EFB Dengue Literature Review

Priyamvada2016 - Cross-Reactive Memory Plasmablasts in Secondary Dengue

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Priyamvada2016 - Cross-Reactive Memory Plasmablasts in Secondary Dengue

Full citation: Priyamvada L, Cho A, Onlamoon N, Zheng NY, Huang M, Kovalenkov Y, Chokephaibulkit K, Angkasekwinai N, Pattanapanyasat K, Ahmed R, Wilson PC, Wrammert J. B cell responses during secondary dengue virus infection are dominated by highly cross-reactive, memory-derived plasmablasts. J Virol. 2016;90(12):5574-5585. doi:10.1128/JVI.03203-15

Raw file: [[raw/priyamvada2016.pdf]]

Summary

This study provides the most detailed functional characterisation of plasmablast-derived monoclonal antibodies from acute secondary dengue infection. Four Thai patients with secondary DHF-grade DENV2 infection were sampled during acute disease (days 3–6 post-fever onset). Plasmablasts constituted 51–80% of peripheral B cells. The authors generated 53 mAbs from single-cell sorted plasmablasts and characterised their binding, neutralisation, and ADE activity against all four DENV serotypes.

The central finding is that secondary dengue plasmablasts are highly affinity-matured (average 18.1 VH mutations, range 5–39), with CDR R:S ratios >2.9 indicating antigenic selection, and 23% clonal relatedness — all strongly supporting memory B cell origin. The mAb panel revealed that 70% of mAbs targeted envelope protein (E), with all E-reactive mAbs being cross-reactive to 2–4 serotypes. Critically, 45/53 mAbs enhanced DENV infection by ADE regardless of neutralisation potency, and several mAbs from two patients preferentially neutralised DENV1 over the infecting DENV2 — evidence of original antigenic sin (OAS) in the acute B cell response.

This paper is from the Wrammert laboratory and directly extends Wrammert2012 - Plasmablast Responses in Acute Dengue by adding mAb functional data and BCR repertoire analysis to the magnitude/kinetics data from the earlier study. It uses the same Siriraj Hospital (Bangkok) clinical platform.

Study Design

  • Type: Observational cohort with single-cell mAb cloning
  • Sample size: n=4 patients; 53 mAbs generated
  • Setting: Siriraj Hospital, Bangkok, Thailand; hospitalised DHF patients
  • Population: Adults (ages 14–58), all secondary DENV2 infection (IgM/IgG ratio <1.7), all clinically diagnosed DHF by WHO 1997 criteria. Blood collected days 3–6 post-fever onset.

Key Findings

  • Massive plasmablast expansion in secondary DHF: Plasmablasts (CD19⁺CD3⁻CD20⁻/low CD27^high CD38^high) constituted 51–80% of peripheral CD19⁺ B cells across all four patients (days 3–6 post-fever onset). Cells were primarily IgG-secreting and largely DENV-specific by ELISpot (36–95% of total IgG ASCs were DENV-specific).
  • High SHM strongly supports memory origin: Per-patient average VH mutations ranged from 14.5 to 21.7 (overall mean 18.1, range 5–39 per sequence). SHM was significantly higher than IgG⁺ GC B cells (p<0.005) and comparable to influenza recall responses. CDR R:S ratios >2.9 in all four patients confirmed antigenic selection.
  • Clonal expansions in all donors: 23% of all plasmablast VH sequences were clonally related (range 15–28% per patient), further supporting recall of pre-existing memory clones.
  • E protein is the dominant target: 70% of mAbs (37/53) were E-specific (31 by rE ELISA, 6 additional by Western blot). All rE-binding mAbs were cross-reactive to ≥2 serotypes; no serotype-specific rE binding was observed.
  • Cross-neutralisation dominates: 46/53 mAbs neutralised at least one DENV serotype by FFA; 34/46 (74%) were cross-neutralising (2–4 serotypes). DENV4 was least neutralised (20 mAbs).
  • Binding and neutralisation are dissociated: Of 26 mAbs that bound all 4 rE proteins, only 6 neutralised all 4 serotypes. Conversely, 15 mAbs that did not bind rE still neutralised virus — indicating virion-dependent conformational epitopes (E dimer interface) that are absent on monomeric rE.
  • Nearly universal ADE: 45/53 mAbs enhanced DENV infection of U937 cells at 1 µg/ml (5–161-fold enhancement). ADE was independent of neutralisation potency — even the most potently neutralising mAbs caused ADE. Cross-reactive neutralising mAbs caused greater average fold-enhancement than mono-neutralising mAbs. mAb 31.3F03, which neutralised only DENV2, exhibited the highest cross-serotypic ADE in the panel.
  • Original antigenic sin (OAS): In 2/4 patients (Pt. 32, Pt. 33), mAbs preferentially neutralised DENV1 over the infecting DENV2. DENV1-specific mAbs had stronger neutralisation potency (median FRNT₅₀ = 0.16 µg/ml) than DENV2-specific mAbs (median FRNT₅₀ = 1.2 µg/ml). DENV1-specific mAbs did not bind rE but showed clear DENV1 preference by capture virus ELISA — they recognise conformational epitopes. Low-level DENV2 binding was detected, explaining their activation during DENV2 infection. This OAS phenotype was also reflected in serum neutralisation titres.
  • Serum neutralisation: All four patients had high FRNT₅₀ titres against DENV2 and DENV1; DENV3 and DENV4 titres were lower and more variable.

Methods Used

  • Conventional Flow Cytometry (5-color: CD19-FITC, CD38-PE, CD3-PerCP, CD20-PerCP, CD27-APC)
  • FACS Sorting (single-cell sort into 96-well plates using FACSAria III)
  • ELISpot (DENV-specific and total IgG/IgM/IgA)
  • BCR Sequencing (single-cell RT-PCR of VH and VL, IMGT analysis, clonality and SHM quantification)
  • FRNT (focus-forming assay; FRNT₅₀ against DENV1-4)

Entities Mentioned

Concepts Addressed

Relevance & Notes

This paper creates a critical interpretive tension with GodoyLozano2016 - Lower IgG SHM Rates in Acute Dengue. Priyamvada2016 finds high SHM (mean 18.1 VH mutations ≈ ~6.5% nucleotide mutation) in sorted plasmablasts from secondary DHF, while GodoyLozano2016 finds globally low SHM in bulk IgG⁺ B cells from a mix of primary and secondary infections. The reconciliation likely involves two populations: (1) memory-derived plasmablasts with high SHM that dominate secondary infections (captured by Priyamvada2016’s sorted-PB approach); and (2) de novo naive-recruited plasmablasts with low SHM that may be more prominent in primary infections or in the non-DENV-specific fraction (captured by GodoyLozano2016’s bulk approach). Both pathways — memory recall and de novo EF differentiation — likely operate concurrently, with their relative contributions depending on infection history.

The OAS finding is directly relevant to the ADE hypothesis: if secondary infection preferentially recalls memory B cells specific for the prior serotype, the resulting antibodies may cross-react with the current serotype at sub-neutralising concentrations — the precise condition for ADE. This connects the Tph→memory B cell→plasmablast axis (Ansari2025) to a specific immunopathological mechanism.

Limitations: Only 4 patients, all secondary DHF DENV2 — no primary infections, no mild cases, no other serotypes. The prior serotype exposure was not formally determined (inferred from neutralisation bias). The ADE assay used a single mAb concentration (1 µg/ml) — concentration-dependent ADE kinetics were not explored.

Questions Raised

  • Does OAS operate differently in primary vs. secondary dengue? Priyamvada2016 cannot address this (all secondary).
  • What fraction of the high-SHM plasmablasts in secondary dengue originated from GC-matured memory vs. EF-matured memory? Priyamvada’s SHM data are consistent with GC-experienced memory recall, but do not exclude EF-matured memory clones.
  • Are the DENV1-biased (OAS) mAbs the specific subset that mediates ADE in secondary DENV2 infection? They bind DENV2 weakly and do not neutralise it — exactly the profile predicted to enhance.
  • Is the near-universal ADE in vitro (45/53 mAbs) clinically meaningful, or does the U937 assay at 1 µg/ml overestimate in vivo enhancement?
  • How do the SHM levels here compare to what would be found in primary dengue plasmablasts? If primary PBs show low SHM (as GodoyLozano2016 suggests for the bulk IgG pool), this would confirm the memory-recall vs. de novo EF dichotomy.

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