Characterization of B-cell Responses to Zika Virus (ZIKV)

Objective: Most ZIKV infections occur in regions endemic for the related dengue virus (DENV). Anti-DENV antibodies have been demonstrated to cross-react with ZIKV. Some neutralize ZIKV infection while others mediate antibody-dependent enhancement (ADE), exacerbating ZIKV infection and complicating diagnosis of the etiologic agent. We aimed to characterize the humoral immune response in a ZIKV+, DENV- experienced individual in order to explore this anamnestic response and identify antibodies that may be useful in the development of therapeutic agents. Design and Methodology: Peripheral blood mononuclear cells (PBMCs) were collected from an individual (TT66) who was newly infected with ZIKV but had two previous DENV infections. Plasmablasts were isolated and analyses conducted using Atreca's Immune Repertoire CaptureTM technology. Monoclonal antibodies (mAbs) derived from TT66 during their acute and convalescent phase of ZIKV infection were screened in vitro for ZIKV and DENV binding and neutralization activity. Epitopes were then mapped using a shotgun mutagenesis approach. Results: We observed clonal expansion of two distinct antibody lineages representing 70% of total immunoglobulin sequences from TT66. We screened 18 mAbs representing two major lineages and five smaller families for neutralization and ADE between DENV and ZIKV. No highly typespecific mAbs were observed but rather a diverse pattern of neutralization, even within an individual lineage. Shotgun mutagenesis epitope mapping demonstrated epitopes for two of these broadly neutralizing mAb lineages lay within domain II ofE, close to the fusion loop. Conclusions: Results suggest that neutralizing antibody responses to ZIKV are extensively shaped by previous DENV exposure.

Anti-Budding Assay: Development of a high throughput screen to identify neutralizing antibodies that block Chikungunya virus (CHIKV) release

Objective: No licensed CHIKV vaccines or effective therapeutic agents are currently available. However, some CHIKV-specific monoclonal antibodies (mAbs) are highly effective in animal models, both prophylactically and therapeutically. This is thought to be largely mediated via blocking of CHIKV entry into cells. However, we and others have shown that inhibition of viral release/ budding is also a major mechanism for CHIKV control. We aimed to develop a high throughput in vitro screening assay to efficiently identify "antibudding"mAbs. Design and Methodology: An assay for quantification of viral budding from infected cells was optimised by varying cell line, cell density, multiplicity of infection (MOI), incubation periods, NH4Cl concentration and plate type. The assay utilized our novel, fully replication -competent, attenuated CHIKV nano-luciferase (nluc) reporter virus (CHIKV 181/25 E2nluc). The optimised assay was used to screen CHIKV+, Zika virus (ZIKV)+, CHIKV-/ZIKV- sera, and cloned memory B-cells from a CHIKV+ individual. Results: Optimal conditions involved use of rhabdomyosarcoma (RD) cells, bulk-infected at MOI 1 for 2hrs, removal of residual virus, resuspension in media containing 20mM NH4Cl, seeding at 2.5x104cells/well into 96-well plates and luminometry after 18hrs. Inter-plate coefficient of variability CV scores and Z' values remained <15% and >0.5 respectively, indicative of a valid assay. Most CHIKV+ sera displayed potent antibudding activity, two displayed no significant activity, and there was no ZIKV cross-reactivity. Of 800 memory B-cell clones, 13 exhibited significant anti-budding antibody activity. Conclusions: We developed a sensitive, reproducible, Biosafety level (BSL-2) safe, high throughput CHIKV antibudding assay useful for screening both polyclonal sera and monoclonal antibodies.