Secreted trimeric viral envelope proteins as a tool for new vaccine design and biochemical assays

Varies viral envelope proteins are formed by trimeric modules that are highly stable while anchored through the transmembrane domain in the virus membrane. Unfortunately, to raise vaccines against the whole viruses could lead to the production of antibodies against hijacked cellular proteins that can cause adverse effects. Therefore, pure protein preparations are used for vaccines production. It was found that the use of monomeric immunogens is insufficient in induction of neutralising antibodies. Protective antibodies were more often produced when trimeric ENV proteins were used. Here, we describe technical aspects of ENV proteins processing in host cells, the way to construct stable trimeric immunogens, and recent progress with construction of soluble trimers of ENV from Ebola, HIV, rabies, and influenza viruses.

1. Smith, R.A.; Baglioni, C. The active form of tumor necrosis factor is a trimer. J Biol Chem 1987, 262, 6951-6954.
2. Mahmood, N.; Hay, A.J. An elisa utilizing immobilised snowdrop lectin gna for the detection of envelope glycoproteins of hiv and siv. J Immunol Methods 1992, 151, 9-13.
3. Büttner, S.; Koch, B.; Dolnik, O.; Eickmann, M.; Freiwald, T.; Rudolf, S.; Engel, J.; Becker, S.; Ronco, C.; Geiger, H. Extracorporeal virus elimination for the treatment of severe ebola virus disease - first experience with lectin affinity plasmapheresis. Blood Purif 2014, 38, 286-291.
4. Hamilton, B.S.; Whittaker, G.R.; Daniel, S. Influenza virus-mediated membrane fusion: Determinants of hemagglutinin fusogenic activity and experimental approaches for assessing virus fusion. Viruses 2012, 4, 1144-1168.
5. Haim, H.; Salas, I.; Sodroski, J. Proteolytic processing of the human immunodeficiency virus envelope glycoprotein precursor decreases conformational flexibility. J Virol 2013, 87, 1884- 1889.
6. Sanchez, A.; Trappier, S.G.; Mahy, B.W.; Peters, C.J.; Nichol, S.T. The virion glycoproteins of ebola viruses are encoded in two reading frames and are expressed through transcriptional editing. Proc Natl Acad Sci U S A 1996, 93, 3602-3607.
7. Volchkov, V.E.; Feldmann, H.; Volchkova, V.A.; Klenk, H.D. Processing of the ebola virus glycoprotein by the proprotein convertase furin. Proc Natl Acad Sci U S A 1998, 95, 5762-5767.
8. Markova, S.V.; Golz, S.; Frank, L.A.; Kalthof, B.; Vysotski, E.S. Cloning and expression of cdna for a luciferase from the marine copepod metridia longa. A novel secreted bioluminescent reporter enzyme. J Biol Chem 2004, 279, 3212-3217.
9. Knappskog, S.; Ravneberg, H.; Gjerdrum, C.; Trösse, C.; Stern, B.; Pryme, I.F. The level of synthesis and secretion of gaussia princeps luciferase in transfected cho cells is heavily dependent on the choice of signal peptide. J Biotechnol 2007, 128, 705-715.
10. Berger, J.; Hauber, J.; Hauber, R.; Geiger, R.; Cullen, B.R. Secreted placental alkaline phosphatase: A powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 1988, 66, 1-10.
11. Lowe, M.E. Site-specific mutations in the coohterminus of placental alkaline phosphatase: A single amino acid change converts a phosphatidylinositol-glycan-anchored protein to a secreted protein. J Cell Biol 1992, 116, 799- 807.
12. Zhang, L.; Leng, Q.; Mixson, A.J. Alteration in the il-2 signal peptide affects secretion of proteins in vitro and in vivo. J Gene Med 2005, 7, 354-365.
13. Wang, J.Y.; Song, W.T.; Li, Y.; Chen, W.J.; Yang, D.; Zhong, G.C.; Zhou, H.Z.; Ren, C.Y.; Yu, H.T.; Ling, H. Improved expression of secretory and trimeric proteins in mammalian cells via the introduction of a new trimer motif and a mutant of the tpa signal sequence. Appl Microbiol Biotechnol 2011, 91, 731-740.
14. Kober, L.; Zehe, C.; Bode, J. Optimized signal peptides for the development of high expressing cho cell lines. Biotechnol Bioeng 2013, 110, 1164- 1173.
15. Haas, J.; Park, E.C.; Seed, B. Codon usage limitation in the expression of hiv-1 envelope glycoprotein. Curr Biol 1996, 6, 315-324.
16. Wen, B.; Deng, Y.; Guan, J.; Yan, W.; Wang, Y.; Tan, expression and secretion of hepatitis c virus envelope glycoproteins. Acta Biochim Biophys Sin (Shanghai) 2011, 43, 96-102.
17. Tsuchiya, Y.; Morioka, K.; Taneda, I.; Shirai, J.; Yoshida, K. Gene design of signal sequence for the eff ective secretion of recombinant protein using insect cell. Nucleic Acids Symp Ser (Oxf) 2005, 305-306.
18. Arndt, K.; Fink, G.R. Gcn4 protein, a positive transcription factor in yeast, binds general control promoters at all 5‘ tgactc 3‘ sequences. Proc Natl Acad Sci U S A 1986, 83, 8516-8520.
19. Harbury, P.B.; Kim, P.S.; Alber, T. Crystal structure of an isoleucine-zipper trimer. Nature 1994, 371, 80-83.
20. Tao, Y.; Strelkov, S.V.; Mesyanzhinov, V.V.; Rossmann, M.G. Structure of bacteriophage t4 fi britin: A segmented coiled coil and the role of the c-terminal domain. Structure 1997, 5, 789-798.
21. Turki, I.; Hammami, A.; Kharmachi, H.; Mousli, M. Engineering of a recombinant trivalent single-chain variable fragment antibody directed against rabies virus glycoprotein g with improved neutralizing potency. Mol Immunol 2014, 57, 66- 73.
22. Ito, T.; Iwamoto, K.; Tsuji, I.; Tsubouchi, H.; Omae, H.; Sato, T.; Ohba, H.; Kurokawa, T.; Taniyama, Y.; Shintani, Y. Trimerization of murine tnf ligand family member light increases the cytotoxic activity against the fm3a mammary carcinoma cell line. Appl Microbiol Biotechnol 2011, 90, 1691-1699.
23. Sliepen, K.; van Montfort, T.; Melchers, M.; Isik, G.; Sanders, R.W. Immunosilencing a highly immunogenic protein trimerization domain. J Biol Chem 2015.
24. Mason, J.M.; Arndt, K.M. Coiled coil domains: Stability, specifi city, and biological implications. Chembiochem 2004, 5, 170-176.
25. Nielsen, B.B.; Kastrup, J.S.; Rasmussen, H.; Holtet, T.L.; Graversen, J.H.; Etzerodt, M.; Th øgersen, H.C.; Larsen, I.K. Crystal structure of tetranectin, a trimeric plasminogen-binding protein with an alpha-helical coiled coil. FEBS Lett 1997, 412, 388-396.
26. Allen, J.E.; Ferrini, R.; Dicker, D.T.; Batzer, G.; Chen, E.; Oltean, D.I.; Lin, B.; Renshaw, M.W.; Kretz- Rommel, A.; El-Deiry, W.S. Targeting trail death receptor 4 with trivalent dr4 atrimer complexes. Mol Cancer Th er 2012, 11, 2087-2095.
27. Byla, P.; Andersen, M.H.; Holtet, T.L.; Jacobsen, H.; Munch, M.; Gad, H.H.; Thøgersen, H.C.; Hartmann, R. Selection of a novel and highly specifi c tumor necrosis factor alpha (tnfalpha) antagonist: Insight from the crystal structure of the antagonist-tnfalpha complex. J Biol Chem 2010, 285, 12096-12100.
28. Yang, X.; Lee, J.; Mahony, E.M.; Kwong, P.D.; Wyatt, R.; Sodroski, J. Highly stable trimers formed by human immunodefi ciency virus type 1 envelope glycoproteins fused with the trimeric motif of t4 bacteriophage fi britin. J Virol 2002, 76, 4634-4642.
29. Weldon, W.C.; Wang, B.Z.; Martin, M.P.; Koutsonanos, D.G.; Skountzou, I.; Compans, R.W. Enhanced immunogenicity of stabilized trimeric soluble infl uenza hemagglutinin. PLoS One 2010, 5.
30. Lin, S.C.; Huang, M.H.; Tsou, P.C.; Huang, L.M.; Chong, P.; Wu, S.C. Recombinant trimeric ha protein immunogenicity of h5n1 avian infl uenza viruses and their combined use with inactivated or adenovirus vaccines. PLoS One 2011, 6, e20052.
31. Hood, C.L.; Abraham, J.; Boyington, J.C.; Leung, K.; Kwong, P.D.; Nabel, G.J. Biochemical and structural characterization of cathepsin l-processed ebola virus glycoprotein: Implications for viral entry and immunogenicity. J Virol 2010, 84, 2972-2982.
32. Chen, X.; Lu, L.; Qi, Z.; Lu, H.; Wang, J.; Yu, X.; Chen, Y.; Jiang, S. Novel recombinant engineered gp41 n-terminal heptad repeat trimers and their potential as anti-hiv-1 therapeutics or microbicides. J Biol Chem 2010, 285, 25506-25515.
33. Lu, Y.; Welsh, J.P.; Swartz, J.R. Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective infl uenza vaccines. Proc Natl Acad Sci U S A 2014, 111, 125-130.
34. Chen, X.; Zaro, J.L.; Shen, W.C. Fusion protein linkers: Property, design and functionality. Adv Drug Deliv Rev 2013, 65, 1357-1369.
35. Krammer, F.; Margine, I.; Tan, G.S.; Pica, N.; Krause, J.C.; Palese, P. A carboxy-terminal trimerization domain stabilizes conformational epitopes on the stalk domain of soluble recombinant hemagglutinin substrates. PLoS One 2012, 7, e43603.
36. Li, J.; Ulitzky, L.; Silberstein, E.; Taylor, D.R.; Viscidi, R. Immunogenicity and protection effi cacy of monomeric and trimeric recombinant sars coronavirus spike protein subunit vaccine candidates. Viral Immunol 2013, 26, 126-132.
37. Côté, M.; Misasi, J.; Ren, T.; Bruchez, A.; Lee, K.; Filone, C.M.; Hensley, L.; Li, Q.; Ory, D.; Chandran, K., et al. Small molecule inhibitors reveal niemannpick c1 is essential for ebola virus infection. Nature 2011, 477, 344-348.
38. Feng, Y.; McKee, K.; Tran, K.; O‘Dell, S.; Schmidt, S.D.; Phogat, A.; Forsell, M.N.; Karlsson Hedestam, G.B.; Mascola, J.R.; Wyatt, R.T. Biochemically defined hiv-1 envelope glycoprotein variant immunogens display diff erential binding and neutralizing specifi cities to the cd4-binding site. J Biol Chem 2012, 287, 5673-5686.
39. Sissoëff , L.; Mousli, M.; England, P.; Tuff ereau, C. Stable trimerization of recombinant rabies virus glycoprotein ectodomain is required for interaction with the p75ntr receptor. J Gen Virol 2005, 86, 2543-2552. The

PDF