has shortcomings: because the olfactory nerve in the nasal cavity is connected to the central nervous system, there is a risk that the drug will be transferred to the central nervous system through the olfactory nerve when administered via the nasal mucosa. It also has drawbacks such as retention and effects on the lower respiratory tract. Therefore, finding a safe and effective mucosal delivery route for influenza vaccines is particularly important.

[0006] Sublingual immunotherapy (SLIT) offers significant advantages over traditional mucosal administration due to its rapid absorption, avoidance of the first-pass effect, high bioavailability, convenience, and good patient compliance. Unlike injected vaccines, which primarily induce antibody production in the blood, SLIT induces antibodies in both the pulmonary mucosa and the blood. However, SLIT vaccines present several challenges in terms of administration: ① insufficient immunogenicity with sublingual administration; ② the potential for inducing immune tolerance in the mucosal immune system. Effectively addressing these issues is a major concern for industry professionals.

[0007] Currently, the main dosage forms suitable for SLIT administration include drops, films, pellets, and sublingual tablets. For example, a study (Murugappan S, Patil HP, Frijlink HW, et al. Simplifying Influenza Vaccination During Pandemics: Sublingual Priming and Intramuscular Boosting of Immune Responses with Heterologous Whole Inactivated Influenza Vaccine[J]. The AAPS Journal, 2014, 16(2): 342-349.) explored the formulation of influenza vaccines into sublingual fast-disintegrating tablets to enhance the drug's retention time on the sublingual mucosa and provide rapid drug release. However, this study did not provide any information on whether sublingual fast-disintegrating tablets of influenza vaccines could prevent immune tolerance; in other words, there is uncertainty as to whether sublingual formulations of influenza vaccines can effectively enhance the immune effect of influenza vaccines.

[0008] To enhance the sublingual mucosal immune response, adjuvants are often added to vaccine formulations. For example, Chinese invention patent CN101524537A discloses an oral sublingual influenza vaccine, an oral extended-release influenza vaccine, and methods for preparing both. The shortcoming of this approach is that the paper only mentions on page 15 that adjuvants can be added to the vaccine, but does not screen the types and amounts of adjuvants to determine whether the immunogenicity of the vaccine can be improved. Another example is the literature (Spinner JL, Oberoi HS, Yorkensen YM, et al. Methylglycol chitosan and a synthetic TLR4agonist enhance immune responses to influenza vaccine administered sublingually[J]. Vaccine,2015,33(43):5845-5853.) which explores whether adding adjuvants CRX-601, chitosan, or a combination of both to influenza vaccines can improve the immunogenicity of the vaccine. However, its shortcoming is that it does not solve the problem of immune tolerance associated with SLIT administration.

[0009] In summary, current research on vaccines administered via the oral mucosa focuses on enhancing the immunogenicity of the antigen. However, numerous studies have found that oral mucosal administration can induce immune tolerance, which prevents the production of specific antibodies and thus hinders the normal immune response. Unfortunately, current literature on oral administration of viral vaccines has not addressed the issue of immune tolerance arising from oral mucosal administration.

[0010] Through reviewing relevant literature, the applicant learned that dendritic cells (DCs) are immune cells in the oral mucosa. The degree and type of immune response they induce are closely related to the maturation state of DCs. It is generally believed that immature DCs (imDCs) are mainly distributed in non-lymphatic tissues such as the oral mucosa, forming the first line of defense for mucosal immunity and are related to immune tolerance; mature DCs (mDCs) are mainly distributed in secondary lymphatic tissues and organs such as lymph nodes, and are powerful antigen-presenting cells that primarily induce immune activation. Therefore, how to effectively induce the transformation of imDCs into mDCs and increase the content of mDCs is the focus of this invention application.

[0011] In summary, how to provide an influenza vaccine composition that can both avoid immune tolerance and effectively induce an immune response, and achieve effective delivery to the sublingual mucosa, is a technical problem that has not yet been solved by those skilled in the art. Summary of the Invention

[0012] The technical problem to be solved by the present invention is to provide an influenza vaccine composition for sublingual mucosal delivery, which overcomes the shortcomings of the prior art by selecting specific types and amounts of adjuvants.

[0013] Therefore, the present invention adopts the following technical solution:

[0014] An influenza vaccine composition for sublingual mucosal delivery, comprising: a) an effective dose of influenza vaccine; b) FimH adjuvant at a concentration of 15%.

[0015] Preferably, the ratio of influenza vaccine to FimH adjuvant in the influenza vaccine composition is 1:10.

[0016] Preferably, in the influenza vaccine composition, the influenza vaccine targets influenza A and B viruses, and the influenza vaccine is selected from one of trivalent and quadrivalent split vaccines, subunit vaccines, whole virus vaccines, and attenuated vaccines.

[0017] Preferably, the aforementioned influenza vaccine composition is combined with pharmaceutically acceptable excipients to form tablets, drops, films, or pellets for sublingual mucosal delivery.

[0018] More preferably, the aforementioned influenza vaccine composition is combined with pharmaceutically acceptable excipients to form a sublingually disintegrating tablet, which is composed of an influenza vaccine, FimH adjuvant, and tablet matrix material, wherein the tablet matrix material includes, but is not limited to, fillers, excipients, disintegrants, and lubricants.

[0019] More preferably, the components and their mass percentages in the sublingual rapidly disintegrating tablet are as follows: influenza vaccine 1.5%, FimH adjuvant 15%, sucrose 55%, croscarmellose sodium 10%, microcrystalline cellulose 18%, and micronized silica gel 0.5%.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] This invention provides a method for incorporating a specific amount of FimH adjuvant into an influenza vaccine composition for sublingual mucosal delivery. This not only effectively enhances the immune response to the influenza vaccine but also avoids the immune tolerance associated with sublingual mucosal delivery. Furthermore, this invention formulates the influenza vaccine composition into a sublingually disintegrating tablet dosage form, which enhances the drug's retention time on the sublingual mucosa, allowing for effective antigen presentation through the mucosal layer. It also enables rapid drug release, making the administration quick and convenient, simplifying the vaccination procedure, facilitating large-scale influenza vaccination, and promoting large-scale immunization during influenza epidemics.

[0022] Instruction manual illustrations

[0023] Figure 1 The graph shows the average weight gain of each experimental group over 7 days.

[0024] Figure 2 The hemagglutination inhibition potency of each experimental group was measured under different adjuvants.

[0025] Figure 3 The graph shows the hemagglutination inhibition potency of each experimental group at different concentrations of the same adjuvant. Detailed Implementation

[0026] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention. However, the scope of protection of the present invention is not limited to these embodiments. All changes or equivalent substitutions that do not depart from the concept of the present invention are included within the scope of protection of the present invention.

[0027] Based on safety and feasibility considerations, this invention screened out several adjuvants after reviewing relevant literature: MF59, aluminum hydroxide, CRX-601, chitosan, and FimH.

[0028] MF59 and aluminum hydroxide are commonly used adjuvants and are widely added to various vaccines. CRX-601 has also been reported in the literature to synergize with chitosan in improving the immunogenicity of influenza vaccines. FimH is an adhesin component of the tip of type I fimbriae and has been shown to be related to the early stages of dendritic cells, but there are currently very few studies on the use of FimH as an adjuvant.

[0029] Furthermore, there are no literature reports on whether the aforementioned adjuvants, such as MF59, aluminum hydroxide, CRX-601, chitosan, and FimH, can improve the immunogenicity of influenza vaccines while avoiding immune tolerance of the oral mucosa when used alone.

[0030] Therefore, applicants must undergo the following screening tests.

[0031] Example 1: Preparation of Sublingual Disintegrating Influenza Vaccine Tablets

[0032] To facilitate screening, this invention, after preparing the influenza vaccine composition into sublingual disintegrating tablets, investigates the influencing factors on the immunogenicity and immune tolerance of the influenza vaccine. It should be noted that this invention focuses on exploring the influence of adjuvant screening on the immunogenicity and immune tolerance of the influenza vaccine, not on investigating the influence of changing the dosage form of the influenza vaccine composition on the immunogenicity and immune tolerance of the influenza vaccine. Therefore, the sublingual disintegrating influenza vaccine tablet prepared in Example 1 is merely an example of SLIT administration, but the dosage form application of the influenza vaccine composition is not limited to the sublingual disintegrating tablets mentioned in Example 1, and also includes pharmaceutically feasible SLIT dosage forms such as ordinary tablets, drops, films, or pellets.

[0033] The influenza vaccine sublingual rapid-disintegrating tablets to be prepared in this invention are formulated with influenza vaccine, FimH adjuvant, and pharmaceutically acceptable tablet matrix materials. The preparation method is as follows:

[0034] Prepare the influenza vaccine according to the prescription dosage. Take the influenza virus vaccine and adjuvant, mix them evenly, and freeze-dry. Add equal amounts of the freeze-dried product to the tablet matrix material (55% sucrose, 10% croscarmellose sodium, 18% microcrystalline cellulose, and 0.5% micronized silica powder) and mix them together. Compress the mixture into tablets to obtain the influenza vaccine sublingual disintegrating tablets.

[0035] Upon testing, the sublingual disintegrating influenza vaccine tablets prepared in this embodiment all meet the evaluation standards for disintegrating tablets specified in the pharmacopoeia.

[0036] Example 2: The effect of the type of adjuvant on the immunogenicity of influenza vaccine

[0037] The purpose of this embodiment is to investigate the effect of the type of adjuvant on the immunogenicity of influenza vaccines, and to conduct a comparative study on FimH adjuvant, MF59 adjuvant, CRX-601 adjuvant, chitosan adjuvant, and aluminum hydroxide adjuvant.

[0038] The examination method is as follows:

[0039] 1) Take the prescribed amounts of FimH adjuvant, MF59 adjuvant, CRX-601 adjuvant, chitosan adjuvant, and aluminum hydroxide adjuvant for later use;

[0040] 2) The ratio of adjuvant-containing influenza vaccine to tablet matrix material was fixed. Referring to the article (Huang Jihan, Huang Xiaohui, Chen Zhiyang, et al. Equivalent dose conversion between animals and between animals and humans in pharmacological experiments [J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2004, 9(9):1069-1072.), the dose of human sublingual tablets was converted to the dose of mouse sublingual tablets. The preparation was carried out according to the method in Example 1. Blank influenza vaccine samples and different types of adjuvant-containing influenza vaccine sample groups were obtained. A total of 6 experimental groups were prepared, which were named as follows: vaccine group, MF59 adjuvant group, Al(OH)3 adjuvant group, CRX-601 adjuvant group, CRX-601 adjuvant group, and FimH adjuvant group.

[0041] 3) Immunization and Testing: Standard female BALB / c mice (6-8 weeks old) were immunized with SLIT using the six different samples prepared above. Three mice were in each group. The weight of mice in each group was measured every 7 days after the first day of immunization. The average weight gain of each group over 7 days was calculated, and the results are shown below. Figure 1 Tail blood was collected at 14, 21, 28, 35, 42, and 49 days after the first immunization. Serum was separated and hemagglutination inhibition titers were measured. The results are shown in […]. Figure 2 .

[0042] Combination Figure 1 and Figure 2 It can be known that:

[0043] 1) After inoculation of the experimental mice, all six experimental groups showed corresponding weight gain. The influenza vaccine group showed the smallest weight change, followed by the CRX-601 group, while the weight gain of the chitosan and FimH groups was almost identical.

[0044] 2) Compared to the influenza vaccine group without adjuvants, the other 5 groups with different adjuvants all showed an increase in the hemagglutination inhibition antibody titer of their influenza vaccines.

[0045] 3) The potency curves of the MF59 adjuvant group and the Al(OH)3 adjuvant group were very similar before day 21. Before day 28, the overall level of the Al(OH)3 adjuvant group was slightly higher than that of the blank vaccine group. After day 28, the potency curves declined rapidly and then stabilized after day 42.

[0046] 4) Compared to the adjuvants Al(OH)3 and MF59, the FimH group showed a slight lead before day 21, but experienced a jump in titer at days 28, 35, and 42; reaching its highest titer at day 42. In the measurements at days 49 and 56, the FimH group showed a more stable decreasing trend compared to the CRX-601 and chitosan groups.

[0047] The reasons are analyzed as follows: ① The weight change after vaccination was not significant in the adjuvant group compared with the blank vaccine group, which to some extent indicates that the side effects of the five adjuvants were not obvious and the safety was relatively high; ② Compared with the other adjuvant groups, the hemagglutination inhibition titer of the FimH group was also much higher than that of the other groups at its peak, and the decline trend was gradual, indicating that the FimH adjuvant exerted a better immune effect when combined with the influenza vaccine.

[0048] Example 3: The effect of the type of adjuvant on sublingual mucosal immune tolerance

[0049] The purpose of this embodiment is to investigate the effect of different types of adjuvants on sublingual mucosal immune tolerance, and to conduct a comparative study on FimH adjuvant, MF59 adjuvant, CRX-601 adjuvant, chitosan adjuvant, and aluminum hydroxide adjuvant.

[0050] The examination method is as follows:

[0051] 1) Take the prescribed amounts of FimH adjuvant, MF59 adjuvant, CRX-601 adjuvant, chitosan adjuvant, and aluminum hydroxide adjuvant for later use;

[0052] 2) The ratio of adjuvant-containing influenza vaccine to tablet matrix material was fixed, and the corresponding sublingual rapidly disintegrating influenza vaccine tablets were prepared according to the preparation method in Example 2. Mice were SLIT-immunized. Sublingual mucosa was isolated 0 or 2 hours after SLIT immunization, and cells were collected from the mucosa for analysis using flow cytometry. The increase in mDC and the trend of imDC conversion to mDC were used as indicators to evaluate sublingual mucosal immune tolerance. The experimental results are shown in Table 1.

[0053] Table 1. Effects of different adjuvants on mDC (mean ± SD, n = 3)

[0054] experimental group mDC increment (%) The percentage of imDC to mDC conversion. Vaccine Group 1.03±0.24 <0.5 MF59 adjuvant group 1.41±0.18 <0.5 <![CDATA[Al(OH)3 adjuvant 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

[0055] From Table 1, we can see that:

[0056] 1) In terms of mDC increment, the FimH adjuvant group achieved a 12-fold increase in growth rate compared to the blank vaccine group, which was much higher than the other adjuvant groups.

[0057] 2) In the statistics of the amount of imDC to mDC conversion, except for the FimH group, the conversion amount of the other groups was less than 0.5%, while the FimH adjuvant group reached a value of about 5.37%, which far exceeded the other groups.

[0058] The reasons are analyzed as follows: ① MF59, as a commonly used adjuvant in injections, does not have a good effect when added to sublingual preparations, indicating that the effect of adjuvants varies for different administration sites; ② The FimH adjuvant group showed the best effect in increasing mDC and converting imDC to mDC, indicating that FimH, as a mucosal adjuvant, can effectively induce dendritic cell maturation and antigen presentation.

[0059] Example 4: Effect of adjuvant content on the immunogenicity of influenza vaccine

[0060] The purpose of this embodiment is to investigate the effect of adjuvant content on sublingual mucosal immune tolerance. Based on Examples 2 and 3, the content of FimH adjuvant was changed for comparative study.

[0061] The examination method is as follows:

[0062] 1) The adjuvant selected is FimH, with contents of 5%, 10%, 15%, 20%, and 25% respectively, for later use;

[0063] 2) The components of the adjuvant-containing influenza vaccine and tablet matrix material were fixed, and the corresponding sublingually disintegrating influenza vaccine tablets were prepared according to the preparation method in Example 2. Standard female BALB / c mice (6-8 weeks old) were immunized with SLIT. Three mice were in each group, vaccinated once on day 1 and again on day 21. Tail blood was collected at 7, 14, 21, 28, 35, 42, and 49 days after the first immunization, and serum was separated to determine the hemagglutination inhibition titer. The results are shown in […]. Figure 3 .

[0064] from Figure 3 It can be known that:

[0065] 1) When the adjuvant content is 5%, the potency reaches its peak on day 35 and is at its lowest during the test period.

[0066] 2) When the adjuvant content was 10%, the potency level was significantly higher than that at 5%, and reached a peak of about 1500 on day 42, but then declined rapidly.

[0067] 3) When the adjuvant content was 20% and 25%, both peaked on day 35. However, they then declined rapidly and were also lower than the peak at a content of 10%.

[0068] 4) When the adjuvant content was 15%, there was a slight difference compared to the optimal potency group before day 35. After day 35, the potency rapidly increased, reaching an optimal potency value of over 2000 on day 42. Subsequently, the downward trend stabilized.

[0069] The analysis results are as follows: the optimal potency is achieved when the adjuvant content is 15%.

[0070] Example 5: Effect of adjuvant content on sublingual mucosal immune tolerance

[0071] The purpose of this embodiment is to investigate the effect of adjuvant content on sublingual mucosal immune tolerance. Based on Examples 2 and 3, the content of FimH adjuvant was changed for comparative study.

[0072] The examination method is as follows:

[0073] 1) The adjuvant selected is FimH, with contents of 5%, 10%, 15%, 20%, and 25% respectively, for later use;

[0074] 2) The components of the adjuvant-containing influenza vaccine and tablet matrix material were fixed, and the corresponding sublingual rapid-disintegrating influenza vaccine tablets were prepared according to the preparation method of Example 2. Mice were SLIT-immunized. Sublingual mucosa was isolated 0 or 2 hours after SLIT immunization, and cells were collected from the mucosa for analysis using flow cytometry. The increase in mDC and the trend of imDC conversion to mDC were used as indicators to evaluate sublingual mucosal immune tolerance. The experimental results are shown in Table 2.

[0075] Table 2. Effects of different adjuvants on mDC (mean ± SD, n = 3)

[0076] content mDC increment (%) The percentage of imDC to mDC conversion. 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

[0077] As can be seen from Table 2:

[0078] 1) When the adjuvant content was 5%, the mDC increase was only about 7.62%, which was the worst compared to the other four groups.

[0079] 2) When the adjuvant content was 10%, the mDC increment was more than 1.6 times that of the 5% content group, showing a corresponding improvement, but it was still worse than the 20% and 25% groups.

[0080] 3) When the adjuvant content was 15% and 20%, the optimal increase in mDC was achieved, and the difference was not significant. The increase in the 25% content group was not as large as the first two groups.

[0081] 4) When the adjuvant content was 15% and 20%, the amount of imDC converted to mDC was about 8.20%, which was the best effect compared with the other groups.

[0082] The analysis results are as follows: the optimal dendritic cell maturation rate was observed when the adjuvant content was 15% or 20%. However, considering the effect of Example 4 on immunogenicity, it was found that only when the adjuvant content was 15% could both the immunogenicity of the influenza vaccine be improved and immune tolerance be avoided.

[0083] In summary, the optimal composition and weight ratio of each component in the sublingual disintegrating influenza vaccine tablets are as follows: influenza vaccine 1.5%, FimH adjuvant 15%, sucrose 55%, croscarmellose sodium 10%, microcrystalline cellulose 18%, and micronized silica gel 0.5%.

Claims

1. A sublingually disintegrating influenza vaccine tablet, characterized in that, The components and their mass percentages in the sublingual rapid-disintegrating tablets are as follows: influenza vaccine 1.5%, FimH adjuvant 15%, sucrose 55%, croscarmellose sodium 10%, microcrystalline cellulose 18%, and micronized silica gel 0.5%.

2. The sublingual rapidly disintegrating influenza vaccine tablet according to claim 1, characterized in that, The influenza vaccine mentioned targets both influenza A and B viruses.

3. The sublingual rapidly disintegrating influenza vaccine tablet according to claim 1 or 2, characterized in that, The influenza vaccine mentioned is selected from one of the following: trivalent and quadrivalent split vaccines, subunit vaccines, whole virus vaccines, and live attenuated vaccines.

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