Rabies virus (RABV), a single-stranded RNA virus, invades the central nervous system and causes acute, zoonotic diseases worldwide. The glycoprotein (G) of RABV is the only surface antigen responsible for inducing potent neutralizing antibodies, making it a key target for medical countermeasures. In this study, two anti-G protein monoclonal antibodies (mAbs) were prepared using hybridoma cell technology, with their high specificity and robust reactivity validated by indirect enzyme-linked immunosorbent assay (i-ELISA), indirect immunofluorescence assay (IFA) and Western blot. Through systematic epitope mapping using a series of truncated G protein fragments, two mAbs (1G2 and 4E5) were found to recognize a minimal linear epitope, AEDFVEVHLP (amino acids 412–421) through Dot-ELISA and i-ELISA. Alanine-scanning mutagenesis further defined the core binding residues within this epitope as 412Ala-Glu-Asp-Phe-Val416, 418Val, 420Leu, and 421Pro. Bioinformatic analysis with MEGA software revealed highly conserved with conservative substitutions of this epitope across diverse RABV of genotype 1. Homology modeling with SWISS-MODEL localized the epitope “412AEDFVEVHLP421” to a solvent-exposed α-helical conformation on the G protein surface. And this epitope is located in the extracellular C-terminal region, Prior studies have demonstrated that this region plays a role in stabilizing the trimerization of the rabies virus glycoprotein (RVG). In conclusion, our study identifies a novel, highly conserved with conservative substitutions linear B-cell epitope on the RABV glycoprotein, providing a precise molecular target for the rational design of next-generation epitope-based vaccines and specific diagnostic tools.
Human Immunodeficiency Virus (HIV) infection impairs the immune system, diminishing its ability to defend against pathogens and increasing susceptibility to infections and diseases. To gain a comprehensive understanding of the heterogeneity, functions, and regulatory mechanisms of immune cells affected by different HIV viral loads, we investigated the biological impacts of HIV infection on the human body. We reanalyzed a publicly available peripheral blood mononuclear cell (PBMC) single-cell RNA sequencing dataset (GSE157829) comprising three people living with HIV with high viral load (HL-HIV), three people living with HIV with low viral load (LL-HIV), and one healthy control donor. We constructed single-cell immune atlases that represent different viral load profiles. We also analyzed the dynamic changes in specific immune cell subpopulations to gain insights into their signaling pathways, developmental trajectories, and key transcriptional regulators. At the single-cell level, non-classical monocyte subpopulations, specifically ncMono_LYN_TCF7L2 and ncMono_IL32, functioned as immunosurveillance elements but also activated pro-inflammatory pathways, leading to immune activation. B_CD83 subpopulation was critical in B cell development, facilitating B cell differentiation and tolerance. Naive.T_TNFAIP3 subpopulation exhibited anti-inflammatory effects and protected against aberrant apoptosis. Central memory T (TCM)_TCF7_CCR7 subpopulation played a crucial role in regulating the expansion of HIV-specific CD8+ T cells and maintaining the immune response. Additionally, we observed a significant enrichment of IFI44L-positive subpopulations across different cell types, particularly in HL-HIV samples. Our findings demonstrate that HIV infection increases the complexity of immune cell subpopulations. Notably, IFI44L has emerged as a potential disease hub gene in HL-HIV samples, highlighting its potential as a novel biomarker for HIV.
Despite the recent approval of preventive measures, the disease burden of respiratory syncytial virus (RSV) infection in children and older adults remains significant. To improve understanding of RSV disease, this study characterized extracellular microvesicles (EMVs) secreted during in vitro infection with a clinical RSV-A (ON1 genotype) strain (RSV/40G-A), isolated in 2022 from a pediatric pneumonia case. The type and content of EMVs released by A549 and BEAS-2B respiratory epithelial cells infected with this clinical isolate were analyzed up to 96 h post-infection (hpi). RSV infection induced a time-dependent increase in EMV levels compared to uninfected controls. EMVs released by infected BEAS-2B and A549 cells contained increasing levels of RSV nucleocapsid protein (RSV-NP) and viral RNA with a peak at 72 hpi. These findings suggest that the kinetics of vesicular release and EMV content are modulated during RSV infection. EMV secretion by host cells may constitute a potential mechanism for intercellular communication or immune activation.
Granulomatous amoebic encephalitis (GAE) is a rare but highly fatal central nervous system infection caused primarily by Acanthamoeba spp. and Balamuthia mandrillaris. Delayed diagnosis and the absence of standardized treatment protocols contribute to mortality exceeding 90
Microbes frequently exist as biofilm-embedded multi-species communities where their interactions may establish or exacerbate chronic infection. Recently, fungi and bacteria have been associated with various human tumor microenvironments, suggesting that dynamic cross-kingdom interactions may directly or indirectly contribute to tumor-associated processes. Here, we aimed to investigate whether cell-free supernatants from mono- and dual-species biofilms of the commonly associated fungus Candida albicans and bacterium Staphylococcus aureus could alter human monocyte responses that promote a tumor-related genetic signature in dysplastic oral epithelial (DOK) cells. Treatment of THP-1 monocytes with S. aureus cell-free supernatant increased the production of proinflammatory cytokines (IL-8, IL-1β, and TNF) and CD86 expression. However, exposure to cell-free supernatants from dual-species biofilm suppressed these responses. To determine the fungal virulence factors responsible, C. albicans mutants deleted for genes involved in adhesion (als3Δ/Δ), hyphal growth (efg1Δ/Δ cph1Δ/Δ), or candidalysin production (ece1Δ/Δ) were assessed during co-culture. While candidalysin was dispensable, loss of hyphal growth or the adhesin Als3p phenocopied effects of S. aureus mono-culture treatment. Conditioned medium from THP-1 cells initially challenged with mono- or dual-biofilm cell-free supernatants was applied to DOK cells to assess TP53 and BCL2 gene expression. Conditioned medium from S. aureus treated THP-1 cells led to decreased epithelial TP53, but increased BCL2 expression, which was reversed by the presence of wild-type C. albicans. These phenotypes were similarly dependent on C. albicans hyphal growth during dual-biofilm co-culture. Collectively, our results reveal that fungal–bacterial interactions may shape the monocyte–epithelial axis by orchestrating immune responses that enhance tumor-associated gene expression in dysplastic oral epithelial cells.
Phospholipase A2 (PLA2) is a crucial enzyme in lipid metabolism and inflammatory signaling, playing a significant role in macrophage activation and neuroinflammation. However, its role in Japanese encephalitis virus (JEV)-mediated immune responses remains unclear. In this study, we show that PLA2 and its associated receptors, lysophosphatidic acid receptors (LPAR1 and LPAR3), are significantly upregulated at 24 h post-JEV infection, suggesting their involvement in JEV-induced inflammatory responses. To investigate the effect of PLA2 inhibition, we used arachidonyl trifluoromethyl ketone (AACOCF3). Molecular docking using AutoDock Vina 1.1.2, supported by Protein Data Bank and AlphaFold structures, indicated that AACOCF3 interacts with key inflammatory targets including gp91phox (− 7.9 kcal/mol) and LPAR3 (− 7.8 kcal/mol), suggesting its potential to modulate inflammatory and oxidative pathways. In vitro studies in JEV-infected macrophages demonstrated that AACOCF3 reduced activation of calcium-dependent cytosolic (c) PLA2 expression and phosphorylation, LPAR1, 3, MAPK signaling (JNK, ERK1/2, p38), and NF-κB protein levels, along with a reduction in JE viral RNA levels, likely reflecting indirect effects mediated through host pathway modulation. AACOCF3 also decreased proinflammatory cytokines (IL-1β, TNF-α) and autophagy-related markers (pAkt, LC3-II/I, SQSTM1/p62), and reduced expression of the ROS-generating enzyme gp91phox (NOX2), indicating partial attenuation of inflammatory and oxidative stress–associated pathways. Oxidative stress was assessed via gp91phox expression, and NF-κB was evaluated at protein level. These findings suggest that PLA2 contributes to JEV-induced inflammatory signaling, and that its inhibition modulates host responses during infection in vitro, rather than exerting a definitive therapeutic effect.
Infection with the protozoan parasite Trypanosoma cruzi can lead to Chagas disease, and diagnosis of infection mostly relies on serological testing, although no gold standard exists. The evaluation of treatment efficacy is also challenging as seronegativization takes decades to be detected. The Multi-Cruzi platform, which provides a serological profile against 15 parasite antigens, has been proposed as promising alternative for confirmatory diagnostic and monitoring of treatment response. To further evaluate this platform, we performed epitope mapping of the Multi-Cruzi antigens with peptide microarrays, using confirmed T. cruzi positive samples, as well as serologically discordant samples with positive T. cruzi PCR. Epitope conservation among parasite strains was also assessed. We identified multiple linear epitopes among Multi-Cruzi antigens with samples from patients with confirmed serology, although some redundancy in epitopes may limit the breadth of the antibody profile evaluated. On the other hand, this antigen panel showed very limited reactivity with serodiscordant samples with confirmed T. cruzi infection, with weaker recognition of fewer epitopes. Epitope conservation ranged from highly conserved to more variable. The usefulness of this platform may be limited to a fraction of patients and parasite strains. The addition of alternative antigens may help improve the monitoring of treatment response of serodiscordant patients.
Recent research indicates that the composition of serum anti-SARS-CoV-2 Spike IgG subclass antibodies changes upon repeated exposure to viral Spike. These antibody subclass dynamics were suggested to impact adaptive immune responses and protection against infection. To characterize the role of the IgG subclass composition in the immune response to SARS-CoV-2, assays that measure virus variant-specific anti-Spike IgG subclass concentrations are needed. Here, we describe an easy-to-use serological method for quantifying anti-Spike IgG subclass antibodies against various SARS-CoV-2 variants in multiplex from less than 10 µL of serum. Our assay enables antigen-independent quantification, making it easily adaptable to emerging variants or other pathogens. We validated our method with 20 sera collected from 10 infection-naïve individuals after the second and third COVID-19 mRNA vaccination. We observed increasing anti-Spike IgG2 and, especially, IgG4 concentrations after the third immunization. More recent Spike variants preserved this pattern with decreased levels of total anti-Spike IgG. Our results showed a strong positive correlation with those from an established flow cytometry-based approach. Our method might help deepen our understanding of immune responses to SARS-CoV-2 and, moreover, help gauge the individual’s protection from severe disease.
The pathogenesis of dengue is complex and requires further study. Infection with the dengue virus can lead to severe dengue, in which patients experience severe plasma leakage and excessive bleeding. The dengue virus non-structural protein 1 (DENV NS1) is among several factors that affect the outcome. The secreted form of this protein induces severe cases through direct and indirect immunopathogenesis mechanisms. Previously, we discovered that the stimulation of blood monocytes by NS1 upregulates transcripts involved in inflammation, and suppresses transcripts involved in antiviral processes. In this study, we examined the specific responses unique to high-dose NS1 exposure of cultured human monocytes. We found that genes involved in hemostasis were altered following NS1 exposure and were associated with platelet activation, fibrinolysis, and hemostatic regulation. Some of these expression changes were also observed in patients with dengue fever (DF) or dengue hemorrhagic fever (DHF). The altered expression of these genes suggests that fibrinolysis is more likely balanced during DF, whereas coagulation may be impaired in DHF.
The mechanism of non-/hypo-response to hepatitis B vaccine among infants born to Hepatitis B surface Antigen (HBsAg) positive mothers has not been fully clarified. Intrauterine exposure to HBsAg may alter the innate and adaptive immunity of the offspring, further complicating vaccine response. One potential factor is myeloid differentiation factor 88 (MyD88), which is a key adaptor protein linking innate and adaptive immunity and may be regulated by microRNAs (miRNAs). The study aimed to determine the impacts of intrauterine exposure to HBsAg on the immune response to hepatitis B vaccine in the offspring and the potential mechanisms. In the cohort of infants born to HBsAg positive mothers, three pairs of hepatitis B vaccine non-responsive/responsive infants were matched. And miRNA sequencing and bioinformatics were performed to identify miRNAs that are involved in regulating MyD88. Moreover, to mimic human intrauterine exposure to HBsAg, female Hepatitis B Virus-Transgenic (HBV-Tg) mice were mated with male C57BL/6J mice. After birth, pups including HBV-Tg+ and HBV-Tg− pups were administered 1 µg hepatitis B vaccine according to the 0-2-4week vaccination schedule. Among infants born to HBsAg positive mothers, microRNA-155-5p (miR-155-5p) expression was higher in non-responders than responders. miRNA sequencing and bioinformatics suggested miR-155-5p may regulate MyD88 expression in these neonates. In the animal experiment, pups with intrauterine exposure to HBsAg produced less anti-HBs after vaccination. Flow cytometry showed that both HBV-Tg− pups and HBV-Tg+ pups had less generation of CD4+ T cells, B cells, dendritic cells (DCs), and Germinal Center B (GC B) cells in comparison with wild type (WT) mice pups. Cytokine analysis demonstrated that Interleukin-6 (IL-6) and Interleukin-12 (IL-12) were expressed at lower levels in the pups with intrauterine exposure to HBsAg. Further investigation of MyD88 signaling pathway and miR-155-5p pointed that in DCs of pups with intrauterine exposure to HBsAg, there were lower expression of MyD88 and Nuclear Factor-kappa B (NF-κB), while miR-155-5p was elevated. There is a potential link between intrauterine HBsAg exposure, compromised DC generation and activation, and subsequent suboptimal vaccine response. The current data implied that intrauterine HBsAg exposure may have an inhibitory effect on the MyD88 signaling pathway in DCs, with miR-155-5p potentially negatively regulating MyD88.
Virus-like particles (VLPs) are considered as promising components of vaccines. We studied the response of human dendritic cells (DCs) and naïve CD4+ T cells in vitro and the response of murine T cells in vivo to VLPs derived from VP1N, from a fragment of this protein containing the shell domain and hinge region (SN), or from chimeric molecules obtained by genetic fusion of SN with the VP1, VP2, or VP3 polypeptides of echovirus 30 (SN-VP1E30, SN-VP2E30, and SN-VP3E30). Norovirus VLPs were efficiently endocytosed and stimulated the maturation of monocyte-derived DCs and conventional blood-borne CD1c+CD141− DCs. Chimeric SN-VP2E30 VLPs strongly stimulated DC maturation, SN-VP3E30 VLPs had a weak effect, but a mixture of these VLPs potently induced DC maturation. VLP-treated DCs enhanced cytokine production and increased the ability to induce differentiation of human naïve CD4+ T cells into T helper type 1 cells. Immunization of mice with VP1N VLPs, SN-VP2E30 VLPs or a mixture of SN-VP1E30, SN-VP2E30 and SN-VP3E30 VLPs increases the number of CD4+ splenocytes capable of proliferating in response to the antigens of all particles administered to the mouse, as well as CD8+ splenocytes capable of proliferating in response to VP2E30 or SN-VP1E30. Immunization of mice with VLPs resulted in increased IFN-γ production by splenocytes stimulated ex vivo with VP1N, VP2E30, and SN-VP3E30. In addition, immunization with SN-VP2E30 VLPs resulted in increased IL-5 production by splenocytes in response to specific antigens. The results indicate the ability of norovirus VLPs and chimeric VLPs to induce T cell responses.
The CCR5Δ32 polymorphism is a 32-base pair deletion in the CCR5 gene that has been associated with slower HIV disease progression. However, its role in immune reconstitution during antiretroviral therapy (ART) remains unclear. We analyzed 236 virologically suppressed people living with HIV (PLHIV) after 24 months of ART, comprising 217 individuals homozygous for the wild-type CCR5 allele and 19 heterozygous carriers of CCR5Δ32. Heterozygotes exhibited a significantly higher frequency of central memory CD4+ T cells compared with wild-type homozygotes (40.33 ± 8.79 vs. 32.71 ± 7.06; p = 0.0196), along with a tendency toward increased effector CD4+ T cells (5.100 [1.818–7.588] vs. 2.260 [1.485–3.503]; p = 0.0459). In contrast, longitudinal follow-up revealed that wild-type homozygotes achieved higher absolute CD4+ T cell counts at both 18 and 24 months of ART (p < 0.05). These results suggest that CCR5Δ32 contributes to qualitative preservation of CD4+ T cell subsets while limiting quantitative immune reconstitution, thereby providing novel insights into the long-term immunological impact of this polymorphism in virologically suppressed PLHIV.
Tick-borne Neoehrlichia mikurensis is the cause of neoehrlichiosis, an infectious disease that features fever and vascular events. Compromised B-cell immunity is a risk factor for severe neoehrlichiosis, indicating the importance of antibodies in host defense. The development of serological assays has been hampered by the difficulty of culturing these intracellular bacteria. Here we present the first serological test for N. mikurensis, an ELISA for human IgM and IgG antibody responses to a P44/Msp2 protein of N. mikurensis. Serum or plasma from immunocompetent (n = 44) and immunosuppressed (n = 60) Swedish adults infected with N. mikurensis, with and without symptoms, were analyzed and compared with blood samples from non-infected immunocompetent individuals (n = 17). Sera from non-infected children (n = 23) and plasma from cord blood (n = 10) were also analyzed. Immunocompetent neoehrlichiosis adults had higher IgM and IgG antibody levels compared with immunosuppressed neoehrlichiosis patients treated with the B-cell suppressive agent rituximab. There were no significant differences in the IgM or IgG antibody levels between immunocompetent individuals with symptomatic versus asymptomatic N. mikurensis infection. Sera from healthy children contained high levels of IgM antibodies, despite no evidence of current infection with N. mikurensis nor previous exposure to tick bites as reflected by negative Borrelia serology. This IgM response was absent in cord blood plasma (n = 10), indicating it was not due to natural IgM antibodies. The high constitutional IgM antibody levels to N. mikurensis in children may explain why there have been no reports of pediatric neoehrlichiosis.
Dengue virus (DENV) infection remains a major global health concern, with clinical manifestations ranging from mild febrile illness to severe, life-threatening complications. In the absence of specific antiviral therapies, the development of novel treatment strategies is crucial. This study evaluates the in vivo antiviral efficacy of 2-BFU, a 2′-α-fluoro,2′-β-bromouridine monophosphate prodrug, in a murine model of DENV Serotype 2 (DENV-2) infection. Adult A129 mice were infected subcutaneously with DENV-2 and treated intraperitoneally with 2-BFU at 15 mg/kg, starting 12 h post-infection. Treatment with 2-BFU significantly reduced viral titers in plasma, spleen, and liver, demonstrating potent antiviral activity. Moreover, 2-BFU effectively attenuated DENV-2 -induced thrombocytopenia at 72- and 120-h post-infection. However, the treatment did not significantly affect the production of inflammatory mediators, nor did it prevent infection-associated weight loss or mortality. These findings suggest that 2-BFU holds promise as an antiviral candidate by lowering viral burden and ameliorating thrombocytopenia, although it may require adjunctive anti-inflammatory strategies to improve overall clinical outcomes. Further investigation is warranted to optimize its therapeutic potential for dengue.
Species outside the Acinetobacter calcoaceticus–baumannii (Acb) complex have recently emerged as new clinically relevant carbapenem-resistant pathogens, such as A. haemolyticus. A new mechanism that could contribute to the dissemination of this resistance is the secretion of outer membrane vesicles (OMVs). We investigate OMVs secreted by A. haemolyticus AN54, a Mexican clinical strain carrying a plasmid-borne blaNDM−1 gene and by its plasmid-cured derivative AN54Δe. Active secretion of OMVs formed by a lipid membrane, measuring 10–50 nm in both strains, was observed by TEM. The OMVs from AN54 possess hydrolytic capacity against carbapenem. The OMV protein profile varied depending on the antibiotic concentration during induction, with additional protein bands detected at 8 and 32 µg/ml of antibiotic exposure. Mass spectrometry identified proteins involved in different metabolic pathways and in resistance-related processes. The porins, efflux pumps, and ADC enzyme were more abundant in the OMVs from AN54Δe than in those from AN54 harboring the active NDM-1 enzyme. The OMVs protected the sensitive strains from the same and different genera against the antibiotic action for a limited period. However, the OMVs transferred the blaNDM−1-carrying plasmid (pAhaeAN54e) only to the AN54Δe strain, conferring permanent resistance to carbapenems. Together, these findings suggest that OMVs can help protect surrounding bacteria from antibiotic action and serve as vehicles for disseminating resistance genes. These results show the importance of studying OMVs as a novel mechanism for the dissemination of antimicrobial resistance.
Klebsiella pneumoniae causes severe respiratory-associated infections in healthy individuals, and widespread antibiotic resistance in K. pneumoniae poses a global health threat. Although great efforts have been taken to develop effective vaccines against K. pneumoniae, there are no licensed vaccines against K. pneumoniae available up to date. In the current study, we designed a potent subunit vaccine OmpW, which was obtained from an outer membrane protein (OmpW) to maximize host responses in mice. Antibody-mediated responses and protective effects were determined to compare the immunogenicity of the OmpW subunit vaccine in the vaccinated mice. Immunization experiments showed that mice immunized with recombinant OmpW generated high IgG antibody production, which promoted macrophage opsonophagocytic activity and induced strong serum bactericidal activity. The residual bacterial burdens from the different organs were declined in the immunized mice compared with the control group. Notably, immunization with the OmpW subunit vaccine provided better protection against K. pneumoniae challenge in vaccinated mice. The passive transfer of antiserum from OmpW-immunized mice also provided protection against K. pneumoniae infection. This study constitutes an important step toward developing potent antibacterial vaccines against K. pneumoniae infections and prevents further dissemination of K. pneumoniae strains.
Synergistic polyreactivity of antibodies—where distinct oligo- or polyreactive clones combine to enable broad-spectrum antigen binding—was recently demonstrated for human anti-galactose-α-1,3-galactose (αGal) antibodies. However, it remains unclear whether this phenomenon also occurs in other antigen-defined antibody populations. Here, we investigated synergistic polyreactivity in isohemagglutinins (naturally occurring antibodies against ABO blood group antigens) and its potential role in antimicrobial defense. Isohemagglutinins were affinity-purified from pooled plasma from groups of healthy donors sharing the same ABO blood group, then characterized by solid-phase immunoassays, SDS-PAGE, and Western blotting. Binding to formaldehyde-fixed Streptococcus pneumoniae representing 30 serotypes was assessed by flow cytometry. Specificity was further examined using inhibition assays with soluble pneumococcal polysaccharides. To evaluate clinical relevance, ABO blood group types (used as proxies for isohemagglutinin profiles) were linked to serotype-specific invasive pneumococcal disease (IPD) events using Danish nationwide registry data. The purified isohemagglutinins retained specificity for their cognate ABO antigens. Notably, they bound all tested pneumococcal strains, primarily through reactivity with the common cell wall polysaccharide. Adsorption studies further revealed serotype-specific capsule binding mediated by distinct isohemagglutinin clones, demonstrating synergistic polyreactivity. Despite this, population-level analyses showed only modest protection: most notably, individuals with blood types B or O (producers of anti-A and anti-A,B isohemagglutinins, respectively) had a 19
Legionella pneumophila is a facultative intracellular bacterium that causes Legionnaires’ disease, one of the most severe manifestations of atypical pneumonia. The capacity of L. pneumophila to cause disease relates with its ability to survive and multiply inside a membrane-bound compartment, the Legionella-containing vacuole (LCV). The remodeling of the LCV into a replication-competent compartment requires the Icm/Dot type 4 secretion system, that translocates over 300 bacterial effector proteins into host cells. Their action is concerted spatially and timely in order to modulate a multitude of host cell targets and thus ensure LCV segregation from the phagolysosomal pathway. The L. pneumophila effector VipA associates with early endosomes and promotes the nucleation of new actin filaments in vitro. Additionally, ectopically produced VipA impairs vesicle trafficking in Saccharomyces cerevisiae. However, the mechanisms underlying its association with the endocytic pathway have remained elusive. In this work, we sought the molecular interacting partners of VipA in early endosomes and explored the effects of this association. We found that VipA interacts directly with two key components of these organelles, the membrane lipid phosphoinositide 3-phosphate (PI3P) and the small GTPase Rab5. Binding to Rab5 requires the NH2-terminal region of VipA but not the COOH-terminal/actin-binding region. Furthermore, binding to Rab5 inhibits VipA-mediated actin polymerization by preventing de novo actin filament formation. These findings offer new insights into the mode of action of VipA and underscore the intricate network of interactions between L. pneumophila effectors and their host cell targets, namely in the endocytic pathway.
Development of an effective vaccine against Neisseria gonorrhoeae comes with unique challenges. After five decades of research, the lack of an FDA-approved vaccine underscores the need for novel immunization strategies. Therefore, in this study, we evaluated a bivalent vaccine compared to two single-antigen formulations containing LtgC or TamA. The tamA and ltgC genes were cloned, expressed and purified. Serum IgG levels against recombinant proteins in gonorrhea patients were assessed by the enzyme-linked immunosorbent assay (ELISA). Mice were immunized with TamA and/or LtgC formulations alongside Monophosphoryl-lipid A (MPLA) adjuvant. Serum and vaginal IgG and IgA levels were measured. Then, the serum bactericidal assay (SBA), and opsonophagocytic activity (OPA) assays were performed. The presence of antigen-specific antibodies in patients’ sera indicates that the recombinant proteins elicit immune responses in natural infection. All formulations elicited robust long-lasting systemic IgG responses, with TamA + MPLA inducing the highest IgG titers. The bivalent formulation showed superior IgA responses in serum, while in vaginal wash, its superiority was not evident. TamA + MPLA and the bivalent formulation showed comparable higher SBA and OPA titers compared to the LtgC + MPLA. TamA and LtgC monovalent formulations induced long-lasting antibodies, with desirable bactericidal effects. TamA + MPLA and the combination group exhibited similar SBA and OPA titers. Despite the half-dose of TamA in the combination, inclusion of LtgC appears to enhance serum bactericidal activity. Thus, future investigations should focus on dose optimization to maximize synergistic effects of the bivalent formulation.
Respiratory cilia play a crucial role in clearing pathogens from the airway, and understanding how Pseudomonas aeruginosa (PA) impairs their function is essential for developing targeted therapies to enhance airway epithelial defense and hamper bacterial invasion. Thus, we investigated the mechanisms by which PA impairs respiratory cilia function and identified pharmacological interventions to restore cilia motility. We used mucociliary differentiated human airway organoids expressing motile cilia on the apical side, to model the initial stage of PA infection. We show that co-culturing of organoids with PA down-regulates the expression of genes associated with cilia formation, structure and function. Electron microscopy confirmed ciliary structural damage and membrane disruption in infected organoids and slowed ciliary beating frequency as quantified by slow-motion video recording. Reduction of cilia function resulted in increased bacterial cell invasion, and we identified the bacterial toxin Pyocyanin as one causative cilia damaging factor. We could further demonstrate, that the selective phosphodiesterase-4 inhibitor Roflumilast can maintain ciliary function and beating frequency upon infection of organoids with PA, thereby reducing bacterial invasion of epithelia in our model system. Our findings emphasize the critical role of motile cilia in innate host defense against PA infection, implying that treatment strategies aimed at restoring ciliary function can enhance epithelial robustness during bacterial invasion. Our human organoid infection model enables the evaluation of cellular and functional responses of the airway epithelium to respiratory pathogens and to test potential therapeutic strategies aimed at restoring ciliary function and improving infection control.
