Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay

Abstract

Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units.

FIG 1

Immunofluorescence analysis of MAb 3F4 to DENV-, JEV-, and YFV-infected Vero cells. (A) MAb 3F4 showed strong fluorescence with YF-17D virus-infected Vero cells but not with DENV-2- and JEV-infected Vero cells. (B) MAb 3F4 showed strong reactivity to wild-type YFV strains Baringo 1, Baringo 2, and 17D. Similarly, all seven of the remaining MAbs showed strong fluorescence with YFV strains and no fluorescence with DENV-2- or JEV-infected Vero cells (images not shown).

FIG 2

SDS-PAGE analysis of purified YFV-E protein fragments attached to MBP-fusion proteins. YFV-E protein fragments were expressed and purified as MBP-fusion proteins in E. coli. Lanes 1 to 6 indicate individual fragments; MW represents the prestained protein marker.

FIG 3

Epitope mapping of YFV MAbs. Indirect ELISA using YFV-E protein fragments as coating antigen was done to identify the epitopes of the generated YFV MAbs. Three MAbs (3F4, 4C9, and 4H10) showed strong reactivity to fragment DoIR1 (amino acids 1 to 51), while two MAbs (5B6 and 5H2) showed strong reactivity to fragment DoIIR1 (amino acids 52 to 135). Three MAbs (3B6, 4A1, and 8H3) did not show strong reactivity to any of the six fragments.

FIG 4

Antigen detection ELISA using newly developed YFV MAbs. (A) Antigen detection using YF vaccine virus 17D-infected culture fluid (YF-17D ICF). The antigen detection ELISA could detect up to 1.0 × 10³ FFU/ml of YF-17D virus. (B) The antigen detection ELISA could detect up to 2 ng of YFV-E protein. The dotted line represents the cutoff value (calculated as twice the mean absorbance value of the negative control). Uninfected Vero cell culture supernatants and PBS were used as negative controls for panels A and B, respectively.

FIG 5

Application of MAbs in IgM capture ELISA. (A and B) Six serially diluted YF vaccinee serum samples (Pos1 to Pos6) were analyzed using MAb 8H3 (A) or MAb 3F4 (B). (C and D) The sensitivity of the IgM capture ELISA was further verified using six serially diluted patient serum samples (Kd1 to Kd6) and analyzed with MAb 8H3 (C) and MAb 3F4 (D).