The impact of existing total anti-toxin B IgG immunity in outcomes of recurrent Clostridioides difficile infection.

Late anti-toxin-B humoral immunity acquired after treatment is important for preventing recurrent Clostridioides difficile infection. We prospectively-measured anti-toxin-B IgG and neutralization titers at diagnosis as potential early predictors of recurrence. High anti-toxin-B-IgG/neutralizing antibodies were associated with short-lasting protection within 6-weeks, however, no difference in recurrence risk was observed by 90-days post-infection.

Novel Biomarkers, Including tcdB PCR Cycle Threshold, for Predicting Recurrent Clostridioides difficile Infection.

Traditional clinical models for predicting recurrent Clostridioides difficile infection do not perform well, likely owing to the complex host-pathogen interactions involved. Accurate risk stratification using novel biomarkers could help prevent recurrence by improving underutilization of effective therapies (i.e., fecal transplant, fidaxomicin, bezlotoxumab). We used a biorepository of 257 hospitalized patients with 24 features collected at diagnosis, including 17 plasma cytokines, total/neutralizing anti-toxin B IgG, stool toxins, and PCR cycle threshold (CT) (a proxy for stool organism burden). The best set of predictors for recurrent infection was selected by Bayesian model averaging for inclusion in a final Bayesian logistic regression model. We then used a large PCR-only data set to confirm the finding that PCR CT predicts recurrence-free survival using Cox proportional hazards regression. The top model-averaged features were (probabilities of >0.05, greatest to least): interleukin 6 (IL-6), PCR CT, endothelial growth factor, IL-8, eotaxin, IL-10, hepatocyte growth factor, and IL-4. The accuracy of the final model was 0.88. Among 1,660 cases with PCR-only data, cycle threshold was significantly associated with recurrence-free survival (hazard ratio, 0.95; P < 0.005). Certain biomarkers associated with C. difficile infection severity were especially important for predicting recurrence; PCR CT and markers of type 2 immunity (endothelial growth factor [EGF], eotaxin) emerged as positive predictors of recurrence, while type 17 immune markers (IL-6, IL-8) were negative predictors. In addition to novel serum biomarkers (particularly, IL-6, EGF, and IL-8), the readily available PCR CT may be critical to augment underperforming clinical models for C. difficile recurrence.

Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis.

The disease severity of Entamoeba histolytica infection ranges from asymptomatic to life-threatening. Recent human and animal data implicate the gut microbiome as a modifier of E. histolytica virulence. Here we have explored the association of the microbiome with susceptibility to amebiasis in infants and in the mouse model of amebic colitis. Dysbiosis occurred symptomatic E. histolytica infection in children, as evidenced by a lower Shannon diversity index of the gut microbiota. To test if dysbiosis was a cause of susceptibility, wild type C57BL/6 mice (which are innately resistant to E. histiolytica infection) were treated with antibiotics prior to cecal challenge with E. histolytica. Compared with untreated mice, antibiotic pre-treated mice had more severe colitis and delayed clearance of E. histolytica. Gut IL-25 and mucus protein Muc2, both shown to provide innate immunity in the mouse model of amebic colitis, were lower in antibiotic pre-treated mice. Moreover, dysbiotic mice had fewer cecal neutrophils and myeloperoxidase activity. Paradoxically, the neutrophil chemoattractant chemokines CXCL1 and CXCL2, as well as IL-1ß, were higher in the colon of mice with antibiotic-induced dysbiosis. Neutrophils from antibiotic pre-treated mice had diminished surface expression of the chemokine receptor CXCR2, potentially explaining their inability to migrate to the site of infection. Blockade of CXCR2 increased susceptibility of control non-antibiotic treated mice to amebiasis. In conclusion, dysbiosis increased the severity of amebic colitis due to decreased neutrophil recruitment to the gut, which was due in part to decreased surface expression on neutrophils of CXCR2.

Neutralization of macrophage migration inhibitory factor improves host survival after Clostridium difficile infection.

Clostridium difficile is an important cause of nosocomial diarrhea in the western world. Toxins (A, B, and binary toxins) generated by C. difficile bacteria damage intestinal epithelial cells. Hallmarks of host response to C. difficile infection (CDI) include upregulation of inflammatory mediators and tissue infiltration by immune cells. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that is known to enhance the host immune response to infectious pathogens. Additionally, MIF can adversely impact host survival to numerous infections. The role of MIF in the pathogenesis of CDI remains poorly understood. Here, we show that patients with CDI had significantly higher circulating MIF compared to patients who had diarrhea but tested negative for C. difficile (non-CDI controls). Similarly, in a mouse model, C. difficile challenge significantly increased levels of plasma and tissue MIF. Antibody-mediated depletion of MIF decreased C. difficile-induced inflammatory responses, clinical disease, and mortality. Together, these results uncover a potential role for MIF in exacerbating CDI and suggest that use of anti-MIF antibodies may represent a therapeutic strategy to curb host inflammatory responses and improve disease outcomes in CDI.

Entamoeba histolytica-Encoded Homolog of Macrophage Migration Inhibitory Factor Contributes to Mucosal Inflammation during Amebic Colitis.

Understanding the mechanisms by which Entamoeba histolytica drives gut inflammation is critical for the development of improved preventive and therapeutic strategies. E. histolytica encodes a homolog of the human cytokine macrophage migration inhibitory factor (MIF). Here, we investigated the role of E. histolytica MIF (EhMIF) during infection. We found that the concentration of fecal EhMIF correlated with the level of intestinal inflammation in persons with intestinal amebiasis. Mice treated with antibodies that specifically block EhMIF had reduced chemokine expression and neutrophil infiltration in the mucosa. In addition to antibody-mediated neutralization, we used a genetic approach to test the effect of EhMIF on mucosal inflammation. Mice infected with parasites overexpressing EhMIF had increased chemokine expression, neutrophil influx, and mucosal damage. Together, these results uncover a specific parasite protein that increases mucosal inflammation, expands our knowledge of host-parasite interaction during amebic colitis, and highlights a potential immunomodulatory target.

Immunolocalization of TAR DNA-binding protein of 43 kDa (TDP-43) in mouse seminiferous epithelium.

TAR DNA-binding protein of 43 kDa (TDP-43) is an evolutionarily conserved, ubiquitously expressed, multi-functional DNA/RNA-binding protein with roles in gene transcription, mRNA splicing, stability, transport, micro RNA biogenesis, and suppression of transposons. Aberrant expression of TDP-43 in testis and sperm was recently shown to be associated with male infertility, which highlights the need to understand better the expression of TDP-43 in the testis. We previously cloned TDP-43 from a mouse testis cDNA library, and showed that it functions as a transcriptional repressor and regulates the precise spatiotemporal expression of the Acrv1 gene, which encodes the acrosomal protein SP-10, during spermatogenesis. Here, we performed immunoblotting and immunohistochemistry of the mouse testis using four separate antibodies recognizing the amino and carboxyl termini of TDP-43. TDP-43 is present in the nuclei of germ cells as well as Sertoli cells. TDP-43 expression begins in type B/intermediate spermatogonia, peaks in preleptotene spermatocytes, and becomes undetectable in leptotene and zygotene spermatocytes. Pachytene spermatocytes and early round spermatids again express TDP-43, but its abundance diminishes later in spermatids (at steps 5-8). Interestingly, two of the four antibodies showed TDP-43 expression in spermatids at steps 9-10, which coincides with the initial phase of the histone-to-protamine transition. Immunoreactivity patterns observed in the study suggest that TDP-43 assumes different conformational states at different stages of spermatogenesis. TDP-43 pathology has been extensively studied in the context of neurodegenerative diseases; its role in spermatogenesis warrants further detailed investigation of the involvement of TDP-43 in male infertility.

The macrophage migration inhibitory factor homolog of Entamoeba histolytica binds to and immunomodulates host macrophages.

The host inflammatory response contributes to the tissue damage that occurs during amebic colitis, with tumor necrosis factor alpha (TNF-α) being a key mediator of the gut inflammation observed. Mammalian macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays an important role in the exacerbation of a wide range of inflammatory diseases, including colitis. We identified a MIF gene homolog in the Entamoeba histolytica genome, raising the question of whether E. histolytica MIF (EhMIF) has proinflammatory activity similar to that of mammalian MIF. In this report, we describe the first functional characterization of EhMIF. Antibodies were prepared against recombinantly expressed EhMIF and used to demonstrate that EhMIF is expressed as a 12-kDa protein localized to the cytoplasm of trophozoites. In a manner similar to that of mammalian MIF, EhMIF interacted with the MIF receptor CD74 and bound to macrophages. EhMIF induced interleukin-6 (IL-6) production. In addition, EhMIF enhanced TNF-α secretion by amplifying TNF-α production by lipopolysaccharide (LPS)-stimulated macrophages and by inhibiting the glucocorticoid-mediated suppression of TNF-α secretion. EhMIF was expressed during human infection, as evidenced by the presence of anti-EhMIF antibodies in the sera of children living in an area where E. histolytica infection is endemic. Anti-EhMIF antibodies did not cross-react with human MIF. The ability of EhMIF to modulate host macrophage function may promote an exaggerated proinflammatory immune response and contribute to the tissue damage seen in amebic colitis.

Inflammatory biomarkers and physiomarkers of late-onset sepsis and necrotizing enterocolitis in premature infants.

Background: Early diagnosis of late-onset sepsis (LOS) and necrotizing enterocolitis (NEC) in very low birth weight (VLBW, <1,500 g) infants is challenging due to non-specific clinical signs. Inflammatory biomarkers increase in response to infection, but non-infectious conditions also cause inflammation. Cardiorespiratory data contain physiological biomarkers, or physiomarkers, of sepsis that may be useful in combination with inflammatory hematologic biomarkers for sepsis diagnosis. Objectives: To determine whether inflammatory biomarkers measured at the time of LOS or NEC diagnosis differ from times without infection and whether biomarkers correlate with cardiorespiratory sepsis physiomarkers in VLBW infants. Methods: Remnant plasma sample collection from VLBW infants occurred with blood draws for routine laboratory testing and suspected sepsis. We analyzed 11 inflammatory biomarkers and a pulse oximetry sepsis warning score (POWS). We compared biomarker levels obtained at the time of gram-negative (GN) bacteremia or NEC, gram-positive (GP) bacteremia, negative blood cultures, and no suspected infection. Results: We analyzed 188 samples in 54 VLBW infants. Several biomarkers were increased at the time of GN LOS or NEC diagnosis compared with all other samples. POWS was higher in patients with LOS and correlated with five biomarkers. IL-6 had 78% specificity at 100% sensitivity to detect GN LOS or NEC and added information to POWS. Conclusions: Inflammatory plasma biomarkers discriminate sepsis due to GN bacteremia or NEC and correlate with cardiorespiratory physiomarkers.

Immunogenicity and safety of an Entamoeba histolytica adjuvanted protein vaccine candidate (LecA+GLA-3M-052 liposomes) in rhesus macaques.

Entamoeba histolytica, the causative agent of amebiasis, is one of the top three parasitic causes of mortality worldwide. However, no vaccine exists against amebiasis. Using a lead candidate vaccine containing the LecA fragment of Gal-lectin and GLA-3M-052 liposome adjuvant, we immunized rhesus macaques via intranasal or intramuscular routes. The vaccine elicited high-avidity functional humoral responses as seen by the inhibition of amebic attachment to mammalian target cells by plasma and stool antibodies. Importantly, antigen-specific IFN-γ-secreting peripheral blood mononuclear cells (PBMCs) and IgG/IgA memory B cells (BMEM) were detected in immunized animals. Furthermore, antigen-specific antibody and cellular responses were maintained for at least 8 months after the final immunization as observed by robust LecA-specific BMEM as well as IFN-γ+ PBMC responses. Overall, both intranasal and intramuscular immunizations elicited a durable and functional response in systemic and mucosal compartments, which supports advancing the LecA+GLA-3M-052 liposome vaccine candidate to clinical testing.

Binary Toxin Expression by Clostridioides difficile Is Associated With Worse Disease.

BACKGROUND: The incidence of Clostridioides difficile infection (CDI) has increased over the past 2 decades and is considered an urgent threat by the Centers for Disease Control and Prevention. Hypervirulent strains such as ribotype 027, which possess genes for the additional toxin C. difficile binary toxin (CDT), are contributing to increased morbidity and mortality. METHODS: We retrospectively tested stool from 215 CDI patients for CDT by enzyme-linked immunosorbent assay (ELISA). Stratifying patients by CDT status, we assessed if disease severity and clinical outcomes correlated with CDT positivity. Additionally, we completed quantitative PCR (PCR) DNA extracted from patient stool to detect cdtB gene. Lastly, we performed 16 S rRNA gene sequencing to examine if CDT-positive samples had an altered fecal microbiota. RESULTS: We found that patients with CdtB, the pore-forming component of CDT, detected in their stool by ELISA, were more likely to have severe disease with higher 90-day mortality. CDT-positive patients also had higher C. difficile bacterial burden and white blood cell counts. There was no significant difference in gut microbiome diversity between CDT-positive and -negative patients. CONCLUSIONS: Patients with fecal samples that were positive for CDT had increased disease severity and worse clinical outcomes. Utilization of PCR and testing for C. difficile toxins A and B may not reveal the entire picture when diagnosing CDI; detection of CDT-expressing strains is valuable in identifying patients at risk of more severe disease.

Development of COVID-19 vaccine using a dual Toll-like receptor ligand liposome adjuvant.

We developed a SARS-CoV-2 spike subunit vaccine formulation containing dual TLR ligand liposome adjuvant. The vaccine-induced robust systemic neutralizing antibodies and completely protected mice from a lethal challenge. Two immunizations protected against lung injury and cleared the virus from lungs upon challenge. The adjuvanted vaccine also elicited systemic and local anti-Spike IgA which can be an important feature for a COVID-19 vaccine.

Optimizing a Multi-Component Intranasal Entamoeba Histolytica Vaccine Formulation Using a Design of Experiments Strategy.

Amebiasis is a neglected tropical disease caused by Entamoeba histolytica. Although the disease burden varies geographically, amebiasis is estimated to account for some 55,000 deaths and millions of infections globally per year. Children and travelers are among the groups with the greatest risk of infection. There are currently no licensed vaccines for prevention of amebiasis, although key immune correlates for protection have been proposed from observational studies in humans. We previously described the development of a liposomal adjuvant formulation containing two synthetic TLR ligands (GLA and 3M-052) that enhanced antigen-specific fecal IgA, serum IgG2a, a mixed IFNγ and IL-17A cytokine profile from splenocytes, and protective efficacy following intranasal administration with the LecA antigen. By applying a statistical design of experiments (DOE) and desirability function approach, we now describe the optimization of the dose of each vaccine formulation component (LecA, GLA, 3M-052, and liposome) as well as the excipient composition (acyl chain length and saturation; PEGylated lipid:phospholipid ratio; and presence of antioxidant, tonicity, or viscosity agents) to maximize desired immunogenicity characteristics while maintaining physicochemical stability. This DOE/desirability index approach led to the identification of a lead candidate composition that demonstrated immune response durability and protective efficacy in the mouse model, as well as an assessment of the impact of each active vaccine formulation component on protection. Thus, we demonstrate that both GLA and 3M-052 are required for statistically significant protective efficacy. We also show that immunogenicity and efficacy results differ in female vs male mice, and the differences appear to be at least partly associated with adjuvant formulation composition.

IL-13 is a driver of COVID-19 severity.

Immune dysregulation is characteristic of the more severe stages of SARS-CoV-2 infection. Understanding the mechanisms by which the immune system contributes to COVID-19 severity may open new avenues to treatment. Here, we report that elevated IL-13 was associated with the need for mechanical ventilation in 2 independent patient cohorts. In addition, patients who acquired COVID-19 while prescribed Dupilumab, a mAb that blocks IL-13 and IL-4 signaling, had less severe disease. In SARS-CoV-2-infected mice, IL-13 neutralization reduced death and disease severity without affecting viral load, demonstrating an immunopathogenic role for this cytokine. Following anti-IL-13 treatment in infected mice, hyaluronan synthase 1 (Has1) was the most downregulated gene, and accumulation of the hyaluronan (HA) polysaccharide was decreased in the lung. In patients with COVID-19, HA was increased in the lungs and plasma. Blockade of the HA receptor, CD44, reduced mortality in infected mice, supporting the importance of HA as a pathogenic mediator. Finally, HA was directly induced in the lungs of mice by administration of IL-13, indicating a new role for IL-13 in lung disease. Understanding the role of IL-13 and HA has important implications for therapy of COVID-19 and, potentially, other pulmonary diseases. IL-13 levels were elevated in patients with severe COVID-19. In a mouse model of the disease, IL-13 neutralization reduced the disease and decreased lung HA deposition. Administration of IL-13-induced HA in the lung. Blockade of the HA receptor CD44 prevented mortality, highlighting a potentially novel mechanism for IL-13-mediated HA synthesis in pulmonary pathology.

IL-13 is a driver of COVID-19 severity.

Immune dysregulation is characteristic of the more severe stages of SARS-CoV-2 infection. Understanding the mechanisms by which the immune system contributes to COVID-19 severity may open new avenues to treatment. Here we report that elevated interleukin-13 (IL-13) was associated with the need for mechanical ventilation in two independent patient cohorts. In addition, patients who acquired COVID-19 while prescribed Dupilumab had less severe disease. In SARS-CoV-2 infected mice, IL-13 neutralization reduced death and disease severity without affecting viral load, demonstrating an immunopathogenic role for this cytokine. Following anti-IL-13 treatment in infected mice, in the lung, hyaluronan synthase 1 (Has1) was the most downregulated gene and hyaluronan accumulation was decreased. Blockade of the hyaluronan receptor, CD44, reduced mortality in infected mice, supporting the importance of hyaluronan as a pathogenic mediator, and indicating a new role for IL-13 in lung disease. Understanding the role of IL-13 and hyaluronan has important implications for therapy of COVID-19 and potentially other pulmonary diseases.

Immune Profiling To Predict Outcome of Clostridioides difficile Infection.

There is a pressing need for biomarker-based models to predict mortality from and recurrence of Clostridioides difficile infection (CDI). Risk stratification would enable targeted interventions such as fecal microbiota transplant, antitoxin antibodies, and colectomy for those at highest risk. Because severity of CDI is associated with the immune response, we immune profiled patients at the time of diagnosis. The levels of 17 cytokines in plasma were measured in 341 CDI inpatients. The primary outcome of interest was 90-day mortality. Increased tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), C-C motif chemokine ligand 5 (CCL-5), suppression of tumorigenicity 2 receptor (sST-2), IL-8, and IL-15 predicted mortality by univariate analysis. After adjusting for demographics and clinical characteristics, the mortality risk (as indicated by the hazard ratio [HR]) was higher for patients in the top 25th percentile for TNF-α (HR = 8.35, P = 0.005) and IL-8 (HR = 4.45, P = 0.01) and lower for CCL-5 (HR = 0.18, P ≤ 0.008). A logistic regression risk prediction model was developed and had an area under the receiver operating characteristic curve (AUC) of 0.91 for 90-day mortality and 0.77 for 90-day recurrence. While limited by being single site and retrospective, our work resulted in a model with a substantially greater predictive ability than white blood cell count. In conclusion, immune profiling demonstrated differences between patients in their response to CDI, offering the promise for precision medicine individualized treatment.IMPORTANCEClostridioides difficile infection is the most common health care-associated infection in the United States with more than 20% patients experiencing symptomatic recurrence. The complex nature of host-bacterium interactions makes it difficult to predict the course of the disease based solely on clinical parameters. In the present study, we built a robust prediction model using representative plasma biomarkers and clinical parameters for 90-day all-cause mortality. Risk prediction based on immune biomarkers and clinical variables may contribute to treatment selection for patients as well as provide insight into the role of immune system in C. difficile pathogenesis.

Development of the Gateway system for cloning and expressing genes in Entamoeba histolytica.

The early branching eukaryote Entamoeba histolytica is a human parasite that is the etiologic agent of amebic dysentery and liver abscess. The sequencing of the E. histolytica genome combined with the development of an E. histolytica microarray has resulted in the identification of several distinct gene expression profiles associated with virulence. The function of many modulated transcripts is unknown and their role in pathogenicity is unclear. They however represent a pool of potential virulence factors that could be targets for the development of novel therapeutics. Efficient tools and methods to characterize these novel virulence-associated genes and proteins would be beneficial. Here we report the use of the Gateway((R)) cloning system to generate the E. histolytica expression vector pAH-DEST. To test the usefulness of this system, the vector was used to construct a plasmid containing a recombinant version of the locus EHI_144490, which encoded a protein of unknown function. The recombinant gene was expressed and the recombinant protein, which was strep-myc-tagged, showed a cytoplasmic localization in transfected trophozoites. This expression vector with the Gateway((R)) system should facilitate investigation into the functions of novel proteins in E. histolytica.

Characterization of an Entamoeba histolytica high-mobility-group box protein induced during intestinal infection.

The unicellular eukaryote Entamoeba histolytica is a human parasite that causes amebic dysentery and liver abscess. A genome-wide analysis of gene expression modulated by intestinal colonization and invasion identified an upregulated transcript that encoded a putative high-mobility-group box (HMGB) protein, EhHMGB1. We tested if EhHMGB1 encoded a functional HMGB protein and determined its role in control of parasite gene expression. Recombinant EhHMGB1 was able to bend DNA in vitro, a characteristic of HMGB proteins. Core conserved residues required for DNA bending activity in other HMGB proteins were demonstrated by mutational analysis to be essential for EhHMGB1 activity. EhHMGB1 was also able to enhance the binding of human p53 to its cognate DNA sequence in vitro, which is expected for an HMGB1 protein. Confocal microscopy, using antibodies against the recombinant protein, confirmed its nuclear localization. Overexpression of EhHMGB1 in HM1:IMSS trophozoites led to modulation of 33 transcripts involved in a variety of cellular functions. Of these, 20 were also modulated at either day 1 or day 29 in the mouse model of intestinal amebiasis. Notably, four transcripts with known roles in virulence, including two encoding Gal/GalNAc lectin light chains, were modulated in response to EhHMGB1 overexpression. We concluded that EhHMGB1 was a bona fide HMGB protein with the capacity to recapitulate part of the modulation of parasite gene expression seen during adaptation to the host intestine.

Colitis-Induced Th17 Cells Increase the Risk for Severe Subsequent Clostridium difficile Infection.

Clostridium difficile infection (CDI) is the number one hospital-acquired infection in the United States. CDI is more common and severe in inflammatory bowel disease patients. Here, we studied the mechanism by which prior colitis exacerbates CDI. Mice were given dextran sulfate sodium (DSS) colitis, recovered for 2 weeks, and then were infected with C. difficile. Mortality and CDI severity were increased in DSS-treated mice compared to controls. Severe CDI is dependent on CD4+ T cells, which persist after colitis-associated inflammation subsides. Adoptive transfer of Th17 cells to naive mice is sufficient to increase CDI-associated mortality through elevated IL-17 production. Finally, in humans, the Th17 cytokines IL-6 and IL-23 associate with severe CDI, and patients with high serum IL-6 are 7.6 times more likely to die post infection. These findings establish a central role for Th17 cells in CDI pathogenesis following colitis and identify them as a potential target for preventing severe disease.

IL-33 drives group 2 innate lymphoid cell-mediated protection during Clostridium difficile infection.

Clostridium difficile (C. difficile) incidence has tripled over the past 15 years and is attributed to the emergence of hypervirulent strains. While it is clear that C. difficile toxins cause damaging colonic inflammation, the immune mechanisms protecting from tissue damage require further investigation. Through a transcriptome analysis, we identify IL-33 as an immune target upregulated in response to hypervirulent C. difficile. We demonstrate that IL-33 prevents C. difficile-associated mortality and epithelial disruption independently of bacterial burden or toxin expression. IL-33 drives colonic group 2 innate lymphoid cell (ILC2) activation during infection and IL-33 activated ILC2s are sufficient to prevent disease. Furthermore, intestinal IL-33 expression is regulated by the microbiota as fecal microbiota transplantation (FMT) rescues antibiotic-associated depletion of IL-33. Lastly, dysregulated IL-33 signaling via the decoy receptor, sST2, predicts C. difficile-associated mortality in human patients. Thus, IL-33 signaling to ILC2s is an important mechanism of defense from C. difficile colitis.

Adjuvant composition and delivery route shape immune response quality and protective efficacy of a recombinant vaccine for Entamoeba histolytica.

Amebiasis caused by Entamoeba histolytica is the third leading cause of parasitic mortality globally, with some 100,000 deaths annually, primarily among young children. Protective immunity to amebiasis is associated with fecal IgA and IFN-γ in humans; however, no vaccine exists. We have previously identified recombinant LecA as a potential protective vaccine antigen. Here we describe the development of a stable, manufacturable PEGylated liposomal adjuvant formulation containing two synthetic Toll-like receptor (TLR) ligands: GLA (TLR4) and 3M-052 (TLR7/8). The liposomes stimulated production of monocyte/macrophage chemoattractants MCP-1 and Mip-1ß, and Th1-associated cytokines IL-12p70 and IFN-γ from human whole blood dependent on TLR ligand composition and dose. The liposomes also demonstrated acceptable physicochemical compatibility with the recombinant LecA antigen. Whereas mice immunized with LecA and GLA-liposomes demonstrated enhanced antigen-specific fecal IgA titers, mice immunized with LecA and 3M-052-liposomes showed a stronger Th1 immune profile. Liposomes containing GLA and 3M-052 together elicited both LecA-specific fecal IgA and Th1 immune responses. Furthermore, the quality of the immune response could be modulated with modifications to the liposomal formulation based on PEG length. Compared to subcutaneous administration, the optimized liposome adjuvant composition with LecA antigen administered intranasally resulted in significantly enhanced fecal IgA, serum IgG2a, as well as systemic IFN-γ and IL-17A levels in mice. The optimized intranasal regimen provided greater than 80% protection from disease as measured by parasite antigen in the colon. This work demonstrates the physicochemical and immunological characterization of an optimized mucosal adjuvant system containing a combination of TLR ligands with complementary activities and illustrates the importance of adjuvant composition and route of delivery to enhance a multifaceted and protective immune response to amebiasis.