Multiplexed fluorospot for the analysis of dengue virus- And zika virus-specific and cross-reactive memory B cells

Awadalkareem Adam, Marcia Woda, Sonia Kounlavouth, Alan L. Rothman, Richard G. Jarman, Josephine H. Cox, Julie E. Ledgerwood, Gregory D. Gromowski, Jeffrey R. Currier, Heather Friberg, Anuja Mathew

Research output: Contribution to journalArticlepeer-review


Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne pathogens that have a significant impact on human health. Immune sera, mAbs, and memory B cells (MBCs) isolated from patients infected with one DENV type can be cross-reactive with the other three DENV serotypes and even more distantly related flaviviruses such as ZIKV. Conventional ELISPOTs effectively measure Ab-secreting B cells but because they are limited to the assessment of a single Ag at a time, it is challenging to distinguish serotype-specific and cross-reactive MBCs in the same well. We developed a novel multifunction FluoroSpot assay using fluorescently labeled DENV and ZIKV (FLVs) that measures the cross-reactivity of Abs secreted by single B cells. Conjugation efficiency and recognition of FLVs by virus-specific Abs were confirmed by flow cytometry. Using a panel of DENV immune, ZIKV immune, and naive PBMC, FLVs were able to simultaneously detect DENV serotype-specific, ZIKV-specific, DENV serotype cross-reactive, and DENV/ZIKV cross-reactive Abs secreted by individual MBCs. Our findings indicate that the FLVs are sensitive and specific tools to detect specific and cross-reactive MBCs. These reagents will allow the assessment of the breadth as well as the durability of DENV/ZIKV B cell responses following vaccination or natural infection. This novel approach using FLVs in a FluoroSpot assay can be applied to other diseases such as influenza in which prior immunity with homosubtype- or heterosubtype-specific MBCs may influence subsequent infections. The Journ Al of Immunology, 2018, 201: 3804-3814.

Original languageEnglish (US)
Pages (from-to)3804-3814
Number of pages11
JournalJournal of Immunology
Issue number12
StatePublished - Dec 15 2018
Externally publishedYes

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology


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