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Mouse CD1d is a nonclassical MHC molecule able to present lipids and glycolipids to a specialized subset of T cells known as natural killer T cells. The antigens presented by CD1d have been shown to cover a broad range of chemical structures and to follow precise rules determining the potency of the antigen in the context of T cell activation. Together with lipids, initial reports suggested that CD1d can also bind and present hydrophobic peptides with (F/W)XX(I/L/M)XXW. However, the exact location of peptide binding and the molecular basis for the required motif are currently unknown. Here we present the crystal structure of the first peptide identified to bind CD1d, p99, and show that it binds in the antigen-binding groove of CD1d in a manner compatible with its presentation to T cell receptors. Interestingly, the peptide adopts an α-helical conformation, which orients the motif residues toward its deep binding groove, therefore explaining the molecular requirements for peptide binding. Moreover, we demonstrate that a lipopeptide version of the same peptide is able to bind CD1d in a similar conformation, identifying another class of molecules binding this antigen-presenting molecule., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Aim: To develop an oral nanovaccine delivery system for lipopeptide-based vaccine candidate against group A Streptococcus., Materials & Methods: Lipid-core peptide-1-loaded nanoliposomes were prepared as a template and coated with opposite-charged polyelectrolytes to produce particles with size <200 nm. Efficacy of this oral nanovaccine delivery system was evaluated in mice model., Results: Polymer-coated liposomes produced significantly higher antigen-specific mucosal IgA and systemic IgG titers in comparison to vaccine formulated with a strong mucosal adjuvant upon oral immunization in mice. Moreover, high levels of systemic antibody titers were retained even at day 185 postprimary immunization., Conclusion: Efficient oral delivery platform for lipopeptide-based vaccines has been developed.

TLRs represent an attractive target for the stimulation of myeloid cell production by HSPCs. We have previously demonstrated that HSPCs use TLR2 to sense Candida albicans in vivo and induce the production of macrophages. In this work, we used an in vitro model of HSPCs differentiation to investigate the functional consequences for macrophages of exposure of HSPCs to various PAMPs and C. albicans cells. Mouse HSPCs (Lin(-) cells) were cultured with M-CSF to induce macrophage differentiation, in the presence or absence of the following PRR agonists: Pam3CSK4 (TLR2 ligand), LPS (TLR4 ligand), depleted zymosan (which only activates Dectin-1), or C. albicans yeasts (which activate several PRRs, but principally TLR2 and Dectin-1). Our data show that these PAMPs differentially impact the anti-microbial function of the macrophages produced by the exposed HSPCs. Pure TLR2 and TLR4 ligands generate macrophages with a diminished ability to produce inflammatory cytokines. In contrast, HSPCs activation in response to C. albicans leads to the generation of macrophages that are better prepared to deal with the infection, as they produce higher amounts of inflammatory cytokines and have higher fungicidal capacity than control macrophages. Therefore, the tailored manipulation of the differentiation process may help to boost the innate immune response to infection., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

The SV-40-transformed MH-S cell line maintains some, but not all, features of primary alveolar macrophages (AMs) from BALB/c mice. We show here that MH-S cells produce inflammatory cytokines IL-6 and CXCL10 in response to challenge with Gram-positive Lactobacillus reuteri, and to TLR2 and NOD2 ligands Pam3CSK4 and MDP, respectively. In contrast, although wild-type AMs are infected in vivo by pneumonia virus of mice (PVM), no virus replication was detected in MH-S cells. Interestingly, the surface immunophenotype of MH-S cells (CD11c(+)Siglec F(-)) differs from that of wild-type AMs (CD11c(+) Siglec F(+)) and is similar to that of immature AMs isolated from granulocyte macrophage-colony stimulating factor (GM-CSF) gene-deleted mice; AMs from GM-CSF(-/-) mice also support PVM replication. However, MH-S cells do not express the GM-CSF receptor alpha chain (CD116) and do not respond to GM-CSF. Due to these unusual features, MH-S cells should be used with caution as experimental models of AMs., (Published by Elsevier B.V.)

A new class of highly antigenic, MHC-II-restricted mycobacterial lipopeptides that are recognized by CD4-positive T lymphocytes of Mycobacterium tuberculosis-infected humans has recently been described. To investigate the relevance of this novel class of mycobacterial Ags in the context of experimental bacille Calmette-Guérin (BCG) vaccination, Ag-specific T cell responses to mycobacterial lipid and lipopeptide-enriched Ag preparations were analyzed in immunized guinea pigs. Lipid and lipopeptide preparations as well as complex Ag mixtures, such as tuberculin, mycobacterial lysates, and culture supernatants, all induced a similar level of T cell proliferation. The hypothesis that lipopeptide-specific T cells dominate the early BCG-induced T cell response was corroborated in restimulation assays by the observation that Ag-expanded T cells specifically responded to the lipopeptide preparation. A comparative analysis of the responses to Ag preparations from different mycobacterial species revealed that the antigenic lipopeptides are specific for strains of the M. tuberculosis complex. Their intriguing conservation in pathogenic tuberculous bacteria and the fact that these highly immunogenic Ags seem to be actively released during in vitro culture and intracellular infection prompt the urgent question about their role in the fine-tuned interplay between the pathogen and its mammalian host, in particular with regard to BCG vaccination strategies., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Withdrawal of iron from serum (hypoferraemia) is a conserved innate immune antimicrobial strategy that can withhold this critical nutrient from invading pathogens, impairing their growth. Hepcidin (Hamp1) is the master regulator of iron and its expression is induced by inflammation. Mice lacking Hamp1 from birth rapidly accumulate iron and are susceptible to infection by blood-dwelling siderophilic bacteria such as Vibrio vulnificus. In order to study the innate immune role of hepcidin against a background of normal iron status, we developed a transgenic mouse model of tamoxifen-sensitive conditional Hamp1 deletion (termed iHamp1-KO mice). These mice attain adulthood with an iron status indistinguishable from littermate controls. Hamp1 disruption and the consequent decline of serum hepcidin concentrations occurred within hours of a single tamoxifen dose. We found that the TLR ligands LPS and Pam3CSK4 and heat-killed Brucella abortus caused an equivalent induction of inflammation in control and iHamp1-KO mice. Pam3CSK4 and B. abortus only caused a drop in serum iron in control mice, while hypoferraemia due to LPS was evident but substantially blunted in iHamp1-KO mice. Our results characterise a powerful new model of rapidly inducible hepcidin disruption, and demonstrate the critical contribution of hepcidin to the hypoferraemia of inflammation., (© 2016 S. Karger AG, Basel.)

Background: Group A streptococcus (GAS) is an exclusively human pathogenic bacteria. A delay in treatment of GAS infection often lead to severe diseases such as rheumatic heart disease which attributes to hundreds of thousands deaths annually. For the past few decades, the quest for a commercial GAS vaccine has been futile. Currently one of the most investigated strategies to develop vaccine against GAS includes the use of conserved epitopes from major virulent factor of GAS, M-protein., Methods: In this study, cationic liposomes of various sizes (70 nm to 1000 nm) were prepared with dimethyldioctadecylammonium bromide (DDAB) encapsulating lipopeptide bearing M-protein derived B-cell epitope (J14)., Results: Smaller liposomes induced higher antibody titres, though the differences between groups were not statistically significant., Conclusion: Nonetheless, all mice which were immunized with liposome-lipopeptide delivery system elicited high levels of systemic (IgG) and mucosal antibodies (IgA), which were discernably higher than those induced with the help of commercial adjuvant (cholera toxin B subunit)., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

In addition to being key elements in hemostasis and thrombosis, platelets amplify neutrophil function. We aimed to gain further insight into the stimuli, mediators, molecular pathways, and regulation of neutrophil extracellular trap formation mediated by human platelets. Platelets stimulated by lipopolysaccharide, a wall component of gram-negative bacteria, Pam3-cysteine-serine-lysine 4, a mimetic of lipopeptide from gram-positive bacteria, Escherichia coli, Staphylococcus aureus, or physiologic platelet agonists promoting neutrophil extracellular trap formation and myeloperoxidase-associated DNA activity under static and flow conditions. Although P-selectin or glycoprotein IIb/IIIa were not involved, platelet glycoprotein Ib, neutrophil cluster of differentiation 18, and the release of von Willebrand factor and platelet factor 4 seemed to be critical for the formation of neutrophil extracellular traps. The secretion of these molecules depended on thromboxane A(2) production triggered by lipopolysaccharide or Pam3-cysteine-serine-lysine 4 but not on high concentrations of thrombin. Accordingly, aspirin selectively inhibited platelet-mediated neutrophil extracellular trap generation. Signaling through extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, and Src kinases, but not p38 or reduced nicotinamide adenine dinucleotide phosphate oxidase, was involved in platelet-triggered neutrophil extracellular trap release. Platelet-mediated neutrophil extracellular trap formation was inhibited by prostacyclin. Our results support a role for stimulated platelets in promoting neutrophil extracellular trap formation, reveal that an endothelium-derived molecule contributes to limiting neutrophil extracellular trap formation, and highlight platelet inhibition as a potential target for controlling neutrophil extracellular trap cell death., (© Society for Leukocyte Biology.)

TLR-ligands are frequently chosen as candidates for vaccine or adjuvant development because they can primarily bridge innate signaling with adaptive immune responses. Since the adjuvant action of porin, the major outer membrane protein commonly present on Gram-negative bacteria, has been tested on several antigen-presenting cells, we investigated its role in driving systemic immunity which is considered a benchmark for a successful adjuvant. Here, we show porin differentially regulated splenic marginal zone (MZ) and follicular zone (FO) B cell responses in contrast to other classical TLR2-ligands FSL-1 and Pam3CSK4. The protein up-regulated TLR2 and TLR6 and stimulated the activation and costimulatory molecules on FO B cells skewing the cells toward TLR-dependent type-1 cytokine response. However, porin could not up-regulate the TLRs and activate MZ B cells. These cells responded to porin by expressing toll-interacting protein (TOLLIP), the TLR2 and -4 signaling inhibitor along with stimulation of the intracellular pathogen recognition receptor NLR caspase recruitment domain containing protein 5 (NLRC5). The CD1d(hi) MZ B cells released IL-10 unequivocally demonstrating regulatory B cell feature. Immunization with porin also resulted in transient IL-10 expression by the CD19(+)CD21(hi) B cells prior to plasma cell formation. Moreover, the plasma cells developed from the B-2 cell subsets show marked variation in generation of immunoglobulin subclasses. The work delineates multi-faceted role of B cell subsets induced by porin for robust immunity without compromising with the checks and controls., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Innate immune engagement results in the activation of host defenses that produce microbe-specific inflammatory responses. A long-standing interest in the field of innate immunity is to understand how varied host responses are generated through the signaling of just a limited number of receptors. Recently, intracellular trafficking and compartmental partitioning have been identified as mechanisms that provide signaling specificity for receptors by regulating signaling platform assembly. We show that cytokine activation as a result of TLR2 stimulation occurs at different intracellular locations and is mediated by the phagosomal trafficking molecule adaptor protein-3 (AP-3). AP-3 is required for trafficking TLR2 purified ligands or the Lyme disease causing bacterium, Borrelia burgdorferi, to LAMP-1 lysosomal compartments. The presence of AP-3 is necessary for the activation of cytokines such as IL-6 but not TNF-α or type I IFNs, suggesting induction of these cytokines occurs from a different compartment. Lack of AP-3 does not interfere with the recruitment of TLR signaling adaptors TRAM and MyD88 to the phagosome, indicating that the TLR-MyD88 signaling complex is assembled at a prelysosomal stage and that IL-6 activation depends on proper localization of signaling molecules downstream of MyD88. Finally, infection of AP-3-deficient mice with B. burgdorferi resulted in altered joint inflammation during murine Lyme arthritis. Our studies further elucidate the effects of phagosomal trafficking on tailoring immune responses in vitro and in vivo., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Unlabelled: The continual threat to global health posed by influenza has led to increased efforts to improve the effectiveness of influenza vaccines for use in epidemics and pandemics. We show in this study that formulation of a low dose of inactivated detergent-split influenza vaccine with a Toll-like receptor 2 (TLR2) agonist-based lipopeptide adjuvant (R4Pam2Cys) provides (i) immediate, antigen-independent immunity mediated by the innate immune system and (ii) significant enhancement of antigen-dependent immunity which exhibits an increased breadth of effector function. Intranasal administration of mice with vaccine formulated with R4Pam2Cys but not vaccine alone provides protection against both homologous and serologically distinct (heterologous) viral strains within a day of administration. Vaccination in the presence of R4Pam2Cys subsequently also induces high levels of systemic IgM, IgG1, and IgG2b antibodies and pulmonary IgA antibodies that inhibit hemagglutination (HA) and neuraminidase (NA) activities of homologous but not heterologous virus. Improved primary virus nucleoprotein (NP)-specific CD8(+) T cell responses are also induced by the use of R4Pam2Cys and are associated with robust recall responses to provide heterologous protection. These protective effects are demonstrated in wild-type and antibody-deficient animals but not in those depleted of CD8(+) T cells. Using a contact-dependent virus transmission model, we also found that heterologous virus transmission from vaccinated mice to naive mice is significantly reduced. These results demonstrate the potential of adding a TLR2 agonist to an existing seasonal influenza vaccine to improve its utility by inducing immediate short-term nonspecific antiviral protection and also antigen-specific responses to provide homologous and heterologous immunity., Importance: The innate and adaptive immune systems differ in mechanisms, specificities, and times at which they take effect. The innate immune system responds within hours of exposure to infectious agents, while adaptive immunity takes several days to become effective. Here we show, by using a simple lipopeptide-based TLR2 agonist, that an influenza detergent-split vaccine can be made to simultaneously stimulate and amplify both systems to provide immediate antiviral protection while giving the adaptive immune system time to implement long-term immunity. Both types of immunity induced by this approach protect against vaccine-matched as well as unrelated virus strains and potentially even against strains yet to be encountered. Conferring dual functionality to influenza vaccines is beneficial for improving community protection, particularly during periods between the onset of an outbreak and the time when a vaccine becomes available or in scenarios in which mass vaccination with a strain to which the population is immunologically naive is imperative., (Copyright © 2015 Chua et al.)

Necator americanus (hookworm) infects over half a billion people worldwide. Anthelminthic drugs are commonly used to treat the infection; however, vaccination is a more favorable strategy to combat this parasite. We designed new B-cell peptide epitopes based on the aspartic protease of N. americanus (Na-APR-1). The peptides were conjugated to self-adjuvanting lipid core peptide (LCP) systems via stepwise solid-phase peptide synthesis (SPPS) and copper catalyst azide-alkyne cycloaddition (CuAAC) reactions. The LCP vaccine candidates were able to self-assemble into nanoparticles, were administered to mice without the use of additional adjuvant, and generated antibodies that recognized the parent epitope. However, only one LCP derivative was able to produce a high titer of antibodies specific to Na-APR-1; circular dichroism analyses of this compound showed a β-sheet conformation for the incorporated epitope. This study provides important insight in epitope and delivery system design for the development of a vaccine against hookworm infections., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Macrophage-activating lipopeptide-2 (MALP-2) is a potent inducer of proinflammatory cytokine secretion by macrophages, monocytes, and dendritic cells. MALP-2 was reported to be involved in natural killer (NK) cell activation and ensuing tumor rejection. However, the mechanism of MALP-2-mediated NK cell activation remained unclear. Therefore, we studied the effects of MALP-2 on cultured human NK cells. We found that MALP-2 had no direct effect on NK cells. Instead, MALP-2 acted on monocytes and triggered the release of different molecules such as interleukin (IL)-1β, IL-6, IL-10, IL-12, IL-15, interferon gamma-induced protein (IP-10), and prostaglandin (PG)-E2. Our data show that monocyte-derived IP-10 could significantly induce NK cell cytotoxicity as long as the immunosuppression by PGE2 is specifically inhibited by cyclooxygenase (COX)-2 blockade. In summary, our results show that MALP-2-mediated stimulation of monocytes results in the production of several mediators which, depending on the prevailing conditions, affect the activity of NK cells in various ways. Hence, MALP-2 administration with concurrent blocking of COX-2 can be considered as a promising approach in MALP-2-based adjuvant tumor therapies.

Stable vaccines administered to the lungs by inhalation could circumvent many of the problems associated with current immunizations against respiratory infections. We earlier provided proof of concept in mice that pulmonary delivered whole inactivated virus (WIV) influenza vaccine formulated as a stable dry powder effectively elicits influenza-specific antibodies in lung and serum. Yet, mucosal IgA, considered particularly important for protection at the site of virus entry, was poorly induced. Here we investigate the suitability of various Toll-like receptor (TLR) ligands and the saponin-derived compound GPI-0100 to serve as adjuvant for influenza vaccine administered to the lungs as dry powder. The TLR ligands palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4), monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG ODN) as well as GPI-0100 tolerated the process of spray freeze-drying well. While Pam3CSK4 had no effect on systemic antibody titers, all the other adjuvants significantly increased influenza-specific serum and lung IgG titers. Yet, only GPI-0100 also enhanced mucosal IgA titers. Moreover, only GPI-0100-adjuvanted WIV provided partial protection against heterologous virus challenge. Pulmonary immunization with GPI-0100-adjuvanted vaccine did not induce an overt inflammatory response since influx of neutrophils and production of inflammatory cytokines were moderate and transient and lung histology was normal. Our results indicate that a GPI-0100-adjuvanted dry powder influenza vaccine is a safe and effective alternative to current parenteral vaccines., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Inflammatory immune responses are mediated by signaling molecules that are both produced by and recognized across highly heterogeneous cell populations. As such, the study of inflammation using traditional immunostimulants is complicated by paracrine and autocrine signaling, which obscures the origin of a propagating response. To address this challenge, we developed a small-molecule probe that can photosensitize immune cells, thus allowing light-mediated inflammation. This probe was used to control the origin of inflammation using light. Following this motif, inflammation was initiated from fibroblasts or dendritic cells. The contributions of fibroblasts and dendritic cells in initiating inflammation in heterogeneous co-culture are reported, thus providing insights into the future development of vaccines and treatment of inflammation., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We evaluated the immunogenicity of a prime/boost vaccine strategy combining 5 lipopeptides (HIV-Lipo-5) and a recombinant modified vaccinia virus Ankara (rMVA-HIV) in cynomolgus macaques. Both of these vaccine components deliver HIV LAI Gag, Pol, and Nef antigens. Systemic and local safety was excellent in all groups. Immunization with HIV-Lipo-5 alone induced significant serum anti-HIV antibody titers which were not modified by rMVA-HIV immunization. However, induction of T-cell responses, as measured by IFNγ and IL-2 producing cells upon short-term stimulation with HIV peptide pools, required combined immunization with rMVA-HIV. Responses were preferentially observed against Gag antigen. Interestingly, HIV-Lipo-5 efficiently primed HIV induced T-cell responses upon the injection of rMVA-HIV, which may help to reduce the required number of vector injections. Our results provide a rationale for the use of a strategy involving HIV-Lipo-5 priming followed by rMVA-HIV booster immunization as a prophylactic or therapeutic vaccine approach against HIV infection and AIDS., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Intestinal barrier dysfunction is associated with chronic gastrointestinal tract inflammation and diseases such as IBD and IBS. Serum-derived bovine immunoglobulin/protein isolate (SBI) is a specially formulated protein preparation (>90%) for oral administration. The composition of SBI is greater than 60% immunoglobulin including contributions from IgG, IgA, and IgM. Immunoglobulin within the lumen of the gut has been recognized to have anti-inflammatory properties and is involved in maintaining gut homeostasis. The binding of common intestinal antigens (LPS and Lipid A) and the ligand Pam3CSK4, by IgG, IgA, and IgM in SBI was shown using a modified ELISA technique. Each of these antigens stimulated IL-8 and TNF-α cytokine production by THP-1 monocytes. Immune exclusion occurred as SBI (≤50 mg/mL) bound free antigen in a dose dependent manner that inhibited cytokine production by THP-1 monocytes in response to 10 ng/mL LPS or 200 ng/mL Lipid A. Conversely, Pam3CSK4 stimulation of THP-1 monocytes was unaffected by SBI/antigen binding. A co-culture model of the intestinal epithelium consisted of a C2BBe1 monolayer separating an apical compartment from a basal compartment containing THP-1 monocytes. The C2BBe1 monolayer was permeabilized with dimethyl palmitoyl ammonio propanesulfonate (PPS) to simulate a damaged epithelial barrier. Results indicate that Pam3CSK4 was able to translocate across the PPS-damaged C2BBe1 monolayer. However, binding of Pam3CSK4 by immunoglobulins in SBI prevented Pam3CSK4 translocation across the damaged C2BBe1 barrier. These results demonstrated steric exclusion of antigen by SBI which prevented apical to basal translocation of antigen due to changes in the physical properties of Pam3CSK4, most likely as a result of immunoglobulin binding. This study demonstrates that immunoglobulins in SBI can reduce antigen-associated inflammation through immune and steric exclusion mechanisms and furthers the mechanistic understanding of how SBI might improve immune status and reduce inflammation in various intestinal disease states.

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that exhibit potent immunosuppressive activity. They are increased in tumor-bearing hosts and contribute to tumor development. Toll-like receptors (TLRs) on MDSCs may modulate the tumor-supporting properties of MDSCs through pattern-recognition. Pam2 lipopeptides represented by Pam2CSK4 serve as a TLR2 agonist to exert anti-tumor function by dendritic cell (DC)-priming that leads to NK cell activation and cytotoxic T cell proliferation. On the other hand, TLR2 enhances tumor cell progression/invasion by activating tumor-infiltrating macrophages. How MDSCs respond to TLR2 agonists has not yet been determined. In this study, we found intravenous administration of Pam2CSK4 systemically up-regulated the frequency of MDSCs in EG7 tumor-bearing mice. The frequency of tumor-infiltrating MDSCs was accordingly increased in response to Pam2CSK4. MDSCs were not increased by Pam2CSK4 stimuli in TLR2 knockout (KO) mice. Adoptive transfer experiments using CFSE-labeled MDSCs revealed that the TLR2-positive MDSCs survived long in tumor-bearing mice in response to Pam2CSK4 treatment. Since the increased MDSC population sustained immune-suppressive properties, our study suggests that Pam2CSK4-triggered TLR2 activation enhances the MDSC potential and suppress antitumor immune response in tumor microenvironment., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Designing a lipopeptide (LP) vaccine with a specific asymmetric arrangement of epitopes may result in an improved display of antigens, increasing host-cell recognition and immunogenicity. This study aimed to synthesise and characterise the physicochemical properties of a library of asymmetric LP-based vaccine candidates that contained multiple CD4(+) and CD8(+) T-cell epitopes from the model protein antigen, ovalbumin. These fully synthetic vaccine candidates were prepared by microwave-assisted solid phase peptide synthesis. The C12 or C16 lipoamino acids were coupled to the N or C terminus of the OVA CD4 peptide epitope. The OVA CD4 LPs and OVA CD8 peptide constructs were then conjugated using azide-alkyne Huisgen cycloaddition to give multivalent synthetic vaccines. Physiochemical characterisation of these vaccines showed a tendency to self-assemble in aqueous media. Changes in lipid length and position induced self-assembly with significant changes to their morphology and secondary structure as shown by transmission electron microscopy and circular dichroism., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Delivering vaccine antigens to mucosal surfaces is potentially very attractive, especially as protection from mucosal infections may be mediated by local immune responses. However, to date mucosal immunization has had limited successes, with issues of both safety and poor immunogenicity. One approach to improve immunogenicity is to develop adjuvants that are effective and safe at mucosal surfaces. Differences in immune responses between mice and men have overstated the value of some experimental adjuvants which have subsequently performed poorly in the clinic. Due to their closer similarity, non-human primates can provide a more accurate picture of adjuvant performance. In this study we immunised rhesus macaques (Macaca mulatta) using a unique matrix experimental design that maximised the number of adjuvants screened while reducing the animal usage. Macaques were immunised by the intranasal, sublingual and intrarectal routes with the model protein antigens keyhole limpet haemocyanin (KLH), β-galactosidase (β-Gal) and ovalbumin (OVA) in combination with the experimental adjuvants Poly(I:C), Pam3CSK4, chitosan, Thymic Stromal Lymphopoietin (TSLP), MPLA and R848 (Resiquimod). Of the routes used, only intranasal immunization with KLH and R848 induced a detectable antibody response. When compared to intramuscular immunization, intranasal administration gave slightly lower levels of antigen specific antibody in the plasma, but enhanced local responses. Following intranasal delivery of R848, we observed a mildly inflammatory response, but no difference to the control. From this we conclude that R848 is able to boost antibody responses to mucosally delivered antigen, without causing excess local inflammation.

Current split influenza virus vaccines that induce strain-specific neutralising antibodies provide some degree of protection against influenza infection but there is a clear need to improve their effectiveness. The constant antigenic drift of influenza viruses means that vaccines are often not an exact match to the circulating strain and so levels of relevant antibodies may not be sufficiently high to afford protection. In the situation where the emergent influenza virus is completely novel, as is the case with pandemic strains, existing vaccines may provide no benefit. In this study we tested the concept of a combination vaccine consisting of sub-optimal doses of split influenza virus vaccine mixed with a cross-protective T-cell inducing lipopeptide containing the TLR2 ligand Pam2Cys. Mice immunised with combination vaccines showed superior levels of lung viral clearance after challenge compared to either split virus or lipopeptide alone, mediated through activation of enhanced humoral and/or additional cellular responses. The mechanism of action of these vaccines was dependent on the route of administration, with intranasal administration being superior to subcutaneous and intramuscular routes, potentially through the induction of memory CD8+ T cells in the lungs. This immunisation strategy not only provides a mechanism for minimising the dose of split virus antigen but also, through the induction of cross-protective CD8+ T cells, proves a breadth of immunity to provide potential benefit upon encounter with serologically diverse influenza isolates.

TLR ligands are critical activators of innate immunity and are being developed as vaccine adjuvants. However, their usefulness in conjunction with NOD-like receptor agonists remains poorly studied. In this study, we evaluated a new ligand that targets both TLR2 and NOD2 receptors. We assessed its ability to enhance dendritic cell maturation in vitro in addition to improving systemic and mucosal immune responses in mice. The chimeric NOD2/TLR2 ligand induced synergistic upregulation of dendritic cell maturation markers, costimulatory molecules, and secretion of proinflammatory cytokines compared with combinations of separate ligands. Furthermore, when coadministered with biodegradable nanoparticles carrying a model Ag, the ligand was able to induce high Ag-specific IgA and IgG titers at both systemic and mucosal sites after parenteral immunizations. These findings point out the potential utility of chimeric molecules TLR/NOD as adjuvants for vaccines to induce systemic and mucosal immune responses., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Aim: Utilize lipopeptide vaccine delivery system to develop a vaccine candidate against Group A Streptococcus., Materials & Methods: Lipopeptides synthesized by solid-phase peptide synthesis-bearing carboxyl (C)-terminal and amino (N)-terminal Group A Streptococcus peptide epitopes. Nanoparticles formed were evaluated in vivo., Results: Immune responses were induced in mice without additional adjuvant. We demonstrated for the first time that incorporation of the C-terminal epitope significantly enhanced the N-terminal epitope-specific antibody response and correlated with forming smaller nanoparticles. Antigen-presenting cells had increased uptake and maturation by smaller, more immunogenic nanoparticles. Antibodies raised by vaccination recognized isolates., Conclusion: Demonstrated the lipopeptidic nanoparticles to induce an immune response which can be influenced by the combined effect of epitope choice and size.

Skin is constantly exposed to bacteria and antigens, and cutaneous innate immune sensing orchestrates adaptive immune responses. In its absence, skin pathogens can expand, entering deeper tissues and leading to life-threatening infectious diseases. To characterize skin-driven immunity better, we applied living bacteria, defined lipopeptides, and antigens cutaneously. We found suppression of immune responses due to cutaneous infection with Gram-positive S. aureus, which was based on bacterial lipopeptides. Skin exposure to Toll-like receptor (TLR)2-6-binding lipopeptides, but not TLR2-1-binding lipopeptides, potently suppressed immune responses through induction of Gr1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs). Investigating human atopic dermatitis, in which Gram-positive bacteria accumulate, we detected high MDSC amounts in blood and skin. TLR2 activation in skin resident cells triggered interleukin-6 (IL-6), which induced suppressive MDSCs, which are then recruited to the skin suppressing T cell-mediated recall responses such as dermatitis. Thus, cutaneous bacteria can negatively regulate skin-driven immune responses by inducing MDSCs via TLR2-6 activation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Peptides are of great interest to be used as vaccine antigens due to their safety, ease of manufacturing and specificity in generating immune response. There have been massive discoveries of peptide antigens over the past decade. However, peptides alone are poorly immunogenic, which demand co-administration with strong adjuvant to enhance their immunogenicity. Recently, fibril-forming peptides such as Q11 and lipoamino acid-based carrier have been identified to induce substantial immune responses when covalently linked to peptide epitope. In this study, we have incorporated either Q11 or lipoamino acids to a peptide epitope (J14) derived from M protein of group A streptococcus to develop self-adjuvanting vaccines. J14, Q11 and lipoamino acids were also conjugated together in a single vaccine construct in an attempt to evaluate the synergy effect of combining multiple adjuvants. Physicochemical characterization demonstrated that the vaccine constructs folded differently and self-assembled into nanoparticles. Significantly, only vaccine constructs containing double copies of lipoamino acids (regardless in conjugation with Q11 or not) were capable to induce significant dendritic cells uptake and subsequent J14-specific antibody responses in non-sizes dependent manners. Q11 had minimal impact in enhancing the immunogenicity of J14 even when it was used in combination with lipoamino acids. These findings highlight the impact of lipoamino acids moiety as a promising immunostimulant carrier and its number of attachment to peptide epitope was found to have a profound effect on the vaccine immunogenicity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The host inflammatory response to the Onchocerca volvulus endosymbiont, Wolbachia, is a major contributing factor in the development of chronic pathology in humans (onchocerciasis/river blindness). Recently, the toll-like pattern recognition receptor motif of the major inflammatory ligands of filarial Wolbachia, membrane-associated diacylated lipoproteins, was functionally defined in murine models of pathology, including mediation of neutrophil recruitment to the cornea. However, the extent to which human neutrophils can be activated in response to this Wolbachia pattern recognition motif is not known. Therefore, the responses of purified peripheral blood human neutrophils to a synthetic N-terminal diacylated lipopeptide (WoLP) of filarial Wolbachia peptidoglycan-associated lipoprotein (PAL) were characterized. WoLP exposure led to a dose-dependent activation of healthy, human neutrophils that included gross morphological alterations and modulation of surface expressed integrins involved in tethering, rolling and extravasation. WoLP exposure induced chemotaxis but not chemokinesis of neutrophils, and secretion of the major neutrophil chemokine, interleukin 8. WoLP also induced and primed the respiratory burst, and enhanced neutrophil survival by delay of apoptosis. These results indicate that the major inflammatory motif of filarial Wolbachia lipoproteins directly activates human neutrophils in vitro and promotes a molecular pathway by which human neutrophils are recruited to sites of Onchocerca parasitism., (© 2014 The Authors. Parasite Immunology Published by John Wiley & Sons Ltd.)

We describe herein the synthesis and immunological evaluation of self-adjuvanting mucin 1 (MUC1)-macrophage activating lipopeptide 2 (MALP2) (glyco)peptide vaccine candidates. Vaccine constructs were shown to induce high titres of class-switched IgG antibodies in C57BL/6 mice after four immunisations despite the lack of a helper T cell epitope.

Active immunisation against gonadotropin releasing hormone (GnRH) is a potential alternative to surgical castration. This study focused on the development of a GnRH subunit lipopeptide vaccine. A library of vaccine candidates that contained one or more (up to eight) copies of monomeric or dimeric GnRH peptide antigen, an adjuvanting lipidic moiety based on lipoamino acids, and an additional T helper epitope, was synthesised by solid phase peptide synthesis. The candidates were evaluated in vivo in order to determine the minimal components of this vaccine necessary to induce a systemic immune response. BALB/c mice were immunised with GnRH lipopeptide conjugates, co-administered with or without Complete Freund's Adjuvant, followed by two additional immunisations. Significant GnRH-specific IgG titres were detected in sera obtained from mice immunised with four of the seven lipopeptides tested, with an increase in titres observed after successive immunisations. This study highlights the importance of for epitope optimisation and delivery system design when producing anti-hapten antibodies in vivo. The results of this study also contribute to the development of future clinical and veterinary immunocontraceptives., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Chemical conjugates comprising synthetic Toll-like receptor ligands (TLR-L) covalently bound to antigenic synthetic long peptides (SLP) are attractive vaccine modalities, which can induce robust CD8(+) T-cell immune responses. Previously, we have shown that the mechanism underlying the power of TLR-L SLP conjugates is improved delivery of the antigen together with a dendritic cell activation signal. In the present study, we have expanded the approach to tumor-specific CD4(+) as well as CD8(+) T-cell responses and in vivo studies in two nonrelated aggressive tumor models. We show that TLR2-L SLP conjugates have superior mouse CD8(+) and CD4(+) T-cell priming capacity compared with free SLPs injected together with a free TLR2-L. Vaccination with TLR2-L SLP conjugates leads to efficient induction of antitumor immunity in mice challenged with aggressive transplantable melanoma or lymphoma. Our data indicate that TLR2-L SLP conjugates are suitable to promote integrated antigen-specific CD8(+) and CD4(+) T-cell responses required for the antitumor effects. Collectively, these data show that TLR2-L SLP conjugates are promising synthetic vaccine candidates for active immunotherapy against cancer., (©2014 American Association for Cancer Research.)

Dendritic cells (DCs) are the major sentinel, antigen-presenting and regulatory components of the immune system. One of the central DC functions is to rapidly sense and alert host immune system of a pathogen invasion. In the present study, we investigated the role of DC exosomes (DCex) in this sentinel function. We demonstrated that DCex could bind bacterial Toll-like-receptor ligands (TLR-Ls), and acquire their ability to strongly activate bystander DCs. Consequently, bystander DCs enhance the expression of transmembrane tumor necrosis factor, secretion of proinflammatory cytokines and cross-talk with natural killer cells leading to the elevated secretion of IFNγ. These findings newly show that DCex can bind and cross-present TLR-Ls to innate-immunity effector cells, and indicate a potent mechanism to systemically alert the host immune system of pathogen invasion. They also suggest a potential novel strategy to generate effective vaccines by binding TLR-L-immune adjuvants to DCex., (Published by Elsevier Inc.)

A20 functions to terminate Toll-like receptor (TLR)-induced immune response, and play important roles in the induction of lipopolysacchride (LPS)-tolerance. However, the molecular mechanism for Pam3CSK4-tolerance is uncertain. Here we report that TLR1/2 ligand Pam3CSK4 induced tolerance in monocytic THP-1 cells. The pre-treatment of THP-1 cells with Pam3CSK4 down-regulated the induction of pro-inflammatory cytokines induced by Pam3CSK4 re-stimulation. Pam3CSK4 pre-treatment also down-regulated the signaling transduction of JNK, p38 and NF-κB induced by Pam3CSK4 re-stimulation. The activation of TLR1/2 induced a rapid and robust up-regulation of A20, suggesting that A20 may contribute to the induction of Pam3CSK4-tolerance. This hypothesis was proved by the observation that the over-expression of A20 by gene transfer down-regulated Pam3CSK4-induced inflammatory responses, and the down-regulation of A20 by RNA interference inhibited the induction of tolerance. Moreover, LPS induced a significant up-regulation of A20, which contributed to the induction of cross-tolerance between LPS and Pam3CSK4. A20 was also induced by the treatment of THP-1 cells with TNF-α and IL-1β. The pre-treatment with TNF-α and IL-1β partly down-regulated Pam3CSK4-induced activation of MAPKs. Furthermore, pharmacologic inhibition of GSK3 signaling down-regulated Pam3CSK4-induced A20 expression, up-regulated Pam3CSK4-induced inflammatory responses, and partly reversed Pam3CSK4 pre-treatment-induced tolerance, suggesting that GSK3 is involved in TLR1/2-induced tolerance by up-regulation of A20 expression. Taken together, these results indicated that A20 is a critical regulator for TLR1/2-induced pro-inflammatory responses.

Synthetic lipopeptides have been widely used as vaccine adjuvants to enhance immune responses. The present study demonstrated that the tryptic N-terminal fragment of the lipoprotein rlipo-D1E3 (lipo-Nter) induces superior antitumor effects compared to a synthetic lipopeptide. The lipo-Nter was purified and formulated with protein or peptide vaccines to determine if lipo-Nter could be used as a novel adjuvant and could induce antitumor immunity in a cervical cancer model. Purified lipo-Nter activated the maturation of bone marrow-derived dendritic cells (BM-DCs), leading to the secretion of TNF-α through TLR2/6 but not TLR1/2. A recombinant mutant HPV16 E7 (rE7m) protein was mixed with lipo-Nter to immunize the mice; the anti-E7 antibody titers were increased, and the T helper cells were skewed toward the Th1 fate (increased IL-2 and decreased IL-5 secretion). Single-dose injection of rE7m and lipo-Nter inhibited tumor growth, but the injection of rE7m alone did not. Accordingly, lipo-Nter also enhanced the antitumor immunity of the E7-derived peptide but not the synthetic lipopeptide (Pam3CSK4). We demonstrated that the lipo-Nter of a bacterial-derived recombinant lipoprotein is a novel adjuvant that could be used for the development of a new generation of vaccines.

Novel and more effective immunization strategies against many animal diseases may profit from the current knowledge on the modulation of specific immunity through stimulation of innate immune receptors. Toll-like receptor (TLR)2-targeting formulations, such as synthetic lipopeptides and antigens expressed in fusion with lipoproteins, have been shown to have built-in adjuvant properties and to be effective at inducing cellular and humoral immune mechanisms in different animal species. However, contradictory data has arisen concerning the profile of the immune response elicited. The benefits of targeting TLR2 for vaccine development are thus still debatable and more studies are needed to rationally explore its characteristics. Here, we resume the main features of TLR2 and TLR2-induced immune responses, focusing on what has been reported for veterinary animals.

It has been reported that lipopeptides can be used to elicit cytotoxic T lymphocyte (CTL) responses against viral diseases and cancer. In our previous study, we determined that mono-palmitoylated peptides can enhance anti-tumor responses in the absence of adjuvant activity. To investigate whether di-palmitoylated peptides with TLR2 agonist activity are able to induce anti-tumor immunity, we synthesized a di-palmitic acid-conjugated long peptide that contains a murine CTL epitope of HPV E749-57 (Pam2IDG). Pam2IDG stimulated the maturation of bone marrow-derived dendritic cells (BMDCs) through TLR2/6. After immunization, Pam2IDG induced higher levels of T cell responses than those obtained with its non-lipidated counterpart (IDG). In the prophylactic model, Pam2IDG immunization completely inhibited tumor growth, whereas IDG immunization was unable to inhibit tumor growth. However, Pam2IDG immunization could not effectively inhibit the growth of established tumors. Therefore, we further investigated whether the depletion of immunosuppressive factors could improve the therapeutic effects of Pam2IDG. Our data indicate that treatment with Pam2IDG combined with clodronate/liposome delays tumor growth and increases the survival rate. We also observed that the therapeutic effects of Pam2IDG are improved by diminishing the function of tumor-associate macrophages (TAMs) and through the use of an IL10 receptor blocking antibody or a Cyclooxygenase 2 (Cox-2) inhibitor. In conclusion, the depletion of TAMs may enhance the anti-tumor immunity of a TLR2 agonist-conjugated peptide.

Toll-like receptors (TLRs) are pattern recognition receptors that mediate host responses to pathogens by promoting cellular activation and the production of cytokines. Ligands for TLRs are conserved structural motifs of pathogens termed pathogen-associated molecular patterns. In the case of TLR2, these ligands include peptidoglycan, lipomannan and lipopeptides. In mammals, it has been shown that different TLR2 ligands induce distinct cytokine responses. However, whether a similar phenomenon occurs in chickens remains to be determined. To this end, chicken splenocytes were stimulated with three different TLR2 ligands: Pam3CSK4, FSL-1 and lipomannan, and the relative gene expression of several cytokines was quantified at 2, 6 and 18h post-stimulation. The results suggest that Pam3 and FSL-1 modulate the kinetics of the pro-inflammatory cytokine response differently, as Pam3 induced a robust interleukin (IL)-1β response, while FSL-1 induced an early and prolonged up-regulation of IL-8. Furthermore, it appears that all three TLR2 ligands induce a mixed T-helper (TH) 1 and 2-like response, as characterized by the up-regulation of IFN-γ, IL-12, IL-4 and IL-13. In conclusion, we have demonstrated that different TLR2 ligands may induce different cytokine responses in chicken splenocytes. Future studies may be aimed at examining the immunomodulating effects of these ligands in vivo., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Human CD1a mediates foreign Ag recognition by a T cell clone, but the nature of possible TCR interactions with CD1a/lipid are unknown. After incubating CD1a with a mycobacterial lipopeptide Ag, dideoxymycobactin (DDM), we identified and measured binding to a recombinant TCR (TRAV3/ TRBV3-1, KD of ≈100 μM). Detection of ternary CD1a/lipid/TCR interactions enabled development of CD1a tetramers and CD1a multimers with carbohydrate backbones (dextramers), which specifically stained T cells using a mechanism that was dependent on the precise stereochemistry of the peptide backbone and was blocked with a soluble TCR. Furthermore, sorting of human T cells from unrelated tuberculosis patients for bright DDM-dextramer staining allowed recovery of T cells that were activated by CD1a and DDM. These studies demonstrate that the mechanism of T cell activation by lipopeptides occurs via ternary interactions of CD1a/Ag/TCR. Furthermore, these studies demonstrate the existence of lipopeptide-specific T cells in humans ex vivo.

Background: Toll-like receptors (TLRs) are crucial for recognition of pathogen-associated molecular patterns by cells of the innate immune system. TLRs are present and functional in CD4⁺ T cells. Memory CD4⁺ T cells, predominantly central memory cells (TCM), constitute the main reservoir of latent HIV-1. However, how TLR ligands affect the quiescence of latent HIV within central memory CD4⁺ T cells has not been studied., Results: We evaluated the ability of a broad panel of TLR agonists to reactivate latent HIV-1. The TLR-1/2 agonist Pam3CSK4 leads to viral reactivation of quiescent HIV in a model of latency based on cultured TCM and in resting CD4⁺ T cells isolated from aviremic patients. In addition, we investigated the signaling pathway associated with Pam3CSK4 involved in HIV-1 reactivation. We show that the transcription factors NFκB, NFAT and AP-1 cooperate to induce viral reactivation downstream of TLR-1/2 stimulation. Furthermore, increasing levels of cyclin T1 is not required for TLR-mediated viral reactivation, but induction of viral expression requires activated pTEFb. Finally, Pam3CSK4 reactivates latent HIV-1 in the absence of T cell activation or proliferation, in contrast to antigen stimulation., Conclusions: Our findings suggest that the signaling through TLR-1/2 pathway via Pam3CSK4 or other reagents should be explored as an anti-latency strategy either alone or in combination with other anti-latency drugs.

A radical lipidation: Application of a novel thiol-ene lipidation enables the one-step synthesis of self-adjuvanting antigenic peptides as vaccine candidates. The resultant monoacyl lipopeptides are shown to activate monocytes in a robust manner., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: We have shown that the intradermal (ID) administration of an HIV-1 lipopeptide candidate vaccine (LIPO-4) is well tolerated in healthy volunteers, with one fifth the IM dose delivered by this route inducing HIV-1-specific CD8(+) T-cell responses of a magnitude and quality similar to those achieved by IM administration. In this long-term follow-up, we aimed to investigate the sustainability and epitopic breadth of the immune responses induced., Methods: In a prospective multicentre trial, 68 healthy volunteers were randomised to receive, at weeks 0, 4 and 12, either a 0.5 ml IM (500 μg of each lipopeptide; 35 volunteers) dose or a 0.1 ml ID (100 μg of each lipopeptide; 33 volunteers) dose of the LIPO-4 vaccine, in the deltoid region of the non-dominant arm. All 68 volunteers received the first two vaccinations, and 44 volunteers in the ID group and 22 in the IM group received the third. We describe here the long-term CD8(+) and CD4(+) T-cell immune responses, up to 48 weeks after the first immunisation., Results: Response frequency was highest at week 14 for CD4(+) T cells, at 85% (28/33) for the IM group and 61% (20/33) for the ID group (p=0.027), and at week 48 for CD8(+) T cells, at 36% (12/33) for the ID group and 31% (11/35) for the IM group (p=0.67). Response rates tended to be lower for volunteers receiving the third vaccination boost, whether IM or ID. Finally, we also observed a striking change in the specificity of the CD8(+) T-cell responses induced shortly (2 weeks) or several months (48 weeks) after LIPO-4 vaccination., Conclusion: Lipopeptide vaccines elicited sustainable CD4(+) and CD8(+) T-cell responses, following IM or ID administration. CD8(+) T-cell responses had shifted and expanded to different epitopes after one year of follow-up. These results should facilitate the design of the next generation of prime-boost trials with repeated doses of lipopeptide vaccines., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

CD1 proteins evolved to present diverse lipid Ags to T cells. In comparison with MHC proteins, CD1 proteins exhibit minimal allelic diversity as a result of nonsynonymous single nucleotide polymorphisms (SNPs). However, it is unknown if common SNPs in gene regulatory regions affect CD1 expression and function. We report surprising diversity in patterns of inducible CD1a expression on human dendritic cells (DCs), spanning the full range from undetectable to high density, a finding not seen with other CD1 isoforms. CD1a-deficient DCs failed to present mycobacterial lipopeptide to T cells but had no defects in endocytosis, cytokine secretion, or expression of costimulatory molecules after LPS treatment. We identified an SNP in the 5' untranslated region (rs366316) that was common and strongly associated with low CD1a surface expression and mRNA levels (p = 0.03 and p = 0.001, respectively). Using a CD1a promoter-luciferase system in combination with mutagenesis studies, we found that the polymorphic allele reduced luciferase expression by 44% compared with the wild-type variant (p < 0.001). Genetic regulation of lipid Ag presentation by varying expression on human DCs provides a mechanism for achieving population level differences in immune responses despite limited structural variation in CD1a proteins.

WH1fungin, a surfactin lipopeptide from Bacillus amyloliquefaciens WH1, can be used as an adjuvant for eliciting strong immune response by parenteral immunization. In this study, WH1fungin was firstly reported as an oral immunoadjuvant. In mice, WH1fungin markedly enhanced the immune response to co-administered protein antigens (OVA or GST), similar to levels elicited by CTB, but no immune response was elicited to itself. Both IgG1 and IgG2a antibodies elicited from the immunizations indicating a mixed Th1/Th2 response. Splenocytes from mice immunized with OVA plus WH1fungin responded to OVA CTL peptide stimulation resulting in an increase in CD8(+)TNF-α(+) and CD8(+)IFN-γ(+) T cell populations. These results further suggested that WH1fungin helps to elicit both humoral and cellular responses to OVA. More studies revealed that the potential mechanism as oral immunoadjuvant was that WH1fungin could form co-precipitates with antigens in a pH value similar to gastric juice. The precipitation protected the antigens from degradation by pepsin providing an explanation for the antigens to withstand the acidic and proteolytic environments of the gastrointestinal tract when co-administered with WH1fungin. Moreover, WH1fungin promoted the uptake of OVA by the intestine and by cultured DC2.4 cells, and increased the expression of cell surface markers and cytokines in DC2.4 cells. Taken together, WH1fungin is a potent oral immunoadjuvant with the ability of protecting protein antigens from acidic and proteolytic degradation, suggesting its potential usage in oral vaccine development., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Heme oxygenase-1 (HO-1) is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury and performs a vital function in the maintenance of cell hemostasis. Increasing numbers of reports have indicated that mycoplasma-derived membrane lipoproteins/lipopeptides, such as macrophage-activating lipopeptide-2 (MALP-2), function as agents that stimulate the immune system by producing various inflammatory mediators, such as cytokines and cyclooxygenase 2 (COX-2), which play roles in the pathogenesis of inflammatory responses during mycoplasma infection. Here, we report that MALP-2 induced HO-1 mRNA and protein expression and upregulated HO-1 enzyme activity in THP-1 cells. Specific inhibitors of mitogen-activated protein kinases (MAPKs), SB203580, PD98059, and SP600125, significantly abolished HO-1 expression. In addition, MALP-2 also induced NF-E2-related factor 2 (Nrf2) translocation, and the silencing of Nrf2 expression in THP-1 cells decreased the levels of MALP-2-mediated HO-1 expression. Furthermore, COX-2 protein expression levels were upregulated in THP-1 cells in response to MALP-2, and transfection with small interfering RNAs of HO-1 significantly increased COX-2 accumulation. These results demonstrate that MALP-2 induces HO-1 expression via MAPKs and Nrf2 pathways and, furthermore, that MALP-2-induced COX-2 expression was modulated by HO-1 in THP-1 cells.

Background and Aims: Presence of serum antibodies against Mycobacterium avium paratuberculosis (MAP) in Crohn's Disease (CD) as a disease characteristic remains controversial. In the present work, we assessed antibody reactivity of serum and intestinal fluid against four distinct MAP-antigens, including the recently identified MAP-specific lipopentapeptide (L5P)., Methods: Immunoglobulin concentrations and specificity against 3 non MAP-specific antigens: glycosyl-transferase-d (GSD), purified protein derivative from MAP (Johnin-PPD), heparin binding haemagglutinin (MAP-HBHA) and one MAP-specific antigen: synthetic L5P were determined by ELISA in gut lavage fluids from adult controls or patients with CD, and in sera of children or adult controls or patients with CD, ulcerative colitis or celiac disease., Results: Total IgA and IgG concentrations were increased in sera of children with CD but were decreased in sera of adults with CD, thereof specificity against MAP antigens was assessed by normalizing immunoglobulin concentrations between samples. In CD patients, IgG reactivity was increased against the four MAP antigens, including L5P in gut lavage fluids but it was only increased against L5P in sera. By contrast, anti-L5P IgG were not increased in patients with ulcerative colitis or celiac disease., Conclusions: A significant increase in anti-L5P IgG is observed in sera of children and adults with CD but not in patients with other intestinal inflammatory diseases. Anti-L5P antibodies may serve as serological marker for CD.

Biological control of plant diseases has gained acceptance in recent years. Bacillus subtilis UMAF6639 is an antagonistic strain specifically selected for the efficient control of the cucurbit powdery mildew fungus Podosphaera fusca, which is a major threat to cucurbits worldwide. The antagonistic activity relies on the production of the antifungal compounds iturin and fengycin. In a previous study, we found that UMAF6639 was able to induce systemic resistance (ISR) in melon and provide additional protection against powdery mildew. In the present work, we further investigated in detail this second mechanism of biocontrol by UMAF6639. First, we examined the signalling pathways elicited by UMAF6639 in melon plants, as well as the defence mechanisms activated in response to P. fusca. Second, we analysed the role of the lipopeptides produced by UMAF6639 as potential determinants for ISR activation. Our results demonstrated that UMAF6639 confers protection against cucurbit powdery mildew by activation of jasmonate- and salicylic acid-dependent defence responses, which include the production of reactive oxygen species and cell wall reinforcement. We also showed that surfactin lipopeptide is a major determinant for stimulation of the immune response. These results reinforce the biotechnological potential of UMAF6639 as a biological control agent., (© 2013 The Authors. Published by Society for Applied Microbiology and Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)

Traditional vaccines, based on the administration of killed or attenuated microorganisms, have proven to be among the most effective methods for disease prevention. Safety issues related to administering these complex mixtures, however, prevent their universal application. Through identification of the microbial components responsible for protective immunity, vaccine formulations can be simplified, enabling molecular-level vaccine characterization, improved safety profiles, prospects to develop new high-priority vaccines (e.g. for HIV, tuberculosis, and malaria), and the opportunity for extensive vaccine component optimization. This subunit approach, however, comes at the expense of decreased immunity, requiring the addition of immunostimulatory agents (adjuvants). As few adjuvants are currently used in licensed vaccines, adjuvant development represents an exciting area for medicinal chemists to play a role in the future of vaccine development. In addition, immune responses can be further customized though optimization of delivery systems, tuning the size of particulate vaccines, targeting specific cells of the immune system (e.g. dendritic cells), and adding components to aid vaccine efficacy in whole immunized populations (e.g. promiscuous T-helper epitopes). Herein we review the current state of the art and future direction in subunit vaccine development, with a focus on the described components and their potential to steer the immune response toward a desired response., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The inability to generate broadly neutralizing antibody (bnAb) responses to the membrane proximal external region (MPER) of HIV-1 gp41 using current vaccine strategies has hampered efforts to prevent the spread of HIV. To address this challenge, we investigated a novel hypothesis to help improve the anti-MPER antibody response. Guided by structural insights and the unique lipid reactivity of anti-MPER bnAbs, we considered whether amino acid side chain modifications that emulate hydrophilic phospholipid head groups could contribute to the generation of 2F5-like or 4E10-like neutralizing anti-MPER antibodies. To test this hypothesis, we generated a series of chemically modified MPER immunogens through derivatization of amino acid side chains with phosphate or nitrate groups. We evaluated the binding affinity of the chemically modified peptides to their cognate monoclonal antibodies, 2F5 and 4E10, using surface plasmon resonance. The modifications had little effect on binding to the antibodies and did not influence epitope secondary structure when presented in liposomes. We selected five of the chemically modified sequences to immunize rabbits and found that an immunogen containing both the 2F5 and 4E10 epitopes and a phosphorylated threonine at T676 elicited the highest anti-peptide IgG titers, although the high antipeptide titers did not confer higher neutralizing activity. These data indicate that side chain modifications adjacent to known neutralizing antibody epitopes are capable of eliciting antibody responses to the MPER but that these chemically modified gp41 epitopes do not induce neutralizing antibodies.

In this study, we examined the reactivity of human peripheral blood mononuclear cells to a panel of influenza A virus (IAV) CD8(+) T-cell epitopes that are recognised by the major human leukocyte antigen (HLA) groups represented in the human population. We examined the level of recognition in a sample of the human population and the potential coverage that could be achieved if these were incorporated into a T-cell epitope-based vaccine. We then designed a candidate influenza vaccine that incorporated three of the examined HLA-A2-restricted influenza epitopes into Pam2Cys-based lipopeptides. These lipopeptides do not require the addition of an adjuvant and can be delivered directly to the respiratory mucosa enabling the generation of local memory cell populations that are crucial for clearance of influenza. Intranasal administration of a mixture of three lipopeptides to HLA-A2 transgenic HHD mice elicited multiple CD8(+) T-cell specificities in the spleen and lung that closely mimicked the response generated following natural infection with influenza. These CD8(+) T cells were associated with viral reduction following H3N1 influenza virus challenge for as long as 3 months after lipopeptide administration. In addition, lipopeptides containing IAV-targeting epitopes conferred substantial benefit against death following infection with a virulent H1N1 strain. Because CD8(+) T cell epitopes are often derived from highly conserved regions of influenza viruses, such vaccines need not be reformulated annually and unlike current antibody-inducing vaccines could provide cross-protective immunity against newly emerging pandemic viruses.

We have recently isolated a rhesus macaque cytotoxic T cell line, 2N5.1, that specifically recognizes an N-myristoylated 5-mer peptide (C(14)-Gly-Gly-Ala-Ile-Ser [C14nef5]) derived from the simian immunodeficiency virus (SIV) Nef protein. Such C14nef5-specific T cells expand in the circulation of SIV-infected monkeys, underscoring the capacity of T cells to recognize viral lipopeptides; however, the molecular basis for the lipopeptide antigen presentation remains to be elucidated. Here, functional studies indicated that the putative antigen-presenting molecule for 2N5.1 was likely to have two separate antigen-binding sites, one for interaction with a C(14)-saturated acyl chain and the other for anchorage of the C-terminal serine residue. Mutants with alanine substitutions for the second glycine residue and the fourth isoleucine residue were not recognized by 2N5.1 but interfered with the presentation of C14nef5 to 2N5.1, indicating that these structural analogues retained the ability to interact with the antigen-presenting molecules. In contrast to the highly specific recognition of C14nef5 by 2N5.1, an additional cytotoxic T cell line, SN45, established independently from a C14nef5-stimulated T cell culture, showed superb reactivity to both C14nef5 and an N-myristoylated Nef 4-mer peptide, and therefore, the C-terminal serine residue was dispensable for the recognition of lipopeptides by the SN45 T cells. Furthermore, the mutants with alanine substitutions were indeed recognized by the SN45 T cells. Given that N-myristoylation of the Nef protein occurs in the conserved motifs and is critical for viral pathogenesis, these observations predict that the lipopeptide-specific T cell response is difficult for viruses to avoid by simply introducing amino acid mutations.

Intestinal epithelial cells (IECs) normally promote the development of gut resident tolerogenic dendritic cells (DCs) and regulatory T cells, but how this process is altered in inflammatory bowel disease is not well characterized. Recently, we published that the cell injury signal ATP modulates IEC chemokine responses to the TLR5 ligand flagellin and exacerbates colitis in the presence of flagellin. We hypothesized that ATP switches these IECs from tolerogenic to proinflammatory, enhancing DC activation and immune responses to commensal antigens. Here, we report that ATP enhanced murine IEC production of KC, IL-6, TGF-β, and thymic stromal lymphopoietin in response to TLR1/2 stimulation by Pam(3) CSK(4) (PAM). Moreover, supernatants from IECs stimulated with ATP+PAM enhanced expression of CD80 on bone marrow derived dendritic cells, and increased their production of IL-12, IL-6, IL-23, TGF-β, and aldh1a2, suggesting a Th1/Th17 polarizing environment. DCs conditioned by stressed IECs stimulated an enhanced recall response to flagellin and supported the expansion of IFN-γ(+) and IL-17(+) memory T cells. Lastly, colonic administration of nonhydrolysable ATP increased production of IL-6 and Cxcl1 (KC) by IECs. These findings indicate that ATP influences the response of IECs to TLR ligands and biases the maturation of DCs to become inflammatory., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

WH1fungin, a surfactin cyclopeptide from Bacillus amyloliquefaciens WH1, is firstly reported as a novel immunoadjuvant, which can markedly enhance the immune response when given in mixture with antigens. After intramuscular or subcutaneous immunization, WH1fungin can help to induce both of durable humoral and cellular immune response, even as strong as Freund's adjuvant. Both IgG1 and IgG2a antigen-specific antibodies were elicited from the immunizations indicating a mixed Th1/Th2 response. Splenocytes from mice intramuscularly immunized with OVA plus WH1fungin responded to OVA CTL peptide stimulation resulting in an increase in CD8(+)TNF-α(+) and CD8(+)IFN-γ(+) T cell populations, and also an increase in CD4(+)TNF-α(+) T cells and CD4(+)IFN-γ(+) T cell populations was found from mice subcutaneously immunized with OVA plus WH1fungin when responded to OVA Th peptide stimulation. These results further suggest that WH1fungin helps to elicit humoral and cellular responses to OVA. The potential mechanism of WH1fungin as an immunoadjuvant was investigated. In vitro assays showed that WH1fungin could enter into RAW 264.7 cells, induce ROS accumulation, and increase the expression of cell surface markers and cytokines in cells. Further investigation suggested that WH1fungin might exert its adjuvant activity by ligating with TLR-2 in antigen present cells such as RAW 264.7. Taken together, WH1fungin is very potent as a novel adjuvant for development of vaccines in the future., (Copyright © 2012 Elsevier Inc. All rights reserved.)