Dengue viruses (serotypes 1-4) (DENV-1-4) and several other insect-borne flaviviruses, including Japanese encephalitis virus (JEV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV) are important human pathogens. Passive immunization using monoclonal antibodies (MAbs) from humans or nonhuman primates represents an attractive alternative to vaccine for prevention of infections. We have identified a number of flavivirus-reactive MAbs from infected chimpanzees by repertoire cloning, including DENV-4-specific MAb 5H2, JEV-specific MAbs A3, B2 and E3. These MAbs neutralize the respective virus at a high titer in vitro and protect against infection using a mouse model. MAb 5H2 has also been shown to protect against DENV-4 infection when passively administered to rhesus monkeys as human surrogates. MAb 5H2 recognizes an epitope involving determinants mapped to amino acids Lys174 and Pro176 in domain I of E. Epitope analysis of JEV-neutralizing MAb A2 has identified a determinant at Lys179 of JEV E, which aligns with Lys174 of DENV-4 E. Thus, these two antibodies recognize analogous epitopes, indicating that these epitopes elicit antibodies contributing importantly to host immunity. In 2009, crystals of the DENV-4 E/Fab 5H2 complex that diffracts X-ray well enough to collect data at 3.2 resolution have been obtained. Determination of the crystal structure has revealed information about the antibody-antigen contacts involving critical determinants at Lys174, Glu180 and Arg293 in E. Of interest is the involvement of Arg293 in the linker region between domain I and domain III which participates in the dimer-to-trimer shift during membrane fusion, suggesting that MAb 5H2 might interfere with this event in a manner similar to that of WNV-specific MAb E16, which recognizes domain III. The stage is set to further investigate the mechanisms of fusion inhibition by MAb 5H2 and other MAbs using liposomes in vitro. A phage library was also constructed from bone marrow of a TBEV-infected chimpanzee. Fab 2E6 was the most potent neutralizer among several Fabs isolated. Humanized MAb 2E6 neutralized TBEV at a titer of 0.02-0.04 μg/ml in the same range of highly-neutralizing MAbs against other flaviviruses isolated thus far. The major epitope determinant of MAb 2E6 was localized to Lys202 in E domain I by analysis of neutralization escape mutants. Titration of MAb 2E6 against intracerebral (ic) infection with 50 lethal dose50 (LD50) of TBEV/DENV-4 24 hr later showed a dose-dependent response in terms of the survival rate. The 50% protective dose of MAb 2E6 was calculated at 18 μg/ml per mouse. The protective capacity of MAb 2E6 was also analyzed using three-week-old Swiss-Webster mice against the TBEV-related LGTV by intra-peritoneal challenge. The peripheral route of infection is highly relevant to TBEV infection in humans. Using the mouse model, the 50% protective dose of MAb 2E6 against challenge with 50 LD50 of LGTV was calculated at 0.18 μg/ml per mouse or lower. Results also showed that MAb 2E6 administered at a single dose one day after LGTV infection had a 50% protective dose at 8.2 μg/ml per mouse. These experiments demonstrate proof of concept for the therapeutic potential of MAb 2E6 against TBEV infection.
| Anez, German; Men, Ruhe; Eckels, Kenneth H et al. (2009) Passage of dengue virus type 4 vaccine candidates in fetal rhesus lung cells selects heparin-sensitive variants that result in loss of infectivity and immunogenicity in rhesus macaques. J Virol 83:10384-94 |
| Rodrigo, W W Shanaka I; Block, Olivia K T; Lane, Christopher et al. (2009) Dengue virus neutralization is modulated by IgG antibody subclass and Fcgamma receptor subtype. Virology 394:175-82 |
