Abstract
Domain III of the dengue virus envelope protein (EDIII, aa295-395) has an immunoglobulin fold and is the proposed receptor-binding domain of the virus. Previous studies have shown that monoclonal antibodies against EDIII can be neutralizing and have therapeutic potential. Here, cloned Fab-phage libraries of human and mouse origin were screened for DENV specific antibodies. Firstly, bacterially expressed EDIII or whole virus particles were used as bait in biopanning against a large naïve human Fab-phage library (>10 billion independent clones). Multiple panning strategies were employed, and in excess of 1000 clones were screened, but all of the antibodies identified bound the envelope in regions outside EDIII suggesting EDIII antibodies are virtually absent from the naïve human repertoire. Next, a chimeric Fab-phage library was constructed from a panel of EDIII specific mouse hybridomas by pooling the VH and VL chain sequences from the hybridomas and cloning these into the pComb3X phagemid vector with human CH and CL encoding sequences. Biopanning against EDIII identified a unique antibody (C9) that cross-reacts with EDIII from DENV1-3 and, in the IgG format, binds and neutralizes DENV2 in cell-based assays. Sequence analysis and saturation mutagenesis of complementary determining regions (CDR) in the C9 light chain suggest an antigen recognition model in which the LCDR3 is a key determinant of EDIII specificity, while modifications in LCDR1 and LCDR2 affect DENV serotype cross-reactivity. Overall, this study supports the current prevailing opinion that neutralizing anti-EDIII monoclonal antibodies can be readily generated in murine systems, but in humans the anti-DENV immune response is directed away from domain III.
Highlights
Dengue virus (DENV) is a mosquito-borne Flavivirus responsible for at least 100 million symptomatic infections each year
Correct folding of the proteins was confirmed by size exclusion chromatography (SEC), which showed each of the evolved its receptor-binding domain (EDIII) proteins to be monomeric, and circular dichroism (Figure 1)
This lack of EDIII specific clones is unlikely to be a reflection of library quality, but rather extremely low levels of anti-EDIII antibodies in the naïve human repertoire since DENV positive/EDIII negative Fab were identified when whole virus particles were used as antigen
Summary
Dengue virus (DENV) is a mosquito-borne Flavivirus responsible for at least 100 million symptomatic infections each year. A primary infection provides effective long-term protection against re-infection with the same serotype, but can increase disease severity upon infection with a different serotype. This process, termed antibody dependent enhancement (ADE), is thought to be facilitated by poorly neutralizing cross-reactive antibodies generated in the primary infection that promote virus entry via Fcγ receptor bearing cells such as monocytes [2,3]. Structural analysis of the flavivirus E protein has identified three beta barrel domains: EDI, EDII and EDIII, with the native protein forming a head-to-tail homodimer [7]. The reorganized E trimer exposes the hydrophobic fusion loop in EDII to mediate cellular fusion [11]
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