CN114272367B

CN114272367B - Oral rabies virus-like particle vaccine and preparation method thereof

Info

Publication number: CN114272367B
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: 2023-07-07
Anticipated expiration: 2041-12-03
Also published as: CN114272367A

Abstract

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

L100 and PLGA are used as main materials to prepare the oral acid-resistant slow microsphere. The mucosa immune adjuvant LTB is used for improving the antigenicity of the oral RVLPs and enhancing the immune effect. By designing a protein drug carrier suitable for oral delivery to intestinal administration, various physiological disorders of the gastrointestinal tract can be successfully overcome, and the protein drug carrier can be directly delivered to the intestinal tract, and the immune effect caused by the protein drug carrier is subjected to preliminary immunological evaluation in response to antigen release under the pH condition of the intestinal tract. The oral rabies virus-like particle vaccine can improve the actual utilization efficiency of protein medicines, improve the treatment or prevention effect of the vaccine, and is hopeful to lay an important foundation for expanding the rabies vaccine inoculation range in China, eliminating rabies from animal sources, improving the safety of the vaccine and researching and 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 neurotropic zoonotic disease initiated by rabies virus (RABV), 99% of cases of human rabies death are mediated by dogs, vaccination is the most effective means of preventing transmission of RABV (Zhang W J, zheng X, cheng N, et al Isatis Indigotica Root Polysaccharides as Adjuvants for an Inactivated Rabies Virus Vaccine [ J ]. International Journal of Biological Macromolecules,2016, 87:7-15.). The control of the number of puppies, large-scale or forced parenteral vaccination campaigns on domestic dogs and wild animals, and advances in oral vaccination of wild animals have successfully destroyed land predator-mediated human rabies in several developed countries. However, thousands of people die annually in developing countries due to financial, logistical, and other impediments, inability to control dog numbers and to 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, gargard M S, et al Rabies Vaccination in Dogs Using a Dissolving Microneedle Patch [ J ] Journal of Controlled Release, 2016, 239:19-26.).

ORV plays a decisive role in global elimination of dog-mediated human rabies, and specific practical activities should be urgently developed to promote rapid and large-scale use of ORV that is safe and cost-effective. Although several self-replicating biologics, including modified live viruses, attenuated live viruses and recombinant viruses, have been evaluated orally in canine populations, there is currently no suitable ORV developed due to safety concerns (Cliquet F, guiot A L, aubert M, et al Oral Vaccination of Dogs: A Well-Studied and Undervalued Tool for Achieving Human and Dog Rabies Elimination [ J ]. Veterinary Research,2018,49 (1): 61).

The oral administration has the advantages of convenience, rapidness, safety, good patient compliance and the like compared with injection administration, and no special inoculating personnel are needed. Second, oral vaccines can stimulate systemic and mucosal immune responses, establishing a broader and lasting protection. However, oral administration is challenging, especially for protein drugs, and it is desirable to overcome the harsh gastrointestinal environment, avoiding induction of immune tolerance, to achieve effective protection (renus, han Y, dhakal S, et al, chitosan-Adjuvanted Salmonella Subunit Nanoparticle Vaccine for Poultry Delivered through Drinking Water and Feed [ J ]. Carbohydrate Polymers,2020, 243:116434.).

Protein drugs have absolute safety compared with viruses, wherein VLPs have higher stability and immunogenicity than polypeptides, and are therefore hot spots for protein drug development, and suitable delivery vehicles are the best means for achieving oral targeting and release of such drugs (Chen X Y, butt A M, amin M C I M.enhanced Paracellular Delivery of Vaccine by Hydrogel Microparticles-Mediated Reversible Tight Junction Opening for Effective Oral Immunization [ J ]. Journal of Controlled Release,2019,311-312:50-64.).

PLGA and EL100 are FDA approved materials that can be used in humans. PLGA NPs have many applications in the delivery of injectable vaccines (Kole S, qadiri S N, shin S M, et al PLGA Encapsulated Inactivated-visual Vaccine: formulation and Evaluation of Its Protective Efficacy against Viral Haemorrhagic Septicaemia Virus (Vhsv) Infection in Olive Flounder (Paralichthys Olivaceus) Vaccinated by Mucosal Delivery Routes [ J)].Vaccine,2019,37(7):973-983.Umerska A, Gaucher C,Ampuero F O,et al.Polymeric Nanoparticles for Increasing Oral Bioavailability of Curcumin[J]Antioxidants (Basel), 2018,7 (4): 46); eudragit protects the drug from acidic conditions, rajasreee (Rajasreee P H, paul W, shalma C P, et al Eudragit Encapsulated Cationic Poly (Lactic-Co-Glyclic Acid) Nanoparticles in Targeted Delivery of Capecitabine for Augmented Colon Carcinoma Therapy [ J ] ]Journal of Drug Delivery Science and Technology,2018,46:302-311), etc

S100 is coated on the surface of PLGA NPs carrying drugs, and is used as a novel targeting drug delivery system for treating colon cancer (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.) etc. loaded with BSA>

Oral immunization of L100-capped mannosylated chitosan NPs was found to elicit better systemic IgG and mucosal IgA antibody responses.

At present, the related research on the rabies virus-resistant oral vaccine is less, and the related oral vaccine which can cope with the severe gastrointestinal tract environment is not seen.

Disclosure of Invention

The invention aims at the problems, aims at developing an oral rabies virus-like particle vaccine, and provides a design idea for oral administration of protein drugs.

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

the invention firstly prepares oral rabies virus-like particle vaccine RVLPs/EPLGA MPs, and after in vitro characterization, the freeze-dried RVLPs/EPLGA MPs are subjected to immunogenicity evaluation on stomach-perfused mice, and preliminary evaluation is carried out on cell immune response generated by the vaccine.

In a first aspect of the invention, there is provided an oral rabies virus-like particle vaccine comprising a broad spectrum rabies virus-like particle antigen and a pharmaceutical 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 prepared from PLGA and

l100-composed hybrid vector,>

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 is1:5～1:10。

Preferably, the method comprises the steps of,

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 more preferably the concentration of PLGA is 50mg/mL.

Preferably, the oral rabies virus-like particle vaccine also comprises a mucous membrane 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 described above, comprising the steps of:

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

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

2) Dripping the obtained colostrum into 1% PVA with 10-12 times of volume under the action of magnetic stirring to form W2/O/W1 type compound emulsion;

3) Continuously stirring the compound emulsion at the temperature of 4 ℃ for at least 4 hours, and volatilizing the organic solvent to obtain cured particles RVLPs/EPLGA MPs;

4) Centrifuging and collecting solidified RVLPs/EPLGA MPs at 4 ℃ and 300rpm, washing with ultrapure water, cleaning PVA and non-coated antigen on the surface of the RVLPs/EPLGA MPs, and collecting all the centrifuged supernatant;

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

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

The concentration of L100 was 25mg/mL.

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

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

The preparation conditions are obtained through orthogonal experiments, and experimental results show that when the PLGA concentration 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 (Encapsulation efficieny, 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 above method were sequentially subjected to in vitro characterization, stability analysis, in vitro release experiments and analysis of the residence time of the intestinal tracts of mice.

In vitro characterization results showed that the as-prepared RVLPs/EPLGA MPs were in regular spheres and had good size uniformity (FIG. 2A). SEM results showed that the RVLPs/EPLGA MPs prepared were surface-flattened after vacuum freeze-drying, and had a relatively regular spherical shape with an average particle size in the range of 168.20.+ -. 39.28. Mu.m (FIG. 2B).

Stability results showed that LTB and RVLPs were successfully encapsulated in EPLGA MPs, that RVLPs particle size and morphology were unchanged, and that the natural secondary structure of LTB and RVLPs was not changed. After 3 months of storage at 4 ℃, the morphology and the encapsulation efficiency are not changed obviously.

The results of simulating the drug release in the gastric (pH 1.2) and intestinal (pH 6.8) environments in vivo 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 (phosphate buffered saline) with pH6.8, well overcome the denaturation effect of the severe gastric environment on the proteins, and smoothly deliver the target proteins to intestinal sites (figure 4).

In the analysis of the intestinal residence time of mice, about 20% of mCherry is released in the stomach in a cumulative way after 5 hours of administration, wherein about 10% of mCherry is released in the stomach to be denatured and deactivated, and mCherry released in the intestinal tract is also partially deactivated by the action of proteases in the intestinal tract; after 10h of oral administration, the remaining undegraded microspheres directly leave the small intestine to the large intestine where the protease content is low. The EPLGA MPs prepared by the invention can overcome the acidic environment of the stomach and successfully deliver proteins to the intestinal tract.

Oral immunization of Balb/c mice showed that RVLPs induced a corresponding antigen-specific humoral, cellular and mucosal immune response, and that combined administration of RVLPs and LTB loads in EPLGA MPs induced a stronger immune response. The immune response reached the highest level at immunization 3. The body weight of the mice and the HE staining results of the main organs show that the vaccine components of the experiment have good safety.

The beneficial effects of the invention are as follows:

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

The L100 and PLGA are used as main materials to prepare the oral acid-resistant slow microsphere, RVLPs can be slowly released in the intestinal tract, and the RVLPs with the nano structure can be quickly absorbed by intestinal M cells and other APC. RVLPs with high-density antigen epitope are used together with a mucous membrane adjuvant LTB, so that antigenicity of oral RVLPs can be improved, and immune effect can be enhanced.

The invention can smoothly overcome various physiological disorders of the gastrointestinal tract, directly deliver the protein drug carrier to the intestinal tract by designing the protein drug carrier which is suitable for oral delivery to intestinal tract administration, and respond to the release of antigen under the pH condition of the intestinal tract. Preliminary immunological evaluation is carried out on the immune effect initiated by the RVLPs, and the result shows that the RVLPs can induce the organism to generate corresponding antigen-specific humoral, cellular and mucosal immune responses, and the combined administration of the RVLPs and the LTB load in the EPLGA MPs can induce the organism to generate stronger immune responses; immune response reached the highest level at immunization 3; the body weight of the mice and the HE staining results of the main organs show that the vaccine components of the experiment have good safety.

The oral rabies virus-like particle (Rabies virus like particles, RVLPs) vaccine prepared by the research can improve the actual utilization efficiency of protein medicines, improve the treatment or prevention effect of the vaccine, and is hopeful to lay an important foundation for expanding the inoculation range of rabies vaccine in China and eliminating 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 developing novel virus-like particle oral subunit vaccine.

Drawings

Figure 1 is the preparation of oral rabies virus-like particle (RVLPs) vaccine RVLPs (LTB)/EPLGA MPs and its induced mucosal immune response mechanism.

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

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

Figure 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.

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

FIG. 6 is an in vitro image of the gastrointestinal tract of mice following oral administration of mCherry/EPLGA MPs.

Figure 7 is the content of anti-RVLPs IgG antibody subtype in serum, wherein: (A) IgG; (B) IgG1; (C) IgG2a; (D) IgG1/IgG2a ratio.

Figure 8 is the content of anti-RVLPs sIgA antibodies in faeces and intestinal fluid, 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 serum of various dosed mice, wherein: (a) INF- γ; (B) IL-4.

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

Detailed Description

The present invention will be described in detail with reference to the drawings and examples thereof, which are provided on the premise of the technical solution of the present invention, and the detailed embodiments and specific operation procedures are given, but the scope of the present invention is not limited to the following examples.

For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages, and other values used in the present application are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, 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. 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 prepared from PLGA and

l100-composed hybrid vector,>

the mass ratio of L100 to 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 mucous membrane immune adjuvant LTB, and RVLPs are used together with the mucous membrane adjuvant LTB, so that the antigenicity of the oral RVLPs can be improved, and the immune effect can be enhanced.

2. Vaccine preparation

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

1) Adding broad spectrum rabies virus-like particle antigen alone or together with LTB to PLGA-dissolved ethyl acetate and dissolved

In the mixed oil phase of the ethanol of L100, O/W1 type colostrum is formed after vortex oscillation;

3) Continuously stirring the compound emulsion at 4 ℃ for at least 4 hours, and volatilizing the organic solvent to obtain cured particles RVLPs (LTB)/EPLGA MPs;

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

5) Re-suspending RVLPs (LTB)/EPLGA MPs with proper amount of sterile PBS, freezing and setting 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. Component optimization

An orthogonal experiment was designed at a 4 factor 3 level (table 1) by optimizing the concentration of PLGA, the mass ratio of PLGA to EL100, the volume of 1% pva, and the stirring speed of the magnetic stirrer using the encapsulation efficiency (Encapsulation efficieny, EE) as a standard. The concentration of free RVLPs in the supernatant was measured by BCA method and EE was calculated as follows:

EE (%) = (total amount of RVLPs dosed-amount of RVLPs in supernatant)/total amount of RVLPs dosed x 100%

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 1% PVA volume is 20mL, the mass ratio of PLGA to EL100 is 2: the best EE is obtained at 1. The encapsulation efficiency of RVLPs/EPLGA MPs after optimization was 97.14.+ -. 1.37%.

When the PLGA concentration is 12.5mg/mL, EE is very low, regardless of other conditions, probably because the low concentration of PLGA cannot saturate with the aqueous protein solution; when the volume of 1% PVA is too large, the obtained EE is also not ideal, and the concentration of PLGA is greatly reduced due to the possibly excessive external water phase; when the mass ratio of PLGA to EL100 is 1, EE is unlikely to be high because, after an excessive amount of EL100 is added, it forms a film together with PLGA to precipitate, so that the oil phase and the water phase cannot mutually saturate.

TABLE 1 factors and levels of orthogonal designs

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

TABLE 2 orthogonal experiment L ₉ (3 ⁴ ) Results

EXAMPLE 2 RVLPs/EPLGA MPs in vitro characterization

After RVLPs/EPLGA MPs were prepared, the morphology was observed using an inverted microscope. After lyophilization, morphology and size were observed using a scanning electron microscope (Scanning electron microscope, SEM).

The RVLPs/EPLGA MPs just prepared were in the form of regular spheres and had good size uniformity (FIG. 2A). SEM results showed that the RVLPs/EPLGA MPs prepared were surface-flattened after vacuum freeze-drying, and had a relatively regular spherical shape with an average particle size in the range of 168.20.+ -. 39.28. Mu.m (FIG. 2B).

EXAMPLE 3 RVLPs LTB/EPLGA MPs stability

1. Structural stability

Fourier infrared (Fourier transform infrared spectrometer, FITR) and circular dichroism (Circular dichroism, CD) spectra were used to analyze whether RVLPs were successfully loaded into EPLGA MPs and whether their secondary structures were intact. To assess the pH responsiveness of the EPLGA MPs, EGFP/EPLGA MPs were incubated with PBS at pH1.2 and pH6.8 for 2, 4 and 8 hours, respectively, and samples were taken to observe the integrity of EGFP/EPLGA MPs. To further determine the storage stability of lyophilized EGFP/EPLGA MPs, the RVLPs/EPLGA MPs after vacuum freeze drying were stored at 4, 25 and 37 ℃ respectively, photographed at regular intervals, and the RVLPs/EPLGA MPs under different storage conditions were lysed to calculate EE while the integrity of the RVLPs was checked using a transmission electron microscope (Transmission electron microscope, TEM).

To confirm successful encapsulation of LTB and RVLPs in EPLGA MPs, FTIR spectroscopy was used to analyze the presence of amide bonds in the protein. As shown in FIG. 3A, the bonds detected at 1544.7cm-1 and 1654.7cm-1 by the LTB/EPLGA MPs confirm the encapsulation of the LTB protein within the EPLGA MPs, as compared to the EPLGA MPs; the bonds detected at 1543.5cm-1 and 1654.5cm-1 by RVLPs/EPLGA MPs confirm the encapsulation of RVLPs proteins within the EPLGA MPs. These two bonds are generated by bending vibrations of the proteinamide I (C=O) and II (N-H) bonds, respectively (Roy K, kanwar R K, subramann, et al, comparative Inhibition of Survivin Using a Cell-Permeable Recombinant Protein Induces Cancer-Specific Apoptosis in Colon Cancer Model [ J ]. International Journal of Nanomedicine,2015, 10:1019-1043.). FTIR spectroscopic results showed that LTB and RVLPs were successfully encapsulated in EPLGA MPs.

To further confirm that the natural secondary structures of LTB and RVLPs within the EPLGA MPs were not altered, the secondary structures of LTB/EPLGA MPs and RVLPs/EPLGA MPs were determined using CD spectroscopy. Wherein the alpha-helices (negative peaks: 222nm and 208 nm), beta-sheets (positive peaks: 195-198nm, negative peaks: 217-218 nm) and random coils (positive peaks: 220nm, negative peaks: 198 nm) constitute a protein secondary structure (Fan K L, jiang B, guan Z, et al, nanobody: A Ferritin-Displayed Nanobody with High Apparent Affinity and Half-Life Extension [ J ]. Analytical Chemistry,2018,90 (9): 5671-5677 ]). As shown in fig. 3B, LTB includes 3 structures of alpha helix, beta sheet and random coil; RVLPs are formed by self-assembly of RVMP and RVGP, RVMP mainly based on beta-sheet and random coil, RVGP mainly based on alpha-helix, beta-sheet and random coil. LTB/EPLGA MPs and RVLPs/EPLGA MPs are consistent with the CD spectral trends of natural LTB and RVLPs, indicating that encapsulation of LTB and RVLPs into EPLGA MPs does not affect their natural secondary structure.

In addition, TEM was used to analyze the integrity of RVLPs encapsulated in EPLGA MPs. After the RVLPs/EPLGA MPs were lysed, the supernatant was concentrated and observed by TEM, which showed that the RVLPs particle size and morphology in the supernatant were not changed, being round or oval NPs of 180-200nm (FIG. 3C). Thus, the EPLGA MPs prepared in this study successfully encapsulate LTB and RVLPs and maintain their structural integrity.

2. Storage stability

RVLPs/EPLGA MPs after vacuum freeze drying are stored under different temperature conditions and photographed periodically. As shown in figure 4B, the RVLPs/EPLGA MPs exhibited significant shrinkage and aggregation after 1 week of storage at 37℃indicating that the storage of RVLPs/EPLGA MPs was not suitable at 37℃and the morphology remained substantially unchanged after 3 months of storage at 4℃and 25 ℃. Thus 10mg of RVLPs/EPLGA MPs were taken for different storage periods at 4℃and 25℃respectively, and after lysis in PBS, protein content was determined using the BCA kit and EE was calculated. The results are shown in figure 4A, where RVLPs/EPLGA MPs were only slightly down-regulated by EE after 3 months of storage at 4℃and were not statistically different from day 0. However, during storage of RVLPs/EPLGA MPs at 25℃EE gradually decreases with time until 3 months, EE decreases by about 15%. Therefore, 4℃is more suitable for preservation of RVLPs/EPLGA MPs.

EXAMPLE 3 RVLPs/EPLGA MPs in vitro Release assay

10mg RVLPs/EPLGA MPs were added to 1mL of release medium and incubated at 100rpm at 37℃and supernatants were 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 gastric lavage, it is necessary to mimic drug release in the gastric (pH 1.2) and intestinal (pH 6.8) environments in vivo. As shown in FIG. 5, the EPLGA MPs delivery system has a pH dependent drug controlled release profile compared to PLGA MPs, RVLPS/EPLGA MPs and LTB/EPLGA MPs release less than 10% of the protein of interest within 2 hours at pH1.2, whereas RVLPS/PLGA MPs burst approximately 70% of the protein of interest within 2 hours at pH 1.2. Although two MPs in simulated intestinal fluid accumulate and release about 95% of target proteins in 24 hours, EPLGA MPs effectively realize the slow release of most of target proteins in PBS with pH of 6.8, well overcome the denaturation effect of the gastric harsh environment on the proteins, and smoothly deliver the target proteins to intestinal tract parts.

EXAMPLE 4 intestinal residence time in RVLPs/EPLGA MPs mice

EPLGA MPs loaded with cherry red fluorescent protein (mCherry) (excitation/emission wavelength 580nm/610 nm) -mCherry/EPLGA MPs were prepared. Balb/c mice were fasted for 12h but normally supplied with water prior to gavage, then were gavaged with PBS resuspended in mCherry/EPLGA MPs (100. Mu.L), and were dissected at 0.5, 1.5, 3, 5, 10h after gavage, gastrointestinal organs were dissected, and photographed using KODAK living animal imaging system.

After mCherry/EPLGA MPs lavaged mice, the mice were dissected at specific time points and taken out of the gastrointestinal tract for photography. As shown in FIG. 6, when administered for 0.5h, both mCherry/EPLGA MPs concentrated in the stomach, after 1.5h, mCherry/EPLGA MPs began to flow slowly from the stomach to the duodenum until 3h, the microspheres were all left from the stomach, reached the small intestine, and gradually flowed from the duodenum to the ileum. Since mCherry/EPLGA MPs are degraded due to the neutral environment of the intestinal tract, mCherry is released and taken up by M cells, the fluorescence intensity at the jejunum and ileum is slightly reduced at 5 h. About 20% of mCherry has been released cumulatively at 5 hours, wherein about 10% of mCherry is released in the stomach to denature and deactivate, while mCherry released in the intestine is also partially deactivated by proteases in the intestine, leaving the non-degraded microspheres directly out of the small intestine to the large intestine with low protease content after 10 hours 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 method

(1) RVLPs/EPLGA MPs were inoculated by gavage into SPF-class female Balb/c mice 6-8 weeks old. Mice were randomly grouped and labeled according to table 3. Mice were fasted but free-drinking 12h after one week post-immunization, gastrinated on

days

7, 21 and 35, and their immune responses were evaluated.

(2) Determination of body weight of mice: the mice were weighed 1 day before immunization, 1 day at fixed time after immunization, 6 times after immunization was completed, and analyzed for changes in body weight.

(3) Collection of mouse faeces and detection of 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 ⁺ Detection of T cells.

(5) Collection of mouse intestinal fluid and detection of antigen-specific sIgA antibodies.

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

TABLE 3 animal experimental grouping and dosing

Sampling operation:

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

(1) and (3) collecting: before immunization and 1 week after each immunization, 15 mice faeces were randomly collected using clean forceps, placed in sterilized EP tubes, marked, and kept at-80 ℃ for later use in triplicate;

(2) And (3) treatment: taking out mouse feces from-80deg.C, grinding feces with syringe core, adding 500 μl of sterilized PBS, continuously blowing to dissolve thoroughly, centrifuging at 12000rpm for 5min at 4deg.C, collecting supernatant, labeling, and preserving at-80deg.C for RVLPs specific sIgA analysis.

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

(1) and (3) collecting: blood was collected by puncturing the mouse orbit with a capillary glass tube 1 week before immunization and after the first two immunizations; 1 week after the 3 rd immunization, the eyeballs were rapidly picked up with sterilized forceps to collect blood;

(2) and (3) treatment: blood samples were allowed to stand at 4℃for 12h, then centrifuged at 1000rpm for 30min at 4℃and the serum was drawn into sterilized EP tubes, labeled, stored at-80℃for analysis of RVLPs specific antibodies IgG, igG1 and IgG2a, serum 1 week after 3 rd immunization was also used for IL-4 and IFN-gamma analysis, and 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 mice comprises the following steps:

(1) and (3) collecting: sterilizing mice killed by cervical dislocation with 75% alcohol 1 week after 3 rd immunization, aseptically cutting small intestine segments, cutting with a scalpel to expose the inner wall of the intestine, and placing in an aseptic EP tube;

(2) And (3) treatment: to the small intestine-loaded EP, 1mL of sterile PBS solution was added, vortexed for 15min, and centrifuged at 12000rpm at 4℃for 10min, and the supernatant was collected and stored at-80℃for RVLPs-specific sIgA analysis.

Detection aspect:

RVLPs specific antibodies IgG, igG1, igG2a and sIgA were detected as follows:

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

(2) closing: 200. Mu.L of 1% BSA was added to the reaction wells and incubated at 37℃for 2h;

(3) sample adding: 100. Mu.L of fecal extract and intestinal juice and serum diluted with 1% BSA (1:100) were added, respectively, and incubated at 37℃for 2h;

(4) adding enzyme-labeled secondary antibodies: to the reaction wells were added 100. Mu.L of HRP conjugated goat anti-mouse polyclonal antibody diluted with 1% BSA (1:10000) and incubated at 37℃for 1h;

(5) adding a substrate solution for color development: adding 100 mu L of color development liquid, and incubating for 20min at 37 ℃ in dark place;

(6) terminating the reaction: after adding 100. Mu.L of 1M H2SO4 and mixing well, the OD value (490 nm) was measured.

(7) The steps (1) - (4) were each completed and washed 6 times with PBST.

The detection of the cytokines IFN-gamma or IL-4, according to the instructions of the IL-4 and IFN-gamma kit, is as follows:

(1) before use, all the reagents are fully and uniformly mixed, so that foam is avoided;

(2) The number of strips required was determined based on the number of experimental wells (blank and standard). Both the sample (containing standard) and the blank should be re-perforated;

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

(4) adding a detection antibody: 50. Mu.L/well of Biotinylated antibody working fluid was added. After mixing, covering a sealing plate film, and incubating for 90min at 37 ℃;

(5) washing the plate: the liquid in the hole is removed, and 300 mu L/well is added with 1X Washing buffer working solution; after 1min of residence, the liquid in the wells was discarded. Repeating for 4 times, and buckling on the filter paper for drying each time;

(6) adding enzyme: 100. Mu.L/well of strepavidin-HRP working solution was added. Covering a sealing plate film, and incubating for 30min at 37 ℃;

(7) washing the plate: repeating the step 5;

(8) color development: 100. Mu.L/well of TMB was added and incubated at 37℃in the absence of light for 5-30min, and termination of the reaction was judged by the depth of color (dark blue) in the wells;

(9) terminating the reaction: 100. Mu.L/well was rapidly added to Stop solution to terminate the reaction.

2. Analysis of results

2.1 serum-specific IgG antibody subtype

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

No specific IgG antibodies were detected in the sera of all groups after immunization 1 (fig. 7A). Following booster immunization, the other groups induced significantly enhanced specific IgG antibody responses, except for the group of Rabisin (i.g.) where no specific IgG antibodies were detected. Wherein, the content of the IgG antibody of the RVLPs group is 2.27 times (P < 0.001) of that of the Saline group; there was no statistical difference in IgG antibody content between rvlps+ltb, RVLPs/EPLGA MPs and rvlps+ltb/EPLGA MPs 3 groups, but 1.43, 1.56 and 1.66 times (P < 0.001), respectively, of RVLPs groups; there was no statistical difference in IgG antibody content between RVLPs+LTB/EPLGA MPs and (RVLPs+LTB/EPLGA MPs). Times.2 groups, but the (RVLPs+LTB/EPLGA MPs). Times.2 groups were 1.21 times (P < 0.01) that of the RVLPs/EPLGA MPs groups; the IgG antibody content of the Rabisin (i.m.) group was highest, 1.67 times (P < 0.001) that of the (rvlps+ltb/EPLGA MPs) x 2 group. After immunization 3, RVLPs+LTB/EPLGA MPs and (RVLPs+LTB/EPLGA MPs) x 2 groups had the highest levels of specific IgG antibodies, but there was no statistical difference between the two groups. The IgG antibody content of the (rvlps+ltb/EPLGA MPs) ×2 group was 1.35 and 4.07 times (P < 0.001) that of the Rabisin (i.m.) and Rabisin (i.g.) groups, respectively. The above results indicate that oral RVLPs are capable of eliciting specific humoral immune responses in the body, and that the adjuvant LTB is capable of enhancing the level of immune responses, which are higher than the group of Rabisin (i.m.) and Rabisin (i.g.) when RVLPs and LTB loads are co-administered in EPLGA MPs (P < 0.001), but without dose dependence.

The results of RVLPs specific IgG1 antibody analysis are shown in figure 7B. Following primary immunization, only the Rabisin (i.m.) group induced a significantly enhanced IgG1 antibody response (P < 0.001). After two booster immunizations, none of the Rabisin (i.g.) groups induced an IgG1 antibody response in the body, while each of the other groups induced a significantly enhanced IgG1 response. After immunization 2, the results of antigen-specific IgG1 antibody content analysis showed that the RVLPs+LTB/EPLGA MPs group was 1.37 times (P < 0.001) than the RVLPs/EPLGA MPs group, and that the (RVLPs+LTB/EPLGA MPs). Times.2 group was 1.37 times (P < 0.001) than the RVLPs+LTB/EPLGA MPs group. After immunization 3, the specific IgG1 antibody content of (rvlps+ltb/EPLGA MPs) ×2 groups was 1.17 (P < 0.01) and 1.59 times (P < 0.001) that of rvlps+ltb/EPLGA MPs and Rabisin (i.g.), respectively. However, after two boosts, the IgG1 antibody content was highest in the Rabisin (i.m.) group, 1.39 and 1.59 fold (P < 0.001) of (rvlps+ltb/EPLGA MPs) x 2 groups, respectively.

In RVLPs specific IgG2a antibody detection (fig. 7C), after primary immunization, specific IgG2a antibody levels were significantly increased for rvlps+ltb/EPLGA MPs, (rvlps+ltb/EPLGA MPs) ×2, rabisin (i.g.) and Rabisin (i.m.) 4 groups only, and there was no significant difference in IgG2a content between rvlps+ltb/EPLGA MPs and (rvlps+ltb/EPLGA MPs) ×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). Times.2 group is 1.13 times (P < 0.01) the RVLPs+LTB/EPLGA MPs group, 1.73 times (P < 0.001) the Rabisin (i.g.) group, and 2.01 times (P < 0.001) the Rabisin (i.m.) group is the (RVLPs+LTB/EPLGA MPs). Times.2 group.

Figures 7B and C demonstrate that the (rvlps+ltb/EPLGA MPs) ×2 group stimulated the organism to produce the most pronounced IgG1 and IgG2a antibody responses compared to the other experimental groups. The levels of IgG1 and IgG2a reflect Th2 and Th1 type cellular immune responses, demonstrating that RVLPs/EPLGA MPs and LTB/EPLGA MPs prepared in this experiment, when administered in combination at twice the dose, are able to induce a significantly enhanced cellular immune response in the body, while not as effective as the commercial inactivated rabies vaccine Rabisin (i.m.) group, but significantly higher than the Rabisin (i.g.) group.

The ratio of IgG1 to IgG2a was calculated after the 3 rd immunization (fig. 7D). The ratio of IgG1 to IgG2a reflects the propensity of Th2 to react with Th 1-type cellular immune responses, and if the ratio is close to 1, it is suggested that the more balanced Th1 and Th 2-type immune responses are induced, and the RVLPs and Rabisin (i.m.) groups belong to this class; if the ratio is greater than 1, it indicates that the IgG1 antibody subtype is dominant and a Th2 type immune response is favored, namely RVLPs+LTB, RVLPs/EPLGAMPs, RVLPs +LTB/EPLGA MPs and (RVLPs+LTB/EPLGA MPs) x 2 group; if the ratio is less than 1, it is stated that the IgG2a antibody subtype is dominant and favors a Th1 type immune response, namely the Rabisin (i.g.) group.

2.2 specific sIgA antibodies in faeces and intestinal juice

The level of mucosal immune response by oral vaccine can be judged by analysis of RVLPs specific sIgA antibody content in mouse feces and intestinal fluid. As can be seen from FIG. 8, the EPLGA MPs, LTB/EPLGA MPs and Rabisin (i.m.) groups, like the Saline group, were unable to stimulate the production of mucosal-specific sIgA antibodies.

RVLPs-specific sIgA antibody levels in feces from all experimental groups and Rabisin (i.g.) groups increased significantly with increasing number of immunizations (figure 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 RVLPs+LTB and RVLPs/EPLGA MPs groups, 1.26 and 1.23 times (P < 0.01) than the RVLPs group, respectively; the sIgA content of RVLPs+LTB/EPLGA MPs group is 1.22 times (P < 0.01) than that of RVLPs/EPLGA MPs group; 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.), respectively. After immunization 2, there was no difference in sIgA content of RVLPs, RVLPs+LTB and RVLPs/EPLGA MPs group, but 1.8, 2.03 and 2.39 times (P < 0.001) of the Saline group; there was no difference between the RVLPs/EPLGA MPs and RVLPs+LTB/EPLGA MPs groups, which were 1.46 times (P < 0.01) that of the RVLPs+LTB group; there was no difference between the RVLPs+LTB/EPLGA MPs and the (RVLPs+LTB/EPLGA MPs) ×2 groups, and the (RVLPs+LTB/EPLGA MPs) ×2 groups were 1.32 and 1.35 times (P < 0.01) the RVLPs/EPLGA MPs and Rabisin (i.g.) groups. After immunization 3, there was no statistical difference in the sIgA content of RVLPs and RVLPs+LTB groups, but 2.1 and 2.16 times that of Saline group (P < 0.001), respectively; while the RVLPs/EPLGA MPs group is 1.44 times (P < 0.001) that of the rvlps+ltb group, the rvlps+ltb/EPLGA MPs group is 1.73 times (P < 0.001) that of the RVLPs/EPLGA MPs group, the (rvlps+ltb/EPLGA MPs) ×2 group is 1.87 and 4.07 times (P < 0.001) that of the rvlps+ltb/EPLGA MPs and the rabin (i.g.) group.

After immunization 3, specific sIgA antibody levels were significantly enhanced in all experimental groups intestinal fluids (P < 0.001), with the specific mucosal immune response induced by (RVLPs+LTB/EPLGA MPs). Times.2 being most pronounced, 1.16 (P < 0.01) and 1.70 times (P < 0.001) for the RVLPs+LTB/EPLGA MPs and Rabisin (i.g.) groups (FIG. 8B).

The experimental results show that the oral RVLPs can excite organisms to generate mucous membrane specific sIgA antibodies, the LTB adjuvant can enhance the level of mucous membrane immune response induced by antigens, and the immune effect generated after the EPLGA MPs are wrapped is better. The best immune effect was produced when RVLPs/EPLGA MPa and LTB/EPLGA MPs were co-orally delivered at 2-fold doses, analyzed as sustained stimulation of antigen was achieved due to the slow release effect of EPLGA MPs, resulting in a longer lasting immune response, which produced a significantly higher mucosal immune response than in the Rabisin (i.g.) group.

2.3 detection of INF-gamma and IL-4 in serum

After immunization 3, the trends of INF-gamma and IL-4 in the serum of mice were substantially identical (FIG. 9). EPLGA MPs also induced significantly enhanced IFN-gamma and IL-4 levels (P < 0.001) compared to LTB/EPLGA MPs compared to Saline group. Wherein there is no difference in IFN- γ levels in the group of Rabisin (i.m.) and Rabisin (i.g.), but the IL-4 level in the group of Rabisin (i.m.) is 1.78 times (P < 0.001) that in the group of Rabisin (i.g.). Although RVLPs and RVLPs+LTB induced organisms produced IFN-gamma 1.44 and 1.46 times (P < 0.001) that produced by the LTB/EPLGA MPs group, they induced no statistical difference in IL-4 levels from the LTB/EPLGA MPs group. IFN- γ and IL-4 levels of the RVLPs/EPLGA MPs group were 1.15 (P < 0.01) and 1.17 times (P < 0.001) respectively of the RVLPs+LTB group. The RVLPs+LTB/EPLGA MPs and (RVLPs+LTB/EPLGA MPs). Times.2 groups induced the highest levels of IFN-gamma and IL-4 in the organism, but there was no significant difference between the two groups. The INF-gamma levels of 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) of the RVLPs/EPLGA MPs, rabisin (i.m.) and Rabisin (i.g.) groups, respectively; IL-4 levels were 1.43 times (P < 0.001) for the (RVLPs+LTB/EPLGA MPs). Times.2 group compared to the RVLPs/EPLGA MPs group, and no difference was found from the Rabisin (i.m.) group to 1.78 times (P < 0.001) for the Rabisin (i.g.) group.

The above results indicate that RVLPs can enhance the expression level of serum cytokines, which stimulate the body to produce cytokine levels that are not dose-dependent when RVLPs/EPLGA MPa and LTB/EPLGA MPs are administered in combination, but IFN- γ and IL-4 levels are higher than that of the group of Rabisin (i.g.) group (P < 0.001), while IL-4 levels produced are not statistically different from that of the group of Rabisin (i.m.), IFN- γ levels are significantly higher than that of the group of Rabisin (i.m.) group (P < 0.001).

2.4 peripheral blood CD4 ⁺ And CD8 ⁺ Variation of T cell content

After immunization 3, CD4 in the peripheral blood of mice ⁺ And CD8 ⁺ T cell content was analyzed (Table 4) for CD4 ⁺ And CD8 ⁺ The ratio of T was increased, indicating that a reporter T lymphocyte immune response was 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, CD4 in peripheral blood of Saline, EPLGA MPs, LTB/EPLGA MPs, RVLPs and Rabisin (i.g.) groups ⁺ T and CD8 ⁺ The T ratio was not statistically different, indicating that oral RVLPs and Rabisin were unable to stimulate a significant cellular immune response in the body. Although CD4 of RVLPs+LTB group ⁺ And CD8 ⁺ The T ratio was not different from that of the RVLPs group, but was 1.06 times higher than that of the Saline group (P<0.01). CD4 of RVLPs+LTB group and RVLPs/EPLGA MPs group ⁺ And CD8 ⁺ Nor did the T ratio have statistical differences, but 1.02 and 1.08 times (P <0.01). There was no statistical difference between RVLPs/EPLGA MPs and RVLPs+LTB/EPLGA MPs groups, but CD4 ⁺ And CD8 ⁺ T ratio is 1.06 and 1.11 times (P<0.01 Indicating that RVLPs, when encapsulated with EPLGA MPs, are able to stimulate a significant cellular immune response in the body and that the adjuvant LTB has the ability to enhance the cellular immune response. CD4 of group (RVLPs+LTB/EPLGA MPs) ×2 ⁺ And CD8 ⁺ The T ratio increased most significantly, 1.09 times (P<0.01 It was shown that co-administration of double doses of antigen and adjuvant with EPLGA MPs encapsulation induced a stronger cellular immune response. However, the cellular immune response induced by the Rabisin (i.m.) group was most pronounced, 1.14 times (P) than that of the (rvlps+ltb/EPLGA MPs) x 2 group<0.001)。

TABLE 4 post-immunization CD4 ⁺ T and CD8 ⁺ Average and ratio of T cell content

3. Mouse biosafety assessment

To assess whether vaccine components would affect normal vital activity in 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 remained normal, and the decrease in weight of some mice was probably due to the damage to the esophagus of the mice caused by gastric lavage, which reduced the intake of food into the mice, but the weight was quickly restored to normal growth, indicating that the prepared vaccine did not significantly affect the normal growth of the mice. After the immunization, the major organs of each group of mice were aseptically removed, and the morphology of the major organs was observed by HE staining. Compared to the Saline group, the major organs of mice in each dosing group had no obvious histomorphology change (fig. 10B), which fully demonstrated that the vaccine components did not adversely affect the normal vital activity of the mice.

In summary, in the study, we used EPLGA MPs loaded with RVLPs and LTB, respectively, to evaluate the mucosal and systemic immune effects generated after their co-administration. The RVLPs/EPLGA MPs prepared are spherical or elliptic with smooth surface and uniform size (168.20 +/-39.28 mu m). After mCherry/EPLGA MPs were lavaged, the intestinal tracts of mice were taken out at different time points for imaging, and fluorescence distribution showed that the prepared EPLGA MPs were able to smoothly leave the stomach to reach the intestinal tracts and release antigen. In vitro release experiments showed that EPLGA MPs were pH responsive, substantially intact at pH1.2 for 2h, releasing less than 10% of the antigen, whereas RVLPs were slowly cleaved and released at pH6.8 with an accumulated release rate of about 95% over 24 h. Most importantly, FTIR and CD spectroscopic results indicate that the natural structure of the EPLGA MPs loaded RVLPs and LTB is not altered and can be stably stored for at least 3 months at 4 ℃.

Oral immunization of Balb/c mice showed that RVLPs induced a corresponding antigen-specific humoral, cellular and mucosal immune response, and that combined administration of RVLPs and LTB loads in EPLGA MPs induced a stronger immune response. The immune response reached the highest level at immunization 3. First, there was no statistical difference in antigen-specific IgG content induced by rvlps+ltb/EPLGA MPs and (rvlps+ltb/EPLGA MPs) ×2 groups, but significantly higher than the other experimental groups and 1.36 and 1.35 times (P < 0.001) that of the Rabisin (i.m.) group. Second, the antigen-specific IgG1 and IgG2a antibody content of the (rvlps+ltb/EPLGA MPs) ×2 group was significantly higher than that of the other experimental groups and the Rabisin (i.g.) group (P < 0.001), and the IgG1 to IgG2a ratio was greater than 1, indicating a propensity to a Th2 type immune response; whereas the IgG1 and IgG2a antibody content of the Rabisin (i.m.) group was 1.59 and 2.01 times (P < 0.001) that of the (rvlps+ltb/EPLGA MPs) x 2 group. In addition, the cd4+ to cd8+ T ratio of the (rvlps+ltb/EPLGA MPs) x 2 group was significantly increased (P < 0.001) over the other experimental and Rabisin (i.g.) groups, but the cd4+ to cd8+ T ratio after immunization of the Rabisin (i.m.) group was 1.14 times (P < 0.001) that of the (rvlps+ltb/EPLGA MPs) x 2 group. It was demonstrated that (rvlps+ltb/EPLGA MPs) ×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 immunization 3, the sIgA content was significantly increased in all experimental and in the Rabisin (i.g.) group, except in the Rabisin (i.m.) group, where the antigen-specific sIgA titers were highest in faeces and intestinal fluids of the (RVLPs+LTB/EPLGA MPs) x 2 group. The sIgA content in the (RVLPs+LTB/EPLGA MPs). Times.2 group of faeces was 1.87 and 4.07 times (P < 0.001) that in the RVLPs+LTB/EPLGA MPs and Rabisin (i.g.) group, while the sIgA antibody content in intestinal fluid was 1.16 (P < 0.01) and 1.70 times (P < 0.001) that in the RVLPs+LTB/EPLGA MPs and Rabisin (i.g.) group. The above results indicate that oral RVLPs can stimulate the production of mucosal specific sIgA antibodies in the body, and that optimal immune effects can be produced when RVLPs/EPLGA MPa and LTB/EPLGA MPs are co-orally delivered at twice the dose. Second, RVLPs+LTB/EPLGA MPs and (RVLPs+LTB/EPLGA MPs). Times.2 groups induced the highest levels of IFN-. Gamma.and IL-4 (P < 0.001) in the organism compared to the other experimental groups. The INF-gamma levels of 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 a difference from the Rabisin (i.m.) groups. The results show that oral RVLPs+LTB/EPLGA MPs are capable of strengthening the expression level of body cytokines and have higher effect than that of available vaccine, but have no dose dependence. Finally, the body weight of the mice and HE staining results of the major organs indicate good safety of the vaccine components of the experiment.

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

MVPQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFS YMELKVGYILAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYN WKMAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVADLDPYDRSLHSRVFPSGK CPGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYK SLKGACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPDQLVNLHDFRSDEIEHLVVE ELVRKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVR TWNEILPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLESSVI PLVHPLADPSTVFKDGDEAEDFVEVHLPDVHNQVSGVDLGLPNWGKYVLLSAGALTA LMLIIFLMTCCRRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESHKSGGQTRL

RVMP amino acid sequence (SEQ ID NO. 2)

MNFLRKIVKNCRDEDTQKPSPVSAPLDDDDLWLPPPEYVPLKELTSKKNMRNFCIDGG VKVCSPNGYSFRILRHILKSFDEIYSGNHRMIGLVKVVIGLALSGSPVPEGMNWVYKLR RTFIFQWADSRGPLEGEELEYSQEITWDDDTEFVGLQIRVIAKQCHIQGRIWCINMNPRA CQLWSDMSLQTQRSEEDKDSSLLLE

While the preferred embodiments of the present invention have been illustrated and described, the present invention is not limited to the embodiments, and various equivalent modifications and substitutions can be made by one skilled in the art without departing from the spirit of the present invention, and these equivalent modifications and substitutions are intended to be included in the scope of the present invention as defined in the appended claims.

Sequence listing

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<120> an oral rabies virus-like particle vaccine and method for preparing the same

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Claims

1. An oral rabies virus-like particle vaccine, which is characterized by comprising a broad-spectrum rabies virus-like particle antigen, a mucosal immune adjuvant LTB and a drug carrier;

the broad-spectrum rabies virus-like particle antigen comprises CVS strain rabies virus glycoprotein RVGP and matrix protein RVMP, wherein 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 prepared from PLGA and

l100-composed hybrid vector,>

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 virus-like particle vaccine according to claim 1, characterized in that:

wherein,,

3. The oral rabies virus-like particle vaccine according to claim 1, characterized in that: 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 virus-like particle vaccine according to claim 1, characterized in that: wherein the concentration of PLGA is 50mg/mL

5. The oral rabies virus-like particle vaccine according to claim 1, characterized in that: wherein the content of the mucosal immune adjuvant LTB is 20-100 ug/mL.

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

1) Adding broad spectrum rabies virus-like particle antigen and LTB simultaneously to ethyl acetate dissolved with PLGA and dissolved with

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

7. The method of manufacturing according to 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 25mg/mL.

8. The method of manufacturing according to claim 6, wherein:

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

9. The method of manufacturing according to claim 6, wherein:

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