Epitope Determinants of a Chimpanzee Fab Antibody That Efficiently Cross-Neutralizes Dengue Type 1 and Type 2 Viruses Map to Inside and in Close Proximity to Fusion Loop of the Dengue Type 2 Virus Envelope Glycoprotein

Abstract

The epitope determinants of chimpanzee Fab antibody 1A5, which have been shown to be broadly reactive to flaviviruses and efficient for cross-neutralization of dengue virus type 1 and type 2 (DENV-1 and DENV-2), were studied by analysis of DENV-2 antigenic variants. Sequence analysis showed that one antigenic variant contained a Gly-to-Val substitution at position 106 within the flavivirus-conserved fusion peptide loop of the envelope protein (E), and another variant contained a His-to-Gln substitution at position 317 in E. Substitution of Gly 106 Val in DENV-2 E reduced the binding affinity of Fab 1A5 by approximately 80-fold, whereas substitution of His 317 Gln had little or no effect on antibody binding compared to the parental virus. Treatment of DENV-2 with ␤-mercaptoethanol abolished binding of Fab 1A5, indicating that disulfide bridges were required for the structural integrity of the Fab 1A5 epitope. Binding of Fab 1A5 to DENV-2 was competed by an oligopeptide containing the fusion peptide sequence as shown by competition enzyme-linked immunosorbent assay. Both DENV-2 antigenic variants were shown to be attenuated, or at least similar to the parental virus, when evaluated for growth in cultured cells or for neurovirulence in mice. Fab 1A5 inhibited low pH-induced membrane fusion of mosquito C6/36 cells infected with DENV-1 or DENV-2, as detected by reduced syncytium formation. Both substitutions in DENV-2 E lowered the pH threshold for membrane fusion, as measured in a fusion-from-within assay. In the three-dimensional structure of E, Gly 106 in domain II and His 317 in domain III of the opposite E monomer were spatially close. From the locations of these amino acids, Fab 1A5 appears to recognize a novel epitope that has not been mapped before with a flavivirus monoclonal antibody.The four dengue virus serotypes (DENV-1 to DENV-4) constitute the dengue virus complex within the Flavivirus genus of the Flaviviridae. Dengue outbreaks and epidemics continue to pose a public health problem in most tropical and subtropical countries. Dengue illnesses range from mild dengue fever to severe dengue, characterized by dengue hemorrhagic fever and dengue shock syndrome, which has a high mortality rate. According to one estimate, approximately 50 to 100 million dengue virus infections and up to 250,000 dengue hemorrhagic fever cases occur every year worldwide (12, 34). Despite six decades of research, a safe and effective dengue vaccine has not been developed, nor is a specific, short-term preventive measure available. Currently, prevention of dengue is carried out by mosquito vector control, which is rather ineffective. Several other arthropod-borne flaviviruses are also important human pathogens, including the yellow fever virus (YFV), tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), and West Nile virus (WNV), which has recently emerged in North America (23,25). Vaccines against all of these viruses except WNV have been developed.The flavivirus genome contains a positive-strand RNA with one ...

Monoclonal antibody-mediated enhancement of dengue virus infectionin vitroandin vivoand strategies for prevention

“…This possibility was explored with DENV-4 antigenic variants of IgG 1A5 (11). A higher level of enhancement was detected in infection with DENV-4 v1 containing Leu 107 Phe substitution compared with the parental DENV-4.…”

“…This mutation moderately reduced the IgG 1A5-binding affinity (Ϸ10-fold), but there were sufficient immune complexes to facilitate infection of Fc receptor-bearing cells. The IgG 1A5-mediated enhancement was not observed with variant DENV-4 v2 containing Gly 106 Val substitution, which has a low binding affinity for the antibody (11). If the mutation in the flavivirus-conserved sequence proves to be a major determinant of an ADE epitope, such a DENV-4 variant can be explored to address vaccine safety related to ADE.…”

“…This possibility was explored by ADE analysis using DENV-4 antigenic variants containing Gly 106 Val or Leu 107 Phe substitution in E, which reduced their antibodybinding affinity (11 (Fig. 1C).…”

“…These antibodies remain detectable for a long period and rise rapidly during a subsequent heterotypic infection as a result of an anamnestic response. A major subset of these cross-reactive antibodies is directed to immuno-dominant epitopes involving determinants mapped to the flavivirus-conserved fusion peptide in the envelope glycoprotein (E) (11)(12)(13). The functional activities of these cross-reactive antibodies are not well characterized.…”

West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody

“…Indeed, the E glycoprotein is the principal antigen that elicits neutralizing antibodies against flaviviruses. Many of the neutralizing epitopes are located in DIII of E and in close proximity to the conserved fusion loop in DII (20)(21)(22)(23)(24)(25)(26)(27)(28). E16 is a strongly neutralizing mAb against WNV that recognizes four polypeptide loops at the tip of DIII and inhibits infection primarily subsequent to cell attachment.…”