Intranasal Immunization with Zika Virus Envelope Domain III-Flagellin Fusion Protein Elicits Systemic and Mucosal Immune Responses and Protection against Subcutaneous and Intravaginal Virus Challenges
Abstract
:1. Introduction
2. Materials and Methods
2.1. Expression and Purification of Recombinant ZDIII and FliC-ZDIII Fusion Proteins
2.2. Expression and Purification of Recombinant LTIIb-B5 Protein
2.3. NF-ĸB-Dependent Luciferase Reporter Assay
2.4. Mouse Immunizations
2.5. ELISA IgG and IgA Titer Assay
2.6. Plaque Reduction Neutralization Test (PRNT)
2.7. T Cell Cytokine ELISA Assay
2.8. Subcutaneous Virus Challenge
2.9. Intravaginal Virus Challenge
2.10. Statistical Analysis
3. Results
3.1. Expression, Purification, and Characterization of ZDIII and FliC-ZDIII Fusion Proteins
3.2. Intranasal Immunization with ZDIII and FliC-ZDIII Fusion Proteins plus LTIIb-B5 Mucosal Adjuvant
3.3. Protective Immunity in Immunized Mice by Subcutaneous and Intravaginal ZIKV Challenges
3.4. Immune Responses Elicited by the Second-Generation FliCΔD3-2ZDIII and FliCΔD2ΔD3-3ZDIII Fusion Proteins to Reduce FliC-Specific Adaptive Response
3.5. Intranasal Immunization with the Use of LTIIb-B5 Adjuvant for the Second-Generation FliCΔD3-2ZDIII Fusion Proteins
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
- Lindenbach, B.D.; Rice, C.M. Molecular biology of flaviviruses. Adv. Virus Res. 2003, 59, 23–61. [Google Scholar] [PubMed]
- Campos, G.S.; Bandeira, A.C.; Sardi, S.I. Zika Virus Outbreak, Bahia, Brazil. Emerg. Infect. Dis. 2015, 21, 1885–1886. [Google Scholar] [CrossRef] [PubMed]
- Schuler-Faccini, L.; Ribeiro, E.M.; Feitosa, I.M.; Horovitz, D.D.; Cavalcanti, D.P.; Pessoa, A.; Doriqui, M.J.; Neri, J.I.; Neto, J.M.; Wanderley, H.Y.; et al. Brazilian Medical Genetics Society–Zika Embryopathy Task Force. Possible Association between Zika Virus Infection and Microcephaly—Brazil, 2015. MMWR 2016, 65, 59–62. [Google Scholar] [PubMed]
- Fauci, A.S.; Morens, D.M. Zika Virus in the Americas—Yet Another Arbovirus Threat. N. Engl. J. Med. 2016, 374, 601–604. [Google Scholar] [CrossRef]
- Ferguson, N.M.; Cucunuba, Z.M.; Dorigatti, I.; Nedjati-Gilani, G.L.; Donnelly, C.A.; Basanez, M.G.; Nouvellet, P.; Lessler, J. EPIDEMIOLOGY. Countering the Zika epidemic in Latin America. Science 2016, 353, 353–354. [Google Scholar] [CrossRef] [Green Version]
- WHO. WHO Director-General Summarizes the Outcome of the Emergency Committee Regarding Clusters of Microcephaly and Guillain-Barré Syndrome, 1 February 2016. Available online: http://www.who.int/mediacentre/news/statements/2016/emergency-committee-zika-microcephaly/en/ (accessed on 30 August 2017).
- Faye, O.; Freire, C.C.; Iamarino, A.; Faye, O.; de Oliveira, J.V.; Diallo, M.; Zanotto, P.M.; Sall, A.A. Molecular evolution of Zika virus during its emergence in the 20(th) century. PLoS Negl. Trop. Dis. 2014, 8, e2636. [Google Scholar] [CrossRef] [Green Version]
- Wikan, N.; Smith, D.R. Zika virus: History of a newly emerging arbovirus. Lancet Infect. Dis. 2016, 16, e119–e126. [Google Scholar] [CrossRef] [Green Version]
- D’Ortenzio, E.; Matheron, S.; Yazdanpanah, Y.; de Lamballerie, X.; Hubert, B.; Piorkowski, G.; Maquart, M.; Descamps, D.; Damond, F.; Leparc-Goffart, I. Evidence of Sexual Transmission of Zika Virus. N. Engl. J. Med. 2016, 374, 2195–2198. [Google Scholar] [CrossRef]
- Mead, P.S.; Hills, S.L.; Brooks, J.T. Zika virus as a sexually transmitted pathogen. Curr. Opin. Infect. Dis. 2018, 31, 39–44. [Google Scholar] [CrossRef]
- Barzon, L.; Pacenti, M.; Franchin, E.; Lavezzo, E.; Trevisan, M.; Sgarabotto, D.E.; Palù, G. Infection dynamics in a traveller with persistent shedding of Zika virus RNA in semen for six months after returning from Haiti to Italy, January 2016. Euro Surveill. 2016, 21, 30316. [Google Scholar] [CrossRef] [Green Version]
- Matheron, S.; d’Ortenzio, E.; Leparc-Goffart, I.; Hubert, B.; de Lamballerie, X.; Yazdanpanah, Y. Long-lasting persistence of Zika virus in semen. Clin. Infect. Dis. 2016, 63, 1264. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Prisant, N.; Bujan, L.; Benichou, H.; Hayot, P.H.; Pavili, L.; Lurel, S.; Herrmann, C.; Janky, E.; Joguet, G. Zika virus in the female genital tract. Lancet Infect. Dis. 2016, 16, 1000–1001. [Google Scholar] [CrossRef] [Green Version]
- Visseaux, B.; Mortier, E.; Houhou-Fidouh, N.; Brichler, S.; Collin, G.; Larrouy, L.; Charpentier, C.; Descamps, D. Zika virus in the female genital tract. Lancet Infect. Dis. 2016, 16, 1220. [Google Scholar] [CrossRef] [Green Version]
- Wira, C.R.; Rodriguez-Garcia, M.; Patel, M.V. The role of sex hormones in immune protection of the female reproductive tract. Nat. Rev. Immunol. 2015, 15, 217–230. [Google Scholar] [CrossRef] [PubMed]
- Tang, W.W.; Young, M.P.; Mamidi, A.; Regla-Nava, J.A.; Kim, K.; Shresta, S.A. Mouse Model of Zika Virus Sexual Transmission and Vaginal Viral Replication. Cell Rep. 2016, 17, 3091–3098. [Google Scholar] [CrossRef] [Green Version]
- Yockey, L.J.; Varela, L.; Rakib, T.; Khoury-Hanold, W.; Fink, S.L.; Stutz, B.; Szigeti-Buck, K.; Van den Pol, A.; Lindenbach, B.D.; Horvath, T.L.; et al. Vaginal Exposure to Zika Virus during Pregnancy Leads to Fetal Brain Infection. Cell 2016, 166, 1247–1256.e4. [Google Scholar] [CrossRef] [Green Version]
- Abbink, P.; Stephenson, K.E.; Barouch, D.H. Zika virus vaccines. Nat. Rev. Microbiol. 2018, 16, 594–600. [Google Scholar] [CrossRef]
- Rey, F.A.; Heinz, F.X.; Mandl, C.; Kunz, C.; Harrison, S.C. The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature 1995, 375, 291–298. [Google Scholar] [CrossRef]
- Modis, Y.; Ogata, S.; Clements, D.; Harrison, S.C. Structure of the dengue virus envelope protein after membrane fusion. Nature 2004, 427, 313–319. [Google Scholar] [CrossRef]
- Zhao, H.; Fernandez, E.; Dowd, K.A.; Speer, S.D.; Platt, D.J.; Gorman, M.J.; Govero, J.; Nelson, C.A.; Pierson, T.C.; Diamond, M.S.; et al. Structural Basis of Zika Virus-Specific Antibody Protection. Cell 2016, 166, 1016–1027. [Google Scholar] [CrossRef] [Green Version]
- Stettler, K.; Beltramello, M.; Espinosa, D.A.; Graham, V.; Cassotta, A.; Bianchi, S.; Vanzetta, F.; Minola, A.; Jaconi, S.; Mele, F.; et al. Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection. Science 2016, 353, 823–826. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Yang, M.; Dent, M.; Lai, H.; Sun, H.; Chen, Q. Immunization of Zika virus envelope protein domain III induces specific and neutralizing immune responses against Zika virus. Vaccine 2017, 35, 4287–4294. [Google Scholar] [CrossRef] [PubMed]
- Yang, M.; Lai, H.; Sun, H.; Chen, Q. Virus-like particles that display Zika virus envelope protein domain III induce potent neutralizing immune responses in mice. Sci. Rep. 2017, 7, 7679. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Qu, P.; Zhang, W.; Li, D.; Zhang, C.; Liu, Q.; Zhang, X.; Wang, X.; Dai, W.; Xu, Y.; Leng, Q.; et al. Insect cell-produced recombinant protein subunit vaccines protect against Zika virus infection. Antivir. Res. 2018, 154, 97–103. [Google Scholar] [CrossRef] [PubMed]
- Tai, W.; He, L.; Wang, Y.; Sun, S.; Zhao, G.; Luo, C.; Li, P.; Zhao, H.; Fremont, D.H.; Li, F.; et al. Critical neutralizing fragment of Zika virus EDIII elicits cross-neutralization and protection against divergent Zika viruses. Emerg. Microbes Infect. 2018, 7, 7. [Google Scholar] [CrossRef] [PubMed]
- Hayashi, F.; Smith, K.D.; Ozinsky, A.; Hawn, T.R.; Yi, E.C.; Goodlett, D.R.; Eng, J.K.; Akira, S.; Underhill, D.M.; Aderem, A. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 2001, 410, 1099–1103. [Google Scholar] [CrossRef]
- Honko, A.N.; Mizel, S.B. Effects of flagellin on innate and adaptive immunity. Immunol. Res. 2005, 33, 83–101. [Google Scholar] [CrossRef]
- Kofoed, E.M.; Vance, R.E. Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity. Nature 2011, 477, 592–595. [Google Scholar] [CrossRef]
- Mizel, S.B.; Bates, J.T. Flagellin as an adjuvant: Cellular mechanisms and potential. J. Immunol. 2010, 185, 5677–5682. [Google Scholar] [CrossRef] [Green Version]
- Hajam, I.A.; Dar, P.A.; Shahnawaz, I.; Jaume, J.C.; Lee, J.H. Bacterial flagellin-a potent immunomodulatory agent. Exp. Mol. Med. 2017, 49, e373. [Google Scholar] [CrossRef]
- Tang, N.; Lu, C.Y.; Sue, S.C.; Chen, T.H.; Jan, J.T.; Huang, M.H.; Huang, C.H.; Chen, C.C.; Chiang, B.L.; Huang, L.M.; et al. Type IIb heat labile enterotoxin B subunit as a mucosal adjuvant to enhance protective immunity against H5N1 avian influenza viruses. Vaccines 2020, 8, 710. [Google Scholar] [CrossRef] [PubMed]
- Lin, H.H.; Yang, S.P.; Tsai, M.J.; Lin, G.C.; Wu, H.C.; Wu, S.C. Dengue and Zika virus domain III-flagellin fusion and glycan-masking E antigen for prime-boost immunization. Theranostics 2019, 9, 4811–4826. [Google Scholar] [CrossRef] [PubMed]
- Wen, J.; Elong Ngono, A.; Regla-Nava, J.A.; Kim, K.; Gorman, M.J.; Diamond, M.S.; Shresta, S. Dengue virus-reactive CD8(+) T cells mediate cross-protection against subsequent Zika virus challenge. Nat. Commun. 2017, 8, 1459. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Guo, Q.; Chan, J.F.; Poon, V.K.; Wu, S.; Chan, C.C.; Hou, L.; Yip, C.C.; Ren, C.; Cai, J.P.; Zhao, M.; et al. Immunization with a Novel Human Type 5 Adenovirus-Vectored Vaccine Expressing the Premembrane and Envelope Proteins of Zika Virus Provides Consistent and Sterilizing Protection in Multiple Immunocompetent and Immunocompromised Animal Models. J. Infect. Dis. 2018, 218, 365–377. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Liang, S.; Hosur, K.B.; Nawar, H.F.; Russell, M.W.; Connell, T.D.; Hajishengallis, G. In vivo and in vitro adjuvant activities of the B subunit of Type IIb heat-labile enterotoxin (LT-IIb-B5) from Escherichia coli. Vaccine 2009, 27, 4302–4308. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Liang, S.; Hajishengallis, G. Heat-labile enterotoxins as adjuvants or anti-inflammatory agents. Immunol. Investig. 2010, 39, 449–467. [Google Scholar] [CrossRef] [Green Version]
- Kiyono, H.; Fukuyama, S. NALT-versus Peyer’s-patch-mediated mucosal immunity. Nat. Rev. Immunol. 2004, 4, 699–710. [Google Scholar] [CrossRef]
- Czerkinsky, C.; Holmgren, J. Topical immunization strategies. Mucosal Immunol. 2010, 3, 545–555. [Google Scholar] [CrossRef]
- Woodrow, K.A.; Bennett, K.M.; Lo, D.D. Mucosal vaccine design and delivery. Annu. Rev. Biomed. Eng. 2012, 14, 17–46. [Google Scholar] [CrossRef] [Green Version]
- Lu, Y.; Swartz, J.R. Functional properties of flagellin as a stimulator of innate immunity. Sci. Rep. 2016, 6, e11021. [Google Scholar] [CrossRef] [Green Version]
- Lightfield, K.L.; Persson, J.; Brubaker, S.W.; Witte, C.E.; von Moltke, J.; Dunipace, E.A.; Henry, T.; Sun, Y.H.; Cado, D.; Dietrich, W.F.; et al. Critical function for Naip5 in inflammasome activation by a conserved carboxy-terminal domain of flagellin. Nat. Immunol. 2008, 9, 1171–1178. [Google Scholar] [CrossRef] [PubMed]
- Halff, E.F.; Diebolder, C.A.; Versteeg, M.; Schouten, A.; Brondijk, T.H.; Huizinga, E.G. Formation and structure of a NAIP5-NLRC4 inflammasome induced by direct interactions with conserved N- and C-terminal regions of flagellin. J. Biol. Chem. 2012, 287, 38460–38472. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Yonekura, K.; Maki-Yonekura, S.; Namba, K. Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy. Nature 2003, 424, 643–650. [Google Scholar] [CrossRef] [PubMed]
- Smith, K.D.; Andersen-Nissen, E.; Hayashi, F.; Strobe, K.; Bergman, M.A.; Barrett, S.L.; Cookson, B.T.; Aderem, A. Toll-like receptor 5 recognizes a conserved site on flagellin required for protofil-ament formation and bacterial motility. Nat. Immunol. 2003, 4, 1247–1253. [Google Scholar] [CrossRef]
- Yoon, S.I.; Kurnasov, O.; Natarajan, V.; Hong, M.; Gudkov, A.V.; Osterman, A.L.; Wilson, I.A. Structural basis of TLR5-flagellin recognition and signaling. Science 2012, 335, 859–864. [Google Scholar] [CrossRef] [Green Version]
- Biedma, M.E.; Cayet, D.; Tabareau, J.; Rossi, A.H.; Ivičak-Kocjan, K.; Moreno, G.; Errea, A.; Soulard, D.; Parisi, G.; Jerala, R.; et al. Recombinant flagellins with deletions in domains D1, D2, and D3: Characterization as novel immunoadjuvants. Vaccine 2019, 37, 652–663. [Google Scholar] [CrossRef]
- Alexander, K.L.; Zhao, Q.; Reif, M.; Rosenberg, A.F.; Mannon, P.J.; Duck, L.W.; Elson, C.O. Human Microbiota Flagellins Drive Adaptive Immune Responses in Crohn’s Disease. Gastroenterology 2021, 161, 522–535. [Google Scholar] [CrossRef]
- Cook, L.; Lisko, D.J.; Wong, M.Q.; Garcia, R.V.; Himmel, M.E.; Seidman, E.G.; Bressler, B.; Levings, M.K.; Steiner, T.S. Analysis of Flagellin-Specific Adaptive Immunity Reveals Links to Dysbiosis in Patients with Inflammatory Bowel Disease. Cell. Mol. Gastroenterol. Hepatol. 2020, 9, 485–506. [Google Scholar] [CrossRef] [Green Version]
- Torres, J.; Petralia, F.; Sato, T.; Wang, P.; Telesco, S.E.; Choung, R.S.; Strauss, R.; Li, X.J.; Laird, R.M.; Gutierrez, R.L.; et al. Serum Biomarkers Identify Patients Who Will Develop Inflammatory Bowel Diseases Up to 5 Years Before Diagnosis. Gastroenterology 2020, 159, 96–104. [Google Scholar] [CrossRef]
- Gupta, N.K.; Tomar, P.; Sharma, V.; Dixit, V.K. Development and characterization of chitosan coated poly-(ɛ-caprolactone) nanoparticulate system for effective immunization against influenza. Vaccine 2011, 29, 9026–9037. [Google Scholar] [CrossRef]
- Moon, H.J.; Lee, J.S.; Talactac, M.R.; Chowdhury, M.Y.; Kim, J.H.; Park, M.E.; Choi, Y.K.; Sung, M.H.; Kim, C.J. Mucosal immunization with recombinant influenza hemagglutinin protein and poly gamma-glutamate/chitosan nanoparticles induces protection against highly pathogenic influenza A virus. Vet. Microbiol. 2012, 160, 277–289. [Google Scholar] [CrossRef] [PubMed]
- Lai, C.H.; Tang, N.; Jan, J.T.; Huang, M.H.; Lu, C.Y.; Chiang, B.L.; Huang, L.M.; Wu, S.C. Use of recombinant flagellin in oil-in-water emulsions enhances hemagglutinin-specific mucosal IgA production and IL-17 secreting T cells against H5N1 avian influenza virus infection. Vaccine 2015, 33, 4321–4329. [Google Scholar] [CrossRef] [PubMed]
- Uematsu, S.; Fujimoto, K.; Jang, M.H.; Yang, B.G.; Jung, Y.J.; Nishiyama, M.; Sato, S.; Tsujimura, T.; Yamamoto, M.; Yokota, Y.; et al. Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5. Nat. Immunol. 2008, 9, 769–776. [Google Scholar] [CrossRef] [PubMed]
- Van Maele, L.; Carnoy, C.; Cayet, D.; Songhet, P.; Dumoutier, L.; Ferrero, I.; Janot, L.; Erard, F.; Bertout, J.; Leger, H.; et al. TLR5 signaling stimulates the innate production of IL-17 and IL-22 by CD3(neg)CD127+ immune cells in spleen and mucosa. J. Immunol. 2010, 185, 1177–1185. [Google Scholar] [CrossRef] [Green Version]
- Kinnebrew, M.A.; Buffie, C.G.; Diehl, G.E.; Zenewicz, L.A.; Leiner, I.; Hohl, T.M.; Flavell, R.A.; Littman, D.R.; Pamer, E.G. Interleukin 23 production by intestinal CD103(+)CD11b(+) dendritic cells in response to bacterial flagellin enhances mucosal innate immune defense. Immunity 2012, 36, 276–287. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ramos, H.C.; Rumbo, M.; Sirard, J.C. Bacterial flagellins: Mediators of pathogenicity and host immune responses in mucosa. Trends Microbiol. 2004, 12, 509–517. [Google Scholar] [CrossRef] [Green Version]
- Van Maele, L.; Fougeron, D.; Janot, L.; Didierlaurent, A.; Cayet, D.; Tabareau, J.; Rumbo, M.; Corvo-Chamaillard, S.; Boulenouar, S.; Jeffs, S.; et al. Airway structural cells regulate TLR5-mediated mucosal adjuvant activity. Mucosal. Immunol. 2014, 7, 489–500. [Google Scholar] [CrossRef]
- Sanos, S.L.; Kassub, R.; Testori, M.; Geiger, M.; Pätzold, J.; Giessel, R.; Knallinger, J.; Bathke, B.; Gräbnitz, F.; Brinkmann, K.; et al. NLRC4 Inflammasome-Driven Immunogenicity of a Recombinant MVA Mucosal Vaccine Encoding Flagellin. Front. Immunol. 2018, 8, 1988. [Google Scholar] [CrossRef] [Green Version]
- Grant, A.; Ponia, S.S.; Tripathi, S.; Balasubramaniam, V.; Miorin, L.; Sourisseau, M.; Schwarz, M.C.; Sánchez-Seco, M.P.; Evans, M.J.; Best, S.M.; et al. Zika Virus Targets Human STAT2 to Inhibit Type I Interferon Signaling. Cell Host Microbe 2016, 19, 882–890. [Google Scholar] [CrossRef] [Green Version]
- Tripathi, S.; Balasubramaniam, V.R.; Brown, J.A.; Mena, I.; Grant, A.; Bardina, S.V.; Maringer, K.; Schwarz, M.C.; Maestre, A.M.; Sourisseau, M.; et al. A novel Zika virus mouse model reveals strain specific differences in virus pathogenesis and host inflammatory immune responses. PLoS Pathog. 2017, 13, e1006258. [Google Scholar] [CrossRef] [Green Version]
- Lazear, H.M.; Govero, J.; Smith, A.M.; Platt, D.J.; Fernandez, E.; Miner, J.J.; Diamond, M.S. A Mouse Model of Zika Virus Pathogenesis. Cell Host Microbe 2016, 19, 720–730. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kaushic, C.; Ashkar, A.A.; Reid, L.A.; Rosenthal, K.L. Progesterone increases susceptibility and decreases immune responses to genital herpes infection. J. Virol. 2003, 77, 4558–4565. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Chen, C.-H.; Chen, C.-C.; Wang, W.-B.; Lionel, V.; Liu, C.-C.; Huang, L.-M.; Wu, S.-C. Intranasal Immunization with Zika Virus Envelope Domain III-Flagellin Fusion Protein Elicits Systemic and Mucosal Immune Responses and Protection against Subcutaneous and Intravaginal Virus Challenges. Pharmaceutics 2022, 14, 1014. https://doi.org/10.3390/pharmaceutics14051014
Chen C-H, Chen C-C, Wang W-B, Lionel V, Liu C-C, Huang L-M, Wu S-C. Intranasal Immunization with Zika Virus Envelope Domain III-Flagellin Fusion Protein Elicits Systemic and Mucosal Immune Responses and Protection against Subcutaneous and Intravaginal Virus Challenges. Pharmaceutics. 2022; 14(5):1014. https://doi.org/10.3390/pharmaceutics14051014
Chicago/Turabian StyleChen, Chi-Hsun, Chung-Chu Chen, Wei-Bo Wang, Vania Lionel, Chia-Chyi Liu, Li-Min Huang, and Suh-Chin Wu. 2022. "Intranasal Immunization with Zika Virus Envelope Domain III-Flagellin Fusion Protein Elicits Systemic and Mucosal Immune Responses and Protection against Subcutaneous and Intravaginal Virus Challenges" Pharmaceutics 14, no. 5: 1014. https://doi.org/10.3390/pharmaceutics14051014
APA StyleChen, C.-H., Chen, C.-C., Wang, W.-B., Lionel, V., Liu, C.-C., Huang, L.-M., & Wu, S.-C. (2022). Intranasal Immunization with Zika Virus Envelope Domain III-Flagellin Fusion Protein Elicits Systemic and Mucosal Immune Responses and Protection against Subcutaneous and Intravaginal Virus Challenges. Pharmaceutics, 14(5), 1014. https://doi.org/10.3390/pharmaceutics14051014