The association between rLiHyp1 protein plus adjuvant and amphotericin B is an effective immunotherapy against visceral leishmaniasis in mice.

Treatment of visceral leishmaniasis (VL) is compromised by drug toxicity, high cost and/or the emergence of resistant strains. Though canine vaccines are available, there are no licensed prophylactic human vaccines. One strategy to improve clinical outcome for infected patients is immunotherapy, which associates a chemotherapy that acts directly to reduce parasitism and the administration of an immunogen-adjuvant that activates the host protective Th1-type immune response. In this study, we evaluated an immunotherapy protocol in a murine model by combining recombinant (r)LiHyp1 (a hypothetical amastigote-specific Leishmania protein protective against Leishmania infantum infection), with monophosphoryl-lipid A (MPLA) as adjuvant and amphotericin B (AmpB) as reference antileishmanial drug. We used this protocol to treat L. infantum infected-BALB/c mice, and parasitological, immunological and toxicological evaluations were performed at 1 and 30 days after treatment. Results showed that mice treated with rLiHyp1/MPLA/AmpB presented the lowest parasite burden in all organs evaluated, when both a limiting dilution technique and qPCR were used. In addition, these animals produced higher levels of IFN-γ and IL-12 cytokines and IgG2a isotype antibody, which were associated with lower production of IL-4 and IL-10 and IgG1 isotype. Furthermore, low levels of renal and hepatic damage markers were found in animals treated with rLiHyp1/MPLA/AmpB possibly reflecting the lower parasite load, as compared to the other groups. We conclude that the rLiHyp1/MPLA/AmpB combination could be considered in future studies as an immunotherapy protocol to treat against VL.

Structural differences in bacterial lipopolysaccharides determine atherosclerotic plaque progression by regulating the accumulation of neutrophils.

BACKGROUND AND AIMS: Gut microbial lipopolysaccharide (LPS) induces endotoxemia, an independent risk factor for cardiovascular disease (CVD). However, no studies have demonstrated how structural differences in each bacterial LPS contribute to endotoxemia. Here, we investigated the effects of different acyl chains in the lipid A moiety of LPS on endotoxemia and the subsequent immune response and atherosclerotic plaque formation. METHODS: Apoe-/- mice were intraperitoneally administered 2 mg/kg of Escherichia coli-derived LPS (E. LPS, as a representative of hexa-acylated lipid A), Bacteroides-derived LPS (B. LPS, as a representative of penta- or tetra-acylated lipid A), or saline (control) once a week, six times. An immunohistological assessment was performed on plaque sections. RESULTS: E. LPS administration induced endotoxemia, but B. LPS and saline did not. In E. LPS-treated mice, total plaque areas in the aortic root were significantly increased, and neutrophil accumulation and increased formation of neutrophil extracellular traps (NETs) were observed at the plaque lesions, but not in B. LPS-treated mice. A single dose of E. LPS significantly increased the accumulation of neutrophils in plaque lesions on day 3, and NET formation on day 7. E. LPS also increased interleukin-1 beta (IL-1ß) production in plaque lesions on day 7. Furthermore, NET formation and IL-1ß production were also observed in human coronary plaques. CONCLUSIONS: We identified a previously unknown link between structural differences in LPS and atherosclerosis. Lowering microbial LPS activity may reduce NET formation in plaques and prevent CVD progression.

Manufacturing-dependent change in biological activity of the TLR4 agonist GSK1795091 and implications for lipid A analog development.

A phase I trial (NCT03447314; 204686) evaluated the safety and efficacy of GSK1795091, a Toll-like receptor 4 (TLR4) agonist, in combination with immunotherapy (GSK3174998 [anti-OX40 monoclonal antibody], GSK3359609 [anti-ICOS monoclonal antibody], or pembrolizumab) in patients with solid tumors. The primary endpoint was safety; other endpoints included efficacy, pharmacokinetics, and pharmacodynamics (PD). Manufacturing of GSK1795091 formulation was modified during the trial to streamline production and administration, resulting in reduced PD (cytokine) activity. Fifty-four patients received GSK1795091 with a combination partner; 32 received only the modified GSK1795091 formulation, 15 received only the original formulation, and seven switched mid-study from the original to the modified formulation. Despite the modified formulation demonstrating higher systemic GSK1795091 exposure compared with the original formulation, the transient, dose-dependent elevations in cytokine and chemokine concentrations were no longer observed (e.g., IP-10, IL10, IL1-RA). Most patients (51/54; 94%) experienced ≥1 treatment-emergent adverse event (TEAE) during the study. Safety profiles were similar between formulations, but a higher incidence of TEAEs associated with immune responses (chills, fatigue, pyrexia, nausea, and vomiting) were observed with the original formulation. No conclusions can be made regarding GSK1795091 anti-tumor activity due to the limited data collected. Manufacturing changes were hypothesized to have caused the change in biological activity in this study. Structural characterization revealed GSK1795091 aggregate size in the modified formulation to be twice that in the original formulation, suggesting a negative correlation between GSK1795091 aggregate size and PD activity. This may have important clinical implications for future development of structurally similar compounds.

Variation in blood microbial lipopolysaccharide (LPS) contributes to immune reconstitution in response to suppressive antiretroviral therapy in HIV.

BACKGROUND: In HIV infection, even under long-term antiretroviral therapy (ART), up to 20% of HIV-infected individuals fail to restore CD4+ T cell counts to the levels similar to those of healthy controls. The mechanisms of poor CD4+ T cell reconstitution on suppressive ART are not fully understood. METHODS: Here, we tested the hypothesis that lipopolysaccharide (LPS) from bacteria enriched in the plasma from immune non-responders (INRs) contributes to blunted CD4+ T cell recovery on suppressive ART in HIV. We characterized plasma microbiome in HIV INRs (aviremic, CD4+ T cell counts < 350 cells/µl), immune responders (IRs, CD4+ T cell counts > 500 cells/µl), and healthy controls. Next, we analyzed the structure of the lipid A domain of three bacterial species identified by mass spectrometry (MS) and evaluated the LPS function through LPS induced proinflammatory responses and CD4+ T cell apoptosis in PBMCs. In comparison, we also evaluated plasma levels of proinflammatory cytokine and chemokine patterns in these three groups. At last, to study the causality of microbiome-blunted CD4+ T cell recovery in HIV, B6 mice were intraperitoneally (i.p.) injected with heat-killed Burkholderia fungorum, Serratia marcescens, or Phyllobacterium myrsinacearum, twice per week for total of eight weeks. FINDINGS: INRs exhibited elevated plasma levels of total microbial translocation compared to the IRs and healthy controls. The most enriched bacteria were Burkholderia and Serratia in INRs and were Phyllobacterium in IRs. Further, unlike P. myrsinacearum LPS, B. fungorum and S. marcescens LPS induced proinflammatory responses and CD4+ T cell apoptosis in PBMCs, and gene profiles of bacteria-mediated cell activation pathways in THP-1 cells in vitro. Notably, LPS structural analysis by mass spectrometry revealed that lipid A from P. myrsinacearum exhibited a divergent structure consistent with weak toll-like receptor (TLR) 4 agonism, similar to the biological profile of probiotic bacteria. In contrast, lipid A from B. fungorum and S. marcescens showed structures more consistent with canonical TLR4 agonists stemming from proinflammatory bacterial strains. Finally, intraperitoneal (i.p.) injection of inactivated B. fungorum and S. marcescens but not P. myrsinacearum resulted in cell apoptosis in mesenteric lymph nodes of C57BL/6 mice in vivo. INTERPRETATION: These results suggest that the microbial products are causally associated with INR phenotype. In summary, variation in blood microbial LPS immunogenicity may contribute to immune reconstitution in response to suppressive ART. Collectively, this work is consistent with immunologically silencing microbiome being causal and targetable with therapy in HIV. FUNDING: This work was supported by the National Institute of Allergy and Infectious Diseases (NIAID; R01 AI128864, Jiang) (NIAID; P30 AI027767, Saag/Health), the Medical Research Service at the Ralph H. Johnson VA Medical Center (merit grant VA CSRD MERIT I01 CX-002422, Jiang), and the National Institute of Aging (R21 AG074331, Scott). The SCOPE cohort was supported by the UCSF/Gladstone Institute of Virology & Immunology CFAR (P30 AI027763, Gandhi) and the CFAR Network of Integrated Clinical Systems (R24 AI067039, Saag). The National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR001450 (the pilot grant, Jiang). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Yeast Shells Encapsulating Adjuvant AS04 as an Antigen Delivery System for a Novel Vaccine against Toxoplasma Gondii.

Toxoplasma gondii (T. gondii) infection causes severe zoonotic toxoplasmosis, which threatens the safety of almost one-third of the human population globally. However, there is no effective protective vaccine against human toxoplasmosis. This necessitates anti-T. gondii vaccine development, which is a main priority of public health. In this study, we optimized the adjuvant system 04 (AS04), a vaccine adjuvant constituted by 3-O-desacyl-4'-monophosphoryl lipid A (a TLR4 agonist) and aluminum salts, by packing it within natural extracts of ß-glucan particles (GPs) from Saccharomyces cerevisiae to form a GP-AS04 hybrid adjuvant system. Through a simple mixing procedure, we loaded GP-AS04 particles with the total extract (TE) of T. gondii lysate, forming a novel anti-T. gondii vaccine GP-AS04-TE. Results indicated that the hybrid adjuvant can efficiently and stably load antigens, mediate antigen delivery, facilitate the dendritic uptake of antigens, boost dendritic cell maturation and stimulation, and increase the secretion of pro-inflammatory cytokines. In the mouse inoculation model, GP-AS04-TE significantly stimulated the function of dendritic cells, induced a very strong TE-specific humoral and cellular immune response, and finally showed a strong and effective protection against toxoplasma chronic and acute infections. This work proves the potential of GP-AS04 for exploitation as a vaccine against a range of pathogens.

Immunomodulation Through Beta-D-glucan in Chemically-induced Necrotizing Pancreatitis.

BACKGROUND: Although the ability of ß-D-glucan and monophosphoryl lipid A (MPLA) to modulate immune responses has been studied in human primary cells, their effect on sterile inflammation models such as necrotizing pancreatitis has never been investigated. MATERIALS AND METHODS: 85 male New Zealand rabbits were assigned into following groups: A: control, B: pretreatment with ß-D-glucan 3 d before pancreatitis, C: pretreatment with MPLA 3 d before pancreatitis, D: pretreatment with ß-D-glucan and laminarin 3 d before pancreatitis, E: treatment with ß-D-glucan 1 d after pancreatitis, and F: MPLA 1 d after pancreatitis. Pancreatitis was induced by sodium taurocholate injection into the pancreatic duct and parenchyma. Survival was recorded for 21 d. On days 1, 3, and 7, blood was collected for amylase measurement. Peripheral blood mononuclear cells were isolated and stimulated for tumor necrosis factor alpha and interleukin 10 production. Pancreatic necrosis and tissue bacterial load were assessed. RESULTS: 21-d survival was prolonged after pretreatment or treatment with ß-D-glucan; this benefit was lost with laminarin administration. At sacrifice, pancreatic inflammatory alterations were more prominent in the control group. Bacterial load was lower after pretreatment or treatment with ß-D-glucan and MPLA. Tumor necrosis factor alpha production from stimulated peripheral blood mononuclear cells was significantly decreased, whereas interleukin 10 production remained unaltered after pretreatment or treatment with ß-D- glucan. CONCLUSIONS: ß-D-glucan reduces mortality of experimental pancreatitis in vivo. This is mediated through attenuation of cytokine production and prevention of bacterial translocation.

Shaping Modern Vaccines: Adjuvant Systems Using MicroCrystalline Tyrosine (MCT®).

The concept of adjuvants or adjuvant systems, used in vaccines, exploit evolutionary relationships associated with how the immune system may initially respond to a foreign antigen or pathogen, thus mimicking natural exposure. This is particularly relevant during the non-specific innate stage of the immune response; as such, the quality of this response may dictate specific adaptive responses and conferred memory/protection to that specific antigen or pathogen. Therefore, adjuvants may optimise this response in the most appropriate way for a specific disease. The most commonly used traditional adjuvants are aluminium salts; however, a biodegradable adjuvant, MCT®, was developed for application in the niche area of allergy immunotherapy (AIT), also in combination with a TLR-4 adjuvant-Monophosphoryl Lipid A (MPL®)-producing the first adjuvant system approach for AIT in the clinic. In the last decade, the use and effectiveness of MCT® across a variety of disease models in the preclinical setting highlight it as a promising platform for adjuvant systems, to help overcome the challenges of modern vaccines. A consequence of bringing together, for the first time, a unified view of MCT® mode-of-action from multiple experiments and adjuvant systems will help facilitate future rational design of vaccines while shaping their success.

Peptide vaccine with glucopyranosyl lipid A-stable oil-in-water emulsion for patients with resected melanoma.

Aim: We tested the safety and immunogenicity of a novel vaccine in patients with resected high-risk melanoma. Patients & methods: HLA-A2-positive patients with resected Stage II-IV melanoma were randomized to receive up to three vaccinations of melanoma-associated peptide (MART-1a) combined with a stable oil-in-water emulsion (SE) either with the Toll-like receptor 4 agonist glucopyranosyl lipid A (GLA-SE-Schedule 1) or alone (SE-Schedule 2). Safety and immunogenicity of the vaccines were monitored. Results: A total of 23 patients were registered. No treatment-related grade 3 or higher adverse events were observed. Increases in MART-1a-specific T cells were seen in 70 and 63% of Schedule 1 and Schedule 2 patients, respectively. Conclusion: Both vaccine schedules were well-tolerated and resulted in an increase in MART-1a-specific T cells. Clinical Trial registration: NCT02320305 (ClinicalTrials.gov).

Monophosphoryl Lipid a Attenuates Multiorgan Dysfunction During Post-Burn Pseudomonas Aeruginosa Pneumonia in Sheep.

BACKGROUND: Monophosphoryl lipid A (MPLA) is a TLR4 agonist that has potent immunomodulatory properties and modulates innate immune function to improve host resistance to infection with common nosocomial pathogens in mice. The goal of this study was to assess the safety and efficacy of MPLA in a sheep model of burn injury and Pseudomonas aeruginosa pneumonia. The sheep provides a favorable model for preclinical testing as their response to TLR4 agonists closely mimics that of humans. METHODS: Twelve chronically instrumented adult female Merino sheep received 20% total body surface area, third-degree cutaneous burn under anesthesia and analgesia. At 24 h after burn, sheep were randomly allocated to receive: MPLA (2.5 µg/kg i.v., n = 6), or vehicle (i.v., n = 6). At 24 h after MPLA or vehicle treatment, Pseudomonas aeruginosa pneumonia was induced. Sheep were mechanically ventilated, fluid resuscitated and cardiopulmonary variables were monitored for 24 h after induction of pneumonia. Cytokine production, vascular barrier function, and lung bacterial burden were also measured. RESULTS: MPLA infusion induced small and transient alterations in core body temperature, heart rate, pulmonary artery pressure, and pulmonary vascular resistance. Pulmonary mechanics were not altered. Vehicle-treated sheep developed severe acute lung injury during Pseudomonas aeruginosa pneumonia, which was attenuated by MPLA as indicated by improved PaO2/FiO2 ratio, oxygenation index, and shunt fraction. Sheep treated with MPLA also exhibited less vascular leak, lower blood lactate levels, and lower modified organ injury score. MPLA treatment attenuated systemic cytokine production and decreased lung bacterial burden. CONCLUSIONS: MPLA was well tolerated in burned sheep and attenuated development of acute lung injury, lactatemia, cytokinemia, vascular leak, and hemodynamic changes caused by Pseudomonas aeruginosa pneumonia.

Nanoparticle Encapsulation of Synergistic Immune Agonists Enables Systemic Codelivery to Tumor Sites and IFNß-Driven Antitumor Immunity.

Effective cancer immunotherapy depends on the robust activation of tumor-specific antigen-presenting cells (APC). Immune agonists encapsulated within nanoparticles (NP) can be delivered to tumor sites to generate powerful antitumor immune responses with minimal off-target dissemination. Systemic delivery enables widespread access to the microvasculature and draining to the APC-rich perivasculature. We developed an immuno-nanoparticle (immuno-NP) coloaded with cyclic diguanylate monophosphate, an agonist of the stimulator of interferon genes pathway, and monophosphoryl lipid A, and a Toll-like receptor 4 agonist, which synergize to produce high levels of type I IFNß. Using a murine model of metastatic triple-negative breast cancer, systemic delivery of these immuno-NPs resulted in significant therapeutic outcomes due to extensive upregulation of APCs and natural killer cells in the blood and tumor compared with control treatments. These results indicate that NPs can facilitate systemic delivery of multiple immune-potentiating cargoes for effective APC-driven local and systemic antitumor immunity. SIGNIFICANCE: Systemic administration of an immuno-nanoparticle in a murine breast tumor model drives a robust tumor site-specific APC response by delivering two synergistic immune-potentiating molecules, highlighting the potential of nanoparticles for immunotherapy.

Early toll-like receptor 4 blockade reduces ROS and inflammation triggered by microglial pro-inflammatory phenotype in rodent and human brain ischaemia models.

BACKGROUND AND PURPOSE: Ischaemic stroke is a leading cause of death, disability, and a high unmet medical need. Post-reperfusion inflammation and an up-regulation of toll-like receptor 4 (TLR4), an upstream sensor of innate immunity, are associated with poor outcome in stroke patients. Here, we identified the therapeutic effect of targeting the LPS/TLR4 signal transduction pathway. EXPERIMENTAL APPROACH: We tested the effect of the TLR4 inhibitor, eritoran (E5564) in different in vitro ischaemia-related models: human organotypic cortex culture, rat organotypic hippocampal cultures, and primary mixed glia cultures. We explored the therapeutic window of E5564 in the transient middle cerebral artery occlusion model of cerebral ischaemia in mice. KEY RESULTS: In vivo, administration of E5564 1 and 4 hr post-ischaemia reduced the expression of different pro-inflammatory chemokines and cytokines, infarct volume, blood-brain barrier breakdown, and improved neuromotor function, an important clinically relevant outcome. In the human organotypic cortex culture, E5564 reduced the activation of microglia and ROS production evoked by LPS. CONCLUSION AND IMPLICATIONS: TLR4 signalling has a causal role in the inflammation associated with a poor post-stroke outcome. Importantly, its inhibition by eritoran (E5564) provides neuroprotection both in vitro and in vivo, including in human tissue, suggesting a promising new therapeutic approach for ischaemic stroke.

Safety of uSCIT-MPL-4: prevalence and risk factors of systemic reactions in real life.

Aim: We assessed the safety of allergoid adjuvanted by monophosphoryl lipid A (uSCIT-MPL-4) in a real-life setting. Materials & methods: Patients treated with uSCIT-MPL-4 were followed-up for 1 year. Systemic reactions (SRs) were registered and the association with potential risk factors was evaluated. Results: 2929 patients were included. Grade 0, 1, 2, 3 and 4 SR reactions were observed respectively in 3.3, 1.5, 0.31, 0.07 and 0.07% of patients. A significant association was detected between Grade ≥1 SRs and: female gender, number of administrations, previous local reactions. Conclusion: uSCIT-MPL-4 is safe. Local reactions should be accurately assessed as they may represent a risk factor for Grade ≥1 SRs, together with gender and number of doses/year.

Preconditioning with toll-like receptor agonists attenuates seizure activity and neuronal hyperexcitability in the pilocarpine rat model of epilepsy.

Neuroinflammation plays an important role in epileptic disorders. Toll-like receptors (TLRs) are the key signal transduction tools by which neuroinflammation may promote epileptogenesis. Depending on the stimulus nature, TLRs may engage a distinct signaling pathway. We examined the impact of early minor activation of TLR4 and TLR2 on the severity of seizure in the pilocarpine rat model of temporal lobe epilepsy (TLE). One µg of Lipopolysaccharides (LPS), Monophosphoryl lipid A (MPL), Pam3Cysor or vehicles were microinjected into the right lateral ventricle of the male Wistar rats. 24 h later, seizures were induced by intraperitoneal injection of pilocarpine, and seizure-related behaviors were monitored. 24 h after seizure induction, the hippocampal level of pro/anti-inflammatory mediators and electrophysiological properties of the dentate gyrus (DG) granular cells were investigated by western blot and whole cell patch clamp techniques, respectively. Pretreatment with TLR ligands resulted in decreased seizure severity, lower hippocampal pro-inflammatory (IL-1ß and IL-6) cytokines and higher anti-inflammatory (IL-10 and TGF- ß) mediators in the pilocarpine-treated rats. Pilocarpine induced profound hyperexcitability in the DG granule cells accompanied by potentiated excitatory postsynaptic currents (EPSCs) and dampened inhibitory postsynaptic currents (IPSCs), in contrast to the control group. However, pretreatment with TLR ligands preserved almost normal excitability and synaptic transmission against the pilocarpine. In conclusion, early activation of TLR4 and TLR2, probably through preserving normal hippocampal cytokine profile and neuronal function attenuates seizure severity in the rat model of TLE.

Intratumoral G100, a TLR4 Agonist, Induces Antitumor Immune Responses and Tumor Regression in Patients with Merkel Cell Carcinoma.

PURPOSE: G100 is a toll-like receptor 4 (TLR4) agonist that triggers innate and adaptive antitumor immune responses in preclinical models. This pilot study assessed the safety, efficacy, and immunologic activity of intratumoral (IT) administration of G100 in patients with Merkel cell carcinoma (MCC). PATIENTS AND METHODS: Patients with locoregional MCC (n = 3; cohort A) received neoadjuvant IT G100 (2 weekly doses at 5 µg/dose) followed by surgery and radiotherapy; patients with metastatic MCC (n = 7; cohort B) received 3 doses in a 6-week cycle and could receive additional cycles with/without radiotherapy. RESULTS: IT G100 was safe and feasible in both neoadjuvant and metastatic settings. Treatment-related adverse events were mostly grade 1 or 2 injection-site reactions. IT G100 led to increased inflammation in the injected tumors with infiltration of CD8+ and CD4+ T cells and activation of immune-related genes. These proinflammatory changes were associated with local tumor regression and appeared to promote systemic immunity. All 3 cohort A patients successfully completed therapy; 2 patients remain recurrence free at 44+ and 41+ months, including 1 with a pathologic complete response after G100 alone. In cohort B, 2 patients achieved sustained partial responses, both lasting 33+ months after 2 cycles of therapy. CONCLUSIONS: In this first-in-human study, IT G100 induced antitumor immune responses, demonstrated acceptable safety, and showed encouraging clinical activity.See related commentary by Marquez-Rodas et al., p. 1127.

Comparative study of adjuvants for allergen-specific immunotherapy in a murine model.

AIM: To compare the immunological and clinical changes induced by allergen-specific immunotherapy (AIT) using different adjuvants. MATERIALS & METHODS: Olea europaea pollen-sensitized mice were treated with olea plus aluminum hydroxide, calcium phosphate, monophosphoryl lipid A (MPL) or immunostimulatory sequences (ISS). RESULTS: Aluminum hydroxide seems to drive initially to a Th2-type response. Bacteria-derived adjuvants (MPL and ISS) skew the immune response toward Th1 and Treg pathways. Specific-IgE production was lower after AIT with MPL and ISS. Moreover, IgG2a production significantly increased in ISS-treated mice. Bacteria-derived adjuvants also improved the Th1 cytokine response due to IFN-γ higher secretion. In addition, they improved bronchial hyper-reactivity and lung inflammation. CONCLUSION: Bacteria-derived adjuvants may enhance the efficacy of AIT.

TLR4-Based Immunotherapeutics in Cancer: A Review of the Achievements and Shortcomings.

Toll-like Receptor 4 (TLR4) agonists have had a long journey in the field of cancer immunotherapy. Nevertheless, despite the remarkable number of the TLR4 ligands that have gone through various preclinical and clinical stages, only two (Bacillus Calmette-Guérin (BCG) and monophosphoryl lipid A (MPLA)) have hitherto obtained the FDA approval for clinical application in cancer treatment. This paper provides a comprehensive review of the TLR4 agonists' journey as cancer active immunotherapeutics. Following a brief discussion of the rationale behind the use of TLR ligands in cancer immunotherapy, we will initially focus on the forerunner of the TLR4 agonists, bacterial lipopolysaccharide (LPS). Within this context, the potentials and shortcomings of immunotherapy with this agent will be addressed, the strategies that have been devised to enhance the associated therapeutic outcome will be discussed, and the consequent achievements and shortcomings will be summarized. Subsequently, further and perhaps less well-known, molecular, bacterial, and viral TLR4 agonists with potential for cancer immunotherapy will be introduced, and if present, the outcome of the preclinical and clinical investigations of these agents will be reviewed. Finally, a look will be cast upon the promising souvenirs of the relatively new arena of nanotechnology, where TLR4 activating nanoparticulate systems will be proposed as potential candidates for the future development of this field.

Immunization with recombinant truncated Neisseria meningitidis-Macrophage Infectivity Potentiator (rT-Nm-MIP) protein induces murine antibodies that are cross-reactive and bactericidal for Neisseria gonorrhoeae.

Neisseria meningitidis (Nm) and N. gonorrhoeae (Ng) express a Macrophage Infectivity Potentiator (MIP, NMB1567/NEIS1487) protein in their outer membrane (OM). In this study, we prepared independent batches of liposomes (n = 3) and liposomes + MonoPhosphoryl Lipid A (MPLA) (n = 3) containing recombinant truncated Nm-MIP protein encoded by Allele 2 (rT-Nm-MIP, amino acids 22-142), and used these to immunize mice. We tested the hypothesis that independent vaccine batches showed similar antigenicity, and that antisera could recognise both meningococcal and gonococcal MIP and induce cross-species bactericidal activity. The different batches of M2 rT-Nm-MIP-liposomes ±â€¯MPLA showed no significant (P > 0.05) batch-to-batch variation in antigenicity. Anti-rT-Nm-MIP sera reacted equally and specifically with Nm-MIP and Ng-MIP in OM and on live bacterial cell surfaces. Specificity was shown by no antiserum reactivity with Δmip bacteria. Using human complement/serum bactericidal assays, anti-M2 rT-Nm-MIP sera killed homologous meningococcal serogroup B (MenB) strains (median titres of 32-64 for anti-rT-Nm-MIP-liposome sera; 128-256 for anti-rT-Nm-MIP-liposome + MPLA sera) and heterologous M1 protein-expressing MenB strains (titres of 64 for anti rT-Nm-MIP-liposome sera; 128-256 for anti-rT-Nm-MIP-liposome + MPLA sera). Low-level killing (P < 0.05) was observed for a MenB isolate expressing M7 protein (titres 4-8), but MenB strains expressing M6 protein were not killed (titre < 4-8). Killing (P < 0.05) was observed against MenC and MenW bacteria expressing homologous M2 protein (titres of 8-16) but not against MenA or MenY bacteria (titres < 4-8). Antisera to M2 rT-Nm-MIP showed significant (P < 0.05) cross-bactericidal activity against gonococcal strain P9-17 (expressing M35 Ng-MIP, titres of 64-512) and strain 12CFX_T_003 (expressing M10 Ng-MIP, titres 8-16) but not against FA1090 (expressing M8 Ng-MIP). As an alternative to producing recombinant protein, we engineered successfully the Nm-OM to express M2 Truncated-Nm-MIP, but lipooligosaccharide-extraction with Na-DOC was contra-indicated. Our data suggest that a multi-component vaccine containing a select number of Nm- and Ng-MIP type proteins would be required to provide broad coverage of both pathogens.

Dual TLR agonist nanodiscs as a strong adjuvant system for vaccines and immunotherapy.

Recent studies have shown that certain combinations of Toll-like receptor (TLR) agonists can induce synergistic immune activation. However, it remains challenging to achieve such robust responses in vivo in a manner that is effective, facile, and amenable for clinical translation. Here, we show that MPLA, a TLR4 agonist, and CpG, a TLR9 agonist, can be efficiently co-loaded into synthetic high-density lipoprotein nanodiscs, forming a potent adjuvant system (ND-MPLA/CpG) that can be readily combined with a variety of subunit antigens, including proteins and peptides. ND-MPLA/CpG significantly enhanced activation of dendritic cells, compared with free dual adjuvants or nanodiscs delivering a single TLR agonist. Importantly, mice immunized with physical mixtures of protein antigens ND-MPLA/CpG generated strong humoral responses, including induction of IgG responses against protein convertase subtilisin/kexin 9 (PCSK9), leading to 17-30% reduction of the total plasma cholesterol levels. Moreover, ND-MPLA/CpG exerted strong anti-tumor efficacy in multiple murine tumor models. Compared with free adjuvants, ND-MPLA/CpG admixed with ovalbumin markedly improved antigen-specific CD8+ T cell responses by 8-fold and promoted regression of B16F10-OVA melanoma (P < 0.0001). Furthermore, ND-MPLA/CpG admixed with E7 peptide antigen elicited ~20% E7-specific CD8+ T cell responses and achieved complete regression of established TC-1 tumors in all treated animals. Taken together, our work highlights the simplicity, versatility, and potency of dual TLR agonist nanodiscs for applications in vaccines and cancer immunotherapy.

Improved Immune Responses in Young and Aged Mice with Adjuvanted Vaccines against H1N1 Influenza Infection.

Elderly people are at high risk for influenza-related morbidity and mortality due to progressive immunosenescence. While toll-like receptor (TLR) agonist containing adjuvants, and other adjuvants, have been shown to enhance influenza vaccine-induced protective responses, the mechanisms underlying how these adjuvanted vaccines could benefit the elderly remain elusive. Here, we show that a split H1N1 influenza vaccine (sH1N1) combined with a TLR4 agonist, glucopyranosyl lipid adjuvant formulated in a stable oil-in-water emulsion (GLA-SE), boosts IgG2c:IgG1 ratios, enhances hemagglutination inhibition (HAI) titers, and increases protection in aged mice. We find that all adjuvanted sH1N1 vaccines tested were able to protect both young and aged mice from lethal A/H1N1/California/4/2009 virus challenge after two immunizations compared to vaccine alone. We show that GLA-SE combined with sH1N1, however, also provides enhanced protection from morbidity in aged mice given one immunization (based on change in weight percentage). While the GLA-SE-adjuvanted sH1N1 vaccine promotes the generation of cytokine-producing T helper 1 cells, germinal center B cells, and long-lived bone marrow plasma cells in young mice, these responses were muted in aged mice. Differential in vitro responses, dependent on age, were also observed from mouse-derived bone marrow-derived dendritic cells and lung homogenates following stimulation with adjuvants, including GLA-SE. Besides enhanced HAI titers, additional protective factors elicited with sH1N1 + GLA-SE in young mice were observed, including (a) rapid reduction of viral titers in the lung, (b) prevention of excessive lung inflammation, and (c) homeostatic maintenance of alveolar macrophages (AMs) following H1N1 infection. Collectively, our results provide insight into mechanisms of adjuvant-mediated immune protection in the young and elderly.