Immunological Memory Transferred with CD4 T Cells Specific for Tuberculosis Antigens Ag85B-TB10.4: Persisting Antigen Enhances Protection

BACKGROUND:High levels of death and morbidity worldwide caused by tuberculosis has stimulated efforts to develop a new vaccine to replace BCG. A number of Mycobacterium tuberculosis (Mtb)-specific antigens have been synthesised as recombinant subunit vaccines for clinical evaluation. Recently a fusion protein of TB antigen Ag85B combined with a second immunodominant TB antigen TB10.4 was emulsified with a novel non-phospholipid-based liposomal adjuvant to produce a new subunit vaccine, investigated here. Currently, there is no consensus as to whether or not long-term T cell memory depends on a source of persisting antigen. To explore this and questions regarding lifespan, phenotype and cytokine patterns of CD4 memory T cells, we developed an animal model in which vaccine-induced CD4 memory T cells could transfer immunity to irradiated recipients. METHODOLOGY/PRINCIPAL FINDINGS:The transfer of protective immunity using Ag85B-TB10.4-specific, CD45RB(low) CD62L(low) CD4 T cells was assessed in sub-lethally irradiated recipients following challenge with live BCG, used here as a surrogate for virulent Mtb. Donor T cells also carried an allotype marker allowing us to monitor numbers of antigen-specific, cytokine-producing CD4 T cells in recipients. The results showed that both Ag85B-TB10.4 and BCG vaccination induced immunity that could be transferred with a single injection of 3x10(6) CD4 T cells. Ten times fewer numbers of CD4 T cells (0.3x10(6)) from donors immunised with Ag85B-TB10.4 vaccine alone, transferred equivalent protection. CD4 T cells from donors primed by BCG and boosted with the vaccine similarly transferred protective immunity. When BCG challenge was delayed for 1 or 2 months after transfer (a test of memory T cell survival) recipients remained protected. Importantly, recipients that contained persisting antigen, either live BCG or inert vaccine, showed significantly higher levels of protection (p<0.01). Overall the numbers of IFN-gamma-producing CD4 T cells were poorly correlated with levels of protection. CONCLUSIONS/SIGNIFICANCE:The Ag85B-TB10.4 vaccine, with or without BCG-priming, generated TB-specific CD4 T cells that transferred protective immunity in mice challenged with BCG. The level of protection was enhanced in recipients containing a residual source of specific antigen that could be either viable or inert

Original Encounter with Antigen Determines Antigen-Presenting Cell Imprinting of the Quality of the Immune Response in Mice

BACKGROUND:Obtaining a certain multi-functionality of cellular immunity for the control of infectious diseases is a burning question in immunology and in vaccine design. Early events, including antigen shuttling to secondary lymphoid organs and recruitment of innate immune cells for adaptive immune response, determine host responsiveness to antigens. However, the sequence of these events and their impact on the quality of the immune response remain to be elucidated. Here, we chose to study Modified Vaccinia virus Ankara (MVA) which is now replacing live Smallpox vaccines and is proposed as an attenuated vector for vaccination strategies against infectious diseases. METHODOLOGY/PRINCIPAL FINDINGS:We analyzed in vivo mechanisms triggered following intradermal (i.d.) and intramuscular (i.m.) Modified Vaccinia virus Ankara (MVA) administration. We demonstrated significant differences in the antigen shuttling to lymphoid organs by macrophages (MPhis), myeloid dendritic cells (DCs), and neutrophils (PMNs). MVA i.d. administration resulted in better antigen distribution and more sustained antigen-presenting cells (APCs) recruitment into draining lymph nodes than with i.m. administration. These APCs, which comprise both DCs and MPhis, were differentially involved in T cell priming and shaped remarkably the quality of cytokine-producing virus-specific T cells according to the entry route of MVA. CONCLUSIONS/SIGNIFICANCE:This study improves our understanding of the mechanisms of antigen delivery and their consequences on the quality of immune responses and provides new insights for vaccine development

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α

International audienceThe main aim of this protocol is to describe the development and validation of an interferon (IFN)-α single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-α protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN. The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-α single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-α subtypes. This new methodology allows the detection and quantification of IFN-α protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation

Activation of NLRP3 Inflammasome in the Skin of Patients with Systemic and Cutaneous Lupus Erythematosus

NLRP3 inflammasome is suggested to contribute to the complex pathogenesis of systemic lupus erythematosus, but its role in cutaneous lupus erythematosus has not been addressed. This study investigated the expression of NLRP3 inflammasome components and levels of type I interferons in the skin of 20 patients with cutaneous lupus erythematosus. Expression of NLRP1/3, adaptor protein ASC (apoptosis-associated speck-like protein), caspase-1, interferon-alpha (IFN-alpha), myxovirus resistance protein (MxA), and interferon-induced proteins 1 and 2 (IFIT 1/2) in the skin was assessed using reverse transcription quantitative real-time PCR (RT-qPCR), western blotting and immunohistochemistry. Serum interferon-a protein levels from 12 patients were measured using digital enzyme-linked immunoassay (ELISA). Interleukin-1 beta expression was significantly upregulated in the lesional skin of patients with cutaneous lupus erythematosus compared with their uninvolved skin. However, NLRP1/3, ASC and caspase-1 were not significantly upregulated compared with the skin of control persons. IFN-alpha and IFN-induced proteins MxA and IFIT1/2 were strongly expressed in cutaneous lupus erythematosus skin. Variability in the expression of NLRP3 inflammasome components among patients suggests heterogeneity of pathological pathways in cutaneous lupus erythematosus.Peer reviewe

Delineating the Healthy Human Skin UV Response and Early Induction of Interferon Pathway in Cutaneous Lupus Erythematosus

Non peer reviewe

Development of a Bead-Based Multiplex Assay for the Analysis of the Serological Response against the Six Pathogens HAV, HBV, HCV, CMV, T. gondii, and H. pylori

The spread of infectious diseases and vaccination history are common subjects of epidemiological and immunological research studies. Multiplexed serological assays are useful tools for assessing both current and previous infections as well as vaccination efficacy. We developed a serological multi-pathogen assay for hepatitis A, B and C virus, cytomegalovirus (CMV), Toxoplasma gondii, and Helicobacter pylori using a bead-based multiplex assay format. The multi-pathogen assay consisting of 15 antigens was utilized for the analysis of the serological response in elderly individuals of an influenza vaccination study (n = 34). The technical assay validation revealed a mean intra-assay precision of coefficient of variation (CV) = 3.2 ± 1.5% and a mean inter-assay precision of CV = 8.2 ± 5.3% across all 15 antigens and all tested samples, indicating a robust test system. Furthermore, the assay shows high sensitivities (ranging between 94% and 100%) and specificities (ranging between 93% and 100%) for the different pathogens. The highest seroprevalence rates in our cohort were observed for hepatitis A virus (HAV; 73.5%), followed by CMV (70.6%), T. gondii (67.6%) and H. pylori (32.4%). Seroprevalences for hepatitis B virus (HBV, 8.8%) and hepatitis C virus (HCV, 0%) were low. The seroprevalences observed in our study were similar to those from other population-based studies in Germany. In summary, we conclude that our multiplex serological assay represents a suitable tool for epidemiological studies

Deconvolution of the Response to Bacillus Calmette–Guérin Reveals NF-κB-Induced Cytokines As Autocrine Mediators of Innate Immunity

Bacillus Calmette–Guérin (BCG) is used as a vaccine and diagnostic test for tuberculosis, as well as immunotherapy in the treatment of bladder cancer. While clinically useful, the response to mycobacterial stimulation is complex and the induced protein signature remains poorly defined. We characterized the cell types directly engaged by BCG, as well as the induced cytokine loops that transmit signal(s) to bystander cells. Standardized whole-blood stimulations and mechanistic studies on single and purified cell populations identified distinct patterns of activation in monocytes as compared to neutrophils and invariant lymphocyte populations. Deconvoluting the role of Toll-like receptor 2/4 and Dectin-1/2 in the inflammatory response to BCG, we revealed Dectin-1/2 as dominant in neutrophils as compared to monocytes, which equally engaged both pathways. Furthermore, we quantified the role of NF-κB and NADPH/reactive oxygen species (ROS)-dependent cytokines, which triggered a JAK1/2-dependent amplification loop and accounted for 40–50% of the induced response to BCG. In sum, this study provides new insight into the molecular and cellular pathways involved in the response to BCG, establishing the basis for a new generation of immunodiagnostic tools

JAK Inhibition in the Aicardi-Goutières Syndrome

International audienc

Decreased Type I Interferon Production by Plasmacytoid Dendritic Cells Contributes to Severe Dengue

International audienceThe clinical presentation of dengue virus (DENV) infection is variable. Severe complications mainly result from exacerbated immune responses. Type I interferons (IFN-I) are important in antiviral responses and form a crucial link between innate and adaptive immunity. Their contribution to host defense during DENV infection remains under-studied, as direct quantification of IFN-I is challenging. We combined ultra-sensitive single-molecule array (Simoa) digital ELISA with IFN-I gene expression to elucidate the role of IFN-I in a well-characterized cohort of hospitalized Cambodian children undergoing acute DENV infection. Higher concentrations of type I IFN proteins were observed in blood of DENV patients, compared to healthy donors, and correlated with viral load. Stratifying patients for disease severity, we found a decreased expression of IFN-I in patients with a more severe clinical outcome, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). This was seen in parallel to a correlation between low IFNα protein concentrations and decreased platelet counts. Type I IFNs concentrations were correlated to frequencies of plasmacytoid DCs, not DENV-infected myloid DCs and correlated inversely with neutralizing anti-DENV antibody titers. Hence, type I IFN produced in the acute phase of infection is associated with less severe outcome of dengue disease

Inactive disease in patients with lupus is linked to autoantibodies to type I interferons that normalize blood IFNα and B cell subsets

Systemic lupus erythematosus (SLE) is characterized by increased expression of type I interferon (IFN)-regulated genes in 50%-75% of patients. We report that out of 501 patients with SLE analyzed, 73 (14%) present autoantibodies against IFNα (anti-IFN-Abs). The presence of neutralizing-anti-IFN-Abs in 4.2% of patients inversely correlates with low circulating IFNα protein levels, inhibition of IFN-I downstream gene signatures, and inactive global disease score. Hallmarks of SLE pathogenesis, including increased immature, double-negative plasmablast B cell populations and reduction in regulatory B cell (Breg) frequencies, were normalized in patients with neutralizing anti-IFN-Abs compared with other patient groups. Immunoglobulin G (IgG) purified from sera of patients with SLE with neutralizing anti-IFN-Abs impedes CpGC-driven IFNα-dependent differentiation of B cells into immature B cells and plasmablasts, thus recapitulating the neutralizing effect of anti-IFN-Abs on B cell differentiation in vitro. Our findings highlight a role for neutralizing anti-IFN-Abs in controlling SLE pathogenesis and support the use of IFN-targeting therapies in patients with SLE lacking neutralizing-anti-IFN-Abs