RESUMEN
BACKGROUND: Plasmodium vivax represents the most geographically widespread human malaria parasite affecting civilian and military populations in endemic areas. Targeting the pre-erythrocytic (PE) stage of the parasite life cycle is especially appealing for developing P. vivax vaccines as it would prevent disease and transmission. Here, naturally acquired immunity to a panel of P. vivax PE antigens was explored, which may facilitate vaccine development and lead to a better understanding of naturally acquired PE immunity. METHODS: Twelve P. vivax PE antigens orthologous to a panel of P. falciparum antigens previously identified as highly immunogenic in protected subjects after immunization with radiation attenuated sporozoites (RAS) were used for evaluation of humoral and cellular immunity by ELISA and IFN-γ ELISpot. Samples from P. vivax infected individuals (n = 76) from a low endemic malaria region in the Peruvian Amazon Basin were used. RESULTS: In those clinical samples, all PE antigens evaluated showed positive IgG antibody reactivity with a variable prevalence of 58-99% in recently P. vivax diagnosed patients. The magnitude of the IgG antibody response against PE antigens was lower compared with blood stage antigens MSP1 and DBP-II, although antibody levels persisted better for PE antigens (average decrease of 6% for PE antigens and 43% for MSP1, p < 0.05). Higher IgG antibodies was associated with one or more previous malaria episodes only for blood stage antigens (p < 0.001). High IgG responders across PE and blood stage antigens showed significantly lower parasitaemia compared to low IgG responders (median 1,921 vs 4,663 par/µl, p < 0.05). In a subgroup of volunteers (n = 17),positive IFN-γ T cell response by ELISPOT was observed in 35% vs 9-35% against blood stage MSP1 and PE antigens, respectively, but no correlation with IgG responses. CONCLUSIONS: These results demonstrate clear humoral and T cell responses against P. vivax PE antigens in individuals naturally infected with P. vivax. These data identify novel attractive PE antigens suitable for use in the potential development and selection of new malaria vaccine candidates which can be used as a part of malaria prevention strategies in civilian and military populations living in P. vivax endemic areas.
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Antígenos de Protozoos , Malaria Vivax , Plasmodium vivax , Proteínas Protozoarias , Plasmodium vivax/inmunología , Perú/epidemiología , Humanos , Malaria Vivax/inmunología , Malaria Vivax/epidemiología , Adulto , Masculino , Adulto Joven , Adolescente , Femenino , Persona de Mediana Edad , Proteínas Protozoarias/inmunología , Antígenos de Protozoos/inmunología , Inmunoglobulina G/sangre , Anticuerpos Antiprotozoarios/sangre , Ensayo de Inmunoadsorción Enzimática , Niño , Anciano , Ensayo de Immunospot Ligado a EnzimasRESUMEN
HIV develops single nucleotide polymorphisms (SNPs), some of which lead to drug resistance mutations (DRMs) that prevent therapeutic viral suppression. Genomic sequencing enables healthcare professionals to select effective combination antiretroviral therapy (ART) to achieve and maintain viral suppression. However, sequencing technologies, which are resource-intensive, are limited in their availability. This report describes the first step toward a highly specific ligation-based SNP discrimination method with endpoint PCR detection, which is more suitable for resource-limited clinics. The approach is based on magnetic bead processing to maximize reaction product transfer and minimize the carryover of incompatible buffer for three consecutive enzymatic reactionsâreverse transcription (RT), oligonucleotide ligation assay (OLA), and PCR. The method improved PCR detection following RT â OLA by 8.06 cycles (â¼250-fold) compared to direct pipette processing and detected between 103 and 104 RNA copies per reaction. In studies with synthesized nucleic acids based on the well-studied HIV mutation, K103N, the assay successfully differentiated between wild-type and mutant for RNA targets with high specificity. With further development, this design provides a pathway for SNP detection with more accessible PCR instrumentation and is a step toward a self-contained processing approach that incorporates the SNP specificity of the ligation reaction for more effective clinical management of DRMs in resource-constrained settings.
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Fármacos Anti-VIH , Farmacorresistencia Viral , Infecciones por VIH , VIH-1 , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Farmacorresistencia Viral/genética , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Fenómenos Magnéticos , MutaciónRESUMEN
With the approach of respiratory virus season in the Northern Hemisphere, clinical microbiology and public health laboratories will need rapid diagnostic assays to distinguish severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from influenza virus and respiratory syncytial virus (RSV) infections for diagnosis and surveillance. In this study, the clinical performance of the Xpert Xpress SARS-CoV-2/Flu/RSV test (Cepheid, Sunnyvale, CA, USA) for nasopharyngeal swab specimens was evaluated in four centers: Johns Hopkins Medical Microbiology Laboratory, Northwell Health Laboratories, NYC Public Health Laboratory, and Los Angeles County/University of Southern California (LAC+USC) Medical Center. A total of 319 nasopharyngeal swab specimens, positive for SARS-CoV-2 (n = 75), influenza A virus (n = 65), influenza B virus (n = 50), or RSV (n = 38) or negative (n = 91) by the standard-of-care nucleic acid amplification tests at each site, were tested using the Cepheid panel test. The overall positive percent agreement for the SARS-CoV-2 target was 98.7% (n = 74/75), and the negative agreement was 100% (n = 91), with all other analytes showing 100% total agreement (n = 153). Standard-of-care tests to which the Cepheid panel was compared included the Cepheid Xpert Xpress SARS-CoV-2, Cepheid Xpert Xpress Flu/RSV, GenMark ePlex respiratory panel, BioFire respiratory panel 2.1 and v1.7, DiaSorin Simplexa COVID-19 Direct, and Hologic Panther Fusion SARS-CoV-2 assays. The Xpert Xpress SARS-CoV-2/Flu/RSV test showed high sensitivity and accuracy for all analytes included in the test. This test will provide a valuable clinical diagnostic and public health solution for detecting and differentiating SARS-CoV-2, influenza A and B virus, and RSV infections during the current respiratory virus season.
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COVID-19/diagnóstico , Gripe Humana/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Humanos , Nasofaringe , SARS-CoV-2 , Sensibilidad y EspecificidadRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic has created a precipitous increase in the need for molecular diagnostics. Unfortunately, access to RNA extraction reagents can represent a bottleneck for quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR)-based methodologies, stemming from both extraordinary supply-chain stresses and the global reach of the virus into resource-limited settings. To provide flexible diagnostic options for such environments, we report here an "unextracted modification" for qRT-PCR using the Centers for Disease Control's (CDC's) widely utilized primers/probe sets for severe acute respiratory syndrome coronavirus 2 (N1/N2/N3 targeting viral nucleocapsid and RP-control targeting human RNase P). This approach replaces RNA extraction/purification with a heat-inactivation step of viral transport media (VTM), followed by direct inoculation-with or without VTM spin concentration-into PCR master mixes. Using derivatives of care from our clinical workflow, we compared traditional and unextracted CDC methodologies. Although some decrease in analytic sensitivity was evident (by higher Ct values) without extraction, in particular for the N2 primer/probe-set, we observed high categorical positive agreement between extracted and unextracted results for N1 (unconcentrated VTM-38/40; concentrated VTM-39/41), N3 (unconcentrated VTM-38/40; concentrated VTM-41/41), and RP (unconcentrated and concentrated VTM-81/81). The negative categorical agreement for N1/N2/N3 was likewise high. Overall, these results suggest that laboratories could adapt and validate unextracted qRT-PCR protocols as a contingency to overcome supply limitations, with minimal impact on categorical results.
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Prueba de COVID-19/economía , Prueba de COVID-19/métodos , COVID-19/economía , COVID-19/epidemiología , Países en Desarrollo/economía , SARS-CoV-2 , HumanosRESUMEN
BACKGROUND: Recent gains in reducing the global burden of malaria are threatened by the emergence of Plasmodium falciparum resistance to artemisinins. The discovery that mutations in portions of a P. falciparum gene encoding kelch (K13)-propeller domains are the major determinant of resistance has provided opportunities for monitoring such resistance on a global scale. METHODS: We analyzed the K13-propeller sequence polymorphism in 14,037 samples collected in 59 countries in which malaria is endemic. Most of the samples (84.5%) were obtained from patients who were treated at sentinel sites used for nationwide surveillance of antimalarial resistance. We evaluated the emergence and dissemination of mutations by haplotyping neighboring loci. RESULTS: We identified 108 nonsynonymous K13 mutations, which showed marked geographic disparity in their frequency and distribution. In Asia, 36.5% of the K13 mutations were distributed within two areas--one in Cambodia, Vietnam, and Laos and the other in western Thailand, Myanmar, and China--with no overlap. In Africa, we observed a broad array of rare nonsynonymous mutations that were not associated with delayed parasite clearance. The gene-edited Dd2 transgenic line with the A578S mutation, which expresses the most frequently observed African allele, was found to be susceptible to artemisinin in vitro on a ring-stage survival assay. CONCLUSIONS: No evidence of artemisinin resistance was found outside Southeast Asia and China, where resistance-associated K13 mutations were confined. The common African A578S allele was not associated with clinical or in vitro resistance to artemisinin, and many African mutations appear to be neutral. (Funded by Institut Pasteur Paris and others.).
Asunto(s)
Artemisininas/farmacología , Resistencia a Medicamentos/genética , Lactonas/farmacología , Mutación , Plasmodium falciparum/genética , Polimorfismo Genético , Proteínas Protozoarias/genética , Algoritmos , Artemisininas/uso terapéutico , Asia Sudoriental , China , Enfermedades Endémicas , Genotipo , Humanos , Lactonas/uso terapéutico , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Plasmodium falciparum/efectos de los fármacos , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Rapid diagnostic tests based on histidine-rich protein 2 (HRP2) detection are the primary tools used to detect Plasmodium falciparum malaria infections. Recent conflicting reports call into question whether α-HRP2 antibodies are present in human host circulation and if resulting immune complexes could interfere with HRP2 detection on malaria RDTs. This study sought to determine the prevalence of immune-complexed HRP2 in a low-transmission region of Southern Zambia. METHODS: An ELISA was used to quantify HRP2 in patient sample DBS extracts before and after heat-based immune complex dissociation. A pull-down assay reliant on proteins A, G, and L was developed and applied for IgG and IgM capture and subsequent immunoprecipitation of any HRP2 present in immune complexed form. A total of 104 patient samples were evaluated using both methods. RESULTS: Immune-complexed HRP2 was detectable in 17% (18/104) of all samples evaluated and 70% (16/23) of HRP2-positive samples. A majority of the patients with samples containing immune-complexed HRP2 had P. falciparum infections (11/18) and were also positive for free HRP2 (16/18). For 72% (13/18) of patients with immune-complexed HRP2, less than 10% of the total HRP2 present was in immune-complexed form. For the remaining samples, a large proportion (≥ 20%) of total HRP2 was complexed with α-HRP2 antibodies. CONCLUSIONS: Endogenous α-HRP2 antibodies form immune complexes with HRP2 in the symptomatic patient population of a low-transmission area in rural Southern Zambia. For the majority of patients, the percentage of HRP2 in immune complexes is low and does not affect HRP2-based malaria diagnosis. However, for some patients, a significant portion of the total HRP2 was in immune-complexed form. Future studies investigating the prevalence and proportion of immune-complexed HRP2 in asymptomatic individuals with low HRP2 levels will be required to assess whether α-HRP2 antibodies affect HRP2 detection for this portion of the transmission reservoir.
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Complejo Antígeno-Anticuerpo/inmunología , Antígenos de Protozoos/inmunología , Malaria Falciparum/inmunología , Plasmodium falciparum/fisiología , Proteínas Protozoarias/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Malaria Falciparum/epidemiología , Prevalencia , Sensibilidad y Especificidad , Zambia/epidemiologíaAsunto(s)
Brotes de Enfermedades , Mpox , Humanos , Ciudad de Nueva York/epidemiología , Mpox/epidemiologíaRESUMEN
Herpesviruses morphogenesis occurs stepwise both temporally and spatially, beginning in the nucleus and concluding with the emergence of an extracellular virion. The mechanisms by which these viruses interact with and penetrate the nuclear envelope and subsequent compartments of the secretory pathway remain poorly defined. In this report, a conserved viral protein (VP1/2; pUL36) that directs cytoplasmic stages of egress is identified to have multiple isoforms. Of these, a novel truncated VP1/2 species translocates to the nucleus and assists the transfer of DNA-containing capsids to the cytoplasm. The capsids are handed off to full-length VP1/2, which replaces the nuclear isoform on the capsids and is required for the final cytoplasmic stages of viral particle maturation. These results document that distinct VP1/2 protein species serve as effectors of nuclear and cytoplasmic egress.
Asunto(s)
Cápside/metabolismo , Herpesvirus Suido 1/fisiología , Proteínas Virales/metabolismo , Ensamble de Virus , Animales , Transporte Biológico , Línea Celular , Núcleo Celular/virología , Citoplasma/virología , Isoformas de Proteínas/metabolismo , PorcinosRESUMEN
Following their assembly, herpesvirus capsids exit the nucleus by budding at the inner nuclear membrane. Two highly conserved viral proteins are required for this process, pUL31 and pUL34. In this report, we demonstrate that the pUL31 component of the pseudorabies virus nuclear egress complex is a conditional capsid-binding protein that is unmasked in the absence of pUL34. The interaction between pUL31 and capsids was confirmed through fluorescence microscopy and Western blot analysis of purified intranuclear capsids. Three viral proteins were tested for their abilities to mediate the pUL31-capsid interaction: the minor capsid protein pUL25, the portal protein pUL6, and the terminase subunit pUL33. Despite the requirement for each protein in nuclear egress, none of these viral proteins were required for the pUL31-capsid interaction. These findings provide the first formal evidence that a herpesvirus nuclear egress complex interacts with capsids and have implications for how DNA-containing capsids are selectively targeted for nuclear egress.
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Transporte Activo de Núcleo Celular , Cápside/metabolismo , Herpesvirus Suido 1/fisiología , Proteínas Virales/metabolismo , Liberación del Virus , Western Blotting , Microscopía Fluorescente , Unión Proteica , Mapeo de Interacción de ProteínasRESUMEN
Recombination is an evolutionary process by which many pathogens generate diversity and acquire novel functions. Although a common occurrence during coronavirus replication, detection of recombination is only feasible when genetically distinct viruses contemporaneously infect the same host. Here, we identify an instance of SARS-CoV-2 superinfection, whereby an individual was infected with two distinct viral variants: Alpha (B.1.1.7) and Epsilon (B.1.429). This superinfection was first noted when an Alpha genome sequence failed to exhibit the classic S gene target failure behavior used to track this variant. Full genome sequencing from four independent extracts reveals that Alpha variant alleles comprise around 75% of the genomes, whereas the Epsilon variant alleles comprise around 20% of the sample. Further investigation reveals the presence of numerous recombinant haplotypes spanning the genome, specifically in the spike, nucleocapsid, and ORF 8 coding regions. These findings support the potential for recombination to reshape SARS-CoV-2 genetic diversity.
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COVID-19 , Sobreinfección , Genoma Viral/genética , Humanos , Ciudad de Nueva York/epidemiología , Recombinación Genética , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
The neuroinvasive property of several alpha-herpesviruses underlies an uncommon infectious process that includes the establishment of life-long latent infections in sensory neurons of the peripheral nervous system. Several herpesvirus proteins are required for replication and dissemination within the nervous system, indicating that exploiting the nervous system as a niche for productive infection requires a specialized set of functions encoded by the virus. Whether initial entry into the nervous system from peripheral tissues also requires specialized viral functions is not known. Here we show that a conserved deubiquitinase domain embedded within a pseudorabies virus structural protein, pUL36, is essential for initial neural invasion, but is subsequently dispensable for transmission within and between neurons of the mammalian nervous system. These findings indicate that the deubiquitinase contributes to neurovirulence by participating in a previously unrecognized initial step in neuroinvasion.
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Endopeptidasas/fisiología , Herpesvirus Suido 1/enzimología , Seudorrabia/virología , Células Receptoras Sensoriales/virología , Ubiquitina/metabolismo , Proteínas Estructurales Virales/fisiología , Animales , Cámara Anterior/virología , Transporte Axonal/fisiología , Chlorocebus aethiops , Endopeptidasas/genética , Infecciones Virales del Ojo/virología , Herpesvirus Suido 1/genética , Masculino , Seudorrabia/fisiopatología , Ratas , Ratas Long-Evans , Ratas Sprague-Dawley , Proteasas Ubiquitina-Específicas , Células Vero , Proteínas Estructurales Virales/genéticaRESUMEN
Nasal and nasopharyngeal swab specimens tested by the Cepheid Xpert Xpress SARS-CoV-2 were analyzed by whole-genome sequencing based on impaired detection of the N2 target. Each viral genome had at least one mutation in the N gene, which likely arose independently in the New York City and Pittsburgh study sites.
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COVID-19/epidemiología , COVID-19/virología , Proteínas de la Nucleocápside de Coronavirus/genética , SARS-CoV-2/genética , Ciudades/epidemiología , Bases de Datos Genéticas , Genoma Viral , Humanos , Mutación , Fosfoproteínas/genética , Estados Unidos/epidemiologíaRESUMEN
Nucleic acid-based diagnostic tests often require isolation and concentration of nucleic acids from biological samples. Commercial purification kits are difficult to use in low-resource settings because of their cost and insufficient laboratory infrastructure. Several recent approaches based on the use of magnetic beads offer a potential solution but remain limited to small volume samples. We have developed a simple and low-cost nucleic acid extraction method suitable for isolation and concentration of nucleic acids from small or large sample volumes. The method uses magnetic beads, a transfer pipette, steel wool, and an external magnet to implement high-gradient magnetic separation (HGMS) to retain nucleic acid-magnetic bead complexes within the device's steel wool matrix for subsequent processing steps. We demonstrate the method's utility by extracting tuberculosis DNA from both sputum and urine, two typical large volume sample matrices (5-200 mL), using guanidine-based extraction chemistry. Our HGMS-enabled extraction method is statistically indistinguishable from commercial extraction kits when detecting a spiked 123-base DNA sequence. For our HGMS-enabled extraction method, we obtained extraction efficiencies for sputum and urine of approximately 10 and 90%, whereas commercial kits obtained 10-17 and 70-96%, respectively. We also used this method previously in a blinded sample preparation comparison study published by Beall et al., 2019. Our manual extraction method is insensitive to high flow rates and sample viscosity, with capture of â¼100% for flow rates up to 45 mL/min and viscosities up to 55 cP, possibly making it suitable for a wide variety of sample volumes and types and point-of-care users. This HGMS-enabled extraction method provides a robust instrument-free method for magnetic bead-based nucleic acid extraction, potentially suitable for field implementation of nucleic acid testing.
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Técnicas Bacteriológicas/métodos , ADN Bacteriano/aislamiento & purificación , Imanes/química , Mycobacterium tuberculosis/aislamiento & purificación , Ácidos Nucleicos/aislamiento & purificación , ADN Bacteriano/análisis , ADN Bacteriano/orina , Humanos , Ácidos Nucleicos/análisis , Ácidos Nucleicos/orina , Reacción en Cadena en Tiempo Real de la Polimerasa , Manejo de Especímenes , Esputo/química , Esputo/microbiología , Tuberculosis/diagnósticoRESUMEN
Infection with HIV-1 perturbs homeostasis of human T cell subsets, leading to accelerated immunologic deterioration. While studying changes in CD4(+) memory and naïve T cells during HIV-1 infection, we found that a subset of CD4(+) effector memory T cells that are CCR7(-)CD45RO(-)CD45RA(+) (referred to as TEMRA cells), was significantly increased in some HIV-infected individuals. This T cell subset displayed a differentiated phenotype and skewed Th1-type cytokine production. Despite expressing high levels of CCR5, TEMRA cells were strikingly resistant to infection with CCR5 (R5)-tropic HIV-1, but remained highly susceptible to CXCR4 (X4)-tropic HIV-1. The resistance of TEMRA cells to R5-tropic viruses was determined to be post-entry of the virus and prior to early viral reverse transcription, suggesting a block at the uncoating stage. Remarkably, in a subset of the HIV-infected individuals, the relatively high proportion of TEMRA cells within effector T cells strongly correlated with higher CD4(+) T cell numbers. These data provide compelling evidence for selection of an HIV-1-resistant CD4(+) T cell population during the course of HIV-1 infection. Determining the host factors within TEMRA cells that restrict R5-tropic viruses and endow HIV-1-specific CD4(+) T cells with this ability may result in novel therapeutic strategies against HIV-1 infection.
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Infecciones por VIH/inmunología , VIH-1/inmunología , Receptores CCR5/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/virología , Vacunas contra el SIDA/inmunología , Apoptosis/inmunología , Complejo CD3/metabolismo , Antígenos CD4/metabolismo , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , División Celular/inmunología , Humanos , Memoria Inmunológica/inmunología , Inmunofenotipificación , Antígenos Comunes de Leucocito/metabolismo , Receptores CCR5/inmunología , Receptores CCR7 , Receptores de Quimiocina/metabolismo , Subgrupos de Linfocitos T/metabolismoRESUMEN
Monitoring of antimalarial resistance is important to prevent its further spread, but the available options for assessing resistance are less than ideal for field settings. Although molecular detection is perhaps the most efficient method, it is also the most complex because it requires DNA extraction and PCR instrumentation. To develop a more deployable approach, we designed new probes, which, when used in combination with an inhibitor-tolerant Taq polymerase, enable single-nucleotide polymorphism genotyping directly from whole blood. The probes feature two strategic design elements: locked nucleic acids to enhance specificity and the reporter dyes Cy5 and TEX615, which have less optical overlap with the blood absorbance spectra than other commonly used dyes. Probe performance was validated on a traditional laboratory-based instrument and then further tested on a field-deployable Adaptive PCR instrument to develop a point-of-care platform appropriate for use in malaria settings. The probes discriminated between wild-type Plasmodium falciparum and the chloroquine-resistant CRT PF3D7_0709000:c.227A>C (p.Lys76Thr) mutant in the presence of 2% blood. Additionally, in allelic discrimination plots with the new probes, samples clustered more closely to their respective axes compared with samples using minor groove binder probes with 6-FAM and VIC reporter dyes. Our strategy greatly simplifies single-nucleotide polymorphism detection and provides a more accessible alternative for antimalarial resistance surveillance in the field.
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Antimaláricos/farmacología , Resistencia a Medicamentos/genética , Malaria/diagnóstico , Malaria/genética , Polimorfismo de Nucleótido Simple , Alelos , Genotipo , Humanos , Hidrólisis , Malaria/tratamiento farmacológico , Malaria/parasitología , Reacción en Cadena de la PolimerasaRESUMEN
A T-cell subset, defined as CD4(+)CD25(hi) (regulatory T-cells [Treg cells]), was recently shown to suppress T-cell activation. We demonstrate that human Treg cells isolated from healthy donors express the HIV-coreceptor CCR5 and are highly susceptible to HIV infection and replication. Because Treg cells are present in very few numbers and are difficult to expand in vitro, we genetically modified conventional human T-cells to generate Treg cells in vitro by ectopic expression of FoxP3, a transcription factor associated with reprogramming T-cells into a Treg subset. Overexpression of FoxP3 in naïve human CD4(+) T-cells recapitulated the hyporesponsiveness and suppressive function of naturally occurring Treg cells. However, FoxP3 was less efficient in reprogramming memory T-cell subset into regulatory cells. In addition, FoxP3-transduced T-cells also became more susceptible to HIV infection. Remarkably, a portion of HIV-positive individuals with a low percentage of CD4(+) and higher levels of activated T-cells have greatly reduced levels of FoxP3(+)CD4(+)CD25(hi) T-cells, suggesting disruption of the Treg cells during HIV infection. Targeting and disruption of the T-cell regulatory system by HIV may contribute to hyperactivation of conventional T-cells, a characteristic of HIV disease progression. Moreover, the ability to reprogram human T-cells into Treg cells in vitro will greatly aid in decoding their mechanism of suppression, their enhanced susceptibility to HIV infection, and the unique markers expressed by this subset.
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Ingeniería Genética , Infecciones por VIH/patología , Linfocitos T Reguladores/virología , Linfocitos T CD4-Positivos/virología , Línea Celular , Proliferación Celular , Separación Celular , Transformación Celular Viral , Clonación Molecular , Citocinas/metabolismo , Progresión de la Enfermedad , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Humanos , Leucocitos Mononucleares/virología , Datos de Secuencia Molecular , Fenotipo , Receptores CCR5/metabolismo , Receptores de Interleucina-2/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Reguladores/metabolismoRESUMEN
HIV infection of primary human T cells requires T cell activation signals. However, how strength, duration, and quality of TCR signals affect susceptibility of resting human T cells to HIV infection remains poorly understood. We found that the same threshold and duration of antigen signals that lead to optimal T cell activation are required for HIV to progress beyond the level of reverse transcription within resting T cells. Remarkably, sustained cytokine signaling from the IL-2 receptor following TCR triggering was critical in establishing productive infection. While blockade of TCR signaling pathways with inhibitors of the phosphatidylinositol 3-kinase pathway caused a partial pre-integration block, another inhibitor, rapamycin, completely suppressed the infection. In contrast, cyclosporin A or FK506, inhibitors of NFAT, failed to block infection if the T cells were pre-activated. Collectively, these results bring to light significant parallels between successful HIV infection and optimal thresholds of T cell activation. Furthermore, our results underscore the critical role of IL-2 signaling in establishing productive HIV infection. These findings have important implications for our understanding of the complex interplay of HIV with host factors induced upon T cell activation.