Application of phosphatidylethanolamine as a metabolic adjuvant for respiratory syncytial virus vaccine

Abstract

The present invention relates to the technical field of vaccines and adjuvants, and specifically to the use of phosphatidylethanolamine as a metabolic adjuvant for respiratory syncytial virus (RSV) vaccines. Phosphatidylethanolamine, as a metabolic adjuvant for RSV vaccines, can modulate T cell immune responses, prevent the occurrence of VED, and generate balanced immune memory in vaccine recipients. When infected with RSV, it provides protection while preventing the occurrence of vaccine-enhanced inflammatory diseases, achieving both safety and effectiveness.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccines and adjuvants, and in particular to the application of phosphatidylethanolamine as a metabolic adjuvant for respiratory syncytial virus vaccines. Background Art

[0002] Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants, the elderly, and immunocompromised individuals. Vaccines are the most effective means of combating pathogen infections. However, clinical trials of the formalin-inactivated respiratory syncytial virus (FI-RSV) vaccine, developed in the 1960s, revealed that natural RSV infection in vaccinated infants and young children resulted in more severe pneumonia and two deaths, a condition known as vaccine-enhanced disease (VED). The lungs of the deceased individuals showed extensive inflammatory cell infiltration and a Th2 immune response. This incident delayed the development of RSV vaccines for decades, and safety concerns, particularly VED, have become a primary concern in RSV vaccine development.

[0003] Traditionally, only adaptive immunity has been considered to possess immune memory properties. Recently, it has been discovered that innate immune cells, such as macrophages (MΦ), natural killer cells (NK), and dendritic cells (DC), can, after stimulation, produce enhanced or tolerant responses to the same or different stimuli. 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 post-stimulation epigenetic modifications, transcriptional changes, and metabolic reprogramming to produce memory. However, its role has been recognized in the development of inflammatory diseases, tumors, and vaccines.

[0004] Adjuvants are crucial components of vaccines, enhancing or modifying the immune response to vaccines. Rational adjuvant design can enhance the protective efficacy of vaccines and improve safety by modulating the immune response, thereby suppressing toxic side effects. Developing ideal RSV vaccine adjuvants that regulate immune balance and inhibit VED is a crucial step in addressing RSV vaccine safety concerns. Traditional adjuvants are still primarily developed through trial-and-error methods and lack targeted responses. The development of novel adjuvants utilizes systems vaccinology, conducting mechanistic studies on inactivated RSV vaccines, and designing targeted adjuvants, such as metabolic adjuvants and signaling pathway adjuvants.

[0005] Based on the above background, the present application proposes that phosphatidylethanolamine can be used as a combined metabolic adjuvant of FI-RSV+aluminum salt adjuvant, in order to develop a safe and reliable RSV vaccine.

[0006] In the prior art, there are no reports or studies on using phosphatidylethanolamine as a metabolic adjuvant for RSV vaccines. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an application of phosphatidylethanolamine as a metabolic adjuvant for respiratory syncytial virus vaccine.

[0008] In order to solve the above problems, the technical solution adopted by the present invention is:

[0009] Technical Topic 1

[0010] Application of phosphatidylethanolamine as a metabolic adjuvant for RSV vaccines.

[0011] As a further improvement of the present invention, the RSV vaccine contains or does not contain an aluminum salt adjuvant.

[0012] As a further improvement of the present invention, the RSV vaccine is an inactivated RSV vaccine or a subunit RSV vaccine that induces a Th2 response.

[0013] As a further improvement of the present invention, the inactivated RSV vaccine is a formalin-inactivated RSV vaccine or a β-propiolactone-inactivated RSV vaccine.

[0014] As a further improvement of the present invention, the application is to inject phosphatidylethanolamine into the body in advance or simultaneously with the vaccine to induce balanced immune memory.

[0015] As a further improvement of the present invention, the application is that the phosphatidylethanolamine adjuvant and the vaccine are administered at the same or different times and routes.

[0016] The beneficial effects of adopting the above technical solution are:

[0017] The present invention provides the use of phosphatidylethanolamine as a metabolic adjuvant for respiratory syncytial virus (RSV) vaccines. Phosphatidylethanolamine, as a metabolic adjuvant for RSV vaccines, modulates T cell immune responses, prevents the development of VED, and generates balanced immune memory in recipients. When infected by the corresponding pathogen, it provides protection while preventing the development of vaccine-enhanced inflammatory diseases, achieving both safety and effectiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1Figures 1 and 2 show the changes in lung tissue pathology (H&E staining and PAS staining) of BALB / c mice treated with PBS, FI-RSV, and FI-RSV plus low (L, 2 μg / mouse), medium (M, 10 μg / mouse), and high (H, 50 μg / mouse) doses of PE, as described in Example 1 of the present invention, four days after intranasal instillation. In the figures, the upper column indicates H&E × 200, and the lower column indicates PAS × 200. FI-RSV is formalin-inactivated RSV vaccine (containing aluminum salt adjuvant); PE is phosphatidylethanolamine; L-PE is low-dose phosphatidylethanolamine; M-PE is medium-dose phosphatidylethanolamine; and H-PE is high-dose phosphatidylethanolamine.

[0019] Figure 2 BALB / c mice were treated with PBS, FI-RSV, and FI-RSV + low (L), medium (M), and high (H) doses of PE as described in Example 1 of the present invention. Statistical graphs of lung tissue pathological H&E staining and PAS staining changes in each group 4 days after intranasal instillation are shown. The left figure shows the inflammation score, and the right figure shows the mucus score. In the figure, FI-RSV is formalin-inactivated RSV vaccine (containing aluminum salt adjuvant); L-PE is low-dose phosphatidylethanolamine; M-PE is medium-dose phosphatidylethanolamine; H-PE is high-dose phosphatidylethanolamine (*: P <0.05,**: P <0.01,***: P <0.001);

[0020] Figure 3 BALB / c mice were treated with PBS, FI-RSV, and FI-RSV + low (L), medium (M), and high (H) doses of PE, as described in Example 1 of the present invention, and the white blood cell counts in the bronchoalveolar lavage fluid of each group were recorded 4 days after nasal instillation. In the figure, FI-RSV is formalin-inactivated RSV vaccine (containing aluminum salt adjuvant); L-PE is low-dose phosphatidylethanolamine; M-PE is medium-dose phosphatidylethanolamine; H-PE is high-dose phosphatidylethanolamine (*: P <0.05,**: P <0.01,***: P <0.001);

[0021] Figure 4 BALB / c mice were treated with PBS+RSV, FI-RSV, and FI-RSV+low (L), medium (M), and high (H) doses of PE as described in Example 1 of the present invention. Four days after RSV challenge, the expression of RSV-N in the lung tissues of each group was detected by real-time quantitative PCR. In the figure, FI-RSV is formalin-inactivated RSV vaccine (containing aluminum salt adjuvant); L-PE is low-dose phosphatidylethanolamine; M-PE is medium-dose phosphatidylethanolamine; H-PE is high-dose phosphatidylethanolamine (*: P <0.05,**:P <0.01,***: P <0.001);

[0022] Figure 5 BALB / c mice were treated with PBS, FI-RSV, and FI-RSV+low (L), medium (M), and high (H) doses of PE as described in Example 1 of the present invention. 4 days after intranasal instillation, the expression of cytokine mRNA in the lung tissue of each group was detected by real-time quantitative PCR. In the figure, FI-RSV is formalin-inactivated RSV vaccine (containing aluminum salt adjuvant); L-PE is low-dose phosphatidylethanolamine; M-PE is medium-dose phosphatidylethanolamine; H-PE is high-dose phosphatidylethanolamine (*: P <0.05,**: P <0.01,***: P <0.001). DETAILED DESCRIPTION

[0023] In order to make the objectives, technical solutions and advantages of the present invention more clear, the invention is clearly and completely described below in conjunction with specific embodiments.

[0024] The FI-RSV vaccine used in this application was prepared by the steps described in Preparation Example 1.

[0025] Preparation Example 1

[0026] (1) Inactivation: Take the virus solution prepared by RSV infection of Vero cells, add 5 μl of formalin (final dilution 1:4000) to the centrifuge tube containing the RSV virus solution, pipette and aspirate several times, and incubate at 37°C on a shaker for 72 hours. (Add 25 μl of 40% formaldehyde solution to every 100 ml of virus cell solution)

[0027] (2) Impurity removal: After 72 hours, remove the centrifuge tube and centrifuge at 4°C, 4100 rpm (1800 g) for 10 min; take the supernatant and proceed to step (3).

[0028] (3) Ultracentrifugation: Take the supernatant, centrifuge at 4°C, 49943g, for 60 min, retain the precipitate, and discard the supernatant.

[0029] (4) Ultracentrifugation: Resuspend the precipitate from step (3) with PBS and perform ultracentrifugation at 4°C, 49500g, for 60 min. Keep the precipitate and discard the supernatant.

[0030] (5) Vaccine Collection: Resuspend the precipitate from step (4) in PBS (1 / 25 of the original volume) and mix with aluminum hydroxide solution at a final concentration of 4 mg / ml. The volume of PBS used in this application is 4 ml, and the aluminum hydroxide solution has a concentration of 10 mg / ml and a volume of 2.67 ml. Vortex mix thoroughly and shake at 250 rpm at room temperature overnight. Then centrifuge at 2800 rpm, discard the supernatant, and resuspend the precipitate in 1 / 4 of the original volume of PBS. Aliquot into EP tubes (1 ml / tube) and store at -80°C until use.

[0031] Example 1 Study on Phosphatidylethanolamine as a Metabolic Adjuvant for RSV Inactivated Vaccine

[0032] Experimental study on immunization of BALB / c mice with formalin-inactivated RSV vaccine (FI-RSV, containing aluminum salt adjuvant) plus low (L, 2μg / mouse / dose), medium (M, 10μg / mouse / dose), and high (H, 50μg / mouse / dose) doses of phosphatidylethanolamine (hereinafter referred to as PE) adjuvant

[0033] 1.1 BABL / c mice grouping (6 mice / group):

[0034] ①PBS group;

[0035] ②PBS+RSV group;

[0036] ③FI-RSV group;

[0037] ④FI-RSV+L-PE group;

[0038] ⑤FI-RSV+M-PE group;

[0039] FI-RSV+H-PE group;

[0040] 1.2 Vaccination and RSV challenge protocol for BABL / c mice:

[0041]

[0042] 6-8 week old BALB / c mice were collected and intramuscularly inoculated with the vaccine and adjuvant of each group on days 0, 14, and 28, respectively. During the injection, the PE adjuvant was first injected intramuscularly, and the formalin-inactivated RSV vaccine (FI-RSV, containing aluminum salt adjuvant) was injected 15 minutes later. The injection volume of formalin-inactivated RSV vaccine was 50 μl / mouse / time. The group without vaccine or adjuvant injection was injected with an equal amount of PBS. On day 42, the PBS group was intranasally inoculated with PBS, and the other groups were intranasally inoculated with RSV. On day 47, the mice in each group were sacrificed and the white blood cell count of the alveolar lavage fluid was performed. The lung tissue was obtained, embedded in paraffin, sectioned, and stained with H&E and PAS. Part of the lung tissue was obtained, RNA was extracted, and real-time quantitative PCR was performed.

[0043] The results show

[0044] 1. The inflammatory pathology of lung tissue in the adjuvant group was significantly inhibited (e.g. Figure 1 and Figure 2 )

[0045] H&E staining of lung tissue pathological sections showed that there was a small amount of inflammatory cell infiltration in the bronchus of the FI-RSV+L-PE group, a small number of alveolar fusions, and the inflammation score was significantly lower than that of the FI-RSV group ( P <0.01), significantly higher than that of the PBS group ( P <0.01); there was a small amount of inflammatory cell infiltration in the bronchus of the FI-RSV+M-PE group, and occasional alveolar fusion was observed. The inflammation score was significantly lower than that of the FI-RSV group ( P <0.001), and there was no significant difference between the two groups ( P >0.05); there was a small amount of inflammatory cell infiltration in the bronchus of the FI-RSV+H-PE group, and occasional alveolar fusion was observed. The inflammation score was significantly lower than that of the FI-RSV group ( P <0.001), and there was no significant difference between the two groups ( P <0.01); there was no statistical difference in inflammation scores among the FI-RSV+L-PE group, FI-RSV+M-PE group, and FI-RSV+H-PE group ( P >0.05).

[0046] PAS staining of lung tissue pathological sections showed that there was no obvious mucus secretion around the bronchi in the FI-RSV+L-PE group, and the mucus secretion score was significantly lower than that in the FI-RSV group ( P <0.01), and there was no significant difference compared with the PBS group ( P >0.05); there was no obvious mucus secretion around the bronchi in the FI-RSV+M-PE group, and the mucus secretion score was significantly lower than that in the FI-RSV group ( P <0.001), and there was no significant difference between the PBS group and the FI-RSV+L-PE group (P >0.05); there was no obvious mucus secretion around the bronchi in the FI-RSV+PE group, and the mucus secretion score was significantly lower than that in the FI-RSV group ( P <0.001), and there was no statistical difference between the PBS group, FI-RSV+L-PE group, and FI-RSV+M-PE group ( P >0.05).

[0047] It was demonstrated that phosphatidylethanolamine as a metabolic adjuvant can significantly inhibit lung tissue inflammation and mucus secretion when RSV is reinfected after vaccination.

[0048] 2. The number of inflammatory cells in the alveolar lavage fluid of the adjuvant group was significantly reduced (e.g. Figure 3 )

[0049] The BALF white blood cell count in the FI-RSV+LP group was significantly lower than that in the FI-RSV group ( P <0.001), significantly higher than that of the PBS group ( P <0.01), the BALF white blood cell count in the FI-RSV+M-PE group was significantly lower than that in the FI-RSV group ( P <0.001), significantly higher than that of the PBS group ( P <0.01). The BALF white blood cell count in the FI-RSV+H-PE group was significantly lower than that in the FI-RSV group ( P <0.001), significantly higher than that of the PBS group ( P <0.01). There was no statistical difference among the FI-RSV+L-PE group, FI-RSV+M-PE group, and FI-RSV+H-PE group ( P >0.05).

[0050] It was demonstrated that phosphatidylethanolamine as a metabolic adjuvant could significantly inhibit BALF leukocytosis when RSV was reinfected after vaccination.

[0051] 3. PE as a metabolic adjuvant in the expression of RSV-N in mouse lung tissue (e.g. Figure 4 )

[0052] The RSV viral load in the lung tissue of the low, medium and high dose PE groups was significantly lower than that of the PBS group and FI-RSV group. The RSV housekeeping gene RSV-N expression in the lung tissue of the mice in the FI-RSV+H-PE group was significantly lower than that in the PBS group ( P <0.001) and FI-RSV group ( P <0.01).

[0053] It was demonstrated that phosphatidylethanolamine as a metabolic adjuvant can also significantly inhibit the proliferation of RSV in the lungs when RSV is reinfected after vaccination.

[0054] 4. The balance of Th1 and Th2 responses in lung tissues of the medium and high dose adjuvant groups ( Figure 5 )

[0055] In the lung tissue cytokine mRNA of the low, medium and high dose PE groups, the Th1 cytokine IFN-γ mRNA was significantly lower than that of the FI-RSV group and the PBS group, and the Th2 cytokine IL-4 mRNA expression was significantly lower than that of the FI-RSV group ( P <0.01), and there was no significant difference compared with the PBS group ( P >0.05), and IL-13 mRNA expression was significantly lower than that in the FI-RSV group (P<0.01), and had no significant difference with that in the PBS group ( P >0.05); the expression of Th17 cytokine IL-17 mRNA in the FI-RSV+H-PE group was significantly lower than that in the FI-RSV group ( P <0.001), which had no statistical difference compared with the PBS group.

[0056] It was demonstrated that when RSV is re-infected after vaccination, phosphatidylethanolamine as a metabolic adjuvant can balance and regulate T cell immune responses, prevent the occurrence of VED, and produce balanced immune memory in vaccinated mice.

[0057] All the above experimental results show that PE as a metabolic adjuvant of FI-RSV can improve its protection and safety.

[0058] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements 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 use of phosphatidylethanolamine in the preparation of a metabolic adjuvant for respiratory syncytial virus vaccine, characterized in that: The respiratory syncytial virus vaccine is a formalin-inactivated respiratory syncytial virus vaccine; the adjuvant is injected into the body before the vaccine is injected.

2. The use according to claim 1, characterized in that The respiratory syncytial virus vaccine contains an aluminum salt adjuvant.

3. The use according to claim 1, characterized in that The application is that the phosphatidylethanolamine metabolic adjuvant is administered with the same or different times and routes as the vaccine.

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