NS1 antibody levels in patients with DHF are indicated in red, and in those with DF in blue

NS1 antibody levels in patients with DHF are indicated in red, and in those with DF in blue. and be of potential Entecavir benefit in aiding vaccine and treatment design. The antibody response during infection with dengue virus is a key component involved in the pathogenesis during secondary infection. Here the authors show antibodies targeting NS1 and the epitopes targeted can be associated with disease severity during human infection. == Introduction == Dengue virus (DENV) is one of the most rapidly emerging viral infections worldwide, infecting 390 million individuals annually. Despite such obvious need, there is currently no licensed specific drug for the treatment of this potentially fatal disease1. A tetravalent live attenuated yellow fever LAMNA and dengue chimeric vaccine has been recently licensed in some countries, Entecavir however, it has poor efficacy in naive individuals and efficacy depends on DENV serotype2. In addition, the vaccine manufacturer recently suggested that this vaccine should be avoided in dengue naive individuals, due to the likelihood of disease enhancement3. One of the major challenges faced in the development of an efficacious dengue vaccine is our poor understanding of what constitutes a protective immune response. DENV infections result in a spectrum of disease ranging from subclinical inapparent presentation, through mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF), which is characterized by an increase in vascular permeability resulting in shock and organ dysfunction4. Understanding the molecular pathway that leads to development of vascular leak would provide a major step forward in the development of effective dengue treatments. One such potential target is dengue nonstructural protein 1 (NS1), a secreted glycoprotein used as a diagnostic marker of dengue infection, appearing early in the serum before antibodies are generated5. NS1, synthesized as a monomer, forms a dimer in the ER lumen and hexamer in the serum. NS1 is thought to function as a cofactor for viral RNA replication6. NS1 has been shown to trigger cytokine release and contributes directly to vascular leak through binding TLR4 and engaging the endothelial glycocalyx7,8. In addition, in vitro data show that some antibodies against NS1 cross-react with endothelial cells and induce apoptosis, which is suggested to contribute to endothelial dysfunction and vascular leak9. Interestingly, NS1 is important for viral evasion of complement activation by binding mannose-binding Entecavir lectin and preventing neutralization of DENV via the lectin pathway10. However, some studies have shown that NS1 activates complement, a process that is further enhanced by NS1-specific antibodies11. Secondary dengue infections incur an increased risk of developing DHF, which supports the hypothesis that NS1-specific antibodies derived from primary infection may, upon expansion following secondary challenge, play a role in disease pathogenesis12,13. However, apoptosis of the endothelium and deposition of immune complexes in the endothelium have not been demonstrated in autopsy studies of fatal dengue14. In contrast to the data in the above studies, some investigations in dengue mouse models have shown that mice injected with sera from dengue-infected mice or monoclonal antibodies to NS1, have significantly reduced vascular leak, implying a protective role for antibodies to NS115. It is currently not clear if antibodies targeted to specific regions of dengue NS1 protein may play differential roles in the protection or pathogenesis of dengue fever in humans. The majority of human DENV-specific antibodies are directed against DENV envelope and PrM proteins, and these responses have been relatively well-characterized1619. However, NS1-specific antibodies have not been thoroughly investigated despite constituting 27% of antibody responses20. There are limited data on the target, isotype and function of anti-NS1 antibodies in acute secondary dengue infections, which account for most cases of severe dengue infection. Interestingly, Dengvaxia, the only registered dengue vaccine currently available, does not generate DENVNS1-specific antibodies21, and some have speculated that the failure to raise NS1-specific antibodies may contribute to the lower-than-expected efficacy of this vaccine22. In order to address these questions, here we investigate NS1-specific antibody responses in a cohort of patients with acute secondary dengue (DENV1 and DENV2) infection and study how NS1-specific antibody responses evolve during the course of illness in relation to clinical disease severity. We further characterize anti-NS1 antibody responses in patients and healthy individuals with varying severity of past dengue infection to identify epitope regions within the NS1 protein that associate with disease progression or protection. == Results == == Dengue patient cohort == Prior to investigating the NS1 antibody responses in a patient cohort, we evaluated the NS1 antibody levels in a healthy population to determine NS1 antibody levels at baseline. Entecavir The NS1 antibody levels were assessed by an in-house ELISA, in DENV-seronegative individuals (n= 20), in.