Mutations were mapped onto the DENV2 EDIII structure using the atomic coordinates of DENV2 EDIII (RCSB accession number 1OAN) and displayed using PyMOL Molecular Graphics System, Version 1

Mutations were mapped onto the DENV2 EDIII structure using the atomic coordinates of DENV2 EDIII (RCSB accession number 1OAN) and displayed using PyMOL Molecular Graphics System, Version 1.3 (24S)-24,25-Dihydroxyvitamin D3 (Schrdinger, LLC). == Results == The objective of the current study was to characterize the primary human antibody response to DENV by comparing immune sera and MAbs derived from two individuals previously exposed to primary DENV infections. Moreover, many hMAbs bound to the viral pre-membrane protein and other sites on the virus that were not preserved when the viral envelope protein was produced as a soluble, recombinant antigen (rE protein). Nonetheless, by (24S)-24,25-Dihydroxyvitamin D3 modifying the screening procedure to detect rare antibodies that bound to rE, we were able to isolate and map human antibodies that strongly neutralized the homologous serotype of DENV. Our MAbs results indicate that, in these two individuals exposed to primary DENV infections, a small fraction of the total antibody response was responsible for virus neutralization. == Author Summary == Dengue is a mosquito-borne viral disease of humans. The dengue virus complex is made up of four viruses designated as serotypes. People experiencing their first infection develop immune responses that prevent re-infection with the same serotype only. People experiencing a second infection with a new serotype face a greater risk of developing a severe disease known as dengue hemorrhagic fever. Although studies indicate that antibodies can prevent or enhance disease caused by DENV, few studies have explored the specific properties of human antibodies against DENV. The objective of this study was to conduct a detailed analysis of the antibody response of two individuals who had recovered from primary infections. Human antibodies bound to sites on the dengue virus particle including the viral pre-membrane (prM/M) and envelope (E) proteins. Our studies indicate that the human antibody response consists of a minor population of strongly neutralizing antibody and a major population of DENV serotype cross-reactive, non-neutralizing antibody with potential for enhancement of virus and disease. Further studies with more DENV-immune subjects are needed to determine if our findings are broadly applicable to primary infections. == Introduction == Dengue virus (DENV) complex consists of 4 serotypes. People exposed to primary DENV infections (24S)-24,25-Dihydroxyvitamin D3 develop robust antibody responses that cross-react with all serotypes (Reviewed in[1]). Despite the extensive cross-reactivity, individuals only develop long term, protective immunity against the homologous serotype responsible for the primary infection[2],[3]. Indeed, the risk of progressing to DHF is greater during secondary compared to primary infection[4]. A prevailing theory that explains severe dengue during secondary infection is that pre-existing, non-neutralizing dengue specific antibodies enhance DENV entry and replication in Fc-receptor-bearing cells, which leads to a higher viremia and more severe disease[4]. Antibodies have been demonstrated to enhance DENV in cell culture[5],[6]and in animal models of dengue pathogenesis[7][9]. Our current understanding of how antibodies interact with DENV and other flaviviruses is primarily based on studies utilizing mouse monoclonal antibodies (MAbs) (Reviewed in[10]). The (24S)-24,25-Dihydroxyvitamin D3 DENV envelope (E) protein is the principle target of neutralizing antibodies. Antibody neutralization occurs by blocking critical functions of the E protein, including attachment to host cells and low pH-dependent fusion of the viral and host cell membranes[11]. The crystal structures of the E protein of several flaviviruses have been solved[12][15]. Individual subunits of E protein consist of three beta-barrel domains designated domains I (EDI), II (EDII) and III (EDIII), with the native protein forming a head-to-tail homodimer. Mouse MAbs that bind to all three domains of DENV E have been generated and characterized[16][23]. Although neutralizing mouse MAbs have been mapped to all three domains of E, the most strongly neutralizing MAbs recognize epitopes on the lateral ridge and A strand of EDIII[24]. Following a primary DENV infection, humans develop antibodies that cross-react with all 4 serotypes, but mainly neutralize the homologous serotype responsible for the infection (Reviewed in[3]). Studies with human immune sera and, more recently, human monoclonal antibodies have demonstrated that the dominant antibody response is cross-reactive and weakly neutralizing[25][30]. Multiple viral antigens including E protein, pre-membrane (prM/M) protein and nonstructural protein 1 (NSP1) are recognized by the human humoral response[25][30]. Nonetheless, few studies have defined the actual epitopes of DENV recognized by type-specific and cross-reactive human antibodies at the structural level and compared this to the epitopes defined using mouse antibodies. The target(s) of dengue type-specific, Rabbit polyclonal to PFKFB3 strongly neutralizing human antibodies remain unknown. The goal of this study was to study two subjects in-depth to define the major antigens and epitopes recognized by antibodies that develop following primary human DENV infection. Defining the human B-cell epitopes on DENV is a key step towards understanding how antibodies can both enhance and inhibit the severity of DENV infections. == Materials and Methods == == Viruses, recombinant proteins and immune sera == DENV1 WestPac-74, DENV2 S-16803, DENV3 CH-53489, and DENV4 TVP-360,.