JP2020176221A - Membrane vesicle - Google Patents

Abstract

To provide a membrane vesicle (MV) capable of carrying an exogenous protein antigen, applied to a vaccine body and discharged into the outside of a cell.SOLUTION: The bacterial MV having a 9 type secretion mechanism includes exogenous protein anchored to lipopolysaccharide served as the component part of an outer membrane. Antigen antibodies can be produced and induced to each portion of in-blood, a nasal cavity, a mouth and a lung by medicating vaccine including the MV mounting the exogenous protein in the nasal cavity.SELECTED DRAWING: Figure 1

Description

The present invention relates to a membrane vesicle capable of carrying an exogenous protein antigen and applicable to a vaccine body.

Bacteria release nano-sized vesicles called membrane vesicles (hereinafter sometimes referred to as MV: membrane vesicles) extracellularly. Historically, a similar structure in Bacteroides ruminicola, a gram-negative, obligately anaerobic non-spore-forming bacillus, was reported in 1963 on electromicrographs (Non-Patent Document 1), and various gram-negative bacteria were membrane vesicles. It has been clarified that it produces (Non-Patent Document 2). In addition, Escherichia coli, which is a model organism, was also reported in 1966 as an abnormal morphology when a lysine-requiring mutant was cultured under lysine restriction (Non-Patent Document 3). In addition, a method for increasing the production of MV derived from bacteria by adding 1.0 to 1.2 w / v% of glycine to the culture solution during bacterial culture (hereinafter, this method may be referred to as a glycine induction method). .) Has been reported (Non-Patent Documents 4 and 5).

Membrane vesicles are composed of cell membrane-derived phospholipids, membrane proteins, lipopolysaccharides (sometimes referred to as LPS in the present specification), etc., and also contain various substances such as nucleic acids and enzymes. Therefore, it has multifaceted functions and is expected to be applied to new vaccine antigens. For example, it is used as a simple vaccine production using MV of Escherichia coli carrying a target protein as an antigen (Non-Patent Document 6). It has been reported that MV vaccine application in which foreign sugar chains are localized on the surface layer can be applied (Non-Patent Document 7). However, a technique for localizing an exogenous protein on the surface layer of this bacterium using a bacterium having a type 9 secretory mechanism has not been established. In addition, there is no report that MV of a bacterium having this type 9 secretory mechanism was used as a vaccine body.

H.A.Bladen & J.F.Waters: J.Bacteriol., 86, 1339 (1963). Jain S, Pillai J. Int J Nanomedicin. Bacterial membrane vesicles as novel nanosystems for drug delivery. 12: 6329-6341 (2017) K.W.Knox, M.Vesk & E.Work: J.Bacteriol., 92, 1206 (1966). Satoru Hirayama, Ryoma Nakao., Budapest, Hungary. Glycine strongly enhances immunoactive membrane vesicle production from flagella-deficient E. coli., 12th Vaccine Congress. 9 / 16-19 (2018) Satoru Hirayama, Ryoma Nakao, Japan Society for Bioscience and Biotechnology 2018 Nagoya. Increase in E. coli membrane vesicle production by glycine and its characteristic analysis. 3 / 15-18 (2018) D.J.Chen, N.Osterrieder, S.M.Metzger, E.Buckles, A.M.Doody, M.P.DeLisa & D.Putnam: Proc.Natl.Acad.Sci.USA, 107, 3099 (2010). Price NL, Goyette-Desjardins G, Nothaft H, Valguarnera E, Szymanski CM, Segura M, Feldman MF. Glycoengineered Outer Membrane Vesicles: A Novel Platform for Bacterial Vaccines. Sci Rep. 22; 6: 24931 (2016).

An object of the present invention is to provide an MV capable of carrying an exogenous protein antigen and applicable to a vaccine body.

The MV according to the present invention is a MV of a bacterium having a type 9 secretory mechanism, and is characterized in that an exogenous protein is anchored to lipopolysaccharide, which is a component of the outer membrane.

According to the present invention, an MV capable of carrying an exogenous protein antigen and applicable to a vaccine body can be obtained.

It is a figure explaining the anchoring of a protein to the outer membrane by T9SS. It is a figure explaining the preparation of the chimeric membrane vesicle which introduced GFP. It is a photograph figure of the ELISA analysis which confirmed that the target plasmid was introduced by PCR or the like. It is a figure explaining the immunogenicity of MV of Porphyromonas gingivalis introduced with GFP.

Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings, but the embodiments are for facilitating understanding of the principles of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiment, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.

The MV according to the present embodiment is a bacterial membrane vesicle having a type 9 secretory mechanism, and is characterized in that an exogenous protein is anchored to lipopolysaccharide, which is a component of the outer membrane (FIG. 1). ).

LPS has a structure in which a sugar chain consisting of multiple molecules of sugar is bound to a lipid called lipid A. The sugar chain portion is composed of a portion called a core polysaccharide (or core oligosaccharide) and a portion called an O polysaccharide (O antigen).

According to the present inventor, the C-terminal domain protein carried via the type 9 secretory mechanism is concentrated and produced as MV, and the toxin activity of LPS contained in the MV of bacteria having the type 9 secretory mechanism is extremely low. , MV of a bacterium having a type 9 secretory mechanism was successfully used as a vaccine body, and the present invention was completed. For example, the endotoxin (Lipid A) activity of LPS contained in the MV of Porphyromonas gingivalis is about 0.1% of the endotoxin (Lipid A) activity of LPS contained in the MV of Escherichia coli, and the foreign protein is anchored in lipopolysaccharide. The MV of a bacterium having a ringed type 9 secretory mechanism is very safe, and it is very beneficial when this MV is used as a vaccine body.

In MV, a membrane of Gram-negative bacteria or the like is produced by budding or lysis, but there are various pathways for inducing MV formation, and the MV formation pathway according to the present embodiment is not particularly limited.

The bacterium that obtains MV is not particularly limited as long as it is a bacterium of the phylum Bacteroodetes having a type 9 secretion mechanism, and is, for example, of the genus Porphyromonas, Bacteroides, Prevotella, Tannerella, Chlorobium, or Flavobacterium. It is a bacterium. In the invention according to the present embodiment, the bacterium having a type 9 secretory mechanism is preferably Porphyromonas genus Zingivalis or the like.

Rgp is encoded by two genes (rgpA and rgpB), Kgp is encoded by one gene (kgp), and rgpA and kgp encode adhesin domains such as Hgp44 and HbR (Hgp15) in addition to the protease domain. The four genes in which the adhesin gene hagA is added to rgpA, rgpB, and kgp, which encode gindipine, are called the gindipine gene group, and how the products of these genes are secreted on the cell surface and outside the cell. Was unknown. The rgpA rgpB kgp mutant has been shown to form non-black colonies on blood agar. A mutant library was prepared by the transposon (Tn) mutation introduction method, and mutant strains forming non-black colonies on blood agar were isolated. In one of these non-black mutant strains, the product of the gingipain gene group was peripulus. It is found that this mutant gene (porT) is involved in secretion. A search for the homologue gene of the porT gene revealed that it was present in Cytophaga hutchinsonii and Flavobacterium johnsoniae in the phylum Bacteroidetes, but not in Bacteroides thetaiotaomicron and Bacteroides fragilis. Therefore, Venn diagram analysis was performed on these genes, mutant strains were prepared by P. gingivalis for genes showing the same existence mode as porT, and 11 genes showing the same properties as the porT mutant strain were discovered. It has been suggested that nine genes, excluding the two genes of the two-component regulatory system, encode proteins that are directly involved in the secretory pathway of the products of the gindipine gene group, and the secretory mechanism by these proteins is It is a type 9 secretory mechanism (T9SS).

According to the present invention, it is possible to safely induce antibody production by localizing an exogenous protein on the surface layer of a bacterium having a type 9 secretory mechanism and using the MV of this bacterium as a vaccine antigen. .. This advantage is an important effect that cannot be obtained with a vaccine body using MV having strong endotoxin activity such as Escherichia coli carrying an exogenous protein as an antigen.

In the present embodiment, the exogenous protein anchored to the lipopolysaccharide is not particularly limited as long as it is a protein having antigenicity.

The vaccine according to the present embodiment is the MV according to the present embodiment (that is, the MV of a bacterium having a type 9 secretory mechanism, in which an exogenous protein is anchored to lipopolysaccharide which is a component of the outer membrane. It is characterized by including MV). The vaccine according to the present embodiment can be a lyophilized vaccine preparation containing the membrane vesicle according to the present embodiment. For example, it is dissolved at the time of use and used as an injection or spray solution in vivo or on the epidermal surface / mucosa of the living body. Administered to the face.

(1) Method for Producing a GFP Expression Vector The N-terminal and C-terminal regions are added by the N-terminal motif portion consisting of the nucleotide sequence set forth in SEQ ID NO: 1 and the C-terminal motif portion consisting of the nucleotide sequence set forth in SEQ ID NO: 2. A GFP expression vector was prepared by introducing the entire nucleotide sequence of the GFP sequence (including the promoter sequence upstream and the terminator sequence downstream) into the pTCB vector (Fig. 2). The sequence information of the GFP expression vector is shown by SEQ ID NO: 3.

(2) Introduction into cells This GFP expression vector was introduced into Porphyromonas gingivalis by the electroporation method. Selection was performed on an agar medium containing tetracycline at a concentration of 0.7 micrograms / ml, and it was confirmed that the target plasmid had been introduced by PCR or the like (Fig. 3). This confirmed the completion of the introduction of the GFP expression vector into the cells.

(3) Immunogenicity of MV into which GFP was introduced Nasal immunization was performed on mice using a GFP expression vector consisting of the sequence shown in SEQ ID NO: 3. An Enzyme-Linked ImmunoSorbent Assay (ELISA) in which the GFP protein was immobilized was performed. 0.02% Tween 20 of serum obtained by immunizing mice with Pg MV (Pg MV) introduced with a negative standard plasmid (empty vector) and Pg MV (Pg MV -GFP) introduced with a GFP expression vector. The serum antibody solution was diluted 200-fold with the contained PBS, and ELISA was performed as usual using the above ELISA plate. As the secondary antibody, AP-labeled anti-mouse IgG (manufactured by Invitrogen), which is an alkaline phosphatase (ALP) -labeled secondary antibody, is used, and the ALP substrate paranitrophenyl phosphate is used to develop color at an absorption value of A405. Detected with a plate reader. FIG. 4 shows the results of analysis of GFP antibody expression in mouse sera immunized with a membrane vesicle of Porphyromonas gingivalis by an Enzyme-Linked ImmunoSorbent Assay (ELISA). As shown in FIG. 4, nasal-immunized mice using the GFP expression vector consisting of the sequence shown in SEQ ID NO: 3 had significantly increased production of GFP antibody as compared with the control.

It can be used to create a vaccine body.

SEQ ID NO: 1: N-terminal motif SEQ ID NO: 2: C-terminal motif SEQ ID NO: 3: Vector

Claims (3)

A MV of a bacterium having a type 9 secretory mechanism,
An MV characterized by anchoring foreign proteins in lipopolysaccharide, which is a component of the outer membrane. The MV according to claim 1, wherein the bacterium having the type 9 secretion mechanism is a bacterium of the genus Porphyromonas, Bacteroides, Prevotella, Tannerella, Chlorobium, Flavobacterium, or the like. The MV according to claim 2, wherein the bacterium having the type 9 secretory mechanism is Porphyromonas gingivalis.