Vaccine For The Prevention And Treatment Of C. Difficile Infections And The Use Thereof

Abstract

The present invention relates to a veterinary vaccine containing nanoadjuvants in the form of emulsion and Clostridium difficile antigens, as well as the use of the vaccine in the preventions and treatment of a C. difficile infection, especially in birds and mammals. The object of the invention is also the use of this vaccine to produce C. difficile-specific antibodies.

Claims

1. A vaccine comprising conjugates of epitopes of surface proteins 71 kDa of SEQ ID No. 1 and aminopeptidase M24 of SEQ ID No. 2 from Clostridium difficile suspended in emulsion, comprising additional immunostimulating components and Clostridium difficile toxins.

2. The vaccine according to claim 1, wherein a synthetic oil is a component of the emulsion.

3. The vaccine according to claim 1, wherein the 71 kDa protein epitope has amino acid sequence defined in SEQ ID No. 8, 9 and 10.

4. The vaccine according to claim 1, wherein the M24 protein epitope has amino acid sequence defined in SEQ ID No. 14 and 15.

5. The vaccine according to claim 1, wherein conjugates are selected from K2 ATGKKGSETPTGKTKV (SEQ ID. No. 16), K3 VNKIKNRPYYKGNIPG (SEQ ID. No. 23) and K4 KKGIK (SEQ ID. No. 14)

6. The vaccine according to claim 1, wherein the Clostridium difficile toxins are the TcdA and TcdB toxin or their inactivated versions.

7. The vaccine according to claim 1, wherein, apart from synthetic oil, the emulsion comprises an organic solvent, a non-ionic detergent, a cationic detergent and ultrapure water.

8. The vaccine according to claim 2, wherein the synthetic oil is dimethylpolysiloxane.

9. The vaccine according to claim 7, wherein the organic solvent is selected from ethanol or acetone.

10. The vaccine according to claim 7, wherein the non-ionic detergent is tyloxapol.

11. The vaccine according to claim 7, wherein the emulsion is 1-et-tyl-03, 1-ac-tyl-03, or 1-et-tyl-02, wherein the components of nanoadjuvant in the form of emulsion are combined in the ratio of 60-70% oil, 4-8% non-ionic detergent, 1-3% cationic detergent, 5-10% organic solvent, and 15-25% ultrapure water.

12. The vaccine according to claim 11, wherein 1-et-tyl-03 is a combination of 65% dimethylpolysiloxane, 5% tyloxapol, 1% benzyldimethyldodecylammonium chloride, 8% ethanol and 21% water; 1-ac-tyl-03 is 65% dimethylpolysiloxane, 5% tyloxapol, 1% benzyldimethyldodecylammonium chloride, 8% acetone and 21% water; and 1-et-tyl-02 is 65% dimethylpolysiloxane, 5% tyloxapol, 1% cetylpyridinium bromide, 8% ethanol and 21% water.

13. The vaccine according to claim 1, wherein the immunostimulating components are antigens of probiotic bacteria, such as polysaccharides, teichoic acids, lipoteichoic acids, proteins, peptidoglycan, glycolipids, lipopolysaccharides, monophosphorylic lipid A, glycoproteins, bacteriocins, DNA, RNA, enzymes, peptides and other molecules secreted to the medium.

14. The vaccine according to claim 1, wherein the vaccine is administered parenterally and/or intramucosally, preferably intranasally.

15. A method for use in treating C. difficile infections in animals, the method comprising administering the vaccine of claim 1 to said animals.

16. The method of claim 15, wherein the treated animals are birds and mammals.

17. A method for production of a C. difficile-specific antibody, the method comprising administering the vaccine of claim 1 to an animal.

18. The method of claim 17, wherein the C. difficile-specific antibody is an IgY antibody and wherein the vaccine is administered intramuscularly to birds, optionally to chickens.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0092] FIG. 1. shows the change of physicochemical properties of the nanoadjuvant during twelve months of observation. Changes of sizes and zeta potential of nanoadjuvant vesicles were analysed.

[0093] FIG. 2. shows pictures of bands after polyacrylamide gel electrophoresis (12% SDS-PAGE) with samples of the nanoadjuvant with the model OVA antigen applied, illustrating the durability of the antigen in nanoadjuvant after 6 months of storing under various temperature conditions.

[0094] FIG. 3. shows a graph of antigen release kinetics from nanoadjuvant drops after 0.5 h and 4 h.

[0095] FIG. 4. shows a graph illustrating the cytotoxicity of nanoadjuvants against BMDM line after 8 h and 24 h of incubation. The result was shown as control % which consisted of living BMDM cells not treated with the nanoadjuvant.

[0096] FIG. 5. shows a graph illustrating the effect of nanoadjuvant on absorbing Clostridium difficile by BMDM line cells. Positive control included formaldehyde-inactivated C. difficile cells and negative control included thermally inactivated C. difficile cells. Line cells were incubated with bacterial cells for 4 h and were subsequently analysed using a cytometer.

[0097] FIG. 6. shows a graph illustrating the induction of TNFα in macrophage cells by the nanoadjuvant of various concentration and subactivating concentration of LPS (1 ng/ml). The increase of TNFα production in macrophages after 24 h of incubation with a formulation of nanoadjuvant+LPS relative to samples treated with LPS alone was shown.

[0098] FIG. 7. shows the effect of nanoadjuvant on antigen absorption by RMPI2650 cells. Cells were incubated for 4 h with DQ-OVA with or without the addition of the nanoadjuvant (0.06%). The level of antigen absorption was studied using a flow cytometer. White histograms—DQ-OVA only, grey histograms—DQ-OVA with the nanoadjuvant.

[0099] FIG. 8. shows the result of studying nanoadjuvants tolerance in mice. 8A—change of mice weight with nanoadjuvant administered intramuscularly; 8B—change of mice weight with nanoadjuvant administered intranasally; 8C—mice survival rate.

[0100] FIG. 9. The result of 71 kDa protein mapping. 9A—a list of peptides selected for the synthesis; 9B—the result of immunoreactivity test (ELISA) carried out on synthesised peptides.

[0101] FIG. 10. The result of mapping a highly immunoreacitve region of 71 kDa protein. 10A—a list of peptides selected for the synthesis; 10B—the result of immunoreactivity test (ELISA) carried out on synthesised peptides.

[0102] FIG. 11. selecting the shortest immunoreactive sequences (epitopes) of 71 kDa protein. The upper panel shows truncation from N-terminus; the lower panel from C-terminus. 11A—the truncation of NNKLVKEFRVATGKKSETP peptide; 11B—the truncation of QTGWQEKNGKKYYLGS peptide; 11C—the truncation of GTYQKNSWLKVNGKMY peptide.

[0103] FIG. 12. The result of protein M24 mapping. 12A—a list of peptides selected for the synthesis; 12B—the result of immunoreactivity test (ELISA) carried out on synthesised peptides.

[0104] FIG. 13. selecting the shortest immunoreactive sequences (epitopes) of M24 protein. The upper panel shows truncation from N-terminus; the lower panel from C-terminus. 13A—the truncation of REGATLAEKLSKKGIK peptide; 13B—the truncation of LREKMSEKGTSTHVIT peptide.

[0105] FIG. 14. The result of conjugate immunogenicity testing together with the model adjuvant for intraperitoneal administration. The result is shown for individual epitopes.

[0106] FIG. 15. Total IgG antibodies. 15A—the amount of induced IgG antibodies; 15B—the comparison between the number of total IgG antibodies and the route of administration of the formulation.

[0107] FIG. 16. Total IgA antibodies. 16A—the amount of induced IgA antibodies; 16B—the comparison between the number of total IgA antibodies and the route of administration of the formulation.

[0108] FIG. 17. A comparison of the epitope-specific response. 17A—a comparison of the epitope-specific response for intranasal administration of the formulation; 17B—a comparison of the epitope-specific response for intramuscular administration of the formulation.

[0109] Using the nanoadjuvant with specific antigens as a vaccine against Clostridium difficile to obtain therapeutic antibodies is an entirely new, non-obvious solution and has numerous advantages. Nanoadjuvants are prepared in a very easy way in terms of techniques, and the whole procedure is easy to adapt for the use on an industrial scale. In the manufacturing of nanoadjuvants, commonly known compounds are used, such as synthetic oil (e.g. dimethylpolysiloxane), cationic detergents widely used industrially and organic compounds. All used compounds are on a so-called GRAS (Generally Recognized As Safe) list.

[0110] Nanoadjuvants are very stable under cooling conditions and even at room temperature, and in the case of short-term storage, they do not require storing in a refrigerator. Storing in a refrigerator up to one year does not show effects typical for emulsion ageing, namely creaming, sedimentation, flocculation, phase inversion, or coalescence. They do not change their physicochemical properties for at least 12 month of storage under cooling conditions. Nanoadjuvants interact with an antigen which spontaneously reaches the inside of drops right after its addition, which has been observed in the form of the increase of drop sizes and decrease in the zeta potential. Nanoadjuvants load protein antigen efficiently, retaining them inside and therefore act perfectly as a carrier of a potential vaccine and act as an adjuvant using the so-called depot effect. The protein antigen closed in a lipid encapsulation is separated from harmful environmental conditions, such as oxidating agents, as well as proteases. Consequently, an intact structure is maintained for a long time.

[0111] The surface of mucosa is coated by a layer of protective mucus, in which negatively charged mucin is present apart from enzymes and other proteins. Electrostatic binding to mucin is an effective way of increasing bioadhesion of the vaccine carrier. Nanoadjuvants being a component of the vaccine of the invention bind to mucin, which has been analysed using Zetasizer Nano ZS. Binding depends on the content of the nanoadjuvant and potential zeta resulting from it.

[0112] It is a condition of effective immunisation to induce the so-called danger signals, which are usually created during bacterial infection or in a process of eukaryotic cell death [13]. It is therefore preferred that the vaccine carrier acts to some extent in a cytotoxic and proinflammatory way. Nanoadjuvants with low concentrations and short periods of contact are not cytotoxic and by increasing their concentration it is possible to adjust the cytotoxicity level depending on needs. Moreover, nanoadjuvants show proinflammatory activity. When administered together with an antigen, they induce the secretion of TNFα by macrophages. They are not immunogenic by themselves. Nanoadjuvants also induce the antigen uptake by antigen-presenting cells and influence its presentation by increasing the protein expression responsible for that, namely MHC class II proteins. In animal testing, nanoadjuvants increase the titer of specific antibodies in serum of immunized animals, especially for intranasal administration. They can be administered intramucosally and do not exhibit toxic activity even in high concentrations, e.g. 20%.

[0113] It should be highlighted that nanoadjuvants are very suitable for intramucosal, especially intranasal, administration. Their advantage lies not only in interaction with mucin present in the mucosa but also in induction of absorption of the delivered antigen by respiratory epithelial cells. It was presented not only by using whole Clostridium difficile but also DQ-OVA. Furthermore, mice vaccinated intranasally with formulation consisting of the conjugate and nanoadjuvant showed an increased production of total IgA antibodies and epitope-specific antibodies IgG. Surprisingly, for intranasal administration, the nanoadjuvant significantly enhances the epitope response with simultaneous decrease of the carrier protein response. The example of intramuscular vaccine using commonly used alum presented above shows that the strong response to carrier protein can be a very big problem.

[0114] Nanoadjuvant in conjunction with Clostridium difficile antigens in the form of epitope conjugates with carrier protein, A and B toxins and additional immunostimulats allow for obtaining a high titer and a large amount of specific therapeutic antibodies in a relatively short time. Such antibodies used in humans as auxiliary means during infection or for its prevention act on the early stage of the infection and protect the mucous membrane of bowels by neutralizing the effect of toxins.

[0115] Basing on the above embodiments, the preferred composition of vaccine formulation is a 10% solution of one of nanoadjuvants: 1-et-tyl-03, 1-ac-tyl-03, 1-et-t80-03, 1-et-tyl-05, 1-et-t80-05 and 1-et-tyl-02 with suspended CD antigens in the form of epitope conjugates with a carrier protein (in a concentration of 10 μg for a mouse, 100 μg for a hen). Amino acid sequences of kDa protein epitopes: ATGKKGSETPTGKTKV, VNKIKNRPYYKGNIPG, SRKNTLGYFVNNKLVK, GTYQKNSWLKVNGKMY, QTGWQEKNGKKYYLGS, TGWKTENGKKYYVKSD, NKKYYLGTDGARVSGW, FDTAKKISSVGNWNAD, EFRVAT, KVNGKM and WQEKNGKKYY and M24 proteins: FISGFNGSAGTVIVTK, REGATLAEKKLSKKGIK, KKGIKIEYQYDLIDGI, LREKMSEKGTSTHVIT, MGIDYQCGTGHGIGFV, KKGIK and KGTSTHVIT. Inactivated toxins TcdA and TcdB of Clostridium difficile (1 μg for mouse, 10 μg for hen) can be added to the formulation, as well as other immunomodulating agents, including probiotic bacteria antigens of Lactobacillus, Bifidobacterium, Akkermansia or Faecalibacterium species such as: polysaccharides, teichoic acids, lipoteichoic acids, proteins, peptidoglycans, glycolipids, lipopolysaccharides, monophosphorylic lipid A, glycoproteins, bacteriocins, DNA, RNA, enzymes, peptides and other particles secreted to the medium. Other immunomodulating components not derived from bacteria, including cytokines and modifications thereof, nonimmunogenic peptides, lipids, polar proteins.

[0116] The use of formulation for vaccinating hens in order to obtain therapeutic antibodies is one of the uses of the invention. Manufacturing of IgY-class antibodies is used on an industrial scale. To produce IgY antibodies, a very small dose, as much as micrograms of antigen is needed, and a high titer of antibodies is maintained from a few weeks to a few months. During that time, a high amount of protective IgY antibodies is transferred to an egg. The use of IgY has many advantages: (1) IgY do not react with human complement system, therefore the non-specific inflammatory reaction can be avoided, (2) during the manufacturing, no toxic compounds were used, neither for vaccination nor in the time of cleaning the IgY antibodies, (3) in a method that is very easy to scale, as we are able to discard from the formulation all components apart from IgY. The use of passive immunization in the form of IgY formulation has a range of additional advantages: the activity is virtually immediate and focused on a specific area, namely the digestive system, it is highly specific, it can be used in humans of all ages and those with immunodeficiencies, it is entirely non-toxic because it includes components, which are normally present in our diet.

[0117] Owing to the production technology used, it is very easy to manipulate the composition of the formulation for vaccinating hens in order to obtain antibodies adjusted for changing strains, should they develop resistance. It would be possible to use another epitope from a group of already described epitopes. The obtained product will be administered orally in the form of a water-soluble powder. A novel formulation for the delivery of unchanged IgY antibodies to bowels of the patient where it will act therapeutically will be developed. As a result, it is possible to decrease the dosage.

REFERENCES

[0118] 1. Adjonu, R., Doran, G., Torley, P. and Agboola, S. 2014. Whey protein peptides as components of nanoemulsions: A review of emulsifying and biological functionalities. Journal of Food Engineering. 122, (February 2014), 15-27. [0119] 2. Bielinska, A. U., Gerber, M., Blanco, L. P., Makidon, P. E., Janczak, K. W., Beer, M., Swanson, B. and Baker, Jr., J. R. 2010. Induction of Th17 Cellular Immunity With a Novel Nanoemulsion Adjuvant. Critical Reviews™ in Immunology. 30, 2 (2010), 189-199. [0120] 3. Bielinska, A. U., Janczak, K. W., Landers, J. J., Markovitz, D. M., Montefiori, D. C. and Baker, J. R. 2008. Nasal immunization with a recombinant HIV gp120 and nanoemulsion adjuvant produces Th1 polarized responses and neutralizing antibodies to primary HIV type 1 isolates. AIDS research and human retroviruses. 24, 2 (February 2008), 271-281. [0121] 4. Bielinska, A. U., Makidon, P. E., Janczak, K. W., Blanco, L. P., Swanson, B., Smith, D. M., Pham, T., Szabo, Z., Kukowska-Latallo, J. F. and Baker, J. R. 2014. Distinct Pathways of Humoral and Cellular Immunity Induced with the Mucosal Administration of a Nanoemulsion Adjuvant. The Journal of Immunology. 192, 6 (March 2014), 2722-2733. [0122] 5. Makidon, P. E., Bielinska, A. U., Nigavekar, S. S., Janczak, K. W., Knowlton, J., Scott, A. J., Mank, N., Cao, Z., Rathinavelu, S., Beer, M. R., Wilkinson, J. E., Blanco, L. P., Landers, J. J. and Baker, J. R. 2008. Pre-Clinical Evaluation of a Novel Nanoemulsion-Based Hepatitis B Mucosal Vaccine. PLoS ONE. 3, 8 (August 2008), e2954. [0123] 6. Wong, P. T. et al. 2015. Formulation, High Throughput In Vitro Screening and In Vivo Functional Characterization of Nanoemulsion-Based Intranasal Vaccine Adjuvants. PLoS ONE. 11, (2015). [0124] 7. Pechine et al. 2018. Targeting Clostridium difficile surface components to develop immunotherapeutic strategies against Clostridium difficile infection [0125] 8. Rodriguez-Palacios A, Borgmann S, Kline T R et al (2013) Clostridium difficile in foods and animals: history and measures to reduce exposure. Anim Health Res Rev 14:11-29 [0126] 9. Elixhauser, A. and Jhung, M. Clostridium difficile Diseases in US Hospitals; AHRQ, Centre for Delivery, Organization and Markets, Healthcare Cost and Utilization Project. 2008. [0127] 10. Lai, K. K., Melvin, Z. S., Menard, M. J., Kotilainen, H. R., and Baker, S. Clostridium difficile associated diarrhea: epidemiology, risk factors, and infection control. Infect. Control. Hosp. Epidemiol., 18 (1997), 628-632. [0128] 11. Pepin, J., Routhier, S., Gagnon, S., and Brazeau, I. Management and outcomes of a first recurrence of Clostridium difficile-associated disease in Quebec, Canada. Clin. Infect. Dis., 42 (2006), 758-764. [0129] 12. Hryniewicz W., Martirosian G., Ozorowski T.: Zakażenia Clostridium difficile. Diagnostyka, terapia, profilaktyka. Narodowy Program Ochrony Antybiotyków. Ministerstwo Zdrowia, Warszawa 2011, http://www.antybiotyki.edu.pl/pdf/Clostridium difficile v6_10.pdf.