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Activation of human primary PBMCs by BECC-derived TLR4 ligands. Primary human PBMCs from three separate donors were incubated for 36 h with 1,000 ng/ml of LOS Ec , LOSYp44@26, LOSYp440@26, or PHAD. Cytokine secretion data, as measured by Luminex assay, are shown in pg/ml.
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Adjuvant properties of bacterial cell wall components like MPLA (monophosphoryl lipid A) are well described and have gained FDA approval for use in vaccines such as Cervarix. MPLA is the product of chemically modified lipooligosaccharide (LOS), altered to diminish toxic proinflammatory effects while retaining adequate immunogenicity. Despite the vi...
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... isolated from venipuncture- collected whole blood from three separate donors were stimulated at 1 g/ml for 36 h with LOSYp44@26, LOSYp440@26, PHAD, or LOS Ec , followed by multiplex analysis of cytokines in the supernatant, including TNF-, IL-6, IL-8, IL-10, and MIP-1. All of the TLR4L compounds induced TNF-and IL-6 production at similar levels (Fig. 4). It is of note that two of the donors exhibited strong proinflammatory IL-8 responses, while the third donor initiated a higher IL-10 response; IL-10 is a cytokine classically associated with immune response dampening. This heterogeneous immune response over even a small cohort of human samples is not surprising, due to genetic and ...
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... MPLA exhibits some drawbacks due to its heterogenous makeup, consisting of several lipid A congeners with a 4′ phosphoryl group and 3-7 acyl chains, each with variable activity. Penta-acyl and especially hexaacyl chain variants present in MPLA have been identified as primarily responsible for adjuvant activity [6]. However, Wang et al. revealed that MPLA contains at least two structural variants that are in fact competitive antagonists of hTLR4 [7]. ...
... BECC438b and BECC470b were purified from their corresponding Y. pestis KIM6 + strains as has been described previously [6,8]. Briefly, BECC-engineered strains were cultured at 26 • C for 18 h to an OD 600 of 1.0-1.4. ...
... The lipid A portion was then prepared by mild acid hydrolysis [6]. MALDI-TOF (matrix-assisted laser desorption/ionization time of flight) mass spectrometry was used to confirm the structure of extracted BECC438b and BECC470b. ...
... TLR4 and TLR5 activity assays were performed as described previously with minor modifications (14,15). Briefly, HEK-Blue-hTLR4 cells and HEK-Blue-hTLR5 cells carrying a secreted embryonic alkaline phosphatase (SEAP) reporter construct were obtained from InvivoGen (CA). ...
To address the problem of increased antimicrobial resistance, we developed a glycoconjugate vaccine comprised of O-polysaccharides (OPS) of the four most prevalent serotypes of Klebsiella pneumoniae (KP) linked to recombinant flagellin types A and B (rFlaA and rFlaB) of Pseudomonas aeruginosa (PA). Flagellin is the major subunit of the flagellar filament. Flagella A and B, essential virulence factors for PA, are glycosylated with different glycans. We previously reported that while both rFlaA and rFlaB were highly immunogenic, only the rFlaB antisera reduced PA motility and protected mice from lethal PA infection in a mouse model of thermal injury. Since recombinant flagellin is not glycosylated, we examined the possibility that the glycan on native FlaA (nFlaA) might be critical to functional immune responses. We compared the ability of nFlaA to that of native, deglycosylated FlaA (dnFlaA) to induce functionally active antisera. O glycan was removed from nFlaA with trifluoromethanesulfonic acid. Despite the similar high-titered anti-FlaA antibody levels elicited by nFlaA, rFlaA, and dnFlaA, only the nFlaA antisera inhibited PA motility and protected mice following lethal intraperitoneal bacterial challenge. Both the protective efficacy and carrier protein function of nFlaA were retained when conjugated to KP O1 OPS. We conclude that unlike the case with FlaB O glycan, the FlaA glycan is an important epitope for the induction of functionally active anti-FlaA antibodies.
... We hypothe size that the use of a vaccine formula containing an engineered lipid A mimetic could confer the ideal aspects of both types of pertussis vaccines: the protective immunity of wP and the safety of aP. For these studies, we utilized a novel lipid A structure TLR4 agonist (BECC438b) that was engineered using Bacterial Enzymatic Combinatorial Chemistry (BECC) technology (52). An attenuated strain of Yersinia pestis (Yp) is grown at different temperatures-either 26°C or 37°C-resulting in the synthesis of variable lipid A structures (42,49,52,53). ...
... For these studies, we utilized a novel lipid A structure TLR4 agonist (BECC438b) that was engineered using Bacterial Enzymatic Combinatorial Chemistry (BECC) technology (52). An attenuated strain of Yersinia pestis (Yp) is grown at different temperatures-either 26°C or 37°C-resulting in the synthesis of variable lipid A structures (42,49,52,53). The end result is a breadth of lipid A molecules which have been shown to induce either a highly biased Th1 response, a Th2 response, or a balanced Th1/Th2 response (54). ...
... BECC adjuvants are TLR4 agonists (52). As such, it is expected that some degree of inflammation will occur from their use. ...
The protection afforded by acellular pertussis vaccines wanes over time, and there is a need to develop improved vaccine formulations. Options to improve the vaccines involve the utilization of different adjuvants and administration via different routes. While intramuscular (IM) vaccination provides a robust systemic immune response, intranasal (IN) vaccination theoretically induces a localized immune response within the nasal cavity. In the case of a Bordetella pertussis infection, IN vaccination results in an immune response that is similar to natural infection, which provides the longest duration of protection. Current acellular formulations utilize an alum adjuvant, and antibody levels wane over time. To overcome the current limitations with the acellular vaccine, we incorporated a novel TLR4 agonist, BECC438b, into both IM and IN acellular formulations to determine its ability to protect against infection in a murine airway challenge model. Following immunization and challenge, we observed that DTaP + BECC438b reduced bacterial burden within the lung and trachea for both administration routes when compared with mock-vaccinated and challenged (MVC) mice. Interestingly, IN administration of DTaP + BECC438b induced a Th1-polarized immune response, while IM vaccination polarized toward a Th2 immune response. RNA sequencing analysis of the lung demonstrated that DTaP + BECC438b activates biological pathways similar to natural infection. Additionally, IN administration of DTaP + BECC438b activated the expression of genes involved in a multitude of pathways associated with the immune system. Overall, these data suggest that BECC438b adjuvant and the IN vaccination route can impact efficacy and responses of pertussis vaccines in pre-clinical mouse models.
... MPLA is an attenuated analogue of lipopolysaccharide (LPS) and a potent TLR4 ligand, making it an ideal vaccine antigen and suitable lipid species for incorporation into SLN formulations [23]. It has been studied extensively as an immunomodulatory agent and vaccine adjuvant and is now an FDA approved adjuvant which is used in the human HPV vaccine Cervarix [24]. We have previously demonstrated that a plasmid vector encoding Helicobacter pylori candidate antigen urease subunit A, pcDNA-UreA, was incorporated into lipoplexes synthesised from solid lipid nanoparticles (MPLA nanoparticles) [25]. ...
In this study, novel solid lipid particles containing the adjuvant lipid monophosphoryl lipid A (termed ‘SLN-A’) were synthesised. The SLN-A particles were able to efficiently bind and form complexes with a DNA vaccine encoding the urease alpha subunit of Helicobacter pylori. The resultant nanoparticles were termed lipoplex-A. In a mouse model of H. pylori infection, the lipoplex-A nanoparticles were used to immunise mice, and the resultant immune responses were analysed. It was found that the lipoplex-A vaccine was able to induce high levels of antigen-specific antibodies and an influx of gastric CD4⁺ T cells in vaccinated mice. In particular, a prime with lipoplex-A and a boost with soluble UreA protein induced significantly high levels of the IgG1 antibody, whereas two doses of lipoplex-A induced high levels of the IgG2c antibody. In this study, lipoplex-A vaccination did not lead to a significant reduction in H. pylori colonisation in a challenge model; however, these results point to the utility of the system for delivering DNA vaccine-encoded antigens to induce immune responses and suggest the ability to tailor those responses.
... The immunogenicity of bacterial wall components is well-used in antitumor therapy (53). For example, monophosphorylate lipid A (MPLA) was designed to eradicate toxic proinflammatory effects while maintaining sufficient immunogenicity (54). Emerging data show that beta-glucan and muramyl peptides from bacterial walls specifically recognize receptors to stimulate immune cells (55,56). ...
Immunotherapy has been emerging as a powerful strategy for cancer management. Recently, accumulating evidence has demonstrated that bacteria-based immunotherapy including naive bacteria, bacterial components, and bacterial derivatives, can modulate immune response via various cellular and molecular pathways. The key mechanisms of bacterial antitumor immunity include inducing immune cells to kill tumor cells directly or reverse the immunosuppressive microenvironment. Currently, bacterial antigens synthesized as vaccine candidates by bioengineering technology are novel antitumor immunotherapy. Especially the combination therapy of bacterial vaccine with conventional therapies may further achieve enhanced therapeutic benefits against cancers. However, the clinical translation of bacteria-based immunotherapy is limited for biosafety concerns and non-uniform production standards. In this review, we aim to summarize immunotherapy strategies based on advanced bacterial therapeutics and discuss their potential for cancer management, we will also propose approaches for optimizing bacteria-based immunotherapy for facilitating clinical translation.
... To overcome the congener distribution of MPL, a synthetic variant of MPL termed phosphorylated hexa-acyl disaccharide (PHAD®) was produced and tested in clinical trials [7][8][9][10]. To identify novel and improved MPL analogues and overcome product heterogeneity with more potent immune protection, Bacterial Enzymatic Combinatorial Chemistry (BECC) was used to rapidly generate novel lipid A structures with adjuvant potential and low toxicity in an attenuated Yersinia pestis (Yp) isolate [11]. Among the BECC molecules produced, two BECC molecules, BECC438b and BECC470b, exhibited promising adjuvant properties as compared to MPL and PHAD [11] (see Suppl. ...
... To identify novel and improved MPL analogues and overcome product heterogeneity with more potent immune protection, Bacterial Enzymatic Combinatorial Chemistry (BECC) was used to rapidly generate novel lipid A structures with adjuvant potential and low toxicity in an attenuated Yersinia pestis (Yp) isolate [11]. Among the BECC molecules produced, two BECC molecules, BECC438b and BECC470b, exhibited promising adjuvant properties as compared to MPL and PHAD [11] (see Suppl. Fig. S1). ...
... BECC438b and BECC470b were purified from their corresponding Y. pestis KIM6+ strains as has been described in great detail previously [11,12]. The strain was cultured at 26 • C for 18 h to an OD600 of 1.0-1.4. ...
Toll-like receptor (TLR) agonists are recognized as potential immune-enhancing adjuvants and are included in several licensed vaccines. Monophosphoryl lipid A (MPL®, GlaxoSmithKline) is one such TLR4 agonist that has been approved for use in human vaccines, such as Cervarix and Shingrix. Due to the heterogeneous nature of biologically derived MPL and the need for safer and more potent adjuvants, our groups have developed the novel TLR4 agonist candidates, BECC438 and BECC470 using the Bacterial Enzymatic Combinatorial Chemistry (BECC) platform. BECC438 and BECC470 have been included in studies to test their adjuvant potential and found to be effective in vaccines against both viral and bacterial disease agents. Here, we report detailed biophysical characterization of BECC438 and BECC470 purified from a biological source (BECC438b and BECC470b, respectively) and synthesized chemically (BECC438s and BECC470s, respectively). Both BECC438s and BECC470s have identical acyl chain configurations, BECC438s is bis-phosphorylated and BECC470s is mono-phosphorylated with the removal of the 4′ phosphate moiety. We determined the phase transition temperatures for the acyl chains of BECC438b and BECC470b and found them to be different from those exhibited by their synthetic counterparts. Furthermore, the phosphate groups of BECC438b and BECC470b are more highly hydrated than are those of BECC438s and BECC470s. In addition to exploring the BECC molecules’ biophysical features in aqueous solution, we explored potential formulation of BECC438 and BECC470 with the aluminum-based adjuvant Alhydrogel and as part of an oil-in-water emulsion (Medimmune Emulsion or ME). All of the lipid A analogues could be fully absorbed to Alhydrogel or incorporated onto ME. Surprisingly, the BECC470s molecule, unlike the others, displayed a nearly baseline signal when monitored using a Limulus amebocyte lysate (LAL) endotoxin detection system. Despite this, it was shown to behave as an agonist for human and mouse TLR4 when tested using multiple cell-based systems. This work paves the way for further formulation optimization of two chemically defined TLR4 agonists that are showing great promise as vaccine adjuvants.
... MPLA is the only licensed TLR agonist approved for human use and is currently used as part of the AS04 adjuvant in hepatitis B and human papillomavirus vaccines [62,63]. Engagement of TLRs by agonists such as lipopolysaccharides (LPS), Poly I:C, and CpG dinucleotides leads to a cascade of intracellular signaling and, thus, to the induction of factors and cytokines which enhance immunity [64]. We investigated the addition of MPLA to QAC to enhance the protection observed with pQAC/MVA-N. ...
Infectious bronchitis (IB) is an acute respiratory disease of chickens caused by the avian coronavirus Infectious Bronchitis Virus (IBV). Modified Live Virus (MLV) vaccines used commercially can revert to virulence in the field, recombine with circulating serotypes, and cause tissue damage in vaccinated birds. Previously, we showed that a mucosal adjuvant system, QuilA-loaded Chitosan (QAC) nanoparticles encapsulating plasmid vaccine encoding for IBV nucleocapsid (N), is protective against IBV. Herein, we report a heterologous vaccination strategy against IBV, where QAC-encapsulated plasmid immunization is followed by Modified Vaccinia Ankara (MVA) immunization, both expressing the same IBV-N antigen. This strategy led to the initiation of robust T-cell responses. Birds immunized with the heterologous vaccine strategy had reduced clinical severity and >two-fold reduction in viral burden in lachrymal fluid and tracheal swabs post-challenge compared to priming and boosting with the MVA-vectored vaccine alone. The outcomes of this study indicate that the heterologous vaccine platform is more immunogenic and protective than a homologous MVA prime/boost vaccination strategy.
... Older adults display significantly reduced influenza-specific antibody responses compared with young adults and/or Results Protection in aged mice with BECC-adjuvanted vaccine. Using young adult (6-8-week-old) mice, we previously reported protection from IAV challenge after vaccination with H1 (A/California/04/2009) formulated with BECC438 and BECC470 16 ; 0.04 μg rHA elicited protection from homologous challenge in formulations adjuvanted with 50 μg BECC438 or BECC470, unlike those adjuvanted with 100 μg of alum or 50 μg PHAD 16 . As a follow-up to these experiments, we wanted to assess adjuvant induced protection in aged (~ 12-month-old) mice to determine if immunosenescence observed in aging could be recovered through the use of the BECCbased adjuvant system. ...
... Older adults display significantly reduced influenza-specific antibody responses compared with young adults and/or Results Protection in aged mice with BECC-adjuvanted vaccine. Using young adult (6-8-week-old) mice, we previously reported protection from IAV challenge after vaccination with H1 (A/California/04/2009) formulated with BECC438 and BECC470 16 ; 0.04 μg rHA elicited protection from homologous challenge in formulations adjuvanted with 50 μg BECC438 or BECC470, unlike those adjuvanted with 100 μg of alum or 50 μg PHAD 16 . As a follow-up to these experiments, we wanted to assess adjuvant induced protection in aged (~ 12-month-old) mice to determine if immunosenescence observed in aging could be recovered through the use of the BECCbased adjuvant system. ...
... In our prime-boost vaccine study in aged mice, we showed robust protection from weight loss, superior antibody production, and decreased lung viral titers with the BECC-adjuvanted vaccine (Fig. 1). The BECC470 and other BECC adjuvants have previously been evaluated in vitro for their ability to stimulate a balanced immune response, leading to their selection as adjuvants for analysis 16 . We have found that using BECC470 as an adjuvant in an Influenza HA vaccine model leads to high protection levels in adult aged mice 18 . ...
Influenza A virus (IAV) is a leading cause of respiratory disease worldwide often resulting in severe morbidity and mortality. We have previously shown that the Bacterial Enzymatic Combinatorial Chemistry (BECC) adjuvants, BECC438 and BECC470, formulated with an influenza virus hemagglutinin (HA) protein vaccine, offer greater protection from influenza virus challenge in mouse respiratory models using adult mice than standard HA:adjuvant combinations. In this study, we determined that immunization with HA + BECC adjuvants also significantly broadened the epitopes targeted on HA as compared with other adjuvants, resulting in increased titers of antibodies directed against the highly conserved HA stalk domain. Importantly, we demonstrate that BECC470 combined with an influenza virus HA protein antigen in a prime-only immunization regimen was able to achieve complete protection from challenge in a ~ 12-month-old mouse aged model. Together, this demonstrates the heightened protection provided by the BECC470 adjuvant in an influenza virus vaccine model and shows the enhanced immune response, as compared to other adjuvants elicited by the formulation of HA with BECC470.
... We have used the Bacterial Enzymatic Combinatorial Chemistry (BECC) technology to generate novel lipid A based adjuvants [14,15]. More speci cally, BECC438 and BECC470 were derived from the backbone structure of non-immunogenic Y. pestis lipid A and screened using reporter cell lines and ow cytometry for the ability to activate NFkB and cytokine production [13,14]. ...
... We have used the Bacterial Enzymatic Combinatorial Chemistry (BECC) technology to generate novel lipid A based adjuvants [14,15]. More speci cally, BECC438 and BECC470 were derived from the backbone structure of non-immunogenic Y. pestis lipid A and screened using reporter cell lines and ow cytometry for the ability to activate NFkB and cytokine production [13,14]. BECC438 is bis-phosphorylated (1 and 4' position) with two secondary C16 acyl-chains at the 2 and 2' positions [14,15]. ...
... More speci cally, BECC438 and BECC470 were derived from the backbone structure of non-immunogenic Y. pestis lipid A and screened using reporter cell lines and ow cytometry for the ability to activate NFkB and cytokine production [13,14]. BECC438 is bis-phosphorylated (1 and 4' position) with two secondary C16 acyl-chains at the 2 and 2' positions [14,15]. BECC470 is monophosphorylated (1' position) and has a C14 secondary acyl-chain added at the 4' position along with a secondary C16 acyl-chain at the 2 position. ...
Influenza A virus (IAV) is a leading cause of respiratory disease worldwide often resulting in severe morbidity and mortality. We have previously shown that the Bacterial Enzymatic Combinatorial Chemistry (BECC) adjuvants, BECC438 and BECC470, formulated with an influenza virus hemagglutinin (HA) protein vaccine, offer greater protection from influenza virus challenge in mouse respiratory models using adult mice than standard HA:adjuvant combinations. In this study, we determined that immunization with HA + BECC adjuvants also significantly broadened the epitopes targeted on HA as compared with other adjuvants, resulting in increased titers of antibodies directed against the highly conserved HA stalk domain. Importantly, we demonstrate that BECC470 combined with an influenza virus HA protein antigen in a prime-only immunization regimen was able to achieve complete protection from challenge in a ~ 12-month-old mouse aged model.
Together, this demonstrates the heightened protection provided by the BECC470 adjuvant in an influenza virus vaccine model and shows the enhanced immune response, as compared to other adjuvants elicited by the formulation of HA with BECC470.
... We have used the Bacterial Enzymatic Combinatorial Chemistry (BECC) technology to generate novel lipid A based adjuvants [14,15]. More speci cally, BECC438 and BECC470 were derived from the backbone structure of non-immunogenic Y. pestis lipid A and screened using reporter cell lines and ow cytometry for the ability to activate NFkB and cytokine production [13,14]. ...
... We have used the Bacterial Enzymatic Combinatorial Chemistry (BECC) technology to generate novel lipid A based adjuvants [14,15]. More speci cally, BECC438 and BECC470 were derived from the backbone structure of non-immunogenic Y. pestis lipid A and screened using reporter cell lines and ow cytometry for the ability to activate NFkB and cytokine production [13,14]. BECC438 is bis-phosphorylated (1 and 4' position) with two secondary C16 acyl-chains at the 2 and 2' positions [14,15]. ...
... More speci cally, BECC438 and BECC470 were derived from the backbone structure of non-immunogenic Y. pestis lipid A and screened using reporter cell lines and ow cytometry for the ability to activate NFkB and cytokine production [13,14]. BECC438 is bis-phosphorylated (1 and 4' position) with two secondary C16 acyl-chains at the 2 and 2' positions [14,15]. BECC470 is monophosphorylated (1' position) and has a C14 secondary acyl-chain added at the 4' position along with a secondary C16 acyl-chain at the 2 position. ...
Influenza A virus (IAV) is a leading cause of respiratory disease worldwide often resulting in severe morbidity and mortality. We have previously shown that the Bacterial Enzymatic Combinatorial Chemistry (BECC) adjuvants, BECC438 and BECC470, formulated with an influenza virus hemagglutinin (HA) protein vaccine, offer greater protection from influenza virus challenge in mouse respiratory models using adult mice than standard HA:adjuvant combinations. In this study, we determined that immunization with HA+BECC adjuvants also significantly broadened the epitopes targeted on HA as compared with other adjuvants, resulting in increased titers of antibodies directed against the highly conserved HA stalk domain. Importantly, we demonstrate that BECC470 combined with an influenza virus HA protein antigen in a prime-only immunization regimen was able to achieve complete protection from challenge in a ~12-month-old mouse aged model.
Together, this demonstrates the heightened protection provided by the BECC470 adjuvant in an influenza virus vaccine model and shows the enhanced immune response, as compared to other adjuvants elicited by the formulation of HA with BECC470.
