Development of a Self-Adjuvanting, Cross-Protective, Stable Intranasal Recombinant Vaccine for Shigellosis

Abstract

With the acquirement of antibiotic resistance, Shigella has resulted in multiple epidemics of shigellosis, an infectious diarrheal disease, causing thousands of deaths per year. Unfortunately, there are no licensed vaccines, primarily due to low or serotype-specific immunogenicity. Thus, conserved subunit vaccines utilizing recombinant invasion plasmid antigens (Ipa) have been explored as cross-protective vaccine candidates. However, achieving cross-protection against Shigella dysenteriae 1, which caused multiple pandemics/epidemics in the recent past, has been difficult. Therefore, a rational approach to improve crossprotection in the preparation for a possible pandemic should involve conserved proteins from S. dysenteriae 1 (Sd1). IpaC is one such conserved immunogenic protein that is less explored as an independent vaccine due to its instability/aggregation. Therefore, to improve cross-protection and potential immunogenicity and to be prepared for a future epidemic/pandemic, herein, we stabilized recombinant Sd1 IpaC, expressed without its chaperone, using a previously reported stabilizing detergent (LDAO) in a modified protocol and assessed its vaccine potential without an adjuvant. The protein assembled into heterogeneous complex spherical structures in the presence of LDAO and showed improved stability at storage temperatures of −80, −20, 4, 25, and 37 °C while providing enhanced yield and concentration. The protein could also be stably lyophilized and reconstituted, increasing the convenience of transportation and storage. Upon intranasal administration in BALB/c mice, the stabilized-IpaC-immunized groups generated significant antibody response and were not only protected against a high intraperitoneal dose of homologous S. dysenteriae 1 but also showed 100% survival against heterologous Shigella flexneri 2a without an adjuvant, while the control animals showed visible diarrhea (bloody-Sd1 challenge), lethargy, and weight loss with 0% survival. Overall, this work demonstrates that stabilized IpaC can be explored as a minimalist, self-adjuvanting, cross-protective, intranasal, single-antigen Shigella vaccine.

Stable Recombinant Invasion Plasmid Antigen C (IpaC)-Based Single Dose Nanovaccine for Shigellosis

“…22,38−42 In a previous study, we demonstrated the self-adjuvanting protective efficacy of stabilized recombinant IpaC of Shigella dysenteriae 1 origin, which showed 100% survival against a high dose of heterologous Shigella flexneri 2a in BALB/c mice, in three intranasal doses. 43 In the current work, to further reduce the number of doses and increase patient compliance, the minimum protective dose of stabilized recombinant IpaC protein, determined in the earlier study, was encapsulated in biodegradable polymeric PLGA NPs, which was referred to as a single-dose nanovaccine. Because the gradual degradation of NPs is known to facilitate prolonged antigen presentation to immune cells, it is expected to raise a robust immune response.…”

“…17,47 ■ MATERIALS AND METHODS Bacterial Strains. As described in our previous report, 43 Escherichia coli DH5α and BL21 (DE3) Rosetta cells were procured from Microbial Type Culture Collection (MTCC), Chandigarh, India. Shigella strains, S. flexneri 2a (B294) (Sf2a) and S. dysenteriae serotype 1 (NT407) (Sd1), were obtained from the National Institute of Cholera and Enteric Diseases (NICED), Kolkata, India.…”

“…The cloning, expression, purification, and characterization of Sd1 IpaC were performed as described in our earlier report. 43 Briefly, S. dysenteriae 1 ipaC nucleotide sequence (1092 bp) was retrieved from National Center for Biotechnology Information (reference sequence: NC_007607.1) and, at each terminal, restriction sites were incorporated and synthesized by GenScript (USA). The polymerase chain reaction-amplified gene was cloned into the pET28a vector and transformed into E. coli DH5α for cloned plasmid selection which was finally transformed into E. coli BL21 (DE3) Rosetta cells for protein expression (plasmid mini-preparation kit, Qiagen, Germany).…”

“…IpaC was stabilized and purified as previously reported. 43 Briefly, lauryldimethylamine N-oxide (LDAO), a mild zwitterionic detergent, previously reported to be beneficial for the storage of purified IpaC protein after removal of the chaperone 48 was used at various stages of purification (3 different processes) of Sd1 IpaC expressed without the chaperone to obtain stable IpaC, which could be stored at a range of temperature. The obtained stable, affinity chromatography and size exclusion chromatography purified IpaC in Tris buffer (50 mM Tris−HCl, 0.5 M NaCl, 10 mM imidazole, 10% glycerol, pH�8.3) was resolved using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).…”

“…For quantification of generated antibody response, enzyme-linked immunosorbent assay (ELISA) was performed as described earlier. 43 Briefly, the Sd1 culture was centrifuged, and the resuspended cell pellet was sonicated (10 min Percentage Survival against Shigella Challenge. The protective efficacy of the single-dose nanovaccine against an intraperitoneal injection of a high dose of 1 × 10 9 CFU (100 μL volume) of heterologous virulent Sf2a/mouse was studied on the 43rd and 56th day of immunization.…”