RESUMEN
Vaccines that efficiently target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent for coronavirus disease (COVID-19), are the best means for controlling viral spread. This study evaluated the efficacy of the COVID-19 vaccine S-268019-b, which comprises the recombinant full-length SARS-CoV-2 spike protein S-910823 (antigen) and A-910823 (adjuvant). In addition to eliciting both Th1-type and Th2-type cellular immune responses, two doses of S-910823 plus A-910823 induced anti-spike protein IgG antibodies and neutralizing antibodies against SARS-CoV-2. In a SARS-CoV-2 challenge test, S-910823 plus A-910823 mitigated SARS-CoV-2 infection-induced weight loss and death and inhibited viral replication in mouse lungs. S-910823 plus A-910823 promoted cytokine and chemokine at the injection site and immune cell accumulation in the draining lymph nodes. This led to the formation of germinal centers and the induction of memory B cells, antibody-secreting cells, and memory T cells. These findings provide fundamental property of S-268019-b, especially importance of A-910823 to elicit humoral and cellular immune responses.
Asunto(s)
COVID-19 , Vacunas , Ratones , Animales , Humanos , Glicoproteína de la Espiga del Coronavirus/genética , SARS-CoV-2 , Vacunas contra la COVID-19 , COVID-19/prevención & control , Anticuerpos Neutralizantes , InmunidadRESUMEN
Invasive fungal infections (IFIs) are fatal infections, but treatment options are limited. The clinical efficacies of existing drugs are unsatisfactory because of side effects, drug-drug interaction, unfavorable pharmacokinetic profiles, and emerging drug-resistant fungi. Therefore, the development of antifungal drugs with a new mechanism is an urgent issue. Herein, we report novel aryl guanidine antifungal agents, which inhibit a novel target enzyme in the ergosterol biosynthesis pathway. Structure-activity relationship development and property optimization by reducing lipophilicity led to the discovery of 6h, which showed potent antifungal activity against Aspergillus fumigatus in the presence of serum, improved metabolic stability, and PK properties. In the murine systemic A. fumigatus infection model, 6h exhibited antifungal efficacy equivalent to voriconazole (1e). Furthermore, owing to the inhibition of a novel target in the ergosterol biosynthesis pathway, 6h showed antifungal activity against azole-resistant A. fumigatus.
Asunto(s)
Antifúngicos/farmacología , Ergosterol/antagonistas & inhibidores , Guanidina/farmacología , Infecciones Fúngicas Invasoras/tratamiento farmacológico , Tiazoles/farmacología , Antifúngicos/síntesis química , Antifúngicos/química , Aspergillus fumigatus/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ergosterol/biosíntesis , Guanidina/análogos & derivados , Guanidina/química , Humanos , Infecciones Fúngicas Invasoras/metabolismo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/químicaRESUMEN
Potent fungicidal activity is one of the key factors of antifungals to overcome invasive pulmonary aspergillosis (IPA). To date, quantification of Aspergillus DNA in the lungs and galactomannan (GM) in serum or bronchoalveolar lavage fluid have been developed as general methods for measuring fungal burden in IPA animal models. However, GM quantification is not supposed to be a suitable method for precise evaluation of the fungicidal effects of antifungals, because killed Aspergillus hyphae can release GM for a certain period until they are eliminated by the host. Therefore, in terms of detecting viable fungal burden of Aspergillus, quantification of Aspergillus DNA has been thought to be a suitable method. Here, to obtain a method with much higher sensitivity, we applied reverse transcription quantitative PCR (RT-qPCR) for A. fumigatus 18S ribosomal RNA to measure the viable fungal burden in murine IPA models. Prior to in vivo tests, we confirmed that the sensitivity of 18S rRNA was nearly 50-fold higher than that of 18S ribosomal DNA in vitro. This highly sensitive method made it possible to evaluate the fungicidal effects of antifungals in a low-inoculation murine IPA model. In this model, single administrations of higher doses of voriconazole and posaconazole, which have fungicidal activity, were able to display fungicidal effects with ≥1 log10 reductions by 18S rRNA quantification, whereas significant reductions in serum GM were not observed. These results suggest that 18S rRNA quantification is a powerful tool for screening novel antifungals with potent fungicidal activity only after a single administration.
Asunto(s)
Aspergillus fumigatus/crecimiento & desarrollo , Aspergillus fumigatus/genética , Aspergilosis Pulmonar Invasiva/microbiología , ARN Ribosómico 18S/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Animales , Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/aislamiento & purificación , ADN de Hongos/genética , Humanos , Aspergilosis Pulmonar Invasiva/diagnóstico , Masculino , Ratones , Ratones Endogámicos DBA , Viabilidad Microbiana , Sensibilidad y EspecificidadRESUMEN
Aspergillus flavus is the second most significant pathogenic cause of invasive aspergillosis; however, its emergence risks and mechanisms of voriconazole (VRC) resistance have not yet been elucidated in detail. Here, we demonstrate that repeated exposure of A. flavus to subinhibitory concentrations of VRC in vitro causes the emergence of a VRC-resistant mutant with a novel resistance mechanism. The VRC-resistant mutant shows a MIC of 16 µg/ml for VRC and of 0.5 µg/ml for itraconazole (ITC). Whole-genome sequencing analysis showed that the mutant possesses a point mutation in yap1, which encodes a bZIP transcription factor working as the master regulator of the oxidative stress response, but no mutations in the cyp51 genes. This point mutation in yap1 caused alteration of Leu558 to Trp (Yap1Leu558Trp) in the putative nuclear export sequence in the carboxy-terminal cysteine-rich domain of Yap1. This Yap1Leu558Trp substitution was confirmed as being responsible for the VRC-resistant phenotype, but not for that of ITC, by the revertant to Yap1wild type with homologous gene replacement. Furthermore, Yap1Leu558Trp caused marked upregulation of the atrF ATP-binding cassette transporter, and the deletion of atrF restored susceptibility to VRC in A. flavus These findings provide new insights into VRC resistance mechanisms via a transcriptional factor mutation that is independent of the cyp51 gene mutation in A. flavus.
Asunto(s)
Antifúngicos/farmacología , Aspergillus flavus/genética , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/genética , Mutación Puntual/genética , Regulación hacia Arriba/genética , Voriconazol/farmacología , Transportadoras de Casetes de Unión a ATP/genética , Aspergillus flavus/efectos de los fármacos , Estrés Oxidativo/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Gpx2, one of three glutathione peroxidase homologs (Gpx1, Gpx2, and Gpx3) in Saccharomyces cerevisiae, is an atypical 2-Cys peroxiredoxin that prefers to use thioredoxin as a reducing agent in vitro. Despite Gpx2 being an antioxidant, no obvious phenotype of gpx2Δ mutant cells in terms of oxidative stress has yet been found. To gain a clue as to Gpx2's physiological function in vivo, here we identify its intracellular distribution. Gpx2 was found to exist in the cytoplasm and mitochondria. In mitochondria, Gpx2 was associated with the outer membrane of the cytoplasmic-side, as well as the inner membrane of the matrix-side. The redox state of the mitochondrial Gpx2 was regulated by Trx1 and Trx2 (cytoplasmic thioredoxin), and by Trx3 (mitochondrial matrix thioredoxin). In addition, we found that the disruption of GPX2 reduced the sporulation efficiency of diploid cells.