Production of a Shigella sonnei Vaccine Based on Generalized Modules for Membrane Antigens (GMMA), 1790GAHB

Abstract

Recently, we developed a high yield production process for outer membrane particles from genetically modified bacteria, called Generalized Modules of Membrane Antigens (GMMA), and the corresponding simple two step filtration purification, enabling economic manufacture of these particles for use as vaccines. Using a Shigella sonnei strain that was genetically modified to produce penta-acylated lipopolysaccharide (LPS) with reduced endotoxicity and to maintain the virulence plasmid encoding for the immunodominant O antigen component of the LPS, scale up of the process to GMP pilot scale was straightforward and gave high yields of GMMA with required purity and consistent results. GMMA were formulated with Alhydrogel and were highly immunogenic in mice and rabbits. In mice, a single immunization containing 29 ng protein and 1.75 ng of O antigen elicited substantial anti-LPS antibody levels. As GMMA contain LPS and lipoproteins, assessing potential reactogenicity was a key aspect of vaccine development. In an in vitro monocyte activation test, GMMA from the production strain showed a 600-fold lower stimulatory activity than GMMA with unmodified LPS. Two in vivo tests confirmed the low potential for reactogenicity. We established a modified rabbit pyrogenicity test based on the European Pharmacopoeia pyrogens method but using intramuscular administration of the full human dose (100 μg of protein). The vaccine elicited an average temperature rise of 0.5°C within four hours after administration, which was considered acceptable and showed that the test is able to detect a pyrogenic response. Furthermore, a repeat dose toxicology study in rabbits using intramuscular (100 μg/dose), intranasal (80 μg/dose), and intradermal (10 μg/dose) administration routes showed good tolerability of the vaccine by all routes and supported its suitability for use in humans. The S. sonnei GMMA vaccine is now in Phase 1 dose-escalation clinical trials.

Emerging Technologies for Low‐Cost, Rapid Vaccine Manufacture

“…A) GMMA vaccine production using gram‐negative bacteria. GMMA are released constantly during bacterial culture and are separated using two sequential TFF steps . B) RNA vaccine production using in vitro transcription.…”

“…OMVs are spherical entities of endocytic origin with diameters normally ranging between 20–250 nm . These virus‐sized lipid bilayer vesicles with embedded proteins can be highly immunogenic . OMV‐based vaccines can be produced by: a) culturing wild‐type bacteria which naturally secrete OMVs; b) detergent extraction using sodium deoxycholate in the presence of EDTA; and c) culturing genetically altered bacteria for enhanced OMV production .…”

“…Generalized Modules for Membrane Antigens (GMMA) are OMVs engineered to enhance native OMV formation (by deletion of the gna33 in meningococcus and tolR in Shigella sonnei and Salmonella ), reduce reactogenicity and toxicity (by modifying the acylation pattern of lipid A) and over‐express immunogenic antigens . GMMA contain pathogen‐associated molecular patterns, including toll‐like receptor ligands, which can act as self‐adjuvants in the immune responses they elicit .…”

“…GMMA contain pathogen‐associated molecular patterns, including toll‐like receptor ligands, which can act as self‐adjuvants in the immune responses they elicit . GMMA are highly immunogenic, effective, have low reactogenicity, are safe, and can be manufactured at low cost in a scalable process . The main limitation of GMMA is that it cannot perform human‐like post‐translational modifications (e.g., glycosylation).…”

Current Trends In Research, Development And Production Of Prophylactic Vaccines: Report of Vaccipharma 2015 Congress

“…These observations served as inspiration for further studies of the OMV-concept and use of NOMV as a "platform" for various other antigens. This approach might also well provide benefits in the fight against diseases that are in need for better-performing vaccines, like Bordetella pertussis [96] or where so far there are no other vaccine alternatives in existence, against like S. sonnei [97].…”