OFFLU AVIAN INFLUENZA
MATCHING (OFFLU-AIM) REPORT
23 Oc obe 2023
OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 2
Con en s
Summa y ..................................................................................................................................................... 3
In oduc ion ................................................................................................................................................ 5
Findings ...................................................................................................................................................... 7
Clade 2.3.4.4b......................................................................................................................................... 7
Clade 2.3.2.1a......................................................................................................................................... 9
Clade “2.3.2.1c” ...................................................................................................................................... 9
In luenza A(H7N6) in Sou h A ica ......................................................................................................... 10
Conclusions............................................................................................................................................... 10
Re e ences ................................................................................................................................................. 10
Annex 1 (A guide o assessing an igenic cha ac e is ics o a ian in luenza i uses a
na ional/sub-na ional le el labo a o ies) ................................................................................................ 13
Annex 2 (An igenic Ca og aphy Resul s) ............................................................................................. 15
Summa y ................................................................................................................................................ 15
In oduc ion ............................................................................................................................................ 16
Vaccine seed s ains .............................................................................................................................. 16
Me hods, analyses and esul s .............................................................................................................. 16
Gene a ion o chicken an ise a .............................................................................................................. 16
Hemagglu ina ion Inhibi ion (HI) assay .................................................................................................. 18
An igenic Ca og aphy ........................................................................................................................... 18
Chicken an ise a panel .......................................................................................................................... 19
An igenic maps ...................................................................................................................................... 20
Table o an igenic dis ances .................................................................................................................. 21
Main indings .......................................................................................................................................... 22
2.3.2.1a .................................................................................................................................................. 22
2.3.4.4b .................................................................................................................................................. 22
Recommenda ions and nex s eps o OFFLU AIM............................................................................... 23
Re e ences............................................................................................................................................. 24
Acknowledgemen s .................................................................................................................................. 26
OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 3
Summa y
E ec i eness o accina ion o poul y agains high pa hogenici y a ian in luenza (HPAI) depends on usage
o app op ia e accines a he ecommended doses and age. An igenic a ia ion in ield s ains may ende
accines less e ec i e. This is pa icula ly ue o accines ha depend p ima ily on he humo al immune
esponse, such as killed an igen (inac i a ed) adju an ed accines.
OFFLU-AIM is designed o p o ide in o ma ion on possible an igenic changes in HPAI i uses ha could
educe hei e ec i eness. This epo ocuses on goose/Guangdong/1/96-lineage A(H5Nx) i uses
(gsGD). The in o ma ion in his epo is de i ed om mul iple sou ces including an igenic s udies
conduc ed a OFFLU e e ence labo a o ies.
Clade 2.3.4.4b A(H5N1) HPAI i uses pose he g ea es a ian in luenza h ea globally.
They a e ci cula ing ac oss Eu asia, pa s o A ica, and he Ame icas. Mos ac i i y a p esen (Oc obe
2023) has been epo ed om No he n Eu ope wi h a ecen inc ease in cases in No h Ame ica. These
i uses a e s ill p esen in o he egions, causing disease in wild bi ds, wild mammals and, in some places,
poul y. They a e expec ed o expand hei ange o o he pa s o Eu ope, No h Ame ica and A ica in he
au umn o 2023, la gely ia ca iage o i uses by wild bi ds.
A p esen , he e is no in o ma ion a ailable o sugges ha signi ican an igenic a ian clade 2.3.4.4b
i uses a e ci cula ing ha would in e e e wi h accina ion. This equi es use o accines based on ela ed
clade 2.3.4.4 i uses (including an igens de i ed om 2.3.4.4b and 2.3.4.4c i uses) and hose based on
o he clades/an igens ha ha e been demons a ed o be e icacious agains clade 2.3.4.4b i uses in
expe imen al s udies. These accines a e likely o p o ide a high deg ee o p o ec ion, as long as su icien
doses a e gi en, and o he diseases do no esul in immunosupp ession a he ime o accina ion.
Di e ences in esponse o accines in ypes o poul y o he han chickens need o be ecognized.
Vaccine ials conduc ed in F ance and he Ne he lands demons a ed p o ec ion om disease and
ansmission in poul y agains clade 2.3.4.4b i uses isola ed in hese wo loca ions in 2021. The F ench
s udy in ol ed mule ducks accina ed wi h ei he a sel - eplica ing RNA i us accine o a subuni accine.
The accines p o iding p o ec ion in laye s in he Ne he lands we e HVT ec o accines. These
expe imen al s udies may no e lec he si ua ion in he ield whe e o he ac o s can impac on accine
e ec i eness bu did demons a e good p o ec ion unde labo a o y condi ions. Elsewhe e, upda ed
accine an igens we e in oduced o clade 2.3.4.4b i uses in China (e.g., e-14) and Egyp based on
s ains om 2020 and 2018, espec i ely. Addi ional s udies ha e been conduc ed in u keys bu ha e no
been epo ed o mally. Fo hose in ending o accina e u keys, we ecommend discussions wi h OFFLU
o o he expe s when de e mining app op ia e me hods and candida e p oduc s o conside o accina ion.
Killed an igen accines de eloped agains ea lie gsGD clades, such as clade 2.2, clade 2.1, 2.3.2.1, clade
1 o clade 7, and hei de i a i es, a e unlikely o a o d consis en p o ec ion agains clade 2.3.4.4b i uses
and should only be used as a componen o accines in places whe e i uses in hese o he clades pe sis .
An igenic di e ences also exis be ween clade 2.3.4.4h and 2.3.4.4b i uses ha would educe e icacy o
inac i a ed accines based on he o me .
Clade 2.3.2.1a i uses a e p esen in Sou h Asia whe e accina ion is used o icially in Bangladesh, wi h
some in o mal usage o accina ion elsewhe e. Up o 2018, i uses om Bangladesh demons a ed
easonable an igenic s abili y bu insu icien e idence is a ailable on ecen i uses. Tes s should be
done agains ecen s ains om Sou h Asia o assess whe he an igenic changes ha e occu ed ha migh
a ec e icacy o exis ing a ian accines. Inac i a ed accines based on clade 2.3.4.4b would no be
expec ed o p o ide app op ia e p o ec ion agains clade 2.3.2.1a i uses.
OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 4
Clade “2.3.2.1c”
1
i uses a e s ill ci cula ing in SE Asia, including Cambodia, Laos and Vie nam. Fu he
s udies a e needed on he an igenic cha ac e is ics o ecen i uses, howe e gene ic in o ma ion sugges s
hese i uses a e likely o ha e emained an igenically s able. Inac i a ed accines based on Clade 2.3.4.4b
would no be expec ed o p o ide consis en p o ec ion agains clade 2.3.2.1c i uses.
O he i uses e e ed o as clade “2.3.2.1c” in Indonesia, and mo e ecen ly as 2.3.2.1e, a e s ill p esen
in Indonesia and Timo Les e bu ew ha e been isola ed o ully cha ac e ized ecen ly. Again, u he
s udies a e needed o assess an igenic cha ac e is ics o hese ecen i uses. As wi h o he clade 2.3.2.1
i uses, inac i a ed accines based on clade 2.3.4.4b i uses would no be expec ed o p o ide consis en
p o ec ion agains hese i uses.
Vi uses belonging o mul iple gsGD HA clades, including ecen ly in oduced clade 2.3.4.4b i uses, a e
co-ci cula ing in some coun ies. I is s ill no known whe he p e-exis ing s ains will pe sis in hese places
once clade 2.3.4.4b becomes es ablished. Ne e heless, i appea s ha clade 2.3.4.4h has la gely been
eplaced by clade 2.3.4.4b in Asia. I wo an igenically dis inc s ains pe sis , conside a ion should be gi en
o in oducing a bi alen accine, as has been done in some coun ies in he pas , o ensu e op imal
p o ec ion agains bo h ci cula ing s ains.
When accines a e used, pos - accinal an ibody esponses should be moni o ed o ensu e an app op ia e
esponse o accina ion. I using killed an igen accines, locks should be moni o ed h oughou he li e o
he accina ed lock. Boos e doses should be p o ided i i es all below le els expec ed o p e en
disease. An ibody i es a e less eliable indica o s o likely p o ec ion when using ec o accines alone.
I is also essen ial o moni o o in ec ion in accina ed locks. Any i uses de ec ed in accina ed locks
should be cha ac e ised and assessed o any po en ial adap a ions ha migh e lec an igenic changes.
No hing in his epo should be cons ued as an endo semen o a speci ic p oduc and he in o ma ion
p o ided is gene al in na u e. Addi ional suppo on app op ia e accines can be ob ained om FAO,
WOAH and OFFLU.
1
Clade 2.3.2.1c i uses ha e e ol ed in o di e en sublineages since hey we e las o mally classi ied in 2014. An
upda ed, s anda dised clade nomencla u e o i uses ha p e iously ell wi hin clade 2.3.2.1c is being de eloped
bu has no ye been accep ed o mally. Ne e heless, i uses om Indonesia ha we e p e iously e e ed o as
2.3.2.1c ha e been e e ed o as 2.3.2.1e in OFFLU and WHO documen s o se e al yea s (see oo no e 2), based
on ad ice om molecula gene icis s, gi en hey di e om hose ound in mainland SE Asia. Fu he changes in
classi ica ion a e expec ed o i uses wi hin clade 2.3.2.1c and OFFLU will ad ise once he new nomencla u e has
been accep ed and adop ed.
OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 5
In oduc ion
Cu en ly, some coun ies using accines agains a ian in luenza ha e mechanisms in place o moni o
an igenic changes in ield s ains. Howe e , his in o ma ion is no always sha ed in e na ionally o made
publicly a ailable. The goal o OFFLU A ian In luenza Ma ching (AIM) p ojec (OFFLU-AIM) is o p o ide
imp o ed in o ma ion on an igenic cha ac e is ics o a ian in luenza i uses o suppo accina ion
p og ams agains a ian in luenza. I will also assis coun ies u ilising accines o s ay in o med abou
po en ially signi ican changes in he an igenic cha ac e is ics o ci cula ing s ains. This in o ma ion is also
o alue o coun ies conside ing in oduc ion o accina ion as an addi ional p e en i e/con ol measu e.
OFFLU-AIM se es as a pla o m whe e cu en epidemiological, gene ic, and an igenic in o ma ion on AI
i uses ci cula ing in di e en geog aphic egions is analysed and made eely accessible. In o ma ion
comes om a ange o sou ces including published in o ma ion, da a om coun ies moni o ing o a ian
in luenza i uses including po en ial an igenic a ian s, accine challenge ials, speci ic an igenic
ca og aphy and o he in elligence ha wa an s conside a ion by coun ies using o conside ing
accina ion.
Mul iple coun ies a e using accina ion agains gsGD HPAI i uses, gi en he di icul ies encoun e ed in
de ec ing and p e en ing all in ec ions and he h ea o zoono ic in ec ions. Ea ly adop e s o accina ion
include China, Egyp , Indonesia, Vie nam and Bangladesh – all places whe e he i us was well en enched
in poul y be o e accina ion commenced. Se e al o he coun ies used accines b ie ly in poul y and
un egis e ed accine is being applied in some o he coun ies, a p ac ice ha OFFLU does no ecommend.
Some places (e.g., F ance and Hong Kong SAR) use p e en i e accina ion, despi e he i us no being
p esen in poul y, because o he high isk o in ec ion.
The inc eased h ea posed by clade 2.3.4.4b i uses since 2020 has been a ca alys o inc eased
conside a ion o adop ion o accina ion. In 2023, F ance ( accina ion o mule ducks used o oie g as
p oduc ion) and se e al coun ies in Sou h and Cen al Ame ica ha e in oduced accina ion.
Mos o he accines used a p esen a e adju an ed killed an igen accines con aining whole i us, usually
p oduced by e e se gene ics. O he accines a e also in use o unde de elopmen , including i al ec o
accines such as hose using he pes i us o u keys (HVT) as he ec o , subuni accines (used cu en ly
in F ance), sel -ampli ying RNA accines, DNA accines, i us-like pa icles, and eplicon pa icles. Some
o hese accines, especially hose con aining a li e i us can p oduce b oade c oss p o ec ion agains
he e ologous s ains o A(H5Nx) HPAI i us because hey also s imula e cell media ed immuni y (see o
example Palya e al 2018). This means ha an igens used in hese accines do no necessa ily equi e
upda ing as equen ly as hose in killed an igen accines; hey can ole a e some changes in an igenici y.
Ne e heless, i is s ill c i ical o moni o locks accina ed wi h hese accines o de ec in ec ion and o
cha ac e ise any i uses ound in accina ed locks, by unde aking a ho ough assessmen o hei
an igenic cha ac e is ics.
The ocus o his i s OFFLU-AIM epo is on HPAI i uses o he H5Nx sub ype in he
goose/Guangdong/1/96 lineage (gsGD). These HPAI i uses eme ged in China in 1996 and ha e been
ci cula ing as high pa hogenici y i uses since ha ime. They ha e e ol ed h ough poin mu a ions in o
mul iple 5 h o de HA clades and ha e easso ed o p oduce mul iple geno ypes. They ha e unde aken
se e al in e con inen al wa es o ansmission since 2005 due mainly o ca iage by wild bi ds, p esumably
a e spillback o in ec ion om domes ic poul y in places whe e wild bi ds and poul y mix eely. O he
ini ial 10 clades ha eme ged in he ea ly 2000s only i uses wi hin clade 2 and i s de i a i es ha e
pe sis ed and e ol ed wi h he i uses de ec ed ecen ly belonging o clades 2.3.4.4b (widesp ead globally
excep Oceania), 2.3.2.1a (Sou h Asia), “2.3.2.1c” (lowe Mekong including Cambodia, Laos and Vie nam
wi h ela ed s ains in Indonesia and Timo Les e) and possibly 2.3.4.4h. Vaccines a ge ing each o hese
clades a e being used in a leas one coun y (2.3.4.4b – mul iple coun ies, 2.3.2.1a – Bangladesh,
“2.3.2.1c” – Vie nam; “2.3.2.1c” – Indonesia; 2.3.4.4h - China).

OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 6
This epo should be ead in conjunc ion wi h he OFFLU and WHO accine composi ion mee ing epo s
co e ing a ian in luenza and o he po en ially zoono ic in luenza A i uses de ec ed om Feb ua y o
Sep embe 2023
2
. The ocus o he VCM epo is p epa a ion o candida e accine an igens o use in
humans in he e en o an in luenza pandemic bu i p o ides impo an in o ma ion on i uses de ec ed
ecen ly. I also p o ides in o ma ion on an igenic and gene ic cha ac e is ics o i uses wi h he o me
pe o med using e e an ise a p oduced agains human candida e accine e e ence an igens, no chicken
se a and a ian accine an igens. Se a om e e s can p oduce di e en esul s o hose aised in
chickens.
OFFLU-AIM is building capaci y o es i uses using an ise a aised in chickens agains a ian accine
an igens, o su oga e an igens when he accine s ain is no a ailable. In his i s epo some ini ial
s udies we e conduc ed using i uses de ec ed in 2020, 2021 and 2022, p edominan ly belonging o clade
2.3.4.4b (see Annex 2). This wo k will be expanded in subsequen yea s.
Two ypes o e en s a e ele an when conside ing an igenic changes in a ian in luenza i uses. The i s
is he e olu ion o i uses agains which accines a e al eady being used wi hin he coun y o egion.
Since gsGD HPAI i uses eme ged hey ha e unde gone conside able an igenic a ia ion, especially in
coun ies whe e hese i uses emain en enched. Examples include he an igenic a ia ion ha occu ed
in Egyp ian clade 2.2 i uses and Indonesian clade 2.1 i uses, and hei de i a i es (A a a e al 2012,
Swayne e al, 2015). An igenic change has occu ed apidly in some places whe eas in o he places he
same accine an igen has been used and emained app op ia e o e mul iple yea s (e.g., accines used
in Vie Nam agains clade 1 i uses and hei de i a i es om 2005 onwa ds un il his clade was eplaced
by clade 2.3.2.1c).
The second is he in oduc ion o a s ain o i us ha di e s signi ican ly om exis ing i uses and accine
an igens. In o he wo ds, an igenic changes de eloped elsewhe e, and he no el s ain was impo ed,
usually, bu no always, ia wild bi ds. This is exempli ied by he ecen in oduc ion o A(H5N1) clade
2.3.4.4b o Indonesia whe e accines in use a e designed o p o ec agains A(H5N1) clade “2.3.2.1c”
i uses ha s ill ci cula e he e. In some coun ies he new s ain eplaced he exis ing s ain(s) bu in o he s
bo h p e-exis ing and new s ains co-ci cula e o he new s ain is es ic ed o only pa o he coun y (e.g.,
clade 2.3.4.4b in Indonesia).
The in o ma ion in his documen is gene ic in na u e and depends on epo s o in o ma ion om indi idual
coun ies on ci cula ing i uses. I does no imply p e e ence o speci ic accines o p oduc s. All coun ies
conside ing accina ion should use he in o ma ion in his documen as a supplemen o da a collec ed
locally on ci cula ing s ains and on a ailable accines when de e mining he mos app op ia e accines
and accina ion s a egies o apply.
We u ge all coun ies o con inue sha ing in o ma ion on no el s ains o i us, especially hose suspec ed
o being an igenic a ian s (such as i uses ha a e ci cula ing in well accina ed poul y) so ha OFFLU
can con inue o p o ide up- o-da e in o ma ion on i uses in ci cula ion. Whe e possible, and especially i
he e a e suspicions o an igenic a ian s, i uses and gene sequences should be sha ed wi h OFFLU
e e ence labo a o ies o allow ull an igenic and gene ic cha ac e iza ion.
Much o he in o ma ion in his documen is o g ea e ele ance o killed an igen accines (s ill he mos
commonly used a ian in luenza accines globally) han accines ha a e based on a li e ec o i us o
o he s ha also s imula e cell media ed immuni y and do no ely p ima ily on an ibody media ed esponses
2
A ailable a h ps://www.o lu.o g/wp-con en /uploads/2023/10/A ian-OFFLU-VCM-S23-OFFLU-V6-VCM-OFFLU.pd .
The WHO epo ha includes hese da a is a ailable a h ps://cdn.who.in /media/docs/de aul -sou ce/in luenza/who-
in luenza- ecommenda ions/ cm-sou he n-hemisphe e- ecommenda ion-
2024/202309_zoono ic_ accin i usupda e.pd ?s sn=e78676a0_5
OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 7
o accina ion o p o ec ion. The o me a e mo e likely o equi e upda ing because o an igenic changes
gi en he la e can o en p o ide co e age agains mo e an igenically dis an gsGD HPAI i uses s ains.
Ne e heless, he adju an in killed an igen accines can o e come some an igenic changes, which is one
eason why accines o poul y do no usually equi e annual upda es, as is he case o human In luenza
A accines. Se ological moni o ing o accina ed locks o e idence o immuni y is s ongly ecommended
especially o accines ha gene a e a s ong humo al esponse.
An ibody moni o ing can be o limi ed alue as an indica o o p o ec ion o accines ha gene a e cell
media ed immuni y such as HVT ec o accines, especially when he HA inse is only dis an ly ela ed o
ield s ains. Ne e heless, es ing o accina ed locks should be unde aken o ensu e he HVT ec o has
eplica ed in accina ed bi ds and in si ua ions whe e boos e inac i a ed accines ha e been used
ollowing ini ial p iming wi h he ec o accine, an ibody es ing should be done o moni o he esponse.
Findings
Clade 2.3.4.4b
A p esen , he majo HPAI h ea globally is om gsGD A(H5Nx) i uses, mainly A(H5N1), in clade
2.3.4.4b. In he pas 3 mon hs, mos i al ac i i y has cen ed on no he n Eu ope and Sou h Ame ica,
wi h cases again pe sis ing in wild bi ds du ing he summe and occasional spillo e o poul y. New cases
in comme cial poul y a ms ha e also been epo ed in No h Ame ica in la e Sep embe and ea ly Oc obe
a e a pause du ing he summe .
3
Wild bi d and mammalian cases we e s ill being de ec ed in No h
Ame ica h oughou 2023.
An igenic assessmen s using e e ence chicken an ise a (see Annex 2) ha e demons a ed some a ia ion
in esponse by s ains om 2020 o 2022 bu he e was no appa en inc ease in he an igenic dis ances
o e all o i uses de ec ed be ween he di e en yea s. Some published pape s ha e epo ed changes a
pu a i e an igenic si es (e.g., Yang e al 2023). Howe e , he changes a e p obably no enough o in e e e
wi h p o ec ion om a ecen well-ma ched killed an igen adju an ed accine.
In some coun ies clade 2.3.4.4b i uses now co-exis wi h o he endemic s ains. I may be necessa y o
in oduce mul i alen accines co e ing all an igenically dis inc s ains as has been he case in some
coun ies (e.g., China wi h a i alen accine co e ing gsGD clade 2.3.4.4b ( e-14 an igen), clade 2.3.4.4h
( e-13) and H7N9 ( e-4)). Howe e , i is al eady e iden in some coun ies ha clade 2.3.4.4b has la gely
eplaced some o he s ains (e.g., o he 2.3.4.4 clades in Vie nam).
Eu ope
Recen HPAI e en s in Eu ope ha e been desc ibed in de ail elsewhe e (EFSA 2023). Vi uses de ec ed
in Eu ope om 2023 ha e no been subjec ed o an igenic cha ac e isa ion using e e ence se a in OFFLU
e e ence labo a o ies bu gene ic analysis o s ains sugges s ew signi ican changes a pu a i e o
possible an igenic si es. Ne e heless, i no el s ains o i us a e iden i ied, especially in accina ed locks,
addi ional cha ac e isa ion as discussed in Annex 1 is s ongly ecommended.
Vaccine challenge and ansmission s udies ha e been conduc ed in se e al Eu opean coun ies. Vaccine
ials conduc ed in F ance and he Ne he lands ha e demons a ed p o ec ion om disease and
ansmission in poul y agains clade 2.3.4.4b i uses isola ed in hese wo loca ions in 2021. The F ench
3
Fo epo s om he USA sin poul y see h ps://www.aphis.usda.go /aphis/ou ocus/animalheal h/animal-disease-
in o ma ion/a ian/a ian-in luenza/hpai-2022/2022-hpai-comme cial-backya d- locks. Fo Canada see
h ps://inspec ion.canada.ca/animal-heal h/ e es ial-animals/diseases/ epo able/a ian-in luenza/la es -bi d- lu-
si ua ion/in es iga ions-and-o de s/eng/1688503773556/1688503774196
OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 8
s udy
4
in ol ed mule ducks accina ed wi h ei he a sel - eplica ing RNA i us o a subuni accine. The
accines p o iding p o ec ion in laye s in he Ne he lands we e HVT ec o accines
5
. These expe imen al
s udies may no e lec he si ua ion in he ield whe e o he ac o s can impac on accine e ec i eness
bu demons a ed good p o ec ion and p e en ion o ansmission unde labo a o y condi ions. S udies on
accina ion in u keys ha e also been unde aken in I aly wi h esul s s ill o be published. Resul s om
ials wi h chickens may no be di ec ly applicable o o he species, including u keys (EFSA AHAW Panel
2023b).
A ica
A ica has expe ienced mul iple in oduc ions o gsGD H5Nx clade 2.3.4.4b i uses om Eu asia since
2016. These in oduc ions ha e led o he main enance o hese i uses in poul y popula ions, in Wes and
sou he n A ica, and Egyp wi h he eplacemen o p e iously ci cula ing i uses and in some cases
easso men wi h locally ci cula ing endemic i uses (including A(H9N2). Many o hese i uses ha e
con inued o ci cula e in poul y in geog aphically seg ega ed egions, wi h some new in oduc ions and
i us de ec ed in wild sea bi ds and poul y (Abolnik e al 2023, Meseko e al 2023). An igenic
cha ac e iza ion o ecen (2023) i uses has no been epo ed. Some o he A(H5N8) i uses in A ica
ha e los a glycosyla ion si e in he HA glycop o ein bu his does no appea o be ela ed o changes in HI
i es when es ed agains e e ence an ise a.
Egyp also has endemic in ec ion wi h clade 2.3.4.4b i uses. Po en ial new clade 2.3.4.4b expe imen al
accines (based on A/chicken/Egyp /F71-S86C/2022(H5N8) and A/chicken/Egyp /F71-
F114C/2022(H5N1) we e de eloped in Egyp using e e se gene ics. The killed an igen, adju an ed
accines p oduced om hese i uses p o ided p o ec ion agains clade 2.3.4.4b A(H5N1) and A(H5N8)
i uses ( om 2021) bu we e less e ec i e agains a clade 2.2.1 i us as expec ed (Mahmoud e al 2023).
T ials we e conduc ed wi h exis ing accines in Egyp agains clade 2.3.4.4b i uses when hey i s
eme ged (Kandeil e al 2018) and demons a ed impe ec p o ec ion by mos o he comme cially a ailable
accines and he need o upda e accine an igens. Ano he s udy has also con i med ha accines agains
clade 2.2 de i a i es we e no longe ully e ec i e agains clade 2.3.4.4b i uses (Salaheldin e al 2022).
A new accine an igen was in oduced by a comme cial company o comba clade 2.3.4.4b i uses
(Ib aham e al 2021) based on a 2018 i us. An igenic cha ac e isa ion o A(H5N8) i uses was unde aken
(Kandeil e al 2022) and some an igenic changes we e de ec ed in locks ha had been accina ed wi h
poo ly ma ched accines, demons a ing he impo ance o only using app op ia ely ma ched accines.
In o ma ion on an igenic cha ac e is ics o ecen (2023) clade 2.3.4.4b i uses om Egyp is no a ailable.
No h Ame ica
In he pas h ee mon hs i al ac i i y emained low in No h Ame ica wi h only a small numbe o wild bi d
cases epo ed, p edominan ly om he no hwes o he US and occasional posi i e cases in li e bi d
ma ke s in he no heas o he coun y (New Yo k and New Je sey). Since la e Sep embe , mul iple cases
in comme cial poul y ha e been epo ed om Albe a, Canada and Sou h Dako a and U ah in he USA.
The e a e no epo s ega ding he an igenic cha ac e is ics o hese ecen i uses, bu ea lie i uses
appea ed o ha e ew i any mu a ions in known an igenic si es.
4
h ps://www.anses. /en/ esul s- accina ion-ducks-%20a ian-in luenza
5
h ps://www.wu .nl/en/ esea ch- esul s/ esea ch-ins i u es/bio e e ina y- esea ch/show-b / wo- accines-e ec i e-
agains -bi d- lu.h m
OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 9
Somewha su p isingly, clade 2.3.4.4b i uses in he US emained ela i ely simila o clade 2.3.4.4.c
an igenically, based on esul s using e e ence an ise a (see Annex 2) despi e se e al di e ences a
pu a i e an igenic si es. Simila esul s we e ob ained using e e an ise a (Kandeil e al 2023).
Cen al and Sou h Ame ica
A(H5N1) gsGD clade 2.3.4.4b i uses we e i s de ec ed in Cen al and Sou h Ame ica in 2022 and ha e
sp ead o he sou he n ip o Sou h Ame ica, wi h cases in wild bi ds and sea lions epo ed in sou he n
Chile and A gen ina. They con inue o ci cula e in wild bi ds and poul y al hough some coun ies ha e
decla ed eedom om in ec ion in poul y.
Vi uses epo ed so a a e simila o hose de ec ed in No h Ame ica wi h mul iple incu sions (Jiminez-
Bluhm e al 2023, Ruiz-Saenz e al 2023) Recen isola es a e no a ailable om coun ies whe e accines
ha e been deployed and i will be impo an o con inue o moni o i uses in a eas whe e accina ion is
being used and o ully cha ac e ize hem an igenically.
Asia
Clade 2.3.4.4b i uses a e widesp ead ac oss much o Asia. Limi ed in o ma ion is a ailable on he
an igenic cha ac e is ics o ecen i uses, howe e hose o which sequence da a a e a ailable ha e ew
changes a likely an igenic si es. China upda ed accine an igens in ea ly 2022 ollowing he e-in oduc ion
o clade 2.3.4.4b i uses (Shi e al 2023). Clade 2.3.4.4b i uses appea o ha e la gely eplaced o he
clades in China. S udies pe o med on wild bi d isola es om 2021 in China demons a ed ha he Re-14
accine an igen was an app op ia e ma ch o hese i uses (Tian e al 2023).
An igenic assessmen s on clade 2.3.4.4b i uses om Cambodia om 2021 demons a ed di e ences ha
canno be explained eadily by changes a pu a i e an igenic si es (see Annex 2). Fu he s udies will be
unde aken on hese i uses.
Clade 2.3.2.1a
Vi uses in his clade ha e been ci cula ing in Sou h Asia since 2011. They ha e pe sis ed despi e se e al
incu sions o clade 2.3.4.4 i uses. A s udy was conduc ed on e ec i eness o accines agains clade
2.3.2.1a i uses om Bangladesh ha sugges ed some an igenic a ia ion in i uses om c ows bu less
so in poul y (Kwon e al 2021). No ecen esul s o an igenic cha ac e iza ion a e a ailable al hough some
a ia ion a pu a i e an igenic si es has been epo ed in he OFFLU VCM epo . Fu he s udies a e
needed o assess accine e icacy agains ecen clade 2.3.2.1a i uses de ec ed in Sou h Asia. Killed
an igen accines con aining an igens de i ed om clade 2.3.4.4 i uses would likely p o ide sub-op imal
p o ec ion agains clade 2.3.2.1a i us based on he an igenic dis ance be ween he wo i uses (see esul s
in Annex 2).
Clade “2.3.2.1c”
Vi uses e e ed o in-coun y as clade 2.3.2.1c and in OFFLU and WHO publica ions as clade 2.3.2.1e2,
a e ci cula ing in Indonesia and Timo Les e bu ew ecen i uses ha e been de ec ed o ully
cha ac e ised. Indonesia has a sys em in place o upda ing accines when an igenic a ian s eme ge
(Ha aningsih e al 2015). Vaccines a ge ing hese i uses would be unlikely o p o ide app op ia e
p o ec ion agains clade 2.3.4.4.b i uses now p esen in Indonesia i hese become mo e widesp ead.
Fu he s udies a e needed on he an igenic cha ac e is ics o ecen clade “2.3.2.1c” i uses om
Cambodia, Vie Nam and Laos, howe e gene ic in o ma ion sugges s hese i uses a e likely o ha e
emained an igenically s able. Inac i a ed accines based on Clade 2.3.4.4b would no be expec ed o
p o ide consis en p o ec ion agains clade 2.3.2.1c i uses.
OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 16
An igenic ca og aphy enables he in eg a ion o pheno ypic da a om mul iple labo a o ies, p o iding a
s anda dised assessmen and compa a i e analysis o globally ci cula ing gsGD lineage H5 i uses agains
commonly used accine an igens. La ge an igenic dis ances om an ise a aised agains su oga e accine
an igens o cu en ly ci cula ing s ains migh indica e limi ed p o ec ion om accina ion in he ield.
OFFLU will con inue o aise chicken an ise a agains mo e accine an igens and es addi ional clades o
con empo a y ci cula ing s ains. In he nex epo his will explici ly ensu e con empo aneously ci cula ing
i uses in 2023/24 a e included.
In oduc ion
This annex p o ides in o ma ion on one o he key goals o OFFU-AIM which was o es ablish s anda dized
sys ems o an igenic ca og aphy o gsGD HPAI A(H5Nx) i uses ele an o poul y. This wo k
summa ises p og ess made in his a ea including in e p e a ion o he indings.
Vaccine seed s ains
S ains o i us we e selec ed based on accines ha a e being o ha e been used in he pas . The
ollowing able p o ides in o ma ion on accine seed s ains and also su oga e s ains ha we e used
when he accine s ain was no a ailable.
Me hods, analyses and esul s
Gene a ion o chicken an ise a
A s anda dised panel o chicken se a and homologous an igens we e gene a ed a IZSVe and APHA,
immunising bi ds wi h adju an as his likely mos closely e lec s he chicken immune esponse when
accina ed. Se a we e gene a ed using an igens om i uses which a e gene ically simila o accine seed
s ains, which we e e o h oughou he documen 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 inac i a ed by be a-p opiolac one (BPL). Ba ches had a minimum an igen con en
equal o 120.000 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 emby ona ed chicken eggs.
Speci ic pa hogen ee (SPF) chickens (n=10/an igen) we e inocula ed ia he in amuscula ou e wi h 1ml
BPL inac i a ed a ian in luenza an igen (non-in ec ious an igen) plus Mon anide as an adju an (inocula ion
1). (Vi us/adju an [30/70%]).
A 14 days (+/- 3 days) pos p ime 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 i s 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 an ibody i es we e below use ul le els (usually <1/16 HAIUs/25 ul) bi ds we e boos ed again (inocula ion
3). Bleeding and boos ing was epea ed ano he 1 ime 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, hal he olumes we e shipped be ween IZSVe and APHA.

OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 17
Table 1: Seed s ains o accines which a e cu en ly unde s ood o be in use o ha e been used in he
pas . Whe e a ailable clade in o ma ion and e e ences ha e been included. As e isks (*) deno e s ains
o which su oga e an igens ha e been included in AIM.
Seed s ain
Clade
Re e ence
*Su oga e included in
AIM
A/duck/Po sdam/1402/86
LPAIV
Fe eidouni e al., 2009
A/chicken/Vie nam/C58/2004*
1
Bol z e al., 2009
A/Vie nam/1194/2004/1
A/ u key/ u key/2005-like*
2.2
Palya e al., 2018
A/Tu key/ u key/2005
A/chicken/Egyp /Q1995D/2010
2.2.1.1
Kayali e al., 2016
A/chicken/Egyp /D10552B/2015
2.2.1.2
Gomaa e al., 2019
A/chicken/Egyp /M2583D/2010
2.2.1.2
A/Chicken/Egyp /RG-173CAL/2017
2.2.1.2
Ib ahim e al., 2022
A/duck/China/E319-2/2003
2.3.2
Ca alcan i e al., 2017
A/duck/Guangdong/S1322/2010*
2.3.2.1
Shi e al., 2022
A/chicken/Nepal/T360/20
14
A/duck/Sukoha jo/BBVW-1428-
9/2012*
“2.3.2.1c”
Ind iani e al., 2014
A/mynah/Indonesia(Aus
iaQ)/13064792-010/2013
A/Hubei/1/2010
2.3.2.1a
A chicken/Tanggamus/031711076-
65/2017
2.3.2.1c
A/Duck/Vie Nam/QB7412
“2.3.2.1c”
gCA2/2.3.2.1d
2.3.2.1d
Kang e al., 2022
A/Chicken/Liaoning/SD007/2017
2.3.2.1
Shi e al., 2022
A/duck/Anhui/1/2006*
2.3.4
Shi e al., 2022
A/Anhui/1/2005
A/Wa e owl/Ko ea/S57/2016
2.3.4.4
Ku uppua achchi e al.,
2022
A/duck/F ance/161108 h/2016*
2.3.4.4b
G asland e al., 2023
A/malla d/Geo gia/DT093
82/2017
A/g een-winged eal/Egyp /877/2016*
2.3.4.4b
Goma e al., 2019
A/Mu e
Swan/C oa ia/102/2016
A/whoope swan/Shanxi/4-1/2020*
2.3.4.4b
Shi e al., 2022
A/duck/Cambodia/ 4k241
D3/2021
A/chicken/ME-2018*
2.3.4.4b
Ib ahim e al., 2022
A/malla d/Geo gia/DT093
82/2017
A/gy alcon/Washing on/40188-
6/2014-like*
2.3.4.4c
Be an e al., 2017
A/gy alcon/Washing on/
41088/6/2014
A/chicken/Guangzhou/3/2013
2.3.4.4g
Shi e al., 2022
gES3/2.3.4.4h
2.3.4.4h
Kang e al., 2022
A/duck/Guanzou/S4184/2017
2.3.4.4h
Shi e al., 2022
A/duck/Fujian/S1424/2020
2.3.4.4h
Shi e al., 2022
OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 18
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 and IZSVe 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 speci ic pa hogen
ee chickens we e also es ed o each assay.
An igenic Ca og aphy
An igenic ca og aphy me hods as desc ibed by Smi h e al., (2004), we e p e iously used o cha ac e ise
equine, swine and human in luenza A i uses and also a ian H5 HPAI and LPAI i uses (Smi h e al., 2008;
Lewis e al., 2011; Lo usso e al., 2010). Ca og aphy was used o quan i y and isualise he HI da a and
o cha ac e ise he ela ionships be ween su oga e accine seed s ains and ci cula ing s ains. Map
es ing de e mined ha he da a we e mos obus ly isualised in 3 dimensional an igenic maps, as
desc ibed by Lewis e al., (2021). Maps we e iewed and analysed in R S udio using he Racmacs
package e sion 1.1.35). Inconsis en HI i e s we e excluded om analysis. Fo in o ma ion ega ding map
gene a ion and in e p e a ion please con ac an app op ia e e e ence labo a o y.
Gene ic analysis was ca ied ou using manually cu a ed da ase s using Ma 7.490 (Ka oh and S andley,
2013) o align nucleo ide and amino acid sequences using de aul se ings. Alignmen s we e immed om
he H5 s a codon o he s a o he clea age si e. Phylogene ic analysis was unde aken gene a ing
maximum likelihood phylogene ic ees in iq ee2 V2.2.2.6 (Nguyen e al., 2015) o in e H5 clade
designa ions. S ains in an igenic maps we e colou ed acco ding o i al clades. T ee ime 0.9.2 was used
o in e ances al sequences Sagulenko e al., (2017). The NADC IAV bioin o ma ics oolki (Zhang e al.,
2017) was used o analyse and compa e sequences o di e ences in pu a i e an igenic si es.
OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 19
Chicken an ise a panel
Table 2: Vi uses used o gene a e pos in ec ion chicken an ise um an ise a. Clade in o ma ion is included.
As e isks (*) deno e an igens which ha e been used as a su oga e ep esen a i e o a accine an igen.
Vi us Isola e Name
Clade
A/Vie nam/1194/2004/1*
1
A/ u key/ u key/2005*
2.2
A/chicken/Nepal/T360/2014*
2.3.2.1a
A/mynah/Indonesia(Aus iaQ)/13064792-010/2013*
“2.3.2.1c”
A/Anhui/1/2005* (RG)
2.3.4
A/chicken/Czech Republic/1175-1-20VIR465-1/2020
2.3.4.4b
A/chicken/Bulga ia/722-1-22VIR778-1/2021
2.3.4.4b
A/malla d/Geo gia/DT09382/2017*
2.3.4.4b
A/mu e Swan/C oa ia/102/2016*
2.3.4.4b
A/duck/Cambodia/ 4k241D3/2021*
2.3.4.4b
A/g ea skua/Sco land/B07779/2021
2.3.4.4b
A/gy alcon/Washing on/41088-6/2014*
2.3.4.4c
OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 20
An igenic maps
Figu e 2: A 3 dimensional an igenic map showing he e olu iona y ela ionships o H5 HPAI Gs/Gd lineage
i uses. Each an ise um is ep esen ed by a cube: black cubes ep esen an an ise um aised using a i us
which ep esen s a su oga e accine seed s ain, pink cubes ep esen an ise a aised o quan i y di e si y.
An igens a e ep esen ed as balls and a e colou ed by clade acco ding o he key. Each squa e ep esen s
one an igenic uni . One an igenic uni is ep esen a i e o a 2 old di e en in HA assay i e . The
co esponding able o an igenic dis ances can be ound in able 4.
OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 21
Table o an igenic dis ances
Table 3: A able o an igenic dis ances gene a ed om he an igenic maps. One an igenic uni is equal
o a wo old dec ease in HA i e . Dis ances we e colou ed using a hea map. Vaccine an igens a e
labelled nex o hei su oga e chicken an ise a. An igens a e o de ed by clade, yea and sub ype.

OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 22
Main indings
The ocus was on clade 2.3.4.4b i uses om 2020 (7 i uses), 2021 (23 i uses) and 2022 (15 i uses).
Two clade 2.3.2.1a i uses we e also assessed. No e ha one an igenic uni ep esen s a wo old di e ence
in HI assay i e.
The e we e no an ise a es ed ha we e <3 an igenic uni s om gene ic clade ep esen a i e 2.3.4.4b and
gene ic clade ep esen a i e 2.3.2.1a i uses. This indica es ha i may be necessa y o use mul i alen
H5 accines in places whe e mul iple an igenically di e en i uses a e co-ci cula ing.
2.3.2.1a
Con empo a y i uses om Asia we e >6 uni s om bo h gene ic clade ep esen a i e 2.3.4.4b and gene ic
clade ep esen a i e 2.3.4.4c chicken an ise a.
Bo h an igens we e <3 an igenic uni s om chicken an ise a aised agains gene ic clade ep esen a i e
2.3.2.1 i uses. An igens we e 4-6 an igenic uni s om clade 2.3.4 an igens.
Bo h an igens we e 5 an igenic uni s om he clade 2.2 an ise a.
An igens we e be ween 3-5 an igenic uni s om he clade 1 an ise a.
This indica es ha inac i a ed accines using an igens om clades 2.3.4.4b, 2.3.4.4c, 2.3.2, 2.2, and 1
i uses may no p o ide p o ec ion agains clade 2.3.2.1a i uses.
2.3.4.4b
Clade 1, 2.2 and 2.3.4 an ise a we e a leas 4 an igenic uni s om gene ic clade ep esen a i e 2.3.4.4b
i uses. Clade 2.3.2.1 an ise a was a leas 3 an igenic uni s om gene ic clade ep esen a i e 2.3.4.4b
i uses. This indica es ha inac i a ed accines using hese an igens would p obably no p o ide
su icien p o ec ion agains he con empo a y gene ic clade ep esen a i e 2.3.4.4b i uses, as has been
demons a ed in accine challenge s udies.
Some an igenic di e si y is seen among he con empo a y gene ic clade ep esen a i e 2.4.4.4b i uses.
All we e <6 an igenic uni s om gene ic clade ep esen a i e 2.3.4.4b chicken an ise a wi h mos i uses
showing <3 an igenic uni s om gene ic clade ep esen a i e 2.3.4.4b chicken an ise a.
An igens es ed we e >4 an igenic uni s om clade 1, 2.2 and 2.3.4 an ise a.
Mos an igens es ed we e <4 an igenic uni s om he clade 2.3.4.4c chicken an ise um.
All an igens es ed ha e >3 an igenic uni s om clade 2.3.2.1 chicken an ise a.
Al hough he e is some an igenic a ia ion in clade 2.3.4.4b i uses, he e did no appea o be signi ican
changes when compa ing isola es om 2020 o hose om 2021 and 2022, sugges ing he i uses ha e
been ela i ely s able an igenically du ing his pe iod (see Table 4).
Table 4 p o ides he GMT alues o an igenic di e ence agains h ee e e ence 2.3.4.4b s ain and one
e e ence 2.3.4.4c s ain.
OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 23
Table 4 An igenic dis ance – Clade 2.3.4.4b i uses by yea o isola ion
Yea
Coun
An ise um A*
(2.3.4.4b)
An ise um B*
(2.3.4.4b)
An ise um C*
(2.3.4.4b)
An ise um D*
(2.3.4.4c)
2020 GMT
7
3.14
2.43
3.14
2.71
2020 Min
2
1
2
2
2020 Max
4
3
4
4
2021 GMT
23
3.13
2.04
2.74
3
2021 Min
1
0
1
1
2021 Max
6
4
4
6
2022 GMT
15
2.73
1.87
2.56
2.33
2022 Min
1
1
2
1
2022 Max
4
3
4
4
*An ise um A A/Mu e Swan/C oa ia/102/2016 which is a su oga e o A/g een-winged eal/Egyp /877/2016 (one o
he ea lies clade 2.3.4.4b i uses om Eu ope/Middle Eas )
An ise um B A/malla d/Geo gia/DT09382/2017 which is a su oga e o A/chicken/ME-2018(an an igen used in some
Egyp ian-o igin accines)
An ise um C A/duck/Cambodia/ 4k241D3/2021 which is a su oga e o A/whoope swan/Shanxi/4-1/2020 (Chinese
Re-14 an igen)
An ise um D A/gy alcon/Washing on/41088/6/2014 (ea ly clade 2.3.4.4c i us om No h Ame ica)
A/malla d/Geo gia/DT09382/2017 appea ed o be sligh ly close o es isola es han o he clade 2.3.4.4b
e e ence s ains.
A/gy alcon/Washing on/41088/6/2014 (clade 2.3.4.4c) appea s simila an igenically o clade 2.3.4.4b
i uses in e ms o an igenic dis ance.
The eason o he la ge an igenic dis ance o he i us om Cambodia is being examined gi en i does
no appea o be due o mul iple changes a pu a i e an igenic si es.
Recommenda ions and nex s eps o OFFLU AIM
1. Vi uses om a b oade geog aphical ep esen a ion should be es ed including o he clades which a e
ci cula ing in poul y such as clade “2.3.2.1c”.
2. Se a should be aised agains i al s ains om o he sub ypes which ep esen accine an igen seed
s ains including H9 i uses.
Lyophilised eagen s (an ise a and inac i a ed i uses) gene a ed by APHA and IZSVe can be sha ed,
upon eques , wi h selec ed pa ne s, a ge ing o he WOAH and FAO in e na ional e e ence labo a o ies
o enable he gene a ion o compa a i e da a assessing po en ial al e a ion in i us an igenici y using
s anda dised eagen s and me hodologies unde a de ined quali y amewo k. I should be no ed howe e
ha ca e ul be ween labo a o y s anda diza ion needs o be unde aken be o e da a om addi ional
labo a o ies can be eliably ep esen ed in he analyses. Resul s will allow o con inuous moni o ing o
OFFLU a ian in luenza ma ching epo , Oc obe 2023 Page 24
an igenic changes in cu en ly ci cula ing i uses and in o m he con inuous expansion and upda ing o
se a panels wi h global ep esen a ion and ele ance.
Fu he o es ablishing ha monised me hods ac oss de ined labo a o ies, an igenic da a gene a ed h ough
his ac i i y will complemen da a gene a ed o p esen ed by FAO o WOAH membe s and OFFLU pa ne s
including egional su eillance da a, epidemiological da a including e idence o accine b eakdown and
expe imen al s udies.
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