Cross-inducing agent, and cosmetics, foods, and quasi-drugs containing cross-inducing agent

Abstract

To provide an environmental symbiotic type immunostimulating agent that is administered intranasally, intratracheally, or orally and induces diverse immunoglobulins.SOLUTION: The present invention provides an immunostimulating preparation that is a fusion protein of a bacterial heat shock protein and a viral peptide antigen, which can induce a wide variety of immunoglobulins by simultaneously stimulating the nasal, respiratory, and oral administration. The present invention provides feed, feed additives, and environmental symbiotic preparations for livestock, or cosmetics, foods, and quasi-drugs for humans, which contain the immunostimulating preparation and enable symbiosis with environmental microorganisms. The present invention provides preparations, as well as cosmetics, foods, and quasi-drugs for humans, which enable symbiosis with environmental microorganisms by utilizing livestock-derived IgA, IgG, and IgY from among the immunoglobulins.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an environmentally symbiotic immune activating agent that induces diverse immunoglobulins for nasal, airway, and oral administration, and its uses.

Background Art

[0002] In recent years, technologies in life sciences have advanced from various perspectives. Omics analysis technologies for evaluating complex microorganisms and the composition of complex metabolites have been established, and the understanding of various life phenomena has progressed through the use of machine learning and artificial intelligence. On the other hand, countermeasures against various problems in ecosystems caused by environmental destruction and various unknown infectious diseases whose relevance to environmental destruction has been pointed out are urgent issues not only for humans but also for livestock, and comprehensive responses are desired.

[0003] As a method for preventing infectious diseases, vaccines are used. Generally, live vaccines and vaccines in which the functions of microorganisms are inactivated are mainstream. In recent years, new methods such as RNA vaccines and DNA vaccines that utilize the genetic information of microorganisms, and plant-derived vaccines expressed in plants by utilizing genetic information have also emerged (Non-Patent Documents 1, 2, 3, 4). These vaccines mainly aim to selectively induce IgG that has a specific effect on the target pathogenic microorganism.

[0004] On the other hand, there are nasal vaccines that aim to induce secretory IgA, which has a weak titer but is effective against pathogenic microorganisms. Some of them are commercially available (Non-Patent Documents 1, 5). And some of them are commercially available (Non-Patent Documents 1, 5). (Non-Patent Documents 1, 5).

[0005] An oral vaccine has been devised that utilizes M cells in Peyer's patches of intestinal lymphoid tissue (Patent Document 1, Non-Patent Document 6). M cells express cell surface molecules such as GP-2 and PrP, and are known to take up bacteria and serve as the entry point for the immune response via dendritic cells (Non-Patent Documents 7, 8). Subsequently, several studies have been conducted, suggesting that bacterial-derived Heat shock protein 60 (HSP60) plays an important role in binding to the said surface molecules (Non-Patent Documents 7, 8, 9). Also, it has been suggested that a similar antigen uptake system by M cells is also observed in nasal mucosa and airway mucosa (Non-Patent Documents 10, 11).

[0006] In the activation of the immune system in the airway, it is known that bacterial-derived Heat shock protein 65 (HSP65), particularly HSP65 derived from Mycobacterium leprae, has an immunostimulatory effect (Non-Patent Documents 12, 13). On the other hand, attempts have also been made to develop vaccines against Mycobacterium tuberculosis by utilizing these effects (Non-Patent Documents 14, 15). Thus, in the mucosal system, it has been suggested that HSP60 and HSP65 may regulate host defense by having cell-binding or adjuvant-like activities.

[0007] On the other hand, looking at the symbiotic relationship between animals and bacteria, in recent years, the relationship between the gut microbiota and the physiological functions and pathologies of the host has been rapidly clarified. For example, it is known that more than 1,000 species and over 40 trillion intestinal bacteria inhabit the human intestine, forming a complex intestinal ecosystem, and they interact with the host's tissues to contribute to maintaining health. Therefore, when the balance of these intestinal ecosystems is disrupted, so-called dysbiosis leads to the development of various diseases (Non-Patent Documents 16, 17), and the need for probiotics and prebiotics to control the intestine has been recognized.

[0008] Note that the inventors have successfully developed probiotics that affect the living bodies of animals by utilizing thermophilic Bacillus flora, which is one of the extreme environmental microorganisms that are difficult to grow in the normal temperature range. In particular, it has been suggested that it can control the intestinal flora of livestock such as chickens, pigs, and cows, fish, and rodents as model animals, namely mice and rats, and can have a beneficial effect on the living body in the process (Patent Documents 2 and 3, Non-Patent Document 18).

[0009] Furthermore, turning our attention to the symbiotic relationship between animals and viruses, it has been suggested that phages play an important role in the biological defense system in the intestinal tract (Non-Patent Document 19), but it cannot necessarily be said that it has advanced more than the research on intestinal bacteria.

[0010] However, for example, as a virus that can infect the mucosal immune system of animals, the spread of porcine epidemic diarrhea virus (Alphacoronavirus) has been regarded as a problem in the pig farming industry. The virus has the characteristic that it causes non-lethal infections in sows and fattening pigs, but is highly lethal when young pigs are infected (Non-Patent Document 20). Although the same coronavirus, SARS-CoV, which is a Betacoronavirus, has a different infection pattern from SARS-CoV-2 (Non-Patent Document 21), they have similar properties in that they have Spike proteins (Non-Patent Document 22). However, the development research on these vaccines has been independently advanced in the medical field and the livestock field, and has not progressed in a form that takes into account the characteristics of similar viruses. On the other hand, Edward Jenner, the pioneer who gave birth to the basic concept of vaccines, showed in 1796 that the onset of smallpox was suppressed by utilizing the pus of cowpox that infects cows (Non-Patent Document 23), and later it was shown that the DNA sequences of cowpox virus and smallpox virus are very similar (Non-Patent Document 24). From such the background of vaccine development, there are many points where the current vaccine development is deviated.

Prior Art Documents

Patent Documents

[0011] [Patent Document 1] Japanese Patent Application No. 2006-331950 [Patent Document 2] Japanese Patent No. 5578375 [Patent Document 3] Japanese Patent No. 5041228 [Non-Patent Document]

[0012] [Non-Patent Document 1] Report on Vaccine Research and Development by the Research and Development, Production, and Distribution Subcommittee of the Vaccination and Vaccine Subcommittee of the 22nd Council for Health Sciences, December 25, 2019, "Regarding Vaccine Research and Development" https: / / www.mhlw.go.jp / content / 10906000 / 000580437.pdf [Non-Patent Document 2] DNA Vaccine https: / / ja.wikipedia.org / wiki / DNAワクチン [Non-Patent Document 3] RNA Vaccine https: / / www.t.utokyo.ac.jp / shared / press / data / setnws_201710121450382322891478_608693.pdf [Non-Patent Document 4] Plant-Derived Vaccine https: / / www.mt-pharma.co.jp / ir / meeting / pdf / presen170927_M.pdf [Non-Patent Document 5] Nasal Vaccine https: / / www.amed.go.jp / news / release_20190104.html [Non-Patent Document 6] Oral Vaccine https: / / shingi.jst.go.jp / past_abst / abst / p / 14 / 1407 / riken2-5.pdf [Non-Patent Document 7] Hiroshi Ohno, Biology and Biochemistry of Special Intestinal Epithelial Cells, M Cells, Journal of Biochemistry, Vol. 83, No. 1, pp. 13-22, 2013 http: / / www.jbsoc.or.jp / seika / wp-content / uploads / 2013 / 05 / 83-01-03.pdf

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

非特許文献16

非特許文献17

Non-Patent Document 18

Non-Patent Document 19

Non-Patent Document 20

Non-Patent Document 21

Non-Patent Document 22

Non-Patent Document 23

Non-Patent Document 24

Summary of the Invention

Problems to be Solved by the Invention

[0013] Generally, vaccine development is advanced by targeting the pathogen itself or epitope molecules characteristic of the pathogen. Also, regarding the selection of epitopes, there are many technical constraints such as avoiding complex structures such as alpha-helices and beta-sheets, or selecting hydrophilic regions. Therefore, despite the fact that zoonotic infectious diseases are known to exist in livestock as well as in humans (including pet animals), vaccine development that takes into account the target infectious pathogen and the universal gene sequences common among the pathogen and pathogens genetically related to it has hardly been carried out.

[0014] Thus, in conventional vaccine development, although it may be useful for increasing antibody titers as it localizes the characteristics of the antigen (epitope region) that becomes the vaccine to a specific virus, it is not necessarily efficient when viewed from the perspective of countermeasures against zoonotic infectious diseases and the symbiosis of the entire ecosystem.

[0015] Also, for pathogens that are prone to genetic mutation like RNA viruses, the conventional vaccine development methods have weaknesses. Even if a vaccine is developed, it is assumed that it may not be effective against the mutated virus at the stage of approval.

[0016] Furthermore, in order to enhance the function of the vaccine, the immune system of the original host needs to be healthy. However, currently, research and development considering the synergistic effects with drugs, nutritional agents, supplements, etc. to elicit the effects of the vaccine have not progressed easily. Means for solution

[0017] In modern times, the manufacturing technique of conventional vaccines focuses on the specificity of the target pathogenic bacteria, while the characteristic of the technique using the pus of cowpox by Edward Jenner, who gave birth to the concept of the vaccine itself, is, as described above, to take measures against smallpox virus from different cowpox viruses that are similar in DNA sequence but different after later detailed analysis. That is, as a result, it is a direction of vaccine development considering the genetic similarity of the virus itself, and there are points worthy of attention again regarding these differences.

[0018] Therefore, in developing this preparation, by narrowing down the target to a genetically common sequence among relatively closely related viruses, using them as epitope antigens, and fusing them with bacterial-derived HSP, not necessarily in a hydrophilic region, taking advantage of the characteristics of M cells in mucosal tissues that can take up bacterial-derived HSP, immune system stimulation via dendritic cells is induced.

[0019] Furthermore, by moving the entire mucosal system through nasal, airway, and oral administration, and by improving the gut microbiota in conjunction with probiotics and prebiotics, the overall biological defense function is enhanced.

[0020] The globally well-known hygiene hypothesis (Strachan DP. Hayfever, hygiene, and household size. BMJ 299: 1259 - 1260, 1989) is a hypothesis that shows that differences in exposure to environmental factors during infancy lead to differences in the incidence of subsequent allergies, and that a non-hygienic environment reduces the incidence of subsequent allergies. This hypothesis is now widely recognized, and a lot of data supporting this hypothesis has been accumulated, and relationships with other diseases have also been pointed out in relation to the formation of the gut microbiota. That is, it suggests that it is important to construct an environment in which animals can coexist with environmental microbiota.

[0021] On the other hand, since this preparation does not adhere to the microorganisms already in the living environment and targets molecules common to related microorganisms, it is possible to stimulate the mucosal immune system to artificially construct a symbiotic relationship with unknown environmental microorganisms. As a result, it can also have a defense against unknown viruses. Therefore, when considering the whole animal, it is a novel preparation for constructing a symbiotic relationship with environmental microorganisms. For livestock, it can be used as a functional feed additive, a novel type of environmental purifying agent, and when replaced with humans, it is more preferably assumed to be used in daily necessities such as cosmetics, aromatherapy, and food for the purpose.

[0022] Therefore, provided is an immunopotentiating agent which is a protein obtained by fusing a bacterial-derived heat shock protein (HSP: heat shock protein) and a peptide antigen of a virus, and which enables the induction of highly diverse immunoglobulins by simultaneously stimulating nasal, airway, and oral administration.

[0023] Furthermore, provided are feeds, feed additives, environmental symbiotic agents for livestock, or cosmetics, foods, and quasi-drugs for humans that enable symbiosis with environmental microorganisms containing the immunopotentiating agent.

[0024] Furthermore, provided are agents that enable symbiosis with environmental microorganisms by utilizing IgA, IgG, and IgY derived from livestock among the immunoglobulins, as well as cosmetics, foods, and quasi-drugs for humans.

Advantages of the Invention

[0025] The present invention is a vaccine-like immunopotentiator for forming immunoglobulins having a broad binding ability although with a weak titer. When a pandemic problem caused by an unknown virus occurs, it is necessary to develop a vaccine as powerful as possible. However, since a period of one year is required for the certification of conventional vaccines, at the previous stage or at the stage where the development has failed, it is a preparation aimed at reducing the risk of infection and preventing an increase in the number of patients with severe conditions, although with a weak titer.

[0026] In particular, it will induce secretory IgA that plays a major role in mucosal immunity. For example, since RNA viruses are prone to mutation, by utilizing IgA that has a broad binding ability even when mutated, it is possible to exert a certain degree of preventive effect even with a weak titer, which is the key point and solves the conventional problems. Also, the present invention is expected to enable efficient defense against viruses prone to mutation if there is a highly safe, mutation-resistant, and highly versatile technology. It is expected that the immunopotentiator will be more effective when used in combination with conventional vaccines.

Brief Description of the Drawings

[0027]

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Modes for Carrying Out the Invention

[0028] Next, embodiments of the present invention will be described, but the present invention is not limited to these embodiments. That is not the case.

[0029] The immunostimulant of the present invention is utilized as part of cosmetics, quasi-drugs, or foods targeted at nasal mucosa, airway mucosa, and intestinal mucosal tissues, feed additives for livestock, and environmental purification agents. It is a methodology prepared in case there are weaknesses such as the antibody titer not persisting for more than a year, like the antibodies against seasonal influenza, even if a vaccine that induces IgG is developed like conventional vaccines. By using this preparation regularly, it aims to induce cross-reactive or highly diverse IgA antibodies that recognize relatively widely against viruses that are prone to mutation, such as RNA viruses, as long as there is a certain degree of similarity. Also, along with the synergistic effects of the transnasal vaccine, transairway vaccine, and oral vaccine that have been developed independently until now, it aims for a synergistic effect by controlling the intestinal flora that affects immunostimulation.

[0030] As the gene sequence used in the present invention, among viral surface proteins, it is based on epitope sequences that are common or highly similar to pathogenic viruses and non-pathogenic viruses that are closely related. However, unlike conventional antigen epitopes, it is not necessarily limited to hydrophilic regions. Instead of peptides of about 6 - 20 amino acids as the epitope, in order to make the amino acid sequence as long as possible, a fusion protein is made by binding candidate epitopes with a histidine tag or the like, which will be described later.

[0031] Furthermore, as a bacterial-derived protein, Heat shock protein60 (HSP60) and Heat shock protein65 (HSP65) are fused as candidate adjuvant proteins that are expected to be easily taken up by M cells in mucosal cells to construct a hybrid antigen.

[0032] Examples of HSP60, a candidate protein for an adjuvant in a hybrid antigen, include Brucella aboritus, Clostridium difficle, Salmonella typhimurium, Streptococcus suis, etc. Examples of HSP65 include HSP65 derived from Mycobacterium leprae, which is suggested to contribute to the activation of respiratory immunity. All of them are heat shock proteins derived from bacteria classified as BCL2 or BCL3 in terms of biosafety level. However, this itself is not the cause of pathogenicity, and the important point is that it is easily taken up by M cells. It is known that glycoprotein2 (GP-2) and prion protein (PrP) are expressed in M cells and play a role in the step of taking up antigen molecules and presenting antigens.

[0033] Therefore, in the present invention, it is expected that by utilizing the HSP described in paragraph number

[0029] so that the surface antigen of the virus can be reproduced as much as possible and can be easily taken up by M cells in mucosal tissues, antigen presentation can be efficiently achieved (Figure 3).

[0034] In addition, in chickens (Non-Patent Document 7) having M cells and with M cells densely packed, by exposing the hybrid antigen, it becomes possible to efficiently obtain IgY derived from eggs with high cross-reactivity or high diversity. By utilizing this diverse IgY and incorporating it as part of cosmetics, quasi-drugs, foods, feed additives for livestock, or environmental purification preparations, it is expected to further expand the range of applications (Figure 1).

[0035] In addition, in order to enhance the effect of the immunostimulant of the present invention by controlling the intestinal flora, Bacillus hisashii (International Deposit Number BP-863), bacterial groups of the Bacteroidetes phylum, lactic acid bacteria, yeasts, etc., which are known to enhance the production ability of IgA, are expected to be important.

[0036] (Example 1) Design a fusion protein in which the epitope of the amino acid sequence described in Table 1 is bound to HSP via histidine. Express it in a wheat cell-free system, a silkworm cell-free system, a yeast system, or a plant (such as tomato or rice) capable of protein synthesis, and utilize the crude product. After mixing it with any spreading agent or liquid, perform nasal, respiratory, or oral administration. In the case of producing the fusion protein in food crops, it is promising because it can contribute to reducing production costs. However, under current laws, since it falls into the category of genetically modified crops, generally, it takes time for approval. However, unlike the development of general genetic modification applications, it is not something that introduces molecules that disrupt the ecosystem, and it is expected to be quickly approved due to its utilization as a medical application. Regarding the hybrid antigen, in particular, as shown in No. 3 of Table 1, a fusion protein in which several short-chain epitopes are linked is recommended. Also, as the epitope, it is better to add regions that are not necessarily appropriate before and after, and bind a peptide of 50 amino acids or less in total with amino acids selected from histidine, etc., described later. Not adhering to short-chain epitopes is to make it difficult for immune tolerance by short-chain epitopes to occur, but this point is not the object of the development of the present invention and is based on known reports.

Table 1

[0037] Note that the epitope sequences in Table 1 are the common sequences of the Spike proteins of batacoronavirus with accession codes MN908947, MN996532, AY278741, KY417146, and MK211376 in the NCBI gene bank. It is common to SARS-cov and SARS-cov-2 that are infectious to humans, but it is characterized by being a region that does not affect infectivity.

[0038] (Example 2) Comparative genomic analysis was performed on the gene sequences of the spike protein of alphacoronavirus and batacoronavirus (Figs. 5 and 6). The solid line range encloses the common nucleotide sequences among different viruses. As a result, the sequence region within the dotted frame in Fig. 5 was part of the spike protein of coronavirus and was common or similar between the two viruses. Also, in the sequence region within the dotted frame in Fig. 6, it was also part of the spike protein of coronavirus and was common or similar between both the alpha and beta viruses.

[0039] These sequences are as described in Figs. 7 and 8. Respectively, for alphacoronavirus, they are based on the sequences published as porcine epidemic diarrhea virus (PED) (Literature name: Virus Genes (2013) 54:215 - 224. https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC7088687 / pdf / 11262_2017_Article_1528.pdf). Each accession no. is as described in the figure.

[0040] Therefore, using these arrays as epitopes, a fusion protein was designed that binds via histidine downstream of the HSP in Fig. 9, and the hybrid antigen protein was synthesized by expressing it in a wheat cell-free system, a silkworm cell-free system, a yeast system, or a plant that can synthesize proteins. As alternatives to the Histidine tag, amino acids with medium hydrophobicity such as the HQ tag (HQHQHQ), HN tag (HNHNHNHNHNHN), HAT tag (KDHLIHNVHKEEHAHAHNK), cysteine tag (CCPGCC), cysteine itself tag (C or CCCCCC), methionine (M), and depending on the material for spreading the symbiotic preparation, asparagine (N), glutamic acid (E), threonine (T) as hydrophilic amino acids, and alanine (A), tyrosine (Y), etc. as hydrophobic amino acids are assumed.

[0041] This makes it possible to provide the host with highly diverse spike antigen stimulation common to alphacoronavirus and batacoronavirus, enabling various immune responses. Therefore, in this example, it is particularly expected to induce resistance to unknown alphacoronavirus and batacoronavirus. Regarding the epitope candidate sequence region (sequences shown in Figs. 5 and 6), it is expected to be a usable base sequence for realizing immune induction at least commonly in alphacoronavirus and betacoronavirus even as an invasive vaccine antigen including a DNA vaccine.

[0042] This method is not limited to alphacoronavirus and betacoronavirus in particular, and is also applicable to future unknown infectious diseases such as arteriviruses of the family Arteriviridae, genus Arterivirus, which are involved in porcine reproductive and respiratory syndrome (PRRS), Simian hemorrhagic fever virus (SHFV), etc., or the genus Influenza virus of the family Orthomyxoviridae, which requires various types. Naturally, the sequences will be different from those of the present application, and common sequences among closely related viruses of the target viral family will be used. That is, similar to the present method, by preparing hybrid antigens by fusing multiple common regions and similar regions such as surface antigens of pathogenic microorganisms closely related to the target pathogen, and administering these hybrid antigens, it is expected that the comprehensive biological defense function will be enhanced and symbiosis with various pathogenic microorganisms will become possible.

[0043] In addition, in order to facilitate the binding to carrier proteins, adding cysteine to the N-terminus to make it easier to bind to carrier proteins using sulfide groups is recommended as one of the possibilities in Example 1 as well as Example 2.

[0044] Note that by utilizing the similarity of viruses and the like and binding similar epitopes as a plurality of types of long-chain peptides instead of short-chain peptides, it is expected to prevent the possibility of immune tolerance induced by epitopes of short-chain peptides. At the same time, it is expected to avoid the possibility of antibody-dependent enhancement of immunity (ADE: Antibody-dependent enhancement) due to incomplete antigen presentation. Furthermore, although long-chain peptides in which helper T cell epitopes and CTL epitopes are bound are known to have a high vaccine effect, it is expected that the vaccine effect will be further enhanced by binding them in combination with the fusion protein of the virus gene. In addition, by nasal, airway, and oral administration, non-invasively stimulate mucosal tissues, bind heat shock protein 60 (HSP60) or heat shock protein 65 (HSP65) derived from bacteria to M cells of mucosal cells, and activate the immune system, it is expected to induce highly cross-reactive or highly diverse immunoglobulins against a wide range of environmental microorganisms. If ADE is induced in the form of such a fusion protein, it is expected to be utilized as a tool for elucidating the mechanism of action of ADE, which has an unclear mechanism.

[0045] The etymology of the word "vaccine" is a coinage related to cowpox used in the aforementioned research by Edward Jenner and is derived from the Latin word "Vacca" (female cow). Therefore, for the preparation of the present invention, although inheriting the original concept of the vaccine, from the perspective of emphasizing symbiosis with environmental microorganisms and considering the ecosystem, as a preparation considering the ecosystem, the concepts of Ecoccin (Ecosin or Echocin), and Symbioccin (Symbiosin or Symbiotin) are proposed from Symbiosis, which means symbiosis.

Industrial Applicability

[0046] In addition, the fermented product containing the thermophilic bacterium (NITE International Deposit Number: BP-863) and the thermophilic bacterium complex (ATCC International Deposit Number: PTA-1773) that are utilized as probiotic candidates has been suggested to induce the expression of interferon, a cytokine involved in virus infection defense, after improving the intestinal flora (Journal of Bioscience and Bioengineering, 114(5): 500-505, 2012; the Gene Expression Omnibus (GEO) database (access ID: GSE37732)). Therefore, it is expected that the combined use with the HSP fusion protein described in this specification may enhance the immunostimulatory effect. Accordingly, the development of applications as a novel formulation mixed with the fusion protein is expected.

Deposit Number

[0047] BP-863 (NITE) PTA-1773 (ATCC)

Claims

**Claim 1** A protein obtained by fusing a heat shock protein (HSP) derived from bacteria and a peptide antigen of a virus, which is characterized by enabling the induction of highly diverse immunoglobulins by simultaneously stimulating nasal, respiratory, and oral administrations. An immunopotentiating preparation. **Claim 2** Livestock feed, feed additives, environmental symbiotic preparations, or cosmetics, foods, and quasi-drugs for humans that enable symbiosis with environmental microorganisms containing the immunopotentiating preparation. **Claim 3** Preparations, cosmetics, foods, and quasi-drugs for humans that enable symbiosis with environmental microorganisms by utilizing livestock-derived IgA, IgG, and IgY among the immunoglobulins.

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