Immunoproteomic and Immunopeptidomic Analyses of Histoplasma capsulatum Reveal Promiscuous and Conserved Epitopes Among Fungi With Vaccine Potential

As there are more than 6 million human deaths due to mycoses each year, there is an urgent need to develop fungal vaccines. Moreover, given the similarities among pathogenic fungi, it may be possible to create a multi-fungi vaccine. In this study, we combined immunoproteomic and immunopeptidomic methods, for which we have adapted a technique based on co-immunoprecipitation (Co-IP) that made it possible to map Histoplasma capsulatum epitopes for the first time in a natural context using murine dendritic cells (DCs) and macrophages (Mφ). Although polysaccharide epitopes exist, this research focused on mapping protein epitopes as these are more immunogenic. We used different algorithms to screen proteins and peptides identified by two-dimensional electrophoresis (2-D) and Co-IP. Seventeen proteins were revealed by 2-D gels, and 45 and 24 peptides from distinct proteins were presented by DCs and Mφ, respectively. We then determined which epitopes were restricted to MHC-I and II from humans and mice and showed high promiscuity, but lacked identity with human proteins. The 4 most promising peptides were synthesized, and the peptides with and without incorporation into glucan particles induced CD4+ and CD8+ T cell proliferation and produced a Th1 and Th17 response marked by the secretion of high levels of IFN-γ, IL-17 and IL-2. These epitopes were from heat shock protein 60, enolase, and the ATP-dependent molecular chaperone HSC82, and they each have a high degree of identity with proteins expressed by other medically important pathogenic fungi. Thus, the epitopes described in this study have the potential for use in the development of vaccines that could result in cross-protection among fungal species.

Acquired Hemophilia a Relapse Is Related to Th2 Response, and Increased Expression of B-Cell Activating Factor (BAFF)

Acquired hemophilia A (AHA) is a rare autoimmune disorder caused by the development of autoantibodies against the factor (F)VIII of coagulation with higher incidence in elderly individuals. The treatment includes immunosuppressive alternatives, and the majority of patients reach complete remission of symptoms. Although the role of immune T and B cells in the physiopathology of several autoimmune diseases is widely established, few studies have explored the T and B cells immune response pathway in AHA. This study aimed to determine the role of T cells through their cytokines expression and B cells through their expression of proteins that activate the B cell receptor (BCR), such as B-cell activating factor (BAFF) and A-proliferation inducing ligand (APRIL), in a longitudinal evaluation of patients with AHA. We included in this study 11 patients, 4 (36%) male, with the diagnosis of AHA, based on the occurrence of low FVIII levels (median FVIII level at the diagnosis was 0.10 IU/dL; range 0-18.8) and anti-FVIII inhibitor titer positive (≥0.6 Bethesda units (BU)/mL) (median inhibitor titer at the diagnosis was 60BU/mL; range 7.6-1000.2). The median age at the diagnosis was 59 years (range 9-75). Idiopathic AHA was evident in 72.7% of cases, while autoimmune diseases (18.2%), and pregnancy (9.1%) were the other underlying etiologies. All patients received immunosuppressive therapy (IST). Eight (72.7%) patients used corticosteroid (CS) plus cyclophosphamide (CP), one (9.1%) received only CP, one (9.1%) used CS plus rituximab and one (9.1%) received CS, CP and three cycles of rituximab. The response to IST was defined as complete remission (CR) with anti-FVIII < 0.6BU/mL and FVIII >50IU/dL, without further IST. Relapse was defined when anti-FVIII titer became >0.6BU/mL after previous remission. In this cohort, we observed 64% of sustained CR and 4(36%) AHA patients had at least one relapse, including two AHA patients considered idiopathic and two with other autoimmune diseases associated. Peripheral blood mononuclear cells (PBMC) were collected from all AHA patients at the diagnosis previously IST (baseline), when patient achieved CR, and at the occurrence of relapse. PBMCs were maintaining frozen in liquid nitrogen. Cells were taw and cultivated with RMPI-1640 medium in 7.5x10 5 cells/well in a 48-well plate. After 24h, cells were stimulated with a 1IU/well of a full-length recombinant FVIII concentrate (rFVIII) or phorbol myristate acetate (PMA) (1µM) with ionomycin (1µM). After 24h incubation, cells were analyzed by flow cytometry at BD FACSCalibur™. To determine T CD4 + cells' cytokine production profile, we stained T cells with anti-CD4, anti-interleukins (IL)-17a, IL-4, and IL-21, anti-TGF-b, and anti-TNF-a. We observed a significant increase of IL-17a production in the rFVIII-stimulated cells isolated at baseline from all patients compared to cells isolated from healthy individuals (P=0.01). However, IL-17a expression at baseline was not different between cells from patients who sustained CR to those who relapsed. Interestingly, when we compared cells isolated at baseline from patients that relapsed after different immunosuppressive attempts, we observed a significant difference for IL-4 (P=0.009), IL-21 (P=0.02), and TGF-b (P=0.01) expression in comparison to cells from healthy individuals. However, this difference was not observed in cells from sustained CR vs. cells from healthy individuals (figure 1). Thus, suggesting that all patients present Th17 response, while only patients that relapsed also showed Th2 response at the diagnosis. We also evaluated B cells' expression of BAFF and APRIL. The cells were stained with anti-CD19, BAFF, and APRIL. Only cells isolated at baseline from AHA relapsed patients after rFVIII stimulation or without any stimuli presented increased expression of BAFF (P=0.01 with rFVIII-stimulated and P=0.007 for unstimulated cells) when compared to PBMCs isolated from healthy individuals (figure 1). BAFF is responsible for controlling B-cell maturation and is associated with autoantibody production of different autoimmune conditions. Our finds reveal an increase in the Th17 response in patients with AHA at the diagnosis. However, the presence of Th2 response and increase of BAFF expression was observed only at the diagnosis of patients with recurrence of autoimmune response to FVIII, which suggests a potential biomarker for AHA evaluation. Figure 1 Figure 1. Disclosures Ozelo: BioMarin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer: Speakers Bureau; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Grifols: Other: Grant review.

Inhibition of keratinocyte ferroptosis suppresses psoriatic inflammation

Psoriasis is a common, chronic, and recurrent inflammatory disease. It is characterized by hyperproliferation and abnormal differentiation of keratinocytes. Keratinocyte death is also involved in many pathophysiological conditions and amplifies the inflammatory cascade. As a newly recognized form of cell death, ferroptosis is involved in several inflammatory diseases. In this study, we aimed to investigate a previously unrecognized role for ferroptosis in psoriasis. Ferroptosis is mediated by lipid peroxidation and iron overload. Compared with normal lesions, the mRNA expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), prostaglandin-endoperoxide synthase 2 (PTGS2), and transferrin receptor (TFRC) were highly expressed in psoriatic lesions, with decreased levels of glutathione peroxidase 4 (GPX4), ferritin light chain (FTL), and ferritin heavy chain 1 (FTH1). The protein levels of ACSL4 and GPX4 were consistent with their mRNA levels. A similar tendency of ferroptosis was also observed in erastin-treated human primary keratinocytes and the Imiquimod (IMQ)-induced model of psoriasis. To investigate the correlation between inflammation and peroxidation, we analyzed single-cell RNA-sequencing data and identified 15 cell types. There was a high correlation between the activity of the lipid oxidation and the Th22/Th17 response in keratinocytes at a single-cell level. Moreover, ferrostatin-1 (Fer-1), a potent inhibitor of lipid peroxidation, suppressed ferroptosis-related changes in erastin-treated keratinocytes and alleviated psoriasiform dermatitis of IMQ-induced models. Additionally, Fer-1 blocked inflammatory responses in vitro and in vivo, reducing the production of cytokines including TNF-α, IL-6, IL-1α, IL-1β, IL-17, IL-22, and IL-23. This study revealed an expression pattern of ferroptosis in which specific molecules enhance inflammatory reactions in psoriasis.

An Advax-Adjuvanted Inactivated Cell-Culture Derived Japanese Encephalitis Vaccine Induces Broadly Neutralising Anti-Flavivirus Antibodies, Robust Cellular Immunity and Provides Single Dose Protection

ccJE+Advax is an inactivated cell culture Japanese encephalitis (JE) vaccine formulated with Advax, a novel polysaccharide adjuvant based on delta inulin. This vaccine has previously shown promise in murine and equine studies and the current study sought to better understand its mechanism of action and assess the feasibility of single dose vaccine protection. Mice immunised with ccJE+Advax had higher serum neutralisation titres than those immunised with ccJE alone or with alum adjuvant. ccJE+Advax induced extraordinarily broad cross-neutralising antibodies against multiple flaviviruses including West Nile virus (WNV), Murray Valley encephalitis virus (MVEV), St Louis encephalitis virus (SLEV) and Dengue virus-1 and -2 (DENV-1 and -2). Notably, the DENV-2 cross-neutralising antibodies from ccJE+Advax immunised mice uniquely had no DENV-2 antibody-dependent infection enhancement (ADIE) activity, in contrast to high ADIE activity seen with DENV-1 cross-reactive antibodies induced by mbJE or ccJE alone or with alum adjuvant. JEV-stimulated splenocytes from ccJE+Advax immunised mice showed increased IL-17 and IFN-γ production, consistent with a mixed Th1 and Th17 response, whereas ccJE-alum was associated with production of mainly Th2 cytokines. In a mouse lethal challenge study against highly virulent JaTH160 JEV strain, ccJE+Advax conferred complete protection in a two-dose schedule with 50 ng of vaccine antigen and near complete protection after a single 200 ng dose of vaccine antigen. There is an ongoing lack of human vaccines against particular flaviviruses, including WNV, SLEV and MVEV. Given its ability to provide single-dose JEV protection and induce broadly neutralising antibodies devoid of ADIE activity, ccJE+Advax vaccine could be useful in situations where rapid protection is desirable, e.g., during a local outbreak or for use in travellers or armies requiring rapid deployment to JEV endemic regions.

A TLR5 mono-agonist restores inhibited immune responses to Streptococcus pneumoniae during influenza virus infection in human monocytes

Influenza A virus (IAV) predisposes individuals to often more severe secondary bacterial infections with Streptococcus pneumonia (S. pneumoniae). The outcomes of these infections may be made worse with the increase in antimicrobial resistance and a lack of new treatments to combat this. Th17 responses are crucial in clearing S. pneumoniae from the lung. We previously demonstrated that early IAV infection of human monocytes significantly reduced levels of S. pneumoniae-driven cytokines involved in the Th17 response. Here, we have further identified that IAV targets specific TLRs (TLR2, TLR4, TLR9) involved in sensing S. pneumoniae infection resulting, in a reduction in TLR agonist-induced IL-23 and TGF-β. The effect of IAV is more profound on the TLR2 and TLR9 pathways. We have established that IAV-mediated inhibition of TLR9-induction is related to a downregulation of RORC, a Th17 specific transcription factor. Other studies using mouse models demonstrated that TLR5 agonism improved the efficacy of antibiotics in the treatment of IAV/S. pneumoniae co-infections. Therefore, we investigated if TLR5 agonism could restore inhibited Th17 responses in human monocytes. Levels of pneumococcus-driven cytokines, which had previously been inhibited by IAV were not reduced in the presence of the TLR5 mono-agonist, suggesting that such treatment may overcome IAV inhibition of Th17 responses. The importance of our research is in demonstrating the IAV directly targets S. pneumoniae-associated TLR pathways. Additionally, the IAV-inhibition of Th17 responses can be restored by TLR5 agonism, which indicates that there may be a different Th17 signalling pathway which is not affected by IAV infection.

A rapid qualitative review of sarcoidosis: Clinical manifestations, Immunopathogenesis, Diagnosis, and Treatment

: Sarcoidosis is a worldwide inflammatory disorder of unknown etiology that is characterized by the formation of non-caseating immune granulomas in involved organs, most commonly in the lungs and eyes. Although clinical manifestations of sarcoidosis depend on the organs involved, the most common symptoms include fatigue, fever, weight loss, eye pain, dyspnea, and chest pain. Sarcoidosis usually undergoes spontaneous regression, yet its chronic form progressively threatens the involved organs through the induction of fibrotic damage. Despite decades of medical research, the etiology of sarcoidosis still remains unclear. Nevertheless, a combination of contributors, including genetic factors, environmental exposures, and microbial agents, is believed to trigger the inflammatory state observed in this disease. Furthermore, a highly polarized Th1 and Th17 response with diminished immunomodulatory mechanisms constitute the most significant immunological event associated with this disorder. Indeed, sarcoid granulomas, which consist of highly activated antigen-presenting cells (APCs) and lymphocytes, maintain a robust specialized niche to facilitate antigen presentation and exaggerated immune responses. Both the unknown etiology and multisystem nature of the disease have hampered the development of specific therapeutics and definitive diagnostic assays for sarcoidosis. Consequently, its diagnosis and treatment still represent a challenging task for clinicians. In this article, we aim to summarize contemporary findings of sarcoidosis and its etiology, pathogenesis, and treatment.

An Advax-adjuvanted Inactivated Cell-culture Japanese Encephalitis Vaccine Induces Broadly Neutralising Anti-flavivirus Antibodies and T-cell Immunity and Provides Single Dose Protection

ccJE+Advax is an inactivated cell culture Japanese encephalitis (JE) vaccine formulated with Advax™, a novel polysaccharide adjuvant based on delta inulin. This vaccine has previously shown promise in murine and equine studies and the current study sought to better understand its mechanism of action and assess the feasibility of single dose vaccine protection. Mice immunised with ccJE-Advax had higher serum neutralisation titres than those immunised with ccJE alone or with alum adjuvant. ccJE+Advax induced extraordinarily broad cross-neutralising antibodies against multiple flaviviruses including West Nile virus (WNV), Murray Valley Encephalitis Virus (MVEV), St Louis Encephalitis virus (SLE) and Dengue-1 and -2 viruses. Notably, the DENV-2 cross-neutralising antibodies from ccJE+Advax immunised mice uniquely had no DENV-2 antibody dependent enhancement (ADE) activity, by contrast to high ADE activity seen with DENV-1 cross-reactive antibodies induced by mbJE or ccJE alone or with alum adjuvant. JEV-stimulated splenocytes from ccJE+Advax immunised mice showed increased IL-17 and IFN-γ production, consistent with a mixed Th1 and Th17 response, whereas ccJE-alum was associated with production of mainly Th2 cytokines. There is an ongoing lack of human vaccines against particular flaviviruses, including WNV, SLE and MVEV. Given its ability to provide single-dose JEV protection as well as to induce broadly neutralising antibodies free of ADE activity, ccJE+Advax vaccine could be highly useful in all situations where rapid protection is desirable but ADE needs to be avoided, e.g. during a local outbreak or for use in travellers or the military requiring rapid travel to JEV endemic regions.

Interaction Between Chronic Endometritis Caused Endometrial Microbiota Disorder and Endometrial Immune Environment Change in Recurrent Implantation Failure

ObjectiveTo investigate the Interaction between chronic endometritis (CE) caused endometrial microbiota disorder and endometrial immune environment change in recurrent implantation failure (RIF).MethodTranscriptome sequencing analysis of the endometrial of 112 patients was preform by using High-Throughput Sequencing. The endometrial microbiota of 43 patients was analyzed by using 16s rRNA sequencing technology.ResultIn host endometrium, CD4 T cell and macrophage exhibited significant differences abundance between CE and non-CE patients. The enrichment analysis indicated differentially expressed genes mainly enriched in immune-related functional terms. Phyllobacterium and Sphingomonas were significantly high infiltration in CE patients, and active in pathways related to carbohydrate metabolism and/or fat metabolism. The increased synthesis of lipopolysaccharide, an important immunomodulator, was the result of microbial disorders in the endometrium.ConclusionThe composition of endometrial microorganisms in CE and non-CE patients were significantly different. Phyllobacterium and Sphingomonas mainly regulated immune cells by interfering with the process of carbohydrate metabolism and/or fat metabolism in the endometrium. CE endometrial microorganisms might regulate Th17 response and the ratio of Th1 to Th17 through lipopolysaccharide (LPS).