Efficient production of immunologically active Shigella invasion plasmid antigens IpaB and IpaH using a cell-free expression system

Abstract

Shigella spp. invade the colonic epithelium and cause bacillary dysentery in humans. Individuals living in areas that lack access to clean water and sanitation are the most affected. Even though infection can be treated with antibiotics, Shigella antimicrobial drug resistance complicates clinical management. Despite decades of effort, there are no licensed vaccines to prevent shigellosis. The highly conserved invasion plasmid antigens (Ipa), which are components of the Shigella type III secretion system, participate in bacterial epithelial cell invasion and have been pursued as vaccine targets. However, expression and purification of these proteins in conventional cell-based systems have been challenging due to solubility issues and extremely low recovery yields. These difficulties have impeded manufacturing and clinical advancement. In this study, we describe a new method to express Ipa proteins using the Xpress+TM cell-free protein synthesis (CFPS) platform. Both IpaB and the C-terminal domain of IpaH1.4 (IpaH-CTD) were efficiently produced with this technology at yields > 200 mg/L. Furthermore, the expression was linearly scaled in a bioreactor under controlled conditions, and proteins were successfully purified using multimode column chromatography to > 95% purity as determined by SDS-PAGE. Biophysical characterization of the cell-free synthetized IpaB and IpaH-CTD using SEC-MALS analysis showed well-defined oligomeric states of the proteins in solution. Functional analysis revealed similar immunoreactivity as compared to antigens purified from E. coli. These results demonstrate the efficiency of CFPS for Shigella protein production; the practicality and scalability of this method will facilitate production of antigens for Shigella vaccine development and immunological analysis. Key points • First report of Shigella IpaB and IpaH produced at high purity and yield using CFPS • CFPS-IpaB and IpaH perform similarly to E. coli–produced proteins in immunoassays • CFPS-IpaB and IpaH react with Shigella-specific human antibodies and are immunogenic in mice. Graphical abstract

Shigella virulence protein VirG is a broadly protective antigen and vaccine candidate

“…The immunogenicity and protective efficacy of VirGα were also investigated using a mucosal route and a clinically advanced mucosal adjuvant, the E. coli double mutant heat-labile toxin (dmLT). Mice were immunized IN with 2.5, 5, or 10 µg of VirGα admixed with dmLT on days 0, 14, and 28; this schedule was selected based on results from previous studies 19 , 45 . VirGα-specific serum IgG responses after the 1st and 2nd vaccinations were modest but increased dramatically after the 3rd immunization.…”

A Novel Shigella O-Polysaccharide–IpaB Conjugate Vaccine Elicits Robust Antibody Responses and Confers Protection against Multiple Shigella Serotypes

“…IpaB was produced using Vaxcyte’s proprietary Xpress + CFPS platform ( 22 ). The native protein sequence was used as a template to generate multiple IpaB single-site variants that efficiently incorporated nnAA p -azidophenylalanine (pAMF) ( Fig.…”

“…We report here for the first time the successful development of a Shigella OPS-conjugate vaccine that employs IpaB as a carrier antigen. IpaB is known to be potently immunogenic ( 17 , 18 , 22 , 26 ). IpaB-specific antibodies are present in high levels in sera from individuals living in areas of endemicity ( 27 ).…”

Addition of Lauryldimethylamine N-Oxide (LDAO) to a Copper-Free Click Chemistry Reaction Improves the Conjugation Efficiency of a Cell-Free Generated CRM197 Variant to Clinically Important Streptococcus pneumoniae Serotypes

“…Intriguingly, the addition of LDAO during the purification process dramatically improved the recovery yields of nonaggregated antigens. 23 − 25 Since we are interested in utilizing these target antigens as carrier proteins for generating novel conjugate vaccines (CVs) using click chemistry, we wanted to further investigate the impact of LDAO on the copper-free SPAAC reaction.…”

“…However, recent studies have shown that surfactants like sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), and sodium decanoate can have a meaningful impact on the kinetics of both copper-catalyzed and copper-free click chemistry reactions. − In a recent study, Schneider et al showed that the addition of SDS and N -lauroylsarcosine (NLS) enhanced the rate of a copper-catalyzed SPAAC reaction, resulting in higher yields of conjugated materials, while elsewhere, the presence of certain surfactants at concentrations above the critical micelle concentration (CMC) in click chemistry reactions was found to improve both conjugation efficiency and reaction kinetics. , Recently, we also successfully utilized a zwitterionic detergent, lauryldimethylamine N -oxide (LDAO), to purify a protein vaccine antigen that showed high propensity for aggregation and precipitation by itself. Intriguingly, the addition of LDAO during the purification process dramatically improved the recovery yields of nonaggregated antigens. − Since we are interested in utilizing these target antigens as carrier proteins for generating novel conjugate vaccines (CVs) using click chemistry, we wanted to further investigate the impact of LDAO on the copper-free SPAAC reaction.…”