RESUMO
The pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not completely understood. SARS-CoV-2 infection frequently causes significant immune function consequences including reduced T cell numbers and enhanced T cell exhaustion that contribute to disease severity. The extent to which T cell effects are directly mediated through infection or indirectly result from infection of respiratory-associated cells is unclear. We show that primary human T cells express sufficient levels of angiotensin converting enzyme 2 (ACE-2), the SARS-CoV-2 receptor, to mediate viral binding and entry into T cells. We further show that T cells exposed to SARS-CoV-2 particles demonstrate reduced proliferation and apoptosis compared to uninfected controls, indicating that direct interaction of SARS-CoV-2 with T cells may alter T cell growth, activation, and survival. Regulation of T cell activation and/or turnover by SARS-CoV-2 may contribute to impaired T cell function observed in patients with severe disease.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Linfócitos T/metabolismo , Humanos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação ViralRESUMO
Air pollution particulate matter (PM) is associated with SARS-CoV-2 infection and severity, although mechanistic studies are lacking. We tested whether airway surface liquid (ASL) from primary human airway epithelial cells is antiviral against SARS-CoV-2 and human alphacoronavirus 229E (CoV-229E) (responsible for common colds), and whether PM (urban, indoor air pollution [IAP], volcanic ash) affected ASL antiviral activity. ASL inactivated SARS-CoV-2 and CoV-229E. Independently, urban PM also decreased SARS-CoV-2 and CoV-229E infection, and IAP PM decreased CoV-229E infection. However, in combination, urban PM impaired ASL's antiviral activity against both viruses, and the same effect occurred for IAP PM and ash against SARS-CoV-2, suggesting that PM may enhance SARS-CoV-2 infection.
Assuntos
COVID-19 , Coronavirus Humano 229E , Imunidade Inata , Material Particulado/efeitos adversos , População Urbana , Antivirais/farmacologia , COVID-19/prevenção & controle , COVID-19/transmissão , Humanos , Reação em Cadeia da Polimerase , SARS-CoV-2 , Saúde da População UrbanaRESUMO
The COVID-19 pandemic has caused a high demand for respiratory protection among health care workers in hospitals, especially surgical N95 filtering facepiece respirators (FFRs). To aid in alleviating that demand, a survey of commercially available filter media was conducted to determine whether any could serve as a substitute for an N95 FFR while held in a 3D-printed mask (Stopgap Surgical Face Mask from the NIH 3D Print Exchange). Fourteen filter media types and eight combinations were evaluated for filtration efficiency, breathing resistance (pressure drop), and liquid penetration. Additional testing was conducted to evaluate two filter media disinfection methods in the event that the filters were reused in a hospital setting. Efficiency testing was conducted in accordance with the procedures established for approving an N95 FFR. One apparatus used a filter-holding device and another apparatus employed a manikin head to which the 3D-printed mask could be sealed. The filter media and combinations exhibited collection efficiencies varied between 3.9% and 98.8% when tested with a face velocity comparable to that of a standard N95 FFR at the 85 L min-1 used in the approval procedure. Breathing resistance varied between 10.8 to >637 Pa (1.1 to > 65 mm H2O). When applied to the 3D-printed mask efficiency decreased by an average of 13% and breathing resistance increased 4-fold as a result of the smaller surface area of the filter media when held in that mask compared to that of an N95 FFR. Disinfection by dry heat, even after 25 cycles, did not significantly affect filter efficiency and reduced viral infectivity by > 99.9%. However, 10 cycles of 59% vaporized H2O2 significantly (p < 0.001) reduced filter efficiency of the media tested. Several commercially available filter media were found to be potential replacements for the media used to construct the typical cup-like N95 FFR. However, their use in the 3D-printed mask demonstrated reduced efficiency and increased breathing resistance at 85 L min-1.
Assuntos
COVID-19/prevenção & controle , Desinfecção/normas , Contaminação de Equipamentos/prevenção & controle , Teste de Materiais/normas , Respiradores N95/virologia , Exposição Ocupacional/prevenção & controle , Pandemias/prevenção & controle , Poluentes Ocupacionais do Ar/análise , Análise de Falha de Equipamento/estatística & dados numéricos , Guias como Assunto , Humanos , Exposição por Inalação/análise , SARS-CoV-2RESUMO
BACKGROUND: Human pegivirus (HPgV) is a single-strand RNA virus belonging to the Flaviviridae. Although no definitive association between HPgV infection and disease has been identified, previous studies have suggested an association of HPgV viremia with risk of lymphomas. METHODS: We conducted a systematic review and meta-analysis, including 1 cohort study and 14 case-control studies, assessing the association of HPgV viremia with adult lymphomas. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using a random-effects model, overall and by geographic region and lymphoma subtype. RESULTS: The overall OR for lymphoma was 2.85 (95% CI, 1.98-4.11), with statistically significantly elevated ORs observed in 8 of 15 studies. There was a small amount of heterogeneity among studies (I2 = 28.9%; Q = 18.27, P = .16), and the funnel plot provided no evidence for publication bias. The strongest association with lymphoma risk was observed for studies from Southern Europe (OR, 5.68 [95% CI, 1.98-16.3]), whereas weaker ORs (with 95% CIs) were observed for studies from North America (2.24 [1.76-2.85]), Northern Europe (2.90 [.45-18.7), and the Middle East (2.51 [.87-7.27]), but all of similar magnitude. Participants with HPgV viremia had statistically significantly increased risks (OR [95% CI]) for developing diffuse large B-cell (3.29 [1.63-6.62]), follicular (3.01 [1.95-4.63]), marginal zone (1.90 [1.13-3.18]), and T-cell (2.11 [1.17-3.89]) lymphomas, while the risk for Hodgkin lymphoma (3.53 [.48-25.9]) and chronic lymphocytic leukemia (1.45 [.45-4.66]) were increased but did not achieve statistical significance. CONCLUSIONS: This meta-analysis supports a positive association of HPgV viremia with lymphoma risk, overall and for the major lymphoma subtypes.
Assuntos
Infecções por Flaviviridae , Linfoma , Adulto , Estudos de Coortes , Europa (Continente) , Humanos , Linfoma/epidemiologia , Oriente Médio , América do Norte , Pegivirus , Prevalência , RNA ViralRESUMO
To study the effect of dietary supplementation of Bacillus licheniformis FA6 on the growth, survival and intestinal health of grass carp, we assessed the antioxidant capacity, intestinal barrier, expression levels of immune genes, and the resistance to Aeromonas hydrophila AH-1 infection. Experimental setup comprised three groups (90 specimens each; average initial weight = 16.5 g): the control group was fed the basal diet without B. licheniformis, the low-dose (LD) group was supplemented with B. licheniformis at the concentration of 1 × 105 cfu/g, and the high-dose (HD) group with 1 × 106 cfu/g. After 56 days of growth trial, the challenge test with A. hydrophila AH-1 was conducted for 14 days. The results revealed that the grass carp in LD group and HD group had significantly (p < 0.05) improved percent weight gain (PWG) and specific growth rate (SGR) parameters. Additionally, the antioxidant status was improved, which included increased superoxide dismutase (SOD) activity and decreased malondialdehyde (MDA) levels in the serum, and upregulated mRNA levels of antioxidant enzymes MnSOD and catalase (CAT) in the intestine. Meanwhile, B. licheniformis FA6 supplementation groups exhibited a decreased mRNA expression of proinflammatory cytokines (such as IL-1ß and TNF-α) and increased the expression of anti-inflammatory cytokine IL-10. Histological (villi length was increased) and gene expression (qPCR: upregulated ZO-1, occludin, and claudin-c) analyses suggested improved functioning of the intestinal barrier. Post-challenge mortality rates in LD and HD groups were significantly lower (56.6% and 70% respectively) than in the control group (100%). Overall, these results indicated that dietary supplementation of B. licheniformis FA6 can improve growth, antioxidant capacity, intestinal barrier functions and disease resistance of grass carp.
Assuntos
Antioxidantes/metabolismo , Bacillus licheniformis/química , Carpas/imunologia , Intestinos/imunologia , Probióticos/farmacologia , Ração Animal/análise , Animais , Carpas/crescimento & desenvolvimento , Carpas/metabolismo , Dieta/veterinária , Resistência à Doença/fisiologia , Relação Dose-Resposta a Droga , Doenças dos Peixes/imunologia , Probióticos/administração & dosagem , Distribuição AleatóriaRESUMO
HIV vaccine development is focused on designing immunogens and delivery methods that elicit protective immunity. We evaluated a combination of adenovirus (Ad) vectors expressing HIV 1086.C (clade C) envelope glycoprotein (Env), SIV Gag p55, and human pegivirus GBV-C E2 glycoprotein. We compared replicating simian (SAd7) with nonreplicating human (Ad4) adenovirus-vectored vaccines paired with recombinant proteins in a novel prime-boost regimen in rhesus macaques, with the goal of eliciting protective immunity against SHIV challenge. In both vaccine groups, plasma and buccal Env-specific IgG, tier 1 heterologous neutralizing antibodies, and antibody-dependent cell-mediated viral inhibition were readily generated. High Env-specific T cell responses elicited in all vaccinees were significantly greater than responses targeting Gag. After three intrarectal exposures to heterologous tier 1 clade C SHIV, all 10 sham-vaccinated controls were infected, whereas 4/10 SAd7- and 3/10 Ad4-vaccinated macaques remained uninfected or maintained tightly controlled plasma viremia. Time to infection was significantly delayed in SAd7-vaccinated macaques compared to the controls. Cell-associated and plasma virus levels were significantly lower in each group of vaccinated macaques compared to controls; the lowest plasma viral burden was found in animals vaccinated with the SAd7 vectors, suggesting superior immunity conferred by the replicating simian vectors. Furthermore, higher V1V2-specific binding antibody titers correlated with viral control in the SAd7 vaccine group. Thus, recombinant Ad plus protein vaccines generated humoral and cellular immunity that was effective in either protecting from SHIV acquisition or significantly reducing viremia in animals that became infected, consequently supporting additional development of replicating Ad vectors as HIV vaccines.IMPORTANCE There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV infection and limits in vivo viral replication and associated pathogenesis. Although replicating virus vectors have been advanced as HIV vaccine platforms, there have not been any direct comparisons of the replicating to the nonreplicating format. The present study directly compared the replicating SAd7 to nonreplicating Ad4 vectors in macaques and demonstrated that in the SAd7 vaccine group, the time to infection was significantly delayed compared to the control group, and V1V2 Env-specific binding antibodies correlated with viral outcomes. Viral control was significantly enhanced in vaccinated macaques compared to controls, and in infected SAd7-vaccinated macaques compared to Ad4-vaccinated macaques, suggesting that this vector may have conferred more effective immunity. Because blocking infection is so difficult with current vaccines, development of a vaccine that can limit viremia if infection occurs would be valuable. These data support further development of replicating adenovirus vectors.
Assuntos
Adenoviridae , Vetores Genéticos , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia , Vacinas Sintéticas , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Contagem de Linfócito CD4 , Linhagem Celular , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Genótipo , HIV/imunologia , Humanos , Imunidade Humoral , Imunização/métodos , Estimativa de Kaplan-Meier , Macaca mulatta , Masculino , Ligação Proteica/imunologia , Vacinas contra a SAIDS/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteínas do Envelope Viral/imunologia , Carga ViralRESUMO
Among human RNA viruses, hepatitis C virus (HCV) is unusual in that it causes persistent infection in the majority of infected people. To establish persistence, HCV evades host innate and adaptive immune responses by multiple mechanisms. Recent studies identified virus genome-derived small RNAs (vsRNAs) in HCV-infected cells; however, their biological significance during human HCV infection is unknown. One such vsRNA arising from the hepatitis C virus (HCV) E2 coding region impairs T cell receptor (TCR) signaling by reducing expression of a Src-kinase regulatory phosphatase (PTPRE) in vitro. Since TCR signaling is a critical first step in T cell activation, differentiation, and effector function, its inhibition may contribute towards HCV persistence in vivo. The effect of HCV infection on PTPRE expression in vivo has not been examined. Here, we found that PTPRE levels were significantly reduced in liver tissue and peripheral blood mononuclear cells (PBMCs) obtained from HCV-infected humans compared to uninfected controls. Loss of PTPRE expression impaired antigen-specific TCR signaling, and curative HCV therapy restored PTPRE expression in PBMCs; restoring antigen-specific TCR signaling defects. The extent of PTPRE expression correlated with the amount of sequence complementarity between the HCV E2 vsRNA and the PTPRE 3' UTR target sites. Transfection of a hepatocyte cell line with full-length HCV RNA or with synthetic HCV vsRNA duplexes inhibited PTPRE expression, recapitulating the in vivo observation. Together, these data demonstrate that HCV infection reduces PTPRE expression in the liver and PBMCs of infected humans, and suggest that the HCV E2 vsRNA is a novel viral factor that may contribute towards viral persistence.
Assuntos
Hepatite C/imunologia , Evasão da Resposta Imune/imunologia , Ativação Linfocitária/imunologia , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/imunologia , Linfócitos T/imunologia , Ensaio de Imunoadsorção Enzimática , Hepacivirus/imunologia , Humanos , Immunoblotting , RNA Viral/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , TransfecçãoRESUMO
We evaluated the association of Human Pegivirus (HPgV) viraemia with risk of developing lymphoma, overall and by major subtypes. Because this virus has also been associated with better prognosis in the setting of co-infection with human immunodeficiency virus, we further assessed the association of HPgV with prognosis. We used risk factor data and banked plasma samples from 2094 lymphoma cases newly diagnosed between 2002 and 2009 and 1572 frequency-matched controls. Plasma samples were tested for HPgV RNA by reverse transcription polymerase chain reaction (RT-PCR), and those with RNA concentrations <5000 genome equivalents/ml were confirmed using nested RT-PCR methods. To assess the role of HPgV in lymphoma prognosis, we used 2948 cases from a cohort study of newly diagnosed lymphoma patients (included all cases from the case-control study). There was a positive association of HPgV viraemia with risk of lymphoma overall (Odds ratio = 2·14; 95% confidence interval [CI] 1·63-2·80; P < 0·0001), and for all major subtypes except Hodgkin lymphoma and chronic lymphocytic leukaemia/small lymphocytic lymphoma, and this was not confounded by other lymphoma risk factors. In contrast, there was no association of HPgV viraemia with event-free survival (Hazard ratio [HR] = 1·00; 95% CI 0·85-1·18) or overall survival (HR = 0·97; 95% CI 0·79-1·20) for lymphoma overall, or any of the subtypes. These data support the hypothesis for a role of HPgV in the aetiology of multiple lymphoma subtypes.
Assuntos
Infecções por Flaviviridae/complicações , Linfoma/etiologia , Idoso , Infecções por Flaviviridae/mortalidade , Humanos , Pessoa de Meia-Idade , Prognóstico , RNA Viral/sangue , Risco , Fatores de Risco , Análise de SobrevidaRESUMO
The Flavivirus genus within the Flaviviridae family is comprised of many important human pathogens including yellow fever virus (YFV), dengue virus (DENV), and Zika virus (ZKV), all of which are global public health concerns. Although the related flaviviruses hepatitis C virus and human pegivirus (formerly named GBV-C) interfere with T-cell receptor (TCR) signaling by novel RNA and protein-based mechanisms, the effect of other flaviviruses on TCR signaling is unknown. Here, we studied the effect of YFV, DENV, and ZKV on TCR signaling. Both YFV and ZKV replicated in human T cells in vitro; however, only YFV inhibited TCR signaling. This effect was mediated at least in part by the YFV envelope (env) protein coding RNA. Deletion mutagenesis studies demonstrated that expression of a short, YFV env RNA motif (vsRNA) was required and sufficient to inhibit TCR signaling. Expression of this vsRNA and YFV infection of T cells reduced the expression of a Src-kinase regulatory phosphatase (PTPRE), while ZKV infection did not. YFV infection in mice resulted in impaired TCR signaling and PTPRE expression, with associated reduction in murine response to experimental ovalbumin vaccination. Together, these data suggest that viruses within the flavivirus genus inhibit TCR signaling in a species-dependent manner.
Assuntos
Vírus da Dengue/genética , RNA/genética , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genética , Replicação Viral/genética , Vírus da Febre Amarela/genética , Zika virus/genética , Vírus da Dengue/patogenicidade , Humanos , Vírus da Febre Amarela/patogenicidade , Zika virus/patogenicidadeRESUMO
T cell receptor (TCR) signaling is required for T-cell activation, proliferation, differentiation, and effector function. Hepatitis C virus (HCV) infection is associated with impaired T-cell function leading to persistent viremia, delayed and inconsistent antibody responses, and mild immune dysfunction. Although multiple factors appear to contribute to T-cell dysfunction, a role for HCV particles in this process has not been identified. Here, we show that incubation of primary human CD4+ and CD8+ T-cells with HCV RNA-containing serum, HCV-RNA containing extracellular vesicles (EVs), cell culture derived HCV particles (HCVcc) and HCV envelope pseudotyped retrovirus particles (HCVpp) inhibited TCR-mediated signaling. Since HCVpp's contain only E1 and E2, we examined the effect of HCV E2 on TCR signaling pathways. HCV E2 expression recapitulated HCV particle-induced TCR inhibition. A highly conserved, 51 nucleotide (nt) RNA sequence was sufficient to inhibit TCR signaling. Cells expressing the HCV E2 coding RNA contained a short, virus-derived RNA predicted to be a Dicer substrate, which targeted a phosphatase involved in Src-kinase signaling (PTPRE). T-cells and hepatocytes containing HCV E2 RNA had reduced PTPRE protein levels. Mutation of 6 nts abolished the predicted Dicer interactions and restored PTPRE expression and proximal TCR signaling. HCV RNA did not inhibit distal TCR signaling induced by PMA and Ionomycin; however, HCV E2 protein inhibited distal TCR signaling. This inhibition required lymphocyte-specific tyrosine kinase (Lck). Lck phosphorylated HCV E2 at a conserved tyrosine (Y613), and phospho-E2 inhibited nuclear translocation of NFAT. Mutation of Y613 restored distal TCR signaling, even in the context of HCVpps. Thus, HCV particles delivered viral RNA and E2 protein to T-cells, and these inhibited proximal and distal TCR signaling respectively. These effects of HCV particles likely aid in establishing infection and contribute to viral persistence.
Assuntos
Hepatite C/imunologia , Ativação Linfocitária/imunologia , RNA Viral/imunologia , Linfócitos T/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Bases , Sequência Conservada , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Hepacivirus , Humanos , Immunoblotting , Imunoprecipitação , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T/imunologia , Vírion/imunologiaRESUMO
OBJECTIVE: To investigate the effects of Cordyceps sinensis (CS) on cellular apoptosis and Sirt1 expression in HK2 cells followed by ischemia-reperfusion (I/R).â© Methods: HK2 cells were incubated with different concentrations of CS (10, 20, 40, 80, 160, 320 mg/L) for 24 hours, and the optimal concentration of CS was selected by measuring cell proliferation. The confluent HK2 cells were incubated with 0.01 µmol/L antimycin A for 2 hours to induce ischemia in vitro, and then the reperfusion was achieved by incubating cells with glucose-replete complete growth medium for 24 hours. HK2 cells were divided into 4 groups: a control group, an I/R group, an I/R+CS (160 mg/L) group, and an I/R+CS (160 mg/L)+Sirtinol (25 µmol/L) group. Twenty-four hours later, total RNA and protein were collected. The cell proliferation was evaluated by MTT assay; the mRNA and protein expression of Sirt1 and the cleaved caspase-3 were measured by qRT-PCR and Western blot, respectively. The cellular apoptosis rate was determined by Annexin V-FITC/PI double staining and flow cytometry.â© Results: Certain concentrations (10-160 mg/L) of CS did not show effect on the proliferation of HK2 cells (P>0.05), while 320 mg/L of CS inhibited cell proliferation significantly (P<0.01); compared with the control group, the mRNA and protein expressions of Sirt1 and the cleaved caspase-3 in the I/R group were up-regulated (P<0.01) and the apoptosis rate was extremely high; compared with the I/R group, CS significantly up-regulated Sirt1 mRNA and protein expression (P<0.01) while down-regulated cleaved caspase-3 mRNA and protein levels (P<0.01), and reduced apoptosis rate (P<0.05). The effects of CS were blocked in the presence of sirtinol, an inhibitor of CS.â© Conclusion: CS protects HK2 cells from I/R injury through activation of Sirt1 pathway.
Assuntos
Apoptose , Cordyceps , Traumatismo por Reperfusão/prevenção & controle , Sirtuína 1/metabolismo , Antifúngicos , Antimicina A , Benzamidas/farmacologia , Caspase 3/genética , Caspase 3/metabolismo , Proliferação de Células , Células Cultivadas , Cordyceps/efeitos dos fármacos , Humanos , Isquemia/induzido quimicamente , Naftóis/farmacologia , RNA Mensageiro/metabolismo , Traumatismo por Reperfusão/metabolismo , Sirtuína 1/genéticaRESUMO
Viruses enter into complex interactions within human hosts, leading to facilitation or suppression of each other's replication. Upon coinfection, GB virus C (GBV-C) suppresses HIV-1 replication in vivo and in vitro, and GBV-C coinfection is associated with prolonged survival in HIV-infected people. GBV-C is a lymphotropic virus capable of persistent infection. GBV-C infection is associated with reduced T cell activation in HIV-infected humans, and immune activation is a critical component of HIV disease pathogenesis. We demonstrate that serum GBV-C particles inhibited activation of primary human T cells. T cell activation inhibition was mediated by the envelope glycoprotein E2, because expression of E2 inhibited TCR-mediated activation of Lck. The region on the E2 protein was characterized and revealed a highly conserved peptide motif sufficient to inhibit TCR-mediated signaling. The E2 region contained a predicted Lck substrate site, and substitution of an alanine or histidine for the tyrosine reversed TCR-signaling inhibition. GBV-C E2 protein and a synthetic peptide representing the inhibitory amino acid sequence were phosphorylated by Lck in vitro. The synthetic peptide also inhibited TCR-mediated activation of primary human CD4(+) and CD8(+) T cells. Extracellular microvesicles from GBV-C E2-expressing cells contained E2 protein and inhibited TCR signaling in bystander T cells not expressing E2. Thus, GBV-C reduced global T cell activation via competition between its envelope protein E2 and Lck following TCR engagement. This novel inhibitory mechanism of T cell activation may provide new approaches for HIV and immunoactivation therapy.
Assuntos
Vírus GB C/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Proteínas do Envelope Viral/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Vírus GB C/metabolismo , Hepatite Viral Humana/imunologia , Hepatite Viral Humana/metabolismo , Humanos , Immunoblotting , Células Jurkat , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismoRESUMO
GB virus type C (GBV-C) viremia is associated with reduced CD4+ T cell expansion following IL-2 therapy and with a reduction in T cell activation in HIV-infected individuals. The mechanism(s) by which GBV-C might alter T cell activation or IL-2 signaling have not been studied. In this study, we assess IL-2 release, IL-2R expression, IL-2 signaling, and cell proliferation in tet-off Jurkat cells expressing the GBV-C envelope glycoprotein (E2) following activation through the TCR. TCR activation was induced by incubation in anti-CD3/CD28 Abs. IL-2 release was measured by ELISA, STAT5 phosphorylation was assessed by immunoblot, and IL-2Rα (CD25) expression and cell proliferation were determined by flow cytometry. IL-2 and IL-2Rα steady-state mRNA levels were measured by real-time PCR. GBV-C E2 expression significantly inhibited IL-2 release, CD25 expression, STAT5 phosphorylation, and cellular proliferation in Jurkat cells following activation through the TCR compared with control cell lines. Reducing E2 expression by doxycycline reversed the inhibitory effects observed in the E2-expressing cells. The N-terminal 219 aa of E2 was sufficient to inhibit IL-2 signaling. Addition of purified recombinant GBV-C E2 protein to primary human CD4+ and CD8+ T cells inhibited TCR activation-induced IL-2 release and upregulation of IL-2Rα expression. These data provide evidence that the GBV-C E2 protein may contribute to the block in CD4+ T cell expansion following IL-2 therapy in HIV-infected individuals. Furthermore, the effects of GBV-C on IL-2 and IL-2-signaling pathways may contribute to the reduction in chronic immune activation observed in GBV-C/HIV-coinfected individuals.
Assuntos
Vírus GB C/imunologia , Interleucina-2/antagonistas & inibidores , Interleucina-2/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/imunologia , Proteínas do Envelope Viral/fisiologia , Internalização do Vírus , Vírus GB C/genética , Humanos , Interleucina-2/biossíntese , Células JurkatRESUMO
Hepatitis C virus (HCV) and GB virus type C (GBV-C) are associated with impaired T cell function despite the fact that HCV replicates in hepatocytes and GBV-C in a small proportion of lymphocytes. Recently, we showed that HCV and GBV-C E2-envelope proteins reduce T cell activation via the T cell receptor (TCR) by competing for phosphorylation with a critical kinase in the TCR signaling cascade (Lck). E2 interfered with TCR signaling in E2 expressing cells and in bystander cells. The bystander effect was mediated by virus particles and extracellular microvesicular particles (exosomes). Multiple kinase substrate sites are predicted to reside on viral structural proteins and based on bioinformatic predictions, many RNA virus pathogens may interfere with TCR signaling via a similar mechanism. Identification of T cell inhibitory effects of virus structural proteins may provide novel approaches to enhance the immunogenicity and memory of viral vaccines.
Assuntos
Vírus GB C/imunologia , Hepacivirus/imunologia , Hepatite/imunologia , Evasão da Resposta Imune , Ativação Linfocitária , Linfócitos T/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Vírus GB C/metabolismo , Vírus GB C/patogenicidade , Hepacivirus/metabolismo , Hepacivirus/patogenicidade , Hepatite/epidemiologia , Hepatite/história , Hepatite/metabolismo , Hepatite/virologia , História do Século XX , História do Século XXI , Interações Hospedeiro-Patógeno , Humanos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Linfócitos T/virologia , Proteínas do Envelope Viral/metabolismoRESUMO
There is a critical need to understand the effectiveness of serum elicited by different SARS-CoV-2 vaccines against SARS-CoV-2 variants. We describe the generation of reference reagents comprised of post-vaccination sera from recipients of different primary vaccines with or without different vaccine booster regimens in order to allow standardized characterization of SARS-CoV-2 neutralization in vitro. We prepared and pooled serum obtained from donors who received a either primary vaccine series alone, or a vaccination strategy that included primary and boosted immunization using available SARS-CoV-2 mRNA vaccines (BNT162b2, Pfizer and mRNA-1273, Moderna), replication-incompetent adenovirus type 26 vaccine (Ad26.COV2·S, Johnson and Johnson), or recombinant baculovirus-expressed spike protein in a nanoparticle vaccine plus Matrix-M adjuvant (NVX-CoV2373, Novavax). No subjects had a history of clinical SARS-CoV-2 infection, and sera were screened with confirmation that there were no nucleocapsid antibodies detected to suggest natural infection. Twice frozen sera were aliquoted, and serum antibodies were characterized for SARS-CoV-2 spike protein binding (estimated WHO antibody binding units/ml), spike protein competition for ACE-2 binding, and SARS-CoV-2 spike protein pseudotyped lentivirus transduction. These reagents are available for distribution to the research community (BEI Resources), and should allow the direct comparison of antibody neutralization results between different laboratories. Further, these sera are an important tool to evaluate the functional neutralization activity of vaccine-induced antibodies against emerging SARS-CoV-2 variants of concern. IMPORTANCE: The explosion of COVID-19 demonstrated how novel coronaviruses can rapidly spread and evolve following introduction into human hosts. The extent of vaccine- and infection-induced protection against infection and disease severity is reduced over time due to the fall in concentration, and due to emerging variants that have altered antibody binding regions on the viral envelope spike protein. Here, we pooled sera obtained from individuals who were immunized with different SARS-CoV-2 vaccines and who did not have clinical or serologic evidence of prior infection. The sera pools were characterized for direct spike protein binding, blockade of virus-receptor binding, and neutralization of spike protein pseudotyped lentiviruses. These sera pools were aliquoted and are available to allow inter-laboratory comparison of results and to provide a tool to determine the effectiveness of prior vaccines in recognizing and neutralizing emerging variants of concern.
Assuntos
Vacina de mRNA-1273 contra 2019-nCoV , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacina BNT162 , Vacinas contra COVID-19 , COVID-19 , Testes de Neutralização , SARS-CoV-2 , Humanos , SARS-CoV-2/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , COVID-19/prevenção & controle , COVID-19/imunologia , COVID-19/virologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Vacina de mRNA-1273 contra 2019-nCoV/imunologia , Vacina BNT162/imunologia , Vacina BNT162/administração & dosagem , Glicoproteína da Espícula de Coronavírus/imunologia , Padrões de Referência , Imunização Secundária , Vacinação , Ad26COVS1/imunologiaRESUMO
GB virus type C (GBV-C) is a lymphotropic virus that can cause persistent infection in humans. GBV-C is not associated with any disease, but is associated with reduced mortality in human immunodeficiency virus type 1 (HIV-1)-infected individuals. Related viruses have been isolated from chimpanzees (GBV-Ccpz) and from New World primates (GB virus type A, GBV-A). These viruses are also capable of establishing persistent infection. We determined the nucleotide sequence encoding the envelope glycoprotein (E2) of two GBV-Ccpz isolates obtained from the sera of captive chimpanzees. The deduced GBV-Ccpz E2 protein differed from human GBV-C by 31â% at the amino acid level. Similar to human GBV-C E2, expression of GBV-Ccpz E2 in a tet-off human CD4(+) Jurkat T-cell line significantly inhibited the replication of diverse HIV-1 isolates. This anti-HIV-replication effect of GBV-Ccpz E2 protein was reversed by maintaining cells in doxycycline to reduce E2 expression. Previously, we found a 17 aa region within human GBV-C E2 that was sufficient to inhibit HIV-1. Although GBV-Ccpz E2 differed by 3 aa differences in this region, the chimpanzee GBV-C 17mer E2 peptide inhibited HIV-1 replication. Similarly, the GBV-A peptide that aligns with this GBV-C E2 region inhibited HIV-1 replication despite sharing only 5 aa with the human GBV-C E2 sequence. Thus, despite amino acid differences, the peptide region on both the GBV-Ccpz and the GBV-A E2 protein inhibit HIV-1 replication similar to human GBV-C. Consequently, GBV-Ccpz or GBV-A infection of non-human primates may provide an animal model to study GB virus-HIV interactions.
Assuntos
Linfócitos T CD4-Positivos/virologia , Vírus GB A/fisiologia , Vírus GB C/fisiologia , HIV-1/fisiologia , Proteínas do Envelope Viral/metabolismo , Interferência Viral , Replicação Viral , Animais , Vírus GB A/isolamento & purificação , Vírus GB C/isolamento & purificação , Humanos , Células Jurkat , Dados de Sequência Molecular , Pan troglodytes , Análise de Sequência de DNA , Proteínas do Envelope Viral/genéticaRESUMO
Human immunodeficiency virus (HIV) disease progression is associated with a helper T cell 1 (Th1) to helper T cell 2 (Th2) cytokine profile switch. Persistent GB virus type C (GBV-C) infection is associated with survival and a serum Th1 cytokine profile in HIV-infected individuals. We found that GBV-C infection increased gene expression of Th1 cytokines and decreased Th2 cytokine expression in peripheral blood mononuclear cells. Furthermore, expression of GBV-C NS5A protein in a CD4(+) cell line resulted in upregulation of Th1 cytokines (tumor necrosis factor α) and downregulation of Th2 cytokines (interleukin 4, interleukin 5, interleukin 10, interleukin 13). GBV-C-induced modulation in T-cell cytokines may contribute to the beneficial effect of GBV-C in HIV-infected individuals.
Assuntos
Citocinas/genética , Infecções por Flaviviridae/genética , Vírus GB C/genética , Hepatite Viral Humana/genética , Fosfoproteínas/biossíntese , Células Th1/imunologia , Proteínas não Estruturais Virais/biossíntese , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/biossíntese , Regulação para Baixo , Infecções por Flaviviridae/imunologia , Infecções por Flaviviridae/metabolismo , Infecções por Flaviviridae/virologia , Vírus GB C/imunologia , Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/metabolismo , HIV-1/genética , Hepatite Viral Humana/imunologia , Hepatite Viral Humana/metabolismo , Hepatite Viral Humana/virologia , Humanos , Leucócitos Mononucleares/metabolismo , Fosfoproteínas/genética , Células Th2/metabolismo , Regulação para Cima , Proteínas não Estruturais Virais/genéticaRESUMO
TRPA1 is pivotal in cold hypersensitivity, but its regulatory mechanisms in inflammatory cold hyperalgesia remain poorly understood. We show here that the upregulation of SUMO1-conjugated protein levels in a complete Freund's adjuvant (CFA)-induced inflammatory pain model enhances TRPA1 mRNA stability, ultimately leading to increased expression levels. We further demonstrate that hnRNPA1 binds to TRPA1 mRNA, and its SUMOylation, upregulated in CFA-induced inflammatory pain, contributes to stabilizing TRPA1 mRNA by accumulating hnRNPA1 in the cytoplasm. Moreover, we find that wild-type hnRNPA1 viral infection in dorsal root ganglia neurons, and not infection with the SUMOylation-deficient hnRNPA1 mutant, can rescue the reduced ability of hnRNPA1-knockdown mice to develop inflammatory cold pain hypersensitivity. These results suggest that hnRNPA1 is a regulator of TRPA1 mRNA stability, the capability of which is enhanced upon SUMO1 conjugation at lysine 3 in response to peripheral inflammation, and the increased expression of TRPA1 in turn underlies the development of chronic inflammatory cold pain hypersensitivity.
Assuntos
Dor Crônica , Sumoilação , Animais , Camundongos , Dor Crônica/metabolismo , Adjuvante de Freund , Gânglios Espinais/metabolismo , Hiperalgesia/metabolismo , Inflamação/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Canal de Cátion TRPA1/genética , Canal de Cátion TRPA1/metabolismoRESUMO
Broadly neutralizing Abs to HIV-1 are well described; however, identification of Ags that elicit these Abs has proven difficult. Persistent infection with GB virus type C (GBV-C) is associated with prolonged survival in HIV-1-infected individuals, and among those without HIV-1 viremia, the presence of Ab to GBV-C glycoprotein E2 is also associated with survival. GBV-C E2 protein inhibits HIV-1 entry, and an antigenic peptide within E2 interferes with gp41-induced membrane perturbations in vitro, suggesting the possibility of structural mimicry between GBV-C E2 protein and HIV-1 particles. Naturally occurring human and experimentally induced GBV-C E2 Abs were examined for their ability to neutralize infectious HIV-1 particles and HIV-1-enveloped pseudovirus particles. All GBV-C E2 Abs neutralized diverse isolates of HIV-1 with the exception of rabbit anti-peptide Abs raised against a synthetic GBV-C E2 peptide. Rabbit anti-GBV-C E2 Abs neutralized HIV-1-pseudotyped retrovirus particles but not HIV-1-pseudotyped vesicular stomatitis virus particles, and E2 Abs immune-precipitated HIV-1 gag particles containing the vesicular stomatitis virus type G envelope, HIV-1 envelope, GBV-C envelope, or no viral envelope. The Abs did not neutralize or immune-precipitate mumps or yellow fever viruses. Rabbit GBV-C E2 Abs inhibited HIV attachment to cells but did not inhibit entry following attachment. Taken together, these data indicate that the GBV-C E2 protein has a structural motif that elicits Abs that cross-react with a cellular Ag present on retrovirus particles, independent of HIV-1 envelope glycoproteins. The data provide evidence that a heterologous viral protein can induce HIV-1-neutralizing Abs.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus GB C/imunologia , HIV-1/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Separação Celular , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunoprecipitação , Camundongos , Mimetismo Molecular/imunologia , Coelhos , Vírion/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologiaRESUMO
GB virus C (GBV-C) is a common, non-pathogenic human virus that infects lymphocytes. Persistent GBV-C infection of humans with coexistent human immunodeficiency virus (HIV) infection is associated with prolonged survival, and GBV-C replication inhibits HIV replication in vitro. A GBV-C virus variant was identified in chimpanzees in 1998 and was named GBV-C(trog) or GBV-C(cpz). The prevalence and natural history of GBV-C in chimpanzees remains uncharacterized. We examined the sera from 235 captive chimpanzees for the presence of GBV-C viraemia, viral persistence and clearance, E2 antibody kinetics and RNA sequence diversity. Sequences from six isolates shared more sequence identity with GBV-C(cpz) than with human GBV-C. The prevalence of GBV-C(cpz) viraemia and E2 antibody in chimpanzees (2.5 and 11â%, respectively) was similar to human GBV-C prevalence in healthy human blood donors (1.8 and 9â%, respectively). Persistent GBV-C(cpz) infection occurred in two of the six viraemic animals and was documented for 19 years in one animal. Host subspecies troglodyte GBV-C isolates and published verus GBV-C isolates shared a high degree of sequence identity, suggesting that GBV-C in chimpanzees should be identified with a chimpanzee designation (GBV-C(cpz)). The prevalence and natural history of chimpanzee GBV-C variant (GBV-C(cpz)) appears to be similar to human GBV-C infection. The chimpanzee could serve as an animal model to study HIV-GBV-C co-infection.