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. 2022 Sep 14;10(9):1520.
doi: 10.3390/vaccines10091520.

Influenza A (N1-N9) and Influenza B (B/Victoria and B/Yamagata) Neuraminidase Pseudotypes as Tools for Pandemic Preparedness and Improved Influenza Vaccine Design

Kelly A S da Costa  1 , Joanne Marie M Del Rosario  1   2 , Matteo Ferrari  2 , Sneha Vishwanath  3 , Benedikt Asbach  4 , Rebecca Kinsley  2   3 , Ralf Wagner  4   5 , Jonathan L Heeney  2   3 , George W Carnell  3 , Nigel J Temperton  1
Affiliations

Influenza A (N1-N9) and Influenza B (B/Victoria and B/Yamagata) Neuraminidase Pseudotypes as Tools for Pandemic Preparedness and Improved Influenza Vaccine Design

Kelly A S da Costa et al. Vaccines (Basel). .

Abstract

To better understand how inhibition of the influenza neuraminidase (NA) protein contributes to protection against influenza, we produced lentiviral vectors pseudotyped with an avian H11 hemagglutinin (HA) and the NA of all influenza A (N1-N9) subtypes and influenza B (B/Victoria and B/Yamagata). These NA viral pseudotypes (PV) possess stable NA activity and can be utilized as target antigens in in vitro assays to assess vaccine immunogenicity. Employing these NA PV, we developed an enzyme-linked lectin assay (pELLA) for routine serology to measure neuraminidase inhibition (NI) titers of reference antisera, monoclonal antibodies and post-vaccination sera with various influenza antigens. We also show that the pELLA is more sensitive than the commercially available NA-Fluor™ in detecting NA inhibition in these samples. Our studies may lead to establishing the protective NA titer that contributes to NA-based immunity. This will aid in the design of superior, longer lasting and more broadly protective vaccines that can be employed together with HA-targeted vaccines in a pre-pandemic approach.

Keywords: ELLA; antisera; immunity; influenza; inhibition; monoclonal antibody; neuraminidase; pseudotype; vaccine.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Illustration of main reactions in the pseudotype virus enzyme-linked lectin assay (pELLA). The substrate fetuin is coated on each well of a 96-well plate. Neuraminidase, added via the NA PV we generated, then cleaves the terminal sialic acid (SA) residues of the substrate fetuin, giving rise to galactose residues. In the presence of substances that can impede NA such as mAbs and anti-NA antisera, the action of neuraminidase is inhibited, and this can then be measured indirectly via pELLA. The terminal galactose residues that are present due to the action of neuraminidase are then specifically recognized by the substrate lectin peanut agglutinin conjugated to horseradish peroxidase (HRP). Addition of a peroxidase substrate such as TMB results in a detectable color change that can be measured at OD450.
Figure 2
Figure 2
H11_NA(X) PV production and assessment of functionality. (A) Schematic representation of the production of influenza NA pseudotypes by quadruple plasmid transfection to produce pseudotypes expressing HA and NA on the PV surface. Protease is only required when HA is expressed, as per previous optimization [41]. Images created using Biorender. (B) Phylogenetic tree of representative IAV NA from the PV library constructed. Influenza A Group I NA PV are shown in blue and IAV Group II PV in red. Accession numbers are reported with the subtype on the tree tips. Nodes are shown at the ends of branches which represent sequences or hypothetical sequences at various points in evolutionary history. Branch lengths indicate the extent of genetic change. The tree generated was constructed with PhyML on the Influenza Research Database (IRD) [46] and graphically elaborated with Archaeopteryx.js. (C) Titration of influenza A NA (1–9) and influenza B NA (B/Victoria-like and B/Yamagata-like lineages) PV. Titration was carried out via pELLA. Readout is NA enzymatic activity expressed as %OD (450 nm) of highest dilution tested. (D) Titration of influenza A NA (1–9) and influenza B NA (B/Victoria-like and B/Yamagata-like lineages) PV expressing H5 (A/Indonesia/05/2005) hemagglutinin. Readout is expressed in relative luminescence units (RLU). For (C,D), each point represents the mean and standard deviation of two replicates per dilution (n = 2). Additionally, influenza A (IAV) Group I NA PV are shown in blue (N1, N4, N5 and N8), IAV Group II PV in red (N2, N3, N6 and N9) and influenza B NA PV (B/Victoria-like and B/Yamagata-like lineages) are shown in black.
Figure 3
Figure 3
In vitro inhibition of NA pseudotypes by antisera and monoclonal antibodies. (A) In vitro inhibition of representative influenza A and influenza B NA pseudotype viruses (PV) which have previously caused human infection: N1 (A/England/195/2009), N2 (A/South Australia/34/2019), B/Victoria-like lineage NA (B/Colorado/6/2017) and B/Yamagata-like lineage NA (B/Phuket/3073/2013), by reference antisera obtained from NIBSC. Reference antisera were serially diluted five-fold from a starting dilution of 1:10 and PV diluted to 90% OD450 as determined previously from titration (Figure 2C). Inhibition of NA activity was determined via pELLA. Each point represents the mean and standard deviation of two replicates per dilution (n = 2). (B) Half-maximal inhibitory dilution of reference antisera as calculated from dose response curves (A). (C) In vitro inhibition of representative N1 (A/England/195/2009) PV by monoclonal antibodies CR9114 and CD6. mAbs were diluted two-fold from 1000 to 2 ng/mL. (D) In vitro inhibition of representative N1 (A/England/195/2009) PV by N1-directed mAbs: 3A2, 1H5, 7E9 and 3H10. mAbs were diluted 2-fold from 32 µg/mL to 32 ng/mL. For (C,D), inhibition was determined via pELLA, and each point represents the mean and standard deviation of two replicates per dilution (n = 2). (E) IC50 concentration values as determined from (C,D) using non-linear regression.
Figure 4
Figure 4
Binding and anti-NA activity of post-NA vaccination sera. (A) Binding of representative post-NA vaccination sera to HEK293T/17 transfected with pEVAC encoding homologous neuraminidase sequences of N1, N2, N3, N4, N8 and N9 was determined via FACS and reported as median fluorescence intensity (MFI) in a heatmap. Readings were performed in duplicate (n = 2). (B) In vitro inhibition of representative IAV (homologous subtype) and IBV (homologous lineage) pseudotypes by mouse sera vaccinated with influenza A HA from A/Brisbane/2/18 (N1), A/Kansas/14/17 (N2), A/duck/Cambodia/b0116502/17 (N3), A/chicken/NSW/1688/1997 (N4), A/yellow-billed pintail/Chile/C14831/16 (N5), A/yellow-billed teal/Chile/8/13 (N6), A/swine/England/191973/1992 (N7), A/gyrfalcon/Washington/41088-6/14 (N8) and A/Shanghai/2/13 (N9) and influenza B HA from B/Colorado/6/17 (B/Vic) and B/Phuket/3073/13 (B/Yam). Inhibition was determined via pELLA and reported as IC50 dilution values (IC50 is half-maximal inhibitory serum dilution). For mice vaccinated with N3-N6, N8-N9, n = 6; for N1, N2, N7 and both B lineages, n = 5. Group I NA is indicated in blue and Group II NA in red. Dashed line indicates an arbitrary IC50 dilution value of 1 for samples that were unable to inhibit NA. Plot shows the median and interquartile range of all samples tested.
Figure 5
Figure 5
Determination of NA activity of H11_NA pseudotypes and NA inhibition activity of reference antisera and mAbs via NA Fluor™. (A) Titration of representative IAV (N1-N9) and IBV (B/Victoria-like and B/Yamagata-like lineages) PV. Titers are reported in relative fluorescence units (RFU) (n = 2). Dotted line at 10,000 RFU indicates value used for NA activity normalization (Figure S2). (B,C) Inhibition of A/England/195/09 (N1) (blue), A/South Australia/34/19 (N2) (red), IBV (black) B/Colorado/6/17 (B/Vic NA) and B/Phuket/3073/13 (B/Yam NA) H11_NA PV by reference antisera. (B) Reference antisera were serially diluted five-fold from a starting dilution of 1:10, similar to Figure 2A. The NA PV at a dilution that would give 10,000 RFU as determined in (A) was then added to each well. (C) IC50 dilutions for reference antisera against homologous subtype/lineage PV are shown. For (A–C), Group I NA PV are shown in blue, Group II NA PV in red and IBV (both lineages) in black. (D,E) Inhibition of A/England/195/09 (N1) PV by N1-specific monoclonal antibodies. (D) Monoclonal antibodies were serially diluted two-fold from a starting concentration of 32 µg/mL to 0.0625 ng/mL, and IC50 values are summarized in (E). For plots (A,B,D), each point represents the mean and standard deviation of two replicates per dilution. For (C,E), “n.c.” indicates values not computed by GraphPad Prism.
Figure 6
Figure 6
Comparison of inhibition of H11_NA PV by N1 and N2 post-vaccination mouse sera using enzyme-linked lectin assay (ELLA) and NA Fluor™. Sera from mice vaccinated with A/Brisbane/2/18 (N1) (blue) (n = 5) and A/Kansas/14/17 (N2) (red) (n = 5) were diluted two-fold starting from 1:20 for both assays. They were then tested for their ability to inhibit homologous NA subtype PV. Percent NA inhibition is shown as a function of serum dilution. For all plots, each point represents the mean and standard deviation of two replicates per dilution.
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References

    1. Liu C., Eichelberger M., Compans R., Air G. Influenza type A virus neuraminidase does not play a role in viral entry, replication, assembly, or budding. J. Virol. 1995;69:1099–1106. doi: 10.1128/jvi.69.2.1099-1106.1995. - DOI - PMC - PubMed
    1. Cohen M., Zhang X.Q., Senaati H.P., Chen H.W., Varki N.M., Schooley R.T., Gagneux P. Influenza A penetrates host mucus by cleaving sialic acids with neuraminidase. Virol. J. 2013;10:321. doi: 10.1186/1743-422X-10-321. - DOI - PMC - PubMed
    1. Yang X., Steukers L., Forier K., Xiong R., Braeckmans K., Van Reeth K., Nauwynck H.A. Beneficiary Role for Neuraminidase in Influenza Virus Penetration through the Respiratory Mucus. PLoS ONE. 2014;9:e110026. doi: 10.1371/journal.pone.0110026. - DOI - PMC - PubMed
    1. Russell C.J., Hu M., Okda F.A. Influenza Hemagglutinin Protein Stability, Activation, and Pandemic Risk. Trends Microbiol. 2018;26:841–853. doi: 10.1016/j.tim.2018.03.005. - DOI - PMC - PubMed
    1. Kirkpatrick E., Qiu X., Wilson P.C., Bahl J., Krammer F. The influenza virus hemagglutinin head evolves faster than the stalk domain. Sci. Rep. 2018;8:10432. doi: 10.1038/s41598-018-28706-1. - DOI - PMC - PubMed

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