Intranasal Treatment of Ferrets with Inert Bacterial Spores Reduces Disease Caused by a Challenging H7N9 Avian Influenza Virus

Joe James, Stephanie M Meyer, Huynh A Hong, Chau Dang, Ho T Y Linh, William Ferreira, Paidamoyo M Katsande, Linh Vo, Daniel Hynes, William Love, Ashley C Banyard, Simon M Cutting, Joe James, Stephanie M Meyer, Huynh A Hong, Chau Dang, Ho T Y Linh, William Ferreira, Paidamoyo M Katsande, Linh Vo, Daniel Hynes, William Love, Ashley C Banyard, Simon M Cutting

Abstract

Background: Influenza is a respiratory infection that continues to present a major threat to human health, with ~500,000 deaths/year. Continued circulation of epidemic subtypes in humans and animals potentially increases the risk of future pandemics. Vaccination has failed to halt the evolution of this virus and next-generation prophylactic approaches are under development. Naked, "heat inactivated", or inert bacterial spores have been shown to protect against influenza in murine models. Methods: Ferrets were administered intranasal doses of inert bacterial spores (DSM 32444K) every 7 days for 4 weeks. Seven days after the last dose, the animals were challenged with avian H7N9 influenza A virus. Clinical signs of infection and viral shedding were monitored. Results: Clinical symptoms of infection were significantly reduced in animals dosed with DSM 32444K. The temporal kinetics of viral shedding was reduced but not prevented. Conclusion: Taken together, nasal dosing using heat-stable spores could provide a useful approach for influenza prophylaxis in both humans and animals.

Keywords: Bacillus subtilis; bacterial spores; influenza; innate immunity; prophylaxis.

Conflict of interest statement

S.M.C. is the CEO of SporeGen Ltd. W.L. and D.H. are employees of Destiny Pharma plc. H.A.H. is a shareholder of Sporegen Ltd. C.D. and L.V. are employees of SporeGen Ltd. W.F. is now an employee of Meridian Bioscience. H.T.Y.L. is an employee of HURO Biotech JSC.

Figures

Figure 1
Figure 1
Clinical signs, change in body temperature, and weight loss exhibited by ferrets treated with DSM 32444K (panel B) or mock-treated (panel A) with PBS following infection with H7N9 AIV. Ferrets were treated with 0.5 mL of DSM 32444K or PBS on four separate occasions, 7 days apart, followed by infection with H7N9 AIV 7 days after the last treatment. Individual values were plotted per animal, and lines indicate the mean values per group. All ferrets were scored for clinical signs daily following infection according to the clinical score system presented in Supplementary Table S1. The cumulative daily clinical scores were represented graphically.
Figure 2
Figure 2
Change in body temperature and weight loss exhibited by ferrets treated with DSM 32444K or mock treated with PBS following infection with H7N9 AIV. Ferrets were treated with 0.5 mL of DSM 32444K (red line) or PBS (blue line) on four separate occasions, 7 days apart, followed by infection with H7N9 AIV 7 days after the last treatment. Individual values were plotted per animal, and lines indicate the mean values per group. Body temperatures and weights taken 1 day prior to infection were used to configure a baseline, and then, temperatures (panel A) and weights (panel B) were taken daily following infection with H7N9 AIV. Statistical significance was determined using a two-tailed Mann-Whitney U test; panel A, * p < 0.05; panel B, ** p < 0.01, *** p < 0.001.
Figure 3
Figure 3
Viral shedding exhibited by ferrets treated with DSM 32444K or mock-treated with PBS following infection with H7N9 AIV. Viral RNA was quantified in RNA extracted from nasal wash samples using an influenza A rRT-qPCR. Relative equivalency units (REUs) were calculated and displayed based on Cq values obtained extrapolated from a standard curve of a known titre of A/Anhui/1/13 (H7N9).

References

    1. Permpoonpattana P., Hong H.A., Phetcharaburanin J., Huang J.M., Cook J., Fairweather N.F., Cutting S.M. Immunization with Bacillus spores expressing toxin A peptide repeats protects against infection with Clostridium difficile strains producing toxins A and B. Infect. Immun. 2011;79:2295–2302. doi: 10.1128/IAI.00130-11.
    1. Reljic R., Sibley L., Huang J.M., Pepponi I., Hoppe A., Hong H.A., Cutting S.M. Mucosal vaccination against tuberculosis using inert bioparticles. Infect. Immun. 2013;81:4071–4080. doi: 10.1128/IAI.00786-13.
    1. Song M., Hong H.A., Huang J.M., Colenutt C., Khang D.D., Nguyen T.V., Park S.M., Shim B.S., Song H.H., Cheon I.S., et al. Killed Bacillus subtilis spores as a mucosal adjuvant for an H5N1 vaccine. Vaccine. 2012;30:3266–3277. doi: 10.1016/j.vaccine.2012.03.016.
    1. de Souza R.D., Batista M.T., Luiz W.B., Cavalcante R.C., Amorim J.H., Bizerra R.S., Martins E.G., Ferreira L.C. Bacillus subtilis spores as vaccine adjuvants: Further insights into the mechanisms of action. PLoS ONE. 2014;9:e87454. doi: 10.1371/journal.pone.0087454.
    1. Huang J.M., La Ragione R.M., Nunez A., Cutting S.M. Immunostimulatory activity of Bacillus spores. FEMS Immunol. Med. Microbiol. 2008;53:195–203. doi: 10.1111/j.1574-695X.2008.00415.x.
    1. Wang X., Hu W., Zhu L., Yang Q. Bacillus subtilis and surfactin inhibit the transmissible gastroenteritis virus from entering the intestinal epithelial cells. Biosci. Rep. 2017;37:BSR20170082. doi: 10.1042/BSR20170082.
    1. Barnes A.G., Cerovic V., Hobson P.S., Klavinskis L.S. Bacillus subtilis spores: A novel microparticle adjuvant which can instruct a balanced Th1 and Th2 immune response to specific antigen. Eur. J. Immunol. 2007;37:1538–1547. doi: 10.1002/eji.200636875.
    1. Iuliano A.D., Roguski K.M., Chang H.H., Muscatello D.J., Palekar R., Tempia S., Cohen C., Gran J.M., Schanzer D., Cowling B.J., et al. Estimates of global seasonal influenza-associated respiratory mortality: A modelling study. Lancet. 2018;391:1285–1300. doi: 10.1016/S0140-6736(17)33293-2.
    1. WHO . Influenza (Seasonal) Fact Sheet. World Health Organization; Geneva, Switzerland: 2018.
    1. Mostafa A., Abdelwhab E.M., Mettenleiter T.C., Pleschka S. Zoonotic Potential of Influenza A Viruses: A Comprehensive Overview. Viruses. 2018;10:497. doi: 10.3390/v10090497.
    1. Belser J.A., Katz J.M., Tumpey T.M. The ferret as a model organism to study influenza A virus infection. Dis. Model. Mech. 2011;4:575–579. doi: 10.1242/dmm.007823.
    1. Maassab H.F., Kendal A.P., Abrams G.D., Monto A.S. Evaluation of a cold-recombinant influenza virus vaccine in ferrets. J. Infect. Dis. 1982;146:780–790. doi: 10.1093/infdis/146.6.780.
    1. Fan S., Gu C., Kong H., Guan L., Neumann G., Kawaoka Y. Influenza Viruses Suitable for Studies in Syrian Hamsters. Viruses. 2022;14:1629. doi: 10.3390/v14081629.
    1. Lowen A.C., Mubareka S., Tumpey T.M., Garcia-Sastre A., Palese P. The guinea pig as a transmission model for human influenza viruses. Proc. Natl. Acad. Sci. USA. 2006;103:9988–9992. doi: 10.1073/pnas.0604157103.
    1. Li Q., Zhou L., Zhou M., Chen Z., Li F., Wu H., Xiang N., Chen E., Tang F., Wang D., et al. Epidemiology of human infections with avian influenza A(H7N9) virus in China. N. Engl. J. Med. 2014;370:520–532. doi: 10.1056/NEJMoa1304617.
    1. Huesca-Espitia L.C., Suvira M., Rosenbeck K., Korza G., Setlow B., Li W., Wang S., Li Y.Q., Setlow P. Effects of steam autoclave treatment on Geobacillus stearothermophilus spores. J. Appl. Microbiol. 2016;121:1300–1311. doi: 10.1111/jam.13257.
    1. Nguyen V.A., Huynh H.A., Hoang T.V., Ninh N.T., Pham A.T., Nguyen H.A., Phan T.N., Cutting S.M. Killed Bacillus subtilis spores expressing streptavidin: A novel carrier of drugs to target cancer cells. J. Drug Target. 2013;21:528–541. doi: 10.3109/1061186X.2013.778262.
    1. Bhat S., James J., Sadeyen J.R., Mahmood S., Everest H.J., Chang P., Walsh S.K., Byrne A.M.P., Mollett B., Lean F., et al. Coinfection of Chickens with H9N2 and H7N9 Avian Influenza Viruses Leads to Emergence of Reassortant H9N9 Virus with Increased Fitness for Poultry and a Zoonotic Potential. J. Virol. 2022;96:e0185621. doi: 10.1128/jvi.01856-21.
    1. de Jonge J., Isakova-Sivak I., van Dijken H., Spijkers S., Mouthaan J., de Jong R., Smolonogina T., Roholl P., Rudenko L. H7N9 Live Attenuated Influenza Vaccine Is Highly Immunogenic, Prevents Virus Replication, and Protects Against Severe Bronchopneumonia in Ferrets. Mol. Ther. 2016;24:991–1002. doi: 10.1038/mt.2016.23.
    1. Jonges M., Liu W.M., van der Vries E., Jacobi R., Pronk I., Boog C., Koopmans M., Meijer A., Soethout E. Influenza virus inactivation for studies of antigenicity and phenotypic neuraminidase inhibitor resistance profiling. J. Clin. Microbiol. 2010;48:928–940. doi: 10.1128/JCM.02045-09.
    1. Nagy A., Cernikova L., Kunteova K., Dirbakova Z., Thomas S.S., Slomka M.J., Dan A., Varga T., Mate M., Jirincova H., et al. A universal RT-qPCR assay for "One Health" detection of influenza A viruses. PLoS ONE. 2021;16:e0244669. doi: 10.1371/journal.pone.0244669.
    1. Iwasaki A., Pillai P.S. Innate immunity to influenza virus infection. Nat. Rev. Immunol. 2014;14:315–328. doi: 10.1038/nri3665.
    1. Schmitz N., Beerli R.R., Bauer M., Jegerlehner A., Dietmeier K., Maudrich M., Pumpens P., Saudan P., Bachmann M.F. Universal vaccine against influenza virus: Linking TLR signaling to anti-viral protection. Eur. J. Immunol. 2012;42:863–869. doi: 10.1002/eji.201041225.
    1. Jiang T., Zhao H., Li X.F., Deng Y.Q., Liu J., Xu L.J., Han J.F., Cao R.Y., Qin E.D., Qin C.F. CpG oligodeoxynucleotides protect against the 2009 H1N1 pandemic influenza virus infection in a murine model. Antivir. Res. 2011;89:124–126. doi: 10.1016/j.antiviral.2010.11.013.
    1. Abdul-Careem M.F., Firoz Mian M., Gillgrass A.E., Chenoweth M.J., Barra N.G., Chan T., Al-Garawi A.A., Chew M.V., Yue G., van Roojen N., et al. FimH, a TLR4 ligand, induces innate antiviral responses in the lung leading to protection against lethal influenza infection in mice. Antivir. Res. 2011;92:346–355. doi: 10.1016/j.antiviral.2011.09.004.
    1. Mifsud E.J., Tan A.C., Jackson D.C. TLR Agonists as Modulators of the Innate Immune Response and Their Potential as Agents Against Infectious Disease. Front. Immunol. 2014;5:79. doi: 10.3389/fimmu.2014.00079.
    1. Huang J.M., La Ragione R.M., Cooley W.A., Todryk S., Cutting S.M. Cytoplasmic delivery of antigens, by Bacillus subtilis enhances Th1 responses. Vaccine. 2008;26:6043–6052. doi: 10.1016/j.vaccine.2008.09.024.
    1. Chadwick S., Kriegel C., Amiji M. Nanotechnology solutions for mucosal immunization. Adv. Drug Deliv. Rev. 2010;62:394–407. doi: 10.1016/j.addr.2009.11.012.
    1. Maines T.R., Belser J.A., Gustin K.M., van Hoeven N., Zeng H., Svitek N., von Messling V., Katz J.M., Tumpey T.M. Local innate immune responses and influenza virus transmission and virulence in ferrets. J. Infect. Dis. 2012;205:474–485. doi: 10.1093/infdis/jir768.
    1. Skarlupka A.L., Ross T.M. Immune Imprinting in the Influenza Ferret Model. Vaccines. 2020;8:173. doi: 10.3390/vaccines8020173.
    1. Oh J.E., Song E., Moriyama M., Wong P., Zhang S., Jiang R., Strohmeier S., Kleinstein S.H., Krammer F., Iwasaki A. Intranasal priming induces local lung-resident B cell populations that secrete protective mucosal antiviral IgA. Sci. Immunol. 2021;6:eabj5129. doi: 10.1126/sciimmunol.abj5129.
    1. Fiorini G., Cimminiello C., Chianese R., Visconti G.P., Cova D., Uberti T., Gibelli A. Bacillus subtilis selectively stimulates the synthesis of membrane bound and secreted IgA. Chemioterapia. 1985;4:310–312.
    1. Ciprandi G., Tosca M.A., Milanese M., Caligo G., Ricca V. Cytokines evaluation in nasal lavage of allergic children after Bacillus clausii administration: A pilot study. Pediatr. Allergy Immunol. 2004;15:148–151. doi: 10.1046/j.1399-3038.2003.00102.x.
    1. Elshaghabee F.M.F., Rokana N., Gulhane R.D., Sharma C., Panwar H. Bacillus As Potential Probiotics: Status, Concerns, and Future Perspectives. Front. Microbiol. 2017;8:1490. doi: 10.3389/fmicb.2017.01490.
    1. Ricci A., Allende A., Bolton D., Chemaly M., Davies R., Girones R., Herman L., Koutsoumanis K., Lindqvist R., Norrung B., et al. Scientific Opinion on the update of the list of QPS-recommended biological agents intentionally added to food or feed as notified to EFSA. EFSA J. 2017;15:e04664. doi: 10.2903/j.efsa.2017.4664.
    1. Burrows S.M., Elbert W., Lawrence M.G., Poschl U. Bacteria in the global atmosphere—Part 1: Review and synthesis of literature data for different ecosystems. Atmos. Chem. Phys. 2009;9:9263–9280. doi: 10.5194/acp-9-9263-2009.

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