Debaki Ranjan Howlader

Pseudomonas aeruginosa is a leading cause of severe opportunistic infections and a "critical priority" pathogen due to increasing antimicrobial resistance. We evaluated the immunogenicity and protective efficacy of an intranasally delivered subunit vaccine, L-PaF, formulated with a squalene-based nano-emulsion and the TLR4 agonist BECC438b in a rabbit model. Immunization elicited robust antigen-specific IgG and IgA responses, in addition to significant opsonophagocytic activity. Longitudinal metabolomic profiling across multiple platforms showed that vaccination elicited a coordinated systemic response, characterized by early decreases in circulating amino acids followed by later remodeling of lipid-associated metabolites.,To evaluate functional relevance, animals received a virulent intratracheal challenge. Protective efficacy was observed with the 50 µg dose, which promoted bacterial clearance while preserving body weight and normal physiological regulation. Despite generating similarly robust antibody responses, the 100 µg dose did not provide protection. Integration of these data revealed that the protective 50 µg dose produced a tightly regulated, uniform metabolic ”landing zone,” whereas the nonprotective 100 µg dose was associated with greater inter-animal variability and signatures of metabolic stress. This discordance indicates that standard measures of immunogenicity are insufficient to predict protective outcomes. These findings emphasize that vaccine efficacy is dose-dependent and reflects qualitative systemic differences, suggesting that integrated immunological and metabolic profiling is essential to define true correlates of protection against Pseudomonas aeruginosa.

Pseudomonas aeruginosa (Pa) is a ubiquitous, opportunistic nosocomial pathogen that poses a significant threat due to its innate and acquired multidrug resistance. Novel vaccine strategies are urgently needed for vulnerable populations, many of which harbor pre-existing immunity from prior encounters with Pa. Here, we evaluated a multivalent subunit vaccine combining type III secretion system (T3SS) antigens and exolysin A (ExlA) in a nanoemulsion formulation using a clinically relevant murine pulmonary pre-exposure model. This approach allowed us to determine whether vaccination could overcome the limitations of the host’s initial, ineffective immune response. Vaccination fundamentally transforms suboptimal baseline memory into a potent, multi-faceted Th1/Th17-polarized response. This globally transformed signature was characterized by significantly enhanced antigen-specific IFN- γ and IL-17A production, both locally and systematically in the lung, with exceptionally large biological effect sizes (Cohen’s d values reaching 21.35). By utilizing log10 transformation to accurately reflect pathogen growth kinetics, we demonstrated that this vaccine-augmented immunity conferred statistically significant protection following heterologous challenge. In the twice-exposed cohort, vaccination promoted superior bacterial clearance of the T3SS-positive strain, though clearance of the ExlA-positive strain was not enhanced, potentially due to immunological interference from pre-existing T3SS memory. In the thrice-exposed cohort, a functional protective threshold was observed, where high levels of natural immunity matched the vaccine-induced clearance levels. Our findings established that our vaccine formulation can effectively boost and redirect pre-existing immunity, offering a promising approach to overcome the limitations of natural exposure and protect at-risk individuals from diverse Pa infections.

Pseudomonas aeruginosa (Pa) is a Gram-negative pathogen causing severe pneumonia, especially in immunocompromised and hospitalized patients. The absence of a licensed vaccine and increasing antibiotic resistance highlight an urgent need for new prophylactic strategies. Here, we explore the immune mechanisms of protection offered by the multicomponent nanoemulsion subunit vaccine, L-PaF/E/ME/BECC. Using single-cell RNA sequencing, we mapped the lung immune landscape post-vaccination, revealing coordinated activation of innate and adaptive responses. This included αβ and γδ T cells, macrophages, neutrophils, and dendritic cells. Fibroblasts and endothelial cells also preconditioned the lung microenvironment for infection control. Type I and II interferon signaling played a central role, driving antigen presentation via MHC-II upregulation on alveolar macrophages and promoting macrophage recruitment. These findings were validated in interferon receptor–deficient mice, which showed impaired antigen presentation, reduced bacterial clearance, and diminished protective immunity. Vaccinated animals also displayed accelerated restoration of lung homeostasis post-infection, mediated by balanced cytokine and chemokine profiles orchestrated by stromal–immune interactions, a response absent in unvaccinated controls. These results demonstrate that L-PaF/E/ME/BECC vaccination primes both immune and structural lung compartments for rapid, coordinated defense, limiting bacterial burden and tissue injury, and supporting its strong potential for clinical development.

Shigellosis remains a major global health burden, and the increasing prevalence of multidrug-resistant (MDR) Shigella strains is complicating effective antibiotic therapy. Bacteria may survive antibiotics by transitioning into cell wall-deficient L-forms, which are intrinsically resistant to β-lactams and can revert to a virulent state, potentially causing relapsing infections. Here, we characterized a clinical MDR isolate Shigella sonnei HK8, a.k.a. PD552A, whose genome contains key resistance ( gyrA , PBP3) and virulence ( icsA ) genes. Exposure to ceftriaxone induced a transition into a viable L-form state that was hyper-adhesive to macrophages in vitro . However, this survival adaptation was linked to a profound loss of pathogenicity. Using murine and guinea pig models, the L-form variant was shown to be severely attenuated, failing to cause keratoconjunctivitis, diarrheal disease, or significant histopathology characteristic of the wild-type strain. These findings reveal a critical virulence-survival trade-off, positioning the L-form as a “stealth” phenotype that enables bacterial persistence at the expense of acute virulence. This offers a potential mechanism for asymptomatic carriage and recurrent infections, highlighting a previously underappreciated mechanism by which antibiotic treatment may resolve acute symptoms while permitting the persistence of a cryptic bacterial reservoir capable of driving recurrent infection. IMPORTANCE: Our research provides critical insight into the challenge of antibiotic treatment failure in shigellosis. By integrating experimental validation of reversible L-form transitions with mathematical modeling, we uncover a crucial virulence-survival trade-off. We show that multidrug-resistant Shigella sonnei survives ceftriaxone by adopting a “stealth” phenotype, quantified by a high stealth index—maintaining bacterial burden while evading host inflammatory detection. These findings imply that standard antibiotics may resolve acute symptoms while inadvertently selecting for a cryptic, persistent reservoir poised for relapse. This work challenges the conventional view of therapeutic success and highlights an urgent need to develop novel diagnostic and therapeutic strategies capable of identifying and eliminating these resilient, “stealth” L-form persisters to achieve true bacterial clearance and prevent chronic infections.

Pseudomonas aeruginosa (Pa) is ubiquitous in nature and is an opportunistic, nosocomial pathogen that poses a significant threat due to innate and acquired multidrug resistance. As such, novel vaccine strategies are urgently needed for vulnerable populations, many of whom have had prior exposures to Pa. Here, we evaluated a multivalent subunit vaccine that combines type III secretion system (T3SS) antigens and exolysin A (ExlA) in a nano-emulsion formulation using a clinically relevant murine pulmonary pre-exposure model. This approach allowed us to determine whether the vaccine could overcome the limitations of the host's initial, ineffective immune response. Vaccination transformed a suboptimal baseline memory elicited by prior exposure to Pa into a potent, multi-faceted Th1/Th17-polarized response that included significantly enhanced antigen-specific IFN-γ and IL-17A production, both locally in the lung and systemically. This globally transformed immune signature conferred significant in vivo protection, promoting superior bacterial clearance following challenge with both T3SS-positive and ExlA-positive heterologous strains. Our findings establish that our vaccine formulation can effectively boost and redirect pre-existing immunity, offering a promising approach to overcome the limitations of natural exposure and protect at-risk individuals from diverse Pa infections.

Diarrheal diseases remain a leading cause of global morbidity and mortality, and treatment options are increasingly compromised by the global rise in multidrug-resistant (MDR) pathogens. Furthermore, the absence of effective vaccines against many causative agents renders large populations vulnerable. This critical gap necessitates the development of novel alternatives that can function both prophylactically to prevent infection and therapeutically to treat established diseases. Here, we investigated the commensal Escherichia coli strain HK220822 (HK5), which was isolated from a healthy human, as a potential live biotherapeutic. Comprehensive genomic and phenotypic characterization established a robust safety profile, confirming the absence of key virulence genes while demonstrating essential probiotic traits, including high tolerance to gastrointestinal stressors, such as acid and bile. The functional efficacy of Escherichia coli HK5 has been validated in murine models of infection. Prophylactic administration before pathogen exposure significantly inhibited intestinal colonization by Salmonella Typhimurium, Shigella flexneri 2a , and Vibrio cholerae (El Tor O1 strain). Moreover, HK5 demonstrated therapeutic potential by significantly reducing pathogen shedding when administered to previously infected animals. These findings establish commensal E. coli (HK5) as a potent candidate with dual preventative and therapeutic efficacy, offering a promising non-antibiotic strategy to combat diarrheal diseases.

In vaccine development, the ELISA (Enzyme-Linked Immunosorbent Assay) is commonly used to compare the antibody titers of samples from several treatment groups. This often requires extensive sample preparation, manual labor, and long incubation and processing times. Biolayer Interferometry (BLI) has emerged as an alternative to the ELISA for the detection and quantification of antigen-specific antibodies in biological samples. However, the implementation of BLI as a replacement for the ELISA in vaccine development requires that experimental parameters are established for accurate and reproducible results. Here we give a general protocol for a biolayer interferometry immunosorbent assay (BLI-ISA) for the comparison of antigen-specific antibody levels in treatment group sera that uses secondary antibody binding responses as replacement for ELISA endpoint titers. We also validate that this BLI-ISA yields the same results as the ELISA endpoint titer while requiring far less time and effort.

Shigellosis remains a major global health burden, and the increasing prevalence of multidrug-resistant (MDR) Shigella strains is complicating effective antibiotic therapy. Bacteria may survive antibiotics by transitioning into cell wall-deficient L-forms, which are intrinsically resistant to β-lactams and can revert to a virulent state, potentially causing relapsing infections. Here we characterized a clinical MDR isolate Shigella sonnei HK8, a.k.a. PD552A, whose genome contains key resistance ( gyrA , PBP3) and virulence ( icsA ) genes. Exposure to ceftriaxone induced a transition into a viable L-form state that was hyper-adhesive to macrophages in vitro . However, this survival adaptation was linked to a profound loss of pathogenicity. Using murine and guinea pig models, the L-form variant was shown to be profoundly attenuated, failing to cause the keratoconjunctivitis, diarrheal disease, or significant histopathology characteristic of the wild-type strain. These findings reveal a critical virulence-survival trade-off, positioning the L-form as a "stealth" phenotype that enables bacterial persistence at the expense of acute virulence. This offers a potential mechanism for asymptomatic carriage and recurrent infections, highlighting a previously underappreciated mechanism by which antibiotic treatment may resolve acute symptoms while permitting the persistence of a cryptic bacterial reservoir capable of driving recurrent infection. Importance: Our research provides critical insight into the challenge of antibiotic treatment failure in shigellosis. By integrating experimental validation of reversible L-form transitions with mathematical modelling, we uncover a crucial virulence-survival trade-off. We show that MDR S. sonnei survives ceftriaxone by adopting a “stealth” phenotype, quantified by a high Stealth Index – maintaining bacterial burden while evading host inflammatory detection. These findings imply that standard antibiotics may resolve acute symptoms while inadvertently selecting for a cryptic, persistent reservoir poised for relapse. This work challenges the conventional view of therapeutic success and highlights an urgent need to develop novel diagnostic and therapeutic strategies capable of identifying and eliminating these resilient, "stealth" L-form persisters to achieve true bacterial clearance and prevent chronic infections.

Shigella spp. are responsible for bacillary dysentery or shigellosis transmitted via the fecal–oral route, causing significant morbidity and mortality, especially among vulnerable populations. There are currently no licensed Shigella vaccines. Shigella spp. use a type III secretion system (T3SS) to invade host cells. We have shown that L-DBF, a recombinant fusion of the T3SS needle tip (IpaD) and translocator (IpaB) proteins with the LTA1 subunit of enterotoxigenic E. coli labile toxin, is broadly protective against Shigella spp. challenge in a mouse lethal pulmonary model. Here, we assessed the effect of LDBF, formulated with a unique TLR4 agonist called BECC470 in an oil-in-water emulsion (ME), on the murine immune response in a high-risk population (young and elderly) in response to Shigella challenge. Dual RNA Sequencing captured the transcriptome during Shigella infection in vaccinated and unvaccinated mice. Both age groups were protected by the L-DBF formulation, while younger vaccinated mice exhibited more adaptive immune response gene patterns. This preliminary study provides a step toward identifying the gene expression patterns and regulatory pathways responsible for a protective immune response against Shigella. Furthermore, this study provides a measure of the challenges that need to be addressed when immunizing an aging population.