ML-RR-cGAMP-D elicits serotype-specific and cross-reactive anti-NS1 T-cell responses in WTB6 andIfnar/mice. development. Keywords:dengue virus, NS1, cyclic dinucleotide, vaccine, adjuvant == INTRODUCTION == The four dengue virus serotypes (DENV14) are transmitted byAedes aegyptiandAe. albopictusmosquitoes, causing up to an estimated 390 million infections and 96 million cases of dengue annually (1). DENV is an enveloped flavivirus with 3 structural (C, capsid; prM/M, membrane; E, envelope) and 7 non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) proteins. Human DENV infections can range from asymptomatic to dengue fever (DF) to the potentially lethal dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). During a secondary infection with DENV, antibodies to the E protein may trigger antibody-dependent enhancement (ADE), where they facilitate entry of DENV into Fc receptor-bearing cells, leading to increased viral load, immune activation, and more severe disease (24). In contrast, since NS1 is not a structural component of the DENV virion, it is reasoned that antibodies to NS1 should not enhance viral uptake. As an immunogen, formulations based Araloside V on recombinant NS1 induce strong immune responses and confer protection in rodent models of dengue disease (58). Further, passive Araloside V transfer of polyclonal sera from NS1-immunized mice or anti-NS1 monoclonal antibodies into nave mice conferred protection against vascular leak disease, likely by promoting lysis of infected cells and/or by blocking the pathogenic effects of secreted NS1 (5,9,10). Importantly, NS1 is approximately 6479% conserved across the four DENV serotypes (11,12), and immunodominant regions of this protein have been identified in both NS1-immunized and DENV-infected mice, as well as in naturally infected humans (10,1316). The role of Araloside V NS1-specific CD4+T cells against DENV infection is a topic of active investigation, and studies have provided evidence of the likely protective effect of these cells (1720). Taken together, these findings support further research of NS1 as an important antigen for dengue vaccine development. One of the main challenges of nonreplicating vaccines is the use of adjuvants capable of eliciting strong memory T cells and protective antibodies (21,22). Cyclic dinucleotides (CDNs) are ubiquitous second messengers synthesized by bacteria, which are capable of activating the cytosolic receptor stimulator of interferon genes (STING), resulting in the activation of different immune pathways (2327). Due to their immunostimulatory properties, CDNs were initially used as vaccine adjuvants to elicit protective antibody and T-cell responses against pathogenic extracellular bacteria (2830). However, more recent studies have shown that CDN compounds also have a significant capacity to induce potent anti-tumor responses (3133) and to elicit protective Th1 and Th17 cellular immune responses againstMycobacterium tuberculosisinfection (34). In this study, we evaluated the immunogenicity of DENV NS1 proteins together with CDN compounds in comparison to monophosphoryl lipid A (MPLA) adjuvant, a TLR4 agonist capable of eliciting strong Th1 responses and high antibody titers (3538) that has been approved for human use in vaccines for hepatitis B virus and human papillomavirus infection (39,40). Using both wild-type C57BL/6 and IFN-/ receptordeficient C57BL/6 (Ifnar/) mice, we measured IgG titers and T-cell responses after immunizations with each NS1 from all four DENV serotypes. We found that NS1-CDN vaccinations induced balanced antibody responses against all DENV NS1 serotypes, which were comparable or higher in magnitude to those elicited by NS1-MPLA immunizations. Further, U2AF1 NS1-CDN immunized mice developed serotype-specific and cross-reactive T-cell responses, greater than MPLA-adjuvanted NS1, underscoring the ability of CDN compounds to induce cellular immunity. Finally, using a mouse model of lethal DENV infection, we show that NS1 combined with CDNs confers significant protection against DENVinduced morbidity and mortality. == MATERIALS AND METHODS == == Ethics statement. == All experimental procedures involving the use of animals were pre-approved and performed according to the guidelines of the Institutional Animal Care and Use Committee of the University of California, Berkeley. == Mice. == Wild-type C57BL/6 (WTB6) and IFN-/ receptordeficient C57BL/6 (Ifnar/) mice were bred and co-housed in specific pathogen-free conditions at the University of California, Berkeley Animal Facility. Five- to 8-week-old male and female mice were used for all experimental procedures. == Recombinant NS1 proteins. == Recombinant NS1 proteins from DENV serotypes 1 (Nauru/Western Pacific/1974), 2 (Thailand/16681/84), 3 (Sri Lanka D3/H/IMTSSA-SRI/2000/1266) and 4 (Dominica/814669/1981) were purchased from the Native Antigen Company (Oxford, UK). == Viruses. == DENV2 D220 was generated in our laboratory from the parental strain DENV2 PL046.