Competitive detection of influenza neutralizing antibodies using a novel bivalent fluorescence-based microneutralization assay (BiFMA)

Abstract

Avian-derived influenza A zoonoses are closely monitored and may be an indication of virus strains with pandemic potential. Both successful vaccination and convalescence of influenza A virus in humans typically results in the induction of antibodies that can neutralize viral infection. To improve long-standing and new-generation methodologies for detection of neutralizing antibodies, we have employed a novel reporter-based approach that allows for multiple antigenic testing within a single sample. Central to this approach is a single-cycle infectious influenza A virus (sciIAV), where a functional hemagglutinin (HA) gene was changed to encode either the green or the monomeric red fluorescent protein (GFP and mRFP, respectively) and HA is complemented in trans by stable HA-expressing cell lines. By using fluorescent proteins with non-overlapping emission spectra, this novel bivalent fluorescence-based microneutralization assay (BiFMA) can be used to detect neutralizing antibodies against two distinct influenza isolates in a single reaction, doubling the speed of experimentation while halving the amount of sera required. Moreover, this approach can be used for the rapid identification of influenza broadly neutralizing antibodies. Importantly, this novel BiFMA can be used for any given influenza HA-pseudotyped virus under BSL-2 facilities, including highly pathogenic influenza HA isolates.

Development and applications of single-cycle infectious influenza A virus (sciIAV)

“…The sciIAV ΔHA is also advantageous in that two identical sciIAV varying only in their reporter gene could be quickly pseudotyped with HA to develop flexible multivalent screening assays (Baker et al, 2015). Additionally, the HA cell lines generated could theoretically be used in a virus-free ELISA to determine HA-binding Abs with monoclonal or polyclonal samples (our unpublished results).…”

“…All ΔPB2, ΔPB1, and ΔNA sciIAVs would require de novo rescue to change the HA antigenicity, which is disadvantageous to screen against diverse isolates. Reporter-based NAb screening using sciIAV can also be utilized to identify stalk-reactive broad spectrum NAbs (Baker et al, 2015), which would be advantageous over plaque reduction neutralization assays (Lee et al, 2012). Although sciIAV has been used only to evaluate the presence of NAbs, showing a comparable sensitivity to traditional assays, we cannot discard their limitations to detect other inhibitory Abs, such as those that prevent viral egress.…”

Replication-competent fluorescent-expressing influenza B virus

“…Vaccines and antivirals are available to combat influenza viruses (Baker et al, 2015b;Burnham et al, 2013;Jackson et al, 2011a;Krammer et al, 2015;Nguyen et al, 2010;Seibert et al, 2010). Historically, influenza vaccines contain viral antigens corresponding to the prevalent H3N2 and H1N1 IAVs as well as and one lineage of influenza type B (Victoria or Yamagata) (Belshe et al, 2007;Yang, 2013).…”

Host Single Nucleotide Polymorphisms Modulating Influenza A Virus Disease in Humans

“…Seasonal influenza infections are prevented and controlled through annual vaccination campaigns to decrease IAV infections and viral transmission as well as to reduce their negative impact in the global economy. However, although vaccination remains the most effective approach to protect the population from seasonal infections, the effectiveness of current vaccination approaches is suboptimal [16,[31][32][33][39][40][41][42][43][44]. Thus, the production of improved prophylactic approaches, including universal vaccines, are highly desired.…”