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1.
J Clin Microbiol ; 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33795412

RESUMO

Serological testing of large representative populations for antibodies to SARS-CoV-2 is needed to estimate seroprevalence, transmission dynamics, and the duration of antibody responses from natural infection and vaccination. In this study, a high-throughput SARS-CoV-2 multiplex microsphere immunoassay (MMIA) was developed for the receptor binding domain (RBD) and nucleocapsid (N) that was more sensitive than ELISA (98% vs 87%). The MMIA was then applied and validated in 264 first responders in Colorado using serum and dried blood spot (DBS) eluates, compared to ELISA and evaluated for neutralizing antibodies. Four percent (11/264) of first responders were seropositive in July-August 2020. Serum and DBS were highly correlated for anti-RBD and anti-N antibodies (R=0.83, p<0.0001 and R=0.87, p<0.0001, respectively) by MMIA. The MMIA accurately predicted SARS-CoV-2 neutralizing antibodies using DBS (R=0.76, p=0.037). On repeat antibody testing three months later, anti-RBD IgG decreased less rapidly than anti-N IgG measured by MMIA, with a median change in gMFI of 62% vs 79% (p<0.01), for anti-RBD and anti-N IgG respectively. This novel MMIA using DBS could be scalable for rapid and affordable SARS-CoV-2 serosurveillance in the U.S. and globally.

2.
J Control Release ; 331: 213-227, 2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33378692

RESUMO

Primaquine and tafenoquine are the two 8-aminoquinoline (8-AQ) antimalarial drugs approved for malarial radical cure - the elimination of liver stage hypnozoites after infection with Plasmodium vivax. A single oral dose of tafenoquine leads to high efficacy against intra-hepatocyte hypnozoites after efficient first pass liver uptake and metabolism. Unfortunately, both drugs cause hemolytic anemia in G6PD-deficient humans. This toxicity prevents their mass administration without G6PD testing given the approximately 400 million G6PD deficient people across malarial endemic regions of the world. We hypothesized that liver-targeted delivery of 8-AQ prodrugs could maximize liver exposure and minimize erythrocyte exposure to increase their therapeutic window. Primaquine and tafenoquine were first synthesized as prodrug vinyl monomers with self-immolative hydrolytic linkers or cathepsin-cleavable valine-citrulline peptide linkers. RAFT polymerization was exploited to copolymerize these prodrug monomers with hepatocyte-targeting GalNAc monomers. Pharmacokinetic studies of released drugs after intravenous administration showed that the liver-to-plasma AUC ratios could be significantly improved, compared to parent drug administered orally. Single doses of the liver-targeted, enzyme-cleavable tafenoquine polymer were found to be as efficacious as an equivalent dose of the oral parent drug in the P. berghei causal prophylaxis model. They also elicited significantly milder hemotoxicity in the humanized NOD/SCID mouse model engrafted with red blood cells from G6PD deficient donors. The clinical application is envisioned as a single subcutaneous administration, and the lead tafenoquine polymer also showed excellent bioavailability and liver-to-blood ratios exceeding the IV administered polymer. The liver-targeted tafenoquine polymers warrant further development as a single-dose therapeutic via the subcutaneous route with the potential for broader patient administration without a requirement for G6PD diagnosis.

3.
J Occup Environ Med ; 2020 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-33298759

RESUMO

OBJECTIVES: Define seroprevalence and risk factors for SARS-CoV-2 antibodies in Arapahoe County, Colorado first responders (e.g. law enforcement, human services, fire departments). METHODS: 264 first responders were enrolled June-July 2020. SARS-CoV-2 seropositivity was defined as detection of IgG antibodies to both spike receptor binding domain and nucleocapsid in venous blood by validated enzyme-linked immunosorbent assay. We compared risk factors for being seropositive versus seronegative. RESULTS: 4%(11/264) were SARS-CoV-2 seropositive. Seropositive participants were significantly more likely to have lung disease [% seropositive,% seronegative; p-value] (36%,8%;p = 0.01), prior SARS-CoV-2/COVID-19 testing (36%,8%;p≤0.01), a prior positive result (18%,<1%), and to believe they previously had COVID-19 (64%,15%;p<0.01). Only 15% of those believing they had COVID-19 had anti-SARS-CoV-2 antibodies. CONCLUSIONS: Human services employees and individuals with lung disease are at SARS-CoV-2 exposure risk. Few individuals believed they had COVID-19 had prior exposure.

4.
Infect Agent Cancer ; 15(1): 71, 2020 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-33292357

RESUMO

BACKGROUND: Kaposi sarcoma-associated herpesvirus (KSHV) seroprevalence in sub-Saharan African children can range up to 50% by age 2 years but factors affecting early age of KSHV infection are not well understood. Malaria during pregnancy has been associated with hindered transplacental transfer of antibodies to several pathogens but whether it affects transplacental transfer of KSHV antibodies is unknown. We aimed to determine if in utero malaria exposure reduced the transfer of KSHV antibodies across the placenta. METHODS: A cohort study in Kisumu, Kenya enrolled pregnant women at their first antenatal clinic (ANC) visit and followed them through delivery. We included 70 KSHV-positive, HIV-negative mothers and their children. KSHV antibody levels were measured by ELISA (K8.1, ORF73) and multiplex assay (K8.1, ORF73, K10.5, ORF38, ORF50). Transplacental transfer of antibodies was measured by the cord to maternal blood ratio (CMR) of KSHV antibodies. Malaria during pregnancy was defined as detection of Plasmodium falciparum (Pf) DNA at any ANC visit or delivery. Among women with malaria during pregnancy, we examined time of last malaria infection prior to delivery (< 27 vs. 27+ weeks gestation) and malaria incidence rate (MIR) (episodes/100 person-weeks). RESULTS: KSHV seroprevalence (positive for K8.1 or ORF73 by ELISA) among pregnant women was 88%. Neither malaria during pregnancy, malaria infection timing, nor MIR were associated with maternal delivery KSHV antibody blood levels. Maternal delivery and cord blood KSHV antibody levels were highly correlated but these correlations did not differ by malaria during pregnancy. KSHV transplacental antibody transfer was not associated with malaria during pregnancy, malaria infection timing, nor MIR. CONCLUSIONS: Malaria during pregnancy does not appear to affect transfer of KSHV antibodies across the placenta.

5.
J Infect Dis ; 2020 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-33249494

RESUMO

BACKGROUND: We aimed to determine whether Plasmodium falciparum (Pf) infection affects age of Kaposi sarcoma-associated herpesvirus (KSHV) seroconversion in Kenyan children. METHODS: Kenyan children (n=144) enrolled at age one month, from two sites with different levels of malaria transmission (stable/high malaria vs. unstable/low malaria transmission) were followed through 24 months. Plasma was tested for KSHV antibodies using enzyme-linked immunosorbent assay (ELISA) (K8.1 and LANA) and a multiplex bead-based assay (K8.1, K10.5, ORF38, ORF50, and LANA) and whole blood tested for Pf DNA using quantitative-PCR. Cox proportional hazards models were used to assess associations between Pf DNA detection, malaria annualized rate (Pf detections/person-years), and enrollment site (malaria-high vs malaria-low) with time to KSHV seroconversion. RESULTS: KSHV seroprevalence was 63% by 2 years of age when assessed by multiplex assay. Children with Pf were at increased hazards of earlier KSHV seroconversion and among children with malaria, the hazard of becoming KSHV seropositive increased significantly with increasing malaria annualized rate. Children from the malaria-high transmission region had no significant difference in hazards of KSHV seroconversion at 12 months but were more likely to become KSHV seropositive by 24 months of age. DISCUSSION: Malaria exposure increases the risk for KSHV seroconversion early in life.

6.
J Virol ; 94(11)2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32238579

RESUMO

Epstein-Barr virus (EBV) is associated with a number of T-cell diseases, including some peripheral T-cell lymphomas, hemophagocytic lymphohistiocytosis, and chronic active EBV disease. The tropism of EBV for B cells and epithelial cell infection has been well characterized, but infection of T cells has been minimally explored. We have recently shown that the EBV type 2 (EBV-2) strain has the unique ability to infect mature T cells. Utilizing an ex vivo infection model, we sought to understand the viral glycoprotein and cellular receptor required for EBV-2 infection of T cells. Here, using a neutralizing-antibody assay, we found that viral gp350 and complement receptor 2 (CD21) are required for CD3+ T-cell infection. Using the HB5 anti-CD21 antibody clone but not the Bly-4 anti-CD21 antibody clone, we detected expression of CD21 on both CD4+ and CD8+ T cells, with the highest expression on naive CD4 and CD8+ T-cell subsets. Using CRISPR to knock out CD21, we demonstrated that CD21 is necessary for EBV entry into the Jurkat T-cell line. Together, these results indicate that EBV uses the same viral glycoprotein and cellular receptor for both T- and B-cell infection.IMPORTANCE Epstein-Barr virus (EBV) has a well-described tropism for B cells and epithelial cells. Recently, we described the ability of a second strain of EBV, EBV type 2, to infect mature peripheral T cells. Using a neutralizing antibody assay, we determined that EBV uses the viral glycoprotein gp350 and the cellular protein CD21 to gain entry into mature peripheral T cells. CRISPR-Cas9 deletion of CD21 on the Jurkat T-cell line confirmed that CD21 is required for EBV infection. This study has broad implications, as we have defined a function for CD21 on mature peripheral T cells, i.e., as a receptor for EBV. In addition, the requirement for gp350 for T-cell entry has implications for EBV vaccine studies currently targeting the gp350 glycoprotein to prevent EBV-associated diseases.


Assuntos
Linfócitos B/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Receptores de Complemento 3d/metabolismo , Linfócitos T/metabolismo , Internalização do Vírus , Adulto , Linfócitos B/patologia , Linfócitos B/virologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/patologia , Feminino , Deleção de Genes , Herpesvirus Humano 4/genética , Humanos , Masculino , Receptores de Complemento 3d/genética , Linfócitos T/patologia , Linfócitos T/virologia
8.
Front Immunol ; 10: 2367, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31681275

RESUMO

Cytometry by Time-Of-Flight (CyTOF) uses antibodies conjugated to isotopically pure metals to identify and quantify a large number of cellular features with single-cell resolution. A barcoding approach allows for 20 unique samples to be pooled and processed together in one tube, reducing the intra-barcode technical variability. However, with only 20 samples per barcode, multiple barcode sets (batches) are required to address questions in robustly powered study designs. A batch adjustment procedure is required to reduce variability across batches and to facilitate direct comparison of runs performed across multiple barcodes run over weeks, months, or years. We describe a method using technical replicates that are included in each run to determine and apply an appropriate adjustment per batch without manual intervention. The use of technical replicate samples (i.e., anchors or reference samples) avoids assumptions of sample homogeneity among batches, and allows direct estimation of batch effects and appropriate adjustment parameters applicable to all samples within a batch. Quantification of cell subpopulations and mean signal intensity pre- and post-adjustment using both manual gating and unsupervised clustering demonstrate substantial mitigation of batch effects in the anchor samples used for this adjustment calculation, and in a second validation set of technical replicates.


Assuntos
Citometria de Fluxo/métodos , Citometria de Fluxo/instrumentação , Humanos
9.
Infect Agent Cancer ; 14: 17, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31388351

RESUMO

Background: Burkitt lymphoma (BL) is a relatively common cancer of childhood in tropical Africa, although its precise incidence and continent-wide geographic distribution have not been previously systematically studied. Methods: Using the methods employed to produce national estimates of cancer incidence for the "Globocan" series of the International Agency for Research on Cancer, along with detailed information on cancer incidence by histological subtype from cancer registries in Africa, we estimate the numbers and rates of incidence by sex, age group, country and region of Africa. Results: We estimate that the number of new cases that occurred in 2018 to be about 3900, two thirds in males, and 81% in children aged 0-14. On a national basis, the geographic distribution of incidence rates among children in sub-Saharan Africa resembles that of the prevalence of infection with Falciparum malaria. An estimated 81% of cases are associated with infection with Epstein Barr virus (EBV). Conclusions: BL comprises almost 50% of childhood of non-Hodgkin lymphoma in Africa, almost all of which are associated with EBV, with the geographic distribution - at least in sub Saharan Africa - mediated by infection with malaria.

10.
Open Forum Infect Dis ; 6(6): ofz237, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31214627

RESUMO

Background: Altered neonatal immune responses may contribute to the increased morbidity observed in HIV-exposed but uninfected (HEU) infants compared with HIV-unexposed uninfected (HUU) infants. We sought to examine the effects of prenatal HIV and malaria exposure on maternal and neonatal plasma cytokine profiles and transplacental antibody transfer. Methods: Forty-nine HIV+ and 50 HIV- women and their HIV-uninfected neonate pairs from Kenya were assessed. All HIV+ mothers received combination antiretroviral therapy. Maternal plasma and cord blood plasma samples at delivery were tested for 12 cytokines, total IgG, and IgG specific to 4 vaccine antigens and 14 Plasmodium falciparum antigens. Results: HIV+ mothers had lower levels of all 12 plasma cytokines at delivery compared with HIV- mothers, but there were no differences between HEU and HUU neonates. There were no differences in the cord-to-maternal ratios (CMRs) of vaccine-specific IgG between HIV+/HEU and HIV-/HUU maternal-neonate pairs. HIV+/HEU maternal-neonate pairs had significantly lower CMRs for 3 antimalarial IgGs-merozoite surface protein 9, circumsporozoite protein, and erythrocyte binding antigen 181-which remained statistically significant after adjustment for malaria in pregnancy. Conclusions: In a cohort of optimally treated HIV-infected pregnant women, maternal HIV infection was associated with reduced transplacental transfer of antimalarial antibodies.

11.
PLoS Pathog ; 15(6): e1007849, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31166996

RESUMO

Virus-host interactions are frequently studied in bulk cell populations, obscuring cell-to-cell variation. Here we investigate endogenous herpesvirus gene expression at the single-cell level, combining a sensitive and robust fluorescent in situ hybridization platform with multiparameter flow cytometry, to study the expression of gammaherpesvirus non-coding RNAs (ncRNAs) during lytic replication, latent infection and reactivation in vitro. This method allowed robust detection of viral ncRNAs of murine gammaherpesvirus 68 (γHV68), Kaposi's sarcoma associated herpesvirus and Epstein-Barr virus, revealing variable expression at the single-cell level. By quantifying the inter-relationship of viral ncRNA, viral mRNA, viral protein and host mRNA regulation during γHV68 infection, we find heterogeneous and asynchronous gene expression during latency and reactivation, with reactivation from latency identified by a distinct gene expression profile within rare cells. Further, during lytic replication with γHV68, we find many cells have limited viral gene expression, with only a fraction of cells showing robust gene expression, dynamic RNA localization, and progressive infection. Lytic viral gene expression was enhanced in primary fibroblasts and by conditions associated with enhanced viral replication, with multiple subpopulations of cells present in even highly permissive infection conditions. These findings, powered by single-cell analysis integrated with automated clustering algorithms, suggest inefficient or abortive γHV infection in many cells, and identify substantial heterogeneity in viral gene expression at the single-cell level.


Assuntos
Gammaherpesvirinae/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Infecções por Herpesviridae/metabolismo , RNA Mensageiro/biossíntese , RNA não Traduzido/biossíntese , RNA Viral/biossíntese , Replicação Viral/fisiologia , Animais , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/patologia , Humanos , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , RNA não Traduzido/genética , RNA Viral/genética
12.
J Exp Med ; 216(6): 1255-1267, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31040184

RESUMO

The pleiotropic actions of interleukin-2 (IL-2) are essential for regulation of immune responses and maintenance of immune tolerance. The IL-2 receptor (IL-2R) is composed of IL-2Rα, IL-2Rß, and IL-2Rγ subunits, with defects in IL-2Rα and IL-2Rγ and their downstream signaling effectors resulting in known primary immunodeficiency disorders. Here, we report the first human defect in IL-2Rß, occurring in two infant siblings with a homozygous IL2RB mutation in the WSXWS motif, manifesting as multisystem autoimmunity and susceptibility to CMV infection. The hypomorphic mutation results in diminished IL-2Rß surface expression and dysregulated IL-2/15 signaling, with an anticipated reduction in regulatory T cells. However, in contrast to the IL-2Rß-/- animal model, which lacks NK cells, these siblings demonstrate an expansion of NK cells, particularly the CD56bright subset, and a lack of terminally differentiated NK cells. Thus, the early-onset autoimmunity and immunodeficiency are linked to functional deficits arising from altered IL-2Rß expression and signaling in T and NK cells.


Assuntos
Subunidade beta de Receptor de Interleucina-2/genética , Células Matadoras Naturais/imunologia , Mutação/genética , Linfócitos T/imunologia , Autoimunidade/genética , Compartimento Celular , Proliferação de Células/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Homozigoto , Humanos , Imunofenotipagem , Interleucina-15/metabolismo , Interleucina-2/metabolismo , Subunidade beta de Receptor de Interleucina-2/química , Modelos Moleculares , Fenótipo , Irmãos , Transdução de Sinais , Resultado do Tratamento
14.
Malar J ; 18(1): 19, 2019 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670032

RESUMO

BACKGROUND: Studies of the association between the level of anti-malarial antibody and protection from malaria infection can yield conflicting results if they fail to take into account differences in the malaria transmission rate. This can occur because high malaria exposure may drive high antibody responses, leading to an apparent positive association between immune response and infection rate. The neonatal period provides a unique window to study the protective effects of antibodies, because waning maternally-derived antibodies lead to different levels of protection with time. METHODS: This study uses data from two well-defined infant cohorts in Western Kenya with different burdens of malaria transmission. Survival models were used to assess how the magnitude of maternally derived malaria-specific IgG antibody (to 24 malaria antigens measured using Luminex beads) affected the time-to-first Plasmodium falciparum infection (detected by PCR). In addition, mathematical models were used to assess how the frequency of malaria infection varied between the cohorts with different exposure levels. RESULTS: Despite differences in underlying malaria incidence in the two regions, there was no difference in time-to-first malaria infection between the cohorts. However, there was a significant period of protection observed in children with high initial MSP1 (42 kDa fragment)-specific antibody levels, but this protection was not observed in children with low antibody levels. Children from the high transmission cohort had both longer initial periods of protection from malaria (attributable to higher initial antibody levels), but more rapid time-to-first-infection once malaria specific maternal antibodies declined below protective levels (attributable to higher exposure rates). CONCLUSION: This study demonstrates the complex interaction between passive (maternally-derived) immunity and the degree of malaria exposure in infants. Children from regions of high malaria transmission had higher levels of maternally-derived antibodies in early life, which led to a significant protection for several months. However, once this immunity waned, the underlying higher frequency of infection was revealed. A better understanding of the interaction between malaria exposure, immunity, and transmission risk will assist in identifying protective immune responses in P. falciparum infection.


Assuntos
Anticorpos Antiprotozoários/imunologia , Imunidade Materno-Adquirida , Imunoglobulina G/imunologia , Malária Falciparum/imunologia , Antígenos de Protozoários/imunologia , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Incidência , Lactente , Recém-Nascido , Quênia/epidemiologia , Estudos Longitudinais , Malária Falciparum/epidemiologia , Masculino , Plasmodium falciparum/imunologia
15.
J Infect Dis ; 219(6): 955-963, 2019 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-30312417

RESUMO

BACKGROUND: The Epstein-Barr virus (EBV) viral glycoprotein gp350 has been proposed as a candidate antigen for an EBV vaccine. However, the proposed formulations of these vaccines have not taken into account the presence of 2 unique EBV strains (EBV-1 and EBV-2) present in areas of high incidence of the EBV-associated cancer, Burkitt lymphoma. METHODS: In this study, we analyze the kinetics of EBV-1 and EBV-2 infection in an asymptomatic infant cohort from Kisumu, Kenya. We also analyzed the kinetics of the antibody response against 5 EBV antigens, gp350 (IgG and IgA), VCA (IgG), EBNA-1 (IgG), EAd (IgG), and Zta (IgG). RESULTS: We observed a high frequency of coinfection with both EBV types over time, with the only observable defect in the antibody response in infants coinfected being a significantly lower level of anti-gp350 IgA at peak response. Gp350 IgA levels were also significantly lower in coinfected infants 2.5 months postinfection and at the time of coinfection. CONCLUSIONS: These results suggest that anti-gp350 IgA antibodies may be important for sterilizing immunity against secondary infection. These findings have implications for the development of an efficacious EBV vaccine to prevent both EBV-1 and EBV-2 infection in a population at high risk for Burkitt lymphoma.


Assuntos
Anticorpos Antivirais/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Imunoglobulina A/imunologia , Glicoproteínas de Membrana/imunologia , Adulto , Idoso , Criança , Pré-Escolar , Coinfecção/imunologia , Infecções por Vírus Epstein-Barr/classificação , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Feminino , Humanos , Lactente , Quênia , Masculino , Pessoa de Meia-Idade , Carga Viral
17.
Clin Cancer Res ; 24(20): 5085-5097, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30084838

RESUMO

Purpose: Kaposi sarcoma (KS) is a vascular tumor initiated by infection of endothelial cells (ECs) with KS-associated herpesvirus (KSHV). KS is dependent on sustained proinflammatory signals provided by intralesional leukocytes and continued infection of new ECs. However, the sources of these cytokines and infectious virus within lesions are not fully understood. Here, mast cells (MCs) are identified as proinflammatory cells within KS lesions that are permissive for, and activated by, infection with KSHV.Experimental Design: Three validated MC lines were used to assess permissivity of MCs to infection with KSHV and to evaluate MCs activation following infection. Biopsies from 31 AIDS-KS cases and 11 AIDS controls were evaluated by IHC for the presence of MCs in KS lesions and assessment of MC activation state and infection with KSHV. Plasma samples from 26 AIDS-KS, 13 classic KS, and 13 healthy adults were evaluated for levels of MC granule contents tryptase and histamine.Results: In culture, MCs supported latent and lytic KSHV infection, and infection-induced MC degranulation. Within KS lesions, MCs were closely associated with spindle cells. Furthermore, MC activation was extensive within patients with KS, reflected by elevated circulating levels of tryptase and a histamine metabolite. One patient with clinical signs of extensive MC activation was treated with antagonists of MC proinflammatory mediators, which resulted in a rapid and durable regression of AIDS-KS lesions.Conclusions: Using complimentary in vitro and in vivo studies we identify MCs as a potential long-lived reservoir for KSHV and a source of proinflammatory mediators within the KS lesional microenvironment. In addition, we identify MC antagonists as a promising novel therapeutic approach for KS. Clin Cancer Res; 24(20); 5085-97. ©2018 AACR.


Assuntos
Infecções por Herpesviridae/complicações , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8 , Mastócitos/imunologia , Sarcoma de Kaposi/etiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Citocinas/metabolismo , Suscetibilidade a Doenças , Feminino , Humanos , Imuno-Histoquímica , Masculino , Mastócitos/metabolismo , Metilistaminas/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patologia , Pele/metabolismo , Pele/patologia , Triptases/metabolismo
18.
J Virol ; 92(21)2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30089703

RESUMO

Epstein-Barr virus (EBV) has been classified into two strains, EBV type 1 (EBV-1) and EBV type 2 (EBV-2) based on genetic variances and differences in transforming capacity. EBV-1 readily transforms B cells in culture while EBV-2 is poorly transforming. The differing abilities to immortalize B cells in vitro suggest that in vivo these viruses likely use alternative approaches to establish latency. Indeed, we recently reported that EBV-2 has a unique cell tropism for T cells, infecting T cells in culture and in healthy Kenyan infants, strongly suggesting that EBV-2 infection of T cells is a natural part of the EBV-2 life cycle. However, limitations of human studies hamper further investigation into how EBV-2 utilizes T cells. Therefore, BALB/c Rag2null IL2rγnull SIRPα humanized mice were utilized to develop an EBV-2 in vivo model. Infection of humanized mice with EBV-2 led to infection of both T and B cells, unlike infection with EBV-1, in which only B cells were infected. Gene expression analysis demonstrated that EBV-2 established a latency III infection with evidence of ongoing viral reactivation in both B and T cells. Importantly, EBV-2-infected mice developed tumors resembling diffuse large B cell lymphoma (DLBCL). These lymphomas had morphological features comparable to those of EBV-1-induced DLBCLs, developed at similar rates with equivalent frequencies, and expressed a latency III gene profile. Thus, despite the impaired ability of EBV-2 to immortalize B cells in vitro, EBV-2 efficiently induces lymphomagenesis in humanized mice. Further research utilizing this model will enhance our understanding of EBV-2 biology, the consequence of EBV infection of T cells, and the capacity of EBV-2 to drive lymphomagenesis.IMPORTANCE EBV is a well-established B cell-tropic virus. However, we have recently shown that the EBV type 2 (EBV-2) strain also infects primary T cells in culture and in healthy Kenyan children. This finding suggests that EBV-2, unlike the well-studied EBV-1 strain, utilizes the T cell compartment to persist. As EBV is human specific, studies to understand the role of T cells in EBV-2 persistence require an in vivo model. Thus, we developed an EBV-2 humanized mouse model, utilizing immunodeficient mice engrafted with human cord blood CD34+ stem cells. Characterization of the EBV-2-infected humanized mice established that both T cells and B cells are infected by EBV-2 and that the majority of infected mice develop a B cell lymphoma resembling diffuse large B cell lymphoma. This new in vivo model can be utilized for studies to enhance our understanding of how EBV-2 infection of T cells contributes to persistence and lymphomagenesis.


Assuntos
Linfócitos B/virologia , Carcinogênese/genética , Infecções por Vírus Epstein-Barr/patologia , Herpesvirus Humano 4/patogenicidade , Linfoma Difuso de Grandes Células B/virologia , Linfócitos T/virologia , Animais , Linfócitos B/patologia , Modelos Animais de Doenças , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/classificação , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Linfócitos T/patologia , Tropismo Viral/fisiologia , Ativação Viral/genética , Latência Viral/genética
19.
PLoS Pathog ; 14(7): e1007179, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30052684

RESUMO

Latent Epstein-Barr virus (EBV) infection contributes to both B-cell and epithelial-cell malignancies. However, whether lytic EBV infection also contributes to tumors is unclear, although the association between malaria infection and Burkitt lymphomas (BLs) may involve excessive lytic EBV replication. A particular variant of the viral promoter (Zp) that controls lytic EBV reactivation is over-represented, relative to its frequency in non-malignant tissue, in EBV-positive nasopharyngeal carcinomas and AIDS-related lymphomas. To date, no functional differences between the prototype Zp (Zp-P) and the cancer-associated variant (Zp-V3) have been identified. Here we show that a single nucleotide difference between the Zp-V3 and Zp-P promoters creates a binding site for the cellular transcription factor, NFATc1, in the Zp-V3 (but not Zp-P) variant, and greatly enhances Zp activity and lytic viral reactivation in response to NFATc1-inducing stimuli such as B-cell receptor activation and ionomycin. Furthermore, we demonstrate that restoring this NFATc1-motif to the Zp-P variant in the context of the intact EBV B95.8 strain genome greatly enhances lytic viral reactivation in response to the NFATc1-activating agent, ionomycin, and this effect is blocked by the NFAT inhibitory agent, cyclosporine, as well as NFATc1 siRNA. We also show that the Zp-V3 variant is over-represented in EBV-positive BLs and gastric cancers, and in EBV-transformed B-cell lines derived from EBV-infected breast milk of Kenyan mothers that had malaria during pregnancy. These results demonstrate that the Zp-V3 enhances EBV lytic reactivation to physiologically-relevant stimuli, and suggest that increased lytic infection may contribute to the increased prevalence of this variant in EBV-associated malignancies.


Assuntos
Infecções por Vírus Epstein-Barr/genética , Transativadores/genética , Ativação Viral/genética , Variação Genética/genética , Herpesvirus Humano 4/genética , Humanos , Regiões Promotoras Genéticas/genética
20.
Artigo em Inglês | MEDLINE | ID: mdl-29941635

RESUMO

The 2-aminopyridine MMV048 was the first drug candidate inhibiting Plasmodium phosphatidylinositol 4-kinase (PI4K), a novel drug target for malaria, to enter clinical development. In an effort to identify the next generation of PI4K inhibitors, the series was optimized to improve properties such as solubility and antiplasmodial potency across the parasite life cycle, leading to the 2-aminopyrazine UCT943. The compound displayed higher asexual blood stage, transmission-blocking, and liver stage activities than MMV048 and was more potent against resistant Plasmodium falciparum and Plasmodium vivax clinical isolates. Excellent in vitro antiplasmodial activity translated into high efficacy in Plasmodium berghei and humanized P. falciparum NOD-scid IL-2Rγ null mouse models. The high passive permeability and high aqueous solubility of UCT943, combined with low to moderate in vivo intrinsic clearance, resulted in sustained exposure and high bioavailability in preclinical species. In addition, the predicted human dose for a curative single administration using monkey and dog pharmacokinetics was low, ranging from 50 to 80 mg. As a next-generation Plasmodium PI4K inhibitor, UCT943, based on the combined preclinical data, has the potential to form part of a single-exposure radical cure and prophylaxis (SERCaP) to treat, prevent, and block the transmission of malaria.

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