In-depth analysis of the antibody response of individuals exposed to primary dengue virus infection

Abstract

Humans who experience a primary dengue virus (DENV) infection develop antibodies that preferentially neutralize the homologous serotype responsible for infection. Affected individuals also generate cross-reactive antibodies against heterologous DENV serotypes, which are non-neutralizing. Dengue cross-reactive, non-neutralizing antibodies can enhance infection of Fc receptor bearing cells and, potentially, exacerbate disease. The actual binding sites of human antibody on the DENV particle are not well defined. We characterized the specificity and neutralization potency of polyclonal serum antibodies and memory B-cell derived monoclonal antibodies (hMAbs) from 2 individuals exposed to primary DENV infections. Most DENV-specific hMAbs were serotype cross-reactive and weakly neutralizing. Moreover, many hMAbs bound to the viral pre-membrane protein and other sites on the virus that were not preserved when the viral envelope protein was produced as a soluble, recombinant antigen (rE protein). Nonetheless, by modifying the screening procedure to detect rare antibodies that bound to rE, we were able to isolate and map human antibodies that strongly neutralized the homologous serotype of DENV. Our MAbs results indicate that, in these two individuals exposed to primary DENV infections, a small fraction of the total antibody response was responsible for virus neutralization.

Figure 1. Antigens recognized by hMAbs produced from donor 033.

DENV3 virions were purified and the viral proteins were separated by SDS-polyacrylamide gel electrophoresis. Western blots were performed to identify the viral antigens recognized by hMAbs from donor 033. The figure displays Western blot results for selected hMAbs that bound to E protein (64.31) and prM protein (DV38.1, 59.3, 65.5, 4.3, 11.12, and 18.5). 4G2 and 2H2 are control mouse MAbs that bind E and prM proteins, respectively.

Figure 2. DENV3 neutralization by donor 033 human MAbs.

The 50% neutralization titers against DENV3 were determined for all 16 hMAbs from donor 033 using a flow cytometry based neutralization test that utilizes U937 cells expressing DC-SIGN. (A) Dengue antigen recognized and the 50% neutralization titer for each hMAb. The hMAbs for which a specific antigen has still not been identified are designated with a *?*. (B) Neutralization curves for three representative prM antibodies. Note that prM antibodies have shallow neutralization curves, which plateau when ∼60% of the virions are neutralized. (C) Neutralization curve of the only E protein reactive antibody obtained from donor 033. This antibody has a steeper neutralization curve compared to prM antibodies.

Figure 3. Binding and neutralization properties of donor 013 hMAbs.

Ten hMAbs from donor 013 were tested for binding dengue virus, recombinant E (rE) and EDIII from DENV2. Cross-reactivity was determined by using whole virus antigen from all four serotypes. For each hMAb the 50% neutralization titer was determined using flow cytometry and U937 cell expressing DC-SIGN. (A) Summary of the binding and neutralization data for all hMAbs from donor 013. B, C and D display representative neutralization curves for DENV2 type-specific (B), subcomplex- specific (C) and complex-specific (D) hMAbs.

Figure 4. Epitope mapping of anti-DENV2 hMAbs binding to EDIII.

To identify antibody binding sites, DENV2 was serially passaged in the presence of neutralizing hMAbs DV3.7 or DV10.16. Viruses growing in the presence of hMAbs were plaque purified and expanded. Neutralization escape was confirmed by growing the parental and the cloned antibody selected viruses in the presence of each hMAb 3.7 (A) or hMAb 10.16 (B). (C) Localization of neutralizing human antibody epitopes on the structure of DENV2 EDIII (strain 16681) using residues identified by neutralization escape selection (blue) or yeast surface display screening (see Table 3) (orange). Ribbon diagram of DENV-2 EDIII was generated from a published X-ray crystallographic structure. The disulfide bond is highlighted in yellow. Human MAbs 3.7 and 25.5 are type-specific antibodies that bind to epitopes centered on the lateral ridge while 10.16 is sub-complex-specific and bind to an epitope centered on the A strand of EDIII.