OFFLU Avian Influenza Matching (AIM) Technical Report - September 2025

1 OFFLU A ian In luenza Ma ching (AIM) Technical Repo Sep embe 2025 Sugges ed Ci a ion: OFFLU. (2025). OFFLU A ian In luenza Ma ching (AIM) Technical Repo , Sep embe 2025. Zenodo. h ps://doi.o g/10.5281/zenodo.16638584. 2 Table o Con en s 1. Guiding In o ma ion ...................................................................................................................... 3 2. Execu i e Summa y ...................................................................................................................... 4 3. Backg ound .................................................................................................................................. 5 3.1. Vaccine Seed S ains ................................................................................................................. 7 3.2. Panel o Chicken An ise a .......................................................................................................... 8 3.3. Phylogene ic Rela ionships ........................................................................................................ 9 4. Main Findings ............................................................................................................................. 12 4.1. An igenic Dis ances .................................................................................................................. 12 4.2 An igenic Ca og aphy .............................................................................................................. 16 5. Conclusions and Fu u e Di ec ions ............................................................................................. 18 Links o Key AIM Documen s ......................................................................................................... 19 Re e ences ...................................................................................................................................... 19 3 1. Guiding In o ma ion Acknowledgemen s This epo was p oduced by OFFLU ne wo k, he join Wo ld O ganisa ion o Animal Heal h (WOAH) and he Food and Ag icul u e O ganiza ion o he Uni ed Na ions (FAO) ne wo k o expe ise in animal in luenza. This wo k was led by OFFLU’s A ian In luenza Vaccina ion echnical ac i i y, wi h con ibu ions om Ian B own (The Pi b igh Ins i u e, Uni ed Kingdom), Ashley Banya d, Joe James, and Joshua Lyn on-Jenkins (Animal and Plan Heal h Agency, Uni ed Kingdom), F ancesco Bon an e (Is i u o Zoop o ila ico Spe imen ale delle Venezie, I aly), F ank Wong and Joanne G imsey (CSIRO Aus alian Cen e o Disease P epa edness, Aus alia), Amelia Coggon and Nicola Lewis (Royal Ve e ina y College, Uni ed Kingdom), Lo can Ca negie (OFFLU Scien is , FAO), Amelia Coggon, and Fabien Filai e (FAO). An igenic ca og aphy was unde aken by Amelia Coggon and Nicola Lewis (Royal Ve e ina y College, Uni ed Kingdom). Hemagglu ina ion inhibi ion (HI) da a was gene a ed by he In e na ional Re e ence Labo a o y o A ian In luenza eam, led by Joshua Lyn on-Jenkins a he Animal and Plan Heal h Agency (Uni ed Kingdom), and by F ancesco Bon an e and Sil ia Manie o a he Is i u o Zoop o ila ico Spe imen ale delle Venezie (I aly). We g a e ully acknowledge Leslie Sims (OFFLU) o hei ole in ini ia ing his p ojec and p o iding s a egic guidance. Addi ional HI da a we e con ibu ed by F ank Wong and Joanne G imsey a he CSIRO Aus alian Cen e o Disease P epa edness (Aus alia). Vi al isola es we e kindly sha ed wi h he In e na ional Re e ence Labo a o ies o A ian In luenza om he ollowing coun ies: Bangladesh, Belgium, Benin, Bulga ia, Bu kina Faso, Canada, Cambodia, Came oon, C oa ia, Czech Republic, Falkland Islands, Finland, Geo gia, Ghana, Guinea, Republic o Indonesia, I an, I eland, Is ael, I aly, I o y Coas , Koso o, La ia, Moldo a, Nepal, Nige , Nige ia, No way, Philippines, Poland, Romania, Sain Helena, Ascension and T is an da Cunha, Slo akia, Slo enia, Sou h Geo gia and he Sou h Sandwich Islands, Spain, Swi ze land, Togo, Tu key, Uk aine, Vie Nam and he Uni ed Kingdom. We would like o hank all he OFFLU ne wo k expe s, FAO and WOAH o hei suppo wi h his p ojec . Bene i s Sha ing The OFFLU AIM p ojec ope a es on a p inciple o mu ual bene i , ensu ing ha con ibu o s o i us samples and associa ed da a ecei e imely, meaning ul suppo . Pa icipa ing labo a o ies and submi ing ins i u ions can eques echnical assis ance, including da a analysis, in e p e a ion o an igenic and gene ic indings, and con ex -speci ic ecommenda ions. This suppo is pa icula ly aluable whe e accina ion is being conside ed o implemen ed. In such cases, es ing ep esen a i e local isola es agains mul iple wi hin-clade an ise a is ecommended o de ec an igenic d i which migh impac accine ma ch. In se ings whe e i uses a e enzoo ic in poul y o whe e mul iple clades o subclades co-ci cula e, accine challenge s udies using local s ains a e ad ised o e alua e accine e ec i eness. Reagen s (such as an ise a and inac i a ed i uses) p oduced unde he OFFLU AIM amewo k by APHA and IZSVe can also be sha ed, upon eques , wi h selec ed WOAH and FAO e e ence labo a o ies. This enables gene a ion o compa a i e an igenic da a using s anda dized eagen s and p o ocols unde a de ined quali y amewo k. Howe e , app op ia e in e -labo a o y s anda diza ion and in e p e a ion a e essen ial be o e da a om addi ional sou ces can be eliably in eg a ed in o analyses. Fo u he in o ma ion o echnical dialogue, please con ac : sec e a ia @offlu.o g. Disclaime s This epo p o ides he poin o iew o independen OFFLU expe s and does no necessa ily e lec he posi ion o he pa en o ganisa ions FAO and WOAH. Addi ionally, he e has been a ecen upda e o he H5 nomencla u e by he WHO/FAO/WOAH H5 Nomencla u e Wo king G oup o e lec he gene ic di e si ica ion o he A(H5) i uses, pa icula ly clade 2.3.2.1c, o add 2.3.2.1d, e, , and g (O e al., 2025). Whe e ele an , upda ed clade nomencla u es ha e been adop ed in his epo . 4 2. Execu i e Summa y OFFLU has es ablished a sys em o guide coun ies in how well hei accine seed s ains a e an igenically ma ched o con empo a y H5Nx Goose/Guangdong/1/96 (Gs/Gd) lineage high pa hogenici y a ian in luenza i uses (HPAIVs). Re e ence an ise a a e aised in chickens using ei he clade- ep esen a i e ield isola es o isola es ha gene ically mi o exis ing accine seed s ains (su oga e accine seed s ains). Con empo a y ield i uses a e hen es ed agains hese an ise a wi h haemagglu ina ion‑inhibi ion (HI) assays, and he esul ing i es a e analysed and isualised h ough an igenic ca og aphy o quan i y he dis ance be ween cu en i al an igens and hose used o gene a e he e e ence an ise a. La ge an igenic dis ances indica e an igenic d i , which means he abili y o an ibodies o neu alise s ains may be educed, ha in u n may comp omise accine e icacy. This may necessi a e upda ing accine seed s ains. Backg ound and me hods appea in Module 1 (Backg ound Repo )1 and Module 2 (Guidance Repo )2, while his documen con ains he echnical indings. As o July 2025, clade 2.3.4.4b H5N1 i uses accoun o mos poul y ou b eaks globally, bu H5Nx i uses om clades such as 2.3.4.4h, 2.3.2.1a, 2.3.2.1e, and 2.3.2.1g con inue o widely ci cula e in pa s o Sou h and Sou heas Asia. To cap u e hei combined impac , his OFFLU AIM assessmen applies HI es ing and an igenic ca og aphy o quan i y and isualise an igenic ela ionships be ween ield isola es om hese clades and wi hin-clade accine seed s ains. Fo hese analyses, he pos - in ec ion chicken an ise a panel was expanded o include h ee ep esen a i es om p e iously un es ed clades and wo addi ional clade 2.3.4.4b i uses selec ed o be e e lec con empo a y H5 di e si y in A ica. The es i us da ase comp ises new HI da a om 85 H5N1 i uses ep esen ing clades 2.3.2.1a, 2.3.2.1c, 2.3.2.1e, 2.3.4.4b, 2.3.4.4e, and 2.3.4.4h, co e ing wi h mul iple neu aminidase sub ypes (N1, N2, N5, N8, and o he s). These i uses we e collec ed be ween 2016 and 2025 om Eu ope, No h Ame ica, Sou h and Sou heas Asia, Eas Asia, and An a c ica, wi h es ing conduc ed by in e na ional e e ence labo a o ies a APHA (Uni ed Kingdom), IZSVe (I aly), and ACDP (Aus alia). Analyses conduc ed du ing his pe iod indica e ha he mos ecen H5 i uses om clade 2.3.4.4b, collec ed in 2024 and 2025, a e expec ed o be sui able candida e an igens o inac i a ed whole i us adju an ed accines a ge ing hese s ains, al hough some sub ype-speci ic he e ogenei y was obse ed, consis en wi h p e ious epo s. In con as , accine an igens om ou side clade 2.3.4.4, including con empo a y 2.3.2.1a i uses and ea lie ances al clades such as clades 1, 2.2.1, and 2.3.4 showed limi ed an igenic c oss- eac i i y and a e unlikely o o e e ec i e p o ec ion agains cu en ly ci cula ing clade 2.3.4.4b i uses. No ably, H5N1 i uses om clade 2.3.2.1a, pa icula ly hose de ec ed in Sou h Asia in 2024, exhibi ed educed eac i i y o bo h clade 2.3.4.4 and 2.3.2.1 an ise a, con inuing a end o inc easing an igenic di e gence obse ed since 2021. This da a demons a es ha con inued moni o ing o gene ic and an igenic changes, pa icula ly in coun ies whe e he i us emains enzoo ic in poul y, as well as egions ha pe iodically become ee o ci cula ion bu a e a isk o e- in oduc ion om nea by endemic a eas, is essen ial o he ea ly de ec ion o an igenic d i and he imely upda ing o accine composi ions. 1 h ps://www.offlu.o g/index.php/offlu-a ian-in luenza-ma ching-offlu-aim-in oduc ion-and-backg ound/ 2 h ps://www.offlu.o g/index.php/offlu-a ian-in luenza-ma ching-offlu-aim-guide- o-assessing-an igenic-cha ac e is ics-o -a ian-in luenza- i uses-june-2024/ 5 3. Backg ound Since 2020, H5Nx A/Goose/Guangdong/1996 (Gs/Gd) lineage high pa hogenici y a ian in luenza i uses (HPAIVs) ha e shi ed om being a seasonal h ea o poul y popula ions o a pe sis en global h ea o bo h poul y p oduc ion and wild bi d popula ions (EFSA, 2025). Wild bi d mig a ion has acili a ed hei sp ead o mos egions, igge ing unp eceden ed ou b eaks in domes ic poul y and causing la ge-scale mo ali y e en s in wild bi ds. These e en s ha e had subs an ial inancial impac s on poul y p oduc ion and ad e sely a ec ed a ian biodi e si y, including se e al species o conse a ion conce n. In a eas expe iencing high en i onmen al i al p essu e due o la ge-scale bi d mo ali y caused by HPAIV, he i us has also spilled o e spo adically in o e es ial and aqua ic mammalian species causing mass die-o e en s. The p ima y clade d i ing cu en global ou b eaks is 2.3.4.4b H5Nx, bu i uses om clades 2.3.2.1a, 2.3.2.1e, 2.3.2.1g, and 2.3.4.4h con inue o ci cula e in coun ies in Asia (Figu e 1). In esponse, global calls o he use o accina ion ha e spu ed a se ies o ini ia i es highligh ing he impo ance o moni o ing haemagglu inin (HA) e olu ion o in o m op imal accine s ain selec ion. Figu e 1. Global dis ibu ion o H5 clades acco ding o he upda ed nomencla u e be ween Sep embe 2022-Feb ua y 2025, based on in o ma ion om WHO epo s on ‘Gene ic and an igenic cha ac e is ics o zoono ic in luenza A i uses and de elopmen o candida e accine i uses o pandemic p epa edness’ om Feb ua y 2023-Feb ua y 2025. Geog aphic loca ions o de ec ions a e shown based on publicly a ailable sequence da a and in o ma ion. One key componen o e ec i e accina ion is main aining close an igenic ela edness be ween ci cula ing i uses and accine s ains, which can signi ican ly educe he impac o in ec ion in accina ed locks. Vaccina ion has been used in some coun ies o many yea s, and in ligh o he cu en si ua ion, many mo e coun ies a e now conside ing o implemen ing accina ion agains Gs/Gd-lineage i uses. Vaccina ion is suppo ed and ac i ely ecommended in ce ain con ex s o con ol HPAI (see Clade 2.3.4.4b Clade 2.3.2.1a 2.3.2.1e 2.3.2.1g 2.3.4.4h All o he clades 6 FAO/WOAH Global s a egy o he p e en ion and con ol o high pa hogenici y a ian in luenza (2024–2033)) 3 . Howe e , he con inued e olu ion o HPAIVs poses a pe sis en isk o an igenic d i and escape om an ibody-media ed neu alisa ion (Wang e al., 2025), unde sco ing he need o ongoing su eillance and imely pheno ypic cha ac e isa ion o in o m accine e ec i eness. The OFFLU A ian In luenza Ma ching (AIM) p ojec is a collabo a i e ini ia i e led by he Animal and Plan Heal h Agency (Uni ed Kingdom) wi h suppo om pa ne s in I aly (IZSVe), he USA (USDA Sou heas Poul y Resea ch Labo a o y), and Aus alia (CSIRO ACDP). I s p ima y goal is o gene a e and egula ly upda e a s anda dised panel o chicken se a and homologous an igens, se ing as an FAO–WOAH in e na ional s anda d o he an igenic cha ac e isa ion o ci cula ing a ian in luenza i uses o moni o i al e olu ion. The g oup ope a es eac i ely, con inuously moni o ing eme ging s ains and ob aining ele an isola es h ough global pa ne ships o ensu e an igenic ele ance. An igenic di e si y is assessed using s anda dised HI es ing (see p o ocols he e), wi h esul s isualised h ough an igenic ca og aphy o suppo accine s ain e alua ion in a cons an ly e ol ing landscape. The aims o he o e all and ongoing p ojec a e o: I. Gene a e a con inually upda ed panel o eagen s (an ise a and inac i a ed i uses), p oduced by APHA and IZSVe using local and in e na ionally eques ed a ian in luenza i uses (AIVs). II. E alua e hese eagen s a APHA and IZSVe, alongside eme ging i al s ains, o gene a e p elimina y haemagglu ina ion inhibi ion (HI) da a using s anda dised HI assays. III. Collabo a e wi h o he WOAH and FAO in e na ional e e ence labo a o ies in s a egically selec ed loca ions by sha ing p o ocols and eagen s o b oaden an igenic assessmen co e age. IV. P oduce quali y assu ed compa a i e HI da a ac oss labo a o ies and me ge hese in o an igenic dis ance da a o assess he an igenic cha ac e is ics o con empo a y i uses as hey eme ge globally. V. S anda dise labo a o y p ocedu es by sha ing p o ocols and gene a ing ha monised eagen s and me hodologies wi hin de ined quali y amewo ks, enabling global mul i-cen e compa isons. In addi ion o es ablishing ha monised me hods ac oss designa ed labo a o ies, an igenic da a gene a ed h ough AIM complemen s da a om FAO and WOAH membe coun ies, OFFLU pa ne s, and egional su eillance ne wo ks. This includes epidemiological da a, e idence o accine b eak h ough, and expe imen al s udies. Toge he , hese da a could help o in o m need o upda e accine an igens and may suppo alignmen among accine manu ac u e s unde aking simila analyses. By iden i ying a eas o inc easing an igenic di e si y, his wo k helps posi ion esponses o exis ing accines (Table 1) wi hin an igenic maps o HA glycop o ein a ia ion. While AIM does no di ec ly assess accine e icacy, he combined da a p o ide a obus basis o conduc ing challenge s udies in accina ed bi ds o assess p o ec ion, upda ing poul y accine an igens, and e ining accina ion s a egies. 3 h ps://openknowledge. ao.o g/i ems/4c4 67c1-c8c7-401b-9d04-720493406c19 7 3.1. Vaccine Seed S ains Table 1. Seed s ains o H5 accines which a e ei he cu en ly unde s ood o be in use o used p e iously. Whe e a ailable clade in o ma ion and e e ences ha e been included. An as e isk (*) indica es ins ances whe e he clade nomencla u e may di e om he la es designa ion o he WHO/FAO/WOAH H5 Nomencla u e Wo king G oup. Seed S ain / HA Gene Sou ce Clade Re e ence Rep esen ed in AIM A/duck/Po sdam/1402/86 non-Gs/Gd DEFRA Vaccina ion Task o ce4 No A/Goose/Guangdong/96 0 (Shi e al., 2023) No A/chicken/Vie nam/C58/04 1 (EFSA Panel on Animal Heal h and Animal Wel a e (AHAW) e al., 2023) Yes A/Vie nam/1194/2004 1 (EFSA Panel on Animal Heal h and Animal Wel a e (AHAW) e al., 2023) Yes A/chicken/Legok/2003 2.1.1 (EFSA Panel on Animal Heal h and Animal Wel a e (AHAW) e al., 2023) Pending A/Swan/Hunga y/4999/2006 2.2 DEFRA Vaccina ion Task o ce Yes A/CK/Egyp /ME1010/2016 2.2.1.1 (EFSA Panel on Animal Heal h and Animal Wel a e (AHAW) e al., 2023) Pending A/Chicken/Egyp /Q1995D/2010 2.2.1.2 (EFSA Panel on Animal Heal h and Animal Wel a e (AHAW) e al., 2023) No A/Chicken/Egyp /RG-173 CAL/2017 2.2.1.2 (EFSA Panel on Animal Heal h and Animal Wel a e (AHAW) e al., 2023) Pending A/Duck/EGYPT/M2583D/2010 2.2.1.2 (EFSA Panel on Animal Heal h and Animal Wel a e (AHAW) e al., 2023) Yes A/chicken/Wes -Ja a/Pw -Wij/2006 2.3.1.2 (EFSA Panel on Animal Heal h and Animal Wel a e (AHAW) e al., 2023) No A/duck/China/E319-2/2003 2.3.2 DEFRA Vaccina ion Task o ce No A/duck/Anhui/SI246/2014 2.3.2.1 (Shi e al., 2023) No A/Hubei/1/2010 2.3.2.1a (EFSA Panel on Animal Heal h and Animal Wel a e (AHAW) e al., 2023) Yes A/duck/Guangdong/S1322/2010 2.3.2.1b (Shi e al., 2023) Yes A/chicken/Tanggamus/031711076-65/2017 2.3.2.1c* (EFSA Panel on Animal Heal h and Animal Wel a e (AHAW) e al., 2023) No A/chicken/Liaoning/SD007/2017 2.3.2.1d (Shi e al., 2023) Pending gCA2/2.3.2.1d 2.3.2.1d (Kang e al., 2022) No A/chicken/Vie nam/NCVD-KA435/13 2.3.2.1e* (EFSA Panel on Animal Heal h and Animal Wel a e (AHAW) e al., 2023) Pending A/duck/Sukoha jo/BBVW-1428- 2.3.2.1g* (Ind iani and Dha mayan i, 2014) Yes A/Duck/Anhui/1/2006 2.3.4 (Shi e al., 2023) Yes A/Wa e owl/Ko ea/S57/2016 2.3.4.4 Ku uppua achchi e al., 2022 No A/chicken/Egyp /ME-2018/2018 2.3.4.4b (Ib ahim e al., 2021) Yes A/g een-winged eal/Egyp /877/2016 2.3.4.4b (EFSA Panel on Animal Heal h and Animal Wel a e (AHAW) e al., 2023) Yes A/whoope swan/Shanxi/4–1/2020 2.3.4.4b (Shi e al., 2023) Yes A/ u key/Indiana/22-003707-003/2022 2.3.4.4b DEFRA Vaccina ion Task o ce No A/gy alcon/Washing on/41088-6/2014 2.3.4.4c (EFSA Panel on Animal Heal h and Animal Wel a e (AHAW) e al., 2023) Yes A/duck/Ko ea/ES2/2016 2.3.4.4e (EFSA Panel on Animal Heal h and Animal Wel a e (AHAW) e al., 2023) Pending A/chicken/Guizhou/4/2013 2.3.4.4g (Shi e al., 2023) No A/duck/Guizhou/S4184/2017 2.3.4.4h (Shi e al., 2023) Yes A/duck/Fujian/S1424/2020 2.3.4.4h (Shi e al., 2023) Pending gES3/2.3.4.4h 2.3.4.4h (Kang e al., 2022) No A/duck/Guanzou/S4184/2017 2.3.4.4h (Shi e al., 2023) Pending A/Duck/Vie Nam/QB7412 unknown (EFSA Panel on Animal Heal h and Animal Wel a e (AHAW) e al., 2023) No A/Chicken/Shanxi/2/2006 7.2 (Shi e al., 2023) No A/Chicken/Liaoning/S4092/2011 7.2 (Shi e al., 2023) No 4 h ps://www.go .uk/go e nmen /publica ions/ accina ion-o -poul y-agains -highly-pa hogenic-a ian-in luenza/ accina ion-o -poul y-agains -highly- pa hogenic-a ian-in luenza-hpai-join -indus y-and-c oss-go e nmen - accina ion- ask o ce 8 3.2. Panel o Chicken An ise a The AIM pos in ec ion chicken an ise a panel has been expanded o include h ee ep esen a i es om p e iously un es ed clades and wo addi ional 2.3.4.4b i uses ha mo e accu a ely e lec he cu en H5 di e si y (Table 2). Table 2. Vi uses used in OFFLU AIM o gene a e pos -in ec ion chicken an ise a, including hose in he o iginal co e panel and hose selec ed o he expanded panel. A double-dagge (‡) indica es an an igen used as a su oga e o a accine s ain. Clade in o ma ion is p o ided. S ain Sub ype Clade Rele an accine seed s ain A/Vie nam/1194/2004/1‡ H5N1 1 A/chicken/Vie nam/C58/04 A/ u key/ u key/2005* H5N1 2.2 A/swan/Hunga y/4999/2006 A/duck/Egyp /MS2583D/2010‡ H5N1 2.2.1.1 A/Chicken/Egyp /Q1995D/2010 A/chicken/Nepal/T360/2014‡ H5N1 2.3.2.1a A/duck/Guangdong/S1322/2010 A/Hubei/1/2010‡ H5N1 2.3.2.1a A/Hubei/1/2010 A/duck/Bangladesh/17D1012/2018 H5N1 2.3.2.1a A/mynah/Indonesia(Aus iaQ)/13064792-010/2013‡ H5N1 “2.3.2.1c” like A/duck/Sukoha jo/BBVW-1428- 9/2012 A/Anhui/1/2005* (RG) HN51 2.3.4 A/Duck/Anhui/1/2006 A/Guizhou/1/2013 H5N1 2.3.4.2 A/chicken/Czech Republic/1175-1-20VIR465-1/2020 H5N8 2.3.4.4b A/chicken/Bulga ia/722-1-22VIR778-1/2021 H5N1 2.3.4.4b A/duck/Cambodia/ 4k241D3/2021‡ H5N8 2.3.4.4b A/whoope swan/Shanxi/4–1/2020 A/g ea skua/Sco land/B07779/2021 H5N1 2.3.4.4b A/malla d/Geo gia/DT09382/2017‡ H5N8 2.3.4.4b A/chicken/ME-2018 A/mu e swan/C oa ia/102/2016* H5N5 2.3.4.4b A/g een-winged eal/Egyp /877/2016 A/chicken/Nige ia/VRD-21-338/21VIR7423-29/2021 H5N1 2.3.4.4b A/chicken/Nige ia/VRD-011_23VIR8254-67/2023 H5N1 2.3.4.4b A/gy alcon/Washing on/41088-6/2014‡ H5N8 2.3.4.4c A/Gy alcon/WA/41088-6/2014 A/Guangdong/18SF020/2018 H5N6 2.3.4.4h 9 3.3. Phylogene ic Rela ionships Figu e 2. Maximum likelihood phylogene ic ees o he HA1 o H5 Gs/Gd lineage i uses included in his p ojec and accine seed s ains whe e sequences we e a ailable. Tips a e colou ed by es an igens, accine seed s ains, an ise a used in his s udy. Majo clades a e anno a ed along he b anches acco ding o nomencla u e. 16 4.2 An igenic Ca og aphy An igenic ca og aphy was unde aken o u he quan i y and isualise he an igenic dis ances be ween i us’s ep esen a i e o accine seed s ains and con empo a y ci cula ing i uses as desc ibed in he AIM pilo p ojec . Maps we e analysed and isualised using R S udio e sion 2023.03.1+446 and he package ‘Racmacs’ 6 1.2.9 buil unde R 4.3.1 as desc ibed by (Smi h e al., 2004) and p e iously used in (Lewis e al., 2021). Fo in o ma ion ega ding map gene a ion and in e p e a ion please con ac OFFLU expe s ia he sec e a ia email (sec e a ia @offlu.o g). Amino acid changes in he HA1 we e isualised by econs uc ing ances y using ‘T eeTime’ 0.11.1 (Sagulenko, Pulle and Nehe , 2018) and we e compa ed be ween wi hin- clade and wi hin-sub ype es an igens. An igenic maps we e colou ed acco ding o unpublished i al clade nomencla u e and sub ype using he H5Nx da ase in Nex clade (O e al., 2025). These esul s desc ibed he e and abo e (sec ion 4.1) sugges ha o he accine seed s ains and su oga es es ed, we would expec he cu en ly used H5N8 clade 2.3.4.4 an igens o emain sui able candida es o inac i a ed whole- i us adju an ed accines o mos con empo a y clade 2.3.4.4b i uses. An igens which exhibi highe an igenic dis ance om he se a should be p io i ised o es ing in accine e icacy challenge s udies and wo k is equi ed o de e mine o each accine echnology he cu -o an igenic dis ance associa ed wi h loss o p o ec ion. Fu he mo e, sub ype-speci ic he e ogenei y was obse ed, consis en wi h p e ious epo s and his should be conside ed u he . In con as , accine an igens om ou side clade 2.3.4.4 - including con empo a y 2.3.2.1a i uses and ea lie ances al clades such as 1, 2.2.1, and 2.3.4 - showed limi ed c oss- eac i i y and a e unlikely o p o ide e ec i e p o ec ion agains cu en ly ci cula ing 2.3.4.4b i uses. No ably, H5N1 i uses om clade 2.3.2.1a, pa icula ly hose de ec ed in Sou h Asia in 2024, exhibi ed educed eac i i y o an ise a om bo h clades 2.3.4.4 and 2.3.2.1, con inuing a end o inc easing an igenic di e gence obse ed since 2020. These esul s unde sco e he alue o analysing i us ela ionships a a egional le el, pa icula ly in Sou h and Sou h-Eas Asia, whe e accine equi emen s may need o add ess bo h clade 2.3.4.4b i uses and o he ci cula ing clades such as 2.3.2.1a, 2.3.2.1e, and 2.3.2.1g. 6 h ps://gi hub.com/aco g/Racmacs/ 17 Figu e 6. Th ee-dimensional an igenic map illus a ing he ela ionships among Gs/Gd-lineage a ian in luenza i uses cha ac e ized in his s udy. Each sphe e ep esen s a i us (an igen), colou ed by sub ype and clade; squa es ep esen pos - accina ion chicken an ise a. Clade labels a e placed on ep esen a i e accine seed s ains. Panel A: O e iew o he ull an igenic map, wi hou highligh s. Panel B: An igens cha ac e ized om 2024 and 2025 a e highligh ed, including mul iple clade 2.3.4.4b i uses and one clade 2.3.2.1a i us. 18 5. Conclusions and Fu u e Di ec ions In summa y, he OFFLU AIM p ojec con inues o p o ide a c i ical ounda ion o he global moni o ing o an igenic e olu ion in ci cula ing H5 i uses. Resul s om his epo ing pe iod highligh he inc easing di e gence o H5 i uses o e ime, he apid e olu iona y dynamics ha unde sco e he need o ongoing eassessmen , and he impo ance o examining ela ionships wi hin a clade o assess po en ial egional implica ions o an igenic d i . We belie e his da a is i al o he in e na ional communi y and especially hose conce ned wi h decision making o e whe he o accina e and wha accine o use. This p og amme p o ides us ed impa ial science-based e idence ha we hope will shape and in o m all hose conce ned wi h ei he main aining o newly implemen ing accina ion p og ammes. Con inued Su eillance and Tes Vi us Selec ion We will con inue o moni o and cha ac e ise newly eme ging H5Nx i uses om he Gs/GD lineage, wi h a ocus on expanding ep esen a ion o unde ep esen ed clades in he es i us panel, pa icula ly 2.3.2.1a, 2.3.2.1c, 2.3.2.1g, 2.3.2.1e, and 2.3.4.4h, o be e cap u e he di e si y o ci cula ing i uses. Ou pu s will be deli e ed h ough he annual echnical epo , execu i e epo , and a webina in la e 2025 o ea ly 2026 o in e es ed s akeholde s. Gaps in es i us ep esen a ion will be add essed h ough bo h classical and e e se gene ics app oaches. Classical app oaches will include he ongoing acquisi ion and sha ing o li e i us isola es and associa ed eagen s wi h global pa ne s. Fo i uses whe e isola ion is unsuccess ul o no ou inely pe o med, e e se gene ics (RG) will be applied. This p ocess will be conduc ed anspa en ly, wi h sequence da abase con ibu o s sys ema ically con ac ed o eques pe mission o sequence use, ensu ing hey a e acknowledged and kep in o med. Expansion and Re inemen o he Re e ence An ise a Panel Concu en ly, he OFFLU e e ence an ise a panel will be expanded and e ined o emain globally ep esen a i e and ele an . An ise a p oduc ion a ge ing clades 2.3.4.4h and 2.3.2.1a/c/g is unde way, alongside p oduc ion o addi ional pu a i e accine seed s ains no ye ep esen ed in he AIM panel (see Table 2). P io i y will be gi en o a clade 2.3.4.4b H5N1 sub- An a c ic isola e iden i ied as an an igenic ou lie , which is gene ically ela ed o i uses de ec ed in Chile, U uguay, B azil, and se e al sub-An a c ic island popula ions. Ha monisa ion and ing es ing ac oss pa icipa ing labo a o ies will emain cen al o ensu ing consis en p oduc ion and applica ion o e e ence an ise a. B oadening he Scope o he AIM F amewo k To mee e ol ing needs, he AIM p ojec will expand bo h in scope and unc ionali y. Planned de elopmen s include e alua ing no el accine concep s such as compu a ionally op imised b oadly eac i e an igens (COBRA) (Giles and Ross, 2011) o de e mine hei capaci y o induce an ibody esponses agains con empo a y ci cula ing s ains; ex ending he AIM amewo k o addi ional i us sub ypes, beginning wi h H9, using compa able me hodologies o assess global an igenic di e si y and guide accine s ain selec ion; and enhancing epo ing and communica ion h ough he in oduc ion o a mee ing- epo o ma o egula ou pu s. Labo a o ies and in e es ed pa ies will be in i ed o con ibu e da a o suppo e iews o an igenic di e si y, pa icula ly o su ace p o eins, and o assess implica ions o accine pe o mance. Epidemiological de elopmen s ha may necessi a e accine upda es (e.g., he eme gence o a no el i us in a egion wi h exis ing accine use) will also be iden i ied. These ou pu s will complemen echnical epo s and aim o s eng hen anspa ency and in o ma ion sha ing ac oss he ne wo k. Ne wo k G ow h and Capaci y Building 19 Expanding he AIM ne wo k emains a p io i y, wi h a ocus on engaging addi ional global e e ence labo a o ies, pa icula ly in egions o s a egic impo ance. Ta ge ed capaci y-building e o s will be unde aken o suppo labo a o y pa icipa ion and enable b oade e alua ion o an igenic di e si y using ha monised p o ocols. Simul aneously, we will ocus on add essing he echnical challenges o assimila ing da a om mul iple sou ces in o a single ca og aphic ead-ou , e en when common s anda ds a e applied. Links o Key AIM Documen s Concep No e Module 1: OFFLU AIM epo backg ound upda ed 2024 Module 2: OFFLU AIM Annex 1- sub-na ional and na ional le el guidance o coun ies on assessing he an igenic cha ac e is ics OFFLU AIM Webina (English) OFFLU AIM Webina (Spanish) OFFLU AIM Webina Summa y and FAQs OFFLU AIM Pilo Repo (Oc obe 2023) OFFLU AIM Technical Repo (July 2024) OFFLU AIM Execu i e Summa y (Oc obe 2024) Re e ences EFSA (2025) ‘A ian in luenza o e iew Decembe 2024–Ma ch 2025’, EFSA Jou nal, 23(4), p. e9352. A ailable a : h ps://doi.o g/h ps://doi.o g/10.2903/j.e sa.2025.9352. Banya d, A.C. e al. (2024) ‘De ec ion and sp ead o high pa hogenici y a ian in luenza i us H5N1 in he An a c ic Region’, Na u e Communica ions, 15(1), p. 7433. A ailable a : h ps://doi.o g/10.1038/s41467-024-51490-8. EFSA Panel on Animal Heal h and Animal Wel a e (AHAW), E.U.R.L. o A.I. e al. (2023) ‘Vaccina ion o poul y agains highly pa hogenic a ian in luenza – pa 1. 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