A humanized IgG but not IgM antibody is effective in prophylaxis and therapy of yellow fever infection in an AG129/17D-204 peripheral challenge mouse model

Abstract

Yellow fever virus (YFV), a member of the genus Flavivirus, is a mosquito-borne virus found in tropical regions of Africa and South America that causes severe hepatic disease and death in humans. Despite the availability of effective vaccines, YFV is responsible for an estimated 200,000 cases and 30,000 deaths annually. There are currently no prophylactic or therapeutic strategies approved for use in human YFV infections. Furthermore, implementation of YFV 17D-204 vaccination campaigns has become problematic due to an increase in reported post-vaccinal adverse events. We have created human/murine chimeric MAbs of a YFV-reactive murine monoclonal antibody (mMAb), 2C9, that was previously shown to protect mice from lethal YFV infection and to have therapeutic activity. The new chimeric (cMAbs) were constructed by fusion of the m2C9 IgG gene variable regions with the constant regions of human IgG and IgM and expressed in Sp2 murine myelomas. The 2C9 cMAbs (2C9-cIgG and 2C9-cIgM) reacted with 17D-204 vaccine strain in an enzyme-linked immunosorbent assay and neutralized virus in vitro similarly to the parent m2C9. Both m2C9 and 2C9-cIgG when administered prophylactically 24h prior to infection protected AG129 mice from peripheral 17D-204 challenge at antibody concentrations ≥1.27 μg/mouse; however, the 2C9-cIgM did not protect even at a dose of 127 μg/mouse. The 17D-204 infection of AG129 mice is otherwise uniformly lethal. While the m2C9 was shown previously to be therapeutically effective in YFV-infected BALB/c mice at day 4 post-infection, the m2C9 and 2C9-cIgG demonstrated therapeutic activity only when administered 1 day post-infection in 17D-204-infected AG129 mice.

Fig. 1

Nucleotide and deduced amino acid sequences of m2C9 variable regions. (Panel a), Variable region of the κ-chain. (Panel b), Variable region of the γ-chain. +1 denotes start of mature protein; solid line box, CDR 1; dotted box, CDR 2; dashed box, CDR 3; the splice points of Jκ2 to Cκ (A) and of J_H2 to the C region gene (B) are indicated by the arrows.

Fig. 2

End-point titration of purified cMAbs. Symbols: 2C9-cIgG (●); 2C9-cIgM (◆); cIgG 1GD5 (alphavirus cross-reactive) (■); cIgM 1MD11 (alphavirus cross-reactive) (▲). IgM cMAbs were titrated in MAC-ELISA and the absorbance was measured at 450nm; IgG cMAbs were titrated in indirect ELISA and absorbance was measured at 405nm and titration results were superimposed. Insert: YFV neutralization end-point titres of MAbs m2C9, 2C9-cIgG, 2C9-cIgM, and cIgG 1GD5, expressed as PRNT₅₀ reciprocal end-points.

Fig. 3

Survival curves for antibody-treated mice. Panel a, survival of AG129 mice that were passively transferred intraperitoneally 127 μg of m2C9 (■), 2C9-cIgG (●), 2C9-cIgM (◆), or PBS (▲), 24 hours prior to i.p. challenge with 17D-204. Clinical signs began after 11 days p.i. so graphs show surviving animals from days 12 to 31, after which experiments were terminated. Panel b, survival of AG129 mice challenged i.p. with 17D-204 and administered i.p. 127 μg of m2C9 (■) or 2C9-cIgG (●) at 24 h or m2C9 (□) or 2C9-cIgG (○) 72 h p.i. A survival curve for PBS control mice is included for comparison (▲). Mice were monitored as in Panel a.