OFFLU Avian Influenza Matching (AIM) Guide To Assessing Antigenic Characteristics Of Avian Influenza Viruses

OFFLU AVIAN INFLUENZA MATCHING (OFFLU AIM) GUIDE TO ASSESSING ANTIGENIC CHARACTERISTICS OF AVIAN INFLUENZA VIRUSES Upda ed June 2024 Scope: This epo p o ides in o ma ion o na ional/sub-na ional le el labo a o ies on how o assess an igenic cha ac e is ics o a ian in luenza i uses. OFFLU a ian in luenza ma ching (AIM) epo , 2024 Page 2 In he con inuous assessmen o accine e icacy, he challenge o immunized animals ep esen s he gold s anda d. Howe e , his app oach is labou and cos in ensi e and equi es high biosecu i y labo a o ies and animal acili ies. Challenge ials should be ese ed o cases whe e he e is al eady e idence o an igenic a ia ion om o he sou ces sugges i e o educed accine e icacy. Fu he mo e, as expe imen al s udies a e gene ally unde aken in speci ic pa hogen ee bi ds, assessmen o accines in ield si ua ions, e en in he absence o i ulen challenge, bu wi h obus se ological assessmen is highly bene icial o unde s and esponses in he p esence o po en ial inhibi o y ac o s. An igenic ca og aphy in ol es he use o e e ence se a aised agains e e ence s ains and es i uses bu is usually es ic ed o being unde aken in e e ence labo a o ies wi h he acili ies o unde ake li e i us wo k. As such, whils ca og aphy o ms a c i ical analy ical aspec o he OFFLU AIM p ojec , i equi es samples o li e in ec ious i us o be sen o e e ence labo a o ies. O he me hods can also be used o p o ide e idence o possible an igenic a ia ion in ield i uses, including compa a i e se ological es s using se a om accina ed bi ds agains an igens p epa ed om ield i uses and accine an igens in use. Genomic sequencing is being deployed in many coun ies and can be used o de ec changes in known o pu a i e an igenic o glycosyla ion si es ha could a ec esponses o accina ion. Wo k in his a ea on Gs/Gd H5Nx i uses has been conduc ed o o e 15 yea s (see o example Ca oli e al., 2012) wi h he explici aim o moni o ing o an igenic e olu ion apidly. C oss-HI es ing using se a om accina ed bi ds One o he me hods used o assess he an igenic cha ac e is ics in places whe e accines a e being used is o ob ain se a om accina ed bi ds and conduc c oss hemagglu ina ion inhibi ion (HI) es s using he accine an igen (o a closely ela ed p oxy) e sus an an igen p epa ed om a ield isola e o in e es . I is ecommended ha a leas 30 se um samples om accina ed bi ds a e used o he es and accine an igen. I is impo an o no e ha his me hod only indica es an igenic ela edness. I a i us demons a es a ma ked educ ion in i es (a leas 4 log2 di e ences) hen ull an igenic cha ac e isa ion should be unde aken and g ea e a en ion should be paid o accina ed locks o e idence o in ec ion. Fo example: Coun y X is using a accine con aining an igen Y and inds a i us in a well- accina ed lock h ough e idence o clinical disease o sc eening se a as pa o an ac i e su eillance ini ia i e. Vi us Y was hen used as an an igen in HI es s using 30 se a om accina ed bi ds ha had no been exposed o ield i us. The a e age i e o an igen Y was 20 whe eas he a e age i e o samples using he accine an igen (Z) was 160. This >3 old di e ence be ween mean i e s o an igen Y and Z, along wi h he case his o y s ongly sugges s signi ican an igenic a ia ion is occu ing ha could impac p o ec ion p o ided by accina ion. In such cases conside a ion should be gi en o conduc ing challenge expe imen s o unde aking ull an igenic cha ac e isa ion a an app op ia ely ecognised In luenza e e ence labo a o y, such as a WOAH o FAO animal o a ian in luenza e e ence labo a o y. OFFLU a ian in luenza ma ching (AIM) epo , 2024 Page 3 Use o gene sequences o assessing possible an igenic changes Changes in an igenici y a e usually he esul o gene ic changes o he i al haemagglu inin (HA) p o ein in pu a i e an igenic si es, o loss o acquisi ion o glycosyla ion si es. As ull gene ic cha ac e isa ion and an igenic cha ac e isa ion o A(H5Nx) i uses inc eases, mo e in o ma ion on he signi icance o speci ic changes will be ob ained. Whils o en, mul iple gene ic changes a e equi ed o e ec majo an igenic a ia ion (e.g. Ca oli e al., 2011), single amino acid changes may also ha e a p o ound e ec on p o ein s uc u e and he induc ion o glycop o ein eac i e an ibodies. Sc eening o changes in pu a i e an igenic si es can be ca ied ou using sequence da a, howe e , a en ion mus be paid o he haemagglu inin numbe ing sys em which is used o align sequences and in e poin changes as di e en publica ions use di e en HA numbe ing (e.g. H5 s H3). The publica ion Bu ke and Smi h (2014) p o ides a lis o si es using di e en numbe ing sys ems and a e iew by Luczo and Spackman (2024) compiles o-da e in o ma ion on pu a i e an igenic si es o H5 and H7 HPAI i uses including e e ences o H3, H5 and H7 numbe ing. Whe e possible, p oposed an igenic si es should be assessed wi h e e ence o e ol ing in o ma ion in he li e a u e and wi h conside a ion o de ined e e ence i uses. In e p e a ion o an igenic changes can be complex and ideally pheno ypic da a should be a ailable. Some s udies a e conduc ed and alida ed wi h espec o how he human immune sys em ecognises he an igenic componen s o hese i uses. This may di e o how he chicken, o o he a ian species ecognise hese i uses. The e o e, whe e possible, in e ence using gene ic da a should be backed up wi h pheno ypic da a o ield da a. Technical suppo should be sough om an app op ia e FAO o WOAH e e ence labo a o y i equi ed and OFFLU s ongly ecommend ha membe s a es o coun ies sha e in o ma ion and i uses wi h he OFFLU ne wo k, especially i i uses a e de ec ed wi h changes in pu a i e an igenic si es. Me hods o assessing accines o he han hose based on a killed i us an igen Vec o ed accines can po en ially s imula e a g ea e an ibody esponse as hey eplica e o gene a e highe le els o a ge an igen wi hin he accina ed bi d. Howe e , eplica i e abili y o ec o ed accines may di e depending on he ec o s abili y o eplica e in he accina ed bi d. Examples o di e en ec o s include He pes i us o u keys, Newcas le disease i us and Pox i uses. As such, HI i e s de eloped by hese i uses may be lowe han hose p oduced by killed an igen accines, especially agains ield i uses, making i di icul o assess he le els o p o ec ion likely o be a o ded. Expe imen al s udies ha e demons a ed b oade c oss p o ec ion om hese accines agains a ange o i uses in some si ua ions and his in o ma ion should be used as a guide o likely le els o p o ec ion (Palya e al., 2018; Niqeux e al., 2023) Fu he mo e, because ec o ed accines a e based on accina ion wi h li e i al ma e ial exp essing he a ge p o ein o accinal impo ance hese accines will s imula e cell media ed immune esponses as well as humo al esponses. Howe e , me hods o measu ing cell media ed immuni y agains di e en ec o s emain poo ly de ined. Whe e eplica i e es ic ions o ec o s in di e en a ian species a e iden i ied, op ions exis o assess accina ion e icacy (e.g., using ea he ollicles o de ec he HVT i us 3 weeks pos accina ion (Palya e al., 2018)). Such app oaches may p o ide use ul in o ma ion on he need o e accina ion wi h an al e na i e accine. OFFLU a ian in luenza ma ching (AIM) epo , 2024 Page 4 Me hods ha monised be ween pa icipa ing labo a o ies used o he OFFLU AIM p ojec Gene a ion o chicken an ise a A s anda dised panel o H5Nx chicken an ise a and homologous an igens we e gene a ed a IZSVe and APHA. An ise a we e ob ained by inocula ion o speci ic pa hogen- ee (SPF) bi ds wi h emulsions o he an igens in wa e -in-oil adju an . The selec ion o i uses was based on gene ic and molecula analyses aimed a iden i ying s ains ha a e ei he ep esen a i e o ele an ci cula ing clades o gene ically ela ed o accine seed s ains, he eina e e e ed o as su oga e accine seed s ains. Fo each an ise um, a ba ch o he homologous i us was gene a ed in SPF emb yona ed chicken eggs and he allan oic luid was chemically inac i a ed wi h be a-p opiolac one (BPL). Ba ches had a minimum an igen con en equal o 128.000 HAU (e.g., 100 ml wi h a i e o 1:32). Ba ches unde wen innocui y es ing by h ee blind passages in SPF emb yona ed chicken eggs. 10 SPF chickens pe an igen we e inocula ed ia he in amuscula ou e wi h 1ml emulsions o BPL- inac i a ed a ian in luenza an igen plus Mon anide o wa e -in-oil as an adju an (inocula ion 1). (Vi us/adju an [30/70%]). A 14 days (+/- 3 days) pos p ima y inocula ion, bi ds we e boos ed (inocula ion 2) wi h he same an igen p epa a ion and p ocedu e. A ound 20 days a e he p ima y inocula ion, blood was collec ed om he bi ds ia wing bleeds and used o assess an ibody s a us agains he inocula ed an igen. I HI an ibody i es we e insu icien o use in assays (e.g. < 1:16 HAIUs/25µl) bi ds we e boos ed again (inocula ion 3). Blood sampling, se ological assessmen and e-inocula ion was epea ed once mo e i necessa y (inocula ion 4). A a maximum o day 36, all bi ds we e eu hanised and blood collec ed ia hea bleeds unde e minal anaes hesia. The se um was sepa a ed om ed blood cells and hea ea ed a 56°C o 30 min p io o being lyophilized o s o age and dis ibu ion. Fo each ba ch o se um and an igen gene a ed, olumes we e sha ed be ween WOAH and FAO e e ence labo a o ies IZSVe and APHA, ACDP and SEPRL. Hemagglu ina ion Inhibi ion (HI) assay An igenic p ope ies o a ian in luenza A H5 i uses we e cha ac e ised using es ablished ha monised app oaches o HI assays a APHA, IZSVe, ACDP and SEPRL using chicken mono-speci ic an ise a. These HI assays we e conduc ed as desc ibed in he WOAH Manual o Diagnos ic Tes s and Vaccines o Te es ial Animals (WOAH, 2023) wi h a modi ica ion in he s eps in ol ing he incuba ion o chicken ed blood cells wi h an igens (incuba ion ime o 1 hou a a empe a u e o 4°C). The an igen con aining 4 hemagglu ina ing uni s was con i med by back i a ion. In he HI assay, se a we e dilu ed wo- old om a s a ing dilu ion o 1:2 and es ed in duplica e. Posi i e an ise a and nega i e con ol se a ob ained om SPF chickens we e also es ed o each assay. Sequencing o i al isola es Each i al isola e es ed in HI assay was sequenced and sequence da a was compa ed wi h wild ype sequence o ensu e no egg o cell adap i e changes had aken place in an igenic si es wi hin HA o NA du ing p opaga ion. Whole genome sequencing was unde aken using Illumina sequencing. Sequencing lib a ies we e un on a Nex Seq 550 pla o m (illumina) wi h pai ed end eads. Raw sequencing eads we e assembled using cus om sc ip s FluSeqID o deno oAssembly. OFFLU a ian in luenza ma ching (AIM) epo , 2024 Page 5 Links o OFFLU AIM documen s OFFLU A ian in luenza echnical ac i i y accina ion page Concep no e OFFLU AIM p ojec - Ap il 2022 OFFLU AIM p esen a ion – Feb ua y 2023 OFFLU AIM pilo epo – Oc obe 2023 Re e ences Bu ke, D. F., & Smi h, D. J. (2014). A ecommended numbe ing scheme o in luenza A HA sub ypes. PloS one, 9(11), e112302. h ps://doi.o g/10.1371/jou nal.pone.0112302 Ca oli, G., Milani, A., Tempe on, N., Zecchin, B., Bu a in, A., Moles i, E., Aly, M. M., A a a, A., & Capua, I. (2011). An igenic d i in H5N1 a ian in luenza i us in poul y is d i en by mu a ions in majo an igenic si es o he hemagglu inin molecule analogous o hose o human in luenza i us. Jou nal o i ology, 85(17), 8718–8724. h ps://doi.o g/10.1128/JVI.02403-10 Luczo, J. M., & Spackman, E. (2024). Epi opes in he HA and NA o H5 and H7 a ian in luenza i uses ha a e impo an o an igenic d i . FEMS mic obiology e iews, uae014. Ad ance online publica ion. h ps://doi.o g/10.1093/ ems e/ uae014 Niqueux, É., Flod ops, M., Allée, C., Leb as, M. O., Pie e, I., Loubou in, K., Guillemo o, C., Le P ioux, A., Le Bouquin-Lene eu, S., Keï a, A., Amelo , M., Ma eno , C., Massin, P., Che bonnel-Pansa , M., B iand, F. X., Schmi z, A., Cazaban, C., Dauphin, G., Delquigny, T., Lemiè e, S., … E e adossi, N. (2023). E alua ion o h ee hemagglu inin-based accines o he expe imen al con ol o a panzoo ic clade 2.3.4.4b A(H5N8) high pa hogenici y a ian in luenza i us in mule ducks. Vaccine, 41(1), 145–158. h ps://doi.o g/10.1016/j. accine.2022.11.012 Palya V, Ta á -Kis T, Walkóné Ko ács E, Kiss I, Homonnay Z, Ga din Y, Ke ész K, Dán Á. (2018). E icacy o a Recombinan Tu key He pes i us AI (H5) Vaccine in P e en ing T ansmission o He e ologous Highly Pa hogenic H5N8 Clade 2.3.4.4b Challenge Vi us in Comme cial B oile s and Laye Pulle s. J Immunol Res. 21;2018:3143189. h ps://doi.o g/10.1155/2018/3143189