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Discovery and Basic Research

(T0930-03-15) Exploring Bacterial Peptidoglycan as a Vaccine Platform

Tuesday, November 11, 2025
9:30 AM - 10:30 AM CT
Purpose: Successful vaccines are characterized by their ability to generate strong and specific immune responses. These vaccines can include whole attenuated pathogens or inactivated cells that display a multitude of antigens on their surfaces. Alternatively, they may utilize subunit strategies, which involve using only select portions of the pathogen or antigen. This targeted subunit approach not only directs the immune response toward specific antigens but also enhances safety by minimizing potential adverse effects. To elicit robust and long-lasting immunity, subunit strategies often necessitate the use of carriers or adjuvants1. Recent advancements have highlighted the potential of multiple antigen presenting systems to enhance the specificity and magnitude of immune responses by presenting a high number of antigen copies or multiple molecular patterns2,3. In this context, our study investigates the application of isolated bacterial peptidoglycan (PG) as a novel vaccine platform. PG is a macromolecule composed of glycan strands cross-linked by oligopeptides which can be chemically modified to incorporate multiple antigen species. Its layered, repeated structure allows for the loading of sufficient amounts of antigens. Additionally, antigen conjugation on isolated PG can be performed in vitro, allowing for the presentation of various antigens, such as proteins, glycans and lipid conjugates. Furthermore, PG's unique bacterial characteristics, including its particle size and the presence of muramyl dipeptides, may impart inherent adjuvanting effects and bolster immune responses. Our study investigates the use of isolated bacterial PG as a novel platform, aiming to combine the advantages of traditional vaccination methods with the advancements seen in contemporary approaches.

Methods: Protocols were developed to effectively isolate PG while confirming the removal of contaminants, including cellular nuclei and membranes. Bacterial species were selected based on their unique PG structural characteristics. The isolated PG samples were characterized for their size, shape, and absence of contaminants using microscopy. An in vitro NF-κB stimulation assay was used to evaluate the immunostimulatory potential of the isolated PG. This assay assessed the ability of the PG to activate innate immune pathways.  A Cell Titer Glo assay, which quantitatively measures cell viability, was performed to ensure the non-cytotoxicity of the isolated PG and levels of lipopolysaccharides (LPS), a known endotoxin contaminant, were quantified for Gram negative-derived PG. Cytokine profiling in PG stimulated human peripheral blood mononuclear cells (PBMCs), using Meso scale discovery (MSD) assay was used to further characterize the preparations. Isolated PG was functionalized with orthogonal conjugation handles to enable PG decoration with antigen molecules and the attachment of these handles was confirmed via fluorescence labelling. Subsequently, the model antigen ovalbumin (OVA) was similarly modified and incorporated onto PG. The efficacy of this conjugation was assessed through fluorescence blotting and ELISA.  

Results: PG was successfully isolated from four bacterial species, and structural integrity and the absence of contaminants was confirmed through fluorescence microscopy. For fluorescence microscopy, WGA-AF 488 was used to stain PG, DAPI for nuclei and FM-464 for membrane visualization. A modest reduction in size indicated effective membrane removal. In vitro analyses revealed moderate immunostimulatory effects from two of the four bacterial species. All isolated PGs exhibited non-cytotoxicity, with cell viability exceeding 70%. The successful removal of bacterial LPS from Gram-negative bacterial was confirmed by low endotoxin levels detected in the isolated PG samples. All PGs induced cytokine stimulation indicating a pro-inflammatory response, including TNF- alpha, IL-6 and IL-1beta. The isolated PGs were functionalized with three orthogonal conjugation handles through metabolic labeling and enzyme-mediated modifications. The successful incorporation of these conjugation handles was verified via fluorophore labeling. Conjugation handles were attached to ovalbumin (OVA) that corresponded with those on the PG and confirmed through fluorescent blotting, utilizing labeled linkers for visualization. 

Conclusion: PG was successfully isolated from four bacterial species. Isolated PGs induced immunostimulation in vitro, were non-cytotoxic, and bacterial LPS was effectively removed, as evidenced by low endotoxin levels in the PG samples. The isolated PGs stimulated a pro-inflammatory response in human PBMCs. PGs were functionalized with conjugation handles through metabolic labeling and enzyme-mediated modifications and methods were developed to optimize the conjugation process for the model antigen OVA to the PGs, successfully creating antigen-decorated PGs. Ongoing research aims to identify the most effective conjugation candidates and to assess immunogenicity in animal models. This effort seeks to validate PG-conjugation technology as a vaccine platform that can successfully present a high number of antigen copies and multiple molecular patterns to elicit a better immune response. 

References: Lieberman LA (2022) Outer membrane vesicles: A bacterial-derived vaccination system. Front. Microbiol.13:1029146. DOI: 10.3389/fmicb.2022.1029146
Weidenbacher PA et.al. (2022) Chemically Modified Bacterial Sacculi as a Vaccine Microparticle Scaffold. ACS Chem Biol.17(5):1184-1196. DOI: 10.1021/acschembio.2c00140
Zhang F et al. Multiple antigen-presenting system (MAPS) to induce comprehensive B- and T-cell immunity. Proc Natl Acad Sci U S A. 2013 Aug 13;110(33):13564-9. DOI: 10.1073/pnas.1307228110

Acknowledgements: The authors extend their gratitude to Shane Krska and the MRL Postdoctoral Research Program for their valuable support in this work. They also express their appreciation to Tarit Mukhopadhyay, Juan Alvarez, Lan Zhang, Kalpit Vora, Grant Murphy, Thomas Linsky, and Heath Klock, for their insightful contributions and suggestions.