KR20110056172A - A bacterium salmonella expressing hemagglutin protein and antiviral composition comprising the same - Google Patents

Abstract

PURPOSE: An antiviral composition containing Salmonella strain which expresses hemagglutin protein is provided to ensure synergistic antiviral effect. CONSTITUTION: A samonella strain which expresses hemagglutin protein contains a hemagglutin antigen gene. The hemagglutin antigen gene is inserted and transduced to a plasmid which expresses hemagglutin protein. The plasmid is pcDNA3-HA. An attenuated Salmonella strain is Salmonella typhimurium BRD 509. The hemagglutin antigen gene has a base sequence of sequence number 1.

Description

Salmonella strain expressing hemagglutin protein and antiviral composition containing same {A bacterium salmonella expressing hemagglutin protein and antiviral composition comprising the same}

The present invention relates to a Salmonella strain expressing hemagglutin (HA) protein and an antiviral composition containing the same. More specifically, the present invention relates to Salmonella strains expressing hemagglutin protein which exhibits excellent antiviral effect by application or oral administration to the skin, and in particular, inhibits and vaccines against influenza infection, and an antiviral composition containing the same.

Influenza viruses are RNA viruses belonging to the orthomyxovirus and myxovirus genus (influenza virus genus), which are 80-120 µm long and are spherical but also linear. It is largely divided into three types of A, B, and C. A type is divided into subtypes such as A0, A1, and A2, and B type is divided into subtypes such as B1, B1α, B1β, and B2. Hepatitis A virus is characterized by a slight change in antigenic structure, and sometimes a rapid change, which is called a discontinuous change. For example, infection with a subtype of influenza virus of type A acquires immunity against the virus of this subtype, but a new type of virus is caused by a discontinuous change, thereby causing a pandemic.

Currently, attenuated vaccines of influenza viruses are used as a prevention technique against such influenza. However, rapid vaccination against new influenza such as H1N1 influenza or avian influenza, which is a recent threat, has a long virus propagation time and difficult mass production, making it difficult to manufacture and at the same time, it is ineffective against variant viruses. .

In addition, conventional influenza vaccines are mostly administered by injection, which is a complicated procedure, and should be administered only by doctors, nurses, and the like, and has many disadvantages in terms of time and place.

Therefore, there has been a continuous need to develop new functional biologics that provide defense against influenza in order to solve the shortcomings of such influenza prevention techniques.

Salmonella, on the other hand, is a pathogenic microorganism, which is usually a pathogen that causes food poisoning. However, Yale University researchers in the European Journal of Cancer report that the combination of genetically engineered Salmonella and radiation therapy has been very successful in treating melanoma (Antitumour). effects of genetically engineered Salmonella in combination with radiation. Journal of Cancer, 18, 23972402 J. Platt.).

Since then, many studies have been conducted on the anti-cancer therapeutic effects of Salmonella strains, but studies on the prevention and treatment of viruses by Salmonella strains remain at an early stage.

The present invention has been made to solve the problems and demands of the prior art, an object of the present invention is to provide a Salmonella strain that can induce antiviral effects against influenza infection.

Another object of the present invention to provide an antiviral composition containing the Salmonella strain that can induce an antiviral effect against influenza infection.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

In order to achieve the above object, the present invention is attenuated Salmonella typhimurium , characterized in that the hemagglutin antigen gene of influenza virus is inserted and transduced with a plasmid expressing the hemagglutin protein To provide.

The hemagglutin (HA) antigen gene preferably has the nucleotide sequence 1.

The present invention also provides an antiviral composition containing the attenuated Salmonella strain (Salmonella typhimurium).

Hereinafter, the present invention will be described in more detail.

The object of the present invention is to produce the Salmonella of the present invention by transducing the expression vector of the influenza virus hemagglutin (HA) protein to the attenuated Salmonella, Balb / C mouse model infected with influenza virus that can induce flu It was achieved by administering the transduced Salmonella to compare and confirm the antiviral effect thereby.

In more detail, Salmonella bacteria, unlike other bacteria that infect humans and multiply in blood cells, have the characteristics of being infected and propagated into cells like viruses. This mechanism has the characteristic of inducing an immune response similar to that of the virus when the body is infected with this bacterium. In the present invention, by using the function of inducing an immune response similar to the virus of Salmonella bacteria, the immunity induced by Salmonella was to be induced to suppress the infection of influenza virus. In the present invention, in order to induce more effective anti-influenza immunity, Salmonella expressing influenza HA protein was genetically recombined to produce a Salmonella strain having an antiviral effect.

In view of the above, the present inventors conducted a dosing test showing excellent antiviral effect with various types of preparations using the Salmonella strain. In particular, when Salmonella, which is expected to have an antiviral effect, was administered in vivo, it was confirmed that Salmonella itself, as well as early, effectively induced expression of interferon gamma, and the antiviral effect continued after 30 days. At the same time, it was confirmed that the Salmonella strain expressing the HA protein produced using the strain can be used as an influenza vaccine.

Accordingly, the present invention provides an attenuated Salmonella typhimurium , characterized in that the hemagglutin (HA) antigen gene of influenza virus is inserted and transduced into a plasmid expressing the hemagglutin (HA) protein. .

Hemagglutin protein is one of two glycoproteins on the surface of the virus's shell that acts to anchor the virus as if anchored to enter a cell. Without this fixation (or attachment), the virus cannot penetrate into the cell. There are 16 known hemagglutinin proteins.

The hemagglutin (HA) antigen gene can use a variety of known genes, genes having a nucleotide sequence 1 (Ref. GenBank: EU233675.1, Influenza A virus (A / chicken / Korea / IS / 2006 (H5N1)) hemagglutinin (HA) gene).

The Salmonella typhimurium strain is a pathogen belonging to group D Samlmonella that induces typhoid fever in the present invention. It was.

In the present invention, the attenuated Salmonella strain is characterized in that the attenuated vaccine for preventing typhoid. In the present invention, "attenuated" artificially weakens the toxicity of a living pathogen, and does not cause disease in the body by mutating a gene involved in essential metabolism of the pathogen, and when administered to a normal person, it disappears easily after a certain period of time and is immune. Stimulate the system alone to induce immunity.

As a vaccine for preventing typhoid, many injections and oral attenuated vaccines have already been developed in the art. The most attenuated Salmonella strain used in the production of live oral vaccines is the mutant Ty21a which does not have the uridine diphosphate (UDP) galactose-4- epimerase due to a mutation in the galE gene (Levine MM, et al., Progress in vaccinesagainst typhoid fever. Rev Infect Dis 11: 552-567, 1989).

Salmonella strains (hosts) used in specific examples of the present invention are Salmonella typhimurium BRD 509 strains, which are attenuated strains without pathogenicity (J Infect Dis. 1994 Dec; 170 (6): 1508). -17, available from Medeva, London). The attenuating method of the strain was to alter the genes of aro A, aro B to cause abnormalities in the metabolism of the strain, it was used as a vaccine in the present invention using the attenuation results. This effect can be applied to Salmonella typhi strains that use humans as hosts.

In the present invention, the plasmid may be any plasmid having a promoter capable of expressing the HA protein in eukaryotic cells and a multicloning site for cloning, but is preferably pcDNA3-HA shown in FIG. It is characterized by.

The transduced Salmonella strain according to the present invention was prepared and deposited in Korea Microorganism Conservation Center as 2009. 09. 09. and received accession number KCCM-11033P.

On the other hand, the present invention provides an antiviral composition containing the attenuated Salmonella strain (Salmonella typhimurium).

The antiviral composition of the present invention may be used for any viral infection that may exhibit the antiviral effect, but is preferably used for the prevention or treatment of influenza virus infection. Here, it is understood that prevention includes the vaccine effect.

The antiviral composition of the present invention can be administered to the human body through any general route as long as it can reach the desired tissue. Parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration may be, but is not limited to. The composition may also be administered by any device in which the active agent may migrate to the target cell.

In particular, in the present invention, it was confirmed that the administration of Salmonella strains applied to the skin also induces an immune effect, which is the first method that does not exist in the existing vaccine technique.

The composition may be administered with a pharmaceutical carrier generally used for antiviral treatment, such as physiological saline.

The antiviral composition of the present invention is administered in a therapeutically effective amount. The term “therapeutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, with the effective amount being the severity, age, sex, time of administration, administration of the disease. Route and rate of release, duration of treatment, factors including concomitant medications, and other factors well known in the medical arts. For example, 1 mg to 1000 mg of the Salmonella strain of the present invention may be administered once on an adult basis.

Salmonella strain expressing the HA protein according to the present invention not only induces interferongamma expression with antiviral, antiproliferative and immunomodulatory action (see FIG. 2), but also orally or skin administration of the Salmonella strain according to the present invention. It was then shown to have an antiviral effect upon influenza infection (see FIGS. 3A and 3B). In addition, administration of the Salmonella strain according to the present invention was confirmed that there is a vaccine effect that can maintain the immunity against influenza infection to prevent re-infection (see Figs. 4a to 4c).

As described above, according to the present invention, by introducing an influenza HA protein expression vector into an attenuated Salmonella strain, the antiviral effect and synergistic effect of the Salmonella strain itself can be further improved.

Furthermore, administration of the strain according to the present invention showed a vaccine effect that can prevent reinfection by maintaining immunity against influenza infections such as flu after administration. Therefore, the present invention is greatly expected to be used as a preventive agent against influenza virus infections such as the H1N1 virus.

Therefore, the present invention is a very useful invention in the pharmaceutical industry related to the development of antiviral agents and vaccines, and will be referred to as an invention of great industrial applicability as an antiviral prophylactic and therapeutic agent or adjuvant.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1 Preparation of HA Protein Expression Vector

This vector is a pcDNA3-HA vector expressing HA protein was synthesized by the genotech Co., Ltd. (Korea). The vector was prepared using an expression vector for CMV promoter of the pcDNA3 mother vector of Invitrogen. Specifically, the synthesized HA gene (SEQ ID NO: 1) was amplified by PCR amplification of the gene fragment and the expression vector pcDNA3 was prepared in a conventional manner. At this time, PCR conditions were performed at 94 ° C for 30 seconds, thermal annealing temperature at 58 ° C for 45 seconds, and extension reaction at 72 ° C for 30 seconds.

1 is a cleavage map of a plasmid expressing cultured HA in eukaryotic cells.

Example 2 Preparation of Salmonella Strains Containing HA Protein Expression Vector

First, attenuated Salmonella strain BRD509 (Salmonella typhimurium BRD509) was incubated at 37 ° C. in Luria Bertani medium. In order to transduce the HA protein expression vector into the cultured Salmonella strain, the Salmonella strain was further incubated for 24 hours in an amount of 100 mL, and then bacteria were cultured at 5500 rpm at an optical density of 600 nm and an optical absorbance (OD 600) of 0.7. Centrifuge at speed. After centrifugation, only the bacteria settled on the bottom were collected and diluted with cold distilled water having the same volume as the culture volume. The diluted bacteria were again centrifuged in the same manner as described above, diluted in 2 mL of cold 20% glycerol distilled water, and again, the bacteria obtained through centrifugation were diluted in 100 µl of 20% glycerol. 30 μl (2 μg) of the mixture obtained by the above procedure was placed in a minicentrifuge vessel, and 3 μl (2 μg) of HA protein expression vector plasmid was added thereto, followed by induction of transduction with 275-325V. The resulting HA protein-expressing Salmonella was deposited on September 9, 2009 with the Korea Microorganism Conservation Center and received accession number KCCM-11033P.

<Example 3: Confirmation of interferon protein expression by the transgenic Salmonella strain of the present invention>

After administration of Salmonella, it was determined by the ELISA method that the strain secreted interferon protein, an antiviral substance in the body by the strain. The strains were each incubated at 37 ° C. in Luria Bertani 3 ml medium. After incubation, the bacteria were centrifuged at a rate of 3000 rpm. After the centrifugation, the sinking bacteria were collected on the bottom and suspended in PBS solution. The collected bacterial mixture was cultured by measuring the number of bacteria by absorbance measurement and then adding to spleen cell lines isolated from mice at a concentration of 10 ⁸ CFU. After incubation for 24 hours, the cell culture medium was used as a sample, and the expression level of the interferon gamma protein was confirmed using an IFN-g Instant ELISA kit (BenderMedSystems, Austria) according to the manufacturer's instructions. Figure 2 is a numerical representation of the results of measuring the amount of interferon gamma protein in the blood of the mouse after administration of each bacteria to confirm whether the expression of the interferon gamma protein by the prepared Salmonella and transduced Salmonella. In Figure 2, the control represents the measured value of the normal medium (experimental reference value), SAL509 represents a cell culture solution administered Salmonella, SALHA represents a cell culture solution administered HA protein expression Salmonella.

Example 4 Confirmation of Antiviral Effect of Transgenic Salmonella Strains According to the Present Invention

In order to confirm the antiviral effect of the Salmonella strain according to the present invention, after inoculating the influenza A virus after administration of the strains prepared in Balb / C mice, the degree and survival rate of the infection was confirmed. Experimental strains compared the effects of Salmonella strains and Salmonella strains expressing the recombinant HA protein compared to the control group not treated with Salmonella strains compared the characteristics and effects are shown in Figures 3a and 3b. The control group was nasally infected with 10 ⁴ pfu of influenza A virus 3 days after bacterial treatment in normal model mice treated with only PBS solution without any Salmonella strain. Then, the weight change of the mouse due to infection was measured.

Figure 3a is administered to oral and skin twice a week at 10 ⁸ CFU / 50ul each Salmonella strain expressing the Salmonella strain and HA protein to the mouse and after the final administration to the mouse group administered only PBS solution as a control, After 7 days of infection with 10 ⁴ pfu influenza A virus, the degree of infection is shown based on the weight change. That is, in Figure 3a, the weight change due to infection in mice treated with Salmonella strain through oral and skin coating is shown. As a result, all of the experimental groups treated with Salmonella strains of the present invention showed less weight change than the mice in the control group, and in particular, the weight change of mice treated with Salmonella strains expressing HA protein showed little oral change. Both skin and skin coating showed less weight change.

Figure 3b shows a group of mice treated with Salmonella strains at 10 ⁸ CFU / 50ul, twice a week for oral and skin, and a group of mice administered with PBS solution only as a control, 10 days after 7 days of bacterial administration. ⁴ pfu Influenza A virus after infection shows the change in survival rate due to infection. In the Salmonella strain and HA-expressing Salmonella strain of the present invention in Figure 3b it shows a significant reduction in the mortality caused by infectious disease to show the resistance to infection and to prevent infectious diseases. In particular, in this experiment, Salmonella expressing the HA protein tended to further reduce the mortality rate. This shows that the Salmonella strain of the present invention can be an effective biologic for preventing influenza infection.

Example 5 Confirmation of Vaccine Effect of Transduced Salmonella Strains According to the Present Invention

In order to find out whether the Salmonella and Salmonella expressing the HA protein of the present invention had a vaccine effect on the mice prevented infection, the results were compared after re-infection with the virus. After infecting 10 ⁴ pfu of influenza virus into the nasal mice and the control mice obtained in Example 4, the survival rate and weight change were measured to confirm the presence of the infection, the antibody production, and the vaccine effect.

Figure 4a is a diagram showing the results of testing the vaccine effect using the mice survived after administration of the surviving Salmonella strain in Example 4. Specifically, Figure 4a is a view showing the degree of induction of the vaccine effect by comparing the response of the IgG antibody to the anti-influenza virus in each mouse.

Figure 4b is a diagram showing the results of the vaccine test using the mice survived after administration of the surviving Salmonella strain in Example 4. As a control group, mice treated with PBS solution alone were infected with 10 ⁴ pfu influenza A virus. The mice surviving from the infectious disease were then reinfected with influenza virus after 30 days, and the degree of induction of the vaccine effect was shown in comparison with the weight change.

Figure 4c is a diagram showing the results of the vaccine test using the mice survived after administration of the surviving Salmonella strain in Example 4. As a control group, mice treated with PBS solution alone were infected with 10 ⁴ pfu influenza A virus. The mice surviving from the infectious disease were then reinfected with influenza virus after 30 days, and the degree of incidence of the vaccine effect was compared in the figures.

 As a result, as shown in Figure 4a the antibody response was generated by the Salmonella strain according to the present invention, the weight change was almost as shown in Figure 4b and at the same time showed a 100% survival rate as in Figure 4c. These results show that Salmonella expressing Salmonella and HA proteins can not only prevent infection with influenza, but also induce a vaccine effect.

Although specific portions of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. It is therefore intended that the scope of the present invention be defined by the appended claims and their equivalents.

1 is a pcDNA3-HA vector plasmid expressing an HA protein used to prepare Salmonella expressing hemagglutin (HA) protein.

2 is a diagram showing the results of detecting the interferon gamma protein expressed in the splenocytes administered Salmonella and Salmonella expressing the HA protein.

3A and 3B are diagrams showing the influenza infection and antiviral effects thereof after administration of Salmonella expressing HA protein according to the present invention to mice (Balb / C).

Figures 4a to 4c is a view showing that the vaccine effect on the mice suppressed infection after administration of Salmonella expressing the HA protein according to the present invention.

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Claims (11)

Attenuated Salmonella typhimurium , characterized in that the hemagglutin antigen gene of influenza virus is inserted and transduced with a plasmid expressing the hemagglutin protein. The Salmonella strain according to claim 1, wherein the hemagglutin antigen gene has a nucleotide sequence of SEQ ID NO: 1. The Salmonella strain according to claim 1, wherein the plasmid is pcDNA3-HA shown in FIG. The Salmonella strain according to claim 1, wherein the strain is Accession No. KCCM-11033P deposited at the Korea Microorganism Conservation Center. The Salmonella strain according to any one of claims 1 to 4, wherein the attenuated Salmonella strain is Salmonella typhimurium BRD 509. The antiviral composition containing the attenuated Salmonella strain of Claim 1 (Salmonella typhimurium). 7. The antiviral composition according to claim 6, wherein the plasmid is pcDNA3-HA shown in FIG. The antiviral composition according to claim 6, wherein the strain is Accession No. KCCM-11033P deposited at the Korea Microorganism Conservation Center. The antiviral composition according to any one of claims 6 to 8, wherein the composition is used for the prevention or treatment of an influenza virus infection. The antiviral composition according to any one of claims 6 to 8, wherein the composition is applied to the skin and used. An antiviral composition comprising attenuated Salmonella strains (Salmonella typhimurium) and applied to the skin to be used for the prevention or treatment of influenza virus infection.

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