Impact of Detergent on Biophysical Properties and Immune Response of the IpaDB Fusion Protein, a Candidate Subunit Vaccine against Shigella Species

Abstract

c Shigella spp. are causative agents of bacillary dysentery, a human illness with high global morbidity levels, particularly among elderly and infant populations. Shigella infects via the fecal-oral route, and its virulence is dependent upon a type III secretion system (T3SS). Two components of the exposed needle tip complex of the Shigella T3SS, invasion plasmid antigen D (IpaD) and IpaB, have been identified as broadly protective antigens in the mouse lethal pneumonia model. A recombinant fusion protein (DB fusion) was created by joining the coding sequences of IpaD and IpaB. The DB fusion is coexpressed with IpaB's cognate chaperone, IpgC, for proper recombinant expression. The chaperone can then be removed by using the mild detergents octyl oligooxyethelene (OPOE) or N,N-dimethyldodecylamine N-oxide (LDAO). The DB fusion in OPOE or LDAO was used for biophysical characterization and subsequent construction of an empirical phase diagram (EPD). The EPD showed that the DB fusion in OPOE is most stable at neutral pH below 55°C. In contrast, the DB fusion in LDAO exhibited remarkable thermal plasticity, since this detergent prevents the loss of secondary and tertiary structures after thermal unfolding at 90°C, as well as preventing thermally induced aggregation. Moreover, the DB fusion in LDAO induced higher interleukin-17 secretion and provided a higher protective efficacy in a mouse challenge model than did the DB fusion in OPOE. These data indicate that LDAO might introduce plasticity to the protein, promoting thermal resilience and enhanced protective efficacy, which may be important in its use as a subunit vaccine.

Biophysical Characterization of the Type III Secretion System Translocator Proteins and the Translocator Proteins Attached to Bacterium-Like Particles

“…S1A, S2A, and S3A). Thus, as seen previously for a fusion protein consisting of Shigella IpaD (tip protein) and IpaB (called DB fusion or DBF), the presence of LDAO seemed to have a stabilizing effect at low pH for the translocator proteins.…”

“…The translocator proteins IpaB, SipB, and YopB were coexpressed with their chaperones that were removed with the detergent LDAO . The biophysical characterization of these proteins was conducted in the presence of LDAO to maintain each protein's solubility.…”

Chlamydial Type III Secretion System Needle Protein Induces Protective Immunity againstChlamydia muridarumIntravaginal Infection

“…Some T3SS proteins are surface-exposed and can be targeted by neutralizing antibodies. The T cell response to T3SS antigens was recently shown to be associated with protection against C. trachomatis infection in highly exposed women [13], and T3SS components have recently attracted attention as vaccine candidates against other pathogenic bacteria [14–17]. The C. trachomatis T3SS filament protein, CdsF, and its orthologs in other bacteria form the needles of injectisomes and are believed to facilitate the insertion of translocators into the host cell membrane [11, 12, 18].…”

Detergent Isolation Stabilizes and Activates the Shigella Type III Secretion System Translocator Protein IpaC

“…Furthermore, LDAO is an inexpensive, generally regarded as safe (GRAS) detergent that is effective at low levels and has been included in the successful formulation of Shigella vaccines containing a novel IpaD/IpaB fusion complex already. 22,28 Determining the impact of a vaccine a priori is difficult, though the findings presented here together with previous vaccine studies involving Shigella T3SA tip proteins and the detergent LDAO suggest that a vaccine including IpaC-LDAO is not only feasible, but may provide a valuable addition to the currently available antigens used in anti- Shigella vaccine formulation.…”

“…22–27 The Picking lab at the University of Kansas has recently characterized both IpaD and IpaB as pan- Shigella vaccine candidates finding that each provided significant protection when tested using a murine pulmonary infection model, 23 leading to the development of a novel IpaB-IpaD fusion complex that resulted in similar levels of protection and enhanced cytokine responses. 22,28 While the tip proteins IpaD and IpaB are proving to be valuable vaccine components, pioneering Shigella immunogenicity studies showed that IpaC generates a strong immunogenic response during Shigella infections, 8 driving the development of promising multicomponent Shigella vaccines containing IpaC 26,27,29 as well as preliminary studies using urea-isolated IpaC. 25 Despite these advances, however, challenges obtaining and stabilizing recombinant IpaC have thus far prevented necessary vaccine formulation and full characterization of its efficacy.…”