Plant-Produced N-glycosylated Ag85A Exhibits Enhanced Vaccine Efficacy Against Mycobacterium tuberculosis HN878 Through Balanced Multifunctional Th1 T Cell Immunity
Abstract
:1. Introduction
2. Materials and Methods
2.1. Experimental Animals and Ethics Statement
2.2. Abs and Reagents
2.3. Mtb Strain and Culture Conditions
2.4. Expression and Purification of Ag85A
2.5. Plasmid Construction and Transient Expression
2.6. Purification of Recombinant G-Ag85A from the Leaf Extracts of N. Benthamiana
2.7. Deglycosylation of Plant-Produced Ag85A
2.8. Immunization of Mice with Individual Ag85A and Challenge with Mtb Beijing Strain HN878
2.9. Splenocyte and Lung Cell Preparation
2.10. Cytokine Measurement
2.11. Ab Titer Measurement in Serum
2.12. Intracellular Cytokine Staining
2.13. Analysis of Histopathology and Mtb Burden
2.14. Statistical Analyses
3. Results
3.1. Expression and Purification of Glycosylated Ag85A in N. Benthamiana
3.2. Glycosylation of Ag85A Induced an Augmented Ag-specific IFN-γ response during Mtb Infection and Increased T Cell Proliferation in Coculture with Bone Marrow-Derived Dendritic Cells
3.3. Vaccine Experimental Design and Immunogenic Response in the Lung and Spleen of G-Ag85A- and NG-Ag85A-Immunized Mice
3.4. G-Ag85A Immunization Induced an Increased Number of CD4+ Multifunctional Th1 Cells Compared to NG-Ag85A Immunization Prior to Mtb Challenge
3.5. G-Ag85A Immunization Provides Superior Protection Compared With NG-Ag85A in Terms of Bacterial Burden and Pathological Lung Lesions
3.6. Immunization with G-Ag85A Induced a More Protective T cell Response against Infection with a Virulent Mtb HN878 Strain than Did Immunization with NG-Ag85A
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
- WHO. Global Tuberculosis Report 2018. World Health Organization. 2018. Available online: https://www.who.int/tb/publications/global_report/en/ (accessed on 12 June 2019).
- Andersen, P.; Doherty, T.M. The success and failure of BCG-implications for a novel tuberculosis vaccine. Nat. Rev. Microbiol. 2005, 3, 656–662. [Google Scholar] [CrossRef]
- Agger, E.M. Novel adjuvant formulations for delivery of anti-tuberculosis vaccine candidates. Adv. Drug Deliv. Rev. 2016, 102, 73–82. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Zenteno-Cuevas, R. Successes and failures in human tuberculosis vaccine development. Expert Opin. Biol. Ther. 2017, 17, 1481–1491. [Google Scholar] [CrossRef] [PubMed]
- Kaufmann, S.H.; Weiner, J.; von Reyn, C.F. Novel approaches to tuberculosis vaccine development. Int. J. Infect. Dis. 2017, 56, 263–267. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Nemes, E.; Geldenhuys, H.; Rozot, V.; Rutkowski, K.T.; Ratangee, F.; Bilek, N.; Mabwe, S.; Makhethe, L.; Erasmus, M.; Toefy, A.; et al. Prevention of M. tuberculosis Infection with H4:IC31 Vaccine or BCG Revaccination. N. Engl. J. Med. 2018, 379, 138–149. [Google Scholar] [CrossRef]
- Van Der Meeren, O.; Hatherill, M.; Nduba, V.; Wilkinson, R.J.; Muyoyeta, M.; Van Brakel, E.; Ayles, H.M.; Henostroza, G.; Thienemann, F.; Scriba, T.J.; et al. Phase 2b Controlled Trial of M72/AS01E Vaccine to Prevent Tuberculosis. N. Engl. J. Med. 2018, 379, 1621–1634. [Google Scholar] [CrossRef]
- Terpe, K. Overview of bacterial expression systems for heterologous protein production: From molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 2006, 72, 211–222. [Google Scholar] [CrossRef]
- Celik, E.; Calik, P. Production of recombinant proteins by yeast cells. Biotechnol. Adv. 2012, 30, 1108–1118. [Google Scholar] [CrossRef]
- Xu, Y.; Ma, S.; Huang, Y.; Chen, F.; Chen, L.; Ding, D.; Zheng, Y.; Li, H.; Xiao, J.; Feng, J.; et al. Virus-like particle vaccines for poliovirus types 1, 2, and 3 with enhanced thermostability expressed in insect cells. Vaccine 2019, 37, 2340–2347. [Google Scholar] [CrossRef]
- Yusibov, V.; Streatfield, S.J.; Kushnir, N. Clinical development of plant-produced recombinant pharmaceuticals: Vaccines, antibodies and beyond. Hum. Vaccin. 2011, 7, 313–321. [Google Scholar] [CrossRef]
- Tschofen, M.; Knopp, D.; Hood, E.; Stoger, E. Plant Molecular Farming: Much More than Medicines. Annu. Rev. Anal. Chem. 2016, 9, 271–294. [Google Scholar] [CrossRef] [PubMed]
- Edgue, G.; Twyman, R.M.; Beiss, V.; Fischer, R.; Sack, M. Antibodies from plants for bionanomaterials. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2017, 9. [Google Scholar] [CrossRef][Green Version]
- Ma, J.K.; Drossard, J.; Lewis, D.; Altmann, F.; Boyle, J.; Christou, P.; Cole, T.; Dale, P.; van Dolleweerd, C.J.; Isitt, V.; et al. Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants. Plant Biotechnol. J. 2015, 13, 1106–1120. [Google Scholar] [CrossRef] [PubMed]
- Bendandi, M.; Marillonnet, S.; Kandzia, R.; Thieme, F.; Nickstadt, A.; Herz, S.; Frode, R.; Inoges, S.; Lopez-Diaz de Cerio, A.; Soria, E.; et al. Rapid, high-yield production in plants of individualized idiotype vaccines for non-Hodgkin’s lymphoma. Ann. Oncol. 2010, 21, 2420–2427. [Google Scholar] [CrossRef]
- Van Dolleweerd, C.J.; Teh, A.Y.; Banyard, A.C.; Both, L.; Lotter-Stark, H.C.; Tsekoa, T.; Phahladira, B.; Shumba, W.; Chakauya, E.; Sabeta, C.T.; et al. Engineering, expression in transgenic plants and characterisation of E559, a rabies virus-neutralising monoclonal antibody. J. Infect. Dis. 2014, 210, 200–208. [Google Scholar] [CrossRef][Green Version]
- Hull, A.K.; Criscuolo, C.J.; Mett, V.; Groen, H.; Steeman, W.; Westra, H.; Chapman, G.; Legutki, B.; Baillie, L.; Yusibov, V. Human-derived, plant-produced monoclonal antibody for the treatment of anthrax. Vaccine 2005, 23, 2082–2086. [Google Scholar] [CrossRef]
- 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][Green Version]
- Rigano, M.M.; Alvarez, M.L.; Pinkhasov, J.; Jin, Y.; Sala, F.; Arntzen, C.J.; Walmsley, A.M. Production of a fusion protein consisting of the enterotoxigenic Escherichia coli heat-labile toxin B subunit and a tuberculosis antigen in Arabidopsis thaliana. Plant Cell Rep. 2004, 22, 502–508. [Google Scholar] [CrossRef]
- Dorokhov, Y.L.; Sheveleva, A.A.; Frolova, O.Y.; Komarova, T.V.; Zvereva, A.S.; Ivanov, P.A.; Atabekov, J.G. Superexpression of tuberculosis antigens in plant leaves. Tuberculosis 2007, 87, 218–224. [Google Scholar] [CrossRef]
- Uvarova, E.A.; Belavin, P.A.; Permyakova, N.V.; Zagorskaya, A.A.; Nosareva, O.V.; Kakimzhanova, A.A.; Deineko, E.V. Oral Immunogenicity of plant-made Mycobacterium tuberculosis ESAT6 and CFP10. Biomed. Res. Int. 2013, 2013, 316304. [Google Scholar] [CrossRef]
- Pepponi, I.; Diogo, G.R.; Stylianou, E.; van Dolleweerd, C.J.; Drake, P.M.; Paul, M.J.; Sibley, L.; Ma, J.K.; Reljic, R. Plant-derived recombinant immune complexes as self-adjuvanting TB immunogens for mucosal boosting of BCG. Plant Biotechnol. J. 2014, 12, 840–850. [Google Scholar] [CrossRef] [PubMed]
- Kallenius, G.; Pawlowski, A.; Hamasur, B.; Svenson, S.B. Mycobacterial glycoconjugates as vaccine candidates against tuberculosis. Trends Microbiol. 2008, 16, 456–462. [Google Scholar] [CrossRef] [PubMed]
- Goddard-Borger, E.D.; Boddey, J.A. Implications of Plasmodium glycosylation on vaccine efficacy and design. Future Microbiol. 2018, 13, 609–612. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Avery, O.T.; Goebel, W.F. Chemo-Immunological studies on conjugated carbohydrate-proteins: II. immunological specificity of synthetic sugar-protein antigens. J. Exp. Med. 1929, 50, 533–550. [Google Scholar] [CrossRef][Green Version]
- Adams, E.W.; Ratner, D.M.; Seeberger, P.H.; Hacohen, N. Carbohydrate-mediated targeting of antigen to dendritic cells leads to enhanced presentation of antigen to T cells. Chembiochem 2008, 9, 294–303. [Google Scholar] [CrossRef][Green Version]
- Belisle, J.T.; Vissa, V.D.; Sievert, T.; Takayama, K.; Brennan, P.J.; Besra, G.S. Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis. Science 1997, 276, 1420–1422. [Google Scholar] [CrossRef]
- Brennan, M.J.; Clagett, B.; Fitzgerald, H.; Chen, V.; Williams, A.; Izzo, A.A.; Barker, L.F. Preclinical evidence for implementing a prime-boost vaccine strategy for tuberculosis. Vaccine 2012, 30, 2811–2823. [Google Scholar] [CrossRef][Green Version]
- Brennan, M.J.; Thole, J. Tuberculosis vaccines: A strategic blueprint for the next decade. Tuberculosis 2012, 92 (Suppl. 1), S6–S13. [Google Scholar] [CrossRef]
- Xu, Z.Z.; Chen, X.; Hu, T.; Meng, C.; Wang, X.B.; Rao, Y.; Zhang, X.M.; Yin, Y.L.; Pan, Z.M.; Jiao, X.A. Evaluation of Immunogenicity and Protective Efficacy Elicited by Mycobacterium bovis BCG Overexpressing Ag85A Protein against Mycobacterium tuberculosis Aerosol Infection. Front. Cell Infect. Microbiol. 2016, 6, 3. [Google Scholar] [CrossRef]
- Poecheim, J.; Barnier-Quer, C.; Collin, N.; Borchard, G. Ag85A DNA Vaccine Delivery by Nanoparticles: Influence of the Formulation Characteristics on Immune Responses. Vaccines 2016, 4, 32. [Google Scholar] [CrossRef][Green Version]
- Nemes, E.; Hesseling, A.C.; Tameris, M.; Mauff, K.; Downing, K.; Mulenga, H.; Rose, P.; van der Zalm, M.; Mbaba, S.; Van As, D.; et al. Safety and Immunogenicity of Newborn MVA85A Vaccination and Selective, Delayed Bacille Calmette-Guerin for Infants of Human Immunodeficiency Virus-Infected Mothers: A Phase 2 Randomized, Controlled Trial. Clin. Infect. Dis. 2018, 66, 554–563. [Google Scholar] [CrossRef] [PubMed]
- Shin, S.J.; Lee, B.S.; Koh, W.J.; Manning, E.J.; Anklam, K.; Sreevatsan, S.; Lambrecht, R.S.; Collins, M.T. Efficient differentiation of Mycobacterium avium complex species and subspecies by use of five-target multiplex PCR. J. Clin. Microbiol. 2010, 48, 4057–4062. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Ko, A.; Wui, S.R.; Ryu, J.I.; Lee, Y.J.; Hien, D.T.T.; Rhee, I.; Shin, S.J.; Park, S.A.; Kim, K.S.; Cho, Y.J.; et al. Potentiation of Th1-Type Immune Responses to Mycobacterium tuberculosis Antigens in Mice by Cationic Liposomes Combined with De-O-Acylated Lipooligosaccharide. J. Microbiol. Biotechnol. 2018, 28, 136–144. [Google Scholar] [CrossRef] [PubMed]
- Islam, M.R.; Kwak, J.W.; Lee, J.S.; Hong, S.W.; Khan, M.R.I.; Lee, Y.; Lee, Y.; Lee, S.W.; Hwang, I. Cost-effective production of tag-less recombinant protein in Nicotiana benthamiana. Plant Biotechnol. J. 2019, 17, 1094–1105. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Marillonnet, S.; Thoeringer, C.; Kandzia, R.; Klimyuk, V.; Gleba, Y. Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants. Nat. Biotechnol. 2005, 23, 718–723. [Google Scholar] [CrossRef] [PubMed]
- Chen, X.; Zaro, J.L.; Shen, W.C. Fusion protein linkers: Property, design and functionality. Adv. Drug Deliv. Rev. 2013, 65, 1357–1369. [Google Scholar] [CrossRef][Green Version]
- Lee, G.; Na, Y.J.; Yang, B.G.; Choi, J.P.; Seo, Y.B.; Hong, C.P.; Yun, C.H.; Kim, D.H.; Sohn, E.J.; Kim, J.H.; et al. Oral immunization of haemaggulutinin H5 expressed in plant endoplasmic reticulum with adjuvant saponin protects mice against highly pathogenic avian influenza A virus infection. Plant Biotechnol. J. 2015, 13, 62–72. [Google Scholar] [CrossRef]
- Kang, H.; Park, Y.; Lee, Y.; Yoo, Y.J.; Hwang, I. Fusion of a highly N-glycosylated polypeptide increases the expression of ER-localized proteins in plants. Sci. Rep. 2018, 8, 4612. [Google Scholar] [CrossRef][Green Version]
- Li, M.; Huang, D. Purification and characterization of prokaryotically expressed human interferon-lambda2. Biotechnol. Lett. 2007, 29, 1025–1029. [Google Scholar] [CrossRef]
- Spencer, A.J.; Hill, F.; Honeycutt, J.D.; Cottingham, M.G.; Bregu, M.; Rollier, C.S.; Furze, J.; Draper, S.J.; Sogaard, K.C.; Gilbert, S.C.; et al. Fusion of the Mycobacterium tuberculosis antigen 85A to an oligomerization domain enhances its immunogenicity in both mice and non-human primates. PLoS ONE 2012, 7, e33555. [Google Scholar] [CrossRef][Green Version]
- Freeze, H.H.; Kranz, C. Endoglycosidase and glycoamidase release of N-linked glycans. Curr. Protoc. Mol. Biol. 2010, 17, 17.13A. [Google Scholar] [CrossRef][Green Version]
- Caccamo, N.; Guggino, G.; Joosten, S.A.; Gelsomino, G.; Di Carlo, P.; Titone, L.; Galati, D.; Bocchino, M.; Matarese, A.; Salerno, A.; et al. Multifunctional CD4(+) T cells correlate with active Mycobacterium tuberculosis infection. Eur. J. Immunol. 2010, 40, 2211–2220. [Google Scholar] [CrossRef]
- Sayes, F.; Pawlik, A.; Frigui, W.; Groschel, M.I.; Crommelynck, S.; Fayolle, C.; Cia, F.; Bancroft, G.J.; Bottai, D.; Leclerc, C.; et al. CD4+ T Cells Recognizing PE/PPE Antigens Directly or via Cross Reactivity Are Protective against Pulmonary Mycobacterium tuberculosis Infection. PLoS Pathog. 2016, 12, e1005770. [Google Scholar] [CrossRef] [PubMed]
- Nandakumar, S.; Kannanganat, S.; Posey, J.E.; Amara, R.R.; Sable, S.B. Attrition of T-cell functions and simultaneous upregulation of inhibitory markers correspond with the waning of BCG-induced protection against tuberculosis in mice. PLoS ONE 2014, 9, e113951. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Zelada, A.M.; Calamante, G.; de la Paz Santangelo, M.; Bigi, F.; Verna, F.; Mentaberry, A.; Cataldi, A. Expression of tuberculosis antigen ESAT-6 in Nicotiana tabacum using a potato virus X-based vector. Tuberculosis 2006, 86, 263–267. [Google Scholar] [CrossRef] [PubMed]
- Wan, W.; Wang, D.; Gao, X.; Hong, J. Expression of family 3 cellulose-binding module (CBM3) as an affinity tag for recombinant proteins in yeast. Appl. Microbiol. Biotechnol. 2011, 91, 789–798. [Google Scholar] [CrossRef]
- Van Els, C.A.; Corbiere, V.; Smits, K.; van Gaans-van den Brink, J.A.; Poelen, M.C.; Mascart, F.; Meiring, H.D.; Locht, C. Toward Understanding the Essence of Post-Translational Modifications for the Mycobacterium tuberculosis Immunoproteome. Front. Immunol. 2014, 5, 361. [Google Scholar] [CrossRef]
- Birhanu, A.G.; Yimer, S.A.; Kalayou, S.; Riaz, T.; Zegeye, E.D.; Holm-Hansen, C.; Norheim, G.; Aseffa, A.; Abebe, M.; Tonjum, T. Ample glycosylation in membrane and cell envelope proteins may explain the phenotypic diversity and virulence in the Mycobacterium tuberculosis complex. Sci. Rep. 2019, 9, 2927. [Google Scholar] [CrossRef]
- Harth, G.; Lee, B.Y.; Wang, J.; Clemens, D.L.; Horwitz, M.A. Novel insights into the genetics, biochemistry, and immunocytochemistry of the 30-kilodalton major extracellular protein of Mycobacterium tuberculosis. Infect. Immun. 1996, 64, 3038–3047. [Google Scholar] [CrossRef][Green Version]
- Haurum, J.S.; Arsequell, G.; Lellouch, A.C.; Wong, S.Y.; Dwek, R.A.; McMichael, A.J.; Elliott, T. Recognition of carbohydrate by major histocompatibility complex class I-restricted, glycopeptide-specific cytotoxic T lymphocytes. J. Exp. Med. 1994, 180, 739–744. [Google Scholar] [CrossRef][Green Version]
- Ninkovic, T.; Hanisch, F.G. O-glycosylated human MUC1 repeats are processed in vitro by immunoproteasomes. J. Immunol. 2007, 179, 2380–2388. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Cudic, M.; Ertl, H.C.; Otvos, L., Jr. Synthesis, conformation and T-helper cell stimulation of an O-linked glycopeptide epitope containing extended carbohydrate side-chains. Bioorg. Med. Chem. 2002, 10, 3859–3870. [Google Scholar] [CrossRef]
- Carbone, F.R.; Gleeson, P.A. Carbohydrates and antigen recognition by T cells. Glycobiology 1997, 7, 725–730. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Osorio, F.; Reis e Sousa, C. Myeloid C-type lectin receptors in pathogen recognition and host defense. Immunity 2011, 34, 651–664. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Sancho, D.; Reis e Sousa, C. Signaling by myeloid C-type lectin receptors in immunity and homeostasis. Annu. Rev. Immunol. 2012, 30, 491–529. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Wolfert, M.A.; Boons, G.J. Adaptive immune activation: Glycosylation does matter. Nat. Chem. Biol. 2013, 9, 776–784. [Google Scholar] [CrossRef][Green Version]
- Forbes, E.K.; Sander, C.; Ronan, E.O.; McShane, H.; Hill, A.V.; Beverley, P.C.; Tchilian, E.Z. Multifunctional, high-level cytokine-producing Th1 cells in the lung, but not spleen, correlate with protection against Mycobacterium tuberculosis aerosol challenge in mice. J. Immunol. 2008, 181, 4955–4964. [Google Scholar] [CrossRef][Green Version]
- Karp, C.L.; Wilson, C.B.; Stuart, L.M. Tuberculosis vaccines: Barriers and prospects on the quest for a transformative tool. Immunol. Rev. 2015, 264, 363–381. [Google Scholar] [CrossRef][Green Version]
- Coler, R.N.; Day, T.A.; Ellis, R.; Piazza, F.M.; Beckmann, A.M.; Vergara, J.; Rolf, T.; Lu, L.; Alter, G.; Hokey, D.; et al. The TLR-4 agonist adjuvant, GLA-SE, improves magnitude and quality of immune responses elicited by the ID93 tuberculosis vaccine: First-in-human trial. NPJ Vaccines 2018, 3, 34. [Google Scholar] [CrossRef]
- Ladel, C.H.; Daugelat, S.; Kaufmann, S.H. Immune response to Mycobacterium bovis bacille Calmette Guerin infection in major histocompatibility complex class I- and II-deficient knock-out mice: Contribution of CD4 and CD8 T cells to acquired resistance. Eur. J. Immunol. 1995, 25, 377–384. [Google Scholar] [CrossRef]
- Yang, J.D.; Mott, D.; Sutiwisesak, R.; Lu, Y.J.; Raso, F.; Stowell, B.; Babunovic, G.H.; Lee, J.; Carpenter, S.M.; Way, S.S.; et al. Mycobacterium tuberculosis-specific CD4+ and CD8+ T cells differ in their capacity to recognize infected macrophages. PLoS Pathog. 2018, 14, e1007060. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Chen, C.Y.; Huang, D.; Wang, R.C.; Shen, L.; Zeng, G.; Yao, S.; Shen, Y.; Halliday, L.; Fortman, J.; McAllister, M.; et al. A critical role for CD8 T cells in a nonhuman primate model of tuberculosis. PLoS Pathog. 2009, 5, e1000392. [Google Scholar] [CrossRef] [PubMed]
- Moguche, A.O.; Musvosvi, M.; Penn-Nicholson, A.; Plumlee, C.R.; Mearns, H.; Geldenhuys, H.; Smit, E.; Abrahams, D.; Rozot, V.; Dintwe, O.; et al. Antigen Availability Shapes T Cell Differentiation and Function during Tuberculosis. Cell Host Microbe 2017, 21, 695–706.e695. [Google Scholar] [CrossRef] [PubMed][Green Version]
© 2020 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 (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Kim, H.; Kwon, K.W.; Park, J.; Kang, H.; Lee, Y.; Sohn, E.-J.; Hwang, I.; Eum, S.-Y.; Shin, S.J. Plant-Produced N-glycosylated Ag85A Exhibits Enhanced Vaccine Efficacy Against Mycobacterium tuberculosis HN878 Through Balanced Multifunctional Th1 T Cell Immunity. Vaccines 2020, 8, 189. https://doi.org/10.3390/vaccines8020189
Kim H, Kwon KW, Park J, Kang H, Lee Y, Sohn E-J, Hwang I, Eum S-Y, Shin SJ. Plant-Produced N-glycosylated Ag85A Exhibits Enhanced Vaccine Efficacy Against Mycobacterium tuberculosis HN878 Through Balanced Multifunctional Th1 T Cell Immunity. Vaccines. 2020; 8(2):189. https://doi.org/10.3390/vaccines8020189
Chicago/Turabian StyleKim, Hongmin, Kee Woong Kwon, Jaehun Park, Hyangju Kang, Yongjik Lee, Eun-Ju Sohn, Inhwan Hwang, Seok-Yong Eum, and Sung Jae Shin. 2020. "Plant-Produced N-glycosylated Ag85A Exhibits Enhanced Vaccine Efficacy Against Mycobacterium tuberculosis HN878 Through Balanced Multifunctional Th1 T Cell Immunity" Vaccines 8, no. 2: 189. https://doi.org/10.3390/vaccines8020189