Limulus Amebocyte Lysate Research Articles

Endotoxin testing: past and contemporary methods, ecological impact, and developing of alternatives

In the biotechnology industry, endotoxin testing is a ubiquitous procedure that has a fascinating history. It has impacted not only patients, but also the environment, and has the potential to be improved upon in the future. It is crucial that any drugs introduced to the body maintain a high degree of sterility, lest the drug designed to save a life is the cause of a different life-threatening disease. The predominant method used today is Limulus Amoebocyte Lysate, commonly referred to as LAL, which uses horseshoe crab blood. Since LAL is obtained by harvesting the blood of live horseshoe crabs, there is an ecological impact that comes with this type of endotoxin testing. Endotoxin testing with LAL is a vital and effective method of ensuring the safety of drug products, but it is a limited resource. Because of its necessity, it is pertinent that we develop alternatives to LAL in case it is no longer a viable option. Any alternatives should have an efficacy similar to that of LAL, but should not depend as heavily on harvesting the blood of horseshoe crabs since it is unsustainable in the long term

Detection of endotoxins from selected drinking water microbiota using an LAL-based assay and its implications for human health.

Endotoxins are pyrogenic lipopolysaccharides from Gram-negative bacteria that are known to induce fever, septic shock, and multiple organ failure, posing a substantial risk to human health. Drinking water systems are especially prone to home microbiomes containing a large variety of Gram-negative bacteria. Consumption of water from these systems in developed countries is generally regarded as non-hazardous to humans due to the low number of non-pathogenic bacterial cells per milliliter and oral admission. To assess potential risks posed by endotoxins in drinking water systems, we conducted a conventional microbiological investigation on a local community water system in the north of Germany and mined the resulting data to investigate the endotoxin contents of some of the most abundant microbiota found during these analyses. Using a Limulus amoebocyte lysate (LAL) -based endotoxin detection method, average normalized endotoxin content was determined. Although the average culturable amounts of microbiota in the drinking water system were insufficient to exert endotoxin levels critical to human health, peaks and acute contaminations may pose substantial health risks.

Safety Assessment: a Comparative Analysis of Quantitative Content of Bacterial Endotoxins and Evaluation of Pyrogenicity of the Kazakhstan Vaccine QazCovid-in® against COVID-19.

We measured the levels of bacterial endotoxins in the bulk vaccine product (BVP) and finished vaccine QazCovid-in® and evaluated the effect of aluminum hydroxide (adjuvant) on the results of LAL test and pyrogenicity of samples in vivo (in rabbits receiving intravenous injection into the marginal ear vein). Administration of BVP with LPS resulted in a dose-dependent increase in body temperature in rabbits similar to that caused by LPS alone, which suggests that aluminum hydroxide in the vaccine did not affect the pyrogenic response in rabbits. Moreover, the LAL test showed that the aluminum hydroxide did not hinder LPS activity after serial dilution of samples.

Heparin enables the reliable detection of endotoxin in human serum samples using the Limulus amebocyte lysate assay

The determination of lipopolysaccharide (endotoxin) in serum or plasma samples using Limulus amebocyte lysate (LAL)-based assays is currently not sufficiently reliable in clinical diagnostics due to numerous interfering factors that strongly reduce the recovery of LPS in clinical samples. The specific plasma components responsible for the endotoxin neutralizing capacity of human blood remain to be identified. There are indications that certain endotoxin-neutralizing proteins or peptides, which are part of the host defense peptides/proteins of the innate immune system may be responsible for this effect. Based on our finding that several antimicrobial peptides can be neutralized by the polyanion heparin, we developed a heparin-containing diluent for serum and plasma samples, which enables reliable quantification of LPS measurement in clinical samples using the LAL assay. In a preclinical study involving 40 donors, this improved protocol yielded an over eightfold increase in LPS recovery in serum samples, as compared to the standard protocol. This modified protocol of sample pretreatment could make LPS measurement a valuable tool in medical diagnostics.

Rational design of antibodies and development of a novel method for (1-3)-β-D glucan detection as an alternative to Limulus amebocyte lysate assay.

With advances in medicine, increasing medical interventions have increased the risk of invasive fungal disease development. (1-3)-β-D glucan (BDG) is a common fungal biomarker in serological tests. However, the scarcity of Limulus resources for BDG detection poses a challenge. This study addresses the need for an alternative to Limulus amebocyte lysate by using BDG mutant antibody for chemiluminescence detection. The wild-type BDG antibody was obtained by immunizing rabbits. An optimal V52HI/N34L Y mutant antibody, which has increased 3.7-fold of the testing efficiency compared to the wild-type antibody, was first achieved by mutating "hot-spot" residues that contribute to strong non-covalent bonds, as determined by alanine scanning and molecular dynamics simulation. The mutant was then applied to develop the magnetic particle chemiluminescence method. 574 clinical samples were tested using the developed method, with a cutoff value of 95 pg/mL set by Limulus amebocyte lysate. The receiver operating characteristic curve demonstrated an area under the curve of 0.905 (95% CI: 0.880-0.929). Chemiluminescence detected an antigen concentration of 89.98 pg/mL, exhibiting a sensitivity of 83.33% and specificity of 89.76%. In conclusion, the results showed a good agreement with Limulus amebocyte lysate and demonstrated the feasibility of using BDG mutant antibodies for invasive fungal disease diagnosis. The new method based on chemiluminescence for detecting BDG could shorten the sample-to-result time to approximately 30 min, rescue Limulus from being endangered and is resource efficient in terms of equipment and the non-use of a skilled technician.

Current Considerations and Practical Solutions for Overcoming Nanoparticle Interference with LAL Assays and Minimizing Endotoxin Contamination.

Monitoring endotoxin contamination in drugs and medical devices is required to avoid pyrogenic responses and septic shock in patients receiving these products. Endotoxin contamination of engineered nanomaterials and nanotechnology-based medical products represents a significant translational hurdle. Nanoparticles often interfere with an in vitro limulus amebocyte lysate (LAL) assay commonly used in the pharmaceutical industry for the detection and quantification of endotoxin. Such interference challenges the preclinical development of nanotechnology-formulated drugs and medical devices containing engineered nanomaterials. Protocols for the analysis of nanoparticles using LAL assays have been reported before. Here, we discuss considerations for selecting an LAL format and describe a few experimental approaches for overcoming nanoparticle interference with the LAL assays to obtain more accurate estimations of endotoxin contamination in nanotechnology-based products. The discussed approaches do not solve all types of nanoparticle interference with the LAL assays but could be used as a starting point to address the problem. This chapter also describes approaches to prevent endotoxin contamination in nanotechnology-formulated products.

Dual-functional PCN-242 (Fe2Co) MOF for sensitive bacterial endotoxin detection.

Endotoxin detection is paramount for monitoring bacterial contamination in food, pharmaceuticals, and clinical diagnostics. The limulus amebocyte lysate (LAL) test, which relies on horseshoe crab blood, has long been the gold standard for endotoxin detection. However, the widespread adoption of this method is constrained by ethical concerns and the high costs associated with harvesting endangered species. Although nanozyme-based colorimetric methods present a more cost-effective and straightforward alternative, their application is limited by suboptimal selectivity and sensitivity. In this study, we report the synthesis and rigorous characterization of the bimetallic PCN-242 (Fe2Co) metal-organic framework (MOF), synthesized using 2-amino terephthalic acid and a pre-synthesized [Fe2Co(μ3-O)(CH3COO)6] cluster. Steady-state kinetic analyses revealed that PCN-242 (Fe2Co) MOF exhibits a significantly higher affinity for hydrogen peroxide (H2O2) compared to horseradish peroxidase (HRP) and other iron-based MOFs. The development of a PCN-242 (Fe2Co)-based colorimetric sensor demonstrated a low limit of detection (LOD) of 1.36 μg mL-1 for endotoxins, with excellent selectivity and reproducibility, thereby enabling effective detection of bacterial endotoxins. Recognizing the potential of the PCN-242 (Fe2Co) MOF beyond endotoxin detection, we explored its utility in glucose biosensing. Moreover, incorporating glucose oxidase (GOx) into the PCN-242 (Fe2Co) MOF framework further enhanced its peroxidase-like catalytic activity. This integration enabled sensitive glucose detection, achieving LODs of 4.24 μM for glucose and 2.2 μM for H2O2 within a linear range of 1 to 150 μM. The dual functionality of PCN-242 (Fe2Co) MOF as a peroxidase mimic and biosensor platform highlights its potential for advanced catalytic and diagnostic applications, offering a versatile and ethical alternative to conventional methods.

Eliminating Lipopolysaccharide (LPS) Using Lactic Acid Bacteria (LAB).

Gram-negative bacteria such as Escherichia coli, among intestinal bacteria, have lipopolysaccharide (LPS), which induces inflammation of human intestines. However, lactic acid bacteria (LAB) can improve human intraintestinal conditions. One reason is that ingestion of LAB prevents bacterial diarrhea. This chapter describes a method of LPS elimination using lactic acid bacteria (LAB). First, the LPS concentration is assayed using an LPS assay kit with the limulus cascade reaction made by limulus amebocyte lysate. Some LABs, four bacillus strains and one coccus strain, have LPS-elimination activity. Particularly, the coccus strain Pediococcus pentosaceus eliminates LPS to 43%. The cells fractionate and eliminate four fractions: the extracellular fraction, cell membrane fraction, cytoplasm fraction, and cell wall fraction. Only the cell wall digesting fraction eliminates LPS to 45%. Results confirm that the LAB eliminates all LPS having O-antigen under a low-sugar medium condition at temperatures of 15-30°C. This method can be used for assay of LPS elimination by LABs exactly and easily for the probiotics field.

Detection of Beta-Glucan Contamination in Nanoparticle Formulations.

Beta-glucans with diverse chemical structures are produced by a variety of microorganisms and are commonly found in microbial cell walls. β-(1,3)-D-glucans are present in yeast and fungi, and, for this reason, their traces are commonly used as a sign of yeast or fungal infection or contamination. Despite being less immunologically active than endotoxins, beta-glucans are pro-inflammatory and can activate cytokines and other immunological responses via their cognate pattern recognition receptors. Unlike endotoxins, there is no established threshold pyrogen dose for beta-glucans; as such, their quantity in pharmaceutical products is not regulated. Nevertheless, regulatory agencies recognize the potential contribution of beta-glucans to the immunogenicity of protein-containing drug products and recommend assessing beta-glucans to aid the interpretation of immunotoxicity studies and assess the risk of immunogenicity. The protocol for the detection and quantification of β-(1,3)-D-glucans in nanoparticle formulations is based on a modified limulus amoebocyte lysate assay. The results of this test are used to inform immunotoxicity studies of nanotechnology-based drug products.

Demonstrating Results Equivalence of Bacterial Endotoxins Test Methods.

The LAL test has been the "gold standard" for endotoxin testing ever since it was first published as USP Chapter <85> in 1980. Since then, a number of innovative methods have been proposed to augment or replace the LAL test. However novel an alternate test method might be, we must be cognizant of our obligation to patient safety and heed compendial and compliance requirements to demonstrate results equivalence between any candidate (alternate) method and a predicate (compendial) test method. The following discussion explores the concepts of "equivalent", "different" and "not different", proposes the use of a two one sided test (TOST) for the demonstration of equivalence and provides examples of calculations using commercially available software to assess data on alternative endotoxins detection methods that have been published in the public domain1.

Clinical efficacy of endodontic protocols on reducing cultivable bacteria and endotoxin in infected root canal in patients submitted to head and neck radiotherapy: a randomised clinical trial.

Assess the efficacy of biomechanical preparation using a reciprocating system followed by final irrigation protocols, then intracanal medication, on reducing endotoxins and cultivable bacteria of infected teeth in irradiated patients. Twenty-two infected single-rooted canals in patients submitted to head and neck radiotherapy were prepared by reciprocating motion and 2.5% NaOCl. Patients were randomly divided into two groups of 11 patients before the final irrigation protocol: apical positive pressure (APP) or passive ultrasonic activation (PUA). Both groups were treated in two sessions, using Ca(OH)2 as intracanal medication for 14 days. Root canal content sampling was performed after canal access (S1), after biomechanical preparation plus the irrigation protocol (S2), and after intracanal medication (S3). Chromogenic limulus amoebocyte lysate assay measured endotoxin levels (EU/mL), and bacterial load was determined by culture techniques (CFU/mL). Treatment protocols reduced bacterial counts after S2 in both groups (p = 0.01). S3 differed from S1 (p = 0.01), but not from S2 (p = 0.4). Endotoxin levels were reduced in both groups after S2 (P = 0.03) and were lower in S3 than in S2, with significant differences in the APP group (p = 0.03). Biomechanical preparation using a reciprocating system and 2.5% NaOCl in irradiated teeth, followed by the irrigation protocol (APP or PUA), demonstrated efficacy in reducing endodontic contaminants. Ca(OH)2 as intracanal medication should be performed in irradiated patients with infected root canals. This clinical study demonstrated that endodontic treatment in irradiated patients is efficacious at reducing bacterial load and endotoxin levels.

Neisseria meningitidis accumulate in large organs during meningococcal sepsis.

Neisseria meningitidis (Nm) is the cause of epidemic meningitis and fulminant meningococcal septicemia. The clinical presentations and outcome of meningococcal septic shock is closely related to the circulating levels of lipopolysaccharides (LPS) and of Neisseria meningitidis DNA (Nm DNA). We have previously explored the distribution of Nm DNA in tissues from large organs of patients dying of meningococcal septic shock and in a porcine meningococcal septic shock model. 1) To explore the feasibility of measuring LPS levels in tissues from the large organs in patients with meningococcal septic shock and in a porcine meningococcal septic shock model. 2) To evaluate the extent of contamination of non-specific LPS during the preparation of tissue samples. Plasma, serum, and fresh frozen (FF) tissue samples from the large organs of three patients with lethal meningococcal septic shock and two patients with lethal pneumococcal disease. Samples from a porcine meningococcal septic shock model were included. Frozen tissue samples were thawed, homogenized, and prepared for quantification of LPS by Pyrochrome® Limulus Amoebocyte Lysate (LAL) assay. N. meningitidis DNA and LPS was detected in FF tissue samples from large organs in all patients with meningococcal septic shock. The lungs are the organs with the highest LPS and Nm DNA concentration followed by the heart in two of the three meningococcal shock patients. Nm DNA was not detected in any plasma or tissue sample from patients with lethal pneumococcal infection. LPS was detected at a low level in all FF tissues from the two patients with lethal pneumococcal disease. The experimental porcine meningococcal septic shock model indicates that also in porcinis the highest LPS and Nm DNA concentration are detected in lungs tissue samples. The quantification analysis showed that the highest concentration of both Nm DNA and LPS are in the organs and not in the circulation of patients with lethal meningococcal septic shock. This was also shown in the experimental porcine meningococcal septic shock model. Our results suggest that LPS can be quantified in mammalian tissues by using the LAL assay.

Pre-Clinical Safety and Immunogenicity Study of a Coronavirus Protein-Based Subunit Vaccine for COVID-19.

Creating an effective and safe vaccine is critical to fighting the coronavirus infection successfully. Several types of COVID-19 vaccines exist, including inactivated, live attenuated, recombinant, synthetic peptide, virus-like particle-based, DNA and mRNA-based, and sub-unit vaccines containing purified immunogenic viral proteins. However, the scale and speed at which COVID-19 is spreading demonstrate a global public demand for an effective prophylaxis that must be supplied more. The developed products promise a bright future for SARS-CoV-2 prevention; however, evidence of safety and immunogenicity is mandatory before any vaccine can be produced. In this paper, we report on the results of our work examining the safety, toxicity, immunizing dose choice, and immunogenicity of QazCoVac-P, a Kazakhstan-made sub-unit vaccine for COVID-19. First, we looked into the product's safety profile by assessing its pyrogenicity in vaccinated rabbit models and using the LAL (limulus amebocyte lysate) test. We examined the vaccine's acute and sub-chronic toxicity on BALB/c mice and rats. The vaccine did not cause clinically significant toxicity-related changes or symptoms in our toxicity experiments. Finally, we performed a double immunization of mice, ferrets, Syrian hamsters, and rhesus macaques (Macaca mulatta). We used ELISA to measure antibody titers with the maximum mean geometric titer of antibodies in the animals' blood sera totaling approximately 8 log2. The results of this and other studies warrant recommending the QazCoVac-P vaccine for clinical trials.

Effect of Surgery on Postoperative Levels of the Gut Homeostasis-Regulating Enzyme Intestinal Alkaline Phosphatase.

Intestinal homeostasis is a crucial factor for complication-free short- and long-term postoperative recovery. The brush border enzyme intestinal alkaline phosphatase (IAP) is an important regulator of gut barrier function and intestinal homeostasis and prevents endotoxemia by detoxifying lipopolysaccharides (LPSs). As IAP is predominantly secreted by enterocytes in the duodenum, we hypothesized that pancreaticoduodenectomy (PD) leads to a significantly stronger decrease in IAP than other major abdominal surgery. Pre- and postoperative blood, stool, and intestinal samples were collected from patients undergoing PD, as well as other major surgical procedures without duodenectomy. The samples were analyzed using enzyme histochemistry, the para -nitrophenyl phosphate method for IAP, and the limulus amebocyte lysate assay for LPS. Overall, 88 patients were prospectively enrolled in the study. Fecal IAP activity negatively correlated with serum LPS (r = -0.3603, p = 0.0006). PD led to a significant decline in IAP compared to preoperative baseline levels (p < 0.0001). The decline in IAP correlated with the length of proximal small intestinal resection (r = 0.4271, p = 0.0034). Compared to controls, PD was associated with a much more pronounced reduction in IAP-also after adjusting for surgical trauma (operative time, blood loss; r = 0.4598, p = 0.0086). Simultaneously, PD triggered a clearly more prominent increase in serum LPS compared to controls (p = 0.0001). Increased postoperative LPS was associated with an elongated hospitalization (r = 0.7534, p = 0.0062) and more prominent in pancreatic cancer (p = 0.0009). Based upon the functional roles for IAP, supplementation with exogenous IAP might be a new treatment option to improve short- and long-term outcome after PD.

Recovering What Matters: High Protein Recovery after Endotoxin Removal from LPS-Contaminated Formulations Using Novel Anti-Lipid A Antibody Microparticle Conjugates.

Endotoxins or lipopolysaccharides (LPS), found in the outer membrane of Gram-negative bacterial cell walls, can stimulate the human innate immune system, leading to life-threatening symptoms. Therefore, regulatory limits for endotoxin content apply to injectable pharmaceuticals, and excess LPS must be removed before commercialization. The majority of available endotoxin removal systems are based on the non-specific adsorption of LPS to charged and/or hydrophobic surfaces. Albeit effective to remove endotoxins, the lack of specificity can result in the unwanted loss of essential proteins from the pharmaceutical formulation. In this work, we developed microparticles conjugated to anti-Lipid A antibodies for selective endotoxin removal. Anti-Lipid A particles were characterized using flow cytometry and microscopy techniques. These particles exhibited a depletion capacity > 6 ×103 endotoxin units/mg particles from water, as determined with two independent methods (Limulus Amebocyte Lysate test and nanoparticle tracking analysis). Additionally, we compared these particles with a non-specific endotoxin removal system in a series of formulations of increasing complexity: bovine serum albumin in water < insulin in buffer < birch pollen extracts. We demonstrated that the specific anti-Lipid A particles show a higher protein recovery without compromising their endotoxin removal capacity. Consequently, we believe that the specificity layer integrated by the anti-Lipid A antibody could be advantageous to enhance product yield.

If EAA Test Versus LAL Test is Always Clinically Better and Whether Any Test Always Relates to the Therapeutic Value of Hemoperfusion in Septic Shock Patients?

If EAA Test Versus LAL Test is Always Clinically Better and Whether Any Test Always Relates to the Therapeutic Value of Hemoperfusion in Septic Shock Patients?

Indoor Airborne Microbiome and Endotoxin: Meteorological Events and Occupant Characteristics Are Important Determinants.

Airborne bacteria and endotoxin may affect asthma and allergies. However, there is limited understanding of the environmental determinants that influence them. This study investigated the airborne microbiomes in the homes of 1038 participants from five cities in Northern Europe: Aarhus, Bergen, Reykjavik, Tartu, and Uppsala. Airborne dust particles were sampled with electrostatic dust fall collectors (EDCs) from the participants' bedrooms. The dust washed from the EDCs' clothes was used to extract DNA and endotoxin. The DNA extracts were used for quantitative polymerase chain (qPCR) measurement and 16S rRNA gene sequencing, while endotoxin was measured using the kinetic chromogenic limulus amoebocyte lysate (LAL) assay. The results showed that households in Tartu and Aarhus had a higher bacterial load and diversity than those in Bergen and Reykjavik, possibly due to elevated concentrations of outdoor bacterial taxa associated with low precipitation and high wind speeds. Bergen-Tartu had the highest difference (ANOSIM R = 0.203) in β diversity. Multivariate regression models showed that α diversity indices and bacterial and endotoxin loads were positively associated with the occupants' age, number of occupants, cleaning frequency, presence of dogs, and age of the house. Further studies are needed to understand how meteorological factors influence the indoor bacterial community in light of climate change.

A Surface-Enhanced Raman Spectroscopy-Based Biosensor for the Detection of Biological Macromolecules: The Case of the Lipopolysaccharide Endotoxin Molecules.

The development of sensitive methods for the detection of endotoxin molecules, such as lipopolysaccharides (LPS), is essential for food safety and health control. Conventional analytical methods used for LPS detection are based on the pyrogen test, plating and culture-based methods, and the limulus amoebocyte lysate method (LAL). Alternatively, the development of reliable biosensors for LPS detection would be highly desirable to solve some critical issues, such as high cost and a long turnaround time. In this work, we present a label-free Surface-Enhanced Raman Spectroscopy (SERS)-based method for LPS detection in its free form. The proposed method combines the benefits of plasmonic enhancement with the selectivity provided by a specific anti-lipid A antibody (Ab). A high-enhancing nanostructured silver substrate was coated with Ab. The presence of LPS was quantitatively monitored by analyzing the changes in the Ab spectra obtained in the absence and presence of LPS. A limit of detection (LOD) and quantification (LOQ) of 12 ng/mL and 41 ng/mL were estimated, respectively. Importantly, the proposed technology could be easily expanded for the determination of other biological macromolecules.

Application of a novel chemical assay for the quantification of endotoxins in bacterial bioreactor samples

A novel chemical assay, the so-called Kdo-DMB-liquid chromatography (LC) assay, was used for the accurate and cost-effective determination of the endotoxin content in supernatants of Gram-negative bacteria bioreactor samples. During mild acid hydrolysis, the endotoxin-specific sugar acid 3-deoxy-D-manno-oct-2-ulsonic acid (Kdo) is quantitatively released. Kdo is reacted with 1,2-diamino-4,5-methylenedioxybenzene (DMB) to obtain the highly fluorescent derivate Kdo-DMB. It is separated from the reaction mixture by reversed phase-(U)HPLC and detected by fluorescence. From the Kdo content the endotoxin content of the sample is calculated. For three batch cultivations of Escherichia coli K12 and a fed-batch cultivation of Pseudomonas putida KT2440, the evolution of the endotoxin content in dependence on the cultivation time was monitored. Under optimal, constant cultivation conditions a linear correlation between the endotoxin content and the easy-to-access bioreactor parameters optical density at 600 nm and dry cell weight was found for both endotoxin kinds. Under stress cultivation conditions the E. coli K12 cultivation showed a stronger increase of the endotoxin content at harvest in comparison to optimal conditions. Optical density and dry cell weight may be used for production reactors as an economic real-time estimation tool to determine the endotoxin content at different cultivation time points and conditions. The optical density can further be used to establish straightforward sample dilution schemes for endotoxin quantification in samples of unknown endotoxin content. The endotoxin content [ng mL−1] measured by the Kdo-DMB-LC assay and the endotoxin activity [EU mL−1] obtained by the compendial Limulus Amoebocyte Lysate assay show a high correlation for the bacterial bioreactor samples tested.

Immunostimulatory activity and safety evaluation of Bordetella bronchiseptica-derived lipopolysaccharide, a new vaccine adjuvant candidate

Adjuvants are used to elicit strong immune responses for vaccines that show poor immunogenicity. Previously, we demonstrated that a sonicated bacterin of Bordetella bronchiseptica can be used as a safe adjuvant that enhances the antigen-presenting capability of dendritic cells (DCs). In this study, we purified the lipopolysaccharide (LPS) of B. bronchiseptica (Bb-LPS) and investigated its immunogenic effects on DCs compared to those of Escherichia coli O26:B6 (O26)-derived LPS (O26-LPS), a positive control. Bb-LPS was purified using an LPS extraction kit. Limulus amebocyte lysate assay was performed to determine the optimal concentration of Bb-LPS and O26-LPS for treatment. Bb-LPS increased the metabolic activity of DCs at a concentration of 0 to 250 EU/mL, similar to that of O26-LPS. Bb-LPS significantly increased the expression level of CD40 and CD54, related to the immune responses of DCs. Bb-LPS enhanced the antigen-presenting capability of DCs and significantly increased the interferon-gamma/interleukin-4 ratio of CD4+ T cells co-cultured with DCs to 0.95 (p < 0.05). Moreover, Bb-LPS increased the production of pro-inflammatory cytokines in a safer manner than that obtained by O26-LPS. In vivo safety tests revealed that Bb-LPS was less toxic than O26-LPS in mice. This study demonstrated that Bb-LPS showed unique immune characteristics and immunogenic effects on the antigen-presenting capability of DCs, which differed from those of O26-LPS. This study provides valuable information for basic and clinical research for developing safe vaccine adjuvants.