Development of a rapid assay system for detecting antibody-dependent enhancement of dengue virus infection.

Antibody-dependent enhancement (ADE) is one of the pathogenic mechanisms related to disease severity in dengue virus infection. Conventional assays for detecting ADE activity usually require several days. In this study, we established a rapid assay system to evaluate ADE activity in dengue-seropositive samples using single round infectious particles (SRIPs). Human Fc-gamma receptor-bearing cells (K562 and Mylc cells) were infected with SRIP antigen in the presence of human serum samples to measure ADE activity. Two assay protocols were introduced: (i) rapid assay with 5 h of incubation, and (ii) semi-rapid assay with 24 h of incubation. The rapid assay requires a large quantity of SRIP antigen and gives results in half a day. Although the semi-rapid assay requires slightly more than a day, it can be performed using only a small amount of SRIP. Interestingly, the range of the number of Mylc cells required for the semi-rapid assay was wider than that of K562 cells. Significant correlations were observed between the rapid and semi-rapid assays for both cell types. Although it is difficult to judge which protocol best reflects the current immune status in vivo, both assays could rapidly provide valuable information regarding the risk assessment for severe diseases.

Engineered flavivirus vaccines control induction of crossreactive infection-enhancing and -neutralizing antibodies.

Flaviviruses are important human pathogens because of their global distribution and disease severity. The high structural similarity among flaviviruses induces cross-immunity, with individual flaviviruses exhibiting crossreactive infection-enhancing and/or -neutralizing activities against other flaviviruses. Unlike neutralizing antibodies, enhancing antibodies may increase the risk of disease severity. Vaccine-induced enhancement remains a concern in the development of flavivirus vaccines. Here, we immunized mice with DNA vaccine candidates (pcJEME, pcWNME or pcZIKME) against Japanese encephalitis virus (JEV), West Nile virus (WNV) or Zika virus (ZIKV), respectively, and investigated crossreactive neutralizing and enhancing antibody activities against seven flaviviruses. pcZIKME induced higher cross-neutralization against dengue viruses than against JEV and WNV. Moreover, pcZIKME with a single amino acid substitution (D87N) showed an increase in crossreactive neutralizing activity and a decrease in enhancing activities against other flaviviruses. A similar trend was observed in pcWNME. Engineered antigen might contribute to the development of safe and effective flavivirus vaccines.

Direct suppression of human islet dedifferentiation, progenitor genes, but not epithelial to mesenchymal transition by liraglutide.

ß-cell dedifferentiation has been accounted as one of the major mechanisms for ß-cell failure; thus, is a cause to diabetes. We study direct impacts of liraglutide treatment on ex vivo human dedifferentiated islets, and its effects on genes important in endocrine function, progenitor states, and epithelial mesenchymal transition (EMT). Human islets from non-diabetic donors, were purified and incubated until day 1 and day 4, and were determined insulin contents, numbers of insulin (INS+) and glucagon (GCG+) cells. The islets from day 3 to day 7 were treated with diabetic drugs, the long acting GLP-1 receptor agonist, liraglutide. As observed in pancreatic islets of type 2 diabetic patients, ex vivo dedifferentiated islets showed more than 50% reduced insulin contents while number of glucagon increased from 10% to about 20%. ß-cell specific genes: PDX1, MAFA, as well as ß-cell functional markers: GLUT1 and SUR1, were significantly depleted more than 40%. Notably, we found increased levels of glucagon regulator, ARX and pre-glucagon transcripts, and remarkably upregulated progenitor expressions: NEUROG3 and ALDH1A identified as ß-cell dysfunction markers in diabetic models. Hyperglucagonemia was often observed in type 2 patients that could lead to over production of gluconeogenesis by the liver. Liraglutide treatments resulted in decreased number of GCG+ cells, increased numbers of GLP-1 positive cells but did not alter elevated levels of EMT marker genes: ACTA2, CDH-2, SNAIL2, and VIM. These effects of liraglutide were blunted when FOXO1 transcripts were depleted. This work illustrates that ex vivo human isolated islets can be used as a tool to study different aspects of ß-cell dedifferentiation. Our novel finding suggests a role of GLP-1 pathway in beta-cell maintenance in FOXO1-dependent manner. Importantly, dedifferentiated islets ex vivo is a useful model that can be utilized to verify the actions of potential drugs to diabetic ß-cell failure.