LNP-mRNA combined vaccine of respiratory syncytial virus and varicella-zoster virus and application of LNP-mRNA combined vaccine

Abstract

The invention belongs to the technical field of biological medicine, and provides an LNP-mRNA combined vaccine of respiratory syncytial virus and varicella-zoster virus and application of the LNP-mRNA combined vaccine, the combined vaccine comprises mRNA for coding RSVPre-F and mRNA for coding VZV gE-M which are mixed according to a certain proportion, and the mRNA is wrapped by lipid nanoparticles. The combined vaccine has immunogenicity and specificity aiming at the respiratory syncytial virus (RSV) and the varicella-zoster virus (VZV), and can effectively remove the viruses and play a comprehensive immune function. According to the invention, the blank in the field of RSV / VZV combined LNP-mRNA vaccines is filled up, and the clinical actual demand is powerfully met.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and provides an LNP-mRNA combined vaccine of respiratory syncytial virus and varicella-zoster virus and application thereof. Background Art

[0002] Respiratory syncytial virus (RSV) is the leading pathogen causing severe lower respiratory tract disease (LRTD) in infants, young children, and the elderly, posing a major threat to public health. Relevant data show that approximately 336,000 people are hospitalized each year due to RSV infection, of which 14,000 die in the hospital. The burden of LRTD caused by RSV is particularly significant in people aged 65 and above. In addition, due to insufficient detection frequency, the actual incidence of RSV in the elderly and the impact it causes are likely to be underestimated.

[0003] After the varicella-zoster virus (VZV) first infects the human body, it remains dormant in nerve ganglia. Later, the virus can cause herpes zoster (HZ) and, especially in elderly immunocompromised individuals, postherpetic neuralgia (PHN). In the United States, it is estimated that one-third of people will experience HZ in their lifetime, and the incidence of HZ shows a clear upward trend after the age of 50.

[0004] For the elderly, current treatment options for RSV-related LRTD and VZV-related PHN are extremely limited, which urgently requires the development of effective VZV and RSV vaccines. In terms of vaccine development, RSV vaccines and VZV vaccines have many similarities. On the one hand, the elderly and immunocompromised individuals are susceptible to these two viruses, and vaccination is of great significance to them; on the other hand, cell-mediated immunity (CMI) is highly valued in the research and development of these two types of vaccines. Compared with traditional vaccines, both RSV and VZV vaccines require the induction of CMI, and mRNA can induce strong T cell responses, and its immune properties are consistent with this requirement, making the mRNA platform an ideal candidate for the development of VZV and RSV vaccines. On the market, the existing VZV vaccine products are Shingrix developed by GlaxoSmithKline (GSK). TM This is a subunit vaccine. Clinical trial results show that Shingrix TMIn individuals aged 50 to 59 and over 70, it can provide a protective efficacy of over 90%, while significantly reducing the risk of PHN. In contrast, mRNA-1468 developed by Moderna has a short gE sequence and shows good immunogenicity whether 100 - 200 micrograms of mRNA is injected once or 50 micrograms of mRNA is injected twice. Another study shows that in mouse and macaque experiments, the VZV mRNA vaccine ZOSAL induces better vaccine immunity than Shingrix TM .

[0005] In the field of RSV vaccines, there are currently three products on the market, namely Arexvy from GSK, Abrysvo from Pfizer, and mRNA-1345 from Moderna. Among them, the first two are subunit vaccines, while mRNA-1345 is an mRNA vaccine. In addition, there are many RSV mRNA vaccines in the preclinical trial stage, and some are combination vaccines. For example, Sanofi's SP0256 is an RSV-hMPV-PIV combination vaccine undergoing Phase 1 clinical evaluation; Innorna's IN006 is a bivalent RSV mRNA vaccine encoding two F proteins (RSV-A and RSV-B) with a stable pre-fusion conformation, and it is the first RSV mRNA vaccine approved for clinical trials in China

[0006] The fusion glycoprotein (F) of RSV plays a key role in virus fusion, budding, and transmission. Recent studies have confirmed that the stable pre-fusion (pre-F) conformation, as an extremely superior antigen, can stimulate a strong immune response. Similarly, glycoprotein E (gE), as one of the most abundant glycoproteins in VZV, plays an important role in virus replication and transmission between ganglion cells. After mutating the C-terminus of gE to Y569A, S593A, S595A, T596A, and T598A (gE-M), it has been confirmed that gE can be localized to the trans-Golgi network (TGN) or the cell membrane. Previous studies have shown that compared with other forms of gE, gE-M can induce more superior immunogenicity in VZV mRNA vaccines. Although certain progress has been made in the development of RSV and VZV vaccines, there is currently no LNP-mRNA vaccine that combines the two. Developing a combination vaccine is expected to prevent infections by both viruses simultaneously, reduce the number of vaccinations, and improve vaccination efficiency, which is of great significance for susceptible populations Summary of the Invention

[0007] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide an LNP-mRNA combined vaccine against respiratory syncytial virus and varicella-zoster virus, and its application. This vaccine simultaneously has the immunogenicity, specificity and virus-clearing immune functions of respiratory syncytial virus (RSV) and varicella-zoster virus (VZV), fills the blank in the field of RSV / VZV combined LNP-mRNA vaccine, and meets the clinical needs.

[0008] The purpose of the present invention is achieved by the following technical solutions:

[0009] The first aspect of the present invention provides an LNP-mRNA combined vaccine for preventing and / or treating respiratory syncytial virus and varicella-zoster virus. Among them, the combined vaccine contains mRNA encoding RSV Pre-F and mRNA encoding VZV gE-M mixed in a certain proportion, and the mRNA is encapsulated by lipid nanoparticles (Lipid Nanoparticle, LNP).

[0010]

[0011]

[0012] Furthermore, in the combined vaccine, the ratio of the mRNA encoding RSV Pre-F to the mRNA encoding VZV gE-M is 1:0.5-2.

[0013] Furthermore, in the combined vaccine, the mRNA encoding RSV Pre-F and the mRNA encoding VZV gE-M are respectively encapsulated in lipid nanoparticles to form RSV Pre-F mRNA-LNP and VZV gE-M mRNA-LNP, and the two are mixed to obtain the combined vaccine.

[0014] Furthermore, in the combined vaccine, after the mRNA encoding RSV Pre-F and the mRNA encoding VZV gE-M are mixed, they are simultaneously encapsulated in lipid nanoparticles to obtain the combined vaccine.

[0015] Furthermore, the combined vaccine is in the form of an injection formulation, and the administration methods include intramuscular, subcutaneous or intradermal injection.

[0016] The second aspect of the present invention provides the use of the LNP-mRNA combined vaccine described in the first aspect in the preparation of a vaccine for inducing a cellular immune response and a humoral immune response against RSV and / or VZV in a subject.

[0017] The third aspect of the present invention provides the use of the LNP-mRNA combined vaccine described in the first aspect in the preparation of a vaccine for preventing RSV and / or VZV infection.

[0018] The beneficial effects of the present invention compared with the prior art are as follows:

[0019] 1. The LNP-mRNA combined vaccine of respiratory syncytial virus and varicella-zoster virus described in the present application has immunogenicity and specificity against respiratory syncytial virus (RSV) and varicella-zoster virus (VZV), can effectively clear the virus, and exerts a comprehensive immune function. The present invention fills the gap in the field of RSV / VZV combined LNP-mRNA vaccines and effectively meets the actual clinical needs;

[0020] 2. The combined vaccine described in the present application can reduce the pain caused by injection to patients and reduce other inconveniences caused by the injection frequency. Injecting a single dose can achieve the effect of simultaneously preventing the invasion of both RSV / VZV viruses. Description of the Drawings

[0021] The present invention will be further described below with reference to the drawings and embodiments:

[0022] Figure 1Shows the comparison charts of the detection results of different LNP-mRNAs in Examples 1 and 2; among them, A is the stability detection, B is the particle size, C is the dispersion coefficient (PDI), and D is the encapsulation efficiency;

[0023] Figure 2 Shows the IgG responses of each antigen in the mice and the detection results of neutralizing antibodies in the test examples; among them, A: RSV Pre-F specific IgG antibody titer results; B: Serum neutralizing antibody detection results of RSV A2 strain; C: VZV gE-M specific IgG antibody titer results;

[0024] Figure 3 Shows the ELISA detection results described in the test examples; among them, A and B are the concentrations of specific IL-2 and IFN-γ produced by splenocytes after induction with RSV Pre-F antigen respectively; C and D are the concentrations of specific IL-2 and IFN-γ produced by splenocytes after induction with VZV gE-M antigen respectively;

[0025] Figure 4 Shows the ELISPOT detection results described in the test examples; among them, A and C are the numbers of specific IL-2 and IFN-γ spots produced by splenocytes after induction with RSV Pre-F antigen and the photos of each well respectively; B and D are the numbers of specific IL-2 and IFN-γ spots produced by splenocytes after induction with VZV gE-M antigen and the photos of each well respectively;

[0026] Figure 5 Shows the flow cytometry detection results described in the test examples; among them, A and B are the proportions of CD4+ T cells producing IL-2 and IFN-γ in mouse splenocytes after induction with RSV Pre-F antigen respectively; C and D are the proportions of CD4+ T cells producing IL-2 and IFN-γ in mouse splenocytes after induction with VZV gE-M antigen respectively. Detailed implementation manners

[0027] The examples given are for better illustration of the present invention, but the content of the present invention is not limited only to the examples given. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation manners based on the above invention content still fall within the protection scope of the present invention.

[0028] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0029] The present invention will be described in detail below through examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention in an exemplary manner, and are not used to limit the present invention.

[0030] Example 1: Preparation of the combined vaccine Rv+Vv

[0031] This example provides an LNP-mRNA combined vaccine Rv+Vv for preventing and / or treating respiratory syncytial virus and varicella-zoster virus. The specific preparation method is as follows:

[0032] (1) In vitro, the synthesized DNA sequence is transcribed into mRNA to obtain mRNA encoding RSV Pre-F and mRNA encoding VZVgE-M respectively. After purification by magnetic beads, the mRNA concentration is measured. Among them, the nucleotide sequence encoding the RSV Pre-F antigen is shown in SEQ ID NO.1, and the nucleotide sequence encoding the VZV gE-M antigen is shown in SEQ ID NO.2.

[0033] (2) The mRNA encoding RSV Pre-F and the mRNA encoding VZV gE-M are respectively encapsulated in lipid nanoparticles LNP. The volume ratio of mRNA to LNP is 1:3 to form RSVPre-FmRNA-LNP (Rv) and VZV gE-M mRNA-LNP (Vv) respectively. The two are mixed in a ratio of 1:1 to obtain the combined vaccine Rv+Vv.

[0034] In the single vaccines Rv and Vv, the content of mRNA is 6 μg per dose. In the combined vaccine Rv+Vv, the content of mRNA is 12 μg per dose.

[0035] The stability, particle size, polydispersity index (PDI) and encapsulation efficiency of the single vaccines Rv and Vv nanoparticles are measured respectively. The test results are as Figure 1 shown.

[0036] Figure 1In the nucleic acid electrophoresis map of A, bands 1 and 2 are the reverse transcription products of RSV and VZV mRNA respectively. The bands are about 1500bp and 2000bp respectively, which can match the base lengths of RSV and VZV. Band 3 is a commercial Marker, and bands 4 / 5 are the LNP-mRNA of the completed Rv and Vv groups respectively. Nucleic acid electrophoresis cannot disperse its bands, indicating good integrity of LNP-mRNA. Bands 7 / 8 are the products after the LNP-mRNA particles of Rv and Vv are lysed by 1% Triton. It shows that after the LNP particles are lysed, the RNA bands can be located at positions matching bands 1 and 2 respectively, indicating successful encapsulation of mRNA by LNP. The diameters of LNP-mRNA in the Rv and Vv groups are 85.10±0.82nm and 85.56±0.69nm respectively( Figure 1 in B). The PDI of the nanoparticles in the Rv and Vv groups is about 0.15, which can prove that the formed particles are very uniform( Figure 1 in C). The nucleic acid encapsulation rates of the Rv and Vv group vaccines are 85.2% and 82.1% respectively( Figure 1 in D).

[0037] Example 2: Preparation of combined vaccine (R+V)v

[0038] This example provides another form of LNP-mRNA combined vaccine (R+V)v for preventing and / or treating respiratory syncytial virus and varicella-zoster virus. The specific preparation method is as follows:

[0039] (1) In vitro, the synthesized DNA sequence is transcribed into mRNA to obtain mRNA encoding RSV Pre-F and mRNA encoding VZVgE-M respectively. After purification by magnetic beads, the mRNA concentration is measured. Among them, the nucleotide sequence encoding the RSV Pre-F antigen is shown in SEQ ID NO.1, and the nucleotide sequence encoding the VZV gE-M antigen is shown in SEQ ID NO.2.

[0040] (2) The mRNA encoding RSVPre-F and the mRNA encoding VZV gE-M are mixed at a ratio of 1:1 and simultaneously encapsulated in lipid nanoparticles LNP. The volume ratio of mRNA to LNP particles is 1:3 to obtain the combined vaccine (R+V)v. In the combined vaccine (R+V)v, the content of mRNA is 12μg / per injection.

[0041] The stability, particle size, dispersion coefficient (PDI) and encapsulation rate of the combined vaccine (R+V)v nanoparticles are shown as Figure 1 shown.

[0042] In Figure 1In A, band 6 is the LNP-mRNA of the constructed (R+V)v group. Nucleic acid electrophoresis cannot disperse its band, indicating good integrity of the LNP-mRNA. Band 9 is the product after the LNP-mRNA particles of the (R+V)v group are lysed with 1% Triton. After the LNP particles are lysed, the RNA bands of RSV and VZV encapsulated therein can be located at positions matching bands 1 and 2 respectively, indicating that the LNP has successfully encapsulated the mRNAs of both RSV and VZV. The diameter of the LNP-mRNA of the (R+V)v group is 84.91±1.14nm( Figure 1 in B), and the PDI is about 0.15, which can prove that the formed particles are very uniform( Figure 1 in C), and the encapsulation efficiency of nucleic acid is 83.6%( Figure 1 in D).

[0043] Test example:

[0044] To compare the immune levels of the two combined vaccines (Rv+Vv, (R+V)v) and the single vaccines (Rv, Vv) described in Comparative Example 1 and Example 2, the following animal experiment was conducted in this test example.

[0045] Experimental animals: 6-8-week-old pathogen-free female BALB / c mice provided by the Experimental Center of the Institute of Medical Biology, Chinese Academy of Medical Sciences. The mice were randomly divided into 5 groups, with 6 mice in each group, and were raised under SPF conditions with free access to food and water.

[0046] Take the LNP-mRNA vaccines prepared in Example 1 and Example 2, and immunize the mice by intramuscular injection, 50 μL / mouse. The blank group was injected with an equal amount of PBS. Four weeks later, the second dose of the vaccine was injected intramuscularly. Two weeks after the last immunization, the mice were anesthetized by intraperitoneal injection of tribromoethanol, and then blood was collected by cardiac puncture and the spleen was collected. The blood was placed at 4°C overnight and then centrifuged at 3000 rpm for 20 minutes to obtain serum. A single-cell suspension of splenocytes was obtained by grinding the spleen.

[0047] The antibody titers specific for RSVpre-F and VZV gE-M were detected by indirect enzyme-linked immunosorbent assay. As Figure 2As shown in A, after two immunizations, the average titers of Pre-F specific IgG antibodies in the sera of mice in the Rv, Rv+Vv, and (R+V)v groups were 256000, 138667, and 170667, respectively. In the combined vaccine groups, including Rv+Vv and (R+V)v, although the antibody titers were slightly lower than those in the single vaccine group (Rv), there was no significant difference. The average titers of gE-M specific IgG antibodies in the sera of mice in the Vv, Rv+Vv, and (R+V)v groups were 256000, 277333, and 256000, respectively. The two combined vaccines, Rv+Vv and (R+V)v, could induce antibody titers that were not significantly different from those in the single vaccine group.

[0048] The mouse sera were inactivated at 56 °C for 30 minutes. The inactivated sera were first diluted 50-fold and then serially diluted at a ratio of 1:4. The sera were added to the RSVA2 virus strain cultured to the logarithmic phase and incubated at 37 °C for 1 hour. After incubation, 10000 Hep-2 cells were added to each well of the plate with the virus and serum mixture, and co-cultured at 37 °C and 5% CO2 for 4 - 7 days, and the cytopathic effect was observed daily.

[0049] As Figure 2 As shown in B, the neutralizing antibody titers were consistent with the above IgG antibody titer detection results. There was no significant difference between the combined vaccine groups Rv+Vv and (R+V)v and the single vaccine group Rv. Among them, the neutralizing antibody titer in the (R+V)v group was the highest, with an average value of 3597.

[0050] The double antibody sandwich ELISA method was used to detect the amounts of extracellularly secreted cytokines IL-2 and IFN-γ. A single spleen cell suspension was obtained by grinding the mouse spleen, and the spleen cell concentration was adjusted to 1×10 7 cells / mL by adding RPMI 1640 medium. 100 μL of spleen cells (1×10 6 cells per well) were added to a 96-well cell culture plate, and the corresponding stimulants were added to each group. RSV Pre-F protein was added to the cells in the Rv, Rv+Vv, and (R+V)v groups, and VZV gE-M protein was added to the cells in the Vv, Rv+Vv, and (R+V)v groups, with a final working concentration of 10 μg / mL. After culturing the cell plate at 37 °C and 5% CO2 for 24 hours, the supernatant was collected, and the cytokine levels were detected by the standard ELISA method.

[0051] The number of IL-2 and IFN-γ cytokines was detected using an ELISPOT-specific culture plate. The culture plate was activated with 75% ethanol and then washed twice with PBS to remove residual ethanol. Specific IL-2 and IFN-γ primary antibodies at a concentration of 2 μg / mL were added, 50 μL per well, and incubated overnight at 4°C. The next day, the antibody was aspirated, and after washing with complete 1640 medium, fresh complete 1640 medium was added and incubated for 2 hours to block unbound sites. After removing the 1640 medium, 30 μL of cells (3×10 5 spleen cells per well) were added to each well, supplemented with 75 μL of ELISOPT-specific serum-free medium and 50 μL of protein stimulator. RSV Pre-F protein was added to the cells in the Rv, Rv+Vv, and (R+V)v groups, and VZV gE-M protein was added to the cells in the Vv, Rv+Vv, and (R+V)v groups, with a final working concentration of 20 μg / mL for both. The culture plate was incubated overnight at 37°C and 5% CO2. On the third day, after centrifuging at 800 g for 5 minutes, the cells were washed with pre-cooled distilled water and PBS, and then IL-2 and IFN-γ secondary antibodies (1 μg / mL), 50 μL per well, were added and incubated at room temperature for 2 hours. After washing, HRP-Streptavidin (1:1500) was added and incubated at room temperature for 1 hour. After washing with PBS, an ELISPOT detection kit was used to visualize the spots, and the reaction was stopped by rinsing with running water.

[0052] The results of ELISA and ELISPOT are shown in Figure 3 and 4 As shown, both combined vaccines can induce the intensity of IL-2 and IFN-γ cellular immune responses similar to those of the individual vaccines.

[0053] Flow cytometry was used to detect the number of CD4+ T cells that can secrete IL-2 and IFN-γ. The treated spleen cells (1×10 6 cells per well) and protein stimulator (30 μg / mL) were added to a 24-well plate. Among them, the cells in the Rv, Rv+Vv, and (R+V)v groups were stimulated with RSV Pre-F protein, and the cells in the Vv, Rv+Vv, and (R+V)v groups were stimulated with VZV gE-M protein. After incubating the plate at 37°C for 2 hours, Brefeldin A was added to block cytokine secretion and incubated overnight. The next day, the cell suspension was transferred to a 1.5 mL centrifuge tube, and 100 μL of Zombie NIR containing DMSO was added TMThe dye (diluted 1:2000) was incubated for 15 minutes at room temperature in the dark. After washing twice with staining buffer, 50 μL of staining buffer containing 5 μg / mL of CD16 / CD32 antibody was added and incubated for 10 minutes at 4 °C. Then, the surface staining antibodies diluted with 50 μL of staining buffer, the PerCP / Cyanine 5.5-labeled anti-mouse CD4 antibody, was added and incubated for 30 minutes at 4 °C. After washing with staining buffer, the cells were fixed with 4% formaldehyde and incubated for 20 minutes at room temperature in the dark to fix the cell membrane. After washing twice with permeabilization wash buffer and incubating for 5 minutes, 100 μL of intracellular antibodies diluted with permeabilization wash buffer, including PE-conjugated anti-mouse IFN-γ and APC-conjugated anti-mouse IL-2 antibodies, were added for intracellular staining. The cells were incubated for 40 - 60 minutes at room temperature in the dark. Finally, after adding 400 μL of permeabilization wash buffer for washing, 400 μL was added to resuspend the cells.

[0054] Figure 5 The results showed that there was no significant difference in the ability of CD4+ T cells capable of producing IL-2 and IFN-γ in the splenocytes of mice inoculated with the combined vaccines Rv+Vv and (R+V)v and those inoculated with the single vaccines Vv or Rv.

[0055] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention and not to limit it. Although the present invention has been described in detail with reference to the preferred arrangement, those of ordinary skill in the art should understand that the technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A LNP-mRNA combined vaccine for the prevention and / or treatment of respiratory syncytial virus and varicella-zoster virus, characterized in that: The combined vaccine comprises mRNA encoding RSVPre-F and mRNA encoding VZV gE-M mixed in a certain ratio, and the mRNA is encapsulated by lipid nanoparticles.

2. The LNP-mRNA combined vaccine according to claim 1, characterized in that The nucleotide sequence encoding RSV Pre-F antigen is shown in SEQ ID NO.1, and the nucleotide sequence encoding VZV gE-M antigen is shown in SEQ ID NO.

2.

3. The LNP-mRNA combined vaccine according to claim 1, characterized in that The ratio of the mRNA encoding RSVPre-F and the mRNA encoding VZV gE-M in the combined vaccine is 1:0.5-2.

4. The LNP-mRNA combined vaccine according to any one of claims 1 to 3, characterized in that In the combined vaccine, the mRNA encoding RSV Pre-F and the mRNA encoding VZV gE-M are respectively encapsulated in lipid nanoparticles to form RSV Pre-F mRNA-LNP and VZV gE-M mRNA-LNP, which are mixed to obtain the combined vaccine.

5. The LNP-mRNA combined vaccine according to any one of claims 1 to 3, characterized in that In the combined vaccine, the mRNA encoding RSV Pre-F and the mRNA encoding VZV gE-M are mixed and simultaneously encapsulated in lipid nanoparticles to obtain the combined vaccine.

6. The LNP-mRNA combined vaccine according to any one of claims 1 to 3, characterized in that The combined vaccine is in the form of an injectable dosage form, and the administration method includes intramuscular, subcutaneous or intradermal injection.

7. Use of the LNP-mRNA combination vaccine according to any one of claims 1 to 6 in the preparation of a vaccine for inducing a cellular immune response or a humoral immune response against RSV and / or VZV in a subject.

8. Use of the LNP-mRNA combined vaccine according to any one of claims 1 to 6 in the preparation of a vaccine for preventing RSV and / or VZV infection.

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