Development and evaluation of a dual target glycoconjugate vaccine against Shigella sonnei

This study demonstrates the successful development and evaluation of a novel dual-target *Shigella sonnei* glycoconjugate vaccine, generated via in vivo bioconjugation using an oligosaccharyltransferase to link O-antigen with immunogenic carrier proteins, which effectively elicited IgG responses against both the carrier and the O-antigen in mice.

The Gist

Imagine you are trying to build a fortress to protect a city (your body) from a very tricky, shape-shifting enemy called Shigella. This enemy causes severe diarrhea and is becoming harder to kill because it's learning to resist our medicines.

For years, scientists have tried to build a vaccine (a training manual for your immune system) using just the enemy's "uniform" (a sugar coating called O-antigen). But there's a problem: the immune system often ignores these sugar uniforms because they are too simple. It's like showing a child a picture of a cookie; they might look at it, but they won't remember it or get excited enough to fight for it.

To fix this, scientists usually attach the sugar "uniform" to a "bodyguard" protein. This bodyguard grabs the immune system's attention, carries the sugar over, and says, "Hey! Look at this! We need to learn how to fight it!"

The Problem with the Old Bodyguards
In the past, scientists used the same few bodyguards (like Tetanus or Diphtheria proteins) for every vaccine. The problem? If you use the same bodyguard too many times, the immune system gets bored. It stops paying attention to the bodyguard, and eventually, it stops paying attention to the sugar "uniform" attached to it, too. It's like a teacher who always uses the same funny voice to tell jokes; eventually, the students stop laughing, and they stop listening to the lesson.

The New "Double-Hit" Strategy
This paper describes a clever new way to build a vaccine that solves two problems at once. The researchers decided to use a new, native bodyguard that the Shigella bacteria actually carries on its own back.

Think of it like this:

1. The Old Way: You take a generic bodyguard (ExoA) and glue a piece of the enemy's sugar coat to it.
2. The New "Double-Hit" Way: You take a bodyguard that the enemy itself uses (a protein called MdtA), glue the enemy's sugar coat to it, and present it to the immune system.

Now, when the immune system sees this vaccine, it gets a "double hit":

• Hit 1: It learns to recognize the sugar coat (the specific part that identifies Shigella).
• Hit 2: It learns to recognize the bodyguard (MdtA), which is a part of the enemy's own machinery.

This means the vaccine teaches the immune system to attack the enemy from two different angles, making it much harder for the bacteria to escape.

How They Built It (The "Bio-Factory")
Usually, making these vaccines is like a complex chemistry experiment in a lab, involving dangerous bacteria and messy chemical glues. It's expensive and slow.

Instead, the researchers built a tiny, biological factory inside a harmless bacteria called E. coli.

• They gave this factory three blueprints (plasmids):
  1. The blueprint for the enemy's sugar coat.
  2. The blueprint for the new bodyguard (MdtA).
  3. A blueprint for a "glue machine" (an enzyme called PglS).
• The factory runs itself. The glue machine automatically snaps the sugar coat onto the bodyguard protein while they are being built. It's like a 3D printer that assembles the final product perfectly without needing a human to glue the pieces together later.

The Results
They tested this new vaccine on mice. The results were great:

• The mice's immune systems woke up and made antibodies against both the sugar coat and the bodyguard protein.
• The new bodyguard (MdtA) worked just as well as the old, standard bodyguards, proving that we can use the enemy's own parts against them.

Why This Matters
This is a big deal because:

1. It's Cheaper and Easier: Making the vaccine inside bacteria is much faster and cheaper than chemical methods. This is crucial for helping poorer countries where Shigella is a major killer.
2. It's Smarter: By using a "double-hit" approach with a native protein, we avoid the immune system getting bored.
3. It's Flexible: This method can be easily adapted to fight other strains of bacteria or other diseases.

In short, the researchers found a way to build a smarter, cheaper, and more effective vaccine by using the enemy's own tools against them, all assembled by a tiny biological factory inside a harmless bug.

Technical Summary

1. Problem Statement

• Global Burden: Shigellosis is a leading cause of diarrheal disease and mortality, particularly in children under five in low- and middle-income countries (LMICs). The rise of multidrug-resistant strains exacerbates the need for effective vaccines.
• Vaccine Limitations: Currently, there is no licensed Shigella vaccine. Existing candidates often rely on chemical conjugation of polysaccharides (O-antigens) to protein carriers (e.g., Tetanus toxoid, Diphtheria toxoid, or Pseudomonas ExoA).
• Manufacturing Challenges: Chemical conjugation is costly, complex, and requires handling pathogenic bacteria.
• Immunological Challenges: Repeated use of the same carrier protein in multi-valent vaccines can lead to "carrier-induced epitope suppression," where the immune system focuses on the carrier rather than the polysaccharide, or develops tolerance.
• The Gap: There is a need for a cost-effective, scalable production method (bioconjugation) that utilizes novel, conserved Shigella-specific proteins as carriers to provide "dual-hit" immunity (targeting both the O-antigen and the carrier protein) without relying on standard, non-Shigella carriers.

2. Methodology

The study employed a bioconjugation approach using E. coli as a cell factory to synthesize glycoconjugates in vivo.

• Strain and Plasmid Construction:

  • Host: E. coli SDB1 (a specialized strain for bioconjugation).
  • O-Antigen Source: The S. sonnei O-antigen (SSOAg) pathway was cloned from Plesiomonas shigelloides O17 (which shares an identical O-antigen structure) into a plasmid (pBPSO).
  • Carrier Proteins: Three proteins were engineered:
    1. ExoA: A standard bioconjugate carrier (detoxified fragment).
    2. EmrK: A conserved Shigella outer membrane protein (efflux pump component).
    3. MdtA: A conserved Shigella outer membrane protein (efflux pump component).
  • Engineering: Carrier proteins were modified with an N-terminal DsbA signal sequence for periplasmic trafficking and a C-terminal ComP tag (a specific peptide sequon recognized by the OST enzyme).
  • Enzyme: The oligosaccharyltransferase PglS (from Acinetobacter baylyi) was used to transfer the O-antigen to the ComP-tagged carriers. PglS was selected because it recognizes O-linked glycans, suitable for the unique SSOAg structure (D-FucNAc4N-L-AltNAcA).

• Production and Purification:

  • Cultures were grown in LB or Terrific broth, induced with IPTG (for PglS) and L-arabinose (for carrier proteins).
  • Purification Strategy: A two-step process was used:
    1. Ni-NTA Affinity Chromatography: To capture 6xHis-tagged proteins.
    2. Anion Exchange Chromatography (AEX): Crucial for separating glycosylated conjugates from unglycosylated proteins based on charge differences caused by glycan attachment. Multiple rounds of AEX were required to achieve high purity.

• Immunogenicity Studies:

  • Model: C57BL/6J female mice immunized subcutaneously on days 0, 14, and 28 with 10 µg of antigen + Alhydrogel adjuvant.
  • Groups: Mice received SSOAg-ExoAComP, SSOAg-MdtAComP, unglycosylated carriers, or PBS.
  • Assays:
    • Anti-Protein ELISA: Measured IgG against ExoA and MdtA. To avoid cross-reactivity with the ComP tag, untagged versions of the proteins were used as ELISA antigens.
    • Anti-Glycan ELISA: Measured IgG against SSOAg using whole-cell ELISA with E. coli expressing the O-antigen. To prevent interference from anti-MdtA antibodies (since E. coli also has MdtA), the mdtA gene was deleted from the E. coli W3110 strain used for coating.

3. Key Contributions

• First Use of PglS for Shigella: Demonstrated that the O-linked OST PglS can successfully transfer the unique S. sonnei O-antigen (containing D-FucNAc4N) to protein carriers, expanding the bioconjugation toolbox beyond the commonly used N-linked PglB.
• Novel "Double-Hit" Carriers: Successfully utilized MdtA and EmrK, which are conserved Shigella-specific proteins, as glycoconjugate carriers. This addresses the issue of carrier-induced epitope suppression by using species-specific antigens.
• Proof of Dual Immunogenicity: Showed that these bioconjugates elicit robust IgG responses against both the carrier protein and the O-antigen simultaneously.
• Scalable Purification Protocol: Established a robust purification workflow involving sequential AEX to remove unglycosylated contaminants, a critical step for vaccine efficacy.

4. Results

• Bioconjugate Synthesis: PglS successfully conjugated SSOAg to ExoA, EmrK, and MdtA. Western blot analysis confirmed the shift in molecular weight (glycosylated proteins migrated at ~110-120 kDa vs. ~80 kDa for unglycosylated ExoA).
• Yield Differences: ExoAComP and MdtAComP produced significantly higher glycoconjugate yields than EmrKComP. Consequently, only ExoA and MdtA were advanced to immunogenicity studies.
• Anti-Protein Response:
  • Mice immunized with glycosylated carriers produced significant IgG against both ExoA and MdtA.
  • Glycosylation did not abolish the protein-specific immune response; in fact, unglycosylated and glycosylated versions induced comparable responses (though unglycosylated MdtA showed a slightly higher, non-significant mean response).
  • Cross-reactivity to the ComP tag was identified but did not hinder the generation of specific anti-carrier antibodies when using untagged proteins for analysis.
• Anti-O-Antigen Response:
  • Both SSOAg-ExoAComP and SSOAg-MdtAComP elicited specific anti-SSOAg IgG responses.
  • The anti-SSOAg response was significantly higher in the glycosylated groups compared to unglycosylated controls.
  • Comparison: The SSOAg-ExoA conjugate induced an approximately 3-fold higher anti-O-antigen response than the SSOAg-MdtA conjugate, though both were effective.

5. Significance

• Vaccine Design Innovation: This study validates the "double-hit" strategy for Shigella, where a single conjugate vaccine targets the serotype-specific O-antigen and a conserved, species-specific protein carrier. This could broaden protection across serotypes and reduce the risk of carrier tolerance.
• Manufacturing Advantages: The use of bioconjugation in E. coli offers a cheaper, faster, and safer alternative to chemical conjugation, which is vital for deploying vaccines in LMICs where shigellosis is most prevalent.
• Future Potential: The identification of MdtA as a viable carrier opens the door for next-generation vaccines that utilize conserved Shigella antigens. While the ExoA carrier performed slightly better in this study, the MdtA platform demonstrates the feasibility of using native Shigella proteins, potentially offering heterologous protection against different Shigella species.
• Next Steps: The authors suggest that future work should focus on challenge studies in animal models to determine protective efficacy and further optimize glycosylation sites to maximize yield and immune response.