Application of methyltransferase inhibitors as adjuvants for respiratory syncytial virus vaccines and allergy vaccines

Abstract

This invention relates to the application of methyltransferase inhibitors as adjuvants for respiratory syncytial virus (RSV) vaccines and allergy vaccines. Methyltransferase inhibitors, as adjuvants for RSV vaccines or allergy vaccines, can target and regulate the antigen presentation function of vaccine-induced memory dendritic cells (DCs), thereby regulating the balance of T cell responses and balancing the dominant Th2 and / or Th17 cellular immune responses induced by RSV vaccines or allergy vaccines. This allows the vaccine recipient to develop a balanced immune memory, providing protection while preventing the occurrence of vaccine-enhanced inflammatory diseases when RSV infection or allergen stimulation occurs, thus being both safe and effective.

Description

Technical Field

[0001] This invention relates to the field of vaccine and adjuvant technology, specifically to the application of methyltransferase inhibitors as adjuvants for respiratory syncytial virus vaccines and allergy vaccines. Background Technology

[0002] Respiratory syncytial virus (RSV) can cause lower respiratory tract infections in infants, the elderly, and immunocompromised patients, and is one of the important pathogens causing respiratory infections. Vaccines are the most effective means of controlling pathogen infection. However, in clinical trials of the formalin-inactivated respiratory syncytial virus (FI-RSV) vaccine developed in the 1960s, infants who had received the vaccine developed more severe pneumonia when they were naturally infected with RSV, resulting in two deaths—a vaccine-enhanced disease (VED). The deceased patients' lungs showed extensive inflammatory cell infiltration and a Th2 immune response. This event delayed the development of RSV vaccines by several decades, and safety issues, exemplified by VED, became the primary problem to be solved in RSV vaccine development.

[0003] Traditionally, only adaptive immunity is considered to possess immune memory. However, recent studies have shown that innate immune cells, such as macrophages (MΦ), natural killer cells (NK), and dendritic cells (DC), can produce enhanced or tolerant responses to the same or different stimuli after stimulation. This is known as innate immune memory. Its mechanism differs from the recombination and clonal expansion of immunoglobulin family genes in adaptive immunity. Instead, it relies on epigenetic modifications, transcriptional alterations, and metabolic reprogramming after stimulation to generate memory. Nevertheless, its role has already gained attention in the development of inflammatory diseases, tumors, and vaccines.

[0004] Adjuvants are an important component of vaccines, enhancing the body's immune response or altering the type of immune response. Through rational adjuvant design, the protective effect of vaccines can be enhanced, and the toxic side effects of vaccines can be suppressed by regulating the type of immune response, thereby improving their safety. Developing ideal RSV vaccine adjuvants to inhibit the occurrence of vaccine-induced vasculitis (VED) by regulating immune homeostasis is an important measure to address RSV vaccine safety issues; however, due to a lack of in-depth understanding of the mechanisms of action of VED and adjuvants, the development of novel adjuvants still mainly relies on traditional trial-and-error methods, lacking targeted approaches.

[0005] Allergy is a hypersensitive response of the immune system to harmless substances in the environment. Th2 lymphocytes and their secreted cytokines (IL-4, IL-5, IL-13, etc.) play a crucial role in the occurrence and development of allergic reactions. Allergy vaccines (such as house dust mite vaccines) are extracted from allergens, and their use may cause anaphylactic shock. Finding safe and effective adjuvants is both a hot topic and a challenge in this field.

[0006] Methyltransferase inhibitors, such as MTA, are nucleosides produced from S-adenosylmethionine (SAM) during polyamine synthesis. They are a normal component in the body and can inhibit methyltransferases.

[0007] In the current technology, there are no reports or studies on using methyltransferase inhibitors as adjuvants for RSV vaccines or allergy vaccines. Summary of the Invention

[0008] The purpose of this invention is to solve the above-mentioned technical problems and provide a safe and effective targeted adjuvant for RSV vaccines or allergy vaccines.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0010] This invention provides the application of methyltransferase inhibitors as adjuvants for RSV vaccines or allergy vaccines.

[0011] Furthermore, the RSV vaccine or allergy vaccine may or may not contain aluminum salt adjuvants;

[0012] Furthermore, the RSV vaccine is an inactivated vaccine or a subunit vaccine that induces a Th2 response, and the allergy vaccine is a vaccine based on an allergen that induces a Th2 response.

[0013] Furthermore, the subunit vaccine is a subunit vaccine containing a G protein.

[0014] Furthermore, the allergy vaccine is a house dust mite antigen or an egg albumin.

[0015] Furthermore, the methyltransferase inhibitor is MTA.

[0016] Furthermore, the application involves injecting methyltransferase inhibitors into the body before or simultaneously with the vaccine to induce balanced immune memory.

[0017] Furthermore, methyltransferase inhibitor adjuvants may be administered at the same or different times and via different routes as vaccines.

[0018] Furthermore, methyltransferase inhibitors are used as adjuvants in prophylactic or therapeutic RSV vaccines or allergy vaccines.

[0019] Methyltransferase inhibitors, such as MTA, are nucleosides produced from S-adenosylmethionine (SAM) during polyamine synthesis. They are a normal component in the body and can inhibit methyltransferases. As a co-adjuvant of FI-RSV (aluminum salt adjuvant), they can inhibit DC methylation, regulate T cell immune responses, prevent the occurrence of VED, and develop safe and reliable RSV vaccines.

[0020] The TH2 response induced by RSV subunit vaccines (such as those using adhesion protein G as an antigen) and allergy vaccines (such as house dust mite vaccines) is very similar to the TH2 response induced by FI-RSV. Furthermore, they can also lead to increased methylation of memory dendritic cells (DCs), and individuals immunized with these vaccines may exhibit an excessively strong inflammatory response upon challenge. MTA, as an adjuvant, holds promise for developing safe and effective RSV subunit vaccines and allergy vaccines.

[0021] The beneficial effects of adopting the above technical solution are as follows:

[0022] This invention provides the application of methyltransferase inhibitors as adjuvants for RSV vaccines or allergy vaccines. As adjuvants for RSV vaccines or allergy vaccines, methyltransferase inhibitors inhibit the methylation of dendritic cells (DCs), regulate T cell immune responses, and balance the dominant Th2 and / or Th17 cell immune responses induced by the vaccine. This allows the vaccine recipient to develop a balanced immune memory, providing protection while preventing the occurrence of vaccine-enhanced inflammatory diseases when infected by the corresponding pathogen or triggered by allergens, thus achieving both safety and effectiveness. Attached Figure Description

[0023] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0024] Figure 1 The changes in H&E staining and PAS staining of lung tissue in each group 4 days after RSV challenge were compared between BALB / c mice immunized with the RSV inactivated vaccine and methyltransferase inhibitor adjuvant MTA as described in Example 1 of this invention (the above are H&E×200, and the following are PAS×200).

[0025] Figure 2 The following is a statistical chart showing the changes in H&E staining and PAS staining of lung tissue in BALB / c mice immunized with the RSV inactivated vaccine plus methyltransferase inhibitor adjuvant MTA as described in Example 1 of this invention, 4 days after RSV challenge (*:). P <0.05,**: P <0.01, ***: P <0.001); A. Inflammation score; B. Mucus score;

[0026] Figure 3The RSV inactivated vaccine plus methyltransferase inhibitor adjuvant MTA described in this embodiment of the invention was used to immunize BALB / c mice, and the white blood cell count in the bronchoalveolar lavage fluid of each group was measured 4 days after RSV challenge (*: P <0.05,**: P <0.01, ***: P <0.001);

[0027] Figure 4 BALB / c mice were immunized with the RSV inactivated vaccine plus methyltransferase inhibitor adjuvant MTA as described in this embodiment of the invention. Four days after RSV challenge, the expression of RSV-N in the lung tissue of each group was detected by real-time quantitative PCR. P <0.05,**: P <0.01, ***: P <0.001);

[0028] Figure 5 BALB / c mice were immunized with the RSV inactivated vaccine plus methyltransferase inhibitor adjuvant MTA as described in this embodiment of the invention. Four days after RSV challenge, the expression of cytokine mRNA in lung tissue of each group was detected by real-time quantitative PCR. P <0.05,**: P <0.01, ***: P <0.001);

[0029] Figure 6 The allergy vaccine (ovalbumin OVA, house dust mite antigen HDM) + methyltransferase inhibitor adjuvant MTA described in this embodiment of the invention was used to immunize BALB / c mice. Four days after challenged with OVA and HDM, the changes in H&E staining and PAS staining of lung tissue in each group were analyzed (the above figures are H&E × 200, and the following figures are PAS × 200). (***: P <0.001);

[0030] Figure 7 The following is a statistical chart showing the changes in H&E staining and PAS staining of lung tissue in each group 4 days after challenged with OVA and HDM, respectively, using the allergy vaccine (ovalbumin OVA, house dust mite antigen HDM) + methyltransferase inhibitor adjuvant MTA described in this embodiment of the invention. P <0.001); A. Inflammation score; B. Mucus score;

[0031] Figure 8The allergy vaccine (ovalbumin OVA, house dust mite antigen HDM) + methyltransferase inhibitor adjuvant MTA described in this embodiment of the invention was used to immunize BALB / c mice, and the white blood cell counts of the bronchoalveolar lavage fluid of each group were performed 4 days after challenged by OVA and HDM, respectively; A, ovalbumin OVA; B, house dust mite antigen HDM;

[0032] Figure 9 BALB / c mice were immunized with the allergy vaccine (ovalbumin OVA, house dust mite antigen HDM) + methyltransferase inhibitor adjuvant MTA as described in this embodiment of the invention. Four days after challenged with OVA and HDM, the expression of cytokine mRNA in lung tissue of each group was detected by real-time quantitative PCR. P <0.05,**: P <0.01, ***: P <0.001); Detailed Implementation

[0033] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further explains the invention. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0034] Example 1

[0035] Research on the use of methyltransferase inhibitor MTA as an adjuvant for RSV inactivated vaccines: Experimental study on immunization of BALB / c mice with formalin-inactivated RSV vaccine (FI-RSV, aluminum salt adjuvant) + low (L), medium (M), and high (H) doses of MTA adjuvant.

[0036] 1.1 Grouping of BABL / c mice (6 mice / group):

[0037] ①PBS group;

[0038] ②FI-RSV group;

[0039] ③FI-RSV+L-MTA group;

[0040] ④FI-RSV+M-MTA group;

[0041] ⑤ FI-RSV+H-MTA group;

[0042] 1.2 Vaccination and RSV challenge protocol for BABL / c mice:

[0043]

[0044] Note: Intramuscular injection (IM)

[0045] BALB / c mice aged 6-8 weeks were collected and intramuscularly injected with the vaccine and adjuvant at days 0, 14, and 28. On day 42, the PBS group received PBS intranasally, while the other groups received RSV intranasally. On day 47, the mice were sacrificed and tissue samples were collected. White blood cell counts were performed on the bronchoalveolar lavage fluid, and lung tissue was collected, embedded in paraffin, sectioned, and stained with H&E and PAS. RNA was extracted from a portion of the lung tissue and detected by real-time quantitative PCR.

[0046] Statistical results analysis:

[0047] 1. The inflammatory pathology of lung tissue was significantly suppressed in the adjuvant group (e.g., Figure 1 and 2 )

[0048] H&E staining of lung tissue sections showed that the FI-RSV+L-MTA group had a small amount of inflammatory cell infiltration in the local bronchi, a small number of alveoli fused, and the inflammation score was significantly lower than that of the FI-RSV group. P <0.01, significantly higher than the PBS group ( P <0.01); the FI-RSV+M-MTA group showed a very small amount of inflammatory cell infiltration in the bronchi, with occasional alveolar fusion, and the inflammation score was significantly lower than that of the FI-RSV group ( P <0.001, with no significant difference from the PBS group ( P >0.05); the FI-RSV+H-MTA group showed a very small amount of inflammatory cell infiltration in the bronchi, with occasional alveolar fusion, and the inflammation score was significantly lower than that of the FI-RSV group ( P <0.001, with no significant difference from the PBS group ( P >0.05); there was no statistically significant difference in inflammation scores among the three groups: FI-RSV, FI-RSV+L-MTA, FI-RSV+M-MTA, and FI-RSV+H-MTA. P >0.05).

[0049] PAS staining of lung tissue pathological sections showed that: in the FI-RSV+L-MTA group, there was no obvious peribronchial mucus secretion, and the mucus secretion score was significantly lower than that in the FI-RSV group. P <0.001, with no significant difference from the PBS group ( P >0.05); the FI-RSV+M-MTA group had no obvious peribronchial mucus secretion, and the mucus secretion score was significantly lower than that of the FI-RSV group ( P <0.001, with no significant difference from the PBS group ( P >0.05); The FI-RSV+H-MTA group showed no significant peribronchial mucus secretion, and the mucus secretion score was significantly lower than that of the FI-RSV group ( P <0.001, with no significant difference compared to the PBS group (P >0.05); There was no statistically significant difference in mucus secretion scores among the three groups: FI-RSV+L-MTA, FI-RSV+M-MTA, and FI-RSV+H-MTA. P >0.05).

[0050] 2. The number of inflammatory cells in the bronchoalveolar lavage fluid was significantly reduced in the adjuvant group (e.g., Figure 3 )

[0051] The BALF white blood cell count in the FI-RSV+L-MTA group was significantly lower than that in the FI-RSV group. P <0.001, with no significant difference from the PBS group ( P >0.05); the BALF white blood cell count in the FI-RSV+M-MTA group was significantly lower than that in the FI-RSV group ( P <0.001, with no significant difference from the PBS group ( P >0.05); the BALF white blood cell count in the FI-RSV+H-MTA group was significantly lower than that in the FI-RSV group ( P <0.001, with no significant difference from the PBS group ( P >0.05); there was no statistically significant difference in BALF white blood cell count among the three groups: FI-RSV+L-MTA, FI-RSV+M-MTA, and FI-RSV+H-MTA. P >0.05).

[0052] 3. RSV replication in lung tissue was significantly reduced in the medium- and high-dose adjuvant groups (e.g., Figure 4 )

[0053] In the low, medium, and high dose MTA groups, the RSV viral load in lung tissue was significantly lower than that in the PBS group, and the viral load in the FI-RSV+M-MTA and FI-RSV+H-MTA groups was significantly lower than that in the FI-RSV+L-MTA group. The expression of the RSV housekeeping gene RSV-N in the lung tissue of mice in the FI-RSV+L-MTA group was significantly lower than that in the PBS group. P <0.01, significantly higher than the FI-RSV group ( P <0.01); The expression of RSV housekeeping gene RSV-N in the lung tissue of mice in the FI-RSV+M-MTA group was significantly lower than that in the PBS group ( P <0.001), which is also significantly lower than the FI-RSV group ( P <0.01); The expression of RSV housekeeping gene RSV-N in the lung tissue of mice in the FI-RSV+H-MTA group was significantly lower than that in the PBS group ( P <0.001), which is also significantly lower than the FI-RSV group ( P<0.01); There was no statistically significant difference in the expression level of the RSV housekeeping gene RSV-N in the lung tissue of mice between the FI-RSV+M-MTA group and the FI-RSV+H-MTA group. P >0.05).

[0054] 4. Balance of TH1 and TH2 responses in lung tissue of the medium- and high-dose adjuvant groups (e.g.) Figure 5 )

[0055] The mRNA levels of th1-type cytokine IFN-γ in lung tissues of the low, medium, and high-dose MTA groups were significantly lower than those in the PBS group, with the medium and high-dose groups showing better results. The th2-type cytokine levels in the FI-RSV+M-MTA and FI-RSV+H-MTA groups were significantly lower than those in the FI-RSV group, while there was no significant difference between the FI-RSV+L-MTA and FI-RSV groups. The th17-type cytokine IL-17 levels in the FI-RSV+L-MTA group were significantly lower than those in the FI-RSV group, while there was no significant difference between the FI-RSV+M-MTA and FI-RSV+H-MTA groups and the FI-RSV group.

[0056] There was no significant difference in the expression of Th1 cytokine IFN-γ mRNA in lung tissue between the FI-RSV+L-MTA group and the PBS and FI-RSV groups. P >0.05); Th2 cytokine IL-4 mRNA expression was significantly higher than that in the PBS group ( P <0.05, with no significant difference from the FI-RSV group ( P >0.05), IL-5 mRNA expression was significantly higher than that in the PBS group ( P <0.01, with no significant difference from the FI-RSV group ( P >0.05), IL-13 mRNA expression was significantly higher than that in the PBS group ( P <0.001, with no significant difference from the FI-RSV group ( P >0.05); Th17 cytokine IL-17 mRNA expression was significantly higher than that in the PBS group ( P <0.05%, significantly lower than the FI-RSV group ( P <0.05).

[0057] The expression of Th1 cytokine IFN-γ mRNA in lung tissue of the FI-RSV+M-MTA group was significantly lower than that of the PBS group. P <0.001), significantly lower than the FI-RSV group ( P <0.001), significantly lower than the FI-RSV+L-MTA group ( P<0.01); there was no significant difference in Th2 cytokine IL-4 mRNA expression between the PBS group and the PBS group ( P >0.05%, significantly lower than the FI-RSV group ( P <0.001), FI-RSV+L-MTA group ( P <0.05), IL-5 mRNA expression showed no significant difference compared to the PBS group and the FI-RSV group ( P >0.05, significantly lower than the FI-RSV+L-MTA group ( P <0.001), IL-13 mRNA expression showed no significant difference compared to the PBS group ( P >0.05%, significantly lower than the FI-RSV group ( P <0.01) and FI-RSV+L-MTA group ( P <0.01); there was no significant difference in Th17 cytokine IL-17 mRNA expression between the PBS group, FI-RSV group, and FI-RSV+L-MTA group. P >0.05).

[0058] The expression of Th1 cytokine IFN-γ mRNA in lung tissue of the FI-RSV+H-MTA group was significantly lower than that of the PBS group. P <0.001), significantly lower than the FI-RSV group ( P <0.01), significantly lower than the FI-RSV+L-MTA group ( P <0.01%, with no significant difference compared to the FI-RSV+M-MTA group; Th2 cytokine IL-4 mRNA expression showed no significant difference compared to the PBS group ( P >0.05%, significantly lower than the FI-RSV group ( P <0.001), FI-RSV+L-MTA group ( P <0.05, with no statistically significant difference compared to the FI-RSV+M-MTA group ( P >0.05), IL-5 mRNA expression was significantly higher than that in the PBS group ( P <0.01, with no significant difference from the FI-RSV group ( P >0.05, significantly lower than the FI-RSV+L-MTA group ( P <0.001),

[0059] There was no statistically significant difference between the FI-RSV+M-MTA group and the FI-RSV+M-MTA group. P >0.05), IL-13 mRNA expression showed no significant difference compared to the PBS group ( P >0.05%, significantly lower than the FI-RSV group ( P<0.01) and FI-RSV+L-MTA group ( P <0.01, with no statistically significant difference compared to the FI-RSV+M-MTA group ( P >0.05); Th17 cytokine IL-17 mRNA expression was significantly higher than that in the PBS group ( P <0.05), with no significant difference from other groups ( P >0.05).

[0060] All the above experimental results indicate that the methyltransferase inhibitor MTA, as an adjuvant for FI-RSV, can improve its protective effect and safety.

[0061] Example 2

[0062] Research on the use of methyltransferase inhibitor MTA as an adjuvant in allergy vaccines: An experimental study of immunizing BALB / c mice with ovalbumin OVA and house dust mite antigen HDM (containing aluminum salt adjuvant) + MTA adjuvant.

[0063] 1.1 Grouping of BABL / c mice (6 mice / group):

[0064] ①PBS group;

[0065] ②OVA group;

[0066] ③OVA+MTA group;

[0067] ④HDM group;

[0068] ⑤ HDM+MTA group;

[0069] 1.2 Vaccination and RSV challenge protocol for BABL / c mice:

[0070]

[0071] Note: Intramuscular injection (IM)

[0072] BALB / c mice aged 6-8 weeks were collected and intramuscularly injected with the vaccine and adjuvant at days 0, 14, and 28. On day 42, the PBS group was challenged by intranasal drops of PBS, while the other groups were challenged by intranasal drops of OVA or HDM. On day 47, the mice in each group were sacrificed and their tissues were collected. White blood cell counts were performed on the bronchoalveolar lavage fluid, and lung tissue was collected, embedded in paraffin, sectioned, and stained with H&E and PAS. RNA was extracted from a portion of the lung tissue and detected by real-time quantitative PCR.

[0073] Statistical Results Analysis

[0074] 1. The inflammatory pathology of lung tissue was significantly suppressed in the adjuvant group (e.g., Figure 6 and 7 )

[0075] H&E staining of lung tissue sections showed that the OVA+MTA group had a small amount of inflammatory cell infiltration in the local bronchi, a small number of alveoli fused, and the inflammation score was significantly lower than that of the OVA group. P <0.001); the HDM+MTA group had a very small amount of inflammatory cell infiltration in the bronchi, and occasionally alveolar fusion. The inflammation score was significantly lower than that of the HDM group. P <0.001); *** P <0.001

[0076] PAS staining of lung tissue sections showed that: significant peribronchial mucus secretion was observed in the OVA and HDM groups, while no significant peribronchial mucus secretion was observed in the OVA+MTA and HDM+MTA groups. The mucus secretion scores were significantly lower in the OVA and HDM groups than in the OVA and HDM groups. P <0.001).

[0077] 2. The number of inflammatory cells in the bronchoalveolar lavage fluid of the adjuvant group showed no significant change (e.g., Figure 8 )

[0078] There was no statistically significant difference in BALF white blood cell count between the OVA group and the OVA+MTA group, and between the HDM group and the HDM+MTA group. P >0.05); A, OVA; B, HDM.

[0079] 3. The lung tissue of the adjuvant group showed a more balanced TH1, TH2, and TH17 responses (e.g., Figure 9 )

[0080] The relative expression of cytokine mRNA in lung tissues of each group was detected by real-time quantitative PCR. There was no significant difference in the expression of Th1 cytokine IFN-γ among the groups. The OVA+MTA group showed significantly lower levels of Th2 cytokines IL-5 and Th17 cytokines IL-17 compared to the OVA group, while the HDM+MTA group showed a significantly lower level of Th17 cytokine IL-17 compared to the HDM group. P <0.05; ** P <0.01; *** P <0.001

[0081] 4. The expression of eosinophil and neutrophil chemokines in the lung tissue of the adjuvant group was decreased (e.g., Figure 9 )

[0082] The relative expression of cytokine mRNA in lung tissue of each group was detected by real-time quantitative PCR. The eotaxin and neutrophil chemokine GRO-α in eosinophils in the OVA+MTA group were significantly lower than those in the OVA group, and the eotaxin in eosinophils in the HDM+MTA group was significantly lower than that in the HDM group.

[0083] All the above experimental results indicate that methyltransferase inhibitors (MTA) can improve the protective effect and safety of allergy vaccines when used as adjuvants.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of methyltransferase inhibitor MTA in the preparation of RSV vaccine adjuvants containing aluminum salts, wherein the RSV vaccine is an inactivated vaccine or a subunit vaccine of RSV-G protein.

2. The application according to claim 1, characterized in that, The methyltransferase inhibitor MTA is administered via the same number of doses and route as the vaccine.

3. The application according to claim 1, characterized in that, The application involves simultaneously injecting the methyltransferase inhibitor MTA with the vaccine into the body to induce a balanced Th1 and Th2 immune memory.

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