JP7462864B2 - Membrane Vesicles - Google Patents

Description

The present invention relates to a membrane vesicle that can carry foreign protein antigens and can be used in vaccines.

Bacteria release nano-sized vesicles called membrane vesicles (hereinafter sometimes referred to as MV) outside the cell. Historically, a similar structure was reported in 1963 in electron micrographs of Bacteroides ruminicola, a gram-negative, obligately anaerobic, non-spore-forming bacillus (Non-Patent Document 1), and it has been revealed that various gram-negative bacteria produce membrane vesicles (Non-Patent Document 2). In 1966, it was also reported that a lysine-requiring mutant of Escherichia coli, a model organism, exhibited abnormal morphology when cultured under lysine-limited conditions (Non-Patent Document 3). In addition, a method has been reported in which the production of bacterial MV is increased by adding 1.0 to 1.2 w/v% glycine to the culture medium during bacterial culture (hereinafter this method may be referred to as the glycine induction method) (Non-Patent Documents 4 and 5).

Membrane vesicles are composed of phospholipids and membrane proteins derived from cell membranes, lipopolysaccharide (sometimes referred to as Lipopolysaccharide: LPS) and other substances, and contain various substances such as nucleic acids and enzymes, so they have multifaceted functions and are expected to be applied in the form of new vaccine antigens. For example, they are used to easily produce vaccines using Escherichia coli MVs carrying target proteins as antigens (Non-Patent Document 6). The possibility of using MVs with exogenous glycans localized on the surface as vaccines has been reported (Non-Patent Document 7). However, the technology to localize exogenous proteins on the surface of bacteria with type 9 secretion mechanisms has not been established. Furthermore, there have been no reports of the use of MVs from bacteria with type 9 secretion mechanisms as vaccine bodies.

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, The Agricultural Chemical Society of Japan 2018 Nagoya. Enhancement of Escherichia coli membrane vesicle production by glycine and its characterization. 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).

The present invention aims to provide an MV that can carry foreign protein antigens and can be used as a vaccine.

The MV of the present invention is a bacterial MV with type IX secretion system, and is characterized in that a foreign protein is anchored to lipopolysaccharide, which is a component of the outer membrane.

According to the present invention, MVs capable of carrying foreign protein antigens and being applied to vaccines can be obtained.

FIG. 1 illustrates anchoring of proteins to the outer membrane by T9SS. FIG. 1 is a diagram illustrating the preparation of a chimeric membrane vesicle into which GFP has been introduced. FIG. 1 is a photograph of an ELISA analysis that confirmed that the target plasmid was introduced by PCR or the like. FIG. 1 illustrates the immunogenicity of GFP-introduced Porphyromonas gingivalis MV.

The following describes in detail an embodiment of the present invention with reference to the attached drawings. However, this embodiment is intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the embodiment described below. Other embodiments in which a person skilled in the art appropriately replaces the configuration of the embodiment described below are also included in the scope of the present invention.

The MV in this embodiment is a membrane vesicle of a bacterium with type IX secretion system, and is characterized in that a foreign protein is anchored to lipopolysaccharide, which is a constituent part of the outer membrane (Figure 1).

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

The inventors of the present application have succeeded in using MVs of bacteria with a type 9 secretion mechanism as a vaccine body, because C-terminal domain proteins transported via the type 9 secretion mechanism are concentrated and produced as MVs, and because the toxic activity of LPS contained in MVs of bacteria with a type 9 secretion mechanism is extremely low, and have completed the present invention. For example, the endotoxin (Lipid A) activity of LPS contained in MVs of Porphyromonas gingivalis is approximately 0.1% of the endotoxin (Lipid A) activity of LPS contained in MVs of Escherichia coli, and MVs of bacteria with a type 9 secretion mechanism in which a foreign protein is anchored to lipopolysaccharide are very safe, making them very beneficial when used as a vaccine body.

MVs are produced by the membranes of gram-negative bacteria and the like through budding, bacteriolysis, etc., but there are various pathways that induce MV formation, and the MV formation pathway in this embodiment is not particularly limited.

The bacterium from which MV is obtained is not particularly limited as long as it is a bacterium of the phylum Bacteriodetes that has a type 9 secretion system, but may be, for example, a bacterium of the genus Porphyromonas, Bacteroides, Prevotella, Tannerella, Chlorobium, or Flavobacterium. In the invention according to this embodiment, the bacterium having a type 9 secretion system is preferably a bacterium of the genus Porphyromonas, such as P. gingivalis.

Rgp is encoded by two genes (rgpA and rgpB), and Kgp is encoded by one gene (kgp). In addition to the protease domain, rgpA and kgp also encode adhesin domains such as Hgp44 and HbR (Hgp15). The four genes encoding gingipains, rgpA, rgpB, and kgp, plus the adhesin gene hagA, are called the gingipain gene cluster, but it was unclear how the products of these genes are secreted onto the bacterial surface and outside the bacterial cell. It is known that rgpA rgpB kgp mutants form non-black colonies on blood agar medium. A mutant library was created using transposon (Tn) mutagenesis, and mutants that form non-black colonies on blood agar medium were isolated. It was found that in one of these non-black mutants, the products of the gingipain gene cluster were accumulated in the periplasm, and this mutant gene (porT) is involved in secretion. A search for homologous genes of the porT gene revealed that they are present in Cytophaga hutchinsonii and Flavobacterium johnsoniae in the Bacteroidetes phylum, but not in Bacteroides thetaiotaomicron or Bacteroides fragilis. A Venn diagram analysis was then performed on these genes, and mutant strains were created in P. gingivalis for genes that show a similar existence pattern to porT, discovering 11 genes that show similar properties to the porT mutant strain. It was suggested that nine of these genes, excluding the two genes of the two-component control system, code for proteins directly involved in the pathway that secretes the products of the gingipain gene group, just like porT, and that the secretion mechanism by these proteins is the type 9 secretion system (T9SS).

According to the present invention, by using a bacterium with a type IX secretion system, localizing a foreign protein on the surface of the bacterium, and using the MV of this bacterium as a vaccine antigen, antibody production can be safely induced. This is an important advantage that cannot be obtained with a vaccine that uses the MV of E. coli or other bacteria with strong endotoxin activity that carries a foreign protein as an antigen.

In this embodiment, the exogenous protein anchored to the lipopolysaccharide is not particularly limited as long as it is an antigenic protein.

The vaccine according to this embodiment is characterized by containing the MV according to this embodiment (i.e., the MV of a bacterium having a type IX secretion system, characterized in that a foreign protein is anchored to lipopolysaccharide, which is a constituent part of the outer membrane). The vaccine according to this embodiment can be a freeze-dried vaccine preparation containing the membrane vesicle according to this embodiment, which is dissolved at the time of use and administered into the body or to the epidermal or mucosal surface of the body, for example, as an injection or spray solution.

(1) Method for constructing a GFP expression vector A GFP expression vector was constructed by introducing into a pTCB vector the entire nucleotide sequence of the GFP sequence (including the promoter sequence upstream and the terminator sequence downstream) to which the N-terminal and C-terminal regions have been added, using the N-terminal motif portion consisting of the nucleotide sequence shown in SEQ ID NO:1 and the C-terminal motif portion consisting of the nucleotide sequence shown in SEQ ID NO:2 (Figure 2). The sequence information of the GFP expression vector is shown in SEQ ID NO:3.

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

(3) Immunogenicity of MV with GFP Mice were immunized intranasally using a GFP expression vector consisting of the sequence shown in SEQ ID NO: 3. Enzyme-Linked ImmunoSorbent Assay (ELISA) was performed with GFP protein immobilized. Mice were immunized with Pg MV with a negative standard plasmid (empty vector) introduced (Pg MV) and Pg MV with a GFP expression vector introduced (Pg MV-GFP), and the serum obtained was diluted 200-fold with PBS containing 0.02% Tween 20 to prepare serum antibody solutions. ELISA was performed using the above ELISA plate as usual. The secondary antibody was AP-labeled anti-mouse IgG (Invitrogen), which is an alkaline phosphatase (ALP)-labeled secondary antibody, and the color development was detected with a plate reader at an absorbance value of A405 using the ALP substrate paranitrophenyl phosphate. Fig. 4 shows the results of analyzing the expression of GFP antibody in the serum of mice immunized with membrane vesicles of Porphyromonas gingivalis by Enzyme-Linked ImmunoSorbent Assay (ELISA). As shown in Fig. 4, in mice nasally immunized with a GFP expression vector consisting of the sequence shown in SEQ ID NO: 3, the production of GFP antibody was significantly increased compared to the control.

Can be used to create vaccine bodies.

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

Claims (1)

MVs of a bacterium with a type 9 secretion system,
the bacterium is Porphyromonas gingivalis,
The MV is a membrane vesicle released outside the cell ,
MV is characterized in that a foreign protein is anchored to lipopolysaccharide, which is a component of the outer membrane , and the foreign protein has antigenicity.