Development of a Novel Shigella Quadrivalent Conjugate Vaccine Using Shigella O-Polysaccharide and IpaB Carrier Protein

TL;DR

A new Shigella vaccine candidate was developed and shown to trigger strong immune responses in rabbits.

Contribution

A quadrivalent Shigella conjugate vaccine using O-polysaccharides and IpaB protein was developed and tested for immunogenicity.

Findings

The vaccine significantly increased IgG concentrations for all four Shigella serotypes.

Serum bactericidal activity increased up to 224-fold for S. sonnei after vaccination.

The vaccine induced robust serotype-specific immune responses in preclinical models.

Abstract

Background/Objectives: Shigella is the leading bacterial cause of diarrheal disease worldwide. Although multiple vaccine candidates are under development and in clinical trials, no Shigella vaccine is currently available on the market. Shigella comprises four species: S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. S. flexneri has been recognized as the most prevalent species, particularly in low- and middle-income countries (LMICs), and the top serotypes are S. flexneri 2a, 3a and 6. Conversely, S. sonnei has a single serotype and predominates in high-income countries (HICs). Invasion plasmid antigen B (IpaB) is a critical virulence factor of Shigella type III secretion system (T3SS) that is highly conserved across Shigella serotypes. Here, we report the development of a Shigella quadrivalent O-polysaccharide-IpaB conjugate vaccine candidate (IVT Shigella-04). Methods: IVT Shigella-04 contains O-polysaccharides (O-PS) from S. flexneri 2a, 3a, 6, and S. sonnei, each individually conjugated to recombinantly expressed IpaB as the carrier protein using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) chemistry. The immunogenicity of IVT Shigella-04 was evaluated in a rabbit immunization model. Results: Baseline (day 0) IgG concentrations were low for all four Shigella serotypes (<0.5 µg/mL). Following two doses on day 0 and day 28 (2.5 µg of each conjugate per dose; total 10 µg), IgG geometric mean concentrations increased significantly (p < 0.001) by day 42, reaching 67.96 µg/mL (2a), 91.56 µg/mL (3a), 371.31 µg/mL (6), and 11.00 µg/mL (sonnei). Consistently, serum bactericidal activity (SBA) at day 42 increased 13-fold (2a), 34-fold (3a), 63-fold (6), and 224-fold (sonnei) relative to baseline (day 0). Conclusions: IVT Shigella-04 elicited robust serotype-specific humoral and functional immune responses in preclinical models, supporting its further development toward clinical evaluation.

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1. Introduction

Shigella stands as the leading bacterial cause of global diarrheal disease [1,2,3]. Although multiple Shigella vaccine candidates are currently being evaluated at various stages of preclinical and clinical development [4,5,6], no licensed vaccine is yet available for the prevention of Shigella-induced diarrhea. Every year, there are approximately 100 million reported cases and 164,000 annual deaths attributed to shigellosis globally, with children below the age of 5 in low- and middle-income countries (LMICs) being significantly affected [7,8]. In LMIC pediatric populations, early and repeated exposure, malnutrition, co-infections, and limited access to timely care contribute to a broad spectrum of clinical manifestations, ranging from mild diarrhea to severe dysentery [7,9,10,11]. Due to limited diagnostic capabilities for identifying Shigella infection in developing countries, antibiotics are prescribed only to children with dysentery [12]. While Shigella infection is frequently linked to dysentery in studies, it is common for children with Shigella not to exhibit dysentery symptoms. As a result, the majority of children infected with Shigella in LMICs do not receive antibiotic treatment [12]. Furthermore, antibiotic resistance of Shigella is on the rise at a rapid rate [13]. The antibiotic treatment approaches for shigellosis can vary due to both region-specific antimicrobial resistance (AMR) patterns and species-associated differences in disease severity and resistance profiles [14,15,16]. Hence, there is an urgent need for the development of a vaccine against Shigella infections. The Shigella genus contains four species, namely S. flexneri, S. boydii, S. dysenteriae, and S. sonnei, with over 50 serotypes distinguished by lipopolysaccharide (LPS) O-antigen specificity [17,18]. S. flexneri contains over 15 serotypes and is the most prevalent species in LMICs, whereas S. sonnei is the dominant cause of shigellosis in high-income countries (HICs) [19]. LPS resides on the surface of most Gram-negative bacteria, and it contains three major components: O-polysaccharides (O-PS), also known as O-antigen, lipid A, and core fraction [20,21]. Shigella O-PS consists of repetitive sequences of three to six monosaccharide residues, which determines the serotype specificity [20,22]. The serotyping analysis of Global Enteric Multicenter Study (GEMS) of Shigella isolates revealed that a quadrivalent vaccine, covering S. sonnei, S. flexneri 2a, S. flexneri 3a, and S. flexneri 6, has the potential to confer protection exceeding 80% against Shigella infections [20,23].

Historically, glycoconjugate vaccines have achieved success against Haemophilus influenzae type b, meningococcus, pneumococcus, and Salmonella typhi [24,25]. The conjugation of polysaccharides to a carrier protein activates the T-cell-dependent immune pathway and drastically enhances the immunogenicity [26,27]. The O-specific antigen (O-PS) has been identified as a pivotal component influencing immunogenicity and plays a role in conferring protective immune responses against Shigella infections [28,29,30]. All these findings provide the key foundation for taking the approach of using a glycoconjugate vaccine. Regarding the selection of carrier proteins, Pseudomonas aeruginosa exoprotein A (rEPA), cross-reacting material (CRM_197_) and tetanus toxoid (TT) have been mostly employed for conjugation with the polysaccharide [31,32,33] based on their established efficacy and safety profiles in clinical trials and post-licensure studies. However, these carrier proteins have been extensively employed in other existing glycoconjugate vaccines, which may result in a repression of the immune response [34]. In the exploration of alternative carrier proteins, recent Controlled Human Infection Model (CHIM) studies on Shigella have indicated that Invasion plasmid antigen B (IpaB) protein elicited significant IgG and IgA responses [35]. IpaB is a critical component of the Shigella type III secretion system (T3SS), which has been recognized as the key virulence factor [36]. The T3SS apparatus (T3SA) comprises an envelope-spanning basal body and an external needle topped by a tip complex protein called IpaD [36,37]. This nanomachine is used to deliver effector proteins into host cells to promote pathogen entry. IpaB plays a key role as the translocator protein in T3SS needle tip complex [38]. IpaB has been explored as a key antigen in other Shigella vaccine platforms, highlighting its broad applicability in vaccine development. The Walter Reed Army Institute of Research (WRAIR) has developed the Invaplex technology that supplies Ipa proteins (IpaB, IpaC and IpaD) to Shigella O-antigen. The phase 1 clinical study results revealed a positive safety profile for the candidate [39]. More recently, Vaxcyte Inc. reported the development of a Shigella vaccine candidate conjugating S. flexneri 2a and S. sonnei O-PS to in vitro synthesized IpaB that contains non-native amino acids (nnAA), in which IpaB was further demonstrated to confer cross-protective immune responses [40]. Collectively, these development efforts further support the use of IpaB as an alternative carrier protein and a protective antigen in Shigella vaccine development. Building on these advances, the present study employs IpaB recombinantly expressed in E. coli rather than in vitro synthesis, enabling a scalable and manufacturing-efficient production platform [41,42]. Moreover, IpaB is not used solely as a standalone antigen; instead, it is covalently conjugated to Shigella O-polysaccharides, thereby serving a dual role as both the carrier protein and a Shigella antigen. This design avoids reliance on traditional carrier proteins (e.g., CRM_197_ or TT) that have been associated with carrier-induced immune suppression [34].

Here, we present the development of a novel Shigella quadrivalent conjugate vaccine covering S. flexneri 2a, 3a, 6, and S. sonnei. The purified O-polysaccharides were conjugated to IpaB as a carrier protein, which was recombinantly expressed in E. coli. The conjugation process was carried out using cyanylation chemistry involving CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate). The vaccine candidate successfully completed a GLP-compliant toxicology (GLP-TOX) study and showed no test article-related toxicities. In addition, immunogenicity studies demonstrated that conjugates covering all four serotypes elicited robust immune responses in rabbits. The program has now advanced into the clinical stage and has entered a Phase 1 clinical trial (NCT07205926). In summary, the current work strongly supports the potential of this quadrivalent conjugate as a promising Shigella vaccine candidate.

2. Materials and Methods

2.1. Production of Shigella O-PS

All Shigella strains (serotypes S. flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei) were acquired from the Center for Vaccine Development and Global Health, University of Maryland, Baltimore (UMB), MD, USA. Fermentation was initiated in a baffled Erlenmeyer flask using Terrific Broth (TB, Teknova, Hollister, CA, USA) with trace elements at pH 7.2 in a 37 °C shaking incubator. Once the culture reached an OD_600_ (optical density) > 1.0, it was transferred to 9 L of TB media in a 14 L Eppendorf fermenter, maintaining a temperature of 37 °C, pH 7.2 ± 0.2, and 40% dissolved oxygen. The fermenter was operated with a 1 L dextrose feed starting 3 h post-inoculation until growth slows at 8 h. The culture was inactivated by addition of formaldehyde to a final concentration of 1.0% (v/v) followed by incubation at 37 °C and subsequent cooling to 10 °C prior to harvest. The fermentation broth was then centrifuged at ~10,000× g at 4 °C for ~30 min to collect the cell pellet. The harvested cell pellet was resuspended in aqueous solution, and the pH was adjusted to 3.0 using acetic acid. The suspension was then incubated at 98 °C for 2 h in a water bath with periodic manual mixing to lyse the cells. Cell debris and precipitates were removed by two rounds of centrifugation at 10,000× g for 30 min each. The supernatant of hydrolyzed material was concentrated and diafiltered with 20 diafiltration volumes (DVs) of 0.5 M NaCl and MilliQ water using a tangential flow filtration (TFF) system (30 kDa, Repligen, Waltham, MA, USA). The buffer composition of the O-PS was then adjusted to 4.5 M NaCl and 50 mM sodium phosphate, pH 6.8. The material was subsequently loaded onto a hydrophobic interaction chromatography (HIC) membrane (Sartobind^®^ Phenyl, 400 mL, Sartorius, Göttingen, Germany) and the polysaccharide was collected in the flow-through fraction, followed by a TFF diafiltration against 20 DVs MilliQ water. Purified O-PS was filtered using a 0.22 μm filter (Thermo Fisher Scientific, Waltham, MA, USA) and stored at either 2–8 °C if proceeding to next step, or at ≤−60 °C for long-term storage. Concentration of O-PS was determined by anthrone assay [43,44]. Residual protein content was quantified using the Lowry assay [45], nucleic acid impurities were assessed by UV absorbance at 260 nm [46], and endotoxin levels were measured using a chromogenic Limulus amebocyte lysate (LAL) assay [47].

2.2. Production of Recombinant IpaB Protein

The IpaB expression vector was kindly provided by Dr. Wendy Picking during her tenure at the University of Oklahoma. The expression vector construct employs pACYCDuet-1 (MilliporeSigma, Burlington, MA, USA) plasmid as the backbone, and co-expresses IpaB and IpgC. The IpaB coding gene from S. flexneri 2a (580 amino acids, GenBank Accession No.: SVH88885) was employed for construction of the expression vector. A 6x His-tag was cloned to the N-terminus of IpgC for purification of IpaB/IpgC complex. The newly constructed plasmid sequence was verified by Eurofins Genomics (Louisville, KY, USA). For fermentation, 1 mL IpaB-expressing E. coli cells were inoculated to 150 mL LB (Luria–Bertani) media and incubated at 37 °C, 250 RPM. OD_600_ was monitored until it exceeded 3.0 within 4–10 h. The flask inoculum was then inoculated into a 10 L fermentation vessel containing 6 L in-house-prepared fermentation medium (composition provided in Supplementary Table S1) for continued growth. Dissolved oxygen was maintained at 25% with controlled agitation and air/oxygen supply. The temperature was reduced to 21.5 °C at an OD_600_ of 15. Isopropyl β-D-1-thiogalactopyranoside (IPTG, MilliporeSigma, Burlington, MA, USA) was added at 1 mM to induce protein expression. The culture was maintained under fed-batch conditions with fermentation medium supplemented with glycerol as the feed medium. At an OD_600_ > 120, the culture was terminated and centrifuged at approximately 10,000× g at 4 °C for ~30 min to harvest the cell paste. Approximately 2 kg cell paste was subsequently resuspended in 5 L 20 mM Tris, 500 mM NaCl, 5 mM Imidazole (MilliporeSigma, Burlington, MA, USA), 0.5 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, MilliporeSigma, Burlington, MA, USA), pH 7.9 and homogenized (GEA Niro Soavi homogenizer 2000, Düsseldorf, Germany) at 16,000–17,000 psi with double passes. The clarified lysate was centrifuged at 16,000× g for 1.5 h to remove debris, and approximately 4.5 L supernatant was filtered and loaded onto a 1 L Ni-NTA column (HisPur™ Ni-NTA Resin, Thermo Fisher Scientific, Waltham, MA, USA) with a flowrate of 80 mL/min. The column was washed with 20 mM Tris, 500 mM NaCl, 20 mM Imidazole, pH 7.9, and IpaB/IpgC complex was eluted with 20 mM Tris, 500 mM NaCl, 250 mM Imidazole, pH 7.9. The eluted proteins were then loaded onto a 1 L cation exchange column (Tosoh Sulfate-650F, Toyopearl Bioscience, Tokyo, Japan) and processed at a flowrate of 120 mL/min. The column was washed with 20 mM Tris, 100 mM NaCl, pH 6.8 and eluted with 20 mM Tris, 500 mM NaCl, pH 6.8. IpaB/IpgC complex was incubated with 0.2% Lauryldimethylamine oxide (LDAO, MilliporeSigma, Burlington, MA, USA) for 48 h at 4 °C. The protein was loaded onto a second HisPur Ni-NTA affinity chromatography (1 L column with 80 mL/min flowrate). IpaB was collected in the flow-through and subsequently subjected to TFF diafiltration using 10 DVs of 20 mM Tris, 500 mM NaCl, and 0.2% LDAO, employing a 30 kDa TFF system (Repligen, Waltham, MA, USA). The buffer exchanged IpaB was filtered using a 0.22 μm filter (Thermo Fisher Scientific, Waltham, MA, USA) and stored at either 2–8 °C if proceeding to next step, or at ≤−60 °C for long-term storage. Concentration of purified IpaB was determined by bicinchoninic acid (BCA) assay [48].

2.3. SDS-PAGE and Western Blot Analysis

IpaB samples were boiled for 5 min at 95 °C. The SDS-PAGE gel (SurePage, Bis-Tris, 10 × 8, 4–20%, GenScript, Piscataway, NJ, USA) was set up, filled with running buffers, and samples were loaded. The gel was run at 140 V (PowerPac Basic, BioRad, Hercules, CA, USA) for 1 h. The gel was rinsed three times with Milli-Q water for 5 min each, incubated in Coomassie Safe Stain for 60 min with gentle shaking, and subsequently rinsed with Milli-Q water for 1–3 h and overnight. For Western blotting, the gel was transferred onto a nitrocellulose membrane using the Trans-Blot Turbo Transfer System (BioRad, Hercules, CA, USA) and PowerPac Basic power supply (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% non-fat dried milk in 1X PBS and then incubated with a primary anti-IpaB antibody (CUSABIO Biotechnology LLC, Houston, TX, USA) for 1 h, followed by an anti-mouse IgG Fc HRP conjugate secondary antibody. The membrane was washed with PBS-T (PBS plus Tween-20) and developed using TMB membrane substrate (Bio-Rad, Hercules, CA, USA).

2.4. Size Exclusion High-Performance Liquid Chromatography (SEC-HPLC)

Samples were analyzed using an SEC-HPLC system (e2695, Waters Alliance, Waters Corp., Milford, MA, USA) equipped with UV (2489 UV/Vis Detector) and RI (2414 RI Detector) detectors. The mobile phase consisted of 10 mM potassium phosphate buffer, pH 7.0. The system was set up with OHpak SB-804 HQ and SB-805 HQ columns (Shodex^®^, Showa Denko K.K., Tokyo, Japan) in series, with a flow rate of 1.0 mL/min and both column and sample temperatures maintained at ambient conditions (23–25 °C). Injection volumes ranged from 30–100 µL, with a total run time of 30 min. Sample run, data processing and molecular weight analysis were carried out using Empower 3.0 software (Waters Corp., Milford, MA, USA).

2.5. Nuclear Magnetic Resonance (NMR)

^1^H NMR was conducted to elucidate the identity and molecular structure of purified O-PS. The analysis was outsourced to an NMR facility at Washington State University (WSU, Pullman, WA, USA). Samples were prepared by dissolving the lyophilized O-PS powder in D_2_O. The resuspended materials were analyzed by a ^1^H NMR spectrometer with following critical parameters: spectrometer frequency of 500.13 MHz, temperature of 318.0 K, a total of 256 scans, and a pulse width of 12 µs. Signature proton resonances were identified based on characteristic chemical shift regions, including anomeric protons (H-1, ~4.8–5.5 ppm), O-acetyl methyl protons (~2.0–2.2 ppm) arising from O-acetyl substitutions on rhamnose residues, and N-acetyl methyl protons (~2.0 ppm) corresponding to N-acetylated amino sugars (e.g., GlcNAc, GalNAc, or FucNAc4N) present in the O-PS repeating units. H6 methylene protons (~1.2–1.8 ppm) were assigned to rhamnose residues in S. flexneri serotypes or to the FucNAc4N residue in S. sonnei. Peak assignments were made by comparison with previously reported reference spectra for the corresponding Shigella serotypes, and the presence of expected serotype-specific signals was used to confirm O-polysaccharide identity.

2.6. Conjugation

O-PS corresponding to the four serotypes in IVT-Shigella-04 and IpaB were concentrated using TFF (30 kDa, Repligen, Waltham, MA, USA). CDAP (SelectLab, Münster, Germany) dissolved in acetonitrile (MilliporeSigma, Burlington, MA, USA) was added to O-PS (1.0–1.8 mg CDAP/mg O-PS) for activation. IpaB was then added to the activated O-PS under agitation (150 ± 50 rpm) at 18–23 °C. Serotype-specific target concentrations of O-PS and IpaB, CDAP activation pH and duration, and PS: protein input ratios were summarized in Table 1. Based on the conjugate molar mass profile (using SEC-HPLC) from in-process testing, the conjugation reaction was quenched by adding 2 M glycine (pH 7.5 ± 0.5) at 18–23 °C until the target pH 7.0–8.0 was achieved. Conjugates were concentrated using a 300 kDa MWCO cassette to approximately 400–500 mL as one diafiltration volume (DV), followed by a buffer exchange against 5–15 DVs of two diafiltration buffers. (Buffer 1: 20 mM PB, 0.05% LDAO, 500 mM NaCl, pH 7.5 ± 0.5; Buffer 2: 20 mM PB, 0.05% LDAO, 150 mM NaCl, pH 7.5 ± 0.5). Removal of free PS and protein was monitored by SEC-HPLC. Final conjugate products were 0.22 µm filtered and stored at 2–8 °C. Free polysaccharide content was determined using an aluminum phosphate (Adju-Phos^®^, Croda, Søborg, Denmark) adsorption method. Briefly, conjugate samples were incubated with an excess of Adju-Phos^®^ (750 µg Adjuvant per 100 µg IpaB presenting in the conjugate) at pH 4.8 to adsorb polysaccharide–protein conjugates, followed by centrifugation at 10,000× g for 10 min at room temperature to pellet adjuvant-bound material. Unadsorbed (free) polysaccharide in the supernatant was quantified using the anthrone assay. The polysaccharide-to-protein (PS: protein) ratio was determined by measuring total polysaccharide content using the anthrone assay [43,44] and total protein content using the bicinchoninic acid (BCA) assay [48]. The PS: protein ratio was calculated directly from the measured concentrations.

2.7. Animal Study

The rabbit study was conducted by Cocalico Biologicals, Inc. (Denver, PA, USA) using female New Zealand white rabbits (species: Oryctolagus cuniculus) due to their well-characterized immune system and robust antibody responses to conjugate vaccines. Inclusion criteria for rabbits were an average age of 12–14 weeks and average body weight of 2.0–3.5 kg at the time of dosing. The vaccination was conducted intramuscularly (i.m) on day 1 (first dosing) and day 28 (second dosing). The test article was prepared by formulating all four monovalent conjugates with 0.05% LDAO, 2 mM potassium phosphate, 150 mM sodium chloride, pH 5.9–6.4. Aluminum phosphate adjuvant (AdjuPhos^®^, Croda, Søborg, Denmark) containing 250 µg/mL aluminum was added per group design. The regimen design has been revealed in Table 2. Briefly, three formulation groups are included in the study, targeting dose ranging, adjuvant effect, and immunogenicity analysis. Individual serum samples were collected day 0 (Pre-dosing), day 28, and day 42 (euthanasia) for IgG and SBA analysis. Each group consisted of 12 rabbits (36 animals in total), and the group size was based on prior similar immunogenicity studies. The animals were randomly assigned the next available consecutive number and allocated to the specific group in numerical order. Animal housing, including cage location, and all study procedures were conducted in accordance with the standard operating procedures of the Cocalico facility, and no significant confounding factors were identified. Blinding was not performed for this study. Animal husbandry: Study animals were housed individually in wire cages and acclimated for at least 7 days prior to study initiation. Environmental enrichment included PVC pipes, nylon bones, or wood blocks, visual, olfactory, and auditory contact with conspecifics, routine grooming by animal care staff, and background radio noise to minimize stress.

2.8. IgG Antibody Analysis and Serum Bactericidal Activity (SBA)

The immune sera were tested at Inventprise using a Multiplexed Bead-based Immunoassay (MBIA) to quantify IgG antibody concentrations to each serotype of O-PS using an in-house qualified pooled rabbit polyclonal standard reference serum. As no universally accepted reference standard exists for assessing Shigella-specific immunogenicity in animal or human studies, an internal Shigella Standard Rabbit Serum (IVT-Shig-SRS-664) was developed for use in Inventprise immunogenicity assays. IVT-Shig-SRS-664 was generated from serum collected at day 42 following administration of an IVT Shigella-04 vaccine formulation in a preclinical rabbit study, which was clarified, aliquoted, and stored at −80 °C until use. To characterize serotype-specific IgG content in the reference serum, antigen-specific polyclonal antibodies were generated through a 70-day immunization protocol, purified, and used as quantification reagents in a multiplex bead-based immunoassay. The MBIA assay platform uses a set of specific Shigella O-PS-conjugated fluorescent beads where each bead falls into a unique region in the fluorescent spectrum to capture antibody targeted to the analyte of interest. SBA was conducted at Sunfire Biotechnology LLC (Birmingham, AL, USA) using an internally standardized protocol. Data were recorded as killing indexes (KIs), defined as the reciprocal of the interpolated serum dilution that results in the killing of 50% of the target bacteria. Samples that did not achieve at least 50% bacterial killing at the lowest serum dilution tested (1:8) were reported as KI < 8.

2.9. Statistical Analysis

IgG and SBA data were calculated and plotted using GraphPad Prism 10. The geometric mean concentration (GMC) or geometric mean titer (GMT) and 95% confidence interval (CI) were calculated for each respective group from the individual day 0, day 28, and day 42 serum samples. Within-group comparisons were performed using the Wilcoxon matched-pairs signed-rank test, and between-group comparisons were performed using the Mann–Whitney U test (n = 12). Statistical significance: p < 0.01 (), p < 0.001 (*).

3. Results

3.1. Characterization of Shigella O-Polysaccharides

The production of Shigella O-PS involves controlled acid hydrolysis to selectively cleave the linkage between 2-keto-3-deoxyoctonic acid (KDO) and lipid A to release O-antigen from the LPS [49]. The general analytical characteristics for all four serotypes of purified O-PS were summarized in Table 3. The average molecular weight of purified O-PS ranges from 50 to 200 kDa estimated by SEC-HPLC (Table 3), which is consistent with polysaccharide chain lengths suitable for conjugation. The concentration of residual proteins, nucleic acids, and endotoxins has been diminished to less than 3%, 1%, and 1 EU/µg PS, respectively, meeting the typical quality requirements of purified O-PS to be applied for conjugation (Table 3). Structural identity and integrity of the purified O-PS were verified by ^1^H NMR. The spectra confirmed the serotype-specific O-PS repeating unit structures and preserved key functional groups, including N-acetylation and O-acetylation signals observed at approximately 2.0–2.2 ppm for each serotype (Figure 1). All acquired NMR spectra were fully consistent with reported reference profiles [17,50,51,52]. These data confirmed that the O-PS purification process effectively removed impurities and preserved the native serotype-specific carbohydrate epitopes.

3.2. Characterization of Recombinant IpaB

Recombinant expression of IpaB in E. coli requires the presence of its cognate chaperone IpgC, which enhances the solubility of IpaB and keeps the protein in a non-toxic state to E. coli cells [53,54]. Furthermore, a zwitterionic surfactant, Lauryldimethylamine N-oxide (LDAO), is required to cleave IpaB from IpgC and maintain IpaB in a monomeric form [55,56]. Analytical characterization indicated efficient production of monomeric IpaB. SDS-PAGE analysis showed a dominant band with minimal impurities (Figure 2A), and SEC-HPLC also confirmed that greater than 90% of the purified protein was present in a monomeric form (Figure 2C). The identity of the purified IpaB was further verified by Western blot using an IpaB-specific antibody (Figure 2B), confirming the integrity of the recombinant protein prior to conjugation.

3.3. Conjugation of Shigella O-PS and IpaB

Shigella O-PS-IpaB conjugation was conducted using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate), a well-established PS activation strategy that has been extensively employed in the development and commercial production of glycoconjugate vaccines. Shigella O-polysaccharides (O-PS) were conjugated to the carrier protein IpaB via CDAP-mediated activation, in which activated O-PS cyano-ester intermediates were covalently coupled to amine groups on the protein, generating stable O-PS–IpaB conjugates [57]. The molecular weight of monovalent conjugate was characterized by SEC-HPLC. Figure 3 presents overlaid HPLC chromatograms of the O-polysaccharides (O-PS), carrier protein IpaB, and the corresponding O-PS–IpaB conjugates. All four serotypes of O-PS–IpaB conjugates exhibited a substantial shift toward higher molecular weight species, with average molecular weights exceeding 800 kDa, representing a dramatic increase compared with the unconjugated O-PS and carrier protein IpaB (Figure 3 and Table 4). The ratio O-PS to IpaB is provided in Table 4. In addition, free (unconjugated) polysaccharide levels were below 10% for all four conjugates (Table 4).

3.4. Immunogenicity Study: IgG Analysis

IgG antibody concentrations were measured for all sera samples using Multiplexed Bead-based ImmunoAssay (MBIA). The concentrations are expressed as geometric mean concentrations (GMC) ± 95% confidence intervals (CI). The IgG concentrations are detailed in Table 5, and IgG concentration trends across all groups are plotted in Figure 4. Compared to the pre-dosing baseline (day 0), all three formulation groups demonstrated a significant increase (p < 0.001 except G3 of S. flexneri 3a, p < 0.01) in IgG concentrations on day 28 for each serotype (Figure 4). In the 2.5 µg dose group with adjuvant (G1), baseline IgG concentrations were uniformly low across all serotypes on day 0, with values of 0.03 µg/mL for 2a, 0.21 µg/mL for 3a, 0.06 µg/mL for 6, and 0.06 µg/mL for S. sonnei (Table 5). Following the first immunization, IgG concentrations increased markedly (p < 0.001) by day 28 to 32.85 µg/mL (2a), 61.09 µg/mL (3a), 159.71 µg/mL (6), and 1.90 µg/mL (sonnei), which indicated a strong immunogenic response following the first immunization. After the second dose, further increases were observed on day 42, reaching 67.96 µg/mL (2a), 91.56 µg/mL (3a), 371.31 µg/mL (6), and 11.00 µg/mL (sonnei), demonstrating a drastic booster response particularly for S. flexneri 6 and S. sonnei (p < 0.001). Conversely, S. flexneri 2a and 3a exhibited a statistically significant additional increase (p < 0.01) on day 42, although less substantial than S. flexneri 6 and S. sonnei. The dose–effect comparison between the 2.5 µg and 7.5 µg dose groups (G1 vs. G2) did not show substantial differences in IgG concentrations. Compared with the adjuvanted 2.5 µg group (G1), the unadjuvanted Group 3 showed markedly lower IgG responses at both day 28 and day 42 particularly for the S. flexneri 2a, 3a, and 6 (p < 0.05), indicating the stimulating role of adjuvant in enhancing humoral immunity (Figure 4 and Table 5).

3.5. Functional Antibody Responses by Serum Bactericidal Activity (SBA)

In addition to the IgG antibody test, SBA was conducted for all serum samples. The average SBA titers and their fold increases on day 28 and 42 relative to pre-immunization levels (normalized) are shown in Table 6 and Figure 5. Overall, the SBA titer profile strongly aligned with the IgG antibody analysis (Figure 4 and Figure 5). In the 2.5 µg adjuvanted group (G1), SBA titers (day 28 vs. day 0) increased by 18-, 54-, 30-, and 104-fold for S. flexneri 2a, 3a, 6, and S. sonnei, respectively, indicating a robust bactericidal activity of sera following the first immunization (p < 0.001). A notable booster effect (day 42 vs. day 28, p < 0.01) after the second immunization was observed for S. flexneri 6 and S. sonnei (Figure 5C,D). Conversely, S. flexneri 2a and 3a exhibited no statistically significant booster effect on day 42 relative to day 28, indicating a plateaued functional response in this model (Figure 5A,B). Similar to the observations in IgG data, the comparison between the 2.5 µg and 7.5 µg dose groups (G1 vs. G2) did not reveal substantial dose-dependent responses. Interestingly, the SBA data indicate a stronger immune response for S. sonnei versus S. flexneri serotypes on both day 28 and day 42, which had been slightly less immunogenic in the IgG analysis (Figure 4). These data highlight the importance of including functional assays when assessing the immunogenicity of the vaccine candidate.

4. Discussion

According to the bacterial priority pathogens list given by World Health Organization (WHO), Shigella is among the global priority endemic pathogens for which vaccines are urgently needed due to its substantial disease burden and the growing antibiotic resistance [58,59]. Despite over a hundred years of vaccine development, no licensed Shigella vaccine is available. It remains a leading bacterial cause of childhood diarrheal deaths, compounded by poor diagnostics and limited antibiotic access in LMICs [18]. In this study, we report the development of a quadrivalent conjugate Shigella vaccine against S. flexneri 2a, 3a, 6, and S. sonnei, which aimed to target the most epidemiologically significant circulating serotypes [60].

IVT Shigella-04 vaccine represents a novel design by incorporating IpaB, a conserved and immunodominant protein component of the Shigella type III secretion system (T3SS) as the carrier protein [61]. Shigella uses needle-like type III secretion system (T3SS), including Ipa proteins (IpaA, IpaB, IpaC, and IpaD), to invade the intestinal epithelium. These effectors are encoded by a virulence plasmid common to all Shigella species [62]. It is reported that IpaB protein displays >98% sequence conservation among Shigella species, underscoring its promise as a cross-protective vaccine antigen [40,63]. In addition, IpaB has been demonstrated to be actively involved in bacterial invasion and pathogenesis and elicits strong IgG and IgA responses in human challenge models [64]. Because CRM_197_ and TT are broadly used in current conjugate vaccines, their reuse carries a risk of carrier-induced immune suppression [65,66,67]. In contrast, IpaB is not used in licensed infant conjugate vaccines, thereby minimizing the risk of bystander interference as additional conjugates are introduced into routine immunization schedules, and positioning IpaB as a favorable next-generation carrier for Shigella glycoconjugates. Taken together, by using IpaB as a dual-function carrier and antigen, IVT Shigella-04 was designed to provide a serotype-specific protection through anti-O-PS responses with a potential for broader cross-serotype protection through IpaB-specific immunity, which will be evaluated in future studies. This vaccine design construct will have advantages over existing Shigella O-antigen-based vaccine candidates. In the current study, IpaB was recombinantly co-expressed with its chaperone protein, IpgC, in E. coli cells, and analytical studies by SDS-PAGE and SEC-HPLC indicated that >90% of the protein was in the monomeric form (Figure 2A,C), making it suitable for subsequent conjugation.

O-specific polysaccharide domain of LPS is both a key virulence determinant and the principal protective antigen [68,69]. Traditional O-PS preparation involves hot phenol extraction [70,71], which induces significant challenges for manufacturing due to safety and environmental related risk [72]. In this study, we developed a phenol-free process for isolating Shigella O-PS to achieve high quality and yield. Among the flexneri serotypes, 2a and 3a share a conserved repeating unit composed of three α-L-rhamnose residues and one β-D-N-acetylglucosamine (GlcNAc), forming the characteristic backbone of many Shigella flexneri strains. In contrast, S. flexneri 6 features a repeating unit comprising two α-L-rhamnose residues, one β-D-galactose, and one β-D-N-acetylgalactosamine (GalNAc), making it structurally distinct from other flexneri serotypes. S. sonnei is further differentiated by its unique O-PS, which is a disaccharide of 2-acetamido-2-deoxy-L-altruronic acid (AltNAcA) and 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (FucNAc4N) but lacks rhamnose [73,74,75]. Furthermore, there are reports indicating that O-acetylation (OAc) of O-PS may play a crucial role in eliciting immunogenicity [73,76]. However, the specific impact of O-acetylation on immunogenicity in relation to serotypes remains unknown [73,76,77]. In the current study, ^1^H NMR data (Figure 1) demonstrate alignment with documented O-PS structure in the existing scientific literature and shows no substantial loss of O-Acetyl group [17,51,77,78]. The conjugation of Shigella O-PS to IpaB in this study was accomplished using CDAP cyanylation chemistry, a well-established and scalable approach for glycoconjugate vaccine manufacturing. It enables efficient activation of hydroxyl groups on polysaccharides under mild aqueous conditions, promoting controlled covalent linkage to amino groups on carrier proteins while preserving critical antigenic epitopes [57,79]. Notably, CDAP-based conjugation technology has been successfully implemented in multiple licensed human vaccines such as pneumococcal conjugate vaccine (PNEUMOSIL^®^) and quadrivalent meningococcal conjugate vaccines (MENACWY-TT) [80,81].

In immunogenicity studies, all four serotype-specific conjugates elicited robust (p < 0.001) IgG and SBA responses after the first dose (Figure 4 and Figure 5). No significant differences were observed between the 2.5 µg and 7.5 µg dose levels in IgG responses (Figure 4). In the SBA analysis, the higher dose (7.5 µg) across all S. flexneri serotypes even showed a trend toward reduced immunogenicity compared with the lower dose (Figure 5). This pattern is consistent with the frequently observed non-linear, inverted U-shaped (“Goldilocks”) dose–response relationship, in which intermediate antigen doses elicit optimal immune responses, whereas higher doses result in plateaued or diminished responses [82]. The underlying mechanisms of this observation remain incompletely understood. Previous studies suggested that increased antigen exposure may drive T-cells toward promoted terminal differentiation or functional exhaustion, and/or promote the induction of regulatory T-cells, thereby limiting effective immune expansion [83,84]. In the present vaccine candidate, IpaB functions as both the carrier protein and a Shigella antigen, potentially contributing additional antigenic exposure beyond the O-polysaccharide component. In addition, adjuvant-mediated immune stimulation might further contribute to the observed plateaued dose–response, as the non-adjuvanted groups generally exhibited lower immune responses (G1 vs. G3, Figure 4 and Figure 5). However, the current study did not include a higher-dose non-adjuvanted group, limiting direct assessment of dose dependence in the absence of adjuvant and warranting further investigation. Notably, while preclinical studies frequently suggest greater immunogenicity at lower antigen doses, the relevance of this finding has not been comprehensively evaluated in humans [85]. Hence, optimal dose selection will require further evaluation in future clinical studies to establish an appropriate balance between safety and immunogenicity.

Importantly, the SBA data closely mirrored the IgG responses. SBA measures the capacity of vaccine-induced anti-Shigella antibodies to kill live bacteria through complement-dependent, antibody-directed membrane lysis [86,87]. Because baseline exposure and serotype-specific differences can decouple IgG binding from protective function, SBA provides a more biologically relevant measure of complement-mediated killing and is increasingly considered a key functional correlate of protection for Shigella vaccines [87]. Intriguingly, S. sonnei demonstrated a higher fold increment in SBA titers compared to the S. flexneri serotypes (Table 6), which was not revealed in IgG measurement. The IgG assay used in this study measures antigen-specific binding antibodies directed against the O-polysaccharide (O-PS). In contrast, SBA reflects the collective activity of all serum antibodies capable of mediating bactericidal effects. Although O-PS-specific antibodies are the principal mediators of SBA [88], it has been reported that antibodies targeting surface-exposed protein antigens can mediate complement-dependent bacterial killing when S. sonnei O-antigen expression is limited, supporting a role for non-O-PS-specific antibodies in bactericidal activity under defined conditions [88]. In addition, prior work has shown that S. flexneri 2a O-PS-IpaB conjugate can mediate cross-protection against heterologous S. sonnei challenge in mice, whereas O-PS-CRM conjugates only provide minimal protection, suggesting a potential contribution of IpaB-specific immune responses to S. sonnei protection [61]. Collectively, these observations raise the possibility that inclusion of IpaB in the current vaccine may contribute to extra bactericidal activity against S. sonnei through the induction of functional antibodies. This hypothesis remains speculative and will need to be evaluated in future studies designed to define the respective roles of O-PS- and protein-specific antibodies in bactericidal activity. Overall, this observation underscores the importance of including functional assays, such as SBA, alongside IgG quantification when evaluating vaccine efficacy. Relying solely on antibody levels may overlook key differences in the quality and functional capacity of the immune response, particularly when baseline immune activity varies across serotypes.

The role of aluminum phosphate as an adjuvant in the IVT Shigella-04 formulation warrants further reflection, particularly given the distinct immunological profiles observed with and without its inclusion. While all four conjugates induced strong anti-O-PS-specific IgG responses in rabbits regardless of adjuvant use, the inclusion of aluminum phosphate consistently enhanced both the magnitude and consistency of these responses, especially for S. flexneri serotypes. This aligns with the plausible mechanisms of aluminum salts action in promoting depot formation, antigen uptake by antigen-presenting cells, and subsequent T-helper cell activation, all of which are critical for glycoconjugate vaccine responses [89,90,91]. Taken together, these findings suggest that while the adjuvant is not strictly required for immunogenicity, particularly for certain serotypes, it may provide a meaningful enhancement of immune responses that may be critical in low-responder populations or as dose-sparing strategies. Further studies in human subjects will be essential to evaluate the effect of adjuvanted formulations in boosting immunogenicity.

There are several limitations in the current study. Although robust antigen-specific antibody responses and functional serum bactericidal activity were observed, the study did not include a direct evaluation of protective efficacy. While multiple animal models have been employed to evaluate the protective efficacy of Shigella vaccine candidates [92,93], the strong human host specificity of Shigella makes translation of immunogenicity findings to protective efficacy challenging [86]. Consequently, evaluation of protective efficacy in humans is generally considered more appropriate for Shigella vaccines [94], and assessment of protective efficacy in humans by a CHIM study is being considered for later stages of clinical development. Another limitation of the present study is that, although IpaB was intentionally selected as a conserved Shigella antigen and carrier protein to support the potential for broader cross-serotype protection, the study was not designed to directly assess IpaB-specific immune responses or demonstrate IpaB-mediated cross-protection. The contribution of IpaB-specific immune responses to broader protection remains to be investigated. In addition, the immunological assessment in this study focused primarily on humoral responses, and cellular immune parameters were not evaluated. Previous studies have suggested that T helper cell responses and cytokine-mediated mechanisms may play an important role in shaping the quality and durability of protective immunity against Shigella [95]. A comprehensive evaluation of cellular immune responses, including T-cell activation and functional profiling, will be important for fully characterizing vaccine-induced immunity and warrants further investigation in future studies. Finally, the study design did not include an adjuvant-only control or a higher-dose non-adjuvanted group, limiting the ability to disentangle the respective contributions of antigen dose and adjuvant to the observed immune responses. Future studies may address these limitations through expanded immunological characterization, inclusion of appropriate control formulations, and assessment of protective efficacy in later stages of development.

5. Conclusions

This study evaluated IVT Shigella-04, a quadrivalent O-polysaccharide–IpaB conjugate vaccine targeting S. flexneri 2a, 3a, 6, and S. sonnei. Immunization in rabbits elicited strong serotype-specific IgG responses and robust serum bactericidal activity against all four serotypes. The adjuvanted 2.5 µg per serotype (10 µg total conjugates per dose) formulation demonstrated the most favorable immunogenicity profile. While increasing the antigen dose did not confer additional immunological benefit in this model, further dose-ranging studies may be warranted at later stages of development. IpaB was utilized as both the carrier protein and a conserved Shigella antigen in this vaccine design, with the intent of enhancing protective breadth and functional immune responses while providing an alternative to traditional carrier proteins. However, further studies will be required to clarify the contribution of IpaB-specific immune responses to overall vaccine immunogenicity and potential cross-protection. In addition, evaluation of cellular immune responses and protective efficacy in CHIM studies will be important to fully define the protective potential of IVT Shigella-04. Notably, the vaccine candidate has successfully completed a GLP-compliant toxicology study with no test article-related toxicity observed and is currently undergoing evaluation in a Phase 1 clinical trial.