INTRODUCTION Influenza A viruses are classified using the an5genic and phylogene5c characteris5cs of the envelope glycoproteins haemagglu5nin (HA) and neuraminidase (NA). Currently 17 different haemagglu5nin subtypes are recognized and are divided into two lineages: Group 1 and Group 2 (Figure 1). In humans influenza A viruses that harbour on their envelope haemagglu5nin of H1 and H3 subtypes usually circulate, cause seasonal epidemics and are included in the seasonal vaccine. However viruses with other HA subtypes (eg H5, H7, H9) have been shown to infect humans and if these viruses acquire efficient humantohuman transmission they can poten5ally cause pandemics. The study of heterosubtypic an5body responses directed against influenza A haemagglu5nins in human popula5ons is an important facet of pandemic preparedness. For this purpose it is important to study whether seasonal vaccina5on can elicit and/or boost a crossneutralizing an5body response. Classical serological assays, such as haemagglu5na5on inhibi5on and microneutraliza5on, have demonstrated low sensi5vity for the detec5on of crossneutralizing an5bodies, especially those directed against epitopes in the haemagglu5nin HA2 stalk region. Here we use pseudotype par5cle neutraliza5on assays performed using representa5ves of Group 2 influenza viruses to detect the presence of heterosubtypic neutralizing an5body against Group 2 viruses before and aTer seasonal vaccina5on. MATERIALS AND METHODS Sera Sera from 1860 year old people (13) and from elderly (9) collected before and aTer seasonal vaccina5on with A/Wisconsin/67/2005 (H3N2), A/Solomon Island/3/2006 (H1N1) and B/ Malayasia/2506/2004 were used for this study. Pseudotype par;cles produc;on and ;tra;on Len5viral pseudotypes were produced as described previously [4, 5, 6]. Briefly, HIV gagpol plasmid p8.91, firefly luciferase expressing plasmid pCSFLW, HA expressing plasmid and pCAGGSTMPRSS2 [3] were cotransfected into human embryonic kidney HEK293T/17 cells using Fugene6 transfec5on reagent (Roche, UK). ATer 24 hours incuba5on, recombinant neuraminidase from Clostridium perfringens (Sigma) was added to facilitate pseudotype exit from the producer cells. 48 hours posttransfec5on supernatant was harvested, filtered through 0.45μm filters and stored at 80°C. Calcula5on of the pseudotype 5tres in rela5ve luminescence units (RLU) per ml was carried out as described by us elsewhere [6, poster P2493]. Pseudotype par;cles neutraliza;on assay Neutraliza5on 5tres of each sera were evaluated against A/Udorn/307/1972 H3, A/duck/ Czechoslovakia/1956 H4, A/chicken/Italy/1082/1999 H7, A/chicken/Germany/N49 H10, A/ mallard/Astrakhan/263/1982 H14, A/shearwater/West Australia/2576/1979 H15 pseudotypes. Serial dilu5ons (1:401:5120) of pre and post vaccina5on sera and of posi5ve control sera in white 96well flatboiomed 5ssue culture plates (Nunc) in a final volume of 50μl were performed and 1x10 6 RLU/well of pseudotypes were added to the plate. Following 1 hour incuba5on at 37°C, 1x10 4 HEK293T/17 cells were added to each well and the plates were incubated for 48 hours at 37°C. Luminescence was evaluated by luminometry using the BrightGlo assay system (Promega, UK) (Figure 2). Normaliza5on as percentage of inhibi5on of pseudotype virus entry (neutraliza5on), and the half maximal inhibitory concentra5ons (IC 50 ) were calculated with Prism version 6 (GraphPad SoTware) and expressed as reciprocal of the dilu5ons. 1 Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent, Central Avenue, Chatham Mari5me, ME4 4TB 2 Ins5tute of Virology, Philipps University Marburg, HansMeerweinStr. 2, 35043 Marburg, Germany 3 Department of Molecular and Developmental Medicine – University of Siena, Via Aldo Moro 3, 53100 Siena, Italy 4 Ins5tute for Research in Biomedicine, Via Vela 6, 6500 Bellinzona, Switzerland Francesca Ferrara 1 , Eleonora Moles5 1 , Eva BöicherFriebertshäuser 2 , Emanuele Montomoli 3 , Davide Cor5 4 , Simon Scoi 1 and Nigel Temperton 1 RESULTS For each group 2 virus the IC 50 distribu5on of the sera collected before and aTer vaccina5on were reported on BoxandWhisker plots (Figure 3) for comparison. IC 50 distribu5ons before and aTer vaccina5on are different in shape and a shiT of IC 50 medians and IC 50 interquar5le range is observed. All before and aTer vaccina5on IC 50 distribu5ons (with the excep5on of A/chicken/Germany/N49 H10 distribu5ons) are sta5s5cally different (p<0.05) using a nonparametric Wilcoxon matchedpairs signed rank test. Figure 3. BoxandWhisker plots showing IC 50 distribu;ons Figure 1: Phylogene;c rela;onship of influenza A haemagglu;nin subtypes Figure 2: Pseudotype par;cle neutraliza;on assay Group-2 Group-1 CONCLUSIONS In a this study we have shown that:  The pseudotype par5cle neutraliza5on assay has increased sensi5vity for detec5ng neutralizing an5body response.  Performing pseudotype par5cle neutraliza5on assays using a panel of influenza A pseudotypes permits the detec5on of heterosubtypic an5body responses in pa5ents before and aTer seasonal influenza vaccina5on.  The heterosubtypic an5body response is increased and/or can be elicited aTer seasonal vaccina5on. REFERENCES [1] Cor5 D, Voss J, Gamblin SJ et al. 2011. A neutralizing an5body selected from plasma cells that binds to group 1 and group 2 influenza A hemagglu5nins. Science, 333, 850856. [2] Temperton NJ, Hoschler K, Major D et al. 2007. A sensi5ve retroviral pseudotype assay for influenza H5N1 neutralizing an5bodies. Influenza Other Respi Viruses, 1, 105112. [3] Böicher E, Matrosovich T, Beyerle M et al. 2006. Proteoly5c ac5va5on of influenza viruses by serine proteases TMPRSS2 and HAT from human airway epithelium. J Virol, 80, 98969898 [4] Ferrara F, Moles5 E, BöicherFriebertshäuser E et al. 2013. The human Transmembrane Protease Serine 2 is necessary for the produc5on of Group 2 influenza A virus pseudotypes. J. Genet. Mol. Med. 7: 315320 CORRESPONDENCE Simon Scoi: s.d.scoi@kent.ac.uk & Nigel Temperton: n.temperton@kent.ac.uk Web: www.viralpseudotypeunit.info Twiier: @ViralPseudotype The study of heterosubtypic an;body responses against Influenza A viruses elicited by seasonal vaccina;on using a pseudotype neutraliza;on assay + + + Serial dilu5ons of sera Pseudotype par5cles No signal is detected Luciferase signal is detected In the absence of neutralizing an5bodies or in presence of nonneutralizing an5bodies HA pseudotype par5cles enter into cells If neutralizing an5bodies are present pseudotypes do not aiach or enter into the cells 48h 48h !"#$%" &#'"% () *) +,) -.) ,() +.*) ./,) /+.) +).() .)(*) ()0,) 12 34 &56789:5-);5+0;. =->> !"#$%" &#'"% () *) +,) -.) ,() +.*) ./,) /+.) 12 34 &5?@A?BC:51DEFG5+)*.5+000 =;>> !"#$%" &#'"% () *) +,) -.) ,() +.*) ./,) /+.) 12 34 &57H?B52IC?@8JF8KEBAE5+0/, =(>> !"#$%" &#'"% () *) +,) -.) ,() +.*) ./,) /+.) 12 34 &5J@CE9LEDC95MCJD &HJD9EFAE5./;,5+0;0 =+/>> !"#$%" &#'"% () *) +,) -.) ,() +.*) ./,) /+.) 12 34 &5NEFFE975&JD9EB@E:5.,-5+0*. =+(>> !"#$%" &#'"% () *) +,) -.) ,() +.*) ./,) /+.) 12 34 &5?@A?BC:5OC9NE:G5P(0 =+)>>