Analysis of Cross-Reactive Antibodies Recognizing the Fusion Loop of Envelope Protein and Correlation with Neutralizing Antibody Titers in Nicaraguan Dengue Cases

Abstract

Dengue virus (DENV) is the leading cause of arboviral diseases in humans worldwide. The envelope (E) protein of DENV is the major target of neutralizing antibodies (Abs). Previous studies have shown that a significant proportion of anti-E Abs in human serum after DENV infection recognize the highly conserved fusion loop (FL) of E protein. The role of anti-FL Abs in protection against subsequent DENV infection versus pathogenesis remains unclear. A human anti-E monoclonal Ab was used as a standard in a virion-capture ELISA to measure the concentration of anti-E Abs, [anti-E Abs], in dengue-immune sera from Nicaraguan patients collected 3, 6, 12 and 18 months post-infection. The proportion of anti-FL Abs was determined by capture ELISA using virus-like particles containing mutations in FL, and the concentration of anti-FL Abs, [anti-FL Abs], was calculated. Neutralization titers (NT50) were determined using a previously described flow cytometry-based assay. Analysis of sequential samples from 10 dengue patients revealed [anti-E Abs] and [anti-FL Abs] were higher in secondary than in primary DENV infections. While [anti-FL Abs] did not correlate with NT50 against the current infecting serotype, it correlated with NT50 against the serotypes to which patients had likely not yet been exposed (“non-exposed” serotypes) in 14 secondary DENV3 and 15 secondary DENV2 cases. These findings demonstrate the kinetics of anti-FL Abs and provide evidence that anti-FL Abs play a protective role against “non-exposed” serotypes after secondary DENV infection.

Complexity of Neutralizing Antibodies against Multiple Dengue Virus Serotypes after Heterotypic Immunization and Secondary Infection Revealed by In-Depth Analysis of Cross-Reactive Antibodies

“…1C and E) representing the other two sequences of DENV immunizations in this study (Table 1). This trend is in agreement with the notion that anti-E Abs are the major Ab response and GR anti-E Abs are generated after primary and secondary DENV infections (17,21,42,44). Notably, the strongest anti-E or anti-prM Abs after secondary immunization were against the primary immunization serotype in all vaccinees tested ( Fig.…”

“…Postdepletion sera were examined by virion ELISA at a 1:2,000 dilution (A) and by FRNT (C). A human anti-E GR MAb (82.11LALA) was used to verify that comparable amounts of DENV and WNV antigens were loaded (B) (25,44). Data are means and standard deviations from two experiments, each with duplicates.…”

“…DENV virions derived from ultracentrifugation of culture supernatants of virus-infected Vero cells or pellets of mock-infected Vero cells were UV inactivated (48), diluted in coating buffer, and loaded on a flat-bottom 96-well plate at 4°C overnight, followed by blocking and incubation with primary Abs (serum at a 1:2,000 dilution) and secondary Abs. After a final wash and incubation with tetramethylbenzidine substrate and stop solution, the optical density at 450 nm (OD 450 ) was read, with a reference wavelength of 650 nm (23,44). IgG avidity was determined in a modified enzyme-linked immunosorbent assay (ELISA) with an additional washing step with 8 M urea (49)(50)(51).…”

“…Our recent studies showed that GR anti-E MAbs derived from patients after secondary DENV infection had higher binding avidities and neutralizing potencies than those from patients after primary DENV infection (23). Moreover, the concentrations of GR Abs in sera correlated with the neutralizing titers against the nonexposed serotypes after secondary DENV infection (44). Based on these observations, we hypothesize that cross-reactive anti-E Abs (including GR and CR Abs) generated after secondary DENV infection have high avidity and potent neutralizing actviites, and they contribute to the neutralizing activities against the nonexposed serotypes.…”

Virus-Like Particle Secretion and Genotype-Dependent Immunogenicity of Dengue Virus Serotype 2 DNA Vaccine

“…It also possesses important flavivirus epitopes that elicit protective neutralizing (Nt) antibody responses (11,12). Antibodies against prM and the tip of EDII, which contains a highly conserved sequence (i.e., the fusion peptide loop), are immunodominant and believed to constitute a major portion of the total IgG population in the sera of humans naturally infected by dengue viruses (13)(14)(15).…”