# Virus-like Particles and Spectral Flow Cytometry for Identification of Dengue Virus-Specific B Cells in Mice and Humans

## TL;DR

Researchers developed a new method using virus-like particles and spectral flow cytometry to identify dengue virus-specific B cells in mice and humans, offering insights into antibody responses during dengue infections.

## Contribution

A novel spectral flow cytometry assay using biotinylated virus-like particles to identify DENV-specific B cells in both mice and human patients.

## Key findings

- Biotinylated virus-like particles specifically bound to DENV-specific B cells and hybridomas.
- The method successfully identified DENV-specific plasmablasts in mice after secondary infection and in children with dengue.
- The approach can distinguish serotype-specific and cross-reactive B cell responses in primary and secondary infections.

## Abstract

Severe dengue virus (DENV) infections are associated with circulating non-neutralizing antibodies generated during heterotypic infections. Although antibodies are key mediators of both protection and pathogenesis, the specific dynamics of B cells (Bc) and their antibody responses remain insufficiently characterized due to limited methods of identifying DENV-specific Bc (DENV-Bc) and the absence of animal models resembling the human disease. Here, we developed a spectral flow cytometry assay employing biotinylated virus-like particles (VLPs) to detect DENV-Bc in C57BL/6 mice and children hospitalized with dengue. DENV-1 and DENV-2 VLPs were biotinylated, and the efficiency of biotin incorporation was assessed with an HABA-avidin assay and ELISA. Serotype specificity and optimal binding conditions were confirmed using hybridomas 4G2 (pan-flavivirus) and 3H5-1 (DENV-2 specific). Fluorescent agglutimers were subsequently generated by coupling biotinylated VLPs to streptavidin–fluorochrome complexes. Splenocytes from intraperitoneally DENV-infected mice and peripheral blood mononuclear cells (PBMCs) from naturally infected pediatric patients were stained with these VLPs and Bc-lineage markers. Biotinylated VLPs bound specifically to hybridomas, and this binding was competitively inhibited by unlabeled VLPs. After secondary DENV challenge, VLPs identified DENV-specific class-switched plasmablasts in mice. Circulating DENV-specific plasmablasts were also detected in children, with agglutimers enabling the discrimination of serotype-specific and cross-reactive responses in primary and secondary infections. This VLP-based approach represents a scalable platform to investigate the protective and pathogenic roles of DENV-Bc in infection and vaccination.