Cross-reacting antibodies enhance dengue virus infection in humans

Abstract

Dengue virus co-circulates as four serotypes, and sequential infections with more than one serotype are common. One hypothesis for the increased severity seen in secondary infections is antibody-dependent enhancement (ADE) leading to increased replication in Fc receptor-bearing cells. In this study, we have generated a panel of human monoclonal antibodies to dengue virus. Antibodies to the structural precursor-membrane protein (prM) form a major component of the response. These antibodies are highly cross-reactive among the dengue virus serotypes and, even at high concentrations, do not neutralize infection but potently promote ADE. We propose that the partial cleavage of prM from the viral surface reduces the density of antigen available for viral neutralization, leaving dengue viruses susceptible to ADE by antibody to prM, a finding that has implications for future vaccine design.

Figure 1. Specificity of human antibodies (n=301)

Western Blot of infected cell lysates (non-reduced) showing reactivity of antibodies with dengue NS1, E and prM proteins (A). Cross-reactivity of human mAb within the DENV serotypes (B-D) or between the DENV group and JEV (E-G)

Figure 2. Partial neutralisation but potent enhancement by human anti-prM monoclonal antibodies

Neutralisation assays (A) and enhancement assays (B) were performed with the six human anti-prM mAbs (clones 3-147, 58/5, 2F5, 2G4, 5F9 and 135.3), murine anti-E mAb (4G2) and purified Ig from pooled dengue convalescent serum (PCS) and pooled non dengue immune serum (PND) were used as controls; mean±SE from three independent experiments.

Figure 3. Anti-prM can rescue infectivity in virus containing high densities of prM

DENV produced in the presence of NH₄Cl. The density of uncleaved prM and E were measured by ELISA and expressed as the prM:E ratio (A). Infectivity was determined in Vero cells, expressed as FFU, and the amount of total virus-equivalent particles was calculated based upon the concentration of E protein measured by a sensitive sandwich ELISA. Data is presented as virus-equivalent particles/FFU ratio (B). Neutralisation assays with purified human anti-prM mAb (3-147) (C). Enhancement assays of U937 cells read out by FACS based intracellular staining for DENV antigens (4G2) using either a constant amount of infectious virus (D) or constant number of virus particles (E); mean±SE from three independent experiments.

Figure 4. The roles of anti-prM on neutralisation and enhancement of DENV infection of PBMC

PBMC were infected with DENV2 in the presence of human anti-prM mAbs, at 24hrs DENV Ag was stained intracellularly (4G2) and detected by flow cytometry in gated monocytes (A). PCS, PND and irrelevant human mAb were used as control. The density of prM on DENV from C6/36 cells and DC were detected by ELISA and presented as prM:E ratio (B). Neutralisation (C) and antibody-dependent enhancement of infection (D), performed on Vero and U937 cells respectively, of DENV generated from either C6/36 cells or DC in the presence of PCS or anti-prM mAb (3-147); mean±SE from three independent experiments.