TLR4 Agonist Monophosphoryl Lipid Research Articles

Evaluation of different types of adjuvants in a malaria transmission-blocking vaccine

Adjuvants are critical components for vaccines, which enhance the strength and longevity of the antibody response and influence the types of immune response. Limited research has been conducted on the immunogenicity and protective efficacy of various adjuvants in malaria transmission-blocking vaccines (TBVs). In this study, we formulated a promising TBV candidate antigen, the P. berghei ookinete surface antigen PSOP25, with different types of adjuvants, including the TLR4 agonist monophosphoryl lipid A (MPLA), the TLR9 agonist cytosine phosphoguanosine oligodeoxynucleotides (CpG ODN 1826) (CpG), a saponin adjuvant QS-21, aluminum hydroxide (Alum), and two combination adjuvants MPLA + QS-21 and QS-21 + CpG. We demonstrated that adjuvanted vaccines results in elevated elicited antibody levels, increased proliferation of plasma cells, and efficient formation of germinal centers (GCs), leading to enhanced long-term protective immune responses. Furthermore, CpG group exhibited the most potent inhibition of ookinete formation and transmission-blocking activity. We found that the rPSOP25 with CpG adjuvant was more effective than MPLA, QS-21, MPLA + QS-21, QS-21 + CpG adjuvants in dendritic cells (DCs) activation and differentiation. Additionally, the CpG adjuvant elicited more rubust immune memory response than Alum adjuvant. CpG and QS-21 adjuvants could activate the Th1 response and promote the secretion of IFN-γ and TNF-α. PSOP25 induced a higher number of Tfh cells in splenocytes when combined with MPLA, CpG, and QS-21 + CpG; and there was no increase in these cell populations when PSOP25 was administered with Alum. In conclusion, CpG may confer enhanced efficacy for the rPSOP25 vaccine, as evidenced by the ability of the elicited antisera to induce protective immune responses and improved transmission-blocking activity.

The Adjuvant Combination of dmLT and Monophosphoryl Lipid A Activates the Canonical, Nonpyroptotic NLRP3 Inflammasome in Dendritic Cells and Significantly Interacts to Expand Antigen-Specific CD4 T Cells.

Adjuvants are often essential additions to vaccines that enhance the activation of innate immune cells, leading to more potent and protective T and B cell responses. Only a few vaccine adjuvants are currently used in approved vaccine formulations in the United States. Combinations of one or more adjuvants have the potential to increase the efficacy of existing and next-generation vaccines. In this study, we investigated how the nontoxic double mutant Escherichia coli heat-labile toxin R192G/L211A (dmLT), when combined with the TLR4 agonist monophosphoryl lipid A (MPL-A), impacted innate and adaptive immune responses to vaccination in mice. We found that the combination of dmLT and MPL-A induced an expansion of Ag-specific, multifaceted Th1/2/17 CD4 T cells higher than that explained by adding responses to either adjuvant alone. Furthermore, we observed more robust activation of primary mouse bone marrow-derived dendritic cells in the combination adjuvant-treated group via engagement of the canonical NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome complex. This was marked by a multiplicative increase in the secretion of active IL-1β that was independent of classical gasdermin D-mediated pyroptosis. Moreover, the combination adjuvant increased the production of the secondary messengers cAMP and PGE2 in dendritic cells. These results demonstrate how certain adjuvant combinations could be used to potentiate better vaccine responses to combat a variety of pathogens.

Single-cell epitope-transcriptomics reveal lung stromal and immune cell response kinetics to nanoparticle-delivered RIG-I and TLR4 agonists

Lung-resident and circulatory lymphoid, myeloid, and stromal cells, expressing various pattern recognition receptors (PRRs), detect pathogen- and danger-associated molecular patterns (PAMPs/DAMPs), and defend against respiratory pathogens and injuries. Here, we report the early responses of murine lungs to nanoparticle-delivered PAMPs, specifically the retinoic acid-inducible gene I (RIG-I) agonist poly-U/UC (PUUC), with or without the TLR4 agonist monophosphoryl lipid A (MPLA). Using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), we characterized the responses at 4 and 24 h after intranasal administration. Within 4 h, ribosome-associated transcripts decreased in both stromal and immune cells, followed by widespread interferon-stimulated gene (ISG) expression. Using RNA velocity, we show that lung-neutrophils dynamically regulate the synthesis of cytokines like CXCL-10, IL-1α, and IL-1β. Co-delivery of MPLA and PUUC increased chemokine synthesis and upregulated antimicrobial binding proteins targeting iron, manganese, and zinc in many cell types, including fibroblasts, endothelial cells, and epithelial cells. Overall, our results elucidate the early PAMP-induced cellular responses in the lung and demonstrate that stimulation of the RIG-I pathway, with or without TLR4 agonists, induces a ubiquitous microbial defense state in lung stromal and immune cells. Nanoparticle-delivered combination PAMPs may have applications in intranasal antiviral and antimicrobial therapies and prophylaxis.

Spatio-temporal delivery of both intra- and extracellular toll-like receptor agonists for enhancing antigen-specific immune responses.

For cancer immunotherapy, triggering toll-like receptors (TLRs) in dendritic cells (DCs) can potentiate antigen-based immune responses. Nevertheless, to generate robust and long-lived immune responses, a well-designed nanovaccine should consider different locations of TLRs on DCs and co-deliver both antigens and TLR agonist combinations to synergistically induce optimal antitumor immunity. Herein, we fabricated lipid-polymer hybrid nanoparticles (LPNPs) to spatio-temporally deliver model antigen ovalbumin (OVA) on the surface of the lipid layer, TLR4 agonist monophosphoryl lipid A (MPLA) within the lipid layer, and TLR7 agonist imiquimod (IMQ) in the polymer core to synergistically activate DCs by both extra- and intra-cellular TLRs for enhancing adaptive immune responses. LPNPs-based nanovaccines exhibited a narrow size distribution at the mean diameter of 133.23nm and zeta potential of -2.36mV, showed a high OVA loading (around 70.83μg/mg) and IMQ encapsulation efficiency (88.04%). Our data revealed that LPNPs-based nanovaccines showed great biocompatibility to immune cells and an excellent ability to enhance antigen internalization, thereby promoting DCs maturation and cytokines production. Compared to Free OVA, OVA-LPNPs promoted antigen uptake, lysosome escape, depot effect and migration to secondary lymphatic organs. Invivo immunization showed that IMQ-MPLA-OVA-LPNPs with dual agonists induced more powerful cellular and humoral immune responses. Moreover, prophylactic vaccination by IMQ-MPLA-OVA-LPNPs effectively suppressed tumor growth and increased survival efficacy. Hence, the nanovaccines we fabricated can effectively co-deliver antigens and different TLR agonists and realize coordinated stimulation of DCs in a spatio-temporal manner for enhanced immune responses, which provides a promising strategy for cancer immunotherapy.

MyD88-dependent signaling drives toll-like receptor-induced trained immunity in macrophages.

Immunocompromised populations are highly vulnerable to developing life-threatening infections. Strategies to protect patients with weak immune responses are urgently needed. Employing trained immunity, whereby innate leukocytes undergo reprogramming upon exposure to a microbial product and respond more robustly to subsequent infection, is a promising approach. Previously, we demonstrated that the TLR4 agonist monophosphoryl lipid A (MPLA) induces trained immunity and confers broad resistance to infection. TLR4 signals through both MyD88- and TRIF-dependent cascades, but the relative contribution of each pathway to induction of trained immunity is unknown. Here, we show that MPLA-induced resistance to Staphylococcus aureus infection is lost in MyD88-KO, but not TRIF-KO, mice. The MyD88-activating agonist CpG (TLR9 agonist), but not TRIF-activating Poly I:C (TLR3 agonist), protects against infection in a macrophage-dependent manner. MPLA- and CpG-induced augmentation of macrophage metabolism and antimicrobial functions is blunted in MyD88-, but not TRIF-KO, macrophages. Augmentation of antimicrobial functions occurs in parallel to metabolic reprogramming and is dependent, in part, on mTOR activation. Splenic macrophages from CpG-treated mice confirmed that TLR/MyD88-induced reprogramming occurs in vivo. TLR/MyD88-triggered metabolic and functional reprogramming was reproduced in human monocyte-derived macrophages. These data show that MyD88-dependent signaling is critical in TLR-mediated trained immunity.

Assessing Immunogenicity of Vaccines and Adjuvant Combinations for Q Fever

Abstract There is an unmet need for developing a safer, effective, single-dose vaccine for Coxiella burnetii conferring prolonged resistance, due to its high infectivity and biothreat potential. C. burnetii causes Q fever, which acutely causes fever-like symptoms but may manifest chronically and escalate into pneumonia and endocarditis. Prior sensitization has been documented to exacerbate the current vaccine’s reactogenicity and there have been considerable efforts in developing alternatives. Our lab has been studying the effects of combinations of adjuvants and their synergistic effects in eliciting antibody and T cell responses. Here we describe an effective adjuvant combination using synthetic TLR4 agonist monophosphoryl lipid A (MPLA), TLR9 agonist CpG ODN 1018, and Addavax, a squalene oil-in-water emulsion. In vitro assays were performed to assess the immunogenicity of these adjuvants in the context of NFκB activation and inducible nitric oxide synthase (iNOS) production. Model antigens from C. burnetii were selected based on previous protein microarray data and formulated with varying adjuvant combinations. These formulations were administered to C57BL/6 mice and assessed for immunogenicity. Sera collected at regular intervals were screened against protein microarrays to monitor the dynamics and isotypes of the antibody response. Here we observed robust, durable and antigen-specific T cell and antibody responses. Our top vaccine formulations were administered to Hartley guinea pigs in a challenge study. Initial data demonstrate specific IgG production against immunizing antigens. Downstream analysis for this data set is currently underway. Supported by DTRA contract HDTRA1-16-C-0009 DTRA grants HDTRA1-18-1-0035 and HDTRA1-18-1-0036

Combination of dmLT and MPL-A adjuvants synergistically and differentially activates the canonical inflammasome in DCs and macrophages in the absence of pyroptosis

Abstract The initiation of innate immune responses is critical for the formation of a protective adaptive immune response following vaccination. Innate immune cells, such as dendritic cells (DCs) and macrophages, phagocytose and display antigen, produce immune modulators and migrate to lymphoid tissues, leading to the activation of naïve T cells. These professional antigen-presenting cells are activated in response to pathogen or danger associated molecular patterns, a process that is normally not robust with vaccine antigen alone. Adjuvants are able to increase the immunogenicity of vaccine antigens by influencing the activation status of these innate immune cells. Only a few vaccine adjuvants are currently approved for use in the US and, along with the discovery of novel adjuvants, combination adjuvants are likely to have a major impact on future vaccine development. Here, we investigated the ability of both the double mutant Escherichia coli heat labile toxin (dmLT) and the TLR4 agonist monophosphoryl lipid A (MPL-A) to drive innate and adaptative immune responses independently and in combination. We found that the combination adjuvant induces more robust activation of DCs and macrophages than either adjuvant alone, leading to the formation of the NLRP3 inflammasome complex and GSDMD independent secretion of IL-1b in the absence of pyroptosis. Furthermore, RNA sequencing and ingenuity pathway analysis support that the combination adjuvant may allow these DCs to polarize a multi-faceted Th1/2/17 CD4 T cell response. Because macrophages and DCs have distinct roles in the interface between innate and adaptive immunity, understanding the cellular and molecular actions of adjuvants on these cells is critical when developing new vaccines.

Adjuvant combinations activate dendritic cells and contribute to antigen-specific CD4 T cell expansion

Abstract Adjuvants are important mediators of immune responses and enhance the activation of innate immune cells, leading to protective T and B cell responses. Only a few vaccine adjuvants are currently approved for use in the US and, along with the discovery of novel adjuvants, combination adjuvants are likely to have a major impact on future vaccine development. Here, we investigated the ability of both the double mutant Escherichia coli heat labile toxin R192/L211A (dmLT) and the TLR4 agonist monophosphoryl lipid A (MPL-A) to drive innate and adaptative immune responses independently and in combination. We found that the combination adjuvant induces more robust activation of dendritic cells (DCs) than either adjuvant alone, leading to the formation of the NLRP3 inflammasome complex and increased secretion of IL-1β, as well as other hallmark pro-inflammatory cytokines. Furthermore, RNA sequencing and ingenuity pathway analysis support that the combination adjuvant may allow these DCs to polarize a multi-faceted Th1/2/17 CD4 T cell response. To determine how this adjuvant combination might regulate CD4 T cell responses, we intradermally immunized mice with a model T cell antigen in conjunction with dmLT or MPL-A alone or combined and, using MHC class II tetramers, found that vaccination with the combination demonstrated the greatest expansion and activation of endogenous, vaccine-specific CD4+ T cells in all lymphoid tissues assessed which included a Th1/Th17 response. These results reveal how combination adjuvants can be used to not only potentiate better vaccine responses, but to potentially bias the immune response to one that is more capable of combating infection.

Vaccine adjuvant choice regulates antigen-specific B cell activation and phenotype

Abstract There has been a global rise of antibiotic resistant bacteria that are causing a significant increase in morbidity and mortality. Vaccines offer one of the best ways to combat and prevent these infections. Antibody responses are often the most effective outcomes of vaccine-induced immunity; however, the role of different vaccine adjuvants has not been fully explored in terms of antigen-specific B cell responses. Using a B cell antigen tetramer, we sought to determine how a novel adjuvant, double mutant heat labile toxin from enterotoxigenic E. Coli (dmLT), compared with those currently used in vaccines. We show that either intradermal or intramuscular immunization of mice with a model vaccine formulated using dmLT induces greater antigen specific B cell numbers in the injection site draining lymph nodes and spleen compared to antigen alone. These B cells were also antibody isotype switched and were phenotypically germinal center B cells. We further demonstrate that vaccine-specific B cells migrate from the injection site draining lymph nodes to more distal lymphoid organs. When we compared dmLT to the most commonly used human vaccine adjuvant, alum, we found dmLT was better at inducing a B cell response. Lastly, we show that dmLT is a more potent B cell adjuvant compared to the TLR4 agonist monophosphoryl lipid A (MPL-A) and that the combination of these two adjuvants is superior to either adjuvant alone. Interestingly, MPL-A induced greater antigen specific IgG compared to dmLT despite dmLT inducing a more robust B cell expansion. These results demonstrate that dmLT is a potent B cell activating adjuvant and that combining dmLT with existing adjuvants has the potential to greatly improve vaccine responses.

Monophosphoryl lipid A alleviated radiation-induced testicular injury through TLR4-dependent exosomes.

Radiation protection on male testis is an important task for ionizing radiation‐related workers or people who receive radiotherapy for tumours near the testicle. In recent years, Toll‐like receptors (TLRs), especially TLR4, have been widely studied as a radiation protection target. In this study, we detected that a low‐toxicity TLR4 agonist monophosphoryl lipid A (MPLA) produced obvious radiation protection effects on mice testis. We found that MPLA effectively alleviated testis structure damage and cell apoptosis induced by ionizing radiation (IR). However, as the expression abundance differs a lot in distinct cells and tissues, MPLA seemed not to directly activate TLR4 singling pathway in mice testis. Here, we demonstrated a brand new mechanism for MPLA producing radiation protection effects on testis. We observed a significant activation of TLR4 pathway in macrophages after MPLA stimulation and identified significant changes in macrophage‐derived exosomes protein expression. We proved that after MPLA treatment, macrophage‐derived exosomes played an important role in testis radiation protection, and specially, G‐CSF and MIP‐2 in exosomes are the core molecules in this protection effect.

Targeted co-delivery of Trp-2 polypeptide and monophosphoryl lipid A by pH-sensitive poly (β-amino ester) nano-vaccines for melanoma

Dendritic cell (DC)-targeted vaccines based on nanotechnology are a promising strategy to efficiently induce potent immune responses. We synthesized and manufactured a mannose-modified poly (β-amino ester) (PBAE) nano-vaccines with easily tuneable and pH-sensitive characteristics to co-deliver the tumor-associated antigen polypeptide Trp-2 and the TLR4 agonist monophosphoryl lipid A (MPLA). To reduce immunosuppression in the tumor microenvironment, an immune checkpoint inhibitor, PD-L1 antagonist, was administrated along with PBAE nano-vaccines to delay melanoma development. We found that mannosylated Trp-2 and MPLA-loaded PBAE nano-vaccines can target and mature DCs, consequently boosting antigen-specific cytotoxic T lymphocyte activity against melanoma. The prophylactic study indicates that combination therapy with PD-L1 antagonist further enhanced anti-tumor efficacy by 3.7-fold and prolonged median survival time by 1.6-fold more than free Trp-2/MPLA inoculation. DC-targeting PBAE polymers have a great potential as a nanotechnology platform to design vaccines and achieve synergistic anti-tumor effects with immune checkpoint therapy.

TLR4 Agonist Monophosphoryl Lipid A Alleviated Radiation-Induced Intestinal Injury.

The small intestine is one of the most sensitive organs to irradiation injury, and the development of high effective radioprotectants especially with low toxicity for intestinal radiation sickness is urgently needed. Monophosphoryl lipid A (MPLA) was found to be radioprotective in our previous study, while its effect against the intestinal radiation injury remained unknown. In the present study, we firstly determined the intestinal apoptosis after irradiation injury according to the TUNEL assay. Subsequently, we adopted the immunofluorescence technique to assess the expression levels of different biomarkers including Ki67, γ-H2AX, and defensin 1 in vivo. Additionally, the inflammatory cytokines were detected by RT-PCR. Our data indicated that MPLA could protect the intestine from ionizing radiation (IR) damage through activating TLR4 signal pathway and regulating the inflammatory cytokines. This research shed new light on the protective effect of the novel TLR4 agonist MPLA against intestine detriment induced by IR.

Co-delivery of antigen and dual agonists by programmed mannose-targeted cationic lipid-hybrid polymersomes for enhanced vaccination

Exploiting Toll-like receptor (TLR) agonists or their certain combinations can enhance the immune potency of subunit vaccine. Nevertheless, the design of co-delivery systems which can act in a synergistic and spatio-temporal way to achieve effective and durable specific immune response is still challenging. Here we fabricated mannose-functionalized lipid-hybrid polymersomes (MAN-IMO-PS) for co-delivery of ovalbumin antigen both inside the inner core and outside the lipid layer, TLR7/8 agonist imiquimod within the hydrophobic membrane, TLR4 agonist monophosphoryl lipid A in the lipid layer as programmed nanovaccine to synergistically activate immune responses for improving vaccine efficacy. After efficiently internalized by dendritic cells via mannose targeting and TLR4 ligating, MAN-IMO-PS significantly enhanced cross-presentation and cytokine production. In addition, MAN-IMO-PS showed depot effect at the injection site and enhanced migration to draining lymph nodes. Mice immunized with MAN-IMO-PS elicited greater lymphocyte activation, CD4+ and CD8+ T cell response, effector cytokines secretion, and induced Th-1 biased humoral responses. More importantly, prophylactic vaccination by MAN-IMO-PS significantly delayed tumor occurrence, suppressed tumor growth with prolonged survival, and achieved long-term immune effect. The present study demonstrates a rationally designed nanovaccine for combining antigen, different TLR agonists, and targeting moiety in a programmed manner to induce synergistic antitumor immune response.

Toll-like receptor 4 signalling regulates antibody response to adenoviral vector-based vaccines by imprinting germinal centre quality.

Adenoviral vectors (AdV) are considered promising candidates for vaccine applications. A prominent group of Toll-like receptors (TLRs) participate in the adenovirus-induced adaptive immune response, yet there is little information regarding the role of TLR4 in AdV-induced immune responses in recent literature. We investigated the function of TLR4 in both adaptive and innate immune responses to an AdV-based anthrax vaccine. By immunizing wild-type and TLR4 knockout (TLR4-KO) mice, we revealed the requirement of TLR4 in AdV-induced innate responses. We also showed that TLR4 functions are required for germinal centre responses in immunized mice, as expression of the apoptosis-related marker Fas was down-regulated on germinal centre B cells from TLR4-KO mice. Likewise, decreased expression of inducible costimulator on follicular T helper cells was observed in immunized TLR4-KO mice. Moreover, a potent protective antigen-specific humoral immune response was mimicked using an adjuvant system containing the TLR4 agonist monophosphoryl lipid A. Overall, our findings showed that very rapid antigen-specific antibody production is correlated with the TLR4-imprinted germinal centre response to AdV-based vaccine. These results provide additional evidence for the use of the AdV and a TLR agonist to induce humoral responses. Our findings offer new insights into rational vaccine design.

Stable Nanoemulsions for the Delivery of Small Molecule Immune Potentiators

Adjuvants are required to enhance immune responses to typically poorly immunogenic recombinant antigens. Toll-like receptor agonists (TLRa) have been widely evaluated as adjuvants because they activate the innate immune system. Currently, licensed vaccines adjuvanted with TLRa include the TLR4 agonist monophosphoryl lipid, while additional TLRa are in clinical development. Unfortunately, naturally derived TLRa are often complex and heterogeneous entities, which brings formulation challenges. Consequently, the use of synthetic small-molecule TLRa has significant advantages because they are well-defined discrete molecules, which can be chemically modified to modulate their physicochemical properties. We previously described the discovery of a family of TLR7 agonists based on a benzonaphthyridine scaffold. In addition, we described how Alum could be used to deliver these synthetic TLRa. An alternative adjuvant approach with enhanced potency over Alum are squalene containing oil-in-water emulsions, which have been included in licensed influenza vaccines, including Fluad (MF59 adjuvanted) and Pandemrix (AS03 adjuvanted). Here, we describe how to enable the co-delivery of a TLR7 agonist in a squalene-based oil-in-water emulsion, for adjuvant evaluation.

The TLR4 Agonist Monophosphoryl Lipid A Drives Broad Resistance to Infection via Dynamic Reprogramming of Macrophage Metabolism.

Monophosphoryl lipid A (MPLA) is a clinically used TLR4 agonist that has been found to drive nonspecific resistance to infection for up to 2 wk. However, the molecular mechanisms conferring protection are not well understood. In this study, we found that MPLA prompts resistance to infection, in part, by inducing a sustained and dynamic metabolic program in macrophages that supports improved pathogen clearance. Mice treated with MPLA had enhanced resistance to infection with Staphylococcus aureus and Candida albicans that was associated with augmented microbial clearance and organ protection. Tissue macrophages, which exhibited augmented phagocytosis and respiratory burst after MPLA treatment, were required for the beneficial effects of MPLA. Further analysis of the macrophage phenotype revealed that early TLR4-driven aerobic glycolysis was later coupled with mitochondrial biogenesis, enhanced malate shuttling, and increased mitochondrial ATP production. This metabolic program was initiated by overlapping and redundant contributions of MyD88- and TRIF-dependent signaling pathways as well as downstream mTOR activation. Blockade of mTOR signaling inhibited the development of the metabolic and functional macrophage phenotype and ablated MPLA-induced resistance to infection in vivo. Our findings reveal that MPLA drives macrophage metabolic reprogramming that evolves over a period of days to support a macrophage phenotype highly effective at mediating microbe clearance and that this results in nonspecific resistance to infection.

Enhancement of antigen-specific CD4+ and CD8+ T cell responses using a self-assembled biologic nanolipoprotein particle vaccine

To address the need for vaccine platforms that induce robust cell-mediated immunity, we investigated the potential of utilizing self-assembling biologic nanolipoprotein particles (NLPs) as an antigen and adjuvant delivery system to induce antigen-specific murine T cell responses. We utilized OT-I and OT-II TCR-transgenic mice to investigate the effects of NLP-mediated delivery of the model antigen ovalbumin (OVA) on T cell activation. Delivery of OVA with the TLR4 agonist monophosphoryl lipid A (MPLA) in the context of NLPs significantly enhanced the activation of both CD4+ and CD8+ T cells in vitro compared to co-administration of free OVA and MPLA. Upon intranasal immunization of mice harboring TCR-transgenic cells, NLPs enhanced the adjuvant effects of MPLA and the in vivo delivery of OVA, leading to significantly increased expansion of CD4+ and CD8+ T cells in lung-draining lymph nodes. Therefore, NLPs are a promising vaccine platform for inducing T cell responses following intranasal administration.

Proteoliposomal formulations of an HIV-1 gp41-based miniprotein elicit a lipid-dependent immunodominant response overlapping the 2F5 binding motif

The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, cholesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants.

Treatment with TLR4 agonists protect against infection after severe burn injury.

Abstract Infection is the leading cause of death in severely burned patients. Prophylactic treatment with the TLR4 agonist monophosphoryl lipid A (MPLA) induces resistance to subsequent bacterial challenge. Treatment of mice with MPLA enhances bacterial clearance, leading to improved survival in a model of P. aeruginosa burn wound infection. The current study was aimed to define the mechanisms responsible for improved bacterial clearance and survival in MPLA-treated burn-infected mice, and to determine if MPLA could protect against clinically relevant Gram positive and fungal pathogens. Mice underwent severe burn injury, followed by systemic treatment with MPLA or vehicle control for 2 days. Mice were then inoculated with P. aeruginosa topically or intraperitoneally, and responding neutrophils were measured in bone marrow, blood, burn wound and peritoneal cavity. In later experiments, burned mice were challenged systemically with S. aureus or C. albicans. MPLA treatment induced G-CSF production, decreased bone marrow neutrophil numbers and increased neutrophil numbers in the blood, peritoneal cavity and burn wound site. G-CSF was essential for MPLA-mediated survival, bacterial clearance and neutrophil trafficking. MPLA also improved survival against S. aureus and C. albicans systemic infections after burn. This suggests that G-CSF facilitates MPLA-induced trafficking of neutrophils, allowing for a more rapid response to sites of infection. The ability of MPLA to enhance antimicrobial responses and its ability to protect against a variety of clinically relevant pathogens make it an attractive therapeutic candidate for use in burn patients for the prevention of post-burn infections.

Identification of GLA/SE as an effective adjuvant for the induction of robust humoral and cell-mediated immune responses to EBV-gp350 in mice and rabbits

Childhood infection with Epstein–Barr virus (EBV) is often asymptomatic and may result in mild flu-like symptoms, but exposure during adolescence and young adulthood can lead to acute infectious mononucleosis (AIM) with a pathology characterized by swollen lymph nodes, sore throat, and severe fatigue lasting weeks or months. A vaccine targeting the envelope glycoprotein gp350 adjuvanted with aluminum hydroxide complexed with the TLR4 agonist monophosphoryl lipid A (MPLA) achieved a 78% reduction in AIM incidence in a small phase II trial of college-age individuals, but development of this vaccine was halted by the manufacturer. Here, we report the evaluation in mice and rabbits of an EBV-gp350 vaccine combined with an adjuvant composed of the synthetic TLR4 agonist glucopyranosyl lipid A (GLA) integrated into stable emulsion (SE). In mice, GLA/SE-adjuvanted gp350 generated high IgG titers (both IgG1 and IgG2a/c subtypes), elevated EBV-neutralizing antibody titers, and robust poly-functional anti-gp350 CD4+ T cell responses. In addition, GLA/SE routinely demonstrated superior performance over aluminum hydroxide in all immunological readouts, including induction of durable neutralizing antibody titers out to at least 1 year post-vaccination. Both components of the GLA/SE adjuvant were found to be required to get optimal responses in both arms of the immune response: specifically, SE for neutralizing antibodies and GLA for induction of T cell responses. Furthermore, this vaccine also elicited high neutralizing antibody titers in a second species, rabbit. These promising results suggest that clinical development of a vaccine comprised of EBV-gp350 plus GLA/SE has the potential to prevent AIM in post-adolescents.