Synthesizing Vaccines with Microbes

  • Michael WackerEmail author
  • Danilo R. Casimiro


For a long time, viral or bacterial vaccines were developed by generating an attenuated or less virulent form of the pathogen with or without the combined use of chemical or physical inactivation. The emergence of approaches which enable the identification of protective components of the pathogens has led to alternative processes for manufacturing certain vaccines. In particular, advances in biotechnology allow manufacturing these components in recombinant forms by extracting them from heterologous cell production systems. Various prokaryotic or eukaryotic production systems have been explored and developed as manufacturing platforms for different vaccines. These systems can offer safer and less reactogenic products at, often times, high yields. In this chapter, examples of microbial production systems will be described. Different microbial systems which are being used to manufacture licensed vaccines are summarized. In addition, the chapter will give an outline of the potential of new technologies that are currently being tested for manufacturing of novel vaccines. We concentrate on microbial systems that are able to modify proteins with well-defined sugar structures. Pichia pastoris has been engineered to produce viral glycoproteins that are potentially more antigenic and can be produced at higher yield compared to insect or mammalian production systems. Different glycoengineered yeast cells are being used to manufacture viral glycoproteins that are currently in clinical development. A novel Escherichia coli expression system is also described that allows the glycosylation of proteins. This expression system can be used to manufacture conjugate vaccines, allowing for the first time to produce complex glycoconjugate structures in well-defined microbial production systems. Several of these conjugates that are in preclinical and clinical development are being described.


Conjugate Vaccine Invasive Meningococcal Disease Quadrivalent Vaccine Terminal Sialic Acid Vaccine Immunogen 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The authors would like to thank Robert Davidson, Natarajan Sethuraman, John Balliet, Jessica Flynn, Joseph Joyce, Loren Schultz, and the many scientists at the MRL for their contributions on the use of the glycoengineered yeast for vaccine production. The authors would like to thank Michael Kowarik, Fabiana Fernandez, Michael Wetter, Veronica Gambillara, Cristina Alaimo and many scientists at GlycoVaxyn for their contributions on the development of bioconjugates and Skip Waechter for critically reading the manuscript.


  1. Abdel-Motal U, Wang S, Lu S, Wigglesworth K, Galili U (2006) Increased immunogenicity of human immunodeficiency virus gp120 engineered to express Galalpha1-3Galbeta1-4GlcNAc-R epitopes. J Virol 80(14):6943–6951PubMedCrossRefGoogle Scholar
  2. Ada G, Isaacs D (2003) Carbohydrate-protein conjugate vaccines. Clin Microbiol Infect 9(2):79–85PubMedCrossRefGoogle Scholar
  3. Anderson P (1983) Antibody responses to Haemophilus influenzae type b and diphtheria toxin induced by conjugates of oligosaccharides of the type b capsule with the nontoxic protein CRM197. Infect Immun 39(1):233–238PubMedGoogle Scholar
  4. Avery OT, Goebel WF (1929) Chemo-immunological studies on conjugated carbohydrate-proteins. II Immunological specificity of synthetic sugar-proteins. J Exp Med 50:521–533PubMedCrossRefGoogle Scholar
  5. Awasthi S, Lubinski JM, Friedman HM (2009) Immunization with HSV-1 glycoprotein C prevents immune evasion from complement and enhances the efficacy of an HSV-1 glycoprotein D subunit vaccine. Vaccine 27(49):6845–6853PubMedCrossRefGoogle Scholar
  6. Bett AJ, Dubey SA, Mehrotra DV, Guan L, Long R, Anderson K, Collins K, Gaunt C, Fernandez R, Cole S, Meschino S, Tang A, Sun X, Gurunathan S, Tartaglia J, Robertson MN, Shiver JW, Casimiro DR (2010) Comparison of T cell immune responses induced by vectored HIV vaccines in non-human primates and humans. Vaccine 28(50):7881–7889PubMedCrossRefGoogle Scholar
  7. Bryan JT (2007) Developing an HPV vaccine to prevent cervical cancer and genital warts. Vaccine 25(16):3001–3006PubMedCrossRefGoogle Scholar
  8. Choi BK, Bobrowicz P, Davidson RC, Hamilton SR, Kung DH, Li H, Miele RG, Nett JH, Wildt S, Gerngross TU (2003) Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc Natl Acad Sci USA 100(9):5022–5027PubMedCrossRefGoogle Scholar
  9. Cook JC, Joyce JG, George HA, Schultz LD, Hurni WM, Jansen KU, Hepler RW, Ip C, Lowe RS, Keller PM, Lehman ED (1999) Purification of virus-like particles of recombinant human papillomavirus type 11 major capsid protein L1 from Saccharomyces cerevisiae. Protein Expr Purif 17(3):477–484PubMedCrossRefGoogle Scholar
  10. Feldman MF, Wacker M, Hernandez M, Hitchen PG, Marolda CL, Kowarik M, Morris HR, Dell A, Valvano MA, Aebi M (2005) Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli. Proc Natl Acad Sci USA 102(8):3016–3021PubMedCrossRefGoogle Scholar
  11. Findlow J, Borrow R, Snape MD, Dawson T, Holland A, John TM, Evans A, Telford KL, Ypma E, Toneatto D, Oster P, Miller E, Pollard AJ (2010) Multicenter, open-label, randomized phase II controlled trial of an investigational recombinant Meningococcal serogroup B vaccine with and without outer membrane vesicles, administered in infancy. Clin Infect Dis 51(10):1127–1137PubMedCrossRefGoogle Scholar
  12. Fletcher LD, Bernfield L, Barniak V, Farley JE, Howell A, Knauf M, Ooi P, Smith RP, Weise P, Wetherell M, Xie X, Zagursky R, Zhang Y, Zlotnick GW (2004) Vaccine potential of the Neisseria meningitidis 2086 lipoprotein. Infect Immun 72(4):2088–2100PubMedCrossRefGoogle Scholar
  13. Frasch CE (2009) Preparation of bacterial polysaccharide-protein conjugates: analytical and manufacturing challenges. Vaccine 27(46):6468–6470PubMedCrossRefGoogle Scholar
  14. Galili U (1993) Evolution and pathophysiology of the human natural anti-alpha-galactosyl IgG (anti-Gal) antibody. Springer Semin Immunopathol 15(2–3):155–171PubMedGoogle Scholar
  15. Galili U, Clark MR, Shohet SB, Buehler J, Macher BA (1987) Evolutionary relationship between the natural anti-Gal antibody and the Gal alpha 1–3Gal epitope in primates. Proc Natl Acad Sci USA 84(5):1369–1373PubMedCrossRefGoogle Scholar
  16. Garland SM, Hernandez-Avila M, Wheeler CM, Perez G, Harper DM, Leodolter S, Tang GW, Ferris DG, Steben M, Bryan J, Taddeo FJ, Railkar R, Esser MT, Sings HL, Nelson M, Boslego J, Sattler C, Barr E, Koutsky LA (2007) Quadrivalent vaccine against human papillomavirus to prevent anogenital diseases. N Engl J Med 356(19):1928–1943PubMedCrossRefGoogle Scholar
  17. Gellissen G, Melber K (1996) Methylotrophic yeast hansenula polymorpha as production organism for recombinant pharmaceuticals. Arzneimittelforschung 46(9):943–948PubMedGoogle Scholar
  18. Gerngross TU (2004) Advances in the production of human therapeutic proteins in yeasts and filamentous fungi. Nat Biotechnol 22(11):1409–1414PubMedCrossRefGoogle Scholar
  19. Giuliano AR, Palefsky JM, Goldstone S, Moreira ED Jr, Penny ME, Aranda C, Vardas E, Moi H, Jessen H, Hillman R, Chang YH, Ferris D, Rouleau D, Bryan J, Marshall JB, Vuocolo S, Barr E, Radley D, Haupt RM, Guris D (2011) Efficacy of quadrivalent HPV vaccine against HPV infection and disease in males. N Engl J Med 364(5):401–411PubMedCrossRefGoogle Scholar
  20. Hamilton SR, Bobrowicz P, Bobrowicz B, Davidson RC, Li H, Mitchell T, Nett JH, Rausch S, Stadheim TA, Wischnewski H, Wildt S, Gerngross TU (2003) Production of complex human glycoproteins in yeast. Science 301(5637):1244–1246PubMedCrossRefGoogle Scholar
  21. Hamilton SR, Davidson RC, Sethuraman N, Nett JH, Jiang Y, Rios S, Bobrowicz P, Stadheim TA, Li H, Choi BK, Hopkins D, Wischnewski H, Roser J, Mitchell T, Strawbridge RR, Hoopes J, Wildt S, Gerngross TU (2006) Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 313(5792):1441–1443PubMedCrossRefGoogle Scholar
  22. Han DJ, Weiner DB, Sin JI (2010) DNA vaccines against infectious diseases and cancer. Biomol Ther 18:1–15CrossRefGoogle Scholar
  23. Henics T, Winkler B, Pfeifer U, Gill SR, Buschle M, von Gabain A, Meinke AL (2003) Small-fragment genomic libraries for the display of putative epitopes from clinically significant pathogens. Biotechniques 35(1):196–202, 204, 206 passimPubMedGoogle Scholar
  24. Hofmann KJ, Cook JC, Joyce JG, Brown DR, Schultz LD, George HA, Rosolowsky M, Fife KH, Jansen KU (1995) Sequence determination of human papillomavirus type 6a and assembly of virus-like particles in Saccharomyces cerevisiae. Virology 209(2):506–518PubMedCrossRefGoogle Scholar
  25. Hofmann KJ, Neeper MP, Markus HZ, Brown DR, Muller M, Jansen KU (1996) Sequence conservation within the major capsid protein of human papillomavirus (HPV) type 18 and formation of HPV-18 virus-like particles in Saccharomyces cerevisiae. J Gen Virol 77(Pt 3):465–468PubMedCrossRefGoogle Scholar
  26. Huleatt JW, Nakaar V, Desai P, Huang Y, Hewitt D, Jacobs A, Tang J, McDonald W, Song L, Evans RK, Umlauf S, Tussey L, Powell TJ (2008) Potent immunogenicity and efficacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza M2e to the TLR5 ligand flagellin. Vaccine 26(2):201–214PubMedCrossRefGoogle Scholar
  27. Jansen KU, Rosolowsky M, Schultz LD, Markus HZ, Cook JC, Donnelly JJ, Martinez D, Ellis RW, Shaw AR (1995) Vaccination with yeast-expressed cottontail rabbit papillomavirus (CRPV) virus-like particles protects rabbits from CRPV-induced papilloma formation. Vaccine 13(16):1509–1514PubMedCrossRefGoogle Scholar
  28. Joura EA, Kjaer SK, Wheeler CM, Sigurdsson K, Iversen OE, Hernandez-Avila M, Perez G, Brown DR, Koutsky LA, Tay EH, Garcia P, Ault KA, Garland SM, Leodolter S, Olsson SE, Tang GW, Ferris DG, Paavonen J, Lehtinen M, Steben M, Bosch X, Dillner J, Kurman RJ, Majewski S, Munoz N, Myers ER, Villa LL, Taddeo FJ, Roberts C, Tadesse A, Bryan J, Lupinacci LC, Giacoletti KE, Lu S, Vuocolo S, Hesley TM, Haupt RM, Barr E (2008) HPV antibody levels and clinical efficacy following administration of a prophylactic quadrivalent HPV vaccine. Vaccine 26(52):6844–6851PubMedCrossRefGoogle Scholar
  29. Kowarik M, Young NM, Numao S, Schulz BL, Hug I, Callewaert N, Mills DC, Watson DC, Hernandez M, Kelly JF, Wacker M, Aebi M (2006) Definition of the bacterial N-glycosylation site consensus sequence. EMBO J 25(9):1957–1966PubMedCrossRefGoogle Scholar
  30. Kroeff EP, Owens RA, Campbell EL, Johnson RD, Marks HI (1989) Production scale purification of biosynthetic human insulin by reversed-phase high-performance liquid chromatography. J Chromatogr 461:45–61PubMedCrossRefGoogle Scholar
  31. Lepetic A, Biscayart C, Seigelchifer M, Arduino R, Stamboulian D (2003) Persistence of immunity and seroprotection 4 years after a primary vaccination schedule with a Hansenula polymorpha recombinant hepatitis B vaccine. Vaccine 21(27–30):4481–4485PubMedCrossRefGoogle Scholar
  32. Li H, Sethuraman N, Stadheim TA, Zha D, Prinz B, Ballew N, Bobrowicz P, Choi BK, Cook WJ, Cukan M, Houston-Cummings NR, Davidson R, Gong B, Hamilton SR, Hoopes JP, Jiang Y, Kim N, Mansfield R, Nett JH, Rios S, Strawbridge R, Wildt S, Gerngross TU (2006) Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat Biotechnol 24(2):210–215PubMedCrossRefGoogle Scholar
  33. Lindberg AA (1999) Glycoprotein conjugate vaccines. Vaccine 17(Suppl 2):S28–S36PubMedCrossRefGoogle Scholar
  34. Linton D, Allan E, Karlyshev AV, Cronshaw AD, Wren BW (2002) Identification of N-acetylgalactosamine-containing glycoproteins PEB3 and CgpA in Campylobacter jejuni. Mol Microbiol 43(2):497–508PubMedCrossRefGoogle Scholar
  35. Linton D, Dorrell N, Hitchen PG, Amber S, Karlyshev AV, Morris HR, Dell A, Valvano MA, Aebi M, Wren BW (2005) Functional analysis of the Campylobacter jejuni N-linked protein glycosylation pathway. Mol Microbiol 55(6):1695–1703PubMedCrossRefGoogle Scholar
  36. Lockhart S (2003) Conjugate vaccines. Expert Rev Vaccines 2(5):633–648PubMedCrossRefGoogle Scholar
  37. Mach H, Volkin DB, Troutman RD, Wang B, Luo Z, Jansen KU, Shi L (2006) Disassembly and reassembly of yeast-derived recombinant human papillomavirus virus-like particles (HPV VLPs). J Pharm Sci 95(10):2195–2206PubMedCrossRefGoogle Scholar
  38. Malkin EM, Diemert DJ, McArthur JH, Perreault JR, Miles AP, Giersing BK, Mullen GE, Orcutt A, Muratova O, Awkal M, Zhou H, Wang J, Stowers A, Long CA, Mahanty S, Miller LH, Saul A, Durbin AP (2005) Phase 1 clinical trial of apical membrane antigen 1: an asexual blood-stage vaccine for Plasmodium falciparum malaria. Infect Immun 73(6):3677–3685PubMedCrossRefGoogle Scholar
  39. Munoz N, Kjaer SK, Sigurdsson K, Iversen OE, Hernandez-Avila M, Wheeler CM, Perez G, Brown DR, Koutsky LA, Tay EH, Garcia PJ, Ault KA, Garland SM, Leodolter S, Olsson SE, Tang GW, Ferris DG, Paavonen J, Steben M, Bosch FX, Dillner J, Huh WK, Joura EA, Kurman RJ, Majewski S, Myers ER, Villa LL, Taddeo FJ, Roberts C, Tadesse A, Bryan JT, Lupinacci LC, Giacoletti KE, Sings HL, James MK, Hesley TM, Barr E, Haupt RM (2010) Impact of human papillomavirus (HPV)-6/11/16/18 vaccine on all HPV-associated genital diseases in young women. J Natl Cancer Inst 102(5):325–339PubMedCrossRefGoogle Scholar
  40. Neeper MP, Hofmann KJ, Jansen KU (1996) Expression of the major capsid protein of human papillomavirus type 11 in Saccharomyces cerevisae. Gene 180(1–2):1–6PubMedCrossRefGoogle Scholar
  41. Phalipon A, Tanguy M, Grandjean C, Guerreiro C, Belot F, Cohen D, Sansonetti PJ, Mulard LA (2009) A synthetic carbohydrate-protein conjugate vaccine candidate against Shigella flexneri 2a infection. J Immunol 182(4):2241–2247PubMedCrossRefGoogle Scholar
  42. Plotkin SA, Orenstein WA, Offit PA (2008) Vaccines. Saunders, Philadelphia, PAGoogle Scholar
  43. Pozsgay V, Chu C, Pannell L, Wolfe J, Robbins JB, Schneerson R (1999) Protein conjugates of synthetic saccharides elicit higher levels of serum IgG lipopolysaccharide antibodies in mice than do those of the O-specific polysaccharide from Shigella dysenteriae type 1. Proc Natl Acad Sci USA 96(9):5194–5197PubMedCrossRefGoogle Scholar
  44. Prather KJ, Sagar S, Chartrain M (2003) Industrial scale production of plasmid DNA for vaccine and gene therapy: plasmid design, production, and purification. Enzym Microb Technol 33:865–883CrossRefGoogle Scholar
  45. Roggenkamp R, Hansen H, Eckart M, Zbigniew J, Hollenberg CP (1986) Transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors. Mol Gen Genet 202(2):302–308CrossRefGoogle Scholar
  46. Schneerson R, Barrera O, Sutton A, Robbins JB (1980) Preparation, characterization, and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates. J Exp Med 152(2):361–376PubMedCrossRefGoogle Scholar
  47. Schultz LD, Markus HZ, Hofmann KJ, Montgomery DL, Dunwiddie CT, Kniskern PJ, Freedman RB, Ellis RW, Tuite MF (1994) Using molecular genetics to improve the production of recombinant proteins by the yeast Saccharomyces cerevisiae. Ann N Y Acad Sci 721:148–157PubMedCrossRefGoogle Scholar
  48. Sedegah M, Rogers WO, Belmonte A, Belmonte M, Banania G, Patterson N, Ferrari M, Kaslow DC, Carucci DJ, Richie TL, Doolan DL (2006) Vaxfectin enhances immunogenicity and protective efficacy of P. yoelii circumsporozoite DNA vaccines. Vaccine 24(11):1921–1927PubMedCrossRefGoogle Scholar
  49. Sette A, Rappuoli R (2010) Reverse vaccinology: developing vaccines in the era of genomics. Immunity 33(4):530–541PubMedCrossRefGoogle Scholar
  50. Shivananda V, Somani BS, Srikanth MM, Kulkarni PS (2006) Comparison of two hepatitis B vaccines (GeneVac-B and Engerix-B) in healthy infants in India. Clin Vaccine Immunol 13(6):661–664PubMedCrossRefGoogle Scholar
  51. Song L, Zhang Y, Yun NE, Poussard AL, Smith JN, Smith JK, Borisevich V, Linde JJ, Zacks MA, Li H, Kavita U, Reiserova L, Liu X, Dumuren K, Balasubramanian B, Weaver B, Parent J, Umlauf S, Liu G, Huleatt J, Tussey L, Paessler S (2009) Superior efficacy of a recombinant flagellin:H5N1 HA globular head vaccine is determined by the placement of the globular head within flagellin. Vaccine 27(42):5875–5884PubMedCrossRefGoogle Scholar
  52. Stanberry LR, Spruance SL, Cunningham AL, Bernstein DI, Mindel A, Sacks S, Tyring S, Aoki FY, Slaoui M, Denis M, Vandepapeliere P, Dubin G (2002) Glycoprotein-D-adjuvant vaccine to prevent genital herpes. N Engl J Med 347(21):1652–1661PubMedCrossRefGoogle Scholar
  53. Storrs SB, Przybycien TM (1991) Commercial-scale refolding of recombinant methionyl bovine somatotropin. In: DeBernardez-Clark E, Georgiou G (eds) Protein refolding. American Chemical Society, Washington, DC, pp 197–205CrossRefGoogle Scholar
  54. Talbot HK, Rock MT, Johnson C, Tussey L, Kavita U, Shanker A, Shaw AR, Taylor DN (2010) Immunopotentiation of trivalent influenza vaccine when given with VAX102, a recombinant influenza M2e vaccine fused to the TLR5 ligand flagellin. PLoS One 5(12):e14442PubMedCrossRefGoogle Scholar
  55. Treanor JJ, Taylor DN, Tussey L, Hay C, Nolan C, Fitzgerald T, Liu G, Kavita U, Song L, Dark I, Shaw A (2010) Safety and immunogenicity of a recombinant hemagglutinin influenza-flagellin fusion vaccine (VAX125) in healthy young adults. Vaccine 28(52):8268–8274PubMedCrossRefGoogle Scholar
  56. Verez-Bencomo V, Fernandez-Santana V, Hardy E, Toledo ME, Rodriguez MC, Heynngnezz L, Rodriguez A, Baly A, Herrera L, Izquierdo M, Villar A, Valdes Y, Cosme K, Deler ML, Montane M, Garcia E, Ramos A, Aguilar A, Medina E, Torano G, Sosa I, Hernandez I, Martinez R, Muzachio A, Carmenates A, Costa L, Cardoso F, Campa C, Diaz M, Roy R (2004) A synthetic conjugate polysaccharide vaccine against Haemophilus influenzae type b. Science 305(5683):522–525PubMedCrossRefGoogle Scholar
  57. Villa LL, Ault KA, Giuliano AR, Costa RL, Petta CA, Andrade RP, Brown DR, Ferenczy A, Harper DM, Koutsky LA, Kurman RJ, Lehtinen M, Malm C, Olsson SE, Ronnett BM, Skjeldestad FE, Steinwall M, Stoler MH, Wheeler CM, Taddeo FJ, Yu J, Lupinacci L, Railkar R, Marchese R, Esser MT, Bryan J, Jansen KU, Sings HL, Tamms GM, Saah AJ, Barr E (2006) Immunologic responses following administration of a vaccine targeting human papillomavirus types 6, 11, 16, and 18. Vaccine 24(27–28):5571–5583PubMedCrossRefGoogle Scholar
  58. Wacker M, Linton D, Hitchen PG, Nita-Lazar M, Haslam SM, North SJ, Panico M, Morris HR, Dell A, Wren BW, Aebi M (2002) N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298(5599):1790–1793PubMedCrossRefGoogle Scholar
  59. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, Peto J, Meijer CJ, Munoz N (1999) Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 189(1):12–19PubMedCrossRefGoogle Scholar
  60. West DJ (1993) Scope and design of hepatitis B vaccine clinical trials. In: Ellis RW (ed) Hepatitis B vaccines in clinical practice. Dekker, New York, NY, pp 159–177Google Scholar
  61. Wildt S, Gerngross TU (2005) The humanization of N-glycosylation pathways in yeast. Nat Rev Microbiol 3(2):119–128PubMedCrossRefGoogle Scholar
  62. Wu Y, Ellis RD, Shaffer D, Fontes E, Malkin EM, Mahanty S, Fay MP, Narum D, Rausch K, Miles AP, Aebig J, Orcutt A, Muratova O, Song G, Lambert L, Zhu D, Miura K, Long C, Saul A, Miller LH, Durbin AP (2008) Phase 1 trial of malaria transmission blocking vaccine candidates Pfs25 and Pvs25 formulated with montanide ISA 51. PLoS One 3(7):e2636PubMedCrossRefGoogle Scholar
  63. Zauner W, Lingnau K, Mattner F, von Gabain A, Buschle M (2001) Defined synthetic vaccines. Biol Chem 382(4):581–595PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2012

Authors and Affiliations

  1. 1.GlycoVaxynSchlierenSwitzerland
  2. 2.Vaccines R&D, Merck Research Laboratories, Merck & Co.RahwayUSA

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