High yield production process for Shigella outer membrane particles.
Gram-negative bacteria naturally shed particles that consist of outer membrane lipids, outer membrane proteins, and soluble periplasmic components. These particles have been proposed for use as vaccines but the yield has been problematic. We developed a high yielding production process of genetically derived outer membrane particles from the human pathogen Shigella sonnei. Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30-45 in a 5 L fermenter. Proteomic analysis of the purified particles showed the preparation to primarily contain predicted outer membrane and periplasmic proteins. These were highly immunogenic in mice. The production of these outer membrane particles from high density cultivation of bacteria supports the feasibility of scaling up this approach as an affordable manufacturing process. Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles. This work provides the basis for a large scale manufacturing process of Generalized Modules of Membrane Antigens (GMMA) for production of vaccines from gram-negative bacteria.
Novartis Vaccines Institute for Global Health, Siena, Italy.
Francesco
F
Berlanda Scorza
Anna Maria
AM
Colucci
Luana
L
Maggiore
Silvia
S
Sanzone
Omar
O
Rossi
Ilaria
I
Ferlenghi
Isabella
I
Pesce
Mariaelena
M
Caboni
Nathalie
N
Norais
Vito
V
Di Cioccio
Allan
A
Saul
Christiane
C
Gerke
D000818:Animals; D000954:Antigens, Surface / Q000302:isolation & purification; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005285:Fermentation; D055786:Gene Knockout Techniques; D008563:Membrane Lipids / Q000378:metabolism*; D051379:Mice; D008854:Microscopy, Electron; D015202:Protein Engineering / Q000379:methods*; D012764:Shigella sonnei / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014612:Vaccines / Q000096:biosynthesis
D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D008563:Membrane Lipids; D014612:Vaccines