KR101669142B1 - A novel adjuvant of vaccine for eye-drop vaccination - Google Patents

Abstract

The present invention relates to an ophthalmic vaccine adjuvant comprising poly (I: C), and more particularly to an ophthalmic vaccine adjuvant comprising a poly (I: C) polyunosinic acid-polycytidylic acid as an adjuvant to an influenza virus antigen (I: C) was found to be effective as an anti-infective agent for influenza vaccine against influenza virus, which has excellent cytotoxic effect and similar cytotoxic and inflammatory response. You can use it as a bonus.

Description

A novel adjuvant for vaccine for eye-drop vaccination,

The present invention relates to the use of poly (I: C) as an ophthalmic vaccine adjuvant.

Vaccines that induce immune responses by enhancing mucosal immunity are effective methods for targeting pathogens prior to infection (Non-Patent Documents 1 and 2). Mucosal vaccination is of particular interest because it can induce systemic and mucosal immune responses, mainly secretory IgA (sIgA) antibodies, at the entrance of most infectious pathogens, in contrast to parenteral vaccination 3). Vaccine development is lagging behind the rapid spread of disease in the age of travel around the world. Mucosal vaccination is easy to administer and requires no special training, which is a strategy to stop new pathogens before they become pandemic.

These features of mucosal immunity are that the mucosal immune pathway, which consists of the inducible and the active tissues, can induce antigen-specific immune responses in mucosal tissues and their secretions It is a point. Considering that most of the infectious agents in recent years are infiltrating through the mucous membranes of the respiratory, digestive, and reproductive tracts, it is very important to establish an effective immune defense for the invading pathways. Thus, the development of infection prevention measures using the mucosal immune system has received much attention in recent years.

However, in the case of mucosal vaccination, an immune response is mainly induced by secretory IgA (SIgA) antibody. If the vaccine administration rate does not follow the disease progression rate, there is a risk that the vaccine preventive effect can not be sufficiently expressed . When a wide-area epidemic disease is transmitted to a large number of livestock, it is necessary to expedite the administration of the vaccine. For this purpose, it is possible to administer the vaccine easily without any special technique, and at the same time, the effect of the vaccine administration should be sufficiently exhibited. However, nasal inoculation, one of the mucosal routes of administration, is undesirable because of the risk of the antigen penetrating into the central nervous system.

The ocular mucosa is a viable pathway for mucosal vaccines because it is an important site of entry for environmental antigens and infectious agents that occupy most of the external eye surface (Non-Patent Documents 4-6). The conjunctiva, which is part of the ocular mucosa, has immunologic features similar to other mucosal tissues. The conjunctiva has CD8 + T cells in the epithelium, and the proliferation layer has equivalent ratios of CD4 + and CD8 + T cells, B cells and mast cells, and dendritic cells (DC) and Langerhans cells (Non-Patent Documents 7 and 8). These conjunctiva are areas of the mucosal barrier that are exposed to the external environment and share many common immunological features of other mucosal parts. Previous studies have shown successful protection by eyedrop vaccination in new and small models (Non-Patent Documents 9 and 10). However, the systematic comparison of the induction mechanism of the desired immune response and the conjunctival and nasal passages has not been described yet (Non-Patent Documents 11-13).

Until now, the influenza virus vaccine has been inoculated subcutaneously and nasally, and there has been no reported case of influenza virus immunization after eye vaccination with dead vaccine. There is no report on the efficacy and safety of adjuvant due to the use of certain visible vaccines for influenza virus vaccination through the eye. In addition, cholera toxin (CT), which is the most effective adjuvant for mucosal vaccine, has been reported to cause CNS damage and inflammation during nasal mucosal inoculation. Therefore, there is a need to develop an effective, but safe, ocular mucosal adjuvant as CT other than CT.

The present inventors have made intensive efforts to develop a novel ophthalmic vaccine adjuvant for ocular mucosa. As a result, the present inventors have found that poly (I: C) (polyunosinic acid-polycytidylic acid) is used as an adjuvant for influenza virus vaccine (I: C) can be used as an adjuvant for influenza virus vaccines for ocular mucosa by confirming that it has excellent antibody production enhancing effect similar to CT and has almost no inflammatory reaction stability unlike CT The present invention has been completed.

Brandtzaeg, P., and R. Pabst. 2004. Let's go mucosal: communication on slippery ground. Trends Immunol. 25: 570-577. Holmgren, J., and C. Czerkinsky. 2005. Mucosal immunity and vaccines. Nat. Med. 11 (4, Suppl) S45-S53. Yuki, Y., and H. Kiyono. 2003. New generation of mucosal adjuvants for the induction of protective immunity. Rev. Med. Virol. 13: 293-310. Chandler, J. W., and T. E. Gillette. 1983. Immunologic defense mechanisms of the ocular surface. Ophthalmology 90: 585-591. Franklin, R. M. 1989. The ocular secretory immune system: a review. Curr. Eye Res. 8: 599-606. Montgomery, R. D., W. R. Maslin, C. R. Boyle, T. Pledger, and C. C. Wu. 1991. Effects of an Arkansas strain of infectious bronchitis vaccine on the headassociated lymphoid tissue (HALT). Avian Dis. 35: 302-307. Sacks, E. H., R. Wieczorek, F. A. Jakobiec, and D. M. Knowles, II. 1986. Lymphocytic subpopulations in the normal human conjunctiva. A monoclonal antibody study. Ophthalmology 93: 1276-1283. Rodrigues, M. M., G. Rowden, J. Hackett, and I. Bakos. 1981. Langerhans cells in the normal conjunctiva and peripheral cornea of selected species. Invest. Ophthalmol. Vis. Sci. 21: 759-765. Jayawardane, G. W., and P. B. Spradbrow. 1995. Mucosal immunity in chickens vaccinated with the V4 strain of Newcastle disease virus. Vet. Microbiol. 46: 69-77. Zundel, E., J. M. Verger, M. Grayon, and R. Michel. 1992. Conjunctival vaccination of pregnant ewes and goats with Brucella melitensis Rev 1 vaccine: safety and serological responses. Ann. Rech. Vet. 23: 177-188. Nesburn, A. B., T. V. Ramos, X. Zhu, H. Asgarzadeh, V. Nguyen, and L. Ben Mohamed. 2005. Local and systemic B cell and Th1 responses induced following ocular mucosal delivery of multiple epitopes of herpes simplex virus type 1 glycoprotein D together with cytosine-phosphate-guanine adjuvant. Vaccine 23: 873-883. Muneta, Y., I. S. Panicker, A. Kanci, D. Craick, A. H. Noormohammadi, A. Bean, G. F. Browning, and P. F. Markham. 2008. Development and immunogenicity of recombinant Mycoplasma gallisepticum vaccine strain ts-11 expressing chicken IFN-g. Vaccine 26: 5449-5454. Corner, L. A., and B. M. Buddle. 2005. Conjunctival vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette-Gue'rin. N. Z. Vet. J. 53: 133-136.

It is an object of the present invention to provide an ophthalmic vaccine adjuvant composition comprising poly (I: C), and an ophthalmic vaccine comprising said adjuvant.

In order to achieve the above object, the present invention provides an ophthalmic vaccine adjuvant comprising poly (I: C).

The present invention also relates to a composition comprising i) poly (I: C); And ii) a subunit antigen or an inactivated antigen of the pathogen.

The present invention also relates to a composition comprising i) poly (I: C); And ii) administering to the individual's eye a vaccine comprising a subunit antigen or an inactivated antigen of the pathogen.

In addition, the present invention relates to a composition comprising i) poly (I: C); And ii) a vaccine comprising a subunit antigen or an inactivated antigen of a pathogen, and instructions on an ocular administration method, dose, or administration frequency of the vaccine, and the like.

When poly (I: C) is used as an adjuvant in the inoculation of an influenza virus vaccine, the present invention has excellent cytotoxic and cytotoxic and inflammatory response stability similar to that of CT , The poly (I: C) can be used as a useful adjuvant for an influenza A virus vaccine. Therefore, the present invention has been known as the most effective agent of adjuvant which induces immunity through mucous membrane. However, the present invention has a problem in that it has side effects that cause cytotoxicity and inflammation. Instead of CT, poly (I: C) Can be usefully used as adjuvants for a variety of vaccines, including influenza viruses.

FIG. 1 is a graph showing the results of OVA-specific antibody production in serum and mucosal tissue washings after PBS or OVA alone and various adjuvants (CT, poly (I: C), MPLA, And the increase was measured using an ELISA.
FIG. 2 is a graph showing the results of immunohistochemical staining of BALB / c mouse ocular mucosa with PBS or OVA alone and various adjuvants (CT, poly (I: C), MPLA, And the increase was measured using an ELISA.
FIG. 3 shows the results of ELISA for the production of anti-HA antibody after inoculation of 10 ug of poly (I: C) and 0.1 ~ 2 ug of HA vaccine antigen in the eye of BALB / Fig.
FIG. 4 shows the results of immunization of two BALB / c mice with 1 ug HA vaccine antigen (1 ~ 30 ug poly (I: C) Specific IgG and IgA antibody production by ELISA. At this time, the significance of '*': p <0.05, '**': p <0.01, '***': p <0.001, HA + 1ug poly (I: C) '†': p <0.05, and 'ns' indicates no significant difference between the two groups.
Figure 5 shows the results obtained after inoculation of ocular mucosa or nasal mucosa twice a week with 1 ug or 10 ug poly (I: C) of 1 ug HA vaccine antigen at 6 to 8 weeks of age in BALB / c mice , And the results of measurement of the HA specific IgG and IgA antibody production by ELISA in serum and mucosal washing solution two weeks after the last inoculation. In comparison with HA alone group, '*': p <0.05, '**': significance of p <0.01 and '§' as compared with HA + 1 ug poly (I: C) 0.05, '§§§': p <0.001, and 'ns' indicates that there is no significant difference between the two groups.
FIG. 6 is a graph showing the effect of 1 ug of influenza A virus H1N1 split vaccine antigen in 6 to 8 weeks old BALB / c mice with 1 ug or 10 ug of poly (I: C) The results are shown in the graphs showing the results of measurement of the H1N1-specific IgG and IgA antibody production effects in the sera and mucosal washing solution after the inoculation on the mucosa and 2 weeks after the last inoculation by ELISA. Compared to H1N1 alone group, '†': p <0.05, '††': p <0.01 compared to H1N1 alone group, and '§': p <0.05, significance of '§§': p <0.001, and 'ns' represents no significant difference between the two groups.
FIG. 7 shows the results obtained by inoculating PBS, HAs alone, and HAs + poly (I: C) into the ocular mucosa of eyes of 6 to 8 weeks old BALB / c mice and then obtaining cells from conjunctival tissues, &Lt; / RTI &gt; and CD11c, and then analyzed for changes in cell distribution using a flow cytometer.
Fig. 8 shows the results obtained by inoculating 1 ug HA vaccine antigen alone, antigen + 10 g poly (I: C), and antigen + CT into the ocular mucosa, respectively, in 6 to 8 weeks old BALB / , 6h, 12h, 24h), cDNA was synthesized with reverse transcriptase, and then RT-1alpha and TNF-alpha mRNA expression was measured using real-time PCR And FIG.

Hereinafter, terms used in the present invention are defined.

The term "vaccine" as used herein refers to an antigenic suspension or solution, usually comprising an infectious agent or part of an infectious agent, which causes active immunity when administered into the body. The antigenic portion constituting the vaccine can be a natural substance purified from a microorganism (e.g., a virus or a bacterium) or a microorganism, a synthetic or genetically modified protein, a peptide, a polysaccharide or the like. Examples of live vaccines include, but are not limited to, BCG, smallpox vaccines, poliomyelitis, varicella, measles, rubella, mumps, rinderpest, NDV, Marek's disease. The vaccine includes, but is not limited to, pertussis, diphtheria (toxoid), tetanus (toxoid), influenza, Japanese encephalitis and the like.

In the present invention, the term " inactivated antigen "refers to an antigen used as a vaccine antigen as an antigen whose infectivity has been removed. Such antigens include, but are not limited to, virions, virion, incomplete virus particles, virion-constituent particles, virus non-constituent proteins, antigens to prevent infection, neutralization reaction epitopes, and the like. The term "inactivated antigen" refers to an antigen that has been removed from infectivity but retains immunogenicity. When such an antigen is used as a vaccine, it is called "vaccine". Examples of such inactivated antigens include, but are not limited to, those that are inactivated by physical (e.g., x-ray irradiation, thermal, ultrasonic), chemical (formalin, mercury, alcohol, chlorine)

In the present invention, the term "subunit antigen" may be purified from pathogens such as naturally occurring viruses, or may be produced by synthetic or recombinant techniques. Such methods are well known and commonly used in the art and can be performed using commercially available equipment, reagents, vectors, and the like. For example, in the case of influenza virus, the subunit antigen is preferably a molecule exposed to the surface of the particle, such as hemagglutinin (HA), neuraminidase (NA), matrix (M1, M2) Non-structures (NS), polymerases (PB1, PB2: basic polymerases 1 and 2, acidic polymerases (PA)), and nuclear proteins (NP). There are 15 kinds of HA and 9 kinds of NA. It is known that when the kind is changed, a new strain is generated.

The term " adjuvant "in the present invention refers to a substance that increases or changes an immune response when mixed with an administered immunogen. The term " adjuvant "refers collectively to substances that, when mixed with an antigen, increase antibody production and enhance the immune response. To induce a faster, more potent, or sustained response, it is desirable to use adjuvants with conventional vaccine antigens. Thus, adjuvants are useful even when the antigen supply is limited or the cost of antigen production is high. The adjuvant can be classified as inorganic, bacterial, vegetable, synthetic, or host material.

The term "CT" in the present invention refers to an exotoxin produced by V. cholera, which is a causative agent of diarrhea caused by V. cholera infection. Although the cholera toxin is used as an effective adjuvant, no clinical application has been found due to its toxicity. Thus, CT is usually used as a positive control when looking for an effective vaccine adjuvant.

In the present invention, the term " Poly (I: C) "refers to a double-stranded RNA comprising polyinosinic acid (pI) and poly-cytidyl acid (pc).

In the present invention, the term " mucosal administration "refers to a mode of administration through the mucosa. Here, the mucous membrane refers to the inner wall of the hollow organ, particularly the organs communicating with the outside of the body of the vertebrate.

The term "pathogen" as used herein refers to an organism capable of causing disease or disorder in a host. Examples of human pathogens include, but are not limited to, viruses, bacteria, protozoa, rickettsia, chlamydia, fungi, and the like. Pathogens that can be effective with vaccines include, but are not limited to, viruses, bacteria, and the like.

As used herein, the phrase "sufficient concentration to produce secretory IgA" refers to the ability of the adjuvant or vaccine itself, i.e. the concentration of the adjuvant or vaccine itself which produces IgA secreted at the onset of the post-administration immune response . The concentration can be achieved in vitro or in vivo using methods known in the art.

In the present invention, the term "secretory IgA" refers to secreted IgA. IgA is the major immunoglobulin in the secretory fluid and helps prevent infection on the mucosal surface. The IgA is found abundantly in saliva, runny nose, and body fluids secreted from intestines, organs, or colostrum, but also in serum.

The term "administration" in the present invention means supplying the vaccine of the present invention or a pharmaceutical composition containing the vaccine, alone or in combination with another therapeutic agent, to the host to be treated. The vaccine or composition can be administered, for example, in a mixed state, either simultaneously or sequentially.

Hereinafter, the present invention will be described in detail.

The present invention provides an ophthalmic vaccine adjuvant composition comprising poly (I: C).

The present invention also relates to a composition comprising i) poly (I: C); And ii) a subunit antigen or an inactivated antigen of the pathogen.

The pathogen is preferably an influenza virus and more preferably an influenza species of A / New Caledonia / 20/99 (H1N1), A / Panama / 2007/97 (H3N2) or B / Shandong / 7/97 But is not limited thereto.

The subunit is preferably one or more subunits selected from the group consisting of influenza virus subunits HA, NA, M1, M2, NP, PB1, PB2, PA and NS2.

The adjuvant composition or vaccine is preferably a formulation to be administered to the mucous membranes of the eye, but it is not limited thereto and can be used in all the formulations to be administered to the eye area such as eye drops.

In the Examples and Experimental Examples of the present invention, when poly (I: C) is used as an adjuvant when inoculating the mucous membrane of an influenza virus vaccine, it has an excellent antibody production enhancing effect similar to that of CT, It was confirmed that poly (I: C) can be used as a useful adjuvant for ocular mucosal influenza virus vaccine (see FIG. 1 to FIG. 8).

In conclusion, the present invention can confirm that poly (I: C) can be used as a new useful adjuvant for influenza virus vaccine in place of CT since it can solve the cytotoxic and inflammatory side effects of CT Respectively.

The composition or vaccine according to the invention may additionally comprise a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers for use in the compositions or vaccines of the present invention include, but are not limited to, antioxidants, preservatives, colorants, aromatics and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, Vehicles, diluents, excipients, and / or pharmaceutical adjuvants. Typically, the compositions or vaccines of the invention are administered in the form of a composition comprising the vaccine or modifications or derivatives thereof, together with one or more physiologically acceptable carriers, excipients or diluents. The acceptable carriers, excipients or stabilizers used in the present invention are non-toxic to the receptor and preferably inert at the dose and concentration used. Examples of suitable carriers include neutralized buffered physiological saline, or physiological saline mixed with serum albumin. Other standard carriers, diluents and excipients may be included if desired.

A general method of preparing the composition or vaccine of the present invention may be as follows. An animal pharmaceutical composition, a similar drug, aquaculture drug composition, a food composition and a cosmetic composition can be also produced by a known production method. The vaccine or the like of the present invention may be blended with a pharmaceutically acceptable carrier and administered into the eye.

The present invention also relates to a composition comprising i) poly (I: C); And ii) administering to the individual's eye a vaccine comprising a subunit antigen or an inactivated antigen of the pathogen.

The pathogen is preferably an influenza virus and more preferably an influenza species of A / New Caledonia / 20/99 (H1N1), A / Panama / 2007/97 (H3N2) or B / Shandong / 7/97 But is not limited thereto.

The subunit is preferably one or more subunits selected from the group consisting of influenza virus subunits HA, NA, M1, M2, NP, PB1, PB2, PA and NS2.

The administration can be carried out at a concentration sufficient to produce secretory IgA, but is not limited thereto.

The vaccine is preferably a formulation to be administered to the mucous membrane of the eye, but it is not limited thereto, and the vaccine can be used in all the formulations to be administered to the eye area such as eye drops.

The amount of the vaccine of the present invention can be easily determined by those skilled in the art in consideration of the intended use, the target disease (kind thereof, etc.), the patient's age, body weight, The frequency of administration of the subject (or patient) in the treatment method of the present invention can also be easily determined by those skilled in the art considering the purpose of use, the type of the subject (its kind, severity, etc.), the patient's age, body weight, medical history, As an example of the frequency of administration, mention may be made of administration every day to several months (for example, every week to month), or once before every epidemic epidemic. It is preferable to administer weekly to monthly administration with consideration of progress, and it is advantageous to perform additional immunization at least about one week apart. The additional immunization interval is more preferably about 3 weeks or more, but is not limited thereto.

The dose of the vaccine of the present invention varies depending on the age, body weight, symptom or method of administration of the subject, and is not limited. In the case of ocular mucosa administration, the dose is 0.001 mg to 10 mg, preferably 0.1 mg to 1 mg mg.

In addition, the present invention relates to a composition comprising i) poly (I: C); And ii) a vaccine (1) comprising a subunit antigen or an inactivated antigen of the pathogen, and

(2) regarding the administration method, administration amount, or the number of administration of the vaccine to the eyeball.

The pathogen is preferably an influenza virus and more preferably an influenza species of A / New Caledonia / 20/99 (H1N1), A / Panama / 2007/97 (H3N2) or B / Shandong / 7/97 But is not limited thereto.

The subunit is preferably one or more subunits selected from the group consisting of influenza virus subunits HA, NA, M1, M2, NP, PB1, PB2, PA and NS2.

The vaccine is preferably a formulation to be administered to the mucous membrane of the eye, but it is not limited thereto, and the vaccine can be used in all the formulations to be administered to the eye area such as eye drops.

In the above kit, the manual includes information on a method of administering the medicament or the like of the present invention, a method of performing administration such as a physician or a patient, and a method of diagnosis for a person (who may be a patient) Includes documents containing. The instructions may include instructions on the administration procedure of the diagnostic agent, preventive agent, medicament, etc. of the present invention. The description may be made in accordance with the short form prescribed by the national supervisory body in which the invention is carried out and may indicate that it has been approved by the supervisory body. These instructions are referred to as product attachments and are normally provided in writing, but are not limited to these. The instructions may be provided in the form of, for example, a film affixed to a vial, or in the form of an electronic medium (e.g., a web site provided via the Internet or e-mail).

In the kit, the container may optionally be accompanied by a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of a medicament or biological product, which may be manufactured, Use or sale of the product.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples.

However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

< Example  1> Preparation of mouse

Specific pathogen-free BALB / c mice were prepared at 6-10 weeks of age (Orient Bio, Sungnam, Korea). All mice were maintained in laboratory animal facilities under the conditions of a specific pathogen in the International Vaccine Institute (Seoul, Korea), and sterile foods and filtered tap water were provided free-of-charge. All animal experiments were approved by the Animal Research Committee of the International Vaccine Institute (Seoul, Korea). Prior to experimental manipulation, mice were anesthetized by intraperitoneal (ip) injection of ketamine (100 mg / kg body weight) and xylazine hydrochloride (10 mg / kg body weight).

< Experimental Example  1> in the ocular mucosa poly (I: C) As an adjuvant  Verification of effect

OVA (100 ug) and various adjuvants (CT (2 ug), poly (I: C) (10 ug), MPLA (10 ug) and Alum (1 mg) After the application of a pipette for 3 times a total of 3 times a week, various mucosal washing solutions (Tear, Nasal, Fecal, Vaginal) were obtained one week after the last instillation and IgG and IgA were observed by ELISA.

At this time, sample collection was performed as follows. Specifically, Serum was obtained via retro-orbital bleeding. Tear wash samples were obtained by washing with 10 ml PBS per eye. Fecal extracts were obtained by adding feces to PBS containing 0.1% sodium azide. The excrement was stirred by vortexing, then centrifuged and the supernatant was collected for analysis. Vaginal wash samples were collected by washing with 100 ml PBS. The mice were sacrificed and the nasal wash samples were obtained by flushing 100 ml PBS through the internal nares of the nasal cavity using a pipette.

At this time, ELISA analysis was performed as follows. Specifically, ELISA plates (Falcon, Franklin Lakes, NJ) were coated with PBS containing OVA (100 mg / ml) or inactive A / PR / 8 (5 mg / ml) or LPS (1 mg / ml) , &Lt; / RTI &gt; incubated at 4 C overnight. Blocking was performed with PBS containing 1% BSA (Sigma-Aldrich), and a 2-fold diluted sample was applied to the plate. HRP-conjugated goat anti-mouse IgG or IgA antibody (Southern Biotechnology Associates, Birmingham, AL) was added to each well and incubated overnight at 4 ° C. For color formation, a tetramethylbenzidine solution (Moss, Pasadena, MD) was used. The plates were then measured at 450 nm with an ELISA reader (Molecular Devices, Sunnyvale, Calif.) After addition of stop solution (0.5 N HCl). Detectable titers of antigen-specific antibody (Endpoint titer) are shown horizontally reciprocal log ₂ of the inverse final dilution showing a> 0.1 absorbance than the background level. For detection of total IgA levels, plates were coated with chlorine F (ab9) 2 anti-mouse Ig and HRP-conjugated goat anti-mouse IgA antibody (Southern Biotechnology Associates) was used as detection antibody. (R &amp; D Systems, Minneapolis, MN) and HRP-conjugated rabbit anti-goat IgG (Southern &apos; s) for detection of OVA-specific sIgA antibody levels. Biotechnology Associates) was used as a detection antibody.

As a result, as shown in Fig. 1, it was confirmed that most of the antigen-specific antibodies (IgG and IgA) were generated in the CT inoculated group. In serum, antigen-specific IgG antibody was also significantly increased in the groups inoculated with adjuvants other than CT inoculation group. However, antibody-specific IgA antibody secretion in mucosal tissues was significantly increased only in the group inoculated with poly (I: C) other than the group to which CT was added (FIG. 1). This suggests that poly (I: C) is an effective adjuvant to induce immune responses other than CT in ocular mucosal inoculation.

< Experimental Example  2> As an influenza vaccine poly (I: C) As an adjuvant  Check the effect

The antigens were replaced with hemagglutinin (HA) (CJ, Korea), which was used as an influenza vaccine in OVA under the same conditions as in Experimental Example 1, and the effect of each adjuvant on antibody production Respectively.

In this case, the HA was used as the HA of the trivalent influenza vaccine (Biken), A / New Caledonia / 20/99 (H1N1), A / Panama / 2007/97 (H3N2) and B / Shandong / 7/97 Three influenza species were used.

As a result, as shown in FIG. 2, most of the antibody production enhancement effects were confirmed in CT and poly (I: C) -infected groups, and in the poly (I: C) (FIG. 2). This confirms that poly (I: C) is an adjuvant that induces an effective immune response when using influenza vaccine antigens in ocular mucosa other than CT.

< Experimental Example  3> Confirmation of proper dose of vaccine antigen in ocular mucosa

To determine the minimum and proper dose of the vaccine to be used with the poly (I: C) adjuvant in the mucosa of the eye, BALB / c mice were inoculated with HA antigen twice at intervals of 2 weeks, The level of antigen - specific antibody production in mucosal tissue was examined and compared.

Specifically, 0.1 μg to 2 μg of HA (CJ, Korea) was inoculated with 10 μg of poly (I: C) twice at two-week intervals to confirm the optimal vaccination dose of the vaccine antigen. Antigen, especially IgG and IgA, production in serum and mucosal tissue was determined by ELISA. At this time, the amount of IgG antibody was measured in serum and the amount of IgA antibody was measured in each mucosal washing solution.

As a result, as shown in FIG. 3, in the group in which the amount of HA vaccine antigen-specific IgG antibody production in the serum was significantly increased compared to the antigen alone group, 1 μg of HA vaccine antigen was added to 10 μg of poly (I: And the groups inoculated with 0.1 .mu.g or 2 .mu.g of HA antigen showed no significant increase in antibody production (FIG. 3).

In particular, the effect of increasing the antibody production in the 2 ug HA antigen-inoculated group, which was twice the 1 ug antigen group, was lower than that of the 1 ug HA antigen-inoculated group due to the lower concentration of the HA vaccine (1 ug / 11 ul) Respectively. When vaccinated with 1 ug / 11 μl of vaccine antigen at 2 ug / head, the total volume of ocular mucosal vaccine is 26 μl (22 μl of antigen + poly (I: C) 4 μl) The amount of poly (I: C) was diluted by half compared with the case of ocular mucosal vaccine (total amount of ocular mucosal vaccine: 15 μl), and as the volume of the vaccine to be inoculated into the eye of the mouse was increased, It was assumed that the size would be larger and thus the amount lost when moving the anesthetized mouse was greater than the 15 ul vaccine. Therefore, it was estimated that the effect of poly (I: C) was reduced or significantly lost in the 2 ug vaccinated group, so that the effect of 1 ug vaccination was lower than that of the vaccinated group. Therefore, all subsequent experiments were performed by immobilizing HA vaccine antigen at 1 ug.

< Experimental Example  4> In the ocular mucosa poly (I: C) Adjuvant  Identify the correct dose

To determine the minimum and proper amount of poly (I: C) adjuvant in the mucosa of the eye, BALB / c mice were inoculated twice with poly (I: C) The level of antigen - specific antibody production in tissue washes was determined and compared.

Specifically, BALB / c mice were injected with 1 ug HA, 30 ug of poly (I: C) with 1 ug HA vaccine antigen every two weeks in the eyes and then 2 weeks after the last inoculation. The level of HA - specific antibody production in the mucosal washes was confirmed by ELISA.

As a result, as shown in FIG. 4, there was a significant difference in IgA antibody production level between 1 ug and 10 ug poly (I: C) administration groups only in samples of tear and vaginal lavage fluid. : C), there was no significant difference in the level of IgG or IgA antibody production between the groups (Fig. 4).

This means that even a small amount (1 ug) of poly (I: C) can give a similar level of antibody production enhancement to that of poly (I: C). Therefore, when eye was vaccinated with poly (I: C) as an adjuvant, the least effective poly (I: C) dose was 1 ug. The effect of 10 ug of poly (I: C) was similar to that of 10 ug of poly (I: C) and no additional enhancement was observed. Therefore, in the subsequent experiment, 10 ug of poly (I: C) was administered in order to demonstrate the optimum adjuvant effect and to confirm its effect.

< Experimental Example  5> Comparison of efficacy of antibody production in ocular mucosal inoculation and nasal mucosal inoculation

Objective confirmation of the effect of poly (I: C) on the mucosa of the eye was made by comparison with other inoculation routes. On the other hand, nasal vaccination is known to best promote serum and mucosal antibody production. Therefore, the adjuvant properties of poly (I: C) were confirmed by comparing the effects of inoculation with nasal spray.

<5-1> HA  Identification with vaccine antigen

BALB / c mice were inoculated with 1 ug HA or 10 ug of poly (I: C) to the eyes and nose at 2-week intervals, respectively. Two weeks after the last inoculation, HA-specific antibodies Serum and mucosal wash samples were identified by ELISA.

As a result, as shown in Fig. 5, the nasal-inoculated groups in the serum showed HA-specific IgG antibody production levels significantly higher than those in the inoculated groups. First, the effect of poly (I: C) on antibody production in the nasal inoculation group compared to the group treated with poly (I: C) Could know. However, when comparing the levels of HA-specific IgA antibodies in all mucosal flushes except saliva samples compared to the inoculated group and the nasal inoculation groups of two groups with the same amount of poly (I: C) It was confirmed that there was no statistically significant difference in the effect of enhancing antibody production between stars (FIG. 5).

<5-2> H1N1  Identification with split vaccine antigen

In addition, the adjuvant effect of poly (I: C) was not significantly different between instillation and nasal administration, even when an inactivated whole split vaccine antigen was used instead of influenza virus subunit vaccine To confirm the adjuvant nature of the poly (I: C) after intravenous infusion of the H1N1 influenza virus A / California / 7/2009 split vaccine as an antigen.

Specifically, 6 to 8 weeks old BALB / c mice were inoculated with 1 ug or 10 ug of poly (I: C) twice a week with an instillation and nasal route along with 1 ug of H1N1 split vaccine, After two weeks, the H1N1 split vaccine antigen-specific IgG and IgA antibody production levels were determined in serum and eye wash samples by ELISA assay, respectively.

As a result, as shown in Fig. 6, nasal immunization group was higher in antigen-specific IgG antibody production level than the group treated with the same amount of poly (I: C) than in the inoculation group. This suggests that poly (I: C) improves systemic IgG antibody formation in serum more than oocyte inoculation in nasal vaccination, like HA-vaccine. However, the effect of increasing IgA antibody production in the mucosal wash liquor samples was not observed in almost all of the mucosal samples, as there was no difference in the effect of enhancing pathogen-specific antibody production between instillation and nasal inoculation (Fig. 6).

Thus, inoculation of the poly (I: C) adjuvant with the antigen of the HA vaccine or H1N1-split vaccine into the nasal cavity may exhibit efficient adjuvant properties similar to the enhancement of antibody production after inoculation into the nasal passages I could confirm.

< Experimental Example  6> Poly (I: C)

<6-1> Confirmation of cell distribution change

CT is the most effective adjuvant that causes immunity through the mucosa, but it has cytotoxic and inflammatory side effects. However, poly (I: C) is an adjuvant that has already been identified as safe for clinical use. Confirmation was confirmed once more by cell distribution after instillation.

Specifically, CD8α and CD11c cells were obtained from conjunctival tissues after instillation of PBS, 1 μg of HA alone, and 1 μg of HAs + 10 μg poly (I: C) in 6-8 week old BALB / The cells were stained with FITC and APC, respectively, and cell changes in the conjunctival tissue at 24 hours were confirmed using a flow cytometer.

As a result, as shown in FIG. 7, in the group inoculated with CT, unlike the other groups, it was confirmed that the conjunctival tissue was swollen, and thus CD8α + cells were greatly increased (FIG. 7).

Therefore, unlike CT, it was confirmed that poly (I: C) is a safe adjuvant without inflammation after inoculation. In addition, when HAs was used as a vaccine, it was confirmed that there was no significant difference between the vaccine and PBS control.

<6-2> Confirmation of the expression of inflammatory cytokines

In order to confirm the expression of inflammatory cytokines in the eyes after administration of poly (I: C), expression of inflammatory cytokines was confirmed 24 hours after poly (I: C) administration with HA vaccine antigen .

Specifically, 6-8 week old BALB / c mice were instilled with 1 μg of HA vaccine alone, antigen + 10 μg poly (I: C), or antigen + CT, , 12h, 24h), and cDNA was synthesized with reverse transcriptase. Using this as a template, we confirmed the increase of mRNA expression of IL-1alpha and TNF-alpha by real-time PCR.

As a result, as shown in Fig. 8, no increase in expression of IL-1alpha mRNA and TNF-alpha mRNA was observed in all of the groups treated with PBS, HA vaccine antigen alone, and HA antigen + poly (I: C) 8).

Claims (12)

flu vaccine adjuvants comprising poly (I: C).
delete The vaccine according to claim 1, wherein the poly (I: C) enhances antibody production upon ocular mucosal administration, and is free from cytotoxicity and inflammatory reaction.
An influenza virus vaccine comprising:
i) poly (I: C); And
ii) Subunit antigens or inactivated antigens of influenza virus.
delete 5. The composition according to claim 4, wherein the subunit comprises at least one subunit selected from the group consisting of influenza virus subunits HA, NA, M1, M2, NP, PB1, PB2, PA and NS2. Influenza virus vaccine.
delete delete delete An anti-influenza virus vaccine kit comprising:
A) An influenza virus vaccine comprising:
i) poly (I: C); And
ii) subunit antigens or inactivated antigens of influenza virus; And
B) A description of the procedure, including the method, dosage or frequency of administration, for the ocular mucosal administration of the vaccine.
delete 11. The composition of claim 10, wherein the subunit comprises one or more subunits selected from the group consisting of influenza virus subunits HA, NA, M1, M2, NP, PB1, PB2, Influenza virus back credit kit.

Images