A method for detecting hepatitis C envelope specific memory B cells from multiple genotypes using cocktail E2 tetramers

Wu, Bing-Ruey; Eltahla, Auda A.; Keoshkerian, Elizabeth; Walker, Melanie R.; Underwood, Alex; Brasher, Nicholas A.; Agapiou, David; Lloyd, Andrew R.; Bull, Rowena A.

doi:10.1016/j.jim.2019.06.016

Cited by 17 publications

(23 citation statements)

References 46 publications

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“…Recombinant E2 (rE2) was prepared as described previously 33 . In brief, constructs containing the H77 rE2 (gt1a) were expressed in Freestyle‐HEK293 cells (Thermo Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

“…Analysis of HCV‐specific B cells in HESN and Incident subjects was undertaken as previously described 33 . In brief, cryopreserved PBMCs were thawed for flow cytometric staining with HCV E2–specific B cells detected using a rE2 cocktail tetramer with both gt1a (H77) and gt3a (UKN3A13.6) proteins, as well as anti‐CD10, anti‐CD20, anti‐CD38, anti‐IgD, anti‐CD19, anti‐CD27 and anti‐IgG antibodies.…”

Section: Methodsmentioning

confidence: 99%

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Anti‐envelope antibody responses in highly exposed seronegative individuals may be associated with protection from HCV infection

Mina

Underwood

Eltahla

et al. 2020

Journal of Viral Hepatitis

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In rare cases, individuals with a history of long‐term injecting drug use remain seronegative and aviraemic, despite prolonged and likely repeated exposure to Hepatitis C virus (HCV) through high‐risk behaviour. We describe anti‐HCV Envelope (E) antibody responses in a prospective cohort of carefully defined highly exposed but uninfected subjects (HESN) and comparison subjects who were also high risk and uninfected, but rapidly became HCV infected (Incident). Longitudinally collected samples from HESN cases (n = 22) were compared to Incident controls (n = 22). IgG, IgM and IgA from sera were tested by ELISA to genotype 1a and 3a E glycoproteins, and recombinant genotype 1a E2 antigen. IgG subclass isotyping was performed for those positive for IgG. Virus‐neutralizing activity was assessed on HCV pseudoparticles, and HCV E–specific B cells analysed using flow cytometry. A significant minority of HESN cases (n = 10; 45%) had anti‐E, predominantly in the IgG2 subclass, which was not found in the pre‐infection time point of the Incident cases (n = 1; 5%). A subset of the HESN subjects also had neutralizing activity and HCV‐specific B cells detected significantly more than Incident cases pre‐infection. In conclusion, the HESN phenotype is associated with IgG2 anti‐E antibodies, neutralization activity and HCV E–specific memory B cells. These findings suggest that HESN subjects may be resistant to HCV infection through humoral immune‐mediated mechanisms.

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“…Recombinant E2 (rE2) was prepared as described previously 33 . In brief, constructs containing the H77 rE2 (gt1a) were expressed in Freestyle‐HEK293 cells (Thermo Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Anti‐envelope antibody responses in highly exposed seronegative individuals may be associated with protection from HCV infection

Mina

Underwood

Eltahla

et al. 2020

Journal of Viral Hepatitis

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“…The tetramerization method was adapted from a previously published method used for hepatitis C virus tetramers 30…”

Section: Staining Rbd Memory B Cellsmentioning

confidence: 99%

“…Natively paired heavy and light chain variable (VH and VL) region sequences were obtained by amplifying the regions separately from single sorted B cells as previously described 30 . In brief, single sorted RBD-specific B cells (CD19 + CD20 + CD10 -RBD + )…”

Section: Production Of Monoclonal Antibodies From Single-sorted Rbd-smentioning

confidence: 99%

“…Amplicons of the B cell receptor (BCR)-encoding regions were generated from the SmartSeq2 libraries, as described previously 30,32 . Antibodies were generated by cotransfecting Lenti-X 293T cells (Clontech) with µg each of heavy and light chain expression cassette using 20 µL of Polyfect Transfection Reagent (Qiagen) and 600 µL of DMEM supplemented with 2 mM L-glutamine, and 10% heat inactivated fetal calf serum (Sigma).…”

Section: Production Of Monoclonal Antibodies From Single-sorted Rbd-smentioning

confidence: 99%

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Long-term persistence of neutralizing memory B cells in SARS-CoV-2

Bull

Abayasingam

Balachandran

et al. 2020

Preprint

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Considerable concerns relating to the duration of protective immunity against SARS-CoV-2 have been raised, with evidence of antibody titres declining rapidly after infection and reports of reinfection. Here we monitored antibody responses against SARS-CoV-2 receptor binding domain (RBD) for up to six months after infection. While antibody titres were maintained, half of the cohort’s neutralising responses had returned to background. However, encouragingly in a selected subset of 13 participants, 12 had detectable RBD-specific memory B cells and these generally increased out to 6 months. Furthermore, we were able to generate monoclonal antibodies with SARS-CoV-2 neutralising capacity from these memory B cells. Overall our study suggests that the loss of neutralising antibodies in plasma may be countered by the maintenance of neutralising capacity in the memory B cell repertoire.

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Development of a novel flow cytometry method for detecting pneumococcal‐specific B cells

et al. 2022

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Antigen-specific B cell identification by flow cytometry is crucial for investigating their immunophenotype, subset distribution, and kinetics post-infection or immunization. Methods using biotinylated polysaccharide antigens have been described, but there is still room for improvement regarding sensitivity and applicability. The aim of this study was the development and validation of a multimer bead-based method for detecting pneumococcal polysaccharide serotypes (PS)-specific B cells following pneumococcal immunization. PS was chemically biotinylated and mounted on anti-biotin beads, and labeled with phycoerythrin (PE)-conjugated anti-biotin antibody to form a PS-multimer used for cell staining. Labeled beads were washed to remove excess fluorochrome and diminish nonspecific labeling and background noise. Optimal ratios of PS-bead conjugate to PE and PS-multimer to cells were determined with titration assays. Comparison between the PSmultimer and a PS-PE monomer revealed enhanced detection of PS-specific cells and considerable signal amplification, attributed to the multimeric form of the detection probe and increased availability of antigen epitopes. To validate the specificity of the method, a competition assay using unbound PS was performed. Following pre-incubation with increasing PS concentrations, detection of PS-specific B cells with the PS-multimer was inhibited in a stepwise manner. Pre-incubation with excess PS completely blocked the fluorescent signal. This novel bead-based flow cytometry approach is a sensitive method demonstrating high specificity. It generated enhanced signals, provided clear-cut results, and was easily applicable, not requiring B cell pre-enrichment. It could be modified to adapt other antigens of interest, especially polysaccharides and proteins that could be used to probe antigen-specific B cell responses. The study of such responses may elucidate the underlying mechanisms involved in the establishment of long-term protection, provide evidence-based rationale for improving currently available vaccines and vaccination strategies, and pave the way for future vaccine development.

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A method for detecting hepatitis C envelope specific memory B cells from multiple genotypes using cocktail E2 tetramers

Cited by 17 publications

References 46 publications

Anti‐envelope antibody responses in highly exposed seronegative individuals may be associated with protection from HCV infection

Anti‐envelope antibody responses in highly exposed seronegative individuals may be associated with protection from HCV infection

Long-term persistence of neutralizing memory B cells in SARS-CoV-2

Development of a novel flow cytometry method for detecting pneumococcal‐specific B cells

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