Vaccine 40 (2022) 1837–1845

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Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Vaccination with a fowl adenovirus chimeric fiber protein (crecFib-4/11)

simultaneously protects chickens against hepatitis-hydropericardium
syndrome (HHS) and inclusion body hepatitis (IBH)
Carlotta De Luca a,b,⇑, Anna Schachner a, Sarah Heidl a, Michael Hess a,b
a
Christian Doppler Laboratory for Innovative Poultry Vaccines (IPOV), University of Veterinary Medicine, Vienna, Austria
b
Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria

a r t i c l e i n f o a b s t r a c t

Article history: In the past decades, fowl adenovirus (FAdV)-related diseases became an increasing concern for the poul-
Received 21 December 2021 try industry worldwide. Various immunization strategies against FAdVs have been experimentally inves-
Received in revised form 28 January 2022 tigated, with a particular focus on subunit vaccines against hepatitis-hydropericardium syndrome (HHS),
Accepted 30 January 2022
caused by FAdV serotype 4, and inclusion body hepatitis (IBH), caused by serotypes 2, 8a, 8b and 11. In
Available online 10 February 2022
this study, we extended our innovative concept of recombinant chimeric fiber proteins to design a novel
chimera combining epitopes from two distinct serotypes, FAdV-4 and -11, and we investigated its efficacy
Keywords:
to simultaneously protect chickens against HHS and IBH. Specific pathogen-free chickens were vacci-
Fowl adenovirus
Chimeric fiber
nated with the novel recombinant chimeric fiber and subsequently challenged with either a HHS- or
Cross-protection IBH-causing strain. Vaccinated/challenged birds exhibited a reduction of clinical signs, limited hep-
Hepatitis-hydropericardium syndrome atomegaly and lower levels of AST compared to the respective challenge controls. Furthermore, the vac-
Inclusion body hepatitis cine prevented atrophy of HHS-affected lymphoid organs, such as thymus and bursa of Fabricius, and
Subunit vaccine viral load in the target organs was significantly reduced. Clinical protection was associated with high
levels of pre-challenge antibodies measured on ELISA plates coated with the vaccination antigen.
Interestingly, the development of neutralizing antibodies was limited against FAdV-11 and absent against
FAdV-4, indicating that protection granted by such an antigen may be linked to different immunization
pathways. In conclusion, we proved that the concept of chimeric fiber vaccines can be extended across
viral species boundaries and represents the first single-component FAdV subunit vaccine providing com-
prehensive protection against different FAdV-associated diseases.
Ó 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction specie (Fowl aviadenovirus A to Fowl aviadenovirus E (FAdV-A to

FAdV-E)) based on genomic features, and 12 subordinate serotypes
Fowl adenoviruses (FAdVs), from the family Adenoviridae, genus (FAdV-1 to -8a, -8b to -11) defined by cross-neutralization [1,2].
Aviadenovirus, are non-enveloped dsDNA viruses classified into five Though all three fowl adenoviral disease complexes are caused
by different FAdV species, hepatitis-hydropericardium syndrome
(HHS) and inclusion body hepatitis (IBH) share characteristic
Abbreviations: AGE, adenoviral gizzard erosion; AST, aspartate transaminase; pathogenic and immune mechanisms, being overall very distinct
BW, body weight; CEL, chicken embryo liver; dpb, days post booster; dpc, days post from those of adenoviral gizzard erosion (AGE). One of the under-
challenge; dpv, days post vaccination; ELISA, enzyme-linked immunosorbent assay;
lying reasons appears to be a closer molecular relationship
FAdV, fowl adenovirus; HHS, hepatitis-hydropericardium syndrome; IBH, inclusion
body hepatitis; nAbs, neutralizing antibodies; non-nAbs, non-neutralizing antibod-
between the causative types of HHS (FAdV-4, species FAdV-C)
ies; OD, optical density; ORF, open reading frame; PBS, phosphate buffered saline; and IBH (FAdV-2/-11, FAdV-D; FAdV-8a and -8b, FAdV-E) as com-
qPCR, quantitative polymerase chain reaction; SPF, specific pathogen-free; TCID50, pared to the genetically separated FAdV-1 (species FAdV-A)
50% tissue culture infective dose; VNT, virus neutralization test. responsible for AGE [3,4]. Despite worldwide distribution and sev-
⇑ Corresponding author at: Clinic for Poultry and Fish Medicine, University of
ere economic impact of FAdV-associated diseases, commercially
Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria.
E-mail addresses: [email protected] (C. De Luca), anna.schachner@
available immunization strategies rely on a few registered vaccines
vetmeduni.ac.at (A. Schachner), [email protected] (S. Heidl), michael. mainly to prevent HHS with the extension towards autogenous
[email protected] (M. Hess).

https://doi.org/10.1016/j.vaccine.2022.01.060
0264-410X/Ó 2022 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
C. De Luca, A. Schachner, S. Heidl et al. Vaccine 40 (2022) 1837–1845

vaccines in recent years. In addition, this has been tackled by DNA obtained from transformed Escherichia coli DH10Bac (Invitro-
experimental development of alternative vaccination antigens, gen, Vienna, Austria). The construct was then purified via polyhis-
including certain live and inactivated, as well as subunit vaccines, tidine tag on affinity chromatography columns (His GraviTrap, GE
with a particular focus on the latter given their relative ease and Healthcare, Freiburg, Germany) as described previously [7]. Protein
efficiency of production, as recently summarized by Schachner concentration was determined via Bradford assay (Thermo Scien-
et al. [3]. In the majority of studies, structural proteins of the FAdV tific, Vienna, Austria).
capsid were used for subunit vaccine formulation, based on their
immunogenic potential and role in conferring antigenicity to the 2.2. Virus preparation
virus [5]. In particular, recombinant penton base and fiber,
obtained from FAdV-4, proved successful for protecting chickens Field isolates AG234 and 08–18926 (GenBank accession no.
against the cognate disease, HHS [6,7,8,9]. MK572849 and MK572871) were analyzed through Next-
Despite this, vaccines with an extended protection spectrum are generation sequencing and virus neutralization test (VNT) and thus
still pending and highly required due to simultaneous occurrence identified as members of types FAdV-4 and -11, respectively [20],
and mixed infections with diverse FAdV strains in the field to be used as challenge strains during the protection studies. The
[10,11,12,13,14,15,16,17]. However, the variety of etiological FAdV strains were 3-fold plaque purified and propagated on primary
types, concordant with genetically distinct fibers, somewhat antag- chicken-embryo liver (CEL) cells [23]; viral titers were determined
onizes the idea of a broadly efficacious subunit vaccine. by endpoint titration [24].
Dependent on the diversification of fibers within the same spe-
cies, cross-protection is even problematic for targeting a single dis-
2.3. Animal experiment
ease complex, as confirmed for the fibers of types -8a and -8b,
which are each efficient antigens against the homotypic IBH chal-
One hundred and twenty specific pathogen-free (SPF) chicks
lenge while providing no vice-versa protection [18,19]. In addition,
were hatched, individually tagged and divided into six groups
natural recombination of fibers, mainly between those two FAdV
(n = 20) under the following designation: vaccinated-only (v/
types, has been identified as another potential hurdle for vaccina-
crecFib-4/11), vaccinated and challenged with FAdV-4 (v/c/FAdV-
tion strategies [20].
4), vaccinated and challenged with FAdV-11 (v/c/FAdV-11),
In order to address this issue and provide coverage against the
FAdV-4 challenge control (c/FAdV-4), FAdV-11 challenge control
entire spectrum of IBH strains within species FAdV-E, a recombi-
(c/FAdV-11), and negative control (summarized in Table 1). Each
nant chimeric fiber (crecFib) protein containing epitopes from both
group was housed separately in isolator units (HM2500, Montair,
FAdV-8a and -8b was recently designed, and proved to be efficient
The Netherlands). For immunization, a prime-booster vaccination
in protecting chickens from both serotypes [21]. It is not known,
scheme with intramuscular administrations at 1 and 7 days of
however, if this principle can be extended and therefore applied
age was adopted, according to the model proposed by Schachner
to comprehensively protect chickens against different FAdV-
et al. [21]. A dose of 0.5 ml of the vaccine containing 50 mg of
caused diseases, especially given the phylogenetic distance
crecFib-4/11 homogenized in an oil-based adjuvant was injected
between the amino acidic structures of fibers belonging to distinct
intramuscularly in the Musculus tibialis lateralis of 1-day-old birds.
FAdV species [20], as well as the dissimilarities in the immune
Birds of the challenge control groups were only given phosphate
mechanisms associated with FAdV subunit vaccines. In particular,
buffered saline (PBS) mixed with adjuvant, and the negative con-
protection from IBH seems to be linked to the production of neu-
trol group was administered with sterile PBS only. The booster
tralizing antibodies, whereas experimental studies point towards
injection was administered in the same way at 7 days of age (6 days
other mechanisms involved to protect birds against HHS [7,22].
post vaccination, dpv) in the three vaccinated groups, and birds of
Independent of this, the chimeric fiber concept potentially holds
the challenge and negative controls received an injection of adju-
the key to unlock a comprehensive broad protection against the
vant mixed with PBS and PBS only, respectively, as described
different FAdV diseases, although merging epitopes from different
above. Blood was collected in weekly intervals from 14 days of
FAdV species has never been attempted so far. Therefore, this study
age up to challenge, then at each of the following sampling time
represents the first description of a chimeric protein retaining the
points: 3, 5, 7 and 14 days post challenge (dpc). The birds were
individual epitopic identities (-C/4 and -D/11), and its subsequent
challenged intramuscularly at 22 days of age (15 days post booster,
in vivo testing to protect chickens from both HHS and IBH
dpb) using 200 ml of 107 50% tissue culture infectious dose
simultaneously.
(TCID50)/ml of FAdV-4 strain AG234 (groups v/c/FAdV-4 and c/
FAdV-4) or FAdV-11 field strain 08–18926 (groups v/c/FAdV-11
and c/FAdV-11). In the time period after challenge, birds were
2. Materials and methods
monitored daily for clinical signs related to HHS and IBH, such as
depression, ruffled feathers, huddling behavior, and reluctance to
2.1. Design and expression of chimeric fiber
move and take in feed [3]. Up to five birds per group were sacri-

A chimeric fiber protein retaining epitopes from FAdV-4 and

FAdV-11 was designed in this study following the strategy and Table 1
methods described by Schachner et al. [21]. Briefly, the open read- Design of animal experiment
ing frames (ORFs) of fibers from FAdV-4 (fib-2) and FAdV-11 were group designation vaccination challenge strain
divided into an amino (N)- and a carboxy (C)-distal segment, then
v/crecFib-4/11 vaccine-only crecFib-4/11 –a
amplified and fused seamlessly via Gibson assembly cloning. The v/c/FAdV-4 vaccine vs. FAdV-4 crecFib-4/11 AG234 (FAdV-4)
specificity switch was engineered at an intertype consensus motif v/c/FAdV-11 vaccine vs. FAdV-11 crecFib-4/11 08–18926 (FAdV-11)
corresponding to positions aa491-492 in the pairwise sequence c/FAdV-4 FAdV-4 challenge adjuvant only AG234 (FAdV-4)
alignments of FAdV-4 fiber-2 and FAdV-11 fiber. The resulting con- control
c/FAdV-11 FAdV-11 challenge adjuvant only 08–18926 (FAdV-11)
struct was named crecFib-4/11. Detailed information on the clon- control
ing strategy is provided in Supplementary table 1. The neg. contr. negative control – –
recombinant chimeric fiber protein was expressed in Spodoptera a
not applicable
frugiperda Sf9 cells after transfection with recombinant baculovirus
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ficed and submitted to necropsy at 3, 5, 7 and 14 dpc, and individ- 3. Results

uals that were euthanized due to severe clinical affection were
necropsied and sampled immediately. During necropsy, the 3.1. Clinical protection
organ-body weight (BW) ratio of liver, spleen, thymus and bursa
of Fabricius was recorded in order to determine the presence and Out of twenty birds, sixteen individuals belonging to the FAdV-
degree of hepatomegaly, splenomegaly and atrophy of the lym- 4 challenge control showed clinical affection after challenge
phoid organs. (Table 2). In this group, clinical signs started at 2 dpc with mild
All the procedures were discussed and approved by the institu- depression in two birds, and progressed across the group with 13
tional ethics committee and licensed by the Austrian government affected birds at 3 dpc, three of which showing severe depression;
(license number GZ 2020–0.220.316). at 4 dpc there were nine affected birds, and two of them had to be
euthanized due to their inability to move and take feed; at 5 dpc,
three birds were still showing milder clinical signs. In the FAdV-
2.4. Clinical chemistry
11 challenge control, only one bird was affected with mild depres-
sion at 4 dpc. Among the vaccinated groups, only one individual
Immediately before euthanasia, blood was collected from the
challenged with FAdV-4 showed signs of depression (3 dpc), mark-
jugular vein of each bird from the infected groups and negative
ing a strongly significant difference compared to the respective
control into heparin tubes (VACUETTEÒ, Greiner Bio-One,
challenge control (p < 0.001). No clinical signs were observed in
Kremsmünster, Austria) to investigate the aspartate transaminase
the groups v/crecFib-4/11, v/c/FAdV-11, and negative control
(AST) content in plasma as previously described [25].
throughout the whole experiment. The recorded parameters for
the birds euthanized at 4 dpc were included in the 5 dpc cluster
2.5. Quantitative polymerase chain reaction (qPCR) from tissues of for statistical analyses.
target organs Mean liver-BW ratio was significantly increased from 3 to 7 dpc
in c/FAdV-4 compared to the negative control, and from 3 to 5 dpc
Tissue samples for quantification of viral load, including liver, for the v/c/FAdV-4 group, whereas the spleen-BW ratio remained
spleen and bursa of Fabricius, were collected from birds of the affected up to 7 dpc in both groups (Fig. 1i, 1ii). Group c/FAdV-4
infected groups and negative control, and stored at 20 °C until also showed a significant decrease in the organ-BW ratio of bursa
processing. DNA extraction was performed with DNeasy Blood & of Fabricius, compared to the negative control, at 3 and 5 dpc,
Tissue Kit (Qiagen, Hilden, Germany) according to manufacturers and of the thymus-BW ratio at 5 and 7 dpc, both differently from
protocol, and the DNA was subsequently analyzed with an adapted the respective vaccinated group (Fig. 1iii, 1iv). Group c/FAdV-11
qPCR assay based on the 52 K gene [26]. showed a significant increase of the liver-BW ratio at 3 and 7
dpc, and of the spleen-BW ratio from 3 to 7 dpc, whereas group
v/c/FAdV-11 only showed significant affection of the spleen-BW
2.6. Chimeric fiber enzyme-linked immunosorbent assay (ELISA) and
ratio at 5 dpc.
virus neutralization test (VNT)

Sera collected during the experiments were tested on ELISA 3.2. Plasma AST
plates coated with the vaccination antigen crecFib-4/11, and their
OD value was determined following the protocol described by Plasma AST was significantly increased compared to the nega-
Feichtner et al. [27]; the cut-off OD value was calculated from tive control from 3 to 7 dpc in c/FAdV-4, and from 3 to 5 dpc in
the sera of the negative control birds by computing the arithmetic v/c/FAdV-4, although the values of the vaccinated birds were sig-
mean plus three times the standard deviation. Samples from 21, 27 nificantly lower compared to c/FAdV-4 at these time points
and 29-day-old birds (respectively: pre-challenge, 5 dpc and 7 dpc) (Fig. 2). At 7 dpc, AST values from c/FAdV-11 birds were signifi-
were also investigated for neutralizing antibodies (nAbs) as earlier cantly increased compared to those from the negative control,
described [7], with VNT against the strains that served as a tem- whereas values from v/c/FAdV-11 were significantly increased at
plate for fiber expression (template strains) and the field isolates 14 dpc.
used as challenge strains during the protection studies (challenge
strains). 3.3. Viral load in target organs

2.7. Statistical analyses The mean viral load of v/c/FAdV-4 birds was significantly lower
than in c/FAdV-4 in liver at 5 dpc, in spleen at 3 dpc, and in bursa of
Significance between the clinical signs observed within the Fabricius at both time points (Fig. 3). No significant differences
groups challenged with FAdV-4 (v/c/FAdV-4 vs. c/FAdV-4) and were recorded in the analyzed organs between v/c/FAdV-11 and
the ones challenged with FAdV-11 (v/c/FAdV-11 vs. c/FAdV-11) c/FAdV-11 at any time point. At 14 dpc, it was possible to detect
was assessed comparing the numbers of symptomatic birds per viral DNA exclusively in the spleen of birds from FAdV-4-infected
group through chi-square test. For the numeric datasets, a prelim- groups.
inary analysis was carried out using Shapiro-Wilk test together
with a visual inspection of histograms and normal Q-Q plots in 3.4. Antibody response: ELISA and VNT
order to verify the normal distribution assumption. The mean val-
ues for organ-BW ratios, plasma AST and viral load in target organs The cut-off value for crecFib-4/11 ELISA was calculated at OD
of vaccinated groups were compared with the negative control and 0.12. At 14-day-old (7 dpb), the mean OD of all vaccinated birds
their respective challenge control group via unpaired Student’s t- (n = 60: from v/crecFib-4/11, v/c/FAdV-4, v/c/FAdV-11) was OD
test. Mann-Whitney U test was used for the datasets that did not 0.48 ± 0.48, with 81.7% (49/60) of the birds above the cut-off
meet normality assumptions. In each case, p values  0.05 were (Fig. 4). The day before challenge (21-day-old, 14 dpb), the mean
considered statistically significant. Statistical analyses were per- OD value of vaccinated birds raised up to 2.43 ± 0.87 with all the
formed with the software package SPSS Version 26 (IBM SPSS birds being above the cut-off. Adjuvant-injected birds (n = 40: from
Statistics; IBM Corp., Armonk, New York, USA). c/FAdV-4 and c/FAdV-11) had mean OD values of 0.07 ± 0.03 and
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Table 2
Daily clinical signs for each experimental group following challenge. The number of affected individuals is indicated in brackets. No clinical signs were recorded before 2 dpc and
after 5 dpc.

group clinical signs symptomatic/

total birds
2 dpc 3 dpc 4 dpc 5 dpc
v/crecFib-4/11 –a – – – 0/20
v/c/FAdV-4 – depression (n = 1) – – 1/20c
v/c/FAdV-11 – – – – 0/20
c/FAdV-4 mild depression (n = 2) mild depression (n = 9) mild depression (n = 5) mild depression (n = 2) 16/20
depression (n = 1) depression (n = 2) depression (n = 1)
severe depression (n = 3) severe depression, bird unable to
stand or take feed (n = 2)b
c/FAdV-11 – – mild depression (n = 1) – 1/20
neg. contr. – – – – 0/20
a
no clinical signs observed
b
birds were euthanized
c
significant difference (p  0.001) compared to the respective challenge control

Fig. 1. Organ-BW ratios post-challenge. Mean and standard deviation of liver-BW ratio (i), spleen-BW ratio (ii), bursa-BW ratio (iii) and thymus-BW ratio (iv) for each
experimental group in the time period after challenge. Significant difference against negative control is indicated with letter ‘‘a”, whereas significant difference against
respective challenge control is indicated with letter ‘‘b”. Significance was assessed at p  0.05. No significant difference was observed for thymus-BW ratio between v/c/FAdV-
11 and c/FAdV-11 at any measured time point.

0.05 ± 0.01 at 14- and 21-day-old respectively. Mean OD of the v/ 3.39 ± 0.13 at 14 dpc), whereas c/FAdV-11 birds showed low levels
crecFib-4/11 group suffered a decrease at 25-day-old (1.70 ± 1.09) of antibody development (peak at 14 dpc with mean OD 0.28 ± 0.
before continuously increasing throughout the experiment, reach- 13). Sera of negative control birds remained below the cut-off
ing an OD of 3.46 ± 0.01 at 36-day-old (29 dpb). After challenge, value throughout the whole experiment.
mean OD of v/c/FAdV-4 group raised to 3.09 ± 0.42 at 3 dpc and The day prior challenge, only two vaccinated birds displayed
reached the highest end of measurable values at 5 dpc (3.32 ± 0. some degree of nAbs against the FAdV-11 template strain, one of
28), remaining then consistent in its plateau until the end of the them reacting against both template and challenge strain (titers:
experiment. In comparison, a sharp rise in ELISA-measured anti- 3 and 8 log2 respectively), one only against the template strain
bodies was observed in c/FAdV-4 birds from 5 to 14 dpc (mean (titers: 5 log2), and two against the challenge strain (titers: 3 and
OD 0.25 ± 0.15 to 2.57 ± 0.44), although they remained lower than 9 log2) (Fig. 5). In contrast, there was no neutralizing activity
the vaccinated group. Birds from v/c/FAdV-11 quickly reached the against any of the FAdV-4 strains, and the vaccine-only group
plateau after challenge as well (mean OD 3.24 ± 0.13 at 3 dpc to saw no nAbs development at any of the tested time points. The rise

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Fig. 2. AST mean values post-challenge. Mean and standard deviation of plasma AST for each experimental group in the time period after challenge. Significant difference
against negative control is indicated with letter ‘‘a”, whereas significant difference against respective challenge control is indicated with letter ‘‘b”. Significance was assessed
at p  0.05.

Fig. 3. Viral load in main target organs post-challenge. Mean and standard deviation of viral load (expressed in log10) for each experimental group in the time period after
challenge in liver (a), spleen (b) and bursa of Fabricius (c). Asterisks indicate significant difference compared to the respective challenge control. Significance was assessed at
p  0.05.

in nAbs against FAdV-4 started after challenge in v/c/FAdV-4 and c/ 6.67 ± 1.87 log2 vs. 6.10 ± 0.99 log2 at 7 dpc). Experimental groups
FAdV-4, showing similar values against both the challenge strain infected with FAdV-11 showed a similar pattern against the chal-
(mean titer: 5.07 ± 2.09 log2 vs. 4.57 ± 1.83 log2 at 5 dpc, and lenge strain, although titers in sera from v/c/FAdV-11 birds were
7.56 ± 1.51 log2 vs. 6.90 ± 0.99 log2 at 7 dpc) and the template slightly lower than in c/FAdV-11 (mean: 3.79 ± 2.78 log2 vs.
strain (2.53 ± 2.56 log2 vs. 3.64 ± 2.37 log2 at 5 dpc, and 5.36 ± 2.68 log2 at 5 dpc, and 5.90 ± 1.10 log2 vs. 7.00 ± 1.63 log2

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4. Discussion

FAdV diseases, with discrete clinical pictures caused by particu-

lar serotypes, are an economic burden for the poultry industry
worldwide, with an important increase of outbreaks observed in
recent years asking for new protection strategies [28]. As a general
paradigm, the immunity developed against a specific FAdV type
does not confer protection against other types. This conclusion is
supported by observed shifts towards outbreaks with other viral
types after implementation of a vaccination regimen against the
previously dominating ones [29,30,31,32]. An increasing aware-
ness of antigenically diverse FAdV types co-circulating in the field,
and the demand for their control, explains why much research has
been dedicated lately to elucidating cross-protectivity of candidate
antigens, with less promising results so far in regards to recombi-
Fig. 4. Antibody development post vaccination. Mean titers (OD) for experimen- nant proteins. On the contrary, heterotypic efficacy was demon-
tal groups tested on crecFib-4/11 ELISA throughout the protection study. Values strated within the FAdV-E species with live vaccines [33].
given for the time points prior challenge (14 and 21 days of age) are cumulative for Furthermore, inactivated FAdV-C and -E strains were able to
the three vaccinated groups (v/crecFib-4/11, v/c/FAdV-4, v/c/FAdV-11) and the two
induce broad serotype coverage, extending the protection spec-
challenge control groups (c/FAdV-4, c/FAdV-11). The cut-off value is marked in
black. trum outside species boundaries as well [34,35]. Obviously, whole
virus formulations are more likely to produce a synergistic effect
from different antigenic components that cannot be replicated in
at 7 dpc), and mean titers were generally lower against the tem- the same way by subunit vaccines.
plate strain (0.29 ± 1.07 log2 vs. 0.50 ± 1.29 log2 at 5 dpc, and Subunit vaccines based on the fiber protein from FAdV types
2.80 ± 2.10 log2 vs. 2.30 ± 2.11 log2 at 7 dpc). Interestingly, a small causing hepatitis-hydropericardium syndrome (HHS) and inclu-
number of sera from v/c/FAdV-4 birds developed nAbs against sion body hepatitis (IBH) were repeatedly shown to ensure robust
FAdV-11 after challenge, with three individuals displaying nAbs protection against the respective disease conditions, although only
against both strains and one only against the template strain in settings limited to homologous vaccination/challenge systems
between 5 and 7 dpc. Data on the number of individuals exhibiting [7,8,19,36,37]. In fact, the FAdV fiber is a surface antigen that fea-
nAbs for each group are shown in Fig. 5. tures high levels of diversification between FAdV species and sero-

Fig. 5. Neutralizing antibodies development post vaccination. Mean and standard deviation of VNT titers (log2) before challenge (21 days of age) and at 5–7 dpc: v/c/FAdV-
4, v/c/FAdV-11 and c/FAdV-4 against the FAdV-4 challenge (a) and template (b) strain; v/c/FAdV-4, v/c/FAdV-11 and c/FAdV-11 against the FAdV-11 challenge (c) and
template (d) strain. Headers show the number of birds exhibiting nAbs (some individuals could not be tested due to a lack of serum volume). No nAbs were observed in sera
from v/crecFib-4/11 birds at any measured time point.

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types, and type-specificity of fiber-induced neutralization was recombinant protein, and/or the predominance of epitopes with
recently shown to be linked with failure to cross-protect FAdV-4 specific identity on crecFib-4/11.
[16,19,20]. Whereas this is not an issue for HHS and its mono- Clinical protection in absence of vaccine-elicited nAbs has been
type etiology, the diverse etiological nature of IBH requires the described before within the HHS system: FAdV-4 fiber vaccines
pursue of a more broad-protective immunization strategy. have been shown to provide full coverage against HHS despite lack
Recently, cross-protection between different IBH-causing sero- of neutralizing activity [7], a feature that was observed even upon
types was demonstrated with the introduction of a novel chimeric vaccination with live-attenuated FAdV-4 [22]. On the other hand,
fiber protein merging putative epitopes from both FAdV-8a and -8b immunization against IBH has been linked to stimulation of the
fibers [21]. This represents a promising concept towards broad- humoral immune response with subsequent production of nAbs
protection from FAdV-related diseases, but it has to be clarified for whole virus vaccines, either live or inactivated, and subunit
whether this strategy can be applied to simultaneously protect antigens as well [18,19,21,36,43]. However, it must be considered
chickens against IBH and HHS, despite their differences in etiology, that vaccine-induced nAbs against FAdV-11 were only noticed
pathogenesis and related immune mechanisms. In order to tackle after bivalent vaccination with live or inactivated vaccines,
this issue, a novel chimeric fiber retaining molecular characteris- whereas data for recombinant subunits only refer to the FAdV-
tics of both FAdV-4 and -11 (crecFib-4/11) was designed for this 8a/8b system [18,43]. In another study, a vaccine based on inacti-
study. This represents the first instance where a recombinant chi- vated FAdV-8a elicited detectable ELISA antibodies and was found
meric protein presenting combined epitopes from two different protective against FAdV-11 challenge despite the lack of cross-
FAdV species was successfully expressed, despite the important reacting nAbs in most of the experimental birds [35]. These data,
structural differences between fibers of FAdV-C and -D. The new together with the results of the present work, pose questions over
construct was then tested in vivo for its efficacy to immunize chick- the role of non-neutralizing antibodies (non-nAbs), which are
ens against different FAdV-associated diseases, such as HHS and already considered crucial for immunization against certain
IBH. viruses affecting chickens, such as specific subtypes of Avian Influ-
Vaccination of birds with crecFib-4/11 was able to prevent clin- enza [44]. Since non-nAbs are known to contribute in conferring
ical signs and limit pathological affection upon FAdV-4 and -11 coverage against viral diseases [45,46], our results, together with
infection, in contrast with the challenge control groups. The the reported lack of nAbs after immunization against HHS, suggest
reduced clinical signs noticed in the FAdV-4 challenge control that they may play an important role against FAdV-associated dis-
mark a substantial difference compared to a previous study by eases as well, depending on the serotype. Moreover, recent efforts
Schachner et al. [7], which utilized the same challenge strain anal- have highlighted the importance of vaccine-dependent cellular
ogously to the present work. This may be due to underlying differ- immunity when it comes to subunit antigens against both HHS
ences in the batches of SPF chickens between the two experiments, and IBH, marking it as another possible major player in the protec-
keeping in mind that IBH and HHS are strongly affecting the meta- tion from FAdV-related diseases [8,19,36]. Therefore, the present
bolism of the birds [38]. Various FAdV protection studies faced a work highlights the need of further investigations to dissect the
scarcity of severe clinical signs upon challenge, especially given mechanisms of humoral and cellular immune responses associated
the wide variability in pathogenicity among different strains, even with FAdV vaccines, not only systemically, but also on a local level
within the same serotype [19,21,34,35,39]. In general, it must be in regards to target and lymphoid organs.
considered that repeated passaging on embryonic eggs or cell cul- In conclusion, subunit vaccination with crecFib-4/11 protected
ture, necessary for plaque purification and viral propagation, can chickens from clinical signs and severe outcome of HHS, while at
result in virus attenuation, which could explain discrepancies the same time limiting pathological affection from both HHS and
between field observations and experimental trials [40,41]. In the IBH. Therefore, chimeric fibers are confirmed as an efficient protec-
present study, no mortality was noticed in the FAdV-11 challenge tion strategy to provide broad coverage against FAdVs, not only on
control, which is in agreement with a previous study using the heterotypic level, as previously demonstrated, but also across the
same strain [42]. In fact, results from other vaccination studies species boundary. On this basis, now that the merging of such
indicate that susceptibility to FAdV-11 varies according to the diverse molecular structure into a unique chimeric fiber was suc-
age and type of the birds, as well as route and dose of infection, cessfully achieved, the possibility of designing new chimeras
similarly to other FAdVs, as mentioned above [18,35,43]. For these including even more diverse epitopes and further extending the
reasons, a panel of parameters was taken into account to assess spectrum of protection has been unlocked. Although more investi-
protection in the present study, especially in order to counter the gations are needed to elucidate the immune mechanisms behind
mild effects of the FAdV-11 challenge: organ-body weight ratios, said protection, these findings represent a fundamental step
viral load in target organs, and plasma AST, with the latter con- towards reaching comprehensive cross-protection against the
firmed as a valuable indicator of hepatic health in the course of FAdV-mediated diseases affecting the poultry industry worldwide.
IBH infection [25].
Clinical protection provided by crecFib-4/11 against both HHS Declaration of Competing Interest
and IBH was linked to the induction of high and uniform levels
of systemic antibodies against the antigen used for vaccination, The authors declare the following financial interests/personal
measured with in-house fiber ELISA. However, a consistent lack relationships which may be considered as potential competing
of neutralizing antibodies (nAbs) was observed in the vaccinated interests: Michael Hess and Anna Schachner have patent ‘‘Fowl ade-
groups before challenge, as a very small number of birds exhibited novirus subunit vaccine and production method thereof” pending to
neutralization against FAdV-11, and none against FAdV-4. In con- European Patent Office.
trast, challenge control birds consistently showed neutralizing
activity against the respective challenge virus, although antibodies Acknowledgements
from the FAdV-11 challenge control showed a low level of reaction
with the crecFib-4/11 antigen on ELISA. It is therefore concluded The authors thank the Christian Doppler Research Association, in
that antibodies produced in response to the FAdV-11 virus recog- cooperation with Vaxxinova GmbH, Münster, Germany (grant nr.:
nize the chimeric fiber to a lesser degree compared to antibodies 189) for funding the present work. Furthermore, the authors wish
directed against the FAdV-4 challenge. This could be due to the to thank Dr. Erwin Mombarg from VaxxinovaÒ International BV for
conformation of the vaccine antigen, such as the folding of the providing the formulation of the vaccine and mock solutions used
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C. De Luca, A. Schachner, S. Heidl et al. Vaccine 40 (2022) 1837–1845

during the animal experiments. The authors also wish to thank [17] Niu Y, Sun Q, Zhang G, Sun W, Liu X, Xiao Y, et al. Epidemiological investigation
of outbreaks of fowl adenovirus infections in commercial chickens in China.
Prof. Dr. Ilse Schwendenwein and her team from the Clinical
Transbound Emerg Dis 2018;65(1):e121–6.
Pathology Platform, Department of Pathobiology, University of [18] Gupta A, Popowich S, Ojkic D, Kurukulasuriya S, Chow-Lockerbie B,
Veterinary Medicine Vienna, for performing the clinical chemistry Gunawardana T, et al. Inactivated and live bivalent fowl adenovirus (FAdV8b
analysis. Furthermore, the authors thank Amin Mirzazadehghas- + FAdV11) breeder vaccines provide broad-spectrum protection in chicks
against inclusion body hepatitis (IBH). Vaccine 2018;36:744–50.
sab, Mohammad Deyaa Almasri, Attila Sandor and Vesna Stanisavl- [19] De Luca C, Schachner A, Mitra T, Heidl S, Liebhart D, Hess M. Fowl adenovirus
jevic for assisting in the animal experiment. (FAdV) fiber-based vaccine against inclusion body hepatitis (IBH) provides
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Austrian government (license number GZ 2020-0.220.316). aviadenovirus E (FAdV-E) fiber, promoting the optimization of FAdV fiber
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