title: Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies.
abstract: Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013-2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of Biologics Evaluation and Research, with plaque reduction neutralization test performed by Focus Diagnostics, and with hemaglutination inhibition assay performed in-house at Sanofi Pasteur. Taken together, fADI assay appears to be a useful high throughput functional immunoassay for assessment of antibody-related neutralization of the viral infections for which pre-attachment neutralization pathway is predominant, such as polio, influenza, yellow fever and dengue.
authors: Sanofi Pasteur VaxDesign Campus, 2501 Discovery Dr. Suite 3000, Orlando, Florida 32826, United States of America.
authors: Atul
authors: A
authors: Asati
authors: Sanofi Pasteur VaxDesign Campus, 2501 Discovery Dr. Suite 3000, Orlando, Florida 32826, United States of America.
authors: Olga
authors: O
authors: Kachurina
authors: Sanofi Pasteur VaxDesign Campus, 2501 Discovery Dr. Suite 3000, Orlando, Florida 32826, United States of America.
authors: Alex
authors: A
authors: Karol
authors: Sanofi Pasteur VaxDesign Campus, 2501 Discovery Dr. Suite 3000, Orlando, Florida 32826, United States of America.
authors: Vipra
authors: V
authors: Dhir
authors: Sanofi Pasteur VaxDesign Campus, 2501 Discovery Dr. Suite 3000, Orlando, Florida 32826, United States of America.
authors: Michael
authors: M
authors: Nguyen
authors: Sanofi Pasteur VaxDesign Campus, 2501 Discovery Dr. Suite 3000, Orlando, Florida 32826, United States of America.
authors: Robert
authors: R
authors: Parkhill
authors: U. S. Food and Drug Administration, Center for Biologics Evaluation and Research, 10903 New Hampshire Avenue, Silver Spring, MD 20993, United States of America.
authors: Diana
authors: D
authors: Kouiavskaia
authors: U. S. Food and Drug Administration, Center for Biologics Evaluation and Research, 10903 New Hampshire Avenue, Silver Spring, MD 20993, United States of America.
authors: Konstantin
authors: K
authors: Chumakov
authors: Sanofi Pasteur VaxDesign Campus, 2501 Discovery Dr. Suite 3000, Orlando, Florida 32826, United States of America.
authors: William
authors: W
authors: Warren
authors: Sanofi Pasteur VaxDesign Campus, 2501 Discovery Dr. Suite 3000, Orlando, Florida 32826, United States of America.
authors: Anatoly
authors: A
authors: Kachurin
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000737:chemistry* / Q000276:immunology*; D000914:Antibodies, Viral / Q000737:chemistry* / Q000276:immunology*; D002522:Chlorocebus aethiops; D004912:Erythrocytes / Q000276:immunology; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008954:Models, Biological; D011054:Poliovirus Vaccine, Inactivated / Q000276:immunology; D014422:Turkeys; D014611:Vaccination; D014709:Vero Cells; D014765:Viral Vaccines / Q000276:immunology*; D053585:Virus Attachment
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D005456:Fluorescent Dyes; D011054:Poliovirus Vaccine, Inactivated; D014765:Viral Vaccines