Human antibodies in Mexico and Brazil neutralizing tick-borne flaviviruses

Abstract

Flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV) are spread by mosquitoes and cause human disease and mortality in tropical areas. In contrast, Powassan virus (POWV), which causes severe neurologic illness, is a flavivirus transmitted by ticks in temperate regions of the Northern hemisphere. We find serologic neutralizing activity against POWV in individuals living in Mexico and Brazil. Monoclonal antibodies P002 and P003, which were derived from a resident of Mexico (where POWV is not reported), neutralize POWV lineage I by recognizing an epitope on the virus envelope domain III (EDIII) that is shared with a broad range of tick- and mosquito-borne flaviviruses. Our findings raise the possibility that POWV, or a flavivirus closely related to it, infects humans in the tropics.

Keywords: CP: Immunology; antibodies; flaviviruses; tick diseases.

Figure 1.. Serum neutralizing activity against POWV in Mexico and Brazil

(A and B) Screening for serum IgG antibodies binding to the EDIII of POWV (lineage II, A) and TBEV (European subtype, B) in ELISA. Each dot represents the IgG reactivity of an individual participant at 1:500 serum dilution (single well). Samples with optical density above 0.6 (dotted lines) were selected for the neutralization screening in C and D. (C and D) Screening of select sera (n=66) for neutralization of RVPs corresponding to POWV (lineage II, C) and TBEV (European subtype, D). Shown is the rank-ordered luciferase activity relative to “no-serum” control (lower values correspond to higher neutralization). Each bar represents an individual participant sample analyzed at 1:1,200 dilution; average reading of duplicate wells. (E and F) ELISA IgG binding to POWV (E) and TBEV (F) EDIII by serial serum dilutions. Representative of two experiments. (G and H) Neutralization of POWV (G) and TBEV (H) RVPs by serial serum dilutions. Luciferase (Luc) activity relative to “no-serum” control. Mean ± SD of triplicates. Representative of two experiments. (I) Neutralization of authentic POWV lineage I and lineage II by MEX58 serum using a microscopy-based neutralization assay. One experiment, mean ± SD of triplicates. In A to D: black, dark grey and light grey are samples from New York, Brazil, and Mexico, respectively. In red is MEX58, in blue a sample from a TBEV vaccinated individual. The p values in A to D were determined with the Mann-Whitney test.

Figure 2.. Identification of human monoclonal antibodies binding to POWV EDIII from a resident of Mexico

(A) POWV-EDIII binding antibodies from the memory B cells of MEX58. Top, flow cytometry plot identifying human B cells binding to POWV EDIII proteins corresponding to lineage I and II (gate). The frequency of antigen-binding B cells is shown. Bottom, pie chart showing the distribution of the 31 antibodies. Antibodies that share the same immunoglobulin heavy and light chain genes are represented by the colored slices, singlets are in white. (B) Monoclonal antibody binding to the EDIII of the indicated flaviviruses by ELISA. Z039 is a ZIKV antibody that broadly binds EDIII of all four DENV serotypes and was tested alongside as positive control. Average of two independent experiments.

Figure 3.. P002 and P003 human monoclonal antibodies are broadly cross-reactive to flaviviruses and neutralize POWV

(A and B) ELISA binding of recombinant human monoclonal IgG antibodies P002 and P003 to the EDIII of flaviviruses transmitted by ticks or Aedes mosquitoes (A) and Culex mosquitoes (B). Representative of two independent experiments. (C) Neutralization of POWV lineage I by P002 and P003 antibodies. Neutralization was determined with the PRNT assay and values normalized to no antibody control. Two experiments (mean ± SD). In A: KFDV is Kyasanur forest disease virus, OHFV is Omsk hemorrhagic fever virus, LIV is louping ill virus, LGTV is Langat virus. In B: JEV is Japanese encephalitis virus, MVEV is Murray Valley encephalitis virus, SLEV is Saint Louis encephalitis virus, USUV is Usutu virus.

Figure 4.. P003 recognizes an epitope away from the EDIII lateral ridge

(A) Sandwich ELISA shows that P002 and P003 recognize an EDIII epitope distinct from previously reported ZIKV antibodies. His-tagged Fab antibody fragments were assessed for binding to ZIKV EDIII immobilized by a specific IgG. Binding of the Fab suggests a non-overlapping epitope with the IgG; no binding suggests a similar or overlapping epitope with the IgG. Mean ± SD of quadruplicates. (B) Model of the P003-ZIKV EDIII complex from NMR-validated computational simulations. EDIII residues affected by antibody binding are in green on the surface of EDIII (PDB ID: 5OMZ). P003 is in orange cartoon, with the light chain in lighter tones. See also Figures S3A and S3B. (C) Structure of ZIKV EDIII (white) with selected amino acids colored according to the EC₅₀ value of P003 binding to the EDIII with the amino acid change shown in panel D. Contours indicate the P003 epitope. (D) Binding of P003 and P002 to ZIKV EDIII mutated at selected epitope residues, assessed by ELISA. Summary of EC₅₀ binding values. Z015 is a ZIKV antibody binding to a distinct epitope. See also Figures 4C and S3C. (E) P003 (orange) binds to a different EDIII region than Z021 (blue) or Z006 (pink). (F and G) Same as in C and D for validation of the model using POWV (lineage I) EDIII wild type and mutant proteins. T036 is a TBEV antibody that also binds POWV and recognizes a distinct epitope. See also Figure S3E.

Figure 5.. The P003 epitope on the EDIII is highly conserved across flaviviruses, except DENV

(A) Alignment of the P003 contact residues with the sequences of the EDIII proteins used in ELISA (Figures 2 and 3). Highlighted in green are the identical residues and in yellow those that are similar by side chain functionality to the corresponding residues in POWV. Arrow points to P003 epitope at position 329 (ZIKV numbering). Related to Figure S4A. (B) Coldmaps representing on the ZIKV EDIII the frequency of identical or similar residues at the P003 epitope (dashed contour) across the flaviviruses recognized by P003 and indicated in panel A. (C) Rescue of P002 and P003 binding by the K321E substitution in DENV3 (equivalent to E329_ZIKV). Average of two independent ELISA experiments.