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