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Anti-Budding Assay: Development of a high throughput screen to identify neutralizing antibodies that block Chikungunya virus (CHIKV) release
Ramjag, A; Lu, K; Carrington, C V F; Simmons, G; Jin, J.
Afiliação
  • Ramjag, A; The University of the West Indies. Faculty of Medical Sciences. St. Augustine. TT
  • Lu, K; The University of the West Indies. Faculty of Medical Sciences. St. Augustine. TT
  • Carrington, C V F; The University of the West Indies. Faculty of Medical Sciences. Department of Preclinical Sciences. St. Augustine. TT
  • Simmons, G; The University of the West Indies. Faculty of Medical Sciences. St. Augustine. TT
  • Jin, J; Blood Systems Research Institute. San Francisco, CA. US
In. The University of the West Indies, Faculty of Medical Sciences. Faculty of Medical Sciences, Research Day. St. Augustine, Caribbean Medical Journal, March 21, 2019. .
Non-conventional em En | MedCarib | ID: biblio-1024589
Biblioteca responsável: TT5
ABSTRACT

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.
Assuntos
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Coleções: 01-internacional Base de dados: MedCarib Assunto principal: Vírus Chikungunya Tipo de estudo: Prognostic_studies Limite: Female / Humans / Male País/Região como assunto: Caribe ingles / Trinidad y tobago Idioma: En Ano de publicação: 2019 Tipo de documento: Non-conventional País de publicação: Trinidad e Tobago
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Coleções: 01-internacional Base de dados: MedCarib Assunto principal: Vírus Chikungunya Tipo de estudo: Prognostic_studies Limite: Female / Humans / Male País/Região como assunto: Caribe ingles / Trinidad y tobago Idioma: En Ano de publicação: 2019 Tipo de documento: Non-conventional País de publicação: Trinidad e Tobago