Ralf Wagner Paul-Ehrlich-Institut

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Letitia Strickland
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1 Other Assays for the Detection of Neuraminidase (NA)-Specific Antibodies Ralf Wagner Paul-Ehrlich-Institut

2 Overview to presented assays Assay principle based on: Chemical substrates: Protein substrates: Expressed purified NA: MU-NANA AVINA TBA (vs ELLA) ELISA or Western blot Function in replication cycle: Plaque test (Pseudotype viruses)?? Recombinant Flu Viruses VN with specific adaptations

Surface Glycoproteins of Influenza Viruses Schematic Overview to Influenza Virus Surface Antigens: Surface Antigens Internal components

Fusion mediation Lipid bilayer MATRIX PROTEIN M1 Structural component Ribonucleoprotein (RNP) NUCLEOPROTEIN (NP) POLYMERASES PB1, PB2,

analytic NA: RECEPTOR DESTROYING ENZYME far less used for immunogenicity evaluation promotes efficient spread of progeny viruses to

3 Surface Glycoproteins of Influenza Viruses Schematic Overview to Influenza Virus Surface Antigens: Surface Antigens Internal components INDUCTION OF ANTIBODY RESPONSE NEURAMINIDASE NA: Release of progeny viruses M2 PROTEIN Ion channel HEMAGGLUTININ HA: Receptor binding Fusion mediation Lipid bilayer MATRIX PROTEIN M1 Structural component Ribonucleoprotein (RNP) NUCLEOPROTEIN (NP) POLYMERASES PB1, PB2, PA 8 RNA segments HA: initiates virus infection AB can block infection main surface immunogen Primary target of routine serological analytic NA: RECEPTOR DESTROYING ENZYME far less used for immunogenicity evaluation promotes efficient spread of progeny viruses to neighboring cells prevents progeny virus aggregation (late stage of infection) to enhance dissemination helps virus to penetrate lower airways mucus

4 Chemical substrates: MU-NANA approach MU-NANA: 2'-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid MU-NANA OH NANA NA OH MU: 4-methyl umbelliferone FL NA-specific antibodies reduce fluorescents read-out! Fluorescence Pros: Cons: easy, fast, inexpensive highly variable, difficult to standardise, artificial substrate ability to robustly detect anti-na responses unclear/questionable Ref: Yolken et al., J. Infect. Dis. 1980; Lambre et al., Vaccine 1989

5 AVINA: Accelerated Virus Inhibition with NA as Read-Out Combination of neutralisation and MU-NANA read-out (established mostly for NA-inhibitors) but might be also applicable for antisera Infection of cell monolayer with virus -/+ antiserum / inhibitors After incubation period: test supernatants for NA-activity with MU-NANA Ref: Eichelberger et al., Virology Journal 2008

6 Thiobarbituric acid (TBA) Assay Fetuin (or other substrates Such as gangliosides) ELLA Lectin Sia Sia Sia Sia peroxidase-labelled peanut agglutinin NA- activity by virus T Inhibited by NA-specific AB Gal Fetuin Gal Gal Gal Gal Fetuin Gal Gal desialylated protein Gal + Sia Sia Sia Sia released sialic acids TBA Chemical conversion ( WHO manual ) Can be done in 96-well formate conversion to β-formol-pyruvate by periodate oxidation formation of chromophore by TBA extraction of chromophore in organic solvent Quantification with peroxidase substrate (ELISA) spectrometry Warren, J Biol Chem 1959; Sandbulte et al., Infl. other resp. Viruses 2009)

NA-AB Titer in Ferrets by TBA -Assay 50% inhibition in sera from vaccinated ferrets (H1N1) NA-activity % 100 80 60 40 20 0 140 120 100 80 60 40 20 0 50% NA-Inhibition D 0 : none (1:1) 50% NA-activity

7 NA-AB Titer in Ferrets by TBA -Assay 50% inhibition in sera from vaccinated ferrets (H1N1) NA-activity % % NA-Inhibition D 0 : none (1:1) 50% NA-activity D 28: 1:125 50% NA-Inhibition D 0 : none D 28: 1: N1-NA positive control serum 50% Serum dilution Pros: measurement of NAI on protein substrate (As in ELLA) rel. high throughput (96 well), inexpensive Cons: hazardous reagents, variable, difficult to standardise

8 NAI by ELLA and TBA -Assay: not much in common!? Initial results from pilot study on comparability of ELLA- and TBA-titer Serum sample dilution ELLA dilution TBA S001 1: :75 80 S002 1: :75 80 S003 1: : S004 1: :75 40 S005 1: : S006 1: :75 57 S007 1: : S008 1: : S009 1: : S010 1: :75 7 S011 1: :75 28 S012 1: :75 5 Human AB 1: :75 80 Sheep negative. 1:50 5 1: : : : : here: Very low if any correlation??!! Clear need for further investigation and standardisation!! FLUCOP project

9 Methods based on purified or expressed NA protein Several protocols for supply of NA protein: - Purified viruses (with specific HA and/or NA components) - NA-protein purification from virions (numerous approaches: HPLC, octylglucoside extraction, centrifugation, immune affinity, ) - Expression in cell culture (whole protein, selected domains, modified versions) Use in Capture ELISA, Western blot for detection of anti-na antibodies (numerous references with different NA-sources and NA-versions) Strategy for expression in insect cells: For ELISA development, Western blot and immunisation of animals for preparation of reference sera NH 2 CT TM Ectodomain NA COOH NH 2 NA COOH NH 2 NA COOH streptag NH 2 NA COOH soluble

10 Excursion: Vaccine bulks as Antigen Source Protein profile of seasonal vaccine monovalent bulks: H1N1 H3N2 H1N1 H3N2 HA NA/NP Split vaccine Subunit vaccine Use split vaccine as protein substrate for the detection of anti-na antibodies in WB

Detection of NA-AB Titer by Western Blot Could serve as an approach for quantification of NA-specific AB against standard serum (more calibration data needed) Alternative: do with expressed purified

11 Detection of NA-AB Titer by Western Blot Could serve as an approach for quantification of NA-specific AB against standard serum (more calibration data needed) Alternative: do with expressed purified NA Western blot strips (1-11): H3N2 monvalent split vaccine bulk analysed with different dilutions of anti-n2-na serum (NIBSC) (as primary antibody) NA Blot NA-band intensities against serum dilution NA-band intensity arbitrary units Pros: direct interaction with NA-protein / selected domains ELISA: high throughput Cons: Nativity/integrity of NA-protein; cross-reactivity

virus no serum no virus 1.8µl 1.5µl 4.0µl 3.0µl 1.0µl 1.2µl 2.5µl 2.2µl 0.8µl 0.5µl no serum 2.

12 Impact on Replication: Plaque Size Reduction Apply specific serum during plaque test in cell culture (here: MDCK) Utilises crucial function of NA Virus: H5N1/FPV, Serum: anti-n1 (NIBSC 10/218) no serum no virus no serum no virus 1.8µl 1.5µl 4.0µl 3.0µl 1.0µl 1.2µl 2.5µl 2.2µl 0.8µl 0.5µl no serum 2.0µl With growing amount of serum: reduction of total plaque numbers reduction in plaques size (increase in ratio: small/total)

13 Impact on Replication: Plaque Size Reduction - Analysis After counting of large (>1mm) and small (<1mm) plaques Volume of serum Plaque count % Ratio Large d > 1mm Small d < 1mm Small /Total No serum , ,0 no virus ,5µL AS ,0 0,8µL AS ,0 1,0µL AS ,0 1,2µL AS ,4 1,5µL AS ,2 1,8µL AS ,0 2,0µL AS ,0 2,2µL AS ,8 2,5µL AS ,8 3,0µL AS ,4 4,0µL AS ,8 Clear increase in number of small plaques for higher serum volumes Pros: directly reflects NA-activity in replication cycle, authentic functional read-out Cons: very labour-intensive, long-lasting, not for high sample throughput But: could be applicable to generate supportive/supplementary data Jin et al., Virology 2003; Chen et al., Vaccine 2012)

Impact on replication/infection: Pseudotyped Virions Overview to generation of HA+NA-expressing retroviral pseudotypes HA+NA Components: Retroviral core: virion budding Flu HA +

, 2014 BUT: currently not established, as pseudotypes not for multi-cycle replication (NA-effect) Pros: in general well established and powerful approach quantitative read-out,

14 Impact on replication/infection: Pseudotyped Virions Overview to generation of HA+NA-expressing retroviral pseudotypes HA+NA Components: Retroviral core: virion budding Flu HA + NA: entry/exit Reporter: detection (eg GFP) Plasmid: Flu HA+NA Modified from: Trombetta et al., 2014 BUT: currently not established, as pseudotypes not for multi-cycle replication (NA-effect) Pros: in general well established and powerful approach quantitative read-out, standardised Cons: needs to be specifically adapted for the detection of anti-na antibodies (eg HA vs NA) But: if adequately developed could be very promising tool for the future?! Ref: Trombetta et al., Viruses 2014; Garcia and Lai, Expert Rev. Anti Infect. Ther. 2011)

15 Impact on Replication: Recombinant Influenza Viruses Generate recombinant Influenza viruses by reverse genetics from cdna with specific composition for the detection of anti-na antibodies: homologous NA-protein heterologous HA-protein Long-term goal/vision: use such viruses for the specific detection of either anti-ha or anti-na responses by neutralisation assay if possible??!! Part of FLUCOP project. Exemplary outline: H1N2 H1N1 H9N1 Vaccine virus However: Modifications required to MNT-procedure.

16 Impact on Replication: MNT adaptation for anti-na antibodies Conceivable modifications: Longer incubation period (3-7 days) for more replication cycles (impact of NA) Lower infectious dose for inoculation (for multi-cycle replication) Specialised cell lines no trypsin needed for virus growth in cell culture (could affect antibodies) Others..?? Example: CaCo cells HA-Titer [log2] HA-Titer [log2] ohne Virus 10^-4 10^-5 ohne Virus 10^-4 10^-5 ohne Virus 10^-4 10^-5 ohne Virus MDCK 10^-4 10^-5 H1N1-PR8 H1N1-PR8 H1N1-FPV del mut H1N1-FPV del mut CaCo2 1µg TPCK ohne TPCK 1µg TPCK ohne TPCK No Trypsin added 1µg/ml Trypsin Inoculum dose H1N1-PR8 H1N1-FPV

17 That s all Thank you for your attention!! Questions?? Federal Institute for Vaccines and Biomedicines

Min Levine, Ph. D. Influenza Division US Centers for Disease Control and Prevention. June 18, 2015 NIBSC

Workshop on Immunoassay Standardization for Universal Flu Vaccines Min Levine, Ph. D. Influenza Division US Centers for Disease Control and Prevention June 18, 2015 NIBSC 1 Multiple Immune Mechanisms Contribute