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FOXP3+ Tregs are central for the maintenance of self-tolerance and can be defective in autoimmunity. In multiple sclerosis and type-1 diabetes, dysfunctional self-tolerance is partially mediated by a population of IFNγ-secreting Tregs. It was previously reported that increased NaCl concentrations promote the induction of proinflammatory Th17 cells and that high-salt diets exacerbate experimental models of autoimmunity. Here, we have shown that increasing NaCl, either in vitro or in murine models via diet, markedly impairs Treg function. NaCl increased IFNγ secretion in Tregs, and reducing IFNγ - either by neutralization with anti-IFNγ antibodies or shRNA-mediated knockdown - restored suppressive activity in Tregs. The heightened IFNγ secretion and loss of Treg function were mediated by the serum/glucocorticoid-regulated kinase (SGK1). A high-salt diet also impaired human Treg function and was associated with the induction of IFNγ-secreting Tregs in a xenogeneic graft-versus-host disease model and in adoptive transfer models of experimental colitis. Our results demonstrate a putative role for an environmental factor that promotes autoimmunity by inducing proinflammatory responses in CD4 effector cells and Treg pathways.

CD4+CD25+ Tregs regulate immunity, but little is known about their own regulation. We now report that the human 60-kDa heat shock protein (HSP60) acts as a costimulator of human Tregs, both CD4+CD25int and CD4+CD25hi. Treatment of Tregs with HSP60, or its peptide p277, before anti-CD3 activation significantly enhanced the ability of relatively low concentrations of the Tregs to downregulate CD4+CD25- or CD8+ target T cells, detected as inhibition of target T cell proliferation and IFN-gamma and TNF-alpha secretion. The enhancing effects of HSP60 costimulation on Tregs involved innate signaling via TLR2, led to activation of PKC, PI3K, and p38, and were further enhanced by inhibition of ERK. HSP60-treated Tregs suppressed target T cells both by cell-to-cell contact and by secretion of TGF-beta and IL-10. In addition, the expression of ERK, NF-kappaB, and T-bet by downregulated target T cells was inhibited. Thus, HSP60, a self-molecule, can downregulate adaptive immune responses by upregulating Tregs innately through TLR2 signaling.

In this study, we described the generation and immunogenicity of the Zika Virus (ZIKV) envelope protein (E) domain III (DIII) as a protein subunit vaccine candidate. ZIKV EDIII (zEDIII) was rapidly produced in E. coli in inclusion bodies. ZIKV EDIII was solubilized, refolded and purified to >95% homogeneity with a one-step Ni2+ affinity chromatography process. Further analysis revealed that zEDIII was refolded properly and demonstrated specific binding to an anti-zEDIII monoclonal antibody that recognizes a zEDIII conformational epitope. Subcutaneous immunization of mice with 25 and 50μg of zEDIII was performed over a period of 11weeks. zEDIII evoked ZIKV-specific and neutralizing antibody response with titers that exceed the threshold that correlates with protective immunity against ZIKV. The antigen-specific IgG isotypes were predominantly IgG1 and splenocyte cultures from immunized mice secreted IFN-gamma, IL-4 and IL-6. Notably, zEDIII-elicited antibodies did not enhance the infection of dengue virus in Fc gamma receptor (FcγR)-expressing cells. This study provided a proof of principle for the further development of recombinant protein-based subunit vaccines against ZIKV.

► NDV-3 is safe and generally well-tolerated in healthy adults. ► NDV-3 elicits quick and robust B- and T-cell immune responses. ► A single dose induces an anamnestic rather than priming immune response. ► T-cell response includes increases in PBMCs producing IFN-γ and/or IL-17A. ► A second dose further enhances the B- and T-cell immune response. The investigational vaccine, NDV-3, contains the N-terminal portion of the Candida albicans agglutinin-like sequence 3 protein (Als3p) formulated with an aluminum hydroxide adjuvant in phosphate-buffered saline. Preclinical studies demonstrated that the Als3p vaccine antigen protects mice from oropharyngeal, vaginal and intravenous challenge with C. albicans and other selected species of Candida as well as both intravenous challenge and skin and soft tissue infection with Staphylococcus aureus. The objectives of this first-in-human Phase I clinical trial were to evaluate the safety, tolerability and immunogenicity of NDV-3 at two different antigen levels compared to a saline placebo. Forty healthy, adult subjects were randomized to receive one dose of NDV-3 containing either 30 or 300μg of Als3p, or placebo. NDV-3 at both dose levels was safe and generally well-tolerated. Anti-Als3p total IgG and IgA1 levels for both doses reached peak levels by day 14 post vaccination, with 100% seroconversion of all vaccinated subjects. On average, NDV-3 stimulated peripheral blood mononuclear cell (PBMC) production of both IFN-γ and IL-17A, which peaked at day 7 for subjects receiving the 300μg dose and at day 28 for those receiving the 30μg dose. Six months after receiving the first dose of NDV-3, nineteen subjects received a second dose of NDV-3 identical to their first dose to evaluate memory B- and T-cell immune responses. The second dose resulted in a significant boost of IgG and IgA1 titers in >70% of subjects, with the biggest impact in those receiving the 30μg dose. A memory T-cell response was also noted for IFN-γ in almost all subjects and for IL-17A in the majority of subjects. These data support the continued investigation of NDV-3 as a vaccine candidate against Candida and S. aureus infections.

► The recombinant BCG vaccine VPM1002 was tested in PPD+ and PPD− individuals. ► VPM1002 successfully completed a phase I clinical trial. ► VPM1002 was as safe as BCG. ► VPM1002 and BCG induced both shared and differential immune responses. Current vaccination using Mycobacterium bovis bacillus Calmette-Guérin (BCG), fails to prevent pulmonary tuberculosis (TB). New vaccination strategies are essential for reducing the global incidence of TB. We assessed the safety and immunogenicity of VPM1002, a recombinant BCG vaccine candidate. EudraCT (2007-002789-37) and ClinicalTrials.gov (NCT00749034). Healthy volunteers were enrolled in a phase 1 open-label, dose escalation randomized clinical trial, and received one intradermal dose of VPM1002 (Mycobacterium bovis BCG ΔureC::hly HmR) or BCG. Immunogenicity was assessed by interferon-gamma (IFN-γ) production, cellular immune response markers by flow cytometry and serum antibodies against mycobacterial antigens. Eighty volunteers were randomized into two groups according to previous BCG vaccination and mycobacterial exposure (BCG-naïve, n=40 and BCG-immune, n=40). In each group, 30 individuals were vaccinated with VPM1002 (randomized to three escalating doses) and 10 with BCG. VPM1002 was safe and stimulated IFN-γ-producing and multifunctional T cells, as well as antibody-producing B cells in BCG-naïve and BCG-immune individuals. VPM1002 was safe and immunogenic for B-cell and T-cell responses and hence will be brought forward through the clinical trial pipeline.

Background. Current serodiagnostics for Lyme disease lack sensitivity during early disease, and cannot determine treatment response. We evaluated an assay based on QuantiFERON technology utilizing peptide antigens derived from Borrelia burgdorferi to stimulate interferon-gamma (IFN-γ) release as an alternative to serodiagnosis for the laboratory detection of Lyme disease. Methods. Blood was obtained from patients with erythema migrans before (n = 29) and 2 months after (n = 27) antibiotic therapy. IFN-γ release was measured by enzyme-linked immunosorbent assay (ELISA) following overnight stimulation of whole blood with the peptide antigens, and compared to the results of standard serological assays (C6, ELISA, and Western blot). Results. IFN-γ release was observed in pretreatment blood of 20 of 29 (69%) patients with Lyme disease. Following antibiotic treatment, IFN-γ was significantly reduced (P –.0002), and was detectable in only 4 of 20 (20%) initially positive patients. By contrast, anti-C6 antibodies were detected in pretreatment sera from 17 of 29 (59%) subjects, whereas only 5 of 29 (17%) patients had positive Western blot seroreactivity. Antibody responses persisted and expanded following treatment. Conclusions. Our findings suggest that measurement of IFN-γ after incubating blood with Borrelia antigens could be useful in the laboratory diagnosis of early Lyme disease. Also, after antibiotic treatment, this response appears to be short lived.

PURPOSE: To investigate whether breast-milk composition and microbiota differ in healthy mothers and mothers with celiac disease (CD) to ultimately contribute to identify additional factors determining CD risk. METHODS: Breast-milk samples from healthy mothers (n = 12) and mothers with CD (n = 12) were collected. Cytokines and secretory immunoglobulin A (sIgA) were analyzed by bead-arrays and flow cytometry and human milk oligosaccharides (HMOs) were assessed by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. Breast-milk microbiota composition was analyzed by conventional and quantitative real-time PCR. RESULT: Breast milk from CD mothers showed significantly lower levels of interleukin (IL) 12p70 (P < 0.042), transforming growth factor (TGF)-β1 (P < 0.018) and sIgA (P < 0.003) and almost significantly lower levels of interferon (IFN)-γ (P < 0.058). Six mothers in each group belonged to the secretor Le(a−b+) type, one to the secretor Le(a−b−) type and five to the non-secretor Le(a+b−) type. CD mothers of non-secretor Le(a+b−) type showed increased Lacto-N-tetraose content (P < 0.042) compared with healthy mothers. CD mothers’ milk showed reduced gene copy numbers of Bifidobacterium spp. (P < 0.026) and B. fragilis group (P < 0.044). CONCLUSION: CD mothers’ breast milk is characterized by a reduced abundance of immunoprotective compounds (TGF-β1 and sIgA) and bifidobacteria. The reduction in these components could theoretically diminish the protective effects of breast-feeding on the child’s future risk of developing CD.

•PLGA nanoparticles display sustained release of MOG and IL-10.•Subcutaneous vaccination with MOG-PLGA plus IL10-PLGA inhibits EAE.•This subcutaneous vaccination inhibits MOG-induced secretion of IL-17 and IFN-γ. “Inverse vaccination” refers to antigen-specific tolerogenic immunization treatments that are capable of inhibiting autoimmune responses. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), initial trials using purified myelin antigens required repeated injections because of the rapid clearance of the antigens. This problem has been overcome by DNA-based vaccines encoding for myelin autoantigens alone or in combination with “adjuvant” molecules, such as interleukin (IL)-4 or IL-10, that support regulatory immune responses. Phase I and II clinical trials with myelin basic protein (MBP)-based DNA vaccines showed positive results in reducing magnetic resonance imaging (MRI)-measured lesions and inducing tolerance to myelin antigens in subsets of MS patients. However, DNA vaccination has potential risks that limit its use in humans. An alternative approach could be the use of protein-based inverse vaccines loaded in polymeric biodegradable lactic-glycolic acid (PLGA) nano/microparticles (NP) to obtain the sustained release of antigens and regulatory adjuvants. The aim of this work was to test the effectiveness of PLGA-NP loaded with the myelin oligodendrocyte glycoprotein (MOG)35–55 autoantigen and recombinant (r) IL-10 to inverse vaccinate mice with EAE. In vitro experiments showed that upon encapsulation in PLGA-NP, both MOG35-55 and rIL-10 were released for several weeks into the supernatant. PLGA-NP did not display cytotoxic or proinflammatory activity and were partially endocytosed by phagocytes. In vivo experiments showed that subcutaneous prophylactic and therapeutic inverse vaccination with PLGA-NP loaded with MOG35-55 and rIL-10 significantly ameliorated the course of EAE induced with MOG35-55 in C57BL/6 mice. Moreover, they decreased the histopathologic lesions in the central nervous tissue and the secretion of IL-17 and interferon (IFN)-γ induced by MOG35-55 in splenic T cells in vitro. These data suggest that subcutaneous PLGA-NP-based inverse vaccination may be an effective tool to treat autoimmune diseases.

Breast cancer is a leading cause of cancer-related death, and it is important to understand pathways that drive the disease to devise effective therapeutic strategies. Our results show that Toll-like receptor 4 (TLR4) drives breast cancer cell growth differentially based on the presence of TP53, a tumor suppressor. TP53 is mutationally inactivated in most types of cancer and is mutated in 30–50% of diagnosed breast tumors. We demonstrate that TLR4 activation inhibits growth of TP53 wild-type cells, but promotes growth of TP53 mutant breast cancer cells by regulating proliferation. This differential effect is mediated by changes in tumor cell cytokine secretion. Whereas TLR4 activation in TP53 mutant breast cancer cells increases secretion of progrowth cytokines, TLR4 activation in TP53 wild-type breast cancer cells increases type I IFN (IFN-γ) secretion, which is both necessary and sufficient for mediating TLR4-induced growth inhibition. This study identifies a novel dichotomous role for TLR4 as a growth regulator and a modulator of tumor microenvironment in breast tumors. These results have translational relevance, demonstrating that TP53 mutant breast tumor growth can be suppressed by pharmacologic TLR4 inhibition, whereas TLR4 inhibitors may in fact promote growth of TP53 wild-type tumors. Furthermore, using data generated by The Cancer Genome Atlas consortium, we demonstrate that the effect of TP53 mutational status on TLR4 activity may extend to ovarian, colon, and lung cancers, among others, suggesting that the viability of TLR4 as a therapeutic target depends on TP53 status in many different tumor types. Significance This study fundamentally alters our understanding of how TLR4 drives breast cancer. Although TLR4 was previously considered a tumor promoter, we demonstrate a complex, TP53-dependent role for TLR4 in regulating tumor growth. TP53 is a tumor suppressor commonly inactivated across cancer types. In TP53 wild-type cancer cells, TLR4 activation causes secretion of IFN-γ into the microenvironment, resulting in induction of p21 and inhibition of cell growth. Conversely, TLR4 activation in TP53 mutant cells promotes cancer cell growth by regulating CXCL1 and CD154 secretion. In this paper, we identify a previously unidentified role for TLR4 in modulating tumor cell growth and microenvironment. The TLR4–TP53 association likely extends across cancer types, suggesting the need to determine the TP53 status of any tumor before implementing anti-TLR4 therapy.

One year after the occurrence of the first case of infection by the Middle East Respiratory Syndrome coronavirus (MERS-CoV) there is no clear consensus on the best treatment to propose. The World Health Organization, as well as several other national agencies, are still working on different clinical approaches to implement the most relevant treatment in MERS-CoV infection. We compared innate and adaptive immune responses of two patients infected with MERS-CoV to understand the underlying mechanisms involved in the response and propose potential therapeutic approaches. Broncho-alveolar lavage (BAL) of the first week and sera of the first month from the two patients were used in this study. Quantitative polymerase chain reaction (qRTPCR) was performed after extraction of RNA from BAL cells of MERS-CoV infected patients and control patients. BAL supernatants and sera were used to assess cytokines and chemokines secretion by enzyme-linked immunosorbent assay. The first patient died rapidly after 3 weeks in the intensive care unit, the second patient still recovers from infection. The patient with a poor outcome (patient 1), compared to patient 2, did not promote type-1 Interferon (IFN), and particularly IFNα, in response to double stranded RNA (dsRNA) from MERS-CoV. The absence of IFNα, known to promote antigen presentation in response to viruses, impairs the development of a robust antiviral adaptive Th-1 immune response. This response is mediated by IL-12 and IFNγ that decreases viral clearance; levels of both of these mediators were decreased in patient 1. Finally, we confirm previous in vitro findings that MERS-CoV can drive IL-17 production in humans. Host recognition of viral dsRNA determines outcome in the early stage of MERS-CoV infection. We highlight the critical role of IFNα in this initial stage to orchestrate a robust immune response and bring substantial arguments for the indication of early IFNα treatment during MERS-CoV infection.