Engineering Tunable Cyborg Bacterial Vaccines Using Intracellular Hydrogelation

Introduction: : Bacterial pathogens impact global human populations, causing diseases ranging from persistent urinary tract infections to life-threatening respiratory and bloodstream infections. Current whole-bacteria vaccines offer only limited protection, primarily due to the methods used for bacterial inactivation. Heat inactivation, the most common approach, denatures critical surface and cytoplasmic antigens, impairing immunogenicity and reducing the efficacy of memory responses. As a result, vaccine development against bacterial infections has been constrained by poor antigen preservation and limited ability to modulate immune responses effectively. To address these challenges, we have developed a novel platform for generating Cyborg Pathogen vaccines using a tunable intracellular hydrogelation approach. This method allows precise control over the physical and phenotypic state of bacteria, producing non-replicative yet metabolically active cells. We applied this strategy to two clinically relevant Gram-negative pathogens Pseudomonas aeruginosa PA14 and Klebsiella pneumoniae BIDMC 2A, demonstrating the generalizability and scalability of our approach. Our Cyborg Bacteria Vaccine strategy retains functional antigen expression and promotes robust immune activation without the risks associated with live-attenuated or heat-killed vaccines. By tuning intracellular hydrogelation levels, we can further optimize antigen preservation, metabolic activity, and immune engagement, enabling tailored vaccine responses against emerging and drug-resistant pathogens.

Materials and

Methods: : To create Cyborg Bacteria, wild-type bacteria were incubated in a hydrogel buffer composed of polyethylene glycol diacrylate (PEGDA), a photoactivator, and fluorescein diacrylate. After recovery in fresh media, intracellular hydrogelation was induced by ultraviolet (UV) irradiation, which crosslinked the hydrogel within the bacterial cytoplasm, arresting replication while preserving metabolic activity. Two complementary approaches were employed to further purify the Cyborg cell population following hydrogelation. First, fluorescence-activated cell sorting (FACS) was used to purify hydrogelated bacteria based on their fluorescein signal, ensuring a highly purified Cyborg population. Second, to enhance scalability, a post-hydrogelation treatment with pH 10.5 phosphate-buffered saline (PBS) was developed. This high-pH and batch treatment selectively eliminated wild-type replicating bacteria while maintaining the viability of Cyborg Bacteria. Colony-forming unit (CFU) assays confirmed complete loss of proliferation, while metabolic function was verified through flow cytometry. Antigenic integrity was assessed via Western blotting using surface protein OprF and cytoplasmic protein PvdA. Immunological characterization was performed in C57BL/6 mice vaccinated with Cyborg or heat-killed pathogens, followed by wild-type bacterial challenge. Organ bacterial loads were quantified using RT-PCR. Flow cytometry was used to assess APC activation (CD80/CD86) and T-cell responses (CD3+). Serum IgG1 and IgG2 levels were evaluated by ELISA, and cytokines IFN-γ and IL-4 were measured post vaccination. Neutralization assays assessed the inhibitory capacity of vaccinated serum. Finally, the influence of hydrogelation degree on immune activation was analyzed using co-cultures of Cyborg bacteria with macrophages and dendritic cells.

Results, Conclusions, and Discussions:: Cyborg Pathogens exhibited a stable, non-replicating yet metabolically active state as confirmed by CFU absence and preserved metabolic assays. Western blot analysis revealed unaltered expression of key antigens (OprF, PvdA), suggesting the intracellular hydrogelation process preserved antigenic integrity. Immunological studies demonstrated that Cyborg Vaccines effectively activated antigen-presenting cells, with increased CD80/CD86 expression, and promoted T-cell responses, as shown by elevated CD3+ populations. Mice vaccinated with Cyborg Pathogens showed over 80% survival upon wild-type bacterial challenge, significantly higher than those vaccinated with heat-killed bacteria. RT-PCR analyses showed a markedly reduced bacterial burden in lungs and spleens of Cyborg-vaccinated mice. Notably, ELISA quantification showed significantly higher levels of both IgG1 and IgG2 antibodies compared to heat-killed controls, indicating robust humoral immunity. Importantly, cytokine profiling revealed elevated IFN-γ levels from the day of challenge in Cyborg-vaccinated mice, while IL-4 remained unchanged. This cytokine profile, characterized by high IFN-γ and absent IL-4, suggests a TH1-biased immune response, which is critical for effective clearance of intracellular bacterial pathogens. Neutralization assays confirmed that sera from Cyborg-vaccinated mice effectively inhibited wild-type bacterial infection in vitro, underscoring the functional efficacy of the antibody response. Finally, modulation of intracellular hydrogelation levels significantly impacted immune activation. Lower intracellular hydrogelation degrees resulted in weakened antigen presentation and reduced cytokine release, indicating that immune response strength is tunable based on the degree of intracellular hydrogel crosslinking.
In conclusion, our Cyborg Pathogen vaccine platform offers a new paradigm for bacterial vaccine design by integrating tunable intracellular hydrogelation, preserved antigenicity, and strong TH1-skewed immunity. These results support the further development of Cyborg Pathogen vaccines for targeting a broad spectrum of emerging bacterial pathogens with improved efficacy and safety.

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