CN114272367A

CN114272367A - Oral rabies virus-like particle vaccine and preparation method thereof

Info

Publication number: CN114272367A
Application number: CN202111469820.1A
Authority: CN
Inventors: 马兴元; 赵章婷; 郑文云; 邓昌平
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2021-12-03
Filing date: 2021-12-03
Publication date: 2022-04-05
Anticipated expiration: 2041-12-03
Also published as: CN114272367B

Abstract

The invention provides an oral rabies virus-like particle vaccine and a preparation method thereof, which takes RVLPs self-assembled by glycoprotein RVGP of CVS strain rabies virus and matrix protein RVMP as antigens and utilizes

The L100 and the PLGA are used as main materials to prepare the oral acid-resistant slow microspheres. Increasing antigenicity of oral RVLPs and enhancing immunity effect by using mucosa immunologic adjuvant LTB. By designing the protein drug carrier suitable for oral delivery to intestinal administration, the protein drug carrier can successfully overcome various physiological barriers of the gastrointestinal tract and directly deliver the protein drug carrier to the intestinal tract, and responds to and releases antigens under the intestinal pH condition, so as to carry out preliminary immunological evaluation on the immune effect caused by the antigen. The oral rabies virus-like particle vaccine can improve the actual utilization efficiency of protein drugs and the treatment or prevention effect of the vaccine, is expected to expand the rabies vaccine inoculation range, eliminate rabies from animal sources, improve the safety of the vaccine and lay an important foundation for developing novel virus-like particle oral subunit vaccines.

Description

Oral rabies virus-like particle vaccine and preparation method thereof

Technical Field

The invention belongs to the technical field of oral vaccines, and particularly relates to an oral rabies virus-like particle vaccine and a preparation method thereof.

Background

Rabies is a neuropsychotropic human-veterinary co-disease caused by Rabies Virus (RABV), 99% of human Rabies deaths are dog-mediated, and vaccination is the most effective means of preventing the spread of RABV (Zhang W J, Zheng X, Cheng N, et al. The advances in controlling the number of dogs in wave, large scale or forced parenteral vaccination campaigns for domestic dogs and wild animals, and oral vaccination of wild animals have successfully eradicated terrestrial carnivorous-mediated human rabies in several developed countries. However, in developing countries thousands of people die annually from rabies due to financial, logistical and other obstacles, the inability to control the number of dogs and implement large scale vaccination programs. Providing a more manageable and cost-effective vaccine may help achieve elimination of dog-mediated human rabies (Arya J M, Dewitt K, Garrrard M S, et al. Rabies Vaccination in Dogs Using a dispensing MicroneedlePatch [ J ]. Journal of Controlled Release, 2016,239: 19-26.).

ORVs play a crucial role in global elimination of dog-mediated human rabies, and specific practical activities should be urgently pursued to facilitate rapid and large-scale use of safe and cost-effective ORVs. Although several self-replicating biologicals, including modified live viruses, attenuated live viruses and recombinant viruses, have been evaluated orally in canine populations, there is currently no development of a suitable ORV (Cliquet F, Guiot A L, Aubert M, et al. Oral Vaccination of Dogs: A Well-studed and Undervalued Tool for acquiring Human and Dog viruses immunization [ J ]. viral Research,2018,49(1):61.) due to safety concerns.

Compared with injection administration, the oral administration has the advantages of convenience, rapidness, safety, good patient compliance and the like, and does not need professional vaccinees. Second, oral vaccines stimulate systemic and mucosal immune responses, establishing broader and longer lasting protection. However, oral administration is challenging, especially for protein drugs, and requires overcoming the harsh gastrointestinal environment, avoiding induction of immune tolerance, to achieve effective protection (Renu S, Han Y, Dhakal S, et al Chitosan-added saline Vaccine for Poultry delivery through drug delivery Water and Feed [ J ]. Carbohydrate Polymers,2020,243: 116434.).

Protein drugs have absolute safety compared to viruses, where VLPs are more stable and immunogenic than polypeptides, and are therefore the focus of protein drug development, and suitable Delivery vehicles are the best means for achieving Oral targeting and Release of such drugs (Chen X Y, but a M, an M C I M. enhanced particulate Delivery of Vaccine by Hydrogel Microparticles-medical modified light Junction Opening for Effective organic Immunization [ J ]. Journal of Controlled Release,2019, 311-.

PLGA and EL100 are FDA approved materials that can be used in the human body. PLGA NPs as carriers have many applications in the Delivery of injection vaccines (Kole S, Qadiri S N, Shin S M, et al, PLGA Encapsulated Inactivated-Viral Vaccine: Formulation and Evaluation of the present Protective effective Vaccine (Vhsv) Infection in Viral flood apparatus [ J. clinical details ] mediated by Mucosal Delivery route [ J. S. S.N, Shin S M].Vaccine,2019,37(7):973-983.Umerska A, Gaucher C,Ampuero F O,et al.Polymeric Nanoparticles for Increasing Oral Bioavailability of Curcumin[J]Aniioxidants (basel),2018,7(4): 46.); eudragit protects against acidic conditions, Rajasree (Rajasree P H, Paul W, Sharma C P, et aled Cationic Poly (Lactic-Co-Glycolic Acid)Nanoparticles in Targeted Delivery of Capecitabine for Augmented Colon Carcinoma Therapy[J]Journal of Drug Delivery Science and Technology,2018,46: 302-

S100 is coated on the surface of PLGA NPs carrying drugs, and is used as a novel targeted drug delivery system for treating colon cancer, and a drug delivery system (Xu B H, Zhang W J, Chen Y L, et al).

L100-Coated Mannosylated Chitosan Nanoparticles for Oral Protein Vaccine Delivery[J]International Journal of Biological Macromolecules,2018,113:534-542.) and the like with BSA

L100-coated mannosylated chitosan NPs were orally immunized and found to elicit better systemic IgG and mucosal IgA antibody responses.

At present, related researches on the anti-rabies virus oral vaccine are rare, and related oral vaccines capable of coping with severe gastrointestinal tract environments are not found.

Disclosure of Invention

The invention aims at the problems and aims to develop an oral rabies virus-like particle vaccine, and the oral administration for delivering protein medicaments provides a design idea.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention firstly prepares oral rabies virus-like particle vaccines RVLPs/EPLGA MPs, and after the oral rabies virus-like particle vaccines RVLPs/EPLGA MPs are characterized in vitro, then the lyophilized RVLPs/EPLGA MPs are perfused into a stomach mouse for immunogenicity evaluation, and preliminary evaluation is made on the cellular immune response generated by the vaccines.

In a first aspect of the invention, an oral rabies virus-like particle vaccine is provided, which comprises a broad-spectrum rabies virus-like particle antigen and a drug carrier. Wherein, the broad-spectrum rabies virus-like particle antigen comprises CVS strain rabies virus glycoprotein RVGP and matrix protein RVMP, the polynucleotide sequence of the CVS strain rabies virus glycoprotein RVGP is shown as SEQ ID NO.1, the polynucleotide sequence of the matrix protein RVMP is shown as SEQ ID NO.2,

the drug carrier is composed of PLGA and

a mixed carrier consisting of L100, and a carrier,

the mass ratio of the L100 to the PLGA is 1: 5-1: 1; the concentration ratio of the broad-spectrum rabies virus-like particle antigen to PLGA is 1: 5-1: 10.

Preferably, the first and second liquid crystal materials are,

the mass ratio of L100 to PLGA is 1:2, and the concentration ratio of the broad-spectrum rabies virus-like particle antigen to PLGA is 1: 10.

Preferably, the concentration of the broad-spectrum rabies virus-like particle antigen is 5mg/mL, the concentration of PLGA is 25-50 mg/mL, and the concentration of PLGA is more preferably 50 mg/mL.

Preferably, the oral rabies virus-like particle vaccine also comprises a mucosal immune adjuvant LTB, and the content of the LTB is 20-100 ug/mL.

In a second aspect of the present invention, there is provided a method for preparing the oral rabies virus-like particle vaccine, comprising the following steps:

1) adding broad-spectrum rabies virus-like particle antigen and LTB into the ethyl acetate dissolved with PLGA and the mixture dissolved with PLGA simultaneously

Forming O/W1 type primary emulsion after vortex oscillation in a mixed oil phase of L100 ethanol, wherein the volume ratio of the broad spectrum rabies virus-like particle antigen to the ethyl acetate and the ethanol is 2:10: 5;

2) dropwise adding the obtained primary emulsion into 10-12 times of volume of 1% PVA under the action of magnetic stirring to form W2/O/W1 type multiple emulsion;

3) continuously stirring and re-emulsifying at 4 deg.C for at least 4h, and volatilizing organic solvent to obtain solidified granules RVLPs/EPLGA MPs;

4) centrifuging the solidified RVLPs/EPLGA MPs at 4 ℃, 300rpm for collection, cleaning the PVA and the non-coated antigen on the surface of the RVLPs/EPLGA MPs by using ultrapure water, and collecting all centrifuged supernatant;

5) resuspending RVLPs/EPLGA MPs with appropriate amount of sterile PBS, freezing at 4 deg.C, -20 deg.C and-80 deg.C, vacuum freeze drying for 24 hr, sealing, and storing at-20 deg.C.

Preferably, in the step 1), the concentration of the broad-spectrum rabies virus-like particle antigen is 5mg/mL, the concentration of PLGA is 50mg/mL,

the concentration of L100 was 25 mg/mL.

Preferably, in the step 2), the rotation speed of the magnetic stirring is 300-400 rpm.

Preferably, in step 4), the number of times of cleaning with ultrapure water is 3.

The preparation conditions are obtained by orthogonal experiments, and the experimental results show that when the concentration of PLGA is 50mg/mL, the stirring rotation speed is 300rpm, the volume of 1% PVA is 20mL, and the mass ratio of PLGA to EL100 is 2:1, the optimal Encapsulation Efficiency (EE) is obtained, and the Encapsulation efficiency is 97.14 +/-1.37%.

The oral rabies virus-like particle vaccine RVLPs/EPLGA MPs prepared according to the method are subjected to in vitro characterization, stability analysis, in vitro release experiment and mouse intestinal retention time analysis in sequence.

In vitro characterization showed that the freshly prepared RVLPs/EPLGA MPs were regularly spherical and had good size uniformity (FIG. 2A). SEM results show that the prepared RVLPs/EPLGA MPs after vacuum freeze-drying have flat surfaces and relatively regular spherical shapes, and the average particle size is in the range of 168.20 +/-39.28 microns (figure 2B).

The stability results show that LTB and RVLPs were successfully encapsulated in EPLGA MPs, that the RVLPs particle size and morphology did not change, and that the native secondary structure of LTB and RVLPs did not change. After being stored for 3 months at 4 ℃, the shape and the encapsulation efficiency are not obviously changed.

The simulated drug release results in the in vivo stomach (pH1.2) and intestinal tract (pH6.8) environments show that compared with PLGA MPs, the EPLGA MPs delivery system has a pH-dependent drug controlled release mode, and the EPLGA MPs effectively realize the slow release of most of target proteins in PBS with the pH of 6.8, well overcome the denaturation of the proteins caused by the severe stomach environment, and smoothly deliver the target proteins to the intestinal tract (figure 4).

According to the analysis of intestinal retention time of mice, about 20% of mCherry is released in a cumulative way when the mCherry is administered for 5 hours, wherein about 10% of mCherry is released in the stomach and denatured and inactivated, and the mCherry released in the intestinal tract is also partially inactivated due to the action of protease in the intestinal tract; after 10h of oral administration, the remaining undegraded microspheres were left directly to the small intestine to the large intestine, which was low in protease. The EPLGA MPs prepared by the invention can overcome the acidic environment of the stomach and successfully deliver the protein to the intestinal tract.

The results of oral immunization of Balb/c mice show that the RVLPs can induce the body to generate corresponding antigen-specific humoral, cellular and mucosal immune responses, and the RVLPs and LTB loading in the EPLGA MPs can induce the body to generate stronger immune responses after combined administration. The immune response reached the highest level at 3 rd immunization. The results of HE staining of the body weight and major organs of mice indicate that the vaccine components of the experiment have good safety.

The invention has the following beneficial effects:

the invention takes RVLPs self-assembled by glycoprotein (RVGP) of CVS strain Rabies virus (RABV) and matrix protein (RVMP) as antigens and utilizes the RVLPs as antigens

The L100 and PLGA are used as main materials to prepare the oral acid-resistant slow microspheres, RVLPs can be slowly released in intestinal tracts, and the RVLPs with the nano structure can be quickly taken up by intestinal M cells and other APC (APC). RVLPs with high-density antigen epitope are combined with mucosal adjuvant LTB, and can be extractedHigh antigenicity of oral RVLPs and enhanced immune effect.

The invention designs the protein drug carrier suitable for oral delivery to intestinal administration, so that the protein drug carrier can successfully overcome various physiological disorders of the gastrointestinal tract and directly deliver the protein drug carrier to the intestinal tract, and responds to and releases antigens under the pH condition of the intestinal tract. The results of preliminary immunological evaluation on the immune effect triggered by the RVLPs show that the RVLPs can induce the organism to generate corresponding antigen-specific humoral, cellular and mucosal immune responses, and the RVLPs and LTB loaded in the EPLGA MPs can induce the organism to generate stronger immune responses after combined administration; the immune response reaches the highest level at the 3 rd immunization; the results of HE staining of the body weight and major organs of mice indicate that the vaccine components of the experiment have good safety.

The oral Rabies Virus Like Particle (RVLPs) vaccine prepared by the research can improve the actual utilization efficiency of protein drugs and improve the treatment or prevention effect of the vaccine, is expected to expand the vaccination range of Rabies vaccine in China and eliminate Rabies from animal sources, thereby limiting and preventing RABV from spreading among land carnivorous animal populations, reducing the risk of infection spreading to livestock and human populations, improving the safety of the vaccine and laying an important foundation for developing novel virus like particle oral subunit vaccines.

Drawings

FIG. 1 shows the preparation of oral rabies virus-like particles (RVLPs) vaccine RVLPs (LTB)/EPLGA MPs and the mucosal immune response mechanism induced by the vaccine RVLPs (LTB)/EPLGA MPs.

FIG. 2 shows the morphology of RVLPs/EPLGA MPs, and the morphology of RVLPs/EPLGA microspheres is observed by an inverted microscope (A) and a scanning electron microscope (B).

FIG. 3 is an integrity analysis of RVLPs/LTB proteins in EPLGA MPs, wherein: (A) FTIR spectroscopy; (B) CD spectrum; (C) and a TEM.

FIG. 4 is a storage stability analysis of RVLPs/EPLGA MPs, wherein: encapsulation efficiency (A) and appearance (B) of RVLPs/EPLGA MPs under different storage conditions.

FIG. 5 shows the in vitro release results of RVLPs/EPLGA MPs.

FIG. 6 is an image of the gastrointestinal tract of mice imaged ex vivo after oral administration of mCherry/EPLGA MPs.

FIG. 7 shows the content of anti-RVLPs IgG antibody subtypes in serum, wherein: (A) IgG; (B) IgG 1; (C) IgG2 a; (D) IgG1/IgG2a ratio.

FIG. 8 is the content of anti-RVLPs sIgA antibodies in feces and intestinal fluids, wherein: (A) fecal anti-RVLPs sIgA antibodies; (B) anti-RVLPs sIgA antibodies in intestinal fluid after the third immunization.

FIG. 9 shows the expression levels of INF-gamma and IL-4 in sera of mice administered with different doses, wherein: (A) INF-gamma; (B) IL-4.

FIG. 10 shows the change in body weight and histological examination of major organs of mice.

Detailed Description

The following embodiments are implemented on the premise of the technical scheme of the invention, and give detailed implementation modes and specific operation procedures, but the protection scope of the invention is not limited to the following embodiments.

For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

EXAMPLE 1 preparation of oral rabies Virus-like particle vaccine RVLPs/EPLGA MPs

1. Oral rabies virus-like particle vaccine RVLPs/EPLGA MPs

The oral rabies virus-like particle vaccine comprises broad-spectrum rabies virus-like particle antigen and a drug carrier. Wherein, the broad-spectrum rabies virus-like particle antigen comprises CVS strain rabies virus glycoprotein RVGP and matrix protein RVMP, the polynucleotide sequence of the CVS strain rabies virus glycoprotein RVGP is shown as SEQ ID NO.1, and the polynucleotide sequence of the matrix protein RVMP is shown as SEQ ID NO. 2.

The drug carrier is composed of PLGA and

a mixed carrier consisting of L100, and a carrier,

the mass ratio of the L100 to the PLGA is 1: 5-1: 1, preferably 1: 2. The concentration ratio of the broad-spectrum rabies virus-like particle antigen to PLGA is 1: 5-1: 10, preferably 1: 10; in terms of concentration, the concentration of the broad-spectrum rabies virus-like particle antigen is 5mg/mL, and the concentration of PLGA is 25-50 mg/mL.

Preferably, the oral rabies virus-like particle vaccine also comprises a mucosa immunologic adjuvant LTB, and the RVLPs are used together with the mucosa adjuvant LTB, so that the antigenicity of the oral RVLPs can be improved, and the immunologic effect can be enhanced.

2. Vaccine preparation

FIG. 1 shows the preparation of oral rabies virus-like particles (RVLPs) vaccine RVLPs (LTB)/EPLGA MPs and the mechanism of mucosal immune response induced by them. The preparation process of the oral rabies virus-like particle vaccine comprises the following steps:

1) adding broad-spectrum rabies virus-like particle antigen into the PLGA dissolved ethyl acetate and the PLGA dissolved ethyl acetate or LTB

Forming O/W1 type primary emulsion after vortex oscillation in the mixed oil phase of L100 ethanol;

3) continuously stirring and re-emulsifying at 4 deg.C for at least 4h, and volatilizing organic solvent to obtain solidified granules RVLPs (LTB)/EPLGA MPs;

4) centrifuging the solidified RVLPs (LTB)/EPLGA MPs at 4 ℃, 300rpm for collection, cleaning the PVA and the non-coated antigen on the surface of the RVLPs/EPLGA MPs by using ultrapure water, and collecting all centrifuged supernatant;

5) resuspending RVLPs (LTB)/EPLGA MPs with appropriate amount of sterile PBS, freezing at 4 deg.C, -20 deg.C and-80 deg.C, vacuum freeze drying for 24 hr, sealing, and storing at-20 deg.C.

3. Composition optimization

The concentration of PLGA, the PLGA to EL100 mass ratio, the volume of 1% PVA and the stirring speed of the magnetic stirrer were optimized using Encapsulation Efficiency (EE) as a standard, and orthogonal experiments at a 4-factor 3 level were designed (table 1). The BCA method is used for detecting the concentration of free RVLPs in the supernatant, and the calculation formula of EE is as follows:

EE (%) - (total amount of RVLPs dosed-amount of RVLPs in supernatant)/total amount of RVLPs dosed X100%

Table 2 shows that the optimal experimental combination is A3B3C2D2, i.e. when the PLGA concentration is 50mg/mL, the stirring speed is 300rpm, the volume of 1% PVA is 20mL, the mass ratio of PLGA to EL100 is 2: the best EE is obtained at 1. The optimized RVLPs/EPLGA MPs encapsulation rate is 97.14 +/-1.37%.

When the PLGA concentration is 12.5mg/mL, EE is low regardless of other conditions, probably because the low concentration PLGA cannot saturate with the protein water solution; when the volume of 1% PVA is too large, the obtained EE is also not ideal, and the concentration of PLGA may be greatly reduced by the excessive external aqueous phase; when the mass ratio of PLGA to EL100 is 1, EE is not high probably because excessive addition of EL100 causes film-formation and precipitation together with PLGA, so that the oil phase and the water phase cannot be saturated with each other.

Table 1 factors and levels of orthogonal design

Note: the total PLGA amount and the stirring time were kept constant.

TABLE 2 orthogonal experiment L₉(3⁴) Results

Example 2 in vitro characterization of RVLPs/EPLGA MPs

After the RVLPs/EPLGA MPs are prepared, the morphology is observed by using an inverted microscope. After lyophilization, the morphology and size were observed by Scanning Electron Microscope (SEM).

The freshly prepared RVLPs/EPLGA MPs are regularly spherical and have good size uniformity (FIG. 2A). SEM results show that the prepared RVLPs/EPLGA MPs after vacuum freeze-drying have flat surfaces and relatively regular spherical shapes, and the average particle size is in the range of 168.20 +/-39.28 microns (figure 2B).

EXAMPLE 3 RVLPs LTB/EPLGA MPs stability

1. Structural stability

Whether RVLPs were successfully loaded into EPLGA MPs and whether their secondary structure was intact was analyzed using Fourier transform induced spectrometer (FITR) and Circular dichroism spectroscopy (CD). To evaluate the pH responsiveness of EPLGA MPs, EGFP/EPLGA MPs were sampled and observed for their integrity after incubation with PBS at pH1.2 and pH6.8 for 2, 4 and 8h, respectively. In order to further determine the storage stability of freeze-dried EGFP/EPLGA MPs, the vacuum freeze-dried RVLPs/EPLGA MPs were stored at 4 ℃, 25 ℃ and 37 ℃, the RVLPs/EPLGA MPs under different storage conditions were periodically taken out for photographing, the RVLPs/EPLGA MPs were cracked and EE was calculated, and the integrity of the RVLPs was examined by a Transmission Electron Microscope (TEM).

To confirm successful encapsulation of LTB and RVLPs in EPLGA MPs, FTIR spectroscopy was used to analyze the amide bonds present in the proteins. As shown in FIG. 3A, the bonds detected at 1544.7cm-1 and 1654.7cm-1 for LTB/EPLGA MPs compared to EPLGA MPs confirm encapsulation of LTB protein within EPLGA MPs; the bonds detected at 1543.5cm-1 and 1654.5cm-1 for RVLPS/EPLGA MPs confirm the encapsulation of RVLPS proteins within EPLGA MPs. These two bonds are generated by flexural vibrations of the Protein amide I (C ═ O) and II (N-H) bonds, respectively (Roy K, Kanwar R K, Subramanian, et al. comprehensive Inhibition of Survivin use a Cell-Permeable Recombinant Protein inductors-Specific apoliosis in Colon Cancer Model [ J ]. International Journal of Nanomedicine,2015,10: 1019-. FTIR spectroscopy results indicated that LTB and RVLPs were successfully encapsulated in EPLGA MPs.

To further confirm that the native secondary structures of LTB and RVLGPS within the EPLGA MPs were not altered, CD spectroscopy was used to determine the secondary structures of LTB/EPLGA MPs and RVLPs/EPLGA MPs. Wherein the alpha-helix (negative peaks: 222nm and 208nm, positive peaks: 190nm), beta-sheet (positive peaks: 195-198nm, negative peaks: 217-218nm) and random coil (positive peaks: 220nm, negative peaks: 198nm) constitute the protein secondary structure (Fan K L, Jiang B, Guan Z, et al. Fernobody: A Ferritin-Displayed Nanobody with High application Affinity and Half-Life Extension [ J ] Analytical Chemistry,2018,90(9): 5671-5677.). As shown in fig. 3B, the LTB includes 3 structures of alpha helix, beta sheet, and random coil; RVLPs are formed by the self-assembly of RVMPs that predominate in beta-sheet and random curls, and RVGPs that predominate in alpha-helices, beta-sheet, and random curls. LTB/EPLGA MPs and RVLPs/EPLGA MPs are consistent with the CD spectra trends of native LTB and RVLPs, indicating that encapsulation of LTB and RVLPs into EPLGA MPs does not affect their native secondary structure.

In addition, the integrity of the RVLPs encapsulated in the EPLGA MPs was analyzed using TEM. When the supernatant obtained by cleaving RVLPs/EPLGA MPs was concentrated and observed by TEM, the RVLPs in the supernatant were not changed in particle size and morphology and were 180-200nm circular or elliptical NPs (FIG. 3C). Thus, the EPLGA MPs prepared in this study were able to successfully encapsulate the LTB and RVLPs, while maintaining their structural integrity.

2. Stability in storage

The RVLPs/EPLGA MPs after vacuum freeze drying are stored under different temperature conditions and are photographed regularly. As shown in FIG. 4B, significant shrinkage and aggregation occurred after 1 week of storage at 37 ℃ in RVLPS/EPLGA MPs, indicating that 37 ℃ is not suitable for storage of RVLPS/EPLGA MPs, and the morphology remained essentially unchanged after 3 months of storage at 4 ℃ and 25 ℃. Therefore, 10mg of each of RVLPs/EPLGA MPs at 4 ℃ and 25 ℃ in different storage periods is taken, PBS is used for cracking, and then the BCA kit is used for determining the protein content and calculating EE. As shown in FIG. 4A, EE was only slightly down-regulated after 3 months of storage at 4 ℃ in RVLPs/EPLGA MPs, and was not statistically different from day 0. However, EE decreased gradually with time during storage of RVLPs/EPLGA MPs at 25 ℃ until 3 months, with an approximate 15% decrease in EE. Therefore, 4 ℃ is more suitable for the preservation of RVLPs/EPLGA MPs.

EXAMPLE 3 in vitro Release assay of RVLPs/EPLGA MPs

10mg of RVLPs/EPLGA MPs were added to 1mL of release medium and incubated at 100rpm at 37 ℃, the supernatant was collected at 12000rpm for 10min at predetermined time intervals, supplemented with an equal volume of fresh PBS, and the amount of RVLPs in the supernatant was determined.

Since RVLPs/EPLGA MPs are administered by intragastric administration, it is necessary to mimic drug release in the gastric (pH1.2) and intestinal (pH6.8) environments in vivo. As shown in FIG. 5, the EPLGA MPs delivery system has a pH-dependent controlled drug release pattern compared to PLGA MPs, and RVLPS/EPLGA MPs and LTB/EPLGA MPs release less than 10% of the target protein within 2h at pH1.2, while RVLPS/PLGA MPs burst approximately 70% of the target protein within 2h at pH 1.2. Although about 95% of target protein is accumulated and released in 24h by two MPs in simulated intestinal fluid, the EPLGA MPs effectively realize that most of the target protein is slowly released in PBS with pH6.8, well overcomes the denaturation of the protein by the severe environment of the stomach, and smoothly delivers the target protein to the intestinal tract.

Example 4 intestinal residence time in RVLPs/EPLGA MPs mice

Prepare EPLGA MPs loaded with cherry red fluorescent protein (mCherry) (excitation/emission wavelength is 580nm/610nm) -mCherry/EPLGA MPs. Balb/c mice were fasted for 12h but were normally fed before gavage, then gavage was performed with PBS resuspended mCherry/EPLGA MPs (100 μ L), and after gavage the mice were dissected at 0.5, 1.5, 3, 5,10 h, respectively, and the gastrointestinal organs were stripped off and photographed for observation using a KODAK live animal imaging system.

After the mCherry/EPLGA MPs were gavaged, the mice were dissected at specific time points and the gastrointestinal tract was removed for photography. As shown in figure 6, after 0.5h administration, mCherry/EPLGA MPs were all concentrated in the stomach, and after 1.5h mCherry/EPLGA MPs began to flow slowly from the stomach to the duodenum until 3h, and the microspheres all exited the stomach, reached the small intestine and gradually flowed from the duodenum to the ileum. Due to the partially neutral environment of the intestinal tract, mCherry/EPLGA MPs were degraded, released and taken up by M cells, so at 5h the fluorescence intensity in the jejunum and ileum was slightly reduced. About 20% of mCherry was released at 5h, wherein about 10% of mCherry was denatured and inactivated by gastric release, and partially inactivated by intestinal protease, and the remaining undegraded microspheres directly leave the small intestine to reach the large intestine with low protease content after 10h of oral administration. The EPLGA MPs prepared in this study can overcome the acidic environment of the stomach and successfully deliver proteins to the intestinal tract.

Example 5 immunization Effect of RVLPs (LTB)/EPLGA MPs vaccine

1. Experimental methods

(1) RVLPs/EPLGA MPs were intragastrically inoculated into 6-8 week old female SPF grade Balb/c mice. Mice were randomly grouped according to table 3 and labeled. One week after mice were acclimated, fasted but freely drinking water 12h prior to immunization, gavage immunizations were performed on

days

7, 21 and 35, and the immune responses generated were evaluated.

(2) Measurement of mouse body weight: the body weight was weighed 1 time before immunization, 1 time every 7 days at a fixed time, and 6 times were recorded until the end of the 3 rd immunization, and the mice were analyzed for body weight changes.

(3) Collecting mouse feces and detecting antigen-specific sIgA.

(4) Collection of mouse serum and antigen-specific IgG, IgG1 and IgG2a antibodies, cytokines IFN-. gamma.and IL-4, and CD4 in mouse peripheral blood⁺T and CD8⁺And (4) detecting T cells.

(5) Collecting intestinal juice of the mice and detecting the antigen-specific sIgA antibody.

(6) Histopathological examination of the major organs of the mice (heart, liver, spleen, lung, kidney and small intestine).

TABLE 3 animal Experimental groups and dosages

And (3) sampling operation:

the collection of the mouse excrement in the step (3) comprises the following steps:

collecting: before immunization and 1 week after each immunization, randomly collecting 15 mouse feces by using clean forceps, placing the feces into a sterilized EP tube, marking, and storing at-80 ℃ for later use in triplicate;

processing: taking out mouse feces from-80 deg.C, grinding the feces with injector core, adding 500 μ L sterile PBS, continuously blowing and beating to dissolve completely, centrifuging at 4 deg.C and 12000rpm for 5min, collecting supernatant, marking, and storing at-80 deg.C for RVLPs specific sIgA analysis.

In the step (4), the collection of the blood of the mouse comprises the following steps:

collecting: before and 1 week after the two former immunizations, a capillary glass tube is punctured into the orbit of the mouse to collect blood; 1 week after 3 rd immunization, quickly picking up eyeballs by using sterilized forceps and collecting blood;

processing: blood samples were allowed to stand at 4 ℃ for 12h, then centrifuged at 4 ℃ for 30min at 1000rpm, serum was aspirated into sterilized EP tubes, labeled, stored at-80 ℃ and used for analysis of RVLPs-specific antibodies IgG, IgG1 and IgG2a, and serum 1 week after 3 rd immunization was also used for IL-4 and IFN-. gamma.analysis and detection of CD4+ T and CD8+ T cells in peripheral blood of mice 1 week after 3 rd immunization.

In the step (5), the collection of the intestinal juice of the mouse comprises the following steps:

collecting: 1 week after 3 rd immunization, mice killed by cervical dislocation are disinfected by 75% alcohol, intestinal segments of small intestines are aseptically cut, the inner walls of the intestines are exposed by cutting with a scalpel, and the intestines are placed in a sterile EP tube;

processing: to EP containing the small intestine, 1mL of sterile PBS solution was added, vortexed for 15min, and centrifuged at 12000rpm for 10min at 4 ℃, and the supernatant was collected and stored at-80 ℃ for RVLPs-specific sIgA analysis.

And (3) detection:

detection of RVLPs specific antibodies IgG, IgG1, IgG2a and sIgA was performed as follows:

coating: RVLPs were diluted to 5. mu.g/mL with PBS, and the diluted RVLPs (100. mu.L/well) were added to a 96-well plate and coated overnight at 4 ℃;

sealing: adding 200. mu.L of 1% BSA to the reaction well, and incubating at 37 ℃ for 2 h;

adding sample: adding 100 μ L feces extract and intestinal juice and serum diluted with 1% BSA (1: 100), respectively, and incubating at 37 deg.C for 2 hr;

adding an enzyme labeled secondary antibody: adding 100 μ L of goat anti-mouse polyclonal antibody coupled with HRP diluted with 1% BSA (1: 10000) to the reaction well, and incubating at 37 deg.C for 1 h;

adding a substrate solution for color development: adding 100 μ L of color development solution, incubating at 37 deg.C in dark for 20 min;

sixthly, terminating the reaction: add 100. mu.L of 1M H2SO4 and mix well before detecting the OD value (490 nm).

And seventhly, washing the obtained product 6 times by using PBST after each step is finished.

Detection of the cytokines IFN-gamma or IL-4, according to the instructions of the IL-4 and IFN-gamma kits, the method is as follows:

firstly, before use, all reagents are fully and uniformly mixed, so that foam is avoided;

secondly, determining the required number of the battens according to the number of the experimental holes (blanks and standard products). The sample (containing the standard) and the blank are subjected to multiple holes;

adding sample: add diluted Cytokine standard to standard well at 100. mu.L/well, sample to sample well at 100. mu.L/well, Dilution buffer R (1X) to blank well at 100. mu.L/well;

adding a detection antibody: 50 μ L/well was added with Biotinylated antibody working solution. After mixing, covering a sealing plate film, and incubating for 90min at 37 ℃;

washing the plate: deducting liquid in the hole, and adding 1 × Washing buffer working solution into 300 μ L/well; after staying for 1min, the liquid in the pores is discarded. Repeating for 4 times, and drying on the filter paper each time;

sixthly, adding enzyme: 100 μ L/well were added to Streptavidin-HRP working solution. Covering a sealing plate membrane, and incubating at 37 ℃ for 30 min;

seventh, washing the plate: repeating the step 5;

and color development: adding TMB into the well at a volume of 100 mu L/well, incubating for 5-30min in the dark at 37 ℃, and judging the termination reaction according to the shade (dark blue) of the color in the hole;

ninthly, terminating the reaction: the reaction was stopped by adding 100. mu.L/well quickly to Stop solution.

2. Analysis of results

2.1 serum-specific IgG antibody subtypes

Different specific antibody subtypes may reflect to some extent the level of immune response to which the body responds. As shown in FIG. 7, EPLGA MPs and LTB/EPLGA MPs did not produce antigen-specific antibodies after immunization compared to Saline group.

After the 1 st immunization, no specific IgG antibodies were detected in the sera of all groups (fig. 7A). Following the boost, all groups induced a significantly enhanced specific IgG antibody response, except for the Rabisin (i.g.) group, where no specific IgG antibody was detected. Wherein, the content of IgG antibody in RVLPs group is 2.27 times that of Saline group (P < 0.001); there were no statistical differences in IgG antibody content between the RVLPs + LTB, RVLPs/EPLGA MPs and RVLPs + LTB/EPLGA MPs 3 groups, but 1.43, 1.56 and 1.66 times the RVLPs group respectively (P < 0.001); there was no statistical difference in the IgG antibody content between the RVLPs + LTB/EPLGA MPs and the (RVLPs + LTB/EPLGA MPs). times.2 group, but the (RVLPs + LTB/EPLGA MPs). times.2 group was 1.21 times that of the RVLPs/EPLGA MPs group (P < 0.01); the Rabisin (i.m.) group had the highest content of IgG antibodies, which was 1.67 times (P <0.001) that of the (RVLPs + LTB/EPLGA MPs). times.2 group. After 3 rd immunization, the levels of specific IgG antibodies were highest in the RVLPs + LTB/EPLGA MPs and (RVLPs + LTB/EPLGA MPs). times.2 groups, but there was no statistical difference between these two groups. The IgG antibody content of the (RVLPs + LTB/EPLGA MPs). times.2 group was 1.35 and 4.07 times that of the Rabisin (i.m.) and Rabisin (i.g.) groups, respectively (P < 0.001). The above results indicate that oral administration of RVLPs can stimulate the body to generate specific humoral immune response, adjuvant LTB can enhance the level of immune response, and when RVLPs and LTB are loaded in EPLGA MPs for combined administration, the level of specific humoral immune response generated is higher than that of Rabisin (i.m.) and Rabisin (i.g.) groups (P <0.001), but dose-dependency is not existed.

The results of the RVLPs-specific IgG1 antibody analysis are shown in FIG. 7B. After primary immunization, only the Rabisin (i.m.) group induced a significantly enhanced IgG1 antibody response (P < 0.001). After two boosts, none of the Rabisin (i.g.) groups induced the body to produce an IgG1 antibody response, while each of the other groups induced a significantly enhanced IgG1 response. Analysis of the antigen-specific IgG1 antibody content after the 2 nd immunization revealed that the RVLPs + LTB/EPLGA MPs group was 1.37 times (P <0.001) higher than the RVLPs/EPLGA MPs group, whereas the (RVLPs + LTB/EPLGA MPs) x 2 group was 1.37 times (P <0.001) higher than the RVLPs + LTB/EPLGA MPs group. After the 3 rd immunization, the specific IgG1 antibody content in the (RVLPS + LTB/EPLGA MPs). times.2 group was 1.17(P <0.01) and 1.59 times (P <0.001) that in the RVLPS + LTB/EPLGA MPs and Rabisin (i.g.) groups, respectively. However, after two boosts, the IgG1 antibody content was highest in the Rabisin (i.m.) group, 1.39 and 1.59 times (P <0.001) the (RVLPs + LTB/EPLGA MPs) x 2 group, respectively.

In the RVLPs-specific IgG2a antibody assay (fig. 7C), after the initial immunization, only the specific IgG2a antibody levels of the RVLPs + LTB/EPLGA MPs, (RVLPs + LTB/EPLGA MPs) x 2, Rabisin (i.g.) and Rabisin (i.m.)4 groups were significantly increased, and there was no significant difference in IgG2a content between the RVLPs + LTB/EPLGA MPs and the (RVLPs + LTB/EPLGA MPs) x 2, Rabisin (i.g.) and Rabisin (i.m.) groups. The IgG2a antibody content of all experimental groups reached the highest level after the 3 rd immunization. Wherein the (RVLPs + LTB/EPLGA MPs) × 2 group is 1.13 times (P <0.01) the (RVLPs + LTB/EPLGA MPs) group, 1.73 times (P <0.001) the (i.g.) Rabisin group, and the (i.m.) group is 2.01 times (P <0.001) the (RVLPs + LTB/EPLGA MPs) × 2 group.

FIGS. 7B and C show that the (RVLPs + LTB/EPLGA MPs). times.2 group stimulated the body to produce the most significant IgG1 and IgG2a antibody responses compared to the other experimental groups. The levels of IgG1 and IgG2a reflect Th2 and Th1 cellular immune responses, indicating that RVLPs/EPLGA MPs and LTB/EPLGA MPs prepared in this experiment when administered in combination at twice the dose can induce a significantly enhanced cellular immune response in the body, although not as effective as the commercially available inactivated rabies vaccine Rabisin (i.m.) group, but significantly higher than Rabisin (i.g.) group.

The ratio of IgG1 to IgG2a was calculated after 3 rd immunization (fig. 7D). The ratio of IgG1 to IgG2a reflects the propensity of Th2 to Th1 type cellular immune response, and if the ratio is close to 1, it indicates that a more balanced Th1 and Th2 type immune response is induced, to which the RVLPs and Rabisin (i.m.) groups belong; if the ratio is greater than 1, then the IgG1 antibody subtype predominates, favoring a Th2 type immune response, i.e., RVLPS + LTB, RVLPS/EPLGAMPs, RVLPS + LTB/EPLGA MPs, and (RVLPS + LTB/EPLGA MPs). times.2 groups; if the ratio is less than 1, it indicates that the IgG2a antibody subtype predominates, favoring a Th1 type immune response, i.e., Rabisin (i.g.).

2.2 specific sIgA antibodies in feces and intestinal fluids

The mucosal immune response level generated by the oral vaccine can be judged by analyzing the content of RVLPs specific sIgA antibodies in mouse excrement and intestinal fluid. As shown in FIG. 8, the groups EPLGA MPs, LTB/EPLGA MPs and Rabisin (i.m.) did not stimulate the body to produce mucosal-specific sIgA antibodies, as did the Saline group.

The content of RVLPs-specific sIgA antibodies in feces of all experimental groups and Rabisin (i.g.) group increased significantly with the increase of the number of immunizations (fig. 8A). After the primary immunization, the sIgA content of RVLPs group is 1.26 times that of Saline group (P < 0.01); there was no statistical difference in sIgA content between the RVLPs + LTB and RVLPs/EPLGA MPs groups, which was 1.26 and 1.23 times higher than the RVLPs group (P < 0.01); the sIgA content of the RVLPs + LTB/EPLGA MPs group is 1.22 times that of the RVLPs/EPLGA MPs group (P < 0.01); the sIgA content of the (RVLPs + LTB/EPLGA MPs). times.2 group was 1.33 and 1.54 times (P <0.001) that of the RVLPs + LTB/EPLGA MPs and Rabisin (i.g.) groups, respectively. After 2 nd immunization, there was no difference in sIgA content for the RVLPs, RVLPs + LTB and RVLPs/EPLGA MPs groups, but 1.8, 2.03 and 2.39 times (P <0.001) for the Saline group; the RVLPS/EPLGA MPs and the RVLPS + LTB/EPLGA MPs have no difference, and the RVLPS + LTB/EPLGA MPs are 1.46 times of the RVLPS + LTB (P < 0.01); there was no difference between the RVLPs + LTB/EPLGA MPs and (RVLPs + LTB/EPLGA MPs). times.2 groups, (RVLPs + LTB/EPLGA MPs). times.2 groups were 1.32 and 1.35 times (P <0.01) the RVLPs/EPLGA MPs and Rabisin (i.g.). After 3 rd immunization, there was no statistical difference in sIgA content between the RVLPs and RVLPs + LTB groups, but 2.1 and 2.16 times higher than those of the Saline group, respectively (P < 0.001); while the RVLPS/EPLGA MPs group is 1.44 times (P <0.001) the RVLPS + LTB/EPLGA MPs group is 1.73 times (P <0.001), (RVLPS + LTB/EPLGA MPs) × 2 group is 1.87 and 4.07 times (P <0.001) the RVLPS + LTB/EPLGA MPs and Rabisin (i.g.) groups.

After 3 rd immunization, the level of specific sIgA antibodies in intestinal fluid was significantly enhanced (P <0.001) in all experimental groups, with the (RVLPs + LTB/EPLGA MPs) × 2 groups inducing the most significant specific mucosal immune responses, 1.16(P <0.01) and 1.70 times (P <0.001) in the RVLPs + LTB/EPLGA MPs and Rabisin (i.g.) groups (fig. 8B).

The experimental results show that oral RVLPs can stimulate the organism to generate mucosal specific sIgA antibodies, the LTB adjuvant can enhance the antigen-induced mucosal immune response level, and the immune effect generated after the EPLGA MPs are wrapped is better. When RVLPs/EPLGA MPa and LTB/EPLGA MPs are jointly and orally delivered at 2 times of dosage, the optimal immune effect can be generated, and the analysis is that due to the slow release effect of the EPLGA MPs, the continuous stimulation of antigens can be realized, so that a longer-lasting immune response is generated, and the mucosal immune response generated by the EPLGA MPs is obviously higher than that of a Rabisin (i.g.) group.

2.3 serum INF-gamma and IL-4 detection

After 3 rd immunization, the trends of INF-gamma and IL-4 in mouse serum were substantially identical (FIG. 9). EPLGA MPs and LTB/EPLGA MPs also induced significantly enhanced IFN-. gamma.and IL-4 levels (P <0.001) compared to Saline groups. Wherein the IFN-gamma level of the Rabisin (i.m.) group is not different from that of the Rabisin (i.g.) group, but the IL-4 level of the Rabisin (i.m.) group is 1.78 times that of the Rabisin (i.g.) group (P < 0.001). Although RVLPs and RVLPs + LTB induced body production of IFN-. gamma.1.44 and 1.46 times (P <0.001) that of the LTB/EPLGA MPs group, they induced body production of IL-4 at levels not statistically different from the LTB/EPLGA MPs group. IFN-. gamma.and IL-4 levels in the RVLPs/EPLGA MPs group were 1.15(P <0.01) and 1.17 times (P <0.001) those in the RVLPs + LTB group, respectively. The RVLPs + LTB/EPLGA MPs and (RVLPs + LTB/EPLGA MPs). times.2 groups induced the body to produce the highest levels of IFN-. gamma.and IL-4, but there was no significant difference between the two groups. INF- γ levels in the (RVLPs + LTB/EPLGA MPs). times.2 groups were 1.14(P <0.01), 1.18(P <0.001), and 1.14 times (P <0.001) the RVLPS/EPLGA MPs, Rabisin (i.m.) and Rabisin (i.g.), respectively; IL-4 levels in the (RVLPs + LTB/EPLGA MPs). times.2 group were 1.43 times (P <0.001) those in the RVLPs/EPLGA MPs group, and were not different from the Rabisin (i.m.) group and were 1.78 times (P <0.001) those in the Rabisin (i.g.).

The above results indicate that RVLPs can enhance the expression level of serum cytokines, and when RVLPs/EPLGA MPa and LTB/EPLGA MPs are combined, the cytokine level generated by the body is stimulated without dose dependence, but IFN-gamma and IL-4 levels are higher than that of Rabisin (i.g.) (P <0.001), and IFN-gamma level is obviously higher than that of Rabisin (i.m.) (P <0.001) although the IL-4 level is not statistically different from that of Rabisin (i.m.).

2.4 peripheral blood CD4⁺And CD8⁺Changes in T cell content

After 3 rd immunization, mice were immunized with CD4 in peripheral blood⁺And CD8⁺T cell content was analyzed (Table 4) when CD4 was present⁺And CD8⁺An elevated ratio of T indicates that a reporter T lymphocyte immune response is activated (Wahlin B, Sander B, Christensson B,

B,et al.Entourage:The Immune Microenvironment Following Follicular Lymphoma[J].Blood Cancer Journal,2012,2(1):e52.)。

in the present invention, the peripheral blood CD4 in the group of Saline, EPLGA MPs, LTB/EPLGA MPs, RVLPs and Rabisin (i.g.)⁺T and CD8⁺T ratios were not statistically different, indicating that oral RVLPs and Rabisin failed to stimulate a significant cellular immune response in the body. Although the RVLPs + LTB group CD4⁺And CD8⁺The T ratio is not different from RVLPs group, but is 1.06 times higher than that of Saline group (P)<0.01). CD4 of RVLPs + LTB group and RVLPs/EPLGA MPs group⁺And CD8⁺T ratios were also not statistically different, but were 1.02 and 1.08 times higher in the RVLPs groups (P)<0.01). There was no statistical difference between the RVLPs/EPLGA MPs and the RVLPs + LTB/EPLGA MPs, but CD4⁺And CD8⁺T ratio is 1.06 and 1.11 times that of RVLPs + LTB group (P)<0.01), which shows that the RVLPs can stimulate the body to generate obvious cellular immune response after being wrapped by the EPLGA MPs, and the adjuvant LTB has the capability of enhancing the cellular immune response. (RVLPs + LTB/EPLGA MPs). times.2 set of CD4⁺And CD8⁺The T ratio increased most significantly, being 1 in the RVLPs + LTB/EPLGA MPs group.09 times (P)<0.01), indicating that two-fold doses of antigen and adjuvant co-administered encapsulated in EPLGA MPs induced a stronger cellular immune response. However, the Rabisin (i.m.) group induced the most significant cellular immune responses, 1.14 times (P) that of the (RVLPs + LTB/EPLGA MPs) x 2 group<0.001)。

Table 4 post-immunization CD4⁺T and CD8⁺Mean and ratio of T cell content

3. Evaluation of biological safety of mice

To assess whether vaccine components would have an effect on the normal life activities of mice, we measured and recorded the body weight of mice before and after immunization. As shown in FIG. 10A, the weight of each group of mice was kept normally increased, and the weight of some mice was reduced probably because the gavage caused a certain damage to the mouse esophagus, so that the mice had reduced food intake, but the weight quickly recovered to normal increase, indicating that the prepared vaccine did not significantly affect the normal growth of the mice. After completion of immunization, major organs of each group of mice were aseptically taken out, and the morphology of the major organs was observed by HE staining. Compared with the Saline group, the major organs of the mice of each administration group had no significant histomorphological changes (fig. 10B), which fully indicates that the vaccine components did not adversely affect the normal vital activities of the mice.

In conclusion, in the research, the EPLGA MPs are respectively loaded with RVLPs and LTB, and the mucosal and systemic immune effects generated after the combined administration of the EPLGA MPs and the LTB are evaluated. The prepared RVLPs/EPLGA MPs are spherical or elliptical with smooth surface and uniform size (168.20 +/-39.28 microns). After the mCherry/EPLGA MPs are perfused into the stomach, the intestinal tracts of mice are taken out at different time points for imaging, and the fluorescence distribution shows that the prepared EPLGA MPs can smoothly leave the stomach to reach the intestinal tracts and release antigens. In vitro release experiments show that the EPLGA MPs have pH responsiveness, are basically complete within 2h under the condition of pH1.2, only release less than 10% of antigen, slowly crack and release RVLPs under the condition of pH6.8, and have the cumulative release rate of about 95% within 24 h. Most importantly, FTIR and CD spectroscopic results show that the native structure of EPLGA MPs loaded RVLPs and LTB is not altered and is stable for at least 3 months at 4 ℃.

The results of oral immunization of Balb/c mice show that the RVLPs can induce the body to generate corresponding antigen-specific humoral, cellular and mucosal immune responses, and the RVLPs and LTB loading in the EPLGA MPs can induce the body to generate stronger immune responses after combined administration. The immune response reached the highest level at 3 rd immunization. First, the RVLPs + LTB/EPLGA MPs and (RVLPs + LTB/EPLGA MPs) x 2 groups induced no statistical difference in antigen-specific IgG content, but were significantly higher than the other experimental groups and 1.36 and 1.35 times (P <0.001) that of Rabisin (i.m.). Secondly, the content of antigen-specific IgG1 and IgG2a antibodies in the (RVLPs + LTB/EPLGA MPs) multiplied by 2 group is obviously higher than that in other experimental groups and Rabisin (i.g.) group (P <0.001), and the ratio of IgG1 to IgG2a is more than 1, which indicates that the immune response of Th2 type is inclined; whereas the IgG1 and IgG2a antibody contents of the Rabisin (i.m.) group were 1.59 and 2.01 times (P <0.001) as high as those of the (RVLPs + LTB/EPLGA MPs). times.2 group. Furthermore, the ratio of CD4+ to CD8+ T in the (RVLPs + LTB/EPLGA MPs) × 2 group was significantly increased (P <0.001) compared to the other experimental group and the Rabisin (i.g.) group, but the ratio of CD4+ to CD8+ T after immunization in the Rabisin (i.m.) group was 1.14 times (RVLPs + LTB/EPLGA MPs) × 2 group (P < 0.001). It was demonstrated that (RVLPs + LTB/EPLGA MPs). times.2 induced the strongest cellular immune response in all experimental groups, although lower than the Rabisin (i.m.) group, but significantly higher than the Rabisin (i.g.) group.

After the 3 rd immunization, the sIgA content was significantly increased in all experimental groups and in the Rabisin (i.g.) group, except for the Rabisin (i.m.) group, where the antigen-specific sIgA titer was highest in feces and intestinal fluids of (RVLPS + LTB/EPLGA MPs). times.2 group. (RVLPs + LTB/EPLGA MPs). times.2 the sIgA content in feces was 1.87 and 4.07 times (P <0.001) that of RVLPs + LTB/EPLGA MPs and Rabisin (i.g.) groups, while the sIgA antibody content in intestinal fluids was 1.16(P <0.01) and 1.70 times (P <0.001) that of RVLPs + LTB/EPLGA MPs and Rabisin (i.g.). The above results indicate that oral administration of RVLPs stimulates the body to produce mucosal-specific sIgA antibodies, which produces the best immune effect when RVLPs/EPLGA MPa and LTB/EPLGA MPs are co-orally delivered at twice the dose. Second, the RVLPs + LTB/EPLGA MPs and (RVLPs + LTB/EPLGA MPs). times.2 groups induced the body to produce the highest levels of IFN-. gamma.and IL-4(P <0.001) compared to the other experimental groups. The INF-gamma levels of the RVLPs + LTB/EPLGA MPs and (RVLPs + LTB/EPLGA MPs). times.2 groups were 1.11 times (P <0.01) and 1.14 times (P <0.001) the RVLPs/EPLGA MPs and Rabisin (i.m.) groups, respectively, and the IL-4 levels were 1.40 times (P <0.001) and 1.43 times (P <0.001) the RVLPs/EPLGA MPs and Rabisin (i.g.) groups, respectively, without difference from the Rabisin (i.m.) group. The above results show that oral administration of RVLPs + LTB/EPLGA MPs can enhance the expression level of body cytokines, and the effect is higher than that of the commercial vaccine, but the dosage is not dependent. Finally, the results of HE staining of the body weight and major organs of the mice indicate the good safety of the vaccine components of this experiment.

In the present invention, the amino acid sequence of RVGP (SEQ ID NO.1)

MVPQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFS YMELKVGYILAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYN WKMAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVADLDPYDRSLHSRVFPSGK CPGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYK SLKGACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPDQLVNLHDFRSDEIEHLVVE ELVRKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVR TWNEILPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLESSVI PLVHPLADPSTVFKDGDEAEDFVEVHLPDVHNQVSGVDLGLPNWGKYVLLSAGALTA LMLIIFLMTCCRRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESHKSGGQTRL

Amino acid sequence of RVMP (SEQ ID NO.2)

MNFLRKIVKNCRDEDTQKPSPVSAPLDDDDLWLPPPEYVPLKELTSKKNMRNFCIDGG VKVCSPNGYSFRILRHILKSFDEIYSGNHRMIGLVKVVIGLALSGSPVPEGMNWVYKLR RTFIFQWADSRGPLEGEELEYSQEITWDDDTEFVGLQIRVIAKQCHIQGRIWCINMNPRA CQLWSDMSLQTQRSEEDKDSSLLLE

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.

Sequence listing

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<120> an oral rabies virus-like particle vaccine and a preparation method thereof

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Claims

1. An oral rabies virus-like particle vaccine is characterized by comprising broad-spectrum rabies virus-like particle antigen and a drug carrier,

wherein the broad-spectrum rabies virus-like particle antigen comprises a CVS strain rabies virus glycoprotein RVGP and a matrix protein RVMP, the polynucleotide sequence of the CVS strain rabies virus glycoprotein RVGP is shown as SEQ ID NO.1, the polynucleotide sequence of the matrix protein RVMP is shown as SEQ ID NO.2,

the drug carrier is composed of PLGA and

a mixed carrier consisting of L100, and a carrier,

the mass ratio of L100 to PLGA is 1: 5-1: 1,

the concentration ratio of the broad-spectrum rabies virus-like particle antigen to PLGA is 1: 5-1: 10.

2. The oral rabies vims-like particle vaccine according to claim 1, wherein:

wherein,

3. The oral rabies vims-like particle vaccine according to claim 1, wherein:

wherein the concentration of the broad-spectrum rabies virus-like particle antigen is 5mg/mL, and the concentration of PLGA is 25-50 mg/mL.

4. The oral rabies vims-like particle vaccine according to claim 1, wherein:

wherein the concentration of PLGA is 50 mg/mL.

5. The oral rabies vims-like particle vaccine according to claim 1, wherein:

the oral rabies virus-like particle vaccine also comprises a mucosal immune adjuvant LTB, wherein the content of the LTB is 20-100 ug/mL.

6. The method of preparing an oral rabies vims-like particle vaccine according to claim 1, comprising the steps of:

Forming O/W1 type primary emulsion after vortex oscillation in L100 ethanol mixed oil phase, wherein the volume ratio of the broad-spectrum rabies virus-like particle antigen to the ethyl acetate and the ethanol is 2:10: 5;

4) centrifuging the solidified RVLPs/EPLGAMPs at 4 ℃, 300rpm for collection, cleaning the PVA and the non-coated antigen on the surface of the RVLPs/EPLGAMPs by using ultrapure water, and collecting all centrifuged supernatant;

5) resuspending RVLPs/EPLGAMPs with appropriate amount of sterile PBS, freezing at 4 deg.C, -20 deg.C and-80 deg.C, vacuum freeze drying for 24 hr, sealing, and storing at-20 deg.C.

7. The method of claim 6, wherein:

wherein, in the step 1), the concentration of the broad-spectrum rabies virus-like particle antigen is 5mg/mL, the concentration of PLGA is 50mg/mL,

the concentration of L100 was 25 mg/mL.

8. The method of claim 6, wherein:

wherein, in the step 2), the rotating speed of the magnetic stirring is 300-400 rpm.

9. The method of claim 6, wherein:

wherein, in the step 4), the number of times of cleaning by adopting ultrapure water is 3.