EC50 values from independent experiments are shown in the right panel; error bars represent standard error of the mean (n=8, p=0.68). gene variants containing Cytidine substitutions at surface-accessible residues of the envelope (E) protein to match the corresponding DENV2 sequence. Amino acids that contribute to recognition by serotype-specific Cytidine neutralizing antibodies were identified as DENV mutants with reduced sensitivity to neutralization by DENV1 immune sera, but not cross-reactive neutralizing antibodies elicited by DENV2 vaccination. We identified two mutations (E126K and E157K) that contribute significantly to type-specific recognition by polyclonal DENV1 immune sera. Longitudinal Cytidine and cross-sectional analysis of sera from 24 participants of a phase I clinical study revealed a markedly reduced capacity to neutralize a E126K/E157K DENV1 variant. Sera from 77% of subjects recognized the E126K/E157K DENV1 variant and DENV2 equivalently (<3-fold difference). These data indicate the type-specific component of the DENV1 neutralizing antibody response to vaccination is strikingly focused on just two amino acids of the E protein. This study provides an important step towards deconvoluting the functional complexity of DENV serology following vaccination. == Author Summary == Despite decades of research, there remains a critical need for a dengue virus (DENV) vaccine. Vaccine development efforts are complicated by a requirement to protect against four DENV serotypes (DENV1-4), and incomplete immunity as a risk factor for severe disease. Antibodies play a major protective role against DENV. However, they also have been implicated in severe clinical manifestations of DENV infection. The antibody response to DENV is composed of antibodies that neutralize only the infecting DENV serotype (type-specific), as well as those that are cross-reactive. Cross-reactive antibodies are hypothesized to contribute to severe dengue following heterologous infections. Identifying DENV epitopes that are targets of type-specific neutralizing antibodies may facilitate vaccine development and the identification of correlates of protection. In this study, we identified amino acids on DENV1 recognized by type-specific neutralizing antibodies elicited by DENV1 vaccination. Our results indicate that the type-specific DENV1 response is remarkably focused on just two regions of the DENV1 envelope protein. Furthermore, a significant contribution of antibodies with this specificity was a common feature among vaccine recipients. This study identifies targets of neutralizing antibodies elicited by DENV1 vaccination and provides an important first step toward identifying epitopes recognized by each component of a tetravalent vaccine. == Introduction == Dengue virus (DENV) is a mosquito-transmitted flavivirus responsible for 390 million human infections each year[1]. Four related serotypes (DENV1-4) circulate in virtually all tropical and sub-tropical regions of the world[2]. While DENV infection is often subclinical, clinical symptoms of dengue fever (DF) include a self-limiting febrile illness, myalgia, rash, and retro-orbital pain[3]. A more severe clinical illness (dengue shock syndrome/dengue hemorrhagic fever) involving capillary Cytidine leakage, thrombocytopenia, and hemorrhage has been associated with secondary infections by a heterologous DENV serotype and higher viral loadsin vivo[4],[5]. The incidence of severe DENV disease is rising Cytidine globally due to increasing co-circulation of multiple DENV serotypes in endemic areas[2],[6]. Currently, there are no specific treatments or approved vaccines for DENV infection. Flaviviruses encapsidate a single-stranded RNA genome of positive-sense polarity. This 11 kb genomic RNA is translated as a single open reading frame that is cleaved in infected cells by cellular and viral proteases into at least ten proteins[7]. The virus encodes three structural proteins (envelope (E), premembrane (prM), and capsid (C)) that associate with a lipid envelope and the viral genome to form the virion[8]. Flavivirus assembly occurs on virus-induced membranes derived from the endoplasmic reticulum (ER)[9][13], resulting in the budding of non-infectious immature virus particles into the lumen. The E protein of immature virions exists as heterotrimeric spikes in complex with the prM protein; sixty of these spikes are organized on the virion with icosahedral symmetry[14][16]. During egress through the secretory Rabbit polyclonal to AIBZIP pathway, prM is cleaved by a cellular furin-like protease to generate the mature infectious virus particle[17][19]. Mature virions are characterized by a dense array of antiparallel E protein dimers orientated roughly parallel to the surface of the virion[20][22]. In many cases, this arrangement of E proteins imposes steric constraints for epitope recognition by antibodies[23],[24]. The virion maturation process is inefficient for many mosquito-borne flaviviruses, including DENV. Partially mature viruses with structural features of both mature and immature particles may be infectious and differentially interact with antibodies as a function of their prM content (reviewed in[25]). The humoral response plays an important role in protection against flaviviruses (reviewed in[26]). Development of a neutralizing antibody response is an established correlate of protection following vaccination against yellow fever virus (YFV), Japanese encephalitis virus (JEV), and tick-born encephalitis virus.