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Avian Pathology
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Evaluation of the protection induced by avian

influenza vaccines containing a 1994 Mexican H5N2
LPAI seed strain against a 2008 Egyptian H5N1
HPAI virus belonging to clade 2.2.1 by means of
serological and in vivo tests
a a a a a
Calogero Terregino , Anna Toffan , Filippo Cilloni , Isabella Monne , Elena Bertoli
b b c a
, Lilia Castellanos , Nadim Amarin , Marzia Mancin & Ilaria Capua
a
OIE/FAO and National Reference Laboratory for Avian Influenza and Newcastle
Disease , Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe) , Viale
dell'Università 10, 35020, Legnaro, Padova, Italy
b
Boehringer Ingelheim Vetmedica , Calle 30 Num. 2614 Zona Industrial, 44940,
Guadalajara, Jalisco, Mexico
c
CREV, Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe) , Viale dell'Università
10, 35020, Legnaro, Padova, Italy
Published online: 11 Jun 2010.

To cite this article: Calogero Terregino , Anna Toffan , Filippo Cilloni , Isabella Monne , Elena Bertoli , Lilia Castellanos ,
Nadim Amarin , Marzia Mancin & Ilaria Capua (2010) Evaluation of the protection induced by avian influenza vaccines
containing a 1994 Mexican H5N2 LPAI seed strain against a 2008 Egyptian H5N1 HPAI virus belonging to clade 2.2.1 by
means of serological and in vivo tests, Avian Pathology, 39:3, 215-222, DOI: 10.1080/03079451003781858

To link to this article: http://dx.doi.org/10.1080/03079451003781858

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 Avian Pathology (June 2010) 39(3), 215
222

Evaluation of the protection induced by avian influenza

vaccines containing a 1994 Mexican H5N2 LPAI seed
strain against a 2008 Egyptian H5N1 HPAI virus belonging
to clade 2.2.1 by means of serological and in vivo tests
Calogero Terregino1*, Anna Toffan1, Filippo Cilloni1, Isabella Monne1, Elena Bertoli1,
Lilia Castellanos2, Nadim Amarin2, Marzia Mancin3 and Ilaria Capua1
1
OIE/FAO and National Reference Laboratory for Avian Influenza and Newcastle Disease, Istituto Zooprofilattico
Sperimentale delle Venezie (IZSVe), Viale dell’Università 10, 35020 Legnaro, Padova, Italy, 2Boehringer Ingelheim
Vetmedica, Calle 30 Num. 2614 Zona Industrial, 44940 Guadalajara, Jalisco, Mexico, and 3CREV, Istituto Zooprofilattico
Sperimentale delle Venezie (IZSVe), Viale dell’Università 10, 35020 Legnaro, Padova, Italy

Since 2006 Egypt has been facing an extensive epidemic of H5N1 highly pathogenic avian influenza (HPAI)
with a huge number of outbreaks both in rural and intensively reared poultry areas. The use of efficacious
Downloaded by [181.198.171.9] at 20:04 17 February 2015

vaccines in this country has been, and still remains, essential for the control and possible eradication of
HPAI. The present study was performed to establish whether the administration of inactivated vaccines
containing an H5 virus belonging to a different lineage to the Eurasian H5N1 HPAI viruses guarantees
protection from clinical signs, provides significant immune response and is able to achieve a reduction of
viral shedding in the face of a challenge with a contemporary H5N1 virus isolated in Egypt. Despite the
genetic and antigenic differences between the vaccine strain (H5N2/Mexico) and the challenge strain (H5N1/
Egypt), confirmed by molecular and serological (haemagglutination inhibition) tests, it was established that
the immune response induced by these conventional vaccines is sufficient to prevent infection in the majority
of birds challenged with a contemporary H5N1 Egyptian strain. The data reported in this study also indicate
that there may be a low degree of correlation between haemagglutination inhibition titres, clinical protection
and reduction of shedding.

Introduction

Although the use of vaccines against avian influenza
(AI) viruses in birds has been discouraged over the
years, the unprecedented occurrence of outbreaks
caused by AI viruses in recent times has required
review of this policy. A variety of products are now
available on the market, ranging from inactivated
conventional to live recombinant products. The general
consensus on the use of vaccination is that, complying
with good manufactory practice standards and properly
administered, birds will be more resistant to field
challenge and will exhibit reduced shedding levels in
case of infection.
Since 2006 Egypt has been facing an extensive
epidemic of H5N1 highly pathogenic avian influenza
(HPAI) with a huge number of outbreaks both in rural
and intensively reared poultry. The use of vaccines in this
country has been and still remains massive, with millions
of doses of vaccine used in the field. Despite the use of
vaccination, the number of outbreaks in animals and in
humans is increasing.
The use of heterologous inactivated vaccine
challenge
virus combinations has been widely described in the
literature in experimental challenges and also applied
widely in the field (Terregino et al., 2007; Bos et al.,
2008; Van der Goot et al., 2008; Poetri et al., 2009).
These types of vaccines have several advantages,
including their use in differentiating infected from
vaccinated animals (DIVA) strategies. This system was
developed to support the eradication programmes in
the presence of several introductions of AI viruses
(Capua & Alexander, 2004; Capua et al., 2004).
Previous studies have shown that vaccine containing
the H5N2 A/chicken/Mexico/232/94/CPA strain is pro-
tective in chickens and ducks against H5N1 HPAI
viruses of the Eurasian lineage despite the low hae-
magglutination (HA) homology between the vaccine
strain and the field virus (Swayne et al., 2006; Van der
Goot et al., 2008).
The present study was performed to establish whether
administration of an inactivated vaccine containing an
H5 virus belonging to a different lineage to the Eurasian
H5N1 was protective against clinical signs and achieved
a reduction of viral shedding in the face of challenge
with a 2008 H5N1 virus isolated in Egypt.

*To whom correspondence should be addressed. Tel: 39 49 8084371. Fax: 39 49 8084360. E-mail: cterregino@izsvenezie.it
Received 9 October 2009
ISSN 0307-9457 (print)/ISSN 1465-3338 (online)/10/030215-08 # 2010 Houghton Trust Ltd
DOI: 10.1080/03079451003781858
 216 C. Terregino et al.
Materials and Methods
Birds. Five groups of 10 specific pathogen free (SPF) chicks, hatched in
isolation from SPF eggs supplied by Charles River Laboratories† Inc.,
males and females, were used. Chicks were reared in isolation and were
individually identified by means of a numbered wing tag. Chickens were
fed with a commercial compound suitable for their age. Birds had water
and feed ad libitum.
Vaccines and vaccine administration. Volvac† AI KV and Volvac† AI
ND KV are emulsified, inactivated oil vaccines prepared with A/
chicken/Mexico/232/94/CPA (H5N2), which for the bivalent vaccine is
in combination with the La Sota Newcastle disease (ND) virus strain.
Each 0.5 ml vaccine contains a minimum titre of 108.5 median embryo
infective dose (EID50) or 256 haemagglutinating units (HAU) of AI and
108.2 EID50 or 128 HAU of ND viruses (bivalent vaccine only). The
vaccine dose for the two vaccines under study was 0.5 ml per bird,
inoculated in the lower (dorsal) part of the neck by the subcutaneous
route.
Experimental design. Chicks were randomly distributed in five groups
(Group 1 to Group 5) of 10 birds each. Birds of Groups 1 and 2, were
vaccinated with Volvac† AI and birds in Groups 4 and 5 with Volvac†
AIND at 21 days of age. Birds in Group 3 were left as unvaccinated
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controls. Birds were observed daily for clinical signs throughout the
study. Four weeks after vaccination, chickens from Groups 1, 2 and 3
were challenged intranasally (IN) with 0.1 ml viral suspension contain-
ing 106 EID50/ml (corresponding approximately to 10 100% chicken
lethal doses, based on in vivo tests performed prior to the trial; data not
shown) of the challenge HPAI H5N1 virus A/chicken/Egypt/1709-6/08.
Blood samples for serology studies were obtained from each chicken
of each group prior to each vaccination, before challenge and every
week until 4 weeks after challenge (end of the study). In order to
monitor virus shedding after challenge, tracheal and cloacal swabs from
each challenged bird were obtained on days 3, 7, 10, 14 and 21 post
challenge. These samples were used for real-time reverse transcriptase-
polymerase chain reaction (RT-PCR) analyses and at the same time
they were processed for virus isolation in SPF eggs.
Challenge virus. The challenge virus was selected from 10 Egyptian
isolates provided by the Animal Health Research Institute, Egypt.
Viruses were isolated from rural and industrial poultry farms at the end
of 2007 and beginning of 2008. All of the viruses were passaged in
embryonated SPF fowl’s eggs once. Viruses were then titrated in
embryonated SPF fowl’s eggs and the EID50 was calculated according
to the Reed and Muench formula (Reed & Muench, 1938). All isolates
were fully sequenced and A/chicken/Egypt/1709-6/08 was selected. This
virus was isolated in 2008 from a poultry farm in the Quen-Quena
District, Egypt. It clustered together with other 2008 isolates but
separately from the 2007 isolates. This challenge virus belongs to the
phylogenetic clade 2.2.1 (World Health Organization/World Organisa-
tion for Animal Health/Food and Agriculture Organization H5N1
Evolution Working Group, 2008), and was selected because it was
considered the prototype of the viruses circulating in Egypt in 2008
according to the results of the phylogenetic analysis performed using
available sequences of viruses isolated in Egypt between 2006 and 2008
(data not shown).
Preliminary indirect antigenic analysis of the challenge virus was
carried out by cross-haemagglutination inhibition (HI) tests using
serum produced with the vaccine strain A/chicken/Mexico/232/94
(H5N2) and a selection of antigens; namely, the virus contained in
the vaccine (H5N2), a reference H5N1 isolate (A/chicken/Yamaguchi/7/
04), the challenge virus and an Egyptian isolate considered representa-
tive of HPAI H5N1 viruses circulating in Egypt in 2007 (A/chicken/
Egypt/1709-1/2007). These HI tests showed a very low HI cross-
reactivity between the vaccine virus and the other viruses tested,
including the challenge virus. More specifically, the HI titres of the
serum produced with the vaccine strain were 1:2048, 1:512, 1:128 and
1:16 with the vaccine virus, a reference H5N1 isolate (A/chicken/
Yamaguchi/7/04), the H5N1 Egyptian strain of 2007 (A/chicken/Egypt/
1709-1/2007) and the challenge virus, respectively.
The nucleotide similarity between the HA gene of the vaccine strain
and the respective HA gene of the challenge virus and the viruses used
for serological investigations ranged between 72.5% and 74.3%. In
particular, the degree of similarity for the vaccine strain was 72.5% with
A/chicken/Egypt/1709-6/2008, was 73.7% with A/chicken/Egypt/1709-1/
2007 and was 74.3% with A/chicken/Yamaguchi/7/2004.
The amino acid similarity between the HA1 gene of the vaccine strain
and the respective gene of the challenge virus and the viruses used for
serological investigations ranged between 80% and 84%. In particular
the similarity for the vaccine strain was 80% with A/chicken/Egypt/
1709-6/2007, was 83% with A/chicken/Egypt/1709-1/2007 and was 84%
with A/chicken/Yamaguchi/7/2004.
Sampling. Birds were observed daily for clinical signs throughout the
duration of the study. Following challenge, the occurrence of clinical
signs was evaluated twice a day. Samples were collected and identified
individually with a study number, date of collection, an individual bird
identification number and a treatment group number.
Following challenge, tracheal and cloacal swabs were processed for
attempted virus isolation in SPF eggs, and were analysed by real-time
RT-PCR.
After collection, tracheal and cloacal samples were placed in 1 ml
isotonic phosphate-buffered saline solution (pH 7.0 to 7.4). One
hundred microlitres from each sample were used for RNA extraction.
RNA was analysed by real-time RT-PCR.
The remaining sample was mixed with an equal volume of phosphate-
buffered saline (pH 7.0 to 7.4) containing penicillin (2000 u/ml),
streptomycin (2 mg/ml), gentamicin (0.05 g/ml) and mycostatin
(1000 u/ml) for virus isolation attempts.
Serology. At least 2 ml blood were collected by puncture from the wing
vein at each sampling.
Sera were tested immediately after harvesting in HI tests using 4
HAU of the challenge virus and the vaccine strain as antigens (OIE,
2008b). Sera collected from the birds vaccinated with Volvac† AIND
were also tested for NDV antibodies (OIE, 2008a) using the Ulster 2C
strain as antigen. The vaccine virus was identical to the seed virus and
was supplied by the company that produces the vaccine as an antigen
inactivated by treatment with 0.1% formalin (0.037% formaldehyde).
The other viruses used in this study were inactivated by treatment with
0.05% b-propiolactone for 2 h at 378C.
The sera of vaccinated birds were tested by an indirect immuno-
fluorescence (iIFAT) anti-N1 antibody detection test (Capua et al.,
2003).
Virus isolation. Virus isolation was performed in SPF embryonated
fowl’s eggs in accordance with the OIE guidelines (OIE, 2008b). Briefly,
following clarification by centrifugation at 1000 x g, the supernatant
fluid of the samples collected was inoculated into the allantoic sac of at
least five SPF embryonated eggs of 9 to 11 days’ incubation. The eggs
were incubated at 36 to 378C for 7 days. Allantoic fluids from eggs
containing dead or dying embryos, and all embryos remaining viable at
the end of the first passage were tested for HA activity. Fluids yielding a
negative reaction were inoculated into at least one further batch of eggs.
Positive samples collected from vaccinated and unvaccinated birds were
titrated in SPF embryonated fowl’s eggs by inoculation of serial
dilutions, and the EID50 was calculated according to the Reed and
Muench formula.
Real-time RT-PCR. The swabs (tracheal or cloacal) were immersed in
sterile phosphate-buffered saline. A 100 ml volume of supernatant from
each sample was used for RNA extraction by conventional methods
(Machery-Nagel, Duren, Germany) and was analysed immediately. A
fixed amount of RNA was analysed by real-time RT-PCR for type A
influenza virus (Spackman et al., 2002). Samples with a threshold cycle
value 535 were considered positive for influenza type A viral RNA
based on internal validation trials.
Overall evaluation of efficacy. In order to evaluate the efficacy of the
vaccine by comparing the results obtained from testing different groups
(vaccinated and unvaccinated chickens) challenged with the same virus,
a bird was considered infected if it showed specific signs of HPAI; that

is, appeared clearly sick or died and/or exhibited seroconversion
(unvaccinated birds) or a significant increase of antibody titres
(vaccinated birds) after challenge, and was positive for, at least, real-
time RT-PCR or virus isolation from tracheal/cloacal swabs.
Statistical analysis. Serological data generated by HI were subjected to
statistical analysis to evaluate the significance of the differences
observed among and within the experimental groups. The methods
for the assessment of statistical significance were the Wilcoxon

Mann
Whitney rank-sum test on unmatched data and the Wilcoxon
matched-pairs signed-ranks test on matched data. These tests were
applied to establish whether the pre-infection and post-infection HI
titres differed significantly among and between experimental groups.
In order to determine the survival rate among vaccinated and
unvaccinated groups Cox’s regression analysis for the calculation of
the hazard ratio was used. Data were analysed with the test for
proportional hazard assumption before the applications of Cox’s
regression analysis. Finally, the log-rank test for equality of survival
functions was used to compare the survival rate among groups.
Results
Antibody response. All the Volvac† AI-vaccinated and
Volvac† AIND-vaccinated chickens showed high
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serological titres when tested with the homologous
antigen (H5N2). By contrast, the majority of the
vaccinated birds was below the threshold of positivity
(51:16) by HI using the inactivated challenge virus
(H5N1) as antigen. There was no evidence of statistical
significance between pre-challenge and post-challenge
titres generated with either antigen. At the end of the
experimental trial, the uninfected vaccinated groups as
well as the vaccinated and infected birds showed a slight
decrease in antibody titres when tested with the homo-
logous vaccine haemagglutinating antigen (H5N2).
Unexpectedly, even after infection only a few subjects
showed a slight increase of HI titre with the antigen
produced with the challenge virus (H5N1). Details of the
HI results are presented in Tables 1 and 2.
All of the vaccinated birds exhibited negative results
for the iIFAT anti-N1 antibody assay (DIVA test).
Clinical signs. Unvaccinated chickens were clinically ill
after challenge showing signs of HPAI including depres-
sion, ruffled feathers, cyanosis of the combs, hae-
morrhages on legs and shanks, nervous signs
(incoordination, seizures, tremors), dyspnoea and diar-
rhoea starting 48 h post infection. Birds started dying on
day 3 post infection, and by day 5 all chickens were
found dead.
Among vaccinated chickens, in Group 1 (Volvac† AI)
three birds showed slight depression and monolateral
conjunctivitis on day 4 post infection. One bird (the
chicken identified as Bird 4 in Table 1) recovered in 48 h,
while the other two began showing severe depression and
nervous signs and then died on days 5 and 7, respectively.
In some subjects, temporary unspecific signs were
observed (diminished activity and mild periorbital
hyperaemia) for a few days after challenge.
In Group 2 (Volvac† AIND) only one bird showed
clinical signs of HPAI on day 4, and died on day 5 post
infection, presenting severe nervous signs such as
seizures and incoordination.
In three birds of this group, mild non-specific signs
such as lethargy and ruffled feathers were also observed
from day 4 to day 8 after infection.
Vaccine protection against HPAI Egyptian H5N1 217
Chickens of Groups 4 and 5 (vaccinated control
groups) remained healthy throughout the duration of
the study.
Real-time RT-PCR results. All chickens of Group 3
(control group) yielded positive results by real-time
RT-PCR (with threshold cycle values ranging from
19.7 to 29.9) on day 3 post infection or on the day of
death (day 5).
Among the vaccinated chickens only birds showing
clear signs of illness were positive by real-time RT-PCR
(with threshold cycle values ranging values from 25.9 to
32.7). All of the real-time RT-PCR positive birds died
between days 4 and 7 post infection.
Virological results. All infected chickens of the control
group (Group 3) were positive by virus isolation.
Among the vaccinated chickens, virus isolation con-
firmed the results of real-time RT-PCR. In addition, all
of the positive samples from the vaccinated chickens
were titrated in eggs, displaying a level of viral load
ranging from B100.5 to 102.6 EID50/0.1 ml. The level of
viral load in unvaccinated birds ranged from 100.5 to
103.7 EID50/0.1 ml.
Statistical analysis. Statistical analysis on serological
data showed no significant difference (P 0.05) between
vaccinated experimental groups prior to challenge.
No statistically significant difference (P 0.05) was
observed between pre-challenge and post-challenge (28
days post infection) HI titres in either of the vaccinated
and infected groups.
The test for proportional hazard assumption indicated
that all of the groups taken into account showed a
proportional risk (P 0.05) of developing infection.
Cox’s regression analysis showed that there is a statisti-
cally significant difference in the hazard risk of devel-
oping infection between vaccinated groups and
unvaccinated groups (hazard ratio B1). The values
observed were 0.047 and 0.074 for the Volvac† AI-
vaccinated and Volvac† AIND-vaccinated groups,
respectively. This implies that the administration of
either vaccine significantly reduced the risk of develop-
ing infection.
Finally, the log-rank test for equality of survival
functions showed that there is a statistically significant
difference (P B0.05) in the survival rate among vacci-
nated and unvaccinated chickens, while there is no
statistically significant difference (P 0.05) between the
two vaccines tested.
The data on the shedding were analysed by two-
sample Wilcoxon rank-sum (Mann
Whitney) tests.
A statistically significant difference (P B0.05 or P B
0.01) was observed in shedding levels from both tracheal
and cloacal swabs between vaccinated birds and un-
vaccinated birds. In detail, the values were as follows*
for tracheal swabs, vaccinated AI versus unvaccinated,
P 0.0129 and vaccinated AIND versus unvaccinated,
P 0.0129; and for cloacal swabs, vaccinated AI versus
unvaccinated, P 0.0051 and vaccinated AIND versus
unvaccinated, P 0.0332.
No statistically significant differences were observed in
shedding levels between the two vaccines (Volvac† AI
versus Volvac† AIND).
No significant differences were observed between
shedding levels of tracheal and cloacal swabs (although
 218 C. Terregino et al.
Table 1. Serological log2 haemagglutination inhibition titres in vaccinated groups.

21 days post vaccination 7 days post challenge 14 days post challenge 21 days post challenge 28 days post challenge
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Bird H5N2 H5N1 NDV H5N2 H5N1 NDV H5N2 H5N1 NDV H5N2 H5N1 NDV H5N2 H5N1 NDV
†
Group 1: vaccinated with Volvac AI
1 12 2 14 2 11 4 10 4 11 3
2 11 1 14 2 10 1 11 0 11 0
3 12 0 14 0 10 0 10 0 11 0
4 11 0 14 5 9 3 9 4 11 4
5 12 0 Dead Dead Dead Dead Dead Dead Dead Dead
6 11 0 12 1 8 3 9 4 9 3
7 9 0 9 1 Dead Dead Dead Dead Dead Dead
8 12 0 14 0 9 1 10 2 11 2
9 11 0 13 0 9 0 9 1 10 2
10 12 0 14 0 10 0 10 0 10 0
GMT 11.3 0.3 13.11 1.22 9.50 1.50 9.75 1.88 10.50 1.75

Group 2: vaccinated Volvac† AIND

21 12 0 6 10 0 7 9 1 5 7 2 5 8 1 6
22 13 0 7 15 4 8 13 3 6 10 3 6 10 2 6
23 12 0 6 11 0 6 11 0 4 7 0 5 8 0 5
24 12 0 7 11 0 8 10 1 6 10 1 6 9 1 7
25 11 0 6 11 1 7 10 2 4 10 2 5 9 2 5
26 10 0 5 10 0 6 9 0 3 6 0 4 7 0 4
27 12 4 6 13 3 8 11 2 5 10 3 6 9 2 7
28 11 0 6 11 1 8 10 1 4 10 2 5 9 1 6
29 10 0 6 Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead
30 10 0 6 10 1 7 9 1 5 9 1 5 8 1 6
GMT 11.3 0.4 6.1 11.33 1.11 7.22 10.22 1.22 4.67 8.78 1.56 5.22 8.56 1.11 5.78

H5N2 A/chicken/Mexico/232/94 antigen; H5N1 A/chicken/Egypt/1709-6/08 antigen; NDVUlster 2C antigen; GMT geometric mean titre.
 Table 2. Serological log2 haemagglutination inhibition titres in vaccinated (control) groups.

21 days post vaccination 28 days post vaccination 35 days post vaccination 42 days post vaccination 49 days post vaccination

Bird H5N2 H5N1 H5N2 H5N1 H5N2 H5N1 H5N2 H5N1 H5N2 H5N1
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Group 4: Volvac† AI vaccinated and uninfected

11 10 3 13 3 10 3 9 3 9 3
12 11 0 13 0 11 0 9 0 9 0
13 12 1 14 1 13 1 11 1 10 2
14 12 0 14 0 14 1 10 2 10 2
15 12 0 14 0 10 0 10 0 9 0
16 9 0 11 0 14 0 9 0 9 0
17 12 0 13 0 11 0 10 0 10 0
18 12 0 13 0 12 0 9 0 9 0
19 10 0 13 0 12 0 8 0 8 0
20 9 0 13 0 12 0 7 0 6 0
GMT 10.9 0.4 13.1 0.4 11.9 0.5 9.2 0.6 8.9 0.7
Group 5: Volvac† AIND vaccinated and uninfected
31 11 0 6 11 0 8 10 0 5 9 0 5 9 0 5

Vaccine protection against HPAI Egyptian H5N1 219

32 11 0 6 11 0 7 10 0 5 8 0 4 9 0 7
33 10 0 6 10 0 7 10 0 6 9 0 5 9 0 7
34 11 3 7 12 3 8 9 3 6 9 3 5 9 2 6
35 12 0 4 10 0 5 9 1 3 7 0 3 7 0 4
36 10 0 6 12 1 8 8 1 5 9 0 5 10 0 6
37 11 0 6 11 0 7 8 0 5 8 0 4 9 0 6
38 11 0 6 11 0 7 8 0 4 7 0 3 8 0 5
39 11 0 6 11 0 7 10 0 4 8 0 4 8 0 6
40 10 0 7 12 0 9 11 0 6 9 0 5 10 0 7
GMT 10.8 0.3 6 11.1 0.4 7.3 9.3 0.5 4.9 8.3 0.3 4.3 8.8 0.2 5.9

H5N2 A/chicken/Mexico/232/94 antigen; H5N1 A/chicken/Egypt/1709-6/08 antigen; NDV Ulster 2C antigen; GMT geometric mean titre.
 220 C. Terregino et al.
overall tracheal shedding was higher than cloacal shed-
ding).
Discussion
International organizations recommend that vaccines
used for control of AI be of high quality and meet the
standards of international health guidelines. The experi-
ments performed in this evaluation comply with the OIE
standards (OIE, 2008b) and the minimum requirements
indicated by the European Medicine Agency (2006) for
vaccines for use in birds against HPAI viruses.
According to the OIE, potency tests may rely on the
measurement of challenge and assessment of morbidity
(the ratio of diseased to healthy birds in the population)
and quantitative reduction in challenge virus replication
in respiratory (oropharyngeal or tracheal) and intestinal
(cloacal) tracts.
According to the European Medicine Agency, the
efficacy of the inactivated vaccine against AI should be
demonstrated in laboratory conditions by a challenge
model aiming at defining the onset and the duration of
immunity for each of the indicated target species. A high
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degree of protection against mortality and clinical signs
of disease and a significant reduction of excretion and
transmission of the challenge virus must be the major
goals.
In this experiment we established the efficacy of two
commercial products in a standardized challenge model.
The number of birds and the composition of the
experimental groups were in accordance with previous
experiments (Swayne et al., 1997; Capua et al., 2002;
Terregino et al., 2007).
The challenge dose used (105 EID50/0.1 ml) and the
strain chosen are in line with the OIE standards for
vaccine potency tests, which provide that challenged
control birds must all die within the sixth day post
challenge. All unvaccinated birds were positive by real-
time RT-PCR and virus isolation both in the respiratory
(tracheal swabs) and intestinal (cloacal swabs) tract.
Even though some of the birds were found negative
during the first shedding evaluation (day 3 post infec-
tion), all of the birds were already showing clear disease
signs 2 days after the administration of the challenge
virus, therefore the death of all subjects must be related
to the direct administration of the challenge virus.
Volvac† AIND-vaccinated chickens (Group 2)
protected 90% of challenged birds from lethal infection.
The only chicken that showed severe clinical signs died
within a few days. This bird shed virus from days 3 to 5
post infection (when it was found dead). All the other
chickens in the group did not shed detectable RNA or
virus in the days chosen for the collection of the swab
samples.
In the group vaccinated with Volvac† AI (Group 1),
80% of the challenged birds were protected from lethal
infection.
There was no statistically significant difference in the
survival rate between the two vaccinated/challenged
experimental groups.
No significant increase of serological titres was
observed in vaccinated and infected surviving chickens
4 weeks after challenge. This is in accordance with other
studies (Capua et al., 2002; Terregino et al., 2007) and
could possibly represent a lack of replication of the
challenge virus in the surviving vaccinated birds. The
negative results from the DIVA test strengthen this
hypothesis.
The level of detectable antibodies against H5 was
surprisingly high after just one administration of both
vaccines using the homologous strain of the vaccine
virus in HI tests. This could be due to the high quality of
the components of the vaccine batch used and presum-
ably due to the composition of the adjuvant, which has
not been divulged by the producers of the vaccine. The
lower titres detected towards ND virus can be explained
both by the lower concentration of the ND antigen in
the bivalent vaccine with respect to AI virus and the use
of an heterologous strain (Ulster 2C) to the vaccine virus
as HA antigen in the HI assay. In fact, the use of the
homologous La Sota antigen in the HI assay would
have resulted in significantly higher titres than using the
heterologous Ulster virus (Maas et al., 1998). On the
other hand, for the AI viruses, the low genetic and
antigenic similarity between the vaccine virus and the
challenge virus is in keeping with the very low HI titres
observed using the challenge virus as antigen. It must
also be taken into consideration that the challenge virus
emerged after the extended use of field vaccination
against H5 in Egypt and for this reason it could have
emerged as an escape mutant. This would explain the
low levels of cross-reactivity between the challenge virus
and sera obtained from vaccinated birds as measured by
HI tests.
According to the definition reported above in the
evaluation of efficacy section, the only birds that
developed infection according to the first set of criteria
were the birds that developed clinical disease and died of
HPAI.
The data obtained from this study show a significant
increase of the survival rate in vaccinated birds, a
significant reduction in sick/dead birds, and a significant
reduction in the number of birds shedding the challenge
virus in vaccinated birds, which results in an overall
reduction of shedding levels
Another important element for the OIE and the
European Medicine Agency evaluation of the potency
of vaccines against AI is the assessment of the immune
response. For a good-quality vaccine, the onset of
immunity should be as rapid as possible to allow for
the use of the vaccine in emergency conditions. In
addition, the duration of immunity induced by the
vaccine should cover the economic life of the target
species.
Volvac† AI and Volvac† AIND induced a high
titred immune response in chickens vaccinated at
3 weeks of age. A high level of specific antibody against
the H5 protein was seen until the end of the study period
(70 days of age), as shown in Table 2.
The results comply with previous work in which the
ability of genetically distant vaccines against avian
influenza in preventing infection, disease, and transmis-
sion in chickens and ducks was investigated (Swayne
et al., 2006; Van der Goot et al., 2008). In these papers
the authors showed that despite the low level of
homology between the challenge viruses (Asian HPAI
H5N1) and the vaccine strain (Mexican LPAI H5N2)
transmission, the mortality and disease rate were mark-
edly reduced after vaccination.
The efficacy of vaccination even in the absence of
measurable specific HI antibodies against the HA of the
challenge virus could be explained by the generation of

antibodies against conserved proteins (i.e. matrix pro-
teins, nucleoprotein and non-structural proteins) that
could be cross-protective in birds (Imai et al., 2007; Van
der Goot et al., 2008). This may also suggest that cellular
immunity plays a more important role in AI vaccination
efficacy than originally thought (Khalenkov et al., 2009).
Both the former and the latter have been described to
play a role in protecting mammalian species such as mice
(Tompkins et al., 2007; Zhirnov et al., 2007) and ferrets
(Price et al., 2009).
Our findings also suggest that the HI test alone is
likely to underestimate the degree of protection, since
other factors, which are not measurable with this test,
contribute to in vivo protection.
In conclusion, despite the genetic and antigenic
differences between the vaccine strain (H5N2/Mexico)
and the challenge strain (H5N1/Egypt) used in this
experiment, it can be concluded that the immune
response induced by a single administration of both
Volvac† AI and Volvac† AIND vaccines in SPF birds
is sufficient to prevent infection in the majority of birds
challenged with a contemporary H5N1 Egyptian strain
under experimental conditions.
Downloaded by [181.198.171.9] at 20:04 17 February 2015
The results also indicate that while the vaccine
protects 100% of birds against disease and mortality,
some birds are still susceptible to infection and might
contribute to virus spread particularly in populations
that have low vaccination coverage, which is often the
case in countries such as Egypt and Indonesia. Further-
more, inadequate vaccination practices can lead to the
selection of variants exhibiting antigenic drift.
It is known that vaccination alone has not been
successful in achieving AI eradication (Capua &
Marangon, 2004; EU Commission, n.d.). Indeed, it is
our opinion that if vaccination is used and not managed
appropriately, eradication will not be obtained and the
resulting public health threat will not be removed.
Experience has shown that, to be successful in control-
ling and ultimately eradicating AI infection, vaccination
must be part of a wider control strategy that includes
biosecurity and monitoring of the evolution of infection.
Acknowledgements
The present work was funded by Boehringer Ingelheim
Vetmedica. The authors gratefully acknowledge Dr
Mona Aly of the Animal Health Research Institute in
Egypt for the provision of isolates, and Dr William
Dundon and Dr Nadia Micoli for editing the manu-
script.
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