RESUMEN
HIV-infected infants are at an increased risk of progressing rapidly to AIDS in the first weeks of life. Here, we evaluated immunological and virological parameters in 25 SIV-infected infant rhesus macaques to understand the factors influencing a rapid disease outcome. Infant macaques were infected with SIVmac251 and monitored for 10 to 17 weeks post-infection. SIV-infected infants were divided into either typical (TypP) or rapid (RP) progressor groups based on levels of plasma anti-SIV antibody and viral load, with RP infants having low SIV-specific antibodies and high viral loads. Following SIV infection, 11 out of 25 infant macaques exhibited an RP phenotype. Interestingly, TypP had lower levels of total CD4 T cells, similar reductions in CD4/CD8 ratios and elevated activation of CD8 T cells, as measured by the levels of HLA-DR, compared to RP. Differences between the two groups were identified in other immune cell populations, including a failure to expand activated memory (CD21-CD27+) B cells in peripheral blood in RP infant macaques, as well as reduced levels of germinal center (GC) B cells and T follicular helper (Tfh) cells in spleens (4- and 10-weeks post-SIV). Reduced B cell proliferation in splenic germinal GCs was associated with increased SIV+ cell density and follicular type 1 interferon (IFN)-induced immune activation. Further analyses determined that at 2-weeks post SIV infection TypP infants exhibited elevated levels of the GC-inducing chemokine CXCL13 in plasma, as well as significantly lower levels of viral envelope diversity compared to RP infants. Our findings provide evidence that early viral and immunologic events following SIV infection contributes to impairment of B cells, Tfh cells and germinal center formation, ultimately impeding the development of SIV-specific antibody responses in rapidly progressing infant macaques.
Asunto(s)
Progresión de la Enfermedad , Inmunidad Humoral , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/virología , Variación Genética , Centro Germinal/inmunología , Centro Germinal/virología , Humanos , Interferón Tipo I/inmunología , Tejido Linfoide/inmunología , Tejido Linfoide/virología , Macaca mulatta , Fenotipo , Síndrome de Inmunodeficiencia Adquirida del Simio/patología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Carga ViralRESUMEN
De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37-43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing.
Asunto(s)
Diseño de Fármacos , Gripe Humana/tratamiento farmacológico , Gripe Humana/prevención & control , Terapia Molecular Dirigida/métodos , Ingeniería de Proteínas/métodos , Proteínas/química , Proteínas/uso terapéutico , Toxinas Botulínicas/clasificación , Toxinas Botulínicas/metabolismo , Simulación por Computador , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Calor , Humanos , Gripe Humana/metabolismo , Simulación de Dinámica Molecular , Unión Proteica , Estabilidad Proteica , Proteínas/inmunología , Proteínas/metabolismo , TemperaturaRESUMEN
BCG vaccination has been demonstrated to increase levels of activated CD4+ T cells, thus potentially influencing mother-to-child transmission of human immunodeficiency virus (HIV). To assess the risk of BCG vaccination in HIV infection, we randomly assigned newborn rhesus macaques to receive BCG vaccine or remain unvaccinated and then undergo oral simian immunodeficiency virus (SIV) challenges 3 weeks later. We observed elevated levels of activated peripheral CD4+ T cells (ie, HLA-DR+CD38+CCR5+ CD4+ T cells) by week 3 after vaccination. BCG was also associated with an altered immune gene expression profile, as well as with monocyte activation in both peripheral blood and the draining axillary lymph node, indicating significant BCG vaccine-induced immune activation. Despite these effects, BCG vaccination did not increase the rate of SIV oral transmission or disease progression. Our findings therefore identify patterns of T-cell and monocyte activation that occur after BCG vaccination but do not support the hypothesis that BCG vaccination is a risk factor for postnatal HIV transmission or increased pathogenesis in infants.
Asunto(s)
Inmunidad Activa/efectos de los fármacos , Macaca mulatta/inmunología , Retrovirus de los Simios/efectos de los fármacos , Retrovirus de los Simios/inmunología , Vacunas contra el SIDAS/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Animales , Femenino , Masculino , Modelos Animales , Vacunas contra el SIDAS/administración & dosificación , Síndrome de Inmunodeficiencia Adquirida del Simio/fisiopatología , Vacunación/métodosRESUMEN
Heat shock transcription factor 1 (HSF1) is a major transcriptional regulator of the heat shock response in eukaryotic cells. HSF1 is evoked in response to a variety of cellular stressors, including elevated temperatures, oxidative stress, and other proteotoxic stressors. Previously, we demonstrated that HSF1 is activated in naive T cells at fever range temperatures (39.5°C) and is critical for in vitro T cell proliferation at fever temperatures. In this study, we demonstrated that murine HSF1 became activated to the DNA-binding form and transactivated a large number of genes in lymphoid cells strictly as a consequence of receptor activation in the absence of apparent cellular stress. Microarray analysis comparing HSF1(+/+) and HSF1(-/-) gene expression in T cells activated at 37°C revealed a diverse set of 323 genes significantly regulated by HSF1 in nonstressed T cells. In vivo proliferation studies revealed a significant impairment of HSF1(-/-) T cell expansion under conditions mimicking a robust immune response (staphylococcal enterotoxin B-induced T cell activation). This proliferation defect due to loss of HSF1 is observed even under nonfebrile temperatures. HSF1(-/-) T cells activated at fever temperatures show a dramatic reduction in cyclin E and cyclin A proteins during the cell cycle, although the transcription of these genes was modestly affected. Finally, B cell and hematopoietic stem cell proliferation from HSF1(-/-) mice, but not HSF1(+/+) mice, were also attenuated under stressful conditions, indicating that HSF1 is critical for the cell cycle progression of lymphoid cells activated under stressful conditions.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Activación de Linfocitos , Estrés Fisiológico , Linfocitos T/inmunología , Factores de Transcripción/metabolismo , Animales , Ciclo Celular , División Celular , Proliferación Celular , Células Cultivadas , Ciclina A/biosíntesis , Ciclina E/biosíntesis , Proteínas de Unión al ADN/genética , Enterotoxinas/inmunología , Fiebre/inmunología , Regulación de la Expresión Génica , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/genéticaRESUMEN
Hull blackberries were purified using solid phase extraction to obtain anthocyanin-rich methanol fractions. This method concentrated phenolics and anthocyanins, recovering 97% and 76% of the total yield in puree or powder extracts, respectively, which represented a 24-63 fold increase of the total antioxidant capacity when compared with either the water fraction or the original extract. The ability of these fractions to protect primary keratinocytes against UV-induced oxidative damage was assessed. Anthocyanin-rich methanol fractions derived from either blackberry powder or puree exhibited strong antioxidant properties, protecting against UV-induced ROS nearly as efficiently as N-acetyl cysteine. Furthermore, the fractions up-regulated the expression of catalase, MnSOD, Gpx1/2 and Gsta1 antioxidant enzymes. Thus, it is concluded that blackberry extracts may protect keratinocytes against UV-mediated oxidative damage.
Asunto(s)
Antocianinas/farmacología , Antioxidantes/metabolismo , Queratinocitos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/farmacología , Rosaceae/química , Enfermedades de la Piel/prevención & control , Acetilcisteína/farmacología , Animales , Antocianinas/aislamiento & purificación , Antocianinas/uso terapéutico , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Catalasa/metabolismo , Frutas , Glutatión/metabolismo , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Extractos Vegetales/química , Extractos Vegetales/uso terapéutico , Traumatismos por Radiación/tratamiento farmacológico , Traumatismos por Radiación/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Piel/efectos de los fármacos , Piel/metabolismo , Piel/efectos de la radiación , Enfermedades de la Piel/etiología , Enfermedades de la Piel/metabolismo , Superóxido Dismutasa/metabolismo , Rayos Ultravioleta , Regulación hacia ArribaRESUMEN
Heat shock factor 1 (HSF1) is a stress-induced transcription factor that promotes expression of genes that protect mammalian cells from the lethal effects of severely elevated temperatures (>42°C). However, we recently showed that HSF1 is activated at a lower temperature (39.5°C) in T cells, suggesting that HSF1 may be important for preserving T cell function during pathogen-induced fever responses. To test this, we examined the role of HSF1 in clearance of Listeria monocytogenes, an intracellular bacterial pathogen that elicits a strong CD8(+) T cell response in mice. Using temperature transponder microchips, we showed that the core body temperature increased approximately 2°C in L. monocytogenes-infected mice and that the fever response was maintained for at least 24 h. HSF1-deficient mice cleared a low-dose infection with slightly slower kinetics than did HSF1(+/+) littermate controls but were significantly more susceptible to challenges with higher doses of bacteria. Surprisingly, HSF1-deficient mice did not show a defect in CD8(+) T cell responses following sublethal infection. However, when HSF1-deficient mice were challenged with high doses of L. monocytogenes, increased levels of serum tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) compared to those of littermate control mice were observed, and rapid death of the animals occurred within 48 to 60 h of infection. Neutralization of TNF-α enhanced the survival of HSF1-deficient mice. These results suggest that HSF1 is needed to prevent the overproduction of proinflammatory cytokines and subsequent death due to septic shock that can result following high-dose challenge with bacterial pathogens.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Fiebre/metabolismo , Listeria monocytogenes , Listeriosis/metabolismo , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Linfocitos T CD8-positivos/fisiología , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/fisiología , Genotipo , Factores de Transcripción del Choque Térmico , Interferón gamma/sangre , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The ongoing COVID-19 pandemic, caused by infection with SARS-CoV-2, is having a dramatic and deleterious impact on health services and the global economy. Grim public health statistics highlight the need for vaccines that can rapidly confer protection after a single dose and be manufactured using components suitable for scale-up and efficient distribution. In response, we have rapidly developed repRNA-CoV2S, a stable and highly immunogenic vaccine candidate comprised of an RNA replicon formulated with a novel Lipid InOrganic Nanoparticle (LION) designed to enhance vaccine stability, delivery and immunogenicity. We show that intramuscular injection of LION/repRNA-CoV2S elicits robust anti-SARS-CoV-2 spike protein IgG antibody isotypes indicative of a Type 1 T helper response as well as potent T cell responses in mice. Importantly, a single-dose administration in nonhuman primates elicited antibody responses that potently neutralized SARS-CoV-2. These data support further development of LION/repRNA-CoV2S as a vaccine candidate for prophylactic protection from SARS-CoV-2 infection.
RESUMEN
The coronavirus disease 2019 (COVID-19) pandemic, caused by infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is having a deleterious impact on health services and the global economy, highlighting the urgent need for an effective vaccine. Such a vaccine would need to rapidly confer protection after one or two doses and would need to be manufactured using components suitable for scale up. Here, we developed an Alphavirus-derived replicon RNA vaccine candidate, repRNA-CoV2S, encoding the SARS-CoV-2 spike (S) protein. The RNA replicons were formulated with lipid inorganic nanoparticles (LIONs) that were designed to enhance vaccine stability, delivery, and immunogenicity. We show that a single intramuscular injection of the LION/repRNA-CoV2S vaccine in mice elicited robust production of anti-SARS-CoV-2 S protein IgG antibody isotypes indicative of a type 1 T helper cell response. A prime/boost regimen induced potent T cell responses in mice including antigen-specific responses in the lung and spleen. Prime-only immunization of aged (17 months old) mice induced smaller immune responses compared to young mice, but this difference was abrogated by booster immunization. In nonhuman primates, prime-only immunization in one intramuscular injection site or prime/boost immunizations in five intramuscular injection sites elicited modest T cell responses and robust antibody responses. The antibody responses persisted for at least 70 days and neutralized SARS-CoV-2 at titers comparable to those in human serum samples collected from individuals convalescing from COVID-19. These data support further development of LION/repRNA-CoV2S as a vaccine candidate for prophylactic protection against SARS-CoV-2 infection.
Asunto(s)
Alphavirus/genética , Anticuerpos Neutralizantes/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus/inmunología , Neumonía Viral/inmunología , ARN Viral/genética , Replicón/genética , Linfocitos T/inmunología , Vacunas Virales/inmunología , Animales , Formación de Anticuerpos/inmunología , COVID-19 , Vacunas contra la COVID-19 , Infecciones por Coronavirus/prevención & control , Compuestos Inorgánicos/química , Lípidos/química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Nanopartículas/química , Pandemias , Primates , SARS-CoV-2RESUMEN
The POU-homeodomain transcription factor Pit-1 governs ontogeny and cell-specific gene expression of pituitary lactotropes, somatotropes, and thyrotropes. The splice isoform, Pit-1beta, inserts a 26-amino acid (AA) repressor at AA48 in the Pit-1 transcription activation domain (TAD). The Pit-1 TAD contains a basal regulatory subregion, R1 (AA1-45), and a basal and Ras-responsive region, R2 (AA46-80). To precisely map these activities, we generated GAL4-Pit-1/Pit-1betaTAD fusions and, in full-length HA-Pit-1, a series of substitution mutants of R2. Analysis in GH4 cells identified an activation domain at AA50-70, followed by an overlapping, dual-function, Ras-responsive-inhibitory domain, located from AA60-80. In contrast, GAL4-Pit-1betaTAD repressed both basal and Ras-mediated TAD activity. To determine the functional interplay between TAD subregions and the beta-domain, we inserted the beta-domain every 10 AA across the 80-AA Pit-1 TAD. Like wild-type Pit-1beta, each construct retained transcriptional activity in HeLa cells and repressed the Ras response in GH4 cells. However, beta-domain insertion at AA61 and 71 resulted in greater repression of Ras responsiveness, defining a critical R2 TAD spanning AA61-71 of Pit-1. Furthermore, Ras activation is augmented by steroid receptor coactivator 1, whereas cAMP response element binding protein-binding protein is not a Ras mediator in this system. In summary, the Pit-1/Pit-1beta TADs are composed of multiple, modular, and transferable subdomains, including a regulatory R1 domain, a basal activation region, a selective inhibitory-Ras-responsive segment, and a beta-specific repressor domain. These data provide novel insights into the mechanisms by which the Pit-1 TAD integrates DNA binding, protein partner interactions, and distinct signaling pathways to fine-tune Pit-1 activity.
Asunto(s)
Factor de Transcripción Pit-1/fisiología , Activación Transcripcional , Proteínas ras/fisiología , Animales , Sitios de Unión , Células HeLa , Humanos , Modelos Biológicos , Mutagénesis , Fosforilación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ratas , Transducción de Señal , Proteínas ras/metabolismoRESUMEN
Heat shock factor-1 (HSF1) is a transcription factor that serves as the major temperature-inducible sensor for eukaryotic cells. In most cell types, HSF1 becomes activated to the DNA binding form at 42 degrees C and mediates the classical heat shock response, protecting the cells from subsequent lethal temperatures. We have recently demonstrated that HSF1 is activated at a lower temperature in T lymphocytes than in most other cell types (39 degrees C vs 42 degrees C), within the physiological range of fever. In this study, we show that T cell activation at fever temperatures not only activates HSF1 but induces the up-regulation of the HSF1 protein and the HSF1-regulated protein, HSP70i. T cells from HSF1 knockout mice proliferate normally under optimal conditions but are impaired in proliferation at physiological fever temperatures and low CO2 concentrations, conditions that do not impair wild-type T cells. This defect in proliferation appears to be mediated by a block in the G1/S transition of the cell cycle and is independent of HSP70. Elevated temperature and low CO2 concentrations resulted in a dramatic reduction of the intracellular reactive oxygen species (ROS) levels in both normal and knockout T cells. Wild-type T cells were able to restore ROS levels to normal within 5 h, whereas HSF1-/- T cells were not. These results suggest that the proliferation defect seen in T cells from HSF1-/- mice at fever temperatures was because of dysregulated ROS levels and that HSF1 is important in maintaining ROS homeostasis and cell cycle progression under the stressful conditions encountered during fever.