WO2023097652A1

WO2023097652A1 - An engineered cell and application thereof

Info

Publication number: WO2023097652A1
Application number: PCT/CN2021/135329
Authority: WO
Inventors: Guangjun NIE; Xiao Zhao; Yale YUE
Original assignee: National Center For Nanoscience And Technology
Priority date: 2021-12-03
Filing date: 2021-12-03
Publication date: 2023-06-08

Abstract

An engineered cell that is capable of secreting one or more vesicle comprising one or more protein or immunogenic fragment thereof when said cell is induced and application thereof.

Description

An Engineered Cell and Application Thereof

BACKGROUND OF THE INVENTION

The development of vaccines has greatly promoted the progress of human health. At present, most vaccines in clinical trials use subcutaneous or intramuscular injections. The immune stimulation of this local injection is limited to the draining lymph nodes. Compared to injection vaccination, orally administered vaccine delivery system (OAVDS) stimulates the intestinal immune system to produce a corresponding immune response. The intestine is the largest immune organ in the body, which possesses 70%-80%of the body's immune cells. Therefore, oral delivery of vaccines is expected to further generate a powerful immune effect by activating the abundant immune system in the intestine. In addition, better patient compliance and lower medical costs also make oral vaccines more attractive.

Compared with injection vaccines, oral vaccines show better patient compliance, especially for children and the elderly. At present, oral vaccines for infectious diseases mainly include polio live attenuated vaccine (OPV) and rotavirus attenuated Live vaccine (RV) . Compared with oral infectious disease vaccines, the development of oral tumor vaccines is still relatively limited.

Tumor vaccines control or eliminate tumors by presenting tumor antigens generated by gene mutations to the immune system to activate tumor-specific T cell-mediated immune responses. The complex gastrointestinal environment and severe intestinal epithelial barrier are two major challenges faced by oral tumor vaccines. However, the development of oral vaccines in the field of tumor therapy is still very limited.

SUMMARY OF THE INVENTION

The present disclosure provides an oral vaccine system based on genetically engineered bacteria, which can overcome the digestive tract barrier, break through the intestinal epithelial barrier, realize the regulation of the protein expression in vivo and effectively activate antigen-specific T cells and/or B cell immune response. The present disclosure provides a cell, wherein said cell is capable of secreting one or more vesicle comprising one or more protein or immunogenic fragment thereof when said cell is induced, and said protein or immunogenic fragment thereof is capable of generating an immune response.

The present disclosure provides a cell, wherein said cell is capable of expressing one or more protein or immunogenic fragment thereof on one or more vesicle when said cell is induced, and said protein or immunogenic fragment thereof is capable of generating an immune response.

The present disclosure provides a vesicle, wherein said vesicle is obtained by a cell of the present application.

The present disclosure provides a method of preparing a vesicle of the present application, said method comprising providing a cell of the present application.

The present disclosure provides a method of preparing a vaccine, said method comprising providing a cell of the present application.

The present disclosure provides a method of the present application, wherein the vaccine comprises oral vaccine.

The present disclosure provides a composition comprising a cell of the present application, and/or a vesicle of the present application, and optionally a pharmaceutically acceptable carrier.

The present disclosure provides a method of generating immune response and/or long-term immune memory to a protein or immunogenic fragment thereof, the method comprising administering to a subject in need thereof an effective amount of a cell of the present application, a vesicle of the present application, and/or a composition of the present application.

The present disclosure provides a method of preventing, ameliorating and/or treating one or more disease, the method comprising administering to a subject in need thereof an effective amount of a cell of the present application, a vesicle of the present application, and/or a composition of the present application.

The present disclosure provides that OMVs are natural vesicles secreted by Gram-negative bacteria, rich in pathogen-associated molecular patterns (PAMP) . And the present disclosure shows that OMVs derived from intestinal bacteria reach the lamina propria by penetrating the mucus layer and the intestinal epithelial barrier, and interact with immune cells in the lamina propria to participate in immune regulation. The present disclosure also provides that Fc receptor (FcRn) protein is expressed on the surface of DCs, which allows DCs to interact with Fc fragments through affinity. The present disclosure also provides that OMVs show obvious advantages in penetrating the intestinal barrier and presenting antigens to dendritic cells (DCs) . Considering the huge clinical application potential of oral vaccines at present, combined with the unique advantages of genetically engineered bacteria in oral medicine, the present disclosure develops an oral vaccine system based on genetically engineered bacteria.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCES

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWING

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “figure” and “FIG. ” herein) , of which:

FIG. 1 illustrates process flow diagram of genetically engineered Escherichia coli and engineered OMVs;

FIG. 2A-B illustrate the visual view and WB characterization of the induced expression of Luc protein in Escherichia coli transformed into the ClyA-Luc-HA plasmid;

FIG. 3A-B illustrate the in vivo metabolic distribution diagram of Escherichia coli transformed into ClyA-Luc-HA plasmid;

FIG. 4A-B illustrate the morphology, particle size and WB characterization of OMVs expressing ClyA-HA-OVA-mFc, scale bar, 100nm;

FIG. 5 illustrates the localization of the above OMVs and control OMVs through mouse intestinal epithelium and DCs, scale bar, 100μm;

FIG. 6 illustrates the maturation of DC cells in the lamina propria after the mice are stimulated by oral tumor vaccine;

FIG. 7 illustrates the proportion of OVA tetramer-positive cells in the spleen cells of mice stimulated by oral tumor vaccine;

FIG. 8A-B illustrate graph showing the treatment results of the oral tumor vaccine system in the B16-OVA lung metastasis model, scale bar: left, 1 cm; right: 50μm;

FIG. 9A-B illustrate the immune memory caused by the invented oral vaccine system and the preventive effect of B16-OVA lung metastases, scale bar, 1cm;

FIG. 10 illustrates the therapeutic effect of oral tumor vaccine system on MC38 subcutaneous tumors;

FIG. 11 illustrates the secretion of IFN-γ factor detected by ELISASPOT after the spleen lymphocytes of mice are stimulated by Adpgk polypeptide after the treatment is over;

FIG. 12 illustrates the WB characterization of HBsAg-HA protein in bacterial cells and OMVs after DE3 Escherichia coli was successfully transferred into OmpA-HBsAg-HA-mFc plasmid and induced at 42℃;

FIG. 13 illustrates morphological characterization diagram of OMVs expressing OmpA-HBsAg-HA-mFc protein;

FIG. 14 illustrates schematic diagram of the immunogenicity and biosafety experiment of the oral vaccine for infectious disease hepatitis B;

FIG. 15 illustrates the concentration of HBsAg antibody in the serum of mice inoculated with oral hepatitis B vaccine on the 21st day. Mice were taken orally with PBS as a negative control;

FIG. 16 illustrates the results of HBsAg antibody titer in the serum of mice inoculated with oral hepatitis B vaccine on the 21st day;

FIG. 17 illustrates shows the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice 21 days after receiving oral vaccine;

FIG. 18 illustrates schematic diagram of mice infected with hepatitis B virus receiving oral vaccine treatment;

FIG. 19 illustrates the distribution of HBsAg antibody concentration in the serum of mice at different time points after receiving oral vaccine treatment.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

The term “Fc fragment” generally refers to a C-terminal region of an immunoglobulin heavy chain. The “Fc fragment” may be a native sequence Fc fragment or a variant Fc fragment. Although the generally accepted boundaries of the Fc fragment of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue, variants comprise only portions of the Fc region and can include or not include the carboxyl-terminus. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. In some embodiments, variants having one or more of the constant domains are contemplated. In other embodiments, variants without such constant domains (or with only portions of such constant domains) are contemplated.

The term “immune response” generally refers to an integrated bodily response to a target such as an antigen and preferably refers to a cellular immune response or a cellular as well as a humoral immune response. The immune response may be protective/preventive/prophylactic and/or therapeutic.

The term “immunogenicity” generally relates to the relative effectivity to induce an immune response that is preferably associated with therapeutic treatments, such as treatments against cancers. As used herein, the term “immunogenic” relates to the property of having immunogenicity.

The term “immunogenic fragment” generally refers to a polypeptide fragment that retains, at least in part, the immunogenicity of the protein from which the polypeptide fragment is derived. For example, an immunogenic fragment of the influenza virus M2 protein refers to a fragment of the M2 protein that retains at least partially immunogenicity, such as M2e.

The term “induced” coding sequence or functional RNA generally refers to a coding sequence or functional RNA which is selectively expressed in response to the presence of an endogenous or exogenous stimulus. The term “inducible promoter” generally refers to promoters that selectively express a coding sequence or functional RNA in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals. Inducible or regulated promoters include, for example, promoters induced or regulated by light, heat, stress, flooding or drought, salt stress, osmotic stress, phytohormones, wounding, or chemicals such as ethanol, abscisic acid (ABA) , jasmonate, salicylic acid, or safeners.

The term “OMV (outer membrane vesicles) ” generally refers to vesicles released from the outer membranes of bacteria. For example, vesicles isolated from the medium and/or sheared from cells, or proteoliposomic vesicles obtained from the outer membrane of a Gram-negative bacterium

The term “operons” generally refers to a genetic unit that controls gene expression. An operon typically comprises one or more genes that encode one or more polypeptide (s) or RNA (s) and the adjacent regulatory region (or regions) that controls the transcription of the genes. The regulatory region typically comprises a promoter and an operator. The coding region of a prokaryotic gene is historically termed a “cistron. ” Operons that contain multiple cistrons are termed “polycistronic. ” The genes in a polycistronic operon are typically related in function and are typically co-transcribed as a single unit and expressed in a coordinated manner.

The term “vesicle” generally refers to an enclosed compartment that is separated from its surrounding local environment. For example, by a layer of a water-immiscible substance. In biological systems, a vesicle is a small, subcellular enclosed compartment separated from the cytosol by at least one lipid bilayer.

The present disclosure provides a cell, wherein said cell may be capable of secreting one or more vesicle comprising one or more protein or immunogenic fragment thereof when said cell is induced, and said protein or immunogenic fragment thereof may be capable of generating an immune response.

The present disclosure provides a cell, wherein said cell may be capable of expressing one or more protein or immunogenic fragment thereof on one or more vesicle when said cell is induced, and said protein or immunogenic fragment thereof may be capable of generating an immune response.

For an example of the cell, wherein said protein or immunogenic fragment thereof may be derived from bacteria, virus, parasite or tumor.

For an example of the cell, wherein said protein or immunogenic fragment thereof may be derived from melanoma, and/or colon tumor.

For an example of the cell, wherein said protein or immunogenic fragment thereof may be derived from hepatitis virus, influenza virus, and/or Bacillus tetanus.

For an example of the cell, wherein said protein or immunogenic fragment thereof may comprise one or more antigen.

For an example of the cell, wherein said protein or immunogenic fragment thereof may be selected from the group consisting of: OVA (ovalbumin) , CEA (carcinoembryonic antigen) , Adpgk (ADP Dependent Glucokinase) , MUC1 (Mucin-1) , HBsAg (hepatitis B surface antigen) , H3N2 influenza A virus antigen, and tetanus toxin C.

For an example of the cell, wherein said protein or immunogenic fragment thereof may be capable of being expressed on the membrane of said vesicle. For an example of the cell, wherein part of said protein or immunogenic fragment thereof may be capable of being expressed on the membrane of said vesicle. For an example of the cell, wherein part of said protein or immunogenic fragment thereof may be capable of being expressed on the outside of the membrane of said vesicle.

For an example of the cell, wherein said protein or immunogenic fragment thereof may be fused to one or more surface protein.

For an example of the cell, said surface protein may be selected from the group consisting of: ClyA (Cytolysin A) , OmpA (Outer membrane protein A) and Hbp (Hemoglobin-binding protease) .

For an example of the cell, wherein the C-terminus of said surface protein may be fused directly or indirectly to the N-terminus of said protein or immunogenic fragment thereof.

For an example of the cell, wherein said protein or immunogenic fragment thereof may be fused to an Fc fragment.

For an example of the cell, said Fc fragment may comprise a Fc fragment of an immunoglobulin.

For an example of the cell, wherein said immunoglobulin may be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM and IgA.

For an example of the cell, said cell may be capable of expressing said protein or immunogenic fragment thereof in the gastrointestinal (GI) when said cell is induced.

For an example of the cell, expression of said protein or immunogenic fragment thereof may be configured to be induced by pH, temperature, magnetic, heat, and/or chemical.

For an example of the cell, one or more gene encoding said protein or immunogenic fragment thereof may be configured to be induced by pH, temperature, magnetic, heat, and/or chemical.

For an example of the cell, said one or more chemical may be selected from the group consisting of: arabinose, IPTG, tetracycline, and/or doxycycline.

For an example of the cell, expression of said protein or immunogenic fragment thereof may be controlled by one or more inducible promoter.

For an example of the cell, wherein said inducible promoter may be selected from the group consisting of: pH-inducible promoter, temperature-inducible promoter, magnetic-inducible promoter, heat-inducible promoter, and chemical-inducible promoter.

For an example of the cell, wherein one or more said inducible promoter may be selected from the group consisting of: arabinose-inducible promoter, IPTG-inducible promoter, tetracycline-inducible promoter, and doxycycline-inducible promoter.

For an example of the cell, wherein one or more said inducible promoter may be controlled by one or more operons.

For an example of the cell, wherein said operon may be selected from the group consisting of: tetracycline operon, lactose operon, galactose operon, and arabinose operon.

For an example of the cell, wherein said cell may comprise one or more bacterium.

For an example of the cell, wherein said bacterium may comprise one or more gram-negative bacterium.

For an example of the cell, wherein said bacterium may be selected from the group consisting of: Escherichia coli, Salmonella, Staphylococcus, Lactobacillus, Bacillus, Pseudomonas, and Bifidobacterium pseudolongum.

For an example of the cell, said vesicle may comprise a cell-derived vesicle. For an example of the cell, said vesicle may comprise a bilayer structure. For an example of the cell, said vesicle may comprise an outer membrane vesicle (OMV) .

For an example of the cell, said vesicle may have a diameter of about 30 nm to about 150 nm. For an example of the cell, said vesicle may have a diameter of about 10 nm to about 150 nm, about 20 nm to about 150 nm, about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 100 nm to about 150 nm, 10 nm to about 100 nm, about 20 nm to about 100 nm, about 30 nm to about 100 nm, about 40 nm to about 100 nm, about 50 nm to about 100 nm, 10 nm to about 50 nm, about 20 nm to about 50 nm, about 30 nm to about 50 nm, about 40 nm to about 50 nm, or about 10 nm to about 20 nm. For example, the particle size distribution range of ClyA-OVA-mFc OMVs was 15.18-43.68 nm, and the average particle size was about 17.71 nm. For an example of the cell, said diameter may be determined by dynamic light scattering (DLS) and/or transmission electron microscopy (TEM) .

For an example of the cell, wherein said cell may be capable of secreting one or more vesicle comprising one or more protein or immunogenic fragment thereof when said cell is induced, and said protein or immunogenic fragment thereof may be capable of generating an immune response, and said protein or immunogenic fragment thereof may be fused to an Fc fragment.

The present disclosure provides a vesicle, wherein said vesicle may be obtained by a cell of the present application.

For example, wherein the vaccine may comprise oral vaccine.

The present disclosure provides a method of generating immune response and/or long-term immune memory to a protein or immunogenic fragment thereof, the method comprising administering to a subject in need thereof an effective amount of a cell of the present application, a vesicle of the present application, and/or a composition of the present application. For example, administering to a subject in need thereof an effective amount of a cell of the present application, a vesicle of the present application, and/or a composition of the present application could maintain the antibody level and/or antibody titer. For example, said antibody is capable of specially binding to said protein or immunogenic fragment thereof.

The present disclosure provides a cell of the present application, a vesicle of the present application, and/or a composition of the present application, for use in generating immune response and/or long-term immune memory to a protein or immunogenic fragment thereof.

The present disclosure provides a use of cell of the present application, a vesicle of the present application, and/or a composition of the present application in the preparation of a medicament for generating immune response and/or long-term immune memory to a protein or immunogenic fragment thereof.

The present disclosure provides a cell of the present application, a vesicle of the present application, and/or a composition of the present application, for use in preventing, ameliorating and/or treating one or more disease.

The present disclosure provides a use of cell of the present application, a vesicle of the present application, and/or a composition of the present application in the preparation of a medicament for preventing, ameliorating and/or treating one or more disease.

For example, wherein comprising orally administered. For example, further comprising providing one or more condition inducing the expression of said protein or immunogenic fragment thereof. For example, further comprising inducing the expression of said protein or immunogenic fragment thereof when said cell is located in the intestine. For example, the condition inducing the expression may be provided 2 hours or more after said cell is administered. For example, said cell is administered, and after 2 hours or more, 5 hours or more, 12 hours or more, or 24 hours or more of the administration of said cell, the condition inducing the expression may be provided.

For example, said condition may be selected from the group consisting of: pH, temperature, magnetic, heat, and/or chemical.

For example, said one or more chemical may be selected from the group consisting of: arabinose, IPTG, tetracycline, and/or doxycycline.

For example, further comprising providing said condition after administering an effective amount of a cell of the present application, a vesicle of the present application, and/or a composition of the present application.

For example, said subject may be affected by one or more disease and/or condition.

For example, said disease and/or condition may comprise an infectious and/or tumoral disease.

For example, said disease and/or condition may comprise hepatitis B, melanoma, and/or colon tumor.

For example, said disease and/or condition may comprise melanoma, colon tumor, breast tumor, and/or cervical tumor.

For example, said disease and/or condition may comprise infection of hepatitis B virus, pertussis virus, tetanus virus, invasive Haemophilus influenzae type B (Hib) , influenza virus and/or human papillomavirus.

A recombinant plasmid that can fusion express OMV membrane protein ClyA-HA tag-tumor antigen-mouse Fc fragment (ClyA-HA-Ag-mFc) gene or OMV membrane protein ClyA-Luciferase-HA tag (ClyA-Luciferase-HA tag) -HA) to obtain recombinant plasmid 1 (ClyA-Luc-HA) , plasmid 2 (ClyA-HA-OVA-mFc) and plasmid 3 (ClyA-HA-Adpgk-mFc) that can express the fusion protein.

The recombinant plasmid mentioned above uses the pBAD-HisA plasmid of Jinweizhi Company as the backbone plasmid; The recombinant plasmid as described above; the inducible promoter is L-arabinose. Introduce any of the above recombinant plasmids into TOP10 Escherichia coli to obtain genetically engineered TOP10 Escherichia coli.

The specific design of an oral tumor vaccine system includes oral genetic engineering of Escherichia coli and arabinose. Through the in vivo control of the protein expression of genetically engineered bacteria, OMVs displaying the target antigen are produced in situ in the intestine and relying on the natural characteristics OMVs, to achieve crossing the intestinal epithelial barrier, and effectively activate the antigen-specific T cell immune response.

The final concentration of L-arabinose used to induce genetically engineered bacteria to express the target protein in vitro is 2 g/L; The oral concentration of L-arabinose used to induce genetically engineered bacteria to express the target protein in vitro is 20 g/L. In order to induce genetically engineered bacteria to express the target protein in vivo, the mice drank L-arabinose water for 12 hours.

The functional realization of an oral tumor vaccine system includes: genetically engineered bacteria overcome the gastrointestinal barrier to reach the intestine, induce the production of engineered OMVs (ClyA-HA-Ag-mFc OMVs) in the intestine, and OMVs penetrate the intestinal barrier and activate immune system;

In another embodiment, the invention provides an oral infectious disease vaccine for the treatment of hepatitis B.

A recombinant plasmid that can fusion express the OmpA hepatitis B surface antigen-mouse Fc fragment (OmpA-HBsAg-HA-mFc) of the OMV membrane protein to obtain a recombinant plasmid 4 (OmpA-HBsAg-HA-mFc) capable of expressing the fusion protein) . The recombinant plasmid as mentioned above is a plasmid vector pbv220 constructed on the basis of λpL-cI857.

A genetically engineered DE3 Escherichia coli, involving the introduction of any of the above recombinant plasmids into DE3 Escherichia coli. The genetically engineered E. coli strains successfully transfected were screened out with ampicillin. In the above-mentioned genetically modified E. coli, the bacteria will not express the target protein when the normal body temperature is 37℃, and the bacteria will start to express the target protein OmpA-HBsAg-HA -mFc when the ambient temperature exceeds 42℃.

Regarding a genetically engineered DE3 Escherichia coli, the expression of the target protein not only exists on the parental cells but also on the surface of the OMVs membrane.

The specific design of an oral vaccine system for hepatitis B infectious diseases includes oral genetically engineered Escherichia coli, artificially control local temperature, induce the expression of the target protein OmpA-HBsAg-HA-mFc in vivo, and produce OMVs displaying HBsAg in situ in the intestine. With its natural properties, OMVs penetrate the intestinal epithelial barrier and effectively activate the intestinal T cell and B cell related immune response. For example, the target antigen fused with Fc region could show a better effect of penetrating the intestinal epithelial barrier and could effectively activate the immune response.

The above oral hepatitis B vaccine includes an oral dose of 10^8 genetically engineered bacteria. 0The above-mentioned oral hepatitis B vaccine includes that the condition for initiating the expression of the target protein by the genetically engineered bacteria is that the temperature is controlled at 42℃. The above-mentioned oral hepatitis B vaccine includes the local temperature maintained at 42 ℃ for 1 hour when the genetically engineered bacteria are induced to express the target protein in vivo.

Advantages of the present application can comprise:

1. With the help of genetic engineering technology, in the presence of inducers, the target antigen (tumor antigen, infectious disease antigen, etc. ) and Fc peptide are fused and expressed at the end of the OMV surface protein. The interaction between the Fc peptide and FcRn increases the uptake of OMVs by DCs, thereby increasing the presentation of the target antigen peptide.

2. This oral vaccine system design overcomes two major problems of oral vaccines, the complex gastrointestinal environment and severe intestinal epithelial barrier.

3. Design genetically engineered bacteria that can tolerate the complex digestive tract environment and successfully reach the intestinal tract.

4. In the presence of inducers, genetically engineered bacteria produce OMVs expressing the fusion protein ClyA-HA-Ag-mFc in the intestine, which can penetrate the thick mucus layer and the intestinal epithelial barrier, and present antigens to further trigger immune effects.

5. The oral vaccine can effectively inhibit tumor growth in the B16-OVA lung metastasis model and the MC38 subcutaneous tumor model.

6. The oral vaccine can stimulate the body's long-term immune memory and effectively resist the invasion of B16-OVA lung metastatic tumor cells.

7. The oral vaccine can effectively prevent hepatitis B virus infection.

Examples

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair (s) ; kb, kilobase (s) ; pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i.m., intramuscular (ly) ; i. p., intraperitoneal (ly) ; s. c., subcutaneous (ly) ; and the like.

Example 1

Preparation and characterization of Luc-HA genetically engineered bacteria

Design and synthesize the plasmid pBAD-Luc-HA and introduce it into Top10 competent cells. The specific operations are as follows:

(1) After Top10 competent cells (100 μL) are thawed on ice, add 50 ng plasmid, mix gently, and place on ice for 30 min;

(2) The above competent cells were heat shocked in a 42 ℃ constant temperature water bath for 70 seconds. After cooling on ice for 3 min, 900 μL of LB medium was added, and the competent cells were incubated at 37 ℃ 180 rpm for 45 min.;

(3) After the incubation, 50 μL of bacterial solution was evenly spread on LB solid medium (containing 60 ng/mL ampicillin) for the selection of strains successfully introduced into the target plasmid. After culturing at 37 ℃ for 12-16 h, pick a single colony and cultivate in resistant medium to obtain Luc-HA genetically engineered bacteria;

The above-mentioned monoclonal colonies were cultured in LB medium containing 60 ng/mL ampicillin at 37 ℃ and 200 rpm. When the OD600 of the bacterial solution was 0.6-0.8, L-arabinose (2 g/L) was added. Incubate the bacterial solution at 16 ℃ and 160 rpm for 16 hours to induce the expression of the target protein;

The genetically engineered bacteria successfully introduced into the Luc-HA plasmid were induced by arabinose, and then incubated with 15 mg/mL fluorescein potassium salt for bioluminescence imaging. The bacteria without the introduced plasmid were treated the same as a negative control. The results are shown in Figure 2 (A) . The engineered bacteria introduced into the plasmid can express Luc protein only when induced by arabinose, and the bacteria that have not been introduced into the target plasmid do not express Luc protein.

The engineered bacteria induced by arabinose were lysed on ice with the lysate for 30 min, centrifuged at 4℃, 12000 rpm for 30 min, and then the supernatant was denatured by adding protein loading buffer at 100℃ for 10 min. Before protein denaturation, the protein concentration was determined with the BCA kit. SDS-PAGE gel electrophoresis is used for sample protein analysis. 30 μg per well is loaded. After the electrophoresis is completed, the protein is transferred to the PVDF membrane through the membrane transfer process, and incubated with the HA-labeled primary antibody overnight at 4℃, and then the PVDF membrane and was incubated with the secondary at room temperature for 1 hour, and finally the developing solution was used to detect bands. As shown in Figure 2 (B) , only the genetically engineered bacteria transferred into the plasmid had obvious bands after being induced by arabinose, and there was no protein expression in the other control groups.

The above shows that the successful construction of genetically engineered bacteria and the in vitro verification of the arabinose operon induction system.

Example 2

Induced expression and metabolism of Luc-HA genetically engineered bacteria in vivo

The purpose of this example is to verify the controllability of the genetically engineered bacteria induction system in vivo and the metabolism of genetically engineered bacteria.

In this example, 10^9 Luc-HA genetically engineered bacteria were orally administered to C57BL/6 mice. After oral administration of the bacteria, the mice continued to drink 20 g/L of arabinose aqueous solution for 12 hours. The mice were euthanized at 0, 2, 5, 12, and 24 hours after oral administration of bacteria, and the digestive tract (from stomach to colon) was collected. Inject 2 mL of fluorescein potassium salt (15 mg/mL) into the digestive tract of mice and perform bioluminescence analysis with a small animal fluorescence imager.

As shown in Figure 3A and 3B, which are the fluorescence images of the digestive tract removed and the relative quantitative statistics of the fluorescence of each digestive segment. The results show that within 2h-12h after oral administration of engineered bacteria and arabinose, the fluorescence signals are mainly concentrated in the cecum and colon. At 24h, the fluorescence signal of each digestive tract showed a significant attenuation, which may be caused by excretion in the body or exhaustion of the inducer arabinose.

In summary, the above experimental results show that engineered bacteria can successfully overcome the complicated digestive tract environment to reach the intestine, and oral arabinose can successfully induce the expression of the target protein in vivo.

Example 3

Tumor oral vaccine activates the immune system in vivo

Tumor oral vaccines to activate the immune system in vivo mainly include the following three aspects: 1) the construction of ClyA-OVA-mFc genetically engineered bacteria, 2) the expression of ClyA-OVA-mFc in engineered bacteria and OMVs under the induction of arabinose, 3) Tumor oral vaccine successfully activates the intestinal immune system.

Regarding the construction of ClyA-OVA-mFc genetically engineered bacteria, design and synthesize the plasmid ClyA-OVA-mFc, and refer to the above method to obtain ClyA-OVA-mFc genetically engineered bacteria.

ClyA-OVA-mFc genetically engineered bacteria were cultured in LB medium containing 60 ng/mL ampicillin at 37 ℃ and 200 rpm. When the bacterial solution OD600=0.6-0.8, add L-arabinose (2 g/L) . The bacteria solution was incubated at 16 ℃ and 160 rpm for 16 h, and then centrifuged at 4 ℃ and 5000 rpm for 10 min. The cells and supernatant were collected separately.

The above supernatant was first passed through a 0.45 μm filter membrane, the obtained filtrate was ultrafiltered with a 50 kDa ultrafiltration tube, and the obtained ultrafiltrate passed through a 0.22 μm filter membrane. The filtrate was subjected to ultra-high-speed centrifugation at a speed of 150,000 g for 3 hours. The obtained precipitate is OMV, which was resuspended in PBS and characterized by transmission electron microscope (FEI, Tecnai G2 20 S-TWIN, 200 kV, USA) and Malvern particle size potentiometer. On the other hand, the obtained OMVs were boiled and denatured and then used for SDS-PAGE electrophoresis analysis. Use SDS-PAGE for protein analysis after lysis of the above-mentioned bacteria. And, the protein sample is subjected to electrophoresis, transfer to membrane, and the target protein band is analyzed after the exposure process.

The above experimental results are shown in Fig. 4, as shown in Fig. 4A, the obtained ClyA-OVA-mFc OMVs are spherical and have uniform particle size. DLS results showed that the particle size distribution range of ClyA-OVA-mFc OMVs was 15.18-43.68 nm, and the average particle size was about 17.71 nm. Figure 4B is the WB results of the total protein from engineered bacteria and OMVs.

Under the induction of arabinose, the engineered bacterial cells and secreted OMVs successfully expressed the ClyA-OVA-mFc protein.

In order to verify that the oral vaccine system can successfully activate the intestinal immune system, the ability of engineered OMVs to penetrate the intestinal epithelial barrier and contact with lamina propria antigen-presenting cells (mainly dendritic cells, DCs) was first tested.

The specific steps of the above experiment are as follows: After the C57BL/6 mice are anesthetized, a 1cm long colon segment is ligated with surgical sutures at both ends, and engineered OMVs solution (50μg/mouse) is injected into the ligated colon intestine. During the experiment, the body temperature of the mouse was kept stable with a thermostat. After 2 hours of incubation, the ligation section was removed, washed with cold PBS twice, and then frozen sectioned. Intestinal sections were immunofluorescent stained and photographed with a confocal microscope. CD11c-positive DCs were labeled with green fluorescence, the HA label of OMVs was labeled with red fluorescence, and the nucleus was shown in blue. As shown in Figure 5, compared with the control group, the ClyA-OVA-mFc group had significantly more OMVs in contact with DCs, indicating that engineered OMVs can successfully penetrate the intestinal epithelial barrier, and mFc fragments facilitate the contact between OMVs and DCs.

This example further investigates the activation of the intestinal immune system in mice after receiving oral vaccines. The specific implementation is as follows: healthy C57BL/6 mice orally take 10^9 ClyA-OVA-mFc engineered bacteria and drink 20g/L arabinose water for 12 hours. 24 h later, the mouse colon tissue was collected, and the intestinal lamina propria immune cells were extracted, and the DCs maturation status was detected by flow cytometry. As shown in Figure 6, compared with the control groups, the mice that were orally administered the engineered bacteria and arabinose water had the highest percentage of activated lamina propria DCs. Mice were vaccinated with oral vaccines on the 0th, 3rd, and 8th days. The spleens were collected after euthanasia on the 15th day. The proportion of OVA tetramer-positive T cells in the splenic lymphocytes was analyzed by flow cytometry, as shown in Figure 7, the control group The positive ratio is close to 0, while the proportion of OVA tetramer-positive T cells in the ClyA-OVA-mFc group is greatly increased up to 5%, which further illustrates the effectiveness of mFc and the successful application of the oral vaccine system in vivo.

The above results show that the oral tumor vaccine can successfully overcome the complicated digestive tract environment to reach the intestine in vivo, and the OMVs secreted in the intestine can break through the intestinal epithelial barrier and successfully stimulate the maturation of DCs and achieve efficient tumor antigen presentation.

Example 4

The purpose of this example is to verify the anti-tumor effect of oral tumor vaccines

This example was implemented in a mouse melanoma lung metastasis model. C57BL/6 mice were injected intravenously with 200,000 B16-OVA tumor cells on day 0, and received oral vaccine treatment on days 3, 6, and 11. On the 17th day, the mice were euthanized, and the lung tissues and spleen of the mice were collected for further analysis.

As shown in Figure 8A, mice receiving ClyA-OVA-mFc oral vaccine and arabinose treatment had the least number of lung tumor metastases, which benefited from the efficient presentation of tumor antigen OVA and the body's immune activation.

The condition of CD8+T infiltrating cells in lung tissue sections (Figure 8B) further shows the effects of the present disclosure.

Example 5

The purpose of this example is to verify the immune memory caused by oral tumor vaccines

The specific implementation of this example is as follows. C57BL/6 healthy mice received oral tumor vaccine treatment on the 0th, 3rd, and 8th day. Part of the mice (n = 8) were euthanized on the 50th day. Collect mouse spleens and detect the ratio of central memory T cells (CD3 ⁺CD8 ⁺CD44 ⁺CD62L ⁺) and effector memory T cells (CD3 ⁺CD8 ⁺CD44 ⁺CD62L ^-) in the spleen. As shown in Figure 9 (A) , the proportion of effector memory T cells in the spleen of mice receiving ClyA-OVA-mFc + arabinose oral vaccine was significantly higher than that in other groups.

On the 50th day, another part of the mice (n = 8) was injected intravenously with 200,000 B16-OVA tumor cells. The mice were euthanized on the 65th day and the lungs were collected to observe the number of metastases. As shown in Figure 9 (B) , the mice that received the ClyA-OVA-mFc + arabinose oral vaccine had the fewest lung metastases, which was consistent with the proportion of memory T cells in the spleen.

Example 6

The purpose of this example is to investigate the therapeutic effect of the oral tumor vaccine system on subcutaneous tumors.

The specific implementation process of this example is as follows. On day 0, C57BL/6 mice were injected subcutaneously with 1 million MC38 tumor cells, mice received oral tumor vaccine treatment on days 3, 6, and 11, and mice received euthanasia on day 19. Collect tumor tissue to observe the treatment effect. As shown in Figure 10, compared with the control group, the mice treated with the ClyA-Adpgk + arabinose oral tumor vaccine slowed the tumor growth rate to a certain degree. Mice treated with ClyA-Adpgk-mFc + arabinose oral vaccine significantly inhibited tumor growth.

In the above example, after the mice were euthanized on the 19th day, the spleen tissue was collected, the splenic lymphocytes were extracted and incubated with the Adpgk antigen peptides, and then the secretion of IFNγ was detected by the ELISASPOT plate. As shown in Figure 11, the number of IFNγ spots in the ClyA-Adpgk-mFc + arabinose group was much higher than that in the other groups, indicating that the mice receiving the ClyA-Adpgk-mFc + arabinose oral vaccine stimulated stronger tumor killing effect.

Example 7

The purpose of this example is to investigate the immunogenicity and biological safety of oral hepatitis B vaccine.

The specific implementation process of this example is as follows: C57BL/6 mice were randomly divided into two groups, and the genetically engineered DE3 E. coli was taken orally on the 0th, 7th, and 14th days. After each vaccination, the mice receive a local heating treatment for 1 hour.

On the 21st day, the mouse serum was collected, and the concentration of HBsAg antibody in the serum was detected by ELISA. As shown in Figure 15, the antibody concentration in mice treated with oral vaccine was significantly higher than that in the control group. In addition, the results of the titer experiment also confirmed that the oral hepatitis B vaccine can greatly increase the level of antibodies against HBsAg in mice (Figure 16) .

On the 21st day, the mouse serum was collected to detect the levels of AST and ALT in the serum. The results in Figure 17 show that the levels of the two enzymes are not significantly different between the experimental group and the control group, so the oral hepatitis B vaccine will not cause significant liver toxicity.

Example 8

The purpose of this example is to investigate the effect of oral vaccine on the level of antibodies in the body in the hepatitis B model.

The specific implementation process of this example is as follows: mice are injected intravenously with 10^10 vg of AAV-HBV1.3 virus on day 0. The mice received oral vaccination on the 35th, 42nd, and 49th days. After each vaccine, the mice received temperature control for 1 hour. The mouse serum was collected on the 35th, 42, 49, 56, 64, and 72 days, and the HBsAg antibody level in the serum was detected by ELISA. As shown in Figure 19, compared to the control group, the antibody concentration of mice treated with oral vaccines remained at a higher level within 35-72 days, and the antibody level reached a peak one week after the first vaccine.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

A cell, wherein said cell is capable of secreting one or more vesicle comprising one or more protein or immunogenic fragment thereof when said cell is induced, and said protein or immunogenic fragment thereof is capable of generating an immune response.
A cell, wherein said cell is capable of expressing one or more protein or immunogenic fragment thereof on one or more vesicle when said cell is induced, and said protein or immunogenic fragment thereof is capable of generating an immune response.
The cell of any one of claims 1-2, wherein said protein or immunogenic fragment thereof is derived from bacteria, virus, parasite or tumor.
The cell of any one of claims 1-3, wherein said protein or immunogenic fragment thereof is derived from melanoma, and/or colon tumor.
The cell of any one of claims 1-4, wherein said protein or immunogenic fragment thereof is derived from hepatitis virus, influenza virus, and/or Bacillus tetanus.
The cell of any one of claims 1-5, wherein said protein or immunogenic fragment thereof comprises one or more antigen.
The cell of any one of claims 1-6, wherein said protein or immunogenic fragment thereof is selected from the group consisting of: OVA (ovalbumin) , CEA (carcinoembryonic antigen) , Adpgk (ADP Dependent Glucokinase) , MUC1 (Mucin-1) , HBsAg (hepatitis B surface antigen) , H3N2 influenza A virus antigen, and tetanus toxin C.
The cell of any one of claims 1-7, wherein said protein or immunogenic fragment thereof is capable of being expressed on the membrane of said vesicle.
The cell of any one of claims 1-8, wherein said protein or immunogenic fragment thereof is fused to one or more surface protein.
The cell of claim 9, said surface protein is selected from the group consisting of: ClyA (Cytolysin A) , OmpA (Outer membrane protein A) and Hbp (Hemoglobin-binding protease) .
The cell of any one of claims 9-10, wherein the C-terminus of said surface protein is fused directly or indirectly to the N-terminus of said protein or immunogenic fragment thereof.
The cell of any one of claims 1-11, wherein said protein or immunogenic fragment thereof is fused to an Fc fragment.
The cell of claim 12, said Fc fragment comprises a Fc fragment of an immunoglobulin.
The cell of claim 13, wherein said immunoglobulin is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM and IgA.
The cell of any one of claims 1-14, said cell is capable of expressing said protein or immunogenic fragment thereof in the gastrointestinal (GI) when said cell is induced.
The cell of any one of claims 1-15, expression of said protein or immunogenic fragment thereof is configured to be induced by pH, temperature, magnetic, heat, and/or chemical.
The cell of any one of claims 1-16, one or more gene encoding said protein or immunogenic fragment thereof is configured to be induced by pH, temperature, magnetic, heat, and/or chemical.
The cell of any one of claims 16-17, said one or more chemical is selected from the group consisting of: arabinose, IPTG, tetracycline, and/or doxycycline.
The cell of any one of claims 1-18, expression of said protein or immunogenic fragment thereof is controlled by one or more inducible promoter.
The cell of claim 19, wherein said inducible promoter is selected from the group consisting of: pH-inducible promoter, temperature-inducible promoter, magnetic-inducible promoter, heat-inducible promoter, and chemical-inducible promoter.
The cell of any one of claims 19-20, wherein one or more said inducible promoter is selected from the group consisting of: arabinose-inducible promoter, IPTG-inducible promoter, tetracycline-inducible promoter, and doxycycline-inducible promoter.
The cell of any one of claims 19-21, wherein one or more said inducible promoter is controlled by one or more operons.
The cell of claim 22, wherein said operon is selected from the group consisting of: tetracycline operon, lactose operon, galactose operon, and arabinose operon.
The cell of any one of claims 1-23, wherein said cell comprises one or more bacterium.
The cell of claim 24, wherein said bacterium comprises one or more gram-negative bacterium.
The cell of any one of claims 24-25, wherein said bacterium is selected from the group consisting of: Escherichia coli, Salmonella, Staphylococcus, Lactobacillus, Bacillus, Pseudomonas, and Bifidobacterium pseudolongum.
The cell of any one of claims 1-26, said vesicle comprises a cell-derived vesicle.
The cell of any one of claims 1-27, said vesicle comprises a bilayer structure.
The cell of any one of claims 1-28, said vesicle comprises an outer membrane vesicle (OMV) .
The cell of any one of claims 1-29, said vesicle has a diameter of about 30 nm to about 150 nm.
The cell of claim 30, said diameter is determined by dynamic light scattering (DLS) and/or transmission electron microscopy (TEM) .
A vesicle, wherein said vesicle is obtained by a cell of any one of claims 1-31.
A method of preparing a vesicle of claim 32, said method comprising providing a cell of any one of claims 1-31.
A method of preparing a vaccine, said method comprising providing a cell of any one of claims 1-31.
The method of claim 34, wherein the vaccine comprises oral vaccine.
A composition comprising a cell of any one of claims 1-31, and/or a vesicle of claim 32, and optionally a pharmaceutically acceptable carrier.
A method of generating immune response and/or long-term immune memory to a protein or immunogenic fragment thereof, the method comprising administering to a subject in need thereof an effective amount of a cell of any one of claims 1-31, a vesicle of claim 32, and/or a composition of claim 36.
A method of preventing, ameliorating and/or treating one or more disease, the method comprising administering to a subject in need thereof an effective amount of a cell of any one of claims 1-31, a vesicle of claim 32, and/or a composition of claim 36.
The method of any one of claims 37-38, wherein the method comprising orally administered.
The method of any one of claims 37-39, further comprising providing one or more condition inducing the expression of said protein or immunogenic fragment thereof.
The method of claim 40, said condition is selected from the group consisting of: pH, temperature, magnetic, heat, and/or chemical.
The method of claim 41, said one or more chemical is selected from the group consisting of: arabinose, IPTG, tetracycline, and/or doxycycline.
The method of any one of claims 40-42, further comprising providing said condition after administering an effective amount of a cell of any one of claims 1-31, a vesicle of claim 32, and/or a composition of claim 36.
The method of any one of claims 37-38, said subject is affected by one or more disease and/or condition.
The method of any one of claims 38-44, said disease and/or condition comprises an infectious and/or tumoral disease.
The method of any one of claims 38-45, said disease and/or condition comprises hepatitis B, melanoma, and/or colon tumor.