Profiling Human Cytomegalovirus-Specific T Cell Responses Reveals Novel Immunogenic Open Reading Frames.

Despite the prevalence and medical significance of human cytomegalovirus (HCMV) infections, a systematic analysis of the targets of T cell recognition in humans that spans the entire genome and includes recently described potential novel open reading frames (ORFs) is not available. Here, we screened a library of epitopes predicted to bind HLA class II that spans over 350 different HCMV ORFs and includes ∼150 previously described and ∼200 recently described potential novel ORFs by using an ex vivo gamma interferon (IFN-γ) FluoroSpot assay. We identified 235 unique HCMV-specific epitopes derived from 100 ORFs, some previously described as immunodominant and others that were not previously described to be immunogenic. Of those, 41 belong to the set of recently reported novel ORFs, thus providing evidence that at least some of these are actually expressed in vivo in humans. These data reveal that the breadth of the human T cell response to HCMV is much greater than previously thought. The ORFs and epitopes identified will help elucidate how T cell immunity relates to HCMV pathogenesis and instruct ongoing HCMV vaccine research. IMPORTANCE To understand the crucial role of adaptive immunity in controlling cytomegalovirus infection and disease, we systematically analyzed the CMV "ORFeome" to identify new CMV epitopes targeted primarily by CD4 T cells in humans. Our study identified >200 new T cell epitopes derived from both canonical and novel ORFs, highlighting the substantial breadth of the anti-CMV T cell response and providing new targets for vaccine design.

Widespread Tau-Specific CD4 T Cell Reactivity in the General Population.

Tau protein is found to be aggregated and hyperphosphorylated (p-tau) in many neurologic disorders, including Parkinson disease (PD) and related parkinsonisms, Alzheimer disease, traumatic brain injury, and even in normal aging. Although not known to produce autoimmune responses, we hypothesized that the appearance of aggregated tau and p-tau with disease could activate the immune system. We thus compared T cell responses to tau and p-tau-derived peptides between PD patients, age-matched healthy controls, and young healthy controls (<35 y old; who are less likely to have high levels of tau aggregates). All groups exhibited CD4+ T cell responses to tau-derived peptides, which were associated with secretion of IFN-γ, IL-5, and/or IL-4. The PD and control participants exhibited a similar magnitude and breadth of responses. Some tau-derived epitopes, consisting of both unmodified and p-tau residues, were more highly represented in PD participants. These results were verified in an independent set of PD and control donors (either age-matched or young controls). Thus, T cells recognizing tau epitopes escape central and peripheral tolerance in relatively high numbers, and the magnitude and nature of these responses are not modulated by age or PD disease.

cGAS-STING Signaling Regulates Initial Innate Control of Cytomegalovirus Infection.

UNLABELLED: Several innate sensing pathways contribute to the control of early cytomegalovirus (CMV) infection, leading to a multiphasic type I interferon (IFN-I) response that limits viral replication and promotes host defenses. Toll-like receptor (TLR)-dependent pathways induce IFN-I production in CMV-infected plasmacytoid dendritic cells; however, the initial burst of IFN-I that occurs within the first few hours in vivo is TLR independent and emanates from stromal cells. Here we show that primary human endothelial cells mount robust IFN-I responses to human CMV that are dependent upon cyclic GMP-AMP synthase (cGAS), STING, and interferon regulatory factor 3 (IRF3) signaling. Disruption of STING expression in endothelial cells by clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 revealed that it is essential for the induction of IFN-I and restriction of CMV replication. Consistently, STING was necessary to mount the first phase of IFN-I production and curb CMV replication in infected mice. Thus, DNA sensing through STING is critical for primary detection of both human and mouse CMV in nonhematopoietic cells and drives the initial wave of IFN-I that is key for controlling early viral replication in vivo. IMPORTANCE: Cytomegalovirus (CMV) is one of the most common viral pathogens, with the majority of people contracting the virus in their lifetime. Although acute infection is mostly asymptomatic in healthy persons, significant pathology is observed in immunocompromised individuals, and chronic CMV infection may exacerbate a myriad of inflammatory conditions. Here we show that primary human endothelial cells mount robust IFN-I responses against CMV via a cGAS/STING/IRF3 pathway. Disruption of STING expression by CRISPRs revealed an essential role in eliciting IFN-I responses and restricting CMV replication. Consistently, in mice, STING is necessary for the first phase of IFN-I production that limits early CMV replication. Our results demonstrate a pivotal role for the cGAS-STING pathway in the initial detection of CMV infection.

A Novel Live Pichinde Virus-Based Vaccine Vector Induces Enhanced Humoral and Cellular Immunity after a Booster Dose.

UNLABELLED: Pichinde virus (PICV) is a bisegmented enveloped RNA virus that targets macrophages and dendritic cells (DCs) early in infection and induces strong innate and adaptive immunity in mice. We have developed a reverse genetics system to produce live recombinant PICV (strain P18) with a trisegmented RNA genome (rP18tri), which encodes all four PICV gene products and as many as two foreign genes. We have engineered the vector to express the green fluorescent protein (GFP) reporter gene (abbreviated as G in virus designations) and either the hemagglutination (HA [H]) or the nucleoprotein (NP [P]) gene of the influenza A/PR8 virus. The trisegmented viruses rP18tri-G/H and rP18tri-G/P showed slightly reduced growth in vitro and expressed HA and NP, respectively. Mice immunized with rP18tri-G/H were completely protected against lethal influenza virus challenge even 120 days after immunization. These rP18tri-based vectors could efficiently induce both neutralizing antibodies and antigen-specific T cell responses via different immunization routes. Interestingly, the immune responses were significantly increased upon a booster dose and remained at high levels even after three booster doses. In summary, we have developed a novel PICV-based live vaccine vector that can express foreign antigens to induce strong humoral and cell-mediated immunity and is ideal for a prime-and-boost vaccination strategy. IMPORTANCE: We have developed a novel Pichinde virus (PICV)-based live viral vector, rP18tri, that packages three RNA segments and encodes as many as two foreign genes. Using the influenza virus HA and NP genes as model antigens, we show that this rP18tri vector can induce strong humoral and cellular immunity via different immunization routes and can lead to protection in mice. Interestingly, a booster dose further enhances the immune responses, a feature that distinguishes this from other known live viral vectors. In summary, our study demonstrates a unique feature of this live rP18tri vector to be used as a novel vaccine platform for a prime-and-boost vaccination strategy.

Comparative evaluation of the diagnostic potential of recombinant envelope proteins and native cell culture purified viral antigens of Chikungunya virus.

Despite the fact that Chikungunya resurgence is associated with epidemic of unprecedented magnitude, there are challenges in the field of its clinical diagnosis. However, serological tests in an ELISA format provide a rapid tool for the diagnosis of Chikungunya infection. Indeed, ELISAs based on recombinant proteins hold a great promise as these methods are cost effective and are free from the risk of handling biohazardous material. In this study, the performance of recombinant CHIKV antigens was compared in various ELISA formats for the diagnosis of Chikungunya. Two recombinant antigens derived from the envelope proteins of Chikungunya virus were prepared and evaluated by comparing their competence for detecting circulating antibodies in serum samples of patients infected with CHIKV using MAC-ELISA and indirect IgM-ELISA. The efficacy of the recombinant antigens was also compared with the native antigen. The indirect antibody capture IgM microplate ELISA revealed ≥90% concordance with the native antigen in detecting the CHIKV specific IgM antibodies whereas the recombinant antigen based MAC-ELISA showed 100% specificity. The recombinant antigens used in this study were effective and reliable targets for the diagnosis of CHIKV infection and also provide an alternative for native antigen use which is potentially biohazardous.

Detection of α-Synuclein-Specific T Cells in Parkinson's Disease.

Parkinson's disease (PD) is a widely prevalent chronic neurodegenerative disease. The disease is characterized by loss of dopaminergic neurons with abnormal aggregation of α-synuclein (α-syn). The misfolded deposition of α-syn is known to mount robust adaptive immune response by activating T cells. Here, we show that peripheral mononuclear cells when stimulated with a α-syn-derived peptide pool activate α-syn-specific T cells that produce cytokines.

Transcriptional analysis of peripheral memory T cells reveals Parkinson's disease-specific gene signatures.

Parkinson's disease (PD) is a multi-stage neurodegenerative disorder with largely unknown etiology. Recent findings have identified PD-associated autoimmune features including roles for T cells. To further characterize the role of T cells in PD, we performed RNA sequencing on PBMC and peripheral CD4 and CD8 memory T cell subsets derived from PD patients and age-matched healthy controls. When the groups were stratified by their T cell responsiveness to alpha-synuclein (α-syn) as a proxy for an ongoing inflammatory autoimmune response, the study revealed a broad differential gene expression profile in memory T cell subsets and a specific PD associated gene signature. We identified significant enrichment of transcriptomic signatures previously associated with PD, including for oxidative stress, phosphorylation, autophagy of mitochondria, cholesterol metabolism and inflammation, and the chemokine signaling proteins CX3CR1, CCR5, and CCR1. In addition, we identified genes in these peripheral cells that have previously been shown to be involved in PD pathogenesis and expressed in neurons, such as LRRK2, LAMP3, and aquaporin. Together, these findings suggest that features of circulating T cells with α-syn-specific responses in PD patients provide insights into the interactive processes that occur during PD pathogenesis and suggest potential intervention targets.

Differential proteome analysis of Chikungunya virus-infected new-born mice tissues reveal implication of stress, inflammatory and apoptotic pathways in disease pathogenesis.

Chikungunya infection is a major disease of public health concern. The recurrent outbreaks of this viral disease and its progressive evolution demands a potential strategy to understand major aspects of its pathogenesis. Unlike other alphaviruses, Chikungunya virus (CHIKV) pathogenesis is poorly understood. In every consecutive outbreak, some new symptoms associated with virulence and disease manifestations are being reported such as neurological implication, increased severity and enhanced vector competence. In order to unravel the mechanism of the disease process, proteomic analysis was performed to evaluate the host response in CHIKV-infected mice tissues. Comparative analysis of the multiple gels representing the particular tissue extract from mock and CHIKV-infected tissues revealed a drastic reprogramming of physiological conditions through 35 and 15 differentially expressed proteins belonging to different classes such as stress, inflammation, apoptosis, urea cycle, energy metabolism, etc. from liver and brain, respectively. Based on the alterations obtained in the CHIKV mouse model, most of the aspects of CHIKV infection such as disease severity, neurological complications, disease susceptibility and immunocompetence could be defined. This is the first report unravelling the complicated pathways involved in the mechanism of Chikungunya disease pathogenesis employing proteomic approach.

The TCR repertoire of α-synuclein-specific T cells in Parkinson's disease is surprisingly diverse.

The self-antigen α-synuclein (α-syn) was recently shown to be associated with Parkinson's disease (PD). Here we mapped the T cell receptor (TCR) repertoire of α-syn-specific T cells from six PD patients. The self-antigen α-syn-specific repertoire was compared to the repertoire of T cells specific for pertussis (PT), as a representative foreign antigen that most individuals are exposed to, revealing that the repertoire for α-syn was as diverse as the repertoire for PT. The diversity of PT-specific clonotypes was similar between individuals with PD diagnosis and age-matched healthy controls. We found that the TCR repertoire was specific to each PD patient, and no shared TCRs among patients were defined, likely due to differences in HLA expression that select for different subsets of epitope-specific TCR rearrangements. This study provides the first characterization of α-syn-specific TCR clonotypes in individuals with PD. Antigen-specific TCRs can serve as immunotherapeutics and diagnostics, and means to track longitudinal changes in specific T cells, and disease progression.

T Cell Responses to Neural Autoantigens Are Similar in Alzheimer's Disease Patients and Age-Matched Healthy Controls.

Alzheimer's disease (AD), a chronic multifactorial and complex neurodegenerative disorder is a leading cause of dementia. Recently, neuroinflammation has been hypothesized as a contributing factor to AD pathogenesis. The role of adaptive immune responses against neuronal antigens, which can either confer protection or induce damage in AD, has not been fully characterized. Here, we measured T cell responses to several potential antigens of neural origin including amyloid precursor protein (APP), amyloid beta (Aß), tau, α-synuclein, and transactive response DNA binding protein (TDP-43) in patients with AD and age-matched healthy controls (HC). Antigen-specific T cell reactivity was detected for all tested antigens, and response to tau-derived epitopes was particularly strong, but no significant differences between individuals with AD and age-matched HC were identified. We also did not observe any correlation between the antigen-specific T cell responses and clinical variables including age, gender, years since diagnosis and cognitive score. Additionally, further characterization did not reveal any differences in the relative frequency of major Peripheral Blood Mononuclear Cells (PBMC) subsets, or in the expression of genes between AD patients and HC. These observations have not identified a key role of neuronal antigen-specific T cell responses in AD.

Cellular sensing of extracellular purine nucleosides triggers an innate IFN-ß response.

Mechanisms linking immune sensing of DNA danger signals in the extracellular environment to innate pathways in the cytosol are poorly understood. Here, we identify a previously unidentified immune-metabolic axis by which cells respond to purine nucleosides and trigger a type I interferon-ß (IFN-ß) response. We find that depletion of ADA2, an ectoenzyme that catabolizes extracellular dAdo to dIno, or supplementation of dAdo or dIno stimulates IFN-ß. Under conditions of reduced ADA2 enzyme activity, dAdo is transported into cells and undergoes catabolysis by the cytosolic isoenzyme ADA1, driving intracellular accumulation of dIno. dIno is a functional immunometabolite that interferes with the cellular methionine cycle by inhibiting SAM synthetase activity. Inhibition of SAM-dependent transmethylation drives epigenomic hypomethylation and overexpression of immune-stimulatory endogenous retroviral elements that engage cytosolic dsRNA sensors and induce IFN-ß. We uncovered a previously unknown cellular signaling pathway that responds to extracellular DNA-derived metabolites, coupling nucleoside catabolism by adenosine deaminases to cellular IFN-ß production.

Corrigendum: T Cell Responses to Neural Autoantigens Are Similar in Alzheimer's Disease Patients and Age-Matched Healthy Controls.

[This corrects the article DOI: 10.3389/fnins.2020.00874.].

α-Synuclein-specific T cell reactivity is associated with preclinical and early Parkinson's disease.

A diagnosis of motor Parkinson's disease (PD) is preceded by a prolonged premotor phase with accumulating neuronal damage. Here we examined the temporal relation between α-synuclein (α-syn) T cell reactivity and PD. A longitudinal case study revealed that elevated α-syn-specific T cell responses were detected prior to the diagnosis of motor PD, and declined after. The relationship between T cell reactivity and early PD in two independent cohorts showed that α-syn-specific T cell responses were highest shortly after diagnosis of motor PD and then decreased. Additional analysis revealed significant association of α-syn-specific T cell responses with age and lower levodopa equivalent dose. These results confirm the presence of α-syn-reactive T cells in PD and show that they are most abundant immediately after diagnosis of motor PD. These cells may be present years before the diagnosis of motor PD, suggesting avenues of investigation into PD pathogenesis and potential early diagnosis.

Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin αvß5 Axis.

Zika virus (ZIKV) causes microcephaly by killing neural precursor cells (NPCs) and other brain cells. ZIKV also displays therapeutic oncolytic activity against glioblastoma (GBM) stem cells (GSCs). Here we demonstrate that ZIKV preferentially infected and killed GSCs and stem-like cells in medulloblastoma and ependymoma in a SOX2-dependent manner. Targeting SOX2 severely attenuated ZIKV infection, in contrast to AXL. As mechanisms of SOX2-mediated ZIKV infection, we identified inverse expression of antiviral interferon response genes (ISGs) and positive correlation with integrin αv (ITGAV). ZIKV infection was disrupted by genetic targeting of ITGAV or its binding partner ITGB5 and by an antibody specific for integrin αvß5. ZIKV selectively eliminated GSCs from species-matched human mature cerebral organoids and GBM surgical specimens, which was reversed by integrin αvß5 inhibition. Collectively, our studies identify integrin αvß5 as a functional cancer stem cell marker essential for GBM maintenance and ZIKV infection, providing potential brain tumor therapy.

Cytosolic sensing of immuno-stimulatory DNA, the enemy within.

In the cytoplasm, DNA is sensed as a universal danger signal by the innate immune system. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor/enzyme that catalyzes formation of 2'-5'-cGAMP, an atypical cyclic di-nucleotide second messenger that binds and activates the Stimulator of Interferon Genes (STING), resulting in recruitment of Tank Binding Kinase 1 (TBK1), activation of the transcription factor Interferon Regulatory Factor 3 (IRF3), and trans-activation of innate immune response genes, including type I Interferon cytokines (IFN-I). Activation of the pro-inflammatory cGAS-STING-IRF3 response is triggered by direct recognition of the DNA genomes of bacteria and viruses, but also during RNA virus infection, neoplastic transformation, tumor immunotherapy and systemic auto-inflammatory diseases. In these circumstances, the source of immuno-stimulatory DNA has often represented a fundamental yet poorly understood aspect of the response. This review focuses on recent findings related to cGAS activation by an array of self-derived DNA substrates, including endogenous retroviral elements, mitochondrial DNA (mtDNA) and micronuclei generated as a result of genotoxic stress and DNA damage. These findings emphasize the role of the cGAS axis as a cell-intrinsic innate immune response to a wide variety of genomic insults.

Establishment of Bisegmented and Trisegmented Reverse Genetics Systems to Generate Recombinant Pichindé Viruses.

Pichindé virus (PICV), isolated from rice rats in Colombia, South America, is an enveloped arenavirus with a bisegmented RNA genome. The large (L) genomic segment encodes the Z matrix protein and the L RNA-dependent RNA polymerase, whereas the small (S) genomic segment encodes the nucleoprotein (NP) and the glycoprotein (GPC). This article describes the successful development of reverse genetics systems to generate recombinant PICV with either a bisegmented or trisegmented genome. We have successfully demonstrated that these systems can generate high-titered and genetically stable replication-competent viruses from plasmid transfection into appropriate cell lines. These systems demonstrate the power and versatility of reverse genetic technology to generate recombinant arenaviruses for use in pathogenesis studies and as new viral vaccine vectors.

Recombinant Tri-Segmented Pichinde Virus as a Novel Live Viral Vaccine Platform.

Pichinde virus (PICV) is a nonpathogenic arenavirus with a bi-segmented RNA genome (L and S segments) that encodes four viral genes. We have developed a reverse genetics system to generate recombinant tri-segmented PICV (rP18tri) that packages three RNA segments (L, S1, and S2) and can encode up to two foreign genes. Using influenza virus HA and NP as model antigens, we show that the rP18tri vector can induce strong humoral and cell-mediated immunity, which further increases upon a booster dose. We propose that this novel rP18tri vector can be developed into a useful vaccine platform for other antigens, particularly when strong cellular immunity and prime-boost vaccination strategy are desired.

Characterization of chikungunya virus induced host response in a mouse model of viral myositis.

While a number of studies have documented the persistent presence of chikungunya virus (CHIKV) in muscle tissue with primary fibroblast as the preferable cell target, little is known regarding the alterations that take place in muscle tissue in response to CHIKV infection. Hence, in the present study a permissive mouse model of CHIKV infection was established and characterized in order to understand the pathophysiology of the disease. The two dimensional electrophoresis of muscle proteome performed for differential analysis indicated a drastic reprogramming of the proteins from various classes like stress, inflammation, cytoskeletal, energy and lipid metabolism. The roles of the affected proteins were explained in relation to virus induced myopathy which was further supported by the histopathological and behavioural experiments proving the lack of hind limb coordination and other loco-motor abnormalities in the infected mice. Also, the level of various pro-inflammatory mediators like IL-6, MCP-1, Rantes and TNF-α was significantly elevated in muscles of infected mice. Altogether this comprehensive study of characterizing CHIKV induced mouse myopathy provides many potential targets for further evaluation and biomarker study.

Subunit vaccine formulations based on recombinant envelope proteins of Chikungunya virus elicit balanced Th1/Th2 response and virus-neutralizing antibodies in mice.

The recent resurgence of Chikungunya virus in India and Indian Ocean Islands with unusual clinical severity is a matter of great public health concern. Despite the fact that CHIKV resurgence is associated with epidemic of unprecedented magnitude, none of the vaccine candidate has been approved so far. The envelope protein E1 and E2 being the major immunodominant structural proteins with crucial role in virus attachment and entry, can prove to be potential vaccine candidates. In the present study, the immunogenic potential of bacterially expressed CHIKE1 and CHIKE2 recombinant proteins along with various adjuvants is reported. Assessment of the protective efficacy of both the vaccine formulations was further confirmed by both in vitro and in vivo neutralisation tests. Splenocytes from immunized mice, cultured in vitro when stimulated with the vaccine antigens revealed induction of very high levels of both pro- and anti- inflammatory cytokines indicating a balance of Th1 and Th2 response.