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.

Fig 1. Schematic representation of fADI neutralization assay.

Incubation of virus with sera (upper arm) or without sera (lower arm) either blocks or does not block virus attachment to target cells, respectively. Virus attached to the cells is detected by virus specific antibody and fluorescent tag SA-PE followed by additional staining cells with diluted Trypan blue and finally reading on Bioplex. The maximum fluorescent signal without serum corresponds to zero blocking, (lower arm), reduced fluorescent signal indicating blocking of the virus with serum antibody (upper arm).

Fig 2. Schematic representation of Control ELISA to check for false positive effects in the fADI neutralization assay.

Virus is added to ELISA plates instead of using target cells to check for any false positive readings. Addition of sera (upper arm) or no sera (lower arm) should produce similar reporter signals (fluorescence) indicating the absence or low level of competition of virus-specific labeling antibody with antibodies in test serum. The plates are read on the Synergy HT plate reader (BioTek) after applying the secondary detecting antibody and the fluorescent tag SA-PE. More details are in the text.

Fig 3. Proof of principle study for the fADI assay against different classes of viruses.

fADI displayed a significant sera concentration dependent blocking of virus attachment for polioviruses of serotyopes I, II and III (A), Dengue I, II, III and IV (C), Yellow Fever (E) and influenza California H1N1viruses (G) to Vero cells. The sera used in the demo experiments were standard hyper-immune sera specific to the corresponding viruses. Control ELISA (B, D, F and H) tests with those standard sera did not display any significant reduction in fluorescent signal thus showing very low or no competition between the sera antibodies and virus specific labeling antibodies used in the assay.

Fig 4. fADI screening of pre- and post-vaccinated serum samples from donors immunized with IPOL^® vaccine.

The screening was performed in the single dilution mode rather than using sera titration. Donor-to-donor variation in the capacity of donor sera to block virus attachment was observed for all three poliovirus serotypes (A, B and C). The control ELISA tests (D, E and F) did not display significant reduction of the reporter fluorescence signal, thus showing very low or zero competition between test serum antibodies and virus-specific labeling antibodies used in the assay.

Fig 5. Correlation between fADI and MN methods for Polio I, II and III.

fADI₅₀ were calculated from the Virus Attachment Indexes using a mathematical approximation model (see text for details). fADI showed a significant correlation of virus attachment blocking for polioviruses of serotypes I, II and III with the data of microneutralization assay obtained independently at CBER/FDA using the WHO miconeutralization protocol.

Fig 6. Correlation between fADI and PRNT methods for Yellow Fever vaccine virus.

fADI₅₀ were calculated from the Virus Attachment Indexes using a mathematical approximation model, more details in the text. The fADI titers for all the pre-vaccination sera samples were found very low. fADI displayed a considerable donor-to-donor variation in the capacity of post-vaccination donor sera to block attachment of vaccine strain 17DD of Yellow Fever virus to Vero cells (a). PRNT₅₀ data were obtained for the same sera samples independently by a commercial vendor (b). Control ELISA did not reveal a reduction of the reporter fluorescence signal, thus showing zero competition between the sera antibodies and the virus specific labeling antibodies used in the assay (c). A considerable positive correlation between the fADI and PRNT data for post-vaccination sera samples was found (d).