T Cell Immunity to the Alkyl Hydroperoxide Reductase of Burkholderia pseudomallei: A Correlate of Disease Outcome in Acute Melioidosis.

There is an urgent need for a better understanding of adaptive immunity to Burkholderia pseudomallei, the causative agent of melioidosis that is frequently associated with sepsis or death in patients in Southeast Asia and Northern Australia. The imperative to identify vaccine targets is driven both by the public health agenda in these regions and biological threat concerns. In several intracellular bacterial pathogens, alkyl hydroperoxidase reductases are upregulated as part of the response to host oxidative stress, and they can stimulate strong adaptive immunity. We show that alkyl hydroperoxidase reductase (AhpC) of B. pseudomallei is strongly immunogenic for T cells of 'humanized' HLA transgenic mice and seropositive human donors. Some T cell epitopes, such as p6, are able to bind diverse HLA class II heterodimers and stimulate strong T cell immunity in mice and humans. Importantly, patients with acute melioidosis who survive infection show stronger T cell responses to AhpC relative to those who do not. Although the sequence of AhpC is virtually invariant among global B. pseudomallei clinical isolates, a Cambodian isolate varies only in C-terminal truncation of the p6 T cell epitope, raising the possibility of selection by host immunity. This variant peptide is virtually unable to stimulate T cell immunity. For an infection in which there has been debate about centrality of T cell immunity in defense, these observations support a role for T cell immunity to AhpC in disease protection.

Chronic Infection by Mucoid Pseudomonas aeruginosa Associated with Dysregulation in T-Cell Immunity to Outer Membrane Porin F.

RATIONALE: Pseudomonas aeruginosa (PA) is an environmental pathogen that commonly infects individuals with cystic fibrosis (CF) and non-CF bronchiectasis, impacting morbidity and mortality. To understand the pathobiology of interactions between the bacterium and host adaptive immunity and to inform rational vaccine design, it is important to understand the adaptive immune correlates of disease. OBJECTIVES: To characterize T-cell immunity to the PA antigen outer membrane porin F (OprF) by analyzing immunodominant epitopes in relation to infection status. METHODS: Patients with non-CF bronchiectasis were stratified by frequency of PA isolation. T-cell IFN-γ immunity to OprF and its immunodominant epitopes was characterized. Patterns of human leukocyte antigen (HLA) restriction of immunodominant epitopes were defined using HLA class II transgenic mice. Immunity was characterized with respect to cytokine and chemokine secretion, antibody response, and T-cell activation transcripts. MEASUREMENTS AND MAIN RESULTS: Patients were stratified according to whether PA was never, sometimes (<50%), or frequently (≥50%) isolated from sputum. Patients with frequent PA sputum-positive isolates were more likely to be infected by mucoid PA, and they showed a narrow T-cell epitope response and a relative reduction in Th1 polarizing transcription factors but enhanced immunity with respect to antibody production, innate cytokines, and chemokines. CONCLUSIONS: We have defined the immunodominant, HLA-restricted T-cell epitopes of OprF. Our observation that chronic infection is associated with a response of narrowed specificity, despite strong innate and antibody immunity, may help to explain susceptibility in these individuals and pave the way for better vaccine design to achieve protective immunity.

Characterization of human memory CD4(+) T-cell responses to the dog allergen Can f 4.

BACKGROUND: The recently identified dog lipocalin allergen Can f 4 is an important respiratory allergen. OBJECTIVE: We sought to comprehensively characterize the memory CD4(+) T-cell responses of allergic and nonallergic subjects to Can f 4. METHODS: Can f 4-specific CD4(+)CD45RO(+) T-cell lines (TCLs) from allergic and healthy subjects were established and characterized by their functional and phenotypic properties. The epitope specificity of the TCLs was tested with 48 overlapping 16-mer peptides spanning the sequence of Can f 4. HLA restriction of the specific TCLs and the binding capacity of the epitope-containing peptides to common HLA class II molecules were studied. RESULTS: Can f 4-specific memory CD4(+) TCLs were obtained at an 8-fold higher frequency from allergic than from nonallergic subjects. Functionally, the TCLs of allergic subjects exhibited a higher T-cell receptor avidity and expression of CD25 and predominantly produced IL-4 and IL-5. The TCLs of nonallergic subjects mostly secreted IFN-γ and IL-10, with high CXCR3 expression. Several distinct T-cell epitope regions along the allergen were identified. Importantly, the peptides from the region between amino acids 43 and 67 showed promiscuous HLA-binding capacity and induced memory CD4(+) T-cell responses in 90% of the allergic donors. CONCLUSION: Productive TH2-deviated memory T-cell responses to Can f 4 are observed in allergic but not nonallergic subjects. A 19-mer peptide sequence covering the core of the immunodominant region of the allergen is a potential target for the development of peptide-based allergen immunotherapy.

The serodominant secreted effector protein of Salmonella, SseB, is a strong CD4 antigen containing an immunodominant epitope presented by diverse HLA class II alleles.

Detailed characterization of the protective T-cell response in salmonellosis is a pressing unmet need in light of the global burden of human Salmonella infections and the likely contribution of CD4 T cells to immunity against this intracellular infection. In previous studies screening patient sera against antigen arrays, SseB was noteworthy as a serodominant target of adaptive immunity, inducing significantly raised antibody responses in HIV-seronegative compared with seropositive patients. SseB is a secreted protein, part of the Espa superfamily, localized to the bacterial surface and forming part of the translocon of the type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2. We demonstrate here that SseB is also a target of CD4 T-cell immunity, generating a substantial response after experimental infection in human volunteers, with around 0.1% of the peripheral repertoire responding to it. HLA-DR/peptide binding studies indicate that this protein encompasses a number of peptides with ability to bind to several different HLA-DR alleles. Of these, peptide 11 (p11) was shown in priming of both HLA-DR1 and HLA-DR4 transgenic mice to contain an immunodominant CD4 epitope. Analysis of responses in human donors showed immunity focused on p11 and another epitope in peptide 2. The high frequency of SseB-reactive CD4 T cells and the broad applicability to diverse HLA genotypes coupled with previous observations of serodominance and protective vaccination in mouse challenge experiments, make SseB a plausible candidate for next-generation Salmonella vaccines.

Exploring the Potential of the Model Cyanobacteria Synechococcus PCC 7002 and PCC 7942 for the Photoproduction of High-Value Terpenes: A Comparison with Synechocystis PCC 6803.

We have performed the first comparative analysis of the potential of two physiologically-diverse model cyanobacteria, Synechococcus PCC 7002 (S.7002) and Synechococcus elongatus PCC 7942 (S.7942), for the photosynthetic production of four chemically-different high-value terpenes: two monoterpenes limonene and pinene, and two sesquiterpenes bisabolene and farnesene. We showed, for the first time, that S.7002 and S.7942 can produce farnesene and bisabolene, respectively. Both cyanobacteria produced farnesene (S.7942 produced more efficiently than S.7002) more efficiently than the other tested terpenes (especially pinene, the weakest produced terpene). S.7002 produced limonene more efficiently than bisabolene, whereas S.7942 produced bisabolene more efficiently than limonene. These findings suggest that S.7942 is better suited to produce sesquiterpenes than monoterpenes. Interestingly, higher levels of terpenes were produced by S.7942 and S.7002 expressing a terpene-synthase gene from both an RSF1010-derived replicating plasmid and a neutral chromosomal site, as compared to either the plasmid alone or the chromosome alone. These results suggest that in both cyanobacteria, the production of terpenes is more limited by the activity of terpene synthases than the abundance of terpene precursors. Finally, higher levels of terpenes were produced by S.7002 growing on urea (a frequent pollutant) as compared to nitrate or ammonium, the standard nitrogen sources for cyanobacteria.

A general procedure for carbon isotope labeling of linear urea derivatives with carbon dioxide.

Carbon isotope labeling is a traceless technology, which allows tracking the fate of organic compounds either in the environment or in living organisms. This article reports on a general approach to label urea derivatives with all carbon isotopes, including 14C and 11C, based on a Staudinger aza-Wittig sequence. It provides access to all aliphatic/aromatic urea combinations.

Characterization of CD4 T Cell Epitopes of Infliximab and Rituximab Identified from Healthy Donors.

The chimeric antibodies anti-CD20 rituximab (Rtx) and anti-TNFα infliximab (Ifx) induce antidrug antibodies (ADAs) in many patients with inflammatory diseases. Because of the key role of CD4 T lymphocytes in the initiation of antibody responses, we localized the CD4 T cell epitopes of Rtx and Ifx. With the perspective to anticipate immunogenicity of therapeutic antibodies, identification of the CD4 T cell epitopes was performed using cells collected in healthy donors. Nine T cell epitopes were identified in the variable chains of both antibodies by deriving CD4 T cell lines raised against either Rtx or Ifx. The T cell epitopes often exhibited a good affinity for human leukocyte antigen (HLA)-DR molecules and were part of the peptides identified by MHC-associated peptide proteomics assay from HLA-DR molecules of dendritic cells (DCs) loaded with the antibodies. Two-third of the T cell epitopes identified from the healthy donors stimulated peripheral blood mononuclear cells from patients having developed ADAs against Rtx or Ifx and promoted the secretion of a diversity of cytokines. These data emphasize the predictive value of evaluating the T cell repertoire of healthy donors and the composition of peptides bound to HLA-DR of DCs to anticipate and prevent immunogenicity of therapeutic antibodies.

ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments.

Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.

Inhibitors of retrograde trafficking active against ricin and Shiga toxins also protect cells from several viruses, Leishmania and Chlamydiales.

Medical countermeasures to treat biothreat agent infections require broad-spectrum therapeutics that do not induce agent resistance. A cell-based high-throughput screen (HTS) against ricin toxin combined with hit optimization allowed selection of a family of compounds that meet these requirements. The hit compound Retro-2 and its derivatives have been demonstrated to be safe in vivo in mice even at high doses. Moreover, Retro-2 is an inhibitor of retrograde transport that affects syntaxin-5-dependent toxins and pathogens. As a consequence, it has a broad-spectrum activity that has been demonstrated both in vitro and in vivo against ricin, Shiga toxin-producing O104:H4 entero-hemorrhagic E. coli and Leishmania sp. and in vitro against Ebola, Marburg and poxviruses and Chlamydiales. An effect is anticipated on other toxins or pathogens that use retrograde trafficking and syntaxin-5. Since Retro-2 targets cell components of the host and not directly the pathogen, no selection of resistant pathogens is expected. These lead compounds need now to be developed as drugs for human use.

The Translocation Domain of Botulinum Neurotoxin A Moderates the Propensity of the Catalytic Domain to Interact with Membranes at Acidic pH.

Botulinum neurotoxin A (BoNT/A) is composed of three domains: a catalytic domain (LC), a translocation domain (HN) and a receptor-binding domain (HC). Like most bacterial toxins BoNT/A is an amphitropic protein, produced in a soluble form that is able to interact, penetrate and/or cross a membrane to achieve its toxic function. During intoxication BoNT/A is internalized by the cell by receptor-mediated endocytosis. Then, LC crosses the membrane of the endocytic compartment and reaches the cytosol. This translocation is initiated by the low pH found in this compartment. It has been suggested that LC passes in an unfolded state through a transmembrane passage formed by HN. We report here that acidification induces no major conformational change in either secondary or tertiary structures of LC and HN of BoNT/A in solution. GdnHCl-induced denaturation experiments showed that the stability of LC and HN increases as pH drops, and that HN further stabilizes LC. Unexpectedly we found that LC has a high propensity to interact with and permeabilize anionic lipid bilayers upon acidification without the help of HN. This property is downplayed when LC is linked to HN. HN thus acts as a chaperone for LC by enhancing its stability but also as a moderator of the membrane interaction of LC.

Correction: The Translocation Domain of Botulinum Neurotoxin A Moderates the Propensity of the Catalytic Domain to Interact with Membranes at Acidic pH.

[This corrects the article DOI: 10.1371/journal.pone.0153401.].

The Neurotoxic Effect of 13,19-Didesmethyl and 13-Desmethyl Spirolide C Phycotoxins Is Mainly Mediated by Nicotinic Rather Than Muscarinic Acetylcholine Receptors.

Spirolides are a large family of lipophilic marine toxins produced by dinoflagellates that have been detected in contaminated shellfish. Among them, 13,19-didesmethyl and 13-desmethyl spirolide C phycotoxins are widely distributed and their mode of action needs to be clearly defined. In order to further characterize the pharmacological profiles of these phycotoxins on various nicotinic acetylcholine receptor (nAChR) subtypes and to examine whether they act on muscarinic receptors (mAChRs), functional electrophysiological studies and competition binding experiments have been performed. While 13-desmethyl spirolide C interacted efficiently with sub-nanomolar affinities and low selectivity with muscular and neuronal nAChRs, 13,19-didesmethyl spirolide C was more selective of muscular and homopentameric α7 receptors and recognized only weakly neuronal heteropentameric receptors, especially the α4ß2 subtype. Thus, the presence of an additional methyl group on the tetrahydropyran ring significantly modified the pharmacological profile of 13-desmethyl spirolide C by notably increasing its affinity on certain neuronal nAChRs. Structural explanations of this selectivity difference are proposed, based on molecular docking experiments modeling different spirolide-receptor complexes. In addition, the 2 spirolides interacted only with low micromolar affinities with the 5 mAChRs, highlighting that the toxicity of the spirolide C analogs is mainly due to their high inhibition potency on various peripheral and central nAChRs and not to their low ability to interact with mAChR subtypes.

N-methyldihydroquinazolinone derivatives of Retro-2 with enhanced efficacy against Shiga toxin.

The Retro-2 molecule protects cells against Shiga toxins by specifically blocking retrograde transport from early endosomes to the trans-Golgi network. A SAR study has been carried out to identify more potent compounds. Cyclization and modifications of Retro-2 led to a compound with roughly 100-fold improvement of the EC50 against Shiga toxin cytotoxicity measured in a cell protein synthesis assay. We also demonstrated that only one enantiomer of the dihydroquinazolinone reported herein is bioactive.