To develop neutralizing MAbs, mice were infected with the 16007 (genotype 2) prototype strain of DENV-1 and in some cases, boosted with recombinant DIII from the homologous DENV-1 strain

To develop neutralizing MAbs, mice were infected with the 16007 (genotype 2) prototype strain of DENV-1 and in some cases, boosted with recombinant DIII from the homologous DENV-1 strain. explain disparities in neutralizing potential of MAbs among different genotypes. Overall, our experiments define a complex structural epitope on DIII of DENV-1 that can be recognized by protective antibodies with therapeutic potential. Author Summary Dengue virus (DENV) is a mosquito-transmitted virus that infects 25 to 100 NCH 51 million humans annually and can progress to a life-threatening hemorrhagic fever and shock syndrome. Currently, no vaccines or specific therapies are available. Prior studies identified a highly neutralizing monoclonal antibody (MAb) against West Nile virus, a related flavivirus, as a candidate therapy for humans. In this study, we generated 79 new MAbs against the DENV type 1 (DENV-1) serotype, 16 of which strongly inhibited infection in cell culture. Using structural and molecular approaches, the binding sites of these inhibitory MAbs were localized to distinct regions on domain III of the DENV-1 envelope protein. We tested the protective capacity of all of the neutralizing MAbs in mice against infection by a strain of DENV-1 from a distinct genotype. Only two of the MAbs, DENV1-E105 and DENV1-E106, showed efficacy in a post-exposure treatment model, and these antibodies efficiently neutralized all five DENV-1 genotypes. Collectively, our studies define a complex structural binding site on domain III of the envelope protein for MAbs with therapeutic potential against DENV-1. Introduction Dengue virus (DENV) is a member of the family and is related to the viruses that cause yellow fever, and the Japanese, St. Louis, and the West Nile encephalitides [1]. DENV infection after mosquito inoculation causes a spectrum of clinical disease ranging from a self-limited febrile illness (DF) to a life threatening hemorrhagic and capillary leak syndrome (Dengue Hemorrhagic Fever (DHF)/Dengue Shock Syndrome (DSS)). Globally, there is significant diversity among DENV strains, including four distinct serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) that differ at the amino acid level in the viral envelope proteins by 25 to 40 percent. There is additional complexity within a given DENV serotype, as genotypes vary further by up to 6% and 3% at the nucleotide and amino acid levels, respectively [2],[3]. At present, no approved antiviral treatment or vaccine is available, and therapy is supportive. DENV causes an estimated 25 to 100 million infections and 250,000 cases of DHF/DSS per year worldwide, with 2.5 billion people at risk [4],[5]. DENV is an enveloped virus with a single-stranded, positive-sense RNA genome [6]. The 10.7 kilobase genome is translated as a single polyprotein, which is cleaved into three structural MAT1 proteins (C, prM/M, E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). The mature DENV virion has a well-organized outer protein shell, a lipid membrane bilayer, and a less-defined inner nucleocapsid core [7],[8]. The ectodomains of DENV E proteins are assembled as dimers with each subunit comprised of three discrete domains [9]C[11]. Domain I (DI) is a central, eight-stranded -barrel, which contains a single N-linked glycosylation site in most DENV strains. Domain II (DII) is a long, finger-like protrusion from DI and contains a second N-linked glycan that binds to DC-SIGN [12]C[15] and the highly conserved fusion peptide at its distal end. Domain III (DIII), which adopts an immunoglobulin-like fold, has been argued to contain a cell surface receptor recognition site [16]C[19]. Exposure to mildly acidic conditions in the trans-Golgi secretory pathway promotes virus maturation through a structural rearrangement of NCH 51 the flavivirus E NCH 51 proteins and cleavage of prM to M by a furin-like protease [20],[21]. Mature DENV virions are covered by 90 anti-parallel E protein homodimers, which are arranged flat along the surface with quasi-icosahedral symmetry. Many flavivirus neutralizing antibodies recognize the structural E protein (reviewed in [22]). Serotype-specific MAbs against DENV reportedly have the greatest neutralizing activity [23],[24] although some sub-complex specific MAbs, which recognize some but not all DENV serotypes, also are inhibitory [25]C[27]. Protection in animals by antibodies correlates generally with the degree of neutralizing activity in vitro [24], [28]C[31]. Several type-specific strongly neutralizing antibodies against individual flaviviruses (e.g., West Nile virus (WNV), Japanese encephalitis virus (JEV), DENV-2, DENV-4, and yellow fever.