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

Highlights

• We have developed a bivalent fluorescence assay to identify influenza neutralizing antibodies.

• The bivalent nature of the approach reduces by half the time, reagents, and sera needed.

• Direct competition of antibody neutralization against disparate HAs can be evaluated.

• Rapid and safe methodology amenable to facilities lacking BSL-3 capabilities.

• Can be used to identify and characterize influenza broadly neutralizing antibodies.

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.

Introduction

Influenza A viruses reside in the wild aquatic waterfowl reservoir, but humans and other mammals are continuously affected by cross-species infection [1]. Presently two influenza A subtypes are circulating in humans (H1N1 and H3N2), which account for approximately half of the influenza clinical cases and, together with influenza B viruses, cause three to five million cases of severe illness yearly with 250,000 to 500,000 deaths worldwide [2].

Influenza A viruses are enveloped and contain eight single-stranded RNA segments of negative polarity with two major surface glycoproteins: hemagglutinin (HA), which mediates receptor binding and fusion; and neuraminidase (NA), which mediates nascent virion release [3]. Influenza A viruses are classified by their 18 HA (H1-18) and 11 NA (N1-11) antigenic variants or subtypes [4], [5], [6]. However, antigenically distinct isolates can also exist within the same subtype (referred to as drifted variants), as observed in seasonal H1N1 prior to 2009, where the pandemic H1N1 swine-origin virus displayed unique antigenicity [5], [7], [8]. A majority of influenza A virus isolated from people can readily transmit between humans via aerosolized droplets, and because airborne virus spreads so rapidly, the best mechanism to prevent disease spread is through vaccination, recommended for all non-contraindicated persons >6 months of age in a number of countries [9], [10].

Sterilizing immunity against influenza viruses can be achieved through the induction of neutralizing antibodies (NAbs), which can bind HA to prevent virus-receptor binding or virion–endosomal fusion [3]. Indeed, a four-fold vaccine-induced increase in NAbs, or a resulting >1:40 titer of protective antibodies, is clinically relevant [11], [12]. The two standard methods for evaluating humoral influenza virus inhibition are the hemagglutination inhibition (HAI) assay, which has been shown to correlate with protective immunity [13], and the virus neutralization (VN) assay. For both tests, influenza viruses are pre-incubated with serial dilutions of sera (or antibodies) before being added to erythrocytes for the HAI assay and observing red cell agglutination in a few hours [14], or to Madin–Darby canine kidney (MDCK) cell monolayers for the VN assay and observing cytopathic effect two-to-four days post-infection [15]. Both tests require intact influenza virus, which can be problematic for testing highly pathogenic influenza isolates because such viruses require high biosafety containment (e.g. BSL-3+ laboratories), although the HAI assay does not require infectious virus (e.g. can be performed using inactivated virus [16]). Furthermore, the HAI assay requires a significantly higher amount of virus per reaction (the equivalent to approximately 10⁵–10⁶ of egg infectious dose₅₀, EID₅₀) [17], whereas the VN requires less virus per reaction (100–200 EID₅₀) [18], suggesting HAI may be less sensitive because there is more antigen for the antibodies to neutralize. Also, HAI assay readouts vary based on the amount of erythrocytes used and the subjectivity of the laboratory personnel in terms of considering the presence or absence of red cell agglutination, as well as the time when the assay is read [19]. On the other hand, the HAI is much more rapid than the VN, taking 1–2 h rather than the 2–4 days to achieve results [15]. To obtain a VN titer more rapidly, ELISA or Western blot can be performed on infected cells the day following infection, although this adds another step that requires the use of specific antibodies against the viral antigen and qualified personnel, and that is not optimal for a large number of samples [20]. Despite their differences, both HAI and VN can only be performed against one antigenic virus variant at a time, which is disadvantageous amid the rapid drifting of some avian H5 viruses [21]. Having a single virus per reaction also limits the detection of broadly cross-reactive influenza NAbs. Therefore, an assay for the detection of influenza NAbs that avoids the use of infectious-competent virus, is rapid, and can evaluate multiple antigenic variants of virus will help to identify and characterize laboratory-generated therapeutic NAbs and to evaluate humoral responses from influenza vaccination and infection.

An advantageous approach to detect NAbs against influenza virus with diverse HA subtypes is the HA-pseudotyped single cycle infectious influenza A virus (sciIAV) [22]. Other sciIAV engineered to delete the PB2, PB1, or NA genes have also been used to identify influenza NAbs [23], [24], [25], but changing the antigenicity of the test virus requires de novo virus rescue. As opposed to non-influenza virus pseudotypes, sciIAV maintains appropriate HA:NA stoichiometry, virion morphology, and once sciIAV is rescued, the backbone virus can be pseudotyped on diverse HA-expressing cells more rapidly than rescuing new viruses [22], [26], [27]. Here, we show that our previously developed fluorescence-based microneutralization assay for the detection of influenza NAbs [22] can be extended to a multiplex format to evaluate several antigenic variants of influenza virus in a single-well system. To achieve this, identical sciIAV were rescued that differ only in their fluorescent reporter gene (GFP or mRFP). We applied this bivalent fluorescence approach to demonstrate neutralization against different (heterosubtypic) and similar (homosubtypic) HA isolates. Moreover, we present evidence that BiFMA can be used to easily identify influenza broadly cross-reactive NAbs, all under less restricted BSL-2 laboratory settings. These results demonstrate the feasibility of using similar approaches to screen, in a single test, all isolates comprising vaccine formulations or multiple circulating viruses.